Pediatric Endocrinology [3 ed.] 9781437711097, 143771109X, 9781416040903

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PEDIATRIC ENDOCRINOLOGY, THIRD EDITION

ISBN: 978-1-4160-4090-3

Copyright © 2008, 2002, 1996 by Saunders, an imprint of Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier’s Rights Department: phone: (+1) 215 239 3804 (US) or (+44) 1865 843830 (UK); fax: (+44) 1865 853333; e-mail: [email protected]. You may also complete your request on-line via the Elsevier website at http://www.elsevier.com/permissions.

Notice Knowledge and best practice in this field are constantly changing. As new research and experience broaden our knowledge, changes in practice, treatment and drug therapy may become necessary or appropriate. Readers are advised to check the most current information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to verify the recommended dose or formula, the method and duration of administration, and contraindications. It is the responsibility of the practitioner, relying on their own experience and knowledge of the patient, to make diagnoses, to determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions. To the fullest extent of the law, neither the Publisher nor the Editor assumes any liability for any injury and/or damage to persons or property arising out or related to any use of the material contained in this book. The Publisher Library of Congress Cataloging-in-Publication Data Pediatric endocrinology / [edited by] Mark A. Sperling. — 3rd ed. p. ; cm. Includes bibliographical references and index. ISBN 978-1-4160-4090-3 1. Pediatric endocrinology. I. Sperling, M. [DNLM: 1. Endocrine System Diseases. 2. Child. 3. Infant. WS 330 P3712 2008] RJ418.P42 2008 618.92’4—dc22 2007041424

Acquisitions Editor: Judith Fletcher Developmental Editor: Colleen McGonigal Project Manager: Bryan Hayward Design Direction: Ellen Zanolle

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To my parents, who gave and sustained my life; to my wife, Vera, “woman of valor”; and our children, Lisa and Steven, Jonathan and Shoshana, and grandchildren, Jacob, Benjamin, Tzvi, Sydney, Rebecca, and Julian, who provide meaning, joy, and continuity to our lives.

Foreword to the First Edition

The aim of the editor and contributors to this volume is to establish an effective bridge between the surging progress in biomedical science and the clinical practice of pediatric endocrinology. Half a century ago the biochemical elucidation of the structure and subsequent synthesis of steroid hormones provided the basis for a revolution in the diagnosis and treatment of a number of endocrine and nonendocrine disorders; that era was soon followed by a succession of fundamental discoveries: structure of peptide hormones, identification of releasing hormones from the brain, rapid and precise assay methods, and synthesis of peptide hormones by molecular biological techniques, to name but a few. In no field has laboratory science been more effectively translated into clinical progress than in pediatric

endocrinology. A glance at the roster of contributors to this volume may well provide insight into why; many who are responsible for the dramatic advances in the laboratory also pursue active clinical careers. This volume includes many new sections that were not presented in previous texts devoted to clinical pediatric endocrinology. It will serve as a valuable reference for family physicians, internists, pediatricians, and other health professionals, covering as it does the gamut of information from basic molecular biology to practical considerations in the diagnosis and treatment of pediatric endocrine disorders. Solomon A. Kaplan, MD

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Preface

This third edition of Pediatric Endocrinology bears witness to the ongoing advances in this field, bringing together basic and clinical science and scientists for a deeper understanding of endocrine problems in infants and children, their diagnosis and their evidence-based treatment. No area of inquiry has been more impacted by the contribution of molecular biology, sophisticated imaging, and computer science to discover new knowledge, apply it, and manage it. There is not a single topic in this edition in which discoveries in the past five years have not occurred to provide new insights on old problems. To incorporate these comprehensive discoveries without significantly changing scope, size. and cost, it was necessary to realign some of the chapters. Hence, the chapters on “Organization and Integration of the Endocrine System” and “Imaging in Pediatric Endocrinology” have been reluctantly removed in this edition; the former is addressed in detail in more fundamental texts which approach endocrinology as the science of cell-to-cell communication via chemical messengers. In so doing, they blur previous concepts of hormones as chemical messengers produced in a single organ with specific effects on a limited number of other cell types. Instead, the newer understanding of endocrinology incorporates signal transduction cascades in various cell types in response to circulating messengers as diverse as cytokines into the family of “hormones.” The expanding horizons of sophisticated imaging could not be limited to a single chapter without compromising depth and quality; where relevant, appropriate references are provided in specific chapters, best exemplified by the use of 18-[F] L-DOPA positron emission tomography (PET) scanning to distinguish focal from diffuse hyperinsulinemia. Taking the place of these realigned chapters is a new chapter on dyslipidemias and an expanded chapter on energy homeostasis and its disorders. These metabolic topics were deemed to be essential inclusions for this book, reflecting the importance of the epidemic of obesity and its consequences in pediatric populations throughout the developing and developed world, and the significant time commitment to management by

pediatric endocrinologists. Likewise, management of dyslipidemias in children and adolescents is of increasing importance as data accumulate on the effectiveness and safety of lipid lowering agents in reversing intimal changes and delaying or preventing the consequences of atherosclerosis. As in previous editions, the book is divided into seamless sections. First, there is an introductory section on molecular endocrinology and endocrine genetics, receptor transduction cascades, and the complexity of normal homeostasis as exemplified by the paradigm of bone mineral metabolism. Three chapters on endocrine disorders in newborns follow cover the topics of ambiguous genitalia, hypoglycemia, and abnormalities of thyroid function. These, together with calcium disorders, form the most common endocrine problems in the newborn nursery for which endocrine consultation is sought. A series of chapters on specific disorders in childhood follows, including thyroid, growth, posterior pituitary, diabetes mellitus, hypoglycemia, the adrenal cortex, pheochromocytoma and multiple endocrine neoplasias, puberty in the female, Turner syndrome, disorders of the testes, disorders of calcium and bone mineral metabolism, autoimmune polyglandular syndromes, disorders of energy balance, and lipid disorders in children and adolescents. Finally, the chapter on laboratory methods in pediatric endocrinology is included because of the importance of understanding the methodologies used in correct and appropriate interpretation of test results. Each chapter has a comprehensive bibliography, which, like all modern hard paper texts, may be somewhat out of date by the time of publication. From an editor’s point of view, it has been a pleasure to welcome new contributors from Europe and Israel to the list of recognized authorities in their respective fields; the text is enriched by their expertise and so too will be its readers. For any oversight, omissions, or errors, we extend our apologies with request for indulgence and forgiveness from our readers. As always, we welcome your input to improve the value of this book. Mark A. Sperling Pittsburgh, Pennsylvania Spring 2008

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Acknowledgments

I gratefully acknowledge the contributions and support of each of the authors, who are my colleagues and friends. Our post-doctoral trainees, residents, and students provided constructive critique. Ms Kathy Wypychowski

provided invaluable secretarial assistance. I thank Elsevier and their staff for their support; it has been a pleasure working with them to bring this third edition to print.

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Contributors

John C. Achermann, MD Wellcome Trust Senior Research Fellow in Clinical Science Developmental Endocrinology Research Group Clinical and Molecular Genetics Unit UCL Institute of Child Health University College London, United Kingdom Steven D. Chernausek, MD Professor of Pediatrics Department of Pediatrics University of Oklahoma Health Sciences Center Oklahoma City, Oklahoma Pinchas Cohen, MD Professor of Pediatrics CMRI Edith Kinney Gaylord Chair Director, CMRI Diabetes & Metabolic Research Center UCLA School of Medicine Director, Division of Endocrinology Mattel Children’s Hospital at UCLA Los Angeles, California David W. Cooke, MD Associate Professor Department of Pediatrics Division of Pediatric Endocrinology The Johns Hopkins University School of Medicine Baltimore, Maryland Sarah C. Couch, PhD, RD Associate Professor Department of Nutritional Sciences University of Cincinnati Cincinnati, Ohio

Frank B. Diamond, Jr., MD Professor of Pediatrics University of South Florida College of Medicine Tampa, Florida All Children’s Hospital St. Petersburg, Florida Charis Eng, MD, PhD Sondra J. & Stephen R. Hardis Chair in Cancer Genomic Medicine Chair and Director, Genomic Medicine Institute Director, Center for Personalized Genetic Healthcare Cleveland Clinic Foundation Cleveland, Ohio Delbert A. Fisher, MD Professor Emeritus Pediatrics and Medicine David Geffen School of Medicine at UCLA Los Angeles, California Christa E. Flück, MD Assistant Professor Pediatric Endocrinology and Diabetology University Children’s Hospital Bern Bern, Switzerland Russel Grant, PhD Director of Mass Spectrometry LabCorp Calabasas Hills, California Annette Grueters, MD Professor of Pediatrics and Endocrinology Charite Children’s Hospital Humboldt University Berlin, Germany

Steven Daniels, MD, PhD Professor and Chairman Department of Pediatrics University of Colorado School of Medicine The Children’s Hospital Denver, Colorado

Michael J. Haller, MD Assistant Professor of Pediatrics Department of Pediatrics University of Florida Gainesville, Florida

Diva D. De León, MD Assistant Professor of Pediatrics Division of Endocrinology Children’s Hospital of Philadelphia Philadelphia, Pennsylvania

Ieuan A. Hughes, MD, FRCP Professor of Paediatrics University of Cambridge School of Clinical Medicine Department of Paediatrics Addenbrooks Hospital Cambridge, United Kingdom xiii

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CONTRIBUTORS

Sharon J. Hyman, MD Fellow, Pediatric Endocrinology and Diabetes Mount Sinai School of Medicine New York, New York David R. Langdon, MD Clinical Associate Professor of Pediatrics University of Pennsylvania School of Medicine Division of Endocrinology The Children’s Hospital of Philadelphia Philadelphia, Pennsylvania Peter A. Lee, MD, PhD Professor, Department of Pediatrics Penn State College of Medicine Staff Attending Physician, Department of Pediatrics MSHershey Medical Center Hershey, Pennsylvania Professor, Department of Pediatrics Indiana University School of Medicine Attending Physician, Department of Pediatrics Riley Hospital for Children Indianapolis, Indiana Robert H. Lustig, MD Professor of Clinical Pediatrics Division of Endocrinology Department of Pediatrics University of California San Francisco, California Joseph A. Majzoub, MD Chief, Division of Endocrinology Children’s Hospital Boston Thomas Morgan Professor of Pediatrics Professor of Medicine Harvard Medical School Boston, Massachusetts Ram K. Menon, MD Professor of Pediatrics Professor of Molecular and Integrative Physiology Director, Division of Endocrinology Department of Pediatrics University of Michigan Medical School Ann Arbor, Michigan Walter L. Miller, MD Professor of Pediatrics Chief of Endocrinology University of California San Francisco, California Louis J. Muglia, MD, PhD Alumni Endowed Professor of Pediatrics Washington University Medical School Director, Division of Pediatric Endocrinology and Diabetes St. Louis Children’s Hospital St. Louis, Missouri

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Sally Radovick, MD Professor of Pediatrics Director, Division of Pediatric Endocrinology The Johns Hopkins University School of Medicine Baltimore, Maryland Robert Rapaport, MD Emma Elizabeth Sullivan Professor Director, Division of Pediatric Endocrinology and Diabetes Mount Sinai School of Medicine New York, New York Alan M. Rice, MD Assistant Professor and Division Director Pediatric Endocrinology and Diabetes University of Nevada School of Medicine Las Vegas, Nevada Scott A. Rivkees, MD Professor and Associate Chair Chief Section of Developmental Endocrinology and Biology Department of Pediatrics Yale University School of Medicine New Haven, Connecticut Allen W. Root, MD Professor of Pediatrics Department of Pediatrics University of South Florida College of Medicine Tampa, Florida All Children’s Hospital St. Petersburg, Florida Ron G. Rosenfeld, MD Senior Vice-President for Medical Affairs Lucile Packard Foundation for Children’s Health Palo Alto, California Professor of Pediatrics Stanford University Stanford, California Professor of Pediatrics Oregon Health & Science University Portland, Oregon Robert L. Rosenfield, MD Professor of Pediatrics and Medicine The University of Chicago Pritzker School of Medicine Section of Pediatric Endocrinology Chicago, Illinois Paul Saenger, MD Professor of Pediatrics Albert Einstein College of Medicine Bronx, New York

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CONTRIBUTORS

Desmond A. Schatz, MD Professor of Pediatrics Department of Pediatrics University of Florida Gainesville, Florida Mark A. Sperling, MD Professor and Chair Emeritus Department of Pediatrics University of Pittsburgh School of Medicine Division of Endocrinology, Metabolism, and Diabetes Mellitus Children’s Hospital of Pittsburgh Pittsburgh, Pennsylvania Charles A. Stanley, MD Professor of Pediatrics University of Pennsylvania School of Medicine Director, Division of Endocrinology and Diabetes Children’s Hospital of Philadelphia Philadelphia, Pennsylvania Mark Stene, PhD Director, Laboratory Operations Quest Diagnostics West Hills, California Constantine A. Stratakis, MD, DSc Chief, Section on Endocrinology and Genetics (SEGEN) Director, Pediatric Endocrinology Training Program DEB, NICHD, NIH Bethesda, Maryland

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Massimo Trucco, MD Hillman Professor of Pediatric Immunology Head, Division of Immunogenetics Children’s Hospital of Pittsburgh Rangos Research Center University of Pittsburgh Pittsburgh, Pennsylvania Stuart A. Weinzimer, MD Associate Professor of Pediatrics Division of Pediatric Endocrinology and Diabetes Yale Medical School New Haven, Connecticut Ram Weiss, MD Assistant Professor of Pediatrics Hadassah Hebrew University School of Medicine Ein Kerem, Israel William E. Winter, MD Professor Departments of Pediatrics and Pathology, Immunology, and Laboratory Medicine University of Florida Gainesville, Florida Selma Feldman Witchel, MD Associate Professor Department of Pediatrics University of Pittsburgh Children’s Hospital of Pittsburgh Pittsburgh, Pennsylvania

William V. Tamborlane, MD Professor of Pediatrics Yale University School of Medicine Director, Division of Endocrinology and Diabetes Department of Pediatrics New Haven, Connecticut

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C H A P T E R

1 Molecular Endocrinology and Endocrine Genetics Ram K. Menon, MD • Massimo Trucco, MD • Constantine A. Stratakis, MD, DSc

Introduction Basic Molecular Tools Isolation and Digestion of DNA and Southern Blotting Restriction Fragment Length Polymorphism and Other Polymorphic DNA Studies Polymerase Chain Reaction RNA Analysis Detection of Mutations in Genes Direct Methods Indirect Methods Positional Genetics in Endocrinology The Principles of Positional Genetics Positional Cloning of Endocrine Genes Expression Studies: Microarrays and SAGE Chromosome Analysis and Molecular Cytogenetics Outline of Methods Applications Future Developments

Introduction The study of the endocrine system has undergone a dramatic evolution in the last two decades, from the traditional physiologic studies that had dominated the field for many years to the discoveries of molecular endocrinology and endocrine genetics.1,2 At the current time, the major impact of molecular medicine on the practice of pediatric endocrinology relates to diagnosis and genetic counseling for a wide variety of inherited endocrine disorders. In contrast, the direct therapeutic application of this new knowledge is still in its infancy. This chapter is an introduction to basic principles of molecular biology, com-

Molecular Basis of Pediatric Endocrinopathies Defects in Peptide Hormones Genomic Deletions Causing Human Endocrine Disease Point Mutations Defects in Peptide Hormone Receptors Insulin Receptor Growth Hormone Receptor Recombinant DNA Technology and Therapy of Pediatric Endocrine Diseases Concluding Remarks

mon laboratory techniques, and some examples of the recent advances made in clinical pediatric endocrinologic disorders—with an emphasis on endocrine genetics.

Basic Molecular Tools ISOLATION AND DIGESTION OF DNA AND SOUTHERN BLOTTING The human chromosome comprises a long doublestranded helical molecule of DNA associated with different nuclear proteins.3,4 Because DNA forms the starting 1

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point of the synthesis of all protein molecules in the body, molecular techniques using DNA have proven crucial in the development of diagnostic tools for analyzing endocrine diseases. DNA can be isolated from any human tissue, including circulating white blood cells. About 200 micrograms of DNA can be obtained from 10 to 20 ml of whole blood, with the efficiency of DNA extraction being dependent on the technique used and the method of anticoagulation employed. The extracted DNA can be stored almost indefinitely at an appropriate temperature. Furthermore, lymphocytes can be transformed with the Epstein-Barr virus to propagate indefinitely in cell culture as “immortal” cell lines—thus providing a renewable source of DNA. For performing molecular genetic studies, lymphoid lines are routinely the tissue of choice because a renewable source of DNA obviates the need to obtain further blood from the family. It should be noted that because the expression of many genes is tissue specific immortalized lymphoid cell lines cannot be used to analyze the abundance or composition of mRNA for a specific gene. Hence, studies involving mRNA necessitate the analysis of the tissue(s) expressing the gene—as outlined in the section on the analysis of RNA. DNA is present in extremely large molecules. The smallest chromosome (chromosome 21) has about 50 million base pairs, and the entire haploid human genome is estimated to consist of 3 to 4 billion base pairs. This extreme size precludes the analysis of DNA in its native form in routine molecular biology techniques. The techniques for identification and analysis of DNA became feasible and readily accessible with the discovery of enzymes termed restriction endonucleases. These enzymes, originally isolated from bacteria, cut DNA into smaller sizes on the basis of specific recognition sites that vary from two to eight base pairs in length.5,6 The term restriction refers to the function of these enzymes in bacteria. A restriction endonuclease destroys foreign DNA (such as bacteriophage DNA) by cleaving the DNA at specific sites and thereby restricting the entry of foreign DNA in the bacterium. Several hundred restriction enzymes with different recognition sites are now commercially available. Because the recognition site for a given enzyme is fixed, the number and sizes of fragments generated for a particular DNA molecule remain consistent with the number of recognition sites and provide predictable patterns after separation by electrophoresis. Analysis of the DNA fragments generated after digestion usually employs the technique of electrophoresis.7 Electrophoresis exploits the property that the phosphate groups in the DNA molecule confer a negative charge to that molecule. Thus, when a mixture of DNA molecules of different sizes is electrophoresed through a sieve (routinely either agarose or acrylamide) the longer DNA molecules migrate more slowly relative to the shorter fragments. Following electrophoresis, the separated DNA molecules can be located by a variety of staining techniques—among which ethidium bromide staining is a commonly used method. Although staining with ethidium bromide is a versatile technique, analysis of a few hundred base pairs of DNA in the region of interest is difficult when the DNA from

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all human chromosomes are cut and separated on the same gel. These limitations are circumvented by the technique of Southern blotting (named after its originator, Edward Southern) and the use of labeled radioactive (or, more commonly, nonradioactive) probes. Southern blotting involves digestion of DNA and separation by electrophoresis through agarose.8 After electrophoresis, the DNA is transferred to a solid support (such as nitrocellulose or nylon membranes) that enables the pattern of separated DNA fragments to be replicated onto the membrane (Figure 1-1). The DNA is then denatured (i.e., the two strands are physically separated) and fixed to the membrane, and the dried membrane is mixed with a solution containing the DNA probe. A DNA probe is a fragment of DNA that contains a nucleotide sequence specific for the gene or chromosomal region of interest. For purposes of detection, the DNA probe is labeled with an identifiable tag such as radioactive phosphorus (e.g., 32P) or a chemiluminescent moiety. The latter has almost exclusively replaced radioactivity in recent years. The process of mixing the DNA probe with the denatured DNA fixed to the membrane is called hybridization, the principle being that there are only four nucleic acid bases in DNA [adenine (A), thymidine (T), guanine (G), and cytosine (C)] that always remain complementary on the two strands of DNA—A pairing with T, and G pairing with C. Following hybridization, the membrane is washed to remove the unbound probe and exposed to an x-ray film either in a process called autoradiography to detect radioactive phosphorus or processed to detect the chemiluminescent tag. Only those fragments that are complementary and have bound to the probe containing the DNA of interest will be evident on the x-ray film, enabling analysis of the size and pattern of these fragments. As routinely performed, the technique of Southern analysis can detect a single-copy gene in as little as 5 µg of DNA (the DNA content of about 106 cells).

RESTRICTION FRAGMENT LENGTH POLYMORPHISM AND OTHER POLYMORPHIC DNA STUDIES The number and size of DNA fragments resulting from the digestion of any particular region of DNA form a recognizable pattern. Small variations in a sequence between unrelated individuals may cause a restriction enzyme recognition site to be present or absent. This results in a variation in the number and size pattern of the DNA fragments produced by digestion with that particular enzyme. Thus, this region is said to be polymorphic for the particular enzyme tested [i.e., a restriction fragment length polymorphism (RFLP)] (Figure 1-2). The usefulness of RFLP is that it can be used as a molecular tag for tracing the inheritance of the maternal and paternal alleles. Furthermore, the polymorphic region analyzed does not need to encode the genetic variation that is the cause of the disease being studied, but only to be located near the gene of interest. When a particular RFLP pattern can be shown to be associated with a disease, the likelihood of an offspring inheriting the disease can be determined from the offspring’s RFLP pattern by comparing it with the RFLP

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Figure 1-1. Southern blot. Fragments of double-stranded DNA are separated by size by agarose gel electrophoresis. To render the DNA single stranded (denatured), the agarose gel is soaked in an acidic solution. After neutralization of the acid, the gel is placed onto filter paper—the ends of which rest in a reservoir of concentrated salt buffer solution. A sheet of nitrocellulose membrane is placed on top of the gel and absorbent paper is stacked on top of the nitrocellulose membrane. The salt solution is drawn up through the gel by the capillary action of the filter paper wick and the absorbent paper towels. As the salt solution moves through the gel, it carries along with it the DNA fragments. Because nitrocellulose binds single-stranded DNA, the DNA fragments are deposited onto the nitrocellulose in the same pattern in which they existed in the agarose gel. The DNA fragments bound to the nitrocellulose are fixed to the membrane by heat or ultraviolet irradiation. The nitrocellulose membrane with the bound DNA can then be used for procedures such as hybridization to a labeled DNA probe. Techniques for transferring DNA to other bonding matrices, such as nylon, are similar. [Adapted from Turco E, Fritsch R, M. Trucco M (1990). Use of immunologic techniques in gene analysis. In RB Herberman, DW Mercer (eds.), Immunodiagnosis of cancer. New York: Marcel Dekker, 205.]

pattern of the affected or carrier parents. The major limitation of the RFLP technique is that its applicability for the analysis of any particular gene is dependent on the prior knowledge of the presence of convenient (“informative”) polymorphic restriction sites that flank the gene of interest by at most a few kilobases. Because these criteria may not be fulfilled in any given case, the applicability of RFLP cannot be guaranteed for the analysis of a given gene. In the early years of the molecular endocrinology era, the RFLP technique was the mainstay of experimental strategies employed for investigating the genetic basis of endocrine diseases. For example, RFLP-based genomic studies were used to identify mutations in the RET oncogene as the etiology of the multiple endocrine neoplasia type-2 syndrome. However, at present for routine disease mapping and whole genome and even gene-specific association studies the RFLP technique has been supplanted by more powerful and facile techniques such as microsatellite and single-nucleotide polymorphism studies (see material following). At present, RFLP analysis is only used within the context of a specific gene investigation.

POLYMERASE CHAIN REACTION The polymerase chain reaction (PCR) is a technique that was developed in the late 1980s. It has indeed revolutionized molecular biology (Figure 1-3). PCR allows the selective logarithmic amplification of a desired fragment of DNA from a complex mixture of DNA that theoretically contains at least a single copy of the target fragment. In the typical application of this technique, some knowledge of the DNA sequences in the region to be amplified is

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necessary so that a pair of specific short (approximately 18 to 25 bases in length) oligonucleotides (“primers”) can be synthesized. The primers are synthesized in a manner such that they define the limits of the region to be amplified. The DNA template containing the segment to be amplified is heat denatured such that the strands are separated and then cooled to allow the primers to anneal to the respective complementary regions. The enzyme Taq polymerase, a heat-stable enzyme originally isolated from the bacterium Thermophilous aquaticus, is then used to initiate synthesis (extension) of DNA. The DNA is repeatedly denatured, annealed, and extended in successive cycles in a machine called a thermocycler. This machine permits the process to be automated. In the usual assay, these repeated cycles of denaturing, annealing, and extension result in the synthesis of approximately 1 million copies of the target region in about 2 hours. To establish the veracity of the amplification process, the identity of the amplified DNA can be analyzed by electrophoresis, hybridization to RNA or DNA probes, or digestion with informative restriction enzyme(s) or can be subjected to direct DNA sequencing. The relative simplicity combined with the power of this technique has resulted in widespread use of this procedure and has spawned a wide variety of variations and modifications that have been developed for specific applications.9,10 From a practical point of view, the major drawback of PCR is the propensity to obtain cross-contamination of the target DNA. This drawback is the direct result of the extreme sensitivity of the method, which permits amplification from one molecule of the starting DNA template. Thus, unintended transfer of amplified sequences to items used in the

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Figure 1-2. Restriction fragment length polymorphism. (A) Schematic illustration in which A and B represent two alleles that display a polymorphic site for the restriction enzyme EcoR I. EcoR I will cut DNA with the sequence GAATTC, and hence allele B will be cut by EcoR I at three sites to generate two fragments of DNA—whereas allele A will be cut by EcoR I only twice and not at the site (indicated by horizontal bar) where nucleotide G (underlined) replaces the nucleotide A present in allele B. Following digestion, the DNA is size fractionated by agarose gel electrophoresis and transferred to a membrane by Southern blot technique (see Figure 1-1 for details). The membrane is then hybridized with a labeled DNA probe that contains the entire sequence spanned by the three EcoR I sites. Autoradiography of the membrane will detect the size of the DNA fragments generated by the restriction enzyme digestion. In this particular illustration, both parents are heterozygous and possess both A and B alleles. Matching the pattern of the DNA bands of the offspring with that of the parents will establish the inheritance pattern of the alleles. For example, if allele A represents the abnormal allele for an autosomal recessive disease examination of the Southern blot will establish that (from left to right) the first offspring (B/B) is homozygous for the normal allele, the second offspring (A/A) homozygous for the abnormal allele, and the third offspring (A/B) a carrier. (B) RFLP analysis of the DQ-beta gene of the human lymphocyte antigen (HLA) locus. Genomic DNA from the members of the indicated pedigree was digested with restriction enzyme Pst I, size fractionated by agarose gel electrophoresis, and transferred to nitrocellulose membrane by Southern blot technique. The membrane was then hybridized with a cDNA probe specific for the DQ-beta gene, excess probe removed by washing at appropriate stringency, and analyzed by autoradiography. The sizes of the DNA fragments (in kilobases, kb) are indicated on the right. The pedigree chart indicates the polymorphic alleles (a, b, c, d), and the bands on the Southern blot corresponding to these alleles [a (5.5 kb), b (5.0 kb), c (14.0 kb) , d (4.5 kb)] indicate the inheritance pattern of these alleles. [Adapted from Turco E, Fritsch R, Trucco M (1998). First domain encoding sequence mediates human class II beta-chain gene cross-hybridization. Immunogenetics 28:193.]

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Figure 1-3. Polymerase chain reaction (PCR). A pair of oligonucleotide primers (solid red bars), complementary to sequences flanking a particular region of interest (shaded, stippled red bars), are used to guide DNA synthesis in opposite and overlapping directions. Repeated cycles of DNA denaturation, primer annealing, and DNA synthesis (primer extension) by DNA polymerase enzyme results in an exponential increase in the target DNA (i.e., the DNA sequence located between the two primers) such that this DNA segment can be amplified 1 x 106–107 times after 30 such cycles. The use of a thermostable DNA polymerase (i.e., Taq polymerase) allows for this procedure to be automated. Inset: The amplified DNA can be used for subsequent analysis (i.e., size fractionation by agarose gel electrophoresis). [Adapted from Trucco M (1992). To be or not to be ASP 57, that is the question. Diabetes Care 15:705.]

procedure will result in the amplification of DNA in samples that do not contain the target DNA sequence (i.e., a false positive result). Cross-contamination should be suspected when amplification occurs in negative controls that did not contain the target template. One of the most common modes of cross-contamination is via aerosolization of the amplified DNA during routine laboratory procedures such as vortexing, pipetting, and manipulation of microcentrifuge tubes. Meticulous care to experimental technique, proper organization of the PCR workplace, and inclusion of appropriate controls are essential for the successful prevention of cross-contamination during PCR experiments. In general, PCR applications are either directed toward the identification of a specific DNA sequence in a tissue or body fluid sample or are used for the production of relatively large amounts of DNA of a specific sequence— which is then used in further studies. Examples of the first type of application are very common in many fields of medicine; for example, in microbiology, wherein the PCR technique is used to detect the presence DNA sequences specific for viruses or bacteria in a biological sample. Prototypic examples of such an application in pediatric endocrinology include the use of PCR of the SRY gene for detection of Y chromosome material in patients with karyotypically defined Turner syndrome and for the rapid identification of chromosomal gender in cases of fetal or neonatal sexual ambiguity (Figure 1-4).11

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Most PCR applications as research tools and for clinical use are directed toward the production of a target DNA or the complementary DNA of a target RNA sequence. The DNA that is made (amplified) is then analyzed by other techniques, such as RFLP analysis, allele-specific oligonucleotide hybridization, or DNA sequencing.

RNA ANALYSIS The majority (⬎95%) of the chromosomal DNA represents non-coding sequences. These sequences harbor regulatory elements, serve as sites for alternate splicing, and are subject to methylation and other epigenetic changes that affect gene function. However, at present most disease-associated mutations in human genes have been identified in coding sequences. An alternative strategy for analyzing mutations in a given gene is to study its messenger RNA (mRNA), which is the product (via transcription) of the remaining 5% of chromosomal DNA that encodes for proteins. In addition, because the mRNA repertoire is cell and tissue specific the analysis of the mRNA sequences provides unique information about tissue-specific proteins produced in a particular organ or other tissue. There are many techniques used in the analysis of mRNA, but one of the most commonly used methods is called Northern blotting (so named because it is based on the same principle as the Southern blot). In Northern

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Figure 1-4. Detection of SRY-gene specific sequence in Turner’s syndrome by PCR amplification and Southern blot. SRY-specific primers were used in PCR to amplify DNA from patients with 45X karyotype. The amplified DNA was size fractionated by agarose gel electrophoresis and transferred to membrane by Southern blotting. The membrane was then hybridized to labeled SRY-specific DNA and autoradiographed. From left to right: amplified male DNA (lane 1), amplified DNA from patients with 45X karyotype (lanes 2–5), amplified female DNA (lane 6), negative control with no DNA (lane 7), and serial dilution of male DNA (lanes 8–13). [Adapted from Kocova M, Siegel SF, Wenger SL, Lee PA, Trucco M (1993). Detection of Y chromosome sequence in Turner’s Syndrome by Southern blot analysis of amplified DNA. Lancet 342:140.]

blotting, RNA is denatured by treating it with an agent such as formaldehyde to ensure that the RNA remains unfolded and in linear form.12,13 The denatured RNA is then electrophoresed and transferred onto a solid support (such as nitrocellulose membrane) in a manner similar to that described for the Southern blot.8 The membrane with the RNA molecules separated by size is probed with the gene-specific DNA probe labeled with an identifiable tag. As in the case of Southern blotting, the tag is either a radioactive label (e.g., 32P) or more commonly a chemiluminescent moiety. The nucleotide sequence of the DNA probe is complementary to the mRNA sequence of the gene and is hence called cDNA (complementary DNA). It is customary to use labeled cDNA (and not labeled mRNA) to probe Northern blots because DNA molecules are much more stable and easier to manipulate and propagate (usually in bacterial plasmids) than mRNA molecules. The Northern blot provides information regarding the amount (estimated by the intensity of the signal on autoradiography) and the size (estimated by the position of the signal on the gel in comparison to concurrently electrophoresed standards) of the specific mRNA. Although the Northern blot technique represents a very versatile and straightforward method of analyzing mRNA, it has major drawbacks. Northern analysis is a relatively insensitive technique in terms of the concentration of mRNA that can be detected and in terms of the fine structure. Small changes in size or nucleotide composition of the mRNA being analyzed cannot be detected by this technique. Hence, for these types of specialized analyses newer methods [such as solution hybridization and reverse transcriptase-PCR (RT-PCR)] are employed.

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Solution hybridization methods are based on the principle of hybridizing RNA in solution with either radioactively labeled DNA or RNA probes specific for the mRNA of interest. The main advantages of these methods are the enhanced sensitivity and the ability to yield information regarding the fine structure of the mRNA species being investigated. The enhanced sensitivity of these methods accrues from the fact that neither the mRNA being measured nor the probe being used are constrained by immobilization to a solid support, and hence do not require the additional steps of electrophoresis and transfer prior to hybridization (as in Northern blot analysis). In addition, the probe can be added in excess to drive the hybridization reaction to completion. The two main types of solution hybridization use either single-stranded DNA (S1 nuclease protection assay) or RNA (ribonuclease protection assay) as the probe in the hybridization reaction. The greater sensitivity and the recent commercial availability of kits have propelled the popularity of the ribonuclease protection assay (RPA) as the solution hybridization method of choice for analysis of RNA (Figure 1-5). The first step in RPA is the synthesis of a radioactive single-stranded RNA probe. The RNA probe, which is complementary to the mRNA being analyzed, is synthesized by the RNA polymerase-directed transcription of a suitably engineered commercially available RNA expression vector. An excess of this synthesized RNA probe is then mixed with an aliquot of mRNA to be measured such that the RNA probe will hybridize to the complementary mRNA. Separation of the hybridized from the free probe is achieved by exploiting the property of the enzyme RNase, which will only digest single-stranded RNA molecules.

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Figure 1-5. Detection of mRNA splice variants by ribonuclease protection assay (RPA). The mRNA species (100 and 50 nucleotides long) to be measured are represented by bars with cross hatches. A cRNA probe is synthesized with a region complementary to the mRNAs to be measured. This cRNA probe, which is internally labeled with 32P is hybridized to the sample containing the target mRNAs. After hybridization, the unhybridized segments of the cRNA probe are digested with RNase enzyme. RNase enzyme will preferentially digest single-stranded RNA molecules and leave double-stranded RNA molecules intact. Hence, following RNase digestion RNase-resistant radioactivity will represent that segment(s) of the cRNA probe that was “protected” from RNase digestion by virtue of forming a doublestranded molecule with the target mRNA containing the complementary sequence. Following acrylamide gel electrophoresis, the RNaseresistant radioactivity is detected by autoradiography. Because the cRNA probe is always added in excess, the amount of the RNase-resistant radioactivity will be proportional to the concentration of the target mRNA. As depicted in the lower portion of the cartoon, the undigested probe (lane A) will electrophorese with a size distinct from the probe hybridized to mRNA 1 (lane B) or mRNA 2 (lane C).

Double-stranded RNA (such as that formed by the hybridization of the probe with the complementary RNA) is relatively resistant to digestion by RNase (such as RNase T1 or A). The RNase-resistant radioactive material is then size fractionated by electrophoresis and the doublestranded RNA molecules detected by autoradiography. Because RNase is very discriminatory with respect to its ability to digest single-stranded RNA, while permitting double-stranded RNA to remain intact, even a single mismatch in the nucleotide sequence between the probe and the measured RNA will result in the probe RNA being digested at that point of mismatch (because the RNA will be single stranded at that point) and the appearance of additional radioactive bands after RNase digestion. Thus, this method is very useful for the detection and quantitation of internal splice variants wherein significant changes in one splice variant may be masked by the unchanged combined level of all mRNA variants analyzed by techniques such as Northern analysis. The major disadvantage of RPA is that it requires some prior knowledge of the RNA structure for the design of appropriate RNA probes. One of the most sensitive methods for the detection and quantitation of mRNA currently available is the technique of quantitative RT-PCR (qRT-PCR).9 This technique combines the unique function of the enzyme reverse tran-

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scriptase with the power of PCR. qRT-PCR is exquisitely sensitive, permitting analysis of gene expression from very small amounts of RNA. Furthermore, this technique can be applied to a large number of samples and/or many genes (multiplex) in the same experiment. These two critical features endow this technique with a measure of flexibility unavailable in more traditional methods, such as Northern blot or solution hybridization analysis. Whereas the detection of a specific mRNA by this technique is relatively straightforward, the precise quantitation of the mRNA in a given sample is more complicated. The first step in qRT-PCR analysis is the production of cDNA to the mRNA of interest. This is done by using the enzymes with RNA-dependent DNA polymerase activity that belong to the reverse transcriptase (RT) group of enzymes [e.g., Moloney murine leukemia virus (MMLV), avian myeloblastosis virus (AMV) reverse transcriptase, or an RNA-dependent DNA polymerase]. The RT enzyme, in the presence of an appropriate primer, will synthesize DNA complementary to RNA. The second step in the qRT-PCR analysis is the amplification of the target DNA, in this case the cDNA synthesized by the reverse transcriptase enzyme. The specificity of the amplification is determined by the specificity of the primer pair used for the PCR amplification. To establish the veracity of the amplification process, the identity of

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the amplified DNA can be analyzed by electrophoresis, hybridization to RNA or DNA probes, digestion with informative restriction enzyme(s), or subjection to direct DNA sequencing. Whereas the detection of a specific mRNA by this technique is relatively straightforward, the precise quantitation of the mRNA in a given sample is more complicated. Because the production of DNA by PCR involves an exponential increase in the amount of DNA synthesized, relatively minor differences in any of the variables controlling the rate of amplification will cause a marked difference in the yield of the amplified DNA. In addition to the amount of template DNA, the variables that can affect the yield of the PCR include the concentration of the polymerase enzyme, magnesium, nucleotides (dNTPs), and primers. The specifics of the amplification procedure (including cycle length, cycle number, annealing, extension, and denaturing temperatures) also affect the yield of DNA. Because of the multitude of variables involved, routine RT-PCR is unsuitable for performing a quantitative analysis of mRNA. To circumvent these pitfalls, alternative strategies have been developed. One technique for determining the concentration of a particular mRNA in a biological sample is a modification of the basic PCR technique called competitive RT-PCR.9,14,15 This method is based on the co-amplification of a mutant DNA that can be amplified with the same pair of primers being used for the target DNA. The mutant DNA is engineered in such a way that it can be distinguished from the DNA of interest by size or by the inclusion of a restriction enzyme site unique to the mutant DNA. The addition of equivalent amounts of this mutant DNA to all PCR reaction tubes serves as an internal control for the efficiency of the PCR process, and the yield of the mutant DNA in the various tubes can be used for the equalization of the yield of the DNA by PCR. It is important to ensure for accurate quantitation of the DNA of interest that the concentrations of the mutant and target template are nearly equivalent. Because the use of mutated DNA for normalization does not account for the variability in the efficiency of the reverse transcriptase enzyme, a variation of the original method has been developed. In this modification, competitive mutated RNA transcribed from a suitably engineered RNA expression vector is substituted for the mutant DNA in the reaction prior to initiating the synthesis of the cDNA. Competitive RT-PCR can be used to detect changes of the order of two- to threefold of even very rare mRNA species. The major drawback of this method is the propensity to derive inaccurate results due to contamination of samples with the mRNA of interest. In theory, because the technique is based on PCR contamination by even one molecule of mRNA of interest can invalidate the results. Hence, scrupulous attention to laboratory technique and setup is essential for the successful application of this technique. In general, there are two types of methods used for detection and quantitation of PCR products: the traditional “endpoint” measurements of products and the newer “real-time” techniques. Endpoint determinations

Ch01_001-025-X4090.indd 8

(e.g., the competitive RT-PCR technique described previously) analyze the reaction after it is completed, whereas real-time determinations are made during the progression of the amplification process. Overall, the real-time approach is more accurate and is currently the preferred method. Advances in fluorescence detection technologies have made real-time measurement possible for routine use in the laboratory. One of the popular techniques that takes advantage of real-time measurements is the TaqMan (fluorescent 5’-nuclease) assay (Figure 1-6).16,17 The unique design of TaqMan probes, combined with the 5’-nuclease activity of the PCR enzyme (Taq polymerase), allows direct detection of PCR product by the release of fluorescent reporter during the PCR amplification by using specially designed machines (ABI Prism 5700/7700). The TaqMan probe consists of an oligonucleotide synthesized with a 5’-reporter dye (e.g., FAM; 6-carboxy-fluorescein) and a downstream 3’-quencher dye (e.g., TAMRA; 6-carboxy-tertamethyl-rhodamine). When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by Forster-type energy transfer. During PCR, forward and reverse primers hybridize to a specific sequence of the target DNA. The TaqMan probe hybridizes to a target sequence within the PCR product. Because of its 5’- to 3’-nuclease activity, the Taq polymerase enzyme subsequently cleaves the TaqMan probe. The reporter dye and the quencher dye are separated by cleavage, resulting in increased laser-stimulated fluorescence of the reporter dye as a direct consequence of target amplification during PCR. This process occurs in every cycle and does not interfere with the exponential accumulation of product. Both primer and probe must hybridize to the target for amplification and cleavage to occur. The fluorescence signal is generated only if the target sequence for the probe is amplified during PCR. Because of these stringent requirements, nonspecific amplification is not detected. Fluorescent detection takes place through fiber-optic lines positioned above optically nondistorting tube caps. Quantitative data are derived from a determination of the cycle at which the amplification product signal crosses a preset detection threshold. This cycle number is proportional to the amount of starting material, thus allowing for a measurement of the level of specific mRNA in the sample. An alternative machine (Light Cycler) also uses fluorogenic hydrolysis or fluorogenic hybridization probes for quantification in a manner similar to the ABI system.

Detection of Mutations in Genes Changes in the structural organization of a gene that impact its function involve deletions, insertions, or transpositions of relatively large stretches of DNA—or more frequently single-base substitutions in functionally critical regions. The deletion or insertion of large stretches of DNA can usually be detected by Southern blotting and RFLP analysis. However, these analytic methods can be used for detecting point mutations only if the mutation

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9

Properties of Taq polymerase

synthetic

5' nuclease R

Reporter

Q

Quencher

Probe Forward primer

Q

R

5'

3'

3'

5'

Fluorescence detector

R

5'

Reverse primer

Q

3'

3'

5'

Figure 1-6. Fluorescent 5’-nuclease (TaqMan) assay. Three synthetic oligonucleotides are utilized in a fluorescent 5’-nuclease assay. Two oligonucleotides function as “forward” and “reverse” primers in a conventional PCR amplification protocol. The third oligonucleotide, termed the TaqMan probe, consists of an oligonucleotide synthesized with a 5’-reporter dye (e.g., FAM; 6-carboxy-fluorescein) and a downstream 3’-quencher dye (e.g., TAMRA; 6-carboxy-tertamethyl-rhodamine). When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence—primarily by Forster-type energy transfer. During PCR, forward and reverse primers hybridize to a specific sequence of the target DNA. The TaqMan probe hybridizes to a target sequence within the PCR product. Because of its 5’- to 3’-exonuclease activity, the Taq polymerase enzyme subsequently cleaves the TaqMan probe. The reporter dye and the quencher dye are separated by cleavage, resulting in increased fluorescence of the reporter dye as a direct consequence of target amplification during PCR. Both primers and probe must hybridize to the target for amplification and cleavage to occur. Hence, the fluorescence signal is generated only if the target sequence for the probe is amplified during PCR. Fluorescent detection takes place through fiber-optic lines positioned above the caps of the reaction wells. Inset: The two distinct functions of the enzyme Taq polymerase: the 5’-3’ synthetic polymerase activity and the 5’-3’ polymerase-dependent exonuclease activity.

involves the recognition site for a particular restriction enzyme such that the absence of a normally present restriction site or the appearance of a novel site unmasks the presence of the point mutation. More commonly, these techniques cannot be used for such an analysis— necessitating alternative procedures.

DIRECT METHODS DNA sequencing is the current gold standard for obtaining unequivocal proof of a point mutation. However, DNA sequencing has its limitations and drawbacks. A clinically relevant problem is that current DNA sequencing methods do not reliably and consistently detect all mutations. For example, in many cases where the mutation affects only one allele (heterozygous) the heights of the peaks of the bases on the fluorescent readout corresponding to the wild-type and mutant allele are not always present in the predicted (1:1) ratio. This limits the

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discerning power of “base-calling” computer protocols and results in inconsistent and/or erroneous assignment of DNA sequence to individual alleles.18 Because of this limitation, clinical laboratories routinely determine the DNA sequence of both alleles to provide independent confirmation of the absence/presence of a putative mutation. DNA sequencing can be labor intensive and expensive, although recent advances in pyrosequencing (see material following) have made it technically easier and cheaper. Although the first DNA sequences were determined with a method that chemically cleaved the DNA at each of the four nucleotides,19 the enzymatic or dideoxy method developed by Sanger et al. in 1977 is the one most commonly used for routine purposes (Figure 1-7).20 This method uses the enzyme DNA polymerase to synthesize a complementary copy of the single-stranded DNA (“template”) whose sequence is being determined. Single-stranded DNA can be obtained directly from viral

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Figure 1-7. DNA sequencing by the dideoxy (Sanger) method. A 5’-end-labeled oligonucleotide primer with sequence complementarity to the DNA to be sequenced (DNA template) is annealed to a single strand of the template DNA. This primer is elongated by DNA synthesis initiated by the addition of the enzyme DNA polymerase in the presence of the four dNTPs (2’-deoxynucleoside triphosphates) and one of the ddNTPs (2’,3’-dideoxynucleoside triphosphates). Four such reaction tubes are assembled to use all four ddNTPs. The DNA polymerase enzyme will elongate the primer using the dNTPs and the individual ddNTP present in that particular tube. Because ddNTPs are devoid of 3’-hydroxyl group, no elongation of the chain is possible when such a residue is added to the chain. Thus, each reaction tube will contain prematurely terminated chains ending at the occurrence of the particular ddNTP present in the reaction tube. The concentrations of the dNTPs and the individual ddNTP present in the reaction tubes are adjusted so that the chain termination occurs at every occurrence of the ddNTP. Following the chain elongation-termination reaction, the DNA strands synthesized are size separated by acrylamide gel electrophoresis and the bands visualized by autoradiography.

or plasmid vectors that support the generation of singlestranded DNA or by partial denaturing of double-stranded DNA by treatment with alkali or heat.21 The enzyme DNA polymerase cannot initiate synthesis of a DNA chain de novo but can only extend a fragment of DNA. Hence, the second requirement for the dideoxy method of sequencing is the presence of a primer. A primer is a synthetic oligonucleotide 15 to 30 bases long, whose sequence is complementary to the sequence of the short corresponding segment of the single-stranded DNA template. The dideoxy method exploits the observation that DNA polymerase can use both 2’-deoxynucleoside triphosphate (dNTP) and 2’,3’-dideoxynucleoside triphosphates (ddNTPs) as substrates during elongation of the primer. Whereas DNA polymerase can use dNTP for continued synthesis of the complementary strand of DNA, the chain cannot elongate any further after addition of the first ddNTP because ddNTPs lack the crucial 3’-hydroxyl group. To identify the nucleotide at the end of the chain, four reactions are carried out for each sequence analysis— with only one of the four possible ddNTPs included in any one reaction. The ratio of the ddNTP and dNTP in each reaction is adjusted so that these chain terminations occur at each of the positions in the template where the

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nucleotide occurs. To enable detection by autoradiography, the newly synthesized DNA is labeled—usually by including in the reaction mixture radioactively labeled dATP (for the older manual methods) or, most commonly, currently fluorescent dye terminators in the reaction mixture (now in use in automated techniques). The separation of the newly synthesized DNA strands manually is done via high resolution denaturing polyacrylamide electrophoresis or with capillary electrophoresis in automatic sequencers. Fluorescent detection methods have enabled automation and enhanced throughput. In capillary electrophoresis, DNA molecules are driven to migrate through a viscous polymer by a high electric field to be separated on the basis of charge and size. Although this technique is based on the same principle as identical to that used in slab gel electrophoresis, the separation is done in individual glass capillaries rather than gel slabs—facilitating loading of samples and other aspects of automation. Whereas manual methods allow the detection of about 300 nucleotide of sequence information with one set of sequencing reactions, automated methods using florescent dyes and laser technology can analyze 7,500 or more bases per reaction. To sequence larger stretches of DNA, it is necessary to divide the large piece of DNA into

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MOLECULAR ENDOCRINOLOGY AND ENDOCRINE GENETICS

smaller fragments that can be individually sequenced. Alternatively, additional sequencing primers can be chosen near the end of the previous sequencing results— allowing the initiation point of new sequence data to be progressively moved along the larger DNA fragment. One of the seminal technological advances in recent years has been the introduction of microarray-based methods for detection, and analysis of nucleic acids (see material following). For purposes of detection of mutations, the oligonucleotides fixed to the slide/membrane are complementary to all possible base substitutions or a subset of small deletions and insertions. Fluorescent labeled PCR probes derived from the patient and representing the genes to be tested are then hybridized to the microarrays. Following appropriate washing protocols, the retention of particular probes on the slide provide information regarding the presence/absence of a given mutation, deletion, or insertion. There are limitations of microarray-based techniques. For example, similar to direct DNA sequencing methods microarray-based methods also suffer from the disadvantage of not being able to reliably or consistently detect heterozygous mutations. In addition, microarrays cannot be used to detect insertions of multiple nucleotides without exponentially increasing the number of oligonucleotides that must be immobilized on the glass slides. The most exciting new technique in mutation identification is pyrosequencing. This is based on an enzymatic real-time monitoring of DNA synthesis by bioluminescence. This “read as you go” method uses nucleotide incorporation that leads to a detectable light signal from the pyrophosphate released when a nucleotide is introduced in the DNA strand.22 The rapidity and reliability of this method far exceed other contemporary DNA sequencing techniques. However, the major limitation of this technique is that it can only be used to analyze short stretches of DNA sequence.

INDIRECT METHODS Screening for mutations of the thousands of the sequence products provided by human genome analysis has proven to be a daunting task. Although the gold standard for identifying sequence alterations is direct sequencing, this method remains labor intensive and the least cost effective. Since the mid 1980s, the need for rapid, highthroughput, accurate, and economical mutation analysis systems has led to the development of several technologies as alternatives to analysis by direct sequencing that have allowed detection of single mutations in long stretches of DNA (200–600 bp). These techniques include restriction endonuclease digestion of PCR products (PCRRFLP), denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism (SSCP), dideoxy fingerprinting (ddF), and heteroduplex mobility assay (HMA). Most of these methods utilize PCR to amplify a region of the DNA, a physical or chemical treatment of amplified DNA (by restriction digestion or denaturation), separation of the amplicons by gel electrophoresis (by denaturing or nondenaturing), and visualization of the separated sequence strands (by autoradiography or fluorescence-

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based detection). Most recent modifications in some of these techniques allow simultaneous separation and detection of DNA fragments with the use of sophisticated equipment such HPLC and capillary electrophoresis. Originally described in 1989, SSCP analysis has been a widely used method for the detection of mutations because of its simplicity and efficiency. In SSCP, DNA regions with potential mutations are first amplified by PCR in the presence of a radiolabeled dNTP (Figures 1-8 and 1-9). Single-stranded DNA fragments are then generated by denaturation of the PCR products and separated on a native polyacrylamide gel. As the denatured PCR product moves through the gel and away from the denaturant, it will regain a secondary structure that is sequence dependent. The electrophoretic migration of single-stranded DNA is a function of its secondary structure. Therefore, PCR products that contain substitution differences (as well as insertions and deletions) will have different mobility when compared with wild-type DNA. Although SSCP is simple, rapid, and inexpensive, it also has some disadvantages. The major limitations of the technique are lack of sensitivity and the inability to provide information about the location of a mutation in a DNA fragment. Efficacy studies, in which the technique was evaluated against a known mutation, showed that the sensitivity of SSCP can be highly variable in identifying sequence alterations. This variability is seen not only between amplicons but within the same amplicon if examined under different conditions. Overall, SSCP detects previously identified sequence changes in as many as 90% and as little as 60% of the specimens—depending primarily on the sequence, the amplicon’s size, and the location of the mutation. To circumvent the disadvantages of SSCP, an alternative technique was proposed that became known as dideoxy-fingerprinting (ddF). ddF is essentially a hybrid of SSCP and dideoxy sequencing in which primer extension products are generated in the presence of one dideoxynucleotide and subjected to chemical denaturation and electrophoresis on a nondenaturing polyacrylamide gel to exploit differences in secondary structure of single-stranded conformers. The resulting electrophoretic pattern resembles sequencing gels in which the mobility of extension products is determined by both size and conformation. In ddF, single-base and other sequence changes may result in elimination of a normal band or appearance of an extra band (informative dideoxy component) and altered electrophoretic mobility of one or more sequence termination products (informative SSCP component). The intensity of bands may also be used for dosage analysis because detection of “half loss” would indicate heterozygous mutation. There are three major advantages of ddF over conventional SSCP: the relative position of a mutation may be revealed due to addition or deletion of a dideoxy termination product; the sensitivity is increased because, unlike SSCP, there are multiple bands that exhibit altered mobility (making it unlikely that a mutation will be missed because the technique is based on the same extension principle as dideoxy sequencing, in which multiple DNA strands are generated that contain the sequence change); and it permits large PCR

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Figure 1-8. Single-stranded conformational polymorphism (SSCP). Schematic representation of an experiment designed to use SSCP to detect the presence of heterozygous and homozygous single–base-pair mutation (represented as a filled circle). The segment of DNA is amplified using PCR with flanking primers (represented by arrows). 32P is incorporated into the newly synthesized DNA by end labeling the primers or by the addition of 32P-dATP to the PCR reaction. Theoretically, four different types of conformers can be formed: A and B from the sense and anti-sense strand of the wild-type (normal) DNA, and C and D from the sense and anti-sense strands containing the single–base-pair mutation. Following PCR, the DNA is denatured and analyzed by nondenaturing gel electrophoresis. Lane I represents the wild-type conformers, lane II represents the wild-type and mutant conformers from an heterozygous patient, and lane III represents the presence of mutant conformers with the absence of wild-type conformers from a homozygous patient.

fragments to be generated only once and analyzed in smaller subfragments using different primers. The originally described ddF could screen the same size of DNA as SSCP (250-350 bp), but with a significantly higher level of detection. In recent years, modifications of the original procedure have resulted in new and improved variants of ddF. Bidirectional ddF (bi-ddF), in which the dideoxy termination reaction is performed simultaneously with two opposing primers, has allowed larger fragments (⬃600 bp) to be screened with almost 100% sensitivity. RNA ddF (R-ddF), in which RNA is used as starting material, enables identification of mutations that result in splicing errors and allows screening of genes with large intronic regions. Denaturing fingerprinting (DnF), a modification of bi-ddF in which fingerprints are generated by performing denaturing gel electrophoresis on bidirectional “cycle-sequencing” reactions with each of two dideoxy terminators, has allowed screening of DNA regions with high GC content—avoiding the generation of “smearing” bands and thus increasing the sensitivity of the technique in identifying heterozygous mutations. Further modifications have streamlined the previously cited procedures by adapting them to either automated fluorescent sequencers or capillary electrophoresis utilizing fluorescent dNTPs or primers (automated bi-ddf, capillary electrophoresis-ddf). Automated fingerprinting technology allows simultaneous analysis of a larger number of samples, higher reproducibility, faster data pro-

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cessing, and the ability to analyze longer sequences from a single reaction. DGGE is a method used to detect single-base pair substitutions (or small insertions or deletions) in genes.23-25 Like SSCP, DGGE is also a PCR-based method and exploits the observation that when double-stranded DNA migrates through a polyacrylamide gel incorporating a gradient of chemical denaturing agents the mobility of the partially denatured (“melted”) DNA molecule is extremely sensitive to its base composition. Thus, even single-base changes in the nucleotide composition of the DNA will result in altered mobility of the partially denatured DNA. G-C clamp is a modified DGGE procedure wherein the sensitivity of the procedure is enhanced by the incorporation of a G- and C-rich region at one end of the DNA.24,26-28 This manipulation is most readily done by modifying one of the primers used for the PCR. The addition of the G-C–rich region increases the melting point of the DNA fragment and makes it easier to detect changes in the nucleotide composition. Depending on the relative orientation of the chemical gradient and the electrical field during electrophoresis, DGGE could be parallel or perpendicular. The relative sensitivity of these two protocols has to be determined empirically for each application of this method. Denaturing high-performance liquid chromatography (DHPLC) is a related technique that detects a variation in the DNA sequence but uses HPLC instead of gel electrophoresis for separation of DNA

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MOLECULAR ENDOCRINOLOGY AND ENDOCRINE GENETICS

Figure 1-9. Application of SSCP and ASOH to the analysis of the 21-hydroxylase gene in congenital adrenal hyperplasia. Detection of a mutation of codon 281 from valine to leucine (V281L). Upper panel: Pedigree of a family with proband II-1, which was referred for evaluation of hirsutism and secondary amenorrhoea. Middle panel: ASOH results from both mutant and normal alleles at codon 281 show both the father and sister of the proband to be carriers of the V281L mutation, whereas the patient is homozygous. ASOH was not performed on the mother. The asterisk indicated the mutant allele (L281). Lower panel: SSCP analysis revealed that the two additional conformers, representing the L281 conformer, were detected at the top of the gel in this patient and her family. The greater intensity of the conformers in the proband compared to her family members and the disappearance of the normal V281 conformer (adjacent to the L281 conformers) are consistent with her being homozygous for this mutation. L281 indicates the mutant conformer. P indicates normal polymorphism. [Adapted from Siegel SF, Hoffman EP, Trucco M (1994). Molecular diagnosis of 21-hydroxylase deficiency: Detection of four mutations on a single gel. Biochem Med Metab Biol 51:66.]

fragments. The incorporation of HPLC allows for automation of this technique and significantly enhances throughput by this method. Heteroduplex analysis is a variation of the SSCP method and is used to detect single-pair mismatches in double-stranded DNA.29-31 When polymorphic PCR products are denatured and allowed to slowly cool to 37° C before being loaded onto a native gel, some of the polymorphic strands will reanneal to the slightly different complementary strand and form a double-stranded DNA molecule with a small number of mismatches (i.e., a heteroduplex). During electrophoresis, heteroduplexes migrate more slowly than homoduplexes and the resulting pattern can be visualized after autoradiography. Although heteroduplex analysis would not detect abnormal sequences in a homozygous individual, it is pos-

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sible to combine known DNAs with the DNA being tested to form the heteroduplexes and reveal the presence of the variant allele. Because of the technical similarity of the SSCP and heteroduplex analyses, the choice between the two methods can be dependent on which empirically performs best for each application. However, SSCP has the advantage of greater sensitivity because the definitive bands usually represent a major portion of the particular labeled molecule—whereas heteroduplex analysis must detect the fraction of labeled molecules that actually formed heteroduplexes. Allele-specific oligonucleotide hybridization (ASOH) and reverse blot technique analyze DNA after amplification by PCR and detect sequence variations by the success or failure of hybridization of short oligonucleotide probes that either exactly match or mismatch the sequence being tested (Figure 1-10). The amplified target DNA is first denatured and applied to a nylon membrane in the form of a small dot. Once this target DNA is anchored to the membrane either by heating or by brief ultraviolet irradiation, the DNA is hybridized with a labeled (usually with 32P) oligonucleotide that encompasses the variable nucleotides of the DNA sequence of interest. The membrane is then washed with a salt solution whereby the salt concentration and the temperature control the specificity (“stringency”) of the procedure. Following the wash, the probe remaining on the membrane is detected by autoradiography. When several nucleotide variations (i.e., alleles) are known to exist in the same target sequence, several identical membranes are prepared and each is hybridized with a different oligonucleotide probe complementary to one of the known sequence variations. Although the net result is similar to having actually sequenced the DNA region, the ASOH method is considerably less labor intensive and relatively less expensive than DNA sequencing. Hence, ASOH is one of the methods of choice for screening a large number of samples for a particular genetic variation. The major disadvantage of this method is that it requires prior knowledge of the base changes involved in the mutation and the precise stringency parameters for hybridization and washing of the membrane. In effect, these limitations exclude the routine use of this method for the characterization of a new mutation but allow the use of this method for the rapid screening of large number of samples for a previously characterized mutation. A variation of the original method to perform ASOH is called the reverse blot technique (Figure 1-10).32 The difference when compared to ASOH is that the amplified target DNA is labeled and then hybridized to an anchored unlabeled probe. Because the length of the DNA molecules is an important factor facilitating binding to the membrane, a key to the design of this method was the development of a relatively simple means of synthesizing (using the PCR) a stretch of DNA that contained multiple copies (i.e., a polymer) of the relatively short allele-specific oligonucleotide probes.33 In practice, the amplified DNA is nonradioactively labeled by previously tagging the PCR primers with fluorescein or biotin. After hybridization of the denatured PCR product in the presence of a membrane containing

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MOLECULAR ENDOCRINOLOGY AND ENDOCRINE GENETICS CONVENTIONAL DOT BLOT

DNA

A

SSO

Reporter

REVERSE DOT BLOT USING POLYMERIC PROBES

Sense DNA

B

Anti-Sense DNA

Sense Polymer

Anti-Sense Polymer Reporter

Figure 1-10. (A) Allele-specific oligonucleotide hybridization (ASOH). The denatured (single-stranded) target DNA is anchored to a membrane, which is then treated (hybridized) with a solution of a short DNA segment of the gene of interest [sequence-specific oligonucleotide (SSO)]. The SSO is tagged with a reporter molecule, such as 32P. Unbound probe DNA is removed by washing with buffer solutions. Appropriate stringency conditions of hybridization and washes limit the hybridization of the probe specifically to its complementary segment in the target DNA molecule. Depending on the manner in which the DNA is spotted onto the membrane, this procedure is referred to as a dot blot or a slot blot. (B) Reverse dot blot. In this variation of the conventional ASOH procedure, the DNA probes (sense and anti-sense polymer) tagged with the reporter molecule are fixed to a membrane and the denatured target DNA (sense and anti-sense DNA) is then hybridized to the immobilized probe. Similar to the ASOH procedure, appropriate stringency conditions of hybridization and washes limit the hybridization of target DNA to those that contain the complementary segment to the immobilized probe. The advantages of this method include the increased sensitivity derived from the ability to fix multiple copies (polymer) of the probe to the membrane and that both complementary strands of the probe sequence are present on the membrane. [Adapted from Trucco M (1992). To be or not to be ASP 57, that is the question. Diabetes Care 15:705.]

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all of the relevant polymeric probes, and washing, the retained PCR products are revealed by detection via enzyme activity linked to fluorescein antibodies or to streptavidin. This method is especially useful for testing alleles that are highly polymorphic because a large number of probes and controls are easily tested using a single small membrane. Other advantages include the ability to use nonradioactive tracers, better reproducibility, and the potential for automation. Certain classes of mutations are inherently difficult to detect using the traditional methods of detection outlined previously.18 These types of mutations include promoter region, 3’ untranslated region (UTR), or intronic region mutations affecting levels of transcript of mRNA or deletions of entire genes or of contiguous exons. Thus, if the genomic region examined is deleted from the mutant allele, PCR-based methods will be unable to detect this mutation because the PCR product obtained from the genomic DNA will be exclusively derived from the wildtype allele and thus appear to be normal. Promoter regions, 3’UTR, and intronic regions usually span genomic segments several orders larger than the coding exons and are thus not easily accessible for analysis with the methods outlined previously. Different strategies need to be implemented for the analysis and detection of such mutations. Thus, deletions of one or more exons can be detected by quantitative hybridizations, quantitative PCR, Southern blotting, or fluorescence in situ hybridization—with the combined use of such methods enhancing the sensitivity of the testing protocol. A particularly promising and novel technique, termed conversion, exploits the principle that the diploid state of the human genome is converted to a haploid state that is then analyzed by one of the traditional methods.34 The critical advantage of this manipulation is that heterozygous mutations are much easier to detect in the haploid state because of the absence of the normal wild-type sequence.

Positional Genetics in Endocrinology THE PRINCIPLES OF POSITIONAL GENETICS For the purpose of disease gene identification, the candidate gene approach relies on partial knowledge of the genetic basis of the disease under investigation. This process was successful in identifying disease genes whose function was obvious. For example, the genetic defects of most of the hereditary enzymatic disorders [including congenital adrenal hyperplasia (CAH) syndromes] became known in the late 1980s when the introduction of PCR made the tools of molecular biology widely available to the medical and genetic research community. However, at about the same time research on diseases without any obvious candidate genes [e.g., the multiple endocrine neoplasia (MEN) syndromes] and on diseases in which the screening of obvious candidate genes failed to reveal mutations was ongoing. It was in these diseases that the application of “reverse genetics” (or more appropriately termed positional cloning)35,36 yielded information regarding the genetic basis of

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Linkage analysis

Chromosomal localization and mapping

15

Candidate gene screening in the chromosomal locus; contig construction (if needed) and cloning of new genes

Mutation detection

Figure 1-11. The steps of positional cloning (see text).

these disease states. Positional cloning is complemented by the Human Genome Project (HGP) and the Internet in making available in a fast and controlled manner information that would otherwise be inaccessible.37 The process of positional genetics is outlined in Figure 1-11. The first step is the collection of clinical information from families with affected members, the determination of the mode of inheritance of the defect (autosomal dominant or recessive, X-linked, complex inheritance), and the phenotyping of subjects (or tissues) following wellestablished criteria for the diagnosis of the disorder. If inheritance is not known, formal segregation analysis needs to be performed to determine the autosomal or X-linked (and the recessive or dominant) nature of the inheritance.38 Once this determination is made and the penetrance of the disorder is known, appropriate linkage software may be used.39 For more information on currently available computer software packages, the reader may check http://darwin.case.edu and other related links. Linkage is examined with polymorphic markers that span the entire human genome.36 Any marker that shows polymorphism and is known to lie close to or within a putative disease gene may be used. Genetic linkage can be defined as the tendency for alleles close together on the same chromosome to be transmitted together as an intact unit through meiosis. The strength of linkage can then be used as a unit of measurement to find out how close genetically different loci are to each other. This unit of map distance is an approximation of physical distance but is also highly dependent on other factors (e.g., the frequency of recombination is not the same in both genders, differing along the length of chromosomes and through the various chromosomes). The likelihood [logarithm of odds (LOD) score] method is widely used for linkage analysis. Once a locus on a chromosome has been identified, narrowing of the region (this region is usually several thousands of base pairs in length) is accomplished by analyzing informative recombinations in the cohort of

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patients and families available for study. The disease region may harbor already mapped genes. Online databases [Gen Bank, ENSEMBL at www.ensembl.org, and others, and especially for clinicians the Online Mendelian Inheritance in Man (OMIM)]40 may provide the necessary information. If a transcript is a reasonable candidate, mutation screening may identify the disease gene. If, however, these steps fail to identify the disease gene cloning of new sequences from the area may be needed. This is usually done through the construction of a “contig,” a contiguous array of human DNA clones covering the disease region. These DNA clones are maintained in large vectors such as bacterial artificial chromosomes (BACs). P1 and cosmid clones may also be used for coverage of the entire region or a subset of it. Contig construction may take a long time, depending on the genetic distance to be covered and the features of the genomic region. The identification of new genes and the construction of the contig are also called “chromosomal walking.” The building blocks of this “walk” are the individual clones, which are linked by sequence-tagged sites (STSs) that are present in more than one clone—thus providing critical information that allows for the proper aligning of these clones. Polymorphic markers (including those that were used for the linkage part of the process) are the most useful STSs because they provide a direct link between the genetic and the physical mapping data. Individual clones can be sequenced. Genes are identified in this process through their unique sequence features or through in vitro translation. The latter usually provides expressed sequence tags (ESTs), which can then be analyzed through software that is available online to determine the full gene structure and to identify redundancies (multiple ESTs or STSs of the same gene) and other errors. Each of the newly identified genes may be screened for mutations, as long as the expression profile of the identified transcript matches the spectrum of the tissues

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affected by the disease under investigation. Although this is helpful for most diseases, for others the expression profile may even be misleading. Thus, the presence of a transcript in an affected tissue is not always necessary. Complete segregation of the disease with an identified mutation, functional proof, and/or mutations in two or more families with the same disease are usually required as supportive evidence that the cloned sequence is the disease gene.41

POSITIONAL CLONING OF ENDOCRINE GENES A number of genes were identified by positional cloning in endocrinology in the 1990s. It is worth noting that endocrine tumor syndromes, despite their rarity and small overall impact on everyday clinical endocrine practice, are seminal examples of diseases whose molecular etiology was elucidated by positional cloning. Positional cloning played an essential role in unraveling the etiology of these syndromes because for most endocrine tumor syndromes there were no obvious candidate genes. Futhermore, positional cloning of genes responsible for familial tumor syndromes was greatly assisted by the use of neoplastic tissue for studies such as loss of heterozygosity (LOH), comparative genomic hybridization (CGH), and other fluoroscent in situ hybridization (FISH) applications. These techniques narrow the genetically defined chromosomal regions and thus facilitate the identification of the responsible genes. LOH studies were critical to the identification of von-Hippel-Lindau disease (VHLelongin),42 MEN 1 (menin),43 Cowden disease (PTEN),44 Peutz-Jeghers syndrome (STK11/LKB1),45,46 and Carney complex (PRKAR1A)47 genes.

A

Expression Studies: Microarrays and SAGE Advances in biotechnology, instrumentation, robotics, computer sciences, and the completion of genome sequencing initiatives for several organisms (including the human) have resulted in the development of novel and powerful techniques. A seminal example of such a technique is the development of the microarray technology. Microarrays contain thousands of oligonucleotides deposited or synthesized in situ on a solid support, typically a coated glass slide or a membrane. In this technique, a robotic device is used to print DNA sequences onto the solid support. The DNA probes immobilized on the microarray slide as spots can be cloned cDNA or gene fragments (ESTs) or oligonucleotides corresponding to known genes or putative open reading frames. The arrays are hybridized with fluorescent targets prepared from RNA extracted from tissue/cells of interest. The RNA is labeled with fluorescent tags such as Cy3 and Cy5. The prototypic microarray experimental paradigm consists of comparing mRNA abundance in two different samples. One fluorescent target is prepared from control mRNA, and the second target labeled with a different fluorescent label is prepared from mRNA isolated from the treated cells or tissue under investigation. Both targets are mixed and hybridized to the microarray slide, resulting in target gene sequences hybridizing to their complementary sequences on the microarray slide. The microarray is then excited by laser and the fluorescent intensity of each spot is determined with the relative intensities of the two colored signals on individual spots being proportional to the number of specific mRNA transcripts in each sample (Figure 1-12). Analysis of the fluorescent intensity

B

Figure 1-12. (A) cDNA microarray. Fluorescent labeled cDNA targets, ACTH-independent bilateral macronodular adrenal hyperplasia (Cy3), and ACTH-dependent hyperplasia (Cy5) were hybridized to glass slides containing genes involved in oncogenesis. Following laser activation of the fluorescent tags, fluorescent signals from each of the DNA “spots” are captured and subjected to analysis. (B) Magnified view of the microarray platform displaying the fluorescent signals: green (Cy3) and red (Cy5), with yellow representing overlap of these two colors. (See color plates.)

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data yields an estimation of the relative expression levels of the genes in the sample and control sample. Microarrays enable individual investigators to perform large-scale analysis of model organisms and to customize arrays for special genome applications. Most endocrine human genes participate in complex and highly interactive signaling pathways. They not only directly control a number of genes but modulate the functioning of a larger complement of genes by crosstalking with a multitude of signaling pathways. As noted elsewhere,48 this is analogous to the Internet—the relevant genes being “nodes” or “hubs” in this Internet-like structure.49 It was recently stated that “progress in dissecting signaling pathways has begun to lay out a circuitry that will likely mimic electronic integrated circuits in complexity and finesse, where transistors are replaced by proteins (e.g., kinases and phosphatases) and the electrons by phosphates and lipids.”50 Although this statement related to cancer research,50 translational medicine today identifies these complex interactions to be ubiquitous51—making it no surprise that 30,000 to 40,000 genes (rather than the previously predicted 100,000 genes)52 are sufficient to produce the human phenotype. It is not the number of genes that is critical but the regulation of expression of these genes and the number, quality, and temporospatial organization of interactions among their protein products. An excellent example of an endocrine transcriptome is that recently presented by Roberts et al.:53 the description of mitogen-activated protein kinase (MAPK) pathways during yeast pheromone response. Similar analytic strategies are currently being applied to investigations in a wide variety of fields of study, including that of endocrine tumors.54 In this process (investigations by microarrays), simultaneous collection of information for hundreds of genes at a time (or the entire genome, depending on the type of the array used) have become the standard approach. An example from a recent study55 is shown in Figure 1-12. Endocrine concepts and the reemergence of signaling as the chief means of understanding the complex interactions of genes may shape the new bioinformatics tools needed for the analysis of these data, and may direct the development of new drugs tailored to insights provided by these initiatives.56,57 Currently, in addition to the transcriptional profiling of any tissue performed by applying various types of microarrays there is the alternative technology of generating libraries of ESTs. An advanced expansion of EST libraries, especially in terms of high-throughput and transcript quantitation, is serial analysis of gene expression (SAGE). SAGE is based on generating, cloning, and sequencing concatenated short-sequence tags—each representing a single transcript derived from mRNA from target tissue.58 Analysis of the transcriptional data gained by both methods is most commonly performed using clustering algorithms that group genes and samples on the basis of expression profiles, and statistical methods scoring the genes on the basis of their relevance to clinical manifestations. The method of choice for global expression profiling depends on several factors, including technical, labor, price, time and effort involved, and most importantly the type of information sought. When comparing microarrays

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to EST libraries, an appreciable advantage of the latter is its inherent ability to identify transcripts without prior knowledge of the genes’ coding sequences. This is the reason for its important potential for cloning and sequencing novel transcripts and genes. On the other hand, recent technical advances in the development of expression arrays, their abundance and commercial availability, and the relative speed with which analysis can be done are all factors that make arrays more useful in routine applications. In addition, array content can now be readily customized to cover gene clusters and pathways of interest to the entire genome. Some studies examine series of tissue-specific transcripts and/or genes known to be involved in particular pathology. Others directly use arrays covering the entire genome. Another factor that needs to be considered prior to embarking on any high-throughput approach is whether individual or pooled samples will be investigated. Series of pooled samples reduce the price, the time spent, and the number of the experiments down to the most affordable. Investigating individual samples, however, is important for identifying unique expression ratios in a given type of tissue or cell. A requirement of all high-throughput screening approaches is confirmation of findings (expression level of a given gene/sequence) by other independent methods. A select group of genes is tested. These genes are selected from the series of sequences that were analyzed either because they were found to have significant changes or due to their particular interest with regard to their expression in the studied tissue or their previously identified relationship to pathology or developmental stage. The confirmation process attempts to support the findings on three different levels: reliability of the highthroughput experiment (for this purpose, the same samples examined by the EST libraries or microarrays are used), trustfulness of the observations in general (to achieve this, larger number of samples are examined— assessment of which by high-throughput approaches is often unaffordable in terms of price and/or labor), and verification of expression changes at the protein level.59 A commonly used confirmatory technique is quantitative real-time reverse transcription PCR (qRT-PCR). For verification at the protein level, immunohistochemistry (IHC) and Western blot are the two most commonly chosen techniques. IHC is not quantitative but has the advantage of allowing for the observation of the exact localization of a signal within a cell (cytoplasmic versus nuclear) and the tissue (identifying histologically the tissue that is stained). Modern Western blot methods require a smaller amount of protein lysate than older techniques, and have the advantage of offering high-resolution quantitation of expression without the use of radioactivity.

Chromosome Analysis and Molecular Cytogenetics Chromosomes represent the most condensed state in metamorphosis of the genome during a cell cycle. Condensation of the genetic material at metaphase stage is a crucial event that provides precise and equal segregation of chromosomes between the two newborn daughter cells during the

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next step, anaphase. This is followed by relaxation of the genetic content after cell division. This ability of the genome to transform from a molecular level (DNA) to materialistic submicroscopic stage (chromosome) provides a unique opportunity to visualize the genome of an individual cell of an organism. Different chromosomal abnormalities related to particular diseases or syndromes can be detected at this stage by karyotyping chromosomes. Chromosomes can be individually recognized and classified by size or shape (ratio of the short/long arm) or by using differential staining techniques. In the past, identification of chromosomes was restricted to chromosome groups only. The introduction of chromosome banding technique revolutionized cytogenetic analysis.60 The banding patterns are named by the following abbreviations: G (Giemsa), R (reverse), Q (Quinacrine), and DAPI (4’6’-diamino-2-phenylindole). The last two give a pattern similar to G-banding. Further development of high-resolution banding techniques61 enabled the study of chromosomes at earlier stages of mitosis, prophase, and prometaphase. Chromosomes are longer and have enriched banding pattern at those stages, providing great details for identification of chromosomal aberrations.

OUTLINE OF METHODS Preparation of quality chromosomes is an art. Many different methods for chromosome isolation have been developed in cytogenetics within the last half century. The main principle behind all methods is to arrest cells at metaphase by disruption of the cell spindle. Metaphase spindle is a structure composed of tubular fibers (formed in the cell) to which the chromosomes are attached by kinetochors (centrosomes). The spindle separates the chromosomes into the two daughter cells. The agent commonly used for spindle disruption is colcemid. The exposure time to colcemid varies depending on the proliferative activity of cells. Cells with a high proliferative index need a shorter time of exposure to a high concentration of colcemid (0.1–0.07 ug/ml for 10 to 20 minutes). Slow-growing cells require longer exposure (1–4 hours or overnight with a lower concentration of 0.01–0.05 ug/ ml). Prolonged exposure to colcemid or the use of high concentrations increases the proportion of chromosomes at late metaphase, resulting in shortening of the chromosomes. Conversely, a short exposure with a high concentration of colcemid reduces the total yield of metaphases. The optimum strikes a balance of these parameters. There are some additional modifications that allow for the enrichment of long (prometaphase) chromosomes by using agents that prevent DNA condensation, such as actinomycin D, ethidium bromide, or BrDU. Cell synchronization techniques can also significantly increase the total yield of metaphase chromosomes.

APPLICATIONS Chromosomes are invaluable material for the evaluation of genome integrity and its preservation at the microscopic chromosomal level. The areas of application include prenatal diagnostics, genetic testing of multiple familial syndromes (including cancer), positional cloning of the genes,

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and physical mapping (assignment of the genes to chromosomes and subchromosomal regions). The number and morphology of all 23 chromosome pairs in humans can be examined using G-banding differential staining of chromosomes obtained from a peripheral blood sample. Abberations in the number of chromosomes or visible chromosomal alterations (such as translocations, deletions, and inversions involving extended regions) can be detected by this method. Recent advances such as spectral karyotyping (SKY) allow for better visualization of aneuploidy and translocations between different chromosomes.62 Subtle rearrangements, such as submicroscopic deletions or cryptic translocations (an exchange of the small distal telomeric regions between the two nonhomologous chromosomes), can be visualized using specific probes in the fluourescent in situ hybridization (FISH) technique (Figure 1-13).63

FUTURE DEVELOPMENTS Chromosome analysis will remain a powerful analytical tool in clinical and research fields for the foreseeable future. Possible strategies for improving existing methods include automation and linearization of the genetic content by increasing resolution to visualize at the level of the chromosome, chromatin, DNA, and gene. Another possible direction of development is functional analysis of the genome using constitutional chromosomes and labeled expressed sequences from particular tissues mapped directly to their original position on chromosomes (expression profiling).

Molecular Basis of Pediatrc Endocrinopathies The past two decades haves witnessed the increasing application of recombinant DNA technology to the understanding of the pathogenesis of endocrinopathies. Although this new approach to endocrinologic disorders has resulted in the delineation of new syndromes, its major impact has been in facilitating the diagnosis of these disorders. Genetic counseling that includes anticipatory surveillance, as in multiple endocrine neoplasia (MEN) syndromes (see Chapter 13), is also one of the areas of clinical pediatric endocrinology experiencing major impact from this “new” knowledge. For example, it is becoming increasingly clear that phenotypically homogenous clinical syndromes may result from different genotypic abnormalities and that similar genetic abnormalities may have very different clinical manifestations. In contrast, therapeutic implications of such knowledge are still limited. Hence, the earlier hopes of spectacular gains from gene therapy have not been realized and significant problems need to be addressed before gene therapy becomes a reality in routine patient care. However, targeted pharmacotherapy exploiting knowledge gained regarding molecular mechanisms and pathogenesis has been successfully employed in the treatment of some diseases (such as androgen insensitivity and thyroid cancer). The following section explores a couple of seminal examples of clinical endocrine disorders whose molecular

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A

19

B

Figure 1-13. Human metaphase chromosomes. (A) After FISH using the chromosome-X–specific centromeric probe labeled with spectrum orange (SO) and chromosome-Y–specific heterochromatin labeled with spectrum green (SG). (B) With the inverted DAPI banding (similar to G-banding) allowing chromosomal identification. (See color plates.)

basis has been elucidated. Details of the specific disorders are elaborated in the respective chapters of this book.

DEFECTS IN PEPTIDE HORMONES Genomic Deletions Causing Human Endocrine Disease One of the early discoveries regarding the molecular basis of endocrinopathies was the absence of the gene coding for a particular peptide hormone. The entire gene could be missing, or only a part of the gene could be deleted. In either case, this resulted in the inability to synthesize a functional peptide so that the patient presents with clinical features indicative of deficiency of the hormone. A classic example of this type of endocrinopathy relevant to pediatric endocrinology is isolated growth hormone deficiency, type 1A (IGHD 1A).64 In this syndrome the gene for human growth hormone-N (hGH-N) is deleted. Disease results when both alleles of hGH-N are absent (indicative of an autosomal recessive inheritance). In the human, there are two hGH genes (hGH-N and hGH-V)—and both of them, along with the three placental lactogen (chorionic somatomammotropin) genes, are clustered along a 48-kilobase region of DNA. hGH-N is expressed in the pituitary gland and is the source of circulating growth hormone in the human, whereas hGH-V is expressed only in the placenta and its biologic function is not clear at this time. In autosomal recessive type 1A isolated growth hormone deficiency, although hGH-V is intact the absence of the active gene (hGH-N) results in deficiency of circulating growth hormone and the growthhormone–deficient phenotype. In the original case description, the infants were of relatively normal size at birth but developed severe growth failure during the first year

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of life. A distinctive feature of these infants was the propensity to develop antibodies against growth hormone in response to exogenously administered growth hormone. This feature, although common, is not invariably present in patients with IGHD 1A. The state-of-the-art method for screening for a hGH-N deletion is based on PCR amplification of the highly homologous regions of DNA on the long arm of chromosome 17 that flank the hGH-N gene.65,66 The presence of convenient restriction enzyme sites, such as SmaI, in the amplified DNA is exploited to ascertain the presence or absence of deletions in this region of the chromosome. Although this PCR-based approach is useful in most cases, it does not identify all cases with abnormalities in the hGH-N gene. In particular, more rigorous methods are required for the identification of the less common causes of IGHD-1A (such as point mutations). The study of the hGH-hPL gene cluster has also shed light on the function of prolactin because several studies have identified subjects with hPL deletions. These deletions do not result in overt clinical abnormalities, and specifically do not cause clinical effects in pregnant patients—suggesting that despite high levels of hPL found during pregnancy hPL serves no essential function in the human.67

Point Mutations Peptide hormones act by binding to a specific receptor, which then results in the biologic actions attributed to that particular hormone. The binding of the peptide hormone to its receptor, classically described as the lockand-key mechanism, is a very precise mechanism dependent on the complementary structures of the receptor and the site on the hormone involved in binding to the receptor. A change in the nucleotide sequence of the

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gene coding for a peptide hormone that results in altering an amino acid residue of the hormone can affect the function of the hormone if this change interferes with the hormone’s ability to bind or activate the receptor. Historically, point mutations resulting in aberrant protein production were well described with hemoglobin. This was possible because hemoglobin is present in abundant quantities in the blood, enabling purification and analysis of hemoglobin from patients suspected of having hemoglobinopathies. On the other hand, peptide hormones are present in infinitesimally lesser amounts in circulation—making their direct purification and analysis from blood a more daunting task. With the advent of recombinant DNA technology, it became feasible to clone and analyze the gene coding for the hormone directly without having to resort to purification of the protein from blood or tissue. This approach resulted in the identification of several clinical syndromes due to mutant hormones. One of the classic examples of this type of molecular pathology is non-insulin diabetes mellitus (NIDDM) due to mutant insulins.68 A number of patients have been described with point mutations in the insulin gene. In general, these patients present with hyperglycemia, hyperinsulinemia, and normal insulin sensitivity—a clinical picture that is attributed to the production of an abnormal insulin molecule with reduced biologic activity. Thus, Insulin Chicago is characterized by a single nucleotide change (TTC to TTG) that results in the substitution of a leucine for phenylalanine residue at position 25 of the B chain (Phe-B25-Leu). Similarly, the other two well-characterized point mutations are characterized by the change of a single amino acid residue and result in the formation of Insulin Wakayama (Val-A3-Leu) and Insulin Los Angeles (PheB24-Ser). These mutations are located within the putative receptor-binding region of the insulin molecule, and the insulin molecules transcribed from these mutant genes are characterized by low binding potency (⬍5% compared to normal) for the insulin receptor. A separate class of mutations in the insulin gene gives rise to the syndrome of hyperproinsulinemia with or without clinically significant carbohydrate intolerance. Thus, two mutations described involve substitution of a histidine for an arginine at position 65 in one case and histidine at position 10 of the B chain changed to aspartic acid in the other. Although the original descriptions of these mutant insulins relied on the purification and analysis of the abnormal insulin molecule per se, the current availability of PCR-based methods to screen for these mutations has greatly simplified the laboratory diagnosis of this syndrome. Another example of point mutations resulting in a phenotype that is especially relevant to pediatric endocrinologists is hypopituitarism secondary to abnormalities in transcription factors that orchestrate embryologic development of the anterior pituitary gland.69,70 POU1F1 (also known as PIT1 or GHF-1) was the first transcription factor identified as playing a specific role in pituitary development. The POU1F1/PIT-1 gene encodes a 291– amino-acid Pit Oct Unc (POU) homeodomain DNA binding nuclear protein present in somatotrophs, lactotrophs, and thyrotrophs. POU1F1/PIT-1 is necessary for the

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normal development of these pituitary cell types. The first indication that abnormalities in POU1F1/PIT-1 may result in a phenotypic change was derived from studies on strains of mice with genetic forms of dwarfism. The Snell and Jackson strains of dwarf mice are characterized by a deficiency of growth hormone, prolactin, and TSH. In 1990, Li et al. reported that Snell phenotype was caused by a missense mutation and that the Jackson phenotype was caused by a rearrangement in the POU1F1/PIT-1 gene.71 Since this landmark study, several recessive and dominant types of POU1F1/PIT-1 abnormality have been recognized in sporadic cases and in multiplex families with hypopituitarism.72-74 POU1F1/PIT1 activates growth hormone and prolactin gene expression, and can bind to and transactivate the TSH-B promoter. Accordingly, patients with POU1F1/PIT-1 mutations demonstrate growth hormone, prolactin, and variable TSH deficiencies.72 The syndrome can be inherited in an autosomal dominant or recessive manner, but POU1F1/ PIT-1 mutations are not a common cause of combined pituitary hormone deficiencies. HESX1 is a paired-like homeodomain transcription factor expressed in the developing pituitary gland. Mutations in this gene leading to decreased activity have been found in two siblings with panhypopituitarism, absent septum pellucidum, optic nerve hypoplasia, and agenesis of the corpus callosum—implicating HESX1 in the mediation of forebrain development.75,76 Another mutation in HESX1 was recently discovered in a patient with septo-optic dysplasia and isolated growth hormone deficiency.77 Experimental evidence suggests that this particular mutation results in the production of an altered HESX1 protein with enhanced DNA binding activity that abrogates the transcriptional activity of PROP1, another transcription factor necessary for pituitary development. PROP1 is thought to be involved in the differentiation of somatotropes, thyrotropes, lactotropes, and gonadotropes. Mutations in PROP1 leading to reduced DNA binding and transcriptional activity have been identified in patients with combined pituitary hormone deficiency. These patients have a deficiency of TSH, growth hormone, prolactin, LH, and FSH.78 Patients with a demonstrated gonadotropin deficiency may present with failure to enter puberty spontaneously, whereas other patients do enter puberty but have subsequent loss of gonadotropin secretion. Although PROP1 is directly implicated in the differentiation of only four of the five anterior pituitary cell types, some patients have been described with ACTH deficiency. PROP1 mutations are believed to be relatively common (32%–50%) genetic causes of combined pituitary hormone deficiency.79 Rathke’s pouch initially forms but fails to grow in LHX3-knockout mice.80 Humans have been found to have mutations in the LHX3 gene (a LIM-type homeodomain protein), and demonstrate complete deficits of growth hormone, PRL, TSH, and gonadotropins in addition to a rigid cervical spine leading to limited head rotation. LHX4 is a related protein that (similar to LHX3) regulates proliferation and differentiation of pituitary lineages. A patient has been identified with a dominant mutation in this protein, demonstrating deficiencies of

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growth hormone, TSH, and ACTH; a small sella turcica; a hypoplastic anterior hypophysis; an ectopic posterior hypophysis; and a deformation of the cerebellar tonsil into a pointed configuration.81 Finally, Rieger syndrome is a condition with abnormal development of the anterior chamber of the eye, dental hypoplasia, and a protuberant umbilicus associated with growth hormone deficiency. All mutations in RIEG (Pitx2) found thus far have been heterozygous, with an autosomal dominant inheritance.82

DEFECTS IN PEPTIDE HORMONE RECEPTORS Molecular defects resulting in phenotypic abnormalities in humans have been described in a variety of peptide hormone receptors, including growth hormone, LH, FSH, TSH, ACTH, and insulin. It is expected that this list will continue to expand in the future. Mutations in receptors for peptide hormone interfere in the actions of the hormone by altering the binding of the hormone, by altering the number of the receptors available for binding to the hormone, by interfering with the synthesis or intracellular processing of the receptor, or by disrupting the activation of the postreceptor signaling pathways. In general, mutations in the receptor result in decreased actions of the cognate hormone. However, mutations involving G-protein–linked receptors are exceptions to this generalization and can result in a phenotype characterized by “over-activity” of the particular hormone system. Examples of these “gain-of-function” mutations include mutations in the LH receptor responsible for familial testotoxicosis83 and mutations in the TSH receptor causing thyrotoxicosis84 (see Chapter 2).

Insulin Receptor Following the initial reports in 1988, a variety of mutations have been identified in the insulin receptor gene— with the majority of them being in patients with genetic syndromes associated with acanthosis nigricans and insulin resistance.85 Patients with leprechaunism (a congenital syndrome characterized by extreme insulin resistance, fasting hypoglycemia, and intrauterine growth retardation) have two mutant alleles of the insulin receptor gene. Another syndrome associated with acanthosis nigricans and extreme insulin resistance, the RabsonMendenhall syndrome, has been linked to two different mutations within the insulin receptor gene existing in a compound heterozygous state. The syndrome of type A insulin resistance is a heterogeneous collection of conditions defined by the presence of insulin resistance, acanthosis nigricans, and hyperandrogenism in the absence of lipoatrophy or obesity. Molecular analysis of the insulin receptor gene has revealed that several of these patients have mutations in one or both alleles of the insulin receptor gene. The initial expectation that mutations in the insulin receptor would provide the molecular basis for the common type of type 2 (NIDDM) diabetes mellitus has not been fulfilled. And, no alterations in the insulin receptor gene were identified in a study of Pima Indians—an ethnic group

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with a greater than 50% incidence of type 2 diabetes mellitus. It is noteworthy, however, that recent studies have advanced the search for genetic variations that influence the propensity to develop type 2 diabetes mellitus.86,87 Positional-cloning–based analysis suggests that specific polymorphisms in the CAPN10 gene are associated with type 2 diabetes mellitus in the Finnish and MexicanAmerican (Pima Indian) populations. Whether these genetic variations in the CAPN10 gene, a chromosome 2 gene that encodes a widely expressed calpain-like cysteine protease, are causal factors for Type 2 diabetes mellitus or are merely co-segregating markers remains to be established. However, current studies have not excluded the possibility that polymorphisms in the insulin receptor gene may confer a genetic predisposition for the precipitation of the development of NIDDM by obesity and/or hypertension.

Growth Hormone Receptor Genetic abnormalities in the growth hormone receptor result in the primary form of the syndrome of growth hormone insensitivity, also called Laron syndrome.88 The human growth hormone receptor gene located on the proximal part of the short arm of chromosome 5 spans approximately 90 kilobases and includes nine exons (numbered 2 through 10) that encode the receptor protein and additional exons in the 5’UTR region of the gene. The growth hormone receptor protein contains an open reading frame of 638 amino acids that predicts a 246-amino-acid-long extracellular ligand-binding domain, a single membrane-spanning domain, and a cytoplasmic domain of 350 amino acids. In the human, the extracellular portion of the receptor exists in circulation as the growth-hormone–binding protein (GHBP). Exon 2 encodes a signal sequence, exons 3 through 7 the extracellular GHB domain, exon 8 the transmembrane domain, and exons 9 and 10 the cytoplasmic domain and the 3’UTR region. The mutations that have been described in the growth hormone receptor gene include large deletions, nonsense mutations, splice mutations, and frameshift mutations.89 The diagnosis of growth hormone insensitivity due to mutations in the growth hormone receptor gene is considered when patients demonstrate elevated growth hormone levels and low IGF-1 levels. Because the GHBP is the cleaved extracellular portion of the growth hormone receptor, patients with mutations in the growth hormone receptor that results in decreased synthesis of the receptor protein can demonstrate low GHBP levels in circulation. However, mutations in the growth hormone receptor gene that selectively involve the transmembrane or intracellular domains may demonstrate normal or even enhanced circulating levels of GHBP. For example, a patient with a mutation that inhibits dimerization of the receptor had normal GHBP levels because the receptor had a normal GHB site.90 Another set of affected individuals had a mutation of the transmembrane domain of the receptor, leading to a truncated growth hormone receptor product postulated to be more easily released from the cell membrane. In addition, elevated GHBP levels were noted.91

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Recombinant DNA Technology and Therapy for Pediatric Endocrine Diseases From a therapeutic point of view, recombinant DNA technology can be exploited to tailor pharmacotherapy according to the genotype of a patient (i.e., targeted pharmacotherapy), to manipulate genes within the human body (gene therapy), or to engineer prokaryotic or eukaryotic cells to produce proteins (such as hormones) that can then be administered for therapy or diagnosis. Whereas targeted pharmacotherapy and gene therapy are largely restricted to the research arena, the use of hormones produced by recombinant DNA technology is well established in clinical endocrinology. Historically, insulin was the first hormone synthesized by recombinant DNA technology to be approved for clinical use.92,93 At present, a variety of recombinant hormones (including growth hormone, LH, FSH, TSH, PTH, and erythropoietin) are being used clinically or are in advanced stages of clinical trials. On a theoretical basis, it should be possible to synthesize any protein hormone whose gene has been cloned and DNA sequence determined. Thus, recombinant DNA technology makes it possible to insert the gene coding for a particular protein hormone into a host cell such that the protein is produced by the host cell’s protein synthesizing machinery. The synthesized protein is then separated from the rest of the host cell proteins to obtain the pure form of the hormone of interest. Both prokaryotic and eukaryotic cells can serve as the host cell for the production of proteins by this technology. Because post-translational modifications such as glycosylation may be essential for the optimal action of a protein hormone, the choice of the specific cell system utilized for the production of a particular protein hormone is critical. Prokaryotic cell systems such as Escherichia coli are suitable for the production of protein hormones that do not need post-translational modifications, such as growth hormone.94 Eukaryotic cell systems such as CHO (Chinese hamster ovary) cells, which are capable of post-translational modification of the protein, are useful for the production of hormones (such as TSH) that require glycosylation for optimal bioefficacy.95 In addition, eukaryotic cells are capable of synthesizing proteins that undergo the appropriate folding—a step that is not carried out by prokaryotic cells. The advantages of the use of recombinant DNA for the production of these proteins include the possibility of a limitless supply of a highly pure form of a protein and the absence of the risk of contamination with biologic pathogens associated with extraction of proteins from human or animal tissue. In addition, this technology permits the development of hormone analogues and antagonists with much greater ease than conventional protein synthesis protocols. The influence of genetic factors on the metabolism of various drugs is a well-established phenomenon, with the effect of various isozymes of cytochrome p450 on the circulating half-life of drugs such as anticonvulsants being a classic example of this interaction. Another example of the role of genotype on the choice of pharmacother-

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apy is the phenomenon of drug-induced hemolytic anemia in patients with G6PD deficiency. The recent genomic revolution has allowed for the exploitation of computational approaches to identifying polymorphisms of known genes encoding proteins with different functional characteristics. These single-nucleotide variations (also called single-nucleotide polymorphisms or SNPs) occur with varying frequencies in different ethnic populations and are the focus of intense scrutiny at this particular time. The promise these SNPs hold out is that analysis of these SNPs for a given gene will allow the investigator to predict the response of the particular individual to a class of drugs or chemicals. This pharmacogenomic approach to clinical therapeutics has already been successful in demonstrating an association between specific polymorphisms in the beta-adrenergic receptor and response to beta-agonists in patients with bronchial asthma, and polymorphisms in hydroxytryptamine receptors and response to neuroleptic drugs. The widespread application of the tools of molecular biology to unravel the molecular basis of action of hormones has also yielded benefits by allowing for customization of pharmacotherapy of endocrine diseases and syndromes based on the specific individual genetic defect. One such example is the report of directed pharmacologic therapy of an infant with ambiguous genitalia due to a mutation in the androgen receptor.96 In this infant with an M807T mutation in the androgen receptor, in vitro functional studies had indicated that the mutant receptor exhibited loss of binding capacity for testosterone with retention of binding for dihydrotestosterone (DHT). Furthermore, this differential binding was also reflected in the better preservation of the transactivation potential of DHT compared to testosterone. These in vitro findings were exploited to treat the infant with DHT, resulting in restoration of male genital development. This case illustrates that in selected cases in vitro functional assays can help identify subsets of patients with ambiguous genitalia and androgen insensitivity who would respond to targeted androgen therapy. It can be anticipated that in the coming years more examples of such innovative therapeutic strategies and “customized” hormonal treatment protocols will become routinely implemented in the practice of clinical pediatric endocrinology.

Concluding Remarks The application of recombinant DNA technology has resulted in a tremendous increase in our understanding of physiologic processes and pathologic conditions. The U.S. government sponsored Human Genome Project initiative launched in 1992 has catalyzed this revolution, which is currently being fueled by both public sector efforts and private sector entrepreneurship. The central goal of this initiative, which represents collaborative efforts among scientists in the United States and around the world, is to sequence the entire 3 to 4 billion base pairs of the human genome and to construct a detailed genetic and physical map of the entire human genome (i.e., each of the 24 different human chromosomes).

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Such a map, along with advances in communication and computers, will allow a scientist to locate and isolate any human DNA sequence of interest. Recent achievements, such as the draft sequence of the human genome, are major milestones toward the fulfillment of this goal and have resulted in a paradigm shift in the way that novel genes are being discovered and in the analysis of the function of hormones and related proteins.97 In the traditional paradigm, investigators seeking to discover new genes or to analyze the function of known proteins needed to devote a significant part of their time to conducting “bench research” in “wet laboratories.” The new approach in this “post-genomic” era takes advantage of the unprecedented power of computational biology to “mine” nucleotide, protein sequence, and other related databases. In the future, most researchers will deal with abstract models and data sets stored in computer databases. Hence, initial discoveries of novel genes or novel interactions between known proteins or intracellular signaling pathways could be made using the analytic power of computational software tools (functional genomics). These initial insights can then be verified and expanded upon by traditional benchtop methods. The advantages of this new paradigm are obvious, with computational approaches taking a significantly shorter time and with fewer demands on manpower. The new paradigm can also easily expand the scope of the search to include multiple molecules and organisms (phylogenetic profiling). One of the key contemporaneous scientific developments that have enabled the efficient and widespread use of this new paradigm is the expansion of the Internet. The Internet has enabled the capture, storage, analysis, and dissemination of the enormous amounts of data generated by the Human Genome Project in a manner that is efficient, protects the privacy of individuals, and is easily accessible for legitimate use. Several public-domain web-accessible databases are currently serving as the major repositories for this information. GenBank is the major repository for sequence information and is currently supported by the National Institutes of Health. One of the main sources of the physical location, clinical features, inheritance patterns, and other related information of specific gene defects is the Online Mendelian Inheritance in Man (OMIM) operated by Johns Hopkins University in Baltimore, Maryland. Johns Hopkins University also operates the online Genome Database (GDB), which allows scientists to identify polymorphisms and contacts for gene probes and other related research tools. The ever-expanding number of endocrine (and other) disorders that can be attributed to changes in the nucleotide sequence of specific genes has also increased the necessity for the availability of accurate, reliable, and timely genetic tests such as mutation detection. One source for such information is a collaborative website (www.genetests.org) that maintains an up-to-date catalog of commercially available and research-based tests for inherited disorders. With the ubiquitous use of these powerful tools in laboratories around the world, genes are being cloned and genetic diseases being mapped at a very rapid pace. In all of this excitement, one still needs to keep in mind that whereas this “new” science has allowed for hitherto

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inaccessible areas of human biology to be probed and studied, a lot remains to be understood with respect to individual disease processes. Hence, at the present time we have only a rudimentary understanding of the correlations between phenotype and genotype in many of the common genetic diseases (such as congenital adrenal hyperplasia). These lacunae in our knowledge dictate that clinicians should be cautious about basing therapeutic decisions solely on the basis of molecular and genetic studies. This is especially true in the area of prenatal diagnosis and termination of pregnancy based on genetic analysis. As we improve our understanding of the molecular and genetic basis of disease and translate this knowledge into gains at the bedside, it behooves us both as individuals and as a society to be cognizant of critical issues relating to privacy of health data and to remain vigilant against misuse by inappropriate disclosure of this powerful knowledge.

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84. Duprez L, Parma J, Van Sande J, Rodien P, Sabine C, Abramowicz M, et al. (1999). Pathology of the TSH receptor. J Pediatr Endocrinol Metab 12:295–302. 85. Taylor S, Arioglu E (1998). Syndromes associated with insulin resistance and acanthosis nigricans. J Basic Clin Physiol Pharmacol 9:419–439. 86. MT M (2005). Genetics of type 2 diabetes mellitus. Diabetes Res Clin Pract 68:S10–S21. 87. Mercado M, McLenithan J, Silver K, Shuldiner A (2002). Genetics of insulin resistance. Curr Diab Rep 2:83–95. 88. Laron Z, Pertzelan A, Mannheimer S (1966). Genetic pituitary dwarfism with high serum concentrations of growth hormone: A new inborn error of metabolism. Isr J Med Sci 4:883–894. 89. Rosenbloom A (1999). Growth hormone insensitivity: Physiologic and genetic basis, phenotype, and treatment. J Pediatr Endocrinol Metab 135:280–289. 90. Duquesnoy P, Sobrier M, Duriez B, Dastot F, Buchanan C, Savage M, et al. (1994). A single amino acid substitution in the exoplasmic domain of the human growth hormone (GH) receptor confers familial GH resistance (Laron syndrome) with positive GH-binding activity by abolishing receptor homodimerization. Embo J 13:1386–1395. 91. Woods K, Fraser N, Postel-Vinay M, Savage M, Clark A (1996). A homozygous splice site mutation affecting the intracellular domain of the growth hormone (GH) receptor resulting in Laron syndrome with elevated GH-binding protein. J Clin Endocrinol Metab 81:1686–1690. 92. Goeddel D, Kleid D, Bolivar F, Heyneker T, Yansura D, Crea R, et al. (1979). Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc Natl Acad Sci USA 76:106–110. 93. Riggs A (1981). Bacterial production of human insulin. Diabetes Care 4:64–68. 94. Goeddel D, Heyneker H, Hozumi T, Arentzen R, Itakura K, Yansura D, et al. (1979). Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone. Nature 281:544–548. 95. Gesundheit N, Weintraub B (1986). Mechanisms and regulation of TSH glycosylation. Adv Exp Med Biol 205:87–105. 96. Ong Y, Wong H, Adaikan G, Yong E (1999). Directed pharmacological therapy of ambiguous genitalia due to an androgen receptor gene mutation. Lancet 354:1444–1445. 97. Hsu S, Hsueh A (2000). Discovering new hormones, receptors, and signaling mediators in the genomic era. Mol Endocrinol 14:594–604.

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C H A P T E R

2 Receptor Transduction of Hormone Action ALAN M. RICE, MD • SCOTT A. RIVKEES, MD

Introduction G-Protein–Coupled Receptors Class A Receptors That Transduce Hormone Action The Peptide Receptor Group Adrenocorticotropin and Melanocortin-2 Receptors Other Melanocortin Receptors Vasopressin Receptors The Hormone Protein Receptor Group LH Receptors FSH Receptors TSH Receptors HCG and TSH Receptors During Pregnancy HCG and FSH Receptors During Pregnancy The Gonadotropin-Releasing Hormone Receptor Group Gonadotropin-Releasing Hormone Receptors The Thyrotropin-Releasing Hormone and Secretagogue Receptor Group Thyrotropin-Releasing Hormone Receptors Other Class A Receptors That Transduce Hormone Action Free Fatty Acid Receptor 1 GPR54 Orexin Receptors Ghrelin Receptors Melanin-Concentrating Hormone Receptors Class B Receptors That Transduce Hormone Action Growth-Hormone–Releasing Hormone Receptor Gastric Inhibitory Polypeptide Receptors Parathyroid Hormone and ParathyroidHormone–Related Peptide Receptors

Other Class B Receptors That Transduce Hormone Action Class C Receptors That Transduce Hormone Action Calcium-Sensing Receptors G-Protein Gene Disorders Inactivating Mutations of the GNAS1 Gene Activating Mutations of the GNAS1 Gene Cytokine Receptors Structure and Function of Type I Cytokine Receptors Cytokine Receptors That Transduce Hormone Action Growth Hormone Receptors Leptin Receptors Receptor Tyrosine Kinases Insulin Receptor Tyrosine Kinase Family The Insulin Receptor The Insulin-Like Growth Factor-1 Receptor The Fibroblast Growth Factor Receptor Family Fibroblast Growth Factor Receptor 1 Fibroblast Growth Factor Receptor 3 Nuclear Receptors General Structure of the Nuclear Receptors Subfamily 1 Nuclear Receptors: Thyroid Hormone, Vitamin D3, and Peroxisome Proliferator-Activated Receptors Thyroid Hormone Receptors Vitamin D3 Receptor PPAR
Subfamily 2 Nuclear Receptors: Hepatocyte Nuclear Factor and Retinoid X Receptors HNF

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Estrogen Receptors Mineralocorticoid Receptors Subfamily 0 Nuclear Receptors: DAX1 DAX1 Summary

Subfamily 3 Nuclear Receptors: The Steroid Receptors and Glucocorticoid, Androgen, Estrogen, and Mineralocorticoid Receptors Glucocorticoid Receptors Androgen Receptors

Introduction Hormones exert their actions by activating specific receptors. Activation of these receptors stimulates intracellular responses that influence cellular physiology and, at times, gene expression. The specificity of hormone action is thus determined by the affinity of hormones for different receptors, receptor distribution, and effector and genetic responses to ligand occupancy. Over the past two decades, our understanding of hormone action has increased greatly—in part due to the cloning of many receptors that transduce hormone action. Four major receptor superfamilies have been identified that share common structural elements and/or effector systems. These families include the G-protein–coupled receptors (GPCRs), cytokine receptors, tyrosine kinase receptors (RTKs), and nuclear receptors (Table 2-1). This chapter reviews major features of these important receptor families. Mutations influencing receptor function leading to endocrine disorders are also highlighted.

G-Protein–Coupled Receptors More than 1% of the genome of vertebrates encodes GPCRs.1 Most of these GPCRs are odorant and pheromone receptors.1 It is also important to note that most of the receptors that transduce the effects of hormones are GPCRs (Table 2-2). The GPCR superfamily is divided into eight major classes.1,2 These receptors contain an N-terminal extracellular domain that is frequently called the ectodomain or exodomain.3 These receptors also contain seven putative transmembrane helixes (TM-I to TM-VII) connected by three intracellular (i1 through i3) and three extracellular

27

(e1 through e3) loops that are often collectively called the serpentine region (Figure 2-1).3,4 The C-terminal intracellular region is usually referred to as the endodomain.3 GPCRs are activated by a wide variety of signals, including proteins, nucleotides, amino acid residues, Ca2, light, and odorants (Figure 2-1).1 It is postulated that alteration of the conformation of transmembrane domains by ligand binding changes the conformation of intracellular loops, leading to activation of heterotrimeric guanosine nucleotide binding proteins (G proteins) (Figure 2-1).5,6 When a GPCR is activated by a ligand, guanosine 5'-diphosphate (GDP) is converted to guanosine 5'-triphosphate (GTP)— which causes the heterotrimeric G protein to dissociate into active G-GTP and G
subunits (Figure 2-1).5-7 GTPase then converts GTP into GDP, which inactivates G and increases affinity of G for G
leading to reformation of the inactive heterotrimeric G protein.5-7 Active G and G
subunits can alter the activity of transmembrane channels, and the activity of intracellular effector enzymes that include phospholipase C, adenylyl cyclase, and kinases (Figure 2-1).1,7 Specificity in ligand binding is conferred by variations in the primary structures of the extracellular and intracellular domains.1 Specificity of effector responses is conferred by the variations in the primary structure of intracellular domains and isoforms of the G subunits of G proteins.8,9 Some GPCRs couple predominantly with G proteins with Gi /Go subunits that act primarily to decrease adenylyl cyclase activity.9-12 Other GPCRs couple predominantly with G proteins with Gs subunits that act to increase adenylyl cyclase activity, or Gq/G11,14,15,16 subunits that increase phospholipase C activity.9,13,14 Interestingly, data show that cytoskeletal proteins may module receptor–G-protein coupling. For example, the erythrocyte membrane cytoskeletal protein 4.1G can

TA B L E 2 - 1

Major Types of Hormone Receptors Receptor Class G-protein–coupled receptors Type I cytokine receptors Receptor tyrosine kinases Nuclear receptors

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Hormone Receptors ACTH and other melanocortins, V2 vasopressin, LH, FSH, TSH, GnRH, TRH, GHRH, corticotropin releasing factor, somatostatin, glucagon, oxytocin, gastric inhibitory peptide, type 1 PTH, free fatty acid, GPR54, orexin, ghrelin, melanin-concentrating, calcitonin, glucagon-like peptide-1, and calcium-sensing receptors Growth hormone, prolactin, and leptin receptors Insulin, IGF-1, and fibroblast growth factor receptors Thyroid hormone, vitamin D3, PPAR
, HNF-4, glucocorticoid, androgen, estrogen, mineralocorticoid, and DAX1 receptors

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TA B L E 2 - 2

G-Protein–Coupled Receptors and Clinical Conditions Associated with Receptor Mutations Receptor ACTH/melanocortin-2 receptor Melanocortin-4 receptor V2 vasopressin receptor LH receptor

FSH receptor

TSH receptor

GnRH receptor TRH receptor GPR54 Ghrelin GHRH receptor Type 1 PTH receptor

Calcium-sensing receptor

Germ Line Mutation

Activating mutations (heterozygous) Inactivating mutations (homozygous, compound heterozygous)

Inactivating mutations (most homozygous or compound heterozygous, rarely heterozygous) Activating mutations (heterozygous) Inactivating mutations (homozygous or compound heterozygous) Inactivating mutations (compound heterozygous) Inactivating mutations (homozygous, compound heterozygous) Inactivating mutations (homozygous, possible heterozygous) Inactivating mutations (homozygous/compound heterozygous) Inactivating mutations (homozygous, heterozygous) Activating mutations (heterozygous) Inactivating mutations (heterozygous, homozygous)

Activating mutations (heterozygous)

interfere with A1 adenosine receptor signal transduction.15 4.1G also influences mGlu1alpha-mediated cAMP accumulation, increases the ligand-binding ability of mGlu1alpha, and alters its cellular distribution.16 4.1G may also play a role in receptor-receptor dimerization. Receptor agonist-independent and agonist-induced homo- and heterodimerization have increasingly been recognized as important determinants of GPCRs function.17 For example, the GPCR somatostatin receptor 5 (SSTR5) primarily exist as monomers in the absence of an agonist. However, they form homodimers in the presence of an agonist.18 Furthermore, it has been shown that SSTR5 can form heterodimers with type 2 dopamine receptors (DRD2)—another GPCR—in the presence of hsst2 agonist or dopamine.19 Agonist-induced activation of SSTR5-DRD2 heterodimers in China hamster ovary (CHO)

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Endocrine Disorder

Inactivating mutations (homozygous, compound heterozygous) Inactivating mutations (most heterozygous, some homozygous) Inactivating mutations (most X-linked recessive, rarely X-linked dominant) Inactivating mutations (homozygous, compound heterozygous)

Familial glucocorticoid deficiency type 1 Obesity X-linked nephrogenic diabetes insipidus Males: types I and II Leydig cell hypoplasia Females: asymptomatic or hypergonadotropic hypogonadism with primary amenorrhea Males: male limited precocious puberty Females: autosomal recessive hypergonadotropic ovarian dysgenesis or milder hypergonadotropic hypogonadism Males: variable impairment of spermatogenesis Resistance to TSH

Autosomal-dominant inherited nonautoimmune hyperthyroidism/toxic adenomas Isolated hypogonadotropic hypogonadism Central hypothyroidism Normosmic isolated hypogonadotropic hypogonadism Short stature due to decreased growth hormone secretion Isolated growth hormone deficiency Blomstrand’s chondrodysplasia if homozygous and rarely if heterozygous; enchondromatosis if heterozygous Jansen’s metaphyseal chondrodysplasia Familial benign hypocalciuric hypercalcemia typical if heterozygous, neonatal severe hyperparathyroidism rarely if heterozygous, typical if homozygous Autosomal-dominant hypocalcemic hypocalciuria, Bartter syndrome type V

cells expressing SSTR5 and DRD2 is increased when compared to agonist-induced activation of monomers and homodimers in CHO cells expressing only SSTR5 or DRD2.19 Heterodimerization of receptors may also lead to inactivation of one of the receptors in the complex. For example, heterodimerization of somatostatin receptor 2A (sst2A) with somatostatin receptor 3 (SSTR3) appears to lead to inactivation of the heterodimerized SSTR3 without inactivating the heterodimerized SSTR2.20 GPCRs can form heterodimers with nonreceptor transmembrane proteins. Both the calcitonin receptor (CALCR) and the calcitonin receptor-like protein (CALCRL) can form heterodimers with three different receptor-activity– modifying proteins (RAMPs): RAMP1, RAMP2, and RAMP3.21-23 Whereas CALCRs can be activated by ligand in the absence of heterodimerization with a RAMP, CALCRLs

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Light

Ca2+

Odorants Pheromones

Small molecules • Amino acids, amines • Nucleotides, nucleosides • Prostaglandins, PAF • Peptides…

e1 e2

out

e3

NH2

i2

i1 i3

Proteins • TSH • LH • FSH • Interleukins • Wingless • Chemokines • α-latrotoxin

Effector • Enzyme • Channels

Receptor

in

29

COOH GDP α

γ

Intracellular messengers

β G protein Figure 2-1. GPCR structure and function. GPCRs have an N-terminal extracellular domain, seven putative transmembrane domains separated by three extracellular loops (e1-e3) and three intracellular loops (i1-i3), and a C-terminal intracellular domain. Ligand binding results in the exchange of GTP for GDP, which induces dissociation of the G protein into a GTP subunit and a 
subunit. Then these subunits alter the activity of intracellular effector enzymes and transmembrane channels, resulting in the alteration of intracellular levels of second messengers that can include cAMP and calcium. [Adapted with permission from Bockaert J, Pin JP (1999). Molecular tinkering of G-protein–coupled receptors: An evolutionary success. Embo J 18:1724.]

are only activated by ligand if heterodimerized with a RAMP.21,22 RAMPs alter the ligand specificity of the heterodimerized receptor. CALCRs that are not in heterodimers with RAMPS are activated by calcitonin and thus constitute the classic CALCR.21,22 However, CALCRs heterodimerized with RAMP1, RAMP2, and RAMP3 bind amylin and constitute amylin1, amylin2, and amylin3 receptors, respectively.21,22 CALCRLs dimerized with RAMP1 bind calcitonin generelated peptide and constitute the calcitonin gene-related peptide receptor.21,22 CALCRLs dimerized with RAMP2 and RAMP3 bind adrenomedullin, and constitute adrenomedullin1 and adrenomedullin2 receptors, respectively.21,22 RAMPS alter function of other GPCRs that transduce hormone action. The distribution and function of parathyroid hormone 1 and 2 receptors are altered by binding to RAMP2 and RAMP3, respectively.24 The distribution and function of the glucagon receptor is altered by binding to RAMP2.24 Dimerization/heterodimerization may occur in the endoplasmic reticulum (ER) shortly after protein synthesis occurs.25 The ER plays a role in determining whether or not a protein will be expressed elsewhere in the cell, thus protecting the cell from misfolded and (likely) mutant proteins.25 The non-heterodimerized CALCRL is an orphan receptor because the CALCRLs cannot leave the ER for the cell membrane unless heterodimerized with RAMPs.26 Failure of the endoplasmic reticulum to export mutant GPCR homodimers and mutant GPCR wild-type GPCR

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heterodimers to the cell membrane has been unequivocally found to be the cause of two dominant negative endocrine conditions, and to occur without apparent clinical effect in a third endocrine condition. A dominant negative mutation is a heterozygous mutation that results in a phenotype that would only be expected in the presence of a homozygous mutation. Some V2 vasopressin receptor gene mutations that are known to cause nephrogenic diabetes insipidus encode mutant V2 vasopressin receptors that form dimers in the ER that cannot be exported to the cell membrane.27 These mutant receptors also interfere with cell surface expression of wild-type receptors by forming heterodimers with the wild-type receptors that cannot be exported from the ER to the cell membrane.28 This finding explains why females heterozygous for these V2 vasopressin receptor gene mutations do not concentrate their urine with even high doses of desmopressin, a synthetic V2 vasopressin receptor agonist, in spite of being able to produce wild-type V2 vasopressin receptors.29 A similar phenomenon explains dominant transmission of partial TSH receptor resistance in patients heterozygous for some inactivating TSH receptor mutations.30 In these patients, mutant TSH receptors form oligomers with wildtype receptors and prevent export of wild-type receptors from the endoplasmic reticulum to the cell membrane.30 Similarly, misfolding and misrouting of some mutant gonadotropin-releasing hormone (GnRH) receptors in the endoplasmic reticulum (as well as oligomerization of these mutant GnRH receptors with wild-type GnRH receptors)

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decrease cell membrane expression of wild-type GnRH receptors.31-33 This phenomenon, however, has not been found to have clinical implications in relatives of probands homozygous or compound heterozygous for mutations that cause isolated hypogonadotropic hypogonadism (IHH) because individuals heterozygous for these mutations demonstrate an intact GnRH-gonadotropin axis and do not have clinical signs of IHH. Thus, in these individuals enough wild-type GnRH receptors do not oligimerize with mutant GnRH receptors and are transported to the cell membrane to maintain sufficient normal GnRH-GnRH receptor interactions to avoid development of IHH.32 It has been recognized that many GPCRs (including lutenizing hormone, thyroid-stimulating hormone thyrotropinreleasing hormone, glucagon-like peptide-1, melanocortin, and cannabinoid receptors) activate G proteins in the absence of ligand binding.34,35 Thus, these receptors are constitutively active and cellular G-protein activation increases linearly with increased cell surface expression of the receptors.36 It has also been recognized that there are ligands (often called inverse agonists) that decrease the activity of these receptors.37 Receptor ligands that neither increase nor decrease the activity of receptors are now frequently referred to as neutral antagonists.35 The term antagonists is applied to these ligands because they block activation and inactivation of receptors by agonists and inverse agonists, respectively.35 The term agonist only refers to receptor ligands that increase

receptor activity.35 A scale has been formulated to express the continuity in receptor ligand function—from –1 (representing a full inverse agonist), to 0 (representing a neutral antagonist), to 1 (representing a full agonist).35,37 It is possible that inverse agonists play a role in treating medical conditions caused by GPCR mutations that lead to increased constitutional activation of the receptor.35 Receptor desensitization and resensitization play a role in GPCR activity. During the past decade, mechanisms of GPCR desensitization and resensitization have been elucidated. Three processes for receptor desensitization have been described.38,39 The first receptor desensitization process is rapid uncoupling of the G protein from GPCRs.39 This process occurs within seconds to minutes after initiation of the process and occurs as a result of phosphorylation of GPCRs.39 G-protein–receptor kinases (GRKs) have been increasingly recognized as playing a major role when this process involves homologous desensitization.38 Homologous or agonist-dependent desensitization occurs after agonist activation of the receptor that is desensitized.39 GRK-mediated phosphorylation of serine and threonine residues in the third intracellular loop or the C-terminal intracellular domain leads to activation of -arrestins, which in turn inactivate adenylyl cyclase (Figure 2-2).38-41 Second-messenger–dependent protein kinases also contribute to receptor desensitization when

Agonist

Cell membrane Desensitization

H2N

H2N CO2H  GDP 

H2N CO2H

 

GTP

Recycling

H2N

H2N

CO2H



GRK  GTP

CO2H PO4 PO4

-arrestin

PO4 PO4

Functional responses Endosome Endosome pH

Vesicle acidification

Phosphatase

CO2H

pH

-arrestin Internalization

CO2H PO4 PO4

CO2H

Resensitization

Sequestration

-arrestin

-arrestin

Cytosol Protein degradation (downregulation) Figure 2-2. Desensitization and recycling of GPCRs. Shortly after an agonist binds a GPCR, G-protein–receptor kinases phosphorylation of serine and threonine residues in the third intracellular loop or the C-terminal intracellular domain leads to activation of -arrestin. Activation of -arrestin inactivates adenylyl cyclase and initiates sequestration of the GPCR in clathrin-coated vesicles. Dephosphorylation of the sequestered receptor and subsequent disassociation of the receptor from -arrestin is followed by recycling of the GPCR to the cell membrane. Alternatively, once sequestered the GPCR can be destroyed in lysosomes. [Adapted with permission from Saunders C, Limbird LE (1999). Localization and trafficking of 2-adrenergic subtypes in cells and tissues. Pharmacol Ther 84:200.]

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this process involves homologous desensitization, but also participate in receptor desensitization when desensitization involves heterologous desensitization. Heterologous or agonist-independent desensitization occurs as a result of activation of a different receptor from the one that is desensitized.39 The second receptor desensitization process is internalization/sequestration of GPCRs. This process is slower than receptor phosphorylation-induced uncoupling of the G protein from GPCRs and occurs within minutes to hours after initiation of the process. This process is reversible because the receptors can be recycled to the cell surface (Figure 2-2).39 GRKs and -arrestins play a role in initiating internalization/sequestration of 2-adrenergic, LH, FSH, TSH, TRH, vasopressin V2, angiotensin II type 1A, and other G-protein–coupled receptors in clathrin-coated vesicles (Figure 2-2).38,42-49 Dephosphorylation of the sequestered receptor followed by disassociation of the receptor from -arrestin is necessary for the receptor to be recycled to the cell membrane and resensitized (Figure 2-2).14 The third receptor desensitization process is downregulation. With down-regulation the number of intracellular GPCRs decreases due to increased lysosomal degradation and decreased synthesis of the receptors due to alteration of transcriptional and post-transcriptional regulatory mechanisms (Figure 2-2).50,51 Down-regulation is a slow process that occurs within several hours to days after initiation of the processes that lead to its development.52 One of the ways the Arg137His V2 vasopressin receptor mutation interferes with mutant receptor function and causes X-linked nephrogenic diabetes insipidus is by altering desensitization and recycling of the mutant receptor.53 In vitro studies have revealed that the mutant receptor is constitutively phosphorylated. Thus, even in the absence of ligand binding the mutant receptor is bound by -arrestin—which in turn leads to sequestration of the mutant receptor within clathrin-coated vesicles. Recycling of the mutant receptor back to the cell membrane requires the mutant receptor to be dephosphorylated and disassociated from -arrestin. However, the mutant receptor remains constitutively phosphorylated while sequestered and thus cannot be disassociated from -arrestin and recycled to the cell membrane—thereby reducing cell membrane expression of the mutant receptor. Some investigators suggest that most GPCR-inactivating mutations can be classified by the effects of the mutations into one of five classes.54 Class I inactivating mutations interfere with receptor biosynthesis. Class II inactivating mutations interfere with receptor trafficking to the cell surface. Class III inactivating mutations interfere with ligand binding. Class IV inactivating mutations impede receptor activation. Class V inactivating mutations do not cause discernible defects in receptor biosynthesis, trafficking, ligand binding, or activation but may cause medical disorders. There are also inactivating mutations that interfere with receptor function via multiple mechanisms and thus cannot be placed into one class. Of the eight classes of GPCRs, only classes A, B, and C contain receptors for mammalian hormones (Figure 2-3).2 Class A receptors contain the rhodopsin-like receptors

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31

CLASS A

P P F N

Retinal Odorants Catecholamines Adenosine ATP, Opiates Enkephalins Anandamide

P P F N

Peptides Cytokines IL-8 Formyl Met-Leu-Phe PAF-acether Thrombin

P P F N

Glycoproteins Hormones (LH, TSH, FSH)

NH2 SS D D Rγ COOH

NH2 SS D D Rγ COOH NH2

SS D D Rγ COOH CLASS B NH2

Calcitonin α-latrotoxin Secretin PTH VIP PACAP CRF

COOH CLASS C

Glutamate (metabotropic) Ca2+ Pheromones

COOH Figure 2-3. Examples of class A, B, and C GPCRs. The orange oval represents the ligand. These receptors can differ in amino acid sequence, in length of the N-terminal extracellular and C-terminal cytoplasmic domains, and in the receptor regions involved with ligand-receptor interactions. [Adapted with permission from Bockaert J, Pin JP (1999). Molecular tinkering of G protein-coupled receptors: An evolutionary success. Embo J 18:1725.]

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and are divided into at least 15 groups.2,55 Four of these groups contain receptors activated by hormones. These are the peptide receptor, hormone protein receptor, gonadotropin-releasing hormone (GnRH) receptor, and the thyrotropin-releasing hormone (TRH) and secretagogue receptor groups.2 The peptide receptor group includes the angiotensin, adrenocorticotropin hormone (ACTH)/melanocortin, oxytocin, somatostatin, and vasopressin receptors.2 The hormone protein receptor group includes the receptors for glycoprotein hormones, including folliclestimulating hormone (FSH), leutinizing hormone (LH), and thyrotropin (TSH) receptors.2 These receptors have large extracellular N-terminal domains and ligandbinding sites that include the first and third extracellular loops (Figure 2-3).1,2 There is also much similarity in amino acid sequence among these receptors (Figure 2-3).1 The GnRH receptor group only contains the GnRH receptor.2 The TRH and secretagogue receptor group includes the TRH receptor and the growth hormone secretagogue receptor.2 Class B GPCRs are structurally similar to members of the hormone protein receptor group (Figure 2-3).1 However, unlike the glycoprotein hormone receptors class B GPCRs do not share similar amino acid sequences.1 This family contains receptors for high-molecular-weight hormones, including calcitonin, glucagon, gastric inhibitory peptide, parathyroid hormone (PTH), and corticotrophinreleasing factor (CRF).1,2,56 Class C receptors have a very large extracellular domain with two lobes separated by a hinge region that closes on the ligand (Figure 2-3).57 This region has also been called the “Venus’s-flytrap” domain or module due to the trapping mechanism of the hinge region.58 This family includes the calcium-sensing receptor (CASR).1,2

Class A Receptors That Transduce Hormone Action THE PEPTIDE RECEPTOR GROUP Adrenocorticotropin and Melanocortin-2 Receptors It is important to note that a newly accepted name for the ACTH receptor is melanocortin-2 receptor (MC2R) because the ACTH receptor is one of five members of the melanocortin receptor family of GPCRs.54 For the purpose of clarity, when discussing interactions between ACTH and its receptor the older name will be used for the remainder of this chapter. The ACTH receptor gene is located on the short arm of chromosome 18.59 The ACTH receptor has a small extracellular and intracytoplasmic domain. Adrenocorticotropin-induced activation of ACTH receptor in the zona fasciculata and zona reticularis of the adrenal cortex stimulates Gs, resulting in increased intracellular cAMP levels that stimulate steroidogenesis by activating cAMP-dependent kinases.60-62 Hereditary isolated glucocorticoid deficiency, resistance to ACTH, and familial glucocorticoid deficiency (FGD) are

Ch02_026-073-X4090.indd 32

the same names for an autosomal recessive syndrome that consists of glucocorticoid deficiency accompanied by normal mineralocorticoid secretion. FGD has been classified further as FGD types 1 and 2 and the triple A syndrome.63 Patients with FGD type 1 are homozygous or compound heterozygous for point mutations, resulting in ACTH receptors with abnormal function.63-69 In contrast, patients with FGD type 2 have ACTH resistance that is not caused by ACTH receptor mutations.62,63,70 Triple A (Allgrove syndrome) is an autosomal recessive syndrome characterized by ACTH-resistant adrenal insufficiency, achalasia, and alacrima—which is also not caused by ACTH receptor gene mutations.63,67,71 Patients with FGD type 1 usually present during infancy or early childhood with hypoglycemia.66,72-76 Less commonly, patients may present with a severe infection, frequent minor infections, or childhood asthma that resolves with treatment with physiologic doses of glucocorticoids.63,66,72 Hyperpigmentation may be seen as early as the first month of life, but usually becomes apparent after the fourth month of life.64-66,72-74,77 Neonates may also suffer from jaundice.66,74,76,78 Tall stature accompanied by an advanced bone age, in spite of normal age of onset of puberty, appears to be common in children with FGD type 1.63,66,69,72,73,77 Patients with FGD type 2 have normal heights.63,79 At presentation, plasma cortisol, androstenedione, and dihydroepiandrosterone levels are low or low normal— and plasma ACTH levels are elevated.63,66,72-76 When supine, patients with FGD type 1 have renin and aldosterone levels that are near normal.63,73,75,76 Histologically, the zona fasciculata and zona reticularis are atrophied with FGD.72 However, demonstrating the lack of an essential role for ACTH in the embryologic development and maintenance of the zona glomerulosa, adrenal cortices in patients with all types of FGD contains zona glomerulosa cells.63,71-73,77,80 Abnormalities in ACTH receptor expression may be seen in other conditions. Evidence suggests that the ACTH receptor-Gs-adenylyl cyclase-cAMP cascade maintains differentiation of adrenocortical cells, and that impairment of this cascade leads to dedifferentiation and increased proliferation of adrenocortical cells.81,82 Adrenocortical carcinomas from some patients have been found to have a loss of heterozygosity (LOH) for the ACTH receptor gene, resulting markedly in decreased ACTH receptor mRNA expression.82 Growth of the tumors with LOH for the ACTH receptor gene also may be more aggressive than the other tumors. An activating mutation of Gi2 that constitutively suppresses adenylyl cyclase activity has also been found in adrenocortical tumors.81 Thus, decreased ACTH receptor activity may be associated with tumorigenesis. Interestingly, many patients with ACTH-independent macronodular adrenal hyperplasia (AIMAH)—a cause of ACTH-independent Cushing’s syndrome—exhibit increased glucocorticoid levels in response to noncorticotropin hormones that do not normally induce glucocorticoid release.83-88 These hormones include gastric inhibitory peptide, exogenous arginine and lysine vasopressin, lutenizing hormone, human chorionic gonadotropin, angiotensin II, catecholamines, leptin, and

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serotonin receptor agonists.83-88 Increased expression of the receptors for these ligands in the abnormal adrenal glands has been implicated as a possible explanation for the abnormal induction of glucocorticoid release by these non-corticotropin ligands.88 However, receptors for some of these ligands are expressed in normal adrenal glands.88 Thus, the mechanism for this phenomenon remains to be fully elucidated.

Other Melanocortin Receptors Murine studies reveal that melanocortin-3 receptor (MC3R), another of the five members of the melanocortin receptor family, regulates fat deposition.54 The role of the MC3R in humans is less clear. Homozygosity for a pair of single-nucleotide polymorphisms of the MC3R gene that result in production of partially inactive MC3Rs was found to be associated with pediatric-onset obesity in Caucasian American and African American children.89 The melanocortin-4 receptor (MC4R) is another member of the melanocortin receptor family and plays a role in controlling appetite and weight.90 The MC4R has baseline constitutive activity that can be inhibited by the inverse agonist agouti-related peptide (AgRP).90,91 Activation of the MC4R by its natural agonist -melanocyte stimulating hormone (-MSH) produces anorexigenic effects.90,92,93 Naturally occurring inactivating MC4R mutations have been identified in some individuals with hyperphagia, increased lean body mass, obesity, and hyperinsulinism due to insulin resistance and increased linear growth.94-96 Most of these obese individuals were heterozygous for the identified mutations, and an autosomal dominant mode of inheritance was identified in their blood relatives.95,96 However, five obese children from a consanguineous kindred were found to be homozygous for an N62S missense mutation.95 Family members heterozygous for the N62S mutation were noted to be nonobese. These MC4R mutations are thought to be the most common monogenic cause of human obesity. In one study, 5.8% of 500 probands with severe childhood obesity were found to be heterozygous or homozygous for MC4R mutations.96 AgRP gene polymorphisms appear to be associated with anorexia nervosa.97,98 Very little is known about melanocortin-5 receptors (MC5Rs) in animals and humans. There is only weak evidence from a single linkage and association study of families in Quebec suggesting that MC5Rs may also play a role in regulating body weight and fat mass.99 Another member of the melanocortin receptor family, the melanocortin-1 receptor (MC1R), controls skin and hair pigmentation. Activation of MC1Rs in skin and hair follicle melanocytes with the pro-opiomelanocortin (POMC)-derived peptides -melanocyte stimulating hormone (-MSH) and ACTH leads to release of eumelanin, a brown-black pigment, from the melanocytes.100,101 Inhibition of MC1R baseline constitutive activity by agouti protein leads to release of pheomelanin, a red-yellow pigment, from the melanocytes.101 Inactivating homozygous mutations of the POMC gene cause hypoadrenalism, red hair, fair skin, and early-onset

Ch02_026-073-X4090.indd 33

33

obesity due to lack of ACTH production from the POMC precursor, lack of ACTH and -MSH-induced melanocyte release of eumelanin resulting from activation of MC1Rs, and lack of -MSH-induced anorectic effects resulting from activation of MC4Rs, respectively.102 Heterozygosity for Arg236Gly and Tyr221Cys POMC gene mutations are associated with hyperphagia, early-onset obesity, and increased linear growth.102,103 In one study, 5 out of 538 unrelated probands with severe early-onset obesity were found to be heterozygous for the Tyr221Cys POMC gene mutation.103

Vasopressin Receptors Nephrogenic diabetes insipidus (NDI) occurs when the renal response to arginine vasopressin (AVP) is impaired. NDI is characterized by polydipsia and polyuria that is not responsive to vasopressin and vasopressin analogs.104 X-linked NDI is caused by inactivating mutations of the V2 vasopressin receptor (AVPR2) gene located at Xq28.105108 An autosomal recessive variant of NDI is also caused by loss-of-function mutations in the gene for the aquaporin-2 AVP-sensitive water channel.109,110 More than 100 inactivating AVPR2 gene mutations have been identified that cause X-linked NDI.104 Some of these mutations are complete gene deletions or mutations that cause abnormal mRNA splicing. Other mutations result in receptors with abnormal trafficking to the plasma membrane, ligand binding, or Gs activation.104,111 Gain-of-function mutations in the V2 vasopressin receptor have also been reported.112 DNA sequencing of two patient’s V2R gene identified missense mutations in both, with resultant changes in codon 137 from arginine to cysteine or leucine. These mutations resulted in constitutive activation of the receptor and a syndrome of inappropriate antidiuretic hormone secretion (SIADH)like clinical picture, which was termed nephrogenic syndrome of inappropriate antidiuresis.112

THE HORMONE PROTEIN RECEPTOR GROUP The glycoprotein hormones include TSH, FSH, LH, and HCG. These hormones are composed of similar  subunits that dimerize with hormone-specific  subunits. TSH, FSH, and LH bind to the extracellular N-terminal domain of the TSH, FSH, and LH receptors, respectively.1,2,113,114 The effects of hCG are mediated by the LH receptor.115 Glycoprotein hormone receptors have a large (350–400 residues) extracellular N-terminal domain, also known as the ectodomain, that participates in ligand binding (Figure 2-3).3,115 The ectodomain includes leucine-rich repeats that are highly conserved among the glycoprotein hormone receptors.3,115 There is 39% to 46% similarity of the ectodomain and 68% to 72% similarity of the transmembrane or serpentine domain among the three glycoprotein hormone receptors.3 Activated glycoprotein hormone receptors increase Gs coupling to adenylyl cyclase, leading to increased intracellular cAMP levels and protein kinase A (PKA) activation.115 Mutations leading to endocrine dysfunction have been reported for each of the glycoprotein hormone receptors.

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LH Receptors Both inactivating and activating mutations of the LH receptor have been found in humans.115 The LH receptor gene is located in chromosome 2 p21 and consists of 11 exons.116,117 Exon 1 encodes a peptide that directs the LH receptor to the plasma membrane.115 Exons 2 through 10 encode the ectodomain.115 The last exon encodes the transmembrane domains that are also known as the serpentine regions.3,115,116 Single nonsense mutations, amino acid changes, and partial gene deletions have been found that lead to expression of LH receptors with decreased activity.115 Single–amino-acid changes have also been found that lead to activation of Gs in the absence of ligand binding.115 Inactivating mutations of the LH receptor gene require homozygosity or compound heterozygosity to alter endocrine function because the presence of one normal receptor allele in the heterozygous state can compensate for the decreased function of a mutated allele.115 In contrast, activating mutations of the LH receptor gene cause endocrine disorders in the heterozygous state.115 In the fetus, LH receptors are primarily activated by hCG.115 Leydig cells begin to express LH receptors shortly after testicular differentiation at 8 weeks of gestation.115 Thereafter, androgen production due to activation of these receptors by hCG plays an important role in the development of male genitalia and testicular descent.115 Thus, male infants with inactivating mutations of the LH receptor may present with abnormally developed genitalia—including micropenis, cryptorchidism, and intersex..115 Males with mutations that completely inactivate the LH receptor may present with male pseudohermaphroditism accompanied by failure of fetal testicular Leydig cell differentiation. This phenotype, which is known as type I Leydig cell hypoplasia, includes female external genitalia with a blind-ending vagina, absence of Müllerian derivatives, and inguinal testes with absent or immature Leydig cells.118-125 In addition, patients have elevated serum LH levels, normal serum FSH levels, and decreased serum testosterone levels that do not increase in response to HCG administration.118-125 Mutations that lead to this phenotype include a nonsense mutation (Arg545Stop) that results in a receptor that is missing TM4-7, an Ala593Pro change, and a TM7 deletion (Leu608, Val609) that decreases cell surface expression of the LH receptor.123,124,126 These mutant receptors are unable to couple to Gs.123,124,126 Males with mutations that do not completely inactivate the LH receptor present with type II Leydig cell hypoplasia, which is characterized by a small phallus and decreased virilization.122 A mutation that leads to this phenotype includes the insertion of a charged lysine at position 625 of TM7 in place of hydrophobic isoleucine that disrupts signal transduction.127 Another mutation (Ser616Tyr, found in patients with mild Leydig cell hypoplasia) is associated with decreased cell surface expression of the LH receptor.124,127 Other deletion and nonsense mutations have also been found to cause mild Leydig cell hypoplasia.115 Males with inactivating mutations of the LH receptor may also present with a phenotype intermediate in severity between type I and type II Leydig cell hypoplasia. A

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compound heterozygote patient with Ser616Tyr on one allele and an inactivating deletion (exon 8) on the other allele presented with Leydig cell hypoplasia, hypoplastic phallus, and hypospadias.128 The Cys131Arg mutation has also been found in patients with Leydig cell hypoplasia, small phallus, and hypospadias.129 This mutation is located in the leucine-rich repeat segment of the LH receptor extracellular domain and interferes with high-affinity ligand binding.129 Deletion of exon 10 of the LH receptor gene results in an LH receptor that binds LH and HCG normally.130 However, although there is normal intracellular signaling in response to binding of the mutant receptor by HCG there is impaired intracellular signaling in response to binding of the mutant receptor by LH.130 Because HCG is the main in utero LH receptor-activating hormone, and second-messenger response of the mutant receptors to HCG is not impaired, it is not surprising that a male patient found to be homozygous for the mutation was born with normal male genitalia.54,130 Pubertal progression and later gonadal function, however, are dependent on LH activation of the LH receptor.54,130 Because deletion of exon 10 of the LH receptor gene results in a mutant LH receptor with diminished intracellular signaling in response to LH, it is also not surprising that the patient homozygous for this mutation was found to have delayed pubertal development, small testes, and hypergonadotropic hypogonadism when evaluated at the age of 18 years.130 Prolonged HCG therapy resulted in normalization of testicular testosterone production, increased testicular size, and the appearance of spermatozoa in semen.130 Males with mutations that constitutively activate LH receptors present with male-limited precocious puberty (MLPP), also known as testotoxicosis—which may be familial or sporadic.125,131,132 Boys with this condition present with GnRH-independent precocious puberty before the age of 4 years when the Asp578Gly is present, and as early as the first year of life when the Asp578Tyr mutation is present.115,133-135 Patients with this condition may have an enlarged phallus at birth.133 During the first five years of life, patients with MLPP have very low LH and FSH levels and testosterone levels in pubertal ranges.136 During adolescence and adult life, testosterone levels do not increase above age-appropriate concentrations and gonadotropin levels normalize.115,136138 Thus, adolescents and adults with MLPP do not usually manifest signs of androgen excess (such as hirsutism or severe acne).115,136 Most mutations that cause MLPP are located in TM6 and i3, regions that participate in receptorG protein coupling.115 Somatic activating mutations cause sporadic Leydig cell adenomas.90 In contrast to males, the LH receptor is not known to play an important role in females until puberty. During puberty, activation of LH receptors on ovarian theca cells leads to the production of androgens that are converted to estrogens by aromatase in granulose cells.115 LH, along with FSH, also plays a role in inducing the differentiation of follicles into Graaffian follicles and triggers ovulation and release of the oocyte.115 Females with inactivating mutations of the LH receptor may be asymptomatic or present with primary

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amenorrhea.115 Females with complete inactivating LH receptor mutations may present with primary amenorrhea, inability to ovulate, and decreased estrogen and progesterone levels accompanied by elevated LH and FSH levels.124,139 Affected individuals may have signs of low estrogen levels, including a hypoplastic uterus, a thin-walled vagina, decreased vaginal secretions, and decreased bone mass.124,139

FSH Receptors Inactivating and activating FSH receptor mutations have also been described.140 However, FSH receptor mutations are considerably less common than LH receptor mutations.140 The FSH receptor gene is located in chromosome 2 p21 and contains 10 exons.141 The last exon of the FSH receptor gene encodes the transmembrane and intracellular domains.142 FSH is required in females for normal follicle maturation and the regulation of estrogen production by ovarian granulosa cells.140,143,144 FSH is required in pubertal males for Sertoli cell proliferation, testicular growth, and the maintenance of spermatogenesis.140,145 The first inactivating mutation of the FSH receptor was found in Finnish females with autosomal recessive inherited hypergonadotropic ovarian dysgenesis (ODG). ODG is characterized by primary amenorrhea, infertility, and streak or hypoplastic ovaries in the presence of a 46XX karyotype and elevated gonadotropin levels.146 Twenty-two out of 75 Finnish patients with ODG were found to be homozygous for a C566T point mutation in exon 7 of the FSH receptor gene.147 This mutation leads to the production of an FSH receptor with an Ala189Val substitution in an area of the extracellular ligandbinding domain that is thought to play a role in turnover of the receptor or in directing the receptor to the plasma membrane.147 The mutated receptor demonstrates normal ligand-binding affinity but has decreased binding capacity and impaired signal transduction in transfected MSC-1 cells.147 Males homozygous for this mutation have variable impairment of spermatogenesis and low to low-normal testicular volume but are not azoospermic and can be fertile.148 The C556T point mutation is uncommon outside Finland, where the carrier frequency is 0.96%.149 Compound heterozygosity for mutations that cause partial loss of FSH receptor function may cause endocrine dysfunction in women.150,151 Women may present with infertility, secondary amenorrhea, osteoporosis, and a history of delayed onset of puberty accompanied by elevated LH and FSH, low-normal plasma estradiol, low plasma inhibin B levels, slightly enlarged ovaries with immature follicles, and a small uterus.150 This may be caused by FSH receptor gene mutations that result in an Ile160Thr mutation in the extracellular domain that impairs cell surface expression, and an Arg573Cys mutation in e3 that interferes with signal transduction.150 Other women present with primary amenorrhea, and very elevated gonadotropin, low plasma estradiol and inhibin B levels, normal-size ovaries with immature follicles, and a normal-size uterus.151 This condition is associated with an Asp224Val substitution in the extracellular domain leading to impaired cells surface

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35

expression, and a Leu601Val substitution in e3 impairing signal transduction.151 An activating mutation of the FSH receptor has also been described. Surprisingly, a hypophysectomized male was found to be fertile and to have serum testosterone levels above 4.9 nmol/l and normal testis volume in spite of undetectable gonadotropin levels.152 This patient was found to be heterozygote for an A1700G mutation in exon 10 of the FSH receptor gene that resulted in an Asp567Gly substitution in an area of the third intracytoplasmatic loop that is highly conserved among FSH, LH, and TSH receptors.152-154 The same substitution in corresponding areas of the LH and TSH receptors results in constitutively active receptors, and is found in MLPP and thyroid adenomas, respectively.152-154

TSH Receptors The TSH receptor gene is located on chromosome 14 and contains 10 exons, with the first nine exons encoding the large extracellular domain and the tenth exon coding the remainder of the receptor.155-158 At low extracellular TSH concentrations, TSH receptor activation leads to stimulation of Gs—which activates adenylyl cyclase, resulting in increased intracellular cAMP levels.159,160 At higher extracellular TSH concentrations, activation of the TSH receptor also stimulates the Gq and G11 proteins—activating phospholipase C and resulting in the production of diacylglycerol and inositol phosphate.160 TSH receptors differ from the other glycoprotein hormone receptors in that they exist in two equally active forms.161,162 These are the single-chain and two-subunit forms of the TSH receptor (Figure 2-4). The single-chain form of the TSH receptor is made up of three contiguous subunits: the A subunit, C peptide, and B subunit.162-164 The A subunit begins at the N-terminal of the extracellular domain and contains most of the extracellular domain.162-164 The C peptide is connected to the C-terminal of the A subunit and continues the extracellular domain.162-164 The C peptide contains a 50-amino-acid sequence that is only found in TSH receptors.162-164 The B subunit is connected to the C-terminal of the C peptide and contains the TMs and the C-terminal cytoplasmic portion of the receptor.162-164 The two-subunit form of the receptor is missing the C-peptide, which is cleaved from the protein during intracellular processing and consists of the A and B subunits attached by disulfide bonds.165-168 It is surprising that both receptor forms are activated equally by TSH because the C-peptide and nearby regions of the A and B subunits participate in signal transduction.161-163,169,170 Spontaneous single-allele mutations of the TSH receptor gene leading to replacement of Ser-281 (near the C-terminal of the A subunit, with Ile, Thr, or Asn) result in a constitutively active TSH receptor that may cause intrauterine or congenital hyperthyroidism, or toxic adenomas.164,171-173 Activating somatic mutations that cause toxic adenomas have also been found in different transmembrane domains of the TSH receptor.174-181 More specifically, clusters of mutations are located in the i3 and TM6 regions—found to be involved with signal transduction in all glycoprotein hormone receptors.174-176,178-180

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Figure 2-4. The TSH receptor. There are two forms of TSH receptors. The single-chain form is made up of an A subunit, C peptide, and B subunit. Post-translational cleavage of the C peptide from the single chain form results in the two-subunit form. This form consists of the A subunit joined to the B subunit by disulfide bonds between the C-terminal cysteine residues of the A subunit and the N-terminal cysteine residues of the B subunit. [Reproduced with permission from Rapoport B, Chazenbalk GD, Jaume JC, McLachlan SM (1998). The thyrotropin (TSH) receptor: Interaction with TSH and autoantibodies. Endocr Rev 19:676. Copyright 1998, The Endocrine Society.]

The prevalence of activating mutations of the TSH receptor in toxic adenomas has been estimated to range from 2.5% in Japan to 86% in Brazil.177,178,180,182-185 Activating somatic mutations of the TSH receptor have also been found in multinodular goiters.186 Interestingly, different activating mutations have been found in separate nodules in the same individual.186 Some welldifferentiated thyroid carcinomas have activating mutations of the TSH receptor.187-189 Activating mutations of the Gs gene have also been found in toxic adenomas and differentiated thyroid carcinomas.81,190,191 Activating germ line mutations of the TSH receptor can cause sporadic or autosomal dominant inherited nonautoimmune hyperthyroidism that presents in utero, during infancy, or during childhood.173,192-200 These mutations have been found in the N-terminal extracellular and transmembrane domains.173,192-200 Patients with one allele producing constitutively active TSH receptors and one normal allele may present with hyperthyroidism.162 In contrast, homozygosity or compound heterozygosity for mutations resulting in TSH receptors with reduced function is required to cause clinically apparent thyroid disease.162 Most known loss-of-function TSH receptor mutations are located in the N-terminal extracellular domain.201 A spontaneous Asp410Asn substitution, near the carboxy-terminus of the C peptide, results in a TSH receptor with normal ligand binding affinity and impaired Gs-mediated signal transduction.202 Patients homozygous for this type of mutation present with compensated hypothyroidism.202 Patients homozygous or compound heterozygous for loss-of-function mutations of the TSH receptor present with the syndrome of resistance to TSH (RTSH). Loss-offunction mutations of the TSH receptor that cause RTSH have been identified in the N-terminal extracellular

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domain, TM4, i2, e1, and e3.203 Clinical severity of RTSH may range from a euthyroid state accompanied by elevated TSH levels (fully compensated RTSH), to mild hypothyroidism unaccompanied by a goiter (partially compensated hypothyroidism), to congenital thyroid hypoplasia accompanied by profound hypothyroidism (uncompensated RTSH).62,202,204-208 In patients with uncompensated RTSH, a small bilobar thyroid gland is located at the normal site.62 Because the sodium-iodide symporter is TSH dependent,131 iodine and (99m) Pertechnetate uptakes are diminished or absent in patients with RTSH.62,209 Some families have been found to have an autosomaldominant form of RTSH that is not caused by a mutation of the TSH receptor.210,211

HCG and TSH Receptors During Pregnancy Due to its structural similarity with TSH, HCG can activate the TSH receptor.212 During pregnancy, HCG activation of TSH receptors leads to elevation in thyroid hormones seen after the ninth week of gestation—and decreases in TSH levels between the ninth and twelfth weeks of gestation.213 This phenomenon does not usually result in maternal hyperthyroidism (gestational thyrotoxicosis).213,214 However, when HCG levels are abnormally elevated due to a molar pregnancy or choriocarcinoma hyperthyroidism may occur.215-219 A mother and daughter were identified with recurrent gestational hyperthyroidism and normal serum HCG levels.220 These individuals were found to be heterozygous for a point mutation in the TSH receptor gene, resulting in a Lys183Arg substitution in the extracellular domain of the receptor. It is believed that this substitution increases activation of the receptor by HCG, causing gestational hyperthyroidism.

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HCG and FSH Receptors During Pregnancy

37

nonresponders to become fertile in response to pulsatile exogenous GnRH.233,237

Promiscuous activation of wild-type FSH receptors by excessively elevated HCG and TSH levels during pregnancy, and promiscuous activation of mutated FSH receptors by normal levels of HCG and TSH during pregnancy, have been found to cause spontaneous ovarian hyperstimulation syndrome.221-224 Ovarian hyperstimulation syndrome may occur spontaneously or iatrogenically as a result of ovarian stimulation treatment. The ovaries enlarge as multiple ovarian cysts form in association with the extravascular fluid shifts thought to result from increased mesothelial capillary permeability. The extravascular fluid shifts may cause life-threatening pleural and/or pericardial effusions, and/or ascites.225 Surprisingly, the heterozygous promiscuity-inducing FSH receptor gene mutations alter the serpentine domain rather than the ectodomain.221-224 This finding reveals that transmembrane domain plays a role in conferring FSH receptor specificity. Interestingly, these mutations also cause the FSH receptor to be constitutively active.3

Like the GnRH receptor, TRH receptor activation leads to increased phospholipase C activity.238 To date, only inactivating mutations that cause endocrine dysfunction have been reported for the TRH receptor. One patient has been identified with central hypothyroidism due to mutated TRH receptors.239 He presented during the ninth year of life with short stature (–2.6 SD) accompanied by a delayed bone age (–4.1 SD), low plasma thyroxine level, and normal plasma TSH level. Exogenous TRH did not induce an increase in plasma TSH and prolactin levels. He was found to be compound heterozygous for TRH receptor gene mutations, resulting in receptors with decreased binding.

THE GONADOTROPIN-RELEASING HORMONE RECEPTOR GROUP

OTHER CLASS A RECEPTORS THAT TRANSDUCE HORMONE ACTION

Gonadotropin-Releasing Hormone Receptors

Free Fatty Acid Receptor 1

The GnRH receptor gene is located on 4q13 and includes three exons.226,227 Unlike glycoprotein hormone receptors, GnRH receptors lack an intracellular C-terminal domain.228,229 The GnRH receptor also differs from most endocrine GPCRs in that its actions are mediated by Gq/ G11 stimulation of phospholipase C activity.230 Phospholipase C cleaves phosphatidylinositol-4,5-diphosphate (PIP2) to inositol 1,4,5-triphosphate (IP3) and diacylglcerol, leading to increased protein kinase C (PKC) activity.231,232 Some patients with IHH (idiopathic) have been found to be homozygous or compound heterozygous for lossof-function mutations in the gene for the GnRH receptor.233-235 Unlike patients with Kallman’s syndrome, they have a normal sense of smell.233-235 GnRH receptor mutations that cause IHH result in decreased binding of GnRH and/or impaired GnRH receptor signal transduction, or decreased cell GnRH receptor cell membrane expression due to misrouting of GnRH receptor oligomers from the endoplasmic reticulum.32,33,233-235 Female patients with mutations that partially compromise GnRH receptor function may present with primary amenorrhea and infertility associated with a normal or small uterus and small ovaries with immature follicles.233,236 Males with the same mutations may present with incomplete hypogonadotropic hypogonadism (characterized by a delayed and incomplete puberty) or with complete hypogonadotropic hypogonadism, characterized by absent puberty.233,236 Some patients with IHH due to mutated GnRH receptors have partial or normal gonadotropin responses to exogenous GnRH.233,234 However, decreased amplitude in the pulsatile LH secretion can be observed in these patients.233 Females with a partial or normal gonadotropin response to exogenous GnRH are more likely than

At the time of discovery of a new GPCR, the ligand for the newly discovered receptor is often unknown. Thus, until a specific ligand is discovered these GPCRs are known as orphan receptors. According to the Human Genome Organization (HUGO) Gene Nomenclature Committee, these G-protein–coupled orphan receptors should be named alphanumerically GPR followed by a number until their ligand is known. Once a specific ligand is identified, a more specific name is given the receptor. The ligands for GPR40 were unknown when the receptor was first discovered. The HUGO Gene Nomenclature Committee changed the name of the receptor to free fatty acid receptor 1 (FFAR1) when the ligands were identified as medium- and long-chain fatty acids. With rare exceptions that are clearly identified, HUGO Gene Nomenclature Committee recommendations are followed in this chapter (see http://www.gene.ucl.ac.uk/nomenclature/index.html for more information on receptor nomenclature). FFAR1 is one of several GRCRs for lipid mediators. Lipid mediators are intercellular lipid messengers that include sphingosine 1-phosphate, sphingosylphosphorylcholine, dioleoyphosphatidic acid, lysophosphatidic acid, eicosatetraenoic acid, bile acids, and free fatty acids.55 FFAR1 is activated by medium- and long-chain fatty acids, whereas FFAR2 (formerly known as GPR43) and FFAR3 (formerly known as GPR 41) are activated by shorter-chain fatty acids.55 There is now evidence that FFAR1 activation by medium- and long-chain fatty acids has endocrine implication. FFAR1 is expressed in human pancreatic -islet cells.240 Fatty-acid–induced stimulation of FFAR1 in -islet cells leads to activation of the Gq-phospholipase C secondmessenger pathway, which in turn leads to release of

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THE THYROTROPIN-RELEASING HORMONE AND SECRETAGOGUE RECEPTOR GROUP Thyrotropin-Releasing Hormone Receptors

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calcium from the endoplasmic reticulum that augments insulin-mediated increases in intracellular calcium concentrations due to glucose-induced activation of voltage-gated calcium channels.241-244 Because an increased intracellular calcium concentration induces insulin release, FFAR1mediated augmentation of glucose-mediated increases in the intracellular calcium concentration leads to amplification of glucose-stimulated insulin release.241-244 Wild-type mice placed on an 8-week high-fat diet develop glucose intolerance, insulin resistance, hypertriglyceridemia, and hepatic steatosis—whereas FFAR1 knockout mice on the same diet do not develop these conditions.245 The clinical relevance for humans is yet unclear. However, an Arg211His polymorphism in the FFAR1 gene may explain some of the variation in insulin secretory capacity found in Japanese men. Arg/Arg homozygotes had lower serum insulin levels, homeostasis model of insulin resistance, and homeostasis model of beta-cell function than His/His homozygotes.246

orexin 2 receptors.256 There are also two types of orexins, orexin A and orexin B, formed from the precursor peptide preproorexin.256 Orexins are also known as hypocretins, and orexin A is synonymous with hypocretin-1 and orexin B with hypocretin-2.255,256 Orexin A acts on orexin-1 and orexin-2 receptors, whereas orexin B only acts on orexin-2 receptors.254,257,258 Like most class A GPCRs, orexin receptors couple with Gq/11 and Gi/Go to activate phospholipase C and inactivate adenylyl cyclase, respectively.256,259-261 Surprisingly, however, recent evidence suggests that orexin receptors also couple with Gs—which increases adenylyl cyclase activity.261 Orexins increase food intake and duration of wakefulness.254,255,262 Orexin A and activation of the orexin-1 receptor have greater orexigenic effects than orexin B and activation of the orexin-2 receptor.263 The orexin-2 receptor mediates the arousal effect of orexins.263 Most patients with idiopathic narcolepsy have diminished levels of orexins in cerebral spinal fluid and lack orexin-containing neurons.255,264-267

GPR54 Separate groups of investigators from France and the United States simultaneously found homozygous inactivating GPR54 mutations that cause IHH in consanguineous French and Saudi Arabian kindreds, respectively.247-249 The United States group also found an African American patient with IHH due to compound heterozygous inactivating GPR54 mutations.248 Since then, a patient with a Jamaican father and a Turkish-Cypriot mother, and with cryptorchidism and micropenis at birth and undetectable LH and FSH levels at 2 months of age, was found to have compound heterozygous GPR54 mutations.250 Unlike patients with Kallmann syndrome, these patients’ sense of smell is intact. Furthermore, in contrast to patients with isolated hypogonadotropic hypogonadism due to GnRH mutations patients with IHH due to GPR54 mutations also exhibit increases in gonadotropin levels in response to exogenous GnRH. Thus, inactivating homozygous and compound heterozygous GPR54 mutations are a rare cause of normosmic IHH.249,250 Ligands for GPR54 derive from a single precursor protein, kisspeptin-1.251,252 The longest derivative protein that acts as a ligand for GPR54 is metastin, which is named metastin because metastin is a metastasis suppressor gene in melanoma cells.251 Metastin consists of kisspeptin-1 69-121.251,252 However, shorter C-terminal peptides derived from kisspeptin-1 bind and activate GPR54.251 Administration of metastin to adult male volunteers increases LH, FSH, and testosterone levels.253 Recent animal research suggests that metasin and GPR54 play a role in the timing of onset of puberty, in modulating sex steroid feedback on GnRH release from the hypothalamus, and in the timing of onset of puberty.249

Orexin Receptors Orexins act on orexin receptors, located predominantly in the hypothalamus, to control food intake and play a role in the regulation of sleep/wakefulness.254-256 There are two types of orexin receptors, the orexin-1 and the

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Ghrelin Receptors Another class A receptor that transduces hormone action is the ghrelin receptor. The receptor is also known as the growth hormone secretagogue receptor type 1a because the receptor is also activated by a family of synthetic growth hormone secretagogues.90,263 Ghrelin is a product of post-translational modification of the ghrelin gene product proghrelin.268 Ghrelin is mainly produced in the stomach.269,270 Ghrelin activation of ghrelin receptors located in the hypothalamus and pituitary somatotrophs results in growth hormone secretion.271 The ghrelin receptor also has an orexigenic role. Plasma ghrelin levels are elevated just prior to eating, and decrease rapidly after eating.272,273 In addition, intravenous administration of ghrelin to humans increases appetite and food intake.274 Plasma ghrelin levels are elevated in individuals with Prader Willi syndrome.275 Thus, hyperphagia in patients with Prader Willi syndrome may be due at least in part to overactivation of ghrelin receptors by ghrelin. Screening of 184 extremely obese children and adolescents for mutations of the ghrelin receptor gene failed to identify a single mutation likely to cause obesity.276 Short individuals in two unrelated Moroccan kindreds were found to have a C to A transversion at position 611 in the first exon of the ghrelin receptor gene.277 This transversion results in replacement of the apolar and neutral amino acid alanine at position 204 of the receptor by the polar and charged amino acid glutamate. This mutation interferes with normal constitutive activity of the receptor, and decreases cell membrane expression of the receptor. Receptor activation by ghrelin, however, is preserved. Two-thirds of individuals in the kindreds heterozygous for the mutation had height greater than or equal to 2 standard deviations below the mean. One heterozygous individual’s height was 3.7 standard deviations below the mean. Prior to onset of growth hormone therapy, the only individual in the kindreds homozygous for the mutation had a height 3.7 standard deviations below the mean. Whereas the patient homozygous for

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the mutation became overweight during puberty, the weight of the patients heterozygous for the mutation varied from underweight to overweight. Another product of post-translational modification of proghrelin, obestatin, appears to play a role in controlling appetite and weight.268 Activation of the obestatin receptor, previously known as GPR39, in rats results in decreased food intake and weight.

Melanin-Concentrating Hormone Receptors Formerly known as SLC-1 or GPR24, the type 1 melaninconcentrating hormone (MCH) receptor (MCHR1)—and the more recently discovered type 2 MCH receptor (MCHR2), formerly known as SLT or GPR145—may play a role in regulating feeding and energy metabolism in humans.278-281 When activated by MCH, MCHR1 couples with Gq/11 and Gi/o to increase phospholipase C activity and inhibit adenylyl cyclase activity, respectively.278,279,282 When activated by MCH, MCHR2 couples with Gq/11 to increase phospholipase C activity.280,281 Studies in rodents reveal that MCH is an orexigenic hormone, and treatment of rodents with MCHR1 antagonists decreases food intake, weight, and body fat.278,283,284 Little is known about the role of MCHR2 in animals, and both receptors in humans. Analysis of the MCHR1 gene in more than 4,000 obese German, Danish, French, and American children and adolescents revealed several single-nucleotide polymorphisms and gene variations in the German children and adolescents that may be associated with obesity.285 Another study of 106 American subjects with earlyonset obesity failed to definitively identify MCHR1 and MCHR2 mutations as a cause of obesity.286

Class B Receptors That Transduce Hormone Action GROWTH-HORMONE–RELEASING HORMONE RECEPTOR The growth-hormone–releasing (GHRH) receptor gene is located at 7p14.287 GHRH receptors interact with Gs to stimulate adenylyl cyclase, resulting in increased intracellular cAMP levels that lead to somatotroph proliferation and growth hormone secretion.288 Thus, it is not surprising that activating mutations in Gs leading to constitutive activation of adenylyl cyclase have been found in some growth-hormone–secreting pituitary adenomas in humans.289 Some patients with isolated growth hormone deficiency have been found to be homozygous or compound heterozygous for inactivating mutations in the GHRH receptor gene.288 The same mutation was found in three apparently unrelated consanguineous kindreds from India, Pakistan, and Sri Lanka.287,290,291 A different mutation has been identified in a Brazilian kindred.288 Both mutations result in the production of markedly truncated proteins with no receptor activity.288,291 In another family, two sibling with isolated growth hormone deficiency were found to be compound heterozygous for inactivating GHRH receptor gene mutations.292

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Individuals in these kindreds homozygous or compound heterozygous for inactivating GHRH receptor gene mutations experienced severe postnatal growth failure that resulted in proportionate short stature.287,288,291,292 Males have high-pitched voices and moderately delayed puberty.287,288,291 Unlike infants with complete growth hormone deficiency, they do not have frontal bossing, microphallus, or hypoglycemia.287,291,292 Their bone age was delayed with respect to chronologic age but advanced with respect to height age.291 Some patients were found to have pituitary hypoplasia.287,291 Growth velocity increased with exogenous growth hormone therapy.287,288,291,292

GASTRIC INHIBITORY POLYPEPTIDE RECEPTORS The gastric inhibitory peptide receptor (GIPR) gene is located on the long arm of chromosome 19.293 Two functional isoforms exist in humans due to alternate splicing.294 GIPR activation induces Gs activation of adenylyl cyclase.294-296 Gastric inhibitory polypeptide (GIP) is also known as glucose-dependent insulinotropic polypeptide and is released by K cells in the small intestine in response to food. GIP has numerous physiologic actions, including stimulation of glucagon, somatostatin, and insulin release by pancreatic islet cells.297,298 GIP does not normally induce cortisol release from adrenocortical cells.299,300 Circulating cortisol levels in patients with fooddependent or GIP-dependent Cushing’s syndrome rise abnormally in response to food intake.83,84 These patients may have adrenal adenomas or nodular bilateral adrenal hyperplasia that overexpress GIPRs that abnormally stimulate cortisol secretion when activated.299,301 Thus, in these patients postprandial GIP release leads to activation of these abnormally expressed and functioning adrenal GIPRs—resulting in excessive adrenal cortisol secretion.299,300

PARATHYROID HORMONE AND PARATHYROID-HORMONE–RELATED PEPTIDE RECEPTORS Two types of PTH receptors have been identified. The type 1 PTH receptor (PTHR1) is activated by PTH and parathyroid-hormone–related peptide (PTHrP) and mediates PTH effects in bone and kidney.302 In spite of 51% homology to the PTHR1, the type 2 PTH receptor (PTHR2) is only activated by PTH.302-305 The PTHR2 is expressed in the brain and pancreas and its function is largely unknown.302,303 The PTHR1 has a large amino-terminal extracellular domain containing six conserved cysteine residues.302 Ligand binding induces the PTHR1 to interact with Gs and Gq proteins, leading to activation of the adenylyl cyclase/protein kinase A and phospholipase C/protein kinase C second-messenger pathways, respectively.306-311 Interestingly, mutations in i2 interfere with coupling of the PTHR1 to Gq without interfering with coupling to Gs—whereas mutations in i3 disrupt coupling of the receptor to both G proteins.312,313 Loss-of-function PTHR1 gene mutations cause Blomstrand’s chondrodysplasia.302 This lethal disorder is

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characterized by accelerated chondrocyte differentiation, resulting in short-limbed dwarfism, mandibular hypoplasia, lack of breast and nipple development, and severely impacted teeth.302,314 One patient with this rare condition was found to be homozygous for a point mutation that resulted in a Pro132Leu substitution in the N-terminal domain that interferes with ligand binding.315,316 Another patient was found to be homozygous for a frame-shift mutation that results in a truncated receptor lacking TM5-7, and contiguous intracellular and extracellular domains.317 A third patient was found to have a maternally inherited mutation that altered splicing of maternal mRNA, resulting in a PTHR1 with a deletion of residues 373 through 383 in TM5 (which also interferes with ligand binding).318 In spite of heterozygosity for the mutation, the patient was unable to produce normal PTHR1s because for unknown reasons the paternal allele, was not expressed.318 Heterozygosity for an Arg150Cys PTHR1 gene mutation was found in two out of six patients with enchondromatosis.319 Enchondromatosis is an autosomal-dominant condition characterized by benign cartilage tumors and increased risk for the development of osteosarcomas.14 Some cases of Jansen’s metaphyseal chondrodysplasia have been found to be caused by constitutively activating mutations of the PTHR1 gene.320-322 This autosomaldominant disorder is characterized by short-limbed dwarfism due to impaired terminal chondrocyte differentiation and delayed mineralization accompanied by hypercalcemia.320,321 Interestingly, constitutive activation appears to result predominantly in excessive Gs activity because adenylyl cyclase activity is increased and PLC activity is unchanged in COS-7 cells expressing mutated receptors.320-322

OTHER CLASS B RECEPTORS THAT TRANSDUCE HORMONE ACTION Other class B receptors that transduce hormone action include glucagon-like peptide-1, glucagon, calcitonin, and corticotrophin-releasing factor receptors.323 Class B receptors usually couple with heterotrimeric Gs proteins, leading to activation of adenylyl cyclase—which in turn leads to elevated intracellular cAMP levels.323,324 (See Chapter 10 for discussion of the role of GLP1 in promoting insulin secretion and the use of GLP1 analogues or inhibitors of GLP1 breakdown in therapy.)

Class C Receptors That Transduce Hormone Action CALCIUM-SENSING RECEPTORS The calcium-sensing receptor (CaSR) is located on the long arm of chromosome 3 (3q21.1).325 The CaSR has a large amino-terminal domain that contains nine potential glycosylation sites.326 Binding of ionized calcium to the CaSR leads to activation of phospholipase C, presumably via activation of a Gq protein.326,327 The CaSR is an integral component of a feedback system that utilizes parathyroid hormone and renal tubular

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calcium reabsorption to keep the serum concentrations of ionized calcium in a narrow physiologic range.328 Increased extracellular ionized calcium concentrations activate CaSRs in parathyroid chief and renal tubular epithelial cells, leading to decreased PTH release and renal tubular calcium reabsorption.326,329 When ionized calcium concentrations fall, CaSR activation decreases—leading to increased PTH release and renal tubular calcium reabsorption.326,329 Recent evidence suggests that the CaSR also binds another cation, magnesium, and thus may play a role in magnesium homeostasis by altering reabsorption of magnesium in the thick ascending limb of Henle in the kidneys.330,331 It is probable that increased peritubular levels of magnesium activate renal CaSRs, leading to inhibition of reabsorption of magnesium from the thick ascending limb of Henle—which in turn leads to increased renal excretion of magnesium.330,331 Autosomal-dominant familial benign hypocalciuric hypercalcaemia (FBH) and neonatal severe hyperparathyroidism (NSHPT) are caused by loss-of-function mutations of the CaSR gene.332,333 Most of these mutations are located in the N-terminal extracellular domain.334,335 With few exceptions, individuals heterozygous for loss-offunction mutations have FBH—whereas individuals homozygous for such mutations have NSHPT.336,337 Therefore, children of consanguineous FBH parents are at risk for NSHPT.334,338,339 Occasionally, infants with NSHPT are heterozygous for CaSR gene mutations.340 Decreased CaSR function impedes calcium ion suppression of PTH release and renal tubular calcium reabsorption.333 Thus, FBH is characterized by mild hypercalcemia that is accompanied by inappropriately normal or elevated serum PTH levels and by relatively low urinary calcium excretion.336,341,342 Individuals with FBH may also have hypermagnesemia as a result of decreased peritubular inhibition of magnesium reabsorption from the thick ascending loops of the kidneys by the CaSR.331 There are three type of FBH: FBH type 1 (FBH1), FBH type 2 (FBH2), FBH type 3 (FBH3).343 FBH1 is due to heterozygous loss-of-function mutations of the CaSR gene on 3q21.1.343 Two other chromosome loci have been identified in patients with FBH without CaSR gene mutations. FBH2 has been mapped to 19p13.3 and is biochemically and clinically similar to FBH1.343,344 FBH3, which is also known as the Oklahoma variant (FBHOK), has been mapped to 19q13.343,345 Adults with FBH3 have hypophosphatemia, elevated serum PTH levels, and osteomalacia in addition to the clinical and biochemical findings found in individuals with FBH1 and FBH2.343,346 NSHPT is characterized by severe hypercalcemia accompanied by elevated circulating PTH levels, undermineralization of bone, rib cage deformity, and multiple long-bone and rib fractures.336 Activating mutations of the CaSR gene may cause autosomal-dominant hypocalcemic hypercalciuria (ADHH), also known as autosomal dominant hypocalcemia, as increased CaSR function leads to increased calcium ion suppression of PTH release and suppression of renal tubular calcium reabsorption.327,343,347-351 ADHH is characterized by hypocalcemia and hypomagnesemia accompanied by inappropriately normal or increased urinary

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calcium excretion and inappropriately normal or low serum PTH levels.327,349-351 Patients with ADHH may be asymptomatic or may present with tetany, muscle cramps, or seizures during infancy or childhood.349-352 Similar to inactivating mutations, most activating mutations are located in the N-terminal extracellular domain.347,349,350,352 Treatment of patients with ADHH with vitamin D or its metabolites is contraindicated because treatment with these vitamins results in worsening hypercalciuria and nephrolithiasis, in nephrocalcinosis, and in renal impairment.349,350 Bartter syndrome type V, like other types of Bartter syndrome, is characterized by hypokalemic metabolic alkalosis and by hyperaldosteronism due to elevated renin levels.353,354 Patients with Bartter syndrome type V, unlike patients with other types of Bartter syndrome, may also have symptomatic hypocalcemia and are at risk for developing nephrocalcinosis due to hypercalciuria.353,354 Evidence from in vitro functional expression studies suggests that patients with mild or moderate heterozygous gain-of-function mutations of the CaSR develop ADHH, whereas those with severe heterozygous gain-of-function mutations of the CaSR develop Bartter syndrome type V.353-355 Some single–amino-acid polymorphisms of the CaSR gene appear to be predictive of whole-blood ionized and serum total calcium levels, and may increase risk for bone and mineral metabolism disorders in individuals with other genetic and environmental risk factors for these disorders.356-358 Individuals heterozygous or homozygous for a Gln1011Glu CaSR gene polymorphism tend to have higher calcium levels than individuals with the polymorphism.358,359 The 15.4% of 387 healthy young Canadian women with at least one CaSR gene allele with an Ala986Ser polymorphism were found to have higher total calcium levels than the remainder of the women without the polymorphism.357 Another study of 377 unrelated healthy Italian adult males and females found that 24% of study subjects were heterozygous or homozygous for the Ala986Ser polymorphism, and confirmed the finding that individuals without the polymorphism have lower whole-blood ionized calcium levels than individuals with the polymorphism.358 The Ala986Ser polymorphism has also been associated with Paget disease and primary hyperparathyroidism.359-361 Individuals with a less common Arg990Gly polymorphism tend to have lower whole-blood ionized calcium levels than individuals without the polymorphism.358 The Arg990Gly polymorphism has been found to be associated with hypercalciuria and nephrolithiasis.361,362 Auto-antibodies against the CaSR that interfere with binding of calcium to the receptor may cause autoimmune hypocalciuric hypercalcemia.363 These patients have the clinical and biochemical features of patients with FBH.363 Conversely, auto-antibodies that activate the CaSR cause autoimmune-acquired hypoparathyroidism.364 Both conditions may occur in association with other autoimmune conditions (such as autoimmune thyroiditis), with celiac disease in patients with autoimmune hypocalciuric hypercalcemia, and with autoimmune thyroiditis and autoimmune polyglandular syndrome types 1 and 2 in patients with autoimmune-acquired hypoparathyroidism.363-365

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41

Auto-antibodies that activate the CaSR were found in approximately one-third of individuals with acquired hypoparathyroidism.365

G-Protein Gene Disorders Inactivating and activating mutations of the GNAS1 gene that encodes Gs cause endocrine disorders.

INACTIVATING MUTATIONS OF THE GNAS1 GENE Pseudohypoparathyroidism type Ia (PHP-Ia; Albright hereditary osteodystrophy) and pseudopseudohypoparathyroidism (PPHP) are caused by heterozygous inactivating mutations of the GNAS1 gene that encodes Gs.366-370 PHP-Ia is characterized by hypocalcemia, hyperphosphatemia, elevated circulating PTH levels, short stature, obesity, mental retardation, brachycephaly, short fingers, and short fourth and fifth metacarpals.366-369 Some patients may also have a small phallus, reflecting the same defect in LH receptor transduction. FSH and TSH also may be affected. PPHP is characterized by the same somatic features as PHP-Ia in the absence of resistance to PTH.366,367,370 Interestingly, patients who inherit these mutations from their mothers have PHP-Ia and patients who inherit these mutations from their fathers have PPHP.366-370 It is now known that in the renal proximal tubules Gs can only be encoded from the maternally inherited GNAS1 allele, whereas elsewhere in the body Gs can be encoded from either the maternally or paternally inherited GNAS1 allele.371 Thus, patients with maternally inherited inactivating GNAS1 gene mutations are not able to express Gs in the proximal tubules and have PHP-Ia.371 Patients with paternally inherited inactivating GNAS1 gene mutations have wild-type maternally inherited GNAS1 alleles and thus have normal Gs expression in the proximal renal tubules and have PPHP.371 Pseudohypoparathyroidism type Ib (PHP-Ib) is characterized by resistance to parathyroid hormone in the absence of the somatic features of PHP-Ia. It was thought that PHP-Ib is caused by inactivating mutations in the PTHR1 gene.372 However, no deleterious PTHR1 gene mutations have been found in patients with PHP-Ib.373,374 Instead of PTHR1 gene mutations, defective imprinting is now thought to play a role in causing PHP-Ib. In most tissues, Gs is encoded by both GNAS1 alleles.371 However, in the renal proximal tubules Gs is only encoded by the maternal GNAS1 allele.371 The exon 1A region of the GNAS1 gene contains a region that is imprinted in the oocyte by methylation of the DNA.371,375 This region is not imprinted in most PHP-Ib patients.375 Thus, failure to maternally imprint the maternally inherited GNAS1 allele is now thought to play a role in causing most cases of PHP-Ib.368,375 Gs cannot be expressed in the renal proximal tubules of these patients due to lack of a maternal GNAS1 allele. The PHP-Ib syndrome is also limited to renal resistance to PTH rather than (as in Albright’s hereditary osteodystrophy) having other clinical manifestations because Gs expression in nonrenal tissues does

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RECEPTOR TRANSDUCTION OF HORMONE ACTION

not require a maternally imprinted GNAS1 allele.376 One patient with PHP-Ib was found to have paternal uniparental isodisomy for chromosome 20, which contains the GNAS1 gene.377 Thus, the patient was found to have two paternally inherited GNAS1 alleles. Thus, lack of a maternally imprinted GNAS1 allele also appears to have played a role in causing this patient’s PHP-Ib.

ACTIVATING MUTATIONS OF THE GNAS1 GENE When a GPCR is activated by a ligand, GDP is converted to GTP—which causes the heterotrimeric G protein to dissociate into active G-GTP and G
subunits.5-7 GTPase then converts GTP into GDP, which inactivates G and increases affinity of G for G
—leading to reformation of the inactive heterotrimeric G protein.5-7 Arg201 and Gln227 of Gs are critical to GTPase activity.376 GNAS1 mutations that result in substitutions of these amino acid residues by amino acid residues that disrupt GTPase activity prolong the active state of Gs.376 Somatic mutations of these residues that disrupt GTPase activity are present in approximately 40% of growth-hormone–secreting and some ACTH-secreting and nonsecreting pituitary tumors; in some parathyroid, thyroid, and adrenal tumors; and in some intramuscular myxomas.81,378,379 More widespread mosaic Arg201 Gs mutations that decrease GTPase activity cause fibrous dysplasia or (when tissue distribution of the mutation is very widespread) McCune Albright syndrome, which is characterized by the triad of café au lait spots, polyostic fibrous dysplasia, and gonadotropin-independent precocious puberty.380,381 Patients with McCune Albright syndrome may also have excessive growth hormone production, hyperthyroidism, and Cushing’s syndrome—as well as associated nodularity of the pituitary and the thyroid and adrenal glands due to overactive Gs subunits in GHRH, TSH, and ACTH receptors, respectively.381,382 Hypophosphatemia, which is not uncommon in patients with McCune Albright syndrome, appears to be due in yet unknown ways to fibrous dysplasia. Patients with McCune Albright syndrome may also have nonendocrine problems such as hepatobiliary abnormalities, cardiomyopathy, and sudden death due to overactive Gs subunits in nonendocrine GPCRs.382

Cytokine Receptors Cytokines are molecules produced by one cell that act on another cell.383 Thus, the term can apply not only to molecules with immunologic functions but to hormones. Therefore, growth hormone, prolactin, and leptin are classified as type I cytokines.384 These and other type I cytokines, (including ILs 2–9, 11–13, and 15; erythropoietin; thrombopoietin; and granulocyte-colony–stimulating factor) are characterized by a four -helical bundle structure and signaling via type I cytokine receptors.384 Type II cytokines include the interferons and IL-10 and do not include hormones.384 Type I cytokines are divided into long-chain and short-chain cytokines.384 Prolactin, leptin, and growth

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hormone belong to the long-chain subclass of type I cytokines because their helixes are 25 amino acids in length.384 The short-chain type I cytokines, including IL-2 and stem cell factor, have helixes of approximately 15 amino acids in length.384

STRUCTURE AND FUNCTION OF TYPE I CYTOKINE RECEPTORS All type I cytokine receptors have four conserved cysteine residues, fibronectin type II modules, a Trp-Ser-X-Trp-Ser motif in the extracellular domain, and a praline-rich Box 1/Box 2 region in the cytoplasmic domain.384,385 With the exception of stem cell factor, type I cytokine receptors do not contain catalytic domains such as kinases.384 Type I cytokine receptors for long-chain type I cytokines require homodimerization for activation.384,386 First, the ligand binds a monomeric receptor.384,386 Then, the ligand interacts with a second receptor to induce receptor dimerization and activation.384,386 Activated receptors then stimulate members of the Janus family of tyrosine kinases (Jak kinases) to phosphorylate tyrosine residues in itself and the cytoplasmic region of the receptors.384,387 Signal transducers and activators of transcription (STATs) then dock on the phosphorylated cytoplasmic receptor domains or Jak kinases via an SH2 domain and are tyrosine phophorylated.384 The phosphorylated STATs then dissociate from the receptors or Jak kinases, form homoor heterodimers, and translocate to the nucleus.384,388,389 In the nucleus, the STAT dimers bind and alter the activity of regulatory regions of target DNA.384,388,390 There are four Jak kinases.388,391 Jak3 is only expressed in lymphohematopoietic cells, whereas Jak1, Jak2, and Tyk2 are expressed in every cell.384,392,393 There are seven STATs (Stat1, Stat2, Stat3, Stat4, Stat5a, Stat5b, and Stat6), which have different SH2 domain sequences that confer different receptor specificities.384,387-389

CYTOKINE RECEPTORS THAT TRANSDUCE HORMONE ACTION The actions of growth hormone, prolactin, and leptin are mediated via specific type I cytokine receptors.384 Mutations of the growth hormone receptor (GHR) and the leptin receptor have been found that are associated with endocrine disorders (Table 2-3).

Growth Hormone Receptors The GHR gene is located on the short arm of chromosome 5 (5p13.1-p12), and 9 of the 13 exons of the gene encode the receptor.394-397 A secretion signal sequence is encoded by exon 2, the N-terminal extracellular ligand binding domain is encoded by exons 3 through 7, the single transmembrane domain is encoded by exon 9, and the C-terminal cytoplasmic domain is encoded by exons 9 and 10.394-397 Growth hormone binding protein (GHBP) is the product of proteolytic cleavage of the extracellular domain of the GHR from the rest of the receptor.398 Approximately 50% of circulating growth hormone is bound to GHBP.398

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TA B L E 2 - 3

Cytokine Receptors and Clinical Conditions Associated with Receptor Mutations Receptor Growth hormone receptor

Leptin receptor

Germ Line Mutation

Endocrine Disorder

Some inactivating (heterozygous) Inactivating (homozygous, compound heterozygous) Inactivating (homozygous)

Partial growth hormone insensitivity with mild to moderate growth failure Growth hormone insensitivity/ Laron syndrome with moderate to severe postnatal growth failure Obesity and hypogonadotropic hypogonadism

Growth-hormone–induced receptor dimerization stimulates Jak2, resulting in tyrosyl phosphorylation of Stat1, Stat3, and Stat5.399-403 Then the STATs translocate to the nucleus, where they regulate growth-hormone–responsive genes.400-403 In particular, growth hormone indirectly controls growth by regulating production of insulin-like growth factor-1 (IGF-1)—which has direct effects on cell proliferation and hypertrophy.404 Jak2 also activates the mitogen activated protein (MAP) kinase and insulin receptor substrate pathways.405-407 However, the extent to which these pathways contribute to growth hormone action is as yet unknown.397 Patients are considered to have growth hormone insensitivity (GHI) if they do not exhibit appropriate growth and metabolic responses to physiologic levels of growth hormone.398 The phenotype of GHI is variable and ranges from isolated moderate postnatal growth failure to severe postnatal growth failure accompanied by the classic features of Laron syndrome in Ecuadorean patients with GHR deficiency.398,408-411 Features of GHR deficiency include frontal temporal hairline recession, prominent forehead, decreased vertical dimension of face, hypoplastic nasal bridge, shallow orbits, blue sclera, small phallus prior to puberty, crowded permanent teeth, absent third molars, small hands and feet, hypoplastic fingernails, hypomuscularity, delayed age of onset for walking, high-pitched voice, increased total and lowdensity lipoprotein cholesterol, and fasting hypoglycemia.398,411 All patients with GHI have normal or elevated circulating growth hormone levels, markedly decreased circulating IGF-1 levels, and a delayed bone age.398 Patients homozygous or compound heterozygous for deletion of exons 5 and 6—or homozygous or compound heterozygous for numerous nonsense, missense, frame-shift, and splice-point mutations throughout the GHR gene—have been found to have GHI characterized by severe postnatal growth failure and usually low or absent circulating GHBP levels.398,412,413 Patients homozygous or compound heterozygous for the Arg274Thr or the Gly223Gly splice mutations that result in a truncated receptor that cannot be anchored to the plasma membrane (or that result in the Asp152His missense mutation that interferes with GHR dimerization) have normal circulating GHBP levels.398 Patients heterozygous for mutations that alter the GHR have dimerization complexes that consist of two wildtype receptors (a wild-type receptor and a mutant receptor) and two mutant receptors. Thus, heterozygosity for

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loss-of-function GHR gene mutations may have a dominant negative effect because the wild-type receptor/ mutant receptor dimers may not be able to function normally. As expected from this phenomenon, some patients with moderate to severe growth failure have been found to be heterozygous for loss-of-function point or splice mutations of the GHR gene that alter the cytoplasmic or extracellular domains.398,414-417 Some patients with severe short stature and GHI do not have GHR mutations. Rather, they have defects in GHR-mediated intracellular signaling—including impaired STAT activation.418

Leptin Receptors The leptin receptor [LEPR (also known as Ob-R)] gene is located at 1p31. There are five isoforms of LEPR due to alternative splicing of the LEPR gene transcript (Figure 2-5). 419 Only the Ob-Rb isoform contains both the Jak kinase binding and STAT motifs necessary to maximally transduce the effects of leptin.419 The Ob-Ra, Ob-Rc, and Ob-Rd isoforms contain intact extracellular and transmembrane but are missing the STAT motif from their cytoplasmic domains.419 The Ob-Re isoform is missing the transmembrane and cytoplasmic domains.419 Thus, the Ob-Rb isoform is thought to be the main isoform involved in mediating the effects of leptin.419 Three sisters from a consanguineous kindred were found to be homozygous for a splice mutation in the LEPR gene that resulted in expression of an 831 amino acid protein (Ob-Rhd) that lacks transmembrane and cytoplasmic domains.420 They had been hyperphagic and morbidly obese since birth.420 They were found to have elevated circulating leptin levels, decreased TSH and GH secretion, and failure of pubertal development due to hypogonadotropic hypogonadism.420 Heterozygous carriers of the mutation are not morbidly obese and do not have delayed or absent puberty.420 More recently, in studies of 300 obese subjects 3% had nonsense or missense LEPR mutations. Individuals with mutations had hyperphagia, severe obesity, altered immune function, and delayed puberty due to hypogonadotropic hypogonadism. Importantly, circulating leptin levels were within the range predicted by the elevated fat mass, and clinical features were less severe than those of subjects with congenital leptin deficiency.421

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RECEPTOR TRANSDUCTION OF HORMONE ACTION

Gln269

Tyr763

His796

Lys889

Ob-Rb Extracellular domain

TM Box 1

Box 2

Ob-Ra Extracellular domain

TM

Extracellular domain

TM

Extracellular domain

TM

Ob-Rc

Ob-Rd

Ob-Re Extracellular domain Figure 2-5. Leptin receptor isoforms. There are five leptin receptor isoforms. Box 1 represents the Jak kinase binding motif, and box 2 represents the STAT motif. The Ob-Rb isoform is the only isoform that contains Jak kinase binding and STAT motifs, and is thus thought to be the main isoform involved in mediating the effects of leptin. The Ob-Ra, Ob-Rc, and Ob-Rd isoforms are missing the STAT motif. The Ob-Re isoform is missing the transmembrane (TM) and cytoplasmic domains. [Reproduced with permission from Chen D, Garg A (1999). Monogenic disorders of obesity and body fat distribution. J Lipid Res 40:1737.]

Receptor Tyrosine Kinases The receptor tyrosine kinase (RTK) superfamily consists of 15 receptor tyrosine kinase families (Figure 2-6).422 With one exception, these families consist of receptors with one membrane-spanning domain (Figure 2-6).422 The single-membrane–spanning receptors typically contain an N-terminal extracellular portion, a transmembrane helix, a juxtamembrane region, a tyrosine kinase (TK) domain, and a C-terminal region (Figure 2-6).422 These receptors require dimerization to be maximally activated.422-424 Receptors belonging to insulin RTK family

differ from other RTKs, as they contain two membranespanning polypeptide chains linked by disulfide bonds to two intervening extracellular peptide chains and thus do not dimerize (Figure 2-6).421 Activation of RTKs leads to phosphorylation of tyrosine residues in the activation loop (A-loop) in the TK domain(s), resulting in activation of the TK(s).421,425 Activation of the TK(s), in turn, induces the transfer of phosphate from adenosine triphosphate (ATP) to tyrosine residues in the cytosolic portion of the receptor and in cytosolic proteins that serve as docking sites for second messengers.421

L Cysteine-rich Fibronectin type III Immunoglobulin EGF Leucine-rich Cadherin Discoidin Kringle

EGFR ErbB2 ErbB3 ErbB4

InsR PDGFRα Flt1 FGFR1 Ror1 Met IGF1R PDGFRβ Flt4 FGFR2 Ror2 Ron IRR CSF1R Flk1 FGFR3 Sea Kit FGFR4 Flk2

TrkA TrkB TrkC

Axl Tie EphA1 Ret Eyk Tek Tyro3 EphB1 Nyk

Ryk DDR1 Ros DDR2

Tyrosine kinase SAM

Figure 2-6. The fifteen RTK families. Each family has a characteristic extracellular portion, and a cytoplasmic portion that contains a tyrosine kinase domain. [Reproduced with permission from Hubbard SR (1999). Structural analysis of receptor tyrosine kinases. Prog Biophys Mol Biol 71:344.]

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There is a growing body of evidence suggesting that members of the receptor tyrosine kinase superfamily can directly and indirectly interact with heterotrimeric G proteins. The insulin, insulin-like growth factor-1, and insulin-like growth factor-2 receptors appear to directly interact with Gi/o and Gq/11, Gi/o, and Gi, respectively.426 The fibroblast growth factor receptors appear to directly and indirectly interact with Gs.426 Congenital alteration of function of receptors in the insulin and the fibroblast growth factor RTK families leads to endocrine disorders (Table 2-4).

INSULIN RECEPTOR TYROSINE KINASE FAMILY The insulin RTK family includes the insulin receptor (INSR) and the insulin-like growth factor-1 receptor (IGF1R).423 These receptors are heterotetramers consisting of two  and  subunits in a  configuration (Figure 2-7).427-429 The cysteine-rich extracellular  subunits are linked by disulfide bonds, and each  subunit is linked to a plasma membrane-spanning and cytosolic  subunit by disulfide bonds.429,430 Each  subunit contains a TK domain and a C-terminal region that contain tyrosine residues.421 Both insulin and insulin-like growth factor-1 (IGF-1) can bind INSRs and IGF1Rs. However, insulin has greater affinity for the INSR and IGF-1 has greater affinity for the IGF1R. Ligand binding alters the conformation of the receptor, resulting in trans-autophosphorylation of the C-terminal tyrosine residues on one  subunit by the TK on the other  subunit.431,432 The phosphorylated tyrosine residues create motifs that can be bound by Src homology 2 (SH2)-domain-containing proteins, including Shc, Grb-2, SHP2, nck, phosphatidylinositol-3-kinase (PI3K), and Crk.433-437 The receptor TK also phosphorylate tyrosine residues in insulin receptor substrate proteins (IRS), including IRS-1 and IRS-2, that bind INSRs and IGF1Rs.437-440 When phosphorylated, these tyrosine residues create motifs that are bound by SH2-domaincontaining proteins.433,434,436,437,440 Thus, insulin receptor substrates can serve as docking proteins—allowing SH2-domain-containing proteins to indirectly interact with INSRs and IGF1Rs when stearic constraints do not permit direct interactions between the proteins and the

45

receptors.433,434,437 Ultimately, IRSs, SH2-domain-containing proteins, and other proteins (including mSOS) interact to activate the Ras/Raf/MAPKK/MAPK and PI3K/protein kinase B (PKB) cascades (Figure 2-7).436,437,440 Activation of the Ras/Raf/ MAPKK/MAPK cascade increases mitogenesis and proliferation, and activation of the PI3K/PKB cascade increases glucose uptake and glycogen synthesis.429,441-446 Evidence suggests that IGF1 has a greater effect on cell growth than on glucose metabolism because activation of the IGF1R stimulates the Ras/ MAPK cascade more than INSR activation.429,445 Conversely, it appears that insulin has a greater effect on glucose metabolism because INSR activation stimulates the PI3K/PKB cascade more than IGF1R activation.429,446

The Insulin Receptor The INSR gene is located on 19p and contains 22 exons.447  half-receptor precursors are derived from proteolysis of a single proreceptor comprised of  and  subunits in tandem and disulfide linkage of these subunits.437,447,448 These  half-receptor precursors then join to form a single  heterotetrameric insulin receptor.448 Interestingly,  half-receptor precursors encoded by one allele may combine with  half-receptor precursors encoded by the other allele to form a single insulin receptor.449 This phenomenon explains how heterozygote mutations resulting in impaired  subunit tyrosine kinase activity can have a dominant negative effect because activation of the INSR requires trans-autophosphorylation of one  subunit by the other  subunit.449 Patients with “type A syndrome” have acanthosis nigricans and severe inherited insulin resistance in the absence of INSR autoantibodies.450-452 Patients with this syndrome tend to be lean and develop glucose intolerance.452,453 Females with this syndrome also exhibit signs of ovarian hyperandrogenism, including hirsutism, severe acne, oligomenorrhea, and infertility.450-452 Patients with “type B syndrome” present during adulthood with acanthosis nigricans, ovarian hyperandrogenism, and severe insulin resistance in association with signs of autoimmune disease—including alopecia areata, vitiligo, primary biliary cirrhosis, arthritis, and nephritis.450452,454 Surprisingly, these patients may present with fasting

TA B L E 2 - 4

Receptor Tyrosine Kinases and Clinical Conditions Associated with Receptor Mutations Receptor

Germ Line Mutation

Endocrine Disorder

Insulin receptor

Inactivating (heterozygous) Inactivating (homozygous, compound heterozygous) Gene deletion (heterozygous)

Some cases of type A syndrome Rabson Mendenhall, Donohue (leprechaunism), and some cases of type A syndromes Pre- and postnatal growth failure

Inactivating mutation (heterozygous) Activating mutations (heterozygous)

Kallmann syndrome, missing teeth, cleft palate Achondroplasia, severe achondroplasia with developmental delay and acanthosis nigricans, thanatophoric dysplasia types I and II, and platyspondylic lethal skeletal dysplasias (San Diego types)

IGF-1 receptor FGFR1 FGFR3

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RECEPTOR TRANSDUCTION OF HORMONE ACTION

Insulin receptor

α subunits

Pl4(P)

+

pY

pY

Pl 3,4(P2)

+

+

Shc pY

pY

Grb-2

Grb-2

sos

+? +?

β subunits PI-3K pY IRS-1

Glut-4

sos

+

+

Ras Raf-1 MEK MAP K

SH-PTP2 pY nck crk p85β others

Signal

pp70s6k

+

Signal

Signal

Figure 2-7. Insulin receptor signaling. IRS proteins, SH2-domain–containing proteins (including Grb-2 and Shc), and other proteins (including SOS) interact to activate the Ras/Raf-1/MAP K cascade and PI-3K/PKB cascades, and other enzymes—including SH-PTP2 (SHP2) and p70(s6k). [Adapted with permission from White MF (1997). The insulin signaling system and the IRS proteins. Diabetologia 40:S10.]

hypoglycemia that may or may not be accompanied by postprandial hyperglycemia.450-452,454 Hodgkin’s disease and ataxia-telangiectasia are also associated with this syndrome.452 Patients with type B syndrome are distinguished from patients with type A syndrome by the presence of anti-INSR antibodies in the plasma that block insulin binding.450-452,454 The term HAIR-AN (hyperandrogenism, insulin resistance, and acanthosis nigricans) has also been used to describe women with features of types A and B syndromes in association with obesity.452 However, this term is imprecise because many women who have been labeled as having HAIR-AN may actually have type A or B syndrome or severe polycystic ovary syndrome.452 Patients with Rabson-Mendenhall syndrome present during childhood with severe insulin resistance.455-457 Although patients with this disorder may present initially with fasting hypoglycemia, eventually they develop severe diabetes ketoacidosis that is refractory to insulin therapy.457 Patients with this condition also have acanthosis nigricans, accelerated linear growth, dystrophic nails, premature and dysplastic dentition, coarse facial features, and pineal hyperplasia.453,455-457 Patients with leprechaunism or Donohue syndrome are also severely insulin resistant.458,459 Patients present during infancy with severe intrauterine and postnatal growth retardation, lipoatrophy, and acanthosis nigricans.456,458 They also have dysmorphic features that include globular eyes, micrognathia, and large ears.456,458 Affected male infants commonly have penile enlargement, whereas affected female infants often have clitoromegaly and hirsutism.456,458 In spite of hyperinsulinemia associated with glucose intolerance or diabetes

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mellitus, the major glucose metabolism problem for these patients is fasting hypoglycemia.456,458 Unlike patients with Rabson-Mendenhall syndrome, patients with leprechaunism do not present with diabetic ketoacidosis.457 Many patients with this condition do not survive past the first year of life.456,458 Mutations in the INSR have been found in 10% to 15% of patients with type A syndrome, and in all patients with Rabson-Mendenhall syndrome and leprechaunism.452,459,460 These mutations are divided into five classes.452,453,461 Class I mutations are frame-shift or nonsense mutations that prematurely terminate translation and thus interfere with INSR synthesis. Class II mutations interfere with post-translational processing and intracellular trafficking of the INSR. Class III mutations decrease insulin binding to the INSR. Class IV mutations are point mutations usually located on the intracellular region of the  subunit that decrease INSR TK activity. Class V mutations increase INSR degradation by increasing insulin-induced endocytosis and degradation of the receptors. Patients with Rabson-Mendenhall syndrome and leprechaunism are homozygous or compound heterozygous for these mutations.452,462-466 Some patients with type A syndrome have been found to be heterozygous for dominant negative -subunit mutations that reduces TK activity by 75%.461,466-471 Other patients with type A syndrome have been found to be homozygous or compound heterozygous for -subunit mutations that interfere with receptor trafficking to the plasma membrane, -subunit mutations that interfere with TK activity, or mutations that interfere with proreceptor cleavage into  and  subunits. Still other patients have been found to have decreased INSR mRNA levels that may be due to a loss-of-function

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mutation in the INSR gene promoter.453,472-476 Interestingly, one patient with leprechaunism with parents with type A syndrome has been described.466 The proband was found to be homozygous for an INSR mutation that decreases TK activity, and the parents were found to be heterozygous for the mutation.466

The Insulin-Like Growth Factor-1 Receptor The growth-promoting effects of IGF-1 are mediated by IGR1Rs. IGF1R  subunits are encoded by a single gene.448 Like the insulin receptor, an  half-receptor precursor is produced that then joins with a half-receptor precursor that may be encoded from the other allele to form a complete heterotetrameric IGF1R.448 The IGF1R has 100-fold less affinity for insulin than for IGF1.477 Patients who are heterozygous for a ring chromosome 15, resulting in deletion of the IGF1R gene, present with intrauterine growth retardation and postnatal growth failure accompanied by delayed bone age, mental retardation, cardiac abnormalities, cryptorchidism, and dysmorphic features that include microcephaly, triangular face, frontal bossing, hypertelorism, and brachydactyly.478,479 Similarly, IUGR and postnatal growth failure are commonly found in patients heterozygous for deletion of distal 15q that results in deletion of the IGF1R gene. Patients with deletion of distal 15q often have microcephaly, triangular facies, hypertelorism, high-arched palate, micrognathia, cystic kidneys, and lung hypoplasia or dysplasia.477,480-482 However, the ring chromosome and deleted area of distal 15q may also be missing other genes—and it is unknown to what extent absence of the IGF1R gene contributes to the phenotype found in these patients.477,479 It has been suggested African Efe Pygmies are short due to resistance to IGF1.477 T-cell lines established from Efe Pygmies have decreased IGF1R gene expression, cell surface expression, receptor autophosphorylation, and intracellular signaling when compared with T-cell lines established from American controls.477 However, no IGF1R gene mutation has been identified in Efe Pygmies that can account for these findings.477 In addition to defective INSR function, some patients with leprechaunism and Rabson-Mendenhall syndrome are resistant to the glucose lowering or growth promotion of IGF1 and have abnormal IGF1R function—resulting in decreased ligand binding or altered intracellular signaling.477,483-487 No deleterious IGF1R gene mutation has been identified in patients with these syndromes, and many patients with leprechaunism and Rabson-Mendenhall syndrome have normally functioning IGF1Rs and no evidence of IGF1 resistance.477,488

The Fibroblast Growth Factor Receptor Family There are four members of the fibroblast growth factor receptor (FGFR) tyrosine kinase family.421 These are FGFR1, FGFR2, FGFR3, and FGFR4. These receptors consist of a

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47

single polypeptide chain that contains an N-terminal extracellular region, a transmembrane region, and a cytosolic region (Figure 2-6).421 The extracellular region contains three immunoglobulin-like domains: IgI, IgII, and IgIII (Figure 2-6).489 The cytosolic region contains a TK domain split into two segments (TK1 and TK2) by an intervening amino acid segment.489 At least 13 types of fibroblast growth factors (FGFs) have been identified.489 As monomers, FGFs can only bind a single FGFR—forming an inactive 1:1 complex.423 FGFR activation by dimerization occurs when two or more FGF molecules in 1:1 complexes are linked by heparan sulfate proteoglycans.423 Activation of FGFRs increases receptor TK activity.489 Increased TK activity leads to autophosphorylation of a tyrosine residue in the C-terminal region, resulting in a binding site for the SH2 domain of phospholipase C
(PLC
).490,491 Once PLC
is bound to this site, it is phosphorylated and activated.490,491 In chondrocytes, activation of FGFR3 also induces activation of STAT1.492

FIBROBLAST GROWTH FACTOR RECEPTOR 1 In 2003, inactivating FGFR1 gene mutations were identified as a cause of autosomal-dominant Kallmann syndrome (KS).493 Individuals with KS have anosmia and isolated hypogonadotropic hypogonadism.494,495 The FGFR1, which is located on 8p12, plays a role in olfactory and GnRH neuronal migration from the nasal placode to the olfactory bulb and in the subsequent migration of the GnRH neurons to the hypothalamus.493 Prior to identification of these FGFR1 gene mutations, X-linked KS was found to be caused by inactivating KAL1 gene mutations.494,496,497 The KAL1 gene is located on the X chromosome and encodes anosmin-1.494,496,497 Anosmin-1 is a ligand for the FGFR1 receptor.498 Like FGFR1s, anosmin-1 plays a role in olfactory and GnRH neuronal migration to the nasal placode, and in the subsequent migration of GnRH neurons to the hypothalamus.494,498,499 There is a high penetrance for anosmia and signs of hypogonadotropic hypogonadism, (including lack of puberty, microphallus, and cryptorchidism) in the 10% of KS patients with X-linked KS due to KAL1 gene mutations.494 Female carriers of KAL1 gene mutations do not have anosmia or isolated hypogonadotropic hypogonadism.494 In contrast to patients with KS due to KAL1 gene mutations, the approximately 10% of KS patients with FGFR1 gene mutations (even within the same kindred) exhibit variable phenotypes ranging from anosmia and complete hypogonadotropic hypogonadism (characterized by cryptorchidism and microphallus in males and absent pubertal development in both genders) to anosmia and/or delayed puberty.493,500,501 It has also been noted that in most kindreds with FGFR1 gene mutations females present with more mild KS phenotypes than males.493,501 Female carriers may even be asymptomatic.493,501 Because the KAL1 gene is located on the X chromosome, females may produce more anosmin-1 than males.493 Thus, a possible explanation for milder KS phenotypes in females with FGFR1 gene mutations may

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be that the increased anosmin-1 levels in females may lead to increased anosmin-1–induced activation of the mutant FGFR1s that may partially compensate for the mutation.493 Interestingly, missing teeth and cleft palate are not an uncommon finding in individuals with KS due to FGFR1 gene mutations, whereas unilateral renal agenesis and bilateral synkinesia are associated with KS due to KAL1 gene mutations.500

FIBROBLAST GROWTH FACTOR RECEPTOR 3 In addition to variants of Kallmann syndrome, FGFR mutations cause many conditions—including Pfeiffer syndrome (activating mutations of FGFR1 and FGFR2), Crouzon syndrome (FGFR2 mutations), Crouzon syndrome with acanthosis nigricans (an FGFR3 mutation), Apert syndrome (FGFR2 mutations), and craniosynostosis (FGFR3 mutations).489 Several autosomal-dominant shortlimb dwarfism syndromes—including achondroplasia, severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN), hypochondroplasia, and three types of platyspondylic lethal skeletal dysplasias (PLSD) [thanatophoric dysplasia I (TDI), thanatophoric dysplasia II (TDII), and San Diego types (PLSD-SD)]—are often caused by heterozygous constitutively activating FGFR3 gene mutations.502-505 Individuals with achondroplasia have activating mutations in the transmembrane domain of FGFR3, with the Gly380Asn found in 95% of achondroplastic patients.503,506-508 Forty to 70% of individuals with hypochondroplasia have an activating Asn540Lys mutation in the TK1 domain.503,509-512 All individuals with TDII have an activating Lys650Glu mutation in the activating loop of the TK2 domain, and 90% of individuals with TDI and PLSD-SD have FGFR3 mutations.503,505 Patients with SADDAN have an activating mutation in the same codon as patients with TDII.513 Instead of the Lys650Glu mutation associated with TDII, patients with SADDAN have a Lys650Met mutation.513 However, unlike patients with TDII patients with SADDAN do not have craniosynostosis and a cloverleaf skull—and often survive past childhood.513 The FGFR3 gene is primarily expressed in endochondral growth plates of long bones, brain, and skin pre- and postnatally.514,515 Constitutional activation of FGFR3s in chondrocytes leads to growth arrest and apoptosis.492,516,517 In addition, constitutive activation of FGFR3s is also postulated to alter neuronal migration because patients with SADDAN, TDI, and TDII have neurologic abnormalities that may include developmental delay, paucity of white matter, polymicrogyria, dysplastic temporal cortex, dysplasia of nuclei, and neuronal heterotopia.513,518-520 Furthermore, constitutive activation of FGFRs in skin fibroblasts and keratinocytes is thought to cause the acanthosis nigricans seen in patients with SADDAN and Crouzon syndrome with acanthosis nigricans.521 However, it is not yet known why some activating FGFR3 mutations effect the skeletal system, central nervous system, and the skin whereas other activating FGFR3 mutations only effect the skeletal system.513

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Nuclear Receptors Using a phylogenetic tree based on the evolution of two highly conserved nuclear receptor domains (the DNAbinding C domain and the ligand-binding E domain, V), Laudet divided nuclear receptors into six related subfamilies and a subfamily. Subfamily 0 contains receptors, such as the embryonic gonad (EGON) and DAX1 receptors, that do not have a conserved C or the E domain (Figure 2-8).522,523 Subfamily 1 includes the peroxisome proliferator-activated retinoic acid, thyroid hormone, and vitamin D3 receptors. Subfamily 2 includes the hepatocyte nuclear factor-4 (HNF-4) and retinoid X receptors (RXRs). Subfamily 3 contains the steroid receptors. Subfamilies 4 and 5 contain the NGFIB and the FTZ-F1 orphan receptors, respectively. Subfamily 6 consists of the GCNF1 orphan receptor. Recent evidence suggests that subfamily 3 (which includes the glucocorticoid, androgen, progesterone, and mineralocorticoid receptors) rapidly evolved from a common steroid receptor gene about 500 million years ago.524

GENERAL STRUCTURE OF THE NUCLEAR RECEPTORS Nuclear receptors are made up of four domains: A/B, C, D, and E (Figure 2-9).522 Supporting the notion that nuclear receptor subfamilies are derived from a common ancestral orphan receptor, the C and E domains are highly conserved among the subfamilies.522 Mutation of several nuclear receptors are associated with endocrine disorders (Table 2-5). The A/B domain is located at the N-terminal and contains the activation function 1 (AF-1)/ 1 domain.525 The AF-1/ 1 domain regulates gene transcription by interacting with proteins (such as the Ada and TFIID complexes) that induce transcription.526,527 This transactivation function of the AF-1/ 1 domain is not dependent on binding of the nuclear hormone receptor to its ligand and is not specific in its choice of DNA target sequences.522,528-530 Thus, specificity of action of the nuclear hormone receptor is determined by the function of other nuclear hormone receptor domains. The C domain has characteristics that help to confer specificity of action on each nuclear hormone receptor. This domain consists of two zinc-finger motifs responsible for the DNA-binding activity of the receptor and the selection of dimerization partners.531,532 Each zinc-finger module consists of a zinc ion surrounded by the sulfurs of four cysteine residues, resulting in a tertiary structure containing helixes.531,532 The P-box lies near the cysteines of the first zinc finger and contains the three to four amino acids responsible for specificity of binding to response elements.532,533 The D-box consists of a loop of five amino acids attached to the first two cysteines of the second zinc finger that provides the interface for nuclear receptor dimerization.532 The D “hinge” domain contains nuclear localization signals and contributes to the function of the adjacent C and E domains.522 Thus, the N-terminal portion of the domain contributes to DNA binding and heterodimerization and

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Homo TRA 1 A Homo TRB 2 1 Homo RARA 95 100 B 2 Homo RARB 3 Homo RARG 1 Homo PPARA 97 100 C 2 Homo PPARB 3 Homo PPARG 23 29 Homo REV-ERBA 1 100 92 Homo REV-ERBB 2 D 47 3 Drosophila E75 27 E 1 Drosophila E78 1 Homo RORA 98 100 2 Rattus RZRB 74 F 100 3 Homo RZRG 31 96 4 Drosophila HR3 4 Caenorhabditis CNR3 G 1 Caenorhabditis CNR14 1 Drosophila ECR 38 2 Homo UR 100 100 96 H 3 Homo LXR 4 Mus FXR 59 1 Homo VDR 98 2 Xenopus ONR1 54 I 83 100 Homo MB67 3 4 Mus CAR2 71 J 1 Drosophila DHR96 1 Onchocerca NHR1 K 1 Homo NGFIB 88 53 Homo NURR 1 2 100 A 100 3 Rattus NOR 1 4 Drosophila DHR38 4 Caenorhabditis CNR8 1 Homo ER 100 A 99 2 Rattus ERB 1 Homo ERR 1 100 B 97 2 Homo ERR 2 1 43 Homo GR 82 2 Homo MR C 100 3 Homo PR 4 Homo AR 1 Mus SF1 100 100 2 A Mus LRH 1 98 3 Drosophila FTZF 1 52 B 1 Drosophila DHR39 1 Mus GCNF1 A 1 94 Homo HNF4 100 2 54 Homo HNF4G 100 3 Xenopus HNF4B 4 Drosophila HNF4 77 1 Homo RXRA 81 100 2 Homo RXRB 100 3 Mus RXRG 94 4 Drosophila USP 100 Homo TR2 1 57 Homo TR4 2 1 Drosophila DHR78 100 1 Mus TLX 48 2 Drosophila TLL Homo COUPA 1 100 55 96 Homo COUPB 2 100 3 Drosophila SVP 100 Bootstrap 4 89 Xenopus COUPG 100 5 Zebrafish SVP46 6 Homo ER2

49

100

87

1

4

3

5 6 A

B

C

2

D E

F

Figure 2-8. Phylogenetic tree of nuclear receptors based on the evolution of the highly conserved C and E domains. Numbers at the right side of the figure represent subfamilies, and capital letters represent groups of more closely related receptors. The small numbers to the right of the receptor names are used in combination with the subfamily letters and group letters in a proposed nuclear receptor nomenclature. This nomenclature proposes that nuclear receptors should be named NR, followed by subfamily number, group letter, and individual receptor number. Thus, the mineralocorticoid receptor is named NR3C2 and the PPAR
receptor is named NR1C3 according to this nomenclature. Numbers to the left of the receptor names represent bootstrap values. Values that define subfamilies with more than one member are boxed. [Reproduced with permission from the Nuclear Receptors Nomenclature Committee (1999). A unified nomenclature system for the nuclear receptor subfamily. Cell 97:161.]

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RECEPTOR TRANSDUCTION OF HORMONE ACTION

Nuclear receptor dimer

HDAC

E domain AF-2

A/B domain

Sin3

AF-2

D domain

Histone deacetylation

SMRT (N-CoR)

Blocked transcription

X

AF-1

C domain

DNA

Nucleosomes

Ligand

Coactivators recruitment

Corepressors release

CBP/p300 P/CIP SRC-1

Histone acetylation

LX

XL

L

AF-2

AF-2

P/CAF Transcription Ac

Ac

Ac

Ac

Ac

Ac

AF-1

Histone tail Ac

Ac Ac

Ac Ac

Ac

Figure 2-9. Ligand-induced activation of transcription by nuclear receptors. Often, corepressors [including SMRT and nuclear receptor copressor (N-CoR)] bind a nuclear receptor that is not bound by its ligand. These corepressors then associate with Sin3, which in turn associates with a histone deacetylases (HDAC). Then, HDAC represses transcription by deacetylating histone tails—resulting in compaction of the nucleosomes into structures that are inaccessible to transcription factors. Ligand binding induces structural changes in the E domain that result in release of the corepressor/Sin3/HDAC complexes from the receptor, and binding of coactivator complexes that may include steroid receptor coactivator 1 (SRC-1), p300/cAMP responsive element binding protein (CBP), p300/CBP-associated factor (P/CAF), or p300/ CBP cointegrator-associated protein (pCIP) to the LXXLL motif of the AF2-AD. Then, the coactivator complexes induce transcription by acetylating (Ac) the histone tails—resulting in decompaction of the nucleosomes into structures that are accessible to transcription factors. Dashed lines are used to represent coactivator and corepressor complexes because their composition in vivo is yet unknown. [Adapted with permission from Robyr D, Wolffe AP, Wahli W (2000). Nuclear hormone receptor coregulators in action: Diversity for shared tasks. Mol Endocrinol 14:339. Copyright 2000, The Endocrine Society.]

the C-terminal portion contributes to ligand binding.533-536 The nuclear localization signal plays a particularly important role in the function of glucocorticoids and mineralocorticoid receptors because these receptors bind their ligand in the cytoplasm and must then localize to the nucleus to alter gene transcription.525

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The E domain is known as the ligand-binding domain (LBD) or the hormone-binding domain. In addition to ligand binding, the E domain has effects on dimerization and transactivation.522 The LBD consists of 11 to 12  helixes (named H1 through H12) and contains a ligandbinding pocket that is made up of portions of some of

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51

TA B L E 2 - 5

Nuclear Receptors and Clinical Conditions Associated with Receptor Mutations Receptor Thyroid hormone receptor  (TR) Vitamin D3 receptor PPAR
2 HNF-4 Glucocorticoid receptor Androgen receptor Estrogen receptor  (ER) Mineralocorticoid receptor

DAX1

Germ Line Mutation Inactivating mutations (heterozygous and homozygous) Inactivating mutations (homozygous) Inactivating mutations (heterozygous) Inactivating mutations (heterozygous) Inactivating mutations (heterozygous) Inactivating mutations (X-linked recessive) Inactivating mutations (homozygous) Inactivating mutations (heterozygous, homozygous) Activating mutations (homozygous) Inactivating mutations (X-linked recessive)

the different helixes.537-540 For example, the thyroid receptor (TR) LBD has a ligand-binding cavity that includes components from H2, H7, H8, H11, and H12.540 The contribution of different parts of LBD to the ligand-binding pocket accounts for the finding that mutation of single– amino-acid molecules in different helixes of the LBD can interfere with ligand binding.525 Unlike the AF-1/ 1 transcriptional activating factor, the E domain activation factor 2 (AF2-AD) requires ligand binding to function (Figure 2-9).538-545 Often, when the receptor is not bound by its ligand corepressor complexes simultaneously bind the LBD and transcriptional machinery consisting of protein complexes that place transcription factors on nucleosome binding sites (Figure 2-9).538-545 The corepressor complexes then suppress gene transcription by using histone deacetylases to compact the nucleosomes into inaccessible structures (Figure 2-9).546-549 Ligand binding induces structural rearrangements in the E-domain that lead to release of these corepressor complexes from the transcriptional machinery and the LBD, and exposure of the transcriptional machinery and the LXXLL motif of the AF2-AD to coactivator complexes (Figure 2-9).538-545 These coactivator complexes have histone acetyltransferase activity that acts to relax nucleosome structures, enabling transcription factors to access nucleosome binding sites (Figure 2-9).550 Most nuclear receptors are capable of binding their hormone response element and repress transcription when they are not bound by their ligand.551 However, in the absence of ligand steroid receptors are bound to a complex of heat-shock proteins instead of their response element and do not appear to repress transcription.552

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Endocrine Disorder Generalized resistance to thyroid hormones Vitamin D3 resistance Obesity or early-onset type II diabetes mellitus Maturity-onset diabetes of the young (MODY) type I Glucocorticoid resistance Androgen insensitivity syndrome, Kennedy’s disease Tall stature and incomplete epiphyseal fusion Pseudohypoaldosteronism type I Syndrome of apparent mineralocorticoid excess

X-linked adrenal hypoplasia congenita

Agonists and antagonists have different effects upon the interaction between the ligand binding pocket and AF2-AD. For example, when 17-estradiol binds to the estrogen receptor the position of the AF2-AD containing H12 is altered so that coactivators can access the LBDbinding coactivator binding site.537 However, when the estrogen antagonist raloxifene binds at the same site the coactivator binding site on H12 remains blocked by other portions of H12.537 Although some of the nuclear receptors are fully active when bound as monomers to DNA, the hormone receptors in the nuclear receptor superfamily are most active when bound as homodimers or heterodimers (Figure 2-9).522 RXRs, hepatocyte nuclear factor 4, and the steroid receptors can bind DNA as homodimers or heterodimers.522,553,554 The  isoform of the estrogen receptor (ESR1) is particularly promiscuous, and is able heterodimerize with HNF4- and retinoic acid receptors, the  isoform of the estrogen receptor (ESR2), RXR and the thyroid receptors. 553,554 As a homodimer, RXR binds the dINSRect repeat 1 (DR1).555 It can also join the thyroid, vitamin D3, and peroxisome proliferator-activated receptors to form heterodimers.522,556-558 Interestingly, it has also been suggested that some steroid hormones also act on transmembrane receptors—and these interactions may be responsible for the acute cellular effects of steroids.559 Progesterone has been shown to interact with the G-protein–coupled uterine oxytocin, nicotinic acetylcholine, GABAA, NMDA, and sperm cell membrane progesterone receptors.560-565 Cell membrane estrogen and glucocorticoids receptors have also been identified.566-569

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Subfamily 1 Nuclear Receptors: Thyroid Hormone, Vitamin D3, and Peroxisome ProliferatorActivated Receptors THYROID HORMONE RECEPTORS The two thyroid hormone receptor (THR) isoforms— thyroid hormone receptor  (THRA) and thyroid hormone receptor  (THRB)—are encoded by different c-erbA genes on chromosomes 17 and 3, respectively.536,570 THRs that are not occupied by the thyroid hormone triiodothyronine (T3) exist as homodimers or heterodimers with RXRs that are attached to DNA thyroid hormone response elements in association with corepressor proteins.571 Thyroid hormone binding induces the release of the corepressors from the THR.571 A coactivator, steroid receptor coactivator-1 (SRC-1), is then able to attach to the THR—enabling activation of transcription.571 Generalized resistance to thyroid hormones (GRTH) is due to mutations in the THRB gene.570,572,573 Patients with this syndrome have impaired receptor response to triiodothyronine (T3).570 They have elevated T3 and thyroxine (T4) levels with normal TSH levels.570,572,573 The clinical manifestations are variable but may include goiter, attention deficit disorder, hearing defects, learning disabilities, poor weight gain, mental retardation, and delayed boned age.570,572,573 Mutations have been found in the D and E domains of THRBs of GRTH patients.570,574,575 Thus, these mutations alter ligand binding or transactivation.570,575,576 However, most mutant THRBs retain the ability to repress transactivation of target genes through interactions with corepressors.575,576 Some of the GRTH mutant receptors continue to associate with corepressors and are unable to bind the coactivator SRC-1 even when bound by T3.576,577 Thus, mutant THRBs have a dominant negative effect in the heterozygote state because they are able to interfere with the function of wild-type receptors by repressing transcription of target DNA.570,575,576 Patients who are homozygous for deleterious mutations of THRB demonstrate more severe clinical abnormalities than patients who are heterozygous for the mutations. One such patient with a deletion of both THRB alleles presented with deaf mutism, dysmorphic features, and stippled epiphyses.578 This condition is inherited in an autosomal recessive mode because the mutant allele is missing the THRB gene and is therefore unable to produce a THRB with corepressor function.578 Another patient homozygous for a THRB mutant (“kindred S receptor”) with an amino acid deletion in the ligand-binding domain presented with mental retardation, very delayed bone age, and very elevated T3 and T4 levels.574,579 Heterozygous carriers of the kindred S receptor mutation have milder clinical manifestations of GRTH because this mutant THRB retains corepressor activity and thus has dominant negative effects.574,579,580

VITAMIN D3 RECEPTOR Severe rickets, hypocalcemia, secondary hyperparathyroidism, and increased 1,25-dihydroxyvitamin D3 (D3) levels occur in patients with the autosomal recessive syn-

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drome of “vitamin D3 resistance.”581 These patients have defective vitamin D3 receptors (VDRs). Mutations causing this syndrome have been found in the zinc fingers of the DNA-binding domain (C domain), leading to decreased or abolished receptor binding to regulatory elements of target genes.582 Causative mutations have also been found that lead to the production of receptors that have decreased or abolished ability to bind D3 and heterodimerize with retinoid X receptors (RXRs), which are required for the VDR to maximally transactivate target genes.582,583 Less severe mutations in the VDR are associated with decreased gastrointestinal calcium absorption and bone mineral density, even during childhood, and an increased risk for osteoporosis and fractures.584-589 However, it has not been possible to replicate these findings in some ethnic groups.590-594 Thus, other factors (such as estrogen receptor genotype, dietary calcium, and age) probably contribute to the effects of VDR polymorphisms on bone mineral metabolism.587,589,595,596 VDR polymorphisms have been associated with preand postnatal growth failure. Absence of VDR alleles digested by BSMI in the presence of homozygosity for estrogen receptor alleles digested by PVU II estrogen receptor polymorphism is associated with decreased preand postnatal linear growth.597 In addition, girls homozygous for VDR alleles digested by TaqI have been found to be shorter than girls who do not have the polymorphism.598 Other important associations have been found with VDR polymorphisms. Homozygous polymorphisms have been reported to be associated with primary hyperparathyroidism.599 In addition, the presence of VDR alleles digested by TaqI is associated with increased risk for the development of early-onset periodontal disease.600 Conversely, absence of such alleles has been associated with familial calcium nephrolithiasis.601 Absence of alleles digested by BSMI may be a risk factor for the development of sarcoidosis.602 However, the presence of these alleles is associated with hypercalciuria and nephrolithiasis and increased risk in women for the development of metastatic breast cancer.603,604 Absence of alleles digested by ApaI may be a risk factor for the development of psoriasis.605 VDR polymorphisms have also been associated with increased susceptibility to tuberculosis, leprosy, and other infections.606-609

PPAR␥ The orphan nuclear receptor PPAR
2 has a role in regulating adipocyte differentiation and metabolism. This orphan receptor, as the term indicates, does not have a known ligand. Recent evidence suggests that mutations in the gene encoding PPAR
2 may cause obesity. A missense mutation leading to a Pro115Gln substitution near a site of serine phosphorylation at position 114 that suppresses transcriptional activation in PPAR
2 was found in some morbidly obese patients.610 The Pro115Gln substitution interferes with phosphorylation of the serine at position 114, leading to increased transcriptional activation by PPAR
2—which in turn leads to increased adipocyte differentiation and triglyceride accumulation.610

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Two other mutations in the PPAR
2 gene have been identified that cause severe insulin resistance, early-onset type 2 diabetes mellitus, and hypertension by the end of the third decade of life.611 These mutations lead to amino acid substitutions that disturb the orientation of H12 in the E domain leading to decreased ligand-dependent transactivation by AF-2/AD and coactivator recruitment.611 In addition, these mutant receptors interfere with wildtype receptor function in a dominant-negative manner by continuously suppressing target gene transcription.611

Subfamily 2 Nuclear Receptors: Hepatocyte Nuclear Factor and Retinoid X Receptors This subfamily includes the HNF receptors and the RXR. RXRs form heterodimers with other nuclear receptors (including the estrogen, vitamin D, and thyroid hormone receptors) and with PPAR
(RXRs are discussed elsewhere in this chapter).

53

A single Ile 559 to Asp 559 mutation in the DNA-binding domain is responsible for many cases of glucocorticoid resistance.625 This mutation has a dominant negative effect in that it is able to inhibit the function of the wild-type allele in the heterozygote state.625 Thus, glucocorticoid resistance is often an autosomal-dominant familial disease. Glucocorticoid resistance can also be caused by a mutation in the nuclear localization signal consensus that interferes with hormone-induced nuclear translocation of the glucocorticoid receptor.626 According to the HUGO Gene Nomenclature Committee, the glucocorticoid receptor is also known as the nuclear receptor subfamily 3, group C, member 1 (NR3C1). However, the HUGO Gene Nomenclature Committee also acknowledges the alias GR for the glucocorticoid receptor. GR will be used as the abbreviation for glucocorticoid receptor for the remainder of this chapter. ACTH-secreting pituitary macroadenomas can also be caused by a frame-shift mutation in the GR gene that interferes with signal transduction.625 Patients with this mutation manifest the symptoms of glucocorticoid resistance. The tumor develops as a result of impaired negative feedback regulation by glucocorticoids of the hypothalamic-pituitary axis.

HNF Alteration of another orphan nuclear receptor, HNF-4, also causes an endocrine disorder. Mutations of the HNF4 gene on chromosome 20 that alter the ligand-binding domain (E domain) or the DNA-binding domain (C domain) have been found in patients with maturity-onset diabetes of the young type I (MODY1).612-615 Patients with MODY usually develop diabetes mellitus by the end of the third decade of life. They have a defect in glucosemediated stimulation of insulin secretion.613,616 Carriers of a glycine-to-serine substitution in codon 115 in the DNA-binding domain (C domain) appear to be at increased risk for developing low-insulin diabetes mellitus.617 Hepatocyte nuclear factors 3 (HNF-3, -3, and -3
) are also regulators of the early-onset type 2 diabetes genes HNF-1 , HNF-4 , and IPF-1/PDX-1—which are associated with MODY types 3, 1, and 4, respectively.618-621

Subfamily 3 Nuclear Receptors: The Steroid Receptors and Glucocorticoid, Androgen, Estrogen, and Mineralocorticoid Receptors GLUCOCORTICOID RECEPTORS Glucocorticoid resistance is clinically characterized by the presence of elevated plasma cortisol and ACTH levels, accompanied by the effects of hyperaldosteronism and hyperandrogenism in the absence of striae and central fat deposition.536,622,623 Excess adrenal steroids are made in an attempt to make sufficient cortisol to overcome glucocorticoid resistance. Thus, patients with this disorder may also suffer from hypokalemia, hypertension, fatigue, severe acne, hirsutism, irregular menses, and infertility.622,623 Boys may present with isosexual precocity.624

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ANDROGEN RECEPTORS The human androgen receptor (AR) gene is located on the X chromosome. Known disorders characterized by AR dysfunction due to AR gene mutations are only expressed in patients with a 46 XY karyotype.627 Thus, these disorders are either transmitted by X-linked recessive inheritance or are due to sporadic mutations. Numerous mutations of the AR gene have been found to cause androgen insensitivity syndrome (AIS; testicular feminization). The phenotype of this syndrome can vary in severity from partial undervirilization to complete AIS characterized by intraabdominal testes, absence of mullerian structures, absence of androgen-induced body hair such as pubic and axillary hair, and a female appearance.627-629 Incomplete AIS refers to individuals with ambiguous external genitalia with an enlarged clitoris or microphallus and patients with Reifenstein syndrome.628 Reifenstein syndrome is characterized by severe hypospadias with scrotal development and severe gynecomastia.628 The phenotypic heterogeneity of AIS is due to the wide variety of locations for the mutations causing AIS. Functional consequences of each mutation causing AIS relate to the function of the domain in which the mutation is located. However, the degree of impairment of mutated receptor function in in vitro studies does not always correlate with the phenotypic severity of the syndrome.630 Mutations in exons that code for the AR hormonebinding domain decrease hormone-binding affinity.631 However, these mutations do not abolish the hormonebinding capability of the receptor.631 Thus, patients with these mutations usually present with the incomplete AIS or occasionally with complete AIS.631-634 Patients with mutations in the hormone-binding domain do not appear to respond to treatment with high doses of testosterone.631 Mutations in the DNA-binding domain lead to failure of target gene regulation. Thus, patients with these mutations usually manifest the complete AIS.635,636

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Complete AIS is also caused by a point mutation that results in a premature termination condition, leading to transcription beginning downstream of the termination signal and the AF1/tau1 domain.637 Numerous other mutations have been described that cause truncation or deletion of the AR and complete AIS.638-641 Two patients with ambiguous genitalia and partial virilization were found to be mosaic for mutant ARs.642,643 Some patients with Reifenstein syndrome have been found to have a mutation in the DNA-binding domain that abolishes receptor dimerization.644 Other patients have been found to have a mutation in a different area of the DNA-binding domain that does not effect receptor dimerization, or to have mutations in the hormone-binding domain in the E domain.645-648 The Ala596Thr mutation in the D-box area of the DNA-binding domain has been associated with an increased risk of breast cancer.649 AIS is also a feature of Kennedy’s disease, which is an X-linked recessive condition causing spinal and muscular atrophy.632 This condition is caused by extension of a poly-CAG segment in the AR gene exon that codes for the N-terminus of the AR, leading to an increased number of glutamine residues in the A/B domain.650,651 ARs with a polyQ region increased to 48 glutamine residues accumulate abnormally in transfected cells due to misfolding and aberrant proteolytic processing.652 Because polyQ extension does not completely abolish of transactivation, patients with Kennedy’s disease exhibit a mild partial AIS phenotype consisting of normal virilization accompanied by testicular atrophy, gynecomastia, and infertility.

ESTROGEN RECEPTORS Two major full-length estrogen receptor isoforms have been identified in mammals. Estrogen receptor  (ESR1) was discovered first and mediates most of the known actions of estrogens.536 ESR1 is expressed primarily in the uterus, ovaries, testes, epididymis, adrenal cortices, and kidneys.653 Estrogen receptor  (ESR2) was discovered in 1996.654 ESR1 and ESR2 share 95% and 50% homology in the DBD and LBD, respectively.655 There is little homology in the N-terminal between the two isoforms.655 ESR2 is expressed primarily in the uterus, ovaries, testes, prostate, bladder, lung, and brain.653 Although ESR2 has a high affinity for estrogens, it has less transactivating ability than ESR1 and has not yet been found to be involved in any pathologic condition.536,656,657 There is evidence supporting the existence of other functional estrogen receptors.658 Some of these putative estrogen receptors localize to the cell membrane instead of or in addition to the nucleus. A 46-kDa amino terminal truncated product of ESR1, named ER46, localizes to the cell membrane and mediates estrogen actions that are initiated at the cell membrane.659 Another of these putative estrogen receptors has been named ER-X and is postulated to be a G-protein–coupled receptor that localizes to the cell membrane.660 Another putative receptor is the aptly named heterodimeric putative estrogen receptor (pER), which has been found on the cell and nuclear membranes.661 The pER acts as a serine phosphatase.661 Five other estrogen-binding proteins have also been

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identified, and at least three of them localize to the cell membrane.662-665 It had been thought that androgens are the main hormones responsible for closing the epiphyses during puberty. In 1994, however, it was shown from extensive studies of a 28-year-old man with incomplete epiphyseal closure and continued linear growth (in spite of otherwise normal pubertal development) that the estrogen receptor mediates epiphyseal closure.666 He was found to have a homozygous C-to-T point mutation of codon 157 of the second exon in the ESR1 gene, introducing a premature termination signal. Expression of this gene leads to the production of a nonfunctional ESR1 lacking both the DNA- and hormone-binding domains. He was also found to have increased estradiol levels, impaired glucose tolerance with hyperinsulinemia, and decreased bone density. Further strengthening the association between ESR1 and epiphyseal closure is the observation that women with ESR1-positive breast cancer and a mutation in the B domain (B’ allele) of ESR1 have an increased incidence of spontaneous abortion and tall stature.667 These associations were not found in female carriers of the allele without breast cancer or with ESR1-negative breast cancer. Thus, a second (as yet undiscovered) mutation is likely to play a role in the development of tall stature and spontaneous abortions in female carriers with ESR1-positive breast cancer.

MINERALOCORTICOID RECEPTORS Mineralocorticoid resistance is also known as pseudohypoaldosteronism (PHA). Both sporadic and familial cases with either autosomal-dominant or -recessive cases have been reported.668-672 Clinical presentation of patients with PHA ranges from asymptomatic salt wasting; to growth failure; to chronic failure to thrive, lethargy, and emesis; to life-threatening dehydration accompanied by severe salt wasting.669-671,673-675 Patients with the severe forms of PHA typically present within a year of birth and may even present in utero with polyhydramnios due to polyuria.676 Biochemically, the condition is characterized by urinary salt wasting, hyponatremia, elevated plasma potassium, aldosterone, and renin activity and urinary aldosterone metabolism that are unresponsive to the mineralocorticoid treatment.677-679 Two forms of PHA are recognized. PHA type I consists of classical PHA (due to renal tubular mineralocorticoid resistance) and PHA type II with kidney, intestine, salivary and/or sweat gland mineralocorticoid resistance.680 Unlike PHA type I, PHA type II is not due to defective mineralocorticoid function. Rather, it is due to increased chloride reabsorption by the renal tubule. Patients with these conditions present with hyperkalemia that only responds to treatment with non-chloride ions, such as bicarbonate or sulfate, which increase delivery of sodium to the distal tubule.681 A transient form of mineralocorticoid resistance probably due to abnormal maturation of aldosterone receptor function also exists.682 This variant of PHA is known as the syndrome of early-childhood hyperkalemia. Children with this disorder present with failure to thrive or linear

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55

growth failure accompanied by hyperkalemia and metabolic acidosis. This condition resolves spontaneously by the second half of the first decade of life. The cause of PHA type I is not well understood. The HUGO Nomenclature Committee refers to the mineralocorticoid receptor as the nuclear receptor subfamily 3, group C, member 2 (NR3C2) but accepts the alias MR for the mineralocorticoid receptor. MR will be used in the remainder of this chapter. MRs from patients with PHA type I exhibit decreased or absent binding to aldosterone.683 Mutations have been identified in the genes for MR in patients with PHA type I.684 However, these mutations are not thought to be solely responsible for causing this condition because they have also been found in individuals without PHA.684 Altered MR transcription does not appear to cause PHA type I because MR mRNA levels are not decreased in patients with this condition.683 Thus, it has been hypothesized that PHA type I may be due to a defective post-receptor event that may involve cofactors involved in ligand binding.677,684 An interesting syndrome involving overactivation of the MRs is the syndrome of apparent mineralocorticioid excess. Patients with this autosomal-recessive condition can exhibit pre- and postnatal growth failure, hypervolemic hypertension, medullary nephrocalcinosis, and hypokalemic metabolic alkalosis accompanied by hyporeninemic hypoaldosteronism.685-688 Patients with this syndrome may also be asymptomatic and exhibit only biochemical abnormalities.689 Patients with this syndrome also have increased serum and urinary cortisolto-cortisone ratios.688 This syndrome is caused by mutations in the 11 -hydroxysteroid dehydrogenase type 2 (11 -HSD2) genes that reduce the activity of the enzyme.687,688,690-692 11 -HSD2 converts the active glucocorticoid cortisol to inactive cortisone. Thus, decreased activity of 11 -HSD2 increases cortisol levels in MRcontaining tissues, leading to increased binding and activation of MRs by cortisol.687,688

DAX1 gene mutations have been identified that cause X-linked adrenal hypoplasia congenita.694 Patients with this condition have congenital adrenal insufficiency and are therefore deficient in glucocorticoid, mineralocorticoid, and androgen production.694,697 Gonadotropin deficiency and azoospermia also occur in these patients.697,698 Female carriers may have delayed puberty.698 All mutations that have been found to cause X-linked congenital adrenal hypoplasia are either located in or prevent transcription of the area of the E domain that inhibits SF-1mediated transcription.693,699-701 Thus, DAX1 mutations may cause X-linked congenital adrenal hypoplasia by altering SF-1 regulation of gonado- and adrenogenesis.699 DAX1 deletion may also occur in the setting of the contiguous gene deletion syndrome, resulting in complex glycerol kinase deficiency (cGKD) if individuals have deletions extending from the GK gene into the Duchene muscular dystrophy (DMD) gene and/or involving a significant extension telomeric from DAX1.702

Subfamily 0 Nuclear Receptors: DAX1

REFERENCES

Subfamily 0 nuclear receptors include DAX1.523 DAX1 plays a role in the regulation of steroid, mullerian-inhibiting substance, and gonadotropin production.

DAX1 The dosage-sensitive sex-reversal adrenal hypoplasia congenital critical region on the X chromosome gene 1 (DAX1) is an orphan nuclear receptor because its ligand has not yet been identified.693 It has homologies in the E domain to other orphan receptors, including RXRs.694 However, DAX1 has an unusual DNA-binding C domain that contains a tract of amino acid repeats instead of zinc finger motifs.694 DAX1 inhibits steroidogenic factor 1 (SF1)-mediated transcription. SF-1 is another orphan nuclear receptor that regulates transcription of adrenal and gonadal steroid hydroxylases, mullerian-inhibiting substance, and gonadotropin genes.695,696

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Summary Understanding of receptors that transduce or influence hormone action has increased dramatically during the past decade. As molecular biology techniques improve, it is expected that knowledge of receptor action will continue to increase at a rapid pace. It is likely that subtle defects in receptor function (such as regulatory or promoter region mutations that increase or decrease receptor gene expression, or mutations in second messenger proteins) will be found that cause endocrine disorders. It is also likely that new receptors will be discovered that transduce or influence hormone action and that endocrine roles will be found for receptors that were not previously thought to mediate or alter hormone action.

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syndrome. The Journal of Clinical Endocrinology and Metabolism 77:103–107. McPhaul MJ, Marcelli M, Zoppi S, Wilson CM, Griffin JE, Wilson JD (1992). Mutations in the ligand-binding domain of the androgen receptor gene cluster in two regions of the gene. The Journal of Clinical Investigation 90:2097–2101. Wooster R, Mangion J, Eeles R, Smith S, Dowsett M, Averill D, et al. (1992). A germline mutation in the androgen receptor gene in two brothers with breast cancer and Reifenstein syndrome. Nature Genetics 2:132–134. MacLean HE, Warne GL, Zajac JD (1996). Spinal and bulbar muscular atrophy: Androgen receptor dysfunction caused by a trinucleotide repeat expansion. J Neurol Sci 135:149–157. Chamberlain NL, Driver ED, Miesfeld RL (1994). The length and location of CAG trinucleotide repeats in the androgen receptor N-terminal domain affect transactivation function. Nucleic Acids Res 22:3181–3186. Stenoien DL, Cummings CJ, Adams HP, Mancini MG, Patel K, DeMartino GN, et al. (1999). Polyglutamine-expanded androgen receptors form aggregates that sequester heat shock proteins, proteasome components and SRC-1, and are suppressed by the HDJ-2 chaperone. Human Molecular Genetics 8:731–741. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, et al. (1997). Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology 138:863–870. Mosselman S, Polman J, Dijkema R (1996). ER beta: identification and characterization of a novel human estrogen receptor. FEBS Letters 392:49–53. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA (1996). Cloning of a novel receptor expressed in rat prostate and ovary. Proceedings of the National Academy of Sciences of the United States of America 93:5925–5930. McInerney EM, Tsai MJ, O’Malley BW, Katzenellenbogen BS (1996). Analysis of estrogen receptor transcriptional enhancement by a nuclear hormone receptor coactivator. Proceedings of the National Academy of Sciences of the United States of America 93:10069–10073. Pettersson K, Grandien K, Kuiper GG, Gustafsson JA (1997). Mouse estrogen receptor beta forms estrogen response elementbinding heterodimers with estrogen receptor alpha. Mol Endocrinol 11:1486–1496. Toran-Allerand CD (2004). Minireview: A plethora of estrogen receptors in the brain: where will it end? Endocrinology 145:1069–1074. Li L, Haynes MP, Bender JR (2003). Plasma membrane localization and function of the estrogen receptor alpha variant (ER46) in human endothelial cells. Proceedings of the National Academy of Sciences of the United States of America 100:4807–4812. Toran-Allerand CD, Guan X, MacLusky NJ, Horvath TL, Diano S, Singh M, et al. (2002). ER-X: a novel, plasma membrane-associated, putative estrogen receptor that is regulated during development and after ischemic brain injury. J Neurosci 22:8391–8401. Rao BR (1998). Isolation and characterization of an estrogen binding protein which may integrate the plethora of estrogenic actions in non-reproductive organs. The Journal of Steroid Biochemistry and Molecular Biology 65:3–41. Asaithambi A, Mukherjee S, Thakur MK (1997). Expression of 112-kDa estrogen receptor in mouse brain cortex and its autoregulation with age. Biochemical and Biophysical Research Communications 231:683–685. Ramirez VD, Kipp JL, Joe I (2001). Estradiol, in the CNS, targets several physiologically relevant membrane-associated proteins. Brain Res Brain Res Rev 37:141–152. Joe I, Ramirez VD (2001). Binding of estrogen and progesteroneBSA conjugates to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the effects of the free steroids on GAPDH enzyme activity: physiological implications. Steroids 66:529–538. Zheng J, Ramirez VD (1999). Purification and identification of an estrogen binding protein from rat brain: Oligomycin sensitivityconferring protein (OSCP), a subunit of mitochondrial F0F1-ATP synthase/ATPase. The Journal of Steroid Biochemistry and Molecular Biology 68:65–75. Smith EP, Boyd J, Frank GR, Takahashi H, Cohen RM, Specker B, et al. (1994). Estrogen resistance caused by a mutation in the

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estrogen-receptor gene in a man. The New England Journal of Medicine 331:1056–1061. Lehrer S, Rabin J, Stone J, Berkowitz GS (1994). Association of an estrogen receptor variant with increased height in women. Hormone and Metabolic Research 26:486–488. Kuhnle U, Nielsen MD, Tietze HU, Schroeter CH, Schlamp D, Bosson D, et al. (1990). Pseudohypoaldosteronism in eight families: Different forms of inheritance are evidence for various genetic defects. The Journal of Clinical Endocrinology and Metabolism 70:638–641. Chitayat D, Spirer Z, Ayalon D, Golander A (1985). Pseudohypoaldosteronism in a female infant and her family: Diversity of clinical expression and mode of inheritance. Acta Paediatr Scand 74:619–622. Hanukoglu A, Fried D, Gotlieb A (1978). Inheritance of pseudohypoaldosteronism. Lancet 1:1359. Limal JM, Rappaport R, Dechaux M, Morin C (1978). Familial dominant pseudohypoaldosteronism. Lancet 1:51. Bonnici F (1977). Autosomal recessive transmission of familial pseudohypoaldosteronism. Arch Fr Pediatr 34:915–916. Rosler A (1984). The natural history of salt-wasting disorders of adrenal and renal origin. The Journal of Clinical Endocrinology and Metabolism 59:689–700. Shigetomi S, Ojima M, Ueno S, Tosaki H, Kohno H, Fukuchi S (1986). Two adult familial cases of selective hypoaldosteronism due to insufficiency of conversion of corticosterone to aldosterone. Endocrinol Jpn 33:787–794. Keszler M, Sivasubramanian KN (1983). Pseudohypoaldosteronism. Am J Dis Child 137:738–740. Abramson O, Zmora E, Mazor M, Shinwell ES (1992). Pseudohypoaldosteronism in a preterm infant: Intrauterine presentation as hydramnios. The Journal of Pediatrics 120:129–132. Kuhnle U, Keller U, Armanini D, Funder J, Krozowski Z (1994). Immunofluorescence of mineralocorticoid receptors in peripheral lymphocytes: Presence of receptor-like activity in patients with the autosomal dominant form of pseudohypoaldosteronism, and its absence in the recessive form. The Journal of Steroid Biochemistry and Molecular Biology 51:267–273. Arai K, Chrousos GP (1995). Syndromes of glucocorticoid and mineralocorticoid resistance. Steroids 60:173–179. Oberfield SE, Levine LS, Carey RM, Bejar R, New MI (1979). Pseudohypoaldosteronism: Multiple target organ unresponsiveness to mineralocorticoid hormones. The Journal of Clinical Endocrinology and Metabolism 48:228–234. Hanukoglu A (1991). Type I pseudohypoaldosteronism includes two clinically and genetically distinct entities with either renal or multiple target organ defects. The Journal of Clinical Endocrinology and Metabolism 73:936–944. Schambelan M, Sebastian A, Rector FC Jr. (1981). Mineralocorticoidresistant renal hyperkalemia without salt wasting (type II pseudohypoaldosteronism): Role of increased renal chloride reabsorption. Kidney Int 19:716–727. McSherry E (1981). Renal tubular acidosis in childhood. Kidney Int 20:799–809. Komesaroff PA, Verity K, Fuller PJ (1994). Pseudohypoaldosteronism: Molecular characterization of the mineralocorticoid receptor. The Journal of Clinical Endocrinology and Metabolism 79:27–31. Arai K, Tsigos C, Suzuki Y, Irony I, Karl M, Listwak S, Chrousos GP (1994). Physiological and molecular aspects of mineralocorticoid receptor action in pseudohypoaldosteronism: a responsiveness test and therapy. The Journal of Clinical Endocrinology and Metabolism 79:1019–1023. New MI, Stoner E, DiMartino-Nardi J (1986). Apparent mineralocorticoid excess causing hypertension and hypokalemia in children. Clin Exp Hypertens [A] 8:751–772. Muller-Berghaus J, Homoki J, Michalk DV, Querfeld U (1996). Diagnosis and treatment of a child with the syndrome of apparent mineralocorticoid excess type 1. Acta Paediatr 85:111–113. Dave-Sharma S, Wilson RC, Harbison MD, Newfield R, Azar MR, Krozowski ZS, et al. (1998). Examination of genotype and phenotype relationships in 14 patients with apparent mineralocorticoid excess. The Journal of Clinical Endocrinology and Metabolism 83:2244–2254. Morineau G, Marc JM, Boudi A, Galons H, Gourmelen M, Corvol P, et al. (1999). Genetic, biochemical, and clinical studies of

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patients with A328V or R213C mutations in 11betaHSD2 causing apparent mineralocorticoid excess. Hypertension 34:435–441. Ugrasbul F, Wiens T, Rubinstein P, New MI, Wilson RC (1999). Prevalence of mild apparent mineralocorticoid excess in Mennonites. The Journal of Clinical Endocrinology and Metabolism 84:4735–4738. Monder C, Shackleton CH, Bradlow HL, New MI, Stoner E, Iohan F, et al. (1986). The syndrome of apparent mineralocorticoid excess: Its association with 11 beta-dehydrogenase and 5 beta-reductase deficiency and some consequences for corticosteroid metabolism. The Journal of Clinical Endocrinology and Metabolism 63:550–557. Mune T, Rogerson FM, Nikkila H, Agarwal AK, White PC (1995). Human hypertension caused by mutations in the kidney isozyme of 11 beta-hydroxysteroid dehydrogenase. Nature Genetics 10:394–399. Wilson RC, Harbison MD, Krozowski ZS, Funder JW, Shackleton CH, Hanauske-Abel HM, et al. (1995). Several homozygous mutations in the gene for 11 beta-hydroxysteroid dehydrogenase type 2 in patients with apparent mineralocorticoid excess. The Journal of Clinical Endocrinology and Metabolism 80:3145–3150. Bassett JH, O’Halloran DJ, Williams GR, Beardwell CG, Shalet SM, Thakker RV (1999). Novel DAX1 mutations in X-linked adrenal hypoplasia congenita and hypogonadotrophic hypogonadism. Clinical Endocrinology 50:69–75. Zanaria E, Muscatelli F, Bardoni B, Strom TM, Guioli S, Guo W, et al. (1994). An unusual member of the nuclear hormone receptor superfamily responsible for X-linked adrenal hypoplasia congenita. Nature 372:635–641. Lynch JP, Lala DS, Peluso JJ, Luo W, Parker KL, White BA (1993). Steroidogenic factor 1, an orphan nuclear receptor, regulates the expression of the rat aromatase gene in gonadal tissues. Mol Endocrinol 7:776–786.

