X & Y Chromosomal Variations: Hormones, Brain Development, and Neurodevelopmental Performance (Colloquium the Developing Brain) 9781615046904, 9781615046911, 1615046909

Table of contents :
Dedication
Acknowledgments
Introduction
Meiosis and Mitosis
Testosterone and 47, XXY
3.1 Testosterone and XXY
3.2 Summary
Endocrinological Issues and Hormonal Manipulation in Children and Men With Klinefelter Syndrome
4.1 Introduction
4.2 Materials and Methods
4.3 Results
4.3.1 Diagnosis of 47, XXY
4.3.2 Life-Long Principles of Evaluation and Management of 47, XXY (KS) Patients
4.3.3 Postnatal Testosterone Surge
4.3.4 Sexual Development
4.3.5 Leydig Cell Dysfunction in Klinefelter Syndrome
4.3.6 KS and Infertility
4.3.7 Role and Effects of TRT and Aromatase Inhibitors
4.3.8 Partial Androgen Resistance and Compliance
4.3.9 Outcomes with TRT and Aromatase Inhibitors
4.4 Future Developments
4.5 Conclusion
4.6 Acknowledgments
Neurological Functioning in 47, XXY
5.1 Brain Development
5.2 Neurological Examination in Boys with XXY
5.3 Cognitive Profile
5.4 Psychiatric Disorders and X Chromosome Disorders
5.5 Summary
Neuroimaging in XY Chromosomal Disorders
6.1 Neuroimaging
6.2 Gender Differences in Brain Development
6.3 47, XXY and Neuroimaging Findings
6.4 Epigenetic and Genetic Influences on Brain Development
6.5 47, XXX and Neuroimaging Findings
6.6 Neuroimaging Findings and XYY
6.7 49, XXXXY and Neuroimaging Findings
6.8 The Extra X vs. The Extra Y
6.9 Conclusions
The Evolving Perspective on X and Y Disorders
7.1 Early International Studies
7.2 Growth
7.3 Motor Development
7.4 Speech and Language
7.5 Temperament
7.6 Conclusion
Infant Development in X and Y Chromosomal Disorders
8.1  Progression of Neuromotor Skills
8.2  Symmetry and Movement
8.3  Timing of Physical Therapy
8.4  Speech and Language Development
8.5  Speech and Language Dysfunction: Symptoms of XY chromosomal disorder
8.6  Temperament
8.7  Summary
Phenotype and Developmental Progression of X and Y Chromosomal Variations
9.1  47, XXY
9.2  ADHD
9.3  Reading Function
9.4  Speech and Language
9.5  Biological Treatment
9.6  Behavioral Issues
9.7  47, XYY
9.8  47, XXX
9.9  Summary of Developmental Progression in X and Y Chromosomal Disorders
Social Language in X and Y Chromosomal Variations
10.1 Introduction
10.2 Social Language and Cognition within Autistic Diagnoses
10.3 47, XXX (Triple X, Trisomy X)
10.4 48, XXXX (Tetra X)
10.5 Social Language Development in Variant Forms of XY chromosomal disorders
10.6 Conclusions for Communication Improvement in Children with XY Variants
10.7 The Social Profile of 47, XXY
10.8 The Social Profile of 47, XYY
10.9 Conclusions for 47, XXY and 47, XYY
Conclusions
References
Author Biographies

Citation preview

The Developing Brain

Series ISSN: 2159-5194

Series Editor: Margaret M. McCarthy, University of Maryland School of Medicine

X & Y Chromosomal Variations

Hormones, Brain Development, and Neurodevelopmental Performance Carole Samango-Sprouse, Department of Pediatrics, George Washington University School of Medicine and Health Sciences; Director, Neurodevelopmental Diagnostic Center for Children

Andrea L. Gropman, Department of Pediatrics, George Washington University School of Medicine and Health Sciences; Chief, Neurogenetics and Neurodevelopmental Pediatrics, Department of Neurology, Children’s National Health System, Washington, D.C.

LIFE SCIENCES

X & Y CHROMOSOMAL VARIATIONS

This is the first book on X and Y chromosomal disorders to address these common but rarely diagnosed conditions. This book seeks to present the latest in research and clinical care addressing neuroimaging, the interaction between hormones, brain development and neurodevelopmental progression. This book will primarily focus on 47, XXY (Klinefelter syndrome, or KS), 47, XYY ( Jacobs’ syndrome), and 47, XXX (Triple X). More variant disorders such as 48, XXXX, 48, XXXY and 49, XXXXY will be discussed medically and neurodevelopmentally. Topics of interest include neurological functioning, neuroimaging, social language, and the evolving perspectives of these XY disorders. The effects of testosterone supplementation in males with 47, XXY will also be examined.

SAMANGO-SPROUSE • GROPMAN

Colloquium Lectures on

ABOUT MORGAN & CLAYPOOL PUBLISHERS

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morgan & claypool

This volume is a printed version of a work that appears in the Colloquium Digital Library of Life Sciences. Colloquium books provide concise, original presentations of important research topics, authored by invited experts. All books are available in digital & print formats. For more information, visit store.morganclaypool.com

Colloquium Lectures on

The Developing Brain Series Editor, Margaret M. McCarthy

X & Y Chromosomal Variations Hormones, Brain Development, and Neurodevelopmental Performance

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Colloquium Digital Library of Life Sciences The Colloquium Digital Library of Life Sciences is an innovative information resource for researchers, instructors, and students in the biomedical life science community, including clinicians. Each PDF e-book available in the Colloquium Digital Library is an accessible overview of a fast-moving basic science research topic, authored by a prominent expert in the field. They are intended as time-saving pedagogical resources for scientists exploring new areas outside of their specialty. They are also excellent tools for keeping current with advances in related fields, as well as refreshing one’s understanding of core topics in biomedical science. For the full list of available books, please visit: colloquium.morganclaypool.com Each book is available on our website as a PDF download. Access is free for readers at institutions that license the Colloquium Digital Library. Please e-mail [email protected] for more information.

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Colloquium Series on The Developing Brain Editor Margaret M. McCarthy, Ph.D. Professor and Chair, Department of Pharmacology University of Maryland School of Medicine The goal of this series is to provide a comprehensive state-of-the art overview of how the brain develops and those processes that affect it. Topics range from the fundamentals of axonal guidance and synaptogenesis prenatally to the influence of hormones, sex, stress, maternal care and injury during the early postnatal period to an additional critical period at puberty. Easily accessible expert reviews combine analyses of detailed cellular mechanisms with interpretations of significance and broader impact of the topic area on the field of neuroscience and the understanding of brain and behavior. Published Titles (for future titles please see the website, www.morganclaypool.com/toc/dbr/1/1)

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Copyright © 2017 by Morgan and Claypool All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means—electronic, mechanical, photocopy, recording, or any other except for brief quotations in printed reviews, without the prior permission of the publisher. X & Y Chromosomal Variations: Hormones, Brain Development, and Neurodevelopmental Performance Carole A. Samango-Sprouse and Andrea L. Gropman www.morganclaypool.com ISBN: 9781615046904 paperback ISBN: 9781615046911 ebook DOI: 10.4199/C00134ED1V01Y201506DBR013 A Publication in the COLLOQUIUM SERIES ON THE DEVELOPING BRAIN Lecture #13 Series Editors: Margaret M. McCarthy, University of Maryland School of Medicine ISSN 2159-5194 print ISSN 2159-5208 electronic

X & Y Chromosomal Variations Hormones, Brain Development, and Neurodevelopmental Performance Carole A. Samango-Sprouse

Director, Neurodevelopmental Diagnostic Center for Children Associate Clinical Professor, Department of Pediatrics at George Washington University, Washington, D.C. Adjunct Associate Professor, Department of Human and Molecular Genetics, Florida International University

Andrea L. Gropman

Professor, Department of Pediatrics, George Washington University, Washington, D.C. Chief, Division of Neurogenetics and Neurodevelopmental Pediatrics, Department of Neurology, Children's National Medical Center, Washington, D.C.

COLLOQUIUM SERIES ON THE DEVELOPING BRAIN #13

M &C

MORGAN

& CLAYPOOL LIFE SCIENCES

vi

ABSTRACT

This is the first book on X and Y chromosomal disorders to address these common but rarely diagnosed conditions. This book seeks to present the latest in research and clinical care addressing neuroimaging, the interaction between hormones, brain development, and neurodevelopmental progression. This book will primarily focus on 47, XXY (Klinefelter syndrome, or KS), 47, XYY ( Jacobs’ syndrome), and 47, XXX (Triple X). More variant disorders such as 48, XXXX, 48, XXXY and 49, XXXXY will be discussed. Topics of interest include neurological functioning, neuroimaging, social language, and the evolving perspectives of these XY chromosomal disorders. The effects of testosterone supplementation in males with 47, XXY will also be examined.

KEY WORDS

Klinefelter syndrome; X and Y chromosomal disorders; 47, XXY; 47, XYY; 47, XXX; Jacob's syndrome; 48, XXXY; 49, XXXXY; XY chromosomal disorders; XY disorders, sex chromosome aneuploidy

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Dedication The importance of a father in a young girl's life has been well documented in recent research literature, and both Dr. Gropman and I were lucky enough to have very invested fathers who had great vision for their young daughters. My father taught me from early on to dream big, to see every "No" as opportunity for a better “Yes," and, most of all, to follow my own path. His words of encouragement always conveyed the message that: if you want something bad enough, plan to work hard, long, and well—then it will be yours! Carole Samango-Sprouse I would like to dedicate this book to my father, Leon Saperstein, who encouraged me to pursue my desire to take the path less explored in life because, although it is harder, it is more interesting and ultimately more rewarding. Andrea Gropman

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Contents Dedication���������������������������������������������������������������������������������������������������������������������������  vii Acknowledgments ������������������������������������������������������������������������������������������������������������  xiii 1 Introduction��������������������������������������������������������������������������������������������������������������������������� 1 2

Meiosis and Mitosis ������������������������������������������������������������������������������������������������������������� 5

3

Testosterone and 47, XXY��������������������������������������������������������������������������������������������������� 9 3.1 Testosterone and XXY������������������������������������������������������������������������������������ 11 3.2 Summary�������������������������������������������������������������������������������������������������������� 14

4

Endocrinological Issues and Hormonal Manipulation in Children and Men With Klinefelter Syndrome���������������������������������������������������������������������������������������������� 15 4.1 Introduction���������������������������������������������������������������������������������������������������� 16 4.2 Materials and Methods���������������������������������������������������������������������������������� 16 4.3 Results������������������������������������������������������������������������������������������������������������ 17 4.3.1 Diagnosis of 47, XXY������������������������������������������������������������������������ 17 4.3.2 Life-Long Principles of Evaluation and Management of 47, XXY (KS) Patients�������������������������������������������������������������������������������������� 17 4.3.3 Postnatal Testosterone Surge ������������������������������������������������������������ 18 4.3.4 Sexual Development�������������������������������������������������������������������������� 18 4.3.5 Leydig Cell Dysfunction in Klinefelter Syndrome���������������������������� 19 4.3.6 KS and Infertility������������������������������������������������������������������������������ 23 4.3.7 Role and Effects of TRT and Aromatase Inhibitors ������������������������ 24 4.3.8 Partial Androgen Resistance and Compliance���������������������������������� 27 4.3.9 Outcomes with TRT and Aromatase Inhibitors ������������������������������ 27 4.4 Future Developments�������������������������������������������������������������������������������������� 28 4.5 Conclusion������������������������������������������������������������������������������������������������������ 28 4.6 Acknowledgments������������������������������������������������������������������������������������������ 29

