Seeking Cures : Design of Therapies for Genetically Determined Diseases 9780199915873, 9780199915866

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Seeking Cures

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Seeking Cures Design of Therapies for Genetically Determined Diseases

Moyra Smith, MD, PhD, MFA

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3 Oxford University Press is a department of the University of Oxford. It furthers the University’s objective of excellence in research, scholarship, and education by publishing worldwide. Oxford New York Auckland Cape Town Dar es Salaam Hong Kong Karachi Kuala Lumpur Madrid Melbourne Mexico City Nairobi New Delhi Shanghai Taipei Toronto With offices in Argentina Austria Brazil Chile Czech Republic France Greece Guatemala Hungary Italy Japan Poland Portugal Singapore South Korea Switzerland Thailand Turkey Ukraine Vietnam Oxford is a registered trademark of Oxford University Press in the UK and certain other countries. Published in the United States of America by Oxford University Press 198 Madison Avenue, New York, NY 10016

© Oxford University Press 2014 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, without the prior permission in writing of Oxford University Press, or as expressly permitted by law, by license, or under terms agreed with the appropriate reproduction rights organization. Inquiries concerning reproduction outside the scope of the above should be sent to the Rights Department, Oxford University Press, at the address above. You must not circulate this work in any other form and you must impose this same condition on any acquirer. Library of Congress Cataloging-in-Publication Data Smith, Moyra, author. Seeking cures : design of therapies for genetically determined diseases / Moyra Smith. p. ; cm. Includes bibliographical references and index. ISBN 978–0–19–991586–6 (hardback : alk. paper)—ISBN 978–0–19–991587–3 (UPDF ebook)—ISBN 978–0–19–932605–1 (EPUB ebook) I. Title. [DNLM: 1. Genetic Diseases, Inborn—therapy. 2. Genetic Therapy. QZ 50] RB155 616′.042—dc23 2013004975

9 8 7 6 5 4 3 2 1 Printed in the United States of America on acid-free paper

Hope is the name not of a temperament or of an emotion but of a virtue. A virtue is something that you have to work at, something you have to do. And we can think and act as if it is possible to survive and make things better, because hope is a great energizer, a comforter and an inspirer. —Philip Pullman, quoted by Georgina Ferry Nature 459:35 (2009)

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CONTENTS

Preface ix Acknowledgments xi 1. Introduction and History 1 2. Therapy: Design and General Approaches 13 3. Inborn Errors of Metabolism: Progress in Diagnosis and Treatment 39 4. Lysosomal Storage Diseases and Therapies 67 5. Mitochondrial Function, Defects, and Approaches to Treatment 83 6. Protein Misfolding, Endoplasmic Reticulum Stress, and Pathogenesis of Disease 103 7. Transporters and Solute Carriers: Proteins That Transport Molecules Across Membranes 115 8. Advances in Therapy for Specific Monogenic Diseases 137 9. Identifying Therapeutic Targets in Complex, Multifactorial Diseases 157 10. Approaches to Cancer Treatment 185 vii

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11. Gene-Based Molecular Therapies 207 12. Stem Cells and Induced Pluripotent Stem Cells 225 Epilogue (Envoi) 239 References 241 Index 283

PREFACE

Clinicians involved in the care of patients with disorders apparently due to genetic defects frequently confront situations in which the diagnosis is not clear. Fortunately, progress in molecular genetics together with availability of DNA sequence information has expanded possibilities for establishing diagnoses. However, too frequently, even in cases where diagnoses are made, treatment options remain inadequate. Increasingly molecular techniques to analyze gene sequence and gene regulation, knowledge of gene interactions, and downstream effects of gene changes enable identification of therapeutic targets. Many additional steps are, of course, required to move from therapeutic targets to effective therapeutic agents. Fortunately, progress in development of high-throughput methodologies facilitates identification of potential diseasemodifying small molecules. Primary goals of patient care involve taking care of medical emergencies and providing symptom relief. However, there are compelling reasons to provide long-term relief from disease manifestations and to improve well-being of patients with genetically determined diseases when disease is lifelong. In this book I document progress in identification of therapeutic targets and in initiating novel treatments at the levels of the gene, RNA, protein, and ix

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downstream physiology. Also considered are aspects of treatment at the cellular level, for example, with hematopoeitic stem cells or induced pluripotent stem cells. One question that may arise is why I selected specific diseases to review. Patients I have interacted with over the years and situations that emerged when treatments were inadequate or totally lacking in part influenced my choices. Examples included patients with lysosomal storage disease, and patients suffering from muscular dystrophy, tuberous sclerosis, or epidermolysis bullosa, to name a few. Also included for discussion are more recently defined disorders including those that involve specific solute carriers and diseases due to endoplasmic reticulum stress. In addition I chose to explore conditions that are frequent in the population and remain without adequate treatment, for example, Alzheimer’s disease and autism. Also included is a chapter that documents progress in identification of genetic and metabolic changes in cancer that continue to define therapeutic targets and are utilized to design more effective treatments.

ACKNOWLEDGMENTS

I thank Chad Zimmerman, editor at Oxford University Press, for his encouragement and guidance throughout the processes of conception and realization of this project. I wish to express by gratitude to Maria Cusano for valuable assistance with copyediting, and to Kurt Roediger and to Emily Perry for assistance during production. Any errors remaining are my responsibility. I have benefited greatly from the resources of the University of California library system and was privileged to use the Bodleian Library at Oxford University. I wish to acknowledge support from the Institute for Clinical and Translational Sciences at the University of California, Irvine, funded through the National Institutes of Health CTSA grant NIH UL1 TR000153.

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1 INTRODUCTION AND HISTORY

But these great ones form only a small minority and most of those upon our lists were men of lesser stature, but as teachers who handed down to their pupils the treasures of their observations and experience, busy practitioners, keen observers or men of varied culture, few of them can have failed to add something to the sum of human knowledge. — A.E. Garrod, Harveian oration (1924)

Progress in molecular and cellular biology since completion of the Human Genome Project in 2003 has greatly enhanced abilities to accurately diagnose many diseases due to gene mutations and alterations in gene structure. The number of cases of genetic diseases and birth defects with undefined molecular diagnostic criteria grows progressively shorter as next-generation DNA sequencing methods are applied. More recently increased emphasis has been placed on translational research and on searches for treatment in a broad range of genetically determined diseases. This emphasis grows in part from information gathered on underlying molecular pathogenesis of genetic diseases and identification of potential therapeutic targets. The existence of such targets stimulates energies and hopes in patients and families and in the professionals engaged in their care. Current progress in medical genetics builds on the work of many physicians and scientists who preceded the genome sequencing era, and it is worthwhile revisiting examples of their contributions. 1

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Foundations of the discipline Human Genetics were laid by physicians, and biologists and statisticians who provided detailed descriptions of diseases classified as hereditary. Detailed descriptions of many inherited disorders along with extensive examples of pedigrees and analyses of frequency, sex, and age distribution were published almost yearly in the Treasury of Human Inheritance, compiled by members of staff of the Galton Laboratory, University of London, between 1909 and 1958 Julia Bell authored most of the articles. She compiled information from diverse sources and from her own studies. It is interesting to revisit descriptions in the Treasury of Human Inheritance of three genetic diseases that have now been extensively analyzed through molecular genetics. In two of these disorders, hemophilia and Duchenne muscular dystrophy, there are encouraging data from clinical trials of targeted treatments; in the third, Huntington’s chorea, though much is known about the defect and there is valuable information that can be used for diagnosis and prediction, no treatment is currently known. However, studies on model organisms are providing interesting leads to possible therapies. Writing in a Treasury of Human Inheritance article in 1911, Bulloch and Fildes noted that the advent of circulating medical literature in the early 19th century led to an increase in the documentation of families with hereditary diseases. One example was the publication in 1803 by Dr. John C. Otto, a physician in Philadelphia, of “An account of an haemorrhagic disposition existing in certain families.” Otto reported that males only were affected and all males in a family were not affected. Though females were free of disease, they were capable of transmitting it to their children. It is also worth noting that Julia Bell and J.B.S. Haldane in 1937 published evidence of linkage of hemophilia and color blindness through extensive analysis of pedigrees. In the current human gene map, NCBI version 37.5 (2012), red–green color blindness, referred to as Opsin 1, maps to Xq28 between 153,409 kb and 153,424 kb, and hemophilia A (Factor VIII) maps to Xq28 between 154,206 kb and 154,060 kb. Purified Factor VIII protein is now a mainstay of treatment for this disorder. Furthermore, there is evidence that gene therapy is also effective in treatment of hemophilia due to Factor IX deficiency (Nathwani et al., 2011). In an edition entitled “Pseudohypertrophic and Allied Types of Progressive Muscular Dystrophy” published in 1943, Julia Bell wrote, “A glance at the bibliography appended will reveal the names associated with early accounts of the disease including the classical descriptions of Duchenne, Erb, Landouzy and Dejirine. As early as 1875 Poore, an American, provided some analyses of 85 cases of the disease” (pp. 19–20). On the basis of examination of pedigrees of families with muscular dystrophy, Bell distinguished sex-linked forms, and recessive and dominant forms. She provided data on 129 sex-linked pedigrees

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in which 99.3% of affected cases were male. In 93 pedigrees of recessive muscular dystrophy, 49% of cases were male, and in 51 pedigrees of dominant muscular dystrophy, 54.3% of cases were male. Antisense oligonucleotides are proving useful in the treatment of a number of mutations in the Duchenne muscular dystrophy gene (DMD) that maps to Xp21.2. In Part 1 of Volume IV entitled “Nervous Diseases and Muscular Dystrophies” published in 1934, Julia Bell recorded that in 1872 George Huntington published an account of hereditary chorea in patients in East Hampton, New York. He noted that the disease was already established in the community when his grandfather practiced medicine starting in 1787. His grandfather was, however, not called upon to treat the disease; he noted that the sufferers were well aware that there was no treatment for their malady. Describing the disease manifestations, Bell wrote, “the movements in chorea are aimless, often forceful and often complicated, there is nothing rhythmical or orderly about them; they show themselves unexpectedly” (pp. 4–5). Huntington’s disease was mapped to chromosome 4p16.3 by Gusella et al. (1983) and was subsequently shown to be due to a repeat expansion in a gene HTT located in this chromosomal segment (The Huntington’s Disease Collaborative Research Group, 1993). This repeat expansion leads to production of an mRNA that encodes a greatly increased number of glutamate residues. In studies on a mouse model of the disease, Kordasiewicz et al. (2012) demonstrated that transient infusion into the cerebrospinal fluid of an antisense oligonucleotide directed against the repeated sequence in the mutant HTT mRNA catalyzed its degradation and led to reversal of disease manifestations.

METABOLISM ENZYMES AND COFACTORS: HISTORICAL ASPECTS Early studies on the fermentation of sugars led Emil Fischer in 1894 to propose the existence of enzymes that interacted with specific substrates. He used a lock and key analogy to describe the specificity of these reactions. Michaelis and Menten (1913) published information on properties of enzymes and enzyme kinetics. Processes involved in biological oxidation and energy generation were intensely studied between 1900 and 1940. Steps in glucose conversion to pyruvate and lactate and in glucose conversion to glycogen were elucidated through studies by Emden and Lauer (1921), Meyerhof (1930), Cori et al. (1939), and many others. Krebs and Johnson (1937) elucidated steps in intermediary metabolism and citric acid cycle. In the course of studies on metabolism, it became clear that many enzymes involved in metabolism required cofactors. In 1912 Hopkins drew attention to

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the requirements for “organic accessory factors” for metabolism. These factors were subsequently named vitamins. Many studies between 1915 and 1938 led to isolation of vitamins A, B, C, D, E, and K. Detailed analysis of functions of vitamins in enzyme action were presented by Lohman and Shuster in 1937, who demonstrated that vitamin B1, thiamine, acts as a cofactor for a specific enzyme involved in decarboxylation of pyruvate. It is important to note that characterization of vitamin responsiveness of enzymes and mutated forms of these enzymes remains important in the treatment of inborn errors of metabolism.

INBORN ERRORS OF METABOLISM The concept of inborn errors of metabolism was first presented in 1908 by Archibald Garrod based on his discovery of inheritance of alkaptonuria. However, key studies delineating inborn errors of metabolism in humans were reported primarily after 1934 when Asborn Folling discovered phenylketonuria (PKU) as a cause of cognitive impairment and developmental delay in children. In 1954, Bickel et al. published results of dietary therapies they developed for PKU and demonstrated that early dietary treatment of PKU-affected infants prevented mental retardation. These discoveries stimulated research into human metabolic diseases and led to initiation of newborn screening for these disorders. Technical advances in metabolite analyses by means of chromatography and mass spectrometry led to discovery of inborn errors of metabolism associated with quantitative and qualitative changes in organic acids (Tanaka et al., 1966). Later, severe, different forms of organic aciduria were shown to be vitamin responsive; for example, the health of some patients with methylmalonic aciduria improved dramatically on treatment with vitamin B12 (Rosenberg et al., 1968).

CELLULAR ORGANELLES Development of electron microscopy revealed the presence of subcellular organelles including mitochondria, lysosomes, and peroxisomes. Details on mitochondrial structure were elucidated by Pallade (1953). Studies on mitochondrial metabolism and functions of the respiratory chain and oxidative phosphorylation were published by Chance and Williams (1956). Anderson et al. published the complete mitochondrial DNA sequence in 1981. Early

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elucidation of impaired mitochondrial function and resulting diseases derived from studies by Robinson et al. (1980), DiMauro et al. (1980), and Wallace (1982). The mitochondrial DNA encodes only 13 proteins. Most proteins and enzymes required for mitochondrial functions are encoded in the nucleus. Currently 1,013 mitochondrial proteins and enzymes are characterized, and information on these is available in the Mitocarta database: http://www.broadinstitute.org/pubs/MitoCarta/human.mitocarta.html. De Duve (1963) isolated lysosomes and characterized their functions (De Duve and Wattiaux, 1966). Following this, important information emerged about storage diseases due to impaired lysosomal function, starting with studies on Pompe disease (Hers, 1965) and Tay-Sachs disease (O’Brien et al., 1970). Considerable progress has been made in the treatment of a number of lysosomal storage diseases; these are described in Chapter 3. DeDuve first described peroxisomes, and described the first peroxisomal disease, Zellweger syndrome, in 1973.

INDIVIDUAL VARIATION, MUTATIONS, AND POLYMORPHISMS During the past half-century, intense efforts have been directed toward finding key mutations in enzymes and proteins that constitute the primary defects in specific inborn errors of metabolism. Garrod (1902) predicted that inborn errors of metabolism were “merely extreme examples of variations of chemical behavior which were probably present everywhere in minor degrees” (p. 1624). He noted further that “chemical individuality” leads to predisposition to or immunity from specific diseases. Harry Harris (1963) returned to this concept. Harris and his colleagues (1970) made substantial contributions to elucidations of individual variation in proteins and enzymes (see figure 1–1). Writing of inborn diversity in biochemical makeup, Harris (1966) emphasized, “A particular variant may be rare or common. It may result in pathological consequences for the individual or it may lead to no obvious effects on viability or biological fitness” (p. 8, para. 3).

BUILDING THE GENE MAP Enzyme and protein polymorphisms were useful in developing the gene map, both through family linkage studies and in the analysis of chromosome segregation in somatic cell hybrids. Hybrid cells were generated from fusion of mouse or Chinese hamster cell lines with human cells. Human chromosomes were lost

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– ADH3phenotype

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Figure 1–1. This figure illustrates allelic variation of isozymes of alcohol dehydrogenase locus 3.

from these hybrid cells (Weiss and Green, 1967). Combined analyses of the specific human chromosomes present in a specific hybrid clone and identification of human proteins and enzymes present in that clone facilitated the mapping of genes that encoded proteins and enzymes to human chromosomes. Development of methods to analyze DNA polymorphisms, including restriction fragment length polymorphisms and later microsatellite repeat sequence polymorphisms, greatly enhanced development of the human linkage gene map (Dib et al.1996; Jordan and Collins 1996). Development of libraries of cDNA clones that represented expressed genes, (expressed sequence tags ESTs), also facilitated assignment of specific genes to chromosomes and development of the physical map of human chromosomes.

IDENTIFICATION OF SPECIFIC HUMAN CHROMOSOMES Introduction of techniques that demonstrated banding patterns on chromosomes examined microscopically in 1970 greatly improved identification of human chromosomes. Banding techniques involved use of fluorescent dyes (Caspersson, 1970) or trypsin digestion of chromosome spreads followed by Giemsa staining (Seabright, 1972) (see figure 1–2). Availability of cloned segments of DNA and of techniques to fluorescently label DNA led to the development of fluorescent in situ hybridization (FISH), which further enhanced gene mapping and the identification of specific chromosome regions impacted by structural abnormalities including deletion, duplication, translocation, or inversion (see figure 1-3).

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Figure 1–2. This figure illustrates trypsin Giemsa banding of chromosomes in a case of trisomy 13.

Figure 1–3. This figure illustrates use of a fluorescent in situ hybridization with a region-specific probe to demonstrate presence of an extra chromosome, an inverted duplication of 15q11-q13, also illustrated on cover.

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THE GENE MAP AND POSITIONAL CLONING Availability of linkage maps and physical maps of human chromosomes enabled positional cloning of disease-causing human genes. The strategy involved identification of chromosome regions that segregated with the disease in linkage studies in families or regions that were impacted by structural changes (e.g., deletion or translocation in individuals with that disease). Following assignment of a disease gene to a defined chromosome region, DNA clones corresponding to that region could be isolated and sequenced to search for mutations in genes. Lee et al. reported successful positional cloning of the retinoblastoma gene in 1987, and in the following years genes involved in Duchenne muscular dystrophy, cystic fibrosis, neurofibromatosis, and Huntington’s disease were identified through positional cloning. Identification of mutations or structural changes in genes that led to specific diseases opened the way for more accurate diagnoses, including presymptomatic and prenatal diagnoses. One striking example was the identification of the abnormal expansion of triplet nucleotide repeats in Huntington’s disease, which enabled presymptomatic testing (see figure 1–4). Analysis of the downstream mechanisms through which mutations led to impairments of function provided insight into possible therapeutic measures. Early on, however, some geneticists realized that studies of gene defects and downstream functional impairments were likely to be complicated. In 1970 Harry Harris wrote, “the characteristic features that we observe in any one disorder must often represent the consequences of a very complex chain of phenomena involving interaction at many different levels of the biochemical and physiological organization of the organism” (p. 144, para. 3). DNA SEQUENCING AND GENE ANALYSIS Analysis of gene structure was greatly facilitated by implementation of sequencing methods by Sanger and colleagues, who pioneered methods that involved DNA synthesis with nucleotide primers and DNA polymerase (1975) and DNA sequencing with chain termination inhibitors (1977). Availability of DNA sequence information and of reagents such as polymerase and Taq polymerase led to the development of the polymerase chain reaction and application to mutation detection (Mullis et al., 1986). SEQUENCING THE HUMAN GENOME Two different approaches were taken to sequencing the human genome in the Human Genome Project. One utilized DNA clones with known chromosomal

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Figure 1–4. This figure illustrates region of expansion of nucleotide triplets as seen on gel electrophoresis and autoradiography of sequenced DNA, the technique used to determine DNA sequences (circa 1985) before capillary sequencing and next-generation sequencing.

map positions and then moved out to isolate adjacent DNA probes to identify a tiling path of DNA segments. Another approach was to derive sequence from clones, with or without known map positions, and to identify adjacent clones through identification of overlapping DNA sequences. The two different approaches were ultimately mutually beneficial, leading to publication of a “complete” human genome sequence in 2003 (Guttmacher and Collins, 2003). Sequencing methodologies have greatly expanded since 2001. In next-generation sequencing methodologies, genomic DNA is fragmented to yield a library of small segments that are amplified and sequenced in parallel and the sequences are then aligned.

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STRUCTURAL VARIATION AND SEGMENTAL GENOME DOSAGE DIFFERENCES Availability of microarray technologies to examine DNA across all chromosomes led to surprising discoveries between 2004 and 2007. The extent of segmental copy number variation (CNV) in DNA from normal controls was unexpected. Large-scale population studies were required to establish criteria to determine which CNVs represented polymorphisms and which were likely to play roles in disease causation. Lupski (1998) used the term genomic disease to describe disease that arose as a result of segmental dosage changes or genomic rearrangements. In considering the relevance of CNVs to disease, Rosenfeld et al. (2010) reported differences between individuals in the degree of phenotypic variation induced by a specific segmental copy number variant. For example, diverse phenotypes occur in different individuals with a specific deletion with defined size and defined location within chromosome 16p11.2. Stefansson et al. (2008) proposed that rare variants that arise de novo in an individual are more likely to be significant as a cause of disease. There is evidence that rare deleterious structural chromosome changes play roles in the etiology of autism, schizophrenia, and cognitive impairment (St Clair, 2009). Unfortunately, treatments for patients with segmental chromosome imbalances are not likely to be readily developed given that each these changes often encompasses a number of genes.

ANALYSIS OF PHENOTYPE Freimer and Sabatti (2003) outlined goals of the Phenome Project. These included collecting phenotypic information at different resolution, whole-organism, tissue, cellular, molecular, and gene expression levels. They proposed that neuroimaging and other imaging data should be included when available and that digital imaging and quantitative measurements, for example, to define facial features, be carried out since these are important aspects of the phenotype. An important aspect of data collection for inclusion in databases is ontology, that is, structurally controlled vocabulary.

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SHIFT TO THERAPEUTIC EMPHASIS During past decades much emphasis has been placed on accurate diagnosis and delineation of underlying molecular defects in genetically determined diseases. With the exception of inborn errors of metabolism, few therapies were developed that directly targeted the underlying gene and protein defects prior to 2000. Expanded possibilities to identify specific targets have begun to fuel a new emphasis on therapy for genetically determined diseases.

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2 THERAPY Design, and General Approaches

INTRODUCTION Therapies can be classified as primarily targeted toward alleviation of symptoms or designed for disease modification. The search for disease-modifying therapies is a stepwise procedure and may include the following five steps: (1) Assess whether quantitative changes in the production of specific substances occur and determine whether these changes are specific for the disease. Therapies may be designed to restore normal levels. (2) Establish whether the qualitative and functional changes in specific proteins or enzymes specific to the disease are due to germline mutations or to somatic mutations. Therapeutic strategies may be designed to provide normal protein or enzyme or perhaps to neutralize or bypass the specific mutation. (3) Evaluate functional defects to establish whether specific signaling pathways or cellular processes are involved. A number of different diseases may impact functioning at different points in a signaling pathway. For example, multiple disorders are impacted by alterations in the mTOR pathway and may respond to treatment with mTOR inhibitors. 13

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(4) Establish whether functions of specific cellular structures (e.g., receptors), or organelles (e.g., lysosomes or mitochondria), are impaired or whether a specific cellular function is impaired. The same underlying cellular pathology may occur in a number of different diseases; for example, endoplasmic reticulum stress may occur in several diseases that are associated with the presence of protein aggregates. Therapeutic strategies may be designed to alleviate aggregation and promote protein folding. (5) Consider whether downstream manifestations of the disease are therapeutic targets. These could include altered expression of genes other than the primary disease-causing gene, causing secondary effects on metabolism. This chapter includes review of cellular entities (e.g., receptors, signaling pathways) and processes, such as metabolism, that are targets for many of the currently available therapeutic agents. Also included are brief discussions of clinical trials, pharmacogenetics, and personalized medicine.

PRECLINICAL TRIALS Preclinical trials involve analyses of the effects of the proposed therapeutic agent on its target and on physiological functions in a cellular system or model organisms. However, compounds that have been successful in preclinical trails have high failure rates in clinical trials. This failure includes unexpected side effects or lack of efficacy in humans (Duyk, 2003).

CLINICAL TRIALS Randomized clinical trials and double-blind trials have been considered the gold standard over many years. Increasingly, clinical trial processes are being modified. Modifications include introduction of open-label trials, crossover trials, and adaptive clinical trials. Adaptive clinical trials include prospectively planned opportunity for modification of one or more specified aspects of the design based on interim analysis of the data. Examples of such modifications include modification of the dose of the compound, or patient population changes based on characteristics of the patients that show greater response or based on variation of concentrations of specific biomarkers in response to treatments. Advances in computational capabilities and growing sophistication in statistical analysis methodologies facilitate these clinical trial modifications.

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Protein Therapeutics The use of proteins in the treatment of diseases has increased dramatically in recent years due to recombinant DNA techniques that facilitate manufacture of proteins in bacterial, animal, human, and plant systems. Leader et al. (2008) proposed a functional characterization of protein therapeutics. In their classification, Group 1 therapies included functional or regulatory proteins and enzymes used to replace an abnormal or deficient entity or to augment an existing pathway. Examples of protein replacement include therapy with insulin, with growth hormone, insulin-like growth factor, protein C, or coagulation Factor VIII or Factor IX. Examples of enzyme proteins used to replace deficient or defective enzymes include recombinant proteins to treat lysosomal storage disease such as Gaucher, Fabry, and Pompe diseases, and mucopolysaccharidosis (e.g., Hurler and Hunter diseases). Alpha 1-antitrypsin (α1 proteinase) is used to treat deficiency of that component. In cystic fibrosis animal-derived pancreatic enzymes lipase, amylase and protease are used to compensate for secondary pancreatic insufficiency. DNAase is also sometimes used to treat respiratory tract accumulation of degraded cellular material. For treatment of immune deficiency, adenosine deaminase or pooled immunoglobulins are used. Specific proteins are used to augment levels of normal proteins under certain circumstances; examples include use of erythropoietin or the granulocyte-stimulating protein Filigrastim, to enhance hemapoeitic function. Interferon and interleukin II are sometimes provided as immune regulators. Keratinocyte growth factors are provided to counteract the epithelial damage induced by chemotherapy, and calcitonin protein is sometimes used to treat osteoporosis. To expedite healing of specific types of bone fractures and bone injuries, bone morphogenic protein is sometimes used. Leader et al. (2008) defined a second group of functional therapeutic proteins that have specific targeting activities. These include proteins that target and inhibit certain cellular molecules, often receptors. In this group of targeting proteins, they also included monoclonal antibodies or peptides that bind to a target and deliver specific small molecules (e.g., chemotherapeutic agents).

ANTIBODIES Growing numbers of humanized monoclonal antibodies have been developed to target specific receptors in signaling pathways that are important in

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promoting growth of cancer cells. These antibodies include Herceptin, which binds the HER2 receptor that is upregulated in certain forms of breast cancer. Cetuximab binds to EGFR receptors, and Bevacizumab binds to vascular endothelial growth factor. Specific antibodies are also used in the treatment of specific autoimmune diseases. Antibodies that target the tumor necrosis factor receptors are used to treat rheumatoid arthritis and spondylitis. Monoclonal antibodies against integrins are sometimes used to treat relapsing multiple sclerosis. Following transplantation, specific antibodies are used to block the cellular immune response and to avoid graft rejection; these antibodies block Toll receptors (e.g., CD3 and CD25). Antibodies directed against unusual antigens such as foreign blood group antigens are also in clinical use; anti-Rhesus immunoglobulin is in this category. A fourth group of functional proteins with medical application includes proteins used in diagnosis. These include proteins that are radiolabeled and used in imaging studies (e.g., Octreoscan to detect carcinoid tumors).

Drug Discovery In 2000 Jurgen Drews published an article on the history of drug discovery. He noted that the purification of the active ingredients of medicinal plants occurred throughout the 19th century. He reported that by 1870 the foundations of “chemical theory” had been laid. These foundations included the organization of chemical elements into a periodic table, and the emergence of the theory of acids and bases and of theories on the structure of organic molecules. The development of coal tar derivatives and dye chemistry and application of these to the study of biological materials were greatly enhanced through the activities of Paul Ehrlich (1908) and by development of his concept of chemoreceptors. Drews noted that the discipline of Pharmacology rooted in Physiology was greatly expanded after 1918 when Institutes of Pharmacology were established. One example was establishment of such an institute at the University of Strasbourg. Pharmaceutical divisions of companies were also subsequently established. In 1935 Domagh discovered Prontosil, a sulphonamide-like component with antibacterial action. Following isolation of penicillin in 1940 by Chain et al., drug companies investigated heavily in microbiology and in fermentation units. Progress in biochemistry and characterization of enzymes and receptors led to development of a number of drugs including receptor agonists and antagonists. Drews reviewed the impact of molecular biology and molecular biology–based techniques on drug development. A major development was

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the production of recombinant proteins and the development of monoclonal antibodies. Drews noted further that genomic sciences facilitated identification of genes and gene products and enabled elucidation of gene defects in specific diseases. Improvements in the structural analysis of gene products were enabled through progress in methods of protein crystallization, X-ray crystallography, and nuclear magnetic resonance (NMR) spectroscopy. Druggability of a protein is determined by its folding and its capacity to interact with druglike compounds. Determination of druggability is therefore influenced by technologies available to analyze proteins.

COUPLING SMALL-MOLECULE TESTING TO HIGH-THROUGHPUT SCREENING Phenotypic and target-based screening of chemical libraries, high-throughput screening methodologies, are increasingly being applied to the search for new treatments. Phenotypic screening involves analysis of the effects of drugs on gene expression. One approach is analysis of gene expression in cultured cells. Treatment of cells with a specific small molecule is then carried out to determine the effect of the molecules on gene expression. Li et al. (2012) used this approach to screen for drugs that impact androgen gene expression. Androgen receptor gene overexpression is linked to prostate cancer progression. Li et al. determined that Peruvoside, a cardiac glycoside drug used for treatment of heart disease, caused androgen receptor destabilization and decreased expression. Taboureau et al. (2012) reviewed emerging trends in computational drug discovery. In addition to phenotypic screening, computation approaches are used to analyze interactions of specific small molecules with normal and mutant proteins. These methods include in silico virtual screening to examine molecular interactions and analyze metrics of docking of small molecules with proteins based on structural information gathered from X-ray diffraction and NMR spectroscopy analyses. Analysis of networks and metabolic and signaling pathways is increasingly being carried out to predict downstream and off-target effects of small molecules. In order to become useful as drugs, small molecules need to meet additional criteria related to absorption, distribution, metabolism, excretion and toxicity (ADMET criteria). Drug design increasingly involves not only the use of single small molecules, but also the use of fragments. Fragment-based design involves a process in which a low–molecular weight compound that binds to a specific protein cleft or pocket is linked to another small molecule to increase the strength of interaction.

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THREE-DIMENSIONAL TISSUE-BASED SCREENING There is a growing impetus for development of specialized three-dimensional chips for drug screening (Baker, 2011). These chips comprise multicellular architecture fragments representing normal and disease tissue. Examples of multicellular fragments are the spheroids that develop from induced pluripotent stem cells. Enterocytes derived from intestinal stem cells develop into fragments that can be used to monitor drug absorption. Liver cell chips have been used to study drug toxicity. Hepatocytes grown on a silicon scaffold system give rise to three-dimensional structures that have been used to study the impact of specific drugs on liver cells. Microfluidic-based approaches are being utilized to study three-dimensional tissue targets on chips. In this process small volumes of fluid circulate to and from a specific groove or location on the chip.

SYSTEMS BIOLOGY AND PHARMACOLOGY Drug efficacy is dependent not only on the interaction of a small molecule with a target but also on the downstream and system effects of target alteration. There is, therefore, growing emphasis on knowledge of systems biology and on quantitative effects of drugs on biological networks. In their reviews of systems pharmacology, Wist et al. (2009) noted that progress in biochemistry and in molecular biology has enabled more comprehensive identification of targets and that genomic and proteomic technologies have contributed to identification of disease-specific biomarkers. Delineations of gene defects in monogenic diseases have in some cases been followed by development of therapies. Genomic analyses have also identified specific genes and variations related to individual differences in drug responses. Wist et al., however, emphasized that concurrent alterations in a number of different genes constitute the bases for complex diseases, and designing treatment options for these diseases is therefore difficult. They noted that systems to be analyzed in the context of complex diseases exist at the organismal, tissue, cellular, and molecular levels. Organismal-level studies include clinical evaluations and blood chemistry studies. At the tissue and cellular levels, imaging technologies facilitate analysis. Molecular-level studies involve protein and enzyme studies and analysis or protein–protein interactions. Application of genomic and proteomic techniques to analyze cellular processes may lead to identification of key nodes within networks. This network analysis may lead to identification of new targets. Increasingly metabolomic

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analyses are aiding in systems biology approaches to studies of disease perturbations. Mass spectroscopy is frequently used to resolve disease-specific metabolic signatures. Also, the effects of therapy on metabolic profiles can be quantified.

SYSTEMS BIOLOGY AND RELEVANCE TO PREDICTABILITY OF THERAPEUTIC AGENTS Duyk (2003) reported that given the more than 90% failure rate of clinical efficacy of drug candidates that have efficacy in preclinical models, most companies chose to develop compounds similar to known therapeutic compounds. He attributed this failure to incomplete knowledge of pathways and networks and failure to adequately link genetics to physiology and establish chains of causality.

MOLECULAR COMPLEXES THAT FUNCTION AT SIGNALING PATHWAY HUBS Chemotherapeutic agents that impact intracellular signaling continue to be developed primarily for the treatment of cancers. Cellular signaling pathways play key roles in control of growth proliferation. Drugs that impact the function of molecular complexes located at signaling pathway hubs often have broad effects and may be used to treat a number of different diseases, particularly diseases in which abnormal proliferation occurs. One example is the macrolide rapamycin (sirolimus), originally isolated from a species of Streptomyces. The molecular target of rapamycin was identified and isolated by a number of investigators including Sabatini et al. (1994). They named this target mTOR (mammalian target of rapamycin); mTOR acts as a serine protein kinase and is present in two different molecular complexes: mTORC1 with six components and in mTORC2 with seven components; see figures 2–1 and 2–2.

THERAPIES FOR MONOGENIC DISEASES CHARACTERIZED BY SIGNALING PATHWAY DEFECTS Davies et al. (2011) reviewed the use of small-molecule inhibitors as targeted therapeutic agents. They emphasized that these drugs achieve greatest success

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Seeking Cures Receptor Cell membrane

PI3K

PTEN PIP3

AKT AMPK

TSC2 TSC1 mTORC1 complex

RHEB RAPTOR

PRAS40

mTOR

RAPAMYCIN

4EBP1

4EBP1 eiF4E elF4E eiF4G mTOR increase

Figure 2–1. pathway.

S6 KINASE

elF4E

Translation, protein synthesis, cell growth

Tuberous sclerosis gene products and mTOR in mTORC1 signaling

Activated receptor Cell membrane

PI3K

mTORC2

GSK3

AKT

SGK1

Metabolism

PKCA

Cytoskeletal Organization

Figure 2–2. Receptor activation and downstream signaling pathway with mTORC2.

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in the treatment of tumors with a specific major molecular defect. Major defects are more characteristic of single-gene disorders such as tuberous sclerosis, neurofibromatosis, and Von Hippel–Lindau disease. Lesions with abnormal cell proliferation characterize these disorders. These drugs are less effective in treatment of tumors that exhibit molecular heterogeneity. Tuberous sclerosis proliferation of smooth muscle cells in lung tissue leads to lymphangioleiomyomatosis (LAM); this condition occurs primarily in females. Davies et al. (2011) reported that LAM is primarily associated with biallelic mutations in TSC2. Angiomyolipomas in tuberous sclerosis contain abnormal proliferation of muscle cells, fat, and blood vessels, particularly in kidney. Tumors in tuberous sclerosis may be associated with loss of TSC1 or TSC2 gene expression. TSC1 and TSC2 gene expression is required to inhibit mTOR. Sirolimus inhibits primarily mTORC1. It is in clinical trials for treatment of tuberous sclerosis tumors.

MTOR-RELATED SIGNALING PATHWAYS, DEFECTS, AND TREATMENTS The TSC1–TSC2 complex integrates inputs from a number of different signaling pathways to impact the activity of Rheb and mTORC1. The tuberous sclerosis genes TSC1 and TSC2 encode proteins that are key upstream regulators of mTORC1. They function as Rheb GTPase and promote conversion of Rheb GTP to Rheb GDP. Since Rheb GTP is required for kinase activity of mTORC1, the action of the TSC1 and TSC2 proteins is to downregulate mTORC1 activity. There are at least three phosphorylation sites on the TSC2 protein. Phosphorylation sites on TSC2 protein are at serine S1364, S1395/1397, and S1775. TSC1 protein can undergo phosphorylation at serine 505 and may also be phosphorylated on other serine and threonine sites. AKT1 is activated by growth factors, by phosphatidyl 3-kinase (PI3K), and by RAS and RAF activity. Active AKT1-induced phosphorylation of TSC2 blocks TSC1–TSC2 complex activity and inhibits conversion of RHEB GTP to RHEB GDP, thereby promoting mTOR activity. Activation of PI3K, AKT1, and MTOR promotes metabolic and biosynthesis activities and cell proliferation. In contrast, activation of AMPK1 phosphorylates TSC1, and this activates the TSC1–TSC2 complex, which then converts RHEBGTP to the inactive RHEBGDP, which fails to activate mTOR. Energy and nutrient deprivation activate expression of LKB1, also known as STK11 serine threonine kinase, and AMPK. The role of AMPK activation is to turn off mTOR activity and cell proliferation.

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Impaired activity of the TSC1–TSC2 complex leading to increased mTOR activity and increased cell proliferation is characterized by development of hamartomas and tumors in a number of organs. mTOR inhibitors including rapamycin and related compounds are used for treatment of tuberous sclerosis. Everolimus is FDA approved for treatment of unresectable brain tumors in tuberous sclerosis. There is some evidence that Everolimus may be useful in the treatment of epilepsy, which commonly occurs in patients with tuberous sclerosis. One possible pathway through which rapamycin-related compounds work is related to the excess of glutamate and excess activity of the NMDA glutamate receptor that occur in some forms of epilepsy and that lead to increased PI3K and increased AKT and mTOR activity (McDaniel and Wong, 2011).

MTOR-RELATED ACTIVITIES IN THE PATHOGENESIS OF OTHER HAMARTOMATOUS SYNDROMES mTOR activity is increased in Peutz Jeghers syndrome and in Cowden syndrome. LKB1 (STK11) encoded by a gene on 19p13.3 is mutated in Peutz Jehgers syndrome, which is characterized by pigmented macules often on the lips and by polyps in the gastrointestinal tract. Loss of LKB1 activity diminishes AMPK activity and TSC1 phosphorylation so that the TSC1–TSC2 complex functions less efficiently. MTOR inhibitors are in clinical trials for treatment of Peutz Jehgers syndrome.

PTEN DEFECTS AND COWDEN SYNDROME PTEN is deficient in Cowden syndrome, which is characterized by intestinal hamartomas, neurocutaneous lesions, macrocephaly, epilepsy, and an increased frequency of breast, thyroid, and endometrial cancers. PTEN promotes the conversions of phosphatidyl inositol triphosphate (PIP3) to phosphatidyl inositol diphosphate (PIP2). The normal function of PTEN is to act as phosphatidyl 3,4,5 triphosphate phosphatase. In the absence of PTEN, more PI3P is available, leading to increased activity of AKT, and to increased inhibition of the TSC1–TSC2 complex and, subsequently, to increased mTOR activity. Laplante and Sabatini (2012) reported that effects of rapamycin on mTOR are quite complex and are still being elucidated. Furthermore, a growing number of proteins are being identified that interact with mTOR; mTOR is present in two multiprotein complexes, mTORC1 and mTORC2. They reported that mTORC1 integrates upstream signals from growth factors, signals relating to

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energy status, availability of oxygen, amino acid levels, and cellular stress levels. In response to these signals, mTORC1 controls protein and lipid synthesis and metabolism. Active mTORC1 inhibits autophagy. Protein synthesis is enhanced through mTORC1 stimulation and phosphorylation of translation initiation factor EIF4E binding protein (4EBP) and of S6 kinase. Lipid synthesis is promoted through mTORC1 activation of the sterol regulatory element binding proteins SREBP1 and 2 and through increased expression of genes involved in fatty acid and cholesterol metabolism. In addition, mTORC1 regulates cellular metabolism through increased glycolytic flux, and mTORC1 activity leads to positive regulation of mitochondrial biogenesis and oxidative function, in part, through association with PPARgamma coactivator and the YY1 transcription factor (Laplante and Sabatini, 2012). The mTORC2 complex has three main targets: AKT, SGK1, and PKC alpha. It activates these targets by phosphorylation. AKT then activates GSK3B kinase. Laplante and Sabatini (2012) noted that phosphorylation of PKC alpha by mTORC2 impacts the actin cytoskeleton and cell shape. Upstream signaling of mTORC2 occurs through growth factors and PI3K. The phosphoinositide signaling pathway is upstream of mTORC1 and mTORC2. Activity of the mTORC2 complex is less sensitive to rapamycin.

THERAPEUTIC TARGETING OF MTORC1 Rapamycin and Rapamycin analogs (rapalogs) have been FDA approved for the treatment of renal cell carcinoma and for treatment of tumors in tuberous sclerosis. However, in other tumors where metabolic studies indicate that mTORC1 is activated, rapamycin treatment may be indicated. Several investigators have proposed use of mTOR inhibitors in treatment of neurodegenerative diseases associated with protein aggregates, based on the observation that mTORC1 inhibitors promote autophagy. However, Laplante and Sabatini (2012) proposed that development of small molecules that impact autophagy downstream of mTORC1 will likely be more appropriate given the pronounced deleterious effects of mTORC1 inhibition on metabolism.

ANALYSIS OF ACTION: RAPAMYCIN AND OTHER INHIBITORS OF MTOR Rapamycin is a macrocyclic lactone. It binds with high affinity to the protein FKBP12. Studies on rapamycin action revealed that it forms an intracellular complex with FKBP12 and with mTOR (Nyfeler et al., 2011). The interaction

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of rapamycin bound to FKBP2 with mTOR impairs the interaction of mTOR with scaffold protein raptor in the mTORC1 complex. This then limits the ability of mTORC1 to phosphorylate S6K1 and 4EBP1. Nyfeler et al. (2011) reported that additional small-molecule inhibitors of mTOR have been isolated that have a different mechanism of action; they interact with the catalytic domain of mTOR and are therefore ATP competitive inhibitors. One example is Torin and there is evidence that Torin is particularly active in promoting autophagy. See figures 2–1 and 2–2.

TREATMENT OF MONOGENIC DISEASES THAT IMPACT GENES IN SIGNALING PATHWAYS RAS Signaling Pathway and Neurofibromatosis Neurofibromatosis NF1 is characterized by neurofibromin deficiency and development of hamartomatous nodules in the eye (Lisch nodules), axillary freckling, cafe-au-lait spots, plexiform neurofibromas, tibial dysplasia, megalencephaly that may be associated with increased white matter and thalamic lesions, T2 hyperintense signals on MRI, and attention deficit hyperactivity. Neurofibromin deficiency may lead to cognitive impairment. Neurofibromin, the product of the NF1 gene, is a GTPase protein with activity toward RAS. Active NF1 protein converts RAS GTP to RASGDP. RAS GTP leads to increased activity of the kinases MRK and ERK. This serves to phosphorylate and inhibit activity of the TSC2–TSC1 complex, leading to increased RHEB GTPase activity and to decrease mTOR activity (Davies et al., 2011). Neurofibromatosis (NF1) is characterized by reduced neurofibromin, increased Ras GTP and RAS signaling, and increased mTOR activity, which lead to astrocyte proliferation. Reduced neurofibromin also leads to neuronal cytoskeletal defects (Brown et al., 2012). There is evidence that the rapamycin (Sirolimus) blocks NF1 tumors (Franz and Weiss, 2012). Figure 2–3 illustrates the RAS signaling pathway. Davies et al. (2011) reported that farnesyl transferase inhibitors and 3-hydroxy-3-methyl glutaryl Coenzyme A reductase have been used to inhibit Ras activity. The molecular basis for this therapy was blocking of isoprenylation. Active Ras requires farnesylation and GTP. When active, Ras modulates several signaling pathways (Berndt et al. (2011). Studies on NF mutant mice indicated that farnesyl transferase inhibitors would be therapeutically useful, but studies in humans have been less convincing.

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P SHC GRB2 SOS P RASGTP

RASGDP Neurofibromin

RAF

P

MEK P ERK EIF4E protein turnover

ETS transcription

Figure 2–3. Illustration of the RAS signaling pathway.

PHOSPHOINOSITIDE-3-KINASES AND PI3CA Phosphoinositide-3-kinases (PI3K) catalyze the conversion of phosphoinositide 4,5 biphosphate to phosphoinositide 3,4,5 triphosphate. Ligand binding to a specific tyrosine kinase receptor triggers activation of PI3K; this involves the binding of the PI3K regulatory subunit p85 to the catalytic subunit p110a. This activated PI3K then activates AKT1, AKT2, AKT3 (serine threonine protein kinase, sometimes referred to as PKB). PTEN facilitates deactivation of PI3K and promotes generation of phosphoinositol-biphosphate PIBP. Somatic mutations in the catalytic subunit of PI3K have been found in a number of different cancers. AKT1 mutations have been reported in a malformation disorder referred to as Proteus syndrome that is characterized by overgrowth of skin, connective tissue, and brain. AKT2 is expressed in insulin-responsive tissues, liver, skeletal muscle, and fat. Hussain et al. (2011) reported that an activating AKT2 mutation was associated with insulin-independent hypoglycemia with asymmetric mild overgrowth and progressive obesity. The AKT3 gene is primarily expressed in brain and heart. Poduri et al. (2012) described brain overgrowth and hemispheric developmental malformations in

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brain in two infants with trisomy of chromosome 1q associated with an extra copy of the AKT3 gene and in an infant with an activating AKT3 mutation. The downstream effect of AKT activity is enhanced mTOR activity followed by increased cell proliferation and protein synthesis. (Lindhurst et al. (2012) used exome sequencing to identify mutant genes in patients with congenital segmental overgrowth. They identified mutations in the PIKCA gene in cells of affected tissue. They also determined that enhanced phosphoinositol 3,4,5 generation occurred in affected cells following EGF growth factor treatment. Surgical debulking and sometimes amputation have previously been the treatment of the extensive overgrowth of fibroadipose tissue and bone in limbs. However, recent advances in the understanding of the etiology of this overgrowth indicate that treatment with mTOR inhibitors may now be an option. Marsh et al. (2008) described successful use of Rapamycin in the treatment of PTEN mutation leading to Proteus syndrome associated with respiratory impairment due to large tumor size. Hemimegancephaly has been identified in patients with mutations in PIK3CA AKT and mTOR mutations. J. H. Lee et al. (2012) identified a recurrent PIK3CA mutation, c.1266G>A, in affected brain regions in a patient with hemimegancephaly. They also documented increased activity of S6 kinase downstream of mTOR. Riviere et al. (2012) identified postzygotic mutations in PIK3CA and mutations in the regulatory subunit of phosphoinositide kinase PIK3R2 and in AKT1 in patients with hemimegancephaly.

VON HIPPEL–LINDAU SYNDROME Von Hippel–Lindau syndrome is a dominantly inherited disease characterized by the predisposition to the development of retinal and central nervous system hemangiomas, renal carcinoma of the clear cell type, and visceral cysts in the kidney and pancreas. Pheochromocytomas, pancreatic islet cell tumors, may also develop. This syndrome can be diagnosed on the basis of family history and the presence of one tumor of the type described above. The syndrome can also be diagnosed in the absence of family history in individuals with two of the tumors listed above (Maher et al., 2011). This syndrome occurs with a frequency of approximately 1 in 36,000 in Europe. The VHL gene maps to chromosome 3p, and it encodes a full-length protein of 213 amino acids and a shorter protein that does not contain the first 53 amino acids. Disease-causing germ line gene alterations include deletions 0.5 to 250 kb in length and missense, nonsense, and splice site mutations. Tumors

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in patients with germ line VHL mutations have inactivation of the wild-type allele through structural changes, mutations, or gene silencing. Maher et al. (2011) emphasized that each of the tumors that are characteristic of VHL may occur as sporadic nonfamilial events. There is evidence that the VHL protein has several different functions. It plays a key role in regulating proteolytic degradation of the alpha subunits of the hypoxia-inducible transcription factors HIF1 and HIF2. Under normoxic conditions oxygen and proline hydroxylase modify the HIF alpha subunits. Binding of VHL protein and subsequent ubiquitination of the HIF alpha subunits, a process that targets them for proteolytic degradation, follow this modification. In the absence of VHL, the HIF1 alpha subunits are stabilized. HIF transcription factors then activate downstream targets that include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and transforming growth factor (TGF). In treating tumors in VHL patients, some of the downstream effects of the absence of VHL can be mitigated with tyrosine kinase inhibitors, such as sorafenib and sunitinib, that block VEGF and PDGF receptors. Baldewijns et al. (2010) reported that HIF expression can be targeted with histone deacetylase inhibitors and that this treatment can be beneficial in patients with VHL-related tumors. A second function of VHL protein has come to light through analysis of type 2C VHL mutations that do not impact HIF functions. These mutations occur most frequently in pheochromocytomas that develop from neuronal progenitors in the adrenal gland or in the sympathetic nervous system. Maher et al. (2011) reported that the absence of full-length VHL might impair apoptosis of neuronal progenitors. They reported that another phenotype occurs in individuals homozygous for a specific VHL missense mutation, c.598 C>T (Arg200Trp). This phenotype is characterized by polycythemia and vertebral hemangiomas, and it occurs in a specific population in Russia. TGF Beta Signaling Pathway and Marfan Syndrome Marfan syndrome affects between 1 in 5,000 to 1 in 10,000 individuals. Clinical features include abnormalities of the eye; ectopia lentis; skeletal changes including overgrowth and long, thin limbs and fingers; weakness of the aorta; thickening of the heart valves; dural ectasia; and myopathy. This syndrome is caused by heterozygous mutations in the Fibrillin 1 gene (FBN1 on chromosome 15) (Dietz et al., 1991). Fibrillin 1 protein is a component of extracellular microfibrils and contributes to their elasticity. Many different mutations give rise to this syndrome, and most occur only in one or a few families. In

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approximately 10% of patients with Marfan syndrome, FBN1 mutations are not found. There may be other mutational mechanisms in these patients, for example, large gene deletions, or regulatory mutants. It is also possible that mutations in other genes may give rise to a Marfan-like syndrome (Li-Wan-Po et al., 2011). In addition to its structural function, fibrillin interacts with and regulates TGF beta growth factor (Neptune et al., 2003). Mutant fibrillin binds poorly to TGF, and this defective binding leads to increased TGF beta activity (Chaudhry et al., 2007). Increased TGF beta activity leads to inhibition of maturation of satellite cells, and this contributes to myopathy and reduced mass in individuals with Marfan syndrome (Li-Wan-Po et al., 2011). Blocking of increased TGF beta antibody was shown to rescue the aortic root dilatation associated with Marfan syndrome (Bolar et al., 2012). Angiotensin is also known to increase TGF beta production. One of the downstream effects of angiotensin following binding to the angiotensin II type 1 receptor is increased production of thrombospondin, which then increases TGF beta formation. Losartan is a small-molecule inhibitor that inhibits binding of angiotensin to its receptor and thereby decreases TGF beta production. Losartan has proven effective in reducing the complication of Marfan syndrome (Möberg et al., 2012). Blockers of the beta-adrenergic receptors have also been used to prevent hypertension and reduce risk of aortic rupture in Marfan syndrome. Proteinases, especially metalloproteinase MMP9, degrade arterial elastin. Doxycycline reduces expression of MMP9 and its posttranslational activation, and there is evidence that treatment with this agent will be beneficial in patients with Marfan syndrome (Li-Wan-Po et al., 2011).

MARFAN-RELATED DISORDERS Loeys-Dietz syndrome, arterial tortuosity syndrome, and aneurysmsosteoarthritis syndrome are associated with thoracic aneurysms or intracranial aneurysms. Akhurst et al. (2012) reported that these syndromes are all associated with dilatation of the aortic root, dysfunctional smooth muscle cells in the tunica media of vessels, fragmentation and loss of fibers, and increased extracellular matrix. In addition, these syndromes are all characterized by abnormal function in the TGF beta signaling pathway. There is evidence that the thoracic aorta aneurysm syndrome is associated with loss of function mutations in a TGF beta ligand. Mutations in other TGF beta pathway genes can give rise to diseases associated with thoracic

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aneurysms in relatively young individuals. These include mutations in the genes that encode TGF beta receptors TGFBR1, TGFBR2, and SMAD3. The Loeys-Dietz syndrome shares many features in common with Marfan syndrome including aortic aneurysm, arachnodactyly. In addition, Loeys-Dietz syndrome patients manifest easy bruising and atrophic skin. This syndrome is due to mutation in the TGF beta receptors, TGFBR1 or TGFBR2. There is increased signaling through these receptors, and mutations often occur in the kinase domain of the gene. The aneurysms-osteoarthritis syndrome is associated with aneurysms, arterial vessel tortuosity, craniofacial anomalies, arachnodactyly, scoliosis, and velvety skin. In this syndrome mutations occur in SMAD3, also a component of the TGF signaling pathway. Disorders characterized by increased TGF beta signaling are treated with Losartan. This compound inhibits TGF beta signaling and acts as an angiotensin II type I receptor antagonist. In a review of heritable connective tissue disorders associated with vascular involvement, Van Laer et al. (2012) emphasized that in most of these disorders the causative genes encoded structural components of connective tissue (e.g., collagens, fibrillin, fibronectin, or enzymes involved in the biosynthesis or processing of these proteins). Analyses of the downstream effects of these gene mutations have revealed additionally the role of the genes in control of extracellular matrix functions. These functions include control of cell divisions and reservoir functions; extracellular matrix acts as a reservoir for growth factors and cytokines.

ANALYSIS OF DOWNSTREAM EFFECTS OF MUTATIONS AND DEVELOPMENT OF THERAPIES The product of the androgen receptor gene encoded on the X chromosome is a steroid hormone binding protein. Following ligand binding this protein translocates to the nucleus; there it acts as a transcription factor to enhance expression of androgen response genes. Expansion of a CAG repeat that encodes polyglutamine leads to Kennedy’s disease, spinal and bulbar muscular atrophy (SBMA). This is a late-onset motor neuron disease that leads to progressive weakness and atrophy of muscles in the limbs and face. The abnormal polyglutamine-containing protein accumulates in neurons. Minamiyama et al. (2012) carried out studies on the spinal cords of mouse models of this disease. These investigators analyzed gene expression using microarrays. They identified 124 genes that showed altered expression. One gene found to be

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significantly upregulated was Calca, which encodes calcitonin gene-related peptide (Cgrp1). This gene is normally expressed in dorsal horn cells in the spinal cord. Human CGRP1 is expressed at higher levels in spinal motor neurons of patients with SBMA. Minamiyama et al. demonstrated in neuronal cell cultures that overexpression of CGRP1 reduced cell viability. They demonstrated further that increased levels of CGRP1 activated the JNK signaling pathway. The drug Naratriptan inhibits the JNK pathway. Downregulation of the JNK pathway mitigated the toxicity of the pathogenic androgen receptor protein. Oral administration of Naratriptan attenuated symptoms and improved histopathology. The drug Naratriptan is in use to treat migraines.

THERAPEUTIC AGENTS THAT TARGET REGULATORS OF METABOLISM Nuclear Hormone Receptors The increased frequency of type II diabetes, obesity, and metabolic syndrome has led to studies aimed at identifying drugs that target nuclear hormone receptors that play key roles in metabolism. Nuclear hormone receptors, sometimes referred to as nuclear receptors, are ligand-activated transcription factors. Members of this family were identified as ligands for hormones including steroid hormones and thyroid hormone. However, they are now known to bind to a number of other ligands including fatty acids, cholesterol, retinoic acid, and vitamins. Members of the nuclear hormone superfamily have conserved structural and functional domains. Schulman (2010) reported that they contain three main domains: a heterogeneous aminoterminal domain, a highly conserved central DNA binding domain, and a ligand binding carboxyterminal domain that is functionally complex. This carboxyterminal domain plays important roles in ligand binding and in dimerization. Following identification of the hormone binding nuclear receptors, other members of this family were identified. Initially specific ligands for these nuclear receptors were not known and they were classified as orphan receptors. Ligands for these orphan receptors were subsequently identified. Previously defined orphan receptors now include peroxisome proliferator activated receptors (PPAR), Farnesoid FXR, Retinoic acid receptor (RXR) (ROR), and liver X receptor (LXR). Schulman (2010) emphasized that PPAR, LXR, FXR, and ROR constitute metabolic nuclear receptors through their fatty acid and

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cholesterol-level sensor functions and their regulation of downstream genetic networks. The estrogen-related receptors constitute a separate class of receptors involved in energy utilization and mitochondrial function. There are different classes of PPAR receptors: alpha, beta, delta, and gamma. PPAR monomers form heterodimers with retinoid X receptor subunits. The specific gene targets for PPAR alpha include genes that encode proteins and enzymes involved in fatty acid uptake, beta-oxidation of fatty acid, and ketogenesis. Expression of the fibroblast growth factor 21–encoding gene is also induced by PPAR alpha. PPAR gamma regulates lipid storage and adipogenesis. Troglitazone-related insulin-sensitizing drugs designed to treat type II diabetes are PPAR gamma agonists. However, they also promote fat storage, weight gain, and increased risk for myocardial infarction. PGC1 alpha is a coactivator of PPAR gamma. Ligands for the PPAR delta nuclear receptors include fatty acids, eicosanoid, and prostaglandins. The gene that encodes the ABCD1 ATP binding cassette transporter is a target of PPAR delta; ABCD1 transporter plays a role in the transport of cholesterol out of cells. ABCA1 transports cholesterol out of cells onto high-density lipoprotein complexes (proHDL). Tangier disease occurs in individuals who have defective function or low levels of ABCA1. Cholesterol-laden macrophages accumulate in the tonsils and spleen and in the arterial walls, and patients have severe atherosclerosis. There is evidence that exercise activates expression of PPAR alpha and delta. All three PPAR nuclear receptors, on activation, increase antiinflammatory activity and particularly impact macrophage infiltration. The LXR alpha and beta genes encode the LXR nuclear receptors. LXR subunits form heterodimers with the RXR. Activated LXR receptors bind to two repeats in DNA that are separated by four nucleotides, AGGTCA 4NT AGGTCA. Schulman (2010) reported that the ligands for LXR include hydroxycholesterols. Transporter genes are also targets for LXR receptors; these include ABCG1, ABCG4, and APOE. Another LXR target is CYP7A1, which encodes cholesterol 7a hydroxylase involved in the conversion of cholesterol to bile acids. Agonist-bound LXR receptors increase expression of proteins and enzymes involved in the regulation of fatty acid synthesis (e.g., SREBP1c, the sterol response element binding protein, and the enzyme fatty acid synthase). LXR expressed genes involved in gluconeogenesis including phosphoenolpyruvate carboxykinase and glucose-6-phophatase. Schulman (2010) emphasized the challenges in identifying pharmacologically useful targets for these nuclear receptors. Useful targets will separate beneficial therapeutic activities from unwanted side effects.

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PLASMA MEMBRANE–BOUND RECEPTORS G-Protein-Coupled Receptors and Medicinal Chemistry In 2012 the Nobel Chemistry Prize was awarded to Robert Lefkowitz and Brian Kobilka for their elucidation of structure and function of G protein– coupled receptors. It is estimated that almost 40% of pharmaceutical agents in use impact these receptors. G protein–coupled receptors (GPCR) represent one of the largest families of proteins encoded in the mammalian genome. Family members are defined by their similarities in structure and function. The family comprises more than 1,000 different proteins. Ligands for these receptors include protein and peptides, lipids, nucleotides, and amines. Fredriksson et al. (2003) used DNA sequence information to define members of this family. Five different subfamilies have been identified including rhodopsin, glutamate, secretin, frizzled/ smoothened, and adhesion. GPCRs are homologous proteins composed of domains that traverse phospholipid cell membranes seven times. Three extracellular and three intracellular loops link the 7-transmembrane domains. The extracellular domains contain ligand binding sites, and ligand binding results in activation of intracellular signaling on the cytoplasmic side of the membrane. Intracellular loops interact with G proteins. Inactive G proteins comprise alpha, beta, and gamma subunits bound to GDP (guanosine diphosphate). Following ligand activation of the receptor, the G protein subunits exchange GDP for GTP on the alpha subunit. The GTP-bound alpha subunit is then free to interact with downstream effectors including adenyl cyclase, phosphodiesterase, phospholipase, and tyrosine kinases. A specific protein regulator of G proteins accelerates GTPase activity and deactivates G proteins. Lefkowitz and coworkers (Whalen et al., 2011) discovered that in addition to interacting with intracellular G proteins, the 7-transmembrane receptors interact with intracellular beta arrestins. These molecules play key roles in regulation of receptor function in intracellular signaling. GPCR are considered attractive pharmaceutical targets since they bind many neurotransmitters and hormones. However, Shoichet and Kobilka (2012) reported that progress in identification of effective therapeutics has been slower than expected. They attribute this to the fact that diverse downstream signaling pathways are activated following ligand binding to 7-transmembrane receptors. High-throughput screening assays are designed to assess ligand binding but not downstream effects. Nevertheless, elucidation of the structure of G protein coupled–receptors and docking screens that take structure into account have led to identification of many useful pharmacological products.

