Emery's Elements of Medical Genetics, 13th Edition

13t* EDITION

EMERY'S

E FRcpcH FRcp, MB, chB, Peter D. Turnpenny, BSc, Consuttaht Ctinicat Geneticist RoyalDevonandExeterHospitat & Peninsula Medicat School SeniorCtinicat Lecturer. Exeter, UK

PhD, MRCPath Sian E[lard, BSc. Molecular Geneticist Consuttant Ctinicat RoyalDevonandExeterHospitat

& SchooI Medical Genetics, Peninsuta Professor of HumanMotecutar Exeter: UK

13t* EDITION

EMERY'S

AtanE.H.Emery Emeritus Professor of HumanGenetics & Honorary Fettow University of Edinburgh

Dedication - sources Toourfathers of encouragement andsupport whowoutdhavebeenproudofthiswork.

TheElements wasfirstpubtished in the UnitedStatesin 1968underthe titleHeredity,Diseose, ondManby the University of California Press. WhenProfessor Emeryreturned to theUKhepersuaded Churchitt Livingstone in Edinburgh to pubtish it underthetitleE/ernenfs of Medicol Genetics. Underhisauthorship it subsequentty evotved intomanyeditions, taterwithco-authorship of BobMuetter andthenlanYoung. lt seemsappropriate to commemorate this13theditionto hisindustry andto hiseffortsovermanyyearsto estabtish Ctinicat Genetics asa speciatity in its ownright.

Commissioning Edifor:KateDimock DevelopmentEditor:HeatherMcCorm ick Editori oI Assistont:KirstenLowson ProjectMonoger:GemmaLawson Designer:ErikBigtand IllustrotionMonoger:BruceHogarth /{lustrotor:Antbits Marketing Monogers(USA/UK).AtysonSherby/lanJordan

CHURCHILL LIVINGSTONE

British Library Cataloguing in Publication Data A cataloguerecord for this book is availablefrom the British Library

EISEVIER

An imprint of ElsevierLimited O Harcourt PublishersLtd , 200i @ ElsevierScienceLtd , 2002 O 2005,ElsevierLtd O 2007,ElsevierLimited All rights reserved First edition 1968 Secondedition 1971 Third edition 1974 Fourth edition 1975 Fifth edition 1979 Sixth edition 1983 Seventhedition 1988 Eighth edition 1992 Ninth edition 1995 Tenth edition 1998 Eleventhedition 2001 Twelfth edition 2005 This edition 2007 The rights of Peter Turnpenny and Sian Ellard to be identified as authors of this work has been assertedby them in accordancewith the Copyright, Desiqnsand PatentsAct 1988 No part of this publication may be reproduced, stored in a retrieval system,or transmitted in any form or by any means,electronic,mechanical, photocopying,recording or otherwise, without the prior permission of the PublishersPermissionsmay be soughtdirectly from Elsevier'sHealth SciencesRights Department, l600John E KennedyBoulevard,Suite 1800, Philadelphia,PA 19103-2899, USA: phone:(+1) 215 239 3804; fax: (+1) 215 239 3805;or, ermail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://wwwelsevier com), by selecting'support and contact'and then 'Copyright and Permission'.

Library of Congress Cataloging in Publication Data A catalogrecord for this book is availablefrom the Library of Congress Notice Medical knowledgeis constantly changing. Standard safety precautions must be followed, but as new researchand clinical experiencebroaden our knowledge, changesin treatment and drug therapy may become necessary or appropriate Readersare advised to check the most current product information providedby the manufacturerof eachdrug to be administered to verify the recommendeddose,the methodand durationofadministration, and contraindicationsIt is the responsibilityof the practitioner,relying on experienceand knowledgeof the patient, to determine dosagesand the best treatmentfor eachindividual patient Neither the Publishernor the authors assumeany liability for any injury and/or damageto personsor property arisingfrom this publication The Publisher

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Cettdivision40 43 Gametogenesis 45 Chromosomeabnormatities

Dedication ii Preface ix Acknowtedgementsx

SECTION

PRINCIPLES OF HUMANGENETICS T h e h i s t o r ya n d i m p a c to f g e n e t i c si n m e d i c i n e GregoM r e n d eal n dt h e l a w so f i n h e r i t a n c e3 DNAas the basisof inheritance 5 The fruit fty 6 T h e o r i g i n so f m e d i c agl e n e t i c s 7 T h ei m p a co t f g e n e t i cd i s e a s e 9 Majornew developments 9 T h e c e [ [ u t a ra n d m o l e c u l a rb a s i so f i n h e r i t a n c e 12 The cetl 12 y aterial 12 D N A : t h eh e r e d i t a rm C h r o m o s o m e s t r u c t u r1 e4 T y p e s o f D N A s e q u e n c1e4 Transcriotion 18 Translation 18 T h eg e n e t i c o d e 2 0 R e g u l a t i oonf g e n ee x p r e s s i o n 2 0 DNAsynthesis 22 RNA-directed Mutations 22 M u t a t i o nas n d m u t a g e n e s i s2 6

andce[[division 30 Chromosomes 30 Humanchromosomes analysis 5 Z Methods ofchromosome 34 Motecularcytogenetics 38 Chromosomenomenclature

andapptications55 DNAtechnotogy 55 DNActoning of DNAanatysis59 Techniques of DNAtechnotogy70 hazards Biotogicat genesfor monogenic andidentifying Mapping disorders 73 of humandisease identification Position-independent genes 73 74 Positionatctoning Project 75 TheHumanGenome 82 Developmentalgenetics andgastrutation82 Fertitization genefamities 83 DevelopmentaI model 93 Thetimbasa developmental genesandcancer 94 DevetopmentaI genes 95 anddevetopmentaI effects Positionat motes 96 Hydatidiform anddetermination96 differentiation SexuaI anddevetopment98 Epigenetics Twinning101 Patternsof inheritance 103 studies 103 Famity 103 inheritance Mendetian traits 113 andcomptex attetes Muttipte Anticipation114 Mosaicism114 disomy 115 UniparentaI imprinting115 Genomic 120 Mitochondriatinheritance genetics 122 andpoputation Mathematicat in populations122 Attelefrequencies 128 Geneticpotymorphism 129 Segregationanatysis [inkage 130 Genetic

CONTENTS

M e d i c aat n ds o c i e t ai In t e r v e n t i o n1 3 3 Conctusion 134 9

P o t y g e n i ca n d m u t t i f a c t o r i aiIn h e r i t a n c e 1 3 6 P o l y g e n iicn h e r i t a n caen dt h e n o r m a Id i s t r i b u u o n t J o - the tiabitity/threshotd MuttifactoriaI inheritance mode[ 138 Heritabitity 139 ldentifyinggenesthat causemultifactoriaI disorders 140 Conctusion 143

SECTION

GENETICS IN MEDICINE 1 0 H e m o g t o b i na n d t h e h e m o g l o b i n o p a t h i e s1 4 7 Structureof hemoglobin 147 DevelopmentaI expression of hemogl.obin 147 Gtobinchainstructure 148 Synthesisand controlof hemogtobin expression 149 D i s o r d e ros f h e m o g t o b i n 1 5 0 C t i n i c a t v a r i a t ioofnt h e h e m o g t o b i n o p a t h i e1s5 6 B i o c h e m i c a l g e n e t i c s1 5 8 I n b o r ne r r o r so f m e t a b o l i s m 1 5 8 D i s o r d e r os f a m i n o - a c i d metabotism158 D i s o r d e ros f b r a n c h e d - c h aai nm i n o - a c i d metabotism 163 Ureacycledisorders 153 Disordersof carbohydrate metabotism 164 Disordersof steroidmetabotism 165 Disordersof tipidmetabotism 167 LysosomaI storagedisorders 168 Disordersofpurine/pyrimidine metabolism 171 Disordersof porphyrinmetabolism 171 0 r g a n i c - a c iddi s o r d e r s 1 7 2 Disordersof coppermetabotism 172 P e r o x i s o m a I d i s o r d e r1s7 3 D i s o r d e r s a f f e c t im n gi t o c h o n d r i a t f u n c t i o1n7 4 Prenataldiagnosisof inbornerrorsof metabolism 1i6

12 P h a r m a c o g e n e t i c s1 7 7 Definition 177 Drug metabotism 177 Geneticvariationsreveatedsotetyby the effectof drugs 178 VI

F h a r m a c o g e n e t i c 1s 8 . licogenetics 182

13 fmmunogenetics184 lrnmunity 184 I r r n a t iem m u n i t y 1 8 4 c cquired Sp e c i f i a immunity 185 l r r h e r i t eidm m u n o d e f i c i e ndcivs o r d e r s 1 9 0 Eloodgroups 192

1 1 Cancergenetics 196 Differentiation betweenqeneticand environmentaI f.rctorsin cancer 196 0ncogenes 198 Trmor suppressorgenes 201 E p i g e n e t i casn dc a n c e r 2 0 5 G e n e t i cosf c o m m o nc a n c e r s 2 0 7 G e n e t i c o u n s e t i nign f a m i t i act a n c e r 2 1 2

1 5 Geneticfactors in common diseases 219 Geneticsusceptibitity to commondisease 219 Diabetesmeltitus 221 C- o h nd i s e a s e 2 2 4 Hypertension 225 Crrronary arterydisease 225 E p i l e o s i e s2 2 7 Artism 228 S,:hizophrenia228 A z h e i m e rd i s e a s e 2 2 9 H:mochromatosis 23O Vrrnousthrombosis 231 A t o p i cd i s e a s e 2 3 2 A r ; e - r e t a t em d a c u t a r d e g e n e r a t i o 2n 3 2

sECTION

CLINICAL GENETICS 16 C on g e n i t a Ia b n o r m a l i t i e sa n d d y s m o r p h i c syndromes 237 l n < : i d e n c e2 3 7 Definitionand classification of birthdefects 238 Geneticcausesof matformations 243 EnvironmentaI agents(teratogens) 2/+8 M:rtformations of unknowncause 251 Counsetino 252

17 Geneticcounseling 253 Definition 253 Estabtishing the diagnosis 253 Catcutating and presentingthe risk 254 the options 255 Discussing Communication and support 255 - directiveor non-directive? 256 Geneticcounseting 0 u t c o m e si n g e n e t i c o u n s e t i n g2 5 6 S p e c i apl r o b l e m si n g e n e t i c o u n s e l i n g 2 5 7

1 8 Chromosomedisorders 261 l n c i d e n coef c h r o m o s o maeb n o r m a t i t i e s2 6 1 Disordersof the sex chromosomes 271 C h r o m o s o mdei s o r d e r as n db e h a v i o r a I phenotypes 275 Disordersof sexuaIdifferentiation2]5 C h r o m o s o m a t b r e a k asgyen d r o m e s 2 7 7 l n d i c a t i o nf so r c h r o m o s o m aaIn a l v s i s 2 7 9

1 9 Singte-genedisorders 282 Huntingtondisease 282 Myotonicdystrophy 284 Hereditarymotorand sensoryneuropathy 286 Neurofibromatosis287 Marfansyndrome 289 Cysticfibrosrs 291 I n h e r i t e cd a r d i a a c r r h y t h m i aasn d c a r d i o m y o p a t h i e 2s 9 4 S p i n am I u s c u t aar t r o p h y 2 9 6 Duchennemuscutardystrophy 297 Hemoohitia 299 2 0 S c r e e n i n gf o r g e n e t i cd i s e a s e 3 0 3 S c r e e n i ntgh o s ea t h i g hr i s k 3 0 3 andX-tinked Carriertestingfor autosomaIrecessive disorders 303 P r e s y m p t o m a tdi ci a g n o s iosf a u t o s o m adto m i n a n t disorders 305 in carrierdetectionand predictive Ethicatconsiderations testing 308 P o p u t a t i o n s c r e e n i n3g0 8 Criteriafor a screeningprogram 309 N e o n a t asIc r e e n i n g 3 1 0 Populationcarrierscreening 311 Geneticregisters 313

genetics 315 21 PrenataItestingandreproductive diagnosis315 usedin prenataI Techniques screening318 PrenataI 321 lndicationsforprenatatdiagnosis prenatal 323 diagnosis probtems in Speciat pregnancy 325 of Termination genetic diagnosis325 Preimptantation forgenetic imptications and conception Assisted 326 disease circulation328 offetatcetlsin thematernaI Detection 328 treatment PrenataI

22 Riskcalculation 330 theory 330 Probabitity 331 Autosomatdominantinheritance 333 AutosomaIrecessiveinheritance inheritance334 recessive Sex-tinked Theuseof tinkedmarkers 336 screening337 andprenatal Bayes'theorem risks 337 Emoiric

23 Treatmentof geneticdisease 340 of genetic to treatment approaches ConventionaI disease340 DNA of recombinant appLications Therapeutic technotogy342 Genetherapy 342 24 Ethicatandtegatissuesin medicalgenetics 354 principtes354 GeneraI 356 Ethicatditemmas in a widercontext 359 dilemmas Ethicat 362 Conclusion Appendix- Websitesandctinicatdatabases 354 Gtossary 366 questions 378 Muttipte-choice 390 Case-basedquestions answers 395 Muttipte-choice answers 407 Case-based lndex 413

vll

EFA

A man ought to read.just as inclination lead.shirn; for what he read.s as a task will d,ohim little good.' Dr SamuelJohnson Advances and breakthroughs in genetic science are continually in the news, attracting great interest because of the potential, not only for diagnosing and eventually treating disease,but also for what we learn about humankind through these advances.In addition, almost every new breakthrough raises a fresh ethical, social and moral debate about the uses to which genetic science will be put, particularly in reproductive medicine and issues relating to identity and privacy. Increasingly today's medical graduates must be equipped to integrate genetic knowledge and

doctors and scientists seeking to equip themselves with enough medical genetics to know the basics well, there have to be some limits - in order that the wood is not obscured by the trees. At the same time, however, we have tried to provide sufficient detail for those wanting a little more, for example in the area of epigenetics and imprinting. As before, we have sought to provide a comprehensive basic text for those who seeka work ofreference through which they can swim in calm waters, rather than one in whose rapids they will quickly drown. We continue to be grateful to our predecessors in this work, namely Bob Mueller, Ian Young and Alan Emery to whom we are indebted. In this edition we acknowledge and celebrate Alan's colossal contribution to this well established book, and medical genetics in general, by

scienceappropriately into all areasofmedicine in order to deliver a dimension of practice and patient care that has hitherto largely been the domain of a small breed of specialists. In this 13th edition of Emery's Elementsof Med,ical Genetics we have provided some much needed updates from the l2th

including a portrait in the frontispiece. We hope this text proves to be a friendly companion both for those who require familiarity with medical genetics and for those who wish to make it their career, as similar texts once did for us.

edition and are conscious that there is so much more that could be included. However, for those undergraduate and postgraduate

PeterD. Turnpennyand SianEllard October2006

Exeter,UK

ACKN

For this edition more of our patients were approached for consent to publish their pictures for the first time and we are grateful for their universal willingness, especially those whose pictures have not made it into print this time. Several new fetal ultrasonographic images have been included and we are grateful

to Dr Helen Liversedge for these.Peter Turnpenny is again gratefulto DebbieBristowfor secretarialhelp,and we rhankour colleagues and familiesfor putting up with papersscatteredover deskand floorspaceboth at work and at homewhile the updating wasunder way.

CHAPTER

Thehistoryandimpact in medicine ofgenetics

'It's just a little trick, but there is a long story connectedwith it which it would take too long to tell.' Gregor Mendel, in conversation with C W Eichling 'It has not escapedour notice that the specificpairing we havepostulatedimmediately suggestsa possiblecopying mechanism for the geneticmaterial.' Watson & Crick (April 1953) Presentinghistorical truth is at leastaschallengingasthe pursuit of scientilictruth and our view of human endeavorsdown the ages is heavily biasedin favor of winners - those who haveconquered on military, political or, indeed, scientific battlefields. The history of geneticsin relation to medicine is one of breathtaking discovery from which patients and families already benefit hugely, but in today's world successwill be measured by continuing progressin the treatment and prevention of disease.

and has become an important and integral component of the undergraduatemedical curriculum. In order to put the exciting developmentand growth of genetic scienceinto context we start with a brief overview of some of the most notable milestones in the history of medical genetics. The importance of understanding its role in medicine is then illustrated by reviewing the overall impact of genetic factors in causingdisease.Finally, new developmentsof mafor importance are discussed. It is not known precisely when Homo sapiensfirst appearedon this planet, but according to current scientilic consensusit may havebeen anything from 50 000 to 200 000 yearsago.It is reasonable to supposethat our first thinking ancestorswere as curious as we are about matters of inheritance and, iust as today, they would have experiencedthe birth of babieswith all manner of physical defects.Engravings in Chaldea in Babylonia (now lraq) dating back at least 6000yearsshow pedigreesdocumenting the transmission of certain characteristicsof the horse's mane. However, any early attempts to unravel the mysteries of geneticswould have

G R E G OM R E N D EA L N DT H EL A W S O FI N H E R I T A N C E EARLY BEGINNINGS Developments in genetics during the twentieth century have beentruly spectacularIn 1900Mendel's principles were awaiting rediscovery,chromosomeswere barely visible, and the science of molecular geneticsdid not exist. By contrast, at the time of writing in the year 2006, chromosomescan be analyzedto a very high level of sophisticationand the sequenceof the entire human genome has been published. Nearly 11000 human genes with known sequenceare listed and nearly 6000 genetic diseasesor phenotypeshave been described,of which the molecular genetic basisis known in some 2200. This revolution in scientific knowledgeand expertisehas led to the realizationthat geneticsis an areaof major importance in almost every medical discipline. Recent discoveriesimpinge not iust on rare genetic diseasesand syndromes,but also on many 'acquired' of the common disorders of adult life that may be predisposedby genetic variation, such as cardiovasculardisease, psychiatric illness and cancer. Consequently genetics is now widely accepted as being at the forefront of medical science

been severelyhampered by a total lack ofknowledge and understandingofbasic processessuch as conceptionand reproduction. Early Greek philosophers and physicians such asAristotle and Hippocrates concluded, with typical masculine modesty, that important human characteristics were determined by semen' utilizing menstrual blood as a culture medium and the uterus as an incubator. Semen was thought to be produced by the whole body; hence bald-headedfathers would beget bald-headedsons. These ideasprevaileduntil the seventeenthcentury,when Dutch scientists such as Leeuwenhoek and de Graaf recognized the existenceofsperm and ova,thus explaininghow the femalecould also transmit characteristicsto her offspring. The blossomingof the scientific revolution in the eighteenth and nineteenth centuries saw a revival of interest in heredity by both scientistsand physicians,among whom two particular names stand out Pierre de Maupertuis, a French naturalist' studied hereditary traits such as extra digits (polydactyly) and lack of pigmentation (albinism), and showed from pedigree studies that these two conditions were inherited in different ways. JosephAdams (175G1818), a British doctor, alsorecognizedthat different mechanisms of inheritance existed and published I Treutiseon the Su\plsetl Heretlitary Prlperties of Diseases,whrch was intended as a basisfor geneticcounseling.

1

T H EH I S T O R Y A N I MDP A COTFG E N E T I CI N SM E D I C I N E

Our present understanding of human genetics owes much to the u'ork of the Austrian monk Gregor Mendel (1822-1884; Fig. Ll) who, in 1865, presentedthe results of his breeding experiments on garden peas to the Natural History Society of Briinn in Bohemia (now Brno in the Czech Republic) Shortly afterwards Mendel's observations were published by this associationin the Transactions of the Society, where they remained largely unnoticed until 1900, some l6years after his death, when their importance was first recognized. In essence Mendel's work can be consideredas the discovery of genesand how they are inherited. The term'gene'was first coined in 1909 by a Danish botanist, Johannsen,and was derived from the term 'pangen' introduced by De Vries This term was itself a derivative of the word 'pangenesis',coined by Darwin in 1868. In acknowledgementof Mendel's enormous contribution, the term mendeliaz is now applied both to the different patterns of inheritanceshown by single-genecharacteristicsand to disorders found to be the result of defectsin a single gene. In his breeding experiments Mendel studied contrasting charactersin the garden pea, using for eachexperiment varieties that differed in only one characteristic.For example, he noted that when strains that were bred for a feature such as tallness were crossed with plants bred to be short all of the offspring in the firsty'/ial or F7 generation were tall. If plants in this F1 generationwere interbred, this led to both tall and short plants in a ratio of three to one (Fig. 1.2). Characteristics that were manifest in the Fl hybrids were referred to as tlominant,whereas those that reappearedin the F2 generation were described as betng recessiae. On reanalysisit has been suggestedthat Mendel's results were'too good to be true', in that the segregationratios

Firstfilial cross Purebred Tall

F1

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Purebred Short

x Tt

Tt

Tt

Tall

Secondfilial cross

Hybrid Tall

Hvbrid

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f2' TT

Tt

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Fis.1.2 An rLlustration ofoneof Mendelsbreeding experiments andhow he cnrrectlv internreted theresults

he derived were suspiciouslycloser to the value of 3:1 than the laws of statisticswould predict. One possibleexplanationis that he may have published only those results that best agreed with his preconceivedsingle-genehypothesis.Whatever the truth of the matter, eventshaveshown that Mendel's interpretation of his results was entirely correct. Mendel's proposal was that the plant characteristics being studied were each controlled by a pair of factors, one of which was inherited from each parent The pure-bred plants, with two identical genes,used in the initial cross would now be referred to as hrmlzlgous. The hybrid F1 plants, each of which has one gene for tallness and one for shortness,would be referred to as heterozygous. The genesresponsiblefor these contrasting characteristics are referred to as allelomorphs,or alleles for short. An alternativemethod for determining genrqlpesin offspring involvesthe construction of what is known as a Punnett's square (Fig. 1 3). This is utilized further in Chapter 7 when considering how genessegregatein large populations. On the basis of Mendel's plant experiments, three main principles were established.These are known as the laws of uniformity, segregationand independent assortment

T H EL A WO FU N I F O R M I T Y The lan oJ'uniformityreferstothe fact that when two homozygotes with different allelesare crossed,all of the offspring in the Fl generation are identical and heterozygous.In other words, the

Fi9.1.1 (Reproduced Gregor Mendel withpermission fromBMJBooks )

characteristicsdo not blend, as had beenbelievedpreviously,and can reappearin later generations.

IN MEDICINE IN4PACT OFGENETICS THEHISTORYAND

Hybrid Tall

E o

Fig.1.3 A Punnett's square showing thedifferent waysinwhichgenes c a ns e g r e g aat en dc o m b i nient h es e c o nfdr l r a l c r ofsr so mF i g1 2 Consiruction ofa Punneit's squareprovides a simplemethodfor [ ea m e t ceo m b i n a t i ornnd s h o w r ntgh ep o s s i b g s r f f e r em n ta t r n g s

TH EL A WO FSE GR E GA T ION The law of segregationrefersto the observationthat eachindividual possessestwo genes for a particular characteristic,only one of which can be transmitted at any one time Rare exceptions to this rule can occur when two allelic genesfail to separatebecause of chromosome non-disjunction at the lirst meiotic division

(p.as).

in 1902 that Walter Sutton, an American medical student, and Theodour Boveri, a German biologist, independently proposed that chromosomes could be the bearers of heredity ( F i g . 1 . 4 ) . S u b s e q u e n t l y ,T h o m a s M o r g a n t r a n s f o r m e d Sutton's chromosome theory into the theory of the gene, and Alfons Janssensobserved the formation of chiasmata between homologouschromosomesat meiosis.During the late 1920sand 1930sCyril Darlington began to emerge as the world's leading cytologist of his time, helping to clarify chromosomemechanics by the use of tulips collected on expeditions to Persia. It was 'genome' entered the scientific during the 1920sthat the term 'genom'(German for gene) and vocabulary, being the fusion of 'ome'from chromosome the connection between mendelian inheritance and When chromosomes was first made, it was thought that the normal chromosome number in humans might be 48, although various papers had come up with a range of figures. The figure of 48 was settled upon largely as a result of a paper in 1921 from Theophilus Painter, an American cytologist who had been a student of Boveri. In fact, Painter himself had some preparations clearly showing 46 chromosomes,even though he finally settled on 48. These discrepancieswere probably due to the poor quality of the material at that early stage of the science, and even into the early 1950s cytologists were counting 48 chromosomes.It was not until 1956that the correct number of 46 was established by Tjio and Levan, 3years after the correct structure of DNA had been proposed.Within a few years it was shown that some disorders in humans could be causedby loss or gain of a whole chromosome as well as by an abnormality in a single gene' Chromosome disorders are discussedat length in Chapter 18. Some chromosomeabnormalities,such astranslocations'can run in families (p.47), and are sometimessaid to be segregatingin a

mendelian fashion. T H EL A WO FI N D E P E N D E N T A S S O R T M E NT The larp of independentassortmentrefers to the fact that members of different genepairs segregateto offspring independentlyof one another.In reality, this is not alwaystrue, as genesthat are close together on the samechromosometend to be inherited together, 'linked' (p. becausethey are 130). There are a number of other rvaysby which the laws of mendelian inheritance are breached but, overall, they remain foundational to our understanding of the science.

T H EC H R O M O S O MBAAL S I S O FI N H E R I T A N C E As interest in mendelian inheritance Breq there was much speculation as to how it actually occurred. At that time it was also known that each cell contains a nucleus within which

D N A A ST H EB A S I SO FI N H E R I T A N C E Whilst JamesWatson and Francis Crick are justifiably credited with discovering the structure of DNA in 1953, they were attracted to working on it only becauseofits key role asthe genetic material, as establishedin the 1940s.Formerly many believed that hereditary characteristics were transmitted by proteins, until it was appreciatedthat their molecular structure was far too cumbersome.Nucleic acids were in fact discoveredin 1849. In 1928 Fred Griffith, working on two strains of steptoclccus, reahzedthat characteristicsof one strain could be conferred on the other by something that he called the transformingprinciple'

In 1944, at the Rockefeller Institute in New York, Oswald Avery, there are several thread-like structures known as chromosomes, Maclyn McCarty and Colin Macleod identified DNA as the Even then, genetic material whilst working on the Pneumocoraas. so called becauseof their affinity for certain statns(chroma= coIor, many in the scientific community were skeptical;DNA was only slma=body). These chromosomeshad been observed since a simple molecule with lots of repetition of four nucleic acids the second half of the nineteenth century as a result of - very boring! The genius of Watson and Crick, at Cambridge, the development of cl,tologic staining techniques. Human was to hit on a structure for DNA that would explain the very mitotic hgures were observed from the late 1880s,and it was

1

1

THE HISTORYANDIMPACTOFGENETICSIN MEDICINE

B

Fis.1.4 C h r o m o s o mdeisv i d i ni g n t ot w o daughter cettsatdifferent stagesof ceLL d i v i s i oAn,m e t a p h a sBe,:a n a p h a s e : C t e l n n h a s eT h c h e h a v i o r o f

c h r o m o s o m iensc e l d [ i v i s i o(nm i t o s i s ) i sd e s c r i b eadtt e n g t ihn C h a p t e3r (Photographs courtesy of Dr K Ocraft, CityHospitat. Nottingham )

essenceof biological reproduction, and their elegant double helix has stood the test of time. Crucial to their discovery was the X-ray crystallographywork of Maurice Wilkins and Rosalind Franklin at King's College,London. The relationship between the sequenceof basesin DNA and the sequence of amino acids in protein - the geneticcnde- w^s unravelledin someelegantbiochemicalexperimentsin the 1960s. Thus it becamepossibleto predict the basechangein DNA that led to the amino-acid change in the protein. However, direct confirmation of that prediction had to wait until DNA sequencing methods becameavailableafter the advent of recombinant DNA techniques. Interestingly; however, the first genetic trait to be characterizedat the molecular level had already been identified in 1957by laborioussequencingof the purified proteins.This was sickle-cell anemia,in which the mutation affectsthe amino-acid sequenceof the blood protein hemoglobin.

THEFRUIT FLY Before returning to historical developmentsin human genetics, it is worth a brief diversion to consider the merits of this unlikely creature, which has proved to be ofgreat value in genetic research. The fruit fly, Drosophila, possessesseveral distinct advantagesfor the study of genetics: l. It can be bred easilyin a laboratory. 2. It reproducesrapidly and prolifically at a rate of 20 to 25 generatlonsper annum. 3. It has a number ofeasily recognizedcharacteristics,such as curly wings and,ayellom body,thtt follow mendelian inheritance. 4. Drosophilamelanogaster, the speciesstudied most frequently, has only four pairs of chromosomes,eachof which has a distinct appearanceso that they can be identified easily.

IN MEDICINE IMPACT OFGENETICS THEHISTORYAND

5. The chromosomes in the salivar-vglands of Drosophila larvaeare among the largest known in nature, being at least 100 times bigger than those in other body'cells.

Increasingly,it is becoming clear that the interplay of different genes(polygenic inheritance) is important in disease,and that a somatic genetic disease- should also further category- acquired, be included.

In view ofthese unique properties,fruit flieswereusedextensivelv in earlv breeding experiments.Today their study is still proving of great value in fields such as developmental biology where knowledgeof genehomolog-vthroughout the animal kingdom has enabledscientiststo identify families of genesthat are important in human embryogenesis(Ch. 6). When considering major scientific achievementsin the history of genetics it is notable that sequencingof the 180 million basepairs of the Drosophila genomewas completed towards the end of 1999. melunogaster

T H EO R I G I NO S FM E D I C AG L ENETICS In addition to the afore-mentioned Pierre de Maupertuis and JosephAdams, whose curiosity was aroused by polydact-vlyand albinism, there rvere other pioneers John Dalton, of atomic theorv fame, observed that some conditions, notably color blindness and hemophilia, show what is now referred to as sexor X-linked inheritance, and to this day color blindness is still occasionallvreferred to as daltonism. Inevitably,these founders of human and medical geneticscould only speculateon the nature of hereditary mechanisms In 1900 Mendel's work resurfaced.His papers were quoted

S I N G L E . G E NDEI S O R D E R S In addition to alkaptonuria,Garrod suggestedthat albinism and cvstinuria could also show recessiveinheritance. Soon other examples followed, leading to an explosion in knowledge and diseasedelineation. By 1966 almost 1500 single-genedisorders or traits had been identified, prompting the publication by an American physician, Victor McKusick (Fig. 1.5), of a catalog of all known single-gene conditions. By 1998, when the l2th edition of this catalog was published, it contained more than 'McKusick's Catalog' has 8500 entries (Fig. 1.6).The growth of now accessiblevia the internet as Online beenexponentialand it is MentlelianInheritancein Man (OMIM) (seeAppendix). By mid2006 OMIM containeda total of 16808 entries.

EM ABNORMALITIES CHROMOSO Improved techniques for studying chromosomes led to the demonstrationin 1959that the presenceof an additional number 2l chromosome (trisomy21) results in Down syndrome. Other similar discoveries followed rapidly - Klinefelter and Turner syndromes - also in 1959. The identification of chromosome abnormalitieswas further aided by the developmentof banding

almost simultaneouslyby three European botanists De Vries (Holland), Correns (Germany) and Von Tschermak (Austria) and this marked the real beginning of medical genetics,providing an enormous impetus for the study of inherited disease Credit for the first recognition of a single-genetrait is sharedby William Bateson and Archibald Garrod, rvho together proposed that alkaptonuriawasa rare recessivedisorder.In this relativelybenign condition urine turns dark on standing or on exposureto alkali becauseof the patient'sinability to metabolizehomogentisicacid (p. 162). Young children show skin discoloration in the napkin (diaper) area and affected adults may develop arthritis in large joints Realizing that this was an inherited disorder involving a chemical process, Garrod coined the term inborn error of metabolismin 1908 However, his work was largely ignored until the mid-trventieth centurl', when the advent of electrophoresis and chromatography revolutionized biochemistry. Several hundred such disorders have now been identified, giving rise to the field of study known as biochemical Benetics(Ch. l1). The historl' of alkaptonuria neatly straddles almost the entire trventieth centurli, starting with Garrod's original observations of recessiveinheritancein 1902and culminating in cloning of the relevantgeneon chromosome3 in 1996. During the course of the twentieth centur]' it gradually became clear that hereditary factors ryere implicated in many conditions and that different geneticmechanismswere involved. Traditionallli hereditary conditions havebeen consideredunder the headings of singlegene, chromosomaland multifactorial.

Fis.1.5 havebeenso andcatalogs whosestudies VictorMcKusicktn1994. r - p o ' r a ntto m e d i c agle n e t i c s

1

THE HISTORYANDIMPACTOFGENETICSIN MEDICINE

11000 10000 o 9000 c 8000 7000 6000 c 5000 4000 E 3000 = z. 2000 1000 0'a '68 1966

include congenitalmalformationssuch ascleft lip and palate,and Iate-onsetconditions such ashypertension,diabetesmellitus and Alzheimer disease.The prevailing view is that genesat several loci interact to generate a susceptibility to the effects of adverse environmental trigger factors. Recent researchhas confirmed that many genesare involved in most of these adult-onset disorders, although progress in identifying specific susceptibility loci has

'71

',7s ',78

'83 '86'88'90'92'94 '97 2003 Year

been disappointingly slow. It has also emerged that in some conditions, such as type I diabetes mellitus, different genes can exert major or minor effects in determining susceptibility (p. 219). Overall, multifactorial or polygenic conditions are now known to make a major contribution to chronic illness in adult life (Ch. 15).

Fis.1.5 Histogramshowingthe rapidincreaseIn recognition of conditions (traits)showing sing [e-gene inheritance and characteristics ( A d a p t e fdr o m M c K u s i c k 1 9 9a8n d 0 M l [ / - s e e A p p e n d i x )

A C Q U I R ESDO M A T IG C E N E T ID CI S E A S E Not all geneticerrors are presentfrom conception.Many billions of cell divisions (mitoses)occur in the course of an average

techniquesin 1970(p. 32).These enabledreliableidentificationof individual chromosomesand helped confirm that loss or gain of

human lifetime. During each mitosis there is an opportunity for both single-genemutations to occur, becauseof DNA copy errors, and for numerical chromosome abnormalities to arise

even a very small segmentof a chromosomecan havedevastating effectson human development(Ch. l8). Most recently it has been shown that severalrare conditions featuring learning difliculties and abnormal physical features

as a result of errors in chromosome separation.Accumulating somatic mutations and chromosome abnormalities are now known to play a major role in causing cancer (Ch. l4), and they probably alsoexplain the rising incidencewith ageof many other

are due to loss of such a tiny amount of chromosome material that no abnormality can be detected using even the most highpowered light microscope. These conditions are referred to

seriousillnesses,as well as the aging processitself. It is therefore necessaryto appreciatethat not all diseasewith a geneticbasisis hereditary.

as microdeletion syndromes (p. 264) and are diagnosed using a technique known as FISH (fluoresc€ntin,situ hybridization), which combines conventional chromosome analysis(cytogenetics) with much newer DNA diagnostic technology (moleculargenetics) (p. 3a). Increasingly, in the future, it is likely that rhe new technique of microarray CGH (comparatittegenomichybridization) will play a large part in diagnosingtheseconditions (p 37).

Before considering the impact of hereditary diseaseit is necessaryto introduce a few definitions.

Incidence Incid.encerefers to the rate at which new casesoccur. Thus, if the birth incidenceof a particular condition equalsI in 1000, then on average I in every 1000 newborn infants is affected

M U L TFI AC T OR IADLIS OR D E R S Francis Galton, a cousin of CharlesDarwin, had a long-standing interest in human characteristicssuch as stature, physique and intelligence. Much of his research was based on the study of identical twins, in whom it was realized that differencesin these parametersmust be largely the result of environmental influences. Galton introduced to genetics the concept of the regression cofficient as a means of estimating the degree of resemblance between various relatives.This concept was later extended to incorporate Mendel's discovery of genes,to try to explain how parameterssuch as height and skin color could be determined by the interaction of many genes,each exerting a small additive effect. This is in contrast to single-genecharacteristicsin which the action ofone geneis exertedindependently,in a non-additive fashion. This model of quantitatiaeinheritanceis now widely accepted and has been adapted to explain the pattern of inheritance observedfor many relatively common conditions (Ch. 9). These

Prevalence This refers to the proportion of a population affected at any one time. The prevalenceof a genetic diseaseis usually less than its birth incidence, either because life expectancy is reduced or becausethe condition sholvsa delayedageof onset.

Frequency Frequencyis a general term that lacks scientific specificity although the word is often taken as being synonymous with incidence when calculatinggene 'frequencies'(Ch. 8).

Congenital Congenitalmeansthat a condition is present at birth Thus, cleft palate representsan example of a congenital malformation. Not all genetic disordersare congenital in terms of age of onset (e.g.

INMEDICINE IMPACT OFGENETICS THEHISTORYAND

Huntington disease),nor are all congenitalabnormalitiesgenetic in origin (e.9.fetal disruptions, as discussedin Ch. l6).

T H EI M PA C T OFGE N E T IC D IS E A S E During the twentieth century improvements in all areas of medicine, most notably public health and therapeutics,resulted in changing patterns of disease,with increasing recognition of the role of genetic factors in causing illness at all ages.For some parameters,such as perinatal mortality, the actual numbers of caseswith exclusively genetic causeshave probably remained constant but their relatiae contribution to overall figures has increasedas other causes,such as infection, have declined. For other conditions, such as the chronic diseasesof adult life, the overall contribution of genetics has almost certainly increased as greater life expectancy has provided more opportunity for adversegeneticand environmental interaction to manifest itself, for examplein coronary heart diseaseand diabetesmellitus. Consider the impact of genetic factors in diseaseat different agesfrom the follouing observations.

Spontaneous miscarriages A chromosome abnormality is present in 40-50o/o of all recognizedfirst-trimester pregnancyloss.Approximately I in 6 of all pregnanciesresults in spontaneousmiscarriage,thus around 5-7oloof all recognizedconceptionsare chromosomallyabnormal (p. 261). This value would be much higher if unrecognized pregnanciescould alsobe included, and it is likely that a significant proportion of miscarriageswith normal chromosomesdo in fact havecltastrophicsubmicroscopic geneticerrors.

Newborninfants Of all neonates, 2-3olo have at least one major congenital abnormalitl', of which at least 50o/oare caused exclusively or partially by genetic factors (Ch. l6). The incidences of chromosomeabnormalitiesand single-genedisordersin neonates are approximately I in 200 and I in 100, respectively.

Chitdhood Genetic disorders account for 50o/oof all childhood blindness, 50o/oof all childhood deafnessand 50o/oof all casesof severe learning difficulty. In developed countries genetic disorders and congenital malformations together also account for 30o/oof all childhood hospital admissionsand 40-50o/oof all childhood deaths.

Adutt tife Approximately 1oloof all malignancy is caused by single-gene inheritance, and between 5oloand l0o/o of common cancers such

as those of the breast, colon and ovary have a strong hereditary component. By the age of 25years, 5oloof the population will have a disorder in which genetic factors play an important role. Thking into account the genetic contribution to cancer and cardiovasculardiseases,such as coronary artery occlusion and hypertension, it has been estimated that more than 50o/oof the older adult population in developed countries will have a geneticallydetermined medical problem.

NEWDEVELOPMENTS MAJOR The study of geneticsand its role in causing human diseaseis now widely acknowledgedas being among the most exciting and influential areasof medical research.Since 1962 when Francis Crick, JamesWatson and Maurice Wilkins gained acclaim for their elucidation of the structure of DNA, the Nobel Prize for Medicine and/or Physiology has been won on 19 occasionsby scientists working in human and molecular genetics or related frelds (Thble Ll). These pioneering studies have spawned a thriving molecular technology industry with applications as diverse as crops' the dcvelopment of geneticallymodilied disease-resistant produce therapeutic to animals the use of geneticallyengineered drugs, and the possible introduction of DNA-based vaccines for conditions such as malaria. Pharmaceutical companies are investing heavily in the DNA-based pharmacogenomics - drug therapy tailored to personalgeneticmake-up.

ER O J E C T T H EH U M A NG E N O M P With DNA technology rapidly progressing,a group of visionary scientists in the USA persuaded Congress in 1988 to fund a coordinated international program to sequencethe entire human genome.The program would run from 1990to 2005 and 3 billion US dollars were initially allocatedto the project. Some 5oloof the budget was allocatedto study the ethical and socialimplications of the new knowledge, in recognition of the enormous potential to influence public health policies, screening programs and personal choice. The proiect was likened to the Apollo moon mission in terms of its complexity, although in practical terms the long-term benefits are likely to be much more tangible. The draft DNA sequenceof 3 billion basepairs was completed successfully in 2000 and the complete sequence was published ahead of schedule in October 2004. Before the closing stages of the project it was believed that there might be approximately 100000 coding genesthat provide the blueprint for human life. It has come as a surprise to many that the number is much lower, with current estimatesat between 25000 and 30000. However, many genes have the capacity to perform multiple functions, which in some casesis challenging traditional concepts of disease classilication. The immediate benefits of the sequence data are being realized in researchthat is leading to better diagnosisand counseling for families with a genetic disease.A number of large, long-term, population-based studies are under way in the wake of

1

1

THE HISTORYANDIMPACTOFGENETICSIN MEDICINE

Table1.1 Genetic discoveries thathaveledto the awardof the NobelPrizefor Medicine and/orPhysiotogy 1962-2006 Year

Prize-winners

Discovery

1962

FrancisCrick JamesWatson Maurice Wrtkins

Themotecuiar srrucrure of DNA

1965

FrangoisJacob Jacques Monod And16Lwoff

Genetic regulatron

1966

PeytonRous

Oncogenic viruses

1968

RobertHotley Gobind Khorana Marshatt Nireberg

Deciphering ofthegenetic code

DavidBattimore Renato Dutbecco HowardTemrn

Interaction between tumor vtruses anonuctear DNA

1978

WernerArber Daniet Nathans Hamrlton Smith

Restrictron endonucteases

l9EU

BarulBenacerraf JeanDausset George Sne[[

Genetic controtof immunologrc responses

1983

Barbara McClrntock

genes Mobite (transposons)

1985

MichaelBrown JosephGoldstein

Cel[receptors in famrtiat hypercholesterotemia

1987

SusumuTonegawa

Genetic aspects oI antibodies

1989

Michaet Bishop Harotd Varmus

Studyofoncogenes

T H EI N T E R N E T

1993

Rrchard Roberts Phittip Sharp

Sp[itgenes'

The availability of information in genetics has been enhanced greatly by the developmentof severalexcellentonline databases,

1995

EdwardLewis Homeotrc andother ChristianeNusstein-Volhard devetopmentatgenes EricWreschaus

1915

In the longer term an improved understanding of how genes are expressed will hopefully lead to the development of new strategiesfor the prevention and treatment of both single-gene and polygenic disorders. Rapid DNA sequencing technologies currently under development will in due course greatly extend the range and ease of genetic testing, although many such applicationshaveimportant ethical and socialimplications.

1997

Stantey Prusrner

Prions

1999

GiinterBtobel

Proteintransportsignating

2000

ArvidCarlsson Pau[Greengard EricKandel

Signattransductron Inthe nervoussystem

2001

LelandHartwett Timothy Hunt PauINurse

Regulators ofthece[[cycte

2002

SydneyBrenner RobertHorritz JohnSutston

Genetrc regulation rn development and programmed cettdeath (apoptosrs)

AndrewFrre CraigMetto

RNAinterference

2006

10

the successfulHuman Genome Project, including, for example, UK Biobank, which aims to recruit 500000 individuals aged 40-69 to study the progressionof common disease,lifestyle and geneticsusceptibility.

THERAPY GENE Most genetic diseaseis resistant to conventional treatment so that the prospect ofsuccessfully modifying the genetic code in a patient's cells is extremely attractive. Despite major investment and extensiveresearch,successin humans has so far been limited to a few very rare immunologic disorders. For more common conditions, such as cystic fibrosis, maior problems have been encountered, such as targeting the correct cell populations, overcoming the body's natural defensebarriers and identifying suitably non-immunogenic vectors. However, the availability of mouse models for genetic disorders, such as cystic fibrosis (p. 291), Huntington disease(p. 282) and Duchenne muscular dystrophy (p.297), has greatly enhancedresearchopportuniries, particularly in unraveling the cell biology ofthese conditions. In recent yearsthere has been increasing optimism for novel drug therapiesand stem cell treatment (p.347), besidesthe prospects for gene therapy itself(Ch. 342).

a selectionof which is listed in the Appendix. These are updated r e g u l a r l y a n d p r o v i d e i n s t a n t a c c e s st o a v a s t a m o u n t o f contemporary information. For the basicscientist,facilities such asthe Genome Databaseand GenBank enablerapid determination ofwhether a particular genehas beencloned,togetherwith details ofits sequence,location and pattern ofexpression. For the clinician, OMIM offers a full account of the important genetic aspectsof all mendelian disorders,together with pertinent clinical details and extensivereferences.Although it is unlikely that more traditional sourcesof information, such as this textbook, will becometotally obsolete,it is clearthat only electronic technology can hope to match the explosivepaceof developmentsin all areasof genetic research.

FURTHER READING Baird P A, AndersonT W, NewcombeH B, Lowry R B 1988Geneticdisorders in childrenandyoungaduls:a populationstudy.AmJ HumGenet4Z:677493 A comprehennxe studyofthe incidenceofgeneticdiseasein a large Western urbanpopulation

1 Dunham I, Shimizu N, Roe B A et al 1999The DNA sequenceof human chromosome22. Nature,[02: 489495 Thefr* reportofthe completesequencing of a humanchromosome. Emery A E H 1989Portraits in medical genetics-Joseph Adams 175G1818.J Med Genet 26: I 16-l l8 An accountof the life of a Londond,octorwho rnaderemarkableobsentations about hereditary diseasein hispatients. Garrod A E 1902The incidence of alkaptonuria: a study in chemical individuality. Lancet ii: 1916-1920 A landmarhpaper in phich Garrod,proposed. that alhaptonuriacouldshow mendelianinheritance and aho noted that 'the mating offirst cousinsgiaes exactly the conditionsnost likelJ to enablea rare, and usually recessiue, characterto showitself'. McKusickV A 1998Mendelian inheritance in man, I I th edn. Johns Hopkins University Press,Baltimore An exhaustioethree-tsolutne catalogof all knoon contlitionsail traits shooing mend.elian in herit ance. Online Mendelian Inheritance in Man, OMIMru. Johns Hopkins University, Baltimore. Online. Available:http://www3.ncbi.nlm.nih. gov/omim/ Regularfu updated,online ztersionofmendelian inheitance in man. OrelV 1995Gregor Mendel: the first geneticist.Oxford University Press, Oxford A detailed biography of the ffi and oorh of the Moratsian monk Dho Das describedby his abbot as heing 'zteryd.iligentin the study ofthe sciencesbut muchlessfittedforwork as a parishpriest'. Ouellette F 1999Internet resourcesfor the clinical geneticist.Clin Genet 56:179-185 A guide to how to accesssomeof the mostuseful online d,atabases. Shapiro R 1991The human blueprint: the race to unlock the secretsofour genetic script. St Martin's Press,New York WatsonJ 1968The double helix. Atheneum, New York The storlr of the discoaeryrof the stucture of DNA, through the eyesof Watson himself.

manifest in a hybrid (heterozygote) @ A "hrr..teristic is dominant. A recessive characteristic is expressed only in an individual with two copies of the gene (i.e. a homozygote). @ Mendel proposed that each individual has two genes for each characteristic: one is inherited from each parent and one is transmitted to each child. Genes at different loci act and segregateindependently. @ Chto-orome separation at cell division facilitates gene segregation. @ Genetic disorders are present in at least 2o/o of all neonates, account for 50o/oof childhood blindness, deafness,learning difficulties and deaths, and affect 5oloof the population by the age of 25 years. @ Molecular genetics is at the forefront of medical research. The Human Genome Project and the prospect of gene therapy represent maior new initiatives that will revolutionize the management and treatment of genetic diseases.

11

CHAPTER

Thece[[ular andmolecutar basis of inheritance

'There

is nothing, Sir, too little for so little a creatureas man It is by studying little things that we attain the great arr of having as little misery and as much happinessas possible.'

in the degradation and disposal of cellular waste material and toxic molecules.

SarnuelJohnson The hereditary material is present in the nucleus of the cell, whereas protein synthesis takes place in the cvtoplasm. What is the chain of events that leads from the gene to the linal product? This chapter covers basic cellular biology outlining the structure of DNA, the processof DNA replication, the tvpes of DNA sequences,gene structure, the genetic code, the processes of transcription and translation, the various types of mutations, mutagenicagentsand DNA repair

T H EC E L L Within each cell of the body, visible with the light microscope, is the c.ytoplasmand a darkly staining body, the nur;leus,the latter containing the hereditary material in the form of chromosomes (Fig. 2.1). The phospholipid bilayer of the plasma membrane protects the interior of the cell but remains selectivelypermeable and has integral proteins involved in recognition and signaling between cells. The nucleus has a darkly staining area, the nucleolusThe nucleus is surrounded by a membrane, the nuclear enaelope,which separatesit from the cvtoplasm but still allows communication through nuclearpores The cytoplasm contains the cytosol, which is semifluid in consistency;containing both soluble elements and cytoskeletal structural elements.In addition, in the cytoplasm there is a complex arrangement of very fine, highly convoluted, interconnecting channels,the endoplasmic reticulum.The endoplasmicreticulum, in associationwith the ribosomes, is involved in the biosynthesisof proteins and lipids. Also situated within the cytoplasm are other

12

even more minute cellular organellesthat can be visualizedonly with an electron microscope.These include the Golgi appararus, which is responsiblefor the secretion of cellular products, the mitochondria,which are involved in energy production through the oxidative phosphorylation metabolic pathways(p.226), and the peroxisomes(p. 173) and lysosomes, both of which are involved

DNA:THEHEREDITARY MATERIAL c0MP0stil0N Nucleic acid is composed of a long polymer of individual molecules cilled nucleotides.Each nucleotide is composed of a nitrogenous base, a sugar molecule and a phosphate molecule. The nitrogenous basesfall into two types, 1,1rinesandp.yrimid,ines. The purines include adenine and guanine; the pyrimidines include cytosine,thymine and uracil. There are two different types of nucleic acid, ribonucleic arll (RNA), which contains the five carbon sugar ribose, and deoxyribonuclercacid (DNA), in which the hydroxyl group at the 2' position ofthe ribose sugar is replaced by a hydrogen (i.e. an oxygen molecule is lost, hence 'deoxy'). DNA and RNA both contain the purine basesadenineand guanineand the pyrimidine cytosine, but thymine occurs only in DNA and uracil is found only in RNA. RNA is present in the cytoplasm and in particularly high concentrations in the nucleolus of the nucleus. DNA. on the other hand, is found mainly in the chromosomes.

STRUCTURE For genesto be composedof DNA it is necessarythat the latter should have a structure sufficiently versatile to account for the great variety of different genesand yet, at the sametime, be able to reproduce itself in such a manner that an identical replica is formed at each cell division. In 1953, Watson and Crick, based on X-ray diffraction studies bv themselves and others, proposed a structure for the DNA molecule that fulfilled all the essentialrequirements.They suggestedthat the DNA molecule is composed of two chains of nucleotidesarranged in a double helix. The backboneof each chain is formed by phosphodiester bonds between the 3' and 5' carbons of adjacent sugars,the two chains being held together by hydrogen bonds between the nitrogenousbases,rvhich point in towardsthe center of the helix.

2 Golgicomplex Smooth endoplasmic reticulum Chromatin Rough endoplasmic reticulum Ribosomes Nucleolus Lysosome

EachDNA chainhasa polarity determinedby the orientationof The chainendterminatedby the backbone. the sugar-phosphate 5' carbonatomof the sugarmoleculeis referredto asthe 5' end, andthe endterminatedby the 3' carbonatomis calledthe 3'end. In the DNA duplex the 5' end of one strand is oppositethe 3' end ofthe other,that is, they haveoppositeorientationsand are saidto be antiparallel. The arrangementof the basesin the DNA moleculeis not random.A purine in one chainalwayspairswith a pyrimidine in the other chain,with specificpairing ofthe basepairs: guanine in one chain alwayspairs with cytosinein the other chain, and adeninealwayspairs with thymine, so that this basepairing forms complementarystrands(Fig.2.2). For their work Watson and Crick, alongwith MauriceWilkins, wereawardedthe Nobel Prize for Medicineor Physiologyin 1962(p. 9).

Nucleus Centriole

Fig.2.1 Diagrammatic representation ofan animalcett.

REPLICATION The process of DNA replica'tiln provides an answer to the question of how genetic information is transmitted from one generation to the next. During nuclear division the two strands

3 -hydoxyl

Flg.2.2

thymine: adenine:T, hetix(P phosphate:A. pairing oftheDNAdoubLe DNAdoubtehelixA,Sugar-phosphate backbone andnucteotide hetix G,guanine; C,cytosine). B,Representation oftheDNAdoubLe

13

2

THECELLULARAND MOLECULAR BASISOFINHERITANCE

of the DNA double helix separatethrough the action of enzyme DNA helicase,each DNA strand direcing rhe synrhesis of a complementary DNA strand through specific base pairing, resulting in two daughter DNA duplexes that are identical to the original parent molecule. In this way, when cells divide, the genetic information is conservedand transmitted unchanged to each daughter cell. The process of DNA replication is termed semiconseraatiue, as only one strand of each resultant daughter molecule is newly synthesized. DNA replication, through the action of the enzyme DNA polymerase, takes place at multiple points known as origins of replitation, forming bifurcated Y-shaped structures known as replicationforks.The synthesisof both complementary antiparallel DNA strandsoccurs in the 5'to 3' direction. One strand, known as the leatling strand, is synthesized as a continuous process.The other strand, known as the laggingstranl, is synthesized in pieces called Okazaki fragments, which are then joined together as a continuous strand by the enzyme DNA ligase(Fig'.2.3A). DNA replication progressesin both directions from these points of origin, forming bubble-shaped structures, or replicationbubbles(Fig. 2.38). Neighboring replicarion origins are approximately 50-300kilobases (kb) apart and occur in clustersor replicationunitsof 20 to 80 origins of replication.DNA replication in individual replication units takesplace at different times in the S phaseofthe cell cycle (p. 4l), adfacentreplication units fusing until all the DNA is copied, forming rwo complete identical daushter molecules.

C H R O M O S OSMTER U C T U R E The idea that eachchromosomeis composedof a singleDNA double helix is an oversimplification.A chromosomeis very much wider than the diameterof a DNA doublehelix. In addition, the amount of DNA in the nucleus of each cell in humans means that the total length of DNA contained in the chromosomes,if fully extended, would be several meters long! In fact, the total length of the human chromosomecomplement is lessthan half a millimeter. The packaging of DNA into chromosomes involves several orders of DNA coiling and folding. In addition to the primary coiling of the DNA double helix, there is secondarycoiling around spherical histone 'bead,s', forming what are called nucleosomes. There is a tertiary coiling of the nucleosomesto form the chromatinfbers that form long loops on a scaffoldof non-histone acidic proteins, which are further wound in a tight coil to make up the chromosome as visualized under the light microscope (Fig. 2.4), the whole structure making up the so-calledsolenoid model of chromosomestructure.

TYPES OFDNASEOUENCE DNA, if denatured, will reassociateas a duplex at a rate that is dependent on the proportion of unique and repeat sequences

;l:i-iivr'!rii.L.:i:l::i*,{;,,L-__./r.,t*,,-.*+\;ft..J,-."rt,

New strands

Old strands

Fis.2.3 14

DNArepticatron A,Detarted diagram of DNArepLication atthesiteof origininthereplication forkshowing asymmetrrc strandsynthesis withthecontinuous synthesis oftheleading strandandthedisconiinuous synthesis oftheLagging strandwithtigation oftheOkazaki fragmentsB,Muttrpte pointsoforiginandsemiconservative modeof DNArepticatron

BASISOFINHERITANCE THE CELLULARANDMOLECULAR

D N Ad o u b l e helix

Nucleosomes

Chromatin fi ber

Extended section of chromosome

Loopsof c h r o m a t i nf i b e r

2

M e t ap h a s e cnromosome

Fis.2.4 Simptifie dd iagram o fp r o p o s esdo L e n om i do d eoLfD N Ac o i t i ntgh a tl e a d st ot h ev i s r b tset r u c t u roeft h ec h r o m o s o m e

present,the latter occurring more rapidly.Analysis of the results of the kinetics of the reassociationof human DNA have shown that approximately 60-700lo of the human genomeconsists of single or low copy number DNA sequences.The remainder of the genome, some 30-40o/o,consistsof either moderately or highly repetitiaeDNA sequencesthat are not transcribed. This latter portion consistsof mainly satelliteDNA and interspersedDNA (Box 2.1). sequences

N U C L E AG RE N E S It is estimatedthat there are between 25000 and 30000 genesin the nuclear genome.The distribution of these genesvaries greatly between chromosomal regions. For example, heterochromatic and centromeric (p 35) regions are mostly non-coding' with the highest gene density observedin sub-telomeric regions (p. 36). Chromosomes 19 and 22 are gene rich, whereas 4 and 18 are relatively genepoor. The size of genesalso shows great variability: from small geneswith single exons to geneswith up to 79 exons (e.g.dystrophin, which occupies2 5 Mb of the genome).

Box2.1 Typesof DNAsequence

Uniquesingle-copygenes

Nuctear(-3x lOebp) (-30000) Genes

Most human genes are unique single-copy genes coding for polypeptidesthat are involved in or carry out a variety of cellular functions. These include enzymes, hormones, receptors, and structural and regulatory proteins.

Ll n r n r a < r n n l o r n n r r

famities Muitigene l^la<
Genesuperfamities Extragenic DNA(unique/lowcopynumberor moderate/ highly repetitive) Tandem repeat Satettite M n satetlite Tetomeric Hyperva riabte Mrcrosatetlite Inrersperse0 Sho.r.ntcrsnersed n.rrlearetements I nrn rn'er
(15.6kb,37 genes) Mitochondria[ TworRNAgenes 22tRNAgenes

Muttigenefamilies Man-v genes have similar functions, having arisen through gene duplication eventswith subsequentevolutionary divergencemaking up what are knownasmubigenefamilies.Some are found physically close together in clusters,for example the cx- and B-globin gene clusterson chromosomesl6 and l1 (Fig. 2.5), whereasothers are widely dispersedthroughout the genomeoccurring on different chromosomes,such as the HOXhomeobox genefamily (p. 86). Multigene families can be split into two types, classicgene fumilies that show a high degree of sequence homology and genesupeffimiliesthat have limited sequencehomology but are functionally related,having similar structural domains

15

THE CELLULAR AND MOLECULAR B A S I SO F I N H E R I T A N C E

--3

C h r o m o s o m1e6

j

C h r o m o s o m1e1

Fi1.2.5 Representation oftheq,-andB-globn regions on chTomosomes 1 6a n d ' l ' l

of introns in various genes in humans are extremely variable, although there is a general trend that the larger the gene, the greater the number and size ofthe exons.Individual introns can be far larger than the coding sequencesand some have been found to contain coding sequencesfor other genes(i.e. genesoccurring within genes).Genes in humans do not usually overlap, being separatedfrom each other by an averageof 30 kb, although some of the genesin the HLA complex (p. 189) have been shown to be overlapping.

Pseudogenes ClassicAenefamilies Examples of classic gene families include the numerous copies of genes coding for the various ribosomal RNAs, which are clustered as tandem arravs at the nucleolar organizing regions on the short arms of the five acrocentric chromosomes(p. 3l), and the different transfer RNA (p. l8) gene families, which are dispersedin numerous clusters throughout the human genome

Particularly fascinating is the occurrence of genes that closely resembleknown structural genesbut which, in general, are not functionally expressed:so-calledpseudogenes (p. 149). These are thought to have arisen in two main ways: either by genes undergoing duplication events that are rendered silent through the acquisition of mutations in coding or regulatory elements,or as the result of the insertion of complementaryDNA sequences, produced bv the action of the enzyme ret)ersetransrriptaseon a naturally occurring messengerRNA transcript, that lack the promoter sequencesnecessaryfor expression

Genesuperfamilies Examples of gene superfamilies include the HLA (human leukocyte antigen) genes on chromosome 6 (p. 189) and the T:cell receptor genes,which have structural homology with

EXTRAGENIC DNA

the immunoglobulin (Ig) genes(p. 185). It is believed that these are almost certainly derived from duplication of a precursor gene, with subsequentevolutionarv divergence forming the Ig

The estimated 25000 to 30000 unique single-copy genes in humans representlessthan 2o/oof the genomeencodingproteins. The remainder of the human genome is made up of repetitive

superfamily.

DNA sequencesthat are predominantly transcriptionally inactive. It has been described asjunk DNA, but some regions show evolutionary conservationand may play a role in the regulation of geneexpression.

Genestructure The original concept of a geneas a continuous sequenceof DNA coding for a protein rvasturned on its head in the early 1980sby detailed analysisof the structure of the human B-globin gene It was revealedthat the gene was much longer than necessaryto code for the B-globin protein, containing non-coding intervening sequences, or introns, that separatethe coding sequencesor exons (Fig. 2.6). It appearsto be the exceprionfor genesin humans ro consistof uninterrupted coding sequences. The number and size

TandemlyrepeatedDNAsequences Thndemlyrepeated, DNA sequences consist of blocks of tandem repeatsof non-coding DNA that can be either highly dispersed or restricted in their location in the genome.Thndemly repeated DNA sequencescan be divided into three subgroups: satellite, minisatelliteand microsarelliteDNA.

Transcription

Transcription termination

CAATTATAiniti'tion

o?" b?* C

a

lt

Promoter r e gr on

3

I

Intron1

I

T r an s l a t i o n i ni t i a t i o n codon

IATG)

Fis.2.6 16

Representation ofa typicat humanstructuratgene

Intron2

A

II

A

II Translation Polyadenylation termination signal co00n

ITAA)

BASISOFINHERITANCE N/OLECULAR THECELLULARAND

SotelliteDNA Satellite DNI accounts for approximately 10-l5o/o of the repetitive DNA sequencesof the human genome and consists of very large seriesof simple or moderately complex, short, tandeml.v repeated DNA sequencesthat are transcriptionally inactive and are clustered around the centromeres of certain chromosomes.This class of DNA sequencescan be separated on density-gradientcentrifugation as a shoulder,or'satellite', to the main peak of genomic DNA, and has thereforebeen referred to as satelliteDNA.

Minisotellite DNA

repetitiveDNA Hightyrepeatedinterspersed sequences Approximatel-vone-third of the human genome is made up of two main classesof short and long repetitive DNA sequences that are interspersedthroughout the genome.

Short i nterspe rsed nucleor eIem e nts About 5oloof the human genome consists of some 750000 copies of short interspersednuclear elementsor S/ly'Es. The most common are DNA sequencesof approximately 300bp that har.e sequence similarity to a signal recognition particle involved in protein synthesis.They are called Alu

consists of two families of tandemlr. repeated short DNA sequences:telomeric and hypervariable m i n i s a t e l l i t e D N A s e q u e n c e st h a t a r e t r a n s c r i p t i o n a l l . v lnactlve

rel)eutsbecause they contain tn AluI restriction enzyme recognitton site.

Telorneric DNA The terminal portion of the telomeres of the chromosomes (p 31) contains 10-15kb of tandem repeatsof a 6-base pair (bp) DNA sequencc known as telomericDNA. The tclomeric

About 5o/crof the DNA of the human genome is made up of long interspersednuclear elementsor LINEs. The most commonly occurring LINE, known as LINE-I or an L1 element, consists of morc than 100000copiesof a DNA sequenceof up to 6000bp

repeat scquencesare necessarvfor chromosomalintegrity in replication and are added to the chromosome by a specialized enzyme known as telomerase(p. 31)

that cncodesa re\ ersetranscriptase. The function of these interspersed repeat sequencesis not clear at present Members of the Alu repeat family are f l a n k e d b y s h o r t d i r e c t r e p e a t s e q u e n c e sa n d t h e r e f o r e resemble unstable DNA sequencescalled transposable Transposons, originally identified in elements or transp0s0ns.

tl/linisutellite DNI

Hypervariable rninisatellite DNA H l p e r L ^ a r i a b l em i n i s a t e l l l r e D N A i s m a d e u p o f h i g h l y ' polymorphic DNA sequencesconsistingof short tandem repeats of a common core sequence,the highly variable number of repeat units in different hypervariable minisatellites forming the basisof DNA hngerprinting used in forensic and paternity testing (p. 69). Although often located near the telomeres, hl,pervariableminisatellite DNA alsooccurs at other locationsin the chromosomes.

MicrosotelliteDNA Microsatellite DNA consists of tandem single, di-, tria n d t e t r a - n u c l e o t i d e r e p e a t b a s e - p a i r s e q u e n c e sl o c a t e d throughout the genome. Microsatellite repeats rarell'' occur within coding sequencesbut trinucleotide repeats in or near genes are associatedwith certain inherited disorders (p.62). DNA microsatellites are highly polymorphic (p. 69), i.e. the number of CA repeats varies between persons, and can be used in linkage studies in gene mapping (p 74) This variation in repeat number is thought to arise by incorrect pairing of the tandem repeats of the two complementary DNA strands during DNA replication, or what is known as slippetl strand mispairing. Duplications or deletions of longer sequencesof tandemly repeated DNA are thought to arise through unequal cross-over of non-allelic DNA sequenceson chromatids of homologous chromosomes or sister chromatids (p.31).

2

Long i nterspersed nucleor elem e nts

maize by Barbara McClintock (p. 10), move spontaneously throughout the genome from one chromosome location to another and appear to be ubiquitous in the plant and animal kingdoms. It is postulated that Alu repeats could promote unequal recombination,which could lead to pathogenic mutations (p. 22) or provide selective advantagein evolution b-v gene duplication Both Alu and LINE-1 repeat elements have been implicated as a cause of mutation in inherited human disease.

M I T O C H O N D R IDANLA In addition to nuclear DNA, the severalthousand mitochondria o f e a c h c e l l p o s s e s st h e i r o w n 1 6 . 6 k b c i r c u l a r d o u b l e stranded DNA, mitochontJrialDNA or mtDNA (Fig. 2.7). The mitochondrial DNA genomeis verS-compact,containing little repetitive DNA, and codes for 37 genes, which include two types of ribosomal RNA, 22 transfer RNAs (p' l8) and l3 protein subunits for enzymes, such as cytochrome E and cytochrome oxidase, which are involved in the energy producing oxidative phosphorylation pathways. The genetic code of the mtDNA differs slightly from that of nuclear DNA. The mitochondria of the fertilized zygote arc inherited almost cxclusively from the oocyte, leading to the maternal pattern of inheritance that characterizesmany mitochondrial disorders

(p.174).

17

2

THE CELLULAR AND N/OLECULAR BASISOFINHERITANCE

spliced together to form a shorter mature mRNA before its transportation to the ribosomesin the cytoplasm for translation. The processis known as nRNA splicing(Fig. 2.8). The boundary between the introns and exons consists of a 5' donor GT dinucleotide and a 3' acceptor AG dinucleotide. These, along with surrounding short splicing consensussequences,another intronic sequenceknown asthe splicing branch site,small nuclear RNA molecules and associatedproteins, are necessaryfor the splicing process.

165 rRNA

5'capping

e pIi c a t i o n

LVII

ND4L

ATPS

Fis.2.7 T h e h u m a nm i t o c h o n d r i a l g e n o mHei s t h e h e a v ys t r a n da n d L the tightstrand

TRANSCRIPTION

Shortly after transcription, the nascent mRNA is modified by the addition of a methylated guanine nucleotide to the 5'end of the molecule by an unusual 5' to 5' phosphodiesterbond, the so-called5' cap.'[he 5' cap is thought to facilitate transport of the mRNA to the cytoplasm and attachment to the ribosomes, as well as to protect the RNA transcript from degradation by endogenouscellular exonucleases.

Polyadenylation The cleavageof the 3' end of the mRNA moleculefrom the DNA involves the addition of approximately 200 adenylateresidues, the so-calledp oly (A) tail, after cleavageof the RNA transcript at a site downstream from a specific six-nucleotide sequence.The addition of the poly(A) tail is thought to facilitatetransport of the mRNA to the cytoplasm and translation.

The process whereby genetic information is transmitted from DNA to RNA is called transcription.The information stored in the genetic code is transmitted from the DNA of a gene to messenger RNA or mRNA Everv base in the mRNA molecule is complementaryto a correspondingbasein the DNA of the gene, but with uracil replacing thymine in mRNA. mRNA is single stranded, being synthesized by the enzyme RNA polymerase, which addsthe appropriatecomplementaryribonucleotideto the 3' end of the RNA chain In any particular gene only one DNA strand of the double helix actsasthe so-calledtemplutestrand The transcribedmRNA moleculeis a copy of the complementarystrand, or what is called the sensestrand of the DNA double helix. The template strand is sometimescalled the antisense strantl.The particular strand of the DNA double helix used for RNA synthesisappearsto differ

18

TRANSLATION Tianslationis the transmission of the genetic information from mRNA to protein. Newly processedmRNA is transported from the nucleus to the cytoplasm, where it becomesassociatedwith the ribosomes, which are the site of protein synthesis.Ribosomes are made up of two different sized subunits, which consist of four different types of ribosomalRNI (rRNA) moleculesand a largenumber of ribosomalspecificproteins.Groups of ribosomes associatedwith the same molecule of mRNA are referred to as polyribosomes or polysomes.In the ribosomes,the mRNA forms

throughout different regions of the genome.

the template for producing the specificsequenceof amino acids of a particular pofupeptide.

R N AP R O C E S S I N G

TRANSFER RNA

Before the primary mRNA molecule leaves the nucleus it undergoesa number of modihcations,or what is known as Rly'l processing. This involvessplicing, capping and polyadenylation.

In the cytoplasm there is anorher form of RNA calledtransferRNA or tRNA. The incorporation of amino acids i nto apolypeptidechain requires the amino acids to be covalentlybound by reacting with ATP to the specificIRNA moleculeby the activity of the enzyme

mRNAsplicing

aminoacyl IRNA synthetase.The ribosome, with its associated rRNAs, moves along the mRNA, the amino acids linking up by the formation of peptide bonds through the action of the enzyme peptidyl transferaseto form a polypeptide chain (Fig. 2.9).

After transcription, the non-coding introns in the primarv mRNA are excised, and the non-contiguous coding exons are

2 ption Transcri initiation

ption Transcri termination

+

+

JJ

Translation Poly(A)signal stop

Translation start

I I I

Transcription Polyadenylation Capping

PrimaryRNA Poly(A)tail

5'cap---J

Fi s . 2 . 8 post-transcriptionaI Transcription, processing, processing translation andpost-translationaI

DNA

UUUUUUUUUUUUUUU

AAACTCCACTTCTTC

UUUGAGGUGAAGAAG mRNA

Nuclear membrane

Ribosome

mRNA (template)

UUU

AAGAAG

cA

C

I

Peptide

Fis.2.9 Representation ofthewayinwhichgenetic information istranslated intoprotein

19

2

T H EC E L L U L A R A M ND OLECULA BR ASIS O FI N H E R I T A N C E

POST.TRANSLATI ONAL MODI FICATION

genetic code is non-overlapping The order ofthe triplet codons in a gene is known as the translational readingfrd,me. Howeye\

Many proteins, before they attain their normal structure or functional activitl-, undergo posl-t ranslational motlifc ation, which

some amino acids are coded for by more than one triplet, so (Thble 2.1). Each IRNA species the code is said to be degenerate for a particular amino acid has a specilic trinucleotide sequence called the unticodon,which is complementary to the codon of

can include chemical modilication of amino-acid side-chains (e.9. hydroxylation, methylation), the addition of carbohydrate or lipid moieties (e.g. glycos-vlation),or proteolytic cleavageof polypeptides(e.g.the conr.ersionofproinsulin to insulin).

the mRNA. Although there are 64 codons, there are only 30 cy'toplasmictRNAs, the anticodons of a number of the tRNAs recognizing codons that differ at the position of the third base, with guanine being able to pair with uracil as well as cytosine. Termination of translation of the mRNA is signaled by the presenceof one of the three stopor terminationcod,ons. The genetic code of mtDNA differs from that of the nuclear

This post-translationalmodification, along with certain short amino-acidsequencesknown aslocalizationsequentes in the newly synthesizedproteins, results in transport to specihc cellular locations(e.9.the nucleus),or secretionfrom the cell.

genome.Eight of the 22 tRNAs are able to recognizecodons that differ only at the third baseofthe codon, l4 can recognizepairs of codons that are identical at the first two bases,with either a purine or pyrimidine for the third base,the other four codons acting as stop codons (seeTable 2. I ).

T H EG E N E T C I CO D E Thent-v different amino acids are fbund in proteins; as DNA is composed of four different nitrogenous bases,obviousl-va single basecannot specify one amino acid. If two baseswere to specify one amino acid, there would onll be 42 or 16 possible combinations.If, however,three basesspecifiedone amino acid then the possiblenumber of combinationsof the four baseswould be ,13or 64. This is more than enough to account for all the 20 known amino acids and is known as the senetic codc.

R E G U L A T I OONFG E N EE X P R E S S I O N Nlanv cellular processes,and therefore the genesthat are expressed, are common to all cells, for example ribosomal, chromosomal and cvtoskeleton proteins, constituting what are called the genes.Somecellsexpresslargequantitiesofa specific housekeelting protein in certaintissuesor at specifictimes in development,such ashemoglobinin red blood cells(p. 147) This differential control of geneexpressioncan occur at a variety of stages.

TRIPLEC T ODONS The triplet of nucleotide basesin the mRNA that codes for a particular amino acid is called a cotlon.Each triplet codon in sequencecodes for a specific amino acid in sequenceand so the

qenomes Tabte2.1 Genetic codeof the nuctearandmitochondrial First base

C

A

G

20

Secondbase A

U

c

Phenyla[anine Phenytalanine Leucine

Serine Serine Serine

Tyrosine Tyrosine

Leucine

Serine

Slop

Stop lTryptophon) Tryptophan

L e u cnr e L e u cni e Leucine Leucrne

Protine Pro[ine Pro[ine Proiine

Histidine Hrstrdine Gtutamine Gtutamine

Arginine Arginine Arginrne Arginine

A G

Isoteuct ne Isoteuci ne lsoteucine (Methionine) Methionine

Threonine Threonine Threonine

Asparagine Asparagine Lysine

Serine Serine Arginine

A

Threonrne

Lysrne

Arginine

G

Atanine Atanine Atanine Atanine

A^^^-r:^^:! H>Pdt LtL dLru

Gtycine Gtycine Gtycine Gtycine

A G

Valine Va[ine Valine Vatine

(tnn

G

Third base

U C A

G

a a

( Sinnl

Differencesin the mitochondriatgeneticcode are in itolics

A-^--ri-^,f, H>Pdr LrL dL'U

G t u t a m i ca c i d G t u t a m i ca c i d

a

ANDMOLECULAR BASISOFINHERITANCE THECELLULAR

C O N T R OOLFT R A N S C R I ION PT The control of transcription can be affected permanently or reversibly by a variety of factors, both environmental ( e . 9 . h o r m o n e s ) a n d g e n e t i c ( c e l l s i g n a l i n g ) .T h i s o c c u r s through a number of different mechanisms which include s i g n a l i n g m o l e c u l e s t h a t b i n d t o r e g u l a t o r y s e q u e n c e si n the DNA known as resplnseelements,intracellular receptors knorvn as hormonenuclear receptlrs,and receptors for specific ligands on the cell surface involved in the process of signal transduction All of these mechanismsaffect transcription through the binding of transcription factors to short specific DNA promoter elements located within 200bp 5' or upstreamof most eukaryotic genes in the so-called promoter region that l e a d s t o a c t i v a t i o n o f R N A p o l v m e r a s e( F i g . 2 . 1 0 ) . T h i s

usuallv mediated through a helical protein motifs, and are known as transcriptionfactors. These gene regulatory proteins have a transcriptional activation domain and one of four main types of DNA-binding domains.The most common type of gene regulatory protein are the helix-turn-helit proteins, so named becausethey are made up of two a helices connected 'turn'. Perhaps b.va short chain of amino acids that make up the not surprisingly, structural analysis of the homeodomain sequenceof the homeotic genes (p. 86) has revealed that they contain a helix-turn-helix motif. Analysis of other generegulatory proteins has shown them to contain one of three other types of DNA-binding motif: the zinc Jinger, leucine zipper or helixloop helix motifs, so named as a result of specific structural features

includes the TATA (or Hogness), the GC (or GGGCGGG clnsensus sequeme)and the CAAT boxes.The TATA box, which is about 25 bp upstream ofthe transcription start site, is involved

CONTRO L POST- TRANSCRIPTIONAL O FG E N EE X P R E S S I O N

in the initiation of transcription at a basal constitutive level and mutations in it can lead to alteration of the transcription start site. The GC box, which is about 80bp upstream, and the CAAT box increasethe basal level of transcriptional activity of the TATA box. The regulatory elements in the promoter region are said to be cis-acting,that is, they only affect the expression of the adjacentgeneon the sameDNA duplex, whereasthe transcription

Regulation of expression of most genes occurs at the level of transcription but can also occur at the levels of RNA processing,RNA transport, mRNA degradationand translation. For example,the G to A variant at position 20210 in the 3' untranslated region of the prothrombin gene increasesthe stability of the mRNA transcript, resulting in higher plasma

factors are said to be trans-acting,acting on both copies of a geneon eachchromosomebeing synthesizedfrom genesthat are locatedat a distance.DNA sequencesthat increasetranscriptional

R N A . M E D I A T ECDO N T R OOLFG E N E EXPRESSION

activity,such as the GC and CAAT boxes,are known asenhuncers. There are also negative regulatory elements or silencersthat

RNA-mediated silencing was first described in the early 1990s but it is only in the last few yearsthat its key role in controlling post-transcriptional gene expressionhas been both recognized and exploited (Ch 23). Small interfering RNAs (siRNAs) were discovered in 1998 and are the effector molecules of the RNA interference pathway (RNAi). These short doublestranded RNAs (21 to 23 nucleotides)bind to mRNAs in a

inhibit transcription. In addition, there are short sequences of DNA, usually 500bp to 3kb in size and known as boundar.y elemenls, that block or inhibit the influenceof regulatoryelements of adjacentgenes.

TR A N S C R I P T ION F A C T OR S An increasingnumber of genesare being identified that encode proteins involved in the regulation of gene expression Thev have DNA-binding activity to short nucleotide sequences,

prothrombin levels.

sequence-specificmanner and result in their degradation via a r i b o n u c l e a s e - c o n t a i n i n gR N A - i n d u c e d s i l e n c i n g c o m p l e x (RISC). MicroRNAs (miRNAs) also bind to mRNAs in a manner, but they block translation rather than sequence-specific destroying the mRNA.

-

Trans-acti ng elements

2

CAAT box

80bp+: TATA

, TranscriPtion

--*ry* ption Transcri factors enhancers cis-acting

Fig.2.10 Diagrammatic representation geneexpression ofihefactors thatregulate

21

2

THECELLULARAND MOLECULAR BASISOFINHERITANCE

A L T E R N A T IVSEP L IC IN G

MUTATIONS

The majority of human genes(at leastT4o/o)undergo alternatite splicingand thereforeencodemore than one protein. Some genes have more than one promoter, and these alternatiuepromoters may result in tissue-specific isoforms. Alternative splicing of exons is also seen with individual exons present in only some isoforms. The extent of alternative splicing in humans may be inferred from the finding that the human genome includes only 25000 to 30000 genes,far fewer than the original prediction of more than 100000.

RNA.DIRECTED DNASYNTHESIS The process of the transfer of the genetic information from DNA to RNA to protein has been called the central tlogma.It was initially believed that genetic information was transferred only from DNA to RNA and thence translated into protein. However, there is evidence from the study of certain types of virus - retroviruses- that genetic information can occasionally

A mutation is defined as a heritable alteration or change in the genetic material. Mutations drive evolution but can also be pathogenic.Mutations can arise through exposureto mutagenic agents(p. 26), but the vastmajority occur spontaneouslythrough errors in DNA replication and repair. Sequencevariantswith no obvious effect upon phenotype may be termed polymorphisms. Somatic mutations may cause adult-onset diseasesuch as cancer but cannot be transmitted to offspring. A mutation in gonadal tissueor a gametecan be transmitted to future generations unlessit affectsfertility or survival into adulthood. It is estimated that eachindividual carriesup to six lethal or semilethalrecessive mutant alleles that in the homozygous state would have very serious effects.These are conservativeestimatesand the actual figure could be many times greater.Harmful allelesof all kinds constitute the so-calledgeneticload ofthe population There are also rare examples of 'back mutation' in patients with recessivedisorders. For example, reversion of inherited deleteriousmutations has been demonstratedin phenotypically normal cells present in a small number of patients with Fanconi

flow in the reversedirection, from RNA to DNA (p. 197).This is referred to as RNA-directedDNA rynthesh.It has been suggested

anemla.

that regions of DNA in normal cells serve as templates for the synthesis of RNA, rvhich in turn then acts as a template for the synthesisof DNA that later becomesintegrated into the nuclear DNA of other cells Homology between human and retroviral

TYPESOFMUTATION

oncogene sequencescould reflect this process (p. 198), which could be an important therapeutic approachfor the treatment of inherited diseasein humans.

Mutations can range from single base substitutions, through insertions and deletions of single or multiple bases to loss or gain of entire chromosomes(Table 2.2). Base substitutions are most prevalent (Thble 2.3) and missensemutations account for nearlv half of all mutations.A standardnomenclatureto describe

Tabte2.2 Mainclasses, groups product andtypesofmutation andeffects onprotein Class

Group

5ubstrtution Synonymous Non-synonymous

Detetion

Largedetetion

Muttipte of3 (codon) Notmultipteof 3 Largeinsertio n Expansion oftrinucteotide repeat

22

Effecton protein product

Sitento Missenseo Nonsenseo Snlirc
S a m ea m i n oa c i d Atteredaminoacid- mayaffectproteinfunction or stability Stopcodon- lossoffunction or expression dueto degradation o fm R N A - exonskrpping Aberrant sp[icing or intronretention

Promoter

Alicred

Framesh rft Partialgenedeletion

- mayaffect protein In-frame deieiion of oneoTmoreaminoacid(s) function or stabitrty Likelyto resu[t in premature termrnatron withlossoffunctionor expressl0 n Mayresuttin premature termination withlossoffunction or expression

\.il,/figlg nono

I ncc nf g1p;ggEl6Tl

Multipte of3 (codon) Noimultipleof 3

Insertion

Type

"Somehavebeenshownto causeaberrant soticino

doloirnn

ncnc evnrc<
- mayaffect In-frame insertion ofoneor moreaminoacid(s) proteinfunction or stability Frameshift Liketytoresultin premature termination wrthlossoffunctron or expresSron Partialgeneduptication Mayresultin premature termination withlossoffunction or expression Whotegeneduptication Mayhaveaneffect genedosage dueto rncreased Dynamic mutation geneexpression protein Attered or altered stabilrty or function

BASISOFINHERITANCE N4OLECULAR THECELLULARAND

Tabte2.3 Frequency ofdifferent typesofmutation

2

are known as CpG dinucleotides (p representsthe phosphate) frequentl-vbeing methylatedin genomic DNA with spontaneous deaminationof methylcytosineconverting them to thymine. CpG 'hotspots' for mutation' dinucleotideshavebeen termed

Typeof mutation

Percentageof totat

Mrssense or nonsense

51

Spticing

10

Detetions

Regutatory

1

Smatldeletions insertions or indets"

24

Grossdetetions or insertions

1

A deletioninvolves the loss of one or more nucleotides.If this occurs in coding sequencesand involves one, two or more nucleotides that are not a multiple of three, the reading frame rvill be disrupted (p. 25). Larger deletions may result in partial

0 t h e r ( c o - n o t e rxe a r r a n g e r n e n tosr r e p e a t variations)

<1

or whole gene deletions and may arise through unequal crossover betweenrepeat sequences(e g hereditary neuropathy with liabilitl' to pressurepalsies;p.287).

Datafromhttp://www.hgmd org olndets aremutations thatinvolve anda detetron ol bothaninsertion nucteoti des

lnsertions

mutations (Thble 2.1) has been agreed(see http:/,/www.hgvs orglmutnomen/), although it is not used universally.Examples of chromosomeabnormalitiesare discussedin Chaoter 3.

Substitutions A substitutionis the replacement of a single nucleotide by another These are the most common type of mutation. If the substitution involvesreplacementby the sametype of nucleotide a pyrimidine for a pyrimidine (C for T or vice versa) or a purine for a purine (A for G or vice versa); this is termed a transition.Substitution of a pyrimidine by a purine or vrce versa is termed a transrer.sion. tansitions occur more frequently than transversions.This may be due to the relatively high frequenc.v of C to T transitions, which is likely to be the result of the nucleotides cytosine and guanine occurring together, or what

An insertioninvolvesthe addition of one or more nucleotidesinto a gene Again, if an insertion occurs in a coding sequenceand involves one, two or more nucleotidesthat are not a multiple of three, it will disrupt the reading frame (p. 25). Large insertions can also result from unequal crossover(e.g' hereditary sensory a n d m o t o r n e u r o p a t h yt y p e 1 a ; p . 2 8 6 ) o r t h e i n s e r t i o n o f transposableelements(p. 17). In 1991, expansion of trinucleotide repeat sequenceswas identified as a mutational mechanism. A number of singlegene disorders have subsequentlybeen shown to be associated with triplet repeat expansions(Thble 2 5). These are described as dynamic mutations becausethe repeat sequencebecomes more unstable as it expands in size. The mechanism by which amplilication or expansionof the triplet repeat sequenceoccurs is not clear at present.Triplet repeatsbelow a certain length for eachdisorder are faithfully and stablytransmitted in mitosis and meiosis.Above a certain repeatnumber for eachdisorder they are more likell' to be transmitted unstably' usually with an increase or decreasein repeat number. A variety of possibleexplanations

Tabte2.4 Mutationnomenctature: exampLes of CFTRqenemutations Typeof mutation

Nucleotide designation

Protein description

Consequence

Missense

c482G>A

p Arg117His

to histidine Arginine

Nonsense

c1756G>f

p Gty542X

to Stop Gtycine

Spticing

c 621+ 1G>T

(1bp) Detetion

c10787

p Vat358TyrfsXll

mutation Frameshift

(3bp) Detetion

c1652l654detCTT

p Phe508det

l n - f r a m ed e l e t i o no f o h e n v l a [ a n t n e

tnsefIron

c3905 3906rnsT

p Leul25BPhefsXT

F r a m e s h i fm t utation

donorsitemutatton Sptice

u eAn) c e a n d a r e p r e f i x e d boy rgc r e s p e c t i v e t y . T h e f i r s t b a s e o f t h e s t a r t M u t a t i o n s c a n b e d e s i g n a t e d a c c o r d i n g t o t h e g e n o m i c o r c D N A (smeRq N c o d o n( A T G i)s c 1 H o w e v e 6f o r h i s t o r i c arIe a s o n st h i s i s n o t a t w a y st h e c a s e ,a n d t h e f i r s tb a s eo f t h e C F I R c D N Ai s a c t u a t l yn u c t e o t i d1e3 3

23

2

THE CELLULARANDMOLECULAR BASISOFINHERITANCE

Tabte2.5 ExampLes of diseases arisingfromtripletrepeatexpansions Disease

Repeat sequence

Normal range (repeats)

Pathogenic range (repeats)

Repeat location

(HD) Huntington disease

CAG

o2R

36-100

Coding

Myotonic dystrophy type1(DM1)

CTG

tr ?f

50-4000

3'UTR

FragiteXsiteA(FRAXA)

CGG

10-50

200-2000

5'uTR

(SBMA) Kennedy disease

CAG

13-30

40-62

Coding

Spinocerebeltar 1(5CAl) ata."ia

CAG

6-38

39-80

Coding

Spinocerebettar ataxia2 (SCA2)

CAG

16-30

36-52

Coding

M a c h a d o - J o s e pdhi s e a s e( M J D S C A 3 )

CAG

14-40

60->85

Coding

Spinocerebertar ataxra 6 (SCA6)

CAG

5-20

21-28

Coding

Spinocerebettar ataxia7 (SCA7)

CAG

/19

37,220

Codrng

Sprnocerebeltar ataxra B (SCA8)

CTG

16-31

100->500

3'UTR

Spinocerebe[ar ataxia 12(SCA12)

CAG

a_/,q

55-78

5'UTR

Spinocerebettar ataxia17(SC417)

CAG

25-42

47-55

Codrng

(DRPLA) Dentatorubrat-pattidoluysian atrophy

CAC

7-23

49->75

Coding

Friedrerch ataxia(FA)

GAA

100-900

rnlronrc

FragiteXsrteE(FRAXE)

CCG

L)E

>200

Promoter

OcutopharyngeaI muscutar dystrophy

GCG

b

B-13

Coding

UTR.untranslated region

has been offered as to how the increasein triplet repeat number occurs. These include unequal cross-over or unequal sister chromatid exchange(pp. 45, 278) in non-replicaring DNA,

DMPK allele somehow interferes with the cellular processing of RNA produced by a variety of other genes The expandedDAIPK transcripts accumulatein the nuclei of cells and this is believed

and slipped-strand mispairing and polymerase slippage in replicating DNA. tiplet repeat expansionsusually take place over a numbcr of generationswithin a family, providing an explanation for some unusual aspects of patterns of inheritance as well as possibly being the basis of the previously unexplained phenomenon of anticipation(p ll4).

to havea gain-of-function effect through its binding with a CUG RNA-binding protein (CUG-BP). Excess CUG-BP has been shown to interfere with a number of genes relevant to MD, and CUG repeatsare known to exist in various alternatively spliced muscle-specificenzymes(p. 285). The spectrum of repeat expansion mutations also includes

The exact mechanisms by which triplet repeat expansions causediseaseare not known [Jnstabletrinucleotide repeatsmav be within coding or non-coding regions of genesand hence vary in their pathogenicmechanisms.Expansionof the CAG repeatin the coding region of the I1D geneand someSCI genesresultsin a protein with an elongatedpolyglutamine tract that forms toxic

24

aggregateswithin certain cells. In fragile X the repeat expansion in the 5' untranslated region (UTR) results in methylation of promoter sequencesand lack of expressionof the FMRI protein. In myotonic dystrophy (MD) it is believed rhar rhe expandcd

a dodecamer repeat expansion upstream from the cystatin B gene that causesprogressivemyoclonus epilepsy (EPM1), a tetranucleotiderepeat expansion in intron 1 of the ZNFg gene which causesa secondtype of myotonic dystrophy (type 2 MD, formerly known as proximal myotonic myoparhy - PROMM) and a pentanucleotiderepeat expansionin intron 9 of the ATXNI0 gene shown in families with spinocerebellarataxia type 10. Spinocerebellar ataxia is an extremely heterogeneousdisorder and, in addition to the dynamic mutarions shown in Table 2.5, non-repeat expansion mutations have been reported in four additional eenes.