5

Neurological Functioning in 47, XXY ��������������������������������������������������������������������������� 31 5.1 Brain Development���������������������������������������������������������������������������������������� 32 5.2 Neurological Examination in Boys with XXY������������������������������������������������ 32

x

5.3 Cognitive Profile ���������������������������������������������������������������������������������  33 5.4 Psychiatric Disorders and X Chromosome Disorders �����������������������  34 5.5 Summary���������������������������������������������������������������������������������������������  35 6

Neuroimaging in XY Chromosomal Disorders ����������������������������������������������� 37 6.1 Neuroimaging �������������������������������������������������������������������������������������  37 6.2 Gender Differences in Brain Development�����������������������������������������  38 6.3 47, XXY and Neuroimaging Findings�������������������������������������������������  42 6.4 Epigenetic and Genetic Influences on Brain Development�����������������  43 6.5 47, XXX and Neuroimaging Findings�������������������������������������������������  45 6.6 Neuroimaging Findings and XYY�������������������������������������������������������  46 6.7 49, XXXXY and Neuroimaging Findings �������������������������������������������  47 6.8 The Extra X vs. The Extra Y ���������������������������������������������������������������  48 6.9 Conclusions �����������������������������������������������������������������������������������������  49

7

The Evolving Perspective on X and Y Chromosomal Disorders������������������� 51 7.1 Early International Studies �����������������������������������������������������������������  52 7.2 Growth�������������������������������������������������������������������������������������������������  53 7.3 Motor Development ���������������������������������������������������������������������������  55 7.4 Speech and Language �������������������������������������������������������������������������  57 7.5 Temperament���������������������������������������������������������������������������������������  58 7.6 Conclusion�������������������������������������������������������������������������������������������  59

8

Infant Development in X and Y Chromosomal Disorders �������������������������� 61 8.1  Progression of Neuromotor Skills �������������������������������������������������������  63 8.2  Symmetry and Movement�������������������������������������������������������������������  65 8.3  Timing of Physical Therapy�����������������������������������������������������������������  67 8.4  Speech and Language Development���������������������������������������������������  67 8.5  Speech and Language Dysfunction: Symptoms of XY Chromosomal Disorder�����������������������������������������������������������������������  68 8.6  Temperament���������������������������������������������������������������������������������������  70 8.7  Summary���������������������������������������������������������������������������������������������  70

9

Phenotype and Developmental Progression of X and Y Chromosomal Variations������������������������������������������������������������������������������������������������������������������� 73 9.1  47, XXY ���������������������������������������������������������������������������������������������  73 9.2  ADHD �����������������������������������������������������������������������������������������������  74 9.3  Reading Function���������������������������������������������������������������������������������  75 9.4  Speech and Language �������������������������������������������������������������������������  75

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9.5  Biological Treatment�������������������������������������������������������������������������������������� 76 9.6  Behavioral Issues�������������������������������������������������������������������������������������������� 77 9.7  47, XYY���������������������������������������������������������������������������������������������������������� 78 9.8  47, XXX���������������������������������������������������������������������������������������������������������� 80 9.9  Summary of Developmental Progression in X and Y Chromosomal Disorders�������������������������������������������������������������������������������������������������������� 81 10

Social Language in X and Y Chromosomal Variations����������������������������������������������� 83 10.1 Introduction �������������������������������������������������������������������������������������������������� 83 10.2  Social Language and Cognition within Autistic Diagnoses ������������������������ 84 10.3  47, XXX (Triple X, Trisomy X) �������������������������������������������������������������������� 85 10.4  48, XXXX (Tetra X)�������������������������������������������������������������������������������������� 86 10.5  Social Language Development in Variant Forms of XY Chromosomal Disorders�������������������������������������������������������������������������������������������������������� 86 10.6  Conclusions for Communication Improvement in Children with XY Variants ���������������������������������������������������������������������������������������������������������� 87 10.7  The Social Profile of 47, XXY������������������������������������������������������������������������ 87 10.8  The Social Profile of 47, XYY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ��  88 10.9  Conclusions for 47, XXY and 47, XYY �������������������������������������������������������� 89

11 Conclusions�������������������������������������������������������������������������������������������������������������������������� 91 References����������������������������������������������������������������������������������������������������������������������������� 93

Author Biographies����������������������������������������������������������������������������������������������������������  121

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Acknowledgments We would like to acknowledge all of the families of our patients who were invested in their children with a great optimism for the future, entrusted them to our care, and were extremely generous with their time and energy. This allowed us to learn about these extraordinary children with X and Y chromosomal variations. We are also very appreciative of The Focus Foundation, for their dedication to our scientific endeavors, their support of our “outside of the box” thinking, and most of all, their support for our quest to bring optimal care for children with X and Y variations everywhere in the world. Without the parents of our patients and The Focus Foundation, this book would never have come to fruition.

1

CHAPTER 1

Introduction Although X and Y chromosomal disorders are among the most common neurogenetic disorders, they are relatively unrecognized as a significant cause of neurodevelopmental disturbances. They occur with equal frequency among children and adults in all ethnic groups and races throughout the world (Abramsky and Chapple, 1997). These disorders are more common than Down syndrome (1:650), Williams syndrome (1:7500), or Duchenne muscular dystrophy (1.3:10,000), which are characteristically identified early on in an individual’s life. In contrast, 75‒90% of children with X and Y disorders will remain undiagnosed during their lifetime (Visootsak et al., 2013). While there are five primary chromosomal disorders, our focus in this book will be on 47, XXY (Klinefelter syndrome, or KS), 47, XYY ( Jacob’s syndrome), and 47, XXX (Triple X, a neurogenetic condition that only affects girls). XO (Turner syndrome) and Fragile X will not be discussed. Additionally, the medical and neurodevelopmental characteristics of boys with variant forms of less commonly occurring X and Y disorders, including 48, XXXY and 49, XXXXY, and of girls with 48, XXXX (Tetra X), will be examined. X and Y chromosomal variations were first identified more than 70 years ago, when the study of chromosomal complements in humans and their impact on medical care was in its infancy ( Jacobs and Strong, 1959). In the decades since, these disorders have been referred to by many names, including sex chromosomal disorders, sex chromosomal aneuploidy, sex chromosome aberrations, X and Y chromosomal disorders, and disorders of sexual development (DSD) (Radcliffe, 1982a; Robinson et al., 1991; Re and Birkhoff, 2015). It is critical to establish current and scientifically based explanations to fully understand this family of additive X and Y chromosomal disorders and to dispel some common and persistent misinformation regarding their phenotypic presentations. Throughout these pages we will present cutting-edge facts that have emerged from both recent research studies and our extensive clinical practice with children and young adults who possess these chromosomal variations. Each disorder is unique, yet there is often commonality in the natural history of neurodevelopmental progression in these individuals because of their shared sex chromosomal complement. Comprehensive investigation of these disorders illuminates our understanding of biological differences arising from gender, as well as the influence of additional X or Y chromosomes on brain development, neurodevelopment, and behavioral progression. The major features and intriguing aspects of each disorder throughout the childhood years will be highlighted. The influences of such familial learning and language disorders as reading disorders, attention deficit hyperactive disorder

2

X & Y CHROMOSOMAL VARIATIONS

(ADHD) or math disabilities, time of diagnosis, presence or absence of biological treatment, and the impact of these factors on neurodevelopmental outcome will be considered. The role of androgen deficiency and supplementation on brain function, behavior, growth, and development in the treatment of XXY (KS) will be explored in greater detail. Today, X and Y chromosomal disorders remain relatively unknown to most professionals in the fields of medicine, education, and ancillary healthcare, in spite of the fact that their symptoms present within the first year of life (Simpson et al., 2003b). Early detection of these disorders has remained elusive for many reasons. First, XY chromosomal disorders typically do not come to the attention of medical personnel during the newborn period, since affected infants have few major congenital malformations when compared to those with more recognized chromosomal disorders like Down Syndrome (Trisomy 21) or Williams Syndrome (deletion of chromosome 7) (Abramsky et al., 2001). XY chromosomal disorders often do have some minor congenital malformations such as clinodactyly (curved pinky finger), scoliosis, and pectus excavatum (sternal notch depression). However, these minor differences are so common in the general pediatric population that these symptoms typically do not generate suspicion of a chromosomal etiology, or, specifically, one of the XY chromosomal disorders. Second, the salient developmental delays associated with XY variations are not singular to these disorders. These symptoms are characteristic of many neurodevelopmental disorders. Typically, children identified postnatally have been previously diagnosed with ADHD, autism spectrum disorder (ASD), behavioral disturbances, or various other mental health disabilities before XY chromosomal disorders are even entertained as a possible etiological cause (van Rijn et al., 2014; Bishop et al., 2011). Third, boys and girls with X and Y chromosomal disorders do not have significant dysmorphic features that would alert medical professionals to consider possible chromosomal cause (Ratcliffe, 1982a). Their normal physical appearance and often mild-mannered and attractive personalities may be contributory factors in the delay in appropriate detection for years—or even over the course of a lifetime. With so few defining features, the diagnostic odyssey for children with XY chromosomal disorders and their families can be torturously slow and discouraging for everyone. The creation of this book was spurred by our growing awareness that more light needs to be focused on the prevalence of these chromosomal disorders before detection rates and early, efficacious interventions can change the developmental trajectory of these children. Our work has repeatedly demonstrated how families and children benefit from such targeted treatment and syndrome-specific care. While this introductory chapter presents a brief historical perspective on our understanding of these XY chromosomal disorders, Chapter 2 discusses the importance of the biologic processes that lead to the evolution of the additive Xs and Ys. It frames our perspective that these disorders

INTRODUCTION

and their biological influences exert a significant impact on neural development from conception onward, and reveals the complexity of this process. Chapter 3 discusses the vital importance played by testosterone in boys diagnosed with XXY (Klinefelter Syndrome) and that hormone’s powerful effect on growth and well-being, as well as on intellectual and behavioral development. The role of testosterone therapy and the nuances of treatment, timing, and dosage in the child with XXY is explored in great depth by Drs. Woznitzer and Paduch in Chapter 4. Chapter 5 explores neurological function in XY chromosomal disorders and the intricate relationship between the brain, behavior, and outcome. Chapter 6 further expands on the neurology of XY chromosomal disorders and delves into MRI brain imaging involving them. This chapter leads to intriguing hypotheses regarding variations occurring in various regions of the brain and associated behavioral outcomes exhibited by children with XY chromosomal disorders. Chapters 6, 7, 8, and 9 discuss how perspectives on these disorders have evolved over time and their general characteristics; neurodevelopmental progressions in the early years of life; phenotypic features of XY chromosomal disorders, and social language and the impact of language learning dysfunction on children with XY chromosomal disorders. The latter is an area not yet well understood, and the little that has been written has been confounded by ascertainment bias and small studies. Social language development in XY chromosomal disorders needs extensive study before any realistic and appropriate conclusions can be determined. Chapter 10 summarizes, to the best of our knowledge, the characteristic theories and findings on boys and girls with X and Y chromosomal disorders and plausible hypotheses for future investigations. The great variability seen in children with XY chromosomal disorders is underscored throughout this book. These children, with their atypical learning patterns and irregular growth, continually surprise us with their amazing abilities. The breadth and complexity of their chromosomal disorders require us to conduct the kind of extensive and thoughtful research that will lead to effective and timely treatments that will unlock each child’s enormous potential.