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In 2011 Lappano and Maggiolini reported that only a small number of all known GPCRs are targeted by pharmaceuticals. In addition, many of the GPCRs have no identified ligands and are referred to as orphan receptors. They noted that GPCRs are often overexpressed in cancer. In addition, cross talk between GCPRs and growth factor receptors often occurs in tumors. Of particular importance is cross talk between the epidermal growth factor receptor EGFR and GCPRs.

ESTROGEN RECEPTORS AND TISSUE-SPECIFIC MODULATION Natural estrogens have beneficial cardiovascular effects including promotion of vasodilation and enhanced myocardial perfusion. The major human estrogen is 17 beta-estradiol synthesized by the ovaries. This estrogen and estrone are also derived from androgens through activity of the enzyme aromatase. Humans are also exposed to environmental factors with estrogen activity. These include plant substances, phytoestrogens, isoflavones (genistein), and synthetic estrogen like compounds including pesticides and plastic monomers. Estrogen receptors alpha and beta bind estrogen and act as transcriptional regulators. Meyer et al. (2011) reported that a third estrogen receptor was cloned in 1997. This is a seven-transmembrane G-coupled receptor, GPER, which binds estrogen and then activates signaling cascades including ERK, the extracellular signal-related kinase, and phosphatidylinositol-3-kinase. Drugs that downregulate estrogen receptors were developed to treat breast cancer, and many of these block signaling through estrogen receptors. Selective estrogen receptor modulators (SERMs) are a different class of drugs that have tissue-specific effects. SERMs are agonists for estrogen receptors in bone, heart, and liver and antagonists for estrogen receptors in breast. SERMs include tamoxifen, raloxifene, and lasofoxifene. The latter is reported to have the most favorable cardiovascular risk-to-benefit ratio. Postmenopausal estrogen treatment with conjugated estrogens and medroxyprogesterone acetate did not show evidence of cardiovascular benefit; in fact, cardiovascular disease risk was increased, as was breast cancer risk (Meyer et al., 2011). PHARMACOGENETICS, PHARMACOGENOMICS, AND PERSONALIZED MEDICINE In the context of medicinal therapy, Khoury et al. (2012) defined the goal of precision medicine as treatment of patients with the correct dose of the appropriate medicine based on their individual demographic and genomic makeup.

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Pharmacogenetics deals primarily with individual gene variation that leads to altered responses to specific medications, and the variants are usually of large effect. Important milestones in pharmacogenetics include the discovery of variants in glucose-6-phosphate dehydrogenase (G6PD) that lead to hemolytic anemia on exposure to antimalarial therapies and a number of other drugs (Beutler, 1964; Prankerd, 1964). Studies on variation in metabolism of a number of drugs, including debrisoquine (a guanidine derivative used as an antihypertensive), led to discovery of common genetic variation in the cytochrome P450 CYP2D6. Variants in this gene that lead to altered drug metabolism include mutations, deletions, and duplications (Frank et al., 2007). In pharmacogenetic studies specific candidate genes were often examined to identify variants that led to aberrant drug responses. Important relatively common variants lead to altered responses to drugs used in cancer therapy (e.g., thiopurine-S-methyl-transferase and dihydropyrimidine dehydrogenase) (Lennard, 1999). Figure 2–4 illustrates metabolic enzymes with variants associated with abnormal drug responses currently included in drug labels. In pharmacogenomics genome-wide association studies were carried out to identify locations in the genome associated with abnormal drug responses. The association of genetic variants in the transporter SLCO1B1 with statin-induced 70% 60% 50% 40% Series1

30% 20% 10% 0% CYP2D6 CYP2C19 CYP2C9

CYP1A2 UGT1A1

TPMT

METABOLIC ENZYMES INCLUDED IN DRUG LABELS OF FDA APPROVAL DRUGS Adapted from Wei et al., 2012.

Figure 2–4. Metabolic enzymes with variants associated with abnormal drug responses currently included in drug labels.

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myopathy was identified in genome-wide association studies (GWASs) (Link et al., 2008; SEARCH Collaborative Group, 2008). GWASs led to identification of association of the HLAB*5701 allele and flucloxacillin liver injury (Daly et al., 2009). Life-threatening hypersensitivity to the antiretroviral agent abacivir in Caucasians is associated with the HLAB*5701 allele (Phillips and Mallal, 2009). The HLAA*3101 allele was found to be associated with sensitivity to carbamazepine (Tegretol) in European populations (McCormack et al., 2011). In Taiwan carbamazepine sensitivity and Steven Johnson syndrome with epidermal necrolysis occurred in individuals with the HLAB*1502 allele (P. Chen et al., 2011). Identification of specific HLA alleles is facilitated by interrogation of the HLA complex with high-throughput genomics techniques (de Bakker and Raychaudhuri, 2012). Figure 2–5 illustrates chromosome 6 and HLA loci; specific HLA alleles are associated with abnormal drug responses (see table 2–1). An important online resource for information is the pharmacogenomics knowledge base (www.pharmgkb.org) and a resource for information on population frequencies of variant alleles associated with abnormal drug responses is www.findbase.org.

PHARMACODYNAMICS AND GENOMIC COPY NUMBER VARIANTS Copy number variants occur frequently in the human genome. Rasmussen and Dahmcke (2012) identified 1,721 genomic structural variants that encompass Class I HLA-A

29 Mb

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Class III HLA-C HLA-B

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Class II HLA-DR HLA-DP HLA-DQ

32

33

33.4 Mb

Figure 2–5. Illustration of chromosome 6 and HLA loci; specific HLA alleles are associated with abnormal drug responses (see table 2–1).

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Figure 2–6. Illustration and example of copy number variation encompassing the CYP2A6 gene demonstrated on microarray analysis of genomic DNA from one individual. The enzyme encoded by CYP2A6 is involved in pharmacodynamics of a number of genes including the antimalarial artemisin and the anticoagulant coumadin.

Table 2–1. HLA Alleles and Adverse Drug Reactions population

hla antigen

drug

use

adverse reaction

European

HLA-B*7301

Oxicams

Anti-inflammatory SJS/TEN

European European Euro./ Japanese Caucasian

HLA-B*3802 HLA-B*3801 HLA-B*5801 HLA-A*0201

Sulfamethoxazole Lamotrigine Allopurinol Amoxicillin/ clavulanate

Antibiotic Antiseizure med. Gout/arthritis Antibiotic

SJS/TEN SJS/TEN SJS/TEN DILI

Caucasian Several Han Chinese Thai JAP Korean Several

HLA-B* 5701 HLA-B*5701 HLA-B*1502 HLA-B*1502 HLA-A*3101 HLA-B*5901 HLA-Cw8

Flucloxacillin Abacavir Carbamazepine Phenytoin Carbamazepine Methazolamide Nevirapine

Antibiotic Antiretroviral Antiseizure med. Antiseizure med. Antiseizure med. Anti-infection AIDS

DILI HSR SJS/TEN SJS/TEN SJS/TEN SJS/TEN DHS

SJS/TEN: Steven Johnson syndrome/toxic epidermal necrolysis. DILI: drug-induced liver injury; DHS: delayed hypersensitivity reaction based on Pavlos et al. (2012) and Wei et al., (2012).

Therapy

37

495 different genes that are classified as encoding drug targets. They proposed that copy number variants in genes that play roles in drug metabolism and pharmacodynamics likely lead to variations in drug responses. Figure 2-6 illustrates an example of copy number variation encompassing the CYP2A6 gene demonstrated on microarray analysis of genomic DNA from one individual. The enzyme encoded by CYP2A6 is involved in pharmacodynamics of a number of genes including the antimalarial artemisin and the anticoagulant coumadin.

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3 INBORN ERRORS OF METABOLISM Progress in Diagnosis and Treatment

INTRODUCTION The key concept of inborn errors of metabolism formulated by Garrod (1902) was that each of these conditions arose as a result of deficiency of a specific enzyme and that this deficiency led to a block at a specific step in metabolism. Success in the treatment of phenylketonuria and prevention of cognitive impairment through dietary management (Bickel et al., 1954) served as a catalyst for research in diagnosis and treatment of inborn errors of metabolism. Subsequent development of techniques for automated phenylalanine analysis in blood led to initiation of newborn screening in the United States in 1961. Information on the role of inborn errors of metabolism in causation of severe mental and physical impairments and in acute, life-threatening illnesses in children grew steadily throughout the 20th century. By 1978, when Stanbury, Wyngaarden, and Frederickson published their landmark text The Metabolic Basis of Inherited Disease, at least 180 disorders of metabolism due to specific enzyme deficiencies were known. Many of these diseases were found to be associated with quantitative or qualitative changes in metabolites detectable in plasma or urine. Mass spectrometry led to discovery of additional inborn errors 39

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of metabolism, particularly disorders associated with abnormal organic acids and abnormalities of fatty acid metabolism. Improvements in protein and enzyme isolation techniques and increasing availability of biochemical reagents including substrates and coenzymes facilitated discovery and diagnosis of causes of inborn errors of metabolism not associated with readily detectable metabolic abnormalities in plasma or urine. Elucidation of metabolic processes and the roles of coenzymes, frequently vitamin based, led to design of specific therapies for a number of different inborn errors. Cornerstones of treatment of inborn errors came to include restriction of substrate through dietary manipulation, supplementation with coenzymes or cofactors, replacement of deficient product, and in some cases removal of excess harmful substances (e.g., chelation therapy to remove excess iron or copper). Subsequently, approaches such as enzyme replacement and gene therapy evolved. This chapter includes descriptions of more recently discovered inborn errors of metabolism and approaches to treatment of those disorders.

NEWBORN SCREENING AND TANDEM MASS SPECTROMETRY In 2001 tandem mass spectrometry was introduced into newborn-screening programs. Wilcken (2011) reviewed the dramatic changes that occurred in newborn screening with the availability of tandem mass spectrometry (MS/ MS). She noted that MS/MS was initially applied to screen for inborn errors of amino acid, organic acid, and fatty acid metabolism. Although analytical and clinical validity of these testing methods is established, there are still questions concerning the clinical follow-up of results. Following introduction of MS/ MS screening, the incidence of specific inborn errors (e.g., medium chain acyl dehydrogenase deficiency) has greatly increased. One question that arises is how many of the cases detected on newborn screening would develop clinical symptoms if left untreated. Wilcken published data on outcomes for individuals with rare inborn errors in screened and unscreened cohorts. This study included data on less rare conditions such as phenylketonuria and medium chain acyl CoA dehydrogenase deficiency. In an unscreened cohort born between 1994 and 1998, 40% of cases died before the age of 6 years. In the unscreened cohort born between 1998 and 2002, 57% died before the age of 6 years. In the screened cohort born between 1998 and 2002, 7% died before the age of 6 years. In considering outcomes, Wilcken (2010) reported that in cases with medium-chain acyl CoA dehydrogenase deficiency, the risk of death in the first

Inborn Errors of Metabolism

41

72 hours of life is 4%, and there is a further 5–7% fatality in the first 6 years in undiagnosed patients. She noted that with early diagnosis and treatment, initiation, and good management, the prognosis for these patients is greatly improved. Wilcken emphasized that newborn screening opens new ways to diagnose inborn errors and to initiate early treatment and prevent long-term consequences. Newborn screening for lysosomal disorders is under consideration. Levy (2010) summarized problems related to expanded newborn screening that need to be addressed. These included lack of knowledge about the natural history of certain disorders and the absence of effective therapy for others. He also noted that parental anxiety is created by findings that may turn out to be benign. Newborn screening is carried out on blood. A number of investigators have advocated that urine samples from newborns be saved for analysis and follow-up of abnormal blood studies.

FIRST-LINE METABOLIC SCREENS IN SUSPECTED CASES OF INBORN METABOLIC ERRORS It is important to emphasize that all patients with inborn errors of metabolism are not necessarily detected on newborn screening, and clinical suspicion of these disorders is important. First-line metabolic screens in suspected cases include determination of blood glucose, lactate, pyruvate, ammonia, plasma amino acids, free carnitine and acylcarnitine, and urinary organic acids and glycosaminoglycans.

ORGANIC ACIDURIAS Organic acidurias occur in inborn errors of metabolism of amino acids, carbohydrates, and lipids. They also occur in disorders in mitochondrial metabolism including abnormalities of the tricarboxylic acid cycle and defects in oxidative phosphorylation. Metabolic profiling of organic acids in urine can be carried out using several different methodologies (e.g., liquid chromatography, gas chromatography [GC], mass spectrometry [MS], nuclear magnetic resonance [NMR] spectrometry) (Wajner and Goodman, 2011). Analysis of organic acids and creatinine levels in urine are usually carried out simultaneously to take urine concentration variations into account.

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Specific organic acids may be elevated in particular inborn errors of metabolism. However, abnormal levels of a specific organic acid may occur in a number of different inborn metabolic errors. Abnormal levels of orotate are found in several different urea cycle defects, and orotic aciduria and elevated urine levels of methyl citrate occur in propionic aciduria and methylmalonic aciduria. Increases in urinary levels of dicarboxylic acids are suggestive of fatty acid oxidation defects and specific mitochondrial disorders. Kouremenos et al. (2010) reported that in their studies using gas chromatography or combinations of GC/MS in inborn errors of metabolism organic acid levels in urine were usually increased to levels 10-fold above normal. Analysis of organic acids in urine can serve to facilitate accurate diagnosis and to monitor response to treatment.

CARNITINE ANALYSIS AND CARNITINE PROFILE Within cells carnitine reacts with fatty acids bound to acetyl coenzyme A. Acyl carnitine then transfers fatty acids across membranes, and this is an essential step in the beta-oxidation of fatty acids (see figure 3–1). Amino acid metabolism gives rise to organic acids that esterify with carnitine. Analysis of free carnitine and of the acyl carnitine levels and profile in blood through mass spectrometry has facilitated diagnosis of fatty acid oxidation defects and organic acidurias (Santra and Hendriksz, 2010). In fatty acid oxidation defects, free carnitine is low and acyl carnitine levels are elevated. The acyl carnitine profile represents the conjugation of carnitine with different length fatty acids. Increased urinary levels of dicarboxylic fatty acids also often characterize fatty acid oxidation defects.

INBORN ERRORS OF CARNITINE AND FATTY ACID METABOLISM Carnitine Carnitine is synthesized in the body and is derived from nutrients. Primary carnitine deficiency results in some cases from defects in transport of carnitine across cell membranes in the intestine. Carnitine is filtered through glomeruli. However, carnitine in the glomerular filtrate undergoes reabsorption, and this is controlled by transporters OCTN2 (SLC22A5) and by SLC16A9 (Suhre et al., 2011).

Inborn Errors of Metabolism

43

O R−CH2−CH2−C−OH Fatty acid ATP Mg

Acyl-CoA synthetase O

R−CH2−CH2−C−O−−−S−CoA ACYL-CoA mitochondrial membranes Acyl-CoA dehydrogenase

FAD FADH

Trans Enoyl CoA H20 EnoylCoA dehydratase B-hydroxyacyl CoA B-hydroxy-acyl CoA dehydrogenase

Thiolase

NAD NADH

BKetoacylCoA CoA

Fatty acylCoA + Acetyl CoA

Figure 3–1. Illustration of beta-oxidation of fatty acids.

CONJUGATION OF FATTY ACIDS TO CARNITINE The conjugation of carnitine to fatty acids involves the activity of three different carnitine palmitoyl transferases, each with different tissue distribution. CPT1A, encoded by a gene on chromosome 11q13, is expressed primarily in liver. CPT1B encoded on chromosome 22qter is expressed primarily in muscle. CPT1C encoded on chromosome 9q13 is expressed in brain. Longo et al. (2006) reported that CPT deficiency primarily occurs due to mutations in CPT1A. CPT1 is located on the inner side of the outer mitochondrial membrane, and this enzyme links carnitine to acyl CoA fatty acids. Cases of CPT1A deficiency may present soon after birth with impaired consciousness, or coma, hepatomegaly, and hypoglycemia. Total carnitine levels are not reduced; they may be elevated. Levels of free fatty acids and ketones are increased. Levels of long-chain acylcarnitines are reduced. Dicarboxylic aciduria may be present but is frequently absent. Dicarboxylic acids include C12 dodecanoic acid and 3OH glutaric acid. CPT1A is particularly involved

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in the conjugation and transfer of long-chain fatty acids (C6-C12) across the mitochondrial membranes (see figure 3–2). Less harmful mutations lead to disorders that present primarily with muscle involvement and muscle pain, and strenuous exercise may lead to myoglobinuria. Levels of serum creatine phosphokinase are elevated. TREATMENT OF CPT1 DEFICIENCY This involves increased carbohydrate feeding, low fat intake, increased intake of medium-chain triglycerides, and avoidance of fasting. CARNITINE PALMITOYL TRANSFERASE 2 (CPT2) CPT2 is anchored to the inner mitochondrial membrane and faces toward the mitochondrial matrix. CPT2 frees carnitine from the acyl CoA, and fatty acids can then undergo beta-oxidation; three different forms of this disorder occur. The severest form presents in infancy with respiratory distress, arrhythmias, and cardiomegaly. Dysmorphic features may be present. Renal abnormalities and brain malformation occur. The most common form presents during adolescence and manifests with muscle pain. In 2008 Isackson et al. reported that 68 different disease-causing CPT2 mutations had been identified. Of these 22 were null mutations where enzyme activity was absent. CARNITINE ACYL-CARNITINE TRANSLOCASE (CACT) CACT encoded by SLC25A20 translocates carnitine across the mitochondrial membranes. Acyl carnitine enters mitochondria in exchange for free carnitine

FATTY ACID ACYL COA + CARNITINE

carnitine palmitoyl transferase l ACYLCARNITINE

Outer mitochondrial membrane

translocase carnitine palmitoyl transferase lI

Inner mitochondrial membrane

ACYLCARNITINE + CARNITINE + ACYL-COA

Beta oxidation

Figure 3–2. Illustration of acylcarnitine and transfer across mitochondrial membranes.

Inborn Errors of Metabolism

45

through the activity of CACT translocase. Deficiency of activity of this translocase leads to hypoketotic hypoglycemia, cardiac arrhythmias, hepatomegaly, impaired liver function, and hyperammonemia. This deficiency may result in sudden infant death.

THERAPY Key aspects of therapy for carnitine palmitoyl transferase deficiency and for carnitine acyl translocase deficiency are avoidance of fasting, and a high-carbohydrate diet, especially rich in complex carbohydrates. Diet should be low in fat but with inclusion of medium-chain fatty acids (MCT oil). Carnitine supplementation is also included. In CPT1 and CPT2 deficiency, a similar diet should be followed. In the myopathic form of CPTII deficiency, vigorous exercise should be avoided. The neonatal forms of CPT and CACT (translocase) deficiency often respond poorly to therapy. Pierre et al. (2007) reported outcome in siblings with CACT deficiency. The index patient was not diagnosed in the newborn period, and diagnosis followed acute metabolic illness and the patient suffered profoundly. The younger sibling was prospectively treated from birth on with low-fat diet and supplementation with medium-chain triglycerides (MCTs) and carnitine and had normal development.

CARNITINE OCTANOYL TRANSFERASE Very long–chain fatty acids are oxidized and shortened in peroxisomes in beta-oxidation. Carnitine octanoyl transferase carries out esterification of carnitine and fatty acids bound to coenzyme A. This enzyme also promotes the transfer of long–chain and medium–chain fatty acids out of peroxisomes into the cytosol (Le Borgne et al., 2011)

FATTY ACID OXIDATION DEFECTS Fatty acid dehydrogenases are classified according to the chain length of fatty acids that serve as substrates. ACADS (also known as SCAD; short-chain specific acyl CoA dehydrogenase), encoded on chromosome 12q24.31, is responsible for mitochondrial beta-oxidation of C2-C4-length fatty acids. Deficiency

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of this enzyme leads to increased levels of C4 acyl carnitine in plasma and increased urinary excretion of ethylmalonic acid. Van Maldegem et al. (2010) reported that symptoms in patients with SCAD deficiency include hypotonia, hypoglycemia, and developmental delay. However, they noted that patients detected with SCAD deficiency on newborn screening often remain asymptomatic. Specific analysis of mutations will likely facilitate recognition of genotype–phenotype correlations. It is also important to note that additional acyl CoA dehydrogenases are present in humans. These include ACAD9, encoded on 3q21.3, and ACAD10, encoded on 12q24.31.

MEDIUM-CHAIN ACYL COENZYME DEHYDROGENASE (MCAD) DEFICIENCY MCAD, medium-chain specific acyl CoA dehydrogenase, encoded on chromosome 1p31, acts in mitochondria to oxidize straight-chain fatty acids of length C4 to C12. Wilcken (2010) reported that this is the most frequently occurring fatty acid oxidation defect. She noted that in cases diagnosed on the basis of clinical manifestations, 80% were homozygous for a specific mutation in the MCAD gene A to G mutation at 985. Clinically diagnosed cases most commonly presented with hypoketotic hypoglycemia. The mortality in these cases was high; 16 out of 25 cases died. Furthermore, the morbidity in survivors of acute episodes was high, and 20–25% of such cases had intellectual disability. The rate of occurrence of MCAD deficiency doubled since introduction of newborn screening, and Wilcken reported that the mutation spectrum in these cases is broader. Individuals who have known common MCAD disease-causing mutations benefit from early detection and appropriate management. However, a number of patients have mutations that lead to few problems. Diagnosis can be made on the basis of excretion in urine of high amounts of dicarboxylic acids: C6 adipic, C8 suberic, and sebacic C10 and glycine conjugates. Acyl carnitine profiles reveal high levels of C8 octanoyl carnitine.

TREATMENT Nyhan et al. (2005) emphasized the importance of absence of fasting and high carbohydrate intake and carnitine supplementation.

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47

LONG-CHAIN FATTY ACID DISORDER The enzyme long-chain acyl CoA dehydrogenase LCAD (ACADL) catalyzes the initial step in mitochondrial beta-oxidation. This enzyme encoded by a gene on chromosome 2q34 oxidizes long-chain fatty acids. Symptoms in long-chain fatty acid disorders include nonketotic hypoglycemia and are exacerbated by fasting and during febrile illness. Tandem mass spectrometry newborn screening has identified many infants who are asymptomatic. Some of these cases may develop symptoms under severe catabolic conditions. In these conditions the long-chain fatty acid content of the diet must be reduced, and increased levels of medium-chain fatty acids must be added (omega3 and omega 6). The carbohydrate content of diet should be increased with glucose polymers such as cornstarch.

VERY LONG–CHAIN ACYL COA DEHYDROGENASE (VLCAD) VLCAD (ACADVL), encoded on 17p13, acts on fatty acid esters of chain length C14 to C20. This enzyme is bound to the inner mitochondrial membrane. Deficiency of VLCAD may lead to sudden infant death or to cardiomyopathy with fatty infiltration of the liver. Episodes of rhabdomyolysis and muscle pain may be present in older patients. In these patients acylcarnitine profiles reveal elevation of levels of C14, C16, and C18 acylcarnitine. Treatment includes avoiding fasting, low-fat diet (5–10% of calories from fat), and supplementation with medium-chain triglycerides and carnitine.

LONG-CHAIN L3 HYDROXYACYL-COA DEHYDROGENASE AND TRIFUNCTIONAL PROTEIN DEFICIENCY LCHAD is a component of the trifunctional protein that is bound to the inner mitochondrial membrane. This protein is an octomer composed of alpha encoded by the HADHA locus and beta subunits encoded by HADHB. These loci map adjacent to each other on chromosome 2p23.3 in a head-to-tail configuration. LCHAD has three distinct functions and acts as a dehydrogenase, a hydratase, and a thiolase. The thiolase activity is primarily a function of the HADHB subunits and is sometimes referred to as long-chain 3-keot COA thiolase (LKAT). LCHAD deficiency leads to deficiency of mitochondrial trifunctional protein (MTP).

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Various forms of MTP deficiency occur. Boutron et al. (2011) reported that in one form of MTP deficiency both the alpha and beta subunits are deficient, leading to impaired function of all three enzyme activities. The most common MTP defect is a c.1528 G>C mutation in the alpha subunit. MTP mutations lead to a range of different phenotypes that include severe neonatal disease with cardiomyopathy and high mortality and infant hepatic disease with episodes of hypoketotic hypoglycemia. A milder, late-onset form is characterized by neuropathy and episodic rhabdomyolysis. Diagnosis is made on the basis of abnormal blood carnitine profile with elevated levels of 3OH long-chain acylcarnitine levels, and reduced activity of the LCHAD and LKAT enzyme levels in fibroblasts and abnormal palmitate and myristate oxidation in fibroblasts. Boutron et al. studied 52 patients with MTP deficiency, including 12 patients with the severe neonatal form of the disease, 35 with infantile hepatic phenotype, and 5 with mild-onset neuromuscular disease. They identified 24 different mutations in HADHA in patients with MTP deficiency. The most common mutation occurred in homozygous form in 19 patients and in compound heterozygous form in 16 patients. Eight mutations occurred in HADHB. Boutron et al. reported that maternal liver disease during pregnancy occurred in 11 out of 49 cases in which the mother carried fetuses with MTP deficiency. Liver disease in mothers was associated with elevated liver enzymes, hemolysis, and low platelets. In some cases mothers had fatty liver disease. Wilcken (2010) reported that high rates of early infant mortality occur in complete MTP deficiency. However, cases with isolated mutations in the alpha HADHA and LCHAD deficiency can be treated and managed to prevent hypoketotic hypoglycemia.

GLUTARIC ACIDURIA Abnormally high levels of glutaric acid in urine occur in cases with type I and type II glutaric aciduria. Type 1 is due to deficiency of the enzyme glutaryl CoA dehydrogenase, an enzyme involved in the catabolism of L-lysine, L-hydroxylysine, and L-tryptophan. Diagnosis is made on the basis of abnormal acylcarnitine profiles in blood, especially elevated glutaryl carnitine. It is also made on the basis of finding abnormal organic acid levels in urine, including elevations of glutaric acid, 3-OH-glutaric acid, and glutaconic acid (Kölker et al., 2011). Glutaryl CoA dehydrogenase is encoded by a gene on chromosome 19q13.2, GCDH. It is a mitochondrial matrix protein that uses flavin adenine

Inborn Errors of Metabolism

49

dinucleotide as cofactor. Diagnosis of the disease is confirmed by enzyme analysis and GCDH mutation analysis. Figure 3–3 illustrates amino acids and metabolism impacted in glutaric aciduria type 1. The frequency of this autosomal recessive disease in the general population is 1 in 100,000. There are, however, population isolates in which it occurs at much higher frequency (e.g., in the Amish in Canada, in Oji Cree, in Lumbee in North Carolina, and in the Irish Travelers). Kölker et al. (2011) reported that untreated patients develop neurological disease between 3 and 36 months and that acute encephalopathic crises sometimes occur in these patients following acute illness, febrile episodes, and immunizations. The most prominent neurological feature is a complex movement disorder, dystonia. Dystonia is due to striatal damage. Encephalopathic crises are due to acute striatal damage and hemorrhage. Initially there is axial hypotonia. Later this is followed by rigidity. TREATMENT STRATEGIES IN GLUTARIC ACIDURIA TYPE 1 These include dietary management and reduced intake of lysine, and tryptophan with increased glucose intake and carnitine supplementation. Careful management is necessary to avoid malnutrition. Plasma albumin levels, liver function, and alkaline phosphatase levels must be monitored. Specific treatments for dystonia and movement disorders are sometimes prescribed. Intense emergency treatments are required for encephalopathic crises. TRYPTOPHAN

LYSINE

HYDROXYLYSINE

2-OXOADIPIC ACID Glutaryl carnitine Glutaric acid—GLUTARYL COA

Glutaconic acid—GLUTACONYL COA

Bifunctional enzyme glutaryl CoA/ glutaconylCoA dehydrogenase

CROTONYL COA 3HYDROXYBUTYRYL COA ACETO-ACETYL COA

Figure 3–3. Illustration of amino acids and metabolism impacted in glutaric aciduria type1.

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GLUTARIC ACIDURIA TYPE II This condition is also known as multiple CoA dehydrogenase deficiency (MADD) or ethylmalonic adipic aciduria. Gordon (2006) emphasized that this condition is particularly amenable to treatment and early diagnosis is very important. Manifestations include stress-induced episodes of metabolic decompensation with acidosis, hypoglycemia, and organic acidemia. Patients may develop fatty degeneration of the liver, recurrent pancreatitis, and kidney disease. Multiple CoA dehydrogenase deficiency (glutaric aciduria type II) has at least three different clinical presentations. In the neonatal form, congenital anomalies may be present. Later-onset forms present with myopathy and episodes of cyclical vomiting, metabolic acidosis, and hypoglycemia. Olsen et al. (2007) reviewed findings on patients with multiple CoA dehydrogenase deficiency in 11 pedigrees. Index cases in each pedigree presented with encephalopathy or muscle weakness. Some had episodes of cyclic vomiting. Olsen et al. reported that in this disorder there is defective transfer of electrons from primary flavoprotein dehydrogenases to the mitochondrial respiratory chain. The affected dehydrogenases all use flavin adenine dinucleotide as cofactor. Impacted dehydrogenases are involved in fatty acid oxidation or in oxidation of amino acid derivatives dimethylglycine and sarcosine. The primary defects occur in electron transport flavoprotein (ETF), and in the ETFQO, the electron transfer flavoprotein ubiquinone oxidoreductase reaction. ETF and ETFQO are imported into the mitochondria from the cytosol. ETF is composed of two subunits: ETFA encoded on chromosome 15q24 and ETFB encoded on 19q13.3. A gene on 4q32-q35 encodes ETFQO. ETF shuttles electrons between flavoprotein dehydrogenases and the mitochondrial membrane-bound electron transfer flavoprotein ubiquinone dehydrogenase (ETFQO). ETFQO is sometimes referred to as ETFDH. Watmough and Frerman (2010) reported that the ETF/ETFQO system serves as the electron acceptor for different mitochondrial flavoprotein dehydrogenases. These include SCAD, MCAD, LCAD, VLCAD, isovaleryl dehydrogenase, methylbutyrylCoA dehydrogenase, isobutyryl CoA dehydrogenase, and glutarylCoA dehydrogenase. The ETF/ETFQO system also accepts electrons from sarcosine dehydrogenase and dimethyl glycine dehydrogenase. Figure 3–4 illustrates metabolites and the electron transfer pathway impacted in glutaric aciduria type II. Wang et al. (2011) reported results of DNA analysis in 51 unrelated patients with ETFDH (ETFQO) deficiency. They identified a specific mutation in ETFDH c.250G-A. The carrier frequency for this mutation in the population of South China was 1.35%. They emphasized

Inborn Errors of Metabolism

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MULTIPLE−ACYL COA DEHYDROGENASE DEFICIENCY GLUTARIC ACIDURIA TYPE II Fatty acids VLCAD LCAD MCAD SCAD

Lysine Hydroxylysine Tryptophan

Leucine

Fatty acid CoA Glutaryl CoA IsovalerylCoA

R−CH2−CH2−C=O

Isoleucine

3-Methylbutyryl CoA

FAD

IsobutyrylCoA

ETFred ETF dehydrogenase

FADH

ETFox

QH2 −

acylCoA dehydrogenase

Q

−

R−CH=CH=O

Valine

S − CoA

Figure 3–4. Illustration of metabolites and the electron transfer pathway impacted in glutaric aciduria type II.

the importance of screening for this mutation. Patients homozygous for the mutation respond to riboflavin treatment.

TREATMENT OF GLUTARIC ACIDURIA TYPE II Treatment includes a high-carbohydrate, low-fat, and low-protein diet, and vitamin and carnitine supplementation. Some patients respond well to riboflavin in pharmacological doses (Gordon 2006).

BIOTIN-RESPONSIVE INBORN ERRORS OF METABOLISM Biotin and Biotinidase Deficiency Biotin is also known as vitamin H; it acts as a cofactor for carboxylases. Biotin is present in certain foods; however, biotin homeostasis in humans is dependent on recycling and particularly on the release of free biotin from the amide biocytin through activity of the enzyme biotinidase (see figure 3–5). Wolf (1983) described patients with late-onset, multiple-carboxylase deficiency due to deficiency of biotinidase.

52

Seeking Cures O HN

NH O

Biocytin

−(CH2)4−C−NH

S

CH2 CH2 CH2

Biotinidase

CH2 H3−N−C−COOH H

CH2

O

Biotin

NH2

CH2 HN

NH −(CH2)4COOH S

Lysine

CH2 CH2 H3−N−C−COOH H

Figure 3–5. Illustration of biotinidase function, and cleavage of biocytin to yield free biotin and lysine.

Biotinidase deficiency may present in infancy; however, it often presents later in childhood. Clinical manifestations include patchy skin changes, alopecia, and neurologic manifestations. The latter include myoclonic seizures, ataxia, intention tremor, and limb spasticity. Impaired vision and neurosensory hearing loss may be present in many patients. Occasionally acidosis may be precipitated by acute infections. The manifestations of biotinidase deficiency can be prevented by adequate intake of exogenous biotin, 5–10 mg per day. Biotinidase deficiency is screened for in many newborn screening programs. The gene that encodes biotinidase maps to chromosome 3p25. Pindolia et al. (2010) reported that 140 mutations occur throughout the biotinidase gene, most frequently leading to enzyme levels less than 10% of normal. Mutations types leading to deficiency include nonsense and missense mutation and insertion deletion mutations. They noted that the biotinidase gene is relatively short with four exons and three introns; it encodes 543 amino acids. Pindolia et al. reported that most patients are compound heterozygotes. However, in some cases homozygous biotinidase mutations were found.

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Cowan et al. (2012) reported increased incidence of biotinidase deficiency among Hispanic newborns compared with other populations groups in California. They determined that a specific mutation at position 528, G-to-T, occurred in 43% of cases. Thodi et al. (2011) reported that five specific biotinidase mutations are most frequently encountered in Greek patients detected as biotinidase deficient on newborn screening. These same mutations also occur in Turkish patients. They developed a high-throughput PCR genotyping assay to screen for these mutations.

Biotin and Multiple-Carboxylase Deficiency Biotin serves as the coenzyme for four carboxylases: propionyl CoA carboxylase (PCC), acetyl CoA carboxylase (ACC), pyruvate carboxylase (PC), and 3-methyl crotonyl carboxylase (MCC). These carboxylases catalyze reactions in gluconeogenesis (PC), fatty acid synthesis (MCC and ACC), and catabolism of amino acids (PCC). The carboxylases occur as inactive apocarboxylases, and each requires activation that is carried out by holocarboxylase synthase in the presence of biotin and ATP (see figure 3–6). Biotin is subsequently regenerated when holocarboxylase undergoes proteolytic degradation that releases biotin bound to lysine (biocytin). Biotinidase then releases biotin from lysine.

BIOTIN-RESPONSIVE MULTIPLE-CARBOXYLASE DEFICIENCY DUE TO HOLOCARBOXYLASE SYNTHETASE DEFICIENCY Patients with this disorder present in infancy with life-threatening acidosis, ketosis, and widespread erythrematous skin disease that may be exudative. There is frequently superimposed monilia infection of the skin. Loss of hair may also be a prominent feature. During the acute crisis, organic aciduria is often present. Abnormal quantities of the following organic acids are present: 3-methylcrotonylglycine, 3-OH-isovalericacid, methylcitric acid, and hydroxypropionic acid (Sweetman et al., 1977). Enzyme studies reveal deficiency of at least three carboxylases: propionyl CoA carboxylase (PCC), 3-methyl crotonyl CoA carboxylase (MCC), and pyruvate carboxylase (PC). Burri et al. (1981) reported that the underlying defect in these patients was deficiency of the enzyme holocarboxylase

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Seeking Cures O N

N BIOTIN + APOCARBOXYLASES

acetyl coA carboxylase pyruvate coA carboxylase propionyl coA carboxylase methylcrotonyl coA carboxylase

S

O OH

Holocarboxylase synthase

O

N

N

S

O Holocarboxylases

Figure 3–6. This figure illustrates the function of holocarboxylase synthase in linking biotin and apocarboxylases.

synthase, which catalyzes the binding of biotin to the carboxylases. The HLCS gene on chromosome 21q22 encodes this enzyme. Holocarboxylase synthase impacts gluconeogenesis, fatty acid synthesis, and branched-chain amino acid metabolism. There is evidence that it may also function as a ligase that links biotin to histone. Ingaramo and Beckett (2011) reviewed posttranslational modification of carboxylases and their cell location. Acyl CoA carboxylase encoded by ACC1 is located in the cytosol. ACC2-encoded acyl carboxylase is located on the cytosolic side of the outer mitochondrial membrane. Carboxylases that function in the mitochondria include propionyl CoA carboxylase, pyruvate CoA carboxylase, and methylcrotonyl CoA carboxylase. They characterized the kinetics of biotinylation of the different carboxylases. They determined that carboxylases destined for the mitochondria are biotinylated by HCS at faster rates than the cytosolic carboxylases. This is advantageous since the mitochondrial carboxylases are only transiently in the cytosol.

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TREATMENT OF PATIENTS WITH HOLOCARBOXYLASE SYNTHETASE DEFECTS LEADING TO MULTIPLE CARBOXYLASE DEFICIENCY Although the majority of patients with holocarboxylase synthetase deficiency respond to biotin therapy, there are patients who have inadequate responses and a poor prognosis. Bailey et al. (2008) carried out studies on fibroblast from two such patients. These patients were homozygous for the c.647T>G pL212R mutation. They reported that this mutation substantially decreased the half-life of HCS in cells, indicating instability of the protein. Activity of the mutant enzyme was only marginally increased following addition of biotin to the culture medium.

BIOPTERIN METABOLISM ABNORMALITIES AND MONOAMINE NEUROTRANSMITTER DISORDERS Abnormal biopterin levels and metabolism sometimes occur in association with phenylketonuria and hyperphenylalaninemia. However, some forms of impaired biopterin metabolism lead to neurological complications in the absence of raised levels of phenylalanine. A number of different monoamine neurotransmitter disorders are due to defects in the biosynthesis; degradation or transport of dopamine, norepinephrine, epinephrine, and serotonin; and to defects in interaction with biopterin cofactors. The age of onset of monoamine neurotransmitter disorders may be during infancy or later. Neurological manifestations include movement disorders (dystonia) and pyramidal or extrapyramidal motor disorders, seizures, developmental delay, and sometimes encephalopathy. Symptoms may sometimes be paroxysmal. Kurian et al. (2011), in reviewing monoamine neurotransmitter disorders, emphasized that a high index of clinical suspicion is important since these disorders are often misdiagnosed. Correct diagnosis is important since replacement with monoamine precursors or monoamine analogs and enzyme cofactors can greatly improve patient functions and prognosis.

NEUROTRANSMITTERS AND METABOLITES Dopamine is involved in control of motor function, and decreased concentrations of dopamine in the striatum lead to extrapyramidal movement disorders (e.g. Parkinson’s disease). Kurian et al. noted that dopaminergic neurons occur

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particularly in the substantia nigra pars compacta and project to several brain structures including hippocampus and hypothalamus. Functions served by dopaminergic neurons include locomotion, neuroendocrine processes, cognitive processes, emotion, and affect. Manifestations of dopamine deficiency include Parkinsonian-type movement disorders, dystonia, chorea, ptosis, and epilepsy and oculogyric crises that involve abnormal eye and face movements. Serotonergic neurons are located in the midbrain reticular formation and project to a number of cortical areas including motor and premotor cortex, cerebellum, and spinal cord. Kurian et al. emphasized the importance of the serotonergic system in motor control, autonomic control, and mood. Serotonin deficiency leads to autonomic dysfunction, dystonia. Diagnosis of monoamine neurotransmitter disorders is made on the basis of clinical history, physical exam, and biochemical analysis based on the pattern of neurotransmitter metabolites in the cerebrospinal fluid (Kurian et al., 2011). In some cases enzyme studies and/or analysis of DNA for specific mutations are carried out. Specific neurotransmitter metabolites measured in cerebrospinal fluid (CSF) include homovanillic acid derived from dopamine and 5-OH indole-acetic acid derived from serotonin. Pterin compounds measured in snap-frozen CSF include tetrahydrobiopterin, neopterin, and biopterin, and folate is also determined. Pterin metabolites in urine are also measured. Clinical work-up requires assessment of plasma amino acids, and determination of phenylalanine levels is particularly important. Pterin pathway–related enzymes that can be measured in fibroblasts include GTP cyclohydrolase (GTPCH) and seprapterin reductase (SR).

PTERIN PATHWAY AND TETRAHYDROBIOPTERIN (BH4) SYNTHESIS Tetrahydrobiopterin acts as a cofactor for three hydroxylases, namely, phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase (see figures 3–7 and 3–8). There is evidence that tetrahydrobiopterin is also a cofactor for nitric oxide synthase. Hyperphenylalaninemia occurs as a manifestation of biopterin deficiency that results from defects in BH4 synthesis due to mutations in GTP cyclohydrolase and 6-pyruvoly-tetrahydrobiopterin synthase (PTPS). Hyperphenylalaninemia also occurs as a result of defects in BH4 regeneration due to mutations in dihydropteridine reductase (DHPR), or in pterin-4a-carbinolamine dehydratase (PCD) deficiency. Patients with sepiapterin reductase deficiency do not present with hyperphenylalaninemia. Opladen et al. (2012) emphasized that since BH4

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TYROSINE BH4

BH4 TETRADIHYDROBIOPTERIN QBH2 QUINOID DIHYDROBIOPTERIN

QBH2 L-DOPA

ADRENALINE NORADRENALINE

DOPAMINE

3-METHOXYTYRAMINE HOMOVANILLIC ACID

TRYPTOPHAN BH4 QBH2 5HYDROXYTRYPTOPHAN SEROTONIN

Figure 3–7. This figure illustrates the role of biopterin compounds in metabolism of tyrosine and tryptophan.

Phenylalanine CH2

COO− CH

BH4 Tetrahydrobiopterin

NH3+ Phenylalanine Hydroxylase CH2

COO− CH

QBH2 Quinonoid dihydrobiopterin

NH3+ OH Tyrosine

Figure 3–8. This figure illustrates the role of tetrahydrobiopterin in phenylalanine metabolism.

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deficiencies are treatable it is important that appropriate diagnostic studies be carried out on infants who present with hyperphenylalaninemia, in newborn screening or later. Impact of BH4 loading on phenylalanine levels is often a useful diagnostic test. In a database of 657 patients with BH4 deficiency, Opladen et al. determined that approximately 54.3% cases were due to PTPS deficiency, 33% had DHPR deficiency, GTPCH occurred in 4.7%, SR deficiency in 4.7%, and PCD deficiency in 3.3%. GTP cyclohydrolase produces dihydroneopterin triphosphate. Autosomaldominant and autosomal-recessive mutations in this gene lead to disease. Disease resulting from autosomal-dominant mutations is referred to as Segawa disease. Clot et al. (2009) analyzed mutations in 37 cases of GTP cyclohydrolase deficiency. The range of mutations included nonsense, missense, and splicing mutations, and small and large deletions. Deficiency of GTPCH impacts the basal ganglia and leads to dystonia with diurnal fluctuations and to movement disorders. GTPCH is one of the rate-limiting enzymes in the synthesis of tetrahydrobiopterin (BH4). BH4 deficiency impacts activity of tyrosine hydrolase and leads to dopamine deficiency. Concentration of homovanillic acid, biopterins, and neopterins are reduced in the cerebrospinal fluid. Patients respond well to treatment with levodopa or cardiodopa and with tetrahydrobiopterin supplementation. Deficiency of 6-pyruvoyltetrahydrobiopterin synthase (PTP synthase, or PTPS) is the most common biopterin synthesis defect (Kurian et al., 2011). PTP synthase is responsible for the conversion of dihydroneopterin triphosphate to 6-pyruvoyltetrahydrobiopterin. This enzyme is encoded by a locus on chromosome 11 q22.3-q23.2. The degree of deficiency may be severe or mild. Neurological features in patients with deficiency include dystonia, athetosis, hypotonia, hyperkinesia, rigidity, tremor, and developmental delay. Levels of phenylalanine and neopterin are increased in blood and biopterin levels are decreased. Allelic heterogeneity occurs in patients with deficiency of PTP synthase. However, two specific mutations predominate in Asian populations.

TETRAHYDROBIOPTERIN AND DIHYDROBIOPTERIN SYNTHESIS Several different enzymes can carry this out the conversion of 6-pyruvoyltetrahydrobiopterin (PTP) to tetrahydrobiopterin, and the predominant enzyme used differs in different tissues. Conversion of PTP to sepiapterin is carried out by aldose reductase and carbonyl reductase. The enzyme sepiapterin reductase

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then converts sepiapterin to 7–8 dihydrobiopterin. In sepiapterin reductase deficiency, phenylalanine levels are increased in blood; in cerebrospinal fluid, levels of homovanillic acid (HVA) and 5-hydroxyindole acetic acid (5-HIAA) are reduced. Friedman et al. (2012) reported that sepiapterin reductase deficiency is an underrecognized levodopa-responsive disorder. They noted that this disorder is often misdiagnosed as cerebral palsy. In the 43 patients studied, 16 different SPR gene mutations occurred; 11 of these occurred in exon 2. In a study of 43 individuals with this deficiency, they reported that most cases have motor delays, hypotonia, and oculogyric crises (abnormal eye and face movements) with diurnal fluctuation. Friedman et al. noted that there was significant phenotypic variability even among patients with the same mutation and in siblings. The enzyme dihydropteridine reductase plays an important role in the tetrahydrobioterin regeneration pathway and it transforms dihydrobiopterin to tetrahydrobiopterin. Kurian et al. (2011) reported that DHPR deficiency is present with severe phenotypic manifestation during infancy. These manifestations include microcephaly, bulbar dysfunction, delayed motor and cognitive milestones, hypertonia, and tremor. Blood levels of phenylalanine are usually elevated. Tyrosine hydroxylase and tryptophan hydroxylase activity is reduced. The reduction in tyrosine hydroxylase activity and the impaired dopamine synthesis likely lead to dystonia. Brain-imaging studies reveal white matter abnormalities and brain calcifications.

TREATMENT This includes supplementation with tetrahydrobiopterin (BH4), and 5-hydroxytryptophan and levodopa. In all cases with hyperphenylalaninemia, dietary intake of phenylalanine must be reduced. Patients frequently respond well to treatment with dopamine and serotonin precursors. Dill et al. (2012) reported that inclusion of 5-hydroxytryptophan and tetrahydrobiopterin is advantageous.

TYROSINE HYDROXYLASE AND DOPAMINE SYNTHESIS Tyrosine hydroxylase deficiency may also lead to dystonia. Deficiency of this enzyme is associated with low levels of homovanillic acid. Levels of hydroxyindole acetic acid are normal. Through activity of this enzyme with BH4 (tetrahydrobiopterin) as cofactor and in the presence of iron, L-tyrosine is converted to L-dihydroxyphenylalanine (L-DOPA).

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L-dihydroxyphenylalanine is converted to its decarboxylated form, dopamine, through activity of aromatic acid decarboxylase in the presence of vitamin B6 as cofactor. Aromatase decarboxylase deficiency is a rare cause of neuromuscular disease associated with choreoathetosis and dystonia. Dopamine is converted to norepinephrine through the activity of dopamine beta hydroxylase and subsequently to epinephrine.

DOPAMINE TRANSPORTER DEFECTS Homozygotes or compound heterozygotes for loss-of-function mutations in the dopamine transporter gene SLC6A3 encoded on chromosome 5p15.3 lead to a complex motor disorder with dyskinesia, dystonia, chorea, and abnormal eye movements, referred to as DTDS syndrome. Dopamine accumulates at synapses, and its catalysis leads to increased levels of HVA in CSF. Levels of 5HIAA are normal. Kurian et al. (2011) reported that treatment in these patients is unsatisfactory. However, some improvement in symptoms may be achieved with dopamine agonists.

SEROTONIN SYNTHESIS Tryptophan hydroxylase in the presence of tetrahydrobiopterin converts tryptophan to 5-hydroxytryptophan, which then gives rise to serotonin (5-hydroxtryptamine). Serotonin is subsequently metabolized to 5-hydroxy indole acetic acid.

Congenital Disorders of Glycosylation A wide range of clinical presentations occurs in patients with inherited glycosylation disorders (Freeze et al., 2012). There may be a history of intrauterine growth retardation in these patients. Patients sometimes present with hypotonia, seizures, and strokelike episodes. Speech is often delayed and motor development is impaired. Patients may have dysmorphic facial features including a long, thin face with protruding ears. Retinitis pigmentosa and adult-onset polyneuropathy and cardiomyopathy have been reported. Freeze et al. reported that by 2012, 70 different genetic glycosylation disorders were known. The nomenclature for congenital glycosylation disorders (CGDs) was altered to include the name of the defective enzyme followed

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by CGD. In most cases diagnosis can be established by a relatively simple biochemical test: the measurement of serum levels of glycosylated transferrin. Freeze et al. noted that one to two percent of the genome is involved in encoding proteins involved in glycosylation pathways, and eight distinct such pathways are involved in the glycosylation of proteins and lipids. It is important to note that some enzymes play roles in more than one pathway. Also, glycans from different pathways can modify a single protein. Glycosylation pathway reactions take place in the endoplasmic reticulum or in the Golgi. Defects are known to occur in seven different steps. The first step in the glycosylation pathway involves the addition of an activated monosaccharide or a saccharide chain. Specific pathways include N-linked glycosylation and O-linked glycosylation that involves monosaccharides (see figure 3–9). O-linked glycans include the following monosaccharides: xylose, mannose, fucose, glucose, and N-acetylgalactosamine. O-linked glycans bind to serine or threonine residues. In this process N-acetylgalactosamine linkage to serine, for example, is followed by the sequential addition of sugars, and galactose, fucose, sialic acid, and glucuronic acid may be added. O-linked glycans include glycosaminoglycan, heparin, chondroitin, and dermatan sulphate. Linkage of glucose to ceramide leads to generation of glycoshingolipids.

H OH

+ C−CH2−− NH2 ASPARAGINE

SUGAR O

H O N−C−CH2

GLUCOSE − −

O

O

FRUCTOSE-6-PHOSPHATE phosphomannose isomerase MANNOSE-6-PHOSPHATE phosphomannomutase

− H

MANNOSE-1-PHOSPHATE

N-GLYCOSIDIC BOND O H + OH−CH2−− OH SUGAR

GDP MANNOSE DOLICHOL p-MANNOSE

GDP FUCOSE

SERINE O H OH CH2−

O-GLYCOSIDIC BOND

Figure 3–9. This figure illustrates O and N glycosylation of amino acids and the glycosylation pathway.

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N-linked glycosylation involves the binding of N-acetylglucosamine complex to asparagine. This complex contains 14 sugars, nine mannose, three glucose, and two N-acetylglucosamine residues. The glycoprotein may subsequently be remodeled with the addition of galactose or sialic acid. Freeze et al. reported that with the exception of albumin, all proteins that pass through the endoplasmic reticulum or Golgi undergo N-glycosylation. That process impacts protein folding, stability, and trafficking. N-glycanase enzymes catalyze the detachment of glycan chains from glycoproteins.

SPECIFIC ENZYME DEFECTS IN CONGENITAL DISORDERS OF GLYCOSYLATION Mutations in enzymes involved in the biosynthesis of nucleotide sugars are important causes of congenital disorders of glycosylation. Hennet (2012) reported that enzymes involved in the generation of active mannose are often impacted in CGDs. One such enzyme is phosphomannomutase 2, which converts mannose-6-phosphate to mannose-1-phosphate. This enzyme is encoded by the PMM2 gene.

PHOSPHOMANNOMUTASE 2 DEFICIENCY AND PHOSPHOMANNOSE ISOMERASE DEFICIENCY Freeze et al. (2012) reported that mutations in phosphomannomutase account for 80% of diagnosed cases of congenital disorders of glycosylation. Disease manifestations occur primarily in the central and peripheral nervous systems; however, multisystem abnormalities may also occur. The disease usually presents in infancy, and common manifestations are hypotonia and impaired eye movements, and later seizures and strokelike episodes occur. Later ataxia and limb atrophy occur due to demyelinating peripheral neuropathy. The systemic abnormalities include impaired liver function, bleeding or thrombosis, and endocrine defects. Phosphomannose isomerase catalyzes the conversion of fructose-6phosphate to mannose-6-phosphate. Patients with phosphomannose isomerase deficiency present primarily with disease of the liver and digestive system, including chronic diarrhea. These patients also have chronic hypoglycemia and may also present with hyperinsulinemia. Sharma et al. (2011) reported that in deficiency of phosphomannomutase 2 (PMM2) or of phosphomannose isomerase, the metabolic flux of mannose-6-phosphate into glycosylation is reduced.

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Mutations in the ALG 6 gene, which encodes the asparagine-linked glycosylation 6 form of glucosyltransferase, lead to the second most common congenital disorder of glycosylation, CDG 1c. Freeze et al. (2012) reported that manifestations of this disorder are milder than those found in cases with PMM2 mutations. ALG6 mutations lead to developmental delay, strabismus, axial hypotonia, and seizures. Some patients manifest with enteropathy and skeletal dysplasia.

THERAPEUTIC DESIGN Intensive efforts are in progress to design effective therapies for congenital disorders of glycosylation. Partially successful treatments have been designed for treatment of phosphomannomutase, which catalyzes the conversion of mannose-6-phosphate to mannose-1-phosphate. This is the most common form of CDG and accounts for 80% of cases worldwide. Approaches to treatment of this disorder have been identified. Thiel and Korner (2012) reported that these include oral mannose supplementation. Administration of acetylated mannose-1-phosphate has also been proposed as treatment. Other treatments successful in PMM2-deficient culture systems include metformin (a drug used for treatment of type 2 diabetes), and inhibitors of mannose-1-phosphate isomerase (Sharma et al., 2011). De Lonlay and Seta (2009) reported that phosphomannose isomerase deficiency can be treated with oral mannose and that this treatment improves the general condition of patients.

DEFICIENCY IN SIALIC ACID SYNTHESIS: HEREDITARY INCLUSION BODY MYOPATHY (HIBM) This disorder is characterized by progressive muscle weakness that begins in the upper and lower limbs in young adults. Histological studies on muscle reveal characteristic rimmed vacuoles. These nonstorage vacuoles are thought to be autophagic in origin. HIBM is an autosomal-recessive disease first described in Jews of Persian descent. It occurs worldwide and has been studied intensively in Japan, where it is sometimes known as Nonaka myopathy, and in the United States (Huizing and Krasnewich, 2009). Following linkage studies and the mapping of this gene for this disorder to 9p12-p13, Eisenberg et al. (2001) determined that the mutant gene in HIBM is GNE, a gene involved in the synthesis of sialic acid (N-acetylneuraminic acid).

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UDP-Glc-NAc UDP-GlcNac-2epimerase

GNE gene encoded

ManNac ManNac kinase ManNac-6P Sialic acid 9-P synthase Sialic acid 9-P phosphatase Sialic acid (Neu5Ac) CMP sialic acid synthase +CTP CMP-sialic acid sialyl transferase Sialylglycoconjugate Glycoprotein

Figure 3–10.

Illustration of the sialic acid synthesis pathway.

Sialic acid is present in glycoconjugates. It is biosynthesized in the cytosol in five consecutive reactions starting with the nucleotide sugar N-acetyl UDP glucose (UDPglucose-Nac) (see figure 3–10). The first step in sialic acid synthesis involved removal of UDP and epimerization of glucose to mannose to produce N-acetyl-D-mannosamine. The enzyme involved in this reaction is UDPGlcNac-2-epimerase. The next step involves the kinase activity at the carboxyl terminal end of the same enzymes to produce N-acetyl-D mannose-6 phosphate. The next step involves condensation between N-acetyl-D mannose-6 phosphate and phosphoenolpyruvate to produce N-acetyl neuraminic-9-phosphate. Hinderlich et al. (1997) demonstrated that UDP GlcNac 2 epimerase and ManNac-kinase are parts of a bifunctional enzyme, and both activities are present on a 75-kilodalton polypeptide. Assembly of this polypeptide as a hexamer generates a protein with both enzyme activities. The dimeric form of the polypeptide has only ManNac-kinase activity.

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TREATMENT A mouse model of hereditary-inclusion body myopathy utilized in preclinical studies revealed that treatment with sialic acid metabolites increased muscle strength. The sialic acid metabolite tetra-O-acetylated N-acetyl mannosamine led to increased sialation and to measurable improvements in muscle phenotype (Malicdan et al., 2010). Mitrani-Rosenbaum et al. (2012) developed a human GNE transgene in an AAV vector. Systemic delivery through intravenous injection into a normal mouse was followed by expression of the transgene in muscle tissue, and there was no evidence of focal or general toxicity following treatment.

METABOLIC INDIVIDUAL VARIATION AND THE METABOLOME Following his investigations of alkaptonuria and demonstration that this was an inherited metabolic disorder, Archibald Garrod conceived the idea of biochemical individuality, and he regarded alkaptonuria as an extreme example of this (Harris, 1963). Suhre et al. (2011) carried out metabolite analyses in conjunction with genome-wide association studies and reported that these studies provided information on genetically determined metabotypes or intermediate phenotypes. They carried out nontargeted analyses of more than 250 metabolites in serum samples from two different populations, including 2,820 individuals. Metabolite analyses were carried out using high-performance liquid phase chromatography (HPLC), gas chromatography, and tandem mass spectrometry. They determined ratios between metabolite concentrations. They then correlated the ratios with genotypes. Their studies led to identification of 37 loci associated with specific metabolic traits. Suhre et al. reported that at 30 of these loci the sentinel nucleotide variant (SNP) mapped to a protein directly linked to the metabolite and responsible for synthesis, metabolism, or degradation of the metabolite. Literature searches revealed that 15 of the 37 loci were associated with specific diseases or drug reactions. Suhre et al. reported a highly significant association (5 × 10–252) of blood levels of N-acetylornithine and an SNP variant rs13391552 in the NAT8 locus that encodes N-acetyltransferase. They determined that higher levels of N-acetylornithine were associated with a lower glomerular filtration rate. Serum levels of N-acetylornithine will serve as a biomarker for renal disease.

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LC Liquid chromatography ES Electrospray Mass spectroscopy 78 unique compounds

NMR Nuclear magnetic resonance

TLC Thin-layer chromatography Gas chromatography Flame ionization

20 unique compounds

3316 unique compounds

GC/MS Gas chromatography Mass spectroscopy 70 unique compounds

DFI/MS Direct-flow injection Mass spectroscopy 80 unique compounds

The serum metabolome: 5 different platforms used to identify 3,564 compounds Psychogios et al., 2011 The cerebrospinal fluid metabolome: 5 different platforms used to identify 78 metabolites Mandal et al., 2012

Figure 3–11. This figure indicates the extent of the serum metabolome and the cerebrospinal fluid metabolome investigated using five different platforms.

A specific SNP rs2066938 in the gene that encodes the ACADS (short-chain acyl CoA dehydrogenase) enzyme, which is involved in the beta-oxidation of fatty acids showed highly significant association with the butylcarnitine propionylcarnitine ratio. Of particular interest was the finding that the ratio of levels of specific unphosphorylated and phosphorylated fibrinogen peptides correlated with SNPs at three loci: the ABO blood group locus, the liver alkaline phosphatase locus, and the fucosyltransferase 2 locus. They noted that alteration in these fibrinogen peptide ratios might provide an explanation for the association of ABO blood group variants with venous thromboembolism. They identified genetically determined metabotypes associated with toxicity or adverse responses to medication. Specific variants in SLC2A9, SLC22A1, and SLCO1B1 correlated with sensitivity to metformin and etoposide and predisposition to statin-induced myopathy. They determined that a specific SNP in SLC2A9 rs7094971 correlated with serum urate levels.

GENETIC VARIANTS AND UNUSUAL METABOLIC PROFILES Loss-of-function alleles and, in particular, homozygous loss-of-function alleles, at particular loci lead to in inborn errors of metabolism. However, specific variants in these same genes may lead to qualitative or quantitative enzyme alteration and unusual metabolic profiles. Illig et al. (2010) reported that the finding of specific metabolic profiles associated with variants at a specific locus leads to the generation of new hypotheses regarding functions of the product of that locus (see figure 3–11).

