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Genetic Diagnosis of Endocrine Disorders
Genetic Diagnosis of Endocrine Disorders
Genetic Diagnosis of Endocrine Disorders
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Genetic Diagnosis of Endocrine Disorders

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Genetic Diagnosis of Endocrine Disorders, Second Edition provides users with a comprehensive reference that is organized by endocrine grouping (i.e., thyroid, pancreas, parathyroid, pituitary, adrenal, and reproductive and bone), discussing the genetic and molecular basis for the diagnosis of various disorders.

The book emphasizes the practical nature of diagnosing a disease, including which tests should be done for the diagnosis of diabetes mellitus in adults and children, which genes should be evaluated for subjects with congenital hypothyroidism, which genetic tests should be ordered in obese patients or for those with parathyroid carcinoma, and the rationale behind testing for multiple endocrine neoplasias.

  • Offers a clear presentations of pharmacogenetics and the actual assays used in detecting endocrine diseases
  • Teaches the essentials of the genetic basis of disease in each major endocrine organ system
  • Offers expert advice from genetic counselors on how to use genetic information in counseling patients
  • Includes new chapters on the genetics of lipid disorders and glycogen storage diseases, genetics of hypoglycemia, and whole genome/exome sequencing
LanguageEnglish
Release dateOct 9, 2015
ISBN9780128011348
Genetic Diagnosis of Endocrine Disorders

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    Genetic Diagnosis of Endocrine Disorders - Roy E. Weiss

    Genetic Diagnosis of Endocrine Disorders

    Second edition

    Edited by

    Roy E. Weiss

    Department of Medicine, University of Miami Miller School of Medicine, Miami, FL, USA

    Samuel Refetoff

    Departments of Medicine, Pediatrics and Committee on Genetics, University of Chicago, Chicago, IL, USA

    Table of Contents

    Cover

    Title page

    Copyright

    List of Contributors

    Preface to the First Edition

    Preface to the Second Edition

    I: Introduction

    Chapter 1: Mechanisms of Mutation

    Abstract

    Introduction

    The types of mutation

    The mechanisms of mutation

    The role of technology

    Double-strand break repair-related mechanisms

    Mobile insertion elements

    The human mutation rate

    The phenotypic effect of mutations

    Conclusion and summary

    II: Pancreas

    Chapter 2: A Clinical Guide to Monogenic Diabetes

    Abstract

    Introduction

    Clinical presentation

    Genetic testing

    Conclusions

    Chapter 3: Hypoglycemia

    Abstract

    Introduction

    Genetic pathophysiology

    Summary

    III: Pituitary

    Chapter 4: Functioning Pituitary Adenomas

    Abstract

    Introduction

    Genetic pathophysiology of pituitary adenomas

    Genetic screening in functioning pituitary adenomas

    MEN1

    MEN1-related pituitary tumors

    Carney complex (CNC)

    Multiple endocrine neoplasia 4 (MEN4)

    Familial isolated pituitary adenomas (FIPA)

    Chapter 5: Diabetes Insipidus

    Abstract

    Introduction

    Types of diabetes insipidus

    Familial types of diabetes insipidus

    Clinical diagnosis

    Genetic testing

    Chapter 6: States of Pituitary Hypofunction

    Abstract

    Introduction

    Genetic pathophysiology

    Diagnosis, genetic testing, and interpretation

    Treatment

    IV: Thyroid

    Chapter 7: Congenital Defects of Thyroid Hormone Synthesis

    Abstract

    Introduction

    Pathophysiology and genetics of specific dyshormonogenesis defects

    Availability of genetic testing

    Conclusions

    Acknowledgment

    Chapter 8: Developmental Abnormalities of the Thyroid

    Abstract

    Introduction

    TSH receptor gene mutations (loss of function)

    PAX8 gene mutations

    TTF1/NKX2-1 gene mutations

    TTF2 (FOXE 1 or FKHL15) gene mutations

    GLIS3 gene mutations

    NKX2-5 gene mutations

    Syndromes associated with CH from thyroid dysgenesis

    TSHR gene mutations (gain of function)

    Treatment

    Conclusions

    Chapter 9: Syndromes of Impaired Sensitivity to Thyroid Hormone

    Abstract

    Introduction

    Overview of described and putative defects in syndromes of impaired sensitivity to thyroid hormone

    Resistance to thyroid hormone (RTH)

    Thyroid hormone cell transporter defect

    Thyroid hormone metabolism defect

    Chapter 10: Molecular Diagnosis of Thyroid Cancer

    Abstract

    Introduction

    Oncogene rearrangements

    Gene mutations

    Other genetic alterations

    Application of molecular findings to the clinical diagnosis of thyroid cancer

    miRNAs in thyroid lesions

    Microarray

    mRNA expression

    Conclusion

    V: Parathyroid/bone

    Chapter 11: Genetics of Hyperparathyroidism Including Parathyroid Cancer

    Abstract

    Introduction

    Hyperparathyroidism-jaw tumor syndrome (HPT-JT), HRPT2, and parathyroid carcinoma

    Familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT)

    Autosomal dominant hypoparathyroidism

    Familial isolated hyperparathyroidism

    Summary

    Chapter 12: Genetic Diagnosis of Skeletal Dysplasias

    Abstract

    Introduction

    Sclerosing bone disorders

    Disorders of defective mineralization

    Dysplasias of bone and cartilage with normal or low bone mass

    Chapter 13: Vitamin D Disorders

    Abstract

    Introduction

    Calcium, phosphorus, and vitamin D metabolism

    Vitamin D deficiency and rickets

    Genetic causes of rickets – osteomalacia: disorders in vitamin D metabolism and recognition

    Genetic causes of rickets: hypophosphatemic disorders

    Genetic causes of hypercalcemia associated with alterations in vitamin D metabolism

    VI: Adrenal

    Chapter 14: Congenital Adrenal Hyperplasia

    Abstract

    Introduction

    Genetic pathophysiology

    Diagnosis: genetic testing and interpretation

    Treatment

    Resources

    Chapter 15: Genetics of Adrenocortical Tumors (ACT) and Hypersecretory Syndromes

    Abstract

    Introduction

    Conclusions

    Acknowledgments

    Chapter 16: Hereditary Syndromes Involving Pheochromocytoma and Paraganglioma

    Abstract

    Multiple endocrine neoplasia type 2

    Von Hippel–Lindau syndrome (VHL)

    Neurofibromatosis type 1

    Hereditary paraganglioma/pheochromocytoma syndromes

    Genetic risk assessment in patients with apparently sporadic pheochromocytoma

    Summary

    Chapter 17: Genetic Conditions Associated with Congenital Adrenocortical Insufficiency or Glucocorticoid and/or Mineralocorticoid Resistance

    Abstract

    Introduction

    Genetics of embryology and function of the adrenal glands

    Genetic defects causing CAI: an overview and a comment on treatment

    Specific genetic conditions associated with CAI

    Genetic conditions associated with resistance to glucocorticoids or mineralocorticoids