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696. Ito M, Yu RN, Jameson JL (1998). Steroidogenic factor-1 contains a carboxy-terminal transcriptional activation domain that interacts with steroid receptor coactivator-1. Mol Endocrinol 12:290–301. 697. Muscatelli F, Strom TM, Walker AP, Zanaria E, Recan D, Meindl A, et al. (1994). Mutations in the DAX-1 gene give rise to both Xlinked adrenal hypoplasia congenita and hypogonadotropic hypogonadism. Nature 372:672–676. 698. Seminara SB, Achermann JC, Genel M, Jameson JL, Crowley WF Jr. (1999). X-linked adrenal hypoplasia congenita: A mutation in DAX1 expands the phenotypic spectrum in males and females. The Journal of Clinical Endocrinology and Metabolism 84:4501–4509. 699. Ito M, Yu R, Jameson JL (1997). DAX-1 inhibits SF-1-mediated transactivation via a carboxy-terminal domain that is deleted in adrenal hypoplasia congenita. Molecular and Cellular Biology 17:1476–1483. 700. Zhang YH, Guo W, Wagner RL, Huang BL, McCabe L, Vilain E, et al. (1998). DAX1 mutations map to putative structural domains in a deduced three-dimensional model. American Journal of Human Genetics 62:855–864. 701. Hamaguchi K, Arikawa M, Yasunaga S, Kakuma T, Fukagawa K, Yanase T, et al. (1998). Novel mutation of the DAX1 gene in a patient with X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism. American Journal of Medical Genetics 76:62–66. 702. Zhang YH, Huang BL, Niakan KK, McCabe LL, McCabe ER, Dipple KM (2004). IL1RAPL1 is associated with mental retardation in patients with complex glycerol kinase deficiency who have deletions extending telomeric of DAX1. Hum Mutat 24:273.

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C H A P T E R

3 Disorders of Bone Mineral Metabolism: Normal Homeostasis ALLEN W. ROOT, MD

Introduction Calcium Calcium-Sensing Receptor Phosphate Phosphatonins Magnesium Alkaline Phosphatase Parathyroid Hormone and ParathyroidHormone–Related Protein Parathyroid Hormone Parathyroid-Hormone–Related Protein

Introduction Calcium (Ca), phosphorus [as phosphate (HPO42-) because this substance does not exist in the free state in living tissues], and magnesium (Mg) are elements essential to the structural integrity of the body and to the function of each of its cells1 (Table 3-1). This chapter examines the genetic and physiologic mechanisms that regulate normal mineral homeostasis and bone development, composition, and strength from the prenatal period through adolescence. Table 3-2 lists some of the many genes that direct these processes. Figure 3-1 schematically depicts the factors that regulate serum concentrations of calcium and phosphate.

Calcium Calcium is an essential component of the mineral portion of bone and is necessary for the function of each of the body’s cells. Together, calcium and phosphate form the hydroxyapatite crystal [Ca10(PO4)10(OH)2] of bone. Hydroxyapatite accounts for 65% of bone weight and provides its mechanical and weight-bearing strength.

Parathyroid Hormone and ParathyroidHormone–Related Protein Receptors Calcitonin Vitamin D Vitamin D Receptor Skeleton: Cartilage and Bone Effects of Hormones and Growth Factors on the Skeleton Assessment of Bone Mass and Strength Concluding Remarks

Hydroxyapatite also serves as a reservoir for calcium that may be quickly needed for homeostatic and functional purposes. Although 99% of total body calcium is present in the slowly exchangeable deeply deposited skeletal crystal, it is the rapidly exchangeable 1% of body calcium in recently accumulated surface bone and in vascular, extracellular, and intracellular (soft tissues) spaces with which it is in equilibrium that modulates intercellular communication and intracellular signal transduction; neural transmission; cell-to-cell adhesion; clotting; striated, smooth, and cardiac muscular contraction; cardiac rhythmicity; enzyme action; synthesis and secretion of endocrine and exocrine factors; and cellular proliferation.2 Approximately 50% of total serum calcium is bound to albumin and globulin; 5% is complexed/chelated to citrate, phosphate, lactate, bicarbonate, and sulfate; and 45% is present as biologically active and closely regulated ionized Ca2
e. Serum total and ionized calcium concentrations are related to levels of albumin, creatinine, parathyroid hormone (PTH), phosphate, and serum pH. Approximately 75% of the variability in total serum calcium concentrations is accounted for by genetic factors.3 The measured Ca2
e level is dependent on the serum pH (normal adult range

74

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TA B L E 3 - 1

Abbreviations 1,25(OH)2D3

1,25-Dihydroxyvitamin D3 (calcitriol)

24R,25(OH)2D3 25OHD3 aa ADHR

24,25-Dihydroxyvitamin D3 25-Hydroxyvitamin D3 (calcidiol) Amino acid Autosomal-dominant hypophosphatemic rickets Activating function Adenosine monophosphate Adenosine triphosphate Adenosine triphosphatase Bone mineral apparent density ⬃ volumetric BMD Bone mineral content Bone mineral density Bone morphogenetic protein Bone remodeling unit Bone transmission time Calcium (ionized, extracellular) Calcium (ionized, intracellular) Calcium-sensing receptor Chloride 1,2-Diacylglycerol Vitamin D binding protein Dual energy x-ray absorptiometry Deoxyribonucleic acid Deoxypyridinoline Extracellular fluid Estrogen receptor Fibroblast growth factor Fibroblast growth factor receptor Frizzled related protein-4 Gamma () amino butyric acid Glial cell line-derived neurotrophic factor Alpha subunit of inhibitory GTP-binding protein Alpha subunit of stimulatory GTP-binding protein Alpha subunit of another stimulatory GTP-binding protein Growth hormone G-protein–coupled receptor Guanosine monophosphate Guanosine triphosphate Hydrogen ion, proton

AF AMP ATP ATPase BMAD BMC BMD BMP BRU BTT Ca2
e Ca2
i CaSR ClDAG DBP DEXA DNA Dpd ECF ER FGF FGFR FRP4 GABA GDNF Gi Gs Gq GH GPCR GMP GTP H
ICTP IGF Ihh IL IP3 K
LRP

Carboxyl terminal cross-link telopeptide of collagen type I Insulin-like growth factor (somatomedin) Indian hedgehog Interleukin Inositol-1,4,5-trisphosphate Potassium Low-density lipoprotein receptor-related protein

MAPK MARRS M-CSF MEPE Mg2
MMP Na
NFB NPT2 NTx OMIM OPG P3H1 PHEX PICP PIIINP PKA PLC PO4 PTG PTH PTHrP PTHR1 Pyr QCT QUS RANK RANKL RNA RXR SOS SOX STAT TALH TBP-2 TGF TIP TLIMP TNF TNSALP TR TRAF TRANCE TRAP TRP VDR VDRE VEGF XHR

Mitogen activated protein kinase Membrane-associated rapid response steroid binding protein (see TBP-2) Macrophage colony stimulating factor Matrix extracellular phosphoglycoprotein Magnesium Matrix metalloproteinase Sodium Nuclear factor-B Sodium/phosphate transporter 2, kidney Amino terminal cross-link telopeptide of collagen type I Online Mendelian Inheritance in Man Osteoprotegerin Prolyl 3-hydroxylase-1 Phosphate-regulating endopeptidase on the X chromosome Carboxyl terminal propeptide of collagen type I Amino terminal propeptide of collagen type III Protein kinase A Phospholipase C Phosphate (HPO42-) Parathyroid gland Parathyroid hormone PTH-related protein PTH/PTHrP receptor (PTH/PTHrP-R) Pyridinoline Quantitative computed tomography Quantitative ultrasonography Receptor activator of NFB RANK ligand Ribonucleic acid Retinoic acid X receptor Speed of sound SRY-Box Signal transduction and transcription Thick ascending loop of Henle Thioredoxin binding protein-2 (see MARRS) Transforming growth factor Tuberoinfundibular protein (hypothalamic) TBP-2-like inducible membrane protein Tumor necrosis factor Tissue nonspecific alkaline phosphatase Thyroid (hormone) receptor TNF receptor associated factor(s) TNF-related activation induced cytokine Tartrate-resistant acid phosphatase Transient receptor potential (channel) Vitamin D receptor Vitamin D response element Vascular endothelial growth factor X-linked hypophosphatemic rickets

a. A  adenine, C  cytidine, G  guanine, T  thymidne.

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TA B L E 3 - 2

Human Genes Involved in Mineral Homeostasis and Bone Metabolism Protein Aggrecan 1 Alkaline phosphatase Axis inhibitor 1 1-Catenin Bone morphogenetic protein-2 Bone morphogenetic protein-4 Bone morphogenetic protein-7 BMP Receptor 1A BMP Receptor 2 C-type natriuretic protein Calbindin 3 (9 kDa) Calcitonin Calcitonin receptor Calcium-ATPase-channel Calcium release-activated calcium modulator 1 Calcium sensing receptor Calcium transport protein-5 Calcium transport protein-1 Calcium channel, L-type, subunit 1 Calmodulin 1 Cartilage-associated protein -Catenin Cathepsin K Chloride channel 5 Collagen type I(1) Collagen type I(2) Collagen type II(1) Collagen type III(1) Collagen type IV(1) Collagen type IX(1) Collagen type X(1) Collagen type XI(1) Cyclophilin B Cytochrome P450, III A, 4 Dikkopf Disheveled 1 Distal-less 5 Fibroblast growth factor-1 Fibroblast growth factor-2 Fibroblast growth factor-5 Fibroblast growth factor-7 Fibroblast growth factor-23 Fibroblast growth factor receptor-1 Fibroblast growth factor receptor-2 Fibroblast growth factor receptor-3 Fibroblast growth factor receptor-4 Frizzled receptor Frizzled related protein-4 Glial cell missing 2 (PTG) Guanine nucleotide binding protein, alpha stimulating Hairless Heterogeneous nuclear ribonucleoprotein D Indian hedgehog Inositol trisphosphate receptor Integrin v Integrin 3 Insulin-like growth factor I Insulin-like growth factor 1 receptor Klotho

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Gene

Chromosome

OMIMa

AGC1 ALPL AXIN1 CTNNB1 BMP2 BMP4 BMP7 BMPR1A BMPR2 NPPC CABP1 CALCA CALCR ATP2B1

15q26.1 1p36.1-p34 16p13.3 3p22-p21.3 20p12 14q22-q23 20 10q22.3 2q33 2q24-qter Xp 11p15.2-p15.1 7q21.3 12q21-q23

155760 171760 603816 116806 112261 112262 112267 601299 600799 600296 302020 114130 114131 108731

CRACM1 CASR TRPV5 TRPV6 CACNA1C CALM1 CRTAP CTNNB1 CTSK CLCN5 COL1A1 COL1A2 COL2A1 COL3A1 COL4A1 COL9A1 COL10A1 COL11A1 PIPB CYP3A4 DKK1 DVL1 DLX5 FGF1 (acidic) FGF2 (basic) FGF5 FGF7 FGF23 FGFR1 FGFR2 FGFR3 FGFR4 FZD1 FRP4 GCM2

12q24 3q13.3-q21 7q35 7q33-q34 12p13.3 14q24-q31 3p22 3p22-p21.3 3q Xp11.22 17q21.31-q24 7q22.1 12q13.11-q13.2 2q31 13q34 6q13 6q21-q22.3 1p21 15 7q22.1 10q11.2 1p36 7q22 5q31 4q25-q27 4q21 15q15-q21.1 12p13.3 8p11.2-p11.1 10q25.3-q26 4p16.3 5q35→qter 7q21 7p14-p13 6p24.2

610277 601199 606679 606680 114205 114180 605497 116806 603959 300008 120150 120160 120140 120180 120130 120210 120110 120280 123841 124010 605189 601365 600028 131220 176943 165190 148180 605380 136350 176943 134934 134935 603408 606570 603716

GNAS1 HR HNRPD IHH ITPR1 ITGAV ITGB3 IGF1 IGF1R KL

20q13.2 8p21.1 4q21.1-q21.2 2q33-q35 3p26-p25 2q31 17q21.32 12q22-q24.1 15p25-q26 13q12

139320 602302 601324 600726 147265 193210 173470 147440 147370 604824

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TA B L E 3 - 2

Human Genes Involved in Mineral Homeostasis and Bone Metabolism (Continued) Protein Low density lipoprotein receptor-related protein 5 Low density lipoprotein receptor-related protein 6 Macrophage-colony stimulating factor M-CSF receptor Matrix extracellular phosphoglycoprotein Matrix GLA-protein Mitochondrial RNA-processing endoribonuclease Naturetic peptide precursor C Naturetic peptide receptor B Nuclear factor-B, Subunit 1 Nuclear factor-B, Inhibitor Osteocalcin Osteonectin Osteopontin Osteoprotegerin Osterix Paracellin-1 (Claudin 16) Parathyroid hormone Peroxisome proliferator-activated receptor  PTH related protein PTH (PTH/PTHrP) receptor 1 PTH receptor 2 Patched 1 Phosphate-regulating gene with homologies to endopeptidases on the X chromosome Pregnane X receptor Prolyl 3-hydroxylase 1 Receptor activator of NF-B (RANK) RANK-Ligand Retinoid X receptor  Runt-related transcription factor 2 Sclerostin Sirtuin 1 Smoothened Sodium calcium exchanger Sodium phosphate cotransporter Solute carrier family 34, member 1 Sodium phosphate cotransporter Solute carrier family 34, member 2 Sodium phosphate cotransporter Solute carrier family 34, member 3 SRY-box 9 T-cell factor/ lymphoid enhancement factor Transmembrane protein 142A 25-Hydroxylase 25OHD-1 hydroxylase 25OHD-24 hydroxylase Tuberoinfundular peptide 39 Vascular endothelial growth factor Vitamin D binding protein Vitamin D receptor Voltage-gated calcium channel 1 Wingless 1 Wingless 9A

Gene

Chromosome

OMIMa

LRP5

11q13.4

603506

LRP6 CSF1 CSF 1R MEPE MGP

12p13.3-p11.2 1p21-p13 5q33.2-q33.3 4q21.1 12p13.1-p13.2

603507 120420 164770 605912 154870

RMRP NPPC NPR2 NFB1 NFKB1A BGLAP SPARC SPP1 TNFRSF11B SP7 CLDN16 PTH

9p21-p12 2q24-qter 9p21-p12 4q23-q24 14q13 1q25-q31 5q31.3-q32 4q21-q25 8q24 12q13.13 3q27 11p15.3-p15.1

157660 600296 108961 164011 164008 112260 182120 166490 602643 606633 603959 168450

PPARG PTHLH PTHR1 PTHR2 PTCH1

3p25 12p12.2-p11.2 3p22-p21.1 2q33 9q22.3

601487 168470 168468 601469 601309

PHEX NR112 LEPRE1 TNFRSF11A TNFSF11 RXRA RUNX2 SOST SIRT1 SMOH SLC8A1

Xp22.1 3q13-q21 1p34 18q22.1 13q14 9q34.3 6p21 17q12-q21 10 7q31-q32 2p23-p22

307800 603065 610339 603499 602642 180245 600211 605740 604479 601500 182305

SLC34A1

5q35

182309

SLC34A2

4p15.3 1-p15.2

604217

SLC34A3 SOX9

9q34 17q24.3-q25.1

609826 608160

LEF1 TMEM142A CYP2R1 CYP27B1

4q23-q25 11p15.2 12q13.1-q13.3

153245 610277 608713 609506

CYP24A1 TIP39 VEGF GC VDR CACNA1C WNT1 WNT9A

20q13.3-q13.3 19q13.33 6p12 4q12 12q12-q14 12p13.3 12q12-q13 1q42

126065 608386 192240 139200 601769 114205 164820 602863

Source: www3.ncbi.nlm.nih.gov/htbin-post/Omim. a. OMIM = Online Mendelian Inheritance in Man.

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Phosphatonin–FGF23

Kidney

Low Ca2+ in blood

Low PO43in blood

PTH


1OHase 25(OH)D3

1,25(OH)2D3(1,25D)

 Ca2+ PO43-

FGF23

 Reabsorption Blood Ca PO4

·

Absorption

Osteoblast Mineralization

1,25D VDR

Resorption PO43-

Ca2+

1,25D VDR

Ca2+ Osteoclast

PO43Small intestine

Blood

Figure 3-1. Regulation of calcium and phosphate homeostasis. Calcium is absorbed from the intestinal tract, kidney tubule, and bone in response to calcitriol [1,25(OH)2D3] and parathyroid hormone (PTH). Calcitonin inhibits resorption of calcium from bone. The Ca2
-sensing receptor modulates the activity of the parathyroid glands and the renal tubules. Hypocalcemia and hypophosphatemia enhance renal tubular generation of calcitriol and absorption of intestinal phosphate. PTH inhibits renal tubular reabsorption of phosphate. Fibroblast growth factor-23 (FGF23), a phosphatonin secreted by osteoblasts, inhibits renal tubular reabsorption of phosphate and synthesis of calcitriol. [Reproduced with permission from Kolek OI, et al. (2005). 1,25-Dihydroxyvitamin D3 upregulates FGF23 gene expression in bone: The final link in a renal-gastrointestinal-skeletal axis that controls phosphate transport. Am J Physiol Gastrointest Liver Physiol 289:G1036–G1042.]

1.15-1.35 mmol/L at pH 7.4). An increase in alkalinity (higher pH) raises calcium binding to albumin, thus decreasing Ca2
e—whereas acidic changes (lower pH) decrease binding and thus increase Ca2
e. The relationship between pH and Ca2
e is best described by an inversely S-shaped third-degree function.4 The serum concentration of Ca2
e is maintained within narrow limits by an integrated system involving the plasma membrane Ca2
e sensing receptor (CaSR), PTH and its receptor [PTH and PTH-related protein (PTHrP)-R], the thyroidal parafollicular C-cell product calcitonin and its receptor, and the vitamin D hormone system acting upon the intestinal tract, bone, and kidney5 (Figure 3-1). With increase in serum Ca2
e concentration, the CaSR on the chief cell of the parathyroid gland (PTG) is activated, which depresses PTH secretion instantly—whereas the CaSR in the distal renal tubule is activated, decreasing reabsorption of calcium filtered through the glomerulus and increasing urinary calcium excretion. When the serum Ca2
e concentration falls, signaling through the CaSR also declines—thereby increasing PTH secretion and renal tubular reabsorption of filtered calcium, activating osteoclastic bone reabsorption, and somewhat later increasing synthesis of 1,25 dihydroxyvitamin D3 (calcitriol) and intestinal absorption of ingested calcium.1

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The intracellular concentration of cytosolic free Ca2
i is 10,000-fold less than that in serum and extracellular fluid, a gradient maintained by extrusion of the cation through energy-dependent sodium-calcium exchangers.2 Within the cell, Ca2
i is primarily (99%) within mitochondria, associated with the endoplasmic reticulum, or bound to the inner plasma membrane—from which sites it can be released by chemical signals [e.g., inositol-1,4,5trisphosphate (IP3)]. IP3 acts upon IP3 receptors (encoded by ITPR1; see Table 3-2 for gene locus and OMIM site) located in the membrane of the endoplasmic reticulum to effect rapid egress of Ca2
from storage and thereby quickly increase Ca2
i levels.6 Ca2
i serves as a second-messenger signal transducer that controls many cellular activities, including cell movement, secretion of synthesized products, transcription, and cell division and growth. Ca2
enters the cell through transmembrane protein “pores,” such as voltage-gated calcium channels that “open” in response to depolarization of the plasma membrane—permitting rapid influx of Ca2
into the cell cytoplasm and leading to further depolarization of the membrane and activation of cell function.7,8 These channels are widely distributed in the plasma membranes of all cells (e.g., neurons; cardiac, skeletal, and smooth muscle; endocrine glands; gastric mucosa; white

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blood cells; and platelets) and in cytoplasmic organelles such as mitochondria and the endoplasmic reticulum. They may be activated by high or low electrical voltage. The skeletal muscle high-voltage–activated calcium channel consists of five subunits: 1, 2, , , and . The voltage-sensing pore-forming Ca2
-binding 1 subunit has four repeated domains, each with six transmembranespanning regions (or helixes) and intracytoplasmic amino and carboxyl terminals. Transmembrane helix 4 serves as the voltage sensor. The 1 subunit also has a sequence of amino acids between transmembrane helixes 5 and 6 that is partially inserted into the membrane to serve as a “selectivity filter.”8 The assembly, intracellular movement, interaction with other proteins, activation, and kinetic properties of the 1 subunit are modified by the extracellular glycosylated 2 subunit, the  subunit (a cytoplasmic globular protein), a small membrane-spanning subunit that is disulfide linked to form dimeric 2 , and a second transmembrane subunit .7-9 (The subunit structure of the low-voltage–activated voltage-gated calcium channels is as yet unknown.) The voltage-gated calcium channels are currently designated in accord with their cloned specific 1 subunits and have been termed Cav 1.1, Cav 1.2 (CACNA1C), and Cav 1.3 to Cav 3.3. Formerly, they were designated in accord with the high- or low-voltage strength required for activation and for their sensitivity to specific inhibitors (e.g., L, N, P, Q, R, and T subtypes).7,8,10,11 The high-voltage–dependent L-type calcium channels (Cav 1.1 through Cav 1.4) are present in skeletal, cardiac, and vascular smooth muscle; endocrine cells; neurons; and fibroblasts. L-type calcium channels are activated by the guanine triphosphate (GTP)-binding q subunit of Gq proteins through stimulation of phospholipase C (PLC), leading to phosphorylation of the channel protein. L-type calcium channels modulate the growth and proliferation of fibroblasts and smooth muscle cells, the synthesis of extracellular matrix collagen proteins, and the activation of specific transcription factors.9 In addition to traversing rapidly across the cell’s plasma membrane through voltage-gated channels, Ca2
enters the cytosol but at a markedly slower rate through bifunctional membrane-associated IP3 receptors that also serve as calcium channels.6,12 Encoded by ITPR1, the tetrameric IP3 receptor has six transmembrane domains and a poreforming loop between the fifth and sixth transmembrane segments. Whereas many IP3 receptors/channels are expressed in the endoplasmic reticulum, only one to two such channels are expressed in the plasma membrane of each cell. A third site of Ca2
entry into the cytosol is through a plasma membrane protein important to “storeoperated” Ca2
entry termed CRACM1 [or TMEM142A (transmembrane protein 142A) or ORAI1].6 Store-operated Ca2
entry is triggered after Ca2
release from storage sites has depleted intracellular stores of Ca2
i and is mediated by CRACM1, a protein with four transmembrane-spanning domains. Multiple copies of CRACM1 are expressed in each cell, permitting large amounts of Ca2
to cross the plasma membrane through this route.13 Loss-of-function mutations in ORAI1 have been identified in patients with severe combined immune deficiency.14 Ca2
is translocated not only through

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79

specific Ca2
channels but through paracellular transport channels.15 Ca2
i is extruded from cell cytoplasm by calcium and energy-dependent adenosine triphosphate (ATP)-driven calcium pumps and in exchange for sodium (Na
) through H
-ATPase and Na
-Ca2
exchangers.1,2 There are many intracellular calcium-binding proteins (e.g., calcium-binding proteins 1 and 4, calbindin, sorcin, calmodulin, and so forth). Calmodulin 1 (CALM1) is a widely distributed 149-aa protein with an amino terminal “lobe” linked to a carboxyl terminal lobe that can assume more than 30 three-dimensional conformations. It is a member of a large family of calcium-modulated proteins.8 Each lobe has two calcium-binding motifs. Calcium binding exposes hydrophobic “pockets” that allow calmodulin to bind to and regulate the activity of target proteins. While bound to a target protein, calmodulin can assume any of an exceedingly large number of conformations. This versatility enables calmodulin to act as a calcium sensor for many different proteins subserving distinct processes within a single cell, including the voltage-gated calcium channels and calcium/calmodulin-dependent protein kinases. Gastrointestinal absorption of calcium is primarily an active and saturable process (stimulated by calcitriol) that regulates the availability of transmembrane calcium “pumps” and channels on the luminal and basolateral surface membranes of duodenal and jejunal enterocytes.1 The ileum and colon are also able to absorb calcium when dietary intake is low or demand increases. Calcitriol acting through the vitamin D receptor (VDR) increases duodenal expression of calcium transport protein-1 (epithelial calcium channel 2 encoded by TRPV6-transient receptor potential-vanilloid 6), a luminal calcium channel with six transmembrane domains and intracytoplasmic amino and carboxyl terminals that form a homotetramer or heterotetramer combined with TRPV5. [There are six subfamilies of the transient receptor potential (TRP) family of ion channels, many of which are permeable to calcium.16] After entering the enterocyte, calcium traverses its interior within the cytosol or within a lysosomal vesical bound to calbindin9kd. After fusion of the vesicle with the basolateral plasma membrane, Ca2
is extruded. Ca2
is also released into the circulation through a basolateral Na
-Ca2
exchanger (SLC8A1) or a Ca2
-Mg2
dependent ATPase calcium channel (ATPB21). When present in high amounts, luminal calcium may also be absorbed by diffusion along paracellular channels. PTH indirectly increases intestinal calcium absorption by enhancing renal 25-hydroxyvitamin D-1 hydroxylase activity and calcitriol synthesis. Growth hormone (GH) and estrogens also increase intestinal absorption of calcium. Glucocorticoids and thyroid hormone inhibit this process. Intestinal calcium absorption is increased during adolescence, pregnancy, and lactation—and is depressed in patients with nutritional or functional vitamin D deficiency, chronic renal disease, and hypoparathyroidism. Calcium is excreted into the intestinal tract in the ileum and in pancreatic and biliary secretions. The amount of calcium ingested influences the net amount of calcium absorbed. The lower the calcium intake the greater the efficiency of its absorption. In the adult, when the dietary

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DISORDERS OF CALCIUM METABOLISM

calcium intake is 200 mg/day fecal calcium excretion exceeds intake. Thus, active absorption of calcium cannot compensate for very low intake. As dietary calcium increases from 200 to 1,000 mg/day, active calcium absorption increases but at a progressively decreasing rate. When dietary calcium exceeds 1,000 mg/day, active calcium absorption remains relatively constant at 300 mg/ day but passive absorption of calcium through paracellular channels continues to increase.1 Thus, hypercalcemia may result from dietary calcium excess (as in the milk-alkali syndrome). The availability of calcium for bone mineralization and cellular function is determined by its intake, absorption, excretion, and turnover. Intestinal calcium absorption is influenced by vitamin D status, its food source (the bioavailability of calcium in cow milk formulas is 38%, that in human breast milk is 58%; leafy green vegetables are also a good source of dietary calcium), the form of the calcium salt in supplements, and the presence in food of inhibitors of calcium absorption such as phytates, oxalates, or phosphates (e.g., cola beverages).17 It is currently recommended that infants (including those who are breast fed), children, and adolescents receive a minimum of 200 IU (5.0 g) of cholecalciferol (vitamin D3) daily, but this amount may be too low (vide infra).18,19 Low calcium intake appears to be associated with increased fracture rate in children and adolescents, and therefore adequate dietary intake of calcium during infancy, childhood, and particularly adolescence has been considered necessary to attain a peak bone mass that may lessen the risk of fracture and the later development of osteopenia.20 In an effort to ensure optimal mineralization of the developing skeleton, age-related dietary intakes of elemental calcium for infants, children, and adolescents have been recommended (Table 3-3).20 However, critical review of these recommendations suggests that after basal levels of calcium intake have been achieved ( 500 mg/day in children and adolescents) increased calcium intake in dairy foods or supplements has only transient effects on indices of bone mineralization and no documented positive long-term effects on fracture rate or

TA B L E 3 - 3

Recommended Dietary Calcium Intake in Infants, Children, and Adolescents Age (Years)

0-0.5 0.5-1 1-3 4-8 4-5 6-10 9-18 11-18

Dietary Intake (mg/day) NAS

NIH

210 270 500 800

400 600 800 800 1,000

1,300 1,350

From American Academy of Pediatrics (1999). Calcium requirements of infants, children, and adolescents. Pediatrics 104:1152–1157. NAS  National Academy of Science (1997 data), NIH  National Institutes of Health (1994 data).

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future risk of osteoporosis.17 Nevertheless, adherence to these guidelines has been strongly advocated by the American Academy of Pediatrics.20 In addition to dietary intake of calcium and vitamin D status, the most significant modifiable determinant of bone mineralization is weight-bearing physical activity (vide infra).21 Calcium is primarily excreted by the kidney. Ultrafiltrable serum calcium (both Ca2
e and that complexed or chelated) crosses the renal glomerular membrane. Ninety-eight percent of filtered calcium is reabsorbed by the renal tubule [70% in the proximal renal tubule primarily by a paracellular mechanism, 20% in the thick ascending loop of Henle (TALH) by the paracellular route through generation of a lumen-positive voltage differential by a sodium-potassiumchloride (Na
K
-2Cl-) transporter, and 8% in the distal convoluted tubule by active transcellular transport regulated by PTH and calcitriol].1,22 In the TALH, calcium and magnesium are reabsorbed through voltage-driven paracellular channels (in part through paracellin-1, a tight-junction protein channel instrumental in renal reabsorption of filtered calcium and magnesium). Cells of the TALH also express the CaSR on their basolateral membrane. When activated by peritubular Ca2
, this G-protein–coupled receptor (GPCR) decreases renal tubular reabsorption of calcium through the paracellular channels by inhibiting activity of the Na
-K
-2Cl- transporter and lowering lumen-positive voltage. In active transcellular calcium reabsorption, Ca2
crosses the luminal or apical surface of the renal distal convoluted tubular cell from the tubular lumen through calcium transport protein-5 (renal epithelial calcium channel 1 encoded by TRPV5)—whose expression is increased by calcitriol, estradiol, and a low-calcium diet. The multifunctional -glucuronidase transmembrane protein klotho (KL) is able to de-glycosylate TRPV5, thereby trapping it in the renal tubular cell membrane and prolonging its activity.23 PTH increases calcium reabsorption in the distal tubule by increasing chloride efflux from the basolateral membrane of the distal renal tubular cell, thus increasing the transmembrane voltage gradient.2 Calcium exits the basolateral serosal surface of the renal tubular cell against a chemical gradient in exchange for Na
through the Na
Ca2
exchanger with the aid of Mg2
-dependent Ca2
ATPase. Increased glomerular filtration and/or decreased renal tubular reabsorption increase renal excretion of calcium, phosphate, and magnesium. Urinary excretion of calcium is augmented by increased dietary intake, hypercalcemia of diverse pathophysiology (with the exception of that associated with familial hypocalciuric hypercalcemia), expansion of extracellular volume, metabolic acidosis, and loop diuretics (furosemide) (Table 3-4).1 PTH and PTHrP increase renal tubular reabsorption of Ca2
, whereas glucocorticoids, mineralocorticoids, and Ca2
itself suppress its reabsorption. In utero, fetal serum calcium concentrations are quite high (12-13 mg/dL), because calcium is transported across the placenta against a chemical gradient—likely under the influence of placentally synthesized calcitriol. The high calcium levels in umbilical cord blood (12 mg/ dL) decline rapidly postnatally to a nadir of 9 mg/dL between 24 and 48 hours of age, increase to approximately 10 mg/dL, stabilize, and then decline marginally

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81

TA B L E 3 - 4

Factors Affecting Renal Excretion of Calcium, Phosphate, and Magnesium in Normal Subjects (Sites of Action) Calcium Glomerular Filtration Increased Decreased

Hypercalcemia Hypocalcemia Renal insufficiency

Renal Tubular Reabsorption Increased ECF volume depletion Hypocalcemia Phosphate administration Thiazide diuretics Metabolic alkalosis PTH PTHrP Calcitriol Decreased ECF volume expansion Hypercalcemia Phosphate deprivation Metabolic acidosis Loop diuretics Cyclosporin A

Phosphate

Magnesium

Hyperphosphatemia Mild hypocalcemia Hypophosphatemia Renal insufficiency Moderate hypercalcemia

Hypermagnesemia

ECF volume depletion Hypercalcemia Phosphate deprivation Chronic metabolic alkalosis

ECF volume depletion Hypocalcemia Hypomagnesemia PTH Metabolic alkalosis

PTH/PTHrP Hypocalcemia Phosphate excess Metabolic alkalosis Thiazide diuretics FGF23 Calcitriol

ECF volume expansion Hypercalcemia Phosphate depletion Hypermagnesemia Loop diuretics Cyclosporin A Cisplantin Ethanol

Hypomagnesemia Renal insufficiency

Adapted from Favus MJ, Bushinsky DA, Lemann J Jr. (2006). Regulation of calcium, magnesium, and phosphate metabollism. In Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 76–83. ECF  extracellular fluid.

over the next 18 months. In children and adolescents, serum calcium levels are slightly higher than in adults (8.5–10.5 mg/dL). In preterm infants or full-term ill infants, hypocalcemia is often present because the PTH secretory response to hypocalcemia is blunted and calcitonin secretion exaggerated and prolonged.

CALCIUM-SENSING RECEPTOR Plasma Ca2
e concentrations are detected by the CaSR, a 1,079-aa cell membrane protein with seven transmembrane domains whose extracellular domain binds Ca2
, Mg2
, and specific aromatic amino acids.24 Through its binding of Ca2
e, the CaSR finely regulates the concentration of this anion by modulating the secretion of PTH and the renal tubular reabsorption of calcium. The CaSR is encoded by CASR and is a member of the C family of GPCRs with extremely long extracellular domains (500- to 600-aa residues) and homologies with receptors that bind glutamate, -amino butyric acid, and pheromones.25,26 The very long extracellular domain is heavily glycosylated, a post-translational modification essential to efficient movement of the receptor to the cell surface. This domain forms “pockets” into which the ligand binds. Interestingly, this GPCR is biologically active in the dimeric form linked by cysteine residue numbers 129 and 131.24 Homodimerization of the CaSR takes place in the endoplasmic reticulum after core N-linked glycosylation.

Ch03_074-126-X4090.indd 81

Homodimeric CaSR is then packaged within the Golgi apparatus, where it is further glycosylated and then transported to the cell membrane.27 The CaSR is expressed on the plasma membrane of parathyroid chief cells, at the apical or basolateral membranes of most renal tubular cells (particularly the TALH and the collecting ducts), on the cell membranes of the parafollicular (C) cells of the thyroid, in cartilage, and in bone, lungs, adrenals, breast, intestines, skin, lens, placental cytotrophoblasts, and nervous tissue.26,28 The serum Ca2
e concentration is related to polymorphic variants of the CaSR. Seventy percent of individuals are homozygous for alanine at aa position 986 within the intracellular domain, 3% are homozygous for serine, and the remainder are heterozygous for the two aa. In heterozygous Arg986Ser and homozygous Ser986 subjects, Ca2
e concentrations are significantly higher than in those homozygous for Ala986.29,30 In addition, there are cellular Ca2
sensors unrelated structurally to the CaSR or to subtypes of the CaSR itself.26 The CaSR also serves as a magnesium sensor and perhaps as a moderator of nutrient availability. By binding to the CaSR, aromatic L-amino acids appear to “sensitize” the receptor to a given level of Ca2
e.24 The CaSR also modulates renal tubular reabsorption of magnesium, decreasing its reabsorption when serum cation values rise. Although in the kidney the CaSR is expressed in greatest abundance in the basolateral (plasma) membranes of

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DISORDERS OF CALCIUM METABOLISM

the medullary and cortical TALH, it is also found in glomerular cells and other segments of the renal tubule.26 There too, increasing peritubular concentrations of Ca2
e and Mg2
e inhibit renal tubular reabsorption of filtered Ca2
. Acting through the CaSR, rising serum Ca2
e concentrations stimulate release of calcitonin from thyroid C cells. The CaSR is expressed throughout the (rat) intestinal tract, where it may modulate the changes in intestinal motility that accompany low (increased) and high (depressed) serum Ca2
e values. Cell membrane CaSRs are present in (mouse, rat, bovine) articular and hypertrophic chondrocytes of the epiphyseal growth plate and to a lesser extent in proliferating and maturing chondrocytes.31 Osteoblasts, osteocytes, and osteoclasts also express the CaSR—and agonist (Ca2
, neomycin, gadolinium) activation of the CaSR stimulates intracellular signal transduction.31 The CaSR may mediate recruitment of osteoblast precursor cells to sites of high Ca2
levels, the residue of local osteoclast activity, thus linking the boneremodeling processes of resorption and formation.20 In vitro Ca2
inhibits bone reabsorptive activity of (rabbit) osteoclasts by causing the osteoclast to decrease secretion of acid and catabolic enzymes and to withdraw from the site of bone resorption.32 Through its intracellular carboxyl terminal, the CaSR is linked to G proteins that couple the ligand-receptor message to intracellular signal transduction pathways.24,26,33,34 After binding of Ca2
to the extracellular domain of the CaSR, Gq/11 dissociates from its  subunit complex and activates membrane-bound PLC-1. In turn, this enzyme hydrolyzes membrane-bound phosphatidylinositol 4,5-bisphosphate to diacylglycerol and IP3—the latter leading to increased cytosolic concentrations of Ca2
i. Activation of the CaSR also stimulates activity of phospholipases A2 and D and mitogen-activated protein kinases (MAPK) but inhibits that of adenylyl cyclase, the latter through stimulation of adenylate-cyclase–inhibitory Gi activity.24 Within the PTG, increases in plasma Ca2
e and consequently cytosolic Ca2
i concentrations suppress expression, synthesis, and secretion of PTH and inhibit proliferation of chief cells. Decline in Ca2
e leads to increased PTH secretion, thus enabling the CaSR to exercise minute-to-minute control over the release of PTH and hence of the Ca2
e. In the kidney, binding of Ca2
e to the CaSR decreases not only transcellular transport of filtered Ca2
but its paracellular transport in the TALH.28 Inactivating mutations in CASR result in familial hypocalciuric hypercalcemia, whereas gain-of-function mutations in this gene are associated with autosomal-dominant hypoparathyroidism (“familial hypercalciuric hypocalcemia”).24 The Ca2
/CaSR complex inhibits antidiuretic hormoneinduced renal tubular permeability to water by decreasing the number of apical water channels in the inner medullary collecting ducts, thus leading to polyuria. The CaSR also binds Mg2
and regulates its urinary excretion in a manner similar to that of Ca2
. The CaSR is widely expressed throughout the gastrointestinal tract, where it may regulate secretion of gastrin and gastric acid, intestinal motility, and nutrient absorption.35 Expression of

Ch03_074-126-X4090.indd 82

the CaSR in the brain suggests a mechanism whereby Ca2
may influence neural function locally by modulating neurotransmitter and neuroreceptor function (such as the metabotropic glutamate receptor, which also recognizes Ca2
). Calcimimetics are agonistic drugs that activate the CaSR. Calcilytics are antagonists of the CaSR.35 As noted, the CaSR binds not only Ca2
e but Mg2
e, selected L-amino acids, and some antibiotics. The latter are designated type I calcimimetics. Synthetic compounds that bind to the transmembrane domains of the CaSR and increase the sensitivity of the CaSR to ambient Ca2
e are designated type II calcimimetics. The most widely employed of the calcimimetics is cinacalcet [N-[1-(R)-(-)-(1-naphthyl)ethyl]3-[3-(trifluoromethyl)phenyl]-1-aminopropane hydrochloride], which has been effective in decreasing secretion of PTH in patients with primary or secondary hyperparathyroidism.36 Calcilytics decrease the sensitivity of the CaSR to Ca2
e and thus increase the secretion of PTH and depress renal tubular reabsorption of Ca2
. These compounds remain investigational at present but might in the future be useful for the treatment of some forms of metabolic bone disease.

Phosphate Eighty-five percent of body phosphate is deposited in bone as hydroxyapatite. The remainder is intracellular (in the cytosol or mitochondria in the form of inorganic phosphate esters or salts, membrane phospholipids, and phosphorylated metabolic intermediate compounds) or in interstitial fluid or serum (0.1%), where it circulates as free orthophosphate anions HPO42- and H2PO4- (55%), bound to proteins (10%), or complexed to calcium, magnesium, or sodium (35%).1,22 At pH 7.4, serum HPO42- and H2PO4- are present in a molar ratio of 4:1. In alkalotic states, the ratio increases—and with acidosis it declines. (At pH 7.4, 1 mmol/L of orthophosphate  1.8 mEq/L  3.1 mg/dL.) Intracellularly, cytosolic free phosphate concentrations approximate those in serum (i.e., 3–6 mg/dL). Phosphate is an integral and absolutely essential component of cellular and intracellular membrane phospholipids, ribonucleic (RNA) and deoxyribonucleic (DNA) acids, energy-generating ATP, and intracellular signal transduction systems.2,37 The serum phosphate concentration is regulated by intake, intestinal absorption, excretion, and renal tubular reabsorptive mechanisms and fluctuates with age, gender, growth rate, diet, and serum calcium levels.1,38 Inasmuch as phosphate is found in all cells and foods, dietary deficiency is unusual. Dietary phosphate is absorbed across the intestinal brush border as HPO42 in direct proportion to its intake, principally in the duodenum and jejunum. It is absorbed by both passive paracellular diffusion related to the luminal concentration of this anion and by an active transcellular mechanism stimulated by calcitriol. The latter is an energy-requiring transport process with sodium through a Na
-HPO42- cotransporter protein (SLC34A2, solute carrier family 34, member 2) maintained by a calcitriol-dependent Na
, K
-ATPase. Phosphate is also secreted into the intestinal tract. When dietary

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DISORDERS OF CALCIUM METABOLISM

phosphate intake falls below 310 mg/day in the adult, net phosphate absorption is negative. At low phosphate intakes, absorption is active in the duodenum, jejunum, and distal ileum—whereas at high intakes 60% to 80% of ingested phosphate is absorbed primarily by diffusion. Phosphate absorption can be impaired by its intraluminal precipitation as an aluminum or calcium salt and by intestinal malabsorption disorders. Phosphate is filtered in the renal glomerulus and reabsorbed in the proximal (85%) and distal convoluted tubules. It is actively transported across the luminal membrane against an electrochemical gradient through specific Na
-HPO42- cotransporter proteins with the aid of Na
, K
ATPase.1,22 Expression of the renal Na
-HPO42- cotransporter protein (SLC34A1) is regulated by serum phosphate levels (hypophosphatemia increases expression), PTH, PTHrP, and fibroblast growth factor (FGF)-23. Phosphate exits the renal tubular cell with sodium through cation exchange for potassium. The maximal tubular reabsorption of phosphate approximates the filtered load. Tubular phosphate reabsorption is increased by low phosphate intake and hypophosphatemia (due to decrease in filtered load), hypercalcemia (by decrease in the glomerular filtration rate), depletion of extracellular fluid volume, and metabolic alkalosis (Table 3-4). Renal tubular reabsorption of phosphate is depressed by high phosphate intake and by PTH- and PTHrP-mediated down-regulation of SLC34A1. Calcitriol, glucocorticoids, and thiazide diuretics decrease renal tubular reabsorption of phosphate. Phosphate is deposited in bone as hydroxyapatite dependent on local levels of calcium, phosphate, and alkaline phosphatase activity and is reabsorbed by osteoclasts whose activity is stimulated by PTH, calcitriol, and other osteoclast-activating factors. Serum concentrations of phosphate are highest in infancy and early childhood (4–7 mg/dL) and then decline during mid-childhood and adolescence to adult values (2.5–4.5 mg/dL).

PHOSPHATONINS Renal tubular reabsorption of phosphate is regulated by several substances collectively termed phosphatonins (Figure 3-1). Phosphaturic agents have been identified in the serum of normal subjects and in patients with X-linked hypophosphatemic rickets (XHR), which is due to loss-of-function mutations in the membrane-bound 749-aa endopeptidase encoded by PHEX (phosphateregulating gene with homologies to endopeptidases located on the X chromosome). They have also been identified in patients with autosomal-dominant hypophosphatemic rickets (ADHR-OMIM 193100), due to activating mutations in FGF23, and in patients with tumor-induced osteomalacia in which increased production of FGF23 and other phosphaturic agents has been identified.38,39 By inhibition of phosphate transport in the kidney, phosphatonins lead to hyperphosphaturia and hypophosphatemia. They also inhibit activity of 25-hydroxyvitamin D-1 hydroxylase, resulting in decreased synthesis and thus inappropriately normal or low serum concentrations of calcitriol and in impaired intestinal absorption of phosphate.40 Among the best characterized of the phosphato-

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83

nins is FGF23, generated as a 251-aa with a 24-aa signal sequence that is post-translationally glycosylated. It is expressed primarily by osteoblasts and osteocytes and to a lesser extent by the brain, thyroid, PTG, thymus, cardiac/skeletal muscle, liver, and intestines. In osteoblasts, expression of FGF23 is enhanced by calcitriol acting through the VDR and modified by a chondrocytederived secreted factor yet to be characterized.41,42 FGF23 induces renal phosphate wasting by downregulating expression of SLC34A1, the Na
-HPO42- cotransporter expressed in the apical or luminal membrane of the proximal renal tubule. It also down-regulates expression of a related renal tubular Na
-HPO42- cotransporter encoded by SLC34A3, and of 25-hydroxyvitamin D-1 hydroxylase encoded by CYP27B1. FGF23 acts through binding to the c isoform of tyrosine kinase FGF receptors (FGFR) 1, 2, and 3. The genes encoding the FGFRs consist of 19 exons that may be alternatively spliced to include or to exclude exon 8 or exon 9 (encoding the third extracellular immunoglobulin-like domain of the FGF receptor, which helps to specify the ligand bound by the receptor). When exon 8 is included in the mRNA transcript, the b isoform of the FGFR is formed. When exon 9 is included in the transcript, the c isoform is produced. Although it is likely that FGF23 binds to multiple FGFR c isoforms, the multifunctional protein klotho has been reported to convert FGFR1(IIIc) into a specific FGF23 receptor in renal tissue.38,43 Highly sulfated glycosaminoglycans facilitate ligand-receptor interaction. FGF23 is measurable in normal adult sera with a mean concentration of 29 pg/mL that does not correlate with age or gender. Its concentration is inversely related to that of phosphate, and values rise when dietary phosphate increases and decline with phosphate restriction.38 Serum values of FGF23 are increased in patients with XHR, ADHR, tumor-induced osteomalacia, and fibrous dysplasia associated with hypophosphatemia. In patients with ADHR, gain-of-function mutations (e.g., Arg179Trp) in FGF23 result in resistance to degradation of the protein that is normally cleaved between Arg179 and Ser180. In subjects with tumor-induced osteomalacia, the production of FGF23 is greatly increased. Serum FGF23 concentrations are also elevated in the Hyp mouse model of XHR. FGF23 may be a substrate for PHEX enzymatic activity, but it is unclear if it is the endogenous substrate for this enzyme.40,44 In the Hyp mouse, biallelic “knockout” of Fgf23 reverses the hypophosphatemia and relative calcitriol deficiency—suggesting that FGF23 is of fundamental pathogenic importance in XHR. In familial tumoral calcinosis (OMIM 211900), loss-of-function mutations in FGF23 lead to accelerated intracellular degradation of FGF23 that prevents secretion of intact protein— resulting in decreased renal excretion of phosphate, in hyperphosphatemia, in relatively increased calcitriol levels, and in diffuse ectopic calcification.38,45 Tumors associated with hypophosphatemic osteomalacia also express FRP4 (frizzled related protein-4), MEPE (matrix extracellular phosphoglycoprotein), and FGF7— all of which have phosphaturic properties.38,46 FRP4 is a secreted 346-aa glycosylated protein that shares the structure of the extracellular domain of transmembrane

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DISORDERS OF CALCIUM METABOLISM

frizzled receptors. The natural ligands of frizzled receptors are Wnt proteins, and their coreceptors are the cell surface low-density lipoprotein receptor-related proteins (LRP-5/6). These heterotrimeric complexes stabilize intracellular -catenin and the attendant signal transduction systems and are essential for bone formation (vide infra). Secreted FRP4 serves as a “decoy” receptor competing with the frizzled receptor for binding to Wnt, thus inhibiting the function of this receptor. FRP4 is expressed in bone cells and in large amounts by tumors with associated osteomalacia. In normal adults, the mean serum FRP4 concentration is 34 ng/mL. FRP4 inhibits sodiumdependent renal tubular phosphate reabsorption by inhibition of Wnt signaling, leading to hypophosphatemia. It also reduces expression of the gene encoding 25hydroxyvitamin D3-1.hydroxylase.40 MEPE is primarily expressed by osteoblasts, osteocytes, and odontoblasts— as well as by tumors associated with hypophosphatemic osteomalacia. It encodes a 525-aa 58-kDa protein, a member of the short-integrin-binding ligand-interacting glycoprotein family that also includes osteopontin. MEPE modulates osteoblast and osteoclast function and may both inhibit and support bone mineralization.38 Although MEPE is able to inhibit sodium-dependent renal tubular phosphate reabsorption, its role in phosphate metabolism may be more complex inasmuch as knockout of MEPE in mice results in increased bone mass without altering serum phosphate or calcitriol values.47 MEPE is associated with and may serve as a substrate for PHEX on the osteoblast surface.44

Magnesium Magnesium is the fourth most abundant of the body cations. Two-thirds of body Mg is found in bone (primarily on the surface of the hydroxyapatite crystal, where 50% is freely exchangeable), one-third is intracellular, and 1% is in the ECF compartment.1,2 In blood, magnesium (1.7-2.4 mg/dL  0.7-1.0 mmol/L) is partially bound to proteins (30%), complexed to phosphate and other anions (15%), and found as free Mg2
e (55%). As with Ca2
e, Mg2
e levels rise as pH falls (increased acidity).22 Intracellularly, magnesium (0.5 mmol/L) is bound to ATP and other molecules. Ten percent is in the ionic form, and 50% within mitochondria. Mg2
e is a cofactor in many enzymatic reactions, including those that consume or produce ATP. Mg2
e alters free radicals and influences nitric oxide synthase activity, cyclic guanosine monophosphate generation, endothelin production, and immune function. Mg2
e decreases membrane excitability in nerve and muscle cells and blocks the excitatory N-methyl D-aspartate receptor. It is a necessary cofactor for the regulation of neuromuscular excitability, nerve conduction, enzyme activity, oxidative metabolism by mitochondria, glycolysis, phosphorylation, transcription, and translation. It is essential to the secretion (but not the synthesis) of PTH by the parathyroid chief cell. Net intestinal magnesium absorption is directly related to dietary intake and independent of calcitriol.1 Magnesium is passively absorbed primarily by diffusion through paracellular channels in proportion to the intestinal lumi-

Ch03_074-126-X4090.indd 84

nal concentration of this cation. In addition, there is a small component of active transcellular magnesium absorption. Magnesium is also excreted into the intestinal tract, where its absorption may be impaired by high phosphate intake, significant intestinal disease, or chronic laxative abuse. Seventy percent of serum magnesium is ultrafiltrable and passes through the renal glomerular membrane. Ninety-five percent is reabsorbed: 15% in the proximal convoluted and straight tubules, 70% in the cortical TALH, and 10% in the distal convoluted tubule.1,22 In the TALH, Mg2
reabsorption occurs through a paracellular pathway that is impermeable to water.48 Mg2
is conducted from the lumen of the TALH to the interstitial space and vasculature by paracellin-1 (CLDN16, also termed claudin 16), a 305-aa tight-junction protein whose encoding gene is expressed only in the renal cortical TALH and distal convoluted tubule. Paracellin-1 has four transmembrane domains with intracellular carboxyl and amino terminals and is a member of the claudin family of proteins that bridge intercellular gaps within the tight junctions.15,48 Paracellin-1 is also utilized for Ca2
reabsorption in the TALH. Loss-of-function mutations of CLDN16 lead to familial autosomal-recessive renal hypomagnesemia because of renal wastage of magnesium in association with hypercalciuria and renal calcification (OMIM 248250).48,49 In the distal convoluted tubule, Mg2
reabsorption is related to activity of a Na
-Cl- cotransporter and perhaps to paracellin-1. PTH increases magnesium reabsorption in the renal TALH and distal convoluted tubule, perhaps by regulating paracellin-1. On the other hand, hypermagnesemia and hypercalcemia (acting through the CaSR) decrease renal tubular magnesium reabsorption—as do expansion of extracellular fluid volume, metabolic alkalosis, phosphate depletion, loop diuretics, aminoglycoside antibiotics, and impaired renal function (Table 3-4).1

Alkaline Phosphatase The gene (ALPL) encoding tissue-nonspecific alkaline phosphatase (TNSALP) is expressed in bone (synthesized and secreted by the osteoblast), liver, kidney, and skin fibroblast. ALPL is a 507-aa protein that by alternative processing during transcription and translation permits the osteoblast to synthesize and secrete a reasonably specific bone form. Although TNSALP circulates as a homodimer, in tissue it is a homotetrameric ectoenzyme (ectophosphatase) located on the cell surface anchored through its carboxyl terminal to cell membrane phosphatidylinositol-glycan. In bone, alkaline phosphatase binds to collagen type I and prepares skeletal matrix for mineralization, hydrolyzes organic phosphates (thus increasing the local concentration of phosphate to a value that exceeds the calcium X phosphate product, encouraging deposition of calcium phosphate as hydroxyapatite), transports inorganic phosphate and Ca2
into the cell, and inactivates pyrophosphate and other inhibitors of mineralization by removing their phosphate moieties. Among the endogenous substrates for TSNALP are phosphoethanolamine and pyridoxal-5’-phosphate. The

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hepatic form of TNSALP is formed by alternative splicing of exon 1 of ALPL. Hepatic alkaline phosphatase converts pyridoxal-5’-phosphate to pyridoxal, a compound essential for normal synthesis of neural -amino butyric acid (GABA)—an inhibitory neurotransmitter. Without pyridoxal-5’-phosphate, central nervous system levels of GABA are low and seizures may occur.50 Loss-of-function mutations in ALPL lead to infantile, childhood, and adult forms of hypophosphatasia. Three genes encoding tissue-specific intestinal, placental, and germ cell alkaline phosphatase isoenzymes are clustered at chromosome 2q34-q37.

Parathyroid Hormone and Parathyroid-Hormone–Related Protein PARATHYROID HORMONE PTH is secreted by the chief cells of four paired PTGs that are derived from the endoderm of the dorsal segments of the third (paired inferior glands) and fourth (paired superior glands) pharyngeal pouches. Occasionally, there may be a fifth PTG embedded within the substance of the thyroid gland or in the mediastinum.24 The thymus is formed by the endoderm of the third pharyngeal pouch and calcitonin-synthesizing parafollicular (C) cells of the thyroid by that of the fourth pharyngeal pouch. The mRNA of PTH has also been demonstrated in rodent thymus and hypothalamus.51 A critical factor for the development of the PTGs is the nuclear DNA binding transcription factor encoded by Gcm2 (glial cell missing, drosophila, homolog of, 2). Although essential to neural glial cell development in insects, GCM is not expressed in mammalian brain but is expressed primarily in the rodent placenta and thymus (Gcm1) and in the PTG (Gcm2). The pharyngeal pouch expression of Gcm2 is maintained by a number of homeobox and transcription factors, including those encoded by Hoxa3, Pax1, Pax9, and Eya1. The human homolog of Gcm2 (Gcm2 or GCMB) is expressed in the PTG and in intrathymic PTH-secreting adenomas but not by normal human thymus.52 Intragenic deletion or missense mutations of Gcm2 have been identified in subjects with familial autosomal-recessive hypoparathyroidism (Table 3-5). Chief cells synthesize, store, and secrete PTH—a hormone that increases serum concentrations of calcium, lowers phosphate values, and exerts both anabolic and catabolic effects on bone [primarily in response to ambient Ca2
e concentrations that either enhance (when low) or repress (when high) transcription of PTH and secretion of PTH and regulate the rate of chief cell proliferation, responses mediated by the CaSR]. Calcimimetic drugs have inhibitory effects on PTH secretion. Higher phosphate values enhance transcription of PTH, secretion of PTH, and chief cell replication. 1,25dihydroxyvitamin D3 (calcitriol) and its synthetic analogues inhibit transcription of PTH, secretion of PTH, and proliferation of chief cells.24 In response to acute hypocalcemia, PTH stored in secretory vesicles is rapidly

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released. When hypocalcemia is prolonged, the secretion of PTH1-84 is augmented by a decrease in its intracellular degradation and an increase in transcription of PTH. When hypocalcemia is extended, it is augmented by an increase in chief cell number. The three exons of PTH encode the prepro-PTH sequence of 115 aa. The amino terminal 25-aa signal sequence (encoded by exon 2) is removed by furin, a prohormone convertase, as it leaves the endoplasmic reticulum [forming pro-PTH (90 aa)]. It is then further processed in the Golgi apparatus by furin to mature human PTH (84 aa). PTH is stored in secretory vesicles and granules. The stability, translation, and intracellular translocation of PTH mRNA are regulated by binding of cytosolic proteins to the 3’-untranslated region of PTH mRNA. These proteins include a member of the dynein complex that also binds to microtubules within the parathyroid chief cell and to HNRPD (heterogeneous nuclear ribnucleoprotein D or AUF1), which directs mRNA into the proteasomal pathway of degradation.53

TA B L E 3 - 5

Parathyroid Hormone–Related Protein: Sites of Expression and Proposed Actions Site Mesenchyma Periarticular cells

Bone Smooth muscle Vascular system Myometrium Urinary bladder Cardiac muscle

Action

PTH-rP depresses the rate of differentiation of late-proliferating chondrocytes to hypertrophic chondrocytes, thus permitting increased proliferation and delay of ossification. Enhances or depresses bone resorption. Relaxation.

Positive chronotropic and inotropic effects.

Skeletal muscle Epithelia Skin Breast

Teeth Endocrine system Parathyroid glands Pancreatic islets Placenta Central nervous system

Perhaps regulates proliferation of keratinocytes. Induces ductal branching, secreted into milk, and drives mobilization of calcium from maternal bone for transfer to nursing infant. Stimulates resorption of overlying bone enabling eruption. Stimulates transport of calcium. Stored and co-secreted with insulin. Enhances calcium transport. Neuroprotective by antagonizing excessive calcium-related excitotoxicity.

Compiled and adapted from Broadus AE, Nissenson RA (2006). Parathyroid hormone-related protein. In Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 99-106.

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The full biologic activity of PTH1-84 is found in its first 34-amino-acid sequence, and all of its renal effects are localized within the segment PTH1-31. Amino acid numbers 1 and 2 (serine-valine) comprise an activation sequence essential to the bioactivity of the amino terminal portion of PTH1-84. The PTGs secrete intact PTH1-84, a phosphorylated form of PTH1-84, and carboxyl terminal PTH fragments of varying length but do not secrete amino terminal fragments of PTH1-84.54,55 Intraglandular PTH1-84 is degraded by cathepsins B and H (proteases colocalized with PTH1-84 in secretory granules). PTH degradation represents an important mechanism regulating the release of bioactive PTH1-84 and is accelerated when the Ca2
e concentration is elevated. The half-life of circulating PTH1-84 is approximately 2 minutes. It is rapidly metabolized by the liver and excreted by the kidney. In the hepatic Kupffer cells, PTH1-84 is cleaved usually after either aa 33 or aa 36 to carboxyl terminal PTH peptides with half-lives of approximately 15 to 20 minutes. In the kidney, intact and carboxyl terminal fragments of PTH are filtered by the glomerulus, reabsorbed by the renal tubule, and then degraded to small fragments. Megalin (a multifunctional receptor expressed in coated pits on the luminal/apical surface, endocytic vacuoles, and lysosomes of proximal renal tubular cells; in the PTG; and in other epithelial structures) specifically recognizes intact PTH1-84 and amino terminal fragments of PTH and mediates the renal tubular endocytosis of intact PTH1-84 that has been filtered through the glomerulus.28 The classic functions of PTH1-84 as well as its shorter peptide derivatives PTH1-34 and PTH1-31 upon regulation of calcium and phosphate homeostasis are carried out through the seven-transmembrane G-protein–coupled PTH/PTHrP receptor in the renal tubule and osteoblast. Thus, with equal potency PTH1-84, PTH1-34, and PTH1-31 increase urinary excretion of phosphate by inhibiting its renal tubular reabsorption, the renal tubular and osteoclastic reabsorption of calcium thereby raising serum calcium concentrations common and the renal synthesis of calcitriol by enhancing expression of CYP27B1 (encoding 25-hydroxyvitamin D3-1 hydroxylase), thereby augmenting intestinal absorption of calcium. Mediated by the Gs subunit of the G-protein and adenylyl cyclase, these actions involve generation of cyclic adenosine monophosphate (AMP) and signaling through protein kinase A (PKA). Whereas the first two residues of PTH1-84 (serinevaline) are essential to activation of adenylyl cyclase, residues 15 through 34 are needed for high-affinity binding to its receptor. In addition to cyclic AMP, PTH1-84 activates other signal transduction pathways in skeletal and kidney cells—including those involving PLC and cytosolic Ca2
flux, PKC, and MAPK.55 As noted, multiple species of carboxyl terminal peptides derived from PTH1-84 circulate. They are secreted directly by the PTG or returned to the circulation after metabolism of intact PTH1-84 by hepatic Kupffer cells.55 Indeed, carboxyl terminal fragments of PTH1-84 are secreted in greater abundance from the PTG than is intact PTH1-84, and the proportion of carboxyl terminal fragments secreted increases as the ambient Ca2
concentration rises. Many of the circulating carboxyl terminal frag-

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ments of PTH are generated by hepatic uptake and degradation of PTH1-84. Amino terminal fragments generated by hepatic degradation of PTH1-84 are degraded further within the liver and do not recirculate. PTH1-84 and the carboxyl terminal fragments of PTH are filtered by the kidneys, and the carboxyl terminal fragments are reabsorbed by the renal tubules and further degraded intracellularly. The kidneys are not a major source of circulating carboxyl terminal fragments of PTH. Among the carboxyl terminal fragments of PTH found in circulation are PTH7-84, 24-84, 34-84, 37-84, 41-84, 43-84 . They are extracted by the kidneys, muscle, and bone. The presence of as yet structurally uncharacterized receptors for these carboxyl terminal peptides of PTH1-84 has been demonstrated by their biologic actions. In addition to their classic effects on mineral homeostasis, several nonclassic actions of PTH1-84 have been identified—including rapid and direct stimulation of intestinal calcium absorption independent of its effects on vitamin D metabolism, stimulation of hepatic gluconeogenesis, acute natriuresis and calciuresis, and enhancement of neutrophil movement in vitro.55 Inasmuch as many of these nonclassic biologic effects of intact PTH are not replicated by the amino terminal fragment PTH1-34, it has been suggested that they may be related to the carboxyl terminal portion of the protein. Indeed, specific effects of carboxyl terminal PTH fragments have been observed [e.g., PTH7-84 lowers serum calcium levels in parathyroidectomized rats (maintained eucalcemic by diet) and antagonizes PTH1-84 -stimulated increase in calcium concentrations, urinary phosphate excretion, and bone turnover.55 PTH7-84 directly lowers the rate of bone resorption and suppresses the bone resorbing effects of PTH1-34, calcitriol, prostaglandin E2, and interleukin (IL)-11. PTH7-84 also antagonizes osteoclastogenesis. On the other hand, PTH39-84 and PTH53-84 augment the biologic effects of PTH1-34. PTH7-84 does not bind to the PTH/PTHrP receptor, nor does it inhibit PTH1-84-mediated increase in cyclic AMP generation—implying that PTH7-84 acts through a unique receptor. Because there are multiple circulating forms of PTH, its immunologic measurement is dependent on the specificity of the antibody or antibodies employed in the assay. When a polyclonal PTH radioimmunoassay is utilized, intact and carboxyl terminal fragments of PTH are usually measured. Use of dual monoclonal antibodies and immunometric assays has improved the specificity of immunologic assays. Nevertheless, most assays detect intact and selected fragments of PTH and are consequently rather poor predictors of bone turnover—particularly in patients with chronic renal insufficiency.56 The comparability of PTH assays from different commercial sources is limited.57 Employing a two-site immunochemiluminescent assay, serum PTH concentrations average approximately 11 to 13 pg/mL and range between 2.3 and 24.5 pg/mL in children and adolescents 2 to 16 years of age. Values do not vary with age but are a bit higher in girls than in boys.58 The synthesis and secretion of PTH1-84 and its various fragments are modulated for the most part by the serum Ca2
e concentration acting through the CaSR

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expressed on the plasma membrane of the parathyroid chief cell. Because a change in serum Ca2
e concentration sensed by the parathyroid chief cell CaSR is quickly reflected in changes in cytosolic Ca2
i levels, the release of PTH is regulated on a minute-to-minute basis. Rapidly declining and steady-state low serum concentrations of Ca2
e increase PTH secretion by accelerating its release from storage sites in secretory granules. Hypocalcemia also raises PTG levels of PTH mRNA by increasing the transcription rate of PTH and enhancing the stability of PTH mRNA by its posttranscriptional binding to cytosolic proteins.2,24 Hypercalcemia slightly deceases PTH transcription and cellular levels of PTH mRNA. The serum Ca2
e concentration also determines the form of PTH released by the PTG. During hypocalcemia, PTH1-84 is the predominant form secreted. In hypercalcemic states, carboxyl terminal fragments of PTH are released. Low serum phosphate concentrations exert an independent and direct inhibitory effect on the transcription of PTH, post-transcriptional PTH mRNA stability, PTH secretion, and proliferation of parathyroid chief cells. Hyperphosphatemia enhances PTH secretion.24 Prolonged hyperphosphatemia may contribute to the PTG hyperplasia frequently encountered in patients with chronic renal disease. Calcitriol directly inhibits PTH transcription acting through the VDR and a vitamin D response element (VDRE) in the 5’-untranslated region of PTH. Calcitriol also controls expression of CASR and of VDR and decreases proliferation of parathyroid cells. However, chronic hypocalcemia overcomes the suppressive effects of calcitriol on PTH transcription by decreasing VDR number in the PTG.2 Hypomagnesemia and hypermagnesemia inhibit release but not synthesis of PTH. Other agents that increase PTH release include -adrenergic agonists, dopamine, prostaglandin E, potassium (by decreasing cytosolic Ca2
i levels within the parathyroid chief cell), prolactin, lithium (by “resetting” the set point for PTH release), glucocorticoids, estrogens, and progestins. Prostaglandin F2, -adrenergic agonists, and fluoride suppress PTH release by increasing Ca2
i values. PTH regulates the serum concentration of Ca2
e directly by stimulating its reabsorption in the distal renal tubule and from the skeleton, and indirectly by augmenting the intestinal absorption of calcium by increasing the synthesis of calcitriol. In bone, PTH enhances osteoclast activity indirectly by acting on and through the osteoblast. When administered intermittently, PTH1-84 and amino terminal PTH1-34 exert anabolic effects upon skeletal mass. They augment bone formation by increasing osteoblast number by accelerating their differentiation from progenitor cells and from inactive bone-lining cells and by reducing their rate of death.24 The anabolic effect of PTH may be mediated further by release of matrixembedded growth factors and by local generation of insulin-like growth factor I (IGF-I). PTH stimulates calcitriol synthesis by increasing renal tubular expression of CYP27B1, the gene encoding 25-hydroxyvitamin D31-hydroxylase (the enzyme that catalyzes the synthesis of calcitriol from calcidiol). PTH depresses proximal and

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distal renal tubular reabsorption of phosphate by decreasing expression of SLC34A1, a Na
-HPO4 cotransporter protein, thus increasing the urinary excretion of this anion.

PARATHYROID HORMONE–RELATED PROTEIN PTH and PTHrP have evolved from a common ancestor. They share 8 of their first 13 amino acids (the site of the activating domain for PTHR1), but their structures diverge thereafter. Both peptides bind with equal affinity to a common PTH/PTHrP receptor (PTHR1), but their receptor binding domains are distinct. PTHrP was initially identified as a primary mediator of hypercalcemia of malignancy. However, PTHrP is normally synthesized in many fetal and adult tissues (cartilage, bone, smooth, cardiac and skeletal muscle, skin, breast, intestines, PTGs, pancreatic islets, pituitary, placenta, and central nervous system) and plays a crucial role in chondrocyte differentiation and maturation, formation of the mammary gland and eruption of teeth, epidermal and hair follicle growth, and other developmental events.59,60 Whereas PTH acts as an endocrine hormone on tissues distant from the PTG, PTHrP is synthesized locally and acts primarily as a paracrine or juxtacrine (and perhaps intracrine) messenger. (A nuclear localization sequence is present in the latter half of the PTHrP molecule.59) Although the secretion of PTH is regulated by ambient calcium levels and fluctuates rapidly, the production of PTHrP is constitutive and is controlled at the point of expression of its encoding gene. The six exons of the gene encoding PTHrP (PTHLH) are transcribed and translated into 108-, 139-, 141-, and 173-aa isoforms employing coding sequences from exon 4 alone or from exons 4 and 5 or 6.24 In addition, amino and carboxyl terminal and mid-region products of PTHLH are also formed by post-translational processing. The predominant isoform of PTHrP is the 141-aa sequence. In specific tissues, PTHrP1-139 is cleaved by prohormone convertases to smaller peptides (PTHrP1-36, 37-94, 107-139) that may act as paracrine factors or be secreted into the circulation. PTHrP secreted by fetal PTGs and a mid-region fragment of PTHrP37-94 synthesized by the placenta increase placental calcium transport. PTH107-139 inhibits bone resorption (and has been termed osteostatin).60 Serum concentrations of PTHrP are low except when it is secreted by tumors, leading to humoral hypercalcemia of cancer. There are high concentrations of PTHrP in breast milk. Although the effects of PTHrP1-36 on calcium, phosphate, and vitamin D metabolism are similar to those of PTH, the major roles of this and other segments of PTHrP in many developmental processes (including those of breast, teeth, cartilage, and endochondral bone) distinguish PTHrP from PTH. In the cartilaginous growth plate, proximal periarticular proliferative chondrocytes synthesize PTHrP in response to Indian hedgehog (Ihh), a protein secreted by chondrocytes in their late proliferative and early prehypertrophic phases (Figure 3-2). PTHrP then diffuses into and through the growth plate and signals prehypertrophic chondrocytes through PTHR1 to slow their rate of differentiation to hypertrophic

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Perichondrium

Particular proliferating chondrocytes

PTHrP

Columnar proliferating chondrocytes

Bone collar

Prehypertrophic and hypertrophic chondrocytes

Ihh

Primary Spongiosa Figure 3-2. The epiphyseal cartilage growth plate consists of zones of proliferating, transitional, and hypertrophic chondrocytes. Indian hedgehog is synthesized by prehypertrophic chondrocytes. Receptors for parathyroid-hormone–related protein are expressed by proliferating and transitional chondrocytes. Indian hedgehog stimulates secretion of parathyroid-hormone–related protein from periarticular cells, and this in turn blocks further differentiation and maturation of late proliferating chondrocytes to hypertrophic chondrocytes—thus prolonging the period of cartilage growth. [Reproduced with permission by the American Society for Bone and Mineral Research from Broadus A (2006). Parathyroid hormone-related protein. In Favus M (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 99–106.]

chondrocytes, thus prolonging the stage of proliferation and delaying ossification.59,60 Biallelic loss of PTHrP in knockout mice is lethal due to bony malformations. In these mice (Pthlh-/-), there is a decrease in the number of resting and proliferating chondrocytes, disruption of the columnar organization of the growth plate, premature acceleration of chondrocyte maturation and apoptosis, and inappropriate ossification resulting in a dwarfing phenotype (a domed and foreshortened cranium, short limbs, small thorax) similar to that of Blomstrand chondrodysplasia (a disorder associated with loss-of-function mutations of PTHR1).61 Most of these mice die at birth. Mice in which expression of Pthlh has been maintained only in chondrocytes survive, but display small stature, cranial chondrodystrophy, and failure of tooth eruption.60 In the heterozygous state (Pthlh
/-), mouse fetal development is normal but by 3 months of age the trabeculae of the long bones are osteopenic. A similar bone phenotype is noted when loss of Pthlh expression

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is confined to osteoblasts.59 For comparison, in mice in which Pth has been “knocked out” there is decreased mineralization of cartilage matrix, expression of vascular endothelial growth factor (VEGF) and neovascularization, osteoblast number, and trabecular bone volume.62 Thus, PTH and PTHrP are necessary to normal fetal endochondral bone development. PTHrP has also been identified in the nucleus of chondrocytes and other cells, where it may regulate cell proliferation and act as a survival factor.59,63 Cortical thickness of long bones is increased in Pth- and Pthlh-null mouse models, indicating that the regulation of endochondral and periosteal osteoblast function differs. The placental transport of calcium is dependent on PTHrP produced by the placenta itself because the maternal-fetal calcium gradient is lost in its absence and may be restored by administration of a mid-molecular fragment.59 PTH and PTHrP are required for normal mineral homeostasis in utero. In mice that lack PTGs, fetal serum calcium and magnesium concentrations are low and phosphate levels are elevated. To a lesser extent, similar changes occur with loss of expression of Pthlh.64 PTH does not regulate placental calcium transfer, whereas PTHrP is essential to this process. During lactation, mammary expression of PTHLH and secretion of PTHrP increases—whereas production of estrogens declines, permitting unopposed PTHrP-induced mobilization of maternal skeletal calcium necessary for the breast-fed infant but substantially decreasing maternal bone mineral content (a process reversed when lactation ceases).65,66

PARATHYROID HORMONE AND PARATHYROID-HORMONE–RELATED PROTEIN RECEPTORS PTH and PTHrP utilize a common receptor (PTHR1), through which most of the “classic” physiologic functions of these peptides are exerted. PTHR1 is a 585-aa protein that shares its structure with that of the B family of GPCRs (calcitonin, GH-releasing hormone, secretin, glucagon, vasoactive intestinal polypeptide, corticotropin-releasing hormone) that are characterized by long extracellular amino terminal domains (⬃100-aa residues) with multiple cysteine residues forming disulfide bridges.25 As a result of alternative mRNA splicing, there are several isoforms of the PTH/PTHrP receptor. PTHR1 recognizes the amino terminal sequences of PTH1-84 and PTHrP as essential to activation (aa 1–9) and binding (aa 15–34).2,55 The amino terminal extracellular domain of PTHR1 contains six conserved cysteine residues that form three disulfide bonds; clustered near the first transmembrane domain are four glycosylated asparagine residues. PTH1534 binds to the extracellular domain and loops of the transmembrane domains of PTHR1, whereas the amino terminal of PTH1-84 interacts with the transmembrane domains and their connecting extra- and intracellular loops (termed the J or juxtamembrane domain) to activate Gsand Gq-proteins and their respective signal transduction pathways.67 PTHR1 is expressed in renal tubular cells and osteoblasts, skin, breast, heart, and pancreas, among other

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tissues—the latter sites reflecting the paracrine targets of PTHrP. It is coupled primarily through Gs-protein to adenylyl cyclase, cyclic AMP, and PKA—the initiating steps in intracellular transduction of the PTH signal. PTHR1 also activates Gq-proteins, thereby stimulating PLC activity and leading to hydrolysis of membrane phosphatidylinositol 1,4,5-trisphosphate to IP3 and diacylglycerol, activation of PKC, release of Ca2
from intracellular storage sites, and stimulation of the MAPK signal transduction pathway. PTH and calcitriol decrease expression of PTHR1. Targeted loss of PTHR1 is accompanied by impaired proliferation of chondrocytes and acceleration of chondrocyte maturation and calcification, an outcome mimicked by targeted loss of Gs in chondrocytes.55,59 Constitutively activating mutations of PTHR1 leads to hypercalcemia and Jansen metaphyseal chondrodysplasia, whereas inactivating mutations results in hypocalcemia and Blomstrand chondrodysplasia. The abnormalities of chondrocyte maturation seen experimentally with inactivating mutations of Pthr1 are mimicked to an extent by loss of PTHrP function as well. Loss of PTH results in aberrant formation of primary spongiosa of long bone and in defective mineralization.62 After activation of the G-protein is complete, PTHR1 is phosphorylated by a GPCR kinase. It then associates with -arrestin proteins and undergoes endocytosis. By an alternate pathway, carboxyl terminal PTH peptides may also promote endocytosis of PTHR1.55 Once internalized, PTHR1 may be degraded, recycled to the cell membrane, or directed to the nucleus by importins-1 and - where it is found in the nucleoplasm.68 The role of nuclear PTHR1 in relaying the many biologic effects of PTH and PTHrP is unknown at present, but one can speculate that it might interact with DNA directly to regulate gene transcription. A second PTH receptor (PTHR2) is selectively activated by PTH but does not recognize PTHrP. PTH specificity is determined by Ile5 and Trp23 in native PTH, sites that affect activation and binding, respectively.55 PTHR2 encodes a 539-aa GPCR with 70% homology to PTHR1 that activates adenylyl cyclase. It is expressed predominantly in brain, testis, placenta, and pancreas, but not in bone or kidney. Its physiologic role is uncertain.69 In response to PTH1-84, PTHR2 enhances both cyclic AMP generation and Ca2
mobilization. However, the naturally occurring endogenous ligand for PTHR2 is not PTH but is likely to be the 39-aa PTH-related hypothalamic tuberoinfundibular peptide (TIP39). This protein is also expressed in the testis and various central nervous system regions. TIP39, PTH, and PTHrP may have evolved from a common ancestral protein. A third PTH receptor that recognizes amino terminal sequences of PTH has been identified in zebra fish and is termed the type-3 zPTH receptor (zPTHR3). Rat PTH binds to this receptor and activates adenylyl cyclase.55 This species also expresses a PTHR1like protein. A human homolog for zPTHR3 has not been identified to date. Specific receptors recognizing the amino terminal sequences of PTHrP have been found in brain and skin. PTHrP stimulates release of arginine vasopressin from the supraoptic nucleus in vitro.55

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Although as yet not specifically characterized, receptors for carboxyl terminal fragments of PTH1-84 have been identified as renal and bone cell binding sites for PTH1-84 from which intact hormone can be only partially displaced by PTH1-34 but can be further displaced by PTH53-84 and PTH6984 55 . In addition, in osteocytes, osteoblasts, and chondrocytes from which the PTH/PTHrP receptor has been knocked out and to which intact PTH1-84 but not PTH1-34 binds, labeled PTH1-84 can be displaced by carboxyl terminal PTH19-84, 28-84, 39-84 fragments. Important determinants for binding of PTH to the carboxyl terminal selective receptor(s) appear to be aa 24 through 27 (Leu-Arg-Lys-Lys) and aa 53 and 54 (Lys-Lys). Furthermore, carboxyl terminal fragments of PTH exert biologic effects in intact and PTHR1-null cells—such as regulation of alkaline phosphatase activity in osteosarcoma cells and osteoblasts (but not generation of collagen type I), stimulation of Ca2
uptake by osteosarcoma cells and chondrocytes, and cell survival in vitro. Thus, the physiologic actions of PTH likely reflect the integrated sum of the individual functions of the intact hormone and its carboxyl terminal fragments. Because carboxyl terminal fragments of PTHrP are not recognized by the membrane sites that bind carboxyl terminal fragments of PTH, a means of specifying cellular response to these closely related proteins potentially exists.

Calcitonin Calcitonin is a 32-aa peptide that in mammals is secreted by the neural-crest–derived parafollicular (C) cells of the thyroid gland. It inhibits osteoclastic bone resorption, thus lowering blood calcium concentrations.70 It is encoded by a six-exon gene (CALCA) that by alternative transcription and translation can form two products: a 141-aa protein from which calcitonin (exons 1-4) and katacalcin (a 21-aa hypocalcemic peptide adjacent to the carboxyl terminus of calcitonin) are derived and a 128-amino-acid protein from which is gleaned the 37-aa calcitonin-gene-related peptide- (exons 1-3, 5, 6)—a vasodilator and neurotransmitter that also interacts with the calcitonin receptor. Calcitonin is also expressed by cells in the adenohypophysis and brain, and by neuroendocrine cells in the lung and elsewhere. Calcitonin is produced in abundance by medullary carcinoma of the thyroid, and at times by other neuroendocrine tumors. The calcitonins of multiple species share similar structures, including five of the first seven amino terminal aa, a disulfide bridge between aa 1 and aa 7, glycine at aa residue 28, and a proline amide residue at carboxyl terminal aa 32. In the interior of the peptide, species other than human have several basic amino acids that make them more stable and easily recognized by the human calcitonin receptor and thus more biologically potent (e.g., therapeutically useful salmon calcitonin). Calcitonin secretion is stimulated primarily by increasing serum concentrations of Ca2
e transduced by the CaSR expressed on the plasma membrane of the parafollicular cell.2 Members of the gastrin-cholecystokinin intestinal peptide hormone family (gastrin, glucagon, pancreozymin) are also potent calcitonin secretagogues.70

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Calcium, pentagastrin, and glucagon are effective stimuli employed to assess calcitonin secretion clinically. Somatostatin, calcitriol, and chromogranin A1-40 inhibit (and chromogranin A403-428 stimulates) calcitonin secretion. Calcitonin secretion falls as the ambient Ca2
e concentration declines. The half-life of calcitonin is brief. It is metabolized primarily by the kidney but also by liver, bone, and thyroid gland. Serum levels of calcitonin are high in the fetus and newborn, fall rapidly after birth as serum Ca2
e values decline, and then fall more slowly (until three years of age)—remaining relatively constant thereafter ( 12 pg/mL). After 10 years of age, serum concentrations of calcitonin are higher in males than in females. The physiologic role of calcitonin is unclear because serum calcium concentrations are normal in patients with both decreased (primary congenital or acquired hypothyroidism) and increased (medullary carcinoma of the thyroid) secretion of this peptide. However, disposal of a calcium load is slower in the calcitonin-deficient subject. Immunoassayable concentrations of calcitonin are increased in patients with medullary carcinoma of the thyroid, chronic renal insufficiency, and pycnodysostosis.70 Individual commercial immunoassays for calcitonin may detect differing epitopes, have altered intra-assay dynamics, and provide inconsistent measurements.71 The biologic effects of calcitonin are mediated through its 490-aa GPCR encoded by CALCR, a member of the B family of GPCRs. Intracellular signaling of the CALCR is transduced through the adenylyl cyclase-cyclic AMPPKA signal transduction pathway. Alternative splicing of CALCR results in two isoforms of the calcitonin receptor, one of which has an additional 16-aa inserted into its first intracellular loop between transdomains I and II. Accessory proteins that modulate function of the calcitonin receptor have also been described.70 The calcitonin receptor is expressed in osteoclasts. When exposed to calcitonin, the osteoclast shrinks and bone resorbing activity declines quickly. Thus, calcitonin lowers serum calcium and phosphate levels—particularly in patients with hypercalcemia. Polymorphic variants of CALCR have been related to variations in bone mineral density (BMD). In subjects heterozygous at aa 463 (Pro/Leu) in the third intracellular domain, BMD is greater than in individuals who are homozygous for either amino acid.72 However, for the most part there has been little evidence that calcitonin plays a major role in mineral and skeletal homeostasis. In lactating women, serum levels of calcitonin rise and calcitonin is excreted in breast milk. Women who are breastfeeding their infants lose 5% to 10% of their trabecular bone mineral during 6 months of lactation. This is recouped rapidly when lactation ceases—much more quickly than when bone mass is lost because of other problems (e.g., glucocorticoid excess, bed rest). Experimentally, in the female mouse in which the gene encoding calcitonin has been eliminated loss of calcitonin has no effect on maternal bone mineralization during pregnancy or on the rate of skeletal remineralization after weaning of pups. However, 21 days of lactation are associated with much more marked demineralization

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of the spine of the nursing mother without calcitonin than in the wt female—a response that is reversible by the administration of exogenous calcitonin during the interval of lactation.73 Thus, in mammals calcitonin may be essential to the protection of maternal skeletal mass during lactation.

Vitamin D Cholecalciferol (vitamin D3) is synthesized in skin from cholesterol through 7-dehydrocholesterol. It is also present in oily fish such as salmon and mackerel. Ergocalciferol (vitamin D2) is a plant and yeast sterol74 (Figure 3-3). Vitamin D2 differs from vitamin D3 by the presence of a double bond between carbons 22 and 23 and a methyl group on carbon 24 in vitamin D2. Both forms of vitamin D undergo similar chemical modifications to bioactive metabolites. However, physiologically vitamin D2 is 3- to 10-fold less biologically effective in man than is vitamin D3 because its product (25hydroxyvitamin D2) is cleared much more rapidly from serum than is 25-hydroxyvitamin D3.75 In skin, provitamin D3 7-dehydrocholesterol is transformed to previtamin D3 and then isomerized to vitamin D3 by exposure to ultraviolet B-photon radiation (290–315 nm) and heat (37o C).74,76 The latitude, season of the year, and time of day influence the rate of synthesis of vitamin D3 stimulated by exposure to sunlight. In higher latitudes, the path through which ultraviolet B photons from the sun travel is longer and fewer reach the target. Exposure of the back of a white adult to intense summer sun (mid July) for 10 to 12 minutes in the northeastern United States generates ⬃10,000 to 20,000 IU of vitamin D3 over the next 24-hour interval.19 (For black persons, 30 to 120 minutes of exposure to sunlight may be required for comparable effects.) Sun-screening agents and aging also decrease the cutaneous formation of cholecalciferol in response to sunlight. Orally ingested vitamin D is packaged into chylomicrons and absorbed into the intestinal lymphatic system. It then enters the circulation and is transported to the liver by vitamin-D–binding protein (DBP), a polymorphic variant of the serum 2-globulin termed Gc (group-specific component) encoded by GC. In the liver, vitamin D is hydroxylated to 25-hydroxyvitamin D (25OHD; calcidiol is 25OHD3) by vitamin D-25 hydroxylase—a 501-aa class I mitochondrial cytochrome P450 enzyme encoded by CYP2R1. In addition, there are other hepatic 25hydroxylases that carry out this reaction (CYP27A1, CYP2J3).74 Calcidiol exerts only a minimal inhibitory effect on its production. Thus, serum concentrations of 25OHD reflect body stores of vitamin D. There are substantial data indicating that because of decreased exposure to sunlight and marginal dietary intake of vitamin D3 body stores of this vitamin are deficient or insufficient in many North American subjects.19 In addition, currently established normal values for serum concentrations of 25-hydroxyvitamin D (25OHD, 10– 55 ng/mL) have likely been derived from populations that are not completely vitamin D sufficient. In populations

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Solar UVB radiation Skin Dietary sources of vitamin D2 and D3

7-dehydrocholesterol Via chylomicrons and lymphatic system

PreD3 Heat

Vitamin D3

Vitamin D

DBP Vitamin D

DBP

Lipoprotein

Vitamin D

Circulation Vitamin D 25-OHase

Liver Pi, Ca2+, and other factors +

1,25(OH)2D

-

1-OHase

Preosteoclast

25(OH)D

Kidney

1-OHase

1,25(OH)2D

24-OHase

Calcitroic acid

Urine

PTH

RANK RANKL

Osteoblast PTH Mature osteoclast Bone Ca2+, and HPO42release

Intestine Parathyroid glands Calcium and phosphorus Blood

Ca2+, and HPO42absorption

Bone mineralization Metabolic Neuromuscular function functions Figure 3-3. Metabolism of vitamin D. Cholesterol is metabolized to cholecalciferol in skin, and hydroxylated in the liver to calcidiol and in the kidney to calcitriol. Factors that regulate these processes are depicted (see text). [Reproduced with permission from Holick MF (2006). Resurrection of vitamin D deficiency and rickets. J Clin Invest 116:2062-2072.]

living in sun-rich environments, the lower normal concentration of serum 25OHD is 32 ng/mL—with a range to 100 ng/mL. Physiologic data such as the relationship between serum concentrations of 25OHD and those of PTH, intestinal absorption of calcium, and optimal bone mineralization support the concept that a serum 25OHD level of 32 ng/mL is the minimum normal value in humans and that 25OHD concentrations between 10 and 30 should be considered insufficient. Although the current officially recommended minimal supplemental dose of vitamin D for breast and formulafed infants and older children and adolescents is 200 IU/ day, it is likely that 400 IU/day (10 g) is more appropriate. It has been suggested that children, adolescents,

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adults, and pregnant and nursing women should receive 400 to 600 IU (10-15 g) of supplemental vitamin D daily. Some data suggest that the daily required amount of supplemental vitamin D may be closer to 2,000 IU/day (50 g), and even more in pregnant and lactating women.19,76,77 Bound to DBP, calcidiol is transported to cells in the proximal convoluted and straight renal tubules. There it is further hydroxylated to the biologically active metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3 or calcitriol] by the cytochrome P450 mitochondrial monooxygenase 25OHD-1-hydroxylase encoded by CYP27B1.78,79 CYP27B1 has nine exons that encode a 508-aa protein with a mitochondrial signal sequence at

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its amino terminal and ferredoxin- and heme-binding sites within its structure. As a class I mitochondrial cytochrome P450 enzyme, 25OHD-1-hydroxylase requires for catalytic activity electrons from NADPH that are ferried to the enzyme protein by the electron transport proteins ferredoxin and ferredoxin reductase.80 Although CYP27B1 is expressed primarily in renal proximal convoluted and straight tubular cells, it may also be expressed by keratinocytes and hair follicles, osteoblasts, placental decidual and trophoblastic cells, the gastrointestinal and central nervous systems, testes, breast, and pancreatic islets. In monocytes and macrophages, expression of CYP27B1 may be induced by inflammatory cytokines such as interferon-.81 In the kidney, expression of CYP27B1 in the proximal convoluted tubules is stimulated by PTH through cyclic AMP and in the straight tubules by calcitonin by a pathway independent of cyclic AMP. In addition, activity of 25OHD1-hydroxylase is increased by hypocalcemia and hypophosphatemia, PTHrP, 24R,25(OH)2D3, GH, insulin-like growth factor-I (IGF-I), and prolactin.82 Increased serum and tissue levels of Ca2
and phosphate directly suppress expression of CYP27B1 and thus depress 25OHD-1-hydroxylase activity. Calcitriol exerts an indirect inhibitory effect upon renal CYP27B1 expression and thus upon its own synthesis by inhibition of PTH synthesis in the PTG.76 In activated monocytes, calcitriol actually enhances transcription of CYP27B1.81 FGF23 depresses 25OHD-1-hydroxylase activity, whereas calcitriol up-regulates FGF23 expression—effectively establishing an auto-control system for these compounds.41 Inactivating mutations of CYP27B1 result in vitamin-D–dependent rickets type I or 1-hydroxylase deficiency (OMIM 264700), a disorder associated with hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, severe rickets, and often alopecia. Calcitriol is inactivated in bone, intestine, liver, and kidney by glucuronidation, sulfation, multisite (carbons 23, 24, and 26) hydroxylation, and lactone formation to watersoluble compounds (such as calcitroic acid) excreted in urine and bile.76 In the kidney, 25OHD and 1,25(OH)2D are converted to 24R,25(OH)2D and 1,24R,25(OH)3D, respectively, by 25OHD-24 hydroxylase encoded by CYP24A1—the first in a series of degradative hydroxylations. The expression of this gene is up-regulated by hypercalcemia, hyperphosphatemia, and calcitriol and is suppressed by hypocalcemia and PTH. Cleavage of the side chain between carbons 23 and 24 [which have been hydroxylated by hepatic cytochrome P450-3A (CYP3A4)-dependent enzymes] generates water-soluble calcitroic acid.74 Many drugs (e.g., phenobarbital, phenytoin, carbamazepine, rifampicin) that are known to impair bone mineralization by inactivating calcitriol do so by increasing its state of hydroxylation by binding to and activating the nuclear pregnane X receptor (NR112), which in turn increases expression of CYP3A4.83 Calcitriol may also induce expression of hydroxylases that utilize cytochrome P450-3A, thereby enhancing its own hydroxylation and degradation.84 Bound to DBP, calcidiol is filtered through the glomerular membrane and reabsorbed

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by cells in the proximal renal tubule by the multifunctional receptor megalin (gp330)—expressed in coated pits on the luminal/apical surface, endocytic vacuoles, and lysosomes of proximal tubular cells.28 After uptake, the megalin-calcidiol complex enters the lysosome—where calcidiol is released, enters the cytosol, and is metabolized to calcitriol in mitochondria. The bulk of circulating calcitriol is bound to DBP, but it is its free fraction that is biologically active. Approximately 0.04% of calcidiol and 0.4% of calcitriol are present in free form in serum. Normal ranges of calcitriol concentrations are 8 to 72 pg/mL for neonates, 15 to 90 pg/mL for infants and children, and 21 to 65 pg/mL for adults. Once calcitriol is synthesized, its three-dimensional configuration is flexible rather than fixed—enabling it to exert both genomic and nongenomic (rapid-response) actions.85 The nuclear VDR serves as a transactivating transcription factor after binding to its ligand, calcitriol. The VDR is also associated with caveolae of the cell plasma membrane, flask-shaped invaginations of the membrane composed to a large extent of sphingolipids and cholesterol.85,86 Through binding to the nuclear VDR, calcitriol regulates the expression of many genes involved in mineral and bone metabolism. These effects take place over hours to days as the processes of transcription, translation, and post-translational modifications to the encoded protein(s) occur in multiple cytosolic compartments. Calcitriol also generates rapid responses evident within seconds to minutes after its contact with the cell. Examples of rapid cellular responses to calcitriol include immediate intestinal absorption of calcium (transcaltachia), opening of voltage-gated calcium and chloride channels in osteoblasts, endothelial cell migration, and pancreatic  cell secretion of insulin.85 The three-dimensional flexibility of calcitriol structure is enabled by rotation of side chain carbon pairs 17-20, 20-22, 22-23, 23-24, and 24-25; by rotation of the carbon 6-7 bond around the B ring, and by A-ring chair-chair interconversion with formation of either an - or -configuration of the cyclohexane-like A ring (Figure 3-4). Rotation about the carbon 6-7 bond of the B ring allows calcitriol to assume an extended 6-s configuration or a 6-s-cis conformation. It is the trans form of calcitriol that is employed by the VDR for its genomic responses and the cis form for its rapid actions. For binding to DBP, calcitriol assumes yet another shape.85 Calcitriol primarily regulates intestinal and renal absorption of calcium and phosphate by enhancing expression of genes encoding calcium transporters and channels [e.g., calbindin-D28K, calbindin-D9K, TRPV5 (expressed primarily in the kidney) and TRPV6 (expressed predominantly in the intestinal tract)]. Calcitriol also promotes endochondral bone formation and increases length of the long bones by amplifying epiphyseal volume, proliferation, and differentiation of chondrocytes and mineralization of cartilage matrix.87 Furthermore, this sterol increases trabecular and cortical bone formation by augmenting osteoblast number and function—including alkaline phosphatase activity, osteocalcin synthesis, and type I collagen formation—and by

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Figure 3-4. Cis and trans structures of calcitriol. [Reproduced with permission from Norman AW (2006). Vitamin D receptor: New assignments for an already busy receptor. Endocrinology 147:5542-5548.]

repressing bone resorption by osteoclasts. It does so by a direct cellular effect independent of endogenous PTH, evidenced by its activity in the mouse in which both CYP27B1 and Pth have been knocked out.87 Calcitriol directly suppresses transcription of PTH in the PTG, acting through the VDR. Vitamin D is also important for normal muscle development and is an integral component of the system requisite for achievement of optimal skeletal integrity and strength.88 Mediated by the VDR, calcitriol stimulates the absorption or reabsorption of calcium in the intestines, bone, and kidney. In the duodenum and proximal small intestine, calcitriol increases the efficiency of calcium uptake from the intestinal lumen by increasing the number of epithelial calcium transport channels (TRPV6) in enterocytes, its movement through the cytoplasm, and its transfer across the basal lateral membrane into the circulation—in part by the induction of calbindin9k (a calcium-binding protein), alkaline phosphatase, Ca-ATPase, calmodulin, and other proteins.74 Calcitriol also increases jejunal and ileal absorption of phosphate through a transcellular mechanism utilizing the type II Na
-HPO42- cotransporter protein (NPT2, encoded by SLC34A1) expressed on the luminal surface of the enterocyte. When vitamin D stores are replete, 40% of dietary calcium and 80% of dietary phosphate may be absorbed. Even greater efficiency of mineral absorption is realized during growth spurts, pregnancy, and lactation. A major task of calcitriol is to maintain calcium and phosphate concentrations in blood at levels sufficient to sustain mineralization of osteoid-collagen-containing bone matrix synthesized by osteoblasts. Paradoxically, in states of calcium deficiency calcitriol acts indirectly within bone to stimulate monocytic stem cell differentiation into osteoclasts by stimulating osteoblast/stromal cell synthesis of osteoclast-activating factors such as the ligand for the receptor activator of nuclear factor B (RANKL).

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During periods of calcium deficiency, calcitriol is also able to promote bone resorption through osteoblast production of osteopontin—a bone matrix noncollagenous protein to which osteoclast cell surface integrin receptors bind.89 Calcitriol stimulates osteoblasts to produce osteocalcin, bone-specific alkaline phosphatase, osteoprotegerin, osteonectin, and various cytokines.74 In addition to the classic effects of calcitriol upon mineral and skeletal metabolism, vitamin D and synthetically constructed ligands of the VDR have many non-calcemic actions.90 Among the several synthetic vitamin D3 analogues are alfacalcidiol [1-(OH)D3], calcipotriol [1,25-(OH)2-24-cyclopropyl-D3], maxacalcitrol [1,25-(OH)2-22-oxa-D3], and talcitol [1,24R(OH)2D3]. These vitamin D analogues have been engineered to retain the non-calcemic actions of the parent compound and reduce calcemic properties. Calcitriol and vitamin D3 analogues exert many immunomodulatory effects. In animal models of autoimmune diseases such as systemic lupus erythematosis, multiple sclerosis, type I diabetes mellitus, and inflammatory bowel disease, these agents inhibit T-lymphocyte differentiation into Th1 [IL-2, tumor necrosis factor (TNF)-, and interferon- secreting] cells and thereby modify disease induction, course, and severity. Calcitriol and analogues also exert differentiating and antiproliferative effects on a variety of cells. Thus, calcitriol induces differentiation of promyelocytes into monocytes and macrophages. These agents enhance differentiation of keratinocytes, and when administered orally or topically to patients with psoriasis vulgaris effectively ameliorate the disease. They are particularly effective when coadministered with a topical glucocorticoid. In vitro, calcitriol and its analogues inhibit growth of prostate, breast, and colon cancer cell lines—and clinical trials of these agents in patients with these neoplasms appear promising. Calcitriol

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and several of its analogues have been employed successfully in the treatment of osteoporosis, secondary hyperparathyroidism, and arthritis.90 Calcitriol also enhances secretion of insulin and down-regulates activity of the renin-angiotensin system.74

VITAMIN D RECEPTOR Calcitriol acts primarily by binding to the VDR, a 427-aa protein encoded by 11-exon VDR.91 Because of two potential start site codons for transcription of VDR in exon 2, there is a second isoform of VDR with 424 aa. At its 5’ end, the VDR has three noncoding exons (1A, 1B, 1C)— followed by exons 2 through 9, which encode the active protein [enabling transcription of three unique mRNA isoforms, depending on the splicing pattern of exons 1B and 1C (Figure 3-5A)]. As a member of the steroid/thyroid/vitamin-D receptor gene superfamily of nuclear-transcription–activating factors, the VDR has five domains: a short amino terminal segment of 24 aa (domains A/B) that houses a ligandindependent transactivation function termed activation function-1 (AF-1), a DNA-binding domain (C) with two zinc fingers (exons 2 and 3), a “hinge” region (D), and a

long carboxyl terminal ligand-binding domain (E) (exons 7, 8, 9). Structurally, the E domain consists of 12 helixes (H1–H12) and has two ligand-dependent transactivating regions (E1 between aa 232 and 272 and AF2 between aa 416 and 424) that recruit transcriptional cofactors when the VDR is activated by binding to calcitriol.91 Among the stimulants to the transcription of VDR are calcitonin, retinoic acid, estrogen, the transcription factor SP1, and -catenin. In part, estrogens increase expression of VDR through binding to estrogen receptors present in the caveolae of the cell membrane and then through activation of the MAPK signal transduction pathway.92 The VDR is expressed in the intestinal tract, distal renal tubule, osteoblast, keratinocyte, hair follicle, fibroblast, smooth and cardiac muscle, lung, bladder, thyroid, parathyroid, pancreas, adrenal cortex and medulla, pituitary, placenta, uterus, ovary, testis, prostate, activated T and B lymphocytes, macrophages, monocytes, spleen, thymus and tonsil, brain, spinal cord, and sensory ganglia. Allelic polymorphisms (random variations in gene composition not known to affect structure or function) of VDR have been related to bone mineralization and linear growth (Figure 3-5B). These gene variants have been identified by examining restriction fragment length

Figure 3-5. (A) Structure of mRNA and protein of the vitamin D receptor (VDR) derived from the 9-exon gene structure of VDR. The five domains of the VDR are depicted. Exons 2 and 3 encode the DNA-binding domain, and exons 7 through 9 encode the ligand-binding domain. Sites E1 and AF2 are subregions within the E domain that serve transactivating functions. Regions of homology shared with other members of the nuclear receptor steroid/thyroid hormone/vitamin-D superfamily are indicated by the gray and black areas. (B) Polymorphic variants of VDR. By convention, uppercase letters denote the absence (and lowercase letters the presence) of a polymorphic restriction site. The site of the start codon polymorphism (SCP) is depicted above the gene structure. It generates a FokI polymorphism and generates vitamin D receptor proteins of different lengths. The site of the BAT polymorphism is indicated above intron 10/exon 9. The BsmI, ApaI, and TaqI restriction sites are placed below the VDR gene structure. These polymorphic variants do not change the amino acid structure of the VDR. In exon 9, there is a poly A microsatellite of variable length. [Reproduced with permission from Malloy PJ, et al. (1999). The vitamin D receptor and the syndrome of hereditary 1,25-dihydroxyvitamin D-resistant rickets. Endocrine Reviews 20:156-188.]

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polymorphisms (DNA fragments of varying lengths) defined by endonuclease digestion of VDR DNA by BsmI (B), TaqI (T), FokI (F), and ApaI (A). For example, in VDR there are BsmI cut sites in the 3’ region and in exon 10. By convention, BB indicates absence of a BsmI cut site on both alleles, bb the presence of the cut site on both alleles, and Bb the heterozygous state.91,93 The BB VDR genotype has been variably associated with lower BMD in postmenopausal women and adult men, whereas the aa, bb, and TT genotypes have been linked to higher BMD in prepubertal girls.94 Polymorphic variants of the VDR have also been associated with differences in intestinal calcium absorption (subjects with the bb genotype on a low-calcium diet absorb ingested calcium to a greater extent than do those with the BB pattern), in birth length (BB neonatal males are shorter than Bb and bb male infants), in growth in infancy (at 2 years of age BB girls are longer and heavier than bb girls, BB boys weigh less than bb boys although of similar length, and at 1 year of age tt infant females are heavier than TT or Tt subjects), in pubertal growth (boys with the BB genotype are smaller than their Bb and bb peers through puberty), and in adult stature (the adult stature of subjects with the bT haplotype is on average 1.6 cm greater than that of individuals with other haplotypes).95,96 Polymorphic variants of VDR have also been linked to development of hyperparathyroidism and parathyroid tumors. The mechanism(s) by which polymorphic variants might exert these effects (or even if the associations are meaningful) is unclear. However, it has been suggested that the polymorphisms may influence the expression of the VDR or its function. Whereas most members of the superfamily of nuclear-receptor–transactivating factors pair as homodimers to bind to their specific hormone response elements in the 5’-untranslated region of the target gene, the calcitriol-VDR complex teams through its E (ligandbinding) domain with its obligate partner [unliganded retinoid X receptor  (RXRA)] to form a heterodimer that then binds to a VDRE. The endogenous ligand for RXR is 9-cis-retinoic acid. When unliganded, the bulk of the VDR is cytoplasmic. Binding of calcitriol to the VDR leads to heterodimerization with RXR and translocation of the tripartite complex to the nucleus. However, the unliganded VDR can also be guided to its target gene within the nucleus by a multi-protein chromatin-remodeling complex termed WINAC (where it remains inactive until bound by calcitriol).97 The generic VDRE, located in the 5’ upstream promoter region of the target gene, is composed of two directly repeated hexanucleotide sequences (5’-AG-G/T-TCA-3’) separated by three base pairs (bp). RXR binds to the 5’ half of the VDRE, and the VDR to its 3’ segment. After binding to the VDRE, the calcitriol-VDR-RXR complex recruits coactivating or corepressing comodulating proteins (e.g., SRC/p160 family, TRIP/SUG1, CPB/p300, TIF1, NcooR-1RIP13) into the promoter site and the general transcription apparatus of the target gene (TF-IIA, -B, TAF family).90,91 Gene activation by the VDR complex is initiated by recruitment of a histone acetyltransferase complex to the promoter segment of the gene that destabilizes the region and leads to unwinding of DNA, thereby granting

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access to basal transcription factors and RNA polymerase II that bring about RNA modeling of the target gene. In addition to positive and negative regulation of target gene expression through binding to the VDRE, the VDR complex may also affect transcription by modifying the effect of other transcription factors.90 Corepressors cause chromatin to compact, thereby silencing gene expression. Genes whose expression is regulated by the VDR are listed in Figure 3-6. Calcitriol acting through the VDR stimulates transcription or genes encoding calcium transport proteins (TRPV5/6), bone matrix proteins (osteopontin, osteocalcin), bone reabsorption factors (RANKL), and 25OHD-24-hydroxylase—and represses those that encode PTH and PTHrP. The calcitriol-VDR complex represses expression of multiple cytokines (IL-2, interferon-, granulocyte macrophage-colony stimulating factor) by negatively interacting with transcription factors that enhance their transcription (e.g., NF-B). Although the VDR is necessary for the actions of calcitriol in the intestines, kidney, bone, skin, and elsewhere, its loss does not interfere with embryogenesis and fetal development in humans with loss-of-function mutations in Vdr and vitamin D resistance or in mice in which Vdr has been knocked out. Newborn mice homozygous for targeted deletion of the second zinc finger (exon 3) and resultant truncation of the Vdr survive fetal life, appear normal at birth, and grow well for the first 24 days of life.98 However, after weaning their rates of weight gain and linear growth and serum Ca2
e and phosphate levels decline and serum PTH concentrations and PTG weights increase. In Vdr-/- mice as young as 15 days of age, osteoid surface is increased, bone mineralization decreased, and epiphyseal plate cartilage formation disorganized—with irregular columns of chondrocytes, increased matrix, and excessive vascularity relative to wt-type or heterozygous (Vdr
/-) mice.69,99 In mice in which both Vdr and Rxra have been knocked out, development of the cartilage growth plate is even more disrupted than in Vdr-/- pups.100 In addition, hair loss begins at 4 weeks of age and is complete by 4 months of age in homozygous Vdr-/- animals and is due to a defect in the growth cycle of hair follicles, a phenocopy of the generalized alopecia associated with loss of hairless (HR), a transcriptional corepressor that interacts with the VDR.90 (Vitamin D deficiency alone does not interfere with hair growth.) At approximately 8 weeks of age, Vdr-/- mice are a bit smaller than their wt counterparts and have lower calcium concentrations and cortical bone density and thickness— although trabecular bone density is similar in Vdr-/- and wt animals.101 Vdr-/- mice usually succumb between 4 and 6 months of age. However, maintenance of normal serum concentrations of calcium and phosphate by the feeding of high calcium-phosphate diets to Vdr-/- pups beginning at 16 days of age prevents the development of all of the skeletal abnormalities—suggesting that the main physiologic effects of calcitriol and the VDR are upon the intestinal absorption of calcium and phosphate and the maintenance of normal serum concentrations of these ions.99 In pregnant Vdr-/- 8-week-old mice, fetal (Vdr
/-) epiphyseal cartilage is histologically abnormal and skeleton

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Figure 3-6. Genes regulated by calcitriol and the vitamin D receptor. [Reproduced with permission from Nagpal S, et al. (2005). Noncalcemic actions of vitamin D receptor ligands. Endocrine Reviews 26:662-687.]

mineralization subnormal. Both defects can be reversed by increasing maternal dietary calcium and phosphate intake.101 Thus, absorption of intestinal calcium by non vitamin-D–dependent mechanisms can bypass the absorptive defect caused by loss of the VDR and by inference the deficiency of vitamin D itself. As previously discussed, in addition to acting through the nuclear VDR to regulate gene expression calcitriol also has rapid nongenomic effects mediated through plasma membrane caveolae-associated VDRs (Figure 3-7).85 The classic ligand-binding domain of the VDR recognizes the 6-s-trans configuration of calcitriol, whereas the 6-s-cis shape of the VDR mediates the nongenomic actions of calcitriol. An alternative VDR ligand binding domain that overlaps with the classic domain and accommodates the 6-s-cis conformation of calcitriol has been tentatively identified.85 The mechanism(s) by which the 6-s-trans and 6-s-cis forms of calcitriol recognize the genomic or nongenomic “pockets” of the VDR has not been established at this writing. In vitro, calcitriol induces expression of several membrane-associated rapid-response steroid-binding (MARRS) proteins such as the multifunctional thioredoxin-interacting protein-2 (TXNIP) and its homologue TXNIP-like inducible membrane protein, both of which are situated on the interior of the cell plasma membrane.102-104 After binding of calcitriol to the plasma membrane-associated VDR, its signal is transmitted by several classical intracellular signal transduction systems—including adenylyl cyclase induction of cyclic AMP and PKA; PLC- and PLD-mediated increase in phosphoinositide turnover resulting in generation of 1,2-diacylglycerol and IP3 that increase the permeability of

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Ca2
channels and release Ca2
from cytosolic storage sites; Gq protein through PLC-1 activation and intracellular redistribution of PKC isoforms (, , ); and Jun-activated kinase and the MAPK pathway.102 Within minutes after exposure of a vitamin-D–responsive tissue (e.g., intestine, chondrocyte, osteoblast) to calcitriol, there is increase in the intracellular concentration of Ca2
i (transcaltachia) and activation of PLC, PKC, and MAPK.85 In osteoblasts from VDR-/- mice and in fibroblasts from patients with inactivating mutations of VDR, the rapid actions of calcitriol are lost—as is the association of the VDR with caveolae (evidence of the importance of the VDR to membrane-initiated responses to calcitriol).102 The plasma membrane caveolae-associated VDR may link to Gs and thence to a calcium channel, to adenylyl cyclase, to PLC, or to caveolin—a protein that interacts with the nonreceptor tyrosine kinase Src and in turn with PLC or the kinase h-Ras.102,105 The physiologic role(s) of the rapid actions of vitamin D are not certain. In chondrocytes, there is MARRSdependent enhancement of calcium flux, PKC activity, and matrix vesicle mineralization.106 Vitamin-D3– mediated activation of TLIMP suppresses cell proliferation, perhaps in part by antagonizing the effects of IGF-I by increasing synthesis of IGF-binding protein3.104,107 The rapid effects of vitamin D may optimize its genomic effects by phosphorylation of proteins required by the VDR transcriptional complex. As expected, membrane-related phenomena are often better mimicked by analogs of calcitriol that are in the cis conformation than by calcitriol itself. A membrane-binding protein for 24,25(OH)2D3 has also been reported.108

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Figure 3-7. Genomic and nongenomic (rapid) responses to calcitriol. [Reproduced with permission from Norman AW (2006). Vitamin D receptor: New assignments for an already busy receptor. Endocrinology 147:5542-5548.]

Skeleton: Cartilage and Bone The skeleton is the framework of the body. It consists of cartilage and bone—specialized forms of connective tissue that provide mechanical support for muscle/tendon insertion that enables movement, protective shielding for softtissue organs, repository for bone marrow, and reserve source of calcium, phosphate, and other metabolically important ions.109,110 There are two primary bone shapes: flat bones (e.g., cranium, scapula, pelvis) and long bones (e.g., humerus, femur).111 Flat bones develop by membranous bone formation, whereas long bones develop by endochondral and membranous bone formation. The external surface of bone is enveloped by periosteum (containing blood vessels, nerve terminals, osteoblasts, and osteoclasts), whereas the interior of bone next to marrow is lined by endosteum. Long bones consist of a hollow shaft (diaphysis), distal to which are the metaphyses, cartilaginous growth plates, and epiphyses. The diaphysis consists of cortical bone and the metaphysis/epiphysis of trabecular bone surrounded by cortical bone. Eighty percent of the adult skeleton is dense cortical bone whose primary function is to provide mechanical strength. Twenty percent is cancellous bone, a network of trabeculae with a large surface area and increased turnover of bone constituents. Bone matrix or osteoid is the component of bone that is composed of collagenous (collagen types I and III) and noncollagenous proteins (e.g., osteocalcin, osteopontin) secreted by osteoblasts—upon which the mineral phase of bone is deposited. Modeling of bone is the process that takes place during growth in which the shape and

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size of the bone are determined. Remodeling of bone is a continual process in which formed bone is periodically reabsorbed and replaced by new bone. Remodeling occurs in both the growing child and the adult. Osteoprogenitor pluripotent stromal mesenchymal stem cells provide a continuous supply of bone-forming osteoblasts, the network of osteocytes embedded throughout bone that monitor bone integrity and strength and bonesurface-lining cells. Osteoclasts derived from hematopoietic precursor cells mediate bone resorption. Chondroblasts, osteoblasts, adipocytes, myoblasts, and fibroblasts are derived from a common mesenchymal cell (Figure 3-8).112 Bone morphogenetic proteins (BMPs) are members of the transforming growth factor- (TGF) superfamily that direct the transformation of a pluripotent mesenchymal cell into the pathway leading to formation of chondrocytes and osteoblasts. BMP-2, -4, and -7 are among the factors important for this differentiation process, although there is substantial redundancy in the system and other BMPs participate also. The BMPs act through designated cell membrane threonine/serine kinase receptors (e.g., BMPRIA, BMPR2). Depending on the interaction of the BMP receptors involved, intracellular signaling is transduced by the SMAD (mothers against decapentaplegic homolog) and/or MAPK pathways and induces synthesis of specific transcription factors that further the differentiation process.113,114 During early embryogenesis, bone is formed by condensation of mesenchymal cells in genetically determined patterns of position, arrangement, size, and shape.115 Individual mesenchymal cells then differentiate either into chondrocytes that secrete a matrix of collagen

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Osteoblastogenesis

A

Decreasing Proliferation Increasing Differentiation CFU-Fs sx

O 2,

Limited self renewal

Unlimited self renewal

nx

Ru

2 PPAR

Sox9 Asymmetric division

Osteoblasts Runx2

PPAR2

Adipocytes

Chondroblasts

My oD Multipotential daughter cell

Myoblasts Tri- of bipotential progenitor cells

Stem cell Fibroblasts Figure 3-8. Commitment of stem cells to the osteoblast lineage. Many factors guide the differentiation of chondroblasts, osteoblasts, myoblasts, fibroblasts, and adipocytes from a common mesenchymal stem cell (see text). Bone morphogenetic proteins are involved in the earliest steps, leading to the differentiation of the common mesenchymal precursor cell. RUNX2 is necessary for the initial differentiation of the common progenitor cell of chondrocytes and osteoblasts and for further delineation of osteoblasts in association with osterix. PPAR2 (peroxisome proliferator-activated receptor 2) stimulates the differentiation of adipocytes. Osteoblasts and adipocytes may be interconverted, depending on the active transcription factor. MyoD is a muscle-specific transcription factor necessary for the development of myoblasts. [Reproduced with permission from Aubin JE, et al. (2006). Bone formation: Maturation and functional activities of osteoblast lineage cells. In Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 20–29.]

type II in the anlagen of endochondral bones or directly into osteoblasts in precursor regions of intramembranous bone, where they secrete a matrix rich in collagen type I. Differentiation of the pluripotent mesenchymal cell into an osteoblast is under the guidance of the canonical Wnt signaling pathway.116 (The name Wnt is derived from combining and contracting the Drosophila gene Wingless with the corresponding mouse gene Int.) Binding of secreted Wnt cytokines, a family of lipidmodified signaling glycoproteins with 350 to 400 aa and a conserved sequence of 22 cysteine residues (e.g., Wnt1, Wnt9A), to GPCR frizzled receptors (FZD1) and to the LRP 5/6 coreceptors also situated on the mesenchymal stem cell’s plasma membrane leads to intracellular accumulation of -catenin (CTNNB1).117 Frizzled receptors act through the Gq-protein and PLC signal transduction pathway. LRP 5/6 (LRP5, LRP6) are long-chain proteins with a single transmembrane domain whose function is to prevent degradation of cytoplasmic -catenin through the ubiquitinationproteasomal pathway. When a Wnt ligand binds to its frizzled receptor, the intracellular protein disheveled-1 (DVL1) is phosphorylated and inhibits glycogen-synthasekinase-3-mediated phosphorylation of -catenin—which slows its rate of degradation and releases it from binding to axin (AXIN1), another intracellular protein. The intracellular domain of LRP5 then binds axin, permitting further increase in cytoplasmic levels of -catenin.118 In designated mesenchymal cells, increased levels of -catenin acting as a transcriptional cofactor with T-cell factor/lymphoid enhancement factor (LEF1)

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stimulates expression of runt-related transcription factor 2 (RUNX2, also termed core-binding factor alpha subunit 1 or CBFA1). The product of RUNX2 is itself a transcription factor that stimulates production of osterix (encoded by SP7). RUNX2 and osterix, a 431-aa protein, are essential for differentiation of mesenchymal cells into osteoblasts and for the synthesis of osteocalcin and collagen type I(1) by osteoblasts.116 Osteocalcin (BGLAP) plays an important role in normal mineralization of bone matrix. Other gene targets of RUNX2 include those encoding BMP4, FGFR1, Dickkopf, Wnt10a, and Wnt10b.119 Thus, RUNX2 is not only central to initiation of osteoblastogenesis but to its maintenance. Wnt signaling also increases production of osteoprotegerin, a protein that inhibits osteoclastogenesis—thereby further increasing bone mass vide infra. When intracellular stores of -catenin are depleted, mesenchymal cells differentiate as chondrocytes rather than osteoblasts.120 Through changes in its state of phosphorylation, -catenin also affects cell-to-cell adhesion and cell migration.117 The Wnt signaling pathway is opposed by inhibitors of LRP5 function [including secreted frizzled-related proteins, Dickkopf, and sclerostin (SOST)] that ensure orderly and normal bone mineralization. The axin-binding function of LRP5 is inhibited by binding of its extracellular domain to Dickkopf. Osteocytes are derived from osteoblasts after they have been embedded within mature bone. They form an interconnected network of longcell processes within canaliculi that link deep osteocytes with newly formed osteocytes and with surface-lining

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cells.118 Osteocytes detect mechanical strain by movement of fluid within these channels and respond by secreting growth factors and sclerostin. Because sclerostin inhibits Wnt signaling by binding to the extracellular domain of LRP5, when new bone formation is needed the secretion of sclerostin declines. Cortical or compact bone is present in the cranium, scapula, mandible, ilium, and shafts of the long bones and has periosteal and endosteal surfaces—both of which are lined with layers of osteogenic cells. Cancellous (trabecular or spongy) bone is located in the vertebrae, basal skull, pelvis, and ends of the long bones. Because only 15% to 25% of trabecular bone volume is calcified (compared to 80%-90% of cortical bone volume) and thus has a far greater surface area, trabecular bone is metabolically quite active. It has a high turnover rate, making it more vulnerable to disorders that affect bone mineralization. In flat bones (skull, ilium, mandible), intramembranous ossification begins with the local condensation of mesenchymal cells that then differentiate directly into preosteoblasts and osteoblasts and initiate the formation of irregularly calcified (woven) bone that is then replaced by mature lamellar bone.109 Membranous bones grow by apposition, a process supported by development of new blood vessels induced by VEGF (a protein that also enhances bone formation).121 The periosteum is a fibrous network in which osteoblasts synthesize peripheral compact bone. Cortical bone reinforces bone strength and complements and extends that provided by trabecular and endosteal bone. Tendons and ligaments insert and are fixed into cortical bone. Chondrocyte differentiation and cartilage formation occur in mesenchymal regions in which activity of the canonical Wnt signaling pathway and the expression of VEGF are low but not totally absent. This results in increased expression of SOX9 and related SOX family members, transcription factors that direct differentiation of mesenchymal cells into chondrocytes that are characterized by synthesis and secretion of collagen types II, IX, and XI. The expression of SOX9 is stimulated by FGF signaling through the MAPK pathway. SOX9 is a 509-aa protein with an SRY homology domain that is also expressed in the testis, where it is responsible for differentiation of Sertoli cells. Inactivating mutations of SOX9 lead to campomelic dysplasia and sex reversal in males (OMIM 114290). Target genes of SOX9 include those that encode collagen type II(1) and aggrecan, a chondroitin sulfate proteoglycan core protein. A mutation in the gene encoding aggrecan leads to an autosomal-dominant form of spondyloepiphyseal dysplasia associated with premature degenerative arthropathy (OMIM 608361). SOX9 is expressed not only in chondrocytes in the resting phase but in those in the proliferative phase but not in chondrocytes in the hypertrophic phase of maturation.114 The pattern of endochondral bone development is directed by factors that are independent of bone formation (e.g., in transgenic mice in which Runx2 is inactivated, the cartilage “skeleton” forms normally but is not ossified).116 [In humans, heterozygous inactivating mutations in RUNX2 result in cleidocranial dysplasia (OMIM

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119600), manifested by growth retardation, hypoplasia of the clavicle and pelvis, delayed closure of cranial sutures, and defective tooth eruption.] Vertebrae evolve from the condensation and segmentation of paraxial mesoderm into somites under the direction and control of multiple genes, including Notch1, Sonic hedgehog, and Pax1 and Notch ligands encoded by DLL3 and JAG1.116 The appearance of limb buds, proliferating mesenchymal cells that grow out from the lateral body wall and are capped by an apical ectodermal ridge, heralds development of the cartilage anlagen of the long bones—a process of segmentation directed by homeobox genes (HOXA13, HOXD13), Sonic hedgehog, WNT7a, GLI3, TGF␤, FGF4, FGFR1, FGFR2, BMP2, BMP4, BMP6, BMP7, LMX1B, PITX1, TBX4, TBX5, TWIST, and other signaling, receptor, and transcription-regulating factors involved in differentiation, paracellular communication, and cellto-cell interaction.116,122,123 Mitochondrial RNA-processing endoribonuclease encoded by RMRP is a ribonucleoprotein essential to assembly of ribosomes and cyclindependent cell cycle activity as well as chondrocyte proliferation and differentiation. Inactivating mutations of RMRP result in anauxetic dysplasia (OMIM 607095), a spondylometaepiphyseal dysplasia characterized by intrauterine and postnatal growth retardation (with adult stature 85 cm).124 Histologically, the growth plates of these patients are depleted of chondrocytes. Different mutations of RMRP result in varying clinical manifestations [e.g., cartilage hair hypoplasia (OMIM 250250) and metaphyseal dysplasia without hypotrichosis (OMIM 250460)]. In bones of cartilaginous origin (long bones, vertebrae), mesenchymal stem cells differentiate initially into prechondrocytes and chondrocytes that secrete collagen type II into a matrix in which chondrocytes are embedded as the cartilage mold enlarges. In the center of the mold, chondrocyte proliferation ceases. The chondrocytes hypertrophy and begin to synthesize collagen type X and VEGF.114 Blood vessels from the perichondrium together with cartilage-resorbing cells (chondroclasts) and osteoblasts and osteoclasts invade the hypertrophic region, destroy and reabsorb cartilage matrix and apoptotic chondrocytes, and deposit the primary spongiosa of bone that is later replaced by mature bone—a process dependent on normal function of the VDR.42 At the ends of the long bones, the cartilaginous epiphyseal growth plate develops—separating the epiphysis (a secondary ossification center at the end of a long bone) from the central metaphysis and diaphysis. Orderly columnar proliferation of chondrocytes in isogenous groups permits longitudinal growth of the long bone. Differentiation of resting chondrocytes from the reserve (resting, germinal, or stem cell) zone of the cartilage growth plate into the proliferating zone is initiated by BMP-6 and GH. Locally synthesized IGF-I induced by GH increases proliferation of chondrocytes (Figure 3-9).125,126 The perichondrium contributes chondrocytes to the growth plate and supports its appositional growth. Sequentially, proliferating chondrocytes evolve into prehypertrophic chondrocytes and then into hypertrophic chondrocytes that secrete collagen type X into the matrix in which they are

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Epiphysis

Resting zone

Proliferative zone

Hypertrophic zone Metaphysis

A

GH


B IGF-1 Androgens


IGF-1





Ihh 

Sox9




GC









Estrogens





BMPs







T3 / T4


FGFs

PTHrP




Vitamin D

Runx2 Figure 3-9. Hormones and growth factors affecting differentiation and proliferation of the cartilage growth plate. [Reproduced with permission from De Luca F (2006). Impaired growth plate chondrogenesis in children with chronic illness. Pediatr Res 59:625-629.]

embedded—thus contributing to interstitial growth of cartilage. Central to the maturational development of chondrocytes are PTHrP and Indian hedgehog (Ihh). The latter is a 45-kDa protein synthesized by prehypertrophic and early hypertrophic chondrocytes whose expression is increased by BMP-6. Ihh acts through its receptor Patched 1 (PTCH1) and coreceptor Smoothened (SMOH) to stimulate transcription of genes that are essential to chondrocyte proliferation and maturation. Ihh directly stimulates chondrocytes in the reserve zone to differentiate into proliferating chondrocytes, thus increasing the number of proliferative chondrocytes and lengthening the cartilage growth plate.127 It promotes cell division and growth by increasing expression of cyclins D and E, Wnts, and IGF-I. Ihh also regulates the generation of PTHrP by perichondrial cells on the articular surface, periarticular chondrocytes at the ends of the long bones, and most abundantly by early proliferative chondrocytes.59,114 PTHrP (through PTHR1) decreases the rate of differentiation of proliferating chondrocytes to prehypertrophic chondrocytes, thus antagonizing the effect of BMP-6. It prolongs the stage of chondrocyte proliferation by preventing premature hypertrophic differentiation and delays the generation of Ihh in a negative feedback loop.127 PTHrP also enhances phosphorylation of SOX9 in prehypertrophic chondrocytes in the cartilage growth plate and thereby enhances its transcriptional activity. SOX9 contrib-

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utes to the decelerating effect of PTHrP on the rate of differentiation of prehypertrophic to hypertrophic chondrocytes while maintaining the rate of chondrocyte proliferation and therefore continued elongation of the growth plate and long bone.60,128 Thus, Ihh and PTHrP interact to determine the height of the growth plate and the length of the long bone. In mice in which Ihh, Pthlh (the gene encoding PTHrP), or Pthr1 has been eliminated (knocked out), chondrocyte proliferation is decreased and there is accelerated differentiation into terminal hypertrophic chondrocytes and rapid replacement of cartilage by mineralized bone.129 In humans, inactivating mutations of PTHR1 result in rapid chondrocyte maturation and Blomstrand chondrodystrophy (OMIM 215045)—whereas activating mutations in PTHR1 lead to impaired chondrocyte maturation and Jansen metaphyseal chondrodysplasia (OMIM 156400). FGFs have dual effects upon chondrocyte proliferation and maturation. In part they enhance proliferation by increasing expression of SOX9 and decreasing that of Ihh.114 On the other hand, FGF18 inhibits chondrocyte proliferation and antagonizes the effects of BMPs acting through its cell surface tyrosine kinase receptor (FGFR3).114,116 Furthermore, spontaneous activating mutations of FGFR3 decrease the rate of chondrocyte proliferation and disturb their organization and are associated with achondroplasia and other chondrodysplasias. In hypertrophic chondrocytes, the expression of Runx2 and Vegf is

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enhanced—contributing to the invasion from the perichondrium into hypertrophic cartilage of blood vessels, cartilage resorbing cells, and osteoblast precursor cells.116 Ihh also enhances osteoblast differentiation in perichondrium. Subsequently, a primary ossification center is formed—and cartilage is replaced by trabecular bone and marrow (endochondral bone formation). Stromal mesenchymal stems cells differentiate not only into osteoblasts [through Wnt signaling of Runx2, Dlx5 (another gene capable of stimulating osteoblastogenesis), and osterix] but into chondroblasts (through Sox9), adipocytes [through peroxisome proliferatoractivated receptor 2 (PPAR2)], myoblasts, and fibroblasts (Figure 3-8). Committed osteoblasts and adipocytes appear to be able to re-differentiate one into the other cell type, depending on whether the expression of Runx2 or Pparg is paramount. This process is directed by Wnt10b, which enhances expression of Runx2, Dlx5, and Sp7 while suppressing that of Pparg.112 The nuclear NAD-dependent protein deacetylase encoded by Sirt1 also inhibits the adipocyte-differentiating effects of PPAR2 by docking corepressors to this transcription factor, thereby diverting mesenchymal stem cells into the osteoblastogenic pathway.130 BMP-2, -4, and -7 induce osteoblastogenesis acting through their heterodimeric cell surface receptors and transduce their intracellular signals through receptor-regulated SMADs 1, 5, and 8 (which heterodimerize with DNA-binding SMAD 4 to induce expression of Runx2, Dlx5, and Sp7).131,132 Osteoblasts have a life span of 3 months. They secrete type I collagen and many noncollagenous proteins that form osteoid into which calcium and phosphate are deposited as hydroxyapatite. In mature osteoblasts, Runx2, Dlx5, and osterix promote the synthesis of osteoblast-restricted proteins—including collagen type I(1) and noncollagenous matrix proteins such as bone-specific alkaline phosphatase, osteocalcin, fibronectin, osteonectin, and osteopontin. Several factors (including TGF, platelet-derived growth factor, FGF, and IGF) enhance the proliferation and further differentiation of committed osteoblast precursors, but they cannot initiate this process. Osteoblasts are heterogeneous and express diverse genes independent of the stage of the cell cycle and extent of differentiation. An early marker of the osteoblast is expression of bone alkaline phosphatase.112 The heterogeneity of osteoblasts may relate to the variety of bone architectures and microenvironments. Actively bone-forming osteoblasts have an enlarged nucleus, plentiful Golgi apparatus, and abundant endoplasmic reticulum. When the rate of bone formation is low, osteoblasts are small and quiescent and incorporated into the endosteum separating bone mineral from marrow or into the undersurface of the periosteum. Once differentiated, mature osteoblasts secrete collagenous and noncollagenous proteins—including collagen type I, bone-specific alkaline phosphatase, and calcium and phosphate binding proteins (osteocalcin, osteopontin, and osteonectin)—thus making bone matrix competent for mineralization. They also secrete many other proteoglycans and glycoproteins.133 Osteocalcin, osteonectin, and various phosphoproteins ac-

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count for approximately 10% of the noncollagenous proteins of bone matrix.110 Osteoblasts control mineralization of matrix by regulating local concentrations of phosphate through synthesis of cell-membrane–bound alkaline phosphatase and by reducing levels of inhibitors of bone formation such as pyrophosphates. Calcium and phosphate precipitate in bone matrix as hydroxyapatite crystals. After completing matrix synthesis and local bone formation, mature osteoblasts are embedded in bone as osteocytes (the most abundant cell in bone). Osteocytes are interconnected with each other and with surface osteoblasts by extension of plasma membranes through narrow canaliculi. They function as sensors of bone strength and mechanical integrity and act to identify sites of bone damage requiring repair (i.e., remodeling).112 Osteocytes synthesize osteocalcin and sclerostin but not alkaline phosphatase. Osteocytes may live for several decades in quiescent bone but ultimately die. Receptors for PTH1-84 (PTHR1) and calcitriol (VDR) are expressed by osteoblasts.134 PTH stimulates growth of osteoblast progenitor cells and inhibits apoptosis of osteoblasts and osteocytes, thereby enhancing bone formation and accounting for its therapeutic usefulness in treatment of osteopenic states. However, PTH1-84 also promotes bone resorption via osteoblast production of an osteoclast-activating factor that increases osteoclastogenesis. In osteoblasts, carboxyl terminal fragments of PTH affect generation of alkaline phosphatase, procollagen I, and apoptosis—some of which effects may be opposite those of PTH1-84. There is a high density of binding sites for carboxyl fragments of PTH on osteocyte membranes, suggesting that these sequences may play a role in the mechano-sensory activity of the osteocyte network.55 Calcitriol increases synthesis of several noncollagenous matrix proteins, including osteocalcin. Glucocorticoids increase differentiation of osteoprogenitor cells but also accelerate apoptosis of osteoblasts and osteocytes.112 Estrogens stimulate osteoblast proliferation and synthesis of collagen type I and inhibit apoptosis of osteoblasts and osteocytes. They enhance osteoclast apoptosis. Estrogens stimulate trabecular and endosteal bone growth and are thought to exert a biphasic effect on periosteal bone growth. Thus, in prepubertal boys and girls low amounts of estrogens enhance periosteal bone growth—whereas in pubertal and adult subjects estrogens oppose this process. Estrogens also accelerate growth plate fusion and inhibit bone resorption. Androgens primarily promote mineralization by conversion to estrogens, as evidenced by the osteopenia noted in adult males with aromatase deficiency or loss-of-function mutations in estrogen receptor (ER).135 However, androgens also have a direct effect on bone mineralization because acting through the androgen receptor they increase periosteal bone growth during puberty in both males and females. However, the stronger bones of men compared to women reflect not increased volumetric bone mineral density but rather increased bone size due to greatly expanded periosteal bone width.136 Increased periosteal bone width in males is due in part to the androgen-induced effect of increased muscle mass, strain, and mechanical loading on bones. Nevertheless,

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estrogens too are necessary for normal periosteal bone growth because despite normal testosterone secretion the aromatase-deficient male has decreased periosteal bone width—a situation that can be reversed by administration of estrogen acting through ER. A portion of the anabolic effects of estrogens on bone mineralization may be mediated through their stimulation of the GH/IGF-I axis. GH enhances proliferation and differentiation of osteoblast precursors, whereas IGF-I increases osteoblast function and trabecular and cortical bone volume and protects osteoblasts and osteocytes from apoptosis.112 IGF-I also mediates (some of) the stimulatory effects of PTH on osteoblast function.137 The endogenous GH secretagogue ghrelin promotes proliferation and differentiation of osteoblasts and bone mineralization in vivo.138 Leptin secreted by adipocytes or osteoblasts acting locally in bone exerts anabolic effects and promotes bone formation, whereas centrally active (hypothalamic ventromedial nucleus) leptin both impairs and promotes bone formation.110 In addition to their pivotal role in bone formation, osteoblasts and stromal cells regulate bone resorption by controlling the differentiation, maturation, and function of osteoclasts (Figure 3-10). They do so by expressing RANKL (an osteoclast activator) and a decoy acceptor protein for RANKL that inhibits osteoclastogenesis [osteoprotegerin (OPG)] in response to PTH, calcitriol, interleukins, and other cytokines (e.g., TNF) and in response to prostaglandins. RANKL [also termed osteoprotegerin-ligand

(OPGL)] is a member of the TNF ligand superfamily and is encoded by TNFSF11. It is expressed on the surface of (and its extracellular domain secreted by) bone marrow stromal cells and osteoblasts. RANKL binds to RANK expressed on the surface of primitive osteoclast progenitor cells, where it induces their further differentiation and activation (Figure 3-11).139,140 (Transcription factors important in the very early commitment of the mesenchymal stem cell to the osteoclast lineage are those encoded by PU.1, c-Fos, and MI.141) RANKL is a 317-aa protein composed of cytoplasmic (48 aa), transmembrane (21 aa), and extracellular (248 aa) domains—with the binding site for RANK extending between aa 137 and aa 158. In the promoter region of TNFSF11 is a response element for RUNX2, the osteoblast-differentiating transcription factor. TNFrelated activation-induced cytokine (TRANCE) is the soluble extracellular domain of RANKL released by a specific metalloprotease. RANKL is also expressed in skeletal and lymphoid tissue, striated and cardiac muscle, lung, intestines, placenta, thyroid, pre-chondroblast mesenchymal cells, and hypertrophic chondrocytes. In addition to furthering osteoclast differentiation, RANKL enhances function of the mature osteoclast and inhibits its apoptosis. RANKL stimulates transcription of osteoclast-specific proteins such as tartrate-resistant acid phosphatase (TRAP), cathepsin K, 3-integrin, and the calcitonin receptor. It also stimulates development of calcium resorption lacunae and pits. In

Osteoclastogenesis Prostaglandins, multiple hormones, cytokines, ILs and vitamin D E2 Stromal/osteoblastic cells

GCs

T

T

TGF

IFN

T

OPG, RANKL, M-CSF

TNF, IL-1, IL-6, IL-7, other ILs

RANKL TNF

HSC M-CSF c-FmsRANK-

c-Fms+ RANK-

c-Fms+ RANK+

M-CSF
RANKL

OPG Figure 3-10. Regulation of osteoclast differentiation from the pluripotent mesenchymal stem cell. Osteoclasts differentiate from precursor cells of monocyte/macrophage lineage in response to osteoblast/stromal cell secretion of macrophage-colony–stimulating factor (M-CSF) stimulated by parathyroid hormone (PTH) and other osteoclast-activating cell factors acting through its receptor (c-Fms) and stromal cell/ osteoblast derived ligand for the receptor for activation of nuclear factor B (RANKL). [Reproduced with permission from Ross FP (2006). Osteoclast biology and bone resorption. In Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 30-35.]

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Effect of RANKL and OPG on osteoclast differentiation

B

Osteoblast precursor RANKL M-CSF RANK

c-fms Osteoclast precursor

Fusion and differentiation of osteoclast precursors

C

Mature osteoclast

Osteoclast precursor RANKL M-CSF

OPG RANK

c-fms Inhibition of Osteoclast osteoclast formation Mature osteoclast precursor Figure 3-11. Interaction of RANK, RANKL, and osteoprotegerin in osteoclastogenesis. Osteoprotegerin serves as a pseudoreceptor for RANKL, thus preventing its association with RANK and thereby inhibiting osteoclastogenesis. [Reproduced with permission from Rogers A, Eastell R (2005). Circulating osteoprotegerin and receptor activator for nuclear factor kB ligand: Clinical utility in metabolic bone disease assessment. J Clin Endocrinol Metab 90:6323-6331.]

RANKL knockout mice, loss of osteoclasts leads to osteopetrosis. Because RANKL also affects differentiation and function of the immune system, these animals have thymic hypoplasia and lymph node agenesis. BMPs too stimulate osteoclast formation and function.142 OPG is a member of the TNF receptor superfamily and is synthesized and secreted by the stromal cell/osteoblast. It acts as a “decoy” receptor by binding to RANKL, thus inhibiting the interaction of RANKL and RANK and thereby osteoclastogenesis.134,139 The 5 exon gene (TNFRSF11B) encoding human OPG is expressed also in the lung, liver, heart, kidney, intestinal cells, brain, thyroid, lymphocytes, and monocytes. Human OPG is synthesized as a 401-aa propeptide. After cleavage of the 21-aa signal peptide, the mature protein of 380 aa contains four cysteine-rich amino terminal domains and two carboxyl terminal “death” domains. It is glycosylated and released into the paracellular space as a disulfide-linked homodimer. The synthesis of OPG is enhanced by IL-1 and -1, TNF, TNF, BMP-2, TGF, and estrogen—and is antagonized by calcitriol, glucocorticoids, and prostaglandin E2. Through binding of RANKL, OPG inhibits the osteoclast-activating and bone-absorbing effects of calcitriol, PTH, and the interleukins. Overexpression of OPG in transgenic mice leads to osteopetrosis, whereas

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its knockout is associated with loss of cortical and trabecular bone and osteoporosis, multiple fractures, and hypercalcemia. The latter model is the experimental counterpart of juvenile Paget disease.143,144 In response to macrophage colony-stimulating factor (M-CSF) secreted by both mature osteoblasts and bone marrow stromal cells that acts through its receptor c-Fms expressed on the membrane of preosteoclasts, osteoclast progenitor cells further proliferate and differentiate.134,140,141 Also expressed on the plasma membrane of the preosteoclast is transmembrane RANK (TNFRSF11A), a member of the TNF receptor superfamily. It is a 616-aa protein with a signal peptide (28 aa), cytoplasmic domain (383 aa), transmembrane domain (21 aa), and extracellular domain (184 aa) that is expressed in osteoclasts, fibroblasts, and B and T lymphocytes (Figure 3-11). By cell-to-cell interaction, RANK on the surface of the prefusion osteoclast binds to RANKL expressed on the membrane of the osteoblast and/or stromal cell (or secreted by a monocyte) to induce osteoclast fusion and activation, leading to bone resorption. After binding of its ligand, RANK signals [through TNF receptorassociated factors (TRAFs)] a family of intracellular adaptor proteins that bind to the intracellular domain of RANK and activates several intracellular signaling cascades—including the classical pathway of nuclear factor B (NFB) activation, JNK-AP1, c-src-PI3K-AKT/ PKB, and Ca2
-calmodulin-calcineurin.140 In turn, there is enhanced generation of c-Fos, c-Jun, NFAT1c, and NFB—transcription factors essential for osteoclast formation and function. Synthesis of interferon- is also increased. This cytokine limits expression of c-Fos and consequently osteoclast differentiation, thus establishing an autoregulatory system for osteoclast production.145 Osteopetrosis develops in transgenic mice in whom RANK is overexpressed or in whom NF-B or TRAF-6 has been knocked out. In TRAF-6-depleted mice, osteoclasts develop normally but are not activated and do not form ruffled borders (structures essential to osteolysis). NFB subunit 1 (NFKB1) is a component of a widely expressed “master” transcription factor complex essential for cell differentiation, growth, and function within the hematopoietic and immune systems and the key regulatory element in osteoclastogenesis. The NFB complex is composed of five subunits: NKB1, NFB2, RELA, RELB, and c-REL.146 The subunits form homodimers or heterodimers with one another (e.g., NFB1/RELA and NFB2/ RELB), which are trapped in the cytoplasm bound to specific inhibitors of NFBs or IBs (e.g., NFKB1A). After cell stimulation, phosphorylation of serine residues on IB proteins targets them for ubiquitination and proteasomal destruction—thereby freeing the NFB dimer and allowing it to enter the nucleus and activate target genes. Within the nucleus there are DNA-binding motifs for NFB (e.g., 5'-GGGA/GNNC/TC/TCC-3') that control the transcription of several hundred genes involved in cell replication and apoptosis, including the interleukins, TNFs, VEGF, and colony-stimulating factors. The intricate relationship between osteoblasts and osteoclasts provides the pathway(s) through which bone formation and

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bone resorption are linked. Thus, BMP and other factors induce osteoblast differentiation and production of RUNX2—leading to synthesis of RANKL that binds to RANK of the osteoclast precursor and enhances further differentiation and function of mature osteoclasts.132 The effects of modifying agents and of disease processes on bone formation and bone resorption are mediated to a large extent by their influence on the RANKL-RANK-OPG system. Particularly at the level of the osteoblast/stromal cell production of RANKL and OPG may these effects be exerted, with the relative proportion of the two products being synthesized (or their ratio) regulating osteoclastogenesis and bone resorption.139 Mature osteoclasts are short-lived (approximately 2 weeks) bone-reabsorbing multi-nucleated giant cells. When attached to bone, the inferior surface of the osteoclast forms the ruffled border—a series of villus projections that bind to underlying bone at the site of bone resorption, creating an isolated microenvironment between the inferior surface of the cell membrane and the outer bone surface (Figure 3-12).140,141 These villus structures anchor to the bone surface through interaction of dimeric v3 integrins expressed on the osteoclast cell membrane and matrix-embedded osteopontin and other components that contain the amino acid sequence Arg-Gly-Asp (RGD). Beneath this shield and into the isolated sealed zone the osteoclast pumps acid (H
or protons) generated from carbon dioxide by carbonic anhydrase II and trans-

Osteoclast Function HCO3- CI-

Cath K

H+

HCO3- CI-

v3 H+

CI-

Bone Figure 3-12. Differentiated osteoclasts form a ruffled border by adhering to bone surface through v3 integrin receptors. A sub-osteoclast lacuna is formed by the dissolution of bone mineral and the resorption of organic bone matrix by osteoclast secreted acid and cathepsin K, respectively. Subsequently, osteoblasts are attracted to this pit (perhaps by the high local Ca2
concentration) as new bone is formed in the continuing process of bone remodeling. [Reproduced with permission from Ross FP (2006). Osteoclast biology and bone resorption. In Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 30-35.]

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ported via an electrogenic proton pump and chloride ions transmitted through a chloride channel to form a highly acidic (pH 4.5) milieu that dissolves hydroxyapatite, the mineral phase of bone. The osteoclast also pumps lysosomal proteolytic enzymes [such as the cysteine proteases cathepsins K, B, and L and matrix metalloproteinases (MMP) such as MMP-9 a collagenase] that digest osteoid, the protein matrix of bone. Mutations in the genes controlling carbonic anhydrase II or the chloride channel result in osteopetrosis, whereas mutations in the gene encoding cathepsin K lead to pycnodysostosis (OMIM 265800). The heterodimeric v3 integrin (vitronectin receptor) consists of two transmembrane proteins (v integrin, 3 integrin) with extra- and intracellular domains that attach the osteoclast to bone surface. They are crucial to normal osteoclast function. The integrins also transmit signals between the interior and exterior of the osteoclast. After the osteoclast contacts bone, it functionally polarizes into two realms. The inferior portion of the osteoclast above the ruffled membrane transports protons, chloride ions, and enzymes from the interior of the cell into the subcellular space and reabsorbs the products degraded by these agents. The superior portion of the osteoclast processes and excretes the reabsorbed materials. The absorptive lacuna is surrounded and isolated by a “sealing zone,” whereas the osteoclast is encircled by an actin ring.140 In the 3 integrin knockout mouse, the ruffled membrane is abnormal and osteoclast function impaired. Osteoclasts withdraw from the sites of bone resorption in response to the high local concentration of Ca2
.32 PTH1-84, PTHrP, calcitriol, thyroid hormone, IL-1, -3,-6, and -11, TNF, prostaglandin E2, and glucocorticoids stimulate expression of RANKL and M-CSF and depress that of OPG and hence favor osteoclast development and bone resorption. Calcitonin, estrogen, interferon-, IL-4, -10, and -18, TGF, glucocorticoids, and bisphosphonates antagonize these processes.140 Estrogen maintains and augments bone mass by inhibiting its dissolution by suppressing T-cell production of osteoclast-activating cytokines such as IL-1, -6, and TNF and by suppressing expression of RANKL and increasing that of TGF. Glucocorticoids decrease bone mineralization by depressing osteoblast differentiation, function, and life span and by prolonging the life span of osteoclasts. Receptors for calcitonin and to a limited extent for PTH are expressed by osteoclasts. Calcitonin antagonizes formation and activity of osteoclasts and accelerates their death. Interestingly, carboxyl terminal fragments of PTH1-84 can inhibit osteoclast formation and function and antagonize the osteoclast-stimulating effects of PTH1-84, calcitriol, prostaglandins, and interleukins.55 Thus, they may exert a protective effect upon the skeleton and perhaps work in concert with intact PTH1-84. Bone modeling is accomplished by the independent action of osteoblasts and osteoclasts and is not dependent on prior bone resorption.111 On the other hand, during bone remodeling (the process during which the strength, structure, and function of bone are renewed) bone resorption and deposition are sequentially linked. Bone remodeling is accomplished within the bone remodeling unit (BRU) of designated osteoclasts and osteoblasts. It is

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DISORDERS OF CALCIUM METABOLISM

a continuous process in which old cancellous and cortical bone is reabsorbed and replaced by new bone and takes place in the growing as well as the mature skeleton. The BRU is 1 to 2 mm in length, 0.2 to 0.4 mm in width, led by osteoclasts, and trailed by osteoblasts. In the adult skeleton, the life span of the BRU is 6 to 9 months—and 10% of the skeleton is turned over each year. The site selected for remodeling may be random or may be targeted by osteocytes sensing a mechanical or stress defect. Bone remodeling occurs in four stages. Stage 1 is activation, in which osteoclast precursor cells target a resting bone surface, evolve into osteoclasts, convert the area into a BRU composed of a sub-osteocytic bone resorption compartment, and initiate stage 2. Stage 2 is bone resorption, during which the mineral phase of bone is solublized by acid and the protein component degraded by proteases. When complete, the osteoclast dies. This phase is followed by stage 3, reversal—in which monocytes, osteocytes, and osteoblasts perhaps attracted to the BRU through their detection of the high Ca2
concentrations in the resorption lacunae or by growth factors (IGF-I and -II, TGF, BMP) released from the matrix or secreted by osteoclasts enter the area of reabsorbed bone and initiate the fourth stage. Stage 4 is renewed bone formation, in which osteoid is secreted, increase local concentrations of calcium and phosphate to levels that exceed their solubility, and are degraded pyrophosphates and proteoglycans that inhibit mineralization. The majority of osteoblasts and osteoclasts in the BRU are eliminated by programmed cell death (apoptosis), whereas some osteoblasts develop into osteocytes.132 Organic matrix proteins comprise 35% of bone, and type I collagen makes up 90% of these proteins.133 Type I collagen is composed of a coiled triple helix of two polypeptide chains of collagen type I(1) and one of (2) that are cross-linked intramolecularly by disulfide bonds and intermolecularly at the amino (N) and carboxyl (C) telopeptides by pyridinium compounds that permit bun-

105

dling of collagen molecules into fibrils and fibers (Figure 3-13).147,148 Glycine occupies every third aa position in the collagen  peptides and permits the chains to coil. Proline, hydroxyproline, and hydroxylysine are also incorporated in large quantities. Proline is hydroxylated to 4-hydroxyproline and 3-hydroxyproline by prolyl 4-hydroxylase and prolyl 3-hydroxylase-1 (P3H1), respectively. Lysine is hydroxylated by lysyl hydroxylase and some hydroxylysine residues further glycosylated. P3H1 (also termed leprecan, LEPRE1) specifically hydroxylates the proline residue at codon 986 in bone collagen type I(1), a reaction that requires interaction of P3H1 with cartilage-associated protein (CRTAP) and cyclophilin B (PIPB). CRTAP is expressed in the proliferative zone of developing cartilage and at the chondroosseous junction. Cyclophilin B is a peptidyl-prolyl cis-trans isomerase. That post-translational modifications of collagen type I(1) are essential to normal bone formation is evidenced by the association of inactivating mutations of CRTAP with osteogenesis imperfecta types IIB (OMIM 610854) and VII (OMIM 610682) and of LEPRE1 with osteogenesis imperfecta type VII.149-151 Amino and carboxyl terminal extensions of the propeptides of collagen are removed by proteolysis during formation of the mature collagen molecule and are partially secreted into extracellular space and serum. Pyridinoline (Pyr, hydroxylysyl-pyridinoline) and deoxypyridinoline (Dpd, lysyl-pyridinoline) form nonreducible pyridinium cross-links between mature collagen fibers, thus making them insoluble (Figure 3-14). Type I collagen predominates in bone but is also present in ligaments, tendons, fascia, and skin. Type II cartilage is composed of three procollagen type II(1) chains and is primarily deposited in cartilage. Type III collagen [three procollagen type III(1) chains] is present in bone, tendons, arteries, and intestine, and type IV [three procollagen type IV(1)] cartilage is a component of cell basement membranes.

Type I Collagen



3000 A B

C

Globular Triple domain helix

F

D

E

Telopeptide

Collagen



15A

G

Telopeptide

Triple-helical domain Amino-terminal propeptide

Carboxyl-terminal propeptide Procollagen

Figure 3-13. Type I collagen is a coiled triple helix of two polypeptide chains of collagen 1(I) and one of 2(I) that are cross-linked intramolecularly by disulfide bonds and intermolecularly at the amino and carboxyl telopeptides by pyridinium compounds. In the process of collagen formation, carboxyl and amino terminal propeptides are removed. [Reproduced with permission from Byers PH (1995). Disorders of collagen biosynthesis and structure. In Scriver CR, Beaudet AL, Sly WS, Vale D (eds.),,The metabolic and molecular bases of inherited disease, Seventh edition. New York: McGraw-Hill 4029-4077.]

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DISORDERS OF CALCIUM METABOLISM

Crossed-Linked Telopeptides Type I collagen molecules within bone matrix

N-telopeptides

C

N

Pyr or D-Pyr C-telopeptides

Serum NH

20% free CH CH

CH

80% peptide bound

COOH

COOH NH CH CH2

C-telopeptides

N-telopeptides

OH

renal degradation CH2 CH (OH) CH2 CH1 CH NH2

COOH

Free Pyr and D-Pyr

Pyr or D-Pyr NH2

COOH CTX

NH2

COOH NTX

Crosslinked C and N-telopeptides

Urine 60% peptide bound 40% free Figure 3-14. Pyridinium and telopeptides of collagen. Pyridinoline (Pyr, hydroxylysyl-pyridinoline) and deoxypyridinoline (D-Pyr or Dpd, lysyl-pyridinoline) form nonreducible pyridinium cross-links between mature collagen fibers, rendering them insoluble. Amino (N-) and carboxyl (C-) terminal extensions (NTx, CTx) of the propeptides of collagen are proteolytically removed during formation of mature collagen and secreted into extracellular space and serum. [Reproduced with permission from Garnero P, Delmas PD (1998). Biochemical markers of bone turnover: Applications for osteoporosis. Endocrinol Metab Clin NA 27:303-323.]

Measurement of serum concentrations of the procollagen I extension peptides such as the C-terminal extension of procollagen type I (PICP) and the N-terminal extension of procollagen type III (PIIINP) provides information about bone and collagen formation (as does determination of the osteoblast products, osteocalcin and bone-specific alkaline phosphatase). Degradation of mature bone matrix by osteoclasts catabolizes type I collagen and releases Pyr, Dpd, and the N- and C-telopeptides [NTx; ICTP (CTx)  amino or carboxyl terminal cross-link telopeptide of type I collagen, respectively]. The urinary excretion of hydroxyproline, hydroxylysine, Pyr, Dpd, and NTx reflects catabolism of collagen type I and bone resorption. Serum/urine levels of markers of bone formation and resorption are higher in the fetus than mother152 (Table 3-6). Fetal umbilical cord plasma PICP concentrations are highest in mid-gestation and decline in the last trimester to term values. After birth, PICP values fall in preterm neonates during the first 3 days of life and then increase steadily to peak values at 36 weeks postconceptual age. PICP levels in cord plasma are higher in males than females and correlate with gestational age and birth weight.153 Urine levels of NTx at 2 days of age are higher in preterm than term neonates. Markers of bone turnover increase during the first 3 weeks of life in preterm infants.154 Values of bone formation and resorption markers are highest in infants and decline during childhood and adolescence to adult levels155-160 (Tables 3-7A and B). In the normally menstruating young adult female, urine Pyr and Dpd levels fall during the first half of the cycle and increase during the second half. Serum concentrations of PICP have the reverse pattern. These data suggest that

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the fluctuations in estrogen secretion during the menstrual cycle alter the dynamics of bone formation and resorption.161 Osteoprotegerin and RANKL are also measurable in serum, but their utility in children and adolescents has yet to be assessed.139 The adult skeleton is composed of mineral (50%70%), organic matrix (20%-40%), water (5%-10%), and lipids ( 3%). Ten percent to 15% of bone matrix is composed of noncollagenous peptides secreted by the osteoblast, including proteoglycans (chondroitin sulfate, heparan sulfate), glycoproteins, growth-stimulating proteins (BMP, TGF, IGF-I, and IGF-II), cell attachment peptides (integrin ligands such as osteopontin, osteonectin, fibronectin), and -carboxylated proteins or proteins derived from serum (e.g., albumin).133 Macromolecular proteoglycans are composed of glycosaminoglycans (acidic polysaccharide side chain) linked to a core protein and are important to normal synthesis of collagen and bone development. Osteonectin is a phosphorylated 35- for 45-kDa glycoprotein that binds Ca2
and is necessary for growth and survival of osteoblasts and osteoclasts and for normal mineralization of matrix. Alkaline phosphatase is an 80-kDa glycoprotein essential to bone mineralization. Osteopontin (also termed bone sialoprotein or secreted phosphoprotein 1) is an 85-kDa sulfated and phosphorylated glycoprotein that contains the aa sequence [Arg-Gly-Asp (RGD)] necessary for linkage to integrins and hence for attachment of osteoclasts to bone. It also binds Ca2
and hydroxyapatite and may play a role in the initiation of bone matrix mineralization. Osteopontin is secreted by osteoblasts in response to calcitriol. Osteocalcin is a 49-aa -carboxylic acid-containing 6-kDa peptide that likely

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DISORDERS OF CALCIUM METABOLISM

TA B L E 3 - 6

Markers of Bone Formation and Resorption in Mothers and Neonates

Bone Formation Osteocalcin ng/mL ±SD Bone Alkaline Phosphatase ± IU/L

Maternal

Full-term Cord

19.61 2.6 2731 11

3.31 0.3 691 12

24-29 wk

30-34 wk

Cord

Cord

M

F

29WK

M

F

14.12 9.4

M

F

822 (475-1420)

8243 246-1450)

1950 (402-3372)

1103 (400-1808)

1392 (4732939)

6733 (1282277)

PICP µg/L Bone Resorption ICT µg/L Urine Pyridinoline Total Free Deoxypyridinoline Total Free N-Telopeptide (nmolBCE/mmol Creatinine)

2.71 ±0.2

4.61 0.1 623 ± 2352 217 ± 92

645 ± 2272 248 ± 96

101 ± 41 33 ± 14 1302 ± 790

106 ± 43 34 ± 14 3008 ± 1185

1. Yamaga A, et al. (1999). Comparison of bone metabolic markers between maternal and cord blood. Horm Res 51:277–279. 2. Naylor KE, et al. (1999). Bone turnover in premature infants. Pediatr Res 45:363–366. 3. Seibold-Weiger K, et al. (2000). Plasma concentrations of the carboxyterminal propeptide of type I procollagen (PICP) in preterm neonates from birth to term. Pediatr Res 48:104-108.

regulates osteoclast activity and the interface between bone resorption and formation, whose synthesis is dependent on vitamin K activation of -carboxylases. Calcium and phosphate deposit in extracellular matrix around the osteoblast. Type I collagen and osteopontin are important for the nucleation of hydroxyapatite and the determination of bone structure.162 In the first phase of bone formation, hypertrophic chondrocytes and/or osteoblasts initiate bone crystal formation by generating 100 nm subsurface matrix vesicles containing among other components calcium, phosphate, alkaline phosphatase, calbindin-D9K, carbonic anhydrase, pyrophosphatases, osteocalcin, and osteopontin. These vesicles are extruded from the osteoblast and attach to adjacent bone matrix—in cartilage to collagen types II and X and in bone to collagen type I. Ca2
and H2PO42- enter the vesicle through ion channels and possibly first form rudimentary crystals of calcium phosphate that enlarge, attain the structure of hydroxyapatite, and penetrate the vesicle wall. In the matrix, these crystals enlarge further and deposit about collagen type I. Mineralization of bone is partially dependent on the extent of phosphorylation of osteopontin. When 40% of the phosphorylation sites of osteopontin are phosphorylated, bone mineralization is inhibited. When 95% of its

Ch03_074-126-X4090.indd 107

sites are phosphorylated, hydroxyapatite formation is promoted—a process further enhanced by a complex of osteopontin and osteocalcin.133,163 Osteocalcin is also synthesized by osteoblasts in response to calcitriol acting through the VDR/RXR heterodimer. Inhibition of calcification is an active process as well. In addition to osteopontin, matrix GLA protein (an 84-aa vitamin K-dependent peptide containing -carboxylated glutamate that is related to but distinct from osteocalcin and encoded by MGP) has great affinity for Ca2
and inhibits precipitation of calcium and phosphate. MGP is expressed in arteries and chondrocytes but not in osteoblasts. In patients with biallelic loss-of-function nonsense mutations in MGP (Keutel syndrome, OMIM 245150), there is extensive calcification of cartilage and its experimental deletion in mice leads to calcification of both cartilage and arteries and early demise.162 Bone strength is determined by its size (height, width, depth), mineral mass, macro- and microarchitecture, and material properties (e.g., elasticity) of collagen that in turn are regulated not only by endocrine hormones and paracrine growth factors but by mechanical forces exerted upon the skeleton by the environment (gravity) and by the muscular system.164,165 Bone mass and strength are determined by the loads placed on bone by biomechanical

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TA B L E 3 - 7 A

Normal Values of Markers of Bone Deposition and Resorption in Male Children and Adolescents Bone Formation Bone-specific alkaline phosphatase1,7 (g/L) CA 1 2 103 62 (89-114) (52-72) P1* P2 CA 10.5 12.1 41.3 54.0 SD ± 19.2 27.2 P1* P1* CA 3-6 6-12 Osteocalcin2 74 56 (g/L) (49-93) (29-88) PICP2,3 372 (g/L) (193-716) (264-556) PIIINP2 593 (g/L) (435-941) (223-1,000)

5 74 (62-86) P3 13.2 59.8 33.1 P2

10 88 (74-102) P4 13.8 53.6 25.1 P3

12 90 (74-106) P5 15.1 33.3 36.7 P4

14 102 (82-122)

43 (28-45) 380 (244-650) 704 (428-1,000)

41 (23-56) 398 (384-1,066) 920 (720-1,000)

50 (31-67) 561 (312-692) 882

50 (43-55) 536

P3

P4

P5

15.8 (14.1-35.4)

23.5 (18.4-25.0)

22.6

16 96 (78-114)

18 62 (38-86)

P5

Bone Resorption, P1* 3-6

P1* 6-12 9.3 (10.7-30.3) 11-17 2-118 17-410

P2 CA ICTP2,3 13.6 (g/L) (5.1-17) (11.6-30.2) CA 2-10 Adults Dpd4 40-120 6-26 Pyr4 150-400 23-65 (nmol/mmol creatinine/2 hours) CA 4 6 8 Dpd5 20 20 14 (17-24) (15-22) (12-16) Pyr5 69 68 58 (59-85) (53-77) (46-63) (median - nmol/mmol creatinine - first morning void) P1* P2 P3 7 Dpd 258.8 313.0 273.1 SD ± 68.4 106.1 73.0 Pyr7 70.3 78.2 71.0 SD ± 21.9 29.3 22.1 (mean - nmol/mmol creatinine/2 hours) CA

1 5 10 NTx6 1988 640 425 (507-4942) (189-1056) (150-773) (nmol BCE**/mmol creatinine/2 hours)

10 17 (13-19) 57 (46-67) P4 245.1 94.3 61.8 25.8

P5 141.9 83.3 40.4 32.3

15 376 (133-1146)

>20 71 (43-154)

CA  chronologic age (years). Data presented as mean or median (range or SD). * Pubertal stage. ** Bone collagen equivalent units. 1. Tobiume H, et al. (1997). Serum bone alkaline phosphatase isoenzyme levels in normal children and children with growth hormone (GH) deficiency: A potential marker for bone formation and response to GH therapy. J Clin Endocrinol Metab 82:2056-2061. 2. Sorva R, et al. (1997). Serum markers of collagen metabolism and serum osteocalcin in relation to pubertal development in 57 boys at 14 years of age. Pediatr Res 42:528-532. 3. Crofton PM, Wade JC, Taylor MRH, Holland CV (1997). Serum concentrations of carboxyl-terminal propeptide of type I procollagen, amino-terminal propeptide of type III procollagen, cross-linked carboxy-terminal telopeptide of type I collagen, and their interrelationships in school children. Clin Chem 43:1577-1581. 4. Nichols Institute/Quest #432-04/93. 5. Hussain SM, et al. (1999). Urinary excretion of pyridinium crosslinks in healthy 4-10 year olds. Arch Dis Child 80:370-373. 6. Bollen A-M, Eyre DR (1994). Bone resorption rates in children monitored by the urinary assay of collagen type I cross-linked peptides. Bone 15:31-34. 7. Mora S, et al. (1999). Biochemical markers of bone turnover and the volume and the density of bone in children at different stages of sexual development. J Bone Miner Res 14:1664-1671.

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109

TA B L E 3 - 7 B

Normal Values of Markers of Bone Deposition and Resorption in Female Children and Adolescents Bone Deposition CA 1 2 Bone-specific alkaline phosphatase1,7 (g/L) 84 78 (70-98) (66-90) P1* P2 CA 9.8 11.8 47.4 49.8 SD ± 22.7 26.8 P1* P1* CA 3-6 6-12 Osteocalcin 69 44 (g/L) (33-88) (24-70) P1* P2 PICP2 374 408 (g/L) (242-406) (258-558) Bone Resorption CA 4-8 9-13 9.2 12.0 ICTP3 (g/dL) (5.7-14.9) (7.2-20) CA 2-10 11-17 Dpd4 40-120 2-118 Pyr4 150-400 17-410 (nmol/mmol creatinine/2 hours) CA 4 6 Dpd5 21 19 (17-25) (14-21) (13-18) Pyr5 77 67 (64-85) (58-78) (50-67) (median - nmol/mmol creatinine - first morning void) P1* P2 Dpd7 247.1 337.7 SD ± 249.5 120.4 Pyr7 69.8 97.6 SD ± 75.8 45.6 (mean - nmol/mmol creatinine/2 hours) CA

1 1 NTx6 2218 1207 (872-45,702) (477-2,752) (nmol BCE/mmol creatinine)

5

10

12

14

16

70 (58-82) P3 12.2 40.4 15.8 P2

78 (62-94) P4 12.9 38.3 32.0 P3-P4

96 (84-108) P5 14.2 41.3 27.2 P5

49 (33-67)

30 (22-38)

88 (50-134) P3 442 (307-577)

62 (33-86) P4 401 (190-612)

35 (11-77) P5 203 (100-306)

22 (12-38)

14-15 7.4 (4.1-13.3) Adults 6-26 23-65

16-18 5.0 (2.9-8.5)

8 17 (13-18) 61 (46-71)

10 14

P3 250.0 96.7 68.5 31.1

P4 213.2 91.1 59.8 29.0

P5 124.0 43.3 33.8 19.1

5 728 (335-1,615)

10 515 (116-12,410)

15 217 (107-653)

Adult

50

20 67 (13-137)

1. Tobiume H, et al. (1997). Serum bone alkaline phosphatase isoenzyme levels in normal children and children with growth hormone (GH) deficiency: A potential marker for bone formation and response to GH therapy. J Clin Endocrinol Metab 82:2056-2061. 2. Hertel NT, et al. (1993). Serum concentrations of type I and III procollagen propeptides in healthy children and girls with central precocious puberty during treatment with gonadotropin-releasing hormone analog and cyproterone acetate. J Clin Endocrinol Metab 76:924-927. 3. Crofton PM, et al. (1997). Serum concentrations of carboxyl-terminal propeptide of type I procollagen, amino-terminal propeptide of type III procollagen, cross-linked carboxy-terminal telopeptide of type I collagen, and their interrelationships in school children. Clin Chem 43:1577-1581. 4. Nichols Institute/Quest #432-04/93. 5. Hussain SM, et al. (1999). Urinary excretion of pyridinium crosslinks in healthy 4-10 year olds. Arch Dis Child 80:370-373. 6. Bollen A-M, Eyre DR (1994). Bone resorption rates in children monitored by the urinary assay of collagen type I cross-linked peptides. Bone 15:31-34. 7. Mora S, et al. (1999). Biochemical markers of bone turnover and the volume and the density of bone in children at different stages of sexual development. J Bone Miner Res 14:1664-1671.

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forces exerted by muscles (the “mechanostat” model).166 In this model, bone tissue monitors the stresses and strains (deformations) that are the result of mechanical forces placed upon it.167 In part, mechanical forces are recognized by osteocytes, whose rate of apoptosis declines in response to these dynamic changes. In addition, stromal cells modify their expression of RANKL—and thus the process of osteoclastogenesis. Deformation of the osteoblast cell membrane induced by mechanical forces (e.g., stretch, gravity, vibration) generates signals that are transmitted through alterations in configuration of its cytoskeleton that act upon cytoplasmic organelles and within the nucleus itself, increasing expression of transcription factors such c-Fos and c-Myc. In response to mechanical stress, multiple growth factors likely generated by the osteoblast itself (FGF, IGF-I, TGF) act in an autocrine/paracrine manner upon their tyrosine kinase receptors expressed in the cell membrane of the osteoblast to activate phosphoinositide 3-kinase, PKB, and the MAPK signal transduction system. Prostaglandins activate G-protein–coupled receptors, adenylyl cyclase, PKA, and the cyclic AMP responsive transcription factor. PLC generation leads to increase in cytosolic Ca2
i and osteoblast function, as does influx of Ca2
through L-type calcium channels in the cell membrane.168 One of the target genes affected by mechanical stimulation is Runx2, whose product is essential to osteoblast differentiation and expression and synthesis of osteoblastrelated proteins such as collagen type I, bone-specific alkaline phosphatase, osteopontin, and collagenase-3.169 Repetitive bone strain (an applied deforming force that might be compressive, lengthening, or angulating) leads to enhanced quantity and quality of bone (bone strength). It is this property that enables various exercises to increase bone mineralization at all ages and states of mobility. The mechanisms that lead to increased bone mass are inactivated by decreased weight bearing, such as immobilization or decreased gravity (e.g., space flight), and lead to bone loss (“disuse osteoporosis”). Although bone strength is in part dependent on bone mass, it is the size of the bone that primarily determines its strength.170 Clinically, this is illustrated by the increased rate of fractures in children with osteopetrosis (“marble bone disease”) despite extremely dense bones with thick cortices and trabeculae. Skeletal accretion of calcium begins early in fetal life and progresses through childhood and puberty. The skeleton accumulates 25 to 30 grams of calcium in utero and accrues 1,300 grams by adulthood.171 The average total body bone mineral content (BMC) of the adult male is 2,800 g, and that of the adult female 2,200 g. Approximately 60% of total adult bone calcium is acquired during adolescence (26% in the two years prior to and after the peak velocity of BMC accrual) (Figure 3-15). Thirty-two percent of the mean BMC of the adult female lumbar spine (60 g) is deposited during this interval.172 It is recommended that the adolescent ingest 1,300 to 1,600 mg of elemental calcium daily in order to absorb and retain the 220 mg per day of calcium needed to achieve optimal adult bone mineral mass. Although calcium supplementation has been reported to increase radial and femoral BMC in children, extensive review of

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the effects of calcium supplementation in approximately 3,000 children and adolescents (3–18 years of age) did not support this conclusion. Nor did supplementation decrease fracture rate.173,174 As evidenced by the close concordance of bone mineral status between mothers and daughters, identical and fraternal twins and siblings, 60 to 80% of the peak or maximum adult bone mass is determined by genetic factors.175 There are numerous candidate genes through which this parental relationship might be exercised, including those encoding the vitamin D, calcium-sensing, estrogen, low-density lipoprotein-related, leptin and -adrenergic receptors, cytokines (e.g., IL-6, TGF) and growth factors (BMPs, IGF-I), and bone matrix proteins such as type I collagen and osteocalcin—emphasizing the fact that the regulation of bone mineralization is genetically complex and heterogeneous.176 In addition to intrafamilial factors, race, sex, body size, and composition are important genetic determinants of bone calcium content. In black males and female youths, there is higher whole-body, lumbar spine, total hip, and femoral neck BMD and bone mineral apparent density (BMAD) than in white, Asian, or Hispanic youth.177 Asian female children and adolescents have lower whole-body and femoral neck BMD than do white and Hispanic subjects. Hispanic males have lower lumbar spine BMD than do white and Asian youth. Radial and femoral neck (peripheral) and vertebral (axial) BMDs correlate with sex, age, height, weight, body mass index, pubertal and postpubertal hormonal status, calcium intake, and exercise in children, adolescents, and adults.178,179 In young women, only 16% to 21% of peak vertebral and femoral bone mass can be accounted for by weight, height, physical activity as an adolescent, and the VDR genotype—emphasizing once more the essential role of multiple genetic factors in this process. Adult peak bone mass is inversely related to the risk of osteopenia and osteoporosis in later adulthood. In adults, a 10% increase in BMD reduces the risk of femoral neck fracture by 50%. Thus, it is essential that bone mass be maximized during childhood and adolescence.180 The effects of diminished physical activity on bone growth and strength are dramatically illustrated by those children with neurologic insults that prevent normal motion (Erb paresis, hemiplegia, spinal cord insults) and thus restrain limb growth.166 During prolonged bed rest, the BMC of the peripheral skeleton substantially declines—whereas that of the cranium increases. Thirty minutes of programed exercise thrice weekly increases BMC of the femoral neck and lumbar spine in prepubertal boys and girls.181,182 High-impact weight-bearing exercises (ballet, tennis, volleyball, gymnastics, soccer, rugby) increase mass of weight-bearing bones, particularly in children and adolescents.172,183,184 Exercise does so in part by augmenting periosteal bone acquisition and increasing the cortical thickness of long bones, particularly of the legs at their most distal extremes closest to the ground where weight bearing is maximal. Active boys and girls accrue more bone mineral than do relatively inactive youth at similar intakes of calcium (1,150 mg/day). At peak BMC velocity (Figure 3-15), the annual gain and total accumulation

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111

Total Body Bone Mineral Content–Accrual Rate

TB BMC Velocity Curve Cubic Spline

450

Boys

400

TB BMC velocity in grams per year

350

Age PHV 13.44 yr

300

Age of peak Peak value Size adjusted

14.05 409 394

Girls Age of peak Peak value Size adjusted

12.54 325 342

Age PHV 11.77 yr 250

200

150

100

50

0

9

10

11

12

13

14

15

16

17

18

19

Age in years Figure 3-15. During adolescence, the peak rate of accrual of total body bone mineral content (BMC) occurs in boys and girls 0.7 years after attainment of peak height velocity (PHV). [Reproduced with permission from Bailey DA, et al. (1999). A six-year longitudinal study of the relationship of physical activity to bone mineral accrual in growing children: The University of Saskatchewan bone mineral accrual study. J Bone Miner Res 14:1672-1679.]

(over 2 years) of total body bone mineral in active boys and girls are 80 g/year and 120 g/yr (respectively) greater than in inactive adolescents. One year after peak BMC velocity, the total body, femoral neck, and lumbar spine BMCs are 9% to 17% greater in active than in relatively inactive subjects.

EFFECTS OF HORMONES AND GROWTH FACTORS ON THE SKELETON Systemic hormones and growth factors (GH, IGF-I, PTH, leptin, thyroid and sex hormones, glucocorticoids) have substantial effects on chondrocyte proliferation, matura-

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tion, and function.185 GH increases the synthesis of BMPs, directly enhances differentiation of prechondrocytes, supports proliferation of chondrocytes in the reserve or resting zone, and increases expression of IGF1. IGF-I then ensures the clonal expansion of committed chondrocytes.126 In utero, both IGF-I and IGF-II are essential for normal fetal growth as denoted by the in utero growth retardation experienced by the fetus with a lossof-function mutation in IGF1, IGF2, or IGFR1.185,186 GH and IGF-I receptors are expressed in chondrocytes in the reserve, proliferative, and hypertrophic zones— whereas IGF-I and IGF-II are expressed predominantly in proliferating chondrocytes.187 GH mediates chondrocyte

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proliferation and maturation and IGF-I synthesis through the Janus kinase-2 signal transduction and transcription 5b (JAK2-STAT5b) signal transduction system.188 IGF-I coordinates chondrocyte proliferation and inhibits their apoptosis. It modulates their differentiation and maturation and their synthesis of matrix heparan sulfate proteoglycan, a matrix component necessary for efficient signaling of FGFs and their receptors.126,186 IGF-I also influences the interaction of Ihh and PTHrP. IGF-binding proteins (IGFBP) 1 through 6 are synthesized by growth plate chondrocytes, where they regulate levels of bioactive IGF-I as well as exert direct stimulatory/inhibitory effects on chondrocyte proliferation (depending on the stage of chondrocyte differentiation).126,189 Neither GH nor IGF-I is necessary for patterning of the skeleton, however. Systemic loss of GH secretion or IGF-I production or inactivation of the IGF-I receptor greatly impairs linear growth of long bones postnatally. Selective loss of hepatic IGF-I production lowers total circulating levels of IGF-I to 25% of normal but does not adversely affect growth in transgenic mice, indicating that it is IGF-I synthesized by the cartilage growth plate that affects chondrocyte division by a paracrine mechanism.190 In patients with inactivating mutations of the GH receptor or deletion of the gene encoding IGF-I, administration of IGF-I enhances linear growth—indicating that this growth factor is able to stimulate cartilage proliferation without the initial differentiating effect of GH but does so to a lesser extent than does GH in the GH deficient subject. Thus, sufficient numbers of differentiated prechondrocytes are necessary for optimal IGF-I effect.191 The GH secretagogue ghrelin is also synthesized and secreted by chondrocytes and affects their intracellular metabolism, but its physiologic significance in this region is unknown at present.192 Through expression of the GH receptor by osteoblasts, GH stimulates their differentiation, proliferation, and function—enhancing synthesis and secretion of osteocalcin, bone-specific alkaline phosphatase, and type I collagen. GH also increases expression of IGF-I by the osteoblast, as do estrogen, PTH, cortisol, calcitriol, and other factors. IGF-I is essential for GH-induced osteoblast proliferation in vitro. It also decreases expression of the GH receptor, whereas estrogen stimulates its expression.193 In response to GH, IGFBP-3 and IGFBP-5 are synthesized by (rat) osteoblasts. IGFBP-4 expression is decreased by GH in rat and human osteoblasts. The IGFBPs may augment or restrict activity of IGF-I and IGF-II. Thus, IGFBP-5 binds to bone cells, matrix, and hydroxyapatite and enhances the actions of IGF-I on bone. Expression of the GH receptor by the osteoblast is up-regulated by IGFBP inhibition of IGF-I activity. Acting through the osteoblast, GH increases osteoclast proliferation and activity. Human osteoclasts express receptors for IGF-I, which also enhances osteoclast formation and activation. In children with GH deficiency and adults with childhood-onset or adult-onset GH deficiency, BMC and areal and volumetric BMDs are decreased—and increase when GH is administered.193,194 Administration of GH to the GH-deficient subject is followed by increase in serum and urine levels of markers of bone

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formation and resorption (osteocalcin, bone-specific alkaline phosphatase, PICP, PIIINP, ICTP, Pyr, Dpd, and NTx), with maximal values achieved 3 to 6 months after beginning treatment. There is a biphasic response of bone mass during GH treatment. For approximately the first 6 months of GH administration, BMD declines as bone resorption exceeds formation. Thereafter, BMD increases steadily to positive values over the next 6 to 12 months.193 In subjects with GH receptor deficiency or IGF-I gene deletion, BMC and BMD are decreased relative to control subjects but volumetric BMD is not—suggesting that bone size but not bone mineral acquisition is impaired by isolated IGF-I deficiency.195,196 Nevertheless, administration of IGF-I to subjects with deletion of IGF1 enhances osteoblast function as documented by increased serum concentrations of osteocalcin and bone-specific alkaline phosphatase, BMC, and areal and volumetric BMDs.196 In acromegalic patients, there is increased bone turnover—and variably increased lumbar spine and femoral neck BMD and iliac crest cortical and trabecular bone mass.193 Thyroid hormone receptors (TR, TR) are expressed in the reserve and proliferative zones of the growth cartilage. Triiodothyronine, acting primarily through TR, enables the differentiation of resting chondrocytes and their entrance into the proliferative phase. However, there thyroid hormone inhibits further chondrocyte proliferation and promotes differentiation to terminal hypertrophic chondrocytes and secretion of collagen type X.126 They do so in part by disrupting the reciprocal interaction of Ihh and PTHrP, thereby accelerating chondrocyte maturation (effects mediated by FGFR3 and the STAT signaling pathway), and by down-regulation of IGF-I expression in chondrocytes.126,197 Thyroid hormone is essential also for invasion of the growth plate’s hypertrophic zone by blood vessels and induction of metaphyseal trabecular bone formation. Thyroid hormones are necessary for fusion of the epiphyseal cartilage plate, although fusion may occur in the absence of thyroid hormone through the action of sex hormones. Through osteoblast-expressed receptors for thyroid hormone, triiodothyronine increases osteoblast production of osteocalcin, bone-specific alkaline phosphatase, and IGF-I. Thyroid hormones increase the rate of bone remodeling by expanding the number of osteoclasts and sites of bone resorption and the amount of bone resorptive surface. Urinary calcium excretion is increased by thyroid hormones. In excess, thyroid hormones can lead to net bone loss. Estrogen and androgens promote chondrocyte maturation.185 Although many of the effects of androgens are mediated by their conversion to estrogens, nuclear androgen receptors are expressed by chondrocytes and nonaromatizable androgens stimulate chondrocyte proliferation and long bone growth. Estrogens acting through ER and ER expressed in chondrocytes exert dual effects on long bone growth—stimulatory at low levels and inhibitory at high values. Estrogens decrease chondrocyte proliferation and accelerate differentiation and senescence. Complete maturation and fusion of the growth plate are mediated solely by estrogens, as evidenced by the failure

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DISORDERS OF CALCIUM METABOLISM

of growth plate fusion in young adult males with inactivating mutations of the genes encoding aromatase (the enzyme that converts androgens to estrogens) or ER despite adult levels of testosterone. Chondrocytes may also be capable of synthesizing estrogens from androgens because aromatase activity has been found in growth plate chondrocytes.185 This observation suggests that locally produced (as well as systemic) estrogens may contribute to chondrocyte maturation and growth plate fusion. Sex hormones play major roles in the accretion of bone mineral in both females and males because the bulk of adult bone calcium stores are deposited during puberty, when the peak rate of accrual of total body BMC occurs in boys and girls 0.7 years after attainment of peak height velocity (PHV) and 0.4 to 0.5 years after peak accrual of lean body mass (a surrogate measurement of muscle mass)172,198 (Figure 3-15). After controlling for size, total body mass, and femoral neck peak, BMC velocity and BMC accrual over 2 years around the PHV are greater in males than in females. There is no gender effect on accrual of BMC of the lumbar spine, however. Both androgens (in part by conversion to estrogen) and estrogens markedly influence rates of bone formation and resorption, although it is the effect of estrogen that predominates—as evidenced by the marked osteopenia of adult males with androgen sufficiency but estrogen deficiency related to loss-of-function mutations in the genes encoding aromatase and ER, by the beneficial effects of estrogen but not of testosterone on BMD in males with aromatase deficiency, by the very close association of BMD and serum levels of bioavailable estrogen in elderly men, and by the significant correlation in adult men treated with testosterone between changes in BMD and increases in serum concentrations of estradiol but not of testosterone.132,199 Nevertheless, the osteopenia of adult (46XY) females with complete androgen insensitivity due to loss-offunction mutations of the X-linked androgen receptor despite elevated serum testosterone and (endogenous or exogenous) estradiol concentrations and the fragile bone structure of the (Tfm) mouse counterpart of this disorder indicates that androgens, too, increase bone mineralization.200 Furthermore, non-aromatizable dihydrotestosterone has a direct anabolic effect on bone because it stimulates the proliferation and maturation of osteoblasts, increases the production of procollagen I(1), and prevents bone loss in orchidectomized rats.201 Estrogens have a biphasic effect on chondrocyte proliferation—increasing it at low doses and reducing it at higher doses. By accelerating their rate of maturation and apoptosis, estrogens lead to epiphyseal fusion.199 Both males and females with systemic aromatase deficiency have delayed bone age, absent adolescent growth spurt, and marked osteopenia. Males are tall and eunuchoid, whereas females are short and hyperandrogenic (cliteromegaly, hirsutism). Administration of low doses of estrogens results in bone maturation in both genders. Estrogens increase bone mass primarily by suppressing bone resorption. They do so through inhibition of osteoclastogenesis and down-regulating osteoblast production of osteoclastactivating factors such as IL-6 (and its receptor), TNF, and

Ch03_074-126-X4090.indd 113

113

M-CSF; increasing production of osteoprotegerin; and accelerating apoptosis of mature osteoclasts.132 Estrogens also prolong the life span of osteoblasts and osteocytes. During adolescence, in the female not only does the rate of bone deposition increase but that of bone resorption declines.202 Serum levels of IGF-I are positively correlated with lumbar spine BMD and metacarpal length and cortical thickness in early and mid-pubertal females. There is maturation-related increase in BMC and areal and volumetric BMD, and in metacarpal length, width, and cortical thickness—due in part to decline in width of the marrow cavity. These data suggest that the pubertal increase in production of GH and IGF-I (attributable specifically to increased estrogen synthesis in females) mediates longitudinal and periosteal skeletal growth and mineral acquisition during puberty. Estrogen may decrease endosteal bone resorption through inhibition of IL-6 generation—both systems contributing to increase in cortical bone mass. Estrogens likely account for part of the pubertal growth spurt of girls and boys, acting both indirectly (by increasing the secretion of GH and the systemic and local production of IGF-I) and directly on the chondrocyte. Other gonadal hormones also influence bone quality.203 Experimentally, inhibin A (a member of the TGF superfamily) increases total body BMD and bone volume and tibial biomechanical strength in transgenic mice through enhancement of osteoblast differentiation and function.204 The nuclear glucocorticoid receptor is expressed in chondrocytes in the proliferative and hypertrophic zones. Cortisol exerts an inhibitory effect on chondrocyte proliferation and maturation and hastens the death of hypertrophic chondrocytes.126 Glucocorticoids act in part by depressing expression of the genes encoding the GH receptor, IGF-I, and IGF1R in growth plate chondrocytes and by regulating synthesis of IGFBPs and thereby indirectly function of IGF-I. However, glucocorticoids also increase expression of SOX9 and the earliest phase of chondrocyte differentiation. Glucocorticoids suppress osteoblastogenesis and accelerate the rate of apoptosis of osteoblasts and osteocytes, in part by suppressing expression of BMP-2 and RUNX2.132 Secondarily, glucocorticoids decrease osteoclastogenesis but delay osteoclast death. In excess, glucocorticoids decrease trabecular bone and osteoid volumes and the rate of bone formation—contributing to bone weakness and collapse. Transcripts of C-type natriuretic peptide (NPPC) and its receptor (NPR2) are expressed by chondrocytes. These peptides stimulate the growth of proliferative and hypertrophic chondrocytes, stimulate osteoblast function, and induce endochondral ossification.205 Plasma concentrations of the amino terminal pro-C-type natriuretic peptide are positively related to growth velocity in normal children and adolescents.206 Biallelic loss-of-function mutations in NPR2 have been identified in patients with acromesomelic dysplasia [Maroteaux type (OMIM 602875)].207 In this disorder, there is shortening and deformation of the forearms, forelegs, and vertebrae— resulting in severely compromised adult stature. After genetic influences, the factor to which bone mass is most closely related is weight. Although obese children,

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adolescents, and adults may have greater BMC and BMD than do slimmer subjects, it is primarily lean body mass (i.e., muscle) to which bone mass is related in these subjects.208 Because osteoblasts and adipocytes arise from a common mesenchymal stem cell and can be interconverted, it is not surprising that the fat cell synthesizes and secretes a number of adipokines that exert effects on bone accumulation. Among these products is leptin, a 16-kDa protein that enhances osteoblast differentiation and maturation (but not proliferation) and inhibits adipocyte differentiation in human bone marrow stromal cells in vitro.209 However, leptin concentrations are not correlated with whole-body BMD in either obese or nonobese late prepubertal or early pubertal subjects.210 Furthermore, in mice with loss-of-function mutations in leptin or its receptor bone mass is increased before onset of obesity due to increased osteoblast activity in the presence of normal osteoclast function. Bone mass can be reduced in these and wt animals by intracerebroventricular infusion of leptin, indicating that leptin can act centrally to inhibit bone formation.211 Central infusion of leptin also decreases bone formation in sheep.212 These and other data demonstrate that in addition to peripheral modulating factors there is central (hypothalamic) regulation of bone remodeling that is thought to be exerted through the sympathetic nervous system.213 Adiponectin, another fat cell product, stimulates osteoclastogenesis by inducing osteoblast expression of RANKL and inhibiting that of osteoprotegerin.214

ASSESSMENT OF BONE MASS AND STRENGTH Bone mass is determined by the three-dimensional size (volume) of the bone, its mineral content, and its material properties such as elasticity. Bone mineralization may be assessed by bone biopsy and histomorphometric analysis of bone formation and resorption.215-217 Undecalcified transiliac biopsies permit limited assessment of bone modeling (changes in bone size, shape, mass), but detailed analysis of remodeling (bone renewal) as the iliac crest biopsy is primarily composed of trabecular bone with a limited amount of cortical bone. Although in children bone biopsies are usually reserved for research studies or for the evaluation of disorders of bone formation/mineralization not readily understood by noninvasive procedures (e.g., osteogenesis imperfecta, fibrous dysplasia), they have substantial clinical utility when applied appropriately by experienced personnel.216 During bone modeling, osteoblasts and osteoclasts are active on opposite bone surfaces across from one another. Thus, the bone surface may change position, size, or mass during the modeling process. Usually, modeling is associated with gain in bone mass because the rate of osteoblastic deposition of bone is more rapid than is that of osteoclastic resorption. During bone remodeling, osteoclastic resorption of bone is followed by osteoblastic replacement of the reabsorbed bone at the same surface with a net change of zero in bone mineral at the remodeling site under normal circumstances. Histomorphometry enables quantitation of structural parameters of bone size and amount (cortical width, tra-

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becular number, and thickness), static bone formation (thickness and surface of osteoid or unmineralized bone matrix, osteoblast surface), dynamic bone formation after labeling with a fluorochrome such as tetracycline (mineral apposition and bone formation rates), and static resorption (osteoclast number and appearance and extent of eroded surfaces). Iliac trabecular thickness but not trabecular number increases substantially between 2 and 20 years of age, whereas remodeling activity peaks in young children, declines, and then increases again during puberty.215 Noninvasive methods of assessment of skeletal mineralization include bone x-rays (of limited value), radiographic absorptiometry or photodensitometry, singlephoton or single–x-ray absorptiometry, dual-photon or dual-energy x-ray absorptiometry (DEXA), spinal and peripheral quantitative computed tomography (pQCT), quantitative ultrasonography (QUS), quantitative magnetic resonance, and magnetic resonance microscopy.170 DEXA has become the most frequently employed method of quantifying axial and peripheral bone mass and bone area (as well as body composition) because of its relatively low radiation dosage (5–10 Sv), ease of use and applicability for infants, rapidity (4 minutes for total body scan), accuracy, precision, and reproducibility under controlled circumstances.170,218 The ratios of attenuation of x-rays of two energies (70 kV, 140 kV) traversing the same pathway through the patient reflect the “density” and mass of the tissue through which the x-rays have crossed. Computer analysis of these captured energies then reconstructs the boundaries, density, and mass of the tissue. Two methods of imaging geometry have been employed in DEXA instruments: the earlier pencil beam DEXA instruments utilized a pinhole collimator linked to a single detector, whereas the newer fan beam DEXA units apply a slit collimator coupled with a multidetector array.219 Fan beam DEXA has the advantages of more rapid scan acquisition and improved resolution, making it especially useful for small children and infants.220 Because of variability between instruments and analytical software programs (infant, pediatric, adult) employed for DEXA, the report of the DEXA scan should include not only the recorded data but the type of DEXA instrument and the software version employed for analysis. Because relatively low BMDs in children make it more difficult to distinguish clearly bone edges, specific infant and pediatric software must be used to address this problem.221 DEXA is employed to quantify bone mass and bone area in the axial (head, spine) and appendicular (limbs) skeleton. DEXA assesses a two-dimensional image, and therefore does not measure “true” BMD (the mass within a volume of uniform composition). DEXA measures the area(l) or surface mass of mineral within a region of bone of nonuniform composition (cortex, trabeculae, osteoid, marrow). It is expressed as g/cm2.222 Because DEXA does not take into account the depth of a bone, it underestimates BMD in small children and overestimates it in large subjects.223 In children and adolescents, bone size/volume increases with growth and maturation—and the larger the

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DISORDERS OF CALCIUM METABOLISM

three-dimensional structure of bone the greater the recorded areal BMD [even though the actual or volumetric (v) BMD may not change substantially].222 Therefore, calculated or “apparent” (v) BMDs (BMAD) (g/cm3) data have been generated in an attempt to correct this problem. Volumetric BMD increases as the cortex thickens, the number and width of trabeculae per unit volume rises, and the amount of hydroxyapatite per unit of trabecular volume accrues. Although during childhood and adolescence areal BMD of the femoral shaft increases with age, its vBMD remains relatively constant.224 On the other hand, vBMD of the lumbar spine increases in late puberty and early adulthood because of increasing thickness of trabeculae that is not gender specific but is greater in blacks than in whites after puberty.222,225 Cross-sectional areas of cortical and trabecular bone increase with age, with males achieving greater increase in periosteal apposition than females during puberty. Cortical cross-sectional area is similar in white and black subjects.226 Included in measurement of whole-body areal BMD is the head. This structure characteristically has twice the areal BMD of that of the rest of the skeleton and may comprise as much as 20% to 50% of the total BMD in children. Whole-body areal BMD correlates better with body height when the head BMD is excluded.227,228 Whole- or total body BMC and BMD, lumbar spine BMC and BMD, and femoral BMD are the most commonly measured indices of bone mass in childhood by DEXA and are reported as gm/cm2. Data are often reported as a

115

Z score, the number of standard deviations about the mean of peers matched for chronologic age and gender. (In adults, the DEXA T score is commonly reported. The T score is the number of standard deviations about the mean of maximal or peak bone mass recorded in healthy young adults aged 20 to 29 years. The T score should not be utilized in children and adolescents.) In neonates and infants, it is recommended that whole-body BMC be the primary measurement when assessing bone mineralization.229 In neonates with appropriate weight for gestational age (AGA), whole-body BMC by pencil-beam DEXA doubles between 32 and 40 weeks and increases 3.5-fold between birth weights of 1,000 and 4,000 grams. Neonates small for gestational age (SGA) have lower whole-body BMC than do AGA neonates of comparable gestational age, but they are similar to those of AGA infants with the same birth weights (Table 3-8).230,231 In young adults (16-19 years) born prematurely with birth weights 1.5 kg, total body BMC is less than that of subjects born at term with normal birth weights but appropriate for their smaller stature.232 Utilizing fan-beam DEXA (QDR 4500A, Hologic Inc, Bedford, MA), whole-body BMC of 73 full-term neonates (birth weights 2,720 to 3,982 g) of both genders and of multiple ethnicities has been reported as 89.3 ± 14.1 (SD) grams with BMD of 0.240 ± 0.022 g/cm2.220 Areal BMD of the lumbar spine (L2-L4) is low in prematurely born infants but “catches up” to that of full-term infants by

TA B L E 3 - 8

Whole-Body Bone Mineral Content (BMC g) and Density (g/cm2) by Pencil Beam Dual-Energy X-ray Absorptiometry in Prematurely Born and Full-term Appropriate- or Small-for-Gestational-Age Infants (AGA, SGA) GA

BMC AGA

32-33 34-35 36-37 38-39 Birth Weight

21.9 32.0) 39.4 45.5

BMD SD

(9.7)1 (11.3 (15.9) (18.4) Range

SGA

SD

24.9 27.0

(7.4) (11.1)

AGA

Range 2

1001-1500 1501-2000 2001-2500 2501-3000 3001-3500 3501-4000 Postnatal Age

22.9 31.8 42.2 54.6 66.9 77.6

(21.7-24.1) (30.9-32.8) (41.7-43.2) (53.9-55.3) (66.0-67.9) (76.3-78.8)

0.146 0.162 0.178 0.199 0.220 0.234

(0.141-0.150) (0.158-0.165) (0.175-0.181) (0.196-0.202) (0.217-0.224) (0.229-0.238)

9-90 91-150 150-270 271-390

103 137 196 253

(10)3 (20) (27) (41)

0.238 0.259 0.302 0.335

(0.022) (0.024) (0.018) (0.029)

GA  gestational age (weeks), birth weight (g), postnatal age (days) (95% range). 1. Lapillone A, et al. (1997). Body composition in appropriate and in small for gestational age infants. Acta Paediatr 86:196–200. 2. Koo WWK, et al. (1996). Dual-energy x-ray absorptiometry studies of bone mineral status in newborn infants. J Bone Miner Res 11:1997-1002. 3. Koo WWK, et al. (1998). Postnatal development of bone mineral status during infancy. J Am Coll Nutr 17:65-70.

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2 years of age.233 Particular attention to detail must be exercised when assessing bone mineralization in very small subjects by DEXA because even apparently minor changes (position on the scanning table, prone or supine posture, number of blankets overlaid or wrapped around the subject, administration of a bolus of intravenous fluid prior to scanning, presence of a radiographic contrast agent) may affect measurement of BMC and BMD.234,235 It is important to reemphasize that infant software be employed for data analysis of DEXA measurements in small subjects because adult and pediatric software underestimate bone area and BMC but overestimate BMD in this group.234 Whole-body (or total body) BMC is the more accurate and reliable of the measurements provided by DEXA compared to regional measurements, particularly in growing children and adolescents.228 Whole-body BMC increases three- to fourfold between 6 and 17 years of

age177,236-238 (Tables 3-9A and B). Whole-body BMC is greater in adult males than in females because of the larger size of male bones. The gender difference in whole-body BMC appears after 15 years of age. Lumbar vertebral BMD approximately doubles between 3 and 15 years of age in boys (0.454 to 0.885 g/cm2) and girls (0.444 to 0.960 g/cm2); lumbar spine BMD increases rapidly between birth and 5 years in both sexes (between 13 and 15 years in boys, and between 12 and 14 years in girls)236,238-240 (Table 3-9B and Table 3-10). BMD of the femoral neck increases 1.5-fold during puberty. In females, the rate of maximal increase in wholebody BMC occurs in the year of menarche and follows the year of peak height velocity (Figure 3-15).172,241 Peak BMC and BMD are reached at 18 to 20 years in both sexes, although there may be slight accrual of additional bone mass in the third decade of life. In girls with menarche

TA B L E 3 - 9 A

Whole-Body Bone Mineral Content (BMC g) and Areal(a) Bone Mineral Density (BMD g/ cm2) by Pencil Beam Dual-Energy X-ray Absorptiometry in Children and Adolescents Males BMC1

CA 4-5 6-7 8-9 10-11 12-13 14-15 16-17 18-20 23-263

Females aBMD2 0.781 0.843 0.870 0.894 0.961 1.030 1.133 1.202 1.128

674 (149) 909 (187) 1186 (224) 1540 (226) 1965 (308) 2395 (336) 2599(511)

BMC

(0.05) (0.03) (0.04) (0.05) (0.07) (0.09) (0.11) (0.11) (0.11)

aBMD 0.795 0.828 0.860 0.863 0.974 1.081 1.143 1.159 1.088

669 (152) 896 (185) 1203 (223) 1622 (267) 2043 (294) 2183 (268) 2308 (383)

(0.02) (0.06) (0.05) (0.07) (0.09) (0.07) (0.09) (0.07) (0.08)

CA  chronologic age (years). Mean (SD). 1. Molgaard C, et al. (1997). Whole body bone mineral content in healthy children and adolescents. Arch Dis Child 76:9–15. 2. Boot AM, et al. (1997). Bone mineral density in children and adolescents: relation to puberty, calcium intake, and physical activity. J Clin Endocrinol Metab 82:57-62. 3. Bachrach LK, et al. (1999). Bone mineral acquisition in healthy Asian, Hispanic, black, and Caucasian youth: A longitudinal study. J Clin Endocrinol Metab 84:4702-4712.

TA B L E 3 - 9 B 2

Bone Mineral Density (g/cm ) by Fan-Beam Dual-Energy X-ray Absorptiometry in White Children in the United States (Hologic QDR 4500 Analyzed with Software Version 12.1) (mean ± SD) Males CA

Whole Body

3 5 7 9 11 13 15 17 19

0.582 0.655 0.717 0.791 0.866 0.945 1.041 1.133 1.205

(0.039) (0.046) (0.052) (0.060) (0.068) (0.077) (0.089) (0.101) (0.111)

AP Spine 0.454 0.492 0.530 0.578 0.648 0.750 0.885 0.995 1.067

(0.047) (0.052) (0.058) (0.066) (0.084) (0.112) (0.125) (0.123) (0.120)

Females Total Hip — 0.572 (0.062) 0.652 (0.070) 0.720 (0.080) 0.786 (0.092) 0.872 (0.110) 0.973 (0.129) 1.064 (0.143) 1.139 (0.152)

Whole Body 0.561 0.637 0.698 0.768 0.856 0.959 1.041 1.084 1.107

(0.039) (0.046) (0.053) (0.060) (0.068) (0.073) (0.075) (0.075) (0.074)

AP Spine 0.444 0.490 0.538 0.597 0.697 0.853 0.960 1.003 1.017

(0.046) (0.054) (0.062) (0.075) (0.099) (0.116) (0.111) (0.107) (0.105)

Total Hip — 0.553 (0.061) 0.602 (0.069) 0.669 (0.081) 0.764 (0.097) 0.869 (0.110) 0.938 (0.112) 0.970 (0.110) 0.986 (0.108)

Zemel BS, et al. (2004). Reference data for whole body, lumbar spine and proximal femur for American children relative to age, gender and body size. J Bone Miner Res 19:(1):S231.

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TA B L E 3 - 1 0

Lumbar Spine Bone Mineral Content (BMC, L2–L4, g), Areal (aBMD - g/cm2), and Volumetric (vBMD-g/cm3) Bone Mineral Densities and Femoral Neck Areal Bone Mineral Density by Pencil Beam Dual-Energy X-ray Absorptiometry in Children and Adolescents Lumbar Spine CA

BMC1

Males 1 3 5 7 9 11 13 15 17 19 21 23-264

3.76 9.37 14.5 19.4 22.2 23.7 31.1 44.5 54.2 68.7 69.0

Females 1 3 5 7 9 11 13 15 17 19 21 23-264

4.61 (1.24) 9.91 (1.58) 13.4 (1.40) 16.4 (2.05) 22.2 (3.65) 26.2 (4.10) 39.1 (6.98) 39.2 (6.47) 46.1 (5.1) 46.0 (5.95) 47.5 (6.59)

Pubertal Stage

Whole-Body BMD2

(1.54) (1.91) (1.72) (3.42) (2.17) (2.22) (8.04) (13.1) (9.46) (4.81) (5.25)

aBMD1,2

vBMD2

0.380 0.565 0.625 0.720 0.755 0.791 0.868 1.058 1.204 1.238 1.220 1.037

(0.06) (0.07) (0.06) (0.06) (0.08) (0.07) (0.13) (0.10) (0.11) (0.10) (0.12) (0.15)

0.261 0.282 0.288 0.282 0.280 0.312 0.347 0.355

0.380 0.565 0.674 0.710 0.745 0.887 1.024 1.239 1.214 1.246 1.200 1.074

(0.06) (0.07) (0.07) (0.03) (0.08) (0.14) (0.15) (0.12) (0.11) (0.15) (0.12) (0.12)

0.288 0.290 0.296 0.313 0.345 0.396 0.390 0.397

(0.03) (0.04) (0.03) (0.03) (0.03) (0.03) (0.04) (0.03)

Femoral Neck3

0.824 0.965 (0.072) 0.910 (0.119) 1.169 (0.132) 1.044 (0.096) 1.258 (0.109) 0.916 (0.136)

(0.02) (0.03) (0.04) (0.03) (0.04) (0.04) (0.03) (0.05)

0.692 0.810 0.924 1.056 0.981 0.962

(0.079) (0.061) (0.101) (0.207) (0.122) (0.072)

0.873 (0.114)

Lumbar Spine BMD2

Males I II III IV V

0.86 0.94 1.02 1.11 1.15

(0.06) (0.07) (0.10) (0.12) (0.09)

0.69 0.82 0.91 1.08 1.15

(0.09) (0.10) (0.15) (0.17) (0.09)

Females I II III IV V

0.84 0.93 0.97 1.08 1.15

(0.05) (0.05) (0.08) (0.09) (0.08)

0.71 0.83 0.96 1.14 1.22

(0.08) (0.07) (0.15) (0.15) (0.14)

CA = chronologic age. Mean (SD). 1. del Rio L, et al. (1994). Bone mineral density of the lumbar spine in white Mediterranean Spanish children and adolescents: Changes related to age, sex, and puberty. Pediatr Res 35:362-366. 2. Boot AM, et al. (1997). Bone mineral density in children and adolescents: relation to puberty, calcium intake, and physical activity. J Clin Endocrinol Metab 82:57-62. 3. Kroger H, et al. (1993). Development of bone mass and bone density of the spine and femoral neck: A prospective study of 65 children and adolescents. Bone Mineral 23:171-182. 4. Bachrach LK, et al. (1999). Bone mineral acquisition in healthy Asian, Hispanic, black, and Caucasian youth: A longitudinal study. J Clin Endocrinol Metab 84:4702-4712.

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before 12 years, there is greater peak BMD (at age 18-19 years) than in females with menarche after 14 years.242 Similarly, in adult males with a history of delayed puberty there is lower lumbar spine BMD than in those with pubertal onset at 11 to 12 years.243 A major drawback to the use of DEXA for determination of bone mineralization in children is the reliance of reference data on chronologic age independent of body size. Thus, a short child may appear to have decreased bone mineralization relative to chronologic age but a satisfactory value relative to height age. Accordingly, assessment of bone mass relative to height is essential (Figure 3-16). DEXA measurements of bone mineralization are also affected by the composition of soft tissues that surround the axial skeleton. Thus, variations in fat about the bone may significantly influence the recorded DEXA measurements—limiting the use of DEXA in extremely thin or obese children.244 Generally, BMC, areal BMD, and/or BMAD below –2 SDs for age and gender are considered abnormally low. However, DEXA measurements must always be interpreted in relationship to the patient’s clinical findings.223 QCT measures volumetric BMD of both trabecular and cortical bone at any site but has been commonly applied to the lumbar spine.225 However, the radiation dose delivered to the spine by this method is high (⬃30 Sv). During childhood, lumbar spine vBMD measured by QCT is simi-

lar in black and white youth. During puberty, black males and females gain twice the vBMD recorded in whites (with no gender difference). In peripheral (p)QCT, bone mineralization is quantified in distal and/or proximal radial, femoral, or tibial sites (regions of cortical and trabecular bone). The delivered radiation dose (10 Sv) is low. Peripheral QCT may be examined in the radius twothirds of the distance between its proximal and distal ends and distally at a point that is 4% of forearm length, with a reference line being drawn through the most distal aspect of the cartilage growth plate when it is open or through the middle of the ulnar border of the articular cartilage when the growth plate is closed.245-247 pQCT permits measurement of total and cortical bone area, cortical thickness, BMC (mg/mm), cortical and trabecular vBMD, periosteal and endosteal circumferences, and marrow area. Proximal and distal radial bone pQCT data between 6 and 40 years of age and pubertal stages I through V are recorded in Table 3-11. Interestingly, at all ages total cross-sectional and cortical areas, cortical thickness, BMC, and BMD are greater in males than in females. As mirrored by the proximal radius, pQCT documents the prolonged period of periosteal expansion in males that ultimately accounts for their larger bone size. Widespread application of pQCT awaits standardization of protocols for data acquisition and

Males 2.5 2.0 1.5 1.0 140

160

A

Height (cm)

Females

1.0

Height (cm)

1.5

2.0

Total body bone area (cm2)/1000

Females

1.5

160

0.5

B

2.0

140

1.5

1.0

95 75 50 25 5

120

95 75 50 25 5

2.5

180

Total body BMC (g)/1000

120

Total body BMC (g)/1000

Males 95 75 50 25 5

Total body bone area (cm2)/1000

Total body bone area (cm2)/1000

Bone Area–Bone Mineral Content Total Body

1.5

0.5 1.0

180

95 75 50 25 5

2.5

1.5

2.0

Total body bone area (cm2)/1000 Figure 3-16. Reference curves for total body bone area for height (A, C) and total body bone mineral content (BMC) for bone area (B, D) in white male and female children and adolescents 6 to 17 years of age employing fan-beam dual-energy X-ray absorptiometry. [Reproduced with permission from Ward KA, et al. (2007). UK reference data for the Hologic QDR Discovery dual-energy x-ray absorptiometry scanner in healthy children and young adults aged 6-17 years. Arch Dis Child 92:53-59.]

C

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D

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119

TA B L E 3 - 1 1 A

Peripheral Quantitative Computed Tomography Non-dominant Proximal Radius (Mean ± SD) Total1

Cortical1

Cortical2 Area

BMC3 Area

vBMD4 Thickness

Male 6-7 8-9 10-11 12-13 14-15 16-17 18-23 Adult

73 (13) 86 (14) 98 (16) 108 (19) 123 (21) 128 (20) 151 (22) 158 (27)

37 (8) 47 (6) 52 (8) 57 (8) 72 (16) 83 (13) 95 (14) 97 (12)

1.45 (0.31) 1.71 (0.21) 1.78 (0.26) 1.85 (0.28) 2.27 (0.46) 2.63 (0.35) 2.75 (0.41) 2.74 (0.44)

52 (8) 61 (6) 68 (9) 73 (9) 91 (17) 105 (16) 121 (16) 125 (15)

579 (70) 601 (57) 596 (68) 589 (82) 645 (92) 720 (66) 719 (82) 711 (102)

Pubertal Stage I II III IV V

87 (18) 108 (19) 114 (11) 118 (19) 141 (23)

45 57 59 70 88

1.64 1.88 1.84 2.22 2.61

61 (11) 74 (9) 75 (8) 87 (16) 111 (19)

593 597 574 640 700

Female 6-7 8-9 10-11 12-13 14-15 16-17 18-23 Adult

78 (12) 78 (13) 92( 18) 100 (18) 109 (18) 114 (15) 111 (16) 117 (20)

31 (8) 43 (8) 51 (11) 61 (16) 70 (9) 73 (10) 71 (10) 75 (9)

1.15 (0.39) 1.68 (0.35) 1.84 (0.40) 2.14 (0.51) 2.38 (0.35) 2.42 (0.31) 2.42 (0.34) 2.48 (0.34)

47 (6) 57 (9) 65 (12) 77 (19) 89 (11) 94 (12) 93 (12) 99 (11)

517 (84) 615 (90) 619 (89) 662 (97) 722 (88) 717 (66) 749 (78) 763 (86)

Pubertal Stage I II III IV V

81 (15) 90 (15) 89 (14) 102 (17) 113 (16)

40 50 53 66 74

1.50 1.81 1.98 2.33 2.49

55 65 68 82 95

577 607 648 713 739

Age

(10) (8) (8) (13) (17)

(10) (12) (7) (8) (11)

(0.32) (0.31) (0.35) (0.32) (0.47)

(0.43) (0.39) (0.30) (0.34) (0.34)

(10) (14) (10) (9) (13)

(70) (79) (80) (66) (90)

(93) (102) (72) (85) (75)

1

mm2 2mm 3mg/mm 4mg/cm3 Neu CM, et al. (2001). Modeling of cross-sectional bone size, mass and geometry at the proximal radius: A study of normal bone development using peripheral quantitative computed tomography. Osteoporos Int 12:538-547.

analysis and larger reference databases. By direct comparison of bone mass data gathered by DEXA and tibial metaphyseal pQCT examinations in the same subjects (6-21 years), it has been observed that DEXA whole-body bone area and BMC for height correlate well with pQCTderived cross-sectional area for tibial length.248 Although BMD values of the lumbar spine acquired by DEXA and QCT are reasonably well related, osteopenia is “identified” in children far more often by DEXA than by QCT because DEXA measurements do not account for body height and bone size.244 Clearly, pQCT would appear to be the preferred method for quantitation of bone mineralization in children at this time. Quantitative ultrasonography (QUS) measures the speed of a longitudinal sound (SOS) wave as it is propagated along a bone.170 The rate of movement of sound through bone is dependent on its microstructural and macrostructural characteristics, mineral density, and elasticity—and is thought to be a measure of bone strength. It is an attractive method for assessment of bone because it does not utilize

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radiation, is low in cost, and the equipment is portable. Transmitter and receiver ultrasound transducers placed on either side of the examination site (os calcis, patella, tibia, radius, phalange) quantitate transmission velocity or signal attenuation and convert these observations to SOS. In a study of 1,085 children and adolescents, SOS increased steeply at the tibia and radius during the first 5 years of life, more slowly between 6 and 11 years of age, and then again more rapidly during pubertal development.249 In 3,044 healthy subjects ages 2 to 21 years, phalangeal QUS SOS and bone transmission time (BTT) increased over time and with advancing adolescent development—and were related to gender, age, height, and weight.250 However, the overlap of QUS data between various ages makes interpretation of a single SOS measurement problematic. Serial assessment may be useful. Thus, in a cohort of 29 preterm infants tibial SOS values declined over time in neonates whose gestational ages were less than 29 weeks—suggesting progressive loss of bone strength in this population

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TA B L E 3 - 1 1 B

Peripheral Quantitative Computed Tomography Non-dominant Distal Radius (Mean ± SD) Age

vBMD-tot1

vBMD-Trab1

vBMD-cort1

CSA2

Males 6-7 8-9 10-11 12-13 14-15 16-17 18-23 Adults

306 294 290 292 293 349 401 438

(34) (34) (33) (38) (35) (56) (60) (56)

206 189 194 201 201 217 220 224

(32) (34) (32) (36) (33) (30) (42) (46)

388 380 368 366 369 458 549 594

(42) (41) (41) (47) (47) (86) (83) (81)

174 211 245 289 351 358 377 374

(31) (31) (37) (47) (70) (49) (64) (45)

Pubertal Stage I II III IV V

299 288 286 296 361

(32) (40) (33) (42) (72)

198 186 197 210 215

(31) (31) (36) (35) (40)

381 (41) 372 (52) 359 (37) 367 (50) 481 (109)

212 269 293 334 377

(47) (43) (44) (65) (59)

Females 6-7 8-9 10-11 12-13 14-15 16-17 18-23 Adults

290 283 281 295 303 350 371 395

(36) (22) (36) (39) (37) (57) (50) (46)

191 186 191 197 179 186 195 182

(31) (23) (36) (32) (25) (26) (35) (34)

370 362 355 376 407 483 516 569

(45) (32) (44) (54) (53) (95) (74) (69)

164 185 237 260 297 300 295 281

(30) (25) (39) (55) (32) (45) (42) (37)

Pubertal Stage I II III IV V

284 277 288 291 347

(30) (34) (44) (43) (54)

187 190 204 197 190

(29) (34) (44) (32) (28)

363 348 356 375 476

(39) (37) (50) (56) (87)

188 239 250 282 295

(38) (57) (47) (30) (43)

1

mg/cm3 2mm2 Neu CM, et al. (2001). Bone densities and bone size at the distal radius in healthy children and adolescents: a study using peripheral quantitative computed tomography. Bone 28:227-232. Rauch F, Schonau E (2005). Peripheral quantitative computed tomography of the distal radius in young subjects: New reference data and interpretation of results. J Musculoskelet Neuronal Interact 5:119-126

consistent with the development of osteopenia of prematurity.251 Although there is marginal correlation between vBMD determined by pQCT and SOS measurements in children and adolescents, QUS may complement but is unlikely to replace assessment of mineralization by radiographic methods at this time.252,253 Magnetic resonance imaging of the skeleton is being examined as a method of assessment of bone geometry and strength.254

Concluding Remarks Calcium, phosphate, and magnesium are vital to normal moment-to-moment function of all cells, and calcium and phosphate compose the mineral phase of the skeleton.

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This structure is essential to protection of body organs and to movement, and as a warehouse for these minerals. To an extent, the complex genetic and physiologic mechanisms that require vitamin D, PTH, and PTHrP to regulate calcium, phosphate, magnesium, and skeletal homeostasis have been deciphered—thereby permitting application of this information to the management of patients with derangements in this system (as detailed in an accompanying chapter). In regard to this chapter, two manuscripts of interest have been published. The physiology and genetic regulation of bone formation and resorption have been reviewed in detail.258 The most current data on whole-body and regional bone mineral content and density in North American children and adolescents analyzed by age, gender, and race have been presented259 (Figure 3-17).

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121

Bone Mineral Content Age, Gender, Race

Whole Body BMC-For-Age Percentiles 7 to 17 Years: Non-Black Boys

Whole Body BMC-For-Age Percentiles 7 to 17 Years: Black Boys 4000

3500 97

3000 2500

75 50 25

2000

3

1500 1000 500

Bone mineral content (g)

Bone mineral content (g)

4000

7

8

9

A

75 50 25

2500

3

2000 1500 1000 500

10

11 12 13

14

15

16 17

7

8

9

10

Age (years)

11 12 13

14

15

16 17

Age (years)

Whole Body BMC-For-Age Percentiles 7 to 16 Years: Non-Black Girls

Whole Body BMC-For-Age Percentiles 7 to 16 Years: Black Girls 4000

3500 3000 2500

97 75 50 25 3

2000 1500 1000 500 0

Bone mineral content (g)

4000

Bone mineral content (g)

97

3000

0

0

3500 3000 97

2500

75 50 25 3

2000 1500 1000 500 0

7

B

3500

8

9

10

11 12 13

14

15

16 17

Age (years)

7

8

9

10

11 12 13

14

15

16 17

Age (years)

Figure 3-17. Whole-body bone mineral content for non-black and black boys (A) and girls (B) employing fan-beam dual-energy x-ray absorptiometry. [Reproduced with permission from Kalkwarf HJ, Zemel BS, Gilsanz V, et al. (2007). The bone mineral density in childhood study: Bone mineral content and density according to age, sex, and race. J Clin Endocrinol Metab 92:2087-2099.]

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189. Cohen P (2006). Overview of the IGF-I system. Horm Res 65(1):3–8. 190. Yakar S, Liu J-L, Stannard B, et al. (1999). Normal growth and development in the absence of hepatic insulin-like growth factorI. Proc Natl Acad Sci 96:7324–7329. 191. Savage MO, Attie KM, David A, et al. (2006). Endocrine assessment, molecular characterization and treatment of growth hormone insensitivity. Nature Clin Prac Endocrin Metab 2:395–407. 192. Caminos JE, Gualillo O, Lago F, et al. (2005). The endogenous growth hormone secretagogue (ghrelin) is synthesized and secreted by chondrocytes. Endocrinology 146:1285–1292. 193. Ohlsson C, Bengtsson B-A, Isaksson OGP, et al. (1998). Growth hormone and bone. Endocrine Reviews 19:55–79. 194. Baroncelli GI, Bertelloni S, Ceccarelli C, Saggese G (1998). Measurement of volumetric bone mineral density accurately determines degree of lumbar undermineralization in children with growth hormone deficiency. J Clin Endocrinol Metab 83:3150–3154. 195. Bachrach LK, Marcus R, Ott SM, et al. (1998). Bone mineral histomorphometry and body composition in adults with growth hormone receptor deficiency. J Bone Miner Res 13:415–421. 196. Woods KA, Camacho-Huber C, Bergman RN, et al. (2000). Effects of insulin-like growth factor I (IGF-I) therapy on body composition and insulin resistance in IGF-I gene deletion. J Clin Endocrinol Metab 85:1407–1411. 197. Barnard JC, Williams AJ, Rabier B, et al. (2005). Thyroid hormones regulate fibroblast growth factor receptor signaling during chondrogenesis. Endocrinology 146:5568–5580. 198. Rauch F, Bailey DA, Baxter-Jones A, et al. (2004). The “musclebone unit” during the pubertal growth spurt. Bone 34:771–775. 199. Couse JF, Korach KS (1999). Estrogen receptor null mice: What have we learned and where will they lead us? Endocrine Reviews 20:358–417. 200. Marcus R, Leary C, Schneider DL, et al. (2000). The contribution of testosterone to skeletal development and maintenance: Lessons from the androgen insensitivity syndrome. J Clin Endocrinol Metab 85:1032–1037. 201. Vanderschueren D, Bouillon R (1995). Androgens and bone. Calcif Tissue Int 56:341–346. 202. Libanati C, Baylink DJ, Lois-Wenzel E, et al. (1999). Studies on potential mediators of skeletal changes occurring during puberty in girls. J Clin Endocrinol Metab 84:2807–2814. 203. Martin TJ, Gaddy D (2006). Bone loss goes beyond estrogen. Nature Med 12:612–613. 204. Perrien DS, Akel NS, Edwards PK, et al (2007). Inhibin A is an endocrine stimulator of bone mass and strength. Endocrinology 148:1654–1665. 205. Potter LR, Abbey-Hosch A, Dickey DM (2006). Natriuretic peptides, their receptors, and cyclic guanosine monophosphatedependent signaling functions. Endocrine Reviews 27:47–72. 206. Prickett TMCR, Lynne AM, Barrell GK, et al. (2005). Amino-terminal proCNP: A putative marker of cartilage activity in post natal growth. Pediatr Res 58:334–340. 207. Bartels CF, Bukulmez H, Padayatti P, et al. (2004). Mutations in the transmembrane natriuretic peptide receptor NPR-B impair skeletal growth and cause acromesomelic dysplasia, type Maoteaux. Am J Hum Genet 75:27–34. 208. Janicka A, Wren TAL, Sanchez MM, et al. (2007). Fat mass is not beneficial to bone in adolescents and young adults. J Clin Endocrinol Metab 92:143–147. 209. Thomas T, Gori F, Khosla S, et al. (1999). Leptin acts on human marrow stromal cells to enhance differentiation to osteoblasts and to inhibit differentiation to adipocytes. Endocrinology 140:1630–1638. 210. Klein KO, Larmore KA, de Lancey E, et al. (1998). Effect of obesity on estradiol level, and its relationship to leptin, bone maturation, and bone mineral density in children. J Clin Endocrinol Metab 83:3469–3475. 211. Ducy P, Amling M, Takeda S, et al. (2000). Leptin inhibits bone formation through a hypothalamic relay: A central control of bone mass. Cell 100:197–207. 212. Pogoda P, Egermann M, Schnell JC, et al. (2006). Leptin inhibits bone formation not only in rodents but also in sheep. J Bone Miner Res 21:1591–1599. 213. Baldock PA, Allison S, McDonald MM, et al. (2006). Hypothalamic regulation of cortical bone mass: Opposing activity of Y2 receptor and leptin pathways. J Bone Miner Res 21:1600–1607.

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214. Luo X-H, Guo L-J, Xie H, et al. (2006). Adiponectin stimulates RANKL and inhibits OPG expression in human osteoblasts through the MAPK signaling pathway. J Bone Miner Res 21:1648–1656. 215. Glorieux FH, Travers R, Taylor A, et al. (2000). Normative data for iliac bone histomorphometry in growing children. Bone 26:103–109. 216. Rauch F (2006). Watching bones at work: What we can see from bone biopsies. Pediatr Nephrol 21:457–462. 217. Recker RR, Barger-Lux MJ (2006). Bone biopsy and histomorphometry in clinical practice. In Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 161–169. 218. Bachrach LK (2005). Osteoporosis and measurement of bone mass in children and adolescents. Endocrinol Metab Clin N Am 34:521–535. 219. Koo WWK, Hammami M, Hockman EM (2004). Validation of bone mass and body composition measurements in small subjects with pencil beam dual energy x-ray absorptiometry. J Am Coll Nutr 23:79–84. 220. Hammami M, Koo WW, Hockman EM (2003). Body composition of neonates from fan beam dual energy x-ray absorptiometry measurement. J Parenteral Enteral Nutr 27:423–426. 221. Leonard MB, Propert KJ, Zemel BS, et al. (1999). Discrepancies in pediatric bone mineral density reference data: Potential for misdiagnosis of osteopenia. J Pediatr 135:182–188. 222. Seeman E (1998). Growth in bone mass and size: Are racial and gender differences in bone mineral density more apparent than real? J Clin Endocrinol Metab 83:1414–1419. 223. Ward KA, Ashby RL, Roberts SA, et al. (2007). UK reference data for the Hologic QDR Discovery dual-energy x ray absorptiometry scanner in healthy children and young adults aged 6-17 years. Arch Dis Child 92:53–59. 224. Lu PW, Cowell CT, Lloyd-Jones SA, et al. (1996). Volumetric bone mineral density in normal subjects aged 5-27 years. J Clin Endocrinol Metab 81:1586–1590. 225. Gilsanz V, Skaggs DL, Kovanlikaya A, et al. (1998). Differential effects of race on the axial and appendicular skeletons of children. J Clin Endocrinol Metab 83:1420–1427. 226. Miller PD, Leonard MB (2006). Clinical use of bone mass measurements in children and adults for the assessment and management of osteoporosis. In Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 150–161. 227. Brismar TB, Lindgren A-C, Ringertz H, et al. (1998). Total bone mineral measurements in children with Prader-Willi syndrome: The influence of the skull’s bone mineral content per area (BMA) and of height. Pediatr Radiol 28:38–42. 228. Maynard LM, Guo SS, Chumlea WC, et al. (1998). Total-body and regional bone mineral content and areal bone mineral density in children aged 8-18 y; the Fels Longitudinal Study. Am J Clin Nutr 68:1111–1117. 229. Koo WWK (2000). Body composition measurements during infancy. NY Acad Sci 904:383–392. 230. Koo WWK, Walters J, Bush AJ, et al. (1996). Dual-energy x-ray absorptiometry studies of bone mineral status in newborn infants. J Bone Miner Res 11:997–1002. 231. Lapillone A, Braillon P, Claris O, et al. (1997). Body composition in appropriate and in small for gestational age infants. Acta Paediatr 86:196–200. 232. Weiler HA, Yuen CK, Seshia MM (2002). Growth and bone mineralization of young adults weighing less than 1500 grams at birth. Early Hum Devel 67:101–112. 233. Yeste D, Aslmar J, Clemente M, et al. (2004). Areal bone mineral density of the lumbar spine in 80 premature newborns. A prospective and longitudinal study. J Pediatr Endocrinol Metab 17:959–966. 234. Hammami M, Koo WW, Hockman EM (2004). Technical considerations for fan-beam dual-energy x-ray absorptiometry body composition studies in pediatric studies. J Parenteral Enteral Nutr 28:328–333. 235. Koo WW, Hockman EM, Hammami M (2004). Dual energy X-ray absorptiometry measurements in small subjects: conditions affecting clinical measurements. J Am Coll Nutr 23:212–219. 236. Boot AM, de Ridder MA, Pols HA, et al. (1997). Bone mineral density in children and adolescents: relation to puberty, calcium intake, and physical activity. J Clin Endocrinol Metab 82:57–62.

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237. Molgaard C, Thomsen BL, Prentice A, et al. (1997). Whole body bone mineral content in healthy children and adolescents. Arch Dis Child 76:9–15. 238. Zemel BS, Leonard MB, Kalkwarf HJ, et al. (2004). Reference data for whole body, lumbar spine and proximal femur for American children relative to age, gender and body size. J Bone Miner Res 19(1):S231. 239. del Rio L, Carrascosa A, Pons F, et al. (1994). Bone mineral density of the lumbar spine in white Mediterranean Spanish children and adolescents: Changes related to age, sex, and puberty. Pediatr Res 35:362–366. 240. Kroger H, Kotaniemi A, Kroger L, Alhava E (1993). Development of bone mass and bone density of the spine and femoral neck: A prospective study of 65 children and adolescents. Bone Mineral 23:171–182. 241. McKay HA, Bailey DA, Mirwald RL, et al. (1998). Peak bone mineral accrual and age at menarche in adolescent girls: A 6-year longitudinal study. J Pediatr 133:682–687. 242. Takahashi Y, Minamitani K, Kobayashi Y, et al. (1996). Spinal and femoral bone mass accumulation during normal adolescence: Comparison with female patients with sexual precocity and with hypogonadism. J Clin Endocrinol Metab 81:1248–1253. 243. Finkelstein JS, Neer RM, Biller BMK, et al. (1992). Osteopenia in men with a history of delayed puberty. N Engl J Med 326:600–604. 244. Wren TALH, Liu X, Pitukcheewanont P, Gilsanz V (2005). Bone densitometry in pediatric patients: Discrepancies in the diagnosis of osteoporosis by DXA and CT. J Pediatr 146:776–779. 245. Neu CM, Rauch F, Manz F, Schonau E (2001). Modeling of crosssectional bone size, mass and geometry at the proximal radius: A study of normal bone development using peripheral quantitative computed tomography. Osteoporos Int 12:538–547. 246. Neu CM, Manz F, Rauch F, et al. (2001). Bone densities and bone size at the distal radius in healthy children and adolescents: A study using peripheral quantitative computed tomography. Bone 28:227–232. 247. Rauch F, Schonau E (2005). Peripheral quantitative computed tomography of the distal radius in young subjects: New reference data and interpretation of results. J Musculoskelet Neuronal Interact 5:119–126. 248. Leonard MB, Shults J, Justine E, et al. (2004). Interpretation of whole body dual energy X-ray absorptiometry measures in children: Comparison with peripheral quantitative computed tomography. Bone 34:1044–1052. 249. Zadik Z, Price D, Diamond G (2003). Pediatric reference curves for multi-site quantitative ultrasound and its modulators. Osteoporos Int 14:857–862. 250. Baroncelli GI, Federico G, Vignolo M, et al. (2006). Cross-sectional reference data for phalangeal quantitative ultrasound from early childhood to young-adulthood according to gender, age, skeletal growth, and pubertal development. Bone 39:159–173. 251. Ashmeade T, Pereda L, Chen, Carver JD (2007). Longitudinal measurements of bone status in preterm infants. J Pediatr Endocrinol Metab 20:415–424. 252. Fielding KT, Nix DA, Bachrach LK (2003). Comparison of calcaneus ultrasound and dual x-ray absorptiometry in children at risk of osteopenia. J Clin Densit 6:7–15. 253. Fricke O, Tutlewski B, Schwahn B, Schoenau E (2005). Speed of sound: Relation to geometric characteristics of bone in children, adolescents, and adults. J Pediatr 146:764–768. 254. Loud KJ, Gordon CM (2006). Adolescent bone health. Arch Pediatr Adolesc Med 160:1026–1032. 255. American Academy of Pediatrics (1999). Calcium requirements of infants, children, and adolescents. Pediatrics 104:1152–1157. 256. Hertel NT, Stoltenberg M, Juul A, et al. (1993). Serum concentrations of Type I and III procollagen propeptides in healthy children and girls with central precocious puberty during treatment with gonadotropin-releasing hormone analog and cyproterone acetate. J Clin Endocrinol Metab 76:924–927. 257. Koo WWK, Bush AJ, Walters J, Carlson SE (1998). Postnatal development of bone mineral status during infancy. J Am Coll Nutr 17:65–70. 258. Zaidi M (2007). Skeletal remodeling in health and disease. Nature Med 13:791–801. 259. Kalkwarf HJ, Zemel BS, Gilsanz V, et al. (2007). The bone mineral density in childhood study: Bone mineral content and density according to age, sex, and race. J Clin Endocrinol Metab 92:2087–2099.

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4 Ambiguous Genitalia SELMA FELDMAN WITCHEL, MD • PETER A. LEE, MD, PhD

Introduction Ambiguous Genitalia: Talking with the Parents Sex Determination Development of the Reproductive System Urogenital Differentiation Testicular Differentiation Ovarian Differentiation Germ Cell Differentiation Differentiation of Internal Genital Structures Differentiation of External Genital Structures Sexual Differentiation of the Brain Lessons Learned from Transgenic Models Disorders of Gonadal Differentiation Wilms’ Tumor Gene WNT4 Gene 46,XY Disorders of Sexual Differentiation (Gonadal Dysgenesis) SOX9 DAX1 Desert Hedgehog Chromosome 9p Monosomy Xq28 Deletion ATR-X Syndrome Vanishing Testes Multiple Congenital Anomalies Ovotesticular Disorder of Sexual Differentiation (True Hermaphroditism) 46,XX Testicular Disorder of Sexual Differentiation FOXL2 Disorders of Androgen Synthesis

Introduction Under the auspices of the Lawson Wilkins Pediatric Endocrine Society (USA) and the European Society for Pediatric Endocrinology, an international consensus statement was formulated that recommended a revised clas-

SF1/NR5A1 Gene Luteinizing Hormone Choriogonadotropin Receptor Gene Smith-Lemli-Opitz Syndrome Congenital Lipoid Adrenal Hyperplasia Side Chain Cleavage Cytochrome P450 Enzyme Virilizing Congenital Adrenal Hyperplasias 3␤-hydroxysteroid Dehydrogenase Deficiency 17␤-hydroxysteroid Dehydrogenase Deficiency 5␣-reductase Deficiency Cytochrome P450 Oxidoreductase Deficiency Placental Aromatase Deficiency Maternal Hyperandrogenism Disorders of Androgen Action Mullerian Duct Abnormalities Persistent Mullerian Duct Syndrome Mullerian Duct Abnormalities in 46,XX Individuals Hypogonadotropic Hypogonadism Cryptorchidism Diagnosis History Physical Examination Laboratory Studies Treatment Sex of Rearing Considerations with Regard to Surgery Medical Treatment Psychological and Genetic Counseling and Support Conclusions

sification of the medical terminology used for disorders of sex development to avoid confusing and derogatory terms.1 This descriptive classification attempts to be sensitive to concerns of parents and flexible enough to incorporate novel molecular genetic information. The updated classification system integrates molecular genetic 127

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considerations into the nomenclature for “disorders of sexual differentiation (DSD).”1 Terms such as pseudohermaphrodite, intersex, and gender labeling in the diagnosis should be avoided.2 To accommodate all DSD, the classification system is broad and includes some conditions that do not present with obvious abnormalities of genital development (Table 4-1). The DSD categories are sex chromosome DSDs such as 45,X/46,XY (formerly mixed gonadal dysgenesis) and ovotesticular DSD (formerly true hermaphroditism); 46,XY

DSDs such as disorders of testicular development, disorders of androgen synthesis and action (replacing and expanding the former category of male pseudohermaphroditism), and XY sex reversal; and 46,XX DSDs such as masculinization of the XX individual (replacing female pseudohermaphrodite) and XX sex reversal. Because of the complexities of chromosomal and gonadal development, some diagnoses can be included in more than one of the three major categories. Nevertheless, despite many recent advances the specific cause of genital ambiguity cannot always be identified—particularly among those with a 46,XY karyotype.

TA B L E 4 - 1

Summary of Disorders Associated with Ambiguous Genitalia Sex Chromosome DSD • 45,X Turner syndrome • 45,X/46,XY gonadal dysgenesis • 46,XX/46,XY gonadal dysgenesis 46,XY DSD (Disorders of Testicular Differentiation) • Denys-Drash syndrome (WT1 mutations) • Frasier syndrome (WT1 mutations) • XX gonadal dysgenesis • XY gonadal dysgenesis • SRY positive • SRY negative • Ovotesticular DSD • Campomelic dwarfism (SOX9 mutations) • Dosage sensitive sex reversal (duplications of DAX1) • Desert hedgehog (DHH) mutations • Monosomy distal chromosome 9p • Xq28 deletion • Vanishing testes • ATR-X syndrome • ARX • Persistent Mullerian duct syndrome 46,XY (Disorders of Androgen Synthesis or Action) • Steroidogenic factor-1 (SF-1 mutations) • Leydig cell hypoplasia (LHR mutations) • Smith-Lemli-Opitz syndrome (DHCR7) • 3␤-hydroxysteroid dehydrogenase deficiency (HSD3B2) • 17␣-hydroxylase/17,20-lyase deficiency (CYP17) • Lipoid adrenal hyperplasia (StAR) • 17␤-hydroxysteroid dehydrogenase deficiency (HSD17B3) • 5␣-reductase deficiency (SRD5A2) • Androgen insensitivity (AR) • Hypogonadotropic hypogonadism • P450 oxidoreductase deficiency (POR) 46XX, DSD • 21-hydroxylase deficiency (CYP21) • 11␤-hydroxylase deficiency (CYP11B1) • Placental aromatase deficiency (CYP19) • Maternal hyperandrogenism • XX males • Ovotesticular DSD Other • IMAGe syndrome • Environmental endocrine disrupters • VACTERL syndrome • MURCs syndrome • Cloacal extrophy • Aphallia

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Ambiguous Genitalia: Talking with the Parents For parents, the birth of a child is a long-anticipated, much desired, and exciting event. However, today the scenario has largely changed. In the past, the typical first question or statement after a child’s birth related to the sex of the child: Is it a boy or a girl? Now, with the increased frequency of prenatal ultrasound examinations parents have usually been told the sex of their child and often have selected names for their son or daughter. Thus, when confronted with a newborn with genital ambiguity the issues occur in a different context. This immediately necessitates full disclosure, which allows parents to be actively involved in the medical decisionmaking process. The parents need to be quickly equipped to deal emotionally with these issues for themselves and to enable them to appropriately interact with their infant, family members, friends, and colleagues. Initially, the parents need to hear that there has been a problem in the complex system that directs genital development—which makes it impossible to tell the sex of their child simply by examining the external genitalia. It is important to acknowledge that such development is not a consequence of anything that they, as parents, did or did not do. Despite openness and full disclosure, the parents may harbor guilt and negative feelings. Cultural attitudes, preexisting expectations, and family support systems influence the parents’ responses to their child. The medical team needs to promote an open and caring network to provide support for the parents. The initial treatment goal is to determine if there is an underlying or associated life-threatening condition that requires specific urgent treatment. If the child’s gender remains unclear, information needs to be obtained to assist the parents in determining the most appropriate sex of rearing. Usually, this can be accomplished within a matter of hours or days. In more complex instances, the diagnostic process may take longer. In situations in which it is impossible to identify the specific etiology, the general DSD category provides a basis for decision making. These considerations include the extent of external and internal reproductive system development, evidence of gonadal functionality (potential hormone secretion and fertility), and hormone responsiveness. In some instances, these factors are more relevant than the karyotype. When

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consensus has been reached regarding a diagnostic category, available outcome information for that diagnosis should be reviewed. Knowledge of the specific etiology, including details of the diagnosis, enables planning therapeutic interventions and genetic counseling for future pregnancies. The first interview with the parents should set a positive and optimistic tone to promote parental bonding with their infant. Indeed, the emotional tone of this initial interaction is usually more meaningful than the factual information provided and is recalled by parents for many years. Respect for the family and individual perspectives together with a willingness to repeat or defer detailed explanations are crucial. In the midst of the emotional distress associated with the uncertainty of their infant’s gender, parents cannot be expected to assimilate the vast amount of information that eventually needs to be shared. Factual explanations regarding the process of sexual differentiation with a focus on their infant’s situation should be initially outlined. Detailed explanations and discussion can be repeated multiple times as the child ages. The use of simple sketches and provision of pictures and diagrams can be helpful to explain the embryology of genital development to the parents. The primary goal at this point is to provide the parents with a basic understanding that the internal and external genital structures for both boys and girls develop from the same primordial tissues. It may be helpful to explain that there are not exclusively male and female hormones, but rather the environments in which male and female fetuses develop are characterized by differing relative amounts of these hormones. Thus, incomplete male or female development represents the consequences of the prenatal hormonal environment. Too much or too little androgen effect is reflected in the degree of prenatal virilization. During this initial dialogue, showing the parents the specific physical findings of their infant is often beneficial. This can alleviate apprehension, increase their comfort to look at their infant’s genitalia, and promote the goal of fostering their perception of their child as a human having the needs of any other infant. This approach allows for information to be presented in a manner that will minimize anxiety and better equip parents to participate in the decision-making process. Before parents can provide the best support for their infant, they must each personally reach a resolution with a commitment to a positive perspective concerning this situation. Discussion of many concerns (particularly those related to gender identity, pubertal development, sexual orientation, sexual function, and fertility) may be helpful. The intent is that honest discussions will engender positive feelings that enhance positive interactions and enable the parents to promote their child’s self-esteem. Unless the gender assignment is clear at this point, delay in naming the infant, announcing the baby’s birth, and registering the birth can be recommended until more information becomes available. The message should be clear that there will be an appropriate sex of rearing for their child and that it is the parents’ privilege and responsibility to participate in the process leading to a gender assignment. Until sex of rearing is established, it is best

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to refer to the infant as “your baby” or “your child.” Terms such as he, she, and it should be avoided.

Sex Determination Sex determination is the binary switch that launches the developmental destiny of the embryonic gonads to become testes or ovaries. Sexual differentiation refers to the process through which male or female phenotype develops. The gonads, internal genital ducts, and external genital structures all develop from bipotential embryologic tissues. Each cell in the developing gonad has the potential to differentiate into either a testicular or ovarian cell. However, “the fate decisions in individual cells are highly coordinated such that cells of discordant fate are rarely seen.”3 Thus, differentiation as male or female depends on regulated orchestration of the expression and interaction of specific genes and gene products. Through Alfred Jost’s experiments with fetal rabbits in the 1940s and 1950s, the requirements for a testis and testosterone for male sexual differentiation were established.4 Chromosomal composition of the embryo, XX or XY, determines gonadal sex. The genetic locus primarily responsible for this binary switch, the sexdetermining region on the Y (SRY) gene on the Y chromosome, was identified through studies of patients with disorders of sexual differentiation. Analysis of transgenic mice confirmed the essential role of SRY and provided further molecular understanding of testicular differentiation.5,6 Whereas the karyotype (46,XY or 46,XX) of the primordial gonad determines whether it differentiates into a testis or ovary, respectively, local factors (such as hormones secreted by the developing gonads or tissuespecific transcription factors) influence the ensuing differentiation of the internal and external genital structures.7 Divergence from the normal sequence of events leads to disorders of sexual differentiation that can manifest as abnormal gonadal differentiation, inconsistent internal genital differentiation, or ambiguity of the external genitalia. Although genital ambiguity is usually not considered to be a medical emergency, it is highly distressing to the parents and extended family. Hence, prompt referral and evaluation by a team with expertise in disorders of sexual differentiation is beneficial. Information regarding our current knowledge of sexual differentiation is presented, followed by a discussion of the various causes and treatment for ambiguous genital development.

Development of the Reproductive System UROGENITAL DIFFERENTIATION In humans, at 4 to 6 weeks of gestation the urogenital ridges develop as an outgrowth of coelomic epithelium (mesothelium).8 The gonads, adrenal cortex, kidney, and reproductive tract derive from the urogenital ridge (Figure 4-1). Physical contact with the mesonephros appears to be important for

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Figure 4-1. Cartoon of the genes involved in the process of sexual differentiation. Wilms’ tumor (WT1), EMX2, LIM1, and steroidogenic factor-1 (SF1) play roles in differentiation of gonad from urogenital ridge. Genes involved in testicular differentiation include SF-1, SOX9, sex-determining region on Y (SRY), and anti-Mullerian hormone (AMH). The dosage-sensitive sex-adrenal hypoplasia congenital critical region on X (DAX1) appears to function as an anti-testis factor. Wnt4 promotes development of the Mullerian ducts, whereas Wnt7a promotes expression of the receptor for AMH (AMH-RII). Sertoli cells secrete AMH, which [acting through its cognate receptor (AMH-RII)] promotes regression of the Mullerian ducts. Leydig cells secrete testosterone and insulin-like hormone-3 (INSL3). Testosterone stabilizes the Wolffian ducts and is converted to DHT by 5␣-reductase in target tissues to promote differentiation of the prostate and development of male external genitalia. INSL3 is involved in transabdominal testicular descent.

subsequent gonadal differentiation.9 Due to their origin as part of the developing urogenital system, ovaries and testes are initially located high in the abdomen near the kidneys. One of the earliest morphologic changes is increased proliferation and size of developing 46,XY gonads.

TESTICULAR DIFFERENTIATION The SRY protein is a nuclear high-mobility group (HMG) domain protein expressed in pre-Sertoli cells, where it triggers a molecular switch to induce Sertoli cell differentiation and initiate the process of male sexual differentiation. The HMG domain of the SRY protein binds to the minor DNA groove, where it functions as a transcription factor by bending DNA to presumably permit other proteins access to regulatory regions and to promote assembly of nucleoprotein transcription complexes.10 A threshold SRY level must be achieved at a critical time to establish male sexual differentiation. Otherwise, the ovarian differentiation pathway is activated.11 Yet, the factor(s) upregulating SRY expression remains elusive. SRY expression is independent of the presence of germ cells. In addition to SRY, sequential expression of several other genes is required for normal male sexual differentiation. These genes include SRY-related HMG box-containing-9 (SOX9), anti-Mullerian hormone (AMH), dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on X (DAX1), steroidogenic factor-1

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(SF1), Wilms’ tumor 1(WT1), GATA-binding-4 (GATA4), desert hedgehog (DHH), patched (PTC), wingless-related MMTV integration site 4 (WNT4), and WNT7a. Using immunohistochemistry, SF1 and SOX9 proteins can be detected in human embryonic gonadal tissue at 6 to 7 weeks of gestation. At this time, SOX9 expression becomes limited to nuclei of Sertoli cells in a 46,XY fetus but remains cytosolic in a 46,XX fetus. SF1 and SOX9 protein expression precede that of AMH. Only after AMH protein expression and onset of overt testicular differentiation do Wilms’ tumor (WT1) and GATA-4 protein expression increase in the fetal testis.12 GATA4 belongs to a family of zinc finger transcription factors known as GATA-binding proteins because they bind to a consensus sequence in the promoter and enhancer regions of target genes. Testicular differentiation occurs earlier than ovarian development. The testis consists of five cell types: supporting or Sertoli cells, endothelial cells, peritubular myoid cells, steroid-secreting Leydig cells, and germ cells. The first evidence of testicular differentiation is the appearance of primitive Sertoli cells at 6 to 7 weeks gestation in the human fetal testis. Cells, mostly endothelial cells, migrate from the mesonephros and interact with the pre-Sertoli cells to promote development of the testicular cords.13 The testicular cords are precursors of the seminiferous tubules that will contain Sertoli and germ cells. In addition to the migration of mesonephric cells, testes show an increased rate of cell proliferation.14

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SOX9 induces expression of prostaglandin D synthase (Pgds), an enzyme involved in prostaglandin synthesis.15 In a positive feedback loop, developing Sertoli cells secrete prostaglandin D2—which binds to its cognate receptor to upregulate SOX9 expression and recruit additional Sertoli cells.16,17 Phenotype-genotype studies of humans and mice demonstrate that SOX9 expression is a crucial step, downstream of SRY, in testis development. Fibroblast growth factor 9 (FGF9) is another signaling molecule important for testis development. In the mouse, the absence of FGF9 expression is associated with a premature decline in SOX9 expression—leading to arrested Sertoli cell differentiation, upregulation of Wnt4 expression, and maleto-female sex reversal of germ cells.18 Vascular development in the gonad is sexually dimorphic with the endothelial cells in the developing testis, forming a characteristic pattern consisting of a prominent coelomic vessel on the antimesonephric surface with branches between the testis cords.19 This blood vessel is absent in the ovary. The signaling molecule, WNT4 suppresses formation of this coelomic vessel without affecting the development of its side branches. Peritubular myoid cells are testis-specific smoothmuscle-like cells important to structural integrity and development of the testis cords. Factors relevant to peritubular myoid cell differentiation include desert hedgehog (DHH), which is secreted by Sertoli cells and its receptor Patched (Ptch1)—which is expressed on peritubular myoid cells.20,21 The peritubular myoid cells surround the Sertoli cells, separating them from the Leydig cells—which are then sequestered in the interstitium. Leydig cell differentiation depends on paracrine signals, including platelet-derived growth factor receptoralpha [(PDGFR-␣), DHH, PTCH1, and Aristaless-related homeobox (ARX)]. SF1 is expressed in Leydig cells to promote steroidogenic enzyme genes expression. The number of fetal Leydig cells reflects gonadotropin stimulation because the number is decreased in anencephalic male fetuses and increased in 46,XY fetuses, with elevated gonadotropin concentrations secondary to complete androgen insensitivity.22 Differentiation of adult Leydig cells occurs postnatally.23

OVARIAN DIFFERENTIATION Although ovarian differentiation has been considered the default pathway that occurs in the absence of SRY gene expression, accumulating evidence indicates that specific genes influence ovarian differentiation. Genes involved in cell cycle regulation were found to be overexpressed in developing XX gonads. Persistent DAX1 expression appears to play a role in ovarian differentiation. Other genes identified in developing ovaries include follistatin (Fst), Iroquois-3 (Irx3), Wnt4, and bone morphogenic protein-2 (Bmp2).24 The functions of Wnt4 include suppression of the androgen-secreting interstitial cells, inhibition of coelomic vascularization, and support of Mullerian derivatives—suggesting that it plays a major role in ovarian differentiation. Another gene postulated to influence ovarian differentiation is Forkhead L2 (FOXL2), a forkhead transcription factor.23

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GERM CELL DIFFERENTIATION Despite the requirement for germ cells in the postnatal ovary to maintain its structure, germ cells are not required for the initial development of ovaries or testes.3 Primordial germ cells migrate from their origin in the hindgut to the developing gonadal ridges. Factors important for germ cell migration and colonization of the genital ridges include fragilis proteins 1 and 3 (IFITM1 and IFITM3) and stromal-cell–derived factor 1 (SDF1, also known as CXCL12) and its receptor CXCR4.25,26 The local environment directs the fate of the primordial germ cells.3 Germ cell differentiation into a sperm or an egg is closely linked to the cell cycle decision between mitosis and meiosis.27 In a human XX gonad, the germ cell meiosis begins at 10 to 11 weeks of gestation. This process appears to depend on retinoic acid, which is derived from mesonephric cells. Retinoic acid induces meiosis in an anterior-to-posterior wave and upregulates Stra8 (stimulated by retinoic acid gene 8) expression.28 In mice, Stra8 is necessary to premeiotic DNA replication. Thus, premeiotic DNA replication and the ensuing meiotic prophase are sexually dimorphic regulated steps of terminal differentiation characteristic of germ cells resident in the embryonic ovary.29 After the first meiotic division, the primary oocyte becomes associated with granulosa cell precursors to constitute the primary follicle. In humans, primordial follicles are evident at 20 weeks of gestation. From a peak of 6.8 million oocytes at approximately 5 months of gestation, approximately 2 million are present at birth due to follicular atresia.30 Accelerated follicular atresia contributes to the degeneration observed in streak gonads in X monosomy. The developing somatic cells in the testis actively inhibit meiosis through expression of CYP26B1, a cytochrome P450 enzyme that degrades retinoic acid.28,31 The germ cells in the testis enter a state of mitotic arrest. It has been suggested that fetal germ cells are programmed to enter meiosis and initiate oogenesis unless this process is inhibited.32 Maternal and paternal alleles are differentially imprinted such that monoallelic expression of specific genes occurs. During this process of imprinting, mature oocytes and sperm are differentially marked reflecting “parent-of-origin”–specific methylation patterns. In the primordial immature germ cells, inherited imprints are erased shortly after the germ cells enter the gonadal ridge. Sexually dimorphic methylation imprinting is subsequently reestablished in male and female gametes. This process occurs late in fetal development in the male and postnatally in female germ cells.33,34 The importance of this imprinting process has been elucidated through study of parent-of-origin–dependent gene disorders such as Beckwith-Wiedemann, Prader-Willi, and Angelman syndromes and neonatal diabetes mellitus.