4 LYSOSOMAL STORAGE DISEASES AND THERAPIES

INTRODUCTION Lysosomal storage diseases have traditionally been classified according to the type of material that accumulates in lysosomes in abnormal levels. In lipid storage diseases, sphingolipids and gangliosides accumulate. Mucopolysaccharide storage occurs in Hurler and Hunter disease (Fratantoni et al., 1968a and b). Carbohydrate-rich compounds accumulate in glycogen storage diseases. An alternate method of classification is based on the functional characteristics of the protein that is deficient in specific storage disorders (e.g., phosphotransferases, sulfatases). These diseases are also sometimes classified according to the predominant functional impairment (e.g., lysosomal biogenesis defects or lysosomal trafficking defects). Deficiency can primarily involve specific lysosomal enzymes, cofactors and posttranslation modification factors, membrane transport proteins, and transmembrane proteins (Winchester et al., 2000). In this chapter examples of progress in understanding lysosome biology and in development of treatments for lysosomal disorders are presented. Treatment developments include FDA-approved clinical treatments and also preclinical and proposed treatments based on studies in model organisms and/or cell cultures. Gene therapy approaches to treatment of lysosomal storage diseases are also being investigated (Byrne et al., 2012). 67

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LYSOSOMAL BIOLOGY AND PATHOLOGY IN LYSOSOMAL STORAGE DISEASES De Duve first described lysosomes in 1963. An early key discovery in lysosomal biology made by Elizabeth Neufeld and colleagues (Willingham et al., 1981) was that proteins destined for the lysosome have a defined signal, namely, mannose-6-phosphate. They also reported that lysosomal enzymes pass from cells into the interstitial fluid. Deter and De Duve (1967) first described autophagosomes and lysosomes. Autophagy and specifically macroautophagy are important in clearance of organelles and long-lived cellular proteins in a process that involves engulfment of cytoplasmic components into double-membrane structures. Autophagy plays critical roles in degradation processes and in processes designed to buffer starvation. Cox and Cachon-Gonzalez (2012) reviewed the cellular pathology of lysosomal storage diseases. They reported that 70 different germ line gene defects lead to abnormal lysosomal storage. The combined frequency of lysosomal storage diseases is 1 in 7,500. These authors emphasized that scientific understanding of lysosomal function and the roles of lysosomes in cell biology were greatly advanced by explorations of defects in patients with lysosomal storage diseases. They noted that these advances constitute examples of “the debt of science to medicine.” Normal functioning of lysosomes is particularly important in neurons. Lysosomal activity is key to membrane recycling throughout the neurons and their extended processes. Cox and Cachon-Gonzalez (2012) reported that investigation of defects in patients led to identification of lysosomal enzyme activators, cofactors, and modifiers. Saposins are examples of activators and are important in the function of beta glucosidase (beta glucosyl ceramidase). A specific sulfatase-modifying factor (SUMF1) induces activating cysteine modification in lysosomal phosphatases. Deficiency of SUMF1 leads to a form of lysosomal storage disease, multiple sulfatase deficiency.

SORTING OF PROTEINS TO LYSOSOMES The synthesis and transport of soluble proteins and transmembrane proteins to lysosomes involve sorting signals and recognition receptors; Braulke and Bonifacino (2009) reviewed this process. Lysosomal hydrolases are modified by the addition of mannose-6-phosphate residues. In the endoplasmic reticulum, mannose-rich oligosaccharides (glucose-mannose-N-acetylglucosamine) are transferred to specific asparagine residues on lysosomal enzymes. In the Golgi the

Lysosomal Storage Diseases and Therapies

MAN

MAN

MAN

MAN MAN

GLCNac

MAN

MAN

MAN

MAN

MAN -P-GLCNac

GLNac-P- MAN MAN

MAN MAN

2UDP+GLCNac GLCNac phosphotransferase

MAN

69 MAN MAN -P

P- MAN MAN

MAN MAN

N-acetylglucosaminidase− GLCNac uncovering enzyme GLCNac

GLNac

GLNac

GLNac

-ASN-

-ASN-

-ASN-

ASN in lysosomal hydrolase

Figure 4–1. This figure illustrates roles of GlcNac-phosphotransferase and uncovering enzyme in modification of lysosomal hydrolases. Constructed by the author based on information contained in texts by Fernandes et al. (2000), Voet and Voet (1995), and Nyhan et al. (2005).

oligosaccharide residues are modified. This modification requires two enzymes, GlcNac-phosphotransferase, and NAGPA (N-acetylglucosamine-1-phospho diester-alpha-N-acetylglucosaminidase, sometimes referred to as uncovering enzyme). The enzyme GlcNac-phosphotransferase (N-acetylglucosamine-phosp hotransferase) is composed of alpha and beta subunits encoded by the GNPTAB gene and a gamma subunit encoded by GNPTG. This enzyme catalyzes linkage of GlcNac-1-phophate derived from uridine diphosphate-N-acetylglucosamine to the terminal mannose on the N-linked oligosaccharides on lysosomal enzymes. The NAGPA enzyme then removes the terminal GlcNac from this linkage, and the mannose-6-phophate residue that acts as a targeting signal, is exposed. Mannose-6-phosphate receptors occur in the trans-Golgi network and in endosomes. At least two forms of mannose 6-phosphate receptors are known; the MPR300 (MPR1) receptors are larger and are cation independent while the MPR46 residues are cation dependent. Mannose-6 phosphate signals corresponding receptors in the Golgi complex and ensures transport to the endosomal/lysosomal system. Additional modifications that occur along the transfer pathway include proteolytic cleavage of some lysosomal enzymes and associated proteins. The prosaposins undergo proteolytic cleavage to generate saposins, which serve as activator factors for a number of lysosomal enzymes. There is evidence that subsets of hydrolases and nonenzyme proteins undergo mannose-6-phosphate independent transfer into lysosomes and require specific lysosomal membrane receptors LIMP2 or sortilin.

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Endoplasmic reticulum

Clathrin-coated vesicles with M6P tagged lysosomal hydrolases

Endosome

Lysosomal hydrolases

Low pH in lysosomes leads to release of M6P receptor mannose-6-phosphate receptors

M6P receptors recycled to Golgi

Figure 4–2. This figure illustrates mannose-6-phosphate targeting to lysosomes. Constructed by the author based on information contained in texts by Fernandes et al. (2000), Voet and Voet (1995), and Nyhan et al. (2005).

MUCOLIPIDOSIS II The lysosomal storage diseases mucolipidosis II and mucolipidosis IIA result from defective activity of N-acetylglucosamine-phosphotransferase (GlcNac phosphotransferase) due to mutations in the GNPTAB gene. Mucolipidosis IIIC (III gamma) results from mutations in the GNTABG gene. Deficiency of GlcNac-phosphotransferase leads to impaired targeting of lysosomal hydrolases. Lysosomes become filled with undigested complex molecules, phospholipids, and mucopolysaccharides. These compounds and distended lysosomes give rise to histologically observable inclusion bodies in cells. Clinical manifestations include developmental delay, and severe bone and growth abnormalities.

APPROACHES TO TREATMENT Otomo et al. (2011) demonstrated that lysosomal storage components in fibroblasts of mucolipidosis type II patients could be cleared using a partially purified total enzyme mixture from conditioned medium of normal cultured skin fibroblasts. There is evidence that the flavonoid genistein decreases storage of heparan sulfate in mucolipidosis (Otomo et al., 2012).

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Membrane proteins endocytosed Multivesicular body Early endosome

Iysosome

Figure 4–3. This figure illustrates passage of material from endosomes to multivesicular bodies and then to lysosomes; figure constructed by author on the basis of information in Luzio et al. (2009) and Metcalf and Isaacs (2010).

ENDOSOMES, LYSOSOMES, AND AUTOPHAGOSOMES The endosome system takes up material imported into cells via the plasma membrane. This material includes receptors and ligands. Imported material is sorted, and receptors may be recycled to the cell surface. Substances destined for degradation pass from early endosomes to late endosomes and multivesicular bodies. In reviewing this process, Luzio et al. (2009) reported that key proteins involved in sorting and transport include ESCRT (endosomal sorting complex required for transport) and signaling molecules RAB5 and RAB7. Late endosomes subsequently deliver their material to lysosomes. Fusion of endosomes and lysosomes requires the SNARE protein complex that includes syntaxin 7 and 8 and VAMP7 (vesicle-associated membrane protein 7). The ESCRT complex is also involved in the fusion of multivesicular bodies with lysosomes and with the fusion of autophagosomes and lysosomes. Other proteins important in autophagosome–endosome lysosome fusion include LAMP2 (lysosome-associated membrane protein 2) RAB11, the ubiquitin binding protein ubiquilin and VCP (valosin-containing protein). Autophagosomes are double-membrane structures that engulf cytoplasmic components including organelles. Microautophagy differs from microautophagy. In microautophagy lysosomes take up cytosolic components directly. There is evidence that SNARE proteins play a key role in early phagosome (phagophore) formation. Snare proteins are also required for the fusion of autophagosomes to lysosomes. Microtubule transport of organelles occurs and is facilitated by dynein proteins (Metcalf and Isaacs, 2010). More than 30 genes are involved in autophagosome biogenesis and function. Autophagosomes evolve from simple membrane structures. Proteins, lipids, and membrane structures necessary for phagophore and autophagosome formation are derived from the endoplasmic reticulum, the Golgi, from plasma membranes,

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and mitochondrial membranes. Autophagy is considered to be a cytoprotective process induced by physiological stresses including nutrient starvation, oxidative stress, and accumulation of unfolded proteins (Weidberg et al., 2011). In studies on the cellular effects of starvation, Settembre et al. (2011) discovered a transcriptional program that controls autophagosome formation, fusion with lysosomes, and substrate degradation. They established that a specific transcription factor, TFEB, controls lysosomal biogenesis and autophagosome formation. Movements of TFEB from cytoplasm to nucleus are modulated by phosphorylation of a serine residue in the TFEB protein. This phosphorylation is dependent on activity of a specific kinase encoded by the ERK2 gene.

LYSOSOMAL EXOCYTOSIS AND TREATMENT RELEVANCE In lysosomal exocytosis lysosomes fuse with the plasma membrane of the cell and empty their contents outside the cell. Medina et al. (2011) reported that this process is regulated by the TFEB (transcription factor EB). Previous studies had revealed that lysosomal exocytosis is also dependent upon intracellular calcium levels and on calcium activation of MCOLN1, the calcium channel of lysosomes. Medina et al. demonstrated that lysosomal exocytosis could be exploited to promote clearance of stored material in lysosomal storage diseases utilizing the function of TFEB. This factor enhances release of calcium from lysosomes and facilitates exocytosis. The structure and function of MCOLN1 (mucolipin 1) was intensively studied following discovery that this gene is mutated in a severe lysosomal storage disease. MCOLN1 protein acts as a transient receptor potential calcium channel that impacts lysosomes and the late endosome pathway. Its function is modulated by changes in the calcium concentration. MCOLN1 also plays a role in determining lysosomal pH. The MCOLN1 gene maps to chromosome 19p13.2. MCOLN1 deficiency leads to mucolipidosis IV, a disorder characterized by neurodevelopmental defects and cerebral palsy–like manifestations; visual impairments may occur, due to corneal clouding strabismus and retinal degeneration. Patients with this disorder often have achlorydia or hypochlorydia due to defective function of gastric parietal cells. In some cases manifestations are milder. At least 21 different nucleotide mutations are known to cause this disorder. In addition, a large deletion occurs in MCOLN1 in some patients. Mucolipidosis is a panethnic disorder; however, 70–80% of patients are of Ashkenazi Jewish ancestry (Wakabayashi et al., 2011).

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TREATMENT POSSIBILITIES Medina et al. (2011) proposed that pharmacologic induction of the transcription factor TFEB, activation of the TFEB gene, or gene delivery of TFEB could be potential therapeutic measures for mucolipidosis IV.

Mucopolysaccharide Storage Diseases Proteoglycans in the extracellular matrix are proteolytically cleaved and give rise to glycosaminoglycans (mucopolysaccharides) that are taken up into lysosomes for further digestion. Coutinho et al. (2012) reviewed enzymes involved in glycosaminoglycan (GAG) cleavage and mucopolysaccharide storage diseases. Enzymes involved in GAG cleavage include glycosidases sulfatases and transferases. Progressive storage of undigested material leads to lysosomal expansion. Typical symptoms of mucopolysaccharide storage diseases include bone abnormalities, joint stiffening, organomegaly, cardiovascular impairments, hearing loss, and eye findings including corneal clouding and facial coarsening. Diagnosis may be established through finding abnormal levels of specific glycosaminoglycans in urine. Enzyme deficiency characteristic of a specific subtype of mucopolysaccharide storage disease can be identified through cell-based assays. Subsequent DNA studies are frequently used to establish a specific diagnosis. Substantial progress has been made in development of therapies for several of these diseases. Therapeutic intervention includes primarily enzyme replacement therapy for at least six forms of mucopolysaccharide storage disease (see table 4–1). At the time of writing, enzyme replacement therapies for Sanfilippo MPS III are not available. Clinical manifestations in the four types of Sanfilippo disease are quite similar. Central nervous system impairment is usually quite severe while somatic manifestations may be mild. There is increased urinary excretion of heparan sulfate in all forms.

Niemann-Pick Disease Type C Niemann-Pick disease type C is most commonly associated with neurological symptoms, including abnormal eye movements (characteristically, paralysis of vertical gaze), and with ataxia, speech impairments, swallowing difficulties, psychiatric manifestations, and sometimes seizures. Onset of symptoms may occur in childhood but often begin in adult life (Patterson et al., 2012). In

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Table 4–1. Treatment of Lysosomal Storage Diseases disease name

missing enzyme

treatment

MPSI/H Hurler

Alpha L-iduronidase

HSCT or ERT laronidase

MPSI/HS Hurler/ Scheie

Alpha L-iduronidase

HSCT or ERT laronidase

MPSI/S Scheie

Alpha L-iduronidase

HSCT or ERT laronidase

MPSII/Hunter syndrome

Iduronate-2-sulfatase

ERT idursulfase Elaprase

MPSVI (Maroteux-Lamy)

N-acetylglucosamine sulfatase

ERT galsulfase Naglazyme

MPSIV MorquioA

Galactosamine 6 sulfosulfatase

ERT clinical trial

MorquioB

Beta galactosidase

Preclinical chaperone

MPSIII Sanfilippo A

N-sulfoglucosamine sulfohydralase

Flavones Genistein trial

MPSIII Sanfilippo B

N-acetylglucosaminidase

Preclinical ERT

MPSIII Sanfilippo C

N-glucosaminide acetyltransferase

MPSIII Sanfilippo D

N-acetylglucosamine6-sulfatase

MPS VII Sly syndrome

Beta glucuronidase

Preclinical modified ERT

MPS IX Juvenile arthritis

Hyaluronidase deficiency

—

Gaucher

Acid beta-glucosidase

ERT imiglucerase; chaperone

Fabry

Alpha-galactosidase A

ERT agalsidase, Fabrazyme

Pompe

Acid alpha glucosidase A

ERT Myozyme; reduce substrate

HSCT: hemopoietic stem cell transplant ERT: enzyme replacement therapy

95% of cases, this disease is due to mutations in the NPC1 gene on chromosome 18q11-q12. In 5% of cases, it is due to mutations in the NPC2 gene on 14q24.3. NPC1 and NPC2 encode proteins that transport unesterified cholesterol across membranes in the lysosomal-endosomal system to the cytosol. In Niemann-Pick disease, endosomes and lysosomes continue to take up Apo B lipoproteins and Apo E lipoproteins into endosomes and lysosomes. Defective function of NPC1 leads to accumulation of lipids and cholesterol in lysosomes. Furthermore, the low cytosol concentrations of cholesterol impair feedback control so that cells continuously synthesize cholesterol and there is continuous generation of lipoproteins.

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O

SO

Hurler mucopolysaccharidosis alpha iduronidase deficiency

O

OS

alpha iduronidase

NAc

IDURONATE SULFATE PRESENT IN DERMATAN SULFATE AND HEAPRAN SULFATE

O

SO

Hunter mucopolysaccharidosis iduronate sulfatase deficiency

O

NAc OS Iduronate sulfatase

Figure 4–4. This figure illustrates enzyme defects leading to abnormal accumulation of mucopolysaccharides in Hurler and Hunter diseases. Constructed by the author based on information contained in texts by Fernandes et al. (2000), Voet and Voet (1995), and Nyhan et al. (2005).

TREATMENT POSSIBILITIES Ramirez et al. (2010) reported results of studies on a murine model of NPC1 deficiency in which treatment with a cholesterol binding agent cyclodextrin (2-hydroxypropyl-beta-cyclodextrin) was carried out. This compound was administered subcutaneously, and treatment led to stimulation of cellular release of cholesterol from organs. This release resulted in suppression of the target genes of the sterol regulatory element binding transcription factor SREBF2 and suppression of cholesterol synthesis. Treatment with cyclodextrin led to improvement of neurological symptoms and decrease in liver manifestations of the disease.

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Niemann-Pick Disease Types A and B These diseases result from deficiency of sphingomyelinase (sphingomyelin phosphodiesterase 1) SMPD1 on chromosome 11.p15.1-p15.4. Niemann-Pick type A is characterized by neurological deterioration, psychomotor regression, hepatomegaly, and early death. In Niemann-Pick disease type B, the manifestations are primarily nonneurologic. Most mutations that lead to this disease are private (Desnick et al., 2010). Mutations that lead to total loss of enzyme activity are primarily found in type A. Missense mutations that lead to less marked deficiency of the enzymes occur in type B and are associated with less severe neurologic abnormalities.

HSP70 CHAPERONE PROTEIN AND LYSOSOME STABILIZATION IN NIEMANN-PICK DISEASE Nylansted et al. (2004) demonstrated that HSP70 protein is present on the plasma membrane of cells and on membranes of the endolysosomal system under stress conditions. There is evidence that ectopic HSP protein can bind to lysosomes and reduce their permeabilization. Kirkegaard et al. (2010) targeted recombinant HSP70 to cells. They reported that HSP70 binds to the endolysosomal anionic phospholipid bismonoacylglycerophosphate (BMP). This is an essential cofactor for sphingomyelinase, the enzyme deficient in Niemann-Pick type disease types A and B. Kirkegaard et al. reported that stabilization of lysosomes subsequent to HSP70 binding of BMP led to correction of the cellular phenotype in Niemann-Pick disease.

ENZYME REPLACEMENT AND PHARMACOLOGICAL CHAPERONES IN LYSOSOMAL STORAGE DISEASE THERAPY Recombinant lysosomal enzymes, produced in vertebrate cells or plant cells, are injected intravenously and most efficiently target to reticulo-endothelial cells and to liver and spleen, and they also target kidneys and heart and, to a slightly lesser extent, lung. Bone is generally not well targeted and brain is excluded (Lachmann, 2011). ERT has worked particularly well in treatment of the nonneuronopathic form of Gaucher disease (Desnick and Schuchman, 2012). Usually frequent infusions of enzyme are needed, infused enzyme may be immunogenic, and eventually tolerance to the infused protein is impaired. Another consideration is that ERT is extremely costly.

Lysosomal Storage Diseases and Therapies CH2OH

O

–

HO H

OH

– –

O = C

H

C- -H CH

H

H

OH

H- C

H

H

OH

O H

(CH2)12 CH3

– –

R

O = C R

C-- H CH C

–

– –

Beta-D-glucoside residue

Sphingosine

H2C-C NH

OH

– – ––

OH

–

Fatty acid residue

– – –

– – H2C- C NH

77

(CH2)12 CH3

CERAMIDE

GLUCOCEREBROSIDE

Figure 4–5. Structures of ceramide and glucocerebroside; the latter is present as the abnormally stored material in Gaucher disease. Constructed by the author based on information contained in texts by Fernandes et al. (2000), Voet and Voet (1995), and Nyhan et al. (2005).

Enzyme replacement therapy (ERT) does not reduce storage in the nervous system or manifestations of nervous system impairments since enzyme does not cross the blood–brain barrier. There are, therefore, continuous efforts to identify pharmacological chaperones that cross the blood–brain barrier. Y. O N

O B

N

Ceramide

GLUC

O B

N

Glucocerebrosidase Gaucher disease Galactocerebrosidase Krabbe disease

GAL

Arylsulfatase A metachromatic leukodystrophy

O B

N

gal-3-sulphate

O B

B

A

alpha-galactosidase Fabry disease

N GAL

GLUC

N

O B

B

GAL

B NAGA

GLUC

OSO3

Beat hexosaminidase Tay-Sachs disease

GAL NANA

Figure 4–6. Schematic representations of stored sphingolipids and enzymes deficient in five lysosomal storage diseases. Constructed by the author based on information contained in texts by Fernandes et al. (2000), Voet and Voet (1995), and Nyhan et al. (2005).

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Sun et al. (2012) reported that the pharmacological chaperone isofagomine enhances beta glucosidase function by promoting enzyme folding. In studies on a mouse model of neuronopathic Gaucher disease, they determined that this compound crossed the blood–brain barrier. Chaperone therapy is an option for treatment in several lysosomal storage diseases if the mutation leads to impaired folding of the enzyme or to reduced stability. However, defining chaperones that primarily affect the mutant protein is particularly important since nonspecific protein binding may have deleterious effects. Potential pharmacologic chaperones must, therefore, be screened for selectivity, biochemical action, impact on cells, and pharmacokinetic and pharmacodynamics properties and must also be evaluated in animal models of specific lysosomal storage diseases. Many chaperones used in therapy have structural similarity to the substrate enzyme protein and are reversible competitive inhibitors of the enzyme. For pharmacologic chaperones designed to treat lysosomal storage diseases, it is important that they bind to the enzyme protein and stabilize it in the endoplasmic reticulum at neutral pH and remain bound until the protein reaches the lysosome. There the bound chaperone should be released in order for the enzyme to function adequately (Valenzano et al., 2011).

POMPE DISEASE AND TREATMENT Glycogen storage disease type II results from deficiency of lysosomal acid alpha glucosidase (acid maltase). This deficiency leads to impairments in skeletal and cardiac muscle, hypotonia, and hypertrophic cardiomyopathy. There is some evidence that abnormal glycogen accumulation occurs in nerve ganglia. Treatment of the disease symptoms includes nutritional modification; high-protein, low-carbohydrate diet; and exercise therapy. Richard et al. (2011) reported that more striking improvements in Pompe disease manifestations have been achieved through enzyme replacement, enzyme enhancements, and substrate reduction therapy. Mouse models of glycogen storage disease type II have facilitated investigation of the disease pathogenesis. Richard et al. reported that the exact mechanisms leading to muscle dysfunction are not known. However, there is evidence that the lysosomes that are greatly distended as a result of glycogen storage may rupture and release content into the cells, leading to alterations in mitochondria and to disruption of architecture of contractile units in muscle. Enzyme replacement therapy in Pompe’s disease involves administration of recombinant human acid alpha glucosidase produced in cultured cells

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(e.g., Chinese hamster ovary cells or recombinant enzyme produced in rabbit milk). Induction of immune response in treated patients is particularly a problem in CRIMM-negative patients (i.e., patients who produce no enzyme). Richard et al. reported that glycogen clearance in liver and cardiac muscle result from this treatment. However, glycogen clearance from skeletal muscle is modest. ERT treatment of Pompe disease includes use of recombinant alpha glucosidase (Myozyme, Lumizyme) that is intravenously infused. Expenses are a major consideration. Other forms of therapy for Pompe disease include gene therapy. Richard et al. (2011) reviewed studies on mouse models of Pompe disease where retroviral, lentiviral, or adenoviral vectors containing the human acid alpha glucosidase gene were used and disease manifestations studied. These treatments did not lead to complete reversal of glycogen storage in muscle. Treatment of mouse models of Pompe disease with hematopoietic stem cells transfected with human acid alpha glucosidase led to significant clearance of glycogen in liver, cardiac muscle, and diaphragm, and skeletal muscle strength improved. Chaperone treatment with 1-deoxynojirimycin (DNJ) or the N-butyryl form of this compound led to improvement of disease manifestations in patients with acid alpha glucosidase mutations that did not involve the active site of the enzyme (Richard et al., 2011). Shimada et al. (2011) reported that ERT was approved for Pompe disease treatment but that in many treated patients glycogen removal from skeletal muscle was not adequate to improve function. Furthermore, formation of antibodies proved to be problematic in some cases. They demonstrated that acid alpha glucosidase containing the c546 G>T mutation induces endoplasmic reticulum stress and that the chaperone N-butyl-deoxynojirimycin can improve enzyme stability, maturation, and trafficking of the mutant protein. Shimida et al. reported that treatment with the chaperone N-butyl-deoxynojirimycin, together with a proteosome inhibitor, induced stabilization of acid alpha glucosidase enzyme with a disease-causing mutation. Proteosome inhibitors used included bortezomib and ME132. They used immunofluorescence analysis and the lysosomal marker LAMP and demonstrated increased localization of acid alpha glucosidase in lysosomes.

NEURONAL CEROID LIPOFUSCINOSES These are fatal storage diseases that are classified together since they are all characterized by accumulation of distinctive autofluorescent pigmented

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Table 4–2. Genes Mutated in Ceroid Lipofuscinoses and Functions of Encoded Proteins gene

product

function

chromosome

CLN1

PPT1

Palmitoyl protein thioesterase

1p32

CLN2 CLN3 CLN4B

TPP1 BTS DNAJC5A

11p15 16p12.1 20q13.33

CLN5 CLN6 CLN7 CLN8

CLN5 protein CLN6 protein MFSD8 EMPR

CLN9

Protein

Tripeptidyl peptidase Battenin lysosomal function Membrane trafficking and protein folding Lysosomal protein degradation Lysosomal protein degradation Localizes to lysosomal membrane Transmembrane protein lipid transport Regulator of dihydroceramide synthase

CLN10

CTSD

Lysosomal cathepsin D proteinase

11p15.5

13q21.1-q32 15q23 4q28.3 8p23

material in lysosomes, particularly in brain. Clinical features of these diseases include decline in cognitive and motor abilities, seizures, blindness, and early death. Mutations in any one of at least 10 different genes may lead to neuronal ceroid lipofuscinoses (Cooper, 2010)). Four of these genes encode soluble lysosomal proteins. Six genes that lead to this disorder when mutated encode transmembrane proteins (see table 4–2). The blood–brain barrier constitutes a significant impediment to treatment of these diseases with enzyme replacement therapy.

TREATMENT POSSIBILITIES FOR CEROID LIPOFUSCINOSES Roberts et al. (2012 investigated the efficacy of central nervous system–directed adeno-associated virus AAV2 mediated gene therapy with the gene CLN1, which encodes the lysosomal enzyme palmitoyl protein thioesterase PPT1 in a mouse model with PPT1 deficiency. They reported that this gene therapy resulted in histological, biochemical, and functional improvements. These investigators also carried out a study of treatment of the PPT1-deficient mice with phosphocysteamine only. They reported that this treatment led to incremental improvements in rotarod performance and to slight decrease in stored material. However, the combination of gene therapy with AAV-Ppt1 led to greater improvement. Zhang et al. (2001) reported the therapeutic potential

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of phosphocysteamine and N-acetyl cysteine that disrupts thioester linkages in palmitoylated proteins. They reported that application of these drugs to cultured cells from patients with palmitoyl protein thioesterase deficiency decreased lysosomal storage of palmitoylated proteins. Treatment possibilities for CLN2 include upregulation of tripeptidyl peptidase by fibrate drugs gemfibrozil and fenofibrate (Ghosh et al., 2012).

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5 MITOCHONDRIAL FUNCTION, DEFECTS, AND APPROACHES TO TREATMENT

INTRODUCTION Mitochondria play key roles in energy generation through ATP production. These organelles are also important for thermogenesis and ion homeostasis. They are responsible for generation of reactive oxygen species. Key to these functions are the multisubunit complexes I–V located in the inner mitochondrial membrane and the electron carriers coenzyme Q and cytochrome C. Reducing equivalents NADH and FADH2 derived from the tricarboxylic acid cycle enter the electron transport chain through complex I (NADH ubiquinone oxidoreductase). They may also enter at complex II succinate dehydrogenase. Complex II succinate CoA reductase carries reducing equivalents from FADH2 to coenzyme Q and is composed of succinate dehydrogenase forms and iron sulfur complexes. In complex III electrons are passed to cytochrome C. Complex IV cytochrome C oxidase transfers electrons from cytochrome C to molecular oxygen and protons to the mitochondrial intermembrane space (Wallace, 1999). Mitochondria also play roles in the innate immune response and in programmed cell death (Koopman et al., 2012). Mitochondrial DNA and nuclear DNA each encode specific subunits of the electron transfer complexes and of ATP synthase. Diseases may arise as 83

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a result of DNA deletions, point mutations, or rearrangements in the mitochondrial DNA or in the nuclear DNA. Nuclear proteins control mitochondrial DNA replication, repair, transcription, and translation. Nuclear DNA mutations may lead to multiple deletions in mitochondrial DNA, to defects in specific proteins that function in mitochondria, and to impaired assembly of mitochondrial respiratory complexes. Defects in nuclear genes may also lead to impaired mitochondrial fissions and fusion and may impact mitochondrial quality control mechanisms (Schapira, 2012). The goal of this chapter is to review information on the mitochondrial proteome and on mitochondrial functional impairments derived from high-throughput proteomic analysis and DNA sequencing and advances in the treatment of mitochondrial diseases.

MITOCHONDRIAL DNA MUTATIONS, HETEROPLASMY CLONAL EXPANSION, AND PHENOTYPES Mitochondrial DNA mutations may be associated with oligosymptomatic disorders (e.g., deafness) or with multisystem pathologies. DiMauro (2010) emphasized that heteroplasmy and threshold effects are the best criteria to explain phenotypic variability in disease due to mitochondrial DNA mutations. An example is the case of mutations in mitochondrial DNA at position m.8993T>G. Tatuch et al. (1992) reported that 70% heteroplasmy in skeletal muscle was associated with adult disease, including exercise intolerance, while 90% heteroplasmy in skeletal muscle was associated with severe metabolic disease and encephalopathy, manifestations of Leigh syndrome. DiMauro (2010) noted that specific mitochondrial DNA mutations selectively impact specific structures in the brain. For example, the MERF mutation m8344A>G in tRNAlys impacts the dentate nucleus of the cerebellum. Schapira (2012) emphasized that the consequences of mitochondrial mutations must be considered in the context of heteroplasmy and clonal expansion, which lead to differences in cells and tissues with respect to the admixture of mutant or wild-type mitochondria. Cells and tissues also differ in mutation threshold (i.e., the percentage of mutant mitochondria that lead to pathology). Mitochondrial fusion and fission increase the opportunity for cells to have larger numbers of wild-type mitochondria. Enns et al. (2012) reported that mitochondrial disease due to defects in the mitochondrial DNA occurs in approximately 1 in 5,000 individuals. They noted further that specific diagnoses were lacking in many individuals who exhibit signs and symptoms of mitochondrial disease. There is growing evidence that many of these cases are likely due to nuclear DNA mutations.

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ADP

NAD

FMNH2

Cyt bc III Fe2 Fe3

COQ I COQH2 NADH

FMN II FADH2 FAD

V F1 IV Cyt c oxidase

ATP synthase V

ETF-QO ETFr

ETFo

ANT

F0 ATP

ADP + Pi

Figure 5–1. A schematic representation of the mitochondrial complexes that constitute the electron transfer system. Drawn by the author and constructed based on information in texts by Nyhan et al. (2005), Voet and Voet (1995), and Scheffler (2008).

Pathogenesis of Manifestations Due to Defective Mitochondrial Function It is important to emphasize that both mitochondrial DNA mutations and nuclear DNA mutations contribute to mitochondrial disease. Furthermore, the phenotype associated with a specific mitochondrial disease is modified by additional genotypic variation in nuclear and mitochondrial genomes (Koopman et al., 2012). ATP demand and the rate of oxidative phosphorylation differ in different cell types and tissues. Mutations that impact the function of mitochondrial proteins lead, in some cases, to initiation of adaptive processes. These include compensation for reduced ATP production by increased glycolysis, increased biogenesis of mitochondria, and upregulation of processes to detoxify reactive oxygen species. Somatic mitochondrial DNA mutations result from increased oxidative stress. Defense mechanisms against mitochondrial damage include fission and fusion, mitophagy, and ubiquitin proteosomal degradation of damaged mitochondria. Specific proteins important for fusion and fission include mitofusins encoded by the nuclear genes, MFN1, MFN2, and DRP1. Proteosomal degradation involves binding and activation of ubiquitin ligases including Parkin and PINK1. Important factors that control apoptosis include AIF (apoptosis-initiating factor) and apoptosis inhibitory factors. Saada (2011) classified mitochondrial functional defects into three categories: defects that impacted a single complex (e.g., I, II, III, IV, or V), defects that impacted multiple complexes, and secondary impairments of mitochondrial complex functions. Defects in specific mitochondrial genes or nuclear genes may lead to deficiencies in single complexes. Such deficiencies may also result from defects in specific assembly factors or chaperones.

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Organic acids Lactic Succinic 2-hydroxyglutaric 3-hydroxybutyric 3- hydroxyisovaleric 3-hydroxy-3-methylglutaric

Amino acids Alanine Glycine Glutamic Serine Tyrosine Alpha aminoadipic acid

Other Creatine Carnitine Acylcarnitine

Based on data from Smuts et al., Metabolomics, Sept. 2012

Figure 5–2. This figure outlines metabolic biosignature determination in mitochondrial disease as presented by Smuts et al. (2012).

Combined OXPHOS I, II, III, IV, and V functional defects may result from nuclear gene mutations that impact translation factors, ribosomal proteins, or tRNA-modifying factors. Combined defects may also result from defects in mitochondrial tRNA or ribosomal RNA–encoding genes or from structural abnormalities, deletions, or duplications in the mitochondrial genome. Combined functional defects may result from impaired communication between nuclear and mitochondrial genomes or from replication defects due to impaired nucleotide transfer. Blood cell DNA

Mitochondrial DNA Deletion analysis sequencing gel electrophoresis

Depletion/deletion PCR-specific genes e.g., TK2, DGUOK

Nuclear DNA sequencing specific genes, or exome capture, wholegenome sequencing

Muscle Biopsy

Enzyme assays electron transport complexes

Assays specific enzymes, nuclear or mitochondrial encoded

Histology Cytochemistry

RNA, or DNA for PCR analysis of specific genes or genomes mt/nc

Figure 5–3. This figure outlines studies on blood cell DNA and studies on muscle biopsy samples to diagnose the underlying defects in patients with suspected mitochondrial diseases.

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F1 ATPase 3 alpha subunits 3 beta subunits

OSCP Outer stalk F6 d subunit 2 b subunits a subunit

Inner stalk

F0 ATPase

1 gamma subunit 1 epsilon subunit 1 delta subunit A6L Mitochondrial membranes

C ring ATP5G1 ATP5G2 ATP5G3

Figure 5–4. This figure illustrates components of mitochondrial complex V ATP synthase (F1F0-ATPase). Drawn by the author and constructed based on information in texts by Nyhan et al. (2005), Voet and Voet (1995), and Scheffler (2008).

SECONDARY DEFECTS IN MITOCHONDRIAL FUNCTION DiMauro (2010) emphasized that deficiency in one or more mitochondrial complexes might occur due to toxic effects. One example is the deficiency of cytochrome c oxidase, which occurs secondary to mutations in a nuclear gene, ETHE1 (ethylmalonic encephalopathy), which encodes an enzyme involved in the disulfide relay system. (This is discussed further later in this chapter). These mutations lead to toxic accumulation of sulfides that secondarily impair a number of enzyme complexes, including cytochrome c oxidase (COX).

MITOCHONDRIAL PROTEIN SYNTHESIS Rotig (2011) reviewed translation and protein synthesis in mitochondria and reported that about 100 different proteins are involved in carrying out translation of the messenger RNAs derived from 13 protein-coding genes in mitochondrial DNA. The proteins required for mitochondrial mRNA translation are nuclear encoded. Rotig reported that they include ribosome assembly protein aminoacyl tRNA synthetases, tRNA modifier factors, including tRNA methylation factor, and in addition, translation initiation, elongation, and termination factors. Mutations also occur in specific transfer RNAs (tRNAs) that are encoded in the mitochondrial genome. MELAS syndrome is characterized by lactic

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acidosis and strokelike episodes. It results from a specific mutation A3243G in the mitochondrial tRNAleugene. Strokelike episodes include headache, vomiting, limb weakness, and visual impairment. Other mitochondrial tRNA mutation syndromes include MERFF, myoclonus with ragged red fibers and epilepsy. The A8344G mutation in the mt-tRNAile gene leads to cardiomyopathy and external ophthalmoplegia. Aminoacyl-tRNA synthases are required to ligate specific amino acids to their corresponding tRNAs. These enzymes are all nuclear encoded. Deficiency of these enzymes frequently leads to early death in infants who manifested brain abnormalities. Known aminoacyl-tRNA synthetase deficiencies occur in the following genes: RARS (arginyl-tRNA synthetase), DARS2 (aspartyl tRNA synthetase), SARS (seryl-tRNA synthetase), YARS (lysyl-tRNA synthetase), and HARS (histidinyl-tRNA synthetase). Specific translation elongation factor deficiencies required within mitochondria may result from nuclear gene mutations. Nuclear-encoded mitochondrial translation elongation factors GFM1, GFM2, and TUFM play roles in transporting aminoacyl tRNAs to ribosomes. There is also evidence that TUFM protein acts as a chaperone and enhances protein folding (Suzuki et al., 2007). A locus on chromosome 12 (mTERFD3) encodes a mitochondrially expressed translation termination factor. Disease in mitochondrial protein translation may lead to defective function of a specific mitochondrial respiratory complex. However, combined dysfunction of several respiratory complexes is more common.

MITOCHONDRIAL DISORDERS DUE TO IMPAIRED NUCLEOTIDE METABOLISM Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is due to recessive mutations in the TYMP gene that encodes thymidine phosphorylase. Hirano et al. (2012) reported that they had identified TYMP mutations in 102 MNGIE patients in 79 families. They reported that all patients with this syndrome had activity levels of TYMP enzyme that were less than 10% of normal. Individuals with TYMP levels at least 15% of normal had milder phenotypes and presented later in life. Carriers of the TYMP mutations had levels approximately 35% of normal. Chromatography studies (HPLC: high-performance liquid chromatography) revealed that patients had high levels of thymidine and deoxythymidine in plasma. In the absence of adequate levels of thymidine phosphorylase, there are imbalances in the deoxynucleotide phosphate pool. Hirano et al. postulated

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that these imbalances lead to mitochondrial DNA instability. They observed that multiple mitochondrial deletions and duplications are present in these patients. Hirano et al. reported that clinical improvement occurred in 5 of the 11 patients who underwent hematopoietic stem cell transplant.

ANALYSIS OF MITOCHONDRIAL FUNCTION Koopman et al. (2012) noted that suspensions of isolated mitochondria are frequently analyzed to assess function. They emphasized, however, that mitochondrial metabolism is closely linked to the cellular milieu, including the cytosol and other organelles. Isolated mitochondria may, therefore, differ in function from mitochondria located within cells. They emphasized the value of assessment of cellular mitochondrial function with fluorescent reporter molecules and live cell morphology. In their analysis of mitochondrial complex I function, for example, they compared 26 physiological variables in cells from 24 patients and 14 controls. Assessments of these variables revealed decreased expression of complex I, depolarization of the mitochondrial membrane potential, accumulation of NADH, increased reactive oxygen species, aberrant concentrations of cytosol and mitochondrial calcium, and ATP and abnormal mitochondrial structure in specific patients. Koopman et al. (2012) emphasized that identification of alterations in these parameters can be used to identify specific drug targets and, subsequently, to analyze the effects of lead compound and drugs. The latter are then tested in animal models. Koopman et al. (2012) described treatment intervention approaches in monogenic mitochondrial diseases. These include metabolic manipulation, diet, exercise, small-molecule therapy, and genetic therapy.

DISEASES DUE TO DEFECTS IN PROTEINS LOCATED IN MITOCHONDRIA, THOUGH NOT IN RESPIRATORY COMPLEXES Ethylmalonic acidemia is an autosomal-recessive disease that is frequently fatal. It occurs most frequently in infants with Mediterranean or Arabic ancestry. The disease is characterized by chronic diarrhea, seizures, and encephalopathy, and patients often manifest petichiae and acrocyanosis. Elevated levels of lactate and C4, C5 acylcarnitines occur in blood, and high levels of ethylmalonic acid are excreted in urine. Levels of cytochrome c oxidase (COX) are reduced in muscle and brain.

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In 2004 Tiranti et al. used homozygosity mapping in a consanguineous family with multiple cases of this disorder to map the disease gene to a region on chromosome 19q13.2. They subsequently sequenced genes in the region and identified the ethylmalonic encephalopathy gene ETHE1. Analysis of the function of this gene revealed that it acts as a sulfur dioxygenase. Studies in humans and studies on a mouse model of ETHE1 deficiency revealed that this deficiency leads to the presence of high levels of thiosulfate and of sulfides in tissues. Tiranti et al. (2009) established that these substances inhibit COX activity, and they also inhibit short-chain fatty acid oxidation. Inhibition by thiosulfate and sulfides therefore leads to low COX levels and to the elevated acylcarnitine levels in patients. Tiranti et al. noted further that elevated sulfide levels led to vascular damage and to acrocyanosis and petichiae in patients. In the absence of ETFE1, patients are not able to detoxify sulfides produced by intestinal anaerobes and produced in tissue. Viscomi et al. (2010) used the bactericide metranidazole to curb intestinal sulfide production. In addition, N-acetyl cysteine, a cell permeable precursor of glutathione, was used as a buffer of sulfides. Initial studies of these compounds were carried out on the mouse model of ETHE1 deficiency. Subsequently, Viscomi et al. applied this treatment to patients with ETHE1 deficiency. Brain imaging on the first patient enrolled in the treatment protocol studied prior to treatment showed atrophy, leukodystrophy, and patchy lesions in the basal ganglia. The patient also manifested petichiae and acrocyanosis. Treatment with oral metranidazole led to disappearance of diarrhea, petichiae, and acrocyanosis and to neurological improvement, and reduction in seizure frequency. The only side effect observed was occasional gastroesophageal reflux after N-acetylcysteine ingestion. This side effect was successfully managed with ranitidine. Viscomi et al. reported that five patients responded well to treatment.

HIGH-THROUGHPUT STRATEGIES TO IDENTIFY MITOCHONDRIAL PROTEINS The mitochondrial genome was completely sequenced in 1981. This genome encodes only 13 proteins. DNA sequencing has led to identification of a number of mitochondrial DNA changes that cause diseases with characteristic a maternal transmission pattern and phenotypic heterogeneity (Calvo and Mootha, 2010). Analysis of proteins through two-dimensional electrophoresis revealed that there are between 1,000 and 1,500 proteins present in mitochondria.

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High-throughput strategies to identify nuclear-encoded proteins destined for mitochondria involve analysis of specific mitochondrial targeting sequences on proteins. One algorithm, MitoProt, identifies proteins that have a 15–20 amino acid target sequence (Gaston et al., 2009). One example is a defined sequence that directs proteins to the mitochondrial matrix, and from that location specific proteins may subsequently migrate to other locations in the mitochondria. A specific carboxy-terminal sequence is important to locate proteins to the outer mitochondrial membrane. There is evidence that a specific internal sequence that gives rise to a protein loop determines location to the inner mitochondrial membrane. Translocases of the inner membrane (TIMs) proteins are small cysteine-rich proteins that occur in the intermembrane space. Calvo and Mootha (2010) noted that data on signal sequences alone could not be used to identify all mitochondrial proteins. They consider protein tandem mass spectrometry to be the most useful technique for this purpose, though uncertainties in subcellular assignment of proteins remain. Microscopy of proteins tagged with specific antibodies is used to confirm location. Integrative analysis that combines information derived using the techniques described above has been used to identify mitochondrial sequences in different organisms. Online catalogs of mitochondrial proteins include MitoCarta (Broad Institute), MitoPred, MitoMiner, and Mitophenome. Current databases identify more than 1,000 human proteins that function in mitochondria. Proteins are rated on the basis of the degree of certainty that they are located in mitochondria. There are proteins that have locations in more than one cellular compartment; 15% of mitochondrial proteins have dual locations. In their review of the mitochondrial proteome, Calvo and Mootha noted that the recent growth of information resources regarding the mitochondrial proteome has led to expansion of information on interactive pathways and metabolic processes that take place in mitochondria and to new insights concerning mitochondrial function. The roles of mitochondria in oxidative phosphorylation (OXPHOS), in the tricarboxylic acid (TCA) cycle, and in the urea cycle are well studied. New information has come to light in other processes that take place in mitochondria, including fatty acid oxidation, amino acid oxidation, heme biosynthesis, fatty acid synthesis, and pyrimidine biosynthesis. In addition, there is a growing body of information on mitochondrial calcium import and export and on factors that determine calcium homeostasis in mitochondria. Mootha et al. (2003) first published data that revealed tissue-specific differences in the mitochondrial proteome. Specific examples included electron transport complex IV in which the relative abundance of the specific subunit

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components differed in different tissues. The specific subunit composition of mitochondrial encoded ribosomes also varied in different tissues. The identification of nuclear genes that when mutated lead to mitochondrial disease has greatly accelerated since publication of the human genome sequence and since expanded information became available on the mitochondrial proteome. Two specific examples presented by Calvo and Mootha (2010) include discovery of mutations in the LRPPRC gene (leucine-rich pentatricopeptide repeat–containing) in patients with the French Canadian variants of Leigh’s disease and discovery of the ETHE1 (ethylmalonic encephalopathy protein 1) defects in patients with ethylmalonic encephalopathy. Information on the mitochondrial proteome integrated with family studies and exome sequencing data led to identification of mutations in the dihydro-orotate gene (DHODH) in Miller’s syndrome (Ng et al., 2010; Roach et al., 2010). This is an autosomal-recessive syndrome characterized by micrognathia, cleft lip and palate, limb abnormalities, coloboma of the eyelids, and supernumerary nipples. DHODH maps to chromosome 16q22.2. The protein encoded by DHODH catalyzes the fourth enzymatic step, the ubiquinone-mediated oxidation of dihydro-orotate to orotate, in de novo pyrimidine biosynthesis. The DHODH protein is located on the outer surface of the inner mitochondrial membrane. It is not clear how defects in this gene lead to the specific congenital malformations.

SEQUENCE ANALYSIS ON PATIENTS WITH EVIDENCE OF MITOCHONDRIAL DISEASE Homozygosity mapping in families with cutis laxa and premature aging led to identification of a region on chromosome 17q25.3 that segregated with the disease. Sequencing of genes in this region led to identification of mutations in PYCR1 (pyrroline-5-carboxylate reductase) (Guernsey et al., 2009; Reversade et al., 2009). This gene encodes an enzyme that catalyzes the NAD(P) H-dependent conversion of pyrroline-5-carboxylate to proline. This enzyme may also play a physiologic role in the generation of NADP(+) in some cell types. The protein forms a homopolymer and localizes to the mitochondrion. Calvo et al. (2012) carried out next-generation sequencing on mitochondrial and nuclear DNA on patients with clinical and biochemical evidence of mitochondrial disease. For analysis of nuclear genes, these investigators used their Mitoexome capture protocol for 1,031 genes. In analyzing the sequence data, they specifically searched for mutations that disrupted protein function.

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They prioritized further studies of cases in which specific patients harbored deleterious mutations in both copies of a specific gene. In analyzing mitochondrial DNA for pathogenic changes, Calvo and Mootha searched for deletions and structural rearrangements and also for mutations documented to be pathogenic in databases and in the literature. They considered variants present in more than 10% of mitochondrial sequences. Biochemical studies on these patients included analysis of mitochondrial electron transport complexes I, III, and IV ascertained in up to four different tissues, including skeletal muscle, liver, fibroblasts, and/or heart. Enzyme levels were assayed relative to citrate synthase and complex II. Levels of enzyme deficiency were characterized as markedly deficient, moderately deficient, equivocal, or normal. Calvo and Mootha reported that 23 of the 42 patients with evidence of severe disease suggestive of oxidative phosphorylation defects harbored at least one pathologic mutation in a gene. In 10 cases mutations occurred in nuclear genes known to cause oxidative phosphorylation defects. In 12 patients they identified mutations in nuclear genes not previously described as being involved in disease. They identified a 7.2-kb mitochondrial DNA deletion in one patient with developmental delay, failure to thrive, microcephaly, lactic acidosis, and terminal metabolic decompensation. Genes found to be mutated in the Calvo study that were also reported to be mutated in other patients with mitochondrial disease included the following: acyl- CoA dehydrogenase 9 (ACAD9); POLG, catalytic subunit of mitochondrial DNA polymerase; BCS1L, which is involved in assembly of respiratory complex III; GFM1 and TSFM, which encode mitochondrial translation elongation proteins; mitochondrial alanyl-tRNA synthetase (AARS2); and thymidine phosphorylase (TYMP). Calvo and Mootha reported that DNA sequence variants found in 13 of their patients and not previously described as mutated in mitochondrial disease required further studies to confirm pathogenicity. Such studies were carried out for two of the genes: acylglycerol kinase (AGK) and NADH dehydrogenase (ubiquinone) 1 beta subcomplex 3 (NDUFB3). The two patients with acylglycerol kinase deficiency had combined deficiency of complexes I, III, and IV and had evidence of mitochondrial depletion. They manifested myopathy and cataracts. Deficiency of the NDUFB3 subunit of complex I occurred in a patient with lethal mitochondrial disease. Calvo demonstrated through in vitro studies that introduction of the wild-type form of NDUFB3 complemented the deficiency and restored complex I activity. In 24 of the 47 patients, Calvo and Mootha found no sequence changes that met established criteria for pathogenicity. They emphasized that in these cases mutations may be present in regions not targeted in their exome capture

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studies. They also suggested the possibility that sporadic cases of oxidative phosphorylation defects could result from the combined action of weak alleles with incomplete penetrance. Heterogeneity in Phenotype and Heterogeneity of Genotype With a Specific Phenotype Expanded opportunities for genomic sequencing and analysis of the nuclear genes with mitochondrial function, in combination with analysis of mitochondrial DNA sequence, have revealed that diseases with features consistent with mitochondrial disease (e.g., Leigh syndrome) may be due to defects in a number of different genes. In addition to overlap in gene defects for a specific mitochondrial disease, there is significant overlap in the range of phenotypes in patients with the same mutation. Leigh syndrome most commonly occurs in patients with complex I deficiency; however, it has been reported in families with mutation in the mitochondrial encoded ATP6 gene, 9185T>C. In another family with this same mutation, there are patients with Leigh syndrome and others with NARP (neurogenic muscle weakness, ataxia, and retinitis pigmentosa). It will be important to have data on both mitochondrial and nuclear DNA sequences to explain some of this phenotypic heterogeneity. Complex I is composed of seven different genes encoded in mitochondrial DNA, and 38 different nuclear genes. Deficiency of complex I may therefore be due to a number of different genetic defects. In addition, complex I deficiency may be associated with phenotypic heterogeneity with varying neurological features and varying degrees of cardiomyopathy. Three different mitochondrial genes that encode subunits of complex I are mutated in cases of Leber’s hereditary optic neuropathy (LHON). Fraser et al. (2010) reported that in 69% of LHON cases mutation occurs at 11778; in 14% of LHON cases mutation occurs at nucleotide position 14484 in ND6, and in 13% of cases mutation occurs at position 4360 in ND1. MELAS, mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes syndrome, is caused by mutations in mitochondrial DNA at position 8344 in a transfer RNA in 80% of cases. There is evidence that a number of other different mutations may play roles in the other 20% of cases. Approaches to the Treatment of Mitochondrial Disease In many cases treatment is primarily directed to symptoms (e.g., to treatment of compromised cardiac function). Schon et al. (2010) reviewed treatment

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modalities used to enhance mitochondrial respiratory complex function through cofactor supplementation, and removal of noxious metabolites such as excess lactic acid or excess reactive oxygen species. Newer approaches to treatment include measures to enhance mitochondrial biogenesis, and transplantation to provide cells with normal mitochondrial function. Other methods to enhance normal mitochondrial function include heteroplasmic shifting to increase the proportion of wild-type mitochondria. Gene therapy methods are also under consideration, and there the goal is to eliminate the mutant forms of genes. In some disorders specific treatment strategies have first been applied to mouse models of a specific mitochondrial disease, and when shown to be effective in the model the same strategies were applied to patients with that disease. One example is the treatment of ethylmalonic encephalopathy with metranidazole and N-acetylcysteine as noted previously. SUPPLEMENTATION Vitamins and cofactor supplementation are frequently used to treat mitochondrial dysfunction. A number of investigators have noted that carefully designed clinical trials to adequately confirm efficacy of specific supplementation protocols have not been reported (Kerr, 2010). Antioxidants such as vitamin E tocopherol, trollox (a water-soluble form of vitamin E), vitamin C, and cofactors such as carnitine, riboflavin, thiamine, coenzyme Q, and related compounds are used in treatment of mitochondrial oxidative phosphorylation defects. Schiff et al. (2011) reviewed aspects of nutrition modification for treatment of mitochondrial diseases. They noted that plasma carnitine levels are frequently decreased in patients with primary OXPHOS dysfunction and that these patients may benefit from oral carnitine supplementation. A number of other therapies for mitochondrial disease have been proposed, including arginine, N-acetylcysteine, thiamine, and riboflavin.

COENZYME Q (COQ, COQ10) In a review of coenzyme Q (CoQ) biosynthesis and function, Bentinger et al. (2010) reported that there is now evidence that CoQ participated in a number of metabolic processes in addition to serving as a key molecule in the mitochondrial respiratory chain and as a lipid soluble antioxidant. CoQ inhibits opening of the transition pore in the mitochondrial membrane, thus counteracting the negative consequences of the opening of this pore

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that include loss of the electropotential proton gradient and impaired ATP production. Loss of the proton gradient can occur through activity of uncoupling proteins in the inner mitochondrial membrane. Coenzyme Q protects low-density lipoproteins from oxidation and release of lipid peroxides. As an antioxidant, CoQ protects lipids, proteins, and bases in DNA from oxidation. Adequate quantities of CoQ are particularly important to protect DNA from damage by reactive oxygen species. CoQ also enhances nitric oxide release from endothelial cells and promotes endothelial function. Bentinger et al. reported that CoQ is produced by all cells and is present in cell membranes. Intake through the diet is limited. CoQ levels in blood reflect release of this molecule from liver cell membranes and do not reflect whole-body levels of CoQ. CoQ synthesis initiates from coenzyme A and continues through HMGCoA and through the mevalonate pathway. Bentinger et al. (2010) noted that the complete details of CoQ synthesis were not yet defined.

DEFICIENCIES IN COQ SYNTHESIS DUE TO GENE MUTATIONS Mutations in any one of six genes are known to cause CoQ deficiencies in humans. The genes PDSS1 and PDSS2 encode polyprenyldiphosphate synthetase, which act in the transformation of farnesyl pyrophosphate early on in the CoQ synthesis pathway. Genetic defects in PDSS1 and PDSS2 have been described in newborn infants with encephalopathy, lactic acidosis, and nephrotic syndrome (Quinzii and Hirano, 2010). Mutations in other genes involved in coenzyme A synthesis have been found in infants with multisystemic syndromes. Quinzii and Hirano reported that early renal impairment is a hallmark of CoQ biosynthetic defects. Importantly, there is evidence that early supplementation with CoQ alleviates symptoms at least in some patients. Deficiency of the enzyme ADCK3, a mitochondrial kinase involved in CoQ synthesis, was described in patients with juvenile-onset cerebellar ataxia, exercise intolerance, dystonia, and cognitive impairment. CoQ supplementation led to improvement of symptoms (Lagier-Tourenne et al., 2008). Secondary CoQ deficiency may occur in patients with mitochondrial disease, and these patients may benefit from CoQ supplementation. However, there are questions of efficacy of different CoQ forms and efficacy of gastrointestinal absorption.

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SMALL MOLECULES USED IN THE TREATMENT OF MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION FUNCTION IMPAIRMENTS Metabolites and chemical chaperones used in treatments include nucleosides and nucleotides Small molecules used include phenyl butyrate, ursolic acid, AIRCAR, captopril, and bezafibrate. Phenylbutyrate functions as a histone deacetylase inhibitor, and there is evidence that it enhances mitochondrial biogenesis (Brose et al., 2012). Ursolic acid is a lipophilic triterpene present in the waxy coat of apples, fruits, and herbs. Kunkel et al. (2011) reported that this compound increases muscle strength and increases muscle activity of the serine threonine protein kinase AKT. Bezafibrate is an agonist of PPA, the peroxisome proliferator–activated receptor. Yatsuga and Suomalainen (2012) carried out studies on a mouse model of human mitochondrial disease due to Twinkle helicase deficiency and associated with multiple mitochondrial deletions. They determined that bezafibrate treatment significantly delayed accumulation of multiple mitochondrial DNA deletions and the occurrence of Cox-negative muscle fibers in these animals. Captopril is an acetylcholinesterase inhibitor used to treat high blood pressure. There is evidence that it increases mitochondrial oxygen consumption (Kojic et al., 2011). TARGETING OF SMALL MOLECULES TO MITOCHONDRIA There are concerns that some small molecules proposed for use in mitochondrial diseases are poorly targeted to these organelles. Targeting can be achieved by coupling the molecule to lipophilic cations. Variants of coenzyme Q and quinone are targeted in this way. Specific peptides are imported into mitochondria (e.g., the Szeto-Schiller antioxidant peptide). Mitoporters are vesicles that carry substances into mitochondria. Nakamura et al. (2012) reported that many types of materials can be packaged into this liposome-based nanocarrier and then delivered to mitochondria via membrane fusion mechanisms. However, clinical trials using these carriers have not yet been reported. IDEBENONE TREATMENT OF MITOCHONDRIAL DISEASE Koopman et al. (2012) reported that the drug idebenone, related to coenzyme Q10, is approved for treatment of Friedreich’s ataxia and is also being

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Mitochondrial protein import

ANT nucleotide transport Porin VDAC

Outer mitochondrial membrane Translocase of outer membrane (TOM) Inner mitochondrial membrane

Calcium uniporter

Translocase of inner membrane (TIM)

Pyruvate

Fatty acids

Products of more than 1,000 nuclear genes are imported into and function in mitochondria

Figure 5–5. This figure represents mitochondrial membranes and transport across these membranes. Drawn by the author and constructed based on information in texts by Nyhan et al. (2005), Voet and Voet (1995), and Scheffler (2008).

investigated for treatment of Leber’s hereditary optic neuropathy. Idebenone is a short-chain synthetic benzoquine. Leber’s hereditary optic neuropathy arises as a result of mutations that impact mitochondrial complex I (NAD ubiquinone oxidoreductase). Reports of two studies indicate that this treatment likely delays the onset of loss of visual acuity in this disease (Carelli et al., 2011; Klopstock et al., 2011). Idebenone is also in clinical trials for treatment of MELAS .

COENZYME Q10–RELATED COMPOUND EPI-743 In a review of clinical trials for treatment of mitochondrial diseases, Kerr (2010) reported that treatment with CoQ10 has a marginal but positive effect on manifestations of these diseases. A number of chemical modifications of the CoQ10 molecule have been carried out to improve its bioavailability. One such modification was reduction of the length of the side chain and development of idebenone. Additional modification of CoQ10, including side chain length reduction and generation of a parabenzoquinone analog, led to synthesis of the compound Epi-743. Enns et al. (2012) reported that this compound is 1,000 to 10,000 times more potent than idebenone in protecting cells from oxidative stress. The safety and efficacy profile proved favorable, and FDA approval was obtained

Mitochondrial Function, Defects, and Approaches to Treatment Tyrosine/phenyl alanine

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Acetyl CoA

Hydroxymethylglutaryl-CoA Polyprenyldiphosphate Mevalonate synthetase 4-OH benzoate DecaprenylPDSS1 pyrophosphate Farnesylpyrophosphate PDSS2 Geranylgeranylpyrophosphate COQ2 prenyl transferase

Methylation, decarboxylation, hyrdoxylation Decaprenyl4-OH- modifications benzoate

Coenzyme Q10

Figure 5–6. This figure illustrates steps in the biosynthesis of coenzyme Q10. Drawn by the author and constructed based on information in texts by Nyhan et al. (2005), Voet and Voet (1995), and Scheffler (2008).

to treat patients with severe respiratory chain disease who were considered to be within 90 days of the end of life. Enns et al. carried out clinical evaluation of the effects of treatment. In addition, they carried out single-photon emission tomography (SPECT) radionuclide imaging using technetium-99-hexamethylpropylenenamine-oxime (HMPAO). This compound can reveal alterations in brain redox state. They treated 14 patients with severe mitochondrial disease over a period of 3 months. Mitochondrial functional defects in some of these patients were due to mitochondrial genome defects (MELAS, Pearson syndrome, Leigh syndrome), or to defects in the nuclear genome (POLG, SURF1, FRDA). After a 13-week emergency treatment, 12 of the 14 patients survived, 11 patients showed clinical improvement, and 10 showed improvements on the Newcastle quality-of-life scores. Enns et al. reported that the SPECT brain scans correlated with the clinical response. In patients who responded well to treatment, whole-brain HMPAO uptake was increased. Treatment of the mitochondrial disease Leber’s hereditary optic neuropathy (LHON) with Epi-743 was carried out by Sadun et al. (2012) for 90 days in five patients with genetically confirmed disease and acute vision loss. In five of the patients, there was arrest of disease progression and reversal of vision loss. Total recovery of visual acuity was achieved in two patients. No adverse effects of treatment were reported.

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Martinelli et al. (2012) used Epi-743 to treat 10 patients with Leigh syndrome (subacute necrotizing encephalopathy due to different gene defects). All treated children manifested improvements as measured on the Movement Disorder Childhood Rating Scale. No drug-related adverse effects occurred. Epi-743 (alpha tocotrienolquinone) is a metabolite of tocotrienol; it mitigates oxidative stress. There is evidence that it targets the enzyme NAD/NADP quinone oxidoreductase 1 (NQO1) (Martinelli et al., 2012). This enzyme reduces quinones to hydroquinones using FAD as cofactor.