3

5

CHAPTER 2

Meiosis and Mitosis Carole A. Samango-Sprouse and Patrick Lawson It is important to turn to basic science to understand how X and Y disorders occur, and to employ correct terminology for the processes and additive chromosomes that result in neurogenetic variations. So let's begin at the beginning. Aneuploidy is a general term referring to a number of chromosomes besides 46, the standard number in humans. These chromosomes are grouped in pairs, known as homologous pairs, or sometimes homologs. Twenty-two of these pairs of chromosomes are autosomes; in other words, they do not differ between genders. The 23rd pair of chromosomes, however, are the sex chromosomes. Males have one X and one Y, while females have two X’s. Aneuploidy is the leading cause of moderate-to-severe intellectual deficits and developmental dysfunction. One such well-known aneuploidy, Down syndrome, is a caused by a trisomy of the 21st homologous pair (Trisomy 21), and occurs in 1 in 625 live births (Weijerman et al., 2008). Trisomies are a specific subset of aneuploidy in which there are 3 copies of a chromosome instead of 2. While much less well known, sex chromosome aneuploidies (alternately refered to as sex (X and Y) chromosome variations, or SCVs) are more common and occur collectively in 1 out of every 450 live births (Nielsen and Wohlert, 1991). The SCVs include many varieties of aneuploidies, including trisomies and monosomies (1 copy of a chromosome), as well as, far less frequently, tetrasomies (4 copies) and pentasomies (5 copies). 45, X, also called Turner syndrome (TS), is the only non-lethal monosomy. (In all other cases of full monosomy a developing fetus will not reach maturity.) One in 330 live births (0.3%) have some variety of aneuploidy (Hassold et al., 1996). Many of these aneuploidies can prove fatal for the fetus, resulting in early-term spontaneous abortions. Among stillborn fetuses the chance of an aneuploidy is more than an order of magnitude greater than that occurring in live births—roughly 4% (Hassold et al., 1996). Across all pregnancy stages, it is estimated that 1 in 3 miscarriages are attributable to an aneuploidy, making it the most common cause of miscarriage (Hassold and Hunt, 2001). The most common SCVs include 47, XXY (Klinefelter syndrome, or KS) and 47, XYY ( Jacob’s syndrome) in males, and 47, XXX and 45, X in females (Turner syndrome, or TS). The leading number presented in the name of each disorder denotes an affected individual’s total number of chromosomes. So, for example, 47, XXY describes a genotypic male, as evidenced by the presence

6

X & Y CHROMOSOMAL VARIATIONS

of a Y chromosome, who has 1 extra X chromosome, which raises his total number of chromosomes to 47. 47,XXY is the most common aneuploidy, occurring in roughly 1:650 live births (Nielsen and Wohlert, 1991). Other SCVs—47, XYY, 47, XXX, and 45, X—occur with respective live-birth incidences of 1:850, 1:950, and 1:1900, although other studies have found slightly lower incidences (Nielsen and Wohlert, 1991; Hook and Hamerton, 1977). The rarer SCVs include 48, XXYY, with an incidence of between 1:18,000 and 1:40,000 live male births (Sorensen et al., 1978). 48, XXXY and 48, XXXX do not have well-documented incidences but it is suspected that they occur between 1:50,000 and 1:85,000 live male births. The rarest variant, 49, XXXXY, is thought to occur in 1:100,000 live male births (Kleczkowska et al., 1988; Gropman et al., 2010).

FIGURE 2.1: The nondisjunction process.

Now let us direct our attention to the etiology of these aneuploidies. Because of the difficulty in tracking the origin of additional chromosomes, we will focus on the three 47 chromosome variants, as well as on 45, X, in which the total chromosomal number is only 1 less than the normal complement of 46. In the vast majority of autosomal trisomies, maternal non-disjunction errors

MEIOSIS AND MITOSIS

occurring during Meiosis 1 (M1) are responsible for the extra chromosome (Hassold and Hunt, 2001). 47, XXX is the only sex chromosome variation that shares this pattern in which maternal errors predominate; other SCVs involve paternal errors in greater numbers. Figure 2.1 shows how non-disjunction error can result in zygotes with 1 chromosome too few or too many. It also demonstrates how non-disjunction can occur either during Meiosis I or Meiosis II (M2). M1 non-disjunction errors, as seen in Figure 2.1, occur when the homologous pair fails to separate. M2 non-disjunction errors occur when the sister chromatids (identical copies created during replication) fail to separate. Post-zygotic mitotic (PZM) errors occur after the ovum and sperm fuse. PZM errors result in mosaicism, where some, but not all, of an individual’s cells are aneuploid. The research literature has described great variability in central nervous function and dysfunction in mosaicism. In those instances when children with XY chromosomal disorders evidence less impairment, it is reasonable to determine if mosaicism may be the explanation for the milder presentation. In our clinical experience, we have found that mosaicism is effected by many additional factors, such as familial learning disorders or time of diagnosis. The earlier the PZM error occurs, the greater the proportion of aneuploid cells there will be. In 47, XXX, as many as 90% of meiotic errors leading to this genotype are maternal in origin. Of this 90%, the majority of non-disjunction errors (roughly 60%) occur during M1. The remaining 30% are equally divided between M2 and PZM (Hall et al., 2006). The remaining SCVs differ from 47, XXX and autosomal trisomies in that a much larger percentage (not necessarily the majority) of the errors leading to the SCV are paternal in origin. In 45, X, for instance, between 70-80% of cases result from the loss of the paternally derived X ( Jacobs et al., 1997). In other words, the sole remaining X in these females is maternal in about 75% of cases. It is not currently possible to determine the stage at which these errors occurred, however, as only the added chromosomes can have the stage of error identified. In 47, XYY, the error must be paternal. This is, of course, because only the father can contribute an extra Y. Roughly 85% of these errors occur during M2, with the rest occurring during PZM (Hall et al., 2006). In 47, XXY, half the meiotic errors are paternal, half maternal (Hall et al., 2006). Of the paternally derived errors, all occur during M1. This logically follows because, if the error occurred during M2, the gamete would either be XX or YY (or void). Of the maternally derived errors, the breakdown of M1, M2, and PZM errors are, respectively, 24%, 14%, and 8%, with the remaining 4% unknown (Hall et al., 2006). A 47, XXY genotype also severely limits sperm production, with affected individuals showing either azoospermia (lack of sperm) or cryptozoospermia (extremely low levels of sperm in ejaculate), as well as hypogonadism (Paduch et al., 2009). This is important to recognize, especially when considering ascertainment bias, as the majority of individuals with 47,

7

8

X & Y CHROMOSOMAL VARIATIONS

XXY are identified in adulthood after presenting with either infertility or hypogonadism (Graham et al., 1988). As many as 97% of 47, XXY men are infertile (Krausz and Forti, 2006), although it is important to note that assisted reproductive technologies (such as testicular sperm extraction) can enable as many as 50% of affected individuals to have children with reproductive assistance (Schiff et al., 2005). Women with 45, X (TS) also show greatly diminished fertility: 90% are infertile due to gonadal dysgenesis and oocyte loss (Sybert and McCauley, 2004). However, 30% may achieve pregnancy using donor oocyte and in vitro fertilization (Foudila et al., 1999). 47, XXX women may show somewhat diminished fertility, as well as an increased risk for premature ovarian failure (POF), although the odds of reproductive success are greater than those experienced by 45, X women (Villanueva and Rebar, 1983). On the other hand, 47, XYY males do not typically exhibit impaired fertility, despite the fact that sperm levels may be below their 46, XY counterparts. Beyond direct concerns of reproductive ability, adults with 47, XXY are at a greater risk of reproducing a fetus with aneuploidy. This is evidenced by the fact that significantly more meiotic errors leading to aneuploidy were observed in 47, XXY men than in 46, XY men with unobtrusive azoospermia, who were used as a control (Vialard et al., 2012). Thus, a 47, XXY male is more likely to have aneuploid offspring, though his offspring will not necessarily share the 47, XXY genotype. It appears that 47, XXX women are also at greater risk of having aneuploid offspring, although the literature regarding this is sparse and complicated by serious ascertainment bias (Dewhurst, 1978) Now that the basic science underlying the creation of XY chromosomal disorders on a cellular level has been briefly covered, it is time to focus on the complex neurological presentation of aneuploidy. This is critical to our understanding of the variances of each and every XY chromosomal disorder.

9

CHAPTER 3

Testosterone and 47, XXY Carole A. Samango-Sprouse and Patrick Lawson Testosterone is a steroid hormone belonging to the androgen group of hormones, and is the primary male sex hormone. These androgen hormones are synthesized from cholesterol, and include dehydroepiandrosterone (DHEA), androstenedione (Andro), and dihydrotestosterone (DHT), among others. Cholesterol is converted into testosterone via the pathways illustrated below. Unused testosterone is converted into estrogen (i.e., aromatized), by the aromatase enzyme. Testosterone has both organizational and activational effects in the human body (Arnold and Breedlove, 1985). Organizational effects refer to the gonadal hormone’s ability to permanently alter the structure or function of a body region (especially the brain) through brief exposure during critical periods, including prenatal development. These effects persist in the absence of circulating hormone, in contrast to activational effects, which require circulating hormone. The tremendous effect of testosterone has been widely examined in animal studies (Verdile et al., 2015). These studies support the known powerful effect on central nervous system development and specifically on the development of genitals. This striking organizational effect of testosterone can be seen in in an animal study conducted more than 20 years ago: A 2-hour exposure of female salmon to an aromatase inhibitor (aromatase converts testosterone to estradiol) results in female salmon with fully developed testes, despite remaining genotypically female (Pifferer and Donaldson, 1994). It is difficult to observe organizational effects in controlled experiments in humans due to the nature of such study designs. Nonetheless, some organizational effects may still be observed through normal testosterone level variance in the population. For instance, fetal testosterone levels correlate with the level of asymmetry in the brain region around the isthmus, which is within the corpus callosum. Fetal testosterone levels can account for as much as 36% of the asymmetry in this area (Chura et al., 2010). This is reflected in research showing a left-ward asymmetry in the male brain, including overall area (Witelson and Pallie, 1973) and greater left hemispheric gray matter density (Good et al., 2001).

10 X & Y CHROMOSOMAL VARIATIONS

FIGURE 3.1: Pathway for the hormonal process.