    Acknowledgment

    VII: Reproductive

    Chapter 18: Genetic Considerations in the Evaluation of Menstrual Cycle Irregularities

    Abstract

    Introduction

    Ovarian disorders

    Adrenal disorders

    Clinical and laboratory evaluation

    Conclusions

    Chapter 19: Disorders of Sex Development

    Abstract

    Disorders of sex development

    Disorders of sex determination

    Disorders of sex differentiation

    High-throughput sequencing in the diagnosis of disorders of sex determination

    Conclusions

    Chapter 20: Androgen Insensitivity Due to Mutations of the Androgen Receptor

    Abstract

    Male phenotypic development is controlled by androgens

    The androgen receptor

    Measurements of AR and its function

    The modulation of gene expression by the AR

    Androgen insensitivity: a spectrum of abnormalities caused by defects of the AR

    The genetic basis of androgen insensitivity

    Disruption of the primary amino acid sequence

    Alterations of the DBD

    Alterations of LBD structure

    Mutations within the amino terminus

    Mutations that cause decreased levels of ligand binding

    Phenotype and genotype in patients with various forms of androgen insensitivity

    Spinal and bulbar muscular atrophy and prostate cancer

    Diagnostic resources

    VIII: Adipocyte

    Chapter 21: Obesity

    Abstract

    Introduction

    Genetic pathophysiology

    Diagnosis genetic testing and interpretation

    Treatment

    Chapter 22: Syndromes of Severe Insulin Resistance and/or Lipodystrophy

    Abstract

    Introduction

    Genetic pathophysiology

    Diagnosis, genetic testing, and interpretation

    Treatment

    Chapter 23: Lipodystrophies

    Abstract

    Introduction

    Autosomal recessive lipodystrophies

    Autosomal dominant lipodystrophies

    IX: Multisystem disorders

    Chapter 24: Multiple Endocrine Neoplasia Type 1 (MEN1)

    Abstract

    Introduction

    Genetic pathophysiology of MEN1

    MEN1 mutation analysis

    Treatment

    Recommendations for the future

    Conclusions

    Acknowledgment

    Chapter 25: Genetics of Polyglandular Failure

    Abstract

    Definition, incidence, prevalence

    Clinical spectrum

    Genetic pathophysiology

    Diagnosis, genetic testing, and interpretation

    Management

    X: Growth

    Chapter 26: Genetic Diagnosis of Growth Failure

    Abstract

    Introduction

    Genetic pathophysiology

    Diagnosis: genetic testing and interpretation

    Treatment

    XI: Miscellaneous

    Chapter 27: Cost-Effectiveness of Genetic Testing for Monogenic Diabetes

    Abstract

    Cost of diabetes care

    Heterogeneity of diabetes mellitus

    Monogenic diabetes

    Precision medicine in monogenic diabetes

    Considerations in genetic testing for monogenic diabetes

    The role of cost-effectiveness analysis in healthcare

    Cost-effectiveness analysis of monogenic diabetes

    Future studies of cost-effectiveness analysis in monogenic diabetes

    Conclusions

    Chapter 28: Genetic Counseling: The Role of Genetic Counselors on Healthcare Provider and Endocrinology Teams

    Abstract

    Introduction

    The genetic counseling profession

    The role of genetic counselors on the healthcare provider team

    The genetic counseling process

    The pedigree: medicine and art

    Pedigree analysis and risk perception

    Summary

    Chapter 29: Setting Up a Laboratory

    Abstract

    Introduction

    Regulations for diagnostic genetic laboratories

    The preanalytic phase

    DNA preparation

    Analytic phase

    Methods – general PCR

    Methods – real-time and digital PCR

    Methods – microarrays

    Methods – methylation analysis

    Methods – sequencing

    Bioinformatics for NGS

    Quality assurance for NGS

    General hardware and software considerations

    Postanalytic phase

    Summary

    Chapter 30: Introduction to Applications of Genomic Sequencing

    Abstract

    Overview

    From human genetics to genomics

    Excess of rare variation in the human genome

    Data sharing becomes essential

    PNPLA6 gene identification – an example for a number of trends in genomics

    Conclusions

    Index

    Copyright

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    Medicine is an ever-changing field. Standard safety precautions must be followed, but as new research and clinical experience broaden our knowledge, changes in treatment and drug therapy may become necessary or appropriate. Readers are advised to check the most current product information provided by the manufacturer of each drug to be administered to verify the recommended dose, the method and duration of administrations, and contraindications. It is the responsibility of the treating physician, relying on experience and knowledge of the patient, to determine dosages and the best treatment for each individual patient. Neither the publisher nor the authors assume any liability for any injury and/or damage to persons or property arising from this publication.

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    List of Contributors

    Milad Abusag,     University of Pittsburgh Medical Center – Horizon (UPMC-Horizon), Pittsburgh, PA, USA

    Valerie Arboleda,     Department of Pathology and Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA, USA

    Andrew Arnold,     Center for Molecular Medicine and Division of Endocrinology and Metabolism, University of Connecticut School of Medicine, Farmington, CT, USA

    Guillaume Assié

    Département Hospitalo-Universitaire, Faculté de Médecine Paris Descartes, Université Paris, Paris, France

    Service des Maladies Endocriniennes et Métaboliques, Centre de Recherche des Maladies Rares de la Surrénale, Hôpital Cochin, India

    Albert Beckers,     Department of Endocrinology, Centre Hospitalier Universitaire de Liège, University of Liège, Domaine Universitaire du Sart-Tilman, Liège, Belgium

    Graeme I. Bell,     Section of Endocrinology, Diabetes and Metabolism, The University of Chicago, Chicago, IL, USA

    Xavier Bertagna

    Département Hospitalo-Universitaire, Faculté de Médecine Paris Descartes, Université Paris, Paris, France

    Service des Maladies Endocriniennes et Métaboliques, Centre de Recherche des Maladies Rares de la Surrénale, Hôpital Cochin, India

    Jérôme Bertherat

    Département Hospitalo-Universitaire, Faculté de Médecine Paris Descartes, Université Paris, Paris, France

    Service des Maladies Endocriniennes et Métaboliques, Centre de Recherche des Maladies Rares de la Surrénale, Hôpital Cochin, India

    Gemma V. Brierley,     Metabolic Research Laboratories, University of Cambridge Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge, UK

    Silvia Cantara,     Department of Medical, Surgical and Neurological Sciences, University of Siena, Siena, Italy

    David Carmody,     Section of Endocrinology, Diabetes and Metabolism, The University of Chicago, Chicago, IL, USA

    Jane Hvarregaard Christensen,     Department of Biomedicine, Aarhus University, Aarhus, Denmark

    Karine Clément

    Institute of Cardiometabolism and Nutrition (ICAN), Nutriomique, University Pierre et Marie Curie-Paris, Pitie-Salpêtrière Hospital, Paris

    Nutrition Department, Pitié-Salpêtrière Hospital, Assistance Publique Hôpitaux de Paris, Paris, France