DIFFERENTIATION OF INTERNAL GENITAL STRUCTURES The Wolffian duct originates as the excretory duct of the mesonephros and develops into the epididymis, vas deferens, ejaculatory duct, and seminal vesicle. The epididymis

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consists of four functional portions: initial segment, caput, corpus, and cauda. Sperm mature in the caput and corpus, whereas the cauda is primarily for storage. The Mullerian or paramesonephric duct originates as an invagination of the coelomic epithelium and develops into the Fallopian tubes, uterus, and upper third of the vagina. In the male fetus, the Sertoli cells secrete antiMullerian hormone (AMH), also known as Mullerian inhibitory hormone (MIH). In human 46,XY fetuses, AMH expression can be detected by 7 weeks of gestation, is not dependent on the presence of germ cells within the testis, and promotes regression of the Mullerian ducts.35 AMH, a member of the transforming growth factor-␤ (TGF-␤) family, undergoes proteolytic cleavage to become biologically active. AMH binds to its receptor, AMH-RII, on the surface of the Mullerian duct mesenchymal cells to induce increased matrix metalloproteinase 2 expression.36,37 The net result is degeneration and loss of basement membrane integrity of the epithelial and mesenchymal Mullerian cells, leading to regression of the Mullerian ducts.38 AMH expression is highly regulated because inappropriate expression in a 46,XX fetus would lead to uterine agenesis. In the 46,XX fetus with absence of both AMH and testosterone, the Mullerian duct derivatives persist and the Wolffian ducts regress. When a female fetus is inappropriately exposed to AMH (as in freemartin cattle), Mullerian duct regression and ovarian masculinization occur.39,40 The fetal hypothalamic-pituitary-gonadal (HPG) axis is active by mid-gestation, with peak fetal testosterone concentrations occurring at approximately 15 to 16 weeks of gestation. Prior to this time, placental hCG stimulates testosterone production by the fetal Leydig cells. Secretion of testosterone by the fetal Leydig cells stabilizes the Wolffian ducts in 46,XY fetuses. Region-specific signaling molecules such as bone morphogenic proteins (BMPs), homeobox genes (HOXA10 and HOXA11), growth differentiation factor 7 (GDF7), relaxin, an orphan Gprotein–coupled receptor (LGR4), platelet-derived growth factor A (PDGFA), and its cognate receptor (PDGFRA) influence the development of the epididymis and seminal vesicle.19 The prostate, a male accessory sex gland, contributes to seminal fluid plasma and develops from the urogenital sinus. After the initial testosterone-dependent induction of prostate differentiation, subsequent development involves epithelial-mesenchymal interactions that lead to cell differentiation and branching morphogenesis. The requisite signaling molecules FGFs, sonic hedgehog (SHH), BMPs, HOXA13, and HOXD13 are similar to those required for external genital differentiation.41,42

DIFFERENTIATION OF EXTERNAL GENITAL STRUCTURES The genital tubercle, urethral folds, and labioscrotal swellings give rise to the external genitalia. Under the influence of circulating androgens that are converted to dihydrotestosterone in the target tissues, the urethral folds fuse to form the corpus spongiosum and penile urethra, the genital tubercle develops into the corpora cavernosa of the penis, and the labioscrotal folds fuse to form the scrotum.

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In the normal 46,XY human fetus, a cylindrical 2-mm phallus with genital swellings has developed by 9 weeks of gestation. By 12 to 14 weeks of gestation, the urethral folds have fused to form the cavernous urethra and corpus spongiosum. By 14 weeks, the external genitalia are clearly masculine apart from testicular location. The high incidence of hypospadias in humans suggests that urethral fusion is a delicate and finely regulated process. In the 46,XX fetus, in the absence of androgens the urethral folds and labioscrotal swellings do not fuse and develop into the labia minora and labia majora, respectively. The genital tubercle forms the clitoris, and canalization of the vaginal plate creates the lower portion of the vagina.43 By 11 weeks of gestation, the clitoris is prominent and the lateral boundaries of the urogenital sulcus have separated. Minimal clitoral growth, welldefined labia majora, hypoplastic labia minora, and separate vaginal and urethral perineal openings are present by 20 weeks of gestation. By 33 days postconception, the human fetal adrenal cortex is distinct from the developing gonad. Due to its role as the source of DHEAS for placental estrogen biosynthesis, the fetal adrenal cortex grows rapidly. By 50 to 52 days postconception, expression of several steroidogenic enzymes, steroidogenic acute regulatory protein (StAR), 11␤-hydroxylase (CYP11B1), 17␣-hydroxylase/ 17,20-lyase (CYP17), and 21-hydroxylase (CYP21) in the fetal adrenal cortex have been demonstrated immunohistochemically.44 Recent data indicate that transitory cortisol biosynthesis peaks at 8 to 9 weeks gestation.44 This early cortisol biosynthesis coincides with transient adrenal expression of both nerve growth factor IB-like (NGFI-B) and 3␤-hydroxysteroid dehydrogenase-2 (HSD3B2).44 Concommitantly, ACTH can be detected in the anterior pituitary— suggesting the presence of negative feedback inhibition during the first trimester.44 During the time male sexual differentiation begins, this negative feedback inhibition may serve to prevent virilization of female fetuses to ensure normal female sexual differentiation.44,45

SEXUAL DIFFERENTIATION OF THE BRAIN Clinical investigations suggest that the brain is sexually dimorphic and that testosterone is a masculinizing hormone in human. Males with aromatase deficiency manifest male psychosexual behavior and gender identity. Alternatively, 46,XY individuals with complete androgen insensitivity syndrome (CAIS) develop female gender identity.46,47 Preliminary data implicate genetic differences, independent of sex steroid exposure, as the molecular basis for some aspects of sexual dimorphism of the brain.48 Postmortem histologic examination demonstrated that women have more synapses in the neocortex, whereas men have more neurons in this region.49,50

Lessons Learned from Transgenic Models Sexual differentiation is a complex process in which precise spatiotemporal coordination and regulation of gene expression are crucial to achieve full reproductive

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capability.51 Despite greater knowledge of the molecular details of sexual differentiation, the precise sequence of biologic events and all component factors remain to be elucidated. Investigation of normal and transgenic mice confirmed the crucial role of the sex-determining region on Y (SRY) gene in male differentiation when XX mice carrying only a 14-kb fragment of the Y chromosome showed a male phenotype.52 In some instances, sexreversal phenotypes have been fortuitous observations. Although investigation of transgenic mouse models has provided much information about sexual differentiation (Table 4-2), the mouse and human phenotypes may differ. For example, humans with a 45,X karyotype develop gonadal dysgenesis associated with infertility—whereas XO mice are fertile. The following brief discussion of the spatiotemporal expression of specific genes and phenotypes of transgenic mice reviews the current understanding of sexual differentiation. Abnormal gonadal development has been described in mice homozygous for targeted deletions of genes involved in urogenital differentiation (such as Wt1, Sf1, Emx2, M33, and Lim1). In mice, these genes are expressed earlier in gestation than Sry, which is expressed transiently at 10.5 to 11 days postcoitum (d.p.c).53 For example, Wt1 is expressed throughout the intermediate mesoderm at 9 d.p.c. Subsequently, Wt1 is expressed in the developing gonad. The phenotype of Wt1 knockout mice includes embryonic lethality, failure of gonadal and kidney development, and abnormal development of the mesothelium, heart ,and lungs.54 Unlike humans, as discussed later, heterozygous Wt1 mutations in mice are not associated with kidney tumors or genito-urinary anomalies. Steroidogenic factor 1 (Sf1), also known as NR5A1, is an orphan nuclear hormone receptor that functions as a transcription factor. In mice, Sf1 is expressed from the earliest stages of gonadogenesis at 9 d.p.c. and regulates expression of steroidogenic enzyme genes in gonads and adrenals. At the onset of testicular differentiation, Sf1 expression becomes sexually dimorphic with increased expression in fetal testis and decreased expression in fetal ovaries.55 The pathologic findings in mice homozygous for targeted deletion of Sf1 include absence of gonads, adrenal glands, and ventromedial hypothalamus— with decreased number of gonadotropes in the anterior pituitary.56 Sf1 knockout mice have female internal and external genitalia irrespective of genetic sex and die shortly after birth secondary to adrenal insufficiency.57 Pituitary-specific Sf1 knockout mice manifest hypogonadotropic hypogonadism, confirming the essential role of Sf1 in pituitary function.58 The protein encoded by the Sox9 gene contains a DNAbinding HMG domain and plays a major role in testicular differentiation. Following the onset of testicular differentiation, Sox9 expression increases in the testis and decreases in the ovary by 11.5 d.p.c. Ectopic expression of Sox9 in XX mice leads to testicular differentiation.59 Mice with homozygous targeted deletion of the Sox9 gene die during mid-gestation.60 Sox9 also plays a major role in chondrocyte differentiation and cartilage formation.61 Homozygous deletion of Emx2, a homeodomain transcription factor, results in an embryonic lethal phenotype associated with absence of kidneys, ureters, gonads—and

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in Wolffian and Mullerian duct derivatives.62,63 As Wt1 expression is initially normal in the metanephric blastema of Emx2 knockout mice, Emx2 is likely downstream of Wt1. Interestingly, adrenal gland development is normal in Emx2 knockout mice. Lim1, also known as Lhx1, encodes a homeobox protein—which is important in the differentiation of intermediate mesoderm and the urogenital system.64 Homozygous deletion of Lim1 is associated with absence of kidneys and gonads and with anterior head structures.65 Dax1 is first expressed in the genital ridge at 10.5 to 11 d.p.c. With differentiation of the testicular cords, Dax1 expression becomes sexually dimorphic—with decreased expression in the fetal testis. It continues to be expressed in the fetal ovary, where it appears to inhibit gonadal steroidogenesis.66 Dax1 may interfere with steroidogenesis by inhibiting StAR expression and/or Sf1-mediated transactivation.67,68 Dax1 functions as an adaptor molecule to recruit the nuclear receptor corepressor N-CoR to the Sf1 promoter, thus interfering with transactivation.69,70 Rather than functioning to promote ovarian differentiation, it appears that Dax1 acts as an anti-testis factor. In male mice with targeted disruptions of the Dax1 gene, abnormalities of testicular germinal epithelium and male infertility develop despite normal testicular appearance at birth.71 Female mice homozygous for targeted Dax1 mutation showed normal adult reproductive function.72 Testicular development is delayed in XY mice carrying extra copies of mouse Dax1, but sex reversal is not observed unless the mouse also carries weak alleles of the SRY gene.72 At 11.5 days, Fgf9 is expressed in gonads of both sexes. By day 12.5, Fgf9 is detected only in testes. Mice homozygous for Fgf9-targeted deletions show maleto-female sex reversal with disruption of testis differentiation.73 Loss of Fgf9 does not interfere with Sry expression, but prevents persistence of Sox9 expression in the developing testis. As discussed previously, Fgf9 inhibits Wnt4 expression.18 At 11.5 to 12.5 d.p.c, in both sexes Mullerian ducts arise from coelomic epithelium in the mesonephric region under the influence of Wnt4.74 Male mice with homozygous targeted deletions of Wnt4 show normal testicular and Wolffian duct development, but Mullerian ducts never develop. The phenotype of female mice with homozygous deletions of Wnt4 includes absence of Mullerian duct derivatives, retention of Wolffian duct derivatives, and decreased oocyte development. In addition, the large coelomic blood vessel (typical of testicular differentiation) develops in the ovaries of female mice homozygous for targeted deletion of Wnt4.75 Wnt4 appears to be involved in differentiation of Mullerian ducts, repression of endothelial migration from the mesonephros into the gonad, impeding migration of adrenal cells into the developing gonad, and maintenance of oocyte development. Persistence of Mullerian duct derivatives and infertility were noted in male mice homozygous for targeted deletion of Wnt7a. One consequence of Wnt7a deletions is absence of Mullerian hormone receptor (AMH-RII) expression by Mullerian ducts. In females, although Mullerian duct derivatives develop they are abnormal—with loss of uterine glands, reduction in uterine stroma, and deficient coiling and elongation of the Mullerian duct. Thus, Wnt7a appears

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TA B L E 4 - 2

Consequences of Loss-of-Function Mutations in Genes Associated with DSD Gene WT1

Human Locus 11p13

Human Phenotype Denys-Drash and Frasier syndrome 46,XY Gonadal dysgenesis Campomelic dysplasia and gonadal dysgenesis

SRY SOX9

Yp11.3 17q24-25

DHH

12q13.1

Gonadal dysgenesis

ATRX/XH2

Xq13.3

ARX

Xp22.13

␣-thalassemia, mental retardation, genital abnormalities, short stature Lissencephaly, absence of the corpus callosum, microcephaly 46,XY sex reversal with or without adrenal insufficiency Schizencephaly

SF1/NR5A1

9q33

EMX2

10q26.1

FGF9 WNT4

13q11-q12 1p35

WNT7A

DAX1/NROB1

3p25

Xp21.3

GATA4 FOXL2

8p23.2-p22 3q23

LHGCR DHCR7 StAR

2p21 11q12-q13 8p11.2

CYP11A1

15q23-q24

CYP19A1

15q21.1

POR AMH

Adrenal hypoplasia congenita; duplication associated with male-to-female sex reversal Congenital heart disease Blepharophimosis/ptosis/epicanthus inversus syndrome Leydig cell hypoplasia Smith-Lemli-Opitz syndrome Congenital lipoid adrenal hyperplasia Male-to-female sex reversal, adrenal insufficiency Aromatase deficiency

INSL3

19p13.2

Antley-Bixler syndrome Persistent Mullerian duct syndrome Persistent Mullerian duct syndrome Associated with cryptorchidism

LGR8

13q13.1

Associated with cryptorchidism

AMH-RII

7q11.2 19p13.3-p13.2

?? 46,XX, Uterine agenesis 46,XY, Male-to-female sex reversal Limb malformation syndromes

12q13

to function as an epithelial-to-mesenchymal signal important in the sexually dimorphic differentiation of Mullerian duct derivatives. 76 The phenotype associated with targeted disruption of c-kit ligand, a receptor tyrosine kinase also known as

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Mouse Phenotype Homozygous: embyronic lethal with absence of kidney and gonads. “Knock-in” female-to-male sex reversal. Overexpression in XX mice results in female-to-male sex reversal. Heterozygous null associated with perinatal death, cleft palate, and skeletal abnormalities. Abnormal peripheral nerves, infertility due to impaired spermatogenesis in males. Phenotype of null is embyronic lethal. Overexpression associated with growth retardation, neural tube defects, and embryonic death. Males have abnormal CNS development and abnormal testicular differentiation. Absent adrenal glands, absent gonads, abnormal pituitary differentiation. Absence of kidneys, ureters, gonads, and genital tracts. Male-to-female sex reversal, lung hypoplasia. Males are normal. Females manifest female-to-male sex reversal. Persistent Mullerian duct derivatives in males. Abnormal Mullerian duct differentiation in females. Male infertility.

Embyronic lethal, heart defects. Small, absence of eyelids, craniofacial anomalies, female infertility. NA. IUGR, cleft palate, neurologic abnormalities. All with female external genitalia. Neonatal death due to adrenal insufficiency. Male-to-female sex reversal. Neonatal death due to adrenal insufficiency. Immature infertile female. Males are initially fertile, but develop disrupted spermatogenesis. Embryonic lethal. Male has persistent Mullerian duct derivatives and infertility. Male has persistent Mullerian duct derivatives and infertility. Cryptorchidism in males. Overexpression in females associated with development of gubernaculum and aberrant ovarian location. Cryptorchidism.

Steel factor, is complete lack of germ cells in the gonads.77 Affected animals are sterile, but show normal sexual differentiation.78 Both male and female mice homozygous for targeted deletion of the Stra8 gene show no overt phenotype apart from infertility.29

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Mullerian inhibitory hormone (Amh) is first expressed at 12 d.p.c. Although direct control of Amh expression by the Sry gene product originally appeared to be an attractive hypothesis,79 subsequent investigations have refuted this theory.80 Rather, transcriptional regulation of Amh appears to involve both protein-protein and protein-DNA interactions—with Sf1 and Sox9 being two of the proteins involved.81-83 Amh action is mediated by a heteromeric-signaling complex consisting of type I and type II serine/threonine kinase receptors. The Amh type II receptor binds Amh and recruits a type I receptor. SF1 appears to regulate expression of the Amh type II receptor (Amh-rIII) gene.84 The phenotypes of the Amh and Amh-rII knockout mice are identical. In XY mice, both male and female internal genital structures are found. Male mice are infertile because the retained uterus blocks passage of sperm through the vas deferens. The phenotype of male mice with targeted deletion of desert hedgehog (Dhh) includes infertility and impaired spermatogenesis.85 These mice also showed abnormal peripheral nerves with extensive minifascicles within the endoneurium.86 During development, expression of Dhh is limited to Sertoli cells and Schwann cells in peripheral nerves.87 In mice, other key molecules in the development of male external genital structures include sonic hedgehog (Shh), fibroblast growth factors, Wnts, Bmps, Hoxa13, and Hoxad13. Specifically, Shh increases expression of Bmp4, Hoxa13, Hoxd13, and Ptc expression.19 Expression of 5␣-reductase in the genital tubercle mesenchyme occurs. Male mice homozygous for targeted deletions of the insulin-like hormone-3 (Insl3), Hoxa10, or leucine-rich repeatcontaining Lgr8 genes show bilateral cryptorchidism.88-91 Investigations involving normal and transgenic mice have provided much information about the genes and gene products involved in sexual differentiation. Nevertheless, differences do exist between rodents and humans. Thoughtful examination of patients with disorders affecting sexual differentiation has elucidated many of the factors involved in human sexual differentiation.

Disorders of Gonadal Differentiation WILMS’ TUMOR GENE The Wilms’ tumor suppressor (WT1) gene, located at chromosome 11p13, plays an important role in both kidney and gonadal differentiation.92 Although Wilms’ tumor and genitourinary abnormalities can be associated with heterozygous WT1 deletions, only 6% to 15% of sporadic Wilms’ tumors are associated with WT1 mutations.93 Heterozygous deletions at chromosome 11p13 can be part of a contiguous gene deletion syndrome known as WAGR syndrome (Wilms’ tumor, aniridia, genito-urinary anomalies, gonadoblastoma, and mental retardation). Denys-Drash syndrome (characterized by genito-urinary anomalies, Wilms’ tumor, and nephropathy) is due to mutations in the WT1 gene. Typically, the nephropathy begins during the first few years of life, manifests with proteinuria, and results in end-stage renal failure due to focal or diffuse mesangial sclerosis.94-97 Among affected 46,XY individuals, the external genitalia can range from ambiguous to normal

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female. Affected 46,XX individuals show normal female external genital development. Internal genital differentiation varies because persistence of Wolffian and/or Mullerian structures is inconsistent. Typically, the gonads are dysgenetic in 46,XY individuals. The heterozygous missense mutations associated with Denys-Drash syndrome are believed to act in a dominant negative fashion.98 The features of Frasier syndrome include gonadal dysgenesis, progressive glomerulopathy, and an increased risk for gonadoblastoma. Wilms’ tumor is extremely rare in Frasier syndrome.99 The typical renal lesion is focal glomerular sclerosis. The majority of cases are associated with a specific point mutation in intron 9 of WT1. In the fetal kidney, WT1 induces the mesenchymalepithelial transition—leading to nephron formation.100 Depending on cellular context, WT1 can function as a transcriptional activator, a transcriptional repressor, or tumor suppressor. The carboxyl terminal domain of the WT1 protein contains four zinc fingers that serve as the nucleic acid binding domain. Downstream target genes include WNT4 and AMHRII.101 Through alternative splicing, multiple translation start sites, and post-translational RNA editing, multiple isoforms can be derived from this one gene. Distinct isoforms may have unique functions. Alternative splicing between the third and fourth zinc fingers generates two isoforms differing by the presence or absence of three amino acids [lysine, threonine, and serine (KTS)]. Subnuclear localization studies have shown that the –KTS form colocalizes predominantly with transcription factors, whereas the ⫹KTS form colocalizes mainly with splicing factors.102 The ratio of the ⫹KTS/–KTS isoforms appears to be tightly regulated. The mutation associated with Frasier syndrome alters splicing patterns, leading to decreased amounts of the ⫹KTS isoform.

WNT4 GENE WNT4 is a secreted molecule that binds to members of the frizzled family of receptors, resulting in transcriptional regulation of target genes. WNT4 increases follistatin expression, which inhibits formation of the coelomic vessel (anti-testis action) and supports ovarian germ cell survival (pro-ovarian action).103 Duplication of the WNT4 gene, located at chromosome 1p31-1p35, has been associated with 46,XY male-to-female sex reversal. One such patient presented with ambiguous external genitalia accompanied by severe hypospadias, fibrous gonads, remnants of both Mullerian and Wolffian structures, cleft lip, microcephaly, and intrauterine growth retardation.104 Loss of function WNT 4 mutations have been detected in two women with primary amenorrhea secondary to Mullerian duct abnormalities and androgen excess.105,106 The phenotypes of these patients support the hypothesis that WNT4 plays a role in ovarian differentiation.

46,XY DISORDERS OF SEXUAL DIFFERENTIATION (GONADAL DYSGENESIS) Phenotype/genotype studies of 46,XY sex-reversed patients played a major role in the localization of the Y chromosome gene responsible for the initial signal-promoting

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testicular differentiation. Subsequently, this gene was identified to be the sex-determining region on Y (SRY) gene located at Yp11.3 near the pseudoautosomal region on Yp. Identification of mutations in SRY in 46,XY females confirmed the vital role of SRY in testicular differentiation. SRY is a gene with a single exon that codes for a 204-amino-acid protein and that contains an HMG DNAbinding domain flanked by nuclear localization signals (NLS). The majority of sex-reversing SRY mutations are located in the HMG/NLS domain and affect DNA-binding affinity, DNA bending ability, or nuclear localization.107 Yet, only 15% to 20% of cases of 46,XY DSD due to gonadal dysgenesis can be attributed to SRY mutations.108 Paternal mosaicism for SRY mutations in which different cells carry different SRY genes has been described.109,110 More puzzling are the pedigrees in which fathers and unaffected brothers carry the identical mutant SRY allele as the propositus.111,112 These paradoxical findings implicate involvement of other genes, gene-gene interactions, and gene-environment interactions in the process of sexual differentiation. Individuals with sex chromosome DSDs due to partial or mixed gonadal dysgenesis usually present with asymmetric genital ambiguity (Figure 4-2). Somatic features of Turner syndrome such as short stature, webbed neck, cubitus valgus, and gonadal failure may be present. Multiple cell lines, including a monosomic X cell line, may be detected. The most common karyotype is 45,X/46,XY. However, there is much phenotypic heterogeneity associated with 45,X/46,XY karyotype in that internal and external genital differentiation ranges from normal male to ambiguous to female.113,114 Whereas the typical histologic features consist of poorly developed seminiferous tubules surrounded by wavy ovarian stroma, gonadal differentiation can range from normal testis to streak gonads. At the time of puberty, virilization can occur. Individuals with sex chromosome DSDs due to gonadal dysgenesis have an increased risk of developing gonadal tumors such as gonadoblastoma or dysgerminoma because a dysgenetic gonad carrying a Y chromosome has an increased risk for neoplastic changes.115,116 Although gonadal tumors typically do not develop until the second decade of life, they can occur earlier.117

SOX9 Heterozygous loss of function mutations in the SOX9 gene are associated with autosomal-dominant campomelic dwarfism and male-to-female sex reversal.118,119 Features of campomelic dwarfism include congenital bowing of long bones, hypoplastic scapulae, 11 pairs of ribs, clubfeet, micrognathia, and cleft palate. SOX9 mutations do not cause sex reversal in the absence of skeletal malformations.120 Although the severity of the bone malformations varies, most affected individuals die shortly after birth due to respiratory failure. Approximately 75% of affected 46,XY fetuses show sex reversal, with external genital differentiation ranging from ambiguous to female. Gonadal dysgenesis and persistence of Mullerian duct derivatives are typical. Phenotypic heterogeneity with

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Figure 4-2. Genital ambiguity with asymmetry in patient with 46, XY disorder of sexual differentiation (mixed gonadal dysgenesis).

differing phenotypes (ovotesticular DSD or true hermaphroditism and complete sex reversal) in affected siblings has been described.121 SOX9 is a member of the SRY-related HMG domain gene family located at chromosome 17q24.3-17q25.1.122 It is a 508-amino-acid protein containing an 80-amino-acid HMG domain involved in DNA binding and bending; a 41-amino-acid proline, glutamine, and alanine motif; and a C-terminal transactivation domain. Mutations in SOX9 can affect DNA-binding affinity, DNA bending ability, nuclear import, transactivation, and nuclear export.123 An interstitial deletion of SOX9 in an affected patient provides the strongest evidence to date that haploinsufficiency is the mechanism responsible for the effects of SOX9.124 Somatic cell mosaicism, de novo germ-line mutations, and mitotic gene conversion events have been described.125

DAX1 DAX1 is an orphan nuclear receptor that lacks a typical zinc finger DNA-binding domain. The gene (NROB1) coding for DAX1 is located on the short arm of the X chromosome and consists of two exons. Duplication of the DAX1 locus is associated with male-to-female sex reversal.126 External genital differentiation ranges from female to ambiguous. Descriptions of internal genitalia

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include presence of Mullerian and Wolffian structures. Gonads are typically described as streaks. A submicroscopic interstitial duplication of the DAX1 gene was discovered during thorough evaluation of two sisters. The older sister had presented with primary amenorrhea, female external genitalia, 46,XY karyotype, and gonadal dysgenesis.127 Loss-of-function mutations are associated with X-linked adrenal hypoplasia congenita (AHC). In this disorder, development of the fetal adrenal cortex is normal. However, the adult or definitive adrenal cortex fails to develop— leading to postnatal adrenal insufficiency. Although the symptoms of adrenal insufficiency generally manifest in infancy or early childhood, phenotypic heterogeneity in severity and age at presentation occurs.128 Unilateral or bilateral cryptorchidism can also occur.129 At the age of expected puberty, hypogonadotropic hypogonadism due to hypothalamic and pituitary dysfunction may occur among affected males.130 Delayed puberty has been recognized in heterozygous females.131 One female homozygous for DAX1 mutations has been reported. Her phenotype was hypogonadotropic hypogonadism.132 As part of a contiguous gene deletion syndrome, X-linked AHC can be associated with glycerol kinase deficiency, Duchenne muscular dystrophy, ornithine transcarbamylase deficiency, and mental retardation.133 DAX1/NROB1 is expressed throughout the HPG axis. The DAX1 protein functions as a transcriptional repressor of many genes, including SF1 and some steroidogenic enzyme genes. Nonsense mutations have been identified throughout the gene. Missense mutations account for 20% of mutations associated with AHC. These mutations tend to cluster in the carboxyl terminal of the protein corresponding to the putative ligand-binding domain and impair the transcriptional repression activity of the protein.134,135 One missense point mutation, located in the hinge region of the protein, was identified in an 8-year-old girl with clinical and laboratory features indicative of adrenal insufficiency. Additional studies showed that this mutation hindered nuclear localization of the protein. Curiously, the hemizygous father and heterozygous younger sister of the proband did not manifest the AHC phenotype.136 Recently, an alternatively spliced form of DAX1 (DAX1A) has been identified and found to have a more ubiquitous expression pattern than DAX1. DAX1A is expressed in adrenals, gonads, and pancreas.137,138 The deduced protein sequence of DAX1A contains 400 amino acids, compared with 470 amino acids for DAX1. Another member of the NROB family is small heterodimer partner (SHP). Among its actions, SHP influences expression of cholesterol, bile acid, and glucose homeostasis.139 The protein domain structures of DAX1, DAX1A, and SHP lack the canonical DNA-binding domain, AF-1 modulator domain, and the hinge region—which are characteristics of nuclear hormone receptors. All three proteins can form homodimers or heterodimers with one other, as well as with other nuclear receptors. As transcriptional repressors, DAX1 and SHP repress gene transcription by direct protein-protein interactions with target genes and recruit additional corepressor proteins such as nuclear receptor corepressor

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Alien.140 DAX1, DAX1A, and SHP may have novel and complex interactions.141

DESERT HEDGEHOG One patient with 46,XY DSD associated with a dysgenetic gonad was found to be homozygous for a single nucleotide substitution at the initiation codon, ATG→ ACG, of the DHH gene. This variation was predicted to abolish initiation of translation at the normal start site. This patient’s phenotype consisted of female external genitalia, polyneuropathy, a testis on one side, and a streak gonad on the other side. Histologic analysis of the sural nerve revealed extensive formation of minifascicles within the endoneurium.142 Mutations in the DHH gene have been reported in several additional 46,XY patients with either complete or mixed gonadal dysgenesis.143,144 The DHH gene is located on chromosome 12q12→q13.1 and encodes a protein consisting of 396 amino acids.145

CHROMOSOME 9p MONOSOMY Monosomy for distal chromosome 9p has been reported in male-to-female sex reversal. External genitalia have been described as ambiguous or female. Differentiation of internal genitalia is highly variable, with the presence of Mullerian and Wolffian remnants being reported.146 Description of the gonads has ranged from streak gonads to hypoplastic testes. In addition to sex reversal, clinical features include mental retardation, low-set ears, trigonocephaly, wide nasal bridge, and single palmar creases.147,148 Neither the size of the deletion nor parental origin influences the degree of sex reversal.149-151 Two genes related to double-sex (dsx) and mab-3 regulators of sexual differentiation in Drosophila melanogaster (DMRT1 and DMRT2)152 have been mapped to 9p24. It has been suggested that these human genes, DMRT1 and DMRT2, play roles in testis differentiation.153 Although, to date no, mutations have been detected in either gene,154 a submicroscopic deletion at 9p24.3 was identified in two 46,XY sex-reversed sisters. This deletion was also present in their fertile mother.155 This small deletion (less than 700 kilobases) mapped 5’ of both DMRT1 and DMRT2, but within 30 kilobases of DMRT1. Thus, the deletion could include a previously unidentified upstream exon or regulatory sequence(s) of DMRT1. Because karyotypes for most 9p deletion patients with sex reversal have been 46,XY, DMRT1 may be involved in testicular differentiation. Investigation of sexual differentiation in other species shows higher DMRT1 expression in testis compared to ovaries, whereas DMRT2 is not expressed in adult testis or in developing gonadal ridges.156,157 Sexually dimorphic expression of DMRT1 was found in 6- and 7-week-old human fetuses, with expression limited to male fetuses.158

Xq28 DELETION Sex reversal and myotubular myopathy are associated with interstitial deletions at Xq28. One family has been described in which there were two affected 46,XY infants in

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whom MTM1 and F18 (now known as CXorf6) genes were deleted.159,160 Three nonsense mutations in CXorf6 were identified in three Japanese males with hypospadias.161

ATR-X SYNDROME ATR-X (␣-thalassemia, mental retardation, X-linked protein) syndrome is an X-linked disorder characterized by mild alpha-thalassemia, severe mental retardation, and genital abnormalities. The urogenital anomalies consist of ambiguous genitalia, cryptorchidism, hypoplastic scrotum, hypospadias, shawl scrotum, and small penis.162,163 Other typical features include short stature, microcephaly, seizures, talipes equinovarus, and gastrointestinal problems. Facies are described as coarse, with midface hypoplasia, short nose, and widely spaced incisors. The hemoglobin H inclusions can be demonstrated on brilliant cresyl blue stained peripheral blood smears.164 The typical presence of Wolffian duct and absence of Mullerian duct structures indicates at least partial Sertoli and Leydig cell function. Histologic studies of testes suggest aberrant Leydig cell development.165 The molecular basis of this entity is mutations in the ATRX (also known as XH2 or XHP) gene located at Xq13.3.166,167 The ATRX gene product is a member of the SWI/SNF DNA helicase family and contains functional domains involved in protein-protein and protein-DNA interactions. Based on in vitro evidence and its functional domains, the protein appears to play a role in chromatin remodeling.168,169 Phenotype-genotype correlations are inconsistent. Urogenital anomalies are associated with mutations that truncate the protein and with mutations located in the plant homeodomain-like domain.170 Skewed X-inactivation is typical of carrier females. Greater than 75% of cases are inherited from carrier mothers.171 Carpenter-Waziri, JuberMarsidi, and Smith-Fineman-Myers syndromes and X-linked mental retardation with spastic paraplegia are also associated with mutations in the ATRX gene.172

VANISHING TESTES The terms testicular regression syndrome and vanishing testes are used to describe testicular absence in boys with undescended testes. In some instances, this situation is associated with ambiguous genitalia and under-virilization— which presumably represents regression of testicular tissue occurring between 8 and 14 weeks of gestation. Physical findings reflect duration of testicular function. At operation, a rudimentary spermatic cord and nubbin of testicular tissue may be identified. Histologic examination of the testicular nubbins often reveals hemosiderin-laden macrophages and dystrophic calcification. It has been suggested that an antenatal vascular accident associated with antenatal testicular torsion is a cause of testicular regression.173 Although usually sporadic, familial testicular regression has been described.174

MULTIPLE CONGENITAL ANOMALIES In affected 46,XY infants, the IMAGe syndrome is characterized by intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia, cryptorchidism, and

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micropenis in the absence of DAX1 or NR5A1/SF1 mutations.175 The Pallister-Hall syndrome is associated with micropenis, hypospadias, hypothalamic hamartoma, postaxial polydactyly, and imperforate anus.176 Features of the autosomal dominant Robinow syndrome include small penis, cryptorchidism, bulging forehead, hypertelorism, depressed nasal bridge, short stature, short limbs, and hemivertebrae.177 Genital ambiguity in affected 46,XY individuals in association with X-linked lissencephaly, absence of the corpus callosum, and hypothalamic dysfunction (including temperature instability) has been described. This constellation of features has been associated with mutations in the ARX gene.178-180

OVOTESTICULAR DISORDER OF SEXUAL DIFFERENTIATION (TRUE HERMAPHRODITISM) Ovotesticular DSD is defined as presence of ovarian tissue with follicles and testicular tissue with seminiferous tubules in the same individual. Although an ovotestis is the most commonly identified gonad, there can be an ovary on one side and a testis on the other. In most ovotestes, ovarian and testicular tissue show distinct separation in an end-to-end arrangement. Karyotypes are usually 46,XX. Mosaic karyotypes (46,XX/46,XY and 46,XX/47,XXY) have been described.181 In some instances, Y chromosomal material such as the SRY gene can be detected by PCR amplification. However, ovotesticular DSD in the absence of Y chromosomal material has been reported.182 In one patient in whom the peripheral blood karyotype was 46,XX, molecular genetic analysis showed a deletion of the promoter region of the SRY gene in the testicular tissue of an ovotestis.183 Several pedigrees in which both XX males and XX true hermaphrodites coexist have been described.184,185 These families likely represent incomplete penetrance of mutations of genes involved in sexual differentiation.186 Although most patients present in infancy or childhood, two phenotypic males who presented with bilateral gynecomastia have been reported.187,188 Outcome regarding fertility has been disappointing. In a series of 33 patients followed longitudinally, germ cells identified in the testicular tissue during infancy degenerated—resulting in azoospermia. Although normal menstrual cycles have been reported in a few females, no pregnancies were documented in one series.182 However, pregnancies have been reported in some females with ovotesticular DSD.189-191

46,XX TESTICULAR DISORDER OF SEXUAL DIFFERENTIATION The frequency of the XX male syndrome is approximately 1 in 25,000 males.192 The majority of 46,XX males are found to carry the SRY gene, have normal male genitalia, and often present with infertility.193 In most instances, the SRY gene is located on an X chromosome due to illegitimate recombination between the X and Y chromosomes. An incidental finding was reported for a 61-year-old man with a history of small testes and azoospermia. His karyotype showed 46,XX, with the

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insertion of the SRY gene on the terminal end of chromosome 16q.194 Rare instances of 46,XX males with absence of the SRY gene have been described.195

FOXL2 Mutations in the FOXL2 gene have been associated with the autosomal dominant blepharophimosis ptosis epicanthus inversus syndrome (BPES). The phenotype of BPES type 1 involves premature ovarian failure and malformations of the eyelid [which consist of small palpebral fissures (blepharophimosis)], ptosis, epicanthus inversus, and a broad nasal bridge.196 FOXL2 is a forkhead transcription factor. Curiously, in goats mutations in this locus are responsible for the autosomal dominant phenotype characterized by the absence of horns in male and female goats (polledness). It is also associated with XX female-to-male sex reversal in a recessive manner.197

Disorders of Androgen Synthesis Genital ambiguity can be a manifestation of alterations in sex steroid biosynthesis, which most commonly are secondary to mutations in a steroidogenic enzyme gene.

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During fetal life, steroidogenic enzymes are expressed in placenta, testis, and adrenal (Figure 4-2). The fetal testis secretes testosterone that is converted to dihydrotestosterone in target tissues such as the prostate and external genitalia. Inborn errors of testosterone biosynthesis can lead to ambiguous genitalia in 46,XY fetuses. Specific proteins necessary to testosterone biosynthesis include SF1, LH receptor, steroidogenic acute regulatory peptide (StAR), 17␣-hydroxylase/17,20-lyase, 3␤-hydroxysteroid dehydrogenase type 2, 17␤-hydroxysteroid dehydrogenase type 3, P450-oxidoreductase, and 5␣-reductase type 2 (Figure 4-3). Inborn errors of glucocorticoid biosynthesis are often associated with the virilizing congenital adrenal hyperplasias. The fetal adrenal cortex is derived from coelomic epithelium and consists of two major zones: the fetal zone and the adult zone. Because the human placenta does not express 17␣-hydroxylase/17,20-lyase, it cannot directly convert progesterone to estrogens. The fetal zone is primarily responsible for DHEA synthesis, which is then sulfated to provide substrate for placental estrogen biosynthesis (Figure 4-3). The adult zone, which after birth differentiates into the three zones of the adult adrenal cortex, is primarily responsible for cortisol biosynthesis. By 10 weeks of gestation, the adrenal is secreting DHEAS and the hypothalamic-pituitary-adrenal axis is functional.

Figure 4-3. Diagram of classical steroidogenic pathways. Substrates, products, and genes involved in adrenal, ovarian, testicular, and placental steroidogenesis are indicated. Genes are 17␣-hydroxylase/17,20-lyase (CYP17), 3␤-hydroxysteroid dehydrogenase (HSD3B2), 21hydroxylase (CYP21), 11␤-hydroxylase (CYP11B1), aldosterone synthase (CYP11B2), aromatase (CYP19), 17␤-hydroxysteroid dehydrogenase type 1 (HSD17B1), 17␤-hydroxysteroid dehydrogenase type 3 (HSD17B3), 5␣-reductase type 2 (SRD5A2), sulfotransferase (SULT2A1), and steroid sulftase/arylsulfatase C (ARSC1). CYP3A7 is a cytochrome P450 enzyme expressed in fetal liver, where it catalyzes the 16␣-hydroxylation of estrone (E1) and DHEA. Its expression decreases postnatally. Steroidogenic enzymes that utilize P450 oxidoreductase, a flavoprotein encoded by POR, to transfer electrons are indicated by hatched arrows.

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SF1/NR5A1 GENE The SF1/NR5A1 gene, located at chromosome 9q33, codes for a 461-amino-acid protein. One 46,XY patient heterozygous for an NR5A1/SF-1 mutation presented in the newborn period with adrenal insufficiency and was initially considered to have lipoid adrenal hyperplasia. The patient had female external genitalia, normal Mullerian structures, and streak gonads. Reevaluation prior to induction of puberty revealed normal gonadotropin response to GnRH stimulation. Functional studies showed that this mutation, G35E, fails to transactivate a known SF1 responsive reporter gene.198 A 46,XX female with adrenal insufficiency, adrenal hypoplasia, and an SF1/NR5A1 mutation showed normal ovarian morphology.199 With identification of more patients heterozygous for missense mutations in the SF1/ NR5A1 gene, it has become apparent that haploinsufficiency of SF1 can manifest a predominantly gonadal phenotype characterized by undervirilization of affected 46,XY fetuses without overt adrenal insufficiency.200 Females heterozygous for missense mutations appear to lack an obvious phenotype.

LUTEINIZING HORMONE CHORIOGONADOTROPIN RECEPTOR GENE Leydig cell hypoplasia is an autosomal recessive disorder characterized by failure of testicular Leydig cell differentiation secondary to inactivating LHCGR mutations and target cell resistance to LH.201-203 The inability to respond to hCG or LH results in decreased Leydig cell testosterone biosynthesis. The phenotype of affected 46,XY infants ranges from undervirilization to female external genitalia. Affected individuals raised as females often seek medical attention for delayed breast development. Mullerian duct derivatives are absent because AMH is secreted by the unaffected Sertoli cells. Laboratory studies show elevated LH, low testosterone, and normal FSH concentrations. There is no significant testosterone response to hCG stimulation. The LHCGR gene is mapped to chromosome 2p21.204 The 674-amino-acid protein is a seven-transmembrane domain G-protein–coupled receptor. Specific mechanisms through which the loss-of-function mutations induce LH resistance include decreased receptor protein, decreased ligand binding, altered receptor trafficking, and impaired ability to activate Gs. Undervirilization with hypospadias, micropenis, and cryptorchidism has been described with incomplete loss-of-function missense mutations.205 Genetic females, sisters of affected 46,XY individuals, who carry the identical mutations show normal female genital differentiation and normal pubertal development but have amenorrhea and infertility.206,207 Genetic analysis may be helpful to distinguish LHCGR mutations from other disorders affecting testosterone biosynthesis (such as isolated 17,20-lyase deficiency).208

SMITH-LEMLI-OPITZ SYNDROME Clinical features of this autosomal recessive disorder include multiple malformations, urogenital anomalies, mental retardation, failure to thrive, facial abnormalities,

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developmental delay, and behavioral abnormalities. The most common urogenital abnormalities include male-to-female sex reversal, hypospadias, and cryptorchidism. The facial abnormalities consist of a broad nose, upturned nares, micrognathia, short neck, and cleft palate.209 Typical limb anomalies comprise short thumbs, syndactyly of the second and third toes, and post-axial polydactyly. Several enzymes catalyze the conversion of lanosterol to cholesterol. Decreased activity of these enzymes leads to cholesterol deficiency. The enzyme, 7-dehydrocholesterol reductase, encoded by the 7-dehydrocholesterol reductase (DHCR7) gene catalyzes the last step in cholesterol biosynthesis. Smith-Lemli-Opitz (SLO) is due to mutations in the 7-dehydrocholesterol reductase (DHCR7) gene located at chromosome 11q12-q13.210,211 Mutations in the DHCR7 gene are associated with elevated 7-dehydroxycholesterol concentrations. Demonstration of elevated 7-dehydroxycholesterol concentrations is required to confirm the diagnosis. To date, more than 80 mutations have been reported.212 As anticipated, mutations in this cholesterol biosynthetic pathway are associated with decreased cholesterol and accumulation of sterol intermediates proximal to the defective enzyme. Decreased cholesterol concentrations lead to decreased steroid concentrations because cholesterol serves as the precursor for glucocorticoid, mineralocorticoid, and sex steroid biosynthesis. In addition to its role as the precursor for steroid biosynthesis, cholesterol modification of sonic hedgehog protein (SHH) is necessary for normal signaling through its cognate receptor, Patched (PTCH), which contains a sterol sensing domain. In fibroblasts from patients with SLO, SHH signaling is impaired. Using transgenic mouse models, available data indicate that accumulation of sterol intermediates rather than cholesterol deficiency interferes with midline fusion of facial structures. These observations shed light on the molecular pathophysiology responsible for the cleft palate associated with SLO.213,214 Prenatal diagnosis can be performed by measurement of amniotic fluid DHCR7 concentrations.215 Low plasma estriol and elevated 16␣-hydroxy-estrogens concentrations are found in women pregnant with affected fetuses, presumably due to impaired fetal cholesterol production.216 The incidence of biochemically confirmed SLO is estimated at 1/20,000 to 1/60,000 live births.217 With the identification of the molecular basis for this disorder, a surprisingly high heterozygote carrier rate for DHCR7 mutations has been found. Because the prevalence of SLO at 16 weeks of gestation is comparable to the prevalence at birth, early fetal loss and/or reduced fertility of carrier couples may be occurring.218

CONGENITAL LIPOID ADRENAL HYPERPLASIA This autosomal-recessive disorder is characterized by a severe defect in the conversion of cholesterol to pregnenolone, leading to impaired steroidogenesis of all adrenal and gonadal steroid hormones. Impaired testosterone biosynthesis in utero prevents male sexual differentiation. Hence, all affected fetuses (46,XY or 46,XX) have female

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external genitalia. Low or undetectable steroid hormone levels are consistent with this diagnosis. The hormone determinations to distinguish congenital lipoid adrenal hyperplasia from 3␤-hydroxysteroid dehydrogenase deficiency should include 17-hydroxypregnenolone or pregnenolone. These hormones will be low in the former but elevated in the latter. Milder forms of this disorder with presentation beyond the newborn period have been described.219 Following cloning of the gene for steroidogenic acute regulatory protein (StAR), mutations in the StAR gene were identified among patients with congenital lipoid adrenal hyperplasia.220-224 The StAR protein facilitates cholesterol transport across the mitochondria to P450scc.225,226 In congenital lipoid adrenal hyperplasia, impaired cholesterol transport into mitochondria leads to accumulation of cholesterol esters and sterol autooxidation products. Ultimately, the lipid accumulation alters the cell cytostructure—provoking cell destruction and complete loss of StAR-dependent steroidogenesis.227 Yet, the presence of low levels of StAR-independent steroidogenesis allows for preservation of Wolffian duct remnants in affected 46,XY fetuses and spontaneous pubertal mutation in affected 46,XX girls.228,229

SIDE CHAIN CLEAVAGE CYTOCHROME P450 ENZYME The side chain cleavage enzyme (also known as cholesterol desmolase) is a cytochrome P450 enzyme encoded by the CYP11A1 gene mapped to chromosome 15q23q24. This enzyme converts cholesterol to pregnenolone and is essential to steroidogenesis. Despite the crucial role of this enzyme for placental progesterone synthesis, rare fetuses with loss-of-function mutations on both alleles may be viable. Children affected with this autosomal-recessive disorder have female external genitalia irrespective of the karyotype and adrenal insufficiency. Postnatally, sizes of adrenals and gonads on ultrasound or magnetic resonance imaging have been variable but not enlarged. The absence of adrenal/gonadal tissue has been speculated to result from lipid accumulation similar to the cell destruction observed in patients with StAR mutations.230-232

VIRILIZING CONGENITAL ADRENAL HYPERPLASIAS The virilizing congenital adrenal hyperplasias are a group of disorders due to mutations in the steroidogenic enzyme genes involved in cortisol biosynthesis. These genes are 3␤-hydroxysteroid dehydrogenase type 2 (HSD3B2), 21-hydroxylase (CYP21), and 11␤-hydroxylase (CYP11B1). The common pathophysiology is decreased negative feedback inhibition due to insufficient cortisol concentrations, but the specific manifestations and laboratory abnormalities vary depending on which enzyme gene is involved. Steroid intermediates proximal to the deficient enzyme accumulate. Overall, relative cortisol deficiency leads to a diminution of negative feedback inhibition with subsequent increased ACTH secretion. The adrenal cortex responds by

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proportionally increasing adrenal androgen production. Thus, the clinical signs and symptoms reflect androgen excess in addition to glucocorticoid and mineralocorticoid deficiencies. Indeed, the magnitude of glucocorticoid and mineralocorticoid deficiencies varies—generally in proportion to the severity of the enzyme deficiency. The most common type of congenital adrenal hyperplasia (accounting for 90% to 95% of cases) is 21-hydroxylase deficiency due to mutations in the 21-hydroxylase (CYP21A2) gene located at chromosome 6p21.3 in the HLA class III region.233-237 The reported incidence of 21-hydroxylase deficiency ranges from 1 in 5,000 to 1 in 15,000, with variation among ethnic/racial backgrounds.238,239 Decreased 21-hydroxylase activity impairs conversion of 17-hydroxyprogesterone to 11-deoxycortisol in the zona fasciculata (the primary site of cortisol biosynthesis) and conversion of progesterone to deoxycorticosterone in the zona glomerulosa, the primary site of aldosterone biosynthesis. In addition to the shunting of 17-OHP to androstenedione, DHEA, and DHEAS resulting in increased androgen concentrations in the affected female fetus, 17-OHP can be converted through an alternate route to DHT. This alternate route (the “backdoor pathway”) involves 5␣-reduction of 17-OHP to 5␣-pregnane-17␣-ol-3,20-dione, ultimately generating androstanediol—which is the substrate for 3␣reduction and conversion to DHT240 (Figure 4-4). During fetal life, accumulation of 17-OHP due to mutations in CYP21A2, CYP11B2, or P450-oxidoreductase (POR) may increase flux through this “backdoor pathway”—leading to elevated DHT concentrations.241 Infant girls with classic salt-losing 21-hydroxylase deficiency usually present in the immediate neonatal period due to genital ambiguity (Figure 4-5). When the diagnosis is delayed, affected girls develop dehydration, hyponatremia, and hyperkalemia due to glucocorticoid and mineralocorticoid deficiencies. Among affected female infants, virilization of the external genitalia ranges from clitoromegaly to perineal hypospadias—with chordee to complete fusion of labiourethral and labioscrotal folds. The magnitude of external genital virilization may be so extensive that affected female infants appear to be males with bilateral undescended testes.242,243 Unless identified by neonatal screening, infant boys typically present at 2 to 3 weeks of age with failure to thrive, poor feeding, lethargy, dehydration, hypotension, hyponatremia, and hyperkalemia. When the diagnosis is delayed or missed, congenital adrenal hyperplasia is potentially fatal. Newborn screening programs decrease the morbidity and mortality associated with acute adrenal insufficiency. In affected infants, random 17-hydroxyprogesterone concentrations are usually elevated. Concentrations are greater than 5,000 ng/dL, and often much higher.244 Androstenedione and progesterone concentrations are also typically elevated. In some instances, plasma renin activity (PRA) can be helpful to assess mineralocorticoid status. Measurement of 21-deoxycortisol is extremely helpful, but availability of this hormone assay is limited.245,246 For female infants, a normal uterus is present and can be identified on ultrasound. Ovaries may be too small to be readily identified on ultrasound. Despite excessive antenatal androgen exposure, ovarian position is normal and internal Wolffian structures are not retained.

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Figure 4-4. Steroidogenic pathways relevant to fetal androgen biosynthesis. The classic steroidogenic pathway relevant to the testis (light background) and the “backdoor pathway” (dark background) are indicated. Both pathways can generate DHT through the actions of target tissue enzymes capable of converting substrates, testosterone, and androstanediol into DHT. In the presence of elevated ACTH and 17-OHP concentrations due to CYP21, CYP11B1, or POR mutations, the backdoor pathway may contribute to the excessive androgen concentrations responsible for virilization of XX fetuses.

Figure 4-5. Genital ambiguity in virilized female with 21-hydroxylase deficiency. The labioscrotal folds are fused, and the clitoris is enlarged.

The spectrum of impaired 21-hydroxylase activity ranges from complete glucocorticoid and mineralocorticoid deficiencies to mild deficiencies manifested principally by compensatory excessive adrenal androgen secretion. Infants capable of adequate aldosterone synthesis do not

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usually manifest overt salt loss. Female infants capable of adequate aldosterone synthesis may still have sufficient androgen exposure in utero to virilize their external genitalia. In the absence of newborn screening programs, affected males capable of aldosterone biosynthesis may not be identified until they present with genital overgrowth or premature pubarche. Infants with the milder forms of congenital adrenal hyperplasia are generally not identified by most newborn screening programs.247 CYP21A2 is located approximately 30 kilobases from a highly homologous pseudogene, CYP21A1P. The tenascin-XB (TNXB) gene encoding an extracellular matrix protein is located on the DNA strand opposite CYP21A2.248 At this time, more than 100 CYP21A2 mutations have been reported. However, only a few mutations account for the majority of affected alleles.249,250 Most of the common mutations represent gene conversion events in which CYP21A2 has acquired deleterious CYP21A1P sequences. The frequency of specific mutations varies among ethnic groups.251 Molecular genotyping can be a useful adjunct to newborn screening.252,253 Caveats to bear in mind are that multiple mutations can occur on a single allele and that different CYP21A2 mutations can occur in one family.254-256 Congenital adrenal hyperplasia due to 11␤-hydroxylase deficiency is characterized by glucocorticoid deficiency, excessive androgen secretion, and hypertension.

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This enzyme is expressed in the zona fasciculate, where it converts 11-deoxycortisol to cortisol. This form of congenital adrenal hyperplasia is rare (3%–5% of cases) apart from the high incidence among Moroccan Jews, for whom the incidence approaches 1 in 6,000.257 Despite the presence of the identical mutation, phenotypic heterogeneity for the magnitude of virilization and hypertension occurs even in a single family. Affected females may present with ambiguous genitalia. Typical laboratory findings are elevated serum concentrations of 11-deoxycortisol. Serum concentrations of 17hydroxyprogesterone, androstenedione, and testosterone may be mildly elevated. PRA concentrations are low or suppressed. Although ACTH-stimulated hormone responses among heterozygotic carriers are usually normal, elevated 11-deoxycortisol and 11-deoxycorticosterone have been reported.258,259 Mutations in the 11␤-hydroxylase (CYP11B1) gene have been identified in patients with 11␤-hydroxylase deficiency.260-262 The CYP11B1 gene is located at chromosome 8q22 in close proximity to a highly homologous gene CYP11B2, which codes for aldosterone synthase.263 CYP11B1 is expressed in the zona fasciculate, whereas CYP11B2 is expressed primarily in the zona glomerulosa.264

3␤-HYDROXYSTEROID DEHYDROGENASE DEFICIENCY Congenital adrenal hyperplasia due to 3␤-hydroxysteroid dehydrogenase type 2 deficiency leads to virilization of the external genitalia of 46,XX fetuses due to increased DHEA synthesis. Affected 46,XY fetuses have ambiguous genitalia characterized by undervirilization of the external genitalia secondary to testosterone deficiency. Despite decreased testosterone synthesis, affected 46,XY fetuses usually have intact Wolffian duct structures (including vas deferens). The NAD⫹-dependent enzyme 3␤-hydroxysteroid dehydrogenase/⌬5-⌬4-isomerase catalyzes the conversion of the ⌬5 steroid precursors, pregnenolone, 17-hydroxypregnenolone, and DHEA into the respective ⌬4-ketosteroids, progesterone, 17-hydroxyprogesterone, and androstenedione.265 Two isozymes encoded by two different highly homologous genes have been identified and mapped to chromosome 1p13.1.266,267 The type 1 (HSD3B1) gene is expressed primarily in skin, placenta, prostate, and other peripheral tissues. The type 2 (HSD3B2) gene is the predominant form expressed in the adrenal cortex and gonads. Mutations in HSD3B2, but not HSD3B1, have been detected in patients with 3␤-hydroxysteroid dehydrogenase deficiency congenital adrenal hyperplasia.268-270 Acute adrenal insufficiency occurs in the newborn period when complete loss of function mutations impair biosynthesis of mineralocorticoids, glucocorticoids, and sex steroids. Typical presentations for the non–salt-losing forms include premature pubarche and (in 46,XY infants) perineal hypospadias.271 Confirmatory laboratory findings include elevated pregnenolone, 17hydroxypregnenolone, and DHEA concentrations with elevated ratios of ⌬5 to ⌬4 steroids. Because enzymatic activity of the type 1 isozyme is unimpaired, elevated

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17-hydroxyprogesterone and androstenedione concentrations may be found.

17␤-HYDROSTEROID DEHYDROGENASE DEFICIENCY Saez and colleagues first described ambiguous genitalia in 46,XY fetuses due to 17␤-hydroxysteroid dehydrogenase deficiency.272 In this autosomal recessive disorder, external genitalia range from female with perineal hypospadias and a blind-ending vaginal pouch to ambiguous with labioscrotal fusion to hypospadias. Testes are present and may be palpable in the labio-scrotal folds or incompletely descended. Despite the presence of female external genitalia, Wolffian structures are typically present. In The Netherlands, incidence was estimated to be 1:147,000.273 The 17␤-hydroxysteroid dehydrogenase type 3 gene (HSD17B3) is located at chromosome 9q22 and is expressed in the testis, where it converts androstenedione to testosterone. Loss-of-function mutations result in testosterone deficiency and subsequent undervirilization of 46,XY fetuses.274,275 When unrecognized, patients are usually considered to be female at birth. The clinical features are similar to those of 5␣-reductase deficiency and androgen insensitivity. Phenotypic heterogeneity occurs.276 Diagnostic laboratory features include increased basal and hCGstimulated androstenedione to testosterone ratios. At puberty, progressive virilization attributed to extratesticular conversion of androstenedione to testosterone occurs.277 With progressive virilization, affected individuals may choose to change gender identity from female to male. Increased conversion of androstendione to estrogen may cause gynecomastia. Appropriate male gender assignment can be made in infancy when the diagnosis is suspected and confirmed.278 Affected females show no phenotype.279,280

5␣-REDUCTASE DEFICIENCY In this autosomal-recessive disorder, affected males have genital ambiguity characterized by differentiation of Wolffian structures, absence of Mullerian-derived structures, small phallus, urogenital sinus with perineal hypospadias, and blind vaginal pouch. At puberty, progressive virilization occurs with muscular development, voice change, and phallic enlargement. Facial hair tends to be scanty and the prostate is smaller than normal. Despite pronounced virilization at puberty, semen tends to be viscous and with low amount of ejaculate. The ducts to mature and transport sperm are inadequately developed, so that most affected men are unable to father children. In one case report, intrauterine insemination with sperm from an affected male resulted in two pregnancies.281 Deletions and mutations of the 5␣-reductase type 2 gene (SRD5A2) have been identified in affected individuals.282 The SRD5A2 gene is located at chromosome 2p23 and is expressed primarily in androgen target tissues. An isozyme (SRD5A1, located at chromosome 5p15) is expressed in skin and scalp. Clusters of individuals with SRD5A2 mutations have been described in regions of the Dominican Republic, Papua New Guinea, Turkey, and the Middle East.283

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CYTOCHROME P450 OXIDOREDUCTASE DEFICIENCY In 1985, a disorder with biochemical evidence suggesting decreased 17␣-hydroxylase and 21-hydroxylase activity was initially reported.284 Clinical features include genital ambiguity, craniosynostosis, midface hypoplasia, and radiohumeral synostosis. At birth, genital ambiguity occurs in both males and females. However, there is no progressive postnatal virilization. During pregnancy, some mothers develop signs associated with androgen excess such as acne, hirsutism, and clitoromegaly.285 Typical laboratory findings include elevated 17-OHP, low sex steroid, and normal mineralocorticoid concentrations. Glucocorticoid deficiency can occur. However, despite the apparent resemblance to combined steroidogenic enzyme deficiencies mutations were not identified in the CYP17A1 or CYP21A2 genes. Rather, this disorder is associated with mutations in the cytochrome P450 oxidoreductase (POR) gene. The POR gene codes for a protein involved in electron transfer from NADPH, which functions as a cofactor for steroidogenic and hepatic cytochrome P450 enzymes. The POR gene, mapped to chromosome 7q11-12, consists of 15 exons. Loss-of-function mutations appear to be scattered throughout the gene, without an apparent hot spot. The skeletal malformations resemble those found in the Antley-Bixler syndrome, which is an autosomal dominant disorder associated with mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. The molecular basis of the skeletal anomalies is unclear but may reflect impaired sterol biosynthesis.286,287 Patients with Antley-Bixler syndrome due to FGFR2 mutations have normal steroidogenesis, whereas patients with POR mutations have abnormal steroidogenesis.288 The paradox in this disorder is the virilization of the female fetus and undervirilization of the male fetus. It has been suggested that the alternative backdoor pathway for androgen synthesis (Figure 4-4) occurs with conversion of 17-OHP to 5␣-pregnane-3␣,17␣-diol-20-one through the sequential activity of 5␣-reductase type 1 and 3␣-hydroxysteroid dehydrogenase. Subsequently, 5␣-pregnane-3␣,17␣-diol-20-one can be converted to androstenedione by the 17,20-lyase activity of CYP17A1.289,290

PLACENTAL AROMATOSE DEFICIENCY Placental aromatase deficiency is a rare autosomal recessive disorder. During pregnancies with affected fetuses, progressive maternal virilization characterized by hirsutism, clitoral hypertrophy, acne, and frontal balding occurs. During pregnancy, testosterone, DHT, and androstenedione concentrations are elevated and estradiol, estrone, and estriol concentrations are low. In the postpartum period, some clinical features of androgen excess regress and the elevated androgen concentrations return to normal levels. At birth, 46,XX infants are variably virilized with labioscrotal fusion, clitoromegaly, and perineal scrotal hypospadias.291,292 Affected 46,XX individuals generally manifest delayed puberty characterized by minimal or absent breast development, primary amenorrhea, hypergonado-

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tropic hypogonadism, multi-cystic ovaries, and decreased bone mineral density.293,294 At birth, affected 46,XY infants have normal internal and external genital development. Affected males have generally presented after puberty with tall stature, skeletal pain, delayed skeletal maturation, and infertility.295 Investigation of aromatasedeficient men suggests that estrogen deficiency is associated with abdominal obesity, insulin resistance, dyslipidemia, and relative infertility.296 Patients with less severe phenotypic features have been described.297 Aromatase is a cytochrome P450 enzyme that plays an important role in the biosynthesis of estrogens (C18 steroids) from androgens (C19 steroids). The aromatase gene, CYP19A1, maps to chromosome 15q21.2 and codes for a 503-amino-acid protein.298 Inactivating mutations of CYP19A1 impair conversion of androgens to estrogens, leading to increased androgens.299,300 In addition to its role in estrogen biosynthesis in adolescents and adults, aromatase located in the human placenta converts fetal adrenal androgens to estrogens and protects the mother from the potential virilizing effects of the fetal androgens. Tissue-specific aromatase expression is governed by several different promoters associated with alternative first exon usage.301

Maternal Hyperandrogenism Maternal hyperandrogenism during gestation can be due to luteomas of pregnancy, androgen secreting tumors, and exposure to exogenous androgen. The excessive maternal androgen concentrations can cause virilization of the external genitalia of 46,XX fetuses. Organochlorinepesticides, polychlorinated biphenyls (PCBs), and alkylpolyethoxylates are considered to be “endocrine disruptors” because of their estrogenic and/or antiandrogenic properties. In addition, some pesticides can inhibit placental aromatase activity.302 Genital ambiguity, described in three 46,XY infants born in heavily agricultural areas, was attributed to fetal exposure to endocrine disruptors (especially because no mutations were detected in the SRY or SRD5A2 genes).303 Prenatal treatment with diethylstilbesterol (DES), a nonsteroidal synthetic estrogen, is also associated with urogenital abnormalities of both male and female fetuses. Cryptorchidism has been noted in 46,XY fetuses.304 It has been speculated that the frequency of cryptorchidism and poor semen quality is increasing because exposure to endocrine disruptors in the environment has increased.305-308 One potential mechanism is that environmental hydroxylated PCB metabolites can bind to estrogen sulfotransferase, some with greater affinity than estradiol, leading to increased estrogen levels and cryptorchidism.309

Disorders of Androgen Action During the process of sexual differentiation, androgen action is essential to retention of Wolffian duct derivatives, development of the prostate, and differentiation of male external genitalia. Complete androgen insensitivity

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(CAIS) is characterized by female external genitalia, absence of Mullerian duct derivatives, sparse sexual hair, inguinal masses, spontaneous pubertal breast development due to aromatization of androgens to estrogens, and a 46,XY karyotype. Partial androgen insensitivity (PAIS) is characterized by clinical features suggestive of a partial biologic response to androgens.310 Androgen insensitivity is an X-linked recessive disorder secondary to mutations in the androgen receptor (AR) gene, which is located near the centromere on Xq11-12.311,312 Approximately 30% of cases represent de novo mutations. Somatic cell mosaicism can occur when the mutation arises in the post-zygotic stage and is associated with a lower recurrence risk.313,314 Clinical features of CAIS include inguinal or labial masses in an otherwise normal-appearing female infant or primary amenorrhea in an adolescent girl. Among patients with CAIS, Wolffian duct derivatives (e.g., vas deferens and epididymides) are absent because of deficient androgen action. Although rare exceptions have been described, Mullerian-derived structures are usually absent because Sertoli cell function is normal with in utero AMH secretion. It has been suggested that 1% to 2% of girls with bilateral inguinal herniae may have androgen insensitivity. The finding of a gonad within the hernia sac should prompt cytogenetic studies.315 The expected LH surge in testosterone concentrations during the first few months of life may be absent in some infants with CAIS.316 Phenotypic features associated with PAIS include ambiguous genitalia with perineoscrotal hypospadias, microphallus, and bifid scrotum. Testicular position is variable, ranging from undescended to palpable in the scrotum. Infants with PAIS generally manifest the expected neonatal testosterone surge, suggesting that prenatal androgen responsiveness plays a role in imprinting of the HPG axis.317 Features of mild androgen insensitivity (MAIS) include gynecomastia and infertility in otherwise normal males. Older chronological age at presentation is typical. In all instances, karyotype is 46,XY. Typical laboratory findings are elevated LH and testosterone concentrations because testicular testosterone synthesis is unimpeded and there is loss of negative feedback inhibition of gonadotropins. LH is usually higher than FSH because testicular inhibin secretion is not impeded. FSH concentrations may be elevated or normal. Infants with PAIS may require dynamic endocrine tests to assess hCG-stimulated Leydig cell testosterone secretion and, more importantly, end-organ responsiveness to androgens. The risk for gonadal tumors is increased in the presence of a Y chromosome. Tumors associated with AIS include carcinoma-in-situ (CIS) and seminoma. In one series, only 2 of 44 subjects with CAIS had CIS. Both subjects were postpubertal.318 Androgen action is mediated by the androgen receptor, a member of the steroid/thyroid hormone family of hormone receptors. In common with other members of this receptor family, the androgen receptor is a liganddependent transcription factor with a characteristic modular structure. The major modules of the 110-kD protein include the amino-terminus transactivation (AF1), DNAbinding, and ligand-binding domains. Other features include a nuclear localization signal and another trans-

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activation domain (AF2) in the carboxy terminus ligandbinding domain. Although the androgen receptor is usually described as containing 910 amino acids, the actual number varies because of three polymorphic trinucleotide repeat regions [CAG, GGN (N ⫽ any nucleotide), and GCA] located in the amino terminal domain encoding polyglutamine, polyglycine, and polyproline repeat regions, respectively.319,320 The normal range for the polyglutamine repeat is 8 to 31, with an average length of 20 repeats. In vitro, AR transcriptional activity varies inversely with the number of repeats. Variation in the length of the CAG repeat, even within the normal range, appears to influence AR function—with long normal repeat lengths associated with infertility secondary to decreased spermatogenesis.321,322 The role of CAG repeat length in male sexual differentiation is controversial at this time and awaits more data.323 One possible mechanism is a cell-specific effect on transactivation.324 The usual range for the polyglycine repeat is 10 to 30. The functional significance of the polyglycine repeat is unclear, but complete deletion reduces transactivation in vitro.325 Reports regarding the role of CAG repeat length in females influencing ovarian hyperandrogenism, acne, and premature pubarche have been inconsistent. Phosphorylation, acetylation, ubiquitylation, and sumoylation are post-translational modifications that influence AR transactivational function. The physiologic roles of these modifications remain to be established.326,327 The DNA-binding domain (DBD) contains two zinc fingers that interact with DNA. X-ray crystallographic studies indicate that the three-dimensional structure of the ligandbinding domain (LBD) consists of 12 ␣-helixes that form the ligand-binding pocket. Kinetic and biochemical assays with molecular dynamic simulations of mutations identified in patients with CAIS indicate that the position of helix 12 is crucial to AR function.328 In the absence of ligand, the receptor is located primarily in the cytoplasm—where it is bound to chaperone proteins. Upon ligand binding, the conformation of the androgen receptor changes. The ligand-receptor complexes dimerize and move to the nucleus.329 A key feature of androgen receptor dimerization is the intramolecular interaction between the N-terminal and C-terminal domains.330 Binding of ligand stabilizes the androgen receptor and slows its degradation.331 In the nucleus, the complex binds to androgen response elements (AREs) and alters target gene transcription. Nucleotide sequences of AREs in conjunction with specific AR amino acids confer greater specificity for transcriptional regulation of specific genes.332 Additional proteins (such as coactivators and other transcription factors) are involved in transcription. These other proteins presumably provide a physical bridge linking the basal transcription machinery, the ligand-receptor complex, and chromatin. Once the ligand dissociates from the receptor, the receptor dissociates from the DNA. The most important physiologic ligands are testosterone and dihydrotesterone. The increased potency of dihydrotestosterone is attributed to the greater stability of the dihydrotestosterone-receptor complex compared to the testosterone-receptor complex.

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Numerous AR mutations have been described in affected individuals. Complete loss-of-function mutations (such as deletions, insertions, and deletions associated with frame-shifts) and premature termination codons are typically associated with complete androgen insensitivity.333 Partial loss-of-function mutations are typically missense mutations. Receptors with mutations in the DBD bind ligand normally but fail to transactivate target genes. Mutations in the LBD of the AR gene can be associated with decreased affinity for ligand, increased instability of the hormone-receptor complex, or increased susceptibility of the receptor to thermal denaturation.334 In addition to hormone determinations, diagnostic evaluation may include DNA sequence analysis of the AR gene (www. genetests.org and www.androgendb.mcgill.ca). In general, the phenotype correlates with degree of impaired androgen action. However, clinical features can vary despite the presence of the identical mutation (even within the same family). Complete and partial androgen insensitivity associated with the same AR mutation can occur in siblings.335 Different missense mutations at the same position can also be associated with differing phenotypes.336,337 Factors potentially responsible for this phenotypic heterogeneity include individual variation in ligand concentration, variations within the AR or in other genes influencing androgen action and metabolism, variations in mRNA concentrations, and epigenetic phenomenon.338 In addition to androgen sensitivity, Kennedy’s disease (also known as spinal and bulbar muscular dystrophy) is mapped to the androgen receptor locus. Kennedy’s disease is a progressive neurodegenerative disorder with onset in the thirties or forties. This disorder is associated with excessive expansion of the CAG polyglutamine trinucleotide repeat region in exon 1 of the androgen receptor.339 Repeat lengths greater than 35 are associated with spinal and bulbar muscular atrophy. In Kennedy’s disease, aberrant degradation of misfolded AR generates insoluble aggregates— leading to cellular toxicity. In a mouse model, castration improved the neurologic phenotype—suggesting that the androgen-AR signaling pathway influences the phenotype of Kennedy’s disease.340 Mild symptoms of androgen insensitivity can be detected with slight decreases in AR mRNA and protein concentrations.341

Mullerian Duct Abnormalities PERSISTENT MULLERIAN DUCT SYNDROME The typical clinical features of PMDS include cryptorchidism, testicular ectopia associated with inguinal hernia, and hernia uteri inguinalis. Testicular differentiation is usually normal, but the male excretory ducts may be embedded in the Mullerian duct remnants or incompletely developed. Infertility may ensue secondary to cryptorchidism, intertwining of vas deferens and uterine wall, or lack of proper communication between the testes and excretory ducts. Testicular torsion is not uncommon because the testes may not be anchored properly to the bottom of the processus vaginalis.342

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AMH is a member of the TGF-␤ family and signals through two different interacting membrane-bound serine/ threonine receptors. The ligand, AMH, binds to the type II receptor—which leads to recruitment and phosphorylation of a type I receptor. The type II receptor is specific for AMH, whereas there are multiple subtypes of the type 1 receptors. Inheritance is autosomal recessive and associated with mutations in the AMH gene or in the Mullerian inhibitory hormone receptor (AMH-RII) gene.343 AMH concentrations are low among patients with mutations in the AMH gene. Among patients with AMH-RII mutations, AMH concentrations are normal or elevated. The phenotypes of patients with AMH or AMH-RII mutations are comparable. Females who carry mutations on both AMH alleles appear to have normal fertility.

MULLERIAN DUCT ABNORMALITIES IN 46,XX INDIVIDUALS Mayer-Rokitansky-Küster-Hauser syndrome refers to congenital absence of the vagina associated with uterine hypoplasia or aplasia. Primary amenorrhea is the typical presentation. Renal anomalies and skeletal malformations may be present. Unilateral renal agenesis was found in 29.8% of cases of Mullerian duct anomalies on magnetic resonance imaging (MRI).344 The aggregation of Mullerian duct aplasia, renal aplasia, and cervico-thoracic somite dysplasia has been labeled MURCS syndrome.345 Mullerian duct hypoplasia has been associated with facio-auriculo-vertebral anomalies such as Goldenhar syndrome.346 Transverse vaginal septa can occur sporadically or in association with other features, such as polydactyly in the McKusick-Kaufman syndrome.347 Because of the high frequency associated anomalies, careful physical examination for skeletal malformations and renal sonography should be included in the diagnostic evaluation of women with abnormal development of the Mullerian duct system.348

Hypogonadotropic Hypogonadism Hypogonadotropic hypogonadism may present with microphallus and/or cryptorchidism in male infants (see chapter 16). Genital ambiguity would not be anticipated because.placental hCG secretion is unaffected. Decreased gonadotropin secretion presumably results in decreased testosterone secretion such that microphallus and cryptorchidism can occur.349,350 Kallmann syndrome is the eponym used for the X-linked recessive form of hypogonadotropic hypogonadism associated with anosmia due to failed migration of GnRH neurons from the olfactory placode into the forebrain along branches of the vomeronasal nerve.351,352 Olfactory tract hypoplasia or aplasia has been found on MRI.353 The molecular basis of this X-linked form is mutations in the Kallmann (KAL) gene (located at Xp22.3). This gene escapes X-inactivation, codes for a 680-amino-acid protein, and helps target GnRH neurons to the hypothalamus.354,355

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Mutations in the GnRH receptor (GNRHR), fibroblast growth factor receptor 1 (FGFR1), and GPR54 genes are also associated with hypogonadotropic hypogonadism.356-359 More recently, mutations in the genes coding for prokineticin receptor-2 (PROKR2) and its ligand [prokineticin-2 (PROK2)] have been associated with hypogonadotropic hypogonadism.360

Cryptorchidism Cryptorchidism (undescended testes) is the most common disorder of sexual differentiation, affecting 3% of male infants. Because spontaneous descent often occurs during infancy, the prevalence decreases to 1% by 6 months of age.361 Cryptorchidism has been associated with hypothalamic hypogonadism, aberrant testicular differentiation, impaired testosterone biosynthesis, androgen insensitivity, holoprosencephaly, abnormal AMH production or action, and abnormalities affecting INSL3/LGR8 function. Other associations include prune belly syndrome, bladder exotrophy, and renal anomalies. Cryptorchidism is also a feature of many syndromes (Table 4-3). Maternal diabetes mellitus, including gestational diabetes, may be a risk factor.362 During sexual differentiation, the gonads are positioned between two structures: the cranial suspensory ligament and the gubernaculum. Testicular descent is divided into two phases: intraabdominal and inguinoscrotal. Factors involved in gubernacular development during the intraabdominal phase include INSL3 and its receptor, LGR8. INSL3 is secreted by Leydig cells. Its receptor, LGR8, is a leucine-rich G-protein–coupled receptor expressed by the gubernaculum. By 13 or 14 weeks of gestation, the gubernaculum anchors the testis to the internal inguinal ring.363Androgen action during the intraabdominal phase appears to be limited to regression of the cranial suspensory ligament. In females, the cranial suspensory ligament persists as the suspensory ligament of the ovary. Testicular descent through the inguinal canal is usually accomplished by the end of seventh month of gestation, with completion of the inguinoscrotal phase by the end of week 35.363 Heterozygous missense INSL3 mutations have been identified in patients with cryptorchidism.364 Mutations have also been identified in the LGR8 gene in males with cryptorchidism. Sequence variants have been identified in the HOXA10 gene in boys with cryptorchidism.365 Typically, the testes of patients with androgen insensitivity have completed the intraabdominal phase but fail to undergo inguineoscrotal descent because this second phase is androgen dependent. However, the more complete the androgen insensitivity the greater likelihood of finding abdominal testes. Exposure of XX fetuses to androgen does not promote significant ovarian descent in humans, as evidenced by normal ovarian position in females with congenital adrenal hyperplasia.366,367 Cryptorchidism can be associated with decreased number of germ cells, impaired germ cell maturation, and decreased number of Leydig cells.368 In some instances of unilateral cryptorchidism, abnormal histology is apparent

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TA B L E 4 - 3

Disorders Associated with Small Penis or Cryptorchidism • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

Aarskog syndrome Börjeson-Forssman-Lehman syndrome Carnevale syndrome Cornelia de Lange syndrome Deletion 4p, 5p, 9p, 11q, 13q, 18q Duplication 3q, 10q, 15q Faciogenitopopliteal syndrome Goldenhar syndrome Holoprosencephaly Juberg-Marside syndrome Johanson-Blizzard syndrome Lenz-Majewski hyperostosis syndrome Lowe syndrome Malpeuch facial clefting syndrome McKusick-Kaufman syndrome Meckel-Gruber Miller-Dieker syndrome Multiple pterygium syndrome, Escobar variant Myotonic dystrophy Najjar-syndrome Noonan syndrome Pallister-Hall syndrome Pfeiffer Prader-Willi syndrome Robinow syndrome Rubinstein-Taybi syndrome Seckel syndrome Shprintzen-Goldbert Simpson-Golabi-Behmel, type 1 Townes-Brocks syndrome Varadi-Papp syndrome VATER syndrome Weaver

in the contralateral normally descended testis.369 However, it is unclear if these features represent consequences or causes of cryptorchidism.

Diagnosis HISTORY A detailed family history should be obtained. The family history should include ascertainment of unexplained infant deaths, consanguinity, and infertility. Infants with congenital adrenal hyperplasia may have died prior to diagnosis. Many DSDs are inherited as autosomal-recessive disorders. Infertility and gynecomastia may represent milder phenotypes for some DSDs. For X-linked disorders such as androgen insensitivity, there may be affected maternal family members (i.e., either amenorrheic or infertile aunts or partially virilized uncles). Pertinent questions include prenatal exposure to exogenous or endogenous androgens, estrogens, or potential endocrine disruptors. Maternal virilization during pregnancy should be queried.

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PHYSICAL EXAMINATION DSDs encompass a spectrum of physical findings. The specific physical findings range from micropenis, hypospadias, undescended testes, minimal clitoromegaly, and scrotalized labia to more extensive forms of genital ambiguity. Severe clitoromegaly with posterior labial fusion in an 46,XX patient may be difficult to distinguish from perineal hypospadias, undescended testes, and a bifid scrotum in an 46,XY individual. During the physical examination, attention should be focused on phallic size, symmetry of the external genitalia, presence and location of palpable gonads, and any additional anomalies. The extent of virilization should be carefully documented, recording the configuration, stretched dorsal length, and diameter of the phallus (including the glans penis). The location of the urethral opening, degree of labiourethral fold fusion, and extent of labioscrotal fold fusion should also be noted. Labioscrotal folds fuse from posterior to anterior such that the appearance extends from posterior labial fusion, a partially fused hemiscrota, to completely fused scrotum with labiourethral fusion extending to a midline urethral opening. The position of the urethra should be noted, as well as whether one or two perineal openings are present. Gonadal or adnexal structures may be identified upon careful palpation for content of the labioscrotal structures, scrotum or labia majora, inguinal region, and the lower abdomen. The groin area may be “milked” to maneuver the testis into the scrotum. The absence of palpable testes may indicate a genetic female with viriliza-

tion, as occurs with adrenal hyperplasia or a genetic male with undescended or absent testes. Although structures palpated within the labioscrotal folds are usually testes, ovaries or even the uterine cervix can be found within the labioscrotal folds. Testes typically have a characteristic ovoid structure. Symmetry or asymmetry of external genital differentiation may provide clues to the etiology of the genital ambiguity (Figure 4-6). Unilateral structures with asymmetry of other genital structures suggests ovotesticular or 45X/46,XY DSDs and is often associated with unilateral gonadal maldescent. Asymmetry implies differing local influences, which often reflect abnormalities in gonadal differentiation (Figures 4-6 and 4-7). Repeated examinations may be beneficial for diagnostic precision. Penile length measurements extend from the tip of stretched penis from the pubic ramus. Normal length depends on gestational age, the lower limit (approximately –2.5 SD) at term being 2.0 cm. An isolated micropenis can be a consequence of decreased testosterone exposure in the second half of gestation due to Leydig cell failure, LH deficiency, androgen insensitivity syndrome, LHCGR mutations, or GH deficiency. A micropenis with hypospadias suggests more severe DSDs. Clitoral length is usually less than 1.0 cm, although rare variations exist. Measurement of the clitoris requires a careful estimate of the proximal end and careful exclusion of overlying skin. The location of the urethral opening should be ascertained by visualization, witnessing the urinary stream, or with careful insertion of a firm catheter. If urination is observed, force, diameter, and direction

Figure 4-6. Algorithm for the approach to the child with symmetric genital ambiguity. The configuration of the labio-scrotal folds and presence/absence of palpable gonads is comparable on both sides. The presence or absence of palpable gonads directs the initial laboratory evaluation. Ultrasound examination to determine whether a uterus is present is helpful. For example, symmetric fusion of the labioscrotal folds, nonpalpable gonads, and presence of a uterus provide strong circumstantial evidence for the diagnosis of a virilized female with congenital adrenal hyperplasia.

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Figure 4-7. Algorithm for the approach to the child with asymmetric genital ambiguity. In this instance, the labio-scrotal folds may appear different or a gonad is palpable only on one side.

of the urinary stream should be noted. The position of the inserted catheter may also provide crucial initial information. If directed toward the anal opening and palpable under the perineal skin, the catheter is likely in a urogenital sinus—as occurs often with virilization of a 46,XX fetus secondary to 21-hydroxylase deficiency. However, a penile urethra is anticipated if the catheter is directed anteriorly and is nonpalpable. The anogenital ratio is measured as the distance between the anus and the posterior fourchette divided by the distance between the anus and the base of the phallus. If the ratio is ⬎0.5, this suggests a component of female differentiation and hence virilization with posterior labial fusion.370 Because pelvic ultrasound is part of the initial laboratory assessment, a rectal examination may not be necessary. If present, a midline uterine cervix can often be palpated upon rectal exam. The Prader scale is often used to classify the appearance of the external genitalia: (1) normal female genitalia with clitoromegaly, (2) partial labial fusion and clitoromegaly, (3) labioscrotal fusion so that there is a single opening from the urogenital sinus and clitoromegaly, (4) fusion of labioscrotal folds so that the single opening is at the base of the phallic structure, and (5) complete male virilization with penis-size phallus, complete labial fusion, and meatus on the glands. A recent description extends this traditional Prader classification to include urogenital sinus characteristics by defining the vaginal confluence in relation to the bladder neck and the meatus.371 In addition to the genital examination, the examination should include weight, length, and other features to ascertain whether findings are consistent with gestational age—particularly in the apparent female because the clitoris is more prominent in preterm infants in that there

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is scant subcutaneous fat and clitoral growth is completed before the last trimester of fetal life.372 A careful examination includes inspection for additional dysmorphic features because genital ambiguity may occur in association with other anomalies. These include midline facial defects, head size, and ear and digital anomalies. Infants with congenital adrenal hyperplasia may manifest hyperpigmentation of the genitalia and nipples due to adrenal insufficiency and ACTH hypersecretion.

LABORATORY STUDIES Initial laboratory studies to assess genital ambiguity should include an abdominal/pelvic ultrasound and karyotype. The ultrasound will provide information regarding the presence or absence of a uterus. Information regarding the size and location of the gonads and adrenals may be obtained on ultrasound. When gonads are not palpable, the external genitalia are symmetrically ambiguous, and a uterus and possibly ovaries are present, the most likely diagnosis is a virilized 46,XX fetus with congenital adrenal hyperplasia. However, the possibility of markedly dysgenetic testes cannot be excluded. If the differential diagnosis based on the presentation includes congenital adrenal hyperplasia, the initial laboratory studies should include electrolytes, plasma renin activity, and serum 17-hydroxyprogesterone and cortisol levels. The karyotype is essential to determine chromosomal sex even if prenatal chromosome testing was performed. In general, peripheral blood karyotypes are sufficient. However, the patient may be a mosaic with one or more additional cell lines restricted to gonadal tissue.373 Other initial studies depend on the physical findings. If the external genitalia are symmetrically virilized to any degree in the absence of palpable gonads, particularly if a normal

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uterus is present, additional studies should be directed toward causes of virilization of a female infant. Because 21-hydroxylase deficiency is the most common cause of virilization and genital ambiguity in 46,XX infants, initial laboratory studies should include determination of 17hydroxyprogesterone concentrations. If one or both gonads can be palpated, the intent of screening studies is to determine the adequacy of androgen synthesis and androgen action in a male infant. Determination of LH, FSH, and testosterone concentrations in infancy provides information regarding the function of the testes and the HPG axis (Table 4-4). The pattern of steroid hormone concentrations provides evidence for specific defects in steroidogenesis (Table 4-4). The diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency is confirmed by finding elevated 17-hydroxyprogesterone concentrations. Typically, 17-hydroxyprogesterone concentrations are greater than 10,000 ng/dL (300 nmol/L) in the affected neonate. For 11␤-hydroxylase deficiency, 11-deoxycortisol and 17-hydroxyprogesterone concentrations are elevated. For 3␤-hydroxysteroid dehydrogenase deficiency, pregnenolone, 17-hydroxypregnenolone, and DHEA concentrations are typically elevated. When salt-losing forms of congenital adrenal hyperplasia are included in the differential diagnosis, serum electrolytes and plasma renin activity should be monitored. Typically, hyponatremia and hyperkalemia are not present at birth and develop during the first week of life. Newborn screening programs have been established in many states and countries to identify infants with classic congenital adrenal hyperplasia. Many screening programs measure whole-blood 17-hydroxyprogesterone concentrations eluted from a dried filter paper blood spot.374 Whereas false negative 17-hydroxyprogesterone results are uncommon, slightly increased whole-blood 17-OHP concentrations are detected often enough (especially in

preterm infants) to complicate clinical decision making regarding affected status and need to initiate glucocorticoid therapy. Etiologies of slightly increased 17-OHP concentrations include prematurity, cross-reacting steroids, sampling prior to 36 hours of age, heterozygosity for 21-hydroxylase deficiency, and late-onset congenital adrenal hyperplasia. To avoid an excessive number of false positive screening results, the cutoff levels are typically selected to identify all infants with classic salt-losing or simple virilizing forms—often missing those infants with late-onset congenital adrenal hyperplasia. Improved specificity can be achieved by use of additional procedures such as organic extraction, chromatography, or GC/MS analysis. Prenatal or neonatal treatment with glucocorticoids can result in a false negative screening result.375 If the differential diagnosis includes CAH, specific laboratory testing is warranted even if the newborn screen results are reported as negative for 21-hydroxylase deficiency. For the milder forms of congenital adrenal hyperplasia, ACTH stimulation testing may be necessary to confirm the diagnosis. After a basal blood sample has been drawn, synthetic ACTH (0.25 mg) can be administered by intravenous bolus or intramuscular injection. A second blood sample to measure ACTH-stimulated hormone response can be obtained 30 or 60 minutes later. The milder forms of congenital adrenal hyperplasia generally do not affect external genital differentiation and are therefore not usually associated with genital ambiguity. Infants with late-onset congenital adrenal hyperplasia are generally not detected through newborn screening programs, presumably because the whole-blood 17-OHP concentrations determined from the newborn screening filter paper are lower than the values used as cutoffs. In addition to the diagnostic evaluation for disorders of steroidogenesis, hormone measurements in the immediate neonatal period provide an index to the function of

TA B L E 4 - 4

Normal Values (Mean and SD) for Full-Term and Preterm Infants from 2 Hours to 7 Days of Postnatal Life. 2 Hours PT Progesterone (ng/dl) Mean 3,900 SD 640 17-Hydroxyprogesterone Mean 713 SD 96 Cortisol (␮g/dl) Mean 8.2 SD 2.5 Deoxycorticosterone (ng/dl) Mean 114 SD 13

24 Hours

4 Days

7 Days

FT

PT

FT

PT

FT

PT

FT

5,730 690

679 149

1,250 286

56 11

88 26

53 18

50 12

886 203

286 44

94 16

214 40

79 9

237 58

124 20

10.4 2.6

3.7 0.9

2.7 1.2

4.5 1.4

5.7 1.7

3.2 1.0

3.5 1.6

360 65

38 7

116 38

9 2

13 7

6 2

11 7

PT ⫽ preterm (n ⫽ 8), FT ⫽ full-term (n ⫽ 12). Data from Sippel et al. (1980). Plasma levels of aldosterone, corticosterone, 11-deoxycorticosterone, progesterone, 17-hydroxyprogesterone, cortisol, and cortisone during infancy and childhood. Pediatr Res 14:39; and from Doerr et al. (1988). Plasma mineralcorticoids, and progesterins in premature infants: Longitudinal study during the first week of life. Pediatr Res 23:525.

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the HPG axis. Low testosterone and elevated gonadotropin concentrations in a 46,XY infant with ambiguous genitalia suggest inadequate testosterone biosynthesis. Elevated testosterone and gonadotropin concentrations in an infant with female external genitalia, bilateral labial masses, and 46,XY karyotype are consistent with the diagnosis of androgen insensitivity (Table 4-5). Measurement of AMH may provide another assessment of testicular function because AMH concentrations reflect Sertoli cell function. AMH concentrations are sexually dimorphic, with high values in boys (20–80 ng/mL) during the first six years of life and low values in girls.376 Thus, in the patient with nonpalpable gonads and absence of Mullerian structures AMH concentrations may help distinguish between anorchia and cryptorchidism.377 In addition, AMH concentrations can be helpful in disorders of testicular dysgenesis or in virilized females to determine the presence of testicular tissue.378 Inhibin B concentrations, lower in females than in males, provide another marker of Sertoli cell function.379 Assessment of the ability of a gonad to secrete testosterone may be helpful, especially for patients with evidence of testicular tissue by palpation or ultrasound and AMH levels indicating testicular tissue. This can be done by administering hCG and measuring hormone responses. To assess hormone responses, doses of 1,000 to 1,500 units can be injected subcutaneously either daily or every other day for one to five days—with blood sampling on the day after the last injection. Hormone determinations should include androstenedione, testosterone, and dihydrotestosterone.380 Testosterone concentrations should more than double, and the T/DHT ratio should be ⬍10:1. Intermittent injections can be given for up to three weeks to stimulate penile growth to demonstrate both testosterone secretion

TA B L E 4 - 5

Normal Values (Ranges) From 2 to 12 Months by Sex Male Pregnenolone (ng/dl) 17-Hydroxypregnenolone (ng/dl) Progesterone (ng/dl) 17-Hydroxyprogesterone (ng/dl) DHEA (ng/dl) DHEAS (␮g/dl) Androstenedione (ng/dl) Deoxycorticosterone (ng/dl) Aldosterone (ng/dl) 11-Deoxycortisol (ng/dl) Cortisol (␮g/dl) Testosterone (ng/dl)

(n ⫽ 14) 10 –137 14 – 766 5 – 80 11 – 173 26 – 236 2 – 38 6 – 54 7 – 57 2 – 129 10 – 200 3 – 21 0.6 – 501

Female (n ⫽ 8) 18 – 87 62 – 828 5 – 53 13 – 106 32 – 584 4 – 111 12 – 78 7 – 52 6 – 71 10 – 156 4 – 23 0.3 – 8.1

Data from Lashansky et al. (1991). Normative data for adrenal steroidogenesis in a healthy pediatric population: Age- and sex-related changes after adrenocorticotropin stimulation. J Clin Endocrinol Metab 73:674; and from Lashansky et al. (1992). Normative data for the steroidogenic response of mineralocorticoids and their precursors to adrenocorticotropin in a healthy pediatric population. J Clin Endocrinol Metab 75:1491.

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and target tissue responsiveness. Total dosage should not exceed 15,000 units of hCG. Molecular genetic analyses have become increasingly available to determine and confirm the molecular basis of the genital ambiguity. Knowledge regarding the specific mutation enables more accurate genetic counseling for inherited disorders of sexual differentiation. The genetics laboratory can often perform fluorescent in situ hybridization (FISH), for the SRY gene, or other detailed chromosomal tests. For some DSDs, molecular genetic analysis of specific genes is available through commercial laboratories. Information regarding the particular details can be obtained from an NIH-funded web-based resource, Genetests (http://www.genetests.com). In some instances, genetic testing is available through research laboratories. Endoscopic studies can be used to locate the vaginalurethral confluence in relation to the bladder neck and the single opening of the urogenital sinus. Using a cystoscope and catheter placement, these distances can be determined before or in conjunction with retrograde contrast studies performed to outline the urethra and to demonstrate (if present) the vagina, uterus, cervix, and uterine cavity. Such information is needed to plan for reconstructive surgery, to assess the risk of medical complications, and in certain instances to provide information to determine the sex of rearing. If such procedures are unhelpful, laparoscopy may be necessary to visualize and biopsy gonadal and internal genital structures. MRI may be helpful to define anatomic relationships.

Treatment While awaiting results of the initial laboratory studies, attention is focused on the main decision: the gender of rearing. In the ideal situation, there is a specific DSD team comprised of a pediatric surgeon or pediatric urologist with expertise in urogenital reconstructive surgery, a behavioral science professional (psychologist, social worker, or psychiatrist), a pediatric endocrinologist, and a neonatalogist. Initially, the neonatalogist may facilitate coordination of care and communication with the parents. Long-term management needs to be delineated. In addition to the appointments necessary to the patient’s medical, surgical, and psychological needs, additional sessions may benefit the parents to address their questions and concerns about their child.381 These evaluations should be performed promptly and often necessitate transfer of the infant to a tertiary care facility. The parents need to participate in discussions and decision making regarding the options for sex of rearing and possible surgical interventions. Honest, sensitive, and candid discussions with the parents can only benefit the child. As the child matures, honest explanations regarding the medical condition are essential. The child should progress from a silent partner to a full participating member in this decision-making process, with the child’s wishes taking precedence where appropriate. Once the diagnosis is confirmed as specifically as possible, therapy is instituted. When a specific diagnosis has been confirmed, decision making and therapy can be guided accordingly. Considerations for medical care

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include sex of rearing, possible need for surgery and a timeline for planned surgery, plan for medical treatment, and timely and appropriate psychological counseling and support.

SEX OF REARING Decisions regarding the appropriate sex of rearing are based on the specific pathophysiology, prognosis for spontaneous pubertal development, capacity for sexual activity and orgasm, and potential for fertility.382 With the use of assisted techniques, the potential for fertility is more likely than in the past. Although current surgical techniques spare more of the neurovascular bundle of the phallus, surgery should be avoided except in those instances of severe ambiguity or markedly discordant genitalia to preserve sexual responsiveness. In instances where a decision regarding sex of rearing is necessary, each child evaluated for genital ambiguity warrants careful consideration of the physical findings, laboratory information, and available outcome data. In most instances, children are raised in the gender consistent with the karyotype—acknowledging that there are rare exceptions of sex reversal. Limited longitudinal outcome studies are available regarding adult function and gender identity. Virtually all 46,XY CAIS patients identify themselves as female. About a quarter of PAIS patients are dissatisfied with the gender assignment, regardless of whether they are raised as male or female. The majority of patients with 5␣-reductase deficiency identify as male, whereas about half of those diagnosed with 17␤-hydroxysteroid dehydrogenase self-reassign from female to male.383,384 The majority of virilized 46,XX individuals with CAH identify as female. Of the few estrogen-deficient males identified, all are heterosexual and report normal libido and sexual function.385 Thus, in spite of the accumulating evidence that androgen exposure to the CNS alters general and cognitive behavior much remains to be learned about the influences of nature and nurture on gender identity.386 Because the majority of 46,XX virilized females (primarily those with CAH) identify as female, this sex of rearing is usually recommended if the question arises in infancy. For virilized 46,XX patients with Prader stage 4 or 5 genitalia, the recent consensus conference felt that the outcome data was insufficient to support a sex of rearing as male despite the anecdotal data suggesting good adjustment when this has occurred with late diagnosis of CAH. For virilized females with CAH, the primary issue is not gender assignment but questions regarding genital surgery. To date, more information and knowledge is available about physical sexual development than about psychosexual cognitive development. The primary factors, critical steps, and sequence of psychosexual development are unclear. Traditionally, psychosexual development has been viewed as having three components: gender identity, gender role, and sexual orientation. The development of gender identity begins as an infant with the self-recognition that one is a boy or girl. Gender role develops during childhood as a result of society’s expectations concerning behavior and is influenced by prenatal hormonal exposure.

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Gender roles are defined in part by the messages society relays concerning appropriate and inappropriate male and female behavior. Gender role may subsequently shift, depending on the individual’s reaction to the expectations of society. How prenatal factors such as hormones and environmental exposures impact gender role has been difficult to ascertain. Sexual orientation may be apparent prior to puberty or not expressed until late in adult life, underlying the unknown influencing factors. The effects of prenatal androgen exposure on gender identity are uncertain.387,388 Prenatal androgen levels apparently influence gender-related behavior and cognitive function during childhood.387 Yet, the impact on degrees of femininity and masculinity and cognitive function (spatial and verbal abilities) and handedness does not appear to persist into adult life.389,390 Data obtained from some studies of the 46,XX CAH patient suggest that prenatal androgen exposure may be associated with reduced satisfaction with a female sex assignment.391 Among virilized 46,XX individuals with CAH, an increased rate of homosexual orientation has been reported—primarily based on self-reported sexual imagery and sexual attraction, with reports of actual homosexual involvement being less well documented.392-394 Some females with classic CAH are more likely to question their female gender,395 and the reported incidence of sex reassignment from female to male is greater than in the general population.396 Among 46,XX CAH patients with gender self-reassignment, it was judged that factors contributing to this change were not genotype or phenotype but gender-atypical behavioral self-image and body image and development of erotic attraction to women.397 Other studies concluded that psychological adjustment was comparable between females with CAH and their unaffected siblings.398 In another study, gender identity of girls with CAH was comparable to the control group.399 In these studies, the impact of environmental influences cannot be separated from the effects of prenatal hormonal exposures. Female gender of rearing is appropriate for 46,XY individuals with complete androgen insensitivity because virilization and fertility are unattainable. The lack of androgen responsiveness limits androgen effect on the developing CNS of individuals with CAIS. Conversely, the decision regarding gender of rearing for individuals with partial androgen insensitivity may be problematic because the extent of androgen impact on CNS development and the androgen responsiveness of the genital development are uncertain. The clinical response to exogenous testosterone may benefit this decision-making process. Testosterone responsiveness may be ascertained by assessing penile growth following one or more intramuscular injections of 25-mg testosterone depot formulations. Among individuals with DSD associated with gonadal dysgenesis, hCG-stimulated testosterone secretion and clinical response facilitates the decision regarding gender of rearing. The genital phenotype, ability of the Leydig cell to secrete testosterone, and extent of genital virilization in response to androgen stimulation are indicators regarding the potential for spontaneous pubertal development and need for hormone replacement therapy. Nevertheless, in gonadal dysgenesis the impact of prenatal

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androgen exposure on gender development processes is often indeterminate and cannot be reliably predicted from routine diagnostic studies in the newborn period. Generally, with the exception of CAIS, 46,XY individuals with fetal androgen exposure manifested as partial genital masculinization should be raised as males unless there are extenuating individual circumstances. Caution, reflection, and dialog are appropriate before those with a 46,XY, karyotype are assigned female because fetal androgen exposure appears to impact strongly on selfconcept as male despite compromised external genital development.

CONSIDERATIONS WITH REGARD TO SURGERY For the virilized female patient with female sex of rearing, the extent of ambiguity in combination with the magnitude of clitoromegaly and posterior fusion must be carefully evaluated to determine whether genitoplasty, including clitoral reduction or clitoroplasty, should be considered. Another factor in the decision-making process is the location of the urethral outlet. If located high in the urogenital sinus, early surgery may be indicated to decrease the risk of recurrent urinary tract infections by providing a direct urinary outflow path. The current perspective is that girls with mild to moderate degrees of clitoromegaly do not need surgery because of the potential risk of compromising genital sensitivity. One important point to share with parents of a virilized girl with CAH is that the stimulated genital tissues will regress after glucocorticoid therapy is begun. On one hand, the parents need to be informed that some (including patient advocacy) groups discourage genital surgery until the child is old enough to make her own decision. On the other hand, they must be empowered to make the choice with which they will be comfortable. This must be done with the understanding that their daughter may criticize them for this decision when she is older. Most parents of daughters with severe ambiguity still choose surgery.400 If there is agreement between the interdisciplinary medical team and the parents for surgery, the surgery is generally performed as soon as feasible. If surgery is anticipated, the operation should be described in a manner understandable to the parents. An experienced surgeon should discuss the options, risks, and benefits of surgery— including the innervation of the clitoris or penis and the surgical approach to be used to attempt to spare the neurovascular supply.401,402 Although opinions vary as to whether surgery should be done in one or more stages and whether vaginal reconstruction should be attempted during infancy, the parents need to be informed that subsequent vaginoplasty is almost always required after puberty. Among patients who underwent vaginal reconstructive surgery during infancy, the frequency of postoperative vaginal stenosis has been reported to range from 0% to 77%.403 Therapeutic goals for vaginal reconstruction surgery include adequate sexual function with minimal need for continual dilatation or lubrication.404 For the under-virilized male, decisions concerning genital surgery and when it occurs belong to the parents until the patient reaches adolescence and adulthood.

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Most decide for surgery, except in the instances of minimal to moderate hypospadias. When discussing surgery with the parents, pertinent topics include common male concerns about the importance of being able to stand to urinate, adequacy of genital development, and capability of sexual activities. Because correction of hypospadias and chordee is generally performed in stages, this discussion also needs to cover the details of the surgical approach, the proposed schedule for follow-up visits, the likely number of surgical procedures, and the optimal age for each surgical stage. The surgeon should review the options, risks, and anticipated outcomes. If the precise location and differentiation status of the testes are unknown, the parents should be aware that exploration and biopsy may be performed. Another consideration regarding the need for and timing of surgery is the relative risk for gonadal tumors. The aberrant fetal gonadal environment and subsequent anomalous germ cell differentiation is associated with the development of germ cell tumors. In general, CIS or gonadoblastoma are the most common germ cell tumors and precede the development of the more invasive neoplasms such as dysgerminoma, seminoma, and nonseminoma. The presence of Y chromosome material increases the propensity risk for gonadal tumors.405 Increased and prolonged expression of immunohistochemical markers, OCT3/4 and testis-specific protein Y-encoded (TSPY), is common in CIS and gonadoblastoma.406 In one series, TSPY was abundantly expressed in germ cells within dysgenetic testes and undifferentiated gonadal tissue—suggesting up-regulation when germ cells are located in an unfavorable environment.406 It is hypothesized that the normal germ cell maturation process is interrupted, resulting in prolonged expression of OCT3/4, erased genomic imprinting, and subsequent immortalization of the cell.407 The reported prevalence of gonadoblastoma ranges from 15% to 30%, depending on age of the patient, gonadal histology, and diagnostic criteria used for CIS/ gonadoblastoma. In one series of patients with Turner syndrome, 14/171 (8%) were positive for Y-chromosomal material. Among these 14 patients, the prevalence of gonadoblastoma was 33%.408 A meta-analysis of 11 studies, all using PCR methodology, found that 5% of patients with Turner syndrome showed positive results for Y chromosome material.407 Frasier syndrome is also associated with gonadoblastoma. The prevalence of germ cell tumors is lower in androgen insensitivity and disorders of androgen biosynthesis. Nevertheless, limited sample size, ascertainment bias, inconsistent diagnostic criteria for malignant cells, and confusing terminology leave many questions to be answered. For example, which patients with DSD benefit from gonadal biopsy to assess risk for neoplasia? Another consideration is how many biopsies are necessary to be representative of the histology of the gonad. Laparoscopy and video-assisted gonadectomy are invaluable techniques when the risk for malignancy is high. Ultimately, the decision regarding gonadectomy involves consideration of the patient’s phenotype (internal and external genital anatomy), karyotype, gender of rearing, psychosocial factors, and gonadal histology.

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MEDICAL TREATMENT With the exception of disorders affecting glucocorticoid and mineralocorticoid biosynthesis, most DSD conditions do not require specific medical therapy in infancy. At the time of expected puberty, patients with hypogonadism will need appropriate hormone replacement therapy. In general, hormone replacement therapy is initiated using low doses of the appropriate sex steroid hormone—with incremental increases designed to mirror spontaneous pubertal development. It is helpful to review with families the anticipated frequency of outpatient visits and how the adequacy of replacement therapy will be assessed. For females, induction of puberty involves low-dose estrogen therapy—usually initiated between 10.5 to 12 years of age to avoid excessive acceleration of skeletal maturation. The initial estrogen dosage is usually the lowest available, such as 0.3 mg of conjugated estrogens every other day, 5 ug of ethinyl estradiol daily, or transdermal estrogen preparations (0.025 mg) weekly. Transdermal patches may be used only at night in an effort to mimic spontaneous puberty.409 Matrix transdermal patches can also be cut into smaller pieces to provide a lower estrogen dosage. Based on clinical response and the patient’s perception, the dose of estrogen can be increased in 6- to 12-month intervals such that complete replacement doses and development are achieved within 3 years. Therapy involves the addition of a progestational agent after 12 months of estrogen therapy or when withdrawal bleeding occurs, whichever occurs sooner. Thereafter, cyclic estrogen-progesterone therapy should be used. Once full pubertal development has been reached, the estrogen dosage should be the minimum that will maintain normal menstrual flow and prevent calcium bone loss (equivalent to 0.625 conjugated estrogen or 20 ug ethinyl estradiol). Options for cyclic estrogenprogesterone therapy include low-dosage estrogen birth control pills or estrogen-progesten transdermal patches. Another regimen involves a daily oral estrogen regimen or the transdermal form for 21 days—with the addition of progesterone, 5 to 10 mg of medroxyprogesterone acetate, or 200 to 400 mg of micronized progesterone daily added for 12 days (day 10 to day 21). This is followed by a week of no hormones. At this point, the replacement regimen may be extended so that less frequent withdrawal bleeding occurs. In the absence of a uterus, progesterone therapy becomes optional. Among patients with low circulating androgen levels, sexual hair growth and libido may be improved by administering small doses of DHEA or methyltestosterone. Gonadotropins hCG and/or hMG (human menopausal gonadotropin/recombinant FSH) are only used to stimulate ovulation or during assisted fertility attempts. For males, testosterone hormone replacement typically begins at 12.5 to 14 years. In instances where the psychological impact is felt to be needed, therapy may be initiated one or two years earlier. If therapy is begun earlier to assure the patient concerning physical changes, the rate of skeletal maturation should be carefully monitored. Conversely, treatment may be delayed to allow for psychological or emotional maturity or catch-up growth. Testosterone therapy may be given by depot intramuscular

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injections or topically by a patch or gel. Depot testosterone (such as enanthate or cyprionate) is begun at a dosage 50 mg IM every four weeks, followed by increased dosing and frequency over about 3 years to a full replacement dosage of approximately 200 mg every 2 weeks. Availability of the gel in metered-dose pumps allows gradual increases of dosage from 1.25 g daily upward. Among those with differentiated testes and gonadotropin deficiency, assisted fertility techniques may involve intratesticular germ cell retrieval or hFSH/hLH stimulation. For patients with CAH, carefully monitored hormone replacement therapy is essential. The goal of glucocorticoid therapy is suppression of excessive adrenal androgen secretion while maintaining normal growth and development. Typically, oral cortisol doses range from 8 to 20 mg/m2/day. This range is based on provision of 1.5 to 2 times the daily cortisol production rate, 7 to 12 mg/m2/ day. Oral fludrocortisone (e.g., Florinef) is commonly used for mineralocorticoid replacement for patients with salt-losing CAH. Patients with simple virilizing CAH may benefit from mineralocorticoid replacement. The typical dose is 0.1 mg administered as a single daily dose. Neonates and infants may require higher fludrocortisone doses as well as salt supplementation because of relative mineralocorticoid resistance, higher aldosterone production rates, and relatively lower sodium intakes. Topics of discussion with parents include how to crush and administer tablets, what to do if doses are accidentally missed, and when to administer “stress” dosages. For children with CAH, adequacy of replacement therapy is monitored by periodic reassessment of growth velocity, extent of virilization, and salt craving. Laboratory monitoring may include serum androgen and 17-OHP concentrations, skeletal maturation, and 24-hour urinary 17-ketosteroid excretion. Androstenedione concentrations are useful to assess adequacy of glucocorticoid replacement, whereas 17-hydroxyprogesterone concentrations are useful to assess for overtreatment. Determination of testosterone concentrations is helpful in girls and prepubertal boys. In pubertal and postpubertal girls, menstrual cyclicity is a sensitive indicator of hormone replacement therapy. Adequacy of mineralocorticoid replacement can be judged using plasma renin activity. At times of physiological stress (such as fever greater than 101° F, persistent vomiting, significant trauma, and surgical procedures), the glucocorticoid dose should be increased. In general, two to three times the usual dose is sufficient to prevent adrenal insufficiency. Higher doses may be necessary for surgical procedures. All families should have at home and be able to administer injectable hydrocortisone (e.g., Solu-Cortef) intramuscularly in case of medical emergencies. Recommended intramuscular doses are 25 mg for infants, 50 mg for children less than 4 years of age, and 100 mg for all others. During surgical procedures, additional hydrocortisone can be administered either as continuous intravenous infusion or intramuscular injection. Intravenous normal saline (0.9% NaCl) can be used if oral mineralocorticoid replacement is not tolerated by the patient. While receiving glucocorticoid replacement therapy (and occasionally in the newborn period), physiologically stressed individuals with

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11␤-hydroxylase deficiency may develop hyponatremia and hyperkalemia and benefit from mineralocorticoid therapy.

PSYCHOLOGICAL AND GENETIC COUNSELING AND SUPPORT Longitudinal continuity of care and provision for support systems are essential because of the medical and psychological aspects of DSDs. If an experienced social worker, psychologist, or psychiatrist is available, careful assessment and counseling are valuable throughout childhood and adolescence. Although most such cases of 46,XY DSD do not require medical therapy during childhood, clinical visits with an endocrinologist are helpful intermittently to address the patient’s and parents’ concerns, update parents concerning new therapy and outcome data, and ensure that appropriate psychological support needs are being addressed. Some parents may find disorderspecific support groups (e.g., AIS, www.aissg.org; CAH, www.caresfoundation.org; Turner syndrome, www. turnersyndrome.org) to be helpful. Many DSD conditions are inherited. The most common inheritance patterns are autosomal recessive or Xlinked traits. Genetic counseling is indicated because parents are often interested to learn about recurrence risks. Because phenotypic heterogeneity occurs in some DSD disorders (i.e., partial androgen insensitivity), hormone determinations and genetic analyses (when available) for other family members may be beneficial.

Conclusions Identification of genes involved in sexual differentiation has elucidated some of the molecular events responsible for normal and abnormal sexual differentiation. Knowledge of the genetic, hormonal, and environmental factors that influence sexual differentiation benefits the affected children, their parents, and their health care providers. This information enables better parental education regarding the etiology, natural history, and prognosis for their child. With understanding of factors that affect sexual differentiation, recurrence risks can be estimated. Despite the advances in characterizing the molecular basis of ambiguous genitalia, the most important consideration in the management of an infant with genital ambiguity remains the sensitivity and compassion demonstrated by health care professionals in their interactions with the family.

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306. Ministry of Environment and Energy, Denmark (1995). Male reproductive health and environmental chemicals with estrogenic effects. Copenhagen: Danish Environmental Protection Agency, Miljoprojekt No 290. 307. Sharpe RM (1995). Another DDT connection. Nature 375:538. 308. Weidner IS, Moller H, Jensen TK, et al. (1998). Cryptorchidism and hypospadias in sons of gardeners and farmers. Environ Health Perspect 106:793. 309. Kester MHA, Bulduk S, Tibboel D, et al. (2000). Potent inhibition of estrogen sulfotransferase by hydroxylated PCB metabolites: a novel pathway explaining the estrogenic activity of PCBs. Endocrinol 141:1897. 310. Hughes IA, Deeb A (2006). Androgen resistance. Best Pract Res Clin Endocrinol Metab 20:577. 311. Migeon BR, Brown TR, Axelman J, et al. (1981). Studies of the locus for androgen receptor: Localization on the human X chromosome and evidence for homology with the Tfm locus in the mouse. Proc Natl Acad Sci USA 78:6339. 312. Brown CJ, Goss SJ, Lubahn DB, et al. (1989). Androgen receptor locus on the human X chromosome: Regional localization to Xq11-12 and description of a DNA polymorphism. Am J Hum Genet 44:264. 313. Hiort O, Sinnecker GH, Holterhus PM, Nitsche EM, Kruse K (1998). Inherited and de novo androgen receptor gene mutations: Investigation of single-case families. J Pediatr 132:939. 314. Kohler B, Lumbroso S, Leger J, Audran F, Grau ES, Kurtz F, et al. (2005). Androgen insensitivity syndrome: Somatic mosaicism of the androgen receptor in seven families and consequences for sex assignment and genetic counseling. J Clin Endocrinol Metab 90:106. 315. Deeb A, Hughes IA (2005). Inguinal hernia in female infants: A cue to check the sex chromosomes. BJU International 96:401. 316. Bouvattier C, Carel JC, Lecointre C, David A, Sultan C, Bertrand AM, et al. (2002). Postnatal changes of T, LH, and FSH in 46,XY infants with mutations in the AR gene. J Clin Endocrinol Metab 87:29. 317. Quigley CA (2002). The postnatal gonadotropin and sex steroid surge-insights from the androgen insensitivity syndrome. J Clin Endocrinol Metab 87:24. 318. Hannema SE, Scott IS, Rajpert-De Meyts E, Skakkebaek NE, Coleman N, Hughes IA (2006). Testicular development in the complete androgen insensitivity syndrome. J Pathol 208:518. 319. Brinkman AO, Faber PW, van Rooy HCJ, et al. (1989). The human androgen receptor: Domain structure, genomic organization and regulation of expression. J Steroid Biochem Molec Biol 34:307–310. 320. Lumbroso R, Beitel LK, Vasiliou DM, et al. (1997). Codon-usage variants in the polymorphic (GGN)n trinucleotide repeat of the human androgen receptor gene. Hum Genet 101:43. 321. Tut TG, Ghadessy FJ, Trifiro MA, et al. (1997). Long polyglutamine tracts in the androgen receptor are associated with reduced transactivation, impaired sperm production, and male infertility. J Clin Endocr Metab 82:3777. 322. Dowsing AT, Yong EL, Clark M, et al. (1999). Linkage between male infertility and trinucleotide repeat expansion in the androgenreceptor gene. Lancet 354:640. 323. Lim HN, Chen H, McBride S, et al. (2000). Longer polyglutamine tracts in the androgen receptor are associated with moderate to severe undermasculinized genitalia in XY males. Hum Molec Genet 9:829. 324. Beilin J, Ball EM, Favaloro JM, et al. (2000). Effect of the androgen receptor CAG repeat polymorphism on transcriptional activity: Specificity in prostate and non-prostate cell lines. J Mol Endocrinol 25:85–96. 325. Lee DK, Chang C (2003). Endocrine mechanisms of disease: Expression and degradation of androgen receptor, mechanism and clinical implication. J Clin Endocrinol Metab 88:4043. 326. Poukka H, Karvonen U, Janne OA, Palvimo JJ (2000). Covalent modification of the androgen receptor by small ubiquitin-like modifier 1 (SUMO-1). Proc Natl Acad Sci USA 97:14145. 327. Faus H, Haendler B (2006). Post-translational modifications of steroid receptors. Biomed Pharmacother 60:520. 328. Elhaji YA, Stoica I, Dennis S, Purisima EO, Trifiro MA (2006). Impaired helix 12 dynamics due to proline 892 substitutions in the androgen receptor are associated with complete androgen insensitivity. Hum Mol Genet 15:921.

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329. Wong CI, Zhou ZX, Sar M, Wilson EM (1993). Steroid requirement for androgen receptor dimerization and DNA binding. Modulation by intramolecular interactions between the NH2-terminal and steroid binding domains. J Biol Chem 268:19004. 330. He B, Gampe RT Jr., Kole AJ, Hnat AT, Stanley TB, An G (2004). Structural basis for androgen receptor interdomain and coactivator interactions suggests a transition in nuclear receptor activation function dominance. Mol Cell 16:425. 331. Kemppainen JA, Lane MV, Sar M, et al. (1992). Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation. Specificity for steroids and antihormones. J Biol Chem 267:968. 332. Schoenmakers E, Verrijdt G, Peeters B, et al. (2000). Differences in DNA binding characteristics of the androgen and glucocorticoid receptors can determine hormone-specific responses. J Biol Chem 275:12290. 333. Quigley CA, Friedman KJ, Johnson A, et al. (1992). Complete deletion of the androgen receptor gene: Definition of the null phenotype of the androgen insensitivity syndrome and determination of carrier status. J Clin Endocr Metab 74:927. 334. McPhaul MJ, Griffin JE (1999). Male pseudohermaphroditism caused by mutations of the human androgen receptor. J Clin Endocrinol Metab 84:3435. 335 Rodien P, Mebarki F, Moszowicz I, et al. (1996). Different phenotypes in a family with androgen insensitivity caused by the same M708I point mutation in the androgen receptor gene. J Clin Endocrinol Metab 81:2994. 336. Elhaji YA, Wu JH, Gottlieb B, Beitel LK, Alvarado C, Batist G, (2004). An examination of how different mutations at arginine 855 of the androgen receptor result in different androgen insensitivity phenotypes. Mol Endocrinol 18:1876. 337. Kazemi-Esfarjani P, Beitel LK, Trifiro M, et al. (1993). Substitution of valine-865 by methionine or leucine in the human androgen receptor causes complete or partial androgen insensitivity, respectively with distinct androgen receptor phenotypes. Mol Endocrinol 7:37. 338. Holterhus P-M, Sinnecker GHG, Hiort O (2000). Phenotypic diversity and testosterone-induced normalization of mutant L712F androgen receptor function in a kindred with androgen insensitivity. J Clin Endocrinol Metab 85:3245. 339. La Spada AR, Wilson EM, Lubahn DB, et al. (1991). Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature 352:77. 340. Katsuno M, Adachi H, Kume A, Li M, Nakagomi Y, Niwa H, et al. (2002). Testosterone reduction prevents phenotypic expression in a transgenic mouse model of spinal and bulbar muscular atrophy. Neuron 35:843. 341. Choong CS, Kemppainen JA, Zhou ZX, et al. (1996). Reduced androgen receptor gene expression with first exon CAG repeat expansion. Molec Endocr 10:1527. 342. Hutson JM, Davidson PM, Reece L, Baker ML, Zhou B (1994). Failure of gubernacular development in the persistent Mullerian duct syndrome allows herniation of the testes. Pediatr Surg Int 9:544. 343. Josso N, Belville C, di Clemente N, Picard JY (2005). AMH and AMH receptor defects in persistent Mullerian duct syndrome. Hum Reprod Update 11:351. 344. Li S, Qayyum A, Coakley FV, Hricak H (2000). Association of renal agenesis and mullerian duct anomalies. J Comput Assist Tomogr 24:829. 345. Duncan PA, Shapiro LR, Stangel JJ, et al. (1979). The MURCS association: Müllerian duct aplasia, renal duct aplasia, and cervicothoracic somite dysplasia. J Pediatr 95:399. 346. Wulfsberg EA, Grigsby TM (1990). Rokitansky sequence in association with the facio-auriculo-vertebral sequence: Part of a mesodermal malformation spectrum? Am J Med Genet 37:100. 347. Chityat D, Hahm SYE, Marion RW, et al. (1987). Further delineation of the McKusick-Kaufman hydrometrocolpospolydactyly syndrome. Am J Dis Child 141:1133. 348. Oppelt P, von Have M, Paulsen M, Strissel PL, Strick R, Brucker S, et al. (2007). Female genital malformations and their associated abnormalities. Fertil Steril 87:335. 349. Caron P, Chauvin S, Christin-Maitre S, et al. (1999). Resistance of hypogonadic patients with mutated GnRH receptor genes to pulsatile GnRH administration. J Clin Endocrinol Metab 84:990.

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350. Semple RK, Achermann JC, Ellery J, Farooqi IS, Karet FE, Stanhope RG, et al. (2005). Two novel missense mutations in g proteincoupled receptor 54 in a patient with hypogonadotropic hypogonadism. J Clin Endocrinol Metab 90:1849. 351. Schwanzel-Fukuda M, Pfaff DW (1990). The migration of luteinizing hormone-releasing hormone (LHRH) neurons from the medial olfactory placode into the medial basal forebrain. Experientiat 46:956. 352. Schwanzel-Fukuda M, Bick D, Pfaff DW (1989). Luteinizing hormone releasing hormone (LHRH)-expressing cells do not migrate normally in an inherited hypogonadal (Kallmann) syndrome. Mol Brain Res 6:311. 353. Bick DP, Ballabio A (1993). Bringing Kallmann syndrome into focus. Am J Neuroradiol 14:852. 354. Franco B, Guioli S, Pragliola A, et al. (1991). A gene deleted in Kallmann’s syndrome shares homology with neural cell adhesion and axonal path-finding molecules. Nature 353:529. 355. Maya-Nuñez G, Zenteno JC, Ulloa-Aguirre A, et al. (1998). A recurrent missense mutation in the KAL gene in patients with X-linked Kallmann’s syndrome. J Clin Endocrinol Metab 83:1650. 356. De Roux N, Young J, Brailly-Tabard S, et al. (1999). The same molecular defects of the gonadotropin releasing hormone receptor determine a variable degree of hypogonadism in affected kindred. J Clin Endocrinol Metab 84:567. 357. de Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E (2003). Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci USA 100:10972. 358. Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno JS Jr., Shagoury JK, et al. (2003). The GPR54 gene as a regulator of puberty. N Engl J Med 349:1614. 359. Dode C, Levilliers J, Dupont JM, De Paepe A, Le Du N, SoussiYanicostas N, et al. (2003). Loss-of-function mutations in FGFR1 cause autosomal dominant Kallmann syndrome. Nat Genet 33:463. 360. Dode C, Teixeira L, Levilliers J, Fouveaut C, Bouchard P, Kottler ML, et al. (2006). Kallmann syndrome: Mutations in the genes encoding prokineticin-2 and prokineticin receptor-2. PLoS Genet 2:e175. 361 Virtanen HE, Bjerknes R, Cortes D, Jorgensen N, Rajpert-De Meyts E, et al. (2007). Cryptorchidism: Classification, prevalence and long-term consequences. Acta Paediatr 96:611. 362. Virtanen HE, Tapanainen AE, Kaleva MM, Suomi AM, Main KM, Skakkebaek NE, et al. (2006). Mild gestational diabetes as a risk factor for congenital cryptorchidism. J Clin Endocrinol Metab 91:4862. 363. Virtanen HE, Cortes D, Rajpert-De Meyts E, Ritzen EM, Nordenskjold A, Skakkebaek NE, et al. (2007). Development and descent of the testis in relation to cryptorchidism. Acta Paediatr 96:622. 364. Tomboc M, Lee PA, Mitwally MF, et al. (2000). Insulin- like3/ relaxin-like factor gene mutations are associated with cryptorchidism. J Clin Endocrinol Metab 85:4013. 365. Kolon TF, Wiener JS, Lewitton M, et al. (1999). Analysis of homeobox gene HOXA10 mutations in cryptorchidism. J Urol 161:275. 366. Scheffer IE, Hutson JM, Warne GL, et al. (1988). Extreme virilization in patients with congenital adrenal hyperplasia fails to induce descent of the ovary. Pediatr Surg Int 3:165. 367. Lee SMY, Hutson JM (1999). Effect of androgens on the cranial suspensory ligament and ovarian position. Anat Rec 255:306. 368. Cortes D, Thorup JM, Visfeldt J (2001). Cryptorchidism: Aspects of fertility and neoplasms. A study including data of 1,335 consecutive boys who underwent testicular biopsy simultaneously with surgery for cryptorchidism. Horm Res 55:21. 369. Huff DS, Fenig DM, Canning DA, Carr MG, Zderic SA, Snyder HM III (2001). Abnormal germ cell development in cryptorchidism. Horm Res 55:11. 370. Callegari C, Everett S, Ross M, Brasel JA (1987). Anogenital ratio: Measure of fetal virilization in premature and full-term newborn infants. J Pediatr 111:240. 371. Rink RC, Adams MC, Misseri R (2005). A new classification for genital amniguity and urogenital sinus anomalies. BJU Inter 95:638–642. 372. Riley WJ, Rosenbloom AL (1980). Clitoral size in infancy. J Pediatr 96:918. 373. Kocova M, Siegel SF, Wenger SL, et al. (1995). Detection of Y chromosome sequences in a 45X/46XXq- patient by Southern

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blot analysis of PCR-amplified DNA and fluorescent in situ hybridization (FISH). Am J Med Genet 55:483. Torresani T, Grüters A, Scherz R, et al. (1994). Improving the efficacy of newborn screening for congenital adrenal hyperplasia by adjusting the cut-off level of 17␤-hydroxyprogesterone to gestational age. Screening 3:77. Van der Kamp HJ, Wit JM (2004). Neonatal screening for congenital adrenal hyperplasia. Eur J Endocrinol 151:U71. Lee MM, Donahoe PK, Hasegawa T, et al. (1996). Mullerian inhibiting substance in humans: Normal levels from infancy to adulthood. J Clin Endocrinol Metab 81:571. Guibourdenche J, Lucidarme N, Chevenne D, Rigal O, Nicolas M, Luton D, et al. (2003). Anti-Mullerian hormone levels in serum from human foetuses and children: Pattern and clinical interest. Mol Cell Endocrinol 211:55. Rey RA, Belville C, Nihoul-Fekete C, et al. (1999). Evaluation of gonadal function in 107 intersex patients by means of serum antimullerian hormone measurement. J Clin Endocrinol Metab 84:627. Bergada I, Milani C, Bedecarras P, Andreone L, Ropelato MG, Gottlieb S, et al. (2006). Time course of the serum gonadotropin surge, inhibins, and anti-Mullerian hormone in normal newborn males during the first month of life. J Clin Endocrinol Metab 91:4092. Ng KL, Ahmed SF, Hughes IA (2000). Pituitary-gonadal axis in male undermasculinization. Arch Dis Child 82:54–58. Jurgensen M, Hampel E, Hiort O, Thyen U (2006). “Any decision is better than none” decision-making about sex of rearing for siblings with 17beta-hydroxysteroid-dehydrogenase-3 deficiency. Arch Sex Behav 35:359. Lerman SE, McAleer IM, Kaplan GW (2000). Sex assignment in cases of ambiguous genitalia and its outcome. Urol 55:8. Imperato-McGinley J, Miller M, Wilson JD, Peterson RE, Shackleton C, Gajdusek DC (2001). A cluster of male pseudohermaphrodites with 5 alpha-reductase deficiency in Papua New Guinea. Clin Endocrinol (Oxford) 34:293. Cohen-Kettenis PT (2005). Gender change in 46,XY persons with 5alpha-reductase-2 deficiency and 17beta-hydroxysteroid dehydrogenase-3 deficiency. Arch Sex Behav 34:399. Carani C, Granata AR, Rochira V, Caffagni G, Aranda C, Antunez P, et al. (2005). Sex steroids and sexual desire in a man with a novel mutation of aromatase gene and hypogonadism. Psychoneuroendocrinology 30:413. Glassberg KI (1999). Gender assignment and the pediatric urologist. J Urol 161:1308. Meyer-Bahlburg HF, Dolezal C, Baker SW, Carlson AD, Obeid JS, New MI (2004). Prenatal androgenization affects gender-related behavior but not gender identity in 5-12-year old girls with congenital adrenal hyperplasia. Arch Sex Behav 33:97. Berenbaum SA, Bailey JM (2003). Effects on gender identity of prenatal androgens and genital appearance: Evidence from girls with congenital adrenal hyperplasia. J Clin Endocrinol Metab 88:1102. Long DN, Wisniewski AB, Migeon CJ (2004). Gender role across development in adult women with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Pediatr Endocrinol Metab 17:1367. Malouf MA, Migeon CJ, Carson KA, Petrucci L, Wisniewski AB (2006). Cognitive outcome in adult women affected by congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Horm Res 65:142. Hines M, Brook C, Conway GS (2004). Androgen and psychosexual development: Core gender identity, sexual orientation and recalled childhood gender role behavior in women and men with congenital adrenal hyperplasia (CAH). J Sex Res 41:75. Dittmmann RW, Kappes ME, Kappes MH (1992). Sexual behavior in adolescent and adult females with congenital adrenal hyperplasia. Psychoneuroendocrinology 17:153. Zucker KJ, Bradley SJ, Oliver G, Blake J, Fleming S, Hood J (1996). Psychosexual development of women with congenital adrenal hyperplasia. Horm Behav 30:300. Meyer-Bahlburg HF (2001). Gender and sexuality in classic congenital adrenal hyperplasia. Endocrinol Metab Clin North AM 30:155. Wisniewski AB, Migeon CJ, Malouf MA, Geargart JP (2004). Psychosexual outcome in women affected by congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Urol 171:2497.

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396. Meyer-Bahlberg HF, Dolezal C, Baker SW, Ehrhardt AA, New MI (2006). Gender development in women with congenital adrenal hyperplasia as a function of disorder severity. Arch Sex Behav 35: 667. 397. Meyer-Bahlburg HF, Gruen RS, New MI, Bell JJ, Morishima A, Shimshi M, et al. (1996). Gender change from female to male in classical congenital adrenal hyperplasia. Horm Behav 30:319. 398. Berenbaum SA, Korman Bryk K, Duck SC, Resnick SM (2004). Psychological adjustment in children and adults with congenital adrenal hyperplasia. J Pediatr 144:741. 399. Berenbaum SA, Bailey JM (2003). Effects on gender identity of prenatal androgens and genital appearance: Evidence from girls with congenital adrenal hyperplasia. J Clin Endocrinol Metab 88:1102. 400. Dayner JE, Lee PA, Houk CP (2004). Medical treatment of intersex: parental perspectives. J Urol 172:1762. 401. Baskin LS, Erol A, Li YW, Liu WH, Kurzrocl E, Cunha GR (1999). Anatomical studies of the human clitoris. J Urol 162:1015. 402. Akman Y, Lui W, Li YW, Baskin LS (2001). Penile anatomy under the pubic arch: Reconstructive implications. J Urol 166:225. 403. Creighton SM, Minto CL, Steele SJ (2001). Objective cosmetic and anatomical outcomes at adolescence of feminising surgery for ambiguous genitalia done in childhood. Lancet 358:124.

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404. Gollu G, Yildiz RV, Bingol-Kologlu M, Yagmurlu A, Senyucel MF, Aktug T, et al. (2007). Ambiguous genitalia: an overview of 17 years’ experience. J Pediatr Surg 42:840. 405. Fallat ME, Donahoe PK (2006). Intersex genetic anomalies with malignant potential. Curr Opin Pediatr 18:305. 406. Cools M, van Aerde K, Kersemaekers AM, Boter M, Drop SL, Wolffenbuttel KP, et al. (2005). Morphological and immunohistochemical differences between gonadal maturation delay and early germ cell neoplasia in patients with undervirilization syndromes. J Clin Endocrinol Metab 90:5295. 407. Cools M, Drop SL, Wolffenbuttel KP, Oosterhuis JW, Looijenga LH (2006). Germ cell tumors in the intersex gonad: Old paths, new directions, moving frontiers. Endocr Rev 27:468. 408. Mazzanti L, Cicognani A, Baldazzi L, Bergamaschi R, Scarano E, Strocchi S, et al. (2005). Gonadoblastoma in Turner syndrome and Y-chromosome-derived material. Am J Med Genet A 135:150. 409. Ankarberg-Lindgren C, Elfving M, Wikland KA, Norjavaara E (2001). Nocturnal application of transdermal estradiol patches produces levels of estradiol that mimic those seen at the onset of spontaneous puberty in girls. J Clin Endocrinol Metab 86:3039.

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C H A P T E R

5 Hypoglycemia in Neonates and Infants DIVA D. DE LEÓN, MD • CHARLES A. STANELY, MD • MARK A. SPERLING, MD

Introduction Physiology of Perinatal Glucose Homeostasis Integration of Glucose Homeostasis in the Fetus: Substrates, Enzymes, Hormones, and Receptors Changes at Birth Hormonal and Metabolic Systems of Fasting Adaptation Definition of Hypoglycemia in Neonates and Infants Clinical Symptoms and Signs Associated with Hypoglycemia Classification of Hypoglycemias Transient Neonatal Hypoglycemia Developmental Immaturity of Fasting Adaptation Hypoglycemia in Normal Infants and Children (Ketotic Hypoglycemia) Transient Hyperinsulinism Due to Maternal Factors Prolonged Neonatal Hyperinsulinism: Perinatal Stress-Induced Hyperinsulinism Endocrine System Disorders Congenital Hyperinsulinism KATP-Channel Hyperinsulinism SCHAD-HI Hyperinsulinism Other Forms of Hyperinsulinism Counter-Regulatory Hormone Deficiencies

Glycogen Storage Disorders Glucose 6-Phosphatase Deficiency (GSD Type I) Amylo-1,6-Glucosidase Deficiency (Debrancher Deficiency, GSD Type III) Glycogen Phosphorylase Deficiency (GSD Type VI) and Phosphorylase Kinase Deficiency (GSD Type IX) Glycogen Synthase Deficiency (GSD Type 0) Diagnosis of Glycogen Storage Disorders Disorders of Gluconeogenesis Glucose 6-Phosphatase Deficiency (GSD Type I) Fructose 1,6-Diphosphatase Deficiency Pyruvate Carboxylase Deficiency Phosphoenolpyruvate Carboxykinase Deficiency Galactosemia Hereditary Fructose Intolerance Disorders of Fatty Acid Oxidation Drug-Induced Hypoglycemia Defects of Glucose Transporters GLUT1 Deficiency GLUT2 Deficiency Systemic Disorders Diagnosis Treatment Conclusions

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Introduction One of the most important genetic and metabolic events that marks the transition from fetal to neonatal life is the adaptation from an environment that has a readily available and continuous source of glucose-maternal blood-to an environment in which glucose is provided in a limited and intermittent supply. The complex events involved in the maintenance of plasma glucose concentration must coordinate in their operation to avoid hypoglycemia and resultant damage to the central nervous system. A newborn or infant with hypoglycemia presents an urgent diagnostic and therapeutic challenge. The clinical features must be rapidly assessed and a plan of action developed based on the infant’s age, maternal and parturition history, severity and persistence of hypoglycemic state, and all other relevant clinical clues.

PHYSIOLOGY OF PERINATAL GLUCOSE HOMEOSTASIS A systematic approach to hypoglycemia in the newborn, infant, or child requires an appreciation of the central role of glucose in the body’s fuel economy.1 Glucose metabolism accounts for approximately half of basal daily energy needs. Glucose can be stored for energy in the form of glycogen and fat, and its carbon can be used for synthesis of protein and for structural components (such as cell membranes). The aerobic oxidation of glucose yields high energy by producing 38 mol of adenosine triphosphate (ATP) for each mole of glucose. Glucose is the principal metabolic fuel of the human brain. All glucose extracted by the brain is oxidized, and thus cerebral glucose utilization parallels cerebral oxygen uptake. In infants of 5 weeks of age, cerebral glucose utilization already represents 71% to 93% of the adult level in most brain regions (ranging from 13 to 25 ␮mol/ 100g/min). At that age, the areas with highest metabolic rates for glucose are the sensorimotor cortex, thalamus, midbrain, brain stem, and cerebellar vermis. By 3 months, metabolic rates for glucose increase in the parietal, temporal, and occipital cortices, as well as in basal ganglia. By 8 months, subsequent increases occur in the frontal cortex and various associative regions, concordant with the appearance of higher cortical and cognitive functions. Adult levels of cerebral glucose utilization (19 to 33 ␮mol/100g/min) are reached by 2 years of age, and continue to increase until 3 to 4 years of age—when they reach values ranging from 49 to 65 ␮mol/100g/min that are maintained to approximately 9 years of age. They then begin to decline, reaching adult levels by the end of the second decade.2 Glucose uptake by the brain occurs by means of a carrier-mediated facilitated diffusion process that is glucose concentration dependent, as well as energy-, Na⫹-, and insulin-independent.3 This process is mediated by facilitative glucose transporter (GLUT) proteins. The human genome contains 14 members of the GLUT family. Characterization of the different members has provided new insights into the regulation and significance of glucose transport and its disorders in various tissues3-5 (Table 5-1). Several

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members of the GLUT family have been detected in the brain. GLUT1 is located at the blood-brain barrier,6 and although some neurons express GLUT2 and GLUT4 the majority use GLUT3 as their primary transporter.7 Of major importance is the confirmation at a molecular level of biochemical evidence that glucose entry into brain cells and its subsequent metabolism are not dependent on insulin but rather are dependent on circulating arterial glucose concentration.8 Therefore, a decrease in arterial glucose concentration or a defect in the glucose transport mechanism of the brain will result in intracerebral glucopenia and low cerebrospinal fluid glucose concentration (hypoglycorrhachia)—with attendant symptoms and signs of cerebral glucopenia as subsequently described.8-10 To prevent circulating arterial blood glucose from decreasing precipitously under normal physiologic conditions, and therefore to prevent impairment of vital function that depends on cerebral glucose metabolism, an elaborate defense mechanism has evolved.1 This defense against hypoglycemia is integrated by the autonomic nervous system and by hormones that act synergistically to enhance glucose production through enzymatic modulation of glycogenolysis and gluconeogenesis while simultaneously limiting peripheral glucose use.11,12 Thus, hypoglycemia is the result of a defect in one or several of the complex interactions that maintain a normal range of glucose concentration, preventing its fall to less than 70 mg/dL during fasting and its rise to more than 140 mg/dL during feeding. These mechanisms are not fully developed in neonates, in whom there is an abrupt transition from intrauterine life (characterized by dependence on transplacental glucose supply) to extrauterine life (characterized ultimately by the autonomous ability to maintain precise glucose balance).13 A neonate delivered prematurely whose enzymatic machinery mechanisms are not yet fully developed and expressed or one whose placental insufficiency resulted in intrauterine growth retardation with limited tissue nutrient deposits may be particularly vulnerable to hypoglycemia,14 often with consequences to subsequent cerebral development or function.15,16 Genetically determined defects in enzyme function or hormones are also relatively common,17 and therefore hypoglycemia is an important cause of morbidity in the newborn period.18 The key element in the transition to extrauterine life is separation from the placenta and the adaptations that follow.13,19

INTEGRATION OF GLUCOSE HOMEOSTASIS IN THE FETUS: SUBSTRATES, ENZYMES, HORMONES, AND RECEPTORS Under normal nonstressed conditions, fetal glucose is derived entirely from the mother through placental transfer.20,21 In humans, the evidence remains indirect in that infusion of stable nonradioactive glucose isotopes to steady-state-specific activity in the mother during labor yields an indistinguishable glucose specific activity in fetal arterial blood at birth.22 This indicates that maternal and fetal glucose concentrations behave as a single pool, with no endogenous glucose production in the fetus.

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167

TA B L E 5 - 1

Characterization of Glucose Transporters Subfamily Class I

Class II

Name GLUT1

Erythrocytes, brain (bloodbrain barrier), placenta

GLUT2

Liver, islet cells, kidney, small intestine

GLUT3 GLUT4

Brain (neuronal), testis, placenta Adipocytes, muscle

GLUT14 GLUT5

Testis Testis, small intestine, kidney

GLUT7

Small intestine (enterocytes brush border membrane), colon, testis, prostate Liver, kidney. Expressed at preimplantation stage in mouse Pancreas, kidney, placenta, heart, skeletal muscle

GLUT9

GLUT11

Class III

GLUT6 GLUT8

GLUT10 GLUT12 GLUT13 (HMIT)

Glucose Transport Activity

Tissue Distribution

Brain, spleen, leukocytes Testis, brain (neuronal), adipocytes, preimplantation embryos Liver, pancreas Heart, prostate, breast cancer Brain

Regulation by Insulin

Erythrocyte:asymmetric carrier with exchange acceleration; Km⬃5-30mmol/L (variable):Vmax (influx) ⬍ Vmax (efflux) Low-affinity. Liver:simple, symmetric carrier; Km⬃60mmol/L;intestine: asymmetric carrier: Vmax (efflux) ⬍ Vmax (influx) High-affinity. Exchange Km⬃10mmol/L High-affinity. Adipocyte:simple, symmetric carrier; Km⬃2-5mmol/L ? No glucose transport activity, fructose transporter High-affinity transport for glucose (Km⫽0.3mM)

1p35-p31.3

Zero to minimal

3q26.1-q26.2

Zero to minimal

12p13.3

Zero

17p13

Dependent on insulin

12p13.31 1p36.2

? ?

1p36.2

?

Proven glucose transport activity

4p16-p15.3

?

Low affinity for glucose (possible fructose transporter)

22q11.2

Low affinity for glucose High-affinity glucose transporter (possible multifunctional transporter) Glucose transport activity with Km⬃3mmol/L Proven glucose transporter activity No glucose transport activity, myo-inositol and related isomers transporter

9q34 9q33.3

Mediates insulinstimulated glucose uptake in embryos ? ?

20q13.1-q12

?

6q23.2

?

12q12

?

There are important clinical consequences implicit in these findings. For example, acute hypoglycemia in a mother with diabetes will result in acute hypoglycemia in the fetus—with no ability to acutely compensate for the abrupt reduction in blood glucose supply. The transfer of glucose across the placenta to the fetus occurs by facilitated diffusion along a concentration gradient. GLUT1 has been identified as the major isoform in the human placenta,23 although other members of the family are expressed. Of note is the fact that the placental GLUTs are not regulated by insulin, suggesting that maternal glucose concentration and (more specifically) the maternal-fetal glucose gradient is the major determinant of placental glucose transfer independent of the maternal insulin concentration perfusing the placenta.24,25 Observations in infants with mutations of glucokinase, the glucose sensor of pancreatic beta cells, indicate that

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Location of Human Gene

the physiologic “set point” for plasma glucose in the fetus is identical to that of older children and adults. Fetal glucose metabolism depends directly on simultaneous effects of fetal plasma glucose and insulin concentrations, which in experiments in near-term sheep have been shown to act additively to enhance fetal glucose utilization and oxidation to CO2 according to saturation kinetics. The relative proportion of glucose oxidized in short-term studies does not change significantly over the physiologic range of fetal glucose utilization rates, indicating little effect of glucose or insulin on intracellular pathways of glucose metabolism.26 Although basal plasma concentrations of insulin and glucagon in the fetus are similar to those in the mother, their regulation differs. Acute hypoglycemia or hyperglycemia does not markedly affect fetal insulin or glucagon secretion. Long-term exposure to hyperglycemia is required

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to augment insulin secretion and promote glucagon suppression.27,28 Conversely, chronic starvation of the mother will depress maternal and fetal glucose concentrations and cause diminished insulin but greater glucagon release in the fetus. This sluggishness of intrauterine pancreatic hormone secretion is in part related to immaturity of the cyclic adenosine monophosphate (AMP)-generating system, which occasionally extends into the newborn period.28 Studies in animals, principally in fetal sheep, indicate that a proportion of glucose used is oxidized and the remainder is used for tissue accretion.29 Hyperinsulinemia in the fetus increases total glucose use, whereas the proportions used for oxidation and tissue accretion remain constant.29 A large proportion of glucose use appears to be relatively independent of insulin and is markedly glucose concentration dependent.30 Likewise, glucagon in physiologic doses does not increase fetal hepatic glucose output—and pharmacologic doses are required to demonstrate an effect.31,32 In contrast to glucagon, at relatively physiologic concentrations catecholamines mobilize fetal glucose and free fatty acids by adrenergic mechanisms— reflecting the existence of functionally linked adrenergic receptors in fetal liver and adipose tissues.33 In addition, in high doses catecholamines can exert appropriate modulation of fetal pancreatic hormone secretion by inhibiting insulin and stimulating glucagon release.34,35

CHANGES AT BIRTH The acute interruption of maternal glucose transfer to the fetus at delivery imposes an immediate need to mobilize endogenous glucose. At least three related events facilitate this transition: changes in hormones, changes in their receptors, and changes in key enzyme activity. In all mammalian species, there is a threefold to fivefold abrupt increase in glucagon concentration within minutes to hours of birth.19 Insulin, on the other hand, usually falls initially and remains in the basal range for several days without demonstrating the usual brisk response to physiologic stimuli such as glucose. A dramatic surge in spontaneous catecholamine secretion is also characteristic of several mammalian species.35 These changes in epinephrine, glucagon, and insulin may be interrelated because epinephrine is capable of stimulating glucagon and suppressing insulin release.35 In addition, epinephrine can augment growth hormone secretion by ␣-adrenergic mechanisms—and growth hormone levels are considerably elevated at birth. Acting synergistically, these hormonal changes at birth (characterized by high epinephrine, high glucagon, high growth hormone, and low insulin levels) would be expected to mobilize glucose through glycogenolysis and gluconeogenesis to activate lipolysis and promote ketogenesis.1 This appears to be true because after birth in the human newborn the plasma glucose concentration declines, reaching a nadir by 1 hour of age and then rising spontaneously to plateau levels reached between 2 and 4 hours.36 Liver glycogen stores become rapidly depleted within hours of birth, and gluconeogenesis from alanine (a major gluconeogenic amino acid) can account for approximately 10% of glucose turnover in the human newborn within several hours of age.37 Free fatty acid concen-

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trations also rise sharply in concert with the surges in glucagon and epinephrine and are followed later by an increase in ketone bodies. In this way, glucose can be spared for brain use—whereas free fatty acids and ketones provide alternative fuel sources for muscle and provide essential gluconeogenic cofactors such as acetyl-CoA and NADH from the hepatic fatty oxidation required to drive gluconeogenesis.38 In the early postnatal period, responses of the endocrine pancreas favor glucagon secretion at the relative expense of insulin secretion—perhaps because there is little need for acute insulin responses (AIRs) and so that blood glucose concentration can be protected. In the fetus, the high insulin receptor number tends to facilitate anabolic processes and growth while limiting gluconeogenesis—whereas a low glucagon receptor number (with incomplete functional linkage to cyclic AMP) further limits catabolic events in utero. In contrast, in the newborn there is a fall in insulin receptor number associated with a fall in insulin at a time when glucagon secretion rises abruptly and glucagon receptors become functional. These coordinate changes serve to mobilize glucose and other fuel stores after the interruption of the maternal supply. The surge in epinephrine secretion and its coupling with appropriate receptors also augment glucose production and lipolysis. Key enzymes involved in glucose production change dramatically in the perinatal period. Thus, there is a rapid fall in glycogen synthase activity—whereas that of phosphorylase rises sharply after delivery.14,39-41 The rate-limiting enzyme for gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK), rises dramatically after birth—activated in part by the surge in glucagon and fall in insulin.42 This framework is consistent with the insulin and with other hormonal and enzyme responses to acute hypoglycemia in the adult.1 Failure of these changes to occur may lead to failure of extrauterine autonomy in newborn glucose metabolism.

HORMONAL AND METABOLIC SYSTEMS OF FASTING ADAPTATION Hypoglycemia in neonates, infants, and children is essentially always a problem with fasting adaptation. Postprandial (reactive) hypoglycemia is exceedingly rare and is limited to a few unusual situations, such as postprandial hypoglycemia after Nissen fundoplication or hereditary fructose intolerance. Therefore, a consideration of the major hormonal and metabolic pathways that maintain fuel homeostasis during fasting provides an important framework for understanding the causes, diagnosis, and treatment of different forms of hypoglycemia. The physiology of fasting homeostasis in infants is identical to that of adults, although the timing of adaptation is faster owing to the greater metabolic demands of infants and children. Table 5-2 outlines the sources of potential fasting fuels in an adult man with a metabolic rate of 1,800 kcal/day.43 Note that hepatic glycogenolysis is sufficient to meet energy requirements for only a few hours. Beyond that time, glucose must be produced by hepatic gluconeogenesis from precursors such as amino acids, glycerol, and lactate recycled from glycolysis.

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TA B L E 5 - 2

Fuel Composition of Normal Adult Fuel

Kg

Calories

Tissues Fat (adipose triglyceride) Protein (mainly muscle) Glycogen (muscle) Glycogen (liver)

15 6 0.150 0.075

141,000 24,000 600 300 165,900

Circulating Fuels Glucose (extracellular fluid) Free fatty acids (plasma) Triglycerides (plasma)

0.020 0.0003 0.003

80 3 30 113

From Cahill GF (1970). Starvation in man. N Engl J Med 282:668.

The major source of gluconeogenic precursors is muscle protein. Although the pool of muscle protein is large, it is all required for body function and thus in contrast to stores of glycogen and fat there are no “reserves” of protein to draw on during fasting. To spare the use of essential protein during extended fasting, glucose consumption must be suppressed by switching on the mobilization and oxidation of fatty acids from adipose triglyceride stores. Glucose homeostasis is very limited in children compared with adults, in part because of their smaller reserves of liver glycogen and muscle protein but also because of their relatively larger rates of glucose consumption due to their larger brain-to-body-mass ratio. For example, the fuel stores of a 10-kg infant are only 15% of those of an

169

adult. However, the caloric needs are 60% of those of an adult and glucose turnover rates per kilogram are two- to threefold greater. Major organs (such as skeletal muscle) can readily oxidize free fatty acids, released from adipose tissue lipolysis, in place of glucose and thus limit the need for gluconeogenesis. Brain cannot directly use free fatty acids, because they do not pass the blood-brain barrier. However, the brain can substitute glucose consumption with the ketones acetoacetate and ␤-hydroxybutyrate—which are released by the liver as the end product of hepatic fatty acid oxidation. Thus, as shown in Figure 5-1 the essential metabolic pathways of fasting adaptation are hepatic glycogenolysis, hepatic gluconeogenesis, adipose tissue lipolysis, and fatty acid oxidation and ketogenesis.43 The key enzymatic steps in these pathways are shown in Figure 5-2. A defect in any one of these four pathways impairs fasting adaptation and can lead to fasting hypoglycemia. The four metabolic systems of fasting adaptation (glycogenolysis, gluconeogenesis, lipolysis, and ketogenesis) are regulated by a fifth system: endocrine control. The most important hormone for control of fasting adaptation is insulin, which acts to inhibit all four of the metabolic systems (Table 5-3). These inhibitory effects of insulin are opposed by several counter-regulatory hormones, which as shown in Table 5-3 have overlapping effects on specific fasting metabolic pathways. Excessive secretion of insulin or deficiency of one or more counter-regulatory hormones can result in fasting hypoglycemia. The operation of the metabolic and endocrine systems of fasting adaptation is evidenced by the changes in circulating levels of metabolic fuels and hormones during a fast. As shown in Figure 5-3, in young infants a 24-hour fast is accompanied by a gradual fall in glucose levels as hepatic glycogen stores are depleted, a progressive fall in

Fasting (24 hours, basal: 1800 calories) Origin of fuel

Fuel consumption O2 ~P

Nerve Muscle

Liver

CO2 + H2O

144 g Glycogen

Protein 75 g

Glucose 180 g

Amino acids Gluconeogenesis Glycerol 16 g

O2

RBC, WBC, etc. ~P

H2O

36 g

Adipose tissue Triglyceride 160 g

36 g

Lactate + Pyruvate Fatty acid 160 g

40 g

~P

Ketone 60 g

O2 Heart, kidney, muscle, etc.

~P

CO2 + H2O (Fatty acid) 120 g Figure 5-1. General scheme of fuel metabolism in a normal human who has fasted. Shown are the two primary sources of fuel (muscle and adipose tissue) and the three types of fuel consumers that use fatty acids and ketones: nerve, pure glycolyzers [e.g., red blood cells (RBCs) and white blood cells (WBCs), and the remainder of the body (e.g., heart, kidney, and skeletal muscle). ⬃P represents energy production. [From Cahill GF (1970). Starvation in man. N Engl J Med 282:668. Copyright © 1970 Massachusetts Medical Society. All rights reserved.]

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Figure 5-2. Key metabolic pathways of intermediary metabolism. Disruption of the elements of these pathways may be pathogenetic in the development of hypoglycemia. Not shown is the hormonal control of these pathways. Indicated are (1) glucose 6-phosphatase, (2) glucokinase, (3) amylo-1,6-glucosidase, (4) phosphorylase, (5) phosphoglucomutase, (6) glycogen synthetase, (7) galactokinase, (8) galactose 1-phosphate uridyl transferase, (9) uridine diphosphogalactose-4-epimerase, (10) phosphofructokinase, (11) fructose 1,6diphosphatase, (12) fructose 1,6-diphosphate aldolase, (13) fructokinase, (14) fructose 1-phosphate aldolase, (15) phosphoenolpyruvate carboxykinase, and (16) pyruvate carboxylase. UDP is uridine diphosphate. [From Pagliara AS, et al. (1973). Hypoglycemia in infancy and childhood. J Pediatr 82(3): 365-79 and 82(4):558-77.]

TA B L E 5 - 3

Hormonal Regulation of Fasting Metabolic Systems Counter-Regulatory Hormone

Hepatic Glycogenolysis

Hapatic Gluconeogenesis

Adipose Tissue Lipolysis

Hepatic Ketogenesis

Insulin Glucagon Cortisol Growth Hormone Epinephrin

Inhibits Stimulates

Inhibits

Inhibits

Inhibits Stimulates

Stimulates Stimulates

Stimulates

concentrations of gluconeogenic substrate (e.g., lactate, alanine) as they are used for hepatic gluconeogenesis, a brisk rise in free fatty acids as lipolysis is activated, and a dramatic rise in ␤-hydroxybutyrate (the major ketone) as hepatic ketogenesis is turned on.1,44 Levels of insulin diminish to less than 2 ␮U/mL, whereas there is a rise in the four major counter-regulatory hormones (catecholamines, glucagon, cortisol, and growth hormone) as the plasma glucose level falls. These hormonal changes

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Stimulates Stimulates

Stimulates

lead to a reversal of metabolic pathways from synthesis to breakdown in order to maintain glucose homeostasis. When insulin concentration is 2 ␮U/mL or greater at a time when blood glucose concentration is 50 mg/dL or less, there is a hyperinsulinemic state reflecting failure of the mechanisms that normally result in suppression of insulin secretion during fasting or hypoglycemia.45-47 Thus, a snapshot of the integrity of the metabolic and endocrine fasting systems can easily be obtained by

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171

Figure 5-3. Changes in major metabolic fuels during fasting in a normal infant. Note that plasma glucose declines toward hypoglycemic values by 24 hours as hepatic glycogen reserves are depleted. The level of lactate, a representative gluconeogenic substrate, declines gradually during the fast. Late in fasting, levels of plasma free fatty acids (FFA) increase as lipolysis is activated—followed by an increase in ␤-hydroxybutyrate as rates of hepatic fatty acid oxidation and ketogenesis increase.

measuring the plasma levels of the major fuels and hormones at the end of a fast, when the blood glucose approaches hypoglycemic levels (“critical samples”). The use of these critical samples in diagnosing the cause of hypoglycemia is discussed at the end of the chapter. The key hormone governing glycogen synthesis during and immediately after meals is insulin. Likewise, glycogen breakdown (and later, gluconeogenesis) depends on a low insulin concentration. The control of insulin secretion, its metabolic effects, and its mechanisms of action are detailed in Chapter 10. In this chapter, the following requires emphasis. The neural, hormonal, and metabolic changes (including the rise in blood glucose associated with feeding) lead to a rise in insulin. This rise in insulin serves to modulate the rise in blood glucose concentration and eventually to lower it through the activation of glycogen synthesis, enhancement of peripheral glucose uptake, and inhibition of gluconeogenesis. In addition, insulin stimulates lipid synthesis—simultaneously curtailing lipolysis and ketogenesis. Table 5-3 summarizes the synergistic effects of the counter-regulatory hormones on key metabolic pathways and insulin action. There is a hierarchic redundancy in the interaction of these counter-regulatory hormones that provides a margin of safety (“fail-safe mechanism”) if only one counter-regulatory hormone is impaired. Epinephrine and glucagon are quick acting, each signaling its effects by activation of cyclic AMP. Deficiencies of glucagon, as occurs in long-standing type I diabetes mellitus, can be largely compensated for by an intact autonomic nervous system with appropriate ␣- and ␤-adrenergic effects. Conversely, autonomic failure can be largely compensated for if glucagon secretion remains intact.1 Nevertheless, the defense against insulin-induced hypoglycemia is impaired by deficiency of glucagon or catecholamines—with hypoglycemia ensuing quite rapidly. Similarly, growth hormone deficiency can be compensated for in part by intact cortisol secretion (and vice versa). However, deficiency of either impairs the defense against insulin-induced hypoglycemia. Congenital

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or acquired deficiencies in these hormones may result in hypoglycemia, which will occur when endogenous glucose production cannot be mobilized to meet energy needs in the postabsorptive state (i.e., 8 to 12 hours after meals or during fasting). Combined deficiency of several hormones, as occurs in hypopituitarism, may result in a hypoglycemia that is more severe or appears earlier during fasting than might occur with isolated hormone deficiency. The intricate physiologic regulation that maintains glucose homeostasis provides a diagnostic framework with which to anticipate the phenotypes of infants with hypoglycemia of varying causes. The deficiency of substrates, an excess of insulin or a deficiency of hormones, and the absence, delay, deficiency, or missed timing of the development of activity of enzymes or transporters will affect the presentation of infantile hypoglycemia. Thus, this detailed presentation of the events leading to, involving, and after parturition allows rapid and specific diagnostic considerations to be entertained and to evolve to an appropriate treatment.

DEFINITION OF HYPOGLYCEMIA IN NEONATES AND INFANTS The classic definition of symptomatic hypoglycemia is “Whipple’s triad” (i.e., symptoms typical of hypoglycemia, a blood glucose value less than 50 mg/dL during symptoms, and relief of symptoms with treatment to raise the blood glucose level into the normal range). These three criteria were originally used for diagnosing insulinomas in adults but apply well to diagnosis of any form of hypoglycemia. In infants, children, and adults, the normal range for plasma glucose in the postabsorptive state equals plasma glucose levels between 70 and 100 mg/dL. A plasma glucose level of 50 mg/dL is conventionally used for diagnosing hypoglycemia. Note that this value is a very conservative threshold, well below the threshold for hormonal and cognitive deficits to avoid overdiagnosing hypoglycemia. Falling glucose levels elicit a typical

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sequence of responses: plasma insulin levels decrease when plasma glucose falls to the range of 80 to 85 mg/ dL; glucagon secretion increases when plasma glucose levels are in the range of 65 to 70 mg/dL; epinephrine, cortisol, and growth hormone responses are activated in the range of 65 to 70 mg/dL; acute symptoms may appear in the range of 50 to 55 mg/dL; and cognition is impaired when levels fall below 50 mg/dL.1 In some disorders, such as defects in ketogenesis, signs and symptoms may begin to appear during fasting at plasma glucose levels of 60 mg/dL. On the other hand, some patients (those with glucose 6-phosphatase deficiency) may have few symptoms of neuroglycopenia at plasma glucose levels as low as 20 to 30 mg/dL because their high plasma levels of lactate provide an alternative substrate for the brain. Thus, plasma glucose levels between 50 and 70 mg/dL should be regarded as suboptimal and below the goal for therapy for hypoglycemia. Pediatric endocrinologists should be aware that there continues to be controversy about whether the previously cited standards for normal and abnormal plasma glucose levels should apply in the newborn period.48,49 Because the risk for low blood glucose levels is high in neonates, especially in the first day after birth, it has been traditional to accept lower standards for hypoglycemia in newborns. The major rationale for this is statistical (i.e., low glucose is so frequent it must be “normal”). However, the authors do not recommend using lower standards for glucose in neonates. Instead, we use the same definition for hypoglycemia and the same targets for treatment of hypoglycemia in neonates as in older children. This is especially true for the diverse groups of neonates who have congenital hyperinsulinism (e.g., birth asphyxia, small for gestational age birth weight, maternal diabetes, genetic defects) because they lack alternative fuels, such as ketones, for brain metabolism when glucose levels are low. There are a number of potential artifacts that can interfere with measuring glucose levels in neonates and infants (Table 5-4). Whole-blood glucose concentrations are 10% to 15% lower than plasma glucose levels because of lower erythrocyte versus plasma glucose concentrations. Blood samples that are not processed promptly can have erroneously low glucose levels, owing to glycolysis by red and white blood cells. At room temperature, the decline of whole-blood glucose can be 5 to 7 mg/dL/hr. The use of inhibitors, such as fluoride, in collection tubes avoids this problem.

TA B L E 5 - 4

Factors Affecting Measurement of Blood Glucose Concentration • Whole-blood versus plasma glucose concentration (plasma is ⬃10%-15% higher) • Duration between sample collection and sample measurement • Presence or absence of glycolytic inhibitors in collection tubes • Sample collection from indwelling lines without adequate flushing Compiled from Sacks DB (1994). Carbohydrates. In Burtis CA, Ashwood ER (eds.), Tietz textbook of clinical chemistry, Second edition. Philadelphia: WB Saunders.

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Figure 5-4. Glucose production versus body weight determined in 19 newborns with the use of stable isotopic techniques. These studies provide support for the calculated rates of glucose administration required to correct hypoglycemia. [From Bier DM, Leake RD, Haymond MW, et al. (1977). Measurement of “true” glucose production rates in infancy and childhood with 6,6-dideuteroglucose. Diabetes 26:1016.]

Hospital bedside glucose meters and similar home glucose meters are less precise than clinical laboratory methods and can be expected to have an error range of 10% to 15%. These methods are also prone to errors, such as outdated strips or short-sampling—most of which result in falsely low glucose values. For this reason, bedside monitors can be used for screening purposes—but any glucose value below 60 mg/dL should be verified in the clinical laboratory. Falsely low (or high) glucose values may occur with samples drawn from indwelling lines without adequate flushing of the saline (or glucose) infusate. If a plasma glucose value below 50 mg/dL is verified, treatment should immediately be provided. Plasma glucose values below 60 mg/dL should be rechecked and treatment considered, if confirmed to be below 60 mg/dL. Symptomatic infants and neonates should be treated with intravenous dextrose (0.2 g/kg bolus, followed by infusion at 5 to 10 mg/kg/min). These rates approximate the normal hepatic glucose production rates in neonates and young infants (Figure 5-4). Asymptomatic neonates with plasma glucose levels below 50 mg/dL may be treated with oral glucose, but only if there is good reason to believe the problem is a transient one that will not recur. This applies essentially only to otherwise normal neonates during the first 12 to 24 hours after birth who have delayed feedings, such as with initiation of breastfeeding. Beyond the first day of life, all neonates with verified plasma glucose below 60 mg/dL should be suspected of having a hypoglycemic disorder.

CLINICAL SYMPTOMS AND SIGNS ASSOCIATED WITH HYPOGLYCEMIA The clinical features of hypoglycemia in infants may be associated with both adrenergic and neuroglycopenic components (Table 5-5). Symptoms are often quite subtle,

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TA B L E 5 - 5

Symptoms of Spontaneous Hypoglycemia and Hypoglycemia in Infancy Spontaneous Hypoglycemia Symptoms Due in Part to Activation of Autonomic Nervous System Associated Epinephrin Release (Usually Associated with Rapid Decline in Blood Glucose Level) Sweating Shakiness, trembling Tachycardia Anxiety, nervousness Weakness Hunger Nausea, vomiting

Symptoms Due to Decreased Cerebral Glucose and Oxygen Use (Usually with Slow Decline in Blood Glucose Level and/or Severe Prolonged Hypoglycemia) Headache Visual disturbances Lethargy, lassitude Restlessness, irritability Difficulty with speech and thinking, inability to concentrate Mental confusion Somnolence, stupor, prolonged sleep Loss of consciousness, coma Hypothermia Twitching, convulsions, “epilepsy” Bizarre neurologic signs Motor Sensory Loss of intellectual ability Personality changes Bizarre behavior Outburst of tempor Psychological disintegration Manic behavior Depression Psychoses Permanent mental of neurologic damage

and a high index of clinical suspicion must be maintained. Therefore, any alteration in clinical status in a newborn that suggests a change in neurologic behavior, fall in temperature, change in feeding pattern, or presence of tremors must be considered a possible initial presentation of a hypoglycemic episode. Seizures must always be considered a possible manifestation of hypoglycemia.

Classification of Hypoglycemias A logical approach to diagnosis and treatment must analyze a hypoglycemic event as a maladaptation to fasting. The classification scheme represented in Table 5-6 is based on this approach.

TRANSIENT NEONATAL HYPOGLYCEMIA Developmental Immaturity of Fasting Adaptation During the first day of life, normal infants are susceptible to hypoglycemia—especially if fasted after birth for any length of time. As shown in Figure 5-5, 10% of normal appropriate-for-gestational-age term neonates can develop

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Hypoglycemia in Infancy

Cyanotic episodes Apnea “Respiratory distress” Refusal to feed Brief myoclonic jerks Wilting spells Nausea, vomiting Somnolence Subnormal temperature Sweating

plasma glucose values below 30 mg/dL when the first feeding is delayed for 6 hours after birth.50 This high risk of hypoglycemia reflects immaturity of several of the fasting systems immediately after birth. As reported by Lubchenco and Bard, whereas plasma glucose levels fell below 50 mg/dL in one-third of term infants in the first 6 hours after delivery by the second day of life the frequency of glucose levels below 50 mg/dL was less than 0.5% and only occurred in small-for-gestational-age infants.50 As these observations illustrate, the fasting systems mature very quickly after birth. Studies in normal infants during the first postnatal fast indicate that their high susceptibility to hypoglycemia is associated with developmental lags in the capacity for both hepatic gluconeogenesis and ketogenesis.51 This is consistent with studies in the guinea pig and rat, which show that hepatic phosphoenolpyruvate carboxykinase (one of the four gluconeogenic enzymes) as well as carnitine palmitoyl-transferase-1 (CPT-1) and ␤-hydroxy␤-methylglutaryl-CoA (HMG-CoA) synthase (the first and last steps in hepatic ketogenesis) are not expressed for up to 12 hours after birth52-54 (Figure 5-6). The developmental lags in capacity for ketogenesis mean that during the first day of life, normal neonates lack the protection of generating alternative fuel for the brain during fasting

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TA B L E 5 - 6

Grams

Classification of Hypoglycemia in Infants and Children

4500

Transient Neonatal Hypoglycemia Developmental immaturity of fasting adaptation associated with inadequate substrate or immature enzyme function: • Prematurity • Normal newborn Transient hyperinsulinism due to maternal factors: • Maternal diabetes • Intravenous glucose administration during labor and delivery • Medications: oral hypoglycemics, terbutaline, propranolol Prolonged Neonatal Hypoglycemia Prolonged neonatal hyperinsulinism: • Intrauterine growth retardation • Prematurity • Birth asphyxia • Maternal toxemia/preeclampsia Neonatal, Infantile, or Childhood Persistent Hypoglycemias Hormonal Disorders Hyperinsulinism ATP-sensitive potassium channel congenital hyperinsulinism: • Diffuse KATP channel HI • Focal KATP channel HI • Dominant KATP channel HI Glutamate dehydrogenase congenital hyperinsulinism (hyperinsulinism/hyperammonemia syndrome): • Glucokinase congenital hyperinsulinism • Short-chain L-3-hydroxyacyl-CoA dehydrogenase congenital hyperinsulinism • Congenital disorders of glycosylation • Beckwith-Wiedemann syndrome • Acquired islet adenoma • Insulin administration (Munchausen by proxy) • Oral sulfonylurea drugs Counter-Regulatory Hormone Deficiency • Panhypopituitarism • Isolated growth hormone deficiency • Adrenocorticotropic hormone deficiency • Primary adrenal insufficiency • Epinephrine deficiency Glycogenolysis Disorders • Amylo-1,6-glucosidase (debranching enzyme) deficiency (GSD 3) • Liver phosphorylase deficiency (GSD 6) • Phosphorylase kinase deficiency (GSD 9) • Glycogen synthetase deficiency (GSD 0) Gluconeogenesis Disorders • Glucose 6-phosphatase deficiency (GSD 1a) • Glucose 6-phosphate translocase deficiency (GSD 1b) • Fructose 1,6-diphosphatase deficiency • Pyruvate carboxylase deficiency Lipolysis Disorders • Propranolol Fatty Acid Oxidation Disorders • Carnitine transporter deficiency (primary carnitine deficiency) • Carnitine palmitoyl-transferase 1 deficiency • Carnitine translocase deficiency • Carnitine palmitoyl-transferase 2 deficiency • Very long-chain acyl-CoA dehydrogenase deficiency

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3500

38% (6/16)

4% (2/48)

7% (1/14)

10% (12/126)

5% (2/40)

2500 15% (9/60) 1500

25% (11/44)

18% (2/11)

90% 67% (10/15)

10% 500 26

30

34

38

42

46

Week of gestation Figure 5-5. Incidence of plasma glucose level being less than 30 mg/dL before first feeding at 3 to 6 hours of age in newborns, classified by birth weight and gestational age. [From Lubchenko LO, Bard H (1971). Incidence of hypoglycemia in newborn infants by birth weight and gestational age. Pediatrics 47:831.]

10 9 mRNA (absorbance units)

174

8 7 6 5 4 3 2 1 0 0

5

10 15 20 60 Age (Days) Figure 5-6. Levels of mRNA in newborn, suckling, and adult rat liver for phosphoenolpyruvate carboxykinase (triangles), mitochondrial ␤-hydroxy-␤-methylglutaryl CoA synthase (circles), and carnitine palmitoyltransferase I (solid squares) and II (open squares). [From Hegardt FG (1999). Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase: A control enzyme in ketogenesis. Biochem J 338:569.]

hypoglycemia. Fatty acids provided by the first feeding appear to play a key, possibly direct, role in activating transcription of these two important fatty acid oxidation and ketogenesis enzymes.52,53 In terms of fasting systems, the normal newborn is highly dependent on hepatic glycogen stores to maintain normoglycemia in the first 12 to 24 hours but rapidly acquires the full complement of fasting systems thereafter.

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Normal term infants who are being breast fed unsuccessfully are at highest risk for hypoglycemia because of developmental immaturity of fasting adaptation and may require supplementation until milk production is adequate. Symptomatic hypoglycemia is rare in normal term infants who are fed early and is easily prevented by ensuring that feedings are given. As shown in Figure 5-5, the risk of fasting hypoglycemia in the immediate postnatal period is considerably higher in preterm appropriate-for-gestational-age infants compared with normal full-term neonates. This increased risk may be explained by the fact that premature infants have the same developmental immaturity of ketogenesis and gluconeogenesis as term infants but lower glycogen reserves. In addition, hormonal responses are initially limited in preterm infants.55 As discussed later in the chapter, the higher risk of fasting hypoglycemia on day 1 of life in infants who are large or small for gestational age most likely involves additional factors (especially hyperinsulinism).

Hypoglycemia in Normal Infants and Children (Ketotic Hypoglycemia) The process of fasting adaptation is accelerated in infants and children compared with adults because of their relatively larger ratio of brain weight to body weight. Thus, normal infants may develop fasting hyperketonemia before 24 hours of fasting, whereas adults usually require more than 48 hours of fasting to reach the same point. For this reason, otherwise normal infants and children (as discussed in Chapter 11) are susceptible to fasting hypoglycemia during intercurrent illnesses (such as gastroenteritis)—which interfere with feeding. Beyond acute treatment of the hypoglycemia, these infants need no therapy. However, because of this increased susceptibility to hypoglycemia precautions to avoid prolonged fasting beyond 12 hours should be taken in children with ketotic hypoglycemia. Early intervention with intravenous dextrose should be considered during intercurrent illnesses that might interrupt normal eating. Ketotic hypoglycemia has often been considered a specific diagnosis, but in most cases may simply represent normal infants who have accelerated fasting adaptation (e.g., smaller infants with less fuel reserve). A diagnosis of ketotic hypoglycemia should only be made after other conditions have been excluded (e.g., glycogen synthase deficiency, pituitary deficiency). Because the timing of hypoglycemia in patients with fatty acid oxidation disorders [such as medium-chain acyl-CoA dehydrogenase (MCAD) deficiency] is similar to that of ketotic hypoglycemia, specific measurements of plasma ketones are essential to diagnosis.

Transient Hyperinsulinism Due to Maternal Factors Transient hyperinsulinism is a well-recognized complication in infants of diabetic mothers. Gestational diabetes affects approximately 2% of pregnant women, and approximately 1 in 1,000 pregnant women have insulindependent diabetes. At birth, infants born to these mothers

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may be large and plethoric—and their body stores of glycogen, protein, and fat are replete. Thus, in contrast to the transient hypoglycemia of the infant who is small for gestational age (whose body size and tissue nutrient content reflect diminished placental transfer) infants born to diabetic mothers are examples of nutrient surfeit and represent the opposite extreme of the spectrum. The classic clinical description of the effect of hyperinsulinism relates to the infant of the diabetic mother56: These infants are remarkable not only because like fetal versions of Shadrack, Meshack, and Abednego, they emerge at least alive from within the fiery metabolic furnace of diabetes mellitus, but because they resemble one another so closely they might well be related. They are plump, sleek, liberally coated with vernix caseosa, fullfaced, and plethoric. The umbilical cord and placenta share in the gigantism. During their first 24 or more extrauterine hours, they lie on their backs, bloated and flushed, their legs flexed and abducted, their lightly closed hands on either side of the head, the abdomen prominent and their respiration sighing. They convey a distinct impression of having had such a surfeit of both food and fluid pressed upon them by an insistent hostess that they desire only peace so that they may recover from their excesses. On the second day their resentment of the slightest noise improves the analogy when their trembling anxiety seems to speak of intrauterine indiscretions of which we know nothing.

Hypoglycemia in infants of diabetic mothers is related chiefly to hyperinsulinemia and in part to diminished glucagon secretion. Hypertrophy and hyperplasia of their islets have been documented, as has their brisk, biphasic, and typically adult insulin response to glucose. This insulin response is absent in normal infants. Infants born to diabetic mothers also have a subnormal surge in plasma glucagon immediately after birth, subnormal glucagon secretion in response to stimuli, and (initially) excessive sympathetic activity that may lead to adrenomedullary exhaustion because urinary excretion of epinephrine is diminished. Thus, despite their abundance of tissue stores of available substrate the normal plasma hormonal pattern of low insulin, high glucagon, and catecholamines is reversed. Their endogenous glucose production is inhibited and glucose utilization is increased compared with that in normal infants, thus predisposing them to hypoglycemia. Mothers whose diabetes has been well controlled during pregnancy in general have near-normal-sized infants who are less likely to develop neonatal hypoglycemia and other complications formerly considered typical of maternal diabetes. Nevertheless, treatment of infants born to mothers with diabetes commonly requires provision of intravenous glucose for a few days until the hyperinsulinemia abates. For these infants, glucose should be provided at a rate of 5 to 10 mg/kg/min. However, the appropriate dosage for each patient should be individually adjusted. During labor and delivery, maternal hyperglycemia should be avoided because it may result in fetal hyperglycemia—which predisposes to rebound hypoglycemia when the glucose supply is interrupted at birth. Other maternal factors that can result in transient neonatal hyperinsulinism include oral hypoglycemics (such as

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sulfonylureas) or other medications (terbutaline or propranolol). By definition, transient hyperinsulinism as a cause of neonatal hypoglycemia in an infant of a diabetic mother should abate in 1 or 2 days. If the condition persists, organic hyperinsulinism must become a prominent consideration and the index of suspicion must remain high until it is ruled out. The potential risk of brain damage in an infant suffering from hyperinsulinism is high, placing this diagnosis in a critical position in the diagnostic evaluation of neonatal hypoglycemia.

PROLONGED NEONATAL HYPERINSULINISM: PERINATAL STRESSS-INDUCED HYPERINSULINISM As shown in Figure 5-5, the risk of postnatal hypoglycemia is increased in neonates who are small for gestational age. Although this may sometimes be caused by poorer stores of glycogen, there is increasing evidence that prolonged hypoglycemia in some neonates exposed to perinatal stress such as birth asphyxia, maternal toxemia, prematurity, or intrauterine growth retardation or other peripartum stress is due to hyperinsulinism.57-59 The estimated incidence of prolonged neonatal hyperinsulinism is 1:12,000 live births.59 The clinical presentation of perinatal stress-induced hyperinsulinism is characterized by high glucose utilization, and the response to fasting hypoglycemia shows an elevated plasma insulin level (although it may be normal in some), low ␤-hydroxybutyrate and free fatty acid levels, and a glycemic response to glucagon. Unlike the transient hyperinsulinism seen in the infant of the diabetic mother, perinatal stress-induced hyperinsulinism may persist for several days to several weeks. In a series of neonates diagnosed after 1 week of age, the median age of resolution was 6 months.59 The mechanism responsible for the dysregulated insulin secretion is not known. AIR testing shows that in general the patterns of insulin response to secretagogues (calcium, tolbutamide, glucose, and leucine) in infants with prolonged neonatal hyperinsulinism resembled those of normal controls.59 This suggests that the defect does not involve the ATP-dependent potassium (KATP) channel or glutamate dehydrogenase (GDH) sites. Infants with prolonged neonatal hyperinsulinism usually respond very well to medical therapy with diazoxide.57-59 Previously, it was common practice to use pharmacologic doses of glucocorticoids to treat such neonates with persistent hypoglycemia. Glucocorticoids are not effective in controlling hyperinsulinism, however. Their use as nonspecific therapy for neonatal hypoglycemia is not recommended.

Endocrine System Disorders CONGENITAL HYPERINSULINISM Congenital hyperinsulinism is the most common and most difficult to manage form of persistent hypoglycemia in neonates and infants.60 Advances in defining the genetic

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basis of hyperinsulinism during the past decade have been rapid, and improvements in diagnosis and treatment can be expected to occur quickly in the future. As shown in Table 5-6, both recessive and dominantly expressed genetic forms of congenital hyperinsulinism have been described. In addition, a sporadic form of congenital hyperinsulinism associated with focal adenomatosis of the pancreas has been delineated. Unlike adults, insulinomas are not a cause of hyperinsulinism in infancy; these lesions and surreptitious insulin administration are discussed in Chapter 11. Figure 5-7 outlines the major pathways regulating insulin release by pancreatic beta cells. Glucose-stimulated insulin secretion involves glucose uptake through the GLUT2 glucose transporter and phosphorylation by glucokinase (GK), leading to glucose oxidation and an increased ATP/ADP ratio that results in inhibition of a plasma membrane ATP-dependent potassium (KATP) channel. The ␤-cell KATP channel is a hetero-octameric complex consisting of two subunits: a K⫹-selective poreforming subunit (Kir6.2) and a regulatory subunit (SUR1). Four Kir6.2 subunits form the central pore, coupled to four SUR1 subunits. The KATP channel is inhibited by sulfonylurea drugs (used therapeutically to stimulate insulin secretion in type 2 diabetes) and activated by diazoxide (the main medical treatment for congenital hyperinsulinism). In the unstimulated state, the ß-cell ATP-sensitive potassium channels are open—keeping a resting membrane potential of approximately –65 mV. Following the uptake and metabolism of glucose, an increase in the intracellular ATP/ADP ratio results in closure of ATP-sensitive potassium channels, depolarization of the cell membrane, and subsequent opening of voltage-dependent Ca2⫹ channels. The resulting increase in cytosolic Ca2⫹ concentration triggers release of stored insulin granules. Stimulation of insulin secretion by amino acids occurs through an allosteric activation of glutamate dehydrogenase (GDH) by leucine, which results in increased oxidation of glutamate—leading to increased ATP/ADP ratio, inhibition of KATP-channel activity, and membrane depolarization. Genetic defects in these pathways associated with congenital hyperinsulinism include loss-of-function mutations of SUR1 (encoded by ABCC8) or Kir6.2 (encoded by KCNJ11) and gain-of-function mutations of glucokinase (encoded by GCK) or glutamate dehydrogenase (GDH; encoded by GLUD1).60,61 Mutations of SUR1 and Kir6.2 are usually recessively expressed, however, a few dominantly expressed mutations of ABCC8 and KCNJ11 have been reported.62-64 Mutations of GCK and GLUD1 are dominantly expressed; sporadic, de novo mutations of the latter have been diagnosed more commonly than familial cases.65 Loss-offunction mutations of ABCC8 or KCNJ1 transmitted from the paternal side have also been demonstrated to be responsible for congenital hyperinsulinism in infants with focal hyperinsulinism.66 Recently, loss of function mutations in HADHSC, the gene encoding the mitochondrial enzyme short-chain L-3-hydroxyacyl-CoA (SCHAD),67-69 was found to be associated with recessive congenital hyperinsulinism.

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Figure 5-7. Current model of mechanisms of insulin secretion by the beta cell of the pancreas. Glucose transported into the beta cell by the insulin-independent glucose transporter GLUT2 undergoes phosphorylation by glucokinase and is then metabolized, resulting in an increase in the adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio. The increase in the ATP/ADP ratio closes the KATP channel and initiates the cascade of events characterized by increase in intracellular potassium concentration, membrane depolarization, calcium influx, and release of insulin from storage granules. Leucine stimulates insulin secretion by allosterically activating glutamate dehydrogenase (GDH) and by increasing the oxidation of glutamate, thereby increasing the ATP/ADP ratio and closure of the KATP channel. x ⫽ inhibition; (? ⫽ stimulation; and GTP ⫽ guanosine triphosphate. [From Sperling MA, Menon RK (1999). Hyperinsulinemic hypoglycemia of infancy. Endocrinol Metab Clin North Am 28:695.]

The clinical presentations of congenital hyperinsulinism vary with the severity of the disorder as well as the site of defect. The most severe cases have been associated with recessive or focal KATP channel defects and are often associated with macrosomia caused by fetal hyperinsulinemia. These infants may be confused with infants of diabetic mothers. They usually present with hypoglycemia in the first hours or days after birth. The hypoglycemia is difficult to control and often requires glucose infusion rates greater than 20 mg/kg/min. Less severe phenotypes of congenital hyperinsulinism are associated with defects of GDH, GK, SCHAD, and dominant KATP mutations. Episodes of hypoglycemia may not be recognized until 1 to 2 years of age or later. It is often not easy to diagnose hyperinsulinism based solely on measurements of plasma insulin. This is because standard insulin assays are not sufficiently sensitive in the low range to distinguish a normal from an inadequately suppressed insulin concentration. Plasma insulin levels are rarely dramatically elevated in congenital hyperinsulinism; rather there is inadequate suppression of insulin at low plasma glucose concentrations. Therefore, the diagnosis of hyperinsulinism in infants is most frequently based on evidence of excessive insulin action at the time of hypoglycemia, such as inappropriate suppression of plasma ␤-hydroxybutyrate and free fatty acid levels and an inappropriate glycemic response to glucagon. As shown in Table 5-7, at a plasma glucose level below 50 mg/dL, evidence of hyperinsulinism includes plasma

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TA B L E 5 - 7

Criteria for Diagnosing Hyperinsulinism Based on “Critical” Samples (Drawn at a Time of Fasting Hypoglycemia: Plasma Glucose ⬍50 mg/dL) 1. 2. 3. 4.

Hyperinsulinemia (plasma insulin ⬎2 mU/mL)* Hypofattyacidemia (plasma FFA ⬍1.5 mmol/L) Hypoketonemia (plasma BOB ⬍2.0 mmol/L) Inappropriate glycemic response to glucagon, 1 mg IV (delta glucose ⬎30 mg/dL)

*Depends on sensitivity of insulin assay. BOB, ␤-hydroxybutyrate; FFA, free fatty acids.

insulin greater than 2 ␮U/mL; plasma ␤-hydroxybutyrate (BOB) less than 2.0 mmol/L; plasma free fatty acids (FFA) less than 1.5 mmol/L; and glycemic response to glucagon, 1 mg given intravenously, greater than 30 mg/ dL within 15 to 30 minutes.70,71 Plasma concentrations of insulin-like growth factor binding protein-1 (IGFBP1) may be an additional indicator of insulin action on the liver.72 Children with hyperinsulinism fail to show a rise in circulating IGFBP1 concentration during fasting, which is in contrast to normal subjects, who show a 10-fold increase during fasting.72 In neonates, hypoglycemia due to hypopituitarism can mimic hyperinsulinism and may need to be formally excluded. Note that plasma levels of cortisol and growth hormone are frequently not elevated with

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hypoglycemia, hence the need for separate testing to rule out cortisol or growth hormone deficiency. Additional tests for specific forms of congenital hyperinsulinism include plasma ammonia levels (GDH-HI), plasma acyl-carnitine profile (elevated 3-hydroxybutyrylcarnitine) and urine organic acids (3-hydroxyglutarate) (SCHAD-HI). Genetic testing is available for four of the five genes known to be associated with congenital hyperinsulinism through commercial laboratories (ABCC8, KCNJ11, GLUD1, GCK). For research purposes, AIR tests have been useful in phenotypic characterization. Patients with KATP-HI display abnormal positive responses to calcium, abnormal negative response to the KATP-channel antagonist, tolbutamide, as well as impaired responses to glucose73,74 (Figure 5-8). In contrast, infants with GDH-HI exhibit increased responses to leucine.75

KATP-Channel Hyperinsulinism Recessive KATP-HI Infants with this form of congenital hyperinsulinism frequently present as neonates who are large for gestational age with very severe hypoglycemia immediately after delivery. The genetic defects in these infants are loss of function of either the SUR1 or the Kir6.2 components of the ATP-sensitive potassium channel complex. These two genes are located on chromosome 11p, immediately adjacent to each other. More than 100 mutations of ABCC8 and 20 mutations of KCNJ11 have been found. Heterozygous carrier parents are normal. Common SUR1 founder mutations have been reported in Ashkenazi Jews and in Finland. The incidence of the severe hyperinsulinism phenotype seen with recessive KATP-HI has been estimated at 1 in 40,000 in Northern Europe and Finland, but it is as

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high as 1 in 2,500 in Saudi Arabia—where the frequency of consanguinity is high. As shown in Figure 5-7, because the mutations disrupt the KATP channel these patients are usually unresponsive to diazoxide treatment. Octreotide is helpful in short-term management. However, because of tachyphylaxis, octreotide is often not successful as the sole therapy for long-term management.76 The use of calcium-channel blockers to decrease voltage-dependent calcium-channel activity has been proposed as an alternative medical therapy. A few reports of successful treatment with nifedipine are published,77 but most centers have not had success with this drug. Because of the lack of effective medical therapy, most infants with severe hypoglycemia due to KATP-HI require treatment with near-total (95% to 98%) pancreatectomy. Near-total pancreatectomy is associated with a high risk of later development of diabetes mellitus.78 This most likely reflects not only the effects of pancreas resection but the fact that the loss of channel activity renders the beta cells unresponsive to hyperglycemia as well as to hypoglycemia.73 Histologically, in diffuse hyperinsulinism beta cells throughout the pancreas are functionally abnormal and have characteristic enlarged nuclei in about 2% to 5% of cells.79 Previously, this form of hyperinsulinism was termed nesidioblastosis, although it is now recognized that islet neogenesis from ductal epithelium is a normal feature of early infancy.80 The term nesidioblastosis should therefore be abandoned as a synonym for hyperinsulinism.

Focal KATP-HI (Focal Adenomatosis) From 40% to 60% of the cases of KATP-HI (which require surgery) have focal disease.81 Histologically, these lesions are small (usually less than 10 mm in diameter) and are

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characterized by the presence of confluent proliferation of endocrine cells (adenomatous hyperplasia). In contrast to true adenomas, the focal adenomatous hyperplasia includes exocrine acinar cells intermixed within the lesion. The morphology of islets away from the focal lesion is normal.79 Focal lesions arise by a “two-hit” mechanism of focal loss of heterozygosity for the maternal 11p15 region, leading to a somatic reduction to homozygosity (or hemizygosity) of a paternally inherited mutation of the ABCC8 or KCNJ11 gene. The 11p15 region, which carries the ABCC8 and KCNJ11 genes, contains several imprinted tumor suppressor genes (H19 and CDKN1C, also known as p57kip2) that are only expressed on the maternal chromosome. Loss of these growth-suppressing genes may play an important permissive role in the clonal expansion of the lesion.82 Clinically, infants with focal lesions are indistinguishable from those with diffuse KATP-HI and nearly always require surgery. The focal lesions are potentially curable by surgery, whereas diffuse KATP-HI is not. Efforts to diagnose and localize focal lesions in infants with congenital

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hyperinsulinism are therefore worthwhile. Interventional radiology studies, such as transhepatic portal venous insulin sampling83 and selective pancreatic arterial calcium stimulation,84 have only modest success and are technically difficult and highly invasive. Recently, position emission tomography (PET) scans with flurorine-18 L-3, 4-dihydroxyphenylalanine (18F-fluoro-L-DOPA) have been found to accurately discriminate focal from diffuse hyperinsulinism.85-87 Pancreatic ␤-cells take up L-DOPA,88 and DOPA decarboxylase is active in pancreatic islet cells.89 In children with focal hyperinsulinism, there is local accumulation of 18F-fluoro-L-DOPA. Coregistration of PET and computed tomography (CT) images allows the anatomic localization of the lesion (Figure 5-9).

Dominant KATP-HI A few cases of dominantly expressed mutations of ABCC8 and one of KCNJ11 have been reported.62-64,90 The hypoglycemia in these patients is less severe than the recessive KATP-HI just described. Although birth weight in affected individuals is increased, the onset of

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A Figure 5-9. Computed tomography and positron emission tomography (CT/PET) using 18Fluoro-L-Dopa. (A) Coregistration coronal view of a diffuse lesion and (B) focal lesion located in the head of the pancreas. L-DOPA uptake can be appreciated in the liver, in the kidneys, throughout the pancreas in the diffuse form (A) and in a focal area corresponding to the head of the pancreas (B). [Used with permission from Hardy OT, Hernandez-Pampaloni M, Saffer J, et al. (2007). Diagnosis and localization of focal congenital hyperinsulinism by 18F-fluorodopa PET scan. J Pediatr 150(2): 140-45.] (See color plates.) Continued

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hypoglycemic symptoms is often later in infancy or childhood. The dominant KATP defects retain responsiveness to diazoxide. These dominant KATP mutations presumably exert a dominant negative effect in the heteroctameric KATP channel complexes.

GDH-HI Hyperinsulinism associated with gain-of-function mutations of GLUD1 (encoding glutamate dehydrogenase, GDH) is also a dominant disorder.65,91-94 Affected patients present with symptomatic hypoglycemia together with a characteristically persistent but asymptomatic elevation of plasma ammonia. This unusual hyperinsulinism/hyperammonemia (HI/HA) syndrome represents the second most common form of congenital hyperinsulinism after recessive KATP-HI. In GDH-HI, size at birth is normal. Episodes of symptomatic hypoglycemia are often not recognized until 1 to 2 years of age, and occasionally may not be detected until adulthood. Most cases are sporadic, owing to de novo mutations. Familial cases showing autosomal-dominant patterns of inheritance comprise 20% of the identified probands.

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Plasma ammonia levels in HI/HA patients usually range from 60 to 150 ␮mol/L. Ammonia levels are quite constant, and in contrast to urea cycle enzyme defects do not increase with protein feeding. The hyperammonemia does not appear to cause symptoms and does not require treatment. GDH is a mitochondrial matrix enzyme that is a key regulator of amino acid and ammonia metabolism in pancreatic beta cells, liver, and brain. As shown in Figure 5-7, GDH functions in the beta cell pathway of leucine-stimulated insulin secretion. Leucine is an allosteric activator of the enzyme, causing increased oxidation of glutamate to ␣ ketoglutarate and increased ATP production—which results in insulin release. The HI/HA mutations impair allosteric inhibition of GDH by high-energy phosphates (GTP and ATP), thus leading to excessive insulin release. Isolated islets from transgenic mice expressing a mutated human GDH exhibit normal glucose-stimulated insulin secretion but enhanced leucine- and amino-acid-stimulated insulin secretion.95 In the liver, increased GDH activity leads to hyperammonemia by overproduction of ammonia owing to increased glutamate oxidation and by depression of ammonia detoxification due to the depletion of tissue

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glutamate (because glutamate is the substrate for synthesis of N-acetylglutamate, a required allosteric activator of carbamoyl-phosphate synthetase, the rate-controlling step in ureagenesis). The consequences of increased GDH enzyme activity in the brain are less clear, but might explain the lack of hyperammonemic symptoms in affected individuals. The normal toxic effect of hyperammonemia is thought to be a consequence of increased levels of glutamate and glutamine in brain. Increased GDH activity in HI/HA patients may protect against this effect of hyperammonemia by depleting brain concentrations of glutamate. Patients with GDH-HI have fasting hypoglycemia, which may be relatively mild. Patients may be able to fast for 8 to 12 hours before becoming hypoglycemic. However, these patients have dramatic protein-sensitive hypoglycemia—becoming severely hypoglycemic within 30 to 90 minutes of ingesting a protein meal96 (Figure 5-10). Children with GDH-HI may present with an unusual pattern of absence seizure with EEG pattern of generalized epilepsy.97 Patients with GDH-HI have been shown to have leucine-sensitive insulin secretion.98 The diagnosis of GDH-HI can be suggested by the persistent mild elevation of plasma ammonia. Unlike urea cycle enzyme defects, plasma amino acid and urinary amino acid levels are normal in GDHHI. Plasma ammonia concentrations are not affected by protein feeding, fasting, or plasma glucose levels. GDH enzyme activity can be measured in cultured lymphoblasts to demonstrate impaired responsiveness to allosteric inhibition by GTP. Disease-causing missense mutations have been reported to occur in specific regions of the enzyme involved in GTP inhibition, including exons 6, 7, 10, 11, and 12. Diazoxide therapy, 5 to 10 mg/kg/day, is usually effective in controlling both fasting and protein-induced hypoglycemia in GDH-HI. Carbohydrate preloading may be helpful in avoiding the latter.

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GK-HI A less frequent form of congenital hyperinsulinism is due to activating mutations in GCK (encoding glucokinase). Glucokinase is a hexokinase that serves as the glucose sensor in pancreatic beta cells99 (Figure 5-7). In GK-HI, activating mutations result in increased affinity of glucokinase for glucose, closure of KATP channels, and inappropriate insulin secretion. The beta cell glucose threshold for glucosestimulated insulin secretion in children with GK-HI may be less than 2 mmol/L (38 mg/dL), whereas the normal glucose threshold is maintained close to 5 mmol/L (90 mg/dL). Five dominantly inherited mutations have been reported to date.100 The age of onset and severity of symptoms vary markedly.101-104 Some mutations seem to have a mild phenotype, with fasting hypoglycemia responsive to diazoxide. Others seem to lower the glucose threshold further and may be more difficult to treat.

SCHAD-HI HYPERINSULINISM Recently, a mutation in HADHSC [the gene encoding the mitochondrial short-chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD)] was found to be associated with congenital HI.67-69 SCHAD-HI is an autosomal-recessive disorder characterized by fasting hypoglycemia due to inappropriate insulin regulation. The biochemical hallmark, in addition to markers of increased insulin action, is increased levels of plasma 3-hydroxybutyrylcarnitine and increased levels of 3-hydroxyglutarate in urine. In contrast to all other defects in fatty acid oxidation, children with SCHAD-HI have no signs of hepatic dysfunction or cardiomyopathy, or of affects on skeletal muscle. The clinical presentation of SCHAD-HI is heterogeneous, ranging from late onset of mild hypoglycemia to severe early onset of hypoglycemia in the neonatal period. Affected patients have been responsive to medical

Figure 5-10 Blood glucose responses to fasting (open squares) and protein feeding (solid diamonds) in a 16-year-old girl with the hyperinsulinism/hyperammonemia syndrome caused by a dominantly expressed R269H regulatory mutation of glutamate dehydrogenase. [From Hsu BY, Kelly A, Thornton PS, et al. (2001). Protein-sensitive and fasting hypoglycemia in children with the hyperinsulinism/hyperammonemia syndrome. J Pediatr 138:383.]

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therapy with diazoxide. The cause of dysregulated insulin secretion in SCHAD deficiency remains to be elucidated.

OTHER FORMS OF HYPERINSULINISM Hyperinsulinism can be associated with complex syndromes such as Beckwith-Wiedemann syndrome (BWS) and the congenital disorders of glycosylation (CDG). BWS is a clinically and genetically heterogenous disorder characterized by fetal overgrowth, hypoglycemia in up to 50% of patients, and a predisposition to childhood tumors. Among cases of BWS, 85% are sporadic and 15% are dominant. Genetic abnormalities in the imprinted region of chromosome 11 have been described in patients with BWS. These often lead to isodisomy for the paternal 11p, a region that is highly imprinted—including some maternally expressed growth-suppressing genes and the paternally expressed fetal growth-promoting IGF2 gene. The hypoglycemia in BWS may be mild and transient, although in some cases it can be severe and persistent.105 Some BWS patients respond to diazoxide, whereas others require partial pancreatectomy to control the hypoglycemia. At least two theories have been proposed to explain the hypoglycemia. One involves the insulin-like actions of IGFII. The second involves dysregulated insulin secretion due to loss of KATP channel genes on 11p.106 Hypoglycemia due to hyperinsulinism has been reported in patients with a few of the congenital disorders of glycosylation.107-109 These inherited metabolic diseases result in hypoglycosylation of different extracellular glycoproteins. They usually affect multiple systems, such as the brain, liver, gastrointestinal system, and the skeleton.110 Hypoglycosylation of the sulfonylurea receptor has been speculated to be the responsible mechanism for the dysregulated insulin secretion, but the mechanism of hyperinsulinism remains to be proven. Other forms of hyperinsulinism include islet adenoma or carcinoma (both very rare in childhood), surreptitious insulin administration (Munchausen syndrome by proxy), and ingestion of insulin secretagogues such as sulfonylureas. The latter has occasionally been described in neonates as being caused by drug administration to the mother shortly before delivery. Exogenous insulin produces the usual features of hyperinsulinism (increased glucose use, suppression of lipolysis and ketogenesis, inappropriate glycemic response to glucagon). However, plasma levels of C-peptide are low— indicating suppression of endogenous insulin secretion. Surreptitious insulin administration can be suspected when plasma insulin concentrations at times of hypoglycemia are unusually high (100 ␮U/mL or greater). Note, however, that biosynthetic forms of insulin such as lispro insulin are not detectable by standard insulin assays.

Counter-Regulatory Hormone Deficiencies Hypoglycemia associated with endocrine deficiency is usually caused by glucocorticoid or growth hormone deficiency. In patients with panhypopituitarism, isolated

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growth hormone deficiency, or a combination of ACTH deficiency and growth hormone deficiency the incidence of hypoglycemia is as high as 20%. In the newborn period, hypoglycemia may be the presenting feature of hypopituitarism. In males, a microphallus may provide a clue to a coexistent deficiency of gonadotropin111,112 (Figure 5-11). Newborns with hypopituitarism may also have liver dysfunction resembling cholestatic liver disease, and some have midline malformations such as the syndrome of septo-optic dysplasia.113 Although older infants and children with pituitary deficiency present with ketotic hypoglycemia, in the neonatal period the hypoglycemia may mimic hyperinsulinism. These infants may represent a subset of perinatal stressinduced hyperinsulinism. However, their hypoglycemia is not responsive to diazoxide and only remits with replacement of deficient hormones (including thyroxine, as well as growth hormone and cortisol). When adrenal disease is severe (as in congenital adrenal hyperplasia caused by enzyme defects in cortisol synthesis, adrenal hemorrhage, or congenital absence or hypoplasia of the adrenals,114,115), disturbances in serum electrolytes with hyponatremia and hyperkalemia or ambiguous genitalia may provide diagnostic clues. Abnormalities of the ACTH receptor or adrenal agenesis may also be phenotypically difficult to distinguish from cortisol deficiency of other causes, with the exception of the marked elevations of serum ACTH concentration noted if the ACTH receptor or adrenal gland is malfunctioning.116 However, all states with ACTH elevations can be clinically suspected by virtue of the associated hyperpigmentation (see Chapter 12). These cases of isolated adrenal insufficiency very rarely cause

Figure 5-11. Micropenis and undescended testes in an infant with congenital hypopituitarism. The infant was hypoglycemic at 12 hours of age (glucose, 24 mg/dL). At 72 hours of age, he was jaundiced—and a liver biopsy demonstrated neonatal hepatitis. His endocrine evaluation was positive for hypothyroidism, low cortisol level, undetectable growth hormone level, and an elevated prolactin level (confirming hypothalamic hypopituitarism).

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hypoglycemia in neonates, whereas in older infants and children treated congenital adrenal hyperplasia and isolated ACTH deficiency can cause profound stress-induced hypoglycemia. The mechanism of hypoglycemia in growth hormone deficiency may be the result of decreased lipolysis. The mechanism of hypoglycemia with cortisol deficiency may be reduced liver glycogen reserves plus diminished gluconeogenesis, owing to a failure to supply endogenous gluconeogenic substrate in the form of amino acids from muscle proteolysis. Deficiency of either of these hormones can resemble the syndrome of ketotic hypoglycemia. Investigation of a child with hypoglycemia therefore requires exclusion of ACTH, cortisol, or growth hormone deficiency—and if diagnosed appropriate replacement with cortisol or growth hormone. Although glucagon deficiency117,118 has been described, this disorder is exceedingly rare or nonexistent. Epinephrine deficiency is also rare, but must be a consideration in familial dysautonomia or in children treated with beta blockers.

Glycogen Storage Disorders The glycogen storage disorders (GSDs) may present primarily as hepatic manifestations or muscular and cardiac manifestations. Hypoglycemia is a consistent feature of certain forms (e.g., glucose 6-phosphatase deficiency). Affected children may display a remarkable tolerance to their chronic hypoglycemia. Blood glucose values in the range of 20 to 50 mg/dL (1.1 to 2.7 mmol/L) are often not associated with the classic symptoms of hypoglycemia, possibly reflecting the use of ketones or lactic acid as alternative fuels by the central nervous system. These disorders are uncommon, but they present with distinct phenotypes and are important to diagnose correctly to avoid long-term sequelae.119 Glycogen is a globular structure with a variable molecular weight. It is found primarily in liver and muscle but is present in other cells. The arborized structure consists of straight-chain glucose residues attached through amylo-1,4 linkages, with branch points every 12 to 18 residues attached through a 1,6 linkage. Glycogen is synthesized from incorporation of sequential glucose-1-phosphate residues that have been converted to uridine diphosphoglucose and then incorporated by glycogen synthase. When the elongating glycogen chain consists of at least 11 residues, a branching enzyme transfers a chain of seven molecules to another chain by an ␣1-6 bound. Thus, glycogen synthase elongates the glycogen chain and the branching enzyme produces new branches—creating a molecule with a helical structure of 12 concentric tiers. Glycogen degradation is the result of the activities of glycogen phosphorylase and a debranching enzyme. Glycogen phosphorylase catalyzes the rate-limiting step of glycogenolysis. It cleaves ␣1-4 linkages to remove glucose molecules from the glycogen chain as glucosel-phosphate. The debranching enzyme has both transferase and glucosidase activity. When four glucose units remain before a branch point, the transferase activity of the debranching enzyme catalizes the transfer of three

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glucose residues to an adjacent branch of the glycogen chain. Through a second enzymatic glucosidase component, the debranching enzyme next cleaves the ␣1-6 bond to release a free glucose moiety from the branch point. Glycogen phosphorylase is then able to continue removal of glucose residues from the glycogen chain.120 This complex system offers many specific points of regulation and many sites for mutation.121

GLUCOSE 6-PHOSPHATASE DEFICIENCY (GSD TYPE I) Glucose 6-phosphatase catalyzes the terminal steps of both hepatic gluconeogenesis and glycogenolysis. The phenotype of infants with type I GSD is characteristic, despite the fact that the glucose 6-phosphatase complex possesses a variety of sites for different mutations and several subtypes of GSD I have been described.119 The most striking feature is the massive hepatomegaly that may fill the entire abdomen. Affected infants have profound elevations of plasma triglycerides, and their serum may be creamy. Patients are often tachypneic secondary to respiratory compensation for their metabolic acidemia as a result of dramatic elevation in plasma lactate concentration. Although ketosis is often present, it is minimal compared to the dramatic lactic acidosis seen in untreated patients. Other consistent features are hyperuricemia to plasma concentrations that may precipitate gouty crises, hypophosphatemia, a bleeding diathesis secondary to impairment of platelet adhesiveness, and growth retardation. Hypoglycemia may occur anytime these children are exposed to even brief periods of fasting. They are completely dependent on the provision of glucose from exogenous sources, with the exception of the small amount of free glucose—which is released as part of the process of debranching glycogen. Because less than 10% of glycogen consists of branch points, this mechanism provides little protection against hypoglycemia during fasting. Affected infants may be diagnosed soon after birth with hepatomegaly, and then hypoglycemia may be documented during the diagnostic evaluation. On the other hand, because lactate and ketones may provide adequate brain substrate to protect central nervous system function (and because in early infancy regular feedings are consistently provided) the diagnosis may be delayed for months until massive hepatomegaly brings the infant to medical attention. Hepatomegaly, in the absence of splenomegaly or other signs of a generalized storage disorder, should suggest glycogen as the likely storage component causing liver enlargement. After infancy, affected patients may be seen walking with a waddling gait secondary to their prominent abdomen and muscle weakness—with blood glucose levels of less than 40 mg/dL and lactate levels of more than 8 to 10 mmol/L (apparently in total disregard of their hypoglycemia and with deep respirations reflecting their respiratory compensation for metabolic acidosis). Glucose 6-phophatase is a multicomponent system consisting of a catalytic unit with its active site located on the luminal side of the endoplasmic reticulum, and transmembrane-spanning translocases that allow the entry of

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glucose 6-phophate to the catalytic subunit and the exit of Pi and glucose.122 The gene for the catalytic unit has been cloned and located in a single copy on chromosome 17,121 whereas the gene for the glucose 6-phosphate translocase is located on chromosome 11.123 GSD type I is an autosomal-recessive disease. Molecular genetic evidence has unequivocally demonstrated that GSD type Ia124 is caused by mutations in the catalytic unit, whereas mutations in glucose 6-phosphate translocase cause GSD type Ib.123 The clinical hallmark of patients with GSD type 1b is their susceptibility to infection as a consequence of neutropenia, mouth ulcers, and occasionally chronic enteritis (which is similar to Crohn disease).119 Treatment with granulocyte-macrophage colony-stimulating factor to augment neutrophil production has been shown to ameliorate mouth ulcers and the enteritis.125 Although two additional types have been reported [GSD type Ic126 (presumably caused by a mutation in the putative Pi translocase) and GSD type Id119 (due to a defect in the glucose translocase)], there is insufficient available evidence to sustain the existence of these defects. Rapid onset of hypoglycemia is the hallmark of GSD type I, which occurs 2 to 3 hours after a meal immediately after intestinal absorption of carbohydrates is complete. Renal disease is a frequent complication of GSD type I (with an estimated prevalence of 30%).127 Manifestations include proximal renal tubular dysfuntion (Fanconi-like syndrome), distal tubular acidification defect, and hypercalciuria. The widespread prevalence and serious prognosis of kidney involvement is manifested by severe glomerular hyperfiltration and microalbuminuria over time, systemic arterial hypertension, and consequently renal failure in a considerable number of patients.128-130 The early implementation of treatment with angiotensin-converting enzyme inhibitors has been shown to delay the progression of renal damage.127 The pathologic findings include focal segmental glomerulosclerosis with interstitial fibrosis. The etiology of this renal involvement is unclear, but it seems to correlate negatively with metabolic control. It has been proposed that the dyslipidemia contributes to the kidney injury.131 In addition to the characteristic hepatomegaly, the liver undergoes adenomatous changes. Ultrasound of older patients with glucose 6-phosphatase deficiency will frequently show multiple adenomas.132,133 Reports of malignant degeneration of these lesions are noted.134 Other complications of GSD I include osteopenia and growth retardation. The goal of treatment of children with glucose-6phosphate deficiency is to completely eliminate hypoglycemia and suppress secondary metabolic decompensation. Continuous nasogastric or intragastric feeding during the night or total parenteral nutrition has demonstrated either a reduction or an elimination of the metabolic and clinical findings through complete avoidance of hypoglycemia.135 Oral uncooked cornstarch supplementation (1.6 g/kg per dose every 4 hours in infants and 1.7 to 2.5 g/kg per dose every 6 hours in older patients) has also been applied in treatment regimens.136,137 A typical regimen institutes daytime feedings every 3 to 4 hours that are calculated to provide adequate

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carbohydrate calories to suppress hepatic glucose output. Most of these calories consist of carbohydrates, primarily providing pure glucose as an energy source and avoiding disaccharides containing fructose or galactose. At night, the regimen consist of an intragastric infusion of glucose with or without protein designed to infuse at rates of about 125% calculated hepatic glucose output138 for young infants. For older children, a regimen of uncooked cornstarch can be implemented. Meticulous dietary control of blood glucose levels can lead to a significant clinical and metabolic improvement and prevention of complications. Adjunctive therapies should include careful monitoring of the uric acid level and treatment with allopurinol if the uric acid level remains elevated. With increasing awareness of the renal tubular dysfunction, treatment of the hyperfiltration state with an angiotensin-converting enzyme inhibitor should be initiated promptly.

AMYLO-1,6-GLUCOSIDASE DEFICIENCY (DEBRANCHER DEFICIENCY, GSD TYPE III) The debraching enzyme, together with phosphorylase, is required for complete degradation of glycogen. The lack of activity of this enzyme results in incomplete breakdown of glycogen and glycogen accumulates. The human debranching enzyme gene is a large singlecopy gene located on chromosome 1p21.139 Debrancher deficiency GSD type III is an autosomal-recessive disease. The phenotype of debrancher deficiency, although similar, can be distinguished clinically from that of glucose 6-phosphatase deficiency (e.g., particularly in regard to lack of lactic academia and renal disease). During infancy and childhood, hepatomegaly, hypoglycemia, hyperlipidemia, and short stature are the predominant features. The hepatomegaly may be quite marked in GSD type III. Although these individuals also share a propensity for developing hypoglycemia, it usually tends to be less severe. Moreover, these individuals have the capacity to undergo gluconeogenesis—whereas individuals with glucose 6-phosphatase deficiency lack this capacity. The general presentation of debrancher deficiency is hepatomegaly noted with growth retardation. Hypoglycemia is less often the presenting finding than in patients with glucose 6-phosphatase deficiency. The abnormality in liver function is less profound, and these individuals also may have muscle weakness and myotonia. Approximately half of affected patients may have progressive skeletal muscle weakness and/or cardiomyopathy (type IIIa). In about 15% of patients, GSD III appears to involve only the liver (type IIIb). The patient’s serum creatine kinase level can be used to determine muscle involvement. The debranching enzyme is active in leukocytes and erythrocytes, and thus these present easily accessible tissues for biochemical analysis.140 Because of genetic heterogeneity in enzyme activity, leukocyte or erythrocyte enzyme testing has not been effective in determining the heterozygote state. Frequent feedings with high protein content have been successful in treatment of debrancher deficiency.141,142

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GLYCOGEN PHOSPHORYLASE DEFICIENCY (GSD TYPE VI) AND PHOSPHORYLASE KINASE DEFICIENCY (GSD TYPE IX) Glycogen storage disease due to reduction in liver phosphorylase activity is a heterogeneous group of disorders. Deficiency of phosphorylase kinase (GSD type IX) resulting in failure of hepatic phosphorylase activation is the most common of these disorders, whereas deficiency of liver glycogen phosphorylase (GSD type VI) is very rare. Phosphorylase kinase of liver and muscle is a complex enzyme consisting of four subunits (␣, ␤, ␥, and ␦), of which the ␥ subunit is catalytically active. Mutations in three different genes for PHK subunits can result in deficient activity of hepatic phosphorylase. X-linked GSD type IX caused by mutations in the gene encoding the liver isoform of the ␣ subunit is the most common variant. Other less common variants of GSD type IX are autosomal recessive (affecting only liver) or are a form that affects both liver and muscle. GSD type VI is recessive and caused by mutations in liver glycogen phosphorylase. The clinical phenotype is similar in GSD types VI and IX. Classically, the physical hallmark is hepatomegaly without splenomegaly. Although some patients have been reported with growth retardation and hypoglycemia, this tends to be the exception. The impaired glycogen breakdown in GSD types VI and IX leads to mild hypoglycemia after prolonged fasting. Unlike in GSD type I, blood levels of lactic acid and uric acid are normal. Mild elevation of triglycerides, cholesterol, and serum transaminase may be present. Muscular hypotonia can result in delayed motor development in patients with X-linked GSD type IX. Prolonged fasting should be avoided in these patients. A bedtime snack is usually sufficient to prevent hypoglycemia in the morning. Clinical and biochemical abnormalities gradually improve with age, and most adult patients are asymptomatic.

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glycolytic pathway—leading to postprandial hyperlactatemia and hyperlipidemia. Short stature and osteopenia are common in untreated children, but improve with appropriate metabolic control. The goal of treatment is to prevent hypoglycemia and to minimize metabolic acidosis. Dietary recommendations include a high-protein diet with complex carbohydrates. Uncooked cornstarch is used to prevent fasting ketotic hypoglycemia overnight.

DIAGNOSIS OF GLYCOGEN STORAGE DISORDERS The diagnosis of glycogen storage disorders is based in grant part on the clinical presentation. The combination of clinical features, biochemical analysis, and mutation analysis allows for the specific types of GSD to be differentiated. Clinically, GSD type I can be easily differentiated by the severity of hypoglycemia (which occurs after a short period of fasting) and the association with lactic academia and hyperuricemia. As previously discussed, hypoglycemia is milder and usually happens after prolonged fasting in patients with other forms of GSD. Hepatomegaly is a common clinical feature in all the types except GSD type 0. A fed glucagon stimulation testing with simultaneous assessment of blood glucose and lactate is also helpful in the diagnosis. In GSD type I, glucagon cause a rise in lactate levels (whereas blood glucose levels remain unchanged). Biochemical studies of leukocytes provide information on all glycogen storage diseases except glucose 6-phosphatase deficiency. The diagnosis of glucose 6-phosphatase deficiency may be made through enzymatic studies on liver biopsy tissue, but the availability of mutation analysis has made the need for liver biopsy obsolete. Mutation analysis is now available for GSD types Ia and Ib, as well as GSD type 0.

Disorders of Gluconeogenesis GLYCOGEN SYNTHASE DEFICIENCY (GSD TYPE 0) Glycogen storage disease type 0 is caused by deficiency of the hepatic isoform of glycogen syntase.143 Glycogen synthase catalizes the formation of ␣-1,4 linkages that elongate chains of glucose molecules to form glycogen. Autosomal-recessive mutations in the GYS2 gene located on chromosome 12p12.2 cause GSD 0.144 In contrast to other forms of glycogenoses, in GSD type 0 there is marked decreased in liver glycogen content. GSD type 0 is the only GSD not associated with hepatomegaly. Children with GSD 0 are usually asymptomatic during early infancy, but experience fasting ketotic hypoglycemia when weaned from overnight feedings. They can be relatively asymptomatic because the increased plasma ketones are used as alternative brain fuel. This condition mimics the syndrome of ketotic hypoglycemia and should be considered in the differential diagnosis. In addition to fasting hypoglycemia, deficiency of glycogen synthase results in postprandial hyperglycemia after ingestion of a carbohydrate-containing meal. After uptake by the liver, glucose is shunted into the

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GLUCOSE 6-PHOSPHATASE DEFICIENCY (GSD TYPE I) Although this disorder is often classified among the glycogen storage diseases, it should be considered primarily a defect of gluconeogenesis.

FRUCTOSE 1,6-DIPHOSPHATASE DEFICIENCY Fructose 1,6-diphosphatase is a key regulatory enzyme of gluconeogenesis. A deficiency of this enzyme results in a block of gluconeogenesis from all possible precursors below the level of fructose-1,6-diphosphate (i.e., fructose, glycerol, lactate, amino acids) (see Figure 5-2). Infusion of these gluconeogenic precursors results in lactic acidosis without a rise in glucose, and acute hypoglycemia may be provoked by inhibition of glycogenolysis. Normally, however, glycogenolysis remains intact and glucagon elicits a normal glycemic response in the fed but not in the fasted state. Hypoglycemia does not develop until fasted beyond glycogen reserves. In affected

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families, there may be a history of siblings with known hepatomegaly who died in infancy with unexplained metabolic acidosis. Hepatomegaly in individuals with fructose 1,6diphosphatase deficiency is due to lipid storage. The biochemical hallmark consists of lactic acidosis, ketosis, hyperlipidemia, and hyperuricemia. Their pathogenesis is related to the severity and duration of hypoglycemia and the resultant low levels of insulin and high levels of counter-regulatory hormones. Therapy in these infants consists of avoidance of fasting longer than 8 to 10 hours. A diet high in carbohydrates (56%, excluding fructose, which cannot be used), low in protein (12%), and normal in fat composition (32%) has permitted normal growth and development. Continuous nocturnal provision of calories through the intragastric infusion system described for type I glycogen storage disease is also applicable to children with fructose 1,6-diphosphatase deficiency. During intercurrent illnesses with vomiting, intravenous glucose infusion is necessary to prevent severe hypoglycemia and lactic acidemia.

PYRUVATE CARBOXYLASE DEFICIENCY Pyruvate carboxylase is a mitochondrial enzyme that catalyzes conversion of pyruvate to oxalacetate, a key metabolite in gluconeogenesis. Clinical manifestations of pyruvate carboxylase deficiency include lactic acidosis and hypoglycemia. Other biochemical markers include elevated pyruvate and alanine.145 In addition to lactic acidosis and hypoglycemia, a subgroup of patients develops hyperammonemia and elevation of plasma citrulline, lysine, and proline levels. Mental retardation and seizures may be part of the presentation in some patients.146 The severity of this condition varies from mild intermittent lactic acidosis without mental disability to severe, rapidly progressive, and often fatal disease. The diagnosis of pyruvate carboxylase deficiency is confirmed by measurement of enzyme activity in fibroblasts. Treatment is primarily symptomatic, with correction of the metabolic acidosis. Replacement of Krebs cycle intermediates (citrate, aspartate, or odd-chain fatty acid compounds) has been used, as well as supplementation with coenzymes of pyruvate dehydrogenase complex (thiamine and lipoic acid).147,148

PHOSPHOENOLPYRUVATE CARBOXYKINASE DEFICIENCY This is a potential gluconeogenic disorder, and a few reported cases have been suggested to have a deficiency of this enzyme. None has been confirmed, however. Therefore, the existence of this defect remains uncertain.

Galactosemia Galactose metabolism progresses through phosphorylation (galactose-1-P) and then through conjugation with uridine to form uridine diphosphate (UDP) galactose. UDP galactose may undergo epimerization to form UDP glucose (Figure 5-2). The clinical syndrome of galactosemia results

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from a deficiency of galactose 1-phosphate uridyl transferase. UDP galactose-4-epimerase deficiency may result in a similar syndrome, however.149 Galactose 1-phosphate uridyl transferase is essential in infants consuming lactose as their primary carbohydrate source. In infants with classic galactosemia, exposure to dietery galactose results in acute deterioration of multiple organ systems—including liver dysfunction, coagulopathy, poor feeding and weight loss, renal tubular dysfunction, cerebral edema, vitreous hemorrhage, neutropenia, and Escherichia coli sepsis.150 Most state screening programs have included galactosemia in their newborn screening. The important clinical caveat is that hypoglycemia in an infant who has vomiting or diarrhea and jaundice (with or without hepatomegaly) should raise the diagnostic consideration of galactosemia. However, hypoglycemia is not a common feature unless severe hepatic failure has already developed. A neonate with these findings and concomitant Escherichia coli sepsis should also be considered as possibly affected with galactosemia, because neonatal E. coli sepsis is increased in this disorder (often leading to death). The toxic accumulation of galactose 1-phosphate is proposed as a mechanism to explain the deleterious effects on certain tissues resulting in intellectual impairment, cataracts, hepatic dysfunction, renal tubular defects with Fanconi syndrome, and ovarian (but not testicular) failure. A galactose-restricted diet will effectively reverse many of the listed abnormalities and almost certainly will eliminate the likelihood of hypoglycemia. Long-term effects on mental function as well as on speech and ovarian function may persist despite appropriate dietary therapy, however.151

Hereditary Fructose Intolerance Hereditary fructose intolerance (HFI) is an autosomalrecessive disorder caused by a deficiency in fructose-1phosphate aldolase. This aldolase is the primary isoenzyme used during the incorporation of diet-derived fructose into the hepatic glycolytic and gluconeogenic pathways. HFI may be a diagnostic challenge. In the absence of fructose ingestion, patients are entirely normal. Ingestion or infusion of fructose, sucrose, and sorbitol results in the accumulation of fructose 1-phosphate in the liver and a depletion of intracellular phosphate and ATP pools. These metabolic disturbances result in decreased gluconeogenesis and glycogenolysis, which precipitate hypoglycemia.152 The severity of the clinical phenotype varies with the quantity of the dietary fructose exposure. Breast-fed or milk-based formula-fed infants are normal until fruits and juices are introduced into the diet. If a fructose- or sucrose-containing formula is fed during the neonatal period to an affected individual, the consequences may be lethal. Because many of the symptoms after fructose ingestion suggest gastrointestinal system disturbances, a soy or elemental formula containing sucrose may be tried— resulting in progressive deterioration of the infant. The worsening of symptoms and hypoglycemia with feedings should raise clinical suspicion. Biochemical confirmation

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of the diagnosis may be ascertained by enzyme assay of liver or small intestinal biopsy. Although intravenous fructose tolerance testing was used in the past, the risks to the patient and the availability of molecular diagnosis have made this method obsolete. Symptoms after acute exposure to fructose include those of hypoglycemia, as well as nausea, abdominal pain, and vomiting. Chronic exposure results in failure to thrive and a clinical spectrum suggestive of chronic liver disease. Hypoglycemia may be missed in patients with HFI because the fall in blood glucose concentration may be transient.153 Sorbitol ingestion causes biochemical abnormalities similar to fructose ingestion without fructosemia.154 Treatment of this disorder involves strict dietary avoidance of fructose, sucrose, and sorbitol. This avoidance is often learned and self-imposed by patients if they reach 1 year of age.

Disorders of Fatty Acid Oxidation At least 25 enzymes and transport proteins are involved in mitochondrial fatty acid metabolism, some of which have been recognized very recently. Defects in 22 of these have been shown to cause disease in humans, and although the first defects were identified 30 years ago most have been identified only in the past 15 years.155 The inborn errors of

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metabolism associated with deficiencies of fatty acid oxidation (Figure 5-12) are inherited in autosomal-recessive fashion. All of these disorders may be provoked with fasting and exhibit life-threatening events with varying degrees of hypoglycemia associated with a relative deficiency in the generation of ketone bodies.156 Disorders of fatty acid oxidation may be divided according to the site of the defect: defects of fatty acid and carnitine transport, ␤-oxidation defects, electron transport chain defects, and defects of ketone body synthesis and utilization. The disorders of fatty acid oxidation share many clinical features and tend to be exacerbated with fasting because metabolism of fatty acids is maximal during fasting. Common events (such as immunization or intercurrent infections) will often decrease oral intake and result in symptoms and signs of a defect in fatty acid oxidation. The wide spectrum of clinical presentation includes hepatic, cardiac, and muscle manifestations.157 The most important of these is the hepatic presentation characterized by acute life-threatening attacks of coma precipitated by fasting. This manifestation occurs in nearly all of the defects, and is more clearly exemplified by medium-chain acyl-coenzyme A dehydrogenase deficiency (MCAD). Attacks are associated with hypoketotic hypoglycemia with little or no academia; elevation of serum urea, ammonia, and uric acid; liver function abnormalities; and hepatic steatosis. The risk of severe complications and

Figure 5-12. The pathways of mitochondrial fatty acid oxidation and ketone body synthesis. ACD ⫽ acyl-CoA dehydrogenase, CPT-1 and CPT-2 ⫽ carnitine palmitoyltransferase I and II, ETF 5 electron-transferring flavoprotein, ETF-DH ⫽ electron-transferring flavoprotein dehydrogenase, and HAD ⫽ hydroxyacyl-CoA dehydrogenase. [From Stanley CA, Hale DE (1994). Genetic disorders of mitochondrial fatty acid oxidation. Curr Opin Pediatr 6:476.]

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death is very high unless appropriate treatment to reverse the catabolic state is implemented. The diagnosis of these cases may be confused with Reye’s syndrome. Cardiac involvement frequently accompanies acute hepatic presentations, especially in defects affecting long-chain fatty acid oxidation.158 The cardiac presentation can be acute or chronic, with dilated or hyperthrophic cardiomyopathy. This presentation can evolve to progressive heart failure at 2 to 3 years of age (or later in patients with muscle-kidney plasma membrane carnitine transporter defect).159 The third manifestation of fatty acid oxidation disorders is acute or chronic muscle presentations. The least severe cases can be seen in adults with the mild form of carnitine palmitoyl-transferase 2 deficiency who present with acute rhabdomyolysis and renal failure after strenuous exercise.160 Some manifestations of fatty acid oxidation disorders, such as pigmentary retinopathy and peripheral neuropathy (which occur in long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency), may be explained by toxic effects of fatty acid metabolites. Less common manifestations of fatty acid oxidation disorders involve congenital malformations161 or the acute fatty liver of pregnancy or the HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome in mothers heterozygous for long-chain 3-hydroxy-acyl-coenzyme A dehydrogenase deficiency (LCHAD) mutations.162 Because fatty acid oxidation is reduced with loss-offunction mutations in this pathway, a decreased ability to generate ketones during normal fasting and acute illness is common. Thus, these defects as a class are associated with hypoketotic hypoglycemia. Although hypoglycemia may be a prominent late feature of the severe mutations seen with MCAD, the phenotype of a defect of a fatty acid oxidation disorder may not manifest if a fasting state is avoided. A high index of suspicion for fatty acid oxidation defects is important because appropriate therapy may result in an interruption and prevention of these potentially life-threatening episodes. The most commonly presenting deficiencies involve the mitochondrial acyl-CoA dehydrogenases with very long-, long-, medium-, and short-chain length specificities (VLCAD, LCAD, MCAD, and SCAD, respectively).163 MCAD is the most frequent. Neonatal screening in Pennsylvania has shown an incidence of 1 in 9,000 live births.164 Although there is significant heterogeneity in presentation of MCAD, the most frequent clinical presentation is one of intermittent hypoketotic hypoglycemia. Mild hyperammonemia and coma may be present with a Reye-like syndrome. A distinct association of the symptoms with fasting or substrate deprivation is consistent with this defect. Affected patients have also been misdiagnosed with idiopathic sudden infant death syndrome.165,166 Decreased plasma carnitine levels and an increase in the ratio of esterified to free carnitine is a frequently associated laboratory finding. VLCAD deficiency is commonly associated with cardiac and skeletal muscle myophaty, but hypoketotic hypoglycemia, hyperammonemia, and hepatocellular failure can also occur.167 Although putative LCAD deficiency cases have been reported, all of the patients described before 1992 were subsequently proven to have VLCAD.168 Hypoglycemia is not usual in SCAD deficiency.

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Deficiencies of several steps of carnitine, acylcarnitine, and fatty acid transport into cells and mitochondria have been described.169,170 A genetic defect of the plasma membrane carnitine transporter represents the only true entity of primary carnitine deficiency. Decreased carnitine accumulation in tissue and renal carnitine losses result in impairment of long-chain fatty acid metabolism, which can lead to severe hypoglycemia and dilated cardiomyophaty in infancy or childhood. This disorder (and no others) responds to carnitine supplementation. Liver carnitine palmitoyl transferase I (CPT I) deficiency is characterized by early-onset episodic hypoketotic hypoglycemia, sometimes with hyperammonemia and multiorgan system failure. These patients can have elevated levels of plasma carnitine. Deficiency of the carnitine-acylcarnitine translocase presents with severe hypoketotic hypoglycemia, hyperammonemia, and cardiac arrhytmias in the neonatal period. Plasma carnitine levels are extremely low. Cardiomyopathy related to secondary carnitine deficiency may be present. CPT II deficiency is the most common of this group of disorders (with mostly muscle involvement), although a more severe variant similar to CPT I deficiency has also been reported.171 The carnitine transporter defect may be effectively treated with oral carnitine supplementation, and a rapid reversal of the clinical syndrome (including cardiomyopathy) may be seen over weeks to months.172,173 Defects of other mitochondrial matrix enzymes involved in ␤-oxidation include deficiency of long-chain 3-hydroxy-acyl-coenzyme A dehydrogenase (LCHAD). LCHAD is one of the more severe fatty acid oxidation disorders, with a wide phenotypic spectra and diverse manifestations—including hypoglycemia, cardiomyopathy, skeletal myopathy, hepatocellular disease, pigmentary retinopathy, peripheral neuropathy, and sudden death. In the last decade, many patients with LCHAD deficiency have been reported who were born following pregnancies complicated with severe maternal liver disease (acute fatty liver of pregnancy and HELLP syndrome). Thus, a detailed prenatal history may be key to the diagnosis.174 The vulnerability to maternal complications of pregnancy can also be seen in other fatty acid oxidation disorders. Hypoketotic hypoglycemia can be part of the presentation in defects of ketone body production due to deficiency of 3-hydroxy 3-methylglutaryl-coenzyme A (HMGCoA) lyase and HMG-CoA synthase deficiency. These two disorders can be differentiated by the large urinary excretion of 3-hydroxy-3-methylglutaric acid, which is pathognomonic for HMC-CoA lyase deficiency. Evaluation of suspected errors in fatty acid oxidation should first include determination of the profile of plasma acyl-carnitines by mass spectrometry and measurement of plasma total, esterified, and free carnitine. Most, but not all, of the fatty acid oxidation disorders are associated with specific abnormalities of plasma acyl-carnitines—such as octanoyl-carnitine, which is diagnostically elevated in MCAD deficiency (Table 5-8). Determinations of urinary organic acids with assessment of the presence or absence of dicarboxylic aciduria are also very useful.

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TA B L E 5 - 8

Fatty Acid Oxidation Disorders with Distinguishing Metabolic Markers Disorder

Plasma Acylcarnitines

VLCAD

Tetradecenoyl-

MCAD

Octanoyl

SCAD LCHAD

Butyryl3-Hydroxy-palmitoyl3-Hydroxy-oleoyl3-Hydroxy-linoleoylDodecadienoylButyrylIsovalerylGlutarylMethylglutaryl-

DER ETF and ETF-DH

HMG-CoA lyase

Urinary Acylglycines

HexanoylSuberylPhenylpropionylButyryl-

IsovalerylHexanoyl-

Urinary Organic Acids

Ethylmalonic 3-Hydroxydicarboxylic

Ethylmalonic Glutaric Isovaleric 3-Hydroxy-3-methylglutaric

DER, 2,4-dienoyl-coenzyme A reductase; ETF, electron-transferring flavoprotein; ETF-DH, ETF dehydrogenase; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; LCHAD, long-chain 3-hydroxyacyl-coenzyme A dehydrogenase; MCAD, medium-chain acyl-coenzyme A dehydrogenase; SCAD, short-chain acyl-coenzyme A dehydrogenase; VLCAD, very-long-chain acyl-coenzyme A dehydrogenase. From Stanley CA (1990). Disorders of fatty acid oxidation. In Fernandes J, Bremer E, Saudubray J-M (eds.), Inborn metabolic diseases: Diagnosis and treatment. New York: Springer-Verlag 394-410.

Patients whose disorder cannot be identified by these tests may require further evaluations, including assays of fatty acid oxidation and specific enzyme assays in cultured skin fibroblasts or lymphoblasts. Since the early 1990s, the use of tandem mass espectometry has made newborn screening possible for most fatty acid oxidation disorders based on the acyl-carnitine profile in blood spots. Presymptomatic identification of these individuals can prevent catastrophic events such as sudden death. Direct DNA mutational analysis can be performed for many of these defects, which is particularly useful in MCAD and LCHAD deficiencies in which most cases are due to single common mutations. The primary treatment of disorders of fatty acid oxidation is a devoted avoidance of fasting. For infants younger than 1 year old, 6 to 8 hours of fasting may be sufficient to precipitate an episode. On the other hand, as children become older they appear to be able to withstand periods of fasting of as long as 10 to 12 hours without decompensation. A high index of suspicion and rapid institution of intravenous glucose will often reverse an evolving episode. The presence of hypoglycemia is usually an event that occurs late in the evolution of an episode of metabolic decompensation. High-fat diets should be avoided, although normal amounts of dietary fats do not appear to be toxic. An adjunct approach may involve the use of cornstarch (as used for the treatment of type I glycogen storage disease) in doses of 1.5 to 2 g/kg. This may delay the adaptation from the fed to the fasted state. Riboflavin has been reported to be an adjunct in rare patients.175 The use of carnitine has been advocated.176 However, a systematic assessment of various treatment regimens in these defects has not been available.

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The expansion of neonatal screening programs based on determination of blood acyl-carnitine profiles by mass spectrometry allows earlier identification of these disorders and allows preventive counseling to parents to significantly decrease the occurrence of life-threatening events in these syndromes. Some chemical agents (such as valproic acid, hypoglycin A, and atractyloside) mimic the fatty acid oxidation disorders outlined in Figure 5-12.177 Valproic acid can block ␤-oxidation. Treatment of epilepsy with valproic acid has been associated with Reye-like syndromes, including hypoketotic hypoglycemia.178 Some investigators have advocated treatment with carnitine for infants receiving valproic acid, but there is not universal agreement on this point.178 Jamaican vomiting sickness is caused by the toxin hypoglycin A, a component of the unripened ackee fruit (a Jamaican food staple). This chemical acts as an inhibitor of fatty acid oxidation and can produce a syndrome similar to that of a patient with MCAD deficiency with hypoketotic hypoglycemia.179 Last, rare ingestions of plant atractyloside (such as found in the Mediterranean species Atractylis gummifera) have been associated with a syndrome of vomiting and hypoketotic hypoglycemia.180 Atractyloside is an inhibitor of mitochondrial oxidative phosphorylation and prevents translocation of adenine nucleotides across the mitochondrial membrane.181

Drug-Induced Hypoglycemia Drug-induced hypoglycemia is rare in neonates and young infants. The administration of medications to infants may represent the Munchausen by proxy syndrome. Rarely, it may represent a pharmaceutical dispensing

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error with substitution of an insulin secretogogue (e.g., glyburide) for another medication. Hypoglycemia can also occur as a side effect of therapy (i.e., beta-blockers). Toxic substances such as ethyl alcohol in various beverages or salicylates may be accidentally or deliberately used in detrimental ways in infants. As noted in the discussion of hyperinsulinism, injectable insulin is readily available—as are potent oral hypoglycemic agents. Occasionally, an infant will present with unexplained hypoglycemia and not fit readily into any diagnostic algorithm. In these rare instances, a careful history of drug or alcohol availability in the environment must be undertaken and pharmacy dispensing errors considered.

Defects of Glucose Transporters

neurologic development. The genetic inheritance of the condition behaves as an autosomal dominant trait, although spontaneous heterozygous mutation in the SLC2A1 gene can present sporadically in families.182,183 The phenotypic presentation is variable, from the classic presentation of developmental encephalopathy with seizures to nonclassic presentations with mental retardation, dysarthric speech, and intermittent ataxia without clinical seizures. It can also manifest as a movement disorder characterized by choreoathetosis and dystonia.183 The biochemical hallmark is the finding of hypoglycorrhachia (low cerebrospinal fluid glucose concentration) despite normal plasma glucose concentrations. Treatment efforts have been based on providing alternative brain fuel sources by a ketogenic diet.9,184 The ketogenic diet effectively controls the seizures and other paroxysmal activities, but is has less effect on the cognitive function.

GLUT1 DEFICIENCY GLUT1 deficiency has now been diagnosed in a growing number of patients since the first reports in 1991.9 The cloning of the human SLC2A1 (encoding GLUT1) gene confirmed the initial speculation involving a defect in glucose transport across the blood-brain barrier as the underlying mechanism for the classic presentation of infantileonset epileptic encephalopathy associated with delayed

GLUT2 DEFICIENCY Fanconi-Bickel syndrome (characterized by hepatomegaly, glucose and galactose intolerance, and renal tubular dysfunction) is due to a recessive mutation of the GLUT2 plasma membrane transporter for glucose (encoded by SLC2A2).185 GLUT2 is expressed in liver, renal tubular cells, enterocytes, and pancreatic beta cells. The clinical

TA B L E 5 - 9

Differential Diagnosis of Hypoglycemia in Neonates and Infants PLASMA FUELS AT END OF FAST (mmol/L) Length of Fast (hr)

Glucose

Lactate

Free Fatty Acids

␤-Hydroxybutyrate

Normal Infants Endocrine System Hyperinsulinism Cortisol deficiency GH deficiency Panhypopituitarism

24-36

2.8

0.7-1.5

1.5-2.5

2.0-4.0

Varies 10-16 10-16 10-16

2.8 2.8 2.8 2.8

N N N N

⬍1.5 N N N

⬍2.0 N N N

Epinephrine deficiency (beta-blocker) Glycogenolysis Debrancher deficiency (GSD3) Phosphorylase deficiency (GSD6) Phosphorylase kinase deficiency (GSD9) Glycogen synthase deficiency (GSD0) Gluconeogenesis Glucose 6-phosphatase deficiency (GSD1a and 1b) Fructose 1,6-diphosphatase deficiency Pyruvate carboxylase deficiency Lypolysis Congenital lipodystrophy, familial dysautonomia, beta-blockers Fatty Acid Oxidation Carnitine transporter, CPT-1, Translocase, CPT-2, VLCAD, MCAD, SCAD, LCHAD, MADD, HMG-CoA synthase, HMG-CoA lyase deficiency

10-16

2.8

N

1.5 cm) is an objective indicator of overall estrogen effect.78 Androgen levels are appropriate for the stage of pubarche in true precocity. Third-generation monoclonal-antibody-based immunoassays for gonadotropins are important to the differential diagnosis. Daytime basal and GnRH-stimulated peak LH levels exceding 0.6 and 6.9 U/L in girls have been found to be respectively 70 and 92% sensitive for the

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diagnosis of central precocious puberty.468 FSH levels are not as helpful diagnostically. A GnRH agonist test yields similar diagnostic information and permits assessment of the ovarian secretory capacity. In response to these tests, children with unsustained pseudopuberty as variants of normal will have a minimal gonadotropin response— whereas children with gonadotropin-independent precocity will have suppressed responses.81,82,560 Demonstration of a sleep-related rise of plasma LH is an alternative diagnostic procedure. Table 14-6 gives criteria for the diagnosis of the rapidly progressive type of complete sexual precocity that requires treatment.561,562 Determination of the plasma androgen pattern is useful in discriminating among the causes of premature pubarche and virilization. Androgen determinations are most accurately performed by a specialty laboratory.339 Premature adrenarche is characterized by a pubertal level of DHEAS, whereas plasma testosterone and androstenedione are at most marginally elevated above the prepubertal range. A greatly elevated level of DHEAS is characteristic of adrenal tumors. Androstenedione and 17-hydroxyprogesterone levels are disproportionately elevated compared to testosterone or DHEAS levels in congenital adrenal hyperplasia and many ovarian tumors. Dexamethasone suppression testing and other means of determining the source of androgen excess are discussed in the section on hyperandrogenemia. Ultrasonography is indicated to rule out abdominal or pelvic masses when feminizing or virilizing disorders are suspected. The ovaries of girls with true sexual precocity resemble those of normal pubertal girls.75,562 A cyst of 10 mm or more in diameter is usually due to a transient preovulatory follicle. However, the differential diagnosis of a persistant cyst or of multicystic ovaries includes McCune-Albright syndrome,534 tumor,563 and premature ovarian failure.564 Magnetic resonance imaging (MRI) of the hypothalamic-pituitary area is indicated in rapidly progressive true sexual precocity, especially for those less than 6 years old or those at risk of organic causes by

TA B L E 1 4 - 6

Laboratory Criteria for Rapidly Progressive Complete Precocious Puberty • Sex hormone level pubertal (diurnal early) • Estrogen (girls, cyclical): E2 9 pg/mL, vaginal cornification • Testosterone (boys): 20–1200 ng/dL • DHEAS normal for age height age

• Sex hormone excess is sustained: • Bone age height age chronologic age • LH and FSH pubertal • Sleep-associated LH rise initiates puberty • Basal: LH.0.6 and FSH.2.0 IU/L or more (monoclonal RIA)␣ • Post-GnRH LH 4.2 IU/L␣ • Exclude: tumor, hypothyroidism, gonadotropin-independent precocity a

. Exact values vary among laboratories. DHEAS, dehydroepiandrosterone sulfate; E2, estradiol; FSH, folliclestimulating hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; and RIA, radioimmunoassay.

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571

virtue of their underlyng condition or neurologic symptoms and signs.558,565 The situation of a girl presenting with the onset of breast development or pubic hair between 6 and 8 years of age warrants special consideration. Breast and pubic hair development have been reported in 15 to 20% of U.S. black girls in office practice, in contrast to less than 5% of white girls.483 Subsequent studies have indicated that breast development between 7 and 8 years of age may be normal in blacks and Hispanics, whereas sexual pubic hair is not (Table 14-4). However, pubertal development in girls in the 6- to 8–year-old age range may be associated with pathology—with excessive adiposity an important factor in many.204,205,464,566 Those with rapidly progressive precocity benefit from GnRHag therapy.567-569 On the other hand, many girls in this age range have slowly progressive precocity—with a normal timing of menarche—and are not at risk of short adult stature. Such girls achieve no increase in stature from GnRHag therapy.503,569,570 We conclude that a less comprehensive investigation may be warranted in selected girls presenting with thelarche between 6 and 8 years of age. For most such girls, a complete history and physical exam (including obesity evaluation and a bone age determination) may be all that is needed—along with careful longitudinal follow-up.472,503,570 However, 6- to 8-year-old girls with a suggestion of rapidly progressive or excessive androgenization or feminization, neurologic symptoms, linear growth acceleration, or significant bone age advancement should be more completely evaluated (as outlined previously).

Management The guidelines for management are to rule out an organic disorder that requires treatment in and of itself and to ascertain whether sexual precocity is either rapidly compromising height potential or resulting in important secondary emotional disturbances in the child. Intracranial lesions must be treated by appropriate measures, such as neurosurgery or irradiation. Shunting for hydrocephalus may stop the precocity. Granulosa cell tumors confined to the ovary have a good prognosis for cure by unilateral oophorectomy. Recurrence of tumor may occur up to 20 years after the initial operation, however. Biopsy of the opposite ovary is indicated in unilateral ovarian neoplasms. Compensatory ovarian hypertrophy can be expected at any age after removal of a single ovary.571 The only permanent physical complication of true isosexual precocity, all else being normal, is short adult height. Excessive sex hormone production in the first decade of life causes early maturation of the epiphyses, resulting in their premature closure. About half the girls with this disorder reach an adult height of 53 to 59 inches, and the remainder are more than 60 inches tall.484,503 The mismatch between physical, hormonal, and psychological development may cause behavior changes ranging from social withdrawal to aggression or sexuality. However, frank behavioral problems are unusual in girls and are therefore by themselves seldom indications for treatment.

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When true sexual precocity is rapidly progressive to the point of significantly compromising height potential and its cause cannot be directly alleviated, the best mode of treatment is with GnRH agonists. The downregulating effect of these agents on pituitary gonadotropin release inhibits gonadotropin secretion. Suggested criteria for the use of these drugs are presented in Table 14-7.561 The most widely used agents in the United States are leuprolide acetate (ordinarily given as Lupron depot/ ped 7.5–15 mg/mo IM) and nafarelin acetate (Synarel 800 µg bid IN). A long-acting histrelin implant was recently introduced.571a These doses are larger than the usual adult dosage in order to avoid worsening the status by an agonist effect. Dosage can be adjusted later as necessary. It is unclear whether the longer-acting depot forms of leuprolide are as effective.572 Treatment is adequate if the estradiol level becomes prepubertal573 or LH is below 6.6 U/L 2 hours after GnRH agonist574 1 month after institution of therapy. A withdrawal period may occur at that time, but none should be expected thereafter. Puberty and the pubertal growth spurt are arrested. Concomitantly, epiphyseal closure is delayed and adult height potential is improved because a type of catch-up growth occurs in which height age catches up to bone age. Adult height is greatest when treatment is started soon after onset at an early age, yielding an average height gain above pretreatment height prediction of about 1.4 cm for each year of therapy.503, 568,569 Prolonging treatment beyond a chronological or bone age of 12.0-12.5 years of age generally leads to little further increase in adult height potential, regardless of the prediction of residual height potential from bone age. Coincident GH-deficiency must be treated for optimal growth.575 GH-sufficient patients with central precocity who are started on treatment relatively late and whose height velocity falls below the prepubertal normal range after 2 to 3 years appear to gain an average of 2 cm per year when GH therapy is added.576 Treatment is ordinarily continued until the normal age for puberty to begin.568

TA B L E 1 4 - 7

Suggested Indications for Gonadotropin-Releasing Hormone Agonist Therapy of Precocious Puberty Complete precocious puberty Pubertal sex hormone levels (in sensitive assays) plus Abnormal height potential (predicted adult height falling significantly or below 5th percentile) or Psychosocial considerations, individualized Menses in the mentally or emotionally immature Other (e.g., behavorial or emotional disturbance) Modified from Rosenfield RL (1994). Selection of children with precocious puberty for treatment with gonadotropin releasing hormone analogs. J Pediatr 124:989.