Testosterone (and the estradiol it is converted to in the brain) is responsible for much, if not the majority, of masculinization leading to the sexual dimorphism between males and females. To observe this effect, we can look to male fetuses with complete androgen insensitivity (Figure 3.1), meaning they are entirely unable to respond to testosterone. These individuals are genotypically male (XY), but phenotypically female, with feminine features and external genitalia. Because testosterone works to masculinize male fetuses from the “default” female phenotype, when testosterone is unable to exert an influence on human development it results in fetuses without Wolffian structures (epididymis, vas deferens, seminal vesicles). These individuals still have active Mullerian

TESTOSTERONE AND 47, XXY 11

inhibiting hormone (MIH), however, which causes the Mullerian ducts to regress. This results in fetuses that typically have a shallow vagina and no female reproductive organs (uterus, fallopian tubes, cervix). Again, they are genotypically male, yet strongly identify as females, and have normal female sexual orientation (Breedlove, 1994; Arcari et al., 2007). This sexual differentiation extends well beyond external genitalia and reproductive organs, however. Testosterone is crucial to appropriate bodily function, playing a role in developing and supporting muscle mass and bone density. Although correlational in nature, it is observed that non-elderly hypogonadal men share many ailments with elderly men, including decreased bone mineral density, muscle mass, strength, aerobic capacity, and an increase in abdominal fat (Harman et al., 2001). This is significant, as it is well documented that testosterone levels in males decline steadily with age, the results of which can mimic symptoms of hypogonadism (Harman et al., 2001). Interestingly, very healthy older men were not found to exhibit this decline in testosterone levels (Harman and Tsitouras, 1980). One reason proposed by the authors to explain this observation is that it is not aging in itself that results in decreased testosterone, but aging-related factors, such as accumulated illnesses, medication, exercise levels and the like, which elicit this response. However, this stance has minimal experimental support. Androgens are also crucial to proper brain development, as briefly mentioned in fetal testosterone’s lateralizing effects. For one, lower levels of available testosterone have been found to be associated with greater separation anxiety (Perrin et al., 2008). Testosterone may also drive the steep growth of white matter volume in males, which occurs before and into early adolescence (Perrin et al., 2008). This is supported by the fact that white matter volume increase is found to be highly correlated with the type of androgen receptor (AR) gene present in an individual. The AR gene contains a string of CAG repeats; the more repeats, the less effective the AR will be. That is, longer AR genes (defined here as ≥ 23 repeats) bind androgens less effectively than shorter AR genes. Testosterone’s influence on white matter volume is greater in individuals with the short gene, which binds more testosterone (Perrin et al., 2008). Unlike males, the growth of white matter volume in females follows a less steep, more homogenous trajectory (Giedd, 2003).

3.1

TESTOSTERONE AND XXY

Let us now turn our attention to 47, XXY males. 47, XXY is characterized by low levels of testosterone, as well as high levels of estradiol (Paduch et al., 2008; Chapter 4; Wosnitzer and Paduch, 2013). Affected (untreated) males frequently present with hypogonadism, gynecomastia, abdominal obesity, and hypotonia (decreased muscle tonus that impacts on neuromotor development). Additionally, when untreated these individuals have decreased body hair and more feminine body shape (wide hips, narrower shoulders). They are almost always azoospermic.

12 X & Y CHROMOSOMAL VARIATIONS

Hypergonadotropic hypogonadism is a hallmark of the untreated XXY male. This is a condition in which the testes do not fully respond to the gonadotropins, namely follicle-stimulating hormone (FSH) and leutenizing hormone (LH). This results in a lack of sex hormone creation, especially testosterone. It also commonly results in an increase in circulating gonadotropins, attempting to compensate for the weak response by the gonads to the gonadotropin. Somewhat surprisingly, infantile studies encompassing a relatively broad age-range show 47, XXY boys tending to have normal levels of testosterone and a normal response to gonadotropins (Forti et al., 2010). When specifically looking at the period of the male postnatal testosterone surge occurring between 4‒10 months (known as “mini-puberty”), however, evidence suggests 47, XXY individuals do indeed show a lower testosterone peak than normally developing peers, although this is subject to individual variability (Lahlou et al., 2004). It is for this reason that HRT (hormone replacement therapy) is initiated in the first year of life, when possible. Males treated with testosterone (HRT) show a great alleviation of all of many characteristic features of XXY, including abdominal obesity, hypotonia, and general feminization (Lanfranco et al., 2004; Samango-Sprouse et al., 2013a). HRT significantly helps alleviate the feminization effects, as well as improves many aspects of neurodevelopment, including cognition, emotional regulation, and motor development. For instance, when compared to their untreated peers, children treated with testosterone in infancy show significantly better outcomes in the realms of auditory comprehension, verbal ability, full scale IQ (FSIQ), and verbal IQ (Samango-Sprouse et al., 2013a). These benefits are apparent at both 3 years and 6 years. Furthermore, treated individuals show improved bilateral coordination, strength, and total motor composite as measured by the BOT-2, when compared to their peers (Samango-Sprouse et al., 2013a). When this same cohort was followed up at 9 years of age, they showed significantly improved social communication and social cognition skills, as well as decreased school problems, when compared to non-HRT peers, demonstrating the sustained positive effects of HRT (Samango-Sprouse et al., 2013a). HRT is administered at two critical periods: “mini-puberty” (ideally between 4 and 10 months of age) and again near the onset of puberty, when XXY boys are around 11‒12 years old. (Each boy has to be individually assessed to best determine their onset of pubertal development as well as the need for hormonal treatment.) Administration occurs during these periods to keep pace with typically developing boys, who exhibit testosterone spikes at these times, which in turn lead to rapid physiological development. XXY boys simply can’t replicate these hormonally driven periods of rapid change unassisted, and thus HRT is required (Paduch et al., 2008; Mehta and Paduch, 2012; Wosnitzer and Paduch, 2013). 47, XXY males exhibit distinct morphological differences in multiple brain areas; this is, presumably, the root of any pre-treatment deficits, as compared to neurotypical peers. Some of these morphological differences include decreased frontal, caudate, and temporal lobe volume (especially

TESTOSTERONE AND 47, XXY 13

left), decreased gray matter density in the insula, decreased total brain volume, and a thinner cortex in the temporal and frontal regions (Patwardhan et al., 2000; Nagai et al., 2007; Giedd et al., 2007). The left temporal lobe is associated with language ability, the insula with social and emotional processing (Nagai et al., 2007), and the frontal lobe with executive function, all of which are notably impaired in untreated 47, XXY males. Interestingly, parietal regions are relatively unaffected (as measured by cortical thickness and volume) (Giedd et al., 2007). This is supported by XXY males’ enhanced spatial cognitive skills, relative to those of controls (Samango-Sprouse and Law, 2001).

FIGURE 3.2: Brain imaging differences between 47, XXY and 46, XY. (A) Left hippocampal for-

mation. (B) Left superior temporal gyrus. (C) Cingulate region. (D) Left insula. (E) Right amygdala. (F) Left middle temporal gyri. (G) Right parietal lobe WM. Courtesy of Shen et al., 2004.

14 X & Y CHROMOSOMAL VARIATIONS

Figure 3.2 is a visual summary of the significant group differences in brain development between boys with 46, XY and 47, XXY. The underlying image is the template that we used to normalize individual brains. Color-coding was based on the values of the T-test (Figure 3.2). Only the voxels with significant group differences, i.e., the corrected P values exceeding a significance threshold 0.005, are shown. Although there are few MRI studies comparing 47, XXY individuals receiving HRT versus non-HRT, one such study (with an admittedly small sample size of 5 per group) found that temporal gray matter was preserved in the HRT group (i.e., not significantly different from that present in 46, XY males), but diminished in the non-HRT group (Patwardhan et al., 2000). Further studies are warranted to expand on these findings and to investigate whether other abnormal brain areas, as described above, show similar normalization after HRT.

3.2

SUMMARY

The importance of hormonal influences in boys and girls cannot be overstated since these hormones affect well-being and health, as well as motor skills and neurodevelopment. We will be exploring HRT’s additional impact on mental health outcomes in Chapter 4. It is becoming increasingly apparent that, in boys with XXY, EHT and androgen replacement beginning at 11 years may have a powerful influence on outcomes from multiple perspectives. Additional research is warranted to better understand the complex relationship between androgens and XXY, to further facilitate personalized and targeted care. Girls with 47, XXX and boys with XYY have not been investigated from the hormonal perspective, but it seems realistic to consider that additive X in girls or Y in boys could be impactful in multiple ways that have yet to be investigated. Children with X and Y chromosomal disorders deserve greater study to determine the relationship that exists between additional X or Y chromosomes and hormonal development. This may allow us to understand the natural history of these disorders, as well as develop creative and innovative treatments that will enable each of these boys and girls to realize their optimal potential.

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CHAPTER 4

Endocrinological Issues and Hormonal Manipulation in Children and Men With Klinefelter Syndrome Matthew S. Wosnitzer1 and Darius A. Paduch2 This chapter was originally printed in American Journal of Medical Genetics Part C (Seminars in Medical Genetics) 163C:16‒26 (2013) 47, XXY or Klinefelter syndrome (KS), the most common chromosomal aberration in males, is characterized by either absolute or relative hypogonadism with a frequent decline in serum testosterone (T) following the onset of puberty. Decreased T levels are the result of testicular dysfunction with a decrease in size of Leydig cells, and loss of germs and Sertoli cells leading to tubular hyalinization. An increase in estradiol results from over-expression of aromatase CYP19. Deficient androgen production and observed varied response of end-organs to T leads to delayed progression of puberty with decreased facial/body hair, poor muscle development, osteoporosis, and gynecomastia. It is possible that hypogonadism and excessive estradiol production contribute to emotional and social immaturity, and specific learning disabilities in KS. Based on the authors’ experience and literature review, early fertility preservation and hormonal supplementation may normalize pubertal development, prevent metabolic sequelae of hypogonadism, and have a positive effect on academic and social development. No randomized clinical trials are available studying the effects of T supplementation on reproductive or cognitive issues in KS. Aggressive T supplementation (topical gel) and selective use of aromatase inhibitors may be considered at the onset of puberty with careful follow-up and titration to reach age-specific high-normal physiologic serum values. The decision to institute hormonal 1

Matthew S. Wosnitzer, M.D. is a Male Reproductive Medicine Fellow in the Department of Urology, Weill Cornell Medical College in New York, NY. His research work focuses on genetics and molecular biology of male infertility and the role of opioids and novel immunomodulators in iatrogenic hypogonadism. 2 Darius A. Paduch, M.D., Ph.D. is Associate Professor of Urology and Reproductive Medicine at Weill Cornell Medical College in New York, NY. He is an internationally recognized expert in genetics and reproductive endocrinology of male infertility and hypogonadism, Klinefelter syndrome, sexual medicine, and virology of genital tract.

16 X & Y CHROMOSOMAL VARIATIONS

therapy should be part of a multidisciplinary approach including physical, speech, behavioral, and occupational therapy. © 2013 Wiley Periodicals, Inc.