    Shelly Cummings,     Myriad Genetic Laboratories, Inc., Salt Lake City, UT, USA

    Adrian F. Daly,     Department of Endocrinology, Centre Hospitalier Universitaire de Liège, University of Liège, Domaine Universitaire du Sart-Tilman, Liège, Belgium

    Johnny Deladoëy

    Endocrinology Service and Research Center, Sainte-Justine Hospital and Department of Pediatrics, Universite de Montreal

    Department of Biochemistry, Universite de Montreal, Montreal, Quebec, Canada

    Koen M. Dreijerink,     Department of Clinical Endocrinology, University Medical Center, Utrecht, the Netherlands

    Béatrice Dubern

    Institute of Cardiometabolism and Nutrition (ICAN), Nutriomique, University Pierre et Marie Curie-Paris, Pitie-Salpêtrière Hospital, Paris

    Nutrition and Gastroenterology Department, Armand-Trousseau Hospital, Assistance Publique Hôpitaux de Paris, Paris, France

    Alexandra M. Dumitrescu,     Department of Medicine, The University of Chicago, Chicago, IL, USA

    David A. Ehrmann,     Section of Endocrinology, Diabetes, and Metabolism, The University of Chicago, Maryland, Chicago, IL, USA

    Douglas B. Evans,     Medical College of Wisconsin, Milwaukee, WI, USA

    Murray J. Favus,     Section of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Chicago, Chicago, IL, USA

    Abhimanyu Garg,     Division of Nutrition and Metabolic Diseases, Department of Internal Medicine, Distinguished Chair in Human Nutrition Research, Center for Human Nutrition, UT Southwestern Medical Center, Dallas, TX, USA

    Jennifer L. Geurts,     Medical College of Wisconsin, Milwaukee, WI, USA

    Helmut Grasberger,     Department of Medicine, University of Michigan, Ann Arbor, MI, USA

    Siri Atma W. Greeley

    Section of Endocrinology, Diabetes and Metabolism, The University of Chicago

    Departments of Pediatrics and Medicine, Section of Adult and Pediatric Endocrinology, Diabetes and Metabolism, The University of Chicago, Chicago, IL, USA

    Lionel Groussin

    Département Hospitalo-Universitaire, Faculté de Médecine Paris Descartes, Université Paris, Paris, France

    Service des Maladies Endocriniennes et Métaboliques, Centre de Recherche des Maladies Rares de la Surrénale, Hôpital Cochin, India

    Jo W. Höppener,     Department of Molecular Cancer Research, Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, the Netherlands

    Leslie Hoffman,     Columbus Endocrinology, Columbus, OH, USA

    Michael F. Holick,     Department of Medicine, Section of Endocrinology, Nutrition, and Diabetes, Vitamin D, Skin and Bone Research Laboratory, Boston University Medical Center, Boston, MA, USA

    Elbert S. Huang,     Department of Medicine, Section of General Internal Medicine, The University of Chicago, Chicago, IL, USA

    Hélène Huvenne

    Department of Pediatrics, Saint-Vincent de Paul Hospital, GHICL, Lille

    Institute of Cardiometabolism and Nutrition (ICAN), Nutriomique, University Pierre et Marie Curie-Paris, Pitie-Salpêtrière Hospital, Paris, France

    Vivian Hwa,     Cincinnati Children’s Hospital, Cincinnati, OH, USA

    Andrea L. Jones,     Division of Pediatric Endocrinology, Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Loren J. Joseph,     Molecular Diagnostics Laboratory, The University of Chicago Medical Center, The University of Chicago, Chicago, IL, USA

    George J. Kahaly,     Department of Medicine I, Johannes Gutenberg University Medical Center, Mainz, Germany

    Dorit Koren,     Section of Adult and Pediatric Endocrinology, Diabetes and Metabolism, University of Chicago, Chicago, IL, USA

    Kelly Lauter,     Department of Medicine, Endocrine Division, Massachusetts General Hospital, Boston, MA, USA

    Rossella Libé

    Département Hospitalo-Universitaire, Faculté de Médecine Paris Descartes, Université Paris, Paris, France

    Service des Maladies Endocriniennes et Métaboliques, Centre de Recherche des Maladies Rares de la Surrénale, Hôpital Cochin, India

    Thera P. Links,     Department of Clinical Endocrinology, University Medical Center, Groningen, Groningen, the Netherlands

    Cornelis J. Lips,     Department of Clinical Endocrinology, University Medical Center, Utrecht, the Netherlands

    Michael J. McPhaul,     Division of Endocrinology, University of Texas Southwestern Medical Center, Dallas, TX

    Rochelle N. Naylor,     Departments of Pediatrics and Medicine, Section of Adult and Pediatric Endocrinology, Diabetes and Metabolism, The University of Chicago, Chicago, IL, USA

    Maria I. New,     Mount Sinai School of Medicine, Department of Pediatrics, New York, NY, USA

    Sarah M. Nielsen,     Center for Clinical Cancer Genetics and Global Health, Department of Medicine, University of Chicago, Chicago, IL, USA

    Saroj Nimkarn,     Bumrungrad International Hospital, Department of Pediatrics, Bangkok, Thailand

    Stephen O’Rahilly,     Metabolic Research Laboratories, University of Cambridge Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge, UK

    Furio Pacini,     Department of Medical, Surgical and Neurological Sciences, University of Siena, Siena, Italy

    Andrew Palladino,     Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Louis H. Philipson,     Section of Endocrinology, Diabetes and Metabolism, The University of Chicago, Chicago, IL, USA

    Joachim Pohlenz,     Pediatric Endocrinology, Department of Pediatrics, Johannes Gutenberg University, Mainz, Germany

    Sally Radovick,     Division of Pediatric Endocrinology, Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Margarita Raygada,     Section on Endocrinology & Genetics, Program on Developmental Endocrinology & Genetics, (PDEGEN), Eunice Kenney Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, MD, USA

    Samuel Refetoff

    Departments of Medicine, Pediatrics and Committee on Genetics, University of Chicago

    Department of Medicine, The University of Chicago, Chicago, IL, USA

    Thereasa A. Rich,     Myriad Genetic Laboratories, Denver, Co, USA

    Inne Borel Rinkes,     Department of Surgery, University Medical Center, Utrecht, the Netherlands

    Søren Rittig,     Department of Pediatrics, Aarhus University Hospital, Aarhus, Denmark

    Christopher J. Romero,     Division of Pediatric Endocrinology, Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Ron G. Rosenfeld,     Oregon Health & Science University, Portland, OR, USA

    Liliya Rostomyan,     Department of Endocrinology, Centre Hospitalier Universitaire de Liège, University of Liège, Domaine Universitaire du Sart-Tilman, Liège, Belgium

    David B. Savage,     Metabolic Research Laboratories, University of Cambridge Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge, UK

    Robert K. Semple,     Metabolic Research Laboratories, University of Cambridge Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge, UK

    Julie Støy,     Department of Clinical Medicine – Medical Research Laboratory, Aarhus University, Aarhus, Denmark