BASISOFINHERITANCE ANDMOLECULAR THECELLULAR

STRUCTURAL EFFECTS OFMUTATIONS O NT H EP R O T E I N Mutations can alsobe subdividedinto trvo main groups according to the effect on the polypeptide sequenceof the encodedprotein, being either s.ynonym0us or nln-s.ynlnymlus.

2

results in the loss of an important functional domain(s) of the protein. mRNA transcriptscontaining premature termination codons are frequentlv degradedby a processknown as nlnsensemediuteddecay.This is a form of RNA surveillance that is believed to haveevolved to protect the body from the possibleconsequences of truncated proteins interfering with normal function.

Synonymous or silentmutations If a mutation doesnot alter the pol,vpeptideproduct of the gene, it is termed a synonymousor silent mutatiln. A single base-pair substitution, particularly if it occurs in the third position of a codon becauseof the degeneracyof the genetic code, will often result in another triplet that codesfor the sameamino acid with no alteration in the properties of the resulting proteln

Non-synonymous mutations

Fromeshift If a mutation involvesthe insertion or deletion of nucleotidesthat arc not a multiple of three, it will disrupt the reading frame and constitute what is known as r frameshift mutation. The aminoacid sequenceof the protein subsequentto the mutation bears no resemblanceto the normal sequenceand may have an adverse effect on its function. Most frameshift mutations result in a premature stop codon downstream to the mutation. This may lead to expressionof a truncated protein, unless the mRNA is

If a mutation leads to an alteration in the encoded polypeptide, it is known as a nln-s.ynonymous mutution. Non-synonymous

degraded b1'nonsense-mediateddecay.

mutations are observedto occur lessfrequently than synonymous mutations. Synonvmous mutations are selectively neutral, whereas alteration of the amino-acid sequenceof the protein product of a geneis likely to result in abnormal function, which is

M U T A T I O NI N S N O N - C O D I NDGN A

usually associatedwith disease,or lethality, which has an obvious selectivedisadvantage. Non-synonymous mutations can occur in one of three main ways.

In general,mutations in non-coding DNA are lesslikely to have a phenotypic effect Exceptions include mutations in promoter sequencesor other regulatory regions that affect the level of gene expression.With our new knowledge of the role of RNA interference in gene expression, it has become apparent that mutations in miRNA or siRNA binding sites within UTRs are alsolikely to result in disease.

Missense A single base-pairsubstitution can result in coding for a different amino acid and the synthesis of an altered protein, a so-called mutation If the mutation codes for an amino acid that missense is chemically dissimilar, for example has a different charge,the structure of the protein will be altered. This is termed a nontonserxu,tiae substitutionand can leadto a grossreduction, or even a complete loss,of biological activity. Single base-pairmutations can lead to qualitative rather than quantitative changes in the function of a protein, such that it retains its normal biological activity (e.9.enzymeactivit-y)but differs in characteristicssuch as its mobility on electrophoresis,its pH optimum, or its stability so that it is more rapidly broken down in uiao. Many of the abnormal hemoglobins(p. l5l) are the result of missensemutations. Some single base-pairsubstitutions,although resulting in the replacementby a different amino acid if it is chemically similar, often have no functional effect. These are termed Lons(rl)attre

Spticingmutations Mutations of the highly conservedsplice donor (GT) and splice acceptor (AG) sites (p. l8) usually result in aberrant splicing This can result in the loss of coding sequence(exon skipping) or retention of intronic sequence,and may lead to frameshift mutations. Cryptic splice sites, which resemble the sequence of an authentic splice site, may be activated when the conserved splice sites are mutated. The spectrum of splicing mutations has recentll, been extended with the observation that base substitutionsresulting in apparentsilent, missenseand nonsense mutations can causeaberrant splicing through mutation of exon sltlidng enhancersequences.These purine-rich sequencesare required for the correct splicing of exons with weak splice-site consensussequences.

substitutions.

OFMUTATIO N S EFFECTS FUNCTIONAL O NT H EP R O T E I N

Nonsense

N,Iutations exert their phenotypic effect in one of two ways, through either loss or gain of function

A substitution that leads to the generation of one of the stop codons (see Table 2 l) rvill result in premature termination of translation of a peptide chain, or what is termed a nonsense mutation In most casesthe shortenedchain is unlikely to retain

mutations Loss-of-function

normal biological activity, particularly if the termination codon

mutations can result in either reduced activity or Loss-oJ-Junction complete loss of the geneproduct. The former can be the result

25

2

THECELLULAR ANDMOLECULAR BASISOFINHERITANCE

of reduced activity or of decreasedstability of the gene product and is known as a h.ypomorpl2, the latter being known as a null

or function, as a consequenceof the mutant gene product interfering with the function of the normal gene product of

alleleor umorph.Loss-of-function mutations in the heterozvgous statewould, at worst, be associatedwith half normal levelsof the protein product. Loss-of-function mutations involving enzymes are usually inherited in an autosomal or X-linked recessive manner, becausethe catalytic activity of the product of the

the corresponding allele. Dominant-negative mutations are particularly common in proteins that are dimers or multimers,

normal allele is more than adequateto carry out the reactionsof most metabolicpathwa-\ s.

GENOTYPE.PH ENOTYPECORRELATI ON

Hoplo-insufficiency

Many geneticdisordersare well recognizedas being very variable in severity,or in the particular featuresmanifested by a person with the disorder (p 105) Developments in molecular genetics

Loss-of-function mutations in the heterozygousstate in which half normal levels of the gene product result in phenoty.pic

increasingll, allow identification of the mutational basis of the specificfeaturesthat occur in a person with a particular inherited

effects are termed hapltt-insfficienc.ymutations.The phenotypic manifestationssensitiveto gene dosageare a result of mutations occurring in genesthat code for either receptors,or more rarely enzymes,the functions of which are rate limiting, for example familial hypercholesterolemia (p 167) and acure inrermittent porph-vria(p.172)

disease,or what is known as the phenotype.This has resulted in attempts to correlatethe presenceof a particular mutation, which is often called the genotype,with the specificfeaturesseenin a person with an inherited disorder, this being referred to as gent4ype phenotypecorrelation.This can be important in the management

In a number of autosomaldominant disordersthe mutational basis of the functional abnormalitv is the result of haploinsufficiencv in which, not surprisingly; homozvgousmutations result in more severephenotypic effects;examplesareangioneurotic edema(p. 190) and familial hypercholesterolemia(p 167).

Gain-of-function mutations Gain-o.f-functiozmutations, as the name suggests,result in either increased levels of gene expression or the development of a new function(s) of the gene product. Increasedexpression levelsdue to activatingpoint mutations or increasedgenedosage are responsiblefor one type of Charcot-Marie-Tooth disease, hereditary motor and sensory neuropathy type I (p. 286) The expanded triplet repeat mutations in the Huntington gene cause qualitative changesin the gene product that result in its aggregationin the central nervous system leading to the classic clinical featuresof the disorder (p.282). Mutations that alter the timing or tissue specificity of the expressionof a genecan alsobe consideredto be gain-of-function mutations. Examples include the chromosomal rearrangements that result in the combination of sequencesfrom two different genesseenwith specifictumors (p 199).The novel function of the resulting chimeric genecausesthe neoplasticprocess. Gain-of-function mutations are dominantly inherited and the rare instances of gain-of-function mutations occurring in the homozygous state are often associatedwith a much more severe phenotvpe,which is often a prenatallylethal disorder,for example homozygous achondroplasia(p. 91) or Waardenburg syndrome type I (p 89).

Dominant-negative mutations 26

for instancestructural proteins such as the collagens,mutations in which can lead to osteogenesis imperfecta (p. 103).

A dominant-negatiTemntation is one in which a mutant gene in the heterozygousstate results in the loss of protein activity

of a patient.One exampleincludesthe associationof mutations in the BRCAI genewith the risk of developing ovarian canceras well as breast cancer (p. 2l l). Particularly striking examplesare mutations in the receptor tyrosine kinasegeneREIwhich, depending on their location, can lead to four different syndromesthat differ in the functional mechanism and clinical phenotype. Loss-offunction nonsensemutations lead to lack of migration of neural crest-derived cells to form the ganglia of the myenteric plexus of the large bowel, leading to Hirschsprung disease,whereas gain-of-function missensemutations result in familial medullary thyroid carcinomaor one of the two types of multiple endocrine neoplasiatype 2 (p. 95) Mutations in the LMNA gene are associatedwith an even broader spectrum ofdisease (p. 105).

MUTATIONS ANDM UTAGENES IS Naturally occurring mutations are referred to as splnteneous mut&tionsand are thought to arise through chance errors in chromosomaldivision or DNA replication Environmental agents that causemutations are known as mutasens.

MUTAGENS These include natural or artificial ionizinq radiation and chemical or physicalmutagens.

lonizingradiation Ionizing ratlia,tionincludes electromagneticwavesof very short wavelength (X-rays and y rays) and high-energy parricles (o particles, B particles and neutrons). X-rays, y rays and neutrons have great penetrating power, but CX, particles can penetratesoft tissuesto a depth of only a fraction of a millimeter and B particles only up to a few millimeters.

BASISOFINHERITANCE AND MOLECULAR THE CELLULAR

2

Meosuresof rodiotion The amount of radiation received by irradiated tissuesis often referred to as the 'dose', which is measuredin terms of the rad,iationabsorbed doseor rad.The rad is a measureof the amount of any ionizing radiation that is actually absorbedby the tissues, I rad being equivalentto l00ergs ofenergy absorbedper gram of tissue The biological effectsofionizing radiation depend on the volume of tissue exposed.In humans, irradiation of the whole body with a dose of 300-500rads is usually fatal, but as much as l0000rads can be given to a small volume of tissue in the treatment of malignant tumors without seriouseffects Humans can be exposedto a mixture of radiation, and the rem (roentgenequit:alentforman)is a convenientunit asit is a measureof any radiationin terms ofX-rays. A rem ofradiation is that absorbed dosethat producesin a given tissuethe samebiological effect as I rad of X-rays. Expressingdosesof radiationin terms of rems permits a comparisonof the amountsof different typesof radiationto which humansareexposed.A millirem (mrem) is one-thousandthof arem; l00rem is equivalent to lsie"^ert(Sv), and 100rad is equivalent to I gray (Gy) in SI units. For all practical purposes,sievertsand grays are approximately equal. In this discussion sieverts and millisieverts (mSv) are used as the units of measure

averagedosesof ionizing Tabte2.5 Approximate to the gonadsof the general fromvarioussouTCes radiation OODULATION

Averagedoseper year (mSv)

Averagedoseper 3OYears(mSv)

Natural radiation Cosmrc

025

1.5

y radiationo External

150

450

Sourceof radiation

Internalyradiation 030

90

Artificiat radiology Medrcat

0 30

90

fallout Radioactive

0 01

03

and Occupationat miscellaneous

0 04

12

Totat

240

720

olnctuding radonin dwelting T R E 1989Risksfrom ionizing Datafrom CtarkeR H, Southwood Nature338:197-198 radiation.

Dosimetry Dosimetryis the measurementof radiation.The doseof radiation is expressedin relation to the amount received by the gonads becauseit is the effects of radiation on germ cells rather than somaticcellsthat are important asfar astransmissionof mutations to future progenv is concerned. The gonad doseof radiation is often expressedas the amount received in 30years This period of time has been chosen becauseit correspondsroughly to the generationtime in humans.

the dose:the larger the dose,the greaterthe number of mutations produced. It is believed that there is no threshold below which irradiation has no effect - even the smallest dose of radiation can result in a mutation. The geneticeffectsof ionizing radiation are also cumulative, so that each time a person is exposed to radiation the dose received has to be added to the amount of radiation alreadyreceived.The total number of radiation-induced mutations is directly proportional to the total gonadaldose.

Sourcesof rodiotion The various sourcesand averageannual doses of the different types of natural and artificial ionizing radiation are listed in Thble 2.6. Natural sourcesof radiation include cosmic ra1,-s, external radiation from radioactive materials in certain rocks, and internal radiation from radioactive materials in tissues Artificial sources include diagnostic and therapeutic radiology, occupationalexposureand fallout from nuclear explosions. The averagegonadaldoseofionizing radiationfrom radioactive fallout resulting from the testing of nuclear weaponsis lessthan that from an-vof the sourcesof background radiation. However, the possibilit-vof serious accidents involving nuclear reactors, as occurred at Three Mile Island in the USA in 1979, and at Chernobl-l in the Soviet Union in 1986,with widespreadeffects, alwaysmust be borne in mind.

Geneticeffects Experiments with animals and plants have shown that the number of mutations produced by irradiation is proportional to

dose Permissible The hazard from mutations induced in humans by radiation is not so much to ourselvesas to our descendants.Unfortunately, in humans there is no easyway to demonstrategenetic damage causedby mutagens.Nevertheless,the International Commission on RadiologicalProtection (ICRP), working in close liaison rvith various agenciesof the United Nations - World Health Organization (WHO), United Nations Educational, Scientific and Cultural Organization (UNESCO)' International Atomic Energl- Agency (IAEA) - has been mainly responsible for defining rvhat is referred to as the maximum permissibledoseof radiation. This is an arbitrary safety limit and is probably very much lower than that which would causeany significant effect on the frequency'of harmful mutations within the population. It has beenrecommendedthat occupationalexposureshould not exceed 50mSv per year.However, there is currently much controversy over exactll' rvhat the permissible dose should be and some countries,such asthe USA, set the upper limit significantlylower than many others. In the UK the Radiation Protection Division

27

2

THECELLULAR ANDMOLECULAR BASISOFINHERITANCE

of the Health Protection Agency advisesthat occupational exposureshould not in fact exceedl5 mSv in a 1ear.To put this into perspective,I mSv is roughly 50 times the dose receivedin

DNA strand by an endonuclease,removalof the damagedregion by an exonuclease,insertion of new basesby the enzyme DNA polymerase,and sealingof the break by DNA ligase.

a single chest X-ray and 100 times the doseincurred when flying from the UK to Spain in a jet aircraft!

Nucleotide excisionrapair (NER) removes thymine dimers and large chemical adducts. It is a complex processinvolving more than 30 proteins that remove fragments of approximately 30 nucleotides. Mutations in at least eight of the genes encoding these proteins can cause xeroderma pigmentosum (p.277),

There is no doubting the potential dangers,both somatic and genetic,of exposureto ionizing radiation. In the caseof medical radiology, the dose of radiation resulting from a particular procedurehas to be weighedagainstthe ultimate beneficialeffect to the patient. In the caseof occupationalexposureto radiation, the answerlies in defining the risks and introducing and enforcing adequate legislation. With regard to the dangers from fallout from nuclear accidentsand explosions,the solution would seem obvious.

Chemicalmutagens

characterized by extreme sensitivity to ultraviolet light and a high frequency of skin cancer.A different set of repair enzymes is utilized to excisesingle abnormal bases(baseercisionrepair or BER), with mutations in the geneencoding the DNA glycosylase MYH having recently been shown to causean autosomal recessive form of colorectalcancer(p.207). Naturally occurring reactive oxygen speciesand ionizing radiation induce breakageof DNA strands.Double-strand breaks

In humans, chemical mutagenesismay be more important than radiation in producing genericdamage.Experiments haveshown that certain chemicals, such as mustard gas, formaldehyde,

result in chromosome breaks that can be lethal if not repaired. Post-replicationrepair is required to correct double-strand breaks and usually involves homologous recombination with a sister DNA molecule. Human genesinvolved in this pathway include

benzene,some basic dyes and food additives, are mutagenic in animals.Exposure to environmental chemicalsmay result in the formation of DNA adducts, chromosomebreaks or aneuploidy-.

NBS, BLilI and BRCAI /2, mutated in Nijmegen breakage s-vndrome,Bloom syndrome (p. 277) and hereditary breast cancer (p. 2ll), respectivell'.Alternatively, the broken ends may

Consequently all new pharmaceuticalproducts are subject to a battery of mutagenicity tests that include both in dtro and in cit:o studiesin animals.

be rejoined by non-homologous end-joining, which is an errorprone pathway.

DNAREPAIR The occurrence of mutations in DNA, if left unrepaired, would have serious consequencesfor both the individual and subsequentgenerations.The stability of DNA is dependentupon continuous DNA repair by a number of different mechanisms (Table2.7) Some types of DNA damagecan be repaired directly. Examples include the dealkylation of O6-alkyl guanine or the removal of thymine dimers by photoreactivation in bacteria. The majority of DNA repair mechanismsinvolve cleavageof the

Mismatch repair (MMR) corrects mismatched basesintroduced during DNA replication. Cells defectivein MMR havevery high mutation rates(up to 1000times higher than normal). Mutations in at least six different MMR genes cause hereditary nonpoly'posiscolorectalcancer(HNPCC; p. 209). Although DNA repair pathways have evolved to correct DNA damage and hence prorect the cell from the deleterious consequencesof mutations, some mutations arise from the cell's attempts to tolerate damage. One example rs translesion DNA 1ynthesis, where the DNA replication machinery bypasses sites of DNA damage, allowing normal DNA replication and gene expression to proceed downstream Human diseasemay

Tabte2.7 DNArepairpathways, genesandassociated disorders

28

Typeof DNArepair

Mechanism

Genes

Disorders

B a s ee x c i s i o nr e p a i r( B E R )

R e m o v aol f a b n o T m aD r ases

MYH

CotorectaI cancer

Nucteotide excision repair(NER)

Removat ofthymrne dimersandlarge chemicat adducts

Xp genes

piqmentosum Xeroderma

Post-replication repair

RemovaI of doubte-strand breaks byhomotogous recombination or non-homotogous end-joining

,ryB5 BLM BRCA\/2

Nijmegen breakage syndrome Btoomsyndrome Breastcancer

Mismatch repair(MMR)

Corrects mismatched basescaused bymistakes in DNAreptication

MSHondMLH genes

(HNPCC) CotorectaI cancer

H N P C Ch , e r e d i t a r yn o n- p o t y p o s i sc o i o r e c t acIa n c e r

E OFINHERITANC BASIS ANDMOLECULAR THECELLULAR

also be causedby defective cellular responsesto DNA damage. Cells have complex signaling pathways that allow cell cycle arrest to provide increasedtime for DNA repair. If the DNA damage is irreparable, the cell may initiate programmed cell death (apoptosrs).The ATM protein is involved in sensing DNA 'guardian of the genome'. damageand has been describedas the Mutations in the ATM gene cause ataxia telangiectasia(AT; p.192), characterizedby hypersensitivityto radiation and a high risk of cancer.

FURTHER READING Alberts B, JohnsonA, Lewis J et al 2002 Molecular biology of the cell, 4th edn Garland,London pell pritten arul laaishfuillustratedcomprehensiae text oJ' hry atcessible, problemsbookand,CD ROI4 using molecularbiologywith accompanying ssmen I multimedia rexiem and,selJ:asse DawkinsR 1989The selfishgene,2nd edn Oxford University Press, Oxford An int eresting controrersial concep t EpsteinRJ 2003Human molecularbiology:an introductionto the molecularbasisof healthand diseaseCambridgeUniversity Press, Cambridge A moderntextboobaboutmolecules and their role in humandiseaseFire A, Xu S, Montgomery M K, KostasS A, Driver S E, Mello C C 1998 Potent and specific genetic interferenceby double-strandedRNA in Caenorhubdins elegansNature 391:80G8l l Landmarkpaperdescribingthe discoxeryof RNAi. Lewin B 2004GenesVIII,8th edn. Oxford University Press,Oxford The eighthedition oJ'thisetcellenttextbllk of moletularbiolog.ywith nlor diagramsandfgures Hard Io tmproaeupon Mettler F A, Upton A C 1995Medical effectsof ionisingradiation, 2nd edn WB Saunders,Philadelphia oJ ioniang radiation Cood otertiep of all uspectsoJ'themedicalconsequences Schull WJ, NeelJV 1958Radiationand the sexratio in man Ser ratio amongchildrcn of survivorsof atomicbombingssuggestsinducedsexlinked lethalmutations Science228:134 ,138 The original reportof posible et;itlenterl the eJJicnof atomicradiation StrachanT, ReadA P 200.1Human molecular genetics,3rd edn Garland Science,London An uf to-datecomlrehensiae textbolk 0fall asfettsoJmolecularand cellular in humans biolog-7' s5 h relatesto inheriteddisease Tirrner J E 1995Atoms, radiationand radiationprotection John Wiley, Chichester BusisoJ'thephysicsof radiation,applicationsand harmful effects \lhtson J D, Crick F H C 1953Molecularstructureof nucleicacids- a nucleicacid Nature 171:737-738 structurefor deoxJ'ribose in thislaper, lresentedinjust o.^er,ne fage, resultedin the authors The c7ncepts recei'^ingthe Nobel PrizeI

2

ELEMENTS Q Genetic information is stored in DNA (deoxyribonucleicacid) as a linear sequenceof two types of nucleotide, the purines (adenine [A] and guanine [G]) and the pyrimidines (cytosineIC] and thymine [T])' linked by a sugar-phosphate backbone. @ A molecule of DNA consists of two antiparallel strands held in a double helix by hydrogen bonds between the complementary G-C and A-'l base pairs. pNA replication has multiple sites of origin and is €) semiconservative,each strand acting as a template for synthesisof a complementarystrand. @ G.n.t coding for proteins in higher organisms (eukaryotes) consist of coding (exons) and non-coding (introns) sections. @ T.anscription is the synthesis of a single-stranded complementarycopy of one strand of a genethat is known as messengerRNA (mRNA). RNA (ribonucleic acid) differs from DNA in containing the sugar ribose and the baseuracil insteadof thymine. @ mRNA is processedduring transport from the nucleus to the cytoplasm, eliminating the non-coding sections.In the cytoplasm it becomesassociatedwith the ribosomes, where translation (i.e. protein synthesis)occurs. 'universal' and consistsof triplets @ fn. geneticcode is (codons)of nucleotides,eachof which codesfor an amino acid or termination of peptide chain synthesis.The code is degenerate,as all but two amino acids are specifiedby more than one codon. @ fn. major control of gene expressionis at the level of transcription by DNA regulatory sequencesin the 5' flanking promoter region of structural genesin eukaryotes. General and specifictranscription factorsare alsoinvolved in the regulation of Benes. @ Mutations occur both spontaneouslyand as a result of exposureto mutagenicagentssuch asionizing radiation. Mutations are continuously corrected by DNA repair enzvmes.

29

CHAPTER

Chromosomes andce[[division

'Let

us not take it for granted that life existsmore fully in what is commonly thought big than in what is commonly thought small.'

eachhuman cell contained48 chromosomesand that human sex was determined by the number of X chromosomespresent at conception. Following the developmentin 1956of more reliable techniques for studying human chromosomes it was realized

Vireinia Woolf At the molecularor submicroscopiclevel DNA can be regardedas the basictemplate that providesa blueprint for the formation and maintenance of an organism DNA is packaged into chromosomes and at a very simple level these can be considered as being made up of tightly coiled long chains of genes.Unlike DNA, chromosomescan be visualizedduring cell division using a light microscope,under which they appear as thread-like structures or 'colored bodies'. The word chromosome is derived from the Greek chroma(= color) and soma(= body). Chromosomesare the factorsthat distinguish one speciesfrom another and that enablethe transmissionof genetic information from one generationto the next. Their behavior at somatic cell division in mitosisprovidesa meansof ensuring that eachdaughter cell retainsits own completegeneticcomplement. Similarly, their behaviorduring gameteformation in meiosisenableseachmature ovum and sperm to contain a unique single set of parental genes. Chromosomes are quite literally the vehicles that facilitate reproduction and the maintenanceof a species. The study of chromosomesand cell division is referred to as cytogenetics. Prior to the 1950sit was believed, incorrectly, that

that the correct chromosomenumber in humans is 46 (p. 5) and that malenessis determined by the presenceof a Y chromosome regardlessof the number of X chromosomes present in each cell. It was also realized that abnormalities of chromosome number and structure could seriously disrupt normal srowth and development. Thble 3.1 highlights the methodological developmenrs rhar have taken place during the past five decadesthat underpin our current knowledeeof human cvtosenetics.

H U M A NC H R O M O S O M E S MORPHOLOGY At the submicroscopiclevel chromosomesconsistof an extremely elaboratecomplex, made up of supercoils of DNA, which has been likened to the tightly coiled network of wiring seen 1n a solenoid (p. 30). Under the electron microscope chromosomes can be seento have a rounded and rather irregular morphology

Tabte3.1 Devetopment of methodotogies for cytogenetics

30

Decade

Development

Examplesof application

1950s

Reliabte methods forchromosome preparations

Chromosome numberdetermined as46(1956)

1970s

Giemsa chromosome banding

Phitadetphia chromosome identified as t(9:22)(1973)

1980s

(FISH) Ftuorescent rn-sduhybridization

Interphase (1994) FISHforrapiddetection of Downsyndrome SpectraI karyotyping (i996) forwhole-genome chromosome anatysis

1990s

(CGH) Comparative genomic hybridization

genomic Mapping imbalances in sotidtumors(1992)

2000s

ArrayCGH

Analysis of consitutionaI rearrangements: e g identification of -5Mbdetetion in a patient withCHARGE syndrome thatledto identification oftheqene(2004)

CHARGE' cotobomaof the eye,heart defects,otresiaof the choanae,retardationof growth and/or deve[opment,genitaIand/or urinary abnormalities. a n d e a r a b n o r m a l i t i eas n d d e a f n e s s

ANDCELLDIVISION CHROMOSOMES

(Fig. 3.1). However, most of our knowledge of chromosome structure has been gained using light microscopy.Special stains selectively taken up by DNA have enabled each individual chromosome to be identified. These are best seen during cell division, when the chromosomesare maximally contracted and the constituent genescan no longer be transcribed. At this time each chromosome can be seen to consist of two identical strands known as chromatid,s,or sisterchromatids,which are the result of DNA replication having taken place during the S (synthesis)phaseof the cell cycle (p. 4l). These sisterchromatids can be seento be joined at a primary constriction known as the centrrmere.Centromeres consist of several hundred kilobases of repetitive DNA and are responsible for the movement of chromosomes at cell division. Each centromere divides the chromosome into short and long arms, designatedp (= petite) and q ('g' = grande), respectively. The tip of each chromosome arm is known as the telomere. Telomeresplay a crucial role in sealingthe ends of chromosomes and maintaining their structural integrity. Telomeres have been highly conserved throughout evolution and in humans they consist of many tandem repeatsof a TTAGGG sequence. During DNA replication an enzymeknown as telomerase replaces the 5' end of the long strand, which would otherwise become progressivelyshorter until a critical length was reachedwhen the

3

cell could no longer divide and thus becamesenescent.This is in fact part of the normal cellular aging process,with most cells being unable to undergo more than 50 to 60 divisions. However, in sometumors increasedtelomeraseactivity hasbeen implicated as a causeof abnormally prolonged cell survival. Morphologically chromosomes are classified according to the position of the centromere. If this is located centrally, the chromosome is metacentric,if terrninal it is acrocentric,and,if the centromere is in an intermediate position the chromosome is submet acenuic(Fig. 3.2) Acrocentric chromosomessometimes have stalk-like appendagescalledsatellitesthat form the nucleolus of the resting interphasecell and contain multiple repeat copies of the genesfor ribosomal RNA.

CLASSIFICATION Individual chromosomesdiffer not only in the position of the centromere but also in their overall length. Based on the three parameters of length, position of the centromere, and the presenceor absenceof satellites,early pioneers of cytogenetics were able to identify most individual chromosomes,or at least subdivide them into groups labelledA-G on the basisof overall morphology(A, l-3; B, 4-5; C,6-12 * X; D, l3-15; E, 1Gl8; F, 19-20; G,21-22 + Y). In humans the normal cell nucleus and contains 46 chromosomes,made up of 22 patrs of autosomes a single pair of sex chromosomes - XX in the female and XY in the male. One member of each of these pairs is derived from each parent. Somatic cells are said to have a d'iploid complement of 46 chromosomes,whereas gametes (ova and sperm) have a haploid complement of 23 chromosomes.Members of a pair of chromosomes are known as homologs. The development of chromosome banding @. 32) enabled very precise recognition of individual chromosomes and the detection of subtle chromosome abnormalities.This technique

Chromatids Iites Satel

Fis.3.1 E t e c t r omni c r o g r a pohf h u m a nc h r o m o s o m sehs o w i ntgh e (Courtesy andwetldefined chromatids of centromeres Dr Christine HarrisonReproduced fromHarrison etal1983 Cytogenet CettGenet35:21-27wtlhpermission ofthepubtisher; S K a r g eB r asel)

M e t ac en t r i c

Submetacentric

Acrocentric

Fi9.3.2 as metacentric, aredescribed chromosomes MorphoLogicatly ofthe ontheposition depending oracrocentric, submetacentric centr0mere

31

3

CHROMOSOM AE NSDC E L LD I V I S I O N

alsorevealedthat chromatin,thecombinationof DNA and histone proteins that comprise chromosomes,exists in trvo main forms Euchromatinstainslightly and consistsof genesthat are actively expressed.In contrast, heterochromatiz stainsdarkly and is made up largelv of inactive,unexpressed,repetitive DNA The total number of chromosomes in different organisms varies considerably but is constant for any particular species. Whereas the marmoset and certain monkeys resemblehumans in having 46 chromosomes,higher primates more closelvrelated

E G

to humans, such as the chimpanzee, gorilla and orangutan, have 48 chromosomes.Among these primates the chromosomes of the chimpanzee most closely resemble those of humans. This is consistent u'ith the fact that there is a difference of only lolo between human and chimpanzee DNA. There is general agreement that the human number 2 chromosome is the product of fusion of two chimpanzee chromosomes, with

N

E .L

Fis.3.3 P u n n e t ts' sq u a r seh o w i nsge xc h r o m o s o mceo m b i n a t r of o n rs --

^

-^!

f^--l^

^--^+^-

many other differencesbetween the chromosome complements in the two speciesbeing due to paracentric and pericentric inversions (p. 52) These observationshave been exploited bymolecular biologiststo help map and clone genesin humans.

T H ES E XC H R O M O S O M E S The X and Y chromosomesare knorvn as the sex chromosomes b e c a u s eo f t h e i r c r u c i a l r o l e i n s e x d e t e r m i n a t i o n . T h e X chromosome rvas originall-v labeled as such because of uncertaintl, as to its function when it was realized that in some insects this chromosome is present in some gametes but not in others. In these insects the male has only one sex chromosome(X), whereasthe female has two (XX) In humans, and in most mammals, both the male and the female have two sex chromosomes- XX in the female and XY in the male. The Y chromosome is much smaller than the X and carries only a few genes of functional importance, most notablv the testisdetermining factor, known as SRY (p. 89). Other genes on the Y chromosome are known to be important in maintaining spermatogenesls. In the femaleeachovum carriesan X chromosome,whereasin the male each sperm carries either an X or a Y chromosome.As there is a roughly equal chanceof either an X-bearing sperm or a Y-bearing sperm fertilizing an ovum, the numbers of male and female conceptions are approximately'equal (Fig. 3.3). In fact, slightly more male babiesare born than females,although during childhood and adult life the sex ratio evensout at I :1 The processof sex determination is consideredin detail later

(p.e6).

modifications, are nolv universally employed in cytogenetic laboratories to analyze the chromosome constitution of an individual, which is known as a karyotype.This term is alsoused to describea photomicrograph of an individual's chromosomes, arrangedin a standardmanner.

CHROM OSOM E PREPARATION Any tissue rvith living nucleatedcells that undergo division can be used for studying human chromosomes.Most commonly circulating lymphocytesfrom peripheralblood are used,although samples for chromosomal analysis can be prepared relatively easily using skin, bone marroq chorionic villi or cells from amniotic fluid (amniocytes). In the caseof peripheral (venous)blood, a sampleis added to a small volume of nutrient medium containingphytohemagglutinin, which stimulatesT lymphocytesto divide. The cells are cultured under sterile conditions at 37"C for about 3da1's,during which they divide, and colchicine is then added to each culture. This drug has the extremely useful property of preventing formation of the spindle, thereby arresting cell division during metaphase, the time when the chromosomesare maximally condensedand therefore most visible. Hypotonic saline is then added, which causesthe red blood cells to lyze and results in spreadingof the chromosomes, which are then fixed, mounted on a slide and stainedready for analysis(Fig. 3.a).

CHROMOSOM ENDING BA Several different staining methods can be utilized to identify individual chromosomes.

M E T H O DS OFC H R OMOS OME A N ALYSIS

32

It was generallybelievedthat eachcell contained48 chromosomes until 1956,whenTjio and Levan correctly concluded on the basis of their studies that the normal human somatic cell contains only 46 chromosomes(p. 5). The methods they used,with cerrain

G ( G i e m s ab) a n d i n g This is the method used most commonly.The chromosomesare treated rvith trypsin, which denaturestheir protein content, and then stained with a DNA-binding dye known as Giemsa, which

AE NSDC E L LD I V I S I O N CHROMOSOM

3

frHHM HH HH HX HfiHH HH XHfrfrXHHH fiH ffi$ frHHfr X$ ffiHfrH"Xfr ffiH Karyotype

A n al y z e

sprea0

withtrypsin Digest andstain withGiemsa

Addphytohemagglutinin a n dc u l t u r em e d i u m

S p r e a dc e l l so n t o s l i d eb y d r o p p i n g

A d dc o l c h i c i n e and h y p o t o n i cs a l i n e

C e l l sf i x e d

Fis.3.4 Preparation ofa karyotype

giveseachchromosomea characteristicand reproducible pattern oflight and dark bands(Fig. 3.5).

banding 0 (quinacrine) This gives a banding pattern similar to that obtained with Giemsa, and requires examination of the chromosomeswith an ultraviolet fl uorescencemlcroscoDe

R (reverse)banding The chromosomes are heat-denaturedbefore staining with Giemsa, yielding light and dark bands which are the reverseof those obtained using conventionalG banding (Fig. 3.6).

H i g h - r e s o l u t i obna n d i n g G banding generallyprovides high-quality chromosomeanalysis with approximately 400 to 500 bands per haploid set. Each of these bands corresponds on average to approximately 6000-8000kilobases(kb) (i.e. 6-8megabases) of DNA. Highresolution banding of the chromosomes at an earlier stage of mitosis, such as prophase or prometaphase, provides greater sensitivitywith up to 800 bandsper haploid set,but is much more demanding technically.This involvesfirst inhibiting cell division with an agent such as methotrexateor thymidine. Folic acid or deoxy-cytidineis added to the culture medium, releasingthe cells into mitosis. Colchicine is then added at a specifictime interval, when a higher proportion of cellswill be in prometaphaseand the chromosomeswill not be fully contracted,giving a more detailed banding pattern.

C (centromeric heterochromatin) banding If the chromosomesare pretreated with acid followed by alkali prior to G banding,the centromeresand other heterochromaticregions containing highly repetitive DNA sequencesare stained preferentially.

ANALYSIS KARYOTYPE The next stagein chromosome analysisinvolves first countinB the number of chromosomespresent in a specifiednumber of cells,

33

CHROMOSOM AE NSDC E L LD I V I S I O N

pattern of the individual chromosomes is carried out on both members of eachpair of homologsin approximatelythree to five metaphasespreads,which show high-quality banding. The banding pattern of each chromosome is specific and

12

can be shown in the form of a stylized ideal karyotype known as an id,iogram(Fig. 3.7). The cytogeneticistanalyzeseach pair

3

xtl An

9

11

12

of homologous chromosomes, either while looking down the microscope or, increasingly,on a photograph of the metaphase spread, which can now be produced electronically (Fig. 3.8). Until the advent of banding in 1971, chromosomes could be classified only on the basis of their overall morphology. Now a formally presented karyotype, or karyogram, will show each chromosomepair in descendingorder of size.

MOLECULAR CYTOGEN ETIC5

13

14

15

16

17

18

FLUO RESCENT'N.S'TU HYBRI DIZATI ON This diagnostic tool combines conventional cytogenetics with molecular genetic technology.It is based on the unique ability of a portion of single-strandedDNA (i.e. a probe; p. 35) to

a

f* 19

20

21

22

Fig.3.5 A normaI G-banded malekaryotype

1

10

6789

11

12

14

,r s3 19

20

15

16

45X

X

*e

i

13

3

17

18

6

10

11

12

,;x ** 2122Y

Fig.3.5 (Courtesy A normal R-banded malekaryotype ofH J Evans )

IJ

sometimes referred to Ls meta.phase spreads,followed by careful analysisof the banding pattern of eachindividual chromosomern selectedcells.Usually the total chromosomecount is determined in l0 to l5 cells, but if mosaicism is suspectedthen 30 or more cell counts will be undertaken. Detailed analysisof the banding

19

Fig.3.7 A n i d i o g r a ms h o w i n gt h e b a n d i n gp a t t e r n so f i n d i v r d u a L chromosomesas revealedby f[uorescent and Giemsastaining

AE NS DC E L LD I V I S I O N CHROMOSOM

3

e,tlpa

Oq

l*

Fis.3.8 s yf M r A W i l k r n s oCny, t o g e n e tU A G - b a n d em d e t a p h a ssep r e a d( C o u r t e o i cnsr tC, i t yH o s p i t aNl ,o t t i n g h a)m

anneal with its complementary target sequenceon a metaphase chromosome,interphasenucleusor extendedchromatin fiber. In fluorescentin-srtu h.ybridization(FISH), the DNA probe is labeled with a fluorochrome which, after hybridization with the patient's sample, allows the region where hybridization has occurred to

submicroscopic deletions and duplications (Fig. 3.10) The group of disorders referred to as the microdeletionsyndromes are described in Chapter 18. Another application is the use of

be visualized using a fluorescencemicroscope.FISH is widely used for clinical diagnosticpurposes and there are a number of different types of probes that may be employed.

Differenttypesof FISHprobe probes Centromeric These consistof repetitive DNA sequencesfound in and around the centromereof a specificchromosome.They were the original probes used for rapid diagnosis of the common aneuploid.v syndromes(trisomies 13, 18, 21; seep. 262) using non-dividing cells in interphaseobtained from a prenataldiagnosticsampleof chorionicvilli (Fig. 3.9).

Chromosome- specificunique- seque nceprobes These are specilic for a particular single locus. Locus-specific probes for chromosome 13q14 and the critical region for Down on chromosome2l(21q22.1321q22.2)canbe utilized s-vndrome together with centromeric probes for chromosomes 18, X and Y to provide rapid prenatal diagnosisfor some of the more common numerical chromosomalabnormalities(p. 45). Uniquesequence probes are particularly useful for identifying tiny

Fis.3.9 (FISH) nucleiwrth of interphase ln-situhybrrdizatron FLuorescent 18.X andY (AneuVysion probesforchromosomes centrorneric consistent probessupptied threeaquasignaLs showing byVysrs) (Courtesy BristoLGenetics Detmege. of Catherine withtrisomy'18 Bristot Hospital, Southmead ) Laboratory,

35

3

CHROMOSOM AE NSDC E L LD I V I S I O N

an interphase FISH probe to identify HER2 overexpressionin breast tumors in order to identify patients likely to benefit from herceptin treatment.

probes Telomeric A complete set of telomeric probes has been developed for all 24 chromosomes(i.e. autosomes| 22 plus X and Y). Using these,a method has been devised that enablesthe simultaneous analysis of the subtelomeric region of every chromosome by meansof only one microscopeslide per patient. This has proved to be a particularly useful technique for identifying tiny'cryptic' subtelomericabnormalities,such as deletionsand translocations, in a small but significantproportion of children with unexplained intellectual impairment (p. 269).

Whole- chromosomepoint probes These consist of a cocktail of probes obtained from different parts of a particular chromosome When this mixture of probes is used together in a single hybridization, the entire relevant chromosomefluoresces(i.e. is 'painted') Chromosome painting is extremely useful for characterizingcomplex rearrangements, such as subtle translocations(Fig. 3.11), and for identifying the origin of additional chromosome material, such as small

Fis.3.11 Chromosomepainting showrng a reciprocaItranslocation g involvin chromosomes3 (red)and 20 (qreen)

supernumerary markers or rings. The latest technology, described as multiplex FISH (M-FISH) or spectral karyotyping (SKY), utilizes pools of whole human chromosome paint probes to provide a multicolor human karyotype in which each pair of homologous chromosomescan be identified on the basis of its unique color when studied using computer-basedimage analysis (Fig. 3.12). These approacheshave proved to be extremely useful for detecting subtle chromosome rearrangements, such as deletions and translocations,and for identifying small supernumerarymarkers and ring chromosomes.

Probesderivedfrom flow-sortedchromosomes Because of their differing size and DNA composition, chromosomes bind different amounts of fluorescent dyes, some of which bind specificallyto GC ('gene rich') sequences and others to AT ('gene poor') sequences.This property of differential binding allows chromosomes to be separated by the process of flow c.ytometrl or fluorescent actiaaterLcell sorting (FACS). This involves staining metaphasechromosomes wirh

Fis.3.10

35

(ELN)regionprobe(Vysrs), Metaphase imageofWrllrams chromosome band7q11 23,showing thedeletion assocrated with WiLLiam s ysn d r o mTeh en o r m a t c h r o m o s ohmaess i g n a lfso r thecontroI probe(green) andtheELNgeneprobe(orange), but thedeLeted chromosome showsontythecontrolprobesignaI (Courtesy of Catherine Delmege, BristotGenetics Laboratory, Southmead HospitaL. Bristol)

a fluorescent DNA-binding dye and then projecting them in a fine jet of droplets acrossa focused laser beam, which excites the chromosomes to fluoresce. The fluorescence intensity is measuredby a photomultiplier and the results are analyzedby a computer that draws up a distribution histogram of chromosome size.This is referred to as tf om karyotype. Flow cytometry can be used to ana,lyzean individual's chromosomes, although its clinical application is limited by its expense and the relatively poor separation achieved for

CHROMOSOM AE NSDC E L LD I V I S I O N

3

Fig.3.12 M - F I S Hs h o w i ncgo m p l ecxh r o m o s o mr e a r r a n g e m er nnvt o l v i n nc u l t u r ebdt o o d g r o m o s o m4e s8 1 31 8a n d2 1a so b s e r v ei d ch (Courtesy lymphocytes Harwett. Oxon, UK, of Dr Rhona Anderson, Research Councit Radiation andGenome Unii,MedicaL StabrLrty a n dA p p t i eldm a g i n)g

certain chromosomes,most noticeably those in the C group. It finds greater application in separating preparations of single chromosomes for the construction of chromosome-specific DNA libraries and in the manufacture of chromosome paints for FISH. The reversepainting procedure usesa flow-sorted portion of

the detection of regions of allele loss and gene amplification (p. 199). Tumor or 'test' DNA is labeled with a green paint, and control normal DNA with a red paint. The two samples are mixed and hybridized competitively to normal metaphase chromosomes,and an image is captured (Fig. 3.13). If the test sample contains more DNA from a particular chromosome region

unidentilied chromosome material, such asa small supernumerary marker or ring, as a paint for hybridization to a normal metaphase spread. The origin of the unidentified chromosome segment is then revealed by identifying the chromosome(s)to which it

than the control sample,that region is identified by an increasein the greento red fluorescenceratio (Fig. 3.14).Similarly a deletion in the test sampleis identified by a reduction in the green to red

hybridizes.

The application of CGH has been extended to include the analysis of single cells for prenatal diagnosis following whole-genome amplification to provide sufficient material for analysis. However, its utility is limited by its resolution and technicaldifficulty. Current limits of resolution are l0megabases ( 1 0 0 0 0 0 0 0 b a s e s ,o r l 0 M b ) f o r l o s s e sa n d 2 M b f o r g a i n s , providing a starting point for positional cloning (p. 74) but not

COM PA R A T IV GE E N OMIC H Y B R ID IZ ATION Comparative genomic hybridization (CGH) was originally developedto overcome the difficulty of obtaining good-quality metaphasepreparations from solid tumors. This technique enables

fluorescenceratio.

37

3

CHROMOSOM AE NSDC E L LD I V I S I O N

faster and more sensitivethan conventional metaphaseanalysis for the identification of constitutional rearrangements (with the exception of balanced translocations) and might replace conventional karyotyping if and when the cost of the arrays becomes6nanciallyviable.

C H R O M O S ONMOEM E N C L A T U R E By convention eachchromosomearm is divided into regions and eachregion is subdivided into bands,numbering alwaysfrom the centromereoutwards (Fig. 3.16).A given point on a chromosome is designatedby the chromosomenumber, the arm (p or q), the region and the band (e.9. 15q12).Sometimesthe word region is

Fig.3.13 C o m p a r a t igveen o m ihcy b r i d i z a t (r C o nG Ha)n a t y ssi sh o w i nagr e a s o fg e n ea m p L i f i c a tai n od nr e d u c t i o( d n e l e t i oinn)t u m o rD N A D A P I d i a m i d r n o p h e n y l i nFdI o T tCef l,:u o r e s c ei si no t h i o c y a n (aCt eo u r t e s y of Dr PeterLichter; GermanCancer Research CenterHeidelberq, : n d A n n l i p d l m : n i n n /) "-v"'v

precise localization of genes involved in tumor development. Mitroarra.y, or array, CGH is likely to supersedemetaphase

omitted, so that 15q12would be referred to simply as band l2 on the long arm of chromosome 15. A shorthand notation system exists for the description of chromosomeabnormalities(tble 3.2). Normal male and female karyotypes are depicted as ,l6,XY and 46,XX, respectively.A male with Down syndrome as a result of trisomy 2l would be representedas47,X1 * 21, whereasa femalewith a deletionof the short arm of one number 5 chromosome (cri du chat syndrome; p. 264) would be representedas 46,XX,del(5p). A chromosome report reading 46,Xlt(2;4)(p23;q25) would indicate a male with

CGH.

38

ARRAYCGH

Table3.2 Symbots usedindescribrng a karyotype

Cytogenetic techniques are traditionally based on microscopic analysis.However, the increasing application of microarray technology is also having a major impact on cytogenetics. Although array CGH is a molecular biology technique, it is included in this chapter becauseit has evolved from metaphase CGH and is being used to investigatechromosomestructure. Array CGH also involves the hybridization of patient and reference DNA, but metaphase chromosomes are replaced

Term

Exptanation

p

Shortarm

q

Longarm

cen

Centromere

det

Detetron;eq46.XX,det('1Xq21)

as the target by large numbers of DNA sequencesbound to g l a s ss l i d e s ( F i g . 3 . 1 5 ) T h e D N A t a r g e t s e q u e n c e sc a n b e mapped clones (yeast artificial chromosome [YAC], bacterial artificial chromosome [BAC], Pl-derived artificial chromosome [PAC] or cosmid) or oligonucleotides.They are spotted on to the microscope slides using robotics to create a microarral', in which each DNA target has a unique location. Following hybridization and washingto removeunbound DNA, the relative levels of fluorescenceare measured using computer software. Arrays with 30000 overlapping mapped clones (one clone per megabase)are available,but the highest resolution is achieved with oligonucleotide arrays, which can include up to 500000 probes.

dup

Duptrcatron: e g 46XYdup(13Xq1+)

fra

Fragilesite

i

l s o c h r o m o s o m ee;g 4 6 , X . i ( X q )

inv

Inversion:eg46XX,inv(9xp12q12)

ish

/n-situhybridization

r

Ring,eg 46:XX,r(21)

t

Transtocation; eg 46.XY.t(2:4)(q21:q21)

ter

Termrnat or end;re tipofarm,e g pteror qter

/

Mosaicism: eg 46.XY147,XXY

+ or -

Sometimes usedaftera chromosome armintextto gainor lossof partofthatchromosome; indicate e g 46,XX,5p-

The application of microarray CGH has extended from cancer cytogeneticsto the detection of any t1,peof gain or loss, including the detection of subtelomeric deletions in patients with unexplained intellectual impairment. Array CGH is

AE NSDC E L LD I V I S I O N CHROMOSOM

cnromosomes cell average slide average

Ratio Profile

05075112515

I

i

L.1Jif I

II

5 59;

I

i

I I I

I 1

n=14

6

n=l 5

2

3

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ll=

=17

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5

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7

4

11

n=] 4

n-

12

n,16

r9

B

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n = 1I

n tr

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tt=18

21

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x

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F i s .3 . 1 4 C G H r a t o o . o f i r e s f o r t h e C G H a n a l y s i s s h o w n3i 1 n3 FT r gh e v e r t , c a , [ i n e s a d , a c e " t t o e a c h c h r o m o s o m e s h o w f [ u o r e s c e n c e r a t i o s o f andApptiedlmaging) O 5-15 betweentestand controlDNA (Courtesyof Dr PeterLrchterGermanCancerResearchCenterHeidelberg,

ArrayCGH

C o n v e n t i o n aCl G H

.4.

;

Fis.3.15

e

H y b r i d i s a t i oann d a n a l y s i s

2 1.5 ,l

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andarray of conventtonal Comparison invotve the CGHBothtechniques tabeLed of differentraLLy hybridrzation DNA,butthetargets normalandpatient aremetaphase ofthehybridization ancmlcroarrays, chromosomes Theresultsshowdeletions respectively. 10qanddetetion of chromosome ofthreecloneson a 1-Mbbacterial (BAC) array. chromosone artifrciaL (ArrayCGHdatacourtesy of DrJohn Reference NationaI Genetics Barber, Satisbury.) Laboratory [Wessex],

3

CHROMOSOMES ANDCELLDIVISION

Centrioles

Interphase

Nucleolous N u c l e a rm e m b r a n e

1

s-

Centromere

B i p o l a sr p i n d l ef i b e r

12

Centromere

Prophase

3

Xq

1 1

3 Metaphase

+

2 ,5

o

7

Anaphase

Fig.3.16 X chromosome showing theshortandlongarmseachsubdivrded rntoregions andbands

a reciprocal translocation involving the short arm of chromosome 2 at region 2 band 3 and the long arm of chromosome4 at region 2 band 5. This system of karyotype nomenclaturehas been extendedto include the results of FISH studies. For example, a karyotype that reads 46,XX.ish del(15)(qll.2qll.2XDl5510-) refers to a female with a micro-deletion involving l5qll.2 identified by in-situ hybridization analysis using a probe for the Dl55l0 locus (Dl55l0 = DNA from chromosome 15 site l0). This individual will have either Prader-Willi or Angelman syndrome, as discussedin Chaoter 18.

Fis.3.17 Stagesof mitosis

CELL DIVISION MrT0stS At conception the human zygote consists of a single cell. This undergoes rapid division, leading ultimately to the mature human

40

adult consistingof approximately I x l0ra cells in total. In mosr organsand tissues,such as bone marrow and skin, cells continue to divide throughout life. This processof somatic cell division, during which the nucleus also divides, is known as mitosis. During mitosis each chromosome divides into two daughter chromosomes,one of which segregatesinto each daughter cell.

Consequently the number of chromosomes per nucleus remains unchanged. Prior to a cell entering mitosis, each chromosome consists of two identical sister chromatids as a result of DNA replication having taken place during the S phase of the cell cycle (p. 4l). Mitosis is the process whereby each of these pairs of chromatids separatesand dispersesinto separatedaughter cells. Mitosis is a continuous processthat usually lasts l-2 h, but for descriptive purposes it is convenient to distinguish five distinct stages.These are prophase, prometaphase, metaphase,anaphase and telophase(Fig. 3.U).

ANDCELLDIVISION CHROMOSOMES

3

Prophase During the initial stageof ltrophassthe chromosomescondense and the mitotic spindle begins to form. Tho centriolesform in each cell, from which mirotubulesradiate as the centriolesmove towards opposite poles of the cell.

Prometaphase

Exitfrom cellcycle (non-d ividing

Dving prometaphasethe nuclear membrane begins to disintegrate, allowing the chromosomes to spread around the cell. Each chromosomebecomesattachedat its centromereto a microtubule of the mitotic spindle.

Metaphase ln metaphasethe chromosomes become aligned along the equatorial plane or plate of the cell, where each chromosomers attached to the centriole by a microtubule forming the mature spindle.At this point the chromosomesare maximally contracted and, therefore, most easily visible. Each chromosome resembles the letter X in shape,as the chromatids of each chromosome have separatedlongitudinally but remain attachedat the centromere, which has not yet undergone division.

Anaphase In analthasethe centromere of each chromosome divides longitudinally and the two daughter chromatids separateto opposite poles of the cell.

Telophase By telophasethe chromatids, which are now independent chromosomesconsistingof a single double helix, have separated completely and the two groups of daughter chromosomes each become enveloped in a new nuclear membrane. The cell cytoplasmalsoseparates(cytokinesis),resulting in the formation of two new daughter cells, each of which contains a complete diploid chromosomecomplement.

T H EC E L LC Y C L E The period between successivemitoses is known as the interphase of the cell cycle (Fig. 3.18). In rapidly dividing cells this lasts for between 16 and 24h. Interphase commences with the Gt (G = gap) phase during which the chromosomesbecome thin and extended.This phase of the cycle is very variablein length and is responsiblefor the variation in generation time between different cell populations Cells that have stopped dividing, such as neurons, usually arrest in this phaseand are said to have entered a non-cyclic stageknown as G6. The G1 phaseis followed by the S phase(S = synthesis),when DNA replication occurs and the chromatin of eachchromosome is replicated.This resultsin the formation of two chromatids,giving eachchromosomeits characteristicX-shaped configuration.The

F i s .3 . 1 8 S t a g e so f t h e c e LcLy c t eG 1a n d G 2a r et h e f i r s t a n ds e c o n d ' r e s t i n g ' stagesof interphaseS is the stageof DNA repticatronM, mitosis

processof DNA replication commencesat multiple points on a chromosome(p. l3). Homologouspairsofchromosomesusuallyreplicatein synchrony. However, one of the X chromosomes is always late in replicating. This is the inactive X chromosome (p. 98) that forms the ser chromatinor so-calledBarr body,which can be visualizedin interphase in female somatic cells. This used to be the basis of a rather unsatisfactorymeans of sex determination based on analysisof 'buccal smear'. cells obtained by scraping the buccal mucosa- a Interphase is completed by a relatively short G2 phase during which the chromosomes begin to condense in preparation for the next mitotic division.

MEt0Sls ,L4eiosisis the process of nuclear division that occurs during the final stage of gamete formation. Meiosis differs from mitosis in three fundamental ways: l. Mitosis results in eachdaughter cell having a diploid chromosome complement (46). During meiosis the diploid count is halved so that each mature gametereceivesa haploid complement of 23 chromosomes. 2. Mitosis takesplacein somaticcells and during the early cell divisions in gamete formation. Meiosis occurs only at the final divisionof gametematuration. 3. Mitosis occurs as a single one-stepprocess.Meiosis can be consideredastwo cell divisionsknown asmeiosisI and meiosisII, each ofwhich can be consideredas having prophase,metaphase, anaphaseand telophasestages,as in mitosis (Fig. 3.19).

41

CHROMOSOMES ANDCELLDIVISION

Earlypachytene

Late pachytene

Telophase I

Anaphase ll

Telophase ll

Fis.3.19 Stages of meiosis

42

AE NSDC E L LD I V I S I O N CHROMOSOM

M e i o s i sI

I Anophose

This is sometimesreferred to as the reduction division, becauseit is during the first meiotic division that the chromosomenumber

The chromosomesnow separateto opposite poles of the cell as the spindle contracts

3

is halved.

I Telophose ProphaseI Chromosomesenter this stagealreadysplit longitudinally into two chromatids joined at the centromere.Homologous chromosomes pair and, with the exception of the X and Y chromosomesin

Each set of haploid chromosomeshas now separatedcompletely to oppositeends of the cell, which cleavesinto two new daughter or 1oq/tes. gametes,so-called secondarysqermatoc)/tes

male meiosis,exchangeof homologoussegmentsoccurs between non-sister chromatids, that is, chromatids from each of the pair

M e i o s i sl l

of homologous chromosomes.This exchangeof homologous segments between chromatids occurs as a result of a process known as crossingorer or recombination.The importance of

This is essentiallythe sameas an ordinary mitotic division. Each chromosome,which exists as a pair of chromatids, becomes aligned along the equatorialplane and then splits longitudinally, leading to the formation of two new daughter Bametes,known as

crossingover in linkageanalysisand risk calculationis considered later (pp. 130,336) During prophase I in the male, pairing occurs between homologoussegmentsof the X and Y chromosomesat the tip of their short arms, with this portion of each chromosome being known as the pseudoautosomal regton (p. I l2).

spermatidsor ova.

of meiosis The consequences

Leptotene

When consideredin terms of reproduction and the maintenance of the species,meiosis achievestwo maior obiectives.Firstly it facilitateshalving of the diploid number of chromosomesso that each child receiveshalf of its chromosome complement from each parent. Secondly it provides an extraordinary potential for

The chromosomesbecomevisible as thev start to condense.

generatinggeneticdiversity.This is achievedin two ways:

Zygotene

l. When the bivalentsseparateduring prophaseof meiosisI, they do so independently of one another This is consistentwith Mendel's third law (p. 5). Consequently each gamete recelves a selection of parental chromosomes.The likelihood that any two gametes from an individual will contain exactly the same

The prophasestageof meiosisI is relativelylengthy and can be subdividedinto five stages.

Homologous chromosomes align directly opposite each other, a process known as synapsis,and are held together at several points along their length bv filamentous structures known as synapt onemaI complexes. Pachytene Each pair of homologous chromosomes, known as a biztalent, becomestighdy coiled. Crossingover occurs,during which homologous regions of DNA are exchangedbetweenchromatids. Diplotene The homologous recombinant chromosomes now begin to separate but remain attached at the points where crossing over has occurred. These are known as chiasmata.On average, small, medium and large chromosomeshave one, two and three chiasmata,respectively,giving an overall total of approximately 40 recombinationeventsper meiosisper gamete.

chromosomesis 1 in 223,or approximately1 in 8 million. 2. As a result of crossingover,eachchlomatid usually contains portions of DNA derived from both parental homologous chromosomes.A large chromosome typically consists of three or more segments of alternating parental origin. The ensuing probability-that any two gameteswill have an identical genome is therefore inlinitesimally small. This dispersion of DNA into different gametesis sometimesreferred to as'gene shuffling'.

GAMETOGENESIS

Diakinesis

The processof gametogenesisshowsfundamental differencesin males and females(Table 3.3). These have quite distinct clinical

Separationof the homologouschromosomepairs proceedsasthe

consequencesif errors occur.

chromosomesbecomemaximallv condensed.

OOGENESIS I Metophose The nuclearmembranedisappearsand the chromosomesbecome alignedon the equatorialplaneofthe cell where they havebecome

Mature ova develop from oogonia by a complex series of intermediatesteps.Oogoniathemselvesoriginatefrom primordial germ cells by a processinvolving 20 to 30 mitotic divisions that

attachedto the spindle, as in metaphaseof mitosis.

occur during the lirst few months of embryonic life By the

43

3

CHROMOSOMES ANDCELLDIVISION

the time of ovulation, when a single secondaryoocyte is formed. This receivesmost of the cytoplasm. The other daughter cell

Tabte3.3 Differences in qametoqenesis in malesand females Males

Females

Commences

Puberty

Eartyembryonic tife

Duration

60-65days

10-50years

Numbersof mitoses in gameieformation

30-500

20-30

Gameteproduction peTmerosrS

4 spermatids

1ovum* 3 potarbodies

Gameteproduction

100-200mittion perejacutate

1ovumpermenstrual cycte

from the first meiotic division consistslargely of a nucleus and is known as a polar body. Meiosis II then commences,during which fertilization can occur.This secondmeiotic division resultsin the formation of a further polar body (Fig. 3.20). It is probable that the very lengthy interval between the onset of meiosis and its eventual completion, up to 50years later, accounts for the well documented increasedincidence of chromosome abnormalities in the offspring of older mothers (p. a6). The accumulating effects of 'wear and tear' on the primary oocyte during the dictyotene phase probably damage the cell's spindle formation and repair mechanisms, thereby predisposingto non-disjunction (p. l6).

SPERMATOGENESIS completion of embryogenesisat 3months of intrauterine life, the oogonia have begun to mature into primary oocytes that start to undergo meiosis. At birth all of the primary oocytes have entered a phase of maturation arrest, known as d,ictyltene, in which they remain suspendeduntil meiosis I is completed at

In contrast, spermatogenesisis a relatively rapid process with an averageduration of 60-65 days. At puberty spermatogonia, which will already have undergone approximately 30 mitotic divisions, begin to mature into primary spermatocyteswhich enter meiosisI and emergeas haploid secondaryspermatocytes.These

ncepflon

Conception

Spermatogonia

ff** Fetal life $

Primary spermarocyre

Primary oocyle

$Birth *M

Puberty ffi

o

Dictyotene E.

ul a t i o n

w

Secondary oocyte

P o l a rb o d i e s

60-65 days

zysot.f;D Spermatozoa

Oogenesis

Fis.3.20 44

Stages ofoogenesis andspermatogenesis n.haploid number

Spermatogenesis

ANDCELLDIVISION CHROMOSOMES

then undergo the second meiotic division to form spermatids, which in turn develop without any subsequentcell division into mature spermatozoa,of which 100 to 200 million are present in

Trisomy

each ejaculate. S p e r m a t o g e n e s iiss a c o n t i n u o u sp r o c e s si n v o l v i n g m a n y mitotic divisions, possibly as many as 20 to 25 per annum, so that mature spermatozoaproduced by a man of 50yearsor older

The presence of an extra chromosome is referred to as trisomy. Most casesof Down syndrome are due to the presence of an additional number 2l chromosome; hence Down syndrome is often known as trisomy 21. Other autosomal trisomies compatible with survival to term are Patau syndrome (trisomy 13) (p. 26il and Edwards syndrome (trisomy l8) (p. 264)'

could well have undergone severalhundred mitotic divisions. The observed paternal age effect for new dominant mutations (p. I 13) is consistentwith the concept that many mutations arise as a consequenceof DNA copy errors occurring during mitosis.

Most other autosomal trisomies result in early pregnancy loss, with trisomy 16 being a particularly common finding in firsttrimester spontaneousmiscarriages.The presenceof an additional sex chromosome (X or Y) has only mild phenotypic

AB CHROMOSOM EN O R M A L I T I E S Specific disorders caused by chromosome abnormalities are consideredin Chapter 18. In this section discussionis restricted to a review of the different types of abnormality that may occur. These can be divided into numerical and structural, with a third categoryconsistingof different chromosomeconstitutionsin two or more cell lines (Box 3.1)

N U M E R I C AALB N O R M A L I T I E S Numerical abnorrnalitiesinvolve the loss or gain of one or more chromosomes, referred to as aneuploidlt,or the addition of one or more complete haploid complements, known aspolyp loidy. Loss of a single chromosomeresults in monlslmJ/.Gain of one or two homologous chromosomes is referred to as trisomy a:ndtetrasomlt, respectively.

Box 3.1 Typesof chromosome abnormatity Numerical Aneuptoidy Monosomy Trisomy Tetrasomy Potyptoidy Tdploidy Tetraptoidy StructuraI Transiocatrons procaI Reci Robertso nian Deletions Insertrons lnversrons Paracentric Pericentric Rings tsocnTomosomeS Differentcel[ lines (mixoptoidy) Mosaicism Chimerism

3

effects(p. 99). Tiisomy 21 is usually causedby failure of separationof one of the pairs of homologous chromosomes during anaphaseof maternal meiosis I. This failure of the bivalent to separate is called non-d,isjunction.Less often, trisomy can be caused by nondisjunction occurring during meiosis II when a pair of sister chromatids fails to separate. Either way the gamete receives two homologous chromosomes (disomy)' and if subsequent fertilization occurs a trisomic conceptusresults (Fig. 3.21)'

Theoriginof non-disiunction The consequencesof non-disjunction in meiosis I and meiosis II differ in the chromosomes found in the gamete. An error in meiosis I leads to the gametecontaining both homologs of one chromosome pair. In contrast, non-disiunction in meiosis II resultsin the gametereceivingtwo copiesof one of the homologs of the chromosome pair. Studies using DNA markers have shown that most children with an autosomal trisomy have inherited their additional chromosomeas a result of non-disiunction occurring during one of the maternal meiotic divisions (Thble 3'4). Non-disjunction can also occur during an early mitotic division in the developing zygote. This results in the presence of two or more different cell lines, a phenomenon known as (p.52). mosaicism

originof meioticerrorleadlngto Tabte3.4 Parental aneuptoidY Chromosomeabnormatity

Paternat(%)

Maternat(%)

13 Trisomy

15

85

18 Trisomy

10

90

21 Trrsomy

5

95

80

20

47XXX

5

95

47.XXY

45

55

47XYY

100

0

45,X

45

3

CHROMOSOM AE NSDC E L LD I V I S I O N

Nondisjunction

n

sfl I

N o r m a lm o n o s o m igc a m e t e s

D i s o m i cg a m e t e s

N u l l i s o m igca m e t e s

D i s o m i c N u l l i s o m i c N o r m a lm o n o s o m i c gamete gamete gametes

Fis.3.21 S e g r e g a t i oa nt m e i o s i os f a s i n g t ep a i ro f c h r o m o s o m ei nsA n o r m am I e i o s i sB, n o n - d i s j u n c t iionnm e i o s i Isa n dC n o n - d i s j u n c t iionn m e i o s i lsl .

The causeof non-disjunction The cause of non-disjunction is uncertain. The most favored explanation is that of an aging effect on the primary oocyte, which can remain in a state of suspended inactivity for up to 50years(p. 44). This is basedon the well documentedassociation between advancing maternal age and increased incidence of Down syndrome in offspring (seeTable 18.4;p.263) A maternal ageeffect has also been noted for trisomies 13 and 18. It is not known how or why advancingmaternal agepredisposes to non-disjunction, although researchhas shown that absenceof recombinationin prophaseof meiosisI predisposesto subsequent non-disjunction. This is not surprising, as the chiasmatathat are formed after recombinationare responsiblefor holding eachpair of homologous chromosomestogether until subsequentseparauon occurs in diakinesis.Thus failure of chiasmataformation could allow each pair of homologs to separateprematurely and then segregaterandomly to daughter cells. In the female, however, recombinationoccursbeforebirth whereasthe non-disjunctional event occurs any time between 15 and 50years later. This

46

suggeststhat at leasttwo factors can be involved in causingnondisjunction: an absenceof recombination between homologous chromosomesin the fetal ovary, and an abnormality in spindle formation many years later. An alternative explanation for the associationof advancing maternal agewith increasedrisk of autosomaltrisomy is that survival

of trisomic embryoscould be the result of an age-relatedreduction in 'immunologic'competence. Firm evidencefor this theory is limited. Other factors that have been implicated in causing nondisjunction include radiation and delayed fertilization after ovulation. In animals it has been shown that an increased incidence of aneuploid embryos can result from lengthening of the interval between ovulation and fertilization. It has been suggestedthat this could account for the relationship between maternal age and the incidence of Down syndrome, as with increasingage intercourse is likely to occur lessfrequently, with delayed fertilization therefore being more likely. The story is further complicated by the fact that in some species,such as Drosophila,non-disjunction is under geneticcontrol. This could account for those occasionalfamilies that seem to be prone to recurrent non-disjunction.

Monosomy The absenceof a single chromosomeis referred to asmonosomy. Monosomy for an autosome is almost always incompatible with survival to term. Lack of contribution of an X or a Y chromosome results in a 45,X karyotype,which causesthe condition known as Turner syndrome (p. 272) As with trisomy, monosomy can result from non-disjunction in meiosis.If one gamete receivestwo copies of a homologous

CN ED L LD I V I S I O N CHROMOSOMESA

chromosome(dtsomy),the other correspondingdaughter gamete will haveno copy of the samechromosome(nullisomy).Monosomy can alsobe causedby lossof a chromosomeasit movesto the pole ofthe cell during anaphase,an event known as'anaphaselag'.

Potyptoidy Polyploid cells contain multiples of the haploid number of chromosomessuch as 69, triploidy, or 92, tetruplolly. In humans triploidy is found relatively often in marerial grown from spontaneousmiscarriages,but survival beyond mid-pregnancv is rare. Only a few triploid live births havebeen describedand all died soon after birth. Triploidy can be caused by failure of a maturarion meiotic division in an ovum or sperm, leading,for example,to retention of a polar body or to the formation of a diploid sperm. Alternatively it can be causedby fertilization of an ovum by two sperm: this is known as disperml When triploidy results from the presence of an additional set of paternal chromosomes, the placenta is usually swollen with what are known as hydatidiform changes (p. 96). In contrast, when triploidy results from an additional set of maternalchromosomes,the placentais usually small.Triploid-v usuall-vresults in early spontaneousmiscarriage(Fig. 3.22).The differencesbetweentriploidy due to an additional setof paternal chromosomes or maternal chromosomes provide evidence for important'epigenetic' and'parent of origin' effectswith respect to the human genome. These are discussed in more detail in Chaoter 6

il lfl rtl

11

3

S T R U C T U RA B LNORMALITIES Structural chromosomerearrangementsresult from chromosome breakagewith subsequentreunion in a different configuration. They can be balancedor unbalanced.In balancedrearrangements the chromosome complement is complete, with no loss or gain of genetic material. Consequently,balancedrearrangementsare generallyharmlesswith the exception of rare casesin which one of the breakpoints damagesan important functional gene.However, carriersof balancedrearrangementsare often at risk of producing children with an unbalancedchromosomalcomplement. When a chromosome rearrangement is unbalanced the chromosomal complement contains an incorrect amount of chromosomematerial and the clinical effectsare usually serious.

Translocations A translocationrefers to the transfer of geneticmaterial from one chromosome to another.A reciprocal translocation is formed when a break occurs in each of two chromosomeswith the segments being exchangedto form two new derivative chromosomes. A robertsonian translocation is a particular type of reciprocal translocationin which the breakpointsare locatedat, or closeto, the centromeresof two acrocentricchromosomes(Fig. 3.23).

ReciprocoI tronsIocotions A reciprocal translocation involves breakageof at least two chromosomes with exchangeof the fragments. Usually the

12 R e cpi r o c aI

l8

c!.

.!tq

19

20

r)* 21

*-*s 22

Fis.3.22 Karyotype fromproducts of conception ofa spontaneous m i s c a r r i a sgheo w i ntgr i p t o i d y

Fis.3.23 TypesoftransLocation

47

3

CHROMOSOM AE NSDC E L LD I V I S I O N

chromosome number remains at 46 tnd, if the exchanged fragments are of roughly equal size, a reciprocal translocation can be identified only by detailed chromosomalbanding studies or FISH (see Fig. 3.ll). In general,reciprocaltranslocations are unique to a particular family, although, for reasonsthat are unknown, a particular balancedreciprocaltranslocationinvolving the long arms of chromosomesl1 and 22 is relatively common. The overall incidence of reciprocal translocationsin the general population is approximately 1 in 500.

22 S i t e so f 0reaKage Normal chromosomes

Segregation at meiosis The importance of balancedreciprocaltranslocationslies in their behavior at meiosis,when they can segregateto generatesignificant chromosomeimbalance.This can lead to early pregnancyloss or to the birth of an infant with multiple abnormalities.Problems arise at meiosis becausethe chromosomes involved in the translocation cannot pair normally to form bivalents. Instead they form a cluster known as a pach1tenequadriaalent(Fig. 3.2a).

B al an c e d t r an s l o c a t i o n

D

The key point to note is that each chromosome aligns with homologousmaterial in the quadrivalent. 2:2 segregatiozWhen the constituent chromosomes in the quadrivalent separateduring the later stagesof meiosis I they

AC Pachytene qu a dr i v al e n t r nm e r o s r s

can do so in several different ways (Table 3.5). If alternate chromosomessegregateto each gamete, the gamete will carry a normal or balancedhaploid complement (Fig. 3.25) and with fertilization the embryo will either have normal chromosomes or carry the balanced rearrangement. If, however, adjacent chromosomes segregatetogether, this will invariably result in the gameteacquiring an unbalancedchromosome complement For example, in Figure 3.24, if the gamete inherits the normal number 1l chromosome (A) and the derivative number 22 chromosome(C), then fertilization will result in an embryo with

\ /-.-P l \ 2 =

/_,_____-_f_P

1r.+

tl

D

Fis.3.24 g chromosomes Howa balancedreciprocaL transtocation involvrn 11and22leadsto theformation ofa quadrrvalent at pachytene in r< meiosisI Thequadrivatent isformedto maintain l66pnlnnnr p anr n g

(seeFigs3.24and3.25) Tabte3.5 Patterns ofsegregation ofa reciprocaItranstocation Patternof segregation

Segregatingchromosomes

Chromosomeconstitutioningamete

222 Atternate

A+D

NormaI

B+C

Batanced translocation

(non-homotogous Adjacent-1 centTomeres segregate together)

A+CorB+D

(homotogous Adlacent-2 centromeres segregate together)

A + B o r C+ D

Unbalanced, leading to a combination of partia[ monosomy andpartial trr(nm\/

rn tho

7\/n^fo

3:1 T h r e ec h r o m o s o m e s

A+B+C A+B+D A+C+D B+C+D

Unbatanced, leading to tnsomyinthezygote

0nechromosome

A B

Unbatanced, leading to monosomy inthezygote

c 48

D

ANDCELLDIVISION CHROMOSON4ES

'-+--j =+rr------C€ B llll IJII VV

pachytene qu a dr i v aIen t

D

yieldsnormal Alternate segregation or balanced haploid complement

3

trisomy for the derivative 22 chromosome is the only viable unbalanced product. All other patterns of malsegregationlead to early pregnancy loss. Unfortunately, tertiary trisomy for the derivative 22 chromosome is a serious condition in which affected children have multiple congenital abnormalities and severe learning difficulties. Risks in reciprocal translocations When counseling a carrier of a balanced translocation it is necessaryto consider the particular rearrangementto determine whether it could result in the birth of an abnormal baby.This risk is usually somewhere between lolo and l0o/o. For carriers of the 1l.22 translocationdiscussed,the risk has been shown to be 5olo.

Robe rtsonion tron slocotions AB yieldsunbalanced Adjacent-1 segregation haploid complement

A Robertsonian translocation results from the breakageof two acrocentric chromosomes(numbers 13, 14, 15, 2l and 22) at or close to their centromeres,with subsequentfusion of their long arms (seeFig. 3.23).This is alsoreferred to as centricfusion. The short arms of each chromosome are lost, this being of no clinical importance as they contain genes only for ribosomal RNA, for which there are multiple copies on the various other acrocentric chromosomes.The total chromosome number is reduced to 45. As there is no loss or gain of important genetic

AB yieldsunbalanced Adjacent-2 segregation haploid complement

material, this is a functionally balanced rearrangement. The overall incidence of Robertsonian translocationsin the general population is approximately 1 in 1000, with by far the most common being fusion of the long arms of chromosomesl3 and 1 , 1( l 3 q 1 4 q ) . Segregation at meiosis As with reciprocaltranslocations,the importanceof Robertsonian translocations lies in their behavior at meiosis. For example, a carrier of a l4q21q translocation can produce gametes with (Fig 3.26):

AB

F i g .3 . 2 5 patterns Thedrfferent of2:2segregatron thatcanoccur fromthe quadrivatent shown rnFrg3 24 SeeTabte 35

monosomy for the distal long arm of chromosome 22 and trisomy for the distal long arm of chromosome 11. Another possibility is that three chromosomes 3:l segregation segregateto one gametewith only one chromosomein the other gamete.If, for example,in Figure 3.24 chromosomesl1 (A),22 (D) and the derivative22 (C) segregatetogether to a gametethat is subsequentlyfertilized, this will result in the embryo being trisomic for the materialpresentin the derivative22 chromosome. This is sometimesreferred to as tertiary trisoml. Experiencehas shown that, with this particular reciprocaltranslocation,tertiary

l. A normal chromosome complement (i.e. a normal l4 and a normal 2l). 2 . A b a l a n c e dc h r o m o s o m e c o m p l e m e n t ( i . e . a l 4 q ? l q translocationchromosome). 3. An unbalancedchromosome complement possessinBboth the translocationchromosomeand a normal 21. This will result in the fertilized embryo having Down syndrome. 4. An unbalancedchromosomecomplement with a normal 14 and a missing21. 5 An unbalancedchromosomecomplement with a normal 21 and a missing 14. 6. An unbalanced chromosome complement with the translocationchromosomeand a normal 14 chromosome' The last three combinationswill result in zygoteswith monosomy 21, monosomy 14 and trisomy 14, respectively.All of these combinations are incompatible with survival beyond early pregnancy.

49

3

CHROMOSOM AE NSDC E L LD I V I S I O N

H ll

Normal chromosomes

U

14

14

21

21

B al an c e d 14121carrier

P o s sbil e g am e t e s 21

14121

Normal

Normal carrler

14

0utcome

14121 2 1 Down's syndrome

14

21

14

14121

v

Lethal

Fis.3.26 Formation ofa 14q2'1q robertsonian transtocation gametechromosome andthepossibte patterns thatcanbe produced at meiosis

50

Translocation Down syndrorne The major practical importance of Robertsoniantranslocations is that they can predispose ro the birth of babies with Down syndromeasa result of the embryo inheriting two normal number 21 chromosomes (one from each parent) plus a translocation chromosome involving a number 21 chromosome (Fig. 3.27). The clinical consequencesare exactly the same as those seenin pure trisomy 21. However, unlike trisomy 27, the parents of a child with translocation Down syndrome have a relatively high risk of having further affectedchildren if one of them carriesrhe rearrangementin a balancedform. Consequently the importance of performing a chromosome analysis in a child with Down syndrome lies not only in confirmation of the diagnosis,but also in identilication of those children with a translocation. In roughly two-thirds of these latter children with Down syndrome, the translocation will have occurred as a new (de noao)event in the child, but in the remaining one-third one of the parents will be a carrier. Other relatives might also be carriers. Therefore it is regarded as essentialthat efforts are made to identify all adult translocation carriers in a family so thar rhey can be alerted to possiblerisks to future offspring. This is sometimesreferred to as translocation tracing,or'chasing'.

Fis.3.27 parnting showin Chromosome g a 14q21q Robertsonian translocation tna childwithDownsyndromeChromosome 21is shownInbtueandchromosome 14inyetlow(Courtesy of Meg Heath. CityHospitaL, Nottrngham )

CHROMOSOMESC AN EL DLD I V I S I O N

3

Risks in Robertsonian translocations Studies have shown that the female carrier of either a 13q21qor a l4q2lqRobertsonian translocationruns a risk of approximately 10o/ofor having a baby with Down syndrome, whereasfor male carriers the risk is l-3olo. It is worth sparing a thought for the unfortunate carrier of a 2lqZlq robertsonian translocation AII gametes will be either nullisomic or disomic for chromosome 21. Consequentlyall pregnancieswill end either in spontaneous miscarriageor in the birth of a child with Down syndrome.This is one of the very rare situations in which offspring are at a risk of greater than 50o/ofor having an abnormality. Other examples are the children of a mother with untreated phenylketonuria (p. 162), parents who are both heterozygous for the same autosomaldominant disorder (p. 107), and parentswho are both homozygous for the sa,megenecausing an autosomal recessive disorder,such as sensorineuraldeafness.

Deletions A deletioninvolves loss of part of a chromosome and results in monosomy for that segment of the chromosome A very large deletion is usually incompatible with survival to term, and as a generalrule any deletion resulting in lossof more than 2oloof the total haploid genomewill havea lethal outcome. Deletions are now recognizedasexisting at two levels.A'large'

Fi9.3.28 (Cambro) paintrng chromosome 5 paint using Chromosome whrch 5 (arrowed) showing a smallportionof chromosome 13 SeeFiq329 fromchromosome marksthesiteofan insertion (Courtesy Nottingham CityHospitaL, of MegHeath, )

chromosomal deletion can be visualized under the microscope. Several deletion syndromes have been described, such as the WolfHirschhorn and cri du chat syndromes, which involve loss of materialfrom the short arms of chromosomes4 and 5, respectively (p. 264). More recently submicroscopic microdeletions have been identified with the help of high-resolution prometaphase cytogeneticsaugmented by FISH studies. For example, it has been shown that severalpreviously unexplainedconditions, such as the Prader-Willi and Angelman syndromes,can be causedby microdeletions(p 116, I l7).

lnsertions An insertionoccurs when a segmentof one chromosomebecomes inserted into another chromosome(Figs 3.28 & 3.29). If the inserted material hasmoved from elsewherein another chromosome then the karyotype is balanced. Otherwise an insertion causes an unbalancedchromosomecomplement. Carriers of a balanced deletion insertion rearrangementare at a 50o/orisk ofproducing unbalanced gametes,as random chromosome segregationat meiosis will result in 50o/oof the gametesinheriting either the deletion or the insertion, but not both.

lnversions An inaersionis a two-break rearrangement involving a single chromosomein which asegmentisreversedin position (i.e.inverted) If the inversion segment involves the centromere it is termed a pericentricinaersion(Fig. 3.30A). If it involvesonly one arm of the chromosomeit is known as a paracentricinuersion(Fig. 3.308).

Fig.3.29 pe a i n t r nugs i n gc h r o m o s o m 1e 3p a i n(tC a m b i o ) Chromosom 1e 3o r i g i ni n s e r t eidn t o s h o w r nm g a t e r i a l oc fh r o m o s o m (Courtesy of MegHeath, CityHospital 5 (arrowed) chromosome N o t t r n g h a) m

51

3

CHROMOSOM AE NSDC E L LD I V I S I O N

relatively large.The larger these are, the more likely it is that their effects on the embryo will be so severethat miscarriageensues For a large pericentric inversion the duplicated and deleted segments will be relatively small so that survival to term and beyond becomesmore likely.Thus, in general,the larger the size of a pericentric inversion the more likely it becomesthat it will result in the birth of an abnormal infant. The pooled results of severalstudieshaveshown that a carrier of a balancedpericentric inversion runs a risk of approximately 5-10o/ofor having a child with viable imbalance if that inversion has already resulted in the birth of an abnormal baby.The risk is nearer lolo if the inversion has been ascertainedbecauseof a history of recurrentmiscarriage. Paracentric inversions If a cross-overoccurs in the inverted segment of a paracentric inversion this will result in recombinant chromosomesthat are either acentric or dicentric (Fig 3 3lB). Acentric chromosomes, which strictly speakingshould be known aschromosomalfragments, cannot undergo mitotic division, so that survival of an embryo with such a rearrangementis extremely uncommon. Dicentric chromosomesare inherently unstable during cell division and are, therefore,alsounlikely to be compatible with survival of the embryo. Thus, overall, the likelihood that a balanced parental paracentric inversion will result in the birth of an abnormal baby is exremely low

F i s .3 . 3 0 A Pericentricand B paracentricinversions(Courtesyof DrJ. Dethanty.GattonLaboratory,London)

Inversions are balanced rearrangementsthat rarely cause problems in carriers unlessone of the breakpointshas disrupted an important gene.A pericentricinversioninvolving chromosome number 9 occursasa common structural variantor polymorphism, also known as a heteromorphism,and is not thought to be of any functional importance. Howeveq other inversions,although not causing any clinical problems in balanced carriers, can lead to significant chromosome imbalance in offspring, with important clinical consequences.

Segregotionot meiosis Pericentric inversions An individual who carries a pericentric inversion can produce unbalancedgametesif a cross-overoccurs within the inversion segment during meiosis I, when an inversion Ioop forms as the chromosomes attempt to maintain homologous pairing at synapsis.For a pericentric inversion, a cross-overwithin the loop will result in two complementaryrecombinantchromos<,rmcs, one with duplication of the distal non-inverted segmentand deletion

52

of the other end of the chromosome,and the other having the opposite arrangement(Fig. 3.31A) If a pericentric inversion involves only a small proportion of the total length of a chromosome then, in the event of crossing over within the loop, the duplicated and deletedsegmentswill be

Ringchromosomes A ring chromosome is formed when a break occurs on each arm of a chromosome leaving two 'sticky' ends on the central portion that reunite as a ring (Fig. 3.32). The two distal chromosomal fragments are lost so that, if the involved chromosome is an autosome,the effectsare usually serious. Ring chromosomes are often unstable in mitosis so that it is common to find a ring chromosomein only a proportion of cells. The other cells in the individual are usually monosomic because of the absenceof the ring chromosome.

lsochromosomes An isochromosomeshows loss of one arm with duplication of the other. The most probable explanation for the formation of an isochromosomeis that the centromere has divided transversely rather than longitudinally. The most commonly encountered isochromosomeis that which consistsof two long arms of the X chromosome.This accountsfor up to l5olo of all casesof Turner syndrome(p.272).