4.1

INTRODUCTION

Klinefelter syndrome (KS) is the most common cause of male hypogonadism and chromosomal aberration occurring in 0.2% of the general population, comprising up to 4% of patients in male reproductive practices and 15% of azoospermic males (Bojesen et al., 2003; Ghorbel et al., 2012). KS genotype is caused by meiotic nondisjunction, most commonly resulting in the 47, XXY karyotype (X disomy), with variable phenotype often indistinguishable from boys with normal karyotypes on physical examination. Men with more than two X chromosomes (48,XXXY) are more affected than men with 47,XXY karyotype (Samango-Sprouse, 2001). Given the wide phenotypic spectrum, less than 10% of men are diagnosed before puberty and many men with KS remain undiagnosed. Variation in phenotype may be explained by hormonal and genetic background differences, including androgen receptor polymorphism in the CAGn repeat and skewed inactivation of additional genetic material on the X chromosome (Zechner et al., 2001; Bojesen et al., 2011). Classic KS phenotype typically includes micropenis, small, hard testes with adolescence-onset testicular failure (spermatogenic and steroidogenic), hypergonadotropic hypogonadism with low testosterone, infertility, tall eunuchoid stature (over 50% of males exceeding the 97th centile of height for their age), sparse facial and pubic hair, atypical motor function, behavioral issues, and specific mild to moderate deficits in language-based skills with decreased verbal intelligence quotient, attention, and auditory processing (Klinefelter et al., 1942; Ross et al., 2008; Zahn-Waxler et al., 2008). KS is associated with obesity and hyperestrogenism throughout life as well as increased risk of cancer (breast/germ cell), endocrine complications (diabetes mellitus, growth hormone deficiency, hypothyroidism, hypoparathyroidism), autoimmune diseases, and decreased bone density. Early diagnosis of 47, XXY and proactive testosterone replacement along with a multidisciplinary approach for physical, speech, behavioral, and occupational therapy promotes effective developmental, social, and academic progress (Paduch et al., 2009; Radicioni et al., 2010). It is important to remember that most descriptions of the phenotype and associated comorbidities are derived from studies of older patients who were not treated during puberty and early adulthood.

4.2

MATERIALS AND METHODS

A systematic review was conducted of the relevant literature using the Pubmed NLM database to search for primary and review articles using keywords ‘‘Klinefelter syndrome’’ and ‘‘testosterone,’’ ‘‘testosterone replacement,’’ ‘‘aromatase inhibitors,’’ ‘‘spermatogenesis,’’ ‘‘Leydig cell dysfunction,’’

ENDOCRINOLOGICAL ISSUES AND HORMONAL MANIPULATION 17

and ‘‘partial androgen resistance.’’ In addition, data based on 200 children, adolescents, and young adults seen at our center’s specialized KS clinic was used.

4.3

RESULTS

4.3.1 DIAGNOSIS OF 47, XXY Cytogenetic diagnosis is completed by karyotyping of typically 20 mitotic spreads from peripheral blood. Historically the presence of Barr body in mucosal scraping was used. More recently, fluorescence in situ hybridization (FISH), or molecular techniques like methylation-specific polymerase chain reaction (PCR) based on inactivation pattern differences of genes on the X chromosome: familial mental retardation gene 1 (FMR1) and X chromosome inactivating transcript (XIST) ,are being used to overcome the high cost of cytogenetics and the limited sensitivity of detecting low levels of mosaicism with cytogenetics (Mehta et al., 2012). Peripheral blood cytogenetics in children diagnosed in utero should be performed to confirm prenatal diagnosis. Y chromosome microdeletion analysis is performed in azoospermic men. 4.3.2

LIFE-LONG PRINCIPLES OF EVALUATION AND MANAGEMENT OF 47, XXY (KS) PATIENTS Perinatal evaluation with testosterone treatment from 3 to 6 months after birth is the accepted standard of care. Fifty milligrams of Depo-testosterone (intramuscular injection once/month) for 3 months has been used. Alternatively 1 pump of Androgel 1% (1.25 g/day; 37.5 g/month) may be used for older infants with attention to any skin reaction. Response to T treatment measured as increase in penile length is a good indicator of overall tissue sensitivity to testosterone. During the prepubertal period, focus on physical, occupational, and speech therapy is critical for normal academic progress. Between birth and 10 years of age, androgen treatment is used only for very short periods in children who did not receive the 3-month treatment after birth or if the initial response was not adequate. In our practice we limit the 3-month treatment in prepubertal boys to age 8 to avoid initiation of early puberty. During puberty, physical examination with measurement of testicular volume is performed every 6 months. All men with KS should undergo a complete hormonal profile that includes follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, estradiol, prolactin, inhibin-B, insulin-like growth factor-1 (IGF-1), and cortisol levels every 6 months starting prior to the predicted start of puberty. Thyroid and lipid profiles are obtained once a year. When FSH and LH increase, a multidisciplinary discussion with patient and parents is completed to discuss fertility management options. Morning urine samples can be used to identify sperm, but in adolescents who ejaculate spontaneously, analysis of a semen sample may be considered following discussion with parents and patient. If sperm are identified in semen, semen cryopreservation is of-

18 X & Y CHROMOSOMAL VARIATIONS

fered. Anastrozole 1 mg once a day is used for 6 months, and additional semen samples are obtained for cryopreservation to store at least 4–6 vials of ejaculated sperm. For adolescents without sperm in ejaculate, the option of testicular sperm retrieval during puberty or in adulthood should be discussed. Bone density testing (dual-energy Xray absorptiometry, DEXA) should also be completed routinely given the risk of osteopenia and osteoporosis with low testosterone. If decreased bone density is identified, additional calcium, phosphorus, parathyroid hormone, and vitamin D3 are obtained. Despite conflicting evidence for increased breast carcinoma risk, patients are taught early regarding self-examination to identify abnormal breast nodules or nipple discharge.

4.3.3 POSTNATAL TESTOSTERONE SURGE Testosterone is secreted at adult levels during 3 periods of male life including transiently during the first trimester of pregnancy (male genital tract differentiation) and during early neonatal life as a perinatal androgen surge (minipuberty) between 2 and 6 months of age. The third period starts during puberty and continues throughout the male life span with varied efficiency. It is postulated that decreased testosterone production occurs during all 3 periods in males with KS, although there are studies both in favor and against this in the 47,XXY fetus, during infancy, and during pre-puberty (Salbenblatt et al., 1985; Ratcliffe et al., 1994b; Lahlou et al., 2004; Cabrol et al., 2011). Testosterone levels normally increase up to 10 nmol/L during the first months of life and some studies show that the spike in T production within the first 3 months is decreased in boys with KS. This may be related to the inappropriate function of fetal Leydig cells, but the reason for lower T levels in newborns with KS is not completely understood. In KS patients, confirmatory postnatal karyotype, early evaluation by a physician experienced with management of KS, and measurement of penile size and testosterone level are critical. Failure to exhibit the dramatic changes that occur during normal puberty (approximately 30-fold increase in testosterone levels) is due to testicular abnormalities affecting Leydig cell function. In most KS males, testosterone rises during puberty and subsequently plateaus (Salbenblatt et al., 1985). However, despite the increase, the rate of progression and testosterone levels achieved seem to be less prominent than in non-KS adolescents. 4.3.4 SEXUAL DEVELOPMENT In 47, XXY (KS), testosterone levels characteristically decline during late adolescence and early adulthood. Onset of puberty in KS patients occurs at a predicable time, but decreased androgen production and hyperestrogenism results in delayed progression of puberty with decreased facial/ body hair, muscle development, eunuchoid features, and gynecomastia (Smyth and Bremner, 1998). Based on our experience it seems that men with KS have varied degrees of partial androgen resistance. This manifests as attenuated suppression of SHBG during TRT as compared to non-KS men, and poor androgenization despite high–normal levels of T achieved with TRT. The pubertal

ENDOCRINOLOGICAL ISSUES AND HORMONAL MANIPULATION 19

growth spurt is the same as in 46, XY boys, and prepubertal males with KS have similar testosterone, LH, FSH, inhibin B, and anti-Mullerian hormone (AMH) until the initiation of puberty. Based on our observations (Paduch DA, unpublished work), penile girth may be decreased in some adolescents, and boys with 48, XXXY may have thickening of penile skin with circumference exceeding length. Scrotal development is normal, but testicular size is significantly decreased during puberty (occurs between Tanner stages II–III) secondary to progressive deterioration of germinal epithelium, Sertoli cells, and peritubular fibrosis while the epididymis is spared. In addition to testicular dysfunction, some boys may also demonstrate growth hormone deficiency, which further impairs muscle development and peak pubertal bone mineral density (Rossodivita and Colabucci, 1994; van den Bergh et al., 2001; Bahillo-Curieses et al., 2011).

4.3.5 LEYDIG CELL DYSFUNCTION IN KLINEFELTER SYNDROME Testicular degeneration in KS patients may occur from spatially and ontogenically incorrect gene expression from an additional X chromosome or failure in cell divisions. During meiosis, abnormal paring of sex chromosomes leads to meiotic arrest and subsequent germ cells apoptosis. It is fascinating to notice that most somatic cells proceed through normal mitotic divisions despite the presence of additional X chromosome. However, spermatogonial stem cells, Sertoli cells, and Leydig cells undergo varied degrees of degeneration leading to infertility and hormonal abnormalities (De Sanctis and Ciccone, 2010). There is conflicting information about the timing of degeneration, with some studies describing loss of spermatogonia beginning in infancy (Mikamo et al., 1968). Diminished spermatogonia with normal Leydig and Sertoli cells have also been reported to first occur in pre-pubertal boys with KS (Muller et al., 1995), although the majority of boys have spermatogonia identified in early adolescence (Wikstrom et al., 2007). Patients with KS demonstrate reduced adult dark spermatogonia, with gradual loss mediated by massive apoptosis preceding hypergonadotropic hypogonadism with elevated gonadotropin levels and decreased testosterone levels (Wikstrom et al., 2004, 2007). TEX11, an X-chromosome derived germ-cell-specific protein expressed in spermatgonia and spermatocytes, may lead to spermatogenic defects in KS (Yu et al., 2012). Germ cells in patients with KS are also characterized by maturational arrest occurring either at early stages with type A spermatogonia before meiotic division or during later stages (Lanfranco et al., 2004; Wikstrom et al., 2004; Sciurano et al., 2009). Studies of INSL3 indicate that Leydig cells in male infants with nonmosaic KS are sensitive to LH and such sensitivity is not diminished during the first year of life (Cabrol et al., 2011). However, studies that use very short periods of injections of hCG to test steroidogenic activity of Leydig cells fail to measure Leydig cell activity under pulsatile and continuous release of LH occurring from puberty forward. Therefore, results of hCG stimulation tests have to be carefully interpreted. It is clearly established that T production is regulated by acute response to LH and chronically regulated at the level of gene transcription. Most studies show that, irrespective of age, median T values in men with KS are lower; thus defec-

20 X & Y CHROMOSOMAL VARIATIONS

tive Leydig cell function or excessive conversion of T to estradiol within the testis is a paramount characteristic of KS. Steroidogenesis is initiated by activation of the LH receptor leading to an increase in cAMP, phosphorylation of cAMP-dependent kinase, and activation of steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine (PBR) receptor, a rate-limiting step in steroidogenesis. Cholesterol is transported by StAR and PBR to mitochondria, where cholesterol is converted to pregnenolone by CYP11A1 (a precursor for steroidogenic activity in Leydig cells). Current evidence suggests that hormonal regulation in Leydig cells is also mediated by multiple signal cascades including cAMPprotein kinase A (PKA), serine/threonine AKT kinase (AKT, also called protein kinase B or PKB), phosphatidylinositol 3-kinase (PI-3K), protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and intracellular Ca2þ signaling proteins. In addition, other biologically active agents including growth factors, steroids, prostaglandins, and cytokines can influence Leydig cell response through endocrine, autocrine, or paracrine regulation. This is clinically important since improved understanding of steroidogenic defects in men with KS will enable identification of specific treatment options (Azhar and Reaven, 2007). In adult human Leydig cell culture, the steroidogenic activity after LH stimulation is decreased (Figure 4.1). Experiments performed in our laboratory indicate that the decrease in steroid synthesis occurs downstream from CYP11A1 regulated conversion of cholesterol to pregnenolone. Adult KS testes are characterized by tubular hyalinization with most of the testicular space occupied by Leydig cells, often called ‘‘Leydig cell hyperplasia’’ or ‘‘hypertrophy.’’ Based on morphometric studies performed in our lab, Leydig cell size is decreased and there is no evidence that Leydig cells undergo active cell division or that their total number is higher than in non-KS testis (Figure 4.2; Paduch DA, unpublished work). The majority of Leydig cells in KS have normal morphology (Regadera et al., 1991; Aksglaede et al., 2011). Studies of isolated Leydig cells illustrate that estradiol suppresses testosterone production in up to 40%, and inhibition of estradiol by selective estrogen receptor antagonist reverses this process. Additionally, our group has shown that expression of aromatase CYP19, which converts T to estradiol, is 4 times higher in the testes of KS males (Figure 4.3). Abnormalities of Sertoli cells with failure to mature, and fewer androgen receptors with cytoplasmic rather than cell surface location have been demonstrated (Wikstrom et al., 2004, 2007). Sertoli cell marker secretion and expression are dramatically decreased by the completion of puberty (Wikstrom et al., 2007). Additional study is required to determine whether impaired spermatogenesis in KS results from these changes in germ cells, Sertoli cells, Leydig cells, elevated intratesticular estradiol, or due to interaction between these constituents. Small testicular size is the only consistent physical feature in 47, XXY, but the difference in size of testes between 46, XY and 47, XXY boys does not become evident until at least Tanner stage II.

