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Notice Knowledge and best practice in this field are constantly changing. As new research and experience broaden our knowledge, changes in practice, treatment and drug therapy may become necessary or appropriate. Readers are advised to check the most current information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to verify the recommended dose or formula, the method and duration of administration, and contraindications. It is the responsibility of the practitioner, relying on their own experience and knowledge of the patient, to make diagnoses, to determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions. To the fullest extent of the law, neither the Publisher nor the Editors assumes any liability for any injury and/or damage to persons or property arising out or related to any use of the material contained in this book. The Publisher
Library of Congress Cataloging-in-Publication Data Warner, Thomas T., 1963Practical guide to neurogenetics / Thomas T. Warner, Simon R. Hammans. – 1st ed. p. ; cm. Includes bibliographical references and index. ISBN 978-0-7506-5410-4 1. Nervous system–Diseases–Genetic aspects. 2. Neurogenetics. I. Hammans, Simon R. II. Title. [DNLM: 1. Nervous System Diseases–genetics. 2. Genetics, Medical. WL 140 W284p 2009] RC346.4.W37 2009 2008030067 616.80 0442--dc22
Acquisitions Editor: Adrianne Brigido Developmental Editor: Joan Ryan Project Manager: David Saltzberg Design Direction: Karen O’Keefe Owens Printed in The United States of America Last digit is the print number: 9 8 7 6
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Dedication
We dedicate this book to the late Prof. Anita Harding, who inspired our interest in the field of neurogenetics.
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Contributors
Lucinda Carr, MD, FRCP, FRCPH Honorary Senior Lecturer, Institute of Child Health Consultant Pediatric Neurologist, Department of Neurosciences Great Ormond Street Hospital, London, United Kingdom Susan M. Downes, MD, FRCOphth Honorary Clinical Senior Lecturer, Oxford University Consultant Ophthalmic Surgeon, Oxford Eye Hospital John Radcliffe Hospital, Oxford, United Kingdom Diana M. Eccles, MD, FRCP Professor of Cancer Genetics, Wessex Clinical Genetics Service Princess Anne Hospital, Southampton, United Kingdom Simon R. Hammans, MA, MD, FRCP Consultant Neurologist, Wessex Neurological Centre Southampton General Hospital, Southampton Consultant Neurologist, St. Richard’s Hospital, Chichester Honorary Senior Lecturer, University of Southampton United Kingdom Andrea H. Nemeth, BSc, MD, DPhil (Oxon), FRCP Honorary Senior Lecturer in Clinical (Neuro) Genetics Weatherall Institute of Molecular Medicine, University of Oxford Consultant in Clinical Genetics, Churchill Hospital Oxford, United Kingdom Thomas T. Warner, BA, BM, BCh, PhD, FRCP Reader in Clinical Neurosciences, Department of Clinical Neurosciences UCL Institute of Neurology Consultant Neurologist, Department of Neurology, Royal Free Hospital Honorary Consultant Neurologist, National Hospital for Neurology and Neurosurgery, London, United Kingdom
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Preface
The field of neurogenetics has expanded dramatically in the past 20 years, becoming a recognized separate subspecialty. It is practiced by both neurologists with an interest in genetics and clinical geneticists. Both of these disciplines require extensive knowledge and clinical skills, and for this reason many specialist neurogenetics clinics are run jointly by geneticists and neurologists. However, all practicing neurologists and geneticists will come across neurogenetic disorders as part of their everyday practice, and it is for these individuals that we have produced this book. There are a number of comprehensive texts of neurogenetics available, and increasingly, online resources supplement our knowledge. It was not our purpose to compete with these sources. The main goal of this book is to offer an easy-to-read and pragmatic approach to individuals with, or at risk from, neurogenetic conditions. The first two chapters cover the basic facts concerning molecular genetics and genetic counseling. The subsequent chapters take an approach based on the clinical presentation as it occurs in any clinic, rather than on underlying pathophysiology or genetic mechanisms. Therefore, each chapter focuses on the main symptom complex, such as ataxia, dementia, or movement disorder. The potential diagnoses are discussed, including key clinical hints and investigations, followed by descriptions of the specific conditions and their genetics. Our hope is that the layout of this book will allow rapid reference for clinicians, either before or after they have seen a patient with a potential neurogenetic condition. It is designed to guide the thought process through diagnosis and investigation, genetic counseling, and testing in such individuals. Where it is clear that there is more complexity to the case, the potential diagnoses are listed in tables with key clinical features and cross-references to other chapters. In addition, we have guided the reader to other sources of information. This book does not provide comprehensive lists of genes and mutations, as these lists inevitably become outdated rapidly. This information and more detailed references are increasingly available on the Internet, and we use online resources before, during, and after clinical contact. This book is intended to be an accessible handbook summarizing practical and clinical issues to help patients with the most common forms of neurogenetic disease. xi