MIXOPLOIDY) M O S A I C I SAMN DC H I M E R I S(M Mosaicism Mosaicismcan be defined as the presencein an individual, or in a tissue, of two or more cell lines that differ in their genetic constitution but are derived from a single zygote, that is, they have

CHROMOSOM AE NSDC E L LD I V I S I O N

ED

CBDE

ED

BCA

3

F i s .3 . 3 1 Mechanism over of production inversions by crossing of recombinant unbalanced andB paracentric chromosomes fromA pericentric in an inversion [oop.(Courtesy of DrJ Dethanty, GattonLaboratory, London)

the same genetic origin. Chromosome mosaicismusually results from non-disjunction in an early embryonic mitotic division with the persistenceof more than one cell line. If, for example, the two chromatids of a number 2l chromosome failed to separateat the second mitotic division in a human zygote (Fig. 3.33), this would result in the four-cell zygote having two cells with 46 chromosomes,one cell with 47 chromosomes(trisomy 2l) and one cell with 45 chromosomes(monosomy 2l). The ensuing cell line with 45 chromosomeswould probably not survive,so that the

resulting embryo would be expected to show approximately 33olo mosaicism for trisomy 21. Mosaicism accounts for l-2o/o of all clinically recognizedcasesof Down syndrome. Mosaicism can also exist at a molecular level if a new mutation arisesin a somatic or early germline cell division (p. ll4). The possibility of germline or gonadalmosaicismis a particular concern when counselingthe parentsof a child in whom a condition such as Duchenne muscular dystrophy (p. 297) is an isolatedcase.

Chimerism Chimerismcan be defined as the presence in an individual of two or more geneticallydistinct cell lines derived from more than one zygote,that is, they havea different geneticorigin. The word chimera is derived from the mythological Greek monster that had the head of a lion, the body of a goatand the tail of a dragon. Human chimeras are of two kinds: dispermic chimerasand blood chimeras.

Fis.3.32 PartiaLkaryotype showing a ringchromosome 9 (Courtesy of Meg Heath. CityHospita[, Nottingham )

Dispermicchimeros These are the result of double fertilization whereby two genetically different sperm fertilize two ova and the resulting two zygotes

53

3

CHROMOSOM AE NSDC E L LD I V I S I O N

Rooney D E, Czepulkow'skiB H 1997Human chromosomepreparation. Essentialtechniques John Wiley, Chichester A laboratorj,handboohdescribingthe dtJJbrent methodsaaailablefor chromosome analysts. blurring the SpeicherM R, Carter N P 2006The new cytogenetics: boundarieswith molecularbiology.Nat Rev Gen 6:782-792 An up-to-dateret:ien of the excitingadaancesin FISH and array-based. techniquesTherman E, SusmanM 1993Human chromosomes. Structure,behaviour and effects,3rd edn Springer,NewYork A usefuland comprehensif,^e introductiont0 hum&nc.yt0genetics. Tiio J H, Levan A 1956The chromosomenumber of man Hereditas 42: 1-6 A landmarhpaper that described a reliablemethotlJbrstudltinghuman chromosomes and gaxebirth to the subjectof clinical cJltlgenettcs.

1 c e l lz y g o t e

Website

2ndnitotic

Normal disomy

National Center for Biotechnology Information. Microarrays: chipping awayat the mysteriesof scienceand medicine. Online Available: http://wrvwncbi nlm nih gov/About/primer,/microarrays.html

Monosomy

Fis.3.33 Generation of somaticmosaicismcausedby mitotrcnon-disjunction

fuse to form one embryo. If the tlvo zygoresare of different sex, the chimeric embryo can develop into an individual with true hermaphroditism (p. 275) and an XX/XY karyotype. Mouse chimerasof this type can now be produced experimentallyin the laboratory to facilitate the study of gene transfer.

Bloodchimeros Blood chimerasresult from an exchangeof cells,via the placenta, between non-identical twins in utero. For example, 90o/oof one twin's cells can have an XY karyotype with red blood cells showing predominantly blood group B, whereas 90oloof the cells of the other twin can have an XX karyotype with red blood cells showing predominantly blood group A. It has long been recognizedthat, when twin calvesof opposite sex are born, the female can have ambiguous genitalia. It is now believed that this is becauseof gonadal chimerism in the female calves,which are known asfreemartins.

FURTHER READING

54

Barch M J, Knutsen T, SpurbeckJ L (eds) 1997The AGT cytogenetics laboratory manual, 3rd edn. Lippincott-Raven, Philadelphia A largemultiauthor laboratoryhadbooh produtedby theAssociationof Genetic Tbchnologists. Gersen S L, Keagle M B (eds) 2005The principles of clinical cvrogenerics, 2nd edition Humana Press,Totowa, New Jersey A detailedmultiauthorguideto all aspectsof laboratoryantl clinical rytogenetics. Mitelman F (ed ) 1995An international system for human cytogenetic nomenclature Karger, Basel A reportgixing d.etailsof hon chromosome abnormalities,intlwling the resulnof FISH studies,shouldbedestibed

ELEMENTS @ fn. normal human karyotype is made up of 46 chromosomesconsisting of 22 pairs of autosomesand a pair of sex chromosomes,XX in the female and XY in the male. @ Eactr chromosomeconsistsof a short (p) and long (q) arm ioined at the centromere. Chromosomes are analyzed using cultured cells, and specific banding patterns can be identified by means of special staining techniques. Molecular cytogenetic techniques, such as fluorescence in-situ hybridization (FISH), comparative genomtc hybridization (CGH) and array CGH can be usedto detect and characterizesubtle chromosomeabnormalities. @ During mitosis in somatic cell division the two sister chromatids of each chromosomeseparate,with one chromatid passingto each daughter cell. During meiosis, which occurs during the final stage of gametogenesis, homologous chromosomespair, exchangesegments,and then segregateindependently to the mature daughter gametes. @ Chro-orome abnormalities can be structural or numerical. Numerical abnormalities include trisomy and polyploidy. In trisomy a single extra chromosome is present, usually as a result of non-disjunction in the first or second meiotic division In polyploidy three or more complete haploid sets are present instead of the usual diploid complement. @ Structural abnormalities include translocations, inversions,insertions, rings and deletions.tanslocations can be balanced or unbalanced. Carriers of balanced translocationsare at risk ofhaving children with unbalanced rearrangements;these chidlren are usually physically and mentally handicapped.

CHAPTER

DNAtechnology and app[ications

In the history of medicalgenetics,the'chromosomebreakthrough' in the mid-1950s was revolutionary. In the past three decades, DNA technology has had a profound effect, not only in medical genetics (Fig. 4.1) but also in many areasof biological science ( B o x4 . 1 ) .

mRNA

DNA

cDNA

Restriction

t

I

The identihcation of restriction endonucleases,the development of the DNA cloning vectors,the introduction of the Southernblot technique and Sangersequencingin the 1970s,the development of the polvmerasechain reaction in the 1980s,and the impact of capillarl' sequencing and microarray technolog-vin the 1990s, rank as seminal developmentsin the field. DNA technology can be split into trvo main areas:DNA cloning and methods of DNA analysis.

G e n ei n s e r t i o ni n r e p l i c a t i n cge l l s

Nu c le o t i d e sequence.

D N AC L O N I N G DNA cloning is the selective amplilication of a specific DNA fragment or sequenceto produce relatively large amounts of a homogeneousDNA fragment to enableits structure and function to be analyzedin detail. DNA cloning falls into two main types: techniques that use natural in-aixo cell-basedmechanismsof DNA replication and the more recentlydevelopedcell-freeor in-titro polymerasechain reactlon.

IN-VIVO CELL.BASED DNACLONING

A m i n oa c i d 5 e qu e nc e

AI

l+

Testfor h o m o l o g yb y h y br i di z a t i o n

Linkage Transformed with fragment ceils polymorphism

D i a gn o s i s A n t e n a t a lp, r e c l i n i c a l , carner

F i g .4 . 1 genetics inmedicat ofDNAtechnology Some oftheappLrcations

There are six basic steps in in-t:it:o cell-l.asedDNA cloning.

Although fragments of DNA can be produced by mechanical shearingtechniques,this is a very haphazardprocessproducing fragments that vary in size. In the early 1970sit was recognized

occurring on the two complementary DNA strands when read in one direction of polarity, e.g. 5'to 3' (Table 4.1) The longer the nucleotide recognition sequenceof the restriction enzyme, the lessfrequently that particular nucleotide sequencewill occur bv chance and therefore the larger the averagesize of the DNA

that certain microbes contain enzymes that cleave doublestranded DNA in or near a particular sequenceof nucleotides. These enzymesrestrict the entry of foreign DNA into bacterial

fragmentsgenerated. Well over 300 different restriction enzymeshavebeen isolated lrom various bacterial organisms.Restriction endonucleasesare

cells and were therefore called restrictionenzymesThey recognize a palindromic nucleotide sequenceof DNA of betweenfour and eight nucleotidesin length, i.e. the samesequenceofnucleotides

named according to the organism from which they are derived, e g. EroRI is from Escherichiacoli and was the first restriction enz)me isolatedfrom that organism.

Generation of DNAfragments

55

4

DNATECHNOLOGYAND APPLICATIONS

+

Box 4.1 Applications of DNAtechnotogy

AATTC--

Genestructure/mappi ng/functron genetics Poputation Ctinicatgenetics generic Preimolantatron diagr-osis PrenataI diagnosis Presymptomatic diagnosis Carrierdetection Diagnosis andpathogenesis of disease Genetic - infective. Acquired matignant Biosynthesis growthhormone. e g insutrn, interferon, immunzation Treatment ofgenetic disease Genetherapy Agriculture e g nrtrogen fixaton

--CTTAA

--CCC

GGG--

-_GGG CCC--

t Fis.4.2

Tabte4.1 Someexamotes of restriction endonucteases withtheirnucleotide recoqnition sequence andcleavage sites Enzyme

Organism

Cleavagesite

5',

3',

Bom\l

Boci[LusomyloltquefociensHG.GATCC

EcoRl

Escherichio coliRY13

G r AATT C

Haelll

Hoemopht[usoegyplius

GG.CC

Hindlll

Hoemophtlus influenzoeRd

A. A G CTT

Hpol

Hoemopht[usporotnfLuenzoe

GTT.AAC

Psd

Providencio stuortti

CT G CAo G

Smol

Serrotiomorcescens

CCC.GGG

5oll

StreptomycesolbusG

G.TCGAC

The complementary pairing of basesin the DNA molecule means that cleavageof double-stranded DNA by a restriction endonuclease always creates double-stranded breaks, which, depending on the cleavagepoints of the particular restriction enzyme used, results in either a staggeredor a blunt end (Fig. 4.2). Digestion of DNA from a specific source with a particular restriction enzyme will produce the samereproduciblecollection of DNA fragmentseachtime the processis carried out.

Recombination of DNAfragments

56

DNA from any source,when digestedwith the same restriction enzyme, will produce DNA fragments with identical complementary ends or termini. When DNA has been cleaved by a restriction enzyme that produces staggeredtermini, these are referred to as being'sticky'or'cohesive', becausethey will unite under appropriate conditions with complementary sequences

The staggeredand bLuntendsgeneratedby restrictiondigestof doubte-stranded DNA by EcoRland Srnol Sitesof cte:v:np nf thp DNA strandsare indicatedby arrows

produced by the same restriction enzyme on DNA from any source. Initially the cohesive termini are held together by hydrogen bonding but are covalently attached with the enzyme called DNA ligase.The union of two DNA fragments from different sourcesproduces what is referred to as a recombinant DNA molecule

Vectors A aector, or replicon, is the term for the carrier DNA molecule used in the cloning processwhich, through its own independent replication within a host organism, will allow the production of multiple copies of itself. The incorporation of theforeign or target DNA into a vector allows the production of large amounts of that DNA fragment. For naturally occurring vectors to be used for DNA cloning, they need to be modilied to ensure that the foreign DNA is inserted at a specific location and that recombinant vectors containing foreign inserted DNA can be detected.Many of the early vectors were constructed so that insertion of the foreign DNA in a gene for antibiotic resistanceresulted in loss of that function (Fig'.4.3). The five main types of vector commonly used include plasmids,bacteriophages, cosmid,s, znd bacterial and yeast artfficial chromosomes. The choice of vector used in cloning dependson a number of factors, such as the particular restriction enzyme being used and the size of the foreign DNA to be inserted Some of the early vectors,such as plasmidsand bacteriophages, were severely Iimited in terms of the size of the foreign DNA fragment that could be inserted. Later generationsofvectors, such as cosmids, can take inserts up to approximately 50kb in size. A cosmid is essentiallya plasmid that has had all but the minimum vector DNA necessaryfor propagation removed (i.e. the ras sequence),

DNATECHNOLOGY ANDAPPLICATIONS

dish. Recombinant vectors can be screenedfor by a detection system;for example,loss of antibiotic resistancecan be screened for by replica plating on agar containing the appropriate antibiotic (see Fig. 4.3). Thus, if the enzyme AtI were used to generate DNA fragments and to cut the plasmid pBR322, any recombinant

Ap'. Pstl

p S C 0l 1

4

pBR322

plasmids produced would make the bacterial host cells they transform sensitiveto ampicillin, as this gene would no longer be functional, but they would remain resistant to tetracycline. Replica plating of the master plates from the cultures allows identilication of individual specificrecombinant clones.

Fis.4.3 T w op l a s m i dosr i g i n a lul ys e di n r e c o m b i n aDnN t At e c h n o t o g y genes(,4p1 showing drugresistance ampiciLLin resistance; T-'

-^\ ^^! -t^-..-^^ ^t -^^+, L . +L^c, -L-o,- r, ,y- !l l.^' s^ . ^ - r c ) T->r -L^ o rru c TO D dc t e a v a g e-s.rr t^e- s o f restriction

endonucteases thatarepresent intheDNAontyonceforuseas a ctoning site

to enableinsertion of the largestpossibleforeign DNA fragment and still allow replication. More recently,the developmentof bacterialand.yeastartifcial (BACs and YACs) allows the possibility of cloning chromosomes DNA fragmentsof between300 and 1000kb in size.YACsconsist of a plasmid that containswithin it the minimum DNA sequences necessaryfor centromere and telomere formation plus DNA sequencesknown as autonomous replitati0nsequences, all of which are necessaryfor accurate replication within yeast.YACs have the advantage that they can incorporate DNA fragments of up to 1000kb in size as well as allow replication of eukaryoticDNA with repetitive DNA sequences,which often cannot take place in bacterialcells. Many eukaryotic genesare very large,being up to 2-3 million basepairs (bp) in length (p. 377).YACs allow detailed mapping of genesof this size and their flanking regions,whereas the use of conventional vectors would require an inordinate number of overlappingclones.

Transformationof the host organism After introducing the foreign DNA fragment into the vector, the recombinantvector is introduced into speciallymodified bacterial or yeasthost cells The bacterial cell membrane is not normally permeable to large molecules such as DNA fragments but can be made permeableby a variety of different methods, including exposure to certain salts or high voltage; this is known as becomingcrmpetent.Usually only a singleDNA moleculeis taken up by a host cell undergoing the processknown as transformution. If the transformed cells are allowed to multiply, large quantities of identical copiesof the original singletarget DNA or cloneswill be produced (Fig. 4.4).

Screeningfor recombinant vectors Once the transformed cells have multiplied in culture medium, they are plated out on a master plate of nutrient agar in a Petri

Selectionof specificclones A number of techniques has been developed to detect the presence of clones with specific DNA sequence inserts. The most widely used method is nucleic acid hybridization (p. 60). Colonies of transformed host bacteria with recombinant clones are used to make replica plates that are lyzed and then blotted on to a nitrocellulose filter to which nucleic acid binds. The DNA of the replica blot is then denatured to make the DNA single stranded, which will allow it to hybridize with single-stranded, radioactively labeled DNA or RNA probes (p. 59), which can then be detected by exposure to an X-ray film, or what is known as a,utoradiograpfu.In this way a transformed host bacterial colony containing a sequencecomplementary to the probe can be detectedand, from its position on the replica plate, the colony containing that clone can be identified on the master plate, 'picked' and cultured separately(Fig. 4.5). Alternatively, if an antibody is available to a protein and an expressionvector is used for the DNA cloning, the replica filter clonescan be screenedfor by the presenceor absenceof binding of the antibody to detect clones containing recombinant vectors with the DNA fragment containing the gene (or part of) of lnterest.

D N AL I B R A R I E S Different sources of DNA can be used to make recombinant DNA molecules.DNA from nucleated cells is termed total or genomicDNA. DNA made by the action of the enzyme reverse transcriptaseon messengerRNA (nRNA) is called complementary DNA or cDNA. It is possibleto enrich for DNA sequencesof particular interest by using a specific tissue or cell type asa source of mRNA; for instance,immature red blood cells (reticulocytes) containing predominantly globin nRNA resulted in cloning of the genesfor the globin chains ofhemoglobin (p. 148). The collection of recombinant DNA molecules generatedfrom a specific source is referred to as a DNA library @.9. a genomic or cDNA library). A DNA library of the human genome using plasmids as a vector would need to consist of severalhundred thousand clones to be likely to contain the whole of the human genome.The use ofYACs as cloning vectorswith DNA digested by infrequently cutting restriction enzymes means that the whole of the human genome can be contained in a library of 13000 to 14000clones.

57

4

DNATECHNOLOGYAND APPLICATIONS

D i s r u p t i oann d centrifugation

I

VV

Foreign DNA1

t :hX".',T.tt. Cleavage

ri+AA I

Tc

c f n n rT C

TTTT,TTIT[|[,TTil cTTAAIG cTTAAIG -/ A n

Plasmid c hr m e r a

T

Transformation

I

V

R e pIi c a t i o n

Fig.4.4 (From Generation p[asmid ofa recombinant using EcoRl andtransformation ofthehostbacteriaL A E1981 organism Emery Recombrnant DNAtechnotogy Lancet ti:1406-1409 withpermssion )

C E L L . F R EDEN AC L O N I N G One of the most revolutionary developmentsin DNA technology is the technique first developedin the mid-1980s known as the polymerasechain reattion or PCR. PCR can be used to produce vast quantitiesof a target DNA fragment provided that the DNA sequenceof that region is known.

The polymerasechainreaction DNA sequenceinformation is usedto designtwo oligonucleotide p r i m e r s ( a m p l i m e r s )o f a p p r o x i m a t e l y 2 0 b p i n l e n g t h complementaryto the DNA sequencesflanking the target DNA fragment. The first step is to denature the double-stranded DNA by heating.The primers then bind ro rhe complemenrary DNA sequencesof the single-strandedDNA templates.DNA polymeraseextends the primer DNA in the presenceof the

58

deoxvnucleotidetriphosphates(dATP, dCTR dGTP and dTTP) to synthesize the complementary DNA sequence.Subsequent heat denaturation of the double-stranded DNA, followed by annealingof the same primer sequencesto the resulting singlestranded DNA, will result in the synthesisof further copies of

the target DNA. Some 30 to 35 successiverepeatedcyclesresults in more than I million coptes (amplicons)of the DNA target, sufficient for direct visualizationby ultraviolet fluorescenceafter ethidium bromide staining, without the need to use indirect detection techniques(Fig. 4.6). PCR allows analvsis of DNA from any cellular source containing nuclei; in addition to blood, this can include less invasivesamplessuch as buccalscrapingsor pathologicalarchival material. It is also possible to start with quantities of DNA as small as that from a single cell, as is rhe casein preimplantation genetic diagnosis(p 325). Great care has to be taken with PCR, however,becauseDNA from a contaminatingextraneoussource, such as desquamatedskin from a laboratory worker, will also be amplified. This can lead to false-positiveresults unless the appropriatecontrol studiesare used to detect this possiblesource of error. Another advantageof PCR is the rapid turn-around time of samplesfor analysis.Use of the heat-stablelaa DNA polymerase isolatedfrom the bacterium Thermophilusaquaticus,which grows naturally in hot springs, generatesPCR products in a matter of hours rather than the days or weeks required for cell-basedlzcioa DNA cloning techniques

DAPPLICATIONS DNATECHNOLOGYAN

P l a t eo n

Transformed bacteria

4

Cycle0

DNA Unamplified sequencet Targeted Cycle 1 Denature and anneap l rimers

R e pilc a p l a tn i g

Primer extension

Cycle2

D e n a t u r ea n d

primers anneal

Primer extension

Fis.4.5 ldentrfrcation DNArnserts of recombinant DNAclones withspecrfrc resistance, by[ossofantibiotrc nucterc acidhybrrdizatron and r'

Cycle3

'l^--i'^^r-^hU

ouLUrouru9roPrry.

D e n a t u r ea n d a n n e a lp f l m e r s

Real-time PCR machines have reduced this time to less than t h, and fluorescencetechnology is used to monitor the generationof PCR products during each cycle, thus eliminating the need for gel electrophoresis. DNA cloning by PCR, in contrast to in-ztit-tocell-based techniques, has the disadvantagethat it requires knowledge of the nucleotide sequenceof the target DNA fragment and is best used to amplify DNA fragments of up to lkb, although longrange PCR allows the amplification of larger DNA fragments of up to 20-30 kb.

Primer extension

OFDNAANALYSIS TECHNIOUES Many methods of DNA analysisinvolve the use of nucleic acid probes and the processof nucleic acid hybridization.

NUCLEICAC PIRDO B E S Nucleic acid probes are usually single-stranded DNA sequences that have been radioactively or non-radioactively labeled and can be used to detect DNA or RNA fragments with sequencehomology. DNA probes can come from a variety of sources, including random genomic DNA sequences,specific genes,cDNA sequencesor oligonucleotide DNA sequences produced synthetically based on knowledge of the protein

CYcle4-25

Fig.4.6 seriaI showing chainreaction ofthepotymerase Diagram with andextension of DNA primeranneaLing, denaturation numbersin eachcycle ofthetargetDNAfragment doubling A DNA probecanbe labeledby a variety amino-acidsequence. includingisotopiclabelingwith "'P and nonof processes, i s o t o p i cm e t h o d su s i n g m o d i f i e d n u c l e o t i d e sc o n t a i n i n g fluorophores(e.g.fluoresceinor rhodamine).Hybridization

59

4

DNATECHNOLOGYAND APPLICATIONS

of a radioactively labeled DNA probe with cDNA sequences on a nitrocellulose filter can be detected by autoradiography, whereas DNA fragments that are fluorescently labeled can be detected by exposure to rhe appropriate wavelength of light, for example fluorescent in-situ hybridizatton (FISH)

(p.34)

Restriction enzyme

Electrophoresis gel onagarose

*

Denaturation w i t ha l k a l i

Blotting Drypaper towels Cellulose nitratefilter Gelcontaining denatured DNA Filterpaper Buffer

N U C L E IACC I DH Y B R I D I Z A T I O N Nucleic acid hybridization involves mixing DNA from two sources that have been denatured by heat or alkali to make them single stranded and then, under the appropriate conditions, allowing cornplementary base pairing of homologous sequences.If one of the DNA sourceshas been labeledin some way (i.e. is a DNA probe), this allows identification of specific DNA sequencesin rhe other source.The two main methods of nucleic acid hybridization most commonly used are Southern and northern blotting.

Southernblotting Southern blotting, named after Edwin Southern who developed the technique, involves digesting DNA by a restriction enzyme which is then subjected to electrophoresis on an agarosegel. This separatesthe DNA or restriction fragments by size, the smaller fragments migrating faster than the larger ones. The DNA fragments in the gel are then denaturated with alkali, making them single stranded. A 'permanent' copy of these single-strandedfragments is made by transferring them on to a nitrocellulose filter which binds the single-stranded DNA, the socalled,Southern blot. A particular target DNA fragmenr of interest from the collection on the filter can be visualized by adding a single-stranded 32Pradioactively labeled DNA probe that will hybridize with homologous DNA fragments in the Southern blot, which can then be detected by autoradiography(Fig. 4.7). An exampleof the use of Southern blotting for diagnostictesting in patientswith fragile X is shown in Fig. 4.8.

H y b r i d i z a t i owni t h

X

I 5.2kb (inactive X)

X

Expose to X-rayfilm

1|

,

Autorad iograph showing band(s)

2.8kb(activeX)

Fis.4.8

60

Southernblotto detectmeihyl.ation of theFMRIpromoter in patients wrthfragite X DNAdigested withFcoRlandthe Fis.4.7 methytation sensrtive enzymeBstZlwasprobedwith0x19, Diagram oftheSouthern btottechnique showing sizefractronation whichhybridizes to a CpGislandwithinlheFMR|promoter oftheDNAfragments bygeLetectrophoresis, denaturation ofthe Patient 1 isa fematewitha methylated patrents expansion. 2,3 doub[e-stranded DNAto becomesingtestranded, andtransfer and6 arenormatfemales, patient 4 is anaffected ma[eandpatient to a nitrocettutose fi[terthatis hybrrdized witha 32Pradroactivety 5 isa normalmale(CourtesyofA Gardner: Department tabeted DNAprobe of Molecular Genetics, Southmead Hospitat. Bristot)

DNATECHNOLOGYANDAPPLICATIONS

4

Northernblotting Northern blotting differs from Southern blotting by the use of mRNA as the target nucleic acid in the same procedure; mRNA is very unstablebecauseof intrinsic cellular ribonucleases. Use of ribonucleaseinhibitors allows isolation of mRNA that, if run on an electrophoreticgel, can be transferred to a filter. Hybridizing the blot with a radiolabeled DNA probe allows determination of the size and quantity of the mRNA transcript, a so-called nlrthern blot. With the advent of real-time reversetranscriptase PCR, and microarray technology for gene expression studies (Fig. 4.9), northern blotting is utilized lessoften.

DNAmicroarrays DNA microarraysare basedon the sameprinciple of hybridization but on a miniaturized scale.which allows simultaneous

qo C Q

on

-

!

v

Fif.c-=F=o=o .? ! ^: ,ii

efr

Y= .=

F

,;

Sa =

2 kb 1.5kb 1 kb

o.s kb

F i g ./ * . 1 0 byPCRto test mutation oftheGAArepeatexpansion Ampiificaiion bromide withethidium arestained ataxtaProducts for Fnedreich gel Lanes1andB show ona 15%agarose andelectrophoresed with [anes2 and4 showpatients standards, 500-bpLadder-srze controts, Lanes 3 and6 showunaffected expansions, homozygous 7 is carrierandLane expansion lane5 showsa heterozygous (Courtesy of Department of K.Thomson, controL thenegative Exeter.) DevonandExeterHospitat, Royat Genetics, Molecu|.ar

::t

analysisof tens or hundredsof thousandsof targets'Short, fluorescentlylabeledoligonucleotidesattachedto a glass microscopeslidecan be usedto detecthybridizationof target DNA under appropriateconditions.The color patternof the 9 497 46-

A ti_

2 . 3 -7

1.35300bp 200bp 1 0 0b p

2476p 1 4 0b p 1 1 1b p

0.24-

Fis.4.9 ALrquots representation ofa cDNAmicroarray. Diagrammatic ustnga aregrtdded on a glasstemplate ofcDNAsof interest roboiTestandreference RNAsampLes. computer-controLLed fluorochromes byreverse withdifferently colored Labeled to hybridize withthecDNAs transcription, aremixedandallowed to Laser tightand Thetemptates areexposed onthemicroarray genes patterngenerated identifying is compared, theexcrtation thereference and between in expression thatshowdifferences testsampLes

Fig. t,.11 byRFLPThenormaI C282Y oftheHFEgenemutation Detection of247 withRso/togiveproducts is digested 387-bpPCRproduct recognition anadditional creates mutation and140bp TheC282Y 29bp Lane1showsa of247.111and givingproducts siteforRso/, patients homozygous, 2-4 show Lanes standard ladder-size 1OO-bp respectlvely. mutation, the C282Y normalfor and heterozygous

61

4

DNATECHNOLOGYAND APPLICATIONS

microarray is then analyzed automatically by computer. Four classesofapplication havebeen described:(l) expressionstudies to look at the differential expression of thousands of genes at the mRNA level; (2) analysis of DNA variarion for mutarion detection and single nucleoride polymorphism (SNP) typing ( p . 6 7 ) ; ( 3 ) t e s t i n g f o r g e n o m i c g a i n s a n d l o s s e sb y a r r a y comparative genomic hybridization (CGH) (p. 37); and (4) a combination of the latter two, SNP-CGH, which allows the detection of copy-neutral genetic anomaliessuch as uniparental disomy(p 115).

Sizeanalysisof PCRproducts Deletion or insertion mutations can sometimes be detected simply b-ydetermining the size of a PCR product For example, the most common mutation that causescystic fibrosis, 4F508 (p.Phe508de1),is a 3-bp deletion that can be derecred on a p o l y a c r v l a m i d eg e l S o m e t r i n u c l e o t i d e r e p e a t e x p a n s i o n mutations can be amplified by PCR (Fig. a.l0)

Restriction fragmenttengthpolymorphism If a base substitution createsor abolishesthe recognition site of a restriction enzyme, it is possible to test for the mutation

MUTATION DETECTION The choice of method depends primarily upon whether rhe test is for a known sequencechange or to identify the presenceof any mutation within a particular gene.A number of techniques can be used to screen for mutations that differ in their easeof use and reliabilitrvThe choice of assaydependson manJrfactors, including the sensitivity required, cost, equipmenr, and the size and structure (including number of polymorphisms) of the gene (Table4.2). Identification of a possiblesequencevarianrbv one of the mutation screeningmethods requires confirmation by DNA sequencing.Some of the most common techniquesin current use are describedbelow.

by digesting a PCR product with the appropriare enzyme and separatingthe products by electrophoresis(Fig. 4. I l).

- refractorymutationsystem Amptification (ARMs)PCR Allele-specificPCR utilizes primers specificfor the normal and mutant sequencesThe most common design is a two-tube assay with normal and mutant primers in separatereactions together rvith control primers to ensurethat the PCR reactionhas worked. An example of a multiplex ARMS assayro detect 12 different c1,-stic fibrosis mutations is shown in Fig. 4.12.

Tabte4.2 Methodsfor detectinq mutations Method

Known/unknownmutations

Southernbtot

Known(orunknownrearrangement) Trinucteotideexpanstons nfragile X andmyotonic dystrophy

Sizing of PCRproducts

Known

p Phe5O8det CFIRmutation; trinucleotide Srmprecneap expansions in HD andSCAgenes

ARN4S-PCR

KNOWN

CFIRmutations

possib[e Muttrplex

0tigonucteotide ligatron

Known

CFIRmutations

possible Muttiplex

RealtimePCR

Known

FactorV Lerden

Expensive equipment

Conformation-sensitiveUnknown capittary etectrophoresis (CSCE)

Anygene

H i g h - i h r o u g h o um t e t h o dt h a t c a n u s e c a p i [ [ a r sy e q u e n c e p r tatform

Denaturrng HPLC

Anygene

Unknown

Exampte

Advantages/disadvantages Labolous

Rcnr rrrpc dcdirriod

l-]l--lPl l-

instrument withhighmaintenance High-resotutron mett (HRM)

62

Highsensrtivity; high-throughput method

5equenci ng

Knownor unknown

Anv nonc

Gotdstandard butexpensive

DNAmicroarray

Knownor unknown

Anygene

Highthroughput; expensive equipment Sensitivity forunknown mutations maybelimited

Massspectroscopy

Knownor unknown

Anygene

Highthroughput: expensjve equipment Sensitivity forsome mutation typestimited

DNATECHNOLOGYANDAPPLICATIONS

4

Patient Tube

*

ApoB

+

(N) p.Phe5o8del

1 7 1 7 - 1>GA >

(M)) p.PhesoEdel

+ oDc

Fis.4.12 Detection af CFTRmutations bytwo-tube Patient 2 isa compound ARN4S-PCR Patrent 1 s heterozygous forAF508(pPhe508det) h e t e r o z y g o t e f o r p P h e 5 O S d e t a n d c l TPl Ta-t 1i eGn>t 3Ai s h o m o z y g o u s n o r m a t f o r t h e 1 2 m u t a t i oPnr si m t eesrtsef do r t w o i n t e r n a I c o n t r o l(sA p o Ba n d0 D C )a r ei n c l u d ei d ne a c ht u b e

15 B l u e

l5 Green

15Yellow

Fis.4.13 D e t e c t i oanf C F T R m u t a t i o n s u s i n g agnoonl u c l e o t i d e [ i g a t r o n M a susLat yi p L e x P C R aemsp] 5Lef x o n s o f t h e C F I R g eOn [ei g o n u c l e o t r d e s andarethen to anneaI sequences foreachmutation aredesigned tothePCRproducts to adlacent suchthattwooligonucleotides anneal r n r n ohr r , I ' n : t r nTnh o? ? . n u t a t i o nasr ed i s c r i m i n a tuesdi n ga C O m b , n a tO a tb e l T s h i sp a t i e ni st a z ea - d d r f f e r e . rctol yl o r efdt u o r e s c et n ,O fs n -tesyof M Owens, (Cou Genetics, heterozygote Department of Motecutar compound fortheAF508(pPhe508det) ancp G55lD n utaLions RoyaL DevonandExeterHospitat, Exeter.)

63

4

DNATECH NOLOGYAND APPLICATIONS

0ligonucleotide tigationassay(0LA) A pair of oligonucleotides are designed to anneal to adjacent s e q u e n c e sw i t h i n a P C R p r o d u c t . I f t h e y a r e p e r f e c t l y hybridized, they can be foined by DNA ligase.Oligonucleotides complementarv to the normal and mutant sequences are differentially labeled and the products identified by computer software(Fig. a.l3).

R e a l - t i m eP C R There are multiple hardware platforms for real-time PCR and 'fast' versions that can complete a PCR reaction in under 30 min. TaqManrM and LightCyclerrM use fluorescence technology to detect mutations by allelic discrimination of PCR products. Figure 4 14 givesan exampleof the fluorescenceresonanceenergy transfer (FRET) probe detection of the factorV Leiden mutation.

(0Un'chips') DNAmicroarrays DNA microarrays hold the promise of rapid mutation testing.They involve synthesizing custom-designed20 25bp oligonucleotide sequencesfor both the normal DNA sequenceand known andl or possiblesingle nucleotide substitutions of a gene.These are attachedto a'chip'in a structured arrangementin what is known as a microaruay.The sampleDNA being screenedfor a mutation is amplified by PCR, fluorescentll,labeled and hybridized with the oligonucleotidesin the microarray (Fig. 4.15). Computer analysisof the color pattern of the microarray generatedafter hybridization allows rapid automated mutation testing. The

prospect of gene-specificDNA chip microarrays may lead to a revolution in the speed and reliability of mutation screening, provided the technology is affordable and the technique can be demonstrated to be robust. The detection of known base substitutions, or SNPs, has been very successful,but screening for unknown mutations has oroved more difficult.

tiquid Denaturinghigh-performance (DHPLC) chromatography This technique detects the presence of heteroduplexes owing to their abnormal denaturing profiles. PCR products from patients with heterozygousmutations will form heteroduplexes if denatured by heating (to separatethe double-strandedDNA) and then cooled slowly.Homozygous mutations can be detected by mixing the PCR products with normal sequenceproducts to generateheteroduplexes.DHPLC provides a sensitivedetection platform with a relativelyhigh throughput capacity.

-sensitivecapillary Conformation (CSCE) electrophoresis CSCE is used to detect the presenceof heteroduplexesusing fluorecencetechnology.It is fasterand achieveshigher throughput than DHPLC as PCR products can be multiplexed by using multiple fluorescent dyes.An alteration in the DNA sequence can result in a different conformation, which has a different electrophoreticmobility, and an appropriatepolymer can be used for identification.

PCRproduct

B 3.0 2.8 z.o

2.4 2.2 2.0 U

1 Q

C

n ^

g o

L

r.o t . L

1.0

0.8 0.6 0.4 0.2 0.0

54

460 48.0 s0.0 52.0 s40 560 580 60.0 62.0 64.0 660 680 70.0 72.0 "C Meltinq temperature

Fi1.4.14 PCRto detect ReaL-time theFactor V LeidenmutationA,Whenthetwo probeshybridrze otigonucteotide to the PCRproduct, thedyesarebrought rnto closeproximity andredfluorescent tightis emitted byfluorescence -esonance energyt.ansfer(FRET) B, Melting curveanatysrs separares thetwoatte[es. asthesensorprobe dissocrates fromthemutantatle[e ata lowertemperature Theredlrne showsa normaL controt; thebtueline showsa patient heterozygous for Factor V L e i d e na,n dt h eg r e e nl i n es h o w sa patient homozygous forFactorV Leiden rsothocyanate: FITC, ftuoresceir LC. (Courtesy LightCycler of Dr L Harries. D e p a r t m eonftM o l e c u t G a re n e t i c s , RoyaI DevonandExeterHospitaL, txeter.J

ANDAPPLICATIONS DNATECHNOLOGY

4

F i 9 .4 . 1 5 'HNFIA mutations Detection of HNFIAmutations usinga DNAmrcroarray. normalandmutantprobesfor75different The chip'contains to thechip products spotted intriplrcate whichwerehybndized Patient DNAwasamplrfred labeled bymuttiplex PCRto yietdfluorescentty Institute ofTechnoLogy, A, ControIsamp[e Advanced B, Patrent heterozygous foran HNFlAmutation(Courtesy of N Huh,Samsung SouthKorea )

Homozygous variant1 Heterozygous variant1 Heterozygous vatiant2 F

= f

E

q

o

E

z.

8 2 7 5 8 3 8 3 2 58 3 5 8 3 7 5 8 4 8 4 2 58 4 5 8 4 7 5 8 5 8 5 2 58 5 5 8 5 7 5 8 6 8 6 2 58 6 5 8 6 7 5 8 7 8 7 2 5 Degrees

Fig.4.16 (HRM) toa control afternormalization profLLes samples areshown High-resoLution mettcurve MeLtrng mutant analysis fornormaland c> :o m nlo trrPtc

F : . h , i :vroi t: nt or t t L rhr .o. > o. 6 _l i. f f. p lpn1 mcltinn nrn{ilp _. -. LoLtt

High-resolution melt curveanalysis(HRM)

D N As e q u e n c i n g

This technique employs a new class of fluorescent dyes that intercalatewith double-stranded,but not single-stranded,DNA. The intercalatingdye is incorporatedin the PCR reactionand the products are then heatedto separatethe two strands.Fluorecence levels decreaseas the DNA strands dissociateand this 'melting' profile depends on the PCR product size and sequence(Fig. 4.16). HRM appears to be very sensitive and can be used for high-throughputmutation screenin g

The 'gold standard' method of mutation screening is DNA s e q u e n c i n gu s i n g t h e d i d e o x y c h a i n t e r m i n a t i o n m e t h o d developedin the 1970sby Fred Sanger.This method originally employedradioactivelabelingwith manual interpretation of data' The use of fluorescent labels detected by computerized laser svstemshas improved easeof use and increasedthroughput and accurac]'.Today'scapillary sequencerscan sequencearound I Mb (1 million bases)per day.

65

4

DNATECHNOLOGY ANDAPPLICATI ONS

DNA Polymerase + 5'CCTGCAGGCTGGGCG 3 'G G A C G T C C G A C C C GC C T G T G C5 '

CCTGCAGGCT CCTGCAGGCTG CCTGCAGCCTGG CCTOCAGGCTGGG CCIGCAGGCTGGGT CCTGCAGOCTGGGCG CCTGCAGGCTGGGCGG CCIGCAGGCTGGGCGGA CCT6CAGGCTGGGCOGAC CCTGCAGGCTGGGCGGACA C C T GC A GG C T G GC C GG A C A { ] C C T GC A GG C T G GG C GG A C A C G

Nuc l e o t i d e s e que n c e

A m i n oa c i d s e qu e n c e

M M

D D

R R

R R

L L

G/a

L I L

+

+ OualitV(0-100):44

G C K0 2 N o r m a b l

1,500 G

A

155 G T

G

A

C

160 C G

C

G

G

165 C

C 22

G 16

G 16

165 C C 44 44

C

T

G

170 AG

G

C

T

C 59

T 59

1,000 500 0

Patient_GCK_2_811_047.ab1 OualitV(0-100):45

2,000 G r,500 59 r,000 500 0

65

155 A T G 59 59 59

G 59

A C 59 59

160 C G 44 44

T G 44 45

170 AG G 45 45 45

Fig.4.17 Ftuorescent dideoxy DNAsequencing primer(shownin red)bindsto thetempLate Thesequenctng andprimessynthesrs ofa (A)Thesequencing comptementary DNAstrandinthedirection indicated reactron includes fourdNTPsandfourddNTPs, eachlabeled w i t h a d i f f e r e n t f l u o r e s c e nCtodm y ep e t i t i o n b e t w e e n t h e d N T P s a n d d d N T P s r e s u t t s i n t h e p r o d u c t i o n o f a c o t t e c t i o n o f f r a g m e n t s ( whicharethenseparated (C)A heterozygous byelectrophoresis (GGC>TGC; to generate an etectropherogram pGty44Cys mutation, gtycine>cysteine), is identified bythesoftware

APPLICAT]ONS DNATECHNOLOGYAND

Dideoxl' sequencing involves using a single-stranded DNA template (e.9. denatured PCR products) to s.vnthesizenew complementary strands using a DNA polymeraseand an appropriate oligonucleotide primer. In addition to the four normal deoxynucleotides(dNTPs), a proportion of each of the four respective dideoxynucleotides (ddNTPs) is included, each

Dosage analysisby quantitative fluorescent PCR (qF-PCR) is routinely used for rapid aneuploidy screening,for example in prenatal diagnosis (p. 315). Microsatellites (see below) located on chromosomes 13, l8 and 21 may be amplified within a multiplex and trisomies detected,either by the presenceof three

The dideoxynucleotides labeledwith a different fluorescentd--ve. lack a hydroxvl group at the 3'carbon position; this prevents phosphodiesterbonding, resulting in each reaction container consisting of a mixture of DNA fragments of different lengths

(Fis 4.19)

that terminate in their respective dideorynucleotide, owing to chain termination occurring at random in each reaction mixture at the respective nucleotide When thc reaction products are separatedby polyacrylamide gel or capillary electrophoresis,a ladder of DNA sequencesof differing lengths is produced. The DNA sequencecomplementarv to the single-strandedDNA template is generatedby the computer software and the position of a mutation may be highlighted with an appropriate software package(Fig. a.l7).

Massspectroscopy is a high-resolution technique originallyMass spectroscop-v The development of matrixder,elopedfor protein anal-vsis. assistedlaser desorption/ionization time-of-flight (MALDITOF) technologv allows analysis of hundreds of DNA s a m p l e sr v i t h i n m i n u t e s . H i g h - t h r o u g h p u t S N P t y p i n g i s now a common application of this technolog--qbut detection o f s m a l l i n s e r t i o n s o r d e l e t i o n s i s m o r e c h a l l e n g i n gw h e n DNA scanning for unknown mutations b-v MALDI-TOF sequenclng

ANALYSIS DOSAGE Most of the methods describedabovewill detect point mutations, small insertions and deletions. Deletions of one or more exons are common in boys with Duchenne muscular dystrophy and mal be identified b1,a multiplex PCR that revealsthe absenceof one or morc PCR products. However, these mutations are more difficult to detect in carrier femalesas the normal gene on the other X chromosome'masks'the deletion. Large deletion and duplication mutations have been reported in a number of disorders and may encompassa single exon, severalcxons or an entire gene (e.g. HNPR p. 287, HMSN tvpe l, p. 286). Multiplex ligation-dependent probe amplification (MLPA) is a new high-resolution method used to detect d e l e t i o n sa n d d u p l i c a t i o n s ( F i g . 4 . 1 8 ) . E a c h M L P A p r o b e consists of two fluorescently labeled oligonucleotides that can hybridize, adjacentto eachother,to a target genesequence.When hybridized, the two oligonucleotidesare joined by a ligaseand the probe is then amplified by PCR (eacholigonucleotideincludes a universal primer sequenceat its terminus). The probes include a variable-lengthstuffer sequencethat enablesseparationof the PCR products by capillary electrophoresis.Up to 40 probes can be amplifiedin a singlereaction.

4

allelcs or by a dosageeffect where one allele is overrepresented

A P P L I C A T I OONFD N AS E O U E N C E POLYMORPHISMS There is an enormous amount of DNA sequencevariation in the human genome (p. 12) Two main types, single nucleotide polymorphisms (SNPs) and hypervariabletandem repeat DNA length (VNTR) polymorphisms, are predominantly used in geneticanalvsis

SingtenucleotidePolYmorPhisms Around I in 1000 bases within the human genome shows r.ariation. SNPs are most frequently biallelic and occur in coding and non-coding regions. If a SNP lies within the r e c o g n i t i o n s e q u e n c eo f a r e s t r i c t i o n e n z y m e ' t h e D N A fragments produced by that restriction enzyme will be of different lengths in different people. This can be recognized by the altered mobility of the restriction fragments on gel e l e c t r o p h o r e s i s , s o - c a l l e d r e s t r i c t i o nf r a g m e n t l e n g t h polymorphismsor RFLPs. Early genetic mapping studies used Southern blotting to detect RFLPs, but current technology enables the detection of any SNP. New high-throughput methods such as DNA microarrays have led to the creation of a dense SNP map of the human Benome and will assist g e n o m e s e a r c h e sf o r l i n k a g e s t u d i e s i n m a p p i n g s i n g l e gene disorders (p. 282) and associationstudies in common diseases.

Variablenumbertandemrepeats VNTRs are highly polymorphic and are due to the presenceof variable numbers of tandem repeatsof a short DNA sequence that havebeenshown to be inherited in a mendelianco-dominant fashion (p. 107).The advantageof usingVNTRs over SNPs is the large number of alleles for eachVNTR compared with SNPs, u h i c h a r em o s t l l b i a l l e l i c .

Minisate[[ites 'core' sequencewith Alec Jeffre-vsidentified a short l0-15-bp homology to many highly variable loci spread throughout the human genome (p. 17). Using a probe containing tandem repeatsof this core sequence'a pattern of hypervariableDNA fragments is identified. The multiple variable-size repeat sequences identified by the core sequence are known as minisutellites.These minisatellites are highly polymorphic, and a profile unique to an individual (unless they have an identical

67

4

DNATECHNOLOGY ANDAPPLICATI ON5

DNA

Probehybridization PCRprimer sequence

Probeligation PCRprimer sequence PCRprimer sequence

Amplification of probes using fluorescent-labeled orimers

Separation and quantitation

c

c

c c

c

cc

c c

c

c

2000 1000 M E N 1e x o n 1 -800kb N

oE CG

trtlitt

7 -600kb-200kb

c

cc

cc

CC

€ - 2000 a = 1000 MENl exon -800kb

-5.sMb

7 -600kb-200kb

Fis.4.18 A, lLLustration of muttiptex Ligation-dependent (MLpA) probeampLification methodB, Detection ofa detetion encompassing exons1 and2 oftheMEN1geneC,controlprobe(Courtesy of M Owens, Department of Motecular Genetics, RoyalDevonandExeierHospita; Exeter.)

58

APPLICATIONS DNATECHNOLOGYAND

D21S143s

D2151270

D135634

4

D 18 5 5 3 5

900 600 300

Trisomy 21

120

D21S143s

140

D21S11

180

D2151270

D135634

D18 5 5 3 5

320

Fig.4.19 OF-PaRfnrr:nidnrpn:t:laneup[oidy marken foreachmicrosatettite withtwoaLLeLes testingTheupperpane[showsa normaIcOntrol, (D21S11) (microsatetlites effect or a dosage D2151435.D2151270) ThelowerpaneliLlustrates trisomy21witheitherthreeattetes (Courtesy Genetics, Institute of Medical of ChrisAnderson, Microsatellite markersforchromosomes 13and18showa normaIprofrLe Hospitat ofWaLes. University Cardiff )

twinl) is describedas a DNA fi,ngerprint. The techniqueof DNA lingerprintinghasbeenusedwidely in paternitytesting (Fig.a.20)andfor forensicpurposes.

method: recombination between the microsatelliteand the gene may give an incorrect risk estimate,and the possibility of genetic heterogeneity(where mutations in more than one gene causea disease)should be borne in mind.

Microsate[[ites The human genomecontains some 50000 to 100000 blocks of a variable number of tandem repeatsof the dinucleotide CA:GI so-called CA repeats or mitrosatellites(p. 17). The difference in the number of CA repeats at any one site between individuals is highly polymorphic and these repeats have been shown to be inherited in a mendelian co-dominant manner. In addition, highly polymorphic trinucleotide and tetranucleotide repeats havebeenidentihed, and can be used in a similar way (Fig. 4.21). These microsatellites can be analyzed by PCR and the use of fluorescentdetection systemsallows relatively high-throughput analysis Consequently,microsatellite analysishas largely replaced DNA fingerprinting in the applicationsof paternity testing and establishingzygosity.

of genetracking Ctinicalapplications If a gene has been mapped by linkage studiesbut not identified, 'track' the mutant it is possible to use the linked markers to haplotype within a family. This approach may also be used for known genes where a familial mutation has not been found. Closely flanking or intragenic microsatellitesare used most commonly, becauseof the lower likelihood of finding informative SNPs within families.Figure 4.22 illustratesa family where gene tracking has been used to determine carrier risk in the absence of a known mutation. There are some pitfalls associatedwith this

I CI S E A S E N N O N . G E N E TD DIAGNOSIS DNA technology,especiallyPCR, has found application in the diagnosis and management of both infectious and malignant disease.

lnfectiousdisease PCR can be used to detect the presence of DNA sequences specific to a particular infectious organism before conventional evidencesuch as an antibody responseor the results of cultures is available.An exampleis the screeningof blood products for the presenceof DNA sequencesfrom the human immunodeficiency virus (HIV) to ensurethe safetyof their use (e.g screeningpooled factor VIII concentrate for use in males with hemophilia A). Another exampleis the identification of DNA sequencesspecific to bacterial or viral organisms responsible for acute overwhelming infections,where early diagnosisallowsprompt institution of the correct antibiotic or antiviral agentwith the prospectof reducing morbidity and mortality. Real-time PCR techniquescan generate rapid results,with sometest resultsbeing availablewithin I h of a sample being taken.This methodology is particularly useful in the fi ght against methicillin-resis tanr S t ap hyIococcus aureus (MRS A), as patientscan be rapidly testedon admissionto hospital.Anyone found to be MRSA positive can be isolatedto minimize the risk of infection to other patients.

59

4

DNATECHNOLOGY AND,APPLICATI ONS

kinase inhibitor Imatinib are regularly monitored as resistant clones may develop. Following bone marrow transplantation, microsatellite or SNP markers may be used to monltor the successof engraftment b), analysis of donor and patientspecilicalleles.

m

BIOLOGICAL HAZARDS OFDNA TECHNOLOGY DNA technology has a great deal to offer to medicine but there is genuine concern, among the public in particular, that its potentially serioushazardsshould not be ignored. Recombinant

*{h *;*. w

t

DNA technology is being used to improve animal and plant stocks for food production, and recombinant microbial strains are being engineeredto deal with environmental wasteproducts.

*

x

Some factions suggest that insufficient consideration is being given to the seriouspotential consequencesthat could follow the introduction of thesetechniques.For example,it is claimed that the engineered microbial strains, if releasedinto the environment, could cause far more problems than they solve. These

iiw'

;

iW,r

:!i*

,,&tit&

t,** t:a:g*::

:,

Fis.4.2O Autoradrograph of a minisatettite DNAfingerprintoftwo parents. t h e i r t w oo f f s p r i n a g n da n u n r e l a t e idn d i v i d u aEl a c hb a n di n t h e l y y ee f f s p r r nigs p r e s e n t ' no n e o f t h et w o p a . e n t sa n dt h e d i f f e r , n g patternseen in the two matechiidrendemonstrates thatthey are non- identicaI twins (Courtesvof ProfessorA Markham. StJames's H o s p i t a Ll ,e e d s)

concerns, in many instances,are likely to be more theoretical than practical. The main concern surrounding DNA technology is the danger of producing transgenic organisms that could contain genes for cancer susceptibility or be resistant to all known antibiotics.After all, one of the host organismsthat is commonly used in such experimentsis E. coli,which is ubiquitous, being a normal bowel commensal.These are matters that have been of considerablepublic concern and were the subject of the famous Asilomar Conferences in California in the mid-1970s. The consensusof scientific opinion now; however,seemsto be that these dangershave been greatlv exaggeratedVarious authorities have laid down careful guidelines for the use of recombinant DNA techniques.

TECHNIQUE TS OM I N I M I Z E BIOHAZARDS There have been two main approachesto limiting the potential hazardsofDNA technology.These can be separatedinto physical and biological containment methods.

Physicatcontainment M a l i g n a ndt i s e a s e PCR may assist in the diagnosis of lvmphomas and leukemias by identifying translocarions,for example t(9;22), which is characteristicof chronic myeloid leukemia(CML). The exrreme sensitivity of PCR means that minimal residual diseasema-v be detected after treatment for these disorders, and earlv indication of impending relapsewill inform trearmenr oprions. For example, all patients with CML treated with the tyrosine

70

Here the object is to make sure that any potentially dangerous microorganisms are contained by the use of specially designed laboratorieswith appropriate ventilation systems Laboratories are graded,accordingto the type ofexperimentsbeing conducted, from C I (minimum containment),which involveslittle more than the use of careful techniques,to C IV (maximum containment), rvhere containment is of an exceptionallyhigh order. This high level of containment is used in laboratoriesinvolved in biolosical

NS NOLOGY ANDAPPLICATIO DNATECH

4

Fis.4.21

184,188

188,200

ofa tetranucteotide AnaLysis with markerina famiLy mrcrosateLlite GenotyperrM disorder. a dominant peaks wasusedto Labelthe software withthesizeofthePCRproducts wrth The200-bpalteteis segregating members in theaffected thedisorder (Courtesy of M Owens, ofthefamrly. Genetics. of Molecular Department HospitaL, andExeter RoyaLDevon txeter)

warfare,suchas Porton Down in the UK and Camp Detrick in the USA.

Biotogicalcontainment Host organisms used in recombinant DNA work have been 'crippled', so that they cannot survive outside the attenuated,or confines of specialized laboratory culture conditions. This is achievedby careful selection of particular mutants, which led, for example, to the production of the E. coliKlT strain 1776 (aftet the year of American independence!),which requires many complex growth factors if it is to survive outside the laboratory. At the sametime, complementarywork has produced safervectors.The |1.2

t4

u Fig.4.22 ina fami[y wrthDuchenne muscu|'ar dystrophy Gene tracking proband, llla wherenomutation hasbeenfoundintheaffected of of markers A, B andChasenabted theconstruction Anatysis haplotypes: theaffected hapLotype is shownbyan orangebox sisters wereat 50%priorriskof being Bothoftheproband's the[ow-risk showsthatlll,hasinherited carriersGenetracking to bea carrieIbutlll3hasinherrted hapLotype andis untikeLy LikeLy to bea carrierof thehigh-rrsk haplotype andistherefore riskofrecombination shou[d Duchenne muscular dystrophy.The notbeforgotten

combination of physical containment and the use of attenuated organisms should reduce the risk of any possible hazardFrom the evidence available to date, it seemsthat the hazards of geneticengineeringhave,perhaps,beenoveremphasizedin the past and that the risks are more imaginary than real. Nevertheless, great care will continue to have to be exercisedin this field, if for no other reason than to allay the fears of the general public, as exemplified by the adverse publicity surrounding genetically modihed foods.

READING FURTHER Elles R, Mountford R 2003 Molecular diagnosisof geneticdisease,2nd edn Humana Press,New Jersey in diagnostic usedfor genetictestingof commond'isorders Ke.ytechniques laboratories.

71

4 EmeryA E H, Malcolm S 1995An introduction to recombinant DNA rnedicine,2nd edn. John Wiley, Chichester The second, ed,itionof this text, phich cooersthe techniquesand applications of DNA technolog in clinical med.icine. Strachan! ReadA P 2004 Human molecular genetics,3rd edn. Garland Science,London A comprehensizse textbook of all aspectsofmolecular and cellulor biologt as it relatesto inherited diseasein hurnans. WeatherallDJ l99l The new geneticsand clinical practice,3rd edn. Oxford Medical, Oxford One of the original tetts that prozsidcda lucid atserzsieo of the application of DNA techniquesin clinical medicine.

Q Restriction enzymesallow DNA from any source to be cleavedinto reproduciblefragmentsbasedon the presenceof specificnucleotiderecognitionsequences. These fragmentscan be made to recombine,enabling their incorporationinto a suitablevector,with subsequent transformationof a host organismby the vector,leading to the productionof clonescontaininga particularDNA sequence. @ The polymerasechainreaction(PCR)hasrevolutionized medicalgenetics.Within hours,more than a million copiesof a genecan be amplified from a patient'sDNA sample.The PCR product may be analyzedfor the presenceof a pathogenicmutation, generearrangement or infectiousagent. @ Techniquesincluding Southern and northern blotting, DNA sequencingand mutation screening, real-time PCR and microarray analysiscan be used to identify or analyzespecificDNA sequencesof interest. Thesetechniquescan be usedfor analyzingnormal gene structureand function as well as revealingthe molecular pathologyof inherited disease. This providesa meansfor presymptomaticdiagnosis,carrier detectionand prenatal diagnosis,eitherby directmutationalanalysisor indirectly using polymorphicmarkersin family studies. @ Recombinant DNA technology is potentially biologicallyhazardousbut theseriskscanbe minimizedby physicaland biologicalcontainmentmethods.

72

CHAPTER

genes Mapping andidentifying formonogenic disorders

The identification of the gene associatedwith an inherited single-gene(monogenic) disorder, as well as having immediate clinical diagnostic application, will enable an understanding of the developmentalbasis of the pathology with the prospect of possible therapeutic interventions. The molecular basis for nearly 2000 diseasephenotypesis now known. The first human diseasegenes identilied were those with a biochemical basis where it was possibleto purify and sequence the gene product. The development of recombinant DNA techniquesin the 1980senabledphysicalmapping strategiesand led to a new approach, positional cloning. This describes the identilication of a genepurely on the basisof its location,without any prior knowledgeof its function. Notable early successes were the identification of the dystrophin gene (mutated in Duchenne musculardystrophy),the cysticfibrosistransmembraneregulatory gene and the retinoblastoma gene Patients with chromosome abnormalitiesor rearrangementshave often provided important clues by highlighting the likely chromosomal region of a gene associatedwith disease. In the 1990s a genome-wide set of microsatellites was constructed with approximately I marker per l0centimorgans (cM). These 350 markerscould be amplified by polymerasechain reaction (PCR) and facilitated genetic mapping studies that led to the identification of thousands of genes This approach has been supersededby DNA microarrays or 'single nucleotide polymorphism (SNP) chips'. Although SNPs (p. 67) are less informative than microsatellites,they can be scoredautomatically and microarrays are commercially availablewith up to 500000 SNPs distributed throughout the genome. The common step for all approachesto identify human disease genesis arriving at a candidategene (Fig. 5.1). Candidate genes ma1,be suggestedfrom animal models of diseaseor by homology, either to a paralrgrus human gene (e.9.where multigene families exist) or to an rrthlllgous gene in another species With the sequencingof the human genomenow complete,it is alsopossible to find new diseasegenesby searchingthrough geneticdatabases, r . e . ' i ns i l i n ' . Developments in high-throughput mutation screening techniques have also assistedin the identification of pathogenic mutations in candidate genes Consequently the timescale for identifying human diseasegeneshas decreaseddramaticallyfrom a period of years (e.9. the search for the cystic fibrosis gene in

the 1980s)to weeks or perhaps even days, now that the human genomesequenceis availablein oublic databases.

POSITION.INDEPENDENT OFHUMANDISEASE IDENTIFICATION GENES Before genetic mapping techniques were developed, the first human diseasegeneswere identilied through knowledge of the protein product For disorderswith a biochemical basisthis was a particularly successfulstrategy.

CLL O N I N G FUNCTIONA Funttional clonizgdescribesthe identification of a human disease gene through knowledge of its protein product. From the amino-acid sequenceof a protein, oligonucleotideprobes can be synthesizedto act as probes for screeningcomplementary DNA (cDNA) libraries (p. 57). The difficulty lies with the degeneracy of the genetic code, although a dcgeneruteoligonucleotid'ecanbe designedthat includes variablebasesat eachposition where there are multiple possiblenucleotides. An alternative approach is to generate an antibody to the protein that is then used to screen a cDNA expressionlibrary This cDNA expression library is made by cloning cDNAs into an expressionvector containing the necessaryregulatory sequences such that the host cell is able to produce the encodedproteins.

L ODELS U 5 EO FA N I M A M The recognition of phenotypic features in a model organism, such as the mouse, which are similar to those seen in persons affectedwith an inherited disorder,allowsthe possibility that the cloning of the genein the model organismcan lead to more rapid identification of the gene responsible in humans. An example of this approach was the mapping of the gene responsiblefor the inherited disorder of pigmentation and deafness known as Waardenburg syndrome (p. 89) to the long arm of human chromosome 2. This region of chromosome 2 shows extensive homologl', or what is known as q/nten.y,to the region of mouse

73

5

M A P P I NAGN DI D E N T I F Y IG NE GN E F SO RM O N O G E NDI ICS O R D E R S

L I N K A GA EN A L Y S I S T o m a pr e g i o n

GENETIC DATABASES genes Toidentifli in region

PATIENT WJTHCHROMOSOME TRANSLOCATION ORDELETION

it involvesan approachopposite to that of functional cloning, in uhich the protein is the startingpornt.

LINKAGE ANALYSIS Genetic mapping, or linkageanalysis(p. 131),is basedon genetic distances that are measured in centimorgans (cM). A genetic distanceof I cM is the distancebetweentwo genesthat show lolo recombination, that is, in 1oloof meiosesthe genes will not be co-inherited and is equivalent to approximately 1Mb (1 million bases).Linkage analysisis the first step in positional cloning that definesa geneticinterval for further analysis. Linkage analysiscan be performed for a single, large family or for multiple families, although this assumesthat there is no geneticheterogeneity(p. 370) The use oIgenetic markerslocated throughout the genome is described as a genrme-pidescan. In

PATHOGENIC MUTATION(S)

F i s .5 . 1 Pathwaystowardshuman diseaseqeneidentification

chromosome I to which the gene for the murine pigmentary mutant known as Splotch had been assigned.The mapping of the murine Pax3 gene, which codes for a transcription factor expressedin the developing nervous system, to this region suggestedit as a positionalcandidategenefor the disorder It r,vas suggestedthat the pigmentary abnormalities could arise on the basis that melanocytes,in which melanin synthesistakes place, are derived from the neural crest. Identilication of mutations in PAX3,the human homolog, confirmed it as the generesponsible for Waardenburgsyndrome.

M A P PNI GT R I N U C L E O T IR DEEP E A T DISORDERS An increasing number of human diseasesare attributable to expansionsof trinucleotide repeats,and in particular CAG repeat expansionswhich causeextended polyglutamatetracts in Huntington diseaseand many forms of spinocerebellarataxia. A method has been developedto seeknovel trinucleotide repeat expansionsin genomicDNA from affectedpatients;this resulted in the successfulidentification of a CTG repeat expansion in patients with spinocerebellarataxiatype 8.

PO S I T I ON ACLL ON IN G

74

Positionalcloning describesthe identification of a diseasegene through its location in the human genome, without prior knowledgeof its function It is alsodescribedas ret)erse genetics as

the 1990s, genome-wide scans used microsatellite markers (a commercial set of 350 markers was popular), but microarraysare now commerciallyavailable;they include up to 500000 SNPs and allow rapid genotyping with greaterstatisticalpower. Autoz.ygosit.y mapping(alsoknown as homozygosity mapping) is a powerful form of linkage analysis used to map autosomal r e c e s s i v ed i s o r d e r s i n c o n s a n g u i n e o u sp e d i g r e e s ( p . 2 5 7 ) . Autozygositv occurs when affected members of a family are homozygous at particular loci becausethey are identical by descentfrom a common ancestor. Linkage of cystic hbrosis (CF) to chromosome 7 was found by testing nearly 50 caucasianfamilies with hundreds of DNA markers. The gene was mapped to a region of 500kilobases (kb) between markers .MET md D7S8 at chromosome band 7 q 3 1 - 3 2 , w h e n i t b e c a m ee v i d e n t t h a t t h e m a j o r i t y o f C F chromosomes had a particular set of alleles for these markers (shared haplotype) that was found in only 25o/oof non-CF chromosomes.This finding is describedas linkagedisequilibrium and suggests a common mutation due to a founder effect (p 369). Extensivephysicalmapping studiesevenruallyled to the identification of four geneswithin the geneticinterval identified by linkageanalysis,and in 1989a 3-bp deletion was found within the cystic fibrosis transmembranereceptor QF7]H gene.This mutation (p.Phe508del)was presentin approximately70oloof CF chromosomes and 2-3o/o of non-CF chromosomes,consistent with the carrier frequency of I in 25 in caucasians.

Contiganalysis The aim of linkage analysisis to reduce the region of linkage as far as possiblein order to identify a candidateregion. Before publication of the human genome sequence,the next step was to construct a contig. This contig would contain a series of overlapping fragments of cloned DNA representing the entire candidate region. These cloned fragments were then used to screen cDNA libraries, to search for CpG islands (which are usually located close to genes),for zoo blotting (selectionbased on evolutionary conservation) and exon trapping (to identify coding regions via functional splice sites) The requirement for

I CI S 0 R D E R S S 0 RM 0 N 0 G E N D DD E N T I F Y IG NE GN E F I \ 4 A P P I N G AIN

cloning the region of interest led to the phrase'cloning the gene' for a particular disease.

C H R O M O S OEM ABNORMALITIES Occasionally, individuals are recognized with single-gene disorders who are also found to have structural chromosomal abnormalities. The first clue that rhe gene responsible for Duchenne muscular dystrophy (DMD) (p. 291) was located on the short arm of the X chromosome was the identification of a number of females with DMD who were also found to have a chromosomalrearrangementbetweenan autosomeand a specific region of the short arm of one of their X chromosomes.Isolation of DNA clones spanning the region of the X chromosome involved in the rearrangementled in one such female to more detailed gene-mapping information as well as to the eventual cloning of the DMD or dystrophin gene(p. 298). At the same time as these observations,a male was reported with three X-linked disorders: DMD, chronic granulomatous diseaseand retinitis pigmentosa.He alsohad an unusualXlinked red cell group known asthe Mcleod phenotype.It wassuggested that he could have a deletion of a number of geneson the short arm of his X chromosome,including the DMD gene,or what is now termed a clntiguousgenesyndrome.Detailed prometaphase chromosomeanalysisrevealedthis to be the case DNA from this individual rvas used in vast excessto hybridize in competitire reassociation,under specialconditions, with DNA from persons rvith multiple X chromosomesto enrich for DNA sequencesthat he lacked,the so-calledphenol enhancedreassociation/echnique, or pERl which allowed isolation of DNA clones containing portions of the D,MD gene. The occurrence of a chromosome abnormality and a singlegene disorder is rare, but identification of such individuals is important as it has led to the cloning of severalother important diseasegenesin humans, such as tuberous sclerosis(p. 306) and familial adenomatouspolyposis(p. 208)

C A N D I D A TGEE N E S for geneswith a function likely to be involved Searchingdatabases in the pathogenesisof the inherited disordercan alsosuggestwhat are known as candidategenes.If a diseasehas been mapped to a particular chromosomalregion, any genemapping to that region is a positional candidategene.Data on the pattern ofexpression, the timing and the distribution of tissueand cellstypesmay suggest that a certain positional candidategeneor genesis more likely to be responsiblefor the phenotypic featuresseenin persons affected with a particular single-genedisorder. A number of computer programs have been developed that can search genomic DNA sequencedatabasesfor sequencehomology to known genes,aswell as DNA sequencesspecific to all genes,such as the conserved intron--exon splice junctions, promoter sequences,polyadenltlation sitesand stretchesof open reading frames (ORFs) Identification of a gene with homology to a known gene causing a recognized inherited disorder can suggest it as a

5

possible candidate gene for other inherited disorders with a similar phenot.vpe.For example,the identification of mutations in the connexin 26 gene,which codesfor one of the proteins that constitute the gap junctions betweencells causing sensorineural hearing impairment or deafness,has led to the identification of other connexins responsiblefor inherited hearing impairment/ dcafness.

THATA TESTING CONFIRMATORY E ENE C A N D I D A TGEE N EI SA D I S E A S G Nlutations in candidate genescan be screenedfor by a variety of methods (p. 62) and confirmed by DNA sequencing (p. 65) Finding loss-of-function mutations or multiple different mutations that result in the samephenotype provides convincing evidence that a potential candidate gene is associatedwith a disorder For example, in the absenceof functional data to demonstrate the effect of the p.Phe508delmutation upon the CFTR protein, confirmation that mutations in the C,FIR gene caused cystic librosis was provided by the nonsensemutation p.Gl.v542X. Further support is provided by the observation that the candidategene is expressedin the appropriate tissuesand at the relevant stagesof development.The production of a transgenic animal model by the targeted introduction of the mutation into the homologousgene in another speciesthat is shown to exhibit phenotypic features similar to those seen in persons affected with the disorder, or restoration of the normal phenotype by transfcction of the normal gene into a cell line, provides hnal proof that the candidate gene and the diseasegene are one and the same

T H EH U M A NG E N EM A P The rate at which single-genedisorders and their genesare being mapped in humans is increasingexponentially(seeFig. 1.6' p. 8). Nlany of the more common and clinically important monogenic 'morbid anatomyof disordershavebeen mapped to produce the the human genome'(Fig. 5.2).

ER O J E C T T H EH U M A NG E N O M P NT H E B E G I N N I NAGN DO R G A N I Z A T I O F ER O J E C T H U M A NG E N O M P The concept of a map of the human genome was proposed as long ago as 1969byVictor McKusick (seeFig. 1.5,p' 7), one of the founding fathers of medical genetics.Human gene mapping rvorkshopswere held regularly from 1973to collatethe mapping data.The idea of a dedicatedhuman genomeproiect came from a meeting organized by the US Department of Energy (DOE) at Sante Fe, New Mexico, in 1986. The US Human Genome Project started in 1991 with an estimatedbudget of 200 million

75

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poLyposis, FamitiaL adenomatous syndrome Gardner Marfansyndrome Fibrrltin-1, 15q211 contracturaI 5q23-31 FibriL|.in-2, arachnodactyly 13q12 3 growthfactorreceptor 8p111-112 Fibrobtast syndrome Pfeiffer 1, 11p15 4 growthfactorreceptor Fibrobtast 10q26 19p13 2-133 Apert Pfeiffet, 2.Crouzon, Cnmnlpmpnt f:rtnr 5 9q341 fnmnlomonr {rr+nr A syn0r0me 5p13 growthfactorreceptor l^nmnlomont f:rtnr 7 Fibrob[ast 4p163 FGFR3 5p13 thanatophortc 3, achondropLasia, C n m n l p m p n t f : r t n r 9 5p13 dysptasia hyperpLasia, CongenitaI adrenat 6p213 hypercho[esterotemia 19p131J32 Famitiat FH 21- hydroxytase Fragite X mentaIretardation FRAXA(FMRl) Xq273 21q223 Homocystinuria ataxia 9q13-211 Friedreich porphyria FRDA 10q252-263Congenitalerythropoietic meursacLu t a r F a c i o s c a putohum 4q35 FSHMD fi brosis transmem brane 7q312 Cystrc rnnrlr rrt:neo aaar rla+nr dystrophy Gatactosemia 9p'l3 GAL 10q11 syndrome 2,tateonset Cockayne nevussyndrome, BasalceLL 9q31 GAP 14q12 Hypertrophic obstructive syndrome Gorlin cardiomyopathy type'1 GMg l angLiosidosts 33 3p21 GLBl 1q3 Hypertroph icobstructive -6- phosphate GLucose Xq28 G6PD type2 cardiomyopathy d e h y d r ogenase 15q22 Hypertrophic obstructive tYPe Vll, ridosis Lysa ccha copo M u 7q2111 GUSB type3 cardiomyopathy Stysyndrome 17pl12 disease Charcot-Marie-Tooth 11p15 5 HbB typelA B - G t o b igne n e l - . l rr n t i n n t n n dico:ce 4p163 HD rre-Tooth disease 1q22 Charcot-Ma i ni daseA,Tay-Sachs H m exosa 15q23-24 HEXA type1B drsease disease 1p35-36 Charcot-Marie-Tooth B,Sandhoff Hexosaminidase 5q13 HEXB type2 drsease 17q2131-22CoLLagen typel,c1chain. Hemochromatosis 6p213 HFE imperfecta osteogenesis Hypoxanthg i nuea n i n e Xq26-272 HGPRT type I. chain, 7q221 Co[[agen cr2 se, phosphoribosyltransfera imperfecta osteogenesis syndrome Lesch-Nyhan typell,Stickler syndrome 12q1311J32Cotlagen Locus MajorhistocompatibiLity 6p213 HLA 2q31 C o l t a g et ynp el l l ,q c h a i n , Hotoprosencephaty 7q36 HPE3 type lV syndrome Ehters-Danlos tyPeI. Mucopotysaccharidosis 4p163 hyperptasia, IDUA CongenrtaI adrenaL 8q21 Hurlersyndrome 11B-hydroxylase r Lt iignhct h a i n lmmunogLobu l-)eleted in :znncnprmi: 2p12 IGKC Yq11 '13q12 i , t i ng hct h a i n s l m m u n o g L o b u 22q11 IGLCl N o n - s y n d r o ms iecn s o r i n e u r a I p155 I n s u t i n - d e p e n dder an bt e t e s 11 //Vs third deafness, firstrecessive, type2 metlitus d o m i n a nLto c u s simptex, buLlosa EpidermoLysis 12q1lJ3 KRT5 2-133 Myotonic dystrophy 19q13 eu,

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genetic maps initially involved the production of fairly low-level resolution index, skeletonor framework maps, which were based on polymorphic variable-number di-, tri- and tetranucleotide tandem repeats(p. 16) spacedar approximarely 10-cM inrervals throughout the genome.The most recent high-level resolution maps have polymorphic markers that are, on average,spacedat intervalsof lessthan I cM The mapping information from these genetic maps has been integrated with high-resolution physicalmaps (Fig. 5.3). Access to the detailed information from these high-resolution genetic and physical maps allowed individual research groups, often interestedin a specificor parricular inherited diseaseor group of diseases, rapidly and preciselyto localizeor map a diseasegeneto a specificregion of a chromosome.

Development of new DNAtechnotogies A second major objective was the development of new DNA technologiesfor human genome research For example. at the

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information from the Human Genome SequenceProiect.

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outset of the Human Genome Proiect, the technology involved in DNA sequencing was very time consuming, laborious and relatively expensive.The development of high-throughput automated capillary sequencersand robust fluorescent sequencing kits transformedthe easeand cost of large-scaleDNA sequencing projects. Current developmentsin the use of DNA microarray technologyare further revolutionizinganalyticapplicationsofthe

Iuotort ED3s126s I qTER

Although sequencingof the entire human genome would have been seento be the obvious main focus of the Human Genome Project, initially it was not the straightforward proposal it seemed.The human genomecontainslarge sectionsof repetitive DNA (p. 17) that are often technically difficult to clone and sequence.In addition, it would seem a waste of time to collect sequencedata on the entire genomewhen only a small proportion is made up of expressedsequencesor genes' the latter being most likely to be the regions of greatestmedical and biological importance. Furthermore, the sheer magnitude of the prospect of sequencingall 3 x l()ebasepairs of the human genomeseemed overwhelming. With conventional sequencing technology, as was carried out in the early 1990s,it was estimatedthat a single laboratory worker could sequenceup to approximately 2000bp per day. This meant that it would have taken 1'5X 106persondays to sequencethe whole of the human genome! Projectsinvolving sequencingof other organismswith smaller genomesshowedhow much work is involved as well as how the rate of producing sequencedata increasedwith the development of new DNA technologies.For example, with initial efforts at producing genome sequencedata for yeast,it took an international collaborationinvolving 35 laboratoriesin l7 countries from 1989 until 1995 to sequencejust 315000bp of chromosome3, one of the l6 chromosomesthat make up the 14 million basepairs of the yeast genome. Advances in DNA technologies meant' however,that by the middle of 1995 more than one-half of the yeast genome had been sequenced,with the complete genomic sequencebeing reported the following year. Further advancesin DNA sequencing technology led to publication of the full sequenceof the nematode Caenorhabditis elegansin 1998 and the 50 million base pairs of the DNA sequenceof human chromosome 22 at the end of 1999. As a

79

5

M A P P I NAGN DI D E N T I F Y IG NE GN E F S O RM O N O G E NDI C ISORDERS

consequenceof thesetechnicaldevelopments,the 'working draft' sequence,covering 90oloof the human genome,was published in February 2001.The finished sequence(over 99o/ocoverage)was announced more than 2years ahead of schedule in April 2003, the 50th anniversaryof the discovery of the DNA double helix Researchersnow haveaccessro the full catalogof 25 000 to 30 000 genes,and the human genomesequencewill underpin biomedical researchfor decadesto come Although the Human Genome Sequencing Project is complete, a number of new projects have been initiated as a direct consequence,including the Cancer Genome Project and the HapMap Project. The goal of the International HapMap Project is to develop a haplotype map of the human genome, the HapMap, which will describe the common DNA sequence variantspresentin 270 individuals from variouspopulations.The HapMap is expected to provide a key resource for research into genesaffecting health, diseaseand responseto drugs

Development of bioinformatics Bioinformatics was essentialto the overall successof the Human Genome Project. This is the establishment of facilities for collecting, storing, organizing, interpreting, analyzing and communicating the data from the project, which can be widely shared by the scientific community at large. It was vital for anyone involved in any aspect of the Human Genome Project to have rapid and easy accessto the data,/information arising from it. This dissemination of information was met by the establishmentof a large number of electronic databasesavailable on the World Wide Web on the internet (Appendix) These include protein and DNA sequence databases(e.g. GenBank and EMBL), databasesof genetic maps for humans (such as the GDB, Genethon, CEPH, CHLC and the Whitehead Institute sites)and other species(the Mouse Genome Databaseand the C. elegansdatabase),linkage analysis programs (e.g. the Rockefeller University website),annotatedgenomedata(Ensembland UCSC Genome Bioinformatics) and the catalogof inherited diseasesin humans (Online Mendelian Inheritance in Man, or OMIM). These developmentsin bioinformatics now allow the prospect of identifying coding sequencesand determining their likely function(s) from homologies to known genes, leading to the prospect of identifying a new gene without the need for any laboratory experimental work, or what has been called 'cloning in silico'

Comporotive genomics

80

In addition to the Human Genome Project there are separate genome projects for a number of other species,for what are known as 'model organisms'.These include various prokarvotic organisms such as the bacteria E. coli and,Haemophilusinfluenzae, as well as eukaryotic organisms such as Succharomycescereaisiue (yeast), C. elegans(flatworm), Drosophila melanogaster(fruit fly), Mus musculus(mouse), Rattus noroegicus(rat), Fugu rubripes rupripes (puffer fish), mosquito and zebrafish. Although the

impact of what is called comparatiaegenomicsis not necessarily immediately obvious, it is of vital importance in the Human Genome Project. For example, the availability of the total genome sequence of yeast revealed it to contain a total of approximately 6000 genes, only some 2300 of which had been previously identified from traditional genetic studies in yeast In addition, of the 3700 new genes identified by the sequencingproject, only approximately 2300 sharesignificant sequencehomology with any known genes from any other organism, that is, they are novel genes whose functions are unknown. The sequencing of the fruit fly has also revealedso far some 13000 genes,half of whose functions are unknown. Mapping of the human homologs of these newly identified genes from other species provides new'candidate' genesfor inherited diseasesin humans.

Functionalgenomics The second major way in which model organisms proved to be invaluablein the Human Genome Project was by providing the means to follow the expressionof genesand the function of their protein products in normal development as well as their dysfunction in inherited disorders.This is referred to asfunctional genomics, or what has alsobeen calledpast-genomic genencs. The ability to introduce targeted mutations in specific genes, along with the production of transgenic animals (p. 97), for example in the mouse, allows the production of animal models to study the pathodevelopmental basis for inherited human disorders, as well as serve as a test system for the safety and efficacyof genetherapy and other treatment modalities (p.342). Strategiesusing different model organismsin a complementary fashion,taking into accountfactorssuch asthe easeor complexity of producing transgenic organisms and the generation times of different species,allow the possibility ofrelatively rapid analysis of gene expression, function and interactions in providing an understandingof the complex pathobiologyof inherited diseases in humans.

ETHICAL, LEGALAND SOCIAL ISSUES OFTHE HUMAN G E N O MPER O J E C T The rapid advancesin the scienceand application of developments from the Human Genome Project have presented complex ethical issuesfor both the individual and society.These issuesinclude ones of immediate practical relevance,such as who owns and should control genetic information with respect to privacy and confidentiality; who is entitled to accessto it and how; whether it should be used by employers,schools,etc.; the psychological impact and potential stigmatization of persons positive for genetic testing; and the use of genetic testing in reproductive decision making. Other issuesinclude the concept of disability/ differences that have a genetic basisin relation to the treatment of genetic disordersor diseasesby gene therapy and the possibility of geneticenhancement,i.e. using genetherapy to supply certain characteristics,such as height. Lastly, issuesneed to be resolved

S O RM O N O G E NDI ICS O R D E R S M A P P I N G A NI D DE N T I F Y 1GNEGN E F

with regard to the appropriatenessand fairness of the use of the genetic technologiesthat come out of the Human Genome Project, with prioritization of the use of public resourcesand commercial involvement and property rights, especially with regard to patenting

FURTHER READING BotsteinD, White R L, SkolnickM, Davis R W 1980Construction of a genetic linkage map in man using restriction fragment length polymorphisms.AmJ Hum Genet 32: 314-331 OneoJ'theorigmalpalers describingthe conceptof linked restrictionfragment lengthpolymorphsms. Kerem B, RommensJ M, BuchananJ A et al 1989Identificationof the cysticfibrosisgene GeneticanalysisScience245: 1073-1080 Original paper describingcloningof the c.ystic fibrosisgene. McKusickV A 1998Mendelianinheritancein man, 12th edn Johns Hopkins University Press,London A tomputerizedtatalog oJ'thedominant,recessiu^e and X-linked mendeliantrain and disortlersin humansuith a briefrlinical commenlaryand detaik oJ the mulalional bavs, tJ knonn Also aaailableonline,updatedregularly. B, Kunkel L M, MonacoA P et al 1985Cloning the genefor Royer-Pokora - on the an inheritedhuman disorder- chronic granulomatousdisease basisof its chromosomallocation Nature 322:32 38 genethroughcontiguous Original paperdescribingthe identifcution of a disease deletions. chromosome StrachanT, ReadA P 2004 Human molecular genetics,3rd edn Garland Science,London A comprehensiae textboohofall aspeclsofmolecularand cellularbiologyas it relalesto inheriteddiseasein humans SulstonJ 2002The common thread:a storv of science,politics,ethicsand the human genomeJosephHenry Press,London projectby the man pho led A 2ersonalarcountof the humangenomesequencing the UK teamof vientists

5

ELEMENTS @ Position-independentmethods for the identification of monogenic disorders include functional cloning to identify genes from knowledge of the protein sequence and the use of animal models. A technique to identify novel trinucleotide repeat expansionsled to the identification of the SCl8 diseaselocus. @ Positional cloning describes the identification of a gene on the basis of its location in the human genome. Chromosome abnormalities may assist this approach by highlighting particular chromosome regions of interest. Genetic databaseswith human genome sequence data (in silico' a now make the possibility of identifying genes reality. @ Confirmation that a specific gene is responsible for a particular inherited disorder can be obtained by tissue and developmentalexpressionstudies,in-oitro cell culture studies, or the introduction and analysisof mutations in a homologous gene in another species.As a consequence the 'anatomy of the human genome' is continually being unraveled. @ O". of the goalsof the Human Genome Project was to sequencethe human genome. The sequencing was completed by an international consortium in 2003, and has greatly facilitated the identification of human disease genes.

81

Devetopmentat genetics

CHAPTER

'The history of man for the nine months preceding his birth would, probably,be far more interesting and contarn events of greatermoment than all the three scoreand ten yearsthat follow it.' Sarnuel Thylor Coleridge At fertilization the nucleus from a spermatozoonpenetratesthe cell membrane of an oocyte to form a zygote. This single cell

Tabte6.1 Maineventsin the devetopment of a human infant Stage

Time from conception

Pre-embryonic Firsice[[division Zygotereachesuierrnecavity lmptantatron Formation of brtaminar disc Lyonization in femate Formation of tritaminar discand primitivestreak Embryonicstage Organogenesrs BrarnandspinaI cordareforming Firstsignsof heartandtimbbuds Brain, eyes,heartandlimbs deveLoping rapidty BoweIandtungsbeginnrng to devetop Digitshaveappeared Ears, kidneys, liverandmuscleare devetoping Patate closesandjointsform Sexualdifferentiation almost comptete Fetalstage Fetatmovements felt Eyelids openFetusis nowviable rarrih cnori:lizor'1

82

30 h 4 days 5-6 days 12days 16days 19days

Lengthof embryo/fetus

O2 mm 1mm

4-Bweeks 4 weeks

4 mm

6 weeks

17mm

8 weeks

4 cm

10weeks 12weeks

6 cm 9 cm

and, whilst many mysteries remain, the rate of progress in understandingkey eventsand signaling pathwaysis rapid. A fetus is recognizablyhuman after about 12weeksofpregnancy - the first trimester. Normal developmentrequires an optimum maternal environment but genetic integrity is fundamental; this has given rise to the field of developmental genetics.Most of what we know about the molecularprocessesinevitablycomesfrom the study of animal models,with great emphasison the mouse,whose genomecloselyresemblesour own. Prenatallife can be divided into three main stages'. pre-embryonic, embryonicandfetal (Table 6 l) During the pre-embryonic sragea small collectionof cellsbecomesdistinguishable,first as a doublelayered or bilaminar disc,and then as a triple-layered,or uilaminar d,nc(Fig 6.1) that is destined to develop into the human infant. During the embryonic stage,cranio-caudal, dorso-ventral and proximo-distal axes are established,as cellular aggregationand differentiation lead to tissue and organ formation. The final fetal stageis characterizedby rapid growth and development as the embryo, now known as a fetus, matures into a viable human infant. On average,this extraordinary process takes approximately 38 weeks.By convention pregnancyis usually dated from the first day of the last menstrual period (LMP), which usually precedes conception by around 2weeks, so that the normal period of gestationis often stated(incorrectly) as lasting 40 weeks.