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Use of the depot form of GnRH agonist is complicated by sterile abscesses at injection sites in about 5% of cases. Anaphylaxis is a rare complication.577 No other serious side effects have come to light, but long-term safety remains to be established. The potential risk of bone demineralization has not proven to materialize after GnRH agonist therapy of precocious puberty.504 Girls with slowly progressive idiopathic puberty of onset between 6 and 8 years of age or early fast puberty between 8 and 9 years of age tend to be tall at the onset of puberty, follow an advanced growth pattern, and reach their target height without GnRH agonist therapy.503,570,579-581 Therefore, this treatment is only indicated if there are other compelling reasons to slow the pace of puberty. Those who are obese may have slower pace of puberty if insulin resistance is ameliorated.581a Medroxyprogesterone acetate (Depo-Provera) is useful for stopping menses and as a contraceptive in mentally retarded girls in whom preservation of height potential is not important. It is begun in a dosage commencing at 50 mg/month intramuscularly. Doses as high as 400 mg/month have been used, although Cushingoid side effects may be observed at this level.582 Although this treatment reverses some of the physical changes of premature puberty, it does not reverse the inordinately rapid maturation of the skeleton—possibly because of its inherently weak androgenicity. In addition, use of medroxyprogesterone acetate is associated with a loss of bone mineral density—which must be considered if longterm use is being contemplated.583 A variety of drugs have been used off-label to treat gonadotropin-independent precocity. Antiestrogen and aromatase treatment for McCune-Albright syndrome has met with varying degrees of success.584,585 Ketoconazole, an antifungal agent that inhibits 17,20-lyase activity and other steroidogenic enzymes, has been used to treat a similar condition in boys586 and might be useful in girls as well. Gonadotropin-releasing hormone agonist treatment may be necessary for those in whom true puberty becomes superimposed because the bone age has reached a pubertal level.194,195,587 The fibrous dysplasia of McCuneAlbright syndrome may be amenable to treatment with bisphosphonates,588,589 although this is not a suitable longterm treatment in childhood.590 Patients with premature thelarche or pubarche as variations of normal are counseled as follows. The child’s early development seems to be a matter of a normal stage of puberty occurring early. It is due to an incomplete slow type of puberty or to increased sensitivity to the trace levels of hormones normally present in childhood. Feminization with breast development and eventual menstruation can be expected to occur at an appropriate age. No treatment is indicated. To exclude subtle sex hormone excess or eventual anovulatory syndromes, long-term follow-up is advisable. In addition to dealing with the physical consequences of true isosexual precocity, the physician must be ready to help the family and child cope with the psychological problems that come with early physical maturation. The doctor can help the family by explaining that even though their child looks older and more mature than other children of the same age the child will not behave

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more maturely. The libido of young children with precocity is not increased. The family should be advised to take some precautions to downplay their child’s development (for example, in the choice of clothing and swimsuits). Friendships with children a bit older will help shorten the time affected children spend in social limbo. This may be more easily said than done, however, because the intellectual and social maturity of these patients is not advanced. Early on, any children with these disorders tend to be withdrawn because they feel that they are different from their peers. Later on, they tend to enter into romantic relationships early. It is important to remind the family and child that in a few years the child will not be unique from the standpoint of sexual development.484 The following books may be helpful in explaining precocious puberty: for children, What’s Happening to Me?, by Peter Mayle (Lyle Stuart, Inc., Secaucus, NJ, 1973); for parents, Sex Errors of the Body, Second Edition, by John Money (Paul H. Brookes Publishing Co., Inc., Baltimore, MD, 1994).

TA B L E 1 4 - 8

Differential Diagnosis of Amenorrhea Abnormal Genital Structure • Ambiguous genitalia • Intersex • Pseudointersex • Aplasia␣ • Hymenal • Vaginal • Müllerian • Intersex • Endometrial atrophy Anovulatory Disorders Hypoestrogenism, FSH Elevated • Primary ovarian failure • Congenital - Gonadal dysgenesis - Steroidogenic blocks - Resistant ovaries • Acquired - Oophorectomy - Oophoritis - Radiotherapy or chemotherapy • Bioinactive gonadotropin Hypoestrogenism, FSH Not Elevated • Primary ovarian failure • Complete if BA 11 yr␣ • Incomplete if BA 11 yr • Delayed puberty • Constitutional delay␣ • Growth-retarding disease • Hypogonadotropic hypogonadism • Congenital • Acquired - Organic - Functional • Virilization Estrogenized, FSH Not Elevated • Hypothalamic anovulation • Hypothalamic amenorrhea • Athletic/psychogenic amenorrhea • Post-pill amenorrhea • Nonhypothalamic extraovarian disorders • Pregnancy • Obesity or undernutrition • Cushing syndrome • Hypothyroidism • Hyperprolactinemia • Ectopic gonadotropin secretion • Hyperandrogenism

HYPOGONADISM Causes If hypogonadism is complete and present prepubertally, it causes sexual infantilism in females or disorders of sexual differentiation in genetic males. If it is slightly less severe or of onset in the early teenage years, it may permit too limited a degree of feminization to permit the onset of menses at a normal age (primary amenorrhea). Milder, partial, or incomplete forms of hypogonadism may cause secondary amenorrhea or oligomenorrhea.489a At its mildest, hypogonadism may present with the anovulatory symptoms of dysfunctional uterine bleeding or with excessively frequent periods due to short luteal phase. Consequently, disorders causing hypogonadism appear in the differential diagnosis of disorders of sexual differentiation, sexual infantilism, failure of pubertal progression, and menstrual irregularity. The causes of hypogonadism are listed in the differential diagnosis of amenorrhea in Table 14-8. Primary Ovarian Failure. Primary ovarian failure is characterized by high levels of gonadotropins, particularly FSH. Two exceptions exist to this rule. First, the gonadotropins may not be elevated until CNS maturation has reached a pubertal stage—as indicated by a bone age of approximately 10 to 11 years (Table 14-9).591 Second, patients with partial ovarian failure (as is frequent in women during the menopausal transition) do not have high baseline gonadotropin levels.592-594 FSH may hyperrespond to GnRH and estrogen may hyporespond to GnRH agonist challenge. It seems as if relatively few ovarian follicles (too few to permit the cyclic emergence of preovulatory follicles) suffice to prevent the characteristic rise in basal FSH levels. Primary ovarian failure may occur before or during puberty (causing primary amenorrhea) or after puberty has occurred, causing secondary amenorrhea. The latter is termed premature ovarian failure and resembles premature menopause except that about half of cases sometimes ovulate.595,596

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573

a

. Cause only primary amenorrhea. BA, bone age; and FSH, follicle-stimulating hormone.

Gonadal dysgenesis due to deficiency of genes on the X-chromosome is the most common cause of primary ovarian failure. It is usually due to a relatively large scale deletion of X-chromosomal material, which is associated with a characteristic, but variable, phenotype and is termed Turner syndrome (see Chapter 15). Fetuses with a 45,X karyotype have a normal number of oocytes in the ovary at mid-gestation, but a drastic reduction in the number of follicles,30 which appears to cause gonadal

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TA B L E 1 4 - 9

Bone Age in Workup of Sexually Infantile Girls with Normal FollicleStimulating Hormone Level Bone Age (Years)

Primary hypogonadism Delayed puberty Gonadotropin deficiency

⬍11

11–13

⬎13

Yes Yes Yes

Yes Yes

Yes

Based on Rosenfield RL, Barnes RB (1993). Menstrual disorders in adolescence. Endocrinol Metab Clin North Am 22:491.

streaks via an accelerated rate of apoptosis. However, the gonadal dysgenesis (like other features of the syndrome) is often incompletely expressed.596 Thus, Turner syndrome should be considered in all girls with primary hypogonadism or secondary amenorrhea whether or not they have the typical stigmata of Turner syndrome Specific loci on the X-chromosome are associated with primary ovarian failure. Xp11.2 harbors BMP15, a specific ovarian differentiation factor, a heterozygous mutation of which is a rare cause of gonadal dysgenesis. Xq harbors two independent loci in addition to the fragile X premutation, which is associated with about 7-14% of primary ovarian failure.595,596 Other genes have been incriminated in the ovarian failure of mouse models.597 Gonadal dysgenesis also results from 46, XY complete gonadal dysgenesis and certain forms of autosomal aneuploidy.432,595,598 A variable degree of ovarian dysgenesis occurs in autosomal trisomy 21. Delayed menarche, anovulatory cycles, and primary gonadal failure are occasionally seen.599 However, pregnancy has been reported. Trisomic offspring are common.600 Oocytes are virtually absent in trisomies 13 and 18. Ovarian dysgenesis also occasionally occurs as part of the Denys-Drash syndrome due to a WT-1 mutation.601 There are associations of primary ovarian failure with cerebellar ataxia.602,26 Other autosomal genetic disorders causing premature ovarian failure include blepharophimosis, galactosemia, leukodystrophies, and myotonia dystrophica.597,604 Physical or medical destructive injury to the ovary is a common cause of primary ovarian failure. Irradiation and chemotherapy for childhood neoplasia are becoming increasingly frequent causes of primary ovarian failure now that life is effectively prolonged.604,605 Ionizing radiation and alkylating agents damage DNA whether or not a cell is replicating.606 A radiation dose of 20 Gy or more to the ovaries causes acute ovarian failure in about 85% of children and adolescents.607 A cumulative cyclophosphamide dose of about 100 mg/kg causes equivalent damage. Fertility is even more rare when these modalities are combined. Prepubertal girls are about half as sensitive to these therapies as postpubertal females: among girls who receive 1-10 Gy, acute ovarian failure develops in about 10% of girls under 13 years, but in 25% of those 13. After prepubertal chemotherapy and radiotherapy for leukemia, half of patients can be anticipated to enter puberty and menstruate regularly, and one-quarter to have normal pituitary-ovarian function 7 years later.608 Some with early

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hypergonadotropinism will experience ovarian recovery. However, all will develop premature menopause because of the reduced number of oocytes. Several non-alkylating chemotherapies are also gonadotoxic.609,610 However, data are scarce and the interactions among various classes of chemotherapeutic agents is poorly understood. As a consequence of gonadotropin elevation when gonadal failure begins, puberty may progress rapidly.516 Sterilization by irradiation can be obviated by transposing the ovaries out of the irradiation field if possible. Administration of a GnRH agonist prior to cyclophosphamide administration may decrease ovarian injury.606 Mumps oophoritis is a classic but rare cause of ovarian failure. Functional ovarian failure can arise from autosomal-recessive ovarian mutations of the LH or FSH receptor.595,603,611,612 These have been associated with a spectrum of defects ranging from primary amenorrhea to oligomenorrhea. Ovarian histology in typical resistance to gonadotropin action (Savage syndrome) shows a normal number of primordial follicles but a paucity of growing follicles. Partial gonadotropin resistance is common in the Albright osteodystrophy form of pseudohypoparathyroidism, in association with the generalized defect in G-protein signal transduction.613 The carbohydrate-deficient glycoprotein syndrome reduces gonadotropin bioactivity and causes hypergonadotropic hypogonadism.614 Autoimmune oophoritis is the basis of approximately half of spontaneous premature ovarian failure,618 though estimates vary from 5-85% in various series.595,615,616 It is diagnosed by its association with any of a variety of autoimmune endocrine or nonendocrine disorders, manifest or subclinical, that have in common defects in T cell suppressor function. Autoimmunity may be directed against the granulose cell, oocyte, or theca cell. The clinical picture may resemble relatively selective resistance to FSH or, less frequently, to LH.617 The latter results from lymphocytic infiltration of theca with sparing of primordial follicles. These patients have autoantibodies to steroidogenic cells and have or are at risk for adrenal failure. Usually these antibodies are directed against21-hydroxylase, less frequently to side chain cleavage or 17-hydroxylase, seldom to 3
-hydroxysteroid dehydrogenase. Replacement glucocorticoid therapy may temporarily ameliorate the immune oophoritis in such cases.618 A case with autoantibodies to testosterone has been reported.619 Autoimmune gene regulator (AIRE) gene mutations have been identified as causative of type 1 polyendocrine failure. Ultrasonographic and histologic findings are variable in premature ovarian failure and include large or small ovaries, inactive or polyfollicular ovaries, loss or preservation of primordial follicles, and infiltration by lymphocytes or plasma cells. Functional gonadal failure can also result from specific autosomal-recessive defects in the biosynthesis of androgens and estrogens (or aromatase deficiency, a virilizing disorder). Enzyme blocks can cause gonadal insufficiency in genetic males who are phenotypic females. This occurs in lipoid congenital adrenal hyperplasia (StAR and side chain cleavage mutations, Figure 14-23), 17-hydroxylase deficiency, 17,20-lyase deficiency, 3
-HSI deficiency, and

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17
-HSD3 deficiency. All but the latter are associated with congenital adrenal hyperplasia. Congenital lipoid adrenal hyperplasia is unique in that underlying StAR deficiency has too little direct impact on ovarian function to interfere with the early phases of puberty. However, the gradual buildup of intraovarian lipid deposits resulting from enzyme deficiency (a “second hit”) causes ovarian damage with anovulation and late ovarian failure.620,621 Gonadotropin Deficiency. Congenital gonadotropin deficiency can occur in association with cerebral, hypothalamic, or pituitary dysfunction or as an isolated defect.622 Congenital defects in hypothalamic-hypophyseal formation may be associated with midline facial defects. Congenital hypothalamic dysfunction may be associated with other neurologic or endocrine dysfunction, such as in the Prader-Willi syndrome (congenital hypotonia and neonatal failure to thrive followed by hypothalamic obesity, sometimes with hypopituitarism) or the LaurenceMoon-Biedl syndrome (retinitis pigmentosa, obesity, mental deficiency). The autosomal-recessive form of congenital combined pituitary hormone deficiency due to PROP1 mutation is associated with gonadotropin deficiency. Leptin- and leptin-receptor-inactivating mutations cause gonadotropin deficiency in combination with extreme obesity. Gonadotropin deficiency may be associated with anosmia (olfactory-genital dysplasia or Kallmann syndrome).182 This syndrome is one-fifth as frequent in females as in males.623 Mutations in the KAL-1 gene on the pseudoautosomal region of the X-chromosome cause the X-linked form, are highly penetrant, and account for 10% of cases. This mutation causes deficiency of anosmin-1. Inactivating mutation of the fibroblast growth factor receptor-1c isoform accounts for another 10% of cases, and these are inherited as an autosomal-dominant trait with variable penetrance and occasional association with cleft palate and facial defects. FGFR-1c signaling has been hypothesized to occur at least in part through anosmin-1. Isolated gonadotropin deficiency arises from GnRH receptor mutations in about half of autosomal recessively inherited cases.624 Partial gonadotropin deficiency can rarely result from mutation of the G-protein–coupled receptor GPR54.186 The degree of hypogonadism is variable, even within a family, with delayed puberty and delayed menarche as one presentation.625-627 Isolated hypogonadotropic hypogonadism has also been reported in a woman homozygous for a nonsense mutation of the Xlinked DAX1 gene, which was associated with adrenal insufficiency in her brothers.628 Isolated FSH deficiency due to mutation in the
-subunit has been reported to cause primary amenorrhea in association with a unique test panel of low FSH, elevated LH, and low testosterone levels. Acquired gonadotropin deficiency can be a consequence of tumors, trauma, autoimmune hypophysitis,629,630 degenerative disorders involving the hypothalamus and pituitary,631 irradiation,632 or chronic illness.633 Pituitary adenoma, craniopharyngioma, and dysgerminoma are the most common neuroendocrine neoplasms responsible in children. Most “nonfunctioning” pituitary adenomas are gonadotrope adenomas that secrete gonadotropin subunits in response to thyrotropin-releasing hormone.634 A case of hypothalamic tumor is presented

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575

Figure 14-39 Computed tomography of the brain of a 16-year-old girl with hypothalamic astrocytoma. The low-density tumor mass (arrows) extends superiorly from the hypothalamus, obliterates the third ventricle, and partially compresses the frontal horns of the lateral ventricles (particularly the right). This patient presented with secondary amenorrhea. Menarche had occurred at age 13, and menses were normal until 15.3 years. The patient then became amenorrheic in association with ethargy, episodic headaches, polyuria, and weight gain—despite little change in appetite. Physical examination was negative. The skull radiograph, electroencephalogram, visual fields, and serum prolactin and thyroxine levels were normal—and urine-specific gravity was 1.016. After biopsy of the cyst wall, studies revealed her to have gonadotropin, growth hormone, and partial antidiuretic hormone deficiencies.

with Figure 14-39. Pinealomas most commonly cause sexual infantilism. They may act by secreting an inhibitory substance, rather than by compressing key areas of the hypothalamus.514 Anorexia nervosa is the prototypic form of eating disorder, a common cause of hypogonadotropinism in teenagers. It is a syndrome of undernutrition due to voluntary starvation with a particular psychological dysfunction that results in amenorrhea.635-637 Patients uniformly consider themselves too fat in the face of objective evidence that they are underweight. The psychiatric criteria that distinguish this disorder from food faddism and fear of obesity consist of refusal to gain or maintain body weight to a minimally normal level for height and age (less than the 15th percentile; body mass index  17.5 for postmenarcheal cases) with all of the following being present: intense fear of gaining weight or becoming fat, even though underweight; an inaccurate perception of body weight, such that they have a disturbance in the way that body weight, size, or shape is experienced; undue influence of body shape and weight on self-evaluation or denial of the seriousness of current low body weight; and amenorrhea for 3 or more months in postmenarcheal females.

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Bulimia nervosa, the binge-eating/purging variant eating disorder, is similar in the overevaluation of body shape and weight and the use of extreme weight control behaviors. Physical activity tends to be high. These disorders may be manifest at an early stage as atypical eating disorders, before weight or amenorrhea criteria are met or when the binge is subjective. The cognitive defect that weight can serve as the predominant value in judging self-worth is central to anorexia nervosa. In contrast to other depressive individuals, these patients are generally content with themselves in the areas of intellectual and vocational achievement. The pathogenesis is multifactorial. It involves a genetic predisposition. Concordance rates for the anorexic type are about 50% for monozygous twins, compared with about 5% for dizygotic twins. Many other risk factors have been implicated. Familial factors also include eating disorders of any type, depression, substance abuse, and adverse family interactions. Premorbid experiences (such as sexual abuse or social pressures) or premorbid characteristics (such as low self-esteem, compulsiveness, and perfectionism) are also important. Dieting meets a need

for approval in our culture, with its emphasis on dietary restriction and thinness as goals for women. Anorexia is often precipitated in vulnerable children by a new experience (such as puberty, leaving home, or beginning college) or by adverse life events. The disorder is perpetuated by the complications of starvation, such as depression and reduced gastric emptying. The onset tends to be at 12 years of age or later. Earlier onset is associated with growth arrest, delay of puberty, and primary amenorrhea.638 GH deficiency is common.639,640 The medical complications of anorexia nervosa are serious. The risk of death is approximately tenfold increased. Electrolyte imbalance, hypoglycemia, cardiovascular instability, bone marrow hypocellularity predisposing to silent infection, and renal failure account for about half of the mortality. Suicide accounts for the rest. The weight changes leading to cessation or restoration of menstrual cycles are in the range of 10% to 15% of body weight. Recovery is associated with achieving a critical level of body fat stores above the 10th percentile ( 20% body fat) (Figure 14-40) at a BMI averaging 20.641,642 There is an inverse relationship between body

Figure 14-40 Percentiles of fatness (diagonal lines) for white girls at menarche (left) and after menarche (right) equated with computed percentiles of total water as a percentage of total body weight. The minimal weight necessary at a particular height for the onset or maintenance of menses is very close to the 10th percentile of fatness on these respective charts. Data for anorexia nervosa cases are shown on the right-hand chart: • at presentation; x at resumption of menses. [From Frisch RE, McArthur JW (1974). Menstrual cycles: Fatness as a determinant of minimum weight for height necessary for their maintenance or onset. Science 185:949. Copyright © by the American Association for the Advancement of Science.]

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weight and the maturity of gonadotropin release in these patients. The 24-hour pattern of gonadotropin release tends to be immature (prepubertal or pubertal), and the diurnal LH pattern becomes mature upon recovery from undernutrition.643 Luteinizing hormone pulsatility is low, and may be restored by opiate antagonists.88 The gonadotropin response to GnRH and ovulatory response to clomiphene citrate are blunted in the malnourished state and become normal with weight gain to about 80% of ideal.644,645 Leptin levels are significantly decreased and are a major contributor to the gonadotropin deficiency and to changes in the thyroid and GH axes.646 Mild hypercortisolism is frequent and may contribute to the anovulation by mechanisms discussed further under the section on hypothalamic anovulation.647 Afternoon ACTH and cortisol levels are significantly higher than normal, and the response to CRH is significantly lower than normal. In contrast to Cushing syndrome, DHEAS levels tend to be blunted as a consequence of undernutrition.648 A fundamental neuropsychological flaw or hypothalamic disturbance649 seems necessary to explain the high incidence of GH deficiency, why some patients become amenorrheic before losing weight, and why about half of the cases remain amenorrheic after treatment. The serotonergic systems implicated in the regulation of feeding and mood seem to remain altered even after weight restoration. The authors favor the concept that these psychological problems lead to amenorrhea only in women predisposed to it by a unique preexisting hypothalamic dysfunction. Evidence has recently been obtained for marked individual differences in reactivity of the neuroendocrine system to stress.650 A number of features attributed to hypothalamic dysfunction, such as cold intolerance, may be due to the subtle hypothyroid state secondary to the malnutrition.647 Serum triiodothyronine levels are consistently low, serum thyroxine levels tend to be lower than average (although usually within normal limits), the pattern of TSH release indicates TRH deficiency, and the state of deep tendon reflexes and metabolism is consistent with hypothyroidism. Hypothyroidism may in part complicate malnutrition as a consequence of the interference with IGF-1 generation. Low IGF-I initiates GH excess, compensatory somatostatin release, and subsequent inhibition of the thyrotropin response to thyrotropin-releasing hormone. Undernutrition also diverts the generation of thyroxine metabolites away from triiodothyronine toward reverse triiodothyronine. Hyperprolactinemia can cause functional gonadotropin deficiency.651 Galactorrhea is present in about half of the patients, particularly those with residual estrogen production. The causes of hyperprolactinemia are diverse, including hypothalamic or pituitiary disorders, drugs, hypothyroidism, renal or liver failure, peripheral neuropathy, stress, autoimmunity, macroprolactinemia, and idiopathology.652-654 Elevated serum prolactin levels occur with a variety of tumors that cause functional or anatomic pituitary stalk section, thereby preventing inhibitory pituitary control. About a third of hyperprolactinemic women have an identifiable pituitary adenoma. Prolactinomas less than 1 cm in diameter (microadenomas) cause no problems

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due to local extension. Prolactinoma may be associated with multiple endocrine neoplasia type 1.655 In about a quarter of adult cases, the malfunction is due to the ingestion of drugs such as phenothiazines, estrogen, or cocaine.656 Considerable hyperprolactinemia is idiopathic. Decreased sensitivity to dopaminergic inhibition may underlie such cases.657 Macroprolactinemia is due to a variant molecule or autoantibody formation. In this situation, direct immunoassay indicates elevated levels of prolactin. However, the biologically available or active prolactin level is normal. Thus, there is no physiologic consequence to the macroprolactinemia. Hyperprolactinemia results in luteinizing hormone pulses that tend to be infrequent and LH secretion that is variable in response to GnRH.658 Selective prolactin excess causes variable degrees of gonadotropin deficiency, ranging from severe to partial (hypothalamic amenorrhea). Adrenal hyperandrogenism, hirsutism, and seborrhea are common.266 Frank virilization as a result of very high androgen levels suppresses gonadotropin levels and thus causes defeminization. However, the more common moderately hyperandrogenic disorders discussed in material following are associated with normal estrogenization.

Differential Diagnosis The differential diagnosis of hypogonadism is included in Table 14-8. Investigation should be begun for hypogonadism when the onset of puberty has not begun by the chronologic or bone age of 13 years, if puberty has not progressed as indicated by failure of menses to occur within 4.5 years of the onset of puberty, or if secondary amenorrhea or oligomenorrhea has persisted for 2 years. A family history of delayed puberty is compatible with the delay being constitutional rather than having an organic basis. The history should include a thorough past medical history and systemic review, including systemic, intracranial, visual, olfactory, emotional, abdominal, and pelvic symptoms. It should be kept in mind that chronic endocine, metabolic, or systemic disease of almost any type can lead to delayed puberty. Upon examining the patient, the height and weight should be carefully measured and growth rate and appropriateness of weight for height determined (Figure 14-40). Careful categorization of the stage of breast and sexual hair development are essential. Examination of the mature breast should include an attempt to express milk from the ducts to the nipple. The finding of a structural genital abnormality may indicate that amenorrhea is due to abnormal genital tract development, whereas clitoromegaly659 is a clue to a virilizing disorder. Neurologic examination should include evaluation of eye movements, visual fields, and optic fundi—as well as a search for anosmia and midline defects. An algorithmic approach to the workup of patients with menstrual disorders is shown in Figures 14-41 through 14-43.660 The laboratory workup depends on the degree of estrogenization, as initially assessed from the stage of breast development. It includes a bone age radiograph in adolescents who are not sexually mature

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Figure 14-41 Differential diagnosis of primary amenorrhea. (a) Prime among the causes of primary amenorrhea are growth-retarding or -attenuating disorders. In the absence of specific symptoms or signs to direct the workup, laboratory assessment for chronic disease typically includes bone age radiograph if the adolescent is not sexually mature and a chronic disease panel (complete blood count and differential, sedimentation rate, comprehensive metabolic panel, celiac panel, thyroid panel, cortisol and insulin-like growth factor-I levels, and urinalysis). (b) Breast development ordinarily signifies the onset of pubertal feminization. However, mature breast development does not ensure ongoing pubertal estrogen secretion (see Figure 14-42 and 14-43). (c) Underweight is defined as body fat 20% or less of body mass. Although this generally corresponds to BMI  10th percentile, BMI may not accurately reflect body fat in serious athletes (who have a disproportionately greater muscle mass) or bulimia nervosa. (d) Low body fat is associated with amenorrhea in girls underweight (e.g., anorexia nervosa, a symptom complex consisting of amenorrhea, voluntary starvation, and a self-delusional disturbance in the perception of body fatness), not necessarily underweight (bulimia), and from athletic activity out of proportion to caloric intake (serious athletes). (e) FSH is preferentially elevated over LH in primary ovarian failure. The most common cause of primary amenorrhea due to primary ovarian failure is gonadal dysgenesis due to Turner syndrome, but acquired causes must be considered (such as cytotoxic therapy). The workup of primary ovarian failure is considered in detail in the next algorithm (Figure 14-42, secondary amenorrhea and oligomenorrhea). Lack of FSH elevation virtually rules out primary ovarian failure only when the bone age is appropriate for puberty (11 years or more). (f) A low LH level is more characteristic of delayed puberty and gonadotropin deficiency than a low FSH level. Congenital gonadotropin deficiency is closely mimicked by the more common extreme variation of normal, constitutional delay of puberty (Table 14-10). (g) History and examination may yield clues to the cause of hypogonadotropic hypogonadism, such as evidence of hypopituitarism (midline facial defect, extreme short stature) or anosmia (Kallmann’s syndrome). Random LH levels in hypogonadotropic patients are typically below 0.15 IU/L, but often overlap those of normal pre- and mid-pubertal children. The GnRH test, measuring the gonadotropin response to a 50- to 100-µg bolus, in the premenarchial teenager strongly suggests gonadotropin deficiency if the LH peak is less than 7.0 IU/L by monoclonal assay. However, the GnRH test has limitations because of overlap between hypogonadotropic and normal teenager responses. GnRH agonist testing (e.g., leuprolide acetate injection 10 µg/kg SC) may discriminate better. It may not be possible to definitively establish the diagnosis of gonadotropin deficiency until puberty fails to begin by 16 years of age. (h) Plasma total testosterone is normally about 20 to 60 ng/dL (0.7–2.1 nM), but varies somewhat among laboratories. (i) Androgen resistance is characterized by a male plasma testosterone level (when sexual maturation is complete), male karyotype (46, XY), and absent uterus. External genitalia may be ambiguous (partial form) or normal female (complete form). (j) The differential diagnosis of hyperandrogenism is shown in Figure 14-46. (k) Vaginal aplasia in a girl with normal ovaries may be associated with uterine aplasia (Rokitansky-Kustner-Hauser syndrome). When the vagina is blind and the uterus aplastic, this disorder must be distinguished from androgen resistance. If the external genitalia are ambiguous, it must be distinguished from other disorders of sexual differentiation (intersex). (l) Secondary amenorrhea differential diagnosis is presented in Figure 14-42. [Modified with permission from Rosenfield RL (2003). Menstrual disorders and hyperandrogenism in adolescence. In Radovick S, MacGillivray MH (eds.), Pediatric endocrinology: A practical clinical guide. Totowa, NJ: Humana Press 451–478.]

and generally begins with a chronic disease panel and determining gonadotropins, estradiol, and testosterone level. A pregnancy test is indicated in a sexually mature adolescent. The diagnostic considerations differ in the anovulatory girl without FSH elevation, depending on whether she is hypoestrogenic or well estrogenized (Table 14-8) (Figures 14-41 and 14-42).

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FSH elevation indicates primary ovarian failure (Figures 14-41 and 14-42). Chromosome abnormalities are ordinarily the first consideration because the most common cause is Turner syndrome and its variants. Those individuals with primary ovarian failure that is not due to Turner syndrome and its variants should be investigated for the fragile X premutation.

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Figure 14-42 Differential diagnosis of secondary amenorrhea or oligomenorrhea. (a) Mature secondary sex characteristics are characteristic because the occurrence of menarche indicates a substantial degree of development of the reproductive system. (b) Diverse disorders of many systems cause anovulation. The history may reveal excessive exercise, symptoms of depression, gastrointestinal symptoms, radiotherapy to the brain or pelvis, or rapid virilization. Physical findings may include hypertension (forms of congenital adrenal hyperplasia, chronic renal failure), short stature (hypopituitarism, Turner syndrome, pseudohypoparathyroidism), abnormal weight for height (anorexia nervosa, obesity), decreased sense of smell (Kallmann’s syndrome), optic disc or visual field abnormality (pituitary tumor), cutaneous abnormalities (neurofibromatosis, lupus), goiter, galactorrhea, hirsutism, or abdominal mass. (c) In the absence of specific symptoms or signs to direct the workup, evaluation for chronic disease in a sexually mature adolescent typically includes complete blood count and differential, sedimentation rate, comprehensive metabolic panel, celiac panel, thyroid panel, cortisol and insulin-like growth factor-I levels, and urinalysis. (d) Patients missing only a small portion of an X-chromosome may not have the Turner syndrome phenotype. Indeed, among 45,X patients the classic Turner syndrome phenotype is found in less than one-third (with the exception of short stature in 99%). Ovarian function is sufficient for about 10% to undergo some spontaneous pubertal development and for 5% to experience menarche. If chromosomal studies are normal and there is no obvious explanation for the hypogonadism, special studies for fragile X premutation and autoimmune oophoritis should be considered. (e) Autoimmune ovarian failure may be associated with tissue-specific antibodies and autoimmune endocrinopathies such as chronic autoimmune throiditis, diabetes, adrenal insufficiency, and hypoparathyroidism. Nonendocrine autoimmune disorders may occur, such as mucocutaneous candidiasis, celiac disease, and chronic hepatitis. Rare gene mutations causing ovarian insufficiency include steroidogenic defects that affect mineralocorticoid status (17-hydroxylase deficiency is associated with mineralocorticoid excess and lipoid adrenal hyperplasia with mineralocorticoid deficiency) and mutations of the gonadotropins or their receptors. Ovarian biopsy is of no prognostic or therapeutic significance. (f) The history may provide a diagnosis (e.g., cancer chemotherapy or radiotherapy). Other acquired causes include surgery and autoimmunity. Chromosomal causes of premature ovarian failure include X-chromosome fragile site and point mutations. Other genetic causes include gonadotropin-resistance syndromes such as LH or FSH receptor mutation and pseudohypoparathyroidism. A pelvic ultrasound that shows preservation of ovarian follicles carries some hope for fertility. (g) Withdrawal bleeding in response to a 5- to 10-day course of progestin (e.g., medroxyprogesterone acetate 10 mg HS) suggests an overall estradiol level greatert than 40 pg/mL. However, this is not entirely reliable and thus in the interest of making a timely diagnosis it is often worthwhile to proceed to further studies. (h) A thin uterine stripe suggests hypoestrogenism. A thick one suggests endometrial hyperplasia, as may occur in polycystic ovary syndrome. (i) A single cycle of an OCP containing 30 to 35 µg ethinyl estradiol generally suffices to induce withdrawal bleeding if the endometrial lining is intact. (j) The differential diagnosis of other anovulatory disorders continues in Figure 14-43. [Modified with permission from Rosenfield RL (2003). Menstrual disorders and hyperandrogenism in adolescence. In Radovick S, MacGillivray MH (eds.), Pediatric endocrinology: A practical clinical guide. Totowa, NJ: Humana Press 451–478.]

Lack of FSH elevation in a prepubertal patient does not rule out primary ovarian failure if bone age is below 11 years because neuroendocrine puberty may not have occurred, in which case primary ovarian failure is not hypergonadotropic (Table 14-9).591 If gonadotropins are not elevated and bone age has reached 11 years, in a prepubertal girl without a growth-attenuating or growthretarding disorder one is dealing with constitutional delay of puberty or isolated gonadotropin deficiency (Figure 14-41). “Constitutional” delay of puberty is the most likely diagnosis until the bone age reaches 11 to 13 years

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(Table 14-9).591 Its distinction from isolated gonadotropin deficiency may be difficult. The features that help to distinguish it from isolated gonadotropin deficiency are listed in Table 14-10 and discussed in the footnotes to Figure 14-41. The single most useful test is the LH level in response to GnRH testing because random LH levels in hypogonadotropic patients often overlap those of pre- and midpubertal normal children.95 GnRH agonist testing may discriminate between these disorders better81 and adds the dimension of assessing the gonadal secretory response to the secreted

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Figure 14-43 Differential diagnosis of anovulatory disorders. (a) Anovulatory disorders should be considered in any girl with unexplained amenorrhea or oligomenorrhea, irregular menstrual bleeding, short cycles, or excessive menstrual bleeding. The workup should be initiated as indicated in Figure 14-42, including history and examination, a chronic disease panel, pregnancy test, and gonadotropin levels. (b) Once breast development has matured, the breast contour does not substantially regress when hypoestrogenism develops. Hypoestrogenism is suggested if plasma estradiol is persistently 40 pg/mL in an assay sensitive to 10 pg/mL. However, a single estradiol level may be misleading because of cyclic or episodic variations. (c) Baseline gonadotropin levels may not be low in gonadotropin-deficient patients, especially according to polyclonal antibody-based assays. Gonadotropin-releasing hormone (GnRH) testing is usually performed by assaying LH and FSH before and 0.5 hour after the administration of 1 mcg/kg GnRH intravenously. GnRH agonist testing may alternatively be performed by administering 10 mcg/kg leuprolide acetate subcutaneously and assaying LH and FSH at 4 hours to assess gonadotropin reserve and at 24 hours to assess the ovarian steroid response to endogenous gonadotropin release. (d) Baseline gonadotropin levels may be normal as the ovary begins to fail, as in menopause, but an exaggerated FSH response to GnRH and subnormal E2 response to the gonadotropin elevation induced by acute GnRH agonist challenge are characteristic. The further workup is shown in Figure 14-42. (e) Responses to GnRH may vary from nil to normal in gonadotropin deficiency. Normal LH and FSH responses in the presence of hypoestrogenism indicate inadequate compensatory hypothalamic GnRH secretion. (f) Gonadotropin deficiency may be congenital or acquired, organic or functional. Congenital causes include midline brain malformations or specific genetic disorders such as Prader-Willi syndrome, LaurenceMoon-Biedl syndrome, or Kallmann syndrome. Kallmann’s, the association of anosmia with gonadotropin deficiency, occurs in both the X-linked and autosomal-recessive forms. Special MRI views often demonstrate absence of the olfactory tracts. Acquired gonadotropin deficiency may be secondary to a variety of organic CNS disorders, varying from hypothalamic-pituitary tumor to radiation damage to empty sella syndrome. Autoimmune hypophysitis is a rare disorder, sometimes accompanying a polyendocrine deficiency syndrome. The prototypic form of functional gonadotropin deficiency is anorexia nervosa. Idiopathic hypogonadotropic deficiency may sometimes occur in families with anosmia, suggesting a relationship to Kallmann’s syndrome. (g) Dysfunctional uterine bleeding or menorrhagia not controlled by progestin or OCP therapy additionally requires a pelvic ultrasound examination (for genital tract tumor or feminizing tumor), coagulation workup (which includes platelet count, prothrombin time, thromboplastin generation test, and bleeding time), and consideration of the possibility of sexual abuse. (h) The equivalent of 4 miles per day or more is generally required before body fat stores fall to the point where amenorrhea occurs. Physical or psychosocial stress may cause amenorrhea. (i) Hyperandrogenism differential diagnosis is outlined in Figure 14-46. (j) Mild forms of stress disorders associated with low body fat (anorexia nervosa, bulimia nervosa, and athletic amenorrhea) may cause acquired hypothalamic amenorrhea rather than frank gonadotropin deficiency. The low body fat content of athletic amenorrhea may not be reflected by weight for height because of high muscularity. Dual-photon absorptiometry scan may be useful in documenting body fat below 20%. Patients with anorexia nervosa may become amenorrheic before or when weight loss begins, indicating an important psychological component to the etiology. Obesity is also associated with anovulatory cycles. (k) Hypothalamic amenorrhea is a diagnosis of exclusion. It is a form of partial gonadotropin deficiency in which baseline estrogen secretion is normal but a preovulatory LH surge cannot be generated. It may result from organic CNS disorders. Functional hypothalamic amenorrhea may be stress or undernutrition related or idiopathic. It may be secondary to chronic illness or result from obesity or diverse types of endocrine dysfunction. Research studies show subnormal LH pulsatility or estrogen induceability of the LH surge. (l) Hyperprolactinemia is heterogeneous in its presentation. Galactorrhea is found in half of patients. Some have normoestrogenic anovulation, which may be manifest as hypothalamic anovulation, hyperandrogenism, dysfunctional uterine bleeding, or short luteal phase. On the other hand, some are hypoestrogenic. These do not have galactorrhea. (m) Hyperprolactinemia may be caused by prolactinomas, which secrete excess prolactin, or may be secondary to interruption of the pituitary stalk by large hypothalamic-pituitary tumors or other types of CNS injury. The latter cause variable pituitary dysfunction, which may include complete gonadotropin deficiency and various manifestations of hypopituitarism (including secondary hypothyroidism). (n) Drugs, particularly neuroleptics of the phenothiazone or tricyclic type, may induce hyperprolactinemia. [Modified with permission from Rosenfield RL (2003). Menstrual disorders and hyperandrogenism in adolescence. In Radovick S, MacGillivray MH (eds.), Pediatric endocrinology: A practical clinical guide. Totowa, NJ: Humana Press 451–478.]

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TA B L E 1 4 - 1 0

Features That Distinguish Gonadotropin Deficiency from Constitutional Delay of Puberty In a healthy delayed prepubertal girl with BA 11 yr and prepubertal FSH, gonadotropin deficiency is: • Possible if: • Weight loss greater than 5% to 8% (BMI 10th to15th percentile for height age) • Midline facial defect • CNS dysfunction • CT or MRI brain scan abnormal • Probable if: • BA 13 yr and LH 0.15 U/L in early daytime • Anosmia or panhypopituitarism • Diagnostic if: • Sleep-associated increase in LH lacking • GnRH (agonist) test → flat response • Chronologic age 16 yr Reproduced with permission from Rosenfield RL, Barnes RB (1993). Menstrual disorders in adolescence. Endocrinol Metab Clin N Am 22:491–505.

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up.665 Macroprolactinemia should be considered in the absence of clearly related symptoms, and when MRI is negative or in the setting of autoimmune disease.653,654 Macroprolactinemia is confirmed when the prolactin level measured after precipitation of serum using polyethylene glycol is normal (or substantially reduced compared to the level measured in untreated serum.) Imaging studies are important ancillary measures. Pelvic ultrasound may demonstrate hypoplastic ovaries, endometrial hypoplasia or disorders, and multicystic or polycystic ovaries. MRI of the hypothalamic-pituitary area is important in the workup of gonadotropin deficiency, hyperprolactinemia, and hypothalamic anovulation. Anorexic patients require psychiatric evaluation and consideration of brain tumor and partial bowel obstruction. Diet faddism and athletic addiction may be difficult to distinguish from anorexia nervosa. Constitutional thinness is a variant of normal with normal menses and a distinct hormonal profile.666 It is unclear whether the superior mesenteric artery syndrome is a primary disorder that mimics anorexia nervosa or is a complication of it.667

Management gonadotropins.594 Gonadotropin profiles during sleep have not proven to necessarily distinguish hypothalamic dysfunction from constitutional delay of puberty.661 The assessment of an adolescent’s degree of estrogenization is often difficult. Breast development indicates that there has been estrogen exposure but does not mean that it is current. Determination of plasma estradiol is the simplest test, but diurnal and cyclic variations must be taken into account. Determination of hormonal effects on vaginal cytology is the most indicative of overall estrogen exposure (Figure 14-29), but is less well accepted by patients. A progestin withdrawal test is often helpful. A female who does not experience progestin withdrawal bleeding (Figure 14-42) probably has an ambient estradiol level of less than about 40 pg/mL.662 If bleeding does not occur in response to this maneuver, the integrity of the uterus can be tested by the response to a 3-week course of estrogen-progestin—most conveniently administered in the form of birth control pills. A prolactin level is indicated in the initial workup of normogonadotropic patients, regardless of their estrogen status. The prolactin level correlates with the size of prolactinomas, and a level greater than 200 ng/mL is typical of a macroprolactinoma. A prolactin level that does not correlate with the size of a large pituitary tumor suggests that the tumor is not a prolactinoma and is causing a functional pituitary stalk section or that the tumor is a macroprolactinoma elaborating such high levels of prolactin as to artefactually lower the immunoassayable prolactin level by a “hook effect.”663 Very high blood or cerebrospinal fluid prolactin levels suggest invasiveness. The workup for this should include formal testing of visual fields (Goldman perimetry or evoked response). Pituitary microadenomas may be “incidentalomas” of no clinical significance, judging from an approximate 10% incidence in autopsy material.664 However, they require careful assessment of pituitary function and follow-

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Underlying disorders must be treated appropriately. For example, tumors require surgery and/or radiotherapy. For prolactinoma, dopaminergic treatment is the initial treatment of choice unless the patient’s conditon or eyesight is critical.668 Hyperprolactinemia will be maximally suppressed within 1 month, and the menstrual cycle normalized within 3 months, by an effective dopaminergic agonist regimen. Cabergoline 0.5 to 1.0 mg once or twice weekly will usually control galactorrhea and shrink prolatinomas.652,669 To minimize nausea, it is best to start with a low dose at bedtime. Recent data implicate cabergoline as increasing the risk of cardiac valve regurgitation about fivefold in the elderly, albeit at generally tenfold or higher doses than used to treat hyperprolactinemia.670,671 Bromocriptine does not activate the serotonin 5-HT(2B) receptor, the proposed mechanism through which cabergoline is thought to stimulate valve hypertrophy, and thus bromocriptine should not be associated with an increased risk of cardiac valve regurgitation and may be considered an alternative to cabergoline treatment—albeit a less effective one. The usual bromocriptine maintenance dose is 0.25 to 0.5 mg twice daily. Anorexia nervosa is best managed by an experienced multidisciplinary team. Refeeding is the first priority, and once steady weight gain is evident the psychodynamic issues can be dealt with.672 Family therapy, given on an outpatient basis, for medically uncomplicated cases of anorexia nervosa generally yields the best results—with good improvement in over half of patients. Inpatient intervention for rehydration and metabolic stabilization or failure of weight gain with ongoing cachexia may be required at any time. Menses resume when psychotherapy is effective and body fat is restored to normal (Figure 14-40). The induction of menses by estrogen-progestin replacement is usually injudicious because it provides a false sense of recovery and does not yield the recovery of bone loss that occurs with weight gain.673 Although

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the acute episode can usually be successfully treated, there is a high rate of ongoing psychiatric disability and medical complications. Anorexia nervosa “by proxy” has been described in the offspring of former patients.674 There are two aspects of therapy that are uniformly involved in managing hypogonadism: psychological support and hormone administration. Patients with delayed development that is a variation of normal should be reassured that there is nothing wrong. They are simply among those who are experiencing a delay in timing of the onset of puberty. The wide normal variation in the pattern and time of the pubertal growth spurt should be explained in detail and the girl should be informed of her predicted eventual height. The majority of children with delayed puberty do not have overt psychological symptoms. Complex compensations and sublimations obviously occur. However, peer group pressures may make adjustment to sexual infantilism especially difficult when the age of 13 is approached675—and a poor self-image may lead to social withdrawal and feelings of hopelessness. Physical immaturity may prolong psychological immaturity. A short course of physiologic sex hormone therapy at this time may help alleviate these anxieties. The physician should discuss the fact, when the evidence favors it, that the odds are overwhelmingly in favor of the “timer in the subconscious area of the brain” eventually turning on. When this will happen can be approximated from the skeletal age. One should not hesitate to advise more intensive psychological counseling if it becomes apparent that the concern about puberty is but one aspect of a more general maladjustment. Ultimately, the decision as to whether to undertake treatment for delayed puberty is up to the patient and her family. It is important to assure the teenager with an organic basis for hypoestrogenism that feminization will occur, although in response to appropriate hormone treatment. It should be kept in mind that attainment of normal breast development in the girl with panhypopituitarism requires replacement of GH and cortisol deficits. It is difficult, however, to induce secondary sex characteristics in some patients with systemic chronic inflammatory disease such as lupus erythematosus. In patients in whom short stature is a major concern, as in Turner syndrome, growth potential must be considered before undertaking estrogen replacement. GH therapy improves the adult height potential of patients with Turner syndrome, especially when started as soon as growth failure becomes apparent.676 GH therapy in the United States is generally initiated at the FDA-approved dose of 0.375 mg/kg per week. In older girls, or those with extreme short stature, therapy with oxandrolone [2-oxo-17-methyldihydrotestosterone (Anavar)] 0.05 mg/ kg/day (which augments GH action) can be considered.677 Clitoromegaly is ordinarily negligble on this dosage. Liver function should be monitored. Estrogen replacement therapy starting at about onetenth of the adult dose does not seem to interfere with the positive effect that GH has on adult height.676,678 A physiologic form of treatment is to begin with intramuscular depot E2 0.2 mg/month and to increase the dose by 0.2 mg every 6 months until a midpubertal dose is

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reached, at which time it can be increased by 0.5 mg every 6 months. Midpubertal sex hormone production is approximated by delivering 1.0 to 1.5 mg of depot E2 per month, which alone permits achievement of height potential and typically induces menarche within 1 year. An equivalent starting dose of transdermal E2 appears to be 6.25 µg daily, which requires intermittent or fractionated delivery of the lowest-dose patches currently available. A reasonable alternative regimen begins with 0.25 mg micronized E2 by mouth daily. Adult replacement doses are 100 µg transdermal or 2 to 4 mg oral E2 daily. Progestin should be added to estrogenic regimens after 2 years of estrogen replacement treatment or after withdrawal bleeding occurs. A physiologic replacement regimen consists of micronized progesterone 200 mg daily for 10 days monthly. We typically begin with half of this dose to minimize premenstrual symptoms, but full replacement appears necessary to lower the risk of endometrial hyperplasia and endometrial carcinoma.679,680 Once optimal height is achieved, most patients prefer to switch to birth control pills as a convenient form of estrogen-progestin therapy. The pills containing the lowest dose of estrogen that will result in normal menstrual cycles are advisable. The lowest estrogen dosages currently available in combination contraceptive pills in the United States contain 20 µg (Mircette) to 30 µg (Yasmin) ethinyl estradiol. Hypogonadotropic patients can achieve ovulation with gonadotropin therapy. There has been considerable success in treating those patients with hypothalamic GnRH deficiency by pulsatile GnRH.88,661 Fertility has been achieved by this means. Induction of ovulation is best carried out by a gynecologist specializing in reproductive endocrinology. Patients with primary ovarian failure can successfully achieve pregnancy after oocyte donation and in vitro fertilization.681 Turner syndrome patients are at high risk for obstetrical complications in the areas of uterine anomalies, carbohydrate intolerance, and cardiovascular complications. Oocyte cryopreservation and ovarian tissue cryopreservation and transplantation have been explored to preserve fertility in patients with gonadal dysgenesis and disorders requiring cytotoxic chemotherapy or gonadectomy.682-684 Data are limited, but the modest success with the former outstrips the rare success with the latter. Each technique has limitations and risks. These procedures should currently be considered experimental until greater evidence for efficacy and safety is available.

NORMOESTROGENIC MENSTRUAL DISTURBANCES Hypothalamic Anovulation Hypothalamic anovulation causes menstrual disturbances in sexually mature well-estrogenized women through a deficiency in GnRH secretion too subtle to cause frank hypoestrogenism. The neuroendocrine system stimulates ovarian estrogen secretion to a level normal for an earlyor mid-follicular-phase female, but follicular development is inadequate for a normal dominant follicle to emerge. Amenorrhea or oligomenorrhea may result.

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However, in some patients sufficient estrogenization occurs to cause dysfunctional uterine bleeding (discussed in the next section). Reduced LH pulsatility occurs in the great majority685— failure to generate a midcycle LH surge in the remainder.686,687 The pathophysiology seems to involve varying degrees of undernutrition and/or CRH excess. Negative energy balance may be present even in patients of normal, but lower than average, weight and fat stores.371,646 Leptin deficiency is an important determinant of the decreased LH pulsatility. The anovulation of psychic or physical stress may involve CRH excess.647 In the brain, CRH releases
-endorphin from proopiomelanocortin. This endorphin in turn inhibits GnRH release. This seems to be the major mechanism of anovulation because naloxone blockade of opioid action normalizes gonadotropin secretion.88 In the pituitary, CRH increases the set-point for ACTH release. This brings about a new steady state of increased cortisol secretion. Further ACTH response to CRH is blunted by the negative feedback of this cortisol excess. The result is a mildly Cushingoid cortisol rhythm. Cortisol excess itself can contribute to the amenorrhea by inhibiting the response to GnRH,688 as well as by antagonizing some sex hormone actions. Adrenal androgens are elevated in competitive athletes who maintain body fat stores.689 Causes. Functional hypothalamic amenorrhea is a term commonly used to describe unexplained hypothalamic anovulation. The endocrine features of these patients resemble those of patients with the athletic or psychogenic types of hypothalamic anovulation. A primate model indicates that hypothalamic anovulation develops in stress-sensitive individuals from an innocuous combination of mild stress and mild caloric restriction.689a Athletic amenorrhea is the term used for the hypothalamic anovulation associated with excessive exercise and with low body fat stores. The female athletic triad consists of menstrual disturbance, eating disorder, and osteoporosis.690 Primary or secondary amenorrhea, oligomenorrhea, or short luteal phase are common in athletes.691 Ovarian function decreases approximately in proportion to the amount of physical activity and dietary restriction. Weight-bearing exercise is only partly protective of the effects of hypoestrogenism on weight-bearing bone. There is concern that amenorrheic athletes may be left with a pemanent deficit in bone mass.692 Weight loss to 10% below ideal body weight and body fat less than 10% are risk factors for amenorrhea. Body mass index does not accurately reflect body fat stores in athletes.693 Energy balance seems to be more critical than low body fat stores in mediating the anovulation.691,694 Menarche may occur or menses resume when the athlete’s activity level suddenly decreases and before weight gain occurs. Other factors contribute to cause amenorrhea. Nutritional deficiencies may coexist. Chronic undernutriton may suppress thyroid function, as in anorexia nervosa.647 Delayed menarche may preexist, possibly indicating genetic susceptibility. Athletic amenorrhea resembles anorexia nervosa in patients’ obsession with weight control.691,695 Psychogenic amenorrhea from severe psychic stress has long been known (e.g., “boarding-school amenorrhea”).696 The onset of psychogenic amenorrhea may be

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identified as being associated with a discrete event, but the ovarian dysfunction tends to be long lasting. Subtle nutritional deficits contribute.371 Pseudocyesis is an extremely rare form of psychogenic amenorrhea that is due to persistence of the corpus luteum. This syndrome tends to occur in infertile women with an overwhelming desire for pregnancy and conversion hysteria. Prolactin and LH excess appear to mediate this rare syndrome.697 Post-pill amenorrhea has been a term applied to the amenorrhea that sometimes follows the long-term use of hormonal contraceptives. This in the past was attributed to oversuppression, but oversuppression should not be expected to be the case with the current generation of oral contraceptives.242 About a third of patients with secondary amenorrhea after discontinuation of estrogenand progestin-containing pills have a history of previous menstrual disturbance and ongoing menstrual problems.698 Another third can expect spontaneous remission of the amenorrhea. About half of the remainder of cases will have resolution of their menstrual disturbance after induced pregnancy. The most common cause of post-pill amenorrhea is probably hyperprolactinemia because more than 20% of such cases have galactorrhea. How often this antedates ingestion of the contraceptive pill is unknown. Menses may be restored in normoprolactinemic cases by dopaminergic treatment, which suggests that in such cases there is excessive pituitary prolactin secretion that is too subtle to be detected by measurement of plasma levels.699 The anovulation resulting from depot-medroxyprogesterone acetate contraception is related to the extremely slow rate of absorption and metabolism of this steroid. Menses return when the blood levels of this progestin fall below the threshold for suppression of the LH surge,700 and only rarely has it been associated with disturbed prolactin secretion.701 Differential Diagnosis. Disorders outside the neuroendocrine-gonadal axis may mimic hypothalamic anovulation. These include pregnancy, nutritional disturbance, glucocorticoid excess, disturbed thyroid function, drug abuse, chronic illness, hyperprolactinemia, and ectopic LH secretion. Pregnancy must be excluded in all sexually mature adolescents wtih amenorrhea. An elevation of the plasma
-hCG level is the earliest laboratory sign.702 Constant overproduction of estrogens and progestins by the hCG-driven corpus luteum of pregnancy and the fetoplacental unit leads to the suppression of endogenous pituitary gonadotropin release that underlies the amenorrhea. Overnutrition or undernutrition may cause hypothalamic amenorrhea. Overproduction of estrogen from plasma precursors in adipose tissue appears to mediate the effect of obesity.334 The effect of undernutrition seems to be mediated by factors related to energy balance, as discussed previously. Cushing syndrome (glucocorticoid excess) seems to cause menstrual irregularity by mechanisms discussed earlier in this section. Thyroid hormone deficiency interferes with gonadotropin action on the ovary,703 and may interfere with endometrial function by actions on steroid metabolism704 and action.705 Hypothalamic anovulation may be caused by drug abuse with tetrahydrocannabinol, ethanol, or opiates.706,707

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Cocaine causes menstrual irregularity by suppressing gonadotropin secretion through mechanisms that include depletion of dopaminergic stores (resulting in hyperprolactinemia) and stimulation of CRH release.656,708 Inflammatory illness acutely disrupts the estradiol-induced LH surge709 and chronically causes gonadotropin deficiency, which may be mediated partly by undernutrition and partly by cytokines.633 Disorders as diverse as diabetes mellitus and iron overload all impact GnRH secretion.710,711 Chronic renal failure causes complex dysfunction of the reproductive system, including poor clearance of gonadotropins and prolactin in the presence of inhibition of gonadotropins by a nondialyzable factor.712 Hyperprolactinemia occasionally causes secondary amenorrhea without frank hypoestrogenism.713 This situation probably results from a diminution in FSH secretion that is so marginal as to only inhibit the emergence of a dominant follicle. Ectopic LH or hCG secretion by a tumor can cause normoestrogenic anovulation.714 Sex steroid levels are normal due to ovarian desensitization to LH. Hypothalamic anovulation is ordinarily a diagnosis of exclusion. The medical evaluation should be performed as discussed in the preceding section, with particular attention to the possibilities of emotional disturbances, excessive exercise, the use of birth control pills or other drugs, and state of health. The physical examination should be particularly directed to the state of nutrition, the possibilities of intracranial or systemic disease, galactorrhea, thyroid dysfunction, glucocorticoid excess, hirsutism, and obesity. If this workup is negative, an MRI of the hypothalamic-pituitary area is indicated. The response to a GnRH test may be immature, but it is not necessarily helpful. Hypothalamic anovulation may be documented by demonstrating subnormal LH pulse frequency, but this is not generally practical. Leptin levels tend to be low but nondiagnostic.646 Dysfunctional uterine bleeding from hypothalamic anovulation must be distinguished from that due to other causes (see next section). Management. Many patients with hypothalamic anovulation will benefit from nutritional counseling. Diet faddists and athletes should be advised about the necessity of optimal body energy reserves for the maintenance of normal menstrual cycles (see Figure 14-40). The teleologic significance of this may be pointed out; namely, that inherent in the evolutionary process is the inhibition of pregnancy in times of inadequate food supplies. Ongoing psychological counseling is advisable for patients who cannot change their dietary or exercise patterns because of an abnormal body image. Mature athletes who are hypoestrogenic may benefit from estrogen replacement. Obese girls should be advised that there is a substantial possibility that reduction to a normal weight will result in restoration of menses and improved probability of fertility. Mature teenagers whose amenorrhea is unexplained should be assured that they have a high likelihood of fertility with appropriate endocrinologic treatment. However, such treatment is unlikely to be of any benefit to them until such time as they desire to become pregnant. In the meanwhile, the main objective of therapy is to normalize the endometrial cycle by periodic progestin administration. For this purpose, me-

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droxyprogesterone acetate (up to 10 mg at bedtime for 14 consecutive days) is usually effective in inducing withdrawal periods. During the first few years after menarche, it is reasonable to administer this treatment on alternate months to allow detection of late maturation of a regular menstrual cycle. Induction of an ovulatory cycle has been reported to occasionally result in resumption of spontaneous normal menses.698 An ovulatory cycle can normally be induced by the administration of clomiphene citrate once nightly for five doses. If treatment is successful, bleeding generally occurs about 1 month from commencement of the treatment. One should start with the 50-mg dose because larger doses may cause hyperstimulation of the ovaries with the development of ovarian cysts. For this reason, one should perform an ultrasound examination to rule out cystic ovaries before going successively to 100- to 150-mg dosage. This treatment is not generally recommended in the teenage years, however. Dopaminergic therapy has been reported to be successful in causing the resumption of ovulation in post-pill amenorrhea, modest undernutrition, and other unexplained cases of secondary amenorrhea. Otherwise, induction of ovulation is best left to the endocrinologic gynecologist to supervise at such time as the woman wishes to conceive. The vast majority of patients with no obvious cause for their secondary amenorrhea will become pregnant after appropriate treatment with estrogen, clomiphene, dopaminergic agonist, human menopausal gonadotropins, or pulsatile GnRH therapy.

Dysfunctional Uterine Bleeding Causes. Dysfunctional uterine bleeding (anovulatory bleeding that is too frequent or excessive) is a manifestation of anovulatory cycles in which there is overall excessive estrogen production.591 It is most often a manifestation of physiologic adolescent anovulation. Hyperandrogenism, particularly polycystic ovary syndrome and its variants, is a common cause of dysfunctional bleeding. In some cases it arises from hypothalamic anovulation (discussed previously). Less common are estrogen-producing cysts or tumors, hypothyroidism, hyperprolactinemia, and incipient premature ovarian failure. The workup should therefore include measurement of androgens, prolactin, thyroid function, and FSH blood levels. Corpus luteum insufficiency presents as short (less than 22 days) ovulatory cycles, and thus excessively frequent menses. The immediate cause of an inadequate luteal phase is insufficent progesterone production to sustain a pregnancy.715,716 This in turn may arise from subtle deficiency of FSH during the follicular phase, resultant incomplete emergence of a dominant follicle, and the subsequent formation of an inadequate corpus luteum. Alternatively, the corpus luteum may not be responsive to LH.717 Luteal insufficiency may be the result of hyperprolactinemia,718 obesity, or hyperandrogenism. Differential Diagnosis. Dysfunctional uterine bleeding must be distinguished from the other causes of abnormal genital bleeding listed in Table 14-11.591,719 The possibility that it is pregnancy related must be considered and a pregnancy test performed in a sexually active teenager. Sexual

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TA B L E 1 4 - 1 1

Differential Diagnosis of Abnormal Genital Bleeding • Dysfunctional uterine bleeding (anovulation) • Physiologic anovulation (perimenarcheal) • Hyperandrogenism - Polycystic ovary syndrome • Hyperestrogenism • Hypothyroidism • Hypothalamic anovulation - Hyperprolactinemia • Chronic disease • Incipient premature ovarian failure • Luteal phase defects • Pregnancy-related bleeding • Threatened, missed, or incomplete abortion • Molar pregnancy • Ectopic pregnancy • Vaginal bleeding • Trauma • Tumor • Menorrhagia • Essential menorrhagia • Bleeding diathesis • Uterine tumor, polyp, adenomyosis, intrauterine device Based on Rosenfield RL, Barnes RB (1993). Menstrual disorders in adolescence. Endocrinol Metab Clin North Am 22:491.

abuse is a prime consideration in recurrant vaginal bleeding. Genital tract or feminizing tumors characteristically cause bleeding that cannot be controlled with cyclic progestin or estrogen-progestin therapy. Menorrhagia can be pragmatically considered to exist if prolonged or excessive menses is associated with iron deficiency anemia. Essential or idiopathic menorrhagia is the single most common cause in adolescents. It is theorized to result from imbalance of vasodilating and vasocontricting prostanoid action on the endometrium.720 However, pathologic causes must be considered because bleeding disorders are present in about 20% of adolescents with menorrhagia requiring hospitalization and in 50% of those presenting at menarche. Patients requiring hospitalization for abnormal bleeding should have a platelet count, prothrombin time, partial thromboplastin time, and bleeding time performed. Transvaginal ultrasound, which is not often feasible in the virginal adolescent, is as reliable as hysteroscopy in determining whether or not the endometrial cavity is normal. Failure of serum progesterone to rise above 500 ng/dL during the luteal phase is diagnostic of corpus luteum insufficiency. However, a higher progesterone level may be compatible with inadequacy if not sustained. Management. Estrogen is required to stop an acute episode of dysfunctional bleeding. It can be given together with a progestin as a low-dose oral contraceptive pill, one tablet four times daily for 7 days. Treatment is then stopped for 5 days, and the patient warned that heavy withdrawal bleeding with cramps may occur. Therapy with a low-dose pill, given as for contraception, is then begun to prevent recurrence of dysfunctional bleeding and is continued for about three cycles. If the patient is

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sexually active, oral contraceptive therapy can be continued indefinitely as necessary. A progestin can be used as an alternative to the oral contraceptive pill to prevent recurrent dysfunctional bleeding in a patient who is not sexually active. Medroxyprogesterone acetate 10 mg/day for 1 week is given at 3- to 4-week intervals. After the third month, therapy is stopped and the patient is observed for 1 to 2 months for spontaneous bleeding. If none occurs, the progestin can be given every other month in a dosage of 5 to 10 mg for 7 to 14 days to prevent recurrent dysfunctional bleeding. If in the progestin-free month spontaneous bleeding occurs, progestins are withheld in the subsequent month to determine if the patient has developed regular ovulatory cycles. A patient who is hypovolemic because of rapid and heavy dysfunctional bleeding should be hospitalized and treated with intravenous fluids and blood products as necessary. Premarin can be administered in a dose of 25 mg intravenously every 3 to 4 hours for three to four doses. When medical management fails, a bleeding diathesis or uterine structural abnormality should be considered (see material following). If heavy bleeding persists, curettage should be performed by a gynecologist. Unexplained (“essential”) menorrhagia is treated much the same way as dysmenorrhea. The oral contraceptive pill will decrease menstrual blood loss by about 50% in women with essential menorrhagia. Antiprostaglandins, such as naproxen 500 mg twice a day, decrease blood loss nearly as effectively.

Perimenstrual Symptoms Dysmenorrhea. Pain with menses becomes a source of morbidity in 14% of adolescents.721 When pain is acute and qualitatively different from the usual menstrual pain, ectopic pregnancy must be considered.702,722 An ectopic pregnancy often causes vaginal bleeding that occurs 2.5 weeks later than the time of the expected next menstrual period and is typically light. However, the bleeding may be heavy and thus resemble an episode of dysfunctional uterine bleeding. Ectopic pregnancy is usually diagnosable by a combination of ultrasonography, a serum
-hCG level

1,000 IU/L, and a progesterone level of 2,500 ng/dL. In patients with chronic pelvic pain unresponsive to antiprostaglandins or the oral contraceptive pill, psychological overlay is possible. However, attention should be directed to the possibility of endometriosis, uterine outlet obstruction, or gynecologic tract masses. Ultrasonography and laparoscopy may be indicated to further evaluate these patients. Endometriosis accounts for approximately half of the cases of chronic pelvic pain in teenagers.723 Genetic factors and congenital obstruction of the genital tract predispose to endometriosis, and aberrent estradiol formation in endometrial stroma724 has been incriminated in the pathogenesis. Lack of expression of the cell adhesion molecule integrin in the luteal phase endometrium is associated with a form of endometriosis that is mild but accompanied by infertility.725 Dysmenorrhea may be ameliorated by antiprostaglandin therapy. Naproxen (275 mg qid after a 550-mg loading dose) has been shown to be superior to aspirin

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(650 mg qid) or placebo when begun 2 days before the anticipated onset of menses.726 The oral contraceptive pill is an alternative that will relieve dysmenorrhea in about 90% of cases, presumably by reducing endometrial mass.591 Because smoking, alcohol intake, and excessive weight are risk factors, lifestyle counselling is advisable. Premenstrual Syndrome. Premenstrual syndrome is the term applied when cylic mood changes confined to the second half of the menstrual cycle become debilitating.727 It is often disruptive to women’s personal, social, and occupational function. If symptoms of marked mood swings, depressed mood, anxiety, and irritability occur, it is classified as premenstrual dysphoric disorder.728 Neuropsychiatric symptoms may include epilepsy729 and bizarre behavior.730 These seem to represent aberrant responses to normal cyclic hormonal changes.731 Subnormal activation of the hypothalamic-pituitary-adrenal axis in response to progesterone has been found.732 Some evidence indicates that variation in the degree of progesterone metabolism to neuroactive steroids affects the severity of symptomatology.733 Oral contraceptive therapy with the antimineralocorticoid progestin drospirenone is indicated if psychotropic therapy is unsuccessful. Down-regulation of pituitary gonadotropin secretion by GnRH agonist therapy is efficacious, but its usefulness is limited by the side effects of estrogen deficiency. The relationship of premenstrual syndrome to other luteal phase symptomatology, such as recurrent fever and autoimmune symptoms, is unclear.734,735

HYPERANDROGENISM IN ADOLESCENCE Hyperandrogenism of a mild to moderate degree is the most common cause of normoestrogenic menstrual disturbances. It arises from ovarian or adrenal dysfunction in the vast majority of cases, and abnormal peripheral formation of androgen in most others (Table 14-12).339,736,737 Virilizing tumors are rare (accounting for about 0.2% of hyperandrogenism). These are discussed in the section on precocious puberty.

Causes Polycystic Ovary Syndrome. Polycystic ovary syndrome (PCOS) is the most common cause of hyperandrogenism presenting at or after the onset of puberty. It is a heterogeneous syndrome of unexplained chronic hyperandrogenism and oligo-anovulation, with a polycystic ovary being an alternative diagnostic criterion (Table 14-13).268,736-738 Whether the syndrome can be diagnosed in the absence of hyperandrogenism is controversial. Insulin resistance and LH excess contribute to the pathogenesis of the syndrome, but neither is a criterion for diagnosis. The syndrome is variable both clinically and endocrinologically (Figure 14-44).736 The syndrome in adolescents resembles that in adults. The cardinal symptoms typically begin in the perimenarcheal stage, and it has been documented in children as young as 10 years of age. Adolescents with classic PCOS develop higher LH levels and greater insulin resistance than normal. The classic Stein-Leventhal form of PCOS is characterized clinically by menstrual irregularity, hirsutism (or

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TA B L E 1 4 - 1 2

Causes of Adolescent Hyperandrogenism Functional Gonadal Hyperandrogenism • Primary (dysregulational) functional ovarian hyperandrogenism* • Secondary polycystic ovary syndrome • Poorly controlled classic congenital adrenal hyperplasia • Ovarian steroidogenic blocks • Syndromes of severe insulin resistance • Portohepatic shunting • Epilepsy or valproic acid therapy • Adrenal rests • Ovotesticular disorder of sexual differentiation • Chorionic gonadotropin related Functional Adrenal Hyperandrogenism • Primary (dysregulational) functional adrenal hyperandrogenism** • Congenital adrenal hyperplasia • Prolactin or growth hormone excess • Dexamethasone-resistant functional adrenal hyperandrogenism • Cushing syndrome • Cortisol resistance • Apparent cortisone reductase deficiency Peripheral Androgen Overproduction • Obesity • Idiopathic hyperandrogenism Tumoral Hyperandrogenism Androgenic Drugs * Common form of PCOS. ** Uncommon form of PCOS. Modified with permission from Buggs C, Rosenfield RL (2005). Polycystic ovary syndrome in adolescence. Endocrinol Metab Clin North Am 34:677–705.

TA B L E 1 4 - 1 3

Diagnostic Criteria for Polycystic Ovary Syndrome National Institutes of Health (NIH) Criteria • Clinical and/or biochemical signs of hyperandrogenism • Anovulatory symptoms Rotterdam criteria • NIH criteria • Ultrasonographic evidence of a polycystic ovary as an alternative to either specific NIH criterion Modified with permission from Buggs C, Rosenfield RL (2005). Polycystic ovary syndrome in adolescence. Endocrinol Metab Clin North Am 34:677–705.

hirsutism equivalents such as seborrhea, acne, and alopecia), and obesity (often with acanthosis nigricans). Endocrinologically, they are characterized by hyperandrogenemia and by polycystic ovaries or about twofold elevation of serum LH—although the more obese the patient the less likely serum LH is to be elevated.739,740 However, half the cases are nonclassic or atypical. Nonclassic PCOS patients are hyperandrogenic and have anovulatory symptoms, but lack a polycystic ovary (meeting NIH criteria) or have normal menses yet a polycystic ovary (meeting Rotterdam criteria) (Table 14-13). The broad

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PCOS clinical manifestations

Hirsutism, acne, alopecia

587

PCOS laboratory manifestations

Polycystic ovaries

Irregular menses

Functional Ovarian Hyperandrogenism (FOH)

Insulin resistance LH Excess Central obesity

Functional Adrenal Hyperandrogenism (FAH)

Figure 14-44 The major clinical and laboratory manifestations of PCOS are shown in approximate proportion to their relative incidence and coincidence. [Modified with permission from Buggs C, Rosenfield RL (2005). Polycystic ovary syndrome in adolescence. Endocrinol Metab Clin North Am 34:677–705.]

spectrum of the disorder includes atypical hyperandrogenemic cases with cutaneous manifestations or central obesity that do not fit current diagnostic criteria because they lack clinical or ultrasonographic evidence of ovarian dysfunction, although they have the typical endocrine dysfunction of the ovaries or adrenal glands. Functional ovarian hyperandrogenism (FOH) is the source of the androgen excess in about 80% of cases. It is characterized by 17-hydroxyprogesterone (17PROG) hyperresponsiveness to the gonadotropin stimulation of GnRH agonist (GnRHag) or human chorionic gonadotropin testing and subnormal suppressibility of plasma testosterone upon adrenal suppression by glucocorticoid. About 60% of cases have a typical form of dexamethasonesuppressible functional adrenal hyperandrogenism (FAH) that is characterized by moderate 17-hydroxypregnenolone or DHEA hyperresponsiveness to ACTH. FAH accompanies FOH about half the time, but in atypical PCOS it may be the sole source of androgen excess. Pathogenesis. The central abnormality in primary FOH usually seems to be intraovarian androgen excess (Figure 14-45). The hyperandrogenemia results in the pilosebaceous manifestations. The disproportionately high intraovarian androgen concentration arising from FOH seems to recruit excessive growth of small follicles while hindering the follicular maturation involved in the emergence of a dominant follicle, as well as causing thecal and stromal hyperplasia. Dysregulation of steroidogenesis appears to account for primary FOH and primary FAH. Dysregulation is postulated to result from imbalance among the various intrinsic and extrinsic factors involved in the modulation of trophic hormone action. Within the ovary, there appear to be flaws in the processes that normally coordinate androgen and estrogen secretion (Figure 14-20). This causes the ovaries to hyperrespond to LH, rather than undergoing down-regulation in response to LH stimulation. The majority of cases appear to have an intrinsic theca cell defect that causes widespread overexpression of steroidogenic enzymes, particularly 17-hydroxylase and 17,20-lyase (both properties of P450c17, which are the rate-limiting steps in the biosynthesis of testosterone precursors). As in the ovary, dysregulation of local steroidogenic regulatory processes within the adrenal cortex appears to cause excessive 17-ketosteroid formation as by-products of cortisol secretion. Granulosa cells also exhibit ste-

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Figure 14-45 Model of the pathogenesis of functional ovarian hyperandrogenism. Increased intraovarian androgen concentration is central and causes hyperandrogenemia and follicular maturation arrest. It can also result from follicular atresia. Additional causes of increased intraovarian androgen levels include extraovarian androgen excess, ovarian steroidogenic blocks, and dysregulation of androgen secretion. The latter may result from luteinizing hormone (LH) excess or from escape from desensitization to LH action by insulin, insulin-like growth factors, or other peptides. [Modified from Ehrmann DA, et al. (1995). Polycystic ovary syndrome as functional ovarian hyperandrogenism as dysregulation of androgen secretion. Endocr Rev 16:322. Copyright © 1995 The Endocrine Society.]

roidogenic dysregulation. They are excessively responsive to FSH, particularly at high dosage. This accounts for the tendency of PCOS women to develop the dangerous ovarian hyperstimulation syndrome during fertility treatment. It may also aggravate thecal androgen secretion via a paracrine action.129 The fundamental defect may be a generalized disorder involving specific transcriptional coregulators also involved in the regulation of glucose and fat metabolism. Insulin excess appears to be an important extrinsic factor in dysregulation. As a group, PCOS patients are significantly hyperinsulinemic in association with a state of insulin resistance—and treatments that lower insulin levels reduce the androgen excess moderately. The insulin/IGF system seems to act in synergism with trophic hormones to cause ovarian or adrenal androgen excess. The ovaries and adrenal glands function as if responding to the hyperinsulinemic state in spite of the resistance to the effects of insulin on skeletal glucose metabolism. This paradox remains to be resolved.

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Excessive LH secretion, found in 50% to 75% of cases, was once thought to be central to the pathogenesis of PCOS due to a complex cycle of events in which peripheral conversion of adrenal androstenedione to estrone-sensitized gonadotropes to GnRH.741 However, evidence is accumulating that it results from hyperandrogenemia interfering with progesterone negative feedback effect on LH secretion. 55,252,253 Nevertheless, the possibility of a primary role for LH excess remains—particularly in the PCOS that is secondary to congenital virilizing disorders. Etiology. Increasing evidence suggests that PCOS arises as a complex trait with contributions from both heritable and nonheritable factors. Polygenic influences appear to account for about 70% of the variance in pathogenesis. Hyperandrogenemia and polycystic ovaries each appear to be inherited as independent autosomal-dominant traits. Nearly half of sisters of women with PCOS have an elevated plasma testosterone level, although only half of them are symptomatic. Asymptomatic polycystic ovaries are found in about 10% of women, but they are often accompanied by a subclinical PCOS-type of ovarian dysfunction. Central obesity and insulin resistance seem to play important roles in PCOS, perhaps by accentuating steroidogenic dysregulation—but perhaps more fundamentally because PCOS is closely related to metabolic syndrome in parents. Gestational factors have also been incriminated. The syndrome has been associated with high and low birth weight, and it can develop secondary to fetal virilization. Thus, the syndrome seems to arise from an intrinsic genetic trait that becomes manifest only when interacting with other congenital or environmental factors, excessive adiposity being the most common precipitant. Other Causes of Functional Ovarian Hyperandrogenism. Secondary PCOS can result from several disorders (Table 14-12).742 Extraovarian androgen excess (as in poorly controlled congenital adrenal hyperplasia) and ovarian steroidogenic blocks (such as 3
-hydroxysteroid dehydrogenase, 17
-hydroxysteroid dehydrogenase, and aromatase deficiency) are causes. Excessive stimulation of the LH receptor appears to mediate the hyperandrogenism reported in chorionic gonadotropin-related ovarian dysfunction during pregnancy, and appears to have played a role in a case of FSH-resistant ovarian follicles.743 All known forms of extreme insulin resistance (including hereditary cases of insulin receptor mutations, as well as acromegaly) are accompanied by PCOS, possibly by acting through the IGF-I signal transduction pathway to cause escape from desensitization to LH. Functional ovarian hyperandrogenism may also result from adrenal rests of the ovaries in congenital adrenal hyperplasia and from true hermaphroditism. PCOS has also been reported as a complication of portasystemic shunting. Impaired steroid metabolism has been postulated as the mechanism. Other Causes of Functional Adrenal Hyperandrogenism. The PCOS type of primary (dysregulational) FAH occurs as an isolated entity, not associated with FOH, in about 25% of hyperandrogenic women. This may sometimes be an outcome of exaggerated adrenarche. This type of adrenal dysfunction was previously mistaken for nonclassic 3
-hydroxysteroid dehydrogenase deficiency, which is now known to be a rare disorder.744 Less than

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10% of adrenal hyperandrogenism can be attributed to the more well-understood disorders listed in Table 14-11. Most are due to nonclassic congenital adrenal hyperplasia, which accounts for less than 5% of hyperandrogenism in the general U.S. population, and to the various hyperandrogenic forms of Cushing syndrome. Prolactin excess causes adrenal hyperandrogenism, sometimes in association with polycystic ovaries. Adrenal hyperandrogenism can on rare occasions arise from cortisol resistance or apparent cortisone reductase deficiency. Peripheral Androgen Overproduction. In nearly 10% of hyperandrogenic patients, an ovarian or adrenal source cannot be ascertained by thorough testing. This is idiopathic hyperandrogenemia. Obesity seems to explain some of these cases because adipose tissue has the capacity to form testosterone from androstenedione. Obesity may simulate PCOS by causing amenorrhea, acanthosis nigricans, and hyperandrogenemia. Other idiopathic cases may be due to hereditary quirks in peripheral metabolism of steroids.

Differential Diagnosis Hyperandrogenism should be considered in any girl who presents with hirsutism or cutaneous hirsutism equivalents, menstrual disturbance, or central obesity during puberty. PCOS is by far the most common cause, and its manifestations are variable (Figure 14-44). Hirsutism, acne, and alopecia are inconsistently expressed manifestations of androgen excess. If acne vulgaris is unusually early in its age of onset, if severe acne is persistent, or if Accutane treatment is being considered, the possibility of hyperandrogenemia should be investigated. Androgenetic alopecia is manifest as a diffuse pattern in females. Hyperhidrosis or hidradenitis suppurativa may be the only dermatologic manifestation. On the other hand, some of the cardinal symptoms or signs of androgen excess may not be present. Many cases of hyperandrogenemia are entirely cryptic. Menstrual irregularity of any sort (i.e., ranging from amenorrhea to dysfunctional uterine bleeding) that persists for 2 years has a two-thirds probability of being persistent and thus is an indication for workup for androgen excess. Conversely, a history of normal menstrual cyclicity does not necessarily indicate ovulatory cycles—and some cases are not diagnosed until they present as adults with unexplained infertility745 or with recurrent miscarriages.746 Intractable obesity, large waist circumference ( 88 cm), or acanthosis nigricans should raise concern for PCOS. The possibility of PCOS is heightened if the previously cited symptoms are associated with a history of prepubertal risk factors for PCOS, including congenital virilizing disorders; premature adrenarche, particularly exaggerated adrenarche; atypical sexual precocity; and intractable prepubertal obesity, particularly if associated with pseudoCushing syndrome, pseudo-acromegaly, or a family history of metabolic syndrome. Most hyperandrogenic girls present when sexually mature and normoestrogenic. However, if there is any doubt about whether feminization is adequate and ongoing (as when the presenting symptom is primary amenorrhea) the

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evaluation should include bone age, estradiol, and gonadotropin levels as outlined previously (Figure 14-41). Hirsutism must be differentiated from hypertrichosis, the situation in which vellus hair predominates on “nonsexual” areas of the body. Hypertrichosis is not caused by sex hormone imbalance but appears with heredity, glucocorticoid excess, starvation, and medications such as phenytoins, cyclosporine, and valproic acid.339 The history and examination should also address risk factors for androgenic medications, other endocrinopathies (the most common of which are nonclassic congenital adrenal hyperplasia, Cushing syndrome, hyperprolactinemia, and thyroid dysfunction), and virilizing disorders. A rapid pace of development or progression of hirsutism, evidence of virilization (such as clitoromegaly, genital ambiguity, or increasing muscularity), or an abdominal mass would raise concern for an androgen-secreting neoplasm. However, tumors producing moderately excessive androgen can have indolent presentations. The goals of the laboratory evaluation for PCOS are to attempt to document hyperandrogenism, to determine the specific etiology, and to provide a baseline in case it becomes necessary to reassess because of progression of the disorder. The diagnosis is on the firmest grounds if hyperandrogenism is demonstrated biochemically, rather than relying on hirsutism as a surrogate for it—although documentation of hyperandrogenemia can be problematic. An approach to the workup that depends on assessing the degree of hirsutism and elucidating risk factors for virilizing disorders, androgenic medications, PCOS, and other endocrinopathies is suggested (Figure 14-46).339 If hirsutism is mild and menses are regular with no evidence of risk factors that would suggest a secondary cause, it is reasonable not to pursue laboratory evaluation—given the high likelihood of idiopathic hirsutism (a cutaneous rather than endocrine disorder)—unless the patient is of Asian ethnicity. If hirsutism is moderate or severe or there are features to suggest an underlying disorder, excess androgen production should be ruled out. Risk factor assessment includes follow-up to evaluate response to therapy. Plasma testosterone is the single most important androgen to evaluate. Plasma free testosterone is about 50% more sensitive in detecting excessive androgen production because hyperandrogenic women have a relatively low level of SHBG. Many commercial total and free testosterone assays are not suitable for the evaluation of women and children. Therefore, these assays are best performed by a specialty laboratory that has extensively validated them. Although other androgens are present in blood, their assessment makes little difference in management if plasma free testosterone is normal. However, the variation in androgen levels may miss an occasional case of nonclassic congenital adrenal hyperplasia and therefore further studies are indicated in patients at high risk by virtue of family history or ethnicity. These tests are most clearly interpreted when samples are drawn in the early morning of the mid-follicular (or anovulatory) phase of a menstrual cycle. If androgen elevation is found, the next step in the differential diagnosis is ordinarily to obtain an ultrasound examination of the pelvis. A polycystic ovary is specifically

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defined as one that exceeds a critical volume ( 10.8 cc in adolescents) or is polyfollicular (containing 10 or more follicles in the maximum plane).463 The volume criterion assumes the absence of a dominant follicle ( 10 mm in diameter) or a corpus luteum. Ovarian enlargement is the most solid criterion because multifollicular ovaries, normal in adolescence and present in other anovulatory states, are distinguishable from polycystic ovaries primarily on the basis of whether volume is normal or abnormal (which can be problematic). These findings are not completely specific to PCOS and require excluding disorders that may secondarily cause PCOS. On the other hand, a negative ultrasound examination does not exclude nonclassic PCOS. However, it is useful in ruling out tumor and disorders of sexual differentiation as a cause of the androgen excess.736 Simultaneous ultrasound imaging of the abdomen can be a costeffective screening test for adrenal neoplasm in the hands of an experienced ultrasonographer. Additional testing may include tests to exclude pregnancy and hyperprolactinemia. It may also include measurement of DHEAS and early-morning 17-hydroxyprogesterone if adrenal hyperandrogenism is suspected, and assessment for Cushing syndrome, thyroid dysfunction, or acromegaly if clinically indicated. If this evaluation for the most common disorders that mimic polycystic ovary syndrome is negative, the association of testosterone elevation with anovulatory symptoms or a polycystic ovary fulfills standard diagnostic criteria for polycystic ovary syndrome (Table 14-13). However, it does not exclude some fairly rare hyperandrogenic disorders. The approach to further studies to determine the source of hyperandrogenemia varies among subspecialists and with the needs of the individual patient. Our preference is to use a dexamethasone suppression test to attempt to make a positive diagnosis of the ovarian or adrenal dysfunction of PCOS or to determine whether further workup is necessary for rare forms of CAH or other rare adrenal disorders (Figure 14-47).736 The degree of suppression of plasma androgens and cortisol in response to a low-dose dexamethasone suppression test segregates patients diagnostically. Total testosterone levels of adrenal tumors fail to suppress more than 40% after a 2-day test.747 A more prolonged course of low-dose dexamethasone is normally required to lower adrenal androgens below normal levels, and thus we prefer a “dexamethasone androgen-suppression test” (Figure 14-47). Subnormal androgen suppression with normal adrenocortical suppression indicates a source of androgen other than an ACTH-dependent adrenal one. This is typical of PCOS if tumor or other ovarian pathology has not been found by ultrasound examination. If both cortisol and androgen suppression are subnormal, the androgen excess may be secondary to noncompliance with taking dexamethasone, to Cushing syndrome, or to defective cortisol metabolism or action. If androgen suppression is normal, ACTH (cosyntropin) stimulation testing to assess 17-hydroxyprogesterone and other steroid intermediates is recommended. Further extensive diagnostic studies are seldom indicated unless there is reason to suspect a virilizing tumor

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Figure 14-46 Differential diagnosis of hyperandrogenism and hirsutism. (A) Risk assessment includes assessment for the degree of hirsutism, menstrual irregularity, central obesity, and the other risk factors shown. Acne vulgaris, seborrhea, pattern alopecia, hyperhidrosis, and hidradenitis suppurativa are “cutaneous hirsutism equivalents” and may be alternative signs of hyperandrogenism. A small amount of sexual hair growth is normal (Ferriman-Gallwey score 8). However, if other risk factors are present (even in the absence of hirsutism) androgen excess should be considered. (B) Medications that cause hirsutism include anabolic or androgenic steroids (consider in athletes and patients with endometriosis or sexual dysfunction). Valproic acid, an antiepileptic drug, raises testosterone levels. (C) Mild hirsutism in the absence of the risk factors shown is likely to be idiopathic and to respond to cosmetic or dermatologic therapy. Therefore, it is reasonable to embark on these treatments without an endocrine evaluation. (D) Acne vulgaris of early onset or unresponsive to ordinary dermatologic measures, including antibiotic therapy, and pattern balding are similarly risk factors for hyperandrogenism. (E) Neoplasm risk is suggested by sudden onset, rapid progression, virilization, or an abdominal or pelvic mass. (F) In some hyperandrogenic cases, there is no cutaneous symptomatology (“cryptic hyperandrogenism”) and androgen excess is suspected only because of menstrual irregularity, central obesity, or acanthosis nigricans. (G) The most common hyperandrogenic endocrine disorder other than PCOS in adolescents is nonclassic congenital adrenal hyperplasia (CAH). Risk is increased if family history is positive or in certain ethnic groups, such as Ashkenazi Jews (prevalence 1:27), Hispanics (1:40), and Slavics (1:50). (H) A random sample for plasma total testosterone is a reasonable screening test if reliable assessment of free testosterone is not available. At 8:00 a.m. during the mid-follicular phase of the menstrual cycle (days 4–10), the normal upper limit for plasma total testosterone is typically about 60 ng/dL (2.1 nM) for postmenarcheal adolescents when determined by a specialty laboratory. The upper limit of normal for plasma free testosterone under these conditions is 9 pg/mL (32 pM) in our laboratory. Unfortunately, the methodology for plasma free or bioavailable testosterone yields method-specific norms. (I) A highnormal testosterone level may not reflect a hyperandrogenic state if drawn after the early morning because of diurnal variation—or if only the total testosterone is initially assayed because the plasma free testosterone can be high when total testosterone is normal in that sex hormone binding globulin (SHBG), the major determinant of the bioavailable testosterone, is commonly low in hyperandrogenic women. (J) An early-morning plasma free testosterone determined by a specialty laboratory is indicated on days 4 through 10 of the menstrual cycle or during a period of amenorrhea, when the plasma testosterone is high-normal in patients with moderate or severe hirsutism, if features suggestive of other disorders are present or emerge, or if the response to cosmetic-dermatologic therapy is unsatisfactory. Simultaneous assay of 17-hydroxyprogesterone is indicated in subjects at high risk for CAH, which is suggested by a level greater than 150 ng/dL (4.5 nM)—and is virtually assured by a level greater than 1,200 ng/dL (36 nM). (K) Cosmetic therapies include bleaching, shaving, and waxing. Dermatologic therapies include topical eflornithine and laser treatment. OCP treatment is a useful and effective adjunct if hirsutism is more than minimal. (L) Idiopathic hirsutism may be mimicked by otherwise asymptomatic idiopathic hyperandrogenism, which may be due to atypical polycystic ovary syndrome or abnormal peripheral metabolism of prohormones. (M) Because testosterone undergoes episodic and cyclic changes in addition to diurnal ones, recheck is indicated if the course is progressive or risk factors emerge. (N) Hyperandrogenism with a polycystic ovary or menstrual irregularity in the absence of drug use, neoplasm, or other endocrinopathy meets standard criteria for the diagnosis of PCOS. Therefore, ultrasonic imaging of the ovaries and adrenal glands is usually advisable. See Figure 14-47 for potential further workup to determine the source of androgen excess. [Modified with permission from Rosenfield RL (2005). Clinical practice: Hirsutism. N Engl J Med 353:2578–2588.]

or a disorder of sexual differentiation. Further or alternative workup may include computed tomography for adrenal tumor, acute gonadotropin-releasing hormone agonist administration, or assessment of the response to hormonal suppression treatment to determine the source of androgen.

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Management Management is individualized according to symptoms and patient goals (e.g., hirsutism, acne, and alopecia; menstrual irregularity; obesity and insulin resistance) and the source of androgen excess.339,736,737 Because PCOS is

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Figure 14-47 An approach to determining the source of androgen excess. The association of testosterone elevation with otherwise unexplained anovulatory symptoms (see Figures 14-42 and 14-43) or a polycystic ovary fulfills standard diagnostic criteria for PCOS, which in its various forms accounts for about 80 to 90% of adolescent hyperandrogenism. Determination of the source of excess androgen often permits a positive diagnosis of the characteristic FOH or FAH and rules out rare disorders that mimic PCOS. (A) Our standard dexamethasone androgen suppression test dose is 1 mg/m2 in four divided doses (0.5 mg qid in adult) for 4 days, and plasma cortisol, free testosterone, 17-OHP, and DHEAS are measured on the morning of the fifth day after the final dexamethasone dose. For individuals weighing 100 kg or more, dexamethasone is given for 7 days. (B) Dexamethasone suppression of ACTH-dependent adrenal function normally causes plasma total testosterone to fall below 35 ng/dL (1.2 nM), free testosterone to below 8 pg/mL (28 pM), DHEAS by 75% to below 80 µg/dL (2.1 µM), and 17-OHP to less than 50 ng/dL (1.5 nM). Total testosterone is not as discriminating a criterion as the free testosterone (unfortunately, a method-dependent criterion) or 17-OHP criteria. Subnormal androgen suppression with normal adrenocortical suppression indicates a source of androgen other than an ACTH-dependent adrenal one. (C) Normally, cortisol falls below 1.5 mcg/dL (45 nM). (D) Cushing syndrome is an infrequent cause of hyperandrogenism and requires a more definitive workup. Hyperandrogenism has been reported in the rare conditions of cortisol resistance and cortisone reductase deficiency. (E) Subnormal cortisol suppression is most often due to noncompliance with taking dexamethasone tablets. (F) Ultrasonographic visualization of the ovaries by the vaginal route yields better definition than by the abdominal route, but this is not a generally acceptible technique in the virginal child. An adolescent polycystic ovary is defined as one with a volume greater than 10.8 cc or maximal area greater than 5.5 cm2 or with 10 or more follicles in the maxmum plane. (G) Ovotesticular DSD patients may only have a clearly elevated plasma testosterone level in response to a midcycle LH surge, hCG, or GnRH agonist test. (H) The baseline pattern of plasma androgens may yield a clue to the type of tumor. A DHEAS level greater than 700 mcg/dL (19 µmol/L) is suspicious for adrenal tumor. In the absence of a high DHEAS, disproportionate elevation of plasma androstenedione relative to testosterone or elevated 17-hydroxyprogesterone is typical of a virilizing tumor. Poor dexamethasone suppressibility of testosterone and/or DHEAS is very suggestive of adrenal tumor. CT scan of the abdomen may be indicated in such cases. (I) Polycystic ovary syndrome (PCOS) is a symptom complex with various combinations of hirsutism or its cutaneous equivalents anovulation and central obesity. A polycystic ovary is a classic diagnostic criterion, but is not necessary or specific for the diagnosis. Nonclassic PCOS includes anovulatory hyperandrogenic girls who lack a polycystic ovary. Most have the typical PCOS-type of functional ovarian hyperandrogenism on dexamethasone suppression or GnRH agonist testing. (J) Virilizing adrenal rests of the ovaries may complicate congenital adrenal hyperplasia. They may resemble a polycystic ovary. (K) The standard ACTH test is performed by infusing 250 mcg ACTH1-24 intravenously over a period of 1 minute and obtaining a blood sample before injection 1 hour later. (L) Congenital adrenal hyperplasia (CAH) cannot be confirmed upon mutation analysis unless the steroid intermediates immediately prior to the enzyme block rise 5 standard deviations above average in response to ACTH. For 17-OHP, this is greater than 1,200 ng/dL (36.5 nmol/L). For DHEA, this is greater than 3,000 ng/dL (104 nmol/L) in adolescents. (M) Primary functional adrenal hyperandrogenism (FAH) is a term for idiopathic ACTH-dependent (dexamethasone-suppresible) adrenal hyperandrogenism in which modest rises in DHEA, 17-OHP, and so on do not meet the criteria for the diagnosis of congenital adrenal hyperplasia. It is often found in atypical PCOS. (N) Idiopathic hyperandrogenemia is distinguished from idiopathic hirsutism. About 8% of chronic hyperandrogenemia remains unexplained after intensive investigation, which includes GnRH agonist testing to detect the occasional case of ovarian hyperandrogenism not detected by the dexamethasone test. [Modified with permission from Buggs C, Rosenfield RL (2005). Polycystic ovary syndrome in adolescence. Endocrinol Metab Clin North Am 34:677–705.]

associated with metabolic syndrome, a fasting lipid panel and oral glucose tolerance test are recommended in patients with central obesity or hypertension.748 The twohour blood sugar during an oral glucose tolerance test deteriorates at an average rate of 9 mg/dL/year.749 Because PCOS is closely related to parental metabolic syndrome, we recommend a similar evaluation of primary relatives. Cosmetic measures are the cornerstone of care for hirsutism. Bleaching and shaving suffice for many. Depilitat-

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ing agents and waxing treatments are useful but are prone to causing skin irritation. Eflornithine hydrochloride cream brings about marked improvement of hirsutism in about a third of patients, with the maximal effect by 2 to 6 months. The FDA has permitted marketing of many laser devices, and equivalents such as diode and flashlamp, as effective for permanent hair reduction—for which the criterion is persistant reduction in hair density by 30% or more after three to four treatments of a site.

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Wavelengths between 694 and 1064 nm damage hair follicles by combining relatively selective absorption of heat by dark hairs with penetration into the dermis. Light-skinned individuals are the best candidates, requiring the lower energy pulses. Those with heavily tanned or darker skin require the use of cooling procedures and adjustment of energy levels to minimize the risk of skin side effects. Laser treatment is coming to be preferred to electrolysis, but both types of treatment require trained personnel; are repetitious, expensive, and painful; are practical only for treating limited areas; and may result in local reactions, including burns, dyspigmentation, and scarring. Endocrine therapy is directed at interrupting androgen production or action. This causes the pilosebaceous unit to revert toward the prepubertal vellus type. Endocrinologic treatment of cutaneous symptoms is indicated before undertaking treatment with laser treatment—using Accutane or Rogane if standard cosmetic or topical dermatologic measures are inadequate. The maximal effect on the sebaceous gland occurs within 3 months, but that on sexual hairs requires 9 to 12 months of treatment because of the long duration of the hair growth cycle. All are effective only as long as the patient wishes to maintain her improvement in hirsutism. Combination oral contraceptive pills (OCPs) are the first-line endocrine treatment for women with the dermatologic or menstrual abnormalities of PCOS. They act by suppressing plasma androgens, particularly free testosterone, mainly by inhibiting ovarian function. They also raise SHBG and modestly lower DHEA sulfate levels. They normalize androgen levels within the first month of therapy. All estrogen-progestin combinations generally suffice for women with acne or mild hirsutism, in combination with cosmetic measures. Those with nonandrogenic progestins, such as norgestimate or ethynodiol diacetate combined with 35 µg ethiny E2, have generally favorable risk-benefit ratios and optimize lipid profiles. Those with antiandrogenic progestins in low dose may confer an additional benefit. Drospirenone is available in the United States (with 30 µg ethinyl E2) and in Canada, Mexico, and abroad (with cyproterone acetate 2 mg with 35 µg ethinyl E2). The larger estrogen doses may be necessary in larger girls to provide menstrual regularity. OCPs are also effective in management of menstrual irregularity, which requires treatment because chronic anovulation is associated with increased risk of developing endometrial hyperplasia and carcinoma. There are, however, several potential disadvantages to the use of OCPs in the management of PCOS in adolescents. They will bring growth to end in perimenarcheal girls. OCPs, particularly some third-generation OCPs, may be contraindicated in patients who are at risk for venous thrombosis. In patients with migraine headaches, OCPs should be used with caution and in the lowest estrogen dose possible. Patients may use OCPs as an excuse for not losing weight. The patient may believe that the treatment is curative and defer a definitive diagnostic workup. OCPs do not permit conception if and when it is desired. The long-term consequences of these agents on fertility are unknown. There is the theoretic possibility of post-pill amenorrhea because high-dose estrogen

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begun in early adolescence may increase the risk of infertility.750 It is advisable to recheck patients after 3 months of therapy to assess the efficacy of treatment and normalization of androgen levels. As a general rule, OCP treatment should be continued until the patient is gynecologically mature (5 years postmenarcheal) or has lost a substantial amount of excess weight. At that point, withholding treatment for a few months to allow recovery of suppression of pituitary-gonadal function and to ascertain whether the menstrual abnormality persists is usually advisable. In doing so, however, one must keep in mind that the anovulatory cycles of PCOS lead to relative infertility (not absolute infertility). The need for continued use of OCP for contraceptive purposes must be considered. Progestin monotherapy is an alternative to OCPs for the control of menstrual irregularities. Micronized progesterone (Prometrium) 100 to 200 mg daily at bedtime for 7 to 10 days induces withdrawal bleeding in the majority of patients, but some do not respond—apparently because of an antiestrogenic effect of androgen excess on the endometrium. Breakthough bleeding is more likely with progesterone than with OCPs. Progestin therapy has the appeal of permitting the detection of the emergence of normal menstrual cyclicity. However, it does not normalize androgen levels and is not an adequate treatment if hirsutism or hirsutism equivalents are a problem. The perimenarcheal girl who responds well to progestin therapy can be maintained at approximately 6-week cycles to permit the detection of spontaneous menses. Side effects of progestin include mood symptoms (depression), bloating, and breast soreness. Patients must be informed that oral progestin dosed in this way is not a means of contraception. Antiandrogens generally yield improvement in hirsutism beyond that attainable with OCPs. They can be expected to reduce the Ferriman-Gallwey score by 15% to 40%, although there is considerable variation among individuals. Antiandrogen use for this purpose is off-label because all carry the risk of causing pseudohermaphroditism of the male fetus. Therefore, all antiandrogens should be prescribed with a contraceptive—preferably an OCP. They may have a modest effect on the metabolic abnormalities associated with PCOS.751 Spironolactone in high dosage is the safest potent antiandrogen in the United States. We recommend starting with 100 mg twice a day until the maximal effect has been achieved, and then attempting to reduce the dose to 50 mg twice a day for maintenance therapy. Spironolactone is usually well tolerated, but it is contraindicated in patients with adrenal, hepatic, or renal insufficiency. Women are at risk of hyperkalemia if on potassiumsparing diuretics, potassium supplements, daily nonsteroidal anti-inflammatory drugs, angiotensin-converting enzyme inhibitors, heparin, or such drugs. Therefore, electrolytes should be monitored. Alone, spironolactone tends to cause irregular bleeding. Other antiandrogens used to treat hirsutism and hirsutism equivalents include cyproterone acetate, flutamide, and finasteride. Cyproterone acetate is a potent progestational antiandrogen used with estrogen in a reverse sequential regimen: 50 to 100 mg is given during days 1 to

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10 of cycles in which estrogen is given from days 1 to 21. Flutamide is a more specific antiandrogen with efficacy similar to that of cyproterone and spironolactone, but it is more expensive and rarely causes fatal hepatic failure. Finasteride, a type-1 5-alpha-reductase inhibitor, seems less effective than other antiandrogens in the treatment of hirsutism and less effective in pattern hair loss in females than in males. Although topical minoxidil is the only medication approved for alopecia treatment, antiandrogen-OCP therapy may be superior in those with PCOS. Insulin-lowering treatments, from weight loss to drug treatment, have about a 50% probability of improving menstrual cyclicity and ovulatory status—which seems greater than explicable by the modest reduction in androgen levels that they bring about.268 The effect on hirsutism is neglible. Although weight reduction is indicated in obese PCOS patients, it is typically difficult to achieve. Metformin appears to have more utility than thiazolidines in the management of adolescents because it suppresses appetite and enhances weight loss, albeit to a modest degree. It is most effective in combination with a behavioral weight-reduction program.751 Therapy should start with 500 mg daily of the extended release form before the evening meal, with an increase in the dose by 500 mg per week to a maximal dose of 2,000 mg daily as tolerated. The larger doses are often better tolerated when divided into two daily doses. It is advisable to obtain a comprehensive metabolic panel at baseline and every 3 to 6 months, or as necessary, because of the rare complication of lactic acidosis. Other hormonal manipulations may be useful in specific unusual situations. Prednisone therapy has little utility in the management of the hirsutism or menstrual irregularity of PCOS, although it may potentially be worth a trial in the atypical nonobese hirsute patient with isolated FAH as a single 5% to 7.5-mg bedtime dose. Gonadotropin-releasing hormone agonists are an oral contraceptive alternative when OCPs are contraindicated. They should be used with estradiol replacement therapy.

Future Directions Tremendous advances continue to occur in our understanding of puberty. The identification of genes involved in ovarian differentiation, the discovery of new hormones and hormone receptors, new insights into the regulation of gene transcription and signal transduction, further identification of the role of genetic factors and prenatal imprinting on pubertal disorders, and advances in the application of mass spectrometry to steroid assays can also be anticipated to occur in the next 5 years. We are in the midst of an explosion of information in the biological sciences. It is becoming clear that the body puts a wide but finite repertoire of hormones and growth factors to myriad and unexpected uses. Many concepts we hold dear at this moment are at the best likely to be shown to be oversimplifications; at worst, wrong. New information comes to light faster than we can assimilate it. The understanding of the interactions of the human genome with environmental factors can be expected to

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yield new insights into our understanding of puberty and its disorders.

ACKNOWLEDGMENTS The authors’ studies were supported in part by USPHS grants U54-041859 (RLR, SR) and RR-00055 (RLR).

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737. Rosenfield RL (2006). Polycystic ovary syndrome in adolescents. In Rose BD (ed.), UpToDate.Wellesley, MA: UpToDate. 738. Azziz R, Carmina E, Dewailly D, et al. (2006). Criteria for defining polycystic ovary syndrome as a predominantly hyperandrogenic syndrome: An androgen excess society guideline. J Clin Endocrinol Metab 91:4237–4245. 739. Taylor AE, McCourt B, Martin KA, et al. (1997). Determinants of abnormal gonadotropin secretion in clinically defined women with polycystic ovary syndrome. J Clin Endocrinol Metab 82:2248–2256. 740. Arroyo A, Laughlin GA, Morales AJ, Yen SSC (1997). Inappropriate gonadotropin secretion in polycystic ovary syndrome: Influence of adiposity. J Clin Endocrinol Metab 82:3728–3733. 741. McKenna T (1988). Pathogenesis and treatment of polycystic ovary syndrome. N Engl J Med 318:558. 742. Rosenfield R (1997). Current concepts of polycystic ovary syndrome. Baillière’s Clin Obstet Gynaecol 11:307–333. 743. Meldrum D, Frumar A, Shamonki I, et al. (1980). Ovarian and adrenal steroidogenesis in a virilized patient with gonadotropinresistant ovaries and hilus cell hyperplasia. Obstet Gynecol 56:216. 744. Carbunaru G, Prasad P, Scoccia B, et al. (2004). The hormonal phenotype of Nonclassic 3 beta-hydroxysteroid dehydrogenase (HSD3B) deficiency in hyperandrogenic females is associated with insulinresistant polycystic ovary syndrome and is not a variant of inherited HSD3B2 deficiency. J Clin Endocrinol Metab 89:783–794. 745. Suikkari A-M, MacLachlan V, Montalto J, Calderon I, Healy D, McLachlan R (1995). Ultrasonographic appearance of polycystic ovaries is associated with exaggerated ovarian androgen and oestradiol responses to gonadotropin-releasing hormone agonist in women undergoing assisted reproduction treatment. Hum Reprod 10:513–519.

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746. Okon MA, Laird SM, Tuckerman EM, Li TC (1998). Serum androgen levels in women who have recurrent miscarriages and their correlation with markers of endometrial function. Fertil Steril 69:682–690. 747. Kaltsas GA, Isidori AM, Kola BP, et al. (2003). The value of the low-dose dexamethasone suppression test in the differential diagnosis of hyperandrogenism in women. J Clin Endocrinol Metab 88:2634–2643. 748. Leibel NI, Baumann EE, Kocherginsky M, Rosenfield RL (2006). Relationship of adolescent polycystic ovary syndrome to parental metabolic syndrome. J Clin Endocrinol Metab 91:1275–1283. 749. Ehrmann DA, Barnes RB, Rosenfield RL, Cavaghan MK, Imperial J (1999). Prevalence of impaired glucose tolerance and diabetes in women with polycystic ovary syndrome. Diabetes Care 22:141– 146. 750. Venn A, Bruinsma F, Werther G, et al. (2004). Oestrogen treatment to reduce the adult height of tall girls: Long-term effects on fertility. Lancet 364:1513–1518. 751. Gambineri A, Patton L, Vaccina A, et al. (2006). Treatment with flutamide, metformin, and their combination added to a hypocaloric diet in overweight-obese women with polycystic ovary syndrome: A randomized, 12-month, placebo-controlled study. J Clin Endocrinol Metab 91:3970–3980.

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C H A P T E R

15 Turner Syndrome PAUL SAENGER, MD

Introduction The X Chromosome and the Chromosomal Karyotype X Chromosome Genes Genomic Imprinting Multiple X Chromosomes Turner Syndrome Incidence and Etiology Prenatal Diagnosis Indications for Karyotype Clinical Findings Physical Features Skeletal Growth Disturbances Lymphatic Obstruction Unknown Factors: Eye, Ear, Skin Physiologic Features Skeletal Growth Failure Growth-Promoting Therapy Safety of Growth-Promoting Therapy Otitis and Hearing Loss Germ Cell Chromosomal Defects Gonadal Failure

Introduction It has been more than 60 years since Henry Turner, an internist, reported the clinical characteristics of the seven patients whose phenotype now bears his name.1 Six of these women are shown in Figure 15-1. These women had short stature in association with sexual infantilism, webbing of the neck, low posterior hairline, and increased carrying angle of the elbows (cubitus valgus). In 1930, Ullrich described an 8-year-old girl with short stature; lymphedema of the neck, hands, and feet; subsequent neck webbing, cubitus valgus, and other phenotypic abnormalities (including a high arched palate, ptosis, low-set auricles, and small upwardly curved

Gonadoblastoma Cardiovascular Abnormalities Ongoing Cardiac Care Monitoring for Aortic Dilation Pregnancy and Cardiac Care Growth Hormone Treatment and the Cardiovascular System Renal and Renovascular Abnormalities Unknown Metabolic Factors Autoimmune Disorders Gastrointestinal Disorders Carbohydrate Tolerance Neuropsychological Features Recommendations Management: Pediatric and Adult Evaluation Initial and Follow-up Studies in Adolescence and Adulthood Endocrinologic Management Transition Management The Adult with Turner Syndrome

nails); and several other features that are now associated with Turner syndrome. This gave rise to the less common but more appropriate eponym Ullrich-Turner syndrome. Ullrich later recognized that his patients and those of Turner appeared to have the same condition.2 He also called attention to the work of Bonnevie, who described a group of congenital anomalies in mice consisting of distention of the neck and malformations of the ears, face, and limb buds—all secondary to dissection of the subcutaneous tissues by fluid. This “bleb” mechanism for producing multiple anomalies was suggested by Ullrich as being responsible for the cervical lymphangiectasia noted in some human female abortuses that appeared to produce a scarred webbed neck (pterygium colli). Ullrich

610

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Figure 15-1 Patients exhibiting the syndrome of infantilism, webbed neck, and cubitus valgus. Note the height marker at the left indicating the short stature. [From Turner HH (1938). A syndrome of infantilism, congenital webbed neck and cubitus valgus. Endocrinology 23:566.]

proposed the eponym status Bonnevie-Ullrich to describe the set of specific anomalies arising from a single mechanism (lymphangiectasia) and resulting in the phenotype of Turner syndrome. The links among these phenotypic descriptions, the pathologic evidence of streak ovaries, and the abnormal X chromosomes came with the introduction of the technique for sex chromatin identification by Barr and the demonstration that most patients with Turner syndrome lacked the sex chromatin material.3 Initially, this absence of sex chromatin (or lack of a Barr body) was associated with “maleness” because a similar pattern was found in normal phenotypic males. Only after it was demonstrated that it was the second X chromosome that in the inactivated state constituted the Barr body was it clear that the 45,X karyotype would result in a chromatin pattern similar to the normal 46,XY karyotype. It was not until 1961 that techniques became available for the analysis of the chromosomal constitution and the sex chromosome constitution was shown in a 14-year-old phenotypic female with Turner syndrome to be indeed 45,X.4 Thus, Turner syndrome patients were not XY males but in most cases 45,X females. The original patient of Ullrich was studied in the 1960s and found to have a 45,X karyotype (D. Knorr, personal communication to the author). One of the original seven patients Turner described was also reinvestigated and found to have a 45,X chromosomal karyotype.4

The X Chromosome and the Chromosomal Karyotype A chromosomal karyotype (prepared from peripheral leukocytes, skin fibroblasts, bone marrow elements, or tissue samples) should always be used in making the definitive diagnosis of Turner syndrome. The normal karyotype consists of 22 pairs of homologous autosomes and 1 pair of sex chromosomes. The chromosomes were classified originally into seven groups, A through G, according to the length of the chromosome and the posi-

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tion of the centromere. By convention, the short arms were designated by lowercase p (petit), whereas the long arms were assigned the next letter in the alphabet (lowercase q). The X chromosome has the characteristic of a C-group chromosome. It is similar in size and has a metacentric centromere. The Y chromosome, on the other hand, is a small structure—similar in configuration to the G-group chromosomes.5 If part of the short arm of one of the X chromosomes were missing, the karyotype would be reported as 46,XXp2. Similarly, if part of the long arm were deleted this would be designated 46,XXq2. Should a ring (r) chromosome be identified, composed of material with X-like staining patterns of both the short and long arms, it would be designated 46,X,r(X). In some cases, the ring X chromosome is tiny. Those patients with a small ring X may be much more severely affected than those with a nonmosaic 45,X karyotype. They may have severe mental retardation, developmental delay, profound growth retardation at birth, and multiple congenital anomalies—including dysmorphism (coarse facial features), epicanthal folds, upturned nares, long philtrum, hypertelorism, strabismus, soft-tissue syndactyly of upper and lower limbs, and increased frequency of heart defects (particularly ventricular septal defects and mitral valve stenosis).6-8 Small ring X chromosomes are more detrimental than large ones because they are unable to inactivate, and therefore some genes (those within the ring) are expressed from both the X ring and the normal X chromosome. It has also been shown that the X inactivation specific transcript (XIST) locus on the X chromosome is uniquely transcribed on the inactive X chromosome. It has been proposed that this locus is required for a chromosome to become inactive. Failure of X inactivation has been observed to result from deletion of the XIST gene or deficient transcription of XIST. Ring chromosomes may have a functional disomy for genes present on the ring, leading to the more severe phenotype. The XIST locus is expressed only from the inactive X chromosome residing at the putative X inactivation center. In patients

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with tiny ring X chromosome XIST, transcription is therefore absent and the inability of these rings to be inactive is responsible for the severe clinical phenotype often associated with severe mental retardation.9,10 Another recognized rearrangement is when the entire X chromosome appears to consist of almost two complete long arms with little short-arm material or two short arms with little or no long-arm material. These structurally abnormal chromosomes are called isochromosomes, and the most common one is for the long arm [X,i(Xq)]. Banding and other molecular marker techniques have been used to number all regions of the chromosome, and very specific deletions, translocations, and rearrangements are designated by their numerical region on the short arm or on the long arm.

X CHROMOSOME GENES Concurrent with the development of techniques for chromosome identification, cytogenetic, clinical, and experimental investigations were carried out to attempt to localize specific genes to the X chromosome and to characterize which regions of the chromosome are responsible for the genotypic and phenotypic abnormalities that occur in women with X chromosome disorders. It is beyond the scope of this discussion to review the historical data localizing the genes for such factors as the Xga blood group antigens and the genes for enzymes such as phosphoglycerate kinase and
-galactosidase to the X chromosome, except to mention that genetic studies of families often use the measurement of these gene products in linkage analysis. Similarly, although a complete review of X-linked disorders (such as hemophilia A,11 red-green color blindness, ocular albinism, muscular dystrophy, and ichthyosis) is not feasible here, it is noteworthy that full expression of these disorders has been reported in females with Turner syndrome. Genetic determinants for thyroxine-binding globulin is X linked as well, and thyroxine-binding globulin deficiency has been reported in a 45,X member of an affected kindred.12 With the introduction of molecular techniques for the exploration of the human genome, the X chromosome is being mapped at a rapidly expanding pace. The techniques include classic Southern blot analysis of DNA with X- or Y-specific probes, fluorescence in situ hybridization,13 bivariate flow karyotyping,14 and polymerase chain reaction amplification. Parenthetically, the fluorescence in situ hybridization technique (with both X and Y probes) can be applied to entire cells and tissues—not unlike a molecular version of the historical buccal smear—and is especially useful in identifying low-frequency cell lines and unknown markers.15 Numerous studies have examined the relationship between the chromosomal karyotype and the phenotypic characteristics that appear in Turner syndrome. In a classic article by Ferguson-Smith,16 the karyotypes of 307 patients with various forms of gonadal dysgenesis were correlated with their clinical findings. He noted that short stature was the only clinical finding invariably associated with the 45,X karyotype. Complete gonadal insufficiency was not always present because seven pa-

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tients with a 45,X karyotype demonstrated evidence of spontaneous puberty. He also noted that as a whole patients with mosaic karyotypes, including one normal XX line (i.e., 45,X/46,XX), tended to have fewer phenotypic abnormalities. Save for a very few mosaic patients who were not short, however, on physical examination individual patients with mosaicism could not be readily distinguished from patients with the monosomic karyotype. Finally, he proposed that the area of the X chromosome responsible for the disturbance in growth was localized to the short arm of the X chromosome. Although banding studies were not yet available at the time of this study, the majority of these observations are still valid. A subsequent study of a large group of patients, which included banding,17 confirmed that short stature is the only characteristic present in virtually 100% of patients. Phenotypic abnormalities are most easily understood as resulting from altered dosage of certain genes.18 Genes expected to have an altered dose in Turner individuals are those that escape the process of X inactivation and that have functional homologues on the Y chromosome. Molecular studies to further correlate critical regions with phenotype have tended to confirm the distal pseudoautosomal region (the region that participates in X-Y meiotic pairing and in which recombination takes place) of the short arm as the putative site of at least one stature gene.19 Although this idea was proposed in the pathogenesis of Turner syndrome for many years, only in the past several years have strong candidate genes emerged (Figure 15-2). Zinn et al.20 have argued that there is locus in the interval Xp11.1-p22 encoding for the gene (or genes) pertinent to stature, and have proposed that the transcriuption factor ZFX is the likely candidate gene. To date, there are no genetic data available that would explain the soft tissue and visceral stigmata (such as lymphedema, webbing of the neck, and congenital heart failure) in Turner syndrome… It has been proposed that in primary lymphatic hypoplasia tissues and organs in the vicinity of the affected lymphatic system are secondarily affected.21,22 In analogy to genes affecting skeletal and statural growth, it is inferred that a lymphogenic gene escaping X inactivation is present on the sex chromosomes. It has been suggested that genes residing in the vicinity of Xp11.3 are relevant to this issue. USP9X (DFRX) is a second candidate for the Turner syndrome gonadal dysgenesis gene. DFRX (drosophila fat facets related X) is the human homologue of a fruit fly gene involved in oogenesis and eye development. DFRX escapes X inactivation and maps to Xp11.4, a region of proximal Xp implicated in ovarian failure. Two other candidate genes have been described on Xq. RPS4X encodes an isoform of ribosomal protein S4, which lies within a critical region for the lymphedemarelated Turner syndrome phenotype and may also relate to poor viability in utero. Evidence against such a role has been reported.20,21 DIAPH2 is the second gene and is the human homologue of the drosophila diphanous (DIA) gene. DIAPH2 is required for normal ovarian function.2,22 The prototypes of genes that would escape X inactivation and show expression from the Y chromosome are

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A

B Tel 0

I

SHOX

500 1000 1500 2000 2500 Kbp Chromosomal localization Stature critical interval II III IV V Vla Vib Genomic structure Homeodomain SHOXa cDNA forms SHOXb

C

Turner syndrome

Leri-Weill syndrome Langer dysplasia

Figure 15-2 (A) X and Y chromosomes showing the terminal pseudoautosomal regions (Xp22.3 and Yp11.3) where the SHOX gene has been identified and cloned. (B) The X chromosome with the Turner syndrome critical region and (candidate) genes. [From Zinn AR (1997). Growing interest in Turner syndrome. Nat Genet 16:3.] Reprinted by permission from Macmillan Publisher Ltd: Nature Genetics (16(1): 3-4), copyright (1997)

Idiopathic short stature

genes in the pseudoautosomal region (PAR) of the X and Y chromosomes located at the distal ends of the short and long arms. In 1997, two groups18,24 described independently a pseudoautosomal gene—both using a positional cloning strategy. The gene is 500 kb from the telomere of the sex chromosomes (Figure 15-2). It is located in the pseudoautosmal region 1 (PAR 1) on the distal end of the X and Y chromosomes at Xp22.3 and Yp11.3. Because genes in PAR 1 do not undergo X inactivation, healthy individuals express two copies of the SHOX gene: one from each of the sex chromosomes in 46,XX and 46,XY individuals. Because this gene contained a homeobox, it was named SHOX for “short stature homeobox-containing” gene. There are two isoforms, SHOX A and SHOX B. Both forms were shown to be expressed from the active as well as the inactive X chromosome, with the highest expression levels in bone marrow fibroblasts. The SHOX gene covers a genomic region of approximately 40 kb, consists of seven exons, and encodes two transcripts generated by alternative splicing of its 39 prime exons. It is expressed highly in bone morphogenetic tissues. SHOX mutations have also been described in Leri-Weill dyschondrosteosis, a pseudoautosomal-dominant condition with greater penetrance in females than in males. These patients have shortening and bowing of the radius (with a dorsal subluxation) of the distal ulna, resulting in a dinner-fork-like twist (Madelung deformity) and with distal hypoplasia of the ulna and proximal hypoplasia to aplasia of the fibula (mesial segments). Haploinsufficiency of SHOX is thus the cause of Leri-Weill dyschondrosteosis deformity and probably part of the cause for the short stature seen in Turner syndrome.25

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The rare homozygous (or compound heterozygous) form of SHOX deficiency is referred to as Langer mesomelic dysplasia. It is characterized by extreme dwarfism, profound mesomelia, and severe limb deformity. The elucidation of the role of SHOX in the growth failure of Leri-Weill dyschondrosteosis and Turner syndrome adds to our understanding of the multifactorial nature of growth.26 SHOX is so far the only molecularly characterized growth gene on the X chromosome. Further evidence for SHOX as the short stature gene in Turner syndrome comes from XY females with interstitial Yp deletions and from 45,X/46,X,der (X) patients. These patients have Turner stigmata but are of normal size. The Turner growth gene in the pseudoautosomal region is present in double dosage in these females.22,27 Could SHOX haploinsufficiency also account for additional somatic features in Turner syndrome? SHOX expression also occurs in the first and second pharyngeal arches, ulna, radius, elbow, wrist, and equivalent bones of the leg. This suggests an involvement of SHOX-related growth impairment in the expression of additional somatic Turner syndrome stigmata such as high arched palate, abnormal auricular development, cubitus valgus, genu valgum, and appearance of the carpal bones (including the characteristic short fourth metacarpals). The protein is specifically expressed in the growth plate in hypertrophic chondrocytes undergoing apoptosis, and appears likely to play an important role in regulating chondrocyte differentiation and proliferation.28-31 The state of SHOX haplonsufficency appears to be substantially responsible for the average 20-cm height deficit in untreated women with Turner syndrome. These studies have led to the concept of SHOX

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haploinsufficiency as a common underlying mechanism in the short stature of Turner syndrome, Leri-Weill dyschondrosteosis, and 2% to 15% of children with idiopathic short stature.25,30 Variations in the expression level of the remaining (intact) SHOX gene copy help to explain the variable severity in the manifestations of SHOX mutations. Alternatively, the cause for phenotypic variability might reside in chemical properties of the SHOX encoded transcription factor. An additional area that requires further study is the association of features of Turner syndrome with karyotypes that have no apparent X chromosome abnormality. One group of patients consists of those in whom the phenotype of webbed neck and short stature is associated with normal chromosomes in XX females and XY males. A group of these patients was described by Noonan,32 who noted the frequent occurrence of pulmonic valvular heart disease. The eponym Noonan syndrome is applied to these patients. This syndrome is often confused with Turner syndrome, and when it occurs in males it has been referred to as the male Turner syndrome phenotype. The chromosomes are normal in 46,XX females and 46,XY males who bear this phenotype, however. No point mutations in the SHOX gene have been described in patients with Noonan syndrome.33 Their growth failure must have other causes, and a gene PTPN 11 encoding a tyrosine phosphatase SHP-2 as one of the causes of Noonan syndrome has been identified.34 Noonan syndrome patients with short stature (approximately 50%) respond poorly to growth hormone and may have some form of growth hormone resistance The gonads of the females do not show the early follicular loss or fibrosis characteristic of X chromosome abnormalities, nor is there a chromosomal abnormality in those males in whom cryptorchidism occurs. Therefore, Noonan syndrome in both males and females should be clearly separated from Turner syndrome. The nomenclature, evaluation, genetic counseling, and endocrine management that are appropriate to the female with Turner syndrome do not apply to patients with Noonan syndrome. Another area of confusion arises from the demonstration of gonadal dysgenesis or aplasia in 46,XX females without demonstrable chromosomal abnormalities. In this condition, often called “pure gonadal dysgenesis,” most of these patients tend to have normal phenotypes and normal stature—and some have similarly affected siblings, which is consistent with an autosomal-recessive pattern of inheritance. Finally, there is a group of phenotypic female patients with gonadal dysgenesis and a 46,XY karyotype. These patients (who should not be confused with those with testicular feminization) also have a genetic disorder affecting testicular induction such that gonads do not form. Thus, the concept of gonadal dysgenesis (elegantly reviewed by Opitz and Pallister35) includes a large number of disorders. For the purpose of this chapter, Turner syndrome will be used to describe the patient with an abnormality of the chromosomal karyotype involving loss of part or all of the X chromosome associated with phenotypic abnormalities that include short stature and the potential for or the presence of ovarian failure.

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GENOMIC IMPRINTING The selective silencing of certain alleles depending on the parent of origin is known as genomic imprinting. The putatively imprinted traits in Turner syndrome reflect typical differences between eukaryotic males and females. So far there are three areas in which X-imprinting effects have been found: cognitive function, statural growth, and possible visceral adiposity and lipid metabolism. They are at present rather preliminary and require independent confirmation as well as further work to define the genetic and epigenetic mechanisms.36

MULTIPLE X CHROMOSOMES When there is an excessive rather than deficient number of X chromosomes, ovarian function may also be impaired. The incidence of the 47,XXX (triplo-X) karyotype in newborn phenotypic female infants is approximately 1:1,000.37 The majority of these patients do not have obvious phenotypic or developmental abnormalities and would be unrecognized except for the research-based chromosome screening programs that identified them. When prospective studies are performed on women with the 47,XXX karyotype, however, infertility and gonadal failure are noted more frequently than in the normal population.38 Gonadotropins are increased in the plasma, indicating that the hypogonadism is caused by primary gonadal failure. Histologic data are scanty, but the mechanism of ovarian failure is presumed to be accelerated follicular atresia. Therefore, the manner in which this diagnosis is established is somewhat similar to that of the phenotypically normal patient with Turner syndrome. Primary gonadal failure per se, whether short stature is present or not, necessitates a complete chromosomal karyotype. Should the triplo-X karyotype be identified in a girl before pubertal development, however, assurance may be given that the majority of the patients are fertile. They appear to have a spectrum of neurodevelopmental disorders characterized by gross and fine motor dysfunction,39 speech and language deficiencies, and a high incidence of psychiatric disturbances.40 There is also a low incidence of chromosomal aneuploidy among their offspring.41 Although amniocentesis might be performed during pregnancy, the value of this procedure in prenatal counseling is debatable. The 48,XXXX karyotype is exceedingly rare,42 with fewer than 50 cases reported. These patients are also phenotypic females, but they tend to have more phenotypic abnormalities—including skeletal dysplasias, intellectual impairment, and speech dysfunctions.43 The skeletal abnormalities, including those of carrying-angle varus disturbances and radioulnar synostosis, are similar to those in Klinefelter syndrome. Thus, in contrast to the increased carrying angle of the 45,X patient or the normal angle of the 46,XX patient the angle of the 48,XXXX patient may be decreased to an extent indistinguishable from that of the 47,XXY or 48,XXXY male. In these so-called tetra-X women fertility appears to be decreased to an extent even greater than in triplo-X patients. Again, primary gonadal failure is responsible.

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Plasma gonadotropin concentrations in adolescence are helpful in predicting whether ovarian function and fertility will be normal. We reported one such patient in whom ovarian development was virtually absent. This case represents one extreme within the spectrum of the abnormality.42 The penta-X karyotype 49,XXXXX has also been reported.44,45 It is extremely rare, and the principal phenotypic features are those associated with microcephaly, postnatal growth failure, cardiac defects, and skeletal dysplasia (including radioulnar synostosis). The frequency with which gonadal function is impaired, however, is less well documented because the oldest patient was only 16 years old when reported. This patient was still not fully pubertal, and this suggests that she had ovarian dysfunction. Thus, the evidence confirms that ovarian function may be adversely affected by the presence of multiple X chromosomes. The mechanism is unknown because the precise effect of aneuploidy, be it autosomal or X chromosomal, is unclear. The presence of an additional X chromosome or multiple X chromosomes, however, appears to accelerate the rate of follicular atresia in most patients. In some patients, such as our patient with apparent ovarian agenesis, normal induction of the ovaries may be impaired in the fetus. A possible mechanism for the ovarian failure could be impaired migration of the abnormally constituted germ cells. In no cases without demonstrable or presumptive Y chromosomal material has ovarian neoplasia been documented, and surgical intervention is therefore not indicated.

Turner Syndrome INCIDENCE AND ETIOLOGY The incidence of abnormalities in the sex chromosomal karyotype resulting in the loss of all or part of an X chromosome has been variously reported as 1:2,000 to 1:5,000 in live-born phenotypic females.46,47 Population screenings by Barr body assessment were likely to have underestimated the incidence because mosaic or structural X abnormalities may have been missed. Studies with lymphocyte chromosomal analysis would have detected some mosaic karyotypes such as 45,X/46,XX [as well as some nonmosaic karyotypes such as 46,X,r(X)], but may have missed deletions and rearrangements. Finally, studies with banding techniques applied to the lymphocyte karyotype analysis would tend to detect minor changes in the architecture of the X chromosome (such as partial deletions or an X isochromosome) and should yield a higher incidence. A standard 30-cell karyotype is recommended by the American College of Medical Genetics. This karyotype identifies at least 10% of mosaicism with 95% confidence, although additional metaphases may be counted or fluorescent in situ hybridization (FISH) studies performed if there is suspicion of undetected mosaicism. In rare instances, additional tissues (such as skin) need to be examined if there is strong clinical suspicion of Turner syndrome.48

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Although clinical series using these techniques report a distribution of karyotypes with 45,X approximating 50%,49,50 prospective newborn series might differ. In one prospective study of 34,910 newborn children (17,038 girls) born in Arhus, Denmark,30 cytogenetic analysis detected nine karyotypes consistent with Turner syndrome for an incidence of 1:1,893 live female births. Of note is that only one of the nine had a 45,X karyotype. The other eight were mosaic and included a wide range of abnormalities. A compilation of studies of newborns from centers throughout the world suggests not only that the prevalence might differ in different populations but that within the same center the distribution of karyotypes is shifting toward much higher rates of mosaicism.51 Whether this represents increased technical sophistication in detection or some biologic variable remains to be determined. Our data for a group of 205 patients are shown in Table 15-1. The 45,X karyotype was found in 54.6%, which is consistent with the distribution reported in retrospective clinical series. This percentage, coupled with the prospective incidence data from Aarhus (including the low percentage of 45,X in that series), supports the calculation that the incidence of Turner syndrome in liveborn females is at least 1:2,000. All series, including our own, include a small percentage of patients with cytogenetic evidence of a cell line containing a Y chromosome. For the most part, when those patients are examined they show no evidence of virilization. The same is true for the patients in whom a small marker chromosome, which could be derived from the Y chromosome, is present. The use of molecular techniques has improved the ability to detect Y-specific sequences. Thus, the DNA from Turner syndrome patients can be examined to determine whether the marker chromosome is of Y origin and to detect putative Y sequences in patients with no cytogenetically detectable Y material. The significance of the detection of occult Y chromosomal material is twofold: the association with the risk of gonadoblastoma (see later section on gonadoblastoma for a clinical discussion) and the issue of mosaicism as it relates to fetal survival. With regard to the risk of gonadoblastoma, in one study polymerase chain reaction was used to amplify the gene for the sex-determining region of the Y chromosome (SRY) in a group of 40 patients with Turner syndrome.52 Although the authors detected a very low frequency (1:40) of unrecognized Y material (in an adolescent who had clinical evidence of androgen excess), they suggested that this test may be useful in screening all patients regardless of phenotype. Others,53,54 who reported an even higher frequency of apparent SRY positivity with variable centromeric data, have made the same recommendation. There is no evidence that the SRY gene confers a risk for gonadoblastoma. In fact, there is evidence to the contrary. Deletion analysis of genomic material from numerous patients with gonadoblastoma suggests that the putative gonadoblastoma gene (GBY) is located near the centromere, not on distal Yp.55 A putative candidate gene, TSPY, has been identified by one group.56 In addition, in a report of a girl with Turner syndrome, a gonadoblastoma,57 and a small Y fragment that contained

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TA B L E 1 5 - 1

Summary of the Cytogenetic Findings in 207 Patients with Turner Syndrome No.

%

112 34

54.6 16.6

45,X 46,X,i(Xq)

26

12.6

45,X/46,XX

11 10 5

5.3 4.9 2.4

46,X,r(X) 45,X/46,XY‡ 46,XXq2

4

4 1

Karyotype

1.9

46,XXp2

1.4 0.5

45,X/47,XXX 46,X,t(X;15)

22: 45,X/46,X,i(Xq) 11: 46,X,i(Xq) 1: 45,X/46,X,i(Xq)/ 46,XXp2 24: 45,X/46,XX 1: 45,X/46,XX1mar* 1: 45,X/45,X1mar† All: 45,X/46,X,r(X) 2: 3: 2: 1: 1:

45,X/46,XXq2 46,XXq2 46,XXp2 45,X/46,XXp2 45,X/46,XXp2/ 46,X1mar

(q22.1;q24)

* DNA was examined with Y chromosome–specific probes and found to contain Y sequences. Gonadal streaks were removed, and an in situ gonadoblastoma was found. † Leukocytes examined by fluorescent in situ hybridization with X- and Y-specific pericentromeric probes showed presence of one cell line positive only for X probe and one cell line positive for both probes. Gonadal streaks were removed and were tumor free. ‡ Eight patients had removal of gonadal streaks. One macroscopic calcified gonadoblastoma and three in situ gonadoblastomas were found. Two patients who did not have surgery were lost to follow-up.

pericentromeric material SRY analysis was negative. This supports the assertion of Page58 that in the absence of the centromere the presence or absence of SRY probably has little bearing on the risk of gonadoblastoma. Conversely, if there is a clinical reason to test for occult Y material to assess gonadoblastoma risk, fluorescence in situ hybridization technology with Y-specific centromeric probes may be the applicable method.59 Finally, in one population study of 114 Turner women there were no gonadoblastomas identified among 7 women with occult Y chromosomal material identified only by polymerase chain reaction, and only one gonadoblastoma identified among the 7 women with a karyotypic Y chromosome.60 Thus, in the absence of a demonstrable marker chromosome or clinical evidence of virilization screening of all patients with Turner syndrome for Y sequences is not recommended by the authors at this time.61 Testing for Y chromosome material should, however, be performed in any Turner syndrome patient with a marker61 chromosome. It should be stressed that the prevalence and clinical significance of cryptic Y material detected only by FISH or DNA analysis in patients without virilization or a marker chromosome need additional investigation. False positives may be a problem with highly sensitive PCR-based Y detection methods.62 According to recent analysis of pooled data, true presence of Y chromosome material is associated with an approximately 12% risk of gonadoblastoma.63 The current

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recommendation is for laparascopic prophylactic gonadectomy. It is often assumed that gonads in Turner patients with Y chromosome mosaicism have no reproductive potential, but spontaneous pregnancies in such women have been reported. Thus, preservation of follicles or oocytes may be a future option for some patients undergoing gonadectomy.64,65 The presence of occult chromosomal mosaicism, whether a second cell line contains a second X or Y chromosome, may relate to fetal survival. It is known that the number of affected live births reflects only a small proportion of total conceptuses with X chromosome abnormalities. It is estimated that 99.9% of 45,X conceptuses do not survive beyond 28 weeks’ gestation and that the 45,X karyotype occurs in 1 of 15 spontaneous abortions. It is believed that mosaicism accounts for some of the survival of apparent 45,X fetuses owing to a fetoprotective effect of more than one dose of some loci on the long arm of the X. One piece of evidence to support this hypothesis is that live-born infants with the X,i(Xq) karyotype constitute an unusually large percentage of Turner syndrome individuals compared to the proportion of this karyotype found in early abortuses.46,47 In a cytogenetic and molecular study of 91 patients, low-level mosaicism—attributable to a second cell line (containing small marker chromosomes or small ring chromosomes)—was detected in cell culture of fibroblasts and lymphocytes from a high proportion of individuals originally diagnosed as having 45,X.66 Finally, the expanded use of prenatal chromosomal analysis obtained by chorionic villus sampling or amniocentesis may document mosaicism that is not present at birth. Chorionic villus sampling demonstrates 45,X monosomy at a frequency of 1% compared to 0.08% at amniocentesis. Although part of the discrepancy can be explained by failure of some 45,X pregnancies to survive or a tendency of monosomic cell lines to die out,67 a proportion may be caused by anaphase lag during development of the cytotrophoblast but not the mesenchymal core of the villus.68 Some discrepancies have also been reported between mosaicism detected on amniocentesis and results of postnatal karyotype analysis.69 The significance of mosaicism and the differences between prenatal and postnatal karyotype go beyond issues of mechanism to create a significant genetic counseling dilemma. Termination of pregnancy for false positive 45,X results on chorionic villus sampling has been pointed out as a danger of this method.70 In addition, all infants who have been diagnosed prenatally with Turner syndrome merit a repeat karyotype on delivery because maternal cell contamination, placental mosaicism, and other factors could result in differences between the prenatal and postnatal karyotype. The 45,X/46,XY karyotype present in multiple cultures of amniotic fluid cells presents a different problem because this may be associated with a spectrum of phenotypes from normal female to normal male to genital abnormalities, including the phenotype of mixed gonadal dysgenesis. In a large collaborative international survey of more than 730 cytogenetic laboratories, 92 cases of the 45,X/46,XY mosaicism were identified.71 Seventy-six patients had physical examinations either at birth or at

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termination of pregnancy. Ninety-five percent had normal male genitalia, and only 5% had genital ambiguity or female genitalia with features of Turner syndrome. There was no association between phenotype and the degree of mosaicism. Ultimate gonadal function, stature, and tumor risk remain to be studied. Similar data were reported by Hsu. In her series of some 80 prenatally diagnosed patients, 74 (92%) had normal male external genitalia.72 The role of prenatal ultrasonography as an adjunct in counseling was not evaluated in the series but is advocated by Hsu. These data contrast with a large series of postnatally reported patients with this mosaicism.73 In this series, there was a wide spectrum of abnormal phenotypes that could not be correlated with the degree of mosaicism found in blood. Therefore, with prenatally diagnosed 45,X/46,XY mosaicism the majority of patients will have normally appearing male genitalia at birth. Only determination by physical examination, starting in utero and completed postnatally (not by karyotype), can distinguish patients in whom male gonadal function is sufficient for complete masculinization of external genital development from those with genital ambiguity or normal female genitalia and a Turner phenotype. The high incidence of X chromosome monosomy raises multiple questions about etiology. In abortuses, there is an inverse correlation with maternal age for the 45,X karyotype but not for mosaic or structural X abnormalities.74 There is no correlation with maternal age for viable 45,X infants, however. Thus, young women do not have an increased risk for having a term infant with Turner syndrome—only for having the abnormality in a fetus spontaneously aborted. There are no data to support a positive or negative correlation between the 45,X karyotype in aborted or term pregnancies and paternal age. There is a suggestion that the mechanism responsible for the 45,X conceptus may be different from that responsible for structural X chromosome rearrangements. There is evidence, for example, that the X,i(Xq) karyotype is associated with advanced paternal age.75 If the mechanisms responsible for the loss of the X chromosome are not directly age dependent, the question arises whether there is a relationship between which chromosomes are lost and which are retained. Early studies of the parental origin of the retained X chromosome were performed with Xg blood group linkage but were often uninformative. The introduction of molecular techniques has made it possible to assign parental origin of the single X in almost all affected individuals. With this technique, Mathur et al. and others76-79 have determined that there is no significant parental age effect between the groups retaining the maternal X or the paternal X. In addition, the ratio of maternal X to paternal X is just over 2-3:1, which is consistent with the expected proportion of meiotic or mitotic products—with equal loss at each step, given the nonviability of 45,Y. Finally, Tsezou et al. and others80,81 were unable to find significant differences in physical phenotype between the two groups—providing no evidence for a parental X-imprinting effect. This lack of X imprinting on physical phenotype is also suggested by the finding that there are no significant clinical differences in patients with Klinefelter syndrome between those with

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two maternal X chromosomes and those with one,82 and by the report of mild stigmata of Turner syndrome without any other significant phenotypic disturbances in a patient with 45,X/46,XX mosaicism in whom all X chromosomes are of paternal origin.83 Conversely, there are data to suggest that X-linked imprinted loci may have a subtle effect on neuropsychological function. Skuse and co-workers84 found evidence of an X-linked imprinted locus by comparing females with Turner syndrome who had retained the maternal X (Xm) to those who had retained the paternal X (Xp). In 80 45,X females these authors found 25 to be 45,Xp and 55 to be 45,Xm. Females with a single paternal X chromosome possessed superior social-communicative skills and high-order executive function skills compared with those whose single X was maternal in origin. Neuropsychological and molecular investigation of eight females with partial deletion of the short arm of the X chromosome indicated that the putative imprinted locus escapes X inactivation and probably lies on Xq or close to the centromere on Xp. These preliminary findings, which await confirmation, provide evidence for the evolution of an imprinted X-linked locus that underlies the development of sexual dimorphism in social behavior. The association of a seasonal incidence with a birth abnormality such as Turner syndrome would implicate an environmental cause or an environmental effect on preservation of prenatal viability. When such associations are sought in Turner syndrome, the results are somewhat conflicting. However, when all of the series are reviewed significant peaks do not appear to emerge. Other etiologic factors examined previously include birth order and sibling sex (for which there were no apparent associations) and twinning. In the latter, there appears to be a slightly higher occurrence rate for the 45,X85 and X,i(Xq) karyotypes, although not at a level of statistical significance. At least three cases of Turner syndrome resulting from artificial insemination have been reported.86 In two, studies of blood group antigens suggest that the lost chromosome was of paternal origin. In our series, one patient was the offspring from pregnancy by artificial insemination. The issue of the relationship between autoimmune disorders and chromosomal defects is unresolved. It has been well documented that autoimmune phenomena occur with increased frequency, not only in Turner syndrome but in trisomy 21 and Klinefelter syndrome.87 It has also been suggested that autoimmune disorders occur with increased frequency in first-degree relatives of affected individuals. Preliminary studies of the human leukocyte antigens (HLAs) of Turner syndrome patients and their families failed to show any preponderance of those specific types most commonly associated with autoimmune disorders (HLA B8 and Bw15).88 The role of environmental factors (such as maternal or paternal drug abuse or ethanol consumption, therapeutic medications, cigarette smoking, and so on) in the pathogenesis of Turner syndrome has not been systematically investigated or reported. The lack of evidence for any etiologic mechanisms responsible for the occurrence of Turner syndrome applies equally to the occurrence of the Turner-Down polysomy, or double aneuploidy syndrome.

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In this condition, trisomy 21 is associated with a 45,X karyotype or mosaic cell lines for the X chromosome. Although this is a rare occurrence, its frequency is estimated to be greater than that caused by chance alone.89 Similarly, the double aneuploidy for Down syndrome and Klinefelter syndrome occurs with a frequency greater than that expected from chance alone. There does not appear to be an increased risk for the recurrence of the 45,X karyotype in families. X chromosome structural abnormalities may have an increased risk of recurrence because some deletions, for example, may be transmitted by the carrier of a balanced translocation90 and others may be transmitted on the X by a mother with the Turner phenotype but preserved fertility.91 There also are reports in the literature of trisomy 21 and Turner syndrome occurring in the same sibship.92 We have two such sibships in our series. Taken in conjunction with the evidence that there appears to be an increased risk of a second defect occurring in most other chromosomal disorders, one would have to counsel parents accordingly. In the absence of significantly increased parental age, assignment of a risk figure of 1% may be reasonable.

PRENATAL DIAGNOSIS The use of multiple modalities for prenatal diagnosis and pregnancy monitoring (including chorionic villus sampling, amniocentesis, maternal serum screening, and fetal ultrasound) has increased the number of conditions diagnosed prenatally, including that of Turner syndrome. Although the pitfalls of trying to correlate phenotype with prenatal genotype have been discussed in the previous section, the methodologies that increase the ascertainment of a Turner pregnancy and the issues of outcome based on the diagnosis merit discussion. The measurement of concentrations of human chorionic gonadotropin (hCG), a-fetoprotein (AFP), and unconjugated estriol (uE3) in maternal serum in conjunction with maternal age has been used as a prenatal screen for Down syndrome and trisomy 18. The basis for the utility of this screen is that in fetal trisomy 21 the average hCG is higher than normal and the AFP and uE3 are lower than normal in the presence of fetal trisomy 21. As the screening programs progressed, it was noted that fetuses affected with Turner syndrome were detected among the pregnancies identified at risk for Down syndrome or trisomy 18. In these pregnancies, the abnormality in the screen was most consistently that of high hCG—especially in those with hydropic pregnancies, with more variable findings of normal or low AFP and uE3.93 Subsequent studies of the association of fetal hydrops and increased hCG have concluded that the morphologic defect of hydrops rather than the chromosomal aneuploidy per se may be the reason for the positive detection of the Turner fetus by maternal screening. In addition, second-trimester maternal serum progesterone levels and inhibin A are also elevated in the presence of Turner syndrome with hydrops—further supporting the concept that placental hypersecretion may be a consequence of hydrops rather than aneuploidy.94 Because cystic hygroma, increased nuchal translucency, and overt hydrops can also be detected by ultrasound—

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and ultrasound has detected these abnormalities in some pregnancies identified as at risk for Down syndrome and Turner syndrome by maternal fetal screening—the utility of fetal nuchal translucency as a primary screen for chromosomal abnormalities was evaluated.95 In an unselected population of pregnant adolescents and women, Taipale and co-workers found a 0.8% incidence of nuchal translucency or cystic hygroma. Of the detected fetuses, 24% had an abnormal karyotype and 28% of these had Turner syndrome. All of the fetuses with Turner syndrome had nuchal translucency consistent with hygromas. When fetuses with cystic hygroma are classified by gestational age, autosomal trisomies with cystic hygromas are more often seen in the first trimester and those with Turner syndrome in the second trimester and the fetuses that already demonstrate hygroma in the first trimester are more likely to spontaneously abort.96 A characteristic ultrasound of a 14-week-old Turner fetus with a cystic hygroma is shown in Figure 15-3a and b, compared with a normal fetus in Figure 15-3c. Ultrasound and maternal serum screening are not diagnostic, and to make a prenatal diagnosis of Turner syndrome karyotype confirmation is necessary. Chromosomes should be reevaluated postnatally in all cases. The degree of mosaicism detected prenatally is not generally predictive of the severity of the Turner syndrome phenotype.97 The issue of fetal outcome is affected not only by karyotype and phenotype (hydrops and cardiac abnormalities, in which more 45,X fetuses and those with abnormal phenotypes will spontaneously abort than those with mosaic karyotype) but by decision for elective termination. Buckway and colleagues have reviewed a summary of a series of 24 published studies that reported that only 25% of prenatally diagnosed Turner syndrome pregnancies were continued after counseling, whereas in specific instances in a center with a long-standing developmental focus more than 50% of 45,X pregnancies and more than 80% of mosaic pregnancies were continued.98 They also note that even the hydropic 45,X fetus does not spontaneously abort 100% of the time. Therefore, karyotype and phenotype are not necessarily always a predictor of a poor outcome—and knowledge of the heterogeneity of the phenotype is essential to informed counseling.

INDICATIONS FOR KARYOTYPE The diagnosis of Turner syndrome should be considered in any female with unexplained growth failure or pubertal delay or any constellation of the following clinical findings: edema of the hands and feet, nuchal folds, leftsided cardiac anomalies, low hairline, low-set ears, small mandible, short stature, markedly elevated FSH levels, cubitus valgus, nail hypoplasia, hyperconvex uplifted nails, multiple nevi, characteristic facies, short fourth metacarpal, high-arched palate, and chronic otitis media.61 Newborn screening underdiagnosis and delayed diagnosis remain a problem.99 PCR-based screening methods to detect sex chromosome aneuploidy are feasible but have not been validated on a newborn population sample.

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Clinical Findings Since the original description of Turner, it has been recognized that there are a multiplicity of findings in patients with Turner syndrome—occurring with varying frequencies. More significantly, it has been recognized that the multiple findings may reflect a smaller number of fundamental events. In Table 15-2 and in the following discussion, the common features illustrated by the patients in our series are described—and when possible categorized according to the developmental defects proposed to be fundamentally responsible. What cannot be illustrated are the multiple combinations of findings that occur in any one patient. Thus, it is difficult to appreciate from Table 15-2 that in fact one patient may have only one of the many features described (whereas others may have multiple features).

Physical Features SKELETAL GROWTH DISTURBANCES

Figure 15-3 (A) Fourteen-week-old fetus with Turner syndrome and a cystic hygroma (arrow). (B) A 13-week-old fetus with a normal karyotype and normal nuchal translucency of 1.5 mm (arrow). (C) Same fetus as in view A in transverse plane. Large septated cystic hygroma (arrow) can be seen around the fetal neck. [Courtesy of Pekka Taipale, MD, PhD, Kuppio University Hospital, Finland.]

Again, positive findings will require karyotype confirmation. Screening using FSH levels at 1 to 2 years of age may also be useful.100 Although upholding personal choice about reproduction is a widely embraced ethical principle, decisions to terminate a fetus with Turner syndrome should never be based on misunderstood or unbalanced information.101-103 Outcomes of incidentally detected 45,X/46, XX mosaicism detected by screening on the basis of advanced maternal age, which incidentally is not associated with an increased incidence of Turner syndrome, often tend to be less affected than those diagnosed postnatally on clinical grounds.97,104,105

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The most common physical abnormality associated with Turner syndrome is short stature. The impairment is most pronounced along the longitudinal body axis (the entire problem of the growth disturbance in Turner syndrome is discussed in a subsequent section).106 This gives affected individuals the visual appearance of being stocky or squarely shaped and accounts for the predominantly illusory finding of widely spaced nipples107 and a shieldlike chest. In fact, when chest width and internipple distances are measured and ratios are calculated, the majority of patients do not differ from normal subjects of the same age. What differs is their overall height and the relative width to height of the thorax. Thus, the impaired longitudinal osseous growth is the primary defect. Conversely, the short neck is not illusory but secondary in many cases to hypoplasia of one or more of the cervical vertebrae.108 The osseous abnormalities responsible for the short stature are not limited to the cervical vertebrae. Our own data, and those of others, confirm that long bone growth may be impaired even to a greater degree than vertebral growth. Thus, there is disproportion in the axial segmental measurements resulting in short-leggedness and an abnormal upper-to-lower segment ratio.109 The limbs themselves do not have the radiologic or histologic appearance of a true skeletal dysplasia, however. The low hairline, on the other hand, is secondary to the short neck and to the intrauterine mechanism responsible for the neck webbing (see “Lymphatic Obstruction”). Individual bones appear to be affected to varying degrees. For example, cubitus valgus is commonly appreciated. What this reflects is an increase in the carrying angle. Clinically, this can be measured as the angle of intersection of the long axis of the upper arm with the long axis of the supinated forearm when the elbow is fully extended. In radiographs, it is measured as the acute angle formed between the humerus and the ulna. Normally, in adult women this angle is approximately

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TA B L E 1 5 - 2

Clinical Findings Commonly Described in Patients with Turner Syndrome Primary Defects Physical Features Skeletal growth disturbances

Lymphatic obstruction

Unknown factors

Physiologic Features Skeletal growth disturbances Germ cell chromosomal defects Unknown factors—embryogenic Unknown factors Unknown factors—metabolic

Secondary Features Short stature Short neck Abnormal upper-to-lower segment ratio Cubitus valgus Short metacarpals Madelung deformity Scoliosis Genu valgum Characteristic facies with micrognathia Webbed neck Low posterior hairline Rotated ears Edema of hands/feet Severe nail dysplasia Characteristic ermatoglyphics Strabismus Ptosis Multiple pigmented nevi

100 40 97 47 37 7.5 12.5 35 60 25 42 Common 22 13 35 17.5 11 26

Growth failure Otitis media Gonadal failure Infertility Cardiovascular anomalies Hypertension Strabismus Renal and renovascular anomalies Hashimoto thyroiditis Hyperthyroidism Alopecia Vitiligo Gastrointestinal disorders Carbohydrate intolerance

100 73 90 95 55 7 17.5 39 34 10 2 2 2.5 40

12 degrees—whereas in adult men it is approximately 6 degrees.110 The major determinant of the angle is the depth of the inner lip of the trochlea of the ulna relative to the outer lip. When this relationship is disturbed, abnormalities occur. In many patients with Turner syndrome the angle will be between 15 and 30 degrees (Figure 15-4) as a consequence of developmental abnormalities of the trochlear head. Thus, it is a skeletal abnormality responsible for a physical finding. That the development of the head of the ulna appears to be regulated by the sex chromosomes (and not, as has been previously inferred, primarily by sex hormones during puberty) is suggested by the abnormalities in Turner syndrome as well as by those in other disorders. The carrying angle is most pronounced with the 45,X karyotype, and the angle decreases progressively in association with XX and XY karyotypes. The angle further decreases, approaching cubitus rectus, in patients with an extra X or Y—and frank radioulnar synostosis and radial head

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Incidence (%)

dislocation occur in many patients with multiple extra sex chromosomes.111 The knuckle sign, a depression caused by diminished prominence of the head of the fourth metacarpal so that a straight edge can be placed between the third and fifth metacarpals, is another physical finding that occurs in a large number of patients. It, as well as the so-called knuckle-knuckle-dimple-dimple sign (reflecting a depression of both the fourth and fifth knuckles), is a consequence of abnormally small metacarpals. These signs may be seen on a bone age radiograph and are often clues to the possible diagnosis (Figure 15-5A). Short toes, owing to shortening of the metatarsals, may also be found. Other deformities of the hand and wrist occur commonly. The carpal bones of the first line are frequently arched together abnormally. Superimposed on this is a further deformity (originally described by Madelung in 1898) occurring in 7.5% of our patients. This so-called bayonet deformity is caused by lateral and dorsal bowing

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Figure 15-4 A 16-year-old girl with Turner syndrome and absence of puberty. Note absence of most characteristic stigmata save short stature and an increased carrying angle.

621

of the radius, coupled with the carpal crowding and dorsal dislocation or subluxation of the distal ulna (Figure 15-6).112 It also occurs as part of the autosomal-dominant mesomelic chondrodystrophy, dyschondrosteosis, or Leri-Weill syndrome. We have discussed, in the section on the X chromosome, the SHOX gene on the short arm pseudoautosomal region (PAR 1) of the sex chromosomes. Through other workers identifying the same gene simultaneously, the term PHOG (pseudoautosomal homeobox-containing osteogenic gene) was also introduced. Haploinsufficiency of SHOX has been shown to cause Leri-Weill dyschondrosteosis, as well as other limb skeletal anomalies—such as short fourth metacarpals and cubitus valgus. SHOX deletions or insertions accounted for all Leri-Weill syndrome cases in a recent review.113 Thus, SHOX haploinsufficiency is common to Turner syndrome and Leri-Weill syndrome and accounts for the difficulty in the differential diagnosis. Although gonadal dysgenesis is not a feature of Leri-Weill syndrome, chromosomal analysis may be necessary to distinguish the two—especially in the prepubertal girl without an obviously affected parent. Conversely, gonadal estrogens may play a role in the development of skeletal lesions (in particular, the Madelung deformity) in both conditions.114 The mechanism may be that estrogens exert a maturational effect on skeletal tissues that are susceptible to premature fusion of growth plates because of haploinsufficiency of SHOX, thereby accelerating the development of skeletal lesions such as short fourth metacarpals and Madelung deformity. Both skeletal lesions are associated with premature fusion of epiphyses and may thus become evident in the peripubertal period. This may account for the more pronounced lesions in the girl with

Figure 15-5 Two characteristic hand radiographs. (A) Short fourth metacarpal, the tip falling below a straight line drawn between the third and fifth metacarpals. (B) Generalized lacy (“fish net”) appearance of the carpals and tufting of the distal phalanges, characteristic of the osteoporotic appearance of the bones of patients with Turner syndrome.

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Figure 15-6 A 19-year-old-patient with Turner syndrome and bilateral bayonet-like Madelung deformities of the wrists.

Leri-Weill dyschondrosteosis compared to the girl with Turner syndrome and for the failure of late estrogen therapy in the girl with Turner syndrome to induce Madelung deformity. Scoliosis is reported in a significant number of patients. In our own series, 12.5% had a deformity. The cause was demonstrated radiologically to be secondary to obvious hemivertebrae in three patients and secondary to documented leg-length inequality, with a functional scoliosis occurring secondarily in two patients. In the remaining patients, no obvious bony deformities were noted—and the scoliosis was classified as idiopathic. That severity sufficient to warrant surgical correction has not been reported may be related to the lack of a pubertal growth spurt. As we begin to use more aggressive programs of hormonal treatment for growth acceleration, we may find an increase in clinically significant scoliosis—and appropriate measures should be taken to ensure its early detection. The knock-kneed appearance of many patients described in the literature is caused by abnormalities in the medial tibial and femoral condyles. The medial tibial condyles are enlarged and project medially, the medial femoral condyles are enlarged and project downward below the levels of the lateral condyles, and the epiphyseal plates are deformed and displaced.115 The development of the face is also affected by a number of osseous malformations, contributing in part to the characteristic facies. The high incidences of micrognathia, antimongoloid palpebral fissures, downward droop of the outer corners of the eyes, and epicanthal folds are also consequences of defective facial morphogenesis. The palate is frequently abnormally arched, and when examined using maxillary casts exhibits deformi-

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ties that differ from the more typical gothic or inverted V shape of other syndromes. Instead, an inverted U shape form and a form with a narrow vault and bulges of the lateral alveolar ridges have been described.116 Not manifest clinically but present radiologically is the osteoporotic appearance of the bones. Examination of the hand and wrist for bone age, a procedure frequently performed even before the diagnosis of Turner syndrome is established, often reveals this osteoporotic appearance. In some patients, the carpal bones may be so characteristically affected that the appearance (which we describe as fish net) has been reported as a clue to the diagnosis.117 Others find that ballooning of the tips of the terminal phalanges is a most consistent finding.118 Both findings are illustrated in Figure 15-5B. That this osteoporotic appearance is observed in childhood suggests it may be more related to the developmental localization of SHOX than to primary estrogen deficiency. Both a primary skeletal disorder and estrogen deficiency could compromise bone mass, however. Bone mineral content and bone mineral density are reduced for age in children and adults with Turner syndrome, but these deficits are reduced or diminished when adjustments are made for small bone size and delayed bone maturation.119-121 When looked at in a longitudinal fashion and when compared with control data related to body weight and pubertal status, no advantage was found with early estrogen replacement or growth hormone (GH) therapy in optimizing bone mineralization.119,120 Shaw and associates demonstrated normal bone density in children and adolescents with Turner syndrome using dual-energy x-ray absorptiometry.122 In older adolescents and adults, a normal femoral neck bone mass is not achieved. This may be because of the low-dose estrogen therapy used by some (20 to 30 mg of ethinyl estradiol), which may simply not be enough. Thus, the risk of osteoporosis and fracture appear to be increased— particularly after age 45.123-124 After adolescence, estrogen therapy seems to be the single most important factor in maintaining peak bone mass. In summary, the cause of the apparent early and true late osteopenia in Turner syndrome remains controversial. However, it is most likely the result of an intrinsic bone matrix defect combined with estrogen deficiency or inadequate estrogen replacement.125

LYMPHATIC OBSTRUCTION The appearance of the 45,X fetus illustrated in Figure 15-7 dramatically shows the fetal edema that occurs in many concept uses with Turner syndrome. The edema appears to result from lymphatic malformations and obstruction.126 This type of obstruction may result from a lag in the formation of a communication between the developing jugular lymph sac and the internal jugular vein. This communication normally develops between the fifth and sixth weeks of gestation, and failure to establish it appropriately results in lymphatic distention.127 The search for the gene or genes on the X chromosome responsible for this developmental abnormality is underway. The maldevelopment occurs most often in the nuchal region, with the dilatation of at least two cavities separated

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Figure 15-8 Lateral view of the face of a girl with Turner syndrome, demonstrating low posterior hairline, residual webbing, and micrognathia.

Figure 15-7 A 45,X abortus demonstrating generalized lymphedema. Note the distended cervical region. With resolution of the edema, the redundant skin may cicatrize—resulting in a webbed neck. The edema of the hands and feet may persist and be present at birth. [From Gellis SS, Feingold M (1978). Picture of the month. Am J Dis Child 132:417. Copyright © 1978, American Medical Association.)

by the nuchal ligament. In many cases, each cavity is subdivided by incomplete septa. The cavities extend from the upper part of the occipital bone caudad to the scapular region and medially to beneath the sternocleidomastoid muscle. If the blockage persists, the increased pressure within the lymphatics may alter their development and result in the more generalized malformations of peripheral lymphatics. Peripheral lymphatic hypoplasia or aplasia has also been demonstrated using lymphangiography in adult patients with Turner syndrome.21 Thus, as in the case of the mice with subcutaneous blebs described by Bonnevie a single process may be responsible for a host of the apparently varied physical findings in this syndrome. Webbed neck, or pterygium colli, is perhaps the most obvious consequence of lymphatic obstruction. It results from a scarring process that affects the distended loose skin over the large cystic hygromas that were present in the nuchal region. Some patients may, therefore, have a severe anomaly. Others, however, who never had significant distention or in whom the obstruction was minimal or in whom rupture and decompression occurred early do not have a webbed neck. In fact, in some patients in whom neither webbing nor abnormalities of the cervical vertebrae exist the neck will appear long in proportion to the stature (Figure 15-4). Thus, webbing per se is not a primary anomaly. In our series, only 25% of patients had any webbing at all. The dilatation of the nuchal region is also believed to be responsible for mechanical effects on the pattern of hair direction, resulting in the lower position of the posterior

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hairline (Figure 15-8). This may also be responsible for the heavy extended growth of the eyebrow. Similarly, the dilatation may result in rotation of the axis of the auricle posteriorly and elevation of the lower pinna—resulting in the prominent and low-set appearance of the ears.128 Edema of the dorsum of the hands and feet at birth is an obvious consequence of the incompletely resolved process if it involved peripheral lymphatics. Postnatally, the lymphedema usually resolves—although some patients demonstrate residual involvement. Others may complain of intermittent or recurrent edema, often after institution of estrogen replacement therapy. Less obvious are the mechanical effects the lymphedema may have had on the developing extremities. It is probable that the abnormalities in the nails described in many patients (deeply set into the nail bed with lateral hyperconvexity) are secondary to mechanical distortion of the developing nail bed.129 Severely dysplastic or even absent nails are noted at birth in some patients as a more obvious consequence. The result on the fingertip pads is a predominance of dermal ridge whorl patterning, a characteristic dermatoglyphic feature.129 A compressive or restrictive effect on the developing ossification centers resulting in some of what has been categorized as skeletal defects is also possible.

UNKNOWN FACTORS: EYE, EAR, SKIN The factors responsible for several other common physical features are less clear. Strabismus and unilateral or bilateral ptosis occur more commonly than normal, contributing other features to the characteristic facies. Multiple pigmented nevi, in excess of that expected from familial patterns, occur frequently and were noted in 26% of our patients. Several had surgical excisions of one or more lesions that had been subjected to trauma, and histologic examination did not show evidence of

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Physiologic Features SKELETAL GROWTH FAILURE

Figure 15-9 Unilateral ptosis in a girl with Turner syndrome. Note also significant webbing of the neck on the affected side.

malignant degeneration. The mechanism responsible for the excessive presence of these lesions, especially on the face and arms, is unknown. Because migration of melanocytes into the skin begins at a relatively early stage (10 weeks), it is unlikely that this process is affected by the lymphedema.130

Short stature appears to be the most common phenotypic feature of children and adults with Turner syndrome and has been noted in almost all articles and reviews since Turner’s report in 1938. The first comprehensive assessment of cross-sectional and longitudinal growth and final height did not appear until the report of Ranke and colleagues in 1983.131 Patterns of growth are illustrated in Figure 15-10, which shows cross-sectional height and velocity data from a series of 150 Turner children who had not received therapy for growth promotion. These patterns described by Ranke and colleagues are distinguished by four components: intrauterine growth retardation [with mean birth length 1 standard deviation (SD) below the mean or an average reduction of 2.8 cm] and mean birth weight (not shown in the figure) of 2.18 kg, which is also 1 SD below the mean; a period of what initially appeared to be near-normal growth velocity for 2 to 3 years, which has now been characterized by growth deceleration to a mean of –2.18 SD at 1.5 years132 and a further loss by age 3 years133; after age 3, continued deceleration, so that between ages 3 and 13 years the Turner syndrome girls fall farther and farther away from the normal height curves; and if untreated failure to experience a pubertal growth spurt but continued growth at a slow rate for several more years.

Figure 15-10 Height and height velocity in Turner syndrome. (A) Three hundred eighty-four single measurements of height for 150 children with Turner syndrome. (B) Height velocity from a total of 159 measurements. The normal ranges are shown by the heavy and dashed lines. [From Tanner JM, Whitehouse RH, Takaishi M (1965). Standards from birth to maturity for height, weight, height velocity and weight velocity: British children. Arch Dis Child 41:454, 613; and from Ranke MD, Pfluger H, Rosendahl W, et al. (1983). Turner syndrome: Spontaneous growth in 150 cases and review of the literature. Eur J Paediatr 141:81.]

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A positive correlation is found among height at diagnosis,134 ultimate height achieved, and midparental height.135 The final height deficit, using comparative data on adult heights in patients with different ethnic backgrounds, is approximately 20 cm.136 These authors concluded that the short stature was primarily the result of a generalized abnormality in the growth response of the skeleton but that other genetic factors such as a prolongation of cell generation time associated with the chromosomal disturbance per se137 may also play a part. Lippe et al.138 have shown that disproportionate growth of the lower extremities appears to contribute, in some degree, to the short stature in Turner syndrome. In a group of 16 adult Turner syndrome patients not treated with growth hormone, the mean height was 144.2.0 cm, the sitting height was 79.6 cm, and the sitting height-to-height ratio was 0.55. This observation of a short lower segment has been confirmed by some,139 but not all,140 observers. In addition we found a significant negative correlation between the ratio of sitting height to lower segment and height, indicating that the patients with the greatest proportional reduction in the length of their legs were the shortest. The description of the SHOX gene and its role in statural growth, including mesomelic short stature, may explain the major component of the statural deficit (as discussed previously). After the report of Ranke and colleagues,131 Lyon and associates141 used the data from that report and from three other European centers to synthesize a series of growth curves for Turner syndrome. The curves provided mean height and SD values for age. The mean adult height from these series was 143.1 cm. In the United States, we obtained 3,460 cross-sectional and longitudinal height determinations from 1,363 individuals to validate the applicability of these Western European–derived growth curves to U.S. girls with Turner syndrome.142

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Our data (Figure 15-11, left) demonstrate that U.S. girls with Turner syndrome fit almost exactly the Lyon-Turner curve, with a final height of 144.3 cm, in 584 patients untreated with growth-promoting agents or estrogen before age 18 years. When the smoothed mean values (splines) are compared (right), there is a small but significant increase in the heights of U.S. girls between ages 12 and 16 that might represent the so-called spontaneous growth spurt that has been described by other groups with mathematical models.143,144 With their longitudinal patient data, Lyon and colleagues also noted a very strong correlation (0.95) between the initial height SD on these Turner curves and the adult height SD achieved regardless of whether the patient had received late estrogen replacement therapy and essentially independent of bone age at the time of the first height. Thus, they concluded that one could actually predict, or project, the adult height of a girl with Turner syndrome based on her height at an earlier age. In our study, we validated this projection method with a longitudinal assessment of 56 patients who had not received growth-promoting therapy and for whom we had both childhood heights and heights after the age of 18 years. The results showed a mean difference of less than 1 cm between the projection and the final height achieved. Data such as these have proved invaluable in assessing the efficacy of growth-promoting therapy.

Growth-Promoting Therapy Early studies on the use of growth hormone (GH), dating back to the 1960s, yielded conflicting and inconclusive results.145 Some studies were very short term, others did not use comparable doses of GH—and few patients were followed to adult height. This failure to confirm or preclude a role for GH was confounded by the debate

Figure 15-11 (A) Heights of American girls with Turner syndrome (superimposed on curves of Lyon and associates) representing the 10th, 50th, and 90th percentiles. (B) Spline fit to the American heights (solid curve) together with a dashed curve showing spline fit of Lyon European data. [From Lippe B, Plotnick L, Attie K, et al. (1993). Growth in Turner syndrome: Updating the United States experience. In Hibi I, Takano K (eds.), Basic and clinical approach to Turner syndrome. Amsterdam: Elsevier Science.]

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over the status of GH secretion in Turner syndrome. Although it is clear that girls with Turner syndrome as a group do not have classic GH deficiency, there is no consensus on the nature of their secretory dynamics. In an international workshop on Turner syndrome, four groups reported differing data with regard to spontaneous GH secretion.146-149 These differences in data persist even when age-matched groups are compared. For example, several groups found normal GH secretion in younger girls with Turner syndrome compared with their peers but not in older girls compared with normal adolescents.150-151 Although this might be expected because girls with Turner syndrome do not for the most part experience an estrogen increase with puberty, other groups have not found this difference at puberty.152 In addition, when studied in a longitudinal manner ethinyl estradiol treatment resulted in increased GH pulse amplitude. However, in a cross-sectional study there was no difference in GH secretion between Turner syndrome girls with or without spontaneous breast development or with or without estrogen therapy.153 These data are further confounded by methodologic differences in assessment,154 assay differences,155-156 and biologic variables such as body weight differences157 between Turner syndrome girls and normal girls. Because the consensus is that there is no GH deficiency, however, provocative GH testing is obsolete and need not be performed unless growth velocity is abnormal relative to that expected for Turner syndrome. The use of anabolic steroids in Turner syndrome also dates back to the 1960s,158 but their efficacy in increasing final adult height is still being debated. Some studies indicate that augmented growth velocity may not be accompanied by a concomitant bone age increase and suggest that ultimate height is increased.159-161 Others either found no differences in adult height between androgen- and estrogen-treated girls162 or pointed out that over time changes in stature could make the results difficult to interpret if treated patients were compared with untreated controls of previous years.163 Of the more recent studies, two conclude that androgens alone do have a positive effect on final height165-166 and two conclude that they do not.166-167 Our retrospective analysis of growth data failed to demonstrate that anabolic steroids promoted a statistically significant increase in final adult height. We and others,169,170 however, have confirmed that they do promote marked growth acceleration in the first year or more of therapy—which was perceived as highly beneficial by the patients and their families. The use of a combination of androgen and GH was first reported in 1980, and it was suggested that shortterm combination therapy might promote more rapid growth than either agent alone.170,171 The availability of unlimited recombinant human GH has made it possible to conduct carefully designed studies and to evaluate the growth response to GH and the combination of GH with androgens and/or estrogens. This forms the basis for a large U.S. multicenter study that has now been completed.171 In 1983, this multicenter prospective randomized trial of recombinant human GH alone and in combination with oxandrolone was initiated.

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The mean adult height for the 17 recipients of GH alone was 150.5 cm, which was 8.5 cm above their projected adult heights. For the 43 subjects treated with a combination of GH and oxandrolone, the mean adult height was 152.2 cm—10.4 cm taller than their projected adult heights. Fifty-eight of the 62 GH-treated subjects (94%) attained an adult height greater than their projected adult heights. The mean adult height attained by a retrospective control group as part of this study was 144.3 cm (Figure 15-12). These results compare favorably not only with the matched American control group but with previous reports of final heights in girls with untreated Turner syndrome from the United States and many other countries.131,142,147,172-176 The increment in final height, above the projected adult height, of the group receiving combination therapy was modestly but significantly greater than that in the group receiving GH alone (2.9  0.5 cm). This improved growth response was attained despite the fact that GH administration in the combination group was 1.5 years briefer than in the group receiving GH alone. Combination therapy appears to offer certain advantages over GH alone: increased growth over the first 6 years of treatment (37.3 cm for the combination group versus 31.2 for GH alone), thereby allowing for better normalization of height during childhood; potential for earlier initiation of estrogen replacement because girls will have attained greater heights by adolescence with

Figure 15-12 Most recent height of each subject (or American historical control subject) relative to each subject’s projected adult height (indicated by dotted zero line). Mean increment in height (in centimeters) relative to projected adult height is indicated. Diamond symbols in the combination group indicate the two subjects who discontinued treatment early. [From Rosenfeld RG, Attie KM, Frane J, et al. (1998). Growth hormone therapy of Turner’s syndrome: Beneficial effect on adult height. J Pediatr 132:319.]

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combination treatment; and a briefer requirement for GH. Side effects associated with oxandrolone, 0.0625 mg/kg/ day, were minimal—and skeletal maturation averaged only 1.04 years per year of treatment. Experience with oxandrolone in young girls with Turner syndrome (6 years old) is limited, however, and it remains unclear whether it is better in young patients to initiate therapy with GH alone or in combination with oxandrolone.170 In several other studies in which older Turner syndrome patients were treated with lower doses of GH, improvement of height was not as satisfactory.176 The results of the U.S. study also compare favorably to preliminary data from a second U.S. study177 looking both at the role of GH and at the role of early versus late estrogen therapy. Twenty-nine girls receiving GH alone (plus estrogen replacement at 15 years of age) achieved a mean height of 150.5 cm, which was 8.5 cm above their projected adult heights. This is almost identical to the results of the first U.S. study. The 26 patients in whom estrogen therapy was initiated at age 12 gained only 5.1 cm on average beyond their projected height and achieved a final height of 147.1 cm (Figure 15-13).

Figure 15-13 Height gain for patients treated with growth hormone compared with controls. Data are plotted for individual patients, with the most recent height minus the pretreatment projected height determined as indicated in the text. The mean values for each group are indicated by a dash. Control patients were matched from a historical database (as described in the text). [From Chernausek S, Attie KM, Cara JF, et al. (2000). Growth hormone therapy of Turner syndrome: The impact of age of estrogen replacement on final height. J Clin Endocrinol Metab 85:2439.]

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A multivariate analysis was used to examine several factors that might influence the gain in stature. The number of years of GH therapy before estrogen treatment was a strong predictor. These data show that the early introduction of estrogen has a significant negative impact on adult height. Several published studies have reported that GH therapy results in an average height gain of 5 to 10 cm, a range that represents a surprising variability in outcome.171-182 It appears that at least some of this variability may be caused by differences in the ages in which estrogen therapy was begun in these patients. Some of the reports showed that patients who had more modest apparent gains in final height also had relatively short periods of GH administration before the introduction of estrogen therapy.180,182 The method used to estimate what the adult height would have been without treatment with GH will affect the observed gain in height. Attie and Frane183 compared five methods of predicting adult height in patients with Turner syndrome, using data from patients with Turner syndrome in the United States who had not been given growth-promoting drugs. They found that the Lyon projection method,141 used in the two U.S. studies previously discussed,171,177 was one of the most reliable means of predicting adult height—with the mean error of overprediction being only 0.3 cm. Several studies that did not achieve such reassuring gains in final height180,182 were also utilizing lower GH doses than the U.S. study groups.171,177 This became particularly apparent in elegant studies carried out by the Dutch Advisory Group on GH.184 Their final assessment of 7-year data clearly shows that in a carefully conducted dose-response study the increased dose of GH led to impressive increases in final height exceeding even the data seen in the U.S. studies. These investigators treated 68 patients with chronologic ages between 2 and 11 years and heights below the 50th percentile for healthy Dutch girls. The dosages employed were from 1.3 mg/m2/day (0.23 mg/kg/week) to as high as 2.7 mg/ m2/day (0.63 mg/kg/week). It is clear from this study that there is a dose-response curve and that patients with the higher GH dose grew significantly better. The patients were divided into three groups. In the first year, all three groups received GH at the starting dose of 1.3 mg/m2/day. In the second year, two groups were switched to doses of 2 mg/m2/day. In the third year, one of these two groups was switched to 2.7 mg/m2/day. All were then treated at these doses for the remainder of the study period. After 7 years of GH treatment, mean final heights were 159.1, 161.8, and 162.7, respectively, for the three groups. These investigators concluded that after 7 years of GH treatment most girls with Turner syndrome are growing within the height range for healthy normal girls (Table 15-3, Figure 15-14). In fact, adult height was above 170 cm in 5 girls and above 160 cm in 17 girls. Growth-promoting therapy in these patients was not marked by any major clinical or metabolic side effects (see “Safety of Growth-Promoting Therapy”). Concerns about elevation of IGF-1 levels during GH therapy in Turner syndrome would suggest that inappropriate elevation of IGF-1 levels in the long term should be avoided.185

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TA B L E 1 5 - 3

Final Height in Turner Syndrome Dose U.S. study (Genentech)* GH (n517) GH1oxandrolone (n543) U.S. study (NCGS)† (n5622) Dutch study (dose response)‡ (n568)

0.375 (mg/kg/wk) 0.3751 oxandrolone 50.0625 (mg/kg/day) 0.35 (mg/kg/wk) 0.23 to 0.63 (mg/kg/wk) to 162.3 (154.3-170.3)

Final Height (CM)

Gain (CM)

Duration of Treatment (YR)

150.465.5

8.464.5

7.662.2

152.165.9

10.364.5

6.161.9

148.365.6 158.8 (148.3–172.4) to 16.0 (10.0-24.8)

6.464.9 12.5 (7.8–15.7)

3.761.9 7

* Data from van Es A, Massarano AA, Wit JM, et al. (1991). 24-hour growth hormone secretion in Turner syndrome. In Ranke MB, Rosenfeld RG (eds.), Turner syndrome: Growth promoting therapies. Amsterdam: Elsevier Science 29–33. † Data from Urban MD, Lee PA, Dorst JP, et al. (1979). Oxandrolone therapy in patients with Turner syndrome. J Pediatr 94:823. ‡ Data from Rosenbloom AL, Frias JL (1973). Oxandrolone for growth promotion in Turner syndrome. Am J Dis Child 125:385. GH, growth hormone; and NCGS, Genentech’s National Cooperative Growth Study.

Figure 15-14 Individual heights at the start of the study (s) and after 7 years of growth hormone (d) in groups A, B, and C, respectively. Twelve girls had completed the trial during the 7-year study period (j). Reference curves for healthy Dutch girls (3rd, 10th, 50th, 90th, and 97th percentiles) and for untreated girls with Turner syndrome (North European references for 3rd, 50th, and 97th percentiles) are given. [From Sas TCJ, de Munick Keizer-Schrama SMPF, Stijnen T, et al. (1999). Normalization of height in girls with Turner syndrome after long-term growth hormone treatment: Results of a randomized dose-response trial. J Clin Endocrinol Metab 84:4607.]

In summary, it should be pointed out that final height data reported by many groups in different countries using different study protocols are most likely conservative estimates of the effect of therapeutic intervention with GH on height potential in patients with Turner syndrome. Many of these patients were enrolled at older ages, thus limiting the duration of GH therapy. Doses of GH employed were often well below the mean dose of 0.33 mg/kg/week used in the United States.186 Some investigators began estrogen replacement in patients as early as age 12 years and used relatively high dosages of estrogen. This will only hasten epiphyseal fusion and thus compromise height potential. In general, one can expect only 18 to 24 months continued growth after the initiation of estrogens. If there is still a large height deficit at 12 to 13 years of

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age, estrogen therapy should be delayed. There is no evidence that low-dose estrogen given at any age in growth-promoting dosages before it is used for induction of puberty is advantageous, and it may even curtail final height187 (although spontaneous puberty per se does not appear to reduce final height).188 However, the magnitude of the benefit has varied greatly depending on study designs and treatment parameters. Factors predictive of taller adult stature include a relatively tall height at initiation of therapy, tall parents, young age at initiation of therapy, a long duration of therapy, and a high GH dose.61,189 Therapeutic regimens aiming at early GH treatment initiation and optimizing the dose of GH may well allow for normalization of height during school-age years and thus earlier introduction of estrogens.170,177,189 The recently

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Figure 15-15 Length/height SDS for the nontreatment control group (open symbols) and the growth hormone treatment group (filled symbols) of toddlers with Turner syndrome during the 2year study. Between-group difference at endpoint was 1.6  0.6 SDS (p 0.0001). Mean age at baseline was 24  12.1 months. Published with permission from Davenport ML, Crowe BJ, Travers SH, et al. (2007). Grwoth Hormone Treatment of Early Growth Failure in Toddlers with Turner Syndrome: A Randomized, Controlled, Multicenter Trial. JCEM 93(9): 3406-3416.

completed toddler Turner study in the United States began GH treatment between 9 months and 4 years in 88 girls. GH therapy is effective as early as 9 months of age. Treament with GH should be considered as soon as growth failure (decreasing height percentiles on the normal curve) is demonstrated and its potential risks and benefits have been discussed with the family81 (Figure 15-15).

SAFETY OF GROWTH-PROMOTING THERAPY Human recombinant growth hormone (hrGH) was introduced into the United States in 1985, and worldwide shortly thereafter. Its introduction followed on the heels of the recognition that Creutzfeldt-Jacob disease had been transmitted to patients from some batches of human pituitary-derived GH.190 This serious safety issue, coupled with the fact that hrGH was the first recombinant protein available as a pharmaceutical, has resulted in extensive worldwide scrutiny of this drug. Because it is beyond the scope of this chapter to discuss GH safety in its entirety, we will focus on several specific clinical and metabolic issues related to GH therapy in Turner syndrome. The issue of whether GH alters the body proportions of girls with Turner syndrome has been of interest since the early studies of GH use.191 The report by Sas and associates192 of more than 60 girls observed for up to 7 years on increasingly high doses of GH addresses this issue in depth. Height, sitting height, hand and foot length, and biacromial and bi-iliacal diameters were measured. They note that at the outset Turner syndrome girls have relatively large trunks, hands, and feet and broad shoulders and pelvis compared with height. This is in concert with the reference data,193 illustrating the relative disproportion that these girls have before GH is initiated.

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Long-term GH therapy tends to normalize height, improve the disproportion between height and sitting height, and have little effect on hand length or shoulder or pelvic proportions. Foot length increases disproportionately in a dose-dependent fashion and in their series played a role in the decision of some patients to discontinue GH therapy in the last phases of growth. Scoliosis was not specifically addressed in this paper. The incidence of scoliosis is as high as 35% (Table 15-2) in untreated patients with Turner syndrome. Although it is also reported to be increased after GH treatment compared with GH-deficient patients so treated, it may not be increased compared with untreated patients. In addition, progression to surgical intervention is rare in both the treated and the untreated patient. Sas and associates also evaluated the effect of GH on cardiac left ventricular dimensions and blood pressure in this cohort.193 They found that the growth of the left ventricle was comparable to that of healthy girls, confirming the earlier findings of Saenger and colleagues.192 Sas and associates194 also found that whereas blood pressure in these girls was within the normal range for age at the start of therapy it was significantly higher than in healthy control subjects. Seven years of GH therapy had no significant effect on systolic blood pressure and had a small lowering effect on diastolic blood pressure. Finally, during the course of this 7-year GH trial there was no significant dilation of the aortic root (S.M.P.F. de Muinck Keizer-Schrama, MD, personal communication). Two studies have looked at long-term effects on plasma lipids. van Teunenbroek and co-workers evaluated the effects of GH in two different study groups over time and found that in one group the atherogenic index improved with long-term treatment, whereas in the other it remained the same.196 Querfeld and co-workers evaluated the effect of GH on lipoprotein(a) and found no effect, either alone or in combination with oxandrolone, on lipoprotein(a) levels after a median of 27 months of therapy.197 Lanes and colleagues found normal lipoproteins in the untreated state and a decrease in total and low-density lipoprotein cholesterol during treatment.198 Thus, long-term GH therapy does not appear to have a deleterious effect on cardiac structure or on cardiovascular risk factors and in a high-dose study led to a cardioprotective profile. Other aspects of the safety assessment of GH therapy in these clinical trials showed there were no adverse effects of GH treatment on carbohydrate metabolism. Hgb A1c levels did not change, and glucose levels remained normal. In most studies, fasting and postprandial insulin levels increase during GH therapy but return to normal after GH treatment is stopped.199,200 Liver abnormalities, characterized by transient increases in transaminase and/or gglutamyl transpeptidase concentrations, have been occasionally reported in GH-deficient and Turner syndrome 2 children during GH therapy.201,202 Spontaneous resolution usually takes place within 3 to 6 months of identification. In Turner syndrome specifically, treatment with estrogens or androgens or the presence of autoimmune liver disease was more commonly associated with the increase in liver enzymes than was GH treatment.201 Conversely, in adult women with Turner syndrome hepatic enzyme levels are higher than in normal women but have been found to decrease with estrogen therapy.203 Thus, the

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tendency of liver enzymes to be increased in both girls and women with Turner syndrome appears to reflect both intrinsic and exogenous mechanisms but rarely reflects active dysfunction or disease. Finally, what appears to be hepatic enzymes may be confounded by transient increases in muscle creatine phosphokinase (CK) that has been reported after GH therapy.204 In 1993, Bourguignon and colleagues205 reported that melanocytic nevi may grow more rapidly during GH therapy based on clinical observation and immunoantibody staining for actively growing melanocytes. Subsequently, the issue has been of concern—albeit there are no reports of increased melanoma frequency in patients with acromegaly. Zvulunov and associates evaluated this question in two studies,206,207 concluding that whereas the number of nevi are increased in Turner syndrome GH therapy in GH deficiency is not associated with an increased count or density of nevi (both factors in increasing melanoma risk) and is therefore unlikely to potentiate the risk for melanoma in Turner syndrome. To date, this conclusion is supported by a lack of reports indicating an increased incidence of melanoma in Turner syndrome patients treated with GH. The role of GH as an immunoregulator has been reviewed.208 Although there are data that suggest that GH replacement therapy has a transient effect on B cells,209 clinically significant effects of GH on immune function have not been described. Because Turner syndrome patients are known to have a high incidence of autoimmune thyroiditis, the effect of GH therapy on the incidence of thyroid antibodies was studied.210 No increase in the prevalence of thyroid antibodies was observed. Among adverse events related to GH reported in the Pharmacia International Growth Study database (KIGS), the incidence of pseudotumor cerebri appears to be higher in Turner syndrome patients (and in patients with chronic renal failure and organic GH deficiency) than in other subgroups of patients treated with GH. Scoliosis is also higher in Turner syndrome and in Prader-Willi syndrome patients, conditions known to be associated with scoliosis in the untreated state. Malignancy rates are not increased in Turner syndrome during GH therapy.211

OTITIS AND HEARING LOSS Perhaps the most common medical problem reported by patients with Turner syndrome is bilateral otitis media. In a series of 76 patients, Anderson and co-workers212 recorded medically significant middle ear infection in 68%—with more than one half of these patients reporting not only recurrent episodes but spontaneous perforations, the need for surgical treatment, or both. Hearing problems and ear malformations correlate with karyotype.213-215 In Figure 15-16, the audiogram shows the typical dip in sensorineural hearing paricularly prevalent in higher frequencies. In our series, complete system reviews were recorded for more than 80% of patients. Of these, almost 75% had undergone tonsillectomy and adenoidectomy because of recurrent otitis or had had polyethylene tubes placed for the drainage of serous otitis media or had both procedures performed. Of these, five patients had also under-

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Hearing level (dB)

630

0.125 0 10 20 30 40 50 60 70 80 90 100 110

0.25

0.5

Frequency (kHz) 1 2

4

8

Right ear air conduction Left ear air conduction Right ear bone conduction Left ear bone conduction

Figure 15-16 Audiogram showing the typical dip with the peak of 35 dB in the 1.5-kHz frequency region in a 12-year-old girl with Turner’s syndrome (karyotype 45,X). This girl has no subjective hearing problems.[From Stenberg AE, et al. (1998). Otological problems in children with Turner syndrome. Hearing Research 124:85–90.]

gone mastoid surgery. The cause of otitis does not appear to be related to a specific or generalized immunologic dysfunction. Other infections, as well as other disorders of the mucous membranes, are not reported to occur with increased frequency in Turner syndrome. Although levels of secretory immunoglobulins have not been reported, serum concentrations are not abnormal. IgG and IgA concentrations, however, appear to be in the adult male range—which is significantly lower than the adult female range.216 Instead, frequent occurrence of otitis may be the consequence of abnormalities in growth of the cranial base in Turner syndrome. Cephalometry212,217 has demonstrated that both structural growth of the temporal bone and growth of the condylar cartilage and spheno-occipital synchondrosis are abnormal. The result is that the final development of the facial skeleton reaches a level only corresponding to that of an 11-year-old girl, whereas that of the posterior portion of the cranial base is even less advanced. As a consequence, not only is the position of the external auditory meatus abnormal (giving the appearance of low-set ears) but the relationship of the middle ear to the eustachian tube is disturbed. These factors, coupled with abnormalities in the shape of the palate, create a predisposition to fluid collection and secondary infection. Marked hypocellularity of the mastoid air cells may also be found, further predisposing the individual to acute and chronic suppurative disease.218 The relationship of chronic otitis media to hearing deficit was also explored by Anderson and colleagues.188 Their data suggest that a component of the hearing loss reported to be a common occurrence in Turner syndrome is conductive in nature. A high incidence of eardrum disease and conductive hearing loss (43%) was also reported more recently.219 True sensorineural hearing impairment was also documented in both series. This was generally bilateral and characterized by symmetrical sensorineural dips in the audiogram in the midfrequency range. Audiologic manipulations tended to identify the defect as one of recruitment and specifically localized the defect to the outer hair cells of the organ of Corti. When older patients were compared with children younger than the age of 10, conductive losses were more frequently found in the children and sensorineural losses

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were more frequently found in the adults. Thus, the sensorineural losses appear to develop with age and represent a degenerative rather than a congenital abnormality. In addition to the findings reported previously, highfrequency audiometry also reveals a high prevalence of hearing loss that may precede the mid-frequency dip.220 A relationship between auricular abnormalities and hearing loss and those karyotypes with complete loss of the X or Xp2 is suggested and may confirm the hypothesis that growth dysregulation of the cranial base owing to loss of the growth-regulating genes such as SHOX is in part responsible for the findings.221 Alternatively, growth dysregulation of the cranial base may be secondary to delayed cell cycle caused by chromosomal aberrations per se and not only genes deleted on the X chromosome.222 In older girls and women with Turner syndrome with no history of hearing loss, audiologic surveillance is warranted every 2 to 3 years. The assiduous treatment of ENT problems in childhood and avoiding potential injuries to the inner ear may reduce the risk of hearing loss.61 Removal of the adenoids may exacerbate palatal dysfunction and negatively influence quality of speech, factors that must taken into consideration prior to surgery.

GERM CELL CHROMOSOMAL DEFECTS Gonadal Failure Histologic evidence suggests that the ovary of the fetus with a 45,X karyotype (and presumably the ovary of the fetus with karyotypes with X chromosome deletions, rings, or mosaicism) undergoes an initial phase of differentiation that is the same as that in the 46,XX fetus. If the ovary is examined at 14 to 18 weeks of gestation, no abnormalities are seen. Subsequently, however, when there is a chromosomal defect in the germ cells the process of oocyte loss appears to be accelerated—with a concomitant acceleration of stromal fibrosis. Thus, what is considered the normal process of oocyte loss (beginning prenatally and continuing over 30 to 50 years of postnatal life in normal females) occurs entirely prenatally or in the first few months or years of postnatal life in most females with Turner syndrome.223,224 The triggering mechanism for this premature follicular atresia is postulated to be in part secondary to meiotic pairing anomalies in prophase,225 and may be seen in other chromosomal abnormalities (such as trisomy 13 and trisomy 18).226 The molecular mechanisms presumably involve the acceleration of an oocyte-intrinsic apoptosis defect regulated by members of the Bcl-2 gene family198 and the caspase family of proteases.227 The processes of oocyte loss and fibrosis are, however, neither absolute nor inevitable. Whereas the older literature documents both spontaneous puberty and menarche in a small number of patients with Turner syndrome,228-231 the low percentages reported may be the result of ascertainment of only the most phenotypically obvious Turner syndrome patients. The report of Pasquino and co-workers,188 which includes more than 500 patients older than the age of 12 years, documents a high rate of spontaneous

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puberty—with an incidence of 14% in monosomic X patients and 32% in patients with cell lines with more than one X. In the series of Lippe et al.232 of 141 girls who were in the pubertal age range, 29 (21%) had the onset of breast development to Tanner III or more— with menses occurring at least once in 25 and menses persisting into at least young adulthood in 15 (50% of those with initial ovarian function, or 11% of the entire pubertal age group). Seven of the 29 with some degree of ovarian function had a 45,X karyotype. Of note is that although these 7 girls had some phenotypic features of Turner syndrome they did not have the webbed neck phenotype we associate with the presence of fetal lymphedema. The remaining 22 patients had mosaicism or structural abnormalities, with the highest number of girls with menses in the 45,X/46,XX group. Again, these girls lacked the webbed neck phenotype. Thus, maintenance of some degree of ovarian function can occur in Turner syndrome patients regardless of karyotype—although it is more common in those with cell lines with more than one X, and clearly more common in those who lack the webbed neck phenotype. Pregnancy has been reported in more than 60 patients with Turner syndrome, including those with a 45,X karyotype on multiple tissues.234-238 Although these latter cases remain rare enough to continue to merit individual reports, they illustrate the spectrum of ovarian function in this condition. Counseling about the expectations and future management of the patient with Turner syndrome diagnosed before puberty needs to include the probability of gonadal failure and infertility but not its inevitable occurrence. Even in those instances in which fertility does occur, however, reproductive failure is high and the risk of an abnormal offspring (notably one with trisomy 21) appears significant.237-239 In the series of Pasquino and co-workers,188 there were three patients with spontaneous pregnancies (3.6%). One patient had two pregnancies, one with a normal male karyotype and one female with the same structurally abnormal X as the mother (i.e., Turner syndrome). One patient had twins with normal karyotype but severe cleft palate, and one patient had a normal infant. Ultimately, morethan 90% of indiduals with Turner syndrome will have gonadal failure.61 Physiologic evidence for gonadal failure in Turner syndrome is provided by the response of the hypothalamic-pituitary axis to functional agonadism. It has long been recognized that the negative feedback loop between the gonad and the hypothalamic-pituitary axis is operative in the fetus and demonstrable at birth. In normal infants, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) rise after birth. In the male, the gonadotropin rise is accompanied by a significant surge in plasma testosterone in the first months of life. Gonadotropin and testosterone then gradually decline to prepubertal concentrations by the end of the first year. In the female, although the gonadotropin rise does not appear to be accompanied by as striking a response in estradiol secretion from the ovary the plasma FSH remains slightly elevated for a period of several years. In Turner syndrome, a markedly exaggerated rise in both plasma gonadotropins (but especially in FSH) has

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been demonstrated as early as 5 days of age in one study.240 In a more recent study, however, with the blood spot obtained used for neonatal screening241 a clear-cut elevation of FSH could not be demonstrated in patients with known karyotype abnormalities consistent with Turner syndrome—suggesting that FSH cannot be used for neonatal screening for Turner syndrome. In the series by Lippe et al.,232 the investigators evaluated 14 patients with karyotypes consistent with Turner syndrome at 2 weeks of age or later in the first year of life. Of those who presented with lymphedema at birth, all had FSH determinations of more than 40 mIU/mL. Conversely, of the patients who had the diagnosis of an X chromosome abnormality made by amniocentesis only 50% had evidence of castrate levels of FSH. Thus, although an abnormally high FSH in the neonatal or infancy period is useful to detect gonadal failure a normal level does not preclude the diagnosis of Turner syndrome. Whether the girls with normal FSH levels in infancy will go on to have gonadal failure at adolescence or will turn out to be the 10% to 30% who have some gonadal function remains to be determined. Pelvic ultrasound may have prognostic value in predicting the future sexual development of patients with normal gonadotropins. Data of Mazzanti and associates242 suggest that detection of ovaries bilaterally in patients without castrate levels of gonadotropins and mosaic karyotypes correlated best with preservation of some ovarian function at puberty. When abnormally elevated in infancy, the gonadotropin levels decline again after the first 2 years—although to mean concentrations significantly higher than those in gonadally competent female children. Between ages 4 and 10 to 11 years, a trough is noted, which is followed by the gradual rise of gonadotropins in normal children and a more rapid and exaggerated rise in most children with Turner syndrome. This diphasic pattern of FSH, determined on a large number of patients with gonadal dysgenesis, is illustrated in Figure 15-17A. In Figure 15-17B, serial determinations graphically demonstrate the fall that occurs in the first years of life and the abrupt rise that occurs in early adolescence. The range of values considered normal for a female varies with the assay method and the standard used, and thus the values in the figure are illustrative but will not be normative for most current commercial assays. The mechanisms responsible for the feedback suppression of gonadotropin secretion in the normal child and the similar pattern, although at a higher set point, in the child with gonadal dysgenesis are unclear. It has been presumed that increased sensitivity to circulating steroids of gonadal and adrenal origin results in decreased hypothalamic gonadotropin-releasing hormone (GnRH) release in the prepubertal subject. Studies in primate species suggest that one component of the feedback inhibition may be at the level of the pituitary and that another may be at the hypothalamic level. There may also be significant central neuronal inhibition that is not affected by gonadal steroids. Plasma gonadotropin concentrations can be assessed at the time of diagnosis and again in early adolescence before the institution of steroid hormone therapy and counseling

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Figure 15-17 (A) Plasma follicle-stimulating hormone (FSH) values in patients with Turner syndrome. Triangles (m) indicate patients with 45,X karyotype, and circles (s) denote those with X chromosome mosaicism or structural abnormalities of the X. The curve is a polynomial regression plot. The hatched lines indicate mean plasma FSH values in normal females. Note the very high levels in infancy and adolescence, with lower levels in the mid first decade. FSH is expressed as nanograms per milliliter of standard LER-869. To convert these values to milli-International Units per milliliter of the second IRP, multiply by 3.5. (B) Symbols indicating that serial plasma FSH values in several individuals are connected by straight lines. Note the rise and fall in infancy and the dramatic and abrupt rises in adolescence. [From Conte FA, Grumbach MM, Kaplan SL (1975). A diphasic pattern of gonadotropin secretion in patients with the syndrome of gonadal dysgenesis. J Clin Endocrinol Metab 40:670.]

about ultimate fertility. When GnRH is administered to assess the function of the hypothalamic-pituitary axis, the responses of patients with Turner syndrome with gonadal failure are exaggerated compared with normal subjects.243 This procedure may be used to provide a second test for gonadal integrity in some patients. In one study of two 45,X menstruating adolescents with both documented anovulatory cycles and ovulatory cycles with a prolonged follicular phase and short luteal phases, however, GnRH testing did not show exaggerated responses.244 Nevertheless, in most adolescent patients a single determination of plasma FSH and LH is sufficient to document gonadal failure. In the absence of estrogen replacement therapy, gonadotropin secretion will continue unabated and unmodulated. Although no systemic manifestations are known to result from these excessive concentrations of

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FSH and LH, reactive pituitary abnormalities have been reported in some patients.245 Skull radiographs may show enlargement of the pituitary fossa, which is suggestive of pituitary hyperplasia or microadenoma formation. Although enlargement sufficient to result in organic dysfunction has not yet been reported, the recognition of this phenomenon is important so that the gonadal dysgenesis is not misdiagnosed as being secondary to a pituitary tumor. The potential for progressive pituitary enlargement is an additional reason for instituting estrogen replacement therapy.

Gonadoblastoma Gonadoblastomas are distinctive tumors composed of at least two different cell types. The large cells have the appearance and ultrastructure of oogonial germ cells, and the small cells are ovarian stromal cells that are not clearly differentiated.246 The small cells most closely resemble primitive sex-cord mesenchymal cells and are termed granulosa-Sertoli cells. The tumor arises in a gonad that is dysgenetic; that is, a gonad that has not completely differentiated into a normal ovary or normal testis or that does not behave like a normal ovary or normal testis. The dysgenesis that leads to gonadoblastoma formation almost invariably occurs in patients with Y chromosome material in their karyotype. In the case of Turner syndrome, most karyotypes do not contain a Y. The ovary is induced normally and then usually rapidly degenerates into fibrous streaks with loss of oogonial germ cells. These ovaries are not believed to be at risk for gonadoblastoma formation. There is a small percentage of patients (between 2 and 5% in most series and 5% in our series), however, who fit all of the criteria for Turner syndrome and in whom the karyotype contains a Y chromosome. There is an even smaller percentage with a marker chromosome. Most often, the karyotype is the mosaic 45,X/46,XY. More complex karyotypes (including 46,XX/45,X/46,XY and even 46,XY), however, have been associated with Turner syndrome. Thus, the karyotype per se does not determine the phenotype. What defines these patients as having Turner syndrome (rather than being hermaphrodites or having mixed gonadal dysgenesis) is their short stature, their symmetrical female external genitalia, and their normal female internal genitalia (bilateral fallopian tubes, uterus, and vagina). The only expression of the Y may be its effect on gonadal ridge induction, with the result that a number of cells no longer resemble differentiated ovarian stroma. In this case, all of the oogonial germ cells that develop in these dysgenetic ridges do not necessarily undergo atresia as rapidly as they do in typical Turner syndrome. Instead, some may persist, continue to divide, and develop into a gonadoblastoma. In some patients with a Y in their karyotype, the granulosa/Sertoli cell stroma more closely resembles a testis and produces some androgen. This may occur regardless of whether a gonadoblastoma will eventually develop. These patients, although still having the Turner phenotype, may show some evidence of virilization— including clitoromegaly or partial fusion of the posterior

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labia. Virilization is in itself an indication for gonadectomy, whether a gonadoblastoma is present or not. Because many patients bearing the Y will not virilize, however, these findings are not necessarily helpful in deciding which Turner syndrome patient may be at risk for gonadoblastoma. For this reason, it is essential to obtain a complete chromosomal karyotype even in the most obvious of Turner syndrome patients. If a marker or fragment chromosome is detected that cannot be assigned to a specific chromosome, more specific studies can be performed (see “Incidence and Etiology” for a discussion of the role of Y chromosome-derived genes in gonadoblastoma formation). The risk for the development of the tumor, unilaterally or bilaterally, in the patients with the Y-bearing dysgenetic gonad has been estimated to be as high as 15% to 25%247—although in the recent population study from Denmark by Gravholt and co-workers the risk appears to be much lower.60 In the series by Lippe et al.,232 four of the eight patients with an intact Y chromosome and one of the two with a Y-derived marker had histologic gonadoblastomas. As discussed previously, these data do not pertain to use of molecular markers for any Y-derived genetic material and should not be extrapolated to imply that patients should be screened with molecular markers to detect Y material to monitor them for gonadoblastoma or perform gonadectomy. Although gonadoblastomas usually do not metastasize, local invasion of the surrounding stroma to form microscopic or gross germinomas is seen in about half of cases.248 Gonadoblastomas may also be found in conjunction with malignant germinomas, although these latter tumors tend to occur in conditions other than Turner syndrome. The age at which these tumors are detected varies, but a number have been reported in early childhood.249,250 In some patients, calcification is present— which assists in detection. In others, a tumor mass may be demonstrated with ultrasound, magnetic resonance imaging (MRI), or computed tomography. In many, however, the disease is microscopic. Recommendations for dealing with the problem vary from prophylactic removal of the gonadal ridge streaks in all Turner syndrome patients with an intact Y or marker chromosome in their karyotype to prospective monitoring of the patient at risk with some visualization technique such as ultrasonography. Until the gonadoblastoma gene locus is identified, we do not recommend removal of gonadal streaks based on molecular marker testing in the absence of a cytogenetic Y but do recommend prophylactic removal if a cytogenetic Y or marker chromosome or virilization is present. Gravholt’s data might indicate that even this is too aggressive in all cases, however, and monitoring alone may be sufficient in some. Examples of ultrasonographic studies in a normal pubertal female, a girl with Turner syndrome, and a girl with Turner syndrome and a gonadoblastoma are shown in Figure 15-18.

CARDIOVASCULAR ABNORMALITIES The association of cardiovascular abnormalities, notably coarctation of the aorta, with Turner syndrome has been well documented.16,251-254 The haploinsufficency of the X

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Figure 15-18 Examples of pelvic ultrasound studies. (A) Normal pubertal female demonstrating ovaries of adult size. (B) Patient with Turner syndrome. The corpus of the uterus is seen slightly to the left of the midline. The fallopian tube can be followed into the right adnexa and observed to terminate in a small structure (arrow) believed to be the fimbriated end of the tube. No ovaries are identified. (C) Patient with 45,X/46,XY Turner syndrome previously treated with estrogen. The corpus of the uterus is enlarged to adult size. In the left adnexa, a large gonadal mass (O) is seen. Histologically, this was identified as a gonadoblastoma. The images are transverse, oriented right (R) and left (L) of the midline (ML). The dotted scales are in centimeters. B, BL, bladder; FT, fallopian tube; IP, iliopsoas; O, gonadoblastoma; OV, ovary; Re, rectum; and Ut, uterus.

chromosome causes the most serious life-threatening consequences involving the cardiovascular system. The determination of its frequency, however [as well as the clarification of the conflicting reports of occurrence of other major structural cardiovascular malformations (CVMs)], was impaired by the lack of chromosomal karyotyping in early series. One of the major sources of confusion was the inclusion of patients with normal chromosomes but phenotypic abnormalities, of whom a large number may have had Noonan syndrome.254,255 CVMs occur in approximately 75% of spontaneously aborted Turner fetuses and 30% of living patients. Obstructive lesions of the left side of the heart predominate, ranging in severity from nonstenotic bicuspid aortic valve to aortic stenosis, coarctation of the aorta, and mitral valve anomalies (Table 15-4).256-264 The most severe form of left-sided hypoplasia (hypoplastic left heart syndrome) also occurs, although it is uncommon.265,266 The association of Turner syndrome and the entire spectrum of left-sided cardiovascular malformation is distinctive among malformation syndromes. Only Williams syndrome, commonly associated with supravalvar aortic stenosis, shares this left-sided association. The largest series of patients comes from Italy, where Mazzanti and colleagues259 analyzed the cardiac

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evaluation of 594 Turner syndrome patients ranging from 1 month to 24 years of age. In addition, various other authors260,262 have over the past years published type and frequency of cardiovascular malformations (Table 15-4). The prevalence of congenital heart disease and the relative risk in Turner syndrome patients compared to the general population are shown in Table 15-5. Coarctation per se (especially the preductal adult-type coarctation most characteristic of this condition) is not likely to result from the same mechanisms as other congenital heart defects. Although the final structure of the four chambers of the heart and the orientation of the great vessels are established by 6 to 7 weeks after fertilization, the preductal area of the aorta subject to coarctation may be affected at any time. Moreover, coarctation occurring in this isthmus region has been considered in some cases to be a result of the differential flows through the two fetal circulatory systems that interface at this point (the ductal-placental flow and the systemic-cardiacpulmonary flow). Clark267 elegantly reviewed the embryology of lymphatic sac drainage into the venous system and noted that in the chick disordered drainage implicates a mechanism that distends the cardiac lymphatics. He proposed that this would encroach on the ascending aorta and alter

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TA B L E 1 5 - 4

Cardiovascular Malformation in Turner Syndrome Gotzsche et al.232 Year No. of patients Type and frequency (rounded) All types Among fetuses: 75% Left-sided obstruction, total BAV 6 CVM COA 6 other CVM COA/BAV Aortic stenosis, without BAV Mitral stenosis, hypoplasia Hypoplastic left heart syndrome Other, total Membranous VSD ASD2 PAPVR Mitral valve prolapse

Sybert234

Lin et al.233

Mazzanti et al.231

1994 179 25% 95% 45% 30% 10% 10%

75% 25% 30% 5% 10%

70% 25% 30% 10% 5%

100% 55% 30%

5% 15%-20% 5% 5% .5% 5%

25%-30% .5% .5% .5% 15%

15%-20% .5% .5% 10% 10%

15%

ASD2, atrial septal defect, secundum type; BAV, bicuspid aortic valve; COA, coarctation; CVM, cardiovascular malformation; PAPVR, partial anomalous venous return; and VSD, ventricular septal defect. From Lin AE (2000). Management of cardiac problems. In Saenger P, Pasquino AM (eds.), Optimizing health care for Turner patients in the 21st century: Proceedings of the 5th International Symposium on Turner Syndrome, Naples, Italy. Amsterdam: Elsevier Science 115–123. TA B L E 1 5 - 5

Prevalence of Congenital Heart Disease and Relative Risk in Turner Patients and in the General Population Turner Patients (%) Congenital heart disease Bicuspid aortic valve Coarctation of the aorta Aortic valve disease Partial anomalous pulmonary discharge Ostium secundum atrial septal defect Ventricular septal defect Atrioventricular septal defect

22.9 (136) 12.5 (74) 6.9 (41) 3.2 (19 2.9 (17) 2.2 (13) 0.5 (3) 0.2 (1)

General Population (%) 2 1.28 0.043 0.035 0.009 0.064 0.188

Relative Risk 11.4 9.8 160.5 91.4 320 34.4 2.7

From Mazzanti L, et al. (2000). Italian Turner syndrome study: Cardiac function and complications. In Saenger PH, Pasquino AM (eds.), Optimizing health care for Turner patients in the 21st century: Proceedings of the 5th International Symposium on Turner Syndrome, Naples, Italy. Amsterdam: Elsevier Science 125–136.

intracardiac blood flow. A more recent pathologic examination of 12 fetuses with Turner phenotype of nuchal cystic hygromas found that 75% had left-sided flow defects and aberrations of the lymphatics at the base of the heart.268 These cystic hygromas resolve as lymphatic ducts open in later gestation but often result in residual webbing of the neck. Finally, Miyabara and co-workers269 analyzed 13 fetuses with 45,X karyotypes with cystic hygroma and documented that almost all had bicuspid aortic valves and many tubular hypoplasias of the aortic arch. They postulated that a homeobox gene or genes,

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not SHOX, could be responsible for what appears to be a generalized hypoplasia of tissues of the fourth branchial arch. This further confirms a relationship between the nuchal hygroma/webbed neck phenotype and the cardiac defects found clinically in the 45,X karyotype. Mazzanti and co-workers259 showed that a 45,X karyotype is more likely to be associated with webbing, more severe CVMs, and in particular coarctation and partial anomalous pulmonary venous return. We and others also showed a similar association between the webbed neck phenotype and coarctation.253,269 In contrast, X-structural abnormalities are more likely to be associated with bicuspid

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aortic valve and aortic valve disease. Less frequent than a left-sided obstruction, CVMs are membranous ventricular septal defects, secundum-type atrial septal defects, partial anomalous pulmonary venous return, and mitral valve prolapse.259-262,268 Another cardiovascular abnormality that occurs in Turner syndrome is aortic dilation, dissection, or rupture. Approximately 90% of the cases of dissection or rupture had an associated cardiac malformation, including previous coarctation, “pseudocoarctation,” bicuspid aortic valve, or some degree of hypertension.271 Other large series of Turner syndrome patients have also provided data on aortic root dilation, although the prevalence and natural history remain incomplete.259,260,262 One large series of 244 patients followed in a Turner syndrome clinic in Seattle, Washington, provided both cross-sectional and longitudinal data about cardiac anomalies.262 The frequency of aortic dilation was not reported, but aortic dissection occurred in three patients (1%): one with chest trauma, one with previous coarctation repair, and one with chronic hypertension and obesity. We also analyzed the responses of 245 of 1,000 members of the Turner’s Syndrome Society of the United States.261 Confirmation of karyotype, however, was not obtained. Thirty-five percent were 10 years of age or

younger, and a substantial 14% were 41 years or older. Among all respondents, 15 (6%) reported aortic dilation (8% of those who had specifically been evaluated). Of those reporting dilation, 80% reported having a CVM— usually bicuspid aortic valve, aortic valve stenosis, or coarctation (alone or in combination). About two-thirds of the patients with aortic dilation were younger than 21 years of age, and most had an associated CVM. However, two of the three patients who had aortic dilation without a CVM or other risk factor were also in this younger age group. Despite the limitations of a self-reporting survey, the study provided a conservative estimate of the frequency of aortic dilation. The vast majority of Turner syndrome patients in this survey and the literature have an associated risk factor for aortic dilation. Nevertheless, of concern to patients and health care professionals are the small number in whom there is no risk factor.272,273 Figure 15-19 and Figure 15-20 demonstrates the utility of echocardiography in assessment of the aortic root, as well as of MRI in the imaging of the entire aorta. A prospective study of thoracic cardiovascular MRI of 40 girls with Turner syndrome revealed an overall frequency of anomalies of more than 45%.274 These included not only bicuspid aortic valve (17.5%) and aortic coarctation

Figure 15-19 Cardiac imaging in Turner syndrome. In older children, MRI is the preferred study because it gives better resolution. In younger children, ultrasonography is still the method of choice. This panel shows coractation of the aorta (R) and elongated transverse aortic arch (ETA) (L), which my lead to coarctation. Courtesy of C. Bonoly.

A

B

C

Figure 15-20 Three examples of coarctation of the aorta as seen on MRI studies. Please note the poststenotic dilatation, seen especially in panels B and C. Courtesy of G. Conway.

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(12.5%) but dilated ascending aorta (12.5%). Aortic coarctation and bicuspid aortic valve are each almost fourfold more frequent in patients with webbed necks (e.g., 37% of patients with neck webbing have a BAV, compared to 12% in those without webbing).275 The authors point out that three of five aortic coarctations and four of five ascending aortic dilations were solely detected with MRI and were not evident on echocardiographic examination. Thus, these findings and other series276,278 suggest an MRI is a valuable adjunct in the cardiovascular evaluation of girls with Turner syndrome. In fact, MRI may dectect coarcation missed by echocardiography in infancy.279-281 The risks associated with BAV in Turner syndrome are probably similar to those for nonsyndromic cases. The abnormal valve is at risk for infective endocarditis, and over time it may deteriorate—leading to clinically significant aortic stenosis or regurgitation. BAV can also be associated with aortic wall abnormalirties, including ascending aortic diltation, aneurysm formation, and aortic dissection.282 Recent studies suggest a broader spectrum of CVM than previously recognized. Magnetic resonance angiographic (MRA) screening studies of asymptomatic inviduals with Turner syndrome have identified a high prevalence of vascular anomalies of uncertain clinical significance. Almost 50 have an unusual elongation and angulation of the aortic arch termed elongated transverse arch (ETA) by Ho et al.280 This may reflect an abnormal aortic wall prone to dilation. Additional anomalies are partial anomalous pulmonary connection (PAPVC) and persistent left superior vena cava, affecting 13 versus 1% b in the general population. Whether this defect is clinically significant depends on the degree of the left to right shunt.283-285 There seems to be a generalized dilation of major vessels in women with Turner syndrome, including the brachial and carotid arteries as well as the aorta. The distal extent of this vasculopathy is unknown. Estrogen deficiency contributes to greater intima medial thickness and altered arterial wall dynamics but not the increased caliber of the vessels286-287 (Figure 15-19). The data from this group and another using echocardiography suggest that aortic root diameters are greater in 45,X patients than in matched controls.288 Whereas both studies compared aortic root dimensions to body surface area, which in a condition of short stature may not be the appropriate control group (may be more appropriate to match for age),271 the findings suggest a degree of intrinsic primary abnormality. Pathologic evidence of cystic medial necrosis is reported in some cases of dissection and suggests that the intrinsic disorder might represent an example of a mesenchymal defect in Turner syndrome. This may explain the increased incidence of mitral valve prolapse as well. Alternatively, the tendency toward significant aortic root dilation may be related to the intrauterine hemodynamic events that are a consequence of the lymphatic disorder. The longitudinal survey report that 25% of normal patients had increased aortic root diameters 2 years after the first study277 (coupled with a report that death from aortic dissection was greatly in excess of that expected in a large prospective study of Turner syndrome patients registered with a karyotype registry289) prompted

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us to adopt an aggressive approach to diagnosis, followup, and risk factor management in these patients. Data at present are not adequate to recommend routine prophylaxis with beta-blocking drugs, although this therapy may be appropriate for those with significant aortic root dilation (e.g., Marfan syndrome)—especially those with bicuspid aortic valve.290 Of particular importance is the increased risk of aortic dissection in pregnancy among Turner syndrome women.291 As more Turner syndrome women contemplate pregnancy, it seems prudent to identify those with aortic root dilation before the pregnancy. Those with a previous risk factor (bicuspid aortic valve, coarctation, hypertension) should be monitored very closely. Those women without abnormal risk factors also require close attention.236,291-296 It is also clear from reports of a high incidence of unclassified murmurs and from early patient series297 that the incidence of aortic valvular abnormalities is also increased in Turner syndrome. With the introduction of echocardiography, we were able to undertake a study of the frequency of this association.256 Of 67 patients without coarctation studied with two-dimensional or M-mode echocardiography (or both), 20 (almost 30%) have findings characteristic of bicuspid aortic valve. Some of these valves are anatomically tricuspid but have eccentric closure and are functionally bicuspid, whereas the majority are actually bicuspid. Our data, and the more recent confirmatory data from Mazzanti and co-workers,259 indicate that the percentage distribution of patients with isolated bicuspid valve does not appear to be confined to any karyotype—nor does there appear to be a preponderance of the webbed neck phenotype. Thus, bicuspid aortic valve not only may be more common than coarctation but may represent a more fundamental manifestation of an X chromosome defect in Turner syndrome. We also diagnosed six patients as having mitral valve prolapse (Barlow syndrome), a finding confirmed by a second group.257 The frequency, 10% to 15%, is in excess of that believed to be the occurrence rate in the population at large.298 Mazzanti and co-workers also found a significant number of patients with partial anomalous pulmonary venous return, a rare malformation, and thus the malformation with the highest relative risk of occurrence compared with normals259 (Table 15-5). In a follow-up study of adult Turner women, only 11 of 25 adult women with Turner syndrome had normal cardiac findings using a combined echocardiography and MRI screen of the aorta. Twelve had aortic root dilation, three had bicuspid aortic valve, and five had pseudocoarctation of the aorta.299 Finally, hypoplastic left heart syndrome has been described during prenatal echocardiography266—and four Turner syndrome infants are described in a large outcome study, with limited survival in two.267 Adults with Turner syndrome have a high prevalence of electrcardiographic conduction and repolarization abnormalities, as well as prolonged QT intervals. Therefore, monitoring of echocardiograms (ECGs) in Turner syndrome appears warranted.300 In addition to congenital structural anomalies, there are several acquired cardiac

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problems that have been noted in Turner syndrome women—although their prevalence is not well established. In some series, hypertension occurs in about 15% to 25% of girls (and in a larger percentage of adults with Turner syndrome).259,262,301,302 A threefold relative risk for high blood pressure was noted in Denmark, but patients with repaired coarctation had not been removed from the sample.303 Stratification by age and weight has not been performed. Several large series from different countries also support an increased prevalence of hypertension. These analyses did not consistently analyze patients based on age, weight, and coarctation repair. Nevertheless, in the Danish study of morbidity in Turner syndrome women along with hypertension there were also marked increases in ischemic heart disease and stroke.303 In a Swedish morbidity study, cardiac anomalies and hypertension were common but plasma lipid levels did not differ from those of normal subjects.304 Although a renovascular abnormality is considered the most likely mechanism for development of hypertension, especially in younger patients, obvious structural renal lesions are not always present. Similarly, a history of infectious nephritis or even urinary tract infection is usually absent. Thus, in the majority of patients the cause of the hypertension is unknown. In our series, nine patients with hypertension were identified and in only one did it appear to be of renovascular origin (Figure 15-21). One of the earliest abnormalities in blood pressure regulation is the disappearance of the physiologic nighttime dipping in blood pressure as prodromal state of hypertension61 (Table 15-6). Systemic hypertension is an important risk factor for aortic dilation and dissection. Therefore, blood pressure should be monitored frequently and treated vigorously in all patients with Turner syndrome. If the baseline ECG reveals a significantly prolonged QTc, medications that might further prolong the QT should be avoided.61 There was no apparent relationship

TA B L E 1 5 - 6

Cardiovascular Screening and Monitoring Algorithm for Girls and Women with Turner Syndrome Screening (All Patients at Time of Diagnosis) • Evaluation by cardiologist with expertise in congenital heart disease. • Comprehensive exam, including blood pressure in all extremities. • All require clear imaging of heart, aortic valve, aortic arch, and pulmonary veins. • Echocardiography is usually adequate for infants and young girls. • MRI and echo for older girls and adults. • ECG. Monitoring (Follow-up Depends on Clinical Situation) • For patients with apparently normal cardiovascular system and age-appropriate blood pressure. • Reevaluation with imaging at timely occasions (e.g., at transition to adult clinic), before attempting pregnancy, or with appearance of hypertension. Girls that have only had echocardiography should undergo MRI when old enough to cooperate with the procedure. • Otherwise, imaging about every 5 to 10 years. • For patients with cardiovascular pathology, treatment and monitoring determined by cardiologist. From Bondy CA for the Turner Syndrome Study Group (2007). Care of girls and women with Turner syndrome. J Clin Endocrinol and Metab 92(1):10–25.

to any particular karyotype or phenotype. Other infrequent abnormalities include endocardial fibroelastosis258 and cardiomyopathy.305 In two studies of Turner syndrome girls without major cardiac abnormalities who received GH treatment there was no evidence of left ventricular hypertrophy or hypertension.194,195 In one, after 7 years there was no significant dilation of the aorta (S.M.P.F. de Muinck Keizer-Schrama, MD, personal communication).

Figure 15-21 (A) Radiograph from an intravenous pyelogram demonstrating a horseshoe kidney deformity. (B) Radiograph after an aortic injection, illustrating multiple renal arteries in a patient with Turner syndrome and hypertension.

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Ongoing Cardiac Care For the patient with no identified cardiovascular defects, routine pediatric care is advised with continued monitoring of blood pressure. It seems also prudent to reevaluate aortic dimension at 5- to 10-year intervals. Patients with significant cardiovascular defects need continued monotoring by a cardiologist, with frequency of monitoring determined by circumstances. Children considered at increased risk for hypertension should be educated about this risk, the need for compliance with medical monitoring and treatment, and the possible presenting symptoms (e.g., chest or back pain). Patients with multiple risk factors (BAV, dilated aortic root, hypertension) that put them at high risk for aortic deterioration might want to consider carrrying a medical ID bracelet. They also need to be informed that prophylactic antibiotics should be given before dental procedures or surgery.

MONITORING FOR AORTIC DILATION All measurements of the aorta should be done at end systole. The ascending aorta should be measured at the level of the annulus at the hinge points of the valve, at the level of the sinuses of valsalva perpendicular to the ascending aorta long axis, and at the ascending aorta 10 mm above the sinotubular junction. Normative data for aortic diameters as a function of body surface area are available.282,286,306 Review of available data suggests that unadjusted values greater than 28 to 32 mm will identify patients with diameters greater than 95% of controls, which would clearly be abnormal for women with Turner syndrome who are generally smaller. When aortic root enlargement is found, medical therapy and detailed imaging is recommended. In hypertensive patients with aortic root enlargement who also have resting tachycardia, beta adrenergic receptor blockade is an excellent therapeutic option. Betablockers have been shown to reduce the rate of aortic dilation and dissection in Marfan syndrome,307 although efficacy in treating aortic dilation in Turner syndrome has not yet been investigated. Heart-healthy exercise with moderate aerobic exercise is emphasized and should be encouraged. Eligibility for competitive sports for all those with Turner syndrome should be determined by a cardiologist after a comprehensive evaluation that includes an MRI of the aorta. Experts polled on this issue at the most recent Turner syndrome guidelines meeting agreed that aortic enlargement in Turner syndrome may be defined as an aortic sinus of valsalva or ascending aorta, body size adjusted Z score 2 plus evidence of increasing Z score on a subsequent imaging study of the aorta or a single Z score 3. In those cases, participation in competitive sports is contraindicated.308

PREGNANCY AND CARDIAC CARE Spontaneous or assisted pregnancy in Turner syndrome should be undertaken only after thorough cardiac evaluation. Alarming reports of fatal aortic dissection during

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pregnancy and the postpartum period have raised concern about the safety of pregnancy in Turner syndrome. If pregnancy is being considered, preconception assessment must include cardiology evaluation with imaging of the aorta. A history of surgically repaired cardiovascular defect, the presence of BAV, or current evidence of aortic dilation or systemic hypertension should probably be considered as relative contraindications to pregnancy.309

GROWTH HORMONE TREATMENT AND THE CARDIOVASCULAR SYSTEM Several echocardiographic studies reported normal left ventricular and septal morphology and function in GHtreated girls with Turner syndrome,310 and two recent MR studies found no deleterious effect of GH treatment on aortic diameter311 or compliance.312 It is particularly reassuring that when adult Dutch Turner women were examined for aortic distensibility and the effects of growth hormone on aortic dimensions the patients who had received the higher growth hormones doses (47 mg/kg/ week) had better cardiac health based on the parameters measured.312 In regard to the lymphatic system, abnormalities of cardiovascular and lymphatic development are found in most TS fetuses that fail to survive the first trimester.21,269,313 For those girls who survive, the residua of the fetal lymphedema and cystic hygromas are peripheral lymphedema and webbed neck. Often the newborn lymphedema resolves by 2 years of age without therapy. It may recur at any age and may be associated with initiation of GH or estrogen therapy. Some children and adolescents may require support stockings. Complete decongestive physiotherapy (a four-step process involving skin and nail care, massage for manual lymph drainage, compression bandaging, and a subsequent remedial exercise regimen) is recommended for those with more significant lymphedema.314 Long-term diuretic use should be avoided, as should vascular surgery. Families should be directed to the National Lymphedema Network (http:// www.lymphnet.org) for more information.61

RENAL AND RENOVASCULAR ABNORMALITIES Renal and renovascular abnormalities occur in Turner syndrome with greatly increased frequency. Although the incidence varies from 25% to 70% among different series reported,315-319 the abnormalities tend to be of three specific types: those that primarily involve the pelvocaliceal collecting system, such as complete or partial duplication; those associated with the position and alignment of the organ, such as horseshoe kidney and retrocaval ureter; and those associated with abnormal vascular supply. In a recent study,320 no patient with a normal baseline ultrasound developed renal disease during a follow-up period averaging 6 years. However, some of those with malformations developed hypertension and urinary tract infections. The development of the collecting system begins with the formation of the ureteric bud, its dorsocranial migration, and its penetration of the metanephric blastema. At

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about 5 weeks of gestation it dilates into a primitive pelvis and simultaneously splits into cranial and caudal portions—forming the future major calyces. Thus, duplications of the collecting system are caused by abnormal or early splitting of the ureteric bud at or before 5 fetal weeks and are therefore primary malformations of organogenesis. Conversely, the upward migration of the already preformed kidney from its position in the pelvis to the lumbar region is a somewhat later event. The kidney must pass through the arterial fork formed by the umbilical arteries, and if there are disturbances in either the anatomy of these vessels or the path of migration secondary positional malformations occur. If one kidney fails to traverse the arterial fork, it will remain ectopically in the pelvis. If there is a partial mechanical effect on the kidney during migration, rotational abnormalities may result. The vascular supply of the kidneys may be anomalous (Figure 15-20B), secondary to the multiple budding segments of the kidney, the final positioning, or the presence of an aberrant vessel crossing the upper renal pole. Finally, the horseshoe kidney [which occurs with increased frequency (Figure 15-20A)] may represent a primary defect in embryogenesis resulting in the union of the two metanephric blastemas or a secondary defect caused by malposition of the umbilical arteries. In the experience of Lippe et al.142 patients studied with intravenous pyelography, with contrast medium enhancement at the time of cardiac catheterization, or with ultrasonography we found that 47 patients (33%) had some structural. The lesions we detected covered the spectrum of defects described previously, except that we did not have a case of retrocaval ureter.321,322 Ultrasound, as the initial screening method in recent years, was effective in demonstrating all anomalies previously seen with intravenous pyelography except for mild clinically insignificant rotational abnormalities. Although the overall morbidity of the renal lesions is relatively low (with only four patients requiring surgery, one requiring long-term antibiotic therapy, and four having an absent kidney), the potential for caliceal obstruction, parenchymal infection, and secondary renal impairment is real—and we therefore suggest that all patients should undergo an ultrasound imaging study. The high percentage of horseshoe kidneys (up to 7%320) merits further comment because there may be an increased incidence of Wilms’ tumor in the horseshoe kidney.322 If the increased incidence of Wilms’ tumor results from an abnormal proliferation of the metanephric blastema and if that were the mechanism in Turner syndrome, these patients would be at the same risk as other patients with a horseshoe kidney—and the incidence of Wilms’ tumor in Turner syndrome should be high. Alternatively, if the mechanism in Turner syndrome is vascular these patients should be at no greater risk for Wilms’ tumor than the general population. At present, only one patient with both Wilms’ tumor and Turner syndrome has been reported—suggesting that the latter hypothesis is correct. In a recent paper by Sagi et al.,316 kidney malformations were exclusively found in those patients who had retained the maternal X chromosome.

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UNKNOWN METABOLIC FACTORS Autoimmune Disorders An apparent increased frequency of autoimmune disorders has been noted in patients with Turner syndrome. The reason for this increase is unknown, but it has been theorized that families with a high frequency of autoimmune diseases may be prone to nondisjunctional events. This hypothesis is based on the observations that there is also an increased frequency of autoimmune disorders in other nondisjunctionalchromosomal disorders, such as Down and Klinefelter syndromes, and that when the families of patients with Turner and Down syndromes are investigated autoimmune disease appears to be reported or diagnosed frequently. It should be noted, however, that these studies were retrospective and did not test the parents for antibodies at the time of delivery. A recent prospective study of thyroid antibody positivity as a risk factor for nondisjunction failed to show an association.323 A second theory that autoimmune diseases might result from, or be associated with, genes or gene mutations on the X chromosome is supported by the increased incidence of these disorders in X chromosomal disorders as well as by their increased incidence in women. A third theory, that familial (maternal) autoimmunity may lead to the preferential survival of a fetus with chromosomal aneuploidy (albeit a general risk for pregnancy loss),324 remains to be investigated. The most prevalent autoimmune disorder in Turner syndrome appears to be Hashimoto lymphocytic thyroiditis. Depending on the series reported and on the methods used to measure the antibodies, the prevalence of significant titers may be as high as 50%.210 Although originally described in association with structurally abnormal X chromosomes, notably the isoX,325 increased titers of antithyroid antibodies with or without thyroid failure have been reported in 45,X individuals as well as in patients with mosaic karyotypes without a structurally abnormal X (i.e., 45,X/46,XX).326-328 In our series, approximately 30% of patients have positive antithyroid antibodies at the time of first testing. Of six patients who were overtly hypothyroid, however, two did not have abnormal antibody titers at the time of diagnosis. This is not unexpected because it is well known that children with biopsy-proven Hashimoto thyroiditis often do not have abnormal titers of circulating antithyroid antibodies.329 The clinical picture of overt hypothyroidism in Turner syndrome may be different from the normal population because it has been reported that even severely affected individuals may not show any signs or symptoms of the disease.330 This, coupled with the high frequency, mandates periodic screening of all Turner syndrome patients. There are reports that indicate that Grave’s hyperthyroidism331 may also occur more commonly than previously recognized in girls with Turner syndrome, and we have one affected girl in our series. One report notes that in one patient during the hyperthyroid phase there was a marked increase in growth velocity.332 Final height, however, did not appear to be adversely or positively influenced. In a

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recent study, 24% of 84 children (0–19 years old) with Turner syndrome who were followed (longitudinally mean duration  8 years) developd hypothyroidism (and 2.5% developd hyperthyroidism).333 Thyroid disease has been reported as early as 4 years.334 Therefore, all patients with Turner syndrome should be screened annually for autoimmune thyroid disease with a TSH and a free T4 level from 4 years of age onward. We also have two patients with vitiligo and three with alopecia. Other forms of polyglandular autoimmunity (such as Addison disease, hypoparathyroidism, and pernicious anemia) have not been noted to be increased in frequency in Turner syndrome patients. The association of juvenile rheumatoid arthritis and Turner syndrome had only been reported in single case reports until the study of Zulian and colleagues.335 The authors conducted a survey of 28 pediatric rheumatology centers (15 U.S., 10 European, 3 Canadian) comprising an aggregate patient population of some 15,000 patients. Eighteen cases of juvenile rheumatoid arthritis were found in patients with Turner syndrome, of which 7 were polyarticular and 11 monoarticular. The authors calculated that this represented a sixfold increase over what would be expected, thus strongly suggesting an association between Turner syndrome and the occurrence of juvenile rheumatoid arthritis. The karyotypes of the affected individuals varied, but there appeared to be a predominance of 45,X patients with the more severe polyarticular form.

Gastrointestinal Disorders A number of reports have called attention to gastrointestinal bleeding, often massive, occurring in patients with Turner syndrome.336,337 The bleeding has been ascribed to intestinal telangiectasia, hemangiomatoses, phlebectasia, or dilated veins and venules. These vascular malformations occur without evidence of mesenteric, portal, or hepatic vascular abnormalities—which suggests a developmental rather than acquired cause. They do not appear to be associated with cutaneous hemangiomas reported in some Turner syndrome patients, however.338 It is not known which patients may be at risk for development of vascular bleeding. Whether these vascular abnormalities are a consequence of the same obstructive processes that result in the lymphedema is not known. Because large segments of bowel may be involved, we recommend conservative management when possible to avoid massive resections. If the patient has discontinued estrogen-progesterone replacement therapy, we recommend that it be reinstituted because this therapy appears to be efficacious in treating the bleeding of severe gastrointestinal vascular malformations339 and was reported as appearing to help one affected patient with Turner syndrome.340 A second cause of gastrointestinal bleeding and dysfunction appears to be inflammatory bowel disease. Several reports suggest an increased incidence of Crohn disease and ulcerative colitis in infants and children with Turner syndrome.341-345 Whether this increase may represent another autoimmune phenomenon or is associated with a particular genetic haplotype (HLA type) that may also prove to be increased in Turner syndrome remains

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to be established. Similarly, there is no conclusive evidence for a genetic or familial predisposition to ulcerative colitis in these patients. Because growth retardation and delayed sexual maturation are characteristic manifestations of inflammatory bowel disease and of Turner syndrome, careful attention must be paid to the review of systems in these patients. Conversely, short girls with inflammatory bowel disease and growth and pubertal delay may need assessment of gonadal function (measurement of gonadotropins) before their sexual delay is ascribed to their bowel disease alone. Celiac disease is also being detected in Turner syndrome patients in frequencies that may exceed ethnic population norms.346-348 There is also a suggestion that this may occur in association with immunoglobulin A deficiency, a known risk factor for the development of celiac disease.337 Considering that growth failure and pubertal delay can be manifestations of celiac disease and Turner syndrome, consideration has to be given to testing short girls with celiac disease with or without pubertal delay (especially if they are on dietary management and have not had catch-up growth) for Turner syndrome. In addition, the issue is raised whether screening girls with Turner syndrome for celiac disease is warranted in the absence of clinical symptoms. Based on recent data, the risk of celiac disease is increased in Turner syndrome (up to 6% of individuals are affected). Turner syndrome girls should be screened by measurement of tissue tranglutaminase IgA antibodies. Periodic screening is best begun at age 4, and should be repeated every 2 to 5 years.349,350 Whereas hepatic abnormalities have not in the past been reported to be a typical finding in Turner syndrome individuals, a report by Salerno and co-workers201 references 10 papers that describe a range of hepatic disorders/dysfunction in children and adults with Turner syndrome. A second paper351 describes three additional adult patients with varying forms of hepatic disease. In the pediatric age group, Salerno’s group followed some 70 girls prospectively for a mean of 7.6 years for the development of increased hepatic serum enzymes. They found a significant increase in liver enzymes in 20% over time. The majority appeared to be associated with the institution of oral androgen therapy for growth promotion or oral estrogen therapy for feminization, which were reversed with cessation of drug therapy. That the association was with the estrogen and not the progestational agent is supported by a previous report.352 As previously discussed, GH replacement does not appear to be associated with this finding. Among Salerno’s patients,201 however, were several who were thought to have autoimmune disease (one associated with celiac disease and one with hypergammaglobulinemia and a biopsy consistent with autoimmune hepatitis). In addition, two patients had mild steatofibrosis of unknown cause. Thus, there may be an increase (starting in childhood) in evidence of mild hepatic dysfunction from a variety of causes. Steatosis, steatofibrosis, and steatohepatitis are frequently seen.353 In a review of the Danish Cytogenetic Central Registry, a cohort of more than 500 adult patients with Turner syndrome was identified.303 These were

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cross referenced against the Danish National Registry of Patients to obtain data on first hospitalization diagnoses for these patients. Statistical analysis was done by comparing the patients with population controls to determine the relative risks for the diagnoses given. The relative risk for a diagnosis of cirrhosis of the liver was 5.69 (1.55 to 14.56). Therefore, adult Turner syndrome individuals appear to be at risk for progressive hepatic disease. Regular monitoring of hepatic enzymes, at least in adults, may be indicated. Consideration can be given to using transdermal estrogen therapy rather than oral therapy in girls and women who appear to show liver abnormalities because the transdermal route results in fewer effects on hepatic metabolism than does the oral route.354 Conversely, a study of transdermal estrogen replacement therapy in young healthy women with Turner syndrome showed mildly elevated liver enzymes at the time of estrogen washout—which normalized with oral estrogen replacement.355 Thus, multiple mechanisms for the liver enzyme abnormalities appear to be operative.

CARBOHYDRATE TOLERANCE The high incidence of carbohydrate intolerance, including frank diabetes in patients with Turner syndrome, has been documented over the past 40 years.356-359 In our series, 40% of patients tested with oral glucose tolerance tests showed abnormal responses.360 The clinical features are those of type 2 diabetes mellitus. Patients are non insulin dependent and are not ketosis prone. The abnormality may be reflected in a hyperglycemic response to oral glucose or to a meal, or it may progress to fasting hyperglycemia and polyuria. Insulin levels in the serum may be elevated, in the normal range, or low. Some genetic evidence suggests that the diabetes is the familial type 2 variety because abnormal or borderline glucose tolerance tests have been demonstrated in 51% of parents of patients.360 In that series, however, only one parent had clinical diabetes—which is far fewer than the usual clinical expression in type 2 families. Type 1 diabetes mellitus is infrequent, and in children most reports indicate that it is not in excess of that seen in the childhood population. There are no data on islet cell antibodies or glutamic acid decarboxylase antibodies available in Turner syndrome patients. The metabolic/endocrine abnormality most frequently reported in children and adolescents with Turner syndrome not receiving GH replacement therapy is a greater glucose response to an oral glucose challenge than that noted in age-matched control subjects.361,362 Insulin responses vary from series to series, and when increased in young patients may be related to concomitant androgen or estrogen therapy. Particularly, oxandrolone has been shown to be causing higher insulin levels in glucose tolerance tests than GH alone.363,364 When the metabolic defect was studied with a euglycemic insulin clamp technique to evaluate insulin sensitivity, and with indirect calorimetry to study whole-body glucose and lipid oxidation, insulin resistance was documented.365 The defect appeared to be restricted to nonoxidative pathways of intracellular glucose metabolism and was present in a group of young girls who had never

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received any form of hormone therapy. The authors concluded that the defect is similar to that found in type 2 diabetes mellitus. These data suggest that in naive patients with Turner syndrome insulin resistance is a very early metabolic defect. Other groups have not found this abnormality, however. A possible explanation may be the fact that the clamp studies were methodologically different, employing differing insulin levels.366 A number of physiologic factors are known to be associated with this form of diabetes. Their role in contributing to the insulin resistance of Turner syndrome has not been clearly documented. Obesity per se could be a factor, but when assessed by measurements of skin-fold thickness the majority of the patients are not obese.367 When this method was used in conjunction with standards for ideal body weight appropriate for height and age, we found that the degree of fat or percentage of ideal body weight did nor correlate with their carbohydrate intolerance.360 In the study by Caprio and colleagues,365 body mass index alone was used and there was no correlation between body mass index and insulin-stimulated glucose metabolism. Estrogen-progesterone replacement therapy is known to be associated with changes in carbohydrate tolerance. Data now suggest that it is the chemical structure of the progestins used in oral contraceptives that contributes most to the defect.367 In Turner syndrome, the use of estrogen replacement is probably not an important factor in pathogenesis or progression of insulin resistance. In a 6-month intervention study in adult Turner syndrome patients, a deterioration took place in the glycemic response during treatment with sex hormones (without a change in insulin sensitivity)—although a significant reduction in the level of fasting insulin was seen.368 Others have found a slight improvement in glycemia and insulin when formally testing insulin sensitivity.369 Longer-term treatment with sex hormones may show an improvement in the indices of carbohydrate metabolism, perhaps partly through the expedient effects of sex hormone replacement on physical fitness, body composition, and blood pressure. Hormone replacement therapy did have a beneficial effect on aortic stiffness and cholesterol levels.370 The use of GH alone or in combination with anabolic steroids has prompted a reassessment of the carbohydrate status of girls with Turner syndrome, with specific focus on the effects of these agents. In a study of 71 girls enrolled in the previously discussed GH/oxandrolone protocol of Rosenfeld and colleagues,146 glucose tolerance status was evaluated acutely (before and after 5 days) and after longer-term (2 and 12 months) administration of GH alone, oxandrolone alone, or combination therapy. Pretreatment fasting glucose concentrations were normal in the Turner syndrome patients, but their glucose responses to an oral glucose challenge were higher than in control subjects—with 15% classified as having impaired glucose tolerance. Insulin responses were so varied as to be impossible, as a group, to distinguish from normal. After acute or chronic GH therapy, carbohydrate tolerance as measured by integrated insulin or glucose responses did not change in the GH group.371 In the Dutch Turner syndrome study, in which there was a higher than conventional GH treatment group, Hgb

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A1c levels did not change from baseline yet decreased after discontinuation of GH treatment.200 Although there was clearly a trend toward higher fasting insulin levels with the higher GH doses after 4 years of treatment, no significant differences in the change of fasting insulin level or area under the curve for insulin between the GH dosage groups were found. After a mean period of 7.3 years of treatment, insulin levels decreased to values close to or equal to pretreatment values after discontinuation of GH treatment. Long-term results of the effect of GH treatment on lipid metabolism showed no abnormal effects.196 In another analysis of U.S. patients treated with GH, mean Hgb A1c levels also remained within the low to middle end of the normal range. Fasting insulin levels remained unchanged, whereas postprandial insulin levels rose and yet remained within the normal range at the 5-year mark.372 In a few patients, insulin responses were studied after GH was discontinued. Levels returned to normal except in those patients markedly obese (P.H. Saenger, unpublished data). Although the reversibility of the effects of long-term GH is reassuring, the consequences of hyperinsulinism lasting for several years during GH therapy are still unknown. In summary, glucose-stimulated insulin responses may be increased in children with Turner syndrome whether or not they are treated with GH, but these alterations may be further exaggerated by GH treatment373,374—with hyperinsulinemic responses appearing to compensate for reductions in insulin sensitivity. Data on disease prevalence in adult patients with Turner syndrome suggest an increased incidence of both type 2 and type 1 diabetes mellitus, suggesting an influence of haploinsufficiency of genes on the X chromosome. In adults, the relative risk of type 2 diabetes mellitus was 11.56%. The relative risk of type 1 diabetes mellitus was 4.38%. Because these data are from a medical registry, the risk of misclassification between type 2 diabetes mellitus and type 1 diabetes mellitus cannot be ruled out. The relative risk of diabetes certainly does not seem to be below 2, however. The increased risk for type 2 diabetes mellitus may be secondary to the increased prevalence of obesity in adult women with Turner syndrome.303,368,369 The data on lipid metabolism are more heterogenous and more studies are needed to determine whether or not lipid metabolism is really altrered in Turner syndrome.370

Neuropsychological Features The consensus regarding the currently available evidence is that the intelligence of persons with Turner syndrome is normal. There does not appear to be an increased incidence of moderate or severe mental retardation, nor do the individuals differ from their siblings in overall intelligence.375 The only clearly documented association among karyotype, phenotype, and the occurrence of mental retardation is the presence of a small ring X chromosome.6-10 In these patients, the high risk of mental retardation is most likely caused by the lack of lyonization of the ring X from loss of the X inactivation center—thus creating disomy for some X genes (see section on X chromosome genes).

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Of note is a report from 1973 of two severely retarded institutionalized patients with the Turner syndrome phenotype. In these patients, in addition to the 45,X karyotype extrachromosomal material (believed to be of chromosome 21 in origin) was described.316 Thus, some of the earlier reports of mental retardation16 may be the result of a small ring X or unrecognized coexistent autosomal aneuploidy—a condition recognized to occur in association with Turner syndrome. We have also found a potential physiologic or genetic correlate with IQ. In a group of 33 Turner syndrome patients who underwent neuropsychological testing, we also tested for thyroid autoimmunity.377 The group of 9 girls who had positive antithyroid microsomal antibodies had significantly lower verbal and full-scale IQ scores than the 24 patients in the antibody negative group and the 24 control children. Although overt hypothyroidism was not the mechanism, subclinical or variably transient periods of hypothyroidism could not be ruled out. Alternatively, because thyroid autoimmunity may be familial two other explanations are tenable. One is that the mothers of these patients had subclinical hypothyroidism, a mechanism now documented to account for statistically lower IQ scores in their offspring.378 A second is that there may be a genetic linkage of autoimmunity and learning disability. At this time, we consider that thyroid autoimmunity may be a marker of cognitive impairment in Turner syndrome and recommend early screening; periodic follow-up of thyroid function, including autoantibodies; and careful developmental assessment. Whereas individuals with Turner syndrome have normal intelligence and are characterized by normal verbal skills, they have selective impairments in nonverbal skills—including visual-spatial information processing, arithmetic skills, and the coordination of motor and visual-perceptual skills379-381 (coupled in some with a degree of hyperactivity).382 Studies suggest that these individuals also have an associated movement (motor) problem in daily life that cannot be directly attributed to cognitive dysfunction.383,384 As a group, Turner syndrome children also exhibit delayed emotional maturity, poor relations with peers, timidity, and negative body image.385 This negative body image may also be secondary to the fact that their short stature could negatively affect psychosocial function and self-esteem, although the severity of psychosocial problems associated with short stature is quite variable.386 Although Rovet and Holland reported a positive correlation between height and social competence in patients with Turner syndrome,387 in a more recent study no significant changes in psychosocial function were seen during GH therapy of 2 years.386 Although almost all investigators concur that specific cognitive deficits are present, the areas of presumed cognitive dysfunction differ in different reports—and interpretation of the results depends on numerous variables relating to the testing instrument and data analysis. The discrepancy between verbal and performance IQ appears to be, however, confirmed by many studies and may range between 10 and 15 points—with verbal being higher than performance.379-381,388-390

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In a study dividing patients by karyotype, the 45,X group showed the greatest discrepancy compared with patients with the mosaic or the structurally abnormal X group.388 The performance task was clearly affected and involved mental rotation tasks, particularly of the block design and object assembly type.320,388 Turner syndrome girls are also described as exhibiting deficits in tasks of left/right discrimination, road map skills, mental rotation, line orientation rotation, and integration of motor and visual-perceptual skills.382,389 Abnormalities in the cognitive and psychosocial abilities in persons with Turner syndrome likely reflect underlying atypical brain development and function.391 Therefore, investigators have attempted to use specific tests of brain function in an attempt to more clearly elucidate the neuropathologic basis for these performance differences as well as to examine the more general question of the role of the sex chromosomes and/ or gonadal hormones in brain development and hemispheric specialization. Qualitative and quantitative analyses of electroencephalographic background data suggested transiently appearing hypofunction at the parietal, temporal, and occipital areas.392 Lateralization, however, was inconsistent. Electrical activity, measured as event-related brain potential and reaction time to auditory stimuli, was assessed in groups of young and older Turner syndrome girls.393 Their event-related brain potential data suggested a slower than normal maturational change, whereas their reaction time responses were less than those of control subjects in both age groups. In one study of a pair of prepubertal monozygotic twins discordant for X monosomy, extensive neurobehavioral and anatomic (MRI) data are presented.394 The data demonstrate that although both sisters scored in the superior range of intelligence the affected twin had a wide discrepancy in her performance compared with her verbal scores, which was not present in the unaffected twin. The neuroanatomic findings for both sisters fell in the range of normal, but there were significant differences between the twins—the relevance of which is unknown. In a more extensive MRI study comparing 18 adult women with Turner syndrome and appropriate controls, the subjects had significantly smaller values than the control subjects in MRI-measured volumes of multiple nuclei as well as in bilateral parieto-occipital brain matter.395 The authors postulate that the X chromosome modulates development of gray matter in striatum, diencephalon, and cerebral hemispheres. Others,396 including the group of Lippe et al.,397 have used positron emission tomography in an attempt to correlate regional abnormalities in cerebral metabolic rates with neuropsychological profiles. These initial studies suggest that hypometabolism in the parietal and occipital lobes may be common among Turner syndrome girls with learning disabilities and also may help explain the impaired visual-spatial information processing. In addition, preliminary studies using functional MRI show activation deficits, particularly in the dorsal lateral prefrontal area of the cortex. This area may be related to executive function, another area of deficit observed in women with Turner syndrome,398 and to-

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gether with the other findings may account for the neurobehavioral phenotype in this condition. Recently published results of neurogenetic research have stimulated interest and controversy. Thus, the differential gene expression known to depend on the parental origin of chromosomes (genomic imprinting) is now being associated with the possibility that in Turner syndrome genes may be differentially expressed according to the origin of the X chromosome present. It is known that in 45,X Turner syndrome the X chromosome is of maternal origin in about 70% to 80.5% of cases.76-78 Skuse and associates investigated 80 females with Turner syndrome, of whom 25 (the expected proportion) had an X chromosome of paternal origin (Xp). These girls showed satisfactory social adjustment and had higher verbal and executive functional skills than the larger group of girls with the retained X chromosome of maternal origin (Xm).84 In an extension of these studies, the Skuse group investigated the relationship of verbal and nonverbal memory with origin of the X chromosome.399 They observed that 45,Xp Turner females matched controls in verbal memory, whereas this was not the case in the 45,Xm females. In contrast, the results of 45,Xm patients matched those of controls in visual-spatial memory tests but the 45,Xp group did not. The authors conclude that these data indicate an imprinted locus for social cognition on the X chromosome that is not expressed (silenced) on the maternally derived X (putting males and Xm Turner females at risk for developmental disorders of language and social cognition but protecting girls and Xp Turner females). This defect in social cognition may translate into difficulties or lack of understanding of social and nonverbal cues. For example, a young sibling of an adolescent Turner syndrome patient commented about her sister, “She just doesn’t get the point and that isolates her”—and a mother commented that her daughter “was not street wise.” Many of the girls and women tend not to “read” sarcastic facial expressions or understand double entendres. The differences in cognition associated with parental X chromosome origin, however, have not been reported by all investigators and need to be corroborated by other groups. For example, Haverkamp and co-workers also examined parental origin of the X chromosome in relation to cognition and came to the conclusion that age and familial covariance also influence cognitive function in Turner syndrome patients.400 These factors may not have been controlled for in the previous studies. In other words, social cognition and visual-spatial abilities change with age—and differences were no longer apparent in the Haverkamp cohort of adolescent Turner syndrome patients when compared with the normal population. The role of estrogen deficiency and estrogen replacement therapy also needs to be considered in the context of organic causes for the cognitive, social, and functional profiles of girls and women with Turner syndrome. Indeed, estrogen replacement has been suggested as an explanation for improved motor speed, nonverbal processing, and memory in estrogen-treated Turner syndrome patients compared with placebo-treated patients.401,402 Whether these findings will influence the

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quality of life of females with Turner syndrome is not yet known, although the positive effect on motor function could mitigate the findings of motor impairment described in prepubertal girls. Some of these deficits may be improved by hormonal therapy at puberty.402,403 Difficulties in visual-spatial organization, social cognition, and problem solving (mathematics) and motor deficits describe specific neuropsychological deficits that do not improve with time or estrogens.404-408 As a group, girls and women with Turner syndrome excel at verbal skills, and many adults with Turner syndrome have university level education.408-409 Recent studies do not support the influence of height as influential on dating and initiation of sexual activities, but the role of physical abnormalities is unclear.410-412 The developmental process is likely affected by treatment with GH and estrogen that potentially may influence the child’s perception of herself.61 In open-ended interviews, women with Turner syndrome reported that dealing with premature ovarian failure and loss of fertility was the most difficult part of having the condition.413 Psychiatric disorders have not previously been reported to be increased in Turner syndrome, but there have been a number of reports describing the occurrence of anorexia nervosa in patients with Turner syndrome.414 It is presently unclear whether this is increased in comparison with normal girls. Kron and colleagues414 have pointed out the danger of overlooking an X chromosomal disorder in a patient in whom growth arrest and pubertal delay are attributed to excessive weight loss alone. This is especially relevant because gonadotropins may revert to prepubertal levels.415 One report, in which the literature was reviewed as well as reporting two patients, suggests that schizophrenia may be increased in women with a mosaic chromosomal karyotype416 (but again this remains to be confirmed). Finally, Skuse and co-workers have reported what appears to be a significantly increased risk of autism among Turner syndrome individuals.417 They noted that this appeared to be in excess in those girls who had retained the maternal X chromosome (Xm) and had either a missing or a structurally abnormal second X. A report of a Turner individual with autism and an Xm genotype418 is supportive but not confirmatory of this hypothesis because the majority of patients with Turner syndrome are Xm. The hypothesis of Skuse and co-workers that unmaking of a familial predisposition toward autism may occur by the deletion of the protective paternal X chromosome remains to be tested.419 A final aspect of the neuropsychological profile of girls and women with Turner syndrome might be called personality. The first systematic investigation of the personality of girls and women with Turner syndrome was conducted in the 1970s.420 This study and subsequent studies indicate that some personality traits are common to the majority of women and that Turner syndrome individuals had a high stress tolerance, a tendency toward overcompliance, and a higher degree of dependence and limitations in emotional competence.382,421,422 They also may have an impairment of self-esteem.423 The role of the previously described cognitive differences in affecting adaptive personality traits needs to be

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investigated. Studies indicate that women with Turner syndrome have a typically female pattern of development with unambiguous female gender identification.424 The scant data available indicate that heterosexual romantic fantasies are common but that dating and initiation of sexual activities may be somewhat delayed or infrequent and that they leave home later than their siblings. A Swedish psychosocial report from 22 middleaged women with Turner syndrome notes adequate adjustment as adults but a prolonged and isolated adolescence, concerns about infertility, and poor compliance with hormone replacement therapy.425 Another report from Israel, however, notes a high degree of adjustment that may relate to the more structured environment.426 In the context of psychosexual adaptation, attention should be paid to sex education and orientation in adult sexuality. Because some of these girls mature more slowly than their peers, they may not be ready for or interested in sex education when it is given at school. They may also be more self-conscious in relation to beginning a sexual relationship because of being “different.” More attention to sex education and sex therapy may be necessary to address this area. Information about assisted reproductive techniques should also be provided.