ENDOCRINOLOGICAL ISSUES AND HORMONAL MANIPULATION 21

FIGURE 4.1: Steroidogenic activity after LH stimulation is decreased in adult human Leydig cell culture.

22 X & Y CHROMOSOMAL VARIATIONS

FIGURE 4.2: Morphometric analysis: Leydig cell size is decreased.

FIGURE 4.3: Aromatase CYP19 expression is higher in in 46, XY and 47, XXY.

ENDOCRINOLOGICAL ISSUES AND HORMONAL MANIPULATION 23

FIGURE 4.4: Spermatogenesis in KS males.

4.3.6 KS AND INFERTILITY Men with KS are commonly infertile because of primary testicular failure. While infertility affects 97% of KS patients, KS adolescents (Tanner stages II–III) have few sperm identified in 70% of cases, and less than 10% have adequate sperm in ejaculate (cryptozoospermia or oligospermia) for cryopreservation. There is likely a specific time period in early puberty during which ejaculated sperm or sperm from testicular biopsy may be obtained for cryopreservation (Figure 4.4). Although most adult men are azoospermic, rarely KS mosaic cases have had successful pregnancy without assisted medical technology (Terzoli et al., 1992). Adolescents with KS start masturbation at the same age as 46, XY males, but have delayed ability for ejaculation (mean difference between first masturbation and first ejaculation 2 months in 46, XY vs. 9 months in KS patients), likely secondary to relative testosterone deficiency and delayed development of the spinal cord motor generator, a sexually dimorphic S2–S4 center which is dependent on testosterone. With the widespread use of microdissection testicular sperm extraction (TESE) and intracytoplasmic sperm injection for nonmosaic KS, patients with KS have increased chances of successful sperm recovery (≥50%) and subsequent reproduction considered equivalent to men with nonobstructive azoospermia of other causes (Palermo et al., 1998; Schiff et al., 2005; Ramasamy et al., 2009). Men with normal baseline

24 X & Y CHROMOSOMAL VARIATIONS

testosterone had the highest sperm retrieval rate (86%), while those requiring medical therapy, who responded with a testosterone of 250 ng/dl or greater, had increased sperm retrieval rate (77%) than those who had less optimal testosterone response (55%). This finding indicates that optimal spermatogenesis requires an optimal intratesticular hormonal environment. Serum FSH at baseline did not affect sperm retrieval. Successful TESE has been completed with cryopreservation of rare sperm in 75% of adolescents (Paduch, DA, unpublished work). While some groups recommend more conservative approaches, our group recommends discussion of microsurgical testicular sperm extraction and cryopreservation during puberty and young adulthood (Oates, 2012). To the best of our knowledge, the effects of hormonal manipulation on sperm retrieval rates in adolescents have not been studied outside of our center. When sperm was identified, all adolescents were on testosterone supplementation using topical testosterone and most of them used anastrozole 1 mg a day for 6 months prior to retrieval. The hormonal manipulation in adolescents should result in catchup in progression of puberty, normal strength, agility, muscle mass, and bone mineral density—a goal that can be achieved in most adolescents if treated early. The improvements in academic and social development noted in boys with KS treated with T may be a direct result of normalization of hormonal profile on brain development or secondary to increased attention to academic progress, gain in self-confidence, and selection bias. These three goals of normal academic progress, age adequate psychosocial development, and physical maturity take priority over any effects of hormonal manipulation on fertility. It is critical to recognize that there is a significant difference in FSH and LH suppression between injectable testosterone and topical testosterone. Injectable testosterone suppresses FSH and LH and therapy with injectable testosterone must be stopped prior to any infertility treatment. Topical testosterone, however, may be used as long as there is no significant suppression of FSH and LH. The incorporation of nontestosterone hormonal therapy (clomiphene citrate, aromatase inhibitors, hCG) may be beneficial in circumstances requiring increased testosterone prior to sperm retrieval. The impact of testicular dissection for sperm extraction has been identified to result in serum T decline with recovery in 12–18 months postoperatively, with rare testicular atrophy or persistent hypogonadism (Ramasamy et al., 2005; Takada et al., 2008).

4.3.7 ROLE AND EFFECTS OF TRT AND AROMATASE INHIBITORS Testosterone supplementation in patients with 47, XXY (KS) promotes male phenotype development, increasing penile size, decreasing gynecomastia, abdominal obesity, and improving cognition (Ruvalcaba, 1989; Bojesen et al., 2010). There are isolated reports of androgen therapy during infancy based on androgen therapy for micropenis data, but there is not enough data to draw significant conclusions. Samango-Sprouse (personal communication, 2012) reports on significant increases in cognitive and speech and language development at 36 and 72 months in a cohort of infants with 47, XXY (KS) who were treated with testosterone ethanate once a month for 3 months.

ENDOCRINOLOGICAL ISSUES AND HORMONAL MANIPULATION 25

Randomized clinical trial data of TRT in patients with 47, XXY (KS) are not available (Mehta and Paduch, 2012). Options for TRT for patients with 47, XXY (KS) include initiation early to mid puberty, or at the start of symptomatic hypogonadism (Lanfranco et al., 2004; Bojesen and Gravholt, 2007; Wikstrom and Dunkel, 2011). While specific guidelines do not exist for TRT in KS patients, agespecific testosterone formulations and dosages may be obtained from the Endocrine Society’s Clinical Practice Guidelines (Bhasin et al., 2010). We advocate implementation starting at puberty (approximately age 11 years) with titration to maintain appropriate physiologic serum testosterone levels, gonadotropins (LH, FSH), and estradiol throughout puberty (Winter, 1990). Such a regimen ensures normal completion of puberty and prevents well-established consequences of androgen deficiency. Males with KS may require increased doses of topical testosterone secondary to partial androgen resistance (see below). Topical testosterone is the preferable route of administration since physiologic levels of testosterone are achieved without complete suppression of LH and FSH (unlike injectable testosterone, which is also painful and intimidating for children and adolescents). In pre-adolescent boys, topical testosterone (i.e., Androgel 1%, Abbott, Abbott Park, IL) is applied at one pump (1.25 g gel/day or 37.5 g/month) for 6–12 months, with progressive increase in dosing according to T levels and progression of puberty, assessed every 6 months. Pubic and axillary hairs are much more accurate measures of progression of puberty, as many adolescents have poor facial hair development. Body odor, increase in penile size, nocturnal emissions, and acne are good indicators used to measure response to treatment. For adolescent males, two pumps (2.5 g gel/day) is the recommended starting topical testosterone (Androgel 1%). Once an adolescent starts using three pumps of Androgel 1% (3.75 g/day or 112 g/month), he can switch to more concentrated preparations to decrease the amount of gel applied. Patient preference and compliance are critical in selection of the specific preparation available on the market. Underarm testosterone preparations seem to be preferred in adolescents as it is similar to deodorant application—hence more familiar and less stigma-prone to adolescents. We start at 30 mg of Axiron (Eli Lilly, Indianapolis, IN) and increase every 6–12 months as needed (Table 4.1). It is important to recognize that none of the available topical testosterone preparations in the U.S. are FDA-approved for use in men younger than 18. The goal is to have the serum testosterone level at the upper end of the normal age-specific range. Therapy should be monitored at 6 weeks following initiation and dose-adjusted every 6 months as determined by monitoring puberty and clinical response. Because KS adolescents frequently exhibit decreased response to TRT when compared to an age-matched cohort, vigilant follow-up is required to monitor testosterone response and medication titration to reach desired goal testosterone levels.

26 X & Y CHROMOSOMAL VARIATIONS

TABLE 4.1: Recommended testosterone prepraations for KS patients

Testosterone Formulation Topical gel

Dosage Pre-pubertal: 1 pump (1.25 g gel/day)

Pharmacokinetics Hight DHT:T ratio

Adolescent: 2 pumps (2.5 g gel/day) Once taking ≥.3 pumps/ day, consider switch to more concentrated form of T gel High DHT:T 30 mg daily (1 Axiron 2% ratio (30 mg T/1.5 ml) pump to axilla) (metered-dose Progressive pump with increase in dosing applicator) according to

Androgel 1% (12.5 mg T/1 pump (metereddose pump)

T levels and progression of puberty, assessed every 6 months

Monitoring

Side Effects

Monitor T level 2 hr. after application (after 2 wk. of treatment)

Skin irritation (rare)

Monitor T level 2 hr. after application (after 2 wk. of treatment)

Skin irritation (rare)

Risk of skin-toskin transfer

Risk of skin-toskin transfer

Given the elevation of circulating estrogens associated with increased adipose and aromatase CYP19 activity in KS, an alternative to exogenous testosterone therapy includes the use of aromatase inhibitors such as anastrazole (AstraZeneca, Wilmington DE) (Schlegel, 2012). With decreased T/E ratio, anastrazole 1 mg daily for up to 2 years is helpful in KS adolescents with gynecomastia or central obesity who are nonresponders to maximal doses of topical testosterone (Androgel 1%, 10–12 g/day). This mechanism promotes bone health and may alleviate the suppressive effects of exogenous testosterone on the hypothalamic–pituitary axis gonadotropins. Such therapy has been shown to have positive effects on intratesticular testosterone, testosterone production, and spermatogenesis in KS males (Raman and Schlegel, 2002). Anastrazole has also been used without significant side effects in teenagers with short stature (Faglia et al., 2000). Additional studies will

ENDOCRINOLOGICAL ISSUES AND HORMONAL MANIPULATION 27

be required in order to determine the ideal route and duration of TRT/aromatase inhibitors for 47, XXY (KS) patients. Prior to initiation of hormone supplementation, fertility preservation, ethical, and legal concerns should be discussed with the patient and his parents. Effects of anastrozole on cognition have been suggested by some groups, but there is currently insufficient data to support such a claim. From our experience, many adolescents report feeling worse after stopping anastrozole in terms of ability to concentrate and overall energy level. This clinical observation may explain that high circulation levels of estradiol affect CNS activity. Further research, however, is needed to make an evidence-based recommendation about optimal indications and duration of anti-estrogen therapy. As hyperestrogenism in KS is well-accepted, we often recommend a diet low in naturally occurring phytoestrogens.