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We therefore hope that it will guide the reader to allow even more effective use of these new and powerful online resources in helping their patients. We hope that it will be of use to both general neurologists and geneticists with an interest in neurological disorders, whether fully qualified or still in training. Thomas T. Warner Simon R. Hammans
Acknowledgments
We wish to thank a number of individuals for their invaluable help, comments, and advice regarding various chapters in this book. We are particularly grateful to the clinical geneticists Prof. Diana Eccles and Dr. Andrea Nemeth, and pediatric neurologist Lucinda Carr, who authored a number of the chapters in the book. In addition, Diana also critically reviewed several of the other chapters. Susan Downes coauthored Chapter 5 on Disorders of Vision. We have also been ably assisted by colleagues with expertise in other fields who have reviewed the chapters, given constructive advice, or provided figures: Sarah Tabrizi, Daniela Pilz, Nick Dennis, Anneke Lucassen, Dominic McCabe, Richard Orrell, Lionel Ginsberg, Fiona Norwood, Jane Hurst, Simon Farmer, Jonathan Schott, Ros King, Georgina Burke, and Susan Huson. We are also grateful to Prof. Robert Surtees for his helpful discussion and guidance with Chapter 16. His death before the publication of this book is a great loss to us all. Finally, we would like to thank our respective wives and children (Nisha, Si^an, Rhian, and Sam Warner; Diana, Charlie, Lucy, Rosie, and Harriet Hammans) for their tolerance during the prolonged gestation of this book!
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Chapter 1 DNA, Genes, and Mutations Thomas T. Warner
INTRODUCTION The study of genetic disorders manifesting with neurological disease is a rapidly evolving field. Many neurological disorders are heritable and it is estimated that around one-third of recognizable Mendelian disease traits have phenotypic expression in the nervous system. The isolation of disease genes and subsequent analysis of molecular mechanisms holds the promise of developing new treatments or protective strategies. Gene identification also offers the prospect of more accurate genetic and prognostic advice as well as diagnostic, predictive, and prenatal testing. This chapter describes the basis of heritability in terms of the structure of DNA, genes, and various forms of mutation. It will also describe the fundamental concepts of molecular biology that form the basis of disease gene mapping and isolation.
PATTERNS OF INHERITANCE Patterns of inheritance were recognized long before the identification of DNA as the basic molecule of heredity. Gregor Mendel recognized that physical characteristics were the product of interplay between genetic factors inherited from each parent. Since Mendel laid down the first (the principle of independent segregation) and second (the principle of independent assortment) laws of inheritance it has become clear that there are many cases where a single gene is both necessary and sufficient to express a character. These characters are called Mendelian. A Mendelian character is considered dominant if it manifests itself in a heterozygous individual. If the character is masked it is considered recessive. In other words, a dominant allele exerts its effect despite the presence of a corresponding normal allele on the homologous chromosome, whereas in autosomal recessive inheritance both alleles must be abnormal for the disease trait to be expressed. The four common patterns of inheritance seen in genetic disease are described in Chapter 2. 1
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NUCLEIC ACIDS AND GENES Deoxyribonucleic acid (DNA) is the macromolecule that stores the genetic blueprint for all the proteins of the human body. DNA is the hereditary material of all organisms with the exception of some viruses, which use ribonucleic acid (RNA), and prions, which only contain protein. DNA is made of two antiparallel helical polynucleotide chains wrapped around each other and held together with hydrogen bonds to form a double helix. The backbone of the helices is made from alternating phosphate and deoxyribose sugars. Each sugar molecule is joined to one of four nitrogenous bases, adenine, cytosine, guanine, or thymine. These bases face into the center of the helix and hydrogen bond with their partner on the opposite strand. Adenine can only form hydrogen bonds with thymine, and guanine is only able to hydrogen bond with cytosine. The entire genetic code relies upon these four bases and their specificity of binding. The direction of the helices is described as either 50 to 30 or 30 to 50 depending on which carbon atom in the deoxyribose sugar the chain begins and ends with. DNA is divided into functional units known as genes, and it is believed that the human genome comprises approximately 30,00050,000 genes. A gene is a sequence of bases that determines the order of monomers: i.e., amino acids in a polypeptide, or nucleotides in a nucleic acid molecule. DNA is organized into a three-letter code. Each set of three is called a codon, and, with four possible bases in each position, there are 64 different