RECOMMENDATIONS Whether this complex psychosocial behavior is endogenous or adaptive, as more aggressive and innovative endocrine therapies begin to alter the outlook for stature, timing of feminization, and fertility attention should still be paid to assessment of psychosocial adaptation. Because the factors that affect the quality of life are the same as those that affect the rest of society, psychological care should be provided within the context of helping to prevent difficulties and normalizing the developmental process rather than operating from an illness model. Plans for both medical and psychological intervention should be developed so as to reinforce and support the individual’s self-esteem and to ensure that individuals with Turner syndrome remain in the mainstream of social, educational, and employment activities. A comprehensive psycho-educational evaluation is recommended preceding school entry. Children with Turner syndrome may have other conditions (e.g., dyslexia or attention deficit) that need to be addressed. In view of slower processing speed, untimed testing may be appropriate. Age-appropriate pubertal induction is recommended. It is important to address all issues surrounding sexuality, infertility, and reproductive options in an honest and open manner because “secret-keeping” may have unintended negative consequences and actually amplify the problems.61,427 Many of these issues are discussed in patient-oriented material available through the Turner’s Syndrome Society of the United States web site (www.turner-syndrome-us. org). Similar web sites exist for other national Turner syndrome organizations. Patients with Turner syndrome and their parents need to be well informed about the learning problems associated with Turner syndrome because most individuals are affected by these difficulties,

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if only to a mild degree. They need to understand that even with intervention some of these learning difficulties do not disappear with age but persist throughout adult life. Learning disabilities can be a major impediment to emancipation from family and to career enhancement, although many women with Turner syndrome do achieve high professional status.428

Management: Pediatric And Adult EVALUATION The clinical aspects of which child should be evaluated for Turner syndrome, and at what age, have been addressed in part in this chapter and in the previous chapter on disorders of puberty. Nevertheless, certain clinical aspects warrant review. It is obvious that the female neonate with a webbed neck or edema of the hands and feet merits further investigation. Similarly, the girl with multiple physical stigmata, the girl with coarctation of the aorta, or the girl with radiologic evidence of abnormalities in the urinary collecting system or a horseshoe kidney should also be considered for evaluation. What is less obvious is which girl without obvious stigmata or highly suggestive organic defects should be evaluated (Tables 15-7 and 15-8). Many girls with Turner syndrome do not have obvious physical stigmata, and it is necessary to recognize that the diagnosis may be difficult in such cases. We reviewed the records of 144 of our patients and found that only 41 had been diagnosed as neonates or toddlers because of obvious physical stigmata. The remaining 103 (71%) presented with short stature or short stature and pubertal delay.424 In a report of 100 patients, a similar finding was noted.111 Only 15% were diagnosed at birth. The remaining patients were diagnosed often only after 12 years of age.134 The incidence of Turner syndrome is about 1:2,000 live female births, but the statistics change as one considers stature. For example, among 2,000 girls in childhood only 60 are at or below the third percentile—leaving most of the group at risk (excluding infancy and early childhood, and even if no stigmata are present) much smaller. Given other genetic causes for short stature among this group (as well as other acquired causes, and the possible presence of one or more clinical features), the number of girls who might need evaluation becomes less than 1 in 60. Thus, the rationale for considering performing a complete chromosomal karyotype on a girl with short stature becomes even more obvious. The role and utility of SHOX gene testing as part of a short stature evaluation remains to be evaluated. Once the diagnosis of Turner syndrome has been established and a chromosome analysis has been carried out, additional diagnostic procedures are indicated. These diagnosis and management strategies have been published as recommendations from a consensus workshop. As participants in that workshop, our recommendations tend to be consistent with these recommendations.61,428

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TA B L E 1 5 - 7

Screening at Diagnosis of Turner Syndrome in Children and Adults All Patients • Cardiovascular evaluation by specialistª • Renal ultrasound • Hearing evaluation by an audiologist • Evaluation for scoliosis/kyphosis • Evaluation for knowledge of Turner syndrome; referral to support groups • Evaluation for growth and pubertal development Ages 0-4 Years • Evaluation for hip dislocation • Eye exam by pediatric ophthalmologist (if age 1) Ages 4-10 Years • Thyroid function tests (T4, TSH) and celiac screen (TTG Ab) • Educational/psychosocial evaluation • Orthodontic evaluation (if age  7) Ages ⱖ 10 Years • Thyroid function tests (T4, TSH) and celiac screen (TTG Ab) • Educational and psychosocial evaluations • Orthodontic evaluation • Evaluation of ovarian function/estrogen replacement • LFTs, FBG, lipids, CBC, Cr, BUN • BMD (if age 18) BUN, blood urea nitrogen; CBC, complete blood count; Cr, creatinine; FBG, fasting blood glucose; and LFTs, liver function tests. ª See Table 15-6. From Bondy CA for the Turner Syndrome Study Group (2007). Care of girls and women with Turner syndrome. J Clin Endocrinol and Metab 92(1):10–25.

TA B L E 1 5 - 8

Ongoing Monitoring in Turner Syndrome

From Bondy CA for the Turner Syndrome Study Group (2007). Care of girls and women with Turner syndrome. J Clin Endocrinol and Metab 92(1):10–25.

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Initial and Follow-up Studies in Adolescence and Adulthood Because the clinical manifestations of gradually developing thyroid failure can be subtle and easily overlooked in a short child, routine serum thyroid function tests (including thyroxine, thyroid-stimulating hormone, and antithyroid antibodies) are indicated. Subsequently, thyroxine and thyroid-stimulating hormone or thyroid-stimulating hormone alone should be determined at 1- to 2-year intervals in all patients regardless of whether significant titers of antithyroid antibodies were detected initially. This follow-up should be continued as an adult. Routine testing for other autoimmune glandular failures does not appear necessary unless other evidence of autoimmune disease develops, such as alopecia, vitiligo, or a glandular failure (other than the ovary). Routine renal ultrasound may detect structural abnormalities in renal architecture or collecting system anatomy. If no abnormalities are present, follow-up studies are not routinely indicated. If significant abnormalities are detected, follow-up evaluation and therapy may be indicated and long-term screening for urinary tract infection may be necessary. Infants with Turner syndrome may have an increased risk of congenital hip dislocation,428 and care should be taken to evaluate the infant. Poor management in childhood can result in serious morbidity in the adult woman. Although the other osseous abnormalities of Turner syndrome may be multiple, unless they cause significant morbidity radiologic survey of the entire skeleton is not routinely recommended. Once a skeletal deformity is noted, however, orthopedic consultation may be indicated. In addition, the practical effects of having an increased carrying angle (e.g., interference with activities such as swimming using a backstroke) need to be recognized and discussed. Abnormalities of the fingernails and toenails are usually only of mild cosmetic interest. A report of cellulitis and infection secondary to ingrown toenails warrants calling attention to the toenail deformity (if present) and its predisposing conditions (including intermittent lymphedema) and taking care to prevent the ingrown condition.430 This issue may continue into adulthood. Scoliosis is common but usually does not progress rapidly and can most often be managed conservatively. If growth-promoting regimens are used, careful attention should be paid to detecting progressive scoliosis. If noted, its cause should be determined radiographically. We have observed two patients in whom it was caused by leg-length inequality. Madelung deformity requires recognition so as to exclude other causes of this bony malposition, but it does not require orthopedic correction. It is actually rather infrequent in Turner syndrome.431 Girls with Turner syndrome have higher risks for scoliosis and kyphosis than the general population. Ten to twenty percent of girls with Turner syndrome develop scoliosis and kyphosis. Vertebral wedging also appears to be more common.432,433 Both problems can be accentuated with rapid growth. Phalangeal bone density has been reported to be normal during childhood.434

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Plasma FSH and LH concentrations at the time of diagnosis may or may not be elevated. They could serve as an indication of future gonadal function but should not be used to screen for the diagnosis of Turner syndrome. In addition, we observed two girls with significantly elevated FSH levels in early childhood who went on to spontaneous onset of puberty (although subsequent ovarian failure). Therefore, predictions of gonadal failure based on high infant/childhood FSH may be given, but with care. Pelvic visualization techniques, including ultrasound and MRI, appear to be clinically indicated only in those situations in which there is a question of gonadal anatomy or potential pubertal function. The absolute indication is the patient with a Y chromosome or marker chromosome in the karyotype because she is at risk for the development of gonadoblastoma. The ultrasound may demonstrate an adnexal mass in the patient with the Y chromosome in comparison with the normal female (Figure 15-18). Surgical removal of the bilateral adnexal structures is then indicated. The recommendation for concomitant hysterectomy as a prophylactic measure against endometrial carcinoma in an agonadal individual who will be on long-term estrogen replacement therapy is controversial. Some physicians believe that because it lends little extra morbidity to the indicated pelvic surgery it is a warranted consideration. Others believe that the psychological benefit of monthly menstruation, independent of the artificiality of its method of production and its irrelevance to fertility, precludes the procedure unless there is evidence of local spread of the gonadoblastoma. Finally, oocyte donation, in vitro fertilization, and embryo transfer are being performed successfully in agonadal women— including women with Turner syndrome who have an intact uterus.435-437 Thus, the issue bears complete discussion with the individual patient and physicians involved— as well as informed patient consent. A more difficult management decision concerns the method of follow-up of the patient with a cytogenetically documented Y chromosome or molecularly detected Y-derived marker chromosome in the karyotype in whom the initial radiographic studies are normal. The question of whether prophylactic removal of the adnexal streaks should be performed electively or delayed until the ultrasound or MRI suggests a mass has not been entirely resolved. Data indicating that tumor formation may be present microscopically at a very young age are a strong argument for elective surgery, however, even in the absence of a demonstrable mass.228 However, data from the Danish cancer registry failed to show the presence of gonadoblastoma or dysgerminoma in 29 women with a Y chromosome—suggesting that the risk may be overestimated.60 Pelvic visualization techniques may also be useful in patients with evidence of some gonadal function at puberty.438 In such patients, unilateral or bilateral ovarian structures indistinguishable from normal may be demonstrable. The anatomic findings would lend supportive evidence to the clinical or gonadotropin data and might deter the physician from the institution of hormonal replacement therapy. It is important to point out that spontaneous puberty, even with menses that appear cyclic,

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does not always mean that normal ovulatory cycles are occurring or will continue to occur normally in these patients.232,244 Anovulatory cycles do not result in normal endometrial histology and could impair the capacity for successful ovum implantation and predispose to endometrial hyperplasia or cancer. Although it is clinically impractical to study a complete cycle, efforts should be made to ensure that most cycles are ovulatory. If not, the patient should be either cycled artificially or given periodic courses of progesterone for withdrawal. Otologic abnormalities are very frequent. Deformation of the pinna is most frequent in patients with the lymphedema phenotype. Otitis media is extremely common and should be treated immediately. The high prevalence of hearing loss, either primary or secondary to residual serous otitis media, suggests that otorhinolaryngologic evaluation with audiometry may be indicated in a large number of patients. In infancy, feeding techniques such as those used for cleft palate patients may also be indicated. We have the clinical impression that mild abnormalities in phonation, independent of hearing impairment, are present in a number of our older patients. Because these abnormalities in speech may be a consequence of a palatal deformity, speech evaluation may also be indicated. Myringotomy and polyethylene tube placement are considered the primary modes of therapy for serous otitis media in Turner syndrome, and tonsillectomy or adenoidectomy or both should be avoided if possible. Removal of the tonsils and adenoids may not be advisable because of its dubious value for the treatment of serous otitis media and because the pharyngeal tissue often serves as an anatomic prosthesis for pharyngeal competence. Ophthalmologic abnormalities are common, and an appropriate examination should be performed.130 Ptosis, hypertelorism epicanthal folds, and upward slanting palpebral fissures are common in Turner syndrome. Red-green color deficiency is present in 8% of the population, a percentage similar to that found in most males. Strabismus and hyperopia occur in 25% to 35% of these children, putting them at high risk for amblyopia. To promote early detection and treatment and to prevent visual loss, children with Turner syndrome should be evaluated by a pediatric ophthalmologist at 12 to 18 months of age.61 Hypertension is common in Turner syndrome, and blood pressure should be measured at each visit. Renal arteriography, selective venous catheterization, or both may be indicated in some patients with hypertension. Although the evidence of multiple arterial and venous abnormalities in Turner syndrome suggests that gross or segmental renal vascular disease may be responsible for the hypertension, the disordered anatomy may also render detection of treatable lesions difficult. An example of a study performed in an 11-year-old patient with significant hypertension is shown in Figure 15-20B. Multiple renal arteries and veins were noted, and selective catheterization was virtually impossible. Nevertheless, in some patients surgically treatable lesions may be apparent. The risk of hypertension continues into adulthood and is a major risk factor for aortic dilation. Adult women with Turner syndrome are also at risk for cardiovascular

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disease. Therefore, when hypertension is detected in the child or in the adult it should be treated aggressively to minimize the risk of cardiovascular disease as well as of progressive dilation and aortic dissection.439 Because the detection of cardiovascular abnormalities requires specialized diagnostic and clinical evaluation, and because their presence may indicate the need for cardiac surgery or lifelong antibiotic therapy for subacute bacterial endocarditis prophylaxis, a cardiologic consultation should be obtained for all patients and should include an ECG. A prenatal ECG does not obviate the need for a postnatal examination because bicuspid aortic valve and coarctation may not be detected in utero. A cardiologist skilled in the assessment of congenital heart disease should interpret the ECG. If no abnormalities are detected in childhood, a repeat cardiovascular examination and ECG should be conducted during adolescence because aortic dilation may occur without any other risk factors. If bicuspid aortic valve or mitral valve prolapse is detected, subacute bacterial endocarditis prophylaxis is recommended and follow-up should be more frequent. If aortic root dilation is found, the cardiologist should obtain follow-up ECGs at a frequency based on the severity of the dilation present and the presence or absence of other risk factors for dilation (such as hypertension, bicuspid aortic valve, and previously repaired coarctation). Although we consider the potential morbidity and mortality of this condition (when present) to be similar to that of Marfan syndrome, there are no outcome data to absolutely recommend Marfan management strategies. The role of long-term prophylactic -adrenergic blockade to slow the rate of dilation and lessen the development of aortic complications in some patients needs to be assessed.420 Prophylactic aortic root repair may have to be considered for the patient with marked and progressive dilation, however. If unexplained chest pain occurs, even with initially normal studies, the diagnosis of aneurysm or dissection must always be considered. Coarctation of the aorta is treated surgically in most cases. Although the postoperative risk of the development of mesenteric arteritis is higher in males than in females,441 we have observed this syndrome in a patient with Turner syndrome. Long-term prognosis is generally good, with few reports of recurrence or complications. However, asymptomatic poststenotic aneurysmal dilation of the aorta may be detectable on screening chest radiography or cardiac MRI (Figure 15-19B and C) and should be followed carefully to prevent dissection or rupture. The decision to seek consultation for plastic surgery to correct the webbed neck deformity or the forwardly displaced ears is individual. It must be pointed out to the patient and family that in addition to the webbing the neck may also be short. Therefore, cosmetic surgery may be somewhat disappointing. In some cases, however, satisfactory results are achieved. The apparent predisposition toward the development of keloids in these patients must also be taken into account. There is some evidence to suggest that early surgery may have a better cosmetic result.442 Keloids have also been seen in conjunction with ear piercing, suggesting that this procedure should be avoided or performed with great care.

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If, or when, psychometric testing should be performed is an individual family decision. Nevertheless, a preschool evaluation to rule out major areas of cognitive dysfunction might be advisable. In light of the previous discussion, school performance should be monitored— and specific problems should be attended to with skilled cognitive specialists.

Endocrinologic Management The data show that GH safely increases growth velocity and final adult height in Turner syndrome and have led to regulatory approval for the use of GH in many countries. The criteria for which girls are potential candidates for GH vary. The previously cited guidelines suggest GH should be initiated when the Turner syndrome girl drops below the 5th percentile of the normal curve. This may delay therapy, however, in some girls who could benefit from early therapy and earlier initiation of estrogen replacement (see Turner Toddler study81). It also fails to consider genetic midparental target height. An alternative is to use Lyon curves for Turner syndrome patients as previously described, project their final height if untreated, and initiate therapy if this falls below the 5th through 10th percentiles for normal girls or –1 to –2 SDs below their genetic target midparental height if they are from tall families. A target goal should be set after discussion with the family or with the patient and family, with the emphasis that therapy will be a long process and will not necessarily result in complete normalization of target midparental height. There are data to document that the response to GH in Turner syndrome does not differ between patients with normal GH responses to pharmacologic stimuli and those with insufficient responses.443,444 Therefore, GH-provocative testing is neither required by regulatory agencies for GH use nor recommended unless the patient’s growth velocity is significantly below the Turner velocity for age (suggesting the presence of hypothalamic-pituitary disease). When GH is started in young girls, concomitant anabolic steroids may not be needed—and there might be some theoretical reasons to avoid years of anabolic steroid therapy. In older girls (9 to 12 years) or in girls older than 8 in whom therapy is started at a significant height deficit, consideration should be given to starting combined therapy with GH and a nonaromatizable androgen such as oxandrolone because it is aromatization to estrogen that promotes epiphyseal fusion. The recommended dose of oxandrolone should not be above 0.05 mg/kg/day, significantly lower than that used as the initial dose, and slightly lower than that finally used in an often cited study.171 Dosing with GH has been previously discussed in the section on growth-promoting therapy. The starting dose is 0.05 mg/kg/day. Individualization of dose can be considered, depending on the patient’s growth response. Prediction models may help in this respect.445 GH should be continued until the final target height previously agreed on with the family (or patient and family) is achieved or until near epiphyseal fusion precludes a significant effect. If androgen is being used, it should be

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discontinued when the decision is made to begin estrogen therapy. The issue of when to begin estrogen therapy in this paradigm of GH treatment is less clear. The earlier discussion in this chapter reviews data that suggest that estrogen alone is not useful for augmentation of final height. There are also data that suggest that epiphyseal fusion progresses significantly after 12 to 18 months of estrogen therapy. In addition, when estrogen is used for feminization in conjunction with ongoing GH therapy further growth augmentation may not occur.446 Finally, in one paper it was documented that the longer the estrogen-free years of GH therapy the taller the final height. The data in this report also showed that if GH is begun early, however, estrogen treatment could be begun at a younger more appropriate age and still have a greater gain in projected final height.447 Thus, in aggregate the data suggest that estrogen therapy for feminization should not begin until the patient has reached the estimated height at which she will require only 1 to 2 more years of GH therapy. With the early initiation of GH therapy, that age is likely to be one that is socially acceptable and physiologically normal. In a recent report,448 Quigley et al. report positive data of the synergistic effect of early low-dose estrogen (ethinyl estradiol 25 ng/kg/day from age 5 years on) and growth hormoone on adult height. An additional management issue has developed with respect to girls with spontaneous ovarian function. Although studies done before GH treatment suggest that final height does not appear to differ between those girls with spontaneous and induced puberty,231 there are few data that are applicable to the GH-treated girl. In one study,449 four girls developed spontaneous puberty during the first year of GH treatment. They appeared to have an augmented growth velocity compared with the prepubertal girls that persisted into the second year of therapy. Final height data are not available, however. In the study of Reiter and associates, the relationship to estrogen-free years held whether the estrogen was exogenous or endogenous.447 Thus, if a girl has been treated for several years with GH and has achieved significant height augmentation spontaneous puberty may be of no concern with respect to final height. If it occurs in a girl who is just beginning therapy, however, the initial augmentation could be offset by subsequent epiphyseal closure. Therefore, in a very short girl in whom a significant height augmentation has not yet been achieved and puberty occurs relatively early some consideration may be in order to inhibit puberty with long-acting GnRH analogues. If GH therapy is not available or otherwise contraindicated, consideration of the use of anabolic steroids as a growth promoter before estrogen therapy in girls 10 to 13 years old who have no clinical or hormonal evidence of ovarian function might be appropriate. The rationale for this, recognizing that it may not increase final adult height, is that it could provide a growth spurt so that when feminization is induced the patient is at a height somewhat closer to that which appears appropriate for obvious signs of puberty. Oxandrolone, 0.05 mg/kg/day, could be used—with side effects (including clitoromegaly and facial hair) monitored.

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If the growth velocity begins to decline (usually after 12 months) and if epiphyseal fusion has not taken place, the steroid dose could be increased (0.075 to 0.1 mg/kg/ day) to see whether a second spurt can be induced. After that, the medication is discontinued, gonadal status is reassessed, and estrogen therapy is initiated. Finally, some have considered orthopedic leg lengthening as an alternative approach for correction of short stature in Turner syndrome. Experience with this procedure in this condition is still very limited, however. Although there is controversy over the risks and benefits of hormone replacement therapy in the postmenopausal woman,450,451 the psychological needs for estrogen in the induction and maintenance of secondary sexual characteristics in young women—and the physiologic needs, among others, for induction of peak bone mass and mineral maintenance and metabolism—are well accepted. Compared with the emphasis on developing optimal regimens for postmenopausal hormonal replacement therapy,450-455 however, there are few reviews of the optimum preparation and dose schedule for long-term use in younger agonadal women. With regard to maintenance of bone mineral, the issue is further confounded in Turner syndrome by the difficulty in deciding what standards to apply to the measurements of bone mineral density,121 the presence of an intrinsic appearance of osteopenia,456 and the role GH therapy might have in altering bone mineral content.457 Nevertheless, extrapolation from previous clinical practice and the newer data allow clinicians to develop a rational approach to the principles of therapy. These include longterm cyclic therapy to prevent estrogen-related uterine neoplasia and adequate estrogen to achieve and maintain bone mineral content and preserve the cardioprotective effects on plasma lipids and lipoproteins. Cyclic therapy is recommended, not only for its positive psychological effect in the adolescent but for its uterine protective effect.429,458 Data on steroid receptors in hormone-dependent target tissues suggest that progesterone down-regulates or blocks the estrogen receptors,459 and that when given in conjunction with estrogen may be protective against estrogen-induced neoplasia. Progestins also act to attenuate the action of estrogen by increasing the activities of enzymes that convert estrogen into biologically less active estrone and inactive estrogen sulfate. When endometrial responses were prospectively evaluated in Turner syndrome patients receiving long-term replacement therapy, hyperplastic changes were noted in only a small percentage of those receiving combined therapy compared with those receiving cyclic estrogen alone.460 The goal is the complete conversion of the endometrium from a proliferative to a wholly or predominantly secretory state. The dose of progesterone should be minimized to prevent an adverse effect on carbohydrate tolerance461 and lipid metabolism.462,463 Therefore, the use of traditional-dose oral contraceptives as longterm therapy is not ideal—given that their progesterone content may be higher than is necessary or optimal. The estrogen dose needed to achieve maximum bone mineral and to prevent bone mineral loss in Turner syndrome is not known. There are data in adult women with Turner syndrome, however, to show that there is a posi-

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tive correlation between bone mineral content and the duration of estrogen treatment.464 A short-term study compared the metabolic effects of low-dose oral conjugated estrogen (0.625 mg) with relatively high-dose ethinyl estradiol (30 ug) combined with progesterone in young women with Turner syndrome.465 Whereas both regimens normalized the hypotrophic endometria and suppressed hyperinsulinemia, only the higher dose normalized FSH. Neither agent completely normalized the bone metabolic profile, although the higher-dose ethinyl estradiol regimen was more effective in normalizing bone turnover markers. Future investigations of selective estrogen receptor modulators, as well as calcium and vitamin D, were suggested. Thus, the recommendations for postmenopausal women (0.625 mg of conjugated equine estrogen or its equivalent)—which approximates 60 pg/mL estradiol— may not be adequate long-term replacement for the agonadal woman. Therefore, although this was our previous recommendation for the patient with Turner syndrome it is unlikely that it represents sufficient physiologic longterm replacement. Somewhat higher doses (to achieve the equivalence of 90 to 120 pg/mL) would appear to be more logical. A dose of 2 mg of micronized 17b-estradiol, which is relatively equal to 1.25 mg conjugated estrogen (Premarin), has been shown to have a greater effect in increasing spinal trabecular bone density in postmenopausal women than did the more commonly used 1-mg dose.465 These estrogen doses are still lower than those in most oral contraceptives. As most girls are treated from infancy or early childhood on with GH, they are at a better height percentile by age 12 to 13 years. Based on these considerations, hormone replacement therapy may begin sooner (Table 15-9). To initiate feminization, one can begin with 0.3 to 0.625 mg conjugated estrogen daily for 6 to 12 months. After initiation and progression of breast development and uterine growth, the estrogen may be increased to 0.9 to 1.25 mg and cyclic therapy with progesterone [e.g., 10 mg medroxyprogesterone (Provera)] begun. Alternatively, progressive doses of 17 -estradiol can be used (if available), culminating in a final maintenance dose of 2 mg. A calendar month can be used for convenience, beginning with estrogen on day 1 and continuing it through day 23. Progesterone is started on day 10 and continued through day 23. No medication is ingested for the remainder of the calendar month, when withdrawal menses usually ensue. Estrogen is then restarted on day 1 of the next calendar month, and the cycles are repeated. Follow-up includes monthly breast self-examination. Pelvic examinations and Papanicolaou smears should commence yearly if the patient is sexually active. Otherwise, frequency of these examinations can be determined by the gynecologist to whom the patient is referred as an adult. Cyclic therapy, and maintaining a normal adult uterine endometrium, also enhances the potential for success for reproductive options such as in vitro fertilization. In addition, for those girls with some residual ovarian function advances are now being made in the area of oocyte cryopreservation coupled with later intracytoplasmic sperm injection.189 This potential option also enhances the reasons for preservation of the uterus. When

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Turner women are older, the issue of whether cyclic therapy should be converted to continuous therapy to prevent menopausal symptoms in the “off week” needs to be considered. This can be achieved safely if the progesterone dose is sufficient to prevent hyperplasia. The use of other preparations of estrogen and progesterone should also be assessed for long-term use as replacement therapy. These include oral synthetic ethinyl estradiol, transdermal estradiol-17 , and transvaginal suppositories of estrogen and progesterone. The issues that need to be considered are the route of administration and effects of different preparations on systems (such as the hepatic enzymes) that contribute to the potential adverse affects of estrogen on blood pressure, clotting, and gallstone formation. Transdermal estrogen, for example (unlike oral estrogens), avoids the first-pass effect on the liver and may therefore obviate adverse effects on hepatic proteins.466 Our initial experience with transdermal estradiol was in a Turner syndrome patient who had recurrence of pedal lymphedema with the institution of oral estrogen replacement. She experienced less swelling with the transdermal preparation. Other patients were started on oral estrogen and then switched to patch estrogen with oral progesterone. For this form of therapy, one can use a 100-mg patch—which appears to be similar in potency to 1.25 mg of conjugated estrogen or 2 mg of estradiol. The patient changes the patch after 3.5 days (two per week) for the first 21 days (3 weeks) of the calendar month. Progesterone is taken from days 10 through 21, and no patch or progesterone is used after day 21 until the next month. Follow-up is the same as for oral estrogen therapy. An additional comment should be made about breast development in response to estrogen replacement in Turner syndrome. We have observed that the final size of the breast appears to be more consistent with genetic predisposition than with the dose of estrogen or the time it was initiated. The only clear exception appears to be in the girl in whom there are marked clinical signs of extensive fetal edema of the upper body. This includes severe webbing and/or marked nipple hypoplasia at birth. It appears that some of these girls develop very little breast tissue in response to estrogen and that this may be caused by mechanical damage to the breast primordium in utero. In such patients, breast augmentation in late adolescence may be necessary (if the patient is so inclined and if she has not had a problem with keloid formation). Finally, it must be emphasized that repeated discussion of important issues must take place among family, patient, and clinician. After years of follow-up, one tends to overlook the fact that the patient is no longer a child—and reeducation and new lines of communication need to be established with the patient as an emerging adult.

Transition Management The transition from pediatric to adult health care should occur at the completion of growth and puberty during late-stage adolescence (usually by 18 to 21 years). However, transition should be initiated as a staged process.

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Beginning at approximately 12 years of age, the center of care should be shifted incrementally from the parent to the adolsecent with Turner syndrome.The health care focus also shifts from maximizing height to including feminization, counseling the adolescent with Turner syndrome about the evolving impact of her condition into adulthood, and promoting the development of independent self-care behaviors.61,467

The Adult with Turner Syndrome The number of recent excellent papers and reports that now deal with adult women with Turner syndrome strengthens the fact that awareness is growing that this is a large segment of the adult population and that a comprehensive approach to care is needed.299,425,441,469,470 Most importantly, it needs to be stressed that cardiac care continues. It also must be stressed that women with Turner syndrome receive cylical estrogen and progestin and discontinuation should occur at the age of normal menopause and not before. Recent studies show that women with Turner syndrome become pregnant as easily as other women with other types of infertility and carry their pregnancies to term without an increased miscarriage rate.470,471 Because of their small size and and the narrow android pelvis, most women with Turner syndrome need to deliver by Cesarean section. Most critically, the risk for dilatation and dissection of the aorta appears to increase during pregnancy.471 New data have emerged showing that adolescents with only few signs of spontaneous puberty may still have ovaries with functioning follicles.472 The possibility of using cryopreserved ovarian tissue and immature oocytes, obtained before regression of follicles occurs, is currently under intense investigation and results seem promising.61 With unpredictable changes occurring in health care delivery, however, it is of concern that patients who require a long-term multidisciplinary approach to management may be lost in the medical system. We attempted to reach a consensus in the recently published Guidelines for the Care of Girls and Women with Turner Syndrome,61 which together with previous recommendations430 reflects our desire to make these guidelines for pediatricians and internists more broadly known. The consensus document suggests that puberty should be induced at a physiologically appropriate age to optimize self-esteem, social adjustment, and the potential for initiation of sexual relationships. With the quest for earlier diagnosis and hence earlier introduction of growthpromoting therapy, these recommendations (based on a French survey of 566 Turner women aged 18 to 31 years) should guide the clinician in future therapy.411 Because the parental origin of the missing X chromosome appears to have an impact on renal development and on ocular features, weight, and academic achievement, it may be appropriate to determine parental origin of the X chromosome using polymorphic microsatellite markers on the X and Y chromosomes.317,473 It is clear, however, that the pediatric health care provider will be the major resource for translating the ideas expressed

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into a future management plan for these patients. The strongest ally the practitioner has may be the patient herself. A well-informed and educated patient will become her own best advocate. Information and materials are available from the Turner’s Syndrome Society of the United States (www.turner-syndrome-us.org), regional affiliates, and international societies. With the intervention strategies described, girls and women with Turner syndrome now—more than ever—have the capability of achieving their full potential.

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462. Elkind-Hirsch KE, Sherman LD, Malinak R (1993). Hormone replacement therapy alters insulin sensitivity in young women with premature ovarian failure. J Clin Endocrinol Metab 76:472. 463. Godsland IF, Crook D, Simpson R, et al. (1990). The effects of different formulations of oral contraceptive agents on lipid and carbohydrate metabolism. N Engl J Med 323:1375. 464. Naeraa RW, Brixen K, Hansen RM, et al. (1991). Skeletal size and bone mineral content in Turner’s syndrome: Relation to karyotype, estrogen treatment, physical fitness, and bone turnover. Calcif Tissue Int 49:77. 465. Ettinger B, Genant HK, Steiger P, et al. (1992). Low-dosage micronized 17b-estradiol prevents bone loss in postmenopausal women. Am J Obstet Gynecol 166:479. 466. Porcu E, Fabbri R, Damiano G, et al. (2000). Clinical experience and applications of oocyte cryopreservation. Mol Cell Endocrinol 169:33. 467. Lufkin EG, Ory SJ (1994). Relative value of transdermal and oral estrogen therapy in various clinical situations. Mayo Clin Proc 69:131. 468. Elsheikh M, Conway GS, Wass JAH (1999). Medical problems in adult women with Turner syndrome: Trends in medical practice. Ann Med 31:99. 469. Gravholt CH, Bondy CA (2007). Wellness for girls and women with Turner syndrome. International Congress Series 1298, Amsterdam: Elsevier. 470. Foudila T, Soderstrom-Antitila V, Hovatta O (1999). Turner syndrome and pregnancies after oocyte donation. Hum Reprod 14:532–535. 471. Bodri D, Vernaeve V, Figueras F, et al. (2006). Oocyte donation in patients with Turner syndrome: A successful technique but with an accompanying risk of hypertensive disorders during pregnancy. Hum Reprod 21: 829–832. 472. Hreinsson JG, Otala M, Fridstrom M, et al. (2005). Follicles are found in the ovaries of adolescent girls with Turner syndrome. J Clin Endocrinol Metab 87:3618–3623. 473. Ferraz de Souza B, Lin L, Woodruff TK, Achermann JC (2007). Reproductive endocrinology. In Carel JC, Hochberg Z (eds.), Yearbook of peditaric endocrinology. Basel: Karger 71–86.

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C H A P T E R

16 The Testes: Disorders of Sexual Differentiation and Puberty in the Male IEUAN A. HUGHES, MD, FRCP

Introduction Fetal Male Development Embryology Genetic and Hormonal Control Disorders of Sex Development Defect in Testis Determination Sex Chromosome Anomalies Gonadal Dysgenesis Mixed Gonadal Dysgenesis Disorders of Androgen Synthesis General Leydig Cell Hypoplasia 17␤-Hydroxysteroid Dehydrogenase Deficiency 5␣-Reductase Deficiency Disorders of Androgen Action General Molecular Pathogenesis of Androgen Insensitivity Syndromes Disorder of AMH Persistent Mullerian Duct Syndrome

Introduction Development of the testes is essential for three key components of male reproductive function: sex determination and differentiation, stimulating the somatic components of male puberty, and development of spermatogenesis and acquisition of reproductive capacity. A trio of cells orchestrates these developments: Leydig cells, Sertoli cells, and germ

Other Testis-Related Disorders Anorchia and Cryptorchidism Hypospadias Testis Tumors General Germ Cell Tumors Non Germ Cell (Sex Cord Stromal) Tumors Puberty and the Testis General Disorders of Puberty Genetic Causes of Hypogonadotrophic Hypogonadism Investigating Testis Function General Ambiguous Genitalia Absent Testes Testis Tumors Pubertal Disorders

cells. This chapter focuses on the genetic and hormonal factors that enable a testis to develop, the production of testicular androgens and their mode of action, and how disturbances in these pathways result in disorders seen in pediatric endocrine practice. Endocrine disorders are confined to those resulting from an intrinsic abnormality in testicular function, including a section on testicular tumors. Disorders of male puberty are also covered in this chapter.

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Fetal Male Development EMBRYOLOGY Sex development comprises the dual components of sex determination (the process whereby the bipotential gonad develops as a testis or as an ovary) and sex differentiation (the phenotypic expression of the action of testicular hormones). The summation and amplification of these processes postnatally is manifest at puberty and followed by the acquisition of reproductive capacity. The entire process, extending from fetal life to adulthood, is a dynamic process dependent on the appropriate and timely interaction of a multitude of genes, proteins, signaling molecules, paracrine factors, and endocrine stimuli.1-4 The bipotential primitive gonad arises from a condensation of the mesoderm at the medioventral region of the urogenital ridge (Figure 16-1). This process begins at about 4 to 5 weeks of gestation in humans. The urogenital region is the site of development of the kidney, gonad, and adrenals. Consequently, when disrupted in mice genes (such as WT1 and SF1) that are key to urogenital development result in absent kidney/gonad and adrenal/gonad development (respectively). Inactivating mutations of these genes in humans lead to syndromes [such as Denys-Drash and Frasier (WT1)] and to XY sex reversal with adrenal failure (SF1).5,6 Once the urogenital ridge is formed, the mesonephros becomes essential to the development of the testis as a source of somatic cells—which migrate to encompass the primordial germ cells that have also migrated to this site from the yolk sac. The testis and ovary are morphologically indistinguishable until about 6 weeks of gestation. Then, the appearance of Sertoli cells and seminiferous cords developing adjacent to a prominent coelomic blood vessel are the hallmarks of the developing testis. No such morphologic differentiation occurs in the developing ovary until weeks later. Later, interstitial cells differentiate in the testis into Leydig cells and start producing testosterone for the next stage in development: sex differentiation.

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The adrenal primordium separates from the developing gonad at about 8 weeks of gestation. However, when the testis later migrates trans-abdominally to reach its scrotal position a nidus of adrenal cell rests may also be sited in this position. It is estimated that adrenal rests are found within, or adjacent to, the testis in up to 15% of neonates.7 These rests usually regress in later infancy, although they may persist and form testicular “tumors” in males with congenital adrenal hyperplasia.8 This tissue has characteristics of adrenocortical tissue, such as the production of adrenal-specific steroids and expression of adrenal-specific steroidogenic enzymes. The internal genitalia are also bipotential, with the anlage for development of the male and female internal genital ducts initially present in both sexes. In the male, regression of the Mullerian ducts (destined to form the uterus and fallopian tubes) and stabilization of the Wolffian ducts (destined to form the vas deferens, epididymis, and seminal vesicles) are prerequisites of normal development. This is mediated by the anti-Mullerian hormone (AMH) acting on its type II AMH receptor expressed in the Mullerian mesenchyme.9 Maximum sensitivity to AMH action occurs in a window of 9 to 12 weeks of gestation. Wolffian duct stabilization and differentiation is mediated by testosterone produced in large concentrations by the ipsilateral testis and acting predominantly in a paracrine manner.10 The external genitalia develop from a common anlage, with androgens playing a trophic role to enable the external genitalia to become sexually dimorphic. Thus, under the influence of androgens the genital tubercle differentiates and enlarges to become a penis, the urethral folds form the penile urethra, and labioscrotal swellings fuse to form the scrotum. The 5␣-reduced metabolite of testosterone, dihydrotestosterone (DHT), appears to be essential to this component of sex differentiation based on the anatomic consequences of the human syndrome of 5␣-reductase deficiency.11 The final step in fetal male development is descent of the testis in two stages: trans-abdominal and transinguinal. The

Figure 16-1 Schematic of the embryology of fetal sex development in the male. The continuous line denotes the rise in fetal serum testosterone during the period of sex differentiation.

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first stage, which begins at about 12 weeks of gestation and is completed by the middle of the second trimester, involves contraction and thickening of the gubernacular ligament.12 This phase of descent appears to be controlled by a Leydig cell product [insulin-like 3 (INSL3)] binding to its G-protein– coupled receptor [GREAT, also known as leucine-rich repeatcontaining G-protein–coupled receptor 8 (LGR-8) and as GPR106].13 The phase of transinguinal descent is predominantly androgen dependent.

GENETIC AND HORMONAL CONTROL A panoply of genes is involved in fetal sex determination and sex differentiation. Much of the knowledge gained, particularly in relation to the genetic control of urogenital development and testis determination, has arisen from mouse models of targeted disruption of candidate genes. However, several of these genes (such as Lim1, Emx2, Pdgfr␣, testatin, and SOX3) have not yet been shown to be key components in human sex development based on the effects of inactivating mutations. Figure 16-2 shows a simplified outline of those genes relevant to human fetal sex development. The SRY gene remains the key orchestrator of testis determination, with the most profound evidence illustrated by its expression in more than 90% of phenotypic human males with an XX karyotype14 and the induction of an XX male mouse by transgenesis.15 Inactivating mutations in SRY are found in only 10% to 15% of sex-reversed females with complete XY gonadal dysgenesis. Consequently, genes additional to SRY are required for testis determination. Although SRY is a known transcription factor, little is known of the nature of downstream genes controlled by SRY. It is linked to SOX9, another member of the high-mobility group (HMG) box family of proteins to which SRY belongs, and it is possible that SRY acts by disrupting the binding and function of a repressor—thus allowing activation of downstream genes (such as SOX9).16 The genes involved in mediating sex differentiation are well characterised and are predominantly those that encode for peptide hormones, such as AMH, and for the steroidogenic enzymes required for androgen biosynthesis. For androgen signaling, the ligand-activated nuclear androgen receptor (AR) is a crucial element in the pathway of male sex development.

The pathway of fetal testicular steroidogenesis is shown in Figure 16-3. The production of androgens by fetal Leydig cells occurs as early as 8 to 9 weeks of gestation and is initially autonomous, before becoming dependent on placental hCG secretion.17 Fetal serum testosterone concentrations increase between 10 and 16 weeks of gestation to levels approaching the adult male range. The pathways shown in Figure 16-3 highlight the importance of CYP17 and the POR gene as regulators of androgen biosynthesis. It is possible that the cytochrome P450 oxidoreductase (POR) enzyme is used preferentially to synthesise DHT by an alternative pathway specific to the fetal testis.18 All androgens bind intracellularly with high affinity to a single nuclear AR to mediate androgenic effects, including sex differentiation. The AR is a transcription factor encoded by a gene on chromosome Xq11-q12. The receptor, in common with other members of the large nuclear receptor family, comprises an N-terminal domain (involved in transcriptional activity), a central DNA-binding domain, and a C-terminal domain involved in hormone binding.19 Subdomains are involved in intramolecular interactions, as well as binding to a number of co-regulators that bridge the AR to the general transcriptional machinery.20 The AR is ubiquitously expressed, including the fetal reproductive tract.

Disorders of Sex Development Normal testis development and production of its hormones in an optimal concentration and time-dependent manner is crucial to enable male development to occur against a constitutive background of female fetal development. Consequently, an abnormality of testis function may manifest as a disorder of sex development (DSD). Indeed, it ranks second only to congenital adrenal hyperplasia as the most common cause of ambiguous genitalia of the newborn. This clinical disorder is discussed further in Chapters 4 and 12. The recent changes in terminology and classification of DSD21,22 (which provide a more rational approach to considering causation related to testicular disease) are outlined in Tables 16-1 and 16-2. From an endocrine perspective, the broad categories of causation are threefold: testicular dysgenesis (a defect in testis determination), a defect in testicular hormone production, and a defect in testicular hormone signaling or action.

Defect in Testis Determination SEX CHROMOSOME ANOMALIES

Figure 16-2 Genetic control of sex determination and differentiation in the male related to morphogenetic events.

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Klinefelter syndrome is the male exemplar of the gonadal manifestation of an abnormality in sex chromosomes. This is the most frequent form of sex chromosome aneuploidy. An incidence of about 1 in 600 live births is approximately quadruple the figure obtained in adulthood based on karyotype analysis.23 This mismatch, indicating a significant rate of underdiagnosis, is presumably the result of not recognizing the pathognomonic sign of small firm testes. Not surprisingly, less than 10% of cases are recognized before puberty. The classic 47,XXY karyotype

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Figure 16-3 Pathway of testicular steroidogenesis. Bold arrows indicate a predominant ⌬5 pathway of steroid production. The dotted lines denote a “backdoor” pathway to DHT synthesis postulated to be specific to the fetus. The steroidogenic enzymes and their cognate genes are shown.

is the result of meiotic non-disjunction during gametogenesis, of which 60% occur during oogenesis. About 10% of cases are mosaic (46,XY/47,XXY) and tend to have a milder phenotype. Typically, the external genitalia are normal at birth—although there may be anomalies such as hypospadias, micropenis, and undescended testes.24 The reduced testis size in Klinefelter syndrome is the result of degeneration of the seminiferous tubules. The process has its onset in the fetus, progresses through infancy, and accelerates at puberty.25 Although the number of germ cells is reduced, there is preservation of Leydig cell development—which is reflected in spontaneous onset of puberty in the majority of boys with Klinefelter syndrome.26 Seldom is it necessary to induce puberty with androgens, although supplementary testosterone may be required in adulthood. Fertility has occurred in association with the mosaic forms of Klinefelter syndrome, but with the use of testicular sperm extraction and intracytoplasmic

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sperm injection (ICSI) a significant number of pregnancies can now be attained in men with the 47,XXY karyotype.27 Other variants of Klinefelter syndrome can be associated with 46,XY/47,XXY mosaicism and karyotypes as diverse as 48,XXYY, 48,XXXY, 49,XXXYY, and 49,XXXXY. All have in common small testes, tall stature, some genital anomalies, and varying degrees of abnormal cognitive performance. The XX male syndrome is also similar in nature to Klinefelter syndrome, as characterized by small testes but with some features that differ. The testes are more likely to be undescended, and the incidence of genital anomalies is higher. Adult XX males are shorter, and gynecomastia is more common.28,29 Azoospermia is universal so that infertility is absolute. The incidence is about 1 in 20,000 phenotypic males. About 10% of XX males lack translocation of the Y-chromosomal SRY gene. They have a higher incidence of genital anomalies such as micropenis, hypospadias, and undescended testes.

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TA B L E 1 6 - 1

Nomenclature Relating to Disorders of Sex Development Previous

Proposed

Intersex

Disorders of sex development (DSD)

Male pseudohermaphrodite Undervirilization of an XY male Undermasculinization of an XY male Female pseudohermaphrodite Overvirilization of an XX female Masculinization of an XX female True hermaphrodite XX male or XX sex reversal XY sex reversal

46,XY DSD

It is also possible that XX ovotesticular DSD (true hermaphroditism) is part of this subgroup etiologically, subject to ovarian-tissue-containing follicles having been confirmed in an affected individual. There is yet no molecular explanation for male development in the absence of Ychromosomal material. Proposals include a loss-of-function mutation in a gene that normally inhibits testis formation in the XX female, a gain-of-function mutation in a gene downstream of the SRY transcription factor, and mosaicism for a Y-bearing cell line expressed in the gonads.30,31 These unidentified genes could be autosomal or X-linked.

46,XX DSD

GONADAL DYSGENESIS

Ovotesticular DSD 46,XX testicular DSD 46,XY complete gonadal dysgenesis

The term dysgenesis should only strictly be used where the definition is based on histology of the gonad. In relation to the testis and disorders of sex development, the appearances on histology accepted to define dysgenesis include carcinoma in situ, immature tubules with undifferentiated Sertoli cells, the presence of microliths (sometimes visible on testicular ultrasound), and a Sertoli cell-only pattern.32,33

TA B L E 1 6 - 2

A Proposed Classification of Causes of DSD Sex Chromosome DSD

A: 47,XXY (Klinefelter syndrome and variants)

B: 45,X (Turner syndrome and variants)

C: 45,X/46,XY (mixed gonadal dysgenesis)

D: 46,XX/46,XY (chimerism)

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46,XY DSD A: Disorders of gonadal (testicular) development 1. Complete or partial gonadal dysgenesis (e.g., SRY, SOX9, SF1, WT1, DHH, etc.) 2. Ovotesticular DSD 3. Testis regression B: Disorders in androgen synthesis or action 1. Disorders of androgen synthesis LH receptor mutations Smith-Lemli-Opitz syndrome Steroidogenic acute regulatory protein mutations Cholesterol side-chain cleavage (CYP11A1) 3␤-hydroxysteroid dehydrogenase (HSD3B2) 17␣ hydroxylase/17,20-lyase (CYP17) P450 oxidoreductase (POR) 17␤-hydroxysteroid dehydrogenase (HSD17B3) 5␣-reductase (SRD5A2) 2. Disorders of androgen action Androgen insensitivity syndrome Drugs and environmental modulators C: Other 1. Syndromic associations of male genital development (e.g., cloacal anomalies, Robinow, Aarskog, hand-foot-genital, popliteal pterygium) 2. Persistent Mullerian duct syndrome 3. Vanishing testis syndrome 4. Isolated hypospadias (CXorf6) 5. Congenital hypogonadotropic hypogonadism 6. Cryptorchidism (INSL3, GREAT) 7. Environmental influences

46,XX DSD A: Disorders of gonadal (ovarian) development 1. Gonadal dysgenesis 2. Ovotesticular DSD 3. Testicular DSD (e.g., SRY⫹, dup SOX9, RSP01) B: Androgen excess 1. Fetal 3␤-hydroxysteroid dehydrogenase 2HSD3B2 21-hydroxylase (CYP21A2) P450 oxidoreductase (POR) 11␤-hydroxylase (CYP11B1) Glucocorticoid receptor mutations 2. Fetoplacental Aromatase (CYP19) deficiency Oxidoreductase (POR) deficiency 3. Maternal Maternal virilizing tumors (e.g., luteomas) Androgenic drugs

C: Other 1. Syndromic associations (e.g., cloacal anomalies) 2. Mullerian agenesis/hypoplasia (e.g., MURCS) 3. Uterine abnormalities (e.g., MODY5) 4. Vaginal atresia (e.g., KcKusickKaufman) 5. Labial adhesions

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Gonadal dysgenesis as applied to the testis arises from a defect in sex determination during embryogenesis, which may be associated with a sex chromosome anomaly (as in Klinefelter syndrome) or due to inactivation of a testisdetermining gene such as SRY or SF1. The term gonadal dysgenesis is also used more loosely in association with indirect evidence of gonadal dysfunction such as elevated gonadotrophin and decreased testosterone levels in an XY subject with failure of Mullerian duct regression. A wide range of genital anomalies occurs with gonadal dysgenesis, and some classification systems are all embracing—including Turner syndrome. In the context of this chapter on the testis, Table 16-2 lists the disorders that need to be considered. Complete or pure XY gonadal dysgenesis is also known as Swyer syndrome, and is characterized by complete phenotypic sex reversal in an XY female. There are normal female genitalia at birth, but breast development is delayed at puberty. The uterus and fallopian tubes are present, but Wolffian duct remnants are not found. Gonadotrophin levels are characteristically markedly elevated by the time of puberty. Histology shows “streak” gonads indicating no morphological definition of testis development. There is a high risk of tumor development. The incomplete form is defined by any evidence of some masculinizing effects such as ambiguity of the genitalia at birth or virilization at puberty in the form of clitoromegaly, hirsutism, and deepening of the voice. Histology may reveal some evidence of testis development, but dysgenetic in nature. The molecular pathogenesis of XY gonadal dysgenesis should be explained by a mutation in one of a number of genes involved in testis determination, but only 10% to 15% of individuals with the complete form have a mutation of the SRY gene.34,35 SRY mutations are rarely found in partial XY gonadal dysgenesis. The first reports of SF1 mutations in humans described the expected phenotype of gonadal dysgenesis and primary adrenal failure.36 Now, there are examples identified in which adrenal failure does not appear to be a concomitant component of the syndrome of SF1 deficiency.37 This implies that the yield for identifying mutations in cases of gonadal dysgenesis may be higher if clinicians are more aware of expanded phenotypic spectra associated with testis-determining gene mutations.

MIXED GONADAL DYSGENESIS This form of gonadal dysgenesis is typically associated with a mosaic 45,X/46,XY karyotype, although additional karyotypes include 45,X/47,XXY and 45,X/46,XY/ 47,XYY. The phenotype is highly variable. There is a referral bias toward the spectrum of ambiguous genitalia manifest generally as micropenis, severe hypospadias, and bifid scrotum. Such a clinical presentation can be seen with so many causes of DSD. The clinical phenotype in mixed gonadal dysgenesis may range from almost normal female external genitalia with mild clitoromegaly, through ambiguous genitalia, to isolated hypospadias or normal male external genitalia. Indeed, when a 45,X/46,XY karyotype has been found seren-

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dipitously on prenatal testing most infants have normal male external genitalia at birth.38 There is no information on the longer-term followup of these infants with respect to growth, pubertal development, fertility, and risk of gonadal tumors. It is clear that there are normal men in whom this mosaic karyotype is present but unknown, as rarely this sex chromosome anomaly is identified in an infertility clinic setting. The internal genital ducts in mixed gonadal dysgenesis align in general with the nature of the ipsilateral gonad, with retention of a fallopian tube on the side adjacent to a severely dysgenetic streak gonad. The presence of a 45,X line may manifest with Turnerlike somatic features of nuchal folds and low-set hairline, associated cardiac and renal anomalies, and short stature. The gonads in this disorder are generally a combination of a well-formed testis on one side and a streak dysgenetic gonad on the contralateral side. Their positions are usually inguinal/scrotal and intra-abdominal, respectively. The assignment of gender in the biased referred population can be difficult and depends on several factors. Many are now assigned male and will require careful long-term monitoring at puberty and beyond with respect to malignancy risk. The streak gonad is usually removed during early childhood. Infants with female or only mild clitoromegaly are assigned female. The presence of Mullerian remnants in the form of a uterus or hemi-uterus provides an option later for pregnancy by ovum donation. It is essential to remove dysgenetic/streak gonadal material. Mutations in testisdetermining genes such as SRY are not generally found in this condition.39 Ovotesticular DSD (also previously termed true hermaphroditism) can only be defined on histologic criteria according to the presence of ovarian tissues (containing follicles) and testicular tissue present in the same gonad (ovotestis) or as the morphologic appearance of the contralateral gonad. Often, the internal gonads may be in the form of bilateral ovotestes. The most common karyotype is 46,XX, with about 10% of cases having a 46,XY karyotype. An ovotestis is the most frequent gonad, and about a third of 46,XX cases are SRY positive.40 Rarely, an SRY mutation may be identified in XY ovotesticular DSD.41 The phenotype in ovotesticular DSD is variable, although the predominant presentation is ambiguous genitalia or severe hypospadias. In 46,XX cases with an ovotestis and contralateral ovary, sex assignment is generally female with spontaneous onset of breast development and menses at puberty. All testicular tissue should be removed as completely as possible, and remnants monitored by serum AMH levels and the testosterone response to HCG stimulation. Pregnancies are reported in ovotesticular DSD.42,43 Those raised male will generally require hypospadias repair, orchidopexy, and removal of Mullerian remnants. Any ovarian tissue must be removed before puberty to avoid the risk of breast development. Scrotally sited testicular tissue can be monitored for tumor development, a rare occurrence in this type of DSD. Fertility is uncommon.44

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Disorders of Androgen Synthesis GENERAL The pathway of testicular steroidogenesis is outlined in Figure 16-3. A number of defects in the more proximal intratesticular pathway of steroidogenesis are also manifest as defects in adrenal steroidogenesis (see Chapter 12). These include StAR protein defect (lipoid congenital adrenal hyperplasia), P450 side-chain cleavage deficiency (CYP11A1), 3␤-hydroxysteroid dehydrogenase deficiency, 17␣-hydroxylase/17,20-lyase deficiency (CYP17), and P450 oxidoreductase (POR) deficiency. The predominant mode of presentation is generally adrenal failure with associated varying degrees of genital anomalies. In contrast, the classic presentation of combined CYP17 deficiency is absent puberty in a phenotypic XY female with low renin hypertension and hypokalemic alkalosis. Allied to this enzyme, central to androgen production is the co-enzyme P450 oxidoreductase—which is a key participant in the transfer of electrons to P450 enzymes, including the 17,20-lyase reaction of the CYP17 enzyme.45 Uniquely, deficiency of this enzyme can lead on the one hand to virilization of an affected female (XX, DSD) and on the other to undermasculinization of an affected male (XY,DSD). The underlying mechanism is explained by an apparent combined deficiency of two P450 enzymes (CYP17 and CYP21) and a hitherto unrecognized alternative pathway of androgen biosynthesis specific to the fetus.46 P450-oxidoreductase deficiency is also a feature of a subset of cases of Antley-Bixler syndrome.47 This is characterized by ambiguous genitalia and skeletal dysplasia, the latter comprising craniosynostosis, brachycephaly, midfacial hypoplasia, synostosis of the radioulnar or radiohumeral joints, bowing of the femora, and arachnodactyly. The syndrome also manifests in the absence of a defect in steroidogenesis. This is caused by mutations in the FGFR2 gene (fibroblast growth factor receptor) and is autosomal dominant. The defects in androgen synthesis confined solely to the production and testicular androgens include Leydig cell hypoplasia and deficiencies of the 17␤-hydroxysteroid dehydrogenase and 5␣-reductase enzymes.

LEYDIG CELL HYPOPLASIA Placental HCG during early gestation and pituitary LH thereafter in late fetal and postnatal development stimulate testicular androgen synthesis through binding to the LH receptor expressed in Leydig cells. Both ligands bind with similar affinity. The LH receptor is a glycoprotein hormone receptor and a member of the large group of G-protein–coupled receptors. These receptors are characterized by a large extracellular domain of about 400 amino acids and a transmembrane domain comprising a 7-transmembrane serpentine structure.48 If the receptor is inactivated, the result is a spectrum of undermasculinization in affected males ranging from a complete female phenotype to isolated micropenis. Inactivating mutations of the LH receptor are rare, and usually present with genital anomalies such as severe hypospadias and cryptorchidism.

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More than 30 loss-of-function homozygous or heterozygous mutations of the LH receptor gene (residing on chromosome 2p21) are now described.49,50 Complete resistance may present with primary amenorrhoea and lack of breast development in a phenotypic female who has XY chromosomes. The lack of breast development is a distinguishing clinical feature from the complete androgen insensitivity syndrome. The endocrine profile is as expected: elevated serum LH but normal FSH and low testosterone, which does not respond to HCG stimulation. Mullerian structures are absent, indicative of normal testicular AMH function. However, the epididymis and vas deferens may be present. It is not clear how Wolffian ducts are stabilized in the absence of fetal androgens. Inactivating LH receptor mutations manifest differently in affected females, who develop puberty normally but thereafter have ovarian dysfunction. There is a tendency for partial loss-of-function mutations that result in mild hypospadias or isolated micropenis to localize in the seventh transmembrane domain. Histology of the gonads shows decreased or absent Leydig cells after puberty. These appearances are less definitive in the prepubertal testis.

17␤-HYDROXYSTEROID DEHYDROGENASE DEFICIENCY The 17␤-hydroxysteroid dehydrogenase reaction is mediated by six isoenzymes in humans that convert androstenedione to testosterone, dehydroepiandrosterone to androstenediol, and estrone to estradiol (Figure 16-3). The type 3 enzyme encoded by HSD17B3 located on chromosome 9q22 is testis specific and is key to converting the weak androgen androstenedione to the potent major androgen testosterone. 17␤-HSD deficiency (also termed 17 ␤-hydroxysteroid oxidoreductase or 17-ketosteroid reductase) is now a well-characterized cause of 46,XY DSD.51-53 Most affected males have female external genitalia at birth but may present with inguinal swellings similar to infants with complete androgen insensitivity syndrome. Alternatively, the presentation is at puberty when an affected individual (whose sex is assigned female at birth) becomes profoundly virilized—with deepening of the voice, hirsutism, muscle development, and clitoromegaly. Why such virilization should occur at puberty and yet the fetus not masculinized in utero is not adequately explained. The gonadotrophin rise at puberty increases androstenedione substrate, which can be metabolized to testosterone by extraglandular conversion via alternative 17␤HSD isoenzymes (such as the aldoreductase type 5 17␤-HSD isoenzyme AKRIC3).54 The development of gynecomastia occurs by converson of androstenedione by aromatase enzyme in extraglandular tissues and the action of type 1 or type 2 17␤-HSD isoenzymes. The virilization that occurs at puberty is occasionally followed by gender role reassignment from female to male, similar to that observed in 5␣-reductase deficiency. If the gender remains female, urgent gonadectomy is required—with clitoroplasty and usually vaginoplasty. The deepening of the voice is seldom completely reversible.

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More than 20 mutations in the HSD17B3 gene have now been described.4 Most are missense mutations, with some patients displaying compound heterozygosity. Functional studies of mutant enzymes expressed in vitro generally show complete lack of ability to convert androstenedione to testosterone. Consequently, these studies bear little relationship to predicting the degree of virilization expected at puberty. An inbred population in Gaza has the more frequent mutation causing 17␤-HSD deficiency, resulting from conversion of arginine to glutamine (R80Q) at codon 80.55 This population also serves to illustrate that deficiency of this enzyme in females has no functional consequences because females homozygous for the mutation were normal.

5␣-REDUCTASE DEFICIENCY This cause of XY DSD is also characterized by profound virilization at puberty in an affected individual/raised female. The condition came to prominence through the reporting of a genetic isolate in the Dominican Republic, where gender role changes at puberty are not uncommon.56,57 However, unlike 17␤-hydroxysteroid dehydrogenase deficiency the external genitalia are more ambiguous at birth—with severe hypospadias, micropenis, bifid scrotum/labioscrotal folds, and a urogenital sinus. The testes transcend the abdomen to lie in the inguinal canals or within the bifid/labioscrotal folds. There are no Mullerian structures, and the Wolffian ducts are normally stabilized to form the epididymis, vas deferens, and seminal vesicle. The prostate gland remains hypoplastic at puberty, indicating the specific dependence of this structure on DHT. Furthermore, adult males do not develop acne or temporal hair recession. Fertility is reduced probably as a result of the mal-positioned testes. However, there are reports of fertility—spontaneous and by artificial reproductive techniques.58,59 The enzyme deficiency is caused by mutations in the SRD5A2 gene located on chromosome 2p23. More than 40 mutations have been identified, the majority being missense mutations.4 A genetic isolate affecting the New Guinea population is due to a complete gene deletion. Females homozygous for 5␣-reductase deficiency have normal fertility.60 The biochemical diagnosis centers on demonstrating an elevated ratio of testosterone to dihydrotestosterone (DHT) in serum (following HCG stimulation in prepubertal patients) and a diminished ratio of urinary 5␣ to 5␣-reduced C19 and C21 steroids. Even after gonadectomy, a biochemical diagnosis can still be ascertained because of the role of 5␣-reductase in metabolism of glucocorticoids.

669

levels. This is the paradigm of a hormone resistance syndrome. Formerly called the testicular feminization syndrome, the favored terminology is now androgen insensitivity syndrome (AIS)—subclassified into complete (CAIS) and partial (PAIS) forms.61,62 Male sex differentiation and the subsequent acquisition of secondary sex characteristics at puberty, and the onset of spermatogenesis, are all mediated by androgens binding to a single intracellular androgen receptor (AR) ubiquitously expressed in target tissue. The AR is one of a quartet of nuclear receptors (glucocorticoid, mineralocorticoid, progesterone, and androgen) closely related within a large superfamily. It can activate gene transcription via a common hormone response element. The single-copy gene encoding the AR is located on Xq11-q12 and is made up of 8 exons that encode a protein of 919 amino acid residues. The major functional domains comprise an N-terminal transactivation domain (NTD), a central highly conserved DNA-binding domain (DBD), and a hinge region that connects the DBD to the C-terminal ligand-binding domain (LBD) (Figure 16-4). The DBD contains cysteine residues that coordinate zinc atoms to form the zinc fingers characteristic of all nuclear receptors and many other transcription factors. The main domains comprise subsidiary functions that include dimerization, binding to co-regulator proteins, interaction with heat shock proteins, and transcriptional regulation.63 The two subdomains most involved in activation of transcription are the motif activation function-1 (AF1) in the NTD and the motif activation function-2 (AF2) in the LBD. AF2 also interacts with steroid receptor coactivators such as SRC1, SRC2/TIF2, and SRC3 via their LXXLL motifs—where L is a leucine and X is any amino acid.64 This interaction is weaker in the case of the androgen receptor AF2 subdomain, which uniquely interacts in an intramolecular manner with its cognate AF1 subdomain in the NTD.64 N- and C-terminal interaction stabilizes the AR and slows down the dissociation of the ligand from its receptor.

Disorders of Androgen Action GENERAL The key role of androgens in male sex differentiation is vividly illustrated by the consequence of a total lack of response to androgens in target tissues—a complete female phenotype in a 46,XY individual with normally formed testes producing age-appropriate testosterone

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Figure 16-4 A schematic of the functional domains of the androgen receptor. Numbers inside boxes refer to exons 1 through 8. AF1 is activation function 1. AF2 is activation function 2. (CAG)n encodes for a polyglutamine stretch. (GGT)n encodes for a polyglycine stretch. The three main domains are TAD (transactivation domain), DBD (DNA-binding domain), and LBD (ligand-binding domain).

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cognate receptor. Helix 12 is the outermost ␣-helix, which folds back on top of the ligand hydrophobic pocket like a lid closing on a box. This has been referred to as the “mousetrap effect” to capture the ligand and retain it by slowing the rate of ligand-receptor dissociation. This trapping effect by helix 12 also permits interaction between the LBD and AF2 subdomain and the LXXLL motif in associated co-regulator proteins. Much information about the structural and functional aspects of the AR has been obtained through studying the functional effects of AR mutations that lead to AIS. The phenotype of CAIS is that of a normal female, with a prevalence for this X-linked recessive disorder ranging from 1 in 20,400 genetic males to 1 in 99,000 genetic males.67,68 The typical presentation is primary amenorrhoea in an otherwise normally developed adolescent female. The uterus is absent as a result of normal AMH action. The Wolffian ducts are surprisingly stabilized in many patients.69 The main differential at this age is XY complete gonadal dysgenesis (Swyer syndrome), which is distinguished by poor breast development and a shorter stature. The other typical presentation in early life for CAIS is with bilateral inguinal or labial swellings. This can also occur in 17␤-HSD deficiency. Bilateral inguinal herniae are rare in girls, and it has been estimated that 1% to 2% of such cases have CAIS. Consequently, it is now generally recommended that a CAIS diagnosis be considered in all girls with this type of hernia and the presence of a Y chromosome be checked by FISH analysis and a full karyotype. If the content of the

A sequence of CAG repeats in exon 1 of the AR gene encodes for a homopolymeric stretch of glutamines, which ranges from 11 to 31 repeats in the general population. Another repeat of glycines ranges from 10 to 25. In vitro studies show that the length of the CAG repeat is inversely proportional to the activity of the AR as a transcription factor.65 Figure 16-5 shows a schematic of how androgens interact with the AR on entering target cells. The figure also shows subsequent activation of target genes. A single receptor binds testosterone and DHT, the latter being a biologically more potent androgen because of dissociating from the AR at a slower rate. The AR in the unliganded state is located in the cytoplasm complexed to heat shock proteins (HSPs) such as HSP70 and HSP90. These in turn are also complexed to co-chaperone proteins such as FKBP52.66 Ligand binding initiates dissociation to allow translocation of the AR to the nucleus, where it binds as a homodimer to DNA hormone response elements. The action of the AR is further modulated by interaction with co-regulatory proteins, which function either as coactivators or co-repressors. These proteins act as a physical bridge connecting the receptor to the basal transcription machinery. Coactivators ARA24, ARA55, and ARA70 are AR specific. The three-dimensional structure of a nuclear receptor LBD comprises 12␣ helixes associated with anti-parallel ␤ sheets arranged in the form of a tripartite sandwich. A hydrophobic pocket is formed by helixes, 4, 5, 7, 11, and 12—to which the ligand is bound on contact with its

SHBG

Testosterone

5α-reductase DHT Androgenresponsive cell

Dimerization and phosphorylation

AR

HSP AR

P

P AR

AR

P

P DNA binding

HSP

Ligand binding

AR

AR

Androgen-response element

Co-activator recruitment ARA70

GTA

Target gene activation Biological responses

Figure 16-5 A schematic of the mechanism of androgen activation of the androgen receptor and gene transcription. HSP, heat shock proteins; ARA70, an androgen receptor-specific coactivator; and GTA, general transcriptional apparatus. [From Feldman BJ, Feldman D (2001). The development of androgen-independent prostate cancer. Nature Rev Cancer 1:34–45. Copyright © of and adapted by permission from Macmillan Publishers Ltd.]

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hernial sac contain gonads, a biopsy should be taken in concert with the cytogenetic studies.70 A family history of an older female sibling having had an inguinal hernia repair in infancy but with the diagnosis of CAIS missed is not unusual. Another mode of presentation is mismatch between a prenatal XY karyotype and a female phenotype at birth. The syndrome of PAIS occurs when there is some biologic response to androgens. The external genitalia may be ambiguous at birth, but the prototypic phenotype for PAIS is perineoscrotal hypospadias, micropenis, bifid scrotum, and undescended testes. The more severe form of PAIS presenting as isolated clitoromegaly is only marginally different from CAIS. At the other end of the phenotypic spectrum there may be just isolated hypospadias or even normal male development at birth but gynaecomastia and infertility presenting in adulthood. Surveys of male factor infertility characterized by elevated LH and testosterone levels have revealed a minority to have a mutation in the AR gene.71 High doses of androgens may lead to fertility.72 The list of disorders to consider within the 46,XY DSD category is much larger with the PAIS phenotype. They can be broadly classified as partial gonadal dysgenesis, a defect in androgen biosynthesis (LH receptor, SF1, 17␤-HSD and 5␣-RD deficiencies), and mixed gonadal dysgenesis in association with 45,X/46,XY mosaicism. The hormone profile in AIS shows an increased agerelated testosterone level, increased LH, and only a slightly elevated FSH level. Serum estradiol is increased at puberty from aromatization of testosterone. Concentrations of sex hormone binding globulin (SHBG), a protein produced by the liver, are sexually dimorphic—with levels in CAIS similar to those found in normal females. This hepatic resistance to the action of androgens has been proposed as a biologic marker of androgen responsiveness in AIS.73 Stanazolol, a synthetic nonaromatizable androgen, is administered orally—and the decrement in serum SHBG levels measured. There is normally a 50% reduction, but the change is insignificant in CAIS. There is a moderate response in PAIS, but overlapping with normals. A further biochemical test, available only in research laboratories, used in the investigation of androgen resistance is measurement of androgen binding in genital skin fibroblasts. The AR is ubiquitously expressed, and in greater quantities in genital versus nongenital skin. A small genital skin biopsy collected at genitoplasty or during an examination under anesthetic can be used to generate a cell line to perform a binding assay using radiolabeled androgens. A quantitative and qualitative measure of binding can be determined from a Scatchard plot analysis. Typically, there is absent binding when a nonsense mutation results in a truncated AR. Such a mutation is sufficiently pathogenic to lead to a CAIS phenotype. Alternatively, there may be binding of androgens to the AR—but with lower affinity. A missense mutation is generally associated with such findings, resulting usually in a PAIS phenotype. The cell line is also a source of RNA, which can be analyzed for length if a mutation is suspected to be residing in the noncoding region of the AR gene.

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MOLECULAR PATHOGENESIS OF ANDROGEN INSENSITIVITY SYNDROMES Information about the various mutations that affect the AR and give rise to clinical disease is recorded on an International Mutation Database at McGill University (http://www.androgendb.mcgill.ca). The majority of mutations relate to syndromes of androgen insensitivity, but in addition somatic mutations identified in prostate carcinoma are listed. The database records more than 300 different mutations that can cause AIS. These are distributed throughout the gene, with no specific “hot spot” of mutations but about two-thirds are located in the LBD. The range of mutations identified through the Cambridge DSD database is shown in Figure 16-6. Identifying a mutation, particularly if missense, does not necessarily imply pathogenicity. If the mutation is novel, it is advisable to recreate the mutant AR for functional studies using a reporter gene assay. It is also possible to undertake structureguided modeling of the mutant protein to provide insight into AR dysfunction.74 There is considerable heterogeneity in the phenotypic expression of a particular mutation, sometimes even within families. For example, a missense mutation at codon 703 in exon 4 of the LBD (which changes a serine to a glycine) is reported in four separate individuals on the McGill database. One patient had a normal female phenotype, whereas the other three cases all had ambiguous genitalia consistent with PAIS. However, the degree of androgenization of the external genitalia was sufficiently variable that two were raised male and the other female.75,76 Rarely, two affected members in the same family can be respectively phenotypically CAIS and PAIS. X-linked disorders are associated with a high rate of mutations that are de novo. The rate in AIS is about 30%. Such mutations arise as a single mutational event in a parental germ cell (maternal in the case of AIS) or as a germ cell mosaicism in the maternal gonad. Somatic mosaicism occurs when the mutation arises at the postzygotic stage. Consequently, there is expression of both mutant and wild-type AR in different target tissues, including the external genitalia. About a third of de novo mutations in AIS arise at the post-zygotic stage, thereby explaining some of the variable phenotype in PAIS.77 Other modulatory factors may include differences in 5␣-RD 2 expression and reduced AR transcription and translation.78,79 No AR gene mutation is found in a minority of cases of CAIS or in a significant number of cases of PAIS. Nevertheless, it appears that there is clinical and biochemical evidence of androgen resistance. It is possible that patients with CAIS or PAIS in whom no mutation has been found in the AR gene may have a mutant coactivator protein to explain the androgen resistance. There is increasing evidence that nuclear receptor co-regulators play a role in the formation of mammalian phenotypes and perhaps in human disease.20,81 In a study of the two AR-related coactivators, ARA24 and ARA 70, no substantive variations were found in amino acid residues in a series of patients with PAIS and a normal AR.81,82 Disruption of the steroid receptor coactivator-3 (SRC) in mice results in a phenotype of general hormone resistance, including features consistent with PAIS.83 The SRC3

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A

B Figure 16-6 Androgen receptor mutations recorded on the Cambridge DSD database. (A) Mutations associated with CAIS phenotype. (B) Mutations associated with PAIS phenotype.

gene contains a variable track of CAG/CAA triplets that encode a polyglutamine repeat. The lengths of glutamine repeats were found to be shorter in PAIS subjects compared with controls.84 The large number of co-regulator proteins (more than 300), their array of mechanisms to modulate transcription, and promiscuous binding to nuclear receptors in general makes it unlikely that a single mutant protein in this family would explain the mechanism of androgen resistance in CAIS or PAIS patients who have a normal AR. One patient with CAIS with a normal AR, in whom a subdomain of the AR failed to stimulate transcription of AR target genes, was suggested as evidence for a defect in an AR-specific coactivator.85,86 The polymorphic region in the N-terminal domain of the AR of glutamine repeats has biologic relevance to human disease. A toxic gain-of-function from hyperexpansion (⬎40 repeats) is found in Kennedy’s disease and in spinal and bulbar muscular atrophy (SBMA).87 Affected males have testicular atrophy, decreased spermatogenesis, and gynaecomastia despite elevated androgen levels (in keeping with a degree of androgen resistance). Variations in the length of the polyglutamine tract within the normal range are reported in association with disorders

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such as hypospadias,88 reduced spermatogenesis,89 and the phenotype of Klinefelter syndrome.90 Shorter glutamine repeats and relative hyperandrogenic states are reported in prostate cancer91 androgenetic alopecia and acne,92 and ovarian hyperandrogenism.93 The polymorphic stretch of glycines is also significantly longer in patients with cryptorchidism and in a separate group with hypospadias compared with controls.94 When CAG (glutamine) and GGN (glycine) lengths are analyzed together in the context of a missense mutation in the AR gene causing AIS, the combined effects appear to modulate the phenotypic expression of a given AR mutant in different affected individuals.95

Disorder of AMH PERSISTENT MULLERIAN DUCT SYNDROME Anti-Mullerian hormone (AMH) is a Sertoli-cell-produced glycoprotein homodimer encoded by a 2.75-kb gene on chromosome 19p13.3. The AMH type II receptor is a serine/threonine kinase with a single transmembrane

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THE TESTES: DISORDERS OF SEXUAL DIFFERENTIATION AND PUBERTY IN THE MALE

domain encoded by a gene located on 12q13.96 Persistent Mullerian duct syndrome (PMDS, herniae uteri inguinale) is a condition affecting males who have normally developed testes, internal male ducts, and external genitalia but persistent Mullerian duct derivatives. The diagnosis is often made when a fallopian tube and uterus are found during an inguinal hernia repair or an orchidopexy procedure. The hernia contains a partially descended or scrotal testis, as well as an ipsilateral tube and uterus. Sometimes the contralateral testis is also present in the hernial sac. Such transverse testicular ectopia is virtually pathognomonic of PMDS. PMDS in normally differentiated males may result from failure of the Sertoli cells to synthesize AMH or from resistance to the action of AMH because of an AMH type II receptor defect. There is an even distribution of genetically proven cases of PMDS being the result of AMH or AMH type II receptor gene mutations.97 AMH gene mutations are most common in Mediterranean, Northern African, and Middle Eastern countries and are usually familial homozygous mutations.98 In contrast, mutations in the AMH type II receptor gene are more common in France and Northern Europe and are often compound heterozygous in nature.99 Serum AMH measurement is a useful marker of the likely gene affected. Thus, low or undetectable levels are indicative of a mutation in the AMH gene—whereas a normal or increased serum concentration of AMH points to a defect in the AMH type II receptor gene.

Other Testis-Related Disorders ANORCHIA AND CRYPTORCHIDISM The term vanishing testis syndrome was coined for the phenotype of bilateral anorchia in an otherwise normally developed male infant. It recognizes the presence of normal testes in early gestation functioning to induce Mullerian duct regression, stabilize Wolffian duct development, and differentiate male external genitalia. Any ambiguity of the external genitalia suggests a variant of the syndrome related to some form of XY gonadal dysgenesis. Bilateral anorchia with normal differentiated but small phallus (micropenis) is also a recognized variant of the syndrome.100 A rare finding in such cases is a heterozygous partial loss-of-function mutation in SF1.101 Otherwise, the cause is unknown other than invoking an interruption to the testis blood supply from a torsion or vascular occlusion event in utero. Surgical exploration and histologic findings typically show nubbins of fibrous tissue as remnants of gonads, associated with a vas deferens in the majority and some epididymal tissue.102,103 The presence of hemosiderin-laden macrophages and dystrophic calcification is in keeping with the vascular accident hypothesis. Establishing complete anorchia is based on a combination of biochemical tests, imaging studies, and surgical exploration.104 An undetectable serum AMH level is a reliable marker when evaluating infants with nonpalpable gonads.105 This, coupled with elevated serum gonadotrophins and an absent testosterone response to hCG stimulation, is predictive of the absence of testes. A low inhibin

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673

B level is also confirmatory. Imaging with CT or MRI may be useful prior to laparoscopy. Most centers would currently still undertake surgical exploration to remove testicular remnants, even though there is usually no evidence of malignancy found. Such a procedure could be deferred until the time of insertion of testicular prostheses. Testes that have not descended at birth (cryptorchidism) is the most common congenital abnormality in boys, affecting 2% to 9% male live births.106 The strong association with low birth weight is well recognized, as are disorders of the pituitary-gonadal axis [such as hypogonadotrophic hypogonadism (HH) and the androgen insensitivity syndrome]. The association with disorders of androgen production or action is in keeping with the role of androgens in the inguinoscrotal phase of testis descent. Mutations in INSL3 and GREAT (also known as LGR8) are reported in a minority of boys with cryptorchidism.107-109 Greater exposure to pesticides has been reported in cryptorchid boys based on analyses of breast milk samples as a proxy for fetal exposure.110 Epidemiologic evidence for secular trends in the prevalence of cryptorchidism, and geographic variations in birth prevalence, point to environmental chemicals having an adverse effect on the genetic and hormonal control of testis descent.111-115 Cryptorchidism may be associated with hypospadias. In turn, both are coupled to an increased risk for abnormal spermatogenesis and testicular cancer— two male reproductive tract disorders also increasing in prevalence.116,117 This quartet of disorders constitutes a testicular dysgenesis syndrome (TDS) postulated to be of fetal origin and triggered by environmental chemicals disrupting an androgen/estrogen balance critical to normal sex development.118 The clearest evidence that certain chemicals (such as herbicides, fungicides, bisphenol A, and phthalates) can act as endocrine disruptors is derived from observations of wildlife and laboratory animal experiments. It remains uncertain whether there is also a new and emerging public health problem.119 Direct evidence of toxic effects in humans has yet to be established. The previous practice of using diethyl stilbestrol (DES) to prevent recurrent miscarriages has resulted in transgenerational effects in the form of urogenital anomalies, including cryptorchidism in male offspring.120 Sons of agricultural workers have an increased risk of cryptorchidism, which is related to levels of pesticide exposure.121 The anogenital distance is a measurement used by reproductive toxicologists as a sensitive marker of prenatal exposure to androgen action in rodent studies.122 In a human study, there was an inverse relationship between the anogenital distance in male infants and the levels of several phthalates measured in maternal urine.123 Furthermore, the anogenital distance was shorter in boys with cryptorchidism.

HYPOSPADIAS Hypospadias is incomplete fusion of the penile urethra defined by an arrest in development of the urethral spongiosum and ventral prepuce.124 The normal embryologic correction of penile curvature is also interrupted. It is a common congenital anomaly, with birth prevalence estimated at about 3 to 4 per 1,000 live

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THE TESTES: DISORDERS OF SEXUAL DIFFERENTIATION AND PUBERTY IN THE MALE

births. Despite extensive effort at investigation, the cause is unknown in the majority of cases.125,126 An occasional case of isolated hypospadias has been identified with a mutation in WT1, SF1, LHR, CYP17, or the AR gene. The CXorf6 gene on chromosome Xq28 has been found to be mutated in some cases of hypospadias.127 The mouse homologue is expressed in Leydig and Sertoli cells coincident with the period of sex differentiation (E12.5-E14.5). Because expression of this gene is insignificant in the external genital anlagen, this suggests a transient Leydig cell dysfunction as an explanation for hypospadias in these cases. A mutation screen of BMP4, BMP7, HOXA4, and HOXB6 identified mutations in 14 of 90 cases of hypospadias in a Chinese population (all in the heterozygous state).128 Mutations in HOXA13 are found in the hand-foot-genital syndrome, which includes hypospadias.129 There is generally a familial clustering of cases in hypospadias, with a 7% incidence of one or more additional affected family members.130 Furthermore, the twinning rate is higher than in the general population—of which two-thirds are monozygotic. Associations observed in hypospadias include increased maternal age, mother exposed to DES in utero, paternal subfertility, maternal vegetarian diet, maternal smoking, assisted reproductive techniques, paternal exposure to pesticides, and fetal growth restriction.131 The association with low birth weight suggests a link between factors operating to cause fetal growth restriction and the process of completing fusion of the urethral folds by the early second trimester. Gene expression profiles during urethral development in the mouse indicate that a number of signaling pathways are involved.132

Testis Tumors

Table 16-3. The teratoma/yolk sac tumor presents in infancy and childhood and is not confined to the testis. Other sites include the mediastinum, retroperitoneum, sacral region, and midline brain. Yolk sac tumor is the most common testis tumor occurring before puberty, followed by embryonal carcinoma.138 Type II tumors (comprising seminoma, nonseminoma, and dysgerminoma) originate from primordial germ cells or gonocytes. A premalignant lesion develops initially, termed intratubular germ cell neoplasia unclassified (IGCNU) or carcinoma in situ (CIS). When the gonad is dysgenetic in nature, the premalignant condition is in the form of a gonadoblastoma. They progress to an invasive tumor manifest as a seminoma or nonseminoma and as a dysgerminoma in a dysgenetic gonad or an ovary.139 The existence of a locus on the Y chromosome predisposes a gonadoblastoma to form in dysgenetic gonads. The TSPY (testis-specific protein Y-encoded) gene located on the short arm of the Y chromosome is one such oncogene that is abundantly expressed in the germ cells of XY individuals with DSD. Gonocytes and CIS cells share similarities in morphology and in the expression of certain fetal proteins.140 It is hypothesized that CIS represents delayed maturation of germ cells, which gives rise to an increased risk of germ cell tumors. This is found not only in XY DSDs but in trisomy 21, where there is a fiftyfold higher incidence of seminoma.141 CIS is generally associated with germ cells developing in a depleted androgen environment, whether it be the result of insufficient androgen production or resistance to the action of androgen (as in syndromes of androgen insensitivity). The propensity for gonadoblastomas to develop in gonadal dysgenesis (92%, as opposed to only 8% incidence of CIS) may be explained by the undifferentiated state of dysgenetic gonadal tissue. Invasive germ cell tumors are

GENERAL Testis cancer is rare and has the highest prevalence in young men in the third and fourth decades of life.133 The majority are germ cell in origin, manifesting as seminoma or nonseminoma tumors. The World Health Organization (WHO) classification system is the basis on which histologic typing of testis tumors is practiced.134-136 Tumors arise from the three principal cell types that comprise the formation of the testis: germ cells derived from primitive gonocytes, the supporting cells that differentiate as Sertoli cells, and the stromal interstitial cells that differentiate as Leydig cells. Rarely, tumors can arise from nonspecific testis tissue such as muscle (rhabdomyosarcoma), blood vessels (hemangioma), fibrous tissue (fibroma), and as a result of infiltration (leukemias, lymphoma). Germ cell tumors account for the vast majority, some of which manifest in early life. Leydig cell tumors also occur in childhood.

GERM CELL TUMORS There are five categories of germ cell tumors described, of which types I through III involve the testis.137 The WHO histologic classification of germ cell tumors is shown in

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TA B L E 1 6 - 3

Classification of Germ Cell Tumors Type I

II

Site

Phenotype

Testis/ovary/ retroperitoneum Mediastnum/ midline brain Testis

Teratoma/ yolk sac tumor

Neonates Children

Seminoma/ non-seminoma Dysgerminoma

Young adult

Ovary

III

Dysgenetic gonad Testis

IV

Ovary

V

Placenta/uterus

Dysgerminoma/ non-seminoma Spermatocytic seminoma Dermoid cysts Hydatidiform mole

Age

Childhood → young adult congenital ⬎50 yr Children/young adults Fetal life

Adapted from Cools M, Drop SLS, Wolffenbuttel KP, Oosterhuis JW, Looijenga LHJ (2006). Germ cell tumors in the intersex gonad: Old paths, new directions, moving frontiers. Endocr Rev 27:468-484.

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THE TESTES: DISORDERS OF SEXUAL DIFFERENTIATION AND PUBERTY IN THE MALE

rare before puberty in CAIS. Thereafter, with the rise of estrogens at puberty there may be estrogen-stimulated induction of the c-kit ligand—which can stimulate primordial germ cell growth.142 In contrast, germ cell tumors occur at an earlier age in gonadal dysgenesis as a result of progressive genetic instability.137,140,143 A number of tumor markers can be used for immunohistochemical analysis to identify a premalignant state. These include placental-like alkaline phosphatase (PLAP), KIT (a membrane tyrosine kinase receptor for stem cell factor), AP2 gamma (activator protein-2 gamma), and OCT ¾.139 These protein markers are normally expressed by fetal germ cells (and occasionally in postnatal life) until about 12 months of age. Thereafter, any expression in germ cells is abnormal and indicative of CIS or IGCNU. These tumor markers are not detected in serum, but have been identified in semen samples.144 The risk of gonadal tumor in various causes of DSD can now be quantified reasonably well, particularly for gonadal dysgenesis and AIS (Table 16-4).

NON GERM CELL (SEX CORD STROMAL) TUMORS Other tumors of the testis of relevance to infancy and childhood include Sertoli cell, Leydig cell, and adrenal rest tumors—which are stromal in origin and generally benign. Sertoli cell tumors can be bilateral, large and calcified, and associated with syndrome complexes such as the Peutz-Jeghers syndrome145 and the Carney complex.146 A feature of such tumors is associated breast development from increased estrogen production. Inhibin B, a product of the Sertoli cell, may be elevated in serum as a marker of Sertoli cell tumors.147 Leydig cell tumors are rare and comprise only 1 to 3% of testis tumors.148 They are most common in the prepubertal age group, where they are always benign. In adults, 10% of cases are malignant. Leydig cell tumors are characterized clinically by the manifestations of their increased steroid production, which is generally

675

androgenic. Consequently, precocious puberty is the hallmark in childhood. Estrogens can also be produced, either directly by the tumor or via peripheral aromatization. A testicular mass may be palpable, but the tumor can be occult and only visible on ultrasound examination. Characteristic histologic features include abundant cytoplasmic lipofuscin pigment and Reinke crystals.149 Immunohistochemical markers specific for Leydig cell tumors include ␣-inhibin (particularly useful to distinguish from germ cell tumors), calretinin, and Melan-A. Aberrant adrenal “tumors” may develop from the rest cells found within the testis in up to 15% of neonates.7 The aberrant cells remain ACTH responsive and express adrenal-specific steroidogenic enzymes.150 Consequently, the cells can become hyperplastic to form testicular adrenal rest tumors (TARTs) under states of ACTH hypersecretion in congenital adrenal hyperplasia (CAH), Addison’s disease, and Nelson’s syndrome.151-153 There is almost uniform prevalence of TART in adult male patients with CAH when screened routinely by testicular ultrasound examination.151 Even in male children with CAH, the prevalence is 24%.154 The tumors are usually bilateral and benign. Histologically, they are sometimes difficult to separate from Leydig cell tumors—although the absence of Reinke crystals is a distinguishing feature.155 The tumors are sited adjacent to the mediastinum testis, which can cause obstruction of the seminiferous tubules. This is invoked as one possible reason for gonadal dysfunction and infertility associated with TART.156 An endocrine explanation of infertility centers on the inhibitory role elevated endogenous levels of adrenal steroids may have on gonadotrophin secretion in males with inadequately treated CAH.157,158 Fertility may be restored with improved medical control.159 Testis-sparing surgery by tumor enucleation does not appear to improve pituitary-gonadal function.160 Due recognition must be given to a testicular mass in males with CAH as most likely being a TART. Orchidectomy is not the preferred treatment. There are other causes of testis enlargement that are not tumor related. Macro-orchia is a feature of the fragile

TA B L E 1 6 - 4

Estimated Risks of Gonadal Tumors in Various Forms of DSD

Risk Group

Disorder

Malignancy Risk (%)

Recommended Action

Numbers: Studies (n)

Patients (n)

High

GD (⫹Y) intra-abd. PAIS non-scrotal Frasier Denys-Drash (⫹Y)

15-35 50 60 40

gonadectomy gonadectomy gonadectomy gonadectomy

12 2 1 1

⬎350 24 15 5

Intermediate

Turner (⫹Y) 17␤-HSD GD (⫹Y) scrotal PAIS scrotal gonad CAIS ovotest. DSD Turner (⫺Y) 5␣-reductase Leydig cell hypoplasia

12 28 unknown unknown 2 3 1 0 0

gonadectomy monitor biopsy and irrad. biopsy and irrad. biopsy and testis tissue removal None unresolved unresolved

11 2 0 0 2 3 11 1 2

43 7 0 0 55 426 557 3

Low

No

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X syndrome, which is associated with mental retardation.161 The cytogenetic fragile site on Xq27.3 harbors the FMRI gene, which has a tandem repeat of CGG trinucleotides in its 5' untranslated region. Expansion of this repeat is responsible for the syndrome, with its onset occurring before germ line segregation based on analysis of allele expansions in early fetal ovaries and testes.162 The FMRP protein is widely expressed and is predominant in the brain and testes (mainly Sertoli cells), consistent with the key clinical features of the syndrome. Macro-orchidism is most obvious after puberty, with testicular volume greater than 30 mL characteristically present. However, anthropometric measurements of males with fragile X syndrome from birth to adulthood shows an increase in testicular volume already in childhood—with the 50th percentile equivalent to the normal 95th percentile by 6 years of age.163 Indeed, macro-orchidism has been documented as early as 5 months of age postnatally164— and in the fetus by 24 weeks of gestation.165 Information on testicular histology is scanty. The mass of Sertoli cells is the primary determinant of testis volume. Biopsy studies of macro-orchid adult testes showed interstitial edema, increased lysosomal inclusions in Sertoli cells, and abnormal spermatogenesis.166 In the male Fmr1 mouse knockout model, the rate of Sertoli cell proliferation is increased and is independent of any change in FSH signaling.167 The dominance of Sertoli cells in constituting testis volume is further illustrated by the observation of testis enlargement in males with the McCune-Albright syndrome.168,169 In contrast to affected females, however, precocious signs of hyperandrogenism are often absent. The Gs-alpha-activating mutation, the cause of this syndrome, is manifest in somatic cells and occurs early in development.170 The prevalence of this apparent sex dimorphism in sexual precocity is explained by the observation that in one affected patient of the typical Gs-alpha mutation (Arg201His) the GNAS1 allele was present only in Sertoli cells—explaining the lack of Leydig cell hyperfunction.171 Macro-orchidism may occur without any known cause, including the exclusion of activating mutations in the FSH receptor gene.172 Severe hypothyroidism of long standing can paradoxically result in precocious puberty, particularly in girls (VanWyk-Grumbach syndrome). The manifestation when it occurs in boys is with macro-orchidism.173,174 It has been postulated that the markedly elevated TSH levels have additional gonadotrophin-stimulating effects by binding promiscuously to the FSH receptor.175 Restoring a euthyroid state with thyroxine replacement leads to resolution of the early signs of puberty. There is no evidence to indicate that early puberty in primary hypothyroidism is due to activating mutations in the FSH receptor gene.176

Puberty and the Testis GENERAL The testis is clearly an essential conduit in producing the sex steroid endpoint for translating activation of the GnRH pulse generator into the somatic features characteristic of male puberty. Although most pubertal disorders

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are not the result of a primary testis dysfunction, an overview of puberty and its disorders in the male is briefly provided in this chapter. The control of the onset of puberty remains an enigma because the primary mechanism that awakens the dormant neuroendocrine machinery remains unclear.177 The physiologic paradigm of puberty centers on a period of “restraint” on gonadotrophin secretion during childhood that when released via genetic and extrinsic mechanisms allows puberty to be reactivated. This is based on evidence of prior increased gonadotrophin activity during fetal life and early infancy. Placental gonadotrophin (HCG) and fetal pituitary gonadotrophins (FSH/LH) secretion are essential to sufficient production of testosterone for male genital differentiation and descent of the testes. Placental HCG secretion rises abruptly after 6 weeks, reaching a peak at about 12 weeks of fetal life and then declining to low levels by 18 to 20 weeks of gestation. The rise in fetal HCG is mirrored by a sharp rise in fetal circulating testosterone, essential to the normal development of the internal and external male genitalia. The second half of gestation is characterized by increasing FSH and LH secretion, which stimulate testicular size, descent, and penile growth. At birth, after separation of the umbilical cord there is a rapid rise in testosterone—reaching levels as high as 400 ng/dL in the first day of life. Testosterone levels then decline rapidly during the first week to levels of 20 to 50 ng/dL but then increase again to levels between 60 and 400 ng/dL between 20 and 60 days of life. Thereafter, levels decline to the prepubertal range of less than 10 ng/dL by the end of the first year of life. These surges in testosterone are induced by gonadotrophins. A GnRH stimulation test evokes a greater rise in FSH than LH at these times. What purpose is served by the gonadotrophin-induced surge in testosterone in the first hours and days of neonatal life is less clear. Penile length increases during infancy, particularly during the first 3 months, and is positively correlated with serum concentrations of testosterone.178 Inhibin B, a marker of Sertoli cell mass, is also elevated at 3 months of age and remains so for about another year.179 Sertoli cell numbers proliferate during fetal and early postnatal life, the total number ultimately being a determinant of sperm quantity in adulthood.180 That these changes are FSH dependent is suggested by the observation of larger testis volume at 3 months of age in normal Finnish versus Danish male infants, and related to higher FSH and inhibin levels.181 Furthermore, boys with cryptorchidism have higher FSH levels compared with controls.182 Collectively, these observations indicate that the testis is responsive to a surge in gonadotrophin secretion during infancy. However, FSH and LH secretion abate (and serum levels are quite low) in the prepubertal years. Puberty is heralded by the appearance of nocturnal-sleep-related FSH and LH pulses, a reflection of the reawakening of the GnRH pulse generator (which has been “restrained” by an as yet unidentified mechanism). If puberty is indeed the result of removing a restraint mechanism, it is not initiated by the gonads because a rise in gonadotrophins occurs at the expected time of puberty in girls with Turner syndrome, boys with Klinefelter syndrome, and boys with anorchia.183 Studies in agonadal

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nonhuman primates show similar findings.184 In addition, severe head injuries and irradiation of the skull may be associated with precocious puberty (Table 16-5)—implying damage to the region that regulates the restraint of puberty. Thus, the restraint of puberty is orchestrated in the brain (not the gonad).185-186 The first sign of puberty in boys is an increase in testis volume from a prepubertal value of 3 to 4 mL. This occurs on average between 11 and 11.5 years of age. The timing and tempo of puberty are related to a host of interacting factors comprising GnRH, neuropeptide-Y,

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gamma aminobutyric acid (GABA), leptin, and transforming growth factor-alpha (TGF␣). A recent addition to this list is a G-protein–coupled receptor (GPR54) and its ligand (kisspeptin 54), which act upstream of the GnRH pulse generator (Figure 16-7). The kisspeptins appear to be a gatekeeper to puberty and to play a key role in the negative and positive feedback control of gonadotrophin secretion by sex steroids.187,188 Mutations in the GPR54 gene in humans results in delayed puberty due to HH.189,190 Treatment with exogenous kisspeptin induces a rapid increase in LH and FSH levels in humans and can induce puberty in primates.184,191,192

TA B L E 1 6 - 5

General Classification of Causes of Precocious and Delayed Puberty in the Male Precocious Puberty Gonadotrophin-Dependent • Idiopathic central precocious puberty • Organic central precocious puberty • Hypothalamic hamartoma • Post head trauma • Post cranial radiotherapy • Neurofibromatosis • HCG-secreting germ cell tumor Gonadotrophin-Independent • Activating LH receptor mutation (testotoxicosis) • McCune-Albright syndrome • Leydig cell tumor • Extratesticular hyperandrogenism • Congenital adrenal hyperplasia • Adrenal tumor Delayed Puberty Hypogonadotrophic Hypogonadism • Genetic • KAL1, FGFR1, GnHR, GPR54, SF1, DAX1, Leptin R, STAT5b • Organic • Craniopharyngioma • Post head trauma • Cranial irradiation • Multiple pituitary hormone deficiency • Isolated gonadotrophin deficiency • Syndromal • Prader-Willi syndrome • Lawrence-Moon-Biedl syndrome • Kallmann syndrome • Chronic disorders • Inflammatory bowel disease • Renal failure • Thalassemia • Emotional deprivation • Cystic fibrosis Hypergonadotrophic Hypogonadism • Syndromal • Klinefelter syndrome • XX male • Noonan syndrome • Frasier syndrome • Primary testicular • Gonadal dysgenesis • Androgen biosynthetic defects • Anorchia/cryptorchidism • Chemotherapy

DISORDERS OF PUBERTY It is generally accepted that the age boundaries for the first sign of puberty (Tanner stage G2) in normal males extends from 9 years to 13.5 years. Thus, a boy starting puberty before 9 years of age is deemed precocious in development—whereas delayed puberty warrants investigation if G2 is not reached by 13 to 14 years of age. By far the most common clinical scenario to investigate is constitutional delay in pubertal development, a condition that implies normal but delayed onset of puberty in otherwise healthy boys. There is often a positive family history of similar delay in pubertal onset in the mother or father, suggesting a genetically regulated timing in the awakening of the GnRH pulse generator. This is not a disorder per se, but can cause significant problems relating to self-esteem. It should be distinguished from HH, but this distinction is not easy because low basal FSH and LH levels with a prepubertal low response to a pulse of GnRH will occur in both. Some advocate a short course of exogenous testosterone (100-200 mg IM monthly for 4 to 6 injections). Such treatment improves self-image, promotes linear growth without impairing final adult height, and may result in the initiation of puberty— reflected in part by growth of the testis, which occurs in constitutional delay but not in disorders of HH.193 Because the disorders of puberty are primarily extratesticular in origin, reference to Tables 16-5 and 16-6 will suffice to summarize a general classification for disorders

Figure 16-7 GPR54 as a gate to puberty.

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of precocious and delayed puberty. Precocious puberty involves the gonads (gonadarche) and must not be equated with premature adrenarche, a condition in which adrenal androgens induce premature pubic hair development without virilization or advanced bone age (as discussed in detail in Chapters 4 and 12). It is logical and practical to base classification on the status of gonadotrophin secretion, the prime driver of normal puberty. Some testis-specific disorders are mentioned in further detail. Constitutive activation of mutant LH receptors leads to precocious puberty. This condition is limited to males (testotoxicosis) and is familial in a heterozygous (autosomaldominant) manner.50 Puberty occurs in early childhood and is characterized by elevated serum testosterone and suppressed serum LH levels. Missense mutations are confined to exon 11 of the LH receptor gene, which encodes for the signal-transducing transmembrane domain of the receptor. This contrasts with inactivating LH receptor mutations causing DSD, which are distributed throughout the gene. Mutant LH receptors causing testotoxicosis, when expressed in vitro, show basal constitutive activation of the cAMP signaling pathway in transfected cells in the absence of agonist compared with wild-type receptor. Mutant receptors also demonstrate a dose-response relationship with added LH or HCG.194 It is a curious observation that females heterozygous for LH receptor mutations do not develop early puberty. The explanation lies with the observation that LH receptor expression in the ovary is dependent on FSH levels. Because FSH levels are low in childhood, ovarian follicles do not differentiate and hence LH receptor expression is also not enacted at this time. However, it is not clear why mothers of sons with LH-receptor-activating mutations have no discernible phenotype. There is a relative hot spot of mutations at codon 578 in the sixth transmembrane helix, where aspartic acid (Asp) can be changed to glycine, tyrosine, or glutamic acid. A mutation at this codon (Asp578His) is found in somatic form only, and results in precocious puberty due to a Leydig cell adenoma.195,196 This particular mutation is highly active basally when expressed in transfected cells but does not respond to LH or HCG. Furthermore, the mutant LH receptor constitutively activates an alternative signal transduction pathway involving the phospholipase C pathway.197 The management of precocious puberty due to germline-activating mutations of the LH receptor is a challenge because the conventional approach of using a GnRH analogue to desensitise the gonadotrophes is not applicable. The mainstay of current treatment is to use a combination of an antiandrogen to block the direct biologic effects of androgens and an aromatase inhibitor to reduce the accelerating effects on growth plate closure from estrogens produced from aromatization of increased androgens. An effective combination appears to be bicalutamide (a nonsteroidal antiandrogen) and anastrozole, a third-generation aromatase inhibitor.198 This combination has produced a reversal of signs of hyperandrogenism, as well as reduction in growth velocity and rate of skeletal maturation. A similar gonadotrophin-independent increased sex steroid secretion is the cause of precocious puberty in the McCune-Albright syndrome.170 In this case, the pathophysiology relates to an activating mutation in the ubiquitously expressed G-protein ␣-subunit (Gs␣), a

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major product of the GNAS gene.199 The classic features of the syndrome comprise gonadotrophin-independent precocious puberty, café-au-lait lesions of the skin of a specific distribution, and polyostotic fibrous dysplasia. The pubertal manifestation occurs mainly in affected females. When it occurs in males, there is asymmetric enlargement of the testes. The Gs␣ mutation is somatic and is invariably a substitution of glycine for arginine at codon 201. Treatment for pubertal precocity associated with the McCune-Albright syndrome is similar to that used for LH-receptor-activating mutations. Treatment of central precocious puberty relies primarily on the use of long-acting GnRH agonists that suppress the ability of endogenous GnRH to bind to its cognate receptor and evoke an appropriate increase in FSH and LH. It is based on the discovery that a single pulse of GnRH evokes an appropriate gonadotrophin response, whereas continuous infusion of GnRH down-regulates and abrogates the response.200 Until recently, such GnRH long-acting agonists were administered by monthly IM injections—adjusting the dose to ensure suppression of response to exogenous GnRH. A long-acting form of GnRH that can be implanted subcutaneously and last for 1 year has recently been introduced.201 In addition to suppressing GnRH secretion, a form of reversible “medical castration,” a primary underlying cause must be excluded by appropriate investigation—including cranial imaging. “Idiopathic” precocious puberty is more common in girls than in boys. The causes of delayed puberty can also be classified according to the status of gonadotrophin secretion. Table 16-6 outlines a general classification of causes in males.

GENETIC CAUSES OF HYPOGONADOTROPHIC HYPOGONADISM The genetic causes of HH have been the subject of interest and investigation, resulting in the discovery of novel genes involved in the regulation of puberty via the hypothalamic-pituitary-gonadal axis. Kallmann syndrome consists of isolated HH plus anosmia. An X-linked form and several variants encoded by autosomal genes are described. The gene for the X-linked form KAL1 encodes the protein anosmin, which is a major regulator of the migration of GnRH neurones and olfactory nerves from the olfactory placode to the hypothalamus. Males with this condition have agenesis of the olfactory lobes, which may be detected on MRI imaging, as well as hypogonadism secondary to deficiency of hypothalamic GnRH. Transmitting females may have partial or complete anosmia. Renal agenesis, high-arched palate, cerebellar ataxia, and mirror movements of the hands (synkinesia) have been described. The X-linked form due to KAL1 is listed as Kallmann syndrome 1 in the Online Mendelian Inheritance in Man (OMIM) database (www.ncbi.nlm.nih.gov). Kallman syndrome 2, KAL2, is an autosomal-recessive form due to a mutation in the gene encoding fibroblast growth factor receptor-1 (FGFR-1) and is associated with cleft lip or palate plus tooth agenesis in addition to the anosmia and hypogonadism. KAL 3 is caused by a mutation in the PROKR2 gene, and KAL 4 is caused by a mutation in PROK 2.

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Together, these gene abnormalities account for about a third of all cases of Kallmann syndrome. Treatment with GnRH injected subcutaneously as pulses via a programmable pump can restore normal gonadal development and function. In some, the treatment can be discontinued while the patient retains normal gonadal function.202 The role of Kisspeptin and its cognate receptor in regulating GnRH secretion and hence puberty has been described.189,191,192 The DAX1 and SF1 genes interact in a complex manner in the formation of the GnRH region of the hypothalamus, as well as the formation of the adrenal gland. They are discussed in detail in Chapter 12. Leptin secreted by fat tissue also regulates gonadotrophin secretion. Inactivating mutations in the genes for leptin or its receptor lead to obesity.203,204 Leptin may be a signal in the initiation of puberty, indicating a link between nutritional status and reproductive capacity and the secular trend of pubertal onset in wellnourished societies. That leptin may be regarded as a metabolic gate to puberty is illustrated by the effect of leptin facilitating appropriately timed puberty when administered to children with congenital leptin deficiency.205 Leptin signaling may play an important role in the HH of chronic wasting illnesses, including anorexia nervosa and the chronic disorders listed in Table 16-6. The Prader-Willi syndrome is typically characterized by hypotonia, developmental delay, hypogonadism, and obesity. Stereotypic behavioral abnormalities also occur. The defect is encoded by a gene or genes on the paternal short arm of chromosome 15. Most disorders primarily testicular in nature have been discussed. Management of delayed puberty in the male is dependent on the underlying etiology. The overriding practical issue is to induce somatic signs of puberty in the event of this not occurring spontaneously from endogenous androgen production. A commonly used treatment is Sustanon, a mixture of testosterone esters, given by monthly intramuscular injections at a starting dose of 25 mg and thereafter increasing to adult doses of 250 mg monthly. A wide variety of injectable, oral, implantable, cutaneous patch, and gel preparations of androgens are available.206,207 Although the low-dose Sustanon modality is suited to replicate pubertal changes in a slow and timely fashion, regular hormone replacement in adulthood for persistent hypogonadism is increasingly employing topical delivery systems that appear to provide serum testosterone and DHT profiles similar to those found in normal males.208,209

Investigating Testis Function GENERAL There is no algorithmic-based panacea on how to investigate testis function. The choice of tests should be dictated by the clinical problem to be addressed and the nature of the questions posed. A number of sources provide recipes for investigation of the endocrine system.210-212

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TA B L E 1 6 - 6

General Classification of Causes of Delayed Puberty in the Male Hypogonadotrophic Hypogonadism229,230 Genetic KAL1, FGFR1, GnRH, GPR54, SF1, DAX1, Leptin R, STAT5b Organic craniopharyngioma post-head trauma cranial irradiation multiple pituitary hormone deficiency isolated gonadotrophin deficiency Syndromal Prader-Willi syndrome231 Lawrence-Moon-Biedl syndrome232 Kallmann syndrome233 Chronic disorders inflammatory bowel disease renal failure thalassaemia emotional deprivation cystic fibrosis Hypergonadotrophic Hypogonadism Syndromal Klinefelter syndrome234 XX male Noonan syndrome235 Frasier syndrome236 Primary testicular gonadal dysgenesis androgen biosynthetic defects anorchia/cryptorchidism chemotherapy237

AMBIGUOUS GENITALIA (also see Chapter 14) The approach to the investigation of a newborn infant with ambiguous genitalia requires a two-tierd approach to the choice of tests (Table 16-7). Establishing the karyotype, together with confirming or excluding CAH (the most common cause of ambiguous genitalia of the newborn), is the first task to be undertaken. The clear presence of a uterus/cervix on ultrasound and a markedly elevated serum 170H-progesterone level in a full-term infant with a 46,XX karyotype confirms a diagnosis of CAH due to 21-hydroxylase deficiency. If the karyotype is 46,XY (or 45X/46,XY), one determines whether testes are present—and if present whether they produce normal age-related amounts of testosterone. Table 16-7 does not contain an exhaustive list for this second-tier level of investigation but should allow resolution of the determinations cited. An understanding of the DSD classification shown in Table 16-2 is a guide to a logical pathophysiologic approach to investigation. The HCG stimulation test is the bedrock of investigation for the XY DSD infant. The protocol and timing for this test varies considerably among centers.213 The author’s center uses as a standard three daily injections of HCG (1,500 units by intramuscular injection). A post-HCG

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TA B L E 1 6 - 7

A Two-Tiered Approach to Investigation of Ambiguous Genitalia Tier 1: Establish DSD Category • FISH and full karyotype • Pelvic ultrasound • Serum 17-OH progesterone (save serum) • Blood sugar, electrolytes • Save urine for steroid metabolites Tier 2: In the Event of an XY or XO/XY DSD Category • Serum AMH, LH, FSH (save serum/DNA) • HCG stimulation test (see text for details) • Urinary steroid analysis • Imaging (MRI) • Laparoscopy/gonadal biopsy

completely normal (for example, an associated micropenis), the anorchia may be a form of gonadal dysgenesis in which there is occasionally a mutation in the SF1 gene.220

TESTIS TUMORS Imaging, together with histology and immunohistochemical analysis of a testicular biopsy, is the key investigation for a testis mass. The relevant tumor markers have been listed previously. Ultrasound distinguishes among cystic, solid, or complex scrotal masses and is useful in longitudinal screening of patients with testicular microlithiasis.221 However, microlithiasis is present in 5.6% of the young adult male population—and most men with such an isolated finding do not develop testis cancer.222 Regular self-examination of the testes is the recommended surveillance procedure.

PUBERTAL DISORDERS blood sample is collected 24 hours after the last injection for analysis of testosterone (coupled with androstenedione and DHT tests if indicated). An acute LHRH stimulation test may also be undertaken just prior to the first HCG injection. Occasionally, the HCG stimulation test is extended over 3 weeks (twice-weekly injections). This protocol has the potential for coupling investigation and management where the clinical problem is testis maldescent.214 The timing of an HCG test should be planned to coincide with the endogenous LH-induced rise in testosterone production at about 4 to 8 weeks of postnatal age, but local circumstances may dictate that the test be performed shortly after birth. This may create difficulties with the interpretation of neonatal serum steroid levels if nonextraction or non-chromatographic assays are performed.215 Analysis of urinary steroid metabolites by sensitive highly specific chromatographic techniques may delineate defects in androgen biosynthesis such as P450oxidoreductase deficiency and 5␣-reductase deficiency.

ABSENT TESTES When testes are not palpable at birth in an otherwise normal male infant, a karyotype is still mandatory. The phenotype may be the result of a fully virilized 46,XX infant with CAH. The main question asked of an endocrine investigation of a 46,XY infant with nonpalpable testes is whether testes are present. The HCG stimulation test and baseline LH and FSH measurements will generally answer the question. In addition, serum AMH is proving to be a reliable additional test—measuring levels otherwise almost undetectable in the case of anorchia.216219 Measurement of serum inhibin B levels appears to be equally effective as a marker of loss of Sertoli cells. Undetectable levels of testosterone after HCG stimulation, elevated gonadotrophins (particularly FSH), and very low or undetectable levels of AMH are strongly suggestive of absent testicular tissue. However, efforts are usually made to locate testicular tissue and imaging is not sufficiently reliable for this purpose. Laparoscopic exploration generally reveals just nubbins of fibrous tissue at the end of a vas deferens.102,103 If the genitalia are not otherwise

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A basal and acute LHRH-stimulated LH and FSH level, with interpretation of age-appropriate serum testosterone measurements, is the mainstay of the investigation of pubertyrelated testis disorders. The original term, testotoxicosis, for the clinical syndrome due to LH-receptor-activating mutations is affirmation that elevated testosterone with totally suppressed LH levels is akin to the TSH/FT4 profile in thyrotoxicosis (Grave’s disease). Assessment of LH/FSH levels provides the division between hyper-/hypogonadotrophic states and gonadotrophin dependency, as outlined in Tables 16-4 and 16-5. In pituitary-gonadal interrelationships, the negative feedback effect appears to be more profound on FSH levels than on LH levels. In gonadal dysgenesis, for example, basal FSH levels are usually more elevated than LH levels. Primary hypogonadism may be the result of chemotherapy for cancer in childhood. The germ cells are more susceptible to damage than Leydig cells, and thus boys often develop puberty spontaneously. Fertility is a later problem, with lower than normal testosterone levels for young adulthood subsequently found.223 Whereas the basal FSH level is usually a reliable indicator of germ cell damage and is a predictor of abnormal spermatogenesis, the basal LH is not as predictive of androgen deficiency. It is generally not appropriate to undertake seminal fluid analysis in pubertal boys as a measure of testis function, although obtaining a semen sample for cryopreservation is offered to pubertal boys undergoing cancer treatment.224 Inhibin B levels increase markedly at puberty and correlate with measurements of testis volume and testosterone, LH, and FSH levels.225 The level of inhibin is inversely correlated with FSH levels at mid-puberty (G3-G4), when spermatogenesis is becoming established. This concurs with the results of indirect semen analysis using the observation that spermatozoa can be identified in early-morning urine samples from pubertal boys.226 Spermaturia as an index of the onset of spermatogenesis (spermarche) is possible at a median age of 13.4 years, although there is a large variation between boys of the same pubertal stage.227 Nevertheless, this minimally invasive method of assessing developing germ cell function in adolescent boys has applicability in epidemiologic

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studies—including the effects of changes in the environment on male reproductive function. Thus, the effects of exposure to toxic chemicals before birth may be measurable by spermaturia analysis at puberty.228 The assessment of testis function needs to be designed not only to determine the cause of intrinsic disorders related to the pituitary-gonadal interplay but to account for the increasing evidence that disorders of the testis may have a causation in part environmental in origin.

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163. Butler MG, Brunschwig A, Miller LK, Hagerman RJ (1992). Standards for selected anthropometric measurements in males with the Fragile X syndrome. Pediatrics 89:1059–1062. 164. Carmi R, Meryash DL, Wood J, Gerald PS (1984). Fragile-X syndrome ascertained by the presence of macro-orchidism in a 5-month-old infant. Pediatrics 74:883–886. 165. Rudelli RD, Jenkins EC, Wisniewski K, Moretz R, Byrne J, Brown WT (1983). Testicular size in fetal fragile X syndrome. Lancet 1:1221–1222. 166. Johannisson R, Rehder H, Wendt V, Schwinger E (1987). Spermatogenesis in two patients with the fragile X syndrome: I. Histology: light and electron microscopy. Hum Genet 76:141–147. 167. Slegtenhorst-Eegdeman KE, De Rooij DG, Verhoef-Post M, et al. (1998). Macroorchidism in FMR1 knockout mice is caused by increased Sertoli cell proliferation during testicular development. Endocrinology 139:156–162. 168. Coutant R, Lumbroso S, Rey R, et al. (2001). Macroorchidism due to autonomous hyperfunction of Sertoli cells and Gs␣ gene mutation: An unusual expression of McCune-Albright syndrome in a prepubertal boy. J Clin Endocrinol Metab 86:1778–1881. 169. Arrigo T, Pirazzoli P, De Sanctis L, et al. (2006). McCune-Albright syndrome in a boy may present with a monolateral macroorchidism as an early and isolated clinical manifestation. Horm Res 65:114–119. 170. Spiegel AM, Weinstein LS (2004). Inherited diseases involving G proteins and G protein coupled receptors. Annu Rev Med 55:27– 39. 171. Rey RA, Venara M, Coutant R, et al. (2006). Unexpected mosaicism of R201H-GNAS1 mutant-bearing cells in the testis underlie macroorchidism without sexual precocity in McCune-Albright syndrome. Hum Mol Genet 15:3538–3543. 172. Velaga MR, Wright G, Crofton PM, et al. (2005). Macro-orchidism in two unrelated prepubertal boys with a normal FSH receptor. Horm Res 64:1–2. 173. Castro-Magana M, Angulo M, Canas A, Sharp A, Fuentes B (1988). Hypothalamic-pituitary gonadal axis in boys with primary hypothyroidism and macroorchidism. J Pediatr 112:397–402. 174. Jannini EA, Ulisse S, D’Armiento M (1995). Macroorchidism in juvenile hypothyroidism. J Clin Endocrinol Metab 80:2543–2544. 175. De Leener A, Montanelli L, Van Durme J, et al. (2006). Presence and absence of follicle-stimulating hormone receptor mutations provide some insights into spontaneous ovarian hyperstimulation syndrome physiopathology. J Clin Endocrinol Metab 91:555–562. 176. Suarez EA, d’Alva CB, Campbell A, et al. (2007). Absence of mutation in the follicle-stimulating hormone receptor gene in severe primary hypothyroidism associated with gonadal hyperstimulation. J Pediatr Endocrinol Metab 20:923–931. 177. Parent AS, Teilmann G, Juul A, Skakkebaek NE, Toppari J, Bourguignon JP (2003). The timing of normal puberty and the age limits of sexual precocity: Variations around the world, secular trends, and changes after migration. Endocr Rev 24:668–693. 178. Boas M, Boisen KA, Virtanen HE, et al. (2006). Postnatal penile length and growth rate correlate to serum testosterone levels: A longitudinal study of 1962 normal boys. Eur J Endocrinol 154:125–129. 179. Andersson AM, Toppari J, Haavisto AM, et al. (1998). Longitudinal reproductive hormone profiles in infants: Peak of inhibin B levels in infant boys exceeds levels in adult men. J Clin Endocrinol Metab 83:675–681. 180. Sharpe RM, McKinnell C, Kivlin G, Fisher JS (2003). Proliferation and functional maturation of Sertoli cells, and their relevance to disorders of testis function in adulthood. Reproduction 125:769– 784. 181. Main KM, Toppari J, Suomi AM, et al. (2006). Larger testes and higher inhibin B levels in Finnish than in Danish newborn boys. J Clin Endocrinol Metab 91:2732–2737. 182. Suomi AM, Main KM, Kaleva M, et al. (2006). Hormonal changes in 3-month old cryptorchid boys. J Clin Endocrinol Metab 91:953–958. 183. Conte FA, Grumbach MM, Kaplan SL (1975). A diphasic pattern of gonadotropin secretion in patients with the syndrome of gonadal dysgenesis. J Clin Endocrinol Metab 4:670–674. 184. Plant TM (2006). The male monkey as a model for the study of the neurobiology of puberty onset in man. Mol Cell Endocrinol 254/255:97–102.

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185. Acerini CL, Tasker RC (2007). Traumatic brain injury induced hypothalamic-pituitary dysfunction: A paediatric perspective. Pituitary (E-pub ahead of print). 186. Darzy KH, Shalet SM (2005). Hypopituitarism as a consequence of brain tumors and radiotherapy. Pituitary 8:203–211. 187. Seminara SB (2007). Kisspeptin in reproduction. Semin Reprod Med 25:337–343. 188. Kauffman AS, Clifton DK, Steiner RA (2007). Emerging ideas about kisspeptin-GPR54 signalling in the neuroendocrine regulation of reproduction. Trends Neuroscience 30:504–511. 189. Seminara SG, Messager S, Chatzidaki EE, et al. (2003). The GPR54 gene as a regulator of puberty. N Engl J Med 349:1614–1627. 190. Semple RK, Achermann JC, Ellery J, et al. (2005). Two novel missense mutations in G protein-coupled receptor 54 in a patient with hypogonadotrophic hypogonadism. J Clin Endocrinol Metab 90:1849–1855. 191. Dhillo WS, Chaudhri OB, Patterson M, et al. (2005). Kisspeptin-54 stimulates the hypothalamic-pituitary gonadal axis in human males. J Clin Endocrinol Metab 90:6609–6615. 192. Shahab M, Mastronardi C, Seminara SB, Crowley WF, Ojeda SR, Plant TM (2005). Increased hypothalamic GPR54 signalling: A potential mechanism for initiation of puberty in primates. Proc Natl Acad Sci USA 102:2129–2134. 193. Kaplowitz PB (1989). Diagnostic value of testosterone therapy in boys with delayed puberty. Amer J Dis Child 143:116–120. 194. Kosugi S, Van Dop C, Geffner ME, et al. (1995). Characterisation of heterogeneous mutations causing constitutive activation of the luteinising hormone receptor in familial male precocious puberty. Hum Mol Genet 4:183–188. 195. Richter-Unruh A, Wessels HT, Menken U, et al. (2002). Male LHindependent sexual precocity in a 3.5-year old boy caused by a somatic activating mutation of the LH receptor in a Leydig cell tumor. J Clin Endocrinol Metab 87:1052–1056. 196. d’Alva CB, Brito VN, Palhares HMC, et al. (2006). A single somatic activating Asp578His mutation of the luteinising hormone receptor causes Leydig cell tumor in boys with gonadotrophin-independent precocious puberty. Clin Endocrinol 65:408–410. 197. Liu G, Duranteau L, Carel JC, Monroe J, Doyle DA, Shenker A (1999). Leydig-cell tumors caused by an activating mutation of the gene encoding the luteinising hormone receptor. N Engl J Med 341:1731–1736. 198. Kreher NC, Pescovitz OH, Delameter P, Tiulpakov A, Hochberg Z (2006). Treatment of familial male-limited precocious puberty with bicalutamide and anastrozole. J Pediatr 149:416–420. 199. Weinstein LS, Liu J, Sakamoto A, Xie T, Chen M (2004). GNAS: Normal and abnormal functions. Endocrinology 145:5459–5464. 200. Wildt L, Marshall G, Knobil E (1980). Experimental induction of puberty in the infantile female rhesus monkey. Science 207:1373–1375. 201 Eugster EA, Clarke W, Kletter GB, et al. (2007). Efficacy and safety of histrelin subdermal implant in children with central precocious puberty: A multicenter trial. J Clin Endocrinol Metab 92:1697–1704. 202. Ravio T, Falardeau J, Dwyer A, et al. (2007). Reversal of idiopathic hypogonadotropic hypogonadism. N Engl J Med 357:863–873. 203. Farooqi S, O’Rahilly S (2006). Genetics of obesity in humans. Endocr Rev 27:710–718. 204. Farooqi IS, Wangensteen T, Collins S, et al. (2007). Clinical and molecular genetic spectrum of congenital deficiency of the leptin receptor. N Engl J Med 356:237–247. 205. Farooqi IS, Matarese G, Lord GM, et al. (2002). Beneficial effects of leptin on obesity, T cell hyporesponsiveness, and neuroendocrine/metabolic dysfunction of human congenital leptin deficiency. J Clin Invest 110:1093–1103. 206. Richmond EJ, Rogol AD (2007). Male pubertal development and the role of androgen therapy. Nat Clin Pract Endocrinol Metab 3:338–344. 207. Nieschlag E (2006). Testosterone treatment comes of age: New options for hypogonadal men. Clin Endocrinol 65:275–281. 208. Swerdloff RS, Wang C, Cunningham G, et al. (2000). Long-term pharmacokinetics of transdermal testosterone gel in hypogonadal men. J Clin Endocrinol Metab 85:4500–4510. 209. Mazer N, Bell D, Wu J, et al. (2005). Comparison of the steadystate pharmacokinetics, metabolism, and variability of a transdermal testosterone patch versus a transdermal testosterone gel in hypogonadal men. J Sex Med 2:213–226. 210. Ogilvy-Stuart AL, Brian CE (2004). Early assessment of ambiguous genitalia. Arch Dis Child 89:401–407.

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211. Ranke MB (2003). Diagnostic of endocrine function in children and adolescents, Second edition. Basel: Karger. 212. Hochberg Z (2007). Practical algorithms in pediatric endocrinology, Second edition. Basel: Karger. 213. Ng KL, Ahmed SF, Hughes IA (2000). Pituitary-gonadal axis in male undermasculinisation. Arch Dis Child 82:54–58. 214. Dixon J, Wallace AM, O’Toole S, Ahmed SF (2007). Prolonged human chorionic gonadotrophin stimulation as a tool for investigating and managing undescended testes. Clin Endocrinol (E-pub ahead of print). 215. Tomlinson C, Macintyre H, Dorrian CA, Ahmed SF, Wallace AM (2004). Testosterone measurements in early infancy. Arch Dis Child Fetal Neonatal Ed 89:F558–F559. 216. Rey RA, Belville C, Nihoul-Fékété C, et al. (1999). Evaluation of gonadal function in 107 intersex patients by means of serum antimüllerian hormone measurement. J Clin Endocrinol Metab 84:627–631. 217. Lee MM, Misra M, Donohoe PK, MacLaughlin DT (2003). MIS/ AMH in the assessment of cryptorchidism and intersex conditions. Mol Cell Endocrinol 211:91–98. 218. Josso N, Picard JY, Rey R, di Clemente N (2006). Testicular antiMullerian hormone: History, genetics, regulation and clinical applications. Pediatr Endocrinol Rev 3:347–358. 219. Bergadá I, Milani C, Bedecarrás P, et al. (2006). Time course of the serum gonadotrophin surge, inhibins, and anti-Mullerian hormone in normal newborn males during the first month of life. J Clin Endocrinol Metab 91:4092–4098. 220. Philibert P, Zenaty D, Lin L, et al. (2007). Mutational analysis of steroidogenic factor 1 (NR5a1) in 24 boys with bilateral anorchia: A French collaborative study. Hum Reprod (E-pub ahead of print). 221. Pearl MS, Hill MC (2007). Ultrasound of the scrotum. Semin Ultrasound CT MR 28:225–248. 222. Costabile RA (2007). How worrisome is testicular microlithiasis? Curr Opinion Urol 17:419–423. 223. Greenfield DM, Walters SJ, Coleman RE, et al. (2007). Prevalence and consequences of androgen deficiency in young male cancer survivors in a controlled cross-sectional study. J Clin Endocrinol Metab 92:3476–3482. 224. Müller J, Sønksen J, Sommer P, et al. (2000). Cryopreservation of semen from pubertal boys with cancer. Med Pediatr Oncol 34:191–194. 225. Radicioni AF, Anzuini A, De Marco E, Nofroni I, Castracane VD, Lenzi A (2005). Change in serum inhibin B during normal male puberty. Eur J Endocrinol 152:403–409. 226. Nielsen CT, Skakkebaek NE, Richardson DW, et al. (1986). Onset of the release of spermatozoa (spermarche) in boys in relation to age, testicular growth, pubic hair, and height. J Clin Endocrinol Metab 62:532–535. 227. Pedersen JL, Nysom, K, Jørgensen M, et al. (1993). Spermaturia and puberty. Arch Dis Child 69:384–387. 228. Mol NM, Sørensen N, Weihe P, et al. (2002). Spermaturia and serum hormone concentrations at the age of puberty in boys prenatally exposed to polychlorinated biphenyls. Eur J Endocrinol 146:357–363. 229. Layman LC (2007). Hypogonadotropic hypogonadism. Endocrinol Metab Clin N Am 36:283–296. 230. Trarbach EB, Silveira LG, Latronio AC (2007). Genetic insights into human isolated gonadotropin deficiency. Pituitary (E-pub ahead of print). 231. Burman P, Ritzen EM, Lindgren AC (2001). Endocine dysfunction in Prader-Willi syndrome: A review with special reference to GH. Endocr Rev 22:787–799. 232. Blaque OE, Leroux MR (2006). Bardet-Biedl syndrome: An emerging pathomechanism of intracellular transport. Cell Mol Life Sci 63:2145–2161. 233. Bhagavath B, Layman LC (2007). The genetics of hypogonadotropic hypogonadism. Semin Reprod Med 25:272–286. 234. Lanfranco F, Kamischke A, Zitzmann M (2004). Klinefelter’s syndrome. Lancet 364:273–283. 235. Allanson JE (2007). Noonan syndrome. Am J Genet C Semin Med Genet 145:274–279. 236. Melo KF, Martin RM, Costa EM, et al. (2002). An unusual phenotype of Frasier syndrome due to IVS9 ⫹4C⬎T mutation in the WT1 gene: Predominantly male ambiguous genitalla and absence of gonadal dysgenesis. J Clin Endocrinol Metab 87:2500–2505. 237. Rutter MM, Rose SR (2007). Long-term endocrine sequelae of childhood cancer. Curr Opin Pediatr 19:480–487.

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C H A P T E R

17 Disorders of Mineral Homeostasis in the Newborn, Infant, Child, and Adolescent ALLEN W. ROOT, MD • FRANK B. DIAMOND, JR., MD

Introduction Mineral Homeostasis: Conception Through Adolescence Pregnancy and Lactation Fetus Neonate and Infant Child and Adolescent Disorders of Mineral Homeostasis in the Neonate and Infant Hypocalcemia Early Neonatal Hypocalcemia Late Neonatal Hypocalcemia Hypoparathyroidism Evaluation and Management Hypercalcemia Etiology Evaluation and Management Disorders of Bone Mineralization Low Bone Mass and Rickets Increased Bone Mass Disorders of Magnesium Metabolism Hypomagnesemia Hypermagnesemia Disorders of Mineral Homeostasis in the Child and Adolescent

Introduction Disorders of calcium, magnesium, and phosphate metabolism (and of bone formation, accrual, and maintenance) during the first two decades of life result from suboptimal ingestion, absorption, or retention of constit686

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Hypocalcemia Etiology Evaluation Management Hypercalcemia Etiology Evaluation Management Disorders of Bone Mineralization and Formation Rickets Calciopenic Rickets Phosphopenic Rickets Disorders of Alkaline Phosphatase Renal Osteodystrophy Low Bone Mass Osteogenesis Imperfecta Fibrous Dysplasia High Bone Mass Ectopic Calcification and Ossification Osteochondrodysplasias Concluding Remarks

uent nutrients; abnormal vitamin D metabolism or bioactivity; disorders of parathyroid hormone (PTH) synthesis, secretion, or function; and intrinsic aberrations in cartilage and bone cells. For an integrated overview of calcium, mineral, and skeletal homeostasis, the reader is referred to Chapter 3. 686

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Mineral Homeostasis: Conception Through Adolescence PREGNANCY AND LACTATION The fetus is entirely dependent on the mother for its calcium and phosphate requirements for skeletal formation and cell and tissue growth and function. The fetal-placental unit actively extracts calcium from the maternal circulation, as the mother doubles her rate of intestinal calcium absorption.1-3 During the first trimester of pregnancy, maternal serum concentrations of total calcium decline and remain low through gestation due to a fall in albumin values and expansion of extracellular fluid volume—whereas serum concentrations of ionized calcium (Ca2⫹e) and phosphate remain relatively constant (Figure 17-1). PTH values decline to 10% to 30% of the nonpregnant range in the first trimester of pregnancy, and then rise to mid nonpregnant levels in the latter half of gestation. The secretion of PTH-related peptide (PTHrP) by the placenta, amnion, decidua, umbilical cord, breast, and fetal parathyroid glands increases severalfold beginning early in the first trimester, and maternal levels rise throughout gestation. Maternal calcitonin values also increase during gestation. Serum concentrations of calcidiol do not change, but levels of calcitriol (synthesized primarily by the maternal kidney but also in part by the placenta, decidua, and fetal kidneys) increase more than twofold as pregnancy advances and substantially accelerate the rate (and augment the amount) of calcium absorbed by the maternal small intestine. Increased renal synthesis of calcitriol is stimulated primarily by PTHrP but also by prolactin, estrogen, and human chorionic somatomammotropin. Maternal urinary calcium excretion rises (⫹125%), reflecting the increased absorption of ingested calcium.4 The rate of maternal bone resorption increases in the first trimester of gestation, as determined by histomorphometric analysis of bone biopsies and by the increase in the urinary excretion rates of pyridinoline (Pyr), desoxypyridinoline (Dpd), N-telopeptide (NTx), and hydroxyproline that occur in early gestation and continue to rise through gestation. Markers of osteoblast activity and bone formation (bone-specific alkaline phosphatase, osteocalcin, carboxyl, and amino terminal propeptides of type I collagen) decline during the first trimester but increase in the third trimester.4,5 During a normal 40-week gestation, maternal wholebody bone mineral density (BMD) does not change. Cortical BMD increases (arms ⫹2.8%, legs ⫹1.8%), whereas trabecular BMD declines (vertebrae –4.5%, pelvis –3.2%). However, pregnancy does not appear to have any long-term adverse effects on maternal bone mineralization or fracture risk.3 Maternal serum values of insulin-like growth factor-I (IGF-I) increase 67% by the third trimester of pregnancy, and the increment correlates positively with the increase in markers of bone turnover and inversely with the change in maternal vertebral BMD. Thus, early in gestation the pregnant woman meets fetal demand for calcium by increasing the rate of resorption of stored bone calcium—whereas the calcium requirement of the more mature fetus is met

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Figure 17-1 Schematic changes in mineral and calciotropic hormone values during pregnancy. The shaded areas represent normal adult ranges. To convert calcium in mmol/L to mg/dL, multiply by 4; to convert phosphate in mmol/L to mg/dL, multiply by 3.097; to convert PTH in pmol/L to pg/mL, multiply by 9.50; and to convert calcitriol in pmol/L to pg/mL, multiply by 0.4166. [Reproduced with permission from Kovacs CS, Kronenberg HM (1997). Maternal-fetal calcium and bone metabolism during pregnancy, puerperium, and lactation. Endocrine Reviews 18:832–872.]

by a substantially increased rate of maternal intestinal calcium absorption. During lactation, the nursing mother daily transfers to her suckling infant 280 to 400 mg of calcium mobilized from her skeleton in response to PTHrP secreted primarily by the breast.3 Calcium concentrations are low in colostrum, being approximately 25 mg/dL in breast milk during the first 6 months of lactation and 21 mg/dL during months 6 to 12 of nursing.6 Maternal total calcium, calcitriol, and calcitonin values are normal, while Ca2⫹e, phosphate, and PTHrP levels are increased during lactation. Urinary levels of markers of bone resorption and serum values of markers of bone formation are elevated

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during lactation, implying rapid turnover of maternal bone mineral during nursing. Maternal bone mineralization declines 3% to 10% during lactation, only to reaccrue rapidly after weaning.7 As there is an increasing incidence (or awareness) of vitamin D deficiency in breast-fed infants, supplementation of the breast-fed infant with 400 IU of vitamin D daily and higher than recommended intake (200 IU/day) of vitamin D (up to 2,000 to 4,000 IU/day) have been suggested for pregnant and lactating women.8-10

FETUS During gestation, the fetus accrues 30 to 35 g of calcium. Approximately 80% of calcium accretion occurs in the third trimester.1,3 At 28 weeks of gestation, calcium is deposited into the fetal skeleton at the rate of approximately 100 mg/day—whereas at 35 weeks calcium is deposited at the rate of approximately 250 mg/day.2,5,11,12 At birth, whole-body bone mineral content (BMC) is positively related to gestational age, to body length, and most closely to body weight—as are lumbar spine (L1-L4) BMC and BMD (Figures 17-2 and 17-3).12,13 The fetal skeleton serves two roles: it is a metabolically important source of calcium mobilized by fetal PTH and/or PTHrP acting through the PTH/PTHrP receptor (PTHR1) when the supply of calcium from the mother is limited, and it provides a rigid structural and protective framework for fetal soft tissues.2 Fetal serum calcium levels are established independently of, and are not directly related to, maternal calcium concentrations. From at least 15 weeks of gestation, serum concentrations of total calcium (and particularly Ca2⫹e) are substantially higher in the human fetus and other mammals (rat, sheep) than in the mother (1.4:1). The physiologic significance of this finding is unknown. In addition, fetal serum concentrations of magnesium and phosphate are greater than maternal values. The parathyroid glands (PTG) are essential to maintenance of normal fetal calcium concentrations. By the tenth week of gestation, they secrete PTH and possibly PTHrP—and both peptides function additively to maintain fetal serum calcium levels. PTH does not stimulate placental calcium transport, but it is secreted by the fetal PTG in response to hypocalcemia. Fetal mice in which the expression of PTH has been ablated (e.g., Hoxa3 null mice) are hypocalcemic, and their skeletal mineralization is impaired.1,14 Maternal hypercalcemia suppresses, and maternal hypocalcemia stimulates, secretion of fetal PTH. Both amino and mid-molecule fragments of PTHrP (e.g., PTHrP1-86, PTHrP67-86) produced by the fetal PTG (possibly), placenta (primarily), amnion, chorion, and umbilical cord maintain high fetal serum calcium concentrations by stimulating active maternal-to-fetal transport of calcium across the placental syncytiotrophoblast against a concentration gradient. This PTHrP effect is mediated in part by receptors that recognize mid-molecule and/or carboxyl terminal fragments of PTHrP, as evidenced in fetal mice in which Pthr1 has been ablated (placental calcium transport remains active, whereas fetal serum calcium levels are low). In fetal mice in which the expression of Pthrp itself has been impaired, serum calcium levels are lower than control

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Figure 17-2 Whole-body bone mineral content of the preterm and term neonate relative to body weight, body length, and bone area as determined by dual X-ray absorptiometry near birth. [Reproduced with permission from Rigo J, et al. (2000). Bone mineral metabolism in the micropremie. Clin Perinatol 27:147–170.]

values and are maintained by fetal PTH at values comparable to those of the mother. In Pthrp null fetal mice, placental calcium transfer is decreased—and chondrocyte maturation and bone development are abnormal.1 Although serum concentrations of PTHrP are quite high in the human fetus (term cord blood 2–5 pmol/L), PTH levels are lower than those in maternal serum. In utero, calcitonin concentrations are quite high (an appropriate response to the increased serum calcium levels of the fetus)—but this peptide does not have a major impact on fetal calcium homeostasis.1 Calcium and magnesium regulate fetal calcium levels through the calcium-sensing receptor (CaSR) that controls the synthesis and secretion of fetal PTH. Thus, in fetal mice in which Casr has been knocked out the serum calcium concentration is further elevated—as are values

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juxta-articular cells of the long bone regulates the orderly rate of chondrocyte maturation, whereas fetal PTH maintains serum calcium and phosphate values appropriate to bone mineralization. Magnesium is actively transported across the placenta.19 In utero fetal magnesium concentrations exceed maternal values and are inversely related to gestational age, reflecting the third trimester decline in maternal magnesium concentrations.

NEONATE AND INFANT

Figure 17-3 Lumbar spine (L1-L4) bone mineral content (BMC). (A) Bone mineral density (BMD). (B) BMD of preterm and term neonates relative to body weight as determined by dual X-ray absorptiometry near birth. [Reproduced with permission from Koo WWK, Hockman EM (2000). Physiologic predictors of lumbar spine bone mass in neonates. Pediatr Res 48:485–489.]

of PTH and calcitriol.1,15 The CaSR is also expressed in the human placenta in the first trimester and is involved in placental calcium transport.16 Calcium-selective ion channels (TPRV5, TPRV6) located at the apical surface of trophoblast cells facilitate maternal-fetal transplacental transfer of calcium.17 Fetal serum calcitriol concentrations are a bit lower than those of the mother. Experimentally, fetal serum calcium values and mineralization of the fetal skeleton are normal in the presence of maternal vitamin D deficiency or an inactive vitamin D receptor (VDR). In the VDR-null fetal mouse, if the mother ingests a diet enriched with calcium and phosphate, indicating that the fetus does not have an absolute requirement for calcitriol or the VDR for normal mineral metabolism.1,18 In man, the fetal cartilaginous skeleton is present by the eighth week of gestation. Primary ossification centers appear in the long bones and vertebrae by the twelfth week, and secondary centers at the femoral ends are noted by the thirty-fourth week.1 Fetal PTHrP secreted by

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Calcium levels in cord blood correlate with gestational age and exceed maternal values by 1 to 2 mg/dL as a result of an active placental calcium pump. When the neonate is abruptly removed from the transplacental infusion of maternal calcium, total calcium and Ca2⫹e concentrations decline rapidly in the first 6 to 12 hours after delivery to nadir values (from 12 to 9 mg/dL and 1.45 to l.20 mmol/L, respectively) by 24 to 72 hours of age.1,2,20 Calcium levels are a bit lower and PTH values higher in neonates delivered by cesarean section than in those delivered vaginally.21 After birth, PTHrP values decline rapidly. Thus, to maintain mineral homeostasis the neonate becomes dependent on endogenous PTH, exogenous vitamin D, ingested and absorbed calcium, renal tubular reabsorption of calcium, and bone calcium stores for its calcium needs. In response to the fall in Ca2⫹e values, serum levels of PTH begin to increase on the first day of life—resulting in normal calcium values (8.8–11.3 mg/dL) at 48 hours of age, followed by rise in calcitriol concentrations and slow decline (after an initial postnatal rise) in calcitonin values (Figure 17-4).22 For the first 2 to 4 weeks after birth, there is increased efficiency of intestinal calcium absorption by passive means that are independent of vitamin D—perhaps due to the lactose content of milk, which affects paracellular transport of Ca2⫹e (1,2). Later in the neonatal period, vitamin-D-dependent intestinal calcium absorption increases. Renal tubular handling of calcium and the response to PTH mature during the first several weeks of life. Bone calcium accretion continues at the rate of 150 mg/kg/day for several months after birth, a vitamin-D-dependent process. Due to decreased glomerular filtration and increased tubular reabsorption, serum concentrations of phosphate are maximal in the neonate. After delivery, serum phosphate concentrations increase from cord values of 3.8 to 8.1 mg/dL to levels that range between 4.5 and 9.0 mg/dL during the first week of life and that then stabilize at values between 4.5 and 6.7 mg/dL through the first year of life.23 In preterm or acutely ill neonates, the fall in calcium values is often exaggerated and more prolonged—and bone mineralization is frequently impaired in very preterm newborns and infants. Maternal hypercalcemia suppresses, and maternal hypocalcemia stimulates, secretion of fetal PTH—effects that may carry over to the neonate for several days. In infants and children, serum magnesium concentrations are quite stable—ranging between 1.5 and 2.2 mg/dL through 4 months of age and between 1.7 and 2.3 mg/dL through 5 years of age. Human breast milk contains (on average) calcium at 28 mg/dL, phosphate at 13 mg/dL, and vitamin D 15 to

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Phytate (inositol hexaphosphate) present in soy formulas and infant cereals chelates calcium, and oxalate (a constituent of spinach) precipitates calcium—thereby reducing intestinal absorption of this mineral, a difficulty overcome by increasing dietary calcium content. Optimizing calcium, vitamin D intake, and bone mineralization during infancy (and childhood) may substantially contribute to achievement of an adult peak bone mass of sufficient density to avoid osteopenia and osteoporosis and their adverse consequences in later life.6

CHILD AND ADOLESCENT

Figure 17-4 Schematic changes in mineral and calciotropic hormone values during the first 4 days of life in normal full-term neonates. The shaded areas represent normal adult ranges. The PTHrP values are depicted by a dashed line to indicate their speculative nature. To convert calcium in mmol/L to mg/dL, multiply by 4; to convert phosphate in mmol/L to mg/dL, multiply by 3.097; to convert PTH in pmol/L to pg/mL, multiply by 9.50; and to convert calcitriol in pmol/L to pg/mL, multiply by 0.4166. [Reproduced with permission from Kovacs CS, Kronenberg HM (1997). Maternal-fetal calcium and bone metabolism during pregnancy, puerperium, and lactation. Endocrine Reviews 18:832–872.]

50 IU/L. Thus, vitamin D supplementation (200–400 IU/ day) is necessary during breast feeding.6 Cow’s milk formulas contain calcium at ⬃40 to 60 mg/dL, phosphate at ⬃30 to 40 mg/dL, and vitamin D at ⬃30 to 40 IU/dL. The bioavailability of calcium (that which is accessible for normal metabolic usage) varies dependent on its source and the formulaic content and source of protein, fat, and carbohydrates. For example, infants fed formulas that contain palm olein absorb less calcium than those receiving a formula fortified with another form of fat or human breast milk—and these infants have lower total body bone mineral content.24

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During childhood and adolescence, serum concentrations of total calcium (8.8–10.8 mg/dL, depending on the analytical laboratory) and magnesium (1.7–2.2 mg/dL) remain relatively constant.22 Serum phosphate levels are higher in children (4.5–6.2 mg/dL) than in adults (2.5–4.5 mg/dL), and attain maximum values several months before the peak height velocity of adolescence is achieved. The rise has been attributed to augmentation of the renal tubular reabsorption of phosphate due to the combined effects of the increased secretion of growth hormone (GH), IGF-I, and sex hormones—factors that contribute to the pubertal growth spurt.25 Total serum alkaline phosphatase activity is also higher in children than in adults, and increases transiently during the pubertal growth spurt. Levels of bone-specific alkaline phosphatase (the major isoform of this enzyme in normal adolescents) increase between 9 and 12 months before peak height velocity and reach maximum at Tanner male genital stage III. These levels are directly related to the secretion of testosterone in boys.26 In females, serum bone-specific alkaline phosphatase activity peaks at Tanner stage III breast development and correlates with serum osteocalcin concentrations. Serum concentrations of PTH fluctuate very little during adolescence, whereas levels of calcitriol rise transiently. Serum values of osteocalcin, a bone matrix ␥-carboxylglutamic acid whose concentration reflects osteoblast activity, are higher during infancy than childhood and increase during adolescence—reaching peak values at 12 years of age in girls and at 14 years in boys.27,28 Mean 24-hour levels of osteocalcin are not related to mean 24-hour GH concentrations or to growth rates in prepubertal children, but random and 24-hour mean osteocalcin concentrations are substantially lower in GH-deficient children than in children of normal stature. Serum levels of the carboxyl-terminal propeptide of procollagen type I (PICP) also reflect osteoblast activity. These levels are highest in infancy, fall during childhood, increase briefly during puberty, and then decline to adult values. Serum levels of the amino-terminal extension of procollagen type III (PIIINP) reflect soft-tissue growth and to a limited extent bone formation. Values decline between infancy and childhood, increase during adolescence, and then fall to adult concentrations.29,30 Mean 24-hour concentrations of PICP and PIIINP correlate with mean 24-hour GH values and growth rates. Their levels are significantly lower in GH-deficient children than in normal children.31

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

Serum concentrations of cross-linked carboxyl-terminal telopeptide of type I collagen (ICTP) and urinary excretion of the nonreducible pyridinium compounds and the carboxyl- (CTx) and amino-terminal (NTx) telopeptides of type I collagen are markers of bone resorption. Values of ICTP peak between 9 and 13 years in girls at Tanner stage III breast development and at 14 years and Tanner male genital stage IV in boys. In both cases, they then decline.32 Pyr (hydroxylysyl-pyridinoline; present in bone and cartilage) and Dpd (lysyl-pyridinoline; found primarily in bone) are derivatives of the amino acids lysine and hydroxylysine, respectively—which cross-link adjacent telopeptide regions of collagen fibers in mature bone.33 The urinary excretion of both compounds is independent of diet and reflects collagen degradation. The urinary excretion of the pyridinium cross-links fluctuates diurnally with highest values in the morning. The 24-hour urinary excretion of both pyridinium compounds is greatest in the child 2 to 10 years of age, declines between 11 and 17 years, and still further thereafter as values approach adult levels.28 Urinary levels of NTx, a reliable index of bone resorption, may be determined in spot or 24-hour urine specimens and are expressed as nmol of bovine cartilage equivalent (BCE) units/mmol of creatinine. Values are highest in infancy and decline steadily with age.34 The urinary excretion of hydroxyproline is a less specific marker of bone resorption because it is present in the collagen of connective tissue in many sites other than bone—particularly skin, as well as in dietary protein. Urinary excretion increases during puberty, and in girls peaks (150 mg/day) in the year before menarche coincident with maximal growth rate.29 Tartrate-resistant acid phosphatase activity (TRAP), a marker of osteoclast activity, is released when bone collagen is degraded. In adolescents, markers of bone formation (bone-specific alkaline phosphatase, osteocalcin, and PICP) and of bone resorption (TRAP, ICTP, and urine excretion of NTx and the pyridinolines) increase to a similar extent and then decline in tandem—reflecting the tight link between bone formation and resorption. In females, these measurements correlate inversely with serum concentrations of estradiol—reflecting the maturational effect of estrogen on epiphyseal cartilage and its inhibitory effect on growth.35 The rate of bone accretion is steady throughout childhood, and increases substantially during adolescence— the physiologic interval in which up to 50% of adult bone mass is acquired. Indeed, in the 2 years that bracket the peak growth rate of puberty 25% of peak bone mineral mass is accumulated.36 Approximately 90% of peak bone mass is accrued by 18 years of age in normal subjects, and peak BMC is achieved between 20 and 25 years of age. During puberty, trabecular bone volume and mineralization increase appreciably in response to GH, IGF-I, estrogens, and androgens and are maximal by the end of the second decade of life in females (and perhaps in the male). However, the timing of peak bone mass of the axial skeleton is more variable—being attained between 17 and 35 years of age. Most often this occurs near the age when adult sexual maturity is achieved. At all ages, vertebral

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cross-sectional size is smaller in females than in males— even when controlling for height. However, femoral dimensions are similar in both genders when matched for height and weight. Trabecular bone mass becomes greater in black than in white subjects at adolescence, when trabecular bone mineral density increases 34% in black and 11% in white subjects. The difference persists thereafter. The cross-sectional area of the femur is also greater in black than in white adolescents and adults.

Disorders of Mineral Homeostasis in the Neonate and Infant HYPOCALCEMIA Clinical manifestations of hypocalcemia (total calcium ⬍7.5 mg/dL; Ca2⫹e ⬍1.20 mmol/L) in neonates include irritability, hyperacusis, jitteriness, tremulousness, facial spasms, neuromuscular excitability (tetany), laryngospasm, and focal or generalized seizures.20 Nonspecific symptoms such as apnea, tachycardia, cyanosis, emesis, and feeding problems may also occur. Traditionally, neonatal hypocalcemia develops after 3 days of age in offspring born in the late winter to early spring of the year to multiparous women of lower socioeconomic status with inadequate intake of vitamin D or exposure to sunlight. Causes of neonatal hypocalcemia may be considered in relation to the age of onset (before or after 72 hours of life, Table 17-1).

Early Neonatal Hypocalcemia In the absence of hypoproteinemia, hypocalcemia occurring within the first 72 hours after birth is considered “early neonatal hypocalcemia.” It occurs most commonly in premature or small-for-gestational-age, low birth weight, or asphyxiated neonates—or in those born to women with gestational or permanent forms of diabetes mellitus. It occurs as a consequence of suppression of PTH secretion, prolonged secretion of calcitonin, and/or hypomagnesemia.20 Total calcium and Ca2⫹e concentrations decline more rapidly and to lower nadir values in preterm than in term neonates. In premature infants, early neonatal hypocalcemia has been attributed to blunting of the physiologic postnatal rise in PTH secretion and to the relative resistance of the renal tubule to PTH-mediated phosphate excretion—leading to hyperphosphatemia. Prolonged elevation of circulating levels of calcitonin also contributes to early neonatal hypocalcemia. In low birth weight (LBW) neonates, hypocalcemia may be further attributed to the rapid accretion of skeletal calcium in the presence of relative resistance to the calcium absorptive and reabsorptive effects of calcitriol on the intestinal tract and bone, respectively. Offspring of severely vitamin-D-deficient mothers may become hypocalcemic shortly after birth. Hypocalcemia develops in approximately 33% of asphyxiated newborns who are products of complicated and compromised deliveries. In these infants, increased phosphate load due to cellular injury, reduced calcium intake, and hypercalcitonemia

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TA B L E 1 7 - 1

Causes of Hypocalcemia I. Neonatal A. Maternal Disorders 1. Diabetes mellitus 2. Toxemia 3. Hyperparathyroidism 4. High intake of alkali or magnesium sulfate B. Neonatal 1. Low birth weight, intrauterine growth retardation 2. Asphyxia, sepsis 3. Hyperbilirubinemia, phototherapy, exchange transfusion 4. Hypomagnesemia, hypermagnesemia 5. Acute/chronic renal insufficiency 6. Nutrients/medications: high dietary phosphate, fatty acids, phytates, bicarbonate, citrated blood, anticonvulsants, aminoglycosides 7. Hypoparathyroidism 8. Vitamin D deficiency or resistance 9. Osteopetrosis type II II Hypoparathyroidism A. Congenital 1. Transient neonatal 2. Dysgenesis/agenesis of the parathyroid glands a. Isolated hypoparathyroidism (GCM2, PTH, SOX3) b. Hypercalciuric hypocalcemia (CASR) c. DiGeorge syndrome (TBX1) d. Sanjad-Sakati syndrome (short stature, retardation, dysmorphism - HRD) Kenny-Caffey syndrome 1 (short stature, medullary stenosis) (TCBE) e. Barakat syndrome (sensorineural deafness, renal dysplasia HDR) (GATA3) f. Lymphedema-hypoparathyroidism-nephropathy, nerve deafness g. Mitochondrial fatty acid disorders (Kearns-Sayre, Pearson, mitochondrial encephalopathy, lactic acidosis, stroke-like MELAS) 3. Insensitivity to parathyroid hormone a. Blomstrand chondrodysplasia (PTHR1) b. Pseudohypoparathyroidism - type IA (GNAS) • Pseudohypoparathyroidism - type IB • Pseudohypoparathyroidism - type IC • Pseudohypoparathyroidism - type II • Pseudopseudohypoparathyroidism c. Hypomagnesemia

4. Dyshormonogenesis B. Acquired 1. Autoimmune polyglandular syndrome - type I (AIRE1) 2. Activating antibodies to the calcium-sensing receptor 3. Post surgical, radiation destruction 4. Infiltrative [excessive iron (hemochromatosis, thalassemia) or copper (Wilson disease) deposition; granulomatous or neoplastic invasion; amyloidosis, sarcoidosis] 5. Maternal hyperparathyroidism 6. Hypomagnesemia III. Vitamin D Deficiency IV. Other Causes of Hypocalcemia A. Calcium Deficiency 1. Nutritional deprivation 2. Hypercalciuria B. Hypomagnesemia/Hypermagnesemia 1. Congenital a. Malabsorption b. Hypermagnesuria • Primary (CLDN16) • Bartter syndrome (3) • Renal tubular acidosis 2. Acquired a. Acute renal failure b. Chronic inflammatory bowel disease, intestinal resection c. Diuretics C. Hyperphosphatemia 1. Renal failure 2. Phosphate administration (intravenous, oral, rectal) 3. Tumor cell lysis 4. Muscle injuries (crush, rhabdomyolysis) D. Miscellaneous 1. Hypoproteinemia 2. Hyperventilation 3. Drugs - furosemide, bisphosphonates calcitonin, anticonvulsants, ketoconazole, anti-neoplastic agents (plicamycin, asparaginase, cisplatinum, cytosine arabinoside, doxorubicin), citrated blood products 4. Hungry bone syndrome 5. Acute and critical illness - sepsis, acute pancreatitis, toxic shock a. Organic acidemia - propionic, methylmalonic, isovaleric

Modified from Thakker RV (2006). Hypocalcemia: Pathogenesis, differential diagnosis, and management. In Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 213–215; from Goltzman D, Cole DEC (2006). Hypoparathyroidism. In Favus MJ (ed). Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 216–219; from Levine MA (2006). Parathyroid hormone resistance syndromes. In Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 220–224; and from Carpenter TO (2006). Neonatal hypocalcemia. In Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 224–229.

are important pathogenetic factors in the development of hypocalcemia. Fifty percent of infants of mothers with diabetes mellitus develop early neonatal hypocalcemia. The incidence may be reduced by strict maternal glycemic control.2,20 Its causes are multifactorial and include reduced placental transfer of calcium due to substantial maternal urinary

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excretion of calcium and magnesium, decreased neonatal secretion of PTH, hypercalcitonemia, hypomagnesemia (occurring in 40% of offspring), and limited intake and impaired absorption of ingested calcium.37 Maternal hypercalcemia due to unsuspected hyperparathyroidism leads to increased transfer of calcium to the fetus, and still further increase in fetal calcium

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

concentrations that suppress fetal PTH synthesis and release and stimulate calcitonin secretion—aberrations in homeostatic mechanisms that persist postpartum and may result in hypocalcemic tetany/seizures in the mother’s offspring. Suppression of PTH secretion may persist for several months and be undetected until symptomatic hypocalcemia develops after weaning of the infant from breast milk to higher-phosphate-containing cow’s milk formula. Maternal ingestion of large quantities of calcium carbonate in antacids has also led to neonatal hypocalcemia.38 Hypocalcemia has occurred in neonates with hyperbilirubinemia undergoing exchange transfusion and in those exposed to phototherapy.39 Neonates with acute rotavirus infection and severe diarrhea may present with hypocalcemic seizures.40 Aminoglycoside antibiotics (e.g., gentamycin) increase urinary excretion of calcium and magnesium and facilitate the development of neonatal hypocalcemia. Compounds that complex with and sequester calcium—such as citrate (present in transfused blood), phosphates (that alter the calcium X phosphate product), and fatty acids (given as caloric supplements)—lower Ca2⫹e levels. Bicarbonate administered to correct acidosis increases calcium binding to albumin and thus lowers Ca2⫹e values. Hypocalcemia may also occur in hyperventilated infants with severe respiratory alkalosis, as well as in those with other causes of metabolic alkalosis. Phytates in soy milk bind calcium and phosphate and interfere with their absorption. Neonates with osteopetrosis type II and impaired osteoclastogenesis may present with early or late neonatal manifestations of hypocalcemia.41,42

Late Neonatal Hypocalcemia Hypocalcemia developing after 72 hours of postnatal age may be due to increased intake of phosphate, hypomagnesemia, hypoparathyroidism, or vitamin D deficiency (Table 17-1). One of the most frequent causes of late neonatal hypocalcemia is excessive phosphate intake in evaporated milk or modified cow’s milk formulas. Phosphate forms poorly soluble calcium salts and limits the intestinal absorption of calcium while raising serum phosphate values. Premature introduction of fibercontaining cereals into the infant’s diet also decreases calcium absorption. Affected infants may have an associated defect in renal phosphate excretion or a coexisting deficiency in vitamin D. Hyperphosphatemia and hypocalcemia may initially suggest hypoparathyroidism, but serum PTH concentrations are high in infants with excessive phosphate loading in response to reciprocal reduction in serum calcium values. Newborns and infants with chronic renal insufficiency due to renal hypoplasia or obstructive nephropathies are often hypocalcemic and hyperphosphatemic, with elevated serum PTH levels as well. However, they are also azotemic. Hypomagnesemia leads to impaired secretion of PTH and decreased peripheral responsiveness to PTH, and may be transient or related to congenital abnormalities of intestinal absorption or renal tubular reabsorption of magnesium.20 Hypermagnesemia may occasionally be associated with neonatal hypocalcemia.

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Hypocalcemia due to fetal/neonatal deficiency of vitamin D occurs in offspring of mothers deprived of vitamin D (for cultural or socioeconomic reasons). Impaired renal 25-hydroxyvitamin D-1␣ hydroxylase activity and loss-of-function mutations of the vitamin D receptor also lead to hypocalcemia (and hypophosphatemia). Hypovitaminosis D may develop in the breastfed infant of a vegetarian mother who shields herself from sunlight and ingests a diet low in vitamin D. However, marginal deficiency of vitamin D in neonates and infants is likely much more common than has been recognized heretofore.8,9,11,43 Late neonatal hypocalcemia occurs in premature infants with osteopenia at 3 to 4 months of age in whom the intake of calcium, phosphate, and vitamin D has been marginal. It is perhaps due to avid deposition of all available calcium into bone.20 Hypocalcemia due to vitamin D deficiency may develop acutely, and in the absence of clinical or radiographic signs of rickets, in the older infant and young child ingesting an elimination diet low in vitamin D because of severe allergies and/or maintained indoors with limited exposure to sunlight.

Hypoparathyroidism Hypoparathyroidism presenting in infancy is most often transient and related to delayed developmental maturation of PTG function. It frequently resolves within the first several weeks of life. When prolonged, hypoparathyroidism is often due to an error in the embryogenesis of the PTGs. Occasionally, it may be due to defects in the synthesis of PTH or to peripheral unresponsiveness to PTH. Hypercalciuric hypocalcemia is a form of autosomaldominant hypoparathyroidism (OMIM 146200) due to gain-of-function mutations in CASR (Table 17-2) that result in enhanced sensitivity to Ca2⫹e. A lowered set-point enables PTH secretion to be suppressed, and renal tubular reabsorption of calcium to be depressed, by extremely low concentrations of Ca2⫹e. This disorder may present in the newborn period.44,45 Mutations may be scattered throughout the gene, but occur predominantly in the extracellular domain of the CaSR. Activating mutations (Cys141Trp) of the CaSR may also inhibit function of the renal outer medullary potassium channel (KCNJ1, OMIM 600359), leading to a Bartterlike syndrome with hypokalemic metabolic alkalosis, hyperreninemia, and hyperaldosteronism as well as hypercalciuric hypocalcemia. The paired metabolic defects are in part responsive to treatment with hydrochlorothiazide and low doses of calcitriol.46 Children with hypercalciuric hypocalcemia due to gain-of-function mutations in CASR are very sensitive to even low doses of calcitriol that can lead to even more marked hypercalciuria and to nephrocalcinosis. Thus, management of these patients has been quite difficult. Administration of recombinant human PTH1-34 (0.7 ␮g/ kg/day) to a 14-month-old hypocalcemic male infant with a de novo nonsense mutation in CASR (Leu727Gln) for 17 months in part restored calcium homeostasis, with increased but still subnormal serum levels of calcium (whereas urinary excretion of calcium decreased into the normal range).47 During treatment, the child was

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TA B L E 1 7 - 2

Genetic Origins of Disorders of Mineral, Cartilage, and Bone Metabolism Gene

Chromosome

OMIM

ACVR1 AIRE1 ALPL

2q23-q24 21q22.3 1p36.1-p34

102576 607358 171760

CA2 CASR

8q22 3q13.3-q21

259730 601199

CLCN5

Xp11.2

300008

CLCN7

16p13

602727

CLDN16 CLDN19

3q27 1p34.2

603959 610036

COL1A1

17q21.31-q22

120150

COL1A2

7q22.1

120160

CRTAP

3p22

605497

CTSK CYP2R1 CYP27B1

601105 608713 609506

DMP1

1q21 11p15.2 12q13.1q13.3 4q21

ELN FGF23

7q13.23 12p13.3

120160 605380

600980

GALNT3

2q24-q31

601756

GATA3

10p13-14

131320

GCM2 GNAS

6p24.2 20q13.2

603716 139320

GNPTAB HNRPA1 HRPT2

12q23.2 12q13.1 1q24-q31

607840 164017 607393

IKBKG

Xq28

300248

KCNJ1 LEPRE1 LRP5

11q24 1p34 11q13.4

600359 610339 603506

MEN1

11q13

131100

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Disease Fibrodysplasia ossificans progressiva Autoimmune polyendocrine syndrome, type I Hypophosphatasia, infantile Hypophosphatasia, childhood Hypophosphatasia, adult Osteopetrosis - renal tubular acidosis Hereditary hypocalciuric hypercalcemia Neonatal severe hyperparathyroidism Hypercalcemic hypercalciuria Hypoparathyroidism, familial isolated Acquired hypocalciuric hypercalcemia X-linked recessive hypophosphatemic rickets Dent disease Nephrolithiasis, X-linked recessive Osteopetrosis, autosomal recessive Osteopetrosis, autosomal dominant Primary hypomagnesemia Hypomagnesemia, hypercalciuria, visual impairment Osteogenesis imperfecta type I Osteogenesis imperfecta type IIA Osteogenesis imperfecta type III Osteogenesis imperfecta type IV Osteogenesis imperfecta type IIA Osteogenesis imperfecta type III Osteogenesis imperfecta type IV Osteogenesis imperfecta type IIB Osteogenesis imperfecta type VII Pycnodysostosis 25-Hydroxylase deficiency, selective 25␣-Hydroxyvitamin D-1␣-hydroxylase deficiency (Vitamin D-dependent rickets, type I) Hypophosphatemic rickets, autosomal recessive Williams-Beuren syndrome Hypophosphatemic rickets, autosomal dominant Familial tumoral calcinosis Hyperostosis hyperphosphatemia syndrome Familial tumoral calcinosis Hyperostosis hyperphosphatemia syndrome Hypoparathyroidism, sensorineural deafness, renal disease (HDR/Barakat syndrome) Hypoparathyroidism, familial isolated Pseudohypoparathyroidism, type 1A Pseudohypoparathyroidism, type 1B Osseous heteroplasia, progressive Fibrous dysplasia/McCune-Albright Mucolipidosis type II Vitamin D-dependent rickets type II Familial hyperparathyroidism 2 jaw tumor syndrome Osteopetrosis, anhidrotic ectodermal dysplasia, lymphedema Antenatal Bartter syndrome type 2 Osteogenesis imperfecta type VIII Osteoporosis-pseudoglioma syndrome Idiopathic juvenile osteoporosis High bone mass variation Van Buchem disease, type 2 Multiple endocrine neoplasia type I

OMIM 135100 240300 241500 241510 146300 259730 145980 239200 601199 146200 145980 300554 300009 310468 259700 166600 248250 248190 166200 166210 259420 166220 166210 259420 166220 610854 610682 265800 600081 264700 241520 194050 193100 211900 610233 211900 610233 146255 146200 103580 603233 166350 174800 252500 600785 145001 300301 600839 610915 259770 259750 601884 607636 131100

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

695

TA B L E 1 7 - 2

Genetic Origins of Disorders of Mineral, Cartilage, and Bone Metabolism—Cont’d Gene

Chromosome

OMIM

NPR2 OSTM1 PHEX

9p21-p12 6q21 Xp22.2-p22.1

108961 607649 300550

PTH

168450

PTHR1

11p15.3p15.1 3p22-p21.1

RET

10q11.2

164761

SLC12A1 SLC34A3

15q15-q21.1 9q34

600839 609826

SLC7A7 SOST

14q11.2 17q12-q21

603593 605740

SOX3 STX16 TBX1 TBCE

Xq26.3 20q13.32 22q11.12 1q42-q43

313430 603666 602054 604934

TGFB1 TCIRG1

190180 604592

TNFRSF11A

19q13.1 11q13.4q13.5 18q22.1

TNFRSF11B TRPM6 VDR

8q24 9q22 12q12-q14

602643 607009 601769

168468

603499

Disease

OMIM

Acromesomelic dysplasia (Maroteaux) Osteopetrosis, autosomal recessive Hypophosphatemic rickets, X-linked dominant Hypoparathyroidism, familial isolated

602875 259700 307800

Blomstrand osteochondrodysplasia Murk-Jansen metaphyseal chondrodysplasia Enchondromatosis (Ollier disease) Multiple endocrine neoplasia type IIA Multiple endocrine neoplasia type IIB Familial medullary carcinoma of thyroid Antenatal Bartter syndrome type I Hypophosphatemic rickets with hypercalciuria, hereditary Lysinuric protein intolerance Sclerosteosis Hyperostosis corticalis generalisata (Van Buchem disease type 1) Hypoparathyroidism, X-linked Pseudohypoparathyroidism, type 1B DiGeorge syndrome Sanjad-Sakati (HRD) syndrome Kenney-Caffey syndrome, type 1 Progressive diaphyseal dysplasia Osteopetrosis, autosomal recessive

215045 156400 166000 171400 162300 155240 601678 241530

Polyostotic osteolytic dysplasia, hereditary (familial) expansile Paget disease, juvenile Familial hypomagnesemia with hypocalcemia Vitamin-D-dependent rickets, type II

174810

146200

222700 269500 239100 307700 603233 188400 241410 244460 131300 259700

239000 602014 277440

See Table 17-12 for genes associated with osteochondrodysplasias.

clinically asymptomatic, did not develop nephrocalcinosis, and tolerated the drug well. The most common form of dysgenesis of the PTGs in neonates and infants is that associated with the DiGeorge syndrome (DGS, OMIM 188400), a disorder that occurs with a frequency of 1:4,000 to 1:8,000 births and is present in approximately 70% of children with isolated hypoparathyroidism.48,49 The DGS is a neurocristopathy, the result of disturbed migration of cervical neural crest cells and consequent maldevelopment of tissues of neural crest origin derived from the third and fourth pharyngeal pouches and first to fifth branchial arches. It is usually associated with microdeletions of chromosome region 22qll.2 (del22q11.2, DGCR) and is thus a contiguous gene syndrome (a disorder caused by deletion of several adjacent genes that when individually mutated may result in a distinctive clinical feature and when collectively lost lead to a group of apparently unrelated clinical findings). Although the clinical severity and phenotype of patients with this chromosomal anomaly are variable, characteristically subjects with DGS have the triad of hypocalcemia due to hypoplasia of the PTGs (often manifest in

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the neonatal period but which may not be detected until an older age), defective T-lymphocyte function and impaired cell-mediated immunity due to partial or complete absence of thymic differentiation (leading to increased frequency of viral and fungal infections), and conotruncal defects of the heart or aortic arch (Tetralogy of Fallot, truncus arteriosus, interrupted or right aortic arch, aberrant right subclavian artery).20,48,50 Indeed, delq2211 has been associated with three overlapping disorders—DGS and the conotruncal anomaly face and velocardiofacial syndromes. Collectively, these syndromes are associated with a quite typical face (ocular hypertelorism, lateral displacement of inner canthi, short palpebral fissures, swollen eyelids, dysmorphic segmented nose, small mouth, lowset ears with abnormally folded pinnae, short philtrum, micrognathia, malar hypoplasia, velopharyngeal insufficiency (with/without cleft palate), olfactory dysfunction, short stature, nonverbal learning disabilities, and various psychological maladies.49,51,52 DGS may occur sporadically or be transmitted as an autosomal-dominant characteristic. Takao velocardiofacial syndrome (included in OMIM 188440) consists primarily of the typical cardiac

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defects described previously, which may also be associated with hypocalcemia. Shprintzen velocardiofacial syndrome (OMIM 192430) is characterized by craniofacial and palatal defects and cardiac anomalies. Cayler cardiofacial syndrome (OMIM 125520) is associated with partial unilateral facial paresis (due to hypoplasia of the depressor angularis muscle) and with anomalies of the heart and aorta. These syndromes have been grouped as the CATCH-22 syndromes of Cardiac defects, Abnormal face, Thymic hypoplasia, Cleft palate, and Hypocalcemia. In addition to the anomalies and clinical findings listed, a litany of abnormalities may be seen in patients with del22q11.2.53 Disparate manifestations of these syndromes may be observed in different members of the same family, indicating the variable clinical expressions that accompany del22q11.2.54 A two-megabase microdeletion at chromosome 22q11.2 leads to loss of several contiguous genes within this region, including HIRA (histone cell cycle regulation, OMIM 600237)—a transcription factor (expressed in developing heart and upper body neural crest elements) necessary to normal cardiac development.55 Also within this region is TBX1 encoding T-BOX-1, a transcription factor with a highly conserved DNA-binding sequence (the T-box) essential for organogenesis and pattern formation and expressed in the pharyngeal arches and pouches. Experimental disruption of Tbx1 impairs development of the pharyngeal arch arterial vasculature, whereas introduction of null mutations in Tbx1 results in anomalies of the cardiac outflow track and hypoplasia of the thymus and PTGs.56 Evaluation of patients with clinical characteristics of the CATCH-22 syndromes but intact 22q11.2 has revealed heterozygous loss-of-function mutations in TBX1 in patients with the DGS (Phe148Tyr, Gly310Ser) and Shprintzen velocardiofacial syndrome.49,57 Thus, haploinsufficiency of TBX1 alone can account for the cardiac defects, abnormal face, thymic and parathyroid hypoplasia, and velopharyngeal insufficiency with cleft palate but not for the developmental delay characteristic of CATCH-22. Another candidate gene for the DGS sited at chromosome 22q11.2 is UFD1L (ubiquitin fusion degradation 1-like, OMIM 601754), whose product is important in the posttranslational processing of proteins and/or their degradation by interaction with the ubiquitin fusion protein. Experimentally, the DGS and related disorders have also been linked to genes encoding endothelin-1, vascular endothelial growth factor, and fibroblast growth factor-8 (a target gene for TBX1)—as well as to genes within the DGCR at chromosome 22q11.2 [CRKL (OMIM 602007) and DGCR6 (OMIM 601279)]. In the mouse hypomorphic for Fgf8, there are cardiovascular, craniofacial, parathyroid, and thymic defects—an experimental phenocopy of the human del22q11.2 syndrome.58 Fgf8 functions through stimulation of transcription of Crkl. Its product is an adaptor protein that transduces intracellular signals from several tyrosine kinase receptors, one of which is the receptor for Fgf8, Interestingly, Fgf8 interacts with Tbx1 as well. The DGS has also been associated with microdeletions of chromosomes 10p13, 18q21.33, and 4q21.2-q25—indicative of the cascade of genes likely involved in the generation of this phenotype.

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There are several other syndromes with multisystem involvement and hypoparathyroidism. The Barakat or HDR syndrome (OMIM 146255) of hypoparathyroidism, sensorineural deafness, and renal disease (steroid-resistant nephrosis with progressive renal failure) has been attributed to haploinsufficiency of GATA3—a zinc-finger transcription factor important in the embryonic development of the PTGs, kidneys, and inner ear and in normal function of the immune system as an essential T-cell receptor enhancer.59 The PTGs of these children are dysgenetic (hypoplastic or absent). Hypocalcemia may be present in the newborn period or unrecognized until later childhood.60 Heterozygous deletions, insertions, and missense and nonsense mutations in GATA3 have been identified in patients and families with HDR.61,62 Interestingly, subjects with isolated loss of GATA3 function do not have other features common to patients with larger terminal deletions of 10p—such as growth and developmental retardation, dysmorphic facial features, and congenital heart disease. Biallelic mutations in the gene encoding tubulinspecific chaperone E (TBCE) have been identified in the Sanjad-Sakati syndrome of hypoparathyroidism mental retardation dysmorphism (HRD, OMIM 241410) and the Kenny-Caffey syndrome type 1 of hypocalcemia, cortical thickening, and medullary stenosis (KCS1, OMIM 244460). Children with HRD are short, developmentally delayed, and seizure prone. They have medullary stenosis and other skeletal anomalies. They are microcephalic, with faces characterized by deeply recessed eyes or microphthalmia, depressed nasal bridge, beaked nose, long philtrum, thin upper vermillion border, micrognathia, and large earlobes. The HRD syndrome often presents in infancy with symptomatic hypocalcemia associated with low serum concentrations of PTH and normal phosphaturic response to exogenous PTH. The cardiovascular system of these patients is intact, but as infants they are susceptible to life-threatening pneumococcal infections.63 Neonates with KCS1 are severely hypocalcemic early in the neonatal period. As children they are short, with craniofacial anomalies (due to absence of diploic space in the skull), osteosclerosis, and thickening of the cortices of the long bones with narrowing of the medullary compartment and normal or mildly delayed development. They too are susceptible to recurrent bacterial infections. TBCE is essential for formation of microtubules—cytosolic structures composed of heterodimeric ␣- and ␤-tubulin subunits that form the cytoskeleton, mitotic apparatus, cilia, and other cellular components. This chaperonin assists in the correct folding of ␣- and ␤-tubulin subunits and the formation of ␣- ␤tubulin heterodimers. The ␣- and ␤-tubulin subunits and TCBE are necessary to normal embryogenesis of the PTGs. Mutations in TCBE result in lowered microtubule formation and consequently in decrease in subcellular components such as the golgi apparatus and endosomal compartments required for normal intracellular movement of proteins. Interestingly, the identical mutation in TCBE (a homozygous 12 bp deletion in exon 2) may result in the HRD or KCS1 phenotype in a specific family.64 A child with autosomal-recessive HRD syndrome and intact TCBE has been identified, suggesting

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that this disorder is likely to be genetically heterogeneous.65 In KCS type 2 (OMIM 127000), the phenotype is similar to that of KCS1 but transmission is as an autosomal-dominant trait for which no gene mutation has been identified to date. Familial isolated congenital or later-onset hypoparathyroidism may be transmitted as an autosomal-recessive, autosomal-dominant, or X-linked recessive trait. It has been associated with loss-of-function mutations of GCM2, PTH, and possibly SOX3, and gain-of-function mutations in CaSR. GCM2 encodes a 506-aa DNA-binding protein whose expression is restricted to the PTGs. Knockout of Gcm2 in mice leads to agenesis of the PTGs and hypoparathyroidism. Biallelic intragenic deletion or homozygous missense mutations of GCM2 result in hypoparathyroidism in humans.66-68 Homozygous loss-of-function mutations in PTH have been detected in neonates with autosomal-recessive familial isolated hypoparathyroidism. In one family, substitution of proline for serine at the –3 position of the signal peptide of prepro-PTH likely prevented its normal post-translational processing and accelerated protein product degradation within the endoplasmic reticulum.69 As previous described, hypoparathyroidism due to a heterozygous activating mutation in CASR have been found in a hypocalcemic neonate. One mutation (Phe806Ser) occurred in the sixth transmembrane domain of this G-protein–coupled receptor (GPCR) near its site of interaction with Gs␣.44 X-linked hypoparathyroidism (OMIM 307700) is associated with agenesis of the PTGs. The disorder has been mapped to Xq27 and may involve a deletion-insertion mutation that adversely affects the position of SOX3.70 Because Sox3 is expressed in embryonic mouse PTGs, it is likely an important transcription for normal embryologic development of the PTGs. In the neonate with Blomstrand osteochondrodysplasia (OMIM 215045), hypocalcemia is secondary to loss-offunction mutations in PTHR1 and hence insensitivity to the calcemic effects of PTH.71 Transmitted as an autosomalrecessive trait, its clinical characteristics include polyhydramnios, hydrops fetalis, short-limbed dwarfism, facial anomalies, aberrant tooth development, aplasia of the nipples and breasts, hypoplastic lungs, and preductal aortic coarctation. Serum concentrations of PTH are elevated. Skeletal maturation is advanced. Histologically, the proliferative zone of the cartilage growth plate is narrowed— with relatively few resting and proliferating chondrocytes— whereas the hypertrophic zone is composed of irregular columns of chondrocytes. Although Blomstrand osteochondodysplasia has been lethal to date, skeletal malformations may be more (type I) or less severe (type II). Mutations in PTHR1 that result in complete absence of product (e.g., Arg104Ter) lead to type I, whereas mutations that permit some PTHR1 synthesis (Pro132Leu) result in type II.72 Loss-of-function mutations in GNAS, the gene encoding the G␣s subunit of the activating G protein, lead to PTH insensitivity and pseudohypoparathyroidism (PHP)—a disorder that may be suspected in the neonate with hypocalcemia in whom hyperthyrotropinemia has been detected in the neonatal screening study for congenital hypothyroidism.73 As a

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consequence of the loss-of-function mutation in GNAS in the thyroid gland, the G␣s subunit required by the thyrotropin GPCR is also impaired. In some neonates with elevated serum concentrations of PTH and phosphate and blunted phosphaturic response to PTH, neonatal pseudohypoparathyroidism is transient and resolves within the first few months of life.74 Abnormalities of the mitochondrial genome have also been associated with hypoparathyroidism. In addition, patients with the Kearns-Sayre syndrome (OMIM 530000) manifest ophthalmoplegia, retinal pigmentation, and cardiomyopathy. Point mutations, duplications, and deletions of various length of the mitochondrial genome (16,569 bp) have been found in these patients. Hypoparathyroidism has been observed in patients with the syndrome of mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and the mitochondrial trifunctional protein deficiency syndrome (MTPDS)—a disorder of fatty acid oxidation.71,75

Evaluation and Management Evaluation of the neonate with hypocalcemia begins with review of the maternal, gestational, peripartum, postnatal, and family histories and physical examination (Figure 17-5). Historical data include those related to maternal parity and complications of pregnancy such as maternal diabetes mellitus (gestational, types I or II), toxemia of pregnancy or ingestion of excessive alkali, or abnormalities of delivery, low birth weight, neonatal sepsis, or other early postpartum illnesses. The family history is examined for members with abnormalities of mineral metabolism such as renal calculi, rickets, or hypocalcemia (e.g., seizure disorders). The social history provides information about the socioeconomic status of the mother and her cultural beliefs that may have impacted on maternal diet and exposure to sunlight during gestation. The physical examination (abnormal face, cardiac murmur) may suggest a complex form of hypocalcemia. Determination of a complete blood count; serum concentrations of total calcium, Ca2⫹e, magnesium, phosphate, creatinine, intact PTH, calcidiol, and calcitriol; and urinary calcium and creatinine concentrations in a spot urine should precede initial therapy of the hypocalcemic newborn whenever possible. Decreased serum concentrations of PTH are common in neonates with early-onset hypocalcemia, but persistently low PTH levels suggest impaired PTH secretion. High PTH concentrations are present in patients with vitamin D deficiency or insensitivity, PTH resistance due to loss-of-function mutations in PTHR1 or GNAS, or impaired renal function. Low levels of calcidiol signify decreased maternal (and hence fetal) vitamin D stores or rarely a defect in vitamin D-25 hydroxylase, whereas calcitriol concentrations are inappropriately low in subjects with severely compromised renal function, hypoparathyroidism, or deficiency of 25OHD-1␣-hydroxylase. Elevated calcitriol values suggest vitamin D resistance due to an abnormality in VDR, a disorder that may be associated with alopecia. Skeletal radiographs may disclose osteopenia, whereas chest X-ray may not identify a thymic shadow (an unreliable sign in a severely ill or

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History Physical Examination ↓ Serum Ca, Ca2⫹e

Urine Ca Activating mutation of CASR or Primary hypomagnesemia iPTH

↓MG

Serum Phos

Hypomagnesemia

250HD

Creatinine

Renal failure

Normal

Dietary/D deficiency Malabsorption

Normal

Anticonvulsants

Hypoparathyroidism

CYP27B1 (M)

GNAS (M)

Pseudohypoparathyroidism

Normal

PTH (M)

PTHR1 (M)

Dyshormonogenesis

↑1,25(OH)2D3

VDR (M)

Excessive phosphate load (intake, cell lysis)

PTH resistance

Figure 17-5 Evaluation of hypocalcemia. Abbreviations: Ca (serum total calcium), Ca2⫹e (extracellular ionized calcium), Phos (serum phosphate), Mg (magnesium), PTH (parathyroid hormone), 25OHD [25-hydroxyvitamin D (calcidiol)], 1, 25 (OH)2 D3 [1, 25-dihydroxy vitamin D3 (calcitriol)] PTHR1 (PTH receptor), VDR (vitamin D receptor), D (vitamin D), and M (mutation).

stressed neonate). Serum levels of calcium, Ca2⫹e, phosphate, and intact PTH should be measured in the mothers of neonates with unexplained hypocalcemia. In neonates with hypocalcemia not otherwise explained, evaluation for possible DGS should be undertaken—particularly when physical examination reveals an abnormal face and a congenital anomaly of the outflow tract of the heart. The white blood and T (CD4) lymphocyte counts are low in DGS, and the thymic shadow often absent. The diagnosis of the DGS is confirmed by the presence of a microdeletion of chromosome 22q11.2 as demonstrated by fluorescent in situ hybridization (FISH). Occasionally, sequence analysis of TBX1 may be needed to establish this diagnosis if the FISH study is normal. Because the DGS may be heritable, examination of the karyotype of the parents of a DGS infant is indicated (as well as those of the siblings if the parent also has delq22.11). It should be noted that the majority of neonates and infants with DGS are recognized primarily because of cardiac anomalies and that subjects without

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these lesions may not be identified until mid or late childhood or adolescence.76 Neonates with PHP type I usually do not have the characteristic skeletal phenotype (brachymetacarpals) of Albright hereditary osteodystrophy, but if this diagnosis is suspected analysis of GNAS is indicated. Early neonatal hypocalcemia is often asymptomatic, but nevertheless treatment is indicated when the total serum calcium concentration is below 6 mg/dL in the preterm infant and less than 7 mg/dL in the term infant.20 Asymptomatic neonates are most easily managed by increasing the oral intake of calcium and establishing an overall ratio of calcium:phosphate intake of 4:1 (including that in feedings with a low phosphate formula such as Similac PM 60/40R, calcium:phosphate ratio 1.6:1), with calcium glubionate or calcium carbonate administered in divided doses every 4 to 6 hours (Table 17-3). Eucalcemia is almost always restored in these subjects within 3 weeks after birth, and often earlier. In the hypocalcemic infant with tetany or frank seizures, 10% calcium gluconate (elemental calcium 9.3 mg/mL) at a dose of

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699

TA B L E 1 7 - 3

Preparations of Calcitriol, Calcium, Magnesium, and Phosphate Medication Vitamin D Vitamin D Calcidiol Calcitriol Dihydrotachysterol Calcium Calcium gluconate (iv) Calcium glubionate (solution) Calcium carbonate Calcium carbonate (suspension) Calcium citrate Magnesium Magnesium sulfate (50% im soln) Magnesium oxide Mg gluconate Magnesium chloride Phosphorus Sodium phosphate (phospha-soda) Sodium/potassium phosphate (Neutraphos) Potassium phosphate (Neutraphos-K) Potassium phosphate (K-Phos Original) Sodium/Potassium phosphate (K-Phos MF) Sodium/Potassium phosphate (K-Phos #2) Sodium/Potassium phosphate (K-Phos Neutral)

Content

Elemental Mineral

8,000 IU/mL 25,000 or 50,000 IU/tablet 20 or 50 ␮g/tablet 1 ␮g/mL 0.25 ␮g or 0.5 ␮g/casule 0.2 ␮g/5 mL 01.25, 0.2, 0.4 mg/tablet 93 mg/g 64 mg/g 400 mg/g 1,250 mg/5 mL 210 mg/g

93 mg/10 mL 115 mg/5 mL 500 mg/tablet 500 mg/5 mL 200 mg/tablet

603 mg/g 54 mg/g 120 mg/g

49 mg/mL 241 mg/tablet 27 mg/tablet 64 mg/tablet 127 mg/mL 250 mg/packet (powder) 250 mg/packet (powder) 114 mg/tablet 126 mg/coated tablet 250 mg/coated tablet 250 mg/tablet

Compiled from Alon US (2006). Hypophosphatemic vitamin D-resistant rickets. In Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 342–345; from Bringhurst FR, et al. (2003). Hormones and disorders of mineral metabolism. In Larsen PR, Kronenberg HM, Melmed S, Plonsky KS (eds.), Williams textbook of endocrinology, Tenth edition. Philadelphia: Saunders/ Elsevier 1303–1371; from Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 496; and from Levine B-S, Carpenter TO (1999). Evaluation and treatment of heritable forms of rickets. The Endocrinologist 9:358–365.

1 to 3 mg/kg and rate of less than 1 mL/minute and a total dose not to exceed 20 mg of elemental calcium/kg may be administered by intravenous infusion. Often seizures will cease after 1 to 3 mL of 10% calcium gluconate has been administered.20,22 Cardiac rate and rhythm must be carefully monitored to prevent bradycardia and asystole. Further intravenous bolus doses of calcium (⬃10 mg/ kg at 6-hour intervals) should be used sparingly because they result in wide excursions in serum calcium values. These infants too should receive supplemental oral calcium. Depending on the cause of the hypocalcemia, supplemental vitamin D or calcitriol may also be needed. Serum and urine calcium and creatinine levels should be determined frequently, and treatment modified to maintain eucalcemia and the urine calcium/creatinine ratio ⬍0.2 in an effort to avoid iatrogenic hypercalcemia, hypercalciuria, nephrocalcinosis, and renal insufficiency. After restoration of eucalcemia in the infant with DGS, the other components of this disorder must be addressed. Cardiac anomalies usually require surgical correction, as do palatal clefts. In DGS infants with immunocompromise and recurrent infections due to thymic aplasia, ap-

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propriate anti-infectious therapy is mandatory. Transplantation of fetal or cultured postnatal thymic tissue, bone marrow, or peripheral blood mononuclear cells has restored immune function in infants with the DGS.77,78 Supplemental calcitriol (20–60 ng/kg/day) and calcium are necessary for restoration and maintenance of eucalcemia in infants with hypoparathyroidism. Poor growth due to feeding difficulties and learning disabilities due to developmental delay must be managed on an individual basis and illustrate the need for a multidisciplinary approach to the care of DGS patients.76 Hypocalcemia due to hypomagnesemia is managed acutely by the intravenous infusion or intramuscular injection of 50% magnesium sulfate at a dose of 0.1 to 0.2 mL/kg while monitoring cardiac status.

HYPERCALCEMIA Although hypercalcemia in neonates and very young infants is defined as total blood calcium concentration ⬎10.8 to 11.3 mg/dL (depending on the analytical laboratory), substantial symptoms (anorexia, gastroesophageal

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reflux and emesis, constipation, lethargy and hypotonia, or irritability and seizures) usually do not occur until the total calcium level exceeds 12 to 13 mg/dL.22,79 These infants frequently have polyuria, and may become dehydrated due to renal resistance to antidiuretic hormone if oral fluid intake is restricted. Due to the vasoconstrictive effect of calcium, the hypercalcemic infant may be hypertensive. Hypercalcemia also shortens the S-T segment and can lead to heart block and ultimately to asystole. In older infants and young children with chronic hypercalcemia, poor growth and failure to thrive are often presenting manifestations. Hypercalcemia also leads to hypercalciuria and the complications of nephrocalcinosis and nephrolithiasis.

Etiology Neonatal/infantile hypercalcemia may be iatrogenic in origin (e.g., administration of excessive calcium or vitamin D) at times in the breast milk of mothers ingesting large amounts of cholecalciferol. It may also be due to maternal ingestion of thiazide diuretics that increase renal tubular absorption of calcium or purposeful restriction of phosphate that leads to hypophosphatemia and reciprocal increase in serum calcium concentrations as hypophosphatemia stimulate renal tubular 25OHD-1␣-hydroxylase activity, calcitriol synthesis, and increased intestinal absorption of calcium (Table 17-4). Hypercalcemia, hypophosphatemia, hyperphosphatasemia, and radiographic evidence of rickets develop in premature infants receiving intravenous alimentation deficient in phosphate or in those fed only human breast milk—the phosphate content of which is low. The problem may be prevented or treated by increasing the amount of parenteral phosphate administered to the extent possible or by the use of breast milk fortified with phosphate.80 Adequate extrauterine mineralization of the preterm skeleton requires intakes of calcium and phosphate of approximately 200 mg/kg/day. Extracorporeal life support may also be associated with hypercalcemia in neonates.81 Hypervitaminosis D may be due to prolonged feeding of an improperly prepared formula or commercial dairy milk containing excessive vitamin D; iatrogenic prescription of vitamin D, calcidiol, or calcitriol; or increased endogenous production of calcitriol from inflammatory sites.79 In infants with severe birth trauma or perinatal asphyxia, subcutaneous fat necrosis may develop in tissues that have sustained direct trauma and be manifested by indurated extremely firm violaceous nodules on the cheeks, trunk, buttocks, and legs.22 Hypercalcemia may be present when the lesions first appear or develop as the nodules resolve several weeks later. Histologically, the skin lesions are composed of adipocytes, an inflammatory lymphohistiocytic infiltrate, and multinucleated giant cells in a bed of calcium crystals. The hypercalcemia of subcutaneous fat necrosis is attributable not only to reabsorption of precipitated calcium but to extrarenal synthesis of calcitriol by local macrophages and resultant hyperabsorption of ingested calcium. The 25OHD-1␣-hydroxylase activity of these inflammatory macrophages is not under the control of PTH, calcium, or phosphate levels but is suppressible by glucocorticoids.22

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TA B L E 1 7 - 4

Causes of Hypercalcemia I. Neonate/Infant A. Maternal Disorders 1. Excessive vitamin D ingestion, hypoparathyroidism, pseudohypoparathyroidism B. Neonate/Infant 1. Excessive intake of calcium or vitamin D 2. Phosphate depletion 3. Subcutaneous fat necrosis 4. Williams-Beuren syndrome 5. Familial hypocalciuric hypercalcemia/neonatal severe hyperparathyroidism 6. Metaphyseal chondrodysplasia, Jansen type 7. Persistent parathyroid hormone related protein 8. Bartter syndrome with excessive production of prostaglandin E 9. Lactase/disaccharidase deficiency 10. Infantile hypophosphatasia 11. Mucolipidosis type II 12. Post bone marrow transplantation for osteopetrosis 13. Idiopathic 14. Endocrinopathies: primary adrenal insufficiency, severe congenital hypothyroidism, hyperthyroidism II. Hyperparathyroidism A. Sporadic B. Familial 1. Neonatal severe hyperparathyroidism (CASR) 2. Multiple endocrine neoplasia, type I (MEN1) 3. Multiple endocrine neoplasia, type Iia (RET) 4. McCune-Albright syndrome (GNAS) 5. Familial hyperparathyroidism 2 -jaw tumor syndrome (HRPT2) 6. Jansen’s metaphyseal dysplasia (PTHR1) C. Secondary/Tertiary 1. Postrenal transplantation 2. Chronic hyperphosphatemia D. Ectopic Production of PTHrP III. Familial Hypocalciuric Hypercalcemia (CASR) A. Loss-of-Function Mutations in CASR B. Inhibitory Autoantibodies to the Calcium-Sensing Receptor IV. Excessive Intake of Calcium or Vitamin D 1. Nutritional - milk-alkali syndrome 2. Exogenous ingestion (vitamin D) or topical application (calcitriol or analog) 3. Ectopic production of calcitriol associated with granulomatous diseases: sarcoidosis, inflammatory bowel disease; tuberculosis, histoplasmosis, coccidioidomycosis, leprosy; human immunodeficiency virus; neoplasia: lymphoma, dysgerminoma V. Immobilization VI. Other Causes A. Neoplasia: Osseous metastases, production of PTHrP, cytokines/osteoclast activating factors B. Hypophosphatasia C. Drugs: thiazide diuretics, lithium, vitamin A and analogs, calcium, alkali, anti-estrogens, aminophylline D. Total parenteral nutrition E. Endocrinopathies: hyperthyroidism, hypoadrenocorticism, pheochromocytoma F. Vasoactive intestinal polypeptide-secreting tumor G. Acute or chronic renal failure/administration of aluminum H. Juvenile rheumatoid arthritis, cytokine mediated

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Excessive production of prostaglandin E and interleukins (IL) -1 and -6 further contributes to hypercalcemia in this disorder by increasing the rate of bone turnover.82 Hypercalcemia attributable to subcutaneous fat necrosis is managed by the ingestion of a low-calcium formula, avoidance of vitamin D, and administration of fluids, furosemide, calcitonin, glucocorticoids, or bisphosphonate (etidronate, pamidronate) as needed.83 Hypercalcemia due to subcutaneous fat necrosis has also been observed in older children with major trauma or disseminated varicella.79 Congenital lactase deficiency has been associated with infantile hypercalcemia, perhaps due to increased intestinal absorption of calcium directly promoted by lactose. Neonatal severe hyperparathyroidism (NSHPT) is a potentially lethal form of familial (hereditary) hypocalciuric hypercalcemia (HHC), with very high serum calcium concentrations. NSHPT is most commonly due to homozygous or compound heterozygous inactivating mutations of CASR that greatly increase the serum concentration of Ca2⫹e needed to suppress PTH synthesis and secretion. However, in several infants with NSHPT there have been heterozygous inactivating mutations of CASR (e.g., Arg185Gln, Arg227Leu)—suggesting that the products of these mutations possibly exert a dominantnegative effect on the normal allele, perhaps by interfering with migration of wt receptors to the cell surface or inactivation of wt receptor by linking to the mutated CaSR once embedded in the cell membrane, or by sequestration of G-proteins.84 In addition, the heterozygous fetus who has inherited only one inactivated CaSR allele from an affected father and is delivered by a normal mother may have been relatively hypocalcemic in utero, leading to hyperplasia of the fetal PTGs that persists after birth. Conversely, homozygous mutations near the amino terminal of CASR (e.g., Leu13Pro) may not become manifest until mid childhood or even adulthood.85,86 The clinical spectrum of NSHPT ranges from mild—(constipation, polyuria) with calcium concentrations ranging from 12 to 13 mg/dL—to severe and life-threatening (dysrhythmia, respiratory distress due to hypotonia, demineralization and fractures of the ribs) when calcium levels exceed 15 mg/dL.87 NSHPT may present within the first few days of life to several months of age, depending on the degree of hypercalcemia. Search of the family history may identify members with autosomal-dominant HHC1. The serum calcium concentration is usually markedly elevated (⬎14–17 mg/dL), as is the PTH value. There are hypermagnesemia, normal to low serum phosphate levels, hyperphosphatasemia, elevated calcitriol values, low renal tubular reabsorption of phosphate, and relative hypocalciuria. Radiographically, osteopenia, metaphyseal widening and irregularity, subperiosteal resorption, and varus angulation of the hips may be seen in response to the bone resorptive effects of PTH. Treatment of NSHPT includes induction of sodium diuresis (fluids, furosemide) in order to increase urinary calcium excretion and intravenous administration of a bisphosphonate (pamidronate, zoledronic acid) to acutely lower serum calcium values. Calcimimetic drugs that act directly on the CaSR have also been effective in lowering

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serum calcium values in infants with NSHPT.88,89 Parathyroidectomy may be a requisite life-saving measure at times. Children with NSHPT who remain hypercalcemic are anorectic, fail to thrive, and are at risk for developmental delay.22 Secondary hyperparathyroidism in the neonate may be the result of maternal hypocalcemia due to hypoparathyroidism or PHP.22 Maternal hypocalcemia reduces placental transport and net delivery of calcium to the fetus, resulting in relative fetal hypocalcemia and leading to hyperplasia of the fetal PTGs and secondary hyperparathyroidism proportional to the maternal calcium deficit. Although 25% of infants of hypocalcemic mothers are hypercalcemic, most have skeletal changes that reflect PTH excess that vary from severe demineralization with fractures to osteopenia detectable only by absorptiometry. Secondary hyperparathyroidism usually resolves within a few weeks after birth as the infant ingests adequate calcium and phosphate. Another cause of neonatal secondary hyperparathyroidism is mucolipidosis type II (OMIM 252500). This Hurler-like disorder is characterized by facial abnormalities (asymmetry, flat nasal bridge), hepatosplenomegaly, skeletal deformities (dysostosis multiplex), and developmental delay and is due to inactivating mutations in a gene (GNPTAB- N-acetyglucosamine-1-phosphotransferase, ␣/␤ subunits) encoding a phosphotransferase required for synthesis of mannose 6-phosphate.90 In this disease, maternal calcium concentrations are normal but placental histology is abnormal—suggesting impaired placental transport of calcium and fetal hypocalcemia, leading to compensatory increase in in utero PTH generation. In turn, skeletal evidence of PTH excess (osteopenia, fractures) develops. Secondary hyperparathyroidism and its adverse effects remit within the first several weeks to months after birth. Murk-Jansen metaphyseal chondrodysplasia (OMIM 156400) is an autosomal-dominant chondrodystrophy associated with marked hypercalcemia as a consequence of heterozygous mutations that lead to constitutive ligandindependent activation of PTHR1 expressed in the kidney, bone, and growth plate chondrocytes.91 Phenotypically, there are marked short-limbed dwarfism; deformities of the long bones, digits, spine, and pelvis; choanal atresia; highly arched palate; micrognathia; widely open cranial sutures (in infancy); sclerosis of the basal cranial bones; disorganization of the metaphyses (delayed chondrocyte differentiation, irregularly calcified cartilage protruding into the diaphysis); and excessive loss of cortical bone but normal trabecular bone. Interestingly, birth length and physical appearance are usually normal in these neonates—although radiographic evidence of the chondrodysplasia is present. In affected neonates and infants, there are hypercalcemia, hypophosphatemia, increased serum concentrations of calcitriol, and elevated urinary excretion of nephrogenous cyclic adenosine monophosphate but low or undetectable serum levels of PTH and PTHrP. Constitutively activating mutations of PTHR1 in these subjects include His223Arg at the junction of the first intracellular loop and second transmembrane domain and Thr410Pro in the sixth transmembrane domain—sites specifically

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important in conferring ligand-independent activity upon PTHR1.79,92 Excessive secretion of PTHrP and resultant hypercalcemia have been observed in infants with neonatal iron storage disease and embryonal renal tumors (Wilms’, mesoblastic nephroma). In these infants, intravenous pamidronate (0.5 mg/kg in 30 mL of normal saline over 4 hours repeated once or twice) has restored eucalcemia.93 The Williams-Beuren syndrome (OMIM 194050) is characterized by intrauterine and postnatal growth retardation; hypercalcemia in infancy in 15% of patients that usually resolves by 1 year of age but may occasionally persist into adulthood; consistent hypercalciuria; supravalvular aortic stenosis in 30% of subjects; stenoses of the pulmonary, renal, mesenteric, and celiac arteries; microcephaly; elfin face (epicanthal folds, stellate iris pattern, esotropia, short nose with full nasal tip, arched upper and prominent lower lips, long philtrum, full cheeks with flattened malar eminences, dental malocclusion); hoarse voice; radioulnar synostosis; renal hypoplasia or unilateral agenesis; hypertension; and developmental delay (poor visual-motor integration, attention deficit disorder, IQ 20–106).22 Although most patients with the Williams-Beuren syndrome are developmentally challenged, they have unique and proficient verbal skills with a large vocabulary and enhanced auditory memory (particularly for names), adept social language skills, and exceptional musical aptitude—including the ability to memorize and sing many musical compositions and play many instruments.79 The pathophysiology of hypercalcemia in this syndrome is unknown. No consistent abnormalities of vitamin D metabolism or of PTH or calcitonin secretion have been found in patients with the Williams-Beuren syndrome. Interestingly, many patients with this disorder have hypoplasia of the thyroid gland and elevated serum concentrations of thyrotropin. However, the significance of these findings to the pathophysiology of the syndrome itself is unclear.94 The Williams-Beuren syndrome may be transmitted as an autosomal-dominant trait. It has been localized to chromosome 7q11.23, where microdeletions of 0.9 to 2.5 Mb and hemizygous loss of perhaps as many as 17 contiguous genes combine to produce the syndromic phenotype. The microdeletions may arise in the maternal or paternal seventh chromosome. Hemizygous deletions of ELN have been found in more than 90% of patients with the Williams-Beuren syndrome and likely account for the abnormalities of vascular connective tissue present in these subjects, but not the other manifestations of this disorder. Singular deletion and intragenic loss-of-function mutations of ELN lead to isolated supravalvular aortic stenosis without other features of the Williams-Beuren syndrome. Hemizygosity for ELN leads to compensatory increase in the number of rings of smooth muscle and elastic lamellae, resulting in arterial thickening and increased risk of obstruction.95 Additional genes at chromosome 7q11.23 whose haploinsufficiency may be responsible for aspects of the Williams-Beuren phenotype include RFC2 (OMIM 600404, encoding a subunit of a DNA replication factor whose loss might account for disturbed growth), LIMK1

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(OMIM 601329, encoding LIM kinase-1, a protein expressed in the brain that may be important in visualspatial constructive cognition), GTF2I (OMIM 601679, a constituent of a growth factor signal transduction pathway whose hemizygous loss may contribute to developmental delay), GTF2IRD1 (OMIM 604318, a transcription factor whose heterozygous loss may lead to abnormalities of neurologic development and craniofacial and somatic growth). CYLN2 (OMIM 603432) is a cytoplasmic protein that links membranous organelles and microtubules and whose haploinsufficiency results in mild growth retardation and neural defects.96 Microdeletions of chromosome 7q11.23 arise by unequal crossing over of chromosomal segments between homologous seventh chromosomes during meiosis or by intrachromosomal recombination. The Williams-Beuren phenotype has also been associated with interstitial deletion of chromosome 6q22.2q23 as well as defects in chromosomes 4, 11, and 22, implying that the syndrome is quite genetically heterogeneous (involving a number of genetic cascade systems). The diagnosis of the Williams-Beuren syndrome is suspected on the basis of the characteristic clinical phenotype (with/without hypercalcemia) and confirmed by demonstration of the microdeletion at chromosome 7q11.23 or of ELN by FISH, although a normal chromosome analysis does not entirely eliminate this diagnosis. Hypercalcemia is managed by ingestion of a low-calcium, vitamin-D-free formula. Occasionally, short-term glucocorticoid therapy may be necessary to restore eucalcemia. Idiopathic infantile hypercalcemia is clinically similar to the Williams-Beuren syndrome (hypertension, hyperacusis, strabismus, radioulnar synostosis), and the distinction between the two entities is sometimes difficult.79 Prolonged hypercalcemia and elevated serum concentrations of the amino-terminal fragment of PTHrP have been recorded in some children with this disorder in the absence of a neoplasm, but in the majority of patients the pathophysiology of this disorder is not known. In most patients, hypercalcemia resolves within the first several years of life (but may persist to older ages). Avoidance of vitamin D, low dietary calcium intake, and glucocorticoids to reduce intestinal absorption of calcium are therapeutic modalities for this disorder. Antenatal Bartter syndromes, type 1 (OMIM 601678) and type 2 (OMIM 600839), are quite similar clinically and biochemically and are due to loss-of-function mutations in genes controlling transepithelial transport of chloride and potassium (respectively) across the renal tubular thick ascending limb of the loop of Henle (TALH). Affected fetuses develop polyhydramnios, leading to premature delivery with postnatal salt wasting, hypokalemic metabolic alkalosis, hypercalciuria (and occasionally hypercalcemia), failure to thrive, and often death. The type 1 disorder is due to inactivating mutations of the gene encoding sodium/potassium/chloride cotransporter2 (SLC12A1)—the mediator of active reabsorption of sodium chloride in the TALH. The type 2 syndrome is due to an inactivating mutation in the gene encoding an inwardly rectifying potassium channel (KCNJ1).97 Hypokalemia (occasionally transient hyperkalemia in the type 2 syndrome), reduced intravascular volume, and

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increased levels of angiotensin sum to increase renal and systemic production of prostaglandin E2 that further inhibits sodium and chloride reabsorption in the TALH and enhances juxtaglomerular renin release. Hypochloremic, hypokalemic alkalosis, hyperprostaglandin E, hypercalciuria leading to nephrocalcinosis and osteopenia, and hypercalcemia suggest neonatal Bartter syndrome. Replacement of fluid and electrolytes and administration of potassium-sparing diuretics and the cyclooxygenase inhibitors (indomethacin or a specific inhibitor of cyclooxygenase type 2) are usually effective in ameliorating the biochemical and clinical manifestations of the disease. Perinatal hypophosphatasia is a lethal disorder manifested during gestation by marked skeletal hypomineralization. At times only the base of the skull may be calcified. Usually, the calvarium and vertebrae are partially mineralized, rachitic changes are present at the distal ends of the long bones, and fractures are common.98 Infantile hypophosphatasia (OMIM 241500) is an often fatal autosomal-recessive disorder recognized clinically by 6 months of age and characterized by rickets, demineralization of the calvarium and peripheral skeleton, increased intracranial pressure, flail chest, hypercalcemia, and hypercalciuria. Radiographically, spurs of cartilage and bone extend from the sides of the knee and elbow joints. The disease is due to defective osteoblast synthesis of tissue non-specific alkaline phosphatase because of loss-of-function missense, nonsense, and donor splice site mutations of ALPL. The lethal homozygous or compound heterozygous mutations of ALPL are located within or near the enzyme domain and/or the homodimer and tetramer interfaces.99,100 Decreased alkaline phosphatase activity leads to a deficit in phosphate ions for combination with calcium at the site of hydroxyapatite formation, whereas continued intestinal calcium absorption results in hypercalcemia. Inappropriately low serum bone alkaline phosphatase activity differentiates this condition from other rachitic states in which alkaline phosphatase activity is usually elevated. Increased urine phosphoethanolamine and serum inorganic pyrophosphate and pyridoxal-5’-phosphate values are consistent with the diagnosis of hypophosphatasia, whereas analysis of ALPL identifies the gene mutation(s) itself. Hypophosphatasia can be diagnosed prenatally by ALPL genotyping.98 The hypercalcemia of infantile hypophosphatasia is managed by hydration, diuretics that act at the TALH (e.g., furosemide), and administration of bisphosphonates (pamidronate), calcitonin, or glucocorticoids as necessary. Dietary calcium intake should be restricted, and vitamin D and its metabolites avoided. Bone marrow transplantation and stem cell boosts of transfused donor osteoblasts have also been used to treat affected patients.

Evaluation and Management After completing the historical review (during which the family history is explored for members with mineral disorders and the patient’s intake of calcium, phosphate, and vitamin D are estimated) and physical examination (searching for the facial and cardiovascular signs of

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Williams-Beuren syndrome, the subcutaneous nodules associated with subcutaneous fat necrosis, or the deformities of metaphyseal chondrodysplasia and infantile hypophosphatasia), evaluation of the hypercalcemic neonate and infant continues with total calcium and Ca2⫹e, phosphate, alkaline phosphatase, PTH, calcidiol, and calcitriol measurements as indicated and appropriate (Figure 17-6). In infants with suspected Williams-Beuren syndrome, FISH analysis of chromosome 7q11.23 or for ELN should be undertaken. Treatment of hypercalcemia in neonates and infants must be directed to its cause and severity. Use of a formula low in calcium and avoidance of vitamin D (excessive intake or sunlight) are helpful in the majority of neonates with modest hypercalcemia.22 Significantly elevated serum calcium levels that must be rapidly decreased may require infusion of 0.9% sodium chloride (10–20 mL/kg over 1 hour), followed by an intravenous bolus injection of furosemide (1–2 mg/kg) when adequate urine flow has been established. Hydrocortisone (l mg/kg intravenously every 6 hours) reduces intestinal calcium absorption, and salmon calcitonin (10 units/kg subcutaneously) inhibits calcium mobilization from bone. Currently, bisphosphonates (analogs of pyrophosphate that adsorb to the surface of hydroxyapatite crystals in bone and inhibit osteoclast function and bone resorption) are the agents of choice for the treatment of

History Physical Examination ↑ Serum Ca, Ca2⫹e Urine Ca

Familial (autoimmune) hypocalciuric hypercalcemia

250HD

Vitamin D intoxication

Normal

PTH

↓Phos

Primary Hyperparathyroidism

Hypophosphatemia

1,25(OH)2D

Intoxication Ectopic production

Normal PTHrP

Neoplasia

Other hypercalcemic factor (e.g., Prostaglandin E)

Figure 17-6 Evaluation of hypercalcemia. Abbreviations: Ca (serum total calcium), Ca2⫹e (extracellular ionized calcium), Phos (serum phosphate), PTH (parathyroid hormone), 25OHD [25-hydroxyvitamin D (calcidiol)], and 1,25(OH)2 D-[1,2,5-RTHnP RTH related protein dihydroxyvitamin D (calcitriol)].

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substantial hypercalcemia in infants. Etidronate (5 mg/kg twice daily orally) and pamidronate (0.5–2.0 mg/kg in 30 mL normal saline intravenously over 4 hours) have been successfully employed in infants with hypercalcemia due to vitamin D intoxication, subcutaneous fat necrosis, and other causes.83,101-103 Parathyroidectomy may be urgently required in the newborn with NSHPT and life-threatening hypercalcemia.

DISORDERS OF BONE MINERALIZATION Low Bone Mass and Rickets Approximately 80% of total bone calcium in the full-term neonate is accrued in the last trimester of pregnancy as the in utero rate of calcium deposition increases more than twofold between 28 and 36 weeks of gestation. LBW (⬍1,500 g) and very low birth weight (VLBW ⬍1,000 g) infants are particularly vulnerable to the development of bone disease of prematurity because they are unable to maintain the in utero rate of synthesis of organic bone matrix (osteoid) or the rate of calcium and phosphate deposition into osteoid from the minerals provided via the gastrointestinal tract or by parenteral nutrition.104 Decreased calcification of bone matrix results in low bone mineral content (BMC), whereas depressed calcification of the cartilage growth plate leads to rickets and its characteristic deformities. In general terms, osteopenia may be defined as too little bone tissue with decreased thickness of bone cortex and/or decreased thickness or number of bone trabeculae. Osteoporosis is present when bone mass is so low that fractures occur after minor trauma.104 (Thus, in this population of LBW and VLBW neonates these terms are not necessarily defined by the same quantitative measurements of bone density as employed in adults.) Postpartum, hypocalcemia, and decrease in spontaneous movement against the force exerted by the muscular wall of the uterus depress the rate of bone mineral acquisition— whereas an increased rate of bone resorption further decreases skeletal mass in premature infants.105,106 Approximately 30% of preterm infants with birth weights ⬍1,500 g develop bone disease.107 Birth weight and rate of postnatal weight gain, as well as umbilical cord concentrations of IGF-I, are important determinants of bone mass in premature infants.108,109 Necrotizing enterocolitis, a disorder that affects approximately 10% of LBW infants, increases the rate of bone resorption as assessed by measurement of serum (ICTP) and urinary (Dpd) markers of this process.110 Interestingly, neonatal sepsis is not associated with increase in levels of bone resorption markers.110,111 Malrotation of the intestinal tract or catastrophic necrotizing enterocolitis leading to intestinal infarction requiring extensive small bowel resection substantially increases the risk of malabsorption and subsequent low bone mass. Parenteral alimentation of LBW or VLBW neonates restricts administration of fluid, calcium, and phosphate (in part, because of the incompatibility of the coinfusion of these ions in high concentrations). Excessive aluminum in parenteral fluids also adversely affects bone formation. Another factor that may contribute to the bone disease of

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prematurity is decreased synthesis of glucagon-like peptide 2 (GLP2, OMIM 138030, chromosome 2q36-q37), a 33-aa post-translational derivative of proglucagon whose production is depressed by small intestinal resection or atrophy during prolonged parenteral alimentation. GLP-2 promotes intestinal calcium absorption and depresses osteoclastic activity.110 Increased maternal parity, male gender, severe systemic disease (bronchopulmonary dysplasia), immobility, and pharmacologic agents (glucocorticoids, methylxanthines such as theophylline, diuretics such as furosemide) also adversely impact bone formation in these neonates.106,112 Theophylline and furosemide increase urinary excretion of calcium.113 A polymorphic variant of estrogen receptor ␣ with multiple TA repeats (⬎18) at 1174 bp upstream appears to protect LBW neonates from development of bone disease of prematurity, and a variant with a low number of TA repeats increases the risk of bone disease. The mechanism of this effect is not known at present.107 Two prenatal factors that contribute to low bone mass in VLBW and LBW neonates include intrauterine growth retardation (possibly by reducing placental transport of calcium and decreasing the rate of bone formation) and prenatal exposure to large amounts of magnesium sulfate administered repeatedly to the mother in preterm labor that leads to hypocalcemia and osteopenia by suppressing PTH secretion through its interaction with the CaSR and by competing with calcium for deposition at bone surfaces, respectively.114 In preterm neonates, serum levels of total and bonespecific alkaline phosphatase, PICP, and osteocalcin (markers of osteoblast activity and bone formation) are elevated relative to full-term neonates and older infants and continue to rise over the first 10 weeks of life.115 Urinary hydroxyproline and Pyr/Dpd values (markers of osteoclast activity and bone resorption) are also increased, although serum concentration of ICTP (another marker of bone resorption) decline during the first 10 weeks after premature delivery. Overall, the data indicate that intrauterine and postnatal rates of bone turnover in preterm newborns are rapid and persistently elevated through 40 weeks postconceptual age—a conclusion confirmed by bone histomorphometry.105,112,116 By photon absorptiometry and quantitative ultrasonography, bone mass of preterm infants appears to decline during the first several weeks after birth.116a Serum levels of alkaline phosphatase and osteocalcin and urinary excretion of Pyr and calcium may remain elevated in LBW infants with bone disease of prematurity relative to values in LBW infants without bone disease for the first year of life, even though radiographic improvement in skeletal mineralization is usually evident by 6 months of age.107 Because standard roentgenograms may not detect low bone mineralization before deficits of 20 to 30% or greater have occurred in the second month of life, estimation of BMC by dual-energy X-ray absorptiometry (DEXA) has become the preferred method of assessing bone mineralization in infants because of its accuracy, reproducibility, rapidity of performance, and low radiation exposure (2–3 mrems).12,117 Mean total body BMC in the first 2 days of life ranges from 21.7 g in newborns with birth weights of 1,001 to 1,500 g to 78.8 g in neonates

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with birth weights of 3,501 to 4,000 g, whereas BMD varies from 0.146 mg/cm2 (1,001–1,500 g) to 0.234 gm/ cm2 (3,501–4,000 g). In full-term healthy neonates, the mean whole-body BMC measured by newly introduced fan beam DEXA is 89.3 (SD ᭙ 14.1) g.118 BMC and BMD increase through the first year of life and beyond as measured by DEXA (see Chapter 3, Table 8). No racial, gender, or seasonal factors affect bone mineralization at this age. Body weight is best correlated with bone mass.119 There is increasing use of quantitative ultrasound (QUS) to assess bone integrity and strength in preterm and other LBW infants because the study may be performed at the crib side, there is no exposure to radiation, and it may be repeated as frequently as necessary.120 QUS measures the speed of sound (SOS) through a bone (humerus, tibia, radius, patella, os calcis, metacarpal, phalanx), a measurement correlated with the strength of the bone. Mineral content is but one of several skeletal components (elasticity, cortical thickness, microstructure) that collectively contribute to bone strength. QUS also permits calculation of bone transmission time, a measurement that determines the difference in the velocity of sound as it travels through bone and surrounding soft tissue. There is considerable overlap of SOS values at various ages and somatic sizes. However, bone transmission times may discriminate to a greater extent between these parameters. Humeral QUS measurements are lower in preterm than term infants and correlate positively with gestational age, length, and weight.121 In neonates with intrauterine growth retardation, tibial SOS levels may be appropriate for gestational age or may even be elevated.122,123 With prolonged deprivation of calcium, phosphate, and vitamin D, not only does the LBW infant lag in accumulation of bone mass but clinical and radiographic evidence of rickets also develops—usually between the sixth and twelfth postnatal weeks—and fractures may occur in as many as 24% of VLBW infants.116a The LBW neonate at risk for low bone mass and/or rickets is best managed preventively by the daily enteral administration of as much of the needed amounts of calcium (140–160 mg/100 kcal), phosphate (95–108 mg/100 kcal of formula), and vitamin D (400 U)—as well as protein (for collagen synthesis) and energy (carbohydrates, lipids)—as possible. When parenteral administration of nutrients is necessary in the LBW or VLBW neonate, an attempt should be made to administer the maximum amounts of calcium and phosphate safely attainable. The solubility of calcium and phosphate depends not only on their quantities but on the forms selected for infusion (i.e., calcium chloride, gluconate, glycerophosphate, monobasic phosphate, or dibasic phosphate). Utilizing monobasic phosphate and glycerophosphate, it is possible to infuse as much as 86 mg of calcium/kg/dL and 46 mg/kg/dL of phosphate.12 Enteral feeding should begin as soon as possible in the LBW infant. Fortification of human breast milk with calcium glycerophosphate (calcium 170 mg/kg/day, phosphate 87 mg/kg/day) permits 57% absorption and 91% retention (88 mg/kg/day) of calcium and 94% absorption and 61% retention (50 mg/kg/day) of phosphate.12 Prepared formulas for feeding of LBW neonates can also provide up to 90 mg/kg/day of retained calcium and

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40 mg/kg/day of retained phosphate. The type of prepared formula (sources of protein, fat, and carbohydrates) and its lipid and mineral additives determine the rate of intestinal calcium absorption and retention. Therefore, the choice of formula must be carefully considered before it is selected.6 Nevertheless, parenteral nutrition, fortified human milk, and current preterm formulas are unable to provide the amounts of calcium and (in particular) phosphate that would normally accrue to the fetus in utero. Vitamin D 400 IU/day should also be provided to the preterm infant either enterally or parenterally. Monitoring of serum levels of calcium, phosphate, creatinine, and alkaline phosphatase (and urinary excretion of calcium, phosphate, and creatinine) is essential in order to prevent hypercalcemia, hypercalciuria, and nephrocalcinosis. It is also important to avoid hypocalcemia because of the avidity of bone matrix for calcium once remineralization has commenced (hungry bone syndrome). Passive physical activity (daily range of motion with extension/flexion of all joints of each extremity in the supine infant for 4 weeks, beginning after the neonate has been stabilized between 2 and 6 weeks of postnatal age) with or without gentle massage of the prone infant from head to toe increases serum levels of markers of bone formation as well as bone mineralization by DEXA and QUS changes consistent with augmented bone strength.116a,124 Administration of estrogen and progesterone to VLBW female infants in sufficient amounts to maintain intrauterine levels of these hormones for 6 weeks postnatally does not increase the retention of calcium or phosphate during this interval or bone mineralization in later infancy.125,126 The efficacy and safety of bisphosphonates or of PTH1-34 in the management of osteopenia of prematurity remain to be examined. Although in the prematurely born infant bone mass may remain low through infancy and early childhood, bone mineralization eventually catches up to the norms.127-130 Osteogenesis imperfecta congenita (OIC) [type II (OMIM 166210)] is a perinatal lethal disorder most commonly associated with heterozygous newly developed loss-offunction mutations in the genes encoding collagen-␣1(I) (COL1A) or collagen-␣2(I) (COL1A2). The mutations may be partial gene deletions resulting in decreased synthesis of type I collagen or missense mutations that lead to amino acid substitutions (e.g., arginine, aspartic acid, cysteine) for the glycine residues essential to the normal three-dimensional conformation of collagen-␣1(I) and synthesis of the triple helix and structural integrity of type I collagen in extracellular matrix of bone upon which hydroxyapatite is deposited. Osteogenesis imperfecta type II may rarely be transmitted as an autosomal-dominant trait by a parent mosaic for a heterozygous mutation in collagen-␣1(I) or -␣2(I).131 Clinical manifestations of OIC are variable and include fractures present at birth (which may also occur in newborns with osteogenesis imperfecta types I and III), deformities of the long bones, osteopenia of the skull with large fontanelles, intrauterine growth retardation, premature delivery, and death (usually in infancy) due to respiratory insufficiency. Radiologically, OIC has been categorized into three subgroups: A (short, broad, and crumpled long

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bones, angulation of the tibia, and continuous beading of the ribs), B (similar femoral and tibial configurations but incomplete beading of the ribs), and C (thin long bones with many fractures and thin beaded ribs). The diagnosis of OIC is most often made clinically by its differentiation from campomelic dysplasia, achondrogenesis type I, thanatophoric dysplasia, and perinatal hypophosphatasia and is confirmed by quantitation of subnormal amounts of collagen synthesis by fibroblasts in vitro and identification of the mutation in COL1A1 or COL1A2 by direct genotyping. The lethal mutations in collagen-␣1(I) are clustered at sites at which the collagen monomer binds integrins, matrix metalloproteinases, fibronectin, and cartilage oligomeric matrix protein. Those in collagen-␣2(I) coincide with binding sites for proteoglycans. Administration of the bisphosphonate pamidronate has been helpful in infants with severe manifestations of osteogenesis imperfecta types III and IV but has been ineffective in the lethal form of osteogenesis imperfecta type II.132,133 Lysinuric protein intolerance (OMIM 222700) is an autosomal-recessive disorder of hepatic and renal tubular transport of dibasic amino acids (lysine, arginine, ornithine) that manifests itself in infancy and childhood by vomiting, diarrhea, failure to thrive, developmental delay, hepatomegaly, and cirrhosis. Affected infants and children have impaired urea synthesis due to decreased hepatic uptake of ornithine but are episodically hyperammonemic with increased urinary excretion of the dibasic amino acids. There is extremely low bone mass due to marked protein deprivation and perhaps due to increase in cytokineinduced bone resorption.134 Administration of citrulline has been reported to increase growth and bone mass in some of these patients. Lysine protein intolerance is due to lossof-function deletion, duplication, missense, nonsense, and splice-site mutations in SLC7A7 encoding an amino acid transporter.135

INCREASED BONE MASS Increased bone mass may be generalized or localized. Osteosclerosis refers to thickening of trabecular bone, and hyperostosis refers to increase in cortical bone mass.136 The infantile malignant form of osteopetrosis (OMIM 259700) is an autosomal-recessive disorder due to abnormal osteoclast differentiation or function resulting in defective resorption of the mineral phase of bone. Affected infants manifest failure to thrive, delayed development, nasal obstruction, loss of sight, hearing and other cranial nerve functions, and intense bone overgrowth leading to pancytopenia and increased susceptibility to infection, hepatosplenomegaly as sites of extramedullary hematopoiesis, increased susceptibility to fracture because of decreased bone strength despite high bone mass, mandibular osteomyelitis, and death (often within the first several years of life) due to sepsis, anemia, or hemorrhage. Physical examination reveals impaired linear growth, enlarged head circumference, nystagmus, and hepatosplenomegaly. The radiographic hallmark of infantile osteopetrosis is relatively uniform increase in bone density of the skull, vertebrae, and axial skeleton, Ehrlenmeyer flask deformity at the distal ends of the long bones

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in older children, and alternating bands of sclerotic and lucent bone in the iliac wings. This usually lethal form of osteopetrosis may be due to homozygous or compound heterozygous loss-of-function mutations in one of three genes: TCIRG1, CLCN7, and OSTM1. TCIRG1 encodes a subunit of the vacuolar proton pump within the osteoclast ruffled border through which hydrogen ions are transported from the cytosol into the subosteoclast resorption lacuna. CLCN7 encodes a chloride channel required for movement of this cation into and acidification of the resorption lacuna. OSTM1 encodes a subunit of CLCN7 necessary to its normal post-translational processing. In selected subjects, bone marrow transplantation effectively supplies sufficient osteoclast precursors to halt the progression of this disorder—albeit often with substantial residual deficits. Transient but substantial hypercalcemia may occur after this procedure. Osteopetrosis (OMIM 259730) due to deficiency of CA2 encoding carbonic anhydrase II may present in infancy with failure to thrive or fracture with insignificant trauma. Disproportionate short stature is the cardinal manifestation of pycnodysostosis (OMIM 265800) and is manifest during infancy or early childhood.136 In subjects with pycnodysostesis, there is relative macrocranium with open fontanelles and cranial sutures, dysmorphic facial features (fronto-occipital prominence, proptosis, bluish sclerae, hypoplastic maxilla, micrognathia, highly arched palate, malocclusion, beaked nose), stubby and clubbed fingers with hypoplastic nails, narrow thorax, pectus excavatum, lumbar lordosis, kyphoscoliosis, and increased fracture risk. Radiographically, there is marked osteosclerosis that increases with age, open fontanelles and cranial sutures, thin clavicles with hypoplastic lateral ends, erosion and hypoplasia of the distal phalanges and ribs, and dense vertebrae yet normal transverse processes. Histologically, there are decreased numbers of osteoblastic and osteoclastic activity. Pycnodysostosis is due to homozygous or compound heterozygous loss-of-function mutations (stop, missense, nonsense) in CTSK, the gene encoding cathepsin K—a lysosomal cysteine protease expressed in osteoclasts. Loss of cathepsin K activity impairs degradation of collagen and the resorption of organic matrix but not that of the mineral component of bone.137 Isolated decreased stimulated secretion of GH has been recorded in some subjects. Administration of GH has improved the rate of linear growth of some patients with pycnodysostosis, but no effective treatment of the abnormal bone mineralization has been reported.138

Disorders of Magnesium Metabolism In serum, magnesium is present complexed to proteins and ionized or free. Approximately 50% of body magnesium stores are deposited within bone adsorbed to the surface of hydroxyapatite. The CaSR binds magnesium as well as calcium. Magnesium regulates the secretion but not the synthesis of PTH and the generation of calcitriol.20 In addition, experimental magnesium deficiency leads to decreased bone mineralization in mice, and magnesium oxide supplements increase BMC in healthy girls.139,140

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HYPOMAGNESEMIA Hypomagnesemia (serum total magnesium concentration ⬍1.5 mg/dL) leads to hypocalcemia by inhibiting the release of PTH and by interfering with its peripheral action and may mimic congenital hypoparathyroidism, which is itself often associated with hypomagnesemia. In neonates and infants, hypomagnesemia may be clinically silent or present with neuromuscular irritability or seizures often in association with hypocalcemia. Hypomagnesemia occurs in infants born to mothers deficient in magnesium, those with preeclampsia or gestational or type 1 diabetes mellitus, and in neonates with low birth weight (prematurity or intrauterine growth retardation).141 Prolonged nasogastric suctioning, malabsorption disorders due to extensive intestinal resection (short gut syndrome), intestinal fistulas, or other diseases associated with chronic diarrhea and steatorrhea also lead to infantile hypomagnesemia. In renal tubular disorders such as Gitelman (OMIM 263800) and Bartter (OMIM 242200) syndromes, as well as in subjects exposed to diuretics and nephrotoxic (cisplatin, cyclosporin, mercury, gentamycin) agents, hypermagnesuria leads to hypomagnesemia.97,142 In children and adolescents, hypomagnesemia is manifested by heightened neuromuscular irritability (carpal pedal spasm, tetany, seizures)—and when prolonged and profound by muscle wasting, weakness, apathy, and tachycardia with prolonged PR and QT intervals by electrocardiography. In these age groups, hypomagnesemia may be primary and due to a specific defect in the intestinal absorption of magnesium or in the renal tubular resorption of filtered magnesium. It may also be secondary and due to gastrointestinal losses (chronic vomiting or diarrhea or malabsorptive states due to inflammatory bowel disease, bowel resection or fistulas, or pancreatitis); to associated renal tubulopathies (Gitelman and Bartter syndromes); to exposure to alcohol, diuretics, and chemotherapeutic agents; or to specific endocrinopathies (diabetes mellitus, primary hyperparathyroidism, hyperaldosteronism).143 Familial hypomagnesemia with hypocalcemia (OMIM 602014) is an autosomal-recessive disorder pathophysiologically due to a selective small intestinal defect in magnesium absorption. The disease presents with hypocalcemic tetany and/or seizures in the neonatal period and can lead to myocardial, renal, and arterial calcinosis. Renal excretion of magnesium is normal in subjects with this disease. Hypocalcemia is attributable to decreased secretion of and peripheral sensitivity to PTH. This disorder is due to biallelic loss-of-function mutations in TRPM6 (transient receptor potential cation channel, subfamily M, member 6), encoding a 2,022-aa bifunctional protein with two domains: a calcium- and magnesium-permeable ion channel domain and a protein tyrosine kinase domain expressed in the kidney and colon.144 For full functional activity, TRMP6 must team with its homolog TRPM7 (OMIM 605692) and form a functional TRPM6/TRPM7 complex at the surface of the cell.145 Although most mutations of TRPM6 associated with this disease (nonsense, deletion) have resulted in extensive loss of product, the naturally occurring missense mutation Ser141Leu specifically disrupts formation of the complex. Oral ingestion of large quantities of magnesium is

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effective therapy for this illness. Primary hypomagnesemia (OMIM 248250) is an autosomal-recessive disorder due to decreased renal tubular resorption of filtered magnesium linked to biallelic loss-of-function mutations in CLDN16.146 This disease often presents in infancy and is associated with tetany, hypermagnesuria, hypercalciuria, mild hypocalcemia, nephrocalcinosis, impaired renal function, and secondary hyperparathyroidism. Claudin 16 (also termed paracellin 1) is a 305-aa protein with four transmembrane domains and intracellular amino and carboxyl terminals that is expressed within the intercellular tight junctions of renal epithelial cells in the TALH and distal convoluted tubule, where it facilitates paracellular transport and reabsorption of magnesium and calcium from the renal tubule. The first extracellular loop of claudin 16 bridges the intercellular space and is the site of paracellular conductance of ions. A number of missense mutations in CLDN16 have been identified in this gene, particularly at leucine 151 (Leu151Phe, Leu151Trp, Leu151Pro).147 Most of the mutations in CLDN16 impair its normal movement to the renal epithelial cell’s lateral surface. In other mutations (Ala62Val, His71Asp), products localize to the tight junctions but are functionally defective.148 Oral administration of 20 times the normal daily requirement of magnesium has been successful therapy in these subjects.149 Hypomagnesemia, hypercalciuria, and visual impairment (macular colobomata, myopia, nystagmus; OMIM 248190) have been associated with lossof-function mutations in CLDN19—a second renal epithelial tight junction protein localized to the distal renal tubule and eye and necessary for paracellular transport of calcium and magnesium.150 Calcium and magnesium are also reabsorbed from the renal tubule by transcellular passage from apical to basolateral surfaces of the renal epithelial cell. The presence of hypomagnesemia is identified by measurement of serum magnesium concentrations, whereas its pathophysiologic etiology is determined by concurrent assay of calcium, phosphate, sodium, potassium, chloride, bicarbonate, creatinine, PTH, and vitamin D levels and assessment of its urinary loss and intestinal absorption.141 Hypomagnesemic hypocalcemic seizures are only transiently responsive and sometimes resistant to parenteral administration of elemental calcium alone. Intravenous or intramuscular administration of a 50% solution of magnesium sulfate (MgSO4.7H20 0.05–0.1 mL/kg, or 2.5–5.0 mg/kg elemental magnesium with cardiac monitoring) is often necessary to control convulsions in the hypomagnesemic neonate.20,141 Oral magnesium supplements may also be helpful (50% MgSO4.7H20, 0.2 mL/ kg/day). Chronic hypomagnesemic states are treated with oral magnesium supplements as tolerated because large doses may lead to diarrhea.

HYPERMAGNESEMIA Hypermagnesemia (⬎2.5 mg/dL) is frequently recorded in the neonatal period because magnesium sulfate has been administered to pregnant women with hypertension, preeclampsia, or toxemia of pregnancy. Most neonates with hypermagnesemia are asymptomatic. However,

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when serum magnesium concentrations are exceptionally high hypotonia and depression of the central nervous system may be present—and when extended,metabolic bone disease may develop.141 Thus, prolonged (9–10 weeks) administration of intravenous magnesium sulfate to women with multiple fetuses who have entered labor prematurely has been associated not only with hypermagnesemia but with marked hypocalcemia in the offspring (and with significant osteopenia).114 Hypermagnesemia may also result from its parenteral administration or oral ingestion of magnesium-containing antiacids or enemas. In large amounts, magnesium sulfate suppresses secretion of PTH and decreases renal tubular reabsorption of calcium—factors contributing to hypocalcemia. The hypermagnesemic neonate is most appropriately managed by adequate hydration to permit urinary excretion of the high magnesium load. If the newborn is also hypocalcemic and osteopenic, administration of calcium and calcitriol is indicated. Hypermagnesemia may also develop in patients with renal insufficiency receiving magnesium-containing antiacids. Magnesium concentrations are modestly increased in patients with familial hypocalciuric hypercalcemia.

Disorders of Mineral Homeostasis in the Child and Adolescent HYPOCALCEMIA Etiology Causes of hypocalcemia in the child and adolescent are listed in Table 17-1. Hypocalcemia is defined by the norms of the analytical laboratory and is dependent on the age of the subject (total calcium concentrations: 1–5 years, 9.4–10.8; 6–12 years, 9.4–10.2; ⬎20 years, 8.8–10.2 mg/dL).22 Total calcium levels are low in the hypoalbuminemic patient. A correction for hypoalbuminemia may be calculated by adding 0.8 mg/dL to the recorded total calcium concentration for every decrease in albumin concentration of 1 g/dL.151 For this and other reasons, it is appropriate to measure total and Ca2⫹e values when evaluating the hypocalcemic child. However, reliance on Ca2⫹e determinations alone is discouraged given the technical difficulties with this assay. Hypocalcemia develops as a consequence of too little inflow of calcium from the gastrointestinal tract, bone, or kidney into the extracellular and vascular spaces or excessive loss of calcium from these spaces into urine, stool, and bone. Thus, hypocalcemia may be due to decreased intake or absorption or excessive loss of calcium, decreased production of bioactive PTH due to congenital abnormalities of PTG development or PTH synthesis or of the CaSR, destruction of PTGs by autoantibodies, metal overload (copper, iron), surgical or radiation insults, granulomatous infiltration, or impaired cellular responsiveness to PTH. Restricted exposure to sunlight or reduced intake, absorption, metabolism, or activity of vitamin D leads to hypocalcemia. Hypomagnesemia impairs the secretion of (but not the synthesis of PTH), and blunts tissue responsiveness to,

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PTH. Hypocalcemia occurs in the very ill child or after exposure to a number of drugs and medications. Hypocalcemic tetany may develop after the administration of phosphate-containing enemas by rectum or laxatives by mouth.152 At times, the hypocalcemic child or adolescent may be asymptomatic and identified by chemical screening for an unrelated problem—or may present with intermittent muscular cramping at rest or during exercise (when the increase in systemic pH due to hyperventilation lowers still further the concentration of Ca2⫹e); paresthesias of fingers, toes, or circumoral regions; tetany (carpopedal spasm, laryngospasm, bronchospasm); or seizures (grand mal, focal, petit mal, adynamic, or syncopal). Physical examination often reveals a positive Chvostek and/or Trousseau sign (carpopedal spasm) and hyperreflexia. However, a Chvostek sign is commonly present in normal adolescents also. Hypoparathyroidism may occur as a solitary disorder, as part of a multidimensional autoimmune polyendocrinopathy, or as one manifestation of a group of complex congenital anomalies (DiGeorge, HRD, Kenny-Caffey, Barakat, Blomstrand, and other syndromes). There are sporadic and familial forms of hypoparathyroidism. When familial, hypoparathyroidism may be transmitted as an autosomal-dominant, autosomal-recessive, or X-linked recessive trait (Tables 17-1 and 17-2). Abnormalities in the development of the PTGs, transcription of PTH, and processing of the translated product have been associated with inherited forms of hypoparathyroidism. In a family with autosomal-dominant dyshormonogenic hypoparathyroidism, a T⌿C transition in codon 18 of the 25-aa signal peptide of prepro-PTH that altered cysteine to arginine (Cys18Arg) has been identified.153 This heterozygous mutation within the hydrophobic region (aa 10 to 21) of the signal sequence (a domain necessary for efficient transport of protein from the ribosome and interaction of prepro-PTH with the signal recognition particle) impairs movement of the precursor peptide into and exit from the rough endoplasmic reticulum, its cleavage by a signal peptidase, and its incorporation into a secretory granule.154 The autosomaldominant transmission of hypoparathyroidism in this family suggests that the mutant pre-pro-PTH exerted a dominant-negative effect on the synthesis of normal PTH directed by the wild-type allele. Autosomal-recessive dyshormonogenic hypoparathyroidism has been associated with a homozygous G⌿C transversion in nucleotide 1 of intron 2 of PTH within the signal sequence that prevented normal cleavage of preproPTH and decreased secretion of PTH.155 However, inactivating mutations in PTH are uncommon in patients with sporadic idiopathic hypoparathyroidism.156 Isolated hypoparathyroidism may be found in patients with del22q11.2 but no other signs or symptoms of the DGS or the velocardiofacial syndrome.157 Autosomal-dominant hypoparathyroidism (OMIM 146200) due to heterozygous gain-of-function mutations in the extracellular, transmembrane, and intracellular domains of CASR (OMIM 600199) that transcribe a CaSR that is not intrinsically constitutively active but is exceptionally sensitive to and easily activated by very low serum Ca2⫹e concentrations may be identified in infancy or in

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709

Figure 17-7 Schematic view of the calcium-sensing receptor (CaSR). Gain(*)- and loss(X)-of-function missense and nonsense mutations associated with autosomal-dominant hypoparathyroidism, familial hypocalciuric hypercalcemia, and neonatal severe hyperparathyroidism are depicted. Abbreviations: NCM (normal amino acid-codon-mutation), SP (signal peptide), and HS (hydrophobic segment). [Reproduced with permission from Brown EM, et al. (1997). Familial benign hypocalciuric hypercalcemia and other syndromes of altered responsiveness to extracellular calcium. In Krane SM, Avioli LV (eds.), Metabolic bone diseases, Third edition. San Diego: Academic Press 479–499.]

older subjects (Figure 17-7).84 Even at hypocalcemic levels, Ca2⫹e binds avidly to the CaSR and activates phospholipase C-␤1—increasing cytosolic levels of inositol phosphate and Ca2⫹i and stimulating the mitogenactivated protein kinase (MAPK) signal transduction pathway in parathyroid chief cells (suppressing PTH synthesis and secretion). In the kidney, it decreases renal tubular calcium and magnesium resorption—leading to urinary wasting of these cations (hypercalciuric hypocalcemia). Urinary concentrating ability is also depressed. Serum levels of phosphate are increased and magnesium values decreased. PTH concentrations are low or inappropriately normal in these subjects. Affected patients frequently have symptomatic hypocalcemia such as tetany and seizures. They are very sensitive to vitamin D, and its administration can lead to hypercalciuria (even when serum calcium levels remain subnormal), nephrocalcinosis, and functional renal insufficiency. Administration of recombinant human (rh)PTH1-34 restores calcium homeostasis in this disorder, although experience with treatment in childhood is limited at present.47,158 There has been reluctance to administer rhPTH to children because there is an increased incidence of bone tumors in young rats receiving very large amounts of this agent. However, primates appear to be less susceptible to PTHinduced bone tumor formation than rodents.159 Development of stimulatory autoantibodies to the CaSR results in an acquired variant of spontaneous hypoparathyroidism that may be isolated or part of a complex

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autoimmune endocrinopathy.160,161 Indeed, in perhaps as many as one-third of patients with acquired isolated idiopathic hypoparathyroidism antibodies directed against epitopes in the extracellular domain of the CaSR may be present. This form of acquired hypoparathyroidism may be reversible, as these antibodies do not destroy the PTGs. In patients with other forms of autoimmune hypoparathyroidism, the antibodies are likely cytotoxic and accompanied by lymphocytic infiltration, atrophy, and fatty replacement of parathyroid tissue. In mid-childhood and adolescence, acquired hypoparathyroidism may be a late manifestation of a congenital abnormality (e.g., DGS). However, it is also likely to be the result of destruction of the PTGs by autoimmune disease or surgical removal or operative trauma to the vascular supply of these structures. Unusual causes of acquired hypoparathyroidism in this age group include infiltration by iron (hemochromatosis, thalassemia) or copper (Wilson’s hepatolenticular degeneration), granulomatous diseases, or radiation (mantle radiation for Hodgkin/non-Hodgkin lymphoma or radioiodine therapy of hyperthyroidism).162,163 Autoimmune hypoparathyroidism may occur as an isolated disorder or as part of the complex of autoimmune polyendocrinopathy syndrome type I.163,164 Development of an autoimmune endocrinopathy begins with presentation of a peptide specific for a target organ to a subgroup of T cells that recognize that peptide. When immunologic tolerance for that peptide is lost, clones of

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CD4 T cells for the peptide expand. Type 1 helper T cells secrete inflammatory cytokines such as interferon-␥, whereas type 2 helper T cells stimulate B-cell function and lead to autoantibody-mediated inflammation. Loss of immune tolerance may be the consequence of the postinfectious inflammatory state due to activation of the innate immune system or due to a gene mutation that depresses immune tolerance and permits expansion of a CD4 T-cell clone after exposure to quantitatively small amounts of antigen. Genetic variations within the major histocompatibility complex (HLA-DQ, HLA-DR) that determines peptide (antigen) presentation to CD4 T cells join with genetic abnormalities in immune regulation to induce autoimmune disease. As noted, in 30% of patients with isolated idiopathic hypoparathyroidism the disorder is due to antibodies to the extracellular domain of the CaSR and thus functionally inhibitory but potentially reversible. In approximately 33% of patients, serum antibodies to other components of the parathyroid chief cell may be present.160,161 Autoimmune polyendocrinopathy syndrome type I (OMIM 240300) is an autosomal-recessive disorder with the classic triad of autoimmune polyendocrinopathy, mucocutaneous candidiasis, and ectodermal dystrophy (APECED).165 In a Finnish cohort of 91 patients with APECED, the cardinal manifestations were mucocutaneous candidiasis involving the nails and mouth occurring in 100% of patients (often in the first two years of life), hypoparathyroidism, and hypoadrenocorticism (both of the latter illnesses developed in 80% to 90% of affected subjects).166 Hypoparathyroidism occurred most often between 2 and 10 years of age, and hypoadrenocorticism developed between 5 and 15 years of age. Almost all females with APECED developed hypoparathyroidism, whereas 80% of affected males did so. Hypomagnesemia, often severe and recalcitrant to therapy, was common in patients with hypoparathyroidism due to APECED. The most frequent presenting manifestations of APECED were mucocutaneous candidasis (60%), hypoparathyroidism (32%), and hypoadrenocorticism (5%). The disease first became apparent between 2 months and 18 years of age. However, 10% of patients presented with another manifestation of APECED. In addition to hypoparathyroidism and hypoadrenocorticism, other endocrinopathies encountered in APECED included autoimmune oophoritis leading to ovarian failure (70%), orchitis resulting in testicular failure (30%), diabetes mellitus (30%), thyroiditis (30%), and hypophysitis (4%). Besides mucocutaneous candidiasis, dermatologic manifestations and complications of APECED in the Finnish cohort included alopecia (40%), vitiligo (30%), and rashes with fever (15%). Keratoconjunctivitis developed in 20% of affected subjects, pernicious anemia in 30%, hepatitis in 20%, and chronic diarrhea in 20%. In a Norwegian population of 36 patients with APECED, 13 had clinical evidence of disease at or before 5 years of age—and an additional 15 subjects presented at or before 15 years of age.167 Clinical manifestations of APECED and their age of onset vary between subjects and even between siblings. Later manifestations of APECED include esophageal and oral

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squamous cell carcinoma, asplenia, and interstitial nephritis. The diagnosis of APECED usually requires the presence of two of three of its primary manifestations (mucocutaneous candidiasis, hypoparathyroidism, hypoadrenocorticism), but occasionally hypoparathyroidism may be its only sign. Autoimmune polyendocrinopathy syndrome type I is due to homozygous or compound heterozygous loss-offunction mutations in AIRE (autoimmune regulator), a 14-exon gene with 1,635 base pairs encoding a 545-aa with two zinc-finger motifs that is expressed in nuclei of thymic medullary epithelial cells and in lymph nodes, spleen, monocytes, and other tissues where it functions as a transcription factor.168 AIRE also serves as an E3 ubiquitin ligase, an essential component of the ubiquitinproteasomal system for protein modification and destruction involved in cellular division and differentiation, protein transport, and intracellular signaling.169 Structurally, AIRE contains two plant homeodomains in an amino acid sequence composed of an octet of cysteines and histidines that coordinate two zinc ions. The first plant homeodomain is essential to the E3 ubiquitin ligase activity of the protein, and the second plant homeodomain is required for its transcription-regulating action. Functionally, AIRE may assist in the elimination of forbidden clones of T cells from the thymus—or it might promote expression of peripheral antigens in the thymus, thereby increasing immune tolerance.164 In the Finnish population with APECED, the most common loss-of-function mutation in AIRE was a homozygous truncating mutation at codon 257 (Arg257Ter). More than 55 pathogenic mutations in AIRE [missense, nonsense (Arg139Ter), insertion, and deletion (e.g., 13 base pair deletion; 964del13, NT 1094, exon 8)] that alter the subcellular distribution of AIRE and/or decrease its transcriptional activation capacity and/or its E3 ubiquitin ligase activity have been detected in patients with APECED.168,169 Pseudohypoparathyroidism (PHP) is a heterogeneous group of disorders associated with resistance to the action of PTH—classified as types IA, IB, IC, and II and pseudopseudohypoparathyroidism (PPHP). PHP is due to inactivating mutations in GNAS (guanine nucleotide-binding protein, ␣-stimulating activity polypeptide 1). PHP type IA (OMIM 103580) is the result of heterozygous inactivating mutations in GNAS, a gene with a complex structure of 13 exons that encodes the ␣ subunit of stimulatory G protein (Gs␣).170,171 After binding of PTH to the PTHR1, the receptor is linked through its carboxyl terminal to a stimulatory signal transduction protein whose Gs␣ subunit then binds guanosine triphosphate (GTP). GTP-binding proteins (G proteins) are heterotrimers with ␣, ␤, and ␥ subunits. Individual genes encode each of 16 ␣, 5 ␤, and 11 ␥ peptides—leading to multiple potential combinations and functions primarily defined by the G␣ subunit.172 After binding of PTH (or PTHrP) to PTHR1, there is a change in the three-dimensional configuration of PTHR1 leading to replacement of guanosine diphosphate (GDP) on Gs␣ by GTP. Gs␣ then dissociates from its ␤␥ companion subunit complex and stimulates membrane-bound adenylyl cyclase activity, generating in turn cyclic adenosine monophosphate (AMP) and activating protein kinase A and phosphorylating serine and tyrosine residues of specific proteins. Interaction of PTH with its receptor

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also leads to activation of Gq␣ and G11␣, with stimulation of phospholipase C and breakdown of membrane phosphoinositides resulting in increase in intracellular Ca2⫹i levels and stimulation of signal transduction pathways that lead to cellular action and the physiologic response(s) to PTH. Gs␣ has two distinct domains. One of these is a GTPase domain that degrades GTP to inactive GDP, thus terminating Gs␣ activity. Within this domain are binding sites for GTP/GDP, the GPCR, and the intracellular second messenger effector protein. The second domain is a helical domain that may be necessary for maintenance of guanine nucleotide binding.173 Upstream (5') of exon 2 of GNAS are four alternative first exons that can link to exons 2 through 13 of GNAS. The most upstream of these promoters is termed NESP55, which generates a transcript for a chromogranin-like protein with a coding region limited to the specific first exon. Exons 2 through 13 form its 3' untranslated region (Figure 17-8). NESP55 is expressed in neuroendocrine tissues and is parentally imprinted. Thus, it is only expressed by the maternally transmitted allele. (There is also a NESP antisense transcript in this region.) The next promoter (XL␣s) yields a Gs␣ isoform also specific for neuroendocrine tissues and identical to Gs␣ except that it has a very long amino terminal sequence of amino acids. XL␣s is also imprinted. It is expressed only by the paternally acquired allele. Exon 1A contains a differentially methylated region (DMR) that is methylated on the maternal allele and unmethylated on the paternal allele and is expressed only by the paternal allele. Exon 1A generates an untranslated mRNA transcript. Exon 1 (and thus Gs␣) is generally expressed in most tissues by both parental alleles. However, in the renal proximal tubules, thyroid, pituitary, and ovaries Gs␣ is expressed primarily by the maternal allele.171 Tissue-specific parental imprinting of Gs␣ is not related to differential methylation within its CpG island but rather with differences in the extent of methylation of histone H3 lysine 4 in the region of exon 1 of GNAS. Thus, GNAS has several independent imprinting

Figure 17-8 Schematic view of the first two of the 13-exon GNAS gene complex. Three alternate exons (NESP55, XLas, and 1A) are upstream of exon 1. The pattern of methylation, and hence the expression of the alternate exons, is dependent on the parent of origin of the allele and the specific tissue in which GNAS is expressed. [Reproduced with permission from Weinstein LS, Liu J, Sakamoto A, et al. (2004). GNAS: Normal and abnormal functions. Endocrinology 145:5459–5464.]