FERTI LIZATION AND GASTRU LATION 16-18weeks 24-26weeks

20 cm 35 cm

28-38weeks

40-50cm

r:ro

Rapidweightgarndueto growth andaccumutation offatas [ungsmature

divides to become two, then four, and when the number has doubled some 50 times the resulting organism comprises more than 200 distinct cell types and a total cell number of about 10000 trillion. This is a fully formed human being with complex biochemistryand physiology,capableof exploring the cosmosand identifying subatomic particles.Not surprisingly, biologists and geneticistsare intrigued by the mechanismsof early development

Fertilization, the processby which the male and female gametes fuse, occurs in the fallopian tube. Of the 100 to 200 million spermatozoa deposited in the female genital tract only a few hundred reach the site of fertilization. Of these, usually only a single spermatozoon succeedsin penetrating first the corona

GENETICS DEVELOPMENTAL

B u c c o p hray n g e a I memora ne Ectoderm Mesoderm Endoderm P r e c h o r d apll a t e mesoderm N e u r apl l a t e

Notocord

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Fig.5.1 A s c h e m a t itcr i l a m i n adr i s c s, e c t i o n eadl o n gt h e r o s t r o - c a u d a L axrs Cettsfrom ihe futureectoderm(mid blue)migratethroughthe prim'tivestreakto fo.rr Lheendoderm(trghtbtue)and -esoderm (gray)Formationof the neutraIptatein the overtyingectoderm, destinedto be the centraInervoussystem,invotvessonic hedgehogsignalrng(p 85)from the notochordand prechordial platemesoderm (Redrawnwith permissionfrom LarsenW-J1998 E s s e n t r a ot sf h u m a ne m b r y o [ o g yC.h u r c h i L t ti v i n g s t o nN e ,e w Y o r k )

5

12 to 16 cells by 3 days, when it is known as a morula. A key concept in developmentat all stagesis the emergenceof polari4r within groups of cells - part of the process of differentiation that generatesmultiple cell types with unique identities. Whilst precisemechanismsremain elusive,observationssuggestthat this begins at the very outset; in the fertilized egg of the mouse, the point of entry of the sperm determinesthe plane through which the first cell cleavagedivision occurs. This seminal event is the first step in the development of the so-called dorso-ventral, or primary body, axis in the embryo. Further cell division leads to formation of a blastocyst,which destinedto form the consistsof an inner cell massor embr.yoblast, embryo, and an outer cell mass or trophlblast,which gives rise to the placenta.The processof converting the inner cell mass into firstly a bilaminar and then a trilaminar disc (seeFig. 6.1) is known as gilsuulation,and takesplace between the beginning of the secondand the end of the third weeks. Between4 and 8 weeksthe body form is established,beginning with the formation of the primitive streakat the caudalend of the embryo. The germinal layers of the trilaminar disc give rise to structures (Box 6.1). The and endod,ermal mesodermal ectod,ermal, neural tube is formed and neural crest cells migrate to form sensory ganglia,the sympatheticnervoussystem,pigment cells,and both bone and cartilagein parts of the face and branchial arches. Disorders involving cells of neural crest origin, such as neurofibromatosis (p. 287), are sometimes referred to as This period between4 and 8 weeksis described neurocristopathies. as it is during this interval that all as the period of organogenesis, of the major organsare formed asregionalspecializationproceeds in a cranio-caudaldirection down the axis of the embryo.

radiata, then the zona pellucida, and finally the oocyte cell membrane, whereupon the oocyte completesits second meiotic division(seeFig. 3.19,p.42). After the sperm has penetrated the oocyte and the meiotic process has been completed, the two nuclei, now known as pronuclei, fuse, thereby restoring the diploid number of 46 chromosomes.Germ cell and very early embryonic development are two periods characterizedby widespread changesin DNA methylation patterns - epigenetic reprogramming (see below, p. 98). Primordial germ cells are globally /emethylated as they mature and are subsequently methylated de noao during gametogenesis, the time when most DNA methylation imprints are established.After fertilization a second wave of change occurs.The oocyte rapidly removesthe methyl imprints from the sperm'sDNA, which has the effect of resettingthe developmental stopwatch to zero. By contrast, the maternal genome is more passively /amethylated in such a way that imprinting marks resist demethylation. A third wave of methylation, de noz,o, establishesthe somatic cell pattern of DNA methylation following implantation. These alternating methylation stateshelp to control which genesare active,or expressed,at a time when two genomes, initially alien to eachother, collide. The fertilized ovum or zygote undergoesa seriesof mitotic divisions to consist of two cells by 30h, four cells by 40h and

FAMILIES GENE DEVELOPMENTAL Information about the genetic factors that initiate, maintain and direct embryogenesis is incomplete. However, extensive

B o x 6 . 1 0 r g a na n dt i s s u eo r i g i n s Ectodermal nervoussystem Central system Perpheralnervous F pd e r r n i Isn c t u d nnga i ra n dr a i l s glands Subcutaneous DentalenameI MesodermaI tissue Connective Cartlageandbone muscte Smoothandstriated system Cardiovascutar taIsystem Urogen Endodermal Thymusandthyroid system Gastrointestinal Liverandpancreas

83

5

DEVELOPMENTAL GENETICS

genetic studies of the fruit fly, Drosophila melanogaster,a;nd vertebratessuch as mouse, chick and zebrafish have identified several genes and gene families that play important roles in the early developmentalprocesses.It has also been possible through painstaking gene expressionstudies to identify several key developmentalpathways,or cascades,to which more detail and complexity is continually being added. The gene families identified in vertebratesusually show strong sequencehomology with developmental regulatory genesin Dra sophila.Recent studies in humans have revealedthat mutations in various members of these genefamilies can result in either isolatedmalformations or multiple congenital anomaly syndromes (seeThble 16.5,p.245). Many developmentalgenesproduce proteins called transcription factors (p. 2l), which control RNA transcription from the DNA template by binding to specific regulatory DNA sequencesto form complexesthat initiate transcription by RNA polymerase. Transcription factors can switch geneson and off by activating or repressing gene expression. It is likely that important transcription factors control many other genes in coordinated sequentialcascadesand feedbackloops involving the regulation of fundamental embryological processessuch as induction (the processin which extracellularsignalsgive rise to a change from one cell fate to anotherin a particular group ofcells), segmentatiln, migration, d.ffirentiation and programmed cell death (known as aprptlsts). It is believed that these processesare mediated by growth factors, cell receptors and chemicals known as morph0gens. Across speciesthe signaling moleculesinvolved are very similar. The protein signals identihed over and over again tend to be members of the transforminggrowthfactor-B (TGF-B) family, the wingless(Wnr) family and the hetlgehog(HI{) famrly.In addition, it is clear within any given organism that the same molecular pathwaysare reused in different developmentaldomarns.

EARLY PATTERNING The emergenceof the mesoderm heraldsthe transition from the stage of bilaminar to trilaminar disc, or gastrulation.Induction of the mesoderm - the initiation, maintenanceand subsequent patterning of this layer- involvesseveralkey families of signaling factors.The Na dal family isinvolved in initiation, -FGFs(fibroblast growth factors) and Wnts are involved in maintenance, and BMPs (bone morphogenetic proteins) are involved in patterning the

84

mesoderm. Signaling pathways are activated when a key ligand binds specificmembrane-bound protein receptors.This usually Ieads to the phosphorylation of a cytoplasmic factor, and this in turn leads to binding with other factor(s) These factors translocate to the nucleus where transcriptional activation of specifictargetsoccurs. In the case of Nodal and BMP pathways,ligand binding of a specific heterotetramer membrane-bound protein initiates the signaling, which is common to all members of the TGF-B family. The cytoplasmic mediators are Smad factors. The embryo appears to have gradients of Nodal activity along the dorsalventral axis, although the sigificanceand role of these gradients in mesoderminduction are uncertain.

The Wnt pathway has two main branches: one that is B-catenin dependent (canonical) and the other independent of B-catenin. In the canonical pathway Wnt ligand binds to a FrizzledllRP heterodimer membrane-bound protein complex and the downstreamintracellular signalinginvolvesa G-protein. The effect of this is to disrupt a large cytoplasmic protein complex that includes Axin, the adenomatouspolyposis coli (APC; see p. 208) protein, and the glycogen synthase kinase30 (G.SK--?p) protein. This prevents the phosphorylation of B-catenin, but when B-catenin is not degraded it accumulatesand translocatesto the nucleus where it activatesthe transcription of dorsal-specific regulatory genes. Binding of the ligand to the FGF receptor results in dimerization of the receptor and transphosphorylation of the receptor'scytoplasmic domain, with activation of Ras and other kinases, one of which enters the nucleus and activates target transcription factors. There is no known human diseasephenotype due to mutated WNT genes, but overexpression of BMP4 has been found in the rare bony condition fibrodyplasia ossificansprogressiva (FOP). FOP has recently been shown to be causedby mutated AC|/RI, which encodes a BMP type I receptor, and mutated BMP receptor 2 has beenshown to be a causeof familial primary pulmonary hypertension.BMPs and Nodal genesare part of the IGF-B superfamily.

S O M I T O G E N EASNI D ST H EA X I A L SKELETON The vertebrate axis is closely linked to the development of the primary body axis during gastrulation, and during this process the presomitic mesoderm (PSM), where somites arise, is laid down in higher vertebrates. Wnt and FGF signals play vital roles in the specification of the PSM. The somites form as blocks of tissue from the PSM in a rostro-caudal direction (Fig. 6.2), each being laid down with a precise periodicity that, in the 1970s, gave rise to the concept of the 'clock and wavefront' model. Since then molecular techniques have given substance to this concept, and the key pathway here is notch-delta signaling and the 'oscillation clock' - a precise, temporally defined, wave of cyclinggeneexpression (c-hairy in the chick, lunatic fringe and,hes genes in the mouse) that sweeps from the tail-bud region in a rostral direction and has a key role in the process leading to the defining of somite boundaries Once again, not all of the components are fully understood, but the nltch rec€ptorand its ligands, d,elta-like-l and tleltalike-3, together with presenilin-l and mesodermposterior-2, work in concert to establish rostro-caudal polarity within the PSM such that somite blocks are formed. Human phenotypes due to mutated genes in this pathway are now well known and include presenile dementia (presenilin-l), which is dominantly inherited, and spondylocostaldysostosis(delta-like-3,mesoderm p0sterilr-2 and, lunatic fringe), which is recessively inherited (Fig. 6.3). Another componenr of the pathway is JAGGEDI, which, when mutated, results in the dominantly inherited and

GENETICJ DEVELOPMENTAL

6

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r o l ei n P S Ms p e cf i c a t r ownh i l stth es e g m e n t a t icol no c kd e p e n d s o no s c rat tf ' o no, r c y c t i . lg e. n e tsh a ta r ei m p o r t a ri n t somite formation boundary wheregenesoftheNotch-Delta pathway polarity estabtish rostro-caudal H0Xgeneshavea gtobalfunction in estab[ishing somiteidentity alongtheentirerostro-caudaL '

l ^

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axrs (Adaptedtrom lrcktet tedl2tJtJ3Parterning in vertebrate devetopmentOxfordUniversityPress,Oxford)

very variable condition known as Alagille syndrome (arteriohepatic dysplasia)(Fig. 6.4). Recently,mutations ln NOTCH2 havebeen shown to causesome casesof Alasille svndrome.

THE S O NI CH E D GE H OG-P A T C H E D- GLI PATHWAY The Sonic hedgehoggene(51111)is as well known for its quirky name as for its function. SHH induces cell proliferation in a tissue-specificdistribution and is expressedin the notochord, the brain, and the zone of polarizing activity of developing limbs. Following cleavageand modification by the addition of a cholesterol moiety, the 51111protein binds with its receptor, Patched (Ptch), a transmembrane protein. The normal action

in patient with deve[opment ofthevertebrae Disrupted inthedeltotype1,dueto mutations spondylocostaLdysostosis (Courtesy pathway of liAe-3gene,parrofthenotchs,gnaLing f l r M e r i eM . c F n t a o aK' 1e.n ^ e d v - G a lC t oenn l r eI o n d n n )

Mole cular defectsin any part of this pathwaylead to a number of apparently diverse malformation syndromes (see Fig. 6.5). N{utations in, or deletions of, SHH (chromosome 7q36) cause holoprosencephaly(Fig. 6.6), in which the primary defect is incomplete cleavageof the developing brain into separate hemispheres and ventricles. The most severe form of this malformation is cyclopia - the presenceof a single central eye. (The complexity of early developmentcan be appreciatedby the fact that a dozen or so chromosomal regions have so far been implicated in the pathogenesisof holoprosencephaly[p. 246].) Nlutations in PTCH (9q22) result in Gorlin syndrome (nevoid basal cell carcinoma syndrome; Fig. 6 7), which comprises multiple basalcell carcinomas,odontogenickeratocysts,bifid ribs, calcihcationof the falx cerebri and ovarianfibromata. Mutations in SMO (7q31) are found in some basal cell carcinomas and medulloblastomas.Mutations in CLI3 (7p13) cause PallisterHall and Grieg syndromes, which are distinct entities with more or less the same body systems affected. However, there

of Ptch is to inhibit another transmembrane protein called Smoothened (Smo), but when bound by Shh this inhibition

are also links to other conditions, in particular the very variable syndrome (SLOS), which may include Smith-Lemli-Opitz holoprosencephalyas well as some characteristicfacial features,

is releasedand a signaling cascadewithin the cell is activated. The key intracellular targetsare the GLI family of transcription factors (Fig. 6.5)

genital anomalies and syndactyly. This condition is due to a defect in the final step of cholesterolbiosynthesis,which in turn may disrupt the binding of Shh with its receptor Ptch. Some,

85

DEVE[OPMENTALGENETLCS

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{"

c o n n e c ro n w L ho i s e a s e D i f ' e ' e n Le i e r e n t s i n t ^ e p a t n w a ya c r a s

(arrows)or inhibitors(bars)The Shh proteinis rnitially activators cleaved t o a n a c t i v eN - t e r m n a l f o r m w , h i c hi s t h e nm o d f r e db y t h ea d d i t l o n o f c h o [ e s t e r oTlh e n o r m a l a c t i o o n f P t c hi s t o i n h i b i t S m o ,b u t w h e nP t c hl s b o u n db y S h ht h s i n h i b i t i oins r e m o v e da n d t h ed o w n s t r e a m s i g n a nl g p r o c e e d sC R E B B Pc A M Pr e s p o n s e e l e m e n t - bn d n g b i n d i n gp r o t en

Fig.6.tr A, BoywithAtagitlesyndromeand confirmedmutationtnJAGGED| -he o ' t h c o n g e n i t ah.e a r td s e a s eB , w h o p r e s e n t ew s a m eb o ya o t h e r f e w y e a r se a r t r ew r t h h ' s p a r e n r sH r s h a sa p i g m e n t a r y retinopathy and was posttivefor the same genemutatron

or all, of the featuresof SLOS may therefore be due to loss of integrity in this pathwav (p. 276). Furthermore, a cofactor for the Gli proteins, CREBBP (16p13) is mutated in RubensteinT[-vbi syndrome.

transcription factors or morphogen signals,as well as operating at many other levels, on genes that mediate cell adhesion, cell division rates,cell death and cell movement.Thev specifycell fate and help to establishthe embryonic pattern along the primary (rostro-caudal) axis as well as the secondarv (genital and limb

In Drosophilaa classof genesknown as the homeotic geneshas been shown to determine segmentidentitl.. Incorrect erpression of thesegenesresults in major structural abnormalities;the Antp gene, for example, which is normally erpressed in the second

bud) axis.The-v therefore play a major part in the development of the central nervous svstem, axial skeleton and limbs, the gastrointestinaland urogenital tracts, and external genitalia Drosophilahas eight 11ar genes arranged in a single cluster, but in humans, as in most vertebrates,there are four homeobox geneclusterscontaining a total of 39 HOX genes(Fig. 6.8). Each

thoracic segment, rvill transform the adult fly's antennae into legs if incorrectly expressedin the head.Homeotic genescontain a conserved180-basepair (bp) sequenceknown asthe homeobox, which is believedto be characteristicof genesinvolved in spatial

cluster contains a series of closely linked genes.In vertebrates such as mice it has been shown that these genesare expressed in segmentalunits in the hindbrain and in global patterning of the somites formed from axial presomitic mesoderm. In each

H O M E O B O X ( H OGXE) N E S

85

pattern control and development.This encodesa 60-amino-acid domain that binds to DNA in Hox-responseenhancers.Proteins from homeobox (or HOX)-containing genes are therefore important transcription factorsthat activateand repressbatteries of downstreamgenes.At least 35 downstreamtargetsare known The Hox proteins regulate other 'executive' genes that encode

ENTALGENETICS DEVELOPM

6

F i s .6 . 6 'F :oerr : jl o r {r oc :or rr u r q r o) <

|, n

r nr c ou n) c- 6r n rn rnul nr un P r Lh c: lP' r r o r y .

T' h o a',oc rrc cyc)

:-o o c

rl^^^ Lru>c

-nna+hor rugcLr rcl

and there is a midlinecLeftLipdue to failureof normaIprotabia development

llOX cluster there is a direct linear correlation between the position of the gene and its temporal and spatial expression. Thesc obscrvationsindicate that these genes plav a crucial role in earlv morphogenesis.Thus, in the developing limb bud (p. 93) HOXA9 is cxpressedboth anteriorlv to, and before, HOX10, and so on. N{utations rn HOXAI3 causea rare condition knorvn as the hand-foot-genital svndrome. This shows autosomal dominant inheritance and is characterizedby shortening of the first and lifth digits, rvith hvpospadiasin males and bicornuate uterus in females.Experiments r'vith mouse Hoxul3 mutants have sholn that exprcssion of another gene, EphA7, is severell' reduced. Therefore, if this geneis not activatedb.t Hoxal3, there is failure to form the normal chondrogenic condensationsin the distal limb primordial Mutations in HOXDI3 result in an equall.vrare limb developmentalabnormality knorvn as s-ynpolydactylyThis also showsautosomaldominant inheritance and is characterized by insertion of an additional digit between the third and fourth

Fig.6.7 G o r ln ( n e v o i db a s a ct e l lc a r c i n o m as)y n d r o m eA . T h i s6 - y e a r - o l d girl from a Largefamilywiih Gortinsyndromehas macrocephaty and a cherubicappearanceB, Her affectedststerdevelopeda (arrowed)in the mandib[e keratocysi rapidlyenlargingodontogenic roots of her teeth the at the age of 9years.disptacing

Iingers and the fourth and fifth toes, which are webbed (Fig. 'fhe phenot,r-pcin homozygotes is more severe,with the 6 9) mctacarpalsand metatarsalsbcing convertedinto short carpaland Reported mutations take the form tarsal-likebones,respectir.el-v of'an increascin the number of residues in a polyalanine tract. This triplet-repeat expansion probably alters the structure and function of the protein, thereby constituting a gain-of-function mutation (p.26). To date, these are the only two 11OX genes proved to be mutated in human malformations. They are each located at the 5' end of their respectiveclusters and cooperate in the dcvelopment of caudal structures along the primary body axis, the most distal part of the limb, and the genital tubercle.

87

6

DEVELOPMENTAL GENETICs

A Drosophila

5'

3' Abd-B Abd-A Ubx

Human bein .q

HOXA chr'7p14

A13

A11

A10

A9

HOXB ch.17q21

HOXC chr'12q13

HOXD chr'7q31

BB

c8

DB

Antp

5cr

Dfd

At

46

A5

A4

87

86

85

84

c6

c5

c4

D4

pb

lob

A3

A2

A1

83

82

81

D3

D1

Fig.6.8 A, Drosophilio haserghtHoxgenesrna singtectuster whereas thereare39 H0Xgenesin humans, arranged rnfourclusters [ocated on chromosom es7p,17q,12q and2qfortheA,B.CandD clusters, patterns respectively B, Expressron of HoxandHOXgenesalongthe rostt'o-caudaI axisin invertebrates andvertebrates, respectively. Invertebrates thec[usters areporotogous andappearto compensaie for (Redrawn oneanother. fromVeraksa A,DetCampoM,McGinnrs patterning W 2OOO genesandtheirconserved DevetopmentaI functions: a i r tnho r m i < < i)n q f r o mm o d e l o r g a n i s tmosh u m a n sM o lG e n eM 6 SR 6 - 1 OrO t etab

Fig.6.9

88

CLrnrcal(A) and (B) radiographic vrewsof the handsin synpoLyoacryLy

GENETICS DEVELOPMENTAL

6

Given that there are 39 ,F1OXgenesin mammals it is surprising that no other recognizedsyndromesor malformations havebeen attributed to HOX gene mutations. One possibleexplanationis that most flOX mutations are so devastatingthat the embrvo cannot survive. Alternatively, the high degree of homology between HOX genes in the different clusters could lead to functional redundancy so that one HOX genecould compensate for a loss-of-function mutation in another.In this context HOX genes are said to be paralogousbecausefamily members from different clusters, such as HOXAI3 and HOXD|3i are more similar than adjacentgenesin the samecluster. Severalother developmentalgenesalso contain a homeoboxlike domain. These include MSX2 and EMX2. Mutations in ,4,ISX2 can cause craniosynostosis- premature fusion of the

F i s .5 . 1 0 canthorum in an infant lrisheterochromia andmarkeddystopia type1,dueto a mutationin PAX3. syndrome withWaardenburg

cranial sutures. Mutations in EMX2 cause a severe cerebral malformation known as schizencephalgin which there is a large full-thickness cleft in one or both cerebralhemispheres.

PA I R E D - B O(P X A X )GE N E S The paired-box is a highly conserved DNA sequence rhat encodesa 130-amino-acidDNA-binding transcription regulator domain. Nine PIX genes have been identified in mice and humans.In mice thesehavebeen shownto play important roles in the developingnervoussystemand vertebral column. In humans loss-of-function mutations infive PAX geneshave been identified in associationwith developmentalabnormalities(Table 6.2). Waardenburgsyndrome type I is causedby mutations in PAX3. It showsautosomaldominant inheritanceand is characterizedby sensorineuralhearing loss, areasof depigmentation in hair and skin, abnormal patterns of pigmentation in the iris, and widely spaced inner canthi (Fig. 6.10). Waardenburg syndrome shows genetic heterogeneity;the more common type 2 form, in which the inner canthi are not widely separated,is sometimescaused by mutations in the human microphthalmh (MITfl gene on chromosome3 The importance of expression of the PAX gene family in eye development is illustrated by the effects of murarions in PAX2 and PAX6. Mutations in PAX2 cause the renal-coloboma

Tabte5.2 DeveLopmental abnormalities associated with P/Xqenemutations Gene

Chromosome location

Developmental abnormatity

PAX2

10q24

Renal-cotoboma syndrome

PAX3

2q35

Waardenburg syndrome type1

PAX6

11p13

Aniridia

PA,X8

2q12

Absentor ectopic thyroidgtand

PAX9

14q12

Otigodontia

F i s .6 . 1 1 A n e y es h o w i n ga b s e n c eo f t h e i r i s( a n i r i d i aT)h e c o r n e as h o w s (Courtesyof Mr R Gregson.Oueen's abnormalvascuLarization M e d i c aCt e n t r eN. o t t i n g h a m )

s-vndrome,in which renal malformations occur in association with structural defects in various parts of the eye, including the retina and optic nerve. Mutations in PAX6 lead to absence of the iris, which is known as aniridia (Fig. 6.11). This is a ke-v feature of the WAGR syndrome (p. 266), which results from a contiguous gene deletion involving the PAX6 locus on chromosome11.

HMGBOX( SOX)GENES SRY- TYPE SRI is the Y-linked gene that plays a maior role in male sex determination (p. 96). A family of genes known as the SOX genes shows homology with SRY by sharing a 79-aminoacid domain known as the HMG (high-mobility group) box. This HMG domain activates transcription by bending DNA in such a way that other regulatory factors can bind with the promoter regions of genesthat encode for important structural proteins. These SOX genes are thus transcription regulators

89

6

DEVELOPMENTAL GENETICS

and are expressedin specific tissues during embryogenesis. For example, SOXI, SOX2 and SOXJ are expressed in the developingmouse nervous system. In humans it has been shown that loss-of-function mutations in SOX9 on chromosome 17 cause campomelic dysplasia. This very rare disorder is characterizedby bowing of the long bones, sex reversal in chromosomal males and verv poor longterm survival In-sttu hybridization studies in mice have shown that SOX9 is expressedin the developing embryo in skeletal primordial tissue,where it regulatestype II collagenexpression, as well as in the genital ridges and early gonads. SOX9 is now thought to be one of several genes that are expressed downstream of SRY in the processof male sex determination (p. 96). Mutations in SOXI0 on chromosome 22 catse a rare form of Waardenburg syndrome in which affected individuals have a high incidence of Hirschsprung disease.Mutations in SOX2 (3q26) have recently been shown to causeanophthalmia or microphthalmia together with esophagealatresiaand genital hypoplasia in males: the anophthalmia-esophageal-genital (AEG) syndrome.

T.BOX(TBX)GENEs The T genein mice playsan important role in specificationof the paraxialmesodermand notochord differentiation Heterozvgotes for loss-of-function mutations have a short tail and malformed sacral vertebrae.This gene, which is also known as Brach.yur.y, encodesa transcription factor that contains both activator and repressor domains. It shows homolog.v with a series of genes through the shared possessionof the T domain, which is also referred to asthe T:box. These Tlbox or IBX genesare dispersed throughout the human genome, with some family members existing in small clusters.One of these clusters on chromosome 12 contains TBX3 md IBX5. Loss-of-function mutations in IBXJ cause the ulnar-mammary syndrome in which ulnar rav developmentalabnormalities in the upper limbs are associated with hypoplasia of the mammarv glands Loss-of-function mutations in TBXS cause the Holt-Oram syndrome. This autosomaldominant disorder is characterizedbv congenitalheart abnormalities,most notably atrial septal defects,and upper limb radial ray reduction defects that can vary from mild hypoplasia (sometimes duplication) of the thumbs to almost complete absenceof the forearms.

Z I N CF I N G E R GENES

90

The term zinc finger refers to a finger-like loop projection consisting of a series of four amino acids that form a complex with a zinc ion. Genes that contain a zinc finger motif act as transcription factors through binding of the zinc finger to DNA. Consequently they are good candidatesfor single-gene developmentaldisorders(Table 6.3). For example, a zinc finger motif-containing gene knorvn as GLI3 on chromosome 7 has been implicated as the cause of two developmentaldisorders. Large deletions or translocations

Tabte5.3 DeveLopmentaL abnormalities associated with genescontaining a zincfingermotrf Gene

Chromosome location

Developmentat abnormality

GLl3

7p13

GreigsyndromeandPattister-Hall syndrome

WTl

1'1p13

Denys-Drash syndrome

ZIO

13q32

Hotoprosencephaly

ZIC3

Xq26

Lateralitydefects

involving GLI3 catse Greig cephalopolysyndactyly,which is characterized by head, hand and foot abnormalities such as polJ'dactylyand syndactyly (Fig 6.12A) In contrast, frameshift mutations in GLI3 have been reported in the Pallister-Hall syndrome (Fig. 6.12B), in which the key featuresare polydactyly, hypothalamichamartomataand imperforate anus. Mutations in anotherzinc finger motif-containing geneknown as WTI on chromosome 1l can causeboth Wilms' tumour and a rare developmentaldisorder, the Denys Drash syndrome, in which the external genitalia are ambiguous and there is progressiverenal failure as a result of nephritis. Mutations in two other zinc finger motif-containing genes, ZIC2 and ZIC3, have recently been shown to cause holoprosencephaly and laterality-defects, respectivel]'.Just as polarity is a key concept in development, so too rs laterality, with implications for the establishment of a normal left-right body axis. In very early development, integrity of man-vof the same gene families previouslv mentioned Nodal, Sonic Hedgehog and Notch - is essentialto the establishmentof this axis.Clinicalll; situssolitusis the term given to normal left-right asymmetry and sr,tus tnrersus to reversalof the normal arrangement.Up to 25o/oof individuals with situs inversus have an autosomal recessivecondition Kartagenersyndrome,or ciliary dyskinesia.Other terms usedare isomerismsequence, heterotaxy,asplenia/polyasplenia,and Iaemark syndrome.Laterality defects are characterized by abnormal positioning of unpaired organs such as the heart, liver and spleen,and more than 20 genesare now implicated from studies in vertebrates,with a number identified in humans by the study of affected families, with all of the main patterns of inheritance represented.

('S S I G N AT LRANSDUCTIO NI G N A L I N G ' ) GENES Signal transduction is the processwhereby extracellulargrowth factors regulate cell division and differentiation by a complex pathwayof geneticallydetermined intermediatesteps.Mutations in many of the genesinvolved in signal transduction play a role in causing cancer (p 198). In some casesthey can also cause derelopmentalabnormalities.

GENETICS DEVELOPMENTAL

5

l gl l l

TK1

]TM :

I a-::::t:

FGFRl

*)

FGFR2

-)

t t t t TD1 P

A C,J,P

FGFR3

t

ACH

t

HCH

tt

ID2 TD1

F i g .5 . 1 3 (FGFR) growthfactorreceptor ofthefibroblast Structure inthecraniosynostosis of mutatrons thelocation Arrowsindicate groupofskeletaL dysptastas andachondropLasia syndromes T;M ,t r a n s m e m b r adnoem a i n ; l g ,i m m u n o g t o b u t i n -dtoi kmea i n Apert syndrome;A, kinase domain; P Pfeiffer TK,tyrosine syndrome; -J. Jackson-Weiss syndrome: syndrome;C.Crouzon s iCa H ; , i aC; Ha, c h o n d r o p t aH T D .t h a n a t o p h odr yi cs p l a sA hypochondropLasta

Fibroblastgrowth factorreceptors FGFs play key roles in embryogenesis,including cell division, migration and differentiation. The transduction of extracellular FGF signals is mediated by a family of four transmembrane tyrosine kinase receptors. These are the fibroblast growth factor receptors (FGFRs), each of which contains three main

Fis.5.12

components:an extracellularregion with three immunoglobulinlike domains, a transmembrane segment, and two intracellular tyrosinekinasedomains(Fig. 6.13) Mutations in the genes that code for FGFRs have been identified in two groups of developmentaldisorders(Thble 6 4). syndromesand the achondroplasia These are the craniosynostosis The craniosynostosissyndromes, familv of skeletal dysplasias. (Fig is the best known' are 6.14) of which Apert syndrome of the cranial sutures,often in premature fusion characterizedby

A,Thefeetof a chitdwrthGreigcepha[opolysyndactyly. Noiethat (extradrgits) potydactyty andsyndactyly theysnowbothpreaxraI (fused digits)B,Thetefthandofa womanwrthPallrster-Hatl rnGLl3Notethepostaxral syndrome anda provenmutation associationwith hand and foot abnormalitiessuch as syndactyly polydactyly andthesurgrcaIscar. wherean extradigitarising polydactyly) (fusion of the digits). Apert syndrome is causedby a mutation frombetween thenormaimetacarpaI rays(mesoaxiat in one of the adjacentFGFR2 residuesin the peptides that link wasremoved

TheREf proto-oncogene The proto-oncogeneRET on chromosomelOql1.2 encodesa cell-surfacetyrosine kinase.Gain-of-function mutations,whether inherited or acquired, are found in a high proportion of thyroid cancers.Loss-of-function mutations in REThave been identified in approximately50o/oof familial casesof Hirschsprung disease, in which there is failure of migration of ganglionic cells to the submucosaland myenteric plexusesof the large bowel. The clinical consequencesare usually apparent shortly after birth when the child presentswith abdominal distention and intestinal obstruction.

the second and third immunoglobulin loops (see Fig. 6 l3). In contrast, mutations in the third immunoglobulin loop can causeeither Crouzon syndrome, in which the limbs are normal, or Pfeiffer syndrome, in which usually only the thumbs and big toes are abnormal. Achondroplasia is the most commonly encountered form of genetic short stature (Fig. 6.15). The limbs show proximal ('rhizomelic') shortening and the head is enlarged with frontal bossing. Intelligence and life expectancy are entirely normal. Achondroplasia is almost always causedby a mutation in, or close to, the transmembrane (TM) domain of FGFR3. The common TM domain mutation leads to the replacement of a glycine amino-acid residue by an arginine - an amino acid that is never normally found in cell membranes' This in turn appears to enhance dimerization of the protein that catalyzes downstream signaling Hypochondroplasia'

91

DEVELOPI'/ENTAI GENETICS

T a b t e6 . 4 D e v e l o p m e n tdai sI o r d e rcsa u s e db y m u t a t i o n s in fibrobLast growthfactorreceptors Gene

Chromosome

Syndrome

Craniosynostosis syndromes FGFRI 8p11 Pferffer FGFR2 10q25 Apert Crouzon Jackson Werss Pfeffer FGFR3 4p16 (withacanthosis Crouzon nigricans) Skeletaldysplasias FGFR3 4p16

Achondroplas a Hypocho ndrop[a sia Thanatophoric dysplasra

a mildcr fbrm of skelctal dvsplasil lvith similar trunk and limb changcs but normal head shlpe and sizc, is caused bv mutations in the prorimal tvrosine kinasedomain (intracellular) of FGI'R3 Finallr, thar.ratophoric dl.splasia,a much morc sc\iere and invariablr lethrl fbrm of skcletaldl.splasia,is causedbr mutations in either thc pcptides linking the secondand third immunoglobulin domrins (extracclir-rlar) of FGlR.l, or the distal F C F R J t r r , s i n e k i n a s cd o m r i n

92

The mechanism b1. x'hich these mutations causc skeletal shortening is not understood at present. The mutations cannot have loss-of-function cffccts as children rvith the WolfHirschhorn svndromc (p. 261), which is duc to chromosome microdeletionsthat includc FGFR3, do not shorvsimilar skeletal abnormalitics Instead, the mutations probablv involve a gain of f unction mediated bv incrcasedligand binding or receptor actlvatl0n.

Thc pharyngeal (or branchial) arches correspond to the gill s l s t e m o f l o r v e r v e r t c b r a t e s .F i v e ( s e g m e n t c d )p h a r v n g e a l archesin humans arisc lateral to the structurcs of the head (Fig 616) and each comprises cells from the thrcc germ la1,.ers and t h e n e n r a l c r c s t T h e l i n i r - r go f t h c p h a r v n r , t h y . r o i d a n d parxthvroids arisesfrom thc enioderrn,and the outcr epidermal la1'er arises from the ectoderm The musculature arises from thc mesoderm,and bon.v structures from neural cra.i/cells. Separating the arches arc the phar-vngealclefts externall-vand the pharvngealpouchcs internallr,;thcsc have important destinics Numbered from the rostral end, the first arch forms the jau', the first clcft is destined to be thc external auditor). mcrtus, and the lirst pouch the middle ear apparatusThe second arch lbrms the hvoid appxratus,whilst the third pouch devclops into the thvmus, and the third and fourth pouches becomc the

Viewsof ihe face(A) hand(B)and foot(C)of a chrldwrthApert svndrome

GENETICS DEVELOPMENTAL

6

MODEL THELIMBA5 A DEVELOPMENTAL Four main phases are recognized in limb development: (1) initiation, (2) specification, (3) tissue differentiation and (4) grorvth. Although none of these stagesis fully understood, insight into the probable underlying mechanisms has been gleanedfrom the study of limb developmentin chicks and mice in particular.

ANDSPECIFICATION INITIATION Limb bud formation is thought to be initiated at around 28days bv a member of the -FG,Ffamily as illustrated by the development of an extra limb if FCFI, FGF2 or FGF{ is applied to the side of a developing chick embryo. During normal limb initiation -FG-F8transcripts have been identified in mesenchymenear the initiation site. .FGF8 expression is probably controlled by HOX genes,which determine limb type (forelimb or hindlimb) and number.

ODNG R O W T H T I S S UD E I F F E R E N T I A TAIN F i 9 .5 . 1 5 A y o u n gc h i t dw i t ha c q o n d r o p t a s i a

parathyroids.The arteries within the arches have important destinies too and, after remodeling, give rise to the aortic and pulmonary arterial systems. The most well known, and probably most common, condition due to disturbed development of pharl'ngeal structures is DiGeorge syndrome (DGS), also known as velocardiofacial syndrome (VCFS), and well describedevenearlier by SedlidkovdL of Praguein 1955.This is describedin more detail in Chapter l8 (p.267); it results from a submicroscopicchromosome deletion of band 22qll with the loss of some 30 genes.Studies in mice (the equivalent,or syntenic,region is on mouse chromosome 16) suggest that the most significant Bene loss is that of Tbxl, s t r o n g l y e r p r e s s e dt h r o u g h o u t t h e p h a r y n g e a l a p p a r a t u s . Heterozygous Tbxl knock-out mice show hypoplastic or absent fourth pharyngeal arch arteries, suggestingthat TBXI in humans is the key.Indeed, mutations in this genehavenow been found in some congenital heart abnormalities and it is possible that TBXI is the key gene for other elements of the phenotype. However, there are still unansweredouestionsin the whole DGS/VCFS/ Sedl6dkovl story. There are many examples of developmental genes in lower organisms whose homologs in humans are linked to malformation syndromes;in the first branchial arch one such is EYAI . ln

Once limb formation has been initiated, a localized area of thickened ectoderm at the limb tip, known as the apical ectodermal ridge (AER), produces growth signals such as FGF4 and FGF8, which maintain further growth and establishthe proximo-distal axis (Fig. 6.17). Expression of the gene TP63 rs crucial for sustainingthe AER and, when this gene is mutated, split handfoot (ectrodactyly)malformations result, often together with oral clefting and other anomalies.Signalsfrom another localizedarea on the posterior margin of the developingbud, known as the zlne of polarizing actiaitl (ZPA), determine the antero-posterioraxis' One of these signals is Sonic Hedgehog 6Hm (p. 85), which acts in concert with other .FGF genes, CLI3 md another gene family, which produces bone morphogenetic proteins (BMPs). Another morphogen, retinoic acid, is believedto play a maior role at this stagein determining development at the anterior margin of the limb bud. Subsequent development involves the activation of genes from the HOXA and HOXD clusters in the undifferentiated proliferating mesenchymalcells beneath the AER. This area is known as the progress zone. Cells in different regions express different combinations of HOX genes that determine local cell proliferation, adhesion and differentiation. Downstream targets of the HOX geneclusters remain to be identified. Other genes that clearly have a key role are those of the T:box family, already discussed, and SALL{, which is mutated in Okihiro syndrome (radial ray defectswith abnormal eye movementsdue

Drosophila this is the eyesabsentgene, but in humans, when mutated, it causesbranchio-oto-renal syndrome, consisting of branchial sinuses,externalear malformationsand abnormal renal

to congenitalpalsy affecting the sixth cranial nerve). FGFs continue to be important during the later stagesof limb development.In this context it becomeseasyto understand why-limb abnormalitiesare a feature of disorders such as Apert svndrome(seeFig 6.14),in which mutations havebeen identified

development.

in the extracellulardomains of FGFR2.

93

5

DEVELOPMENTAL GENETICS

Head

0pticvesicle

Pharyngeal arches 0ticvesicle Pericardial bulge

Fis.5.16 Ectoderm

Firstpharyngeal arch

Endoderm Firstpharyngeal cleft pouch Firstpharyngeal Mesenchyme Archartery

DEVELOPMENTAL GENES ANDCANCER Several genes that play important roles in embryogenesis have also been shown to play a role in causing cancer (Thble 6.5). This is not surprising, given that many developmental genes are expressedthroughout life in processessuch as signal transduction and signal transcription (p. l8). It has been shown that several

94

(orbranchiat) Thepharyngeat (A)shows apparatus Thelateratview thefivepharyngeal archescloseto the embryonic headandthecross-section (B)showsthebasicarrangement from whichmanyheadandneckstructures, aswetlastheheart,devetopHumans andmicedo nothavearchno 5 (Redrawn fromGraham A,SmithA 2OO1 Patterning thepharyngeal arches. permrssron Bioessays 23:54-61.with Inc,a subsidiary ofWi[ey-Liss ofJohn Witey& Sons,Inc)

different mechanisms can account for the phenotypic diversity demonstrated by these so-called teratogenes.

GAIN- OF.FUNCTION VERSUS LOSS.OFFUNCTION MUTATION S Mention has already been made of the causal role of the RET proto-oncogene in familial Hirschsprung disease,as well as in

DEVELOPMENTAL GENETICS

6

In contrast, mutations causing a gain-of-function effect result in either type 2A or type 28 multiple endocrine neoplasia (MEN) These disorders are characterizedby a high incidence of medullary thyroid carcinoma and pheochromocytoma.The

L i m bb u d

Ectoderm A p i c a le c t o d e r m arli d g e (FGF2,4.8)

Ip63,T-80X,SALLa

activating mutations that cause MEN-24 are clustered in five cysteine residues in the extracellular domain. MEN-2B, which differs from MEN-2A in that affected individuals are tall and thin, is usually caused by a unique mutation in a methionine residuein the tyrosine kinasedomain.

Proximal

Distal

Progresszone (HoxA, HoxD WNT7A,GLI,BMP)

Zoneof polarizing activity (SHH)

S O M A T IR CE A R R A N G E M E N T S Activation of the RET proto-oncogenecan occur by a different mechanism whereby the genomic region encoding the intracellular domain is juxtaposedto one of severalactivating genesthat are normally preferentiallyexpressedin the thyroid gland.The newly formed hybrid RCTgene producesa novel protein whoseactivity is not ligand dependent.These somaticrearrangementsare found in a high proportion of papillary thyroid carcinomas,which show a particularly high incidence in children who were exposed to radiation following the Chernobyl accidentin 1986. PAX3 provides another example of a developmental gene that can causecancerif it is fused to new DNA sequencesA specific translocationbetweenchromosomes2 and l3 that resultsin a new chimeric transcript leadsto the developmentin children of a rare

Fis.6.17 Srmplrfiedrepresentation of vertebratetrmb devetopment

lung tumor called alveolarrhabdomyosarcoma. both inherited and sporadic thyroid cancer (p. 9l). The protein product encoded by RET consists of three main domains: an extracellular domain that binds to a glial cell line-derived neurotrophil facto\ a transmembrane domain, and an intracellular tyrosine kinasedomain that activatessignal transduction (Fig. 6.18). Mutations causing loss of funcrion result in Hirschsprung disease.These include whole genedeletions,small intragenic deletions,nonsensemutations and splicing mutations leading to synthesisof a truncated protein.

SP

ECD

TMD

1 28

TKD

636 651 726

r 6 0 9 - 6 3 4r

-

A

'i

I

IVEN24 and FIVTC

PTC

999 1114

T

AND POSITIONAL EFFECTS DEVELOPMENTAL GENES The discovery of a chromosomal abnormality, such as a translocation or inversion, in a person with a single-gene developmental syndrome provides a strong indication of the probableposition ofthe diseaselocus,as it is likely that one ofthe breakpoints involved in the rearrangementwill have disrupted the relevant gene. However, in a few instancesit has emerged that the chromosomebreakpoint actually lies approximately l0I 000kb upstream or downstreamof the genethat is subsequently shown to be mutated in other affected individuals (Thble 6.6). Thc probable explanation is that the breakpoint has separated

M E N2 8

Fig.5.18 TheREfproto-oncogene Themostcommon mutation sitesin thedifferent clinicaLentities associated withREf areindicated N u m b e rrse f e tro a m i n o - a cr eds i d u eSs Ps i g n apt e p t t d E e ;C D , extraceltular domain;TMD.transmembrane domar n;TKD,tyrosi ne k i n a s de o m a i nM; E Nm , u l t i p leen d o c r i naed e n o m a t o sFi M s ;T C , famitial meduLlary thyrod carcinoma ThearrowabovePTC (papiLlary thyroidcarcinoma) indicates thesomatrc rearrangement siiefortheformation of newhybridformsof RET(Adapted from P a s i nBi ,C e c c h e r Ii .nRi o m e o G1 9 9 6R E Tm u t a t i o ni nsh u m a n TrenosGenet12,138-144\ d,sease

genesthatshowa position Tabte6.6 Developmental effect Gene

€hromosome

Developmental anomaly

GLI3

7p13

Greigcephatopotysyndactyty

SHH

7q36

Hotoprosencephaty

PAX6

11p13

Aniridia

)cun4v7o

1l /19^4) +/ ,

a - ^U1l,r-r s t r L1u"y. > ^ lP- .l o . .> t o u -d-r^r ^r P

95

6

DEVELOPMENTAL GENETICS

the coding part of the genefrom contiguousregulatory elements (p. 21).These observationshave created obvious difficulties for thosecarrying out the original researchwhen the putative disease gene in translocation families has been found not to contain an lntragenlc mutatron.

animals such as arthropods but has been reported in a human on only one occasion,this being in the form of chimeric fusion with another cell line that had a normal male-derivedcomplement. The main importance of completemoles lies in their potential to undergo malignant change into invasive choriocarcinoma. This can usually be treated successfullyby chemotherapy,but if untreated the outcome can be fatal. Malignant change is seenonly very rarely with partial moles.

H YD A T I DIF ORMOL M ES Occasionally conception results in an abnormal pregnancy in which the placentaconsistsof a proliferating disorganizedmass known as a hydatidiform mole. These changes can be either partial or complete (Thble 6.7).

P A R T I AL H Y D A T ID IF ORMOL M E Chromosome analysis of tissue from partial moles reveals the presenceof 69 chromosomes,i.e. triploidy (p.269). Using DNA polymorphisms it has been shown that 46 of thesechromosomes are always derived from the father, with the remaining 23 being maternal in origin. This doubling of the normal haploid paternal contribution of 23 chromosomescan be due to either fertilization by two sperm, which is known as disperm.y,or to duplication of a haploid sperm chromosome set by a process known as entloretluplication. In these pregnancies the fetus rarely if ever survives to term. Triploid conceptions survive to term only when the additional chromosome complement is maternally derived, in which casespartial hydatidiform changesdo not occur. Even in thesesituationsit is extremely uncommon for a triploid infant to survive for more than a few hours or davs after birth.

C O M P L E THEY D A T I D I F O R MM OLE Complete moles have only 46 chromosomes, but these are exclusivelypaternal in origin. A complete mole is causedby fertilization of an empty ovum either by two sperm or by a single sperm that undergoesendoreduplication.The opposite situation of an egg undergoing development without being fertilized by a sperm, a process known as parthenogenesis,occurs in lower

EXPRESS ION IN PARENTAL DIFFERENT ANDEMBRYOBLA ST TROPHOBLAST Studies in mice have shown that when all nuclear genes in a zygote are derived from the father the embryo fails to develop, whereastrophoblastdevelopmentproceedsrelativelyunimpaired In contrast, ifall ofthe nuclear genesare maternal in origin, the embryo developsnormally but extra-embryonic development is poor. The observationsoutlined above on partial and complete moles indicate that a comparable situation exists in humans, with paternally derived genes being essential for trophoblast development and maternally derived genesbeing necessaryfor early embryonic development. These phenomena are relevant to the concept of epigenetics (see below, p. 98) and genomic imprinting (p. 115).

AND DIFFERENTIATION SEXUAL DETERMINATION The sexof an individual is determined by theX andY chromosomes (p. 32).The presenceof an intactY chromosomeleadsto maleness regardlessof the number of X chromosomespresent.Absenceof a Y chromosomeresults in female development. Although the sex chromosomesare present from conception, differentiation into a phenotypic male or female does not commence until approximately 6 weeks. Up to this point both the miillerian and wolffian duct systems are present and the embryonic gonads,although consistingof cortex and medulla, are still undifferentiated. From 6 weeksonwards the embryo develops into a female unless the testis-determining factor initiates a sequenceof events that prompt the undifferentiated gonads to develop into testes.

Tabte5.7 Characteristics of oartiaLand com0lete hydatidiform motes

96

Partial mole

Completemole

No of chromosomes

69

46

Parental originof chTomosomes

23- maternal 46* paternat

At[46 paternal

Fetuspresent

Yes- butnotviabte

No

M a l i g n a npl o l e n t r a l

V e r yl o w

High

AG C T O-RS R Y T H ET E S T I S - D E T E R M I NFI N In 1990 it was shown that the testis-determining factor or gene is located on the short arm of the Y chromosome close to the pseudoautosomalregion (p. 112).This geneis now referred to as being locatedin the sex-determiningregion of the Y chromosome (^tRl). It consistsof a single exon that encodesa protein of 204 amino acids that include a 79-amino-acid HMG box (p. 89), indicating that it is likely to be a transcription regulator. Evidence that the SRY gene is the primary factor that determinesmalenesscomesfrom severalobservations:

DEVELOPN4ENTAL GENETICS

l. .tRy sequencesare present in XX males. These are infertile phenotypic males who appear to have a normal 46,XX karyotype. 2. Mutations or deletions in the SRY sequencesare found in many XY females.These are infertile phenotypic females who are found to have a 46,XY karyotype. 3. In mice the SRYgene is expressedonly in the male gonadal ridge as the testesare developingin the embrvo. 4. Transgenic XX mice that have a tiny portion of the Y chromosomecontaining the SRYregion developinto maleswith testes. From a biologicalviewpoint (i.e.the maintenanceof the species), it would clearly be impossiblefor the SRYgene ro be involved in crossing over with the X chromosome during meiosis I. Hence SRY has to lie outside the pseudoautosomalregion. However, there has to be pairing of X and Y chromosomes,as otherwise they would segregatetogether into the same Bamete during, on average,50o/oof meioses.Nature's compromise has been to ensurethat only a small portion of the X and Y chromosomesare homologous,and thereforepair during meiosis I. Unfortunately, the closeproximity of SRYto the pseudoautosomalregronmeans that, occasionally,it can get caught up in a recombinationalevent.

5

This almost certainly accountsfor the majority of XX males,in whom molecular and fluorescent in-situ hybridization (FISH) studies show evidenceofY-chromosome sequencesat the distal end of one X-chromosome short arm (Ftg. 18.22,p.276) Expressionof SRYtriggers off a seriesof eventsthat involves other genes such as SOXg,leading to the medulla of the undifferentiated gonad developing into a testis, in which the Leydig cells begin to produce testosterone(Fig. 6.19).This leads to stimulation of the wolffian ducts, which form the male internal genitalia, and also to masculinization of the external genitalia. This latter step is mediated by dihydrotestosterone,which is produced from testosterone by the action of 5cx-reductase (p. 166, 276).The Sertoli cells in the testesproduce a hormone known as miillerian inhibitorv factor. which causesthe millerian duct systemto regress. In the absenceof normal SRY expression,the cortex of the undifferentiated gonad develops into an ovary. The mrillerian duct forms the internal genitalia The external genitalia fail to fuse and grow as in the male, and instead evolve into normal female external genitalia. This normal process of female development is sometimes referred to rather chauvinistically as the 'default' pathway. Without the stimulating effects of testosterone,the wolffian duct systemregresses.

)

mrnl

-KtsRY)

t--l

a + ,/ Testosterone

*,ql9/

,1

I

il"\Q*,^ \

oinyorotlstosterone

ilt rrellm

F;

99

EI

Epididymis S e m i n avl e s i c l e s V a sd e f e r e n s

Penis Scrotum

Clitoris Labia D i s t a vl a g i n a

F a l l o p i taunb e s Uterus Proximal vagina

F i s .5 . 1 9 Summaryofthemaineventsinvolved in sexdetermination MlF,muLLerian inhibrtor, regionoftheYchTomosome; SRYsexdetermining factor.

97

5

DEVETOPMENTAL GENETICS

Normally sexual differentiation is complete b-v 12-l4weeks' gestation,although the testes do not migrate into the scrotum until late pregnanc]'.Abnormalities of sexual differentiation are uncommon but thel' are important causesof infertility and sexual ambiguity.They are consideredfurther in Chapter 18.

E P I G E N E TA I CNSDD E V E L O P M E N T

The process of XCI occurs early in development at around l5-16 days' gestation,when the embryo consistsof approximately 5000 cells. Normally either of the two X chromosomescan be inactivated in any particular cell. Thereafter the same X chromosomeis inactivatedin all daughter cells (Fig. 6.20).This differs from the case in marsupials, in which the paternally derived X chromosomeis consistentlyinactivated. The inactive X chromosome exists in a condensed form during interphase when it appearsas a darkly staining mass of chromatin known as the sex chromatin, or Barr body. During

The concept of'epigenetics' is not recent. 'Epigenesis'was Iirst mooted as a theme by Conrad Waddington in 1942and referred, in essence,to the unfolding of developmentalprograms and processesfrom an undifferentiated zygote - the very heart of

mitosis the inactiveX chromosomeis late replicating.Laboratory techniques have been developed for distinguishing which of the X chromosomes is late replicating in each cell. This can be useful for confirming that one of the X chromosomes is

embryonic development.This roughly equateswith our modern understanding of the control of developmentalgene expression and signalingpathways.It incorporatedthe concept of epigenetic mechanismsbeing 'wiped clean' and 'reset' at one point in the life cycle Although this is still valid, the term in current usageis extendedto include heritable changesto geneexpressionthat are not due to differencesin the geneticcode. Such geneexpression statesmay be transmitted stablythrough cell divisions- certainly'

structurally abnormal, as usually an abnormal X chromosome will be preferentially inactivated,or, more correctly, only those hematopoietic stem cells in which the normal X chromosome

mitosis but also meiosis (thereby not necessarilysubject to a 'resetting'process). One genotypecan thereforegive rise to more than one phenotype, dcpending on the 'epigenetic state' of a locus,or loci. The most common form of DNA modification- the biochemical mechanism for epigenesis is direct covalent methylution of nucleotides.This appearsto lead to a seriesof steps that alters local chromatin structure In human geneticsthe best recognized epigeneticphenomenaare X-chromosome inactivation,described 6.1611', and parent-of-origin-specilic gene expression (parental

is active will have survived. Apparent non-random inactivation also occurs when one of the X chromosomes is involved in a translocationwith an autosome(p. 110). The epigenetic process of XCI is achieved by differential methylation (a form of imprinting; p. 206) and is initiated by a gene, XLSI ('X inactivation specific transcript'), which maps within the X-inactivation centre at Xql3.3. XISI is expressed only from the inactive X chromosome and produces RNA that spreads an inactivation methylation signal up and down the X chromosome on which it is located. This differential methvlation of the X chromosomeshas been utilized in carrier detection studies for X-linked immunodeficiency diseases, e.g. Wiskott-Aldrich syndrome, using methylation-sensitive restriction enzvmes (p. 192). Not all of the X chromosome is

imprinting), which is realized in Prader-Willi and Angelman syndromes(p 117), and Beckwith-Wiedemann and RussellSilver syndromes(p. 119), i.e. rvhen errors occur.There is much interest, however,in the possibilit--v that epigeneticstatescan be influenced by environmental factors In animal studies there is evidence that the nutritional and behavioral environment may lead to different 'epialleles',and in human populations epidemiological studies have shown convincing correlations of maternal (and in some casesgrandparental) nutritional status with late-onsetcardiovascularand metabolic-endocrinedisease.

X-CHROMOSOM I NEA C T I V A T I O N As techniqueswere developedfor studying chromosomes,it was noted that in femalemice one of the X chromosomesoften differed from all other chromosomesin the extent to which it wascondensed. In 1961 Dr Mary Lyon proposed that this heteropyknotic X chromosomewas inactivated,citing as evidenceher observations on the mosaicpattern of skin coloration seenin mice known to be heterozygousfor Xlinked genesthat influencecoat color. Subsequent events have confirmed the validity of Lyon's hypothesis,

98

and in recognition of her foresight the processof X-chromosome inactivation (XCI) is often referred to as lyonization.

Sex

F i g .5 . 2 0 duringdeveLopment The maternaLly X-chromosomeinactivatLon ' ^ ^ - - ) - - h T n m n c n m ceqr p r t r n r e q e n l .aesdX m -".r d l l U "p .d-t -c-l -l l-d-trt ry U t r l V t r Un L r l :nd Yn rocnortivplri -"' "H.

DEVELOPMENTAL GENETICS

6

inactivated.Genesin the pseudoautosomal region at the tip of the short arm remain active,as do other loci elsewhereon the short and long arms, such as XIST. There are more genesthat escape XCI in Xp compared with Xq. This explains why more severe phenotypic effects are seen in women small Xp chromosome deletions compared with those in women with small deletions in Xq. If all loci on the X chromosome were inactivated then all women would have the clinical features of Tirrner syndrome and the presence of more than one X chromosome in a male (e.9. 47,XXY) or two in a female (e.g. 47,XXX) would have no phenotypic effects.There are,in fact, quite characteristicclinical featuresin these disorders(p.272). XCI provides a satisfactoryexplanation for severalobservations, described below.

Barrbodies In men and women with more than one X chromosome, the number of Barr bodies (p. 99) visible at interphaseis alwaysone less than the total number of X chromosomes. For example, men with a 47,XXY karyotype have a single Barr body, whereas women with a 47,XXX karyotype havetwo Barr bodies.

Dosagecompensation Women with two normal X chromosomeshave the same blood Ievels of X-chromosome protein products, such as factor VIII, as normal men, who of course have only one X chromosome. An exception to this phenomenonof dosagecompensationis the level of steroid sulfatase in blood, which is increased in women compared with men. Not surprisingly it has been shown that the locus for steroid sulfatase(deficiencyof which causesa skin disorder known as ichthyosis)is in the pseudoautosomalregion.

Mosaicism Mice that are heterozygous for X-linked genes affecting coat color show mosaicismwith alternating patchesof different color rather than a homogeneouspattern. This is consistent with patches of skin being clonal in origin in that they are derived from a single stem cell in which one or other of the X chromosomes is expressed,but not both. Thus, eachpatch reflectswhich of the X chromosomes was active in the original stem cell. Similar effects are seen in tissues of clonal origin in women who are heterozygous for X-linked mutations such as ocular albinism (Fig.6.21). Other evidence confirming that X-inactivation leads to mosaicism in females comes from studies of the expression of the enzyme glucose-6-phosphate dehydrogenase(p. I 79) in clonesof cultured fibroblasts from women heterozygousfor variants ofthis gene.Each clone is derived from a single cell and expressesone of the variants,but never both. The clonal origin of tumors can be confirmed in women who are heterozygousfor such variants by demonstrationof the expressionof only one of the variantsin the tumor.

Fi1.6.21 showing ocuLar atbinism Thefundusofa carrterofX- [inked (Courtesy of Mr S J of retrnalpigmentation a mosaicpattern f-h:rlec

The Rnv:l Fvp Hncnit:l

Manr^hester)

Problemsof carrierdetection Carrier detection for X-linked recessivedisorders based only on examination of clinical featuresor on indirect assayof gene function is notoriously difficult and unreliable. Cells in which the X chromosome with the normal gene is active can have a selective advantage,or they can correct the defect in closelyadiacentcells in which the X chromosome with the mutant gene is active. For example, only a proportion of carriers of Duchenne muscular dystrophy (DMD) show evidenceof muscle damageas indicated by measurementof creatine kinasein serum (p. 304). Similarly distorted ratios of very long chain fatty acids (VLCFAs) are seen in many, but not all, carriers ofX-linked adrenoleukodystrophy (XLALD). Fortunately, the development of molecular methods for carrier detection in X-linked disorders can bypass these problems, as techniques such as Southern blotting are not influenced by methylation unless methylation-sensitive restriction enzymes are used. The use of methylation-sensitiveenzymescan in fact provide a means of carrier detection if there has been strong selection against the cell line in which the mutant-bearing X chromosomeis active(Fi1.6.22).

Manifestingheterozygotes Occasionally a woman is encountered who shows mild or even full expression of an X-linked recessivedisorder, such as DMD or XLALD. One possible explanation is that she is a manifesting heterozygotein whom, by chance, the X chromosome bearing

99

5

DEVELOPMENTAL GENETICS

A P

P

o

M

P

IV

G

o c

ooooo ooooo

e c eee c e eee

ooooo ooooo iiffi:l

ooooo

eee eeeee

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V survival

ceeee ce ee eeeee

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(P)andmaternatty (M) A, NormalX-chromosome inactivation resulting in survrvalof roughty equaInumbers of cettswiththepaternalty (*)whichresultsin setectron derived X chromosome activeB, Inthissituation thematernaLLy derived X chromosome hasa mutation againstthecettsinwhichitisactiveThussurvivingcellsshowpreferentiaIexpressionofthepaternaLLyderivedXchromoso

100

the normal gene has been inactivatedin significantly more than 50o/oof relevant cells. This is referred to as skewedX-inactivation (p. I l0). There is someevidencethat X-chromosome inactivation can itself be under genetic control, as families with several manifesting carriers of disorders such as DMD and Fabry diseasehavebeen reported. In a few families, marked skewingof

be causedby functional disomy for those genespresent on their ring X chromosome. Other studies in Turner syndrome, this time on pure 45,X cases,have shown some differencesin social cognition and higher-order executive function skills according to whether their X chromosomewas paternalor maternal in origin. Those with a paternal X scored better, from which the existence

X-inactivation in severalfemaleshas been shown to be associated with an underlying mutation in XIST.

of a locus for social cognition on the X chromosorne can be postulated.If such a locus is not expressedfrom the maternal X, this could provide at least part of the explanation for the excess

The 46,Xr(X)phenotype

difficulty with languageand social skills observedin 46,XY males, as their X is always maternal in origin.

A 46,Xr(X) karyotype is found in somewomen with typical features of Turner syndrome. This is consistent with the ring lacking X sequences,which are normally not inactivatedand which are neededfor a normal phenotype.Curiously a few 46,Xr(X) women havecongenitalabnormalitiesand show intellectual impairment In these women it has been shown that XISI is not expressed on the ring X, so their relatively severephenotype is likely to

Recentresearch Recent researchhas suggestedthat, just as XCI is not an all-ornone phenomenon for the whole chromosome,it is probably not all-or-none for every gene. In a study of skin fibroblasts, which express more than 600 of the 1098 senes identified on the X

GENETICS DEVELOPMENTAL

chromosome, about 20olowere found to be inactivated in some but not all samples.About l5o/oescapedXCI completely,whilst only 650lowere fully silencedand thus expressedin one dose.In addition to non-random XCI, the variable dosageof genesthat escapeXCI may account for variation among normal femalesas well as those who are heterozveousfor Xlinked diseaseeenes.

mother, suggestinga single-genedefect that predisposesto the phenomenon. We tend to think of MZ twins asbeing genetically identical, and basicallythis is of coursetrue. However,occasionallythey can be discordant for structural birth defectsthat may be linked to the twinning process itself - especially those anomalies affecting midline structures.There is probably a two- to three-fold increased risk of congenitalanomaliesin MZ twins, i.e. 5-l0o/o of MZ twins overall. Discordance for single-gene traits or chromosome abnormalities may occur becauseof a post-zygotic somatic mutation or non-disjunction, respectively.One exampleof the latter

TWINNING Twinning occurs frequently in humans, although the incidence in early pregnancy as diagnosedby ultrasonography is greater than at delivery,presumablyas a result of death and subsequent resorptionofone ofthe twins in a proportion oftwin pregnancies. The overall incidence of twinning in the UK is approximately I in 80 of all pregnancies,so that approximatelyI in 40 (i.e 2 of 80) of all individuals is a twin. However, the spontaneoustwinning rate variesenormously,from approximately I in 125 pregnancies in Japanto I in 22 in Nigeria. Twins can be identical or non-identical, i.e. monozygotic(MZ) (uniovular) or dizygotic (DZ) (biovular), depending on whether they originate from a single conception or from two separate conceptions(Thble 6.8). Comparison of the incidence of disease in MZ md DZ twins reared apart and together can provide information about the relative contributions of genetics and environment to the cause of many of the common diseasesof adult life, as discussedin Chapter 15.

M O N O Z Y G O T IW CI N S MZ twinning occursin about 1 in 300births in all populationsthat havebeen studied. MZ twins originate from a single egg that has been fertilized by a single sperm. A very early division, occurring in the zygotebefore separationof the cellsthat make the chorion, results in dichorionic twins Division during the blastocl'st stage from days 3 to 7 results in monochorionic diamniotic twins. Division after the first week leadsto monoamniotic twins. However,the reason(s)why MZ twinning occurs at all in humans is not clear.As an event, the incidence is increasedtwo- to fivefold in babiesborn by in-ritro fertilization. There are rare cases of familial MZ twinning that can be transmitted by the father or

Tabte6.8

5

is the rare occurrenceof MZ twins of different sex:one 46,XY and the other 45,X. Curiously, MZ femaletwins can show quite striking discrepancy in X-chromosome inactivation. There are several reports of femaleMZ twin pairs of which only one is affectedby an X-linked recessivecondition such as DMD or hemophilia. In theserare examplesboth twins havethe mutation and both show non-random X-inactivation, but in opposite directions. MZ twins have traditionally provided ideal researchmaterial for the study of genetic versus environmental influences. In a recent study of 40 pairs of MZ twins, geneticistsmeasuredIevels of two epigenetic modifications, DNA methylation and histone acetylation.Two-thirds of the twin pairs had essentiallyidentical profiles, but significant differences were observedin the remaining third. These differenceswere broadly correlatedwith the age of the twins, with the amount of time spent apart and the differences in their medical histories,suggestinga cumulativeeffect on DNA modification over time. It also suggestsa possible causal link bet',r'eenepigenetic modification and susceptibility to disease. Very late division occurring more than 14days after conception can result in conjoined twins. This occurs in about 1 in 100000 pregnancies,or approximately I in 400 MZ twin births. Conioined twins are sometimesreferred to as Siamese,in memory of Chang and Eng, who were born in 1811 inThailand, then known as Siam, joined at the upper abdomen. Chang and Eng made a successfulliving out of showing themselves at traveling shows in the USA, where they settledand married. They both managedto havelargenumbers of children despiteremaining conioined until they died within a few hours ofeach other at the ageof6l years. The sex ratio for conioined twins is markedly distorted, with about 75olobeing female.The later the twinning event, the more distorted the sex ratio in favor of females, and X-inactivation

twins Summaryof differences betweenmonozygotic anddizygotic Monozygotic

Dizygotic

0rigin

Single eggfertrtized

bya singlesperm Twoeggs.eachfertilized

In c r d e n c e

1in 300pregnancies

to 1in 500preqnancres Varies from1in'1OO

Proportion ofgenesIncommon

100%

50%(onaverage)

F e t a lm e m b r a n e s

7 0 %m o n o c h o r i o n iacn d d i a m n i o t i c3:0 %d i c h o r i o n iac n d d i a m n r o t i cr :a r e [ ym o n o c h o r i o n iacn d m o n o a m n i o t i c

A t w a y sd r c h o r i o n iacn d d i a m n i o t i c

101

6

DEVELOPMENTAL GENETICS

studies suggestthat MZ twinning occurs around the time of Xinactivation,a phenomenon limited to femalezygotes,of course.

DIZYGOTICTWINS DZ twins result from the fertilization of two ova by two sperm and are no more closely related genetically than brothers and sisters,as they share, on average,50o/oof the same genes from each parent. Hence they are sometimesreferred to asfraternal twins. DZ twins are dichorionic and diamniotic, although they can have a single fused placentaif implantation occurs at closely adjacentsites.The incidence variesfrom approximately 1 in 100 deliveriesin Afro-Caribbean populations to I in 500 deliveriesin Asia and Japan.In western European caucasiansthe incidence is approximately I in 120 deliveriesand has been observedto fall with both urbanization and starvation,but increasesin relation to the amount of seasonallight (e.g. in northern Scandanavia during the summer). Factors that convey an increasedrisk for DZ twinning are increasedmaternalage,a positive family history (due to a familial increasein FSH levels)and the use of ovulationinducing drugs such as clomiphene

D E T ER M I NA T ION OFZ Y GOS IT Y Zygosity used to be establishedby study of the placenta and membranes and also by analysisof polymorphic systems such as the blood groups, the HLA antigens and other biochemical markers.Now it is determined most reliably by the use of highly polymorphic molecular (DNA) markers (p. 69).

FURTHER READING

102

Dreler S D, Zhou G, Lee B 1998The long and the short of it: developmental geneticsof the skeletaldysplasiasClin Genet 54: 464473 A short retiep oJ'deulopmentalgenesknopn to causeabnormulsbeletal dexelopment HallJ G 2003Tx'inning Lancet 362: 735-713 HammerschmidtM, Brook A, McMahon A P 1997The world accordingto hedgehog.tends Genet 13: 1,1-20 A comprehensite atcountofthe role oJ the hedgehog geneJitmily in early tertebratedeu lopment. Kleinjan DJ, van HeyningenV 1998Positioneffectin human genetic d i s e a s eH u m M o l G e n e t7 : 1 6 1 I 1 6 1 8 An outlineof the tarious theoriesantl mechunisms that hore beenproposedto positionaleffettsin tlexelolmentalgeneexlression actountJbr obserced Kornak U, Mundlos S 2003Geneticdisordersof the skeleton:a developmental approachAm J Hum Genet 73: 447474 An u!-t7-date summaryof wrent hnowledge. LacombeD 1999Transcriptionfactorsin dysmorphologyClin Genet 55: 137-t43 As the title intlitates,a deuription ofthe role oftranscriptionregulatorytgenesin causingmultiple congenitalabnormaltt.ys.yndromes Lindor N M, Ney J A, Gaffey T A er al 1992A genetic revien of complete and partial hydatidiform moles and nonmolar triploidy. Mayo Clin Proc 67:791-799 A detailedreaiep of the mechanisms that can leadto theformatitn uf hydatidiformmoles Lyon M F 1961Gene actionin the X chromosomeof the mouse(,Mzs muvulusL\ Nature 190:372-373 The originalproposalofX-inattiration ury shortand eusilyunderstood

M, Lyonnet S 1999Geneticsof Manouvrier-HanuS, Holder-Espinasse limb anomaliesin humans tends Genet 15:.409417 A detailedand pell illustratedaccounlofrertebrate limb deaelopment receptormutationsin Muenke M, SchellU 1995Fibroblast-growth-factor human skeletaldisorderstends Genet l1: 308 313 reztiewoJthefunctionsof theJibrobla*growthfactorsand their receptors A concise Muragaki Y, Mundlos S, Upton J, Olsen B R 1996Altered growth and branching patterns in synpolydactylycausedby mutations in HOXDl3 Science272: 548 551 The long-awaitedJirst rellrt 0f a humanmalformationcauserlby u mutationin a HOX gene. Sagal ThkedaH 2001The making of the somite:moleculareventsin vertebratesegmentationNature Rev Genet 2: 835 844 An etcellentreuien of somitedeaelopment. Tickle C (ed ) 2003 Patterning in vertebratedevelopment Oxford University Press,Oxford A detailetl,muln-author collectionhandlingt:ery early deaelopment, from mainly moletu lar p erspectia es.

ELEMENTS Q Seueral developmental gene families first identified in Drosophila and mice also play important roles in human morphogenesis.These include seBment polarity genes, homeobox-containing genes (HOX) and paired-boxcontaining genes (PAX). Many of these genes act as transcription factors that regulate sequentialdevelopmental processes.Others are important in cell signaling. It has recently been shown that several human malformations and multiple malformation syndromes are caused by mutationsin thesegenes. @ Fo. no.-"I developmentahaploid chromosomesetmust be inherited from eachparent.A paternaldiploid complement resultsin a completehydatidiform mole if there is no maternal contribution, and in triploidy with a partial hydatidiform mole if there is a haploid maternal contribution. @ A testis-determining factor on the Y chromosome, known as SRY, stimulates the undifferentiated gonads to developinto testes.This, in turn, setsoff a seriesof events leading to male development. In the absenceof SRY expressionthe human embryo developsinto a female. @ In femalesone of the X chromosomesis inactivated in eachcell in early embryogenesis. This can be either the maternally derived or the paternally derived X chromosome. Thereafter, in all daughter cells the sameX chromosome is inactivated.This process,known as lyonization, explains the presenceofthe Barr body in femalenuclei and achieves dosagecompensationof X-chromosome gene products in males and females. can be monozygotic (identical) or dizygotrc @ f*i"r (fraternal). Monozygotic twins originate from a single zygote that divides into two during the first 2weeks after conception. Monozygotic twins are geneticallyidentical. Dizygotic twins originate from two separatezygotes and are no more geneticallyalike than brothers and sisters.

CHAPTER

Patternsof inheritance

'That the fundamental aspects of heredity should have turned out to be so extraordinarily simple supports us in the hope that nature may,after all, be entirely approachable.' Thornas Morgan (1919)

who is at risk of Huntington diseaseis actually a sra2-childand not a biological relative.

INHERITANCE MENDELIAN FAMILY STUDIES If we wish to investigatewhether a particular trait or disorder in humans is genetic and hereditary, we usually have to rely either on observationof the way in which it is transmitted from one generation to another, or on study of its frequency among relatives. An important reason for studying the pattern of inheritance of disorders within families is to enable advice to be given to members of a family regarding the likelihood of their developing it or passing it on to their children, i.e. genetic counseling (Ch 17).Thking a family history can, in itself, provide a diagnosis. For example,a child could come to the attention of a doctor with a fracture after a seemingly trivial injury. A family history of relatives with a similar tendency to fracture and blue sclerae would suggestthe diagnosisof osteogenesisimperfecta. In the absenceof a positive family history, other diagnoseswould have to be considered.

GN DT E R M I N O L O G Y P E D I G R EDER A W I N A A family tree is a shorthand system of recording the pertinent information about a family. It usually begins with the person through whom the family came to the attention of the investigator. This person is referred to as the index case,proband or prrllsitus, or, if female, the proposita. The position of the proband in the family tree is indicated by an arrow Information about the health of the rest of the family is obtained by asking direct questions about brothers, sisters, parents, and maternal and paternal relatives, with the relevant information about the sex of the individual, affection status and relationship to other individuals being carefullyrecordedin the pedigreechart (Fig. 7.1).Attention to detail can be crucial becausepatients do not always appreciate the important difference between siblings and /zafsiblings, or rnight overlook the fact, for example, that the child of a brother

More than 16000 traits or disorders in humans exhibit single geneunifactorial or mendelianinheritance.However, characteristics such as height, and many common familial disorders, such as diabetesor hypertension,do not usually follow a simple pattern of mendelian inheritance(Ch. 9). A trait or disorder that is determined by a gene on an autosome is said to show a.utlslmal inheritance,whereas a trait or disorder determined by a gene on one of the sex chromosomes is said to show sex - lin k ed in heritunce.

A U T O S O M ADLO M I N A NITN H E R I T A N C E An autosomal dominant trait is one that manifests in the heterozygous state, that is, in a person possessingboth an abnormal or mutant allele and the normal allele. It is often possible to trace a dominantly inherited trait or disorder through many generationsof a family (Fig. 7 .2). In South Africa the vast majority of casesof porphyria variegatacan be traced back to one couple in the late seventeenth century. This is a metabolic disorder characterized by skin blistering as a result of increased sensitivity to sunlight (Fig. 7.3), and the excretion of urine 'port wine' colored on standing as a result of the that becomes presence of porphyrins (p. 172). This pattern of inheritance is sometimesreferred to as'vertical'transmission and is confirmed when male-male (i.e. father to son) transmission is observed.

Geneticrisks Each gametefrom an individual with a dominanttrait or disorderwill contain either the normal allele or the mutant allele.If we represent the dominant mutant allele as W and the recessivenormal allele 'a', then the various possible combinations of the gametescan as be representedin a Punnett's square (Fig. 7.4). Any child born to a person affected with a dominant trait or disorder has a I in 2 (50o/o)chance of inheriting it and being similarly affected.

103

7

ERNS oFTNHERTTANcE Individuals

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PATTERNSOFINHERITANCE

7

Affectedparent(Aa)

f Q nrecteo Fis.7.2

E o

Family treeofan autosomaI dominant traitNotethepresence of -to-matetransmission maLe

Fis.7.4 for an Punnett'ssquareshowtngpossibtegametecombinations autosomaIdominantalteLe

gene, for example the LMNA gene (which encodeslamin A/C) and the X-linked filamin A gene.Mutations in LMNA may cause Emery-Dreifuss muscular dystrophy, a form of limb girdle muscular dystrophy, a form of Charcot-Marie- Tooth disease (p. 286), dilated cardiomyopathy(p.295), Dunnigan-type familial partial lipodystrophy (Fig. 7.6), mandibuloacral dysplasia,and the very rare condition that has always been a great curiosity - Hutchinson-Gilford progeria.These are due to heterozygous mutations, with the exception of the Charcot-Marie-Tooth diseaseand mandibuloacral dysplasia,which are recessive,and therefore homozygous for LMNA mutations. Sometimes an

F i 9 .7 . 3 Blistenng skrnlesions onthehandinporphyria variegata

individual with a mutation is entirely normal. Mutations in the filamin A gene have recently been implicated in the distinct, though overlapping,Xlinked dominant dysmorphic conditions oto-palato-digital syndrome, Melnick-Needles syndrome and frontometaphysealdysplasia.In addition, however,it could not havebeen foreseenthat a form of X-linked dominant epilepsy in \vomen,called periventricular nodular heterotopia,is also due to mutations in this gene.

Pleiotropy Autosomal dominant traits may involve only one organ or part of the body, for example the eye in congenital cataracts.

Variableexpressivity

It is common. however. for autosomal dominant disorders to manifest in different systems of the body in a variety of wa-vs. This is pleiotropy - a single gene that may give rise to two or

The clinical featuresin autosomaldominant disorderscan show striking variation from person to person, even in the same family. This difference between individuals is referred to as ztariable erpressiuity.Inautosomaldominant polycystickidney disease,for

more apparently unrelated effects.In tuberous sclerosisaffected individuals can present with a range of problems including

example, some affected individuals develop renal failure in early adulthood whereasothers have just a few renal cysts that do not

learning difficulties, epilepsy, a facial rash known as adenoma sebaceum(histologicallycomposedof blood vesselsand fibrous

affect renal function significantly.

tissueknown as angiokeratoma)or subungualfibromas (Fig. 7 5); some affected individuals have all features,whereasothers may havealmost none Recently,our conceptualunderstandingofthe term pleiotrolry has been challenged by the remarkably diverse

Reducedpenetrance

svndromesthat can result from different mutations in the same

In some individuals heterozygousfor genemutations giving rise to certain autosomaldominant disordersthere may be no abnormal clinical features,representing so-called reducetlpenetranceor whar

105

7

PATTERNS OF]NHERITANCE

Fis.7.5 aa d e n o msae b a c e u m T h ef a c i arla s h( A )o fa n g i o k e r a t o(m r n)a m a l ew t h t u b e r o ussc t e r o s a i sn, da t y p r c as lu b u n g u a t f i b r oom fa t h en a i b t e d( B )

is commonlv referred to in la1'tcrms as 'skipping a generation'. Rcduced penetranceis thought to be the result of the modif-ving effects of other genes,as rvell as interaction of the gene with environmental factors An individual who has no fcatures of a disorder despite bcing heteroz-vgousfor a particular gene mutation is said to representnon-1)enetrante Reduccd penetranceand variable expressivifi',together with the pleiotropic effectsof a mutant allele,all need to be taken into account uhcn providing geneticcounselingto individuals at risk of autosomaldominantlv inherited disorders.

New mutations In autosomaldominant disordersan affectedpersonusuallyhasan affectedparent. Holever, this is not ahvavsthe caseand it is not unusualfor a trait to appearin an individual r,hen there is no familv historv of the disorder.A striking example is achondroplasia,a form of short-limbeddr,varfism (p. 9l), in u,hichthe parentsusually

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106

have normal stature The sudden unexpected appearanceof a conditionarisingasa resultof amistakeoccurringin the transmission of a geneis calledanewmtfintiaz.The dominantmode of inheritance of achondroplasiacould be confirmed onl1,-b1,the observation that the offspring of pcrsons with achondroplasiahad a 50o/o chanceof having achondroplasiaor being of normal stature.

PATTERNSOFINHERITANCE

7

In less striking examples,other possible explanationsfor the 'sudden' appearanceof a disorder must be considered.One of the parents might be heterozl'gousfor the mutant allele but so mildl-v affected that it was not previously detected, i.e non-penetrance.The possibility of variable expression also needs to be considered, as well as the family relationships not being as stated,i.e. non-paternie (p. 333) (and occasionall,v nonma.terntt.y) New dominant mutations, in certain instances,have been associatedwith an increasedage of the father. It is believedthat this is becauseof the large number of mitotic divisions that male gametestem cells undergo during a man's reproductive lifetime

(p.aa).

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Fig.7.7 trait recessive Fami[y treeofanautosomaI

Co-dominance

Consanguinity

Co-dominanceis the term used for two allelic traits that are both expressedin the heterozygousstate.In persons with blood group AB it is possibleto demonstrateboth A and B blood group

Enquiry into the family history of individuals affectedwith rare rccessivetraits or disorders might reveal that their parents are rarer a recessivetrait or disorder, related,i.e. consunguineous.The among the parentsof of consanguinity greater the frequency the 'serious' librosis, the commonest In cystic affected individuals. autosomal recessivedisorder in persons of western European origin (p. 291), the frequency of parental consanguinity is only slightly greater than that seen in the general population. By contrast, in alkaptonuria, one of the original inborn errors of metabolism (p. 162), which is an exceedingly rare recessive disorder,Batesonand Garrod, in their original description of the disorder,observedthat one-quarter or more of the parents were first cousins.They reasonedthat rare allelesfor disorders such 'meet up' in the offspring of as alkaptonuria are more likely to cousinsthan in the offspring of parentswho are unrelated.

substanceson the red blood cells,so the A and B blood groups are therefore co-dominant (p. 193)

Homozygosity for autosomaIdominanttraits The rarity' of most autosomal dominant disorders and diseases means that they- usually occur onll'- in the heterozJrgous state.There are, however,a few reports of children born to couples where both parents are heterozygousfor a dominantly inherited disorder. Offspring of such couples are, therefore, a t r i s k o f b e i n g h o m o z v g o u s I n s o m e i n s t a n c e sa f f e c t e d individuals appear either to be more severely affected, as has b e e n r e p o r t e d w i t h a c h o n d r o p l a s i a ,o r t o h a v e a n e a r l i e r age of onset, as in familial hypercholesterolemia (p. 167). The heterozygote with a phenotype intermediate between the homozygotes for the normal and mutant alleles is consistent with a haoloinsufficiencv loss-of-function mutation (p. 26). Conversely, with other dominantly inherited disorders, homozygous individuals are not more severely affected than heterozygotes,e.g. Huntington disease(p. 282) and myotonic dystrophy (p. 284).

Geneticrisks 'A' and the If we represent the normal dominant allele as 'a', then each parental gametecarries recessivemutant allele as either the mutant or the normal allele (Fig. 7.8). The various possiblecombinationsof gametesmean that the offspring of two heterozvgoteshave a 1 in 4 (25o/o)chance of being homozygous affected,a 1 in 2 (50o/o)chanceofbeing heterozygousunaffected, and a I in 4 (25o/o)chanceof being homozygousunaffected.

AU T O SO M ARE L C E S S IVIN E H E R IT A NCE

Pseudodominance

Recessivetraits and disordersare manifest only when the mutant allele is present in a double dose,i.e. homozygosity.Individuals

If an individual who is homozygous for an autosomalrecessive disorder has children with a carrier of the same disorder, their offspring have a 1 in 2 (50o/o)chance of being affected. Such a (Fig. 7.9). pedigreeis said to exhibitpseudodominance

heterozygous for such mutant alleles show no features of the disorder and are perfectly healthy; they are describedas carriers The family tree for recessivetraits (Fig 7 7) differs markedly from that seenin autosomaldominant traits. It is not possibleto trace an autosomalrecessivetrait or disorder through the family, as all the affected individuals in a family are usually in a single sibship(i e. brothers and sisters).This is sometimesreferred to as 'horizontal' transmission - an inappropriate and misleading term.

Locusheterogeneity A disorder inherited in the samemanner can be due to mutations in more than one gene,or what is known as locusheter0geneity.For example,it is recognizedthat sensorineuralhearing impairment/ deafnessmost commonlv showsautosomalrecessiveinheritance

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PATTERNSOFINHERITANCE

Normal parent heterozygous

Disorders with the same phenotype due to different genetic loci are known as genocopies,whereasthe samephenotype being the result of environmental causesis known as a phenlclly.

Mutationalheterogeneity Heterogeneity can also occur at the allelic level. In the majority of single-genedisorders, e.g. B-thalassemia,a large number of different mutations has been identified as being responsible (p. 154).There are individuals who havetwo different mutations at the same locus and are known as compoundheterozltgotes, constituting what is known as allelic or mutatirn&l heterogeneitjt. Most individualsaffectedwith an autosomalrecessivedisorderare probably compound heterozygotesrather than true homozygotes, unless their parents are related, when they are likely to be

Fis.7.8 P u n n e t tssq u a r seh o w i npgo s s i b g l ea m e t icco m b i n a t i of no sr heterozygous carnerparents ofan autosomaI recessive attete

homozygousfor the samemutation by descent,having inherited the same mutation from a common ancestor.

S E X .L I N K E DI N H E R I T A N C E Sex-linkedinheritancerefers to the pattern of inheritanceshown by genes that are located on either of the sex chromosomes. Genes carried on the X chromosome are referred to as being X-linked, and those carried on the Y chromosomeare referred to as exhibiting Y-linked or holandric inheritance.

X-linkedrecessiveinheritance

n0m0zy90us Heterozygous

Fis.7.9 A pedigreewith a woman (l) homozygousfor an autosomal Tecessrve disorderwhose husbandis heterozygous for the same disorder. They havea homozygousaffecteddaughterso thatthe pedigree s h o w sp s e u d o d o m i n ainnth e r i t a n c e

108

Deaf persons, by virtue of their schooling and involvement in the deaf community, often chooseto have children with another deaf person. It would be expectedthat, if two deaf personswere homozygous for the same recessivegene, all of their children would be similarly affected Families have been described in which all the children born to parents deaf due to autosomal recessivegenes have had perfectly normal hearing and are what is known as doubleheteroz.yg0tes.The explanation for this must be that the parents were homozygous for mutant alleles at different loci, i.e that a number of different genescan cause autosomalrecessivesensorineuraldeafness.In fact, over the past l0-l 5 years,20 genesand a further I 5 loci havebeen shown to be involved. A very similar story applies to the autosomalrecessive condition retinitis pigmentosa,and there are now six distinct loci for primary autosomalrecessivemicrocephaly.

An X-linked recessivetrait is one determined by a gene carried on the X chromosome and usually manifests only in males. A male with a mutant alleleon his singleX chromosomeis said to be hemizygous for that allele.Diseasesinherited in an X-linked manner are transmitted by healthy heterozygousfemale carriers to affected males, as well as by affected males to their obligate carrier daughters,with a consequentrisk to male grandchildren through these daughters(Fig. 7.10). This type of pedigree is sometimessaid to show'diagonal' or a'knight's move'pattern of transmlsslon.

tl ill

nrected Q Carrier f

Fis.7.10 Fami[ytree of an X-tinkedrecessive trartin whichaffectedmales reproduce

PATTERNS OFINHERITANCE

The mode of inheritancewhereby only malesare affectedby a diseasethat is transmitted by normal femaleswas appreciatedby theJews nearly 2000yearsago.They excusedfrom circumcision

7

C a n i e rf e m a l e ( X hX )

the sons of all the sisters of a mother who had sons with the 'bleeding disease', in other words, hemophilia (p. 299) The sons of the father's siblings were not excused Qreen Victoria was a carrier of hemophilia, and her carrier daughters, who were perfectly healthy,introduced the geneinto the Russianand Spanish royal families. Fortunately for the British royal family, Qreen Victoria's son, Edward VII, did not inherit the gene and so could not transmit it to his descendants.

a

u E G

Geneticrisks A male transmits his X chromosome to each of his daughters and his Y chromosome to each of his sons. If a male affected

Fis.7.12

with hemophilia has children with a normal female, then all of his daughters willbe obligatecarriersbut none of his sons will be affected (Fig 7.1l). A male cannot transmit an X-linked trait to

gametecombinations for A Punnett's squareshowingpossibte disorder recessive theoffspring ofa femalecarrierofanX-[inked (X' rep.esenrs gene) foranX-tinked a mutation

his son, with the very rare exceptionof uniparentalheterodisomy

( p .l l s ) . For a carrier female of an X-linked recessive disorder having children with a normal male, each son has a I in 2 (50o/o)chance of being affectedand each daughter has a I in 2 (50o/o)chanceof being a carrier(Fig. 7.12).

or early20s(Fig 7.13).As affectedboysdo not usuallysurviveto is transmittedalmostentirelyby healthy reproduce,the disease (Fig 7.14). femalecarriers

Some X-linked disorders are not compatible with survival to reproductive age and are not, therefore, transmitted by affectedmales.Duchenne muscular dystrophy is the commonest muscular dystrophy and is a severedisease(p.297). The first signs are a waddling gait, difficulty in climbing stairs unaided,

Voriableexpressionin heterozygousfemales

and a tendency to fall over easily.By about the age of lOyears affected boys usually need to use a wheelchair. The muscle weakness progresses gradually and affected males ultimately become confined to bed and often die in their late teenase vears

Affectedmale (XttY)

In humans, several X-linked disorders are known in which heterozygous females have a mosaic phenotype with a mixture of featuresof the normal and mutant alleles.In X-linked ocular albinism the iris and ocular fundus of affected males lack pigment. Careful examination of the ocular fundus in femalesheterozygous for ocular albinism revealsa mosaicpattern of pigmentation (see Fig. 6.21, p. 99). This mosaic pattern of involvement can be explainedby the random processofX-inactivation (p. 99). In the pigmented areasthe normal geneis on the activeX chromosome, whereas in the depigmented areas the mutant allele is on the activeX chromosome.

disorders FemolesoffectedwithX-linked recessive Occasionally a woman might manifest features of an X-linked recessive trait. There are several explanations for how this can happen. Homozygosity for X-linked recessive disorders A common Xlinked recessivetrait is red-green color blindness - the inability to distinguish between the colors red and green. About 8o/oof males are red-green color blind and, although it is unusual, becauseof the high frequency of this allele in the population about 1 in 150 women are red-green colour-blind by

Fis.7.11 possibte gametecombinations Punnetts squareshowing for theoffspring ofa maleaffected byanX-Linked recessive disorder (Xnrepresents gene) a mutatron foranX-tinked

virtue of both parents having the allele on the X chromosome. Therefore, a female can be affected with an X-linked recessive disorder as a result of homozygosity for an X-linked allele, although the rarity of most X-linked conditions means that the

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PATTERNSOFINHERITANCE

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IV Carrier

Fis.7.11 Fami[ytree of DuchennemuscuLar dystrophywiih the drsorder berngtransmittedby carrierfemalesand affectng mates,who do not surviveto transmitthe drsorder.