BIOMARKER TO ANALYZE EPI 743 TREATMENT EFFECTS Blankenberg et al. (2012) carried out studies on 22 patients with mitochondrial disease, including 7 with Leigh syndrome, 7 with POLG deficiency, 5 with MELAS 2 with Friedreich ataxia, 1 with Kearns-Sayre syndrome, 1 with Pearson syndrome, and 1 with mitochondrial DNA depletion syndrome. The determined that in all cases treatment resulted in significant correlation between cerebellar uptake of HMPAO, as measured on SPECT brain scans, and functional improvement on the Newcastle functional score. The MELAS subgroup showed significant correlation between functional improvement and whole-brain uptake of HMPAO. Kearns-Sayre syndrome, external ophthalmoplegia and multisystem disease, and Pearson syndrome, bone marrow hypoplasia and pancreatic insufficiency, are usually associated mitochondrial functional impairment due to deletions in mitochondrial DNA. POLG encodes polymerase gamma, which plays an important role in mitochondrial DNA maintenance. Recessive POLG mutations lead to neurological symptoms, ataxia, choreoathetotic movements, external ophthalmoplegia, and cachexia (Habek et al., 2012; Van Goethem et al., 2001). Heterozygous missense mutations in POLG lead to a dominant disorder characterized by myopathy and cachexia (Pitceathly et al., 2013). In Friedreich’s ataxia, deficiency of the nuclear encoded protein frataxin leads to impaired iron sulfur cluster assembly in mitochondria proteins and to oxidative stress.

MITOCHONDRIAL GENE DELIVERY Introduction of DNA into mitochondria is complicated by the double-membrane structure of those organelles. Yu et al. (2012a and b) designed a vector to

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introduce DNA into mitochondria. They added a mitochondrial targeting sequence, MTS, to the capsid of an adeno-associated virus. With this vector they were able to deliver the normal ND4 gene to rescue a genetic mutation in ND4 G11778A in cybrid cells. This substitution leads to an arginine-to-histidine substitution. ND4 encodes subunit 4 in ubiquitin oxidoreductase, a component of mitochondrial complex 1. ND4 is one of three mitochondrial genes that, when mutated, lead to Leber’s hereditary optic neuropathy (LHON) and blindness. LHON due to homoplasmic 11778 G-to-A substitution in mitochondrial DNA was first described by Wallace et al. in 1988. Yu et al. also tested the efficiency of the vector they developed in transferring the normal ND4 gene and in restoring normal ND4 function in a mouse model of LHON. They determined that large peptides can be inserted in the capsid protein of the adeno-associated AAV serotype 2 and are tolerated. They introduced a 23-amino-acid Cox8 (cytochrome oxidase) presequence to target the AAV to mitochondria, and they linked ND4 to the plasmid sequence. In the mouse model of LHON, Yu et al. demonstrated that the introduction of the normal ND4 gene prevented early visual loss induced by the mutant ND4. Iyer et al. (2012) introduced normal mitochondrial DNA into cells that contained mitochondrial mutations. They complexed the mitochondrial DNA with recombinant mitochondrial transcription factor A (TFAM) protein and incubated cells with this mixture. They monitored entry of the normal mitochondrial DNA into cybrid cells of fibroblasts. The cells were then placed in normal growth medium. Iyer et al. were able to confirm that introduction of the mitochondrial DNA TFAM protein complex improved mitochondrial respiratory complex function in LHON cells and restored ATP synthase activity in cells from a patient with Leigh syndrome and a 8993T>G mutation in the mitochondrial DNA–encoded ATP synthase component.

CELL AND TISSUE TRANSPLANTATION Mitochondrial complex III deficiency is in some cases associated with severe liver failure. Iwama et al. (2011) proposed that liver transplantation be considered in these cases. Hematopoietic stem cell transfer has been successfully used to treat the thymidine phosphorylase (TYMP) gene deficiency that leads to impaired nucleotide metabolism associated with multisystem neurogastrointestinal leukoencephalopathy, MNFIE syndrome (DiMauro, 2010).

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6 PROTEIN MISFOLDING, ENDOPLASMIC RETICULUM STRESS, AND PATHOGENESIS OF DISEASE

INTRODUCTION Many degenerative diseases are associated with the accumulation of protein aggregates and endoplasmic reticulum (ER) stress. The goal of this chapter is to review cellular processes involved in mitigation of the effects of protein aggregation. Insight into these processes may provide clues to therapeutic interventions.

PROTEIN FOLDING AND MISFOLDING Polypeptide chains fold into defined secondary and tertiary structures. Specific proteins alter their folding structure upon interaction with molecular targets. Protein misfolding occurs at various locations in the cell, including in the cytoplasm, in the endoplasmic reticulum and in mitochondria. A number of proteins have an increased tendency to aggregate. Unfolded proteins often adopt a beta-pleated structure that facilitates fibril formation and aggregation Aggregates may subsequently be incorporated in inclusions. Neef et al. (2011) reviewed protein misfolding and noted that there is an increased tendency for 103

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protein misfolding and aggregation in neurons and aging cells. Protein aggregates in the cell disrupt calcium homeostasis, promote oxidative stress, and bind chaperones, thereby limiting the availability of chaperones for other cellular functions. Response to protein misfolding in the cytosol, sometimes referred to as the heat shock response, leads to activation of the heat shock transcription factor HSF1. HSF1 occurs in cells as a monomer bound to chaperones including HSP90, HSP70, and HSP40. In this chaperone bound, monomeric form the HSF1 is inactive. Under conditions of proteotoxic stress, HSF1 dissociates from chaperones; it forms trimers that then migrate to the nucleus and bind to heat shock elements in stress response genes (Neef et al. 2011).

Unfolded-Protein Response in the Endoplasmic Reticulum Secreted proteins and membrane protein synthesized on polyribosomes enter the endoplasmic reticulum. In the lumen of the endoplasmic reticulum, they undergo folding and maturation. Correctly folded proteins are then delivered to the specific cellular compartments or secreted in to the Golgi. Proteins that do not undergo proper folding accumulate in the endoplasmic reticulum. Sustained endoplasmic reticulum stress leads to impaired cellular function and ultimately to cell death. Increased concentrations of unfolded proteins elicit the unfolded-protein response that is designed to reduce ER stress. Walter and Ron (2011) reviewed the core molecular elements of the unfolded-protein response. Initiation of this response is dependent upon activation of three specific stress sensors, ATF6, IRE1, and PERK1. ATF6 is a transcription factor, PERK is a protein kinase, and IRE1 is an inositol-requiring enzyme. Activation of each sensor leads to production of a specific transcription factor that passes to the nucleus to activate expression of specific subsets of genes. There are three branches of the unfolded-protein response; each is initiated through activation of one of these sensors. ATF6, PERK, and IRE1 are positioned as transmembrane proteins. ATF6 is a globular protein that protrudes into the ER lumen. The binding of unfolded protein to ATF6 causes it to be released from the membrane and to pass to the Golgi apparatus. There it encounters two protease: S1P and S2P. These proteases digest ATF6 to generate an N-terminal fragment, ATF6N. This fragment then enters the nucleus and activates expression of specific genes (e.g., the chaperone encoding BiP, the gene that encodes the glucose regulatory protein GRP44, and genes that encode protein disulfide isomerase).

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ENDOPLASMIC RETICULUM STRESS PATHWAYS

ER membranes

unfolded proteins

Pro-ATF6

Golgi

PERK selective translation activation ATF4

cleavage

P

P eif2a global translation inhibition

ATF6N

P

XBP mrNA altered splicing

XBP1 alpha

Nucleus UPR genes GRP

P IRE

XBP1 antioxidant genes CHOP, GADD4

chaperones lipid synthesis, ERAD

Figure 6–1. Schematic diagram of three branches of the unfolded-protein response. Based on and modified from Walter and Ron (2011).

The PERK sensor is a transmembrane kinase that is activated by the accumulation of unfolded proteins in the ER and initiates the second branch of the unfolded-protein response. Activation of PERK leads single molecules of PERK to form oligomers within the ER membrane. This oligomerization is followed by phosphorylation of PERK and of the translation initiation factor EIF2a. Phosphorylation of EIF2a protein leads to inactivation and inhibition of mRNA translation of many proteins. The effect of PERK activation is, therefore, to decrease the amount of protein produced. However, phosphorylation of EIF2a increases the translation of the transcription factor ATF4. Excess of this transcription factor drives expression of the CHOP gene (C/EBP homologous protein) and GADD34 (growth factor arrest and DNA damage inducible). Both of these gene products play a role in cellular apoptosis. Walter and Ron (2011) emphasized that increased PERK activity triggered by the presence of increased levels of unfolded proteins is protective at modest levels of activation. However, prolonged PERK stimulation and production of phosphorylated EIF2A lead to ATF production and activation of GADD34 that may expedite apoptosis. The IRE1 sensor is a transmembrane protein, and when activated by unfolded proteins it undergoes oligomerization and conformational changes that activate its kinase and endoribonuclease domains. Kinase activation promotes

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phosphorylation. The activated endoribonuclease domain of IRE1 specifically splices out an intron from the mRNA transcribed from the XBP1 gene. The spliced mRNA is translated to give rise to the transcription factor XBP1s. The latter encodes Xbox binding protein, which enhances expression of lipid biosynthetic enzymes and enzymes involved in building the endoplasmic reticulum. Walter and Ron noted that homeostasis of the unfolded-protein response is in part dependent on maintenance of IRE1 and PERK in an inactive state until accumulation of protein requires their activity. The chaperone protein BiP binds to IRE1 and PERK in the ER membrane and impairs their oligomerization. There is also evidence that ATF6, PERK, and IRE1 are maintained in an active state by binding of GRP78 (glucose-regulated heat shock protein) (Minamino et al., 2010). The unfolded-protein response acts as a signaling response to increase expression of genes that encode proteins involved in facilitating protein folding, reducing translation of mRNA, and enhancing development of the ER. In cases in which the UPR (unfolded-protein response) is overwhelming, endoplasmic reticulum stress and ultimately cell death ensue. Protein misfolding diseases include retinitis pigmentosa, in which misfolded rhodopsin accumulates. In specific forms of diabetes mellitus, in which accelerated insulin production occurs, insulin may fail to fold effectively, leading to cell death (Fonseca et al., 2011).

PROTEIN FOLDING IN THE ER AND ER-ASSOCIATED PROTEIN DEGRADATION Many of the proteins in the endoplasmic reticulum are glycosylated. Misfolded glycosylated proteins in the endoplasmic reticulum are recognized by unusual chaperones including calnexin that interact with these proteins to promote folding. A number of different protein disulfide isomerases exist in the endoplasmic reticulum and may facilitate protein folding by promoting interaction between the sulfhydryl groups in cysteine residues. The special environment in the ER that includes the presence of calcium, ATP, and the activity of chaperone GRP78 (glucose-regulatory protein 78) facilitates correct protein folding. Protein folding is also facilitated by the oxidizing environment in the ER created, in part, through activity of ERO1 (endoplasmic reticulum oxidoreductin 1). The oxidizing environment enhances the formation of disulfide bonds. Proteins that are improperly folded undergo a culling process to protect cells; Smith et al. (2011) reviewed aspects of the culling process. They reported

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that proteins that remain unfolded are removed from chaperone protein. They are modified by adaptor proteins and by proteins with E3 ubiquitin ligase function that include membrane-associated proteins such as MARCH4.

ACETYLATION AND THE UPR There is evidence that histone deacetylase activity interfaces with different biologic processes through modulation not only of histones in chromatin but also through deacetylation of nonhistone substrates. Kahali et al. (2012) reported that HDAC regulates the UPR through altered acetylation of GRP78. GRP78 acetylation facilitates its release from PERK, leading to activation of PERK downstream signaling, increased phosphorylation of EIF2a, and decreased protein synthesis. The chaperone protein HSP90 is also a target of histone deacetylase

ER STRESS AND UPR IN DISEASE PATHOGENESIS It is important to consider the consequences of excessive uncontrolled ER stress. Excessive ER stress followed by apoptosis may play roles in a number of different diseases, including ischemic heart disease and neurodegenerative diseases. ER stress and UPR signaling play critical roles in nonalcoholic fatty liver disease (NAFLD). This disease is a manifestation of the metabolic syndrome. In this condition there is excessive hepatic lipid accumulation, inflammation, and fibrosis. Excess of metabolic factors including lipids, saturated fatty acids, and glucose and excess of inflammatory cytokines disrupt protein folding in the ER (Zheng et al., 2010).

ER Stress and Apoptosis There are a number of reports of studies designed to investigate the relationship of the unfolded-protein response and neuronal cell death. Moreno et al. (2012) carried out studies on prion disease in mice where misfolded prion protein, PrP, accumulates. They demonstrated that prion protein accumulation leads to reduction in synapse number, and as disease progresses there are reductions in levels of synaptic proteins SNAP25 (synaptosomal-associated protein kD25), VAMP2 (vesicle-associated protein 2, synaptobrevin 2), and postsynaptic proteins PSD95 and NMDAR1 (NMDA glutamate receptor 1). Moreno et al.

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demonstrated that this decrease in concentration of proteins essential for synaptic function was due to decreased translation. Decreased translation correlated with increased levels of phosphorylated EIF2a likely related to activation of the unfolded-protein response by excess prion protein PrP. They determined that overexpression of GADD34, the phosphatase that specifically removes phosphate from EIF2A, improves neuronal survival. Moreno et al. emphasized that chronic blockade of protein synthesis induced by triggering of the unfolded-protein response may be an important cause of neuronal synaptic loss in other neurodegenerative diseases. In many forms of neurodegenerative diseases, unfolded proteins accumulate. However, there is evidence that concentrations of specific proteins may be reduced in the ER. One example occurs in a form of frontotemporal dementia also known as frontotemporal lobar degeneration, characterized by haploinsufficiency due to mutations in the gene than encodes progranulin. Granulin is a neurotrophic factor and granulin levels are reduced. In this form of frontotemporal lobar degeneration, accumulation of ubiquitin and TAR DNA binding protein occur in inclusions.

APPROACHES TO TREATMENT Capell et al. (2011) carried out studies to identify compounds that stimulate progranulin production. They initially studied fibroblasts, lymphoblasts, and neuroblastoma cells in culture. Subsequently, they studied organotypic slice cultures of mouse neocortex. They determined that the compound bafilomycin A1 increased levels of intracellular and secreted granulin, and granulin levels were increased in the ER and in the Golgi network. Capell et al. determined that the cellular target of bafilomycin A1 is vacuolar ATPase. This enzyme plays a role in determining vesicular pH. Their studies revealed the intravesicular pH changes induced by vacuolar ATPase inhibitors or vacuolar-alkalizing drugs such as chloroquine lead to translational upregulation of granulin. Furthermore, they determined that drug levels required to achieve increased granulin concentrations were therapeutically achievable.

MITOCHONDRIA AND THE UNFOLDED-PROTEIN RESPONSE Homeostasis in mitochondria requires correct protein folding. Pellegrino et al. (2012) reviewed protein folding within mitochondria. They noted that this

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process takes place within a potentially protein-damaging environment due to generation of reactive oxygen species through activity of the electron transport complexes. Promotion of protein folding in mitochondria is facilitated by activity of chaperone proteins located in the mitochondrial matrix and on the mitochondrial inner membrane. HSP60, HSP10, and HSP70, and a specific form of HSP90 (TRAP1), occur in the mitochondria. HSP70 occurs in association with the translocase of the inner mitochondrial membrane channel TIM23 and facilitates transport through this membrane channel. Iron sulfur transport in mitochondria is also facilitated by mtHSP70. Pellegrino et al. reported that specific proteases in the mitochondria act to degrade misfolded and oxidatively damaged proteins. These proteases include LON protease. Unfolded proteins in the mitochondria induce expression of genes HSP60, HSP10, mtDNAJ, and CLPP ATP–dependent protease.

THERAPEUTIC IMPLICATIONS Information on mitochondrial unfolded-protein response has therapeutic implications. There is evidence that efficient UPR protects cancer cells. HSP60 and the HSP90 isoform TRAP1 are highly expressed in different cancers. Pelligrino et al. reported that treatment with gamitrinib inhibits mitochondrial HSP and results in death of tumor cells.

CROSS TALK BETWEEN ENDOPLASMIC RETICULUM AND MITOCHONDRIA Vance (1991) first described contacts between mitochondria and ER and reported evidence that close apposition of ER and mitochondria is important for bioenergetics and cell survival. This interaction is mediated in part through membrane association. Pinton et al. (2011) reported that disruption of associated membranes interrupts intracellular calcium signaling and impacts apoptosis. The endoplasmic reticulum is the main intracellular storage organelle for calcium. Altered calcium homeostasis in the ER may lead to activation of apoptosis pathways (Raghubir et al., 2011). These authors noted that there is close physical contact between the endoplasmic reticulum and mitochondria. There is evidence that cytochrome C translocates from mitochondria to the endoplasmic reticulum and that this leads to calcium efflux from the endoplasmic reticulum.

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THERAPEUTIC STRATEGIES FOR ER STRESS A number of different therapeutic strategies for treatment of endoplasmic reticulum stress have been developed based on manipulation of chaperone protein expression. There is evidence that HSP90 inhibition causes its release from the HSF1 transcription factor. A compound used to achieve HSP90 inhibition is geldanamycin. However, this release of inhibition has limited and short-term effect in reducing cytotoxicity in protein misfolding diseases such as Huntington’s chorea, Alzheimer’s disease, and Parkinsonism. Neef et al. (2011) proposed screening small molecules to identify compounds that directly activate HSF1 gene expression.

THERAPEUTIC APPROACHES TO THE TREATMENT OF ONE FORM OF FAMILIAL AMYLOID POLYNEUROPATHY Familial amyloid polyneuropathies (FAPs) are disorders characterized by deposition of insoluble protein that form beta-pleated sheets and fibrils. Three different proteins are known to give rise to FAP; these are transthyretin, apolipoprotein A1, and gelsolin. FAP is most commonly due to transthyretin deposition; FAP is rarely due to gelsolin deposition or apolipoprotein A deposition. All three forms of FAP are autosomal dominant conditions, and all have onset later than the third decade of life. FAP symptoms due to transthyretin deposition may occur as late as the seventh decade of life. In all three forms, the penetrance varies, and individuals with the same mutations have different degrees of impairment. Planté-Bordeneuve and Said (2011) in their review of FAP noted that because of the late onset and variable penetrance of transthyretin-induced FAP, some cases designated as sporadic might, in fact, have inherited mutations. There is some evidence that transthyretin amyloidosis may occur in cases with the wild-type gene (Johnson et al., 2012). FAP due to transthyretin mutations is endemic in Portugal and occurs frequently in Japan and in Sweden. It also occurs in West Africa, in Brazil, in Australia, and in the USA. The most common mutation is a Val30Met mutation in exon 2. Plante-Bordeneuve and Said reported that this is the most common transthyretin mutation in FAP patients in Portugal, Brazil, and Sweden. However, in Japan more that 30 different transthyretin mutations have been identified in patients with FAP. In West African and in African American FAP patients, the most common mutation is Val122Ile. Transthyretin is a 127-amino acid protein synthesized in the liver. It forms homotetramers that are soluble and circulate in the blood. The chief function of

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circulating transthyretin is the transport of holoretinol binding protein (Johnson et al., 2012). Transthyretin is one of three circulating proteins that bind T4 thyroxin; however, it is the minor T4 thyroxin binding protein since this protein is primarily transported by albumin and by thyroid binding globulin Johnson et al. reported that both wild-type and mutant transthyretin and tetramers composed of both wild-type and mutant protein partially unfold and misassemble to fibrils and aggregates. They reported that wild-type transthyretin misassembles outside and inside cardiomyocytes and causes late-onset sporadic cardiomyopathy. Transthyretin point mutations lead to familial amyloid cardiomyopathy, familial amyloid polyneuropathy, and a rare central nervous system amyloid disorder. Johnson et al. reported that more than 100 different transthyretin mutations occur and give rise to aggregates that must be degraded through activities of the endoplasmic reticulum degradation system (ERAD). They emphasized that patients with familial amyloid polyneuropathy often have cardiac impairment. Mutations that give rise to the cerebral nervous system amyloidosis are the most destabilizing forms. In this condition the abnormal transthyretin is secreted into the brain from the choroid plexus. Johnson et al. noted that the relatively high concentration of T4 thyroxin binding protein in the choroid plexus tend to stabilize wild-type transthyretin and also mutant transthyretin forms except those that are highly unstable. Tissue damage is induced primarily by diffusible transthyretin aggregates. In peripheral nerves, deposits of these aggregates occur in the endoneurium and in endoneurial capillaries. Planté -Bordeneuve and Said (2011) reported that involvement of unmyelinated nerve fibers occurs early in the course of the disease and leads to impaired sensory response beginning in the feet and then spreading upward. Subsequent demyelination occurs, leading to motor deficits, particularly in the lower limbs. Neuropathic pain is a common manifestation of the polyneuropathy. Subsequently life- threatening involvement of the autonomic nervous system occurs, leading to functional impairments in the cardiocirculatory, gastrointestinal, and genitourinary systems.

DESIGNING TRANSTHYRETIN KINETIC STABILIZERS Johnson et al. (2012) reported that over 1,000 aromatic small molecules complementary to the thyroxin T4 binding site of transthyretin were designed and tested. The most effective agents for stabilization included diflunisal, an FDA approved nonsteroidal anti-inflammatory drug. In 2012 this drug entered phase III trials for treatment of familial amyloid polyneuropathy.

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Another small molecule, tafamidis, was shown to be highly selective for one of the two T4 thyroxin-binding sites in transthyretin and to stabilize the transthyretin protein. Clinical trials in patients with the V30M revealed that tafamidis slowed progression of the amyloid deposition. Additional compounds, including stilbenes, are also being studied as amyloid stabilizers. Johnson et al. emphasized that structure-based design principles have led to efficient therapy for familial amyloid polyneuropathy. The only other effective treatment for this disorder, liver transplantation, is highly invasive and costly.

ENDOPLASMIC RETICULUM ASSOCIATED PROTEIN DEGRADATION (ERAD) AND THE PROTEOSOME SYSTEM There is evidence that a specific protein, HERP, with an ubiquitin-like domain plays a role in maintaining ER homeostasis (Belal et al., 2012). HERP (homocysteine-inducible ER stress protein) facilitates degradation of ER calcium release channel proteins. Aberrant accumulation of these proteins promotes ER stress. Agents that increase HERP expression therefore have therapeutic potential.

UBIQUITIN PROTEASOME SYSTEM IN THE CYTOSOL Unfolded proteins in the cytosol are targeted for degradation in the ubiquitin proteasome system (UPS). Protein misfolding may occur as a result of mutations and structural changes in proteins present in the cytosol. It may also result from specific conditions in the cell. Kriegenburg et al. (2012) reviewed ubiquitin-dependent degradation and emphasized the important role of chaperones in recognizing unfolded proteins. They emphasized the importance of this process in prevention of protein aggregation and also noted that premature degradation of proteins is hazardous for cell function. Chaperones involved in recognizing unfolded proteins include heat shock proteins Hsp70, HSP90, HSP110, and HSP97. Interaction with chaperones may ensure that proteins are correctly folded. If this cannot be achieved, proteins are ubiquitin targeted. Ubiquitination of misfolded proteins is a three-step process. The first step involves activation of ubiquitin, a 76-amino-acid protein. Activation requires ATP and the ubiquitin-activating enzyme E1. The second step involves transfer of activated ubiquitin to an ubiquitin-conjugating enzyme E2. The third step is carried out

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by ubiquitin ligase E3, which forms a bond between the ubiquitin-associated E2 enzyme and the substrate to be ubiquinated. E3 mediates the transfer of ubiquitin to a lysine residue in the substrate. After this monoubiquitination reaction, a polyubiquitin chain is formed through binding of additional ubiquitin molecules to lysine residues in the bound ubiquitin molecules. The ubiquitin protein contains lysine residues at positions 6, 11, 27, 29, 33, 48, and 63. Ubiquitinated proteins are then transferred to the proteosome for degradation. The ubiquitin ligase family comprises diverse enzymes, and there are hundreds of members of this family in the human genome. More than 600 genes in the genome encode ubiquitin ligases. Unfolded proteins in the endoplasmic reticulum are sometimes selectively transferred back to the cytoplasm, and they may then be ubiquitinated on the cytosolic side of the endoplasmic reticulum membrane. Following ubiquitination they are then transferred to the proteasome for degradation (Goder, 2012).

O

O ATP

AMP+PPi

Ubiquitin−COO− + E1−SH

Ubiquitin−C−S−E1

O

O

Ubiquitin−C−S−E1 + E2−SH

Ubiquitin−C−S−E2

O

O E3

Ubiquitin−C−S−E2 + Protein---NH2

Ubiquitin−C−NH−Lys−Protein

E1 Ubiquitin-activating enzyme E2 Ubiquitin-conjugating enzyme E3 Ubiquitin ligase

Protein UbUbUb

Ub Ub Ub

ATP-ADP peptides

proteasome

Figure 6–2. Illustration of ubiquitin activation, linkage to protein, and protein degradation in the proteasome. Based on and modified from information in Glickman and Ciechanover (2002).

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UBIQUITIN PROTEASOME SYSTEM AND NEURODEGENERATION Specific forms of familial Parkinson’s disease are due to mutations in the PARK7 gene or to mutations or duplications in the alpha Synuclein gene. Dennissen et al. (2012) reported that the Parkin gene that is mutated in juvenile Parkinson’s disease has ubiquitin ligase function, and it generates polyubiquitinated chains at lysine residues (K) K27, K63. Ubiquitination of proteins at K63 occurs more commonly in neurodegenerative disease, and K63 target proteins do not generally undergo proteasomal degradation. Dennissen et al. concluded that the exact role of the unfolded-protein response in neurodegenerative disease is not yet defined. It may contribute to the disease process.

7 TRANSPORTERS AND SOLUTE CARRIERS Proteins that Transport Molecules Across Membranes

INTRODUCTION Transporters play crucial roles in cellular nutrition and in acid base homeostasis in cells. There are three classes: ATP-dependent transporters, solute carriers, and channels. Channels differ in that they form pores, and translocation of substances through channels is generally passive. ATP hydrolysis is used as energy source to drive transport via ATP-dependent transporters. Solute carriers are transmembrane proteins that undergo sequential changes during transport. In their review of transporters, Bergeron et al. (2008) noted that solute carriers are membrane-embedded proteins and located in all forms of cell membranes with the possible exception of the nuclear membrane. They have transmembrane domains and serve to move substrates across membranes. They move inorganic cations and anions and organic molecules including carboxylates, urea, amines, amino acids, sugars, nucleosides, acetyl coenzyme A, neurotransmitters, and small peptides. Solute carriers likely also play roles in the transport of exogenous substances including drugs. Solute carriers are functionally classified as passive transporters and active transporters. Passive 115

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transporters move molecules down a concentration gradient. Active transporters couple movement of one molecule with the movement of another molecule. They include symporters, which move molecules in the same direction; antiporters move the molecules in different directions. He et al. (2009) reported that there are 55 families of solute carrier (SLC) genes in humans and that within these families there are 362 genes. Solute carriers manifest 20–25% amino acid homology, and most of this occurs in a specific consensus domain. Examples of different diseases due to mutations in transporter genes are presented in this chapter, and approaches to treatment of these diseases are discussed.

SLC9A6 MUTATIONS Mutations in the Xq26.3-linked SLC9A6 gene were identified through linkage analysis positional cloning and sequencing in families in which affected individuals had clinical manifestations similar to those seen in Angelman syndrome. These include autistic behaviors, mental retardation, absent speech, epilepsy ataxia, apparent happy disposition, and episodes of incontrollable laughter. Gilfillan et al. (2008) first reported Angelman syndrome–like behaviors associated with SLC9 mutations. Garbern et al. (2010) reported results of postmortem studies in a family with SLC9A6 mutations who manifested mental retardation, absent speech, epilepsy, ataxia weakness, and dystonia. Results of studies on brain revealed widespread neuronal loss with tau protein deposition in neurons and glia. The SLC9A6 gene encodes sodium hydrogen exchanger 6, Na+/H+6, NHE6. Na+/H+ exchangers are localized to membranes primarily of the Golgi apparatus and endosomes, and they play key roles in determining luminal pH (Ohgaki et al., 2011). NHE6 is located primarily on sorting endosomes. It is interesting to note that the intraluminal pH in sorting endosomes is 6.5; in late endosomes it is 6.0; in lysosomes the pH is 5.5 while cytoplasm has a pH of 7.2. Stromme et al. (2011) reported results of their studies on Slc9a6 knockout mice. They determined that these mice exhibited evidence of endosomal lysosomal dysfunction. Within neuronal late endosomes and lysosomes, there was abnormal accumulation of ganglioside GM2 and unesterified cholesterol. Brain regions particularly involved included basolateral nuclei, amygdala, and hippocampus CA3 and CA4 regions. Extensive degeneration of cerebellar Purkinje cells was also noted. Tau deposition was present in some brain regions.

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Stromme et al. noted that accumulation of ganglioside GM2 and GM3 occurs in lysosomal storage diseases; however, this accumulation occurs not only in defects of glycosphingolipid metabolism. These findings indicate that GM2 and GM3 accumulation also represent markers of endosomal and lysosomal dysfunction.

SLC2 GENE FAMILY Solute carriers in the SLC2 family are involved in glucose transport and encode GLUT protein. A number of glucose transporters have tissue-specific expression (e.g., SLC2A1, encoded on 1p34.2, encodes a product involved in transport across the blood–brain barrier). Defects in this gene lead to microcephaly, seizures, and delayed development.

SLC5 GENE FAMILY One member of the SLC5 gene family encodes a sodium/iodide symporter involved in the transport of iodide across the basolateral membrane of thyroid follicular cells. Mutations in this gene result in goiter and thyroid hormone deficiency. There is evidence that thyroid-stimulating hormone controls expression of the gene encoding this sodium/iodide symporter.

SLC1, 3,6 AND 7 GENE FAMILIES Bergeron et al. (2008) reviewed SLC1, 3, 6, and 7 gene families, which encode proteins involved in amino acid transport, including the neurotransmitter amino acids. The SLC3A1 gene on 2p16.3 encodes a protein sometimes referred to as rBAT, which is expressed in the apical cells of the kidney and small intestine. This protein is involved in the transport of dibasic and neutral amino acids, including cystine. Mutations in SLC3A1 and SLC7A9, encoded on 19q13.1, give rise to cystinuria. In cystinuria levels of cystine in renal tubules and in urine are increased and frequently lead to the formation of kidney stones. The underlying physiological defect in cystinuria is impaired transfer of cystine across epithelial membranes in renal tubules and small intestines. There are significant population differences in the frequency of cystinuria. The average frequency in neonates in France is 1 in 7,000; in Libya the frequency is 1 in 2,500.

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Guanidinoacetate methyltransferase

CREATINE

+

S-adenosylhomocystine

Creatine kinase ATP CREATINE PHOSPHATE Creatine Cell membrane

SLC6A8 creatine transporter

Figure 7–1. Synthesis of creatine and creatine phosphate. Drawn by the author and constructed based on information in texts by Nyhan et al. (2005), Voet and Voet (1995), and Fernandes et al. (2000).

Inheritance of Cystinuria Some forms manifest only in homozygotes. In some forms urinary cystine levels are higher in heterozygotes than controls but not as high as in homozygotes. In yet another form of cystinuria, clinical manifestations occur in homozygotes and heterozygotes and cystinuria is classified as autosomal dominant. There is evidence that some cases are digenic, and defects occur in both solute carrier genes that predispose to cystinuria (i.e., SLC3A1 and SLC7A9). The SLC3A1 gene, which encodes the rBAT protein, maps to chromosome 2q21. Heterozygotes for mutations in this gene have normal urine cystine levels. Homozygotes have high urinary cystine levels. The SLC7A9 locus on chromosome 19q13.1 was found to be mutant in 80% of cases of autosomal-dominant cystinuria. Heterozygotes for SLC7A9 mutations have increased levels of urinary cystine. Different SLC7A9 mutations differ in the extent to which they impact cystine excretion. Formation of kidney stones may begin during childhood. Goodyer (2004) noted that the average cystinuric patient could expect to have undergone seven surgical procedures for nephrolithiasis by middle age. Furthermore, in some cases cystine precipitation is asymptomatic but may still lead to reduced glomerular filtration and eventually to loss of renal parenchymal tissue.

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TREATMENT Conservative therapy for cystinuria involves high fluid intake, especially at bedtime, and use of alkalizing agents (e.g., potassium citrate). Penicillamine has been used to treat cystinuria since the 1960s. Other therapeutic agents used include thiol reagents such as alpha mercaptoproponylglycine (MPG). These agents are not without side effects including hyperlipidemia, myopathy, and jaundice.

CREATINE AND SLC6A8 CREATINE TRANSPORTER Brain creatine deficiency disorders are in some cases due to creatine synthesis defects, and in other cases they are due to defective transfer of creatine into the brain. Primary creatine deficiency syndromes due to defects in creatine synthesis arise as a result of deficiency of the enzymes arginine glycine amidotransferase (AGAT) or guanidinoacetate methyl transferase (GNMT) or as a result of deficiency of the creatine transporter SL6A8. AGAT and GNMT deficiencies are inherited as autosomal-recessive conditions. SLC6A8 is X linked and was reported to account for 2% of cases of X-linked mental retardation in males (Beard and Braissant, 2010). Females with creatine transporter SLC6A8 defects may have mild cognitive impairments, and behavior and learning problems. In creatine transport defects, plasma levels of creatine are normal. The tissue primarily affected by creatine deficiency is brain. There is growing evidence that creatine plays an important role in brain function. It may act as a neurotransmitter. Creatine phosphate acts as an important energy reservoir. In the presence of ATP and through activity of creatine kinase, ATP is generated from creatine phosphate. Longo et al. (2011) reviewed brain creatine deficiency disorders. These disorders can be detected on brain MRI studies. Manifestations in patients include mental retardation, autism, behavioral problems, and seizures. Creatine transfer defects are due to impaired function of the SLC6A8 creatine transporter encoded by a gene on Xq28. Creatine becomes phosphorylated primarily in mitochondria and promotes the transfer of high-energy phosphate groups to the cytoplasmic sites of energy consumption. The phosphocreatine molecule is much smaller than ATP and ADP and is more easily diffusible. The transfer of phosphate residues from ATP to creatine requires the activity of mitochondrial creatine kinase.

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TREATMENT Fons et al. (2008) treated creatine transporter defects with arginine supplementation with limited improvement. Creatine synthesis defects can be treated with creatine supplements and dietary restriction of protein. Creatine transporter deficiency in the Slc6a8 knockout mouse has been successfully treated with the creatine derivative cyclocreatine, a planar molecule that has the capacity to cross membranes (Kurosawa et al., 2012).

SLC25 GENE FAMILY The SLC25 gene family encodes mitochondrial carriers of amino acids. SLC25A22 encodes a mitochondrial glutamate transporter. Deficiency of the product of this gene leads to seizures (Molinari et al., 2005). SLC25A15 encodes a protein that transports ornithine, lysine, arginine, and citrulline across the mitochondrial membrane. This is an exchange transfer and is important in urea metabolism. Mutations in the SLC25A15 gene, which maps to chromosome 13q14, lead to HHH syndrome, characterized by hyperammonemia, hyperornithinemia, and homocitrullinemia. The clinical features of HHH vary. Symptoms may include confusion and lethargy, perhaps related to consumption of high-protein foods. Other patients manifest with mental retardation and spastic paraplegia. There is a founder mutation for this disorder in Quebec Canada. Palmieri (2008) noted that early diagnosis and effective management might prevent mental retardation.

TREATMENT This includes avoidance of high-protein diet and citrulline supplementation. During crises associated with hyperammonemia, patients are treated with sodium benzoate and phenylbutyrate.

MITOCHONDRIA AND TRANSPORT CARRIERS Specific transport proteins play roles in the passage of solutes in and out of mitochondria. Proteins encoded in the nuclear genome are imported into mitochondria. Specific carriers are involved in transfer across the inner mitochondrial membrane. Carrier molecules are involved in the oxidation reduction

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pathway, in homeostasis of the intramitochondrial adenine nucleotide pool, and in transport of cofactors (e.g., thiamine pyrophosphate and carnitine). In a review of mitochondrial transport, Palmieri (2008) noted that the driving forces utilized for transport across membranes include concentration gradients and the H+ electropotential gradient generated across the inner mitochondrial membrane through electron transfer in the respiratory chain. Palmieri reported that 50 different mitochondrial carriers were encoded in the human genome and that nine diseases are attributable to mitochondrial carriers. SLC25 mitochondrial carrier proteins share homology in three tandemly repeated domains, each 100 amino acids in length. For each SLC25-encoded transporter, six helices cross the mitochondrial membrane.

SLC25A4 ANT1 The SLC25A4 gene on chromosome 4q35 encodes a protein that functions as an ADP/ATP carrier. This protein is designated ANT1 or AAC1. Specific uptake of inorganic phosphate and of ADP into mitochondria is necessary for generation of ATP. Mutations in SLC25A4 and ANT1 protein were first described in a patient with exercise intolerance, lactic acidosis, and hypertrophic cardiomyopathy. Defective ADP/ATP transfer also leads to the presence of multiple mitochondrial DNA deletions. Defects in ANT1 function lead to a number of different disorders. EchanizLaguna et al. (2012) reported that autosomal-dominant mutations in ANT1 lead to progressive ophthalmoplegia, while recessive null mutations lead to cardiomyopathy, myopathy, and lactic acidosis. These authors described a patient with ANT1 deficiency who had those manifestations and congenital cataracts. Histological studies on muscle revealed typical ragged red fibers and absence of cytochrome C oxidase. Activities of mitochondrial respiratory complexes I, II, and III were normal, but complex IV activity was 52% of normal. Multiple deletions were present in mitochondrial DNA extracted from muscle. EchanizLaguna noted that survival of these patients is likely determined by whether or not mitochondria numbers can be increased and whether expressed of the homologous ANT3 can be induced. Progressive external ophthalmoplegia may occur as a manifestation of a number of different mitochondrial diseases including defects in ANT1. Other members of the SLC25A family that impact mitochondrial function include SLC25A3, SLC25A33, and SLC25A20. The SLC25A3 gene encoded on 12q23 catalyzes uptake of inorganic phosphate into mitochondria. Defective function of this transporter leads to muscle hypotonia, hypertrophic

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cardiomyopathy, and elevated plasma lactate (Mayr et al., 2007). The SLC25A33 gene encodes a mitochondrial pyrimidine carrier. SLC25A20, which maps to chromosome 3p21.31, encodes a carrier that transfers acylcarnitine across the inner mitochondrial membrane. Within the mitochondria the acyl group is transferred to coenzyme A through activity of the enzyme carnitine palmitoyl transferase II. Abnormalities of carnitine metabolism and their treatment are discussed in Chapter 5 (this volume) on mitochondrial disorders.

SLC25A19 In 2002 Kelley et al. described five infants with severe microcephaly born to Amish parents in Lancaster County, Pennsylvania, who had alpha-ketoglutaricaciduria and recurrent episodes of metabolic acidosis. The gene for Amish microcephaly was mapped to 17q25.3 through linkage and haplotype analysis followed by sequence analysis of candidate genes. Rosenberg et al. (2002) determined that affected infants were homozygous for a mutation in SLC25A19, with glycine to alanine substitution at aminoacid 177. SLC25A19 encodes a protein that acts as a transporter of thiamine pyrophosphate, thiamine monophosphate, and deoxynucleotides. Thiamine pyrophosphate is a cofactor for pyruvate dehydrogenase, alpha ketoglutaric acid dehydrogenase, and transketolase (Lindhurst et al., 2006). In 2010 Siu et al. described an infant with severe microcepahly born to parents in an Old Amish community in Ontario, Canada. At birth the infant’s length and weight were in the 25th percentile, and head circumference was in the 3rd percentile. No other dysmorphic features were noted. Hypertonia of the extremities and irritability were present. The infant had episodes of metabolic crisis during which there were marked elevations of blood lactate and ammonia, modest elevation of liver enzymes, and elevated levels of urine alpha ketoglutarate. When the infant was biochemically stable, there were slight elevations in lactate and ammonia and alpha ketoglutarate levels were normal. DNA analysis revealed that this infant had the same SLC25A19 mutation as the Pennsylvania Amish infants with microcephaly.

TREATMENT Siu et al. treated the SLC25A19-deficient patient with a high-fat diet, 70% fat, 26% carbohydrate, and 4% protein and with supplementation with empiric

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NH2 CH2

C−CH3

N

THIAMINE MONOPHOSPHATE

O

−−CH2−CH2−O−P−OH CH3

OH

S

THIAMINE PYROPHOSPHOKINASE

NH2 CH2

CH3

C−CH3

N

O O THIAMINE PYROPHOSPHATE −−CH2−CH2−O−P−O−O−P−O−

S

OH

O−

SLC25A19

Mitochondrial membranes

transporter

Thiamine monophosphate Thiamine pyrophosphate

Figure 7–2. Structures of thiamine monophosphate and thiamine pyrophosphate and transport into mitochondria through activity of transporter encoded by SLC25A19. Drawn by the author and constructed based on information in texts by Nyhan et al. (2005), Voet and Voet (1995), and Fernandes et al. (2000)

mitochondrial cocktail, thiamine, coenzyme Q, carnitine, vitamin K, riboflavin, vitamin C, and with biotin dichloroacetate. At the time of the report, the infant was 6 years of age with severe developmental impairment.

SLC25A12, SLC25A13 ASPARTATE GLUTAMATE CARRIERS, AGC1 AND AGC2 There is close homology between AGC1 and AGC2. Both proteins catalyze the transfer of glutamate into mitochondria and the exit of aspartate. The SLC25A12 gene maps to chromosome 2q24 and encodes AGC1 (sometimes referred to as Aralar 1). Defective function of this protein was reported in cases of global cerebral hypomyelination. Napolioni et al. (2011) reported that AGC1 plays a key role in synthesis of brain aspartate, which then leads to N-acetylaspartate, a key factor in myelin production. Myelin is synthesized by oligodendrocytes. In AGC1 deficiency, levels of brain aspartate and N-acetylaspartate are low, and there is a decrease in myelin lipids and protein. N-acetylaspartate provides acetyl groups for lipid synthesis and for protein acetylation. AGC1 activity is

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regulated by mitochondrial calcium. AGC1 (Aralar) is an important component of the malate aspartate shuttle. Napoliani et al. proposed that defects in AGC1 are involved in autism pathogenesis.

SLC25A13 AGC2 AND CITRULLINEMIA The SLC25A13 gene on 7q21.3 encodes AGC2 (sometimes referred to as Aralar 2); mutations in AGC2 occur with relatively high frequency in Japan and Southeast Asia and give rise to citrullinemia type II. Aspartate in mitochondria is produced by the transamination of oxaloacetate to aspartate in the presence of glutamate. The transporter encoded by SLC25A13 promotes exit of aspartate from mitochondria. In the cytosol, aspartate conjugates with citrulline in a reaction catalyzed by arginosuccinate synthase, and this is the first step in the urea cycle. Citrullinemia type I is caused by mutations in the arginosuccinate synthase gene on 9q34. Aspartate combines with citrulline in the cytosol to form arginosuccinate in a reaction that requires the enzyme arginosuccinate synthase. AGC2 deficiency leads to defective transfer of aspartate out of mitochondria; citrulline accumulates in the cytosol since cytosolic aspartate levels are low. There is also evidence that AGC2 plays a role in the reaction whereby malate aspartate shuttles in mitochondria and this then impacts the NAD/ NADH. Type II citrullinemia in adults manifests with episodes of hyperammonemia and with neuropsychiatric manifestations. Severe forms of AGC2 arginosuccinate aspartate + citrulline Cytosol

glutamate

aspartate + Na-cetylaspartate SLC25A12 SLC25A13

oxaloacetate + glutamate

Mitochondrial membranes

aspartate GOT transaminase

Figure 7–3. This figure illustrates the mitochondrial glutamate aspartate shuttle and activity of transporters encoded by SLC25A12 and SLC25A13. Drawn by the author and constructed based on information in Napolioni et al. (2011).

Transporters and Solute Carriers NH2

COOH

C=O CITRULLINE

NH

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H2−N−C−H +ATP+

(CH2)3

CH2

ASPARTIC ACID

COOH

HCNH2

Arginosuccinate synthase

COOH NH2 C

COOH H−N−C

ARGINOSUCCINATE NH

CH2

(CH2)3

COOH

HCNH2 COOH

Figure 7–4. Synthesis of arginosuccinate from citrulline and aspartate. Drawn by the author and constructed based on information in texts by Nyhan et al. (2005), Voet and Voet (1995), and Fernandes et al. (2000).

deficiency lead to liver disease, neonatal hepatits, and intrahepatic cholestasis in infancy.

TREATMENT OF CITRULLINEMIA TYPE II Acute episodes of hyperammonemia may be treated with sodium benzoate and hemodialysis. Saheki et al. (2008) reported that patients with citrullinemia type II often self-regulate their diet. They tend to avoid high-carbohydrate substances and have a great liking for beans and peanuts, which contain high amounts of aspartate and asparagine. Yazaki et al. (2012) reported successful treatment of type II citrullinemia with liver transplant.

ENDOSOME-LYSOSOME MEMBRANE TRANSPORTERS CYSTINOSIS Cystinosis is characterized by accumulation of cystine crystals in the liver, spleen, and other tissue including the thyroid and the cornea of the eye. Cystine levels are elevated in leucocytes, and this elevation is used as a diagnostic test (Goodyer, 2011).

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Cystinosis leads to growth retardation in children. In addition, affected children manifest sign of rickets (limb bowing) due to increased urinary excretion of phosphate. The renal manifestations of cystinosis were classified as Fanconi syndrome. There is failure of reabsorption in the proximal renal tubule, and increased quantities of phosphates, urinary amino acids, glucose, and proteins, and organic acids are present in urine. In 1982 Gahl et al. reported defective efflux of cystine from lysosomes in this disorder. They also reported that the compound cysteamine promoted exodus of cystine from lysosomes. Town et al. (1998) mapped the cystinosis gene CTNS to chromosome 17p13. CTNS encodes the protein cystinosin, a specific transporter in lysosomal membranes. The H+ electrochemical gradient across lysosomal membranes drives activity of cystinosin. Anikster et al. (1999) reported that approximately half of the CTNS defects in Western populations involved a 65-kb deletion that extended from the 10th exon of CTNS. This deletion involved two adjacent genes, SHPK (sedoheptulokinase) and TRPV1, which encodes the capsaicin receptor gene. Goodyer (2011) reported that more than 90 different mutations have been described in cystinosis. Patients are sometimes hemizygous for the deletion, and they carry mutations in the CTNS gene on the homologous chromosome.

TREATMENT Treatment of cystinosis includes use of cysteamine and dietary supplementation to compensate for renal losses, especially of phosphate. Patients with cystinosis frequently require treatment for hypothyroidism. Renal transplant is often beneficial in these patients. Even after transplant, treatment with cysteamine is required (Goodyer, 2011).

SLC22A5 ORGANIC CATION AND CARNITINE TRANSPORTER OCTN2 Carnitine plays a key role in the transport of fatty acids from cytoplasm to mitochondrial matrix. Carnitine homeostasis is maintained through dietary absorption of carnitine contained primarily in animal protein and through endogenous synthesis. Carnitine is also reabsorbed through renal tubular function. Vaz and Wanders (2002) reviewed carnitine biosynthesis and disorders

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H

H H 2N

C

COOH

H

C

S

H2N

C

S

COOH

C H H

H cystine

cystine reductase cysteamine

H H2N

C

COOH

H

C

S−H

H cysteine

Figure 7–5. Structures of cystine and cysteine. Drawn by the author and constructed based on information in texts by Nyhan et al. (2005), Voet and Voet (1995), and Fernandes et al. (2000).

of carnitine metabolism. Carnitine is synthesized in a four-step reaction from N6-trimethyllysine. The first step in the reaction involves hydroxylation mediated by trimethyllysine dehydrogenase (TMLD), also know as trimethyllysine hydroxylase. An exonic deletion in the Xq28 gene (TMLHE), which encodes the epsilon subunit of this enzyme, was identified in a male subject with autism (Celestino Soper et al., 2012). Intermediate steps in carnitine synthesis yield butyrylbetaine, and in the final synthesis step, butyrylbetaine is hydroxylated to yield L carnitine. Carnitine biosynthesis occurs in liver, heart, brain, and muscle primarily. Butyrylbetaine and carnitine are efficiently reabsorbed in the kidney. Carnitine transporter deficiency is due to mutation in the SLC22A5 gene on 5q33.1. In this autosomal-recessive disorder, there is wastage of carnitine in the intestine and plasma levels of carnitine are low. OCTN2 protein is a plasma membrane protein involved in the cellular uptake of carnitine. Deficiency of this protein leads to systemic carnitine deficiency with low levels of free and acetylated carnitine and may be associated with hypoketotic hypoglycemia, acute metabolic decompensation, and sometimes sudden death. Later manifestations include skeletal myopathy and cardiomyopathy. Individuals heterozygous for SLC22A5 mutations usually have reduced plasma carnitine levels due to increased urinary loss. Longo et al. (2006) emphasized that primary carnitine deficiency must be distinguished from carnitine deficiency due to defects in fatty acid oxidation or carnitine synthesis

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defects. Treatment with adequate dietary supplements of carnitine results in a good prognosis.

OTHER SOLUTE CARRIERS THAT PLAY ROLES IN CARNITINE TRANSPORT The gene SLC16A9 is highly expressed in kidney. Illig et al. (2010) and Suhre et al. (2011) reported that a variant allele at this locus, rs7094971, is correlated with plasma carnitine concentrations. They also demonstrated that SLC16A9 plays a role in transport of carnitine across membranes. Kolz et al. (2009) reported that an allelic variant, rs12356193, in SLC16A9 was associated with plasma concentrations of DL-carnitine (p = 4.0 × 10–26) and with propionyl-L-carnitine concentrations (p = 5 × 10–8). A transport carrier of carnitine across the blood–brain barrier is encoded by SLC6A14, also known as transporter B0. The protein encoded by SLC6A14 localizes on the apical surface of brain epithelial cells and plays an important role in control of amino acid and carnitine delivery from blood and brain fluid to the brain parenchyma (Czeredys et al., 2008).

URIC ACID LEVELS: TRANSPORTERS AND GOUT In 1909 Garrod wrote, “It is still uncertain how far the accumulation of uric acid in the blood and the deposition of biurate in the tissues, which are the characteristic features of gout, are actually due to derangement of metabolism, as distinct from a mere excretory defect” (p. 11, para. 3). Uric acid from the glomerular filtrate undergoes partial reabsorption in the distal renal tubules. Blood uric acid levels normally range between 2 and 7 milligrams per 100 milliliters. Individuals with significantly raised blood levels of uric acid are likely to have attacks of gout that may be precipitated by dietary factors including alcohol intake. One of the transporters involved in urate reabsorption in the renal tubules is URAT1 (SLCA12). Blocking this activity of this transporter by means of the drug benzbromarone is used as a therapeutic measure to increase excretion of uric acid (Aringer et al., 2008). The transporter SLC2A9 (Glut9) also plays a role in tubular reabsorption of uric acid and may be blocked by benzbromarone. Genome-wide association studies revealed that specific SLC2A9 gene polymorphisms influence the level of expression of the gene and play an important role in gout.

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Loss-of-function mutations in specific transporters involved in uric acid reabsorption lead to lower levels of uric acid in blood and higher levels of uric acid in urine. Homozygous loss-of-function mutations in SLC2A9 predispose to kidney stone formation and to exercise-induced renal failure (Riches et al., 2009). The SLC2A9 polymorphic variant that has the strongest association with gout is rs734553. Dehghan et al. (2008) reported that the allele that represents the minor allele in the general population occurs with higher frequencies in patients with gout. SLC2A9 gives rise to two protein isoforms that differ in their tissue-specific expression. SLC2A9 protein is expressed in chondrocytes and is likely involved in transport of urate into joints in gout (Riches et al., 2009). Additional loci that impact blood uric acid levels include SLCA7 and ABCG2. The latter locus encodes an ATP binding cassette transporter. Variants in members of family of solute carrier genes located on chromosome 6p21 were found to be associated with gout. These include SLC17A3, SLC17A1, and SLC17A4. Hyperuricemia occurs in the metabolic syndrome, and the exact relationship of these two disorders has not yet been clearly defined. Uric acid is generated from the purine xanthine through the activity of xanthine oxidase. One mode of treatment is inhibition of xanthine oxidase activity with allopurinol. Hypericemia is, however, usually not treated with allopurinol unless attacks of gout occur. Dietary factors that play roles in hyperuricemia include intake of purines, fructose, and alcohol. It is interesting to note that the product of the SLC2A9 gene product GLUT9 is also characterized as a glucose/fructose transporter. Hyperuricemia may also occur in specific inborn errors of metabolism, including abnormalities of purine metabolism such as hypoxanthine guanine phosphoribosyl transferase (HGPRT) deficiency and glycogen storage diseases.

THE ASTROCYTE NEURON LACTATE SHUTTLE AND MONOCARBOXYLATE TRANSPORTERS Metabolism in neurons is primarily oxidative while in astrocytes it is primarily glycolytic. Astrocytes derive lactate from metabolism of glycogen stores and glucose. Pellerin and Magistretti (2012) reported that the astrocyte neuron lactate shuttle provides a framework to understand neuronal energetics and activity. There is evidence that release of lactate from astrocytes supports neuronal functions. Monocarboxylate transporters transfer lactate between cells. MCT2 is expressed in neurons, MCT4 is in astrocytes, and MCT1 transfers lactate

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into and out of oligodendrocytes. Lactate in oligodendrocytes is utilized to produce myelin. Following completion of myelination, oligodendrocytes export lactate, which then feeds axons (Y. Lee et al., 2012). The monocarboxylate transporter MCT1 (SLC6A1) is enriched in oligodendroglia, and that knockdown of the MCT1 gene in mice and in cultured cells leads to axonal damage and neuron death. They also demonstrated that MCT1 is reduced in patients with amyotrophic lateral sclerosis. Rinholm et al. (2012) proposed that lactate has therapeutic potential. They suggested that one of the ways that exercise can benefit the brain is that it increases lactate production. There is also evidence that exogenously supplied lactate can be processed by oligodendrocytes. ATP BINDING CASSETTE TRANSPORTERS ATP binding cassette transporters, sometimes referred to as ABC transporters, are members of a large superfamily of proteins. The key function of these transmembrane proteins is to use energy to move substrates across membranes (Kaminski et al., 2006). ABC transporters occur in the plasma membrane of cells, in the membranes of intracellular organelles, and endoplasmic reticulum. These transporters have a common molecular structure with a transmembrane domain and an ATP cassette domain. Molecules transported by the ABC transporters include lipids, proteins, amino acids, carbohydrates, vitamins, glucuronides, and xenobiotics. In 2006 Kaminski et al. reported that mutations in 14 different ABC transporters give rise to specific diseases that include respiratory distress syndrome (RDS), skin abnormalities such as icthyosis, bile transport defects (cholestasis), and eye diseases such as retinitis pigmentosa and cone rod dystrophy. In addition, specific ABC transporter mutations lead to abnormalities of lipid and lipoprotein metabolism, including deficiency of high-density lipoprotein and sitosterolemia that is associated with increased absorption of plant sterols. Members of the ABCB and ABCC subclasses play particularly important roles in the transport of drugs across cell membranes. The ABCD1-encoded transporter is deficient in X-linked adrenoleukodystrophy. ABCA3 TRANSPORTER, SURFACTANT, AND RESPIRATORY DISTRESS SYNDROME ABCA3 plays an important role in the transport of surfactant phospholipids into lamellar bodies. Deficiency of surfactant in newborn infants is most commonly

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due to immaturity, and it leads to respiratory distress syndrome in premature infants. Surfactant deficiency may also arise due to mutations in the surfactant genes or in the ABCA3 transporter (Gower and Nogee, 2011). Alveolar type II epithelial cells synthesize the surfactant lipoprotein complex. This complex is composed of surfactant proteins encoded by the SPA, SPB, SPC, and SPD genes, and it contains dipalmitoylphosphatidylcholine, phospholipids, and cholesterol. It is packaged into lamellar bodies that are derived from lysosomes and is secreted from cells through exocytosis. The surfactant layer forms a surface tension–reducing layer on alveolar cells at the air–liquid interface. Gower et al. reported that mutations within surfactant genes or in ABCA3 transporters lead to respiratory distress syndrome in infants and may lead to interstitial lung disease in children or young adults. Specific mutations in ABCA3 lead to aberrant folding of the enzyme protein and its retention in the endoplasmic reticulum compartment and to endoplasmic reticulum stress.

X-Linked Adrenoleukodystrophy: ATP Binding Cassette Transporter ABCD1 X-linked adrenoleukodystrophy is characterized by neurodegeneration in cerebral white matter and peripheral nerves. Distal axonopathy occurs, leading to demyelination, and induces an inflammatory response. Degenerative changes also occur in the adrenal cortex and in testes. Phenotypic variation, particularly with respect to age of onset, is a feature of this disease. In addition, some patients may manifest primarily adrenal symptoms. Phenotypic variation occurs even in different affected members of a single family. Moser et al. (1981) reported that in X-linked adrenoleukosdystrophy (XLAD) there are increased plasma levels of saturated very long–chain fatty acids VLCFA C24 and C26. Gootjes et al. (2003) confirmed that increased levels of VLCFA constitute the most important biomarker for this disease. VLCFAs, especially C26, also accumulate in fibroblasts and blood cells of patients. In 1999 Braiterman et al. reported that the key functional biochemical abnormality in XLAD is impaired beta-oxidation of very long–chain fatty acids in peroxisomes. Linkage studies in which the XLAD locus was mapped to Xq28, and subsequent positional cloning and sequencing of the XLAD locus, revealed that it encodes an ATP binding cassette transporter ABCD1 (Mosser et al., 1993). This locus encodes a half-ABC transporter, and dimerization with another half-transporter is required for function. ABCD2, ABCD3, and ABCD4 genes frequently encode the latter. The products of these loci show considerable homology. There is evidence that homodimeric proteins may form from the

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product of ABCD1. The protein encoded by ABCD1 is designated adrenoleukodystrophy protein ALDP; it is embedded in the peroxisomal membrane with the ATP binding site facing toward the cell cytoplasm (Roerig et al., 2001). ALDP participates in transport of very long–chain fatty acid co-A esters across the peroxisomal membrane, where they are partially degraded. Defective function of ALDP, the ABCD1 gene product, leads to accumulation of very long–chain fatty acid (VLCFA) in the cytosol of the cell. Kemp and Wanders (2010) reported that in peroxisome, fatty acid chains are shortened and these exit peroxisomes. Full beta-oxidation of fatty acids occurs in mitochondria. VLCFA accumulation in the cytosol also increases oxidative stress and damage through lipid peroxidation.

MOLECULAR GENETIC STUDIES XLAD is due to mutations in the ABCD1 transporter that impair function. In 2010 Kemp and Wanders reported that 1,065 mutations were documented in the XLAD database. They noted that the majority of affected families each had a unique mutation. DNA testing is therefore time consuming and expensive. Of the disease-causing mutations in the XLAD database, 61% are missense, 22% are frame shift, and 10% are nonsense. Mutation and sequence analysis of ABCD1 are also complicated by the fact that there are four paralogs. Exons 7–10 of ABCD1 were duplicated in primate evolution and transferred from the X chromosome to chromosomes 2p11, 10p11, 16p11, and 22q11. Liquid chromatography coupled to tandem mass spectrometry can be effectively used to determine levels of C26 VLCFA and derivatives in dried blood spots, thus facilitating newborn screening for XLAD.

TIME FRAME OF CLINICAL MANIFESTATIONS OF XLAD Symptoms of this disorder usually occur in males in the second decade of life or later. The earliest manifestations may be school difficulties, behavioral problems, impaired vision, and hearing. Subsequently, seizures develop. Some patients present first with adrenocortical insufficiency and/or peripheral neuropathy. The manifestation may occur at any point between the second and fourth decade of life. The AMN (adrenomyeloneuronopathy) type of XLAD is a form that affects the dorsal columns of the cerebrospinal tracts, leading to progressive paresis.

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Clinical studies include measurement of VLCFA and measurement of nerve conduction velocities to determine presence of neuropathy. Brain MRI studies are important in documenting onset and presence of cerebral demyelination. In areas of demyelination, invasion with inflammatory cells occurs. In these areas there is breakdown of the blood–brain barrier. Contrast medium used in tomography scans and gadolinium used in MRI can accumulate in these areas.

THERAPEUTIC INTERVENTIONS IN ALD Patients who manifest adrenocortical insufficiency benefit from appropriate treatment with adrenocortical hormones. The efficacy of Lorenzo’s oil in treatment of X-linked ALD is controversial. Lorenzo’s oil is a mixture of glyceryltrioleate and glyceryltrierucate. There is evidence that administration of Lorenzo’s oil and a low-fat diet lead to lower levels of plasma very long–chain fatty acids. In patients with ALD and normal brain MRI scans, this therapy apparently delayed the onset of neurological symptoms. In a study of 45 men with adrenomyeloneuropathy (AMN), Lorenzo’s oil reportedly slowed progression of the disease (Berger et al., 2010).