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sites and mechanisms. Bioactive isoforms of Gs␣ are also formed by inclusion (long transcript variant of Gs␣ ⫽ Gs␣-L) or exclusion (short transcript variant of Gs␣ ⫽ Gs␣-S) of exon 3 in the mRNA transcript.174 Patients with PHP type IA have the distinct phenotype of Albright hereditary osteodystrophy (AHO), including short stature, husky to obese body habitus, shortening of the third through fifth metacarpal bones and distal phalanx of the first finger (brachydactyly), syndactyly between the second and third toes, round face, flat nasal bridge, short neck, subcutaneous calcifications (heterotopic ossification), and cataracts—and in some subjects developmental delay in association with hypocalcemia, hyperphosphatemia, hyperphosphaturia, and elevated serum concentrations of PTH. Administration of exogenous PTH (rhPTH1-34) does not increase serum concentrations of calcium or urinary excretion of nephrogenous cyclic AMP or phosphate. The phenotype of AHO but with normal serum calcium, phosphate, and PTH concentrations and response to exogenous PTH1-34 is termed PPHP. Normocalcemic PHP (eucalcemia with elevated serum concentration of PTH) has also been described.174 Clinically, subnormal thyroid function with hyperthyrotropinemia is a common presenting manifestation of PHP type IA—particularly in infants less than 2 years of age.175 Subcutaneous calcifications also appear early in life. Hypocalcemia is commonly identified in infancy but may not be recognized until mid-childhood or early adolescence. Heterozygous loss-of-function mutations in GNAS (nonsense, missense, splice site, and base pair deletions) have been identified throughout the genomic structure of GNAS in subjects with PHP-IA and PPHP. These mutations lead to abnormal transcription and translation of GNAS and decreased expression of the mutated allele, resulting in approximately 50% of normal Gs␣ activity in most cells. Because Gs␣ is essential for normal chondrocyte differentiation by mediating the effects of PTH and PTHrP on this process, the skeletal manifestations of AHO reflect partial loss of Gs␣ activity in chondrocytes.176 If the mutated GNAS allele is of maternal origin, the kidney, thyroid, pituitary, and ovary have extremely low levels of Gs␣ activity and function in these tissues is almost nil—resulting in renal tubular resistance to PTH, thyroid insensitivity to thyroid-stimulating hormone, somatotroph resistance to GH-releasing hormone, and ovarian resistance to gonadotropins.173,177,178 Thus, PHP type IA occurs in the offspring when mutated GNAS is transmitted from mother to child. In PPHP, the clinical phenotype is that of AHO but the patient is eucalcemic. Because the mutated GNAS has been transmitted from father to child and the proximal renal tubule, thyroid gland, pituitary somatotroph, and ovary express the intact maternal GNAS allele, nearnormal Gs␣ activity is retained in these tissues. However, chondrocyte differentiation is impaired because in this tissue GNAS is expressed from both the maternal and paternal alleles and levels of Gs␣ activity are only one-half normal. Mutations in Gs␣ can also result in progressive osseous heteroplasia (POH, OMIM 166350)—severe ectopic

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ossification of skin, skeletal muscle, and deep connective tissue—because Gs␣ and cyclic AMP play important inhibitory roles in the differentiation of osteoblasts.171,173 The same mutation in GNAS can manifest itself clinically in the same family as POH or AHO. Localized dermal calcifications are frequently encountered in subjects with AHO/PHP type IA. Olfactory sense may also be decreased in patients with PHP type IA and IB. More than 80 heterozygous loss-of-function mutations in GNAS have been described to date. A four base pair deletion in exon 7 (codons 188 and 189) in GNAS that leads to a frame-shift and premature stop codon has been found in a number of families with PHP type IA and appears to be an amutational hotspot because it impairs DNA polymerization and replication. Other mutations alter intracellular movement of GNAS protein (Leu99Pro, Ser250Arg), increase the rate of release of GDP (Arg258Trp, Ala366Ser), or impair coupling of G protein to PTHR1 (Arg385His). In the only mutation identified in exon 3 to date, insertion of adenosine in codon 85 led to a frame-shift and a stop sequence at codon 87 in exon 4—resulting in isolated deficiency of only the long transcript variant of Gs␣.174 Gs␣ activity was 75% of normal in the tissues of this patient, who had the AHO phenotype, developmental delay, and hyperthyrotropinemia. Serum calcium levels were normal, but PTH values increased (normocalcemic PHP). In patients with PHP type IB (OMIM 603233), the phenotype is normal but Gs␣ activity is deficient in the proximal renal tubule and thyroid. Thus, these patients are resistant to PTH (and in part to thyrotropin) and develop hypocalcemia and hyperphosphatemia.171,173 This disorder is the result of an epigenetic defect: loss of the maternal pattern of GNAS expression due to an imprinting error and failure of methylation of the DMR of maternal exon 1A. Thus, in PHP type IB both GNAS alleles have a paternal imprinting pattern (nonmethylation of exon 1A on both alleles; i.e., a paternal epigenotype). Thus, there is a quantitatively normal amount of Gs␣ in most tissues (including the osteoblast and chondrocyte accounting for the normal skeletal phenotype). When occurring sporadically, the primary abnormality in PHP type IB may lie within the DMRs of antisense NESP and/or exon 1A. When transmitted as a familial autosomal-dominant characteristic, PHP type IB develops only in the offspring of female obligate carriers and might at times be the result of partial deletion of STX16 (Syntaxin 16)—a gene centromeric to NESP55 and exon 1A of GNAS that contains a cis-acting element(s) that regulates imprinting (methylation) of the DMR in maternal exon 1A of GNAS.173,179 Loss of this element within the maternal STX16 perhaps results in failure of methylation of the DMR in exon 1A of maternal GNAS and consequently a paternal pattern of GNAS expression with decreased synthesis of Gs␣ in the proximal renal tubule. As anticipated, uniparental paternal disomy for chromosome 20 or familial mutations within GNAS itself may also result in the PHP type IB phenotype.180 Epigenetic defects in methylation of structurally intact GNAS have also been described in patients with PHP type IA, variable Gs␣ activity in peripheral tissues, and mild skeletal abnormalities (designated PHP type IC).171,181 In PHP

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type II, the phenotype is normal, serum calcium concentrations are low, phosphate and PTH levels elevated, and in response to exogenous PTH urinary cyclic AMP excretion increases but phosphate excretion does not. The pathogenesis of this disorder is unknown but may be related to unsuspected deficiency of vitamin D.171 In several unrelated boys with PHP type IA/AHO, Ala366Ser and Arg231His mutations in GNAS have been associated with gonadotropin-independent isosexual precocity. Their mutated Gs␣ products were quickly degraded at body temperature, leading to PHP type IA, but quite stable at scrotal temperature—resulting in increased Leydig cell function.182,183 Hypocalcemia occurs in patients with deficiency of vitamin D intake, metabolism, or bioactivity.75 In the subject with skeletal demineralization due to marked vitamin D deficiency, serum calcium concentrations may fall precipitously after administration of even small amounts of vitamin D as renewed mineralization of bone matrix due to accelerated osteoblastic activity consumes calcium and phosphate (the “hungry bone” syndrome). After parathyroidectomy for primary hyperparathyroidism, calcium concentrations often decline rapidly by the same mechanism. Drugs that inhibit PTH secretion (excessive magnesium), osteoclast resorption of bone (bisphosphonates), or renal resorption of calcium (furosemide) may lead to hypocalcemia. Intravenous infusion or rectal administration of phosphate (in enemas), acute cellular destruction by tumor cell lysis or rhabdomyolysis, and acute and chronic renal failure increase serum phosphate levels and lead to reciprocal decline in calcium values. Serum calcium concentrations decline in patients receiving multiple transfusions of citrated blood or during plasmapheresis. In subjects with acute pancreatitis, calcium complexed with free fatty acids generated by pancreatic lipase is deposited in necrotic tissue.184 Acute severe illness of diverse pathogenesis is often associated with hypocalcemia. This has been attributed to hypoalbuminemia, functional hypoparathyroidism, hypercalcitonemia, hypomagnesemia, decreased calcitriol synthesis, alkalosis and increased serum concentrations of free fatty acids (the latter increase binding of Ca2⫹e to albumin), and increased cytokine activity.

Evaluation Figure 17-5 outlines the evaluation of the child/adolescent with hypocalcemia. Hypocalcemia may be asymptomatic until detected by a multiassay chemical profile obtained for another purpose. It may be first identified during evaluation of a long QT interval noted by electrocardiography obtained for evaluation of a functional heart murmur or arrhythmia.185 Clinical symptoms that suggest hypocalcemia include paresthesias, muscular cramping (particularly during vigorous exercise), tetany (uncontrollable muscular contractions, including laryngospasm), carpal-pedal spasm (flexion of the elbow and wrist, adduction of the thumb, flexion of the metacarpal/ metatarsal-phalangeal joints, and extension of the interphalangeal joints), or seizure.151 Review of the past medical history may reveal symptoms consistent with or illnesses associated with

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

hypocalcemia (recurrent infections, congenital cardiac anomalies, surgical procedures in the neck, cervical radiation, infiltrative diseases), or the family history may reveal members with hypoparathyroidism, dysmorphic physical characteristics, autoimmune endocrinopathies, or hypomagnesemia. Physical examination will disclose characteristic abnormalities in children with PHP type IA in whom the AHO phenotype is present (short stature, round face, subcutaneous hard nodules, brachymetacarpals), the DGS (typical face, recurrent infections, cardiac murmur), and familial autoimmune polyendocrinopathy syndrome type I (chronic mucocutaneous candidiasis and other ectodermal abnormalities such as vitiligo, alopecia, keratoconjunctivitis)—whereas rachitic deformities in the hypocalcemic child imply the presence of a form of hypovitaminosis D. Most commonly, however, the physical examination reveals no striking abnormality in the hypocalcemic child other than those of increased neuromuscular irritability: hyperreflexia [e.g., positive Chvostek sign (twitching of the circumoral muscles when tapping lightly over the seventh cranial nerve)], Trousseau sign (carpal pedal spasm when maintaining the blood pressure cuff 20 mm Hg above the systolic blood pressure for three minutes), and occasionally cataracts, papilledema, or abnormal dentition. (Tetany also occurs in subjects with hypo- and hypernatremia, hypo- and hyperkalemia, and hypomagnesemia. A positive Chvostek sign may be found in many normal adolescents.) After confirmation of the hypocalcemic state (by measuring serum total calcium and Ca2⫹e levels, the latter to exclude hypoalbuminemia as a cause of a low total calcium value), urine calcium excretion is determined. In most hypocalcemic patients, urine calcium excretion is low (Figure 17-5). If the urine calcium excretion is inappropriately normal or high, disorders such as autosomal-dominant hypoparathyroidism (gain-of-function mutation in CASR) may be considered. CASR may then be analyzed or antibodies to CaSR determined as clinically indicated. The frequency with which antibodies to the CaSR are detected in patients with autoimmune hypoparathyroidism depends on the method employed for their identification. Thus, antibodies to CaSR have been found in 86% of subjects with APECED employing an immunoprecipitation assay with the full-length CaSR expressed in human embryonic kidney cells and in 50% of the same subjects utilizing a flow cytometry assay but in none of these patients applying a radiobinding assay.186 The serum levels of intact PTH, magnesium, phosphate, creatinine, and alkaline phosphatase are to be measured. Determination of the serum concentration of intact PTH (by ultrasensitive assay) permits differentiation between low (or inappropriately anormal) and high PTH secretory states. The child/adolescent with hypocalcemia, hypocalciuria, hyperphosphatemia, and low or undetectable serum PTH concentration (and normal or only slightly low serum magnesium level) likely has hypoparathyroidism due to a primary defect in PTH synthesis or secretion related to congenital malformation or acquired destruction of the PTGs. Patients with hypoparathyroidism often have low serum concentrations of calcitriol, normal levels of calcidiol, decreased excretion of urinary nephrogenous cyclic AMP, and increased renal tubular reabsorption of

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phosphate.163 The DGS may be identified by abnormal FISH analysis of chromosome 22q11.2 or by TBX1 genotyping. Analysis of GCM2 or SOX3 and genes associated with syndromic hypoparathyroidism (TCBE, GATA3) is indicated in the appropriate clinical context (Table 17-1). The diagnosis of autoimmune polyendocrinopathy syndrome type I is based on clinical and laboratory findings and genotyping of AIRE. The presence of two of three of its major manifestations (candidasis and/or ectodermal dystrophy, hypoparathyroidism, hypoadrenocorticism) is accepted for its clinical diagnosis, but isolated hypoparathyroidism may occasionally be its only manifestation. Antibodies to the CaSR or other cellular components of the PTGs, the adrenal glands (side-chain cleavage, 21-hydroxylase, 17␣-hydroxylase enzymes), neurotransmitters (aromatic L-amino acid decarboxylase, tryptophan hydroxylase), and interferons-␣ and -␻ may be determined. Antibodies to interferon-␻ are commonly present in patients with APECED.167 Genotyping of AIRE and identification of the mutations confirm the diagnosis of APECED. There is wide variability in the clinical expression of APECED between families and among siblings because the phenotype is not directly related to the genotype.187 In patients with isolated idiopathic hypoparathyroidism, a search for antibodies to the CaSR and/or to parathyroid tissue is warranted. In hypomagnesemic subjects, magnesium and PTH levels are quite low—and PTH secretion increases rapidly after intravenous administration of magnesium. Primary hypomagnesemia should be considered when hypocalcemia and hypercalciuria coincide and serum PTH and magnesium values are low. Urinary magnesium excretion should then be quantitated and CLDN16 genotyped. Because hypomagnesemia may also be due to a selective small intestinal defect in magnesium absorption, mutations in TRPM6 should be examined as warranted. Primary hypomagnesemia must be differentiated from that due to the Gittleman and Bartter syndromes. An elevated serum concentration of PTH in a hypocalcemic subject suggests that the patient is secreting an abnormal PTH molecule, is resistant to PTH, or is experiencing a compensatory (secondary) PTH secretory response to hypocalcemia (Figure 17-5). Physicochemical characterization of the PTH molecule and analysis of PTH enable one to define the abnormality in PTH synthesis, post-translational processing, secretion, or activity leading to the functionally hypoparathyroid state. Analysis of GNAS and its pattern of imprinting permit identification of the specific genetic defect in the majority of patients with clinical PHP type IA and PPHP. Skeletal and renal responsiveness to PTH may be assessed if warranted by measurement of changes in serum calcium, phosphate, cyclic AMP, and calcitriol concentrations and measurement of urinary nephrogenous cyclic AMP and phosphate excretion following administration of biosynthetic PTH1-34 (Elsworth-Howard test). In the normal subject and in the patient with primary hypoparathyroidism, urinary cyclic AMP excretion increases tenfold to twentyfold and that of phosphate several fold. In the patient with PHP types IA and IB, there is less than a threefold increase in the excretion of cyclic AMP.

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The diagnosis of PHP type IB may be established by examining the imprinting pattern of GNAS (paternal pattern only) and analysis of STX16 as indicated. In the child with vitamin D deficiency, serum levels of calcidiol are subnormal. In patients with decreased renal 25OHD31␣-hydroxylase activity, serum concentrations of calcidiol are normal and those of calcitriol are inappropriately low. Increased concentrations of calcitriol suggest the presence of a defect in its nuclear receptor. The patient with renal failure is recognized by an increased serum creatinine value. Other findings in hypocalcemic subjects include prolongation of the QT interval by electrocardiography and calcification of the basal ganglia by cranial computerized tomography.

Management The primary goal in the care of the hypocalcemic child and adolescent is to increase serum calcium concentrations to levels at which the patient is asymptomatic and as close to the lower range of normal as possible. The secondary goal is to identify the cause of hypocalcemia as quickly as possible in order to provide disease-specific management.75,163 Asymptomatic hypocalcemia (total calcium ⬎7.5 mg/dL) may not require immediate intervention. With lower serum calcium levels or when hypocalcemia is symptomatic (tetany, seizures), acute management may require the intravenous administration of calcium gluconate (93 mg of elemental calcium/10 mL vial) at a slow rate (not greater than 2 mL/kg over 10 minutes) while closely monitoring pulse rate (and the QT interval). Acutely, intravenous administration of calcium is intended to ameliorate the more serious consequences of hypocalcemia such as seizures—not to restore and maintain the eucalcemic state. Intravenously, calcium should not be administered with phosphate or bicarbonate because these salts may co-precipitate. Extravascular extravasation of calcium is to be avoided because it may precipitate and cause local tissue injury. After the acute symptoms have resolved, calcium gluconate (10 mL in 100 mL 5% dextrose/0.25 N saline) may be temporarily infused intravenously at a rate sufficient to maintain calcium levels in the asymptomatic low-normal range while the cause of the hypocalcemia is identified and more specific therapy for persistent hypocalcemia prescribed. The evaluation should proceed as rapidly as possible, and oral therapy begun reasonably quickly. In the child with marked hyperphosphatemia as a cause of hypocalcemia, in addition to parenteral calcium administration infusion of normal saline sufficient to maintain urine output at or above 2 mL/kg per hour is necessary.152 Frequent measurement of serum calcium and phosphate concentrations permits rapid adjustment of fluid and electrolyte therapy. After stabilization, patients with hypoparathyroidism or PHP may be treated with calcitriol (20–60 ng/kg/day) and supplemental calcium (calcium glubionate or calcium citrate 30–75 mg elemental calcium/kg/day in divided doses, Table 17-3) to restore eucalcemia. The serum calcium concentration should be maintained within the low-normal range. Each patient must be carefully monitored to avoid hypercalcemia, hypercalciuria, nephrocalcinosis, and nephrolithiasis. Basal and periodic mea-

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surements of serum concentrations of calcium, phosphate, and creatinine and urinary calcium and creatinine excretion and renal sonography are mandatory. Children with autosomal-dominant hypoparathyroidism due to gain-of-function mutations in CASR are extremely sensitive to vitamin D and its metabolites. Even small doses of calcitriol may lead to hypercalciuria, with minimal increase in serum calcium levels. In this instance, addition of hydrochlorothiazide (0.5–2.0 mg/kg/ day) may increase renal tubular reabsorption of calcium and lower the calcitriol requirement. In adults with primary hypoparathyroidism due to a variety of causes, the use of rhPTH1-34 (0.5 ␮g/kg every 12 hours subcutaneously) together with calcium carbonate (1,000 mg/day of elemental calcium in four equally divided doses) has proven effective and safe for as long as 3 years—perhaps even safer than calcitriol because rhPTH1-34 did not lead to hypercalciuria.158 rhPTH1-34 led to acceleration of bone turnover, as reflected by increases in serum alkaline phosphatase and osteocalcin and urinary Pyr and Dpd values without substantial changes in bone mass. Because PHP type IA is associated with resistance to a number of peptide hormones that act through GPCRs, periodic assessment of pituitary-thyroid and pituitaryovarian function and GH secretion is necessary—and hormone replacement therapy begun as indicated. In general, the short stature of PHP type IA reflects the AHO phenotype and not GH deficiency. Transient hypoparathyroidism of infancy may be the initial manifestation of later-onset hypoparathyroidism. Thus, it is important to assess calcium homeostasis in such subjects throughout childhood. Patients with apparently isolated hypoparathyroidism of unknown etiology should be reevaluated periodically to identify the development of autoimmune disorders in the patient or family. Assessment of thymic function is important in those subjects with findings suggestive of the DGS. When hypomagnesemia is symptomatic, administration of magnesium sulfate parenterally may be necessary (50% solution, 0.1–0.2 mL/kg intramuscularly, repeated after 12–24 hours if needed). The patient with primary hypomagnesemia may require daily parenteral (intramuscular, intravenous) doses of magnesium sulfate in order to prevent tetany, seizures, and other neurological symptoms (slurred speech, choreo-athetoid movements, weakness) and to enable normal growth and development.188 Calcitriol alone raises serum calcium levels in hypomagnesemic subjects but is ineffective in the prevention of tetany. Continuous overnight nasogastric infusion of magnesium may help alleviate the gastrointestinal side effects of multiple large doses of oral magnesium. More mild and transient forms of hypomagnesemia may be treated with oral magnesium gluconate or tribasic magnesium citrate (Table 17-3).

HYPERCALCEMIA Etiology Causes of hypercalcemia in children and adolescents are listed in Table 17-4. In the presence of a normal serum protein concentration, hypercalcemia occurs when the

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

rate of entry of calcium into the extracellular and circulatory compartments exceeds its rate of loss and develops when the set-point for serum Ca2⫹e is increased due to a loss-of-function mutation in CASR, when the resorption rate of bone mineral (e.g., excessive secretion of PTH or PTHrP, constitutive activation of PTHR1, increased production of osteoclast-activating inflammatory cytokines, localized osteolytic processes such as metatastic neoplasms) or the absorption rate of intestinal calcium (hypervitaminosis D) exceeds the renal excretory capacity for calcium, or when there is augmented renal tubular absorption of calcium (e.g., administration of calciumsparing diuretics such as thiazides).151 The total serum calcium concentration is increased in the presence of hyperalbuminemia, whereas the Ca2⫹e concentration is normal. Venous stasis (e.g., by tourniquet) results in spuriously altered local pH and Ca2⫹e values. Familial hypocalciuric hypercalcemia type 1 (HHC1, OMIM 145980) is an autosomal-dominant disorder with 100% penetrance at all ages characterized by PTH-dependent usually asymptomatic, total and ionized hypercalcemia with hypocalciuria, slight hypermagnesemia, hypomagnesuria, and hypophosphatemia due to heterozygous loss-of-function mutations in CASR.189 Serum concentrations of PTH may be normal or slightly elevated, but inappropriately high for the Ca2⫹e level. Calcidiol and calcitriol values are normal. In children, HHC1 is most commonly suspected initially by the presence of unexpected hypercalcemia (11–14 mg/ dL) in a chemistry profile or through family screening of a parent or other relative with hypercalcemia. Older subjects with HHC1 may complain of fatigue, weakness, or polyuria. There is a slightly increased incidence of relapsing pancreatitis, cholelithiasis, chondrocalcinosis, and premature vascular calcification in HHC1. However, bone mass and fracture rate are normal. Because of decreased parathyroid chief cell membrane CaSR number and/or function, the set-point for Ca2⫹e suppression of PTH secretion is reset upward.84,189 The PTGs are slightly hyperplastic. In renal tubular cells, decreased CaSR number and activity lead to increased renal tubular reabsorption of calcium and relative hypocalciuria (ratio of calcium clearance to creatinine clearance ⬍0.01). Renal tubular reabsorption of magnesium is also increased. Urinary concentrating ability and other measures of renal function are normal. In hypercalcemia of other pathogenesis, urinary calcium excretion is increased and renal concentrating ability depressed. Usually, HHC1 requires no therapy— but must be differentiated from mild primary hyperparathyroidism in which hypomagnesemia and hypercalciuria are present. Subtotal parathyroidectomy in HHC1 does not lower calcium levels as the residual PTGs hypertrophy. Total parathyroidectomy is unnecessary except in infants with NSHPT. Approximately 70 nonsense, missense, insertion, and deletion mutations in CASR associated with HHC1 or NSHPT have been identified—mostly in the receptor’s extracellular Ca2⫹ binding domain. These mutations decrease receptor affinity for Ca2⫹ or alter intracellular processing of the CaSR (glycosylation, dimerization; e.g., Arg66His, Asn583Stop) in the endoplasmic reticulum, preventing its translocation to the surface of the cell

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membrane190 (Figure 17-7). Many of the mutations are located in the extracellular domain of CaSR between codons 39 and 300, a region rich in aspartate and glutamate residues in which Ca2⫹ may nestle.71 Glycosylation is necessary for dimerization and trafficking of the CaSR. Missense mutations involving arginine at codon 66 (Arg66His, Arg66Cys) result in a product that is able to be only partly glycosylated. It can form homodimers in the endoplasmic reticulum but cannot enter the golgi apparatus and be transported to the cell plasma membrane.190 Mutations in CASR tend to be unique to the affected family. In approximately one-third of families with HHC, no mutations in the coding region of CASR have been identified. They may have a mutation in a noncoding region of CASR or an abnormality in genes associated with the HHC phenotype that have been identified on chromosomes 19p13.3 (HHC2, OMIM 145981) and 19q13 (HHC3, OMIM 600740).189 Autoantibodies against the amino-terminal extracellular domain of the CaSR that reversibly inhibit receptor activity have been identified in a few patients with acquired HHC. This disorder may be responsive to glucocorticoid therapy.191 In some patients with primary or uremic secondary hyperparathyroidism, there is reduced expression of CASR and an increased setpoint for suppression of PTH secretion.192 Primary hyperparathyroidism is an unusual childhood disorder with an incidence of 2–5/100,000 compared to that in adults of approximately 100/100,000.193,194 In adults with hyperparathyroidism, females outnumber males 3:1. In children, the female/male ratio is closer to one. In older children and adolescents, primary hyperparathyroidism is most often a sporadic disease and usually the result of a single parathyroid adenoma. It also occurs as an autosomal-dominant disorder in familial isolated primary hyperparathyroidism (OMIM 145000) due to germline mutations in the genes responsible for multiple endocrine neoplasia (MEN) type I (MEN1), the hypoparathyroidism-jaw tumor syndrome (HPRT2), or HCC1.75,79,193,195 Many children (80% to 90%) with hyperparathyroidism and hypercalcemia are symptomatic at the time of diagnosis [headache, fatigue, abdominal pain, nausea and vomiting, polydipsia, and behavioral changes (particularly depression)] or present with symptoms reflecting the consequences of this disorder, including flank pain due to renal calculi (hypercalciuria) and pathologic fractures (through areas of osteopenia or lesions of osteitis fibrosa cystica).194 It is unusual to palpate a cervical mass in these patients. Hypercalcemia, hypophosphatemia, and elevated serum concentrations of intact PTH are present in the majority of children with hyperparathyroidism. Ultrasonography, magnetic resonance imaging, computed tomography, and radionuclide scans (99mTc-SestaMIBI) have been employed to localize the abnormal PTG(s). Occasionally, the parathyroid tumor may be located ectopically in the thymus, thyroid gland, or mediastinum. In subjects with primary hyperparathyroidism, hypercalcemia is the result of increased secretion of PTH due to loss of the normal relationship between the set-point of serum Ca2⫹e and PTH synthesis and release and to Ca2⫹-independent (constitutive) PTH secretion related to

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

the mass of parathyroid tissue.193 Pathologically, hyperparathyroidism in children is most often due to a chief cell adenoma involving one PTG. Hyperplasia (particularly in MEN) and rarely carcinoma of the chief cells may occur. The majority of PTG neoplasms are monoclonal in origin. That is, a single mutant cell develops into a tumor. In adolescents, parathyroid tumors may rarely develop after external radiation of the neck for treatment of lymphoma. In some parathyroid tumors, increased expression of cyclin D1 (encoded by CCND1, OMIM 168461, chromosome 11q13) has been demonstrated. Cyclins are intracellular proteins that regulate cyclindependent protein kinases that control the rate of transition of G1 to S in the cycle of cell division.71 Overexpression of CCND1 in parathyroid chief cells is at times the result of a somatic chromosome mutation-inversion (rotation) of regions 11p15 and 11q13 in which the promoter region of PTH is repositioned to serve as a promoter for CCND1, thereby increasing the rate of chief cell division whenever the (hypocalcemic) stimulus for PTH generation is received and leading ultimately to (benign) tumor formation. However, in many parathyroid adenomas there is increased activity of cyclin D1 without this chromosomal rearrangement.193 In patients with parathyroid adenomas and carcinomas, overexpression of the retinoblastoma and p53 tumor-suppressor genes (whose products inhibit the cell cycle at the G1/S step) has been demonstrated.71 Chronic renal insufficiency leads to secondary hyperparathyroidism due to hyperplasia of the PTGs and to monoclonal parathyroid tumors (tertiary hyperparathyroidism) associated with somatic chromosomal deletions. In approximately 17% of sporadic parathyroid adenomas, a somatic loss-of-function mutation of the tumor suppressor factor menin (the germ-line aberration in patients with MEN type I) can be identified.196 Germ-line loss-offunction mutations in MEN1 and CASR have also been detected in patients with isolated hyperparathyroidism in association with multiglandular involvement. In this instance, patients with CASR mutations did not have the typical biochemical findings of HHC1.195,197 Autosomal-dominant familial primary hyperparathyroidism associated with multiple ossifying fibromas of the jaw (HRPT2, OMIM 145001) is due to loss-of-function heterozygous germ-line mutations in the tumor suppressor gene encoding parafibromin (HRPT2).198 Hyperparathyroidism occurs in 80% of subjects with a mutation in HRPT2 at a mean age of 32 years, but may also appear in children before 10 years of age.199 In these patients, the parathyroid lesion may be an atypical potentially premalignant cystic adenoma (65%), hyperplasia (20%), or even carcinoma (15%). The parathyroid tumor may be isolated or associated with maxillary and/or mandibular tumors composed of ossified fibrous tissue. Renal (Wilms’ tumor, papillary renal cell carcinoma, hamartoma, polycystic kidney), pancreatic (carcinoma), and uterine (tumor) lesions may also develop in these patients.71,195 Lesions within the PTGs may develop asynchronously. HRPT2 is a 17-exon gene that encodes parafibromin, a 531-aa protein that is a component of a complex of accessory factors that modulate RNA polymerase II activity, gene transcription, and cell proliferation.

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For a parathyroid tumor to develop, a second hit must occur that results in loss-of-heterozygosity (i.e., the germline inactivating mutation of HRPT2 on one allele must be matched by a mutation in or deletion of HRPT2 in the remaining normal allele).200 When a germ-line mutation in HRPT2 has been detected, screening of family members for this mutation and longitudinal evaluation of affected subjects is recommended because asymptomatic individuals with atypical adenoma or carcinoma of the PTGs may be so identified.201 Somatic mutations in HPRT2 have also been identified in atypical parathyroid adenomas and carcinomas, but are distinctly unusual in the typical sporadic parathyroid adenoma.202 Familial isolated hyperparathyroidism type 3 (HRPT3, OMIM 610071) has been linked to chromosome 2p14-p13.3, but a specific mutated gene in this region has not as yet been identified. The syndromes of multiple endocrine neoplasia (MEN) are familial autosomal-dominant diseases of high penetrance associated with the development of tumors in two of three primary endocrine tissues (PTG, pituitary, pancreas) within the same person and multiple tumors in the same tissue (Table 17-5).195,203-206 Within a single family, there is a high degree of uniformity in the clinical expression of MEN1. Hyperparathyroidism (due to a parathyroid adenoma or tumors within or hyperplasia of all four parathyroid glands) is the most common manifestation of MEN1, occurring in more than 90% of affected patients. It is the most frequent endocrinopathy in children with MEN1, at times appearing before 10 years of age. There are equal numbers of males and females with hyperparathyroidism in MEN1. Pituitary tumors secreting prolactin and/or GH often (30% of MEN1 subjects) develop, as do gastrin(Zollinger-Ellison syndrome), insulin-, and glucagon-secreting tumors of the pancreatic islets (40% of patients). These neoplasms also occur in children and adolescents with MEN1.71,204 The Cushing syndrome that develops in patients with MEN1 may be due to excessive secretion of adrenocorticotropin by a pituitary adenoma or ectopically by a neoplasm or to a primary adrenal tumor. Benign and malignant thyroid tumors occur in 25% of patients with MEN1. Nonendocrine tumors such as facial angiofibroma (90%), collagenomas (72%), lipomas (34%), intestinal and bronchial carcinoids, and other intestinal neoplasms are reasonably common in subjects with MEN1. Indeed, the dermatologic manifestations of MEN1 (more than three angiofibromas and any collagenomas) are extremely sensitive and specific indicators of this disease.207 Patients with MEN1 may also develop a Schwannoma or a pheochromocytoma, the latter a tumor most often present in patients with MEN types IIA and IIB. Although most of the tumors that develop in MEN1 are benign but functionally hyperactive, those of pancreatic, intestinal, and foregut origin may be malignant. Germ-line mutations in MEN1, a 10-exon gene that encodes a 610-aa nuclear protein termed menin, have been demonstrated in the majority of patients with familial and sporadic forms of MEN1.204 Menin localizes to the nucleus through two nuclear localization signals in its carboxyl terminus (aa 479-498, 589-608), where it is involved directly in the regulation of transcription, replication, and the cell cycle.

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TA B L E 1 7 - 5

Multiple Endocrine Neoplasia Syndromes Subtype

Gene

Chromosome

Tumors

MEN I

MEN1

11q13

Parathyroid (90%) Pancreatic (50%) - gastrinoma, insulinoma pancreatic polypeptide, glucagonoma, VIPoma somatostatinoma, nonfunctional Anterior pituitary (35%) - prolactinoma, GH, ACTH, TSH, nonfunctional Adrenocortical (25%) - diffuse and nodular hyperplasia, adenoma, carcinoma Intestinal - foregut carcinoid tumor, gastric enterochromaffin-like Carcinoid - gastric, thymic, bronchial Other: facial angiofibroma, collagenoma, lipoma, pheochromocytoma (rare)

Intron 4 N:5168 G→A (10%) Codons 83-84 (4%) Codons 118-119 (3%) Codons 209-211 (9%) Codon 418 (4%) Codon 516 (7%)

RET

10q11 MCT (100%) MCT (95%) Pheochromocytoma (50%) Parathyroid hyperplasia (30%) Cutaneous lichen amyloidosis Megacolon MCT (100%) Pheochromocytoma (50%), ganglioneuroma Associated: Marfanoid habitus (100%) Mucosal neuromas (90%) Medullated corneal nerves

Codon 618 (⬎50%) Codon 634 (Cys→Arg ⬎80%)

MEN II Familial MCT MEN IIA

MEN IIB

Sites of Frequent Mutations

Codon 918 (Met→Thr ⬎95%)

MCT - Medullary carcinoma of thyroid Compiled from Marx SJ, et al. (1999). Multiple endocrine neoplasia type 1: Clinical and genetic features of the hereditary endocrine neoplasias. Rec Prog Horm Res 54:397–438; from Marx SJ, Simonds WF (2005). Hereditary hormone excess: Genes, molecular pathways, and syndromes. Endocrine Reviews 26:615–661; from Root AW (2000). Genetic disorders of calcium and phosphorus metabolism. Crit Rev Clin Lab Sci 37:217–260; and from Thakker RV (1998). Editorial: Multiple endocrine neoplasia, syndromes of the twentieth century. J Clin Endocrinol Metab 83:2617–2620.

Menin is a tumor suppressor factor. By binding directly to JunD, menin blocks JunD-mediated inhibition of transcription of activating protein-1 (and consequently cell division). Many of the mutations in MEN1 in patients with MEN1 cluster in exon 4 and interrupt the binding of these two proteins (between menin aa 139-142 and 323-428). However, loss of inhibition of JunD is but one of several mechanisms that lead to unrestricted cell growth in targeted MEN1 tissues.208 By interacting with SMAD3 (OMIM 603109, chromosome 15q21-q22), menin inhibits signaling by transforming growth factor (TGF) ␤ and impairs TGF␤-mediated inhibitory control of cell replication.209 Interaction of menin with the SMAD 1/5 complex inhibits signaling by bone morphogenetic protein (BMP) 2. Menin also inhibits the transcription-regulating protein nuclear factor ␬B (NF␬B).71,210 In subjects with heterozygous germ-line loss-of-function mutations in MEN1, unregulated cell growth and tumor formation occur when a second insult leads to loss of MEN1 on the normal allele within susceptible tissues. As with HPRT2, the two-hit hypothesis of tumorigenesis in MEN1 denotes that the patient receives germ-line suscepti-

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bility to neoplasia superimposed on which is a second insult leading to loss of heterozygosity for chromosome segment 11q13 and biallelic loss of MEN1. The second hit may be deletion of a segment of chromosome 11 that includes 11q13 or a mutation (missense, frame-shift) within the wt MEN1 allele itself, an observation that also extends to MEN type I tumors with somatic mutations in MEN1.195,204,211 Several hundred germ-line mutations in MEN1 have been identified in patients with MEN1, of which 25% have been nonsense mutations, 15% missense mutations, and 45% frame-shift insertions or deletions. More than 80% have led to the synthesis of an inactive product due to loss of the nuclear localization signal or the ability to bind to JunD or other downstream factor (Figure 17-9).204,212 Especially susceptible germ-line mutational hot spots in MEN1 are nucleotide 5168 G→A transition that results in a novel splice site in intron 4 (codons 83, 84, 118, 119, 209–211, 418, and 516), where collectively mutations have been identified in 37% of patients with MEN1.213 On either side of many of these sites are segments of repeat DNA sequences of single nucleic acids or of

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1812del5

100 bp

M561T

1650delC, 1650insC (2) R527X

W436X R460X (2) 1484del8 1509ins2 D418N ,D418del W436R F447S*

1280delG* 1279ins11

1202del2*

T344R (3) E363del

A309P

Q260X* W265X* A242V

W183X W198X 713delG 735del4 (2)

K120X* 512delC (6)

(2) K119del H139D, H139Y* F144V A160P A176P

E26K

P12L L22R

313delC* 357del4 (2) 416delC (5)

R108X

Germline Parathyroid

S308X Y312X Y323X 1132delG

934 + 1 (G A)

DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

L286P

718

Figure 17-9 Mutations in MEN1 associated with multiple endocrine neoplasia type 1. Long tethered boxes represent mutations in isolated parathyroid tumors. Nonsense, frame-shift, and splice site mutations are depicted above, and missense and in-frame deletions below the outline of the codons of MEN1. [Reproduced with permission from Marx SJ, et al. (1998). Germline and somatic mutation of the gene for multiple endocrine neoplasia type 1 (MEN1). J Intern Med 243:447–453.]

dinucleotides to octanucleotides. This configuration may lead to increased susceptibility to replication-slippage because of misalignment of the nucleotide repeat segments during DNA replication, permitting deletion or insertion of nucleotides at inappropriate sites. To date, no significant correlation between genotype and phenotype has been recognized in MEN1. The tissue-specific susceptibility to tumor formation with a mutation in MEN1 remains unexplained at present.195 Mutations in MEN1 have been detected in approximately 75% to 95% of patients with familial MEN1. In only 7% of patients with a variant of MEN1 in which only tumors of the PTG and the adenohypophysis are present has a mutation in MEN1 been identified.214 Familial autosomal-dominant isolated primary hyperparathyroidism has been variably associated with germline mutations (e.g., Val184Glu, Glu255Lys, Gln260Pro) in MEN1 as well.215 Somatic mutations in MEN1 have also been identified in patients with sporadic isolated tumors of the PTGs, pancreatic islet cells, anterior pituitary, and adrenal cortex.71 Clinically apparent disease due to mutations in MEN1 increases with advancing age. At 10 years of age, 7% of children with a mutation in MEN1 have a detectable endocrinopathy. Fifty-two percent of affected 20-year-old subjects manifest one or more tumors. Penetrance increases to 87% by 30 years, to 98% by 40 years, and to 100% by 60 years.216 New mutations of MEN1 occur sporadically in 10% of patients with MEN1. In 5% to 20% of families with MEN1, no mutation in the coding region of MEN1 has been identified—suggesting that there may be mutations in the untranslated regions of MEN1 or that another unrecognized gene(s) may be involved in the pathogenesis of the MEN1 phenotype. Medullary carcinoma of the thyroid (MCT) is the most common neoplasm encountered in MEN IIA, occurring in

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95% of patients (OMIM 171400, Table 17-4). These subjects also develop pheochromocytomas, parathyroid hyperplasia or adenoma, localized cutaneous lichen amyloidosis (suprascapular pruritic deposits of subepidermal keratin), and partial or complete megacolon. In addition to MCT and pheochromocytoma, patients with MEN IIB (OMIM 162300) have a Marfanoid habitus, mucosal neuromas of the lips and tongue, and gastrointestinal ganglioneuromas.195,206,217 Familial isolated MCT is a variant of these disorders. Germline gain-of-function mutations in the RET proto-oncogene underlie the pathogenesis of the type II hereditary endocrine neoplasias (Figure 17-10). RET is a 20-exon gene encoding three isoforms of a glycosylated cell membrane tyrosine kinase receptor with extracellular, transmembrane, and intracellular domains that is expressed in tissue of neural crest origin (sympathetic ganglia, adrenal medulla, thyroid parafollicular cells). The natural ligand of this receptor is glial-cell-linederived neurotrophic factor (GDNF, OMIM 600837, chromosome 5p13.1-p12).218 Constitutively activating mutations among five cysteine residues in the extracellular domain of RET, particularly at codon 634 (Cys→ Arg), are present in patients with MEN IIA. Loss of but one cysteine residue facilitates receptor homodimerization without ligand binding, activation of the RET intracellular tyrosine kinase domain, autophosphorylation of critical tyrosine residues (particularly at codons 1015 and 1062), and subsequent signal transduction.195 An activating mutation has been identified at codon 918 (Met→Thr) in more than 95% of patients with MEN IIB. This site lies within the tyrosine kinase domain and this mutation permits signal transduction and neural cell transformation and differentiation in the absence of ligand binding and receptor homodimerization. Missense mutations at codons 618 (Cys→Arg/Gly) or 620 (Cys→ Arg/Ser) permitting receptor dimerization in the absence

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Figure 17-10 Schematic depiction of the domains of the RET proto-oncogene with sites of activating mutations in RET associated with multiple endocrine neoplasia types IIA and IIB and familial medullary carcinoma of the thyroid. [Reproduced with permission from Marx SJ, Simonds WF (2005). Hereditary hormone excess: Genes, molecular pathways, and syndromes. Endocrine Reviews 26:615–661.]

of GDNF have been found in more than 50% of patients with familial MCT. Mutations within the tyrosine kinase domain (Val804Leu) have also been identified in some of these families. Two co-receptors, GFRA1 and GFRA2, interact with Ret protein—but the role of the co-receptors in the pathogenesis of MEN II, if any, has not been identified. In addition to the disorders designated multiple endocrine neoplasia, other familial syndromes associated with multiple tumors of the endocrine system include VonHipple Lindau (adrenal medulla, pancreas, neuroendocrine), Carney complex (adrenal cortex, testes, pituitary, thyroid), McCune-Albright (pituitary, adrenal cortex), and neurofibromatosis type I (adrenal medulla, thyroid).195,217 In children, hyperparathyroidism is an unusual manifestation of the McCune-Albright syndrome due to a postzygotic activating mutation of GNAS.219 Ingestion of excessive amounts of vitamin D or calcitriol for therapeutic reasons (treatment of rickets, hypoparathyroidism, or other causes of hypocalcemia), megavitamin intake, and inappropriate fortification of milk are the major causes of hypervitaminosis D.220-222 Topical application of creams containing vitamin D or an analogue (e.g., 22-oxacalcitriol) for treatment of psoriasis might also lead to hypercalcemia, particularly if the urinary excretion of calcium is compromised.223,224 Patients with granulomatous diseases (noninfectious sarcoidosis, berylliosis, eosinophilic granuloma, subcutaneous fat necrosis, and inflammatory bowel disease and infectious tuberculosis, histoplasmosis, coccidioidomycosis, candidiasis, and cat-scratch disease) and neoplastic disorders (B-cell lymphoma, Hodgkin disease, dysgermi-

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noma) develop hypercalcemia due to associated monocytic (macrophage and other cells) expression of 25OHD31␣-hydroxylase activity and production of calcitriol.225 Unlike renal tubular cells (in which this enzyme is within mitochondria and the transcription of CYP27B1 is closely regulated by PTH, calcitriol, calcium, and phosphate), in monocytes 25OHD3-1␣-hydroxylase is microsomal in location and its gene is constitutively expressed and calcitriol synthesis regulated by the amount of substrate. The monocytic expression of CYP27B1 is very sensitive to stimulation by interferon-␥ and its postreceptor signal transducer nitric oxide, as well as by leukotriene C4. It is easily suppressed by glucocorticoids, ketoconazole, and chloroquine. Subjects with acquired immunodeficiency disease may become hypercalcemic because of being infected with granuloma-forming organisms or by osteoclast-activating cytokines elaborated during the course of this disorder. Elevated serum calcium concentrations have been recorded in children with congenital hypothyroidism, primary oxalosis, congenital lactase deficiency, and trisomy 21.226 Hypercalcemia has occurred in adolescents with juvenile rheumatoid arthritis due to increased synthesis of the osteoclast-activating cytokine interleukin-1␤.227 In some hypercalcemic children, excessive prostaglandin production may be of pathogenetic significance. Hypercalcemia develops frequently in the infantile form of hypophosphatasia, likely a consequence of the dissociation of the rates of low bone formation and normal bone resorption.228 Hypercalcemia may follow successful bone marrow transplantation in infants with osteopetrosis because functional osteoclasts rapidly reabsorb excess bone mineral. Oncogenic hypercalcemia may be the consequence of secretion of PTHrP, (rarely) PTH, or calcitriol—or of direct invasion and destruction of bone by the neoplasm.229 Although hypercalcemia occurs in less than 1% of children with cancer, it may develop in patients with leukemia, Hodgkin and non-Hodgkin lymphoma, rhabdomyosarcoma, hepatoblastoma, neuroblastoma, and Ewing sarcoma.230 Acute immobilization of the rapidly growing child with a femoral fracture or a spinal cord injury results in decreased bone mineral accretion and uncoupling of the interaction of osteoblasts and osteoclasts with increased rate of bone resorption, leading to hypercalciuria and “acute disuse osteoporosis.”231 When the rate of bone resorption exceeds the renal tubular capacity for excretion of calcium, hypercalcemia ensues. Acute disuse osteoporosis and hypercalcemia can even occur in the immobilized hypoparathyroid or vitamin-D-depleted individual. Increased intake of calcium and absorbable alkali (milk or calcium-containing antacids such as calcium carbonate) for peptic ulcer disease or as dietary supplements leads to absorptive hypercalcemia, hypercalciuria, and nephrocalcinosis. Parenteral nutrition with excessive calcium or aluminum or too little phosphate also leads to hypercalcemia. Hypophosphatemia of various etiologies leads to hypercalcemia as the body attempts to maintain the calcium X phosphate product over 30. Drugs causing hypercalcemia include thiazide diuretics (which increase renal tubular resorption of calcium and decrease plasma volume), vitamin D and analogs

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(which increase intestinal absorption of calcium), vitamin A and its retinoic acid analogs (which stimulate bone resorption), and lithium (which increases the set-point for PTH secretion, thereby increasing serum calcium concentrations while lowering urinary calcium excretion and thus mimicking HHC1). In the thyrotoxic subject, hypercalcemia is the result of thyroid-hormone-mediated stimulation of osteoclast function and subsequent increase in the rate of bone resorption. Pheochromocytomas and some islet cell tumors may be associated with hypercalcemia, in some instances because of co-secretion of PTHrP. Hypercalcemia in the hypoadrenal patient is of uncertain pathogenesis but is not related to increased serum concentrations of PTH, calcidiol, or calcitriol—or with augmented bone resorption. During recovery from acute renal failure, serum calcium levels may increase due to mobilization of calcium from ectopic sites in which it had been deposited during the hyperphosphatemic phase of the illness. Hypercalcemia can develop in patients with chronic renal failure due to a combination of factors, including immobilization, aluminum toxicity, excessive ingestion of calcium antacids or vitamin D or its analogs, and hyperparathyroidism. After renal transplantation, hypercalcemia is the result of secondary hyperparathyroidism due to hypertrophy and hyperplasia of parathyroid chief cells that occurred in response to the PTH stimulatory effects of hyperphosphatemia, hypocalcemia, and decreased synthesis of and response to calcitriol during the period of chronic renal insufficiency. In patients with compromised renal function, mild hypocalcemia and calcitriol deficiency develop when the glomerular filtration rate falls below 80 to 60 mL/min/1.73 m2—whereas phosphate retention occurs after the glomerular filtration rate has fallen to 60 to 30 mL/min/1.73 m2.232 The secretion of PTH rises in these patients in an effort to increase the synthesis of calcitriol, raise calcium levels, and lower phosphate values. Prolonged uncontrolled secondary hyperparathyroidism can lead to relatively autonomous parathyroid hyperfunction (“tertiary hyperparathyroidism”) and hypercalcemia, primarily in patients with chronic renal failure. Hyperplasia of chief cells is followed by defects in function of the CaSR and loss of effective down-regulation of PTH secretion refractory to increased serum concentrations of Ca2⫹e. There is an expanded number of monoclonal chief cells in which the expression of CASR and the number of vitamin D nuclear receptors have declined. Secondary and tertiary hyperparathyroidism have occurred in patients with prolonged nutritional vitamin D deficiency rickets and in subjects with X-linked hypophosphatemic rickets receiving large amounts of phosphate.232,233 Enhanced but transient secretion of PTH may accompany the administration of GH to adolescents with chronic renal failure, likely the result of superimposing upon a high basal rate of PTH secretion further increase in the rate of bone remodeling related to GH and sex hormones.234 In acutely ill adults, GH administration has been associated with hypercalcemia as well.231 Isolated hypercalciuria in the eucalcemic child may be idiopathic or due to renal medullary or tubular dysfunction, mutations in the gene encoding the CLCN5 chloride chan-

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nel, demineralizing disorders such as juvenile rheumatoid arthritis, hyperalimentation, metabolic acidosis, excessive protein ingestion, diabetes mellitus, hypomagnesemia, Bartter syndrome, and other disorders.235 Secondary hyperparathyroidism (in which by definition serum calcium concentrations are normal) also develops in patients with inadequate dietary calcium, impaired intestinal absorption of calcium (lactose intolerance, ingestion of phytates, malabsorption syndromes due to pancreatic insufficiency or celiac disease), or excessive calcium loss in the urine or soft tissues.232

Evaluation When hypercalcemia is mild (total calcium concentration ⬍12 mg/dL), there may be few (if any) symptoms. Such children/adolescents are often identified by a multichannel screening study obtained for some other purpose. Hypercalcemia may also be detected during studies for renal calculi, abnormal bone mass, pathologic fractures, or during screening of families for associated problems. It should also be recognized that an elevated serum (total or ionized) calcium concentration in a single specimen may reflect assay variability and must be verified by repeated determinations in a reliable laboratory. Pseudohypercalcemia is the presence of persistently elevated total calcium concentrations while the Ca2⫹e is normal and is found in hyperalbuminemia and other dysproteinemic states.226 Symptoms attributable to hypercalcemia are independent of its cause and are related to the degree of hypercalcemia and include intestinal symptoms [anorexia, nausea, vomiting, abdominal pain (peptic ulceration, acute pancreatitis)] and constipation, urinary symptoms [polydipsia, nocturia, and polyuria (calcium acts as an osmotic diuretic while hypercalcemia impairs the concentrating function of the distal renal tubule)], skeletal symptoms (bone pain), and nervous system symptoms [headache, muscular weakness, impaired ability to concentrate, increased requirement for sleep, altered consciousness (ranging from lethargy and confusion to irritability, delirium stupor, and coma)]. On occasion, depression may be the major presenting concern in an adolescent with hypercalcemia.151,236,237 In the toddler and young child, hypercalcemia is manifested by anorexia, constipation, poor weight gain, and impaired linear growth (failure to thrive). In a series of 52 children and adolescents with hypercalcemia due to primary hyperparathyroidism, 80% were symptomatic. The most common symptoms were fatigue/lethargy (35%), headache (35%), nausea (29%), vomiting (23%), and polydipsia (21%).194 Bone involvement (low bone mass, fractures) was present in 30%, and 14% of these subjects were depressed. All of the children (N ⫽ 17) with nephrolithiasis in this series were symptomatic. Evaluation of the hypercalcemic child begins with the historical review (Figure 17-6), during which the family/ patient is queried not only about symptoms related to hypercalcemia and its consequences (renal calculi) but about possibly excessive intake of vitamin D, vitamin A, and related compounds (such as retin A for treatment of

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

acne); of calcium (perhaps to “prevent” osteoporosis), and of alkali or drugs that affect calcium metabolism (thiazide diuretics may “unmask” hyperparathyroidism by increasing renal tubular resorption of calcium and thereby raising borderline calcium concentrations into the hypercalcemic range). The family history is explored for members with known disorders of calcium metabolism (HHC1, hyperparathyroidism, renal calculi) or familial neoplasms (galactorrhea as a sign of a prolactinoma, and severe peptic ulcer disease as an indicator of a gastrinoma and the Zollinger-Ellison syndrome). Except in extreme instances [when hypertension (if normally hydrated) or bradycardia, dehydration, decreased muscular strength, or altered consciousness may be present or in the subject with MEN IIB], physical examination of the hypercalcemic child and adolescent is usually normal. Rarely is a paratracheal (parathyroid) mass palpable in the hyperparathyroid patient. Subjects with hypercalcemia due to subcutaneous fat necrosis have firm to hard irregular movable masses scattered about the trunk and extremities. Those with the WilliamsBeuren syndrome have a typical face, whereas those with Jansen metaphyseal chondrodysplasia have characteristic skeletal deformities. After confirming the presence of total and ionized hypercalcemia, the urinary excretion of calcium is next measured (Figure 17-6). If calcium excretion is low, it is most probable that the patient has HHC1. This diagnosis can be substantiated by the finding of asymptomatic hypocalciuric hypercalcemia in one of the parents and further defined by identification of the inactivating mutation in CASR. If the patient is hypercalciuric, other causes of hypercalcemia should be sought. With highly sensitive and moderately specific immunoradiometric and immunochemiluminometric assays for intact PTH184 (these assays may also measure larger carboxyl-terminal fragments of PTH; e.g., PTH7-84) in comparison to serum calcium values, separation of patients with hyperparathyroidism from those with other causes of hypercalcemia in whom PTH values are low or normal is usually possible. In the absence of secondary hyperparathyroidism (chronic renal insufficiency, malabsorption syndromes, ingestion of thiazide diuretics or lithium), consistently elevated PTH concentrations in the hypercalcemic, hypophosphatemic, hypercalciuric child or adolescent are consistent with primary hyperparathyroidism. Although the diagnosis of primary hyperparathyroidism is usually quite apparent in children and adolescents, there is an occasional patient in whom serum calcium and/or PTH values may not be elevated in a single specimen and in whom repeated measurements of serum and urine calcium and PTH values are necessary before this diagnosis can be established. In the series of 52 children/ adolescents with hyperparathyroidism previously described, serum calcium values were normal in 10% and PTH levels in 15%. However, in all subjects the PTH concentration was inappropriately increased relative to the calcium level.194 Phosphate values are usually low for age in this group. Osteitis fibrosa cystica, brown tumors (localized non-neoplastic areas of bone resorption composed of

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osteoclast-like multinuclear giant cells, fibroblast-like spindle-shaped cells, and hemorrhagic infiltrates), and subperiosteal and endosteal bone resorption can be detected radiographically in some children with hyperparathyroidism. Cortical (distal radial) bone mineral density is likely to be decreased in these subjects. Nonspecific findings in the hypercalcemic subject of diverse etiology include shortening of the QT interval by electrocardiography because the rate of cardiac repolarization is increased by increased calcium levels, bradycardia, and first-degree atrioventricular block—and nephrocalcinosis and renal calculi detected by abdominal ultrasonography. Serum concentrations of PTHrP should be measured when clinical and laboratory findings are consistent with primary hyperparathyroidism, but PTH values are low and humoral hypercalcemia of malignancy is suspected. When PTH concentrations are low in the hypercalcemic patient, metabolites of vitamin D (calcidiol, calcitriol) should be measured and other causes of hypercalcemia sought. Preoperatively, a parathyroid adenoma may be localized by high-resolution ultrasonography, computed tomography, magnetic resonance imaging, or radionuclide scanning with 99mTc-sestamibi. The latter radionuclide is taken up by the thyroid and parathyroid glands but quickly washed out from the thyroid gland. Thus, two scans obtained 2 hours apart permit differentiation of parathyroid from thyroid tissue.193 Alternatively, a simultaneously administered second radionuclide selectively accumulated by the thyroid (123iodine) may be employed to visualize parathyroid tissue. Scans may be obtained by conventional two-dimensional or computed tomographic techniques, the latter offering a three-dimensional image. Only occasionally is it necessary to undertake selective venous catheterization with sampling of local PTH levels and/or arteriography to identify the site of a parathyroid adenoma. Evaluation for associated endocrine tumors is necessary if the family history suggests the possibility of MEN I or MEN IIA or if there are clinical findings (galactorrhea, excessive growth, hypertension) to suggest the presence of a prolactinoma, somatotropinoma, or pheochromocytoma. Patients at risk for MEN may be screened by determining basal and stimulated serum concentrations of prolactin, GH, IGF-I, gastrin, glucagon, pancreatic polypeptide, calcitonin, catecholamines, and other substances as warranted. Indeed, it is reasonable to consider screening children with hyperparathyroidism preoperatively for an associated pheochromocytoma or tumors of the maxilla and mandible and for mutations in RET, MEN1, and HPRT2.195,204 Prior to surgery, survey of the pattern of bone mineralization for preferential loss of cortical rather than cancellous bone might also be considered. In the patient with hypercalcemia due to ingestion of excessive amounts of vitamin D, serum concentrations of calcidiol are markedly elevated. In those receiving exogenous calcitriol or in hypercalcemic patients with granulomatous, chronic inflammatory and lymphomatous diseases serum levels of calcitriol are increased. Other disorders associated with hypercalcemia (Table 17-4) should be eliminated by appropriate historical findings and laboratory studies.

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Management Appropriate management of the patient with hypercalcemia depends on the severity and the cause of the high calcium levels. It is important to identify the patient with a pathologic cause of hypercalcemia so that effective medical and surgical therapy may be initiated promptly. It is equally important to recognize the child/adolescent with HHC1 so that aggressive therapy is avoided. When the calcium concentration is ⬍12 mg/dL in the asymptomatic subject, treatment may be delayed until the cause of the hypercalcemia is understood. In the interim, it is reasonable to recommend that the patient increase fluid intake, avoid calcium and vitamin D supplements, and discontinue drugs associated with hypercalcemia if possible. The child with HHC1 often has serum total calcium concentrations between 11 and 13 mg/dL but no clinical symptoms and requires no therapy under usual circumstances. In the absence of HHC1, if the total serum calcium concentration exceeds 12 mg/dL or if the child is symptomatic efforts to lower this level are necessary because of the adverse effects of hypercalcemia on cardiac, central nervous, renal, and gastrointestinal function. In these children, diagnostic studies as outlined previously and treatment should begin simultaneously. The first tool of therapy of severe hypercalcemia is hydration [with 0.9% saline (twice maintenance volume over 24-48 hours), which restores intravascular volume, dilutes and decreases serum Ca2⫹e levels, increases glomerular filtration of Ca2⫹, decreases reabsorption of Ca2⫹ in the proximal and distal renal tubules, and promotes calciuresis]. Hydration alone usually lowers the total serum calcium concentration 1 to 3 mg/dL. A second tool is calciuresis. Intravenous infusion of the loop diuretic furosemide (1 mg/kg slowly) initiated only after restoration of extracellular fluid volume with saline further lowers calcium levels by inhibiting resorption of calcium (and sodium) by the TALH. Thiazide diuretics are to be avoided because they increase renal tubular reabsorption of calcium and increase serum calcium concentrations and are clearly contraindicated in the management of hypercalcemia. A third tool is inhibition of bone resorption. If hypercalcemia does not respond to the above measures, specific inhibitors of osteoclast function may need to be employed. Bisphosphonates (pamidronate, etidronate, zoledronic acid) are the agents of choice in the acute management of hypercalcemia in children and adolescents. Bisphosphonates are phosphatase-resistant analogs of pyrophosphate. In vivo, they inhibit osteoclast function by impeding osteoclast differentiation. By binding to and coating the hydroxyapatite crystal beneath the osteoclast, they interfere with its attachment and functional ability to dissolve bone.236 Pamidronate (0.5–1.0 mg/kg/dose by intravenous infusion over 4–6 hours) has effectively lowered serum calcium concentrations in hypercalcemic infants and children.102,103,230 The hypocalcemic effect of bisphosphonates is variable in duration (days to weeks). Transient systemic side effects (fever, myalgia) may accompany the administration of pamidronate and other bisphosphonates. Salmon calcitonin also acts rapidly, but transiently, to lower

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serum calcium concentrations by inhibiting osteoclast activity and increasing urinary calcium excretion. It must be administered by multiple daily subcutaneous injections (2–4 U/kg/injection every 6–12 hours). Calcitonin may be paired with a bisphosphonate at the beginning of treatment of hypercalcemia in order to lower serum calcium levels more rapidly.151 Glucocorticoids do not lower calcium levels in patients with hyperparathyroidism or solid tumor malignancies but are quite effective in the management of hypercalcemia due to excess vitamin D ingestion or calcitriol production by activated monocytes or hematologic malignancies. Rarely, it may be necessary to dialyze (peritoneal or hemodialysis) with low- or zero-calcium dialysate the severely hypercalcemic patient resistant to conventional therapy. During acute treatment of hypercalcemia, Ca2⫹e concentrations may be assessed indirectly by monitoring the (shortened) QT interval by electrocardiography. Although in older adults (⬎50 years) with asymptomatic primary hyperparathyroidism (without bone disease or renal stones) no immediate intervention may be advised at times, in younger adults, children, and adolescents with hyperparathyroidism surgical intervention is recommended when the diagnosis is established. Although hyperparathyroidism in youth is most often (⬎60%) due to an adenoma, hyperplasia of the parathyroid glands is also common (⬃30%).194 In the pediatric population, efforts are undertaken to localize a parathyroid adenoma before its removal by a surgeon with experience and expertise in parathyroid surgery.238 In adults and older adolescents, minimally invasive procedures for removing a parathyroid adenoma(s) may be employed. 99mTc-sestamibi is administered 2 hours before surgery in conjunction with single-photon emission computed tomography. If the scan is consistent with an adenoma, skin and subcutaneous tissue are infiltrated with a local anesthetic, a horizontal 2.5-cm incision is made on the side of and slightly above the suspected parathyroid adenoma, and dissection directed by insertion of a handheld gamma probe until the adenoma is located and removed. Video assistance is an alternative method for localization of a parathyroid adenoma. A complementary technique for assessing the completeness of the removal of the adenoma intraoperatively is to measure peripheral levels of PTH by rapid immunoassay before and 10 minutes after removal of the adenoma. A 50% decline in PTH levels indicates complete excision. If the PTH concentration does not decline following removal of suspected abnormal parathyroid tissue, further exploration is undertaken and additional tissue removed as guided by the serum PTH level. These procedures are reported to be cost effective because they decrease operative time and patient morbidity. Many patients return home within hours after leaving the operating suite. These techniques have been applied to the surgical management of the child and infant with a parathyroid adenoma or hyperplasia of multiple PTGs. If there is parathyroid hyperplasia, subtotal (3.5 glands) or total parathyroidectomy is performed and autotransplantation of small fragments of one gland to a forearm pocket considered. Following removal of a parathyroid adenoma, many patients develop transient hypocalcemia

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

that may be managed by the administration of supplemental oral calcium. When there is severe osteitis fibrosa cystica and marked demineralization, substantial hypocalcemia may occur as a result of the “hungry bone” syndrome. Other complications of surgery include (transient) vocal cord dysfunction and the need for further operations because of development of a second adenoma. If permanent hypoparathyroidism develops postoperatively, it is treated by the administration of calcitriol and supplemental calcium as needed. Calcimimetic agents (phenylalkylamines) bind to and activate the membrane CaSR on the parathyroid chief cell and thereby increase cytosolic levels of Ca2⫹i and depress secretion of PTH in adults with mild primary hyperparathyroidism.193 The use of these agents in children and adolescents with this disorder remains to be assessed, but they offer a potential treatment pathway for subjects with diffuse parathyroid hyperplasia. The secondary hyperparathyroidism of chronic renal disease is best managed by lowering serum phosphate concentrations to the extent possible by limiting intake and by administration of oral phosphate binding agents and by maintaining serum Ca2⫹e levels within the low-normal range by the administration of calcitriol or analogue. Calcimimetic agents (e.g., cinacalcet) may also be useful in this situation.239 Parathyroidectomy may be necessary for effective management of refractory secondary and tertiary hyperparathyroidism as manifested by severe renal osteodystrophy, hypercalcemia, and systemic symptoms such as pruritus and bone pain.232 Hypercalcemia due to hypervitaminosis D or excessive production of calcitriol by granulomatous and chronic inflammatory tissues may be treated with glucocorticoids to suppress activity of 25OHD3-1␣-hydroxylase. Ketoconazole (3–9 mg/kg/day in three divided doses) is an antifungal agent that also inhibits 25OHD3-1␣-hydroxylase activity, and that promptly lowers calcitriol and calcium values in children and adults with similar disorders.240 Side effects of therapy with ketoconazole include nausea, vomiting, abdominal pain, depressed secretion of gonadal steroids, and adrenal production of cortisol. Therefore, careful monitoring of patients receiving ketoconazole is essential. Glucocorticoids ameliorate the hypercalcemia related to excessive interleukin-1␤ production in adolescents with juvenile rheumatoid arthritis.227 An attempt should be made to prevent hypercalcemia in the immobilized child or adolescent by ingestion of a low-calcium diet, avoidance of vitamin D, copious fluid intake, and early mobilization. Serum and urine calcium levels should be monitored frequently, and fluids increased still further if hypercalciuria occurs. Once present, hypercalcemia is best treated by mobilization. Saline diuresis and/or bisphosphonate administration may be necessary until eucalcemia is restored. Restriction of dietary calcium and limitation of exposure to sunlight may be appropriate in the long-term management of some patients with hypercalcemia not amenable to more specific treatment. Antiprostaglandin agents may be useful in the child with hypercalcemia associated with excessive production of these compounds. Specific treatment of diseases accompanied by hypercalcemia (thyrotoxicosis, hypoadrenocorticism) restores the eucalcemic state.

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Disorders of Bone Mineralization and Formation Bone formation may be impaired because of lack of minerals (calcium and/or phosphate) or because of deficient production of bone matrix. Bone mineralization may be excessive because of increase in the rate of mineral deposition or decrease in the rate of resorption of the mineral phase of bone. Ectopic calcification of extraskeletal tissues may occur when local calcium and phosphate levels are high, whereas extraskeletal ossification may ensue when the regulation of bone formation is deranged.

RICKETS Rickets and osteomalacia are disorders that result from decreased mineralization of bone matrix due to deficiencies of calcium and/or phosphate241-243 (Table 17-6). During endochondral bone formation in children, matrix is elaborated and subsequently mineralized. When endochondral osteoid is not fully mineralized, the ends of the long bones (particularly those that are weight bearing) deform and rickets ensues. During the processes of modeling and remodeling of trabecular bone and the periosteal and endosteal surfaces of cortical bone, osteoid is formed by osteoblasts. Failure to mineralize bone matrix in these regions results in osteomalacia. During intervals of calcium and/or phosphate deprivation, the actively growing weight-bearing child with open cartilage growth plates develops rickets and osteomalacia—whereas adults develop only osteomalacia during remodeling as unmineralized bone matrix accumulates. Thus, rickets is the expression of defective endochondral mineralization at the growth plate and osteomalacia is the failure of mineralization of bone cortex and trabeculae. Clinically, rickets is manifested by skeletal deformities such as delayed closure of the fontanelles, craniotabes (reversible compression of the skull’s outer table), frontal bossing (expansion of cranial bones), and occasional craniosynostosis in infants; delayed tooth eruption with poor enamel formation and propensity to caries; pectus carinatum, prominence of the costochondral junctions, and flaring of the lower rib cage; scoliosis and kyphosis; and genu varum and/or valgum, flaring of the metaphyses of the long bones, and tibial or femoral torsion. Radiographically, rickets is characterized by cupping, splaying, and fraying of the metaphyses of long bones and demineralization. Osteomalacia is associated with increased fracture risk as well as limb deformities. Histologically, as a consequence of impaired calcification within the cartilage growth plate the pattern of chondrocyte differentiation and maturation is disrupted and disorganized—whereas osteoid seams widen at other sites of bone formation.241 In subjects deprived of phosphate, it is the trabeculae that are primarily undermineralized. Impaired mineralization of osteoid may be due to dietary deficiencies or depressed intestinal absorption of calcium, phosphate, or vitamin D; inadequate amounts of these nutrients in fluids utilized in total parenteral nutrition; metabolic errors in the metabolism or action of

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

TA B L E 1 7 - 6

Disorders of Bone Mineralization: Rickets I. Vitamin D Deficiency A. Decreased Intake, Endogenous Synthesis, Retention, or Sequestration 1. Maternal vitamin D deficiency - breast feeding 2. Reduced skin synthesis - sunlight deprivation, sunscreen use, increased skin pigmentation 3. Malabsorption - celiac disease, hepatobiliary dysfunction, short gut syndrome, cystic fibrosis, inflammatory bowel disease, gastric bypass surgery 4. Drugs - anticonvulsants, glucocorticoids, cholestyramine 5. Nephrotic syndrome 6. Obesity B. Metabolic Errors 1. Deficiency of 25-hydroxylase a. Loss-of-function mutation of CYP2R1 b. Hepatic dysfunction 2. Deficiency of 25OH-vitamin D3-1-hydroxylase a. Loss-of-function mutation of CYP27B1 b. Decreased renal mass - hypoplasia, chronic renal insufficiency 3. Loss-of-function mutation of vitamin D receptor (VDR) II. Calcium Deficiency A. Nutritional Deprivation B. Hypercalciuria - Hyperprostaglandin E2 Syndromes III. Phosphate Deficiency A. Nutritional Deprivation 1. Low-birth-weight infant 2. Aluminum-containing antacids B. Hyperphosphaturia 1. X-linked-dominant familial hypophosphatemic rickets (PHEX) 2. Autosomal-dominant hypophosphatemic rickets (FGF23) 3. Autosomal-recessive hypophosphatemic rickets with hypercalciuria (SLC34A3) 4. Autosomal-recessive hypophosphatemic rickets (DMP1) 5. X-linked-recessive hypophosphatemic rickets (CLCN5) 6. Oncogenic hypophosphatemic osteomalacia (FGF23, sFRP4, MEPE, FGF7) 7. Renal tubular acidosis a. Renal tubular acidosis - Fanconi syndrome • Heritable - cystinosis, tyrosinemia, hereditary fructose intolerance, galactosemia, idiopathic (AD, AR, XLR) • Acquired - nephrotic syndrome, vitamin D deficiency, renal vein thrombosis, cadmium, lead, bismuth, outdated tetracycline, 6-mercaptopurine, valproic acid, ifosfamide, saccharated ferric oxide IV. Hypophosphatasia A. Perinatal, Infantile, Childhood, and Adult Forms (ALPL) B. Odontohypophosphatasia C. Pseudohypophosphatasia V. Inhibitors of Mineralization A. Aluminum - Parenteral B. Bisphosphonates C. Fluoride

vitamin D; defects in renal tubular conservation of phosphate or calcium; or abnormalities of alkaline phosphatase generation and function (Table 17-6). Broadly, rickets may be considered calciopenic— usually related to nutritional deprivation of vitamin D (or

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rarely to a defect in its metabolism to the active metabolite calcitriol or in its cellular action), to decreased intake of calcium or its excessive loss in urine, or phosphopenic (related to renal phosphate wasting due to primary renal tubular defects in phosphate reabsorption or to generation of excessive amounts of phosphatonins, compounds that inhibit normal renal handling of phosphate). Thus, nutritional rickets may be due to decreased intake of vitamin D (or inadequate exposure to sunlight) or calcium or to marginal intakes of both nutrients. Dietary deficiency of phosphate is unusual, given its wide availability. However, this nutrient may be deficient in parenteral fluids. Bone mineralization may also be directly impaired by abnormalities of alkaline phosphatase generation or by agents such as aluminum or fluoride.241

Calciopenic Rickets Neonates born to severely vitamin-D-deficient mothers may display signs of rickets at birth, including fractures and hypocalcemia. Clinical manifestations of rickets in preambulatory infants include bowing of the forearms, craniotabes, frontal bossing, and delayed closure of the cranial fontanelles.243 In older infants and children, genu varum or valgum (bowed legs or knock knees) or a windswept deformity involving both legs, flaring (widening) of the metaphyses of the long bones with markedly enlarged wrists, prominence of the costochondral junctions (rachitic rosary), and indentation of the lower anterior thoracic wall (Harrison’s groove) are noted. Tooth eruption may be delayed, and the enamel hypoplastic (predisposing to dental caries). Short stature and suboptimal weight are also frequently present.244 Because vitamin D has so many extraskeletal sites of action, there are a number of nonosseous systemic symptoms observed in children with vitamin-D-deficiency rickets. These include muscular weakness (manifested by hypotonia and delay in walking), anorexia, and increased susceptibility to infection—particularly pneumonia (due both to the lack of the stimulatory effect of vitamin D on the immune system and weakness of the thoracic wall).245 Occasionally, reversible cardiomyopathy may develop. Hypocalcemia, tetany, and seizures may occur in a severely vitamin-D-deficient infant without gross clinical or radiographic signs of rickets. Rarely, vitamin D deficiency in an adolescent may be associated with hypocalcemic seizures and fractures.246 Radiographically, osteopenia with cortical thinning and thin stress fracture lines—as well as cupping, widening, and irregularity (fraying) of the distal metaphyses of the long bones—are observed in the rachitic subject.12,242 Areas of osteitis fibrosa cystica associated with secondary hyperparathyroidism may sometimes develop. Bone pain is the most common symptom of osteomalacia in the adult. After the introduction of cod liver oil as a dietary supplement in 1918 (and the later fortication of infant formulas and milk and other foods with irradiated ergosterol) and the discovery in 1919 that exposure to sunlight prevented development of rickets, nutritional deficiency of vitamin D became relatively unusual in North America— only to reemerge seven decades later.247 Presently, deficiency of vitamin D occurs predominantly in darkly

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

skinned black or brown infants and young children who ingest a diet low in vitamin D (breast milk from a vegetarian or poorly nourished mother or one with little or no dietary milk, meat, eggs, or fish) without supplemental vitamin D and who have limited exposure to sunlight because they are confined indoors due to illness, the climate, or parental choice or because they wear clothing that shields the entire body from sunlight.244,245 More than half of 30 infants with nutritional vitamin D deficiency cared for at two North Carolina medical schools between the years 1990 and 1999 were identified in 1998 and 1999, suggesting an increasing incidence of this problem. The median age at diagnosis was 15.5 months. Presenting complaints included failure to thrive, skeletal deformities, and hypocalcemic tetany/ seizures.244 During the same interval, 30 infants with vitamin-D-deficient rickets were identified in Maryland. All were black and had been breast fed for 6 or more months, and 90% had not received vitamin D supplements.248 Vitamin D deficiency is more common in breast-fed black infants than in white infants due in large part to increased maternal skin pigmentation and decreased endogenous synthesis of cholecalciferol that coupled with socioeconomic circumstances leads to lower levels of 25OHD in maternal serum and of vitamin D in breast milk. These infants are also more likely to be weaned to diets low in calcium and vitamin D.241 Subtle forms of vitamin D deficiency or insufficiency are prevalent throughout the North American population, particularly in the winter months when there is little sunlight exposure and the ultraviolet light requisite for endogenous synthesis of vitamin D is limited.12,43 In a group of 307 healthy urban male and female adolescents 11 to 18 years of age in the northeastern United States, 42% had serum concentrations of 25OHD less than 20 ng/mL.249 The prevalence of very low 25OHD values (⬍15 ng/mL) was most common in black (36%) and Hispanic subjects (22%), whereas 6% of white students had such levels. There was an inverse relationship between serum levels of 25OHD and PTH, with secondary hyperparathyroidism present in many subjects and with low 25OHD concentrations implying the likelihood of incipient metabolic bone disease. In addition to skin pigmentation and northern latitude, low 25OHD values were attributable to meager consumption of milk and multivitamins due to a large intake of phosphate-containing soft drinks and to increase in fat mass into which vitamin D had been deposited and thus not bioavailable. Similar findings have been recorded in otherwise healthy adolescent females in the United Kingdom.250 Interestingly, women with low milk intake during childhood are at increased risk for low bone mass and increased fracture rate as adults.251 Inasmuch as there is reasonable evidence that serum values of 25OHD below 30 to 32 ng/mL are suboptimal, the prevalence of vitamin D deficiency or insufficiency may be far greater than is currently acknowledged.43,252 Assuming a reliable assay such as those employing liquid chromatography-tandem mass spectrometry that measure both 25OHD2 and 25OHD3, 25OHD concentrations below 12 to 15 ng/mL in children clearly indicate vitamin D deficiency and values between 15 to 31 ng/mL are consistent with vitamin D insufficiency.241,253 Serum

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levels of 25OHD that exceed 60 to 100 ng/mL are indicative of vitamin D excess. Subtle dietary deficiency of calcium may accentuate the adverse effects of borderline vitamin D stores.254 Vitamin D deficiency may also be the consequence of intestinal malabsorptive disorders (such as celiac disease, biliary obstruction, gastric resection or bypass, or pancreatic exocrine insufficiency), ingestion of calcium-binding agents such as cholestyramine, or the accelerated degradation by anticonvulsant drugs (such as phenytoin) of vitamin D to water-soluble forms that increase its urinary loss.43 In the majority of infants and children with rickets due to vitamin D deficiency, serum concentrations of total calcium are borderline-normal or low, phosphate levels low, and alkaline phosphatase activity and PTH concentrations increased (Table 17-7). Secondary hyperparathyroidism develops as the intestinal absorption of calcium is reduced, resulting in lowered serum levels. PTH increases urinary phosphate loss and lowers serum phosphate concentrations while enhancing the rates of bone resorption and turnover.242 PTH also increases synthesis of calcitriol, which may increase the rate of calcidiol metabolism and further deplete the body’s store of vitamin D.241 Typically, in vitamin D deficiency serum concentrations of calcidiol are low—whereas calcitriol values may be normal, high, or low (depending on whether vitamin D deficiency is modest, moderate, or severe). Serum concentrations of osteocalcin are low. Serum levels of PICP (a marker of bone formation) and ICTP and urinary excretion of NTx (markers of bone resorption) are substantially increased in infants with vitamin D deficiency rickets, indicating increased collagen turnover in this disorder.255 With treatment, these values increase transiently and then fall to age-appropriate norms before radiographic healing of the rachitic lesions is complete. Prevention of vitamin D deficiency is its most effective management. Because the amount of vitamin D in human breast milk is approximately 20 IU/L and vitamin D supplementation of the breast-feeding mother may be inadequate to ensure normal calcidiol levels in her infant (unless she is receiving approximately 2000 IU per day), it is important that all breast-fed infants receive a supplement of vitamin D daily (200–400 IU/day). By extension, this recommendation is also appropriate for infants who are not receiving adequate amounts of vitamin D in their prepared formulas or diet or have suboptimal exposure to sunlight.43,241,243,244,247,256 It is somewhat ironic that an increase in the incidence of vitamin D deficiency in infants and children coincides with well-intentioned recommendations that exposure to sunlight be limited by sunscreen and protective clothing. Vitamin D supplementation (at least 400 IU/day and perhaps as high as 800 to 1,000 IU/day) is appropriate throughout life if sunlight exposure is limited.43,252 In the active lightly dressed white child with skin that normally tans with exposure to sunlight and who plays outdoors 30 minutes thrice weekly, endogenous vitamin D synthesis is usually sufficient to obviate the need for supplementation.241,243 A brown or black-skinned child requires several fold longer sunlight exposure for a comparable biologic effect. The latitude in which the child lives, season of the

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TA B L E 1 7 - 7

Laboratory Data in Rickets of Varying Pathogenesis Type

Calcium

Phosphate

Alkaline Phosphatase

Calcidiol

Calcitriol

PT

Calcium deficiency Phosphate deficiency Vitamin D deficiency Mild Moderate Severe Loss-of-function CYP2R1 (25-hydroxylase) Loss-of-function CYP27B1 (25OHD-1␣-hydroxylase) Loss-of-function VDR (Resistance to calcitriol) Loss-of-function PHEX (X-linked hypophosphatemic rickets)

↓↓ N, ↑

↓ ↓↓

↑↑ ↑↑

N N

↑ ↑

↑ N,↓

N,↓ N,↓ ↓ ↓

N,↓ ↓ ↓ ↓

↑ ↑↑ ↑↑ ↑

↓ ↓ ↓↓ ↓

N ↓,N,↑ ↓ ↓

N ↑ ↑↑ ↑

↓↓

↓↓

↑↑↑

N

↓↓↓

↑↑↑

↓↓

↓↓

↑↑↑

N

↑↑↑

↑↑↑

N

↓

↑

N

N,↓

N

N ⫽ normal, ↓ ⫽ low, ↑ ⫽ high.

year, time of day, local environmental pollutants, amount of clothing, and use of sunscreen affect the time required for sunlight exposure to evoke adequate vitamin D synthesis in the individual child.256 Between November and February, little or no vitamin D can be synthesized above 35 degrees latitude (Atlanta, GA)—but vitamin D insufficiency is also prevalent in lower latitudes.43,257 Once established, vitamin D deficiency in the child or adolescent may be treated by the oral ingestion of vitamin D3 2,000 to 10,000 IU daily for 4 to 6 weeks or 50,000 IU weekly for 8 weeks or by the administration of a single oral (or intramuscular) dose of 150,000 to 600,000 units of vitamin D3—depending on patient age and other individual circumstances.43,220,241 At the beginning of treatment, elemental calcium (40 mg/kg/day in divided doses) must also be administered to the vitaminD-deficient child receiving vitamin D in order to avoid the hypocalcemia that accompanies rapid remineralization of bone matrix (the hungry bone syndrome). Serial measurement of total and/or bone-specific alkaline phosphatase values is an effective tool for monitoring the efficacy of treatment as levels decline progressively in tandem with the roentgenographic healing of the rachitic lesions.255 Care should be exercised to avoid hypercalcemia, hypercalciuria, and nephrocalcinosis. Rickets due primarily to low dietary intake of calcium has been observed in infants who ingest low-calciumcontaining formulas and in children from developing countries receiving a diet with 200 mg (or less) of elemental calcium per day despite normal intake of phosphate and adequate endogenous stores of vitamin D as determined by serum calcidiol levels.241 Calcium intake in these infants and children is well below that recommended (375 mg/day in infants, 500 mg/day in children below 4 years of age, 800–1300 mg/day in older children and adolescents). Histologically, bone biopsies from children with calcium-deficiency rickets reveal widened

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seams of unmineralized osteoid and low bone turnover rates—findings compatible with rickets. In Nigerian infants, rickets due to vitamin D deficiency is most prevalent between 4 and 12 months of age. In 123 older Nigerian children (34–63 months of age) with rickets, low serum concentrations of calcium, normal phosphate levels, normal to low calcidiol, and elevated calcitriol concentrations, administration of calcium alone (1,000 mg daily in divided doses orally over 24 weeks) resulted in more rapid decline in serum levels of alkaline phosphatase and in radiographic healing of rickets than did administration of vitamin D (600,000 IU intramuscularly at inception of the study and at ⫹12 weeks)—data supportive of the concept that calcium deficiency alone was the cause of the rickets in this population.258 Interestingly, calcidiol values rose and calcitriol levels declined with calcium supplementation alone—suggesting that a low-calcium diet and attendant calcium deficiency led to increased secretion of PTH and accelerated conversion of calcidiol to calcitriol. Calcium deficiency rickets also occurs in the United States when after completion of breast feeding infants and children are weaned to low-calciumcontaining foods.254 Calcium deficiency rickets may also develop as a consequence of impaired intestinal absorption of dietary calcium that has been bound by ingestion of high fiber- and phytate-containing cereals. Calcium deficiency is best addressed by its prevention, ensuring adequate intake of this element according to established guidelines for growing children and adolescents. When present, calcium deficiency rickets may be effectively treated by ensuring an intake of 1,000 mg of elemental calcium daily for 6 months—with provision of normal amounts of vitamin D by sunlight exposure or supplementation.242 Dietary phosphate deficiency is unusual because it is present in large amounts in most foods. Phosphate deficiency occurs in patients receiving parenteral nutrition with fluids low in this cation, in those ingesting large

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

amounts of aluminum-containing antiacids as aluminum and phosphate co-precipitate in the intestinal tract, and in premature infants drinking human breast milk without supplemental phosphate.242 In very premature infants receiving long-term parenteral nutrition, development of metabolic bone disease is frequent and related to deficiencies of calcium, phosphate, and vitamin D and to excess aluminum in the infusates. Infants receiving large amounts of aluminum-containing antiacids over prolonged periods for treatment of gastroesophageal reflux may also have substantially low bone mass. Aluminum lowers the rate of bone formation by several mechanisms. Administered orally, aluminum binds intestinal phosphate—thereby impeding its absorption and leading to phosphate depletion. Administered intravenously during total parenteral nutrition or during hemodialysis, aluminum inhibits osteoblastic function and prevents mineralization of osteoid. It also impairs the secretion of PTH and decreases 25OHD-1␣ hydroxylase activity.242 Patients requiring total parenteral nutrition should receive as much calcium and phosphate as can be administered safely—as well as supplemental vitamin D 400 IU/day. Periodic measurements of serum levels of calcium, phosphate, alkaline phosphatase, PTH, and calcidiol (and serial skeletal radiographs and estimations of skeletal mineralization) are recommended in patients receiving total parenteral nutrition. If metabolic bone disease develops in spite of these efforts, serum aluminum levels should be measured. If they are elevated (⬎100 ␮g/L), a search for the source of the aluminum should be initiated and that product eliminated from the infusate if possible. Cadmium, fluoride, and saccharated ferric oxide are also able to impede normal bone mineralization.242 Metabolic and functional defects of vitamin D lead to rare forms of rickets. Rickets due to a defect in 25-hydroxylation has been described in two brothers of Nigerian origin in whom a homozygous loss-of-function mutation (Leu99Pro) in CYP2R1 eliminated hydroxylase activity of this 501aa protein.259,260 Hypocalcemia, hypophosphatemia, skeletal abnormalities of rickets, and low plasma levels of 25OHD were present in these siblings. Vitamin-D-dependent rickets type 1 (OMIM 264700) or pseudovitamin D deficiency rickets (PDDR) type 1 is due to loss-of-function mutations in CYP27B1— the enzyme in the renal proximal tubule that catalyzes 1␣-hydroxylation of 25OHD to 1,25(OH)2D or calcitriol, the biologically active metabolite of vitamin D.261,521 PDDR type 1 is an autosomal-recessive disease whose clinical manifestations [including bone deformities (bowing of the forearms), growth retardation, weakness, and/or seizures] appear in the first year of life. Biochemically, hypocalcemia, hypophosphatemia, hyperphosphatasemia, and markedly elevated serum levels of PTH are typical. Radiographs reveal rachitic deformities of the long bones. The diagnosis of PDDR type 1 is established by finding normal serum concentrations of calcidiol but extremely low calcitriol values that do not increase after administration of vitamin D or calcidiol. The diagnosis is confirmed by identification of the mutation in CYP27B1 (Table 17-7). The clinical, biochemical, and radiographic manifestations of PDDR resolve completely and reason-

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ably rapidly following treatment with physiologic amounts of calcitriol (10–20 ng/kg/day). Serum calcium concentrations often begin to rise within the first 24 hours of treatment. Life-long therapy is necessary, and calcitriol doses usually need to be increased during pregnancy. PDDR type 1 is found with high frequency in a Quebec FrenchCanadian population but occurs in all races and in diverse geographic regions. CYP27B1 encodes a 508aa mitochondrial cytochrome P450 hydroxylase with conserved sites that bind ferrodoxin and heme. Many loss-of-function missense, nonsense, splicing, and duplication or deletion/frame-shift mutations in CYP27B1 lead to inactive or truncated protein products that are unable to bind substrate (calcidiol) or heme—the latter defect preventing electron transfer and inhibiting catalysis (Figure 17-11).262 The most common mutation in CYP27B1 in the Quebec French-Canadian population at risk for PDDR type 1 is deletion of guanine at nucleotide 958 (codon 88, exon 2), which changes the reading frame and results in premature termination of translation and an inactive product (the Charlevoix mutation). A second common mutation in this population is triplication of a normally duplicated sequence in exon 8. However, this mutation is also found in patients of other ethnicities (Asian, Hispanic). Homozygous or compound heterozygous inactivating mutations of VDR, the gene encoding the vitamin D receptor, lead to resistance to the biologic effects of calcitriol (autosomal-recessive vitamin-D-dependent rickets type II or vitamin-D-resistant rickets, OMIM 277440).261,263 In addition to the radiographic findings of rickets, clinical and biochemical manifestations of resistance to calcitriol include severe infantile-onset bony deformities characteristic of rickets, growth retardation, varying degrees of alopecia, hypocalcemia, hypophosphatemia, and extraordinarily high serum concentrations of calcitriol (300–1,000 pg/mL) and PTH. Serum levels of 24,25-dihydroxyvitamin D are often low (Table 17-7).

Figure 17-11 Genetic errors in CYP27B1 leading to absence of P450c1␣ activity, decreased synthesis of calcitriol, and pseudovitamin D deficiency rickets. IVS3 ⫹ 1g6a is guanine-to-adenosine transition in the first nucleotide of intron 3, resulting in retention of intron 3 in the transcribed product and introduction of a premature stop codon. Mutations have been identified in each of the nine exons of CYP27B1—including Q65H (exon 1), W241Ter (exon 4), S323Y (exon 6), R429 P (exon 8), P497R (exon 9)—and in introns 2, 3, 6, and 7. Abbreviations: C (cysteine), D (aspartic acid), E (glutamic acid), G (glycine), H (histidine), L (leucine), N (asparagine), P (proline), Q (glutamine), R (arginine), S (serine), T (threonine), W (tryptophan), Y (tyrosine), and X (termination). [Reproduced with permission from Kitanaka S, et al. (1999). No enzyme activity of 25-hydroxyvitamin D3 1␣-hydroxylase gene product in pseudovitamin D deficiency rickets, including that with mild clinical manifestation. J Clin Endocrinol Metab 84:4111–4117.]

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The high calcitriol values reflect the combined stimulatory effects of hypocalcemia, hypophosphatemia, and secondary hyperparathyroidism on the activity of 25OHD3 1␣-hydroxylase together with decrease in its rate of catabolism due to depressed calcitriol-dependent 1,25␣dihydroxyvitamin D3 24-hydroxylase activity. Alopecia is the result of impaired vitamin D function in epithelial nuclei and those of the outer root-sheath cells of the hair follicle. Loss-of-function mutations (particularly in the DNA-binding region) of VDR result in a phenocopy of the generalized alopecia associated with loss of HR function (Hairless, OMIM 602302, chromosome 8p21.2). Interestingly, the role of the VDR in the maintenance of normal hair growth is not dependent on its binding to ligand.261,264 Although striking in infancy and early childhood, clinical manifestations of this disorder may vary and patients with milder defects in VDR may not be identified until adolescence or adulthood. The VDR consists of DNA-, ligand-, and retinoid-X-receptor-binding domains and a

transactivation domain to which many co-modulators of VDR function are recruited. Loss-of-function mutations have been found in each of these domains. The mutated VDR may be unable to bind calcitriol because of decreased receptor number or affinity for ligand, incapable of forming heterodimers with the retinoid X receptor or translocating to the targeted gene in the nucleus, unable to bind to the vitamin D response element (VDRE) or to initiate gene transcription once bound to the VDRE (Figure 17-12). Spontaneous remission of the rachitic process may occur rarely, most often between 7 and 15 years of age and when the patient enters puberty.265 The diagnosis of vitamin-Dresistant rickets is suggested by the finding of elevated serum concentrations of calcitriol in the rachitic patient and confirmed by identification of the loss-of-function mutation in VDR. In general, patients with vitamin-D-resistant rickets without alopecia may be more responsive to treatment.263 Administration of high doses of calcitriol (1 to 6 ␮g/kg/day) and supplemental calcium (1 to 3 g of elemental calcium

Figure 17-12 Mutations in the zinc-finger DNA-binding (A) and ligand-binding (B) domains of the vitamin D receptor (VDR) in patients with end-organ insensitivity to vitamin D. Shaded amino acids are conserved. Large circled amino acids denote missense mutations. Abbreviations: FS (frame-shift), H (␣ helixes), E1 (helixes within the ligand binding domain involved in transactivation), ␤-turn (change in spatial orientation of the VDR protein). [Reproduced with permission from Malloy PJ, et al. (1999). The vitamin D receptor and the syndrome of hereditary 1,25-dihydroxyvitamin D-resistant rickets. Endocrine Reviews 20:156–188.]

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

daily) has been effective in increasing serum calcium concentrations and healing rickets in patients with nonsense and missense mutations in VDR that lead to decreased affinity for ligand or alter nuclear targeting. It is appropriate to provide a trial of high-dose calcitriol/calcium therapy to each patient with vitamin-D-resistant rickets regardless of the VDR mutation.261,263 During treatment, serum values of calcium, phosphate, alkaline phosphatase, creatinine, and PTH; urinary calcium and creatinine excretion; skeletal radiographs; and renal ultrasounds for development of nephrocalcinosis are monitored serially. In patients refractory to oral therapy, continuous intravenous or intracaval administration of large amounts of calcium (0.4–1.4 g of elemental calcium/m2/day) normalizes calcium, phosphate, alkaline phosphatase, and PTH values; heals rickets; and increases growth rate in selected children. If this mode of therapy is employed, careful monitoring for catheter sepsis and cardiac arrhythmia (as well as hypercalcemia, hypercalciuria, and nephrocalcinosis) is mandatory. After healing of the rickets by parenteral calcium, maintenance therapy with large doses of oral calcium (3.5–9 grams of elemental calcium/m2/day) is appropriate. In younger infants with vitamin-D-resistant rickets prior to development of florid rickets, high doses of oral calcium may ameliorate the rachitic process. These clinical observations indicate that the major defect in patients with vitamin-D-resistant rickets is lack of calcium. With treatment, growth may normalize in patients with vitamin D resistance. However, if alopecia is present the condition is unlikely to resolve.265 Genetically engineered mice in which the VDR has been inactivated by ablation of the second zinc finger in the DNA-binding domain (Vdr-/-) are normal at birth, but develop an expanded zone of hypertrophic chondrocytes in the cartilage growth plate at 15 days, hypocalcemia at 21 days, alopecia at 28 days, and rickets by 35 days of age.266 Except for alopecia, all of these abnormalities can be prevented (including the histomorphometric abnormalities characteristic of calciopenic rickets) by feeding these animals a diet known to prevent rickets in vitaminD-deficient rats that is high in calcium (2% versus 1%), phosphorus (1.5% versus 0.67%), and lactose (20% versus none, lactose being a disaccharide that increases intestinal mucosal transport of calcium) beginning at 16 days of age.266,267 Clinical and experimental observations suggest that despite its known effects on osteoblast and osteoclast differentiation and function the primary role of the vitamin-D/VDR system in bone formation is to increase intestinal absorption of calcium in order to provide sufficient amounts of this cation for hydroxyapatite formation. Furthermore, in the mouse model of vitamin-D-resistant rickets maintenance of eucalcemia prevents secondary hyperparathyroidism—indicating that Ca2⫹ is a more potent regulator of PTH expression than is calcitriol and that secondary hyperparathyroidism is of fundamental pathophysiologic importance in the development of rickets of different etiologies. A second form of vitamin D resistance (OMIM 600785) but with intact VDR and a phenotype similar to that of vitamin-D-dependent rickets type II (except for alopecia)

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is due to inhibition of binding of the VDR-retinoid X receptor heterodimer to the VDRE by a member of a family of heterogeneous nuclear ribonucleoproteins.268,269 HNRPA1 encodes one of several ribonucleoproteins that are able to bind the VDRE and to inhibit its transactivation by the VDR-retinoid X receptor heterodimer. The mechanism by which overexpression of these otherwise normal ribonucleoproteins occurs is unknown at present.

Phosphopenic Rickets Hypophosphatemia in childhood may be due to hereditary or acquired disorders (Table 17-8). Acute hypophosphatemia is accompanied by paresthesias, muscle weakness, and confusion. Chronic hypophosphatemia is often manifested by rickets. One of the most common (1:20,000 TA B L E 1 7 - 8

Disorders of Phosphate Homeostasis in Children I. Hypophosphatemia A. Decreased Intestinal Absorption 1. Decreased intake/absorption - parenteral hyperalimentation, antacid abuse, starvation (anorexia nervosa) 2. Malabsorption - vitamin D deficiency, metabolism, function B. Increased Urinary Excretion 1. Hypophosphatemic rickets - X-linked (PHEX), autosomal dominant (FGF23), autosomal recessive (DMP1), autosomal recessive with hypercalciuria (SLC34A3), X-linked recessive (CLCN5), tumor-induced osteomalacia, fibrous dysplasia (GNAS) 2. Renal tubular defects - Dent and Fanconi syndromes, postrenal transplantation, hypomagnesemia, fructose intolerance, macrophage activation syndrome 3. Hyperparathyroidism, acidosis, respiratory alkalosis 4. Drugs - diuretics, bicarbonate, glucocorticoids, calcitonin 5. Expansion of extracellular fluid volume C. Shift from Extracellular to Intracellular Space 1. Recovery from diabetic ketoacidosis/insulin administration, sepsis, salicylate intoxication 2. Administration of glucose, nutritional repletion, hungry bone syndrome II. Hyperphosphatemia A. Increased Intake 1. Intravenous, oral, rectal B. Decreased Urinary Excretion 1. Renal insufficiency 2. Hypoparathyroidism, acromegaly 3. Pseudohypoparathyroidism (GNAS) 4. Familial tumoral calcinosis (FGF23, GALNT3) a. Hyperostosis hyperphosphatemia syndrome (FGF23, GALNT3) 5. Drugs - growth hormone, bisphosphonates C. Excess Bone Resorption 1. Severe illness - acidosis (metabolic or respiratory), hemolytic anemia, diabetic ketoacidosis, hepatitis, catabolic states, rhabdomyolysis, hyperthermai 2. Drugs - cytotoxic therapy Modified from Hruska KA (2006). Hyperphosphatemia and hypophosphatemia. In Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D. C.: American Society for Bone and Mineral Research 233–242; and from Ward LM (2005). Renal phosphate-wasting disorders in childhood. Pediatr Endocrinol Rev 2:342–350.

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births) forms of clinically evident rickets encountered in developed countries is X-linked hypophosphatemic rickets (XHR, OMIM 307800). XHR is an X-linked dominant disorder manifested in affected hemizygous males and heterozygous females, albeit with substantial inter- and intrafamilial variability in its clinical expression. Physical findings in children with XHR include short stature, genu varum or valgum that develops when the infant begins to walk, flaring of the metaphyses, rachitic rosary, frontal bossing, increased frequency of dental decay and/or periradicular abscesses in teeth free of caries, and bone, muscle, and joint aching and stiffness. Craniotabes, tetany, and muscular weakness are not found in patients with XHR as they are in those with vitamin D deficiency. Adults with XHR have osteomalacia and increased fracture rate, dental abscesses, and bone pain. They may also develop stenosis of the spinal canal. Enesopathy (calcification of tendons, ligaments, and joint capsules) is common in adults and may be present in children with XHR. Whereas serum levels of total calcium and Ca2⫹e are normal, hypophosphatemia is marked due to urinary wastage because of substantially decreased renal tubular reabsorption of filtered phosphate and limited intestinal absorption of this anion. Serum concentrations of PTH and calcidiol are normal, but calcitriol values are inappropriately low for the degree of hypophosphatemia. Serum alkaline phosphatase activity is increased.270 Because the serum Ca2⫹e concentration is normal, secondary hyperparathyroidism does not occur unless the patient receives excessive amounts of supplemental phosphate. Phosphate induces differentiation and death of hypertrophic chondrocytes in the cartilage growth plate by activating the mitochondrial apoptotic pathway that is caspase 9 dependent.261,271 Thus, hypophosphatemia leads to delayed loss and increased numbers of hypertrophic chondrocytes and expansion of the growth plate characteristic of rickets. Histomorphometrically in XHR, unmineralized osteoid accumulates along the trabeculae within cancellous bone. Classic XHR is caused by loss-of-function mutations in PHEX (phosphate-regulating endopeptidase homolog, X-linked).272 PHEX is a 22-exon gene that encodes a 749-aa integral membrane protein with very long extracellular (702 aa), transmembrane (27 aa), and short intracellular (20 aa) domains that structurally resembles several neutral endopeptidases (endothelin-converting enzyme-1, Kell antigen). The extracellular domain has 10 conserved cysteine residues and a pentapeptide motif (His-Glu-Phe-Thr-His) characteristic of zinc metallopeptidases that may either convert propeptides to active forms or degrade and inactivate their substrates. PHEX is expressed in bone (osteoblasts), muscle, lung, liver, testis, and ovary. Its expression by osteoblasts is downregulated by calcitriol.273 In most patients with XHR, inactivating mutations of PHEX have been found primarily in the extracellular domain and include frame-shift deletions (16%), duplications, insertions (8%), deletional-insertional, splice site (15%), nonsense (27%), and missense (34%) mutations— many in exons 15 and 17. A mutation has also been identified in the 5’-untranslated region (A→G transver-

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sion 429 bp upstream of the ATG initiation site, Figure 17-13).272,274,275 Because PHEX is a glycosylated protein, failure of glycosylation leads to its sequestration within the endoplasmic reticulum—thus impeding its movement (trafficking) to the cell membrane. Other mutations interfere with the catalytic function of the protein or its threedimensional conformation.276 Mutations in PHEX arise spontaneously in more than 20% of patients with XHR. There is no correlation between the location or type of mutation in PHEX and the clinical manifestations or severity of the disease.277,278 Fibroblast growth factor (FGF)-23 (OMIM 605380, chromosome 12p13.3), is a phosphate-wasting element present in high concentrations in the serum of patients with XHR and serum concentrations of FGF23 correlate with the degree of hypophosphatemia.279 FGF23 is a 251-aa that inhibits sodium-dependent phosphate uptake by renal proximal tubular cells and depresses 25OHD-1␣ hydroxylase activity and thus synthesis of calcitriol—characteristics that define a phosphatonin (a family of phosphaturic agents that includes in addition to FGF23, matrix extracellular phosphoglycoprotein and serum frizzled related protein-4).280 FGF23 inhibits phosphate reabsorption by down-regulating expression of the genes encoding type II (a and c) sodium-phosphate co-transporters present in the apical membrane of the renal tubule. FGF23 is expressed and secreted primarily by osteoblasts and osteocytes. FGF23 is coexpressed with PHEX in these cells. Normally, FGF23 is cleaved to biologically inactive products between amino acids Arg179 and Ser180 by metallic endopeptidases such as PHEX. Although the natural endogenous substrate for PHEX was thought likely to be FGF23, this peptide is not a substrate for PHEX action.281,282 Rather, FGF23 is cleaved by subtilisin-like proprotein and furin-like convertases. In normal adults, the mean serum FGF23 concentration utilizing an immunometric assay that detects intact FGF23 is 29 pg/mL. FGF23 levels decline in response to phosphate deprivation and increase with phosphate loading, consistent with an important role for FGF23 in the normal regulation of serum phosphate concentrations and phosphate homeostasis.283 In the Hyp mouse model of XHR, there is increased expression of Fgf23 in bone and in osteoblasts in vitro. Because normally PHEX is also expressed in bone, these observations suggest that decreased expression of PHEX in the Hyp mouse (and the patient with XLH) may upregulate expression of Fgf23.282 Although serum levels of matrix extracellular phosphoglycoprotein have been reported to be increased in some patients with XHR, it plays no role in the pathogenesis of XHR in the Hyp mouse (and its role in the human disorder is likely to be limited).280,284 When renal function is normal, rickets of diverse pathogenesis is commonly associated with hypophosphatemia. Secondary hyperparathyroidism as a cause of hypophosphatemia may be suspected by the presence of elevated serum concentrations of PTH (e.g., vitamin D deficiency). Serum PTH values are normal in subjects with primary hypophosphatemia due to an abnormality in renal tubular resorption of phosphate intrinsic to the renal cell or mediated by a circulating phosphatonin.272

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Figure 17-13 Nonsense, splice, frame-shift, and missense mutations in PHEX in patients with X-linked hypophosphatemic rickets. [Reproduced with permission from Sato K, et al. (2000). Three novel PHEX gene mutations in Japanese patients with X-linked hypophosphatemic rickets. Pediatr Res 48:536–540.]

The diagnosis of XHR is established when the typical family history (if the patient is not the initial mutant), clinical findings (deformities of the lower extremities, flaring of the metaphyses), and roentgenographic (rachitic changes) and laboratory data (hypophosphatemia, hyperphosphaturia, inappropriately low serum level of calcitriol, normal serum concentration of PTH, calcium, creatinine, and 25OHD) are present and when other causes of hypophosphatemia and hyperphosphaturia have been excluded (Tables 17-6 through 17-8). The diagnosis of XHR may be confirmed by identification of the PHEX mutation, although occasionally no mutation is detected by current methods.277 Rarely, somatic and germ-line mosaicism for a mutation in PHEX may mimic autosomal-dominant transmission of hypophosphatemic rickets.285 The primary therapeutic agents employed in the treatment of XHR are calcitriol (25–70 ng/kg/day) administered in two doses (with the larger dose given at night when PTH secretion tends to increase) and elemental phosphorus 0.25 to 3 g daily (beginning at a dose of 30 mg/kg/day and increasing to 70 mg/kg/day) administered in four to six divided daily doses depending on age, size, compliance, and response to therapy.272 Table 17-3 lists the preparations of oral phosphate. Infants and young children may tolerate a phospha-soda solution more readily than other preparations. When able, most older children prefer a chewable phosphate tablet to the powder form that is dissolved in water or juice. Acidic potassium phosphate products are preferred

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because they do not increase intravascular volume and phosphate excretion and because they acidify the urine, thereby increasing the solubility of calcium phosphate. Calcitriol (Rocaltrol, Roche Pharmaceuticals) is available as an oral solution at a concentration of 1 ␮g/mL and as capsules with either 0.25 or 0.5 ␮g per capsule. If hypercalcemia or hypercalciuria (urine calcium excretion more than 4 mg/kg/day) occurs during treatment, the dose of calcitriol should be lowered. If that leads to exacerbation of the rachitic process, an agent (e.g., amiloride) that increases renal tubular resorption of calcium may be added cautiously to the therapeutic program. Frequent (every 3 months) clinical evaluation and measurement of serum and urine calcium, phosphate, alkaline phosphatase, and creatinine levels and serum intact PTH values are essential to avoid hypercalcemia, hypercalciuria, nephrocalcinosis, and secondary hyperparathyroidism because high doses of phosphate may lead to counterproductive secondary (and sometimes tertiary) hyperparathyroidism. Two goals of therapy of XHR are to maintain serum phosphate concentrations determined before a daytime dose of phosphate in the low normal range and alkaline phosphatase values within the high normal range. Renal sonograms prior to treatment and at 12-month intervals during therapy to identify early-stage nephrocalcinosis (and yearly skeletal radiographs to assess the degree of healing of the rickets) are recommended. Complete radiologic healing of XHR is often difficult to attain. Development of nephrocalcinosis (and compromise of renal

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function in some patients) is directly related to the amount of phosphate the patient receives. Hyperoxaluria has also been implicated in the pathogenesis of nephrocalcinosis. Co-management with an experienced orthopedist is important because the orthopedist may prescribe braces, or in patients with extreme and progressive deformities perform corrective osteotomies. However, bracing is unpredictable and poorly tolerated—and osteotomies are often followed by complications. Femoral and tibial hemiepiphysiodeses may offer alternative surgical procedures for the correction of lower limb deformities in children with XHR younger than 10 years of age.286 The birth length of children with XHR is normal, but growth rate is slow during the first several years of life—leading to progressive shortening of height. Many children with XHR are significantly short by 5 years of age, although some patients (particularly girls) with minimal involvement may grow normally.287 Treatment of the older child with XHR with calcitriol and phosphate may improve growth rate, in part due to correction of the deformities of the lower extremities.288 During puberty, gain in height is normal in boys (⫹28.2 cm) and girls (⫹24.2 cm) with XHR. Thus, the compromised adult stature in XHR is related to impaired early childhood and preadolescent growth. In a series of 19 closely monitored children with XHR, initiation of treatment with calcitriol and phosphate at a mean chronologic age of 4.2 months (range of 7 weeks to 6 months, N ⫽ 8) resulted in greater adult stature [–0.2 versus –1.2 standard deviations (SD)] than when treatment began at a mean age of 2.1 years (range of 1.3 to 8.0 years, N ⫽ 11)—with no difference in complication rate (secondary hyperparathyroidism, nephrocalcinosis, craniosynostosis) between the two groups.289 The enhanced growth response to early treatment was likely related to the normal length and mild skeletal and biochemical signs of rickets in early infancy and the prevention of more clinically significant bone disease as the child aged. Despite close adherence to treatment, many children with XLH retain mild to moderate radiographic signs of rickets and mildly elevated serum alkaline phosphatase activities—although the extent of lower limb deformities is often ameliorated by good compliance. GH increases glomerular filtration rate, renal tubular resorption, serum concentrations of phosphate, and rate of accrual of bone mineral. In the short term (6–12 months), GH accelerates the velocity of linear growth of children with XHR.290-292 Approximately 90 children with XHR who have been treated with rhGH combined with conventional therapy have been reported. The majority have experienced improved linear growth, and some have achieved normal adult stature.293 Whether GH therapy will increase adult stature in larger series of XHR subjects remains a matter of further clinical investigation. Future treatment of XHR may include development of agents that decrease production of FGF23, accelerate its degradation, or block its action in the renal tubule. The majority of untreated adults with XHR are hypophosphatemic. Serum alkaline phosphatase activity is increased, and osteomalacia is present on bone biopsy.

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Nevertheless, these patients are often clinically asymptomatic except for frequent dental abscesses, degenerative hip disease due to deformities of the lower limbs, and hearing impairment.272 Occasionally, a female with XHR may be clinically well despite isolated hypophosphatemia. Roentgenographic manifestations of XHR in adults include thickening of the spinous processes and fusion of the vertebrae and stenosis of the spinal canal. BMD determined by single and dual-photon absorptiometry tends to be normal in adults with XHR (despite the histomorphologic abnormalities), suggesting that most of these subjects are not at increased risk for osteoporotic fractures. However, in approximately 25% of adults with XHR there is clinical evidence of osteomalacia such as progressive lower limb deformities, bone pain, fractures, and pseudofractures.294,295 Treatment of these patients with calcitriol and phosphate can be beneficial.296 Autosomal-dominant hypophosphatemic rickets (ADHR, OMIM 193100) is a partial phenocopy of XHR with hyperphosphaturia and inappropriately low serum levels of calcitriol that is due to mutations (Arg176Gln, Arg179Trp) in FGF23 that render the product less susceptible to cleavage between amino acid residues 179 and 180 by furin, a pro-protein convertase.297 ADHR may be incompletely penetrant, variable in age of onset (childhood to adult), and rarely self-limiting.298 The clinical, biochemical, and radiographic findings in ADHR are similar to those of XHR except that ADHR subjects manifest muscle weakness as a consequence of hypophosphatemia.299 ADHR may be identified by its pattern of transmission and detection of a mutation in FGF23. Treatment involves administration of calcitriol and phosphate, with close serial monitoring for safety and because hyperphosphaturia may occasionally resolve spontaneously. Excessive production of FGF23 with hypophosphatemia and inappropriately low calcitriol values has also been documented in patients with autosomal-recessive hypophosphatemic rickets, fibrous dysplasia due to a gain-of-function mutations in GNAS, the linear nevus sebaceous syndrome associated with hypophosphatemic rickets (OMIM 163200), osteoglophonic dysplasia (OMIM 166250; craniosynostosis, rhizomelic shortening of the limbs, noncalcifying bone lesions), and opsismodysplasia [OMIM 258480, a spondylo(epi)metaphyseal dysplasia with delayed ossification, micromelia, platyspondyly, and vertebral hypoplasia].280,300 Tumor-induced osteomalacia/rickets is an acquired disorder due to excessive production of one of several phosphatonins by a tumor of mesodermal origin. The majority of such tumors have secreted FGF23, but these neoplasms have also synthesized secreted frizzled related protein-4, matrix extracellular phosphoglycoprotein, and FGF7—all of which increase urinary phosphate excretion and suppress renal synthesis of calcitriol, albeit perhaps not to the same extent as FGF23.280,301 The identification of an FGF23-secreting tumor is dependent on the sensitivity of the FGF23 assay employed.302 Although unusual in children, tumor-induced osteomalacia/rickets has been described in this age group. For example, an 11-year-old girl had significant bone pain and functional limitation associated with biopsy-proven hypophosphatemic osteomalacia/rickets and markedly

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

elevated serum levels of FGF23.303 Following removal of a benign fibro-osseous tumor from a small exostosis on a distal ulnar metaphysis, serum FGF23 concentrations normalized within 7 hours postoperatively and phosphate levels were normal 2 weeks later. Clinical symptoms abated, and radiographic and histomorphometric abnormalities resolved within 1 year after surgery. In an 11-year-old boy with severe bone pain, weakness progressing to confinement to a wheelchair, hyperphosphaturia, and hypophosphatemia, elevated levels of FGF23 (1874 RU/mL) declined to normal values (43 RU/ mL) within 48 hours after removal of a FGF23-containing hemangiopericytoma from his left iliac wing.304 This lesion had not been identified by routine roentgenograms, computed tomographic or magnetic resonance imaging, or technetium bone scan. The tumor was demonstrated only by gradient recall echo magnetic resonance imaging (Figure 17-14). Within 2 weeks after surgery, the lad was walking without assistance—and several weeks thereafter he resumed normal activity. Autosomal-recessive hypophosphatemic rickets (ARHR, OMIM 241520) is clinically, biochemically, and histomorphometrically similar to XHR and ADHR except for its mode of transmission and for the development of osteosclerosis at the base of the skull and in the calvarial bones. Serum concentrations of FGF23 are elevated in these subjects, and calcitriol values are inappropriately normal. Urinary calcium excretion is normal. Familial ARHR has been associated with homozygous loss-of-function mutations in the gene encoding dentin matrix acidic phosphoprotein 1 (DMP1).305,306 DMP1 is a member of a class of tooth and bone noncollagenous matrix proteins termed SIBLING proteins (small integrin-binding ligand, N-linked glycoproteins) that include osteopontin and bone sialoprotein. These substances regulate phosphorylation of proteins essential to initial nucleation of calcium and phosphate, as well as to early formation of hydroxyapatite crystals and hence to mineralization of osteoid.307 DMP1 is ex-

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pressed in osteocytes. Rickets and osteopenia develop in mice in whom Dmp1 has been deleted.305,308 In osteocytes without Dmp1, the expression of Fgf23 is increased and secondarily hyperphosphaturia develops—suggesting that Dmp1 regulates expression of Fgf23. This disorder has permitted identification of a bone-renal axis essential to normal bone mineralization, although the mechanism by which Dmp1 may regulate expression of Fgf23 has not yet been elucidated. Autosomal-recessive hereditary hypophosphatemic rickets with hypercalciuria (OMIM 241530) is due to renal wastage of phosphate and is the consequence of a loss-of-function mutation in SLC34A3 encoding sodium-dependent phosphate co-transporter type IIC (NPT2c). This 599aa protein with eight transmembrane domains is located in the brush border of juxtamedullary proximal renal tubular cells. Because calcitriol synthesis is normal in these subjects, its production is substantially elevated in response to hypophosphatemia. Thus, intestinal absorption of calcium and its urinary excretion are increased. Heterozygotic carriers of inactivating mutations in SLC34A3 also have moderately increased serum concentrations of calcitriol, hyperphosphaturia, and hypercalciuria but do not have identified metabolic bone disease. Lossof-function missense and nonsense mutations have been found throughout the coding region of SLC34A3, as well as deletions in introns 9 and 10.309,310 This disorder may be treated with phosphate salts in conjunction with hydration and avoidance of a high-sodium diet. Supplemental vitamin D is not needed, and may even be detrimental. Increased urinary phosphate excretion due to acquired and heritable disorders of the proximal renal tubule is characteristic of the metabolic bone disease that accompanies various forms of the Fanconi syndrome of renal tubular acidosis, glucosuria, and amino aciduria (heritable:cystinosis, tyrosinemia, galactosemia, oculo-cerebral-renal syndrome, fructose intolerance, and Wilson disease, and acquired renal transplantation,

E

Figure 17-14 Demonstration by gradient recall echo magnetic resonance imaging of a FGF23 producing hemangiopericytoma in the left iliac wing of an 11-year-old boy with tumor-induced rickets. [Reproduced with permission from Shulman DI, et al. (2004). Tumor-induced rickets: Usefulness of MR gradient echo recall imaging for tumor localization. J Pediatr 144:381–385.]

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nephrotic syndrome, renal vein thrombosis, mercury, lead and copper poisoning, and outdated tetracycline).311,312 In addition to hypophosphatemia, acidosis contributes to the pathogenesis of bone disease in Fanconi syndrome by increasing the solubility of the mineral phase of bone, increasing urinary loss of calcium, and impairing conversion of calcidiol to calcitriol. Inactivating mutations in CLCN5 encoding a voltagegated proximal renal tubular chloride channel lead to X-linked recessive hypophosphatemic rickets (XLRH, OMIM 300534); X-linked nephrocalcinosis, nephrolithiasis, and renal failure (OMIM 310468); and Dent disease (aminoaciduria, proteinuria, glycosuria, hypercalciuria, nephrocalcinosis, nephrolithiasis; OMIM 300009).311,313 Mutations in different domains of this 12exon 746aa transmembrane chloride channel result in varying clinical and biochemical manifestations. The Ser244Leu mutation in CLCN5 has ben particularly associated with X-linked recessive hypophosphatemic rickets.314 Causes of hyperphosphatemia are also listed in Table 17-8.

Disorders of Alkaline Phosphatase In its severe forms, hypophosphatasia is an autosomalrecessive disorder due to loss-of-function mutations in ALPL—the gene encoding the isoenzyme of tissuenonspecific (bone/liver/kidney) alkaline phosphatase (TNSALP).98 TNSALP is a homodimeric phosphomonoesterase anchored through its carboxyl terminal to the exterior of the cell membrane (i.e., an ectophosphatase) by a phosphatydylinositol-glycan moiety.315 Pathophysiologically, decreased TNSALP activity leads to accumulation of its endogenous substrates pyrophosphate, pyridoxal 5’-phosphate, and phosphoethanolamine. Pyrophosphate is an inhibitor of osteoid mineralization. Calcification of bone matrix is impaired because pyrophosphate coats the surface of hydroxyapatite crystals, restricting crystal growth. In addition, inability to raise bone matrix phosphate levels to values sufficient to permit normal deposition of hydroxyapatite contributes to decreased bone mineralization. Clinical manifestations of inactivating mutations of ALPL reflect the extent of loss of function of mutant ALPL. The younger the age of onset the more severe the disease is likely to be. The perinatal form is often a lethal disorder (at times in utero) due to marked osteopenia leading to cranial malformation; intracranial hemorrhage; pyridoxine-dependent seizures; short, bowed, and fractured extremities; and fractures of the ribs and deformities of the chest wall resulting in respiratory insufficiency and apnea. Survival is unusual. The infantile form (OMIM 241500) develops in the first 6 months of life and is characterized by anorexia, impaired linear growth and weight gain, deformities of the long bones and rib cage, widely open fontanelles and sutures (calvarial hypomineralization) with functional craniosynostosis with increased intracranial pressure manifested by prominence of the anterior fontanelle, proptosis, papilledema, pyridoxine-dependent seizures, hypotonia, constipation, hypercalcemia, hypercalciuria, and radiographic evidence of rickets with marked skeletal hypomineralization. Approximately 50% of these infants die of respiratory failure before their first birthday.

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During childhood (OMIM 241510), clinical findings range from isolated premature shedding of deciduous teeth to physical and radiographic evidence of low bone mass and rickets. Spontaneous clinical improvement may occur during puberty and evolve into the adult form (OMIM 146300), which may be subclinical and identified when studying the family of an offspring with hypophosphatasia. The parent may have a history of premature loss of teeth, increased susceptibility to recurrent fractures predominantly of the metatarsal bones, pseudofractures of the proximal femur, chondrocalcinosis, or pseudogout due to articular deposition of calcium pyrophosphate dihydrate crystals. Odontohypophosphatasia (OMIM 146300) is manifested only by premature shedding of primary teeth without radiologic abnormalities. Periodontitis may also develop. Pseudohypophosphatasia is phenotypically and biochemically similar to the classic disease, but serum alkaline phosphatase activity in vitro is normal—indicating that in this disorder enzyme activity toward fabricated substrates is preserved but alkaline phosphatase activity toward endogenous substrates is abnormal. The perinatal and infantile forms of hypophosphatasia are usually inherited as autosomal-recessive diseases, whereas childhood and adult forms may be transmitted as autosomal-recessive or autosomal-dominant traits. In addition to the site(s) of mutation of ALPL, the clinical severity of hypophosphatasia is related inversely to the age at which skeletal disease is evident and to individual biologic factors that affect the expression of the disease. In the lethal perinatal form of hypophosphatasia, missense, nonsense, and frame-shift mutations in TNSALP cluster in crucial segments of the protein within (Ala94Thr, Arg167Trp) or near (Gly103Arg, Gly317Asp) its enzyme active site or at the homodimer interface (Ala23Val, Arg374Cys).315 These mutations interrupt binding to the phosphate ligand or destabilize attachment of necessary cofactors. On the other hand, ALPL mutations associated with infantile and childhood forms of hypophosphatasia (Arg119His, Asp361Val) tend to cluster on the three-dimensional surface of the enzyme molecule (at sites that relate to its tethering to the cell surface or its formation of tetramers) and retain substantial residual bioactivity. In some patients with moderately severe hypophosphatasia, heterozygotic mutations in ALPL (Gly46Val, Arg167Trp, Asn461Ile) exert a dominant-negative effect on the intact TNSALP dimer. These mutations have been clustered at the enzyme active site or at the domain(s) involved with dimerization, tetramerization, or membrane anchoring.316 Odontohypophosphatasia has been associated with heterozygotic mutations in ALPL (Pro91Leu, Ala99Thr). In a mouse knockout model of hypophosphatasia with inactivating mutations of Alpl, the clinical, biochemical, radiographic, and histologic findings are similar to those in infants with this disease.317 These animals are small at birth, grow poorly, and succumb within the first several months of life. Bone roentgenograms and histology are normal at birth and through 8 days of age, even though no TNSALP activity can be demonstrated. They become increasingly abnormal thereafter. There is developmental arrest of chondrocyte differentiation, with failure of hypertrophic

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zone differentiation, formation of secondary ossification centers, and marked accumulation of unmineralized bone matrix—leading to skeletal deformities and fractures. In addition, these animals have seizures due to deficiency of pyridoxine—the product of TNSALP dephosphorylation of pyridoxal 5'-phosphate and the cofactor form of vitamin B6 necessary for the synthesis of neural ␥-aminobutyric acid. Diagnostically, in addition to the clinical findings and radiographic features of rickets patients with hypophosphatasia have low serum levels of total and bone-specific alkaline phosphatase activity, increased serum concentrations of pyrophosphate and pyridoxal-5'-phosphate (⬃5,000 nM), and elevated urinary excretion of phosphoethanolamine (0.5–1.5 mmol/g of creatinine, control 0.15 mmol/g) and pyrophosphate (500–1,000 ␮mol/g of creatinine, control 200 ␮mol/g). In contrast to other forms of rickets, serum concentrations of calcium and phosphate are normal or even elevated—likely because intestinal absorption of calcium and renal tubular reabsorption of phosphate are normal, whereas the bone formation rate is depressed. In the perinatal and infantile forms of hypophosphatasia, hypercalcemia is frequent.98,228 There are normal serum levels of PTH and no evidence of secondary hyperparathyroidism. Although serum alkaline phosphatase activity in vitro is normal in patients with pseudohypophosphatasia, it is functionally inadequate in vivo—perhaps because the TNSALP protein is sequestered within the cell due to a defect in its transport to the cell surface or because the protein is active with artificial substrate but not with natural substrates at physiologic pH. There is no specific or effective therapy for hypophosphatasia currently available. Enzyme replacement by infusion of serum from patients with Paget disease and high alkaline phosphatase activity has been of only occasional and limited benefit. Administration of even small doses of vitamin D or its metabolites should be avoided because these patients easily develop hypercalcemia. Hypercalcemia in infants with hypophosphatasia responds to bisphosphonates and transiently to calcitonin.318 Bone marrow transplantation may be effective therapy but requires further evaluation.98 Seizures may be responsive to pyridoxine administration. Milder adult forms of hypophosphatasia may be responsive to teriparatide (rhPTH1-34).318a Serum bone-specific alkaline phosphatase activity is normally increased during puberty and in response to skeletal injuries and reflects osteoblastic activity. Hyperphosphatasemia is encountered transiently in clinically well infants and children less than 5 years of age (median age 16 months). In this situation, serum activities of bone and liver alkaline phosphatases isoforms are increased, but there are no skeletal abnormalities. It is a self-limited process, with alkaline phosphatase values returning to normal within several months.319 Rarely, hyperphosphatasemia may be familial and transmitted as a benign autosomal-dominant trait.320 Occasionally, an adolescent with persistent nonfamilial benign hyperphosphatasemia may be encountered. In one family, familial hyperphosphatasemia has been associated with mental retardation (OMIM 239300). Juvenile Paget disease (OMIM 239000), also termed hyperostosis corticalis deformans juvenilis, often begins

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in early childhood and is characterized clinically by expanded and bowed extremities, nontraumatic fractures of the long bones, kyphosis, macrocephaly, and muscular weakness. This disorder may progress to wheelchair dependence. Serum levels of alkaline phosphatase are markedly increased—a coupled response to increased osteoclastic action. Radiographic skeletal abnormalities include cortical thickening, osteosclerosis and osteopenia, coarse trabeculations, and progressive skeletal deformities. The disorder is due to biallelic loss-of-function mutations of TNFRSF11B encoding osteoprotegerin—a member of the tumor necrosis factor (TNF) receptor superfamily that functions as a decoy acceptor for receptor activator of nuclear factor ␬B-ligand (RANKL)—thereby modulating osteoclastogenesis. Hence, inactivating mutations of TNFRSF11B are associated with enhanced osteoclastic activity. The severity of juvenile Paget disease depends on the site of mutation. Those that result in deletion of the entire gene or those in the ligand-binding domain that involved loss of cysteine residues result in marked clinical disease.321323 Treatment with recombinant osteoprotegerin has resulted in clinical and radiologic improvement.324 Familial expansile osteolysis (OMIM 174810) is an autosomal-dominant disorder pathophysiologically similar to juvenile Paget disease but clinically and etiologically distinct. Focal areas of increased bone turnover in the appendicular skeleton appear in the second decade of life, followed by medullary expansion, pathologic fractures, and skeletal deformities. Deafness and premature loss of dentition may occur. The disorder is due to monoallelic gain-of-function mutations (insertion duplications of 18 and 27 bases in the signal peptide region of exon 1) of the gene (TNFRSF11A) encoding RANK that result in an increase in NF␬B signaling, as a consequence of which there is augmented osteoclastogenesis.325

RENAL OSTEODYSTROPHY Renal osteodystrophy is the metabolic bone disease that accompanies chronic renal failure. It is most commonly associated with a high rate of bone remodeling (increased rates of bone resorption and formation) due to secondary hyperparathyroidism (high-turnover lesions of osteitis fibrosa). Dynamic (low-turnover) bone disease with relatively low PTH secretion and osteomalacia due to accumulation of aluminum may also occur. Generally, areas of low- and high-turnover skeletal abnormalities (termed mixed renal osteodystrophy) are detected by histomorphometry.326,327 The pathogenesis of secondary hyperparathyroidism is multifactorial (Figure 17-15). When the glomerular filtration rate declines to less than 30% of normal, urinary phosphate excretion is impaired— leading to its intracellular and extracellular accumulation, hyperphosphatemia, and very slight hypocalcemia (the latter two leading to secondary increase in PTH generation). In addition, down-regulation of the expression of CASR in uremic PTGs (raising of the set-point) contributes to chief cell hyperplasia and augmented PTH release—as do decline in calcitriol synthesis and relative skeletal insensitivity to PTH. Phosphate may also slow the rate of

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Phosphorus retention ↑ Phosphorus ↓ Renal mass ? Acidoisis ? Other

Low calcitriol levels

Altered PTG growth and function

Hyperparathyroidism ↑ Phosphorus ↓ Calcitriol Calcitriol resistance Skeletal resistance

Hypocalcemia PTG: Parathyroid Gland

Skeletal resistance VDR: Vitamin D Receptor

Hypocalcemia ↓ Calcitriol Calcitriol resistance ↑ Set-point ↓ VDR PTG Hyperplasia ↑ PTG mass ↓ Ca receptor ↑ Phosphorus ⌬ PTH mRNA stability ↑ Phosphorus Desens. to PTH ↓ PTH-R ? ↓ Calcitriol ? Uremic toxins ? PTH inhibitors

Figure 17-15 Pathophysiology of renal osteodystrophy. [Reproduced with permission from Martin KJ, et al. (2006). Renal osteodystrophy. In Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 359–366.]

degradation of PTH mRNA within the PTG and exert a direct enhancing effect on PTG growth. Because calcitriol inhibits PTG growth and function, decrease in its synthesis also results in increased proliferation of parathyroid chief cells and synthesis of PTH. In the presence of elevated PTH secretion and various cytokines [IL-1, -6, and -11; TNF; and macrophage-colony stimulating factor (M-CSF)], osteoclastogenesis and the rates of bone resorption and formation are increased. Acidosis contributes to the dissolution of the mineral phase of bone directly and by impairing osteoprotegerinmediated inhibition of osteoclast generation. Chronic renal disease and osteodystrophy in children may be clinically silent except for failure of linear growth. As the disease progresses, deformities of the extremities, slipped epiphyses, fractures, bone and joint pain, and weakness and lassitude develop. In patients in whom the rate of bone formation is diminished and the volume of unmineralized bone (i.e., osteomalacia) increased, the process has been due to accumulation of aluminum at the mineralization front. However, with discontinuation of aluminum-containing phosphate binders osteomalacia in renal failure is now unusual.326 In the absence of osteomalacia, adynamic bone disease in chronic renal failure is the result of decreased PTH generation due to improved control of serum phosphate levels, increased calcium stores, higher levels of PTH7-84 and other carboxyl terminal fragments of PTH that inhibit bone resorption, and other factors that affect tissue response to PTH. Biochemically, renal osteodystrophy is marked primarily by hyperphosphatemia and increase in serum PTH concentrations and alkaline phosphatase activity. Radiographic signs of rickets, low bone mass, and pseudofractures are often present in children with renal osteodystrophy. Therapeutically, the goals in treating a child with chronic renal failure in an effort to minimize renal osteodystrophy are to maintain (near) normal serum calcium, phosphate, and alkaline phosphatase values and to prevent the development or progression of secondary hyperparathyroidism. To do so, supplemental vitamin D

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and calcium may be needed—and dietary phosphate restriction imposed. Calcitriol is able to decrease the rate of bone formation in patients with chronic renal insufficiency by inhibiting osteoblast differentiation or function, decreasing PTH synthesis, altering intra-PTG degradation of PTH, and decreasing expression of PTHR1. Calcium-containing oral phosphate binders may also be useful. Dialysis fluids must be prepared with aluminum-free water. When indicated, suppression of PTH secretion may be further achieved with the use of calcitriol analogues such as paricalcitol or of a synthetic ligand of the CaSR (cinacalcet hydrochloride).326 After successful renal transplantation, secondary hyperparathyroidism may persist for months or years—its extent and intensity reflecting the severity and duration of chronic renal failure before renal transplantation, development of nodular or monoclonal hyperplasia of the PTGs, and the 20-year life span of the parathyroid chief cell.328 In one study of 47 childhood kidney recipients, 3 years after renal transplantation iliac crest bone biopsies revealed normal bone formation in 65% but persistent mild hyperparathyroidism in 25% and adynamic bone disease in 10%.329 In all children in whom prerenal transplantation iliac crest biopsy revealed normal bone formation rates, the post-transplantation biopsy was also normal. In approximately 50% of those patients with an abnormal pretransplantation biopsy, normal bone formation rates were achieved in the post-transplantation period. However, in many subjects secondary hyperparathyroidism or adynamic bone disease persisted or evolved—and random measurements of serum PTH concentrations did not necessarily correlate with or predict the histologic picture. Five years after renal transplantation during childhood, serum concentrations of PICP, osteocalcin, and ICTP remain significantly increased—indicating accelerated bone turnover rate—whereas areal and volumetric bone mineral densities at the distal third of the nondominant radius are normal for height but subnormal

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

for age.330 Hypercalcemia and persistent secondary or tertiary hyperparathyroidism requiring parathyroidectomy may become apparent in children after renal transplantation.331 The osteopenic effects of glucocorticoids and immune suppressive agents such as cyclosporin are observed in postrenal transplantation patients as well.

LOW BONE MASS Osteopenia and osteoporosis (porous bone) designate states of reduced bone mass and abnormalities of bone microarchitecture that increase the risk for fracture (osteomalacia refers primarily to decrease in the mineral phase of bone).332,333 Osteoporosis in adults is defined by the World Health Organization (WHO) as a BMD at a specific bone site that is –2.5 or more SD below the mean peak young adult value for gender (T score). In adults, osteopenia is present when the BMD lies between –1.1 and –2.4 SD below the mean peak young adult value for gender—and a normal BMD is one that is not more than 1 SD below or above the mean peak young adult value. When the BMD is more than ⫹2 SD above the mean for age and gender, bone mass is considered high. The WHOdesignated categories of low bone mass do not necessarily apply to children and adolescents in whom variability in height, weight, and stage of sexual maturation affect bone mineralization. Use of the terms osteopenia and osteoporosis has been discouraged (although not eliminated) when describing mineralization in children. Presently, children may be identified as those with low bone density (Table 17-9) for chronologic age or height or stage of sexual maturation (reported as less than –2 SD or Z score for gender)—provided the method employed for determination of bone mineralization [DEXA, quantitative computed tomography (QCT), QUS] and the specific instrument, software version utilized for analysis, and ethnic mix of the reference population are stated.334-337 Despite its limitations (provision of areal rather than volumetric BMD data and failure to distinguish between trabecular and cortical bone), DEXA is the most widely employed bone densitometric method in children at this time—although peripheral (p) QCT may become the method of choice in the future. In infants, children, and adolescents, DEXA whole-body bone mineral measurement (BMC, BMD) rather than regional measurements is the currently preferred index of mineralization status. The inclusion or exclusion of head measurements in the determination of whole-body BMC and BMD by DEXA should also be stated.338,339 Relative to bone strength determined by calculation of the stress-strain index of a long bone with data garnered by pQCT, the DEXA whole-body areal BMC (minus the skull) for height appears to afford the most reliable measurement for determining cortical bone strength and hence fracture risk.338 Although heritable factors account for 60 to 80% of optimal bone mineralization, modifiable factors that contribute to the development of osteopenia and osteoporosis in adulthood (weight-bearing exercise, nutrition, body mass, hormonal milieu) have their genesis in utero, infancy, childhood, and adolescence.332,333 In children (as in adults), bone mass, composition, and size determine bone strength. Decreased bone strength is associated with

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TA B L E 1 7 - 9

Disorders of Bone Mineralization: Low Bone Mass I. Primary A. Osteogenesis Imperfecta (Types I–VIII) B. Osteoporosis-Pseudoglioma Syndrome (LRP5) C. Idiopathic Juvenile Osteoporosis D. Marfan Syndrome (FBN1) E. Ehlers-Danlos Syndrome (COL1A1) F. Homocystinuria (CBS) G. Idiopathic Hypercalciuria H. Fibrous Dysplasia (GNAS) I. Glycogen Storage Disease Type I (G6PC) J. Menkes Kinky Hair Syndrome (ATP7A) II. Secondary A. Suboptimal Nutrition 1. Socioeconomic 2. Cultural 3. Excessive exercise 4. Anorexia nervosa 5. Malabsorption - cystic fibrosis, celiac disease, biliary atresia, short gut syndrome, gastric bypass B. Endocrinopathies/Metabolic Diseases 1. Constitutional delay in growth and sexual development 2. Hypogonadism a. Hypergonadotropic - gonadal dysgenesis (Turner, Klinefelter syndromes), aromatase deficiency, estrogen receptor deficiency b. Hypogonadotropic - Kallmann syndrome, excessive physical activity, hyperprolactinemia 3. Diabetes mellitus 4. Hyperglucocorticoidism 5. Hyperthyroidism 6. Hyperparathyroidism 7. Growth hormone deficiency 8. Inborn errors - homocystinuria, lysinuric protein intolerance, propionic aciduria, methylmalonic aciduria C. Disuse/Immobilization 1. Femoral fracture 2. Cerebral palsy 3. Muscular dystrophy 4. Quadriplegia/paraplegia 5. Spina bifida D. Drugs 1. Glucocorticoids, immune suppressants, anticonvulsants, antiretroviral therapy, warfarin, lithium, methotrexate, cyclosporine A 2. Alcohol, tobacco E. Chronic Illness 1. Rheumatologic disease 2. Inflammatory bowel disease 3. Hemoglobinopathies - thalassemia, sickle cell disease 4. Hemophilia 5. Cranial radiation 6. Renal failure, transplantation 7. Malignancy - leukemia, lymphoma 8. Human immunodeficiency virus infection Adapted from Bachrach LK (2005). Osteoporosis and measurement of bone mass in children and adolescents. Endocrinol Metab NA 34:521–535; and from Rauch F, Bishop N (2006). Juvenile osteoporosis. In Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 293–296.

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

a greater risk of forearm fractures in children.340 Because many youths consume excessive amounts of carbonated beverages and diluted fruit juices (thus limiting their intake of milk), most children and adolescents ingest only 55% to 70% of the recommended daily calcium allowance (1,300 mg/day)—although late pubertal males tend to consume more than do pubertal females.341-343 In adolescent and adult females, excessive intake of cola drinks with high phosphoric acid content lower body calcium content because of the sequestration of dietary calcium in the intestinal tract and the needed dissolution of bone mineral to neutralize acid—with consequent development of mild secondary hyperparathyroidism.344,345 Sedentary non weight-bearing activities encouraged by television, video, and computer games also impair bone mineralization.346 It had been suggested that fat may enhance bone mineralization through increased mechanical stress and estrogen production and through the stimulatory effects of leptin on osteoblast differentiation. Although body weight and fat mass have correlated with bone mass in many studies, other data suggested that fat has a negative effect on the accrual of bone mass.347,348 In a study of 300 male and female adolescents and young adults (13–21 years of age) employing DEXA assessment of body composition and QCT measurement of the axial and appendicular skeletons, a positive correlation between lean mass and all bone measurements was demonstrated in both genders—whereas fat mass had an inverse relationship or no relationship to bone mass. These observations strongly suggest that bone mass and strength are determined by dynamic muscular force and not by static load.348 The mechanism(s) through which fat might exert an inhibitory effect on bone mass is unknown but may be related to the synthesis of cytokines that negatively affect bone accrual. An alternative postulated mechanism is through diversion of the mesenchymal stem cell (common to adipocytes and osteoblasts) into the adipogenic pathway. The longer and more intense the weekly sporting activity (soccer, basketball, gymnastics, tennis) in children and adolescents the greater the vertebral and femoral BMDs independently of calcium intake.343,346 In pre- and peripubertal children, simple school physical education programs utilizing jumping, hopping, and skipping exercises two to three times weekly significantly increase areal BMD at the femoral trochanter in as little as 8 months relative to children engaged in a standard physical education curriculum.349-351 Thus, suboptimal nutrition and sedentary activities during childhood and adolescence (as well as consumption of colas and alcohol and smoking of cigarettes) prevent optimal bone mineralization and increase the likelihood of later development of osteoporosis and its complications.342,352 Because the risk of developing an osteoporotic fracture declines by 40% for every 5% increase in peak bone mineral mass, the foundation for the prevention of osteoporosis in the adult must be constructed in the child and adolescent by maintaining adequate calcium intake (1–3 years, 500 mg/day; 4–8 years, 800 mg/day; 9–18 years, 1,300 mg/day; ⬎19 years, 1,000 mg/day), vitamin D stores (serum concentrations of 250 HD

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⬎30–32 ng/mL), and complementary weight-bearing activity during these formative years.341,353 Discouragingly, combined quantitative analysis of multiple trials of calcium supplementation (usually of relatively short duration) in children revealed little effect on BMD or reduction of the risk of fracture.354 However, the effects of sustained calcium supplementation over the many years of childhood and adolescence on future fracture risk has not been systematically examined. Magnesium supplementation for 1 year increased BMC of the hip in healthy girls.139 Sufficient protein, vitamins C and K, and copper must also be consumed for optimal bone matrix synthesis. It may be possible to identify the child or early pubertal subject at (genetic) risk for accrual of low peak bone mass and thus for later development of osteopenia/osteoporosis (e.g., offspring of a mother with osteopenia or osteoporosis). Axial and appendicular BMD and bone size determined by central or pQCT in the normal early pubertal boy and girl may accurately predict these measurements at sexual maturity.355 If so, children at risk for low peak bone mass might benefit by a diet and exercise program during puberty that increases these values. Nutritional deprivation depresses the rate of bone accrual, a process observed most dramatically in subjects with anorexia nervosa. The majority of postmenarchal late adolescent females with anorexia nervosa have significantly decreased total body, vertebral, and femoral neck areal BMDs—although volumetric BMDs may be normal for their small bone size.356,357 In adolescent females with anorexia nervosa, decreased bone mineralization is associated with a slow rate of bone turnover—as demonstrated by lower serum concentrations of osteocalcin, estradiol, free testosterone, IGF-I, leptin, and bone-specific alkaline phosphatase and depressed urinary excretion of Dpd relative to normalweight subjects.356,358 In these patients, serum levels of osteoprotegerin correlate negatively with fat mass and leptin values and with lumbar spine areal and apparent BMDs. (by DEXA) The decline in bone mass in adolescents with anorexia nervosa may be attributed to nutritional deprivation, chronic acidemia, and functional hypogonadism. Thus, the osteopenia encountered in patients with anorexia nervosa is the result of generalized nutritional deprivation with suboptimal intake of protein, calcium, and vitamin D; hypercortisolemia; and lowered IGF-I generation— leading to decreased osteoblast-mediated bone formation. Hypoestrogenism enhances to a limited extent osteoclaststimulated bone resorption.359 The bone loss of the patient with anorexia nervosa is not fully recovered even after return to normal weight, resulting in a several fold increase in fracture risk for these women. In adolescents with anorexia nervosa, administration of estrogen/progestin does not increase bone mass or prevent its loss.360 Bisphosphonates, IGF-I, and dehydroepiandrosterone have been reported to increase or maintain BMD in small series of patients with anorexia nervosa. However, their use should be limited presently to investigational studies.359 Experimentally, in young adult female rats isocaloric restriction of protein alone lowers plasma concentrations of IGF-I—resulting in a decreased

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

rate of periosteal cortical bone formation and impaired osteoblastic responsiveness to IGF-I.361 The athletic triad of suboptimal body fat mass, amenorrhea in women, and osteoporosis is encountered in the highly trained female athlete and in male elite long-distance runners. Acute immobilization of the healthy active child and adolescent leads to sudden reduction in weight bearing, and to consequent decrease in the mechanical load on bones and thus a lowered rate of bone formation. In the presence of continued bone resorption, hypercalciuria and later hypercalcemia and lowered bone mass develop.362 In the chronically partially or fully immobilized child or adolescent (cerebral palsy, spastic quadriplegia, muscular dystrophy), the fracture rate (primarily of the femur) is high during such simple maneuvers as turning, dressing, or feeding. In this group of subjects, not only lack of weight bearing but the severity of the primary illness, body size and pubertal status, state of general nutrition, vitamin D and calcium intake, coexisting inflammatory states, medications (e.g., anticonvulsants, glucocorticoids), and indoor confinement adversely impact bone mass and fracture risk.363-365 In a study group of 117 patients (2 to 19 years of age) with moderate to severe cerebral palsy, distal femoral BMD Z scores were below –2.0 in 77%—and the incidence of low femoral and vertebral BMDs as well as fractures increased with advancing age.363 Distal femoral and lumbar vertebral BMDs increase at slower than normal rates as the child with spastic cerebral palsy ages, resulting in diminution of BMD Z score in the older subject.364 Intravenous administration of pamidronate in a small (N ⫽ 6) selected group of children with quadriplegic cerebral palsy increased distal femoral BMD by ⫹88% over 18 months, with mean Z score increasing from –4.0 to –1.8 over the interval of treatment.366 In 5 lads with spastic cerebral palsy, daily administration of recombinant human GH (0.35 mg/kg/week) increased vertebral BMD assessed by DEXA by ⫹1.17 SD over 18 months of therapy without altering the quality of life of these subjects.367 Assisted standing alone increases BMD in children with severe cerebral palsy.368,369 The fundamental importance of normal gonadal sex steroid secretion during age-appropriate sexual maturation is emphasized by the observation that in adult males with delayed sexual development radial, vertebral, and femoral areal BMDs are lower than in males with normal timing of pubertal maturation.370 Volumetric BMD has been reported to be normal or subnormal in young adult men with a history of delayed adolescence.371,372 In prepubertal children of both genders with constitutional delay in growth, areal vertebral (trabecular) and nondominant radial (cortical) BMC and BMD are decreased relative to values in age and size-matched subjects with familial genetic short stature before and after correction for height, weight, bone age, and sex.373,374 This finding suggests that factors other than sex hormones may also impair bone mineralization in short children with delayed skeletal maturation. Osteoporosis with reduced bone mass and abnormal microarchitecture results in decreased skeletal strength and increased risk of fracture. Histomorphometrically, in sex-steroid-deprived osteoporotic bone there is

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decrease in bone cortical width, trabecular number, osteoid, and mineralization activity.375 As a consequence of estrogen (and androgen) deficiency, there is increase in the production but decline in the life span of osteoblasts and osteocytes—whereas osteoclastogenesis is stimulated and the life span of osteoclasts prolonged. These events reflect the sum of the activities of multiple pro-osteoclastogenic cytokines (including M-CSF, IL-1, IL-6, TNF, RANKL), whose synthesis is regulated by estrogen.375,376 Although osteopenia responsive to estrogen has been recorded in adult males with aromatase deficiency, mature women with complete androgen insensitivity are also osteopenic despite normal to increased estrogen production—clearly indicating that androgens are also important to normal bone mineralization.377 In prepubertal children and in adolescents with Turner syndrome, there is osteopenia with decreased cortical and trabecular bone mass.378 Although decreased relative to chronologic age, areal and volumetric BMDs may be normal relative to height or bone age in girls with Turner syndrome. Nevertheless, the frequency of wrist fractures is increased during childhood—as may be the general risk for fractures in adults with Turner syndrome. Estrogens, GH, and particularly the administration of both agents increase bone calcium deposition and BMD in adolescents with Turner syndrome.378-380 In adults with Turner syndrome, however, there appears to be intrinsic reduction in cortical bone that is independent of sex hormones and might possibly be related to elevated levels of follicle-stimulating hormone—which may have intrinsic proresorptive properties distinct from its effect on estrogen synthesis.307,381 Pubertal subjects with primary (Klinefelter syndrome, galactosemia, postradiation, or chemotherapy) or secondary hypogonadism (anorexia nervosa, excessive physical training, hypogonadotropism) also have decreased bone mineralization. By decreasing estrogen production, even short-term (6 months) use of the intramuscular contraceptive depot medroxyprogesterone acetate (MPA) results in significant loss in bone mass in adolescent females and young women (18–21 years of age) when BMD would ordinarily be increasing. However, bone mass increases over time after this agent is discontinued.359 The adverse impact of depot MPA on BMD may be prevented by concomitant administration of estrogen. Estrogen-/progestin-containing oral contraceptives do not decrease bone mass in adolescent females, although they do slow its rate of acquisition. After treatment of the child with central precocious puberty for 1 to 2 years with a gonadotropin-releasing hormone analogue that suppresses pituitary-gonadal function, there may be arrest or even decline in bone mineral accumulation in the peripheral and axial skeletons—a process that can be prevented or reversed by the co-provision of 1 g of calcium per day during analogue therapy.382 The low bone mass of glucocorticoid excess is the result of inhibition of osteoblastogenesis and increase in the rate of apoptosis of the osteoblast and osteocyte leading to decrease in the rate of bone matrix formation and microfracture repair, and of enhanced osteoclastogenesis and decrease in the rate of apoptosis of the

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

osteoclast—permitting prolonged and excessive bone resorption.383 Thus, during each remodeling cycle the amount of bone replaced is far less than the amount removed and skeletal microarchitecture is degraded— resulting in declining bone strength. At the molecular level, glucocorticoids suppress expression and synthesis of RUNX2 and BMP-2—factors essential to prenatal and postnatal osteoblast differentiation, respectively—and increase osteoblast expression of RANKL and decrease expression of osteoprotegerin, changes that favor osteoclastogenesis.383-385 Glucocorticoids inhibit synthesis of collagen type I and increase its rate of degradation. They impair IGF-I formation and function. To a limited extent, glucocorticoids inhibit normal vitamin D metabolism and thereby vitamin-D-dependent intestinal absorption of calcium. They also increase renal loss of calcium by a direct effect on the renal tubule, leading to secondary hyperparathyroidism.383,384 Glucocorticoids also reduce production of sex hormones in the adolescent and adult. The muscle weakness of chronic glucocorticoid exposure reduces the impact of mechanical forces on bone formation. Finally, the disease for which glucocorticoids have been prescribed may contribute to decreased bone mass by impairing mobility and by elaboration of osteoclastogenic cytokines. The risk of glucocorticoid-induced low bone mass is far greater with oral than with inhaled glucocorticoids in children with asthma.386 However, in young adults with asthma there is an inverse relationship between vertebral and femoral BMDs and cumulative dose of inhaled glucocorticoid—with increasing fracture risk as the dose and duration of glucocorticoid administration increases. A cumulative dose of 5,000 mg leads to a 1 SD decline in vertebral BMD.387 In adult women with 21-hydroxylasedeficient congenital adrenal hyperplasia treated with glucocorticoids, bone mineralization is modestly reduced—in part related to the extent of suppression of adrenal androgen production.388 In children experiencing adverse effects of glucocorticoids on growth and bone mineralization, it is important to lower their steroid dose to the greatest extent possible and to withdraw them if at all feasible. Increased weightbearing exercise (walking) and supplemental calcium and active vitamin D metabolites may be helpful. In 7 of 10 children with juvenile rheumatoid arthritis and other rheumatic disorders and glucocorticoid/illness-mediated low bone mass, pamidronate at a dose to 2 to 4 mg/kg per infusion administered at 6-month intervals was followed by decline in bone pain, improved ambulation, and progressive increase in BMD of the lumbar spine.389 PTH1-34 increases bone mass in adults with glucocorticoid-induced osteoporosis, but its efficacy and safety in children with this problem have not yet been evaluated.390 Low bone mass is often encountered in GH-deficient children and in adults with GH deficiency of childhood or adult onset. In part, low bone mass may be attributed to the small bone size of the short child compared to age peers. It is also due to loss of direct and indirect actions of GH (particularly impaired local generation of IGF-I) on osteoblast differentiation, proliferation, and function.

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GH enhances osteoblastic expression of IGF-I and IGF binding protein-3 and stimulates their synthesis of osteocalcin, bone-specific alkaline phosphatase, and procollagen type I—thus increasing bone matrix formation.391 Secondarily, it increases osteoclastogenesis and bone resorption. Thus, administration of GH to GH-deficient children and adults increases the rates of bone formation and destruction—the latter predominating initially. Over long periods of treatment (12–18 months), GH increases BMD in these patients. To achieve peak bone mass, however, GH therapy must be continued into adulthood. Nevertheless, in many untreated adults with congenital GH deficiency volumetric BMD is often normal— reflecting the smaller size of their bones.392 In normal short children, GH administration also increases areal BMD.393 Thyroid hormone, through direct action on the osteoblast, increases synthesis of osteocalcin, alkaline phosphatase, and IGF-I. It also enhances osteoclastogenesis, and thus the rate of bone resorption—the latter effect predominating.394 With excess thyroid hormone there is increase in the rate of bone turnover but decrease in the length of the bone remodeling cycle (primarily due to shortening of the bone formation phase), resulting in a net loss of mineralized bone. As in adults with thyrotoxicosis, whole-body, vertebral, and femoral BMDs are low in children and adolescents with hyperthyroidism—but substantially increase within the first 12 to 24 months after restoration of the euthyroid state.395 Administration of physiologic replacement doses of thyroxine to children with acquired or congenital hypothyroidism does not adversely affect bone mineralization during childhood, although adults with congenital hypothyroidism have a 10% reduction in radial bone mass.396 In adolescents with type 1 diabetes mellitus, whole-body, axial, and appendicular bone mass assessed by DEXA is decreased relative to control subjects and inversely related to hemoglobin A1c values—reflecting the adverse effects of chronic hyperglycemia and insulin deficiency on bone formation.397,398 Utilizing pQCT in young prepubertal subjects with type 1 diabetes mellitus, cortical bone cross-sectional area and BMD were found to be decreased—implying an increased risk for fracture.399 Bone mass is decreased in 80% of children with acute lymphoblastic leukemia, and 40% sustain a fracture within the first 2 years of treatment.400 Pathogenetic factors involved in the development of low bone mass in these subjects include adverse effects of the disease itself directly on bone; radiation injury of bone; inhibitory effects of glucocorticoid, chemotherapeutic, antibiotic, and immunosuppressive agents on bone formation; decreased caloric, protein, and vitamin D intake; sex hormone deficiency due to delayed or arrested adolescent development; and GH deficiency in children who have received cranial radiation. Cyclosporine A induces bone loss in organ transplant recipients by increasing osteoblast expression of RANKL and decreasing production of osteoprotegerin, thereby augmenting osteoclastogenesis.401 Leukemic patients should receive appropriate calcium and vitamin D supplements, and treatment with GH if they are GH deficient after the primary illness has been in prolonged remission. Adult survivors of childhoodonset acute lymphoblastic leukemia also have significant

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

osteopenia of the lumbar spine, femur, and radius—primarily related to GH deficiency.402 Decreased bone mineralization is common in the post-bone-marrow or postsolid-organ transplant subject. Its diverse pathogenesis includes the primary disease itself and the chronic illness that may accompany it, the use of high-dose glucocorticoids and antirejection medications, and altered intestinal and renal function.403 In addition to provision of adequate nutrition, calcium, and vitamin D, in adults the effects of transplantation may be partially ameliorated by administration of bisphosphonates. Their usefulness and safety in pediatric transplant patients has not yet been fully evaluated. In severely burned patients and in children with hemophilia, sickle cell disease, central diabetes insipidus, Marfan syndrome, homocystinuria, lysinuric protein intolerance, propionic, and methylmalonic aciduria, BMD is also decreased.404,405 Children with cystic fibrosis may have low vertebral and femoral neck BMD Z scores as a consequence of suboptimal nutrition, chronic inflammation, concomitant diabetes mellitus, pubertal delay, and drug therapy. However, approximately one-third of optimally managed cystic fibrosis patients with good clinical control may nevertheless have subnormal BMDs (Z score below –1 but seldom below –2.5)—although this is not necessarily translated into increased fracture risk.406,407 Low bone mass and vertebral collapse may be early manifestations of chronic inflammatory bowel disease.408 Vitamin D deficiency and secondary hyperparathyroidism, as well as the chronic inflammatory state and therapeutic agents (such as glucocorticoids), likely contribute to decreased bone formation and increased bone resorption in this illness. That whole-body BMC in children, adolescents, and young adults with chronic inflammatory bowel disease is reportedly normal relative to lean body mass (although reduced relative to racial, age, and height norms) does not necessarily imply that bone strength in these patients is normal—as evidenced by the increased fracture risk of adults with this disorder.409,410 Low bone mass is common in children and adults with celiac disease.411 In children and adolescents infected with the human immunodeficiency virus, whole-body BMD is decreased as a consequence of the infective agent itself, the chronic inflammatory state it induces, suboptimal nutrition, and the administration of highly active antiretroviral therapy that may have direct effects on osteoblast and osteoclast generation and function.412,413 Despite clinical well-being and normal linear growth, the rate of accrual of bone mass is decreased in these subjects—whereas the rate of bone resorption is increased. Bone mass is reduced in children with a variety of rheumatic diseases (juvenile idiopathic arthritis, systemic lupus erythematosus, juvenile dermatomyositis) due to the chronic inflammatory state, production of pro-osteoclastic cytokines, and therapy with glucocorticoids.414 Idiopathic juvenile osteoporosis (OMIM 259750) is an unusual disorder of generalized low bone mass of unknown pathogenesis that appears in mid to late childhood and often resolves as sexual maturity is achieved.415 In affected subjects, roentgenograms ob-

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tained for evaluation of joint, muscle, and/or back pain; difficulty walking; foreshortening of the trunk; and/or the presence of kyphosis reveal biconcave vertebrae and/or vertebral compression and long bone radiolucent areas and fractures in the metaphyses. Chemical studies are normal. Histomorphometry reveals findings consistent with a low rate of bone turnover, with reduction in cancellous bone volume, trabecular thickness, and bone formation due primarily to decreased osteoblast activity on the endosteal but not the periosteal bone surface. There is no evidence of increased bone resorption. Idiopathic juvenile osteoporosis is quite likely to be genetically heterogenous in origin. Mutation analyses of COL1A1 and COL1A2 have been normal in these subjects. In 15% of patients with juvenile osteoporosis, a familial heterozygous loss-of-function mutation in the gene encoding LDL receptor-related protein 5 (LRP5) has been detected.416 Homozygous loss of LRP5 results in the osteoporosis-pseudoglioma syndrome. The most difficult diagnostic problem is the clinical distinction between idiopathic juvenile osteoporosis and osteogenesis imperfecta type I (Table 17-10). This form of osteogenesis imperfecta is characterized clinically by a positive family history, onset in early infancy, lifelong persistence, diaphyseal fractures, blue sclerae, abnormal dentition, wormian bones, and high bone turnover.131,415 In children with idiopathic juvenile osteoporosis, symptomatic treatment is offered. In some patients, calcitriol or supplemental sodium fluoride has been of benefit. Although the disorder ameliorates and even disappears at puberty, treatment of the prepubertal patient with sex steroids does not seem to accelerate the healing process. Administration of the bisphosphonate pamidronate has been helpful in reducing bone pain and increasing vertebral BMD in a small group of children with idiopathic juvenile osteoporosis.417 The osteoporosis-pseudoglioma syndrome (OMIM 259770) is characterized clinically by congenital or early infantile onset of severe visual impairment due to hyperplasia of the vitreous (pseudoglioma that may be erroneously identified as retinoblastoma), leading to retinal detachment, glaucoma, and blindness; marked osseous fragility with craniotabes and fractures during late infancy, childhood, or adolescence; and variable cognitive impairment, ligamentous laxity, and hypotonia.418 The disorder is transmitted as an autosomal-recessive trait and is due to biallelic (homozygous or compound heterozygous) inactivating [missense (Arg494Gln), nonsense (Arg428Ter), frame-shift, splice-site] mutations in LRP5—primarily located in the extracellular domain of this protein.419 Missense mutations likely prevent normal binding of LRP5 to the product of the mesoderm development gene (OMIM 607783, chromosome 15), a chaperone protein that directs LRP5 to the cell membrane. Although often asymptomatic, heterozygous carriers are usually osteopenic. However, vision is not impaired. Mutations in LRP5 have also been associated with familial exudative vitreoretinopathy (OMIM 133780), a developmental disorder of retinal vasculature that may be transmitted as an autosomal-dominant (Leu145Phe) or autosomal-recessive (Arg570Gly, Arg752Gly) trait. These patients also have reduced

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Few fractures, little deformity, hearing loss in 50%; rarely dentinogenesis imperfecta Many rib & long bone fractures at birth, severe long bone deformities, unmineralized calvarium

Mild

Perinatal lethal

I - 166200

IIA - 166210

Moderately deforming, clinically similar to Type IV

Moderately to severely deforming, clinically similar to Type IV Moderately deforming

V - 610967

VI - 610968

Glycine substitutions in COL1A1 or COL1A2 Unknown

Unknown

Inactivating mutation (duplication) of CRTAP Inactivating mutations of LEPRE1

AD

AD AD

Unknown

AR

AR

Present but lighten with age Greyish or absent Absent

Absent or faint

Absent or faint

Absent

Moderate, variable Mild to moderate

Moderate

Moderate

Severe

B - Inactivating mutations of CRTAP Glycine substitutions in COL1A1 or COL1A2

Severe

AR

AD, parental mosaicism

Present

—

Nonsense & frameshift mutations resulting in premature STOP codons in COL1A1 A - Glycine substitutions in COL1A1 or COL1A2.

AD

Present

Gene defect

Inheritance

Minimal

Growth impairment Blue sclera

Adapted and modified from Barnes AM, Chang W, Morello R, et al. (2006). Deficiency of cartilage-associated protein in lethal osteogenesis imperfecta. N Engl J Med 355:2757–2764; from Marini JC (2006). Osteogenesis imperfecta. In Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 418–421; from Rauch F, Glorieux FH (2004). Osteogenesis imperfecta. Lancet 363:1377–1385; and from Sillence DO, Senn A, Danks DM (1979). Genetic heterogeneity in osteogenesis imperfecta. J Med Genet 16:101–116. AD, autosomal dominant; and AR, autosomal recessive.

VIII- 610915

Severely deforming, overlaps type II & III

Mild to moderate bone fragility, ossification of interosseous membranes of forearm, hypertrophic callus formation at fracture site Onset of fractures in infancy; increased osteoid, fish-scale@ pattern of lamellation Fractures present at birth with frequency declining with age, rhizomelia, limb deformities Phenotype overlaps those of types II and III

Moderately deforming

IV -166220

VII - 610682

Moderate to severe bowing, multiple fractures, dentinogenesis imperfecta, hearing loss Mild to moderate bowing, fractures

Severe, progressive deforming

III - 259420

IIB - 610854

Clinical features

Severity

Type - OMIM

Classification of Osteogenesis Imperfecta

TA B L E 1 7 - 1 0

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bone mass. LRP5 is a membrane protein that transduces the signals of two extracellular ligands: Wnt10b (OMIM 601906, chromosome 12q13) and Norrin (OMIM 310600, chromosome Xp11.4). Wnt signaling increases the accrual of bone by activating ␤-catenin, a transcription factor that enhances differentiation of pluripotential mesenchymal precursor cells into the pathway of chondrogenesis and osteogenesis and impedes their differentiation into the pathway of adipogenesis.420 By stimulating Runx2, ␤-catenin further directs the osteochondroprogenitor cell into the osteoblastic track. In the mature osteoblast, ␤-catenin enhances expression of osteoprotegerin and hence depresses osteoclastogenesis.421 Norrin signaling modulates vitreoretinal formation in the eye. In addition to the primacy of genetic and hormonal factors, the most important considerations for the accrual and maintenance of bone mass are those that relate to diet (sufficient intake of calcium and protein), sustained normal vitamin D stores by exposure to sunlight or ingestion of supplements, and consistent weight-bearing exercise. Therapeutically, when trying to prevent bone loss or restore lost bone initial efforts are directed to the assurance that these basic approaches are being utilized to the fullest extent possible for the specific patient. Therapeutic agents that increase bone mass act by inhibiting resorption (antiresorptive or antiremodeling drugs) or by stimulating bone formation (anabolic medications).390 The most widely employed antiresorptive medications are sex hormones, selective estrogen receptor modulators, calcitonin, and bisphosphonates. Selective estrogen receptor modulators are triphenylethylene-, benzothiophene-, or naphthalene-related compounds (e.g., raloxifene) that bind with high affinity to estrogen receptor ␣ (perhaps to estrogen receptor ␤ as well) in specific tissues—where they alter the three-dimensional configuration of the receptor and recruit tissue-selective cohorts of various coactivating factors, thus inducing receptor function in targeted sites (e.g., bone).422 These compounds decrease osteoclast formation primarily at trabecular bone sites.307 Nasal salmon calcitonin inhibits osteoclast function directly and has modest bone restorative effects. Bisphosphonates are analogues of pyrophosphate, with carbon substituted for the oxygen bridge between two phosphate groups. Also attached to the carbon atom are two side chains. R1 is usually a hydroxyl group that together with the phosphate residues binds tightly to and coats bone surface.423,424 The bisphosphonates impede osteoclast function by hastening their death by one of two mechanisms. After entering the osteoclast by endocytosis, etidronate forms cytotoxic acyclic analogues of adenosine triphosphate (ATP) that interfere with cellular metabolic processes and lead to apoptosis. After endocytosis nitrogen-containing pamidronate and alendronate inhibit the mevalonate pathway and the activity of farnesyl diphosphate synthase, a property shared with statin drugs.425 The resulting failure of transfer of fatty acids (prenylation) to guanosine-triphosphate-binding proteins such as Ras renders them inactive, impairs cell metabolism, and in turn begins the apoptotic process. As osteoclast function declines, bone mass increases. In addition, bisphosphonates are incorporated into the surface of

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hydroxyapatite and thereby block its dissolution. The biologic activity of bisphosphonates on osteoclast function is observed immediately after its administration as serum calcium concentrations decline rapidly. Indeed, this rapid effect has been utilized in the treatment of hypercalcemic infants and children. In adults, the effects of bisphosphonates on bone mass last long after the agent has been discontinued (the residence time)—enabling some compounds (zolendronate) to be given as infrequently as once yearly. Indeed, bisphosphonates remain in bone for extremely long intervals—and their long-term effects appear to be cumulative. Histomorphometric analysis has revealed that bisphosphonates increase bone mineralization by decreasing the number of resorption cavities (and thus the remodeling space), preserving cancellous (trabecular) bone architecture, and decreasing porosity of cortical bone.390 Bisphosphonates have been useful in improving mineralization in children with osteogenesis imperfecta, as well as in those with glucocorticoid induced osteoporosis, osteoporosis-pseudoglioma syndrome, Menkes disease, and cerebral palsy. In most infants and children, intravenous pamidronate (1 mg/kg/day on 3 consecutive days every 3 to 6 months to 2–15 mg/kg/year administered once every 3 to 6 months) has been utilized— although a number of different regimens have been employed with reasonably similar increases in BMD, decline in fracture incidence, and improved well-being.423 Limited data indicate that oral bisphosphonates (pamidronate, alendronate, olpadronate) administered daily also increase BMD in children with osteogenesis imperfecta and connective tissue disease, but with lower efficacy than intravenous administration. Side effects of bisphosphonates have been acute (fever, myalgia, abdominal pain, vomiting, hypocalcemia) and chronic (inflammatory disorders of the eye, osteonecrosis of the jaw in the elderly, and induced osteopetrosis).423,426 Experimentally and in adults receiving long-term therapy, bisphosphonates can suppress bone turnover and contribute to hypermineralization—the latter leading to reduced mechanical strength and increased fracture risk.427 Therefore, when selecting a child for treatment with bisphosphonates one must carefully considered the primary diagnosis and whether the patient’s low bone mass and fracture frequency merit therapy in view of the potential side effects of bisphosphonates. In addition, there are many unanswered questions concerning which bisphosphonate to use, its route of administration, the dose of the drug, the duration of therapy, and the method of outcome analysis. At this time, the use of bisphosphonates should be confined to centers with experience in the care and management of children with bone disease.423,424 Treatment with bisphosphonates several years prior to conception does not appear to have an adverse effect on fetal outcome, but treatment during pregnancy is contraindicated because of possible toxicity.428 Administered intermittently in small amounts, PTH and its analogue PTH1-34 (teriparatide) preferentially accelerate the rates of bone remodeling and of bone formation relative to that of bone resorption by direct stimulatory effects on the osteoblast.390 PTH also acts on the osteocyte to decrease the production of sclerostin, an inhibitor of bone

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synthesis that acts by repressing Wnt- and BMP-mediated bone formation.429 The quantity of bone formed in each remodeling unit is increased, thus augmenting trabecular and cortical bone mass and strength. PTH1-34 increases trabecular thickness and trabecular interconnectivity. By contrast, bisphosphonates preserve but do not alter trabecular architecture. PTH1-34 increases periosteal new bone formation and cortical thickness and diameter (i.e., bone size)—thereby increasing bone strength and reducing fracture risk.390 Teriparatide is also capable of dissociating bone formation from resorption, doing so in part by decreasing the rate of osteoblast apoptosis. Side effects of PTH1-34 administration include transient hypercalcemia, hypercalciuria, and development of antibodies to the peptide—all rather unusual events. Although osteosarcoma has been observed in mice receiving very high doses of PTH and PTH1-34, no malignant disorders have been recorded in adults receiving either agent. To date, the pediatric use of PTH1-34 has been limited to children with hypocalcemia due to a gain-of-function mutation in CASR and consequent hypercalciuric hypocalcemia—one form of familial isolated hypoparathyroidism. In development is an analogue of PTHrP (PTHrP1-36) that increases lumbar spine BMD in postmenopausal women with osteoporosis by stimulating bone formation selectively without inducing hypercalcemia.430,431 Denosumab is a human monoclonal antibody to RANKL that binds tightly to its ligand and prevents its interaction with RANK, thereby inhibiting osteoclastogenesis. Denosumab thus acts as a pseudo-osteoprotegerin. In postmenopausal women with decreased bone mineralization, denosumab decreases bone resorption and increases lumbar spine BMD.432 Strontium renalate is another agent in development for treatment of low bone mass in adults. Strontium is incorporated into the structure of bone mineral.433

OSTEOGENESIS IMPERFECTA Osteogenesis imperfecta (brittle bone disease) is a disorder of increased bone fragility due to low bone mass that varies in clinical severity from lethality in the perinatal period due to respiratory insufficiency to mildly increased susceptibility to fractures in later life.132 The original Sillence classification of four types of osteogenesis imperfecta based on clinical characteristics and disease course has been expanded to include four additional types of this disorder and identification of mutant genes to which the illnesses can be attributed (Table 17-10).132,434 The hallmark of each type is increased bone fragility, but severity varies—with type II being a lethal form and type I being a relatively benign form. In decreasing order of severity, type II ⬎ type VIII ⱖ type III ⬎ types IV, V, VI, and VII ⬎ type I. In osteogenesis imperfecta types I through IV, heterozygous loss-of-function mutations have been identified in one of the two genes (COL1A1, COL1A2) encoding procollagen subunits ␣1(I) and ␣2(I), respectively—which intertwine to form collagen type I in bone, skin, ligaments, tendons, sclerae, and teeth.131,132 [As previously described, the triple helical structure of type I collagen

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consists of two collagen ␣1(I) (COL1A1) peptides and one collagen ␣2(I) peptide—each of which consists of triple repeats of glycine and two additional amino acids (often proline, hydroxyproline, or lysine).] Transmitted as an autosomal-dominant trait, insertion, duplication, frame-shift, or point mutations within COL1A1 or COL1A2 reduce the amount of collagen synthesized or alter its structure and properties—interfering with the assumption of a normal three-dimensional configuration and leading to decreased bone formation, low bone mass, and increased fracture risk (Figure 17-16).435 To an extent, the site of the more than 800 mutations identified within COL1A1 or COL1A2 is related to the clinical phenotype of osteogenesis imperfecta types I through IV. Mutations that lead to a stop codon result in a truncated procollagen product that is rapidly degraded. Thus, only normal collagen type I is produced (but in reduced mounts). Lethal mutations in COL1A1 are those that alter an amino acid with a branched or charged side chain; those within the binding sites of the collagen monomer for integrins, matrix metalloproteins, fibronectin, and cartilage oligomeric matrix protein; and those that result in binding to and degradation of intact procollagen subunits.131 Lethal mutations in COL1A2 are those that interfere with its binding to proteoglycans. Mutations (Arg134Cys) in COL1A1 may also be found in patients with classic Ehlers-Danlos syndrome (OMIM 130000) of hyperextensible skin and laxity of ligaments of the spine and large and small joints. Children with clinical features of osteogenesis imperfecta (osseous fragility) and Ehlers-Danlos syndrome have been described.436 In these patients, the mutations have been concentrated within the first 90 aa of the helical region of collagen␣1(I) and prevent normal post-translational removal of the procollagen amino-propeptide. Although the mutant protein can be incorporated into collagen, the structural integrity of the product is impaired because its fibrils are thin and weak.437 Osteogenesis imperfecta type I (OMIM 166200) is an autosomal-dominant disorder (or new mutation in 33% of patients) due primarily to functionally null alleles—the result of splicing defects or point mutations that lead to insertion errors or truncation (COL1A1: Gly178Cys, Arg963Ter, IVS26DS), which are mutations that result in decreased transport of procollagen-␣1(I) into the cytoplasm or its release into matrix (thereby modestly decreasing production of intact procollagen type I). Its clinical manifestations are relatively benign: intensely blue sclerae present at birth that persist throughout adulthood, modestly low bone mass, infrequent fractures with little deformity (however, 15% of affected children develop deformities and 24% develop kyphoscoliosis before 10 years of age), low normal adult stature, hearing loss in 50%, mitral valve prolapse in 18%, and rarely dentinogenesis imperfecta (osteogenesis imperfecta type IB). Mutations in COL1A2 less frequently lead to the phenotype of osteogenesis imperfecta type I. Subjects with mutations in COL1A1 more frequently have blue sclerae and taller stature than those with mutations in COL1A2. Osteogenesis imperfecta type II (OMIM 166210) is a disorder that is lethal in the perinatal period or in early infancy. It is

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745

α1 Severe disease Moderate disease Mild disease

α2 Mild disease Moderate disease

Alanine Serine

Severe disease

Valine Cysteine 0

200

400

600

800

1000

Arginine Cysteine

Figure 17-16 Mutations in COL1A1 and COL2A1 that result in glycine substitutions associated with osteogenesis imperfecta of variable clinical severity. [Reproduced with permission from Prockop DJ (2005). Type II collagen and avascular necrosis of the femoral head. N Engl J Med 352:2268–2270.]

usually the result of de novo heterozygous mutations in COL1A1 or COL1A2, with alternative amino acid being substituted for glycine in the triple helical domains of the procollagen ␣1(I)/␣2(I) chains (COL1A1: Gly94Cys, Gly391Arg, Gly1003Ser; COL1A2: Gly547Asp, Gly865Ser, Gly976Asp). These mutations lead to the synthesis of abnormal procollagen chains that bind to and thereby inactivate intact procollagen peptides in a dominant-negative manner, severely curtailing the synthesis of intact collagen type I. Clinically, it is manifested by in utero fractures, long bone deformities, very little calvarial mineralization, and death due to respiratory insufficiency. A very similar phenotype (type IIB, OMIM 610854) is associated with homozygous loss-of-function mutations in the gene (CRTAP) encoding cartilage-associated protein. Osteogenesis imperfecta type III (OMIM 259420) is an autosomal-dominant trait due to point or frame-shift mutations in COL1A1 (Gly154Arg, Gly844Ser) and COL1A2 (Gly526Cys). It is characterized by recurrent fractures that lead to progressive bone deformities that are often apparent at birth, and by kyphoscoliosis, extreme short stature, blue sclerae that lighten with age, abnormal dentition (in 80% of children less than 10 years of age), and hearing loss. Osteogenesis imperfecta type IV (OMIM 166220) is an autosomal-dominant disease usually associated with point mutations or small deletions in COL1A2 (Gly586Val, Gly646Cys, Gly1012Arg) and occasionally in COL1A1 (Gly175Cys, Gly832Ser). It is of variable severity, with prolonged survival, mild to moderate bone deformities, short stature, normal sclerae, dentinogenesis imperfecta, and hearing loss. One post-translational modification of type I procollagen is hydroxylation of proline and lysine residues by prolyl and lysyl hydroxylases, an essential step in nor-

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mal collagen folding and stability. Prolyl 3-hydroxylase 1 [P3H1, also termed leprecan (LEPRE1)] specifically hydroxylates the proline residue at codon 986 in COL1A1, a reaction that requires interaction of P3H1 with CRTAP and cyclophilin B (chromosome 15, OMIM 123841). CRTAP is expressed in the proliferative zone of developing cartilage and at the chondro-osseous junction. Cyclophilin B possesses peptidyl-prolyl cis-trans isomerase activity. Crtap knockout mice develop an osteochondrodysplasia (kyphoscoliosis, shortening of the proximal segment of the limbs consistent with rhizomelia) and severe osteopenia, the latter due to reduced production and alteration in the quality of osteoid and consequently decreased rate of mineral deposition.438 Mice deficient in Crtap are unable to 3-hydroxylate the proline residue near the carboxyl terminus of bone COL1A1, leading to increased hydroxylation of lysine residues and resultant abnormal structure of the collagen fibril—changes that result in defective mineralization of bone collagen type I. A recessive form of lethal osteogenesis imperfecta of unknown pathogenesis in which detailed analyses of COL1A1 and COL1A2 have been normal has been described. In 3 out of 11 patients with a recessive clinical form of lethal type II or severe osteogenesis imperfecta type III characterized by multiple fractures of the long bones (resulting in rhizomelic shortening of the limbs with externally rotated and abducted legs, poorly mineralized calvaria and ribs, proptotic eyes, and white or light blue sclerae), nonhydroxylated proline at codon 986 in bone COL1A1 has been demonstrated.439 This proved due to homozygous or compound heterozygous loss-of-function mutations in CRTAP [frame-shift

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(c.879delT), 16 bp duplication in exon 1, nonsense (Gly276Ter), missense (Met1Ile), splice donor site of exon 1 at the first intronic nucleotide (IVS1 ⫹ 1G→C)] that interfered with effective hydroxylation of the proline residue at codon 986 of bone procollagen ␣1(I). Loss-offunction mutations in LEPRE1 lead to impaired P3H1 activity and a severe form of osteogenesis imperfecta designated type VIII.434 It seems likely that a mutation in the gene encoding cyclophilin B as well as in others that post-translationally modify collagen subunits may be identified in different patients with similar clinical manifestations but intact COL1A1 and COL1A2. Currently, there are four forms of osteogenesis imperfecta (types V, VI, VII, VIII) in which extensive analyses of the structures of COL1A1 and COL1A2 have been normal.131,132 Osteogenesis imperfecta type V (OMIM 610967) is phenotypically similar to type IV and is characterized by autosomal-dominant transmission, moderate to severe bone fragility, moderate to mild growth retardation, dislocation of the radial head, mineralization of interosseous membranes, exuberant callous formation at fracture sites, white sclerae, and intact dental development. Histologically, there is irregular arrangement of lamellae. Approximately 5% of patients with osteogenesis imperfecta appear to have type V, the molecular cause of which is as yet unidentified. Osteogenesis imperfecta type VI (OMIM 610968) is also phenotypically similar to type IV. It is characterized by a fish-scale pattern of lamellation of bone on microscopic examination. Type VI is found in approximately 4% of patients with osteogenesis imperfecta. Its inheritance pattern is unknown. Osteogenesis imperfecta type VII (OMIM 610682) has been identified in northern Quebec in a Native American population. Clinically, it is an autosomal-recessive disorder in which fractures are present at birth but the frequency of fractures declines with advancing age—particularly after adolescence. The sclerae are slightly bluish, and there is progressive skeletal deformation that leads to rhizomelic shortening of the limbs and restricted ambulation. In patients with type VII, a homozygous mutation in CRTAP has been identified: specifically, inclusion of 73 bp of intron 1 into the genome of CRTAP due to alteration of one nucleotide (c.472, 1021C→G) that generates a cryptic splice donor site that extends exon 2. However, this alteration results in a frame-shift that permits more rapid degradation of CRTAP and consequently leads to decreased 3-hydroxylation of proline 986 in bone COL1A1. Thus, inactivating mutations in CRTAP can result in at least two clinically distinct forms of osteogenesis imperfecta (types IIB and VII)—as do mutations in COL1A1 and COL1A2. The phenotype of osteogenesis type VIII (OMIM 610915) overlaps with those of types II and III. In addition to osseous fragility, it is associated with substantial growth retardation, white sclerae, and bulbous metaphyses. It is due to loss-of-function mutations in LEPRE1 encoding P3H1.434 In addition to diffusely low bone mass, thin cortices, metaphyseal flaring, and fractures and bone deformities resulting therefrom, radiographic findings in subjects with osteogenesis imperfecta include wormian skull bones (frequent but not pathognomonic of osteogenesis

Ch17_686-769-X4090.indd 746

imperfecta), platybasia that may compress overlying hindbrain, vertebral compression, and a triradiate pelvis.131 Bone densitometry reveals decreased mineralization, the extent of which correlates to a degree with clinical manifestations. The diagnosis of osteogenesis imperfecta is established by clinical criteria and confirmed by genotyping of COL1A1 and/or COL1A2 or other pertinent gene(s)—although failure to detect a genetic mutation does not necessarily rule out this disorder. Occasionally, biopsy of the iliac crest and histologic examination of the bone may be necessary for subclassification of the disorder. Determination of the rate of synthesis and forms of procollagen secreted by dermal fibroblasts in vitro permits identification of the forms and relative amounts of collagen subunits and intact proteins being synthesized (type I, type III, and so forth). Osteogenesis imperfecta type II can be identified prenatally by fetal ultrasonography. Types I, III, and IV can be determined prenatally by analysis of collagen synthesized by cells cultured from chorionic villus biopsies and by analysis of the procollagen genes.440 The basic management of patients with osteogenesis imperfecta is directed to prevention of fractures to the extent possible and to the treatment of fractures that occur by sound orthopedic procedures and by orthopedists familiar with this disorder. Rehabilitative services and physical therapy to improve muscle strength and mobility within the constraints of bone fragility are to be encouraged, as are protected ambulation and exercises such as swimming.131 Introduction of bisphosphonates into the management of infants, children, and adolescents with osteogenesis imperfecta types I, III, and IV has been of substantial benefit. Patients with these disorders have responded to the intermittent intravenous administration of the bisphosphonate (pamidronate), with marked increase in bone mass and decline in fracture rate—as well as symptomatic improvement such as decreased pain and more facile mobility.132,133,441 Infants as young as 2 months of age have safely tolerated 4-hour intravenous infusions of pamidronate (0.5 mg/kg/day for 3 consecutive days every 6 to 8 weeks), realizing clinical improvement such as decline in bone pain (perhaps a placebo effect), increase in lumbar vertebral BMD of 86% to 227%, and decrease in fracture rate after 1 year of therapy.131,442 In children (3–16 years of age), pamidronate administered as a 4-hour infusion (1.5–3.0 mg/kg/ day) for 3 consecutive days every 4 months resulted in increases in lumbar spine BMD of 42% per year and increases in metacarpal cortical width of 27% per year. in decreased vertebral size, decline in vertebral fracture rate, and symptomatic improvement also occurred. During 2 to 4 years of intravenous pamidronate administration, increase in vertebral (trabecular) bone mass and size are accompanied by decline in the extent of vertebral compression and fewer compressed vertebrae than in untreated patients.443 In the iliac crest, bisphosphonates increase cortical bone thickness and trabecular number but not trabecular thickness. In metacarpals, bisphosphonates enhance cortical thickness. In treated subjects, the relative risk of long bone fracture may be a bit reduced—implying a beneficial effect of bisphospho-

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

nates on long bone cortical volume and hence on bone strength.131,132,444 Oral alendronate (1 mg/kg/day) and intravenous pamidronate are reported to be equally effective in children with osteogenesis imperfecta, although data are limited and gastrointestinal side effects of oral bisphosphonate are of concern.445 Near-maximal benefits of bisphosphonates on lumbar vertebral BMD by DEXA (and on mean cortical width, cancellous bone volume, and trabecular bone formation rate by histomorphometric analysis of iliac crest bone biopsies) are achieved within the first 2 to 4 years of treatment, with little further change with more prolonged therapy.446 Currently, because of the persistence of bisphosphonates in bone and their long-term effects it is suggested that these agents be administered to patients with osteogenesis imperfecta for approximately 2 to 4 years. Their positive effects on bone mass gain, reduced fracture rate, and functional well-being are often longer lasting.447 Care must be exercised when selecting a patient with osteogenesis imperfecta for treatment with bisphosphonates. It is currently recommended that these agents be employed only in patients with substantial disease, such as frequent fractures (more than two per year) and deformities of the long bones and vertebrae, irrespective of the specific mutation in the procollagen subunit, clinical type of osteogenesis imperfecta, or BMD.133 Pamidronate has been the bisphosphonate most frequently administered to patients with osteogenesis imperfecta. Recent treatment recommendations utilizing this agent are: ⬍2.0 years (0.5 mg/kg/day intravenously for 3 days every 2 months), 2.0 to 3.0 years (0.75 mg/ kg/day for 3 days every 3 months), and ⬎3.0 years (1 mg/kg/day to a maximum dose of 60 mg/day for 3 days every 4 months).132 Among the complications of bisphosphonate therapy are transient hypocalcemia and flu-like reaction of fever, vomiting, and rash after first exposure managed symptomatically. Although bisphosphonates decrease linear growth in experimental animals, experience in children indicates that over the span of several years these agents improve linear growth rate and height compared with untreated subjects with moderately severe forms of osteogenesis imperfecta.441 Increase in the rate of linear growth has been reported during administration of recombinant human GH to children with osteogenesis imperfecta, but the effect has not been sustained—nor has treatment positively affected fracture risk.132,441,448 Bone marrow transplantation in five children with severe osteogenesis imperfecta has been reported to have been beneficial, but experience with this procedure has been variable.449 As yet untested in osteogenesis imperfecta subjects is the use of PTH, PTH1-34, or PTHrP1-36. Gene therapy of osteogenesis imperfecta remains a goal. Selective inactivation of wt or mutant COL1A1 in mesenchymal stem cells from two patients with severe forms of osteogenesis imperfecta (COL1A1: Gly773Ser, Gly1040Ser) has been accomplished. Cells in which survival and function of the wt gene and destruction of the mutant gene co-occur potentially permits their reintroduction into the affected host and normal collagen production.450,451

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FIBROUS DYSPLASIA Fibrous dysplasia involves the long bones and skull and may be monostotic, polyostotic, or panostotic. It primarily occurs in patients with the McCune-Albright syndrome (OMIM 174800) in association with large café-au-lait pigmentations and various endocrinopathies, including isosexual precocity, hypersomatotropism, thyrotoxicosis, and hyperadrenocorticism—as well as dysfunction in many other tissues (heart, liver, pancreas).452 It is due to mosaicism for postzygotic somatic gain-of-function missense mutations (Arg201 to Cys, His, Ser, Gly) in GNAS, the gene encoding the ␣ subunit of the Gs protein.453 The extent and severity of disease is determined by the point in development at which the mutation occurs and its tissue distribution. As a result of loss of intrinsic guanosine triphosphatase activity within the Gs␣ subunit, the stimulatory effect of Gs␣ on adenylyl cyclase is prolonged. The clones of mutated mesenchymal preosteoblasts proliferate, but their differentiation into mature osteoblasts is incomplete and their secreted matrix is abnormal. Continued expansion steadily erodes contiguous bone. These lesions can also synthesize FGF23 and lead to hyperphosphaturia, hypophosphatemia, and excess unmineralized osteoid and a rickets-like clinical state.454 Fibrodysplastic lesions are initially silent, whereas osteoclasts at the periphery of the lesions actively thin and compress bone cortices—ultimately resulting in bone pain and pathologic fractures of the long bones (particularly the proximal femoral metaphyses). Children between 6 and 10 years of age have the highest fracture rate (0.4 fractures per year). Within the skull base and facial bones, expansion of fibrous dysplastic lesions leads to disfiguration and compression of cranial nerves. Radiographically, the fibrodysplastic lesion is viewed as a cyst-like medullary structure with a ground-glass consistency. Histologically, there are abundant immature bone marrow stromal cells, incompletely differentiated osteoblasts, poorly formed bony trabeculae with excess undermineralized osteoid seams characteristic of osteomalacia, and islands of cartilage. The clinical manifestations of fibrous dysplasia depend on the sites and extent of bone involvement and associated endocrinopathies, particularly GH excess.455 Diagnosis of fibrous dysplasia is based on clinical characteristics and confirmation of the genetic mutation in GNAS. In addition to managing the multiple endocrinopathies and organ defects, attention must be paid to the osseous lesions. Fractures are repaired by standard techniques, including intramedullary nailing when indicated. Occasionally, it may be feasible to evacuate a fibrodysplastic lesion surgically and to fill the cavity with bone grafts. In children with fibrous dysplasia, the bisphosphonate pamidronate has proven useful in ameliorating bone pain (but not the skeletal lesions).456,457

HIGH BONE MASS Abnormally increased bone mass is the consequence of disruption of the normal equilibrium between the velocities of bone formation and resorption. Thus, it may

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be due to decrease in the rate of bone resorption or to an increase in the rate of bone formation. Increase in cortical bone width is termed hyperostosis. Thickening of trabecular bone is termed osteosclerosis.136 Table 17-11 lists selected dysplastic, metabolic, and other diseases associated with increased bone density in children and adolescents. Failure of osteoclast-mediated bone resorption leads to osteopetrosis (“marble bone disease”), a group of heritable disorders with heterogeneous manifestations.458 Histologically, osteopetrotic bone is characterized by quiescent osteoclasts with few ruffled borders and retained calcified cartilage formed during endochondral ossification (primary spongiosa) due to failure of reabTA B L E 1 7 - 1 1

Disorders of Bone Mineralization: High Bone Mass I. Decreased Bone Resorption A. Osteopetrosis 1. Autosomal recessive (infantile) (TCIRG1, CLCN7, OSTM1) a. Transient 2. Intermediate (CLCN7) 3. Autosomal dominant (adult) (LRP5, CLCN7) 4. Immunodeficiency, lymphedema, ectodermal dysplasia (IKBKG) 5. Carbonic anhydrase II deficiency (CA2) 6. Pycnodysostosis (CTSK) 7. Neuronal storage disease 8. Drug-induced - bisphosphonates II. Increased Bone Formation A. Activating Mutations of LRP 1. Autosomal-dominant high bone mass 2. Endosteal osteosclerosis (van Buchem disease type 2) B. Inactivating Mutations of SOST 1. Sclerosteosis 2. Van Buchem disease type 1 III. Osteosclerosis A. Dysplasias 1. Dysosteosclerosis 2. Infantile cortical hyperostosis (Caffey disease) (COL1A1) 3. Juvenile Paget disease (TNFRSF11B) 4. Metaphyseal dysplasia (Pyle disease) 5. Osteopoikilosis (LEMD3) 6. Progressive diaphyseal dysplasia (Camurati-Engelmann disease) (TGFB1) 7. Tubular stenosis, type 1 (Kenny-Caffey syndrome) (TBCE) B. Metabolic Disorders 1. Fluorosis 2. Heavy metal poisoning 3. Hypervitaminosis A, D 4. Hypoparathyroidism, pseudohypoparathyroidism 5. Milk-alkali syndrome C. Other 1. Hepatitis C-associated osteosclerosis 2. Ionizing radiation 3. Sarcoidosis 4. Sickle cell disease (HBB) 5. Tuberous sclerosis (TSC1) Modified from Whyte MP (2006). Sclerosing bone disorders. In Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 398–414.

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sorption of immature bone. Although densely packed with mineral, osteopetrotic bone is quite fragile because the abnormality in bone remodeling due to decreased osteoclastic bone resorption leads to incorporation of weak calcified growth plate cartilage into bone and to delay in repair of microfractures. Radiographically, osteopetrosis is characterized by diffuse increase in bone mass, diaphyseal/metaphyseal widening with an Ehrlenmeyer flask appearance, alternating bands of sclerotic and lucent bone at the ends of the long bones, iliac crest and vertebrae, sclerotic changes at the base of the skull, narrow medullary cavities, and pathologic fractures.136 Cranial computed tomography often reveals narrowing of the bony canals through which cranial nerves (II, III, IV, VII, VIII) pass. Classically, three clinical forms of osteopetrosis have been delineated. However, as knowledge of the genetic mutations responsible for this disease are being identified this classification is being replaced. The infantile form of osteopetrosis (OMIM 259700) is an autosomal-recessive disorder (Albers-Schonberg disease) with attenuated growth (particularly of the limbs), delayed development, increased fracture rate, and failure of tooth eruption. Bony overgrowth leads to maldevelopment of the paranasal sinuses and to symptomatic nasal stuffiness. Narrowing of cranial foramina compromises cranial nerve function (II, III, VII, VIII), with consequent blindness and deafness. Decrease in bone marrow volume leads to depressed intramedullary hematopoiesis, anemia, and leukopenia—partially compensated by extramedullary hematopoiesis and ensuing hepatosplenomegaly, with consequent increased susceptibility to infection and hemorrhage. Retention of teeth within the sclerotic jaw leads to recurrent and persistent mandibular and maxillary osteomyelitis. Physical examination reveals short stature, macrocephaly, frontal bossing, and small facial features. Death usually occurs often within the first decade of life due to sepsis, anemia, or hemorrhage. Autosomal-recessive osteopetrosis may be due to biallelic mutations in a subunit of the osteoclast’s vacuolar proton pump (TCIRG1), its chloride channel (CLCN7), or the osteopetrosis-associated transmembrane protein-1 (OSTM1). The intermediate form of osteopetrosis is transmitted as an autosomal-recessive trait and is associated with short stature, macrocephaly, recurrent fractures, variable compromise of cranial nerve function, abnormal dental development predisposing to osteomyelitis of the mandible or maxilla, and anemia. Pathogenetically, it likely represents the variable penetrance of one of the genetic mutations associated with the infantile form of osteopetrosis—predominantly of CLCN7. There are two clinical and radiographic forms of autosomal-dominant osteopetrosis previously thought to be manifested only in the adult. Type I (OMIM 607634) is characterized by an enlarged and dense cranial vault and diffuse vertebral sclerosis, and is related to activating mutations of LRP5. It is not associated with an increase in fracture rate as bone strength is increased. Type II (OMIM 166600) is typified by thickening of the vertebral end plates, resembling “bone within bone” and resulting in a “rugger jersey spine” and sclerotic

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bands of bone in the pelvis and base of the skull.459 It is a variant of Albers-Schonberg disease that results from heterozygotic mutations in CLCN7.136,460 Affected subjects manifest cranial nerve compromise (16%), mandibular and nonmandibular osteomyelitis (19%), osteoarthritis of the hip (27%), and fractures (78%).459 Clinical evidence of the disease tends to worsen over time. However, the expression of the trait is variable. Thus, one-third of carriers of an inactivating mutation in CLCN7 have no radiographic or clinical manifestations— although they do have significantly higher BMD than do subjects with the wt gene.461 In one-quarter of clinically apparent patients with a heterozygous loss-of-function mutation in CLCN7, the expression of illness (fractures, osteomyelitis, compromised vision) is identifiable at birth or early in infancy or childhood. Patients with radiologic/clinical manifestations of this disorder have elevated serum concentrations of tartrate-resistant acid phosphatase and the BB isoform of creatine kinase elaborated by osteoclasts. These values are normal in unaffected carriers.462 Identification of many of the genes that regulate osteoclastogenesis and osteoclast function has greatly increased our understanding of the molecular mechanisms underlying experimental models of osteopetrosis. There are more than 17 murine models of osteopetrosis involving a litany of molecules that regulate osteoclastogenesis (Figure 17-17).458,463 By mid 2007, five genetic mutations that impeded resorption of bone had been identified in patients with osteopetrosis. As anticipated, they related to abnormalities in osteoclastogenesis or function—particularly the efficiency of acidification of the resorption lacuna beneath the osteoclast’s ruffled membrane and mineral dissolution or enzymatic degradation of organic bone matrix. The syndrome of osteopetrosis, lymphedema, anhidrotic ectodermal dysplasia, and immunodeficiency (OL-EDAID, OMIM 300301) has been linked to an inactivating mutation in a modulator of NF␬B—an essential transcription factor for differentiation and function of osteoclasts.464,465 The inhibitor of the kinase of kappa light polypeptide gene enhancer in B cells gamma subunit (IKBKG) is also termed NF␬B essential modulator (NEMO). IKBKG is a 412-aa component of the I␬B kinase complex that activates NF␬B. The substitution of guanine for adenine at N:1259 (A1259G) results in a change from a stop codon to tryptophan (Ter420Trp), permitting incorporation of an additional 27-aa component at the carboxyl terminus of the protein and resulting in decreased function of the product and 50% to 60% decline in activation of NF␬B and hence in osteoclastogenesis. Carbonic anhydrase II (one of several zinc metalloisoenzymes) is a protein expressed in osteoclasts, erythrocytes, brain, and kidney. It regulates the formation of carbonic acid from water and carbon dioxide (CO2 ⫹ H2O → H2O3), which then dissociates to form proton/ hydrogen (H⫹) and bicarbonate (HCO3-) ions. Loss-of function homozygous or compound heterozygous (Lys17Glu, Tyr40Ter, His107Tyr) mutations in CA2 lead to an autosomal-recessive disease that presents in childhood with failure to thrive, short stature, visual impairment,

Ch17_686-769-X4090.indd 749

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Figure 17-17 Sites at which pharmacologic agents may be targeted to affect osteoclast production and/or function: 1-4 (blockade of the RANK/RANK ligand/OPG signaling pathway of osteoclastogenesis), 5 (inhibition of ␣v␤3 integrin receptor binding to bone surface), 6 (antagonism of cathepsin K protease), 8 (inhibition of p38 kinase, an enzyme important in the inflammatory reaction), 9 (blocking of p60C-SRC kinase, an enzyme important in osteoclast activation), 10 (inhibition of matrix metalloproteinase9), 11 (agonist ligands of the calcitonin receptor with enhanced function), 12 (inhibition of carbonic anhydrase II), and 13 (enhancement of osteoclast death). [Reproduced with permission from Rodan GA, Martin TJ (2000). Therapeutic approaches to bone diseases. Science 289:1508–1514.)

and developmental delay in association with mild proximal and severe distal renal tubular acidosis, cerebral calcifications within the cortex and basal ganglia, and osteopetrosis with increased fracture risk.136 Osteopetrosis is of modest severity and is usually nonprogressive. It may even improve at puberty. After generation by carbonic anhydrase II, H⫹ is extruded from the osteoclast into the subobsteoclastic resorption lacuna through transporters and proton pumps. TCIRG1 (T-cell immune regulator 1) encodes an 822-aa 116-kDa protein that is a subunit of the osteoclast’s vacuolar proton pump (H⫹-ATPase). [By alternative splicing, this gene also encodes a 614-aa protein (TIRC7) essential to activation of T lymphocytes.] Biallelic-inactivating (missense, nonsense, deletion, splice site) mutations in TCIRG1 whose loss impairs transport of H⫹ and thus

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decreases bone mineral resorption have been found in 50% of subjects with the neonatal/infantile form of lethal osteopetrosis (OMIM 259700).466,467 Loss-of-function mutations in CLCN7, a chloride channel expressed in the ruffled membrane of the activated osteoclast, also impair acidification of the subosteoclastic resorption space and hence mineral dissolution. Heterozygotic-inactivating mutations (Arg767Trp) of CLCN7 lead to the autosomal-dominant form of osteopetrosis (OMIM 166600)—whereas homozygous mutations (Leu766Pro) are found occasionally in infants with the lethal autosomal-recessive form of this disease (whose parents may even be clinically normal).460,468 CLCN7 is coexpressed with and complexed to osteopetrosis-associated transmembrane protein 1 (OSTM1) in endosomes and lysosomes and in the ruffled membrane of activated osteoclasts.469 By decreasing post-translational stability of CLCN7, loss-offunction mutations in OSTM1 have been pathogenetically related to autosomal-recessive lethal osteopetrosis in a subset of patients.470,471 The murine homolog of OSTM1 is Gl (Grey-lethal), a mouse model of osteopetrosis. Related to the longacting inhibitory effects of bisphosphonate on bone modeling and remodeling, administration of high doses of intravenous pamidronate (2800 mg) over a 3-year period resulted in an acquired osteopetrosislike disorder in a 12-year-old boy with unexplained hyperalkaline phosphatasemia that persisted for at least several years after this agent was discontinued.426 The metaphyses were extremely dense and clubshaped, the base of the skull sclerotic, and the vertebral endplates thickened. Histologically, iliac crest biopsy revealed bars of calcified cartilage and quiescent osteoclasts. Despite the severity of the radiographic and microscopic findings, the patient was clinically well—with normal growth and without evidence of bone marrow suppression or extramedullary hematopoiesis, although the risk for future fractures may have been increased. A multidisciplinary team skilled in the management of patients with osteopetrosis is essential to the optimal care of infants and children with this disorder. In addition to appropriate orthopedic and neurosurgica input, medical therapy may at times be helpful. Nonspecific treatment with interferon-␥ and high doses of calcitriol has led to arrest of disease progression and even its regression in some osteopetrotic children.136,472 In this setting, calcitriol may act as an osteoclast-activating factor—whereas interferon ␥ indirectly stimulates osteoclast formation and increases generation of superoxide in osteoclasts, an important factor for osteoclast-mediated bone resorption.473 Some children improve after bone marrow transplantation from human leukocyte antigen-identical donors with replacement of defective osteoclasts by normal osteoclast progenitor cells.136 Hypercalcemia may complicate the post-transplantation period as osteoclast function resumes. It can be managed with dietary restriction, calcitonin, and occasionally bisphosphonate administration. With improved treatment, there has been somewhat increased life span and improved developmental progress. Osteopetrosis due to deficiency of

Ch17_686-769-X4090.indd 750

carbonic anhydrase II is not corrected by restoration of normal systemic acid-base balance, but bone marrow transplantation may be useful.136 Identification and correction of the specific underlying gene defect may ultimately be feasible in patients with osteopetrosis.467 Pycnodysostosis (OMIM 265800) is clinically manifested by disproportionate short stature during infancy and childhood, with macrocranium and open cranial sutures, high forehead, small facial features, proptosis, bluish sclerae, beaked and pointed nose, micrognathia, highly arched palate, retained primary teeth, short fingers with hypoplastic nails, narrow thorax, pectus excavatum, and kyphoscoliosis with lumbar lordosis.136 Radiologically, there is increased bone density that becomes progressively worse with age. Susceptibility to fractures is increased. The clavicles are slim and hypoplastic laterally. Ribs, distal phalanges, and hyoid bone may be partially or totally absent. Laboratory data and bone histology are basically normal, although there is evidence of decreased osteoblastic and osteoclastic activity. An abundance of osteoclasts with ruffled borders surrounded by enlarged clear zones suggested that dissolution of bone mineral was normal but that degradation of matrix was abnormal in these patients. Indeed, pycnodysostosis is due to biallelic lossof-function mutations in CTSK. This gene encodes cathepsin K, the osteoclast’s lysosomal cysteine protease that degrades organic matrix after the mineral phase of bone has been reabsorbed.137 Among the genetic abnormalities found in patients with pycnodysostosis have been unipaternal isodisomy for chromosome 1, with paternal CTSK harboring an inactivating Ala277Val mutation; Leu9Pro substitution in the signal peptide of the precursor form of the enzyme protein, preventing completion of its post-translational processing; and Ter330Trp substitution, permitting the addition of 19 amino acids to the carboxyl terminus of this enzyme. In contrast with diseases that increase bone mass by decreasing bone resorption are those disorders that primarily increase bone formation. An example of the latter is the familial relatively benign form of autosomaldominant high bone mass (OMIM 601884) that is associated with a heterozygous gain-of-function mutation (Gly171Val) in LRP5.474 Loss-of-function mutations in the same gene are associated with idiopathic juvenile osteoporosis, the osteoporosis-pseudoglioma syndrome, and familial exudative vitreoretinopathy. LRP5 (and its homolog LRP6) is a co-receptor for Wnt proteins whose primary receptor is Frizzled. After binding to LRP5 and Frizzled, the Wnt glycoprotein activates a canonical pathway that involves repression of glycogen synthase kinase 3 and leads to dephosphorylation of ␤-catenin—permitting its translocation to the nucleus, where it interacts with T-cell factor/ lymphoid enhancing factor to control target genes that divert the mesenchymal stem cell into the track leading to osteoblastogenesis.475 In the mature osteoblast, ␤-catenin stimulates expression of osteoprotegerin—thus impairing osteoclastogenesis. Dickkopf and sclerostin are proteins that bind to the extracellular domain of LRP5 and internalize the receptor complex, thereby blocking Wnt signaling.476

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DISORDERS OF MINERAL HOMEOSTASIS IN THE NEWBORN, INFANT, CHILD, AND ADOLESCENT

Mutations in LRP5 associated with high bone mass are clustered near the amino terminal of its extracellular domain at the sites of binding to Dickkopf and sclerostin.419 The Gly171Val mutation in LRP5 interferes with the binding of LRP5 to Dickkopf and thus prolongs the interaction of LRP5 with Frizzled, thereby augmenting the Wnt signal and increasing bone formation.477,478 Although generally benign, activating mutations of LRP5 may also be associated with neurologic complications such as hearing loss, headaches, and pain in the extremities.419 In some families, heterozygous activating mutations (Ala242Thr) of LRP5 and exuberant bone formation have been associated with autosomal-dominant generalized endosteal osteosclerosis (van Buchem disease type 2, OMIM 607636) or even osteopetrosis (Gly171Arg, Thr253Ile). However, the dense bones encountered in these subjects are not prone to fracture (as in classic forms of osteopetrosis). Sclerosteosis (OMIM 269500) is an autosomal-recessive disorder first manifested in childhood and characterized by very thick peripheral and cranial bones with calvarial overgrowth, leading to facial disfigurement, entrapment of cranial nerves VII and VIII, increased intracranial pressure, and brain stem compression.136 Affected patients also have variable asymmetric cutaneous or bony syndactyly of the index and middle fingers and excessive somatic growth. Sclerosteosis is due to biallelic loss-of-function mutations in SOST, the gene that encodes sclerostin—a 213-aa peptide secreted primarily by osteocytes embedded within bone. Normally, sclerostin inhibits Wnt-mediated bone formation by binding to and internalizing LRP5 (the co-receptor for Wnt).475 When sclerostin activity is decreased, increased bone formation ensues. In heterozygous carriers of inactivating mutations in SOST, bone mass is increased (but not to pathologic levels).479 van Buchem disease type 1 (OMIM 239100), an autosomal-recessive form of generalized osteosclerosis, is due to biallelic deletion of a 52-kb noncoding SOST-specific regulatory region approximately 35 kb downstream of the intact gene itself that results in loss-of-function of SOST.480 Thus, van Buchem disease type 1 and sclerosteosis are allelic disorders. Progressive diaphyseal dysplasia (Camurati-Englemann disease, OMIM 131300) is an autosomal-dominant cranial-peripheral hyperostotic disorder with variable expressivity that presents in children with problems such as limping, waddling gait, and/or leg pain, fatigue, and nonprogressive muscular weakness. Radiographically, there is symmetrical cortical thickening (hyperostosis) due to increased periosteal and endosteal bone formation in the diaphyses of the long bones, axial skeletons, and skull.136,481 Pathogenetically, this disorder is primarily due to missense mutations within the latency-associated peptide domain of the precursor propeptide of TGF␤1 (TGFB1). Normally, after post-translational processing two latency-associated peptides are noncovalently linked to two mature TGF␤1 peptides to form a latency complex. Mutations within the latency-associated peptide domain of TGFB1 (particularly at codon 218, a mutational hot spot) impair this association, resulting in premature activation of TGF␤1 and consequent stimulation of bone formation and repression of bone resorption.482 TGF␤1

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also inhibits myogenesis and adipogenesis. Because of the inhibitory effects of glucocorticoids on bone formation and its stimulatory effects on bone resorption, short courses of these agents have been useful in alleviating many of the clinical symptoms and radiologic abnormalities in patients with progressive diaphyseal dysplasia.481 However, long-term therapy with glucocorticoids is not advised.

ECTOPIC CALCIFICATION AND OSSIFICATION Extraskeletal calcification may occur in a number of hypercalcemic, hyperphosphatemic, or dystrophic states (renal failure, hypo- and hyperparathyroidism, sarcoidosis, after cell lysis induced by cancer chemotherapy, subcutaneous fat necrosis, dermatomyositis, atherosclerosis)—as well as in specific diseases (e.g., PHP type IA, McCune-Albright syndrome).483 Familial tumoral calcinosis (OMIM 211900) presents in childhood with recurrent bone pain, extensive cutaneous, periarticular and vascular calcifications, and periarticular hard and lobulated masses. In some patients, the ectopic calcifications may be confined to the eyelids. It is characterized radiographically by cortical hyperostosis, periosteal reaction, and mineral deposits around large joints (particularly hips and shoulders).484-486 Laboratory studies reveal marked hyperphosphatemia and relative hypophosphaturia due to increased renal tubular reabsorption of phosphate and inappropriately normal or elevated serum calcitriol levels, because despite hyperphosphatemia PTH secretion is not increased and synthesis of calcitriol and intestinal calcium absorption persist. The disorder is due to functional loss of FGF23 action and consequently unhindered renal tubular reabsorption of phosphate. The pathophysiology of this disorder is thus the mirror image of that associated with XHR, ADHR, and tumor-induced osteomalacia in which there is exaggerated FGF23 production and activity leading to hyperphosphaturia and consequent hypophosphatemia, rickets, and osteomalacia. Familial tumoral calcinosis is genetically heterogeneous. Homozygous inactivating mutations (Met96Thr, Ser129Phe) in FGF23 have been identified in a few patients with this disorder.484,487 More commonly detected in patients with familial tumor calcinosis are biallelic lossof-function microdeletions and splice site and missense mutations (Arg162Stop, Thr272Lys) in GALNT3 (UDP-Nacetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 3). The product of GALNT3 is a glycosyl transferase that initiates O-glycosylation in which N-acetylgalactosamine is the first sugar in the side chain, a step essential to secretion of intact and functional FGF23.488 Failure to O-glycosylate FGF23 at Thr178 in the golgi apparatus permits its rapid intracellular cleavage between Arg179 and Ser180 to biologically inactive amino- and carboxyl-terminal fragments.485,489 Serum concentrations of intact FGF23 are low or nondetectable, whereas carboxyl-terminal FGF23 levels are elevated in patients with familial tumoral calcinosis due to either gene mutation. Therapy with an oral phosphate binder and the carbonic anhydrase inhibitor acetazolamide has resulted in hyperphosphaturia and reabsorption of

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ectopic calcifications without change in serum phosphate or calcium concentrations.485,490 The hyperostosis hyperphosphatemia syndrome (OMIM 610233) is a clinical variant of familial tumoral calcinosis and is due to mutations in FGF23 or GALNT3. Symptoms and signs may precede development of the more typical phenotype of familial tumoral calcinosis.489,491 Fibrodysplasia ossificans progressiva (FOP, OMIM 135100) is a disabling disorder of ectopic bone formation that may develop spontaneously or at sites of injury. It leads to ankylosis of all major joints, which severely limits mobility.492 It is characterized by progressive ectopic ossification of muscle and connective tissue, leading to immobility of the mandible, neck, spine, hips, and other joints and to the development of a second skeleton that encases and imprisons the body. The disorder may be present at birth and is often manifest by 5 years of age. It is also associated with monophalangic big toes, deafness, scalp baldness, and mild developmental delay. Microscopically, there is normal endochondral osteogenesis occurring at an ectopic site. Although primarily sporadic because affected subjects rarely reproduce, FOP can be transmitted as an autosomal-dominant trait. This disease is due to a highly specific (Arg206His) mutation in ACVR1 (activin A receptor, type 1), to date the only mutation identified in these patients. Activins are members of the TGF␤ superfamily that includes the BMPs as well as the inhibins and Mullerian-duct-inhibiting factor. The Arg206His mutation in ACVR1 resides at the junction of the receptor’s glycine-serine activation and tyrosine kinase domains and results in a constitutively active receptor product that directs the pluripotent mesenchymal stem cell into the chondrogenic pathway, leading to (ectopic) endochondral new bone formation. BMPs alone are able to stimulate complete endochondral osteogenesis in ectopic sites.493 BMP4 induces osteogenesis but is also required for commitment of the stem cell to the adipocyte pathway of development.494 BMP4 and the genes encoding its coreceptors (e.g., BMPR1A) are not mutated in patients with FOP. BMP4 (but not BMPR1A) is overexpressed in cells from patients with FOP. However, there is an increased number of plasma membrane BMPR1A receptors in their cells due to a slow rate of ligand-mediated receptor internalization and consequently decreased rate

of degradation—which might lead to prolonged BMP4mediated intracellular signaling and function.493 Overall, data suggest that BMP4-directed osteogenic activity is enhanced and the adipogenic pathway circumvented in patients with FOP. However, the role that the mutated ACVR1 plays in the pathogenesis of this disease is unclear at present. Management of these patients is primarily symptomatic to the extent possible, although immunosuppression may diminish the intensity of extraskeletal ossification.495 Progressive osseous heteroplasia (POH, OMIM 166350) is characterized by multiple foci of dermal membranous bone formation (osteoma cutis) beginning in infancy in the absence of any local injury or inflammatory insult. Lesions may develop on the trunk, extremities, or digits and may be asymptomatic or painful. Over time, ossification involving skeletal muscle and deep connective issue evolves. The disorder occurs in both genders. POH is transmitted as an autosomal-dominant trait and is due to inactivating mutations of the GNAS allele inherited from the father.496,497 Identical mutations in GNAS may be clinically manifested as POH or PPHP in different members of the same family.

Osteochondrodysplasias The osteochondrodysplasias form a heterogeneous group of malformations of cartilage and bone that have been grouped according to clinical and radiologic characteristics into those involving long bone growth alone (epiphyseal, metaphyseal, and/or diaphyseal dysplasias), long bones and vertebrae (spondyloepiphyseal and/or spondyloepimetaphyseal dysplasias), and variants thereof (Figure 17-18).498 The genetic skeletal disorders have been classified into 37 groups based on the underlying genetic mutations and/or radiographic manifestations.499 Mutations in one gene may give rise to several clinically defined disorders (Table 17-12). These disorders are of interest not only because of the diagnostic and therapeutic clinical challenges they present but because they have identified many factors that normally regulate cartilage and bone development. Achondroplasia (OMIM 100800), the most common of the human chondrodystrophies (1/15,000–40,000 live births), is due to gain-of-function mutations in FGFR3—the gene

E D

A

B

C

Involvement A⫹D B⫹D C⫹D B⫹E B⫹C⫹E

Disease Category Normal Epiphyseal dysplasia Metaphyseal dysplasia Spondyloepiphyseal dysplasia Spondyloepimetaphyseal dysplasia

Figure 17-18 Anatomic classification of osteochondrodysplasias. [Reproduced with permission from Shohat M, Rimoin DL (2007). The skeletal dysplasias. In Lifschitz F (ed.), Pediatric endocrinology, Fifth edition. New York: Informa Healthcare 145–162.]

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753

TA B L E 1 7 - 1 2

Human Osteochondrodysplasias: Selected Mutated Genes Gene

Chromosome Locus

OMIM

Fibroblast Growth Factor Receptors FGFR1 8p11.2-p11.1 136350 FGFR2

10q25.3-q26

176943

FGFR3

4p16.3

134934

Collagenopathies COL2A1 12q13.11- q13.2

120140

COL9A1

6q13

120210

COL9A2

1p33-p32.2

120260

COL9A3

20q13.3

120270

COL10A1

6q21-q22.3

120110

COL11A1

1p21

120280

COL11A2

6p21.3

120290

Gene Product Function

Clinical Disorder (Syndrome)

Inheritance pattern

OMIM

Transmembrane tyrosine kinase receptor for FGFs

Pfeiffer

AD

101600

Apert Crouzon Jackson-Weiss Pfeiffer Antley-Bixler Beare-Stevenson cutis gyrata Achondroplasia Hypochondroplasia Thanatophoric dysplasia, Types I, II Severe with developmental delay and acanthosis nigricans Muenke nonsyndromic coronal craniosynostosis

AD AD AD AD AD AD AD AD AD

101200 123500 123150 101600 207410 123790 100800 146100 187600

AD

134934

AD

602849

Achondrogenesis type II Spondyloepiphyseal dysplasia congenita Spondylometaphyseal dysplasia Kneist dysplasia Spondyloepimetaphyseal dysplasia Strudwick type Stickler syndrome (1) Stickler syndrome Multiple epiphysea dysplasia Multiple epiphyseal dysplasia (2) Multiple epiphyseal dysplasia (3) Metaphyseal chondrodysplasia Schmid-type Stickler syndrome (2) Marshall syndrome Otospondylomegaepiphyseal dysplasia Stickler syndrome (3)

AD AD

200610 183900

AD

184252

AD AD

156550 184250

AD AR AD AD

108300

Extracellular matrix protein

Sulfation Disorders SLC26A2 5q32-q33.1

606718

Transmembrane sulfate transporter

PAPSS2

10q22-q24

603005

ARSE

Xp22.3

300180

3’-Phosphoadenosine 5’-phosphosulfate synthase 2 Arylsulfatase E

CHST3

10q22.1

603799

Carbohydrate sulfotransferase 3

AD AD

156500

AD AD AR

604841 154780 215150

AD

184840

Diastrophic dysplasia Atelosteogenesis type II Achondrogenesis type IB Multiple epiphyseal dysplasia (4) Spondyloepimetaphyseal dysplasia

AR AR AR AR

222600 256050 600972 226900

AR

603005

Chondrodysplasia punctata (1) Spondyloepiphyseal dysplasia (Omani)

XLR

302950

AR

608637 (Continued)

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TA B L E 1 7 - 1 2

Human Osteochondrodysplasias: Selected Mutated Genes—Cont’d Gene

Chromosome Locus

OMIM

Gene Product Function

Clinical Disorder (Syndrome)

Inheritance pattern

OMIM

Perlecan Group HSPG2 1p36.1

142461

Heparan sulfate proteoglycan

Myotonic chondrodystrophy (Schwartz-Jampel, type 1)

AR

255800

Filamin Group FLNA Xq28

300017

Actin-binding protein

XLD

305620

FLNB

603381

Fronto-metaphyseal dysplasia Otopalataldigital (1,2) Atelosteogenesis (1,3)

3p14.3

XLD AD

108720, 108721

Pseudoachondroplasia Group COMP 19p12-13.1

600310

Cartilage oligomeric matrix protein

Pseudoachondroplasia Multiple epiphyseal dysplasia (Fairbank)

AD AD

177170 132400

Metaphyseal Dysplasias RMRP 9p21-p12

157660

Cartilage-hair hypoplasia Anauxetic dysplasia

AR AR

250250 607095

PTHR1

168468

Mitochondrial RNAprocessing endoribonuclease Transmembrane G-protein receptor for PTH & PTHrP

Murk-Jansen metaphyseal chondrodysplasia Blomstrand chondrodysplasia

AD

156400

AR

215045

Homeobox gene Transcription factor

Leri-Weill dyschondrosteosis Langer dysplasia (Idiopathic short stature) (Turner syndrome)

XLD Biallelic

127300 249700

Osteoblast- specific transcription factor Transcription factor

Cleidocranial dysplasia

AD

119600

Campomelic dysplasia

AD

114290

⌬7-Dehydro cholesterol reductase 3␤-Hydroxysteroid ⌬8,⌬7 isomerase

Smith-Lemli-Opitz syndrome

AR

270400

Chondrodysplasia punctata (2) (Conradi- HunermannHapple) Desmosterolosis

XLD

302960

AR

602398

Hydrops-ectopic calcification moth eaten dysplasia (Greenberg) Congenital hemidysplasia, icthyosiform erythroderma, limb defects

AR

215140

XLD

308050

3p22-p21.1

Mesomelic/Rhizo-mesomelic Dysplasias SHOX Xpter-p22.32 312865

Others RUNX2

6p21

600211

SOX9

17q24.1- q25.1

608160

Defects in Cholesterol Synthesis DHCR7 11q12-q13

602858

EBP

Xp11.22- p11.23

300205

DHCR24

1p33-p33.1

606418

LRB

1q42.1

600024

NSDHL

Xq28

300275

3␤-hydroxysterol-⌬24 reductase 3␤-hydroxysterol-⌬14 reductase (Lamin B receptor) 3␤-hydroxysteroid C-4 sterol demethylase complex

AD, autosomal dominant; AR, autosomal recessive; CD, chondrodysplasia punctata; MED, multiple epiphyseal dysplasia; PTH, parathyroid hormone; PTHrP, PTH-related protein; SED, spondyloepiphyseal dysplasia; XLD, X-linked dominant; and XLR, X-linked recessive. Adapted from Herman GE (2003). Disorders of cholesterol biosynthesis: Prototypic metabolic malformation syndromes. Hum Molec Genet 12:R75– R88; from Superti-Furga A, Unger S, and the Nosology Group of the International Skeletal Dysplasia Society (2007). International nosology and classification of genetic skeletal disorders: 2006 revision. Am J Med Genet 143A:1–18 (www.isds.ch).

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Ig I

Ig II AB

Ig III TM

TKp

TKd

ACH TDII HYP TDI G380R K650E K650Q R248C TDI S249C G370C HYP SADDAN S371C N540G K650M Y373C Figure 17-19 Mutations in fibroblast growth factor receptor-3 leading to achondroplasia (ACH), hypochondroplasia (HYP), thanatophoric dysplasia (TD) types I and II, and severe achondroplasiadevelopmental delay-acanthosis nigricans (SADDAN). [Reproduced with permission from Horton WA (2006). Molecular pathogenesis of achondroplasia. Growth Genet Horm 22:49–54.]

encoding fibroblast growth receptor 3. It is transmitted as an autosomal-dominant disorder but with a high rate of spontaneous mutations (primarily of the paternal allele). Clinically, it is manifested by short limbs but normal trunk length, large head, frontal bossing, and depressed nasal bridge. It is complicated by increased risk for cervical cord compression and spinal stenosis.500,501 FGFR3 is a transmembrane protein with three immunoglobulin domains in the extracellular region of the receptor and two tyrosine kinase domains in its intracellular portion (Figure 17-19). Three additional clinically distinct osteochondrodysplasias are associated with mutations in the same gene. Thanatophoric dysplasia is characterized by severe bony malformations, particularly of the skull, long bones, and ribs—the latter leading to respiratory insufficiency and early death. There are two radiographic forms of this disorder (I and II). Severe achondroplasia— developmental delay—acanthosis nigricans (SADDAN) is a clinical phenotype intermediate between those of thanatophoric dysplasia and achondroplasia. Hypochondroplasia is a clinically less severe manifestation of mild shortlimbed (rhizomelic) short stature of variable severity that presents in mid-childhood. Mutations in FGFR3 correlate with the clinical phenotype. Achondroplasia is most often (98%) associated with a missense mutation (Gly380Arg) in its transmembrane domain. Thanatophoric dysplasia type I is related to mutations (Arg248Cys, Gly370Cys) in the extracellular ligandbinding region next to the transmembrane region of the receptor. Type II is related to a mutation (Lys650Glu) in the distal tyrosine kinase domain. SADDAN is also the result of a Lys650Met mutation. Hypochondroplasia has been associated with mutations within the proximal (Asn540Gly in 60%) and distal (Lys650Gln) tyrosine kinase domains, respectively, as well in the immunoglobulin domains of the extracellular region (Ser84Leu, Arg200Cys) and transmembrane domain (Val381Glu) of FGF23.501,502 It is noteworthy that different mutations at Lys650 result in three distinct clinical phenotypes. Patients with Muenke nonsyndromic coronal craniosynostosis also have a mutation (Pro250Arg) in FGFR3. In chondrocytes, constitutively active FGFR3 initiates ligand-independent autophosphorylation of tyrosine residues within its

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755

cytoplasmic domain that then propagate signal through the MAPK and signal transducer and activator of transcription pathways—resulting in inhibition of mitosis, matrix synthesis, and terminal (hypertrophic) differentiation. Thus, FGFR3 normally functions as a negative regulator of cartilage formation. As a single-gene disorder, it is possible to identify a mutation in FGFR3 in an affected fetus by analysis of fetal cell-free DNA in maternal plasma if the mother is unaffected.503 A loss-of-function mutation (Arg621His within the distal tyrosine kinase domain) in FGFR3 has been identified in a family whose members display the phenotype of tall stature, camptodactyly, and hearing loss.504 Activating mutations in FGFR1 and FGFR2 have been associated with chondrodysplasias complicated by premature craniosynostosis (Pfeiffer, Apert, Crouzon, Jackson-White, and Beare-Stevenson cutis gyrata syndromes). Interestingly, mutations in FGFR1 have also been associated with hypogonadotropic hypogonadism (OMIM 147950), as FGFR1 is an essential neuronal migration factor.505 Mutations in FGFR4 have not been associated with osteochondrodysplasias to date. Defective formation of several types of collagen due to mutations in COL2A1, COL9A1, COL9A2, COL10A1, COL11A1, and COL11A2 result in a large number of skeletal malformations depending on the site and developmental timing of the synthetic error (Table 17-12). Abnormalities of sulfate transport, collagen matrix protein sulfation, and sulfatase activity have resulted in several chondrodysplasias. Spondylepimetaphyseal dysplasia (OMIM 603005) is due to a biallelic loss-offunction mutation (Ser438Ter) in the gene (PAPSS2) encoding 3’-phosphoadenosine-5’-phosphosulfate synthase 2—an enzyme with dual activities. It catalyzes the synthesis of adenosine 5’-phosphosulfate and its phosphorylation to 3’-phospho-adenosine 5’-phosphosulfate, the universal sulfate donor necessary to sulfation of cartilage and bone matrix proteins.506 Clinical manifestations of this disorder include short limbs, kyphoscoliosis, brachydactyly, and enlarged knee joints. In the mouse model of this disease, there is decreased sulfation of matrix proteoglycans—resulting in short chondrocyte columns with irregularly aligned cells and decrease in the number and size of hypertrophic chondrocytes. Loss-of-function mutations (insertions, missense, nonsense) in SOX9, a transcription factor expressed in both developing chondrocytes (where it is coexpressed with COL2A1) and in the genital ridges during gonadal differentiation, cause campomelic dysplasia (OMIM 114290) and male-to-female sex reversal in 75% of affected 46XY subjects. Clinically, this disorder is characterized by prenatal onset of bowing of tubular bones, hypoplastic scapulae, 11 ribs, cleft palate, and micrognathia—leading often to neonatal death. Heterozygous pseudoautosomal microdeletions and intragenic inactivating mutations (Leu 132Val, Arg195Ter) of the X (and Y) -linked pseudoautosomal gene SHOX (short stature homeobox-containing gene) resulting in haploinsufficiency are present in patients with Leri-Weill dyschondrosteosis (OMIM 127300)— typified by mesomelic limb shortening and growth retardation, Madelung deformity of the wrist, bowing of the

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radius, and ulnar dislocation.507 Patients with idiopathic short stature and no identifying skeletal characteristics have also been observed to have heterozygous loss-offunction mutations in SHOX. The Leri-Weill phenotype has been recorded in patients with intact SHOX but with microdeletions of downstream segments of the X chromosome pseudoautosomal region, implying the presence of modifying genes in this region.508 Homozygous loss of SHOX leads to Langer mesomelic dysplasia (OMIM 249700). Endogenous SHOX is primarily expressed in hypertrophic and apoptotic chondrocytes within the cartilage growth plate. It is a transcriptional activator whose gene targets are as yet unidentified but whose expression in osteogenic cells results in arrest of the cell cycle and in apoptosis.509 Gain- and loss-of-function mutations of PTHR1 lead to abnormalities of bone formation and growth.510 Blomstrand chondrodysplasia (OMIM 215045) is an autosomalrecessive disorder that is lethal in utero and that may be identified in the fetus with short extremely dense long bones and markedly advanced skeletal maturation (as well as somatic anomalies such as aortic coarctation and facial anomalies). Pathologically, epiphyseal cartilage is reduced—and there are irregular columns and erratic distribution of chondrocytes within matrix. The abnormality is the result of inactivating mutations (deletions, missense mutations, Pro132Leu) of the gene encoding the PTH/ PTHrP receptor. Patients with Murk-Jansen metaphyseal chondrodysplasia (OMIM 156400) have short limbs and fingers, micrognathia, and deformities of the spine and pelvis but survive to adulthood. The average adult height is 125 cm, and child bearing is possible. Characteristically, these patients have hypercalcemia, hypophosphatemia, and low or undetectable serum levels of PTH or PTHrP. The disorder is due to activating mutations (His223Arg, Thr410Pro) of PTHR1 and is associated with extraordinary delay in chondrocyte differentiation and decreased mineralization due to excessive bone resorption. Both disorders reflect the functional effect of PTHrP in developing cartilage, where it normally acts to slow differentiation and decrease the rate of chondrocyte apoptosis— thus prolonging chondrocyte proliferation and enhancing long bone growth. Anauxetic dysplasia (OMIM 607095) is a spondylometaepiphyseal dysplasia transmitted as an autosomal-recessive disorder characterized by intrauterine growth retardation (birth length ⬍40 cm), severely compromised adult height (⬍85 cm), hypodontia, and mild mental retardation. All bones are malformed, and there are few chondrocytes in the cartilage growth plates. It is due to loss-of-function mutations (insertions) in RMRP (RNA component of mitochondrial RNA-processing endoribonuclease), a gene that encodes the untranslated RNA subunit of the endoribonuclease RNase MRP.511 This enzyme is involved in the assembly of ribosomes (the structural units in which translation and protein synthesis takes place), the generation of RNA primers for replication of mitochondrial DNA, and the regulation of the cyclin-dependent cell cycle. The mutations that result in anauxetic dysplasia impair ribosome assembly and protein synthesis exclusively. Inactivating mutations

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(duplications, insertions) in RMRP that modestly impair ribosomal assembly and regulation of the cell cycle have been found in patients with cartilage hair hypoplasia (OMIM 250250) and metaphyseal dysplasia without hypotrichosis (OMIM 250460). Errors in the biosynthesis of cholesterol have been associated with several abnormalities of bone development (Table 17-12, Figure 17-20).512 In patients with the SmithLemli-Opitz syndrome (OMIM 270400), inactivating mutations of the microsomal enzyme ⌬7-dehydrocholesterol reductase (DHCR7) impair synthesis of cholesterol and lead to intrauterine and postnatal growth retardation, short limbs, syndactyly and polydactyly, characteristic facial features (blepharoptosis, anteverted nares, broad alveolar ridges, cleft palate), congenital malformations of the heart and central nervous system, microcephaly, incomplete virilization of male external genitalia, hypoplastic thumbs, and developmental delay.513 Stippled epiphyses are detected by radiologic examination. Hypocholesterolemia with elevated serum concentrations of 7-dehydrocholesterol are present. Cholesterol covalently binds to the amino terminal and is essential to the function of Indian hedgehog, Sonic hedgehog, and Desert hedgehog—factors necessary to normal development of cartilage and bone, brain, and testes, respectively. However, substitution of 7-dehydrocholesterol for cholesterol does not impair the function of Sonic hedgehog. Therefore, it is likely that lack of cholesterol exerts its teratologic effect by impairing the intracellular signaling response(s) to these factors. Alternatively, the accumulation of precursors of cholesterol may exert toxic effects on the developing fetus. Cholesterol supplementation may improve the health, behavior, and growth of children with the SmithLemli-Opitz syndrome. Chondrodysplasia punctata 2 (OMIM 302960) is an Xlinked dominant disorder (Conradi-Hunermann-Happle syndrome) that is ordinarily lethal in the affected male and characterized clinically in the affected female by short stature with asymmetric short proximal limbs (rhizomelic dwarfism), frontal bossing, icthyosis in children and atrophic pigmentary lesions in adults, coarse hair with alopecia, and cataracts. Reflecting functional Xchromosomal mosaicism in females, the phenotype may vary from stillborn to mildly affected.512 Radiographic examination reveals generalized osteosclerosis, irregular punctate calcification (stippling) of epiphyses in children, and hemivertebrae. This disorder is due to loss-of-function mutations in EBP (emopamil-binding protein), the gene encoding 3␤-hydroxysteroid-⌬8,⌬7isomerase—an enzyme important in sterol biosynthesis. In the serum of affected subjects, plasma concentrations of 8-dehydrocholesterol cholest-8(9)-en-3␤-ol are elevated. This protein also binds many unrelated molecules. Its genetic designation derives from its ability to bind emopamil (i.e., EBP), a calcium ion antagonist. X-linked recessive and autosomal-recessive forms of the Conradi-Hunermann syndrome have also been reported, suggesting genetic heterogeneity for this phenotype. The X-linked recessive form of chondrodysplasia punctata (CDPX1, OMIM 302950) is due to inactivating mutations of the gene (ARSE) encoding aryl-sulfatase E. Affected males may

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757

CH3

Lanosterol

CH3 24 1

9

2

?ABS

HO CH3

HO

3

7

5 4

CH3

CH3

14 8

(1)

CH3

CH3

(1) HO

6

CH3

CH3

CH3 (2)

(2)

HEM Dysplasia 4,4-dimethycholesta-8(9)-dien-3β-ol

HO

4,4-dimethylcholest-8(9)-en-3β-ol

CH3

CH3

HO

(3) CH3

CH3

(3)

Cholesta-8(9),24-dien-3β-ol (Zymosterol)

CHILD syndrome Bpa/Str mice (?5)

HO

(4)

HO

Cholest-8(9)-en-3β-ol Cholesta-7,24-dien-3β-ol HO

(4)

CDPX2, CHILD syndrome, Td mice

HO

(5)

(4)

7-dehydrodesmosterol Lathosterol HO HO (5)

Lathosterolosis SLOS

(6)

(6)

Desmosterolosis HO

Cholesterol HO

7-dehydrocholesterol

(7) HO

Desmosterol

Figure 17-20 Biosynthesis of cholesterol depicting the sites of enzyme activity lost by inactivating mutations that lead to skeletal, genital, and other malformations. The numbers in parentheses denote specific enzymes: (1) lanosterol 14␣-demethylase, (2) 3␤-hydroxysteroid-⌬14reductase, (3) 3␤-hydroxysteroid C-4 sterol demethylase complex, (4) 3 ␤ -hydroxysteroid-⌬8-⌬7-sterol isomerase, (5) 3␤-hydroxysteroid␣5-desaturase, (6) 3␤-hydroxysteroid-⌬7-reductase, and (7) 3␤-hydroxysteroid-⌬24-reductase. [Reproduced with permission from Herman GE (2003). Disorders of cholesterol biosynthesis: Prototypic metabolic malformation syndromes. Hum Molec Genet 12:R75–R88.]

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be stillborn or may survive, whereas female carriers may be asymptomatic or mildly affected. Desmosterolosis (OMIM 602398) is an autosomalrecessive disorder whose skeletal manifestations include osteosclerosis, shortened limbs, macrocephaly, cleft palate, and thick alveolar ridges in addition to congenital heart disease and ambiguous genitalia in the affected female. It is due to inactivating mutations of the gene (DHCR24) encoding 3␤-hydroxysterol-⌬24 reductase, the enzyme that converts desmosterol to cholesterol.513,514 The rare lethal Greenberg dysplasia of hydrops ectopic calcification moth-eaten skeletal dysplasia (OMIM 215140) is transmitted as an autosomal-recessive trait associated with short-limbed dwarfism, polydactyly, and irregularly decreased calcification of the long bones together with calcification of the larynx and trachea. It is due to a lossof-function mutation in the gene (LRB) encoding the lamin B receptor, a nuclear envelope inner membrane protein that not only binds lamin B but has 3␤-hydroxysteroid-⌬14 reductase activity—an enzyme necessary to normal cholesterol biosynthesis.515 The CHILD syndrome (OMIM 308050) of congenital hemidysplasia with icthyosiform erythroderma (or nevus) and limb defects is remarkable for its unilateral distribution of anomalies that are confined to one-half of the body.512 It is an X-linked disorder due to loss-of-function mutations in NSDHL (NADPH steroid dehydrogenase-like) encoding a sterol dehydrogenase or decarboxylase that is part of the 3␤-hydroxysteroid C-4 sterol demethylase complex.

Concluding Remarks In the past decade, our understanding of the complex mechanisms that underlie the pathophysiology of numerous illnesses that adversely affect the regulation of calcium, phosphate, and magnesium metabolism; chondrocyte differentiation and growth; and bone formation, mineralization, and strength has been transformed by the insights advances in genetics and proteomics have brought to these clinical problems. As further clarification of the very most basic mechanisms that underlie these disorders becomes available in the next decade, our ability to identify and specifically manage these problems will almost certainly experience another quantum leap forward.

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462. Waguespack SG, Hui SL, White KE, et al. (2002). Measurement of tartrate-resistant acid phosphatase and the brain isoenzyme of creatine kinase accurately diagnoses type II autosomal dominant osteopetrosis but does not identify gene carriers. J Clin Endocrinol Metab 87:2212–2217. 463. Rodan GA, Martin TJ (2000). Therapeutic approaches to bone diseases. Science 289:1508–1514. 464. Doffinger S, Smahi A, Bessia C, et al. (2001). X-linked anhidrotic ectodermal dysplasia with immunodeficiency is caused by impaired NF-␬B signaling. Nature Genet 27:277–285. 465. Dupuis-Girod S, Corradinin N, Hadj-Rabia S, et al. (2002). Osteopetrosis, lymphedema, anhidrotic ectodermal dysplasia, and immunodeficiency in a boy and incontinentia pigmenti in his mother. Pediatrics 109:1–6. 466. Sobacchi C, Frattini A, Orchard P, et al. (2001). The mutational spectrum of human malignant autosomal recessive osteopetrosis. Hum Molec Genet 10:1767–1773. 467. Susani L, Pangrazio A, Sobachhi A, et al. (2004). TCIRG1-dependent recessive osteopetrosis: Mutation analysis, functional identification of the splicing defects, and in vitro rescue by U1 snRNA. Hum Mutat 24:225–235. 468. Kornak U, Kasper D, Bosl MR, et al. (2001). Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man. Cell 104:205–215. 469. Lange PF, Wartosch L, Jentsch TJ, Fuhrmann JC (2006). ClC-7 requires Ostm1 as a ␤-subunit to support bone resorption and lysosomal function. Nature 440:220–223. 470. Chalhoub N, Benachenhou N, Rajapurohitam V, et al. (2003). Grey-lethal mutation indices severe malignant autosomal recessive osteopetrosis in mouse and human. Nature Med 9:399–406. 471. Ramirez A, Faupel J, Goebel I, et al. (2004). Identification of a novel mutation in the coding region of the grey-lethal gene OSTM1 in human malignant infantile osteopetrosis. Hum Mutat 23:471–476. 472. Key LL Jr., Rodriguez RM, Willi SM, et al. (1995). Recombinant human interferon gamma therapy for osteopetrosis. N Engl J Med 332:1594–1599. 473. Gao Y, Grassi F, Ryan MR, et al. (2007). IFN-␥ stimulates osteoclast formation and bone loss in vivo via antigen-driven T cell activation. J Clin Invest 117:122–132. 474. Boyden LM, Mao J, Belsky J, et al. (2002). High bone density due to a mutation in LDL-receptor-related protein 5. N Engl J Med 346:1513–1521. 475. Ott SM (2005). Sclerostin and Wnt signaling: The pathway to bone strength. J Clin Endocrinol Metab 90:6741–6743. 476. Shoback D (2007). Update in osteoporosis and metabolic bone disorders. J Clin Endocrinol Metab 92:747–753. 477. Patel MS, Karsenty G (2002). Regulation of bone formation and vision by LRP5. N Engl J Med 346:1572–1574. 478. Van Wesenbeeck L, Cleiren E, Gram J, et al. (2003). Six novel missense mutations in the LDL receptor-related protein 5 (LRP5) gene in different conditions with increased bone density. Am J Hum Genet 72:763–771. 479. Gardner JC, van Bezooijen RL, Mervis B, et al. (2005). Bone mineral density in sclerosteosis: Affected individuals and gene carriers. J Clin Endocrinol Metab 90:6392–6395. 480. Loots GB, Kneissel M, Keller H, et al. (2005). Genomic deletion of a long-range bone enhancer misregulates sclerostin in Van Buchem disease. Genome Res 15:928–935. 481. Janssens K, Vanhoenacker F, Bonduelle M, et al. (2007). CamuratiEngelmann disease: Review of the clinical, radiological, and molecular data of 24 families and implications for diagnosis and treatment. J Med Genet 43:1–11. 482. Janssens K, ten Dyke P, Janssens S, Van Hul W (2005). Transforming growth factor-beta 1 to the bone. Endocrine Reviews 26:743–774. 483. Whyte MP (2006). Extracellular (ectopic) calcification and ossification. In Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 436–437. 484. Benet-Pages A, Orlik P, Strom TM, Lorenz-Depiereux B (2005). An FGF23 missense mutation causes familial tumoral calcinosis with hyperphosphatemia. Hum Molec Genet 14:385–390. 485. Ichikawa S, Imel EA, Sorensen AH, et al. (2006). Tumoral calcinosis presenting with eyelid calcifications due to novel missense mutations in the glycosyltransferase domain of the GALNT3 gene. J Clin Endocrinol Metab 91:4472–4475.