    Constantine A. Stratakis,     Section on Endocrinology & Genetics, Program on Developmental Endocrinology & Genetics, (PDEGEN), Eunice Kenney Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, MD, USA

    Bernard S. Strauss,     Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL, USA

    Marc Timmers,     Department of Molecular Cancer Research, University Medical Center, Utrecht, the Netherlands

    Patrick Tounian

    Institute of Cardiometabolism and Nutrition (ICAN), Nutriomique, University Pierre et Marie Curie-Paris, Pitie-Salpêtrière Hospital, Paris

    Nutrition and Gastroenterology Department, Armand-Trousseau Hospital, Assistance Publique Hôpitaux de Paris, Paris, France

    Gerlof D. Valk,     Department of Clinical Endocrinology, University Medical Center, Utrecht, the Netherlands

    Anouk N.A. van der Horst-Schrivers,     Department of Clinical Endocrinology, University Medical Center, Groningen, the Netherlands

    Rob B. van der Luijt,     Department of Medical Genetics, University Medical Center, Utrecht, the Netherlands

    Bernadette P.M. van Nesselrooij,     Department of Medical Genetics, University Medical Center, Utrecht, the Netherlands

    Guy Van Vliet,     Endocrinology Service and Research Center, Sainte-Justine Hospital and Department of Pediatrics, Universite de Montreal, Montreal, Quebec, Canada

    Eric Vilain,     Department of Human Genetics, Pediatrics, and Medical Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA, USA

    Menno Vriens,     Department of Surgery, University Medical Center, Utrecht, the Netherlands

    Tracy S. Wang,     Medical College of Wisconsin, Milwaukee, WI, USA

    Roy E. Weiss,     Department of Medicine, University of Miami Miller School of Medicine, Miami, FL, USA

    Mabel Yau,     Mount Sinai School of Medicine, Department of Pediatrics, New York, NY, USA

    Stephan Zuchner,     Department of Human Genetics and Neurology, Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL, USA

    Preface to the First Edition

    Imagine the skepticism of a physician of the 1950s or 1960s if told that genetic testing would be used to diagnose specific complex endocrine disorders. Until relatively recently major abnormalities of the endocrine system were diagnosed, treated, and monitored with clinical assessment only. It was the clinical acumen of the astute physician that enabled correct diagnosis and determined which gland was responsible for producing too much or too little of a given hormone. The clinically pertinent markers of endocrine diseases were physiological measurements of basal metabolic rates, body weight, and urine output, which we now term the physiologic era of endocrinology.

    Despite the discovery of insulin by Banting, Best, Macleod, and Collip in 1921 and its use for humans in 1923, it was only with Rosalyn Yalow and Salomon Berson’s seminal report on the immunoassay of endogenous plasma insulin in 1960 that the assay period of endocrinology was introduced. This momentous methodological breakthrough enabled the endocrinologist to assay hormones previously impossible to measure at physiologically or pathologically relevant levels. The competitive protein-binding assay facilitated measurement of nanomolar or picomolar concentrations of hormones in plasma and tissues. Adaptation for other compounds further extended the field until 20 years ago, when molecular or genetic endocrinology evolved with the discovery of genes for insulin and growth hormone. Despite Paul Wermer’s 1954 publication of the first clearly inherited endocrine disease familial adenomatosis (American Journal of Medicine, 1954, pp. 363–371), it is only with access to the genetic tools of the new millennium that the clinician can now precisely identify the genetic defects causing a disease and apply rational therapy. In Genetic Diagnosis of Endocrine Disorders we present to the clinician a straightforward, clinically relevant review of important genetic tests currently in use for the diagnosis of endocrine disorders, and practical information as to where these tests are performed.

    Some endocrine disorders follow familial patterns of inheritance, while others may represent sporadic mutations. In both cases, identification of the mutation associated with the particular disease ideally allows the physician to test other family members, who may be asymptomatic. For example, in the case of a mutation with potential for adverse outcome such as medullary thyroid cancer. Physician and patient can now consider prophylactic thyroidectomy, for harboring such a gene prior to actual presentation of clinical disease; an approach possible only with the endocrine genetic revolution. Correlation of phenotype and genotype is frequently concordant, though in some instances the same genotype may cause different subtle or obvious phenotypes. An example would be patients with resistance to thyroid hormone who, despite identical mutations in the thyroid hormone receptor gene, may present with different phenotypes. The contrary example would be where the same phenotype may be due to different genotypes, as is occasionally the case in adrenal hyperplasias. Even in truly monogenic diseases, the genetic background of affected individuals may substantially modulate the phenotype. Thus, while genetic diagnosis is a critical part of the armamentarium of the modern-day Banting and Best, correlation of the genetic abnormality with its physiological manifestations remains crucial. Knowledge of which genetic tests to order must be supported by a full understanding of the genetic information they provide. The health care team responsible for diagnosis and follow-up, should ensure inclusion of the patient’s family/primary care physician and a genetic counselor.

    Genetic Diagnosis of Endocrine Disorders was initially conceived for purely selfish reasons. For our own use we needed a comprehensive clinical practice handbook for the genetic diagnoses of endocrine diseases. We therefore invited world experts to summarize the full range of currently available genetic endocrine diagnoses for our text. Initially we had to justify to ourselves taking time from our research to edit yet another endocrine book. However, the real advantage of editing this compilation of excellent reviews by renowned experts is the knowledge we have acquired in doing so. We hope that the reader will as well.

    Our many thanks to those who have so graciously contributed to Genetic Diagnosis of Endocrine Disorders as well as to Fay, Heather, and our children who have been unswerving in their support of our careers. We would also like to acknowledge the support of the National Institute of Health (grants DK15070, DK07011, DK20595, and RRO4999), the Abrams and Esformes Endowments, and the Sherman family.

    Roy E. Weiss MD, PhD, FACP, FACE

    Samuel Refetoff MD, PhD

    Preface to the Second Edition

    The first edition of Genetic Diagnosis of Endocrine Disorders was published five years ago. Since then, the revolution of Precision Medicine has taken center stage in medical diagnosis, and those treating endocrine diseases need the appropriate ammunition to approach their patient’s problems. There is rarely an article in the medical literature involving endocrine diseases, which does not mention a new gene or mutation. An encyclopedic compilation of the totality of genes involved in endocrine diagnosis does not lend itself to a printed text, which by nature is static. Therefore, the purpose of this book is to present emerging concepts to practicing pediatric and adult endocrinologists, students in the field, and genetic counselors, and review the most common genetic causes for endocrine disorders. Given the increasing affordability and availability of whole exome sequencing, the genetic cause of many more diseases will be identified, and there will be additional genes to know. New genetic conditions will emerge. In addition the role of epigenetics miRNAs, and enhancers in the cause of genetic endocrine disorders is quickly being recognized.

    The purpose of having a genetic diagnosis should enable the patient and physician to understand the basis for the disease and thereby apply rationale and targeted therapy. In addition, based on the genetic information, decisions can be made regarding risks in asymptomatic relatives, allowing for preemptive.