combinations, which are more than enough for the 21 amino acids from which proteins are built. There is approximately 2 meters of DNA in each of our cells and this is achieved by packing the DNA into chromosomes. Humans have 23 pairs of chromosomes in the majority of cells in their body. One of each pair is inherited from each parent, and most cells have diploid status, in that they contain homologous pairs of each chromosome. One of these pairs is the sex chromosomes (XY in males and XX in females) and the remainder are called autosomes. Genes are arranged in linear order on the chromosome, each having a specific position or locus. With the exception of the sex chromosomes, each of a pair of chromosomes carries the same genes as its partner. For any particular character coded for by a gene there may be a number of different forms, which are called alleles. If an individual carries two different alleles for a particular characteristic, this is termed heterozygous; if both alleles are the same this is called homozygous. The human genome is a term used to describe all the DNA in human cells and actually comprises two genomes. First there is the nuclear genome, which accounts for 3300 million base pairs (Mb) of the total genetic makeup of the cell. Second there is the much smaller mitochondrial genome. Mitochondria are cytoplasmic organelles that generate energy in the form of ATP by oxidative phosphorylation. They contain two to 15 copies of mitochondrial DNA, which comprises a 16,539 base-pair circle of double-stranded DNA. This contains 37 genes specifying 13 polypeptides, 22 transfer RNAs (tRNAs), and two ribosomal RNAs. Production of the protein product for a gene consists of two steps. Transcription describes the synthesis of messenger RNA (mRNA) from the original DNA template. Translation is the process by which the mRNA code
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is translated into a polypeptide chain. Controlled synthesis of a gene product is initiated by its promoter, which is the collective name for a number of short sequences, called cis-acting elements, that are usually clustered upstream of the coding sequence of the gene. Transcription factors bind to these sequences and allow the attachment of RNA polymerase. The remainder of the gene can be divided into coding and non-coding regions called exons and introns, respectively. The average exon is between 150 and 250 nucleotides in length. Genes can have a very large number of exons, such as the dystrophin gene (responsible for Duchenne muscular dystrophy) with 79 exons, or just one. The purpose of introns is not known. However, their presence in all eukaryotes and in most genes means there is either no selective disadvantage to having them, or they have a positive function that is not yet clear. It is estimated that up to 97% of the human genome consists of non-coding sequence.
FROM GENE TO PROTEIN Synthesis of a protein begins with an appropriate signaling molecule binding to the promoter of the gene. This initiates transcription, which creates a singlestranded RNA copy of the gene. RNA, like DNA, is composed of a linear sequence of nucleotides, but the sugarphosphate backbone consists of ribose sugar instead of deoxyribose and the base thymine is replaced by a very similar base uracil. Before the RNA molecule leaves the nucleus it undergoes a process known as splicing to create a messenger RNA molecule, mRNA. Splicing removes intron sequences from the RNA, leaving a small molecule containing all the information of the original gene. The expression of the gene can also be modified at this level through a mechanism known as alternative splicing. This is where different forms of mRNA, and hence protein, are produced by altering which sequences are cut from the original transcribed RNA. Once spliced, the mRNA can then move into the cytoplasm to direct protein synthesis. There are two other important molecules that are required for protein synthesis. The first of these are ribosomes. Ribosomes are found free in the cytoplasm and attached to the surface of the rough endoplasmic reticulum. Once the mRNA has entered the cytoplasm these molecules bind to it and read along the sequence until an AUG is reached. These three bases mark the beginning of translation, the process of reading the sequence and turning it into the appropriate protein molecule. The nucleic acid bases are read in sets of three called codons, where AUG is the start signal and also sets the frame for reading the remaining codons. The second molecule required for protein synthesis is transfer RNA (tRNA). For every codon there is a tRNA with a domain of complementary sequence that will selectively bind to it (an anticodon). Each codon codes for a specific amino acid, and the tRNA with the matching anticodon is responsible for bringing it to the ribosome where it will bind to the amino acid from the tRNA molecule attached to the previous codon. Any one of three stop or nonsense codons (UAA, UAG, or UGA) signals the termination of protein synthesis. Further information is contained within the protein sequence itself and signal peptides can direct the newly formed protein to particular cell organelles for post-translational modifications
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PRACTICAL GUIDE TO NEUROGENETICS
(e.g., glycosylation, addition of metal ions or other polypeptides), and allow its insertion into membranes.