PROPOSED MOLECULAR THERAPIES The autosomal gene ABCD2 is highly homologous to ABCD1. Studies on a mouse model of XALD revealed that oveexpression of ABCD2 normalized tissue levels of VLCFA. The mouse study involved transfection of a transgene that increased expression of ABCD2. The question arises whether ABCD2 expression can be upregulated through pharmacological induction. Berger et al. (2010) stated that pharmacological induction would have to target the right cell types and would have to induce robust levels of increased expression. Macrophages and microglia might represent therapeutic targets. In the case of X-linked ALD and treatment in males, the ABCD2 gene is a surrogate for the mutated ABCD1 gene. Efforts to induce increased ABCD2 expression have included use of histone deacetylase (HDAC) inhibitors and fibrates such as fenofibrate. Histone deacetylases remove acetyl groups from histones and promote chromatin condensation and silencing of transcription. HDAC inhibitors increase expression of a number of different genes by promoting hyperacetylation of lysine groups

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in histones. Small-molecule HDAC inhibitors used in therapy for a number of genetic and neurodegenerative diseases include hydroxamic acids, vorinostat (SAHA), and trichostatin A. Small-molecule organic acids useful as HDAC inhibitors include 4-phenylbutyrate and valproic acid. It is interesting to note that HDAC inhibitors are most useful in treatment of autosomal-dominant diseases to upregulate expression from the normal gene.

HEMATOPOIETIC STEM CELL TRANSPLANTATION There is evidence that hematopoietic stem cell therapy is beneficial in treatment of XALD. Cartier and Aubourg (2010) reported that allogeneic stem cell therapy undertaken early in the course of the disease could prevent progressive cerebral demyelination in boys with XALD. However, matched donors are frequently not available. They noted that in adults with this disease, allogeneic stem cell therapy is less successful. Of particular importance is the fact that microglia in the brain are derived from brain bone marrow and are likely derived from myeloid precursors. Cartier and Aubourg (2010) reported that in a growing number of cases, stem cell transplantation had arrested the characteristic neuroinflammatory pathology in XALD. Of interest is the fact that following transplantation demyelination continued for about 2 months, and then by 12–18 months this was arrested. They attribute this to the fact that the transplanted microglial cells slowly replaced the host microglial cells in brain. They cautioned that allogeneic bone marrow transplant is a highly risky procedure with 15–20% mortality in children and 30–40% mortality in adults. They noted that there is no definite evidence that hematopoietic stem cell therapy is advantageous in treating the axonal degeneration characteristic of adrenomyeloneuronopathy.

TRANSPLANTATION OF AUTOLOGOUS, GENETICALLY MODIFIED BONE MARROW STEM CELLS A key issue in genetic modification of stem cells is which vector should be used to correct the gene defect. Early studies in treatment of severe combined immunodeficiency (SCID) revealed that the retrovirus used to introduce the correcting gene has integrated in host chromosomes close to oncogenes, and leukemia occurred posttransplantation in 5 of 20 transplanted patients.

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Recent successes in transfection with adeno-associated viral vectors in hemophilia open the way to use of these vectors in treatment of other genetic diseases. Koerber et al. (2009) reported development of an adeno-associated viral vector especially effective in transduction of glial cells. This has relevance to treatment of leukodystrophies including XALD. In leukodystrophies the white matter of the brain is defective. The white matter is composed of myelinated axons, glial cells, and oligodendrocytes. Glial cells provide trophic support for myelin and axons. There is now evidence that microglia play roles in the maintenance of synaptic integrity. Graeber (2010) reported that microglia are frequently fused to apical dendrites. The degree of accumulation of microglia differs depending on the functional state of the synapse. Microglia likely play roles in eliminating damaged or nonfunctional synapses. In the central nervous system, microglia constitute 20% of all glial cells. Subsets of perivascular microglial cells are likely replaced by hematopoeitic cells, probably monocytes, following bone marrow stem cell transplantation.

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8 ADVANCES IN THERAPY FOR SPECIFIC MONOGENIC DISEASES

. . . diseases once considered quite distinct can share similar molecular pathways; this realization suggests that the entire framework of medical taxonomy requires rethinking and therapeutics of the future will be designed with cellular frameworks in mind . . . —Francis Collins (2011)

INTRODUCTION The goal of this chapter is to present examples of monogenic diseases and reports of approaches to treatment and preclinical studies, designed to impact specific gene defects or the downstream effects of gene mutations. It is also important to take into account the fact that mutations in a number of different genes may lead to highly similar phenotypes. Several examples of genetic heterogeneity in monogenic diseases are presented. Correct diagnosis and identification of the underlying gene defect is, of course, essential for implementation of gene-based therapies.

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Hemoglobinopathies Sickle cell disease and thalassemia are monogenic diseases with autosomalrecessive inheritance. Weatherall (2010) reported that worldwide in excess of 300,000 children are born each year with either sickle cell disease or a form of thalassemia. James Herrick first described sickle cell anemia in 1910. Thomas Cooley (1927) in the United States and Fernando Rietti (1926) in Italy first described thalassemia, a severe form of anemia in patients of Mediterranean ancestry in 1926. Annual worldwide reported birthrates for the most common major hemoglobin disorders in 2008 were 217,331 homozygotes sickle cell disease cases, 22,898 cases of beta thalassemia major, and 19,128 cases of hemoglobin E thalassemia, which leads to severe anemia. Weatherall noted that in 2011 therapy for sickle cell disease was still primarily at the level of hydration, antibiotics, and blood transfusions. Severe thalassemia syndromes are most often treated with blood transfusion, and the iron accumulation that occurs in consequence of multiple transfusions is treated with chelating agents. Orkin and Higgs (2010) noted that bone marrow transplantation using cells from matched donors works and that there have been successes with gene therapy and gene repair in stem cells followed by infusion into patients. Nevertheless, the latter therapies are out of reach for the majority of patients in Africa, the Caribbean, and Asia. There is evidence that reactivation of expression of gamma globin genes can lessen the impacts of thalassemia mutations and sickle gene mutations. Expression of gamma globin is usually silenced in early infancy. Important factors in gamma globin gene expression include specific sequences in the promoter region of gamma globin and in control sites, in particular binding sites for the transcription factor GATA1. In some individuals hereditary persistence of fetal hemoglobin expression occurs due to mutations in the gamma globin gene promoter. Fetal hemoglobin (HbF) contains alpha and gamma globin chains. Hereditary persistence of fetal hemoglobin also occurs in some patients who have a deletion that encompasses the delta globin locus and adjacent locus control region. Additional factors that impact gamma globin gene expression include the transcription factor BCL11A. A multiprotein complex that negatively regulates gamma globin expression contains transcription factors BCL11A and SOX6. This complex does not bind to the gamma or beta globin gene promoters; rather, it binds to an upstream locus control region for beta globin and to intergenic regions between the gamma and beta globin genes. The BCL11A-containing complex also binds to a control element upstream of the delta globin locus.

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Alpha-like globin genes : chromosome 16p13.3 TEL

CEN

Regulatory Zeta region

Pseudogenes Alpha2

Alpha1 Theta

Beta-like globin genes : chromosome 11p15.5-p15.4 CEN

TEL LCR locus control regulatory region

Epsilon

Gamma g a

Delta

Beta

Embryonic

Fetal

Adult

Hb Gower 1 : Zeta2 epsilon2 Hb Gower 2 : Alpha2 epsilon2 Hb Portland : Zeta 2 gamma2

Hb F : alpha2 gamma 2

Hb A : alpha2 beta 2 Hb A2 : alpha2 delta 2

Figure 8–1. This figure illustrates chromosome organization of globin gene loci and regulatory regions. Figure based on and modified from information in Higgs et al. (2012).

The transcription factor KLF1 activates expression of BCL11A. There is now evidence that hereditary persistence of fetal hemoglobin occurs in individuals with deletion in BCL11A or KLF1. Orkin and Higgs (2011) reported that advances in high-throughput molecular screening might facilitate discovery of molecules that induce globin gene reexpression. Pharmacological agents that are effective in induction of gamma globin expression include hydroxyurea that is in clinical use; however, long-term side effects of administration of this compound have raised concerns. As noted previously, the zinc-finger transcription factor BCL11A is a repressor of globin and hemoglobin F expression. Knockdown of BCL11A increases globin transcription. Graslund et al. (2005) designed zinc finger–based transcriptional activators to target sites in the gamma globin gene promoter proximal to position −117. This region is known to serve as binding site for transcription factors. Retroviral delivery of the zinc finger–based transcription factor designed by Graslund et al. into the erythroleukemia cell line K562 resulted in increased production of gamma globin. Costa et al. (2012) reported that this artificial zinc-finger transcription factor enhanced gamma globin gene expression in a mouse model or thalassemia. Gene therapy for globin gene defects is complicated by the intricate regulation required for appropriate expression of globin genes.

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Sebastiano et al. (2011) reported gene correction of the sickle mutation in patient-derived pluripotent stem cells (see also Chapter 12).

Duchenne Muscular Dystrophy (DMD) Clinical trials are in place for treatment of DMD patients who have stop codon mutations in the central gene region (see also Chapter 11 on gene-based therapies). Encouraging results have been achieved with exon-skipping approaches when stop codons are present between exons 42 and 55 (Cirak et al., 2012; Verhaart and Aartsma-Rus, 2012) (see also Chapter 11). In patients who have stop codon mutations in various locations in the DMD gene, it is may be possible to use pharmacological substances to promote stop codon read-through (e.g., ataluren PTC124). Verhaart and Aartsma-Rus reported that clinical trials are in progress using PTC124 in DMD patients; however, they noted that there are indications that achievement of optimal dosage levels is challenging. The large size of the dystrophin gene is a complicating factor in the design of gene therapy experiments. The adeno-associated viral vectors used in gene therapy accommodate 4 kb of inserted DNA, and the dystrophin-processed mRNA encompasses 14 kb. Verhaart and Aartsma-Rus noted that dystrophin contains repetitive and partly redundant domains. Mini-dystrophin genes have been generated and used in gene transfer; however, they have generated an immune response. An additional complication of gene therapy is the difficulty in achieving bodywide delivery.

TREATMENT OF DOWNSTREAM EFFECTS OF THE DMD MUTATIONS Treatments are being devised to enhance muscle mass, and these include use of Follistatin, which acts to inhibit the effects of myostatin. Myostatin is an endogenous protein that inhibits muscle growth. Another growth-promoting treatment approach includes the use of insulin-like growth factor (IGF1) that stimulates muscle growth (Gehrig et al., 2012a). Corticosteroids are frequently used to decrease inflammatory changes present in muscle in DMD. However, steroids have negative side effects. Dystrophin forms part of a complex that is associated with the muscle membrane. One approach to therapy is to explore compounds that impact the strength of this complex and the adhesiveness of the components. Compounds

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that strengthen the complex include Biglycan, which recruits the protein utrophin to the complex. Biglycan also binds to sarcoglycans that form part of the complex. Biglycan is a leucine-rich proteoglycan that is a component of extracellular matrix. Components of the dystrophin-containing complex undergo glycosylation, and specific copolymers are being investigated in treatment. Clinical trials require adequate outcome measures for monitoring. The serum marker MMP (matrix metalloproteinase) and TIMP, inhibitor of metalloproteinase, are useful outcome-monitoring markers. Creatine kinase level is useful as a diagnostic marker but not as a treatment outcome marker (Verhaart and Aartsma-Rus, 2012).

IDENTIFYING SYNERGISTIC TREATMENT MODALITIES, INCLUDING TREATMENTS THAT INCREASE GENE EXPRESSION Increased Expression of HSP72 Chaperone Protein Treatment targets that impact expression or function of proteins downstream of a defective gene and its product may be identified through studies on the pathophysiology of a specific disease. Gehring et al. (2012) reported that in the absence of the stabilizing molecule dystrophin, muscle fibers become susceptible to membrane tears, channel alteration, increased calcium influx, and inflammatory changes that lead to muscle degeneration. They noted further in studies on the mouse model of Duchenne muscular dystrophy that increased expression of the gene SERCA (sarcolemma endoplasmic reticulum calcium pump) improves calcium homeostasis in muscle and suppresses pathophysiological changes. Studies on the inflammatory reactions characteristic of DMD revealed that the tumor necrosis factor TNF alpha plays a key role in activation of NF kappa B (nuclear factor kappa B) and the JNK (Jun N terminal kinase) signaling pathway. Gehrig et al. (2012b) carried out studies on the molecular chaperone HSP72 to further investigate preliminary evidence that increasing the levels of expression of HSP72 protects dystrophic muscle through inhibition of inflammatory mediators, TNF alpha, and JNK and NFK beta, and evidence that HSP72 protects SERCA function. These investigators carried out studies on the dko dystrophin–null mouse. This mouse manifests muscle weakness, kyphosis, and impaired diaphragm function and has elevated serum levels of creatine kinase. They introduced a transgene that overexpressed Hsp72 in the dko mouse. Following insertion

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of the transgene, creatine kinase levels were found to be lower, body strength and endurance were greater, and respiratory function was less impaired than in the untreated mouse. Introduction of the transgene led to increased activity of SERCA. Gehrig et al. (a and b) the explored the effects of treatment with BGP15 or with heat. Both of these treatments are known to increase expression of HSP72. BGP15 is a nicotinic amidoxime derivative that is a pharmacological inducer of HSP72, which is in phase 2 clinical trials for treatment of insulin resistance. BGP15 treatment ameliorated muscle pathology in the dko mice. Gehrig et al. (b) concluded that pharmacological induction of HSP72 delayed disease progression and is likely to benefit patients with Duchenne muscular dystrophy as a synergistic therapy.

Rett Syndrome Classical Rett syndrome is an X-linked, dominant condition and manifests in females. In males MECP2 mutations are usually lethal. In females symptoms begin 6–18 months after birth and following normal initial development. Patients progressively lose motor and cognitive skills and develop abnormal hand movements. Later, autistic symptoms and ataxia develop. In later stages the patients have seizures, hyperventilation, and apnea. Variability in manifestations occurs even in patients with the same MECP2 mutation. This is likely due to the proportion of normal X chromosomes that are inactivated (Zoghbi, 2009). This syndrome results from mutations in MECP2 located on the Xq28 chromosome, which encodes a methyl CpG DNA binding protein. In the Rett syndrome database (Rettbase), more than 600 different MECP2 mutations that give rise to the syndrome are listed.

GENETIC HETEROGENEITY IN RETT SYNDROME Rare patients with clinical manifestations of Rett syndrome have mutations in the CDKL5 gene on Xp22. Characteristic features of Rett syndrome were found in a patient with a translocation between chromosomes 1 and 7 that disrupted the brain-expressed Netrin 1 (NTNG1) gene on chromosome 1 (Borg et al., 2005). Features of Rett syndrome have also been described in patients with mutations in FOXG1 (Bahi-Buisson et al., 2010). FOXG1 encodes a transcription factor that is critical for forebrain development.

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NEUROANATOMICAL ABNORMALITIES IN MECP2-DEFICIENT RETT SYNDROME Brain size is usually reduced, and neuronal soma are unusually small and are densely packed. Synapses are reduced in number, and there are reductions in dendritic spine density (Bauman et al., 1995). There is evidence that Mecp2 functions not only in neurons but also in glia HT (Zoghbi, 2009). Pluripotent stem cells from patients with this syndrome have been derived in an attempt to understand the pathophysiology of the disease (see Chapter 12). In addition, patient-derived cells have been utilized in searches for compounds that neutralize the effects of specific mutations. Mouse models of this syndrome have been developed and intensely studied by a number of investigators. In mouse models of Rett syndrome, neurological manifestations develop in adult mice. In female mice heterozygous for Rett syndrome mutations, progression is relatively slow and life span is almost normal. In homozygous male mouse models of Rett syndrome, symptoms develop early and include tremors and motor impairments; death occurs between 10–20 weeks (Gadalla et al., 2011). There is evidence that microglia play an important role in Rett syndrome. Derecki et al. (2012) transplanted wild-type mouse bone marrow into radiation-conditioned Mecp2-null host mice. This led to engraftment into brain of microglial cells derived from bone marrow myeloid cells. The expression of Mecp2 from the transplant-derived microglia arrested facets of the Rett syndrome pathology. The improvements in locomotor function and breathing were particularly striking in male Mecp2-negative mice and were also seen in female mice. Derecki et al. suggested that bone marrow transplantation might be a feasible therapeutic approach in Rett syndrome. MECP2 PROTEIN FUNCTION The MECP2 gene encodes a protein that binds to methylated cytosine-guanine (CpG) residues in DNA. Recent studies indicate that this protein plays a key role in protein remodeling and that activity of this protein is dynamically regulated by phosphorylation at specific residues in the protein. Cohen et al. (2011) demonstrated through studies in mouse mutants that activity-dependent phosphorylation of Mecp2 S421 (serine 421) plays a key role in synaptic function. Their studies indicate that Mecp2 is bound at sites across the genome and constitutes a core component of chromatin. A consideration in design of therapy for Rett syndrome is that restoring activity of the abnormal gene may lead to abnormally high levels of the protein.

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INDIRECT THERAPY OF RETT SYNDROME: NEUROTROPHIC FACTORS Studies on mouse models of Rett syndrome revealed that increasing brain-derived neurotrophic factor (BDNF) signaling ameliorated disease manifestations. Ogier et al. (2007) reported that BDNF expression is enhanced by CX546, an ampakine that modulates the AMPA receptor (alpha amino-3-hyd roxy-5methylisoxazole-4-propionic acid). Gadalla et al. (2011) reported that treatment with insulin-like growth factor 1 (IGF1) reverses manifestation of Rett syndrome in mouse models of the disorder. The N-terminal peptide of IGF1 has neuromodulatory and neuroprotective effects in Rett mouse models. This peptide increases expression of the postsynaptic density protein PSD 95. It also increases cortical spine density and increases amplitude of excitatory synaptic currents (Tropea et al., 2009). Recombinant IGF is under investigation in clinical trials in Rett syndrome patients. NEUROTRANSMITTER SYSTEMS Glutamatergic synapses and glutamatergic signaling are reduced in Rett syndrome. Maliszewska-Cyna et al. (2010) reported altered subunit distribution of NMDA subunits in glutamatergic synapses in this disorder. Reports indicated that memantine, which acts on the NMDA system, might be useful in treatment. Modulators of GABA-ergic neurotransmission have some evidence of effectiveness in mouse models of Rett syndrome.

MODULATION OF EPIGENETIC FACTORS There are reports that administration of histone deacetylase inhibitors decreased manifestations of Rett syndrome in a mouse model of the disorder.

GENE THERAPY Guy et al. (2007) reported that in a specific mouse model of Rett syndrome, the disease manifestations were reversed when a stop codon mutation was silenced. Aminoglycoside derivatives that promote stop codon read-through (e.g., ataluren PTC124 and NBT54) may be useful in treatment of specific mutations in Rett syndrome patients.

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DOWNSTREAM EFFECTS OF REDUCED LEVELS OF MECP2 PROTEIN ON MITOCHONDRIAL FUNCTION A number of studies have revealed altered mitochondrial function in Rett syndrome. Kriaucionis et al. (2006) carried out studies on Mecp2-deficient mice. They reported altered expression of the mitochondrial respiratory complex III subunit, ubiquinol cytochrome C reductase core protein. Gibson et al. (2010) reported decreased expression of cytochrome C subunit 1 in brain tissue from Rett syndrome patients. De Felice et al. (2012) reported evidence for increased oxidative stress and increased lipid peroxidation in Rett syndrome. They also reported that treatment of patients with omega 3 polyunsaturated fatty acids including eicosapentaenoic acid and docosahexanoic acid (DHA) for 6 months led to improvement in clinical manifestations in patients with stage 1 Rett syndrome. De Felice et al. noted improvement in motor-related symptoms and communication abilities.

MONOGENIC DISEASES THAT DISPLAY GENETIC HETEROGENEITY DESPITE CLINICAL SIMILARITY: ADVANCES IN TREATMENTS Eye Diseases and Therapies Retinitis Pigmentosa The term retinitis pigmentosa is used to define a group of chronic, degenerative diseases of the retina. Characteristic features of the disorder include early loss of night vision (vision in dim light) due to defects in rod photoreceptors. Subsequently, cones degenerate and vision in daylight diminishes. Peripheral vision deteriorates, and later central vision also deteriorates, leading to blindness. Most patients with retinitis pigmentosa are legally blind by 40 years of age. Defects in more than 50 genes have been implicated in retinitis pigmentosa (Hartong et al., 2006). The worldwide prevalence of this disorder is 1 in 4,000. Retinitis pigmentosa may be inherited as an autosomal-recessive disease (50–60%) autosomal-dominant (30–40%) or X-linked (5–15%). The disease may also result from new mutations or structural chromosome changes. Retinitis pigmentosa may be the only defect present in the patient; however, it may also occur as one feature of a syndrome. In Usher syndrome vision and hearing is impaired. Bardet–Biedl syndrome includes retinitis pigmentosa, obesity, cognitive impairment, polydactyly, hypogenitalism, and renal disease.

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METABOLIC DEFECTS THAT LEAD TO RETINITIS PIGMENTOSA It is important to establish whether or not metabolic disease is present in young patients with retinitis pigmentosa since early treatment may prevent progressive vision deterioration (Grant and Berson, 2001). Oral intake of fat-soluble vitamins A, E, and K is effective in the treatment of retinitis pigmentosa that occurs in abetalipoproteinemia (Bassen-Kornzweig disease). Refsum disease has retinitis pigmentosa as a feature and may be treated with diet low in phytol and phytanic acid. RETINAL STRUCTURE Retinal pigment epithelium overlies the outer nuclear layer with rod and cone photoreceptors and an inner nuclear layer that contains rod bipolar cells, cone bipolar cells, horizontal cells, ganglion cells, amacrine cells, synaptic interneurons, and Muller glial cells that provide support and can become stem cells (Bernardos et al., 2007). GENES MUTATED IN RETINITIS PIGMENTOSA Mutations in the light-absorbing protein rhodopsin are present in approximately 25% of cases of autosomal-dominant retinitis pigmentosa. Mutations in the USH2a, the Usher syndrome gene, lead to about 20% of cases of the autosomal-recessive form. Mutations in the retinitis pigmentosa GTPase regulator gene RPGR lead to 70% of X-linked cases. Together mutations in these three genes account for approximately 30% of cases of retinitis pigmentosa (Hartong et al., 2006). Mutations in each of the other approximately 48 genes each account for a small percentage of cases of retinitis pigmentosa. BIOCHEMICAL PATHWAYS Cyclic guanosine monophospate (GMP) phosphodiesaterase alpha and beta subunits encoded by the PDE6A and PDE6B genes impact the concentration of cyclic GMP, which is an important regulator of rod membrane current. Without adequate control, levels of GMP rise and cause excessive opening of cation channels. Excessive cation inflow damages rods. Rhodopsin is a transmembrane light-absorbing protein that undergoes photoexcitation and initiates signal transduction and the visual transduction

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cycle. There is evidence that mutations in the transmembrane domains of rhodopsin lead to the most severe phenotypes. Some rhodopsin mutations lead to abnormal protein folding (Schuster et al., 2005). Mutations in the gene that encodes arrestin, the rhodopsin-deactivating protein, also lead to retinitis pigmentosa. GMP-gated ion channels play key roles in the phototransduction cascade, and mutations in the genes that encode these channels lead to retinitis pigmentosa. Also important in the etiology of this disease are mutations in genes that impact vitamin A and retinaldehyde metabolism (e.g., RPE65, vitamin A trans cis isomerase, and LRAT, lecithin retinal acetyltransferase, which synthesizes vitamin A esters). Membrane and scaffolding protein mutation may impact localization of signaling molecules; these include mutations in USH1C, USH2A, and PCH15 (Hartong et al., 2006).

APPROACHES TO TREATMENT Jacobson and Cideciyan (2010) documented approaches to treatment of patients with retinitis pigmentosa. In cases in which some photoreceptors were intact and a known biochemical defect in the visual cycle exists, such as Leber’s congenital amaurosis due to RPE65 mutation, gene replacement therapy has been successful. This therapy involves subretinal injection of an RPE transgene cloned into an adeno-associated viral vector. A second treatment modality for Leber’s amaurosis involves use of nutritional supplements and application of neurotrophic factors to inhibit photoreceptor degeneration and apoptosis. Other approaches to treatment of retinitis pigmentosa involve transplantation of glial stem cells and stimulation of the visual pathway with electric signals from implants. An additional approach involves cloning of light-sensitive archeabacterial halorhodopsin and injection of this to resensitize degenerating cone cells (Busskamp et al., 2010). Gene therapy of eye diseases is discussed also in Chapter 11.

EVALUATION OF GENE THERAPY FOR RPE65 EFFICACY IN A 3-YEAR FOLLOW-UP Jacobson et al. (2012) carried out a follow-up evaluation on 15 patients (aged 11–30 years) who had four doses of subretinal injection of rAAV2RPE45 into the worse functioning eye. They reported that no systemic toxicity was

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detected. Visual function improved in all patients and to different degrees. They concluded that extrafoveal injection strategies led to improvement in functioning of rods and cones.

PRECLINICAL STUDIES IN OTHER FORMS OF RETINITIS PIGMENTOSA These studies have been greatly facilitated through the availability of mouse models of various gene mutations that lead to this disease. Koch et al. (2012) demonstrated efficacy of gene therapy treatment of a mouse model of CNGB1 deficiency through CNGB1 knockout. CNGB1 encodes a subunit of rod cyclic AMP-gated channel. They injected an AAV vector into which they had cloned CNGB1 cDNA, a short rod promoter, and regulatory elements. Gene replacement was able to restore CNGB channel expression and localization. Furthermore, the treated CNGB1 knockout mice had improved retinal function and vision.

PRECLINICAL STUDIES ON PHOTOCHEMICAL RESTORATION OF VISUAL RESPONSES Polosukhina et al. (2012) carried out studies to investigate strategies to restore light sensitivity in eyes damaged by degenerative diseases such as retinitis pigmentosa and age-related macular degeneration. In blind mice they injected a synthetic small-molecule potassium channel photo switch AAQ (acrylamide azobenzene quaternary ammonium) into the vitreous cavity. Application led to prolonged light sensitivity, pupillary reflex and locomotor activity. The specific AAQ formulation used lasted 24 hours. The investigators noted that second-generation AAQ formulations with longer activity are being investigated.

Pelizaeus-Merzbacher Syndrome Clinical features of this disorder include early development of nystagmus followed within the first 2 years of life by respiratory and swallowing difficulties. There is also evidence of dysfunction of the pyramidal system, including spasticity, brisk reflexes, and myotonia. Upper-body and axial hypotonia are present so that ability to sit unsupported and to lift the head are impaired. Signs

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of cerebellar ataxia may also be present. The key feature of the disease on brain MRI is the presence of diffuse white matter hyperintensities (Boulloche and Aicardi, 1986). The disease is due to defects in myelination, classified as dysmyelination or hypomyelination, indicating defective production of myelin. Myelin sheaths are essential for maximizing conduction velocity. Oligodendrocyte precursor cells play key roles in synthesis of constituents of the myelin sheath, including lipids and membrane-associated myelin proteins. Key myelin components are the myelin basic proteins and the proteolipid protein produced by the PLP1 gene on Xq. PLP1 protein constitutes approximately 50% of the protein in the central nervous system myelin. Bilir et al. (2013) reported that approximately 805 of the patients with Pelizaeus-Merzbacher disease (PMD) carry PLP1 gene duplications that range in size from 100 kb to 4.6 Mb and that the breakpoints of these duplications were variable. Mutations in PLP1 also occur in patients with PMD. Bilir et al. reported that PMD patients with duplications often have a milder phenotype than patients with mutations in the PLP1 gene. Patients with deletion or loss of PLP1 usually have a milder phenotype characterized by peripheral neuropathy. There is evidence that increased PLP1 activity inhibits oligodendrocyte differentiation and that this effect impacts signaling through the ERK-signaling cascade (Miyamoto et al., 2012). These investigators carried out studies on oligodendrocyte cultures from PMD patients. They reported that the dysmyelination in cultures diminished on treatment of cultures with kinase inhibitors that led to ERK inhibition. They established that PLP1 promotes ERK phosphorylation.

THERAPY OF PELIZAEUS-MERZBACHER DISEASE WITH HIGH-CHOLESTEROL DIET Saher et al. (2012) carried out studies on transgenic mice with extra copies of the Plp1 gene. They determined that in these mice dysmyelination and demyelination occurred. This was followed by secondary inflammation and axon damage. Plp1 protein and cholesterol accumulated in endosomes and in lysosomes of oligodendrocytes. Sequestration of cholesterol by increased Plp1 protein and its retention in these vesicles decrease the amount of cholesterol available in the plasma membrane. This, in turn, impairs myelin synthesis. In mice fed on a cholesterol-enriched diet, oligodendrocyte numbers and myelin production improved. Inflammatory changes and secondary astrogliosis

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and microgliosis in the spinal cord diminished. To explain the beneficial results of cholesterol treatment, Saher et al. proposed that the increased cholesterol available at the cellular membrane promotes myelin synthesis. Saher et al. concluded that results in the mouse model have implications in patients.

CELLULAR THERAPY IN PELIZAEUS-MERZBACHER DISEASE Gupta et al. (2012) investigated the value of treatment of this disease with allogeneic fetal neural stem cells. They noted that Pelizaeus-Merzbacher disease is due to PLP1 gene mutations or duplications that lead to hypomyelination of the central nervous system due to abnormal myelin production or trafficking. Since myelin production is a function of one cell type, oligodendrocytes, they defined Pelizaeus-Merzbacher disease as a single-cell disease. Gupta et al. (2012) carried out an open-label phase 1 study on four subjects with the early-onset, severe form of Pelizaeus-Merzbacher disease. Human fetal neural stem cells shown to produce functional myelin were transplanted by injection through frontal burr holes to target the centrum semiovale or corona radiata. Following transplantation immunosuppression was carried out for 9 months with oral tacrolimus, and antibiotic prophylactic treatment was used for 3 to 9 months. Extensive neurologic examinations carried out 12 months after transplantation revealed modest gains in neurological function in three of the four subjects, and no adverse affects were observed that were due to the transplanted cells. Diffusion tension imaging (DTI) findings were consistent with increased myelination in the region of cell transplantation.

GENETIC HETEROGENEITY IN PELIZAEUS-MERZBACHER DISEASE It is now clear that patients with clinical findings consistent with PMD do not always have defects in the PLP1 genes. Bilir et al. (2012) reported a high frequency of mutations in the connexin 47–encoding gene GJA12 (sometimes referred to as GJC2). This gene maps to chromosome 1q42.13, and the disease in these patients is inherited as an autosomal-recessive disorder. Bilar et al. identified five different GJA12 mutations in six families; mutations included short insertion or deletions and single nucleotide changes. Allan-Herndon-Dudley syndrome is an X-linked disorder with many features in common with PMD, including axial hypotonia, limb spasticity, limb clonus, and dystonic movements. However, in their review of this disease

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Boccone et al. (2010) do not mention presence of nystagmus in these patients. This syndrome is characterized by low total and free thyroxin T4 and elevated total and free thyroxin T3. TSH levels are normal. The disorder is due to mutation in the monocarboxylate thyroid hormone transporter MCT8 (SLC16A2).

CHARCOT MARIE TOOTH DISEASE Charcot-Marie-Tooth disease (CMT) includes a number of genetically determined peripheral neuropathies that impact both sensory and motor nerves, leading to weakness and tendon reflex abnormalities. Genetic studies have revealed that clinical manifestations of this syndrome are due to defects in a number of different pathways that impact axonal function, myelination, and communication between axons and myelin-producing cells (Juarez and Palau, 2012). Detailed analysis and understanding of defective molecular function may lead to improved therapy for CMT. The collective population frequency of CMT is 17–40 per 100,000. The disease may be inherited in autosomal-dominant, autosomal-recessive, or X-linked modes. Mutations or structural changes in at least 40 different genes are known to cause CMT. The CMT1 type of the disease arises as a result of defects in genes that impact myelin. Slowed nerve conduction velocities (i.e., below 38 m/sec), characterize CMT1. In the CMT2 form of the disease, the axonal form, nerve conduction velocities are higher. CMT1A is an autosomal-dominant peripheral neuropathy due to 1.4 Mb duplication on chromosome 17p11.2. CMT1B is caused by mutations in the myelin basic protein zero gene, MPZ, which maps to chromosome 1q23.3. Mutations in this gene lead to autosomal-dominant CMT. In rare cases, mutations in other genes may lead to a CMT1 similar phenotype with autosomal-dominant inheritance. These genes include EGR2, early growth response zinc-finger gene on 10q21.2 (CMT1D); LITAF, lipopolysaccharide-induced lysosomal membrane protein–encoding gene on 16p13.3 (CMT1C); and NEFL, neurofilament light protein–encoding gene on chromosome 8p21 (CMT1F), which plays a role in axonal communications and transport. CMT2 includes axonal forms of the disease. In 2011 Shy and Patzkó reported that causative mutations have only been identified in 25–35% of CMT2 patients. There are known mutations in 12 different genes leading to axonal CMT. Genes that impact mitochondrial fission and fusion are mutated in cases of CMT2A; specific mutations in the neurofilament light protein NEFL may

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give rise to CMT2E. CMT2 also arises due to defects in DNA–RNA synthesis or processing. Disrupted chaperone function and inadequate stress responses due to HSP72 or HSP27 mutations may cause CMT2. CMT type 4 includes demyelinating disorders that frequently also involve nerves to the face, eye, and tongue. Genes that play role in regulation of membrane trafficking may play roles in CMT-type neuropathies (Bucci et al., 2012). Membrane trafficking genes encode proteins involved in vesicle formation, phosphoinositide metabolism, lysosomal, and endosomal function and mitochondrial fission and fusion. Membrane-trafficking genes mutated in CMT include the signaling proteins RAB 7, NDRG1 (NMYC downstream), and SH3TC.

PERIPHERAL MYELIN PROTEIN 22 (PMP22) PMP22 is duplicated in the CMT1A-associated copy number change on chromosome 17p11.2. CMT1A is a demyelinating neuropathy that manifests with peroneal muscular atrophy and slowing nerve velocities. Deletion of this protein leads to a specific neuropathy, hereditary neuropathy with liability to pressure palsies (HNLPP). This disorder is characterized by focal neuropathies and nonuniform conduction velocity changes. Specific mutations in the PMP22 gene may also lead to neuropathies. APPROACHES TO MOLECULAR THERAPY OF PMP22 OVEREXPRESSION There is evidence that normal myelination requires that the level of expression of PMP22 must be tightly controlled. Various strategies have been explored to alter levels of expression of PMP22. There is a clinical trial in place that investigates the effects of high doses of ascorbic acid in decreasing PMP22 gene expression. Progesterone antagonists have also been shown to reduce PMP22 expression. Jang et al. (2012) carried out in vitro studies to identify small molecular compounds that can inhibit overexpression of PMP22 that occurs in CMT1A. They developed a transcription-based cellular assay system to investigate PMP22-modulating chemicals. Three drugs reduced the quantity of PMP22 produced in their assay system. These drugs were fenritinide, a retinoic acid analog; olvanil, an agonist for a cation channel; and bortezomib, an anticancer drug. These drugs inhibit expression of a number of myelin-producing genes and they dramatically alter PMP22 gene expression.

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CMT1X The X-linked form of Charcot-Marie-Tooth disease is due to defects in the GJB1 gene, which encodes connexin 32. This protein is expressed in oligodendrocytes and in other cells. Connexin 32 forms gap junctions that facilitate transfer of ions and small molecules. Approximately 400 different connexin 32 mutations have been described in CMT1X patients. This condition is characterized by neuropathies including impaired visual-evoked responses and impaired auditory-evoked responses.

Keratin Disorders: Epidermolysis Bullosa The term epidermolysis bullosa describes inherited mechanobullous disorders of the skin and mucous membrane. Intong and Murrell (2012) reviewed these disorders and reported that four major types are recognized based on location in the dermis of the structural defect and the genes involved. They noted that advances in immunofluorescence mapping of proteins within the epidermis and dermis have greatly improved the specificity of diagnosis. Epidermolysis bullosa simplex results from defect in intraepidermal proteins. Two forms occur; one form defined as supraepidermal is due to mutations in the PKP1 gene that encode plakophilin 1 protein or to mutations in the DSP gene that encodes desmoplakin. In normal epidermis these proteins together form the desmosome. A second form, basal epidermolysis simplex, results from mutations in keratins K5 and K14, or as a result of mutations in PLEC1 that encodes plectin. Mutations in integrins alpha6- or beta4-encoded ITGA6, ITGB4, or in dystonin encoded by DST, may also lead to epidermolysis bullosa. Most forms of epidermolysis bullosa simplex are inherited as autosomal-dominant disorders. Plakophilin 1 and Desmoplakin gene mutations give rise to autosomal-recessive disorders. Epidermolysis simplex usually leads to blistering on hands and feet. However, a severe form of epidermolysis bullosa occurs as a result of specific mutation in keratin K5 and keratin K14 and gives rise to generalized blistering. K14 is a type 1 keratin and K5 is a type 2 keratin; both are expressed in the basal cell layer that is in contact with the basement membrane. McLean and Moore (2011) reported that the vast majority of keratin mutations that lead to epidermolysis bullosa are either missense or small in-frame deletions and are most frequently located in the rod portion of the keratin molecule. They noted that cell proliferation in epithelium is limited to the basal

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cell compartment, and cells there express keratin K5 and K14. Cells migrate upward from the basal layer and become suprabasal cells that switch to expression of keratins K1 and K10. They reported that in epidermolysis simplex, blistering removes the basal cells in the area of the blister. In a separate disorder, bullous icthyosis, the cells in the suprabasal layer are fragile due to K1 or K10 deficiency, and they rupture, releasing cytokines that lead to overproliferation of the epithelium and hyperkeratosis. Keratin K9 is expressed only in the epidermis of the palms and the soles of the feet. Mutations in keratin K9 lead to epidermolytic palmoplantar keratoderma characterized by very painful hyperkeratosis. In humans there are 54 function keratin genes localized in two clusters on 12q and 17q. McLean and Moore (2011) noted that although the genes within each cluster map closely to each other, there are specific patterns of gene expression. The keratin proteins are self-assembling heteropolymers. Each heteropolymer requires type 1 and type 2 keratins.

TREATMENT OF EPIDERMOLYSIS BULLOSA SIMPLEX Small-molecule therapies in development including compounds that induce expression of antioxidant genes have been found to be beneficial in treatment of animals with keratin K14 deficiency. These include sulforophane, a compound found in broccoli. Since most forms of epidermolysis bullosa are dominant-negative mutations, it is necessary to ensure that treatment applications do not block expression of the normal gene. Atkinson et al. (2011) developed short inhibitory RNAs (siRNAs) to two different epidermolysis simplex–causing mutations, K5 p. Ser191Pro and p. Asn193Lys. In a cell-based system, they determined that the siRNAs knocked down expression of the mutant allele but had no effect on the wild-type allele. They emphasized that for clinical application of these treatments, noninvasive delivery systems are required to get small molecules into the appropriate cells.

JUNCTIONAL EPIDERMOLYSIS BULLOSA This disorder is most commonly inherited as autosomal-recessive due to mutations laminin332, collagen XVII, or integrins alpha6 or beta 4. Intong and Murrell (2012) reported that the disorder is due to defect in proteins in the lamina lucida that occurs below the basal cell and above the lamina densa.

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In infants with junctional epidermolysis bullosa, there may be extensive erosions of mucous membranes and of epithelial layers of the gastrointestinal, respiratory, and urinary tracts. In the Herlitz type of junctional epidermolysis bullosa, where laminin 332 is deficient but not absent, there are fewer manifestations and disease is milder. Dystrophic epidermolysis bullosa is due to mutations in collagen VII. It involves the lamina densa, the layer above the dermis. This condition is inherited as autosomal dominant, collagen VII is reduced, and areas of blistering result from trauma and may give rise to scars. Another component of the lamina densa is kindlin. This is deficient in Kindler syndrome associated with blistering, photosensitivity, and mental retardation, and bone abnormalities may be present.

PARONYCHIA CONGENITA This is a rare form of keratoderma due to defects in keratin K6a. It impacts skin, nails, and oral mucosa. Painful plantar hyperkeratosis occurs and frequently leads to inability to walk. Leachman et al. (2010) developed short inhibitory RNAs to target a cytosine to adenine mutation that led to the mutation p. N17K. This was tested in a phase 1 trial. It was found to cause regression of painful hyperkeratosis and calluses on the treated foot. No improvement was found with vehicle treatment of the other foot. No adverse effects were reported during the 17-week-long trial. However, the investigators noted that treatment injection proved very painful and improved methods of application are indicated.

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9 IDENTIFYING THERAPEUTIC TARGETS IN COMPLEX, MULTIFACTORIAL DISEASES

The capacity to blunder slightly is the real marvel of DNA. Without this special attribute, we would still be anaerobic bacteria and there would be no music. —Lewis Thomas (1974)

INTRODUCTION Complex common diseases are most frequently considered to be due to the collective impact of risk variants at a number of different loci. It is, however, important to note that diseases with similar phenotypes to those found in complex common diseases may in some cases be due to monogenic gene defects or to structural chromosome aberrations in specific regions of the genome. One of the main goals of molecular studies in complex common diseases is to identify genetic variants that are associated with these diseases and contribute to disease risk. Additional analyses are then carried out to determine if the disease-associated genes operate in a single or related physiological or biochemical pathways. 157

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Key questions to consider in selecting therapeutic targets for diseases with complex etiology is whether a specific molecule, protein, or gene should be selected as the therapeutic target or whether one or more nodal points in a specific disease pathway represent better targets. Other questions are whether the therapeutic target should be a specific structure (e.g., synapse), or specific subcellular structures (e.g., mitochondria). In some cases functions downstream of the mutation gene are targeted. Another important question is whether monotherapy directed against one target should be instituted or combination therapy directed against several specific targets. Factors that modify the phenotype in cases with known gene mutations could also be considered as therapeutic targets. Another important consideration is whether identification of specific targets in monogenic forms of a specific disease can provide clues for the treatment of multigenic forms of that disease.

CLASSIFICATION OF GENE VARIANTS An initial approach is to classify variants as high-risk, moderate-risk, or lowrisk alleles. High-risk alleles primarily involve sequence changes or structural changes in protein coding regions of genes. Examples of high-risk, rare alleles with Mendelian inheritance include mutations in the amyloid precursor protein–encoding gene APP and mutations in the presenilin gene that lead to familial Alzheimer’s disease, which is frequently of relatively early onset. Shared haplotypes in affected members in a family may serve as proxies for the presence of family-associated rare variants. Moderate-risk, low-frequency alleles have been referred to as “the dark matter” of disease risk (Manolio, 2010). These alleles will most likely to be detected through exome sequencing. Singleton et al. (2010) noted that mutations in beta glucocerebrosidase that lead to Gaucher disease when present in homozygous or compound heterozygous forms are associated with Parkinson’s disease in heterozygotes. This association was discovered through studies that revealed high rates of Parkinson’s disease in relatives of patients with Gaucher disease. Singleton et al. emphasized that obtaining detailed phenotype information and follow-up on family members of parents with children with recessive disease might provide insight into the impact of the heterozygous state of specific variants in the etiology of common diseases. Other examples of moderate-risk, low-frequency alleles that play a role in disease include the variants in the TREM2 gene recently described in cases of Alzheimer’s disease (discussed further later in this chapter). Individuals

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who are homozygous for mutations or deletions in the TREM2 gene develop early-onset dementia and frequently have bone dysplasia (Pekkarinen et al., 1996; Takahashi et al., 2005). Heterozygotes for specific coding mutations in TREM2 have higher risk for Alzheimer’s disease and in some cases for psychiatric manifestations (Guerreiro et al., 2012). Neumann and Daly (2012) noted that low-prevalence coding variants that impact disease risk, such as those found in TREM2, might have been missed in genome-wide association studies (GWASs). Moderate-risk alleles such as copy number variants may also be identified through microarray studies. The E4 allele at the APOE locus represents a moderate risk, relatively high-frequency allele common variant. Low-risk, moderate-to-high frequency alleles are primarily identified in GWASs. Few GWAS-identified alleles double the disease risk. Many of the GWAS-risk alleles impact the expression of genes (Singleton et al., 2010). Most single-nucleotide polymorphic (SNP) alleles identified as disease-associated in GWASs are not directly causal but are in linkage disequilibrium with coding mutations or important regulatory sequences.

EXOME SEQUENCING TO DISCOVER GENE VARIANTS THAT CONTRIBUTE TO THE ETIOLOGY OF COMMON DISEASE It is likely that rare coding variants play roles in the pathogenesis of complex common disease. Do et al. (2012) reviewed key elements in designing and carrying out exome sequencing for complex diseases. They emphasized the importance of having sequence information on populations and especially ethnically matched controls. Study designs that include samples at the phenotypic extremes are often particularly useful. An outstanding example of the use of phenotypic extremes to determine the significance of sequence variants is the study of low-density lipoprotein levels, risk of coronary heart disease, and rare loss of function variants in the PCSK9 gene, which encodes proprotein convertase subtilisin-like/kexin type 9, reported by Cohen et al. (2006). Both loss- and gain-of-function mutations occur in PCSK9. In a study of 3,278 black subjects, Cohen determined that subjects with the lowest levels of LDH cholesterol had the highest frequency of the loss-of-function PCSK9 alleles. PCSK9 protein undergoes autocatalytic cleavage and is then transported to the cell surface. The mature protein binds to the LDL receptor and promotes its degradation. Specific gain-of-function mutations, D374Y, F216L, increase LDLR degradation. These variants lead to genetically determined hypercholesterolemia. Horton et al. (2009) reported that the D374Y PCSK9 mutant protein

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Figure 9–1. This figure illustrates PCSK9 allelic variation and associated LDL levels, based on studies of Cohen et al. (2006).

is 10 times as active as the wild-type protein. Based on these observations, PCSK9 is considered a therapeutic target. Monoclonal antibodies have been developed to reduce activity of gain-of-function mutations.

Approaches to Treatment of Alzheimer’s Disease Familial forms of Alzheimer’s disease are primarily due to mutations in amyloid precursor–processing genes, or to increased production of amyloid precursor proteins. Familial forms and sporadic forms of late-onset Alzheimer’s disease are associated with increased production of the amyloid forms Abeta 42 and 40, with increased production of the enzyme GSK3 (glycogen synthase kinase 3B) and with increased production of phosphorylated forms of Tau. Extensive genome-wide association studies in late-onset Alzheimer’s disease confirm the strong association of Alzheimer’s disease with APOE4; the significance of this association reaches 1 × 10-295. Other risk factors identified in GWASs have average significance values of approximately 2 × 10-14 and are specific alleles in genes in endocytic pathways, PICLAM and BIN1; in lipid-processing pathways, ABCA7; and in inflammation and immune response pathways, CR1, MS4. A specific allele in the clusterin gene CLU is associated with Alzheimer’s disease with significance 5 × 10-16. The product of the CLU

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APPsa APP P3 Alpha secretase

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Figure 9–2. This figure illustrates products of amyloid precursor protein generated through cleavage with alpha, beta, and gamma secretase. Figure drawn by author based on information in Wilquet and de Strooper (2004).

gene is multifunctional; it binds the amyloid Abeta oligomers and possibly promotes their transport (Sullivan et al., 2012). In their review of Alzheimer’s disease, Huang and Mucke (2012) emphasized that interdisciplinary studies have revealed multiple molecular mechanisms that contribute to the development of the disease. Analyses of brain pathology and functional imaging reveal that neuronal loss occurs in specific brain regions, particularly in the entorhinal cortex, temporal lobe, and the CA1 region of the hippocampus. Brain volume loss is primarily due to loss of neuronal processes, and cognitive loss correlates with loss of synapses and dendritic spines. Amyloid typically accumulates in fibrillar plaques; however, plaque load does not correlate strongly with cognitive impairment, and soluble beta amyloid may be damaging. Neurofibrillary tangles derived from Tau protein form, and Tau accumulation correlate strongly with the degree of cognitive impairment. Huang and Mucke consider Alzheimer’s disease to be the result of complex interactions between genetic, epigenetic, and environmental factors. Genetic factors for familial early-onset disease include autosomal-dominant

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Alzheimer’s disease due to duplications of the gene that encodes amyloid precursor protein or mutations in the presenilin genes PS1 and PS2 that encode enzymes involved in processing of amyloid precursor protein APP. Jonsson et al. (2012a) studied coding variants in the amyloid beta precursor gene in 1,795 individuals in the Icelandic population. They identified a specific coding mutation, A673T, which protects against cognitive decline and Alzheimer’s disease in the elderly. They determined that this substitution alters cleavage of amyloid precursor protein (APP) by the beta secretase enzyme. The protective effect of this allele supports the hypothesis that reducing cleavage of APP by beta secretase will be protective against Alzheimer’s disease (Jeppson et al., 2012). A specific allele of the apolipoprotein E gene, APOE4, is a major contributor to late-onset Alzheimer’s disease and should be considered as a semidominant risk allele. There is evidence that APOE4 has an amyloid beta–independent role in Alzheimer’s disease; it impacts brain activity and mitochondrial function before the onset of amyloid beta (Abeta) accumulation. Filippini et al. (2009) investigated effects of the APOE4 allele in healthy young adults. Brain activity at rest and during a memory skill test were analyzed using blood oxygen level– dependent (BOLD) functional magnetic resonance imaging. They determined that the memory produced greater hippocampal activation in APOE4 carriers. They concluded that the APOE4 allele impacts brain function before onset of neurodegeneration. APOE4 protein is more susceptible to proteolytic cleavage than the proteins encoded by the other APOE alleles. Huang (2010) reported that the APOE4 protein increases Tau phosphorylation and generation of neurotoxic Tau species. There is evidence that excess amyloid beta enhances misfolding and accumulation of alpha synuclein in mice. There is also evidence that APOE4 enhances accumulation of the TDP43 (DNA binding protein) in patients with frontotemporal dementia. Higashi (2007) reported that accumulations of alpha synuclein and TDP43 protein are present in a large proportion of patients with Alzheimer’s disease.

TARGETING APOE4 The APOE4 allele is present in 40–65% of cases of Alzheimer’s disease. Huang and Mucke (2012) searched for small molecules that abolish the negative effects of APOE4 on neurite outgrowth and mitochondrial mobility. Using the Chembridge library of small molecules, they identified a phthalazinone

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derivative that restored mitochondrial cytochrome C levels to normal and restored mitochondrial mobility. Another approach they suggested was the use of humanized monoclonal antibodies specific for neurotoxic APOE4 fragments. Chen et al. (2012) reported that specific small molecules corrected the detrimental effects of apoplipoprotein E4 in cultured neurons. They noted that many clinical trials are carried out in Alzheimer’s patients with advanced disease, and treatment at that stage may not be able to reverse the pathologies. Furthermore, heterogeneity within the patient population may obscure beneficial effects. Reiman et al. (2011) proposed carrying out prevention trials in two groups, one group with known presenilin mutations and a second group of APOE4 homozygotes. Treatment in each group would commence close to their estimated average age of onset of symptoms. The best biomarkers to monitor therapeutic efficacy include PET scanning to assess fibrillar amyloid beta burden, and analysis of CSF to search for low-CSF amyloid beta 42 and high levels of CSF phosphotau. Fleisher et al. (2012) investigated the APOE gene and effects of the APOE4 allele and aging effects on the accumulation of fibrillar amyloid using florbetapir F18 PET (positron emission tomography). They carried out studies in 86 young controls; 61 older, healthy volunteers; 53 individuals with mild APOE4

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Figure 9–3. This figure illustrates structural and domain interaction differences between the APOE3 and APOE4 proteins and a small molecule that inhibits the abnormal domain interactions of APOE4. Figure drawn by author based on information in Huang (2010).

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cognitive impairment, and 45 patients with Alzheimer’s disease dementia. They reported that cerebral amyloid deposition is associated with APOE4 carrier status in older healthy controls and in symptomatic Alzheimer’s disease patients. Importantly, they established that amyloid imaging positivity appears to begin around 56 years of age in cognitively intact APOE4 carriers and at 76 years of age in APOE4 noncarriers.

TARGETING TAU, TAU PHOSPHORYLATION, AND GSK3B Lasagna-Reeves (2012) developed an antibody specific for Tau oligomers. They determined that levels of Tau oligomers were 4-fold higher in Alzheimer’s brain tissue than in control brain tissue. The microtubule-associated protein Tau accumulates in neurons in the somatodendritic and axonal domains. Three stages of Tau accumulation occur. The first involves the formation of pretangle phosphotau aggregates, a second phase involves formation of intraneuronal neurofibrillary tangles, and in a later stage extraneuronal tangles form. Hyperphosphorylation of Tau increases its capacity for aggregation. Lasagna-Rees et al. reported that in the Tau protein there are 30 potential phosphorylation sites. In Tau present in neurofibrillary tangles, most of the available sites are phosphorylated. A number of different kinases are involved in Tau phosphorylation, including GSK3B, Map kinase (MAPK), and CDK 2 and CDK5 cyclin-dependent kinase. There are conflicting reports in the literature concerning the importance of Tau aggregates and neurofibrillary tangles in neurodegeneration. Based on their studies, Lasagna-Reeves et al. proposed that Tau oligomers are present in early Alzheimer’s disease brain tissue, and they increase progressively to form neurofibrillary tangles. They proposed that Tau oligomers be considered as therapeutic targets. Takashima et al. (2010) reported that amyloid beta activates GSK3B and that activated GSK3B induced hyperphosphorylated Tau, formation of neurofibrillary tangles, and neuronal death. They proposed that therapeutics be developed that reduce GSK3B activity. Onishi et al. (2011) developed a GSK3B inhibitor, MMBO, that has high selectivity for GSK3B and demonstrated that this compound reaches the brain following oral administration. They demonstrated that MMBO inhibited Tau phosphorylation. In a transgenic mouse model, they demonstrated that MMBO decreased hippocampal Tau phosphorylation and significantly improved memory and cognition.

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MOUSE MODELS OF ALZHEIMER’S DISEASE AND TREATMENT STUDIES LaFerla (2012) emphasized that a number of different Alzheimer’s disease treatments that have been successful in mice have failed in clinical trials in humans. He noted that one possible explanation is that the mutant mice harbor amyloid pathology but lack the other pathologies typical of Alzheimer’s disease. He emphasized that Alzheimer’s disease is a complex disorder and will likely require multiple therapeutic interventions.

NONPHARMACOLOGICAL APPROACHES TO TREATMENT OF ALZHEIMER’S DISEASE Important approaches to prevention and perhaps even to treatment of cognitive impairment associated with aging include exercise to enhance brain health and plasticity. Cotman and Berchtold (2002) in defining the concept of brain plasticity included structural changes in the brain at the cellular, molecular, and systems levels. They emphasized the importance of behavioral stimulation and exercise in maintaining brain health and plasticity. There is evidence that physical exercise from midlife on reduces the risk of age-related cognitive impairment, dementia, and Alzheimer’s disease (Laurin et al., 2001). Studies in humans and animals have demonstrated that exercise and behavioral enrichment increase neuronal survival and vascularization and stimulate neurogenesis (Carro et al., 2000; van Praag et al., 1999, 2002). Cotman and Berchtold (2002) undertook studies on mice to examine the effects of voluntary exercise. They determined that exercise induces expression of genes that play roles in brain plasticity, particularly brain-derived neurotrophic factor (BDNF). In studies on cultured neuronal cells, they determined that BDNF promotes neurite extension and survival of neurons in culture. They reported that intraventricular BDNF infusion protected the hippocampus and cortex from ischemic damage. Furthermore, BDNF potentiates synaptic transmission and increases levels of cyclo-oxygenase 2, vaso-active intestinal peptide, synaptotagmin, syndecan, vascular endothelial growth factor, and mismatch repair protein. They determined that exercise induces synthesis of peripheral factors that also play roles in upregulating BDNF gene expression. These factors include steroid hormones and insulin-like growth factor. Exercise also increased expression of other genes that enhance brain plasticity.

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TREM2 Gene and Dementia Homozygous mutations in the TREM2 gene give rise to an early-onset dementia associated with bone dysplasia and cysts referred to Nasu-Hakola disease. Montalbetti et al. (2005) published evidence of subclinical manifestations, including visuospatial memory deficits, in individuals who were heterozygotes for TREM2 mutations. There is new evidence that heterozygous variants in the coding sequence of this gene lead to increased risk for late-onset Alzheimer’s disease. Pekkarinen et al. (1998) described linkage of Nasu-Hakola disease to chromosome 19q13.1 and to markers that flank the DAP12 (TYROBP) gene. Studies by Paloneva et al. (2002) determined that in other families with this disease, the disease gene segregated with a mutation in the TREM2 gene on chromosome 6p12.2. Takahashi et al. (2005) demonstrated that TREM2 protein is expressed on microglia and that stimulation of this receptor induced phosphorylation of DAP12 and downstream signaling. This signaling led to cytoskeleton reorganization and increased phagocytosis. Knockdown of TREM2 expression in microglia led to inhibition of phagocytosis. On the basis of these findings, Takahashi et al. proposed that TREM2 deficiency led to impaired clearance of apoptotic neurons and played a role in causation of brain degeneration in Nasu Hakola disease, also known as polycystic lipomembranous sclerosing leukoencephalopathy (PLOSL). Klesney-Tait et al. (2006) reviewed structure and function of the TREM receptor family cluster and signal integration. The TREM gene cluster comprises four TREM genes and two TREM-like genes on chromosome 6p21.1, and this cluster lies approximately 6 megabases downstream of the major histocompatibility locus HLADQ. TREM genes have an extracellular and a transmembrane region and a short cytoplasmic region. The intermembrane and short cytoplasmic regions of TREM 1,TREM 2, and TREM 3 associate with DAP12 (TYROBP). TREM receptor activation leads to phosphorylation of tyrosine residues in the ITAM domain of DAP12 through activity of SRC kinases. Phosphorylation of cellular scaffolding molecules, recruitment of phosphatidyl-inositol-3OH-kinase and phospholipase, downstream activation of AKT protein kinase, and rearrangement of the actin cytoskeleton follow DAP12 phosphorylation. TREM1 is expressed on neutrophils and monocytes and amplifies inflammatory signaling. TREM2 is expressed on macrophages and microglia, and activation of TREM 2 potentiates phagocytosis (Klesney Tait et al., 2006). TREM2-related dementia has been reported in cases that do not have bone cysts. Chouery et al. (2008) described a Lebanese family with early-onset

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Figure 9–4. A schematic representation of the TREM2 transmembrane receptor and its association with the DAP2 domain, which is the docking site for a protein kinase. Figure drawn by author on the basis of information in Neumann and Daly (2012).

dementia, and affected members showed deletion of a TREM2 consensus donor splice site in intron 1.

TREM2 VARIANTS IN LATE-ONSET ALZHEIMER’S DISEASE There is evidence from the studies of Jonsson et al. (2012b) and Guerreiro et al. (2012) that rare variants in the coding sequence of TREM2 increase the risk of late-onset Alzheimer’s disease. Jonsson et al. noted that rare variants that increase the risk of late-onset disease are more common in the general population than in the older members of the population who are without that disease. They determined that a coding variant in TREM2 rs75932628T leading to amino acid change R47H occurred with a frequency of 0.63% in the general population in Iceland. In cognitively intact population controls >85 years of age, this variant occurred with a frequency of 0.31%. They then analyzed data in an Alzheimer’s disease cohort of 2,037 individuals and in 9,227 controls from the United States, Germany, Norway, and the Netherlands and determined that there was a highly significant association between rs75932628T and Alzheimer’s disease, p = 2.1 × 10-12. The population frequency of rs75932628T in Iceland, 0.63%, is much lower than the frequency of the APOE4 allele, which is 17.3%. However, the alteration of risk for the two rare alleles is similar. The risk odds ratio is 2.92 for rs75932628T in TREM2 and 3.08 for APOE4. In a comprehensive GWAS sequencing analysis of TREM2 in Alzheimer’s disease patients and controls, Guerreiro et al. discovered significantly more

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coding variants in patients than in controls. The rs75932628T R47H variant was highly associated with Alzheimer’s risk (p < 0.001). They then carried out direct sequencing of TREM2 in 1,887 patients and 4,061 controls and determined that sequence variants in TREM2 (including variants other than R47H) occurred significantly more frequently in cases than in controls. Guerrero et al. analyzed Trem2 expression in brain samples from control mice and in a mouse model of Alzheimer’s disease and determined that expression levels were higher in the Alzheimer’s mice. In 2005 Montabetti et al. reported that heterozygotes for the TREM2 mutations that cause Nasu Hakola disease showed deficits in visuospatial memory. Neumann and Daly (2012) noted that TREM2 is expressed in the cell membrane of dendritic cells, osteoclasts, macrophages, and microglia. They reported that the specific ligand for TREM2 is not known but that TREM2 does bind to lipopolysaccharides of bacteria. Following activation, TREM2 signals through DAP2 (TYROBP) and this signaling promotes phagocytosis. They suggested that dysfunction in microglial phagocytosis or inflammatory pathway dysfunction occurs in Alzheimer’s disease. Guerreiro et al. (2012) reported that TREM2 levels in brain rise in parallel with cortical levels of beta amyloid. They propose that compromised function of TREM2 impacts clearance of cell debris and removal of beta amyloid in Alzheimer’s disease. In 2009 Stefano et al. reported that the chaperone HSP60 bound TREM2 at the surface of cultured neuroblastoma and astrocyte cells. HSP60 stimulated phagocytosis in microglial cells with TREM2 receptors. These findings provide new insight in Alzheimer’s pathogenesis and may open new avenues for treatment. Adult Neurogenesis and Synaptic Plasticity: Relevance to Neurodegenerative Diseases Earlier literature emphasized the importance of synaptic transmission, synaptic contacts, and gene expression as components of brain plasticity in adults. Subsequently, evidence has accumulated that supports neurogenesis in adult brain (van Praag et al., 2002). Winner et al. (2011) reported that adult neurogenesis involves several steps. These include asymmetric division of stem cells in the subventricular zone, olfactory bulb, hippocampus, and dentate gyrus. Following asymmetric division, one of the daughter cells develops into a neuroblast that then migrates and becomes integrated in neural pathways. Winner et al. noted that in a number of neurological diseases, including Alzheimer’s disease, Parkinson’s disease, and Huntington’s chorea, neurogenesis is impaired.