    Another reason for the second edition was based on the favorable reviews of the first edition and being urged by those reviewers to write a new edition.

    We thank our returning and new authors for their outstanding contributions to this second edition.

    Roy E. Weiss

    Miami

    Samuel Refetoff

    Chicago

    I

    Introduction

    Chapter 1: Mechanisms of Mutation

    Chapter 1

    Mechanisms of Mutation

    Bernard S. Strauss    Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL, USA

    Abstract

    Mutation is a sudden, inheritable change in DNA sequence. Single nucleotide variation (SNV), insertion or deletion of a few nucleotides (indels), rearrangements, change in the number of copies of larger stretches of DNA (copy number variation, CNV), change in the structure or number of chromosomes, and insertion or movement of transposable elements are all mutations. Many of these changes occur as a result of replication and recombination. Others result from the alteration of DNA by exogenous agents (mutagens) followed by replication by polymerases of relaxed specificity. The mutation rate is the resultant of the interaction of error producing and repair processes that detect changes and restore DNA to the parental sequence. Studies of the cancer genome have identified new mechanisms for the production of multiple closely linked mutations. A portion of human genetic disease is the result of de novo mutation. However, it is still not possible to predict the precise phenotypic consequence of a particular mutation in a particular individual.

    Keywords

    transition

    transversion

    indel

    single nucleotide variation (SNV)

    copy number variation (CNV)

    excision repair

    mismatch repair (MMR)

    double-strand break repair (DSBR)

    translesion synthesis, recombination

    Introduction

    Darwin realized the need for variation to provide a basis for natural selection, but he had no way of understanding the mechanisms by which such variation arose. Vague ideas of the origin of variation in the late nineteenth century gave way to the term mutation, coined by deVries to describe the discontinuous variation associated with Mendelian traits.¹ Genes were first recognized and defined by mutations with an extreme phenotype (see Table 1.1). Further progress led to conceptualizing the gene as a more complex structure with multiple sites for mutation available within the same gene. The nature of such sites was not clear, nor was the relationship between the physiological effects of gene mutations and the structural change involved. A typical pre-Watson–Crick examination question, what is a gene? could be answered in terms of function, of mutation, or of recombination and the question for geneticists was the relationship between these definitions.

    Table 1.1

    Special Abbreviations and Definitions

    Modern understanding of the mechanism of mutation is based on the Watson–Crick DNA structure. Recognizing the importance of nucleotide sequence followed by the deciphering of the genetic code led to a change from a biological and formalistic or mathematical view of mutation to a more biochemical approach. This chapter presents the problem of mutation as mainly one of biochemistry.

    The immediate response of investigators to the Watson–Crick structure was to focus attention on the base changes that resulted in mutation and on the chemical changes that might alter base pairing.² The specificity of particular mutagenic agents was initially ascribed to chemical changes in either the incoming or template nucleotide, resulting in altered pairing properties, mainly involving hydrogen bonding. Benzer and Freese³ and Brenner et al.⁴ defined mutation in terms of substitutions, additions, and deletions of nucleotides. DNA in eukaryotes is organized into discrete chromosomes. Changes in the structure (rearrangements and translocations) and numerical distribution of these chromosomes that leave a viable organism are also mutation, but it is only recently, with the availability of extensive DNA sequence information, that these changes can even be partially accounted for biochemically.

    Advances in our understanding of the complex biochemistry of DNA replication and its interaction with the various DNA repair and recombination pathways has led to a more mechanistic approach to understanding mutation (Fig. 1.1). The discovery in the late 1990s of a series of DNA polymerases with altered fidelity and ability to replicate past damaged sites in DNA⁵ advanced a view of mutation as an event involving both initial changes in the DNA and the interaction of these changes with the protein complement of the cell. Most recently, the advent of rapid and relatively inexpensive DNA sequencing technology has permitted a direct measurement of normal human mutation rates and the recognition that de novo mutation plays a role in human disease. The identification of thousands of mutational changes in individual tumors, only a small minority of which are involved as drivers in the etiology of the tumors, has permitted recognition of a set of mutational signatures that implicate particular repair processes in the generation of the mutations. The observation of numerous closely linked mutations in tumor cells suggests the operation of unique mutagenic mechanisms whose operation in normal cells remains an open question.

    Figure 1.1   The interactions between repair and mutation.

    Abbreviations: TC-NER, transcription-coupled repair; BER, base excision repair; MMR, mismatch repair; HR, homologous recombination; NAHR, nonallelic homologous recombination; TLS, translesion synthesis; NER, nucleotide excision repair; DSBR double-strand break repair; NHEJ, nonhomologous end joining; MMEJ, microhomology-mediated end joining; CNV, copy number variations. See Table 1.1 for definitions.

    The types of mutation

    Mutations are defined in this chapter as changes in the parental sequence of the DNA (Table 1.1). This definition is not without problems, since it is sometimes difficult to distinguish such changes from the normal process of recombination. Mutations include single nucleotide variation (SNV), insertion or deletion of small numbers of nucleotides (indels, frameshifts), rearrangements of the DNA sequence, change in the number of copies of larger stretches of DNA (copy number variation, CNV), and changes in the structure (inversions and translocations) or number of chromosomes (aneuploidy). The insertion or movement of transposable elements may affect phenotype and be obviously mutagenic. Nucleotide changes may occur outside the exome, the protein coding region of the genome, and these may or may not have an observable effect on phenotype. This view of mutation as a sequence change anywhere in the genome⁶ is a product of the sequencing revolution, since the recognition of mutation in the presequencing era required some observable change in the phenotype. The definition of mutation as any change in DNA sequence results in classifying sequence changes that have no obvious phenotypic effect as mutations. There are about 20,000–25,000 human genes, and the exome comprises somewhere about 2% of the total number of nucleotides.⁷ Much of the remainder of the DNA is transcribed into RNA,⁸ and some of this plays an important regulatory role in gene function, but as yet there is no automatic way to predict whether or what a change in DNA sequence will mean for physiology.