This has been reported in a number of X-linked disorders, including Duchenne muscular dystrophy and hemophilia A (p 300) In addition, there are reports of several X-linked disorders in which there are severalmanifesting carriers in the same family, consistent rvith the coincidental inheritance of an abnormalit-vof X-inactivation (p. 192). Nurnerical X-chrornosorne abnorrnalities A femalecould manifest an X-linked recessivedisorder by being a carrier of an X-linked reccssivcmutation and having only a single X chromosome, i.e. Turner syndrome (p. 272). Women rvith Turner syndrome and hemophilia A or Duchenne muscular dystrophy havebecn reported.

Fis.7.13 Boy with DuchennemuscuLar dystrophyr notethe enlargedcatves a n dw a s t i n go f t h e t h i g hm u s c t e s

phenomenon is uncommon. A female could also be homozvgous if her father was affected and her mother was normal, but a ne$r mutation occurred on the X chromosome transmitted to the daughter; or alternatively if her mother rvas a carrier and her father was normal but a new mutation occurred on the X chromosome he transmitted to his dauehter - but these scenarlosare rare Skewed X-inactivation The process of X-inactivation usuallv occurs randomly, there being an equal chance of either of the trvo X chromosomesrn a heterozygousfemale being inactivated in an-vone cell. After X-inactivation in embrvogenesis,therefore, in roughly half the cells one of the X chromosomes is active, rvhilst in the other half it is the other X chromosomethat is activc. Sometimesthis processis not random, allowing for the possibilitl'that the active

110

X chromosomc in most of the cells of a heterozygous female carrier is the one bearing the mutant allelc If this happens, a carrier female would exhibit some of the symptoms and signs of the diseaseand be a so-calledmaniJbsting heteroz.ygote or turrier.

X-autosome translocations Femaleswith a translocationinvolving one of the X chromosomes and an autosome can be affected rvith an X-linked recessive disorder. If the breakpoint of the translocation disrupts a gene on the X chromosome, then a female can be affected. This is becausethe X chromosome involved in the translocation survives preferentially-so as to maintain functional disomy of the autosomal genes (Fig. 7.15). The observationof females affected with Duchcnne muscular dystrophy with X-autosome translocationsinvolving the sameregion of the short arm of the X chromosomehelped to map the Duchenne musculardystrophy gene (p. 298) This type of observation has been vital in the positional cloning of a number of genesin humans (p. 74).

X-tinkeddominantinheritance Although uncommon, there are disorders that are manifest in the heteroz-vgousfemale as well as in the male rvho has the mutant allele on his single X chromosome This is known as X-linked dominant inheritance (Fig 7 16). X-linked dominant inheritancesuperficiallyresemblesthat of an autosomaldominant trait becauseboth the daughters and sons of an affected female have a 1 in 2 (507o) chanceof being affected.There is, however, an important difference. With an X-linked dominant rrait an affectedmale transmits the trait to all his daushters but to none

PATTERNSOFINHERITANCE

7

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of Charcot-Marie-Tooth disease(hereditary motor and sensory neuropathy)is another example. A mosaic pattern of involvement can be demonstrated in females heterozygous for some Xlinked dominant disorders. An example is the mosaic pattern of abnormal pigmentation

V\

AqJ

of the skin that follows developmental lines seen in females heterozygousfor the X-linked dominant disorder incontinentia pigmenti (Fig. 7.17).This is alsoan exampleof a disorder that is usually lethal for male embryos that inherit the mutated allele. Others include the neurological conditions Rett syndrome and

(N A C -T-

periventricular nodular heterotopia.

I

Y-tinkedinheritance A

ABI^B Normal 6 Xchromosome " N rnactivated

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YY C e l l ss u r v r vw erth b r e a k p o i nat t X p 2 1l e a d i n g t o d e v e l o p m e notf D M D

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Fis.7.15 Generation of an X-autosometranslocation with breakpornt in a femaleand how this resultsin the devetopment of Duchenne muscutardystrophy.

of his sons. Therefore, in families with an X-linked dominant disorder there is an excessof affected femalesand direct maleto-male transmissioncannot occur. An example of an X-linked dominant trait is vitamin Dresistant rickets. Rickets can be due to a dietary deficiency of vitamin D, but in vitamin D-resistant rickets the disorder occurs even when there is an adequatedietary intake of vitamin D. In the X-linked dominant form of vitamin D-resistant rickets,both males and females are affected, although the females usually have less severeskeletalchansesthan the males.The X-linked form

Y-linketl or holandrit inheritance implies that only males are affected. An affected male transmits Y-linked traits to all of his sons but to none of his daughters. In the past it has been suggestedthat bizarre-sounding conditions such as porcupine skin, hairy ears and webbed toes are Y-linked traits. With the possible exception of hairy ears, these claims of holandric inheritance have not stood up to more careful study. Evidence clearly indicates, however, that the H-Y histocompatibility antigen (p. 189) and genes involved in spermatogenesis are carried on the Y chromosome and, therefore, manifest holandric inheritance The latter, if deleted, lead to infertility due to azoospermia(absenceof the sperm in semen) in males. The recent advent of techniques of assistedreproduction, particularly the technique of intracytoplasmic sperm iniection (ICSI), meansthat, if a pregnancywith a male conceptusresults after the use of this technique, the child will also necessarily be infertile.

Partialsex-linkage Partial sex-linkagehas been used in the past to account for certain disordersthat appearto exhibit autosomaldominant inheritance in some families and X-linked inheritancein others.This is now known to be likely to be becauseof genescarried on that portion of the X chromosomesharinghomology with the Y chromosome, and which escapesX-inactivation. During meiosis, pairing

111

7

PATTERNSOFINHERITANCE

effect of male hormones. Gout, for example, is very rare in women beforethe menopausebut the frequency increasesin later life. Baldnessdoes not occur in males who have been castrated. In hemochromatosis(p. 230), the most common autosomal recessivedisorder in Western society, homozygous females are much less likely than homozygous males to develop iron overload and associatedsymptoms; the explanation usually given is that women have a form of natural blood loss through menstruanon.

Sex limitation Sexlimitation refers to the appearanceof certain featuresonly in individuals of a particular sex. Examples include virilization of female infants affected with the autosomal recessiveendocrine disorder,congenital adrenalhyperplasia(p. 165).

ESTABLISHIN TH G EM O D EO F INHERITANC OEF AG E N E T ID CI S O R D E R

Fis.7.17 M o s a ipca t t e ronfs k i np i g m e n t a t iionna f e m a [ w e i t ht h eX - t i n k e d d o m i n a ndti s o r d ei n r ,c o n t i n e npt i g t ih ep a t i e nhta sa a m e nT m u t a t i ornna g e n eo no n eo fh e r Xc h r o m o s o m et hse: p i q m e n t e d a r e a si n d i c a t ies s u e i nw h i c ht h en o r m a l Xc h r o m o s o mnea so e e n inactivated Thisdeve[opmental pattern foftows Blaschko's lines (seeCh 18,p270)

In experimental animals it is possible to arrange specific types of mating to establishthe mode of inheritance of a trait or disorder. In humans, when a disorder is newly recognized, the geneticist approaches the problem indirectly by fitting likely models of inheritance to the observedoutcome in the offspring. Certain features are necessar)to support a particular mode of inheritance.Formally establishingthe mode of inheritanceis not usually possiblewith a single family and normally requires study of a number of families (Box 7 l).

B o x 7 . 1 F e a t u r eLsh ast u p p o rtth es i n g t e - g e noer m e n d e l i apna t t e r nos f i n h e r i t a n c e occurs between the homologous distal parts of the short arms of the X and Y chromosomes,the so-calledpseudoautosomal region.hs a result of a cross-over,a gene could be transferred from the X to the Y chromosome, or vice r.ersa,allowing the possibility of male-to-male transmission. The latter instances would be consistent with autosomal dominant inheritance A rare skeletal dysplasia,Leri Weil dyschondrosteosis,in which affected individuals have short stature and a characteristicwrist deformity (Madelung deformity), has been reported to show both autosomaldominant and X-linked inheritance.The disorder has been shown to be due to deletions ol; or mutations in, the short stature homeobox (S11o)0 gene, which is located in the pseudoautosomalregion.

5ex influence

112

Some autosomal traits are expressed more frequently in one sex than in another so-calledser influence.Gout and presenile baldness are examples of sex-influenced autosomal dominant traits, males being predominantly affected in both cases.The influence of sex in these two examplesis probably through the

Autosomatdominant Matesandfemales affected in equalproportrons generaLions Affected rrdrvduatsin rnrr.tipte Transmiss onby ndividuals of bothsexes, ie mateto mate,femaleto female, ma[eto female, andfemate to mate Autosomalrecessives Vatesandfemates atfected in equaproportiors Alfecteo indrvidrats generaLior rsuat.y,ron.ya sir-gte Parents canberetated, i e consanguineous X-tinked recessive Ontymalesusuatty affected Transmitted throughunaffected femates Matescannottransmitthe disorderto theirsons, i e nomate-ro-mare transmsston X-tinkeddominant l\4ates andlerrales affected butofleranexcess offerraLes lessseve'e.v Females affected rhanmates Affected matescantransmit thedrsorder tother daughters butnotto S ON S

Y-tinkedinheritance Affected malesonty Affected matesmusttransmit itto theirsons

PATTERNS OFINHERITANCE

AutosomaIdominantinheritance In order to determine whether a trait or disorder is inherited in an autosomal dominant manner, there are three specific features that need to be observed. Firstly, it should affect both males and females in equal proportions. Secondly, it is transmitted from one generation to the next. Thirdll; all forms of transmission between the sexes are observed, i e male to male, female to female, male to female and female to male. Male-to-male transmission excludes the possibility of the gene being on the X chromosome.In the caseof sporadicallyoccurring disorders, increased paternal age may suggest a new autosomal dominant mutatlon.

AutosomaIrecessiveinheritance There are three featuresthat suggestthe possibility of autosomal recessive inheritance. Firstly, the disorder affects males and females in equal proportions. Secondly, it usually affects only individuals in one generation in a single sibship (i e brothers and sisters) and does not occur in previous and subsequent generations.Thirdly consanguinity in the parents provides further support for autosomalrecessiveinheritance.

X- linkedrecessivei nheritance There are three main features necessaryto establish X-linked recessiveinheritance Firstly, the trait or disorder should affect males almost exclusively.Secondly,X-linked recessivedisorders are transmitted through unaffectedcarrier femalesto their sons Affected males, if they survive to reproduce, can have affected grandsons through their daughters who are obligate carriers Thirdly, male-to-male transmissionis not observed,i.e. affected males cannot transmit the disorder to their sons.

7

ANDCOMPLEX ALLELES MULTIPLE TRAITS So far, each of the traits we have considered has involved only two alleles, the normal and the mutant. However, some traits and diseasesare neither monogenicnor polygenic. Some genes have more than two allelic forms, i.e. multiple alleles Multiple allelesare the result of a normal genehaving mutated to produce various different alleles, some of which can be dominant and others recessiveto the normal allele In the case of the ABO blood group system (p. 193), there are at least four alleles (A1, A,2,B and O). An individual can possessany two of these alleles, which may be the same or different (AO, A2B, OQ and so on). Alleles are carried on homologous chromosomesand therefore a person transmits only one allele for a certain trait to any particular offspring. For example, a person with the genotype AB will transmit to any particular offspring either the A allele or the B allele,but never both or neither (Thble 7.1). This relatesonly to geneslocated on the autosomesand does not apply to alleles on the X chromosome;in this instancea woman would have two alleles,either of which could be transmitted to offspring, whereas a man only hasone alleleto transmit. The dramatic advancesin genome scanning using multiple DNA probeshas made it possibleto begin investigatingso-called complextraits,i.e.conditions that are usually much more common than mendelian disorders and likely to be due to the interaction of more than one gene. The effects may be additive, one may be rate limiting over the action of another, or one may enhance or multiply the effect of another; this is considered in more detail in Chapter 15. The possibility of a small number of gene loci being implicated in some disorders has given rise to the concept of oligogenicinheritance, examples of which include the followine.

X-linkeddominantinheritance There are three features necessaryto establishXlinked dominant inheritance. Firstly, males and females are affected but affected females are more frequent than affected males. Secondly, females are usually less severely affected than males. Thirdly, although affected females can transmit the disorder to both male and female offspring, affected males can transmit the disorder only to their daughters (except in partial sex-linkage;p. l1l), all of whom will be affected. In the case of X-linked dominant disorders that are lethal in male embryos, only females will be affected and families may show an excessof females over males as well as a number of miscarriaeesthat are the affected male pregnancles

Y-linkedinheritance

phenotypes genotypes, andgametes Tabte7.1 Possibte A',Ar,B and0 at theAB0 tocus formedfromthefouratleles Genotype

Phenotype

Gametes

A,A,

A1

A1

AzA,

A2

A2

BB

B

B

00

0

0

ArAz

Ar

A1orA2

A,B

A,B

,A1OT B

ArO

Ar

41or0

AD

AzB

A2orB

Az0

A2

A2or0

BO

B

Bor0

There are two featuresnecessaryto establishaY-linked pattern of inheritance. Firstly, it affects only males. Secondly, affected males must transmit the disorder to their sons, e.g. male infertility by ICSI (p. 327).

113

7

PATTERNS OFINHERITANCE

D i g e n i ci n h e r i t a n c e This refers to the situation where a disordcr has been shorvn to be due to the additivc effects of heterozl,gousmutations at trvo different gene loci, a concept referred to as digenic inheritante.This is seen in certain transgcnicmice. Mice that are homozvgotesfor rr or Dlll manifest abnormal phenotvpes, rvhereastheir respective hetcrozygotes are normal Howcvcr, mice that are tloubleheteroz.ygotes for rz' (rib-vertebrac) and Dlll (Delta-like-l) shor,vvertebral defects. In humans one form of retinitis pigmentosa,a disorder of progressivevisual impairment, is causedb1--double hcterozl,gositvfor mutations in trvo unlinked genes,RO-441andlteripherin,rvhich both encodeproteins presenr in photoreceptors Individuals with onl-vone of these mutations are not affectcd.

Triatteticinheritance Bardet-Biedl syndrome is a rare dysmorphic condition (though relativelv more common in some inbred communities) with obesitl,',polydactyll, renal abnormalities, retinal pigmentation and learning disabilitv Seven different gene loci have been identihed and, until recenth,;the syndromc wasbelievedto follow

for the severeneonatalform of my'otonicdl,strophv that usuall,v only occursrvhenthe geneis transmittedby the mother (Fig. 7.18) A similar expansionin the CAG cxpansionin the 5'end of the Huntington diseasegene (Fig. 7.19) in paternal meiosis appears to account for the increasedrisk of juvenile Huntington disease n-henthe gene is transmitted by the father. Fragile X syndrome and the inherited spinocerebellarataxia group of conditions are other examDles

M0sAtctsM An individual, or a particular tissue of the body, can consist of more than one cell t-vpeor line, through an error occurring during mitosis at anv stage after conception. This is known as (p. 52) Nlosaicismof either somatic or germ cells can mostticism 2lccountfor some instancesof unusual patterns of inheritance or phenotypic featuresin an affectedindividual.

Somaticmosaicism

straightforward autosomal recessiveinheritance. Horvever,it is now known that one form occurs onlv rvhen an individual lho is homozvgous for mutations at one locus h a/so hctcrozvgous

The possibilitl'of somaticmosaicismis suggestedb1-the features of a single-genc disorder being less severein an individual than is usual, or by- being confined to a particular part of the

for mutation at another Bardet Bicdl locus: this is referred to as triulle lic in heritunce.

body. in a segmental distribution, for example as occurs occasionally-in neurofibromatosistype I (p. 287). Depending on rvhen a mutation arises in dcvelopmcnt, it may or may not be transmitted to thc next generation with full expression,

Other patterns of inhcritance that are not classicallymendelian are alsorecognizedand explain some unusual phenomena.

AN T I C I PA T ION In someautosomaldominant traits or disorders,such asmvotonic dystrophy the onset of the diseaseoccurs at an earlier age in thc offspring than in the parents, or the diseaseoccurs with increasingseverityin subsequentgenerxtionsThis phenomenon is called anticipation.It used to be belicvcd that this effect was the result of a bias of ascertainment,becauseof the rvayin which thc families were collected.It tvasargued that this arosebecausc personsin whom thc diseasebegins earlier or is more sclere are more likeh, to be ascertainedand only those individuals who arc less severelyaffected tend to have children. In addition, it was felt that, becausethe observer is in the same generation as the affected presenting probands, manv individuals who at present are unaffectedwill, by necessitl.,dcvelop the diseaselater in life.

114

Recent studies, however, have shor,n that in a number of disorders,including Hunrington diseaseand m1-otonicdvstrophl; anticipation is, in fact, a real biological phenomenon occurring as a result of the expansionof unstablc triplet repeat sequences ( p . 2 3 ) . A n e x p a n s i o no f t h e C T G t r i p l e t r e p e a t i n t h e 3 ' untranslatcd end of the mvotonic d-ystroph]' gene, occurring predominantlv in maternalmeiosis,appearsto be the explanation

Fis.7.18 Newbornbabywith severehypotoniarequiringvent Lation as a resultof havinginheritedmyotonicdystrophyfrom his mother

PATTERNSOFINHERITANCE

7

DISOMY UNIPARENTAL An individual normall-v inherits one of a pair of homologous chromosomes from each parent (p. al). Over the past decade,with the advent of DNA technology, some individuals have been shown to have inherited both homologs of a chromosome pair from only one of their parents, so-called uniparental dtsomy.If an individual inherits two copies of the same homolog from one parent, through an error in meiosis II (p. 't3), this is called uniparentalisodisomy(Fig. 7.20). It however,the individual inherits the two different homologsfrom one parent through an error in meiosis I (p 43), this is termed In either instance it is presumed that uniparentalheterodisomy. the conceptus would originally be trisomic, with early loss of a 'normal' disomic state.One-third of chromosomeleading to the such chromosomelosses,if they occurred with equal frequency, would result in uniparental disom.v.Alternatively it is postulated that uniparental disomy could arise as a result of a gametefrom

*i&6r! {ia*rd

dlw'ae,

Fis.7.19 S i l v esr t a i n i nogfa 5 %d e n a t u r i ngge t o tf h ep o l y m e r a sc e harn products reaction oftheCAGtriprer Inthe5' unt'anslaled endof genefromanaffected theluntingtondrsease maleandhiswife. s h o w i nhge rt o h a v et w os i m i L a r - s i zr e d p e a tisnt h en o r m a l r a n g e( 2 0a n d2 4c o p i e sa)n dh i mt o h a v eo n en o r m a l - s i z e d l r i p t erte o e a(t1 8c o p i e sa)n da ne x p a n d et rdi p l e-te p e a(t4 4c o p i e s ) .rarl^ers Thebandsi^ theteftlanearestanoard toalLow sizng s yf A l a nD o d g eR, e g i o n D o f t h eC A Gr e p e a(t C o u r t e o a LN A Laboratory, StMary'sHospita[, Manchester.) ,

l^

depending on whether the mutation is alsopresentin all or some of the germline cells.

Gonadalmosaicism There have been many reports of families with autosomal dominant disorders, such as achondroplasia and osteogenesis imperfecta, and X-linked recessivedisorders,such as Duchenne muscular dystrophy and hemophilia, in which the parents are phenotypicallynormal and the resultsofinvestigationsor genetic tests have also all been normal, but in which more than one of their children has been affected.The most favored explanation for these observationsis gonadal or germline mosaicism in one of the parents,that is. the mutation is presentin a proportion of the gonadalcells.An elegantexampleof this was provided by the demonstrationof a mutation in the collagengeneresponsible for osteogenesisimperfecta in a proportion of individual sperm from a clinically normal father who had two affected infants with different partners. It is important to keep germline mosaicism in mind when providing recurrence risks in genetic counseling for apparently new autosomaldominant and X-linked recessive mutations (p. 335)

one parent that does not contain a particular chromosome h o m o l o g , o r w h a t i s t e r m e d n u l l i s o m i c ,b e i n g ' r e s c u e d ' b y fertilization with a gametethat, through a secondseparatechance error in meiosis,is disomic. Using DNA techniques,uniparental disomy has been shown to be the causeof a father with hemophilia having an affected son and of a child with cystic fibrosis being born to a couple in which only the mother was a carrier (with proven paternity!). Uniparental paternaldisomy for chromosome15 may be linked to either Prader-Willi or Angelman syndrome, or for chromosome 1I rvith a proportion of casesof the overgrowth condition known as the Beckwith-Wiedemann syndrome (seebelow).

G E N O MI M CPRINTING phenomenon, referred to in Genomic imprinting is an epigenetic Chapter 6 (p. 98). Epigeneticsand genomic imprinting give the lie to Thomas Morgan's quotation at the start of this chapter! Although it was originally believed that genes on homologous chromosomeswere expressedequally, it is now recognized that different clinical features can result, depending on whether a gene is inherited from the father or from the mother. This 'parent of origin' effect is referred to as genomic imprinting, and meth.ltlationof DNA is thought to be the main mechanism by which expression is modified. Methylation is the imprint applied to certain DNA sequencesin their passagethrough gametogenesis,although only a small proportion of the human genomeis in fact subiectedto this process.The differential allele expression(i.e. maternal or paternal) may occur in all somatic cells, or in specifictissuesor stagesof development.Thus far, at least80 human genesare known to be imprinted and the regions involved are known as differentially methylatedregions (DMRs). These DMRs include imprinting control regions (ICRs) that control geneexpressionacrossimprinted domains.

115

7

PATTERNSOFINHERITANCE

A

ffi /

\

Meiosist

/

\

a-_-.-t..

'riffi) uu..' y'\

/\ ./-t,

H r e i o sy i' \, "

/\

fr..

lHiifli 't''!rj\..!.2' F e r tIi z a t o in

\/

I

Lossof c hr o m o s o m e

Uniparental isodisomy

\

y'

Fertilization

+ $;;;'.,... Uniparental heterodisomy

Fis.7.20 M e c h a n i somfo r i g i no fu n i p a r e n tdarls o mA y ., U n i p a r e n it saoI d i s o moyc c u r r i nt h gr o u ga h d i s o m iAc a m e taer r s i nfgr o mn o n - d i s j u n c tiino n meiosisll fertiLizing gametewithlossofthechromosome a monosomic fromtheparentcontributing thesingtehomotogB, UniparentaL h e t e r o d r s oomcyc u r r i nt hgr o u g a h d i s o m igca m e t ae r i s i nfgr o mn o n - d i s j u n c tirnom e i t hL o sos f n e i o s iIsf e r t i L i z ianm g o n o s o mgi ca m e tw thechromosome fromtheparentcontributino thesinotehomoLoo

Evidence of genomic imprinting has been observed in two pairs of well known dysmorphic syndromes: Prader-Willi and Angelman syndromes (chromosome l5q), and BeckwithWiedemann and Russell-Silver syndromes (chromosome llp). The mechanisms giving rise to these conditions, although complex, reveal much about imprinting and are therefore now consideredin a little detail.

(P WS) P R A D E R -WILS L IY N D R OME Prader-Willi syndrome (p. 266) occurs in approximately I in 20000 births and is characterized by short stature, obesity, hypogonadismand learning difficulty (Fig 7.21).Approximately

deletion, have been shown to hle maternaluniparental disomy. Functionally, this is equivalent to a deletion in the paternally derived chromosome 15. It is now known that only the paternally inherited allele of this critical region of 15q11-q13 is expressed.The molecular organization of the region is shown in Fig. 7.22. PWS is a multigene disorder and in the normal situation the small nuclear ribonucleoprotein polypeptide N (SNRPI/) and adjacent genes (,MKRN3, etc.) are paternally expressed.Expression is under the control of a specific ICR. Analysis of DNA from patients with PWS and various submicroscopic deletions

50-600/o of individuals with PWS can be shown to have an interstitial deletion of the proximal portion of the long arm

enabled the ICR to be mapped to a segment of about 4kb, spanning the first exon and promoter of SNRPN and upstream reading frame (SNt/Rfl. The 3' end of the ICR is required for expression of the paternally expressedgenes and also the

of chromosome15, approximately2Mb at l5ql1-q13, visible by conventional cytogenetic means, and in a further 15oloa submicroscopic deletion can be demonstrated by fluorescent in-situ hybrtdization (FISH; p. 34) or molecular means. DNA analysis has revealed that the chromosome deleted is almost always the paternally derived homolog. Most of the remaining 25-30o/o of individuals with PWS. without a chromosome

origin of the long -INLrRF/SNRPN transcript. The maternally expressed genes are not differentially methylated but they are silenced on the paternal allele, probably by an antisense RNA generated from .!NURF/.SNRPN. In normal cells, the 5' end of the ICR, needed for ma,terna,l expressionand involved in Angelman syndrome (seebelow), is methylated on the maternal allele.

PATTERNS OFINHERITANCE

7

the svndrome can be shown to have arisen through paternal uniparental disomy. Unlike PWS, the features of AS arise through loss of a single gene, U B E 3A. In up to I 0oloof individuals rvith AS, mutations have been identified in UBE3A, one of the ubiquitin genes, which appears to be preferentially or exclusivelyexpressedfrom the maternally derived chromosome l5 in brain. How mutationsin UBE3A lead to the featuresseen in persons with AS is not clear, but could involve ubiquitinmediated destruction of proteins in the central nervous s1,'stem in development,particularly where UBE3A is expressed most strongll', namely the hippocampus and Purkinje cells of the cerebellum. UBE3A is under control of the AS ICR (see Fig.7.22), which was mapped slightly upstream of SNURF/ .INRPN through analysis of patients with AS and various mircodeletions. About 2oloof individuals with PWS and approximately 5olo of those with AS have abnormalities of the ICR itself; these patients tend to show the mildest phenotypes. Patients in this last group, unlike the other three, havea risk of recurrence.In the caseof AS, rf the mothercarries the samemutation as the child, the recurrencerisk is 50olo,but even if she tests negativefor the mutation there is an appreciablerecurrence risk due to gonadal

Fi1.7.21 Femate childwithPrader-W Llsvndrome

AS) A N G E L M AS NY N D R O M(E Angelman syndrome (p. 266) occurs in about I in 15000 births and is characterizedby epilepsS severelearning difficulties, an unsteadv or ataxic gait, and a happy affect (Fig. 7.23). Approximatelv 70o/oof individuals with AS have been shown to have an interstitial deletion of the same 15q11-ql3 region as is involved in PWS, but in this caseon the maternully derived homolog. In a further 5oloof individuals with AS,

mosalclsm. Rare families have been reported in which a translocation of the proximal portion of the long arm of chromosome 15 involved in these two syndromes is segregating.Depending on N'hcther the translocation is transmitted by the father or mother, affected offspring within the family have had either PWS or AS. In approximately llokt of AS casesthe molecular defect is unknown. In man1,'genetics service laboratories a simple DNA test is used to diagnose both PWS and AS, exploiting the differential DNA methylation characteristicsat the l5qll-ql3 locus (Fig.7 .21).

P a t e r n aal l l e l e

Telomere 's)

PWSI C R

SNURF/SNRPN

H,rr[r,r:|

n,),., A )

IVAGE-12

M a t e r n aal l l e l e

Fis.7.22 (smpirfred) (AS)Theimprinting (PWS)andAngelman MoLecular organization at15q'11-q13: controI region Prader-Wilti syndrome syndrome (lCR) forthislocushastwocomponents Themoretelomeric SNURF/ thepromoter of S|VURF/SIVRPN actsasthePWSICRandcontains produces SIVRP/V several Long inhrbitor andcomptex transcripts, to bean RNAantisense of UBE3AThemore oneofwhichis believed l xpressio c e n t r o m e rI iCc Ra c t sa sL r eA SI C Ro n U B E 3 Aw, n i c ^i sr h eo n t yg e n ew h o s e- a l e r n a e i sn. o s itnA S T h eA SI C Ra , s oi n h i b i t sh e (o) PWSICRonthematernalattete ThePWSICRatsoactsontheupstream genesMKRN3. whichareunmethylated MAGE-L2 and,ND/V, (a)onthematernalallele onthepaternalattele butmethytated

117

7

PATTERNSOF INHERITANCE

Fis.7.23 A . F e m a tceh i [ dw r t hA n g e l m asny n d r o m B t a t ew i t hA n g e l m asny n d r o m e e ,A d u tm

MDA N N S Y N D R OEM B E C K W I T H - WEI E (BWS)

234

4 2 k b M a t e r n abl a n d

B W S i s a c l i n i c a l l y h e t e r o g e n e o u sc o n d i t i o n w h o s e m a i n underlying characteristic is overgrowth. First described in 1963-1964, the main featuresare macrosomia(prenatal and/or postnatal overgrorvth), macroglossia(large tongue), abdominal rvall defect (omphalocele, umbilical hernia, diastasis recti) (Fig. 7.25). Hemihyperplasia may and neonatal h1-'poglycemia be present, as well as visceromegah',renal abnormalities, ear anomalies (anterior earlobe creases,posterior helical pits) and cleft palate, and there may be embryonal tumors (particularly Wilms'tumor). BWS is, in a 'w'ar., celebratedin medical geneticsbecauseof the multiple different (and complex) molecular mechanisms t h a t u n d e r l i e i t . C e n o m i c i m p r i n t i n g . s o m a t i cm o s a i c i s m and multiple genes are involved, all within a l-Mb region at c h r o m o s o m e1 1 p 1 5 ( F i g . 7 . 2 6 ) . W i t h i n t h i s r e g i o n l i e t w o

0 . 9 k b P a t e r n abl a n d

Fig.7.24 Southern blottodetect methylations DNAdigested ofSNRP,N with Xbo / and Not /was probedwith KB17which hybridzes to a CpGistandwlthlnexona of S/VRPNPatjentlhas Prader-W[i . i e n t3sa n d c v e d T o m cn a l e n r2 h a sA n' g-s-Lrl-l -l o^ > y l l U l U l l l s , o- l-l .Ur -P -o + l / . - - ^ u, , ^L ^o .I St L^L-C-U^ ^( . ^ , , -t+g >^y - .u. n- ' AG a - d . e reDu e + olc n a r t m p n t r r I r l r , r p u r O fM O l e C u t a \UUUI

118

G e n e t i cSso, u t h m e aHdo s pt a l ,B r s t o t )

independentlyregulatedimprinted domains.The more telomeric (differentially methylated region I IDMRI] under control of ICRl) contains puternally expressed1G,82 (insulin growth factor 2) and maternally expressedHI9.The more centromeric imprinted domain (DMR2, under control of ICR2) contains the muternally expressed KCNU (previouslv known as KaLQTI) and CDKNIC genes, and the paternalfu expressed antisense transcript KCNQ,/ OTI,the promoter for w'hichis locatedwithin the KCNQ,/ gene.

PATTERNS OFINHERITANCE

7

is implicated in 50-600/o of sporadic BWS cases. CDKNIC may be a growth inhibitory gene and mutations have been found in 5-l0o/o of casesof BWS. About 15oloof BWS cases are familial, and CDKNIC mutations are found in about half of these.In addition to imprinting errors in DMR1 and DMR2, other mechanismsmay account for BWS: (1) paternally derived d u p l i c a t i o n s o f c h r o m o s o m e 1 1 p 5 . 5 ( t h e s e c a s e sw e r e t h e first to identify the BWS locus); (2) paternal uniparental disomy for chromosome I I - invariably present in mosaic form, often associatedwith neonatal hypoglycemia and hemihypertrophy, and associated with the highest risk (about 25o/o)of embryonal tumors, particularly Wilms' tumor; and (3) maternally inherited balanced translocations involving rearrangementsof 1lpl 5.

(RSS) sYNDROME RUSSELL-SILVER This well known condition has'opposite'characteristicsto BWS by virtue of marked prenatal and postnatal growth retardation. The head circumference is relatively normal, the face rather small and triangular, giving rise to a'pseudohydrocephalic' appearance(Fig.7 .27), and there may be body asymmetry.About 10o/oof casesappear to be due to maternal uniparental disomy, indicating that this chromosome is subject to imprinting. In contrast to paternally derived duplications of I lpl5, which give

Fig.7.25 Babygirlwrth Beckwith-Wiedemann syndrome Notethelarge tongue andumbiLicaI hernia

Disruption to the normal regulation of methylation can give rise to altered gene expressiondosageand, consequentially,features of BWS In DMRI, gain of methylationon the maternal allele leadsto lossof /1,/9 expressionand biallelic 1G,F2expression,i.e. effectively two copies of the paternal epigenotype.This occurs in up to 7oloof BWS casesand is usually sporadic. In DMR2, lossofmethylatlazresultsin two copiesofthe paternalepigenotype and a reduction in expression of CDKN,/C; this mechanism

DMR2

rise to overgrowth and BWS, maternally derived duplications of this region are associatedwith growth retardation. Recently it has been shown that about a third of RSS casesare due to abnormalities of imprinting at the llp. 15.5 locus. Whereas hypermethylation of DMR1 leads to upregulated IGF2 and overgrowth, hlpomethylation of HI9 leads to downregulated IGF2,the oppositemolecular and biochemicalconsequence,and these patients have featuresof RSS. Interestingly,in contrast to BWS, there are no casesof RSS with altered methylation of the more centromeric DMRZ re$on.

P a t e r n aal l l e l e

DMRI

r-

lcR1

Centromere

Telomere

$, ^ - ^ - ,- l1 LrLr

other CDKNlC

rnE.raTCF+o

KCNOl

a

Enhancer L.lro

9enes KCNOlOTI

M a t e r n a la l l e l e

Fis.7.26 (simptrfied) twoimprinted Theregioncontains syndromes Molecutar organization andRusseLL-Srtver at11p15 5:Beckwith-Wiedemann (. methytated: o unmethytated) (DMR1 methylated domains andDMR2) thatarereguLated independentty. ThelCRsaredifferentiaLLy to expression of lGF2 Leads regulatton coordinated CCCTC-brndrng factor(CTCF) bindsto theunmethylated alleles of bothlCRsIn DMR1. of expression leads to moternoL reguLation paternaLaLLeLe coordrnated alle|.e ln DMR2, oniyonthe andHi9 expression on[yonthemaiernal (anon-coding to KC/VO/) (ptusotherqenes), transcripiion RNAwithantisense KC,N07 andCDKIV1C expression of KCNQ\OTl andpoternol AngledbLack arrowsshowthedirectron ofthetranscnpts

119

7

PATTERNS OFINHERITANCE

Fis.7.28 Fami[ytree consistent with mitochondriaI inheritance

In humans, cytoplasmicor mitochondrial inheritance has been proposed as a possibleexplanationfor the pattern of inheritance observedin some rare disorders that affect both males and females but are transmitted only through females,so-calledmaternal or matrilineal inheritance (Fig. 7.28). A number of rare disorders with unusual combinations of neurological and myopathic features, sometimes occurring in associationwith other conditions such as cardiomyopathy and conduction defects,diabetesor deafness,havebeencharacterized as being due to mutations in mitochondrial genes (p. 174). As

Fis.7.27 GirtwithRussetl-Sitver syndromeNotethebossed forehead, triangu [arfaceand'pseudohyd rocephaLic' appearance

M I T O C H O N D RI N I AHLE R I T A N C E Each cell contains thousands of copies of mitochondrial DNA with more being found in cells that havehigh energy requirements, such as brain and muscle. Mitochondria, and therefore their DNA, are inherited almost exclusively from the mother through the oocyte (p. a3). Mitochondrial DNA has a higher rare of spontaneous mutation than nuclear DNA, and the accumulation of mutations in mitochondrial DNA has been proposed as being responsible for some of the somatic effects seen with aging.

H o m o p l a s m-y n o d i s e a s e

120

Milddisease

mitochondria have an important role in cellular metabolism through oxidative phosphorylation, it is not surprising that the organs most susceptibleto mitochondrial mutations are the central nervous system,skeletalmuscle and heart. In most persons the mitochondrial DNA from different mitochondria is identical, or shows what is termed homoplasmy. If a mutation occurs in the mitochondrial DNA of an individual, initially there will be two populations of mitochondrial DNA, so-called heterlplesmy.The proportion of mitochondria with a mutation in their DNA variesbetweencells and tissues.and this. together with mutational heterogeneity, is a possible explanation for the rangeof phenotypic severityseenin personsaffectedwith mitochondrially inherited disorders(F ig. 7 .29). Whilst matrilineal inheritance applies to disorders that are directly due to mutations in mitochondrial DNA, it is also important to be aware that mitochondrial proteins are encoded

Fis.7.29 Nodisease

Nodisease

S e v e r ed i s e a s e

Prnnroccive

e f f e r t c n f ,h,p, -t t_r r- u ^ ^Pl r -d->- ,t,i l y

on the c[inicatseverity of diseasedue to mutationsin the mitochondriaI genome Low proportionsof mutant mitochondria are toteratedweLL, but as the proportronincreasesdifferent thresholdsfor celuLarand hencetissue dysfunction are breached(mauvecircte representsthe cettnucteus)

PATTERNSOFINHERITANCE

mainly by nuclear genes. Mutations in these genes can have a devastatingimpact on respiratory chain functions within mitochondria. Examples include genes encoding proteins within the cytochrome r (COX) system, which follow autosomal recessiveinheritance,and the G4.5 (fAA gene that is Xlinked and causesBarth syndrome (endocardialfibroelastosis)in males (p. 175). There is even a mitochondrial myopathy following autosomaldominant inheritancein which multiple mitochondrial DNA deletionscan be detected,although the gene(s)mutated in this condition are as yet unknown. Further spaceis devoted to mitochondrial disordersin Chapter l1 (p. 174).

7

ELEMENTS Q Family studies are often necessary to determine the mode of inheritance of a trait or disorder and to give appropriate genetic counseling.A standard shorthand convention exists for pedigree documentation of the family history. @ Mendelian, or single-gene,disorderscan be inherited in five ways: autosomal dominant, autosomal recessive' X-linked dominant, X-linked recessiveand Y-linked inheritance

F U R T H ERRE A D I N G BatesonW, SaundersE R 1902Experimentalstudiesin the physiology of heredity. Royal Society Reports to the Evolution Commirtee, pp 132-134 Early obseraations on mendelianinheritance. BennetR L, SteinhausK A, Uhrich S B et al 1995Recommendations for standardizedhuman pedigreenomenclatureAmJ Hum Genet 56: J+5-tJl

HallJ G 1988Somaticmosaicism:observations relatedto clinicalgenetics. A m J H u m G e n e t 4 3 : 3 5 53 6 3 Cood reaiep offndings arisingfrom somaticmosaicism in clinicalgenetics. HallJ G 1990Genomicimprinting: reviewand relevanceto human diseases Am J Hum Genet46: 857-873 Ettensiaereuiewof eramplesof imprinting in inheriteddiseases in humans Heinig R M 2000The monk in the garden:the lost and found genius of Gregor Mendel Houghton Mifflin, London The hfe and work of GregorMendel as the histor.yof the birth of genetics. Kingston H M 1994AnABC of clinicalgenetics,2ndedn. British Medical Association,London A simplezutlineprimer 0f the basicprinciplesof clinicalgenetics Reik W, Surami A (eds) 1997Genomic imprinting (Frontiers in Molecular Biology) IRL Press,London Detaileddiscussion of examplesand mechanisms of genomicimprinting. Vogel E, Motulsky A G 1996Human genetics,3nd edn Springer, Berlin This text hasdetailedexplanationsofmany ofthe clnceptsin humangenetics outlinedin this chapter.

@ Antosomal dominant alleles are manifest in the heterozygousstate and are usually transmitted from one generationto the next but can occasionallyarise as a new mutation. They usually affect both males and females equally. Each offspring of a parent with an autosomal dominant gene has a I in 2 chance of inheriting it from the affected parent. Autosomal dominant alleles can exhibit reduced penetrance, variable expressivity and sex limitation. @ Autosomal recessivedisorders are manifest only in the homozygousstateand normally only affect individuals in one generation,usually in one sibship in a family. They affect both males and females equally. Offspring of parents who are heterozygous for the same autosomal recessive allele have a 1 in 4 chance of being homozygous for that allele. The less common an autosomal recessiveallele' the greater the likelihood that the parents of a homozygote are consangurneous. @ X-tint"a recessivealleles are normally manifest only in males. Offspring of females heterozygous for an Xlinked recessiveallele have a I in 2 chance of inheriting the allele from their mother. Daughters of males with an X-linked recessiveallele are obligate heterozygotes but sons cannot inherit the allele. RarelS females manifest an X-linked recessivetrait becausethey are homozygous for the allele, have a single X chromosome' have a structural rearrangement of one of their X chromosomes, or are heterozygous but show skewed or non-random X-inactivation @ Th"t" are only a few disorders known to be inherited in an X-linked dominant manner. In X-linked dominant disorders, hemizygous males are usually more severely affected than heterozygous females. @ Unusual featuresin single-genepatterns of inheritance canbe explainedby phenomenasuch asgeneticheterogeneity mosaicism, anticipation, imprinting, uniparental disomy and mitochondrial inheritance.

121

CHAPTER

MathematicaIand poputation genetics

'Do not worry about your difficulties in mathematics. I can assureyou mine are still greater.' Albert Einstein In this chapter some of the more mathematical aspectsof the ways in which genes are inherited are considered, together with how genes are distributed and maintained at particular frequenciesin populations. This subject constitutes what is known as populationgenetics.By its very nature genetics lends itself to a numerical approach,with many of the most influential and pioneering figures in human geneticshaving come from a mathematicalbackground. They were particularly attracted by the challengesof trying to determine the frequenciesof genes in populations and the rates at which they mutate. Much of this early work impinges on the specialtyof medical genetics,and in particular on genetic counseling,and by the end of this chapter it is hoped that the reader will have gained an understanding of the following: l. Why a dominant trait does not increasein a population at the expenseof a recessiveone. 2. How the carrier frequency and mutation rate can be determined, when the diseaseincidence is known. 3. Why a particular genetic disorder can be more common ln one population or community than in another. 4. How it can be confirmed that a genetic disorder shows a particular pattern of inheritance. 5. The concept of genetic linkage and how this differs from linkage disequilibrium

disturbance by outside influences, dominant traits do not increase at the expenseofrecessiveones.In fact, in such a population, the relative proportions ofthe different genotypes(and phenotypes) remain constant from one generation to another. This is known as the Hardy-Weinberg principle, as it was proposed, independently, by an English mathematician, G. H. Hardy, and a German physician,W. Weinberg,in 1908.This is one of the most important fundamental principles in human genetics.

T H EH A R D Y . W E I N B E P RR GI N C I P L E Consider an 'ideal' population in which there is an autosomal locus with two alleles,A and a, that have frequenciesofp and q, respectively.These are the only alleles found at this locus, so that p + q = l00o/o,or L The frequency ofeach genotypein the population can be determined by construction of a Punnett's square, which showshow the different genescan combine (Fig. 8.1). From Fig. 8.1 it can be seen that the frequencies ofthe different genotypes are: Genotype

Phenotype

AA Aa Aa

A A

Frequency p2 2pq qt

6. The effectsof medical intervention.

A L L E L EF R E O U E N C IIENSP O P U L A T I O N S

122

On first reflection it would be reasonableto predict that dominant genes and traits in a population would tend to increaseat the expenseof recessiveones. After all, on average,three-quarters of the offspring of two heterozygoteswill manifest the dominant trait, but only one-quarter will have the recessivetrait. It might be concluded, therefore, that eventually almost everyone in the population would havethe dominant trait. However,it can be shown that in a large randomly mating population, in which there is no

Fis.8.1 Punnett's squareshowing frequencies attete andresutting genotype frequencies fora two-atLeLe systeminthefirst generatron

MATHEMATICAL ANDPOPULATION GENETICS

We have now established that, if there is random mating of sperm and ova, the frequencies of the different genotypes in the first generation will be as shown above Next consider that

frequency of male Genotype

these individuals mate with one another to produce a second generation. Once again a Punnett's square is used to show the different matings and their frequencies(Fig. 8 2). From Fig. 8.2 a table can be drawn up to calculaterhe total frequency for each genotype in the second generarion(Thble 8.1).

c o

o

E

This reveals that the relative frequency or proportion of each genotype is the same in the second generation as in the first. In fact, it can be shown that no matter how many genera-

4p2q2 u

tions are studied the relative frequencieswill remain constant. The actual numbers of individuals with each genotype will change as the population size increasesor decreases,but their relative frequenciesor proportions remain constant This is the fundamental tenet of the Hardy-Weinberg principle. When studies confirm that the relative proportions of each genotype are indeed remaining constant with frequenciesof pz, 2pq and q2, then that population is said to be in a stateof Hurdy Weinberg

8

o o

F i e8 . .2 Punnett's squareshowingfrequenciesof the differentmatingsin t h e s e c o n dg e n e r a t i o n

equilibrium for that particular genotype. 3 Selection 4. Small population size 5. Gene flow (migration).

FA C T O RTSH A TC A ND IS T U R B H A R D Y. W E I N B E RE GO U I L I B R I U M The discussionaboverelatesto an'ideal'population. By definition such a population is large and showsrandom mating with no new mutations and no selection for or against any particular genotype. For some human characteristics,such as neutral genes for blood groups or enzyme variants,these criteria can be fullfilled. However,in geneticdisorders,severalfactorscan disturb HardyWeinberg equilibrium, either by influencing the distribution of genesin the population or by altering the genefrequencies.These factors include:

Non-randommating Randommating, or panmixis, refers to the selectionof a partner regardlessof that partner's genotype. Non-random mating can lead to an increasein the frequency of affectedhomozygotesby two mechanisms,either assortativemating or consanguinity.

Assortotivemoting Assortatioemating is the tendency for human beings to choose partners who share characteristicssuch as height, intelligence

l. Non-random mating 2. Mutation

Tabte8.1 Frequency of thevarioustypesof offspring fromthe matingsshownin Fig B 2 Frequencyof offspring Aa

Matingtype

Frequency

AA

AAXAA

p4

n4

AA x Aa

4ptq

/n'n

tp-q

Aa \ Aa

4p'q'

n2n2

)n2o2

AA x aa

2p'q'

2p'q'

Aa x aa

4pq'

tpq-

aa

p'q'

tpq' n4

q4

TotaI

p2(p2 + 2pq+ q2)

+ 2pq+ q2) 2pq(p2

q2(p2 + 2pq+ q2)

Relative frequency

n2

2pq

q2

123

8

MATHEMATICALAND POPULATION GENETICS

and racial origin. If assortative mating extends to conditions such as autosomal recessive(AR) deafness,which accounts for a large proportion of all congenital hearing loss, this will lead to a small increase in the relative frequency of affected homozygotes.

Consonguinity Consanguinity is the term used to describe marriage between blood relativeswho have at least one common ancestorno more remote than a great-great-grandparent Widespread consanguinity in a community will lead to a relative increasein the frequency of affectedhomozygotesand a relative decreasein the frequency of heterozygotes.

Mutation The validity of the Hardy-Weinberg principle is based on the assumption that no new mutations occur. If a particular locus shows a high mutation rate then there will be a steady increase in the proportion of mutant allelesin a population. In practice mutations do occur at almost all loci, albeit at different rates,but the effect of their introduction is usually balancedby the loss of mutant allelesdue to reduced fitnessof affectedindividuals. If a population is found to be in Hardy-Weinberg equilibrium, it is generallyassumedthat these two opposing factors have roughly

and affected homozygotes. Once again, this will result in a disturbanceof Hardy-Weinberg equilibrium.

Smattpopulationsize In a large population the numbers of children produced by individuals with different genotypes, assuming no alteration in fitness for any particular genotype, will tend to balanceout, so that gene frequencies remain stable. However, in a small population it is possiblethat by random statisticalfluctuation one allele could be transmitted to a high proportion of offspring by chance,resulting in marked changesin allelefrequency from one generation to the next, so that Hardy-Weinberg equilibrium is disturbed. This phenomenon is referred to as randomgeneticd,rift. If one allele is lost altogether, it is said to be extinguishedand the other allele is describedas having becomefixed(Fig. 8.3).

Geneflow (migration) If new allelesare introduced into a population as a consequence of migration with subsequentintermarriage, this will lead to a change in the relevant allele frequencies.This slow diffusion of

L a r g ep o p u l a t i o n

'A' gene

equal effects.This is discussedfurther in the section that follows on the estimation of mutation rates.

08

Selection In the 'ideal' population there is no selection for or againstany particular genotype. In reality, for deleterious characteristics there is likely to be negative selection,with affected individuals having reduced reproductive (= biological = 'genetic') fitness. This implies that they do not have asmany offspring asunaffected control members of the samepopulation. In the absenceof new mutations this reduction in fitnesswill lead to a gradualreduction in the frequency of the mutant gene, and hence disturbance of Hardy-Weinbergequilibrium. Selection can act in the opposite direction by increasing fitness.For some autosomalrecessivedisordersthere is evidence that heterozygotes show a slight increase in biological fitness compared with unaffected homozygotes.This is referred to as heter0zyg0teudrantage.The best understood example is sickle-cell disease,in which affected homozygoteshave severeanemia and often show persistentill-health (p. l5l). However,heterozygotes are relatively immune to infection with Plasmodiumfalciparum malaria becausetheir red blood cells undergo sickling and are rapidly destroyedwhen invadedby the parasite.In areasin which this form of malaria is endemic, carriers of sickle-cell anemia,

124

who are describedashaving the sickle-celltrait, are at a biological advantagecompared with unaffected homozygotes Therefore, in these communities there is a tendency for the proportion of heterozygotesto increaserelative to the proportions of normal

'a'gene

o U u d

u r

S m a l lp o p u l a t i o n F i x a t i o no f 'A'gene

t.u

0.8 06

E x t i n c t i o no f 'a' gene

01234 Generations

Fis.8.3 Possible effects ofrandomgenetic driftrnlargeandsmatlpopulations

POPULATION GENETICS MATHEMATICALAND

allelesacrossa racial or geographicalboundary is known asgaza flow.The most widely quoted example is the gradient shown by the incidence of the B blood group allele throughout the world (Fig. 8.4). This allele is thought to have originated in Asia and spread slowly westward as a result of admixture through invasion

VALIDITY OFHARDY.WEINBERG EQUILIBRIUM It is relatively simple to establish whether a population is in Hardy Weinberg equilibrium for a particular trait if all possible genotypescan be identified. Consider a system with two alleles, A and a, with three resulting genotypes, AA, AalaA and aa. Amongst 1000 individuals selected at random, the following genotypedistributions are observed:

800 185 l5

AA Aa/aA aa

From these figures the incidence of the A allele (p) equals ft2 X 800) + 185l/2000 = 0 8925 and the incidenceofthe a allele (q) equals[85 + (2 x 15)]/2000= 0.1075

These observed and expected values correspond closely and formal statisticalanalysiswith a 12 test would confrrm that the observed values do not differ significantly from those expected if the population is in equilibrium. Next consider a different system with two alleles, B and b. Among 1000 randomly selectedindividuals the observed genotypedistributions are:

BB Bb/bB bb

430 540 30

From these values the incidence of the B allele (p) equals = t(2 x 430) + 5401/2000 0.7 and the incidenceofthe b allele(q)

= 0.3. equals [540+ (2 x 30)]/2000

Using these values for p and q, the observed and expected genotypedistributions can be compared:

Genotype BB Bb/bB bb

Observed

Expected

430 s40 30

490(p2x lo0o) 420(2pqx 1000) 90(q'zx 1000)

Genotype

Observed

Expected

These values differ considerably,with an increasednumber of heterozygotesat the expenseof homozygotes.Deviation such as this from Hardy-Weinberg equilibrium should prompt a search for factors that could result in increased numbers of heterozygotes,such as heterozygote advantageor negative

AA Aa/aA at

800 185 15

(p2x 1000) 796.5 192(2pqx 1000) 115 (q2x 1000)

assortativemating, i.e. the attraction of opposites! Unless there is strong evidenceto the contrary, it is usually assumedthat most populationsare in equilibrium for most genetic

Now consider what the expectedgenotypefrequencieswould be if the population were in Hardy-Weinberg equilibrium, and compare thesewith the observedvalues:

8

Fig.8.r* ofthe distribution K1976The group Distribution ofbLood B throughoutthe world(From MourantA E,Kop6cA C,Domanrewska-Sobczak groups withpermissron London, human blood andotherpotymorphrsms,2nd Press, ednOxford University )

125

MATHEMATICAL ANDPOPULATION GENETICS

traits, despitethe number of factorsthat candisturb this equilibrium, asit is very unusualto find apopulation in which genotypefrequencies show significant deviation from those that would be expected.

its mutation rate can be made relatively easily by counting the number of new casesin a defined number of births. Consider a sample of 100000 children, 12 of whom have a particular autosomal dominant (AD) disorder such as achondroplasia

A P P L I C AO TN I 5 O FH A R D Y - W EB I NE R G EOUILIBRIUM

(p. 9l). Only two of thesechildren havean affectedparent, so that the remaining l0 must have acquired their disorder as a result of new mutations. Therefore l0 new mutations have occurred among the 200000 genesinherited by these children (eachchild inherits two genes),giving a mutation rate of 1 per 20 000 gametes

Estimation of carrierfrequencies If the incidence of an AR disorder is known, it is possible to calculate the carrier frequency using some relatively simple algebra. If, for example, the diseaseincidence is 1 in 10000, then q2 = 1/roooo and q = 1/,00. As p + q = 1, therefore p = eelroo. The carrier frequency can then be calculated as 2 x ee/rrr,, x r/ro.1, i.e. 2pq, which approximatesto 1 in 50. Thus a rough approximation of the carrier frequency can be obtained by doubling the square root of the diseaseincidence Approximate values for gene frequency and carrier frequency derived from the diseaseincidence can be extremely useful when calculating risks for genetic counseling(p. 255) (Thble 8.2). Note that if the diseaseincidence includes casesresulting from consanguineous relationships then it is not valid to use the Hardy-Weinberg principle to calculateheterozygotefrequences,as a relativelyhigh incidenceofconsanguinity disturbs the equilibrium by leadingto a relative increasein the proportion of affectedhomozygotes. For an X-linked recessive(XLR) disorder the frequency of affected males equals the frequency of the mutant allele, q. Thus, for a trait such as red-green color blindness,which affects approximatelyI in 12 male westernEuropean caucasians, g= t/rz 11lrz. and p This meansthat the frequency of affected females (q2)and carrier females(2pq) is t/n+ and 22l1aa, respectively.

Estimationof mutationrates Directmethod If an autosomal dominant disorder shows full penetrance and is therefore always expressedin heterozygotes,an estimate of

Tabte8.2 Approximate vatuesfor genefrequency and carrierfrequency calcutated fromthe diseaseincidence assumingHardy-Weinberg equilibrium Diseaseincidence (q')

Genefrequency (q)

Carrierfrequency (2pq)

'/rooo

1lp

tlra

tlzaoa

llqs

llzz

per generation.In fact, all new mutations in achondroplasiaoccur on the paternally derived chromosome 4, so that the mutation rate is I per 10000 in spermatogenesisand, as far as we know, zero ln oogenesrs

lndirectmethod For an AD disorder with reproductive fitness(f) equal to zero, all casesmust result from new mutations. If the incidence of a disorder is denoted as I and the mutation rate as p, then as each child inherits two alleles,either of which can mutate to causethe disorder,the incidenceequalstwice the mutation rate, i.e. | = 4t. If fitness is greater than zero, and the disorder is in HardyWeinberg equilibrium, then genes lost through reduced fitness must be counterbalancedby new mutations. Therefore,

Ztt= t(t - 0 o. p = lI(1- f)l/Z. Thus, if an estimate of genetic fitness can be made by comparing the average number of offspring born to affected parentscomparedwith controls such as their unaffectedsiblings, it will be possibleto calculatethe mutation rate. A similar approach can be used to estimate mutation rates for AR and XLR disorders. With an AR condition, two genes will be lost for each homozygote that fails to reproduce. These will be balancedby new mutations.Therefore, 2lt = l(t - f) x 2 orp-I(l-0. For an XLR condition with an incidencein malesequal to IM, three X chromosomesare transmitted per couple per generation. Therefore, 3p = It(l * 0 or p = IINI(I - f)]/3.

Why is it helpfulto know mutationrates? At first sight it might seem that knowledge of these formulae Iinking mutation rates with diseaseincidence and fitness is of Iittle practical value. Howevet there are severalways in which this information can be useful.

Estimotionof genesize

126

tlsooo

tlt

1 lze

r/oooo

llno

tlso

'/soooo

llzz+

1hz

'/rooooo

llsa

1l$s

If a disorder has a high mutation rate this could yield information about the structure of the gene that would help expedite its isolation and characterization.For example, the gene could contain a high proportion of GC residues,which are thought to be particularly prone to copy error (p 8), or it could contain a high proportion of repeat sequences(p. 23), which could predisposeto misalignment in meiosis resulting in deletion and duplication, or it could simply be that the geneis very large.

GENETICS ANDPOPULATION N4ATHEN/ATICAL

D eterminotionof mutogenicpotentiol If valuable information is to be gained about the potential mutageniceffectsofnuclear accidentssuch as Chernobyl (p.27), it will be necessaryto have accurate methods for determining mutation rates and how these are related to observedchangesin diseaseincidence

Consequences of treotmentof geneticdisease Knowledge of the relationship betweendiseaseincidence,{itness and mutation rate is necessaryto determine the possibleeffects of improved treatment for serious genetic disorders.This is discussedfurther towardsthe end ofthis chapter.

W H Y A R ES O M EG E N E T ID CI S O R D E R S M O R EC O M M OTNH A NO T H E R S ? It is axiomatic that, if a gene shows a high mutation rate, this will usually result in a high incidence of the relevant disease. However, factors other than the mutation rate and the htness of affected individuals may also be involved. Mention has alread-v been made of someof the mechanismsthat can accountfor a high gene frequency of a particular disorder in a specificpopulation These are now consideredin the context of population size.

Smattpopulations Several rare AR disorders show a relatively high incidence in certain populationsand communities (Table8.3).The most likely explanationfor most of these observationsis that the high allele

Tabte8.3

8

frequcncy has resulted from a combination of a founder ffict coupled with social, religious or geographical isolation of the relevant group. Such Broups are referred to as genetic isolates. In some of the smaller populations genetic drift could also have plaveda role. For example,severalotherwisevery rareAR disordershavebeen found to occur at a relatively high frequency in the Old Order Amish living in Pennsylvania- Christians originating from the Anabaptist movement who fled Europe during religious persecution in the eighteenthcenturJ'.Presumably,by chance,one or two original founders of the group carried abnormal alleles that became establishedat relatively high frequency becauseof the restricted number of partners availableto members of the community' A founder effect can also be observed for AD disorders. Variegateporph.vria,which is characterizedby photosensitivity and drug-induced neurovisceraldisturbance,has a much higher incidencein the Afrikaner population of South Africa than in any other countr).or race.This is thought to be due to one ofthe early Dutch settlershaving the condition and transmitting it to a large (p 103). number ofhis or her descendants One particularly novel explanationfor a high gene frequency in a small population is provided by the Hopi Indians of Arizona, who show a high incidence of albinism. Affected males were protected from outdoor farming activity because of their susceptibility to bright sunlight and it seemsthat this provided them with opportunity for reproductive activity in the absence of their unaffected peers.It is worth noting that severalfamous historical ltgures were affected with albinism, including the RcverendWilliam Spooner,who was famous for transposingthe 'spoonerism'. initial letters of words - hence

Rarerecessive disorders thatare retativety commonin certaingroupsof peop[e

Group Fn i ns

Disorder

Clinicalfeatures

CongenitaI nephroticsyndro me

proteinuria,suscepti brtrtyto infection Edema, coarsefeatures mentalandmotordeterioration, Progressive Muscte. Iiver,brainandeyeinvolvement diarrhea Reduced CFabsorption. andscotiosts withdwarfism dysplasra epiphyseaI Progressive

A - ^ ^ - + . , 1^ 1 . , ^ ^ - ^ - i ^ , , - i H>Pdr Ly19ryLU>dil ilr rurro

Mulibrey nanism chloride diarrhea CongenitaI ni--+-^^Li^ !.,^^l--i^ urd5u upr ilL uy>Prd>rd

Clr rt:rir :ridr rrir tvno 1

andsparsehatr frne.[ight-cotored Dwarfism, heart disease congenrtaI Dwarfism,potydactyty. patsy-tike dystonta andcerebraI Episodic encephalopathy

H o pai n dS a nB t a sl n d i a n s

Atbinism

Lackof pigmentation

Ashkenazi Jews

Tay-Sachsdisease Gauched r isease Dysautonomia

blindness mentaI andmotordeterioration Progressive skinpigmentation bonelesions. Hepatosptenomegaly, [ackoftears,hyperhidrosis io pain,emotionaltabitity, Indiffeence

Karaite Jews

W e r d n r g - H o f f m a ndni s e a s e

l n f a n t i l es p i n a Im u s c u t a ra t r o p h y

Afrikaners

ScLerosteosis Lipoidproteinosis

boneswithcraniaInervepatsies' of craniofactal overgrowth Tallstature, ctyiy synda of skrnandmucousmembranes Thickening

'Ryukyan spinalmuscular atrophy

ctubfoot,scotiosis weakness, Muscte

Amish

(offJapan) istands Ryukyan

hypoptasia Cartilage-hair Crevetd syndrome Ellis-van

127

8

MATHEMATICAL ANDPOPULATION GENETICS

Largepoputations When a seriousAR disorder, which results in reduced fitness in affectedhomozygotes,has a high incidencein a large population, the explanation must lie in either a very high mutation rate or in heterozygote advantage.The latter explanation is the more probablefor most AR disorders(tble 8.4).

Heterozygoteadvantoge For sickle-cell anemia (p. l5l) and thalassemia(p. 152) there is good evidence that heterozygote advantageresults from reduced susceptibility to Plasmodiumfalciparum malaria. The mechanism by which this is thought ro occur is that the red cells of heterozygotesfor sickle-cell diseasecan more effectively expressmalarial or altered self antigens that will result in more rapid rernovalof parasitizedcellsfrom the circulation.Americans of Afro-Caribbean origin are no longer exposed to malaria, so it would be expectedthat the frequency of the sickle-cell allele amongsttheseindividuals would gradually decline.However,the predicted rate of decline is so slow that it will be many generations before it is detectable. For several AR disorders the mechanisms proposed for heterozygote advantageare largely speculative (see Table 8.4). The discovery of the cystic fibrosis gene, with the subsequent

decline in the incidence of cystic fibrosis would be expected. However, ifthis theory is correct one has to ask why cystic fibrosis has not become relatively common in other parts of the world where gastrointestinalinfections are endemic, particularly the tropics; in fact, the opposite is the case,for cystic fibrosis is rare in theseregions. An alternative and entirely speculative mechanism for the high incidence of a condition such as cystic fibrosis is that the mutant allele is preferentially transmitted at meiosis.This type of segregationdistortion, whereby an allele at a particular locus is transmitted more often than would be expectedby chance(i.e. in more than 50o/oof gametes),is referred to as meioticdritse. Firm evidencefor this phenomenon in cystic fibrosis is lacking, although it has been demonstratedin the AD disorder myotonic dystrophy (p.28+). A major practical problem when studying heterozygote advantageis that even a tiny increase in heterozygote fitness compared with the fitness of unaffected homozygotes can be sufficient to sustain a high allele frequency. For example in cystic fibrosis, with an allele frequency of approximately 1 in 50, a heterozygote advantageof between 2o/o and 3olo would be sufficient to account for this high allele frequency.

elucidation of the role of its protein product in membrane permeability (p. 292), supporrs the hypothesis of selective advantage through increased resistance to the effects of gastrointestinal infections, such as cholera and dysentery, in the heterozygote.This relative resisrancecould result from reducedlossoffluid and electrolytes It is likely that this selective advantagewas of greatestvalue severalhundred yearsago when theseinfections were endemic in westernEurope. If so,a gradual

G E N E T IP CO L Y M O R P H I S M Polymorphtsmis the occurrence in a population of two or more geneticallydetermined forms (alleles,sequencevariants)in such frequenciesthat the rarest of them could not be maintained by mutation alone. By convention, a polymorphic locus is one at which there are at least two alleles, each with a frequency greater

Tabte8.4 Presumedincreased resistance in heterozygotes thatcouLd account for the maintenance of variousgenetic disordersin certainDooulations Disorder

Genetics

Region/poputation

Resistanceor advantage

- c e [ [d i s e a s e Srckle

ND

TroprcatAfrica

FaLciparum malaria

cr-andB-thalassemia

AD

South-EastAsiaandtheN4editerranean Fatciparummalaria

G6PDdeficiency

XLR

Mediterranean

Falciparum mataria

Cystic fibrosis

ND

WesternEurope

Tubercu[osis? Tho nlrnr ro?

Chotera?

128

Tay-Sachsdisease

AD

EasternEuropean Jews

Tubercu [osrs?

Congenital adrenal hyperptasia

AR

Yupik Eskrmos

Influenza B

Iype2 diabetes

AD

PimaIndrans andothers

Periodrc starvation

Phenytketonurra

AR

WesternEurope

Spontaneous abortion ratelower?

AR:autosomaI recessive: XLR:X-tinked recessive; AD:autosomaI gtucose dominant; G6PD. 6-phosphate dehydrogenase

GENETICS CALAND POPULATION N4ATH EMATI

than lolo Alleles with frequenciesofless than lolo are referred to as rare vanants. Studies of enzvme and protein variability have shown that, in humans, at least 30o/<-, of structural gene loci are pol-vmorphic, with each individual being hcterozygous at between l0o/o and 20o/oof all structural loci. Knorvn polymorphic protein s)-stems include the ABO blood groups (p 193)and many serum protcins A large number of enz1,-mes exhibit pol-vmorphicelectrophoretic differencesor shat are known as is,tzymes Polymorphism at the DNA level has proved particularly valuable in the positional cloning studies that have led to the isolation of many diseasegenes (p. 74) Polymorphic DNA markers are also of use in gene tracking (p. 69), which can be used in presvmptomaticdiagnosis,prenataldiagnosisand carrier detection for a large number of single-genedisorders.The value of a particular polymorphic systemis assessed by determining its lolymorphic inJbrmarioncontent(PIC) The higher the PIC value, the more likely it is that a polymorphic marker will be of value in linkage analysisand genetracking Finally, mention should be made of the distinction betwecn baluncedand tr&nsient?oljmorl)hisms In abalancedpolymorphism, two or more different forms are maintained by a balancebetwecn the selectiveadvantageof the heterozygoteand the reduced fitnessof the affectedhomozygote.Thus the high incidcnceof the sickle-cellallele in areaswhere malaria is endemic is an example of a balancedpoll,morphism. However, as alreadydiscusscd,the incidenceof the sickle-cellalleleis likely to decline in populations that are no longer exposedto malaria, so that in these situations the pol-vmorphismcan be consideredas transient

8

mode of inheritance. The mathematical aspectsof segregation analysis are extremely complex and fall far beyond the scope of this book - and most doctors! However, it is important that those who encounter families with genetic diseasehave some understanding of the principles involved in segregationanalysis as u,ell as an awarenessof some of the pitfalls and problems.

CE AljT* SSMALt} OMINANTINHERITAN For an AD disorder, the simplest approach is to compare the observednumbers of affected offspring born to affected parents rvith u'hat lvould be expectedbased on the diseasepenetrance, i.c 50(/trif penetranceis complete.A 12 test can be used to see whether the observedand expectednumbers differ significantly. Care must be taken to ensure that a bias is not introduced by excluding parents who r'vereascertained through an affected child.

X;.:' :'$#MAL f INHERITAN C E RECESSIVE For disordersbelievedto showAR inheritance,formal segregation analvsisis much more difficult This is becausesomecoupleswho are both carriers will by chance not have any affected children Usuall,v such couples and their healthy offspring will not be ascertained To illustrate this, consider 64 possible sibships of size 3 in which both parents are carriers, drawn from a large h1-potheticalpopulation (Table 8.5).The sibship structure shown in T[ble 8 5 is that which rvould be expectedon average'assuming that the birth of an affectedchild doesnot deter the parentslrom har.ingfurther children. In this population, on average,27 of the 64 sibships will not contain any affected individuals. This can be calculatedsimply 3/+,i.e.3/+ x 3/a x'/, = t'/,,0 Therefore, when the bl cubing familics are analyzed,these 27 sibships containing only healthy

S E G R H G A T IC AN INA L Y S IS Segregationanalysisrefers to the study of the rvar: in which a disorderis transmitted in families so asto establishthe underh'ine

individuals will not be ascertained.This is referred to as truncate or intompleteascertainment.If this is not taken into account, a

poputation 64 sibshipseachof size3, in whrchboth thatcontains Tabte8.5 Expected sibshipstructurein a hypothetical in ihatthe 27 parentsare carriersof an autosomal is madefor truncateascertainment, recessive lf no aLlowance disorder. (= 0 43)wiLL be obtained ratioof 481111 thena falsetyhighsegregation sibships with no affected caseswiL[notbe ascertained, No.of affectedin sibship

Structureof sibship

No.of sibships

No.of affected

Totalno.of sibs

3

rrr

1

3

3

2

TT! NTT

3 3 3

6 6 6

9 9 9

rnn trnr

9 9 9

9 9 9

27 21 2l

ntr!

21

0

81

oq

48

192

rrr

1

!l!

0 TotaI

129

I

MATHEMATICAL ANDPOPULATION GENETICS

falsely high segregationratio of 0.43 will be obtained instead of the correct value of 0.25. Mathematical methods havebeen devisedto cater for truncate ascertainment, but analysis is usually further complicated by the problems associatedwith achieving full or complete ascertainment. In practice it can be very difficult to 'prove' that a disorder showsAR inheritance unless accuratemolecular or biochemical methods are available for carrier detection. Affected siblings (especiallywhen at least one is female) born to unaffected parents usually suggestsAR inheritance, but somatic and germline parental mosaicism (p 114), nonpaternity (p. 333) and other possibilities need to be considered. There are some good examples of conditions originally reported to follow AR inheritance but subsequently shown to be dominant with germline or somatic mosaicism,for example osteoBenesis imperfecta and pseudoachondroplasia However,a high incidenceofparental consanguinityundoubtedly provides strong supportive evidence for AR inheritance, a point first noted by Bateson and Garrod as long ago as 1902 (p 7,107).

allelesofgenes at different loci segregateindependently. Although this is true for genes on different chromosomes, it is not always true for genesthat are located on the samechromosome, i.e. genes that are sltntenic. If two loci are positioned close together on the same chromosome,so that allelesat these loci are inherited together more often than not, theseloci are said tobe linked,.In effect, the two loci are sufficiently close together for it to be unlikely that they will be separatedby a cross-over,or recombination, during meiosisl(Fig.85) Alleles at linked loci on the samechromosomeare said to be in nupling, whereas those on opposite homologous chromosomes are described as being in repulsion.This is known as the linhage phase.Th,asin the parental chromosomesin Fig. 8.5C, A and B, as well as a and b, are in coupling, whereasA and b, as well as a and B, are in repulsion

RECOMBINATION FRACTION The recombinationfraction, which is usually designated as 0 (Greek theta), is a measureof the distance separatingtwo loci, or more preciselyan indication of the likelihood that a cross-over will occur betweenthem. If two loci are not linked then 0 equals 0.5 as, on average,Benesat unlinked loci will segregatetogether during 50o/oof all meioses.If 0 equals 0.05, this means thar on

G E N E T IL CIN K A GE Mendel's third law - the principle of independent assortment - statesthat members of different gene pairs assort to gametes independently of one another (p. 5). Srared more simply, the

averagethe syntenicalleleswill segregatetogether 19 times out of 20, i.e. a cross-overwill occur betweenthem during, on average, only I in 20 meioses.

ParentaI chromosomes

G am e t e chromosomes

Proportion5[o/6)

25

25

25

2s

25

25

25

25

>45 >45

<5

<5

Fig.8.5 130

Segregatron at metosis ofalleles attwolociIn(A)theLocr areon drfferent chromosomes andin (B)theyareonthesamechromosome b u t w r d e t y s e p a r aHt eedn c e t h e s e t a o rcei n o t L i n k e d a n d t h e r e i s i n d e p e n d e n t a s sI no(rC tm t)heenl ot cai r e c l o s e l y a d j a c e n t s o t h a t separation bya cross-over is unlrkety, i e thelocraretinked

GENETICS AND POPULATION MATHEM,ATICAL

CENTIMORGANS The unit of measurementfor genetic linkage is known as a map unit or centimorgan(cM).If two loci are I cM apart, a cross-over occurs between them during, on average,only 1 in every 100 meioses,i e. 0 = 0.01. Centimorgansare a measureof the genetic or linkage distance between two loci. This is not the same as physical distance,which is measured in basepairs (kb - kilobases: 1000basepairs:Mb - megabases: 1000000basepairs). The human genome has been estimated by recombination studies to be about 3000cM in length in males.As the physical length of the haploid human genomeis approximarell'3 x lOqbp, l cM correspondsto approximately106bp (l Mb or 1000kb). However, the relationship between linkage map units and physical Iength is not linear. Some chromosome regions appear to be particularly prone to recombination, so-called 'hot spots', and for reasonsthat are not understoodrecombinationtends to occur less often during meiosis in males than in females, in whom the genome 'linkage' length has been estimated to be 4200cM Generally in humans there are one or two recombination events betweeneachpair of homologouschromosomesin meiosisI, with a total of around 40 acrossthe entire genome. Recombination events are rare close to the centromeresbut relativelv common in telomeric regions.

LIN KA G AN E A L Y S IS Linkage analysishas proved to be an extremely valuabletool for mapping genes(p. 74). The basic methodology involvesstudy of the segregationof the diseasein large families with polymorphic markers from each chromosome Eventually a marker will be identified that co-segregateswith the diseasemore often than would be expected by chance, i.e. the marker and diseaseloci are linked. The mathematicalanalysistends to be very complex, particularly if many closelyadjacentmarkersare being used,asin multipoint linkage analysri However, the underlying principle is relativelystraightforwardand involvesthe use oflikelihood ratios, the logarithms of which are known as LOD scores(/ogarithm of

8

more likely that the diseaseand marker loci are closelylinked (i.e. 5cM apart) than that they are not linked. It is generally agreed that a LOD score of *3 or more is confirmation of linkage. This would yield a ratio of 1000 to I in favor of linkage, but, becausethere is a prior probability of only I in 50 that any two given loci are linked, a LOD score of *3 means that the overall probability that the loci are linked is approximately 20 to l, i.e. r/r,,1' 1. Th. importance of taking prior probabilities [1000 x into account in probability theory is discussedin the section on Bayes'theorem (p. 330).

A

'simple'example

Consider a three-generationfamily in which several members haveanAD disorder (Fig. 8.6). A and B are allelesat a locus that is being tested for linkage to the diseaselocus. To establishwhether it is likely that these two loci are linked, the LOD scoreis calculatedfor various valuesof 0. The value of 0 that gives the highest LOD score is taken as the best estimate of the recombination fraction. This is known as a maxlmum likelihood method. To demonstrate the underlying principle, the LOD score is calculatedfor a value of 0 equal to 0.05. If 0 equals 0.05 then the loci are linked, in which case the diseasegene and the B marker must be on the samechromosomein II2, as both of these characteristicshavebeen inherited from the mother. Thus in II2 the linkage phaseis known: the diseaseallele and the B allele are in coupling Therefore the probability that IIII will be affected and will alsoinherit the B marker equals0.95, i.e. 1 - 0. A similar result is obtained for the remaining three members of the sibship in generationIII, giving a value for the numerator of (0.95)1.If the loci are not linked, the likelihood ofobserving both the disease and marker B in IIIl equals0.5.A similar result is obtainedfor his three siblings,giving a value for the denominator of (0.5)4. Therefore the LOD score for this family given a value of 0 = 0 . 0 5e q u a l s l o g 1 e 0 . 9 5 4 / 0=. 5l o4 g r 61 3 . 0 3 2= l . l 2 F o r a v a l u e

the orlds).

L O DS C O R E S When studying the segregationof allelesat two loci that could be linked, a seriesof likelihood ratios is calculatedfor different values of the recombination fraction (0), ranging from 0 = 0 to e = 0.5. The likelihood ratio at a given value of 0 equalsthe likelihood of the observeddata if the loci are linked at recombination value of 0 divided by the likelihood of the observeddata if the loci are not linked (0 = 0.5). The logarithm to the base 10 ofthis ratio is known as the LOD score(Z), i.e. LOD (0) = log10[L0lL(0.5)]. Logarithms are used becausethey allow results from different families to be added together. For example,when a researchpaper reports that linkage of a

F i s .8 . 6

diseasewith a DNA marker hasbeen identified with a LOD score (Z) of 4 at recombination fraction (e) 0.05, this means that the results,in the families studied,indicatethat it is l0 000 1l0a)times

pedigreeshowingsegregaiionof an autosomal Three-generation dominantdisorderand alteles(A and B) at a Locusthat may or may not be [inkedto the disease[ocus

AB

131

I

MATHEMATICALAND POPULATION GENETI CS

of e = 0, the LOD scoreequalslogtol{/0.54 = logle16 = 1.20.For = logro a valueof 0 = 0.1, the LOD scoreequalslog,o0.9110.51 10.498= 1.02.The highest LOD scoreis obtained for a value of

this graph the peaks represent possiblepositions of the disease locus,with the tallest peakbeing the most probablelocation.The troughs representthe positions of the polymorphic marker loci.

0 equals0, which is consistentwith the fact that ifthe diseaseand marker loci are linked then there have been no recombinations betweenthe two loci in members of generationIII.

Multipoint linkage analysis is used to define the smallest possible interval in which a diseaselocus is located, so that physical mapping methods can then be applied to isolate the diseasegene(p. 74).

To confirm linkage other families would haveto be studied by pooling all the results until a LOD score of *3 or greater was obtained. A LOD score of -2 or less is taken as proof that the loci are not linked. This less stringent requirement for proof of non-linkage (i.e. a LOD scoreof -2 comparedwith *3 for proof of linkage)is due to the high prior probability of aelso that any two loci are not linked.

M U L T I P OINLTINK A GEA N A L Y S IS Two-point linkage analysisis often used to map a diseaselocus to a specific chromosome region. This gives a rather rough or 'coarse' indication of the location of the diseaselocus.The next step often involves multipoint linkage analysis using a series of polymorphic markers that are known to map to the disease region. This processallows 'fine' tuning of the probableposition

MAPPING AUTOZYGOSITY This is an ingenious form of linkage analysis that has been used to map many rare AR disorders. Autozygosity occurs when individuals are homozygous at particular loci through identity by descent from a common ancestor In an inbred pedigree containing two or more children with a rare AR disorder it is very likely that the children will be homozygousnot only at the diseaselocus but also at closely linked loci. In other words, all affected relatives in an inbred family will be homozygous for markers within the region surrounding the diseaselocus.Thus a searchcan be made for sharedareasofhomozygosity in affected relatives using highly polymorphic markers such asmicrosatellites (p. l7). In a pedigree with a relatively large number of affected

of the diseaselocus within the narrow interval defined by the small number of previously ordered polymorphic marker loci.

individuals only a small number of shared homozygous regions will be identihed; one of these can be expected to harbor the

Using this approach the results of linkage studies with the various markers are analyzed bv a computer program that calculatesthe overall likelihood of the position of the disease locus in relation to the marker loci The results are presentedin

relevant diseaselocus, which can then be isolatedusing physical mapplng strategres. Autozygosity mapping can be applied in both small inbred families (Fie. 8.8) and in genetic isolates(p. 127) with a shared

the form of a likelihood ratio known as a locationsrare.This is calculatedfor different positions ofthe diseaselocus and a graph is drawn up of location scoreagainstmap distance(Fig. 8 7). On

common genetic ancestry, e.g. the OId Order Amish. It is a particularly powerful technique in large inbred families where more than one branch has affected individuals. Several of the genesthat causeAR sensorineuralhearing losshavebeenmapped in this way,aswell as a number of skeletaldysplasiasand primary microcephalies,for example.

L I N K A GD EISEOUILIBRIUM Linkage dtsequilibriumis defined formally as the associationof two allelesat linked loci more frequently than would be expectedby chance, and is also referred to * allelic association.The concept and the term relate to the study of diseasesin populations rather thanfh.milies.Inthe latter, an associationbetween specificalleles and the diseasein question holds true only within an individual family; in a separateaffected family a different pattern of alleles, or markers, at the same locus may show associationwith the disease- becausethe alleles themselves are polymorphic.The

ZXY U n i t so f g e n e t i c( l i n k a g ed) i s t a n c e

132

Fis.8.7 M u l t i p o iLnitn k a qaen a t y s A i s B a n dCr e p r e s e tnht ek n o w n [ i n k a gree t a t i o n s hoi pftsh r e ep o l y m o r p hmi ca r k e r L oXc.i Ya n dZ represent rndescending position orderof likelihood theprobabLe o f t h ed i s e a sleo c u s

rationale for studying allelic associationin populations is based on the assumption that a mutation occurred in a founder case some generationspreviously and is still causativeof the disease. If this is true, the pattern of markersin a small region closeto the mutation will have been maintained and thus constitutes what is termed theJbunderhaplotype.The underlying principles used in mapping are the sameas those for linkage analysisin families, the difference being the degree of relatednessof the individuals under study. In the pedigree shown in Fig. 8.6, support was

POPULATION MATHEMATICALAND GENETICS

I

Fi9.8.8 mapping ina famity with Autozygosity spondylocostal dysostosis Thefather D155117 D 15 5 1s 5 D 15 5 15 3 D15S983 D1sS131 D15S1026 D 1 5 5114 D1sS205 D1551046 D155127 D1551004 D 15 5 13 0 D 15 5 12 0

10 I 10 1

10

I J

1 6 I 5 10 3 1 7

I

2

,|

11 isthebrotherof l2's of individuat grandfather. Theregionof homozygosity is defined bvmarkersD15S155 and Dl55127 A mutation intheME5P2gene

5 1 10 6 16 4 2 11 7 6 4 2 3

r , r i e qq r r h q e n r r p n t l v s h o w n t o b e t h e r:rrco nf cnnndvlncnct:l

drrcnctncic in

t h i sp e d r g r e e

lls D15S117 10

5

Dlsslss 8 1 D 1 5 S 1 s 31 0 1 0 D 15 5 9 8 3 D15S131 D 15 S 10 2 6 D15S114 D155205 D15S1046 D1sS127 D15S1004 D155130 D15S120

1 15 1 6 I 5 10 3 1 1

6 16 4 2 11 7 6 4 2 1

obtained for linkage of the diseasegene with the B marker allele. Assumethat further studiesconfirm linkageofthese loci and that theA and B alleleshave an equal frequency of0.5. It would be reasonableto expect that the diseasegene would be in coupling with allele A in approximately 50o/oof families and with allele B in the remaining 50o/o.If, however, the diseaseallele was found to be in coupling almost exclusivelywith one particular marker allele,this would be an exampleof linkage disequilibrium. The demonstration of linkage disequilibrium in a particular diseasesuggeststhat the mutation causing the diseasehas occurred relatively recently and that the marker locus studied is very closely linked to the disease locus. For example, one of the very small number of the original mutations for sicklecell disease occurred in a B-globin gene that was closely adjacent to a very rare syntenic recombinant fragment length polymorphism (RFLP). Selection for this sickle-cell murarion, with only very rare recombination having occurred between it and the adjacenttightly linked locus, has resulted in a very high proportion of present-day sickle-cell alleles being in coupling with this otherwise rare RFLP. There may be pitfalls, however, in

3 1 7

whereby particular alleles enhance or diminish reproductive Iitness;and population admixture, where population subgroups with different patterns of allele frequencies are combined into a single study. Allowance for the latter problem can be made by using family-based controls and analyzing the transmission of test alleles using a method called the transmission/d.isequilibrium (TDT) (p 142). This uses the fact that transmitted and nontransmitted alleles from a given parent are paired observations, and examines the preferential transmission of one allele over the other in all heterozygousparents. The technique has been applied, amongstothers, to studiesbasedon sibling pairs that are discordant for the diseaseor condition under study.

ANDSOCIETAL INTERVENTION MEDICAL

interpreting haplotype data that suggestlinkage disequilibrium. Other possible reasonsfor linkage disequilibrium include: the rapid growth of genetically isolated populations leading to large

Recent developmentsin molecular biology, such as the human genome project (Ch. 23) and pilot studies using gene therapy (p. 342), have reawakenedconcern that future generations could have to cope with an ever increasingburden of genetic disease. The term eugenicswas first used by Charles Darwin's cousin, Francis Galton, to refer to the improvement of a population

regions of allelic associationthroughout the genome; selection,

by selectivebreeding.The notion that this should be applied to

133

8

MATHEMATICAL ANDPOPULATION GENETICS

human populations became popular during the early years of the twentieth century, culminating in the horrifying and totally unacceptablepractices of Nazi Germany. Ensuing revulsion led to the abandonmentof eugenicprograms in humans,with universal condemnation and agreementthat such programs have no place

that if there is complete selection against an AR disorder, so that no homozygotesreproduce, the number of generations(n) required for the allele frequency to change from q6 to qn equals l/q^- l/qs Therefore, for a condition with an incidence of approximately I in 2000 and an allele frequency of roughly 1

in modern medical practice. Sadly, however, these practices have continued by groups engagedin territorial conflicts - somewhat sanitizedby the term 'ethnic cleansing'.

in 45, if all affected patients refrained from reproduction then it would take more than 500 years (18 generations) to reduce the diseaseincidence by half and more than 1200 years (45 generations)to reduce the gene frequency by half, assuming an

Nevertheless,doctors who care for patients and families with hereditary diseaseare faced with a dilemma. On the one hand, by helping patients with serious genetic diseaseto survive and reproduce they are indeed increasingthe numbers of'bad genes' in society,thereby potentiallyadding to humanity's future genetic load. Such behavior could be interpreted as dysgenic.On the other hand, most medical practitioners would argue that their responsibility to an individual patient overrides their obligation to either contemporary societyor future generations. Before returning to this ethical debateit is worth considering the possible long-term effects of artificial selection for or against geneticdisorders.

averagegeneration time of 27 years. Now considerthe oppositesituation,where selectionoperating againsta seriousAR disorder is relaxedbecauseof improvement in medical treatment. More affectedindividuals will reach adult life and transmit the mutant allele to their offspring. The result will be that the frequency of the mutant allele will increaseuntil a new equilibrium is reached Using the formula p = I(l - 0 (p. 135), it can be shown that, when the new equilibrium is eventuallyreached,an increasein fitness from 0 to 0.9 will have resulted in a tenfold increasein the diseaseincidence.