DNA REPLICATION AND CELL DIVISION The constant replacement of somatic cells occurs by the process of mitosis, which is the simple division of a parent cell into two identical daughter cells. This process is divided into four steps; prophase, metaphase, anaphase, and telophase (see Fig. 1.1). In this process the chromosomes are condensed and pulled to the equatorial plane at metaphase. The centromere splits in anaphase and the two chromatids of each chromosome are pulled to opposite poles. In telophase the chromosomes reach the poles and start to decondense. The nuclear membrane reforms and the cytoplasm starts to divide, yielding the two daughter cells. The other form of cell division is the production of gametes by meiosis. There are a number of important differences between mitosis and meiosis. First, in meiosis the daughter cells produced are not identical to the original parent cell. Meiosis consists of two divisions, but the cellular DNA is only replicated once. This means the daughter cells produced are haploid. Second, during prophase I an important event called crossover occurs. Visible manifestations of this event, called chiasmata, can be seen during metaphase I. Crossover is recombination between two non-sister chromatids, where there is a precise break, swap, and repair of DNA, thus exchanging genetic material. This is a very important process that creates genetic diversity within the gametes and therefore the next generation. This phenomenon is also of great
Figure 1.1. Process of mitosis and meiosis.
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significance in the study of genetics as it is the fundamental concept behind genetic mapping and linkage analysis.
IDENTIFYING DISEASE GENES Numerous neurological disease genes have been identified using molecular biological techniques over the past 20 years. Most have been the cause of single gene (monogenic) disorders and have relied on the process of positional cloning, or identification of the gene based solely on its chromosomal map position. Gene identification by positional cloning starts with the collection of families in which the disease is segregating. Genetic linkage analysis is then used to localize the disease gene to a particular chromosome between two defined markers. The candidate interval is then refined further by study of other genetic markers in this interval. This region of DNA is then cloned, usually in the form of a series of overlapping fragments inserted in vectors (known as a contig). Finally, candidate genes are isolated from within the cloned contig and mutations in these genes sought in affected individuals. The basic premise underlying linkage analysis relies on the fact that if two genetic loci are close to each other on a chromosome, they do not segregate independently during meiosis, and the degree to which this happens is a reflection of their physical proximity. As stated above, during meiosis recombination can occur and is the process whereby genetic information is exchanged at chiasmata when homologous chromosomes pair. For loci close together, the chance of recombination between them is small, and they will tend to be inherited together. Loci further apart are more likely to have a recombination event occurring between them. The probability of a recombination event occurring between two loci during meiosis is termed the recombination fraction (y), which is taken as a measure of the genetic distance between loci. The recombination fraction can vary from y=0.0 for loci right next to each other, to y=0.5 for loci far apart (or on different chromosomes). The function which relates genetic to physical distance is called a mapping function, and translates recombination frequency (in percent) into mapping distance measured in centimorgans (cM). To map a disease gene, therefore, the segregation of the disease locus and a known genetic marker through one large family, or a number of pedigrees, is analyzed to determine whether the loci are linked and then the level of recombination between them is assessed. Using the likelihood method, LOD (likelihood of odds ratio) scores are generated over a range of y. A LOD score is defined as log10 of the odds ratio for cosegregation of the loci versus independent assortment. The value of y at which the LOD score is largest represents the best estimate of genetic distance between the two loci under study (referred to as two-point LOD scores). Linkage is considered significant when the LOD score is >3.0, corresponding to the odds for linkage of at least 1000:1. In practice this correlates with a probability for linkage of 20:1, due to the prior probability that two autosomal loci are linked because they must be on one of the 22 pairs of chromosome. A LOD score of