    The possible single base changes were first cataloged by Ernst Freese and Seymour Benzer.³,⁹ Freese coined the term transition to denote the change from one purine to another, or of one pyrimidine to another. The four possible transitions are cytosine (C) to thymine (T) and its reverse, and adenine (A) to guanine (G) and its reverse. Freese defined transversions as changes from a purine to a pyrimidine or the reverse. Change from an A or a G to a C or a T, and the reverse C or T to A or G was defined as a transversion. These definitions remain even though some of the putative transversions described were actually insertions or deletions of a few nucleotides.⁴ These were later called frameshifts because of the discovery that the genetic code was read in groups of three nucleotides to specify a particular amino acid. The addition (or deletion) of any number of nucleotides not divisible by three results in a change in the reading frame, thereby changing the amino acid composition of all amino acids downstream of the coding change. The details of the genetic code, as elucidated in the 1960s, also indicated that such frameshifts might not only result in major changes in the amino acid composition of a protein but might also produce unexpected termination codons as a result of the shift. Point mutations that resulted in protein terminations were at first termed nonsense mutations, as opposed to missense mutations, that resulted in the substitution of one amino acid for another. The nonsense mutations did not make sense, that is, did not specify any amino acid. There are three such codons, now called termination (ter) codons: UAA, UAG, and UGA. Since messenger RNA is the molecule that is actually read by the protein-synthesizing machinery, the code is an RNA code with U(racil) substituted for T(hymine). One of the stop codons, UGA, is read as tryptophan in mitochondria, and the mitochondrial code includes a few other variations; AGG and AGA are mitochondrial stop codons instead of coding for arginine and AUA codes for methionine instead of isoleucine. There are 64 possible codons but only 20 (or 21 including selenocysteine) natural amino acids incorporated into protein, and the codes are degenerate, or redundant, in that several codons can specify the same amino acid. Selenocysteine is synthesized from a special sertRNA and is coded for by an in-frame UGA stop codon. The mechanism by which particular UGA sites are selected for selenocysteine incorporation requires particular transcription factors and a cis-acting insertion sequence (SECIS) on the mRNA.¹⁰

    Point mutations within genes that do not change the meaning (amino acid coded) of the codon are termed synonymous, or silent, as opposed to nonsynonymous changes. Silent mutations may actually affect physiology when they are part of splice sites and because of the different availability of various tRNAs.¹¹ Synonymous is probably the better term. Although there was some initial confusion about the necessity of punctuation between the triplet codons, it was realized that if reading of the code began at a fixed site, and if the reading frame was designed to read three nucleotides at a time, the correct sequence of amino acids would be automatically produced. This terminology was developed before it was realized that there were large amounts of noncoding DNA. Frameshift has no meaning for mutations within such noncoding regions. A more recent term for small insertions or deletions, regardless of their physiological effect is indel, although the frameshift terminology continues to be used when appropriate.

    Change in the structure of other cellular constituents (e.g., membranes, prions) may also be heritable and alter physiology. Methylation of the DNA base cytosine and modifications of the histones constitute a set of markers that can alter cell physiology and direct patterns of differentiation.¹² Some of these modifications are propagated through mitosis and constitute a mechanism for somatic inheritance and for tissue differentiation. Certain of these markers can survive meiosis,¹² but the situation is complicated in large part because of the massive removal of tissue specific markers in the period between the formation of the gametes and early differentiation followed by their replacement. Cytosine methylation in DNA is carried out by specific maintenance and de novo methylation enzymes.¹³ The signal for maintenance is the presence of a methyl group on the parental strand of a 5′CpG sequence. Methylated promoters are usually associated with gene inactivation. Environmental influences can affect DNA methylation and gene function, and such environmental effects can persist through several generations.¹² Perhaps the most well-known study is the demonstration of low birth weight in offspring of Dutch mothers pregnant during the 1945 famine.¹⁴ The general biological significance of such epigenetic marks (see Table 1.1) is clearly a matter of current interest,¹⁵ since it reintroduces a Lamarckian cast to molecular biology. Epigenetic changes can mimic mutational ones since they affect phenotype. In addition, the rate of somatic mutation can vary as much as 10-fold according to tissue, and the determining factor is apparently the distribution of (epigenetic) markers.¹⁶

    The mechanisms of mutation

    Base Pairing and the Action of Mutagenic Agents

    It was first assumed that the fidelity of normal replication stemmed from the stability of the A:T and G:C base pairs resulting from hydrogen bonding. The early workers on the molecular nature of mutations accounted for the specificity of a variety of base analogs and other mutagenic agents by drawing acceptable alternative base pairings, resulting from the incorporation of these compounds into DNA or by their reaction with DNA nucleotides.² Alkylating agents, such as methylnitrosourea and the chemotherapeutically active mustard gas derivatives, were shown to react with individual nucleotides to produce multiple changes. Production of O⁶-methylguanine by agents such as methylnitrosourea or methylnitronitrosoguanidine was shown to promote mistaken base pairing, making understandable the highly mutagenic characteristics of such compounds. A major development was the discovery that metabolic systems in the host activated ingested compounds, making it possible for them to react with DNA. Carcinogenic polycyclic hydrocarbons, including those present in tobacco smoke and aflatoxins, are converted to epoxide derivatives with the participation of the cytochrome p450 system. These epoxides react directly with DNA, producing mutagenic adducts.²

    We live in an environment that is not friendly to DNA. We are essentially 55.6 M water. Given the law of mass action, hydrolytic reactions are inevitable. It has been estimated that we lose about 18,000 bases per cell in every 24-h period as a result of spontaneous hydrolysis of the glycosidic bond.¹⁷ The abasic sites so created are targets for base excision repair (see Modifiers of Mutation Not Associated with Replication p 11) but those that survive are mutagenic.

    The most reactive mutagen in our environment is undoubtedly oxygen. Breathing, however unavoidable, is inherently dangerous! The electron transport chain, by which adenosine triphosphate (ATP) is generated, results in the generation of reactive oxygen species (ROS) that produce the hydroxyl radical OH. When formed in proximity to DNA, this species produces a variety of oxidation products, of which a guanine with a saturated imidazole ring (8-oxoguanine) and altered hydrogen bonding properties are the most important.

    We are dependent on the sun both as a primary source of energy and for our feeling of well-being. But sunlight is a major source of DNA damage and results in the production of pyrimidine dimers and altered pyrimidines, all of which may be mutagenic and carcinogenic.

    The Role of Enzymes in Mutation

    It is not surprising that organisms have developed a set of enzymes to protect themselves from such damage and from mutation. Most mutations, at least in the exome, may have a deleterious effect, but it is essential that they occur at a sufficient rate in germ cells to ensure the variation on which natural selection is based. The problem organisms have had to solve is to adjust the mutation rate to some presumably optimal rate. A group of enzymes accomplishes this purpose.

    The free energy differences between correct and incorrect base pairs are very small, at most 0.4 kcal/mol. This means that in a water solution, in which there is much competitive hydrogen bonding, a correct base pair is only about twice as likely to form as is a mismatch. The major contribution to specificity is provided by the replicative polymerases: the structural nature of the pockets into which incoming nucleotides fit and the kinetic interactions between elongation of the chain and reversal of the reaction accounting for this specificity. Humans (and other organisms) have developed a variety of polymerases with vastly different specificities (Table 1.2). The free energy differences between correct and mismatched bases for a reaction catalyzed by a replicative polymerase (Drosophila melanogaster polymerase alpha) indicated a difference of 4.9 kcal/mol, equivalent to a discrimination factor of about 1 in 3,000.¹⁸ The in vitro measured error frequency of the different polymerases (Table 1.2) varies from a low of about 1 in 100,000 for the different B family replicative polymerases to more than 3.5 per 100 for human polymerase eta.¹⁹ Polymerase iota (pol iota) confronted with a template T will actually incorporate a G three times more frequently than the correct A!²⁰