X - L I N K E DR E C E S S I VDEI S O R D E R S A U T O S O M ADLO M I N A ND TI S O R D E R S Ifeveryone with anAD disorderwere successfullyencouragednot to reproduce,the incidenceofthat disorderwould declinerapidly, with all future casesbeing the result only of new mutations.This would have a particularly striking effect on the incidence of relatively mild conditions such as familial hypercholesterolemia, in which geneticfitnessis closeto l. Alternatively, if successful treatment became available for all patients with a seriousAD disorder that at present is associated with a marked reduction in genetic fitness, there would be an immediate increase in the frequency of the diseasegene followed by a more gradual leveling off ata new equilibrium level. If, at one time, all those with a seriousAD disorder died in childhood (f = 0) then the incidence of affected individuals would be 2u. If treatment raised the fitness from 0 to 0.9, the incidence of affected children in the next generationwould rise to 2p due to new mutations plus l.8p inherited, which equals3.8p. Eventually a new equilibrium would be reached,by which time the disease incidencewould haverisen tenfold to 20u. This can be calculated relatively easilywith the formula p = U(l - f)l/2 (p. 135), which can be expressedalternativelyas I = 2'p"/(l - f). The net result would be that the proportion of affected children who died would be lower (from 100o/odown to l0olo), but the total number affectedwould be much greater,although the actual number who died from the diseasewould remain unchaneedat 2[.

A U T O S O M ARLE C E S S I VDEI S O R D E R S

134

In contrast to an AD disorder, artificial selectionagainstan AR condition will haveonly a very slow effect. The reason for this difference is that in AR conditions most of the genesin a population are presentin healthy heterozygotes who would not be affectedby euqenicmeasures.It can be shown

When consideringthe effectsof selectionagainstthesedisorders, it is necessaryto take into accountthe fact that a large proportion of the relevant genes are present in entirely healthy female carriers, who are often unaware of their carrier status. For a very serious condition, such as Duchenne muscular dystrophy (p.297), with fitness equal to 0 in affected males, selectionwill have no effect unless female carriers chooseto limit their families. Ifall femalecarriersopted not to haveany children, the incidence would be reduced by two-thirds, i.e. from 3p to p. A much more plausible possibility is that effective treatment will be forthcoming for these disorders. This will result in a steadyincreasein the diseaseincidence.For example,an increase in fitness from 0 to 0.5 will lead to a doubling of the disease incidence by the time a new equilibrium has been established. - f)]/3 This can be calculatedusing the formula p [IM(l

(p.13s).

CONCLUSION In reality it is extremely difficult to predict the long-term impact of medical intervention on the incidence and burden of genetic disease.Non-directive genetic counseling could lead to a reduction in the number of affected children being born, but it is quite likely that many of these children will be 'replaced' by carrier siblings, either becausesuch couples compensateby having large families,or through the use of prenatal diagnosis,so that the overall effects on gene frequency are almost impossible to determine. Although it is true that improvements in medical treatment could result in an increased genetic load in future generations,it is equally possible that successfulgene therapy will easethe overall burden of thesedisordersin terms of human

POPULATToN GENETICS MATHEMATTCALAND

suffering. Some of theseargumentscould have been made many years ago for other major medical developments, such as the discoveryof insulin and antibiotics, that havehad overwhelming financial implications in terms of burgeoning pharmaceutical costs and an increasingly aging population. Ultimately it can reasonablybe argued that it is how a society copes with these challengesthat gives a true measure of the validity of its claim to be civilized.

FURTHER READING Allison A C 1954Protection afforded by sickle-cell trait againstsubtertian malarial infection BMJ i:290J94 A landmarhpaperproxiding cleareaidencethat thesichle-celltrait prouides ?rotectionagainstparasitemiabyfalciparum malaria. Emery A E H 1986Methodologyin medicalgenetics,2nd edn Churchill Livingstone,Edinburgh A usefulhandboohof basicpopulationgeneticsand mathematicalmethods for analyzing the resultsof geneticstudies FrancomanoC A, McKusickV A, BieseckerL G (eds)2003Medical genetic studies in the Amish: historical perspective,Am J Med Genet C Semin Med Genet l2l:14 HaldaneJ B S 1935The rateof spontaneous mutationof a human gene J Genet 31: 317-326 TheJirst estimateof the mutationrateJbr hemolhilia usingan indirectmethod. Hardy G H 1908Mendelianproportionsin a mixed population Science28: 49 50 A short letter in nhich Hardy pointetlout that in a largerandomlymating populationdominanl 'characters'pould not increaseat Ihe expense 0f recesstres Khoury MJ, BeatyT H, CohenB H 1993Fundamentalsof genetic epidemiology.Oxford University Press,New York A nmprehensit:etertbllb 0Jlllulation geneticsand its areasoJ'ouerlapwith epidemiologlt. Ott J 1991Analysisof human geneticlinkage JohnsHopkins University Press.Baltimore A detailedmathematice.l explanatiln of linbageanalysis. Vogel $ Motulsky A G 1997Human genetics,problems and approaches, 3rd edn Springer,Berlin Thedefnitixe textboohof humangeneticswith extensiteroaerageof mathemalicaI d,sp ecIs.

8

ELEMENTS Q According to the Hardy-Weinberg principle, the relative proportions ofthe possiblegenotypesat a particular locus remain constant from one generationto the next. @ Factorsthat may disturb Hardy-Weinberg equilibrium are non-random mating, mutation, selection for or againsta particular genotype,small population size and migration. O If an autosomalrecessivedisorder is in Hardy-Weinberg equilibrium, the carrier frequency can be estimated by doubling the squareroot of the diseaseincidence' @ The mutation rate for an autosomal dominant disorder can be measured directly by estimating the proportion of new mutations among all members of one generation. Indirect estimatesof mutation rates can be made using the formula: = U(1 - f)]/2 for autosomaldominant inheritance = I(l - f) for autosomalrecessiveinheritance = U\l - f)l/3 for X-linked recessiveinheritance. @ Otherwise rare single-genedisorderscan show a high incidencein a small population becauseof a founder effect coupled with geneticisolation. G) Wtt." a serious autosomal recessivedisorder has a relativelyhigh incidence in a large population this is likely to be due to heterozygote advantaBe. @ Ctosely adjacent loci on the same chromosome are regardedas linked if genesat theseloci segregatetogether during more than 50o/oof meioses.The recombination fraction (0) indicates how often two such genes will be separated(recombine)at meiosis. @ fn. LOD score is a mathematicalindication of the relative likelihood that two loci are linked. A LOD score of * 3 or greater is taken as confirmation of linkage. Twopoint linkage analysisis used to map a diseaselocus to a chromosomeregion. Multipoint linkage analysiscan then be used to determine the probable order of polymorphic loci within that region and to narrow down the size of the interval to be studied by physical mapping.

135

CHAPTER

PoVgenic and muttifactorial inheritance

Many disorders demonstrate familial clustering that does not conform to any recognized pattern of Mendelian inheritance. Examples include several of the most common congenital malformations and many of the common acquired diseasesof childhood and adult life (Box 9.1). These conditions show a definite familial tendency but the incidence in close relativesof affectedindividuals is usually around 24o/o,instead.of the much higher valuesthat would be seenif theseconditions were caused by mutations in single genes. As it is likely that many factors, both genetic and environmental, are involved in causing thesedisorders, they are generally referred to as showing muhifactorialinheritance.This doesnot mean that the underlying geneticmechanismsare well understood- in fact they are not! The prevailing view until recently has been that in multifactorial inheritance environmental factors interact with many genes to generate a normally distributed susceptibility. According to this theory, individuals are affectedif they lie at the

distribution generated by many genes, known as polygenes, each acting in an additive fashion, is plausible for physiological characteristicssuch as height and possibly blood pressure. However, for diseasestatessuch as insulin-dependent diabetes mellitus recent researchhas shown that the genetic contribution is not straightforward and probably involves many loci, some of which play a much more important role than others. Sequencingof the human genomehas shown that the 3 billion basepairs are 99.9o/oidentical in every person.This also means that individuals are, on average,0.lolo different geneticallyfrom every other person on the planet. And within this 0.lolo lies the mystery of why some people are more susceptibleto a particular illness, or more likely to be healthy, than other members of the population. Our increased knowledge of genetic variation at the level of single nucleotide polymorphisms (SNPs), togerher with high throughput SNP genotyping platforms, is starting to uncover diseasesusceptibility allelesfor many common diseases.

wrong end of the distribution curve. This concept of a normal

Box 9.1 Disorders whichshowmultifactoriaL inherrtance CongenitaI malformations Ctefttip/patate CongenitaI distocation ofthehip CongenitaI heartdefects NeuraItubedefects Pytoricstenosis Ta[ipes

136

Acquireddiseasesof chitdhoodandadutttife Asthma Autism Diabetes mettitus Epitepsy Glaucoma nyperrensron (Crohn Inftammatory boweIdisease disease andutcerat ve cotitis) lschaemic heartdisease lschaemic stroke Manicdepression Muttipte sc[erosis Parkinson disease Psoriasis Rheumatoid arthritis Schizophrenia

POLYGENIC INHERITANCE ANDTHE N O R M ADLI S T R I B U T I O N Before considering the impact of recent researchin detail, it is necessaryto outline briefly the scientificbasisofwhat is known as polygenicor quantitatiae inheritance. This involves the inheritance and expressionof a phenotype being determined by many genes at different loci, with each gene exerting a small additive effect. 'Additive'implies that the effectsof the genesare cumulative,i.e. no one geneis dominant or recessiveto another. Several human characteristics(Box 9.2) show a continuous distribution in the general population that closely resemblesa

Box 9.2 Humancharacteristics thatshowa continuous normaLdistribution B l o o dp r e s s u r e D e r m a t o g t y p h i c(sr i d g ec o u n t ) H e a dc i r c u m f e r e n c e Height In t e t tgre n c e S k i nc o t o r

INHERITANCE POLYGENIC AND MULTIFACTORIAL

9

normal distribution. This takes the form of a symmetrical bellshaped curve distributed evenly'about a mean (Fig. 9.1). The spread of the distribution about the mean is determined by the standard deviation. Approximately 680/o,9 5o/o and, 99.7o/o of observationsfall within the mean plus or minus one, two or three standarddeviations,respectively. It is possible to show that a phenotype with a normal distribution in the general population can be generated by polygenic inheritance involving the action of many genes at different loci, each ofwhich exertsan equal additive effect.This can be illustrated by considering a trait such as height. If height were to be determined by two equally frequent alleles,a (tall) and b (short), at a single locus, this would result in a discontinuous phenotype with three groups in a ratio of I (tall - aa):2 (average - ablba): I (short - bb). If the sametrait were to be determined by two allelesat each of two loci interacting in a simple additive way, this would lead to a phenotypic distribution of five groups in a ratio of I (4 tall genes):,t (3 tall * I short):6 (2 tall + 2 short):4 (1 tall + 3 short): I (4 short). For a system with three loci eachwith two allelesthe phenotypic ratio would be I : 6 : l5 : 2 0 : 1 5 : 6 : 1( F i g 9 . 2 ) . It can be seen that, as the number of loci increases,the distribution increasingly comes to resemble a normal curve, thereby lending support to the concept that characteristicssuch as height are determined by the additive effects of many genesat different loci. Further support for this concept comes from the study of familial correlations for characteristicssuch as height and, to a lesser extent, intelligence. Correlation is a statistical measureof the degree of associationof variablephenomena,or, in more simple terms, a measure of the degree of resemblance or relationship betweentwo parameters.As first-degreerelatives share, on average,50o/oof their genes (Thble 9.1), it would be reasonableto predict that, if a parameter such as height were

Fis.9.2 suchas height,with of genotypesfor a charactertstic Distribution one,two and three Locieachwith two atlelesof equatfrequency The valuesfor eachgenotypecan be obtainedfrom the binomial e x p a n s i o(np + q ) ( 2 " ) , w h epr e= q = 1 l 2 a n d n i s t h e n u m b e r o fl o c i

polygenic,the correlation between first-degree relativessuch as siblingswould be 0.5. Severalstudieshaveshown that the sib-sib correlation for height is indeed closeto 0.5. In reality, human characteristicssuch as height and intelligence are also influenced by environment, and possibly alsoby genesthat

ofrelationship Tabte9.1 Degrees

t Mean +l1sD+ (=0eo/o) -+25D+

Retationship

Proportionof genesshared

First degree Parenis Sibtings Children

lll2l

Seconddegree Unctes andaunts andnreces Nephews Grandparents Grandchildren Half-sib|.ings

11/41

Third degree Firstcousins Great-grandparents Great-grandchildren

{1/8}

[=950/o)

+ 3Sf) [=gg.zolo)

Fis.9.1 T h en o r m a(Lg a u s s i adni )s t r i b u t i o n

137

9

POLYGENIC ANDMULTIFACTORIAL INHERITANCE

are not additive in that they exert a dominant effect. These factors probably account for the observedtendency of offspring to show what is known as 'regressionto the mean'. This is demonstrated by tall or intelligent parents(the two are not mutually exclusive!) having children rvhoseaverageheight or intelligence is slightl-v lorverthan the averageor mid-parental value Similarlg parentswho are verv short or of low intelligencetend to have children whose averageheight or intelligence is lower than the generalpopulation average,but higher than the averagevalue of the parents.If a trait were to show true polygenic inheritance with no erternal influences,the measurementsin offspring would be distributed evenly around the mean of their parents' values.

. MULTIFACTORIAL INHERITANCE THELIABILITY/THRESHOLD MODEL Efforts have been made to extend the polygenic theory for the inheritance of quantitative or continuous traits to trl to account for discontinuoas multifactorial disorders.According to the liability/threshold model, all of the factors that influence the development of a multifactorial disorder, rvhether genetic or environmental, can be consideredas a single entity kno\\'n as liabilitr.. The liabilities of all individuals in a population form a continuous variable,which has a normal distribution in both the generalpopulation and relativesofaffected individuals.However, the curves for theserelativeswill be shifted to the right, and the extent to which thev are shifted is directll, relatedto rhe closeness oftheir relationship to the affectedindex case(Fig 9.3). To account for a discontinuous phenotype (i.e. affected or not affected) rvith an underlying continuous distribution, it is proposed that a threshold exists above which the abnormal phenotype is expressedIn the generalpopulation the proportion bey-ondthe threshold is the population incidence, and among relatives the proportion beyond the threshold is the familial incidence It is important to emphasizeonce again that liabilit.vincludes all factorsthat contribute to the causeofthe condition. Looked at very simpll; a deleteriousliability can be viewed as consistingof a combination of several'bad' genesand adverseenvironmental factors. Liabilitv cannot be measured,but the mean liabilitv of a group can be determined from the incidence of the diseasein that group using statisticsof the normal distribution. The units ofmeasurement are standarddeviations.and thesecan be used to estimatethe correlation between relatives.

C O N S E O U E N COF E ST H EL IA B LI ITY/ T H R E S H O LMDO D E L

138

Part of the attraction of this model - and it should be emphasized once again that this is a h.ypothesis rather than proven fact - is that it provides a simple explanation for the observedpatterns of familial risks in conditions such as cleft lip,/palate, pyloric stenosisand spina bifida. For example:

Liabilitv

Fis.9.3 HypotheticaI lrability curvesin the generalpopulationand in 'etatrvesfor a hereditarydisorderin whichthe genetic rs muttifactoriaI oredisDosition

l. The incidence of the condition is greatestamong relatives of the most severelyaffected patients, presumably becausethey are the most extreme deviants along the liability curve. For example,in cleft lip,/palatethe proportion of affectedfirst-degree relatives (parents, siblings and offspring) is 60lo if the index patient has bilateral cleft lip and palate,but only 2oloifthe index patient has a unilateral cleft lip (Fig. 9 4) 2. The risk is greatestamong closerelativesof the index case and decreasesrapidly in more distant relatives.For example,in spina bifida the risks to lirst-, second- and third-degree relatives of the index caseare approximately4o/o,lo/o and lessthan 0.57o, respectivell'. 3. If there is more than one affected close relative, the risks for other relativesare increased.In spina bifida, if one sibling is affectedthe risk to the next sibling (if folic acid is not takenby the mother periconceptionally)is approximately4olo;if two siblings are affected, the risk to a subsequent sibling is approximately 10o/o 4. If the condition is more common in individuals of a particular sex, then relatives of an affected individual of the less frequently affected sex will be at higher risk than relatives of an affected individual of the more frequently affected sex. This is illustrated by the condition pyloric stenosis.Pyloric stenosisshorvsa male to female ratio of 5 : 1. The proportions of affected offspring of male index patients are 5.5o/ofor sons and 2.4o/ofor daughters, whereas the risks to the offspring of femaleindex patients are 19.4o/ofor sons and 7 .3o/ofor daughters. The probable explanation for these different risks is that in

INHERITANCE AND MULTIFACTORIAL POLYGENIC

9

Fis.9.4 Severe(A) and mild (B) forms of cleftLrp/palate

order for a female to be affected she has to lie at the extreme of the liability curve, so that her closerelativeswill also havea very high liability for developing the condition. As males are more susceptible to developing the disorder, risks in male offspring are higher than in female offspring regardlessof the sex of the affectedparent. 5 The risk ofrecurrence for first-degreerelatives(i.e. siblings and offspring) approximatesto the square root of the general population incidence.Thus, if the incidence is I in 1000,the sibling and offspring risk will equal approximately I in 32, or 3o/o.

try to disentanglethe possibleeffectsof common environmental factors. Estimates of heritabilitv for some common diseasesare given in Thble 9 2. The degree of familial clustering shown by a multifactorial disorder can be estimated by measuring the ratio of the risk to siblings of affected individuals compared with the general population incidence. This ratio of sib risk to population incidence is known as 1,..For example,in type 1 diabetes,where the UK population incidence is 0.4oloand the risk to siblings is 60lo,1,"is 15. For type 2 diabetesin Europe, 1",is estimatedat a more modest 3.5 (35o/osibling risk; 10o/opopulation risk).

HERITABILITY of variousdisorders of heritabiLrtv Tabte9.2 Estimates Although it is not possibleto assessan individual's liability for a particular disorder, it is possible to estimate what proportion of the etiology can be ascribed to genetic factors as opposed to environmental factors. This is referred to as heritabiliry, which can be defined as the proportion ofthe total phenotypic variance of a condition that is caused by additive genetic variance In statistical terms, variance equals the square of the standard deviation. Heritability is often depicted using the symbol hz and is expressedeither as a proportion of I or as a percentagevalue. Estimates of the heritability of a condition or trait provide an indication of the relative importance of genetic factors in its causation, so that the greater the value for the heritability the greaterthe role of geneticfactors. Heritability is estimated from the degree of resemblance between relativesexpressedin the form ofa correlation coefficient, which is calculated using statistics of the normal distribution. Alternatively, heritability can be calculated using data on the concordance rates in monozygotic and dizygotic twins. In practice it is desirableto try to derive heritability estimates using different types of relatives, and to measure the disease incidence in relatives reared together and living apart so as to

Disorder

Frequency(%)

Heritabitity

Schrzophrenia

1

85

Asthma

4

80

C l P f ltr n + c l e f tn a l a t e

01

76

Pytoric stenosis

03

75

Ankylosing spondylitis

02

70

Ctubfoot

01

68

.L^U- ^rU^ r-r^d,ry d - .r+L^s-ry, ,iu; r- >^ -s^o^) c

3

65

Hvoertenslon fessentrat)

5

62

ofthehip Congenital drslocation

0'l

60

andspinabifida Anencephaty

03

60

Pepticulcer

4

37

Cnnoenith ae l artdisease

05

35

139

POLYGENIC AND MULTIFACTORIAL INHERITANCE

I D E N T I FY INGE G NES T H A TC A U SE M UL T F I A C T OR IADLIS OR D E R S Multifactorial disorders are common and make a major contribution to human morbidity and mortality (p. 8) It is therefore not surprising that vigorous efforts are being made to try to identify genesthar contribute to their etiology.A number of strategieshave been used to searchfor diseasesusceptibility genes.Figure 9.5 illustratessomeof the methods employedin the hunt for genesassociatedwith type 2 diabetes.

2. Many multifactorial diseasesshow a variableageof onset so that the genetic status of unaffected family members cannot be known with certainty. 3. Most families in which a multifactorial diseaseis, or has been, present have only one or two living affected members so that the number of informative meioses' available for studv is usually very small. 4. Some apparent multifactorial disorders, such as coronary artery diseaseand schizophrenia, are probably etiologically heterogeneous, with different genetic and environmental mechanismsinvolved in different subtypesthat cannot be easily distinguished at the phenotypic level. This makes analysis of

L I N KA G E A N A L Y S IS

linkage results very difficult.

Linkage analysishas proved extremely valuablein mapping singlegenedisordersby studying the co-segregationof geneticmarkers

Despite these limitations, progress is being made towards identifying susceptibility loci using modifications of the approachesutilized for mapping single gene loci Some of this progress is discussedin Chapter l5 on the 'common diseases'.

with the disease(p. 13l). However,this type of approachis much more difficult in multifactorial disorders.for the followins reasons: 1 Ifa multifactorial disorder has a true polygenic underlying genetic susceptibility, in theory it is unlikely that alleles at a single locus will make a major contribution. It is extremely difficult mathematically to develop strategiesfor detecting linkage of additive 'polygenes',eachof which makesonly a small contribution to the DhenotvDe.

Biology (insulin

It has been recognized for some time that one of the best approacheswould be to undertake diseaseassociationstudies and linkage analysisutilizing a so-called'ideal'population. Such a population would be relatively large yet historically isolated and therefore geneticallyhomogeneous,with extensivemedical records dating back for many generations,a large tissue bank, good medical servicesand a cooperativewilling citizenship.The

Humanmodels e.9.IPF1 (M0DY 4)

Position e . 9 .N I D D M lC a l p a i n1 0

Animalmodels e . g .L e p t i no b / o bm o u s e

C a n d i d a tgee n e

I

ll

I

I

ll

I

lcandidatevar,ants

Association study

Cases Positiveassociation . R e p l i c a t ree s u l ti n o t h e rc o h o r t s . F u n c t i o n aal n a l y s i os f v a r i a n t

Negativeassociation Repeatthe processover again

Fis.9.5

140

(T2DN4) Strategy tofinddisease genesfortype2 diabetes susceptibrLity genesmaybeselected mettitus Candidate fromhumanmodels ( eg m o n o g e nfiocr m so fd i a b e t e sk )n,o w t e d goefb r o l o g(yi n s u t isne c r e t r oonr a c t i o np) o , s i t i o ncatto n i nogr a n i m am l o d e l sT h ec a n d i d a t e geneis screened to findvariants. whicharethentestedforassociation wrthT2DMbygenotyping cohortsofsubjects withT2DMand controts(Modrfred fromGtoyn A L 2003Thesearch fortype2 diabetes genesAgeingResRev2:111J27 withpermission )

INHERITANCE POLYGENIC ANDMULTIFACTORIAL

270000citizensoflceland havebeendeemedto representsuch an ideal population, and a genomicscompany,known as DeCODE Genetics,has been granted a licenceto set up a national medical databaseand undertake Beneticresearch.Similar initiatives are likely in other populations; recently,for example,the Center for Arab Genomic Studies (CAGS) has been establishedin Dubai. Although on the one hand these initiatives have raised serious concern about the issue of informed consent (p. 355), on the other hand they could lead to the relatively rapid isolation of genesthat make a significant contribution to human morbidity and mortality.

Affectedsibting-pair analysis Standard linkage analysis requires information regarding the mode of inheritance, gene frequencies and penetrance.For multifactorial disordersthis information is not usually available. One solution to this problem is to use a model-free method of linkage analysis that seeks to identify alleles or chromosome regions sharedby affectedindividuals.A common approachis to look for regionsof the genomethat are'identicalby descent'(IBD) in affected sibling pairs. If affected siblings inherit a particular allele more or less often than would be expectedby chance,this indicates that the allele or its locus is involved in some wav in causingthe disease Consider a set of parents with alleles AB (father) and CD (mother) at a particular locus. The probability that any two of their children will haveboth allelesin common is I in 4 (Fig. 9.6). The probability that they will have one allele in common is I in 2, and the probability that they will have no allelesrn common is 1 in 4. If siblings who are affected with a particular disease show deviation from this 1 :2: I ratio for a particular variant, this implies that there is a causalrelationship between the locus and the disease. Many genome-wide scans (p. 74) have been performed for various disorders and, although a number of loci have been mapped, the number of diseasesusceptibility genes identified

9

by this approachis disappointingly small. One reason,probablS is the complex nature of multifactorial disease,with numerous genes of modest effect interacting with one another and the environment. Some studies are simply underpowered and recent efforts have concentrated on large collections of carefully phenotl ped affectedsibling pairs.

L i n k a g ed i s e q u i t i b r i u m a p p i n g Once a chromosomeregion that appearsto confer susceptibility has been identified, the next step is to reduce the geneticinterval by 'frne mapping'. The most powerful method uses linkage (LD) (p. 132) mapping to construct haplotypes diser1uilibrium by genotyping SNPs within the region. Historical cross-over 'blocks' points reduce the genetic interval by defining LD (Fig. 9 7). Candidategeneswithin the region are then sequenced to find DNA variants that can be tested for association with the disease.

SN TUDIES ASSOCIATIO The study of diseaseassociationsis undertaken by comparing the incidence of a particular variant in affectedpatients with the incidence in a carefully matched control group. This approach 'case-control' study. If the incidencesin is often describedas a the two groups differ significantly,this provides evidence for a positrreor negativeassociation. The polymorphic system that has frequently been studied is the HLA (human leukocyteantigen) histocompatibility complex on chromosome 6 (p. 188). One of the strongest known HLA associationsis that between ankylosing spondylitis and the F.27 allele This is present in approximately 90o/oof all patients and in only 5oloof controls. The strength of an HLA associationis indicated by the ratio of the risk of developing the diseasein those with the antigen to the risk of developing the diseasein those without the antigen (Thble 9.3). This is known as the odds ratio and it gives an indication of how much more frequently the diseaseoccurs in individuals with a specificmarker than in those without that marker. One of the maior difficulties with diseaseassociationsis to establishhow they should best be interpreted In particular it is important to try to rule out a chance or spurious observation by ensuring as far as possible that the proposed associationis biologically plausibleand that the patient and control groups are

A l l e l e si n common

Ratio

ACAC21 ADI

F12 BC-I BDOl

Fis.9.6 Theprobabitity thaisibLings wrLL have2,1or 0 parentaI a[[eles in c o m m o nS i g n i f i c adnetv i a t i of rno mt h e1 : 2 : 'rla t i oi n d i c a t e t hsa t related thelocusis causatly to thedisease

closelymatched. If evidencefor a strong associationis forthcoming, this suggests that the allele encoded by the marker locus is directly involved in causingthe disease(i.e. a susceptibilitylocus) or that the marker locus is in linkagedisequilibrium (p. 132) with a closely linked susceptibility locus. When considering disease associationsit is important to remember that the identification of a susceptibility locus doesnot mean that the definitive disease gene has been identified. This is illustrated by the association of HLA-P.Z7 with ankylosing spondylitis Although this is one of the strongest diseaseassociationsknown, only lolo of all individuals with HLA-827 develop ankylosing spondylitis, so

141

9

POLYGENIC AND MULTIFACTORIAL INHERITANCE

)

;

t,

r

i j I

|

):

3\'

:t,

) trBltli

:*.t:*''\:,

l'

&

Fig.9.7 TheLDstructure of glucokinase 12 vatues the84 SNPsacrossa between 116-kb regron arepresented An 12value of1 indicates thattwoSNPsare[inked Therearetwobtocks of LDwithinthe g L u c o k i n agseen e( o u t t i n ei ndr e d )

Table9.3 CalcuLation of oddsratiofor a disease association Allele I

Atlele2

Patients

a

b

Controls

c

d

Oodsratio

= alc : bld = ad/bc

that many other factors, genetic and/or environmental, must be involved in causingthis condition. Positive results from associationstudies require replication in other cohorts.A common reasonfor false-positiveassociation is population stratification, where the population contains a number of subsetsand both the diseaseand the allele happen to be common within that subset.A famous example,reported in a study by Lander and Schork, showed that in a San Francisco population HLA-AI is associatedwith the ability to eat with chopsticks.This associationis simply explained by the fact that HLA-AI is more common amongst Chinese people than caucasians!

Transmission disequilibrium test

142

One way to overcomepopulation stratificationproblems is to use family-based controls. The transmissiondisequilibriumtest (TDT) requires a collection of trios that consist of an affected proband and both parents(regardlessofaffection status).Parentswho are heterozygousfor the marker allele in question are selectedand the number of times this allele is transmitted to their affected

offspring is compared to the number of times the other allele is transmitted. Overtransmissionof the marker allele strengthens the evidence for association,but definitive evidence that a variant is a predisposingallele usually requires functional studies.

WHOLE.GENOM AE SSOCIATIO SN TUDIES In whole-genomeassociationstudies, researchers compare the entire genomein a case-control study, rather than looking at just one variant at a time. This powerful new method can thereforebe used to identify new diseasesusceptibility genes.Technological advancesmean that it is now possiblesimultaneouslyto test up to 500000 SNPs on a single microarray (a 500K 'SNP Chip'). In the UK, the Wellcome tust has funded a large project to perform whole-genomeassociationstudiesin approximately3000 controls and 2000 patients affected with tuberculosis,coronary heart disease,type 1 diabetes, type 2 diabetes,rheumatoid arthritis, Crohn diseaseand ulcerativecolitis, bipolar disorder or hypertension(http :,/,/www.wtccc.org.uk).

InternationalHapMapProject(http://www. hapmap.org) Whilst it is estimatedthat there may be up to l0 million SNPs in the human genome,many SNPs are in linkage disequilibrium (p. 132) and are therefore co-inherited. Regions of linked SNPs are known as haplotypes.A single SNP can be chosenthat 'tags' a haplotype; these are describedas tag,SNA. The International HapMap Project is identifying haplotypes in different populations (Thble9.4) and it is estimatedthat the total number of haplotypetagging SNPs is between 300000 and 600000 depending on the population studied. This means that whole-genomeassociation studies of approximately 500000 tag SNPs can resr for the majority of geneticvariation in the human genome.The HapMap

INHERITANCE AND MULTIFACTORIAL POLYGENIC

Tabte9.4 Pooulations studied intheInternationa-

9

F U R T H ERRE A D I N G

l-.1:nMan Prnioet ""H""ts

Town/country

Ancestry

Samplesanalysed

lbadan. Nigeria

Yoruba

30 trios(adutt andboth parents)

Tokyo.Japan

Japanese

45unretated rndivrduats

Beijing. China

Chinese

45unrelated individuats

USA

Northern and westernFrtrnnean

30 trios(adutt andboth narents)

Project will provide a valuableresourceto learn more about the genetic predispositionsthat underlie common diseasessuch as cardiovascular disease,diabetes, cancer, autoimmune and psychiatric disorders.

C O N C L U SION The term multifactorial has been coined to describethe pattern of inheritancedisplayedby a large number of common disorders that show familial clustering and are probably caused by the interaction of genetic and environmental factors. The genetic mechanismsunderlying thesedisordersare not well understood. The liability/threshold model should be viewed as an attractive hypothesis rather than as proven scientific fact. Researchin molecular biology is beginning to unravel some of the mysteriesof multifactorial inheritance.The past l0years has seen the recruitment of large numbers of patients and controls to create valuable DNA resources for study, and further collections are in progress. For example, the UK Biobank Project (http://www.ukbiobank.ac.uk) aims to collect DNA samplesand information on the health and lifestyle of 500000 volunteers aged between 40 and 69years. Over the next 20-30years, approved researcherswill be able to use these resourcesto study the progression of illnessessuch as cancer, heart disease,diabetes and Alzheimer disease.From this they hope to develop new and better ways of preventing, diagnosing and treating such problems. Technologicaldevelopmentsin SNP typing, together with an increasedunderstandingof geneticvariation, mean that the next few yearsare likely to prove very exciting as these new approaches are applied to polygenicdisease.Examplesof progressto date are describedin Chapter 15. This emphasis on the underlying genetic contribution to multifactorial disorders should not in any way detract from the importance of trying to identify major environmental causal factors. This is amply demonstrated by the beneficial effect of folic acid supplementation in preventing neural tube defects

b.247)

Botstein D, Risch N 2003 Discovering genotypesunderlying human for mendelian disease,future approachesfor phenotypes:past successes complexdiseaseNature Genet Suppl 33:228-237 reaiewarticle that suggests Comprehensiae for identtJyinggenes future strategies underlying comPlex disease. Carlson C S, Eberle M A, Kruglyak L, Nickerson D A 2004 Mapping complex diseaseloci in whole-genomeassociationstudies.Nature 429: 446452 stud,ies to id'entifu Article deuribing the useofohole-genomeassocation polygenes. FalconerD S 1965The inheritance of liability to certain diseasesestimated from the incidenceamongrelativesAnn Hum Genet 29:51J6 The original erpositionofthe liability/threshold modeland hoa correlations betmeenrelatiaescan beusedIo calculateheritability. FraserF C 1980Evolution of a palatablemultifactorial threshold model. Am J Hum Genet 32: 796-813 An amusingand'reader-friendllt' accountofmodek Prllosed to exllain muI t tfact ori aI in heri t ance.

ELEMENTS Q fn" concept of multifactorial inheritance has been proposed to account for the common congenital malformations and acquired disorders that show nonMendelian familial aggregation. These disorders are thought to result from the interaction of genetic and environmental factors. intelligence, @ Hu-an characteristicssuch asheight and which show a normally distributed continuousdistribution in the general population, are probably caused by the additive effectsofmany genes,i.e. polygenic inheritance. @ According to the liability,/threshold model for multifactorial inheritance,the population's geneticand environmental susceptibility, which is known asliability, is normally distributed.Individuals areaffectediftheir liability exceeds a threshold superimposedon the liability curve. @ R"crr.t.ttce risks to relatives for multifactorial disorders are influenced by disease severity, degree of relationship to the index case,number of affected close relativesand, ifthere is a higher incidencein one particular sex,the sex of the index case. @

Heritability is a measureof the proportion of the total of a characteror diseasethat is due to the senetic

;::1il:: @ Loci that contribute to susceptibility for multifactorial disorders can be identified by (a) a search for disease associationswith variants in candidate genes,(b) linkage analysis looking, for example, for chromosomal regions that are identical by descent in affected sibling pairs and (c) whole-genomeassociationstudies to compare genetic variation acrossthe entire genome in large case-control studies.

143

145

CHAPTER

'Blood is very a specialjuice.' Johann Wolfgang von Goethe, in

(Faust

I' (1808)

It has been estimated that more than a quarter of a million people are born in the world eachyear with one of the disorders of the structure or synthesisof hemoglobin (Hb), the so-called hemoglobinopathies. As a consequencethe hemoglobinopathies havethe greatestimpact on morbidity and mortality of any single group of inherited disorders. The mobility of modern society means that appreciable numbers of individuals from areas of high prevalenceoften constitute significantminority populations with a high risk of the hemoglobinopathiesin countries where they are uncommon in the endemic population. This means that it is incumbent upon all clinicians to be familiar with this group of disorders.In addition, during the past 40years the hemoglobinopathieshave also served as a paradigm for our understandingof the pathology of inherited diseasein humans at the clinical, protein and DNA levels In order to understand better the various types of hemoglobinopathiesand their clinical consequences, it is first necessary to consider the structure, function and synthesisof Hb.

S T R U C T U ROEFH E M O G L O B I N Hb is the protein present in red blood cells that is responsible for oxygen transport. There are large quantities of Hb in blood, making it amenableto analysis.

who possessedboth variants had children with normal Hb, offspring who were heterozygousfor only one Hb variant, aswell as offspring who, like their parents, were doubly heterozygous for the two Hb variants. These observationsprovided further support for the suggestionthat at least two different geneswere involved in the production of human Hb. Shortly thereafter,the amino-terminal amino-acidsequenceof human Hb was determined and showedvaline-leucine and valinehistidine sequencesin equimolar proportions, with two moles of eachof thesesequencesper mole of Hb. This was consistentwith human Hb being made up of a tetramer consistingof two pairs of different polypeptidesreferred to as the cr- and B-globin chains. Analysis of the iron content of human Hb revealedthat iron constituted 0.35o/oof its weight, from which it was calculated that human Hb should have a minimum molecular weight of 16000Da. In contrast, determination of the molecular weight of human Hb by physical methods gave values of the order of 64000Da, consistent with the suggestedtetrameric structure, u282,with eachofthe globin chainshavingits own iron-containing group,heme (Fig. 10.1). Subsequentinvestigatorsdemonstratedthat Hb from normal adults also contained a minor fraction, constituting 2-3o/oof the total Hb, with an electrophoretic mobility different from the majority of human Hb. The main component was called Hb A, whereasthe minority component was called Hb 42. Subsequent studies revealedHb A, to be a tetramer of two normal c-globin chains and two other polypeptide chains whose amino-acid sequenceresembled most closely the p-globin chain and was designareddelta (6).

PR O T EI A NN A L Y S IS In 1956, by fractionating the peptide products of digestion of human Hb with the proteolytic enzyme, trypsin, Ingram found 30 discrete peptide fragments.Trypsin cuts polypeptide chains at the amino acids arginine and lysine.Analysis of the 580 amino acids of human Hb had previously shown there to be a total of 60 arginine and lysine residues,suggestingthat Hb was made up of two identical peptide chains with 30 arginine and lysine residues on eachchain. At about the same time a family was reported in which two hemoglobin variants, Hb S and Hb Hopkins II, were both present in some family members.Severalmembers of the family

D E V E L O P M ELNE X P R E S S I O N O FH E M O G L O B I N Analysis of Hb from a human fetus revealedit to consist primarily of a Hb with a different electrophoretic mobility from normal Hb A, which was called fetal Hb or Hb E Subsequentanalysis showed Hb F to be a tetramer of two cr-globin chains and two polypeptidechainswhosesequenceresembledthe p-globin chain and which weredesignatedgamma($. Hb F makesup somewherein the resion of 0.5oloof hemoelobin in the blood of normal adults.

147

10

HEMOGLOBIN ANDTHEHEMOGLOBINOPATHIES

Porphyrin molecule Y ol k s a c

Total Hb [o/o)

36 Prenatal life[months)

Birth 3 6 Postnatal life(months)

Fig.10.2 Hemogtobrn synthesrs duringprenataL andpostnataI development (AfterHuehnsE R, Thereareseveralembryonic hemogtobins Shooter E N41965Humanhaemoglobins J MedGenet2:48-90. withoermission )

Fis.10.1 Diagrammatrc representation of one of the g[obinchainsand a s s o c i a t epdo r p h y r i nm o L e c u [oef h u m a nh e m o g t o b i n

GLOBIN CHAIN STRUCTURE Analysis of the structure of the individual globin chains was initially carried out at the protein level.

Analysis of Hb from embryos earlier in gestationrevealsthere to be a developmentalor ontological successionof different embryonic Hbs, Hb Gower I and II, and Hb Portland, which are produced in varying amounts at different times of gestation. Subsequentanalysishas revealedthat these various Hbs, which are expressedtransiently in development, are in fact tetramers of various combinations of cxor cx-likezeta (() chains with B or B-like y- and epsilon (e)-globin chains (Thble 10.1).Apart from the transient expressionof the ( chain early in embryonic life, the cr-globin chain gene is expressedthroughout development. Similarly, e-globin chain expressionoccurs early in embryonic life, with y-chain expression occurring throughout fetal life followed by increasing levels of expression of the B-globin chain towards the end of fetal life. The ordered expressionof these chains results in the various Hb tetramers seen durins development(Fig. 10.2).

T a b t e1 0 . 1 H u m a nh e m o q l o b i n s

148

Stagein development

Hemoglobin

Embryonic

GowerI Gowerll PortlandI

Structure

Proportionin normatadutt (%)

P R O T E ISNT U D I E S Amino-acid sequencingofthe various globin polypeptides, carried out in the 1960sby a number of different investigators,showed that the a-globin chain was l4l amino acids long whereasthe B chain contained 146amino acids.The cr and p chains were found to have a similar sequenceof amino acids but were by no means identical. Analysis of the amino-acid sequenceof the 6 chain showed it to differ from the B-globin chain by l0 amino acids. Similar analysis of the y-globin chain showed that it also most closely resembled the p-globin chain, differing by 39 amino acids.In addition, it was found that there were two types of fetal Hb in which the y chain contained either the amino acid glycine or alanine at position 136; these were consequentlynamed (G)y and (A)y, respectively. More recently, partial sequence analyses of the ( and e chains of embryonic Hb suggestthat ( is similar in amino-acid sequenceto the cr chain, whereast resemblesthe B chain. Thus, it appearedthat there are two groups of globin chains, the cr-like and B-like. all of which seem to be derived from an ancestralprimordial Hb gene that has undergone a number of geneduplications,and diverged during the courseofevolution.

\202

Ctztz

GLOBIN G E N EM A P P I N G

Fetal

F

(]z^{z

<1

Adutt

A A2

szFz o,zh

97-98 2:3

The first evidence for the arrangement of the various globin structural genes on the human chromosomeswas provided by analysis of the Hb electrophoretic variant, Hb Lepore. Comparison of trypsin digests of Hb Lepore with Hb from

ANDTHEHEMOGLOBINOPATH1ES HEMOGLOBIN

GLOBIN G E N ES T R U C T U R E

normal personsrevealedthat the u chains were normal, whereas the non-o chains appearedto consist of an amino-terminal 6-globin-like sequenceand a carboxy-terminal B-globin-like sequence. It was therefore proposed that Hb Lepore could represent a 'fusion' globin chain that had arisen as a result of a cross-over

Detailed information on the structure of the globin geneshasbeen made possibleby DNA techniques(p. 57). Immature red blood cells, reticulocytes, provide a rich source of globin messenger RNA (mRNA) for the synthesis of complementary DNA (cDNA) becausethey synthesize little else! Use of B-globin cDNA for restriction mapping studies of DNA from normal persons revealed that the non-o(, or B-like, globin genes are located in a 50-kilobase(kb) stretch on the short arm of chromosome l1 (Fig l0 4). The whole ofthis 50-kb stretch has been cloned and the nucleotide sequenceof each of the various globin structural

coincidentalwith mispairing of the 6- and B-globin genesduring meiosis as a result of the sequencesimilarity of the two genesand the close proximity of the 5- and B-globin genes on rhe same chromosome (Fig. 10.3). If this hypothesis was correct, ir \4'as argued that there should also be an 'anti-Lepore' Hb, i.e. a B-6-globin fusion product in which the non-o-globin chains contained $-chain residues at the amino-terminal end and 6chain residues at the carboxy-terminal end. In the late 1960s, a new Hb electrophoretic variant, Hb Miyada, was identilied by investigators in Japan in which analysis of trypsin digests did, indeed, show it to contain B-globin sequenceat the aminoterminal end and 5-globin sequenceat the carboxy-terminalend, as predicted

genesis known. Of particular interest are regions with sequences similar to thoseof the globin structural genesbut which are nonfunctional and do not produce an identifiablemessageor protein product, that is, pseudogenes(p. l6). Similar studies of the cr-globin structural genes have shown that there are, in fact, two u-globin structural genes,cx,1and clZ, locatedon the short arm of chromosome 16 (seeFig 10.4).DNA sequenceanalysishas revealednucleotide sequencedifferences betweenthesetwo structural geneseventhough the cr-globin chains produced havean identicalamino-acidsequence- evidenceofthe 'degeneracy'of the geneticcode In addition, there are pseudo-tx, pseudo-( and ( genesto the 5' side of the cr-globin genes,as well asx recently discoveredtheta (O)-globingeneto the 3' side of the

Further evidenceat the protein level for the physicalmapping of the human globin geneswas provided b1,the report of another Hb electrophoretic variant, Hb Kenya. Amino-acid sequence analysisof this variant suggestedthat it was a y-B fusion product with a cross-over having occurred somewhere between amino acids 8l and 86 in the two globin chains Ir was suggestedthat, in

ul-globin gene This latter gene,whose function is unknown, is ofinterest because,unlike the globin pseudogenes,which are not exprcssed,its structure is compatiblewith expression.It has been suggestedthat it could be expressedin very early erythroid tissue such as the fetal liver and yolk sac.

order for this fusion polvpeptide to have occurred, the y-globin structural Bene must also be in close physical proximitv to the B-globin gene. Little evidence was forthcoming from protein studies abour the mapping of the u-globin genes.The presence of normal Hb A in individuals who, by family studies, should have been homozygous for a particular o-globin chain variant or obligate

ANDCONTROL SYNTHESIS EXPRESSION OFHEMOGLOBIN

compound heterozygotes (p. 103) suggested that there could be more than one cr-globin gene.In addition, the proportion of the total Hb made up by the u-globin chain varianr, in persons heterozvgousfor those variants,was consistentlylower (lessthan 20o/o)than that seen with the B-globin chain variants (usuall-v

From in-aitro translation studies with reticulocyte mRNA from normal persons it is known that c- and B-globin chains are synthesized in roughly equal proportions In-aitro studies of globin-chain synthesis have shown, however, that B-globin

more than 30o/o),suggestingthat there could be more than one a-globin structural gene.

an I l -L e p o r e M i s al i gn m e n t

t-isaro

p

-..-|

Fis.10.3 M e c h a n i s muonfe q u a L c r o s s i n g o v e r w h i c h g e n e r a t e s H b L e p o r e a n( A dd aa np t rt-eLdefpr o m r eW e a t h DeJr, aCtLt e g g J B l 9 B l Thethalassaemia syndromes BLackwelt, Oxford )

149

10

HEMOGLOBIN ANDTHE HEMOGLOBINOPATHI ES

C h r o m o s o m1e6

o

C h r o m o s o m e1 1 5 '

Fis.10.4 T h ec r -a n dB - g l o b irne g i o nosnc h r o m o s o m1e6sa n d1 1s h o w i ntgh es t r u c t u r a l g e naensdp s e u d o g e n(eVs)a n dt h ev a r i o uhse m o g t o b i n s (Adapted produced N (eds)Recent In:Dawson,A M,BesserG,Compsion fromCarrell RW LehmanH 1985Thehaemog[obinopathies pp 223-225) advances in medicine 19Churchill Edinburgh, Livingstone,

mRNA is slightly more efficient in protein synthesis than cxglobin mRNA, and that this differenceis compensatedfor in the red blood cell precursorsby a relativeexcessof cr-globin mRNA.

Typesof mutation

It seemsthat the most important level of regulation of expression of the globin genes,Iike other eukaryotic genes,is likely to occur at the level oftranscription (p. 18).

A point mutation that results in substitution of one amino acid for another can lead to an altered hemoglobin, such asHb S, C or E, which are missensemutations (p. 22).

DNA studies of the B-globin genesand flanking regions have revealedthat, in addition to promoter sequencesin the 5' flanking regions ofthe various globin genes,there are sequences6-20kb 5' to the e-globin genenecessaryfor the expressionof the various BJike globin genes.This region has been called the locuscontrol regionor lcr, and,is involved in the timing and tissue specificity of expressionor switchingof the BJike globin genesin development. There is a similar region 5' to the cr-globin genes involved in the control of their expression.Both are involved in the binding of proteins and transcription factors involved in the control of expressionof the globin genes.

Pointmutotion

Deletion There is a number of Hb variants in which one or more amino acidsof one of the globin chains is missing or deleted(p. 23), e.g. Hb Freiburg.

lnsertion Conversely,there are variants in which the globin chains are longer than normal becauseof insertions (p. 23), such as Hb Grady.

Frameshiftmutotion

D I S O R D E ROSFH E M O G L O B I N The disordersof human Hb can be divided into two main groups: (1) structural globin chain variantssuch assickle-celldisease,and (2) disordersof synthesisof the globin chains,the thalassemias

Frameshift mutations involve disruption of the normal triplet reading frame, i.e. the addition or removal of a number of bases that are not a multiple of three (p. 25). In this instance,translation of the mRNA continues until a termination codon is read 'in frame'. These variants can result in either an elonsated or a shortenedglobin chain

STRUCTURALRIANTS/DISORDERS In 1975, Ingram demonstrated that the difference between Hb A and Hb S lay in the substitution of valine for glutamic acid in the B-globin chain. Since then more than 300 Hb electrophoretic variantshavebeen describeddue to a variety oftypes ofmutation (Table 10.2). Some 200 of these electrophoreticvariants are single amino-acid substitutions resulting from a point mutation. The majority of these are rare and not associatedwith clinical disease.A few are associatedwith diseaseand relativelyprevalent

150

in certain populations.

Chointerminotion A mutation in the termination codon itself can lead to an elongatedglobin chain, e.g.Hb Constant Spring.

Fusionpolypeptides The last type of structural variant arethefusionpolypeptides,Hbs Lepore and Kenya, which result from unequal cross-overevents in meiosis,as detailed previously (p. 149).

PATHIE5 HEMOGLOBIN ANDTHE HEMOGLOBINO

T a b t e1 0 . 2 S t r u c t u r a l v a r i a o n iLhse n o g t o b ; . r Typeof mutation

Examples

Chain/residue(s)/alteration

Point(over200variants)

HbS HbC HbE

B,6 gtuto val B,6 gtuto tys B,26gtuto tys

D e t e t r o n[ s h o r t e n e dc h a i n )

Hb Freiburg Hb Lyon Hb Lerden H bG u nH i i t

t o0 B,23 p,17-18 to0 B,6or7toO p.92-96 to0 or93-97

(elongated Insertion chain)

Hb Grady

(gtu,phe,thr)dupticated o,116-118

Frameshift(insertion ordetetion of oiherthan3 basepairs) mutiiptes

lbTak,Hb Cranston

of2 basepairsin codon codon, insertion lossoftermination B*,+ 11residues, 146f47

HbWayne

codonbysinglebase-pair dueto lossoftermination cr* +5 residues, in codon138/139 detetion

Hb McKees Rock

premature generating termtnatlon pointmutatron in'145, B*,-2 residues, codon

Chain termination

Hb Consiant Spring

codon pointmutation intermination cr*,+31resrdues,

Fusion chain(unequaI crossing over)

residues at C-ierminal endandB-trke at N-terminat residues !b Lepore/anti-Lepore Non-cr,6-tike end,andvlceversa,respectlveiy Hb Kenya/ anti-Kenya

at C-terminal endandp-tikeresidues y-trkeresidues at N-terminal Non-cr. respeciivety endandviceversa.

*Residues areeitheradded(+) or tost(-)

Ctinicat aspects Some of the hemoglobin variantsare associatedwith disease,but many are harmless and do not interfere with normal function, having been identihed only in the courseof population surveysof Hb electrophoreticvariants.A number, however,do interfere in a variety of ways with the normal function of Hb (Thble 10.3). If the mutation is on the inside of the globin subunits,in close proximitJ-to the heme pocketsor at the interchain contact areas, this can produce an unstable Hb molecule that precipitates in the red blood cell, damaging the membrane and resulting in hemolysisof the cell Alternatively,mutations can interfere with the normal oxygentransport function of Hb, leading to either an enhancedor a reduced oxygenaffinity or to a Hb that is stablein its reduced form, so-calledmethemoglobin The structural r.ariantsof Hb identified by electrophoretic techniques probabll' represent a minority of the total number of variants that exist, as it is predicted that only one-third of the possibleHb mutations that could occur will produce an altered charge in the Hb molecule and thereby be detectableby (Fig 10.5). electrophoresis

only in 1940 that the red blood cells from persons with sicklecell diseasewere noted to appear birefringent when viewed in polarized light under the microscope,reflecting polymerization of the sicklehemoglobin, leadingto distortion in the shapeof the

Clinicalfeatures Hemolyticanemia drsorders Sickting

n hemoglob Unstable

Cyanosis M (methemogLobrnemia) !emogtobrn

l o w o x y q e na f f i n i t y

S I C K L E . C EDLILS E A S E Although the severe hereditary hemoll,tic anemia sickle-cell diseasewas first recognizedearl,vin the twentieth centurl; it was

Potycythemia affnity Flrqh oxygen

ExamPles

Hb S HbC HbE Hb Kotn H bG u nF i l HbBristot Hb M (Boston) lb M (rryoePa-r) lb Kansas

Hb Chesapeake Hb Heathrow

151

10

HEMOGLOBIN ANDTHE HEMOGLO BINOPATHI ES

.r' 0riginH b CH b SH b FHbA-

& Fis.10.5 H e m o g t o b ienl e c t r o p h o r e ssi hs o w i n gh e m o g t o b i nAs, C a n d S (Courtesyof Dr D Norfotk,GeneralInfirmary,Leeds)

Fis.10.5 red blood cells under deoxygenatedconditions, so-calledsickling (Fig. 10.6). Pauling, in 1949, analysing Hb from patients with

Btood f i L ms h o w i nsgi c k l i nogf r e dc e t l si ns r c k l e - c d e it s[ e a s e Srckted ceLls arearrowed(Courtesy o'Dr D Norlotk,

sickle-celldiseaseby electrophoresis,demonstratedthat it had a different mobility from Hb A. and called it Hb S for sickle.

Gcnerrl lnfirm:rv

Ctinicataspectsof sickte-ce[[ disease

Sickle-celltrait

Sickle-cell disease,an autosomal recessivecondition, is the most common hemoglobinopathy; the clinical manifestations are manifold, including cerebral symproms, kidney failure, 'pneumonia', heart failure, weaknessand lassitude.All of these are a manifestationof the mutant allele.Hb S is lesssolublethan normal hemoglobin and tends to polymerize, causingthe sickleshapeddeformation of the red cells.A proportion of sickle cells becomesirreversibly sickled becauseof damageto the red cell membrane; these cells are taken up b-v the reticuloendothelial system.The shorter red cell survival time leads to a more rapid red cell turnover, with consequentanemia.In addition the sickle cells have a reduced deformability, tending to obstruct small arteries,resulting in an inadequateoxygen supply to the tissues

The heterozygousor carrier statefor the sickle-cellalleleis known as sickle-celltrait and is not thought, in general,to be associated with any signihcant risk to health. There are, however,reports suggestinga small increasedrisk of sudden death associatedwith

(Fis'.l0 7). Persons with sickle-cell diseasecan present acutely unwell with a sudden onset of chest, back, or limb pain, fever and dark urine, the latter due to the presenceof free hemoglobin in the urine, a so-calledsickle-cellcrisis. When compared to the general population, persons with sickle-cell diseasehave an increasedrisk of early death; earlier recognition and treatment of the known complicationsof sicklecell diseasehave resulted in improved life expectancy. Some simplemeasures,such asprophylacticpenicillin to preventthe risk of overwhelming sepsisdue to splenic infarction, have met with limited successin treating specificcomplications.It is fair to stare

152

that, although there has been an understandingofthe molecular basisof sickle-celldiseasefor more than 30 years,the therapeutic approachesavailableto date to prevent the sickling processhave had limited benefit.

I oedc )

strenuousexercise.In addition, there is continuing controversy about whether there are risks from hypoxia in persons with sickle-celltrait on airplane flights

MutationaIbasisof sickle-ce[[disease In sickle-cell disease,knowledge of the genetic code (p. 6) suggestedthat the substitution of valine at the sixth position of the B-globin chain was due to an alteration in the secondbaseof the triplet coding for glutamic acid, i.e GAG to GTG. Using the restriction enzyme,MsrII,the nucleotiderecognition sequenceof which is abolishedby the point murarion in Hb S, it is possibleto demonstratea differencebetweenpersonslvith sickle-celldisease and normal personsin the restriction fragmentswhen h1'bridized with a radioactivelylabeledB-globin probe. Use of a mutation-specific restriction fragment length polymorphism (RFLP) is possiblewith Hb E using the resrriction en4,mesHphl or MvII. Family studiesusing linked polymorphic DNA sequencevariants are needed for carrier testing and prenatal diagnosisfor the other hemoglobinopathies

D I S O R D E ROSFH E M O G L O BS I NY N T H E S I S The thalassemias are the commonest single group of inherited disorders in humans, occurring in personsfrom the mediterranean

I E N I O G L O BAI N DT H EH E M O G L O B I N O P A T H I E S

- G u t-

A b d o m i n apla i n

- S p l e e -n

S p l e n i ci n f a r c t i o n

L i m bp a i n - E x t r e m i t i e-s l g n t t e n d e r n e s s

Abnorma I B-polypeptide inHbS

HNEUMAIISM

0steomyelitis a"-i^

- Kidney-

Cerebrovascular accr0enr Haematuria H e n aIla r l u r e

- Lung-'Pneumonia' - Heart-

H e a r ft a i l u r e

l-;'i::ffi[:"'' Lassitude I

l - A b n o r msakl u l l I radiographs

Fis.10.7 T h e o l e , o t r o oe i cf f e c t so f r l ' e n e n p[ n ' s i . < l F - . e lnl i . e a s e

Mutotionolbosisof a-tholassemia area, the N'Iiddle East, the Indian subcontinent and South-East Asia. Thel' are a heterogeneousgroup of disorders and are classifiedaccording to the particular globin chain or chains svnthesizedin rcduced amounts,e.g. o-, B-, bB-thalassemia. The pathophysiolog-v is similar in all forms of thalassemia.An

Itr,^itro translation studies using mRNA from reticulocJ'tes from f'etusesrvith hy'drops fetalis shorv no s-Ynthesisof cr-globin Hollelcr, rvhen mRNA from reticulocl-tesof persons rvith the milder fbrm of q-thalasscmia,Hb H disease,is used, a-globin

imbalanceof globin-chain production resultsin the accumulation offrec globin chainsin the red blood cell precursors,rvhich, being insoluble, precipitate, resulting in hemolysis of thc red blood cells, i.e. a hemol.vtic anemia, rvith consequent compensator-y h1'perplasiaof the bone marrow.

s-Thalassemia u-Thalassemiaresults from an underproduction of the cr-globin chains. It occurs most commonly in persons from South-East rvhich differ in Asia There are two main types of cr-thalassemia, their severit,v:the severeform, in rvhich no cr-globin chains are produced, is associatedwith death of the fetus in utero,as a rcsult of massiveedemabecauseof heart failure from the severein-utero anemia,so-calledh),drofsJitalis (Fig. 10.8). Anall''sis of the Hb present in fetuses with hydrops fetalis revealsit to be a tetramer of y-globin chains, s'hich used to be calledHb Barts. In the milder forms of o thalassemiacompatible lr'ith survival, although some u-globin chains are produced, there is still a relative excessof B-globin chains that results in production of the B-globin tetramer Hb H, or what is known as Hb H disease.Both the Hb Barts and Hb H globrn tetramers have an oxygen affinit.vsimilar to that of myoglobin and do not releaseoxygen normallv in the peripheral tissues.In addition, Hb H is unstable and precipitates,resulting in hemolysisof thc red blood cells.

Fis.10.8 L o n g i t u d ru n lat tr a s o n o g r a pshc iacno fa c o r o n asIe c t i oonft h eh e a d (tother ght)andthoraxofa fetuswithhydrops fetalis dueto the pLeuraI a Large HbBarts,showing formofs-thaLassemia, seveTe (Courtesy HospitaL' (arrowed) StJames's MrJ CampbeLl, effusion L e e d)s

153

10

H E M O G L O BAIN NDT H EH E M O G L O B I N O P A T H I E S

is produccd but in rcduced amounts uhen compared to mRNA from reticulocvtcs from normal persons. Studies compirring thc quantitative hl.bridization of radioacrively.labeled u-globin cDNA to DNA from fetuscs rvith hvdrops fetalis, persons with Hb H diseaseand normal persons are consistent uith crthalasscmiabeing due to dcletions of the u-globin genes. Rcstriction mapping studies of the o-globin region of chromosome l6 reveal that there are two u-globin structural geneson the short arm of chromosomc 16.The various forms of cr-thalassemiahave becn shoun to be mostly due to deletions of one or more ofthese structuralgencs(trig. 10.9). Deletions of the a-globin gencsin a thalassemiaare believed to havearisen as a result ofuncqual cross-ovcreventsin meiosis, rvhich are thought to bc more likely-to occur lherc geneswith homologous sequcncesare in close proximitl: Support for this hypothesiscomes from the linding of the other product of such an c\ient,i.e personsrvith three a-globin structural geneslocatcd on one chromosomc. Thesc observationsresulted in the rccognition of t\{o other mildcr furms of ct-thalassemiathat are not associatedl ith anemia and can bc detected onlv bv the transient presencein the immcdiate nervborn period of Hb Barts. The results of the DNA mapping studies of thc cr-globin gcnes have shotn that these milder forms of G,-thalassemia are due to the deletion of one or two of the U-globin gencs.Less commonll,, non-delction mutations that include point mutations in the u-globin genes, and mutationsin the 5'region involvedin the control of their transcription har.ebeen found to causeg-thalassemit. An exception to this classification of the c-thalassemias is the Hb variant Constant Spring, namcd after the torvn in the USA where the original patient came from. This rvas detectcd as an electrophoreticvariant in a person with Hb H diseasc,i.e.

p-Thatassemia B-Thalassemia is caused by underproduction of the B-globin chain of hemoglobin. In-t:itro translation studies using mRNA from personswith B-thalassemiadue to these different types of mutation show either reduced or absentproduction of B-globin chains, 0(*)

B0, respectivell'.Persons homozygous for B0".rd mutations have a severe transfusion-deoendent

;::*::-t"

Mutotionolbosisof B-tholossemio Restriction mapping studies have shol.n that p-thalassemiais rarely due to a deletion, and DNA sequencinghas often been necessaryto rer.eal the molecular patholog].. In excessof 100 different mutations have been shown to cause B-thalassemia. These involve a wide variety of different t1,pesof mutation, including point mutations, insertions and deletionsof one or more bases.These occur at a number of places, s.ithin both the coding and non-coding portions of the B-globin genes as rvell as in the 5'flanking promoter region, the 5'capping sequences(p. l8) and the 3' pol-vadenylationsequences(p. 18) (Fig.10.10). The r.arioustvpes of mutation causingB-thalassemiaare often unique to certain population groups and can be consideredto fall into six main functional types. Transcription rnutations X,futationsin the 5' flanking TATA box or the promoter region of the B-globin genecan result in reduced transcription levelsof the B-globin mRNA.

u-thalassemia Hb Constant Spring is due ro an abnormallv long u chain that is thc result of a mutation in the normal tcrmination codon at position 142 in the u-globin gene.Tianslation of this a-globin mRNA continues until the occurrence of anothcr

rnRNA splicing mutations Nllutationsinvolving the invariant 5' GT or 3' AG dinucleotides of the introns in the B-globin gene or the consensusdonor o r a c c e p t o r s e q u e n c e s( p . l 8 ) r e s u l t i n a b n o r m a l s p l i c i n g rvith consequent reduced levels of B-globin mRNA. In the

termination codon, resulting in an abnormallv long o,-globin chain variant. In addition, this abnormal u-globin mRNA molecule is unstable, with a consequent relative deficiencv of a-globin chains, resulting in the presence of the B-globin tetramer,Hb H.

most common B-thalassemiamutation in persons from the mediterranean region, the mutation leads to the creation of a new acceptor AG dinucleotide splice site sequencein the first intron of the B-globin gene,creatinga so-calledcrvptic splicesite (p. 25). The cry-pticsplice site competeswith the normal splice

r-

I

sa

I

(xcx

Normal

154

o.ct. I

a-

A l p h ah e t e r o z y g o t e

t-

A l p h ah e t e r o z y g o t e

A l p h ah e t e r o z y g o t e

Hb H disease

Hvdrnn<

fpfrlic

Fig.10.9 S t r u c t u r e o f t h e n o r m a l a n d d e t e t e d c r - g l o b n snttrhuecvt a uro au l gsef o n rem sc rs-ot h f a l a s s e( A mdi aa p t e d f r o m E m e r y A E H l g S 4 Anintroduction torecombinant DNAJohn Witey, Chichester.)

N DT H EH E N l O G L O B I N O P A T H I E S I E M O G L O BAI N

, 100bp, Exon1

I

lntron1

Exon2

lntron2

T r a n s c r i p t i o n R N As p l i c i n g f l C a ps i t e ?

I' F r a m e s h i f t

{' N o n s e n s e codon

f Unstable qlobin

Exon3

t R N Ac l e a v a g e f l n i t i a t o rc o d o n r---Small deletion

Fis.10.10 ( Ai ad a p t e d f r o m 0 r k i n S H eB m L o c a t i o n a n d s o m e o f t h e t ym pu e tsaotfi onnt h e B - g L o b i n g e n e a n d f l a n L i n g r e g r o n t h at thraeLsausl tsi n 131-171) RevGenetlB: DNA Annu Kazazian H H 1984The mutatonandpolymorph sm ofthehumanB-globn geneanditssurrounding

site, leading to reduced levels of the normal B-globin mRNA. Mutations in the coding regions of the B-globin rcgion can also lead to cr-rptic splice sites

transfused,compensatoryexpansionof the bone marrow irdcquatel--v rcsultsin irn unusualll'shapedf'aceand skull (Fig. 10.11) Although pcrsons rvith thalasscmiamaior uscd to die in their late tecns or carll' 20s ls a result of complications due to iron overload from

Polyadenylation signal rnutations N{utationsin the 3' end of the untranslatedregion of thc B-globin gene can lead to loss of the signal for cleavageand pol-vaden-vlation of rhe B-globingenetranscript. R\A modifi cation rnutations Nlutations in thc 5' and 3' DNA sequences,involved respectivelv in the capping (p. l8) and poll,adenylation(p 18) of the mRNA, can result in abnormal processingand transportation of the B-globin mRNA to the cvtoplasm, with conscqucnt rcduccd levelsof translation. Chain termination mutations Insertions, deletions and point mutations can all gencratc x nonsenseor chain termination codon, resulting in the premature termination of translationof the B-globin mRNA This rvill result in the majoritl.of instancesin a shortenedB-globin mRNA that is often unstable and more rapidly degraded, rvith consequent reduced levelsoftranslation ofan abnormal B-elobrn. Missense rnutations Missensemutations that lead to a B-globin that is highly-unstable can rarelv result in B-thalassemia. An exampleis Hb Indianapolis.

Clinicolospectsof B-tholossemio Childrcn affectedlvith thalassemiamaior, or Coole-v'sanemia irs it rvasoriginally known, usuallypresentin the lirst.vearof life with a severetransfusion-dependentanemia. Unless the child is

Fig.10.11 F a c i e so f a c h i l dw i t h B - t h a [ a s s e msi ah o wn g p r o m i n e n c oe f t h e foreheadthroughchangesin skullshapeas a resuttof bonemarTow (Courtesyof Dr D Norfolk,GeneralInfirmary.Leeds) hypertrophy.

155

10

HEMOGLOBIN ANDTHEHEMOGLOBINOPATHIES

repeated transfusions, the regular daily use of iron-chelating drugs, such as desferrioxamine,has improved their long-term survival. Individuals heterozygousfor B-thalassemia, Ihalassemia trait or thalassemiaminor, usualh have no s)rmptoms or signs Thev do, however,havea mild hypochromic,microcytic anemia,which can often be confused with iron deficicncv anemia.

6p-Thalassemia ln \ftthalassemiuthere is underproduction of both the 6- and pglobin chains Personshomozygous for 6B-thalassemiaproduce no 6- or B-globin chains. Although one would expecr such persons to have a fairly profound illness, thcy are only mildlv anemic becauseof increasedproduction ofy-globin chains,with Hb F levels being much higher than the mild compensarorv increaseseenin homozygotesfor B-thalassemia.

Mutotionolbosisof 6B-thalossemio 8B-Thalassemia has been shown to be caused br. extensive deletions in the B-globin region involving the 6- and B-globin structural genes(Fig 10.12).Somedeletionsextendto include the Ay-globin geneso that only the Gy-globin chain is synrhesized.

Hereditarypersistence of fetaIhemoglobin Hereditary persistenceof fetal Hb, or HPFH, in rvhich there is persistenceof the production of fetal Hb into childhood and adult life, is included in the thalassemias. Mosr forms of HPFH are,in fact, a form of 8B-thalassemiain which continued y-chain synthesiscompensatesfor the lack of production of 6- and Bglobin chains. Persons with hereditary persistenceof fetal Hb continue to produce significant amounts of fetal Hb after birth,

accounting for 20-30o/o of total Hb in heterozygotes and l00o/o in homozygotes This is not associatedrvith any svmptoms and rvas originally considered more of a scientific curiosity than a medical problem

Mutationolbosisof HPFH Some forms of HPFH havebeen shown to be due to deletionsof the 6- and B-globin genes.Analysis of the non-deletion forms of HPFH has shorvn point mutations in the 5' flanking promoter region of either the Gy or Ay globin genesnear the CAAT box sequences(p. 21) involved in the control of expression of the hemoglobin genes.

HEMOGLOBINOHIES The marked mutational heterogeneity of B-thalassemrameans that affected individuals are often compound heterozygotes (p. 108),i.e. they havedifferent mutations in their B-globin genes, leading to a broad spectrum of severityin the disorder One form of B-thalassemiaof intermediate severity,known as thalassemia intermedia, requires lessfrequent transfusions. In certain populations many of the hemoglobinopathiesare relativelv 'common' and, not unexpectedll',personsare reported who have two different disorders of Hb Understandably,in the past, recognition of such individuals was often quite difficult. Becauseof the high prevalencein certain populations of some of the structuralvariantsof hemoglobin,suchasHb S, individualscan presentwith severeanemiawho are found to be heterozygousfor both Hb S and B-thalassemia,i.e. are compound heterozygotes (p.108)

HPFHtype- 1 H P F Ht y p e - 2

HO KCNVA

y6p-thal

Fis.10.12 156

Some of the deletionsin the B-globn regionthat resuLtin some forms of thalassemiaand heredrtarypersistence of fetathemogLobin

BINANDTHEHEMOGLOBINOPATHIEs HEMOGLO

Finally; certain combinations of hemoglobinopathiescan also result in a previously unexplainedmild form of what is normally thought to be an invariably severe hemoglobinopathy. For example,deletion of one or two of the cr-globin genesin a person h o m o z y g o u s f o r B - t h a l a s s e m i ar e s u l t s i n a m i l d e r i l l n e s s becausethere is lessof an imbalancein globin chain production. Similarly, the presenceof one of the forms of HPFH in a person homozygous for B-thalassemiaor sickle-cell diseasecan contribute to amelioration of the diseaseas the increased production of y-globin chains compensatesfor the deficient B-globin chain production. This finding has been exploited therapeuticallyby the use of drugs, such as hydroxyureaand butyrate, which have been found to be associatedwith increasedHb F levels Recent developmentsin genetherapy(Ch. 23) for the hemoglobinopathies in a mousemodel for sickle-celldisease,in which the introduction of the fetal Hb gene resulted in increasedHb F levels,led to a reduction in the sickling phenomenon This approachholds great promise for a reduction in the morbidity and mortality associated with this disorder.

READING FURTHER Cav J C, Phillips J A, Kazazian H H 1996Haemoglobinopathiesand In: Rimoin D L, ConnorJ M, PyeritzR E (eds)Principles thalassemias and practiceofmedical genetics,3rd edn Churchill Livingstone, Edinburgh,pp 1599-1626 A useJitl,ui) to date,contisesummar1oJ-thehemoglobinopathies in children with Coole.vT B, Lee P 1925A seriesof casesof splenomegaly anemiaand peculiarbonechanges. TransAm PediatrSoc 37: 29-40 The originttl descriptionof p-thalassemia. PaulingL, Itano H A, SingerS J, WellsI C 1949Sickle-cellanaemia, a moleculardiseaseScience110:543-548 Thefrst genetitdiseasein phich a molecularbasismasdescribed, lead.ingto a Nobel Prize 2nd edn. Oxford University Press, SerjeantG R 1992Sicklecell disease, Oxford Euellent, comprehensite text cooeringall aspecnof this importantdisorder.

\\utherall D J, ClcggJ B, Higgs D R, \VoodsW G 1995The In: ScriverC R, BeaudetA L, SIl' W S,VallcD hemoglobinopathies 'I'he 7th edn (cds) mctabolicand molccularbasisof inheriteddisease, \lcGrau' Hill, \ew Ytrrk,pp 3417 3484 and the .1 tcr.1,nmprthensite,detailedtucountoJhemoglobin hemoglohtnoPuthies

ELEMENTS red blood Q Hemoglobin (Hb), the protein present in cclls responsiblefor oxygen transport, is a tetramer made up of two dissimilar pairs of polypeptide chains and the iron-containing molecule heme. it @ Human Hb is heterogeneous.During development are globin that chains of different comprisesa succession expressed differentially during embryonic, fetal and adult life, i e. (\282,a2\2, a"262,u2P2,etc' @ fne disordersof Hb the hemoglobinopathies can be divided into two main groups: the structural disorders, such as sickle-cellHb or Hb S, and disordersof synthesis) the thalassemiasThe former can be subdivided by the way in which they interfere with the normal function of Hb and/or the red blood cell (e.g.abnormal oxygenaffinity or hemolytic anemia).The latter can be subdivided according to which globin chain is synthesizedabnormallS i.e' c[-, B- or 6B-thalassemia Hb @ Family studies of the various disordersof human and analysisof the mutations responsiblefor these at the protein and DNA levels have led to an understanding of the normal structure, function and synthesisof Hb. This has allowed demonstration of the molecular pathology of these disorders,and prenatal diagnosisof a number of the inherited disordersof human Hb is possible.

157

CHAPTER

'Life

... is a relationship betwcen molecules' Linus Pauling

'Thc

existenceof chemical individualitl' follou's of necessitv from that of chcmical specilicit1.,but rve should expectthe differcncesbctween individuals to be still more subtle and difhcult of dctection.' Archibald Garrod (1908) In this chapter we consider single-genebiochemicalor metabolic d i s e a s e si,n c l u d i n g m i t o c h o n d r i a l d i s o r d c r s .T h e r a n g e o f known disorders is vast, so onl-van overview is possible,but it is hopcd that the readcr uill gain a flar.or of this fascinating area of medicine. At the beginning of the rwcntieth centurv Garrod introduced the concept of 'chcmical individuality', leading in turn to the concept of the inhorn ercor of-metabolism Beadle and Tatum later devcloped the idea that metabolic processes,whether in humans or anv other organism,proceedbv steps Thev proposcdthat eachstep wascontrolled bv a particular enzvme and that this, in turn, was the product of a particular gene. This rvas rcferred to as the 'one gene-one cnzvme (or protein) concept'.

INBORN E R R O RO S FM E

PHENYLKETONURIA Children u,ith phenylketonuria(PKU), if untreated,are severely intellectuall.v impaired and often har.e convulsions In PKU there is a deliciency of the enzvme needcd for the conr.ersionof phenylalanincto tvrosine, phenylalaninehl,droxylase(PAH) In othcr words there is a'genetic block' in thc metabolic pathway' ( F i g .1 l . l ) PKU was, in fact, thc first genetic disorder in humans shorvn to be causedby a specific enzvme delicicncy,bv Jcrvis in 1953 As a result of the enzvme defect, phen1,'lalanine accumulatesand is conr.ertedinto pheny'lpyruvicacid and other metabolitesthat are ercreted in the urine. The enzvme block leadsto a deficiency

pigmented, such as the substantianigra, may also lack pigmcnt

BOLISM

e n z v m e i s r a t c l i m i t i n g ( i . e h a p l o i n s u f f i c i e n c vm u t a t i o n , p. 26) or the gene product is part of a multimcric compler (i e dominant-negativc mutation, p 26), thc disordcr can manifcst in thc heterozygousstatc, i.e. be dominantlf inherited

(p.103).

There are a number of disordersof amino-acid metabolism. the best known of rvhich is phenvlketonurra

of tvrosinc, rvith a consequentreduction in melanin formation Affected childrcn therefore often have blond hair and blue e-ves(Fig 11.2). In addition, areasof the brain rhat are usually-

I n e x c e s so f 2 0 0 i n b o r n e r r o r s o f m c t a b o l i s m a r e k n o w n which can be grouped by either the metabolite, metabolic pathwa]',function of thc enzyme or cellular organelle involved (Table 1l 1). Most inborn errors of metabolismare inheritcd in an autosomal recessiveor X-linked manncr rvith only' a fer, being inherited in an autosomaldominant manner. This is bccausethe defective protein in most inborn errors is an enzyme rvhich is diffusible, and there is usuallv sufficient residual activity in the heterozvgousstate (i c loss-of-function mutation, p. 25) for the enzl.mc to function normallv in m o s t s i t u a t i o n s .I f , h o w e v e r ,t h c r e a c t i o n c a t a l y s e db y a n

158

D I S O R D EO RS FA M I N O . A C I D ME BOLISM

Treatmentof PKU An obvious method of treating children rvith PKU would be to replace the missing enzyme, but this cannot be done simpl-vby any conventionalmeansof treatment (p 3a0). Bickel, only 1 year after the cnzvme delicicncv had been identilied, suggestedthat PKU could be treatcd by removalof phenl-lalaninefrom the diet. This has pror,edto be an effectivetrexrment. If PKU is detected earlv cnough in infancr,., intellectualimpairment can be prevented b-vgiving a diet containing a restricted amount of phenylalanine. Phcnvlalanine is an essentialamino acid and thercfore cannot be removed entirely from the diet. 81. monitoring the level of phenvlalaninein the blood, it is possiblero suppll.sufficient amounts to meet normal requirements and avoid levels that rvould result in mental retardation. Once brain development is complete, dietary restriction can be rclaxed - from adolescence onlyards

GENETICS B OCHEN4ICAL

Tabte11.1 Characteristics of somernbornerrorsof metabolism Typeof defect

Genetics

Deficiency

Main clinicatfeatures

Amino-acid metabolism Phenylketonuria A l k a p t o n uar O c u i o c u t a n e o uasl b i n r s m Homocystrnuria

AR AR AR AR

Phenyalan e hydroxylase l o m o g e n t r s i ca c r do x i d a s e Tyrosinase C y s t a t h i o ncer - s y n t h a s e

M e n t a lr e t a r d a t i o nf a, i r s k l n ,e c z e m ae, p l l e p s y A r l h r i ts l a c k o f s k i na n d h a r p i g m e n t e, y e d e f e c t s n f [ e n s ,l h r o m b o s i s , M e n t a l r e t a r d a t t odni,s t o c a l i o o

M a p t es y r u pu T r n ed s e a s e

AR

B r a n c h e d - c h a lB n k e t o a cd 0ecarooxvrase

Ureacycledisorders deficiency Carbamyl synthase transferase 0rnithinecarbamyl

AR XD

Carbamyl synthase transferase 0rnithine carbamyl deficiency acidsynthase Arginosuccinic

Citrullinemia aciduria Argrnrnosuccinic

ND AD

A -^:^ ^^,,--t^t - --it Ar qil ru>uLLil rr ouru

Hyperargininemra

AR

Arginase

t,,--^ rydrs

s k e l e t aaI b n o r m a l t t t e s M e n t a lr e t a r d a t t o n

H y p e r a m m o n e m i ac o m a d e a t h H y p e r a m m o n e m i ad.e a i ht n e a r t yi n f a n c y V a r i a b t ec t i n i c acl o u r s e H y p e r a m m o n e m i am, t t dm e n t a lr e t a r d a t l o n , proteinintolerance H y p e r a m m o n e m i ap,r o g r e s s t vsep a s t l c l t y , i n t e t t e c t u ad le t e r i o r a t i o n

Carbohydratemetabolism Mon osocchoride m etoholism Galactosemra

AR

jntolerance Hereditaryfructose

AR

Glycogenstoragediseases Primorily offecting liver (GSD-l) vonGrerke disease (GSD-lll) Coridisease AnoeTson olsease tu>u-1vl phosphorylase Hepatic -Vl) (GSD deficiency

AR AR AR AR/ X-Linked

Primorily offecting muscle (GSD-V) disease McArdLe (GSD-ll) Pompedrsease

nn AR

phosphorytase Muscle 4-glucosidase Lysosomato-1

cramps Muscte weakness muscte Heartfailure.

Steroidmetabolism Congenital adrenathyperplasia

AR

sattLosing Viritization.

Androgenrnsensitivity

XR

2'1-Hydroxytase,'11B-hydroxylase, 3P-dehydrogenase Androgenreceptor

Lipid metabolism Fami[ialhvoercholesterotemia

AD

receptor tipoprotern Low-density

arterydtsease Eartycoronary

L y s o s o m a ls t o r a g e d i s e a s e s M uco p o lyso ccho r i d oses H u r l e rs y n d r o m e( M P S - | )

AR

cr-L-lduronrdase

H u n t e rs y n d r o m e( N / P S - 1 1 )

XR

l d u r o n a t es u t f a t es u l f a t a s e

S a n f i t i p p soy n d r o m e( N / P S - l l l )

AR

M e n t a l r e t a r d a t i o snk, e t e t a l a b n o r m a t r t i e s , o r. n e a l h e p a t o s p l e n o m e g acl Y c t o u drn g M e n t a lr e t a r d a t l o ns k e t e t aal b n o r m a t i t i e s hepatop sl e n o m e g a l y B e h a v i o r apI r o b t e m sd, e m e n t i af,r t s

lV) Morquosyndrome(MPS

AR

e PS-| A) H e p a r a n S - s u L f a m r n i d a (sM A / a c c r - r - g t u c o s a m i nd a s e( l ' / P S l l l B ) n dase Ac-CoA-cr-g[ucosam N - a c e t y l t r a n s f e r a s( M e PS I C) N a c - g l u c o s a mn n e - 6 s u l f a t e ]) s u l f a t a s e( M P S - l LD G a l a c t o s a m n e - 6 - s u t f a t a s e ( N I P S - l V A )C o r n e a l o p a c i t e s , s h o r t s i a t u r e . s k e t e t a l [/PS lV B) abnormatities B - g a l a c t o s i d a s( e

'

/--n

'r /\

l-phosphate uridyt Gatactose translerase atdotase Fructose 1-phosphate

Clr rcn<e-6- nhncnhAtacP

Amyto-1,6^g[ucosidase enzyme Gtycogen debrancher l'-lon:+ir

nhncnhnrrrl:
cirrhosis mentalretardation Cataracts, convulsions vomiting,Jaundice' to thrive. Faiture

ta hypogLYcem HepatomegaLy, ta hypogtycem Hepatomegaty, ture Abnorma[[iverfunction/fai failureto thrive hypogtycemia, Hepatomegaty,

testes. maLe chromosomes Female externalgenttalia.

Toblecontinued

159

11

B I O C H E M I CGAELN E T I C S

Tabte11.1 Characteristics of someinbornerrorsof metabolismGont'd\ Typeof defect

Genetics

Deficiency

MPS-V(formerty Scheie disease, nowknownto bea mrtdaltetrc formof MP5-ll Maroteaux-Lamy syndrome

AR

(MPs-Vll) Slysyndrome

AR

Arylsutfatase B.N-acetyt-gatactosamine.Corneal clouding. sketetal abnormatities. cardiac (MPS-Vl) cr-4-sulfatesutfatase abnormatitres p-Gtucuronidase presentation, Variabte skeletaI andcardiac abnormalities, hepatosptenomegaty, corneaI ctouding, mentaIretardatjon

Main clinicalfeatures

Sphingolipidoses Tr -o,y, - JCo-L- ra - r i ^ ^ - - ^ rJ ut>Ed5c

Hexosa minidase-A

DevetopmentaI regression btindness, cherry-red cnnt

Gaucher disease

Gtucosytceramide B-Glucosidase Sphingomye[inase

Niemann-Pick disease

Purine/pyrimidinemetabolism Lesch-Nyhan disease

XR

Adenosine deaminase defrcrency AR Purinenucleoside phosphorytase AR

guanine Hypoxanthine phosphoribosyttransferase Adenosine deaminase Purinenucleoside phosphorylase

Mental retardation, uncontrotled movements, setf-mutitation Severe combined immunodeficiency Severe viratinfections dueto impaired T-cetl runcton phosphoribosyltransferase, Orotate Megatobtastic anemia, faitureio thrive. orotidine5'-phosphatedecarboxytase devetopmentatdeLay

Hereditary oroticaciduria

AR

Porphyrinmetabolism Hepotic porphyrios (AlP) Acuteintermittent porphyria Hereditary co-proporphyria Porphyrra variegata

AD AD AD

Uroporphyrrnogen I synthetase Co-proporphyrrnogen oxidase Protoporphyrinogen oxidase

pain,CNSeffects AbdominaI As forAlP photosensitivity Photosensitivity, asforAIP

AR

Uroporphyrinogen lllsynthase

Hemolytic photosensrtivtty anemia,

AD

Ferrochetatase

Photosensitivity. liverdrsease

0rganic-aciddisorders Methylmalonic acidemia

AR

MethytmatonytCoAmutase

Propionic acidemia

AR

Propionyl-CoA carboxytase

Hypotonia. poorfeeding, acidosis. devetopmental oeray Poorfeedingfailure to thrive. vomiting, acidosrs, nypogrycemta

Coppermetabotism Witsondisease

AR

Spasticity, rigrdity. dysphagra, cirrhosis

Menkes disease

XR

ATPase membranecoDner transnort protet n ATPasemembranecoppertransport protein

Peroxisomaldisorders Pe roxisomoI hi og enesisd isorders ZelWeqer svndrome AR

At[peroxisomal enzymes

Dysmorphic features, hypotonia, [argelivenrenal cysts

I soloted peroxi somoI enzyme deficiency Adrenoteukodystrophy XR

Verylong-charn fattyacid-CoA synthase

Mentaldeterioration. fits.behavioral chanqes, adrenaI faiture

Mutation in lysineIRNA(m 8344G>A substitutron, m 8356T>C substitution)

Myoctonus, seizures, optrcatrophy, hearing impairment, dementra, myopathy

Erythropoietic porphyrias Congenitalerythropoietic porphyna Erythropoietic protoporphyria

Disordersinvolvingmitochondria MERFF Mt

160

dcafncc<

Type| - jointandlimbpains, sptenomegaty Typell - spasticity. fits.death Faiture to thrive,hepatomega[y, cherry-redspot. devetopmentaI regression

Faiture to thrive.neurologicaldeterioration

f

Phenylketonuria

albinism @ Oculocutaneous @ Alkaptonuria hypothyroidism @ Congenital

F i g . 1'biochemicat 1.1

btock' in Sitesof phenytketonuria, alkaptonuria, and hypothyrordism congenitaI atbinism ocutocutaneous

The intellectual impairment seen in children with phenylketonuria is most likely the result of increased levels of phenylalanine and/or its metabolites to toxic concentrations, rather than a deficiency of tyrosine, as an adequate amount of the latter amino acid is usually available in a normal diet. It could well be that there are both prenatal and postnatal factors responsible for the mental retardation in persons with untreated

PKU.

of PKU Diagnosis Although PKU only affectsapproximatelyI in 10000personsof westernEuropeanorigin,PKU wasthe first inbornerror routinely screenedfor in newborns.This can be doneby teststhat detect the presenceof the metaboliteof phenylalanine,phenylpyruvic acid, in the urine by its reactionwith ferric chlorideor through increasedlevelsof phenylalaninein the blood. The latter test, known as the Guthrie test, involvestaking blood samplesfrom

151

11

B I O C H E M I CGAELN E T I C S

Mutationalbasisof PKU Although all casesof classic PKU arise from a deficiency of phcnl'lalaninehvdroxvlase,more than 450 different mutations in thc PAH gene have now been identilied. Ccrtain mutations are morc common in persons with PKU from specific population groups. In addition, in personsof western European origin with PKU, the mutations occur on a limited number of DNA haplotypes. Interestinglr,', holvever,a variety of different individual mutations has been found in associationrvith some ofthese haplotvpes.

MaternaIphenylketonuria Childrcn born to mothers rvith phcn1,-lkctonuria havean increased risk of mental retardationeven when their mothers are on closell,controllcd dietar.vrestriction. It has been suggestedthat the reduccd ability of the mother with PKU to deliver an appropriate amount of tvrosine to her fetus in tfiero could result in reduced f'ctalbrain gror'vth.