4.3.8 PARTIAL ANDROGEN RESISTANCE AND COMPLIANCE Patients with KS manifest partial androgen insensitivity or resistance, which likely results from increased adipose tissue, aromatization from upregulation of aromatase CYP19 (with enhanced testosterone to estradiol conversion), and decreased activated androgen receptor trafficking from cytoplasm to nucleus. This resistance blunts the physiologic response of KS patients to TRT, and may lead to discouraged patients, due to lack of rapid changes in physical appearance. For this reason, it is imperative that the urologist treating patients with KS set age-specific goals for patients regarding muscle strength, facial/sexual hair changes, and penile size changes. Attention to the concerns of the patient with KS is integral to their compliance with the treatment regimen and to ultimate outcome. Compliance must also be assessed by pill counting and weighing of the testosterone container weekly by parents to ensure that the adolescent is properly using the medication. Adolescents with KS, similar to any other group of young men with chronic conditions, have poor recognition of the negative consequences of medical noncompliance. Therefore, close interaction with parents, respect for adolescent autonomy, and regular serial follow-ups with serum blood measurements (T, SHBG, E, LH, FSH, CBC) are critical to achieve treatment objectives. During adolescence, more frequent patient visits may be necessary to maintain and assess compliance, to discuss safety, preventive measures, alcohol/drug use, bullying, depression, and anxiety. An alternative TRT regimen using implantable testosterone pellets for noncompliant patients has been utilized in rare occasions by our group and others (Khera et al., 2009). 4.3.9 OUTCOMES WITH TRT AND AROMATASE INHIBITORS TRT is accompanied by normalization of body proportions, decreased obesity, blood pressure, and improved behavior, work/academic performance, and musculoskeletal development. Additional long-term reduction in autoimmune disease, breast carcinoma, and osteoporosis risk occur (Mehta et al., 1993; Landin-Wilhelmsen et al., 1999). The long-term consequences of TRT in KS patients

28 X & Y CHROMOSOMAL VARIATIONS

and the extent of suppressive effects on the hypothalamic pituitary axis are difficult to assess from available literature. The first report of hormonal therapy outcomes in a large cohort of adolescents with KS was conducted by chart review from our center including 110 adolescent patients with KS treated between 2007 and 2012 (Mehta A, Paduch DA, unpublished work). Patients received hormone therapy (topical TRT (n = 110 patients) or aromatase inhibitors (n = 75 patients)) initiated at mean age of 13 years. Good clinical efficacy was achieved in all patients as defined by age-specific serum T levels. Following therapy, the percentage of obese patients decreased from 17% to 11% and mean serum T level improved from 240 to 650 ng/dl. Serum LH and FSH increased with puberty progression (2.6–16.6 mIU/ml, and 7–42 mIU/mL, respectively). No adverse outcomes related to TRT were reported. Topical TRT appears safe and efficacious in adolescents with 47, XXY (KS) and was not associated with suppression of serum LH or FSH. It is unknown whether treatment with testosterone, combined with an exercise program and dietary modifications, will further lower the risk of developing childhood and adult obesity. Bone mineral density in patients with KS has been shown to correlate positively with serum testosterone, with benefits from testosterone treatment prior to age 20 but not after this age (Kubler et al., 1992; Wong et al., 1993). The benefits of bisphosphonate therapy have also been demonstrated in decreasing markers of bone turnover in patients with KS (Stepan et al., 2003).

4.4

FUTURE DEVELOPMENTS

Gradual and consistent evidence accumulates indicating that early diagnosis and multidisciplinary management of children and adolescents with KS, utilizing hormonal supplementation and manipulation to mimic the normal progression of puberty, is not harmful and likely beneficial. Three major challenges exist in the endocrinological management of KS: overcoming partial androgen resistance; developing oral androgen receptor agonists; and enhancing understanding of ideal estrogen levels to facilitate optimal physical and neurobiological development. With improved understanding of the molecular defects leading to hypogonadism, and the tissue-specific requirements of sex steroids and growth factors, we will be able to plan and execute randomized prospective multicenter clinical trials, which will lead our management of men with KS in the future.

4.5

CONCLUSION

KS is the most frequent chromosomal abnormality in infertile males. Physicians should be aware of the variety of issues associated with KS, including testicular changes with seminiferous tubules degenerating during pubertal maturation leading to hypogonadism. Early fertility discussion with detection and appropriate management of low testosterone associated with KS has significant po-

ENDOCRINOLOGICAL ISSUES AND HORMONAL MANIPULATION 29

tential to avoid the perils of hypogonadism and to have somatic and behavioral benefits along with improvement of general health, pubertal progression, academic progress, and social integration.

4.6

ACKNOWLEDGMENTS

This study has been supported by The Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust and Robert Dow Foundation.

31

CHAPTER 5

Neurological Functioning in 47, XXY Andrea L. Gropman In this chapter, we move from a discussion of basic science and hormonal development to an investigation into the neural underpinnings of cognitive and behavioral dysfunctions observed in males with 47, XXY. Also referred to as Klinefelter syndrome (KS), 47, XXY is one of the most common chromosome aneuploidies overall, with an estimated incidence rate of 1 in 650 males (Nielsen and Wohlert, 1991). In addition, 47, XXY provides us with an ideal human model for studying the impact of X-chromosome gene expression and potential androgen effects on brain development and subsequent cognitive function. The presence of 1 or more additional X chromosomes results in testicular dysgenesis, leading to a hypergonadotropic hypogonadism state. Gonadal development begins as early as the first trimester, typically at 9‒10 weeks gestation. Subsequently, the central nervous system, including gonadal development in boys with XXY, is altered early in prenatal growth (Knickmeyer and Baron-Cohen, 2006). Thus, this early lack of testosterone in the developing brain has been associated with a deleterious impact on later neurocognitive development, suggesting that early repletion of testosterone may improve cognitive outcomes (Samango-Sprouse et al., 2011; Samango-Sprouse et al., 2013a). It is our belief that such androgen therapy should be the standard of care in this condition. 47, XXY may be associated with increased psychiatric cognitive comorbidities. The neuropsychological and neurocognitive profiles, however, show a significant amount of variability across the syndrome. This precludes our ability to make sweeping recommendations, and, as a result, clinical management and decision-making should be determined on a case-by-case basis. Additionally, androgen receptor polymorphism (CAG repeat length), skewed X-chromosome inactivation, and parent-of-origin of the extra X-chromosome have all been suggested as influences on the neurocognitive function and psychological traits exhibited by these boys. The contribution, if any, from each of these influences remains unknown—although these factors are actively being researched. In a previous study (Samango-Sprouse et al., 2013a) we suggested that part of the XXY phenotypic profile may be modulated by the presence of familial learning disorders. This may well point to a complex genetic impact on cognitive function and learning from the additive X. Advances in genetics and neuroimaging have substantially expanded our knowledge of potential mechanisms that underlie these XXY phenotypes, including a putative dosage effect of X and Y chromosome genes on neuroanatomical structures and cognitive abilities.

32 X & Y CHROMOSOMAL VARIATIONS

5.1

BRAIN DEVELOPMENT

Sex differences in the brain and behavior are a well-known phenomenon in homo sapiens and elsewhere in the animal kingdom. Neural development already occurs before birth, in utero, and continues thereafter with myelination and the process of synaptic pruning and apoptosis. The role of androgens on sexually dimorphic behaviors was initially described over 50 years ago (Phoenix et al. 1959), yet this topic is being revisited in research studies as attention has focused on the impact of hormonal modulation of brain sexual dimorphisms. Still, in addition to sex differences in hormone secretions, males and females have differences in gene expression arising from genomic imprinting and X and Y chromosomes (van Nas et al., 2009). Androgens influence neurodevelopment, brain function, and behavioral outcomes from as early as 16 weeks gestation, continuing throughout adulthood (Knickmeyer and Baron-Cohen, 2006). Fetal testosterone has been shown to affect the development of the cortex and limbic systems (Hines, 2006). The long-term cognitive effects of infantile androgen deficiency are presently being investigated in boys and men with 47, XXY. Studies suggest that testosterone treatment in adult males with 47, XXY can also result in positive effects (Lanfranco et al., 2004; Patwardhan et al., 2000; Steinman et al., 2009).

5.2

NEUROLOGICAL EXAMINATION IN BOYS WITH XXY

From a neurological standpoint, boys with extra X chromosomes may present because of low tone (hypotonia) and clumsiness. The neurological examination focuses on gross motor skills, which are larger movements made with the arms, legs, or feet, or the entire body. Such gross motor skills as crawling, running, and jumping often show the effect of low testosterone and XXY. Boys with extra X may have delayed motor milestones; they may be late walkers or lack coordination when they run. They may have difficulties climbing on playground equipment or ascending or descending stairs. Generally speaking, in many aspects of motor development boys with XXY appear clumsy when compared to their non-KS peers. Fine motor skills are smaller and more refined actions that pose additional challenges for boys with XXY. When a child picks things up between his finger and thumb or wriggles his toes in the sand he's using his refined motor skills. Poor fine motor skills are often reflected in activities of daily living (ADL), such as having difficulty with the digital manipulation involved in getting dressed—fastening buttons or snaps and zippers. Children with weak fine motor skills may find it difficult to grip a pencil or quickly become fatigued while writing. They often have poor handwriting, and may have a hand tremor when performing writing and other fine motor tasks. Stringing beads or making small objects out of clay may be problematic. All these challenges are often seen in boys with XXY.

NEUROLOGICAL FUNCTIONING IN 47, XXY 33

Motor planning—the ability to conceive of a motor act, plan, and carry it out in the correct sequence from beginning to end—is often difficult for boys with extra X. When given verbal instructions, the required motor action must be correctly identified and then integrated in order to perform, and many boys with extra X have poor neural connections. This lack of motor integration from atypical brain development results in an inability to plan and execute tasks quickly and efficiently, so that the boys can appear clumsy as they execute new tasks. Their difficulty with sensory processing can lead to poor motor planning for both fine and gross motor tasks, such as handwriting, jumping, and other complex actions. These motor planning deficits may impact on all aspects of learning and may be contributory to the behavioral issues associated with XXY. The ability to motor plan is a learned ability that can then be generalized to all unfamiliar tasks so a child does not need to identify the motor plan for each new discrete action over and over again. But, for many boys with extra X, they often have to reinvent the wheel each time they are faced with identifying, planning, and executing even simple motor planning tasks. Just consider how frustrating and taxing it is for the child who has to think and plan the motor actions that come automatically to his peers. The variability and the unpredictability of his planning capacities are very challenging. For instance, a child may know how to skip, but because of his developmental dyspraxia he may be able to skip pretty effortlessly on some days but have trouble doing it on others. He faces real challenges and frustrations because his mastery of motor planning skills remains fickle. Many boys with extra X also have hypotonia, or decreased muscle tone. This is usually detected during infancy and may improve with age. An infant with hypotonia exhibits a floppy quality or feels like a "rag doll" when he or she is held. A hypotonic baby may feel as if it is slipping through your hands when being lifted or held. Infants with hypotonia often are late in achieving fine and gross motor developmental milestones, such as holding up their head when placed in a prone (on their stomach) position or getting and remaining in a sitting position without falling over. Some children with hypotonia may have trouble feeding if they are unable to suck or chew for long periods. A child with hypotonia may also have problems with speech or exhibit shallow breathing.