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486. Whyte MP (2006). Tumoral calcinosis. In Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 437–439. 487. Araya K, Fukumoto S, Backenroth R, et al. (2005). A novel mutation in fibroblast growth factor 23 gene as a cause of tumoral calcinosis. J Clin Endocrinol Metab 90:5523–5527. 488. Kato K, Jeanneau C, Tarp MA, et al. (2006). Polypeptide GalNActransferase T3 and familial tumoral calcinosis: Secretion of fibroblast growth factor 23 requires O-glycosylation. J Biol Chem 281:18370–18377. 489. Frishberg Y, Ito N, Rinat C, et al. (2007). Hyperostosis-hyperphosphatemia syndrome: A congenital disorder of O-glycosylation associated with augmented processing of fibroblast growth factor 23. J Bone Miner Res 22:235–242. 490. Garringer HJ, Fisher C, Larsson TE, et al. (2006). The role of mutant UDP-N-acetyl-alpha-D-galactosamine-polypeptide N-acetylgalactosaminyltransferase 3 in regulating serum intact fibroblast growth factor 23 and matrix extracellular phosphoglycoprotein in heritable tumoral calcinosis. J Clin Endocrinol Metab 91:4037–4042. 491. Narchi H (1997). Hyperostosis with hyperphosphatemia: Evidence of familial occurrence and association with tumoral calcinosis. Pediatrics 99:745–748. 492. Shore EM, Xu M, Feldman GJ, et al. (2006). A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva. Nature Genet 38:525–527. 493. Kaplan FS, Fiori J, Serrano de la Pena L, et al. (2006). Dysregulation of the BMP-4 signaling pathway in fibrodysplasia ossificans progressiva. Ann NY Acad Sci 1068:54–65. 494. Bowers RR, Kim JW, Otto TC, Lane MD (2006). Stable stem cell commitment to the adipocyte lineage by inhibition of DNA methylation: Role of the BMP-4 gene. Proc Natl Acad Sci USA 103:13022–13027. 495. Kaplan FS, Glaser DL, Shore EM, et al. (2007). Hematopoietic stem-cell contribution to ectopic skeletogenesis. J Bone Joint Surg 89:347–357. 496. Chan I, Hamada T, Hardman C, et al. (2004). Progressive osseous heteroplasia resulting from a new mutation in the GNAS1 gene. Clin Exper Dermatol 29:77–80. 497. Shore EM, Ahn J, Jan de Beur S, et al. (2002). Paternally inherited inactivating mutations of the GNAS1 gene in progressive osseous heteroplasia. N Engl J Med 346:99–106. 498. Shohat M, Rimoin DL (2007). The skeletal dysplasias. In Lifschitz F (ed.), Pediatric endocrinology, Fifth edition. New York: Informa Health Care 145–162. 499. Superti-Furga A, Unger S, and the Nosology Group of the International Skeletal Dysplasia Society (2007). International nosology and classification of genetic skeletal disorders: 2006 revision. Am J Med Genet 143A:1–18 (www.isds.ch). 500. Brook CGD, de Vries BBA (1998). Skeletal dysplasias. Arch Dis Child 79:285–289. 501. Horton WA (2006). Molecular pathogenesis of achondroplasia. Growth Genet Horm 22:49–54. 502. Heuertz S, Le Merrer M, Zabel B, et al. (2006). Novel FGFR3 mutations creating cysteine residues in the extracellular domain of the receptor cause achondroplasia or severe forms of hypochondroplasia. Eur J Hum Genet 14:1240–1247. 503. Saito H, Sekizawa A, Morimoto T, et al. (2000). Prenatal DNA diagnosis of a single gene disorder from maternal plasma. Lancet 356:1170–1171. 504. Toydemir RM, Brassington AE, Bayrack-Toydemir P, et al. (2006). A novel mutation in FGFR3 causes camptodactyly, tall stature, and hearing loss (CATSHL) syndrome. Am J Hum Genet 79:935–941. 505. Pitteloud N, Acierno JS Jr., Meysing A, et al. (2006). Mutations in fibroblast growth factor receptor 1 cause both Kallmann syndrome and idiopathic hypogonadotropic hypogonadism. Proc Natl Acad Sci USA 103:6281–6286. 506. Haque MF, King LM, Krakow D, et al. (1998). Mutations in orthologous genes in human spondyloepimetaphyseal dysplasia and the brachymorphic mouse. Nature Genet 20:157–162. 507. Belin V, Cusin V, Viot G, et al. (1998). SHOX mutations in dyschondrosteosis (Leri-Weill syndrome). Nature Genet 19:67–69. 508. Benito-Sanz S, Thomas NS, Huber C, et al. (2005). A novel class of pseudoautosomal region 1 deletions downstream of SHOX is associated with Leri-Weill dyschondrosteosis. Am J Hum Genet 77:534–544.

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509. Marchini A, Marttila T, Winter A, et al. (2004). The short stature homeodomain protein SHOX induces cellular growth arrest and apoptosis and is expressed in human growth plate chondrocytes. J Biol Chem 279:37103–37114. 510. Nissenson RA (1998). Parathyroid hormone (PTH)/PTHrP receptor mutations in human chondrodysplasia. Endocrinology 139:4753–4755. 511. Thiel CT, Horn D, Zabel B, et al. (2005). Severely incapacitating mutations in patients with extreme short stature identify RNAprocessing endoribonuclease RMRP as an essential cell growth regulator. Am J Hum Genet 77:795–806. 512. Herman GE (2003). Disorders of cholesterol biosynthesis: prototypic metabolic malformation syndromes. Hum Molec Genet 12: R75–R88. 513. Opitz, JM (1999). RSH (so-called Smith-Lemli-Opitz) syndrome. Curr Opin Pediatr 11:353–362. 514. Waterham HR, Koster J, Romeijn GJ, et al. (2001). Mutations in the 3 beta-hydroxysterol delta 24-reductase gene cause desmosterolosis, an autosomal recessive disorder of cholesterol biosynthesis. Am J Hum Genet 69:685–694. 515. Waterham HR, Koster J, Mooyer P, et al. (2003). Autosomal recessive HEM/Greenberg skeletal dysplasia is caused by 3 ␤hydroxysterol ⌬14-reductase deficiency due to mutations in the lamin B receptor gene. Am J Hum Genet 72:1013–1017.

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516. Levine B-S, Carpenter TO (1999). Evaluation and treatment of heritable forms of rickets. The Endocrinologist 9:358–365. 517. Hruska KA (2006). Hyperphosphatemia and hypophosphatemia. In Favus MJ (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, Sixth edition. Washington, D.C.: American Society for Bone and Mineral Research 233–242. 518. Sillence DO, Senn A, Danks DM (1979). Genetic heterogeneity in osteogenesis imperfecta. J Med Genet 16:101–116. 519. Brown EM, Bai M, Ollak M (1997). Familial benign hypocalciuric hypercalcemia and other syndromes of altered responsiveness to extracellular calcium. In Krane SM, Avioli LV(eds.), Metabolic bone diseases, Third edition. San Diego: Academic Press 479–499. 520. Kitanaka S, Murayama A, Sakai T, et al. (1999). No enzyme activity of 25-hydroxyvitamin D3 1␣-hydroxylase gene product in pseudovitamin D deficiency rickets, including that with mild clinical manifestation. J Clin Endocrinol Metab 84:4111–4117. 521. Kim CJ, Kaplan LE, Perwad F, et al. (2007). Vitamin D 1␣hydroxylase gene mutations in patients with 1␣-hydroxylase deficiency. J Clin Endocrinol Metab 92:3177–3182.

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C H A P T E R

18 Autoimmune Polyglandular Syndromes MICHAEL J. HALLER, MD • WILLIAM E. WINTER, MD • DESMOND A. SCHATZ, MD

Introduction Mechanisms Underlying Generation of Autoimmunity Introduction Central T-Cell Tolerance Peripheral T-Cell Tolerance B-Cell Tolerance Autoimmune Diseases Defects in Tolerance That Cause Autoimmune Diseases Classification of the Autoimmune Polyglandular Syndromes Clinical Aspects APS I APS II IPEX

Introduction The autoimmune polyglandular syndromes (APS) I and II are uncommon constellations of organ-specific autoimmune diseases characterized by the occurrence of more than one autoimmune disease in an affected individual. More commonly, autoimmune disease of endocrine glands occurs in only a single organ. However, multiorgan involvement of endocrine and nonendocrine organs and tissues may be present. Tolerance is an active state in which the immune system does not mount a reaction against self-antigens.1-3 If tolerance is not established or is lost, autoimmunity and subsequent autoimmune disease can result. Although the breakdown in self-tolerance remains unexplained in most human autoimmunities, our improved understanding of normal

Diagnostic Approach and Follow-up Treatment Genetics of APS I Genetics of APS II Autoantibodies in Autoimmune Polyglandular Syndromes Adrenal Cytoplasmic Autoantibodies Adrenal Enzyme Autoantibodies Adrenal Enzyme Autoantibodies in APS I Adrenal Enzyme Autoantibodies in APS II Adrenal Enzyme Autoantibodies in Isolated Addison Disease Steroidal Cell/Gonadal Autoantibodies Autoantibodies in Hypoparathyroidism Other Autoantibodies in APS I and II Summary

immunologic processes has identified a number of possible mechanisms. To comprehend these mechanisms, a brief overview of how tolerance is maintained is essential.

Mechanisms Underlying Generation of Autoimmunity INTRODUCTION In a normal immune response, the host organism must differentiate self from non-self, generate an immune response to non-self, and eliminate non-self to protect the host from injury, organ dysfunction, and even death.1-3 Endogenous antigens represent self, whereas exogenous antigens represent non-self. The adaptive immune system assumes that

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all exogenous (foreign) antigens are potentially harmful and acts to eliminate non-self. Self/non-self discrimination is carried out by the adaptive (specific) immune system through the use of T- and B-cell surface receptors.4 These receptors recognize distinctive peptides or epitopes and are the keys to the specificity of the adaptive immune response. Whereas B cells and their receptors recognize soluble antigen or antigens on cell surfaces, T cells and their receptors only perceive short polypeptides presented by specialized cell-surface molecules encoded by the major histocompatibility complex (MHC).5-7 The human MHC is termed the human leukocyte antigen (HLA) complex. Class I MHC (e.g., HLA-A, HLA-B, and HLA-C molecules) present peptides derived from the cell cytoplasm to CD8 T cells, whereas class II MHC (e.g., HLA-DP, HLA-DQ, and HLA-DR molecules) present peptides derived from the extracellular space and intravesicular space to CD4 T cells. Regulation of self-tolerance occurs at two major levels. First, through a process of negative selection thymic medullary epithelial cells project an immunologic “self-shadow” and signal the removal or silencing of self-reactive thymocytes (central tolerance).3,8 Negative selection occurs when double positive (CD4⫹, CD8⫹) alpha/beta T cells bind too tightly to dendritic cells and/or macrophages at the corticomedullary border in the thymus and are triggered to undergo apoptosis. Second, in lymphoid and non-lymphoid tissues mature self-reactive T cells are deleted or anergized (peripheral tolerance) when their receptors engage peptides plus MHC molecules in the absence of appropriate co-stimulatory molecules (Figures 18-1 and 18-2). Nevertheless, recent data have demonstrated that proteins Development of Tolerance to Self-Antigens Bone marrow Peripheral tolerance Thymus

Anergy

Self-AG Self-AG Self-AG T cells 99% Death Central tolerance

1%

Nonself antigen Immune response

Figure 18-1 Normal tolerance pathways. T-cell precursors initially arise in the bone marrow. These progenitors enter the thymus and encounter self-antigen. Strong self-antigen stimulation of developing T cells induces apoptosis, with approximately 99% of all developing T cells dying. This is central immunologic tolerance, where strongly anti-self T cells are eliminated. Naive T cells that do leave the thymus can be subsequently tolerized to self-antigens if they encounter self-antigen without the normal co-stimulatory signals (B7-CD28; see Figure 18-3). Induction of tolerance outside the thymus is termed peripheral tolerance (top right), which is a complementary mechanism to central tolerance. Peripheral tolerance is functionally expressed as anergy: autoreactive cells are present but are inactive (top right). If non-self antigen is encountered, a normal immune response ensues (bottom right).

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Autoimmunity: Failure of Tolerance to Self-Antigens Thymus

Peripheral tolerance

Self-AG

T cells

Self-AG Failure

Self-AG Failure Central tolerance

Anergy

Self-AG Anti-self lives

Autoimmunity

Figure 18-2 Autoimmunity: failure of tolerance to self-antigens. With a failure of central tolerance (bottom left), anti-self T cells survive that should not normally survive. When such anti-self T cells leave the thymus, autoimmunity may result (dashed arrows). Alternatively, with a failure of peripheral tolerance (top right) if anergy (solid arrow) does not occur after contact with self-antigen, an autoimmune response can occur (red arrow).

previously thought to be expressed only in peripheral tissues are expressed in the developing thymus and therefore play a critical role in establishing tolerance.9 Although many of the mechanisms involved in establishing tolerance remain poorly understood, recent characterization of the autoimmune regulatory gene (AIRE) has improved understanding of positive and negative T-cell selection. AIRE plays a critical role in the expression of peripheral antigens and provides for the negative selection of autoreactive T-effector cells.10 Deletions in the mouse AIRE homologue result in multiorgan autoimmunity, and mutations in the human AIRE gene result in APS I.11,12 Tolerance is initially developed in utero. During gestation and early life, tolerance is most easily induced by exposure of the host to a specific antigen. Although the thymus atrophies during puberty, residual thymic tissue provides for T-cell development throughout life. T cells do not require exposure to large doses of antigen to achieve tolerance during their thymic development. However, larger doses of antigen are required to induce B-cell tolerance (which is often short lived). B cells are produced by the bone marrow continuously throughout life. Tolerance is immunologically specific, learned or acquired, most easily induced in immature or developing lymphocytes, and potentially induced in mature lymphocytes when co-stimulatory signals are absent at the time of peptide recognition by the T lymphocyte.

CENTRAL T-CELL TOLERANCE T cells are educated to distinguish self and non-self in the thymus.13-15 Central T-cell tolerance is the process by which anti-self T cells are eliminated in the thymus. As many as 99% of developing thymocytes die in the thymus and never reach the periphery. T-cell tolerance is a function of the selection of the T-cell receptor (TCR) repertoire that exits the thymus. In the thymic cortex CD4⫹, CD8⫹ (double positive) T cells bearing alpha/beta TCRs

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that bind to self-MHC initially survive (positive selection). In this way, the thymus initially chooses for survival T cells that bind to self-MHC—as opposed to TCRs that might bind to other self-molecules, leading to ineffective communication. This positive selection resulting from self-peptide-MHC presentation is carried out by thymic nurse epithelial cells in the cortex. At the corticomedullary junction of the thymus, if such saved TCRs bind to self-MHC too tightly autoreactivity is possible and these T cells then undergo negative selection and suffer apoptotic death. The cells inducing negative selection are macrophages and dendritic cells. This process of positive selection for MHC binding in the thymic cortex and negative selection (at the thymic corticomedullary border) against tight binding to self-peptides accounts for central (thymic) immunologic tolerance.3,8,10

PERIPHERAL T-CELL TOLERANCE Once in the circulation and in secondary lymphoid organs (e.g., lymph nodes and spleen), naive T cells still require multiple signals to become activated.16 The initial signal is the presentation of antigen-specific peptides to TCRs by MHC molecules. CD4 and CD8 molecules on these T-cell subsets serve as antigen-nonspecific co-receptors binding to nonpolymorphic portions of the class II MHC molecules and class I MHC molecules, respectively. The second signal is antigen nonspecific and is provided by the B7 molecule of the antigen-presenting cell interacting with the CD28 molecule on the T-cell surface. When both signals are perceived by the T cell, a cascade of intracellular signaling events occurs—leading to T-cell activation. Activated CD4 T cells express cytokines (including IL-2, IL-4, IL-5, and so on), cytokine receptors, and CTLA-4. Activated CD4 T cells then down-regulate TCR expression and acquire class II MHC expression. It is unknown why activated human T cells express class II MHC (activated T cells in mice do not express class II MHC). CTLA-4 expression by the activated T cell and its interaction with B7 provides an immunosuppressive signal to the T cell down-regulating the T-cell immune responses. Thus, CTLA-4 and CD28 act antithetically: B7-CD28 turns on T cells, whereas B7-CTLA-4 down-regulates them (Figure 18-3). Functionally, helper CD4 T cells are predominantly subdivided into Th1 cells (which activate cell-mediated and some antibody responses) and Th2 cells, which predominantly activate antibody-mediated responses.17 Th1 cells can activate macrophages, natural killer cells, and B cells—and they secrete predominantly IL-2, gamma interferon (IFN-␥), and IL-12. Th2 cells elaborate IL-4, IL-5, IL-6, and IL-10. Cross talk between Th1 and Th2 cells occurs: IFN-␥ from Th1 cells suppresses Th2 cells and IL10 from Th2 cells inhibits Th1 cells. Another subset of CD4 T cells has been described as regulatory T cells that secrete the immunomodulatory cytokines IL-10 and/or transforming growth factor-beta (TGF-beta). Regulatory T cells include CD4⫹CD25⫹ T cells and Tr1 and Th3 cells. Tr1 and Th3 cells express CD4 but do not express CD25 (the IL-2 receptor alpha chain). Tr1 cells secrete IL-10 and TGF-beta, whereas Th3 cells secrete IL-4, IL-10, and TGF-beta. Although CD4⫹CD25⫹ T cells can

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Role of B7, CD28, and CTLA-4 in T-Cell Activation Activated APC B7

Up-regulated T cell CD28

MHC T cell Antigen receptor Down-regulated Activated APC CTLA-4 CD-28 T cell B7

MHC T cell Antigen receptor

T cell proliferation and differentiation

T cell downregulation and inactivation

Figure 18-3 Role of B7, CD28, and CTLA-4 in T-cell activation. An activated antigen-presenting cell (APC) presents antigen on the multiple histocompatibility complex (MHC) and expresses B7 costimulators. When B7 is bound by CD28 and MHC is bound by a T-cell receptor, T-cell proliferation and differentiation of naive T cells ensues. Conversely, an activated T cell will now express CTLA-4 and bind to B7—thereby inducing down-regulation and inactivation of T cells.

secrete IL-10 and TGF-beta, their regulatory action on autoreactive T cells appears to occur through cell-to-cell contact. Upon activation, CD8 T cells (often with the help of Th1 cells supplying IFN-␥ to up-regulate B7 expression on antigen-presenting cells) become functional cytotoxic T killer cells. The requirement for two signals to activate naive T cells accounts for peripheral T-cell tolerance. When the naive T cell perceives antigen peptide presented by MHC molecules without the necessary co-stimulatory signal (e.g., B7-CD28), the T cell becomes unresponsive. This state of unresponsiveness is termed anergy. The T cell may also undergo apoptosis (programmed cell death) to be completely removed from the T-cell repertoire. Anergic T cells can generally not be restimulated with antigen peptide displayed by the antigen-presenting cell. Tolerance may also exist because the TCR does not come into contact with the relevant peptide. This has been termed T-cell ignorance. Improvements in the characterization of regulatory T cells have recently allowed for further understanding of the peripheral tolerance pathway. Regulatory T cells play a critical role in suppressing the activity of effector T cells that escape negative selection to self-antigen in the thymus.18 Functional regulatory T cells are able to anergize previously self-reactive T effector cells, resulting in improved tolerance to self. The expression of the forkhead transcription factor, FOXP3, is specific for identification of the CD4⫹CD25⫹ cell (Treg) population. First identified in the Scurfy mouse, a mouse model of immune dysfunction and polyendocrinopathy, abnormal FOXP3 expression is now known to be responsible for the failure in tolerance in humans affected with a similar polyendocrinopathy (discussed in material following).19,20 The absence of normal FOXP3 expression in humans leads to an extremely rare recessively inherited (yet X-linked) and typically fatal autoimmune lymphoproliferative disease

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known as IPEX (immune dysregulation, polyendocrinopathy, enteropathy, and X-linked inheritance). Defects in the forkhead transcription factor responsible for IPEX map to Xp11.23-Xq13.3.

B-CELL TOLERANCE B cells are partially educated in the bone marrow to be anergized or undergo apoptosis in response to self-antigen when they are at the stage of development of the naive immature B cell (central B-cell tolerance). In the bone marrow, naive immature B cells that see multivalent antigens become anergized (unresponsiveness to subsequent stimulation)—whereas exposure to highly polyvalent antigens can induce apoptosis. Naive immature B cells express IgM on their surface but are not yet IgD positive, as observed in naive mature B cells. Anergized B cells do not instantly die, but will live no longer than unstimulated naive immature B cells. Naive mature B cells expressing IgD on their surface require T-cell help for realization of their full potential through affinity maturation and class switching. The absence of T-cell help leads to B-cell tolerance.

AUTOIMMUNE DISEASES The organ-specific nature of many autoimmune diseases results from abnormal immune system recognition of tissue-specific self-antigens. In many autoimmune endocrinopathies, the target molecule is a tissue-specific or tissue-limited (i.e., the protein is not unique to one tissue but is clearly restricted in its distribution) enzyme or cellsurface receptor21,22 (Table 18-1).

TA B L E 1 8 - 1

Autoantigens in Autoimmune Endocrine and Associated Diseases Disease Addison disease Hashimoto thyroiditis Graves’ disease Diabetes

Premature gonadal failure Pernicious anemia Myasthenia gravis Vitiligo

Celiac disease

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Confirmed Autoantigens

Putative Autoantigens

P450c21 Thyroid peroxidase Thyroglobulin Thyrotropin receptor Insulin Glutamic acid decarboxylase-65 IA-2 (ICA 512) IA-2␤

P450c17, P450scc

Proinsulin Carboxypeptidase H ICA69 Glima 38 P450c17 P450scc 3BHSD

H⫹/K⫹ ATPase Intrinsic factor Acetylcholine receptor

Transglutaminase

Tyrosinase Tyrosinase-related protein-2 Reticulin Gliadin Endomyseum

773

The criteria for classification of a disease as autoimmune are not universally agreed upon.23 However, major criteria generally accepted as strong evidence that the disease is autoimmune include detection of autoantibodies or autoreactive T cells, including lymphocytic infiltration of the targeted tissue or organ; disease transfer with antibodies or lymphocytes; disease recurrence in transplanted tissue; and ability to abrogate the disease process with immunosuppression or immunomodulation. Few, if any, human autoimmune diseases meet all four of these criteria. Further information supportive of (but not diagnostic for) an autoimmune disease includes increased disease frequency in women compared to men, presence of other autoimmune diseases in affected individuals, and increased frequencies of particular HLA alleles in affected individuals compared to an unaffected control population.

DEFECTS IN TOLERANCE THAT CAUSE AUTOIMMUNE DISEASES Several hypotheses explaining defects in tolerance have been proposed.24 Theoretically, autoimmunity may develop because tolerance never developed to specific selfantigens or because established tolerance was lost. If selfantigen is not efficiently presented in the thymus, tolerance may not be established during T-cell education within the thymic cortex.25 For example, variations in the insulin gene VNTR (variable number of tandem repeats; ⬃500 base pairs upstream of the insulin gene promoter) influence the extent of insulin gene expression in the thymic cortex. Risk of developing type 1 diabetes is enhanced when certain VNTR alleles are present, which leads to lower mRNA expression of insulin in the thymus. Specifically, class III alleles (greater number of VNTR) are associated with increased thymic expression of insulin and a decreased risk of developing type 1 diabetes—whereas class I alleles (smaller number of VNTR) are associated with decreased thymic expression of insulin and an increased risk of developing diabetes.26 If there is failure to delete an autoreactive clone of T cells, autoimmunity could develop. If autoimmunity does result from defects in thymic tolerance, the defects must be antigen specific because organ-specific autoimmune diseases are usually extremely selective. For example, in type 1 diabetes the beta cells are attacked and ultimately destroyed by a cell-mediated autoimmune process but the remaining islet cells (including alpha cells, delta cells, and pancreatic polypeptide-producing cells) are unscathed. Defects in peripheral tolerance could result from concurrent T-cell stimulation by self-antigen/MHC plus T-cell co-stimulation (e.g., B7-CD28), leading to aberrant T-cell activation and an autoimmune response. If tolerance has not been developed because an antigen is sequestered intracellularly or is not expressed in the thymus during T-cell ontogeny, T-cell reactivity in the periphery would not be abrogated. However, several antigens initially thought to be sequestered intracellularly have now been shown to circulate in low concentrations in normal individuals. Thyroglobulin is such a self-antigen in autoimmune thyroid disease.

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The development of thyroglobulin autoantibodies was believed to follow the release of thyroglobulin from the thyroid gland as part of viral attack, trauma, or some other form of environmental damage. Hypothetically, this immunization then leads to an antithyroglobulin humoral response and autoimmune thyroid disease. However, we now know that thyroglobulin does circulate in low but appreciable quantities in normal individuals who show no serologic evidence of thyroid autoimmunity. Furthermore, thyroid follicular cell destruction in Hashimoto thyroiditis is cell mediated (not humorally mediated). If sequestered antigens do play a role in autoimmune disease, viral infections, trauma, ischemia, or irradiation are all mechanisms that could disturb cellular integrity and lead to release of intracellular antigens.27 Some selfantigens may never normally come into contact with the immune system unless there is a breakdown of anatomic barriers within the body. An example is the occurrence of autoimmunity to the eye following orbital trauma. Although a rare consequence of orbital damage, initiation of an autoimmune response to eye proteins in adjacent lymph nodes can generate autoreactive T cells that can invade and damage the contralateral eye (sympathetic ophthalmia).28 Removal of the inciting damaged tissues and immunosuppression may be required to sustain vision in the undamaged eye. Similarly, transient autoantibody reactivity to cardiac myosin following myocardial infarction has also been described.29 Hypothetically, tolerance may not develop if self-antigen expression is delayed during negative selection. When the self-antigen is ultimately expressed, if tolerance has not previously been established the autoantigen is perceived as foreign and autoreactivity develops. No spontaneous examples of this process have been described. However, in experimental systems in which transgenes are placed under control of promoters that can be turned on by exogenous agents such as metals, autoreactivity can be elicited when gene expression is stimulated after the neonatal period. Alteration of self-antigens as a result of infection or neoplasia is believed to be a plausible theory explaining some types of autoimmunity. As environmental triggers, viral infections could lead to modification of self-proteins and neoantigen expression (e.g., a new antigen is present on self-cells). Alternatively, a self-antigen may be partially degraded—leading to a “new” antigenic target for the adaptive immune system. This new antigen is recognized as foreign by the immune system, and the immune response to these new antigens results in autoimmunity. Some self-cells/tissues may suffer unintended autoimmune damage when substances bind to the cells and elicit an initial immune response. For example, certain drugs bind to red blood cells and result in an immune hemolytic anemia. If an antibody response to the red-cell-bound drug is elicited, the antigen-antibody complex present on the red blood cell can lead to red blood cell destruction. This can occur through red blood cell phagocytosis by the monocyte-macrophage system or via complement-mediated lysis of the red blood cell. Thus, the red blood cell is an innocent bystander to the antidrug humoral immune response. Theoretically, this could also occur with viruses that serendipitously attach to tissues.

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Molecular mimicry is one of the most popular explanations for autoimmunity.27 Due to exposure to a dietary, viral, or bacterial antigen (e.g., infection) and molecular mimicry (similarity) between the self-antigen and the foreign antigen, the immune response to the foreign antigen leads to cross reactivity with self-antigen, autoimmunity, and disease.30-32 For this theory to work, tolerance must not previously exist to the self-antigen. This might be true if the self-antigen is truly sequestered and the immune system has never developed tolerance to the self-antigen. Alternatively, the self-antigen peptides may be present in too low a concentration to elicit an immune response and initial immune system tolerization has not occurred. Only with infection or novel dietary exposure would there be a sufficient degree of self-immunization to develop immune autoreactivity. With immune autoreactivity, self is now recognized as foreign during the response to the cross-reactive pathogen. If the self-antigen is a cell surface antigen, the “pathogen-induced” autoantibodies could fix to self and produce disease via complement fixation—or the antibodies could act as opsonins for fixed or circulating phagocytes (antibody-dependent-cell cytotoxicity). In rheumatic fever, cross reactivity between Streptococcus M protein and cardiac myosin have been described. In ankylosing spondylitis, cross reactions between Klebsiella nitrogenase and HLA-B27 have been described. In rheumatoid arthritis there is cross reactivity between cartilage protein and a mycobacterial proteoglycan wall component. Aberrant class II MHC expression was a theory in vogue in the mid 1980s. In this hypothesis, cells elicit autoimmune reactions by presenting their own self-peptides via selfexpressed class II MHC molecules. Indeed, class II MHC expression has been identified on various cells that are targets of autoimmune-mediated cell destruction. Examples include pancreatic islet beta cells in type 1 diabetes, biliary tract cells in primary biliary cirrhosis, and thyroid follicular cells in Hashimoto thyroiditis. However, there are strong counterarguments to this theory. First, class II MHC molecules do not present intracytoplasmic antigens (which are often targets of attack). Instead, class II MHC molecules present peptides derived from extracellular proteins. Second, accessory molecules are typically needed to activate naive T cells. If, for example, pancreatic beta cells present peptides via their class II MHC molecules without B7 the naïve T cells seeing these peptides in the absence of B7 will actually be tolerized. Indeed, aberrant (or ectopic) class II MHC expression may be a mechanism cells use to actually induce a state of tolerance to down-regulate an immune response. Aberrant class II MHC expression may therefore serve an anti-inflammatory role in modulating an immune response to a lower level of intensity. Costimulator expression (e.g., B7) is highly regulated even among professional antigen-presenting cells. For example, in the basal state neither macrophages nor B cells express B7. Upon phagocytosis of bacteria species, macrophages will express class II MHC and B7. Whereas B cells express class II MHC in their basal state, internalization of bacterial antigen bound to their cell surface receptor antibody molecules will induce B7 expression.

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Some cases of autoimmunity may result from superantigens initiating an anti-self immune response as part of the polyclonal immune activation process. Superantigens are polycolonal T-cell stimulators that have the ability to cross-link TCR beta chains and MHC molecules. Superantigens have been reported to activate as many as a third of all T cells in the body. In such cases, systemic disease can develop from massive cytokine release. This is the case in toxic shock syndrome, wherein a staphylococcal exotoxin acts as a superantigen. Mycobacterial antigens have been promulgated as possible superantigens in Crohn disease.33 This theory presuposes that T cells bearing anti-self TCRs have not been deleted or permanently anergized. T cells with anti-self receptors may be stimulated, and if they encounter self-antigen they may further proliferate to develop an autoimmune response. Similar to polyclonal T-cell activation, polyclonal B-cell stimulation has also been implicated in humoral autoimmunity. Indeed, autoreactive B cells can be found in normal individuals. If an autoreactive clone of B cells encounters self-antigen and a co-stimulator (which might be nonspecific, such as Epstein Barr virus or a bacterial product such as lipopolysaccharide), autoantibodies could be produced—bypassing the need for T-cell help. However, which of these speculative theories applies to the APS is unknown. Human disease most often results from an interaction of environmental and genetic factors.34 Many environmental factors are implicated in various autoimmune diseases: wheat gliadin ingestion and celiac disease, penicillamine exposure and myasthenia gravis, methimazole and autoimmune hypoglycemia from insulin autoantibodies, and amiodarone and thyroiditis. Cytokine exposure is also associated with autoimmune disease in the case of alpha interferon use in hepatitis and the apparent development of thyroiditis. Even cancer can be associated with the development of autoimmunity: thymoma and myasthenia gravis are associated, as are ovarian carcinoma and cerebellar degeneration, and breast cancer and stiffman syndrome. How this interaction among genes, the environment, and the immune system actually leads to autoimmunity and disease clearly needs to be elucidated.

Classification of the Autoimmune Polyglandular Syndromes APS I, also known as APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy), is an autosomal-recessive disorder mapped to a single gene (the autoimmune regulator or AIRE gene) on chromosome 21q22.3.11,12 The presence of two of the following three conditions are prerequisites for diagnosis: adrenocortical failure (Addison disease) or serologic evidence of adrenalitis (e.g., adrenal autoantibodies), hypoparathyroidism, and mucocutaneous candidiasis.35-37 APS II is defined by the coexistence of autoimmune adrenocortical insufficiency or serologic evidence of adrenalitis with autoimmune thyroiditis (Schmidt syndrome) and/or type 1 diabetes mellitus (Carpenter syndrome, which is Schmidt syndrome plus type 1 diabetes) or serologic evidence of thyroid or islet autoim-

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APS I

Hypoparathyroidism

Autoimmune hepatitis

775

APS II

Gonadal failure

Autoimmune thyroid disease

Addison disease

Chronic mucocutaneous candidiasis

Type 1 Diabetes

Pernicious anemia

Diagnostic relationship Association

Figure 18-4 Diagnostic relationship and common associations of APS I and APS II. The solid lines indicate diagnostic relationships. The dashed lines indicate common associations. The diagnosis of APS I depends on the coexistence of Addison disease (or adrenal autoantibodies) and hypoparathyroidism or chronic mucocutaneous candidiasis, or both. The diagnosis of APS II depends on the coexistence of Addison disease (or adrenal autoantibodies) and autoimmune thyroid disease or type 1 diabetes, or both (or their associated autoantibodies).

munity38-41 (Figure 18-4). The presence of thyroiditis without adrenal disease but associated with type 1 diabetes mellitus, pernicious anemia, vitiligo, or alopecia has been referred to as APS III. However, because APS II and III differ only by the presence or absence of adrenocortical disease and share similar susceptibility genes and immunologic features we do not recognize APS III as a unique syndrome and consider it an extension of the APS II constellation.

Clinical Aspects APS I The major disease components, frequencies, and differences between APS I and II are outlined in Table 18-2. APS I (APECED) may occur sporadically or in families. Although the disease is not common, cohorts of patients have been reported from Finland and the United States and among Iranian Jews. Males and females are equally affected in this autosomal-recessive disorder.35,37,42 Persistent mucocutaneous candidiasis is usually the first sign, which commonly appears during the first year or two of life. However, it may also have its onset in adulthood. Candidal infections in the region of the diaper area often present early, with vulvovaginal candidiasis commonly developing at puberty in females. Colonization of the gut can lead to intermittent abdominal pain and diarrhea. The nails may be affected with chronic candidiasis, leading to a darkened discoloration, thickening, or erosion. Retrosternal pain occurring in patients with confirmed oral candidiasis suggests esophageal candidiasis and can be confirmed by esophagoscopy. It is extremely important that the candidiasis be

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TA B L E 1 8 - 2

The Autoimmune Polyglandular Syndromes I and II Disease Characteristic Comparative frequency Onset Heredity Gender Genetics Hypoparathyoidism Mucocutaneous candidiasis Ectodermal dysplasia Addison disease Type 1 diabetes Autoimmune thyroid disease Pernicious anemia Gonadal failure Females Males Vitiligo Alopecia Autoimmune hepatitis Malabsorption

APS I

APS II

Less common

More common

Infancy/early childhood Autosomal recessive Males ⫽ females

Late childhood, adulthood Polygenic

AIRE gene; no HLA association 77-89% 73-100%

Female predominance HLA associated; DR/DQ None None

77% 60-86% 4-18% 8-40%

None 70-100% 41-52% 70%

12-15%

2-25%

30-60% 7-17% 4-13% 27% 10-15%

3.5-10% 5% 4-5% 2% Rare

10-18%

Rare

aggressively treated. Carcinoma of the oral mucosa with its high mortality is well described in APS I patients with chronic mucosal candidiasis. Any patient with refractory mucocutaneous candidiasis should be thoroughly investigated not only for a T-lymphocyte abnormality (absolute lymphocyte count, enumeration of T-cell subpopulations, assessment of T-cell function) but for the presence of a polyendocrinopathy. The largest cohort of longitudinally followed APS I patients resides in Finland. In Perhentupa’s recent review of these 91 APS I patients, 60% presented with candidaisis as the first sign of APS I, 96% developed candidiasis by 20 years of age, and 100% developed candidiasis by 40 years of age.35 Addison disease is found in more than 85% of APS I patients, yet the disease is often missed—with the diagnosis commonly made late or at the time of a life-threatening adrenal crisis. Autoimmune adrenocortical failure usually has its peak onset before adolescence, when it is associated with APS I. However, Addison disease may have its onset in adulthood. Deficiencies of cortisol, aldosterone, and adrenal andogens may present simultaneously or may evolve over months to several years. The initial clinical features of Addison disease are often nonspecific mimicking psychiatric or gastrointestinal disease. These include fatigue, weight loss, myalgias, arthralgias, behavioral changes, nausea and vomiting, abdominal pain, and diarrhea. Hyperpigmentation (due to elevated ACTH) in non sun-exposed areas and postural hypotension can

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usually be found on careful examination. Unexplained hypotonic dehydration should raise the suspicion of Addison disease. Adrenal crises with hyponatremia, hyperkalemia, and hypoglycemia may be fatal unless recognized early and treated appropriately. Like other components of APS I, hypoparathyroidism usually presents before puberty. Severe hypocalcemia as evidenced by seizures, carpopedal spasms, muscle twitching, and laryngospasm may be presenting features. Hyperphosphatemia with a low intact parathyroid hormone (PTH) level is diagnostic. These symptoms may, however, be masked in the presence of adrenal insufficiency. Ectodermal dystrophy unrelated to hypoparathyoidism or to mucocutaneous candidiasis has been well characterized in the Finnish patients with APS I. Dental enamel hypoplasia of permanent (but not deciduous) teeth, as well as nail dystrophy, is commonly found. There may be complete absence of the enamel or transverse hypoplastic bands alternating with zones of well-formed enamel. Dystrophy of nails is manifest by 0.5- to 1-mm pits. Nearly a third of the Finnish patients also had calcification of the tympanic membranes.43 Premature gonadal failure is more common in females and may present in puberty. Less than 30% of men with APS I develop testicular failure, whereas more than 50% of women with APS I develop ovarian failure by 20 years of age.35 In younger female patients, gonadal failure often presents with primary amenorrhea—and in time menstrual irregularities, polycystic ovaries, or infertility may manifest.35,44 As shown in Table 18-2, in contrast to patients with APS II type 1 diabetes and autoimmune thyroiditis occur far less frequently in APS I. When present, thyroiditis is typically atrophic rather than goitrous. Atrophic gastritis occurs in 15% to 30% of APS I cases, with a mean age of onset of 16 years.35,43 Gastric-parietal cell autoimmunity, which leads to atrophic gastritis with resultant achlorhydria and intrinsic factor deficiency, typically presents as iron deficiency anemia or vitamin B12-deficient pernicious anemia. Nonendocrine organ-specific diseases include alopecia, vitiligo, autoimmune hepatitis, and malabsorption. All types of alopecia may occur, but progression to alopecia totalis (total loss of scalp hair) or universalis (total loss of all body hair including eyelashes, eyebrows, and scalp hair)—which are most common—usually occurs before puberty. Vitiligo presents initially as small pale pigment-lacking skin patches. These may be missed unless specifically sought with ultraviolet light examination of the skin. The appearance of clay-colored stools, dark urine, and jaundice confirms the diagnosis of chronic active hepatitis—which is not related to infectious hepatitis. Hepatitis occurs in 10% to 15% of APS I patients and is the leading cause of death in these patients. Consequently, all patients suspected of having APS I should have their liver function regularly monitored. Intermittent malabsorption (typically of fat) has been linked to hypoparathyroidism, bacterial and fungal overgrowth, gluten sensitivity, and IgA deficiency. There have also been rare reports of APS I and associated diabetes insipidus, growth hormone deficiency, ACTH deficiency, rheumatoid arthritis, Sjögren’s disease, and myopathy.45

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APS II APS II is the most common of the polyendocrinopathies, and unlike APS I usually has its onset in adulthood— particularly during the third or fourth decades. APS II is at least three times more common in females than in males. In 1926, Schmidt first described the association of adrenocortical and thyroid gland failure—and Carpenter extended this in 1964 to include insulin-dependent diabetes mellitus.38,39 In 1957, the autoimmune nature of these diseases was suggested by Doniach and Roitt’s discovery of thyroglobulin autoantibodies in patients with Hashimoto thyroiditis.46 APS II was originally defined by the occurrence of adrenocortical insufficiency (Addison disease) in conjunction with autoimmune thyroid disease and/or type 1 diabetes mellitus. Adrenocortical failure is the presentation in approximately 50% of APS II cases. The disease usually has its onset between ages 20 and 50 years, although it is not unusual to find cases before or after these ages.41,47,48 Several of the disease components may be present at diagnosis. Type 1 diabetes coexists in nearly 50% of patients with Addison disease, whereas autoimmune thyroid disease (AITD) coexists in about two-thirds of patients with Addison disease. Thus, type 1 diabetes and AITD should be vigorously sought in any patient presenting with Addison disease. The most common component of the APS II to occur as an isolated condition is AITD. AITD affects nearly 4.5% of the U.S. population,49 and because of a strong female predominance 80% to 90% of all cases occur in females. AITD has an increased incidence during the teen years, with a peak appearing in the fifth and sixth decades. Chronic lymphocytic thyroiditis (Hashimoto disease) is by far the most common form of AITD, although Graves disease or postpartum thyroiditis may also occur. Several studies have reported on the coexistence of anti-islet immunity (3%-8%) or even overt type 1 diabetes and AITD.50,51 Just 1% of patients with otherwise isolated thyroiditis have serologic evidence of adrenal autoimmunity. Although polyglandular involvement in patients with autoimmune thyroid disease is infrequent, thyroid autoimmunity or a family history of thyroiditis is common in patients with pernicious anemia, vitiligo, alopecia, myasthenia gravis, and Sjögren syndrome.52-54 More patients with APS I than APS II have vitiligo, but because APS I is far less common most patients with vitiligo who have another autoimmune disease have APS II. Twenty to 40% of vitiligo patients have another component of APS II, with thyrogastric autoimmunity being the most common.55,56 Thyrogastric autoimmunity is a descriptive term for the high frequency of concurrent AITD and autoimmune lymphocytic gastritis. Many of these patients with vitiligo are asymptomatic, and evidence of their autoimmunity can only be ascertained by autoantibody screening. Segmental vitiligo with involvement of dermatomal regions is not associated with autoimmunity, however.57 Up to 15% of patients with alopecia (areata, totalis, universalis) and 5% of their first-degree relatives have thyroid disease. Nearly 30% of patients with myasthenia gravis (autoimmune disease characterized by muscle weakness wors-

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ening during muscular contraction and caused by antiacetylcholine receptor autoantibodies) have AITD. Both Hashimoto thyroiditis and Graves disease may occur in patients affected with myasthenia gravis.58,59 Interestingly, these subjects with AITD appear more likely to have a milder expression of their myasthenia gravis and a lower incidence of thymic disease or autoantibodies to the acetylcholine receptor ␣-chain than AITD-negative patients. The proportion of ocular myasthenia is higher in patients with Graves disease. Type 1 diabetes mellitus is also a diagnostic component of APS II. The worldwide incidence of type 1 diabetes continues to rise, particularly in children less than 5 years of age. The disease has a peak incidence during the teen years, with a smaller but increasing incidence occurring in the preschool years.60 Nevertheless, the disease may have its onset at any age. Approximately 10% to 15% of diabetes patients with disease onset after 40 years actually have slowly progressive autoimmune disease [latent autoimmune diabetes of adults (LADA)], rather than insulin-resistant forms of diabetes (classic type 2 diabetes).61 A gender bias is not present in patients with isolated type 1 diabetes. However, a female predominance occurs in APS II patients whose disease constellation includes type 1 diabetes. This female bias in APS II is almost certainly related to the coexistence of AITD. The presence of thyroid microsomal (thyroid peroxidase) and/or thyroglobulin autoantibodies documents thyroiditis (often asymptomatic) in 20% to 25% of females with type 1 diabetes.47 A female bias is also found in APS II patients with type 1 diabetes and gastric parietal cell autoimmunity. Gastric parietal cell autoantibodies (PCA) are present in approximately 10% of females and 5% of males with type 1 diabetes.62 Although pernicious anemia typically affects women after the fifth decade, children at increased risk for pernicious anemia should be closely monitored when PCA are detected. Atrophic gastritis may lead to the development of a megaloblastic anemia due an inability to produce intrinsic factor, and consequently to an inability to absorb vitamin B12. Iron deficiency anemia secondary to an inability to absorb iron consequent to decreased acid production (achlorhydria) has also been reported in adolescents and adults.63 Adrenocortical autoimmunity is much less frequent among patients with type 1 diabetes, with serologic evidence reported in 1.5% of cases.64,65 Antibodies suggestive of celiac disease are present in up to 10% patients with type 1 diabetes. Endomysial or transglutaminase autoantibodies can be used to screen for celiac disease.66 Although celiac disease should be suspected in patients with unexplained diarrhea, weight loss, failure to gain weight, or failure to thrive, biopsy-proven celiac disease is seen in less than 6% of type 1 diabetes patients.67,68 Approximately 10% of women with APS II who are less than 40 years of age develop ovarian failure presenting as primary or secondary amenorrhea. A female sex bias also occurs in the relationship between adrenocortical and gonadal autoimmunities. Among females with biopsy-proven lymphocytic oophoritis, adrenocortical failure or subclinical adrenal autoimmunity is often present.69 Progression to gonadal failure is very rare among

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males with Addison disease, even in the presence of the high-risk steroidal cell autoantibodies (SCA) that mark the disease in women. Pituitary involvement is occasionally seen in APS II.70,71 Hypophysitis and empty sella syndrome have been described, usually leading to isolated failure of secretion of GH, ACTH, TSH, FSH, or LH. Several nonendocrinologic conditions have been reported in association with APS II. These include ulcerative colitis,72 primary biliary cirrhosis,73 sarcoidosis,74,75 achalasia,76 myositis,77 and neuropathy.78

IPEX In addition to the classic diagnoses of APS I and II, an improved understanding of abnormal FoxP3 expression and T-cell function has led to the characterization of yet another autoimmune polyendocrinopathy. As previously stated, the absence of normal FoxP3 expression leads to the extremely rare recessively inherited X-linked and typically fatal autoimmune lymphoproliferative disease known as IPEX.79-82 Neonatal onset type 1 diabetes can be observed in IPEX, as well as dermatitis, enteropathy, thyroiditis, hemolytic anemia, and thrombocytopenia. To date, long-term immunosuppression or bone marrow transplantation appears to be the only effective therapy for IPEX. Bone marrow transplantation may be effective due to the normalization of FOXP3 expression in T cells even in patients with only partial chimerism. Increased expression of FOXP3 has recently been shown to reprogram effector T cells to act as regulatory T cells.83,84 Thus, enhancement of FOXP3 expression to augment the number and function of regulatory T cells has significant potential to treat human autoimmune diseases.

Diagnostic Approach and Follow-up The approach to diagnosing polyglandular syndromes is threefold. First, autoantibody screening is used to verify the autoimmune nature of the suspected endocrinopathy and to test for the involvement of other organs and tissues. Second, a full assessment of endocrine function is required in patients with confirmed autoantibodies—as well as in those who may be autoantibody negative but in whom disease may be suspected clinically. Third, mutation analysis can now be used to confirm the diagnosis and to screen siblings and other relatives for their carrier status. Recognition of multiorgan autoimmune diseases prior to their symptomatic phases is the best way to minimize their associated morbidity and mortality. A thorough history and physical examination should always be performed, and a high index of suspicion should be maintained. Another clue to the identification of asymptomatic polyglandular disease may come from the history of a relative who has a different component disease than the proband or has typical multiorgan disease. Any patient with suspected APS should be screened with a panel of autoantibodies (Figure 18-5). These include adrenal cortex cytoplasmic autoantibodies or autoantibodies directed against 21 hydroxylase (markers for autoimmune Addison disease); GADA, IA-2A, IAA, and ICA (for type 1 diabetes); thyroid microsomal/thyroperoxidase and thyroglobulin autoantibodies (for autoimmune thyroid disease); steroidal cell autoantibodies (for ovarian failure); and endomysial or transglutaminase autoantibodies (for celiac disease). Other autoantibodies have been reported in autoimmune adrenal disease and/ or gonaditis, such as those against 17-hydroxylase, the

Figure 18-5 Antibody and end-organ testing in patients with suspected APS. This flow diagram shows which autoantibodies should be obtained when APS is suspected due to clinical signs and symptoms. Following autoantibody positivity, annual testing of end-organ function should be obtained as shown.

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autoantibodies is recommended annually (Figure 18-5). Fasting and/or 2-hour postprandial blood glucose testing and testing of calcium, phosphate, and PTH and TSH levels can effectively assess pancreatic islet, parathyroid, and thyroid function in asymptomatic individuals. Gonadal failure can be diagnosed by the finding of elevated FSH and LH levels with concomitant low sex steroids. Obtaining a hemoglobin and hematocrit together with indices can assess progression to atrophic gastritis in patients with gastric autoimmunity. The findings of a megaloblastic anemia with an elevated mean corpuscular volume (MCV) suggest vitamin B12 deficiency, whereas a microcytic hypochromic anemia confirms iron deficiency. However, vitamin B12 levels should be followed in all patients with parietal cell autoantibodies because neuropathy can develop without anemia. It is useful to obtain a vitamin B12 level and an iron profile prior to starting therapy. Methylmalonic acid levels are not routinely needed in patients with gastric autoimmuinity but can be helpful if the vitamin B12 levels are borderline low.86 Liver function tests and antimitochondrial autoantibodies should be obtained in patients with APS I. Patients testing positive for endomysial or transglutaminase autoantibodies should have an intestinal biopsy to confirm the diagnosis of celiac disease. Low early morning cortisol levels, electrolyte abnormalities (hyponatremia/hyperkalemia), and hypoglycemia represent late changes occurring at or just before the onset of adrenal insufficiency. Just as the natural history of pre type 1 diabetes has now been well described (see Chapter 10), there is now data to predict the subsequent development of adrenocortical insufficiency once adrenal autoantibodies are first recognized (Figure 18-6). During the development of adrenocortical insufficiency in adrenocortical autoantibody positive subjects, a regular progression of sequential findings is exhibited. Stage 1 involves increased plasma renin activity, with normal to low aldosterone. Stage 2 involves Natural History of Autoimmune Endocrinopathies Environmental Humoral trigger autoimmunity

Target gland mass

side-chain cleavage enzyme, and 3-hydroxysteroid dehydrogenase. However, clinical testing is only available for the adrenal cytoplasmic autoantibodies (as detected by indirect immunofluorscence) and 21-hydroxylase autoantibodies. There is a clear link between the presence of organ-specific autoantibodies and the presence of preexisting disease and subsequent progression to disease. However, in patients with APS the number of associated disorders that will develop and their age of appearance are clinically unpredictable. Thus, clinical long-term follow-up is necessary in both autoantibody positive and negative subjects. All patients with a single autoimmune disease must be considered at risk for other autoimmune diseases. Whether and when to screen for other autoantibodies is based on the likelihood of finding another autoimmune disease, on cost effectiveness, and on the likelihood that screening will prevent morbidity and mortality from other diseases (e.g., diabetic ketoacidosis, Addisonian crisis, or hypocalcemia with seizures) in the future. Because of the high incidence of AITD in patients with type 1 diabetes, we recommend that such patients have thyroid microsomal/thyroperoxidase and thyroglobulin autoantibodies measured biannually. This approach is preferred to assessing thyrotropin levels because autoantibody seroconversion is a much earlier event in the evolution of thyroid disease. In addition, measurement of thyroid microsomal/thyroperoxidase and thyroglobulin autoantibodies has close to 90% sensitivity. In those subjects who are positive, thyrotropin levels are measured annually. Most patients with isolated Hashimoto thyroiditis will not develop additional endocrine disease, but when they do the thyroid disease has usually been preceded by the overt failure of another gland. Screening for APS is not recommended in patients with isolated autoimmune thyroid disease. However, several reports have demonstrated an increased incidence of parietal cell autoantibodies in patients with isolated autoimmune thyroid disease.63,85 Thus, screening for parietal cell autoantibodies in children with AITD can be considered. In hypothyroid patients with confirmed APS, evidence for adrenal autoimmunity must also be sought before starting thyroid hormone replacement therapy because thyroid hormone replacement can precipitate an adrenal crisis in patients with marginal adrenocortical function (by increasing metabolism and catabolism of steroid hormones). Delayed diagnoses and even preventable deaths unfortunately still occur in patients with undiagnosed adrenocortical failure. As mentioned previously, the presentation is often vague and nonspecific until an Addisonian crisis ensues. In patients with type 1 diabetes, unexplained hypoglycemia or other unexplained improvement in blood glucose control might be a clue to the diagnosis of Addison disease. Improved glycemia may represent the loss of anti-insulin activity associated with glucocorticoid deficiency. As mentioned previously, any patient with prolonged or unexplained chronic mucocutaneous candidiasis or hypoparathyoidism should be evaluated for APS I. Screening for APS should also be performed in females with premature ovarian failure or young patients with vitiligo. Assessment of end-organ function in any patient with

779

Remission Genetic predisposition Inflammation of target gland

Relapse Biochemical abnormalities Disease

Time Figure 18-6 Proposed model of the natural history of an organspecific autoimmune disease. In genetically susceptible individuals, the onset of autoimmunity (identified by the presence of serum autoantibodies) is thought to be triggered by environmental agents. These organ-specific autoantibodies can be identified months to years before the disease becomes clinically manifest— thus allowing for the prediction and early diagnosis of the disease before the clinical manifestations or metabolic abnormalities can usually be detected.

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TA B L E 1 8 - 3

Stages in the Development of Autoimmune Addison Disease Stage 1 2 3 4

Renin

Aldosterone

Basal Cortisol

Cortisol Post-ACTH

Basal ACTH

Elevated Elevated Elevated Elevated

N or Low N or Low N or Low Low

N N N Low*

N Low Low Low

N N Elevated Elevated

*Clinical Addison disease. ACTH, adrenocorticotropic hormone; and N, normal.

decreased cortisol response after parenteral ACTH administration. Stage 3 involves elevated basal ACTH. Stage 4 involves low basal cortisol (Table 18-3).87,88 In those individuals with adrenocortical autoantibodies, screening with mid-afternoon or later ACTH levels together with a supine renin should be carried out. Complete assessment of adrenocortical function should be carried out in those with ACTH levels ⬎55 pg/mL or in those with elevated renins.

Treatment Hormone replacement or other therapies for the component diseases of APS I and APS II are similar whether the ailments occur in isolation or in association with other conditions. Specific therapies are described in the individual chapters.

Genetics of APS I For some time it had been recognized that APS I could be inherited in an autosomal-recessive fashion.89 Unlike APS II, specific HLA alleles are not associated with APS I.90 APS I was initially mapped to chromosome 21q22.1 in Finnish families, and the AIRE gene was subsequently cloned.11 Individuals with APS I were found to be homozygous (possess two mutated copies) for the AIRE gene, with their parents being heterozygous for the mutated gene. AIRE is expressed in the thymus, lymph nodes, fetal liver, pancreas, adrenal cortex, and testes. The gene spans 11.9 kb and contains 14 exons. The predicted protein contains 545 amino acids.12 Initially, five AIRE mutations were described: one nonsense mutation and four frame-shift mutations.11,90 Multiple shared and unique mutations in the gene have now been defined in Sardinians, Britains, Italians, Finns, Japanese, and North Americans.37 Sequence analysis of the AIRE protein demonstrates that the predicted AIRE protein displays features common to other proteins that function as transcription factors (proteins that bind to the regulatory elements in the DNA regulatory region of genes).3,91 Transcription factors act to modulate the rate of gene transcription, whereas regulatory elements include the promoter region, enhancers, silencers, and metabolic response units. Further understanding of the AIRE protein

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function may provide fundamental insights into the nature of polyglandular and uniglandular autoimmunity. With recognition that APS I can be predicted in families with an index case, the focus of future therapy should be on the diagnosis of affected individuals using direct AIRE gene analysis, prediction of disease using autoantibody measurements, functional assessment (described previously), and the treatment of physiologic deficits prior to their clinical expression. More than 50 different mutations in the AIRE gene have now been reported, and mutations can be confirmed in more than 95% of patients with clinical diagnoses of APS I.36,92-94 Thus, screening for mutations in the AIRE gene can be considered if the diagnosis is unclear. Mutational analysis should be offered to siblings of affected children in order to determine carrier status and possibly to allow for the diagnosis of endocrinopathies before the onset of symptoms. Despite the availability of mutational analysis, more studies are needed to determine the relative sensitivity and cost effectiveness of mutational analysis screening versus primary antibody screening.

Genetics of APS II Whereas APS I displays an autosomal-recessive Mendelian pattern of inheritance, APS II is not inherited as a single gene mutation. APS II is much more typical of other autoimmune endocrinopathies wherein cases occur sporadically or within families. Overall, patients with APS II have HLA associations similar to those of patients with type 1 diabetes—especially having increased frequencies of HLA-DR3 (DQB1*0201) and DR4 (DQB1*0302).95 This is not surprising because type 1 diabetes, a frequent component of APS II, is strongly HLA associated. However, when patients with type 1 diabetes are removed from the APS II subject group the association between HLA-DR alleles and APS II disappears. Thus, in the absence of type 1 diabetes HLA-DR is not a major locus for the development of APS II. Previous studies have shown not only that Addison disease and type 1 diabetes share the major risk DR3/DR4 genotype but that Addison disease is particularly associated with DRB1*0404 and DRB1*0301 haplotypes.96 It is estimated that 1 in 20 patients with type 1 diabetes with this haplotype will have adrenal autoantibodies.97,98 Another high-risk allele for Addison disease is DRB5 (DQB1*0301). Graves disease

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is more often associated with HLA-DR3 as opposed to Hashimoto thyroiditis, which is associated with HLA-DR4 or DR5.99,100 Autoimmune hypoparathyroidism and mucocutaneous candidiasis do not display associations with specific HLA alleles. Because the association of APS II with specific HLA alleles is only modest, other genes must be important in providing susceptibility. To identify non-HLA genes that influence susceptibility to APS II genome-wide scan or linkage studies are necessary. The G allele of the CTLA-4 exon 1 A/G diallelic polymorphism has been associated with Addison disease in APS II, and less strongly associated with isolated Addison disease.101

Autoantibodies in Autoimmune Polyglandular Syndromes Autoantibodies are antibodies (predominantly IgG) that bind to self-antigens (e.g., autoantigens). Autoantibodies may be pathogenic, as observed in Graves disease or myasthenia gravis. In Graves disease, agonistic autoantibodies directed against the thyroid follicular cell TSH receptor stimulate overproduction of thyroid hormone—producing hyperthyroidism.102 In myasthenia gravis, autoantibodies directed against the motor end plate acetylcholine receptor located on myocytes stimulate internalization of the acetylcholine receptor—producing weakness. Alternatively, autoantibodies may serve solely as serologic markers of autoimmunity—as in type 1 diabetes, in which islet cell cytoplasmic autoantibodies (ICA) and glutamic acid decarboxylase autoantibodies (GADA) are indicators of ongoing autoimmunity.103,104 Detection of autoantibodies in relation to autoimmune polyglandular syndromes serves several important functions. First, detection of autoantibodies implicates an autoimmune etiology as the cause of disease and allows a specific diagnosis to be established.105 Second, autoantibody detection in asymptomatic individuals indicates an increased risk for the later development of clinical disease.64 Third, the diagnosis of one autoimmune disease in an individual (or detection of predilection to one autoimmune disease by the presence of autoantibodies) suggests that this individual is at risk for associated autoimmune diseases.

ADRENAL CYTOPLASMIC AUTOANTIBODIES Adrenal cytoplasmic autoantibodies (ACA) were first detected using a complement-fixation technique with saline extracts of adrenal tissue, and soon afterward by indirect immunofluorescence.106 Usually, all layers of the adrenal cortex fluoresce—whereas the medulla does not usually fluoresce.87 The microsomal localization of autoantigens has been confirmed using ultracentrifuged cellular components.107 Other assays for adrenal autoantibodies include solid-phase radioimmunoassays and nonradioactive enzyme-linked immunoabsorbent assays.88,108 Up to 75 to 80% of new-onset subjects with Addison disease exhibit ACA.87 In up to approximately half of

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asymptomatic ACA-positive individuals, Addison disease develops in less than 3 years. In a follow-up of 20 ACApositive children followed for up to 11 years, the cumulative risk for developing Addison disease was 100%.87 ACA are also predictive of the development of Addison disease in adults, although less frequently than in children. Higher titers of ACA and complement-fixing ACA might be associated with an increased risk of displaying clinical disease.109 Autoantibodies to the surface of adrenal cortical cells have been described but are not detected routinely because of the difficulty in obtaining fresh human or animal adrenal tissue that can be used for such assays. However, almost 90% of individuals with Addison disease were reported to exhibit such autoantibodies.110 ACA are detected in all forms of autoimmune Addison disease, be it isolated Addison disease or as part of APS I or APS II. As stated previously, subjects with other forms of organspecific autoimmune disease exhibit increased frequencies of ACA. In such circumstances, ACA predict an increased risk for developing biochemical and clinical evidence of adrenal insufficiency.

ADRENAL ENZYME AUTOANTIBODIES Typical of many organ-specific autoimmune diseases, major autoantigens that serve as targets of autoantibodies in the autoimmune polyglandular syndromes are enzymes. Such enzymes are typically expressed in the tissues that are being targeted for humoral and/or cellmediated autoimmune attack. Examples of various autoimmune diseases where enzymes are targeted are outlined in Table 18-1. (Adrenal hormone synthesis is discussed in Chapter 12.) 21-hydroxylase (P450c21) is a major autoantigen recognized by sera from patients with Addison disease.111 There is a strong correlation between positivity for ACA and P450c21 autoantibodies.112 Other enzymes have been identified as autoantigens in patients with isolated autoimmune Addison disease or in patients with an autoimmune polyglandular syndrome, including P450 cholesterol side-chain cleavage enzyme (P450scc), 17␣-hydroxylase (P450c17), and 3␤-hydroxysteroid dehydrogenase (not a P450 enzyme).112

ADRENAL ENZYME AUTOANTIBODIES IN APS I Although autoantibodies to P450c21 (21-hydroxylase), P450ssc (side-chain cleavage enzyme), and P450c17 (17␣-hydroxylase) have been reported, autoantibodies to P450c21 are most commonly identified in patients with adrenal autoimmunity. Nearly 75% of APS I and APS II patients reportedly have P450c21 autoantibodies present.87 The autoantigenic epitopes of the P450c21 enzyme are located in the C-terminal end and in a central region of the enzyme.113 Peterson et al. found that two of four epitopes recognized by P450c17 autoantibodies cross-reacted with P450c21, indicating that reactivity to one of these autoantigens could actually reflect molecular mimicry between such epitopes.114 Except for the N-terminal amino acids 1-40 and the C-terminal

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amino acids 456 to 521, immunoreactive epitopes have been described throughout P450c21.115 Higher titers of ACA and higher concentrations of P450c21 autoantibodies appear to correlate with more severe impairment of adrenocortical function and the predicted development of Addison disease.87 Autoantibody concentrations appear to rise until the development of clinical Addison disease, at which time autoantibody reactivity disappear. This is consistent with the concept that once an autoantigen is completely destroyed the immune system is no longer stimulated to produce autoantibodies—with autoantibody prevalence and concentration diminishing.

ADRENAL ENZYME AUTOANTIBODIES IN APS II When clinical or preclinical Addison disease is present, there is no unique combination of adrenal or gonadal antibodies that separate APS I from APS II.105,116,117 The differentiation of APS I from APS II is made on clinical grounds or by detecting an autoantibody characteristic of a nonadrenal APS II–associated autoimmune disease. Furthermore, there are no unique epitopes recognized by P450c21 autoantibodies that allow differentiation of Addison disease as to etiology (e.g., isolated autoimmune Addison disease versus APS I versus APS II) other than indicating that the disease is indeed autoimmune.118 Autoantibodies to P450c21, P450scc, and P450c17 are common in APS II. Most patients with APS I or II with Addison disease have autoantibodies to one or more of these enzymes, compared to less than 25% of APS patients without adrenal insufficiency.119 In one study (using an immunoprecipitation assay with 125I-labeled 21hydroxylase) it was demonstrated that nearly all patients with Addison disease and APS II were positive for autoantibodies to P450c21—compared to 92% of patients with APS-I, 72% of patients with isolated Addison disease, and 80% of non-Addisonian patients who were ACA positive.120 Technical differences in the performance of the various assays contribute to the differences in reported frequencies as well as the presence or absence of SCA.

ADRENAL ENZYME AUTOANTIBODIES IN ISOLATED ADDISON DISEASE Autoantibodies to P450c21 are common in Addison disease patients, whether isolated or part of APS I or APS II, and are rare in the absence of Addison disease.87,121 P450c21 appears to be the major adrenal autoantigen in cases of isolated Addison disease, being detected in nearly 75% of patients. Autoantibodies to P450scc and P450c17 have been reported but are less frequently found (⬍30%) than P450c21 antibodies.112

STEROIDAL CELL/GONADAL AUTOANTIBODIES In some individuals with ACA, their sera has also been shown to react with reproductive-steroid-producing tissues such as the theca interna of the graffian follicle,

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Leydig cells of the testis, and/or syncytiotrophoblastic layer of the placenta.98,122 Sera that recognize antigens in adrenal and reproductive-steroid-producing tissues whose immunoreactivity cannot be absorbed with adrenal extracts are considered SCA. Sera may react to all tissues or to just one. The significance of whether a serum reacts with a single tissue or multiple tissues in defining SCA is unknown. Such variability may represent variations in the autoimmune epitopes recognized by the serum or differences in the tissue substrates relating to autoantigen densities or epitope availability. SCA are associated with an increased risk of developing primary autoimmune gonadal failure that is expressed predominantly in women as either primary amenorrhea or premature menopause. Men are usually asymptomatic. When ACA are present in the absence of SCA, gonadal failure is unlikely to develop.98 About a third of individuals with ACA upon initial screening using adrenal as the sole substrate have SCA using ovary, testis, or placenta as the antigen source. SCA can be observed in APS II patients, although gonaditis is more common in APS I than in APS II. It is estimated that 60% of subjects with APS I and Addison disease express SCA, versus 30% of subsets with APS II. Primary hypogonadism can be observed in APS I or APS II manifesting as hypergonadotropic ovarian, or rarely as testicular failure. Premature ovarian failure independent of APS can also occur as a consequence of autoimmunity.123 Nevertheless, Addison disease coexists in approximately 2% to 10% of women with ovarian failure.124 Autoimmune ovarian failure is characterized histologically by ovarian infiltration with inflammatory cells.125 Patients with isolated premature ovarian failure or premature ovarian failure associated with APS often express SCA. As early as 1968, SCA were described using indirect immunofluorescence.126 Five of 5 women with SCA, all of whom also had ACA consistent with an autoimmune polyglandular syndrome, manifested ovarian failure. Autoantibodies reacting strongly to the pig zona pellucida as determined by indirect immunofluorescence were observed in 6 of 22 women with infertility.127 SCA, depending on the assay used, have been reported in 4% to 87% of women with premature ovarian failure.122,128,129 SCA are also predictive of later gonadal failure in APS women with normal menses at the time of initial study. Ahonen et al. reported that 100% (11 of 11) of APS I patients who were positive for SCA developed primary ovarian failure during a follow-up period of up to 12 years.130 The nature of the ovarian autoantigens in premature ovarian failure is controversial. Because the adrenal cortex and gonad share several synthetic pathways common to cells producing adrenocortical steroids and sex steroids, humoral autoimmunity to a shared autoantigen may be observed in adrenalitis and gonaditis. The synthesis of sex and adrenal steroids requires P450scc, 3␤-hydroxysteroid dehydrogenase, and P450c17. Therefore, it would be anticipated that these enzymes could also be autoantigens in some individuals with gonaditis. Data are however contradictory. In one study, SCA activity was removed by preabsorption with recombinant human 3␤-hydroxysteroid dehydrogenase— suggesting that this enzyme was a major autoantigen

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detected by SCA-positive sera.131 Another study suggested that SCA correlated best with reactivity to P450scc and a 51-kilodalton autoantigen that binds to the aromatic L-amino acid decarboxylase present in granulosa cells, placenta, liver, and pancreatic beta cells.132 In the absence of adrenal autoimmunity, autoantibodies to P450c21, P450c17, or P450scc are rarely found in women with premature ovarian failure.122 This suggests that in the absence of APS few cases of idiopathic premature ovarian failure result from autoimmune damage to the ovary. In another study, no autoantibodies to P450c17 or P450scc in 48 women with isolated premature ovarian failure were identified—whereas only 2 of these women had SCA and only 1 had autoantibodies to 3␤-hydroxysteroid dehydrogenase.133 However, in another study autoantibodies to 3␤-hydroxysteroid dehydrogenase were observed in women with premature ovarian failure who did not have coexistent autoimmune diseases.134 An alternative hypothesis attempting to explain premature ovarian failure is the potential existence of antagonist autoantibodies directed against the FSH or LH receptors. Support for this hypothesis is weak, however, because recent studies have not been able to confirm the presence of gonadotropin receptor autoantibodies in the sera of a large group of women with premature ovarian failure.135

AUTOANTIBODIES IN HYPOPARATHYROIDISM Autoimmune hypoparathyroidism is a characteristic disorder essentially unique to APS I. Hypoparathyroidism is absent in subjects with APS II. In Blizzard’s original report of parathyroid autoantibodies detected using indirect immunofluorescence, nearly 40% of patients with autoimmune hypoparathyroidism were found to have parathyroid cytoplasmic autoantibodies—versus 6% of controls.136,137 However, other laboratories did not confirm the initial reports of such parathyroid cytoplasmic autoantibodies.138,139 It was shown that autoantibodies detected by indirect immunofluoresence directed against the parathyroid gland could be preabsorbed with human mitochondria, indicating that such autoantibodies were not tissue specific.139 Autoantibodies have also been identified that are cytotoxic for cultured bovine parathyroid cells in patients with hypoparathyroidism.140 These autoantibodies also bind to cultured bovine endothelial cells.141 Unrelated to APS I or APS II, autoantibodies that bind to anti-PTH antibodies employed in a PTH immunoassay (e.g., antiidiotypic PTH autoantibodies) have also been described in a patient with hypoparathyroidism.142 More recently, autoantibodies to the extracellular domain of the calcium receptor have been described in patients with hypoparathyroidism.143 Similarly, autoantibodies that block the calcium receptor have been described that cause hyperparathyroidism (e.g., autoimmune hypercalcemia).144

OTHER AUTOANTIBODIES IN APS I AND II Autoantibodies to thyroid follicular cell organelles in autoimmune thyroid disease (e.g., thyroid microsomal autoantibodies), follicular cell enzymes (e.g., thyroperoxidase)

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and products (e.g., thyroglobulin), and autoantibodies to pancreatic beta cells in type 1 diabetes (e.g., islet cell cytoplasmic autoantibodies) and beta cell proteins (e.g., glutamic acid decarboxylase and insulinoma-associated antigen-2) and products (e.g., insulin and carboxypeptidase H) are well described in patients with autoimmune polyglandular syndromes. Their frequency is especially high in patients with APS II. However, the biology of these autoantibodies will not be reviewed here. (See the specific chapters that address autoimmune thyroid disease and type 1 diabetes.) Celiac disease is accompanied by mucosal (especially) IgA autoantibodies to reticulin, endomysium, transglutaminase, and jejunum.145 Retrospective studies show that tissue transglutaminase autoantibodies can provide 100% sensitivity and specificity for biopsy-proven celiac disease.146 There is a high prevalence of various autoimmune disorders [including type 1 diabetes (6% of type 1 patients reportedly have antiendomyseal antibodies), autoimmune thyroiditis, dermatitis herpetiformis, autoimmune alopecia, autoimmune hepatitis, and collagen vascular diseases] in patients with celiac disease.145 In one study, 3.6% of new-onset type 1 diabetes patients had celiac disease.147 Autoantibodies to tyrosine hydroxylase have been reported in ⬃40% of patients with APS I and correlate with alopecia areata.148 Antibodies to liver/kidney microsome type 1 (LKM1) are found in nearly 100% of patients with type 2 autoimmune hepatitis.149 Forty percent of such patients have associated autoimmune disease commonly seen in APS I. The hepatic autoantigens P450IA2 and P4502A6 have been reported as targets of these antibodies. Autoantibodies to aromatic L-amino acid decarboxylase have also been recognized in APS I and isolated cases of Addison disease.132 Autoantibodies to the adrenal medulla may also occur in various autoimmune conditions, but especially so in type 1 diabetes.150,151 Adrenal-medullary autoimmunity has been linked to autonomic dysfunction in individuals with diabetes mellitus.152 Pteridin-dependent hydroxylases have also been proposed as autoantigens in APS I.153

Summary The autoimmune polyglandular syndromes result from a loss of tolerance to self-antigens. A thorough understanding of tolerance induction and defects in tolerance is required to fully understand the immunopathogenesis of APS. APS I, an autosomal-recessive disorder mapped to the AIRE gene, is defined by the presence of two of the following: adrenocortical autoimmunity, hypoparathyroidism, and mucocutaneous candidiasis. APS II is defined by the coexistence of autoimmune adrenocortical insufficiency or serologic evidence of adrenalitis with autoimmune thyroiditis and/or type 1 diabetes mellitus. The occurrence of any component disease of an APS may be linked to the occurrence of others through shared autoimmunity background genes that lead to a loss of tolerance. As such, a high index of suspicion should be maintained whenever one autoimmune disorder is diagnosed. For example, in type 1 diabetes it is

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routine to screen for thyroid autoimmunity and celiac disease. Treatment of APS should be aimed at optimal management of the specific underlying diseases. Screening for the presence of associated autoimmune disorders should be performed regularly. An improved understanding of the interaction among susceptibility genes, environmental triggers, and the development of impaired immune tolerance should prove to be the best path to improved diagnostic and therapeutic modalities in the care of patients with APS.

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46. Doniach D, Roitt IM (1957). Auto-immunity in Hashimoto’s disease and its implications. J Clin Endocrinol Metab 17:1293–1304. 47. Betterle C, Lazzarotto F, Presotto F (2004). Autoimmune polyglandular syndrome Type 2: The tip of an iceberg? Clin Exp Immunol 137:225–233. 48. Hugle B, Dollmann R, Keller E, Kiess W (2004). Addison’s crisis in adolescent patients with previously diagnosed diabetes mellitus as manifestation of polyglandular autoimmune syndrome type II: Report of two patients. J Pediatr Endocrinol Metab 17:93–97. 49. Hollowell JG, Staehling NW, Flanders WD, Hannon WH, Gunter EW, Spencer CA, et al. (2002). Serum TSH, T4, and Thyroid Antibodies in the United States Population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab 87:489–499. 50. Jaeger C, Hatziagelaki E, Petzoldt R, Bretzel RG (2001). Comparative analysis of organ-specific autoantibodies and celiac disease: Associated antibodies in type 1 diabetic patients, their first-degree relatives, and healthy control subjects. Diabetes Care 24:27–32. 51. Kordonouri O, Klinghammer A, Lang EB, Gruters-Kieslich A, Grabert M, Holl RW (2002). Thyroid autoimmunity in children and adolescents with type 1 diabetes: A multicenter survey. Diabetes Care 25:1346–1350. 52. Alkhateeb A, Fain PR, Thody A, Bennett DC, Spritz RA (2003). Epidemiology of vitiligo and associated autoimmune diseases in Caucasian probands and their families. Pigment Cell Research 16:208–214. 53. Ruggeri RM, Galletti M, Mandolfino MG, Aragona P, Bartolone S, Giorgianni G, et al. (2002). Thyroid hormone autoantibodies in primary Sjögren syndrome and rheumatoid arthritis are more prevalent than in autoimmune thyroid disease, becoming progressively more frequent in these diseases. J Endocrinol Invest 25:447–454. 54. Tsao CY, Mendell JR, Lo WD, Luquette M, Rennebohm R (2000). Myasthenia gravis and associated autoimmune diseases in children. J Child Neurol 15:767–769. 55. Zettinig G, Tanew A, Fischer G, Mayr W, Dudczak R, Weissel M (2003). Autoimmune diseases in vitiligo: Do anti-nuclear antibodies decrease thyroid volume? Clinical and Experimental Immunology 131:347–354. 56. Kakourou T, Kanaka-Gantenbein C, Papadopoulou A, Kaloumenou E, Chrousos GP (2005). Increased prevalence of chronic autoimmune (Hashimoto’s) thyroiditis in children and adolescents with vitiligo. Journal of the American Academy of Dermatology 53:220–223. 57. Hann SK, Lee HJ (1996). Segmental vitiligo: Clinical findings in 208 patients. J Am Acad Dermatol 35:671–674. 58. Tanwani LK, Lohano V, Ewart R, Broadstone VL, Mokshagundam SP (2001). Myasthenia gravis in conjunction with Graves’ disease: A diagnostic challenge. Endocr Pract 7:275–278. 59. Weetman AP (2005). Non-thyroid autoantibodies in autoimmune thyroid disease: Best practice and research. Clinical Endocrinology and Metabolism Autoimmune Endocrine Disorders 19:17–32. 60. Karvonen M, Viik-Kajander M, Moltchanova E, Libman I, LaPorte R, Tuomilehto J for the Diabetes Mondiale (DiaMond) Project Group (2000). Incidence of childhood type 1 diabetes worldwide. Diabetes Care 23:1516–1526. 61. Stenstrom G, Gottsater A, Bakhtadze E, Berger B, Sundkvist G (2005). Latent autoimmune diabetes in adults: Definition, prevalence, beta-cell function, and treatment. Diabetes 54:S68–S72. 62. Alonso N, Granada ML, Salinas I, Lucas AM, Reverter JL, Junca J, et al. (2005). Serum pepsinogen I: An early marker of pernicious anemia in patients with type 1 diabetes. J Clin Endocrinol Metab 90:5254–5258. 63. Segni M, Borrelli O, Pucarelli I, Delle Fave G, Pasquino AM, Annibale B (2004). Early manifestations of gastric autoimmunity in patients with juvenile autoimmune thyroid diseases. J Clin Endocrinol Metab 89:4944–4948. 64. Barker JM (2006). Type 1 diabetes-associated autoimmunity: Natural history, genetic associations, and screening. J Clin Endocrinol Metab 91:1210–1217. 65. Barker JM, Ide A, Hostetler C, Yu L, Miao D, Fain PR, et al. (2005). Endocrine and immunogenetic testing in individuals with type 1 diabetes and 21-hydroxylase autoantibodies: Addison’s disease in a high-risk population. J Clin Endocrinol Metab 90:128–134. 66. Hansson T, Dahlbom I, Rogberg S, Dannaeus A, Hopfl P, Gut H, et al. (2002). Recombinant human tissue transglutaminase for

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109. Betterle C, Volpato M, Smith BR, Furmaniak J, Chen S, Greggio NA, et al. (1997). Adrenal cortex and steroid 21-hydroxylase autoantibodies in adult patients with organ-specific autoimmune diseases: Markers of low progression to clinical Addison’s disease. J Clin Endocrinol Metab 82:932–938. 110. Khoury EL, Hammond L, Bottazzo GF, Doniach D (1981). Surfacereactive antibodies to human adrenal cells in Addison’s disease. Clin Exp Immunol 45:48–55. 111. Husebye ES, Bratland E, Bredholt G, Fridkin M, Dayan M, Mozes E (2006). The substrate-binding domain of 21-hydroxylase, the main autoantigen in autoimmune Addison’s disease, is an immunodominant T cell epitope. Endocrinology 147:2411–2416. 112. de Carmo Silva R, Kater C, Dib S, Laureti S, Forini F, Cosentino A, et al. (2000). Autoantibodies against recombinant human steroidogenic enzymes 21-hydroxylase, side-chain cleavage and 17alphahydroxylase in Addison’s disease and autoimmune polyendocrine syndrome type III. Eur J Endocrinol 142:187–194. 113. Nikoshkov A, Falorni A, Lajic S, Laureti S, Wedell A, Lernmark K, et al. (1999). A conformation-dependent epitope in Addison’s disease and other endocrinological autoimmune diseases maps to a carboxyl-terminal functional domain of human steroid 21-hydroxylase. J Immunol 162:2422–2426. 114. Peterson P, Krohn KJ (1994). Mapping of B cell epitopes on steroid 17 alpha-hydroxylase, an autoantigen in autoimmune polyglandular syndrome type I. Clin Exp Immunol 98:104–109. 115. Liiv I, Teesalu K, Peterson P, Clemente M, Perheentupa J, Uibo R (2002). Epitope mapping of cytochrome P450 cholesterol sidechain cleavage enzyme by sera from patients with autoimmune polyglandular syndrome type 1. Eur J Endocrinol 146:113–119. 116. Falorni A, Laureti S, Santeusanio F (2002). Autoantibodies in autoimmune polyendocrine syndrome type II. Endocrinol Metab Clin North Am 31:369–389. 117. Betterle C, Lazzarotto F, Presotto F (2004). Autoimmune polyglandular syndrome type 2: The tip of an iceberg? Clinical and Experimental Immunology 137:225–233. 118. Nikoshkov A, Falorni A, Lajic S, Laureti S, Wedell A, Lernmark A, et al. (1999). A conformation-dependent epitope in Addison;s disease and other endocrinological autoimmune diseases maps to a carboxyl-terminal functional domain of human steroid 21-hydroxylase. J Immunol 162:2422–2426. 119. Uibo R, Perheentupa J, Ovod V, Krohn KJ (1994). Characterization of adrenal autoantigens recognized by sera from patients with autoimmune polyglandular syndrome (APS) type I. J Autoimmun 7:399–411. 120. Tanaka H, Perez MS, Powell M, Sanders JF, Sawicka J, Chen S, et al. (1997). Steroid 21-hydroxylase autoantibodies: Measurements with a new immunoprecipitation assay. J Clin Endocrinol Metab 82:1440–1446. 121. Boe A, Bredholt G, Knappskog P, Hjelmervik T, Mellgren G, Winqvist O, et al. (2004). Autoantibodies against 21-hydroxylase and side-chain cleavage enzyme in autoimmune Addison’s disease are mainly immunoglobulin G1. Eur J Endocrinol 150:49–56. 122. Dal Pra C, Chen S, Furmaniak J, Smith B, Pedini B, Moscon A, et al. (2003). Autoantibodies to steroidogenic enzymes in patients with premature ovarian failure with and without Addison’s disease. Eur J Endocrinol 148:565–570. 123. Goswami D, Conway GS (2005). Premature ovarian failure. Hum Reprod Update 11:391–410. 124. Bakalov VK, Vanderhoof VH, Bondy CA, Nelson LM (2002). Adrenal antibodies detect asymptomatic auto-immune adrenal insufficiency in young women with spontaneous premature ovarian failure. Hum Reprod 17:2096–2100. 125. Santoro N (2001). Research on the mechanisms of premature ovarian failure. Journal of the Society for Gynecologic Investigation 8:S10–S12. 126. Irvine WJ, Chan MM, Scarth L, Kolb FO, Hartog M, Bayliss RI, et al. (1968). Immunological aspects of premature ovarian failure associated with idiopathic Addison’s disease. Lancet 2:883–887. 127. Shivers CA, Dunbar BS (1977). Autoantibodies to zona pellucida: A possible cause for infertility in women. Science 197: 1082–1084. 128. Falorni A, Laureti S, Candeloro P, Perrino S, Coronella C, Bizzarro A, et al. (2002). Steroid-cell autoantibodies are preferentially expressed in women with premature ovarian failure who have adrenal autoimmunity. Fertility and Sterility 78:270–279.

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129. Reimand K, Peterson P, Hyoty H, Uibo R, Cooke I, Weetman AP, et al. (2000). 3␤-hydroxysteroid dehydrogenase autoantibodies are rare in premature ovarian failure. J Clin Endocrinol Metab 85:2324–2326. 130. Ahonen P, Miettinen A, Perheentupa J (1987). Adrenal and steroidal cell antibodies in patients with autoimmune polyglandular disease type I and risk of adrenocortical and ovarian failure. J Clin Endocrinol Metab 64:494–500. 131. Uibo R, Aavik E, Peterson P, Perheentupa J, Aranko S, Pelkonen R, et al. (1994). Autoantibodies to cytochrome P450 enzymes P450scc, P450c17, and P450c21 in autoimmune polyglandular disease types I and II and in isolated Addison’s disease. J Clin Endocrinol Metab 78:323–328. 132. Soderbergh A, Rorsman F, Halonen M, Ekwall O, Bjorses P, Kampe O, et al. (2000). Autoantibodies against aromatic L-amino acid decarboxylase identifies a subgroup of patients with Addison;s disease. J Clin Endocrinol Metab 85:460–463. 133. Reimand K, Peterson P, Hyoty H, Uibo R, Cooke I, Weetman AP, et al. (2000). 3beta-hydroxysteroid dehydrogenase autoantibodies are rare in premature ovarian failure. J Clin Endocrinol Metab 85:2324–2326. 134. Arif S, Vallian S, Farzaneh F, Zanone MM, James SL, Pietropaolo M, et al. (1996). Identification of 3 beta-hydroxysteroid dehydrogenase as a novel target of steroid cell autoantibodies: Association of autoantibodies with endocrine autoimmune disease. J Clin Endocrinol Metab 81:4439–4445. 135. Tonacchera M, Ferrarini E, Dimida A, Agretti P, De Marco G, De Servi M, et al. (2004). Gonadotrophin receptor blocking antibodies measured by the use of cell lines stably expressing human gonadotrophin receptors are not detectable in women with 46,XX premature ovarian failure. Clinical Endocrinology 61:376–381. 136. Blizzard RM, Chee D, Davis W (1966). The incidence of parathyroid and other antibodies in the sera of patients with idiopathic hypoparathyroidism. Clin Exp Immunol 1:119–128. 137. Irvine WJ, Scarth L (1969). Antibody to the oxyphil cells of the human parathyroid in idiopathic hypoparathyroidism. Clin Exp Immunol 4:505–510. 138. Chapman CK, Bradwell AR, Dykks PW (1986). Do parathyroid and adrenal autoantibodies coexist? J Clin Pathol 39:813–814. 139. Betterle C, Caretto A, Zeviani M, Pedini B, Salviati C (1985). Demonstration and characterization of anti-human mitochondria autoantibodies in idiopathic hypoparathyroidism and in other conditions. Clin Exp Immunol 62:353–360. 140. Brandi ML, Aurbach GD, Fattorossi A, Quarto R, Marx SJ, Fitzpatrick LA (1986). Antibodies cytotoxic to bovine parathyroid cells in autoimmune hypoparathyroidism. Proc Natl Acad Sci USA 83:8366–8369.

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C H A P T E R

19 Disorders of Energy Balance ROBERT H. LUSTIG, MD • RAM WEISS, MD

Introduction Neuroendocrine Regulation of Energy Balance The Afferent System Alimentary Afferents Metabolic Afferents Central Processing Anorexigenesis, POMC/
MSH, and CART Orexigenesis, NPY, and AgRP Other Neuroendocrine Modulators of Energy Balance Melanocortin Receptors and Central Neural Integration The Efferent System The Sympathetic Nervous System and Energy Expenditure The Efferent Vagus and Energy Storage Negative Feedback Modulation of Energy Balance: The Starvation Response The Hedonic Pathway of Food Reward Leptrin Resistance Energy Excess: Obesity Definition Prevalence and Epidemiology Global Prevalence Racial and Ethnic Considerations Predictive Factors Metabolic Impact of Childhood Obesity Lipid Partitioning Adipocytokines Leptin Adiponectin Inflammatory Cytokines Other Adipocytokines Insulin Resistance Vascular Changes The Metabolic Syndrome Other Co-Morbidities Related to Insulin Resistance

Nonalcoholic Fatty Liver Disease Polycystic Ovary Syndrome Other Endocrine Co-Morbidities Other Co-Morbidities Factors Associated with the Current Epidemic of Obesity Genetics Epigenetics and Fetal and Neonatal Programming Environment Stress and Cortisol Sleep Deprivation Television Viewing and “Screen Time” Dietary Fat Versus Carbohydrate Glycemic Index and Fiber Fructose Calcium and Dairy Trace Minerals Infectious Causes Medications Disorders of Obesity Classic Endocrine Disorders with an Obesity Phenotype Monogenetic Disorders of the Negative Feedback Pathway Leptin Deficiency Leptin Receptor Deficiency POMC Splicing Mutation Prohormone Convertase-1 Deficiency Melanocortin-3 Receptor Mutation Melanocortin-4 Receptor Mutation SIM-1 Mutation Pleiotropic Obesity and Mental Retardation Disorders Prader-Willi Syndrome Bardet-Biedl Syndrome TrkB Mutation Carpenter Syndrome Cohen Syndrome

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Alstrom Syndrome Börjeson-Forssman-Lehmann Syndrome Insulin Dynamic Disorders Hypothalamic Obesity and Insulin Hypersecretion Primary Insulin Resistance Evaluation and Treatment of the Obese Child Workup Lifestyle Modification Dietary Intervention Physical Activity Intervention School Intervention Family Intervention Pharmacotherapy Indications for Pharmacotherapy Reduction of Energy Intake: Sibutramine Reduction of Energy Absorption: Orlistat Improvement of Insulin Resistance: Metformin Suppression of Insulin Hypersecretion: Octreotide Other Targeted Therapies The Future of Pediatric Obesity Pharmacotherapy Bariatric Surgery

Introduction Energy balance is the “final frontier” of endocrinology. Prior to 1994, with the discovery of leptin, the disorders of energy balance were not even considered endocrine diseases. Today, obesity can account for up to 25% of pediatric endocrine practice referrals—and type 2 diabetes accounts for up to 30% of the new referrals for diabetes, virtually all of whom are also obese. Since the discovery of leptin, the negative feedback pathway of energy balance has been elucidated—and endocrinologists have embraced disorders of energy balance as part of their portfolio. Thus, the study of energy balance has become a matter of continuing education for pediatric endocrinologists. The entire field is a “work in progress,” which is problematic because our knowledge, diagnostic armamentarium, and treatment options are still in their infancy. This chapter conveys a clear and up-to-date basic understanding of the energy balance pathway, and provides a clinical rationale and formulation for evaluating and treating patients with energy balance disorders.

Neuroendocrine Regulation of Energy Balance The negative feedback axis of energy balance and its function during homeostasis has been largely delineated through studies in animal models. Human data are presented where available. The axis consists of three arms (Figure 19-1). The first is the afferent arm, which conveys in the form of hormonal and neural

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Indications for Bariatric Surgery Restrictive: Laparoscopic Adjustable Gastric Banding Combination: Roux-en-Y Gastric Bypass Who Should Perform Bariatric Surgery in Children Energy Inadequacy Starvation Versus Cachexia Failure to Thrive Definition Classification and Etiology Diagnosis and Evaluation Management Prognosis Cancer Cachexia The Diencephalic Syndrome Anorexia Nervosa Definition Endocrine Associations Treatment Conclusions

inputs peripheral information on hunger and peripheral metabolism to the hypothalamus. The second is a central processing unit, consisting of various areas within the hypothalamus.The ventromedial hypothalamus [VMH, consisting of the ventromedial (VMN) and arcuate (ARC) nuclei] integrates the afferent peripheral signals, along with other central stimuli. The paraventricular nuclei (PVN) and lateral hypothalamic area (LHA) serve as a gated neurotransmitter system to alter neural signals for changes in feeding and energy expenditure. The third component is the efferent arm, which consists of a complex network of autonomic effectors that regulate energy intake and monitor energy expenditure versus storage.1,2 Anatomic disruptions or genetic or metabolic alterations of the afferent, central processing, or efferent arms can alter energy intake or expenditure in stereotyped ways—which can lead to obesity or cachexia.

THE AFFERENT SYSTEM Alimentary Afferents Hunger. The afferent vagus: The vagus nerve is the primary neural connection between the brain and the gut. The afferent vagus nerve conveys information regarding mechanical stretch of the stomach and duodenum and sensations of gastric fullness to the nucleus tractus solitarius (NTS).3 Of note is that each of the alimentary neuropeptide effects on hunger and satiety discussed in the material following is obviated by concomitant vagotomy, implicating the afferent vagus as the primary mediator of alimentary energy balance signals.4-6

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PVN LHA

VMH

5-HT NE

Me

LC

d.

Ra p

he

Leptin Insulin

DMV

NTS

erve

sn Vagu Ghrelin Adipocytes PYY3-36

Pancreas Small Intestine

Liver

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s vou Sympathetic ner

t sys

em

Figure 19-1 The homeostatic pathway of energy balance. Afferent (gray), central (black), and efferent (white) pathways are delineated. The hormones insulin, leptin, ghrelin, and peptide YY(3-36) (PYY3-36) provide afferent information to the ventromedial hypothalamus regarding short-term energy metabolism and energy sufficiency. From there, the ventromedial hypothalamus elicits anorexigenic (
-melanocyte-stimulating hormone, cocaine-amphetamine-regulated transcript) and orexigenic (neuropeptide Y, agouti-related protein) signals to the melanocortin-4 receptor in the paraventricular nucleus and lateral hypothalamic area. These lead to efferent output via the locus coeruleus, via the nucleus tractus solitarius, which activates the sympathetic nervous system—causing the adipocyte to undergo lipolysis. Alternatively, this is achieved via the dorsal motor nucleus of the vagus, which activates the vagus nerve to store energy by increasing pancreatic insulin secretion and (in rodents) by increasing adipose tissue sensitivity to insulin. 5-HT, serotonin (5-hydroxytryptamine); DMV, dorsal motor nucleus of the vagus; LC, locus coeruleus; LHA, lateral hypothalamic area; NE, norepinephrine; NTS, nucleus tractus solitarius; PVN, paraventricular nucleus; and VMH, ventromedial hypothalamus. [From Lustig RH (2006). Childhood obesity: Behavioral aberration or biochemical drive? Reinterpreting the first law of thermodynamics. Nature Clin Pract Endo Metab 2:447–458. Courtesy of Nature Publishing Group, with permission.]

Ghrelin: Ghrelin, an octanoylated 28-amino-acid peptide, was discovered serendipitiously while looking for the endogenous ligand of the growth hormone (GH) secretagogue receptor (GHS-R).7 Ghrelin induces rat GH release through stimulation of the pituitary GHS-R. The endogenous secretion of ghrelin from the fasting stomach is high, but is decreased by nutrient administration. Volumetric stretching of the stomach wall has no effect. However, ghrelin also binds to the GHS-R in the VMH—which increases hunger, food intake, and fat deposition.8,9 Ghrelin also increases the respiratory quotient (RQ) in rats, suggesting a reduction of fat oxidation and promotion

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of fat storage. Ghrelin appears to tie the lipolytic effect of GH with the hunger signal, and is probably important in the acute response to fasting. In humans, ghrelin levels rise with increasing subjective hunger—and peak at the time of voluntary food consumption.10 This suggests that ghrelin acts on the VMH to trigger meal initiation. Ghrelin infusion increases food intake in humans.11 However, plasma ghrelin levels are low in obese individuals—and increase with fasting,12 suggesting that ghrelin is a response to (rather than a cause of) obesity. Satiety. Peptide YY3-36 (PYY3-36): A recently identified hormonal signal to control meal volume is PYY3-36.13 This

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peptide fragment is secreted by intestinal L cells in response to exposure to nutrient, crosses the blood-brain barrier, and binds to the Y2 receptor in the VMH. Activation of this receptor causes a decrease in NPY mRNA in neurons of the orexigenic arm of the central processing system. In nonobese humans, infusion of PYY3-36 during a 12-hour period decreased the total amount of food ingested from 2,200 to 1,500 k/cal—but without effect on food ingested during the next 12-hour interval.13 Although the pharmacology of this peptide is being elucidated, its specific role in obesity is not yet known. Glucagon-like peptide-1 (GLP-1): Those same intestinal L cells produce GLP-1 through post-translational processing of pre-proglucagon. Two equipotent forms of GLP-1 are generated: a glycine-extended form called GLP-1(7-37) and the amidated peptide GLP-1(7-36) amide.14 GLP-1 acts on the stomach to inhibit gastric emptying. This prolongs the time of absorption of a meal. GLP-1 also activates its receptor on pancreatic -cells to stimulate cAMP production, protein kinase A activation, and insulin secretion (Figure 19-2)—thereby improving glucose tolerance (a mechanism of the “incretin” effect). GLP-1 also acts on  cells to stimulate neogenesis, thereby increasing -cell mass.15 GLP-1 also exerts potent effects on reduction of appetite, through reduction in gastric emptying and through direct decreases of corticotropin-releasing hormone (CRH) signaling in the PVN and increasing leptin signaling in the VMH.16 Cholecystokinin (CCK): CCK is an 8-amino-acid gut peptide released in response to a caloric load. It circulates and binds to CCKA receptors in the pylorus, vagus nerve, NTS, and area postrema to promote satiety.3

Metabolic Afferents Leptin. Energy intake versus expenditure is normally regulated very tightly (within 0.15% per year) by the hormone leptin. Leptin is a 167-amino-acid hormone produced by adipocytes, which transmit the primary long-term signal of energy depletion/repletion to the VMH.17,18 Leptin’s primary neuroendocrine role is to mediate information about the size of peripheral adipocyte energy stores to the VMH. Leptin is a prerequisite signal to the VMH for the initiation of high-energy processes, such as puberty and pregnancy.19,20 Leptin reduces food intake and increases the activity of the sympathetic nervous system (SNS).21 Conversely, low leptin circulating levels infer diminished energy stores (which signal via the VMH to reduce energy expenditure), inhibit metabolic processes and increase appetite. Serum leptin concentrations drop precipitously (in excess of body fat loss) during periods of short-term fasting,22,23 and it seems likely that leptin functions primarily as a peripheral signal to the hypothalamus of inadequate caloric intake rather than specifically as a satiety signal.24 In the fed state, circulating levels of leptin correlate with percentage of body fat.25,26 Leptin production by adipocytes is stimulated by insulin and glucocorticoids,27,28 and is inhibited by -adrenergic stimulation.24 Programming of relative leptin concentrations by early caloric intake may be one mechanism that links early overnutrition with later obesity.29

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Leptin binds to its receptor (a member of the cytokine receptor superfamily) on target VMH neurons. There are four receptor isoforms formed by differential mRNA splicing: ObRa (an isoform with a shortened intracellular domain, which may function as a transporter), ObRb (the intact fulllength receptor), ObRc (also with a short intracellular domain), and ObRe (without an intracellular domain, but which may function as a soluble receptor).30 As leptin binds to its VMH receptor, three neuronal signals are transduced. The first is opening of an ATP-sensitive potassium channel, which hyperpolarizes the neuron and decreases its firing rate.31 The second is the activation of a cytoplasmic Janus kinase 2 (JAK2), which phosphorylates a tyrosine moiety on proteins of a family called signal transducers and activators of transcription 3 (STAT-3).32 The phosphorylated STAT-3 translocates to the nucleus, where it promotes leptin-dependent gene transcription.33 However, leptin also activates the insulin receptor substrate 2/phosphatidyl inositol-3-kinase (IRS2/PI3K) second-messenger system in VMH neurons—which increases neurotransmission of the central anorexigenic signaling pathway.34 Insulin. Insulin plays an extremely important role in energy balance35 because it is part of the afferent and efferent systems. On the afferent side, there is a significant insulin receptor density in a subpopulation of VMH neurons36—and there is coordinated transport of insulin across the blood-brain barrier.37 This suggests a central role for this hormone. In animals, acute intracerebroventricular (ICV) infusions of insulin decrease feeding behavior and induce satiety.38-40 The data on acute and chronic peripheral insulin infusions are less clear. Studies of overinsulinized diabetic rats demonstrate increased caloric intake (to prevent subacute hypoglycemia) and the development of peripheral insulin resistance.41,42 Chronic experimental peripheral insulin infusions decrease hepatic and skeletal muscle glucose uptake by decreasing Glut4 expression, but do not alter adipose tissue glucose uptake.43,44 One study in humans showed that injecting short-term insulin peripherally during meals did not have an effect on satiety.45 Insulin normally activates the insulin receptor substrate 2/phosphatidyl inositol-3-kinase (IRS2/PI3K) secondmessenger system in VMH neurons,46 which increases neurotransmission of the central anorexigenic signaling pathway. The importance of CNS insulin action was underscored by the construction of a brain/neuron-specific insulin receptor knockout (NIRKO) mouse, which cannot transduce a CNS insulin signal.47 Such mice become hyperphagic, obese, and infertile—with high peripheral insulin levels. These data suggest that peripheral insulin mediates a satiety signal in the VMH to help control energy balance.48 Various knockouts of the insulin signal transduction pathway that reduce insulin signaling lead to an obese phenotype,49,50 whereas those that increase insulin signaling lead to a lean phenotype.51,52

CENTRAL PROCESSING The peripheral afferent signals outlined previously reach neurons in the VMH, where they are integrated by a gated neural circuit designed to promote or diminish energy intake and expenditure (Figure 19-2). This circuit consists

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A

B

C

Figure 19-2 Central regulation of leptin signaling, autonomic innervation of the adipocyte and  cell, and the starvation response. (A) The arcuate nucleus transduces the peripheral leptin signal as one of sufficiency or deficiency. In leptin sufficiency, efferents from the hypothalamus synapse in the locus coeruleus—which stimulates the sympathetic nervous system. In leptin deficiency or resistance, efferents from the hypothalamus stimulate the dorsal motor nucleus of the vagus. (B) Autonomic innervation and hormonal stimulation of white adipose tissue. In leptin sufficiency, norepinephrine binds to the 3-adrenergic receptor—which stimulates hormone-sensitive lipase, promoting lipolysis of stored triglyceride into free fatty acids. In leptin deficiency or resistance, vagal acetylcholine increases adipose tissue insulin sensitivity (documented only in rats to date), promotes uptake of glucose and free fatty acids for lipogenesis, and promotes triglyceride uptake through activation of lipoprotein lipase. (C) Autonomic innervation and hormonal stimulation of the  cell. Glucose entering the cell is converted to glucose-6-phosphate by the enzyme glucokinase, generating ATP—which closes an ATP-dependent potassium channel, resulting in cell depolarization. A voltage-gated calcium channel opens, allowing for intracellular calcium influx—which activates neurosecretory mechanisms, leading to insulin vesicular exocytosis. In leptin sufficiency, norepinephrine binds to
2-adrenoceptors on the -cell membrane to stimulate inhibitory G proteins and decrease adenyl cyclase and its product cAMP—and thereby reduce protein kinase A levels and insulin release. In leptin deficiency or resistance, the vagus stimulates insulin secretion through three mechanisms. First, acetylcholine binds to a M3 muscarinic receptor, opening a sodium channel—which augments the ATP-dependent cell depolarization, increasing the calcium influx and insulin exocytosis. Second, acetylcholine activates a pathway that increases protein kinase C—which also promotes insulin secretion. Third, the vagus innervates L cells of the small intestine, which secrete glucagon-like peptide-1—which activates protein kinase A, contributing to insulin exocytosis. Octreotide binds to a somatostatin receptor on the  cell, which is coupled to the voltage-gated calcium channel—limiting calcium influx and the amount of insulin released in response to glucose.
2-AR,
2-adrenergic receptor; 3-AR, 3-adrenergic receptor; AC, adenyl cyclase; ACh, acetylcholine; DAG, diacylglycerol; DMV, dorsal motor nucleus of the vagus; FFA, free fatty acids; Gi, inhibitory G protein; GK, glucokinase; GLP-1, glucagon-like peptide-1; GLP-1R, GLP-1 receptor; Glu-6-PO4, glucose-6-phosphate; Glut4, glucose transporter-4; HSL, hormone-sensitive lipase; IML, intermediolateral cell column; IP3, inositol triphosphate; LC, locus coeruleus; LHA, lateral hypothalamic area; LPL, lipoprotein lipase; MARCKS, myristoylated alanine-rich protein kinase C substrate; NE, norepinephrine; PIP2, phosphatidylinositol pyrophosphate; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PVN, paraventricular nucleus; SSTR5, somatostatin-5 receptor; TG, triglyceride; VCa, voltage-gated calcium channel; VMH, ventromedial hypothalamus; and SUR, sufonylurea receptor. [From Lustig RH (2006). Childhood obesity: Behavioral aberration or biochemical drive? Reinterpreting the first law of thermodynamics. Nature Clin Pract Endo Metab 2:447–458. Courtesy of Nature Publishing Group, with permission, and also reprinted with permission of Springer Science and Business media.]

of two arms: the anorexigenic arm [which contains neurons expressing the colocalized peptides pro-opiomelanocortin (POMC) and cocaine/amphetamine-regulated transcript (CART)] and the orexigenic arm, which contains neurons with the colocalized peptides neuropeptide Y (NPY) and agouti-related protein (AgRP). Ghrelin receptor-

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immunoreactivity colocalizes with NPY and AgRP neurons, whereas insulin and leptin receptors are located on POMC/CART and NPY/AgRP neurons in the VMH53— suggesting divergent regulation of each arm. These two arms compete for occupancy of melanocortin receptors (MC3R or MC4R) in the PVN and LHA.

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Anorexigenesis, POMC/␣-MSH, and CART POMC is differentially cleaved in different tissues and neurons. The ligand
-melanocyte-stimulating hormone (
MSH) is the primary product involved in anorexigenesis. Overfeeding and peripheral leptin infusion induce the synthesis of POMC and
-MSH within the ARC.54
-MSH induces anorexia by binding to melanocortin receptors within the PVN or LHA. CART is a hypothalamic neuropeptide induced by leptin and reduced by fasting. Intrahypothalamic infusion blocks appetite, whereas antagonism of endogenous CART increases caloric intake.55

Orexigenesis, NPY, and AgRP NPY and AgRP colocalize to a different set of neurons within the ARC, immediately adjacent to those expressing POMC/CART.56 NPY has numerous functions within the hypothalamus, including initiation of feeding, initiation of puberty, regulation of gonadotropin secretion, and adrenal responsiveness.57,58 NPY is the primary orexigenic peptide. ICV infusion of NPY in rats rapidly leads to hyperphagia, energy storage, and obesity59,60 mediated through Y1 and Y5 receptors. Fasting and weight loss increase NPY expression in the ARC, accounting for increased hunger—whereas PYY3-36 (through Y2 receptors) and leptin decrease NPY mRNA.13,61 AgRP is the human homolog of the protein agouti, which is present in abundance in the yellow (Aya) mouse.62 This protein is an endogenous competitive antagonist of all MCRs, accounting for the yellow color in these mice. In the presence of large amounts of AgRP at the synaptic cleft in the PVN,
-MSH cannot bind to the MC4R to induce satiety.63

Other Neuroendocrine Modulators of Energy Balance Norepinephrine. Norepinephrine (NE) neurons in the locus coeruleus synapse on VMH neurons to regulate food intake.64 The actions of NE on food intake seem paradoxical because intrahypothalamic NE infusions stimulate food intake through effects on central
2- and -adrenergic receptors,65 whereas central infusion of
1agonists markedly reduces food intake.66 Serotonin. 5-HT has been implicated in the perception of satiety based on many lines of evidence: injection of 5-HT into the hypothalamus increases satiety, particularly with respect to carbohydrate;67 central administration of 5-HT2c receptor agonists increases satiety, whereas antagonists induce feeding;68 administration of selective 5-HT reuptake inhibitors induces early satiety;69 leptin increases 5-HT turnover;70 and the 5-HT2cR-KO mouse exhibits increased food intake and body weight.71 The role of 5-HT in the transduction of the satiety signal may have central and peripheral components because intestinal 5-HT is secreted into the bloodstream during a meal, where it may impact on gastrointestinal (GI) neuronal function and muscle tone and may bind to 5-HT receptors in the NTS to promote satiety.72 Melanin-concentrating Hormone. MCH is a 17-aminoacid peptide expressed in the zona incerta and LHA. MCH

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neurons synapse on neurons in the forebrain and locus coeruleus. MCH appears to be important in conditions such as anxiety and aggression around food.73 Expression of this peptide is upregulated in ob/ob mice. MCH-knockout mice are hypophagic and lean,74 whereas transgenic MCHoverexpressing mice develop obesity and insulin resistance.75 ICV administration of MCH stimulates food intake, similar to that seen with NPY administration.76 Orexins A and B. These 33- and 28-amino acid peptides, respectively, have been implicated in energy balance and autonomic function in mice.77 Orexin-knockout mice demonstrate narcolepsy, hypophagia, and obesity78—suggesting that orexins bridge the gap between the afferent and efferent energy balance systems.79 Orexins in the LHA stimulate NPY release, which may account for their effects on orexigenesis. They also stimulate the corticotropin-releasing factor (CRF) and SNS output to increase wakefulness and energy expenditure, learning and memory, and the hedonic reward system.80 Conversely, orexin neurons in the perifornical and dorsomedial hypothalamus regulate arousal and response to stress. Endocannabinoids. It has long been known that tetrahydrocannibinol stimulates food intake. This observation led to the identification of endogenous ECs and their receptor, termed CB1.81 The CB1 receptor is expressed in corticotropin-releasing hormone (CRH) neurons in the PVN, in CART neurons in the VMN, and in MCH- and orexin-positive neurons in the LHA and perifornical region. Fasting and feeding are associated with high and low levels of ECs in the hypothalamus, respectively. For example, CB1 receptor-knockout mice have increased CRH and reduced CART expression. In the ob/ob mouse, hypothalamic EC levels are increased—whereas leptin infused intravenously reduces these levels, indicating that a direct negative control is exerted by leptin on the EC system. Glucocorticoids increase food intake by stimulating EC synthesis and secretion, whereas leptin blocks this effect.82 Finally, the presence of CB1 receptors on afferent vagal neurons suggests that endocannabinoids may be involved in mediating satiety signals originating in the gut.

Melanocortin Receptors and Central Neural Integration The human MC4R localizes to chromosome 2 and is a 7-transmembrane G-coupled receptor encoded by an intronless 1-kB gene. The binding of hypothalamic
MSH to the MC4R in the PVN and LHA results in a state of satiety, whereas ICV administration of MC4R antagonists stimulate feeding—suggesting that MC4R transduces satiety information on caloric sufficiency. In the MC3R knockout mouse, a different phenotype is seen. These animals are obese, but they are instead hypophagic and have increased body fat for their lean mass. They gain weight on low- or high-fat chow, and do not change caloric oxidation in response to changes in dietary fat content—suggesting a defect in energy expenditure.83 Thus, these two hypothalamic MCRs appear to modulate different aspects of energy metabolism. One hypothesis is that the MC4R modulates energy intake and the MC3R modulates energy expenditure.84

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THE EFFERENT SYSTEM The MCRs in the PVN and LHA transduce the anorexigenic and orexigenic information coming from the VMH in order to modulate activity of the SNS, (which promotes energy expenditure) and of the efferent vagus, which promotes energy storage (Figure 19-2). In this way, peripheral energy balance can be modulated acutely to provide requisite energy for metabolic needs and to store the rest.

The Sympathetic Nervous System and Energy Expenditure Anorexigenic pressure increases energy expenditure through activation of the SNS.85 For instance, leptin adminstration to ob/ob mice promotes increased brown adipose tissue lipolysis, thermogenesis, renovascular activity, and increased movement—all associated with increased energy expenditure, which assists in weight loss.86 Similarly, insulin administration acutely increases SNS activity in normal rats and in humans.87,88 The magnitude of energy expenditure also has a salutary effect on quality of life. Those factors that reduce resting energy expenditure (REE; e.g., hypothyroidism) reduce quality of life, whereas those factors that increase REE (e.g., caffeine) increase quality of life (at least acutely). The SNS increases energy expenditure in four ways: by innervating the hypothalamus and appetite centers in the medulla to reduce appetite, by increasing TSH secretion to increase thyroid hormone release and energy expenditure, by innervating skeletal muscles to increase energy expenditure, and by innervating 3-adrenergic receptors in white adipose tissue to promote lipolysis. Activation of the SNS increases energy expenditure at the skeletal muscle by activating 2-adrenergic receptors,89 which in turn increase the expression of numerous genes in skeletal muscle90—especially those involved in carbohydrate metabolism. SNS activation stimulates glycogenolysis, myocardial energy expenditure, glucose and fatty acid oxidation, and protein synthesis.91 Activation of the SNS in rodents stimulates the 3-adrenergic receptor of brown adipose tissue to promote lipolysis.92 In humans, activation of the 3-adrenergic receptor increases cAMP—which activates protein kinase A (PKA). PKA acts in two separate molecular pathways to increase energy expenditure. First, PKA phosphorylates cyclic AMP response element binding protein (CREB)—which induces expression of PPAR-coactivator-1
(PGC-1
). PGC-1
then binds to enhancer elements on the uncoupling protein-1 (UCP1) gene, which increases the expression and activity of uncoupling proteins (UCPs) 1 and 2.93,94 UCPs reduce the proton gradient across the inner membranes of mitochondria, which thereby diverts protons from storage in the form of ATP to heat production. Originally, UCPs were discovered in brown adipose tissue and were found to be responsible for thermogenesis. UCP1 is an inner membrane mitochondrial protein that uncouples proton entry from ATP synthesis.95 Therefore, UCP1 expression dissipates energy as heat—thus reducing the energy efficiency of the adipose tissue. However, UCP2 has been found in most tissues and UCP3 in skeletal muscle. Second, PKA activation acti-

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vates the enzyme hormone-sensitive lipase (HSL), which is responsible for lipolysis of intracellular triglyceride to its component free fatty acids (FFAs). The FFAs also induce UCP1, further increasing energy expenditure. The FFAs released from the adipocyte also travel to the liver, where they are utilized for energy by metabolizing into two-carbon fragments. Lipolysis reduces leptin expression. Thus, a negative feedback loop is achieved between leptin and the SNS (Figure 19-2).

The Efferent Vagus and Energy Storage In response to declining levels of leptin and/or persistent orexigenic pressure, the LHA and PVN send efferent projections residing in the medial longitudinal fasciculus to the dorsal motor nucleus of the vagus nerve (DMV)— activating the efferent vagus.96 The efferent vagus opposes the SNS by promoting energy storage in four ways: by slowing the heart rate, myocardial oxygen consumption is reduced; the vagus nerve promotes alimentary peristalsis, pyloric opening, and energy substrate absorption; through direct effects on the adipocyte, the vagus nerve promotes insulin sensitivity to increase the clearance of energy substrate into adipose tissue; and through effects on the  cells, the vagus increases postprandial insulin secretion—which promotes energy deposition into adipose tissue.97-100 Retrograde tracing of white adipose tissue reveals a wealth of efferents originating at the DMV.100 These efferents synapse on the M1 muscarinic receptor on the adipocyte, which increases insulin sensitivity of the adipocyte. Denervation of white adipose tissue results in reduction of glucose and FFA uptake and in induction of HSL, which promotes lipolysis—both of which reduce the efficiency of insulin-induced energy storage. Thus, vagal modulation of the adipocyte augments storage of glucose and FFAs by improving adipose insulin sensitivity101 (Figure 19-2). The DMV also sends efferent projections to the  cells of the pancreas.102 This pathway is responsible for the “cephalic” (preabsorptive) phase of insulin secretion, which is glucose independent and can be blocked by atropine.103 Overactive vagal neurotransmission increases insulin secretion from  cells in response to an oral glucose load through the following three distinct but overlapping mechanisms104 (Figure 19-2). • Vagal firing increases acetylcholine availability and binding to the M3 muscarinic receptor on the  cell, which is coupled to a sodium channel within the pancreatic -cell membrane.105 As glucose enters the  cell after ingestion of a meal, the enzyme glucokinase phosphorylates glucose to form glucose6-phosphate—increasing intracellular ATP, which induces closure of the ATP-dependent potassium channel. Upon channel closure, the  cell experiences an ATP concentration-dependent -cell depolarization106,107 and opening of a separate voltagegated calcium channel within the membrane. Intracellular calcium influx increases acutely, which results in rapid insulin vesicular exocytosis. Concomitant opening of the sodium channel by vagally mediated acetylcholine augments -cell depolarization,

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which in turn augments the intracellular calcium influx and results in insulin hypersecretion.108-110 • Vagally mediated acetylcholine increases phospholipases A2, C, and D within the  cell—which hydrolyze intracellular phosphatidylinositol to diacylglycerol (DAG) and inositol triphosphate (IP3).104 DAG is a potent stimulator of protein kinase C (PKC),111 which phosphorylates myristoylated alanine-rich PKC substrate (MARCKS)—which then binds actin and calcium-calmodulin and induces insulin vesicular exocytosis.112 IP3 potentiates release of calcium within  cells from intracellular stores, which also promotes insulin secretion.113 • The vagus also stimulates the release of GLP-1 from intestinal L cells, which circulates and binds to a GLP1 receptor within the -cell membrane. Activation of this receptor induces a calcium-calmodulin-sensitive adenyl cyclase, with conversion of intracellular ATP to cAMP—which then activates PKA. PKA causes the release of intracellular calcium stores and the phosphorylation of vesicular proteins, each contributing to an increase in insulin exocytosis.14,114 In the efferent pathway, insulin is responsible for shunting blood-borne nutrients into adipose for storage. Indeed, the primary hormonal signal for adipogenesis is insulin.115 Within the adipocyte, insulin increases Glut4 expression, acetyl-CoA carboxylase, fatty acid synthase, and lipoprotein lipase.116 Thus, the net effect of insulin on the adipocyte is the rapid clearance and storage of circulating glucose and lipid. Thus, insulin promotes energy storage.

NEGATIVE FEEDBACK MODULATION OF ENERGY BALANCE: THE STARVATION RESPONSE The regulation of the components of the energy balance system is manifest during the starvation response. Everyone has a “personal leptin threshold” (probably genetically set) above which the brain interprets a state of energy sufficiency.117 Thus, the leptin-replete state is characterized by increased physical activity, decreased appetite, and increased feelings of well-being. However, in response to caloric restriction leptin levels decline even before weight loss is manifest22,23—which is interpreted by the VMH as starvation. Gastric secretion of ghrelin is increased, which increases pituitary GH release in order to stimulate lipolysis to provide energy substrate for catabolism. Ghrelin stimulates NPY/AgRP to antagonize
-MSH/ CART. Decline of leptin reduces
-MSH/CART as well. This leads to decreased MC4R occupancy. The resultant lack of anorexigenic pressure on the MC4R results in increased feeding behavior and energy efficiency (with reduced fat oxidation) in order to store energy as fat. In response, the efferent pathway of energy balance coordinates efforts at improving energy efficiency and increasing energy storage. Total and resting energy expenditure decline in at attempt to conserve energy.118 Specifically, UCP1 levels within adipose tissue decline119 as a result of decreased SNS activity in response to starvation.120 However, in spite of decreased SNS tone at the adipocyte there is clearly an obligate lipolysis (due to insulin

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suppression and up-regulation of HSL)—which is necessary to maintain energy delivery to the musculature and brain in the form of liver-derived ketone bodies. In addition, in the starved state vagal tone is increased in order to slow the heart rate and myocardial oxygen consumption, increase -cell insulin secretion in response to glucose, and increase adipose insulin sensitivity—all directed at increasing energy storage.120 These revert back to baseline once caloric sufficiency is reestablished, and leptin levels rise.

THE HEDONIC PATHWAY OF FOOD REWARD The negative feedback pathway delineated previously is not the only site of central regulation of food intake. Complementary to insulin and leptin’s ability to alter energy balance, these hormones also modify the hedonic pathway—the pleasurable and motivating responses to food. This is the same pathway that responds to drugs of abuse, such as nicotine and morphine. The hedonic pathway comprises the ventral tegmental area (VTA) and the nucleus accumbens (NA), with input from various components of the limbic system—including the striatum, amygdala, hypothalamus, and hippocampus. Food intake is a readout of the hedonic pathway. Administration of morphine to the NA increases food intake in a dose-dependent fashion.121 When functional, the hedonic pathway helps curtail food intake in situations in which energy stores are replete. However, dysfunction of this pathway can increase food intake—leading to obesity. The VTA appears to mediate feeding on the basis of palatability rather than energy need. The dopaminergic projection from the VTA to the NA mediates the motivating, rewarding, and reinforcing properties of various stimuli (such as food and addictive drugs). Leptin and insulin receptors are expressed in the VTA, and both hormones have been implicated in modulating rewarding responses to food and other pleasurable stimuli.122 For instance, fasting and food restriction (where insulin and leptin levels are low) increase the addictive properties of drugs of abuse—whereas ICV leptin can reverse these effects.123 In rodent models of addiction, increased addictive behavior and pleasurable response from a food reward (as measured by dopamine release and dopamine receptor signaling) are greater after food deprivation.124 Acutely, insulin increases expression and activity of the dopamine transporter—which clears and removes dopamine from the synapse. Thus, acute insulin exposure blunts the reward of food.125 Furthermore, insulin appears to inhibit the ability of VTA agonists (e.g., opioids) to increase intake of sucrose.126 Finally, insulin blocks the ability of rats to form a conditioned place preference association to a palatable food.127 However, insulin resistance of this pathway may lead to increased reward of food. The role of the hedonic pathway in human obesity is not yet elucidated, but may be surmised.

LEPTIN RESISTANCE Most obese children have high leptin levels but do not have receptor mutations, manifesting what is commonly referred to as leptin resistance. Leptin resistance prevents

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exogenous leptin administration from promoting weight loss.128 The response to most weight loss regimens plateaus rapidly due to the rapid fall of peripheral leptin levels below a personal “leptin threshold,”129 which is likely genetically determined. Leptin decline causes the VMH to sense a reduction in peripheral energy stores, which modulates a decrease in REE to conserve energy— analogous to the starvation response118 but occurring at elevated leptin levels. The cause of leptin resistance is unknown, but may have several etiologies. Leptin crosses the blood-brain barrier via a saturable transporter, which limits the amount of leptin reaching its receptor in the VMH.130,131 Activation of the leptin receptor induces intraneuronal expression of suppressor of cytokine signaling 3 (SOCS-3), which limits leptin signal transduction.51 Dietary fat limits access of peripheral leptin to the VMH, and interferes with leptin signal transduction upstream of STAT-3 (its primary second messenger).132 Two clinical paradigms have been shown to improve leptin sensitivity. After weight loss through caloric restriction, exogenous administration of leptin can then increase REE back to baseline and permit further weight loss133,134—suggesting that the weight loss itself improves leptin sensitivity. Second, suppression of insulin correlates with improvement in leptin sensitivity and promotes weight loss135—suggesting that hyperinsulinemia promotes leptin resistance by interfering with leptin signal transduction in the VMH and VTA.136 Indeed, insulin reduction strategies can be effective in promoting weight loss in children with hyperinsulinemia by improving leptin sensitivity.137 This has led to the hypothesis that chronic hyperinsulinemia functions to block leptin signal transduction at the VMH and VTA, which turns a negative feedback cycle into a vicious feed-forward cycle18 (Figure 19-3). However, this hypothesis remains to be proven.

Energy Excess: Obesity The rise in the prevalence of obesity in children and adolescents is one of the most alarming public health issues facing the world today. Obesity is associated with significant health problems in children and is an early

risk factor for much of adult morbidity and mortality.138 It is also a major contributor to increasing health care expenditures. Importantly, childhood obesity tends to track to adulthood and thus represents an early beginning of a potentially lethal pathologic process.

DEFINITION The theoretical definition of obesity is a degree of somatic overweight that causes detrimental health consequences.139 Based on morbidity and mortality statistics, and with a desire to prevent future risk of morbidity, we practically define obesity as a statistical magnitude of overweight for a population—keeping in mind that morbidity and mortality vary with degree of overweight in different racial, ethnic, and socioeconomic groups140 and indeed among individuals. The World Health Organization141 categorizes adult overweight into four subgroups based on body mass index [BMI; weight (kg) ÷ height (m2)]: BMI 25 to 30 (overweight); BMI 30 to 35, grade 1 (moderately obese); BMI 35 to 40, grade 2 (severely obese); and BMI 40, grade 3 (morbidly obese). Some make a further delineation at BMI 60, denoting this as “superobesity” because even surgical therapies are less effective in this range. The majority of obesity in adulthood has its origins in childhood,142,143 making obesity a pediatric concern—and the prevention and treatment of obesity a pediatric goal. BMI is also the accepted marker in children.144 In childhood, comparison of BMI to normal curves for age145 allows for categorization of BMI above the 85th percentile as overweight and above the 95th percentile as obese (Figure 19-4A and B).

PREVALENCE AND EPIDEMIOLOGY The prevalence of childhood obesity in the United States has increased dramatically during the past 30 years,146,147 although the comparison of longitudinal and crosssectional data is difficult due to different definitions and measurement parameters among epidemiologic studies. The most recent estimates of obesity prevalence in the United States are based on data from the 1999-2000 National Health and Nutrition Examination Survey (NHANES IV).147 NHANES demonstrates that the epidemic of childhood obesity is occurring at earlier ages. Based on the

Figure 19-3 Postulated algorithm describing the role of hyperinsulinemia in the dysfunction of the energy balance pathway by promoting energy storage into adipocytes; by interfering with leptin signal transduction in the hypothalamus, promoting the starvation response; and by interfering with dopamine clearance at the nucleus accumbens, thereby increasing the reward of food. Each of these alterations turns a negative feedback pathway into a “vicious cycle.” [From Isganaitis E, Lustig RH (2005). Fast food, central nervous system insulin resistance, and obesity. Arterioscler Thromb Vasc Biol 25:2451-2462. Courtesy of the American Heart Association, with permission.]

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1999-2000 NHANES, 20.6% of 2- to 5-year-old U.S. children were overweight, defined as BMI for age 85th percentile. The prevalence of obesity in older children was even higher: 30.3% of 6- to 11-year-old children and 30.4% of adolescents (12-19 years old) have now become obese. The prevalence of obesity (defined as weight for length 95th percentile in 2-year-old children or younger, or BMI greater than 95th percentile in older children) among children aged 0 to 23 months, 2 to 5 years, 6 to 11 years, and adolescents were 11.4%, 10.4%, 15.3%, and 15.5%, respectively. The prevalence of obesity among both sexes was not significantly different. It was slightly higher in 2- to 5-year-old females (11.0% versus 9.9%), 6- to 11year-old males (16.0% versus 14.5%), and similar (15.5%) in adolescents. In all age ranges, the prevalence has increased compared with the previous report by NHANES

III (1988-1994)—with the greatest change (from approximately 11% to 15%) among 6- to 19-year-olds.

GLOBAL PREVALENCE Obesity has overtaken AIDS and malnutrition as the number one public health problem in the world.148 The global prevalence of childhood obesity has been increasing worldwide at an alarming rate during the past 20 years. Rates have increased 2.7-fold to 3.8-fold over 29 years in the United States,147 2.0-fold to 2.8-fold over 10 years in England, 3.4-fold to 4.6-fold over 10 years in Australia, and 3.4-fold to 3.6-fold over 23 years in Brazil. In Asia, the prevalence has increased 1.1-fold to 1.4-fold over 6 years in China and 2.3-fold to 2.5-fold over 26 years in Japan. In Africa, the prevalence has increased 3.9-fold over 18 years in Egypt,

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3.8-fold over 6 years in Ghana, and 2.5-fold over 5 years in Morocco.149 Even in Japan, the rate has doubled from 5% to 10% in the last 10 years.150,151 In developed countries, the urban poor are more susceptible to obesity, presumably due to poor dietary practices and limited opportunity for physical activity.152,153 In contrast, obesity is more frequent in the upper socioeconomic class of developing countries— probably due to transition to a more Western lifestyle, with a more energy-dense diet consisting of higher fats and sugar (which tend to be more palatable at a lower cost).154,155 This may be due to specific properties of processed food, which promote leptin resistance.136

RACIAL AND ETHNIC CONSIDERATIONS The NHANES surveys only list prevalence among Caucasians, African Americans, and Hispanics despite the fact that Native Americans, Pacific Islanders, Asians, and other racial/ethnic groups are experiencing rapid in-

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creases in prevalence as well. Across racial groups, there is a marked dichotomy in the prevalence and rate of increase in childhood obesity.147,156 For instance, the prevalence among African-American (23.6%) and Hispanic (23.4%) adolescents is twice that than among white adolescents (12.7%)—and the rate of increase in the prevalence of obesity among African-American and Hispanic adolescents almost doubled between 1988 to 1994 and 1999 to 2000, from 13.4% to 23.6% in African Americans and from 13.8% to 23.4% in Hispanics. Analyses based on the adult definition of obesity (BMI 30 kg/m2) indicated that 11.2% of adolescents in general (but approximately 20% of African-American female adolescents) fit into this category. Within these high-risk groups, female African-American adolescents and male 6- to 19-year-old Hispanics exhibit the highest prevalence of obesity (26.6%-27.5%). In the NHLBI Growth and Health Study,157 the prevalence of obesity in 9-year-old African-American girls was 17.7% and in 9-year-old Caucasian girls 7.7%—and both of these prevalences doubled

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over the 10 years of study. These prevalences are true at younger ages as well. The 1994 Pediatric Nutrition Surveillance System (PedNSS) indicated that 12% of 2- to 4-year-old Native American children were overweight, which is similar to Hispanic children at the same age (12%) but much higher than white children (6%). The prevalence of overweight at 5 to 6 years in Native Americans is twice that in U.S. youth in general, and the prevalence of obesity is three times higher.158 Among infants and toddlers less than 2 years of age, the prevalence of obesity is highest in African Americans (18.5%)—compared to 10.1% in Caucasians and 13.7% in Hispanics. It is possible that different dietary practices may account for some of these differences. For instance, a study of 2-year-old Latino children in California correlated obesity with early consumption of sugar-sweetened beverages.159 Within racial populations, ethnic variability in the prevalence of childhood obesity has also been noted. The United States National Longitudinal Study of Adolescent Health (Add Health) indicated that the BMI 85th percentile in adolescent Hispanics was more common among Mexican Americans (32.1%) and Puerto Ricans (30.3%) compared with Cuban Americans (27.1%) and Central/South Americans (26.2%).153 Only 25% of firstgeneration Hispanic adolescents were overweight based on BMI 85th percentile compared with 32% of secondand third-generation Hispanics. The prevalence of overweight in Asian-American adolescents in this study was 20.6%, with comparable prevalence among Filipinos (18.5%) and Chinese (15.3%). Again, only 12% of first-generation Asian Americans were overweight—compared with 27% and 28%, respectively, of second and third generations. In Native Americans, among the studies performed between 1990 and 2000 there is great variation in the prevalence of obesity (12%77%) based on tribe, age group, measurement tool, and cutoff value.158 These studies indicate that obesity in Native Americans begins very early in childhood.

PREDICTIVE FACTORS The higher the BMI during childhood the more likely adult obesity will manifest. In general, children with a BMI 95th percentile have a very high risk for adult obesity.160 Obesity in adolescence is a primary risk factor for obesity in adulthood, with an increased odds ratio from 1.3 for obesity at 1 to 2 years of age to 17.5 for obesity at 15 to 17 years of age.161 The change of BMI during and after adolescence was the most important predictive variable for adult obesity.162 Long-term studies suggest that between 50% and 75% of all obese adolescents will become obese adults, and more than one-third of 18-year-olds with BMI greater than the 60th percentile will also be overweight as adults. Children and adolescents with BMI 95th percentile have a 62% to 98% chance of being obese at 35 years of age, with a 50% chance in males aged 13 years and 66% chance in girls aged 13 years.163 The age of adiposity rebound, the point of the BMI nadir before the body fatness begins to rise (between 5 and 6 years of age; Figure 19-4A and B), is also an important predictor for adult obesity.164 Girls tend to

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have slightly earlier adiposity rebound than do boys. Children with early adiposity rebound have a fivefold greater chance of becoming obese as adults, compared with those with late adiposity rebound. At the age of adiposity rebound, children already overweight have a sixfold greater risk for adult obesity compared to lean children. Therefore, the earlier the onset of childhood obesity the greater the risk for adult obesity. Infant overnutrition plays an extremely important role in the future development of obesity. Numerous studies have implicated bottle feeding as a specific risk factor.165 The prevalence of obesity in children who were never breast fed was 4.5%, compared with 2.8% in breast-fed children—and a clear time-response effect was identified for the duration of breast feeding on the decline in prevalence of obesity.166 Early overnutrition has been correlated with elevated leptin concentrations in later life.29 Differences in volume and composition of commercial formula versus breast milk have been proposed as etiologic factors. Parental obesity is also an important predictor of childhood obesity. Children with at least one overweight parent at the age of adiposity rebound have a four- to fivefold greater chance of becoming obese adults. Lean children 5 years or younger have a thirteenfold risk of adult obesity if both parents are obese. Conversely, older children (10–14 years of age) who are obese have a 22.3fold increased risk of adult obesity regardless of parental weight.143 Parental obesity is related to early adiposity rebound, suggesting that genetic influences predominate in childhood weight gain.167 Obesity and type 2 diabetes often run in families, particularly in minorities with lower socioeconomic backgrounds.168

Metabolic Impact of Childhood Obesity Many of the metabolic and CV complications of obesity are already evident during childhood, and are closely related to the development of insulin resistance and hyperinsulinemia—the most common biochemical abnormality seen in obesity. The obesity-related co-morbidities that emerge early in childhood are alterations in glucose metabolism, dyslipidemia, and hypertension. Although an accelerated atherogenic process is present in obese children, thrombotic CV events do not usually appear until adulthood. The clustering of these manifestations is termed the metabolic syndrome. In addition, nonalcoholic fatty liver disease (NAFLD) and polycystic ovarian syndrome are often related—as are other nonendocrine morbidities.

LIPID PARTITIONING The term lipid partitioning refers to the distribution of excess body fat in various organs and compartments. The majority of excess fat is stored in its conventional subcutaneous depot, yet other potential storage sites exist— such as the intra-abdominal (visceral) fat compartment and insulin-sensitive tissues such as muscle and liver. One hypothesis to explain the relation between obesity

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and insulin resistance is the “portal-visceral” paradigm.169 Associations among visceral adiposity, insulin resistance, and co-morbidities have been demonstrated across most age groups and ethnicities.170 This hypothesis claims that increased adiposity causes accumulation of fat in the visceral depot, leading to an increased portal and systemic free fatty acid (FFA) flux.171 Increased circulating FFA inhibits insulin action in insulin-sensitive tissues by a “competitive” mechanism, proposed by Randle.172 Of note, studies of in vivo FFA fluxes from the visceral and the subcutaneous truncal and abdominal depots have failed to demonstrate a substantial difference in net fluxes between these depots. Recent studies demonstrate that subcutaneous fat, which does not drain into the portal system, is strongly related to insulin resistance in healthy obese and in diabetic men.173 Similarly, truncal subcutaneous fat mass has been demonstrated to independently predict insulin resistance in obese women. Visceral and subcutaneous fat differ in their biologic responses,174 with visceral fat more resistant to insulin and more sensitive to catecholamines. These observations emphasize that visceral and subcutaneous abdominal fat can contribute to insulin resistance, possibly by different mechanisms.175 An alternative theory to explain the relation between obesity and insulin resistance is the “ectopic lipid deposition” paradigm.176 This theory is based on the observation that lipid content in muscle is increased in obesity and in type 2 diabetes mellitus (T2DM) and is a strong predictor of insulin resistance.177 Moreover, in conditions such as lipodystrophies all fat is stored in liver and muscle due to lack of subcutaneous fat tissue—causing severe insulin resistance and diabetes.178 In obese adults (BMI 30), muscle attenuation on CT (representing lipid content) is a stronger predictor of insulin resistance than is visceral fat.179 Studies performed in vivo using 1H-NMR spectroscopy demonstrated that increased intramyocellular lipid (IMCL) content is a strong determinant of insulin resistance in humans.180 Thus, obesity co-morbidity may begin when the subcutaneous fat reaches its capacity to store excess fat and begins to shunt lipid to ectopic tissues (such as liver and muscle)—leading to peripheral insulin resistance.181 Another postulated cause of intramuscular lipid accumulation is a reduction of fat oxidation,182 related to low aerobic capacity or reduced SNS tone. The effect of IMCL accumulation on peripheral sensitivity is postulated to be due to an alteration of the insulin signal transduction pathway in muscle caused by derivates of fat such as long-chain fatty acyl-CoA and diacylglycerol within the myocyte. These derivates activate the serine/threonine kinase cascade and cause serine phosphorylation of IRS-1, which inhibits insulin signaling.183 A comparable mechanism has been demonstrated in the liver, where accumulation of lipids (in particular, diacylglycerol) activates the inflammatory cascade by inducing c-jun N-terminal kinase (JNK-1)—which causes serine rather than tyrosine phosphorylation of IRS-1, leading to inhibition of hepatic insulin signaling.184,185 Studies in obese children have shown a strong association between IMCL accumulation and peripheral insulin sensitivity.186 In addition, IMCL is greater in those with

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impaired glucose tolerance in comparison to more insulin-sensitive adolescents with normal glucose tolerance.184 Similarly, an inverse relation between the degree of visceral adiposity and insulin sensitivity has been shown in obese children.187 These observations imply that IMCL and hepatic lipid accumulation may play a pivotal role in the development of peripheral insulin resistance.

ADIPOCYTOKINES Leptin The discovery of leptin in 1994 has dramatically changed the view of adipose tissue in the regulation of energy balance.17 Adipocytes secrete several proteins that act as regulators of glucose and lipid homeostasis.188 These proteins have been collectively referred to as adipocytokines because of their structural similarity with cytokines. Circulating leptin levels correlate with the degree of obesity. As stated previously, the primary role of leptin is to serve as an adiposity sensor to protect against starvation. Leptin probably has a permissive role in high-energy metabolic processes such as puberty, ovulation, and pregnancy. However, its role in states of energy excess is less known. In obesity, the development of leptin resistance may result in a breakdown of the normal partitioning of surplus lipids in the adipocyte compartment.189

Adiponectin The cytokine adiponectin is peculiar in obesity because in contrast to the other adipocytokines its level is reduced in obesity.190 The adiponectin gene is expressed exclusively in adipose tissue and codes a protein, a carboxyl-terminal globular head domain, and an aminoterminal collagen domain—which is structurally reminiscent of the complement factor 1q.191 The gene is located on chromosome 3q27, a location previously linked to the development of type 2 diabetes and the metabolic syndrome. Several single-nucleotide polymorphisms (SNPs) in the adiponectin gene have been reported to be associated with the development of type 2 diabetes in populations around the world, suggesting that adiponectin plays a major role in glucose metabolism.192 Adiponectin circulates in plasma in three major forms: a low-molecular-weight trimer, a middle-molecularweight hexamer, and a high-molecular-weight 12- to 18-mer.193 Circulating plasma adiponectin concentrations demonstrate a sexual dimorphism (females have greater concentrations), suggesting a role for sex hormones in the regulation of adiponectin production or clearance. Dietary factors such as linoleic acid or fish oil versus a high carbohydrate diet or increased oxidative stress have been shown to increase or decrease adiponectin concentrations, respectively. These observations suggest that the circulating levels of adiponectin are regulated by complex interactions between genetic and environmental factors.194 The receptors for adiponectin have recently been characterized in rodent models and cloned. Two receptors (ADIPOR1 and ADIPOR2) have been characterized. ADIPOR1 is

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expressed in numerous tissues, including muscle—whereas ADIPOR2 is largely restricted to the liver. Both receptors are bound to the cell membrane, yet are unique in comparison to other G-protein–coupled receptors in the fact that the C terminal is external and the N terminal is intracellular.195 ADIPOR1 and ADIPOR2 are receptors for the globular head of adiponectin and serve as initiators of signal transduction pathways that lead to increased PPAR
and AMP kinase activities, which promote glucose uptake and increased fatty acid oxidation. Adiponectin has been shown to have potent antiatherogenic functions because it accumulates in the subendothelial space of injured vascular walls to reduce the expression of adhesion molecules and the recruitment of macrophages.196 Studies in obese children and adolescents have shown that adiponectin is inversely related to the degree of obesity, insulin sensitivity visceral adiposity, and IMCL—whereas weight loss increases adiponectin. A fall in adiponectin has been shown to coincide with the onset of insulin resistance197 and the development of diabetes in monkeys.198 All of these observations, along with human clinical data, support a pivotal role for adiponectin in the prevention of the co-morbidities of the metabolic syndrome.

Inflammatory Cytokines Recent accumulating evidence indicates that obesity is associated with subclinical chronic inflammation.199 The adipose tissue serves not merely as a simple reservoir of energy stored as triglycerides but as an active secretory organ releasing many peptides, including inflammatory cytokines, into the circulation. In obesity, the balance among these numerous peptides is altered such that larger adipocytes and macrophages embedded within them produce more inflammatory cytokines (e.g., TNF-
, IL-6) and fewer anti-inflammatory peptides such as adiponectin.200 One theory posits that as energy accumulates in adipocytes the perilipin border of the fat vacuole breaks down, causing the adipocyte to die.201 Cell death recruits macrophages in the adipose tissue, especially the visceral compartment, which in the process of clearing debris also elaborate inflammatory cytokines—initiating a proinflammatory milieu that predates and possibly drives the development of systemic insulin resistance, diabetes, and endothelial dysfunction.202,203 Systemic concentrations of C-reactive protein (CRP) and IL-6, two major markers and participants of the inflammatory process, are increased in obese children and adolescents. CRP levels within the high-normal range have been shown to predict CV disease204 and the development of T2DM205 in adults. Elevated levels of CRP correlate with other components of the metabolic syndrome in obese children.206,207 Thus, inflammation may be one of the links between obesity and insulin resistance—and may promote endothelial dysfunction and early atherogenesis.

Other Adipocytokines Several other novel adipocytokines have been identified recently, but their clinical significance in humans is unclear. Among them is visfatin,208 which is secreted exclusively from adipose tissue, is correlated with the amount

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of visceral fat, and has insulin mimetic effects on adipogenesis in vitro. Another novel adipocytokine is resistin, a 12.5-kDa polypeptide hormone produced by adipocytes in rodents and by immunocompetent cells in humans. In rodents, resistin appears to have an important role in the development of hepatic insulin resistance. However, its role in humans is less clear but may be related to involvement in the regulation of inflammatory processes rather than in tissue-specific insulin sensitivity.

INSULIN RESISTANCE Insulin resistance presents in the obese child as altered glucose metabolism manifest as impaired glucose tolerance or overt T2DM, dyslipidemia, vascular changes culminating in hypertension, NAFLD, and/or polycystic ovarian syndrome (PCOS). The development of T2DM is covered in depth in Chapter 10. However, it is worth noting that impaired glucose tolerance (IGT)—known as pre-diabetes—is a relatively common condition in obese children and adolescents. The prevalence of IGT among obese children and adolescents is reported to be greater than 20%.209 Higher prevalence rates of IGT have been reported in obese children from Thailand and the Philippines, in Latino children living in the United States,210 and in Germany211—whereas a lower prevalence rate (15%) was found in obese children in France. IGT in obese youth is typically characterized by obesity with an unfavorable pattern of lipid partitioning, with increased deposition of fat in the visceral and IMCL compartments.212

VASCULAR CHANGES Early stages of the atherosclerotic process may be detected in obese children. In recent years, it has become clear that endothelial dysfunction represents a key early step in the development of atherosclerosis.213 The hallmark and cause of endothelial dysfunction is impairment in nitric oxide (NO)-mediated vasodilatation.214 This is due to decreased NO production by endothelial nitric oxide synthase (eNOS), which has been postulated to result from high levels of FFAs and inflammatory cytokines (IL-6, TNF-
; in insulin-resistant obese individuals), from increased reactive oxygen species, or from increased uric acid (which inhibits eNOS activity).215 Decreased NO bioavailability leads to an imbalance between vasodilating and vasoconstricting factors (such as endothelin), which leads to impaired vascular smoothmuscle relaxation, increased adhesion of inflammatory cells to the endothelium, increased expression of plasminogen activator inhibitor-1 (PAI-1; a prothrombotic molecule), and increased vascular smooth-muscle cell proliferation. Thus, decreased NO bioavailability is thought to create a proinflammatory prothrombotic environment that promotes atherosclerosis.216 Endothelial function represents an integrated index of the overall CV risk burden in any given individual. During the last decade, noninvasive techniques for the assessment of endothelial function [including high-resolution external vascular ultrasound to measure flow-mediated endothelium-dependent dilatation (FMD) of the brachial

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artery during hyperemia] have been developed.217,218 Impaired FMD correlates with arterial wall stiffness, coronary dilatation, and endothelial dysfunction in obese children.219 Similarly, anatomic changes in peripheral arterial vessels [such as increased intimal medial thickness (IMT)] have also been demonstrated in obese children and adolescents.220 These changes mimic early coronary pathology and predict adverse CV outcomes. The landmark Bogalusa heart study demonstrated that CV risk factors present in childhood are predictive of coronary artery disease in adulthood.221,222 Among these risk factors, LDL cholesterol and BMI measured in childhood were found to predict IMT in young adults.223 There is now substantial evidence that the insulin resistance of childhood obesity creates the metabolic platform for adult CV disease.224-226 Moreover, the constellation of peripheral insulin resistance, an unfavorable adipocytokine profile, subacute inflammation, and endothelial dysfunction work in parallel to promote the pathologic processes of aging.

THE METABOLIC SYNDROME The association and clustering of T2DM, hypertension, dyslipidemia, and CV disease in adults has led to the hypothesis that they may arise from a common antecedent. The World Health Organization argues that this antecedent is insulin resistance and defines this association as the metabolic syndrome.227-230 An alternative definition is provided by the National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP) III, which categorizes adults deemed to have the syndrome as meeting at least three of the following five criteria: elevated blood pressure, high triglyceride level, low HDL-cholesterol level, high fasting glucose, and central obesity. The metabolic syndrome affects approximately 25% of the U.S. adult population.231 Because of its wide prevalence, the metabolic syndrome is of enormous clinical and public health importance even at its earliest stages. Although still debated, one scheme of the pathophysiology of the metabolic syndrome is shown in Figure 19-5. According to this paradigm, the impact of obesity is determined by the pattern of lipid partitioning (i.e., the specific depots in which excess fat is stored). This pattern of lipid storage determines the adipocytokine secretion profile on circulating concentrations of inflammatory cytokines and on the flux of FFA. The combined effect of these factors determines the sensitivity of insulin target organs (such as muscle and liver) to insulin and impacts the vascular system by affecting endothelial function. Peripheral insulin resistance and endothelial dysfunction are the early promoters of overt pathology, culminating in T2DM and CVD. Of note is that formal criteria for the diagnosis of the metabolic syndrome in children have not yet been agreed upon.232

OTHER CO-MORBIDITIES RELATED TO INSULIN RESISTANCE Nonalcoholic Fatty Liver Disease NAFLD represents fatty infiltration of the liver in the absence of alcohol consumption.233 The spectrum of NAFLD ranges from pure fatty infiltration (steatosis) to

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Figure 19-5 A hypothesis on the relation between obesity and the metabolic syndrome. The metabolic impact of obesity is determined by the pattern of lipid partitioning. Lipid storage in insulinsensitive tissues such as liver or muscle and in the visceral compartment is associated with a typical metabolic profile characterized by elevated free fatty acids and inflammatory cytokines alongside reduced levels of adiponectin. This combination can independently lead to peripheral insulin resistance and to endothelial dysfunction. The combination of insulin resistance and early atherogenesis (manifested as endothelial dysfunction) drives the development of altered glucose metabolism and of cardiovascular disease.

inflammation (nonalcoholic steatohepatitis, or NASH) to fibrosis and even cirrhosis.234 NAFLD was found in the NHANES III survey to be more prevalent in obese African-American and Hispanic males with T2DM, hypertension, and hyperlipidemia.235 These associations have led to the hypothesis that NAFLD may precede the onset of T2DM in some individuals. NAFLD is now the most common liver disease among children in North America.236,237 NAFLD in children is associated with increased visceral fat deposition,238 and may progress to cirrhosis and related complications.239 The association between abdominal obesity and fatty liver may be partially explained by sustained exposure of the liver to an increased flux of FFA from the visceral depot.175 NAFLD may represent an early manifestation of ectopic lipid deposition in the liver and represents a challenge to the clinician due to the contrast of its minimal early manifestations and its potential serious outcomes. Recent data indicate that insulin plays a key role in regulating transcription factors, such as sterol response element binding protein-1c (SREBP-1c), which are abundantly expressed in the liver.240 SREBP-1c is pivotal in the control of hepatic lipogenesis and is increased in proportion to circulating insulin levels.241 These data raise the possibility that fasting hyperinsulinemia may contribute to hepatic steatosis, rather than vice versa. Alternatively, inflammatory cytokines released by visceral fat or by the hepatic immunoreactive cells may contribute to altered hepatic lipid metabolism.233 The majority of patients probably experience NAFLD without progressing to NASH. It is likely that subsequent inflammation or increased oxidative stress is necessary to promote progression to NASH (the “second

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hit” theory).239 As hepatic imaging modalities improve, noninvasive quantification of hepatic lipid deposition may enable us to use it as a target for intervention. For the time being, NAFLD can be surmised by an elevated ALT or GGT—although the sensitivity and specificity of these enzymes are low.

Polycystic Ovary Syndrome The association of hyperandrogenism and oligomenorrhea or amenorrhea (termed PCOS) in females is a frequent co-morbidity of obesity that can often be traced to childhood. This disorder is covered in detail in Chapter 14. A 2003 consensus statement242 defined the diagnostic criteria for PCOS as two out of the following three (after exclusion of other hyperandrogenic disorders): oligo/anovulation, clinical or biochemical manifestations of hyperandrogenism, and polycystic ovaries by ultrasound. PCOS is the most common cause of infertility due to anovulation and a major risk factor for development of the metabolic syndrome and altered glucose metabolism in females. The antecedents of PCOS have been identified in prepubertal girls, suggesting a developmental lesion.243 About two-thirds of patients with classic PCOS have hirsutism, acne, or male-pattern alopecia—with a similar portion having manifestations of anovulation such as amenorrhea, dysfunctional uterine bleeding, or oligomenorrhea.244 The biochemical manifestations of classic PCOS may include evidence of hyperandrogenism and an increased LH level (or an increased LH/FSH ratio) or the demonstration of polycystic ovaries by ultrasound. The nonclassic form of PCOS includes females with manifestations of hyperandrogenism and anovulation without evidence of alterations of gonadotropin levels or of polycystic ovaries on ultrasound. Obesity characterizes about 50% of women with classic PCOS,245 although it is even more common among adolescents. Increased peripheral insulin resistance occurs in approximately 50% of patients with PCOS, and almost certainly plays a role in the pathogenesis of this condition. On the other hand, almost all forms of severe insulin resistance (such as T2DM or rare lipodystrophy syndromes) are also associated with PCOS. Insulin resistance has not been included as a diagnostic criterion for PCOS mainly because it is difficult to measure or define. Fasting hyperinsulinemia and an increased insulin secretory response to an oral glucose load have been demonstrated in girls with PCOS.246 Indeed, obese adolescent girls with PCOS have been shown to be 50% more insulin resistant than weight-matched controls without PCOS.247 The constellation of metabolic abnormalities typically seen in insulin-resistant individuals is commonly encountered in obese adolescents with PCOS, including NAFLD248 and T2DM.249 The increased prevalence of the metabolic syndrome may be related to the hyperandrogenism independent of obesity-related insulin resistance.250 Early markers of accelerated atherogenesis are already present in young females with PCOS,251 indicating that early intervention aimed at reducing CV risk may be beneficial.

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Metabolic examination of patients with PCOS demonstrates hepatic and muscle resistance, but not ovarian insulin resistance—possibly accounting for insulin stimulation of theca cell androgen production.252 The correlation between insulin resistance and hyperandrogenism begs a unifying hypothesis as to their pathogenesis, which is proferred by the “serine phosphorylation hypothesis”—which suggests that P450c17 and the insulin receptor are aberrantly serine phosphorylated. In the case of P450c17, this leads to excess activity and increased androgen production.253 In the case of the insulin receptor, this leads to tissue-specific insulin resistance.254 However, this hypothesis remains to be proven.

OTHER ENDOCRINE CO-MORBIDITIES Obesity causes changes in other hormonal systems, some of which confer specific morbidities. The age at which pubertal initiation occurs continues to decrease, particularly in African Americans. This phenomenon is explained in part by the increasing overnutrition and BMI seen in this population.255 Infertility in older adolescents and adult women may occur as a manifestation of PCOS due to excessive ovarian androgen production in females or to excessive aromatization of androgen to estrogen by peripheral adipose tissue (with suppression of the hypothalamic-pituitary gonadal axis in both sexes).256 The hyperestrogenemia may also promote gynecomastia in males.257 In addition, the hypercapnia associated with obstructive sleep apnea can suppress hypothalamic GnRH function—leading to a syndrome of delayed puberty.258 Obesity is associated with decreased GH secretion, and indeed most obese subjects (despite normal or excessive statural growth) fail GH stimulation testing. However, caloric restriction for 24 hours can restore normal GH responsivity.259 Despite the functional GH inadequacy, statural growth is accelerated, bone age is advanced, and peripheral total and free IGF-1 levels are normal or elevated in obesity—suggesting normal or accentuated GH sensitivity260 or the suppression of IGFBP-1 and the effects of hyperinsulinemia on activation of the growth plate IGF-1 receptor.261 Free thyroxine levels tend to be lower and TSH higher in obese children, although still within the normal range. The mechanism is unknown. Last, obesity can be associated with increased cortisol exposure— possibly due to conversion of circulating cortisone to cortisol by the enzyme 11-hydroxysteroid dehydrogenase-1 (11-HSD1) located within visceral adipocytes.262

OTHER CO-MORBIDITIES Childhood obesity is associated with numerous other comorbidities. Pseudotumor cerebri263 is a rare and poorly understood condition leading to incracranial hypertension, whose manifestations include papilledema and headache. Treatment includes serial lumbar puncture, acetazolamide to reduce CSF production, and occasionally optic nerve sheath fenestration to save eyesight. Obstructive sleep apnea occurs frequently in morbidly obese children, presumably due to the large amount of retropharyngeal fat (which compresses the upper airway during sleep).264 Affected patients snore, often stop

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breathing for more than 20 seconds during sleep, and wake up during the night with headache. Treatment includes nocturnal positive airway pressure, and when appropriate tonsilloadenoidectomy. However, symptoms often recur. Obese children manifest numerous orthopedic difficulties, including fractures, knee pain, anatomic lower limb malalignment, and impairment in mobility.265 Cholelithiasis occurs in approximately 2.5% of obese adolescents, especially in females, but is not usually seen in prepubertal obese children.266 Last, psychological distress (including clinical depression) is clearly manifested in obese children.267 These various co-morbidities all appear to be associated with BMI Z score in a curvilinear fashion.268 Thus, the more obese the more likely patients will manifest co-morbidity.

Factors Associated with the Current Epidemic of Obesity GENETICS The association between obesity and genetics owes to two separate lines of investigation: the discoveries of monogenic disorders of the energy balance pathway, and studies of specific racial and ethnic groups in which obesity seems prevalent (such as the Pimas and other Hispanics in the Southwest United States).269,270 These observations are combined with an attractive theory (termed the thrifty gene hypothesis271) on the natural selection of individuals in response to drastic environmental/ecologic pressure (e.g., famine) to yield a very strong driving force for the elucidation of specific genetic loci in the pathogenesis of obesity.272 However, the rapid timescale of increased prevalence of childhood obesity cannot possibly reflect a population genetic change. Therefore, the current model is that obesity is a result of gene-environment interactions—an ancient genetic selection to deposit fat efficiently that is maladaptive with our current food overabundance. In the common forms of obesity, relating single-nucleotide polymorphisms with associated risks for obesity is difficult because the effects are uncertain and the results not always confirmed. Despite feverish searches for specific candidate genes, none has thus far been discovered.273

EPIGENETICS AND FETAL AND NEONATAL PROGRAMMING Follow-up studies of newborns born small for gestational age (SGA), large for gestational age (LGA), and premature have noted markedly increased risks for obesity and the metabolic syndrome. The “fetal origins hypothesis”274 states that some aspect of the in utero environment contributes to the development of obesity and chronic disease in later life. The specific developmental aberration that promotes obesity remains unknown. However, each of these three antenatal conditions is associated with insulin resistance. Documentation of the relationship of SGA with adult obesity and CV disease started with studies of the Dutch famine during World War II and its aftermath.275 Several

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studies of newborns born SGA demonstrate that they are hyperinsulinemic and insulin resistant at birth, exhibit rapid catch-up growth in the early postnatal period, and develop obesity in childhood—which remains and promotes persistent insulin resistance and the metabolic syndrome. An analysis of Indian newborns born in India versus in the United Kingdom276 demonstrates that despite those born in India weighing 700 g less at birth, their glucose and insulin levels are markedly elevated. After adjustment for birth weight, the India-born babies demonstrate increased adiposity and four times higher insulin and two times higher leptin levels than the U.K.-born babies. Thus, these babies are insulin resistant even at birth— which translates into increased adiposity. Following such babies into childhood, there are numerous studies documenting insulin resistance during early childhood.277-279 Babies born LGA are hyperinsulinemic at birth.280 Although LGA in most babies is due to gestational diabetes mellitus (GDM) and exposure to hyperglycemia throughout the pregnancy, this is not always the cause. Follow-up of LGA babies without GDM demonstrates a doubling of prevalence of insulin resistance and metabolic syndrome, whereas LGA babies resulting from GDM manifest a threefold increase.281,282 Indeed, the “vertical” transmission of maternal diabetes to the offspring in the form of later obesity and diabetes has been documented in studies of Pima Indians.283,284 Last, weight gain during pregnancy increases the risk for LGA and poor outcomes.285 Although there are no studies documenting hyperinsulinemia at birth in premature infants due to technical reasons, follow-up of these babies into early childhood also demonstrates increased weight gain—as well as insulin resistance and compensatory insulin secretion that are inappropriately high for the degree of weight gain.286 The protective effect of breast feeding against development of future obesity has long been known,166 and there appears to be a dose response (i.e., the longer the breast feeding the more protective).287 However, this may be complicated by confounding factors such as socioeconomic status, maternal smoking in pregnancy, and maternal BMI.288 The mechanism of breast feeding’s antiobesity effect is also unclear. Some think infant feeding self-regulation is most relevant, whereas a recent study suggests that leptin in breast milk may contribute to this protection.289 Concern regarding fructose/sucrose content in infant formula has also received attention.

ENVIRONMENT Numerous environmental factors have also been associated with the obesity epidemic, particularly in children. However, most of these associations are cross-sectional rather than longitudinal—and in many cases a mechanism remains lacking.290

Stress and Cortisol In humans, elevated cortisol or markers of HPA axis dysregulation correlate with abdominal fat distribution and the metabolic syndrome.291 Although circulating cortisol is clearly important in determining visceral adiposity, the recent identification of reduction of circulating cortisone

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to cortisol within visceral fat tissue by the enzyme 11hydroxysteroid dehydrogenase-1 (11HSD1) has also been linked to the metabolic syndrome.262,292 These data suggest that cortisol is important in increasing visceral adiposity and promoting the metabolic syndrome. In adults, job stress and depression stress cause increased cortisol secretion293—which leads to insulin resistance and the metabolic syndrome. Psychosocial stresses correlate with risk of myocardial infarction in adults.294 It is assumed that such patients exhibit increased HPA axis activation.295 Even exogenous glucocorticoid administration is a risk factor for CV events.296 Evidence of associations between elevated cortisol and psychological distress with abdominal fat distribution in adults is compelling. For instance, urinary glucocorticoid excretion is linked to aspects of the metabolic syndrome—including blood pressure, fasting glucose, insulin, and waist circumference.291 Although circulating cortisol is clearly important in determining visceral adiposity, the recent identification of reduction of circulating cortisone to cortisol within visceral fat tissue by 11HSD1 has also been linked to the MetS. The role of cortisol in mediating visceral fat accumulation, insulin resistance, and T2DM has been elegantly demonstrated by the transgenic knockout and overexpression of 11HSD1.262,292 These data suggest that cortisol is important in increasing visceral adiposity and promoting the MetS—equivalent to Cushing syndrome of the abdomen.297 However, the role of stress and cortisol in childhood obesity is currently speculative.

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watching may increase stress levels and cortisol—causing increased food intake and promotion of obesity.305 Second, television watching displaces physical activity and reduces voluntary energy expenditure (VEE). Most studies find inverse correlations between television watching and physical activity and fitness.306,307 Third, television watching increases calorie consumption from eating during viewing or from the effects of food advertising. Television viewing is also associated with increased high-fat food intake, decreased fruit and vegetable consumption, and increased soft drink intake.308 Junk food is the most frequently advertised product category on children’s television. Last, REE and nonexercise associated thermogenesis (NEAT) appears to be decreased during television watching.309 According to NHANES III (1988-1994), the prevalence of childhood obesity is lowest among children watching television 1 hour/day and highest among those watching 4 hours/day.310 The relationship between television watching and obesity has been examined in large number of cross-sectional epidemiologic studies but in few longitudinal studies.304 Several experimental studies of reducing television watching have been conducted, and their results support the suggestion that reduced television watching may help to reduce the obesity risk or help promote weight loss in obese children.311 These studies represent the strongest direct evidence that altering television watching alone is a promising strategy for prevention of childhood obesity. Other forms of “screen time,” such as video games, computers, and cell phones, are also implicated in obesity pathogenesis.

Sleep Deprivation Americans get significantly less sleep than they did three decades ago. Adults in the United States currently average less than 7 hours of sleep per night, which is almost 2 hours less than in 1980 (and about one-third of them get less than 6 hours per night).298 Analyses of data from the first NHANES revealed that adults (ages 32 to 49) who got less than 7 hours of sleep were more likely to be obese 5 to 8 years later than those who got 7 or more hours of sleep.299 Similarly, a 13-year prospective cohort study in which participants were interviewed at ages 27, 29, 34, and 40 years of age found that sleep duration correlated negatively with obesity.300 The link between short sleep duration and obesity has also been observed among children.301 Like adults, increasing numbers of children are chronically sleep deprived. This is especially true of obese children, who have been found to get less sleep than those of normal weight. In addition to its other effects, sleep is one of the most powerful cross-sectional302 and longitudinal303 predictors of childhood obesity in prepubertal children. Although relatively little is known about the mechanism for the sleep-obesity relationship, especially among children, there are reasons to assume increased stress and altered activity of various hormones (such as leptin, ghrelin, and cortisol).

Television Viewing and “Screen Time” Television watching is considered one of the most modifiable causes of childhood obesity.304 There are four possible mechanisms linking television watching and obesity. First, television

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Dietary Fat Versus Carbohydrate Fat is generally considered more obesigenic than other macronutrients, given its more energy-dense, highly palatable, and effective conversion to body fat.312 A high-fat meal induces decreased thermogenesis and a higher positive fat balance than an isocaloric and isoproteic lowfat meal.313 Excessive fat intake is believed to cause weight gain,314 but the relationship between dietary fat intake and childhood adiposity remains controversial.315 The prevalence of overweight in the United States has increased despite a decreased percentage of dietary energy derived from fat. A meta-analysis of 12 studies in overweight or obese adults who were given dietary advice on low-fat diet and followed for 6 to 18 months suggested that low-fat diets are no better than calorierestricted diets in long-term weight loss.316 Similarly, in children total fat consumption expressed as a percentage of energy intake has decreased.317 This decrease in fat consumption is largely due to increased total energy intake in the form of carbohydrates. Much of this imbalance is attributed to changing beverage consumption patterns characterized by declining milk intake and substantial increases in soft drink consumption,318 which may have its own etiopathogenesis. Most interventions with a low-fat heart-healthy diet have not been successful in childhood overweight prevention.319 Reduction in carbohydrate intake is taken to the extreme in the Atkins diet, which restricts adult subjects to less than 25 g/d of ingested carbohydrate. Adult evaluations of the diet for weight control have been disappointing

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long-term,320,321 and the popular diet has been abandoned recently. There are currently no data in children or adolescents. However, it should be noted that the ketogenic diet used for seizure control is similar in composition to the Atkins diet. A 2-year study of the ketogenic diet demonstrated persistent decreases in weight Z scores in children who were above average upon diet initiation, without significant compromise in general nutrition or in height.322

Glycemic Index and Fiber Not all sugars exert the same insulinogenic response. Complex carbohydrates can take two forms: a combination of
1-4 linkages and
1-6 linkages that gives the starch a globular stucture called amylopectin (as seen in bread, rice, pasta, potatoes, and glycogen) or a linear polymer of
1-4 linkages called amylose (as seen in beans, lentils, and other legumes). Digestion and absorption of the former in the intestine is rapid due to the simultaneous actions of
1-4 and
1-6 glucosidases, whereas that of the latter is much slower because the
1-4 glucosidase can only cleave single glucose moieties on either side of the polymer. This phenomenon constitutes the basis of the glycemic index (GI),323 which refers to the glucose area under the curve after consumption. High-GI foods lead to an accentuated insulin response, which can shunt energy substrate to adipose tissue.324 In children, controlled studies with a high-GI diet demonstrate that energy intake is 53% higher than on low-GI diet.325 One adolescent study demonstrated that an ad libitum low-GI diet was more effective in promoting weight loss than an energy-restricted low-fat diet.326 Therefore, the GI may be a simple concept to institute— although the “toxic environment” of American foodstuffs may make it difficult to maintain. Dietary fiber consists of the nonstarch polysaccharide portion of plant foods, including cellulose, hemicellulose, pectins, -glucans, fructans, gums, mucilages, and algal polysaccharides. Major sources of dietary fiber include whole grains, fruits, vegetables, legumes, and nuts. Fiber content accounts for 50% of the variability in glycemic load (GL; GI  volume) among foods. Cohort studies of adults demonstrate that fiber intake is inversely associated with weight gain, fasting insulin levels, and risk of T2DM.327,328 Fiber may influence body weight regulation by several mechanisms involving intrinsic, hormonal, and colonic effects—which eventually decrease food intake by promoting satiation (lower meal energy content) or satiety (longer duration between meals) or by increasing fat oxidation and decreasing fat storage.329 A fiber-rich meal is processed more slowly and has less caloric density, fat, and added sugars. Fiber-containing foods engender slower glucose absorption, which lessens the postprandial insulin surge and decreases lipogenesis.330 In addition, high-fiber meals allow for delivery of undigested triglyceride to the colon— where fermentation to short-chain fatty acids and their absorption improve lipids and insulin sensitivity.331 Archeologists surmise that our ancestors used to consume 100 to 300 g of fiber a day.332 However, the dietary fiber intake throughout childhood and adolescence currently

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averages approximately 12 g/day and has not changed during the past 30 years.333 Therefore, parents and school foodservice personnel should strive to offer fiber-rich foods to children so that their acceptance and consumption of them will be increased.334

Fructose The most commonly used sweetener in the U.S. diet is the disaccharide sucrose (i.e., table sugar), which contains 50% fructose and 50% glucose. However, in North America and many other countries non-diet soft drinks are sweetened with high-fructose corn syrup (HFCS)— which contains up to 55% of the monosaccharide fructose. Because of its abundance, sweetness, and low price, HFCS has become the most common sweetener used in processed foods. It is not that HFCS is biologically more ominous than sucrose. It is that its low cost has made it available to everyone, especially low socioeconomic groups. HFCS is found in processed foods ranging from soft drinks and candy bars to crackers to hot dog buns to ketchup. Average daily fructose consumption has increased by more than 25% over the past 30 years. The growing dependence on fructose in the Western diet may be fueling the obesity and T2DM epidemics.335 Animal models demonstrate that high-fructose diets lead to increased energy intake, decreased resting energy expenditure, excess fat deposition, and insulin resistance336—which suggest that fructose consumption is playing a role in the epidemics of insulin resistance and obesity and T2DM in humans.337 The metabolism of fructose differs significantly from that of glucose (Figure 19-6). Fructose is absorbed in the intestine and enters the liver without insulin regulation. There, fructose is converted to fructose-1-phosphate and enters the glycolytic pathway without regulation. This leads to an excess accumulation of citrate outside the mitochondria, which then undergoes de novo lipogenesis and is reassembled into free fatty acids (which promote insulin resistance), very low-density lipoproteins (which promote atherogenesis and serve as a substrate for obesity),338 and triglycerides (some of which precipitate in the liver, activate the inflammatory pathway, and cause nonalcoholic steatohepatitis).339 Fructose also does not suppress secretion of the “hunger hormone” ghrelin, levels of which correlate with perceived hunger. In sum, fructose consumption has metabolic and hormonal consequences that facilitate development of obesity and the metabolic syndrome.340 The highest fructose loads are soda (1.7 g/oz) and juice (1.8 g/oz). Although soda has received most of the attention,159,341 high fruit juice intake is also associated with childhood obesity—especially in lower income families.342 Nonetheless, the American Academy of Pediatrics recommends that fruit juice consumption be allowed to 4 to 5 oz/day for 1- to 6-year-old children and 8 to 12 oz/day for 7- to 18-year-old children.343

Calcium and Dairy There have been several reports of an inverse relationship between dietary calcium and obesity indices.344,345 Dietary calcium plays an important role in energy metabolism

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A

B Figure 19-6 Hepatic (A) glucose and (B) fructose metabolism. Only 20% of an ingested glucose load is metabolized by the liver, whereas 100% of a fructose load is hepatically metabolized. Thus, in a 120-calorie glucose load (two slices of white bread) 24 calories are hepatically metabolized—whereas in a 120-calorie sucrose load (an 8-oz. glass of orange juice) a bolus of 72 calories reach the liver. In contrast to glucose, fructose induces substrate-dependent hepatocellular phosphate depletion, which increases uric acid and contributes to hypertension through inhibition of endothelial nitric oxide synthase and reduction of nitric oxide (NO); stimulation of de novo lipogenesis and excess production of VLDL and serum triglyceride, promoting dyslipidemia; accumulation of intrahepatic lipid droplets, promoting hepatic steatosis; production of FFA, which promotes muscle insulin resistance; c-jun N-terminal kinase (JNK-1) activation, which serine phosphorylates and the hepatic insulin receptor—rendering it inactive and contributing to hepatic insulin resistance, which promotes hyperinsulinemia and influences substrate deposition into fat; and CNS hyperinsulinemia, which antagonizes leptin signaling (see Figure 19-3) and promotes continued energy intake.

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regulation. Increased calcitriol (1,25-dihydroxyvitamin D) in response to low-calcium diets stimulates Ca2 influx in human adipocytes may lead to stimulation of lipogenic gene expression and lipogenesis, as well as inhibition of lipolysis.345 This may result in an expansion of adipocyte triglyceride stores, which can promote adiposity. Increased dietary calcium reduces calcitriol levels and leads to reduction of fat mass without caloric restriction in mice,346 and this antiobesity effect of dietary calcium is supported by human clinical and epidemiologic studies.347 Vitamin D deficiency correlates with increasing BMI, especially in African Americans.348 However, it is not known if this is due to substitution of soft drinks for dairy, to lactose intolerance, or to other factors. One adult study349 revealed a consistent effect of higher calcium intake on lower body weight and body fat. However, pediatric studies are lacking.

Trace Minerals Chromium and vanadium appear to be involved in the insulin signaling process. In diabetic animals, chromium or vanadium supplementation results in improvement in insulin sensitivity and glycemic control350,351—and there is a suggestion of decreased weight. It is not yet known whether the pathogenesis of insulin resistance in humans involves a deficiency of a trace mineral. However, inadequate dietary intakes of vitamins and minerals are widespread—most likely due to excessive consumption of energy-rich micronutrient-poor refined food. Inadequate intake of micronutrients may result in chronic metabolic disruption, mitochondrial decay, DNA damage, oxidant leakage, and cellular aging associated with late-onset diseases such as obesity and cancer.352

Infectious Causes The pattern of increase in prevalence during the current obesity epidemic is reminiscent of an infectious transmission. Studies in animals implicate adenovirus-36 in the conversion from lean to obese.353 Studies in adults thus far demonstrate correlation between BMI and antibodies to this virus,354 but mechanism is lacking. Alternatively, the predominance of certain human intestinal flora species (firmicutes versus bacteroides) may predispose animals and humans to obesity355—possibly by increasing efficiency of energy absorption. However, factors that determine their predominance are unknown.

Medications Numerous medications promote excessive weight gain in children. The most commonly prescribed are pharmacologic doses of glucocorticoids (e.g., prednisone, methylprednisolone, dexamethasone), used for their antiinflammatory and antineoplastic activities. Patients so treated frequently become obese,356,357 and develop many of the features of Cushing syndrome (e.g., visceral adiposity, hyperlipidemia, hypertension, glucose intolerance)—which typify the metabolic syndrome.296 Sex hormone administration also promotes excessive weight gain, presumably by inducing insulin resistance.358

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In an effort to improve type 1 diabetes glycemic control, many physicians will overinsulinize the patient—but at the expense of excessive weight gain.359 Last, more and more children are being placed on the atypical antipsychotics risperidone, olanzapine, quetiapine, clozepine, aripriprazole, and ziprasidone to affect mood and behavior.360 These medications induce insulin resistance, which foments persistent hyperphagia and weight gain and increases risk for the metabolic syndrome.361

Disorders of Obesity The concept that obesity is a phenotype of numerous pathologies is evident from the examination of specific endocrine disorders leading to obesity in early childhood (Table 19-1). Some involve neural mechanisms, others classical hormonal mechanisms, and still others dysregulation of increased energy intake, decreased energy expenditure, and/or increased energy storage at the adipocyte.

CLASSIC ENDOCRINE DISORDERS WITH AN OBESITY PHENOTYPE In children, linear or statural growth accounts for up to 20% of ingested calories. Endocrine states that allow for normal energy intake for age but inhibit linear growth will of necessity lead to excessive energy storage. This is the case for the four “classic” endocrine disorders associated with obesity. These can be distinguished from other causes of pediatric obesity on the basis of their suboptimal growth rate—as opposed to overnutrition, which tends to increase the rate of growth and skeletal maturation (probably due to excess insulin cross-reacting with the IGF-1 receptor).261 Hypothyroidism (see Chapter 1) results in a lower REE due to insufficient circulating T3, along with decreased VEE due to fatigue. The decrease in total energy expenditure, despite a relatively low caloric intake, promotes persistent energy storage and increases adiposity. Thyroid hormone replacement is sufficient to increase growth, REE, and VEE to resolve the obesity over time. Cushing syndrome (see Chapter 12) results in growth arrest and cortisol-induced hyperphagia,362 with a decrease in REE and VEE due to muscle wasting. Reduction of circulating glucocorticoid through medical or surgical means usually reverses the obesity to some degree. Exogenous glucocorticoid therapy can result in the same obesity phenotype. Hypercortisolism may be more related to obesity than previously realized, due to the transgenic model of 11HSD1 overexpression in visceral adipose tissue—which converts inactive circulating cortisone to cortisol.262 However, its role in human obesity is not clear because correlations between 11HSD1 polymorphisms and BMI or waist:hip ratio were weak at best363—and enzyme activity was not elevated in one human study.364 GH deficiency (see Chapter 8) prevents lipolysis and promotes visceral adiposity, although obesity is usually not severe. GH deficiency is also associated with fatigue and decreased VEE. GH deficiency is often accompanied by other pituitary hormone deficiencies (e.g., central hypothyroidism), which can also decrease REE. GH therapy

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TA B L E 1 9 - 1

Classification of Childhood Obesity Disorders Mongenetic Disorders of the Energy Balance Pathway • Leptin deficiency • Leptin receptor deficiency • POMC mutation (“red” hair, adrenal insufficiency) • Prohormone convertase-1 deficiency • MC3R mutation • MC4R mutation • SIM-1 mutation Syndromic Disorders (Mental Retardation Prominent) • Prader-Willi syndrome • Short stature • Hypogonadism • Hypotonia • Ghrelin overproduction • Bardet-Biedl syndrome • Retinitis pigmentosa • Polydactyly • Hypogonadism • TrkB mutation • Hypotonia • Impaired short-term memory • Decreased nociception • Börjeson-Forssman-Lehmann syndrome • Microcephaly • Large ears • Hypogonadism • Carpenter syndrome • Variable craniosynostosis • Brachdactyly, polydacytly, syndactyly • Congenital heart disease • Hypogonadism • Cohen syndrome • Persistent hypotonia • Microcephaly • Maxillary hypoplasia • Prominent incisors • Alström syndrome • Hypogonadism • Short stature • Neurosensory deficits Classic Endocrine Disorders (Short Stature and Growth Failure Prominent) • Hypothyroidism • Primary • Central • Cushing syndrome (adrenal hypercorticism) • Adrenal adenoma/carcinoma • Adrenal micronodular hyperplasia • Pituitary ACTH-secreting tumor • Ectopic ACTH-secreting tumor • Exogenous glucocorticoid administration • Growth hormone deficiency • Pseudohypoparathyroidism 1a • Maternal transmission (AHO multihormone resistance) • Paternal transmission (pseudopseudohypoparathyroidism, AHO only) Insulin Dynamic Disorders • Hypothalamic obesity (insulin hypersecretion) • Insulin resistance • Leptin resistance

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is able to reverse these defects, increase muscle mass, and promote weight loss. Pseudohypoparathyroidism type 1a (PHP1a) (see Chapters 2 and 17) is an autosomal-dominant mutation of GNAS1, which codes for the Gs
subunit necessary to peptide hormone signal transduction. Maternal transmission leads to PHP1a (multihormone resistance), along with Albright’s heriditary osteodystrophy (AHO)—of which a cardinal feature is obesity, probably due to the inability to stimulate cAMP in response to -adrenergic stimulation within adipocytes (due to defective G-protein signal transduction).365 Paternal transmission leads to AHO without multihormone resistance, also known as pseudopseudohypoparathyroidism. Unfortunately, this form of obesity is not responsive to current medications.

MONOGENETIC DISORDERS OF THE NEGATIVE FEEDBACK PATHWAY The elucidation of the regulation of the energy balance pathway is exemplified by the discovery of specific defects within that pathway leading to early-onset obesity. Numerous obesity syndromes within the pathway have been described over the past 10 years, and are reviewed in detail elsewhere.366,367

Leptin Deficiency Mutations of the leptin gene in humans recapitulate the phenotype of the ob/ob leptin-deficient mouse.368 Approximately 11 such patients have been described, primarily of Pakistani and Turkish descent expressing the products of consanguinity. These patients manifest hyperphagia from birth, with obesity documentable as early as 6 months of age. The lack of leptin induces the starvation response in the form of reduced thyroid hormone levels, lack of sympathetic tone, lack of pubertal progression, and defective immunity.369 Despite the modest hypothyroidism, the concomitant hyperinsulinemia allows excess insulin to cross-react with the IGF-1 receptor in order to maintain growth rate and bone age until the usual time of puberty. However, because of the important role of leptin in initiating and maintaining puberty, untreated patients with leptin deficiency are short due to the lack of a pubertal growth spurt. The diagnosis is made by demonstrating extremely low or unmeasurable serum leptin levels. However, treatment with recombinant leptin is effective in restoring leptin signaling—resulting in reduction of hyperphagia, resolution of obesity, induction of puberty, and restoration of immune regulation.369 Heterozygotes for leptin deficiency assume an intermediate phenotype.370

Leptin Receptor Deficiency Three family members in France of Algerian extraction were found to have a mutation truncating the leptin receptor prior to its insertion in the membrane.371 This family had symptoms and signs similar to those with leptin deficiency, although they had growth retardation, low thyroid levels, and low IGF-1 and IGFBP-3 levels. The reason for this dichotomy is not known. Diagnosis

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hinges on documenting extremely high leptin levels even for the degree of obesity. No other families have been described. Unfortunately, no treatment currently exists.

POMC Splicing Mutation Inability to synthesize POMC due to missense or truncation mutations results in the inability to splice out
-MSH in the brain, leading to defective anorexigenesis at the MC4R and early-onset obesity. In the periphery, this defect leads to “red” hair to due lack of
-MSH action at the skin MC1R. In the pituitary, the defect results in inability to splice out ACTH—leading to secondary adrenal insufficiency.372 Recently, a Turkish patient with this mutation was reported to have dark hair—indicating that pigmentation of the hair is not entirely explained by the mutation’s effect on MC1R.373 Approximately six patients have thus far been reported, but are easily diagnosed due to their unusual phenotype, ACTH levels, and hypocortisolemia. There is currently no treatment for the obesity.

SIM-1 Mutation SIM1 stands for “single-minded,” a drosophila gene involved in neurogenesis—particularly the PVN, which expresses the MC4R. SIM1 appears to act as a signal transduction mechanism integrating information downstream from MC4R activation.378 Heterozygous null mice for SIM1 are obese. The human homolog is on chromosome 6q. A girl with hyperphagia, obesity, and developmental delay with a balanced translocation between 1p22.1 and 6q16.2 was found to have a mutation of SIM1.379 Only a few polymorphisms of SIM1 in obesity have been reported, and their significance is not yet established.380

PLEIOTROPIC OBESITY AND MENTAL RETARDATION DISORDERS Approximately 30 different obesity syndromes have been described, and most (if not all) are associated with mental retardation.381 Each has some other distinguishing phenotype, which makes the diagnosis obvious on clinical grounds. Various genetic linkages have been noted, but the etiologies for the obesity in these disorders remain obscure.

Prohormone Convertase-1 Deficiency Defects in this enzyme lead to the inability to process various preprohormones to their active ligands, such as POMC to ACTH and
-MSH, proinsulin to insulin, and various gut propeptides to active hormones.374 Only two unrelated patients have thus far been reported. They manifest severe early-onset obesity, ACTH deficiency, hypogonadism, hyperproinsulinemia, and small intestinal dysfunction—presumably on the basis of inability to cleave intestinal propeptides to their mature form. The diagnosis can be made by finding extremely high levels of proinsulin. No treatment currently exists for the obesity.

Melanocortin-3 Receptor Mutation Two family members in Singapore with mutations of the MC3R manifested with early-onset obesity.375 This receptor appears to have a slightly different function from the MC4R, as it seems to be involved in regulation of energy expenditure as opposed to energy intake.84 Diagnosis can only be made by gene sequencing. No treatment currently exists.

Melanocortin-4 Receptor Mutation Mutations in the MC4R appear to account for up to 5% of morbid obesity, especially those beginning in childhood.376,377 This mutation is transmitted as a co-dominant inheritance because homozygotes are more severely affected than heterozygotes, and not all carriers exhibit obesity. Patients exhibit early-onset hyperphagia and obesity, although not as severe as those with leptin deficiency. These patients grow rapidly, due to the hyperinsulinemia. Notably, their lean mass and their bone mineral density are increased commensurate with the adiposity.377 The diagnosis is made by gene sequencing. There is currently no treatment for this disorder.

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Prader-Willi Syndrome Prader-Willi syndrome (PWS) is a defect in the paternal allele of chromosome 15q11-13382 from deletion, mutation, defective imprinting, or uniparental disomy of chromosome 15. Subjects affected with PWS present with hypotonia and failure to thrive in the neonatal period, and as they get older they develop the classic findings of hypogonadism, short stature, mental retardation, and severe obesity. PWS patients are classically described as voracious eaters, although they can be easily dissuaded from food if they are removed from behavioral signals for food intake. REE is approximately 60% of normal in PWS, promoting adiposity.383 In addition, ghrelin levels are massively elevated in PWS384—which may be one etiology for their persistent hyperphagia. Others have suggested that the obesity in PWS is a function of GH deficiency, with defective lipolysis.385

Bardet-Biedl Syndrome This syndrome is characterized by obesity, mild mental retardation, dysmorphic extremities (including polydactyly), retinitis pigmentosa, hypogonadism, and renal malformations. Its inheritance is complex, but is usually described as autosomal recessive. Eight different genetic loci have been implicated,386 although their functions continue to remain elusive. The cause of the obesity remains unknown, but it is postulated to be due to failure of development of ciliated hypothalamic neurons.387,388 Diagnosis usually rests on clinical grounds. Although there is no specific treatment available, anecdotal instances of efficacy of metformin exist.

TrkB Mutation The NTRK2 gene codes for the ligand-specific subunit for the receptor TrkB, which has brain-derived growth factor (BDNF) as its ligand. TrkB is important for neural

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development. An 8-year-old boy with early hypotonia, developmental delay, impaired short-term memory, decreased responsiveness to nociception, and severe hyperphagia starting at 6 months culminating in morbid obesity was reported recently.389 Examination of the NTRK2 gene demonstrated a A to G transition in codon 722, substituting cysteine for tyrosine, which inhibited phosphorylation of TrkB. A second patient with obesity, impaired cognitive function, and hyperactivity was found to have an isocentric inversion at the BDNF locus.390 The etiology of the hyperphagia and obesity remain unclear.

Carpenter Syndrome This syndrome consists of obesity plus variable craniosynostosis, brachdactyly, polydacytly, syndactyly, congenital heart disease, and hypogonadism. Its etiology remains obscure.

Cohen Syndrome This disorder presents with persistent hypotonia, microcephaly, maxillary hypoplasia, and prominent incisors. The gene for Cohen syndrome has been mapped to chromosome 8q,391 and it codes for a transmembrane protein of unknown function.

Alström Syndrome This syndrome presents with neurosensory deficits, such as deafness, with various other endocrinopathies that cause an early-onset type 2 diabetes. The ALMS1 gene has been discovered.392 It appears to be important in cilia function,388 and may account for abnormal neuronal migration.

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sense of energy sufficiency and a subjective state of satiety.54,395 Children with hypothalamic obesity exhibit weight gain even in response to forced caloric restriction.401 This seems paradoxical, as one would expect that if hyperphagia were the reason for the obesity caloric restriction would be effective. The reason for this paradox is that similar to the db/db mouse these subjects exhibit “organic leptin resistance” (that is, the inability to respond to their own leptin due to the VMH damage). Numerous assessments of weight gain following cancer therapy in children have been performed. Most of these evaluations have been retrospective, and performed in the acute lymphoblastic leukemia (ALL) survivor population.402 An extremely high frequency of hypothalamic obesity of 30% to 77% has been documented after craniopharyngioma treatment.403 In each of these cancer types, hypothalamic damage is the primary risk factor for development of this syndrome.404 However, the syndrome has also been reported in cases of pseudotumor cerebri, trauma, and infiltrative or inflammatory diseases of the hypothalamus.399 Aside from the symptoms of tumor-induced increased intracranial pressure, patients with hypothalamic obesity classically exhibit signs of limbic system involvement such as hypogonadism, somnolence, rage, and hyperphagia.405 However, such classic presentations are actually rare.396 Hypothalamic obesity is the result of “organic” leptin resistance due to death of VMH neurons. This leads to defective autonomic neurotransmission.99 This is akin to the starvation response in that it manifests defective activation of the SNS (which retards lipolysis and energy expenditure)406,407 and overactivation of the vagus,408 which promotes an obligate insulin hypersecretion and energy storage.409 In animals and in humans, vagal hyperreactivity can be prevented by pancreatic vagotomy.110,410-412

Primary Insulin Resistance Börjeson-Forssman-Lehmann Syndrome This syndrome is characterized by microcephaly, seizures, large ears, and hypogonadism. The defective gene is a zinc-finger protein of unknown function.393

INSULIN DYNAMIC DISORDERS Hypothalamic Obesity and Insulin Hypersecretion It is well known that bilateral electrolytic lesions or deafferentation of the VMH in rats leads to intractable weight gain,109,110,394-396 even upon food restriction.397 Originally, the obesity was felt to be due to damage to a satiety center—which promoted hyperphagia and increased energy storage.398 However, we now understand that dysfunction of leptin signal transduction in the VMH due to hypothalamic damage secondary to CNS tumor, surgery, radiation, or trauma can alter the afferent and efferent pathways of energy balance and lead to severe and intractable weight gain.399,400 In this syndrome of “hypothalamic obesity,” hypothalamic insult prevents integration of peripheral afferent signals. The VMH cannot transduce these signals into a

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Insulin resistance is a primary entity in some pediatric populations. It is associated with the development of the metabolic syndrome, especially in certain racial and ethnic groups.413 For example, the Pima Indians of Arizona exhibit a 50% incidence of obesity and type 2 diabetes.414 Insulin resistance correlates with abdominal adiposity and CV morbidity.415,416 The presence of acanthosis nigricans, a hyperpigmented and hypertrophic patch of skin at extensor surfaces such as the nape of the neck, is a clinical marker of hyperinsulinemia417 due to cross reactivity between insulin and the epidermal growth factor receptor of the skin.418 Fasting hyperinsulinemia, an indicator of inherent insulin resistance in children, is an important predictor of adult obesity.419,420 This is further exacerbated by sex hormones (especially estrogen), contributing to the increased incidence of insulin resistance and obesity in teenage girls. The cause is unknown, but primary insulin resistance may be a manifestation of all three inciting factors (genetic, epigenetic, and environmental). There may be specific genetic predispositions269 that have been enriched by natural selection. In addition, the high incidence of gestational DM in Pima mothers promotes obesity and T2DM in the offspring.284

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Last, the consumption of low-quality processed food136 made available by government subsidies promote continued obesity. It should be noted that in primary insulin resistance dietary and exercise interventions have been notoriously ineffective at reducing the obesity.421,422 The locus of the resistance to insulin is not defined. Reduced numbers and function of insulin receptors may in part be secondary to the hyperinsulinemia itself, rather than due to a primary defect.

Evaluation and Treatment of the Obese Child WORKUP The key to successful obesity therapy is accurate diagnosis. Our diagnostic armamentarium is not yet fully developed, and thus matching treatment to diagnosis is still uncertain. Specific points in the evaluation and their rationale are listed in Table 19-2. In eliciting the history, birth weight, parents’ BMIs, gestational diabetes, prematurity, history of breast feeding, and neonatal complications (especially CNS injury) are all relevant. The earlier the patient’s obesity is noted (i.e., early adiposity rebound) the more likely an

organic reason will be discerned. Neurodevelopmental abnormalities may signify the need for a genetics referral. The medication list must be reviewed, especially for atypical antipsychotics. Orthopedic pain, headache, and snoring must be assessed. Dietary history must include skipping breakfast, daily ingestion of sodas and juices, and frequency and type of snacking. The degree of perceived stress by the patient is also likely a major contributor to visceral adiposity. A corollary is the number of caretakers of the child because this increases stress, family chaos, and lack of child supervision. On physical examination, linear growth is key because classical endocrine evaluation [e.g., hypothyroidism, Cushing syndrome, GH deficiency, pseudohypoparathyroidism (PHP)] is not necessary if linear growth is not attenuated. However, hyperinsulinemia and insulin resistance usually cause accelerated growth due to cross reactivity of insulin with the IGF-1 receptor.261 Important physical features to assess include acanthosis nigricans and waist circumference (both of which are associated with insulin resistance and the metabolic syndrome), fundoscopic examination to rule out pseudotumor cerebri, liver enlargement to suggest hepatic steatosis, hirsutism to suggest PCOS, and muscle tone to evaluate hypotonia and myopathy (which reduce energy expenditure).

TA B L E 1 9 - 2

Diagnostic Workup of Childhood Obesity and Its Co-morbidities Etiology/Co-Morbidity • • • • • • • • • • • • •

Genetic etiology Epigenetic etiology CNS etiology Endocrine etiology Medication etiology Dietary etiology Physical activity etiology Stress etiology Sleep apnea PCOS Type 2 diabetes Orthopedic morbidity Depression

• • • • • • • • •

Insulin resistance Hypertension Pseudotumor cerebri Hepatic steatosis PCOS Precocious/delayed puberty Sleep apnea Myopathy Syndromic obesity

• • • • • • •

Hepatic steatosis Glucose intolerance Type 2 diabetes mellitus Dyslipidemia Insulin resistance Insulin hypersecretion CNS lesion

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Evaluation Histroy • Parent BMI, race, mental retardation • Birth weight, gestational difficulties, prematurity • CNS insult, mental retardation or developmental delay • Slowdown in linear growth, red hair • Medication history, especially atypical antipsychotics • Calorie recall, especially sugar-containing liquids, breast-feeding Hx • Exercise history, television, computer, and cell phone recall • Socioeconomic status, number of caregivers, television recall, sleep status, atypical depression • History of snoring, headache, waking up with headache • Hirsutism, oligomenorhea, amenorrhea • Polyuria, polydipsia, nocturia, recent weight loss • Knee or hip pain, limitation of motion • Affect, activity level, school performance Physical • Acanthosis nigricans, skin tags, waist circumference • SBP or DBP 90th percentile for age • Papilledema • Hepatomegaly • Hirsutism • Gonadal and pubic hair status • Tonsillar hypertrophy • Decreased muscle tone, hyporeflexia • Specific neurocutaneous stigmata (see Table 19-1), retardation Laboratory • ALT, hepatic ultrasound • Fasting glucose 100 or 2-hr glucose 140 • Fasting glucose 125 or 2-hr glucose 200; HbA1c 6.5% • Lipid profile with increased VLDL, TG:HDL 2.5 • Fasting insulin, glucose • 3-hour OGTT with insulin levels • MRI, especially with hypothalamic coned-down views

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Laboratory evaluation includes tests of obesity-related morbidity (e.g., AST, ALT, lipids, fasting glucose and HbA1c, knee and hip x-rays, and sleep studies as necessary). Specific diagnostic studies must be tailored to the individual patient. For instance, growth attenuation requires endocrine evaluation—including thyroid function tests, IGF-1 and IGFBP-3, 24-hour urinary cortisol or midnight serum cortisol, and possibly magnetic resonance imaging (MRI) of the hypothalamus and pituitary. Patients with developmental delay will require karyotype and MRI. Severe obesity in a toddler may require a leptin level and genetic testing for a MC4R mutation. Fasting insulin and glucose allow for the computation of indices of insulin resistance, such as the homeostatic model of insulin resistance (HOMA-IR).423 However, an OGTT may become necessary to evaluate for type 2 diabetes and for insulin hypersecretion.409 Luteinizing hormone (LH), follicle stimulating hormone (FSH), and testosterone levels may be appropriate when evaluating for delayed puberty in males or PCOS in females.

adult health. Third, modifying chronic disease risks in childhood may lead to lower rates and risk factors in adults. Last, modification of children’s behaviors may lead to improved behaviors in adulthood that would protect against chronic diseases. Behavioral-cognitive therapy is designed to deal with both parent and patient, with behavior restructuring and reinforcement. Behavior changes include counseling sessions, teaching parenting skills, praise and contracts, selfmonitoring tools, stimulus control within the home, rolemodeling of behaviors by parents, and vigorous and long-term exercise programs. These programs have been successful in small studies with handpicked subjects by specific investigators431,432 but have not yet been successful when attempted in clinic populations. One new clinical approach involves motivational interviewing,433 a method for helping patients work through their ambivalence about behavior change. This method has been shown to be effective for substance abuse and in adult diabetes. Whether it will be successful in pediatric obesity remains to be seen.434

LIFESTYLE MODIFICATION

Dietary Intervention

Lifestyle modification is and remains the cornerstone of obesity therapy, especially in children. This approach is common sense, and based on a handful of early studies demonstrating efficacy of lifestyle in a select handpicked group of children with intensive follow-up.424 Indeed, the Diabetes Prevention Trial for T2DM demonstrated efficacy of lifestyle on weight loss in adults. However, the cost per patient of administering such an intervention was astronomical.425 Data supporting the efficacy of lifestyle modification in the “real world” is not particularly persuasive. Indeed, one analysis of numerous methodologies concluded that lifestyle modification was effective in modulating reported behaviors but had little effect on BMI or prevalence of pediatric obesity.319 In addition, follow-up studies often find a rapidly diminishing effect once the study is completed. Another meta-analysis concluded that the results of well-controlled studies were too weak to be definitive.426 Finally, a meta-analysis of 39 published intervention studies designed to prevent childhood obesity showed that 40% of the 33,852 participating children had reduction in BMI and that the remaining 60% exhibited no effect.427 Although weight loss is the conventional goal for intervention among adults, weight maintenance is recommended for the majority of children. The prevention of weight gain is easier, less expensive, and more effective than treating obesity itself428—and the prevention of overweight among children prior to the presence of riskrelated behaviors is crucial to stem the obesity epidemic. The primary goal of obesity prevention should be to promote physical activity and healthy diet, with emphasis on improving overall health rather than weight loss.429 There are at least four reasons to promote interventions to improve nutrition and physical activity in children.430 First, the child may receive immediate benefits such as better fitness or energy or micronutrient intake. Second, intervention at critical periods may improve

Dietary intervention is essential to the reduction of caloric intake and to reduction of the insulin response that promotes excessive energy deposition into adipose tissue. A myriad of studies demonstrate an association between consumption of high-calorie high-fat high-carbohydrate low-fiber foods and the development of pediatric obesity.319 Specific maneuvers that have been successful in reducing obesity in children include elimination of sugarcontaining beverages (including soda and juice)435,436 and a shift to a low-glycemic-load diet.326 Other common sense approaches in adults (although pediatric efficacy data are lacking) include eating breakfast, reducing portion sizes, increasing fruit and vegetable consumption, reducing between-meal snacking, and reducing fast-food consumption.437,438 However, dietary intervention alone is not a successful strategy for reducing pediatric overweight unless the treatment is very intensive439—in which case, the majority drop out. Furthermore, studies of nonsupervised dieting have demonstrated the opposite effect. That is, increased attempts in female adolescents predict a greater increase in weight and risk for obesity.440

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Physical Activity Intervention In adults, exercise is not an effective means of inducing weight loss unless combined with reduction of caloric intake. The role of exercise may have a greater impact on weight maintenance, rather than weight loss.441 Similarly, there is question as to whether physical activity regimens for children can stabilize or reduce BMI.442,443 Short-term studies show that vigorous exercise can result in shortterm reductions in adiposity444 and improved insulin resistance.445 This amounts to a minimum of 30 minutes of vigorous exercise 5 days per week. This is still under the recently proposed activity guidelines from the Institute of Medicine.446 However, such interventions eventually plateau in their effectiveness—and

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even short-term cessation rapidly reverses any accrued benefit.447 To succeed, physical activity interventions must be long-term, sustained, and incorporated into behavioral modifications aimed at the individual, at the family, at the school, and within the community in general.444,447 In other words, to make physical activity work it must become a priority. Appropriate interventions include making schoolbased physical education mandatory for every child and school, increasing access to after-school recreation, increasing culturally appropriate activities, reducing the competitive nature of sports so that more children will participate, increasing the incorporation of physical activity into daily life (e.g., stairs, walking to school as appropriate), and increasing participation of parents in physical recreation for their own weight management and as role models for their children. Last, the effects of reducing sedentary behavior by restricting television viewing has been efficacious in small and large studies and in diverse ethnic groups304— although fitness is not improved. Restriction of television also has a secondary effect of reducing caloric intake while watching television. Results using dietary or exercise intervention alone are not encouraging. Those studies that use behavioral, dietary, and exercise components together appear to be more successful319,448—although long-term efficacy is lacking.

School Intervention Numerous school-based interventions have focused on reducing obesity rates, most of which have not been successful—in part because school cafeteria fare in most districts remains problematic. However, interventions that increased time spent in vigorous physical activity (20 minutes in elementary schools and 30 minutes in middle schools) were more successful.449 One approach is to improve self-esteem and self-efficacy of students, as opposed to simply educating them about eating disorders,450 because this increases physical activity.451 Schools can model health-promoting environments, provide appropriate health education, and engender increased selfefficacy in coping with stress—which can empower children to make healthy lifestyle choices.

make healthy dietary choices, increase activity, and reduce perceived stress. A recent meta-analysis of randomized trials of combined lifestyle interventions for treating pediatric obesity yielded a significant although underwhelming decrease in BMI of 1.5 kg/m2, with targeted family intervention and a nonsignificant decrease in BMI of 0.4 kg/m2 in those that targeted the patient alone.452

PHARMACOTHERAPY Indications for Pharmacotherapy Pharmacologic therapies in children must currently be considered adjuncts to standard lifestyle modification. At present, several limitations preclude physicians from early implementation of drug therapies for the treatment of childhood obesity, including: the youngest child for whom any obesity pharmacotherapy is currently Food and Drug Administration (FDA) approved is 10 years; the long-term use of pharmacologic intervention has not always proven to be more efficacious than behavior modification; there exists a limited number of well-controlled studies of safe and effective pharmacologic intervention in obese children; the relative risk for the development of adverse events in children must be weighed against the long-term potential for improvement of morbidity and mortality, which is difficult to estimate in children; and targeting the pathology is still in its infancy. In addition, we cannot forget that many drugs used for treatment of adult obesity resulted in unforeseen complications—which resulted in their restriction (thyroid hormone, amphetamine) or recall (e.g., dinitrophenol, fenfluramine, dexfenfluramine, phenylpropanolamine, ephedra).453-458 Despite these concerns, the negative health impact of childhood obesity may justify long-term medication to control its progression. In the current pharmacopoeia of childhood obesity, nonspecific and specific strategies based on mechanisms of action within the energy balance negative feedback system are employed (Table 19-3). Currently, the approaches are suppression of caloric intake, limitation of the availability or absorption of nutrients, and insulin sensitization or suppression.

Reduction of Energy Intake: Sibutramine Family Intervention Invariably, the patient is not the only obese member of the family. There is frequently family chaos, which promotes obesity in other family members. Various caretakers (e.g., grandmothers, babysitters) will feed children and allow unrestricted television as a method of confining their activities indoors, particularly in dangerous neighborhoods. Some parents will not alter their shopping for junk food because they believe other sibs should not be deprived. Usually it is those same parents who do not want to be deprived. Divorced parents will often use food as a reward for a child’s love and loyalty. Thus, the family itself must be the target of lifestyle intervention. Parental involvement is critical, and the concept of “food is not love” must be emphasized. Families need training to modify behavior to

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Numerous anorexiant drugs have been used to treat obesity in adults. These drugs alter neurotransmission within the VMH in order to reduce caloric intake. Only sibutramine (a nonselective reuptake inhibitor of serotonin and norepinephrine) and dopamine are approved for children as young as 16 years.459,460 Sibutramine effectively inhibits caloric intake461 and stimulates thermogenesis in rats, although data on humans are contradictory.462,463 In adolescents, three randomized controlled trials (RCTs) document the safety and efficacy of sibutramine.464-466 Tolerability and side effects of sibutramine are similar to adults.466 Sibutramine can cause vasoconstriction and increase heart rate and blood pressure that persists even after significant weight loss.454 Other adverse reactions include dry mouth, headache, insomnia, anxiety, nervousness, depression, somnolence or drowsiness, edema, palpitations,

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Decreases in percentage body fat, with increases in absolute lean body mass

250-1,000 mg PO bid

5-15 mcg/kg/d SQ ÷ tid

Titration of dose to serum levels, SQ 96-256 mg PO qd

1-3 mg/m2 SQ qd

20 mg PO qd

Metformin Not FDA approved for treatment of obesity. Approved for 10 years of age for type 2 diabetes mellitus.

Octreotide Not FDA approved for treatment of obesity; otherwise 18 years of age.

Leptin Not approved by FDA.

Topiramate Not FDA approved for treatment of obesity.

Growth hormone Not FDA approved for treatment of obesity.

Rimonabant Not FDA approved in the US. No pediatric data.

Should be considered only after an unsuccessful 6-month trial of lifestyle intervention. All drugs effective only when combined with appropriate lifestyle intervention. RCT randomized controlled trial, MVI multivitamins.

RCT: Wt 4.8 kg over placebo at 12 months

RCT: Wt 8.0% over placebo at 6 months

120 mg PO tid

Orlistat Not FDA approved for 12 years of age.

RCT: Wt 4.6 kg, BMI 4.5%, WC 5.4 cm over placebo at 6 months RCT: Wt 8.1 kg, BMI 2.5 kg/m2 over placebo at 6 months Open-label: Wt 5.4 kg, BMI 2.0 kg/m2 at 6 months; RCT: Wt 2.6 kg, BMI 0.85, WC 2.7 cm over placebo at 12 months RCT: BMI Z score 0.35 SD vs. placebo at 6 months RCT: Wt 2.7% vs. placebo at 6 months; Post hoc analysis: efficacy dependent on degree of insulin resistance; BMI Z score 0.23 SD in first 4 months, 0.12 SD in next year Open-label: Wt 4.8 kg, BMI 2.0 kg/ m2 in 6 months; RCT: 7.6 kg, BMI 2.5 kg/m2 over placebo at 6 months Post hoc analysis: BMI Z score 0.70 SD in 6 months, dependent on insulin secretion and sensitivity Anecdotal: BMI 19.0 kg/m2 over 4 years

Efficacy

5-15 mg PO qd

Dosage

Sibutramine Not FDA approved for 16 years of age.

Drug

Depressed mood, nausea, vomiting, diarrhea, dizziness, headache, anxiety.

Paresthesias, difficulty with concentration/attention, depression, difficulty with memory, language problems, nervousness, psychomotor slowing. Edema, carpal tunnel syndrome, death in patients with preexisting obstructive sleep apnea.

Local reactions.

Gallstones, diarrhea, edema, abdominal cramps, nausea, bloating, reduction in thyroxine concentrations.

Nausea, flatulence, bloating, diarrhea; usually resolves. Lactic acidosis not yet reported in children.

Monitor HR, BP. Do not use with other drugs or MAO inhibitors.

Tachycardia, hypertension, palpitations, insomnia, anxiety, nervousness, depression, diaphoresis. Borborygmi, flatus, abdominal cramps, fecal incontinence, oily spotting, vitamin malabsorption.

Recommended only in Prader-Willi syndrome primarily to increase height velocity. It also decreases fat mass but should only be used in those have been after screening to rule out obstructive sleep apnea. Must closely monitor pulmonary function, glucose, HbA1c. 40% dropout rate. No pediatric data.

No pediatric data.

Useful only in leptin deficiency.

Monitor fasting glucose, FT4, HbA1c. Useful only for hypothalamic obesity. Ursodiol co-administration strongly recommended.

Do not use in renal failure or with intravenous contrast. MVI supplementation is strongly recommended.

Monitor 25OHD3 levels. MVI supplementation is strongly recommended. A lower dose preparation has been approved for over-the-counter sale.

Monitoring and Contraindications

Side Effects

Medications for the Treatment of Pediatric Obesity

TA B L E 1 9 - 3

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diaphoresis, xerostomia, constipation, dizziness, paresthesias, mydriasis, and nausea. Sibutramine cannot be administered in conjunction with monoamine oxidase inhibitors or selective serotonin reuptake inhibitors.453,467 Sibutramine is currently licensed in the United States for use in subjects 16 years of age and older. The FDA has extended the interval of treatment to 2 years.466

Reduction of Energy Absorption: Orlistat This drug is a modified bacterial product that specifically inhibits intestinal lipase and can reduce fat and cholesterol absorption by approximately 30% in subjects eating a 30% fat diet.468 Orlistat irreversibly binds to the active site of the lipase, preventing intraluminal deacylation of triglycerides—resulting in a 16 g/day increase in fecal fat excretion.469 Orlistat does not inhibit other intestinal enzymes. It has minimal absorption and exerts no effect on systemic lipases.470,471 Although there have been several open-label trials of orlistat in adolescents, only two RCTs have been published.472,473 The side effects with orlistat are predictable from its mechanism of action on intestinal lipase.453 Orlistat appears to be well tolerated in adults, with the principal complaints being borborygmi, flatus, and abdominal cramps. The most troubling side effects are fecal incontinence, oily spotting, and flatus with discharge— which are highly aversive in the pediatric population. Orlistat does not affect the pharmacokinetic properties of most other pharmaceutical agents. Absorption of vitamins A and E and -carotene may be slightly reduced, and this may require vitamin therapy in a small number of patients. In one study,474 vitamin D supplementation was required in 18% subjects despite the prescription of a daily multivitamin containing vitamin D—although in the company-sponsored study effects on vitamin levels were minor.472 Orlistat must be taken with each meal, which reduces its attractiveness in children—who are in school during lunchtime. Orlistat is currently approved for treatment of children as young as 12 years. An over-the-counter lower-dose preparation recently obtained FDA approval.

Improvement of Insulin Resistance: Metformin Metformin is a bisubstituted short-chain hydrophilic guanidine derivative used for the treatment of children and adults with T2DM.475-478 Metformin also decreases fasting hyperinsulinemia, prevents T2DM,479 and promotes weight loss in some obese individuals480,481 by improving hepatic and muscle insulin sensitivity. Metformin has little effect on energy expenditure.476 Although some believe that metformin promotes weight loss through a primary anorectic effect (as initial side effects of nausea and GI distress limit caloric intake acutely),482 most believe that the decline in caloric intake observed with metformin is related to its enhancement of glucose clearance through reduction of hepatic glucose output and reduction in fasting hyperinsulinemia.483,484 Metformin improves hepatic insulin resistance by inducing hepatic AMP kinase,485 which reduces hepatic

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gluconeogenesis. Therefore, pancreatic insulin secretion and peripheral insulin levels fall. Metformin also restores PI3-kinase and MAP kinase activity in muscle cells, improving muscle insulin sensitivity.486 Another possible mechanism of metformin action is through stimulation of glucagon-like peptide-1 (GLP-1),14,487 which may inhibit food intake through central actions on the VMH.15 Thus far, two RCTs and an observational prediction study in children and adolescents have been conducted.488,489 Examination of the open-label responses to metformin in a multivariate analysis demonstrated two predictors for efficacy: race (Caucasian  African American) and the degree of insulin resistance prior to therapy.137 Metformin has also been used “off-label” for treatment of polycystic ovarian syndrome and nonalcoholic steatohepatitis, with varying degrees of success.490-495 One particular use for metformin may be to combat the weight gain associated with atypical antipsychotics.496 However, cessation of metformin therapy leads to a rebound hypersinsulinemia and rapid weight gain, which may negate any beneficial effects seen during the medication window. Side effects with metformin include nausea, flatulence, bloating, and diarrhea at initiation of therapy—which appears to be self-limited and resolves within 3 to 4 weeks of initiation of the drug. Approximately 5% of pediatric patients discontinue metformin therapy because of severity of side effects. The most feared complication of metformin in adults is lactic acidosis, which is estimated to occur at a rate of 3 per 100,000 patient-exposure years—primarily in patients with contraindications to the use of metformin. However, no documented cases in children have been reported. Metformin increases the urinary excretion of vitamins B1 and B6, which are important in the tricarboxylic acid cycle and which may hasten lactic acidosis.497 Vitamin B12 deficiency has also been reported in as many as 9% of adult subjects using metformin. Therefore, prophylactic multivitamin supplementation is recommended with metformin use. Contraindications to metformin use include renal insufficiency, congestive heart failure or pulmonary insufficiency, acute liver disease, and alcohol use sufficient to cause acute hepatic toxicity. Metformin should also be withheld when patients are hospitalized with any condition that may cause decreased systemic perfusion, or when use of contrast agents is anticipated.483 It should be noted that metformin is FDA approved for treatment of T2DM in children but is unlikely to be approved for childhood obesity or insulin resistance due to the short exclusivity interval afforded the makers of metformin by the FDA upon its introduction to the United States in 1996.

Suppression of Insulin Hypersecretion: Octreotide It is well known that bilateral electrolytic lesions or deafferentation of the VMH in rats leads to intractable weight gain,109,110,394-396 even upon food restriction.397 In humans, hypothalamic damage due to CNS tumor, surgery, radiation, or trauma can alter the afferent and efferent pathway of energy balance and lead to severe and intractable weight gain.399,400 In this syndrome of hypothalamic obesity, hypothalamic insult confers an “organic leptin resistance” as the

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VMH senses starvation.54,395 Therefore, energy intake is high and expenditure is low.403 Children with hypothalamic obesity exhibit weight gain (even in response to forced caloric restriction401) secondary to overactivation of the vagus (which promotes an obligate insulin hypersecretion and energy storage) and to defective activation of the SNS, which retards lipolysis and energy expenditure.99,402 Insulin hypersecretion with normal insulin sensitivity is noted on oral glucose tolerance testing in these children.409 This same phenomenon of insulin hypersecretion has also been documented in a subset of obese adults without CNS damage.498 The voltage-gated calcium channel of the  cell is coupled to a somatostatin (SSTR5) receptor.499,500 Octreotide binds to this receptor, which limits the opening of this calcium channel, reduces influx of calcium into the  cells, and in turn reduces calmodulin activation and vesicle exocytosis—thereby acutely decreasing the magnitude of insulin response to glucose501 (Figure 19-2), which results in weight loss or stabilization. Two RCTs and an observational prediction study using octreotide for obesity have been performed.502,503 An examination of BMI responses to octreotide in pediatric hypothalamic obesity in a multivariate analysis demonstrated that insulin hypersecretion with concomitant retention of insulin sensitivity prior to therapy augured success.137 Octreotide is usually well tolerated. The most common side effects include diarrhea, abdominal cramps, nausea, and bloating—which are self-limited and usually resolve in 3 to 4 weeks.504,505 Other adverse events include gallstones (which are preventable by coadministration of ursodiol), edema, development of sterile abscess at the injection sites, B12 deficiency, suppression of GH and TSH secretion, and mild hyperglycemia—especially in those with severe insulin resistance.506 At present, octreotide offers a promising approach to the treatment of insulin hypersecretion as seen in hypothalamic obesity but is not FDA approved for this use. The use of octreotide in obese children with acute glucose-stimulated insulin hypersecretion without cranial pathology has not yet been evaluated.

Other Targeted Therapies Leptin. Mutations of the leptin gene in humans recapitulate the phenotype of the ob/ob leptin-deficient mouse.368 Approximately 11 such patients have been described. They manifest hyperphagia from birth, with obesity documentable as early as 6 months of age. Leptin deficiency induces the starvation response,369 with increased energy intake and decreased REE. The diagnosis is made by extremely low or unmeasurable serum leptin levels. In children with leptin deficiency, leptin therapy results in extraordinary loss of weight and fat mass507,508—along with reduction in hyperphagia, resolution of obesity, induction of puberty, and improvement in immunity.369 Although leptin administration in adults did not prove effective by itself due to leptin resistance,128 leptin may serve as an adjunct in combination with other medications after leptin sensitivity is ameliorated through weight loss.133,509 Growth Hormone. GH fosters anabolism and lipolysis. GH therapy has been shown to increase REE, promote linear growth, increase muscle mass, and decrease body fat percentage in Prader-Willi syndrome.385,510 It has also

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been shown to decrease body fat percentage in children with GH deficiency511 due to its effect on lipoprotein lipase.512 However, it is not clear whether these reductions in body fat percentage are primary effects on the adipose tissue compartment or are due to the increase in lean body mass. Obesity results in a state of functional GH insufficiency, which can be ameliorated through weight loss.513 GH therapy also improves the lipid profile in GHdeficient adults.514 Currently, the role of GH therapy in the treatment of nonsyndromic childhood obesity is unclear and not approved.

The Future of Pediatric Obesity Pharmacotherapy In response to the relative lack of efficacy of lifestyle interventions, the ever-expanding knowledge of the physiology of energy balance, and particularly as a business decision of potential financial reward, many pharmaceutical companies have launched obesity research programs. The agents discussed in the following are currently in human study. However, use of any of these new agents in children will depend on proof of safety and efficacy based on experience in adults. Oxyntomodulin is an analog of PYY(3-36), which has been shown in a 4-week RCT to reduce energy intake and weight in adults.515 Topiramate is a novel anticonvulsant that blocks voltagedependent sodium channels, enhances the activity of the GABAA receptor, and antagonizes a glutamate receptor other than the N-methyl-D-aspartate (NMDA) receptor.516 Topiramate promotes weight loss in a dose-dependent fashion.517 A recent RCT in adults demonstrated a 9.1% weight loss in subjects taking topiramate 192 mg/day— along with significant improvements in blood pressure, waist circumference, and fasting glucose and insulin.518 However, almost 33% of the subjects dropped out due to adverse events—which included paresthesias, somnolence, anorexia, fatigue, nervousness, decreased concentration, difficulty with memory, and aggression. There are currently no studies of topiramate in childhood obesity. Rimonabant is an endocannabinoid receptor (CB1) antagonist that reduces the reinforcement and reward properties of drugs of dependence at the level of the nucleus accumbens.519 It also extinguishes the reward properties of food. A 1-year RCT in adults demonstrated a 6.6 ± 7.2 kg weight loss (p  0.001), 6.5 ± 7.4 cm reduction in waist circumference (p  0.001), and improvement in lipid profile.520 Side effects included depressed mood, nausea, vomiting, diarrhea, dizziness, headache, and anxiety in 20% of subjects. Rimonabant was not FDA approved in the US and is under consideration for withdrawal in Europe due to these side effects.

BARIATRIC SURGERY Indications for Bariatric Surgery Conventional treatment of childhood obesity has proven to be time consuming, difficult, frustrating, and expensive. Although numerous short-term successes have been noted, long-term weight reductions are modest—and

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recidivism is the rule. In adolescents with extreme and morbid obesity that may be life-threatening, surgical therapy may be indicated in extreme and defined circumstances.521,522 However, in comparison to adults stricter and more conservative criteria must be applied to adolescents due to the following factors. • Not all obese adolescents will become obese adults.523 • Modestly improved rate of lifestyle and pharmacotherapeutic efficacy. • Longer time interval before co-morbidities become life-threatening. • The inability of adolescents to give legal consent. Therefore, it is virtually impossible to perform RCT surgical studies in children. Efficacy of any given approach will continue to be suspect, and different procedures cannot be compared. For all of these reasons, an expert panel with representation from the American Pediatric Surgical Association and the American Academy of Pediatrics522 suggested that bariatric surgery in adolescence could be justified in situations in which obesity-related co-morbid conditions threaten the child’s health. They provided stringent recommendations that bariatric surgery be limited to those adolescents with BMI 40 kg/m2 with presence of severe co-morbidity, or BMI 50 kg/m2 with a less severe co-morbidity. However, these stringent criteria are undergoing careful scrutiny in an attempt to liberalize them.524 Particular care should be taken to avoid bariatric surgery at very late stages of obesity, when the presence of obesity-related co-morbidities and the inaccessibility of imaging (most MRI scanners have a weight limit of 450 lbs.) may affect surgical outcome. Indeed, a review of eight retrospective studies in adolescents found that bariatric surgery in adolescents can promote durable weight loss in most patients. However, there appears to be a significant complication and mortality rate.525 Therefore, guidance is needed to determine the ideal circumstances in which the balance of risk versus benefit favors health preservation and reversal of complications with the lowest risk of morbidity and mortality from the procedure. Bariatric procedures for weight loss can be divided into malabsorptive, restrictive, and combination procedures. Purely malabsorptive procedures aim to decrease the functional length or efficiency of the intestinal mucosa through anatomic rearrangement of the intestine. These procedures include the jejunoileal bypass and the biliopancreatic diversion with duodenal switch. Due to the high morbidity and mortality of these procedures, they cannot be recommended in children and will not be discussed further. The restrictive procedures reduce stomach volume to decrease the volume of food ingested. They include the bariatric intragastric balloon (no data in children) and laparoscopic adjustable gastric band (LAGB). The Roux-en-Y gastric bypass (RYGB) is a combination procedure.526 Other bariatric procedures, such as gastric pacing, are still research modalities.

Restrictive: Laparoscopic Adjustable Gastric Banding LAGB utilizes a prosthetic band to encircle and compartmentalize the proximal stomach into a small pouch and a large remnant.526 The theoretical advantage of this tech-

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nique is decreased risk of staple line dehiscence. The more recent introduction of a new laparoscopic approach and the use of an adjustable band (allowing the stomach size to change) make this procedure more attractive. Finally, this procedure is reversible (at least theoretically, but there are some surgeons who scoff at this notion)—or can be modified into the RYGB at a later date. Results vary widely in adults. However, several small studies support the safety and efficacy of LAGB in morbidly obese adolescents. In one study of 11 morbidly obese adolescents (11-17 years old), LABG resulted in decreased mean BMI from 46.6 to 32.1 kg/m2 at a mean follow-up of 23 months—with improved co-morbidities. No patient experienced operative or late complications.527 In another study following LAGB, BMI in 7 adolescents (12-19 years old) fell from a preoperative median of 44.7 kg/m2 to 30.2 kg/m2 at 24 months—which corresponded to a 59.3% loss of excess weight.528 In a 3-year follow-up study of 41 adolescents, BMI was reduced from 42 ± 8 to 29 ± 6—with an excess weight loss of 70%.529 Again, complications were minor—although erosion of the band through the stomach serosa is documented. Although this procedure is considered safer than RYGB, it has not yet been approved by the FDA for use in adolescents—despite several studies that now support such use.

Combination: Roux-en-Y Gastric Bypass RYGB involves dividing the stomach to create a small (15-30 mL) stomach pouch into which a segment of jejunum approximately 15 to 60 cm inferior to the ligament of Treitz is inserted—with the proximal portion of the jejunum that drains the bypassed lower stomach and duodenum reanastomosed 75 to 150 cm inferior to the gastrojejunostomy.526 This procedure combines the restrictive nature of gastrectomy with the consequences of dumping physiology as a negative conditioning response when high-calorie liquid meals are ingested. In addition, RYGB is associated with decline in the circulating level of ghrelin—which may be in part responsible for the decrease in hunger associated with this procedure.12 RYGB appears to result in significant early weight reduction in adults.526,530 However, long-term studies demonstrate weight regain in many patients.531,532 Limited data are available regarding the efficacy of these surgical procedures to induce weight loss in severely obese children and adolescents, and most of these are case series from individual surgeons or institutions.467 In a case review, 10 severely obese adolescents (BMI 52.5 10.0 kg/m2) who underwent RYGB were followed for a mean of 69 months (range of 8-144 months).533 In this series, weight loss was significant in 9 of 10 adolescents—and was maintained as long as 10 years. The average weight loss was 53.6 25.6 kg, which represents approximately 59% excess weight lost. Weight loss was also associated with improvement of associated co-morbidities, including sleep apnea and hypertension. Finally, a large retrospective series of 33 obese adolescents534 age 16 ± 1 year with BMI 52 ± 11 (range of 38-91) and obesity co-morbidities followed these subjects up to

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14 years after bariatric surgery (mostly RYGB). There were surgical complications in 13, which were treated. There were 2 sudden deaths, and 5 of the subjects regained their weight. However, the majority experienced significant weight loss—with resolution of their co-morbidities and improvement in quality of life. Adolescents participating in a multicenter study reported by the Pediatric Bariatric Study Group experienced excellent weight loss after laparoscopic RYGB, with mean BMI change from 58 kg/m2 to 35.8 kg/m2 at 1 year.535 Gastrojejunostomy stenosis (21 patients) requiring endoscopic balloon dilation and internal hernia (14 patients) requiring laparoscopic or open reduction were the most common complications. This procedure appears to be safe and effective when candidates are carefully selected and the bariatric surgeon has advanced laparoscopic skills. The most common reported complications of RYGB include iron-deficiency anemia (50%), transient folate deficiency (30%), and events requiring surgical intervention (40%: cholecystectomy in 20%, small bowel obstruction in 10%, and incisional hernia in 10%).526 Because most of the stomach and duodenum is bypassed in this procedure, there is an increased risk for deficiencies in vitamin B12, iron, calcium, and thiamine. Although beriberi has been reported in teenagers after RYGB,536 compliance with daily supplementation and regular monitoring of patients can prevent such nutritional deficiencies.

Who Should Perform Bariatric Surgery in Children Surgical outcomes in adults vary widely among surgeons and institutions.537-539 Furthermore, there is a very clear learning curve because the morbidity of bariatric surgery varies inversely with the number of procedures performed.540 In addition, because RCTs in adolescents are unlikely the only method to validate and refine the use of these procedures will come from following patients carefully and long-term. Last, the increased risk of readmission after bariatric surgery in adults541 argues for close and careful follow-up and monitoring in adolescents. Therefore, it is essential that bariatric surgery in adolescents be performed in regional pediatric academic centers with programs equipped to handle the data acquisition and long-term follow-up involved in (and the multidisciplinary nature of) the treatment of these difficult patients.522 A multidisciplinary team with medical, surgical, nutritional, and psychological expertise should carefully select adolescents who are well informed and motivated to become potential candidates for LAGB or RYGB. Attention to the principles of growth, development, and compliance is essential to avoid adverse physical, cognitive, and psychosocial outcomes following bariatric surgery.522 It must be clear to the subject and the parent that bariatric surgery is in fact an adjunct to a sincere commitment to lifestyle, rather than a “magic bullet.” Indeed, evidence of recidivism in adults after RYGB is now commonplace. Subjects and families must be well informed as to the risks and complications of such surgery. The medical team will require endocrine, GI, cardiology, pulmonary,

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and otolaryngologic support. Prophylactic tracheostomy is rarely required to maintain airway patency and to allow for resolution of the hypercapnia prior to surgery.542 Adolescents undergoing bariatric surgery require lifelong medical and nutritional surveillance postoperatively.521 Extensive counseling, education, and support are required before and after bariatric surgery. Patients left to their own devices tend to regain weight over time. Indeed, studies in adults document an increased risk of hospitalization after RYGB due to difficulties from the procedure.541 Monitoring of long-term weight maintenance, improvements in CV morbidity, and longevity are all necessary to determine the cost effectiveness of bariatric surgery in the pediatric population.

Energy Inadequacy STARVATION VERSUS CACHEXIA Although both are weight loss syndromes, understanding the neuroendocrine mechanisms that distinguish starvation from cachexia is integral to understanding and treating these disorders properly. In starvation, the negative feedback energy balance pathway is intact. The signal of leptin inadequacy from the weight loss is transduced by the VMH neuron into reduced sympathetic activity (to conserve energy) and increased vagal activity (to store energy). However, in cachexia this pathway is shortcircuited by cytokine action on the hypothalamus. The VMH POMC neuron expresses receptors for various cytokines, including IL-1 and TNF-
543 In response to cytokine exposure, POMC neurons are activated—resulting in anorexigenesis, increased sympathetic activity, decreased vagal activity, and energy wastage.544,545 Proinflammatory cytokines increase epinephrine, GH, and cortisol, and reduce insulin. These long-term hormonal changes accelerate muscle proteolysis (cortisol), increase resting energy expenditure (SNS), contribute to insulin resistance (glucagon and cortisol), increase catabolism (cortisol), and suppress appetite and intestinal transit (vagus). This is clearly adaptive in the short term during times of infection (to generate body heat to eradicate the organism) but maladaptive in the long term, when chronic cytokine signaling can lead to cachexia. Thus, even in the situations of leptin decline or inadequacy cytokine activation of POMC neurons will promote continued cachexia and weight loss through persistent SNS activation.

FAILURE TO THRIVE Failure to thrive (FTT) is not a disease per se but rather a sign of multiple organic and nonorganic conditions and the interactions between them that lead to compromised growth at a young age. FTT still represents a common pediatric medical problem largely managed in the outpatient setting. Although FTT can rarely be a manifestation of critical illness, the majority of cases are the result of undernutrition due to the combination of biologic, environmental, and psychological factors. The diagnosis of FTT requires a thorough, prudent, and oriented history

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taking by the caregiver and is not always simple to establish. Moreover, this diagnosis may carry several legal implications (which are not within the scope of this text).

Definition There is no consensus on a single definition of FTT. The condition reflects inadequate physical growth recorded over time using standard growth charts. The commonly used definitions in clinical practice include length and/or weight below the 5th percentile for age and gender, a downward cross of two major percentile lines of the growth chart over time, or a weight per height below the 10th percentile of expected. The practicality of usually using weight and not length curves evolves from the fact that undernutrition and chronic disease tend to primarily affect weight gain while preserving linear growth. Ultimately, linear growth is also affected if these conditions persist. It is important to emphasize that single measurements without longitudinal follow-up growth points are inadequate to make the diagnosis of FTT, and a wrongful diagnosis may be established in infants who were born SGA or prematurely—and in some healthy infants growing along the lower percentiles.

Classification and Etiology The traditional classification of FTT is to segregate organic and nonorganic causes. The nonorganic causes refer to environmental and psychological factors such as sensory deprivation, parental and emotional deprivation, and feeding difficulties of no organic source that occur in infancy. This traditional classification seems to lack the insight that the majority of cases suffer from a combination of the two, reflecting a mixed etiology.546 A different approach is to classify the disorder based on the pathophysiology of the disorder (i.e., inadequate caloric intake, inadequate absorption, excess metabolic requirements, defective utilization of intake, and reduced growth potential).547 Common causes of FTT based on this classification scheme are outlined in Table 19-4. Of note, normal growth variation can confound the diagnosis of FTT because some infants may be born large for gestational age due to intrauterine causes (such as gestational diabetes) and later experience a “catch-down” pattern of growth during infancy toward the actual growth potential curves. Another cause of such negative crossing of percentiles can be constitutional growth delay. It is estimated that up to 25% of children can cross curves by more than 25 percentile lines (representing a cross of two major growth percentile lines) due to the previously cited reasons.548 These infants reach a new point from which they display a normal growth rate and weight gain pattern, yet they do not have FTT. Endocrine causes of FTT are uncommon, as typical hormonal deficiencies such as GH deficiency or hypothyroidism present as growth failure but with preserved or increased weight gain. Hyperthyroidism (representing a state of increased metabolic demands and characteristically manifested by increased linear growth) and disorders of salt metabolism (such as hypoaldosteronism and pseudohypoaldosteronism)

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Differential Diagnosis of Failure to Thrive Inadequate Caloric Intake • Poverty and low food resources • Mechanical feeding difficulties (altered swallowing ability, congenital anomalies, central nervous system damage, severe gastroesophageal reflux) • Wrongful preparation of infant formula (too diluted, too concentrated) • Unsuitable feeding habits by parent • Behavioral problems affecting eating • Child neglect • Poor parent-child interaction Inadequate Absorption of Caloric Intake • Reduced absorption surface area (short bowel syndrome, s/p necrotizing enterocolitis) • Chronic liver disease, biliary atresia • Celiac disease • Cystic fibrosis • Cow’s milk allergy • Chronic diarrhea • Vitamin or mineral deficiencies (acrodermatitis enteropathica) • Vomiting due to CNS abnormalities (tumor, raised ICP) Increased Metabolism • Hyperthyroidism • Chronic infection (due to immune deficiency) • Occult malignancy • Congenital heart defects or acquired heart disease (mainly right to left shunts and heart failure) • Chronic lung disease with hypoxemia (broncho-pulmonary dysplasia) • Burns Defective Utilization of Calories • Renal failure, renal tubular acidosis • Inborn errors of metabolism (storage diseases, amino acid disorders) Reduced Growth Potential • Genetic disorders (trisomies, skeletal dysplasias, Russel-Silver syndrome) • Specific genetic syndromes • Primordial dwarfism

may have FTT as part of their clinical manifestations.549 Hypophosphatemic rickets may also present as FTT.

Diagnosis and Evaluation The key to making the diagnosis of FTT is in plotting anthropometric data (weight and length) during a reasonable follow-up period. Although a prudent and focused history and physical examination are the keys to diagnosis, often the correct diagnosis is made in retrospect. The lack of an organic etiology to explain the findings is not enough to establish a diagnosis of nonorganic FTT. A response to an active intervention, manifested at least as a limited period of adequate growth while altering a behavioral element by the caregiver or child, can help establish a diagnosis of nonorganic FTT. History should focus on the dietary and feeding history, past and present medical history, social environment, and family history. The dietary history is aimed at

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assessing as accurately as possible the actual caloric intake of the patient. An important tool for this assessment can be the use of food logs of several days. The important details are actual amounts of food, the way the food is prepared (specifically, relevant to the dilution technique of infant formulas and to cereals added to the formula), and beverages consumed (with specific emphasis on sweetened juices and formula). These details should allow the practitioner to estimate the caloric intake. The important details regarding feeding begin with the location of the meals and their timing throughout the day. Who feeds the patient or supervises the feeding process is of major importance. The feeding technique should be appropriate to the developmental stage of the child. The timing is relevant in regard to frequent snacking between meals that may cause early satiety during mealtimes. A standard pediatric medical history should be taken from all patients, yet it should be focused on details that may be relevant to the diagnosis of FTT. The pregnancy and birth history are important for differentiation of infants who were born SGA versus those who suffer from FTT. The timing at which poor weight gain began, especially in relation to changes in feeding, is critically important to the diagnosis. Chronic medical conditions such as congenital heart disease, asthma, multiple recurrent infections, and anemia can all be causes of organic FTT. Multiple hospitalizations and a history of injuries can raise the suspicion of parental neglect. GI manifestations of relevant medical conditions such as frequent vomiting (in cases of milk allergy or gastroesophageal reflux) and stool frequency and consistency (to rule out malabsorption, celiac, inflammatory bowel disease, or cystic fibrosis) should be elicited in detail. The social history should focus on identifying the actual caregivers of the patient during the majority of the time and whether there are economic issues that may affect the ability to raise the patient adequately. Potential external and intrafamilial stressors that may affect the supply of food to the child should be sought (any stressor or life event that can affect the functioning of the caregiver in a way that could compromise the well-being of the child). The family history should focus on the body habitus of parents and siblings to obtain clues regarding genetic potential for height and weight. Medical conditions in siblings and relatives can suggest a predisposition to genetic disorders. The caregivers should be asked about mental illnesses (such as depression) that may hamper their ability to provide adequate care for the child. A family history of previous children who suffered from FTT should be investigated as well. A call to the local Department of Family Services may be warranted. The physical examination begins with plotting the child’s length, weight, and head circumference on standard growth charts—along with previous measurements (if available). The severity of the FTT can be estimated by assessing the present weight in comparison to the expected weight for age. If the weight is less than 60% of expected by the 50th percentile for age and length, the condition is severe—whereas a weight between the 60th and 75th percentile of expected is considered moderate FTT. Microcephaly accompanied by neurologic signs may suggest a CNS lesion. It should be remembered that head

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circumference is the last parameter to change in FTT, and only in the severest cases. Detection of dysmorphism may suggest a genetic cause of impaired growth and development. Measures of nutritional status (such as thickness of skin folds and body fat distribution) can be examined. It is important to carefully observe the interaction of the caregiver and the child during feeding. Impaired parentchild interactions can have a major impact on feeding habits, and their identification is critical to the design of effective behavioral interventions tailored to the patient and family. The majority of children with FTT have no laboratory abnormalities, and no hormonal alterations. There is minimal literature available about comprehensive laboratory workups in children with FTT, although a classic manuscript about the workup of more than 180 infants in an in-patient setting found laboratory abnormalities in less than 1.4% of tests taken.550 The choice of tests that may be beneficial should be based on the history and physical examination, and usually should be focused on the assessment of malnutrition in severe cases. A minimal workup (although not cost effective) may include a blood count, chemistry panel (including liver and renal function tests, electrolytes, serum protein and albumin concentrations, and blood acid-base status), and urinalysis with pH. Additional tests should be oriented toward specific findings from the history and physical examination. No hormonal tests are warranted initially unless a clinical suspicion of a specific disorder arises. In children older than 6 months, screening for iron deficiency and lead poisoning is warranted. Hospitalization and in-patient workup does not add any yield to the workup551 unless the degree of FTT is severe or if there are concerns of child safety and neglect.

Management The management of FTT is based on the identification of the underlying cause and its correction. The vast majority of cases are handled by a combination of nutritional and behavioral intervention. Importantly, the intervention should begin before the workup is complete (i.e., from the first evaluation). All medical problems are treated independently of nutritional and behavioral interventions and should not delay or hamper them. The mainstay of treatment of all infants with FTT is a calorie-rich diet accompanied by frequent and close monitoring of weight response. An effective intervention will document a catchup weight and height gain that is maintained over time. Feeding and eating behaviors should be addressed, walking the fine line between encouragement and pressure to promote eating. Timing meals and snacks and eating as a family in a pleasant environment of low stress may be important to the acquisition of improved eating and feeding practices. The feeding intervention is dependent on the infant’s age at presentation. For breast-feeding infants, it is beneficial to attempt to increase breast milk supply552 by pumping milk, treatment with metoclopramide to induce oxytocin secretion,553 improving maternal nutrition and fluid intake, and making adaptations at the home and workplace that can promote and

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simplify the breast-feeding process. Suckling problems in neurologically impaired infants can be solved by providing expressed human milk via bottle feeding. Bottle-fed and older infants allow more interventions to promote increased caloric content of the diet. Infants with FTT should receive ⬃150% of the recommended daily caloric intake based on their expected weight (rather than based on their actual weight).554 The enrichment of formula may be achieved by adding cereals, and toddlers may benefit from the addition of palatable high-energy-density foods (such as cheese and peanut butter) to their diet. High-calorie milk-based drinks (such as PediaSure, which provides 30 calories/oz.; in comparison to whole milk, which provides 19 calories/oz.) can be added alongside vitamin supplementation. Zinc supplementation has been shown to increase IGF-1 levels without affecting IGFBP-3 in infants with nonorganic FTT, yet this effect did not actually promote growth.555

Prognosis The vast majority of infants and children with FTT show improvement with intervention. Others may even show progress when they achieve a more independent stage of development at which they can attain their own food. Those who require gastrostomy feeding due to neurologic dysfunction may require assisted enteral nutrition for life. The cognitive and intellectual function outcomes of those who suffered from FTT seem worse than their peers, although this association has only been well established in cases of iron deficiency anemia.556 It seems conceivable that deficiencies of other elements critical to brain development during infancy may have a similar adverse impact on intellectual properties at later ages, although this has not been studied systematically. The effects of nonorganic factors (such as emotional deprivation) on intellectual development, often coexisting with organic factors, may also contribute to decreased cognitive ability at later ages.

CANCER CACHEXIA Cancer activates a complex set of CNS metabolic pathways, which result in cachexia557 (Table 19-5). Peripheral cytokines gain access to the CNS through the central circumventricular organs that bypass the blood-brain barrier or through stimulation and amplification of CNS microglial cytokine or eicosanoid production. For instance, TNF-
stimulates VMH POMC neurons—which stimulates the SNS, which increases resting energy expenditure, increases cortisol and glucagon levels, and contributes to insulin resistance. IL-1 decreases neuropeptide Y within the VMH and thus decreases appetite. IL-1 also increases CRF, which indirectly inhibits appetite. IL-6 bears striking similarity to ciliary neurotrophic factor (CNTF), which has been shown to reduce weight by activating VMH POMC neurons through a leptinindependent mechanism.558 Conversely, due to reduction in vagal activity, GI motility is impaired in cancer cachexia and is clinically manifest by early and inappropriate satiety—which occurs in 40% to 60% of cancer patients.

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Metabolic Changes in Cachexia Expression of Cytokines • Increased production of acute phase proteins (APP) • Up-regulation of transcription factor NF Kappa B and AP-1 • Increased interleukin-1, IL-6, and tumor necrosis factor-
and interferon- • Increased expression of tumor-specific cachexins (proteolysis-inducing factor, lipid-mobilizing factor, and anemia-inducing substance) • Increased expression of ubiquitin, E1, E2, E3, and proteasome components (cell death and removal) Increased SNS Tone • Increased expression of hormone-sensitive lipase in adipose tissue • Up-regulation of uncoupling proteins (UCP2 and UCP3) in muscle and adipose tissue • Increased hepatic gluconeogenesis Reduced Vagal Tone • Reduced lipoprotein lipase expression in adipose tissue • Reduced intestinal transit • Reduced hunger

In addition, cytokines have adverse peripheral effects. Uncoupling proteins are up-regulated by cytokines and contribute to increased energy expenditure. Cancer cachexia leads to overexpression of UCP1 in brown adipose tissue, UCP2 in brain, skeletal muscle and liver, and UCP3 in skeletal muscle. Levels of UCP2 and UCP3 in the liver and muscle are regulated by prostaglandins, and UCP3 is also regulated by triglycerides—all of which are increased in cancer.559 Cytokines cause insulin resistance in skeletal muscle, liver, and adipose tissue. TNF-
decreases insulin receptor and PPAR activity. In addition, there is an inverse correlation between interleukin-6 levels and insulin sensitivity.560 Adipose tissue insulin resistance increases fat oxidation and decreases lipoprotein lipase activity, resulting in continued lipolysis.561 The insulin resistance of cancer is not related to defective insulin clearance, and is therefore different than other forms of primary insulin resistance.562 Cancers are uniformly anaerobic and depend on glucose for survival. Thus, the glucose manufactured from gluconeogenesis secondary to hepatic insulin resistance is essential to tumor growth. Cancers release large amounts of lactate, which is converted in the liver back to glucose. Such gluconeogenesis consumes ATP, which also increases energy expenditure.563 Additional raw materials for gluconeogenesis are alanine (derived from skeletal muscle proteolysis) and glycerol (from lipolysis). Treatment is difficult. Many methods have been tried (SNS antagonists, prostaglandin inhibitors, omega-3 fatty acids, melatonin, thalidomide, interleukins, anticytokine monoclonal antibodies, IL-1 receptor antagonists, chemotherapy), but all are lacking. One promising new avenue is that of melanocortin antagonists,543 but this is still in preclinical evaluation.

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THE DIENCEPHALIC SYNDROME Originally described by Russell in 1951,564 this rare disorder presents in infants less than age 1 year and is an indication of a hypothalamic lesion—usually an anterior hypothalamic glioma or other neoplasm affecting hypothalamic function. Although the clinical spectrum is variable, emaciation with paucity of subcutaneous fat (but with normal linear growth and head circumference) is inviolate. Other frequent features include hyperalterness, hyperkinesis, nystagmus, and vomiting.565,566 Although numerous patients have been anecdotally reported and characterized, the cause of the emaciation remains unclear. Subjects with diencephalic syndrome have extremely elevated baseline GH levels, but with normal IGF-1 levels, suggesting a modicum of GH resistance.565 It has been suggested that the high GH leads to lipolysis and accounts for the emaciation, but this finding is not consistent in all patients. Only one evaluation of energy balance has been performed, which demonstrated 30% to 50% greater REE in comparison to normal babies and 13% greater energy expenditure compared to intake.567 Treatment recommended is surgical extirpation of the lesion whenever feasible. Radiation is usually reserved for the very young patient. Frequently, these patients postoperatively manifest hypopituitarism—and ultimately develop hypothalamic obesity.566

ANOREXIA NERVOSA Definition Anorexia nervosa (AN) is an eating disorder that typically begins during adolescence and consists of persistent dieting and intense physical activity, usually accompanied by compulsive behavioral traits and sometimes with purging behavior and binge eating. Most subjects manifest a disturbed body image and a persistent fear of fatness, both of which promote further weight loss. The result of this behavior is a pathologic weight loss, with pathophysiologic consequences. The risk of developing AN among females in Western societies is estimated to be between 0.5% and 1%.568 There are two subtypes of anorexia: the food-restrictive type (characterized by very low caloric intake plus excessive exercise) and the purging type (characterized by varying levels of food purging, usually by way of self-induced vomiting and laxative abuse). Alongside the obvious mental elements, the definition in the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) includes anthropometric as well as metabolic components: a significantly low weight (defined as a sustained weight below the 85th percentile of the expected weight per height due to weight loss or failure to gain weight during growth and development) and secondary amenorrhea in pubertal girls and women (defined as no menses in 3 months). Medical complications driven by the chronically reduced caloric intake, purging behavior, and excessive exercise may affect several organ systems. Typically, patients develop a marked loss of subcutaneous fat tissue, impaired menstrual function, bradycardia and orthostatic

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hypotension, hypothermia, and increased hair loss. Importantly, anorexia nervosa that develops during adolescence may create adverse clinical effects that persist into adulthood569—including osteopenia and osteoporosis,570 higher rates of miscarriage, and reduced offspring birth weight.571 Anorexia may also alter cognitive abilities (as during extreme weight loss), and a reduction of gray and white matter occurs. Conversely, during weight restoration white matter returns to premorbid levels but gray matter does not.572 Anorexia carries an increased mortality risk—specifically for suicide, yet also from medical causes such as starvation per se and purging-induced arrhythmias. Full recovery of body habitus and of growth and development occurs in 50% to 70% of treated adolescents, yet achieving a full physiologic and psychological recovery may take a comprehensive treatment intervention that may last as long as 5 to 7 years.573 Greater weight loss, lower sustained weight, and the coexistence of other psychiatric disorders adversely affect the probability of recovery. The outcomes for adults with AN are poorer than those who are diagnosed and treated in adolescence.

Endocrine Associations Obesity and malnutrition usually result in opposing effects on normal physiology and are both associated with changes in the hormonal profile. The majority of these changes represents an adaptive response yet should be considered part of the differential diagnosis of specific hormonal excess or deficiency disorders. Figure 19-7 shows the typical hormonal profile of patients with AN, aimed at energy preservation and the cessation of energetically costly and nonvital processes. Hypothalamic-Pituitary-Thyroid Axis. The starvation status of AN may resemble the sick euthyroid syndrome. In patients with AN, serum total and free T4, total and free T3, TSH, and TBG are significantly lower than normal—whereas rT3 levels are significantly greater than healthy controls.574 Most AN patients have a hyporesponsive or delayed responsiveness of TSH to TRH stimulation. Of note, weight regain reverses the effects of AN on the hypothalamic-pituitary-thyroid axis back to normal. Thus, the reduction in thyroxinemia may actually be a normal adaptive physiologic response to starvation. The differential diagnosis of hypothyroidism should be considered in patients with AN because both disorders are characterized by low T4 and T3 levels. In primary hypothyroidism, TSH levels will be greater than those seen in patients with AN—although in mild secondary hypothyroidism this distinction may be difficult. Obtaining a serum reverse T3 (rT3) level may be helpful in distinguishing a true thyroid disorder from the euthyroid sick syndrome associated with systemic illness. Growth Hormone-IGF-1 Axis. Patients with AN typically have GH hypersecretion accompanied by low IGF-1 levels. Whether this profile is due to a primary hypothalamic dysfunction, peripheral target organ resistance to GH, or an impaired negative IGF-1 central feedback mechanism is unclear. An increased GH response to GHRH has been demonstrated in AN, possibly reflecting an impairment of beta-adrenergic suppression of GH

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Figure 19-7 Hormonal changes in anorexia nervosa. The hormonal profile of the patient with anorexia nervosa represents an adaptive response aimed at conserving energy. Processes that require significant energy such as reproduction and growth are limited by a complete suppression of the gonadotropic axis and by reduced IGF-1 levels respectively. A seemingly chronic stress response characterized by increased growth hormone and cortisol is aimed at efficient utilization of the limited energetic sources present. Reduced leptin serves as a signal for down-regulating the hypothalamic-pituitary-gonadal axis. Reduced leptin also serves as a starvation signal, although this signal appears to be circumvented in this disorder.

secretion.575 The clinical presentation of weight loss, cessation of menses, and cold intolerance alongside low levels of pituitary derived hormones may resemble panhypopituitarism—yet patients with AN present with GH hypersecretion. Hypothalamic-Pituitary-Adrenal Axis. Patients with AN typically present with elevated cortisol levels in the presence of normal ACTH levels.576 The elevated cortisol levels are apparently due to increased cortisol secretion alongside a reduction in cortisol clearance. Elevated CRH levels found in cerebrospinal fluid of patients with AN suggest that AN represents an overall state of activation of the hypothalamic stress response, manifested peripherally by hypercortisolemia. An abnormal response in the dexamethasone suppression test, mainly of reduced suppression, suggests that an element of decreased feedback sensitivity occurs in this disorder. Bone Metabolism. Adolescence represents a critical period for the accumulation of bone mineral, thus building bone strength and density for later years. The achievement of peak bone mass is dependent on several hormonal effects characteristic of puberty (such as estradiol and IGF-1), as well as on being dependent on adequate nutrition—all of which are compromised in patients with AN. Adolescents and adults with AN have a low bone mineral density. Whereas in adults this is due to increased bone resorption and reduced bone formation, adolescents are characterized by an overall reduced bone turnover.577 The reduced bone density is caused by the typical hormonal profile of AN (i.e., reduced estrogens and androgens, low IGF-1, and relative hypercortisolemia). Moreover, the reduced lean body mass and lower mechanical forces acting on long bones may also contribute to the overall reduced BMD. Recent publications also suggest that elevated levels of gut-derived hormones

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PYY(3-36) and ghrelin may also contribute to impaired bone metabolism in AN.578 Importantly, resolution of AN with weight gain often does not bring full recovery of the bone mineral density status. Osteopenia resulting from undernutrition and the typical hormonal dynamics of AN is one of severe long-term complications of AN during adolescence, and thus is a major treatment target. Hypothalamic-Pituitary-Gonadal Axis. Amenorrhea is one of the hallmarks of AN, yet it is not always explained by the severe weight loss. Thus, hypothalamic amenorrhea in AN often precedes weight loss and may persist after re-feeding and achievement of normal weight. Gonadotropin levels are reduced in patients with AN, and GnRH stimulation testing in patients with AN demonstrates a blunted LH response with a preserved FSH response and very low levels of estradiol. LH pulsatility may revert to prepubertal patterns in adolescents who have previously achieved pubertal status. The amenorrhea of AN is also marked by strikingly reduced leptin levels. Leptin serves as a metabolic signal of energy status and nutritional reserve and thus may have a permissive role in the initiation of the complex hormonal dynamics necessary to normal reproductive function.579 Teleologically, conservation of energy for immediate and necessary metabolic demands while suppressing energetically expensive processes such as reproduction serves as a protective measure in severely malnourished individuals (as seen in AN). The rise in leptin upon weight gain is associated with increases in gonadotropin secretion, suggesting that leptin serves a permissive role in the activation of the hypothalamic-pituitary-gonadal axis. Fat-derived Hormones. Adipocytes secrete a wide array of adipocytokines, the normal profile of which is altered in AN. Patients with AN typically have very low plasma leptin concentrations alongside a marked

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disturbance of the leptin diurnal secretion profile.580 The concentration of leptin-binding protein (the soluble isoform of leptin receptor) has been reported to be increased in patients with AN, contributing to a further reduction in concentration of free leptin.581 Leptin has a major role in the neuroendocrine adaptations to the chronic starvation and undernutrition typical of AN, such as reduction in gonadotropins, reduction in thyroid hormone in order to conserve energy, a modified stress response manifested by hypercortisolemia, and elevated GH aimed at mobilizing and utilizing alternative energy sources.582 Weight gain in AN patients can induce relative hyperleptinemia in comparison to controls matched for BMI. Circulating leptin concentrations in AN patients thus traverse from subnormal to supranormal levels within a few weeks. Adiponectin is the only adipocytokine whose plasma concentrations are inversely related to fat mass. Conflicting results regarding adiponectin concentration in AN, ranging from hyperadiponectinemia583 to hypoadiponectinemia,584 have been reported. Interestingly, weight gain in AN is not necessarily associated with reciprocal changes in adiponectin concentration.585

Treatment Indications for hospitalization and in-patient treatment of AN include dehydration, electrolyte disturbances (mainly hypokalemia), arrhythmias, CV instability (significant bradycardia, hypotension, and orthostatic changes), hypothermia, acute food refusal, acute complications of malnutrition (seizures, pancreatitis, cardiac failure), and psychiatric emergencies. Along with psychological interventions that may consist of psychotherapy and/or pharmacotherapy (based on the spectrum of psychiatric pathology), an increase in caloric intake must be part of the treatment protocol. Starting with 1,200 to 1,500 kcal per day and increasing by 500 kcal is aimed at a gain of 0.5 to 1 kg per week.586 There is no superior feeding regimen as long as adequate caloric intake is supplied. Extreme cases may be handled through hospitalization in dedicated in-patient units, and caloric intake in this setting may initially be provided by nasogastric feeding—and in extreme cases by parenteral feeding. One major treatment decision facing the caregiver of patients with AN is whether to utilize estrogen replacement therapy in order to treat amenorrhea and protect the skeleton from osteopenia. There is a lack of a proven efficacious beneficial effect of estrogen replacement therapy in regard to improved bone mass in AN in comparison to placebo,587 although this treatment modality is still commonly used.588 This treatment approach may have several adverse effects on the treatment of adolescents because it masks the beneficial effect of weight gain on the resumption of menses and may provide an erroneous sense of security in patients still at critically low weight status. Increased calcium intake alongside 400 IU of vitamin D to accelerate absorption should be encouraged in all patients with AN as another potential protective measure of bone status.