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Vivar et al. (2012) reported evidence that exercise was highly correlated with enhanced hippocampal and dentate gyrus neurogenesis, increased synaptic activity, increased production of BDNF, and improvements in learning and memory. AMYOTROPHIC LATERAL SCLEROSIS: POSSIBILITIES TO THERAPEUTICALLY TARGET DOWNSTREAM EFFECTS OF SPECIFIC MUTATIONS Mutations in more than 16 different genes lead to amyotrophic lateral sclerosis (ALS) (Otomo et al., 2012). ALS is associated with progressive degeneration of motor neurons in the spinal cord and brain with relative sparing of sensory neurons. In many cases specific gene mutations have not been identified. In 2011, Renton and coworkers discovered a hexanucleotide repeat expansion at a specific open reading frame locus on chromosome 9p21, designated C9ORF72, in sporadic ALS patients. Millecamps et al. (2012) carried out studies on 950 French patients with ALS. They reported that the C9ORF72 hexanucleotide repeat expansions occurred in 46% of cases of familial ALS and 8% of cases of sporadic ALS. Phenotypic comparisons in ALS patients with mutations in different genes revealed that the C9ORF72 repeat expansion patients presented more frequently with bulbar motor insufficiency. Frontotemporal dementia was also more common in this group. Furthermore, disease duration from diagnosis to death was shorter. Studies on brain samples from cases with C9ORF72 repeat expansions revealed that intracytoplasmic inclusions of the protein TDP43 (DNA–RNA binding protein) were present in several brain areas, particularly in the temporal lobe, the parietal lobe, hippocampus, and cerebellum. TDP43 inclusions occur in ALS due to a number of different gene mutations. In rare cases of ALS, disease is due to mutations in the TDP43 gene. In a study of familial ALS patients in the Netherlands, Simon-Sanchez et al. (2012) reported that C9ORF72 repeat expansion were present in 28.7% of cases of familial ALS. Lattante et al. (2012) studied 480 sporadic ALS patients and 48 cases of familial ALS in an Italian referral center. In the sporadic cases, TARDBP (TDP43) mutations were found in 2.7%, C9ORF72 in 2.5%, and SOD1 mutations in 2.1%. In familial cases of ALS, mutations were detected in 43.7%. The downstream effects of these mutations include induction of oxidative stress, endoplasmic reticulum stress, mitochondrial dysfunction, protein misfolding, abnormal protein trafficking, abnormal RNA processing, and neural inflammation.

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Results from initial clinical trials indicate that a mitochondrial modulator, dexpramipexole, is useful in the treatment of amyotrophic lateral sclerosis (Rudnicki et al., 2012). It was well tolerated and shown to be safe in humans studies. This compound impacts mitochondrial membrane conductance and it improves oxidative phosphorylation. Zebra fish are utilized as a model system to identify interacting proteins that modify the effects of a specific mutant gene. Zebra fish have been utilized as a model of ALS. Sakowski et al. (2012) reported effects of the SOD1 (superoxide dismutase) mutation G39A. This mutation occurs in some cases with familial ALS. In Zebra fish it impacts motor neuron outgrowth and axonal branching. Van Hoecke et al. (2012) reported that reduction or pharmacological inhibition of e ephrin type A receptor (EPHA4) signaling increased survival in zebra fish and in mouse and rat models of ALS. They carried out studies on zebra fish with a specific ALS-associated TDP43 mutation and reported that reduction of EPHA4 had a positive effect. EPHA4 encodes a subclass of receptor tyrosine kinases. Ephrin ligands and their cognate receptors have been shown to play key roles at synapses. Hruska and Dalva (2012) reported that interactions of ephrins with their receptors induce synapse formation and spine morphogenesis in the developing nervous system. They reported that in the mature nervous system ephrin signaling modulated synaptic function and synaptic strength. Van Hoeke et al. (2012) reported that loss-of-function mutations in EPHA4 were associated with longer survival in ALS patients. They concluded that expression of the ephrin signaling system increased vulnerability of neurons to degeneration. Pharmacological inhibition of ephrin signaling has also been achieved. Noberini et al. (2008) identified a series of small-molecule inhibitors of binding of ephrin to receptors.

STIMULATION OF EXCITATORY AMINO ACID TRANSPORTER EAAT2 EXPRESSION TO PREVENT GLUTAMATE TOXICITY AND NEURODEGENERATION Glutamatergic neurons occur particularly in the cerebral cortex and limbic region, and glutamate is the primary excitatory amino acid. Kim et al. (2011) reported that the concentration of glutamate in the synaptic cleft must be closely regulated. This regulation is, in part, dependent on the function of astrocytes located in close proximity to synapses. Glutamate binds to ionotropic

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postsynaptic neurons. This binding leads to movement of calcium and sodium into the cells and to depolarization. The concentration of glutamate in the synaptic cleft is regulated in part by the activity of glutamate transporters, including five subtypes of the EAAT-excitatory amino acid transporters. These transporters are located in cell membranes and function as membrane pumps similar to ion channels. They are symporters, and substrate transport is linked to transport or countertransport of ions; EAAT2 and EAAT3 are expressed throughout the brain in humans. EAAT1 and EAAT4 are predominantly expressed in the cerebellum but are also expressed in other brain regions. In a number of different neurodegenerative diseases, there is excessive accumulation of glutamate in the synaptic cleft. EAAT2 is also known as solute carrier SLC1A2. A specific mutation in this gene that leads to decreased expression occurs in some patients with amyotrophic lateral sclerosis. Since EAAT 2 is widely expressed in postsynaptic neuronal terminals and astrocytes, Kim et al. (2010) undertook studies on the regulation of expression in neuron and astrocyte cell cultures. They demonstrated that NFkappaB plays an important role in the control of EAAT2 gene transcription. Activation of EAAT2 transcription was dependent on the binding of the transcriptional regulator NFKB to the gene promoter. Kim et al. then analyzed the effects of 1,000 FDA-approved drugs and nutritionals on EAAT2 gene expression. They demonstrated that 15 different beta lactam antibiotics stimulated EAAT2 expression. In studies on animals, they demonstrated that the beta lactam antibiotic ceftriaxone increased brain glutamate transport and prevented glutamate toxicity. Kim et al. emphasized that their screening paradigm was useful in identification of neuroprotective drugs. Accumulation of Tau Protein in Neurodegenerative Disorders In a number of different neurodegenerative disorders, including Alzheimer’s disease, frontotemporal dementia, and Parkinson’s disease, hyperphosphorylated forms of Tau and Tau aggregates accumulate in the form of paired helical filaments or neurofibrillary tangles. Miyata et al. (2011) reviewed information on Tau physiology and pathology. Abnormal accumulation of Tau occurs in association with neuronal loss. In rare individuals, a point mutation in the Tau gene on chromosome 17 leads to frontotemporal dementia and to Parkinsonism. The normal function of Tau protein is to stabilize microtubules that play key roles in axonal transport. Microtubule binding repeats occur in Tau protein, and the number of these repeats differs in different Tau isoforms. The binding of Tau to microtubules is also impacted by Tau phosphorylation. Tau

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phosphorylation reduces its affinity for microtubules. This modification utilizes a number of different kinases, particularly GSK3B, CGK5, and MARK2. Dephosphorylation of Tau through activity of protein phosphatase enzymes encoded by PP2A and PP5 restores Tau binding to microtubules. Proteolytic processing of Tau generates fragments that are more prone to hyperphosphorylation and aggregation. TREATMENT STRATEGIES FOR TAU ACCUMULATIONS (TAUOPATHIES) Approaches to treatment of abnormal Tau accumulation include use of kinase inhibitors (e.g., GSK3 inhibitors). Another treatment strategy is based on the observation that diminished protein quality control leads to Tau accumulation. HSP70 and HSP 90 heat shock proteins play key roles in protein quality control. HSP expression is regulated by heat shock factor 1 (HSF1). Function of heat shock proteins includes regulation of protein folding, trafficking, and assembly of multiprotein complexes. HSP chaperones, particularly HSP90 and HSP70, also facilitate the clearance of degraded and aggregated protein through the ubiquitin proteosome system and autophagy. HSP70 cochaperones, referred to as HSP40 or as J proteins, encoded by DNAJ genes also play important roles in control of protein folding. Proteins may become unfolded as a result of cellular stress induced by oxygen radicals and nitrosylation. The HSP70 chaperone, working together with its cochaperone, J protein, plays a particularly important role in this refolding (Kampinga and Craig, 2010). A number of different compounds have been identified that impair heat shock protein function and are used in cancer therapy to promote degradation of overexpressed growth factors and oncoproteins. However, proteins that increase HSP activity were not available in 2012.

Identifying Therapeutic Targets in Autism Autism is associated with impairments in communication and reciprocal social interaction and repetitive behaviors and patterns of interest; 70% of autism patients have intellectual disability and 25% have seizure disorders (Zoghbi and Bear, 2012). Key pathologic findings in autism include increased brain volume in early childhood as measured by MRI. Specific brain regions that show

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changes include the hippocampus, anterior cingulate, prefrontal cortex, and cerebellum. In contrast to other areas, the cerebellar vermis is often decreased in size and Purkinje cell loss has been documented. Diffusion tension imaging shows altered white matter volume and altered patterns of connectivity (Darnell et al., 2011). Genes found to be mutated in autism frequently encode proteins that function at synapses. Penzes et al. (2011) reported alteration in dendritic spine morphology and dynamics in autism spectrum disorders. Treatments for autism in 2012 are primarily directed at symptoms and include antipsychotics that often have significant side effects (Hampson et al., 2012).

MOLECULAR GENETICS IN AUTISM The goal of molecular genetics is to identify specific gene mutations present in cases of autism, and to determine how these mutations lead to functional impairments and contribute to specific disease manifestations either through direct effects or through effects on a specific pathway. Causative gene defects have been identified in a number of syndromic forms of autism, including, fragile X syndrome, Rett syndrome, Angelman syndrome, tuberous sclerosis (TSC1, TSC2), neurofibromatosis (NF1), and Phelan-McDermid syndrome. Specific genes mutated in some cases of autism include the postsynaptic density protein–encoding gene SHANK3, neuroligins (NGLN3 and NGLN4), and neurexin (NRXN1) and ion channel genes including CACNA1C. Genes identified in monogenic cases of autism are active at neuronal synapses. The question arises whether nonsyndromic cases of autism have defects in genes that function at synapses. It is important to note that there is consistent evidence for an increased burden of large genomic structural copy number variants in autism. In a study of autistic probands and their unaffected siblings, Sanders et al. (2011) reported that copy number variants occurred in 5.8% of autistic cases and in 1.7% of their siblings. DNA sequencing studies have revealed an increased rate of de novo exonic mutations in autism spectrum cases (Sullivan et al., 2012). On the basis of analysis of genome-wide association studies Klei et al. (2012), proposed that ASD liability is due to the impact of a myriad of common variants; each variant has very small effect and variants act additively.

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SYNDROMIC FORMS OF AUTISM Fragile X mental retardation protein (FMRP) synaptic function and autism Autism is a frequent feature of fragile X syndrome that is associated with decreased expression of the FMRP protein. Darnell et al. (2011) emphasized that understanding FMRP functions and interactions and the consequences of decreased FMRP expands our insight into mechanisms and pathogenesis of autism. FMRP is a neuronal RNA binding protein that is thought to play a key role in translation regulation. Darnell et al. (2011) undertook studies to identify the specific mRNA targets of FMRP. They determined that FMRP interacts with transcripts that encode presynaptic and postsynaptic proteins, including glutamate signaling pathways; CREB, PI3K, and AKT signaling proteins; and reelin and semaphorin signaling pathway proteins. In addition FMRP interacts with mRNA transcripts of specific proteins that have been shown to be mutant in autism, including Neuroligin 3, Neurexin 1, SHANK3, PTEN, TSC2, and NF1. Based on these studies, Darnell et al. suggested that reduction of the synthesis of FMRP and impaired control of translation of several proteins lead to overexpression of proteins. They determined that 25 of the 196 candidate autism proteins present in chromosome duplications associated with autism are FMRP targets. Genes that map in chromosomal regions deleted in autism were found less likely to be FMRP targets. Darnell et al. determined that 66% of FMRP binding occurred in the coding sequence of target mRNAs and that FMRP binding specifically led to ribosome stalling. They noted that ribosome stalling may have particular relevance in the nervous system where mRNA is often translocated great distances from where it is transcribed. In addition to controlling translation of mRNAs for protein that are active at the synapse, FMRP also controls translation of proteins located elsewhere. FMRP targets are potentially useful as treatment targets in autism. Darnell et al. emphasized that the gene balance hypothesis is important to take into account in considering autism and mental retardation. Homeostasis and synaptic function are impaired by excess of gene products due to duplications, or by reduction of products due to deletions. Mental retardation occurs as a result of FMRP reduction in fragile X syndrome, and it also occurs in cases of duplication of the Xq27-q28 duplication that encompasses the FMR1 gene. One treatment modality to fragile X syndrome is to partially inhibit the metabotropic glutamate receptor mGluR5 to reduce effects of increased

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synthesis. Another mode of therapy is to inhibit the enzyme matrix metalloproteinase MMP, which is present in high amounts in cells depleted in FMRP. Inhibition of MMP with minocycline is being investigated in clinical trials (Zoghbi and Bear, 2012). Some cases of fragile X syndrome respond to activators of GABA receptors. The selective GABA receptor agonist AR-baclofen has yielded promising results in early phase 3 trials. AR-baclofen was specifically reported to increase sociability (Henderson et al., 2012). There is evidence that three specific proteins, UBE3A, ARC, and FMRP, interact at the synapse and that increased or decreased expression of any one of these proteins impacts homeostasis and synaptic function. UBE3A (ubiquitin ligase 3A) encoded on chromosome 15q13.2 regulates excitatory synaptic activity through control of the quantity of ARC (activity-associated cytoskeleton-associated protein encoded on 8q24.3). Increased UBE3A leads to increased degradation of ARC. Greer et al. (2010) reported that ARC promotes internalization and endocytosis of AMPA receptors. Decrease in UBE3A leads to increased expression of ARC and to increased AMPA neurotransmitter receptor internalization and impaired synaptic activity.

TUBEROUS SCLEROSIS AND AUTISM Zoghbi and Bear (2012) emphasized evidence of the importance of deletions or mutations in the TSC1 or TSC2 genes in affecting activity at the synapse. Deletions or mutations in these genes lead to increased mTOR activity, which is associated with increased synthesis of synaptic proteins and enhanced long-term potentiation of synaptic activity. There is now evidence that abnormalities of TSC gene function lead to impaired synaptic function and to brain dysfunction independently of tuber formation. Bateup et al. (2011) deleted TSC1 in hippocampal CA1 neurons in postnatal mice using a viral delivery system and gene targeting. They determined that TSC1 is required for specific forms of protein translation–dependent synaptic plasticity. The TSC1 deletions in this study led to aberrations in synaptic plasticity independently of alterations in dendritic spines. They determined that there were increased glutamatergic responses, and increases in AMPA receptor and NMDA receptor currents following TSC1 gene knockout and that these alterations led to hyperexcitability of cortical circuits. Bateup et al. concluded that their findings support the concept that perturbations in protein translation–dependent forms of synaptic plasticity may be common to a number of different forms of autism.

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MECP2 DELETIONS, RETT SYNDROME, AND AUTISM Deletions of the gene that encodes MECP2 (methyl CpG–binding protein) lead to Rett syndrome in females. This syndrome is associated with loss of language and social responsiveness after 2 years of age, with abnormal hand movements, progressive autonomic nervous system malfunctions, and seizures. The MECP2 protein plays an important role in control of gene expression. MECP2 protein silences some genes; however, it activates expression of others (e.g., BDNF). Loss of MECP2 protein impairs electrical activity and passage of calcium ions at synapses (Zoghbi and Bear, 2012). Approaches to the treatment of Rett syndrome include use of drugs to enhance BDNF expression and also use of chromatin-modifying agents.

NEUROTRANSMITTERS AND ION CHANNELS IN AUTISM BDNF controls intracellular chloride levels through activity of K+ CL− cotransporters including KCC2 (Rivera et al., 2002). There is evidence that GABA neurotransmitter functions are dependent on intracellular chloride levels. Furthermore, GABA-ergic neurons play key roles in generating gamma and other high-frequency oscillations that are related to higher cognitive functions. Lemonnier et al. (2012) reported that these oscillations are reduced in autism. They carried out a double-blind clinical trial in children with autism using a chloride import antagonist, bumetanide. This compound is approved for clinical use as a diuretic. They reported that this treatment improved emotional perceptions and social interactions in children with autism. Inborn Errors of Metabolism That Increase Autism Risk It is important to emphasize that inborn errors of metabolism may lead to autism and these disorders may be treatable. In some cases they are diagnosed through analysis of metabolites in blood or urine. More recently exome sequencing in cases of autism has led to discovery of mutations in genes that encode products involved in metabolism. Novarino et al. (2012) carried out whole-exome sequencing in members of two consanguineous families. They refined their search of sequence data from each family using Homozygosity Mapper software. In each family they identified a homozygous null mutation in the branched-chain ketoacid dehydrogenase kinase gene BCKDK. In a broader study, they subsequently identified another family with an autistic proband with BCKDK gene mutations.

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They developed a mouse model of BCKDK deficiency and demonstrated that feeding these mice with chow supplemented with 2% branched-chain amino acids led to normalization of the phenotype. Untreated mice had seizures and hind limb clasping. They also demonstrated that in patients with BCKDK deficiency, supplementation of diet with branched-chain amino acids led to normalization of the levels of branched-chain amino acids. In untreated patients the plasma levels of these amino acids were abnormally low. The branched-chain keto acid dehydrogenase complex is mitochondrially localized. It catalyzes the second step in the catabolism of the branched-chain amino acids leucine, isoleucine, and valine. This complex is composed of three catalytic components: E1, a heterodimeric alpha 2 beta 2 branched-chain alpha keto-acid decarboxylase; E2, a dihydrolipoyltransacylase; and E3, a lipoamide dehydrogenase (see also Chapter 3 of this volume). Branched-chain keto acid dehydrogenase kinase phosphorylates serine and threonine subunits in the E1alpha subunit of the BCKD complex. Phosphorylation inactivates the E1alpha subunit (Harris et al., 2005). Branched-chain amino acids are toxic in excess, but adequate quantities are necessary for protein synthesis. Harris et al. reported that activity of BCKD complex is achieved by phosphorylation and dephosphorylation.

CH3 NH2 CH3 - CH2 - CH - CH - COOH

CH3 O CH3 - CH2 - CH - C - COOH

CH3 CH3 - CH2 - CH - CO - CoA

ISOLEUCINE

2-OXO-3-METHYLVALERIC ACID

2-METHYLBUTYRYLCoA

CH3

CH3

CH3

NH2 CH - CH2 - CH - COOH

CH3

CH3 LEUCINE

CH3

O CH - CH2 - C - COOH

NH2 CH - CH - COOH

CH3

2-OXOCAPROIC ACID

CH3

O CH - C - COOH

CH3 VALINE

CH - CH2 - CO - CoA CH3

2-OXOVALERIC ACID

ISOVALERYLCoA

CH3 CH - CO - CoA CH3 ISOBUTYRYLCoA

Branched-chain ketoacid dehydrogenase complex deficiency impacts metabolism of isoleucine leucine and valine. The complex is composed of E1 with alpha and beta subunits, an alpha ketodehydrogenase, E2, a dihydrolipoyltransacylase, and E3 lipoamide dehydrogenase. Cofactors: thiamine TPP, FAD, NAD, lipoate, and coenzyme A.

Figure 9–5. This figure illustrates the role of the branched-chain keto dehydrogenase complex in metabolism of branched-chain amino acids. Figure drawn by author based on information in Harris et al. (2005) and Voet and Voet (1995).

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If phosphorylation and deactivation of E1A are defective as is the case with BCKDK deficiency, excess activity of the BCKD complex leads to reduced levels of branched-chain amino acids. Celestino-Soper et al. (2012) demonstrated that deficiency of carnitine biosynthesis due to defects in the enzyme trimethyllysine hydroxylase epsilon, TMLHE, also known as trimethyllysine dioxygenase, led to autism in males. They reported that low carnitine levels in the diet make males with TMLHE defects particularly prone to development of autism. TMLHE deficiency may be due in some cases to point mutations. However, deletions of exon 2 of this gene that map to the X chromosome are relatively common in males in the general population, 1 in 366. However, in the population of males in multiplex autism families, the frequency of TMLHE exon 2 deletions was increased 2.82-fold. Celestino-Soper et al. proposed that TMHLE deletion has a low penetrance; however, additional factors related to dietary intake of carnitine, carnitine transport, and carnitine loss through diminished renal tubular reabsorption may combine to increase risk of autism. They proposed that the carnitine pathway might provide a therapeutic target in autism. Carnitine is synthesized from amino acids lysine and methionine. N-methylation of lysine residues utilizes S-adenosylmethionine as methyl donor (Strijbis et al., 2010). Carnitine is also derived from diet. The organic cation transporter OCTN2 (SLC22A5) transports carnitine across cell membranes. Since institution of widespread newborn screening, it has become evident that systemic carnitine deficiency has a range of different clinical presentations, ranging from mild to severe. Severe systemic carnitine deficiency has a clinical presentation similar to the of fatty acid oxidation defects, and patients may present with hypoketotic hypoglycemia, and skeletal and cardiac myopathy. DNA sequencing has revealed a broad spectrum of OCTN2 mutations that lead to systemic carnitine deficiency (Li et al., 2010). Other solute carriers are also involved in carnitine transport.

Identifying Therapeutic Targets in Autism Associated With Predisposing Chromosome Variants A key question that arises in cases in which multiple genes are impacted by deletion or duplication is whether one specific gene is of key importance in the generation of the predominant phenotypic feature (e.g., autism) and whether that gene could represent a phenotypic target. Animal models of specific chromosome abnormalities are utilized to study these questions.

Identifying Therapeutic Targets in Complex, Multifactorial Diseases CH3

179

H O

CH3 - N - CH2 - CH2 - CH2 - CH2 - C - C - OH CH3

NH2

N6 TRIMETHYLLYSINE (TML) TRIMETHYLLYSINE HYDROXYLASE EPSILON (TMLHE) 3-HYDROXY-N6 TRIMETHYLLYSINE (HTML) HTML ALDOLASE

4-N-TRIMETHYLAMINOBUTYRALDEHYDE (TMABA) TMABA DEHYDROGENASE 4-N-TRIMETHYL AMINOBUTYRATE (BUTYRYLBETAINE BBD) BBD DEHYDROGENASE CARNITINE CH3

OH

O

CH3 - N - CH2 - CH - CH2 - C - OH CH3 CARNITINE IS SYNTHESIZED FROM AMINO ACIDS LYSINE AND METHIONINE. N-METHYLATION IS CATALYZED USING S-ADENOSYL METHIONINE AS METHYL DONOR.

Figure 9–6. This figure illustrates the pathway of carnitine synthesis and the role of the TMLHE product (trimethyllysine hydroxylase epsilon). Drawn by author on the basis of information in Vaz and Wanders (2002).

An important location of recurrent copy number variants in autism is chromosome 16p11.2. Deletion of a 600-kb region within 16p11.2 is associated with a number of different psychiatric diagnoses, and in addition with abnormal growth, possibly due to aberrations in energy metabolism (Zufferey et al., 2012). The growth pattern in cases of 16p11.2 deletions is similar to that observed in Prader-Willi syndrome, which results from dosage changes in 15q13.3. Infants frequently have low birth weight; however, at around 3 years of age there is a sudden increase in weight and in body mass index. Infants also manifest increased head circumference. The mirror change of deletion is duplication of this region, and patients with duplication manifest growth retardation and low weight. These patients may also have developmental delay, language delays, speech abnormalities,

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and seizures (Rosenfeld et al., 2010). It is, however, important to note that there are individuals with 16p11.2 deletions and individuals with 16p11.2 duplications who do not manifest symptoms (Shinawi et al., 2010). Detailed studies in patients with an apparently uniform 16p11.2 deletion revealed that they have a range of psychiatric diagnoses. Autism spectrum disorders occur in 11.4% of patients; other DSM (Diagnostic and Statistical Manual of Mental Disorders) diagnoses besides autism include attention deficit disorder, disruptive behavior disorder, anxiety and mood disorders, and substance-related disorders; 20% met DSMIV criteria for intellectual disability, and verbal IQ was often significantly lower than nonverbal IQ. In 15.7% of these patients, there are no psychiatric manifestations. Twenty-seven protein-coding genes map in this 600-kb region on chromosome 16p11.2. It is interesting to note that many of these genes are involved in signal transduction processes and several are particularly abundantly expressed in brain. The KCTD13 gene plays an important part in brain development. Studies in mice have revealed that depletion of the product of this gene leads to increased proliferation of cells in the brain. Studies in zebra fish revealed that KCTN13 depletion led to macrocephaly. In some patients with 16p11.2 deletion, vertebral anomalies occur. The TBX6 gene, which maps in the 600-bp deletion region, is of interest in this regard since polymorphisms in this gene are associated with scoliosis in humans and Tbx6 deficiency leads to vertebral anomalies in mice. Based on their modeling studies in zebra fish, Golzio et al. (2012) reported that KCTD13 is a major driver of the neuroanatomical phenotype in the 600-kb 16p11.2 deletion. Steinberg et al. (2012) identified an SNP variant in 16p11.2 associated with schizophrenia (p = 6.6 × 10-11). This variant maps in the serine threonine kinase protein TAOK2. This gene impacts the function of basal dendrites and axonal projections in cortical pyramidal neurons and is essential for dendrite morphogenesis (de Anda et al., 2012). .

Psychiatric Disorders In recent decades studies have been undertaken to examine genetic and genomic changes in psychiatric disease with the hope that understanding of these changes will lead to better treatments. Key studies have involved genome-wide association studies and studies on larger structural genomic changes. DNA sequencing studies have also been initiated. Sullivan et al. (2012) reported that replicated findings have emerged from comprehensive studies and have revealed that alterations in many areas

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of the genome play roles in the etiology of psychiatric diseases. Important new information that has emerged is that the same genes or gene regions are impacted in a number of different psychiatric disorders. Examples include the association of structural variants in specific regions with both autism and schizophrenia. Another important concept that has emerged is that genomic imbalance due to deletions or duplications in a specific region lead to psychiatric manifestations. Chromosome 15q13.3 duplications lead to autism and to attention deficit hyperactivity disorder (ADHD). Deletion in this same region is often associated with schizophrenia. Duplication or deletion of a 600-kb region in 16p11.2 occurs in autism, and duplication of this region has been reported in schizophrenia. Sullivan et al. (2012) reported that deletions in chromosome17q12 34.8 to 36.2 Mb and deletion in 22q11.2 (18.7 to 21.8 Mb) occur in autism and in schizophrenia.

GENOME-WIDE ASSOCIATION STUDIES IN PSYCHIATRIC DISEASES The Psychiatric Genomics Consortium reported GWASs in schizophrenia. In the first phase of the study, 9,394 cases of schizophrenia and 12,462 controls were studied. From results of these studies, 81 loci yielded significant scores and were selected for analysis in 8,000 cases. Sullivan et al. (2012) reported that 14 of the 81 loci reached genome-wide significance scores of 1 × 10-8. The most significant association was with the major histocompatibility locus MHC 1.1 × 10-13. Other highly significantly associated loci were micro RNA MIR137 on 1p21.2 (p = 1.6 × 10-11) and ZNF804A on chromosome 2q32.1. In studies of bipolar disorder on 7,481 cases and 9,250 controls, 34 statistically significant loci were identified, and these were followed up in 4,500 cases. Sullivan et al. noted that associations with three loci reached significance scores, 1 × 10-8. These loci were within neurocan (NCAN) on chromosome 19, which encodes a proteoglycan involved in neuronal cell migration, in CACNA1C, which encodes a subunit of a calcium ion channel gene and in ODZ4. ODZ4, also designated as TENM4 teneurin transmembrane protein 4, regulates oligodendrocyte differentiation and myelination of small-diameter axons (Suzuki et al., 2012). Combined analysis of the schizophrenia and bipolar cases revealed association with significance greater than 2.4 × 10-8 for four loci. The greatest significance was with locus ZNF804A; this locus encodes a DNA binding protein that controls expression of a number of loci, including COMT (catechol O

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methyl transferase). Other significantly associated loci were ANK3 (ankyrin 3) on 10q21, CACNA1C (calcium ion channel subunit), and ITIH3 (protease inhibitor). Sullivan et al. noted that in bipolar disorder cases there are ongoing clinical trials for therapies directed at calcium ion channels. It is important to note that none of the associated loci were loci of major effect. Sullivan et al. concluded that psychiatric disorders are polygenic and due to variation in multiple loci.

TREATMENT OF PSYCHIATRIC DISORDERS Meyer-Lindenberg and Tost (2012) emphasized that despite the wealth of knowledge of neuroscience and pathophysiology of psychiatric disorders, there are few examples of successful therapeutic interventions. They attributed this to the fact that single facets of biology are favored in studies (e.g., specific receptors or specific genes) and integrating systems are less well studied.

NEW EMPHASIS IN PSYCHIATRY: FUNCTIONAL BRAIN NETWORKS The new emphasis derives from evidence that normal brain function requires coordinated activity of neural networks, and that dysfunction of networks leads to psychiatric illness. Emphasis on neural networks emerged from functional magnetic resonance imaging (fMRI) studies carried out under various conditions including resting states. Other technologies applied to analysis of functional connectivity include electroencephalography (EEG), magnetic electroencephalography (MEGG), optical imaging of energy use in the brain, and diffusion MRI, which maps long-distance connections by following water transfer. The Human Connectome Project, funded by the National Institutes of health NIMH, is designed to identify anatomical and functional connectivity in the brain in normal controls and to compare patterns of connectivity in identical and fraternal twins (http://www.humanconnectomeproject.org). Development during infancy and childhood is associated with regional changes in brain networks. There is some evidence that network connectivity patterns differ in children with attention deficit hyperactivity disorders or autism from patterns seen in normal children. Ameis and Szatmari (2012) noted that much emphasis has been placed on dysfunction at the neuronal synapses

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in the etiology of autism. They reviewed neuroimaging and neuropathological studies in autism and concluded that these studies provided evidence that altered structure, activity, and connectivity within complex neural networks play roles in the pathogenesis of autism. They proposed that risk genes for autism alter neural circuits that are involved in socioemotional, visuospatial, and language processing.

NEURAL OSCILLATIONS, SYNCHRONY, AND COGNITION Hameroff (2010) reported that consciousness is characterized by a specific pattern of EEG activity, gamma wave patterns of 30–90 hertz. He noted that this pattern is associated with transient syncytial formation linking dendritic gap junctions. He demonstrated that coupling of dendrites through gap junctions leads to the formation of electrical synapses. Uhlhaas and Singer (2010) reported that oscillations in the high-frequency range (beta and gamma) established synchronization in local cortical networks, while lower frequency oscillations (alpha and theta) establish synchronization over longer distances. They reported that synchronization of beta and gamma activity was abnormal in patients with schizophrenia. Neural oscillations are thought to play a role in the transfer of information. Oscillations are associated with alterations in neuron action potential and can be observed in EEGs. Resting-brain networks have become important areas of investigation. Networks can be investigated with functional MRI based on changes in blood oxygenation with neural activity. They can also be investigated through correlations of neural oscillatory activity in magnetoencephalography (Brookes et al., 2012).

COGNITIVE BEHAVIORAL THERAPY (CBT) This is a form of psychotherapy that is being increasingly adopted and adapted. Butler et al. (2006) reviewed meta-analyses of treatment outcomes of CBT in a range of different disorders. They obtained evidence for large positive effects of CBT in treatment of unipolar depression, generalized anxiety disorders, panic disorder, social phobias, posttraumatic stress disorders, and childhood depressive and anxiety disorders. They reported that CBT was somewhat superior to antidepressants in treatment of adult depression. Information from the Royal College of Psychiatrists in the United Kingdom states that CBT is helpful in the treatment of anxiety, depression,

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social phobias, stress, obsessive-compulsive disorders, posttraumatic stress disorders, bipolar disorders, and psychosis (Blenkiron and Timms, 2012).

GENE–ENVIRONMENT INTERACTIONS IN PSYCHIATRIC ILLNESS Meyer-Lindenberg and Tost (2012) noted that impairments in social interactions are characteristic of schizophrenia and autism. They also emphasized the importance of social processes in recovery from psychiatric illness and that social interaction should be structured to benefit the client. Meyer-Lindberg and Tost emphasize the importance of interaction between environmental and genetic risk factors in the etiology of psychiatric illness. Urban upbringing and migration are associated with development of schizophrenia. Migration adds a number of stresses including social fragmentation and socioeconomic difficulties.

RESILIENCE AND RESISTANCE TO STRESS AND DEPRESSION Studies of factors that enhance resistance to stress and depression indicate the importance of cognitive reappraisal (i.e., the ability to reframe events in a more positive light) (Aldoa and Nolen-Hoeksema, 2010). Factors that foster resilience to stress include engaging in projects or missions that have purpose, improving health through exercise and diet, and developing social and support networks (Southwick and Charney, 2012).

10 APPROACHES TO CANCER TREATMENT

Even in well-known affections advances are made from time to time that render necessary a revision of our accumulated knowledge, a rearrangement of old positions, a removal even of the old landmarks. —William Osler (1895)

INTRODUCTION Studies carried out over recent decades have revealed that cancer-specific mutations frequently impact cell signaling pathways. A question that arises is whether targeting individual signaling molecules is a practical therapeutic strategy. Signaling pathways involved in tumorigenesis, including the RAS, AKT, mTOR, and HIF pathways, impact glucose uptake and metabolic processes such as glycolysis, glutaminolysis, and fatty acid oxidation (Birsoy et al., 2012). Cairns et al. (2011) proposed that alterations in cellular metabolism be considered as representative of the crucial hallmark in cancer. They noted that the three basic requirements for tumor growth are rapid ATP generation for energy, increased macromolecular biosynthesis, and control of redox status. Many studies are ongoing to determine whether specific genetic alterations lead to altered tumor metabolism and whether these genetic alterations involve mutations. Tumors with the same histology often manifest mutations in different genes or have different structural chromosome abnormalities. 185

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The goal of this chapter is to review metabolic changes in tumors and evidence for altered cell signaling and approaches to treatment of tumors based on these changes.

Altered Metabolism in Tumors Specific mutations that lead to altered metabolism and that in turn promote tumor growth, may represent targets for therapeutic intervention. Specific metabolic alterations characteristic of tumors are also relevant as biomarkers in diagnosis and to monitor impact of specific therapies. Increased glucose uptake by cancer cells is used in imaging (FDG PET) of primary and metastatic tumors. FDG is 2-deoxy-2-(18F) fluoro-D-glucose, a glucose analog, with the isotope fluorine-18 substituted for the normal hydroxyl group at the 2’ position in the glucose molecule. It has a very short half-life. Increased activity through the glycolytic pathway may initially be triggered through the relative hypoxia in tumors.

WARBURG EFFECT Warburg (1927) first reported that in tumors glucose is primarily converted to lactate even when oxygen is present at normal adequate levels. This is in contrast to normal metabolism in which glucose is primarily degraded via glycolysis, and products enter the citric acid cycle and oxidative phosphorylation pathway under those conditions. Metabolism in cancer cells is characterized by increased ATP generation through glycolysis, and increased lactate production. In addition there is increased activity in the pentose phosphate pathway, which generates increased levels of NADPH, which in turn promotes macromolecular synthesis. Increased levels of NADPH and of reduced glutathione (GSH) along with increased thioredoxin counteract the reactive oxygen species generated (Cairns et al., 2011). Underlying the metabolic changes in tumors are mutations not only in oncogenes and tumor suppressor genes but also in genes that encode specific enzymes involved in metabolic processes (e.g., isocitrate dehydrogenases).

SIGNALING PATHWAYS AND GLYCOLYSIS Increased activity through phosphatidyl inositol kinase (PI3K) drives AKT1 activity and glycolytic pathway activity. AKT1 further stimulates mTOR activity. This is achieved in part through AKT1 phosphorylation of the tuberous

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sclerosis gene TSC2, leading to inhibition of TSC and release of the inhibitory effect of TSC on mTOR. Increased mTOR levels and function lead to increased ribosome biogenesis, increase protein and lipid biosynthesis, and activation of HIF1. HIF1 enters the nucleus and amplifies transcription of glucose transporters and enzymes involved in glucose metabolism.

PYRUVATE KINASE AND PKM2 IN TUMORS Pyruvate kinases are involved in the conversion of phosphoenolpyruvate to pyruvate and ATP. Pyruvate kinase enzymes catalyze transfer of phosphate from phosphoenolpyruvate to ATP and generate pyruvate. Pyruvate kinase 1 (PK1) encoded by the PKLR gene on chromosome 1q21 is primarily expressed in liver and red cells. This gene generates multiple transcript variants. The PKL isozyme is expressed in liver, kidney, and intestine. Liver levels of this enzyme are low on fasting and high on feeding. Phosphorylation inactivates the PKL isozyme. PKR is expressed in erythrocytes and is encoded by the same gene as PKL, and different promoters are involved in the generation of the two isozymes. PKL has a low affinity for PEP.

Growth factor receptor activated

Cell membrane

P13K AKT MTOR HIF synthesis HIF stabilization

gene expression increased: VEGF, PDGF, TGF

Figure 10–1. Schematic diagram of growth receptor and downstream signaling pathway activation of PI3K, AKT, MTOR, and HIF. Diagram prepared by author based on information in Davies and Sampson (2010) and Shaw (2009).

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The PKM locus on chromosome 15q22 generates isozymes PKM1 and PKM2, which are primarily expressed in muscle. PKM1 and PKM2 are different splice products derived from the same gene. PKM1 contains exon 9, which is absent from PKM2. PKM2 contains exon 10, which is absent from PKM1. Differences occur within a 56–amino acid region, and alterations in PKM2 primarily impact the region involved in tetramerization (Mazurek, 2011). PKM1 is expressed in tissues with high-energy demand (e.g., muscle and brain), and PKM1 has a high affinity for the substrate phosphoenolpyruvate (PEP). PKM2 is expressed in a few tissues in the adult (e.g., in retina and pancreatic island); however, it is primarily expressed in rapidly proliferating cells (e.g., during development and in stem cells). During tumorigenesis, PKM2 is the primary pyruvate isozyme expressed. PYRUVATE KINASE M2 Proliferation-promoting metabolic characteristics of cancer cells include increased uptake of glucose, increased glycolysis, and conversion of glucose to COO− 3-phosphoglycerate

H−C−OH

3-phosphoglycerate

H−C−O−P H

3-phosphoglycerate mutase

COO− 2-phosphoglycerate

H−C−O−P H−C−OH H Enolase

Phosphoenolpyruvate

COO− C−O−P H−CH ADP ATP

Enolpyruvate

Pyruvate kinase

COO−

COO−

C−OH

C=O

CH2

CH3 Pyruvate

COO− OH−C−H CH3 Lactate

Figure 10–2. Metabolism of phosphoenolpyruvate and activity of pyruvate kinase. Diagram generated by author based on information in Voet and Voet (1995) and in Gao et al. (2012)

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lactate rather than derivation of pyruvate from glucose and subsequent oxidation through the tricarboxylic acid mitochondrial cycle. There is also evidence that tumor cells preferentially express pyruvate kinase M2 (PKM2). Christofk et al. (2008) showed that a single switch in the splice site led to PKM2 production. They used short-hairpin RNAs to knock down this splice site. This led to replacement of PKM2 with PKM1 and reduced tumor proliferation. Gao et al. (2012) reported that PKM2 localizes to the nucleus and that levels of PKM2 expression correlate with rates of cell proliferation. Tetrameric PKM2 acts as a pyruvate kinase. The PKM2 dimeric form plays a role in promoting cell proliferation. Goldberg and Sharp (2012) demonstrated that in multiple cancer cell lines, specific siRNAs that knock down PKM2 result in decreased viability and increased apoptosis. In cancer xenografts they demonstrated that in vivo delivery of siRNAs to target PKM2 caused substantial tumor regression. GLUCOSE METABOLITES IN CANCER CELLS Locasale et al. (2011) used nuclear magnetic resonance (NMR)-based spectroscopy to quantify steady-state levels of glucose metabolites in cancer cell lines. They established that the highest intensity peaks contained lactate; glycine was also abundant in specific cell lines. They noted that glycine is generated from glucose, and the first step in this pathway is oxidation of 3-phosphoglycerate (3PG) to 3-phosphohydroxypyruvate (3PYR) through the activity of the enzyme 3-phosphoglycerate dehydrogenase. 3PG can be metabolized to pyruvate and lactate; 3PG can be converted to 3-phosphopyruvate and then transaminated to phosphoserine. Phosphoserine is then dephosphorylated to give rise to serine, which may then be converted to glycine. Locasale et al. determined that in a number of cancer cell lines substantial fractions of glucose and 3-phosphoglycerate were converted into serine and glycine biosynthesis. They also established that the 3-phosphoglycerate dehydrogenase (PHGDH) locus on chromosome 1p12 showed copy number gain in 16% of cancers. This region of the chromosome contains four genes; none are known oncogenes. Amplification of the PHGDH locus region is also found in cells from an esophageal cell line and occurred most commonly in melanoma. In studies of human melanoma tissue samples, Locasale found increased expression of PHGDH in 21% of cases and copy number gain in 21 of 42 samples. They then analyzed the effect of reduction of PHGDH expression in melanoma cell lines using short-hairpin RNAs. They determined that reduction in PHGDH expression decreased proliferation of these cells.

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3-phosphoglycerate

H−C−O−P H phosphoglycerate PHGDH dehydrogenase COO− 3-phosphohydroxypyruvate C=O

NAD NADH

H−C−O−P Glutamate

H phosphoserine aminotransferase

alphaketoglutarate

COO− H3N−C−H H−C−O−P

Phosphoserine

H

Phosphoserine phosphatase COO− Serine H3N−C−H H−C−OH H

Figure 10–3. Metabolism of 3-phosphoglycerate and generation of serine. Diagram based on information in Voet and Voet (1995) and Locasale et al. (2011).

Locasale et al. carried out studies in 106 breast cancer tumor samples. They determined that high PHGDH expression was associated with specific cancer subtypes, including triple-marker negative types and basal types. Locasale et al. reported that diversion of glycolytic flux into serine biosynthesis has a number of biological consequences with respect to the folate pool, amino acid and lipid intermediates, and redox regulation. In addition this pathway impacts generation of alphaketoglutarate from glutamate. They concluded that metabolic flux out of glycolysis is important in cancer development, beyond increase in lactate. GLUTAMINE AND TUMOR GROWTH Increased glutaminolysis occurs in cancer cells. Glutamine provides carbon for energy and nitrogen and carbon for biosynthetic reactions, and it impacts signal transduction pathways. Daye and Wellen (2012) reviewed glutamine

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metabolism in tumors and noted that it is particularly important as a carbon source in mitochondria in proliferating cells. Enzymes that impact glutamine include glutaminase, glutamine pyruvate transaminase (GPT), which produces alanine, and glutamate oxaloacetate transaminase (GOT), which produces aspartate. Glutamine also participates in synthesis of proline and arginine and plays a role in the generation of glutathione, which reduces reactive oxygen species. Glutamine and alphaketoglutarate are important substrates for enzymes involved in the generation of NADH. Glutamate transporters, including SLC1A5 and SLC7A5, show increased expression in cancer cells. Hassanein et al. (2012) demonstrated that SLC1A5 is important for the growth of lung cancer cells. Furthermore, they demonstrated that a pharmacological inhibitor of SLC1A5 decreased growth and viability of cancer cells and downregulated mTOR signaling. Expression of the MYC oncogene stimulates expression of a number of metabolic enzymes in glycolysis and also stimulates increased glutamine uptake (Daye and Wellen, 2012).

IDENTIFICATION OF METABOLIC GENES ESSENTIAL FOR CANCER GROWTH Possemato et al. (2011) developed a strategy to identify metabolic genes essential for growth proliferation of specific forms of cancer. They started by compiling a list of 2,752 genes that encode enzymes in metabolic pathways and transporters, using the KEGG database. They then used oncogenomic data to identify genes with the following characteristics: genes that showed higher levels of expression in tumor cells than normal cells, genes that showed high levels of expression in breast cancer, and genes that showed higher levels of expression in stem cells. They then developed short-hairpin RNAs (shRNAs) to target genes in these categories. The specific shRNAs cloned into lentivirus vectors were introduced into human breast tumor cell lines. Each cell carried a specific integrated shRNA. These cells were then transplanted into mice. Five gene products were identified as playing important roles in tumor formation. Suppression of these genes reduced capacity of the cells to produce tumors. The genes encoding these products were PHGDH (phosphoglycerate dehydrogenase), GMPS (guanosine monophosphate synthase), SLC16A3 monocarboxylate transporter, PYCR1 mitochondrial pyrroline 5-carboxylate reductase (NADPH generation), and VDAC1 mitochondrial ATP transporter.

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Possemato et al. also used data gathered by Beroukhim et al. (2010) on somatic copy number alterations in cancer. Their analyses identified PHGDH as a gene that maps to a region of human chromosome 1p that is frequently amplified in breast cancer, in melanoma, and other cancers. Studies by Pollari et al. (2011) determined that PDGDH messenger RNA levels were elevated in estrogen-negative breast cancer associated with a poor 5-year survival and with increased incidence of bone metastases and osteolytic bone lesion. PHGDH oxidizes 3-phosphoglycerate to phosphohydroxypyruvate, and this is the first step in the phosphorylated synthesis of serine. Serine is used in the synthesis of proteins, nucleotides, and sphingosine. Possemato et al. (2011) reported that several genes that promote serine biosynthesis and its subsequent metabolism are elevated in estrogen receptor–negative breast cancer. PHGDH overexpression facilitates proliferation of tumor cells, and suppression of its activity reduces tumor cells growth. In cells with high levels of PHGDH expression, the serine synthesis pathway contributed approximately 50% of the glutamate available for conversion to alpha ketoglutarate; therefore, PHGDH expression significantly impacted intermediary metabolism and anaplerosis of glutamate into the tricarboxylic acid (TCA) cycle. PHGDH suppression revealed that alphaketoglutarate levels showed the largest changes whereas cellular serine levels did not change dramatically. Other TCA cycle components were also lower; these included levels of citrate, isocitrate, succinyl CoA, fumarate, malate, and oxaloacetate. Possemato et al. (2011) postulated that targeting PHGDH is potentially valuable in treatment of tumors with elevated expression of PHGDH.

ISOCITRATE DEHYDROGENASES 1 AND 2 Since publication of reports on the presence of a specific mutation in the IDH1 mutation in a specific brain tumor, glioblastoma multiforme (Parsons et al., 2008), a number of studies have confirmed high frequency of IDH1 mutations in brain tumors. Jin et al. (2012) reported that IDH1 amino acid 132 mutation occurs in 70% of grade I and grade II astrocytomas and oligodendrogliomas and secondary glioblastomas. Mutations in the corresponding amino acid in the homologous gene IDH2 were also found. IDH2 encodes an enzyme active in mitochondria. IDH1 and IDH2 mutations were subsequently identified in other cancers, including leukemias, cartilaginous bone tumors, and some liver tumors. The tumor mutations are somatic in origin.

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Wild-type IDH converts isocitrate to alpha ketoglutarate and NAD to NADH. Mutant IDH in gliomas has an additional activity; it converts alpha ketoglutarate to D-2-hydroxyglutarate (also known as 2-hydroxyglutarate) and NADPH to NADP. Mutated IDH therefore generates a specific metabolite, D-2-hydroxyglutarate. This metabolite does not occur in normal metabolism but does occur in patients with germ line IDH2 mutations and the inborn error of metabolism D-hydroxyglutaricaciduria. Mutations at the active site in IDH1 and IDH2 impair the normal reaction, namely, the conversion of isocitrate to alpha ketoglutarate. Analysis of methylation patterns in gliomas revealed that the IDH mutations are associated with abnormal methylation patterns (Jones and Baylin, 2007). The glioma-specific methylation is sometimes referred to as G CIMP (glioma CpG island methylator phenotype) and is associated with distinct patterns of hypermethylation at specific loci. A number of investigators have reported results of studies designed to determine the relationships between IDH1/2 mutations, the G CIMP methylation patterns, and tumor pathogenesis. Turcan et al. (2012) introduced mutant IDH1 R132H or wild-type IDH1 into immortalized astrocyte cell lines. They demonstrated that astrocytes into which the mutant IDH1 was introduced produced the oncometabolite D2-hydroxyglutarate, and in these cells the methylome was remodeled and the transcriptome was altered. They concluded that studies on IDH1 mutations, oncometabolite production and methylome changes highlight genomic, epigenomic interactions in cancer. They also determined that gliomas with IDH mutations and D2-hydroxyglutarate production had a better prognosis than other forms of glioma. Lu et al. (2012) reported that mutant IDH1 or D2-hydroxyglutarate introduction into untransformed cells or neurosphere clones was associated with the repression of genes involved in cell differentiation. Jin et al. (2012) studied a fibrosarcoma-derived cell line, HT1080, with a heterozygous IDH1 mutation, R132C. They determined that knockdown of mutant IDH1 with short-hairpin RNA (shRNA) led to significant decrease in the proliferative capacity of the cells. IDH1 and IDH2 mutations have been identified in cytogenetically normal cells of patients with acute myeloid leukemia (CN-AML). Marcucci et al. (2010) reported that IDH mutations occurred in 33% of AML patients in which cytogenetic abnormalities were absent in diagnostic bone marrow and blood studies; 14% of these patients had IDH1 mutations and 19% had IDH2 mutations. Patients with IDH1 and IDH2 mutations had lower remission rates. These investigators reported altered gene expression profiles in AML patients with IDH1 or IDH2 mutations. Expression of APP amyloid precursor protein and of microRNA miR1

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and miR 133 were increased. Downregulated genes included KYNU (kynurenine hydrolase), which is involved in NAD cofactor biosynthesis, and also SULCG2 (succinate CoA ligase), which is involved in the Krebs cycle.

MONOCARBOXYLATE TRANSPORTERS Monocarboxylate transporters MCT1 (SLC16A1), MCT3, and MCT4 (SLC16A3) transport lactate out of tumor cells. Boidot et al. (2012) reported that loss of p53 in tumors promoted MCT1 expression in part through MCT mRNA stabilization. Breast cancer tumors with p53 mutation had increased MCT1 expression.

HEREDITARY CANCER SYNDROMES ASSOCIATED WITH GERM LINE MUTATIONS IN METABOLIC GENES Fumarate Hydratase Germ Line Mutations Germ-line mutations in fumarate hydratase lead to an autosomal-dominant syndrome associated with leiomyomas of the skin, leiomyomas of the uterus, and aggressive renal cancer (HLRCC syndrome). Sanz-Ortega et al. (2013) reported that up to 77% of women with this syndrome have uterine leiomyomas. Multiple leiomyomas were usually present in affected patients. A high percentage of the leiomyomas studied also had deletion of the region on chromosome 1q43 that encoded the normal fumarate hydratase (FH) gene. Isaacs et al. (2005) carried out studies on renal tumors from patients with HLRCC syndrome and demonstrated that bilateral mutation and/or deletion of FH led to overexpression of hypoxia-inducible factor (HIF). Sudarshan et al. (2009) demonstrated through studies in renal cancer that fumarate hydratase deficiency leads to stabilization of HIF, likely through inhibition of prolyl hydroxylase. In a comprehensive study of 56 families with HLRCC, Gardie et al. (2011) identified 36 different FH germ line mutations including missense, frame shift, nonsense, insertion, deletion, and splice site mutations. They reported that in some members of these families papillary renal carcinoma was the only manifestation of the syndrome.

THERAPEUTIC APPROACHES O’Flaherty et al. (2010) reported that reexpression of extramitochondrial fumarate hydratase in tumor cells corrected impaired prolyl-hydroxylation of

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HIF. In cultures of FH-negative cells, they determined that provision of exogenous 2-oxoglutarate corrected impaired prolyl hydroxylation. SUCCINATE DEHYDROGENASE AND TUMORS Succinate dehydrogenase (SDH) subunits and accessory factors compose complex II of the electron transfer system. Complex II is located at the intersection of the tricarboxylic acid cycle and mitochondrial phosphorylation. SDHA encoded on chromosome 5p15 is the catalytic subunit and SDHB, encoded on 1p36.1 has iron sulfur–related functions. Subunits C and D encoded on 1q23.2 and 19q13.12, respectively, are involved in anchoring the SDH complex to the mitochondrial membrane. SDHAF1 and SDHAF2 encoded on 19q13.12 and 11q12.2, respectively, encode accessory subunits, likely involved in assembly of the complex. Hoekstra and Bayley (2012) reviewed the roles of the SDH complex in diseases. Of particular interest with respect to tumors are the associations of germ line mutations in the SDH subunits and accessory subunits with hereditary paraganglioma and pheochromocytoma syndromes. These are neuroendocrine tumors that arise from sympathetic and parasympathetic branches of the autonomic nervous system. Paragangliomas of the head and neck (e.g., in the carotid body) are usually slow growing. Pheochromocytoms arise in the sympathetic nervous system, particularly in the adrenal medulla. They are not usually malignant but may lead to life-threatening hypertension. Paragangliomas and pheochromocytomas arise as a result of mutations in the SDHA, SDHB, SDHC, and SDHD subunits. Mutations in the SDHAF2 subunits have also been found in paragangliomas. Germ line mutations in SDHB, SDHC, and SDHD occur in some patients with renal carcinoma. Mutations in the SDH subunits have also been described in association with gastrointestinal stromal tumors (GIST) in children and young adults. These tumors are thought to arise in the pacemaker cells that control gastrointestinal peristalsis. Most GIST tumors arise is older individuals and carry gain-of-function mutations in the KIT oncogene and/or in the platelet-derived growth factor receptor PDGFRA. METABOLIC EFFECTS OF SUCCINATE AND FUMARATE ACCUMULATION Kaelin (2011) reported that enzymes in the 2-oxoglutarate-dependent dioxygenase family are inhibited by succinate and fumarate, which accumulate in

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tumors deficient in succinate dehydrogenase or fumarate hydratase. Members of the 2-oxoglutarate dehydrogenase family include prolyl hydroxylase, which facilitates the proteosomal degradation of hypoxia-inducible factor alpha. HIF is composed of a stable subunit beta and an unstable alpha subunit. Stability of alpha is dependent upon the local oxygen concentration and O2 partial pressure. Under hypoxic conditions the alpha subunit is stable. HIF alpha dimerizes with HIF beta, translocates to the nucleus, induces enzymes involved in glycolysis, and decreases transcription of genes involved in oxidative phosphorylation. Kaelin reported that increased mTOR activity also increases HIF activity. When oxygen is abundant, HIF alpha becomes a substrate for prolyl hydroxylase. Hydroxylation of one or more proline residues with HIF alpha creates binding sites for ubiquitin ligase and VHL (Von Hippel–Lindau) protein, and promotes targeting of HIF to proteasomes for degradation. Activity of HIF alpha is also influenced by the local 2-oxoglutarate concentration.

SYNTHETIC LETHALITY Earlier therapies for cancer were based on the concept that cancer cells divide more rapidly than noncancerous cells and that specific agents that target dividing HEREDITARY AUTOSOMAL-DOMINANT TUMOR SYNDROMES WITH MUTATIONS IN METABOLIC ENZYMES COO− CH2

COO− HC Succinate dehydrogenase CH

CH2

COO−

COO− Succinate

Fumarate COO−

COO− HC

Fumarate hydratase +H2O

HCOH

CH

CH2

COO−

COO−

Fumarate

Malate

Figure 10–4. Activities of succinate dehydrogenase and fumarate hydratase. Based on information in Voet and Voet (1995).

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cells would be more likely to kill cancer cells than normal cells. Unfortunately, these strategies also lead to destruction of normal cells that are dividing, particularly cells in bone marrow, intestinal mucosa, and hair follicles. The synthetic lethality concept is based on the concept that when changes leading to function losses occur in different gene products that function in the same pathway, even though each change is not lethal on its own, the two losses together can be lethal. In some cases loss of functional changes in protein or enzyme homologs that have the same function lead to synthetic lethality (Lehner and Park, 2012). The concept of synthetic lethality can also be extended to action of specific drugs. In some tumors a specific driver mutation that impacts a specific function may not be lethal. However, when a drug is administered that impacts that same function, the cell can no longer survive. A key example of the application of synthetic lethality in cancer treatment is the use of PARP1 inhibitors to treat breast cancer tumors with BRCA1 or BRCA2 mutations. PARP1 (polyADP ribose polymerase) plays a role in repair of single-stranded DNA breaks. The normal functions of BRCA1 and BRCA2 are to facilitate repair of double-stranded DNA breaks; these functions are impaired in tumors with BRCA1 or BRCA2 mutations (Farmer et al., 2005). In such tumors treatment with PARP1 inhibitors further impacts repair of DNA damage and results in lethality (Bryant et al., 2005). Muller et al. (2012) proposed that deletions of homologous genes in tumors could expose therapeutic vulnerabilities. They carried out studies in glioblastomas in which the ENO1 (enolase1) gene on 1p36.2 was deleted. They demonstrated that in these tumors inhibition of ENO2, encoded on 12p13, through short-hairpin RNA silencing or through use of a specific inhibitor phosphonoacetohydroxymate was lethal. ENO2 is predominantly expressed in neural tissue. ENO1 deletion in glioblastomas is not lethal since it is supplemented by ENO2 activity. Muller et al. noted that targeted therapies are most often directed at amplified or mutant activated driver oncoproteins and that loss-of-function mutations and gene deletions have received less attention in the development of target therapies. They emphasized that highly homologous products of two or more genes that originated in duplications carry out many essential processes. They proposed that when a member of a pair of duplicated homologous genes is lost the cells become more dependent on the function of the remaining gene. It is interesting to note that the vulnerability targeted, in this case enolase, had a metabolic function and that deletion of the encoding gene was not considered a driver change. Muller et al. defined this as collateral vulnerability and noted that it should be applicable to other passenger gene deletions or mutations that impact cellular functions.

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Normal cells

BRCA1/BRCA2 repair double-stranded DNA breaks by homologous recombination

Parp carries out Base Excision repair through recruitment of XRCC1, Ligase III, and polymerase beta

Breast Cancer

BRCA mutations no repair of double-stranded breaks

Parp carries out Base Excision repair through recruitment of XRCC1, Ligase III, and polymerase beta

Synthetic Lethality

BRCA mutations no repair of double-stranded breaks

Parp inhibitor No Base Excision repair

Figure 10–5. Synthetic lethality of PARP inhibitors and BRCA1 mutations. Diagram prepared by author based on information in Chalmers (2009).

Targeting Protein Kinases for Cancer Therapy The human kinome, a superfamily of more than 500 kinases, includes protein kinases, receptor tyrosine kinases, and nonreceptor and intracellular kinases. In 1992 Edmond Fischer and Edwin Krebs received the Nobel Prize for discoveries concerning reversible protein phosphorylation as a biological regulatory system. Protein kinases transfer phosphate residues to the hydroxyl group of an amino acid within a peptide or protein. In nonreceptor tyrosine kinases, located within the cytoplasm, transfer of phosphate occurs from ATP to tyrosine residues in proteins. ABL is an example of a nonreceptor tyrosine kinase. Nonreceptor tyrosine kinases are coupled to intracellular signaling. RECEPTOR TYROSINE KINASES At least 20 different families of receptor tyrosine kinases (RTKs) occur in humans; these proteins share structural organization. They are comprised of three separate domains: an extracellular domain, a transmembrane domain, and an intracellular domain with kinase function (Zwick et al., 2001). The extracellular regions of receptor tyrosine kinase show significant variability, with presence or absence of immunoglobulin-like domains. Ligand binding may occur at different positions in the extracellular domain in different

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receptors. RTKs have an intracellular domain composed of an N lobe and a C lobe. ATP binds to the N-terminal lobe. The catalytic site of the kinase is at the hinge between the N and the C lobes. In the inactive state, proteins and other molecules are bound to the kinase domain. Ligand binding to the receptor leads to conformation changes and activation. Activation involves dimerization or in some cases inactivation of the inhibitors that are bound to the catalytic site. On activation phosphate transfer occurs from ATP. There is also evidence that a specific inhibitory domain exists in the juxtamembrane region of RTK that controls activation. Specific mutations in the inhibitory domain may increase rates of activation (Medves and Demoulin, 2012). Triggering of kinase activity leads to activation of downstream signaling pathways. Downstream signaling cascades activated by receptor tyrosine kinases include RAS, RAF, MEK, and ERK and also PI3K, AKT, and mTOR. Specific inhibitors have been developed for BRAF (PLX4032), for MEK, for PI3K (PX886), for AKT (MK2206), and for mTOR (rapamycin) (Yarden and Pines, 2012). Receptor tyrosine kinases important for tumor growth include the ERBB network, which comprises four different receptor tyrosine kinases that constitute the epidermal growth factor receptor family. These include EGFR, sometimes referred to as ERB1, HER2/ERB2, HER3/ERB3, and HER4/ERB4. HER2 acts as a coreceptor for other ERB receptors. ERB receptors bind to at least 11 different ligands, including epidermal growth factor (EGF), transforming growth factor (TGF), and neuregulins. Other important growth factor receptors for tumor growth include insulin-like growth factor receptors, vascular epithelial growth factor receptor, platelet-derived growth factor receptor, and hepatic growth factor receptor.