    Table 1.2

    Eucaryotic DNA Polymerases

    DNA synthesis can be considered as a series of steps in which the growing chain is elongated. As a first step, the base to be inserted must fit into the appropriate pocket of the enzyme. The various DNA polymerases can be classified into families based on sequence homology. The Y family polymerases characteristically have pockets that are less restrictive²¹ than those of the replicative polymerases of the B family. The nucleotides may have either a syn or anti configuration, depending on the orientation of the purine/pyrimidine relative to the sugar; the most common orientation is anti. The pocket of pol iota is so constructed that it forces template dA into a syn configuration where it is restricted to pairing with T. Similar pol iota interactions keep template dT in the anti configuration, where structural considerations make it more likely to pair with incoming dG than with the correct dA.²²,²³

    After formation of a phosphodiester bond with an incoming base, the newly elongated chain is proofread to determine if it meets built-in pairing specifications. Terminal bases that do not fit are removed by exonucleolytic action. The requisite 3′→5′ nuclease activity is either built into the structure of B class polymerases or exists as a separate but closely associated protein(s). Y-family polymerase members are devoid of exonuclease activity. Proofreading results in about a 100-fold increase in the fidelity of replication. Mutations in either the exonuclease domains or the separate exonucleases may have mutator properties, producing additional mutations in every round of replication. Organisms can fine tune their proofreading, and mutants (of bacterial viruses) have been isolated in which the rate of spontaneous mutation is lowered because of an increase in the efficiency of proofreading. Such an increase comes at a cost in energy, since the ATPs required to provide the pyrophosphates required for polymerization are wasted. Even replication events with normal bases involve proofreading. Measurements made many years ago²⁴ indicate about 6–13% of the polymerization events result in an excised (proofread) base. The replication process can be depicted as a competition between proofreading and further elongation (Fig. 1.2), since once the chain has been elongated five or six nucleotides beyond a mismatch, it appears immune to proofreading. The elongation step is distinct from the initial addition opposite any particular base. Some of the enzymes of the Y series are relatively efficient in the addition of a nucleotide opposite a nonpairing template, but are unable to elongate the resulting product. It has been suggested that polymerase zeta, a B-family polymerase, has as a function the elongation of mismatched bases inserted by iota and other error-prone polymerases.²⁵–²⁷

    Figure 1.2   Schematic outline of the competition between extension and proofreading in DNA synthesis.

    Many agents that damage DNA block DNA synthesis but are nonetheless mutagenic. Since production of viable offspring requires replication of the DNA, cells must be able to overcome this inhibition. Several mechanisms are available to cells, not all of which are mutagenic. However, one of the mechanisms used is direct translesion synthesis (TLS), in which the damaged base serves as a template for replication. Mistakes in replication are likely to occur during such TLS. As just described, the Y-family polymerases are likely to be involved because of the relaxation in the specificity of their combining site. The events in translesion synthesis can be described as follows: the replicative complex recognizes a mismatch or altered base. TLS requires displacement of the replicative complex followed by recruitment of nonprocessive (generally Y family) polymerase(s) and then, after the bypass, reassociation of the normal replication complex. A key player in this process is proliferating cell nuclear antigen (PCNA). This protein forms a trimeric clamp encircling replicating DNA and is essential for processive DNA synthesis. PCNA binds numerous proteins, and ubiquitination of the PCNA clamp results in dissociation of the replicative complex from the DNA and allows access of a Y-family polymerase to the growing point.²¹,²⁸ A deoxynucleotide is added, and in at least some cases before the replicative complex and its associated proofreading activity can access the mismatch, polymerase zeta replaces the Y-family polymerase and elongates for a few nucleotides.²⁵,²⁷ Polymerase zeta then falls off and is replaced by the normal replication complex. Ubiquitination of PCNA plays a critical regulatory role in the process²⁸ as do other gene products.²¹

    Superimposed on these events must be the availability of the different deoxynucleotides used for synthesis. Alterations in the pool size of the different DNA constituents can affect the selection of bases, and altering relative pool sizes can be mutagenic.²⁹,³⁰ The result of any particular elongation attempt is determined by the various competitions for access to the nucleotide at the growing point.

    Mismatch Repair

    Neither hydrogen bonding, the innate specificity of the replicative polymerase, nor the efficiency of proofreading alone or together can account for the low in vivo mutation rate. Newly synthesized DNA is subject to yet another inspection by the set of proteins constituting the mismatch repair (MMR) system. These proteins detect mismatches in the DNA: both base pair mismatches and mismatches due to small additions or deletions. In bacteria, the detection is carried out by a single protein acting as a homodimer, the mutS protein, which when bound to the mismatch, recruits a second protein dimer, mutL. In the enteric bacteria, this ATP-dependent complex activates the endonuclease activity of a third protein, mutH, which makes a single stranded break in the error-containing strand. The nicked strand is unwound by a helicase encoded by the UvrD gene, and the displaced strand is degraded by an exonuclease. The resulting single-stranded gap is then filled by the replicative polymerase. The key to the successful operation of this scheme is making sure that the newly synthesized strand, including the error, is the one removed. In Escherichia coli, this trick is accomplished by a special methylation mechanism. Adenines at GATC sites are methylated on both strands, but the methylation of the newly inserted adenine is accomplished only after replication. Immediately after replication, the newly synthesized strand is nonmethylated. It is this hemimethylated DNA, which is the substrate for mismatch repair, and it is the nonmethylated, that is, newly synthesized, strand which is removed.¹⁷

    The MMR system of enteric bacteria has served as a paradigm, but eukaryotic cells have a more complex, although clearly similar, mismatch repair mechanism.³¹,³² Instead of a single mutS protein, eukaryotes have five, three of which (MSH2 [MutS homolog], MSH3, and MSH6) form dimers with slightly different specificities. There are four MutL homologs (MLH1, MLH2, PMS1 [postmeiotic segregation protein], and PMS2) that also function as heterodimers. The MSH2:MSH6 heterodimer recognizes base–base mismatches and small insertions or deletions; the MSH2:MSH3 complex specializes in recognition of larger insertions and deletions. As the names indicate, certain of these proteins also play an important role in meiosis. There is no MutH analog. The adenine methylation recognition mechanism appears confined to enteric bacteria. In vitro reconstructions of the eukaryotic mismatch repair system use a free 3′OH end (i.e., a nick in the DNA) to identify the newly synthesized strand, and it appears likely that in vivo, it is the growing point of the DNA (or an unligated Okazaki fragment) that provides the MMR signal. Eukaryotic MMR is more closely tied to replication as compared to the enteric bacteria. The MSH proteins have been shown to bind to PCNA, which locates them at the site of the DNA growing fork.³³