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Conclusions In childhood obesity, the overwhelming majority of cases are not due to a documentable genetic or neuroanatomic lesion. Indeed, although classic endocrinopathies account for less than 2% of childhood obesity in fact every obese patient does manifest an endocrine disturbance (e.g., insulin resistance and/or leptin resistance). We must acknowledge the obvious: that the child eats too much and exercises too little. The question physicians must internally pose when evaluating an obese patient is, “Where in the negative feedback energy balance pathway is this patient’s dysfunction?” Only then can appropriate treatment be proffered. Similarly, in cachexia one must go beyond the lack of appetite to understand the reasons for the wasting and illness. These are endocrine paradigms that may permit modulation, especially with endocrine therapies. Understanding the energy balance pathway, and where these various disorders impair its regulation, is the key to further research and to successful prevention and treatment.

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DISORDERS OF ENERGY BALANCE 524. Inge TH, Xanthakos SA, Zeller MH (2007). Bariatric surgery for pediatric extreme obesity: Now or later? Int J Obes 31:1–14. 525. Apovian CM, Baker C, Ludwig DS, Hoppin AG, Hsu G, Lenders C, et al. (2005). Best practice guidelines in pediatric/adolescent weight loss surgery. Obes Res 13:274–282. 526. Mun EC, Blackburn GL, Matthews JB (2001). Current status of medical and surgical therapy for obesity. Gastroenterology 120:669–681. 527. Abu-Abeid S, Gavert N, Klausner JM, Szold A (2003). Bariatric surgery in adolescence. J Pediatr Surg 38:1379–1382. 528. Dolan K, Creighton L, Hopkins G, Fielding G (2003). Laparoscopic gastric banding in morbidly obese adolescents. Obes Surg 13:101–104. 529. Fielding GA, Duncombe JE (2005). Laparoscopic adjustable gastric banding in severely obese adolescents. Surg Obes Relat Dis 1:399–405. 530. Rubino F, Gagner M, Gentileschi P, Kini S, Fukuyama S, Feng J, et al. (2004). The early effect of the Roux-en-Y gastric bypass on hormones involved in body weight regulation and glucose metabolism. Ann Surg 240:236–242. 531. Sjostrom L, Lindroos AK, Peltonen M, Torgerson J, Bouchard C, Carlsson B, et al. for the Swedish Obese Subjects Study Scientific Group (2004). Lifestyle, diabetes, and cardiovascular risk factors 10 years after bariatric surgery. N Engl J Med 351:2683–2693. 532. Shah M, Simha V, Garg A (2006). Review: Long-term impact of bariatric surgery on body weight, co-morbidities, and nutritional status. J Clin Endocrinol Metab 91:4223–4231. 533. Strauss RS, Bradley LJ, Brolin RE (2001). Gastric bypass surgery in adolescents with morbid obesity. J Pediatr 138:499–504. 534. Sugerman HJ, Sugerman EL, DeMaria EJ, Kellum JM, Kennedy C, Mowery Y, et al. (2003). Bariatric surgery for severely obese adolescents. J Gastrointest Surg 7:102–107. 535. Lawson ML, Kirk S, Mitchell T, Chen MK, Loux TJ, Daniels SR, et al. (2006). One-year outcomes of Roux-en-Y gastric bypass for morbidly obese adolescents: A multicenter study from the Pediatric Bariatric Study Group. Ped Surg 41:137–143. 536. Towbin S, Inge TH, Garcia VF, Roehrig HR, Clements RH, Harmon CM, et al. (2004). Beriberi after gastric bypass surgery in adolescence. J Pediatr 145:263–267. 537. Mason EE, Scott DH, Doherty C, Cullen JJ, Rodriguez EM, Maher JW, et al. (1995). Vertical banded gastroplasty in the severely obese under age twenty-one. Obes Surg 5:23–33. 538. Greenstein RJ, Rabner JG (1995). Is adolescent gastric-restrictive anti-obesity surgery warranted? Obes Surg 5:138–144. 539. Breaux CW (1995). Obesity surgery in children. Obes Surg 5:279– 284. 540. Nguyen NT, Paya M, Stevens M, Mavandadi S, Zainabadi K, Wilson SE (2004). The relationship between hospital volume and outcome in bariatric surgery at academic medical centers. Ann Surg 240:586–594. 541. Zingmond DS, McGory ML, Ko CY (2005). Hospitalization before and after gastric bypass surgery. JAMA 294:1918–1924. 542. Ray RM, Senders CW (2001). Airway management in the obese child. Ped Clin NA 48:1055–1063. 543. DeBoer MD, Marks DL (2006). Therapy insight: Use of melanocortin antagonists in the treatment of cachexia in chronic disease. Nat Clin Pract Endo Metab 2:459–466. 544. Walsh D, Nelson KA (2002). Autonomic nervous system dysfunction in advanced cancer. Support Care Cancer 10:523–528. 545. Barber MD, Ross JA, Fearon KC (2000). Disordered metabolic response with cancer and its management. World J Surg 24:681– 689. 546. Gahagan S, Holmes R (1998). A stepwise approach to evaluation of undernutrition and failure to thrive. Ped Clin North Am 45:169–187. 547. Krugman SD, Dubowitz H (2003). Failure to thrive. Am Fam Phys 68:879–884. 548. Schmitt BD, Mauro RD (1989). Non-organic failure to thrive: an outpatient approach. Child Abuse Negl 13:235–248. 549. Kuhnle U, Lewicka S, Fuller PJ (2004). Endocrine disorders of sodium regulation: Role of adrenal steroids in genetic defects causing sodium loss or sodium retention. Horm Res 61:68–83. 550. Sills RH (1978). Failure to thrive: The role of clinical and laboratory evaluation. Am J Dis Child 132:967–969. 551. Berwick DM, Levy JC, Kleinerman R (1982). Failure to thrive: Diagnostic yield of hospitalization. Arch Dis Child 57:347–351.

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552. Gahagan S (2006). Failure to thrive: A consequence of undernutrition. Pediatr Rev 27:e1–e11. 553. Gabay MP (2002). Galactogogues: Medications that induce lactation. J Hum Lact 18:274–279. 554. Maggioni A, Lifshitz F (1995). Nutritional management of failure to thrive. Ped Clin North Am 42:791–810. 555. Hershkovitz E, Printzman L, Segev Y, Levy J, Phillip M (1999). Zinc supplementation increases the level of serum insulin-like growth factor-I but does not promote growth in infants with nonorganic failure to thrive. Horm Res 52:200–204. 556. Lozoff B, Jimenez E, Wolf AW (1991). Long-term developmental outcome of infants with iron deficiency. N Engl J Med 325:687– 694. 557. Davis MP (2007). Anorexia and cachexia in cancer. In Yeung S, Escalante C, Gagel RF (eds.), Internal medicine care of cancer patients. New York: Decker (in press). 558. Kelly JF, Elias CF, Lee CE, Ahima RS, Seeley RJ, Bjorbaek C, et al. (2004). Ciliary neurotrophic factor and leptin induce distinct patterns of immediate early gene expression in the brain. Diabetes 53:911–920. 559. Argiles JM, Busquets S, Lopez-Soriano FJ (2002). The role of uncoupling proteins in pathophysiological states. Biochem Biophys Res Comm 293:1145–1152. 560. Makino T, Noguchi Y, Yoshikawa T, Doi C, Nomura K (1998). Circulating interleukin 6 concentrations and insulin resistance in patients with cancer. Br J Surg 85:1658–1662. 561. Yoshikawa T, Noguchi Y, Doi C, Makino T, Okamoto T, Matsumoto A (1999). Insulin resistance is connected with the alterations of substrate utilization in patients with cancer. Cancer Lett 141:93–98. 562. Yoshikawa T, Noguchi Y, Doi C, Makino T, Nomura K (2001). Insulin resistance in patients with cancer: Relationships with tumor site, tumor stage, body-weight loss, acute-phase response, and energy expenditure. Nutrition 17:590–593. 563. Tayek JA, Katz J (1997). Glucose production, recycling, Cori cycle, and gluconeogenesis in humans: Relationship to serum cortisol. Am J Physiol 272:E476–E484. 564. Russell A (1951). A diencephalic syndrome of emaciation in infancy and childhood. Arch Dis Child 26:274. 565. Fleischman A, Brue C, Poussaint TY, Kieran M, Pomeroy SL, Goumnerova L, et al. (2005). Diencephalic syndrome: A cause of failure to thrive and a model of partial growth hormone resistance. Pediatrics 115:e742–e748. 566. Brauner R, Trivin C, Zerah M, Souberbielle JC, Doz F, Kalifa C, et al. (2006). Diencephalic syndrome due to hypothalamic tumor: A model of the relationship between weight and puberty onset. J Clin Endocrinol Metab 91:2467–2473. 567. Vlachopapadopoulou E, Tracey KJ, Capella M, Gilker C, Matthews DE (1993). Increased energy expenditure in a patient with diencephalic syndrome. J Pediatr 122:922–924. 568. Hoek HW, van Hoeken D (2003). Review of the prevalence and incidence of eating disorders. Int J Eating Dis 34:383–396. 569. Johnson JG, Cohen P, Kasen S, Brook JS (2002). Eating disorders during adolescence and the risk for physical and mental disorders during early adulthood. Arch Gen Psychiatry 59:545–552. 570. Rigotti NA, Neer RM, Skates SJ, Herzog DB, Nussbaum SR (1991). The clinical course of osteoporosis in anorexia nervosa: A longitudinal study of cortical bone mass. JAMA 265:1133–1138. 571. Bulik CM, Sullivan PF, Fear JL, Pickering A, Dawn A, McCullin M (1999). Fertility and reproduction in women with anorexia nervosa: A controlled study. J Clin Psychiatry 60:130–135. 572. Lambe EK, Katzman DK, Mikulis DJ, Kennedy SH, Zipursky RB (1997). Cerebral gray matter volume deficits after weight recovery from anorexia nervosa. Arch Gen Psychiatry 54:537–542. 573. Strober M, Freeman R, Morrell W (1997). The long-term course of severe anorexia nervosa in adolescents: Survival analysis of recovery, relapse, and outcome predictors over 10-15 years in a prospective study. Int J Eating Dis 22:339–360. 574. Tamai H, Mori K, Matsubayashi S, Kiyohara K, Nakagawa T, Okimura MC, et al. (1986). Hypothalamic-pituitary-thyroidal dysfunctions in anorexia nervosa. Psychother Psychosom 46: 127–131. 575. Gianotti L, Arvat E, Valetto MR, Ramunni J, Di Vito L, Maccagno B, et al. (1998). Effects of beta-adrenergic agonists and antagonists on the growth hormone response to growth hormone-releasing hormone in anorexia nervosa. Biol Psychiatry 43:181–187.

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576. Licinio J, Wong ML, Gold PW (1996). The hypothalamic pituitary adrenal axis in anorexia nervosa. Horm Res 62:75–83. 577. Misra M, Klibanski A (2006). Anorexia nervosa and osteoporosis. Rev Endo Metab Dis 7:91–99. 578. Misra M, Miller KK, Tsai P, Gallagher K, Lin A, Lee N, et al. (2006). Elevated peptide YY levels in adolescent girls with anorexia nervosa. J Clin Endocrinol Metab 91:1027–1033. 579. Welt CK, Chan JL, Bullen J, Murphy R, Smith P, DePaoli AM, et al. (2004). Recombinant human leptin in women with hypothalamic amenorrhea. N Engl J Med 351:987–997. 580. Balligand JL, Brichard SM, Brichard V, Desager JP, Lambert M (1998). Hypoleptinemia in patients with anorexia nervosa: Loss of circadian rhythm and unresponsiveness to short-term refeeding. Eur J Endocrinol 138:415–420. 581. Monteleone P, Fabrazzo M, Tortorella A, Fuschino A, Maj M (2002). Opposite modifications in circulating leptin and soluble leptin receptor across the eating disorder spectrum. Mol Psychiatry 7:641–646. 582. Chan JL, Mantzoros CS (2005). Role of leptin in energy-deprivation states: Normal human physiology and clinical implications for hypothalamic amenorrhoea and anorexia nervosa. Lancet 366:74–85.

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583. Brichard SM, Delporte ML, Lambert M (2003). Adipocytokines in anorexia nervosa: A review focusing on leptin and adiponectin. Horm Metab Res 35:337–342. 584. Tagami T, Satoh N, Usui T, Yamada K, Shimatsu A, Kuzuya H (2004). Adiponectin in anorexia nervosa and bulimia nervosa. J Clin Endocrinol Metab 89:1833–1837. 585. Bosy-Westphal A, Brabant G, Haas V, Onur S, Paul T, Nutzinger D, et al. (2005). Determinants of plasma adiponectin levels in patients with anorexia nervosa examined before and after weight gain. Eur J Nutr 44:355–359. 586. American Psychiatric Association (2006). Treatment of patients with eating disorders, third edition. Am J Psychiatr 163:4–54. 587. Klibanski A, Biller BM, Schoenfeld DA, Herzog DB, Saxe VC (1995). The effects of estrogen administration on trabecular bone loss in young women with anorexia nervosa. J Clin Endocrinol Metab 80:898–904. 588. Robinson E, Bachrach LK, Katzman DK (2000). Use of hormone replacement therapy to reduce the risk of osteopenia in adolescent girls with anorexia nervosa. J Adolesc Health 26:343–348.

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C H A P T E R

20 Lipid Disorders in Children and Adolescents SARAH C. COUCH, PhD, RD • STEPHEN R. DANIELS, MD, PhD

Introduction Metabolism Primary Dyslipidemias Disorders of Cholesterol Metabolism Familial Hypercholesterolemia Autosomal-Recessive Hypercholesterolemia Familial Ligand-Defective apoB-100 Phytosterolemia Disorders of Overproduction of VLDL Disorders of Marked Hypertriglyceridemia Familial Hypertriglyceridemia Familial Lipoprotein Lipase Deficiency Hypolipidemias Low HDL Cholesterol Abetalipoproteinemia Hypobetalipoproteinemia

Introduction Cardiovascular disease (CVD) is a major cause of morbidity and mortality among adults in industrialized countries. Dyslipidemia (specifically elevated LDL cholesterol, low HDL cholesterol, and high triglycerides) has been identified as an independent risk factor in the development of CVD. There is strong evidence that lipoprotein levels track from childhood into adulthood and that abnormal levels of LDL cholesterol and perhaps other lipoproteins are associated with atherosclerosis and therefore with related adverse outcomes. This chapter reviews the evidence for the role of cholesterol abnormalities in the early natural history of atherosclerosis. In addition, a general overview of lipoprotein metabolism is provided—followed by a review of genetic disorders in the metabolism of lipoproteins. Sec-

Disorders with Lipoprotein Clearance via apoE Pathways Secondary Causes Vascular Changes and Dyslipidemia Screening for Lipid Disorders Routine Screening Genetic Testing Diet Therapy in Managing Dyslipidemia Pharmacologic Management Bile Acid Binding Agents HMG-CoA Reductase Inhibitors Inhibitors of Cholesterol Absorption Fibric and Acid Derivatives Niacin Dietary Additives and Supplements Conclusions and Future Directions

ondary causes of high cholesterol are explained, including the increasing prevalence of obesity and metabolic syndrome as a cause of cholesterol abnormalities in the pediatric population. Standards and approaches to screening for hyperlipidemia in childhood are reviewed, as well as current approaches to the dietary and pharmacologic management of pediatric lipid disorders.

Metabolism Lipid disorders in children and adolescents can result from defects in the production, transport, and/or degradation of lipoproteins. To understand the diverse causes of lipoprotein abnormalities, a brief review of lipoprotein structure, function, and metabolism is provided. Table 20-1 summarizes the lipoprotein subclasses, the source of 839

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TA B L E 2 0 - 1

Lipoprotein Subclasses and Associated Apolipoproteins and Lipid Constituents Lipoprotein Chylomicrons

Apolipoprotein

Source

Lipid Constituents

Intestine

Dietary triglycerides

VLDL

apoB-48, apoC-II*, apoC-III, apoE* apoB-100, C-II*, C-III, apoE*

Liver

IDL LDL HDL

apoB-100, apoE apoB-100 apoA-I, apoA-II, apoC-II, apoE

VLDL metabolism VLDL metabolism Liver and intestine

Endogenous cholesterol and triglyceride Cholesterol and triglyceride Cholesterol Cholesterol and phospholipid

* Transferred from HDL.

each, and the constituent lipids and apolipoproteins associated with each particle. Triglycerides, cholesterol esters, phospholipids, and plant sterols within food postingestion are digested to fatty acids, 2-monoglycerides, unesterified cholesterol, lysophospholipids, and unesterified plant sterols. These lipid-soluble products diffuse through the apical surface of the enteric membrane and are reaggregated into lipoproteins through the action of a microsomal triglyceride transfer protein (MTP). MTP (and perhaps an additional transfer protein) conjugate triglycerides with cholesterol ester (a phospholipid monolayer) and apolipoprotein B-48 (apoB-48) to create a mature chylomicron.1 Most of the plant sterol ingested and about half of the absorbed cholesterol are excreted from the intestinal cell back into the lumen by two ATP-binding cassette (ABC) half-transporters, thus limiting the amount of these sterols that are absorbed.2,3 Once formed, chylomicrons are too large to penetrate the capillary membrane. Consequently, they are secreted into the lymphatic system and enter the venous plasma compartment through the thoracic lymph duct. As the nascent particles are released into the plasma, several apolipoproteins (including apoC-II and apoE) are preferentially transferred to the chylomicrons from other lipoproteins (e.g., high-density lipoproteins, HDL).4 Figure 20-1 depicts chylomicron metabolism. Chylomicrons transport dietary triglyceride and cholesterol to sites of storage or metabolism.5 They are rapidly cleared from the circulation through the action of lipoprotein lipase (LPL). LPL is a triglyceride hydrolase found on

the capillary endothelium of various tissues, with highest concentration in muscle and adipose tissues.6 LPL is activated by apo-CII on the chylomicron. As the triglyceride contained within the chylomicron is hydrolyzed, the particle decreases in size. When approximately 80% of the initial triglyceride has been removed, apoC-II dissociates from its surface.4 The triglyceride-depleted chylomicrons, now considered chylomicron remnants, are taken up by the liver through a receptor that recognizes apoE on the particle surface. A smaller fraction of remnants may also be internalized via a low-density lipoprotein (LDL) receptor-like protein (LRP)-mediated endocytosis.7,8 Very-low-density lipoproteins (VLDLs) originate from the liver, and like chylomicrons are triglyceride-rich particles (Figure 20-2). In contrast to the intestinally derived chylomicrons, however, the fatty acids contained within the VLDL triglyceride come from de novo synthesis from dietary carbohydrate, lipoprotein remnants, or circulating fatty acids internalized by the liver from plasma.4 Within the hepatocyte, triglyceride and cholesterol ester are assembled by an MTP and surrounded with a phospholipid membrane associated with apoB-100.1 The mature VLDL particles are released into the lymph and ultimately into the vascular space, where other apolipoproteins (including apoC-II and apoE) adsorb to the VLDL surface. The metabolism of the VLDL particle follows a route similar to that of the chylomicron: apo C-II on its surface activates LPL, LPL hydrolyzes the VLDL triglyceride, the particle decreases in size (80% loss of triglyceride), and ultimately apoC-II dissociates—resulting in the formation

Chylomicron B

LPL

B Intestine

C-⌸ E

Liver

Chylomicron Remnant Figure 20-1 Exogenous lipoprotein metabolism. [Courtesy of Emilie Graham, University of Cincinnati.]

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841

C⌸

LPL

HL

B

VLDL

C-⌸

IDL LDL

LPL

B B

E Oxidation

E

Lipids CETP ⌱ A-

Liver

Cholesterol Pool

HDL A-⌱

AT LC ⌱ C AB

Nascent HDL

Macrophage

Intestine Figure 20-2 Endogenous lipoprotein metabolism. [Courtesy of Emilie Graham, University of Cincinnati.]

of VLDL remnants [also known as intermediate-density lipoproteins (IDLs)]. Approximately half of the IDL is then removed from plasma through the interaction of apoB with the apoB-100/apoE receptor on the surface of liver cells.9 The rest of the IDL is converted to LDL through further hydrolysis of core triglycerides by hepatic triglyceride lipase (HL). ApoE is transferred from IDL to HDL during the transition of the remnant to LDL.10 LDL, the major carrier of cholesterol in plasma, is taken up into peripheral tissues and liver cells by the apoB-100/apoE receptor—which recognizes apoB-100 on the particle surface. Upon receptor binding, the LDLreceptor complex is rapidly internalized into clathrincoated pits by endocytosis. Within the cell, the newly formed endosome becomes acidified through the action of an ATP-dependent proton pump.11 Acidification causes degradation of the clathrin coat, dissociation of the receptor from LDL, and subdivision of the endosomal membranes. The endosome containing the LDL receptor recirculates back to the cell membrane for additional LDL uptake. It then fuses with lysosomes, where the lipoprotein is digested into its component parts: unesterified cholesterol, fatty acids, and free amino acids.11 The amount of cholesterol released from endosomal uptake regulates hepatic synthesis of LDL receptor and cholesterol. When cellular concentration of cholesterol is low, sterol receptor binding protein (SREBP) is released. SREBP is a nuclear factor that enhances the transcription of LDL receptor and hydroxy-methylglutaryl (HM6) CoA reductase, the rate-limiting enzyme of cholesterol biosynthesis.12 In this way, intracellular hepatic cholesterol concentration regulates the amount of cholesterol internalized and synthesized by the cell. When excess LDL is present in the plasma, the capacity of the LDL receptor to remove it is exceeded and LDL

Ch20_839-854-X4090.indd 841

is more susceptible to oxidation. Oxidized LDL can be taken up by scavenger receptors on macrophages in the subendothelium of arteries and may contribute to the formation of atherosclerotic lesions.13 HDL transfers cholesterol and other lipids from peripheral tissues (including arterial atheroma) back to the liver. The particles are synthesized predominantly in the liver (and to a lesser extent in the intestine) as lipid-poor precursor particles (pre beta HDL) containing apoA-I. Nascent HDL interacts with the plasma membrane of cells, collecting lipid through an ATP-binding cassette transporter-A1 (ABCI) mechanism.2,3 The cholesterol and phospholipids transferred through this process adsorb to the HDL, forming a disc-shaped particle referred to as HDL3. Within the plasma, HDL3 interacts with the enzyme lecithin cholesterol acyl transferase (LCAT)—which catalyzes the esterification of particle-associated cholesterol. ApoA-I on the HDL surface activates LCAT. Once formed, the cholesterol ester is more hydrophobic and moves to the interior of the particle—creating a sphereshaped HDL particle known as HDL2.14 As HDL2 increases in size, the particle becomes substrate for cholesterol ester transfer protein (CETP). This enzyme promotes the exchange of esterified cholesterol within HDL2 for triglyceride contained within apoB-100associated lipoproteins.15 This lipid exchange is one mechanism whereby HDL indirectly participates in reverse cholesterol transport from tissues back to the liver. Two direct pathways in which HDL2 can transport cholesterol back to the liver involve holoparticle uptake via the apoB-100/apoE receptor and cholesterol ester transfer from HDL2 to the liver and other steroid-hormone-producing tissues through a scavenger receptor B1 (SRB1). This latter process may require the action of HL.16

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have myocardial infarction in the first decade of life and death from CAD in the second decade.20

Primary Dyslipidemias Lipoprotein synthesis, transport, and metabolism occur in many steps and involve many specialized proteins. A number of genetic defects have been identified in these processes and are referred to as primary dyslipidemias. Most of these genetic defects present in childhood. Table 20-2 provides a summary of pediatric lipoprotein disorders with reference to the characteristic lipoprotein profile of each. The genetic and metabolic etiologies of these disorders are detailed in the material following.

DISORDERS OF CHOLESTEROL METABOLISM Familial Hypercholesterolemia Familial hypercholesterolemia (FH) is the most common single gene disorder of lipoprotein metabolism. FH is inherited as an autosomal-dominant trait with relatively low prevalence in western countries. The heterozygous form is found in 1 in 500 persons and the homozygous form is found in 1 in 1,000,000 persons. The disorder is caused by a mutation in the apoB-100/apoE receptor or LDL receptor (LDLR) gene.17,18 More than 900 mutations in this gene have been identified, including those that affect receptor synthesis, intracellular transport, ligand binding, internalization, and recycling.19 In the heterozygous form, inheritance of one defective LDLR gene results in plasma LDL cholesterol levels two to three times higher than normal.17 Individuals with heterozygous FH are at an increased risk of developing early-onset coronary artery disease (CAD), usually between the ages of 30 and 60 years.17,18 In the homozygous form, individuals inherit a mutant allele for FH from both parents—resulting in plasma LDL cholesterol concentrations that are five to six times higher than normal. Due to the excessively high plasma cholesterol levels in these individuals, cholesterol deposits are common in the tendons (xanthomas) and eyelids (xanthelasmas)—generally by the age of 5 years.20 In the heterozygous form, xanthomas occur only rarely and generally not until older adulthood. Children with homozygous FH have early-onset atherosclerosis, and often

Autosomal-Recessive Hypercholesterolemia Autosomal-recessive hypercholesterolemia (ARH) is another inherited disorder resulting in marked elevations in LDL cholesterol levels. This disorder is caused by a defect in the ARH protein.21 ARH protein binds to the LDLR and clathrin, suggesting a role for ARH in the recruitment and retention of LDLR in clathrin coated pits.22 Several different mutations in this protein have been identified, all leading to a lack of (or suboptimal internalization of) the LDLR.22,23 Cholesterol levels in individuals with ARH are five to six times higher than normal. Children with this disorder are clinically similar to those with homozygous FH. However, their parents usually have normal lipoprotein profiles.23

Familial Ligand-Defective apoB-100 Familial ligand-defective apoB-100 (FDB) is a monogenic disorder resulting in moderate to markedly high plasma LDL cholesterol levels. The disorder is caused by poor binding of the LDL particle to the LDLR due to a mutation in apoB-100.24 Such deficient binding results in decreased clearance of LDL from plasma. The disorder is most common in individuals of European descent (1 per 1,000).9 Patients with FDB are at moderate risk of developing CAD.

Phytosterolemia Phytosterolemia is a rare autosomal-recessive disease caused by a mutation in two genes (ABCG5 and ABCG8) encoding the ABC half-transporters.9,25,26 These proteins limit the absorption of cholesterol and plant sterols (and possibly shellfish sterols) in the gut. They also promote biliary and fecal excretion of cholesterol and phytosterols.26 Defective proteins result in an abnormally high absorption of plant sterols (and to a lesser extent, cholesterol) into the enterocyte and decreased excretion of these sterols from the liver into the bile. Plasma cholesterol can be mildly, moderately, or markedly elevated— whereas plant sterol concentrations in the plasma are markedly increased.25,26 Patients with phytosterolemia

TA B L E 2 0 - 2

Pediatric Lipoprotein Disorders* Lipoprotein Disorder Familial hypercholesterolemia Autosomal recessive hypercholesterolemia Familial ligand-defective apoB-100 Phytosterolemia Familial combined hyperlipidemia Familial hypertriglyceridemia Familial lipoprotein lipase deficiency Hypoalphalipoproteinemia Dysbetalipoproteinemia

Lipoprotein Analysis ↑↑ LDL ↑↑ LDL ↑↑ LDL ↑ LDL ↑ VLDL, ↑ LDL, ↓ HDL ↑↑ VLDL, ↓ HDL ↑↑ Chylomicrons ↓ HDL ↑↑ Chylomicron remnants, ↑↑ IDL

Blood Lipids ↑↑ Cholesterol ↑↑ Cholesterol ↑↑ Cholesterol ↑ Cholesterol ↑ Cholesterol, ↑ triglycerides ↑ Triglycerides ↑↑ Triglycerides Normal ↑↑ Cholesterol, ↑↑ triglycerides

* ↑↑ very high; ↑ moderately elevated; and ↓ decreased.

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develop premature CAD, xanthomas in childhood, and may develop aortic stenosis.25,27

DISORDERS OF OVERPRODUCTION OF VLDL Familial combined hyperlipidemia (FCHL) is an autosomaldominant disorder with variable phenotypic expression, even within members of the same family.28 In most cases of FCHL, the disorder is caused by an overproduction of VLDL in the liver, a reduction in fatty acid uptake and retention by adipose tissue, and a decreased clearance of chylomicron remnants. The most common lipoprotein pattern is high LDL cholesterol, high triglycerides, and low HDL cholesterol.29 LDL particles tend to be small and dense. The prevalence of this disorder is estimated to be 0.5% to 1% of the adult population.21 Several studies identify FCHL as three times as common in clinical practice as FH.30,31 FCHL is diagnosed based on a primary hyperlipidemia, including hypercholesterolemia and/or hypertriglyceridemia; multiple lipoprotein phenotypes within a family; and a positive family history of premature CHD.21 Tendon xanthomas are usually not present in patients with FCHL. Patients with FCHL often have concurrent problems with insulin resistance, central obesity, and hypertension—and are at an increased risk of premature cardiovascular disease.28 Syndromes with a similar phenotype are hyperapobetalipoproteinemia, LDL subclass pattern B, and the metabolic syndrome.29,32-34 Of the three, the latter syndrome is much more prevalent in children. Rates of metabolic syndrome are continuing to rise with the prevalence of obesity in the pediatric population.34 There appears to be a mechanistic link between central obesity, insulin resistance, and dyslipidemia—with central obesity generally preceding both glucose and lipid abnormalities.

DISORDERS OF MARKED HYPERTRIGLYCERIDEMIA Familial Hypertriglyceridemia Familial hypertriglyceridemia (FHTG) follows an autosomal-dominant inheritance pattern expressed predominantly in adulthood. However, the prevalence in children is increasing.9 Obesity is an important factor that can expedite the expression of FHTG, and patients often have concurrent glucose intolerance. The phenotype for FHTG is moderate to markedly high serum triglycerides and low to normal LDL and HDL cholesterol levels. The metabolic cause of the disorder is hepatic secretion of large triglyceride-rich VLDL particles that are catabolized slowly.28 The fundamental genetic defect for FHTG has not been identified.

Familial Lipoprotein Lipase Deficiency Familial lipoprotein lipase (FLPL) deficiency is rare. The disorder is expressed as elevated serum chylomicron levels due to diminished or absent hydrolysis of chylomicron associated triglycerides by lipoprotein lipase.28 In the heterozygous state, patients have low to moderate

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levels of chylomicron catabolism and levels of serum triglycerides that range from 200 to 750 mg/dL.9 In patients in the homozygous state, serum triglycerides can reach 10,000 mg/dL or higher.35 Blood from patients with homozygous FLPL has a viscous, creamy appearance due to the presence of large numbers of chylomicron particles. Risk for pancreatitis is increased in the homozygous state due to the markedly elevated serum triglycerides. In addition, eruptive xanthomas and neurologic symptoms may be apparent. The gene for LPL has been located on chromosome 8p22, and more than 50 different mutations have been identified in patients with FLPL.36

HYPOLIPIDEMIAS Low HDL Cholesterol In clinical practice, patients with low HDL cholesterol levels commonly have concurrent high triglycerides, with or without elevations in small dense LDL cholesterol.37-39 These patients are usually obese, and the mechanistic explanation for this dyslipidemic triad is VLDL overproduction. Less common are familial disorders of HDL, including familial hypoalphalipoproteinemia, mutations of the apoA-1 protein, and Tangier disease. These disorders are characterized by low HDL level with no other lipid abnormality. Familial hypoalphalipoproteinemia follows an autosomaldominant inheritance pattern.9 ApoA-1 levels are also often low due to decreased production of HDL. A number of mutations have been described in the apoA-1 gene and are associated with low HDL cholesterol and low apoA-1.40,41 Tangier disease is due to mutations in the ABCI gene.42,43 Patients affected by this disease are not able to actively withdraw cholesterol from cells onto nascent HDL particles, causing rapid degradation of the nascent HDL. ApoA-1 is rapidly cleared before it is able to acquire cholesterol.43 In Tangier disease, HDL cholesterol levels are close to zero and the apoA-1 levels are less than 5 mg/dL. The risk of premature CAD in these patients is mild to moderate.9

Abetalipoproteinemia Abetalipoproteinemia is associated with low serum cholesterol (⬍50 mg/dL) and triglycerides (2-45 mg/dL). Patients with this disorder present with steatorrhea and fatty liver. Without treatment, ataxia follows (with acanthocytosis and retinitis pigmentosis). Abetalipoproteinemia is caused by a defect in MTP.28 Without MTP, no chylomicrons, VLDL, or LDL appear in the plasma. In these patients, HDL takes over as the primary cholesterol carrier. Thus, the defect is not fatal. Because of significant fat malabsorption, fat-soluble vitamin status is impaired. In particular, because vitamin E absorption and cellular uptake require chylomicron and LDL transport, high doses of vitamin E are required to prevent retinal and sensory neuron degeneration. Additional dietary considerations include restricting long-chain dietary triglycerides to less than 15 g/day to alleviate the steatorrhea. Medium-chain triglycerides (MCT oils) can be used as an alternative source of energy.44

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Hypobetalipoproteinemia Hypobetalipoproteinemia is an autosomal-dominant disorder resulting from a defect in the apoB gene that produces a truncated apolipoprotein B.28 Cholesterol levels in patients with heterozygous hypobetalipoproteinemia are usually 50% of those of an unaffected family member. The heterozygous form of this condition is benign. However, homozygous hypobetalipoproteinemia is associated with severe hypocholesterolemia, significant steatorrhea, fatty liver, acanthocytosis retinopathy, and peripheral neuropathy. Dietary considerations are the same as for patients with abetalipoproteinemia.44

DISORDERS WITH LIPOPROTEIN CLEARANCE VIA apoE PATHWAYS Dysbetalipoproteinemia is characterized by elevated cholesterol and triglyceride levels.28 The disorder results from the presence of a polymorphism of the apoE allele (apoE2, rather than the more common apoE3) along with the gene for FCHL.44 Metabolically, this defect results in poor uptake of remnant particles and abnormal remnant catabolism because of the abnormal apoE. Increased remnants, VLDL, chylomicrons, and apoE are all present. Xanthomas may occur, and premature CAD has been reported. This lipoprotein disorder is rare in children and often presents in young adulthood.28

Secondary Causes Secondary dyslipidemias can result from a variety of diseases and conditions (see Table 20-3 for a list). In the United States, the most prevalent cause of secondary dyslipidemia is overweight and obesity.45 The dyslipidemic triad (namely, elevated triglycerides and small TA B L E 2 0 - 3

Selected Secondary Causes of Pediatric Hyperlipoproteinemia Endocrine • Hypothyroidism • Diabetes • Pregnancy Exogenous • Drugs • Obesity • Alcohol Renal • Nephrotic syndrome • Chronic renal failure Hepatic • Cholestasis • Biliary atresia • Hepatitis • Biliary cirrhosis Immunologic • HIV infection/AIDS

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dense LDL and low HDL cholesterol) is commonly associated with overweight (in particular, with central adiposity).45,46 In addition to dyslipidemia, insulin resistance and elevated blood pressure may be present. This cluster of abnormalities characterizes the metabolic syndrome and is associated with increased cardiovascular disease risk.47,48 The prevalence of metabolic syndrome appears to be increasing in children and adolescents, along with the prevalence of obesity.47 The primary approach to treating this disorder is weight management. Metabolic lipid perturbations in adult patients with type 1 and 2 diabetes mellitus are similar to those found in patients with the metabolic syndrome, but often are more severe.49 Generally in adults with diabetes, triglycerides are elevated and HDL-cholesterol is low—and LDL cholesterol can be normal or mildly or moderately elevated. Diabetes in adults is considered a CHD risk equivalent according to the National Cholesterol Education Program (NCEP). This means that the risk for developing CAD in patients with poorly controlled diabetes is equivalent to those with established CHD.50 For this reason, the NCEP recommends aggressive treatment of dyslipidemia in adult patients with diabetes. Although type 1 diabetes is currently the main form of diabetes seen in children, in the United States a growing number of patients with type 2 diabetes are under the age of 18 years.51 Change in the prevalence of type 2 diabetes in youth is likely related to the growing obesity epidemic occurring in the pediatric population.51,52 Data on lipid concentrations in children and adolescents with diabetes are few, particularly in those with type 2 diabetes. The Search for Diabetes in Youth Study assessed the prevalence of serum lipid abnormalities among a representative sample of U.S. children and adolescents with type 1 and type 2 diabetes.53 Findings from this study showed a substantial number of diabetic children over the age of 10 years with abnormal serum lipids: nearly 50% had an LDL cholesterol level above the optimal level of 100 mg/dL. For children with type 2 diabetes, 37% had elevated triglyceride levels and 44% had low HDL cholesterol. These data highlight the importance of serum lipid screening in children with diabetes. Recommended approaches to managing dyslipidemia in children and adolescents with diabetes are discussed later in the chapter. Other causes of secondary dyslipidemia include hypothyroidism, nephrotic syndrome, other renal diseases, liver diseases, and infection.54 Dylipidemias can also result from the ingestion of a variety of medications. These medications include progestins, estrogens, androgens, anabolic steroids, corticosteroids, cyclosporine, and retinoids. Secondary causes of dyslipidemias should be identified by patient historical data and a careful physical examination. Laboratory tests (including thyroid, renal, and liver function panels) would confirm the diagnosis. The risk of development of atherosclerosis with these conditions is unknown but is likely proportionate to the length of exposure and extent of elevation in serum LDL cholesterol levels. Cardiovascular disease is common in patients with chronic renal insufficiency.55 The treatment of dyslipidemia in patients with secondary causes is

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focused on managing the underlying disease. Diet and physical activity changes may also be recommended to reduce elevated LDL cholesterol and triglyceride levels.

Vascular Changes and Dyslipidemia It is well established that elevated concentrations of total cholesterol and LDL cholesterol in adult life are strong and reversible risk factors for CAD.50 Whether dyslipidemia in childhood contributes to atherosclerotic lesions in coronary and other arteries has been a subject of debate, but accumulating evidence from pathologic and in vivo imaging studies favors a relationship. Atherosclerotic lesions result from deposits of lipid and cholesterol in the intima of the arterial wall.56 Early lesions, called fatty streaks, are formed from the accumulation of macrophages filled with lipid droplets (foam cells). Fatty streaks do not disorganize the normal structure of the intima and do not deform or obstruct the artery, and are in themselves not considered harmful.57 However, some continue to accumulate macrophage foam cells and extracellular lipid and smooth muscle cells— forming raised plaques. From these, more advanced lesions may develop—with further deposition of extracellular lipid, cholesterol crystals, collagen, and potentially calcium.58 It is these raised lesions that result in a myocardial infarction because of their increasing size and obstruction of the arterial lumen or because of rupture of the fibrous plaque, which results in the release of thrombogenic substances from the necrotic core.56 Pathobiologic studies of the coronary arteries of young individuals who died from causes unrelated to heart disease have been useful in documenting the progression of atherosclerosis by age and risk factor determinants. Stary et al. studied more than 500 postmortem samples of coronary arteries from persons younger than 30 years of age and found presence of fatty streaks in the majority of children less than 9 years of age, raised lesions in about half of adolescents, and more advanced lesions in about a third of the young adults studied.59 In 93 autopsies of young adults for whom childhood risk factor data were available, Berenson and colleagues found that the extent of the surface of arteries covered with fatty streaks and fibrous plaques was positively associated with LDL cholesterol, triglycerides, blood pressure, and body mass index and negatively associated with HDL cholesterol levels in childhood.60 The Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study reached similar conclusions from examination of more than 3,000 postmortem samples of coronary arteries of young adults who died from noncardiovascular events and who likewise had a variety of antimortem risk factor measures available.61 In general, pathology studies have made important contributions to the identification of risk factors for early aspects of the atherosclerotic process. In conjunction with findings from longitudinal studies such as the Framingham Heart Study (in which risk factor assessments of participants preceded the development of cardiovascular disease)62 a group of risk factors, often referred to as the traditional risk factors

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for CAD, has been established. A complete list of pediatric risk factors for CAD is found in Table 20-4. Recent advances in vascular imaging technology have provided a means of measuring early pathologic changes and functional abnormalities against coronary and other arteries in response to adverse changes in cardiovascular disease risk factors. The advantage in using this technology is that walls of superficial arteries can be imaged noninvasively in real time at high resolution, and changes to the arterial wall can be measured as a continuous variable from childhood to adulthood in patients with and without presence of risk factors for CVD.63 Computed tomography (CT) scanning is considered one of the most sensitive noninvasive tools for imaging the extent and location of coronary artery calcium present in atheroma.64 The presence of coronary artery calcium has been associated with adverse cardiovascular disease outcomes in adults.65 In adolescents, small studies have shown associations among hypercholesterolemia, BMI, and significant coronary artery calcium. In the Muscatine Study (in which participants were assessed for CVD risk factors during their school-age years and later assessed for cardiovascular changes by CT scan), 31% of men and 10% of women aged 29 to 37 years had significant coronary artery calcification.66 Childhood risk factors associated with calcification were obesity, increased blood pressure, and low HDL cholesterol. Vascular ultrasound imaging has been utilized to assess alterations in brachial artery flow-mediated dilation, which is a measure of endothelial function, and carotid intimamedia thickness (IMT).64 In adults, both measures have been associated with adverse changes in traditional cardiovascular disease risk factors,67 respond to normalization of risk factors,68 and are considered important early markers for the progression of atherosclerotic disease.67-70 Although few studies have used ultrasound technology to evaluate coronary arteries in the young, children with hypercholesterolemia have been assessed using these measures and have been found to have abnormalities of carotid IMT and brachial artery vasodilation.71-73 Further, in young adults aged 33 to 42 years who had comprehensive risk factor assessments performed some 25 years prior Davis et al.74 found an association between mean carotid IMT and elevations in total cholesterol and triglyceride concentrations during childhood. Lavrencic et al.75 found that the mean carotid IMT was significantly TA B L E 2 0 - 4

Pediatric Risk Factors for Coronary Artery Disease • Elevated LDL cholesterol (⬎130 mg/dL) • Family history of premature (aged ⬍55 years) coronary heart disease, CVD, or peripheral vascular disease • Smoking • Hypertension • Low HDL cholesterol (⬍35 mg/dL) • Obesity [⬎95th percentile weight for height on National Center for Health Statistics (NCH) growth chart] • Physical inactivity • Diabetes

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greater in youth with FH compared with those in a control group—as well as being significantly greater in all subjects in regard to total cholesterol, LDL cholesterol, and systolic blood pressure. Similar risk factors have been associated with impaired vasodilation, indicating endothelial dysfunction.76-78 In summary, these studies confirm the utility of vascular imaging for detecting early pathologic and functional changes to coronary vessels and associations with modifiable CVD risk factors in the young. Clinically, vascular imaging by ultrasound may be a valuable means of estimating the benefit of treating multiple CVD risk factors in children and adolescents. CT scans may be less useful in younger patients because calcium depositions are uncommon before young adulthood.

Screening for Lipid Disorders ROUTINE SCREENING Data from pathologic and, more recently, in vivo imaging studies (as discussed previously) support the screening and treatment of children and adolescents with elevated LDL cholesterol levels and other risk factors. For more than a decade, pediatric guidelines established by the NCEP have provided the standard of care with respect to lipid screening and treatment of dyslipidemia in childhood.79 An algorithm summarizing these diagnostic and therapeutic guidelines is presented in Figure 20-3. In brief, the guidelines recommend targeted blood choles-

Risk Assessment Positive Family History of Parental High Blood Cholesterol or Premature CVD

Fasting Lipoprotein Analysis

Acceptable LDL-Cholesterol ⬍110 mg/dl Acceptable LDL-Cholesterol ⬍110 mg/dl

Borderline LDL-Cholesterol 110-129 mg/dl

Repeat Lipoprotein Analysis and Average Previous Measurements

Borderline LDL-Cholesterol 110-129 mg/dl

Repeat lipoprotein analysis within 5 years. Provide education on recommended eating pattern and risk factor reduction.

Risk factor advice Provide Step-One Diet and other risk factor intervention Reevaluate status in one year.

Elevated LDL-Cholesterol ⱖ130 mg/dl

HDL-Cholesterol ⬍35 mg/dl*

Persistently high LDL-Cholesterol ⱖ130 mg/dl

*If low HDL-cholesterol is detected, then patients should be counseled regarding cigarette smoking, low saturated fat diet, physical activity and weight management (if overweight).

Do clinical evaluation (history, physical exam, lab tests) • Evaluate for secondary causes • Evaluate for familial disorders Screen all family members Intensive clinical intervention Step-One, then Step-Two diet** Set goal LDL-cholesterol • Minimal: ⬍130 mg/dl • Ideal: ⬍110 mg/dl

**For patients 10 years old and over and with LDL-C ⬎190 mg/dl (or ⬎160 mg/dl with additional risk factors), if diet does not achieve the goal, then pharmacologic intervention should be considered. Figure 20-3 Child treatment algorithm based on LDL cholesterol levels. [Reprinted with permission from Williams CL, Hayman LL, Daniles SR, Robinson TN, Steinberger J, Paridon S, et al. (2002). Cardiovascular health in childhood: A statement for health professionals from the Committee on Atherosclerosis, Hypertension, and Obesity in the Young (AHOY) of the Council on Cardiovascular Disease in the Young, American Heart Association. Circulation 106:143-160.]

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terol screening if a child or adolescent has a parent with hypercholesterolemia (total cholesterol ⬎240 mg/dL) or if a child has a parent or grandparent ⬍55 years of age who has documented CVD. CVD is defined as angina pectoris, myocardial infarction, established coronary atherosclerosis, peripheral vascular disease, cerebrovascular disease, or sudden cardiac death. Lipid screening of children with multiple risk factors for future CVD (e.g., smoking, hypertension, obesity, diabetes, poor diet quality, and sedentary lifestyle) is recommended by the NCEP. Screening children whose family histories are unknown is discretionary. A fasting (12-hour) lipoprotein analysis is recommended for screening a child. A fasting lipoprotein analysis allows quantification of total cholesterol, HDL cholesterol, triglycerides, and calculation of LDL cholesterol. The Friedewald equation [LDL cholesterol ⫽ (total cholesterol – HDL cholesterol) – triglycerides/5] can be used to calculate LDL cholesterol as long as the serum triglyceride level is ⬍400 mg/dL.80 Direct measurement of LDL cholesterol concentration is available through some commercial laboratories and is indicated for individuals whose fasting triglyceride level is ⬎400 mg/dL. The NCEP defines cut-points for total cholesterol and LDL cholesterol in children based on data from the National Health and Nutrition Examination Survey (NHANES), performed from 1988 to 1994. The 75th and 95th percentiles for lipid values from this survey were used to define “borderline” and “high” risk categories, respectively. Acceptable, borderline, and high risk values for total and LDL cholesterol are outlined in Table 20-5. If a lipoprotein analysis reveals LDL cholesterol to be borderline or high, a repeat test should be performed and the average value of the two tests considered for clinical decision making.46 Clinical evaluation of children and adolescents at high risk for CVD based on LDL cholesterol levels should include careful review of the patient’s medical and family history and physical examination to identify additional risk factors and secondary causes of dyslipidemia. Assessment should include the following: review of past medical or family history for hypertension, diabetes mellitus, medica-

Normal Plasma Lipid and Lipoprotein Concentrations for Children and Adolescents* Total cholesterol LDL cholesterol Triglycerides 0-9 years 10-19 years Category HDL cholesterol

Acceptable ⬍170 ⬍110 ⬍75 ⬍90 Acceptable ⬎45

Borderline

High

170-199 110-129

⬎200 ⬎130

75-99

⬎100

90-129 Borderline

⬎130 Low

35-45

⬍35

LDL, low-density lipoprotein; and HDL, high-density lipoprotein. * Values for plasma lipids and lipoproteins are from the National Cholesterol Education Program Expert Panel on Cholesterol Levels in Children.

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tion use, obesity, poor dietary habits (including excessive intake of saturated fat), sedentary behavior, and tobacco use; measurement of height, weight, and calculation of BMI; Tanner staging to assess pubertal growth; blood pressure measurement; physical inspection of skin, eyes, and tendons for lipid deposition and palpitation of the thyroid gland and liver for signs of enlargement; and laboratory tests (including thyroid, renal, and liver function panels). Glucose and insulin levels should be measured to assess for the presence of metabolic syndrome or diabetes. Several limitations have been identified regarding the NCEP guidelines for the identification of children and adolescents at high risk for CVD. First, several studies have noted that selectively screening children and adolescents for elevated cholesterol based on parental history may be inadequate to identify a substantial proportion of the population with elevated LDL levels.81-83 In general, studies have shown that approximately 30% to 60% of pediatric patients with elevated cholesterol would be missed using the NCEP screening approach based on family history.82,83 A second limitation is that current guidelines give no significant consideration to variation in LDL cholesterol level according to race, gender, age, and level of sexual maturation. There is considerable variation in LDL cholesterol with age during growth and development. This is especially true during the period of puberty.84,85 Total and LDL cholesterol levels tend to decline during puberty, meaning that some adolescents will appear normal when in fact they will have elevated levels after puberty. A third limitation is that guidelines were published at a time when the epidemic of overweight and obesity was not yet obvious, and the guidelines do not address screening for low HDL cholesterol and high triglycerides. Recent data from pathobiologic and vascular imaging studies show adverse changes in vascular structure and function in adulthood related to low HDL cholesterol and high triglyceride levels in childhood. These data support pediatric assessment of triglyceride and HDL cholesterol levels. The American Heart Association86 has recommended that triglyceride levels ⬎130 mg/dL and HDL cholesterol levels ⬍35 mg/dL be considered abnormal for children and adolescents (Table 20-5).

GENETIC TESTING

TA B L E 2 0 - 5

Category

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Increasingly, DNA-based tests are being used to confirm the diagnosis of FH in patients with a family member who has a mutation or in a young patient with high LDL cholesterol with tendon xanthomas or atherosclerotic disease.87 Currently, three genes (LDLR, APOB, PCSK9) have been identified in association with mutations that cause this disorder.88 In addition to LDLR, most laboratories test for the apolipoprotein B gene r500Q mutation. Rapid and relatively inexpensive methods have been developed to test a selected subset of the LDLR mutations. However, more expensive “complete gene scans” are needed for mutation-negative samples.88 Once a mutation is identified, testing of relatives can be performed rapidly and cheaply. At present, it is unclear whether knowledge of specific mutations will lead to improved treatment. Several studies have shown that the degree of cholesterol lowering

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LIPID DISORDERS IN CHILDREN AND ADOLESCENTS

achieved by statins is influenced by the type of mutation (e.g., individuals with the APOB r3500Q mutation showed a strong positive response to statin therapy).89,90 In addition, the diagnosis of FH based on genetic testing improved uptake and adherence to treatment in several studies.91,92 Concerns about long-term benefits and side effects from life-long treatment for identified children remain. As progress in this area continues, current treatment algorithms may need modification to describe the role of genetic testing in clinical practice.

Diet Therapy in Managing Dyslipidemia The NCEP guidelines outline a two-pronged approach to managing pediatric hypercholesterolemia: one geared to the population in general and the second to individualized treatment of elevated LDL cholesterol levels in highrisk patients. The population-based approach stipulates that all children older than 2 years of age should consume a low-fat diet, referred to as the Step 1 diet.79 This recommendation is viewed as a primary preventive measure to reduce serum lipids, thereby reducing risk of CAD in the pediatric population at large. The individual patient approach recommends that all children and adolescents with elevated LDL cholesterol first be treated with the Step 1 diet. If after 3 to 6 months of dietary compliance LDL cholesterol levels remain abnormal further restriction of dietary saturated fat and cholesterol, referred to as the Step 2 diet, is recommended. Repeated studies have shown that this stepwise dietary approach to lowering LDL cholesterol levels is safe and efficacious in children with hypercholesterolemia.93-95 The nutrient composition of the Step 1 diet is 30% or less of calories from total fat, less than 10% of calories from saturated fat, and less than 300 mg/day of cholesterol—with adequate energy to support growth and development.79 The dietary restriction on total fat is not intended to be a daily recommendation but rather averaged over several days.96 Further, these recommendations are not intended for children younger than 2 years of age because infants and young children are thought to require a higher level of dietary saturated fat and cholesterol to support development of the central nervous system. To prevent overzealous implementation of a very-low-fat diet for children with high cholesterol, which could lead to failure to thrive,97 the American Academy of Pediatrics (AAP) recently set a lower limit for percentage of energy from fat at 20% of calories.98 In most cases, with dietary compliance the Step 1 diet should normalize borderline-high LDL cholesterol levels. The reported response to the diet in children with LDL cholesterol levels ⬍130 mg/dL has ranged from 3% to 10%.46,99 For children with persistently high LDL cholesterol levels, further reduction of dietary saturated fatty acids and cholesterol is recommended. Specifically, the Step 2 diet calls for restricting saturated fat intake to ⬍7% of calories and cholesterol to ⬍200 mg/day.79 Based on clinical evidence, these dietary restrictions should achieve an additional LDL cholesterol reduction of 4% to 14%.100,101

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However, the extent to which LDL cholesterol is lowered may depend on previous dietary intake and baseline LDL cholesterol levels. To achieve the NCEP dietary goals for saturated fat and cholesterol, key sources of these nutrients must be the focus of intervention. Foods high in saturated fat and cholesterol include red meats, poultry with skin, wholemilk dairy products, and egg yolks. The NCEP recommends that red meats be limited to modest amounts of lean cuts (e.g., 5-6 oz per day) and that dairy products be limited to skim or low-fat varieties (24-32 oz/day).79 Skin should be removed from chicken and turkey, and intake limited to white meat. Processed meats should contain no more than 3 g of fat per serving, and egg yolks should be limited to 4 per week, on the Step 1 diet (2 egg yolks per week on the Step 2 diet). Oils used in cooking should be unsaturated, and soft margarines used in place of butter. Table 20-6 highlights some additional practical dietary strategies for lowering saturated fat and cholesterol in the diets of the young. To ensure children prescribed low-fat diets consume adequate calories and nutrients to support growth and development, high-fat foods omitted from the diet should be replaced with lower-fat versions of the same. For example, whole-milk dairy products contribute calcium, phosphorus, and protein to the diet—along with substantial amounts of saturated fat and cholesterol. Children should be encouraged to replace whole-milk dairy foods with low-fat or nonfat milk, yogurt, cottage cheese, and frozen desserts to ensure overall nutrient adequacy. Breads, cereals, grains, fruits, and vegetables should comprise the largest proportion of energy in children’s diets. Most choices within these food groups are low in fat, cholesterol free, and high in fiber—and will help displace energy sources containing saturated fat. Children should be encouraged to choose 6 to 10 servings of whole-grain cereals, breads, and pasta and to consume at least five servings of fruits and vegetables per day. TA B L E 2 0 - 6

Practical Dietary Strategies for Lowering Saturated Fat and Cholesterol • Eat 5 to 9 servings of fresh, frozen, or canned fruits and vegetables daily. • Use vegetable oils and soft margarines low in saturated fat and trans fatty acids instead of butter or most other animal fats in the diet. • Eat whole-grain breads and cereals rather than processed grain products. • Use nonfat (skim) or low-fat milk and dairy products daily. • Eat more fish, especially oily fish (broiled or baked). • Eat lean cuts of meat, and trim any obvious fat from red meat before cooking. • Take the skin off chicken or turkey and eat only the white meat. • Avoid processed meats such as hotdogs, sausage, and bologna. • Avoid creams or sauces made with butter or whole-milk dairy products. • Choose low-fat snacks such as ginger snaps, graham crackers, pretzels, plain popcorn, animal crackers, and vanilla wafers. • Use recommended portion sizes on food labels when preparing and serving food.

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Although pediatric dietary recommendations do not include specific guidelines for lowering serum triglycerides or raising HDL cholesterol, diets containing high levels of sucrose and excess calories have been associated with adverse changes in these lipids/lipoproteins.102 For this reason, children should be counseled to reduce intake of simple sugars from processed foods such as cookies and desserts, and limit intake of sugared beverages. Weight management should be a goal of diet therapy for children with a BMI ⬎85th percentile. Weight reduction approaches should focus on decreasing the child’s weight-for-height percentile while maintaining linear growth.101 Although weight loss may temporarily lower HDL cholesterol, weight stabilization at a new lower level will lead to a gradual increase in HDL cholesterol over time.103 Moderate caloric restriction can be achieved by following the Step 1 diet. Increased physical activity should be encouraged, and sedentary activities (such as television viewing and playing computer and video games) should be limited to no more than 2 hours per day.

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age and whose post-dietary LDL cholesterol level is ⬎190 mg/dL or whose LDL cholesterol level is ⬎160 mg/dL with a family history of early CVD, with two or more risk factors for CVD, or with the metabolic syndrome present (Figure 20-3). A follow-up LDL cholesterol assessment is recommended 6 weeks after starting drug therapy, and every 3 months thereafter until LDL cholesterol goals are met. Thereafter, follow-up can be less frequent. For children in general, the goal for LDL cholesterol is ⬍130 mg/dL. For children with diabetes, the goal for LDL cholesterol is ⬍100 mg/dL.104,105 The lower LDL cholesterol goal for children with diabetes reflects a synthesis of pediatric guidelines and treatment recommendations for adults with diabetes that now consider the presence of diabetes a coronary heart disease risk equivalent.50 Table 20-7 summarizes the recommendations of the American Diabetes Association for lipid screening and management of children and youth with diabetes. Medications used in the treatment of specific lipid abnormalities are summarized in Table 20-8 and are reviewed in the material following.

Pharmacologic Management

BILE ACID BINDING AGENTS

For many children with moderately or severely elevated LDL cholesterol, diet alone will not lower their cholesterol levels to the acceptable or even borderline range. Longterm drug therapy is associated with decreased incidence of heart disease and overall mortality in adults.50 However, no studies directly demonstrate the efficacy of administering lipid-lowering drug therapy in children to prevent CAD. In addition, long-term safety studies for some of the newer medications have not been performed. The pediatric panel of the NCEP recommends using medication only in patients who are at least 10 years of

Bile acid binding agents lower serum cholesterol indirectly by binding with bile acids in the gastrointestinal tract. This action prevents their reabsorption into the enterohepatic circulation, resulting in their loss from the body and removal from the cholesterol pool.106 To compensate for this loss, the liver increases endogenous cholesterol synthesis and up-regulates LDL-receptor synthesis—thereby lowering LDL cholesterol levels in the blood. In bile-acid resin trials in children, a dose of 8 g/day while on a cholesterollowering diet resulted in a decrease in LDL cholesterol ranging from 10% to 20%.107,108

TA B L E 2 0 - 7

American Diabetes Recommendations for Management of Children and Adolescents with Diabetes* Type 1 Diabetes Screening

Lipid goals

Treatment strategies

Type 2 Diabetes

After glycemic control ⬎2 years at diagnosis if other CVD risk factors; At diagnosis regardless of age; if otherwise at 12 years; if normal, rescreen normal, rescreen every 2 years every 5 years LDL ⬍100 mg/dL HDL ⬎35 mg/dL Triglycerides ⬍150 mg/dL Glycemic control Diet Physical activity Weight reduction if appropriate Medication indications if initial management fails: • Age ⬎10 years • If LDL ⬎160 mg/dL • If LDL 130–159 mg/dL consider based on CVD risk profile • Statins with or without resins • Fibrates if TG ⬎1,000 mg/dL • Manage other CVD risk factors (Table 20-4) if appropriate

* Recommendations are from the American Diabetes Association. American Diabetes Association (2005). Standards of medical care in diabetes. Diabetes Care 28(1):S4–S36.

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LIPID DISORDERS IN CHILDREN AND ADOLESCENTS TA B L E 2 0 - 8

Drugs Used in the Treatment of Pediatric Lipid Disorders Class of Medication

Common Name

Bile acid binding agents

Cholestyramine Colestipol Atorvastatin Simvastatin Pravastatin Rosuvastatin Lovastatin Fluvastatin Bezafibrate Clofibrate Fenofibrate Gemfibrozil Niaspan

HMG Co-A reductase inhibitors*

Fibrates*

Niacin*

Cholesterol absorption blocker*

Ezetimibe

Starting Daily Dose

Change in Lipid Profile

Adverse Effects

80 mg/kg 5g 10 mg 5 mg 10 mg 5 mg 10 mg 20 mg 200 mg TID 500 mg BID 48 mg 600 mg BID 500 mg @ HS

↓ LDL ↑ TG ↓ LDL ↓ TG ↑ HDL

Constipation Abdominal cramping Dyspepsia ↑ Liver transaminases ↑ CK Myositis

↓ TG ↑ HDL

Constipation Myositis Anemia

↓ LDL ↓ TG ↑ HDL ↓ LDL

Flushing Headache ↑ Liver transaminases Not reported in children or adolescents

10 mg

* Long-term safety and efficacy has not been established in children and adolescents. CK ⫽ creatine kinase; LDL ⫽ low density lipoprotein; TG ⫽ triglycerides; and HDL ⫽ high-density lipoproteins.

Bile-acid resins come in powder and tablet form. The powder is usually taken twice daily (4-g scoop) mixed with water or juice. Resins in this form tend to be gritty in texture and children complain that they are unpleasant to drink. Tablets are more palatable, but are large and difficult for some children to swallow. Overall, studies report poor to fair compliance with the medication.106,109 Side effects are few and are mainly gastrointestinal in nature, including constipation and gas. These can be minimized with increased intake of water and fiber. Resins may increase triglyceride levels and may interfere with the absorption of certain medications and fatsoluble vitamins.110 Supplementation with a multivitamin and folate (1 mg daily) is usually recommended.106

HMG-CoA REDUCTASE INHIBITORS HMG-CoA reductase inhibitors (also known as statins) lower LDL cholesterol in the blood by blocking hepatic HMG Co-A reductase, the rate-limiting enzyme in cholesterol biosynthesis.111 This action depletes the intracellular cholesterol pool, leading to an up-regulation of LDL receptors and a decrease in serum cholesterol. Doses ranging from 5 to 40 mg/day have resulted in a 20% to 40% lowering of LDL cholesterol, and slight increases in HDL cholesterol levels have been reported in children.112-114 In vivo imaging studies have demonstrated improvements in surrogate markers for atherosclerosis with statin therapy. Reversal of endothelial dysfunction and regression of carotid IMT have been reported in children treated with statins over a 2-year period.115,116 This latter finding suggests that initiation of lipid-lowering statins in childhood may inhibit progression or might even lead to regression of atherosclerosis. Adverse effects of the medication are few but have included GI complaints, elevated liver transaminase

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and creatine kinase (CK) levels, and myositits.106,110,115 For this reason, statins would not be recommended for patients with liver disease. Follow-up of patients treated with statins should include laboratory assessment of liver transaminase and CK levels and identification of adverse physical symptoms such as muscle cramps. Because statins are potentially teratogenic, it is essential that physicians determine that adolescent girls are not pregnant or likely to become pregnant before initiating therapy.106 The longest statin trials have been 2 years in length. Thus, whether statins adversely affect long-term growth and development and are safe for lifelong use has not been ascertained. Longer-term safety and efficacy studies are needed, particularly with follow-up of vascular endpoints.

INHIBITORS OF CHOLESTEROL ABSORPTION Ezetimibe is a new cholesterol-lowering agent that prevents the absorption of cholesterol and plant sterols by inhibiting the passage of sterols across the intestinal wall.117 The reduction in cholesterol absorption leads to a decrease in hepatic cholesterol uptake and availability. As a result, there is a compensatory increase in hepatic cholesterol biosynthesis, an up-regulation of LDL receptor expression, and an overall decrease in blood LDL cholesterol levels. In adult trials, a daily intake of 10 mg/day reduced LDL cholesterol by approximately 18%.117-120 Although Ezetimibe has already been approved for children older than 10 years who have FH, it has not been adequately studied in this group of patients. Such studies are needed to confirm adult findings and to assess the safety and efficacy of this medication for use in children and adolescents.

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FIBRIC ACID DERIVATIVES Fibric acid derivatives (also known as fibrates) lower blood triglyceride levels by reducing the hepatic production of VLDL.106,117 The drug also increases the production of apoA-I, resulting in higher HDL cholesterol levels. Side effects are similar to those for statins and include GI complaints, elevated liver transaminase activity, and myopathy.106 For this reason, fibrates are not recommended for used with statins. No cut-points for initiating fibrate therapy in children and adolescents have been established. Because the major risk of elevated triglyceride (pancreatitis) does not increase until triglycerides levels reach 750 to 1,000 mg/dL, many experts recommend withholding fibrate use in children until the triglycerides are persistently ⬎350 mg/dL or a random level is ⬎700 mg/dL.106

NIACIN Niacin decreases hepatic VLDL production, leading to decreased production of LDL cholesterol.106,117 Children with heterozygous FH treated with 1,000 to 2,250 mg of niacin daily over an average of 8 months showed a 23% to 30% reduction in LDL cholesterol.120 However, 76% of the children had adverse effects from therapy (e.g., flushing, headache, nausea, glucose intolerance, myopathy, abnormal liver function) and 38% discontinued the drug. Niacin is generally not used to treat children with FH unless LDL cholesterol is persistently elevated or unusual hypertriglyceridemia and low HDL cholesterol are present.106 Niacin given in combination with statins has been used to treat homozygous FH. Niacin is available in immediate and slow-release forms (Niaspan, Slo-Niacin).

DIETARY ADDITIVES AND SUPPLEMENTS Plant sterols and stanols consumed at levels of 2 g per day have been shown to reduce LDL cholesterol levels by 9% to 20% in adults.50 Foods containing these additives (e.g., margarines and salad dressings) lower serum cholesterol by preventing dietary cholesterol absorption in the gastrointestinal tract.121 In children with familial hypercholesterolemia, use of 2 g/day of plant sterols decreased LDL cholesterol by 14% but did not improve endothelial function.122 This suggests that LDL cholesterol must be reduced to a certain threshold level before improvement of endothelial function can occur. More studies examining the long-term effects of plant sterols on the vascular endothelium are warranted. Concern has been raised about the potential for the malabsorption of fat and fat-soluble vitamins in children consuming plant sterols chronically. The AHA recommends reserving the use of foods supplemented with plant sterols/stanols to children with moderate to severe elevation in cholesterol, and monitoring fat-soluble vitamin status.123 Dietary supplements containing soluble fiber, garlic, and omega-3 fatty acids have been used to treat pediatric hyperlipidemia with unremarkable findings. In a randomized controlled trial, Dennison et al.124 studied the effect of psyllium fiber versus placebo on change in blood cholesterol levels of 5- to 17-year-old children. Psyllium fiber (6 g/d) and the placebo were added to a ready-to-eat

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cereal. Although compliance was excellent, there were no significant effects of the added psyllium on total, LDL, or HDL cholesterol levels over a 5-week period. Garlic extract therapy (300 mg ⫻ 3 doses per day) was studied in a randomized controlled trial for effects on serum lipoproteins in 8- to 18-year-old children with FH.125 Garlic extract was given in 3 daily doses of 300 mg versus a placebo for 8 weeks. No significant effects of garlic treatment versus placebo were noted on total, LDL, or HDL cholesterol. Omega-3 fatty acids, as found in fish oils, are known to reduce serum triglyceride levels in adults.50 In a randomized crossover study, children with FH and FCH were supplemented with either 1.2 g/day of docosahexaenoic acid (DHA) or a placebo for 6 weeks.126 All children were given counseling on the NCEP Step 2 diet. Outcomes studied included triglycerides, LDL and HDL-cholesterol, and endothelial function as measured by flow-mediated dilation (FMD) of the brachial artery. Findings showed that DHA supplementation was associated with increased levels of total, LDL, and HDL cholesterol but no change in triglycerides compared to the placebo group. FMD improved significantly after DHA supplementation compared to baseline in both groups, and the change was greater in the DHA-treated group versus the controls. This finding suggests that in children DHA may not be effective for positively modifying serum lipids. However, the endothelium may be a therapeutic target for DHA in hyperlipidemic children.

Conclusions and Future Directions Increasing evidence indicates that extreme elevation in LDL cholesterol is associated with vascular pathology in youth. Fortunately, appropriate therapy can reverse these changes. Pediatric clinical trials of lifestyle and drug therapy to treat lipid abnormalities suggest similar effectiveness and safety to that observed in adults. The majority of studies, however, have been short term in design. Longer-term safety and efficacy studies are needed, particularly with follow-up of vascular endpoints. Few clinical data are available to document at what age drug therapy should be appropriately and safely started. More work is needed in this regard to support clinical judgments on what age and dosage should be used in children at high risk for CHD. Importantly, current diagnostic and treatment algorithms should be updated to reflect recent advances in the field—including the role of genetic testing in the confirmation and management of genetic hyperlipidemias.

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95. Jacobson MS, Tomopoulus S, Williams CL, et al. (1998). Normal growth in high risk hyperlipidemic children and adolescents with dietary intervention. Prev Med 27:775–780. 96. Couch SC, Daniels SR (year). Current concepts of diet therapy for children with hypercholesterolemia. Prog Pediatr Cardiol 17:179–186. 97. Lifshitz F, Moses N (1989). A complication of dietary treatment of hypercholesterolemia. Am J Dis Child 143:537–542. 98. American Academy of Pediatrics Committee on Nutrition (1998). Cholesterol in childhood. Pediatrics 101:141–147. 99. Tershakovec AW, Shannon BM, Achterberg CL, et al. (1998). Oneyear follow-up of nutrition education for hypercholesterolemic children. Am J Public Health 88:258–261. 100. Kuehl KS, Cockerham JT, Hitchings M, Slater D, Nixon G, Rifai N (1993). Effective control of hypercholesterolemia in children with dietary intervention based in pediatric practice. Prev Med 22:154– 166. 101. Gidding SS, Dennison BA, Birch LL, Daniels SR, Gilman MW, Lichtenstein AH, et al. for the American Heart Association (2006). Dietary recommendations for children and adolescents: A guide for practitioners. Pediatrics 117:544–559. 102. Starc TJ, Shea S, Cohn LC, Mosca L, Gersony WM, Deckelbaum RJ (1998). Greater dietary intake of simple carbohydrate is associated with lower concentrations of high-density lipoprotein cholesterol in hypercholesterolemic children. Am J Clin Nutr 67:1147–1154. 103. Reinehr R, de Sousa G, Toschke AM, Andler W (2006). Long-term follow-up of cardiovascular disease risk factors in children after an obesity intervention. Am J Clin Nutr 84:490–496. 104. American Diabetes Association (2005). Standards of medical care in diabetes. Diabetes Care 28(1):S4–S36. 105. American Diabetes Association (2003). Management of dyslipidemia in children and adolescents with diabetes. Diabetes Care 26:2194–2197. 106. McCrindle BW (2003). Drug therapy of hyperlipidemia. Prog Pediatr Cardiol 17:141–150. 107. McCrindle BW, O’Neill MB, Cullen-Dean G, Helden E (1997). Acceptability and compliance with two forms of cholestyramine in the treatment of hypercholesterolemia in children: A randomized, crossover trial. J Pediatr 130:266–273. 108. Tonstad S, Knudtzon J, Sivertsen M, et al. (1996). Efficacy and safety of cholestyramine therapy in peripubertal and prepubertal children with familial hypercholesterolemia. J Pediatr 129:42–49. 109. McCrindle BW (2006). Hyperlipidemia in children. Thromb Res 118:49–58. 110. McCrindle BW, Helden E, Cullen-Dean G, Conner WT (2002). A randomized crossover trial of combination pharmacologic therapy in children with familial hyperlipidemia. Pediatr Res 51:715–721. 111. Rodenburg J, Vissers MN, Trip MS, Wiegman A, Bakker HD, Kastelein JJP (2004). The spectrum of statin therapy in hyperlipidemic children. Sem Vasc Med 4:313–320. 112. Knipscheer HC, Boelen CC, Kastelein JJ, van Diermen DE, Groenemeijer BE, van den EA, et al. (1996). Short-term efficacy and safety of pravastatin in 72 children with familial hypercholesterolemia. Pediatr Res 39:867–871.

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113. Lambert M, Lupien PJ, Gagne C, Levy E, Blaichman S, Langlois S, et al. (1996). Treatment of familial hypercholesterolemia in children and adolescents: Effects of lovastatin. Canadian lovastatin in children study group. Pediatrics 97:619–628. 114. Stein EA, Illingworth DR, Kwiterovich PO Jr., Liacouras CA, Siimes MA, Jacobson MS, et al. (1999). Efficacy and safety of lovastatin in adolescent males with heterozygous familial hypercholesterolemia: A randomized controlled trial. JAMA 281:137–144. 115. Arambepola C, Farmer AJ, Perera R, Neil HA (2006). Statin treatment for children and adolescents with heterozygous familial hypercholesterolemia: A systematic review and meta-analysis. Atherosclerosis 2006 (in press). 116. Weigman A, Hutten BA, de Groot, et al. (2004). Efficacy and safety of statin therapy in children with familial hypercholesterolemia: A randomized controlled trial. JAMA 292:331–337. 117. Rodenburg J, Vissers MN, Daniels SR, Wiegman A, Kastelein JJ (2004). Lipid-lowering medications. Pediatr Endocrinol Rev 2(1):171–180. 118. Pearson GJ, Francis GA, Romney JS, Gilchrist DM, Opgenorth A, Gyenes GT (2006). The clinical effect and tolerability of ezetimibe in high risk patients managed in a speciality cardiovascular risk reduction clinic. Can J Cardiol 22:939–945. 119. Wierzbicki AS, Doherty E, Lumb PJ, Chik G, Crook MA (2005). Efficacy of ezetimibe in patients with statin-resistant and statinintolerant familial hyperlipidemia. Curr Med Res Opin 21:333– 338. 120. Colletti RB, Neufeld EJ, Roff NK, McAuliffe TL, Baker AL, Newburger JW (1993). Niacin treatment of hypercholesterolemia in children. Pediatrics 92:78–82. 121. Ostlund RE Jr. (2004). Phytosterols and cholesterol metabolism. Curr Opin Lipidol 15:37–41. 122. Jakulj L, Vissers MN, Rodenburg J, Wiegman A, Trip MD, Kastelein JJP (2006). Plant sterols do not restore endothelial function in prepubertal children with familial hypercholesterolemia despite reduction in low-density lipoprotein cholesterol levels. J Pediatrics 148:495–500. 123. Lichtenstein AH, Deckelbaum RJ (2001). Stanol/sterol estercontaining food and blood cholesterol levels: A statement for healthcare professionals from the nutrition committee of the council on nutrition, physical activity and metabolism of the American Heart Association. Circulation 103:1177–1179. 124. Dennison BA, Levine DM (1993). Randomized double-blind, placebo-controlled, two period crossover clinical trial of psyllium fiber in children with hypercholesterolemia. J Pediatr 123:24–29. 125. McCrindle BW, Helden E, Conner WT (1998). Garlic extract therapy in children with hypercholesterolemia. Arch Pediatr Adolesc Med 152:1089–1094. 126. Engler MM, Engler MB, Malloy M, Chiu E, Besio D, Paul S, et al. (2004). Docosahexaenoic acid restores endothelial function in children with hyperlipidemia: Results from the EARLY study. Int J Clin Pharmacol Ther 42:672–679.

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C H A P T E R

21 Laboratory Methods in Pediatric Endocrinology ROBERT RAPAPORT, MD • RUSSELL GRANT, PhD • SHARON J. HYMAN, MD • MARK STENE, PhD

Introduction Hormonal Assay Methods Preanalytic Variables Standardization Reference Ranges Method Validation Immunoassay Techniques Consolidated Analyzers Mass Spectrometry

Introduction Pediatric endocrinology is a specialty that relies greatly on laboratory testing for evaluating and monitoring children with suspected or known endocrine conditions. It is therefore essential that those involved in the care of children understand all variables involved in proper prescribing, sample collecting, performance, and interpretation of laboratory tests. Appropriate use and application of hormonal assays can improve the health care of children by helping to avoid unnecessary detailed and invasive examinations. We hope this chapter will lend sufficient guidance to help clinicians appropriately select and evaluate endocrine hormone tests. The principles of molecular endocrinology testing are discussed elsewhere in this textbook. Endocrine hormone assays have evolved from radioimmunoassay (RIA) technology by using monoclonal antibodies, sensitive detection techniques, extensive automation, and most recently tandem mass spectrometry (MS/MS). With extensive automation, the sudden availability of platforms with a menu of commonly demanded hormone tests rapidly broadened access of endocrine testing to more than 1,600 laboratories today.1

Quality Assurance and Quality Control Case Studies, Common Diagnoses, and Testing 17-OHP Testosterone Summary

The distribution of endocrine testing now likely resembles the overall medical testing industry, in which hospitals perform 55% of testing, commercial laboratories perform 24%, physician office laboratories perform 11%, and others (such as nursing homes) perform 10%. As of 2003, laboratory testing at $40 billion accounted for 2.9% of all American health care expenditures. Specialized testing, including endocrinology, consisted of about $3.7 billion.2 Despite the vast number of testing laboratories, most endocrine testing is concentrated on only a handful of automated analyzers manufactured by a small number of companies. Recent advances in information technology are also changing the physician, hospital, and laboratory interaction. Similar to computer-computer interfaces, laboratory web-based information systems for ordering testing and receiving results are now increasingly available. These systems may reduce certain types of errors while potentially creating others.3 How these systems will reliably populate hospital systems, handheld devices, and electronic medical records or practice management systems is an emerging variable. Changes in the reimbursement environment have influenced the structure of hospital, commercial, and 855

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physician-office laboratories. Today, these laboratories employ only laboratory generalists and depend largely on these simple-to-operate platforms. Although practical, such systems have limited accuracy in certain applications. Their ease of installation and tacit interchangeability are also potential limitations. In most laboratories, appropriate technical knowledge of endocrinology and analytic systems is diminished and is often outsourced to device manufacturers whose continued focus is further integration and consolidation. Although these comments apply primarily to the current situation in the United States, they are likely to reflect trends in all developed countries.

Hormonal Assay Methods PREANALYTIC VARIABLES Normal endocrine physiology, nonendocrine illness,4 sample collection, and handling influence measured hormone levels (Table 21-1). Under normal circumstances in the basal state, many hormones are secreted in an episodic manner. Chronologic age, pubertal stage, emotional and physical stress, nutritional status, and postural effects contribute to the variation. Consequently, some hormones, growth factors, or surrogate markers may have very wide normal basal ranges and large intrasubject variability. Intrasubject variability can be remarkable for 24-hour integrated concentrations5,6 and for dynamic responses.7 Proper collection, documentation, and storage are needed to ensure accurate hormonal determinations.8,9 Most steroid, thyroid, peptide, and protein hormones TA B L E 2 1 - 1

Preanalytic Variables Affecting Hormonal Measurements Variable Episodic secretion Exercise (acute)

Circadian rhythm, diurnal variation

Seasonal variation Postural change Nutrition

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Hormone Pituitary hormones, cortisol Adrenocorticotropic hormone (ACTH), anti-diuretic hormone (ADH), aldosterone, cortisol, epinephrine, glucagon, growth hormone (GH), prolactin, norepinephrine, testosterone ACTH, cortisol, dehydroepiandrosterone sulfate (DHEAS), epinephrine, estradiol, folliclestimulating hormone (FSH) , GH, luteinizing hormone (LH), norepinephrine, prolactin, testosterone Estradiol, prolactin, testosterone Aldosterone, epinephrine, norepinephrine, renin C peptide, estradiol, glucagon, insulin-like growth factor-I (IGF-I), insulin-like growth factor binding protein-1 (IGFBP-1), insulin, proinsulin, thyroxinebinding globulin (TBG)

(and nearly all measured antibodies) are relatively resistant to collection and handling factors—including freezing and thawing. Generally, a serum sample drawn under normal conditions should be allowed to clot at room temperature for an hour, separated, and frozen until analysis is adequate. See Table 21-2 for patient preparation, sample collection, handling requirements, and relevant information regarding certain hormone assays. For best results, always consult with the specific laboratory to understand its collection and handling requirements.

STANDARDIZATION Unlike routine chemistries, most quantitative hormone assays can vary widely in absolute terms. To simplify research and clinical findings, standardization of hormone methods has been a long sought after and continuing goal.10 Standardization of steroid and other small-molecule hormone assays is relatively simple because these hormones are of low molecular weight and are synthesized and highly purified using physical and chemical methods. Such standards are typically available as preparations that can be reliably prepared and stably stored.11 By contrast, peptide hormones, protein hormones, and antihormone antibody standards are of high molecular weight and higher complexity (with many circulating isoforms)—making an absolute assignment more difficult. Recombinant technology and the availability of pharmaceutical-grade hormone are enabling improvement.12 Despite better standards, similarly designed assays often yield different absolute results.13 Some differences happen for proprietary reasons, but others are reflecting the complexity of the circulating and standard hormone preparations and variances in recognition of these hormones by capture and detection antibody systems. Testing proficiency data from automated systems show that identical systems have not reduced variability substantially for all hormones.

REFERENCE RANGES For the clinician, basal and dynamic testing reference data appropriate to the method result is more important than the absolute standardization. As outlined in Tables 21-3 through 21-5, ideally reference data should be available for all patient groups commonly making use of the test.14 These data must be established comprehensively, especially for new analytes, and must account for all factors that partition circulating hormone levels. For existing analytes, a reference method with an extensive normal data set allows for a less costly and more accessible way of establishing reference ranges. A subset of normal samples can be used to confirm the reference database if the newly implemented method is similar in design and the reference population is the same.14 Laboratories increasingly rely on device manufacturers to provide this base data set against which the smaller internally derived normal data are statistically evaluated for transference.15 Current practice may fall short of these requirements because method differences and bias between methods are sometimes unrelated to the differences in reference intervals.16

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TA B L E 2 1 - 2

Hormone Assays: Preanalytic and Other Considerations Patient Preparation, Sample Collection, and Hormone Stability

Hormone ACTH

ADH

Aldosterone Calcitonin

• • • • • • • • • • •

EDTA 6-hr RT, 4° C Ship frozen Avoid FT EDTA Avoid FT Ship frozen Patient: posture and sodium Stability: 1 day at RT, 2 days at 4° C 1 day at RT 1 day at 4° C

Cortisol, urine-free cortisol

• With boric acid 5 days at RT and 14 days at 4° C

C peptide

• • • • • • • • • • • •

EDTA: 1 day at RT RT: 1 day at 4° C Ship frozen Patient preparation: posture and activity Stability: 0 days at 4° C Must freeze sample ASAP 2 days at RT 6 days at 4° C Avoid FT Ship frozen 2 days at RT 2 days at 4° C

• • • • •

EDTA Avoid FT Ship frozen 2 days at RT 2 days at 4° C

Dopamine, epinephrine, norepinephrine Estradiol Free IGF-I GH

Glucagon

IGF-I

Insulin

• 6 hours at RT • 1 day at 4° C • Ship frozen

IPTH, intact parathyroid hormone

• EDTA • 4 hours at RT • 1 day at 4° C

Assay Notes • 1-24 ACTH, used for stimulation studies, and proopiomelanocortin (POMC) fragments can lower ACTH levels as measured by two-site methods. • Solid-phase purification.

• Spironolactone and tetrahydroaldosterone 3-glucuronide may cross-react in some assays. • Large calcitonin and others cross-react in some RIA methods. • High dose suppression of values possible with two-site methods. • Heterophile antibodies and human anti-animal antibodies may give falsely elevated results in two-site methods. • Prednisolone and 6-beta-hydroxycortisol may cross-react.

• C peptide immunoreactivity may be unstable. • Some immunoassays may be affected by anti-insulin antibodies. • Very unstable analytes present in low concentrations.

• RU-486, Efavirez, and steroid-binding proteins may interfere with some assays. • Direct methods controversial. • 20-kD GH react in certain RIA methods. • Human placental lactogen (hPL) may augment or suppress hGH levels. • 44-191 GH in RIA methods. • High hGH concentrations may require sample dilution if accurate results are needed. • After treatment, anti-GH antibodies may suppress GH levels in sandwich assays. • Unstable; keep frozen.

• IGF binding proteins react in methods that do not exclude or block. • Variable reactivity with therapeutic IGFI and IGFI/IGFBP3 complex with some assays. • Variable reactivity with various recombinant insulins. • On treatment, hook effect possible for two-site assays. • Human anti-insulin antibodies may interfere (use free and total measurements to avoid this issue). • Assays typically most reactive with human insulin, less reactive with porcine insulin, and least reactive with bovine insulin. • Assay terminology: second-generation N-terminal anti-1-24, third-generation N-terminal anti-1-6. • Second and third generations typically use C-terminal anti-39-84. • Midrange PTH RIAs (MPTH) recognize PTH molecules containing amino acid sequences 44-68. • C-terminal (CPTH) assays recognize molecules containing amino acids 39-84. • Circulating PTH fragments are not biologically active unless they have key N-terminal residues (1-24) and can lower or raise results from two-site methods. • Third-generation assays do not detect PTH 7-84 that is not biologically reactive. • Fragments are especially high with renal failure. • Human anti-PTH antibodies may interfere with assays. Continued

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TA B L E 2 1 - 2

Hormone Assays: Preanalytic and Other Considerations—cont’d Patient Preparation, Sample Collection, and Hormone Stability

Hormone LH and FSH

• • • • • •

Osteocalcin Proinsulin

2 days at RT 2 days at 4° C EDTA Avoid FT 1 day at RT 1 day at 4° C

• HCG for stimulation or due to tumor may augment RIA values and suppress two-site methods. • Some RIAs more sensitive to degradation.

Prolactin

• 2 days at RT • 2 days at 4° C

PTH-rP, parathyroidhormone-related protein

• Plasma with protease inhibitor • Avoid FT • Ship frozen

Renin

• • • • • • • •

Testosterone Thyroglobulin

EDTA Process at RTsSnap Freeze Ship frozen Do not leave cold 2 days at RT 6 days at 4° C 2 days at RT 2 days at 4° C

TSH, thyroid-stimulating hormone

• 2 days at RT • 2 days at 4° C

Vitamin D 25 OH

• Avoid FT • Ship frozen

Assay Notes

• High insulin levels associated with treatment may suppress proinsulin levels in two-site assays. • Human antibodies specific to insulin or proinsulin. • Macroprolactin or big prolactin a complex of prolactin and IgG lacks biologic activity because not freely available to tissues. • Two-site assays may demonstrate suppression (high-dose hook) at extremely high prolactin levels associated with macroprolactinomas. • Unstable; collect with inhibitor and keep frozen. • PTHrp N terminal contains key PTH sequences and is biologically active. • Also known as the humoral hypercalcemina of malignancy factor. • Process ambient and freeze rapidly to avoid plasma renin activity (PRA) increases due to cold-activated proteases. • Activity assays known as PRA. • Also direct renin assays. • RU-486, testolactone, and steroid binding proteins may interfere in some assays. • Circulating anti-TG antibodies significantly suppress two-site methods. • RIA methods are less likely to be impacted. • ␤HCG or alpha subunit with some methods causing falsely lower results. • Heterophile antibodies have been reported as causing falsely high values. • Controversy about TSH reference ranges. • Assays may or may not efficiently measure D2 form and C-3 epimer.

Notes: Stability: RT ⫽ room temperature. FT ⫽ freeze/thaw cycle. Stability studies using normal sera have shown that all hormone levels except ACTH, ADH, dopamine, epinephrine, glucagon, insulin, norepinephrine, osteocalcin, IPTH, and renin are relatively stable when kept at ambient temperature (20-25° C) for 1 day. Stability may be worse for patient samples and for samples kept at elevated temperatures, and may vary with the method. For most reliable hormone data, patient samples generally should be separated as soon as possible and kept frozen until measurement. Consult your laboratory for proper handling. Circulating antihormone antibodies will usually suppress two-site assay data. Hormone assay precision, the 95% confidence interval for a hormone level, ranges from ±10-20% to ±15-30%— depending on the absolute hormone concentration and the method used.

TA B L E 2 1 - 3

IGF-I (ng/mL) Pediatric Normal Data for an Acid Ethanol Extracted Blocking Method Children and Young Adults 1-2 3-4 5-6 7-8 9-10 11-12 13-14 15-16 17-18 19-20

Male Range

Male Average

Female Range

30-122 54-178 60-228 113-261 123-275 139-395 152-540 257-601 236-524 281-510

76 116 144 187 199 267 346 429 380 371

56-144 74-202 82-262 112-276 140-308 132-376 192-640 217-589 176-452 217-475

Source: Esoterix/LabCorp.

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Female Average 100 138 172 194 224 254 416 403 314 323

Like method-specific normals for basal testing, many dynamic tests can only be interpreted correctly if the method has been validated and normal ranges developed in response to various stimuli.17,18 If these data are not available, a clinician must rely on information from the literature, from similar assay methods, or from their own clinical experience with the assay method and the provocative stimuli.

METHOD VALIDATION Underlying our belief in hormone testing is the assumption that the results correlate to biologic action and clinical findings. Such faith is only possible with analytic and clinical validation. Key elements of analytic validation are: • Sensitivity: What is the lowest concentration of hormone detected by the method with less than 20% variation between assay runs?

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TA B L E 2 1 - 4

Baseline Steroid Levels and Steroid Responses to a 0.25-mg Bolus of ACTH in Normal Females Steroid

Group 1 (⬍1 Year)

Group 2 (1-5 Years)

Group 3 (6-12 Years)

Group 4 (T II- III)

Group 5 (T IV-V)

17 OH preg. (ng/dL) 0 minutes 17 OH preg. (ng/dL) 60 minutes 17 OH prog. (ng/dL) 0 minutes 17 OH prog. (ng/dL) 60 minutes Cortisol (ug/dL) 0 minutes

298 ± 272 61.9-829 1612 ± 801 898-3178 32.4 ± 28.8 12.9-106 142 ± 49.7 85-207 12.8 ± 7.1 4.2-23 40 ± 8.1 32.1-60.1 20.8 ± 13.6 7-52 64.9 ± 39.1 19.9-158

23.6 ± 12.0 10-47 282 ± 233 45-733 21.5 ± 26.5 5-90 186 ± 98 50-354 10.2 ± 4.1 7.3-19 31.8 ± 5.3 24-40 8.3 ± 3.6 4.3-15.9 67.9 ± 33. 8 26.1-143.3

58.6 ± 43.3 11-141 355 ± 187 70-657 27.1 ± 14.9 7.0-55.9 134 ± 52.6 75-218 8.3 ± 3.4 3.0-12 22.4 ± 3.4 17-28 6.94 ± 3.31 1.99-12.9 38.7 ± 14.9 19.2-61.2

137.9 ± 111 58-452 559 ± 171 251-802 56.6 ± 54.6 18.2-220 200 ± 103 88-424 8.8 ± 3.7 4.3-16 22 ± 4.2 16-32 6.62 ± 3.64 1.99-11.9 37.4 ± 14.6 12.9-63.2

221 ± 154 52-543 953 ± 306 503-1604 98.3 ± 62.6 36.1-197.3 166 ± 45 80-226 10 ± 2.8 6.0-15 26 ± 5.1 18-35 7.28 ± 1.66 4.97-9.93 28.5 ± 5.96 23.2-40.0

Cortisol (ug/dL) 60 minutes DOC (ng/dL) 0 minutes DOC (ng/dL) 60 minutes

Note: Selected hormones shown.

TA B L E 2 1 - 5

Baseline Steroid Levels and Steroid Responses to a 0.25-mg Bolus of ACTH in Normal Males Steroid

Group 1 (⬍1 Year)

Group 2 (1-5 Years)

Group 3 (6-12 Years)

Group 4 (T II- III)

Group 5 (T IV-V)

17 OH preg. (ng/dL) 0 minutes

283 ± 284 13.9-767 1243 ± 755 394-3291 59.6 ± 49 10.9-173 197 ± 85 108-468 12.6 ± 5.3 3.0-21 38 ± 4.4 32-40 24.5 ± 14.9 6.95-57.3 78.1 ± 25.8 38.1-110

38.3 ± 28.6 12-103 207 ± 171 55-738 40.4 ± 33.8 3.97-114 146 ± 56.9 65.2-269 12 ± 5.8 5.7-25 28 ± 3.9 22-37 14.6 ± 13.9 3.97-49 79.1 ± 30.1 34.1-139

84.6 ± 59 31-186 261 ± 132 114-498 34.4 ± 18.9 13.9-69.2 144 ± 32.1 115-197 9.2 ± 3.1 5.7-15 24 ± 1.6 22-27 15.9 ± 8.94 8.94-34.1 89.4 ± 42.4 33.1-139

102 ± 115 19.9-364 399 ± 202 88.3-676 47 ± 40.4 11.9-131 160 ± 70.8 69.2-313 8.1 ± 2.7 4-13 21 ± 8.2 15-45 9.93 ± 8.28 3.97-30.1 38.1 ± 14.9 11.9-74.1

120 ± 91.6 32-297 517 ± 190 220-860 101 ± 38.4 51-190 157 ± 41.4 105-230 9.5 ± 2.9 5.0-15 22 ± 3.2 18-27 8.94 ± 2.98 4.97-13.9 31.45 ± 9.93 19.2-46

17 OH preg. (ng/dL) 60 minutes 17 OH prog. (ng/dL) 0 minutes 17 OH prog. (ng/dL) 60 minutes Cortisol (ug/dL) 0 minutes Cortisol (ug/dL) 60 minutes DOC (ng/dL) 0 minutes DOC (ng/dL) 60 minutes

Note: Selected hormones shown.

• Specificity: Does the method measure the intended hormone specifically without interference from other related molecules? • Precision: How variable are data from repeated testing of the same sample? • Accuracy: Does the method yield an absolute value as expected based on adding defined standard to matrix, and does the method render proportional data when the sample is diluted progressively? • Carryover: Is each analysis free from influence by previously measured samples?

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• Reportable range: Over what range are sensitivity and precision sufficient to allow for validated results?19,20 Clinical validation often proves more difficult, and for existing tests is often replaced by testing sets of patient samples having a range of values (for excellent correlation and small, uniform bias).21 Newly developed analytes especially require careful and detailed assessment of each proposed clinical use prior to widespread acceptance, ideally including calculation of clinical sensitivity, clinical specificity, positive predictive value, negative predictive value, and full receiver-operating characteristic (ROC) analysis.18,22

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Immunoassay Techniques Competitive protein binding methods are the basis of some platform methods (Figure 21-1, Table 21-6), but manual RIAs are infrequently used today (yet still have an important role in certain esoteric applications). RIAs use reagents, antibodies, or hormone receptors/binding proteins that bind the analyte in a specific way—commonly called an analyte-specific reagent (ASR).23 The ASR is added in limited quantity and the endogenous hormone competes with radiolabeled hormone for binding with the ASR. The principle advantage of RIAs in a clinical setting is resistance to autoantibody interference and their relatively broad reactivity with the hormone in its various forms. The primary limitation of competitive methods is their broader reactivity, often with related molecules that are not biologically relevant. Despite this limitation, for some applications (such as monitoring for residual tumor) broader reactivity may actually be desired. RIAs,

but not methods using newer sensitive detection techniques, are also relatively insensitive. RIA technology is still commonly used in select situations, depending on the laboratory. Examples are growth hormone (GH), insulin-like growth factor-I (IGFI), insulin-like growth factor binding protein-3 (IGFBP3), leptin, thyroglobulin, urinary gonadotropins, and numerous steroid hormone methods. Noncompetitive protein binding methods are widely used as manual and automated platform methods (Figure 21-2, Table 21-6). The methods are commonly known as sandwich methods and are named in part based on the detection method used: IRMA for immunoradiometric (radioisotope detection) assay, ELISA for enzyme-linked immunosorbent assay (enzyme transformation of a colorless substrate to a colored product-spectrophotometric detection), ICMA for immunochemiluminometric assay (chemiluminescent detection), and IFMA for immunofluorimetric assays (time-resolved fluorescent detection). These methods use complementary antibody pairs to capture and detect the analyte of interest.23

Figure 21-1 Basic hormone radioimmunoassay (RIA). For traditional RIA methods, reagent antibody (Ab) is harvested from the serum of animals. Rabbits or goats are immunized following a prescribed immunization protocol and schedule. Small nonimmunogenic molecules (haptens) such as steroid hormones are first conjugated to irrelevant proteins prior to immunization. Larger human peptide and protein hormones are typically immunogenic in these animals without conjugation. Animals are bled during the schedule, and each lot is evaluated for concentration (titer), affinity, and specificity. The “bleed” with the highest titer, optimal affinity, and specificity is selected for RIA without or with further purification. Hybridoma technology is frequently used to develop monoclonal antibodies (MAbs) from immunized mice. These MAbs are typically used in noncompetitive immunoassay configurations. As shown for RIA (top left), hormone (H) in patient, control, or standard is incubated with purified radiolabeled hormone (*H in this case is 125H) and the reagent antibody (Ab)—shown here in an equilibrium-type reaction. Two key elements for RIA, a competitive immunoassay, is that H and *H are as similar as possible and that they compete for a limiting amount of antihormone antibody (Ab). The antihormone-specific antibody is referred to as the first antibody. For this example, a second antibody (reactive with the animal immunoglobulin of the first antibody) is used along with centrifugation to separate free hormone (H and *H) from hormone bound to the first antibody (HAB and *HAB). The bound fractions (HAB-2nd Ab and *HAB-2nd Ab) precipitate and are then “counted” (counts/min or CPM) in a gamma counter. The bound CPM are converted to % H bound and plotted versus the standard hormone concentration. “Unknown” % H bound from patient samples or controls are converted into hormone concentration. A number of algorithms are used to automatically deduce the most statistically valid standard curve from which unknown responses (% H bound) for patient and control samples are converted into hormone concentration. All hormone assays, including mass spectrometry methods, create some type of dose-response relationship (with standard hormone as a basis for determining hormone levels in patient samples and controls). Most curves are displayed to the operator as a simple linear plot. Shown (top right) is a simple point-to-point plot rarely used today. These data create a linear dose-response curve when plotted as the Logit function versus log of hormone concentration. Based on simple equilibria of binding, the affinity and binding sites (concentration) of an antisera, binding protein, or receptor can also be estimated using Scatchard analysis of data from a set of properly designed experiments.

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861

TA B L E 2 1 - 6

Immunoassay Techniques Separation of Unbound Label (Effectiveness)

Sensitivity

Specificity Varies. More likely to measure multiple forms related to hormone. Some not biologically reactive. Can be excellent. More likely to measure intact biologically active hormone. Can be excellent. More likely to measure intact biologically active hormone.

Method and Principle

ASR

RIA analyte and tracer competition for ASR

Polyclonal antisera limited

Second antibody (good)

⬎0.5 ng/mL

ELISA or IRMA two-site capture and detection, noncompetitive

Paired monoclonal or affinitypurified polyclonal antisera Paired monoclonal or affinitypurified polyclonal antisera

Physical removal of solid phase is manual, gel filtration or magnetic (excellent) Physical removal of solid phase is manual, gel filtration or magnetic (excellent)

⬎0.1 ng/mL

ICMA or IFMA

⬎1 pg/mL

Precision Interassay (% CV) 8-12

7-10

3-9

Ist INCUBATION +

β

β

α

α

Unknown (or known standard) ligand

First antibody (capture)

Wash

β

+ α

β T

α

T

Second tracer antibody Wash

β

α

T

T

Quantitation of bound tracer Figure 22-2 Depiction of an assay system with two antibodies in a sandwich system. The first antibody is solid phase (attached to the wall of the vessel) and binds a specific portion of the ligand. After all of the ligand is bound, the reaction vessel is washed. The second antibody is added, which binds to a specific epitope on the other end of the ligand. This antibody contains the tracer, and thus after washing out the excess second antibody the amount of ligand within the sandwich can be quantified as indicated (T, lower right). The tracer can be radioactive, enzymatic, fluorescent, chemiluminescent, or other. Most direct immunoassays add capture and label antibody at the same time, thus eliminating the requirement for an intermediate wash. The format often depends on the characteristics of the antibody pair and the technical requirements of the assay method. Showing the ␣ and ␤ subunits also allows one to see how high levels of the free subunits could actually suppress the apparent hormone levels. Generally, the capture antibody to label antibody stoichiometry is such that the capture is present at a 10:1 ratio or more than the label. High-level cross reactants that bind the label phase can therefore suppress binding.

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The antibodies are selected against two noninteracting epitopes, hopefully unique within the hormone of interest. Ideally, both epitopes are essential to the biologic activity of the hormone—giving the assay excellent correlation with hormone bioactivity. The two-site design improves the assay specificity by eliminating smaller partially cross-reactive molecules from detection. Variations of these methods are used on many automated platforms. The primary limitations of these methods are greater interference from nonspecific binding of hormone, autoantibodies, heterophile antibodies (human anti-rabbit or anti-goat antibodies), and reactivity or suppression of reactivity by smaller molecules containing a selected epitope.24 Most hormones today are measured by noncompetitive protein binding methods. Examples are adrenocorticotropic hormone (ACTH), follicle-stimulating hormone (FSH), GH, luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and many others. Depending on the analyte, the methods described previously are suitable for direct analyses or may require significant pretreatment (such as solvent extraction and chromatography) prior to immunoassay to remove interfering analytes. Unlike steroid hormones, which are very similar chemically and immunologically, most protein hormones or factors can be measured accurately in relatively direct methods.

CONSOLIDATED ANALYZERS Today, 10 manufacturers are supporting more than 25 immunoassay analyzers—of which 5 are used widely for performing hormone immunoassays (Table 21-7). Nearly all of the devices perform typical thyroid testing. Many add reproductive hormones, but few offer less fre-

quently ordered hormone tests. All of these methods consolidate more than 20 or 30 hormone or other assays on a single platform that can operate in batch or random access mode with unattended capability, timely turnaround, and cost-effective throughput. These methods are calibrated automatically and ideally are designed to maintain calibration for long periods and to have procedures that call for as little as one quality control event every 24 hours. The methods are essentially free from user-related bias and require minimal training to operate. Competitive immunoassays and noncompetitive immunoassays combined with sensitive detection techniques are used to maximize analytic sensitivity and specificity to the extent possible with such simple techniques. Limitations vary, depending on the hormone being measured and the sample population being tested. In general, the methods are subject to the same type of interferences associated with other competitive or noncompetitive manual or semiautomated immunoassays.25

MASS SPECTROMETRY Mass spectrometry is a technique that selects and measures the presence and quantity of molecules within a sample. Steroid hormones, for example, are charged in an interface and then accelerated as gas-phase ions into a mass spectrometer operated under vacuum. In tandem mass spectrometry (MS/MS), molecules (gas-phase ions) of interest are selected and fragmented—and products specific to the analyte are again selected for detection. Filter paper bloodspot testing for 17-hydroxyprogesterone (17-OHP) is a new application for LC-MS/MS methods.26,27 17-OHP is the marker for 21-hydroxylase deficiency, the most common form of congenital adrenal hyperplasia (CAH).

TA B L E 2 1 - 7

Commonly Used Analyzers Analyzer

No. Units

Method(s)

Protein Hormone

Steroid Hormone

Thyroid Hormone

Abbott AxSym Plus

2,400 USA, 10,000 worldwide

Competitive FPIA, noncompetitive MEIA

FSH, hCG, LH, prolactin, TSH, anti-TPO, anti-TG

Cortisol, estradiol, progesterone, testosterone

T3, T4, free T3, free T4, T3 uptake

Bayer Advia Centaur

⬎1,300 USA, ⬎3,100 worldwide

Competitive chemiluminescent with magnetic particle separation

Cortisol, estradiol, progesterone, testosterone

T3, T4, free T3, free T4, T3 uptake

Beckman Coulter Access 2

⬎1,300 USA

Noncompetitive chemiluminescent assay with magnetic particle separation

FSH, hCG, LH, prolactin, TSH, anti-TPO, antiTG, IPTH, C peptide, insulin FSH, hCG, LH, prolactin, TSH, anti-TG, GH, ostase, TG, insulin

T3, T4, T3 uptake

Diagnostic Products Corp. Immunlite 2000

⬎3,600 worldwide

Noncompetitive chemiluminescent assay with bead separation

DHEAS, estradiol, unconjugated estriol, cortisol, progesterone, testosterone Cortisol, estradiol, unconjugated estriol, progesterone, testosterone, androstenedione

Thyroglobulin, TBG, antiTG, anti-TPO, ACTH, IPTH, hCG, FSH, LH, prolactin, GH, IGFI, IGFBP3, calcitonin, IPTH, gastrin, SHBG

T3, T4, FT3, FT4

Notes: This is a partial listing of platforms and available methods. Check with manufacturer for current configurations. FPIA ⫽ fluorescence polarization immunoassay and MEIA ⫽ microparticle enzyme immunoassay.

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MS sensitivity gains made possible serum testing for CAH follow-up, and for measuring other serum steroids.28-34 New-generation LC-MS/MS steroid assays have shown improved accuracy, precision, sensitivity, and selectivity (specificity) over simple direct and complex reference immunoassays.31-34 LC-MS/MS methods using isotope dilution typically use more than five degrees of selectivity to provide quantitative results. Selectivity requirements are greatest when analyzing steroid hormones in pediatric and hormone-deficient samples.

Same process for experiment and control

I.S. for recovery

Remove cross reacting conjugated steroids

Patient samples control samples testosterone calibrators

2H

Supernatant evaporate and reconstitute Remove high M.W. Remove cross reacting steroids Select steroids Select steroids Select steroids ID and quantify steroid

Numerous steroids circulate physiologically that cannot be separated by tandem MS/MS alone—such as cortisol/cortisone,28 estradiol/estrone,33 and 17-hydroxyprogesterone/11-deoxycorticosterone.34 Selectivity in steroid analysis using LC-MS/MS technologies represents the greatest challenge during analysis. For example, estradiol measurement using MS/MS requires resolution of 24 interfering steroids/medications.35 In Figure 21-3, an isotope dilution LC-MS/MS assay that uses additional preparative steps to remove interfering steroids is compared with an automated direct competitive

Different processes for experiment and control

Patient samples control samples

No recovery tracking

testo

Non-polar solvent extraction

863

Discard precipitate

Remove SHBG release steroids Competitive immunoassay testo*** and testo compete for limited antibody. All selectivity based on antibody specificity

Releasing agent

Discard precipitate

Incubation steroids and other

Testo*** ⫹ testo Ab⫹anti-rabbit IgG-Solid phase

Separation free and bound

Discard solution phase

HTLC or HPLC

2nd HPLC Ionization precursor ion selection MS fragment product ion selection

Higher RLU lower concentration

Retain and enumerate solid phase

I

Figure 21-3 Comparison of a direct method of testosterone measurement and an isotope dilution HTC-LC-MS/MS method. LC/MS/MS: The isotope dilution protocols follow the general outline shown (top left). First, a stable isotopically labeled mimic [also known as an internal standard (IS)] of the hormone is added to calibrators, quality control samples, and patient samples. Following sample mixing, extraction of the hormone and internal standard are performed off-line using liquid/liquid extraction, solid-phase extraction, or sample dilution/ precipitation or on-line using turbulent flow chromatography. This type of sample preparation reduces sample complexity prior to LCMS/MS detection by removing unwanted sample constituents such as albumin, lipids, salts, and so on. It also enables concentration of analyte. The IS corrects for potential losses of hormone during these processing steps and during MS/MS. Fractionation of the sample is then usually accomplished by liquid chromatography (LC) separation based on hydrophobic differences between potential cross-reacting hormones. This LC fractionation step is critical to the removal of cross-reacting steroids that would otherwise impact the accuracy of steroid measurements by MS/MS. Following LC separation, the effluent flows into an interface—where the hormone solution and IS are converted to gas-phase ions and solvents are removed. The hormone and IS are then detected using MS/MS. MS/MS is depicted in Figure 21-4. See the American Society of Mass Spectrometry (ASMS.org) for more detail and references on this complex technology. Calibration curves based on standards performed in each assay batch are plotted as concentration (X axis) and hormone/IS ratio (Y axis). The concentration of samples is back-calculated against the calibration curve based on the hormone/IS response ratio observed. In comparison with existing immunoassays, calibration curves are linear over 3 to 4 orders (i.e., 3–4 logs10). When all components of the isotope dilution LCMS/MS experiment are considered, it is apparent that in excess of 5 degrees of selectivity are generally used to provide quantitative results. Direct platform method: This automated competitive immunoassay follows the outline shown (top right). Samples are treated to release testosterone and other steroids from specific binding proteins, such as SHBG (sex-hormone-binding globulin). Then chemiluminescent-tagged testosterone is added to compete with a limit amount of first antibody directed against testosterone and a solid-phase antibody reactive against the antibody species of the first antibody. After incubation, the solid phase is separated and enumerated. The response information is compared to a stored calibration curve. The instrument requires a two-point calibration every 7 days. Two quality controls are evaluated each day.

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Mass Analysis

Fragmentation Chamber

Ion Selection

Mass Analysis

Figure 21-4 Schematic for MS/MS. Two mass analyzers are combined sequentially in the devices known as a tandem mass spectrometry or MS/MS. This method is helpful in identifying and measuring specific hormones within complex mixtures. As shown (top), the first mass analyzer selects ions of a desired mass (more correctly stated, the desired mass/charge ratio) for steroid hormones. Then the selected ions are fragmented into hormone-characteristic ions that are analyzed in the second mass analyzer and enumerated. In this way, the tandem MS device adds analytic specificity to the hormone assay.

immunoassay method for testosterone. A graphical description of MS/MS is provided in Figure 21-4. Note that development, validation, and operation of MS-based hormone methods require expertise that is in short supply. Facilities suitable for MS and ancillary core competencies are not currently available in many commercial testing laboratories in the private or academic setting. At present, only a handful of laboratories have reported using MS for steroid hormone proficiency testing.36 To date, LC-MS/MS steroid tests include androstenedione, dehydroepiandrosterone (DHEA), estradiol, estrone, estrone sulfate, progesterone, testosterone, 17-hydroxypregenolone (17-OPN), 17-OHP, pregnenolone, 25-hydroxy-vitamin D3, and others. Reference methods have been reported for some peptide hormones, such as thyroxine, c peptide, insulin, renin, and others.

Quality Assurance and Quality Control Laboratory quality assurance (QA) programs include internal and external mechanisms to ensure laboratory quality. The overriding goal of QA and quality control systems is to ensure that results meet or exceed expected operating parameters. Key components of an internal program at the method level are method validation, instrument qualification, assay batch or daily quality control, and data review before reporting. Proficiency testing, laboratory certification, and on-site quality audits are important external systems for laboratory quality. Clinicians are most likely to encounter assay inaccuracy, assay imprecision, or sample errors. Repeated measurement data typically have a normal distribution, with the difference from the average value being distributed about both sides of the mean without cumulative bias. As such, these data should be reported with a 95% confidence interval [±2 SD or ±2 * (% CV),

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% CV ⫽ Stdev/Average * 100] representing the likely range of true values for a particular sample. For example, the value 10 ng/mL should be reported as 10 ng/mL ± 2 ng/mL if the assay has a variation of 10% in that analytic range. Because data are not reported with an associated variance, it is important to consider such potential variance—especially when reviewing clinically borderline results. Variance for nearly all assay systems is greatest at low levels of detection. Inaccuracy of a particular assay should always be considered when results are unusual for the clinical presentation, especially when evaluations are being performed on certain patient populations (such as infants and patients with renal impairment) for whom cross reactants are unusually elevated. Although most assay data are reliable, it is estimated that significant medical errors are as frequent as 5%—whereas estimates of laboratory errors, exclusive of blood banking, range from 0.05% to 0.61% (with preanalytic errors accounting for 31%-75%, analytic errors for 13%-32%, and postanalytic errors for 9%-31%).37 Table 21-8 lists common errors associated with laboratory results.

Case Studies, Common Diagnoses, and Testing 17-OHP In the United States, most state governments now have mandatory screening for 21-hydroxylase deficiency—by measuring 17 hydroxyprogesterone on filter paper blood spots taken from newborns. By design, these tests have high clinical sensitivity but low clinical specificity. Most positive screens tests will test negative when properly evaluated by a reference method. Reference methods should be completely free of interference from cross-reacting steroids present during the newborn period. A male patient positive on state screening 17-OHP was

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TA B L E 2 1 - 8

Common Errors Frequency

Type of Error

Error

Example(s)

Very common

Preanalytic

Biological variance

Common

Preanalytic

Wrong patient’s samples

Common

Preanalytic

Incorrect date of birth (DOB) information

Common

Preanalytic

Wrong sample type

Common

Preanalytic

Improper patient preparation

Common

Preanalytic

Improper sample handling

Common

Preanalytic

Systemic illness

Infrequent

Preanalytic

Drug interference

Very common

Analytic

Typical assay variance

Common

Analytic

Test used not sufficiently specific for patient in question

Common

Analytic

Test used not sufficiently sensitive for patient in question

Common

Analytic

Infrequent Infrequent

Analytic Analytic

Infrequent

Postanalytic

Patient sample contains crossreacting substances that raise or lower results for improperly designed or improperly used methods Assay out of calibration Assay reporting reference data that are inappropriate for the method Wrong data

Testosterone, DHEA, prolactin and other pituitary hormones. More consistent results are achieved by pooling multiple draws. Improperly labeled sample, or sample mix-up during draw or in laboratory. With the wrong DOB pediatric samples may not get extraction or other necessary steps and may have incorrect normal data provided with the report. Serum required for most total calcium determinations, sodium fluoride tubes for glucose and EDTA for ACTH. Most assays are not validated for samples other than serum or plasma. Examples; A) Fasting for glucose, cpeptide, insulin, B) Posture for aldosterone, catecholamines and renin. Examples ACTH, plasma catecholamines, IPTH, renin and PTHrp often problematic. Ambient stability can decline at elevated summer temperatures. Hormone levels may be impacted by systemic illness. Most well recognized is the impact of illness on thyroid hormone levels. Many pharmaceuticals directly or indirectly influence hormone levels. Pay attention to time since last dose. Most assays not validated to deal with this type of interference. Tests have inherent variability. Contact the laboratory to discuss borderline results. 95% confidence interval for most hormone immunoassays is at least ±10%-20%. Direct measurement of steroids, 17-OHP, and testosterone not appropriate especially for infants, children, and women. ACTH, calcitonin, GH (acromegaly), estradiol, IGFI, testosterone, and other methods require better analytic sensitivity for certain applications. Stimulation doses of 1-24 ACTH, cortisone, dexamethasone, HCG, antihormone antibodies and others may affect otherwise valid methods. Conjugated steroids and others steroids such as cortisol that circulate at high levels. Method improperly calibrated initially or loses calibration. Reference data used not sufficient or insufficiently validated and inappropriate. Manual or automated data entry error especially a problem for referred testing.

Notes: Errors that can lead to erroneous hormone reports are included. The errors can be preanalytic, analytic, or postanalytic. Other errors (such as improper test or test method selection) are more difficult to track. An assay designed for use in adults may not be suitable for use with samples from infants and children. Review the laboratory directory of service for proper assay selection, patient preparation, sample collection, and handling.

examined and lacked any hallmarks of CAH. A follow-up serum sample was obtained and referred to a commercial laboratory for 17-OHP measurement. The laboratory reported a result more than 10 times the upper limit for the reported age. As the data were not congruent with the clinical picture, another serum sample was obtained and referred to an endocrine reference laboratory that reported the value just at the upper limit of normal. Upon further investigation, the first confirmation sample was discovered as being submitted with a transcription or data entry error—changing the patient

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age from newborn to early childhood. The initial referral laboratory, being aware of cross-reacting steroids present in the newborn, performs extractions prior to 17-OHP determinations on samples from newborns and infants but not from older children.38

TESTOSTERONE An infant with prenatal chromosomes 46XY was born with normal female genitalia. Testosterone by an automated direct immunoassay method was unexpectedly

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measured as being four times the upper limit of normal for newborn females in a reference method. Based on the case presentation and laboratory data, a diagnosis of androgen insensitivity was considered. Later, extracted samples were evaluated in an “in-house” RIA and found to be close to what would be an upper limit of normal for female infants. The diagnosis was reconsidered, and at 10 months of age baseline and human chorionic gonadotropin (HCG) stimulation samples had undetectable testosterone levels. Gonadotropins were markedly elevated after luteinizing-hormone-releasing hormone (LHRH) and a repeat ultrasound revealed an infantile uterus. Based on these data, the diagnosis was changed to 46XY complete gonadal dysgenesis.39

Summary Preanalytic patient and sample-related variables must be considered before hormonal sampling is underway. Coexistent illness (including extreme body mass index, emotional well-being, posture, activity level, and recent or current exposure to therapeutic or nutritional products) may affect hormonal concentrations. Studies on normal subjects have also shown that many endocrine hormones exhibit large intrasubject variation, as reflected in broad cross-sectional normal reference ranges. Sample collection errors are important as well but are largely avoidable by following proper collection and handling procedures and by carefully labeling samples and creating accurate and legible requests. All laboratories must have active and thorough QA programs that include assay validation reports and continuous quality control data. Standard hormone preparations and consolidation of many tests on a few predominant platforms have led to greater comparison of some hormone assays among laboratories. Comparable normal reference data must be confirmed by each laboratory each time they significantly modify or change methods. Pediatric reference data, especially for steroid hormones and insulin-like growth factors, by design must be different for simple methods when compared to complex reference methods. For these reasons, longitudinal test data are most reliable when all testing is done with a single method— preferably at one laboratory location. When testing is controlled in this way, variation is limited but still typically ranges from 10% to 30%—depending on the method and the hormone concentration being evaluated The other common systematic error relates directly to using simple methodology for complex samples, such as those from infants and children and from patients with kidney disease. Newer automated methods, such as LC-MS/MS coupled with other purification techniques, are beginning to correct a decline in the quality of these complex analyses. However, widespread application of such methods awaits improvements in usability, training, and instrument cost. The physician and other health care providers as the final line of quality control can influence testing decisions and can intervene when individual patient data seem inappropriate. Ongoing interaction between laboratories

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and clinicians also contributes to clinical investigation that is integral to the provision of quality care to children with endocrine disorders.

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45. Sadee W, Finn AM, Schmiedek P, Baethmann A (1975). Aldosterone plasma radioimmunoassay interference by a spirolactone metabolite steroids. 25(3):301–311. 46. Uwaifo GI, Remaley AT, Stene M, Reynolds JC, Yen PM, Snider RH, et al. (2002). A case of spurious hypercalcitoninemia: A cautionary tale on the use of plasma calcitonin assays in the screening of patients with thyroid nodules for neoplasia. J Endocrinol Invest 25:197. 47. Leboeuf R, Langlois M, Martin M, Ahnadi C, Fink GD (2006). Clinical case seminar “hook effect” in calcitonin immunoradiometric assay in patients with metastatic medullary thyroid carcinoma: Case report and review of the literature. J Clin Endocrinol Metab 91:361–364. 48. Sinicco A, Raiteri R, Rossati A, Savarino A, Di Perri GE (2000). Favirenz interference in estradiol ELISA assay. Clin Chem 46(5):734–735. 49. Tejada F, Cremades A, Monserrat F, Penafiel R (1998). Interference of the antihormone RU486 in the determination of testosterone and estradiol by enzyme-immunoassay. Clin Chim Acta 275(1):63–69. 50. Markkanen H, Pekkarinen T, Valimaki MJ, Alfthan H, KauppinenMakelin R, Sane T, et al. (year). Effect of sex and assay method on serum concentrations of growth hormone in patients with acromegaly and healthy controls. Clinical Chemistry 52:468–473. 51. Jansson C, Boguszewski C, Rosberg S, Carlsson L, AlbertssonWikland K (1997). Growth hormone (GH) assays: Influence of standard preparations, GH isoforms, assay characteristics, and GHbinding protein. Clinical Chemistry 43(6):950–956. 52. Blum WF, Brier BH (1994). Radioimmunoassays for IGFs and IGFBPs. Growth Regulation 4(1):11–19. 53. Owen WE, Roberts WL (2004). Cross-reactivity of three recombinant insulin analogs with five commercial insulin assays. Clinical Chemistry 50:257–259. 54. Boudou P, Ibrahim F, Cormier C, Chabas A, Sarfati E, Souberbielle J (2005). Third- or second-generation parathyroid hormone assays: A remaining debate in the diagnosis of primary hyperparathyroidism. J Clin Endocrinol Metab 90:6370–6372. 55. Cantor T, Yang Z, Caraiani N, Ilamathi E (year). Lack of comparability of intact parathyroid hormone measurements among commercial assays for end-stage renal disease patients: Implications for treatment decisions. Clinical Chemistry 52:1771–1776. 56. Gibney J, Smith TP, McKenna TJ (2005). The impact on clinical practice of routine screening for macroprolactin. J Clin Endorinol 90:3927–3932. 57. Key TJ, Moore JW (1988). Interference of sex-hormone binding globulin in a no-extraction double-antibody radioimmunoassay for estradiol. Clin Chem 34(6):1357–1358. 58. Sapin R, d’Herbomez M, Schlienger JL, Wemeau JL (1998). Antithyrotropin antibody interference in thyrotropin assays. Clinical Chemistry 44(12):2557–2559. 59. Wartofsky L, Dicker RA (2005). Controversy in clinical endocrinology: The evidence for a narrower thyrotropin reference range is compelling. J Clin Endocrinol Metab 90:5483–5488. 60. Surks ML, Goswami G, Daniels GH (2005). Controversy in clinical endocrinology: The thyrotropin reference range should remain unchanged. J Clin Endocrinol Metab 90:5489–5496. 61. Lensmeyer GL, Wiebe DA, Binkley N, Drezner MK (2006). HPLC method for 25-hydroxyvitamin D measurement: Comparison with contemporary assays. Clinical Chemistry 52:1120–1126. 62. Singh RJ, Taylor RL, Satyanarayana GS, Grebe SKG (2006). C-3 epimers can account for a significant proportion of total circulating 25-hydroxyvitamin D in infants, complicating accurate measurement and interpretation of vitamin D status. J Clin Endocrinol 91:3055–3061. 63. Advia Centaur 111751 Rev. J, 2004-03. 64. American Society for Mass Spectrometry (1998). What is mass spectrometry, Third edition. [city: publisher] 22. 65. Fuqua JS, Sher ES, Migeon CJ, Berkovitz GD (1995). Assay of plasma testosterone during the first sex months of life: Importance of chromatographic purification of steroids. Clinical Chemistry 41:1146–1149.

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Index

A Abdominal ionizing radiation, 409 Abdominal ultrasound, of adrenal adenoma, 569f Abetalipoproteinemia, 843–844 Absorptiometry. See also Dual-energy X-ray absorptiometry of BMC/BMD near birth, 687t of whole-body bone mineral content, 687f Acanthosis nigricans, 46 type A insulin resistance with, 411 Acetyl-CoA dehydrogenase, 187f, 188 Achondroplasia diagnosis of, 282 types of, 282t Acid ethanol extracted blocking method, IGF-1 pediatric normal data for, 858t ACTH. See Adrenocorticotropin hormone Addison disease in APS, 776, 776t autoantigens in, 773t incidence of, 776 isolated, autoantibodies in, 782 stages of, 776t Adenohypophysis, structure of, 264 Adenoma adrenal, 569f in Cushing syndrome, 489–490 hyperinsulinism islet, 182 pituitary, excess GH in, 320–321 Adenomatosis, hyperinsulinism focal, 178–179, 179f Adenylate cyclase, 550f ADHH. See Autosomal dominant hypocalcemic hypercalciuria Adipocytes autonomic innervation of, 792f in osteoblastogenesis, 97, 98f storage of, 796f Adipocytokines, 800–801 Adiponectin, 800–801 Adipose tissue autonomic innervation of, 792f cortisol secretion in, 805 fasting metabolic systems in, 389t leptin secretion in, 679 lipolysis, 170t maturation of, 558 metabolism in, 800 storage in, 796f Adiposity, age of, 799 Adolescence androgens in, 280–281 anovulation in, 563–564, 563f–564f BMC in, 115t, 117t BMD in, 116t–117t bone deposition in, 108t–109t, 111f Ca intake in, 80–81, 80t congenital hypothyroidism in, 228t

Adolescence (Continued) diabetes in, 849t diffuse nontoxic goiter in, 228t estrogens in, 280–281 female reproductive system in, 536–539, 536f–539f, 539t GnRH secretion in, 536–538, 537f hyperandrogenism in, 585–592, 586t, 587f, 589f hypercalcemia in etiology of, 713–719, 715t, 717f evaluation of, 719–720 management of, 720–722 hypocalcemia in etiology of, 706–711, 708t–709t evaluation of, 711–712 management of, 712–713 lipids in, 847t mineral homeostasis in, 689–690 puberty in, 536–539, 536f–539f, 539t sex steroid levels in, 280–281 sexual maturation in, 561–562, 561f, 562t thyroid dysgenesis in, 228t Adrenal adenoma, abdominal ultrasound of, 569f Adrenal cortex development of, 446f fetal formation of, 132, 447 response of, in maturation, 549f Adrenal function test, 560t Adrenal gland. See also Hypothalamicpituitary-adrenal axis androgen secretion in, 457–458, 457f cytoplasmic autoantibodies in, 781 enzyme autoantibodies in, 781–782 excess of, 487–492, 487t–488t function of clinical evaluation of, 459–460 laboratory evaluation of, 460–464, 460t–461t, 463t, 464f hypoplasia congenita of, 483–484 steroids in 60-minute ACTH test of, 463t hormone synthesis in, 449f tumors of, 489–492 weight of, 447f Adrenal insufficiency ACTH causing, 486–487 acute primary, 481–482, 482t causes of, 481, 482t chronic autoimmune disorders of, 482–483 symptoms of, 482t hypothalamic dysfunction causing, 486 long-term steroid therapy for, 487 metabolic disorders causing, 484–486 POMC disorders causing, 486–487 secondary, 486–487 symptoms of, 482t

Adrenalectomy, for CAH deficiency, 478 Adrenarche adrenal androgen secretion in, 548–549, 549f premature, 563 regulation of, 457–458, 457f Adrenergic receptors classification of, 514t function of, 514t pharmacology of, 514t Adrenocorticotropin hormone (ACTH) 60-minute, test, 463t actions of, 456 baseline steroid response to, 859t causing adrenal insufficiency, 486–487 characteristics of, 455 ectopic syndrome, 489 glucocorticoid feedback in, 456–457 -independent bilateral macronodular adrenal hyperplasia, 16f -independent multinodular adrenal hyperplasia, 490 in maturation, 549f in pituitary gland, 455–457, 455f plasma, 462–463 receptors of, 28t, 32–33 resistance syndromes, 484 rhythms of, 456 structure of, 455f Adrenodoxin reductase, 449, 450f Adrenoleukodystrophy, 490 Adults fasting metabolism in, 169f–170f female reproductive system in, 539–543, 539f–542f fuel composition of, 169t GHD in, 310, 310f height of by bone age, 266t prediction of, 261–262, 266t Turner syndrome in, 646, 646t, 651–652 follow-up studies of, 647–649 Afferent system alimentary, 789–791, 790f for energy balance, 789–791, 790f metabolic, 791 vagus nerve, 789–790, 790f Agarose gel electrophoresis, 3f Age of adiposity, 799 aldosterone v., 479f body mass-index-for, 262f–263f, 797f–798f bone, 260 for adult height, 266t serial, assessment of, 309 dependency, sex steroids and, 300 gestational, GH treatment of, 311 head circumference-for-, percentiles, weight-for-length and, 257f, 259f

869

870

INDEX

Age (Continued) iodine distribution space by, 234f length/weight percentiles for, 256f–258f peripheral T4 metabolism with, 234t renal excretion by, 234f standardized incidence by, of diabetes mellitus type 1, 378f thyroid function by, 235, 235f thyroid gland volume variation by, 234t thyroid uptake by, 234f Aging, normal catabolic state of, 313 AgRP, 793 Albumin, 74 Alcohol -induced hypoglycemia, 438 Aldosterone concentrations of, age v., 479f endocrine-angiotensin-, 344–345 glucocorticoid-suppressible hyper-, 480–481 normal levels of, 151t PHA, 54, 492 renin-angiotensin-, 344 secretory rates of, 463 Alimentary afferents, 789–791, 790f Alimentary hypoglycemia, 437 Alkali therapy, 396 Alkaline phosphatase, 84–85 disorders of, 732–734 Allele-specific oligonucleotide hybridization (ASOH), 13–14, 13f–14f Allgrove syndrome, 484 ALS mutations, growth abnormalities from, 296–297 Alstrom syndrome, 811 Ambiguous genitalia algorithm for, symmetric/asymmetric, 148f–149f disorders associated with, 128t genetics of, 22 investigation of, 679–680, 680t karyotype for, 679 laboratory studies for, hormones in, 149–151, 150t–151t parental concerns with, 128–129 pelvic ultrasound in, 149–150 treatment goals for, 128–129 in virilized female, 142f Amenorrhea in APS, 778 in CAIS, 145 causes of, 578f–579f differential diagnosis of, 573t, 578f–579f functional hypothalamic, 582 over/under nutrition causing, 583 post-pill, 583 AMH. See M¸llerian inhibitory hormone Amino acid 164-amino acid preprovasopressin, 338f aquaporin, 344f composition, of natriuretic peptide system, 346f vasopressin, 342f Amylo-1,6-glucosidase deficiency (Debrancher deficiency, GSD type III), 184 with hypoglycemia, 433 Anabolic steroids, 642 Analog-based basal-bolus insulin regimens, 398–399 Analyzers, for immunoassays, 862, 862t Anatomic defects, congenital, 357 Androgen receptors (AR), 53–54 functional domains of, 669f mechanism of, 670, 670f mutations of, 672f in sex differentiation, 669, 669f for skeletal growth, 112–113

Androgens actions of disorders of, 144–146 in men, 669–671, 669f mechanism of, 670, 670f mediation of, 145 mutations, 146 in steroid metabolism, 554f transcription for, 145 adolescent rise in, 280–281 anti-, inhibitors of, 478 insensitivity syndromes of complete characteristics of, 669 phenotype of, 670 molecular pathogenesis of, 671–672, 672f partial causes of, 671 partial characteristics of, 669 overproduction of, in hyperandrogenism, 588, 591f pregnenolone in, 142f secretion of in adrenarche, 548–549, 549f regulation of, 548f in skeletal maturation, 281 synthesis of 46,XY Disorders of sexual differentiation, 128t disorders of, 139–144, 139f, 142f general, 668 types of, 668–669 fetal, 142f steroidogenic pathways in, 139f in Turner syndrome, 629–630 Androstenedione, 457 Anemia, autoantigens in, 773t Angiotensin endocrine-,-aldosterone, 344–345 local renin-, systems, 345–346 renin-, system, 457 renin-aldosterone-, 344 Anions, types of, 236t Anorchia, 673 Anorexia nervosa bone metabolism in, 824 definition of, 823 endocrine associations with, 823–825, 824t fat-derived hormones in, 824–825 growth hormone-IGF-1 axis, 823–824 hormonal regulation in, 824t hypogonadism in, 575 hypothalamic-pituitary-adrenal axis in, 824 hypothalamic-pituitary-gonadal axis in, 824 hypothalamic-pituitary-thyroid axis in, 823 treatment of, 825 Anorexigenics, 793 Anovulation, 563–564, 563f–564f disorders with, differential diagnosis of, 580f hypothalamic, 582–584 testosterone levels in, 591f ANP. See Atrial natriuretic peptide Antiandrogen inhibitors, 478 Antibody in Hashimoto thyroiditis, 237, 237f islet cells, 384t receptor-stimulating TSH antibodies, 230 in RIA, 861f testing, for APS, 778–779, 779f Antidiuretic hormone. See Vasopressin Antigens auto-, 773, 773t in diabetes mellitus, 381f, 381t self-, tolerance and, 771, 771f

Anti-m¸llerian hormone (AMH) characteristics of, 134t, 135 disorders of, 672–673 Antithyroid antibody, in Hashimoto thyroiditis, 237, 237f Aorta coarctation of, 636, 636f, 648 dilation of, 639 Aphallia, 128t apoB-100, familial ligand defective, 842 apoE pathways, 844 Apolipoproteins, 840t Appetite, 789–790, 790f stress and, 804–805 APS. See Autoimmune polyglandular syndrome Aquaporin-2 mutations, 361–362 structure of, 344f studies of, 343–344 AR. See Androgen receptors Arg137His V2 vasopressin receptor mutation, 31 Arginine vasopressin (AVP) function of, 455 in hypothalamus, 455 Aromatase, 144 characteristics of, 453 inhibitors, 478 regulation of, 548f Artifactual hypoglycemia, 438 ARX, 134t ASOH. See Allele-specific oligonucleotide hybridization Aspirin, intoxication of, hypoglycemia from, 438 Astrocytoma, hypothalamic, 575f Asymmetric genital ambiguity, 149f Athyreosis, 228t Atretic follicles, 542f Atrial natriuretic peptide (ANP) biochemistry of, 346, 346f hyponatremia with, 355 secretion of, 347 ATRX syndrome, 138 XH2, 134t Audiogram, in Turner syndrome, 630t Autoantibody adrenal cytoplasmic, 781 adrenal enzyme, 781–782 in APS, 781–783 in celiac disease, 783 in diabetes mellitus type 1, 384f in hypoparathyroidism, 783 in isolated Addison disease, 782 steroidal cell, 782–783 to tyrosine hydroxylase, 783 Autoantigens in Addison disease, 773t in anemia, 773t in autoimmune disorders, 773, 773t in Celiac disease, 773, 773t Autoimmune disorders autoantigens in, 773, 773t of chronic adrenal insufficiency, 482–483 with diabetes mellitus, 403 diabetes mellitus and, 777–778 hypoglycemia, 429 organ-specific, 780f polyendocrine syndrome, 483 in thyroid disease, 228t tolerance defects in, 773–775 in Turner syndrome, 640–640 Autoimmune polyglandular syndrome (APS) Addison disease in, 776, 776t amenorrhea in, 778

INDEX

Autoimmune polyglandular syndrome (APS) (Continued) antibody testing for, 778–779, 779f autoantibodies in, 781–783 classification of, 775, 775f clinical aspects of, 775–778, 776t diagnostic approach to, 778–780, 779f–780f follow-up to, 778–780, 779f–780f genetics of, 780–781 IPEX in, 778 tolerance and, 770 treatment of, 780, 783–784 Autoimmunity mechanisms for, 770–775, 771f–772f, 773t thyrogastric, 777 Autonomic nervous system, in hypoglycemia, 173t Autonomous nodular hyperthyroidism, 244 Autosomal dominant hyperthyroidism, 208t, 219 Autosomal dominant hypocalcemic hypercalciuria (ADHH), 40–41 Autosomal-recessive disorder, 140–141 Autosomal-recessive hypercholesterolemia, 842 AVP. See Arginine vasopressin Axial skeleton, defects of, 282t

B B cells autonomic innervation of, 792f tolerance of, 773 Bardet-Biedl syndrome, 810 Bariatric surgery, in pediatric obesity, 817–819 BAT thermogenesis, 205 Beta cells function of genetic abnormalities in, 406–410, 408t, 409f–410f history of, 379f residual, 401 insulin and resistance of, 375f secretion of, 177f, 410f pancreatic, diagram of, 410f, 426f BGLAP. See Osteocalcin Bile acid binding agents, 849–850 Binding protein. See also Growth-hormonebinding protein abnormalities, types of, 228t IGF, 273f, 277–279, 278f Bipotential gonad, 130f Birth BMC at, 687, 687f BMD at, 687, 687f edema in, 623 fatty acids at, 173–174 glucose changes at, 168 low, weight infants, 702–704 Bisphosphonates, in low bone mass, 740–741 Blepharophimosis ptosis epicanthus inversus syndrome (BPES), 139 Blomstrand osteochondrodysplasia, 696 Blood glucose concentration factors affecting measurement of, 172t in GDH, 181, 181f machines measuring, 172 in neonates, 172, 172t Blood production rate, 552 Blood tests, for hypoglycemia, 192, 192f, 423t Blood vessels, in Turner syndrome, 634, 635t BMC. See Bone mineral content BMD. See Bone mineral density

Body composition, in adults, for fuel, 169t image, in Turner syndrome, 643 mass-index-for-age percentiles for boys, 262f for girls, 263f for obesity, 797f–798f proportions of evaluation of, 258 standard, 259 weight by age/gender, percentiles, 256f–263f glucose production v., 172f, 424f Bone age of for adult height, 266t caveats of, 260 serial, assessment of, 309 in sexually infantile girls, 573t area of, reference curves for, 118f calcium content of, 110 deposition of in female child/adolescent, 109t, 111f in male child/adolescent, 108t, 111f development of, endochondral, 99 formation of, 97–106, 98f disorders of, 722–732, 723t–724t, 726f–727f, 728t, 729f, 732f exercise in, 110–111 increased, 748t in mother/neonate, 107t PTH-rP in, 100–101 mass of, 110 assessment of, 114–120, 115t–117t, 118f, 119t–120t high, 747–751, 748t, 749f increased, in infants, 704–705 increased, physical examination, 705 low, 702–704 bisphosphonates in, 740–741 categorization of, 735–736, 736t glucocorticoid-induced, 738–739 with other conditions, 737–740 risk of, 736–738 treatment of, 741–742 matrix, 97–99 maturation of, 558 metabolism of in anorexia, 824 genetic causes of, 693t–694t mineral homeostasis and, genes involved in, 75t–77t mineralization of disorders in, 702–704 glucocorticoids for, 104 Wnt signaling pathway in, 98–99 morphogenic proteins, 98f remodeling of, sites for, 105–106 resorption of decreased, 748t in mother/neonate, 107t strength of, 107, 110 assessment of, 114–120, 115t–117t, 118f, 119t–120t Bone mineral content (BMC), 111f at birth, 687, 687f in child/adolescent, 115t–117t hormones for, 113 in infants, 115t–117t, 703 in neonates, 691f for non-black/black boys/girls, with DEXA, 121t Bone mineral density (BMD), 114–115 at birth, 687, 687f in child/adolescent, 115t–117t

871

Bôrjeson-Forssman-Lehmann syndrome, 811 BPES. See Blepharophimosis ptosis epicanthus inversus syndrome Brain death, causing central diabetes insipidus, 358 fatty acids in, 169 hypothalamic astrocytoma in, 575f sexual differentiation of, 132 trauma of, growth abnormalities from, 290–291 Breasts development of, 561, 561f pubertal milestone attainment in, 562t Bulimia nervosa, 575

C C cells, production of, 523 Ca. See Calcium Cachexia cancer, 822 cytokines in, 822t metabolic changes in, 822t starvation response v., 819 CAH. See 21-Hydroxylase deficiency CAIS. See Complete androgen insensitivity Calciotropic changes, during pregnancy, 686f Calcipenic rickets, 722–728, 723t–724t, 726f–727f Calcitonin, 89–90 production of, 523 Calcitriol, 92–94, 93f genes regulated by, 96f in osteoblast formation, 101 preparations of, 699t responses to, 97f VDR and, 94–96 Calcium (Ca), 74–82, 75t–77t, 78f, 81t absorption of, 79–80 in bone, 110 in gastrointestinal tract, 79 homeostasis, 78, 78f intake of, recommended, 80–81, 80t metabolism disorders, 686 in muscles, 79 obesity and, 806, 808 preparations of, 699t PTH regulating, 87 in fetus, 687–688 renal excretion of, 81t sensing receptor, 28t, 81–82 Calcium sensing receptor (CASR), 708f as class C hormone receptor, 40–41 CALCR, 29 Cancer. See also Tumors cachexia, 822 risk, long-term, 315, 315t vasopressin and, 355t Carbohydrate -deficient glycoprotein hyperinsulinism, 428 dietary fat v., in obesity, 805–806 tolerance of, in Turner syndrome, 642–643 Carcinoma FTC, 522f, 523 MTC, 24t7–248t, 246–248, 246t, 247f, 522 papillary-follicular carcinoma, 246 Cardiac care pregnancy and, 639 in Turner syndrome, 639–643 Cardiac imaging, in Turner syndrome, 636f Cardiovascular disease for growth abnormalities, 286 metabolism in, 839–841, 840f–841f, 840t morbidity of, 839

872

INDEX

Cardiovascular system abnormalities, in Turner syndrome, 633–638, 634f, 635t, 636f GH treatment and, 639 malformation of, 635t screening of, in Turner syndrome, 638t sex hormones in, 559 Carney complex clinical presentation of, 525 endocrine manifestations of, 525 management of, 525–526 molecular genetics of, 525 Carnitine, elevate levels of, 188 Carpenter syndrome, 811 CART, 793 Cartilage disorders of, genetic causes of, 693t–694t epiphyseal, growth plate, 88f, 99–100, 100f CASR. See Calcium sensing receptor Catabolic states, GH treatment in, 313 Catecholamines actions of, 513–514, 514f biosynthesis of, 513–514, 514f clinical presentation of, 514 metabolism of, 514f Cations, 236t CCK. See Cholecystokinin cDNA. See Complementary DNA Celiac disease autoantibodies in, 783 autoantigens in, 773, 773t gastrointestinal tract in, 640 risk of, 640 in Turner syndrome, 640 Central diabetes insipidus brain death causing, 358 causes of, 356–358 congenital anatomic defects with, 357 drugs causing, 358 fluid therapy for, 358–359 increased vasopressin metabolism causing, 358 infectious disease causing, 357–358 neoplasms with, 357 neurosurgical intervention for, 356–357 primary enuresis causing, 358 trauma and, 356 treatment of, 358–360, 363 vasopressin analogues for, 359–360 Central nervous system (CNS) maturation of, 558–559 thyroid hormone actions in, 205 in development, 233t tumors of, 314 vasopressin and, 355t Central neural integration, melanocortin receptors and, 793 Central processing, in energy balance, 791–793, 792f Cerebral edema, 394–395 Cerebral salt wasting, 355 Chief cells, hyperplasia of, 719 Children BMC in, 115t–117t BMD in, 115t–116t bone deposition in female, 109t male, 108t Ca intake in, 80–81, 80t congenital hypothyroidism in, 228t coronary artery disease in, 845–846, 845t, 846f diabetes in, 849t diencephalic syndrome in, 823 diffuse nontoxic goiter in, 228t

Children (Continued) failure to thrive in, 819–820 glucose metabolism in, 424–425, 424f–425f, 425t Graves disease in, 242–244 growth measurement of, 255 height of, in GHR deficiency, 298f hormone secretion in, 534–536, 534f–536f hypercalcemia in etiology of, 713–719, 715t, 717f evaluation of, 719–720 management of, 720–722 hypocalcemia in, 690–692, 691t etiology of, 706–711, 708t–709t evaluation of, 711–712 management of, 696–697, 712–713 hypoglycemia in causes of, 425–438 classification of, 174t ketotic, 175 hypoparathyroidism in, 692–696, 693t–694t hypophosphatemic rickets in, 728t iodine metabolism in, 233, 234f, 234t LDL cholesterol in, 846f, 851 lipids in, 847t mineral homeostasis in, 689–690 sex steroids in, 460t sexual maturation in, 560t, 561 thyroid neoplasia in, 245t clinical features of, 249t tolbutamide in, with hyperinsulinism, 178f Turner syndrome in, 646, 646t follow-up studies of, 647–649 Chinese hamster ovary cells (CHO), 22 CHO. See Chinese hamster ovary cells Cholecalciferol. See also Vitamin D metabolism of, 91f synthesis of, 90 Cholecystokinin (CCK), 791 Cholesterol absorption of, inhibitors of, 850 in androgen synthesis, 142f biosynthesis of, in mutations, 756, 757f, 758 high diet therapy for, 848–851, 848t–849t dietary additives for, 851 supplements for, 851 LDL, in children, 846f, 851 low HDL, 843 metabolism of, disorders of, 842–843, 842t mitochondrial uptake of, 450 in osteochondrodysplasia, 756 steroid biosynthesis from, 552f Chondroblasts, 97, 98f Chondrocytes in estrogen synthesis, 113 PTHrP developing, 100–101 Chondrodysplasia. See Osteochondrodysplasia Chromosomes analysis of, 17–18 germ cell, in Turner syndrome, 631–633, 632f human metaphase, 19 karyotype of studies of, 612 in Turner syndrome, 611 9p monosomy, 137 sex abnormalities in, with growth retardation, 282–283 anomalies in, 664–666, 665f DSD, 128, 128t X genes, 612–614 monosomy, 617 multiple, 614–615

Chromosomes (Continued) showing SHOX, 613, 613f small ring, 611–612 Circulatory system, in pituitary gland, 268, 268f Cirrhosis, of liver, 642 CNS. See Central nervous system Coarctation, of aorta, 636, 636f, 648 Cockayne syndrome, 285 Coelomic epithelium, 130f Cohen syndrome, 811 COL1A1, 743, 746f in osteochondrodysplasia, 755 Collagen pyridinium of, 106f telopeptides of, 106f type I, 105–106, 105f–106f Compact bone, 99 Complementary DNA (cDNA), 6 microassay, 16 Complete androgen insensitivity (CAIS), 145 Computed tomography and positron emission tomography (CT/PET), using 18 Fluoro-L-Dopa, 179f–180f Computers, for DNA analysis, 23 Congenital adrenal hyperplasia, 143 DHEA in, 141 hormone replacement therapy in, 154 laboratory findings in, 466t lipoid, 465–468 two-hit model of, 467f testing for, 150–151 virilizing, 141–143, 142f Congenital anatomic defects, with central diabetes insipidus, 357 Congenital anomalies, 138 Congenital autosomal nephrogenic diabetes insipidus, 361–362 Congenital heart disease prevalence of, 635t risk of, in Turner syndrome, 635t Congenital hyperinsulinism, 176–178, 177f–178f, 177t Congenital hyperthyroidism, 217–219, 218f, 218t Congenital hypopituitarism, undescended testes in, 182f Congenital hypothyroidism childhood/adolescence, 228t newborn screening for, 206–207 presumptive positive screening tests for, 214–215 transient, 213–214 treatment of, 215–217, 215f–217f, 216t Congenital lipoid adrenal hyperplasia, 140–141 Congenital x-linked diabetes insipidus, 360–361, 361f Conn syndrome, 492 Constitutional delay, of growth, 304–306, 305t Constitutional tall stature, management of, 319–320 Consumptive hypothyroidism, 240 Coronary artery disease in children, 845–846, 845t, 846f pediatric risk of, 845t, 846f Corticosterone methyl oxidase, deficiency of, 480 Corticotropin-releasing factor (CRF) function of, 455 in hypothalamus, 455 testing, 464 Cortisol in adipose tissue, 805 in early postnatal life, 150t

INDEX

Cortisol (Continued) in infants, 2-12 months old, 151t normal levels of, 151t obesity and, 804–805 plasma concentrations of, 460–461, 460t–461t rhythms of, 456 secretory rates of, 463 stress and, 805–806 therapeutic, potency of, 476t Cortisone reductase deficiency, 11bhydroxysteroid dehydrogenase1-apparent, lesions in, 481 Counter-regulatory hormone, 170t deficiency of, 182–183, 182f Craniofacies defects, from GH insensitivity, 297t CRF. See Corticotropin-releasing factor Cross-contamination, in PCR, 5 Cryptorchidism causes of, 673 characteristics of, 147, 147t CT/PET. See Computed tomography and positron emission tomography Cushing syndrome adenoma in, 489–490 causes of, 489–490 characteristics of, 487–489 clinical findings of, 488, 488t differential diagnosis of, 490–491, 491t etiology of, 487t growth abnormalities from, 287–288 CYP11A1, 134t CYP17, 139f CYP19A1, 134t in placental aromatase deficiency, 144 CYP21A2, in virilizing congenital adrenal hyperplasia, 141–143, 142f CYP27B1, 92–93 genetic errors in, 726, 726f Cystic hygroma, 619f Cytochrome b5, 451–452, 451f Cytochrome p450 characteristics of, 448 oxidoreductase deficiency, 144 side chain cleavage, enzyme, 141 Cytogenetics molecular, 17–18 in Turner syndrome, 616t Cytokines adipo-, 800–801 in cachexia, 822t inflammatory, 801 receptors of, 27t, 42, 43t

D Dairy, 806, 808 DAX1, 55 in gonadal dysgenesis, 136–137 latest research with, 137 -NROB1, 134t transgenic models of, 133 dDAVP. See Desamino-d-arginine vasopressin Debrancher deficiency, GSD type III. See Amylo-1,6-glucosidase deficiency Deformity facial, 623f kidney, 638f Madelung, 622, 622f plastic surgery for, 648–649 Dehydration, in hyponatremia, 351 Dehydroepiandrosterone (DHEA) concentration of, 457f in congenital adrenal hyperplasia, 141

Dehydroepiandrosterone (DHEA) (Continued) regulation of, 457–458 response, to adrenal steroid, 463t Deiodination iodothyronine, enzymes, 232, 232t of T4, 202f in thyroid hormone metabolism, 231–232 of thyroxine, 202f Denaturing fingerprinting (DnF), 12 Denaturing high performance liquid chromatography (DHPLC), 12–13 Denys-Drash syndrome, 135 2’-deoxynucleoside triphosphate (dNTP), 10, 10f Deoxycorticosterone levels in 2-12 month infants, 151t in early postnatal life, 150t Deoxypyridinoline, 107t Deoxyribose nucleic acid (DNA) c, 6, 16 digestion of, 1–2, 3f fragments, 2, 4f isolation of, 1–2, 3f computers for, 23 recombinant, pediatric endocrine disease therapy and, 22–23 sequencing of direct methods of, 9–11, 10f indirect methods of, 11–14, 12f–14f zinc-finger, binding, 727f DES. See Diethylstilbestrol Desamino-d-arginine vasopressin (dDAVP), 336, 336f Desert hedgehog, 137 DEXA. See Dual-energy X-ray absorptiometry Dexamethasone, 463 DGGE, 12 DHCR7, 134t in Smith-Lemli-Opitz syndrome, 140 DHEA. See Dehydroepiandrosterone DHH, 134t DHPLC. See Denaturing high performance liquid chromatography Diabetes in adolescence, 849t in children, 849t fibrosis related, 409 fluid requirements in, 391t genetic abnormalities in, insulin and, 411–412 gestational, 412 lipoatrophy, 411 mitochondrial, 407 neonatal, 412–414, 413t research in, future, 415 thiamine-responsive, 407 Diabetes insipidus central brain death causing, 358 causes of, 356–358 congenital anatomic defects with, 357 drugs causing, 358 fluid therapy for, 358–359 increased vasopressin metabolism causing, 358 infectious disease causing, 357–358 neoplasms with, 357 neurosurgical intervention for, 356–357 primary enuresis causing, 358 trauma and, 356 treatment of, 358–360, 363 vasopressin analogues for, 359–360 congenital autosomal nephrogenic, 361–362 congenital x-linked, 360–361, 361f evaluation of, 349f

873

Diabetes insipidus (Continued) nephrogenic, 33 causes of, 360–362, 361f treatment of, 362 Diabetes mellitus in adolescence, 849t antigens in, 381f, 381t autoantigens in, 773t autoimmune disorders in, 403, 777–778 autoimmune polyglandular syndromes in, 777 characteristics of, 374 classification of, 375–376, 376t clinical manifestations of, 391 diagnosis of, 391–392 drug induced, 376t exercise in, 401 fibrosis related, 409 with growth abnormalities, 287 HLA-DQ subtypes in, 380t–381t, 381f hypoglycemia in, 401–402 insulin in action of, 387–389 receptors for, 387–389, 387f–388f secretion of, 386–387 lipoatrophic, 411 living with, 397 management of, 849t medical nutritional therapy, 400–401 mitochondrial, 407 morbidity of, 375 obesity and, 812t outpatient care for, 403–404 peptide hormones in, 385–386, 385f psychosocial problems with, 403 risk of, 377t sick day management in, 402–403 during surgery, 404–405 thiamine-responsive, 407 treatment of, 393–397, 395t goals of, 397 levels of, 401t type 1 age-standardized incidence of, 378f autoantigens in, 384f characteristics of, 376–377, 377t epidemiology of, 377–379, 378f etiology of, 379–383, 379f, 380t–381t, 381f genetics of, 379–383, 379f, 380t–381t, 381f human susceptibility loci for, 380t immune intervention trials in, 383, 383t nonautoimmune, 405 pathophysiology of, 389–391, 389t, 390f, 391t phenotypes in, 380t prevention of, 383–385, 384f, 384t risk of, 381t, 384t type 2 characteristics of, 377, 377t mutations causing, 406t typical, 405–406 variants in, 411 viral infections in, 409 youth-onset, 408t Diabetic heterodimers, 384t Diabetic ketoacidosis, 392–393, 393t treatment of, 393–394, 395t Diarrhea, in hypoglycemia, 435 Dideoxy (Sanger) method, 10f Diencephalic syndrome, in children, 823 Diet. See also Nutrition additives in, cholesterol reducing, 851 fat in, in obesity, 805–806 intervention in, in childhood obesity, 813 therapy, for dyslipidemia, 848–851, 848t–849t

874

INDEX

Diethylstilbestrol (DES), in maternal hyperandrogenism, 144 Diffuse nontoxic goiter in childhood/adolescence, 228t genetics of, 240 Diiodotyrosine (DIT), 230f Disorders of sexual differentiation (DSD) 46,XX, 128t M¸llerian duct abnormalities in, 146 46,XY for androgen synthesis/action, 128t for gonadal dysgenesis, 135–136, 136f, 152–153 sexual rearing of, 152 for testicular differentiation, 128t categories of, 680t causes of, classification, 666t diagnosis of, 147–151, 148f–149f, 150t–151t genetic counseling for, 155 gonadal tumors in, risk of, 675t laboratory studies for, 149–151 mutations of, 134t nomenclature relating to, 666t ovotesticular, 667 phenotypes for, 134–135 psychological counseling for, 155 in puberty, 565 research models for, 132–135, 134t sex chromosomes in, 128, 128t SRY-gene in, 134t surgery for, 153 treatment of, 151–152 medical, 154–155 Distal tubule, structure of, 344f DIT. See Diiodotyrosine DNA. See Deoxyribose nucleic acid DnF. See Denaturing fingerprinting dNTP. See 2’-deoxynucleoside triphosphate Donohue’s syndrome, 411 Dose-response curve, for GH/GV, 307t Down syndrome diagnosis of, 283 GH treatment of, 311 Drosophila melanogaster, 137 Drug central diabetes insipidus from, 358 diabetes mellitus induced by, 376t for dyslipidemia, 849–851, 849t–850t hypercalcemia from, 718 hyperinsulinism and, 718 for GDH, 427t hypoglycemia from, 189–190 impairing free water clearance, 352t insulin resistant, 815t, 816 obesity and, 808 in pediatric lipid disorders, 850t targeting osteoclasts, 749f Dual-energy X-ray absorptiometry (DEXA), 114–118, 115t–117t BMC with, for non-black/black boys/girls, 121t Dwarfism idiopathic, 292 in mice, 20 psychosocial, 293 Dysalbuminemic hyperthyroxinemia, familial, 220–221 Dyslipidemia diet therapy for, 848–851, 848t–849t pharmacologic agents for, 849–851, 849t–850t primary, 842–844, 842t secondary causes of, 844–845, 844t vascular changes and, 845–846, 845t, 846f Dysmenorrhea, 585

E Ears, in Turner syndrome, 623–624 EcoR I, 4f Ectopic ACTH syndrome, 489 Ectopic calcification, 751–752 Ectopic thyroid gland, 207 Edema at birth, 623 cerebral, 394–395 in Turner syndrome, 623 Effector pathways osmotic sensory and, 336–344, 336f–344f volume sensor and, 344–347, 346f Efferent system for energy balance, 794–795 vagus nerve, energy storage and, 794 EGF growth factors, 280 Electrolytes, therapy with, 393t, 394 Electron transport to P450c17, 451–452, 451f to P450scc, 449, 450f–451f Electrophoresis, 10f agarose gel, 3f Embryogenesis, 97–98 thyroid system, 199 Embryology of fetal male development, 2f, 663–664 of Turner syndrome, 446–447, 446f, 446t EMX2, 134t Encoding glucokinase (GCK,GK-HI), 177f, 181 Endocannabinoids, in energy balance, 793 Endochondral bone development, 99 Endocrine disorders autoimmune disorders with, 483 environmental, 128t genomic deletions causing, 19 growth abnormalities from, 287–305, 289f–290f, 289t, 294f–296f, 294t, 297t, 298f–299f, 300t, 302f, 304t–305t with hyperinsulinism, in infants/neonates, 176–183, 178f–182f, 178t nuclear receptor mutations associated with, 51t with obesity, 808–809, 809t pediatric history of, 249–250 recombinant DNA technology in, 22–23 RTK germ line mutations associated with, 45t Endocrine function test, 349f Endocrine genes, positional cloning of, 16 Endocrine hormone assays, 855 Endocrine system in anorexia nervosa, 823–825, 824t developing studies in, 1 management of, of Turner syndrome, 649–651 regulation, of growth, 263–281, 267f–269f, 267t, 272f–273f, 275f, 278f–279f Endocrine-angiotensin-aldosterone system anatomy of, 344–345 biochemistry of, 344–345 Endocrinology. See also Pediatric endocrinology positional genetics in, 14–16, 15f–16f Endocrinopathy, pediatric, 18–21, 19f Energy balance of afferent system for, 789–791, 790f central processing in, 791–793, 792f efferent system for, 794–795 endocannabinoids, 793 ghrelin in, 790, 790f hedonic pathway in, 795 in hyperinsulinemia, 796f leptin resistance in, 795–796

Energy (Continued) melanin-concentrating hormone in, 793 negative feedback modulation of, 795 neuroendocrine regulation of, 789–796, 790f, 792f norepinephrine in, 793 orexin receptor, 793 serotonin in, 793 study of, 789 excess of, in obesity, 796–799, 796f–799f expenditure of, sympathetic nervous system and, 794 inadequacy of, in pediatric obesity, 819–825, 820t, 822t, 824f intake of, in childhood obesity, 814 storage of, efferent vagus and, 794 Enteropancreatic islet cell tumors, characteristics of, 519–520 Enuresis, causing central diabetes insipidus, 358 Environmental factors in endocrine disorders, 128t in iodine deficiency disorder, 230 in obesity, 804–808, 807f in Turner syndrome, 617–618 Enzymes adrenal autoantibody, 781–782 cytochrome p450, 141 in fetus, 166–168, 167t iodothyronine deiodination, 232, 232t iodothyronine monodeiodinase, 229f mitochondrial matrix, 187–188 of steroid hormone synthesis, 448–454 steroidogenic human genes for, 446t types of, 448–454 Epigenetics, fetal/neonatal programming and, 804 Epimutation, 284 Epinephrine in fasting metabolism, 169–170, 170t in hypoglycemia, 173t nor-, in energy balance, 793 Epiphyseal cartilage growth plate, 88f factors affecting, 99–100, 100f Epithelium coelomic, 130f follicular, tumors of, 235t site of, of PTH-rP, 85t vaginal, 557f Escherichia coli, 22 Estradiol in early puberty, 538, 538f in infants, 536f in menstrual cycle, 541–542 regulation of, 548f sources of, 553f testosterone-, binding globulin, 554f Estrogen. See also Normoestrogenic menstrual disturbances adolescent rise in, 280–281 mechanism of, model, 555f -progesterone therapy, 642 regulation of, 548f in skeletal maturation, 281 synthesis, chondrocytes in, 113 in thyroid metabolism, 199f in Turner syndrome, 629–630 Estrogen receptors, 54 hypothalamic, 535f selectivity of, 555 for skeletal growth, 112–113 Estrone, 553f Ethidium bromide, 2 Euthyroid hyperthyrotropinemia, 214

INDEX

Exercise associated hyperinsulinemic hypoglycemia, 428 in bone acquisition, 110–111 in diabetes mellitus, 401 Expression studies, 16–17, 16f External genitalia, virilization of, 470f Extrauterine thyroid adaptation, 200f, 203–204 Eyes, in Turner syndrome, 623–624

F Face appearance of, in GHR deficiency, 299f deformity of, in Turner syndrome, 623f Failure to thrive in children, 819–820 differential diagnosis of, 820t management of, 821–822 Familial benign hypocalciuric hypercalcemia (FBH), 40 Familial combined hyperlipidemia (FCHL), 843 Familial dysalbuminemic hyperthyroxinemia, 220–221 Familial glucocorticoid deficiency, 484 Familial glucocorticoid resistance, 492 Familial medullary thyroid carcinoma (FMTC). See also Medullary thyroid carcinoma clinical presentation of, 523 mechanisms of, 522f Familial short stature, 305 Family history of, for DSD diagnosis, 147 intervention of, in childhood obesity, 813–814 Fanconi-Bickel syndrome, in hypoglycemia, 434–435 Fasting adaptation, 168–171, 169f–171f, 169t–170t fuel metabolism in, 169f–170f in glucose metabolism, 424–425, 425f hormonal regulation in, 170t in infants, 171f Fasting metabolic systems epinephrine in, 169–170, 170t hormonal regulation of, 425t in tissue, 389t Fasting system approach, to hypoglycemia diagnosis, 438–440 Fat -derived hormones, in anorexia nervosa, 824–825 dietary, in obesity, 805–806 fuel composition from, 169t saturated, diet therapy for, 848–851, 848t–849t Fatty acid at birth, 173–174 in brain, 169 free, receptor 1, 37 oxidation clinical manifestations of, 436t defects in, in hypoglycemia, 435–436 disorders of, 187–189, 187f, 189t with distinguishing metabolic markers, 189t neonatal screening for, 189 FBH. See Familial benign hypocalciuric hypercalcemia FCHL. See Familial combined hyperlipidemia Female reproductive system in adolescent, 536–539, 536f–539f, 539t in adults, 539–543, 539f–542f

Female reproductive system (Continued) fetal development of, 532–534, 533f infant development of, 534–536, 534f–537f Feminizing adrenal tumors, 491–492 Ferredoxin reductase, 449 Fetal life glucose homeostasis in, integration of, 166–168, 167t to neonatal life, transition of, 166 thyroid function in, 200–201, 200f–201f thyroid function test parameters in, 218f Fetal-neonatal Graves’ disease, 217–219, 218f, 218t Fetus abnormalities in, intrinsic, 284t adrenal cortex formation in, 136, 447 adrenal steroidogenesis in, characteristics of, 454–455 androgen biosynthesis in, steroidogenic pathways for, 142f female reproductive system in, 532–534, 533f male developed embryology of, 2f, 663–664 genetic control in, 664, 664f normal, 664 sex development disorders in, 664–667, 665f, 666t mineral homeostasis in, 686–688, 687f neuroendocrine unit in, 532 normal sexual maturation of, 559–561, 560t overgrowth in, 317–318, 317t as patient, 221 programming of, obesity and, 804 substrates in, 166–168, 167t testicular steroidogenesis in, 664, 664f Turner syndrome in, 619f FGF growth factors, 280 FGF3. See Fibroblast growth factor 3 FGF9, 134t FGF23. See Fibroblast growth factor 23 FGFR. See Fibroblast growth factor receptor family Fiber, obesity and, 806 Fibric acid derivatives, 851 Fibroblast, 97, 98f Fibroblast growth factor 3 (FGF3), in osteochondrodysplasia, 752, 753t, 755f Fibroblast growth factor 23 (FGF23) in osteochondrodysplasia, 755 in tumor-induced rickets, 731, 732f Fibroblast growth factor receptor family (FGFR), 45t, 47–48 phosphatonins and, 83–84 Fibrosis related diabetes, 409 Fibrous dysplasia, 747 Fluid concentrations of, in steroids, 542f electrolyte therapy, 393t, 394 osmotic regulation of, 339–340, 339f–340f physiology of, 336 of vasopressin secretion, 339–340, 339f–341f requirements of, in diabetes, 391t therapy, for central diabetes insipidus, 358–359 Fluorescence 5’-nuclease assay with, 9f in CT/PET, using 18Fluoro-L-Dopa, 179f–180f detection methods with, 9–11, 9f–10f FMTC. See Familial medullary thyroid carcinoma Focal KATPHI, 178–179, 179f Focal lesions, infants with, 178–179, 179f–180f

875

Follicles abnormal, 542, 542f antral, 545f atretic, 542f autoamplification processes in, 539t classification of, 533f healthy, 542f ovarian of, 533f Follicle-stimulating hormone (FSH) in puberty, 534f–539f receptors, 28t, 35 during pregnancy, 37 secretion of, 547 in sexually infantile girls, bone age in, 573t in Turner syndrome, 632t Follicular epithelium, tumors of, 235t Follicular phase hormone blood production rates in, 551t of menstrual cycle, 540–543, 541f–542f Food intake, glucose concentration and, 409f Food reward, hedonic pathway of, 795 FOXL2 BPES associations with, 139 mutation, 134t Frasier syndrome, 135 Free water clearance. See Renal free water clearance Fructose 1,6-diphosphatase deficiency, 185–186 in hypoglycemia, 435 intolerance to, hereditary, 186–187 hypoglycemia in, 435 metabolism of, 807f obesity and, 806 FSH. See Follicle-stimulating hormone Fuel composition, of normal adult, 169t metabolism in fasting human, 169f–170f in infants, 171f

G Galactosemia, 186 Gastric inhibitory polypeptide receptor (GIPR), as class B receptor, 39 Gastrointestinal tract bleeding in, 640 Ca in, 79 in Celiac disease, 640 in Turner syndrome, 640–642 vitamin D in, 90f GATA4, 134t GCK. See Encoding glucokinase GDH. See Glutamate dehydrogenase Gender assignment, 129 body weight by, 256f–263f labeling, 128 rearing, 152–153 Generalized resistance to thyroid hormone (GRTH), 216, 217f Genes calcitriol regulating, 96f endocrine, positional cloning of, 16 in G-protein disorder, 41–42 for growth hormone, 270–271 for iodotyrosine deiodinase defect, 211 in mineral homeostasis, bone metabolism and, 75–77t for oxytocin/vasopressin expression, 337, 337f for sex determination, 129 models for, 133

876

INDEX

Genes (Continued) for steroidogenic enzymes, 446t for tumors, 135 for VDR, 96f x chromosomes, 612–614 Genetic abnormalities of beta cell function, 406–410, 408t, 409f–410f with diabetes, insulin and, 411–412 of GH/GHD, 292 of lipoatrophy, 412t in ovaries, 138–139 Genetic control, in fetal male development, 664, 664f Genetic counseling, for DSD individuals, 155 Genetic defects decision tree for, for IGF deficiency, 290f in GH-IGF axis diagram of, 289f IGF deficiency from, 289t in hypoglycemia, 434 in insulin action, 411 Genetic errors, in CYP27B1, 726, 726f Genetic lesions, in steroidogenesis, 465–481, 466t, 467f, 470f–471f Genetic testing for lipid disorders, 847–848 for MEN1, 521 for MEN2, 524–525 Genetics of ambiguous genitalia, 22 of APS, 780–781 of CAH deficiency, 472–473, 473f of carney complex, 525 cyto-, 17–18, 616t of diffuse nontoxic goiter, 240 factors of, for obesity, 804 of MEN, 517–518 metabolism and, 22 positional, in endocrinology, 14–16, 15f–16f of type 1 diabetes mellitus, 379–383, 379f, 380t–381t, 381f Genital tract, maturation of, 556–557, 557f Genital tubercle, 132 Genitalia ambiguous algorithm for, symmetric/asymmetric, 148f–149f disorders associated with, 128t genetics of, 22 investigation of, 679–680, 680t karyotype for, 679 laboratory studies for, hormones in, 149–151, 150t–151t parental concerns with, 128–129 pelvic ultrasound in, 149–150 treatment goals for, 128–129 in virilized female, 142f external development of, 663 structures of, differentiation of, 130f, 132 virilization of, 470f internal development of, 663 structures of, differentiation of, 130f, 131–132 Genomic deletions 18q, 283 causing human endocrine disease, 19 Xq28, 137–138 Genomic imprinting, in Turner syndrome, 614 Germ cells chromosomal defects, in Turner syndrome, 631–633, 632f differentiation, 131

Germ cells (Continued) in ovarian follicular development, 533f tumors in characteristics of, 674–675 classification of, 674–675 risk of, 675t Germ-line mutations loss-of-function, 523 in MEN1, 519f, 521–523, 522t in MEN2, 521–523, 522t of RTK, clinical conditions associated with, 45t Gestational age, GH treatment of, 311 Gestational diabetes, 412 GH. See Growth hormone GHBP. See Growth-hormone-binding protein GHD. See Growth hormone deficiency GHR. See Growth hormone receptor Ghrelin, 28t in energy balance, 790, 790f receptors, as class A receptor, 38–39 GHRH. See Growth-hormone-releasing hormone GIPR. See Gastric inhibitory polypeptide receptor GK-HI. See Encoding glucokinase Glandular diseases, in MEN2, 526t Globulin, 74 estradiol-testosterone binding, 554f Glomerular filtration, 81t GLP-1. See Glucagon-like peptide-1 Glucagon-like peptide, 40 Glucagon-like peptide-1 (GLP-1), 791 Glucocorticoid for bone mineralization, 104 familial, deficiency, 484 familial, resistance, 492 feedback, adrenal, 456–457 -induced low bone mass, 738–739 mean concentration of, 461t receptor, 53 -suppressible hyperaldosteronism, 480–481 Glucocorticoid therapy common preparations for, 494–495 high-dose, complications of, 495t mineralocorticoid replacement, 497 pharmacological, 495 replacement, 493–494 stress doses of, 496–497 uses of, 492–493 withdrawal of, 495–496 Glucokinase hyperinsulinism, 428 Gluconeogenesis disorders of, 185–186 with hypoglycemia, 434 hepatic, 170t metabolism of, 432f Glucose concentration, food intake and, 409f metabolism of fasting adaptation in, 424–425, 425f in liver, 807f physiologic adaptation of, in infants/ children, 424–425, 424f–425f, 425t production of body weight v., 172f, 424f changes in, 424, 424f self-monitoring of, 399–400 tolerance, impaired, 414 transporters of, 167t defects of, 190–191, 190t–191t Glucose 6-phosphatase deficiency (GSD type I), 183–184 as gluconeogenesis disorder, 185 hypoglycemia from, 431–433

Glucose homeostasis at birth, 168 in hypoglycemia, 171–172, 172f, 172t integration of, into fetus, 166–168, 167t perinatal, 166 plasma levels of hyperinsulinism diagnosis by, 177t in newborns, 174f GLUD1, 180–181, 181f GLUT1 characterization of, 167, 167t deficiency of, 190 GLUT2, deficiency, 190–191 Glutamate dehydrogenase (GDH) blood glucose concentration in, 181, 181f hyperinsulinism with, 180–181, 181f diagnosis of, 427 drugs for, 427t mechanism of, 428f Glycemic index, obesity and, 806 Glycogen phosphorylase deficiency (GSD type VI), 185 Glycogen storage disorders diagnosis of, 185 hypoglycemia in, 183, 431 types of, 183–185 Glycogen synthase deficiency (GSD type 0), 185 in hypoglycemia, 434 Glycogenolysis hepatic, 170t metabolism of, 432f Gly-Lys-Arg, 337f GNAS -1 gene, mutations of, 41–42 gene complex, first steps in, 709f GnRH. See Gonadotropin releasing hormone Goitrogenic agent exposure to, in acquired juvenile hypothyroidism, 235 types of, 236t Gonadal differentiation, disorders of, 135–139, 136f Gonadal dysgenesis causes of, 573–574 characteristics of, 667 histology of, 667 mixed, 667 sexual differentiation in, 135–136, 136f sexual rearing in, 152–153 Gonadal failure, in Turner syndrome, 631–633, 632t Gonadal tumors, 674–676, 674t–675t Gonadoblastoma, 153 in Turner syndrome, 633 Gonadostat, 532f Gonadotrophin lifetime cycles of, 532f in precocious puberty -dependent, 677t -interdependent, 677t rise, at puberty, 668–669 Gonadotropin deficiency, 574–577, 575f–576f constitutional pubertal delay v., 581t Gonadotropin releasing hormone (GnRH), 28t, 29–30 in adolescents, 536–538, 537f agonist therapy for CAH deficiency, 478 for precocious puberty, 571t in infants, 534–536, 535f–536f location of, 546f in menstrual cycle, 540f pathways mediating, 550f receptor group, 37

INDEX

Gonadotropin releasing hormone (GnRH), (Continued) regulation of, 544f secretion of, 546–547, 546f GPCR. See G-protein-coupled receptors GPCR somatostatin receptor (SSTR5), 28 GPR54 as class A receptor, 38 in puberty, 677t G-protein gene disorders, 41–42 mutations in, activating, in juvenile hyperthyroidism, 245 G-protein-coupled receptors (GPCR), 27–32, 27t–28t, 29f–31f classes of, 31–32, 31f structure/function of, 29f N-terminal extracellular domain for, 29f, 31 G-protein-receptor kinase (GRK), 30–31, 30f Granulosa cells, 548f Graves disease in children clinical features of, 242 laboratory diagnosis of, 242 medical management of, 243 radioactive iodine for, 242–243 surgical treatment of, 242–243 therapeutic approach to, 243–244 treatment of, 242 fetal-neonatal, 217–219, 218f, 218t hyperthyroidism in, 219 thyroid dysfunction in, in neonates, 218t transient hypothyroidism in, 218t GRK. See G-protein-receptor kinase Growth abnormalities in from endocrine disorders, 287–305, 289f–290f, 289t, 294f–296f, 294t, 297t, 298f–299f, 300t, 302f, 304t– 305t IGF for, 275–276 primary classification of, 281t types of, 281–285, 282t, 284t secondary classification of, 281t types of, 285–305, 289f–290f, 289t, 294f–296f, 294t, 297t, 298f–299f, 300t, 302f, 304t–305t of body proportions, 258–259 charts, 255–258, 256f–265f constitutional delay of, 304–305, 305t criteria for, 305t disorders of, 305–306 endocrine system regulation of, 263–281, 267f–269f, 267t, 272f–273f, 275f, 278f–279f excess, 317–321 factors EGF, 280 FGF, 280 new developments in, 280 for skeleton, 111–114 failure biochemical evaluation of, algorithm of, 302f causes of, 313 IGF-1 related to, 296–297 primary, 284t inhibitory peptides, 280 measurement of, 255 new understandings in, 321 normal, 255–263, 256f–253f promotion of, 302f, 629–630 in Turner syndrome, 625–630

Growth (Continued) response, 309–310 retardation of chromosomal abnormalities for, 282–283 classification of, 281, 281t intrauterine, 283–284 etiology of, 284t GH treatment of, 311 maternal factors in, 284t skeletal maturation, 259–260 thyroid hormone effects on, 233t Growth hormone (GH) actions of, 270–271 binding of, model, 272f bio-inactive, 293–294 chemistry of, 268–269 dose-response curve for, 307t dosing of, 307, 307t excess, 281 diagnosis of, 320–321 secretion of, 320 treatment of, 321 genes, 270–271 genetic abnormalities of, 292 -IGF axis, genetic defects from diagram of, 289f IGF deficiency from, 289t IGF peptides for, 271–272 -IGF-1 axis, in anorexia, 823–824 insensitivity, 294–295, 294t, 295f–296f clinical features of, 297–298, 297t, 298f limited accuracy assays of, 300 neurosecretory dysfunction, 293 pituitary, deficiency, growth abnormalities from, 291–292 provocative testing, 300–301 secretion of, 269–270 dual contributions of, 294f obesity and, 803 testing of, 302f side effects of, 313–314 structure of, 269f thyroid hormone interactions with, 281 treatment with for CAH deficiency, 478 cardiovascular system and, 639 in childhood obesity, 817 monitoring of, 308, 308t of short stature, 310–313 side effects of, 314–315 studies of, 649–651 in Turner syndrome, 311, 626–627 Growth hormone deficiency (GHD) acquired idiopathic isolated, 293 diagnosis of, 304 adult, 310, 310f genetic abnormalities of, 292 IGHD 1A, 19 leukemia from, 314 neonatal testing of, 304 novel modalities for, 307–308, 308t physical examination findings for, 304t pituitary, 291–292 risk of, 302f SCFE and, 314 treatment of, 306 adult, 310, 310f goals for, 309–310 Growth hormone receptor (GHR), 19, 21, 42–43, 43t class A, for transducing action, 37–39 class B, for transducing action, 39–40 class C, for transducing action, 40–41 deficiency, 295f–296f diagnosis of, 298–304, 299f, 300t, 302f

877

Growth hormone receptor (GHR) (Continued) facial appearance in, 299f height in, 298f GHBP and, 270–271 signaling defects, 295–296 Growth hormone releasing hormone receptor (GHRHR), molecular defects from, 290 Growth velocity (GV), dose-response curve for, 307t Growth-hormone-binding protein (GHBP) abnormalities in, 21 affinity of, 271 concentrations of, in GHR deficient patient, 295f growth hormone receptors and, 270–271 signaling of, 272f Growth-hormone-releasing hormone (GHRH) as class B receptor, 39 molecular defects from, 290 GRTH. See Generalized resistance to thyroid hormone GSD type 0. See Glycogen synthase deficiency GSD type I. See Glucose 6-phosphatase deficiency GSD type IX. See Phosphorylase kinase deficiency GSD type VI. See Glycogen phosphorylase deficiency Guidelines for the Care of Girls and Women with Turner Syndrome, 651 GV. See Growth velocity

H Hair. See Pubic hair HAIR-AN. See Hyperandrogenism, insulin resistance, and acanthosis nigricans Hamartoma, hypothalamic, magnetic resonance imaging of, 567f Hashimoto thyroiditis in acquired juvenile hypothyroidism, 236–237, 237f antibodies in, 237, 237f diagnosis of, 239 hCG. See Human chorionic gonadotropin HDAC. See Histone deacetylases HDL. See High-density lipoproteins Head circumference-for-age percentiles, weight-for-length and, 257f, 259f Hearing loss, in Turner syndrome, 630–631, 630t Heart disease, congenital prevalence of, 635t in Turner syndrome, 635t Hedonic pathway, of food reward, 795 Height adult, prediction of, 261–262, 266t of adults, by bone age, 266t final, in Turner syndrome, 628f–629f, 628t in GHR deficiency children, 298f target, of parents, 262–263 velocity chart for men, 264f for women, 265f in Turner syndrome, 624f–626f Hematologic disease, for growth abnormalities, 287 Hepatic gluconeogenesis, 170t Hepatic glycogenolysis, 170t Hepatic ketogenesis, 170t Hepatic type I activity, in sheep models, 202 Heredity fructose intolerance (HFI), 186–187, 435

878

INDEX

Hermaphroditism, true, 138 HESX1, 20 Heteroduplex analysis, 13 Heterotrimeric Gs proteins, 40 HFI. See Heredity fructose intolerance hGH-N. See Human growth hormone-N hGH-V. See Human growth hormone-V High bone mass, 747–751, 748t, 749f mutations of, 750–751 High-density lipoproteins (HDL), low, cholesterol, 843 High-mobility group (HMG), 130 Hirschsprung disease, testing for, 525 Hirsutism, 589f Histology, of gonadal dysgenesis, 667 Histone deacetylases (HDAC), 50f HLA-DQ subtypes, in diabetes mellitus, 380t–381t, 381f HMG. See High-mobility group HMG-CoA reductase inhibitors, 850 Homeostasis. See also Glucose homeostasis; Mineral homeostasis Ca, 78, 78f phosphate, 78f PTH in, 86 vasopressin in, 336 Honeymoon period, 401 Hormonal assay methods accuracy of, 859 carryover in, 859 history of, 855 pre-analytic variables for, 856, 856t–858t precision of, 859 reference ranges for, 856, 858 reportable ranges in, 859 specificity in, 859 standardization of, 856 validation for, 858–859, 859t Hormone replacement therapy, in congenital adrenal hyperplasia, 154 Hormones. See also Growth hormone; Peptide hormones; Steroid hormones blood production rates of, in mid-follicular phase women, 551t for BMC, 113 deficiency of, 430–431 fasting adaptation and, 168–171, 169f–171f, 169t–170t in fasting metabolic systems, regulation of, 170t fat-derived, 824–825 in fetus, 166–168, 167t future research for, 592–593 in hypoglycemia, 432 in pubertal disorders, 680–681 radioimmunoassay of, 860–864, 860f receptors of activation of, 27 protein, 33–37, 36f types of, 27t–28t regulation of in anorexia, 824t of fasting metabolic systems, 425t secretion of, in children/infants, 534–536, 534f–536f sensitivity of, 858–859 for skeleton, 111–114 for testing ambiguous genitalia, 149–151, 150t–151t 170HP values, 464, 464f in newborns, 471f HTC-LC-MS/MS method, 863f–864f Human chorionic gonadotropin (hCG) during pregnancy, 36–37 in thyroid metabolism, 199f

Human growth hormone-N (hGH-N), 19 Human growth hormone-V (hGH-V), 19 Human metaphase chromosomes, 19 Hunger, 789–790, 790f 3b-Hydroxysteroid dehydrogenase characteristics of, 450–451 deficiency of, 468 11b-Hydroxysteroid dehydrogenase -1-apparent cortisone reductase deficiency, lesions in, 481 -2-apparent mineralocorticoid excess, lesions in, 481 characteristics of, 454 deficiency of, 479–480 isozymes of, lesions in, 481 17a-Hydroxylase/17,20-lyase, deficiency of, 468 17b-Hydroxysteroid dehydrogenase characteristics of, 452–453 deficiency of, 668–669 17-Hydroxyprogesterone (17-OHP) in 2-12 month infants, 151t in early postnatal life, 150t testing of, 864–865 21-Hydroxylase deficiency (CAH) adrenalectomy for, 478 characteristics of, 142, 142f clinical forms of, 470–472, 471f diagnosis of, 475–476 genetics of, 472–473, 473f hypertensive forms of, 479 incidence of, 472 microconversions causing, 474, 474t missense mutations causing, 474–475 nonclassic, 471–472 p450c21 causing, 472–475, 473f–474f, 474t pathophysiology of, 469–470, 470f point mutations causing, 474, 474f postnatal, treatment of, experimental, 478 prenatal diagnosis of, 475 treatment of, experimental, 477 salt-wasting, 471 simple virilizing, 471 treatment of, 476–477 Hydroxyapatite, 74 3b-Hydroxysteroid dehydrogenase deficiency, 143 17b-Hydrosteroid dehydrogenase deficiency, 143 Hydroxysteroid dehydrogenase, characteristics of, 448 Hyperandrogenism in adolescence, 585–592, 586t, 587f, 589f adrenal, 588 androgen overproduction in, 588, 591f differential diagnosis of, 588–590, 589f functional ovarian, 587–588, 587f management of, 590–592, 591f Metformin for, 592 polycystic ovarian syndrome causing, 585–587, 586t, 587f progestin monotherapy for, 592 Hyperandrogenism, insulin resistance, and acanthosis nigricans (HAIR-AN), 46 Hypercalcemia in adolescence etiology of, 713–719, 715t, 717f evaluation of, 719–720 management of, 720–722 causes of, 698–702, 699t in children etiology of, 713–719, 715t, 717f evaluation of, 719–720 management of, 720–722

Hypercalcemia (Continued) drugs causing, 718 etiology of, 698–702, 699t evaluation of, 702, 702f in infants, 697–702, 699t, 702f management of, 702 in neonates, 697–702, 699t, 702f oncogenic, 718 Hypercholesterolemia autosomal-recessive, 842 familial, 842 Hyperglycemia, biology of, 20 Hypergonadotropic hypogonadism, causes of, 679t Hyperinsulinemia algorithm for, 796f energy balance in, 796f negative feedback pathway in, 796f Hyperinsulinism carbohydrate-deficient glycoprotein, 428 in children, tolbutamide in, 178f congenital, 176–178, 177f–178f, 177t diagnosis of, 177t criteria for, 426 dominant KATPHI, 179–170 endocrine disorders with, in infants/ neonates, 176–183, 178f–182f, 178t exercise-associated, hypoglycemia, 428 factitious, 428–429 focal adenomatosis, 178–179, 179f with GDH, 180–181, 181f, 427, 427t, 428f GK, 181 glucokinase, 428 in infants, 425–427 islet adenoma, 182 KATP channel, 177f, 178, 426–427 perinatal stress-induced, 176 recessive KATPHI, 177f, 178 SCHAD, 181–182, 428 transient, from maternal factors, 175–176 Hypermagnesemia, 705–706 Hypernatremia, 348–350, 349f Hyperosmolality, 340, 340f Hyperparathyroidism characteristics of, 519 neonatal secondary, 700 severe, 40 Hyperphosphatemia, 728t Hyperplasia. See also Congenital adrenal hyperplasia ACTH-independent bilateral macronodular adrenal, 16f ACTH-independent multinodular adrenal, 490 of chief cells, 719 congenital lipoid adrenal, 140–141 Hyperprolactinemia, 577 Hypertension, in Turner syndrome, 638 Hyperthyroidism, 36 autosomal dominant, 208t, 219 congenital, 217–219, 218f, 218t juvenile activating TSH receptor mutations in, 244–245 autonomous nodular hyperthyroidism, 244 G-protein mutations in, 245 Graves’ disease, 242–243 TSH-dependent hyperthyroidism, 244 with pituitary T3 resistance, 244 pituitary tumors with, 244 Hyperthyrotropinemia, euthyroid, 214 Hyperthyroxinemia, dysalbuminemic, familial, 220–221

INDEX Hypertriglyceridemia, disorders of, 843 Hypobetalipoproteinemia, 844 Hypocalcemia in adolescence etiology of, 706–711, 708t–709t management of, 712–713 causes of, 691t in children, 690–692, 691t etiology of, 706–711, 708t–709t evaluation of, 711–712 management of, 696–697, 712–713 in neonates causes of, 691t early, 690 late, 690–692 management of, 696–697 Hypochondroplasia, diagnosis of, 282 Hypoglycemia alcohol-induced, 438 alimentary, 437 artifactual, 438 autoimmune, 429 autonomic nervous system in, 173t blood tests for, 192, 192f, 423t classification of, 173–183, 174f, 174t, 177t–183t in infants/children, 174t clinical examination of, 439 critical sample of, 439–440 Debrancher deficiency, GSD type III in, 433 definition of, 423 in diabetes mellitus, 401–402 diagnosis of, 191–193, 192f fasting system approach to, 438–440 diarrhea in, 435 drug induced, 189–190 emergency treatment of, 440 epinephrine in, 173t evaluation of, 422–423 exercise-associated, hyperinsulinism, 428 from exogenous agents, 438 Fanconi-Bickel syndrome in, 434–435 fasting, 177t, 181 in adults, 169f–170f fatty acid oxidation defects in, 435–436 fructose 1,6-diphosphatase deficiency in, 435 genetic defects in, 434 gluconeogenesis disorders in, 434 glucose homeostasis in, 171–172, 172f, 172t in glycogen storage disorders, 183, 431 GSD type 0 in, 434 GSD type I in, 431–433 in hereditary fructose intolerance, 435 hormonal changes with, 432 in infant/children causes of, 425–438 ketotic, 175 in infant/neonate, 168–169, 171–172, 171t, 172f differential diagnosis of, 190t–191t ketotic, 429–430 malaria in, 435 nonpancreatic tumor, 436–437 in organ failure, 438 reactive, 437–438 from salicylate intoxication, 438 signs/symptoms of, 172–173, 173t spontaneous, symptoms of, 423–424, 423t with systemic disorders, 191 transient neonatal, 173–175, 174f, 174t treatment of, 191, 193 urine tests for, 423t

Hypogonadism anorexia nervosa in, 46 causes of, 572–576, 573t, 575f–576f differential diagnosis of, 577–581, 578f–580f, 581t management of, 581–582 menstrual cycle in, 575–576 Hypogonadotropic hypogonadism, 146–147 causes of, 679t genetic causes of, 678–679 Hypokalemia, 701 Hypolipidemias, 843–844 Hypomagnesemia, 705–706 Hyponatremia, 347–348 ANP with, 355 causes of, 363 organic osmolyte changes in, 354f plasma volume in, decreased effective, 352–353 sodium chloride in, 351 systemic dehydration in, 351 treatment of, 351, 353 emergency, 353, 354f true/factitious, 355–356 vasopressin in abnormal regulation of, 354–356, 355t with decreased regulation, 350–351, 356–362, 361f with increased regulation, 351–353 with normal regulation, 350–351, 352t Hypoparathyroidism autoantibodies in, 783 causing hypocalcemia, 691t in children, 692–696, 693t–694t in infants, 692–696, 693t–694t pseudo-, growth abnormalities from, 288 Hypophosphatasia, 723t Hypophosphatemic rickets, 288 in children, 728t PHEX in, 729, 729f Hypopituitarism, congenital, undescended testes in, 182f Hypoplasia congenita of in adrenal gland, 483–484 X-linked, 483–484 Leydig cell characteristics of, 668 types of, 140 mutations, 228t of pituitary gland, 292 Hypospadias, 674 Hypothalamic anovulation, 582–584 Hypothalamic astrocytoma, in brain, 575f Hypothalamic dysfunction causing adrenal insufficiency, 486 growth abnormalities from, 288–290 Hypothalamic estrogen receptors, 535f Hypothalamic hamartoma, magnetic resonance imaging of, 567f Hypothalamic-pituitary hypothyroidism in acquired juvenile hypothyroidism, 237–238 diagnosis of, 214–215 in infants, 213 types of, 228t Hypothalamic-pituitary portal system, 268, 268f Hypothalamic-pituitary TSH axis, 231f–232f Hypothalamic-pituitary-adrenal axis, 455–457, 455f in anorexia, 824 Hypothalamic-pituitary-gonadal axis, in anorexia, 824 Hypothalamic-pituitary-thyroid axis, in anorexia, 823

879

Hypothalamus AVP in, 455 CRF in, 455 development of, 264–265 inflammation in, 291 irradiation of, 291 trauma to, 290–291 tumors of, 291 vasopressin cells in, 339f Hypothyroidism. See also Juvenile hypothyroidism congenital childhood/adolescence, 228t newborn screening for, 206–207 presumptive positive screening tests for, 214–215 transient, 213–214 treatment of, 215–217, 215f–217f, 216t consumptive, 240 growth abnormalities from, 287–288 hypothalamic-pituitary in acquired juvenile hypothyroidism, 237–238 diagnosis of, 214–215 in infants, 213 types of, 228t sexual precocity caused by, 567f transient, 228t with Graves’ disease, 218t primary, 205–206, 205t Hypothyroxinemia, of prematurity, 206

I IGF. See Insulin-like growth factor IGF-1. See Insulin-like growth factor-1 IGHD 1A. See Isolated growth hormone deficiency type 1A IMAGe syndrome, 128t Imaging studies cardiac, in Turner syndrome, 636f magnetic resonance of hypothalamic hamartoma, 567f in Turner syndrome, 636f in pheochromocytomas, 516t of testes tumors, 680 Immune intervention trials, in diabetes mellitus type 1, 383, 383t Immunoassay techniques. See also Radioimmunoassay analyzers for, 862, 862t basic, 860f examples of, 861t goals of, 860 for mass spectrometry, 862–864, 863–864 Inborn abnormalities growth abnormalities from, 287 from thyroid hormone metabolism, 208t–209t Infantilism diagram of, 611f in girls, 573t Infants age of 2 hour-7 day old, 150t 2-12 month old, 151t BMC in, 115t–117t, 703 bone mineralization disorders in, 702–704 Ca intake in, 80–81, 80t with congenital hypothyroidism, treatment of, 215–217, 215f–217f, 216t estradiol in, 536f female reproductive system in, 534–536, 534f–537f with focal lesions, 178–179, 179f–180f

880

INDEX

Infants (Continued) glucose metabolism in, physiologic adaptation of, 424–425, 424f–425f, 425t GnRH secretion in, 534–536, 535f–536f growth of, measurement of, 255 hormone secretion in, 534–536, 534f–536f hypercalcemia in, 697–702, 699t, 702f hyperinsulinism in, 425–426 endocrine disorders with, 176–183, 178f–182f, 178t hypoglycemia in, 168–169, 171–172, 171t, 172f causes of, 425–438 classification of, 174t differential diagnosis of, 190–191t ketotic, 175 symptoms of, 173f hypoparathyroidism in, 692–696, 693t–694t hypothalamic-pituitary hypothyroidism in, 213 increased bone mass in, 704–705 KATP channel hyperinsulinism in, 177f, 178, 426–427 low birth weight, 702–704 metabolic fuels in, when fasting, 171f mineral homeostasis in, 688–689, 688f disorders of, 690–705 normal thyroid gland in, 216t osteopetrosis in, 747–748 Pendred syndrome in, 211 premature, thyroid dysfunction syndromes in, 205–206, 205t rickets in, 702–704 sex steroids in, mean concentration of, 460t thyroid function in, 201 iodine metabolism, 233, 234f, 234t vitamin D deficiency in, 725–726, 726f Infection causing central diabetes insipidus, 357–358 causing diabetes mellitus, 409 chronic, growth abnormalities from, 287 in obesity, 808 in thyroiditis, 228t Inflammation of brain/hypothalamus, 291 cytokines in, 801 Inner ring monodeiodination, 202f INSL3, 134t Insulin action of acquired defects in, 411 genetic defects in, 411 beta cells and resistance of, 375f secretion of, 177f, 410f biosynthesis of, 385–386, 385f in diabetes mellitus action of, 387–389 receptors, 387–389, 387f–388f secretion of, 386–387 dynamic disorders, in obesity, 811–812 gene, mutations in, 20 genetic abnormalities in, 411–412 hypersecretion of, 811 suppression of, 816–817 in metabolic afferents, 791 pro-, 385f receptors, 21, 45–47, 46f structure of, 387f tyrosine kinase family, 45, 45t regimens for, 398–401, 401t three-dose, 409f resistance to, 801 acanthosis nigricans with, 411

Insulin (Continued) b-cell function relationship to, 375f drugs for, 815t, 816 nonalcoholic fatty liver disease and, 802–803 polycystic ovarian syndrome and, 803 primary, 811–812 secretion of action and, 375 pancreatic beta cells in, 177f therapy, 396–397 types of, 397–398, 397t Insulin-like growth factor (IGF) assay methodologies for, 274–275 axis, 273f binding protein, 273f, 277–279, 278f for GH actions, 271–272 GH-, axis diagram of, 289f genetic defects from, IGF deficiency from, 289t in growth disorders, 275–276 historical background of, 272–273 molecular biology of, 273–274, 273f new sensitive assays for, 301 receptors, 276–277 serum levels of, 275, 275f structure of, 273–274, 273f, 278f targeted disruption of, 279–280 Insulin-like growth factor deficiency (IGFD), 278t diagnosis of, 298–304, 299f, 300t, 302f genetic defect decision tree for, 290f growth abnormalities from, 288, 289f–290f, 289t risk of, 302f treatment of, using IGF-1, 315–317, 316f Insulin-like growth factor-1 (IGF-1), 45t, 47 affinity of, 276 cancer risk with, 315, 315t deficiency, clinical features of, 297–298, 297t, 298f with GHR deficiency, 295f–296f growth failure related to, 296–297 pediatric normal data, for acid ethanol extracted blocking method, 858t rh-, 316f role of, 277 serum levels of, 275, 275f, 309 for skeletal growth, 111–112 splicing of, 274 structure of, 278f treatment with, for IGFD, 315–317, 316f Insulinoma, 429 Intersex, 128 Intrauterine growth retardation GH treatment of, 311 maternal factors in, 284t Iodide, -sodium symporter defects, 209–210 Iodine clearance, in thyroid gland, 229f deficiency syndromes of environmental, 230 T3/T4 in, 240 types of, 228t distribution space, by age, 234f metabolism of, in infants/children, 233, 234f, 234t placental, thyroid metabolism and, 199–200, 199f radioactive, in Graves disease, 242–243 transport defect, 208t Iodothyronine deiodinase, tissues enzymes in, 232, 232t monodeiodinase

Iodothyronine (Continued) deficiency of, 211 in thyroid metabolism, 200 enzymes in, 229f sulfonated, 203, 203f Iodotyrosine, deiodinase genes for, 211 prevalence of, 208t types of, 211 Ionizing radiation, abdominal, 409 IPEX, 778 Irradiation, of brain/hypothalamus, 291 Islet cells adenoma hyperinsulinism, 182 antibody, 384t enteropancreatic, tumors, characteristics of, 519–520 Islet transplantation, pancreas and, 414–415 Isolated growth hormone deficiency type 1A (IGHD 1A), 19

J Jak kinase binding motif, 44f Juvenile hyperthyroidism activating TSH receptor mutations in, 244– 245 autonomous nodular hyperthyroidism, 244 G-protein mutations in, 245 Grave’s disease, 242–244 TSH-dependent hyperthyroidism, 244 Juvenile hypothyroidism, acquired goitrogenic agent exposure in, 235 growth failure in, 238f Hashimoto thyroiditis in, 236–237, 237f hypothalamic-pituitary hypothyroidism in, 237–238 management of, 238–239, 238f miscellaneous causes of, 238 thyroid dysgenesis in, 235–236 thyroid dyshormonogenesis in, 236

K Kallmann syndrome, 146 Karyotype 45X, 6f for ambiguous genitalia, 679 in Turner syndrome, 618 KATP, hyperinsulinism channel in infants, 426–427 recessive KATPHI, 177f, 178 dominant, 179–170 focal, 178–179, 179f Kennedy’s disease, 146 Ketone body synthesis, 187f Ketonemia, 397 Ketonuria, 397 Ketotic hypoglycemia, 429–430 in infant/children, 175 Kidney deformity, in Turner syndrome, 638f vasopressin action in, 342–344, 343f–344f

L L281, 13 Labioscrotal swellings, 132 abnormal, 142f asymmetric, 149f symmetric, 148f Laboratory evaluation, of adrenal gland function, 460–464, 460t–461t, 463t, 464f Laboratory quality assurance, 864

INDEX

Laboratory studies for ambiguous genitalia, 149–151, 150t–151t for DSD, 149–151 for rickets, 724, 724t of uterus, 149–150 Laboratory testing case studies for, 864–866 for MEN2, 248t for pediatric endocrinology general overview, 855–856 specific tests for, 864–866, 865t QA in, 864, 866 Lactation, mineral homeostasis in, 686, 686f Laparoscopic adjustable gastric bending, for pediatric obesity, 818 Late dumping, 437 LBD. See Ligand-binding domain LDL. See Low density lipoproteins Length-for-age percentiles, weight-for-age and, 256f–258f Leprechaunism, 411 Leptin in adipose tissue, 679 central regulation of, 792f deficiency of, 809 in metabolic afferents, 791 receptor, 43, 43t–44t deficiency of, 809–810 in puberty, 679 for skeletal growth, 114 resistance, 795–796 sensitivity, weight loss and, 795–796 therapy, 817 Leukemia, from GHD, 314 Leydig cell, 130f hypoplasia characteristics of, 668 types of, 140 LGR8, 134t LH. See Luteinizing hormone LHGCR, 134t LHX3-knockout mice, 20 Lifestyle modification, in childhood obesity, 813–814 Ligand defective apoB-100, familial, 842 Ligand-binding domain (LBD) of nuclear receptors, 50–51, 50f of VDR, 727, 727f Ligand-induced activation, of transcription, by nuclear receptors, 50t Lipids constituents of, 840t disorders of genetic testing for, 847–848 hypolipidemia, 843–844 routine screening for, 846–847 normal levels of, in children/adolescents, 847t partitioning of, 799–800 Lipoatrophy diabetes, 411 genetic syndromes of, 412t Lipoproteins disorders of, 840t, 843–844 pediatric, 842t, 844t, 850t HDL, 843 LDL, 846f, 851 metabolism of, 839–841, 840f–841f, 840t normal levels of, in children/adolescents, 847t subclasses of, 840t VLDL, 843 Liver cirrhosis of, 642 fasting metabolic systems in, 389t

Liver (Continued) fatty, insulin resistance in, 802–803 glucose metabolism by, 807f phosphorylase deficiency, 434 Low birth weight infants, 702–704 Low density lipoproteins (LDL) cholesterol levels, in children, 846f, 851 new research in, 851 Luminal membrane, 344f Luteal phase ovary, 542–543 Luteinized follicular cyst, precocious puberty caused by, 569f Luteinizing hormone (LH) choriogonadotropin receptor gene, 140 peak from, 540f in puberty, 534f–539f receptor, 28t, 34–35 regulation of, 547–548, 548f Lymphatic obstruction, of Turner syndrome, 622–623, 623f Lymphedema, in Turner syndrome, 623f

M Macrophage-colony-stimulating factor (M-CSF), in osteoclast differentiation, 102f, 103 Madelung deformity, in Turner syndrome, 622, 622f Magnesium (Mg), 84 metabolism disorders, 686 hypermagnesemia, 705–706 hypomagnesemia, 705–706 preparations of, 699t renal excretion of, 81t Magnetic resonance imaging (MRI) of hypothalamic hamartoma, 567f in Turner syndrome, 636f Malabsorption, 286 Malaria, in hypoglycemia, 435 Malignancy, second, recurrence of, 314 Malnutrition, with growth abnormalities, 285–286 Mammary gland, maturation of, 557 MAPK. See Mitogen-activated protein kinase Mass spectrometry, 862–864, 863f–864f Maternal factors in hyperandrogenism, 144 for intrauterine growth retardation, 284t in transient hyperinsulinism, 175–176 M-CSF. See Macrophage-colony-stimulating factor Medullary thyroid carcinoma (MTC). See also Familial medullary thyroid carcinoma causes of, 247, 247t classification of, 246, 246t from germ-line RET mutations, 522 risk groups, management of, 248t Melancortin-4 receptor mutations, 810 Melanin-concentrating hormone as class A receptor, 39 in energy balance, 793 Melanocortin receptors, 28t, 32–33 central neural integration and, 793 Melanocortin-3 receptor, 33 mutation, 810 Melanocortin-4 receptor, 28t, 33 MEN. See Multiple endocrine neoplasia Men. See also Fetus androgen action disorders in, 669–671, 669f androgen synthesis disorders in, 668–669 puberty in delayed/precocious, 677t, 679, 679t disorders of, 677–678, 677f sex development disorders in, 664–667, 665f, 666t

881

MEN1. See Multiple endocrine neoplasia 1 MEN2. See Multiple endocrine neoplasia 2 Menarche pubertal milestone attainment in, 562t in rhesus monkey, 537f weight at, 576f Menstrual cycle abnormalities in, 564f estradiol in, 541–542 failure of, 565 follicular phase of, 540–543, 541f–542f GnRH in, 540f in hypogonadism, 575–576 interval between, 563f luteal phase of, 543 mature, normal, 564–565 normal, 539–540 sex steroids in, 540f steroidogenesis in, 541f Mental retardation disorders, pleiotropic obesity and, 810–811 Mesenchymal sites of PTH-rP, 85t stem cell, 102f Messenger RNA (mRNA) analysis of, 5–8, 7f detection of, reverse transcriptase for, 7 in newborns, levels of, 174f splice variants, 7f VDR, 94f Metabolic afferents insulin in, 791 leptin in, 791 Metabolic changes, in cachexia, 822t Metabolic disorders, causing adrenal insufficiency, 484–486 Metabolic markers, fatty acid oxidation disorders with, 189t Metabolic syndrome, in obesity, 802, 802f Metformin, 815t, 816 for hyperandrogenism, 592 Metyrapone test, 464 Microassays, 16–17, 16f Microconversions, for CAH deficiency, 474, 474t Micropenis, 182f Mineral homeostasis in adolescence, 689–690 in children, 689–690 in fetus, 686–688, 687f genes involved in, 75t–77t bone metabolism and, 75t–77t genetic causes of, 693t–694t in infants, 688–689, 688f disorders of, 690–705 in lactation, 686, 686f in low birth weight infant, 702–704 in mouse models, 687–688 in neonates, 688–689, 688f disorders of, 690–705 in pregnancy, 686, 686f PTH effects on, 86 Mineralocorticoids excess of, 11b-Hydroxysteroid dehydrogenase-2-apparent, lesions in, 481 mean concentration of, 461t receptors of, 54–55 replacement, in glucocorticoid therapy, 497 secretion of, 457 Missense mutations, for CAH deficiency, 474–475 MIT. See Monoiodotyrosine Mitochondrial cholesterol uptake, 450 Mitochondrial diabetes, 407

882

INDEX

Mitochondrial disorders, 490 Mitochondrial matrix enzymes, defects of, 187–188 Mitogen-activated protein kinase (MAPK), 17 MODY syndromes, 406–407, 406t Molecular cytogenetics, 17–18 Molecular defects of GHRH gene, 290 of GHRH receptor, 290 Molecular pathogenesis, of androgen insensitivity syndromes, 671–672, 672f Molecular tools, basic, 1–8, 3f–7f 9p monosomy, 137 Monogenetic disorders mitochondrial diabetes, 407 of negative feedback pathway, 809–810 Monoiodotyrosine (MIT), 230f Morbidity of cardiovascular disease, 839 of diabetes mellitus, 375 Mosaicism, in Turner syndrome, 616 Mother, bone resorption/formation in, 107t Mouse models dwarf, 20 mineral homeostasis in, 687–688 MRI. See Magnetic resonance imaging MS/MS method, 863f–864f MTC. See Medullary thyroid carcinoma M¸llerian ducts characteristics of, 130f, 132–135 in Leydig cell hypoplasia, 668 persistent syndrome, 146, 673 M¸llerian inhibitory hormone (AMH), 134t characteristics of, 135 disorders of, 672–673 Multiple endocrine neoplasia (MEN), 18 characteristics of, 512 classification of, 520t future developments of, 526 genetics of, 517–518 pediatrics and, 526 types of, 715t Multiple endocrine neoplasia 1 (MEN1) age-related onset of, 519f characteristics of, 518 clinical presentation of, 518–519, 521t genetic testing of, 521 mutations in, 519f, 717f pathogenesis of, 518, 518f pituitary tumors with, 520 screening for, 520–521 Multiple endocrine neoplasia 2 (MEN2) B, 523–524, 524f characteristics of, 521 clinical presentation of, 523–525, 524f genetic testing of, 524–525 germ-line RET proto-oncogene mutations causing, 519f, 521–523, 522t glandular diseases in, 526t laboratory tests for, 248t mutated codons of, 247t, 248 pheochromocytomas in, 248 MURCs syndrome, 128t Murk-Jansen chondrodysplasia, 700 Musculoskeletal system Ca in, 79 defects, from GH insensitivity, 297t Mutations. See also Germ-line mutations; Rearranged during transfection ALS, growth abnormalities from, 296–297 androgen, 146, 672f aquaporin-2, 361–362 Arg137His V2 vasopressin receptor, 31 cholesterol biosynthesis in, 756, 757f, 758 detection of

Mutations (Continued) ASOH for, 13–14, 13f–14f DNA sequencing in, 9–11, 10f electrophosphoresis in, 9–10, 10f methods of, 8–9, 9f SSCP for, 11–14, 12f–14f for diabetes mellitus type 2, 406t in DSD, 134t epi-, 284 FOXL2, 134t GNAS-1, 41–42 G-protein, 245 in high bone mass, 750–751 hypoplasia, 228t in insulin gene, 20 in MEN1, 519f, 717f missense, for CAH deficiency, 474–475 of nuclear receptors, 51t in osteogenesis imperfecta, 746, 746t in papillary-follicular carcinoma, 246 point, 19–21, 474, 474f POMC splicing, 810 RMRP, 99 StAR, 134t, 141 TrkB, 810–811 in TSH receptor, 228t, 244–245 Myoblasts, 97, 98f

N Nafarelin, 535f Natriuretic peptide system, 346–347, 346f Nausea, vasopressin secretion and, 340–341 NDI. See Nephrogenic diabetes insipidus Negative feedback pathway in energy balance, 795 in hyperinsulinemia, 796f monogenetic disorders of, 809–810 Neonates blood glucose concentration in, 172, 172t BMC in, 691f bone formation in, 107t diabetes in, 412–414, 413t endocrine disorders in, with hyperinsulinism, 176–183, 178f–182f, 178t epigenetics of, 804 fatty acid oxidation disorders in, 189 fetal life transition in, 166 GHD testing in, 304 Graves disease in, 217–219, 218f, 218t hypercalcemia in, 697–702, 699t, 702f hyperinsulinism in, endocrine disorders with, 176–183, 178f–182f, 178t hyperparathyroidism, 40, 700 hypocalcemia, 690–692, 691t, 696–697 hypoglycemia in, 168–169, 171–172, 171t, 172f, 190t–191t transient, 173–175, 174f, 174t mineral homeostasis in, 688–689, 688f disorders of, 690–705 neuroendocrine unit in, 534 whole-body bone mineral content in, 691f Neoplasms, with central diabetes insipidus, 357 Nephrogenic diabetes insipidus (NDI), 33 causes of, 360–362, 361f treatment of, 362 Nervous system autonomic, in hypoglycemia, 173t central maturation of, 558–559 thyroid hormone actions in, 205 in development, 233t tumors of, 314 vasopressin and, 355t sympathetic, energy expenditure and, 794

Nesidioblastosis, 178 Neuroendocrine unit in fetus, 532 in neonates, 534 regulation in, of energy balance, 789–796, 790f, 792f Neuroendocrine-ovarian axis maturation of, 532–543, 533f–542f, 539t in normal puberty, 531, 531f regulation of, 543–548, 544f–546f, 548f Neuropsychological features, of Turner syndrome, 643–645 Neurosecretory dysfunction, GH-, 293 Neurosurgical intervention, for central diabetes insipidus, 356–357 Newborns glucose plasma levels in, 174f 170HP values in, 471f mRNA levels in, 174f screening of, for congenital hypothyroidism, 206–207 NFKB, 103 Niacin, 851 Non germ cell tumors characteristics of, 675–676 risk of, 675t Nonalcoholic fatty liver disease, insulin resistance and, 802–803 Non-follicular tumors, 235t Non-osmotic regulation of thirst, 340–341, 341f of vasopressin, 340–341, 341f Nonpancreatic tumor hypoglycemia, 436–437 Non-thyroidal illness, 228t thyroid function abnormalities in, 239t Noonan syndrome diagnosis of, 284–285 Turner syndrome v., 614 Norepinephrine, in energy balance, 793 Normoestrogenic menstrual disturbances dysfunctional uterine bleeding, 584–585, 584t hypothalamic anovulation, 582–584 perimenstrual syndromes, 585 Northern blotting, 5–6 NPH-based conventional treatment regimens, 399 Nuclear receptors, 48–51, 49f–50f, 51t LBD of, 50–51, 50f mutations of, 51t phylogenetic tree of, 49f subfamily 0, 55 subfamily 1, 52–53 subfamily 2, 53 subfamily 3, 53–55 thyroid hormone binding to, 232–233, 233f Nutrition deprivation, 723t mal-, with growth abnormalities, 285–286 over/under, causing amenorrhea, 583 Pediatric, Surveillance System, 799 therapy, for diabetes mellitus, 400–401

O Obesity. See also Pediatric obesity body mass-index-for-age percentiles for, 797f–798f Ca and, 806, 808 cortisol and, 804–805 definition of, 796 diabetes mellitus and, 812t dietary fat in, carbohydrate v., 805–806 drugs and, 808 endocrine disorders with, 808–809, 809t

INDEX

Obesity (Continued) energy excess in, 796–799, 796f–799f environmental factors in, 804–808, 807f fetal programming and, 804 fiber and, 806 fructose and, 806 genetic factors of, 804 GH secretion and, 803 glycemic index and, 806 hypothalamic, 811 infectious causes in, 808 insulin dynamic disorders in, 811–812 metabolic syndrome in, 802, 802f neonatal programming and, 804 pleiotropic, 810–811 in Prader-Willi syndrome, 810 predictive factors for, 799 prevalence of, 796–797 global, 797–798 racial/ethnic considerations in, 798–799 risk of, 799 factors associated with, 804–808, 807f sleep deprivation and, 805 stress and, 804–805 television watching and, 805 trace minerals in, 808 vascular changes in, 801–802 Octreotide, 815t, 816–817 17-OHP. See 17-Hydroxyprogesterone Olfactory system, at puberty, 678 Oligomenorrhea, 579f Oligonucleotide primers, 5f OMIM. See Online Mendelian Inheritance in Man Oncogenic hypercalcemia, 718 Online Mendelian Inheritance in Man (OMIM), 23 Orchidism crypto-, 147, 147t, 673 macro-, 676 Orexigenics, 793 Orexin receptor as class A receptor, 38 in energy balance, 793 Organ failure, hypoglycemia in, 438 Organic osmolytes, in hyponatremia, 354f Organification defects, 208t, 210 Orlistat, 815t, 816 Osmotic fluid regulation physiology of, 336 of thirst, 339–340, 339f–340f of vasopressin secretion, 339–340, 339f–341f Osmotic sensory, effector pathways and, 336–344, 336f–344f Ossification, 751–752 Osteoblastogenesis, adipocytes in, 97, 98f Osteoblasts, 97, 98f formation of, calcitriol in, 101 maturation of, 101 Osteocalcin (BGLAP), 98 Osteochondrodysplasia Blomstrand, 696 cholesterol in, 756 classification of, 282, 282t anatomic, 752f COL1A1 in, 755 features of, 281–282 FGF3 in, 752, 753t, 755f FGF23 in, 755 GH treatment of, 312 mutated genes in, 753t–754t PTH in, 755–756 Osteoclast differentiation of, 102–103, 102f–104f maturation of, 104 pharmacologic agents targeting, 749f

Osteoclastogenesis, 103f Osteocytes, 99 Osteogenesis imperfecta classification of, 742–743, 744t–745t mutations in, 746, 746t treatment of, 746–747 Osteolyses, idiopathic, 282t Osteomalacia/rickets, tumor-induced, 731, 732f Osteonectin, 106–107 Osteopetrosis, in infants, 747–748 Osteoporosis, low bone mass in, 740 Osteoprotegerin, 103f Osteosclerosis, 748t Otitis, in Turner syndrome, 630–631, 630t Ovarian follicles, 533f Ovarian function tests, 560t Ovaries. See also Neuroendocrine-ovarian axis diagram of, 533f differentiation of, 130f, 131 fetal development of, 532–534, 533f follicular phase of, 540–543, 541f–542f genetic abnormalities in, 138–139 luteal phase of, 543 secretion of, 547–548, 548f SF1/NR5A1 gene in, 140 steroidogenesis in, in menstrual cycle, 541f steroidogenic pathways in, 139f testosterone secretion in, 552 Overgrowth differential diagnosis of, 317, 317t in fetus, 317–318, 317t Ovotesticular disorder DSD, 667 of sexual differentiation, true hermaphroditism, 138 Ovulation, diagram of, 541f P450 oxidoreductase, 451–452, 451f deficiency of, characteristics of, 478–479 Oxytocin biochemistry of, 336–339, 336f–338f peptide products of, 337f receptors, 342f structure of, 336f

P P450aro, characteristics of, 453 P450c11, isozymes of, lesions in, 479–481 P450c11AS, 452 P450c11b, 452 P450c17 characteristics of, 451 electron transport to, 451–452, 451f P450c21 causing CAH deficiency, 472–475, 473f–474f, 474t characteristics of, 452 genetic map of, 473f mapping of, 473–474 structure/function interferences from, 475 P450scc characteristics of, 448–449 electron transport to, 449, 450f–451f Paget disease, 734 Pancreas beta cells in diagram of, 410f, 426f in insulin secretion, 177f, 410f enteropancreatic islet cell tumors, 519–520 exocrine, disorders of, 409 islet transplantation and, 414–415

883

Pancreatectomy, 409 Papillary-follicular carcinoma incidence of, 246 mutations in, 246 Parathyroid hormone (PTH) in Ca homeostasis, 78 as class B receptor, 39–40 in mineral homeostasis, 86 in osteochondrodysplasia, 755–756 regulating calcium, 87 in fetus, 687–688 Parathyroid-hormone-related protein (PTH-rP), 85t, 87–88 in bone formation, 100–101 receptors related to, 85–89, 85t, 88f types of, 88–89 Parents talking with, about ambiguous genitalia, 128–129 target height of, 262–263 Pars distalis, 264 Pars intermedia, 264 Parvocellular neurons, role of, in vasopressin synthesis, 339 PCR. See Polymerase chain reaction Pediatric endocrine disease history of, 249–250 therapy for, recombinant DNA technology and, 22–23 Pediatric endocrinology. See also Endocrinology case studies for, 864–866 diagnosis of, 864–866, 865t laboratory testing for, 855–856 QA in, 864, 866 testing for, 864–866, 865t Pediatric endocrinopathy, molecular basis of, 18–21, 19f Pediatric Nutrition Surveillance System, 799 Pediatric obesity bariatric surgery for, 817–819 co-morbidities in, 803–804 disorders in, classification, 809f energy inadequacy in, 819–825, 820t, 822t, 824f energy intake in, 814 general overview, 797 GH treatment in, 817 laparoscopic adjustable gastric bending in, 818 lifestyle modification in, 813–814 metabolic impact of, 799–804, 802f pharmacotherapy for, 814–817, 815t future of, 817 Roux-en-Y gastric bypass in, 818–819 workup of, 812–813, 812t Pediatric thyroid disease advances in, 227–228 types of, 228t Pelvic ultrasound in ambiguous genitalia, 149–150 studies, in Turner syndrome, 634f Pendred syndrome, 208t incidence of, 210 in infants, 211 Penis micro-, 182f small, disorders associated with, 147, 147t Peptide hormones defects in, 19–21 in diabetes mellitus, 385–386, 385f metabolism of, 550–551 synthesis of, 21f, 550 types of, 549–550, 550f Peptide YY33-36, 790–791

884

INDEX

Perimenstrual syndromes, 585 Perinatal glucose homeostasis, 166 Perinatal stress-induced hyperinsulinism, 176 Peripheral quantitative computed tomography, non-dominant proximal radius, 119t–120t Peroxidase system defects, 208t, 210 Peroxisome biogenesis disorders, 490 PHA. See Pseudohypoaldosteronism Pheochromocytomas characteristics of, 512–513 diagnosis of, 514–515 genetic differential, 515 evaluation of, 517 genetic causes of, 513t imaging studies in, 516t indications for, 520t management of, 516–517, 516t in MEN2, 248 screening of, 525 surgical treatment of, 516–517 tumor localization of, 515–516, 516t PHEX, in hypophosphatemic rickets, 729, 729f Phosphate administration of, 711 homeostasis, 78f metabolism disorders, 686 preparations of, 699t renal excretion of, 81t renal tubular reabsorption of, 83 in sodium transport, 82–83 Phosphatonins, 83–84 Phosphoenolpyruvate carboxykinase deficiency, 186 Phosphopenic rickets, 728–732, 728t, 729f, 732f Phosphorylase kinase deficiency (GSD type IX), 185 Phylogenetic tree, of nuclear receptors, 49f Physical activity intervention, in childhood obesity, 813–814 Physical examination for DSD diagnosis, 148–149 for GHD, 304t for increased bone mass, 705 Phytosterolemia, 842–843 Pilosebaceous unit, maturation of, 557–558, 558f Pituitary cell lineage, 267f Pituitary gigantism, 320 Pituitary gland ACTH action in, 455–457, 455f circulatory system in, 268, 268f congenital absence of, 292 development of, 264–265 evaluation of, 302f formation of, 263 hypoplasia of, 292 size of, 265, 267 Pituitary growth hormone deficiency of growth abnormalities from, 291–292 multiple, management of, 308 T3 resistance to, hyperthyroidism with, 244 Pituitary thyrotroph cell, in TSH synthesis, 231f Pituitary tumors, 292–293 with hyperthyroidism, 244 with MEN1, 520 Pituitary-specific transcription factors, for development, 265, 267f, 267t Placenta abnormalities of, 284t aromatase deficiency of, 144 iodine in, thyroid metabolism and, 199–200, 199f

Plasma concentrations, of cortisol, 460–461, 460t–461t renin, 461 volume, decreased effective, in hyponatremia, 352–353 Plasma steroids catabolism of, 459 circulating types of, 458–459 nomenclature of, 458 structure of, 458, 458f Pleiotropic obesity, mental retardation disorders and, 810–811 Point mutations, 19–21 for CAH deficiency, 474, 474f Polycystic ovarian syndrome causing hyperandrogenism, 585–587, 586t, 587f insulin resistance and, 803 manifestations of, 587f Polydipsia, determination of, 348–350, 349f Polymerase chain reaction (PCR), 3–5, 5f cross-contamination in, 5 modifications to, 11 qRT-, 7–8, 17 quantification products for, 8 RT-, 7–8 Polymorphic markers, 15 Polyuria, 348–350, 349f POMC differentiation of, 793 disorders, causing adrenal insufficiency, 486–487 gene, 455, 455f peptides, 462–463 splicing mutation, 810 POR, 134t Positional cloning of endocrine genes, 16 steps of, 15, 15f Positional genetics, in endocrinology, 14–16, 15f–16f Post-genomic era, 23 Postnatal life 2 hour-7 days of, hormone levels in, 150t CAH in, 478 statural overgrowth in, tall stature and, 318 PPARg, 52–53 Prader-Willi syndrome diagnosis of, 285 GH treatment of, 311–312 obesity in, 810 Pregnancy calciotropic changes during, 686f cardiac care and, 639 FSH receptors during, 37 HCG receptors during, 36–37 mineral homeostasis in, 686, 686f thyroid function during, fetal, 200–201, 200f–201f TSH receptors during, 36 Pregnenolone in androgen synthesis, 142f normal levels of, 151t regulation of, 548f Premenstrual syndrome, 585 Probes cRNA, 7f TaqMan, 8, 9f Progeria, 285 Progesterone levels. See also 17-Hydroxyprogesterone in androgen synthesis, 142f estrogen-, therapy, 642 normal, 151t in postnatal life, 150t

Progestin monotherapy, for hyperandrogenism, 592 Prohormone convertase-1 deficiency, 810 Prolactin secretion of, 551 in steroidogenesis, 548 structure of, 551 PROP1, 20 Proteins. See also Specific protein i.e. G-protein for binding, 228t, 273f, 277–279, 278f in bones, 98f in hormones, 33–37, 36f in plasma, 247, 247f Proto-oncogene codes for, RET, for plasma protein receptors, 247, 247f germ-line RET mutations of, for MEN2, 519f, 521–523, 522t Pseudohermaphrodite, 128 Pseudohypoaldosteronism (PHA), 54 causes of, 492 Pseudohypoparathyroidism, growth abnormalities from, 288 Pseudotumor cerebri, 314 Pst I, 4f Psychiatric disorders, in Turner syndrome, 645 Psychological aspects counseling, for DSD individuals, 155 neuro-, in Turner syndrome, 643–645 Psychosocial syndromes with diabetes mellitus, 403 dwarfism, 293 PTH. See Parathyroid hormone PTH-rP. See Parathyroid-hormone-related protein Ptosis, in Turner syndrome, 624f Pubarche, 563 Pubertal development constitutional delay of, 563 disorders in, hormone levels in, 680–681 normal, 562 premature, 563 Pubertal milestone attainment, 562t Puberty abnormal, 565 adolescent development of, 536–539, 536f–539f, 539t autoamplification processes in, 539t delayed, 319 classification/causes of, 677t, 679, 679t constitutional, gonadotropin deficiency v., 581t testes in, 677t, 679 disorders of, in men, 677–678, 677f DSD in, 565 early development of, in GH treatment, 649–651 estradiol in, 538, 538f FSH in, 534f–539f future research for, 592–593 in GH insensitivity, 297t gonadotrophin rise at, 668–669 GPR54 in, 677t history of, 531 leptin in, 679 LH in, 534f–539f normal, 531, 531f olfactory system at, 678 onset of, 543–546, 544f–546f precocious, 319 causes of, 565, 566t, 567f, 569f complete of, 565–567 differential diagnosis of, 570–571, 571t

INDEX

Puberty (Continued) GnRH agonist therapy for, 571t gonadotrophin-dependent, 677t gonadotrophin-interdependent, 677t incomplete, 567–570 management of, 571–572 in rhesus monkey, 537f testes and, general characteristics of, 676–677 testosterone levels at, 676 Pubic hair development of, 561, 561f pubertal milestone attainment in, 562t Pulmonary disease, growth abnormalities from, 287 Pyridinium, of collagen, 106f Pyridinoline, urine, 107t Pyrosequencing, 11 Pyruvate carboxylase deficiency, 186

Q QA. See Quality assurance qRT-PCR. See Quantitative real-time reverse transcription PCR Quality assurance (QA), 864, 866 Quality control, 864 Quantitative real-time reverse transcription PCR (qRT-PCR), 7–8, 17

R Rabson-Mendenhall syndrome, 411 Racial considerations, for obesity, 798–799 Radiation exposure ionizing, abdominal, 409 in thyroid neoplasia, 245t Radioactive iodine, for Graves’ disease, in children, 242–243 Radioimmunoassay (RIA) basic, 860f of hormones, 860–864, 860f sandwich system in, two antibodies in, 861f RAMP, 28–29 RANKL, in osteoclast differentiation, 102–103, 102f–104f Rathke’s pouch, 20 Real-time reverse transcription (RT-PCR), 7–8 Rearranged during transfection (RET) mutations activity of, 522f analysis of, 524–525 causing MEN2, 521–523, 522t domains of, schematic depiction of, 717f testing for, 525 proto-oncogene codes for, for plasma protein receptors, 247, 247f Receptor tyrosine kinase (RTK), 44–45, 44f, 45t Receptors. See also Specific hormone i.e. Adrenocorticotropic hormone calcium sensing, 28t, 81–82 in fetus, 166–168, 167t of hormones activation of, 27 types of, 27t–28t nuclear, 48–51, 49f–50f, 51t subfamilies, 52–55 selectivity of, in steroid hormones, 555–556 vitamin D, 94–96, 94f, 96f Receptor-stimulating antibodies, TSH, 230 Recombinant DNA technology, pediatric endocrine disease therapy and, 22–23

5a-reductase characteristics of, 453 deficiency of, 143 causes of, 669 Reference curves, for bone area, 118f Reference ranges, for hormonal assay methods, 856, 858 Renal abnormalities, in Turner syndrome, 639–640 Renal cascade, of vasopressin function, 342–344, 343f–344f Renal disease, for growth abnormalities, 286–287 Renal excretion by age, 234f of Ca, 81t of Mg, 81t of phosphate, 81t Renal failure, GH treatment of, 311 Renal free water clearance decreased, 350–351 drugs impairing, 352t thyroid hormone in, 351 Renal osteodystrophy, 734–735 pathophysiology of, 734f Renal tubular reabsorption, 81t of phosphate, 83 Renin-angiotensin system, 457 local, 345–346 Renin-angiotensin-aldosterone system, basic understanding of, 344 Reproductive system development of, 129–132, 130f female in adolescent, 536–539, 536f–539f, 539t in adults, 539–543, 539f–542f fetal development of, 532–534, 533f infant development of, 534–536, 534f–537f Restriction fragment length polymorphism (RFLP), 2–3, 4f RET. See Rearranged during transfection Retinoid X receptor, 273f Reverse transcriptase, for mRNA detection, 7 RFLP. See Restriction fragment length polymorphism Rhesus monkey, prepubertal, 537f RIA. See Radioimmunoassay Ribonuclease protection assay (RPA), 6, 7f Ribose nucleic acid (RNA), analysis of, 5–8, 7f Rickets calcipenic, 722–728, 723t–724t, 726f–727f FGF23 in, 731, 732f growth abnormalities from, 288 in infants, 702–704 laboratory studies for, 724, 724t phosphopenic, 728–732, 728t, 729f, 732f tumor-induced osteomalacia and, 731, 732f vitamin D-resistant, growth abnormalities from, 288 Risk of cancer, 315, 315t of celiac disease, 641 of congenital heart disease, in Turner syndrome, 635t of coronary artery disease, in children, 845t, 846f of diabetes mellitus, 377t type 1, 381t, 384t of germ cell tumors, 675t of GHD, 302f of gonadal tumors, in DSD, 675t of IGF deficiency, 302f of IGFD, 302f

885

Risk (Continued) of low bone mass, 736–738 of MTC, 248t of non germ cell tumors, 675t of obesity, 799 factors associated with, 804–808, 807f RMRP mutations, 99 RNA. See Ribose nucleic acid Roux-en-Y gastric bypass, in pediatric obesity, 818–819 RPA. See Ribonuclease protection assay RTK. See Receptor tyrosine kinase RT-PCR. See Real-time reverse transcription Russell-Silver syndrome, 284

S S1 nuclease protection assay, 6 SADDAN, 755f SAGE. See Serial analysis of gene expression SAID. See Vasopressin V2 receptor Salicylate intoxication, hypoglycemia from, 438 Salt cerebral, wasting, 355 wasting, in CAH, 471 Sandwich system, RIA in, two antibodies in, 861f Sanger method. See Dideoxy (Sanger) method Satiety, 790–791 Saturated fat, diet therapy for, 848–851, 848t– 849t SCFE. See Slipped capital femoral epiphysis SCHAD. See Short-chain L-3-hydroxyacyl-CoA dehydrogenase School intervention, in childhood obesity, 813–814 Scoliosis, in Turner syndrome, 622 Seckel syndrome, 284 Secondary growth abnormalities, types of, 285–305, 289f–290f, 289t, 294f–296f, 294t, 297t, 298f–299f, 300t, 302f, 304t– 305t Secretagogue receptors, 37 Self-antigens, tolerance and, 771, 771f Self-monitoring, of glucose, 399–400 Sella turcica, 267 Sequence tag sites (STS), 15 Sequence-specific oligonucleotide (SSO), 14f Serial analysis of gene expression (SAGE), 17 Serial bone age assessment, role of, 309 Serotonin, in energy balance, 793 Sertoli cell, 130f, 132 Sex chromosomes abnormalities in, with growth retardation, 282–283 anomalies in, 664–666, 665f DSD, 128, 128t Sex determination, 129, 133 Sex development disorders classification of, 127–128, 128t in testes determination, 664–667, 665f, 666t Sex differentiation androgen receptors in, 669, 669f genetic control in, 664, 664f testosterone during, 663f Sex hormones in cardiovascular system, 559 lifetime cycles of, 532f in menstrual cycle, 540f regulation of, 545f target organs of, maturation of, 556–559, 557f–558f testes and, 534

886

INDEX

Sex of rearing, 152–153 Sex steroids adolescent rise in, 280–281 age dependency and, 300 mean concentration of, in infants/children, 460t testes and, 534 Sexual development, 152 Sexual differentiation. See also Disorders of sexual differentiation for androgen synthesis, 128t for gonadal dysgenesis, 135–136, 136f ovotesticular disorder of, true hermaphroditism, 138 Sexual maturation in adolescence, 561–562, 561f, 562t in children, 560t, 561 normal in fetus, 559–561, 560t in neonate, 559–561, 560t Sexually infantile girls, bone age in, with normal FSH levels, 573t SF1/NR5A1 gene loss-of-function mutations in, 134t in ovarian development, 140 Short stature diagnosis of, 282 familial, 305 genetic causes of, 285 GH treatment of, 310–313 idiopathic classification of, 281t features of, 305 GH treatment of, 312–313 Short-chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD), 181–182 hyperinsulinism, 428 SHOX gene, 613, 613f haploinsufficiency of, 621 Sibutramine, 814, 815t Side chain cleavage cytochrome p450 enzyme, 141 Side effects, of GH treatment miscellaneous, 314–315 specific, 313–314 Signaling defects, GHR, 295–296 Single-strand conformation polymorphism (SSCP), 11–12, 12f Skeletal growth, disturbances of, in Turner syndrome, 619–622, 620t, 621f Skeletal growth failure, in Turner syndrome, 624–625, 624f–625f Skeletal maturation androgens in, 281 disorganized development during, 282t estrogens in, 281 potential of, 259 studies in, 259–260 Skeleton growth factors for, 111–114 hormones for, 111–114 metabolism disorders of, 97–120, 98f, 100f, 102f–106f, 107t–109t, 111f, 115t–117t, 118f, 119t–120t tubular/axial defects in, 282t Skin, in Turner syndrome, 623–624 Sleep deprivation, obesity and, 805 Slipped capital femoral epiphysis (SCFE), GHD and, 314 Small penis, disorders associated with, 147, 147t Smith-Lemli-Opitz syndrome causes of, 490 DHCR7 in, 140

Snell and Jackson dwarf mice, 20 Sodium chloride, in hyponatremia, 351 Sodium/iodide symporter defects, 209–210 Solitary nodule, in thyroid neoplasia, 249, 249t Southern blotting, 1–2, 3f, 6f SOX9, 134t, 136 Spectrum orange, 19f Splice variants IGF-1, 274 mRNA, 7f SRY-gene in DSD, 134t in testicular differentiation, 130 in Turner syndrome, 6f, 283 SSCP. See Single-strand conformation polymorphism SSO. See Sequence-specific oligonucleotide SSTR5. See GPCR somatostatin receptor StAR. See Steroidogenic acute regulatory protein Starvation response cachexia v., 819 diagram of, 792f regulation of, 795 STAT motif, 44f Stature-for-age percentiles, weight-for-age and, 260f–261f Steroid hormones metabolism of, 553–555, 554f–555f origins of, 552 pathways of, 551–552, 552f receptor selectivity in, 555–556 relationship among, 553f Steroidal cell autoantibodies, 782–783 Steroidogenesis adrenal, fetal, 454–455 enzymes of human genes for, 446t types of, 448–454 genetic lesions in, 465–481, 466t, 467f, 470f–471f in menstrual cycle, 541f pathways of in androgen synthesis disorders, 139f for fetal androgen biosynthesis, 142f testicular, 665f prolactin in, 548 regulation of, 455–458, 455f, 458f testicular, fetal, 664, 664f Steroidogenic acute regulatory protein (StAR) in congenital lipoid adrenal hyperplasia, 141 in mitochondrial cholesterol uptake, 450 mutations of, 134t, 141 Steroids. See also Plasma steroids; Sex steroids in adrenal gland 60-minute ACTH test of, 463t hormone synthesis in, 449f anabolic, 642 catabolism of, 459 fluid concentrations of, normal, 542f hormone synthesis of enzymes for, 448–454 steps of, 448 long-term therapy with, for adrenal insufficiency, 487 response, to ACTH, 859t secretory rates of, 463 sulfotransferase, 453 therapeutic, potency of, 476t types of, 458–459 urinary secretion of, 461–462

Stress appetite and, 804–805 cortisol and, 805–806 doses, of glucocorticoid therapy, 496–497 in hyperinsulinism, 176 obesity and, 804–805 STS. See Sequence tag sites Subacute thyroiditis, 241 Substrates, in fetus, 166–168, 167t Sulfatase, 453 Supplements, cholesterol reducing, 851 Suppurative thyroiditis, 241 Sur12/2, 178f Surgery bariatric, 817–819 diabetes mellitus during, 404–405 for DSD, 153 of juvenile hyperthyroidism, 242–243 for pheochromocytomas, 516–517 plastic, for deformity, 648–649 Symmetric genital ambiguity, 148f Sympathetic nervous system, energy expenditure and, 794 Systemic disorders, hypoglycemia with, 191

T T cells activation of, 772f central, tolerance of, 771–772 ignorance, 772 receptors, 381f tolerance of, 771–773, 772f peripheral, 772–773, 772f T3. See Triiodothyronine T4. See Thyroxine Tall stature constitutional/syndromic, management of, 319–320 differential diagnosis of, 317–319, 317t familial, diagnosis of, 319 postnatal statural overgrowth and, 318 TaqMan probes, 8, 9f Target organs, sex hormones, maturation of, 556–559, 557f–558f TBG deficiency of complete, 219–220, 220t–221t partial, 220 excess, 220, 220t normal, 216t Television, watching of, in obesity, 805 Telopeptides of collagen, 106f N-, 107t N-terminal extracellular domain for GPCR, 29f, 31 for TSH receptor, 36, 36f Testes absent, 680 cryptorchidism in causes of, 673 characteristics of, 147, 147t descent of, 663–664 development of, importance of, 662 disorders of, 664–667, 665f, 666t, 673–674 function of, 679 puberty and delayed, 677t, 679, 679t general characteristics of, 676–677 sex steroid/hormones in, 534 tumors in, 674–676, 674t–675t imaging of, 680

INDEX

Testes (Continued) undescended, in congenital hypopituitarism, 182f vanishing, 138, 673 46,XX testicular disorder, 138–139 Testicular differentiation, 130–131, 130f 46,XY for, 128t Testicular regression syndrome, 138 Testicular steroidogenesis, fetal, 664, 664f Testicular steroidogenic pathways, 665f Testosterone in anovulation, 591f estradiol-binding globulin, 554f measurement of, direct method of, 863f normal levels of, 151t in ovaries, 552 at puberty, 676 replacement therapy, 154 during sex differentiation, 663f steroidogenic pathways in, 142f testing for, 865–866 Tg. See Thyroglobulin Theca cells, 548f Thelarche, premature, 562–563, 563f Thermogenesis BAT, 205 thyroid hormone effects on, 233t Thiamine-responsive diabetes, 407 Thirst non-osmotic regulation of, 340–341, 341f osmotic regulation of, 339–340, 339f–340f THR. See Thyroid hormone receptors Thyrogastric autoimmunity, 777 Thyroglobulin (Tg) metabolism of, 229–230 synthesis of, 211 Thyroid adaptation, extrauterine, 200f, 203–204 Thyroid dysfunction syndromes future directions for, 249–250 in neonates, with Graves’ disease, 218t in premature infants, 205–206, 205t prevalence of, 205t Thyroid dysgenesis in acquired juvenile hypothyroidism, 235–236 in childhood/adolescence, 228t prevalence of, 205t, 207 Thyroid dyshormonogenesis in acquired juvenile hypothyroidism, 236 features of, 207 TSH resistance of, 207–209 types of, 228t Thyroid gland ectopic, 207 follicular cell in biosynthesis of, 207f synthesis/secretion in, 230f function of abnormalities in, in non-thyroidal illness, 239t by age, 235, 235f in infants, 216t iodine metabolism in, 233, 234f, 234t regulation of, 230–231, 231f test parameters for, in human fetus, 218f iodine clearance in, 229f Thyroid hormone actions of, 204–205, 232–233 in central nervous system, 233t biosynthesis of, 208f steps of, 229, 230f effects of, 233f GH interactions with, 281

Thyroid hormone (Continued) in growth, 233t membrane transporter defects, 212–213 metabolism of deiodination, 231–232 inborn abnormalities of, 208t–209t maturation of, 202–203, 202f–203f new studies of, 198 pathways of, 231–232, 232t placental iodine and, 199–200, 199f nuclear receptors binding to, 232–233, 233f in renal free water clearance, 351 resistance to, 208t disorders of, 212 GRTH, 216, 217f syndromes of, 228t serum concentrations of, T3/T4 production and, 234–235, 235f, 235t thermogenic effects and, 233t transport of, 202–203, 202f–203f disorders of, 219–221, 220t–221t illustration of, 232f uptake of, by age, 234f volume of, by age, 234t Thyroid hormone receptors (THR), 52 defects of, 212 for skeletal growth, 112 Thyroid neoplasia, 228t in children, 245t clinical features of, 249t classification of, 245 miscellaneous, 249 MTC, 246–248, 246t–248t, 247f papillary-follicular carcinoma in, 246 radiation exposure in, 245t solitary nodule in, management of, 249, 249t Thyroid stimulating hormone (TSH) deficiency of, 213 dependent hyperthyroidism, 244 fetal, concentration levels of, 218f hypothalamic-pituitary axis, 231f–232f in juvenile hyperthyroidism, receptor mutations in, 244–245 receptors, 28t, 35–36, 36f mutations of, 228t N-terminal extracellular domain in, 36, 36f during pregnancy, 36 -stimulating antibodies, 230 in thyroid dyshormonogenesis, 207–209 in thyroid follicular cell, 230f in thyroid regulation, 230–231 Thyroid system embryogenesis in, 199 maturation, 200–201, 200f–201f ontogenesis, 198 Thyroiditis Hashimoto in acquired juvenile hypothyroidism, 236–237, 237f antibodies in, 237, 237f diagnosis of, 239 infectious, 228t subacute, 241 suppurative, 241 Thyroid-releasing hormone (TRH) deficiency of, 213 fetal, 201, 201f receptors, 37 Thyrotoxicosis, 218t Thyrotropin-releasing hormone (TRH), 37 for TSH synthesis, 231f

887

Thyroxine (T4) binding of, 232, 233f biosynthesis of, 208f deiodination of, 202f fetal concentrations of, 218f in iodine deficiency syndromes, 240 metabolism of, 229f peripheral, metabolism of, with age, 234t production of, thyroid hormone serum concentrations and, 234–235, 235f, 235t replacement therapy, 216f in thyroid adaptation, 203–204 in thyroid maturation, 202–203, 202f–203f Tolbutamide, in children, with hyperinsulinism, 178f Tolerance autoimmune polyglandular syndrome and, 770 of b cells, 773 of carbohydrate, in Turner syndrome, 642–643 defects in, causing autoimmune diseases, 773–775 impaired glucose, 414 self-antigens and, 771, 771f of T cells, 771–773, 772f Trace minerals, in obesity, 808 Transcription for androgen action, 145 factors, pituitary-specific, 265, 267f homeodomain, 267t ligand-induced activation of, by nuclear receptors, 50t qRT-PCR, 7–8, 17 RT-PCR, 7–8 Transgenic models, 132–135, 134t Transthyretin variants, 220 Trauma brain, growth abnormalities from, 290–291 central diabetes insipidus and, 356 to hypothalamus, 290–291 TRH. See Thyroid-releasing hormone; Thyrotropin-releasing hormone Triiodothyronine (T3) binding actions of, 232–233, 233f biosynthesis of, 208f fetal concentrations of, 218f gene products of, 233t in iodine deficiency syndromes, 240 metabolism of, 229f pituitary, resistance, 244 production of, thyroid hormone serum concentrations and, 234–235, 235f, 235t r, 200f, 202, 202f–203f metabolism of, 229f in thyroid adaptation, 203–204 in thyroid maturation, 202–203, 202f–203f TrkB mutation, 810–811 TSH. See Thyroid stimulating hormone Tubular skeleton, defects of, 282t Tumors adrenal, 489–490 feminizing, 491–492 of brain/hypothalamus, 291 of CNS, 314 enteropancreatic islet cell, 519–520 of follicular epithelium, 235t genes for, 135 gonadal, 674–676, 674t–675t -induced osteomalacia/rickets, 731, 732f non-follicular, 235t of pheochromocytomas, 515–516, 516t

888

INDEX

Tumors (Continued) pituitary, 244, 292–293, 520 testes, 674–676, 674t–675t, 680 virilizing, 491–492 Turner, Henry, 610 Turner syndrome in adults, 651–652 evaluation of, 646, 646t follow-up studies of, 647–649 anatomy of, 447–448 androgens in, 629–630 audiogram in, 630t autoimmune disorders in, 640–641 body image in, 643 carbohydrate tolerance in, 642–643 cardiac care in, 639–643 cardiac imaging in, 636f cardiovascular abnormalities in, 633–638, 634f, 635t, 636f cardiovascular screening in, 638t celiac disease in, 641 chromosomes in germ cell defects in, 631–633, 632f karyotypes of, 611–612 multiple X, 614–615 clinical findings in, 619, 620t congenital heart disease in, risk of, 635t cytogenetic findings in, 616t diagnosis of, 618–619, 619f edema in, 623 embryology of, 446–447, 446f, 446t endocrinologic management of, 649–651 environmental factors in, 617–618 estrogen in, 629–630 etiology of, 615–618 eye/ear/skin in, 623–624 facial deformities in, 623f in fetus, 619f final height in, 628f–629f, 628t FSH in, 632t gastrointestinal disorders in, 641–642 genomic imprinting in, 614 GH in, 626–627 treatment of, 311 gonadal failure in, 631–633, 632t gonadoblastoma in, 633 growth-promoting therapy for, 625–630 hand radiographs of, 621f hearing loss in, 630–631, 630t height velocity in, 624f–626f history of, 445–446 hypertension in, 638 incidence of, 615–618, 646 karyotype in, 618 kidney deformity in, 638f lymphatic obstruction of, 622–623, 623f lymphedema in, 623f Madelung deformity in, 622, 622f management of, 646, 646t mosaicism in, 616 neuropsychological features of, 643–645 Noonan syndrome v., 614 otitis in, 630–631, 630t pathogenesis of, 612–614, 613f pelvic ultrasound studies in, 634f physiologic features of, 620t, 624–625, 624f–625f prenatal diagnosis of, 618 psychiatric disorders in, 645 ptosis in, 624f recommendations for, 36–37 renal abnormalities in, 639–640

Turner syndrome (Continued) scoliosis in, 622 skeletal growth disturbances of, 619–622, 620t, 621f skeletal growth failure in, 624–625, 624f– 625f SRY-gene sequence in, 6f, 283 stigmata in, 621f therapeutic regimens for, 628–629 transition management of, 651 in women, 651 Type I collagen, 105–106, 105f–106f Type I cytokine receptors, 27t, 42, 43t Tyrosine hydroxylase, autoantibodies to, 783

U Ulrich-Turner syndrome, 610 Ultrasound abdominal, of adrenal adenoma, 569f pelvic in ambiguous genitalia, 149–150 in Turner syndrome, 634f Urethral folds, 132 Urine pyridinoline, 107t steroid excretion in, 461–462 tests, for hypoglycemia, 423t Urogenital differentiation, 129–130, 130f Uterus dysfunctional bleeding of, 584–585, 584t laboratory studies of, 149–150 symmetric development of, 148f

V V281L, 13f VACTERL syndrome, 128t Vaginal epithelium, 557f Vaginal septa, transverse, 146 Vagus nerve afferent, 789–790, 790f efferent, 794 Van Wyk-Grumbach, 239, 287, 568–569, 677 Vanishing testes, 138, 673 Vascular changes dyslipidemias and, 845–846, 845t, 846f in obesity, 801–802 Vasopressin action of, 341–342, 342f analogues of, for central diabetes insipidus treatment, 359–360 AVP, 455 biochemistry of, 336–339, 336f–338f cancer and, 355t cells, in hypothalamus, 339f central nervous system and, 355t CNS and, 355t dDAVP, 336, 336f in homeostasis, 336 hyponatremia with abnormal regulation of, 354–356, 355t decreased, 356–362, 361f normal regulation of, 350–353, 352t in kidney, 342–344, 343f–344f metabolism of, 341 increased, central diabetes insipidus with, 358 nausea and, 340–341 non-osmotic regulation of, 340–341, 341f origin of, 337 osmotic regulation of, 339–340, 339f–341f

Vasopressin (Continued) parvocellular neurons role in, 339 peptide products of, 337f pre-, peptide, structure of, 338f receptors, 28t, 31, 33, 341–342, 342f renal cascade of, 342–344, 343f–344f roles of, 363 SAID, 354–355, 355t, 360–361, 361t structure of, 336f Vasopressin V2 receptor (SAID) causes of, 354, 355t causing NDI, 360–361, 361f treatment of, 354–355 VDR. See Vitamin D receptor Very low density lipoproteins (VLDL), disorders of, 843 Viral infections, in diabetes mellitus, 409 Virilizing congenital adrenal hyperplasia, 141–143, 142f Virilizing tumors, 491–492 Vitamin D, 90–94, 91f, 93f deficiency of causing hypocalcemia, 691t in infants, 725–726, 726f in gastrointestinal tract, 90f metabolism of, 91f repletion of, 93 -resistant rickets, growth abnormalities from, 288 Vitamin D receptor (VDR), 94–96, 94f, 96f calcitriol involvement with, 94–96 genes regulating, 96f LBD of, 727, 727f mRNA, 94f Vitamin D3 receptor, 52 VLDL. See Very low density lipoproteins Volume sensor, effector pathways and, 344–347, 346f

W Water deprivation test, 349, 349f metabolism disorders differential diagnosis of, 347–350, 349f types of, 350–353, 352t, 354f Weight of adrenal gland, 447f body by age/gender, percentiles, 256f–263f glucose production v., 172f, 424f -for-age percentiles length-for-age and, 256f–258f stature-for-age and, 260f–261f -for-length percentiles, head circumferencefor-age and, 257f, 259f loss of leptin sensitivity and, 795–796 lifestyle modification for, 813–814 low birth, infants, 702–704 at menarche, 576f Whipple’s triad, 171 Whole-body bone mineral content absorptiometry of, 687f of neonate, 691f Williams syndrome, 634 Williams-Beuren syndrome, 700–701 Wilms’ tumor gene, 135 Wnt signaling pathway 4, 134t, 135 7A, 134t in bone mineralization, 98–99

INDEX Wolff-Chaikoff blockade, 230 Wolffian duct, 131–132 Wolfram syndrome, 407 Wolman disease, 490 Women. See also Female reproductive system adolescent, bone deposition in, 109t, 111f height velocity chart for, 265f mid-follicular phase, blood production rates in, 551t Turner syndrome in, 651 virilized, ambiguous genitalia in, 142f Workup, of childhood obesity, 812–813, 812t WT1, 134t

X

Y

X chromosomes genes, 612–614 monosomy, 617 multiple, 614–615 showing SHOX, 613, 613f small ring, 611–612 X-linked adrenal hypoplasia congenital, 483–484 X-linked congenital diabetes insipidus, 360–361, 361f Xp22.3, 613, 613f XX male syndrome, 665–666

Yp11.3, 613, 613f

Z Zinc-finger DNA binding, 727f Zona fasciculata, 32 Zona reticularis, 32

889