ALKAPTONURIA Alkaptonuria rvasthe original autosomalrecessiveinborn error of mctabolism describedb-vGarrod In alkaptonuriathere is a block in the breakdownof homogentisicacid, a metabolite of ryrosine, becauseof a dcficiencl.of the enzvme homogentisicacid oxidase (seeFig. 1I . I ). As a conscquence,homogentisicacid accumulates

Fis.11.2 Faciesof a malewith phenylketonuria; notethe faircomplexion

children in the first week of life and comparing the amount of grow-thinduced b1' the sample rvith standardsin a strain of the bacterium Bacillus subtilis, which requires phenylalanine for gro\ th. This technique has been replacedbv the use of a varietv of biochcmical assavsof phcnl,lalaninclcvels.

Heterogeneity of hyperphenytalaninemia Raised phenvlalanine levels in the newborn period can be the result of causesother than PKU A small proportion of ncrvborn infants har.ea condition called benign h1-perphenvlalaninemia, causedby a transient immaturitv of liver cells to metabolize phenvlalanine.Thesc children do not require treatment as thev xre not at risk of developing mental rctardation There are, however, tno othcr rare causes of h1'pcrphenvlalancmiar,yith seriousconsequences; in thcsetrvo disordcrslevelsof the enz-ymc phenvlalaninehvdroxrlase are normal but there is a dehciencv o f e i t h c r d i h y d r o p t e r i d i n e r e d u c t a s eo r d i h v d r o b i o p t e r i n svnthase.These two enzl-mesare involvcd in the svnthesis of tctrahl.drobiopterin,a cofactor necessarvfor normal acti\,itv of phenvlalaninehydroxl-lase Both disordcrs are morc serious

152

than classicPKU becausetherc is I high likclihood of mental handicap despite satisfactory managemcnt of phenvlalanine levels.

and is excretcd in the urine, to which it imparts a dark color on exposureto air Dark pigment is alsodcpositedin certain tissues, such as thc ear wax, cartilage and joints, lvhere it is knorvn as ochronosis,uhich in the latter location can lead to arthritis later in life.

O C U L O C UN E O U S ALBINISM Oculocutaneous albinism (OCA) is an autosomal recessive disorderdue to deficiencvofthe enzymetvrosinase,which is necessary for the formation of melanin from tvrosine (seeFig. l1.l) In persons rvith OCA there is a lack of pigment in the skin, hair, iris and ocular fundus (Fig l1 3). The lack of pigment in the e1'e results in poor visual acuitv and tvpical uncontrolled pendular eve mo\rement(nt-stagmus)The reduced pigmentation appears to lead to underdevelopmentof the part of retina for fine vision, the for.ea,and abnormal projection of the visual pathwaysto the optrc cortcx

Heterogeneity of oculocutaneous albinism OCA is geneticallyand biochemicallvheterogeneous.Cells from persons rvith classicalbinism have no measurabletvrosinase activit)! thc so-calledt.yrosinase-negatioe form. Horvever,cells from somc personsuith albinism sholvreducedbut residualtvrosinase activitl.and are termed t.yrosindse posilrre.This is usually.reflected clinicalll' bv variable developmcnt of pigmentation of their hair and skin rvith age.Both t1.pesare known as tyrosinasegene-related oculocutaneousalbininism t\,pc l. or OCA-1.

GENETIC5 BIOCHEMICAL

Homocvstinuria is caused b1' a deficienc-vof the enz-vme cvstathionine B-s-rnthasc and can be scrccnedfor b-vmeansof a positire cvanidc nitroprusside tcst, rvhich detects thc presence of incrcased levels of homocl'stinc in the urine. The diagnosis is confirmed b1' raised plasma homocl-stine levels. Treatment inr.olrcsa lorv-methioninediet uith cystine supplementation. A proportion of individuals rvith homocvstinuria are responslve to the enzlme cofactor pvridoxine, and have what is knorvn as the pvridoxine-rcsponsivefbrm. A small proportion of affected indir icluals har.e mutations in gencs leading to deficienciesof cnzvmesinvolvcd in the svnthesisof cofactorsfor cystathionine nthasc B-s,-r

FB R A N C H E D . C H A I N D I S O R D EO RS A M I N O . A CM I DE B O L I S M The esscntialbranched-chainamino acids lcucine, isoleucinc and ralinc har,ea part of their metabolic pathrvavsin common. Dclicienci of the enzl,me involved rcsults in maplc svrup urine discrrse .

M A P L ES Y R U PU R I N ED I S E A S E Fis.11.3 0 c u t o c u t a n e o ua sI b l n i s mj n a c h i l do fA f r o C a r i b b e aonr g i n ( C o u r t e soyf D r V A M c K u s i c)k

DNA studics have revealedclassictvrosinase-negati\eand some tvrosinase-positivefamilies u,ith oculocutaneousalbinism to be due to mutations in the tvrosinasegenc on thc long lrm of chromosome ll. Linkagc studies in some of the families with t-vrosinase-positiveoculocutaneousalbinism, hower,er,have excluded the tvrosinasegene as being responsible A number of thcsc familics, interestingll,,have a mutation in thc P gene, thc human homolog of a genein thc mouse called,pinh-eyel dilution, or pink-eve for short, locatedon the long arm ofchromosomc 15 This hasbeen termed oculocutaneous albinism tvpe 2, or OCA-2. In addition, in a proportion of families rvith oculocutaneous albinism, linkrge to both of these two loci has bccn cxcluded, consistentwith the existenceof a third locus rcsoonsiblefbr OCA

Ncutoln infants u'ith this autosomalrecessivedisorder present in thc lirst r eck of life with vomiting, thcn alternating dccreased and increascdtone, proceedingto dcath rvithin a fcrv rveeksif left untrelrtcd.There is a characteristicodor ofthc urine likened to that of mirple svrup. Thc disorder is caused bv a dcficiency of thc producing increased branched-chainketoacid decarbox-vlase, c\crction of the branchcd-chainamino acids r-aline,leucine and isolcucine in thc urine, thc presenceof which suggeststhe diirgnosis,rvhich is confirmed bv the demonstrationof the three esscntialbranchcd-chainamino acidsin blood teatment involves a dict limiting thc intake of thc three branchcd-chainamino acids to the amount nccessar)ifor grorvth. Affected individuals are particularlr,'susceptibleto deterioration, in associationrvith intcrcurrent illnessesresulting from the catabolic degradation of protcins.

DISORDERS UREACYCLE

Homocrjstinuria is a reccssivclvinherited inborn error of sul{ur amino acid metabolism characterizedb1. mental retardation, frts, thrombocmbolic episodes,osteoporosisand n tendencl. to

that takes place Thc urel cvcle is a fivc-step metabolic pathwx,-v prinrlrilf in liver cells for the removalof rvastenitrogen from the amino groups of amino acids arising from the normal turnover of protcin. It converts trvo molecules of ammonia and one of bicarbonatcinto urca (Fig. l1 'l) Deficienciesof enzymesin the

dislocationof the lenses.This last feature,along rvith a tendencv to dcvclop a curvature of the spine (scoliosis),together \\ith a pectusexcavatum,and long lingcrs and toes (arachnodactvll'),can lead to confusion rvith the autosomaldominant disorder Marfan s1'ndrome(p. 289).

urcrrcr cle result in intoleranceto protein due to the accumulation of lrnmonia in thc body; or t'hat is knorvn as h-vperammonemta. Increascdammonia levelsare toxic to the central nervous s.vstem and can lead to coma and, rvith some of the urea cvcle disorders, if left untreatcd, death. The various disorders of the urea c-Ycle

HOMOCYSTINURIA

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11

B I O C H E M I CGAELN E T I C S

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are collectivelv rare and individuall-vverl' rare. Thev are all inherited as autosomal recessivedisorders, except ornithine transcarbamvlasedeficiencvrvhich is X-linked.

found in milk that is broken dolvn into galactose.Early diagnosis and treatment are essentialif the severecomolications are to be prevented

Hereditaryfructosei ntolerance

DISORDERS OFCARBOHYDR E ME BOLISM The inborn errors of carbohvdratemetabolismcan be considered in two main groups: disorders of monosaccharidemetabolism and the glvcogenstoragedisorders.

D I S O R D E ROSFM O N O S A C C H A R I D E ME BOLISM Th,o examples of disorders of monosaccharidemetabolism are galactosemiaand hereditarv fructose intolerance.

Gatactosemia Galactosemiais an autosomalrecessivedisorder due to deliciency of the enzvme galactosel-phosphate uridyl transferase,rvhich is necessarl'for the metabolism of the dietarl, sugar galactose Nervborn infants with galactosemiapresent rvith vomiting, lethargl', failure to thrive and jaundice in the second rveek of life. If untreated,they go on to develop complications that include mental retardation, cataractsand cirrhosis of the liver Galactosemiacan be screened for by the presence of reducing substancesin the urine with specific testing for galactose.The complications of galactosemiacan be prevented 164

by earlJ' diagnosis and feeding of affected infants rvith milk substitutes that do not contain galactoseor lactose, the sugar

Hereditar-v fructose intolerance is an autosomal recessive disorder due to deficienc.vof the enzyme fructose l-phosphate a l d o l a s e .D i e t a r v f r u c t o s e i s p r e s e n t i n h o n e y , f r u i t a n d c e r t a i n v eg e t a b l e s , a n d i n c o m b i n a t i o n w i t h g l u c o s e in the disaccharidesucrose in cane sugar. Individuals with hereditary fructose intolerance present at different ages, depending on when fructose is introduced into the diet. Svmptoms can be minimal but might also be as severe as those seen in galactosemia,which include failure to thrive, v o m i t i n g , j a u n d i c e a n d c o n v u l s i o n s .T h e d i a g n o s i s i s confirmed by the presenceof fructose in the urine and enzyme assa)ron an intestinal mucosal or a liver biopsy sample.Dietary restriction of fructose is associatedlvith a good long-term prognosls.

GLYCOGEN 5TORAGEDISEASES GlJ'cogenis the form in which the sugar glucose is stored in muscle and liver as a polymer, acting as a reserve energy source. In the glycogen storage diseases(GSDs) glycogen accumulates in excessiveamounts in skeletal muscle, cardiac muscle and/or liver due to a varietl-of inborn errors of the enz-vmesinvolved in svnthesisand degradationof glycogen. I n a d d i t i o n , b e c a u s eo f t h e m e t a b o l i c b l o c k , g l y c o g e n i s unavailable as a normal glucose source. This can result in hypoglycemia, impairment of liver function and neurological abnormalities

GENETICS BIOCHEMICAL

In each of the six major types of GSD there is a specific enzyme defect involving one of the steps in the metabolic pathwavs of glvcogen synthesisor degradation.The various types can be grouped accordingto rvhetherthey affect primarily the liver or muscle. All six types are inherited as autosomal recessivedisorders, although there are variants of the hepatic phosphorylasethat are Xlinked

Glycogenstoragediseasesthat primarity affect[iver von Gierkediseose(GSD-l) Von Gierke diseasewas the first describeddisorder of gl,,-cogen metabolism and is due to dehciency of the enzyme glucose6-phosphatase,which is responsiblefor degradation of liver glycogen to release glucose. Affected infants present with an enlarged liver (hepatomegaly) and,/or sweating and a fast heart rate due to hypoglycemia,which can occur after fasting of only 3-4h in duration Treatment is simple: frequent feeding and the avoidance of fasting to maintain the blood sugar concentratron.

Coridiseose(GSD-lll) Cori diseaseis causedby deficiencv of the enzyme amylo-l,6glucosidase,which is also known as the debrancher enzyme Deficiency of the enzvme results in glvcogen accumulation in the liver and other tissues due to the inability to cleavethe 'branching' links of the gl1'cogenpol,"-mer.Affected infants can present rvith hepatomeBalybecauseof gl-vcogenaccumulation a n d / o r m u s c l e r v e a k n e s s .T r e a t m e n t i n v o l v e s a v o i d i n g hypoglycemia by frequent feeding and avoiding prolonged periods of fasting

Anderson diseose(GSD- lV) Anderson diseaseresults from deficiency of glycogenbrancher enzvme leading to the formation of abnormal glycogenconsisting of long chains rvith few branches that cannot be broken dorrn by the enzymesnormallv responsiblefor glycogen degradation. Affected infants present with h1-potonia and abnormal liver function in the first vear of life, the latter progressingrapidly to liver failure. No effective treatment is availableapart from thc possibilit-vof a liver transplant.

Hepoticphosphorylose deficiency(GSD-Vl) Hepatic phosphorylase is a multimeric enzvme complex with subunits coded for by both autosomal and X-linked genes. D e f i c i e n c y o f h e p a t i c p h o s p h o r . v l a s eo b s t r u c t s g l l , c o g e n degradation, rvhich results in children presenting in the lirst 2vears of life with hepatomegalv,hvpoglycemia and failure to thrive. Tieatment is with carbohvdratesupplementsthat improve growth

Glycogen storagediseasesthat primarilyaffect muscle (GSD-ll) Pompediseose Infants with Pompe diseaseusually present in the first few months of life with floppiness(hypotonia) and delay in the gross They then develop motor milestonesbecauseof muscleweakness. failure in the first or second an enlarged heart and die from cardiac vear.\bluntary and cardiac muscle accumulatesglycogendue to the deficienc-vof the lvsosomal enzyme c-1,4-glucosidase, which is needed to break dou'n glycogen.The diagnosiscan be conhrmed b1' enzyme assayof white blood cells or fibroblasts. Earlv reports of enzyme replacementtherapy appearpromising.

McArdIe drseose(GSD- V) Personswith McArdle diseasepresent with muscle cramps on cxcrcise in the teenageyears.The condition is caused by a deficiency of muscle phosphorylase,which is necessaryfor degradation of muscle glycogen.There is no effective form of treatment, although in someaffectedindividuals the muscle cramps tcnd to declineifexerciseis continued,probablyasa result ofother cncrgy sources becoming availablefrom alternative metabolic pathwxl's.

FS T E R O IM DE B O L I S M D I S O R D EO RS The disorders of steroid metabolism include a number of autosomalrecessiveinborn errors of the biosynthetic pathways of cortisol These can result in the virilization of a female fetus along rvith an associatedsalt lossin both affectedmale and female infants duc to deliciencyof the hormone aldosterone.In addition, defects of the androgen receptor result in lack of virilization of chromosomallymale individuals(Fig. 11.5).

C O N G E NL I ADRENALHYPERPLASIA (ADRENOGEN I YNDROME) LS The diagnosisof congenital adrenal hyperplasia(CAH) should be considered in any newborn female infant presenting lvith virilization of the external genitalia, as this is the commonest causc of ambiguous genitalia in female nervborns (p. 277) (Fig ll.6). 21-H-vdroxylasedeliciency accounts for more than 90(lo of cases About one-quarter of affected infants have the salt-losing form, presenting in the second or third week of life u,ith circulatorl- collapse, hyponatremia and hvperkalemia. Less commonly CAH is due to deficiency of the enzymes llB-hvdroxylase or 3B-dehydrogenase,and very rarely occurs as a result of deficienciesof enzymes lTcr-hydroxylaseand Desmolasedeficiency is also ver.vrare, with all the 17,20-l1.ase. pathways blocked The phenotype is reversed with ambiguous genitalia in males, and severe addisonian crises may occur. Males rvith the rare 5o-reductase deficiencv are significantlv

165

11

BIOCHEMICAL GENETICS

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under-masculinizedbut do not suffer other metabolic problems and are likely to be raised asfemales.At puberty, however,the surge in androgen production is sufficient to stimulate growrh of the phallus, making gender identity and assignmentproblematic. Affected females with virilizing (classic) CAH have normal miillerian-derived internal genitalia, the virilization of the external genitalia being caused by an accumulation of the adrenocorticalsteroids proximal to the enzyme block in the steroid biosynthetic pathway,many of which have testosteronelike activity (seeFig. 1I .5) The possibility of CAH should not be forgotten, of course,in male infants presenting with circulatory collapsein the first few weeksof life. Affected infants, in addition to requiring urgent correct

166

assignment of gender, are treated with replacement cortisol, along with fludrocortisone if they have the salt-losing form. Virilized females may require plastic surgery in due course. Steroid replacementis lifelong and needsto be increasedduring

intercurrent illness or times of stress,such as surgery.Menarche in girls with salt-losingCAH is late, menstruation irregular, and they are subfertile.

ANDROGEN INSENSITIVITY SYNDROME Individuals with the androgen insensitivity syndrome (AIS) have female external genitalia and undergo breast development in puberty (p.276). They classicallypresent either with primary amenorrhea,which is the lack of onset of menstrual periods, or with an inguinal hernia containing a gonad that turns out to be a testis. Inguinal hernia is uncommon in girls and if present, especiallybilaterally,the possibility ofAIS should be considered. There is often scanty secondary sexual hair and investigation of the internal genitalia revealsan absent uterus and fallopian tubes with a blind-ending vagina.Chromosomeanalysisrevealsa normal male karyotype,46,XY

Fis.11.5 hastestesinthe whoclearly genitalia A,VirrLrzed B.A maLe babywithhypospadias externaL ina femaLe withcongenitaI adrenaI hyperplasia scrotaI sacs

Androgen production by the testes is normal in affected individuals but they do not bind androgennormally becauseof an abnormal androgenreceptor(seeFig. ll.5); a mutation is present in the androgen receptor gene on the X chromosome.This can be functionally assayedin skin fibroblasts. Some individuals have incomplete or partial androgen insensitivity and undergo variable virilization. Affected individuals usually have a female sexual orientation but obviously will be sterile. They require

or adolescencewith subcutaneousdeposition of lipid, known as xanthomata(Fig. I 1.7).Starting with familieswho presentedwith early coronary artery disease,Brown and Goldstein unraveledthe biology of the low-density lipoprotein (LDL) receptor (p. 226) and the pathologicalbasisof FH.

removal of the testesbecauseof an increasedrisk of developing a testicular malignancy, and should be placed on estrogen for secondarysexualdevelopmentand to prevent osteoporosisin the longer term.

D I S O R D EO RS FL I P I DM E B O L I S M Familial hvpercholesterolemiais the commonest autosomal dominant single-genedisorderin Westernsocietl'andis associated with high morbiditl' and mortalitv rates through premature coronar-varter-vdisease(p.226)

MILIAL HYPERCHOLESTEROLEMIA Persons rvith familial hvpercholesterolemia (FH) have raised cholesterol levels with a significant risk of developing earl1. coronarv arterv disease(p.226). The-v can present in childhood

Fis.11.7 hypercholesteroLemia, forfamiliaI Legsofa personhomozygous (Courtesy of Dr E Wraith, xanthomata muttipte showing Manchester.) s HospitaL, Chitdren Manchester RoyaI

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11

GENETICS

Cells normally derive their cholesterol from either endogenous cholesterol synthesis or by uptake of dietary cholesterol from LDL receptors on the cell surface. Intracellular cholesterol levels are maintained by a feedback system, with free cholesterol inhibiting LDL receptor synthesis as well as reducing the level of de noaoendogenouscholesterolsynthesis. The high cholesterol levels in persons with FH are due to deficient or defective function of the LDL receptors leading to increased levels of endogenous cholesterol synthesis. Four main functional types or classesof muration in the LDL receptor have been identified: reduced or defective biosynthesis of the receptor; reduced or defectivetransport of the receptor from the endoplasmic reticulum to the Golgi apparatus; abnormal binding of LDL by the receptor; and abnormal internalization of LDL by the receptor (Fig. 11.8). Specific mutations are found more commonly in persons with FH from certain ethnic groups, as a result offounder effects(p.127). Dietary restriction of cholesterol intake and drug treatment with agentssuch as cholestyramine,which sequesterscholesterol from the enterohepatic circulation, can lower cholesterol levels and, in part, reducethe risk ofcoronary artery disease. The recent detailed elucidation of the metabolic and biosynthetic pathways of

cholesterol has enabled further therapeutic measuresthrough the development of drugs that inhibit the endogenoussynthesisof cholesterol by inhibiting the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase.These are proving to be more effective in preventing long-term complications.

MALSTORAGE DISORDERS In addition to the inborn errors of metabolism, in which an enzyme defect leads to deficiency of an essential metabolite and accumulation of intermediate metabolic precursors, there is a number of disorders in which deficiency of a lysosomal enzyme involved in the degradation of complex macromolecules leads to their accumulation. This accumulation occurs because macromolecules are normally in a constant state of flux, with a delicate balance between their rates of synthesis and breakdown. Children born with lysosomal storage diseasesare usually normal at birth but with the passageof time commence a downhill course of differing duration owing to the accumulation of one or more of a variety or type of macromolecules.

LDL Protein C h ol e s t e r o l ester

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of LDLreceptor synthesis llJ Abnormality of transport to Golgicomplex @ Abnormality p

Abnormality of LDLbinding oyrecepror

of receptor pit clustering in coated @ Abnormality

Fis.11.8 158

Stagesin chotesteroLbiosynthesis (LDL)receptors. andinthemetabolism of low-density Lrpoprotein indicating thetypesof mutation in (Adapted famitiaL hyperchoLesterotemia fromBrownM S,Gotdstein J L 1986A receptor-mediated pathway forchotesterol homeostasis Science 232,34-47)

B ] O C H E M I CGAELN E T I C S

POLYSACCHARI MUCO DOSES C h i l d r e n r v i t h o n e o f t h e m u c o p o l v s a c c h a r i d o s e(sN { P S s ) present \\,ith skeletal, vascular or central nervous s_\stem findings along t ith coarseningof the facial features These f e a t u r e s a r e d u e t o p r o g r e s s i r , ea c c u m u l a t i o n o f s u l f a t e d polysaccharidcs(also known as glvcosaminogl-vcans) causedb-v defective degradation of thc carbohvdrateside-chain of acid mucopolysaccharidc Six different N{PSs are recognized,based on clinical and genetic differences Each specilic MPS t.vpehas a characteristic pattern of excretion in the urine of the glvcosaminogl-vcans, dermatan,heparan,keratanand chondroitin sulfate.Subsequcnt biochemical investigation has revealed the various tvpes to be due to delicienc,vof different indir,idual enzymes.All but Hunter syndrome,rvhich is X-linked, are inherited asautosomalrecessir.e disorders

Hurlersyndrome(MPS-l) Hurler s-vndromeis the most severeof the MPSs Affected infants present in the first year with corneal clouding, a characteristic curvature of the lorver spine and subsequent poor growth. The-v develop hearing loss, coarsefacial features, an enlargedlivcr and spleen,joint stiffnessand vertebral changcs in the second vear. There is progression of these features along rvith mental deterioration and eventualll' death b-v the mid-tcens from a combination of cardiacfailure and resoiratorv infections. T h e d i a g n o s i s o f H u r l e r s - v n d r o m er v a s i n i t i a l l l . m a d e b-v demonstrating the presence of metachromatic granulcs in the cclls, i e l-vsosomes distended b-v the storagematerial that is primarilv dermatan sulfate. Increased urinary excretion of dermatan and heparan sulfate is commonlv used as a screcning test, but confirmation of the diagnosis involr,es demonstration of reduced activit-v of the lysosomal h v d r o l a s e ,c r - r - i d u r o n i d a s e . L e s s s e v e r ea l l e l i c f o r m s o f Hurler svndrome, causedby varving levels of residual o-1.iduronidase activity, were previously classified separatelv a s S c h e i e d i s e a s e( N , I P S - I S ) a n d H u r l e r / S c h e i e d i s e a s e

(MPS-r H/S). H u n t e rs y n d r o m e( M P S - l l ) Nlales rvith Hunter s-vndromeusually present between the ages of 2 and 5 t.earsrvith hearing loss, a histor.v of recurrent infection, diarrhea and poor growth. Examination reveals a characteristiccoarseningof their facial features(Fig. 11.9),along u.ith enlargement of the liver and spleen, and joint stiffness. Radiographsofthe spine revealabnormalitiesin the shapeofthe vertebrae.There is progressivephysicaland mental deterioration, t ith death usuallv in the teenagevears The diagnosisis confirmed by the presenceof excessamounts of dermatan and heparan sulfate in the urine and deficient or decreasedactivit.v of the enzvme iduronate sulfate sulfatasein serum or white blood cells

Fi9.11.9 Huntersyndrome Faces of a malew th the mucopolysaccharidosis, (Courtesyof Dr E Wraith,RoyalManchesterChildrens Hosprta[, Manchester)

S a n f i t i p psoy n d r o m e( MP S - l l l ) Sanfilippo s,vndrome is the most common MPS. Affected individuals present in their second1'earwith mild coarseningof features,skeletalchangesand progressiveintellectual loss rvith bchar.ioral problems, proceeding to convulsions and death in earlr:.adult life. The diagnosisis confirmed bv the presenceof incrcasedurinar-vheparanand chondroitin sulfateexcretion,and deliciencv of one of four enzvmes involved in the degradation of hcparan sulfate: sulfaminidase (MPS-III A)' N-acetyl-u-o(MPS-III B), acetyl-CoA-o-glucosaminidaseglucr,rsaminidase N-acetyltransferase(MPS-III C) or N-acetyl-glucosamine6-sulfatesulfatase(MPS-III D). Individuals with thesedifferent enzvme deficienciescannot be distinguishedclinicalll''

Morquiosyndrome(MPS-lV) Children rvith N{orquio svndrome presentin the secondor third vear of life rvith skeletalabnormalitiesthat include short stature, thoracic deformity- and curvature of the spine (k-vphoscoliosis). Intelligence is normal and survival is long term, although therc is a risk of spinal-cord compression due to progresslon of the skeletalinr,olvement.The diagnosisis confirmed by the presenccof keratan sulfate in the urine and deficiencl' of either (MPS-IV A) or B-galactosidase galactosamine-6-sulphatase (NIPS-I\r B).

syndrome(MPS-Vl) Maroteaux-Lamy Individuals with Maroteaux-Lamy syndrome present with Hurler-likc features in early childhood. These include coarse facial features,short stature with thoracic deformity, kyphosis and restriction of ioint mobility. In addition they developcorneal clouding and cardiac valve abnormalities,but retain normal intelligence.A less severeform presentslater with survival into

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late adulthood. in contrast to the more severe form in which surr,ival is usuallv onlv until the third decade The diagnosisis confirmed bv the presenceof increasedurinar-vdermatan sulfate excretion and arylsulfataseB dehciencl.in rvhite blood cells or hbroblasts.

S l ys y n d r o m e( M P S - V l l ) Sl-vsvndrome shorvsmarked variabilitv in the scveritv of prescntation. This ranges from skeletal features that includc mild k-vphoscoliosisand hip dysplasia to coarse facial fextures, hepatosplenomegall., corneal clouding, cardiac abnormalities and mental rctardation, rvith death in childhood or adolescencc. I n c r e a s e d u r i n a r v g l v c o s a m i n o g l v c a n se x c r e t i o n a n d B glucuronidasedeficienc.rin serum, u.hiteblood cellsor fibroblasts conlirm the diagnosis.

Treatmentof the mucopolysaccharidoses Treatment of the N{PSs has been attempted bv enzvme replacemcnt, but a numbcr of practical difhculties have been responsible for its lack of succcss(p. 3a0). N,Iore recentll-, holever, treatment bv bone marro\\ transplantation has becn attempted, tith varying reports of success,biochemicallv and clinicalh; in the skeletaland cerebralfcatures.

S P H I N G O L I P I D O( L SIEPSI D STORAGE DISEASES) I n t h e s p h i n g ' o l i p i d o s c st h e r e i s a n i n a b i l i t l ' t o d e g r a d c sphingolipid, resulting in the progressivedeposition of lipid or gl1'colipid, primaril.v in the brain, liver and spleen. Central nervous s.vsteminvolvement results in progressive mental deterioration, often rvith fits, usuallv resulting in death in childhood. Thcre are at least l0 different t1'pcs uith specilic enzvmc dehcicncies:tv-Sachs, Gaucher and Niemann-Pick diseasesarc the most common

Tay-Sachsdisease This is the bcst known sphingolipidosisand affectsapproximatell. I in 3600 personsofAshkenaziJervishanccstrv(p. 304) Affected infants usually present bv 6months of agc rvith poor feeding, lethargy and floppiness. Loss of developmental milestones or devclopmentalregressionusually becomcapparentin the second half of the lirst year.Feeding becomesincreasinglv diflicult and the infant progressir.elvdcteriorates,rvith deafncss,visual impairment and spasticitl',which progressesto rigidir\.. Death usuallv

170

occurs bv the age of 3 vears as a result of respiratorv infection Less severcjur.enile,adult and chronic forms arc also reported. The diagnosis of Tav-Sachs diseaseis further supported clinicall.v bv the presenceof a 'cherrv rcd' spot in the centcr of thc macula of the fundus. Biochemical conlirmation of Thv Sachsdiseascis by dcmonstration of reduced hexosaminidaseA levelsin serum, rvhiteblood cells or cultured fibroblasts.Reduced

hexosaminidaseA activitv is due to dehciencl' of the cxsubunit of the enzvme B-hexosaminidasethat leads to accumulation of the sphingolipid, G\"I2 ganglioside Deficiency-of the B subunit of B-hexosaminidaseleads to reduced activit-vof the isozyme, h e x o s a m i n i d a s cB , c a u s i n g t h e o t h e r G M z g a n g l i o s i d o s i s , Sandhoff disease,in r,vhichaffected individuals present with similar clinical features.

G a u c h edr i s e a s e This is the commonest sphingolipidosis;like Tay-Sachs disease, it occursl'ith an increasedfrequencyamong personsofAshkenazi Jewish ancestry There are two main tvpes based on the age of onset. In t-vpeI, or the adult type, which is the most common form of Gaucher disease,affected persons present rvith febrile episodes and limb, joint or trunk pain and a tendenc.vto pathological fractures. Clinical examination usuallv reveals an enlarged spleen and liver. Affected persons often shor,ymild anemia and radiographic changes in the vertebral bodies and proximal f'emora.The central nervous svstemis spared. In t1.peII, or infantile Gaucher disease,central nervous sl-steminvolvement is a major feature Affected infants usually present between 3 and 6months of age rvith failure ro thrive and hepatosplenomegalrr Bv 6 months of age thev begin to show developmental regression and neurological deterioration, with spasticit--\and lits leading on to recurrent pulmonarv infection and death in the secondvear The diagnosis of Gaucher diseaseis conlirmed bv reduced activit]' of the enzvme glucos-vlceramide B-glucosidasein rvhite blood cclls or cultured libroblasts. Treatment of individuals rvith the adult type of Gaucher diseaseinvolves s1-mptomaticrelief of pain In addition, it is often necessaryto remove the enlargedspleen becauseit causes a secondaryanemia due to premature sequesteringof red blood cells, a condition knoln as hvpersplenism The lack of central ner\rous s].stem involvement in t-vpeI Gaucher diseasemeant that it was an obvious candidate for enzvme replacemcnt therapl; as it vvasnot necessarvfor the enzyme to crossthe blood brain barrier (p. 3a0). Initial attempts to treat adults with Gaucher diseasebv enzyme replacement therapv met rvith little successbecauseof difficulty- in obtaining sufficient quantities of enzvme and in targeting rhe appropriate sites However, modification of B-glucosidaseby the addition of mannose6-phosphate,which targets the enzyme to macrophage lysosomes,has led to dramatic alleviation of symptoms and regression of the organomegaly in affected persons. The treatment is, however,expensive,and regimensusing lorverdoses and altcrnativemethods to target the enzvmemore efficiently are being assessed

Niemann-Pick disease Infants with Niemann-Pick diseasepresentwith failure to thrive and hepatomegall-,and a cherrv-red spot may be found on rheir

BIOCHEMICATGEN !:

macula Developmentalregrcssionprogressesrapidlv b.vthe end of the first 1'ear,with death b-vthe ageo[4vears A characteristic finding is the presenceof what arc called foam cells in the bone marrow duc to sphingomyelin accumulation. Confirmation of the diagnosis is b1,demonstration of de{iciency of the enzvme sphingomyelinase.A less ser.ereform rvithout neurological involvement has been reported. Niemann-Pick disease,likc t1.-Sachs and Gaucher diseases,occurs more commonly in Jews ofAshkenazi easternEuropean ancestr]..

dcaminaseAffected children presentin the first year of life rvith recurrcnt viral and bacterial infcctions and, if untreated, will dic from overrvhelminginfcction in thc first year.The diagnosis is confirmed bv deficient red blood cell adenosinc deaminase activit),. Correction of thc immunodeficiencl' by transfusion of irradiated red blood cells has been reported N{ore recently bone marrow transplantationhas been successfullvcarried out - even antenatalll,in utero.

phosphorylase deficiency Purinenucleoside

DISORDERS OFPURINE/PYRIMIDINE ME BOLISM Gout is the disorder in humans that is classicallr,associated with abnormalities of purine mctabolism Joint pain, swelling and tcndernessare a result of the inflammatory responseof the body to depositsof crystals of a salt of uric acid. In fact, onl.va minority of personswho present rvith gout are found to have an inborn error of metabolism.The causein most instancesresults from a combination of genetic and environmcntal factors; it is, however,alway'simportant to consider disorders that can result in an increasedturnover of purines (e.g a malignancv such as leukemia) or reduced secretionof the metabolites(e.g rcnal impairmcnt) as a possibleundcrlying precipitating cause.

LESCH.NYHAN SYNDROME A particularly disabling disorder of purine metabolismis thc Lesch-N1,han svndrome. This X-linked disordcr is due to the deficiency of the enzyme hypoxanthine guaninc phosphoribosyltransferase that results in increasedlevels of phosphoribosylpyrophosphateThc latter is normallv a ratelimiting chemical in the synthesis of purines Its excessleads t o a n i n c r e a s e dr a t e o f p u r i n e s y n t h e s i s ,r e s u l t i n g i n t h e accumuhtion of excessiveamounts of uric acid and some of its metabolic precursors.Thc main effect is on the central nervous mental s!'stem, resulting in uncontrolled movements,spasticity-, retardationand compulsiveself-mutilation.Although drugs such as allopurinol that inhibit uric acid formation can lower uric acid levels,none vct offers satisfactorvtreatment for the debilitating central nervous svstemeffects.

DC I SYE A S ECSA U S E D IMMUNODEFICIEN B YD E F E C TISN P U R I N E ME BOLISM Two inherited immunodeficiency disorders (p. 190), somervhat surprisingll,,are inborn errors of purine metabolism

A proportion of children susceptiblc to severc)recurrent and potentitrll-vfatal viral infections with isolated impaired T:cell function have been shorvn to have a deficicncv of thc enzlme purinc nucleosidephosphorylase.Trcatment rvith irradiated red blood cells has becn reported to result in a temporarv improvement in immune function.

HEREDI RYOROTICACIDURIA Children rvith hereditary.orotic aciduria present in the lirst vear of lif'e with a megaloblasticancmia, failure to thrive and developmentaldelarrThe.vhavedcficienc.vof one of two enzYmes' or orotidine 5'-phosphate orotate phosphoribosl,ltransferase for the svnthesisof both of which are necessar-Y decarboxy'lasc, py'rimidines,resulting in excretion of large quantities of orotic acid in thc urinc. Treatment rvith the p.vrimidine uridine has beenrcportcd to reduce urinary orotic acid excretion' correct the rncmia and restore growth.

OFPORPHYRIN DISORDERS ME BOLISM Thcrc are scveral different disorders of porphyrin metabolism that are duc to deficiencl-'of enzymes in the biosynthetic pathrvavof the iron-containing group in hemoglobin, heme (p l+7) The-vare all inherited as autosomaldominant disorders, e\ccpt for congenital erythropoietic porphyria, u'hich is an autosomal reccssivedisorder. This is becausethe enzl'-mesare ratc limiting (p. 26), so that haploinsufficiencyresults in clinical discase. Thc different types of porphl'ria are variably associatedu'ith neurological or visceral involvement and cutaneousphotosensitir.itr, due to accumulationof the different porphyrin prccursors in those organs.The porphyrias are divided into two types depcnding on whcther the excessproduction of porphyrins occurs predominantly in the liver or in the erythropoietic SVSTElTr

A d e n o s i n de e a m i n a sdee f i c i e n c y About a half of all children rvith the autosomal recessiveform of severecombined immunodeficiencl'lith impaired B- and T-cell function (p 19l) have deficiencyofthe enzvme adenosine

HE

ICPORPHYRIAS

Thcse include acute intermittcnt porph-vria,hereditar-vcoproporphlria and porphl ria raricgata.

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Acuteintermittentporphyria Acute intermittent porphvria (AIP) is characterizedbv attacksof abdominal pain, .lveakness, vomiting and mental disturbance in

personshavephotosensitivitvand can sometimesdevelopchronic livcr disease.Successful treatment of the photosensitivitl' has been reported with B-carotene.

the form of confusion, emotional upset or hallucinations.Even coma mav occur, and rlomcn are morc severel.vaffected than men, with symptoms sometimesassociatedrvith the menstrual cvcle. Attacks can also be prccipitated by the administration of certain drugs, such as exogenoussteroids, anticonvulsantsand barbiturates It is caused bv a partial deficiencl-of the enzvme uroporphy'rinogenI svnthaseleading to increasedexcretion of the porph,v'.rin precursorsporphobilinogen and 6-aminolevulinic acid in urine

Hereditaryco-proporphyria In hercditarv co-proporphvria, a related condition also inherited as a dominant trait, there is partial deficiencr of the enzyme co-proporphvrinogen oxidase.The disorder is clinicallv indistinguishable from acute intermittent porphvria, although approximately one-third of affected persons also have photosensitivirro[ the sLin.

Porphyriavariegata Persons rvith this form of porphyria, r'hich is particularlv prevalent in those ofAfrikaaner origin in South Africa (p. 103), havevariableskin photosensitivitvl,r'ithneurologicaland visceral findings that can also be triggcred bv drugs Increased fecal excretion of the porphl,rin precursors protoporph-vrin and coproporphvrin can bc demonstrated and the disorder has been sho\\n to be due to delicicnc.vof thc enzvmeprotoporphvrinogen oxidase.

ERYTHROPOIE CP H Y R I A S PT OIR

ORGANIC.ACID DISORDERS Children affectedwith one ofthe organic-aciddisorderspresent rvith periodic episodesof poor feeding, vomiting and lethargy in associationr,vith a sevcre metabolic acidosis, low white cell (neutropenia) and platelet (thrombocytopenia) counts, low blood sugar (hypoglycemia)and high blood ammonia levels (hvperammonemia). These episodes are often precipitated bv intercurrent illncss or increasedprotein intake,and after such an episodeaffectedchildren can lose developmentalskills. Analysis ofblood from children at thc time of theseepisodesrevealshigh levels of glvcine (hvperglvcinemia) It was subsequentll.found that the acidosisin theseepisodeswas due to increasedlevelsof the organic acids,either propionic or methylmalonic acid. T h e t w o a u t o s o m a l r e c e s s i r . eo r g a n i c - a c i d d i s o r d e r s methvlmalonic acidemia (MMA) and propionic acidemia(PPA) are caused by deficiencl' of the enzvmes methylmalonyl-CoA mutase and proprionvl-CoA carboxylase,respectively.The enzvmc deficienc-vresults in accumulation of the toxic organicacid metabolitesderived from deamination of cerrain amino acids,specilic long-chain fatty acids and cholesterolside-chains. Therapl' fbr the acute episode inr.olves the treatment of any infection, fluid replacement,correction of the metabolic acidosis and ccssationofprotein intake.Long-term prophylactictrearment involves rcstriction of protein intake and rapid rccognition and management of anv intercurrent illness. A proportion of individuals affected with PPA arc responsiveto biotin, rvhereas some personswith MMA are sensitiveto vitamin Brz.

The erythropoietic porph.vriasinclude congenitalerythropoietic porphyria and erythropoietic protoporphyria.

DISORDEO RS FC O P P EMRE B O L I S M Congenital porphyria erythropoietic The main feature of congenital ervthropoietic porphyria (CEP) is an extreme photosensitivitywith blistering of the skin leading to extensivescarring, to the ertent that most affected persons are unable to go out in normal dal.light. In addition, man-vhave a hemolytic anemia requiring regular blood transfusion and frequently splenectom]'.Affected individuals have red brown discoloration of the teeth, which shos' red fluorescenccunder ultraviolet light. Congenital erl.thropoietic porphl'ria is due to deficiencl,of the enzyme uroporphyrinogen III synthase.

Erythropoietic protoporphyria

172

Ervthropoietic protoporphvria (EPP) is due to deficiency of the enzyme ferrochelatase,rvhich is responsiblefor the insertion of ferrous iron into the porphyrin precursor to form hemc Affected

There are two inborn errors of copper metabolism: Menkes diseaseand Wilson disease

M E N K ED SI S E A S E Menkes diseaseis an X-linked disorder in rvhich affected males present in the first few months of life with fceding difficulties, vomiting and poor .w.eightgain. Subsequently floppinesss (hypotonia), fits and progressive neurological deterioration ensue,with death duc to recurrent respiratory infection usuall-v occurring b1'the agcof 3 vears.A characteristicfeatureis the hair, uhich lacks pigment, is kinky and brittle, and breakseasily.This rvasnotcd to resemblethe wool of sheep suffering from copper dehciency Serum copper and ceruloplasmin levcls are very low. Cloning of the gene for l,{enkes diseasewas facilitated through an affected fcmale with an X aurosome translocation (p ll0)

BIOCHEM]CAL GENETlf,s

and revealedit to code for an ATPase cation transport protein for copper.Tieatment regimenswith different exogenouscopper sourceshavc had limited benefit to date.

W I L S O ND I S E A S E Persons affccted with the autosomal recessivedisorder Wilson diseasecommonly presentin childhood or the early teenagevears rvith lits and abnormal neurologicalfindings. Thcse can include deterioration of coordination, involuntary movements,abnormal tonc, dysarthria (difficulty in speaking), dy'sphagia(difficult,-v and changesin behavior or frank psychiatric uith sr,vallor,ving) disturbance.Clinical examinationcan revealthe presenceof rvhat is called a Kayser-Fleischer ring, rvhich is a golden brorvn or greenishcollaretteat the cornealmargin. Investigationcan reveal the presenceof abnormal liver function, which can progress to cirrhosis. High copper levelsin the liver, decreasedserum concentrations of the copper transport protein ceruloplasmin, and abnormal copper loading test results are suggestiveof the diagnosis.The

cataractsand an enlargedliver They generallygo on to have fits rvith developmentalregressionand usually die by I year of age. Invcstigations can reveal renai cvsts and abnormal calcification in thc cartilaginousgrowing endsof the long bones(Fig. 11.11). There is a rangeof severityof this disorder,with different clinical diagnosesbeing given to the lessseveretypes.The diagnosiscan be conlirmed by- raised levels of plasma long-chain fatty acids. The gene for Zclhvegersyndrome encodesa protein involved in the peroxisomeassembly. It is unusual for inborn errors of metabolism to be associated lr,ith a dvsmorphic s1-ndrome(p.237).In addition to Zellweger srndrome, the Smith-Lemli Opitz syndrome has recently been found to be caused by an inborn error of cholesterol biosvnthesis.

ADRENOLEUKODYSTROPHY Males r,viththe X-linked disorder adrenoleukodystrophy(ALD) classicallvDresent in late childhood with deteriorating school

gene for Wilson diseasewas cloned on the basis of anticipated homology to the Menkes gene, and the gene product has been shoun to be an ATPase cation transport protein involved in copper transfer from the hepatocytes to the biliary collecting system There are dramatic reports of striking improvement of the neurological features in persons rvith Wilson diseaseusing the chelatingagentso-penicillamine and trientine, although these can causeside-effects.

DISORDERS PEROXISOMAL The peroxisomesare subcellular organellesbound b1' a singlc trilal.er lipid membrane present in all cells; thev are especialll' abundant in liver and renal parench-vmalcells The organelle matrix contains more than 40 enzymesthat carrv out a number of reactions involved in fatty-acid oxidation and cholesterol bios-vnthesisinteracting rvith metabolic pathways outside the perorisomes. The enzymes of the peroxisomal matrix arc

Fis.11.10

a prominent showing syndrorne ofan infantw th Zellweger Facies forehead

svnthesizedon the polyribosomes,enter the cytosol and are transferred into the peroxlsomes. There are trvo main categories of peroxisomal disorder: disordersof peroxisomebiogenesis,such as Zellwegersyndrome, in which there are severelyreduced numbers of peroxisomesin all cells, and single isolated peroxisomal enzyme deficiencies, such as X-linked adrenoleukodystropht.

SYNDROME ZEL EGER Nervborn infants with Zelln-eger syndrome present rvith hypotonia and rveaknessand have mildly d.vsmorphicfacial features(Fig. 11.10),consistingof a prominent foreheadand a large anterior fontanelle ('soft spot'). They ma1-also have

Fis.11.11 ofthekneeofa newbornnfantwithZellweger Radiograph ofthedistal punctate calcification abnormaI showrng syndrome f e m o r aeIp t p h y s e s

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performance, although affectcd malcs can prescnr ar anv age a n d e v e n , o n o c c a s i o n ,b c a s y m p t o m a t i c .A p r o p o r t i o n o f affected malcs can also present in adult lifc uith lcss severc ncurologicalfcaturesand adrenalinsufficiencr,or rvhatis termed adrenomreloneuropath].. ALD has been shor.n to bc associatcd

o w i n g t o t h e p h e n o m e n o n o f h e t e r o p l a s m v( s e e F i g . 7 2 9 , 'fhe p. 120). clinical features are mainly a combination of neurological signs (cncephalopathy dementia, ataxia, dystonia,

with a deficiencv of rhe enzyme ver-vlong-chrin fatt-v acid (VLCFA) CoA svnthase,but is sccondarv to deficienc-vof a peroxisomalmembrane protein.

svmptoms and signs mav include deafness,diabetesmellitus and retinal pigmentation, and acidosis ma.v be a feature. The clinical manifcstations are so variable that a mitochondrial c]-'topathvshould be consideredas a possibilit-vat any age when the prescntingillnesshasa neurologicalor mvopathiccomponent.

teatment of ALD is being undertakcn rvith a diet thar uses as a sourcc of fat an oil that has lorv lo.els of thcVLCFAs. This has beenpopularlv called 'Lorenzo's oil' aftcr a {ilm of that name made about a child rvith adrenoleukodvstrophl,. The eflicac-vof this diet has,holever, been disappointing.

DISORDERS AFFECTING MITOCHONDRIAL FUNCTION N'Iitochondrial diseascr,yasfirst identified in 1962 in a parient r,vhosemitochondria shorvedstructural abnormalities and loss of coupling bcts.eenoxidation and phosphorvlation, although it was not until the late 1970sand early 1980sthat the relevanceof mutated mitochondrial DNA (mtDNA) to human discasebegan to be appreciated The small circular double-strandedmtDNA (see Fig. 2.7, p 18) conrains genes coding for ribosomal RNA (rRNA) production and various rransferRNAs (IRNA) required for mitochondrial protein biosvnthesis,as rvell as some of the proteins inr,olved in elcctron transport There are 5523 codons and a total of37 geneproducts Guanine and cvtosinenucleotidcs are asvmmetricallvdistributed betrveenthe ttvo mtDNA strandsthe guanine-rich strand being calledthe heavv(H) strand and the c.v--tosine-rich the light (L) strand. Replication and transcription i s c o n t r o l l e d b v a 1 1 2 2 - b p s e q u e n c eo f m I D N A k n o w n a s the displacement loop (D-loop). Oxidative phosphorl-lation (OXPHOS) is the biochemicalproccssresponsiblefor generating much of the ATP required for cellular energ\..The process is mediatedb1'.fivc intramitochondrial cnzvme complexes,referred to as complexesI-\ and the mrDNA encodcs 13 OXPHOS subunits,22 tRNAs and 2 rRNAs. The 'complexes'arc aptly named! Anall.sis of complex I, for example, has revcaled approximatell- 'll differcnt subunits, of lr.hich sevenare polvpcptides cncoded b_,-mtDNA genesknotn as .\'D1, ND2, ND3, NDL{, AD4, AD5 and ,\D6, with thc remaining 34 subunits encodedb,r'.nuclcar DNA genes Complcr V compriscs 12 or 13 subunits of which trvo, ATPasc 6 and 8, are encodedb1,mtDNA. N{aximal activit-\ of compler V appears to require tight linking with cardiolipin (see Barth syndrome, p. 175),encodedb1,nuclear DNA.

174

As most mitochondrial proteins, including subunits involved in elcctron transport, are encoded bV nuclear genes,these most often follorv autosomal reccssir.einheritancc As rvith other metabolic autosomal recessivediseases,disordcrs resulting from mutations in these genestend to breed true. Ho\\.ever,the disorders due to mutations in mtDNA are extremelv r.ariablc

neuropathv and scizures) and mvopathic signs (h1'potonia, rveaknessand cardioml''opath1. lr,ith conduction defects). Other

A number of distinct clinical entities have been determined and, although some of them overlapconsiderabll.,there is a degreeof genotvpe-phenotvpecorrelation.

M Y O C L O NEIPCI L E P S Y A R NA DG G ERDE D FIBER D I S E A s(EM E R R F ) N'Ivoclonicepilepsy and ragged red hber (MERRtr) diseasewas Iirst describedin 1973and so called becauseGomori's trichrome staining of muscle revealedabnormal deposits of mitochondria as'raggcd red'; in 1988it rvasappreciatedthat the condition rvas maternallv inhcrited. Thc classic picture is of progressir.e myoclonic epilepsy.,mvopathv and slowly progressivedementia. Optic atrophl,' is frequentll' presenr and the EEG is characteristically' abnormal. Post-mortem brain examination reveals rvidespreadneurodegeneration.In 1990 it rvas reported that N'IERRF results from a point muration in the gene for lysine tRNA.

M I T O C H O N D REI N AC L E P H A L O M YH OY LACTIC ACIDOSIS ANDSTROKE. LIKE E P I s O D E( M S ELAS) First delineatedin 1984,this extremel-vvariablecondition is notr recognized as one of the commoncst mitochondrial disorders. Short stature mav bc a feature,but it is stroke-like episodesthat mark out this particular mitochondrial disorder, although these episodesdo not necessarilyoccur in all affectcd family members. Whcn thev do occur, the,v-ma-vmanifest as vomiting, headache or visual disturbance,and sometimeslead to transient hemiplegia or hemianopia.A common presentingfcature of MELAS is type 2 diabetes mellitus, and a sensorineuralhcaring loss mav also occur (described as maternally inherited diabetesand deafness INIIDD]). Thcse latter clinical fearures are associaredwith the most common mutation, r,vhichis an A>G substitution at nucleotidc m.3243, r,vhichaffects IRNA leucineuuR.This is found in about 80o/oof patients,follorvedby a T>C transition at nucleotide m.3271, also affecting IRNA leucineuuR.

( NARP) ANDRETINITIS PIGM ENTOSA The carlv presenting fcature is night blindness, rvhich may be lbllou.ed vcars later bv ncurological svmptoms. Dementia mav occur in oldcr patients,but seizurescan presentat almost anJrage

B I O C H E M I CGAELN E T I C S

and younger patients shorv do'clopmental delar,-. The majorit,v of casesare due to a single mutation the T>G substitution xt nucleotidem 8993, rvhich occurs in the coding region ofsubunit 6 of ATPase. This change is olten referred to as the NARP mutanon.

LEIGH DISEASE This condition is characterizedb-vits neuropatholog],consisting of tvpical spongiform lesions of the basal ganglia, thalamus, substantianigra and tegmental brainstem. In its severe form death occurs in infancl- or early childhood, and it rvas in such a patient that the m.8993T>G NARP mutation was Iirst identified. In effect, therefore, one form of Leigh disease is simply a sevcreform of NARR and higher proportions of mutant mtDNA have been reported in these cases.I{owever, variabilitl. is again sometimes marked and the author knorvs one famill. lvhere a mother, whose daughter died in earlv childhood, was found to have lorv lcvcls of the 8993 mutation and her only svmptom \ras slorv recor.erv from a general anesthetic The same or very similar patholog)., and a similar clinical course, has now bccn described in patients r'r,ithdifferent molecular dcfccts. C.ytochromer deficiencv has been reported in a number of patients and somc of thcsc havc bccn shorvnto have mutations in S[/RF1, a nuclear gene.These casesfollou autosomal recessiveinheritance Lcigh diseascis thereforc geneticalhheterogeneous.

D I S O R D E ROSFM I T O C H O N D R I A L In the 1970s the first reports appearedof patients rvith skelctal muscle rveakncssand abnormal muscle fatty-acid metabolism associatedlvith decreasedmuscle carnitine. The carnitine c1'cleis a biochemicalpathwa-vrequired for the transport of long-chain fatt-vacids into the mitochondrial matrix, and those less than 10 carbons in length are then activatedto form acll-CoA esters The carnitine c1'clcis one part of the pathrval' of mitochondrial B-oxidation that pla]'s a maior role in energlproduction, especiall-vduring periods of fasting. Carnitine dcficiencr is a secondary feature of the B-oxidation disorders' r,ith thc exception of the carnitine transport defect where it and this rare condition responds dramatically to is prin-rar1:, carnitine replacement.The more common fattl'-acid oxidation disordcrs are outlined.

acyt-CoAdehydrogenase Medium-chain deficiency \''lcdium-chain acy-l-CoA dehvdrogenase(NICAD) deficiency is the commoncst of this group of disorders, presenting most provoked b1' frequentlv as episodic h-vpoketotich1'pogl-vcemia fasting.Thc onsetis often in the lirst 2 yearsof life and' tragicallr; is occasionallvfatal, rcsembling sudden infant death syndrome. Nlanlgcment rests on maintaining adequatecaloric intakc and avoidanceof fasting, which can be challengingin voung children rvith intercurrent illnesses.Inherited as an autosomal recessive disordcr, 90o/oof allelcs result from a single point mutation' lhich has led to discussionthat this could be a candidatedisease I o r n c o n r t a lp o p u l a t i o ns c r e e n i n g

Leber hereditary optic neuropathv (LHON) rvas the first human diseaseto be shor'vnto result from a mtDNA point mutation; about a dozen different mutations have no'w,becn described.The most common mutation occurs at nucleotide m.11,778 (NDl gene), accounting for up to 70o/oof casesin

Long-chain(LCAD)and short-chain(5CAD) and [ong-chain3-hydroxyacy[-CoA acyt-CoA, (LCHAD)dehydrogenase deficiencies

Europc and morc than 90o/oof casesin Japan. It presentslvith acute, or subacute, loss of central visual acuitv rvithout pain, which tJ.pically occurs betwecn 12 and 30years of age. Males in affected pedigrees arc much more likcly to develop visual loss than are f-emalesIn some LHON pedigrees additional

Thesc rare conditions all shorv autosomal recessiveinheritance and prcsentearlf in life with a variablecombinationof skeletaland cardioml-opath.v,hepatocellulardysfunction rvith hepatomegall', Treatment revolves around nutritional and enccphalopath-y. maintenanceand avoidanceof fasting, but is not verl' relvarding

neurologicalproblems occur.

in SCAD.

BARTH SYNDROME

Glutaricacidurias

A l s o k n o r v n a s X l i n k e d c a r d i o s k c l e t a lm v o p a t h y , t h i s i s charactcrized by congenital dilatcd cardiomyopathy(including endocardial fibroelastosis),a generalizedml-opxthv and grorvth

G l u t a r i c a c i d u r i a s t , r - p e sI ( g l u t a r . v l - C o A d e h y d r o g e n a s e deliciency) and II (multiple ac1'l-CoAdeh.vdrogenase deficienc,'-') areincludcd aseramplesof organicaciduriasthat areintermediate oxidation;both showautosomalrecessiveinheritance in tatt,-v-acid In t1'pc I macrocephaly is present at birth and infants suffer cpisodcsof encephalopathywith spasticitl"drstonia' seizures and devclopmentaldelay Treatment is b-vdietarl- restriction of trvptophan and hydroxylysine glutarigenicamino acids- 11,-sine,

retardation.Abnormal mitochondria are found in manv tissues, deficient in cardiolipin, and skelctalmuscle sholvsincreasedlipid levels.A variable and somctimcs fluctuating incrcasein urinarr' levels of 3-methylglutaconic acid mav be useful in achieving a diagnosis,and mutations havebeen identified inthe C4.5 (TAQ gene, but the enzl'me defect leading to 3-meth-ylglutaconic aciduria is currentlv unknonn.

neonatal Common among the Old Order Amish of Penns-vlvania, screeninghas been introduced in the area

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11

BrocHEMrcAL GENErcs Ty'pe II glutaric aciduria is variable, rvith two severeforms having neonatalonset, one of these including urogenital anomalies.In both of theseseveretypes,hvpotonia,hepatomegalr,', metabolic acidosisand hl.pokctotic hvpoglycemiaoccur. The Iate-onsetform ma-vpresent in earlv childhood, rather than the neonatal period, lvith failure to thrive, metabolic acidosis, hl,poglycemiaand encephalopathlr.Treatmcnt of the severe forms is supportive only, but in the mildcr form riboflavin, carnitine, and diets lotv in protein and fat have been more successful.

P R E N L D I A G N O SOI S FI N B O R N E R R O ROSFM E B O L I S M For the majorit-v of inborn crrors of metabolism in r,vhichan abnormal or dcficient gene product can be identilied, prenatal diagnosisis possible.Biochemicalanalvsisof cultured amniocytes obtained at mid-trimester amniocentesisis possible but has largelv given way to earlicr testing using direct or cultured chorionic villi (CV), rvhich allorvs a diagnosis to be made bv l2-l4rvecks' gestation (p. 316) For man-vconditions a biochemical analvsison cultured C\r tissue is thc appropriate tcst but, increasingh,direct mutation analvsisis possible.This avoids the inherent dclav of culturing CV tissue and is of particular value for inborn errors for which the biochemical basis is not clearly identificd, or rvhcre the enzvme is not expressedin amniocvtesor CV Prenataldiagnosisof mitochondrial disordersdue to mtDNA mutations presentsparticular difficulties becauscof the problcm of heteroplasmyand the inability to predict rhc outcome for an-v result obtained, whether positive or negativefor the mutation in question. This presents challenging counseling issuesand also raisesconsiderationofother reproductive options, such as ovum donation and, perhapsin the future, nuclear transfer technologv to circumvent maternal mtDNA.

FURTHER READING BensonP F, Ftnsom A H 1985Geneticbiochemicaldisordcrs Oxford Universitv Press,Orford A goodreJirentesourtefor datatledhusitfilrthat inftrmation on the tnbornerrors o/ menholisnt ClarkcJ T R 1996A clinicalguideto inhcritedmetabolicdiseases Cambridgc Universit.vPress,Canbridge

176

A gool busictexl, lroblem hued nnd ilinrcalfu onented. Cohn R NI, Roth K S 1983Metabolicdisease: a guide to earlyrecognition WB Saunders,Philadclphia A use.fultext as it considers the mborn errorsJron their modeo.fpftsntdtion ruther Ihan startingfrom the diagnosu Garrod A E 1908Inborn errorsof metabolism Lancet li: 1 7.73-79. 1 , t 2 - 1 4 8 . 2 t 12 2 0 ReportsoJ theJirs| inhorn errorsofmetabolism N1'han\\i L, OzandP T 1998Atlas of metabolicdiseasesChapman& Hall, London A detarledtert but xer.yreutlablcmdfull ofexcellentillustrationsand,clinical tm0ges Rimoin D L, ConnorJ \{, Pycritz R E, Korf B R (eds)2001Principles and practiceof medicalgenetics,4th edn Churchill Lir.ingstone, Edinburgh Thesettionon metaholitdisorlcrsinclufusI 3 chapterstoxeringin succinctdetarl the turious grotrpsofmetabolicdisorders. ScriverC R, BeaudctA L, St1'\V S,\''alleD (eds)2000The metabolicbasis of inheriteddisease, 8th cdn McGrarv Hill, Neu.York ,r1hugemulti-author three rolume tomprehensitedetailedtett on gentticsnith an exhaustireret'irencelist md, pith this biochemicul editron. u CD-ROM

ELEMENTS Q Metabolic processesin all speciesoccur in sreps, each being controlled by a particular enzyme which is the product of a specific gene, leading to the one gene-one enzyme concept @ A block in a metabolic pathrvayresults in the accumulation of metabolic intermediatesandlor a deficiencyof the end-product of the particular metabolic pathwav concerned, a so-calledinborn error of metabolism @ fn. majoritlr of the inborn errors of metaboirsmare inherited asautosomalrecessiveor X-linked recessivetraits. A few areinherited asautosomaldominant disordersinvolving rate-limiting enzymes,cell surfacereceptorsor multimeric enzl-mes,through haploinsufficiencvor dominant negative mutatlons. @ A number of the inborn errors of metabolism can be screenedfor in the newborn period and treatedsuccessfullv b,vdietary restriction or supplementation. @ Prenatal diagnosis of many of the inborn errors of metabolism is possibleby either conventionalbiochemical methods,the useof linked DNA markersor direct mutation detection.

CHAPTER

'If it rverenot for the great variability among individuals medicine might as well be a scienceand not an art ' Sir Williarn Osler (1892)

DEFINITION

DRUG ME BOLISM The mctabolism of a drug usuall-vfollows a common sequenceof cvents (Fig. 12.1).A drug is first absorbedfrom the gut, passes into the bloodstream,and so becomesdistributed and partitioned in the various tissuesand tissue fluids. Only a small proportion of the total dose of a drug will be responsiblefor producing a

Some individuals can be especially sensitivc to the effects of a particular drug, whereas others can be quite resistant. Such individual variation can be the result of factors that are not genetic. For example, both the young and the elderl.vare ver-v sensitiveto morphine and its derivativcs,as are personsrvith liver

specificpharmacologicaleffect, most of it being broken down or excrctedunchanged

disease.Individual differencesin responseto drugs in humans are, however,often geneticallydetermined. The term pharmucogenetics wasintroduced by'Vogelin 1959for genetically the stud-vof determined r.ariationsthat are rcvcaled

Thc actual breakdorvnprocess,which usually takes place in the liver, varies with different drugs. Some are oxidized completely to carbon dioxide, which is exhaledthrough the lungs. Othcrs are

solely by the effects of drugs Pharmacogeneticsis nowadays used to describethe influence of geneson the efficacy and sideeffects of d,rugs. Pharmacogenomics describcs the interaction between drugs and the genome (i.e. multiple genes),but the two terms are often used interchangeably.Pharmacogenetics/ pharmacogenomicsis important becauseadversedrug reactions are a major cause of morbiditl. and mortality. It is also likely to be of increasing importance in the future, particularly as a result of the development of new drugs from information that has becomc availablefrom the Human Genome Proiect (Ch. 5). In addition, these developmentswill extend our understanding of the inherited differences that lead to susceptibility for the common diseasesthat are the consequenceof interaction with environmental or occupationalexposures,or what is termed

B I O C H E M I C A L M O D IIFOIN C

ercreted in modified forms either via the kidneys into the urinc, or bv the liver into the bile and thence the feces.Many drugs undergo biochemical modificationsthat increasetheir solubilitl', resulting in their being more readily excreted. One important biochemical modification of many drugs is conjugation, which involves union with the carboh,vdrate glucuronic acid. Glucuronide conjugation occurs primarily in the liver. The elimination of morphine and its derivatives, such as codeine, is dependent almost entirel-Yon this process. Isoniazid, used in the treatment of tuberculosis,and a number of other drugs, including the sulfonamides,are modified by the introduction of an acetyl group into the molecule, a process knorvn as acetylation(Fig. 12.2).

ME BOLISM K I N E T I COSFD R U G

ecogenetrcs The human genomc influences the effects of drugs in at describesthc metabolism of least three wtys. Pharmacokinetics drugs, including the uptake of drugs, their conversion to active metabolites,and detoxificationor breakdown.Pharmacodynamics refers to the interaction between drugs and their molecular targets. An example would be the binding of a drug to its receptor.The third rvav relatesto palliative drugs that do not act

The study of the metabolism and effects of a particular drug usually involvesgiving a standarddoseof the drug and then, after a suitabletime interval, determining the response,measuringthe amount of the drug circulating in the blood or determining the ratc at which it is metabolized. Such studies shorvthat there is considerablevariation in the way different individuals respond to ccrtain drugs. This variability in responsecan be continuous

directl_von the causeof a disease,but rather on its s1'mptoms. Analgesics,for example,do not influence the causeof pain but merely the perception of pain in the brain.

or discontinuous If a dose-responsetest is carried out on a large number of subiects,their results can be plotted. A number of different

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PHARMACOGENETICS

G 5

c

a 0

E z.

Fis.12.1 Stages of metabolism ofa drug

Response to drug

Fis.12.3 Various typesof response to different drugsconsistent with potygenic andmonogenic controLof drugmetaboLism A. Continuous variatron, muttifactorial controlofdrugmetaboLism B ,D i s c o n t i n u obui m s o d av La r i a t i oC n ,D i s c o n t i n u ot rui m s odat vanatron

l s o ni a z i d

Acetyl-ison iazid

F19.12.2 Acetylation oftheantituberculosis druqisoniazid

178

possibleresponsescan be seen(Fig. 12.3).In continuousvariation the results form a bell-shaped or unimodal distribution. With discontinuous variation the curve is bimodal or sometimes even trimodal. A discontinuous response suggeststhat the metabolism of the drug is under monogenic control. For example, if the normal metabolism of a drug is controlled by a dominant gene, R, and if some people are unable to metabolize the drug because they are homozygous for a recessive gene, r, there will be three classesof individual: RR, Rr and rr. If the responsesof RR and Rr are indistinguishable, a bimodal distribution will result. If RR and Rr are distinguishable,a trimodal distribution will result, each peak or mode representing a different genotype. A unimodal distribution implies that the metabolismof the drug in question is under the control of many genes, i.e. is polygenic (p. 136).

I

IONSREVEALED SOLELY

Among the best known examples of drugs that have been responsible for revealing genetic variation in response are isoniazid, succinylcholine, primaquine, coumarin anticoagulants, certain anesthetic agents, the thiopurines, phenylbutazone, debrisoquineand alcohol.

N.ACETYLTRANS FERAS EACTIVITY Isoniazid is one ofthe drugs used in the treatment oftuberculosis. It is rapidly absorbed from the gut, resuhing in an initial high blood level that is slowly reduced as the drug is inactivated and excreted.The metabolism of isoniazid allows two groups to be distinguished: rapid and slow inactivators. In the former, blood levels of the drug fall rapidly after an oral dose; in the latter, blood levels remain high for some time. Family studies have shown that slow inactivators of isoniazid are homozygous for an autosomal recessiveallele of the liver enzyme N-acetvltransferase.

PHARIVlACOGENETICS

with lorver activitv levels.N-acetvltransferaseactir-ity laries in different populations In the USA and rvestern Europe about 50o/oof the population are slorv inactivators,in contrast to the Japanese,who are predominatelvrapid inactivators. In some indir.iduals,isoniazid can cause side-effectssuch as polyneuritis, a systemic lupus erythematosus-like disorder or liver damage.Blood levels of isoniazid remain higher for longer periods in slow inactivatorsthan in rapid inactrvatorson equivalent doses.Slow inactivators have a significantly'greater risk of developing side-effectson doses that rapid inactivators require to ensure adequateblood levels for successfultreatment of tuberculosis.Conversel1,, rapid inactivatorshave an increased risk of liver damagedue to isoniazid A number of other drugs are also metabolized by N-acetyltransferase,and therefore slow inactivatorsofisoniazid are alsomore likely to exhibit side-effects. These drugs include hydralazine,which is an antihvpertensive, and sulfasalazine,which is a sulfonamidederivativeused to treat Crohn disease. Studies in other animal speciesled to the cloning of the genes responsible for N-acetvltransferase activit-v in humans This has revealedthat there are three genes,one of rvhich is not expressedand representsa pseudogene(NATP), one that does not exhibit differencesin activity between individuals (NATI), and a third (NAT2), mutations in rvhich are responsiblefor the inherited polvmorphic variation Thesc inhcrited rariations in NAT2 have been reported to modifv the risk of developing a number of cancers,including bladder,colorectal,breastand lung cancer.'Ihis is thought to be through dilferencesin acetylationof aromaticand heteroctclicaminecarcinogens.

SUCCINYLCHOLINE SENSITIVITY Curare is a plant extract used in hunting bv certain South American Indian tribes that produces profound muscular p a r a l v s i s N 4 e d i c a l l y c, u r a r e i s u s e d i n s u r g i c a l o p e r a t i o n s becauseof the muscular relaxation it produces.Succinvlcholine, also knorvn as suxamethonium, is another drug that produces muscular relaxation, though bv a different mechanism from curarc Suxamethoniumhas the advantageover curare that the relaxation of skeletal and respiratorv muscles and the consequent apnea (cessationof breathing) it induces is only short-lived. Thereforc, it is used most often in the induction phase of anesthesiafor intubation. The anesthetist,therefore,needsto maintain respiration bv artificial meansfor onlv 2-3 min before it returns spontaneously. Horvever,about one patient in every 2000 has a period of apnea that can last t h or more after the use of suxamethonium.It was found that the apnea in such instances could be corrected by transfusion of blood or plasma from a normal person. When a suxamethonium-inducedapnea occurs the anesthetisthas to maintain respiration until the effectsof the drug haveworn off. Succin-vlcholineis normally destro-vedin the body by the plasmaenzvme pseudocholinesterase. In patients who are highly plasma pseudocholinesterase to succinylcholine, the sensitive in their blood destroysthe drug at a markedly slower rate than normal, or in some ver-yrare casesis entirel-vdeficient. Familv

studieshar,eshorvnthat succinl'lcholinesensitivity is inherited as ln autosomalrecessiretrait. A relined method of studying plasma pseudocholinesterase activit-y in thc blood involves determining the percentage inhibition of the enzl'me by the local anestheticdibucaine (syz. cinchocaine).The result is termed the dibucaine number. The frcquencl, distribution of dibucaine number values in families rvith succinvlcholine-sensitiveindividuals gives a trimodal curve The three modes representthe normal homozygotes,the hctcrozvgotesand the affectedhomozygotes. Suxamethonium sensitivity is now known to be determined bv the inheritance of mutations of the CHEI gene, and genetic tcsting mal- be offered to the relatives of a patient in rvhom a geneticpredispositionhas been identified.

G L U C O S6E. P H O S P H E DEHYDROGENASERIANTS For manl, -vears,quinine was the drug of choice in the treatment of malaria Although it has been very effective in acute attacks, it is not effective in preventing relapses.ln 1926 primaquine was introduced and pror,ed to be much better than quinine in prcvcnting rclapses.However, it was not long after primaquine u-as introduced that some people were found to be sensitiveto the drug The drug could be taken for a few days with no apparent ill effects, and then suddenly'some individuals t'ould begin to pass vcry-dark, often black, urine. Jaundice der-elopedand the red cell count and hemoglobin concentration graduallv fell as a consequenceof hemolvsis of the red blood cells. Affected individuals usually recor.eredfrom such a hemol-vticepisode'but occasionallvthe destruction ofthe red cells wasextensiveenough to be fatal.The causeof such casesof primaquine sensitivity was subsequently shot'n to be a deliciency in the red cell enzyme glucose6-phosphatedehydrogenase(G6PD) Famill'studies have shou'n that G6PD dehciencyis inherited asan X-linked recessivetrait (p. I 13) G6PD deficiencf is rare in most caucasians,but affects about 10o/oof Afro-Caribbean males and is also relatively common in males of mediterranean origin. G6PD defrciencyis thought to be relatively common rn these populations as a result of conferring increasedresistance to the malarial parasite The red-cell G6PD levelsin personsof mediterraneanextraction with G6PD deficiency are very much lol er than thosein personsof Afro-Caribbean origin with G6PD delicienc-r,'. Persons with G6PD deficiency are sensitive not only to p r i m a q u i n e b u t a l s o t o m a n y o t h e r c o m p o u n d s ,i n c l u d i n g phenacetin, nitrofurantoin and certain sulfonamides'These drugs should be used with caution in males of Afro-Caribbean and mediterraneanorigin if their G6PD statusis unknown, and in a person known to be G6PD-deficient such drugs are absolutely contraindicated.Drug-induced hemol,vsisis uncommon and, in fact. the main risk of G6PD deliciency is favism' in which a hemol-vticcrisis occurs after eating favabeans.This is thought to be the first recognized pharmacogeneticdisorder, having been describedby Pl,thagorasaround 500nc.

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PHARMACOGENETICS

C O U M AR IN ME T A B OL IS M Coumarin anticoagulantdrugs, such as warfarin, are used in the treatment of a number of different disordersto pre\rentthe blood from clotting, e g. after a deep venous thrombosis. \\'arfarin is metabolized b1' the cy.tochromeP,150enz].me encoded by the CYP2C? gene, and t\ro variants (CYPZC?*Z and CYP2C9*3) result in decreasedmetabolism.Consequentlythesepatientsrcquire a lowerwarfarin doseto maintain their targetinternationalnormalized ratio (INR) range and may be at increasedrisk of bleeding.

D E B R I S O O UM I NEE B O L I S M Debrisoquine is a drug that rvasused frequently in the past for the treatment of hvpertension There is a bimodal distribution in the rcsponseto the drug in the generalpopulation.Approximately 5-10o/oof persons of European origin are poor metabolizers, being homozygotesfor an autosomalrecessivegenewith reduced hJ'drox1'lationactivitlr Nlolecular studies have revealed that the gene involved in debrisoquine metabolism is one of the P450 famil.v of geneson chromosome22, knorvn as CYP2D6. The mutations responsible for the poor metabolizer phenotvpe are heterogeneous;l8 different variantshavebeen describedto date. CYP2D6 r.ariation is important becausethis enzyme is involved in the metabolism of more than 20o/oof prescribed drugs, including the B-blockerspropranolol and metoprolol, the antidepressantsamitrvptiline and imipraminc, the antipsvchotics thioridazine and haloperidol, the painkiller codeine, and anticancerdrug tamoxifen.

M A L I G N ANH T Y P E R TE HR MIA

180

(RYRI) gene.Sevenother candidategeneshave been identilied and variants in these genes mav influence susceptibility within individual families.This observationmay explain the discordant results of the in-dtro contracture test and genoty'pein members of some families that segregateRYRl mutations

THIOPU RINE METHYLTRANSFERASE A group of potentially'toxic substancesknown asthe thiopurines, which include 6-mercaptopurine,6-thioguanineand azathioprine, are used extensivelyin the treatment of leukemia,to suppressthe immune responsein patientswith autoimmune disorderssuch as svstemic lupus ervthematosusand to prevent rejection of organ transplants Thev are \.ery effective drugs clinicalll,' but have seriousside-effects,such as leukopeniaand severeliver damage. Azathiopurine is reported to causetoxicity in l0-1 5oloof patients and it mav be possible to predict those patients susceptibleto side-effectsb-vanaly'zinggenetic variation lithin the thiopurine methvltransferase(TPMT) gene.This gene encodesan enzlrme responsible for methvlation of thiopurines, and approximatelltwo-thirds of patients rvho experiencetoxicit]. have one or more variant alleles.

D I H Y D R O P Y R I M ID E I NHEY D R O G E N A S E Dihl'dropy'rimidine dehvdrogenase(DPYD) is the initial and rate-limiting enz]'me in the catabolismof the chemotherapeutic drug 5-fluorouracil (5FU). Deficiency of DPYD is recognized as an important pharmacogeneticfactor in the etiologl of severe 5FU-associatedtoxicit-v Nlleasurementof DPYD activitv in peripheral blood mononuclear cells or genetic testing for the most common DPYD gene mutation (a splice-sitemutation, IVSI4+1G>A, which resultsin the deletionof exon 14)may be rvarrantedin cancerpatientsbefore the administration of 5FU.

N{alignant hypcrthermia (NIH) is a rare complication of anesthesia.Susceptiblc individuals develop muscle rigidity as well as an increasedtemperature (hyperthermia), often as high as 42.3'C (108'F) during anesthesiaThis usually occurs when halothane is used as the anestheticagent, particularlv rvhen succinvlcholineis used as the muscle relaxant for intubation. If it is not recognizedrapidly'and treated with vigorous cooling, the affectedindividual rvill die. \,IH susceptibility-is inherited as an autosomal dominant trait affecting approximatel-vI in 10000 persons.Susceptibleindividuals occasionallvhavea raisedserum creatinekinaselevcl but this cannot be used as a reliable screening test for at-risk familv members. The most reliable prediction of an individual's susceptibilitl. status requires a muscle biopsl' rvith in-xitro muscle contracture testing in responseto exposureto halothaneand caffeine. A person knou,n to be or suspectedof being susceptible to MH can have a general anestheticpror.ided that recognized precipitating anestheticagentsare avoided Should hvperthermia

trvin studies,rvhich have shown high concordancerates (p. 220), and family- studies, which have shown a high prevalenceof alcohol-relatedproblems among relatives of alcoholics.Clearl-r,; however,behavior patterns r.ithin families could artificially inflate what would appearto be geneticfactors.Similarl-r,; apparentracial

develop during surgerr, it is treated b.vcooling and intravenous administration of procaineor procainamide,but most effectiveh: with dantrolene. Nlalignant hyperthermia is genetically.hererogeneous,but the most common causeis a mutation in the rvanodine receptor

or ethnic differencesin the incidence of alcoholism, such as the high incidence among certain American Indians and Eskimos, could rvell be affectedby socialfactors. Perhaps the most convincing evidence for the possible role of genetic factors in alcoholism comes from the study of the

A L C O H OM LE

BOLISM

Under the heading of pharmacogeneticswe can also include alcoholismand alcoholiccirrhosis,which in terms of their frequency and social implications dwarf all others, although some persons would debatervhetheralcohol should really be considereda drug. Alcoholism is clearly related to the amount consumed as well as to dietarv and various socialand economic factors Nevertheless, evidenceis gradually emerging that clearlv indicatesthat genetic factors can also be involved. Some of this evidenceis based on

FHARMACOGENETICS

enzymesinvolved in alcohol metabolism.Alcohol is metabolized in the liver by alcoholdehydrogenase (ADH) to acetaldehl,de, and then further degradedb1'acetaldehvdedehvdrogenase(ALDH). Human ADH consistsof dimers of various combinations of subunits of three different polypeptide units coded for by three loci ADH I codesfor the cr subunit. ADH2 for the B subunit and ADH3 for the y subunit. ADHI is expresscdprimaril-v in earl-v fetal life, tvhereasADH2 is exprcssed in adult life. Personsof Far East Asian origin toleratealcohol lessrvell than persons of caucasianorigin, and often exhibit an acute flushing reaction to it. This sensitir.itvis due to differencesin the rate of metabolism of acetaldehyde.There are two major acetaldehyde variantsor isozymes:ALDHl, which is presentin deh-vdrogenase the c1'tosol,and ALDH2, which is present in the mitochondria. The acuteflushing reactionto alcoholin Far EastAsianshas been shown, in fact, to be due to absentALDH2 activit-v.It has been suggestedthat this unpleasant reaction could account for the reported lower incidence of alcoholismand alcohol-relatedliver diseasein that population.

PHARMACOGENETICS

triggering insulin secretion High-dose sulfonylurea therap-v results in improved gl,vcemiccontrol rvith fewer hy-poglycemic cpisodesand, for some patients,a Hb Alc level (this is a measure of gl1:cemiccontrol) r'ithin the normal range.

PHARMACOGENOMICS lrr is defrnedas the study of the interaction of an Phurmucogenon individual's genetic make-up and responseto a drug. The key distinction betlveen pharmacogeneticsand pharmacogenomics is that the former describesthe study of variabilit.vin drug responsesattributed to individual genesand the latter describes the stud,-vof the entire genome related to drug response.The expectationis that inherited variation at the DNA level results in functional r.ariationin the geneproducts that play an essential role in determining the variability in responses,both therapeutic and adverse,to a drug. If pol-vmorphicDNA sequencevariation occurs in the coding portion or regulatory-regions of genes,it is likell'to result in variation in the geneproduct through alteration of function, actir,it]' or level of expression.Automated analysis of genome-rvide single nucleotide polymorphisms (SNPs) (p. 67) allorvsthc possibility ofidentiffing genesinvolved in drug mctabolism, transport and receptorsthat are likelv to play a role

Increasedunderstandingof the influenceof geneson the eflicacv and side-effectsof drugs has led to the promise of personalized or indiaidualizedmedicine,where the treatment for a particular diseaseis dependent upon the individual's genotype.

side-effectsand toxicity in determining the variability in efficacy-, of a drug. The availability of rvhole-genomeSNP maps will enablean SNP prolile to bc created for patients who experienceadverseeventsor l'ho rcspond clinically to the drug (efficac-v).An individual's wholegenomeSNP t,r-pehasbeendescribedasan'SNP print' However,

M U R I T Y . O N SDEITA B E T EOSF T H E YOUNG

this rrises issuespertaining to the disclosure of information of uncertain significancethat is later shown to be associatedrvith an adverseoutcome unrelated to the reasonfor the original test. An

Maturitl,-onset diabetesof the voung (N{ODY) is a monogenic form of diabetescharacterizedb-v,voungage of onset (often beforc the ageof 25 years),dominant inheritanceand B-cell dl.sfunction (p. 222). Patients with mutations in the HNFIA or HNF1A genesare sensitiveto sulfonvlureasand ma1'experienceepisodes

erample is apolipoprotein E (ApoE) genotyping, where ApoE e4 was lirst reported to be associatedwith variation in cholesterol ler,elsbut later with ageof onset of Alzheimer disease

of hypogly-cemiaon standard doses. However, this sensitivitr i s a d v a n t a g e o u sa t l o u e r d o s e s ,a n d s u l f o n v l u r e a sa r e t h e recommendedoral treatment in this geneticsubgroup

NEONATAL DIABETES The most frequent causeof permanent neonatal diabetesis an activating mutation in the KCNJII or ABCC8 genes, which encode the Kir6.2 and SUR1 subunits of the ATP-sensitive potassium(K-ATP) channcl in the pancreaticB cell (p.222).The effect of such mutations is to prevent K-ATP channel closure b1' reducing the responseto ATP As channel closure is the trigger for insulin secretion,these mutations result in diabetes. Defining the genetic etiologl. for this rare subtype of diabetes has led to improved treatment, as the maiority of patientscan be treated successfullywith sulfonylurea tablets instead of insulin These drugs bind to the sulfonl,lurea receptor subunits of the K-ATP channel to causeclosure independentl-vof ATP, thereb-v

EVENTS ADVERSE The objectir,eof adverse-eventpharmacogeneticsis to identify a genctic prolile that characterizespatients rvho are more likell' to suffer the adverse event. An example is abacalir, a reverse transcriptaseinhibitor usedto treat human immunodeficiency virus (HIV) infection Approximatell- 5oloof patients show potentially fatal hvpcrsensitivitl.to abacavirand this limits its use.A strong associationwith a human leukocl,te antigen (HLA) haplotype dclined as 8*5701, DR7 and DQJ is known, but the actual gene responsiblefor this effect has not.vet been identified. Thc anti-epileptic drug felbamateis a secondexampleof a drug rvhose use has been limited becauseof adverse reactions that probabl-rresultedfrom interindividual variationin its metabolism. Felbamate is metabolized rapidly in the liver to highl-v toric mctabolites that arc usually-rapidly detoxified by coniugation with glutathione Both overproduction of the toxic metabolites and inadequateconjugation might causeadversereactionsin geneticall]'susceptibleindividuals

181

12

PHARMACOGENETICS

EFFICACY There is no doubt that the cost-effectiveness of drugs is improved if the-vare prescribed onl). to those patients likell' to respond to them. The drug herceptin is an antibodl.that targets overexpressionol HER2/neu protein observedin approximatell' one-third of patients with breast cancer Consequently parienrs are prescribed herceptin only if their tumor has been shorvnto overexpressHER2/neu The epidermal growth factor receptor (EGFR) is a t1'rosine kinase receptor involved in the proliferation and differentiation of normal cells.The receptoris activatedb-vbinding of the ligands epidermal gror,vthfactor (EGF), transforming gronth factor-G (TGFcr), or amphiregulin.This leadsto a chain of eventsresulting in proliferation. EGFRs are normally found on the cells of the skin, cornea,kidneli ovaries,liver, and cardiacconduction s)-srcm. EGFR is often overexpressedon malignant cells, including 40-80o/oof patients with non-smallcell lung razrers (NSCLCs) The consequenceof overexpressionof EGFR in tumors is an uncontrolled signal transduction through the receptor that leads to increased proliferation, tumor growth and metastasis.This understandinghas led to the hypothesisthat blocking EGFR can stop the growth of lung cancers.Not surprisingll,, anti-EGFR

T a b t e1 2 . , | E c o g e n e t , cgse:n e L vr ca ra L i o r- s u s c e pbt ' . i t y t o e n vT o n m e n t a L a g e n t s Environmental agent

Geneticsusceptibility

Disease

UV tight

Faircomptexion

Skincancer

Drugs(seetext) Foods Fats Favabeans Gtuten Salt Mitk Atcohol Oxatates Fortified flour

Hypercholesterotemia G6PDdeficiency Glutensensitivity Na-Koumpdelecrive Lactase deficiency AtypicatADH Hyperoxaturia Hemochromatosis

Atherosclerosis Favism Cetiac disease Hypertension Lactose rntolerance Alcoholism Renalstones lronoverload

lnhalants Dust Attergens Infections

cx1-Antitrypsin deficiency Emphysema Atopy Asthma Defective immunrty ?Diabetes metlitus ?Spondyiitis

treatments such as gefitinib and erlotinib are more effective for treatinq tumors with EGPR murations.

ECOGENETICS An extension of pharmacogeneticsis the stud1. of geneticalll. determined differencesin susceptibilityto the action of phvsical, chemicaland infectiousagentsin the environment.This has been referred to as ecogenetirr, a term first coined by Brewer in l97l Such differences in susceptibility can be either unifactorial or multifactorial in causation(Table 12.1).

Paraoxonaseis an enzyme that catalvzesthe breakdown of organophosphates,which are rvidely used as insecticidesin agriculture. Individuals can have different enzlme activitl levels that result from a trvo-allele poll'morphic system. Those uho are homozygous for the low-activity allele are likel-v to be particularly sensitive to accidental or occupational exposure to organophosphates Reports of individuals experiencing either acute or chronic neurological or psychiatric symptoms through exposure to organophosphatescould be the result of these inheriteddifferencesin paraoxonase acti\it_\.

D I SE A S E SU S C E P T IB IL IT Y

182

An extensionof ecogeneticsthat will be particularll- important is the identificationof individuals at high risk of developingdiseases after environmental exposures,for instance particular cancers after exposureto mutagensor carcinogens(p. 26)

There are reports of an increased risk of bladder cancer in personswho are slow acetylatorsand who havehad occupational exposureto aromatic amines,lrhich are used as industrial dyes. There is alsothe possibility ofan increasedrisk ofbladder cancer for slow acetylatorsin the general population where no specilic hazardousexposure has been recognized.Conversely,there are recent reports suggestingthe possibility of an increasedrisk of colorectalcancerin rapid acetvlators R e c e n t s t u d i e s h a v e s u g g e s t e dt h a t p o o r d e b r i s o q u i n e metabolizer status is less common than would be expected in persons r,vithcancer of the lung This is in contrast to another polymorphism for the enzvme glutathione S-transferase (GSTl41\, which shows an increased incidence of the null phenot-ype(i.e. no activit.y)in persons with adenocarcinomaof the lung rvhencomparedto the generalpopulation. This enzyme is involved in the conjugation of glutathione with electrophilic compounds,including carcinogenssuch as benzopyrene,and could have a protective role againstthe developmentof cancer. This susceptibility-to diseaseis not limited only to cancer. Manv of the common diseasesin humans could be due to genetically determined differencesin responseto environmental agentsor susceptibilities.There are reports of a possible increased risk of developing Parkinson diseasebecauseof differences in the detoxilication of potential neurotoxins in associationr,vitha poor metabolizerphenotl'pe in the hepaticcytochromeP+50 CyP2D6 gene. The abilit-vto screen large numbers of persons for SNPs (p. 67) shouldallow identificationof genesinvolvedin determining the inherited contribution for manv of the common diseases. As well as identifying individuals at high risk of developing a common disease,this rvill allow a better understanding of the

diseasepathways involved, holding the promise of directl.v linking possibletherapeutic interventions for those individuals. The major international/multinational pharmaceutical and biotechnologl,'companies,not surprisingly, are investing heavil-v in thesedevelopments.Although there is the prospectof reducing the likelihood of individuals developing severalof the common diseases,many social and ethical problems are raised when the knowledge of genetic variation and susceptibility is translated into public policy with vestedcommercialinterests(p. 359). Genetic profiling is a step towardspersonalizedmedicine This information can be used to selectthe appropriatetreatment at the correct dosageand to avoid adversedrug reactions.