5.3

COGNITIVE PROFILE

The cognitive phenotype in 47, XXY includes language-based learning disabilities, decreased fine motor skills, and mild deficits in general cognitive ability. Delayed speech development requiring speech therapy (Ratcliffe 1982b; Bishop et al., 2011) has also been observed. Boys with XXY are known to have an increased risk for psychosocial problems and may present with impaired motor development and decreased verbal abilities. As a result, they are more likely than other boys to require physical, occupational, and speech therapy services, as well as educational support (Cohen and Durham 1985b; Nielsen and Wohlert 1990; Nielsen and Pelsen 1987; Ross et al. 2008; Rovet

34 X & Y CHROMOSOMAL VARIATIONS

et al. 1995; Rovet et al. 1996; Sorensen 1992). The cognitive phenotype is typified by impaired performance on measures of language development, attention, and academic abilities. Bruining et al. (2009) found a high incidence of ADHD (63%) and ASD (27%) among 51 47, XXY boys aged 6 to 19 years (Bruining et al. 2009). However, it is important to note that these numbers do not represent the findings from prospective studies of boys who are diagnosed prenatally and given early androgen therapy (Samango-Sprouse et al. 2015c). In younger XXY boys, delays in reaching speech milestones may be readily noted, but signif-icant deficits in higher aspects of expressive language may not be observed until the boys are older. These speech delays in boys with 47, XXY are actually an early presentation of infantile developmental dyspraxia and manifestations of the later language-based learning disorders (Samango-Sprouse and Rogol, 2002). These symptoms are not simple speech and motor delays; they are an early expression of the well-known deficits that are common in XXY. Significantly, these deficits have been minimized and even remediated with early hormonal treatment (EHT) (Gropman and Samango-Sprouse, 2013; Samango-Sprouse et al., 2013a, Samango-Sprouse et al., 2015c)

5.4

PSYCHIATRIC DISORDERS AND X CHROMOSOME DISORDERS

When compared with the general population, both child and adult XXY patients have been reported to exhibit a higher prevalence of psychiatric disorders, including ASD, ADHD, and schizophrenia. However, these studies have had numerous confounding factors, including ascertainment bias, small sample sizes, and inadequate family histories. Studies of XXY males’ vulnerability for psychiatric disorders have been driven by the notion that the X chromosome is enriched with genes involved in neural development and related cognitive and mental functioning. Additionally, future studies should explore the family history of mental illness and other comorbid psychiatric disorders in order to determine the impact of family history vs. additional X on the child's development. Bruining et al. (2009) have reported on a psychiatric screening of a childhood XXY sample (n = 51) with two subgroups: one subgroup was recruited through active follow-up of prenatally diagnosed children; the other subgroup was diagnosed postnatally and recruited with help of pediatricians, endocrinologists, and support groups. Their data showed that 8% met criteria for a psychotic disorder and 45% had isolated psychotic symptoms. There were no significant differences between the two subgroups with regard to risk for psycho-pathology (Bruining et al., 2009). However, the impact and the importance of the psychiatric conditions on functioning within both home and community on boys with XXY warrants further investigation.. Others have reported an association of XXY with schizophrenia (DeLisi et al.,2005; Kumra et al., 1998; Bojesen et al., 2006; Van Rijn et al., 2006a). Although there may be an increased vulnerability to psychiatric disorders in XXY, further research is warranted because many highly impactful

NEUROLOGICAL FUNCTIONING IN 47, XXY 35

variables were not well controlled in these studies. Therefore, before the relationship between psychiatric disorders and XXY can be understood, large unbiased studies without ascertainment bias and with comprehensive assessments should be undertaken.

5.5

SUMMARY

Boys with XXY present with wide variability in their neurological functioning. Although many hypotheses have been postulated, most studies have been confounded by timing of diagnosis, ascertainment bias, and very small cohorts. However, there is general consensus that delayed speech and language milestones are associated with later learning disabilities. Executive dysfunction occurs in a subset of these boys, but it is difficult to discern who is at risk within the general population of boys with XXY prior to 6 years of age. The benefits of hormonal therapy are becoming more apparent with time, and androgen deficiency impacts motor, speech, and language development, as well as learning, executive function, anxiety, and behavioral issues.

37

CHAPTER 6

Neuroimaging in XY Chromosomal Disorders Andrea Gropman, Colleen Keen, and Carole A. Samango-Sprouse

6.1

NEUROIMAGING

Increasingly sophisticated neuroimaging technology has provided us with unique opportunities to view the brain and its structures and simultaneously increased our understanding of the intimate dance that occurs between brain development, neurocognition, and behavioral outcome (Giedd et al., 2007). As a result of extensive neuroimaging studies conducted over the past 20 years, we have become more aware of differences in the neurologic functioning of individuals with XY chromosomal disorders. These advances in neuroimaging are beginning to help inform the link between cognitive variance and underlying structural damage. Similar to neurodevelopmental progression in both typical and atypical individuals, brain development is uneven. Brain growth is commonly non-linear and characterized by regional variations (Giedd et al., 2007). There are, however, some generally accepted facts associated with early brain development. These are: • volumetric growth varies regionally; • white matter develops faster than gray matter; and • brain growth in females occurs earlier than in males in most regions of the brain. As we better understand the natural history of brain development and its relationship to neurodevelopmental progression, our ability to comprehend the impact of specific genetic disorders on developmental outcomes is enhanced. Our growing awareness of the effects of gene dosage on brain development has helped reveal the basis for functional differences observed in affected individuals. Our increased understanding of these factors has enabled us to more readily configure targeted treatments and optimize outcomes for those individuals with XY chromosomal disorders.

38 X & Y CHROMOSOMAL VARIATIONS

6.2

GENDER DIFFERENCES IN BRAIN DEVELOPMENT

Sexual dimorphism in human brain anatomy has become a well-established fact. However, brain growth trajectories and the specific ages for this growth have been more challenging to determine (Giedd et al., 2007). Trajectories of brain growth are affected tremendously by age, gender, and individual variation. These brain and gender variations have confounded the longitudinal studies of brain development in individuals with and without neurogenetic disorders (Giedd et al., 2007). The underlying dilemma can be simply summed up: “Is this cognitive profile due to the disorder, or does it result from individual variation, or is it linked to age?" Previous studies suggest that gender is the single greatest discriminating morphometric factor in brain size in humans. Factors giving rise to sexual dimorphism may be risk factors or protective agents for neurodevelopmental disorders, and understanding the development of sexual dimorphism may provide insights into the pathogenesis of neurodevelopmental disorders. One way to study dosage effects of genes on the X and Y chromosomes is to conduct clinical imaging and cognitive studies of patients with X and Y chromosome aneuploidies (Skakkebaek et al., 2014). Figure 6.1 shows a series of scans serving as a visual summary of several detected regions with significant group differences between XXY brains and normal control brains. The underlying image is the template that we used to normalize individual brains. Color-coding was based on the values of the T-Test statistic. Only the voxels with significant group differences, i.e., the corrected p values exceeding a significance threshold 0.05, are shown. (Voxel-based morphology (VBM) is a neuroimaging technique that allows investigation of focal differences in brain anatomy.)

NEUROIMAGING IN XY CHROMOSOMAL DISORDERS 39

A

E

B

F

C

G

D FIGURE 6.1: Visual summary of the atypical regions of the brain in boys with XXY. The high-

lighted regions are: (A) Left hippocampal formation, (B) Left superior temporal gyrus, (C) Cingulate region, (D) Left insula, (E) Right amygdala, (F) Left middle temporal gyri, and (G) Right parietal lobe. Courtesy of Shen et al., 2004.

Neuroimaging studies of children and adults with 47, XXY show characteristic structural changes when compared to typically developing individuals. There are increases in the grey matter volume of the sensorimotor and parieto-occipital regions, as well as significant reductions in amygdala, hippocampal, insular, temporal, and inferior-frontal grey matter volumes.

40 X & Y CHROMOSOMAL VARIATIONS

The red areas indicated in Figure 6.2 depict brain regions in which 47, XXY boys had increased gray matter volumes, as compared to typically developing 46, XY boys. The blue areas indicate brain regions of decreased gray matter volume in boys with 47, XXY, as compared to typically developing boys. Widespread white matter abnormalities have been reported. Based on volumetric imaging, there are reductions in anterior cingulate bilaterally and increases in left parietal lobe. Mechanisms underlying these developmental anomalies could include an imbalance in gene dosage relative to typical men or women, as well as the potential consequence of endocrinological deficits. However, these factors are still not well understood. The typical cognitive profile of 47, XXY, which includes language delay and language-based learning disabilities, executive and attentional dysfunction, and motor development delays, has been previously well characterized (Geschwind et al., 2000, Wattendorf and Muenke, 2005). These characteristics are associated with neuroanatomical variations using volumetric MRI (Itti et al., 2006). Differences in the neurobehavioral and neuroanatomical phenotype associated with 47, XXY may be relevant to an understanding of typical brain development, as well as augment our understanding of the impact of gender on neuroanatomical structures. There is some discrepancy in the neuroimaging studies. While many studies report reduced total brain volume (TBV) in 47, XXY (Warwick et al., 2003; Shen et al., 2004; DeLisi et al., 2005), some report no significant difference (Patwardhan et al., 2002; Itti et al., 2006). In addition, XXY males typically have reduced frontal and temporal grey matter (Shen et al., 2004, DeLisi et al., 2005). Some studies report differences in white matter (Rezaie et al., 2009), whereas others do not (Giedd et al., 2007). Discrepancies in findings may be due to imaging subjects at different ages, as well as using different neuroimaging acquisition techniques and neuroimaging analysis methods (Steinman et al., 2009). It is important to standardize studies and be cognizant of whether subjects have begun puberty or received testosterone replacement therapy (TRT), both of which are likely to influence brain development (Patwardhan et al., 2000; Wattendorf and Muenke, 2005).

NEUROIMAGING IN XY CHROMOSOMAL DISORDERS 41

FIGURE 6.2: Brain imaging of boys with XXY compared to typically developing boys. Courtesy of Bryant et al., 2012.

42 X & Y CHROMOSOMAL VARIATIONS

6.3

47, XXY AND NEUROIMAGING FINDINGS

Adolescents with X and Y disorders provide a unique opportunity for investigation in neuroimaging studies because they constitute a homogeneous group of juveniles who reflect a possible hormonal influence on brain development. They afford us the opportunity to observe and learn about gender diversity and variable gene expression on brain development. The neuroanatomical phenotype for the disorders of XXY, XYY, and XXX are evolving, but remain hampered by small cohorts of subjects, ascertainment bias, and limited longitudinal follow-up data. Studies may be further compromised because families whose children are more atypical often choose to participate frequently in studies. Conversely, families who have children with milder phenotypes may avoid participation. Finally, the low rate of detection in XXY (