    Organisms deficient in their ability to make one of the mismatch repair proteins have increased mutation rates. The medical interest in MMR dates from the discovery that an inherited colon carcinoma syndrome (Lynch syndrome/hereditary nonpolyposis colorectal cancer) can be traced to a deficiency in the MMR proteins.³⁴,³⁵ The most frequent culprits are the hMLH1 (human mutL homolog) and hMSH2 genes, followed by hPMS2 and hMSH6. Analyses of tumor tissue show that the promoters of these MMR genes are frequent targets of epigenetic inactivation by methylation.³⁶ The absence of a functional MMR system is often signaled by an increase in microsatellite instability. The microsatellites are regions of mono- or dinucleotide repeats (e.g., CACACACACA) that are polymorphic, that is, in which the actual number of repeat units at a particular location differs among individuals. The number of repeat units at each locus is inherited and is the basis of much DNA fingerprinting. Instability is observed as a detectable increase in the number of such repeats, easily demonstrated by gel electrophoresis. Individuals deficient in MMR may have thousands of microsatellite instabilities throughout the genome, but a panel of five selected loci is generally used for testing. Instability at two loci serves as a positive signal.⁶

    Bound MMR proteins may also serve as a signal for apoptosis. Organisms deficient in the O⁶-methylguanine methyltransferase protein are exquisitely sensitive to killing by methylating agents. The cells become much less sensitive when made MMR defective, possibly because of a loss of a signal from the MMR proteins combined at the O⁶-methylguanine:T mismatch. The mechanism is important because MMR deficient cells with mutagenic lesions that should be signals for apoptosis survive, reproduce, and propagate mutations.³⁷

    The MMR repair system not only serves as a suppressor of mutation but also may be a component of systems producing mutation. Error-prone replication of single-stranded regions exposed by MMR is associated with the generation of mutations in the process of somatic hypermutation of immunoglobulin genes³¹,³⁸ (see, Somatic Hypermutation section) and in some not yet precisely defined way in the pathogenic expansion of triplet nucleotide repeats.³¹

    About 30 mostly neurodegenerative diseases including Fragile X syndrome, Huntington’s chorea, myotonic dystrophy, and Friedreich’s ataxia are due to multiple expansions of simple repeats in the genome.³⁹ For example, a CAG sequence, which occurs from six to about 35 times in normal individuals, expands to up to 100 repeats in individuals affected with Huntington’s chorea. In Friedreich’s ataxia, a normal GAA sequence occurring from seven to 22 times in an intron may expand to 200–1700 units. There is a role for the mismatch repair system based on the requirement for mutS and mutL proteins in a mouse model of Huntington’s disease,⁴⁰,⁴¹ but exactly what that role is and what sets off the changes are unknown.

    Modifiers of Mutation Not Associated with Replication

    Most endogenous and exogenous damage suffered by DNA is removed before the passage of the replication complex. This removal is accomplished by the action of one or more of the DNA repair pathways: base excision repair (BER), nucleotide excision repair (NER), and its cousin transcription-coupled nucleotide repair (TC-NER). The general tactic of all excision repair pathways is to use the information conserved in the complementary strand as a guide, an argument for the maintenance of the genome as a double-stranded entity. Cells also need to deal with breaks in DNA, some of which occur as the result of normal physiological processes. Double-strand breaks in the DNA would be lethal if not repaired by either recombination repair or the inherently mutagenic nonhomologous end joining (NHEJ). The details of these repair processes are the subject of an extensive text (see Ref. [17]). Over 175 structural genes have been identified with DNA repair functions.⁴² The excision repair pathways have a general similarity with each other and with the general mismatch repair system, albeit utilizing different components. In general, the damage must be recognized and removed. The removal leaves a single-stranded region of variable length, and this single stranded region serves as a template for DNA synthesis, sometimes using specialized polymerases and leaving single-stranded breaks that must be ligated. Recombination involving double-strand breaks is a consequence of the need for precise segregation of chromosomes in meiosis, and double-strand breaks are necessary intermediates in class switching in the immune response. Repair of double-strand breaks occurs by either NHEJ or recombination repair⁴³,⁴⁴ and, as will be seen, these complex processes can lead to mutations and chromosome aberrations.

    Somatic Hypermutation

    The frequency of new single nucleotide mutations in the human germline is about 1.2 × 10−8.45 During the development of a mature antibody response, the frequency of mutation in the variable (V) region of immunoglobulin may reach 10−3 per nucleotide as a result of a process called somatic hypermutation (SHM).³⁸ Mature B cells, migrating to the dark zone germinal centers of lymphoid organs, become centroblasts. These are the cells in which SHM is observed, mainly in the immunoglobulin genes but also at a lower frequency in a number of genes implicated in oncogenesis.⁴⁶ SHM requires transcription, is limited to a particular region of the affected gene, and is critically dependent on the activity of a cytidine deaminase, activation-induced cytidine deaminase (AID), which is expressed in high amounts in the mature B cells. AID requires single-stranded DNA as a substrate; hence, presumably, the requirement for transcription. The requirement for transcription is complex since SHM appears restricted to portions of the transcribed gene closer to the promoter.⁴⁷

    AID is one of a family of cytidine deaminases. The deaminated site is the target for repair. Base excision repair, error-prone DNA polymerases, and mismatch repair proteins are all involved.³⁸ Although a major protection against mutation, the MMR system plays an error-prone role in SHM and is involved in the production of the double-strand breaks that are intermediates in chromosome translocation.⁴⁸ It is not yet clear how the switch between the mutagenic and antimutagenic roles of the MMR proteins is managed.

    The role of technology

    Advances in DNA technology have made it possible to observe events that were previously inaccessible. New, rapid, and relatively inexpensive sequencing technologies promise to reduce the cost of sequencing individual human genomes to $1,000 or less. However, such rapid sequencing technologies are more prone to error than those based on the Sanger technique and require both numerous repetitions and sophisticated statistical techniques for their interpretation.⁴⁹ Determination of the genetic alterations present in tumors has provided not only information on possible driver mutations involved in the carcinogenic process, but also has illuminated the processes (possibly) operative in all cells by revealing multiple passenger mutations. The recognition of widespread CNVs in individual genomes is one example. Another phenomenon, possibly (but not necessarily⁵⁰) related to SHM because of its dependence on cytosine deaminases, is a phenomenon termed kataegis. It is observed as a region of closely linked mutations at cytidines located in 5′TCW motifs (where W is an A or T).⁵¹,⁵²

    As a result of massive sequencing studies, classifying mutations by base change in the context of 3′ and 5′ bases, transcriptional strand, and dinucleotide mutations, a set of 20 mutational signatures was identified.⁵³ Some of these signatures have been correlated with known mechanisms; e.g., lung cancer mutations with etiologically-known carcinogens and deamination of cytosine. Others represent unknown mechanisms. It is a question whether these phenomena are observed only in cancer cells or whether the examination of the expanded single cell clones that occur in cancer provides insight into normal but very rare processes occurring in normal cells.

    The new data also permit a more nuanced answer to the question of whether cancer genomes are inherently more mutable. The thought that tumors are hypermutable is an old suggestion put in modern terms in a series of papers by L. Loeb.⁵⁴ A parallel series of papers, summarized by W. Bodmer,⁵⁵ argued that the spontaneous human mutation rate is sufficient to account for the mutations involved. Mismatch repair deficiency and its associated increased mutation rate are certainly associated with a subclass of colon carcinomas. Determination

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