IMPORTANCE OF STRUCTURAL CHROMOSOME CHANGES IN CANCER Structural chromosome changes leading to copy number alterations occur commonly in cancers. Genomic duplications potentially increase oncogene expression. Specific genomic regions that are amplified are sometimes present in cells as DNA fragments or episomes. Deletions may silence tumor suppressors. Fusion genes often arise in tumors as a result of genomic rearrangements and may be therapeutic targets. Inaki and Liu (2012) reported that whole-genome sequencing has revealed the existence in some cancers of a tandem duplicator phenotype in which large numbers of different chromosome regions are duplicated. Another complex

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ERBB2/HER2NEU

ERBB2 overexpression ERBB2 homodimers

ERBB2 heterodimers with EGFR or ERBB1

Trastuzumab blocks extracellular domains

Lapatinib inhibits Peptides kinase domain derived from HER2 used as vaccine to generate antibodies

P

P

P

P

PI3K AKT mTOR

Figure 10–6. ERBB2/HER2 receptor tyrosine kinases and downstream signaling. Figure based on information in Weinberg (2007) and Vogel et al. (2010).

phenotype, referred to as chromothripsis, is characterized by chromosome fragmentation and genomic rearrangements. They report that chromothripsis occurs in 2–3% of tumors and is particularly common in bone cancers.

FUSION GENES IN CANCER AND INCLUSION OF TYROSINE KINASE DOMAINS The first tyrosine kinase–related fusion gene identified in cancer was the BCR ABL gene in chronic myeloid leukemia (CML). The fusion gene was identified through sequencing of a cytogenetic rearrangement between chromosomes 9 and 22 that was found to commonly occur in CML. Fusion genes have most frequently been identified in hematologic malignancies. This may in part be due to the fact that chromosome studies can more readily be undertaken in these malignancies than in solid tumors. More recently fusion genes have been identified in solid tumors. In reviewing mechanisms of activation of tyrosine kinase fusion genes, Medves and Demoulin (2012) reported that the two most common switches included “enforced” dimerization and inactivation of kinase inhibitory domains.

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In tumor-related fusion genes, the fusion partner translocates to the N-terminal region of the kinase. The expression of the fusion gene is dependent upon the activity of the promoter of the new fusion gene and regulatory molecules. Fusion partners often have specific domains, such as coiled-coiled domains, that promote oligomerization. The subcellular location of the fusion protein is the same as that of the partner protein. In the case of fusion genes that involve the receptor tyrosine kinase ALK (anaplastic lymphoma kinase), numerous different partners are involved. In lymphomas ALK fusion partners include nucleophosmin, tropomyosin, moesin, and clathrin–encoding genes. In solid tumors the EML gene, which encodes a microtubule-associated protein is a more frequent ALK fusion partner. Receptor tyrosine kinase loci that form fusion genes include the following: PDGFRA, PDGFRB (platelet-derived growth factor receptors), RET (ret proto-oncogene), TRK A, TRKC (NTRK) neurotrophin receptors, FGFT1, FGFR3 (fibroblast growth factor receptor), and ALK (anaplastic lymphoma receptor tyrosine kinase). Fusion genes may, in some cases, be derived from cytoplasmic tyrosine kinase–encoding genes, including ABL, FRK (FYN related kinase), SYK (spleen tyrosine kinase), and JAK2 (Janus kinase). An important additional concept is that fusion proteins may be activated through binding of other proteins and factors. Silencing of activated tyrosine kinases has been achieved by specific adenosine-5-triphosphate competitors such as imatinib (Gleevec). However, resistance to these inhibitors frequently develops through new mutations. The first cancer-related fusion genes described were identified in hematological malignancies. More recently fusion genes have been identified in specific solid tumors. In papillary thyroid cancer, RET-activating fusion genes frequently occur. The EML4-ALK fusion gene occurs in non–small cell lung cancer. EML4-ALK fusion genes occur in approximately 4% of non–small cell lung cancers (NSCLCs). EML4 is a microtubule-associated protein and ALK is a receptor tyrosine kinase. Shaw et al. (2011) reported that this fusion gene occurs in association with specific clinicopathologic findings, namely, adenocarcinomas in younger patients with no or minimal smoking history and in which other oncogenic driver mutations are often absent.

THERAPEUTIC APPROACHES Inhibition of tyrosine kinase with ATP competitors has proven useful in the treatment of a number of different cancers. Competitive inhibitors such

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as imatinib (Gleevec) are useful not only for treatment of fusion genes that involve non–receptor tyrosine kinase (e.g., BCR ABL) but also for treatment of cancers that involve aberrant activity of specific receptor tyrosine kinases (e.g., PDGF receptors). Kinase inhibitors have sometimes proven useful in treatment of other cancers characterized by activated kinases (e.g., KIT). KIT is a transmembrane tyrosine kinase constitutively expressed in some tumors, including small-cell lung cancers and gastrointestinal tumors. Kinase inhibitors have also proven useful in treatment of cancers that involve mutated serine threonine kinases (Medves and Demoulin, 2012). Non–small cell lung cancers due to EML4-ALK fusion genes are sensitive to the kinase inhibitor crizotinib. Shaw et al. (2011) reported a 61% response rate to this treatment and a significantly prolonged survival rate in patients treated with crizotinib compared with other patients. Side effects were reported to be minimal.

RESISTANCE TO KINASE INHIBITORS Wilson et al. (2012) explored the development of acquired resistance to kinase inhibitors in cancer therapy. They emphasized that cancer cells express multiple tyrosine kinases and that signaling from these kinases converges on downstream effectors, particularly on phosphatidyl-3-OH kinase. This redundancy of signaling impacts treatment efficacy. Wilson et al. reported that development of resistance to a specific kinase inhibitor is dependent on activity of newly engaged survival signals. They emphasized growing evidence for heterogeneity in tumor cell populations and that treatment with a specific kinase inhibitor may kill only a subset of tumor cells. Wilson et al. established that hepatocyte growth factor expression plays a role in development of resistance of BRAF-mutant melanoma cells to the kinase inhibitor PLX4032. They determined that resistance could be predicted based on the estimation of plasma levels of hepatocyte growth factor in melanoma patients. There were wide ranges in these levels in different patients. Shi et al. (2011) reported evidence that resistance of BRAF-mutant melanoma cells to PLX4032 was driven in some melanomas by upregulation of platelet-derived growth factor receptor beta. Cells overexpressing this receptor also had increased expression of ERK, AKT, and S6 kinase signal transduction proteins. Shi et al. demonstrated that the combination of MEK1/2, PI3K, and mTORC inhibitors triggered apoptosis of resistant melanoma cells.

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ONGOING DEVELOPMENT OF KINASE INHIBITORS One of the earliest gene-targeted cancer therapies was developed to treat chronic myeloid leukemia, which is characterized by a BCR-ABL fusion gene. The drug imatinib, a modified 2-phenylaminopyrimidine, blocked the abnormal kinase activity of the oncoprotein and normalized hematopoiesis (Druker et al., 1996). However, 25% of the chronic myeloid leukemia patients do not respond to imatinib therapy, or they respond only initially and then become resistant to therapy. Gorre et al. (2001) reported that this resistance was due to BCRABL mutations or amplifications. More than 100 different mutations in the BCR-ABL fusion gene have been reported in imatinib-resistant patients. One frequent mutation that leads to imatinib resistance leads to threonine at position 315 in ABL being replaced with isoleucine. The T315I mutation occurs in the ATP binding site. (Goldman, 2012). A new kinase inhibitor, ponatinib, was developed by O’Hare et al. (2009). Ponatinib inhibits the T315I mutation. Goldman (2012) and Cortes et al. (2012) reported that ponatinib treatment of imatinib-resistant patients led to remissions of chronic myeloid leukemia. Goldman noted further that ponatinib is a multikinase inhibitor and has activity toward KIT, PDFGRA, and FGFRA kinases, which are activated in tumors. UNLIMITED PROPAGATION OF EPITHELIAL CELLS AND DRUG SENSITIVITY TESTING Liu et al. (2012) reported that the use of the Rho kinase inhibitor Y27632 in combination with a feeder layer of irradiated mouse fibroblast cells led to indefinite propagation of epithelial cells from a number of different tumors. They reported that cells from a number of different human epithelial tumors were propagated and proliferated indefinitely using this culture system. Yuan et al. (2012) reported use of this culture system to determine chemosensitivity of a respiratory papillomatosis tumor. They determined in the cultures system that this tumor was sensitive to vorinostat, a histone deacetylase inhibitor. These tumors arise due to actions of the HPV11 papilloma virus. A GENE REGULATOR THAT DETERMINES RESISTANCE TO MULTIPLE DRUGS Emergence of resistance to targeted therapeutic drugs commonly occurs in cancer. One example is non–small cell lung cancers that frequently harbor

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activating mutations of epidermal growth factor receptor (EGFR) and be successfully treated with EGFR inhibitors. However, resistance to treatment often develops and may be explained by secondary mutations in EGFR. Resistance to therapy may frequently involve mutations in downstream signaling pathways. Huang et al. (2012) noted that in at least 30% of cases, resistance to therapy could not be explained. They carried out large-scale loss-of-function screens to identify genes that when suppressed confer resistance to the drug crizotinib, a drug particularly useful in tumors that involve the EML4-ALK translocation. Huang et al. determined that suppression of expression of MED12 led to resistance to crizotinib and to a range of anticancer drugs. MED12 is a key component of the transcriptional MEDIATOR complex, and it plays an important role in regulating transforming growth factor beta receptor (TGFBR). In cells in which tyrosine kinases are inhibited and MED12 is lost, there is a proliferation of TGF beta receptors. This leads cells to be resistant to kinase inhibitors. However, the resistant cells are still sensitive to TGF beta receptor inhibition. MED12 is a subunit of the MEDIATOR complex, which is composed of 26 different subunits. The structure of this complex varies, and this variation impacts its role in regulation of gene expression. MEDIATOR is recruited to regulator sites of the genome. There it stabilizes transcription factors at the promoter and enhances polymerase II activity, leading to transcription initiation and elongation. Components of the MEDIATOR complex were initially though to be restricted to the nucleus; however, there is now evidence that they also occur in the cytoplasm (Guo and Wang, 2012).

RECEPTOR TYROSINE KINASES AND THERAPEUTIC ANTIBODIES Different therapeutic strategies have been adopted to counteract receptor kinase activity in tumors. These include use of small molecules to block receptor tyrosine kinase activity, and development of antibodies to block receptor ligand interactions. ERBB Network Four different receptor tyrosine kinases constitute the epidermal growth factor receptor family. These include EGFR, sometimes referred to as ERB1, HER2/ERB2, HER3/ERB3, and HER4/ERB4. HER2 acts as a coreceptor for other ERB receptors. ERB receptors bind to at least 11 different ligands,

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estrogen ligand Receptor

Cell membrane

Tyrosine kinase P domain

P activated Impair synthesis of estrogen aromatase inhibitors

Block estrogen binding: tamoxifen antibodies to receptor

Tyrosine kinase inhibitors

Figure 10–7. Targeting the estrogen receptor and estrogen synthesis and metabolism in breast cancer treatment. Diagram constructed by author based on information in Weinberg (2007) and Rugo (2008).

including epidermal growth factor (EGF), transforming growth factor (TGF), and neuregulins. Zwick et al. (2001) reported that these receptors are frequently coexpressed and can form homodimers or heterodimers. One of the first ERBB network proteins targeted in specific therapies was HER2/ERB2. This protein can be targeted with the therapeutic antibody Herceptin, first developed in 1998 to treat breast cancer. The antibody cetuximab was developed to target EGFR. Oncogenic alterations in EGFR occur in a number of different cancers. These include deletions, truncations, and point mutations. Specific small-molecule inhibitors of receptor tyrosine kinase include erlotinib and geftinib. However, specific point mutations in the kinase domain lead to resistance to these inhibitors (Yarden and Pines, 2012).

PEPTIDES TO TARGET DOWNSTREAM OF ACTIVATOR COMPLEXES Beta catenin becomes activator of tumor growth in specific colon cancers. Specific peptides have been designed that block the transcription-activating activity of beta catenin.

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LRP

CK1a APC beta catenin axin degradation GCK3b complex beta catenin beta catenin

active receptor complex

CK1a APC axin GCK3b beta catenin beta catenin degradation inhibited beta catenin beta catenin

beta catenin as therapeutic target hydrocarbon-stapled peptide Peptide impairs binding to transcription activator complex nucleus

beta catenin transcription activator complex Therapeutic targeting of downstream signaling component

Figure 10–8. This figure shows therapeutic targeting of downstream signaling activity of beta catenin. Diagram constructed by author based on information in Weinberg (2007), MacDonald et al. (2009) and Grossman et al. (2012).

IMMUNOTHERAPY FOR TUMORS Therapeutic approaches to treatment of malignant melanoma include the use of cell transfer therapies with patient’s cytotoxic T cells, which target the melanoma antigens present in the tumor of a specific patient. Tumors frequently regress with this treatment; however, relapse often occurs. Landsberg et al. (2012) reported that melanomas acquire resistance through inflammationinduced, reversible loss of melanoma antigen expression. They determined that tumor necrosis factor alpha (TNFalpha) produced by macrophages in the tumor microenvironment played an important role in inducing loss of melanoma antigen expression. Their studies indicate the importance of the tumor microenvironment in impacting response to treatment. Landsberg et al. emphasized the importance of combination therapies in targeting melanomas.

USING GENOMIC INFORMATION TO GUIDE CANCER THERAPY The goal of personalized cancer therapy is to determine which mutation profiles correlate with sensitivity to specific therapy. A significant resource that includes relevant information on mutations profiles in various forms of cancer and drug sensitivity is the Cosmic website: http://www.sanger.ac.uk/cosmic.

11 GENE-BASED MOLECULAR THERAPIES

INTRODUCTION Ultimate goals in patient care include counteracting or mitigating the effects of gene defects. Approaches to achieve this goal include provision of the normal gene product, and replacement or repair of the abnormal gene. This chapter includes examples of progress in gene targeting and gene therapy for disease treatment. Identification of optimal treatment targets is dependent upon comprehensive analysis of disease-associated functional impairments and identification of genetic variants that contribute to those functional impairments. Expansions of catalogs of variants and their functional impact will be important steps. However, we need to keep in mind that patients with the same variant in a specific disease gene may vary in the degree of severity of their manifestations depending on the impact of other genes in their genome and on environmental factors. An initial approach is to classify variants as high-risk, moderate-risk, or low-risk alleles. High-risk alleles primarily involve sequence changes or structural changes in protein coding regions of genes. Variants are classified as modifiers only if they have impact in the presence of disease-causing alleles in a specific gene.

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RNA-BASED THERAPEUTICS: USE OF OLIGONUCLEOTIDES RNA-based therapeutics includes the use of oligonucleotides that bind to RNA by base pairing. Kole et al. (2012) reviewed the different mechanisms of action of therapeutic oligonucleotides. They noted that mRNA-targeting strategies are used to treat a number of different genetic diseases. Approaches to the use of oligonucleotides for therapy include RNA interference and antisense oligonucleotides. Kole et al. reported that advances in oligonucleotide modification chemistry have led to greater efficiency in cellular delivery. Antisense oligonucleotides are utilized to bind to mRNA and induce degeneration. In this approach to remove disease-causing mRNA transcripts, specific cellular enzymes (e.g., RNAse H) are required. Another therapeutic application of oligonucleotides involves steric blocking. Oligonucleotides bind to mRNA and block access to cellular machinery and RNA processing.

SPLICING DEFECTS AND OLIGONUCLEOTIDE-BASED THERAPIES In eukaryotes primary transcripts of genes undergo splicing to remove introns. The exons are then joined (e.g., 3′ end of exon 1 to 5′ end of exon 2). Specific sequences in introns close to exon start and end positions are required for introns to be spliced out of primary transcripts. These include GT at the 5′ end of the intron and AG at the 3′ end. Additional sequences adjacent to the splice junctions also play roles in splicing and are conserved. They include short sequences immediately adjacent to the GT splice donor site and the branch Duchenne muscular dystrophy Exon skipping therapy

exon 48

mut. 51

exon 52

exon 48

exon 52

antisense oligonucleotide

Figure 11–1. This figure illustrates use of antisense oligonucleotides to induce exon skipping of mutant exon in Duchenne muscular dystrophy. Diagram based on information in Aartsma-Rus et al. (2010) and Verhaart et al. (2012).

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site approximately 40 nucleotides upstream of the splice acceptor sequences. Conserved splice enhancer sequences occur in introns and exons. Splicing in eukaryotes requires the RNA spliceosome: a large RNA protein complex that contains small nuclear RNAs (snRNAs) and at least 50 different proteins. RNA base pairing occurs between the snRNA molecules and the RNA transcripts. Splice donor sites and branch sites are recognized by the U1 and U2 snRNAs. U4, U5, and U6 snRNAs cause looping out of the intronic RNA. Hammond and Wood (2011) reviewed RNA mis-splicing and disease. They reported that RNA splicing defects occur in 50% of genetic diseases. Splicing defects lead to exon skipping, inclusion of intron sequences within the processed mRNA, alterations from normal alternative splicing patterns, and changes in the balance of alternate transcripts. When splice sites are mutated, alternative weak splice sites may be activated, leading to altered splicing and altered processed mRNA. Ars et al. (2000) reported that 50% of the mutations in the NF1 gene that led to neurofibromatosis in 57 patients involved abnormal splicing. These findings led them to stress the importance of studying mutations at both the genomic and RNA level.

SPECIFIC GENETIC DISEASES DUE TO SPLICING DEFECTS: THERAPEUTIC APPROACHES Rodriguez-Pascau et al. (2009) carried out mutation analysis in Niemann-Pick type C patients. They identified a patient with an intronic mutation in the NPC1 gene that created a cryptic splice donor site. This resulted in inclusion of 194 base pairs of intron 9 in the processed mRNA, creating a pseudoexon in the RNA. They designed a specific antisense morpholino oligonucleotide targeted to this cryptic splice site and transfected this into patient fibroblast cultures. This treatment led to restoration of normal splicing.

FAMILIAL DYSAUTONOMIA Aberrant splicing and alteration in the balance of alternate transcripts occurs in specific diseases. Cuajungco et al. (2003) reported aberrant splicing of the gene IKBAP in the recessively inherited disease familial dysautonomia. The IKAP gene encodes a kinase complex–associated protein. The aberrant splicing occurs at a specific site, mutation at base pair 6 in intron 20, T>C. This mutation is present in 99.5% of patients with familial dysautonomia. The mutation

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ribosome mRNA transcript

stop codon translated protein

ribosome mRNA transcript

premature termination codon shortened protein

ribosome mRNA transcript

premature termination codon

Aminoglycosides Ataluren promotes read-through at premature termination codons

Figure 11–2. This figure illustrates therapeutic use of aminoglycoside-related compounds to induce stop codon read-through. Diagram drawn by author based on information in Torra et al. (2010).

impacts splicing efficiency and leads to skipping of exon 20. They noted that this mutation does not completely block inclusion of exon 20 in all IKAP transcripts. They demonstrated that the ratio of wild-type to mutant transcripts varies in different tissues. The mutant transcripts are predominant in the central and peripheral nervous system. Slaugenhaupt et al. (2004) then explored treatment applications that would change the balance of wild-type to mutant alleles. In 2011 Axelrod et al. reported that oral administration of the compound kinetin was found to achieve the desired alteration in allele frequency in a limited clinical trial in patients with familial dysautonomia.

SPINAL MUSCULAR ATROPHY Spinal muscular atrophy is an autosomal recessive disease that results from deletions or mutations in the survival motor neuron gene SMN1. Defects in the SMN1 gene cannot be compensation for by activity of the closely linked SMN2 gene because a C-to-T transition in a specific splice motif precludes inclusion of exon 7 in SMN2 mRNA. There is evidence that small quantities of full-length mRNA transcripts are produced from SMN2 due to increases in the copy number of SMN2 genes in some patients with SMA and that these patients have a milder phenotype.

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There is evidence that these splice motifs in SMN2 that lead to exon 7 skipping can be blocked with antisense oligonucleotides and that this blockage leads to generation of full-length SMN2 mRNA that contains exon 7. Porensky et al. (2012) utilized intrathecal injection of a morpholino oligomer in mouse models of SMA. Other routes of oligomer administration including subcutaneous and intraperitoneal routes also led to improvement (Hua et al., 2011). The latter treatments with oligonucleotides led to expression of SMN2 in heart muscle and in the central nervous system. Antisense oligonucleotides that block the splice site in SMN2 and induce formation of full-length SMN2 to substitute for mutant SMN1 are now in clinical trials for treatment of patients with SMA. Other therapeutic approaches for SMA involve use of small molecules that impact splicing. One small-molecule splice site modulator, drisapersen, is in use in clinical trials not only for SMA but also for Duchenne muscular dystrophy (Dolgin, 2012a and b). Compounds that promote stop codon read-through, including ALB111, are also being considers as therapeutic agents for SMA in specific patients with stop codon mutations. Preclinical gene therapy trials are also ongoing to treat SMA. The plan is to supply a normal SMN1 gene. Mackenzie (2012) suggested that allele-specific oligonucleotide therapy may overtake gene therapy as an approach to treat spinal muscular atrophy and noted that the concept of a rescuing paralog in genetic disorders is comparatively untested. Pharmacological methods to induce fetal hemoglobin expression to treat hemoglobinopathies are one example (Atweh and Fathallah, 2010).

FUKAYAMA MUSCULAR DYSTROPHY Fukuyama muscular dystrophy is one of the most common autosomal recessive diseases in Japan. The disease is due to an ancient event that involved the insertion of a retrotransposon in the 3’ untranslated gene region of the Fukutin-encoding gene (FKTN) on chromosome 9q31-q33. The presence of the retrotransposon leads to activation of a normally silent donor splice site in the terminal exon, exon 10, and this interacts with an acceptor site in the 3′ retrotransposon. The inserted retrotransposon is approximately 3 kb long. The abnormal splicing leads to deletion of sequence from the carboxy terminal region of exon 10 in FKTN and insertion of sequence that encodes 129 amino acids derived from the retrotransposon (Kobayashi et al., 1998). Fukutin plays a role in the glycosylation of alpha dystroglycan that serves as a link between intracellular and extracellular regions of muscle fibers. In studies on cell lines, these investigators demonstrated that mutant fukutin is displaced from the Golgi.

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Taniguchi-Ikeda et al. (2011) developed an oligonucleotide that targeted the splice acceptor site in the retrotransposon. They injected this oligonucleotide into skeletal muscle to treat a mouse model of the disorder. They determined that this treatment led to production of normal-length fukutin and to restoration of fukutin function. Intravenous injection of oligonucleotide also led to increased production of fukutin in the mouse model of the disease. The greatest recovery of normal fukutin mRNA was achieved using a combination of oligos that targeted the splice donor and splice acceptor sites. This cocktail of oligonucleotides also led to normal fukutin mRNA production in lymphoblastoid cell lines from patients with Fukuyama muscular dystrophy. A general question that arises is whether antisense oligonucleotides can be used to treat other disorders in which there is retrotransposon insertion and altered splicing. The retrotransposon inserted in fukutin is an SVA (Sine-VNTR-ALU).

FRAME SHIFT MUTATIONS: CORRECTION THROUGH EXON SKIPPING Treatment of Specific Mutations in Duchenne Muscular dystrophy (DMD) In patients with Duchenne muscular dystrophy due to frame shift mutations in the DMD gene, immunochemical studies with antibodies to dystrophin revealed that occasional muscle fibers that express dystrophin occur between the large numbers of non-dystrophin-expressing fibers. These findings indicated that in some fibers dystrophin protein was produced, possibly because skipping of the diseased exon led to the production of an in-frame transcript. This observation stimulated the search for therapies that could facilitate skipping of mutant exons and result in production of a protein, shortened but nevertheless retaining some degree of function. Mutations in the dystrophin-encoding gene that lead to disruption of an open reading frame lead to a severe form of muscular dystrophy associated with progressive muscle wasting. DMD mutations that do not alter the reading frame lead to a milder form of the disease, Becker muscular dystrophy. One approach to treatment of DMD is the use of antisense oligonucleotides to cause skipping of exons that contain frame shift mutations. Reading frame correction therapy is aimed at inducing skipping of targeted exons from the pre-mRNA to restore reading frames. This correction could involve skipping exons with specific mutations or skipping exons that flank a deletion or

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duplication (Aartsma-Rus et al., 2003). Reading frame correction and skipping of exons in the central rod region of dystrophin are tolerated since this region is not essential for dystrophin function. DMD occurs with a frequency of 1 in 3,500 and is due to a large number of different mutations. Aartsma-Rus and van Ommen (2009) reported that 70% of mutations in DMD patients cluster between exons 45 and 55. They determined that therapies designed to lead to skipping of exon 51 would be applicable to the largest group (13%) of DMD patients. Antisense oligonucleotides to promote exon 51 skipping have been developed. Goemans et al. (2011) carried out a clinical trial of the systemic administration of PRO051, a 2-methyl-phosphorothioate oligonucleotide, to induce exon 51 skipping in DMD patients. PRO051 had previously been tested in local intramuscular administration and led to production of sarcolemma dystrophin in 64–97% of fibers. The study of systemic administration involved subcutaneous abdominal injection for 5 weeks in 12 patients. Assessment of response involved biopsy of the tibialis anterior muscle and analysis of changes in dystrophin RNA and protein. Goemans et al. demonstrated that following treatment over 12 weeks at doses of 2 mg/kg new dystrophin expression was observed in 60–100% of muscle fibers. They reported that there was a modest improvement in the 6-minute walk test. Side effects included irritation at the injection site and mild proteinuria. Cirak et al. (2012) reported clinical efficacy of an intravenous-administered phosphorodiamidate morpholino oligomer in 19 patients with Duchenne muscular dystrophy. They used the morpholino splice-switching oligonucleotide AVI-4658 (eteplirsen), which induces skipping of dystrophin exon 51 in patients with relevant deletions. Patients selected for therapy had mutations that involved exons 45 through 50. They used a dose escalation trial in six cohorts and doses ranging between 0.5 mg/kg and 20 mg/kg. Seven of the patients who completed the full trial showed a positive response; posttreatment the number of dystrophin positive fibers increased, and the intensity of dystrophin staining exceeded pretreatment levels. Six of the seven patients with significant dose response were in the high-level dose cohort. Cirak et al. also reported that there was a dose-dependent reduction in the inflammatory infiltrate in muscles. They reported that administration of the oligomer was well tolerated. Adkin et al. (2012) reported strategies to bypass intraexonic dystrophin mutations that tailored exon skipping strategies to specific patient mutations. Most recent information indicates it is too early to make statements on the comparative clinical safety of the different forms of antisense oligonucleotides used for treatment of Duchenne muscular dystrophy.

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USE OF ANTISENSE OLIGONUCLEOTIDES TO TREAT NEUROLOGICAL DISEASES DUE TO TRINUCLEOTIDE REPEAT EXPANSIONS In a number of different neurological diseases, expansion of CAG oligonucleotide repeats leads to expansion of glutamate residues in proteins, PolyQ diseases. Nine PolyQ diseases are known; they include Huntington’s chorea, spinal-bulbar muscular atrophy, five different forms of spinocerebellar ataxia, and dentatorubral pallidoluysian atrophy (DRPLA). In these diseases there is an inverse correlation of age of onset and repeat length. Evers et al. (2011) reported results of investigations to treat examples of these diseases with antisense oligonucleotides. They utilized modified 2-O-methylphosphorothioate (CUG)n triplet repeat antisense oligonucleotides to reduce levels of mutant mRNA translation and mutant protein in lymphoblasts and fibroblasts from patients with Huntington’s disease. They also demonstrated that this oligonucleotide reduced levels of mutant ataxin 1 in spinocerebellar ataxia type 1, ataxin 3 in spinocerebellar ataxia type 3, and levels of atrophin 1 in DRPLA. Significantly nonexpanded repeats were not affected by treatment. Gagnon et al. (2010) tested a broad spectrum of modified allele specific antisense oligonucleotides that target the CAG repeat expansion in Huntington’s chorea. Their findings revealed that a range of different allele-specific oligonucleotides successfully targeted the repeat expansion in huntingtin mRNA transcripts. Huntington’s disease manifestations occur primarily in the brain. A number of downstream effects of the gene mutation have been reported (e.g., interactions of mutant huntingtin HTT with PGC1alpha, the mitochondrial regulator) Ramos et al. (2012). However, no definitive downstream targets have been shown to be of therapeutic value. The striatum is particularly susceptible to mutant HTT–induced cell damage. However, multiple cell types in the cortex are also damaged in this disease. Kordasiewicz et al. (2012) demonstrated that antisense oligonucleotides to the HTT repeat sequence infused into the cerebrospinal fluid in ventricles of mouse models of Huntington’s disease delayed progression of the disease and led to reversal of some of the disease manifestations. Kordasiewicz et al. (2012) reported that oligonucleotide pairing with mutant HTT mRNA catalyzed RNAase H–mediated degradation of the mRNA. Transient infusion led to prolonged reversal of symptoms and continuous infusion was not required. Studies on intrathecal injection of oligonucleotides in a primate model of Huntington’s disease had a less dramatic effect.

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Aronin and Moore (2012) proposed that in Huntington’s disease symptoms appear when the brain no longer has the capacity to clear mutant HTT protein. Therapy that reduces the quantities of new mutant HTT mRNA, even if intermittent, can reduce the burden so that the clearance can be effective.

MYOTONIC DYSTROPHY Manifestations of myotonic dystrophy include myotonia (hyperexcitability of muscle), muscle wasting, cardiac conduction defects, insulin resistance, cataracts, and sometimes cognitive defects. In type 1 myotonic dystrophy (DM1, dystrophia myotonica), toxic repeat expansions of CAG occur in the region of the DMPK1 gene that encodes the 3′ untranslated region. These expansions can range in size from 50 to 1,000 repeats. The expanded repeat in the DMPK1 gene leads to production of an mRNA with toxic properties and to dominant genetic disease (J. E. Lee et al., 2012). The DMPK mRNA containing these long CUG repeats accumulates as foci in nuclei that sequester RNA binding proteins, including muscleblind-like 1 (MBNL1) and CUG binding protein CUGBP1 (Jones et al. 2011). This sequestration leads to depletion of these proteins encoded by these RNAs. Muscleblind-like 1 plays a role in splicing regulation. CUGBP protein regulates pre-mRNA alternative splicing and may also be involved in mRNA editing, and translation. Downstream splicing defects include abnormal exon skipping in pre-RNA of a number of genes including the muscle-specific chloride channel gene CLC1, cardiac troponin, and the insulin receptor gene. Dystrophica myotonia type 2 (DM2) is due to repeat a CCTG expansion within intron 1 of the ZNF9 gene. The presence of the CCUG repeat in the pre-mRNA results in nuclear and cytoplasmic aggregation of mRNA and reduction in the amount of ZNF9 protein produced. In addition, the aggregates bind and reduce availability of muscleblind-like1 protein and CUG binding protein (CELF). Wheeler et al. (2012) designed gapmer antisense oligonucleotides to silence mutant Dmpk1 in a mouse model of DM1. These oligonucleotides contain a central stretch of oligonucleotides that induce RNAse H cleavage of mRNA bound to the oligonucleotide. Gapmer oligonucleotides were delivered either by intramuscular or subcutaneous injection. Wheeler et al. determined that the nuclear-retained CUG repeats were usually sensitive to oligonucleotide silencing. They determined that following treatment the number of nuclear aggregates decreased. Furthermore, there was evidence that muscleblind-like proteins were released from nuclear aggregates and RNA splicing defects were reduced.

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Success of the gapmer oligonucleotides in DMPK1 repeat expansions may be due to the nuclear retention of mutant RNA and the fact that targeted RNA and bound oligonucleotides are good targets for digestion by RNAse H, which is abundant in nuclei (Todd and Paulson, 2012).

GAPMER OLIGONUCLEOTIDES: POSSIBILITIES FOR TREATMENT OF OTHER REPEAT EXPANSION DISORDERS Todd and Paulson (2012) noted that gapmer oligonucleotides might also be useful in treatment of other repeat expansion disorders, including amyotrophic lateral sclerosis and frontotemporal dementia in which a repeat expansion occurs in the first noncoding exons of the gene C9ORF72. A number of disease mechanisms are being considered, including aberrant promoter function, aberrant splicing of C9ORF72 primary transcripts, or sequestration of RNA-binding proteins (Brettschneider et al., 2012).

OLIGONUCLEOTIDES THAT PROMOTE DEGRADATION OF MUTANT TRANSCRIPTS Treatment of Homozygous Hypercholesterolemia Individuals with homozygous familial hypercholesterolemia have very high levels of plasma low-density lipoprotein (LDL) cholesterol. Cutaneous and tendinous xanthomata and cardiovascular disease occur early in life. This condition is due to mutations in the hepatic LDL receptor. Therapy with statins, even at maximal doses, fails to reduce high levels of LDL cholesterol in these patients. There is evidence that statins work not only by decreasing cholesterol biosynthesis but also by increasing expression of lipoprotein receptors. Treatment options for these patients include plasma apheresis to remove LDL and portocaval shunting. Both are invasive procedures. Apolipoprotein B is an essential component of atherogenic lipoprotein. Mipomersen is an antisense oligonucleotide designed to inhibit apolipoprotein B synthesis. This oligonucleotide binds to apolipoprotein B mRNA and promotes its degradation through ribonuclease H activity. Raal et al. (2010) reported results of a randomized, double-blind, placebo-controlled phase 3 trial over 26 weeks that was completed by 45 patients. They reported that the mean percentage change in LDL was significantly greater in mipomersen-treated patients than in control patients. Reduction of lipoprotein in mipomersen-treated patients was 24% (range 17.7–31.6%). The treatment was well tolerated; the

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main side effects were discomfort at the injection site and increase in levels of serum alanine aminotransferase. There is evidence that mipomersen impacts hepatic apolipoprotein B synthesis but does not impact synthesis of the different apolipoprotein B splice form that is produced in the intestine.

RNA-BASED THERAPEUTICS: USE OF SIRNAS RNA interference with short inhibitory RNAs has proven problematic is clinical applications due to problems in delivery and because of off-target effects. Clinical trials of siRNA have been carried out to treat eye diseases (e.g., macular degeneration). Skin applications of siRNAs have been used for treatment of rare genetically determined skin diseases (e.g., pachyonychia congenita). Modifications of siRNAs for delivery are also being investigated. These modifications include formulations of siRNA in lipid molecules and formulations with cyclodextrin adamantine polyethylene glycol (Kole et al., 2012). Small interfering RNAs (siRNAs) are processed in the cellular interference pathway that includes Dicer, Argonaute, and RISC complexes. Following targeting of siRNA to the mRNA on Argonaute, targeted RNA is cleaved by the RISC complex.

GENE TRANSFER IN RECOMBINANT VECTORS There are growing numbers of reports on apparently successful gene therapy. Two main vectors currently in use for introducing genes into the genome include adeno-associated vectors and lentiviral vectors. Problems with use of viral vectors remain, however. The lentiviral vectors were considered to be less likely to cause insertional mutagenesis (Montini et al., 2006), though this is now in question. There is, however, a report on gene therapy of a patient with severe beta thalassemia in which the functional beta globin gene cloned into a lentiviral vector was introduced into a patient’s hematopoietic stem cells initially in culture. Subsequently, these cells were transplanted into the patient. The patient was followed for 3 years and no longer needed transfusion, though he remained mildly anemic (Cavazzana-Calvo et al., 2010).

HEMOPHILIA Adeno-associated viruses (AAVs) have been used in gene therapy since the virus most often remains in episomal form in the cell and possibilities for genomic

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integration are reduced. However, patients may develop an immune response to the viral vector. High and Aubourg (2011) reported that therapy of hemophilia A with an adeno-associated virus containing the Factor VIII gene was initially successful; later Factor VIII levels in the patient fell due to immune response. Successful adeno-associated virus treatment of hemophilia B was reported by Nathwani et al. (2011). Hemophilia B results from inadequate levels of coagulation Factor IX, due to deletion, rearrangement, or mutation in the Factor IX gene (FIX). These patients often require intravenous administration of FIX protein concentrate two to three times a week to avoid hemorrhages. Nathwani et al. developed a self-complementary adeno-associated viral vector that contained a codon optimized human Factor IX transgene. The FIX codon cassette was optimized for liver expression and was packaged as complementary dimers within individual AAV particles. In preclinical studies the investigators demonstrated persistence of the vector and FIX expression in mouse liver. Subsequent long-term safety studies were carried out in macaques. In patient studies Nathwani et al. assigned six patients to three groups: low dose, medium dose, and high dose. FIX protein was administered as plasma levels required; low-dose patients in particular continued to receive FIX proteins. Patients received FIX gene therapy intravenously. No immunosuppressive therapy was given. Patients were followed for 6–16 months. Their study demonstrated therapeutic expression of the transferred FIX gene, and the increase in blood levels of FIX protein in patients was approximately dose dependent. Later in the course of therapy, four of the six participants were able to discontinue therapy with intravenous FIX protein concentrate. In the two participants who continued to require protein concentrate to attain adequate FIX protein levels in blood, the intervals between these injections was longer. Nathwani et al. noted that these two participants had a history of preexisting severe hemophilic arthropathy. There was no evidence of immunologic response to the transgene FIX. Ward et al. (2011) reported that shorter constructs of Factor VIII coagulation factor were more easily accommodated in the adeno-associated vectors they developed. These shortened forms did not contain the Factor VIII B domain. The B domain shares no homology with other known proteins. Preclinical studies in mice revealed that this factor retained procoagulant function.

GENE THERAPY FOR EYE DISEASES In reviewing clinical applications of retinal gene therapy, Lipinski et al. (2013) noted the importance of early, correct diagnosis of genetically determined

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blindness and initiation of therapy before the onset of significant cellular pathology and cell death. They emphasized that success in treatment is dependent on the efficiency with which the therapeutic transgene can be delivered to the appropriate retinal cell type and noted that vector-delivered and nonviral delivery are used in experimental studies. Writing in 2012, Lipinski et al. reported that the majority of nonviral delivery systems of genes were not suitable for clinical gene therapy. Lipinski et al. emphasized that the eye is a suitable target for gene therapy because it is an immune-privileged site and is readily accessible. The retinal pigment epithelium and photoreceptors have been the primary targets for gene therapy. Fluid suspensions of the therapeutic targets are usually injected into the subretinal space. The vector most commonly used in eye therapy is the adeno-associated viral vector (AAV). Removal of the Rep and Cap genes from AAV gene allows for insertion of a transgene up to 4.7 kb in size. The occurrence of 146 base pair terminal repeats in the viral DNA facilitates second-strand synthesis, genome replication, and genome packing since the inverted repeats promote loop formation of the DNA. Additional DNA sequences added to the therapeutic vector DNA include tetracycline-responsive elements that allow expression of the transgenes to be controlled by antibiotics. Another viral vector sometimes used in eye gene therapy is the HIV1-based lentiviral vector. Retinitis pigmentosa (RP)–causing gene defects treated by gene therapies involve the following genes: phosphodiesterase 6A, RPGTPase regulator, and cyclic nucleotide-gated channel subunits alpha1 and beta 1. Gene therapy for Leber’s congenital amaurosis was developed to treat defects in MERTK (mertyrosine kinase oncogene), guanylate cyclase 2D, vitamin A trans-cis isomerase (RPE45), and 18 other genes. Treatment of achromatopsia was developed for defects in ion channel genes and genes in the phototransduction cascade. Five different gene therapies have been devised to treat Usher syndrome that is associated with visual and auditory sensory impairment. These include recombinant genes for myosin 7a, harmonin, cadherin23, protocadherin15, and Usher syndrome 5G.

GENE SILENCING AND DELIVERY TO THE BRAIN RNA-silencing techniques involve the use of antisense oligonucleotides and use of short inhibitory RNAs (siRNAs). The blood–brain barrier presents a significant problem when intravascular therapies are used to treat brain diseases. The blood–brain barrier is generated by capillary endothelial cells sealed by tight junctions and by association of capillaries with pericytes and astrocytes.

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However, bone marrow–derived cells generate brain microglia, and microglia precursors must therefore pass through the blood–brain barrier (Cartier and Aubourg, 2010). There is also evidence that exosome microvesicles and adeno-associated viral vectors can pass through this barrier (Aronin and Moore, 2012).

EXOSOME BIOLOGY AND POTENTIAL FOR DRUG DELIVERY Lakhal and Wood (2011) reviewed the biology of exosomes and exosome delivery of drugs and RNAi. Exosomes arise from endocytic vesicles when the membranes of late endosomes and multivesicular bodies fuse with the outer cell membrane. Pan et al. (1985) first described selective externalization of transferrin in exosomes originating in sheep reticulocytes. In 2007 Valadi et al. reported that exosomes mediate transfer of microRNAs. Alvarez-Erviti et al. (2011) purified exosomes from bone marrow dendritic cells and demonstrated that they could be used as vehicles for delivery of siRNAs. Intravenously injected siRNAs accumulate primarily in liver, spleen, and kidney. Exosomes were specifically engineered to target brain cells through cloning of a rabies virus glycopeptide (RVG) to the genes and coexpression with LAMP2B that is expressed on exosomes. This glycopeptide specifically binds to the nicotinic acetylcholine receptor that occurs on neurons in the striatum and on vascular epithelial cells. Following intravenous injection, 60% of siRNA in the RVG-targeted exosomes reached the brain, including midbrain, cortex, and striatum. El-Andaloussi et al. (2012) described protocols for harvesting exosomes and transfecting them to be targeted to specific tissues. They also described techniques for loading siRNAs into exosomes.

THERAPEUTIC DESIGNS BASED ON ALTERING REGULATION OF GENE EXPRESSION Hemoglobinopathies Hemoglobinopathies are due to defects in the structure or synthesis of globin proteins. Herrick first described sickle cell disease due to a mutation in the gene that encodes beta globin in 1910. There is evidence that reactivation of expression of gamma globin genes can lessen the impacts of thalassemia mutations and sickle gene mutations. Expression of gamma globin is usually

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silenced in early infancy. Important factors in gamma globin gene expression include specific sequences in the promoter region of gamma globin and in control sites, in particular binding sites for the transcription factor GATA1. In some individuals hereditary persistence of fetal hemoglobin expression occurs due to mutations in the gamma globin gene promoter. Fetal hemoglobin (HbF) contains alpha and gamma globin chains. Gräslund et al. (2005) designed zinc finger–based transcriptional activators to target sites in the gamma globin gene promoter proximal to position −117. This region is known to serve as binding site for transcription factors. Retroviral delivery of the zinc finger–based transcription factor designed by Gräslund et al. into the erythroleukemia cell line K562 resulted in increased production of gamma globin. Costa et al. (2012) reported that this artificial zinc-finger transcription factor enhanced gamma globin gene expression in a mouse model or thalassemia.

Gene Targeting and Correction In gene therapy for monogenic diseases reported prior to 2011, therapeutic DNA was maintained as an episome in cells, or it was integrated into genomic DNA at a random site. Ellis et al. (2012) emphasized the advantages of newer approaches to gene therapy. In these approaches gene cleavage at specific sites with engineered endonucleases is followed by gene repair by homologous recombination. Correction is most commonly carried out in cultured cells, including stem cells. Bone marrow stem cells and induced pluripotent stem cells are used. Ellis et al. documented advantages of these approaches; they include maintenance of proper gene copy number, preservation of location to endogenous regulatory elements, and reduction of concerns about inappropriate integrations and gene disruption. However, this approach does require, in some cases, introduction into cells of endonucleases for cleavage and introduction of donor sequences. Engineered endonucleases such as zinc-finger nucleases (ZFNs) and transcription activator effector nucleases (TALENs) have two domains. The first recognizes target sequences in DNA through engineered DNA binding domains. The second domain has endonuclease function and cleaves DNA to introduce double-stranded breaks. These reagents can be used for gene disruption, gene correction, and gene addition. The DNA binding domain of ZFNs include units that bind to nucleotides in a single strand of DNA. Each Zn finger binds to three nucleotides; however, neighboring Zn fingers can influence binding. TALEN protein domains have a more specific 1-to-1 code for protein to DNA binding. Databases and

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NI

HD

NN

T

A

C

G

22 Talens bind to specific nucleotides

Double-stranded DNA

Central endonuclease

22 Talens bind to specific nucleotides

Figure 11–3. Talens for gene correction; Talens have DNA binding and endonuclease domains. Figure generated by author on the basis of information in Miller et al. (2011) and N.Sun et al. (2012).

knowledge bases of all reported ZFNs and TALENs engineered to bind to specific DNA sequences have been developed (e.g., EENdb) (Xiao et al., 2013).

ZINC-FINGER NUCLEASES Zinc-finger proteins are unique DNA binding transcription factors rich in cysteine and histidine amino acids (C2H2) bound to zinc ions. Choo and Klug first reported their structures, DNA binding properties, and potential molecular applications in 1994. Each zinc finger recognizes 3 base pairs in DNA, and zinc fingers can be linked in tandem. For example, six zinc fingers can be linked in tandem to recognize 18 base pairs of DNA. Zinc-finger arrays can also be linked with other structures to modify DNA. Kim et al. (1996) created fusion proteins of zinc fingers linked to the restriction endonuclease Fok1. This restriction endonuclease will cleave DNA sequences separated by a few bases. Zinc-finger pairs are delivered to cells by electroporation or transfection. Messenger RNA is sometimes used to introduce zinc-finger pairs. Viral delivery involves adeno-associated viruses or lentiviruses. Ellis et al. (2012) developed strategies for efficient use of a recombinant adenoviral vector in which zinc fingers and 750 nucleotides of repair sequence can be accommodated. DNA cleavage occurs between two regions where zinc fingers are bound to opposite strands of DNA. The double-stranded break is then repaired by

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nonhomologous end joining or by homology-directed repair. The latter repair process is favored using homologous sequence from an undamaged sister chromatid. Homologous-directed repair can also use DNA supplied as a template (e.g., DNA present in an episome or plasmid) (Moehle et al., 2007). There is also evidence that single-stranded oligonucleotide sequence can be used for repair (F. Chen et al., 2011). Zinc-finger nuclease targeting can also be used to cleave DNA and insert larger sequences (Carroll, 2011). It has been used to target a transgene to a specific location in the genome (e.g., into the AAVS1 locus, considered to be a “safe harbor” locus in the genome).

Tale Nucleases for Gene Targeting and Genetic Engineering Tale nucleases, referred to as Talens, are plant-derived transcription factors. In Talens multiple-repeat elements comprised of 34 amino acids are arranged in tandem. Each repeat differs in two highly variable amino acids that determine the binding of the repeat to one specific DNA base pair (Mussolino and Cathomen, 2012). The shorter recognition sequence of zinc-finger nucleases may induce more off-target cleavages. Talens have longer recognition sequences and are less likely to produce off-target cleavages. Talens are engineered to position FOK1 between the left and the right arm of the Talen. N. Sun et al. (2012) engineered custom-designed Talens for correction of the sickle mutation in human cells. Talens were introduced to human cells by means of a donor plasmid. The donor normal globin sequence was introduced with a retroviral vector. They demonstrated efficiency of gene correction. Hockemeyer et al. (2011) designed Talens to target the safe harbor locus PPP1R12C (AAVS1) with a pluripotency-inducing factor such as OCT4. The introduced DNA was flanked by Loxp sites and could subsequently be excised with Cre recombinase. In the Cre-Lox mechanism, the 34-bp loxp sequence is comprised of an 8–base pair core sequence followed by two inverted repeats. In cloning of a sequence to subsequently be excised, the lox P sequences are oriented in the same direction on opposite sides of a gene when Cre recombinase is applied and sequence between the loxp sites is removed. Hockemeyer et al. (2011) also used embryonic stem cells and IPS cells to evaluate the utility of Talens to drive targeted gene modification at five different genomic loci and reported efficient targeting and genetic modification.

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12 STEM CELLS AND INDUCED PLURIPOTENT STEM CELLS

From our vantage point, it appears that the field of regenerative medicine has moved out of the rocky shallows and is rapidly sailing towards the therapeutic mainstream. —Mahendra Rao and Francis Collins (2012)

INTRODUCTION Transplants of hematopoietic stem cells and of bone marrow have been used for several decades to treat specific genetic diseases. Optimal results require HLA (human leucocyte antigen)– matched haploidentical donor cells, and these are frequently difficult to obtain. In addition, despite advances in HLA match determination, graft-versus-host disease is a significant complication. Methods to combat this adverse reaction include processing of donor marrow samples to deplete T-cells and to reduce natural killer cells (Handgretinger, 2012). In 2006 Takahashi and Yamanaka reported that cells from differentiated tissues could be reprogrammed using a limited number of transcription factors to produce pluripotent cells, using a limited number of transcription factors. From such pluripotent cells, a variety of different tissues can be produced. 225

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The factors that induced pluripotency, Oct4, Sox2, Kif4, and cMyc, were introduced using a specific plasmid vector. Following transformation to pluripotency, a variety of different factors were used to induce differentiation to specific cell types. Induced pluripotent stem (IPS) cells are used to study the effect of specific gene mutations. IPS cells derived from a specific patient are also used in gene targeting and gene correction to provide sources of cells with normal function. John Gurdon and Shinya Yamanaka were awarded the 2012 Nobel Prize in Physiology and Medicine for the discovery that mature cells can be reprogrammed to become pluripotent.

INDUCED PLURIPOTENT STEM CELLS: MODIFICATIONS TO ENHANCE SAFETY Studies on the derived pluripotent cells revealed that the vector constructs that carried the transcription factors became integrated in various positions in the genome. Expression of the introduced transcription factors diminished over time; however, the vector sequences remained in the genome (Pera, 2009). Concerns were raised about the risks of insertional mutagenesis that could vary depending on the position of integration into the donor genome. In addition, there were concerns about the possibility of disturbance in donor epigenetic mechanisms. Another concern was that inserted sequences, particularly cMyc, which can act as an oncogene, would be incompletely silenced, raising the possibility of tumor development. Revised methodologies to induce pluripotency have been developed. These include the use of nonintegrating adenoviral vectors to carry pluripotency-inducing factors and the use of vectors that could subsequently be excised. Excisable vectors for transformation were developed by a number of different investigators. Soldner et al. (2009) developed a lentiviral vector in which gene expression of transcription factors was doxycycline inducible and where the transgene could subsequently be excised by Cre recombinase. Another interesting approach to pluripotency induction was use of the transposon named Piggy Bac. Yusa et al. (2009) utilized cationic lipofection to introduce Piggy Bac containing pluripotency-inducing transcription factors other than cMyc into cells. This transposon remains as a mobile nonintegrated element that can subsequently be excised using the enzyme transposase. Yusa et al. reported that this system could be used to derive therapeutically applicable induced pluripotent stem cells (IPSCs).

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Strategies that involved the direct use of transcription factor proteins or of transcription factor mRNA have also been utilized (Desponts and Ding, 2010; Zhou et al., 2009). In addition, studies were carried out to identify small molecules that promote pluripotency. Small molecules used included histone deacetylase inhibitors including valproic acid. The frequency of pluripotency induction has, however, generally been low when proteins and small molecules are used (Warren et al., 2010).

IPS CELL DEVELOPMENT WITHOUT USE OF VIRAL VECTORS There is growing impetus for methodologies to develop IPS cells without using viral vectors. One method involves the use of synthetic mRNAs that encode transcription factors. An additional improvement in methodology has enabled culture of IPS cells without feeder culture layers. Both of these improvements facilitate the use of IPS cells for therapeutic purposes (Okita et al., 2012).

NEURAL STEM CELLS An important potential therapeutic use of pluripotent stem cells is the development of neural stem cells for treatment of neurodegenerative diseases. This will require development of cells that are free of genetic defects and apparently free of hazards that arise from abnormal insertions that they carry. Another important consideration is whether or not the transplanted cells will become functionally integrated in the donor. Stem cells may also be potentially useful as “feeder cells” to provide essential components such as neurotrophic factors. In 2011 Major et al. described a study in which they induced pluripotency in human fibroblasts with a lentiviral vector that produced OCT4, SOX2, and KLF4. Following induction of pluripotency, the vector was excised from the induced pluripotent cells. The induced pluripotent stem (IPS) cell clones were then treated with specific neural induction and differentiation factors. Subsequently, specific IPS clones exhibited differentiation to neuroepithelial, neuronal, and glial cells on exposure to specific growth factors including brain-derived neurotrophic factor (BDNF), platelet-derived neurotrophic factor (PDGFRA), sonic hedgehog protein, and others. The derived neural stem cells were stereotactically transplanted into the striatum of adult rats. The migration pattern of the transplanted cells was analyzed in follow-up studies of brain sections. Human cells were identified using

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human specific markers including antibodies to human nuclear antigens and neural cell adhesion molecule (NCAM). Immunohistochemistry of sections revealed that cells in the core transplant expressed human markers. Importantly Major et al. observed that human cells that had migrated to positions beyond the core took on the phenotype of cells in the local environment and had region-specific immunochemical profiles. Human cells occurred in the rat subventricular zones and had also migrated along the rostral migratory stream toward the olfactory bulb. A few human cells reached the granular and periglomerular layer in the olfactory bulb, where they differentiated into calretinin-positive or gamma amino butyric acid (GABA)–positive interneurons. No teratomas of human cells developed in the rat brain during the 12 weeks that the animals were followed after transplantation of IPS cells. Major et al. (2011) concluded that excision of the vector containing the transforming factors had no impact on long-term survival of the transplanted reprogrammed IPS cells or their ability to acquire brain-regional phenotypes. Hargus et al. (2010) used dermal-derived fibroblasts from Parkinson’s disease patients to derive pluripotent stem cells. They used lentiviral vectors in which expression of transcription factors OCT4, KLF4, and SOX2 was inducible by doxycycline. They specifically analyzed the potential of these cell lines to differentiate into dopamine neurons using a differentiation protocol of Perrier et al. (2004). Neural-specific induction involved initial growth under serum-free conditions on a stromal feeder layer. Subsequently stem cell rosettes were transferred to a polyornithine laminin substrate in the absence of feeder cells. Culture medium was supplemented with growth factors sonic hedgehog (SHH), fibroblast growth factor 8 (FGF8), brain-derived neurotrophic factor 8 (BDNF8), and ascorbic acids. Later cells were cultured with neurotrophic-transforming growth factor B, dibutyryl cyclic adenosine monophosphate, and ascorbic acid. Cell populations were examined for dopamine production. Specifically, the medium in which cell colonies were cultured was analyzed by high-pressure liquid chromatography to detect dopamine production. Hargus and coworkers identified cell cultures that contained neurons that expressed tyrosine hydroxylase, and dopamine beta hydroxylase. They transplanted these neurons into rat brain striatum. Four weeks after transplantation, cells were stained for human-specific NCAM and they were microscopically visualized. The fiber outgrowth of NCAM-positive neurons was studied. In addition to carrying out studies on normal rats, they engrafted dopamine-positive cells into the dorsolateral striatum of lesioned rats that serve as a model of

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Reprogramming factors OCT4, cMYC, SOX2, KIF4 Pluripotent stem cells

Fibroblasts

Patient with specific Neurological disease

Differentiation to specific cell type Neuronal precursor cells Neurons

Identification of therapy Drug screening

Molecular characterization Target identification

Figure 12–1. This figure illustrates use of reprogramming factors to derive neuronal precursor cell from patient fibroblasts and use of these cells for molecular characterization and drug screening. Figure generated by the author on the basis of information in Takahashi and Yamanaka (2006) and Sommer et al. (2010).

Parkinson’s syndrome. Subsequent functional studies on these rats revealed improvements in performance after engraftment. Specifically, improvements were observed in rotational rod performance, and motor asymmetry was decreased. PLURIPOTENT STEM CELLS AND RETT SYNDROME Classical Rett syndrome is an X-linked, dominant condition and manifests in females. In males MECP2 mutations are usually lethal. In females symptoms begin 6–18 months after birth and following normal initial development. Patients progressively lose motor and cognitive skills and develop abnormal hand movements. Later autistic symptoms and ataxia develop. In later stages the patients have seizures, hyperventilation, and apnea. There are several driving factors for development of pluripotent stem cells for neurologic diseases, such as Rett syndrome. The first is that animal models frequently do not recapitulate the full spectrum of manifestations of the human disease. Another factor is that cellular and histological features described as characteristic of a specific human neurological disease often only represent the late stages of the disease. Another important consideration is that differentiated cells derived from patients serve as a resource to analyze the effects of potential therapeutic agents on the disease process.

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Marchetto et al. (2010) reported development of pluripotent stem cells to study a number of different neurodegenerative and neurodevelopmental diseases. A number of interesting observations emerged from studies on Rett syndrome. In the process of becoming pluripotent, cells lost imprinting of the X chromosomes; however, following differentiation to neuronal cells X inactivation was once again present. Specific biomarkers of neural stem cells derived from Rett syndrome pluripotent stem cells included reduced numbers and defective function of glutamatergic synapses. Intracellular calcium movement was also impaired in these cells. Studies on therapeutic agents revealed that insulin-like growth factor (IGF) administration could increase glutamatergic synapse activity. However, the dosage and timing of administration were important. Marchetto et al. demonstrated that low doses of gentamycin restored MECP2 gene expression in a cell line derived from a patient with a MECP2 stop codon mutation. The restored levels of MECP2 protein led to normal glutamatergic responses. These investigators emphasized the importance of development of gentamycin analogs with low toxicity. The NBT aminoglycosides have been shown to be less toxic than gentamycin and to mediate enhanced suppression of premature stop codon mutations (Nudelman et al., 2009). Vecsler et al. (2011) reported that NB4 suppressed MECP2 nonsense mutations in Rett syndrome patients.

USE OF PLURIPOTENT STEM CELLS TO MODEL HUMAN DISEASES AND IDENTIFY THERAPIES In recent years mice have increasingly been used to produce models of human disease. One difficulty that emerges when mouse models are used to investigate the effects of a specific gene mutation is that the mouse develops a different constellation of symptoms (Tiscornia et al., 2011). When specific cystic fibrosis gene (CFTR) mutations that cause respiratory disease in humans are introduced into mice, the mice die of intestinal obstruction before they develop respiratory symptoms. There is also evidence that medications that work well in curing a specific disease in mice fail to cure that disease in humans. As pluripotent stem cell technology develops and as methods of gene targeting improve, pluripotent stem cells are used to study human diseases. Interactions between cells types or organs, complex metabolism, detoxification, and excretion profiles cannot necessarily be tested in a cell-based system. Tiscornia et al. (2011) identified human diseases in which cell-based studies were utilized to study specific diseases and drug responses. A number of these studies are described below.

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Hutchinson-Gilford progeria syndrome is a disease associated with premature aging, degeneration of vascular smooth muscle, and development of atherosclerosis. Cell-based assays revealed nuclear defects, including defects in the dense fibrillar network adjacent to the inner nuclear membrane that constitutes the nuclear lamina and reduction of heterochromatin. This disease is due to a point mutation in the Lamin A gene (LMNA) on chromosome 1q22. This gene gives rise to different protein isoforms. This progeria mutation occurs in one specific isoform and leads to production of a truncated form of the gene product, progerin. Studies in induced pluripotent cells led to discovery of a downstream target of lamin A, DNA-dependent protein kinase A (DNAPK). Decreased expression of the DNAPK holoenzyme serves as a marker of premature aging (Liu et al., 2011). A mouse model of Hutchinson-Gilford progeria syndrome (HGPS) has proven useful in the investigation of therapies (Osorio et al., 2011). The genetically modified mice that carry the HGPS mutation accumulate progerin and develop histological and clinical manifestations of the human disease. Osorio et al. developed an antisense morpholino-based therapy that prevents the abnormal splicing that leads to generation of progerin. Treatment with the antisense oligonucleotides led to marked improvement in the phenotype and prolonged life span in the mutant mice.

CELL-BASED STUDIES OF ARRHYTHMIAS In pluripotent cells developed from fibroblasts derived from patients with arrhythmias and cardiac abnormalities, specific targets and drug effects on these targets have been investigated. Studies on induced pluripotent stem cells differentiated into cardiac myocytes were used to investigate the effects of a specific mutation in the CACNA1C gene in Timothy syndrome. This mutation impacts the function of a calcium ion channel. Yazawa et al. (2011) demonstrated that the abnormal electrophysiological parameters could be reversed by treatment of cultures with roscovitine. In cardiac myocytes derived from patients with abnormalities in the potassium channel gene KCNH2, the electrophysiological abnormalities were evaluated, and their responses to specific drugs were investigated (Itzhaki et al., 2011). Moretti et al. (2010) derived pluripotent cells from fibroblasts from two patients with long QT syndrome and from two control individuals. They then differentiated these cells into functional myocytes with ventricular, atrial, or nodal phenotypes and recorded electrical action potentials on cells. They determined that differentiated ventricular and atrial myocytes derived from patient

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cells demonstrated reduction in current and altered channel activation and deactivation. They also demonstrated increased catecholamine sensitivity in the patient-derived cells and that pharmacological beta blockers had a protective effect against this sensitivity.

INDUCED PLURIPOTENT CELL-DERIVED NEURONS AND ANALYSIS OF PHENOTYPE IN TIMOTHY SYNDROME Derivation of pluripotent stem cells (IPS cells) from skin fibroblasts from patients with Timothy syndrome and their differentiation into neurons were first described by Yazawa et al. (2011). Subsequent studies on the cellular phenotype on IPSC-derived neurons were described by Pas¸ca et al. (2011). Timothy syndrome is due to an autosomal dominant mutation in the CACNA1C gene that maps to chromosome 12p13.3. This gene encodes the L-type calcium channel Cav1.2. The mutation is most commonly Gly406Arg (glycine to arginine), and it occurs in exon 8. A Gly402 Ser (glycine to serine) mutation has also been described in this disorder. The impaired calcium channel function leads to cardiac arrhythmia, hypoglycemia, and developmental delay, and more than half of the Timothy syndrome patients meet criteria for autism diagnosis (Splawski, 2004). In the studies described by Pasca et al., differentiated neural cells derived from IPS cells expressed neuronal markers MAP2 and NCAM. Subsets of these cells (85%) expressed neurotransmitter receptors and/or markers of lower cortical layer neurons FOXP1 and ETV. Cells characteristic of upper cortical layers expressed markers BCL11B, CUX1, SATB, or RELN and constituted 15% of the derived neural cell population. One subset of cells expressed markers characteristic of both lower and upper cortical layers. Pasca et al. considered the latter to be representative of subcortical projection neurons. Electrophysiological studies and calcium-imaging studies revealed that action potentials of Timothy syndrome IPS-derived neurons were 37% wider than those of neurons derived from control IPS cells. The intracellular calcium studies revealed an increased sustained rise in calcium following depolarization. Pasca et al. carried out analysis of gene expression in Timothy syndrome IPS-derived neurons and controls. They determined that Timothy syndrome– related altered gene expression particularly impacted CREB genes (cyclic AMP response element–binding genes) including calmodulin kinase (CAMKII) and other genes that are downstream targets of CREB (e.g., tyrosine hydroxylase and calcyon). These gene products are involved in production of dopamine and noradrenaline. They concluded that a downstream effect of the mutation in Timothy syndrome is a perturbation in catecholamine signaling and that

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this effect has therapeutic implications. Pasca et al. established that the altered tyrosine hydroxylase expression pattern in Timothy syndrome neural cells was reversed by treatment with roscovitine, a cyclin-dependent kinase inhibitor reported, and by treatment with an atypical L-type calcium channel blocker. NON–GENETICALLY MODIFIED STEM CELLS Uchida et al. (2000) directly isolated clonogenic human central nervous system stem cells (hCNS-SC) from fresh human fetal brain tissue using antibodies to specific cell surface markers and fluorescence-activated cell sorting. Neurosphere cultures were generated from single sorted cells. They determined that the progeny of these cells could differentiate into neural or glial cells. They transplanted these stem cells into brains of neonatal mice and demonstrated that these cells responded to host microenvironmental cues and were not neoplastic. Uchida et al. (2012) transplanted human fetal nervous system stem cells (hCNS-SC) differentiated to myelin-producing oligodendrocytes into brains of shiver mice. These mice have a defect in myelin product. Transplantation of hCNS-SC led to functional engraftment and production of donor-derived myelination of brain. Studies on transplantation of hCNS-SC into specific brain regions, frontal centrum semiovale, and corona radiata of patients with severe Pelizaeus-Merzbacher syndrome led to modest gains in neurological function (Gupta et al., 2012). These studies on transplantation of myelin-producing oligodendrocytes have relevance for treatment of dysmyelinating disorders. GENE TARGETING IN STEM CELLS New approaches to gene therapy include cleavage at specific sites followed by gene repair by homologous recombination to facilitate possibilities for correction of gene defects. Key reagents for carrying out targeted gene cleavage include zinc-finger nucleases and Tale nucleases (Talens) (Ellis et al., 2012) (see also Chapter 11).

MUTATION CORRECTION IN PATIENT-DERIVED IPS CELLS AND PROPOSED USE OF THESE CELLS FOR THERAPY Major hurdles in therapeutic application of pluripotent stem cells and derivatives are the safety concerns when retroviral vectors are used to transfer

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pluripotency induction factors. There are also safety concerns about the use of retroviral vectors or episomes to deliver correct DNA for targeting and homologous recombination. Papapetrou et al. (2011) described a strategy to integrate transgenes into safe harbor sites in the genome. These sites are regions where integrated transgenes are expressed but do not interfere with endogenous gene structure or function. Safe harbor sites are defined as sites located at least 30 kb from oncogenes or microRNAs, and located at least 50 kb from 5′ gene ends and outside ultraconserved regions of the genome. A genomic locus on human chromosome 19 constitutes a safe harbor site. It is located at a site at an adeno-associated virus (AAVS1) integration site. This locus is within the PPP1R12C gene that encodes the regulatory subunit 12C of protein phosphatase 1. Papapetrou et al. demonstrated that a lentiviral transgene encoding beta globin could readily be cloned into this safe harbor locus. Furthermore, they determined that the transgene was expressed at high levels under control of the PPP1R12C promoter without perturbation of expression of the surrounding genes. Hockemeyer et al. (2009) used zinc-finger nuclease to induce a double-stranded DNA break in the first intron of the PPP1R12C gene. They then introduced a doxycycline-inducible expression cassette and demonstrated that the transgene expression could readily be controlled by doxycycline treatment. There is evidence that pluripotency-inducing cassettes can be cloned into this safe harbor locus. Another strategy for transgene introduction is use of episomes or transposons. A favored transposon is the moth-derived Piggy Bac. In this transposon transgenes are cloned between inverted repeat sequences, and the transgene can subsequently be excised by Piggy Bac transposase (Yusa et al., 2009). Subsequently, Yusa et al. (2011) used a combination of zinc-finger nuclease digestion and introduction of a Piggy Bac transgene to achieve correction of a point mutation in the SERPINA1 gene that leads to alpha-1-antitrypsin deficiency (discussed further later in this chapter).

CORRECTION OF GLOBIN GENE DEFECTS IN STEM CELLS Sebastiano et al. (2011) used zinc-finger targeting to correct a Sickle hemoglobin mutation in patient-derived stem cells. One important aspect of their study design was the use of a polycistronic lentiviral vector for reprogramming stem cells. In this vector, originally described by Sommer et al. (2010), the pluripotency-inducing factors Oct, Kif4, Sox, and cMyc are flanked by loxp sites. Following reprogramming of pluripotent stem cells, the vector can

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be excised using cre recombinase. Another important aspect of the study was that gene-specific zinc-finger pairs were generated using a publicly available oligomerized pool engineering method (OPEN). To target the correcting sequence, the investigators used a gene cassette that also contained a puromycin selection cassette and lox p sites. Sebastiano et al. determined that gene-corrected cells retained pluripotency and normal karyotype following removal of reprogramming factors. The insertion of the puromycin facilitated selection of gene-modified cells, and the presence of the Lox p sites enables excision of the plasmid sequences by recombinase. In this study Sebastiano et al. achieved correction of the sickle hemoglobin mutation E6V in beta globin gene in induced pluripotent lines derived from two patients. Severe forms of alpha thalassemia include loss of three of the four alpha globin genes, leading to hemoglobin H disease, and loss of all four alpha globin genes, leading to hydrops foetalis. Chang and Bouhassira (2012) obtained homozygous alpha thalassemia fibroblasts from a repository. They derived pluripotent stem cells from these fibroblasts used an episomal vector. Cells that then developed the morphology of stem cells were cultured through several passages to ensure loss of the episome. Pluripotent stem cells were then differentiated to erythroid cells by coculture with fetal hepatocytes. These IPS cell then produced zeta globin. Chang and Bouhassira then used zinc-finger nuclease cleavage of the AAVS1 preferential integration site in intron 1 of the PPP1R12C locus and homologous recombination to integrate a transgene cassette with locus control sequences, promoters, and alpha globin genes into induced pluripotent stem cells. They measured the production of alpha globin and also expression of genes adjacent to the AAVS integration site. They defined an optimal transcript that led to expression of the alpha globin genes and less than 0.5% increase in expression of genes adjacent to the AAVS1 site. They determined that homozygous insertion of the alpha globin–containing transcripts led to complete correction of the globin imbalance in the erythroid IPS cells. Chang and Bouhassira noted that additional studies would be required to determine if alterations occurred elsewhere in the genome. Importantly, their study revealed that large genomic and gene defects could be repaired by endonuclease cleavage and homologous recombination

ALPHA-1-ANTITRYPSIN DEFICIENCY AND GENE CORRECTION Alpha-1-antitrypsin (AAT) deficiency occurs in 1 in 200 to 1 in 5,000 individuals and there are population differences in frequency. In a review of this disease,

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Stoller and Aboussouan (2005) reported that 120 different deficiency-causing mutations occur in AAT. Allelic variants were initially classified on the basis of protein electrophoresis, and the Z allele was found to be most commonly associated with disease. The alpha-1-antitrypsin locus encodes a member of the serine protease family, and the gene is now designated as SERPIN1. In addition to the Z allele, there are a number of variant alleles in SERPIN1 that lead to disease manifestations. Dysfunctional alleles are frequently associated with impaired elastase inhibition. Approximately 1.9% of patients with chronic obstructive pulmonary disease have AAT deficiency. The Z allele leads to abnormal intracellular accumulation of the SERPIN1-encoded protein and, in addition, to defective inhibition of neutrophil elastase. The Z allele is most frequently a Glu342Lys mutation, and homozygosity for this allele leads to emphysema and liver disease. In ZZ homozygotes, polymerized aggregates of AAT occur in hepatocytes. A number of other SERPIN1 mutant alleles including S alleles lead to abnormal inclusions in hepatocytes and may later lead to cirrhosis and hepatocellular carcinoma. Normal AAT produced in the liver enters the circulation and diffuses to the lung; there is also some evidence that AAT is produced by macrophages and by bronchial epithelial cells. In the lung AAT neutralizes elastase and other elastolytic enzymes produced by neutrophils and macrophages. AAT also has anti-inflammatory properties. Lung disease occurs in most but not all individuals with the ZZ genotype. The most common cause of death in these patients is respiratory failure, and the second most common cause of death is liver failure. Current treatments include augmentation with purified AAT (e.g., Aralast). The effectiveness of this treatment is variable; however, treatment is usually well tolerated, and yearly treatment costs range between $28,000 and $65,000. Safe and effective alternate treatment would represent major progress (Sandhaus, 2012). Yusa et al. (2011) carried out gene correction in IPS cells developed from fibroblasts of a patient with this deficiency. The IPS cells were subsequently differentiated to a hepatocyte phenotype. They had derived IPS cells using Sendai virus to introduce pluripotency factors in an integration-free method. Sendai virus is an RNA virus, and it does not integrate into the genome. They designed zinc-finger nuclease to cleave the Z allele mutation in SERPIN1A. Yusa et al. used a Piggy Bac vector that contained the normal SERPIN1 sequence in addition to a puromycin cassette adjacent to Piggy Bac repeats. The targeting vector and zinc-finger expression vector were coelectroporated into the pluripotent stem cells. Following zinc-finger nuclease cleavage and targeting puromycin resistance, colonies were isolated. In these colonies targeted, correction on one

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allele had occurred in 54%, and in 4% of colonies both alleles were corrected. The Piggy Bac repeat–flanked cassette was subsequently removed by digestion with Piggy Bac transposase. Yusa et al. reported that genomic changes were not present in the resulting colonies. These corrected IPSC colonies were then differentiated into hepatocytes that secreted albumin and stored glycogen, and they were negative for the A mutation in SERPIN 1. The corrected IPS cell–derived hepatocytes were then transplanted into mouse liver. Yusa et al. demonstrated that the human hepatocyte cells distributed through the liver and integrated into liver parenchyma.

X-LINKED CHRONIC GRANULOMATOUS DISEASE: ZINC-FINGER CORRECTION Zou et al. (2011) reported results of studies on induced pluripotent stem cells from a patient with X-linked chronic granulomatous disease. Patient-derived fibroblasts were converted to pluripotency using a self-inactivating lentivirus vector. They designed a minigene to correct the deficiency of the NADPH oxidase subunit that is defective in this disease. This minigene was targeted to the safe harbor locus (AAVS1) on chromosome 19 through use of zinc-finger homology arms. Following this targeting, stems cells were differentiated to neutrophils. Zou et al. reported that the NADPH oxidase defect was completely corrected in neutrophil-differentiated pluripotent stem cells.

PLURIPOTENT NEURAL STEM CELLS: EVALUATION AND THERAPEUTIC TESTING IPS cells differentiated to neurons have been used in a number of studies to investigate the effects of specific gene mutations on cellular functions. They have also been used to determine if specific therapeutic agents can restore normal function or enhance function.

IPS CELLS WITH TRISOMY 21 Shi et al. (2012) generated cortical neurons from embryonic stem cells and from induced pluripotent stem cells from cases of Down syndrome with trisomy 21, and from normal controls. In neuronal cells from Down syndrome patients, increased quantities of amyloid precursor protein (APP) are generated. Shi

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et al. monitored the production of APP cleavage products Abeta40 and Abeta42. They reported that cultures of Down syndrome neurons increased their production of Abeta40 to high levels, reaching 233 pg/ml by 28 days. In control neurons Abeta40 remained lower than 100 pg/ml. Extracellular medium from older cultures of Down syndrome neurons contained high levels, 843 pg/ml, of the pathologic Abeta42 form, compared with levels of Abeta42 of 32pg/ml in medium of control cultures. Shi et al. demonstrated that a gamma secretase inhibitor used in late-stage cultures of cells from Down syndrome patients reduced Abeta 40 and Abeta 42 production by half in 4 days, and long-term treatment led to Abeta production below detectable levels. They noted further that Down syndrome IPS-derived neuronal cells generated intracellular and extracellular Abeta 42 aggregates. Extracellular tau protein levels were found to be 4 times higher in medium from Down syndrome IPS cell or embryonic stem cell cultures than in medium from control cells. Phosphorylated forms of tau pSer396 and pThr231 were present in medium from Down syndrome cultures but not in medium from control cultures. It is important to note that tau is phosphorylated at Thr231 by the enzyme GSK3B (glycogen synthase kinase 3B). Amyloid, pTau, and GSK3B all play roles in Alzheimer’s disease.

IPS CELLS TO STUDY LATE-ONSET NEURODEGENERATIVE DISEASE There are examples of studies on IPS cells and differentiated neurons derived from skin cells in patients with neurodegenerative disorders of complex genetic etiology (e.g., Alzheimer’s disease). The goal of these studies was to analyze cellular pathology in derived neurons to determine to what extent it correlated with cellular pathology observed in Alzheimer’s disease in cases with defined, primarily monogenic forms of Alzheimer’s disease. Israel et al. (2012) reported results of studies on IPS cells derived from cases of familial Alzheimer disease, from two cases of sporadic Alzheimer’s disease, both with the ApoE3–3 genotype, and from controls. They observed significantly increased production of phosphotau and GSK3B in the familial Alzheimer’s disease cells and in cells from one of the sporadic cases. Levels of amyloid Abeta40 were also increased in these cells. Soldner et al. (2011) engineered a panel of zinc finger nucleases that specifically induced double-stranded breaks at nucleotide 209 in exon 3 of the alpha synuclein gene. An A53T mutation at this site leads to early-onset Parkinson’s disease.

ENVOI

My aim in writing this book was to explore a comprehensive range of approaches that are currently, in 2012–2013, being investigated and applied to the treatment of genetically determined diseases. I also undertook review of information related to the pretreatment analyses of disease-related pathology and changes in physiology that are required to design appropriate therapies. Possibilities for in-depth analyses exist at the levels of the genome, metabolome, and proteome. Furthermore, there are now enhanced capabilities for image analysis at the tissue and cellular levels. Progress in informatics continues to facilitate accurate diagnosis, therapeutic design, and analysis of the impact of therapies. This is indeed a time for hope! However, designing testing and implementing new therapies require perseverance and courage on the part of physicians, scientists, and patients, and all require support and encouragement from the public and from policymakers.

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INDEX

A53T, 238 A3243G, 88 A8344G, 88 AAQ. See acrylamide azobenzene quaternary ammonium AARS2. See alanyl-tRNA synthetase AAV, 65, 101 AAV2, 80 AAV-Ppt1, 80 AAVs. See adeno-associated viruses AAVSI, 223 ABCA1/3, 31, 130–32 ABCA7, 160 ABCD1/2, 31, 130, 131–32, 133 ABCG1/2/4, 31, 129 abetalipoproteinemia, 146 ABL, 201 ABO blood groups, 66 absorption, distribution, metabolism, excretion and toxicity (ADMET), 17 ACAD. See acyl-CoA dehydrogenase ACADS. See short-chain specific acyl CoA dehydrogenase ACC. See acetyl CoA carboxylase N-acetylaspartate, 123

acetyl CoA carboxylase (ACC), 53, 54 N-acetyl cysteine, 81 N-acetyl-D-mannosamine, 64 N-acetyl-D-mannose-6-phosphate, 64 N-acetylgalactosamine, 61–62 N-acetylglucosamine-1-phosphodiester-alphaN-acetyglucosaminidase (NAGPA), 69 N-acetylneuraminic acid. See sialic acid N-acetylornithine, 65 N-acetyltransferase, 65 N-acetyl UDP glucose (UDPglucose-Nac), 64 acrylamide azobenzene quaternary ammonium (AAQ), 148 acute myeloid leukemia (AML), 193 acylcarnitine, 41, 44f acyl-CoA dehydrogenase (ACAD), 46, 93 acylglycerol kinase (AGK), 93 ADCK3, 96 adeno-associated viruses (AAVs), 217–18, 219, 234 ADMET. See absorption, distribution, metabolism, excretion and toxicity ADP, 121 adrenoleukodystrophy (ALD), 133 adrenoleukodystrophy protein (ALPD), 132

283

284

Index

adrenomyeloneuronopathy (AMN), 132, 133 AG, 208 AGAT. See arginine glycine amidotransferase AGC, 123–25 AGGTCA, 31 AGK. See acylglycerol kinase AHDH. See attention deficit hyperactivity disorder AIF. See apoptosis-initiating factor AIRCAR, 97 AKT, 21, 22, 23, 25–26, 97, 174, 186, 199, 202 alanyl-tRNA synthetase (AARS2), 93 ALB111, 211 ALD. See adrenoleukodystrophy ALG6, 63 ALK. See anaplastic lymphoma kinase alkaptonuria, 65 Allan-Herndon-Dudley syndrome, 150–51 ALPD. See adrenoleukodystrophy protein alpha 1-antitrypsin (α1 proteinase, ATT), 15, 235–37 alpha amino-3-hydroxy-5-methylisoxazole-4propionic acid (AMPA), 144, 175 alpha globin, 235 alpha mercaptoproponylglycine (MPG), 119 alpha synuclein, 114, 238 ALS. See amyotrophic lateral sclerosis Alzheimer’s disease, 158–69, 238 aminoacyl-tRNA synthetase deficiencies, 88 aminoterminal domain, 30 AML. See acute myeloid leukemia ammonia, inborn errors of metabolism and, 41 AMN. See adrenomyeloneuronopathy AMPA. See alpha amino-3-hydroxy-5methylisoxazole-4-propionic acid AMPK1, 21 amyloid precursor protein (APP), 158, 162, 193, 237–38 amyotrophic lateral sclerosis (ALS), 169–70 anaplastic lymphoma kinase (ALK), 201 androgen receptor gene, 17, 29 aneurysms-osteoarthritis syndrome, 28, 29 Angelman syndrome, 173 angiomyolipomas, 21 angiotensin, 28 ankyrin 3 (ANK3), 182 ANT1, 121–22 anti-Rhesus immunoglobulin, 16 antisense oligonucleotides, 214–15 Apo B, 74

apolipoprotein E (APOE), 160, 162–64, 163f, 238 apoptosis-initiating factor (AIF), 85 APP. See amyloid precursor protein ARC, 175 arginine glycine amidotransferase (AGAT), 119 arginosuccinate synthase, 124, 125f arginyl-tRNA-synthetase (RARS), 88 aromatase, 33 aromatase decarboxylase, 60 arrhythmias, 231–32 arterial tortuosity syndrome, 28 ASD, 173 aspartate glutamate carriers, 123–24, 124f aspartyl tRNA synthetase (DARS2), 88 astrocytes, 24, 129–30 ATF6, 104 ATP, 112, 115, 119, 129, 201 cancer and, 185, 186 mitochondria and, 83, 85, 89, 101 RTKs and, 199 SLC25A4 ANT1 and, 121 ATP6, 94 ATPase, 87f, 108 ATP binding cassette transporters, 130, 131–32 ATT. See alpha 1-antitrypsin attention deficit hyperactivity disorder (AHDH), 181 autism, 10, 172–80, 177f autophagosomes, 71–72 bafilomycin A1, 108 Bassen-Kornzweig disease, 146 BCKDK, 176–78 BCL11A/B, 138, 139, 232 BCR-ABL, 203 BCS1L, 93 BDNF. See brain-derived neurotrophic factor Becker muscular dystrophy, 212 Bell, Julia, 2–3 beta arrestins, 32 beta catenin, 205, 206f 17 beta-estradiol, 33 beta globin, 138 Bevacizumab, 16 bezafibrate, 97 BGP15, 142 BH4. See tetrahydrobiopterin Biglycan, 141 BIN1, 160

Index biopterin, 55, 56, 57f biotin, 51–54, 52f, 54f biotin dichloroacetate, 123 biotinidase, 51–54, 52f bismonoacylglycerophosphate (BMP), 76 blood oxygen level-dependent (BOLD), 162 BMP. See bismonoacylglycerophosphate BOLD. See blood oxygen level-dependent bone morphogenic protein, 15 bortezomib, 79 BRAF-mutant melanoma, 202 brain-derived neurotrophic factor (BDNF), 144, 165, 169, 176, 227, 228 BRCA1/2, 197, 198f BREAF, 199 breast cancer, 16 N-butyryl-deoxynojirimycin, 79 C2H2, 222 C9ORF72, 169 C12 dodecanoic acid, 43 C90RF72, 216 CA3/4, 116 CACNA1C, 173, 181, 231, 232 CACT. See carnitine acyl-carnitine translocase CAG, 29, 215 calcitonin gene-related peptide (CGRP1), 30 calcium, 89, 109 calmodulin kinase (CAMKII), 232 CAMKII. See calmodulin kinase cancer, 185–206 chromosomes and, 199–200 FH and, 194–96, 196f fusion genes and, 200–204 germline mutations and, 194–95 glucose and, 189–90 glutamine and, 190–91 IDH1 and, 192–94 immunotherapy for, 206 monocarboxylate transporters and, 194 protein kinases and, 198–99 signaling pathways and, 186–87, 187f synthetic lethality and, 196–97, 198f tyrosine kinases and, 200–204 captopril, 97 carbohydrates, 41, 51, 67 carboxylases, 53 carboxyterminal domain, 30 carnitine, 41, 42–45, 49, 51, 126–28, 179f carnitine acyl-carnitine translocase (CACT), 44–45 carnitine octanoyl transferase, 45

285

carnitine palmitoyl transferases (CPT), 43–45 catechol O methyl transferase (COMT), 181–82 CBT. See cognitive behavioral therapy CDK. See cyclin-dependent kinase CDKL5, 142 C/EBP homologous protein (CHOP), 105 CELF, 215 ceramide, 77f cerebral palsy, 59 cerebrospinal fluid (CSF), 56, 60 ceroid lipofuscinoses, 79–81, 80f cetuximab, 16, 205 CFTR. See cystic fibrosis CGDs. See glycosylation disorders CGRP1. See calcitonin gene-related peptide channels, 115 Charcot-Marie-Tooth disease (CMT), 151–54 cholesterol, 23, 74, 216–17 cholesterol 7a hydroxylase, 31 CHOP. See C/EBP homologous protein chromatin, 143 chromatography, 4 chromosomes, 5–6 autism and, 178–80 cancer and, 199–200 identification of, 6 chronic myeloid leukemia (CML), 200 citrulline, 120 citrullinemia, 124–25, 125f CLC1, 215 CLN1/2, 80–81 CLPP ATP-dependent protease, 109 clusterin (CLU), 160–61 CML. See chronic myeloid leukemia CMT. See Charcot-Marie-Tooth disease cMYC, 228, 229f CNGB1, 148 CNV. See copy number variation coenzyme A reductase, 24 coenzyme Q (CoQ), 83, 95–96, 123 coenzyme Q10 (CoQ10), 98–100, 99f cofactors, 3–4, 40, 48–49, 95 cognitive behavioral therapy (CBT), 183–84 collateral vulnerability, 197 COMT. See catechol O methyl transferase copy number variation (CNV), 10 CoQ. See coenzyme Q CoQ10. See coenzyme Q10 corticosteroids, 140 Cowden syndrome, 22–23 COX. See cytochrome c oxidase

286

Index

CpG. See cytosine-guanine CPT. See carnitine palmitoyl transferases creatine, 118, 119–20 creatine kinase, 141 creatine phosphate, 118, 119 CREB, 174, 232 CRIMM, 79, 160 CSF. See cerebrospinal fluid CTNS, 126 CUG. See 2-O-methylphosphorothioate CUGBP1, 215 CUX1, 232 CX546, 144 cyclic guanosine monophosphate (GMP), 146–47 cyclin-dependent kinase (CDK), 164 cyclocreatine, 120 cyclodextrin, 75 CYP2, 34, 36f, 37 cysteine, 127f, 222 cystic fibrosis (CFTR), 8, 15, 230 cystinosis, 125–26 cystinuria, 118 cytochrome C, 83, 109 cytochrome c oxidase (COX), 87, 89, 90, 121 cytochrome P450, 34 cytosine-guanine (CpG), 143 D374Y, 159 DAP, 166, 168 DARS2. See aspartyl tRNA synthetase delta globin, 138 dentatorubral pallidoluysian atrophy (DRPLA), 215 1-deoxynojirimycin (DNJ), 79 deoxythymidine, 88 depression, 184 desmoplakin, 153 dexpramipexole, 170 DHA. See docosahexanoic acid DHODH. See dihydro-orotate DHPR. See dihydropteridine reductase diabetes, 30, 63 dicarboxylic acids, 46 diffusion tension imaging (DTI), 150, 173 diflunisal, 111 dihydrobiopterin, 58–59 dihydro-orotate (DHODH), 92 dihydropteridine reductase (DHPR), 56, 58 L-dihydroxyphenylalanine (L-DOPA), 59–60 DM. See myotonic dystrophy DMD. See Duchenne muscular dystrophy

DMPK1, 215, 216 DNA-dependent protein kinase A (DNAPK), 231 DNAJ, 172 DNAPK. See DNA-dependent protein kinase A DNA sequencing, 8–9, 9f, 92–95 DNJ. See 1-deoxynojirimycin docosahexanoic acid (DHA), 145 dopamine, 55–56, 59–60 dopamine beta hydroxylase, 228 dopamine transporter defects, 60 Down syndrome, 237–38 doxycycline, 28, 234 Drews, Jurgen, 16 DRP1, 85 DRPLA. See dentatorubral pallidoluysian atrophy DSMN2, 210–11 DSP, 153 DTDS. See dyskinesia, dystonia, chorea, and abnormal eye movements DTI. See diffusion tension imaging Duchenne muscular dystrophy (DMD), 2, 3, 8, 140–42, 212–13 dyskinesia, dystonia, chorea, and abnormal eye movements (DTDS), 60 dystrophic epidermolysis bullosa, 155 E (APOE), 31, 158–59, 162 E2, 112–13 E3, 113 E4, 158–59 EAAT2, 170–71 4EBP. See EIF4E binding protein EEG. See electroencephalography EGF. See epidermal growth factor EGFR. See epidermal growth factor receptor EGR2, 151 Ehrlich, Paul, 16 EIF2A, 105, 108 EIF4E binding protein (4EBP), 23 electroencephalography (EEG), 182 electron transfer flavoprotein ubiquinone dehydrogenase (ETFQO), 50 electron transport flavoprotein (ETF), 50 EML, 201 endoplasmic reticulum (ER), 14, 104–6, 105f mitochondria and, 109 stress, 107–8, 110–11 endoplasmic reticulum degradation system (ERAD), 111, 112

Index endosomes, 71–72, 71f ENO1. See enolase 1 ENO2, 197 enolase 1 (ENO1), 197 enterocytes, 18 enzyme replacement therapy (ERT), 76–79 enzymes, 5, 13 CGDs and, 62–65 EPHA4, 170 ephrin, 170 EPI-743, 98–100 epidermal growth factor (EGF), 26, 204–5 epidermal growth factor receptor (EGFR), 16, 199, 204 epidermolysis bullosa, 153–55 epilepsy, 22 epinephrine, 55, 60 ER. See endoplasmic reticulum ERAD. See endoplasmic reticulum degradation system ERB, 2 ERBB, 200f, 204–5 ERK. See extracellular signal-related kinase erlotinib, 205 ERO1, 106 ERT. See enzyme replacement therapy erythropoietin, 15 ESCRT, 71 estrogen receptors, 33 ESTs. See expressed sequence tags ETF. See electron transport flavoprotein ETFQO. See electron transfer flavoprotein ubiquinone dehydrogenase ETHE1. See ethylmalonic encephalopathy ethylmalonic acidemia, 89 ethylmalonic encephalopathy (ETHE1), 87, 90, 92 ETV, 232 eukaryotes, 208 Everolimus, 22 exome sequencing, 159–60 exon skipping, 212–13 exosomes, 220 expressed sequence tags (ESTs), 6 extracellular signal-related kinase (ERK), 33, 72, 149, 202 eye diseases, 218–19 F216L, 159 Fabry disease, 15 Factor IX (FIX), 2, 15, 218 Factor VIII, 2, 15, 218

287

FADH2, 83 familial amyloid polyneuropathies (FAPs), 110 familial dysautonomia, 209–10 Fanconi syndrome, 126 FAPs. See familial amyloid polyneuropathies Farnesoid (FXR), 30 Farnesyl transferase inhibitors, 24 fatty acid dehydrogenases, 45 fatty acids, 23, 40, 43–46, 43f, 91, 126 FBN1. See Fibrillin 1 FDG PET. See fluoro-D-glucose positron emission tomography fenofibrate, 81 fetal hemoglobin (HbF), 138, 221 FH. See fumarate hydratase Fibrillin 1 (FBN1), 27–28 Filgrastim, 15 Fischer, Edmond, 198 Fischer, Emil, 3 FISH. See fluorescent in situ hybridization FIX. See Factor IX FKBP, 23, 24 FKTN. See Fukutin-encoding gene flavin adenine dinucleotide, 48–49, 50 fluorescent in situ hybridization (FISH), 6, 7f fluoro-D-glucose positron emission tomography (FDG PET), 186 FMR1, 174 fMRI. See functional magnetic resonance imaging FMRP. See fragile X mental retardation protein Folling, Asborn, 4 FOX, 142, 232 fragile X mental retardation protein (FMRP), 174–75 FRDA, 99 Friedreich ataxia, 100 Fukutin-encoding gene (FKTN), 211–12 Fukuyama muscular dystrophy, 211–12 fumarate hydratase (FH), 194–96, 196f functional magnetic resonance imaging (fMRI), 182 fusion genes, 200–204 FXR. See Farnesoid G6PD. See glucose-6-phosphate dehydrogenase G39A, 170 GABA. See gamma amino butyric acid GADD34, 105, 108

288

Index

GAG. See glycosaminoglycan gamma amino butyric acid (GABA), 144, 176, 228 gamma globin, 138–39, 220–21 gapmer oligonucleotides, 216 Garrod, A.E., 1 gas chromatography (GC), 41, 42, 65 gastrointestinal stromal tumors (GIST), 195 GATA1, 138, 221 Gaucher disease, 15, 77f GC. See gas chromatography GCDH. See glutaryl CoA dehydrogenase G CIMP. See glioma-specific methylation GCK5, 172 gefitinib, 205 gemfibrozil, 81 gene-based molecular therapies, 207–23, 208–9, 208f genistein, 33 genome-wide association studies (GWASs), 35, 158–59, 167–68, 181 gentamicin, 230 germline mutations, 13, 194–95 GFM1/2, 88, 93 Giemsa banding, 6, 7f GIST. See gastrointestinal stromal tumors GJA12, 150 GJC2, 150 GlcNac-phosphotransferase, 69, 69f Gleevec. See imatinib glioma-specific methylation (G CIMP), 193 globin, 139, 139f, 234–35 glucocerebroside, 77f glucose, 3, 41, 49, 189–90 glucose-6-phosphate dehydrogenase (G6PD), 34 glucose-mannose-N-acetylglucosamine, 68 GLUT, 117, 129 glutamate, 3, 170–71 glutamate oxaloacetate transaminase (GOT), 191 glutamine, 190–91 glutamine pyruvate transaminase (GPT), 191 glutaric aciduria, 48–51, 49f glutaryl CoA dehydrogenase (GCDH), 48–49 glutathione (GSH), 186 glycogen storage diseases, 67. See also Pompe disease glycogen synthase kinase 3B (GSK3B), 160, 164, 172, 238 glycosaminoglycan (GAG), 41, 73 glycosphingolipids, 61

N-glycosylation, 60–61, 60f glycosylation disorders (CGDs), 60–65 GM2/3, 116, 117 GMP. See cyclic guanosine monophosphate GMPS. See guanosine monophosphate synthase GNE, 63, 65 GNMT. See guanidinoacetate methyl transferase GNPT, 69, 70 GNTABG, 70 GOT. See glutamate oxaloacetate transaminase gout, 128–29 GPCR. See G-protein coupled receptors GPER, 33 G-protein coupled receptors (GPCR), 32–33 GPT. See glutamine pyruvate transaminase graft-versus-host disease, 225 granulin, 108 granulocyte-stimulating protein, 15 growth factors, 21, 22–23 growth hormone, 15 GRP44, 104 GRP78, 106, 107 GSH. See glutathione GSK3B. See glycogen synthase kinase 3B GT, 208 GTPase, 24, 146 GTP cyclohydrolase (GTPCH), 56, 58 guanidinoacetate methyl transferase (GNMT), 119 guanosine monophosphate synthase (GMPS), 191 guanylate cyclase 2D, vitamin A trans-cis isomerase (RPE45), 219 GWASs. See genome-wide association studies HADHA/B, 47, 48 Haldane, J.B.S., 2 hamartomatous syndromes, 22 Harris, Harry, 5, 8 HARS. See histidinyl-tRNA synthetase HbF. See fetal hemoglobin hCNS-SC. See human central nervous system stem cells HCS, 54, 55 HDAC. See histone deacetylases heat shock factor 1 (HSF1), 104, 172 hematopoietic stem cells, 79, 134 hemimegancephaly, 26 hemoglobinopathies, 138–40, 220–21 hemophilia, 2, 217–18

Index HER2/3/4, 16, 199, 200f, 204–5 Herceptin, 16, 205 hereditary inclusion body myopathy (HIBM), 63–65, 64f HERP, 112 HGPRT. See hypoxanthine guanine phosphoribosyl transferase HGPS. See Hutchinson-Gilford progeria syndrome HHH syndrome, 120 5-HIAA. See 5-hydroxyindole acetic acid HIBM. See hereditary inclusion body myopathy HIF. See hypoxia-inducible transcription factors high-performance liquid phase chromatography (HPLC), 65, 88 high-throughput screening, 17, 90–92 histidinyl-tRNA synthetase (HARS), 88 histone deacetylases (HDAC), 107, 133–34 HLA. See human leucocyte antigen HLCS, 54 HLRCC syndrome, 194 HMGCoA, 96 HMPAO. See technetium-99-hexamathylpropylenenamine-oxime holocarboxylase synthetase, 53–54, 54f holoretinol binding protein, 111 homovanillic acid (HVA), 56, 58, 59, 60 HPLC. See high-performance liquid phase chromatography HPV11, 203 HSF1. See heat shock factor 1 HSP10, 109 HSP40, 104 HSP60, 109, 168 HSP70, 76, 109, 112, 172 HSP72, 141–42 HSP90, 104, 107, 109, 112, 172 HSP97, 112 HSP110, 112 HTT, 3, 215–16 human central nervous system stem cells (hCNS-SC), 233 human leucocyte antigen (HLA), 35, 36t, 225 Hunter disease, 15, 75f Huntington, George, 3 Huntington’s disease, 2, 8, 168 Hurler disease, 15, 75f Hutchinson-Gilford progeria syndrome (HGPS), 231 HVA. See homovanillic acid

289

hydroxamic acid, 134 hydroxycholesterols, 31 5-hydroxyindole acetic acid (5-HIAA), 59, 60 L-hydroxylysine, 48 5-hydroxytryptamine. See serotonin hyperammonemia, 120, 125 hypercholesterolemia, 216–17 hyperphenylalaninemia, 56–57 hypoglycemia, 50 hypoxanthine guanine phosphoribosyl transferase (HGPRT), 129 hypoxia-inducible transcription factors (HIF), 27, 185, 187, 194, 196 idebenone, 97–98 IDH1. See isocitrate dehydrogenases 1 IGF. See insulin-like growth factor IKAP, 209–10 imatinib (Gleevec), 201 inborn errors of metabolism, 4, 39–66, 52f, 176–78, 177f induced pluripotent stem cells (IPS), 226–27, 233–34, 237–38 insulin-like growth factor (IGF), 15, 140, 144, 230 ion channels, 173, 176 IPS. See induced pluripotent stem cells IRE1, 104, 105–6 isocitrate dehydrogenases 1 (IDH1), 192–94 ITAM, 166 ITG, 153 Janus kinase (JAK2), 201 JNK. See Jun N terminal kinase junctional epidermolysis bullosa, 154–55 Jun N terminal kinase (JNK), 30, 141 K27, 114 K63, 114 KCNH2, 231 KCTD13, 180 KCTN13, 180 Kearns-Sayre syndrome, 100 KEGG, 191 Kennedy’s disease, 29 keratin disorders, 153–55 keratinocyte growth factors, 15 Kindler syndrome, 155 KIT, 203 KLF1/4, 139, 227, 228, 229f Krebs, Edwin, 198 kynurenine hydrolase (KYNU), 194

290

Index

lactase, 41 lactate, 3, 89, 129–30 LAM. See lymphangioleiomyomatosis lamin A (LMNA), 231 LAMP. See lysosome-associated membrane protein lasofoxifene, 33 LCAD. See long-chain acyl CoA dehydrogenase LCHAD. See long-chain L3 hydroxyacyl-CoA dehydrogenase LDL. See low-density lipoprotein L-DOPA. See L-dihydroxyphenylalanine Leber’s congenital amaurosis, 147 Leber’s hereditary optic neuropathy (LHON), 94, 99, 101 lecithin retinal acetyltransferase (LRAT), 147 Leigh syndrome, 84, 94, 100, 101 leucine-rich pentatricopeptide repeatcontaining (LRPPRC), 92 levodopa-responsive disorder, 59 LHON. See Leber’s hereditary optic neuropathy LIMP2, 69 lipids, 41, 74 lipopolysaccharide-induced lysosomal membrane protein (LITAF), 151 liquid chromatography, 41, 132 LITAF. See lipopolysaccharide-induced lysosomal membrane protein liver X receptor (LXR), 30, 31 LKAT. See long-chain-3 keot COA thiolase LKB1, 21, 22 LMNA. See lamin A Loeys-Dietz syndrome, 28 long-chain-3 keot COA thiolase (LKAT), 47, 48 long-chain acyl CoA dehydrogenase (LCAD), 47 long-chain L3 hydroxyacyl-CoA dehydrogenase (LCHAD), 47–48 LON protease, 109 Lorenzo’s oil, 133 low-density lipoprotein (LDL), 216 LRAT. See lecithin retinal acetyltransferase LRPPRC. See leucine-rich pentatricopeptide repeat-containing LXR. See liver X receptor lymphangioleiomyomatosis (LAM), 21 L-lysine, 48 lysosomal storage diseases, 67–81, 74t, 77f

lysosome-associated membrane protein (LAMP), 71, 79, 220 lysosomes, 4, 68–69, 71–72, 71f lysyl-tRNA synthetase (YARS), 88 MADD. See multiple CoA dehydrogenase deficiency magnetic electroencephalography (MEG), 182 ManNac-kinase, 64 mannose-1-phosphate isomerase, 63 mannose-6-phosphate, 68, 69, 70f MAP2, 232 MAPK, 164 MARCH4, 107 Marfan syndrome, 27–29 MARK2, 172 mass spectrometry (MS), 4, 39–40, 41, 42. See also tandem mass spectrometry matrix metalloproteinase (MMP), 141, 175 MCAD. See medium chain acyl CoA dehydrogenase MCC. See 3-methyl crotonyl carboxylase MCOLN1. See mucolipin 1 MCTs. See medium-chain triglycerides ME132, 79 MECP2, 142, 143, 145, 176, 229–30 MED12, 204 MEDIATOR complex, 204 medium chain acyl CoA dehydrogenase (MCAD), 40–41, 46, 50 medium-chain triglycerides (MCTs), 45, 129–30, 151 MEG. See magnetic electroencephalography MEK, 199 MELAS syndrome, 87–88, 94, 99, 100 MERF, 84 MERFF, 88 MERTK. See mertyrosine kinase oncogene Mertyrosine kinase oncogene (MERTK), 219 messenger RNA (mRNA), 3, 140, 208, 209 metabolome, 65–66, 66f metalloproteinase (MMP9), 28 metformin, 63 methionine, 178 3-methyl crotonyl carboxylase (MCC), 53 methylmalonic aciduria, 4, 42 2-O-methylphosphorothioate (CUG), 215 MFN1/2, 85 MHC, 181 mitochondria, 4, 83–101, 85f, 98f analysis of function of, 89 ATPase and, 87f

Index biosignatures for, 86f cell and tissue transplantation and, 101 CoQ for, 95–96 DNA mutations in, 84–86 DNA sequencing for, 92–95 ER and, 109 gene delivery and, 100–101 heteroplasmy clonal expansion and, 84–86 idebenone for, 97–98 nucleotides and, 88–89 phenotypes and, 84–86, 94 protein and, 87–92 small-molecule testing for, 97 transporters and, 120–25 UPR and, 108–9 mitochondrial neurogastrointestinal encephalopathy (MNGIE), 88 mitochondrial targeting sequence (MTS), 101 mitochondrial trifunctional protein (MTP), 47–48 mitoporters, 97 MMBO, 164 MMP. See matrix metalloproteinase MNFIE syndrome, 101 MNGIE. See mitochondrial neurogastrointestinal encephalopathy monoamine neurotransmitters, 55 monocarboxylate transporters, 129–30, 194 monoclonal antibodies, 15–17 monogenic diseases, 19–21, 24, 137–55 Movement Disorder Childhood Rating Scale, 100 MPG. See alpha mercaptoproponylglycine MPR, 69 MPZ. See myelin basic protein zero mRNA. See messenger RNA MS. See mass spectrometry MS/MS. See tandem mass spectrometry mtDNAJ, 109 mTERFD3, 88 mTOR, 13, 19, 20f, 21–22, 23–24, 26, 185, 187, 191, 199, 202 mTORC1/2, 19, 20f, 21, 22–23 MTP. See mitochondrial trifunctional protein MTS. See mitochondrial targeting sequence mucolipidosis, 70, 72 mucolipin 1 (MCOLN1), 72 mucopolysaccharide storage diseases, 73, 75f mucopolysaccharidosis, 15 multiple CoA dehydrogenase deficiency (MADD), 50–51, 51f MYC, 191

291

myelin basic protein zero (MPZ), 151 myelin sheath, 149 myotonic dystrophy type (DM), 215–16 N17K, 155 NADH, 83, 89, 191 NADH dehydrogenase (ubiquinone) 1 beta subcomplex 3 (NDUFB3), 93 NAD/NADP quinone oxidoreductase 1 (NQO1), 100 NADP, 92 NADPH, 186, 191 NAFLD. See nonalcoholic fatty liver disease NAGPA. See N-acetylglucosamine1-phosphodiester-alpha-Nacetyglucosaminidase Naratriptan, 30 NARP. See neurogenic muscle weakness, ataxia, and retinitis pigmentosa NAT8, 65 NB4, 230 NBT54, 144 NCAM. See neural cell adhesion molecule NCAN. See neurocan ND1/4/6, 94, 101 NDRG1, 152 NDUFB3. See NADH dehydrogenase (ubiquinone) 1 beta subcomplex 3 NEFL, 151 Neufeld, Elizabeth, 68 neural cell adhesion molecule (NCAM), 228, 232 neural oscillations, 183 neural stem cells, 227–29 neuregulins, 205 neurexin (NRX1), 173 neurocan (NCAN), 181 neurofibromatosis (NF1), 8, 24, 173, 209 neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), 94 neurotransmitters, 55–56, 144, 176 newborn screening, 40–41, 52 next-generation sequencing, 9, 9f NF1. See neurofibromatosis NF kappa B. See nuclear factor kappa B NHE6, 116 Niemann-Pick disease, 73–74, 76, 209 nitric oxide synthase, 56 NMDA, 22, 144, 175 NMDA glutamate receptor 1 (NMDAR1), 107 NMR. See nuclear magnetic resonance NMYC, 152

292

Index

Nonaka myopathy, 63 nonalcoholic fatty liver disease (NAFLD), 107 non-small cell lung cancers (NSCLCs), 201, 203–4 norepinephrine, 55, 60 NPC1/2, 74, 209 NQO1. See NAD/NADP quinone oxidoreductase 1 NRX1. See neurexin NSCLCs. See non-small cell lung cancers nuclear factor kappa B (NF kappa B), 141 nuclear hormone receptors, 30–31 nuclear magnetic resonance (NMR), 17, 41 OCT4, 223, 227, 228, 229f OCTN2, 42, 126–28, 178 ODZ4, 181 3OH glutaric acid, 43 oligomerized pool engineering method (OPEN), 235 oligonucleotides, 208–9, 208f O-linked glycosylation, 60, 60f omega 3/6, 47 OPEN. See oligomerized pool engineering method orphan receptors, 33 Otto, John C., 2 oxidative phosphorylation (OXPHOS), 86, 91, 95 PARK7, 114 Parkinson’s disease, 168, 229 paronychia congenita, 155 PARP1, 197, 198f PC. See pyruvate carboxylase PCC. See propionyl CoA carboxylase PCD. See pterin-4a-carbinolamine dehydratase PCSK9, 159–60, 160f PDE6A/B, 146 PDGF. See platelet-derived growth factor PDGFR. See platelet-derived growth factor receptors PDSS1/2, 96 Pelizaeus-Merzbacher disease (PMD), 148–51 penicillamine, 119 penicillin, 16 PEP. See phosphoenolpyruvate peripheral myelin protein 22 (PMP22), 152 PERK, 104–6, 107 peroxisomes, 4, 131 Peruvoside, 17 PET. See positron emission tomography

Peutz Jeghers syndrome, 22 PGC1 alpha, 31 pharmacogenetics, 33–35 pharmacogenomics, 33–35 pharmacology, 18–19 Phelan-McDermid syndrome, 173 phenotypes, 10, 84–86, 94, 232–33 phenylalanine, 57f phenylbutyrate, 97, 120 phenylketonuria (PKU), 4, 39, 40 PHGDH. See 3-phosphoglycerate dehydrogenase phosphatidyl inositol diphosphate (PIP2), 22 phosphatidyl inositol triphosphate (PIP3), 22 phosphocysteamine, 81 phosphoenolpyruvate (PEP), 187, 188, 188f phosphoenolpyruvate carboxykinase, 31 3-phosphoglycerate dehydrogenase (PHGDH), 189–92, 190f 3-phosphohydroxypyruvate (3PYR), 189 phosphoinositide-3-kinases (PI3K), 21, 22, 25–26, 174, 186, 199, 202 phosphoinositol-biphosphate (PIBP), 25 phosphomannomutase 2 (PMM2), 62–63 phosphomannose isomerase, 62–63 phthalazinone, 162–63 phytoestrogens, 33 PI3CA, 25–26 PI3K. See phosphoinositide-3-kinases PIBP. See phosphoinositol-biphosphate PICLAM, 160 Piggy Bac, 226, 234, 236–37 PINK1, 85 PIP2. See phosphatidyl inositol diphosphate PIP3. See phosphatidyl inositol triphosphate PK1. See pyruvate kinase 1 PKB, 25 PKC alpha, mTORC2 and, 23 PKL, 187 PKM. See pyruvate kinase PKP1, 153 PKR, 187 PKU. See phenylketonuria plakophilin 1, 153 plasma membrane-bound receptors, 32–33 platelet-derived growth factor (PDGF), 27 platelet-derived growth factor receptors (PDGFR), 201, 227 PLEC1, 153 PLOSL. See polycystic lipomembranous sclerosing leukoencephalopathy PLP1, 149, 150

Index pluripotent stem cells, 143, 225–38 for arrhythmias, 231–32 Rett syndrome and, 229–30 for Timothy syndrome, 232–33 PLX4032, 202 PMD. See Pelizaeus-Merzbacher disease PMM2. See phosphomannomutase 2 PMP22. See peripheral myelin protein 22 POLG, 93, 99, 100 polycystic lipomembranous sclerosing leukoencephalopathy (PLOSL), 166 polyglutamine, 29 Pompe disease, 5, 15, 78–79 positron emission tomography (PET), 163 FDG PET, 186 PP2A, 172 PP5, 172 PPA, 97 PPAR. See proliferator activated receptors PPARgamma, 23 PPP1R12C, 223, 234, 235 PPT1, 80 Prader-Willi syndrome, 179 presenilin, 158 progranulin, 108 proliferator activated receptors (PPAR), 30–31 Prontosil, 16 propionic aciduria, 42 propionyl CoA carboxylase (PCC), 53 protein, 5, 13–17, 87–92 folding and unfolding, 103–7 lysosomes and, 68–69 mTORC1 and, 23 protein C, 15 protein kinases, cancer and, 198–99 proteosome system, 112 Proteus syndrome, 26 PS1/2, 162 PSD95, 107, 144 psychiatric disorders, 180–84 PTC124, 140, 144 PTEN, 22–23, 26, 174 pterin-4a-carbinolamine dehydratase (PCD), 56, 58 PTP. See 6-pyruvolyltetrahydrobiopterin PTPS. See 6-pyruvoly-tetrahydrobiopterin synthase PYCR1. See pyrroline-5-carboxylate reductase 3PYR. See 3-phosphohydroxypyruvate pyrimidine, 91 pyrroline-5-carboxylate reductase (PYCR1), 92, 191

293

pyruvate, 3, 4, 41 pyruvate carboxylase (PC), 53 pyruvate kinase (PKM), 188 pyruvate kinase 1 (PK1), 187–89, 188f 6-pyruvolyltetrahydrobiopterin (PTP), 58–59 6-pyruvoly-tetrahydrobiopterin synthase (PTPS), 56, 58 QT syndrome, 231 RAB5/7, 71, 152 rabies virus glycopeptide (RVG), 220 RAF, 21, 199 raloxifene, 33 rapamycin, 19, 22, 23–24, 26 RARS. See arginyl-tRNA-synthetase RAS, 21, 24, 25f, 185 rBAT, 117 RDS. See respiratory distress syndrome receptor tyrosine kinases (RTKs), 198–99, 204 recombinant human acid alpha glycosidase, for Pompe disease, 78–79 Refsum disease, 146 RELN, 232 respiratory distress syndrome (RDS), 130–32 retinitis pigmentosa (RP), 60, 145–48, 219 retinoic acid receptor (RXR), 30, 31 Rett syndrome, 142–44, 173, 176, 229–30 RHEB, 21, 24 rhodopsin, 146–47 riboflavin. See vitamin B2 rickets, 126 RP. See retinitis pigmentosa RPE45. See guanylate cyclase 2D, vitamin A trans-cis isomerase RPE65, 147–48 RPGR, 146 RTKs. See receptor tyrosine kinases RVG. See rabies virus glycopeptide RXR. See retinoic acid receptor S1P/S2P, 104 SAHA. See vorinostat Sanfilippo disease, 73 sarcolemma endoplasmic reticulum calcium pump (SERCA), 141–42 SARS. See seryl-tRNA synthetase SATB, 232 SBMA. See spinal and bulbar muscular atrophy SCAD. See short-chain specific acyl CoA dehydrogenase

294

Index

schizophrenia, 10, 181 SCID. See severe combined immunodeficiency SDH. See succinate dehydrogenase sedoheptulokinase (SHPK), 126 sentinel nucleotide variant (SNP), 65, 66, 180 sepiapterin reductase (SR), 56, 58–59 SERCA. See sarcolemma endoplasmic reticulum calcium pump serine, 61–62 serine threonine kinase, 21 serotonin (5-hydroxytryptamine), 55, 56, 60–62 SERPINA1, 234, 236 seryl-tRNA synthetase (SARS), 88 severe combined immunodeficiency (SCID), 134 SGK1, mTORC2 and, 23 SH2TC, 152 SHANK3, 173 SHH. See sonic hedgehog short-chain specific acyl CoA dehydrogenase (SCAD, ACADS), 45–46, 50, 66 short-hairpin RNAs (shRNAs), 191, 197 short inhibitory RNAs. See siRNAs SHPK. See seduloheptokinase shRNAs. See short-hairpin RNAs sialic acid, 63–65, 64f sickle cell disease, 138–40 signaling pathways, 13, 19–22, 20f, 24, 25f cancer and, 186–87, 187f molecular complexes and, 19 monoclonal antibodies and, 15–16 of TGF, 27–28 silicon scaffold system, 18 silico virtual screening, 17 Sine-VNTR-ALU (SVA), 212 single-photon emission tomography (SPECT), 99, 100 siRNAs, 154, 189, 217, 219 SLC. See solute carriers SLC1, 117, 171, 191 SLC2, 66, 117, 128–29 SLC3, 117, 118 SLC5, 117 SLC6, 60, 117, 119–20, 128, 130 SLC7, 117, 118 SLC9A6, 116–17 SLC16, 42, 128, 151, 191 SLC17, 129 SLC22, 42, 66, 126–28 SLC25, 44, 120, 121–25 SLCA7, 129

SLCA12, 128 SLCO1B1, 34–35, 66 SMA. See spinal muscular atrophy SMAD3, 29 small interfering RNAs. See siRNAs small-molecule testing, 17, 97 small nuclear RNAs (snRNAs), 209 SMN1/2, 210–11 SMPD1, 76 SNAP25. See synaptosomal-associated protein kD25 SNARE, 71 SNP. See sentinel nucleotide variant snRNAs. See small nuclear RNAs SOD1, 170 sodium benzoate, 120, 125 solute carriers (SLC), 115–35 sonic hedgehog (SHH), 228 sortilin, 69 SOX2, 227, 228, 229f SOX6, 138 SPB, 131 SPC, 131 SPD, 131 specific sulfatase-modifying factor (SUMF1), 68 SPECT. See single-photon emission tomography sphingomyelinase, 76 spinal and bulbar muscular atrophy (SBMA), 29, 30 spinal muscular atrophy (SMA), 210–11 spleen tyrosine kinase (SYK), 201 SPR, 59 SR. See sepiapterin reductase SRC, 166 SREB, 23, 75 stem cells, 225–38. See also specific types Alzheimer’s disease and, 238 globin and, 234–35 stilbenes, 112 STK11, 21, 22 stress, 184 succinate dehydrogenase (SDH), 195–96, 196f SUMF1. See specific sulfatase-modifying factor SURF1, 99 surfactant, ABCA3 transporter and, 130–32 SVA. See Sine-VNTR-ALU SYK. See spleen tyrosine kinase synaptosomal-associated protein kD25 (SNAP25), 107 synthetic lethality, cancer and, 196–97, 198f

Index T3, 151 T4, 111–12, 151 TALENs. See transcription activator effector nucleases tamoxifen, 33 tandem mass spectrometry (MS/MS), 40–41, 47, 65 TAOK2, 180 Taq polymerase, 8 TARDBP, 169 Tau protein, 161, 164, 171–72 Tay-Sachs disease, 5 TBX6, 180 TCA. See tricarboxylic acid TDP43, 162, 169 technetium-99-hexamathyl-propylenenamineoxime (HMPAO), 99 TENM4, 181 tetrahydrobiopterin (BH4), 56–59, 57f TFAM. See transcription factor A TFEB. See transcription factor EB TGF. See transforming growth factor TGFBR1/2, 29 thalassemia, 138–39 therapeutic agents, systems biology and, 19 thiamine. See vitamin B1 three-dimensional tissue-based screening, 18 thymidine, 88 thymidine phosphorylase (TYMP), 93, 101 thyroid-stimulating hormone (TSH), 117, 151 Timothy syndrome, 232–33 TIMP, 141 TIMs. See translocases of the inner membrane TMLD. See trimethyllysine dehydrogenase TMLHE, 127, 178, 179f TNFalpha. See tumor necrosis factor alpha Toll receptors, 16 transcription activator effector nucleases (TALENs), 221–22, 222f, 223 transcription factor A (TFAM), 101 transcription factor EB (TFEB), 72, 73 transfer RNA (tRNA), 87–88 transforming growth factor (TGF), 27–28, 199, 205 translocases of the inner membrane (TIMs), 91 7-transmembrane receptors, 32 transporters, 115–35 of ATP, 115 mitochondria and, 120–25 transthyretin, 110–12 TRAP1, 109

295

TREM1/2/3, 158–59, 166–68, 167f tricarboxylic acid (TCA), 41, 91, 192 trichostatin A, 134 trifunctional protein deficiency, 47–48 trimethyllysine dehydrogenase (TMLD), 127 trinucleotide repeat expansions, 214–15 tripeptidyl peptidase, 81 tRNA. See transfer RNA troglitazone, 31 TRPV1, 126 tryptophan, 57f L-tryptophan, 48 tryptophan hydroxylase, 60 TSC1/2, 21, 173, 174, 175, 187 TSFM, 93 TSH. See thyroid-stimulating hormone tuberous sclerosis, 20f, 21, 173, 175 TUFM, 88 tumor necrosis factor alpha (TNFalpha), 206 TYMP. See thymidine phosphorylase type II diabetes, 30, 63 TYROBP, 166, 168 tyrosine, 57f L-tyrosine, 59 tyrosine hydroxylase, 59–60, 228 tyrosine kinases, 200–204 UBE3A, 175 ubiquitin proteasome system (UPS), 112–14, 113f UDPGlcNac-2-epimerase, 64 UDPglucose-Nac. See N-acetyl UDP glucose uncovering enzyme. See N-acetylglucosamine1-phosphodiester-alpha-Nacetyglucosaminidase unfolded-protein response (UPR), 104–9, 105f UPS. See ubiquitin proteasome system URAT1, 128 uric acid, 128–29 urinary organic acids, 41 ursolic acid, 97 USH2a, 146 Usher syndrome 5G, 219 V30M, 112 valosin-containing protein (VCP), 71 VAMP. See vesicle-associated membrane protein vascular endothelial growth factor (VEGF), 27 VCP. See valosin-containing protein VDAC1, 191

296

Index

VEGF. See vascular endothelial growth factor very long-chain acyl CoA dehydrogenase (VLCAD), 47, 50 very long-chain fatty acid (VLCFA), 131–32, 133 vesicle-associated membrane protein (VAMP), 71, 107 VHL. See Von Hippel-Lindau disease vitamin A, retinitis pigmentosa and, 146 vitamin B1 (thiamine), 4, 123 vitamin B2 (riboflavin ), 123 vitamin B6, 60 vitamin B12, 4 vitamin C, 123 vitamin E, 146 vitamin H. See biotin vitamin K, 123, 146 VLCAD. See very long-chain acyl CoA dehydrogenase VLCFA. See very long-chain fatty acid

Von Hippel-Lindau disease (VHL), 21, 26–28, 196 vorinostat (SAHA), 134 Warburg effect, 186 wild-type mitochondria, 84 X-linked adrenoleukodystrophy (XLAD), 130, 131–35 X-linked chronic granulomatous disease, 237 X-ray crystallography, 17 YARS. See lysyl-tRNA synthetase Zellweger syndrome, 5 ZFNs. See zinc-finger nucleases zinc finger-based transcriptional activators, 139 zinc-finger nucleases (ZFNs), 221–23, 238 ZNF9, 215 ZNF804A, 181