FURTHER READING BeutlerE 1991Glucose6-phosphatedehydrogcnase deficiencl'.N Engl J l.{ed 324: 169-171 polymorphism Rerien of an imporlanl ethnit pharmacogenetic goes GoldsteinD B, Tate S K, Sisodi.va S M 2003Pharmacogenetics genomic Naturc GenetRev 1:93i-947 Recentretiep of pharmacogenetits / genomics Nebert D \\t 1999Pharmacogenetics and pharmacogenomics: rvhyis this relevantto the clinicalgeneticist? Clin Genet 56:217 258 A goodxtmmarl, of the tno areas Neumann D A, Kimmel C A 1998Human variabilitvin responsein chemicalexposures: measures) modelling,and risk assessment CRC Press,London A detailerldiscussion oJ the inheritedhumanaariability to erl)lsureI0 lhe toxiL ellbctsoJenuronmentalchemicals PearsonE R, FlechtnerI, NjolstadP R et al 2006Switchingfrom insulin to oral sulfonylureas in patientswith diabetesdue to Ki16.2 mutations NeonatalDiabetesInternationalCollaborativeGroup N Engl J Med 3 5 5 : 1 6 71 7 7 Pharmatogenetittrealment0J'm0n0genic diabetes RosesA D 2001Pharmacogenetics Hum Mol Genet 1,0:2261-2261 A retentreriep oJpharmarogenetics andpharmacogenomics Vogel!, BuselmaierW, ReichertW, KellermanG, Berg P (eds)1978 Human geneticvariationin responseto medicaland environmental agents:pharmacogenetics and ecogeneticsSpringer,Berlin One of the early defnitiae outlinesof thefeld of pharmamgenetits \\rendell W 1997Pharmacogenetics(Oxford Monographs on Medical Genetics,32) Oxford University Press,Oxford A detilled, nmprehensixetext 0n philrmarcgenettcs.

ELEMENTS Q Phur-u.ogenetics is defined asthe study of genetically determined variations revealed solely by the effects of drugs. Hereditary disordersin which symptoms can occur spontaneouslJror can be exacerbatedor precipitated by drugs are often also included. @ fn. metabolism of many drugs involvesbiochemical modification, often by conjugation with another molecule, rvhich usually takes place in the liver. This biochemical transformation facilitatesexcretion of the drug. @ fft. wavs in which many drugs are metabolizedvary from person to person and can be geneticallydetermined' In someinstances,the biochemicalbasisis understood.For example,personsdiffer in the rate at which they inactivate the antituberculosis drug isoniazid by acetylation in the liver, being either rapid or slow inactivators.Slow inactivators havean increasedrisk of toxic side-effectsassociatedwith isoniazid therapv. Other examples include sensitivity to the muscle relaxant succinylcholinebecauseof abnormal activit$ and the or reduced plasma pseudocholinesterase developmentof a severehemolytic anemiawhen given the antimalarialdrug primaquine (or a number of other drugs) due to deficiency of the enzyme glucose 6-phosphate dehydrogenasein red blood cells. @ Itr ro-. instances,genetic variation can be revealed exclusively by exposure to drugs. One such example is malignant hyperthermia. This rare disorder is associated rvith the use of certain anesthetic agents and muscle relaxantsin generalanesthesia. @ Knowledge regarding the genetic etiology of disease can lead to tailored treatments. Examples include sulfonylurea therapy for certain monogenic subtypes of diabetes,and herceptin for breast cancers showing HER2 overexpression.Testing for 85701 status before prescribing abacaviris now routine for patients with HIV infection, in order to reduce the risk of potentially fatal hvpersensitivity. @ Ecogeneticsis the term used for the study of genetically determined differencesbetweenpersonsin their susceptibility to the action ofphysical, chemicaland infectious agentsin the environment.

183

CHAPTER

'Medicinal

H U M O R AILN N E I M M U N I T Y

discovery, It movesin mighty leaps, It leapt straight past the common cold And gaveit us for keeps.'

A number of soluble factors are involved in innate immunity; they help to minimize tissue injury by limiting the spread of Pam Ayers

IMMUNITY Microorganisms, insects and other infectious agents are far more numerous than members of the human race, and without effective defensemechanismsagainst their acrivity humankind would rapidly succumb.The immune s-vstemin all its forms is our defensemechanism,and in order to understandthe inherited disordersof immunity, we must hrst understandthe fundamentals of the geneticbasisof immunity. Immune defense mechanisms can be divided into two marn types: innate immuniry, which includes a number of nonspecilic systemsthat do not require or involve prior contacr with the infectious agent, and spectf.cacquiretlor adaptite immunity, which involves a tailor-made immune response that occurs after exposure to an infectious agent. Both tvpes can involve either humoralimmunity,which combatsextracellular infections, or cell-mediatedimmuni4t, which fights intracellular infections.

infectious microorganisms.These are often called the acute-l)hase proteinsand include C-reactiveprotein, mannose-bindingprotein and serum am1,-loid P component.The first two act by facilitating the attachment of one of the components of complement, C3b, to the surface of the microorganism, which becomesopsonized (made ready) for adherence to phagocvtes,whereas the latter binds lysosomalenzymesto connectivetissues.In addition, cells when infected by a virus, synthesizeand secreteinterferon,whtch interferes with viral replication by reducing messengerRNA (mRNA) stability and interfering with translation.

Complement Comltlementis a complex series of 20 or so interacting plasma proteins that can be activatedby the cell membranesof invading microorganisms,in what is termed the alternutit:epathna.y.The various componentsof complement interact in a specificcascade sequence,resulting in a localized acute inflammatory response through the action of mediatorsreleasedfrom mast cellsand tissue macrophages. These result in increasedvascularpermeability and the attraction of phagocvtesin the processknown as chemotaxis. In addition, the later componentsof the complementcascadegenerate a membrane attack complex, which induces defects in the cell membrane,resulting in the lysis of microorganisms(Fig. 13.1). Complement can alsobe acti'r.ated through the clussic pathwu.y, by the binding ofantibody with antigen (seebelorv,p. 185).

INNATE IMMUNITY The first simple type of defenseagainstinfection is a mechanical barrier. The skin functions most of the time as an impermeable barrier but, in addition, the acidic pH of sweat is inhibitorv to bacterial growth. The membranes lining the respiratorv and gastrointestinal tracts are protected bv mucus In the case of the respiratory tract, further protection is provided by ciliary movement) whereas other bodily fluids contain a variety of

184

bactericidal agents,such as lysozymes in tears. If an organism succeedsin invading the body, phagocytosis and bactericidal aqentscome into effect

Phagocytosis Microorganisms are engulfed and digested by tu.o maior tvpes of cell: polvmorphonuclear neutrophils or macrophages. P o l y m o r p h o n u c l e a rn e u t r o p h i l s a r e f o u n d m a i n l y i n t h e bloodstream, rvhereasmacrophagesoccur primarily in tissues around the basementmembrane of blood vesselsin connective tissue,lung, liver and the lining of the sinusoidsof the spleenand the medullary sinusesof the lymph nodes. Surface anrigenson microorganismsresult in their being engulfedand fusing with the

IMMUNOGENETICS

'Alternative' pathway R e c o g n i t i oonf an t i gen - an t ib o d y c omp le x e s

R e c on gi t i o n of 0actefla

li

D ':'r

. {C l q r s C.. _.-.i, C4

C3J B

- a',tt"'

C3 + C3a+ C3b-.::ir:-r:1"M a s ct e l d l egranulation Chemotaxis 0psonization

13

the activation of phagocl,'tesand the initiation of the classicpathwalt of complement, resulting in the generationof the membrane attack complex (see Fig. 13.1). Exposure to a specific antigen results in the clonal proliferation of a small l.vmphocytederived from 'B' ll,mphocytes), resulting in mature the bone marro\\ (hence antibod]'-producingcells or plasmurclls Lvmphocl tes capableof producing antibodiesexpresson their surfacecopiesof the immunoglobulin (Ig) for which they code,which actsasa surfacereceptor for antigen. Binding ofthe antigen results, proteins,in signal in conjunction with other membrane-associated transduction leading to the clonal expansionand production of antibodl,.In the first instancethis results in the primarl response rvith production of IgM and subsequendyIgG. Re-exposureto the sameantigen results in enhancedantibodl'levels in a shorter resplnse,reflecting what is period of time, known as the secondar.y knou'n as antigen-specific immunologicalmemory.

Lysis

Fis.13.1 pathwaysof complementactivation CLass c and atternative ( A d a p t e fdr o m P a u I WE ( e d) 1 9 9 3F u n d a m e n t ai m I munotogy. R a v e nP r e s sN , ewYork)

granules of the phagocvte,subjecting them to the action of the bactericidal agents of thc intracellular granules, which contain h1'drogenpcroride, hydroxyl radicals and nitrous oxide, and leading to their destruction

Extracellularkitting Viralll-infected cells can be killed b-vlarge granular lvmphocvtes, known as natural killer celk These have carbohydrate-binding receptors on their cell surfacethat recognizehigh molecular weight glvcoproteinsexpressedon the surface of the infectcd cell as a result of the virus taking over the cellular replicative functions. Attachment of the natural killer cells to the infected cells results in the releaseof a number of agents;this, in turn, resultsin damage to the membrane of the infected cell, leadine to cell death.

SPECIFIC ACOUIRED IMMUNITY

lmmunoglobulins The immunoglobulins, or antibodies, are one of the major classcsofserum protein. Their function, both in the recognition of antigenicvariability and in effectoractivities,rvasinitially revealed b,vprotein, and more recently DNA, studiesof their structure

Imm unoglobuIin structure Papaine,a proteolytic enzyme, splits the immunoglobulin molecule into three fragments. Two of the fragments are similar, each containing an antibody site capableof combining with a specific antigcn and therefore referred to as the antigen-bind,ing fragment or Fuh The third fragment can be crvstalizedand was therefore called Fc The Fc fragment determinesthe secondarybiological functions of antibod-vmolecules, binding complement and Fc receptors on a number of different cell types involved in the lmmune response. The immunoglobulin moleculeis made up of four polypeptide chains:two'light'(L) and trvo'heavy'(H) chainsof approximately 220 and 440 amino acids in length, respectively.They are held together in a Y shape b-v disulfide bonds and non-covalent interactions.Each Fab fragment is composedof L chains linked to the amino-terminal portion of the H chains,whereaseach Fc fragment is composedonly of the carboxv-terminalportion of the FI chains(trig. 13.2)

Man.v infcctive microorganisms have, through mutation and selectivepressures,developedstrategiesto overcomeor evadethe mechanismsassociatedrvith innate immunity. There is a need, therefore, to be able to generate specilic acquired or adaptive immunitl.. This can, like innate immunitli be separatedinto both humoral and cell-mediatedorocesses.

COUIRE IM DM U N I T Y H U M O R ASLP E C I FAI C The main mediators of humoral specificacquired immunity are immunoglobulins or antibodies.Antibodies are able to recognize and bind to antigens of infecting microorganisms, leading to

Imm unog lobulin isotypes,subclosses ond idiotypes Thcre arefive different types ofheavy chain, designatedrespectively xs Y, l-t,cx,6 and e, one eachrespectivelyfor the five maior antibody IgG, IgM, IgA, IgD and classcs,or what are known as iso1/Pes'. L chains are one of two revealed that the IgE. Further anal-vsishas (1"), two types of L chain (r) the or lambda tvpes, either kappa but with only one type occurring in all five classesof antibody, Thus the antibody. of light chain occurring in each individual of The characteristics r2y2. or molecular formula for IgG is 1"2y2 1 1' in Thble 3. in brief the r.ariousclassesof antibodv are outlined

185

13

IMMUNOGENETICS

I c classes,havealsobeenidentihed.These arethe Gm sJrstemassociated n n - i l n | , - --*'*,|1ilil../l.[[[[,}|,,,.','.,....ffi...,ffi,-,,,.,,-. ntD '-.-,,..,.'1r.r.r-r-Ll-n,-r rvith the heavl'chainof IgG, theln svstemassociated with the IgA S o m a t i cr e c o m b i n a t i o n

I

I

I

Antigen-binding srtes

heavv chain, the Km rnd Int: svstemsassociatedrvith the r light chain, the O: slstem for the )" light chain and the Em allotl'pe for the IgE hear,vchain. The Gm and Km systemsare independent ofcach other and are polvmorphic (p 12lt),the frequenciesofthe different allelesvar]'ing in diffcrcnt ethnic groups.

Generation of antibodydiversity It could seemparadoxicalfor a single protein molecule to exhibit sufficicnt structural heterogeneitvto har.especihcitl'-for a large number of different antigens Different combinations of heavy Fab

and light chainscould, to some extent, account for this dir.ersitrr It lvould, horvcver,require thousandsofstructural genesfor each chain t.vpeto provide sufficient variability for the large number of antibodiesproduced in responseto the equalll' large number of antig;cnsto rvhich indir.iduals can be exposed. Our initial undcrstanding of horv this could occur came from personswith a malignancv of antibody.-producingcells, or tvhat is known as mu/tiplem-yelonta. C a r b o x yt e r m i n a l

Multiplemyelomo

Fie.13.2 M o d eot fa n t i b o dmy o l e c u tset r u c t u r e

Pcrsons with multiple mJtelomltmake a single or monoclonal antibod)'speciesin large abundance,uhich is excretcd in large quantitiesin their urine.This protein,knos.nasBenteJonesprotein,

In addition, therc are four IgG subclasses, IgGl, IgG2, IgG3 and IgG,l, and tu,o IgA subclasses, IgAl and Ig'A2, uhich difl-er in thcir amino-acid sequenceand interchain disulfide bonds Individual antibod-vmoleculcs that recognizc specific antigens are knou,n as idiotl-pes.

consistsof antibodvlight chains.Comparisonsof this protein from different patients vvith mrcloma revealedthe amino-terminal ends of the molecule to be quitc r.ariablein their amino-acid sequence r,hereasthe carboxl'-tcrminal ends were relativelv constant These are called the turiable, or V, a,nd,constLtnt, or C, regions, respectivelriFurthcr detailedanalvsisofthe amino-acidsequence

Imm unogIobulin ollotypes

of thc V regions of diflerent mvcloma proteins showed four regions that varied littlc from one antibody to another, known as .frunteworkregions(FR I 4), and three markedlv variable

Thc fir.eclasscsof immunoglobulinoccur in all normal individuals, but allclic variants,or u'hat arc known as antibod-rullot-1,pes of thcse

regions interspersed betl'een thcse, knorvn as h.ypertariuble (HY I III) (seeFig. 13 2) regions

T a b t e1 3 . 1 C l a s s eosf h u m a ni m m u n o q t o b u l r n

186

Class

Mot.wt (Da)

Serumconcentration Antibodyactivity (ms/mt)

,l n v vl l

1(n nnn

Q 1a

Brndsto m croorganisms andneutratizes bacteriaItoxins +

lgM

900000

O5-2

Produced rnearlyimmuneresponse, especiatty in bacteremia

+

gA

160000

1+-+

Guards mucosaI surfaces

+

ISD

185000

0-04

Ontymphocyte cellsurface, invotved in control of dclivaLion ard slppress,on

ISE

200000

Trace

l^

i+l^ r n ^f -i -d- -rid+ S u c d- ^ r! r o a- l tl ^t-e^ i.- q l c -T^ ^e- a clof s

Complement fixation

Placental transfer

IMMUNOGENETICS

DNAstudiesof ontibodydiversity

Antibodygene reorrongement

As long ago as 1965, Dreyer and Bennett proposed that an antibodl'could be encoded by separate'genes' in germline cells that undergo rearrangement,or, as they termed it, 'scrambling', in l1-mphocytedevelopment.Comparisonof the restriction maps

Thc genes for the r and l, light chains and the heavy chains in humans have been assignedto chromosomes 2, 22 and 14, respectivclv C)nl1'one of each of the relevant t.vpes of DNA segmcntis erprcssedin any single antibody molecule.The DNA coding scgmentsfor the r.ariousportions of the antibod-vchains

of the DNA segments coding for the C and V regions of the immunoglobulin ), light chains in embryonic and antibodyproducing cells revealedthat the-r'rvere far apart in the former but close togcther in the latter. More detailed analJ'sisrevealed that the DNA segmentscoding for the V and C rcgions of the light chain are separatedby some 1500basepairs (bp) in antibodyproducing cells. The intervening DNA segment rvas found to code for a joining, or J, region immediately adjaccnt to the V rcgion of the light chain. The r light chain was shown to have the samestructure. Cloning and DNA sequencingof heav-v-chain genesin germline cells revealedthat they have a fourth region, called diaersit.y,or D, between the V and J regions. There are estimated to be some 60 different DNA segments coding for the V region of the heavl- chain, approximatel-v40 DNA segmentscoding for the V region of the r light chain and 30 DNA segments coding for the )" light chain V region. Six functional DNA segments code for the J region of the heavv chain, five for the J region of the r light chain and four for the J region of the l" light chain. A single DNA segmentcodesfor the C region of the r light chain, sevenDNA segmentscode for the C region of thc l, light chain and I 1 functional DNA segmentscode for the C region of the different classesof heavy chain. There are also 27 functional DNA segmentscoding for the D region of the heavv chain (trig. 13.3). Estimation of the number of DNA segmcntscoding for thcsc various portions of the antibody molecule is confounded b.vthe presenceof a large number of unexpressedDNA sequencesor pseudogenes(p. l6).Although the coding DNA segmentsfor

on thesechromosomesare separatcdby DNA that is non-coding. Somaticrecombinationaleventsinvolved in antibodl''production involvc short conserved rccombination signal sequencesthat flank errchgcrmline DNA segment(Fig. 13.4).Further diversitl' occurs bv variable mRNA splicing at the V-J iunction in RNA processingand b1' somatic mutation of the antibody genes. These rncchanismscan easilvaccount for the antibody diversit--v sccn in naturc. Although this probably involves some form of clonirl sclcction, it is still not cntirelv clear horv particular DNA scgmcntsare selectedto produce an antibody to a specificantigen. 'Gcnc shuffling' of this form is alsoknown to accountfor the marked variabilitl' scen in thc surface antigens of the trvpanosome p:rr:rsitc and thc differentmating l\-pesin veast

Classswitchingof antibodies There is a normal srvitch of antibodl- class produced b1' B cells on continued or further exposureto antigen, from IgM' rvhich is thc initial classof antibody produced in responseto exposure to an irntigen, to IgA or IgG. This process, knorvn as class stnitthing,involvesrctention of the specilicity of the antibody to the sxme antigcn. Analvsis of classswitching in a population of cells dcrived from a single B cell has shown that both classcsof antibod]'har,ethc sameantigen-binding sites,having the sameV

SCp

J

the various regions of the antibody molecule can be referred to as genes,use ofthis term in regard to antibodieshas deliberatcll, been avoided becausethey could be consideredan exception t o t h e g e n e r a lr u l e o f ' o n e g e n e o n e e n z y m e ( o r p r o t e i n ) '

SCcr

IT Somatic r e c o m bni a t io n

(p.ls8).

I

I Variable DiversityJunctionalConstant regron regron regron regron :, K Chromosome 2.,-

V

DJSCtT

SCct

4051

I

Chromosome 22

@-

Class ing switch

I

3047

I V 6

DJSCcT

11

Fis.13.3

Fis.13.4

s N As e g m e n tcso d i nfgo rt h er , E s t i m a t endu m b eor ft h ev a r i o uD

a tn d c l a s s l m m u n o g l o b u [h i ne a v y - c h a igne n er e a r r a n g e m e n swrtchrng

1 ^^! A d|u

..--;^, vd tuu>

L^^.,., |trdvy

^L^;^^ L tdil l>

187

13

IMMUNOGENETICS

region, differing onlv in their C region Class s.n'itchingoccurs b1-a somatic recombination event that involr,csDNA segments designatedS (for slr,itching)that lead to looping our and delction of the intervening DNA. The result is to eliminate the DNA segment coding for the C region of thc hear,vchain of the IgNf molecule and to bring the gene scgment encoding the C region ofthe net classofheavv chain adjacentto the scgmentencoding theV region (seeFig. 13 4)

T h ei m m u n o g l o b u l g i ne n es u p e r f a m i t y Studies of the structure of other molecules involved in the immune responsehave shor,vna number to have structural and DNA sequencehomology to the immunoglobulins.This involves a 110-amino-acidsequencecharacterizedbv a ccntrallv placed disulhde bridge that stabilizesa serics of antiparallel B strands into rvhatis calledan antibodv fold. This group of moleculeswith similar structure has bcen called thc immunoglobulinsuperfamily (p l6). It consists of eight multigene families that, in addition to the K and l, light chains and different classesof heav-vchain, include the chains of the T-cell receptor (p l6), the classI and II major histocompatibility complex (MHC), or human leukocvre (HLA) antigens (p. 371), and B2-microglobulin.The latter is a receptor for transporting certain classcsof immunoglobulin acrossmucosal membranes.A series of other molecules shows homology to the immunoglobulin superfamih-.These include the T:cell CD4 and CD8 cell surfacereceptor molccules,which cooperatervith T:cell reccptors in antigen recognition, and the intercellular adhcsion molccules,ICAN{-1, -2 and -3, which are involved in the interaction of lymphocvtes with antigcnpresentingcells.

A n t i b o d ye n g i n e e r i n g The techniquesof geneticcngineeringhaveled to the prospcct of reshapingor designinghuman antibodiesfor specifictherapeutic or diagnosticpurposes.These are rvhatare knorvn as monoclonal antibodies.Recombinantantibodiescan be constructedusing the human variable region framervork, the human constant regions of the heavy and light chains, and the antigcn-binding sitc of a mouse antibody-.Persons treated with these 'engineered' antibodies do not mount an immune response to them - a problem encountered in the use of rodent hybridoma-derived monoclonal antibodies It is hoped that the use of human-derived myelomacells for expressionof thescrecombinantantibodiesrvill overcomethis difliculty

C E L L . M E D I E DS P E C I F IACC O U I R E D IMMUNITY Certain microorganisms, viruses and parasiteslive inside host cells.As a result, a separateform of specificacquiredimmunitr,has evolvedto combat intracellular infections involving lvmphocltes differentiatedin the thvmus - hence T cells T lvmphocyteshave

188

specialized receptors on the cell surface, knoun as Trcell surjirc

untigen recept0rs, rvhich in conjunction wtth the mujor histocomputihilit.y complexon the cell surfaceof the infected cell result in the involvement of T hell)et'cells and cytotoxic T cellsto combat intracellular infections b1'leading to the death of the infected cell.

T-cettsurfaceantigenreceptor T cells expresson their surfacean antigen receptor.This consists of trvo different polypeptide chains,linkcd by a disulfide bridge, that both contain tr,voimmunoglobulin-like domains, one that is rclatively invariant in structure, the other highlv variable like the Fab portion of an immunoglobulin. The diversity in T:cell receptors requircd for recognition of the range of antigenic r-ariationthat can occur is generatedby a processsimilar to that sccn u'ith immunoglobulins. Rearrangemcntof variable (V), diversitl,-(D), junctional (J) and constant (C) DNA scgmenrs during T-cell maturation, through a similar recombination mechanism as occurs in B cells, results in a contiguousVDJ sequenceBindingofantigen to theT:cell receptor,in conjunction with an associatedcomplex of transmembrancpeptides,resultsin signalingthe cell to differentiateand divide.

The majorhistocompatibitity complex The MHC plays a ccntral role in the immune svstem Association of an antigen rvith the NIHC molecule on the surface of the cells is requircd for recognition of the antigen bv the T-cell rcceptor t h a t , i n c o n j u n c t i o n w i t h t h e c l o s e l - va s s o c i a t e dp r o t e i n B2-microglobulin, rcsults in the recruitment of cytotoxic and helper T cells in thc immune response.N{HC molecules occur in three classes:classI moleculesoccur on virtuallv all cells and are responsiblefor recruiting cytotoxic T cells; classII molecules occur on B cclls and macrophagesand are involved in signaling T hclper cells to recruit furthcr B cells and macrophages; the non-classic class III molecules include a number of other proteins with a varietv of othcr immunological functions. The latter include inflammatorv mediatorssuch asthe tumor necrosis factor, hcat-shock proteins and the various components of complement (p. 184). Structural analysisof class I and II MHC molecules reveals them to bc heterodimers with homologl' to immunoglobulin. The genescoding for the classI (A, B, C, E, F and G), classII (DR, DQand DP) and classIII MHC molccules,or whar is also knorvn as the human leukoc.yte antigen(HLA) s]'stem,are located on chromosome6

genetics Transplantation Rcplacementof diseascdorgans bv transplantation has become routine in clinical medicine. Except for corneal and bone grafts, the successof such transplantsdcpcndson the clegreeof antigenic similaritl.betwcen donor and recipient.The closer thc similarity, the grcatcr the likelihood that the transplanted organ or tissue, nhich is knorvnasa homogra,ti,rvill bc acceptedrather than rejected Homograft rejection does not occur betwccn identical twins or

IMMUNOGENETICS

between non-identical twins whcre there has been mixing of the placentalcirculations before birth (p. 51). In all other instances, the antigenic similaritv of donor and recipient has to be assessed b-ytesting them with suitable antiseraor monoclonal antibodies for antigenson donor and recipient tissues.Thcse rvcrcoriginall-v knorvn astrilnsplxntation antigensand arc nou knorvn to be a result of thc \'IHC As a generalrulc, a rccipient will reject a graft from an\ person rvho has antigensthat the recipient lacks.HLA ty'ping of an individual is carried out using pol-vmcrascchain reaction (PCR)-brsedmoleculartcchniques(p. 58). The HLA s,ystemis highlr, polymorphic (Tablc 13 2) A r,irtuallv infinite number of phcnotvpesrcsulting from different combinations of the various allcles at these loci is theoreticallv possible.'lwo unrelatcd individuals are therefbreverv unlikell, to havc idcntical I-ILA phenotvpes 1'hc closc linkage of the HLA loci means that the-vtend to be inherited en bloc, the term huplot.l,pe being used to indicate the particular HI-A allelesthat an individual carrieson eachof the trvo copiesof chromosome6. Thus an1 individual uill have a 25olochanceof having identical HLA antigcns rvith a sibling, as there are onlv four possiblc combinations of the two patcrnal haplotlpes (say-P and Cl) and thc tu,o matcrnal haplot-vpes(sa1.Rand S), i.e PR, PS, QR and QS. The siblings of a particular recipient are more likelv to be antigenicallv similar than cither of his or her parents, and the latter more than an unrciatcd person. tror this reason,a brother or sister is frequentlr sclcctedas a potential donor for organ or tissuetransplantation. Although crossing or,cr does occur rvithin the HLA region, ccrtrin allclcs tcnd to occur together more frequentll' than rvould bc oipcctcd bv chance,i.e. thel.tend to exhibit linkage disequilibrium(p. 132).An examplcis thc associationof thc HLA antigensAl and 88 in populationsof rvesternEuropeant-rrigin.

H-Yontigen In r numbcr of dilfcrent animal speciesit rvasnoted that tissue grafts lrom mllcs rvere rejected b1. femalesof the same inbred s t r a i n . T h e s e i n c o m p a t i b i l i t i e sr v c r e f o u n d t o b c d u e t o a histocompatibilitv antigen, knol n as the H-Y antigen.The H-Y antigen scems,ho\\e\ier, to pla-vlittle part in transplantation in humans. Although the H-Y antigen seemsto be important for

tissue A separatesex-determiningregion of the Y chromosome (SRD has been isolated, which is now known to be the testisdctermininggene(p 32).

and diseaseassociations HLApotymorphisms A linding that helps to throw light on the pathogenesisof certain diseascsis the dcmonstration of their associationwith certain HLA typcs (Thble 13.3).The best documented is that betrveen ankr losing spondr,litisand HLA-B27. In the caseof narcolepsy; a conclition of unknorvn etiology characterized by a periodic uncontrollable tendcncy to fall asleep, almost all affected :indir,idualshave the HLA-DR2 allele. The possessionof a particular HI-A antigen does not mean that an individual 'rvill ncccssarilr develop the associateddisease,merelv that he or shc has a greater relutiaerisk of being affected than the general population (p. 375). In a family, the risks to first-degreerelatives of those affectedis lo'r'r,usually no more than 5olo. Expianations for the r.ariousHlA-associated disease susceptibilitiesinclude closelinkageto a susceptibilitl-genenear of antibodiesto environmental thc HLA complex,cross-reactivit.v antigensor pathogenswith specificHLA antigens,and abnormal rccognition of'self' antigensthrough defectsin T-cell receptors or antigenprocessingThese conditions are known as uutoimmune liseuses.An example of close linkage is congenital adrenal h1'pcrplasia(CAH) due to 21-hydroxylasedeficiency(p 165). The CYP2I gene(mutated in CAH) lies within the HLA major h i s t o c o m p a t i b i l i t vl o c u s o n c h r o m o s o m e6 p 2 1 . 3 .T h e r e i s a dehcienc-v strong associationbetwcen salt-losing2 1-hydrox-vlase and HLA-A3 /8w47 /DR7 in northern European populations. Non-classical21-hydroxylasedeficiencvis associatedwith HLAassociatedwith is negutitsely ts14/DR1, and HLA-Al/88/DR3 21-hrdroxl'lasc deliciencl'.In general,the mechanismsinvolved in most HlA-disease associationsare not well understood.

T a b t e1 3 . 3 S o m eH L A - a s s o c ; a tdeids e a s e s Disease

HLA

Ankytosing spondytitis

B2l

C e L i adci s e a s e

DR4

defrciency 21-Hydroxyfase

A3lBw4TlDRl

lemochromatosis

A3

Tabte13.2 Alletesatthe HLALocr

Insutindependentdiabetes(type1)

DR3/4

HLA locus

No.of alleles

gravrs Myasthenia

88

A

5l

Narcolepsy

DR2

B

111

arthritis Rheumatoid

DR4

C

34

lupuserythematosus Systemic

DR2/DR3

D

228

(Graves disease) Thyrotoxrcosis

DR3

testiculirr differentiation and function, its expressiondoes not ncccssarih correlirte rvith thc prcsencc or absenceof testicular

13

189

13

IMMUNOGENETICS

INl{ERITED IMMUNODEFICIENCY DISORDERs Inherited immunodeficiencv disorders are uncommon and usualll-associatedwith severemorbidity and mortalitv They can occur as a primarv isolated abnormality or can be a secondarv or associatedfinding. The presentationis variablebut is usuallv in early childhood after the benefits of marernal transplacental immunitl, have declined. Investigation of immune function should be consideredin children with unexplaincd failure to thrive and diarrhea, and recurrent bacterial, chronic and opportunistic infections. Unerplained hepatosplenomegall.may alsobe a feature

of complement involved in the formation of the membraneattack complex also result in susceptibility to a particular bacterial species.primarill Neisseriu Deficiency of the Cl inhibitor results in inappropriate activation of the classicpathway of complement, leading to uncontrolled production of CZa, which is vasoactive,resulting in fluid accumulation in soft tissue and the airways,sometimes leading to life-threatening laryngeal edema This is known as hereditary angioneurotic edema,which is inherited asan autosomal dominant disorder.The serum containsbetween5oloand 30o/oof normal Cl inhibitor levels,C4 levelsare reduced, and C3 levels are normal. Acute attackscan be treatedwith fresh frozen plasma or infusions of purified C1 inhibitor. Long-term preyenrion is achieved b"v daily therapl, with attenuated androgens such as danazol.'Ihis suppressesedemaand leadsto a rise in Cl inhibitor, C4 and C2 levels.

P R I M A RI Y N H E R I T EDDI S O R D E R S O FI M M U N I T Y The manifestationsof at leastsomeof the primarv immunological deficiencydiseasesin humans (Thble 13.,1)can be understood by consideringwhether they are disordersof innate immunity or of

Disordersof innatecell-mediotedimmunity

specificacquired immunitl.. Abnormalities of humoral immunitv are associatedwith reduced resistanceto bacterialinfections that can lead to death in infanoi Abnormalities of specific acquired immunity that are cell mediated are associatedwith increased

An important mechanism in innate cell-mediated immunity is phagocytosis,lhich results in subsequentcell-mediatedkilling

susceptibility to viral infections and are manifest experimentalll. in animalsby prolonged survival of skin homografts

Chronic granulornatous disease Chronicgranulomatousdisease (CGD) is the best known example ofa disorder ofphagocytic function. It can be inherited as either an X-linked or an autosomalrecessivedisorder and is in all instances causedb1'an inability to generatesuperoxideradicals,leading to a lossof antibacterialactivity of the phagocytes(Fig. 13.5).CGD is, therefore, associatedwith recurrent bacterial or fungal infections,often b-vcommensalmicroorganisms.Until the advent

Disordersof innateimmunity Primary disorders of innate immunity involving humoral and cell-mediatedimmunitv havebeen described.

of microorganisms

A variety of defectsof complement can lead to disorderedinnate immunity-.

of supportive treatment in the form of infection-related and prophvlactic antibiotics, it was associatedwith a high childhood mortality rate The gene mutated in CGD was the first human diseasegenecloned b1,positional cloning (p. 74).

Disorders of complernent Defects of the third component of complement, C3, lead to abnormalitiesof opsonizationof bacteria,resulting in difficulties in combatingpyogenicinfections.Defects in the later componenrs

Leukocyte adhesion deficiency Individuals affected with leukocyteadhesiondefcienc-ypresent with life-threatening bacterial infections of the skin and mucous membranesand impaired pus formation.The increasedsusceptibility

Disordersof innatehumorolimmunity

T a b t e1 3 . 4 S o m ei m m u n o l o q i cdael f i c r e n d c vi s e a s easn dt h e r rf e a t u r e s

190

Disorder

Thymusgland

Lymphocytes Cett mediated

Ptasma lg celts synthesis

Genetics Treatment

Severe combrned immunodeficiency

VestigiaI

T

J

J

J

ARiXR

DiGeorge/Sed 15d kov6 syndrome

Absent(parathyroids +

t

+

N

Detetion Transplantationof fetaI

Bruton-type agammagtobu [inemia

+

-a^^^+

-^,^,^tl\

BMT,enzyme replacement forADAdeficency

thymus

+

N

J

J

xn

lginjectronsandantibiotics

AR,autosomal recessive;XR,X-iinked recessive: BMT,bonemarrowtransplant;ADA. adenosine deaminase; N,normal; J, reduced: +. Dresent

IMMUNOGENETES

0 p s o n i z a t i oonf b a c t e r i a -* fixed to cell surface

In9esron

Thc disorder hasbeen shown to result from mutations in a t\-rosine kinasespecificto B cells(Btk) that result in lossofthe signalfor B cells to differentiateto mature antibody-producing plasmacells. Hyper-IgM syndrorne This is an X-linked recessivecondition that includes increased levels of IgM, and also usually of IgD, with levels of the other immunoglobulins being decreased.Patients are susceptible to rccurrent pyogenicinfectionsand the mutated geneencodesa cell surfacemoleculeon activatedT cellscalledCD40 ligand (renamed When the gene is not functioning immunoglobulin classswitches are inefficient, so that IgM production cannot be readill- switched to IgA or IgG. TNFS-F!.

G r a n u l e [sG . dl i s c h a r g e e n z y m e isn t o v a c u o l e tso k i l l a n d d i g e s tb a c t e r i a

Fis.13.5 andthepathways invotved in intrace[[ular killing of Phagocytosis mrcro0rganrsms

to infections occurs becauseof the absenceof the B2chain of the leukocvteintegrins; this resultsin defectivemigration ofphagocytic cells duc to abnormal adhesion-relatedfunctions of chemotaxis and phagocytosis.This disorder is fatal unless antibiotics are given, both for infection and prophylactically,until bone marrow transplantationcan be offered.

Disordersof specificacquiredimmunity Again thesecan be consideredunder the categoriesof disorders of humoral and cell-mediatedspecificacquired immunitv.

Disordersof humorolocquiredimmunity Abnormalities of immunoglobulin function lead to an increased tendenc,vto developbacterialinfections. Bruton-type agarnrnaglobulinernia Boys with this X-linked immunodeficiency usually developmultiple recurrent bacterial infections of the respiratory tract and skin after the first few months of life, having been protected initially by placentally transferred maternal IgG. Treatment of lifethreateninginfectionswith antibioticsand the useof prophylactic intravenousimmunoglobulins have improved survival prospects, but children with this disordercan still die from respiratoryfailure through complicationsof repeatedlung infections.The diagnosis of this type of immunodeficiencyis confirmed by demonstration of immunoglobulin deficiency and absenceof B lymphocytes.

Comrnon variable irnrnunodefi ciency This constitutes the most common group of B-cell deficiencies but is Yer)'heterogeneousand the causesare basicallyunknown. The prcsentation is similar to that for other forms of immune dchciency including nodular lymphoid hyperplasia.The sexes are cqually affectedand presentationcan begin at any age.

Disordersof cell-mediotedspecificacquired immunity The most common inherited disorder of cell-mediatedspeci{ic acquired immunity is severe combined immunodeficiency (SCID) Severe combined irnrnunodefi ciency SCID, as the name indicates, is associatedwith an increased susceptibilit.vto both viral and bacterial infections becauseof profoundly abnormal humoral and cell-mediated immunity. Dcath usually occurs in infancy becauseof overwhelming infection, unless bone marrow transplantation is performed. SCID is genetically heterogeneousand can be inherited as either an X-linked or autosomal recessivedisorder, although all forms havein common a defect in T-cell function or development.The X-linked form (SCIDXI) is the most common form of SCID in males,accountingfor 50-600/ooverall, and has been shown to bc due to mutations in the y chain of the cytokine receptor for interleukin-2. In approximatelyone-third to one-half of children with SCID that is not X-linked. inheritanceis autosomalrecessive(SCIDI) - originally known as Swiss-type agammaglobulinemiaThe conditionsareclassifiedaccordingto whether they areB-cell negative or B-cell positive. The group is genetically heterogeneous, and includes deficiency of the enzymes adenosine deaminase (ADA) and purine nucleosidephosphorylase(PNP) (p. 171)' rvhich affect the immune system through the accumulation of purine degradationproducts that are selectivelytoxic to T cells. In addition, there is the protein tyrosine phosphatasereceptor type C (or CD45) deficiencl'. CD45 suppressesJanus kinases $AK), and there is a specificB-cell positive SCID due to JAK3 deficiency, rvhich can be very variable - from subclinical to life threatening in early childhood. Other rare autosomal recessive

191

13

IMMUNOGENETICS

forms of SCID includc the so-calledbare l'1,m1t11s6.1,te syndrome, due to absenccof the classII moleculesof thc MHC, andRAGI / RAG2 (recombination activating gencs) deficiency; these R4G gcnesare responsibleforVDJ recombinationsthat lead to mature immunoglobulin chains and T:cell receptors.

S E C O N D A R Y O R A S SEODC I IMMUNODEFICIENCY There are a number of hcreditarv disorders in which immunological abnormalitiesoccur as one of a numbcr of associated featuresas part of a svndrome.

Di George/Sedt5ikovi syndrome Children u'ith thc DiGe orge s.ltndrome(also rvell described b1' Sedlhdkovii,l0vears carlier than DiGeorgc) present wirh r c c u r r e n t v i r a l i l l n e s s c sa n d a r c f o u n d t o h a v e a b n o r m a l ccllular immunitv as characterizcdby reduccd numbers of T lvmphocvtes,as wcll as abnormal anribod]. production. This has been found to be associatedr,vithpartial absenceof the thvmus gland, lcading to defectsin cell-mediatedimmunity and T cell-dependent antibodv production. Usuall-v these defects are rclativelv mild and improve rvith age,as the immune s).stcm matures Horvevet it is important for all paticnrs diagnosedto be investigated b-v taking a full blood counr with differential CD3, CD,l and CDU counts, and immunoglobulins. Thc levels of diphtheria and tcranus antibodies can indicate the abilitv of thc immune s].stcm to respond. 'fhcse patients usuallv also have a number of characteristiccongenital abnormalitics,r,hich can include heart diseaseand absent parathlrroid glands. T'hc lattcr linding can result in affectcd individuals presenringin the nelvborn period u'ith tetany due to low serum calcium levels secondarvto lorv parathvroid hormonc levels.This syndromehas beenrccognizedto bc part of the spectrumof phenotvpescaused b1'-abnormalities of the third and fourth phar-vngealpouches (p 92) as a consequenceof a microdeletion of chromosome band 22q11.2(p 26+)

Ataxiatelangiectasia Ataxia telungiettusiais an autosomalreccssivedisordcr in which children present in earlv childhood l'ith difhculty in control of movementand balance(cerebellararaxia),dilatcd blood vcsselsof thc whitesof thc e1,.es (conjunctiva),earsand f'ace(oculocutancous telangiectasia),and a susceptibilitv to sinus and pulmonarv infections.Pcrsonsrvith this disordcr have lorv scrum IgA levels and a hypoplastic thvmus as a resuh of a defcct in the cellular rcsponseto DNA damage.The diagnosisof ataxiatelangicctasia

192

can be conlirmed b1 the dcmonstrltion of lor,vor absent serum IgA and IgG and characteristicchromosome abnormalities on culture of peripheral blood l1'mphocr.tcs,so-calledchromosome instability (p 278) In addition, individuals affected with araxia telangiectasiahar.eln increasedrisk of developing lcukemia or lymphoid malip;nancies.

Wiskott-Aldrichsyndrome Wskott Aldruh syndromeis anX-linked recessivedisorder in rvhich affcctcd bo1-shave eczcma)diarrhea, recurrent infections, a lorv platelet count (thrombocytopenia)and, usuallv. lorv serum IgM levels and impaircd T-cell function and numbers. Mutations in the generesponsiblchavebeen shownto result in lossof cytotoxic T:cell responsesand T:cell hclp for B-cell response,leading to an impaired responscto bacterialinfections. Until the advent of bone marrow transplantation,the majority of affectedboy-sdied by mid-adolescencefrom hemorrhageor B-cell malignancl-.

Carriertestsfor X- tinkedimmunodeficiencies Before the identificationof the gcnesresponsiblefor the WiskottAldrich svndrome, Bruton-type hypogammaglobulinemiaand X-linked SCID, thc availability of closelv linked DNA markers allolr,edcarrier tcsting by studics of the pattern of X-inactivation (p. 98) in the l1'mphocytesof femalesat risk. A female relative of a sporadicall-vaffectedmale with an X-linked immunodeliciencv rvould be confirmed as a carrier by the demonstration of a nonrandom patternof X-inactivationin the T-lymphocyte population, indicating that all her peripheral blood T lymphocytes had the samechromosomeinactivated(Fig'. l3 6). The carrier (C) and non-carrier (NC) are both heterozygous lor an HltuII/.44spI restriction site polymorphism. HpalI and MspI recognize the same nucleotide recognition sequence,but .MspI cuts double-stranded DNA rvhether it is methylated or not, whereasHltalI crtts onlv unmcthylated DNA (i.e. only the active X chromosome). In the carrier female, the mutation in thc.lClD gene is on the X chromosome on which the HpaII/ ,MspI restriction site is prescnt. EcoP.I/MspI double digests of T ly'mphocvtes rcsult in 6, 4 and 2-kilobase (kb) DNA fragmentson gel analysisof thc restriction fragmentsfor both the carrier and non-carrier females. EcoRI/HpuII double digestsof T:l1'mphocyte DNA result, however,in a single 6-kb fragment in the carrier female This is becausein a carrier the only T cells to survive rvill be those in which the normal geneis on the active unmethvlated X chromosome.Thus, inactivation appearsto be non-random in a carrier, although, strictly speaking, it is cell population survival that is non-random. A mixed pattem of X-inactivation in the lymphocyes of the mother of a sporadicall.vaffectedmale is consistentwith the disorder either har.ingarisenasa nervX-linked mutation or being due to the autosomalrecessiveform. SimilarX-inactivation studiesofthe peripheral blood B-lymphocyte population can be used to determine the carricr statusof women at risk for Bruton-type agammaglobulinemia and Wiskott-Aldrich syndrome.'I-histechnique has largely been replacedbv direct idcntificationofmutations in the genesresponsible

BLOOD GROUPS Blood groups reflect the antigenicdeterminantson red cellsand were one ofthe first areasin rvhichan understandingofbasic biology led

I1\/MUNOGENETICS

EcoRllMspl d o u b l ed i g e s t C

Non-ca rri er

Carrier

EcoRUHpall double digest NC

NC

E H/IV

E H/IV] E

E

--. b+

l";.--

sites H/l\4, E= HpolllMsplandEcoRlrestriction E=mutantgene - r=normalgene

Fis.13.5 Non-random inacttvationin T tymphocytesfor carrier testing rnX- tinkedSCID

to significant advances in clinical medicine Our knorvledge of the ABO and Rhesusblood groups has resulted in safeblood transfusion and the prevention of Rhesus hemolytic diseaseof the newborn.

THEABOBLOOD GROUPS The ABO blood groups were discoveredby Landsteinerjust after the turn of the twentieth century'.The transfusion of red blood cells from certain personsto others resulted, in some instances, in rapid hemolysis;in other words, their blood was incompatible Studies revealedthere to be four major ABO blood groups:A, B, AB and O. Personswho are blood group A possessthe antigenA on the surfaceof their red blood cells, personsof blood group B haveantigen B, personswho are AB haveboth A and B antigens, and personswho are blood group O haveneither.Personsofblood group A havenaturally occurring anti-B antibodiesin their blood, persons of blood group B have anti-A, and persons of blood group O haveboth. The allelesat the ABO blood group locus for antigensA and B are inherited in a co-dominant manner but are both dominant to the genefor the O antigen.There are,therefore, six possiblegenotypes.The homozygousand heterozygousstates for antigensA and B (i.e. AA, AO, BB, BO) can be determined only by family studies (Thble 13.5). As individuals of blood group AB do not produce A or B

'H' antigen, into the oligosaccharideantigens'A' is known as the 'B'. The allelesfor blood groups A and B differ in sevensingle or base substitutions that result in different A and B transferase activities, the A allele being associatedwith the addition of Nacetylgalactosaminylgroups and the B allele with the addition ofu-galactosyl groups.The O allele results from a critical single base-pairdeletion that results in an inactive protein incapableof modifl'-ingthe H antigen

Secretorstatus In the majority of persons the ABO blood group antigens, in addition to being expressedon red blood cells, are secretedin various body fluids, including saliva.This is controlled by two alleles at the so-called secretlr locus, persons being either secretrr positi'oeor secretlr negriliae,the former being dominant to the latter.Secretlr statushasbeen associatedwith a predisposition to peptic ulcers.The secretorlocus has alsobeen shown to be linked to the locus for myotonic dystrophy. Before the advent of DNA markcrs, family studies of secretor status were used to predict whether an asymptomaticperson had inherited the genefor this disorder(p. 103).

antibodies,they can receivea blood transfusion from individuals of all other ABO blood groups and are therefore referred to as uniuersulrecipients.On the other hand, as individuals of group O do not expresseither A or B antigenson their red cells,they are referred to as unitersal donors.Antisera can differentiate t\\-o subgroupsof blood groupA,Al and A2, but this is of little practical importance as far as blood transfusionsare concerned.

Motecularbasisof AB0 bloodgroups Individuals with blood groupsA, B and AB possessenzymeswith glycosyltransferase activity that convert the basicblood group, which

193

13

IN4MUNOGENETICS

RHESUS BLOOD GROUP The Rhesus (Rh) blood group srstem involves thrcc sets of closelv linked antigens, Cc, Dd and Ee. D is verv stronglv antigcnic and persons are, for practical purposes,eithcr Rh positire (possessingthe D antigcn) or Rh negarive(lacking the D antigcn).

Rhesushemolyticdiseaseof the newborn A proportion of rvomen r,vhoare Rh negativc have an increased chanccof havinga child who will either die lz atarzor beborn severel-\anemic becauseof hcmoll''sisunlcsstransfused in utero This occurs for the follorvingreason.If Rh-positive blood is gir.cnto personsrho are Rh ncgative,thc majorit.v rill develop anri-Rh antibodies.Such sensitizationoccurswith exposureto verv small quantitiesofblood and, once a pcrson is scnsitized,furthcr exposureresults in the production of r.ery high antibodv titers. In thc caseof an Rh-ncgative mothcr carr.vingan Rh-positive fetus, red cells of fetal origin can cnter the mother's circulation, for cxample after a miscarriageor at the time of delir.errrThis can inducc the formation of Rh antibodies in the mother. In a subsequentpregnancv these antibodies can cross the placenta and enter the fetal circulation.This leadsto hemolr,sisof thc fetal red blood cells if the fetus is Rh ncgative,rvhich can result eithcr in fetal death, knorvn as er.)lthroblustlsh.fetalis, or a.severehemolr,tic anemia of nervborn infants that is called hemol.yticdnease('the nepborn Once a woman hasbeensensitizedthere is a significantl_v greaterrisk that a child in a subsequentpregnancy,if Rh positive, till be more severelr,affectcd. To avoid sensitizingan Rh-negarivcwoman, Rh-compatibleblood must ahvaysbe used in anv blood transfusion. Furthermore, thc development of sensitizationand therefore Rh incompatibilit.vafter delivcrv can be prer.entedbv giving thc mother an injection of Rh antibodies,so-calledanti-D, so rhxr an] fetal cells that have found their rva.vinto the maternalcirculation are destrovedbefore the mother can becomesensitizcd. It is routine to screen all Rh-negative lvomen during pregnancy for the devclopment of Rh antibodies.Despire thesc measures,a small proportion of women do become sensitized. If Rh antibodies appear, tcsts are carried out to sec whether the fetus is affected If so, therc is a delicate balance betrvcen the choice of earlv deliverl', t ith the risks of prematurit-v and exchangetransfusion, and treating the fetus in utero rvith blood transfusions.

Molecularbasisof the Rhesusbtoodgroup Recent biochemical evidencehas shoun there to be two tvpes of Rh red cell membranepol-vpeptideOne correspondsro the D anrigen and the other to the C and E sericsof irntig;ensCloning of the genomic sequencesresponsibleusing Rh complementirr_v DNA

194

(cDNA) from reticulocyteshas revealedthat there are two genes coding fbr the Rh svstem:one for D and d, and a secondfor both C and c and E and e. The D locus is present in most pcrsons and codcs for the major D antigen present in those lrho are Rh positive. Rh-negativeindir.idualsare homozygousfor a deletion of the D gcnc. It is not, perhaps, surprising therefore that an antibodv has never been raised to d! Analvsis of cDNA from reticulocytesin Rh-negativepersons lr'ho rverehomozvgousfor dCe, dcE and dce allowedidentification of the genomic DNA sequencesresponsible for the different antigenic vari:rnts at thc second locus, revealing that they are produced bv altcrnativesplicing of the mRNA transcript.The Ee pol-vpeptidcis a full-length product of thc CcEegene,very similar in sequenceto the D polypeptide.The E and e antigensdiffer by' a point mutation in exon 5. The Cc pol-vpeptidesare, in conrrast, products of a shorter transcript of the same gene har.ing either exons,1,5 and 6 or 4, 5 and 8 spliced out. The differencebetrveen C and c is due to four amino-acid substitutionsin exons I and 2 These lindings hclp to explain rvhat ryasan apparentlv complex blood group svstem.

O T H EB RL O O G DROUPS Therc arc approximatelv a furthcr 12 'common' blood group svstcms of clinical importance in humans, including Duffl', Lcwis, X'IN and S. Thcsc are usualll' of conccrn onlv rvhen cross-matchingblood for persons who, bccauseof repeated transfusions, have developed antibodies to one of these other blood group antigcns Until the advent of DNA fingerprinting (p. 69), thc-vrvere used in linkagc studies (p. l3l) and paternitv testing(p 2.59).

FURTHER READING Bcll J I,'lbdd J A, tr{cDcvitt H O 1989The molccularbasisof HLA disease a s s o c i a t i oA n d r . H u m G e n c t1 8 :1 4 1 ()ood retien o.fthe HLA-liease associations Dre.vcrW J, BennetJ C 196.5 The molecularbasisof antibodvformation: a parador Proc Natl Acad Sci USA 54: 864-869 Theproposalof'thegenerilion o.l'antibodydixersitl,. HunkapillcrT, Hood L 1989Diversitl' of the immunoglobulir gcnc superfamill,. Adr, Immunol 41: I-63 Cood re'tcn: 0.fthe structureoJ'thaimnnmoglobulingenesuper/hmily. Jancwal C A, Travers P, \\ialport M, CapraJ D 1999Immunobiologi,; ,lth edn Current Biologl; London Good,n,ellillustrated,textbookof the hrolq.1,of inmunologl. LachmannP J, PetersK, RosenF S, \\'alport M J 1993Clinical aspcctsof immunolog5 5th edn Blackrvell,Oxford A comprehennt:e lhree-tolurnemultiauthor text uLerin!: both basicand rlinicul tmtnunolog.1, Roitt I 1997Esscntiirlimmunologl',9th edn Blacku'ell,Oxford Iya I lent busicimmtmoI oJlytet thook

I[/MUNOGENETICS

ELEMENTS Q fne immune responsein humans can be divided into two main types, innate and specific acquired or adaptive immunitl.. Both types can be further subdivided into humoral and cell-mediatedimmunity. @ Innate humoral immunity involves acute-phase proteins that act to minimize tissueinjury by limiting the spreadof infective organismsand, through the alternative pathway of complement activation, results in a localized inflammatorv responseand the attraction of phagocytes and opsonizationof microorganisms.Complement, which consistsofa seriesofinactive blood proteins that are activated sequentialllrin a cascade,can also be actiyatedthrough the classicpathway by antibody binding to antigen. @ Inn"t. cell-mediated immunity involves engulfment of microorganismsb1' macrophagesand their destruction b-vintracellular granules @ Specificacquiredhumoral immunity involvesproduction of antibodies by mature B cells or plasma cells in responseto antigen.Antibodies areY-shapedmoleculesand each is composed of two identical heav-v(H) chains and trvo identical light (L) chains.The antibody molecule has two parts that differ in their function: trvo identical antigen-binding sites (Fab) and a single binding site for complement (Fc). There are Iive classesof antibodli IgA, IgD, IgE, IgG and IgM, each with a specificheavv chain. The light chain of any classof antibody can be made up of either kappa (r) or lambda (1,)chains. @ Each immunoglobulin light or heavy chain has a variable(V) region of approximately110 amino acidsat the amino-terminal end. The carboxy-terminalend consistsof a constant (C) region of approximately110 amino acids in the t
r,ariationin both the light and heavy chains occurs within several small hypervariable regions. These are thought to be the sites of antigen binding The immunoglobulin chainsare produced from combinationsofseparategroups of DNA segments.These consist of one from a variable number of DNA segmentscoding for the constant (C), variable (V) and foining (f) regions between the V and C regions for the r and l, light chains and the various types of heavl-chains.The heavy chains also contain a diversitl' (D) region locatedbetween the V and J regions.The total number of possibleantibodiesthat could be produced by variouscombinationsof theseDNA segmentsaccountsfor the antibod,vdiversity seenin humans. @ Cetl-mediated specilic acquired immunity primarily involvesT cells which, through the Tlcell surfaceantigen receptor,in conjunction with the major histocompatibility complex moleculeson the surfaceof infected cells, engage T helper cells and cytotoxic T cells to combat intracellular infections. @ fne major histocompatibility complex (MHC) or human leukocyte antigen (HLA) system consists of a number of closelylinked loci on chromosome6. The many diffcrent allelesthat can occur at each locus mean that a verl- large number of different combinations of these can 'en bloc'as a haplotype. result. The HLA loci are inherited Thc closer the match of HLA antigens between the donor and recipient in organ transplantation,the greater the likelihood of long-term survival of the homograft. Possessionof certain HLA antigens is associatedwith an increasedrelativerisk of developingspecificdiseases. @ nn understanding of the ABO and Rhesus blood groups has resulted in safe blood transfusions and the prevention of Rhesushemolytic diseaseof the newborn.

195

CHAPTER

'All canceris genetic, but some cancersare more genctic than others.' Paraphrased frorn Animal Farz, by George Orwell

eti

DIFFERENTIATION BETWEEN GENETIC IN CANCER

Cell biologv and molccular geneticshave revolutionized our understanding of cancer in recent ,vears;all cancer is a generic diseaseof somaticcells becauseof aberrant cell dir.ision or loss of normal programmed cell dcath, but a small proporrion is strongl-vpredisposedbv inherited germlinemntationsbehavingas mendeliantraits Hot ever,this doesnot contradict our traditional understandingthat, for manv canccrs,environmental factors are of primarv etiologicalimportance, whilst heredity secmsto plav littlc or no part. The latter is certainlv true ofthe 'industrial cancers', rvhich result from prolonged exposureto carcinogenicchemicals Examples include cancer of thc skin in tar workers, cancer of thc bladder in aniline dve rvorkers,angiosarcomaof thc liver in processuorkers making polyvinyl chloride (PVC), and cancerof the lung in asbestosrvorkers.Er.en so, for those lvho har.ebeen exposcdto thesesubstancesand are unfortunate enoughto suffer, it is possible that x significanr proportion may har-ea generic predispositionto the acti\,itv of the carcinogen.The link betrrcen ciBarettesmoking and lung cancer(asrvell as someothcr cancers) has been rccognizedfor nearlv half a centurl', but not all smokers der,clopa tobacco-relatedmalignancti Recentstudieshaveshorvn that smokersu'ith short chromosometelomeres(p 31) appearto bc at substantiallv greater risk for tobacco-relatedcancersthan people uith short telomereswho har.enever smoked,or smokers who have long telomeres. The recognition that a number of rare cancer-predisposing svndromes, as lcll as a small but signihcant proportion of common cancershaving a hercditarv basis,has led ovcr the past 2-5r,earsto an explosion in our understandingof the gcnctic basis and cellular biologv of cancer in humans. As a gcneral principle it is nolv clear that cancers arise as the end result of an accumulationof both inherited and somatic mutxtions in proto-oncogenesand tumor suppressorgenes.A third class of genes the DNA mismatch repair genes- are also important becausetheir inactivation is believedto contribute to the genesis of mutations in other genes directlv affecting the survival and proliferation of cells

195

In manv cancers the differentiation between genetic and environmental etiological factors is not ah\ays obvious In the majorit_vof cancersin humans there is no clear-cut mode of inheritance,nor is there an-vclearh,definedenvironmentalcause. In certain of the common cancers,such as breast and botel, genetic factors plar an important, but not exclusive,role in the etiologl,. Evidence to help differentiate environmental and geneticfactors can come from a combination of epidemiological, famil-vand trvin studies,diseaseassociations, biochemicalfactors and animal studies

E PD I E M I O L O G I CSATLU D I E S Breastcanceris the most common cancerin women. Reproductive and menstrual histories are rvcll recognizedrisk factors.Wbmen rvho havehad childrcn havea lorverrisk of developingbreastcancer than nulliparous\yomen.In addition, the voungerthe ageat which a woman has her first pregnanc\! the lo$'er her risk of developing breastcancer.In addition, the later the ageof onset ofmenstrual periods,the lorverthe risk of developingbreastcancer. The incidenceof breastcancervariesgreatlvbetweendifferent populations, being highest in rvomen in North America and rrestern Europe and up to cight times lor,er in women of Japancscand Chinese origin. Although these differencescould be attributed to genetic diffcrences betrveen these population groups) studl' of immigrant populations moving from an area vr,itha lolr,incidenceto one with a high incidencehas shor,vnthat the risk of developingbreastcancerriseswith time to that of the native population, supporting the vierv that breast cancer has a signilicant enyironmentalcomponent It has long beenrecognizcdthat personsfrom the lesswell-off socioeconomicgroups har,ean increasedrisk of developinggastric cancer.Specific dietarv irritants, such as salts and preservati\es, or potential environmcntal agents, such as nitrates, have been suggestedas being possiblecarcinogens Gastric cancer also

CANCER GENETICS

shorvsvariationsin incidencein different populations,being up to eight times more common in Japanescand Chincse populations than in persons of western European origin N{igration studics have shorvnthat the risk of gastric cancer for immigrants from high-risk populations does not fall to that of the native lorv-risk population for trvo or three generations.It has been suggested previously that this could be due to exposureto environmental factors at an earlv critical age.There is a substantialbodl'of evidenceaccumulating that early infection with Helicobacter?J,lori, which causeschronic gastric inflammation, is associatedrvith a five- to sixfold increasedrisk of developinggastriccancer.

FAMILY STUDIES

B I O C H E M I C AC LTORS B i o c h e m i c a l f a c t o r s c a n d e t e r m i n e t h e s u s c e p t i b i l i t . vt o cnvironmental carcinogens.Eramples include the association betrvecn slow-acett-latorstatus and debrisoquine metabolizer status (p. 180) and a predisposition to bladder cancer (p 182), as rvcll as glutathione S-transferaseactivity (p. 182), which influencesthe risk of developinglung cancerin smokers.

ANIMAL STUDIES Certain inbred strains of mice have been developed that have a high chance of developing a particular t-vpeof tumor. The A (albino)Bittner strain is especiallyprone to developtumors of the lung and breast;the C3H strain is particularly prone to develop

The frequency with which other family members develop the same cancer can provide evidence supporting a genetic contribution. The risk of developing breast cancer for a woman

breast tumors as well as tumors of the liver; the C58 strain is prone to develop leukemia. Breeding experiments with mice have shou'n that the tendency to develop cancer is influenced by

tho lives until her mid-7Osin rvesternEurope is at least I in 10. Family studies have shown that, for a woman rvho has a first-

environmcntal factors. In strains with a high incidence ofbreast tumors, the frequency of these tumors is reduced b1' dietary restrictions and increasedby high temperature.

degree relative with breast cancer,the risk that she will also develop breast canceris between I 5 and 3 times the risk for the generalpopulation. The risk variesaccordingto thc ageof onset in the affcctedfamily member: the earlier the agcat diagnosis,the greaterthe risk to closerelatives(p. 2l 1) Similar studies in gastric cancer have shown that hrst-degree relativesof persons lr,ith canccr of the stomach have a t$o- to threefold increasedrisk compared to the general population of der.elopinggastriccancer.The increasedrisk ofdeveloping gastric cancer in close relatir,esis, however,relatir.elysmall, suggesting that environmental factors are likelv to be more imoorrant

TWINSTUDIES Concordance rates for breast cancer in twins are low for both tvpes of nr,ins,being onl-vslightll' greaterin monoz,vgoricfemale nvins, at l7()ir, than the 13o/ofound in dizy'goticfemale twins, suggestingthat, overall, environmental factors are likel-vto be more important than geneticfactors.Th'in studiesin gastriccancer have not shorvn an increasedconcordancerate in monozvsotic comparedrvith diz-vgotictwrns.

D t S E A SAES 5 0 C t t 0 N S B l o o d g r o u p s a r e g e n e t i c a l l y 'd e t e r m i n e d , a n d t h e r e f o r e associationof a particular blood group with a diseasesuggests a possible genetic contribution to the etiology'.A large number of studies from a variety of countries have shown an association betr,veenblood group A and gastric cancer.It is estimated that persons rvith blood group A have a 20oloincreasedrisk over the generalpopulation of developinggastriccancer.Blood group A is associatedrvith an increasedrisk of developingperniciousanemia, rvhich is also closelvassociatedwith chronic gastritis.It appears, horvever,that pernicious anemia has a separateassociationwith gastric cancer.Personswith pernicious anemia have a three- to sixfold increasedrisk of developinggastriccancer.

VIRAL CTORS Animal studiesundertakenb-vPe-vtonRous earlv in the twentieth centur\j! among others, showed that transmissionof a tumor was possiblein the absenceof body cells. Bittner later showed that susceptibility'to bresst tumors in certain strains of mice dcpcnded on a combination of genetic factors as well as a trans'milk agent'. In missible factor present in the milk, knolvn as the high-incidence strains both genetic susceptibility and the milk agent are involved, but in low-incidence strains there is no milk agent.B-vusing foster mothers from cancer-freestrains to suckle nervbornmicc from strainswith a high cancersusceptibilityit was to possibleto reduce the incidence of breast cancer from 100o/cr an increasedincidence was observed lcss than 50o/cr. Conversel-v, in cancer-freestrains by suckling the newborn mice with foster mothers from high cancer-prone strains The milk agent was sho$,nto be a virus thatlvas usuallv transmitted b--vthe mother's milk, but could alsobe transmitted bv the father's sperm. Subsequentstudieshaveshown that certain viruses are tumor inhttmans. A limited number of DNA viruses fbrming or oncogenic are associatcdr,vithcertain t1-pesof human tumors (Table 14.1), causeneoplasia rvhercasa variet.vof RNA viruses,or retrut'iruses, in animals.The study of the geneticsand replicativeprocessesof oncogcnicretroviruseshasrevealedsomeofthe cellular biological proccsscs inr olr ed in carcinogenesis.

Retroviruses Rctror,iruses have their genetic information encoded in RNA and rcplicate through DNA by coding for an enzyme known as reversetranscriptase(p l6), which makes a double-stranded DNA copl- of the viral RNA. This DNA intermediate integrates into the host cell genome,allorvingthe appropriateproteins to be manufactured,resulting in repackagingof new progenv virions.

197

14

C A N C EG RE N E T I C S

T a b t e1 4 . 1 H u m a nD N A v i r u s e sm p l i c a t eidn c a r c i n o g e n e s i s Virusfamity

Hepadna

Type

Tumor

P a p i t t o na ( F P V )

r n d p e n l e ) .s k r r c a r c e r W a r t s( p r a n L aa' n d g e n t a . ) .r r o g e n r t acl a r c e r s( c e r v i c avt .u l v a a

F p s t e i nB a r r ( E B V )

B u r k i t tl y m p h o m a on, a s o p h a r y n g e ac la r o n o m a ,t y m p h o m a si n i m m u n o c o m p r o m i s ehdo s t s

l lon:ti+ < R ll lR\/l

o f n r i r l . n n r n n p n i l i 1 1 76 6 6 1 p 6 1 . ^ r .t -6^y n B . v v - u ' - ' l L u g s l l s d l t r | s L s > > drl ,ya sn g or r rl q

nncnrlrlcB

accne - -f,1_^_!^_^

t-""'^'^'

Naturally occurring retroviruses have only the three genes necessaryto ensure replication: gag, encoding the structural proteins for the core antigens;pol, coding for reversetranscriptase; and,ents,the genefor the glycoprotein envelopeproteins (Fig. 14.1).

translocation breakpoints, or their amplification in doubleminute chromosomesor homogeneouslystaining regions of chromosomes(p. 199). In addition, a number of oncogenes have also been identified by the ability of tumor DNA to induce

Study of the virus responsible for the transmissible tumor in chickens, the so-called Rous sarcoma virus, identified a fourth gene that results in transformationof cells in culture, a model for malignancy in aiao. This viral gene, which transforms the host

tumors in aitro by DNA transfection.

virus Infecting

cell, is known as an oncogene.

g a gp o le n v

[--r--Tt

Oncogenes are the altered forms of normal genes - protolnclgenes- that have key roles in cell growth and differentiation pathways.In normal mammalian cells there are sequencesof DNA that are homologous to viral oncogenes, and it is these that are named prltl-oncogenesor cellular rncrgenes.Although the terms proto-oncogene and cellular oncogene are often used interchangeably,strictly speaking proto-oncogene is reserved for the normal geneand cellular oncogene,or c-rzr, refers to a mutated proto-oncogene,which has oncogenic properties like the viral oncogenes,or y-lnc. Some 30 oncogeneshavebeen identified.

Hostc e t l

gag pol env

r--r-Tl

+

Reverse IranScnplase

R E L I O N S H IBPE T W E ECN. O N C ANDY.ONC Cellular oncogenesare highly conservedin evolution, suggesting that they have important roles as regulators of cell growth, maintaining the ordered progressionthrough the cell cycle, cell division and differentiation. Retroviral oncogenesare thought to acquire their dominant transforming activity during viral transduction through errors in the replication of the retrovirus genome following their random integration into the host DNA. The end result is a viral gene that is structurally similar to its cellular counterpart but is persistentlydifferent in its function.

I D E N T I F I CI O NO FO N C O G E N E S Oncogenes have been identified by two types of cytogeneric finding in associationwith certain types of leukemia and tumor in

198

humans.These include the locationof oncosenesat chromosomal

A

"

,, gag po env i I--f-T__l

B-,-

C

.. I gag pol envsrc i il--f-T--f-ll

m os ."gag ,' I-TIT-L r \,/ Oete'tiO

I

::l:.:.:1 Fis.14.1 ModeL foracquisition in retroviruses oftransformrng abitity A, NormaLretroviraI replrcation B,TheRoussarcoma virushas integrated neara cetluLar oncogene Thetransforming abiLity ofthis virusisdueto theacquired homotog ofthecellu[ar oncogene, v-src C,A defective transforming viruscarriesan oncogene genes, similarto srcbutis defective inthestructural e g Moloney murinesarcoma virus.whichcarriesmos

C A N C EG RE N E T I C S

ldentification of oncogenes at chromosomal translocation breakpoints Chromosome aberrations are common in malignant cells, which often show marked variation in chromosomc number and structure. Certain chromosomesseemedto be more commonlv involved and it was initialll' thought that these changes rvere secondarvto the transformed state rather than causal This attitude changed rvhen evidence suggestedthat chromosomal s t r u c t u r a l c h a n g e s ,o f t e n t r a n s l o c a t i o n s( p . , 1 7 ) , r e s u l t c d

3

i n r e a r r a n g e m e n t sw i t h i n o r a d j a c e n t t o p r o t o - o n c o g e n e s It has been found that chromosomal translocationscan lead

,\

to nor,el chimeric genes rvith altered biochemical function or level of proto-oncogeneactivitli There are numerous examples

10

of both types, of which chronic myeloid leukemia is an erample of the former and Burkitt lymphoma an erample of the latter. 13

14

16

15

17

18

Chronic myeloid leukemio In 1960, investigatorsin Philadelphia ''rere the hrst to describe an abnormal chromosomein white blood cells from patientsrvith chronic m_r-eloid leukemia The abnormal chromosomc,rcferred to as the Philadclphia, or Phr chromosome, is an acquired abnormalit-vfound in blood or bone marrow cells but not in other tissuesfrom these patients.The Pht is a tinl' chromosome that is norv known to be a chromosome22 from r,vhichmaterial from the long arm has been reciprocallv translocatedto and from the long arm of chromosome9 (Fig. 14.2), i.e. t(9;22)(q31;qll). This chromosomal rearrangement is seen in 90o/oof persons rvith this form of leukemia.This translocation has been found to transfer the cellular ABL (Abelson)oncogene from chromosome 9 into a region of chromosome 22 known as the breakpointcluster,or BCR, region, resulting in a chimeric transcript derived from both the c-ABL (70olo)and the .BCR genes This results in a chimeric geneexpressinga fusion protein consistingof the BCR protein at the amino end and ABL protein at the carboxv end, r,vhichis associatedwith transforming acilvrty-.

Burkittlymphomo An unusual form of neoplasia seen in children in Africa is a l.vmphomathat involves the jaw, known as Burkitt l1'mphoma, so named after Dennis Burkitt, a medical missionary who first describedthe condition in the late 1950s.Chromosomal analvsis has revealed the majoritl, (90o/o)of affected children to have a translocation of the c-MYC oncogene from the long arm o f c h r o m o s o m e8 o n t o h e a v y ( H ) c h a i n i m m u n o g l o b u l i n locus on chromosome 14. Less commonly the MYC oncogene i s t r a n s l o c a t e dt o r e g i o n s o f c h r o m o s o m e 2 o r 2 2 , r ' h i c h encode genes for the kappa (rc) and lambda (1,) light chains, respectivel-v(p. 185). As a consequenceof these translocations ,MYC comesunder the influence of the regulatorv sequencesof the respectiveimmunoglobulin geneand is overexpressed tenfold

&p e& 19

20

*!db 21

* f4. 22\

Fig.14.2 Leukemta showing myeLoid withchronic froma patient Karyotype d r) P h r t a d e L pchhirao m o s o m e . t h ec h r o m o s o m2e2( a r r o w e o w h i c hh a sm a t e r i a l t r a n s l o ctaottehdel o n ga r mo fo n eo ft h e n u m b e9r c h r o m o s o m(easr r o w e d )

0ncogeneamplification Proto-oncogenescan also be activated by the production of multiple copiesof the geneor what is known ^s geneamplificd'tiln, a mechanismknown to have survival value when cells encounter environmental stress.For example, when leukemic cells are exposedto the chemotherapeuticagent methotrexate,the cells acquire resistanceto the drug by making multiple copies of the gene for dihydrofolate reductase, the target enzyme for methotrexate. Gcne amplihcation can increasethe number of copies of the oncogeneper cell several-foldto severalhundred times, leadingto greateramounts of the correspondingoncoprotein In mammals the amplified sequenceof DNA in tumor cells can be recognized bv thc presenceof small extra chromosomesknown as doublestaining regionsof the minute chromlslmesor homogeneously chromosomes.These changesare seenin approximately l0o/oof tumors and are often present more commonly in the later rather than the earlv stagesof the malignant process. Amplification of specific proto-oncogenesappears to be a feature of certain tumors and is frequently seen with the -MIC family of genes. For erample, N-,l{yC is amplified in approximately30o/oof neuroblastomas,but in advancedcasesthe proportion rises to 50o/o,where gene amplification can be up to 1000-fold Human small cell carcinomasof the lung also show amplilrcationof MYC, N-i4yC, andL-MYC.

199

14

C A N C EG RE N E T I C S

Amplification of ERB-82, MYC and c-vclinDl is a feature in20o/oofbreast carcinomas,where it has been suggestedthat it correlateswith a number of rvell establishedprognostic factors such as l.vmph node status, estrogen and progestogenreceptor

Growthfactor recepror ryrosrne krnase

T{:

Cytoplasm ic-membrane tyrosine kinases

status,tumor size and histologicalgrade C e l lm e m b r a n e

Detectionof oncogenes by DNAtransfection studies The ability of DNA from a human bladder carcinoma cell line to transform a well establishedmouse hbroblast cell line called NIH3T3, as demonstratedby the lossof contact inhibition of the cells in culture, or what is known as DNA trunsfection,ledto the discoveryof the human sequencehomologousto the rrs gene of the Harvey murine sarcomavirus. The human R 45 gene family

Cytoplasmic tyrosrne krnases

P r o t eni s with GTPase activity

Cytoplasm

D N A - b i ni d ng n u c l e a rp r o t e i n t r an s c rpi t i on factors

consistsof three closely related members, H-R {S, K-RIS and N-Rl.t. The RAS proteins are closelyhomologousto their viral counterparts and differ from one another only near the carboxv termini. Oncogenicity of the ras proto-oncoBeneshas been shown to ariseb1'acquisitionof point mutations in the nucleotide sequence.In approximately 50o/oof colorectalcancersand 95olo of pancreaticcancers,as well as in a proportion of thl,roid and

Nuclear me mb r an e

Geneexpressron on/off I S i g n aIl I translation

l\ucleus

lung cancers,a mutation rn a ras genecan be demonstrated.The RIS gene famill'has recently been shown to be the ke-vpathr,va-v (RIS-MAPK) in Neurofibromatosis t-vpe I (p. 289) and the Noonan/cardio-facio-cutaneous/costellosvndromes(p. 2+1). DNA transfectionstudieshavealsoled to identificationof other oncogenesthat have not been demonstrated through retroviral studies.These include MET (hereditarl'papillary renal cell carcinoma), IRK, MAS and RET(multiple endocrineneoplasiatype 2).

;

at la

a..

Proliferation

'.

.

Differentiation

Fis.14.3 S r m p t r f , esdc h e m ao rr h es t e p s, n s , g n a I r r a n s d l c l i oa.n d + . ^m - C - ^el tlt .5 U .f lfd^L- c^ + , .r1t r^u,>, . T| hr c^ ; ^I r L- -rd- L^ U l rLUtaf I ?.na5^C. .t .l p ^I l + O;n^ ^ I+t O r u^ ^r u, L

p a t h w a ya m p t i f i etsh e s i g n a b I y a c a s c a d teh a tr n v o t v eosn e o r more of the steps

F U N C T I OO NFO N C O G E N E S Cancershavecharacteristicsthat indicate,at the cellular level,loss ofthe normal function ofoncogeneproductsconsistentwith a role in the control of cellular proliferation and differentiation in the processknown assignaltransduction. Signaltransductionis a complex multistep pathwayfrom the cell membrane,through the cytoplasm to the nucleus, involving a varietv of t-vpesof proto-oncogene product involvedin positiveand negativefeedbackloopsnecessary for accuratecell proliferation and differentiation (Fig. la.3). Proto-oncogeneshavebeenhighly conservedduring evolution, being present in a r,ide variety of different species,indicating that the protein products they encodeare likelv to have essential

lrom membrane-associated tyrosine kinasesto serine threonine kinases The third type involves proteins located in the nucleus that control progressthrough the cell cycle,DNA replication and the expressionof genes.

biological functions Proto-oncogenes act in three main lvays in the process of signal transduction The first is through phosphorylation of serine, threonine and tyrosine residues of proteins b.v the transfer of phosphate groups from ATP This leads to alteration of the configuration activating the kinase

factors stimulate cells to grow by binding to growth factor receptors.The best known oncogenerhat acts as a growth factor is the v-S1S oncogene,which encodespart of the biologically active platelet-derivedgrowth factor (PDGF) B subunit. When v-SIS oncoprotein is added to the NIH 3T3 cultures, the cells are transformed, behaving like neoplasticcells, that is, their growrh

activit-v of proteins and generating docking sites for target proteins, resulting in signal transduction. The Rl.! familv of proto-oncogenes are examples of the second rype, rvhich are GTPases and 'w.hichfunction as molecular srvitches through

200

the guanosine diphosphate-guanosinetriphosphate (GDPGTP) cvcle as intermediates relaving the transduction signal

T Y P E SO FO N C O G E N E Growthfactors The transition of a cell from G6 to the start of the cell cycle (p. 1l) is governedby substancescalled growth factors. Growth

rate increasesand thel'lose contact inhibition. In ztiuothey form tumors when injectedinto nude mice.Oncogeneproductsshowing homolog-vto fibroblast growth factors (FGFs) include -F1SI and INT-L, rvhich are amplified in stomachcancersand in malignant melanomas, respectiveh''.

CANCER GENETICS

Growthfactorreceptors N{anv oncogenes encode proteins that form growth factor receptors, r,vith tvrosine kinase activit-v possessingtr-rosine kinasc domains that allorv cells to b-vpassthe normal control mechanisms. More than ,10 different tvrosine kinases have been idcntificd and can be divided into two main types: those that spanthe cell membrane (growth factor receptor tyrosine kinases) and those that arc located in the cy-toplasm(non-receptor tvrosine kinascs) Eramples of tvrosine kinasesinclude ERB-B, u'hich encodesthe epidermal growth factor (EGFR) receptor, and the related ERB-82 oncogene.Mutations, rearrangements amplification of the ER.B-B2 oncogeneresult in ligandindependent activation, which has been associatedwith cancer of the stomach, pancreasand ovar\..Mutations in KIT occur in the hcrcditarv gastrointestinalstromal tumor syndrome. These oncogenesare not activated by translocation (as in Burkitt ll,mphoma) but rather by point mutations. When germline or inherited, the mutations are not lethal, nor are they suflicient b-v themselvesto causecarcinogenesis.In the caseof MET (located on chromosome7), thc papillar--v renal cell carcinomatumors are trisomic for chromosome7 and two of the three copiesof MET are mutant. A ratio of one mutant to one rvild-t-vpecop-vof MET is not suflicicnt for carcinogenesis. but a 2: I ratio is.

Intrace[[ular signattransduction factors Tfio different tvpes of intracellular signal transduction factor have becn identilied, proteins with guanosinetriphosphatase (GTPase) activit)' and cvtoplasmicserinethreonine kinases

Proteinswith GTPoseoctivity Proteins u'ith GTPase actir.itv are intracellular membrane proteins that bind GTP to bccome active and through their intrinsic GTPasc activitr generate GDP, which inactir.ates the protein. Nlutations in the ra.r genes result in increased or

preventscells from entering a prolonged resting phase,resulting in persistentcellular proliferation

Ce[[cyclefactors Cancer cells can increase in number by increased growth and division,or accumulatethrough decreasedcell death.In aiao,most cells are in a non-dividing state.Progressthrough the cell cycle (p 1l) is regulatedat two points: one in G1 when a cell becomes committed to DNA s-vnthesisin the S phase,and another in G2 for cell division in the M (mitosis) phase,through factors known as cy-clin-dependentkinases.Abnormalities in regulation of the cell c1-clethrough growth factors, growth factor receptors, GTPases or nuclear proteins, or loss of inhibitory factors lead to activation of the cyclin-dependent kinases, such as cyclin D1, resulting in cellular transformation with uncontrolled cell division. Alternatively; loss of the factors that lead to normal programmed cell death, a processknown as uprptlsis (p. 84), can result in the accumulationofcells through prolongedcell survival as a mechanism of development of some tumors. Activation of the btl-2 oncogenethrough chromosomal rearrangementsis associatedwith inhibition of apoptosis,leading to certain tvpes of l-vmphoma.

and phakomatoses Signattransduction 'lentil' (in derivesfrom the Greekphakos,meaning Phdkomatosis this context'lentil-shapedobject'),and originally referred to three that included scatteredbenign lesions:neurofibromatosis, diseases tuberous sclerosisand von Hippel-Lindau disease.To this list hasnorv beenaddednevoid basalcell carcinoma(Gorlin) syndrome, Col'den disease,familial adenomatouspolvposis, Peutz-Jegher svndrome and juvenile poly-posis.The genes for all of these conditions are now known and are normall-v active within intracellular signal transduction, and their protein products are tumor suppressors.

sustainedGTPase actiyit),,leading to unrestrainedgrowth.

Cytoplosmic serine threonine kinoses A numbcr of soluble c-vtoplasmicgene products are recognized to be part of the signaltransductionpath\,va\iThe RIF oncogene product modulates thc normal signaling transduction cascade \llutations in the genc can result in sustained or increased transmissionof a growth-promoting signal to the nucleus.

D N A - b i n d i nngu c l e a p r roteins The IOS, JLIN and ER,B-A oncogenesencodeproteins that are specific transcription factors that regulate gene expression bv activating or suppressingnearby DNA sequencesThe function of MYC and related gencsremains uncertain but appearsto be related to alterationsin control of the cell cvcle. The,MYC and MYB oncoproteins stimulate cells to progress from the G1 into thc S phase of the cell c"vcle(p 41) Their overproduction

GENES SUPPRESSOR TUMOR While the studl- of oncogeneshas revealed much about the cellular biologl' of the somatic genetic events in the malignant process,the study of hereditary cancer in humans has revealed the existence of u'hat are knolvn as tumlr su\Pressrrgenes,which constitute the largestgroup of cloned hereditary cancergenes. Studies carried out bv Harris and colleaguesin the late 1960s, which involved fusion of malignant cells with non-malignant cells in culture, resulted in the suppressionof the malignant phenotype in the hybrid cells The recurrence of the malignant phenot.vpewith loss of certain chromosomes from the hybrid cclls suggestedthat normal cells contain a gene(s)with tumor that, if lost or inactive,can lead to malignancy suppressoractir,it--v and that was acting like a recessivetrait. Such genes were This term was considered initiall,v referred to as anlittncogenas.

201

14

CANCER GENETICS

inappropriate as the)' do not opposethe action of the oncogenes and are more correctlv knolvn as tumor suppressor genes. The paradigm for our understanding of the biologl' of tumor suppressorgenesis the e1-etumor retinoblastoma It is important to appreciate, holer.er, that a germline mutation in a tumor suppressorgene(as r,vithan oncogene)docs not by itself provoke carcinogenesis:further somatic murarion at one or more loci is necessarvand environmental factors, such as ionizing radiation, ma-vbe significant in the process. Some 20 tumor suppressor geneshavebeen identilied.

RETINOBLASTOMA Retinoblastoma (Rb) is a relativelv rare, highll. malignanr, childhood cancer of the developing retinal cells of the e;-ethat usually occurs before the age of 5 years(Fig. la.a). If diagnosed and treatedat an earlv stage,it is associatedwith a good long-term outcome. Retinoblastoma can occur either sporadi.nlll,, the so-called 'non-hereditary' form, or be familial, the so-called 'hereditarv' form, which is inherited in an autosomaldominant manner Nonhereditarv casesusually involve onlv one eye,rvhereashereditarv casescan be unilateral but are more commonlv bilateral or occur in more than one site in one eve (i.e. are multifocal). The familial form alsotends to presentat an earlier agethan the nonhereditary or sporadic form.

'Two-hit' hypothesis In 1971,Knudson carried out an epidemiologicalstud-vof a large number of casesof both tvpes of retinoblastomaand advanceda 'two-hit' hvpothesisto explain the occurrenceof this rare tumor

in patients with and without a positive family history. He proposed that affectedindividuals rvith a positive famill,'historv had inherited one non-functional gene that was present in all cells of the individual, or what is known as a germlinemutatiln, with the second gene at the same locus becoming inactivated somaticallyin a developingretinal cell (Fig'.14.5) The occurrence of a secondmutation was likely given the large number of retinal cells, explaining the autosomaldominant pattern of inheritance. This would also explain the observation that in hereditar-v retinoblastomathe tumors were often bilateral and multifocal. In contrast, in the non-heritable or sporadic form, two inactirating slmatic mutationswould need to occur independentl-vin the same retinoblast (seeFig. 14.5), which rvasmuch less likelv to occur, explaining the fact that tumors in these patients were often unilateral and unifocal, and usuallv occurred at a later age than in the hereditary form Hence, although the hereditarv form of retinoblastomafollows an autosomal dominant pattern of inheritance,at the molecular level it ts recessiae becausea tumor occurs only-after the loss of both alleles. It lr,as also recognized, hower.er,that approximatel.v5oloof children presenting with retinoblastomahad other physical abnormalities along with developmental concerns. Detailed cytogeneticanalvsisofblood samplesfrom thesechildren revealed some of them to have an interstitial deletion involving the long arm of one of their number l3 chromosomepair. Comparison of the regionsdeletedrevealeda common'smallestregion of overlap' invoh'ing the sub-band 13q14 (Fig 14 6). The detection of a specific chromosomal region involved in the etiology of these

A

b

o oo oo oooo ooo o ooo o /\

/\

/\

/\

/\

/\

/\

/\

/\

/\

Fis.14.5

202

Fi1.14.4 Section ofaneyeshowing a retinoblastom a tnsitu

R e t i n o b l a s t oam na dK n u d s o nt w s o - h iht y p o t h essA LcI e t t isn thehereditary form(A)haveonemutated copyofthegene. RBi,i e themutation is inthegermhneInthenon-hereditary form (B)a mutation (somotrc) in RB'larisesasa post-zygotic event sometimeearlyin development Theretinobtastoma tumoroccurs o^tywhe- oothRBlgenesa-emutated.e aftera(nol.^e ) somolic event, whichis moreLikelyto beearlierin lifen thehereditaryform compared withthenon-hereditary form:it is alsomoretikety to giver seto bitateral andmuttifocaltumors

CANTERGETiIETIIS

r*!

of consistent cytogeneticrearrangementsin other malignancies has led to demonstration of LOH in a number of other cancers (Thble 14 2). Subsequent to the observation of LOH, linkage studiesof familial casescan be carried out to determine whether the familial casesof a specific type of malignancy are due to mutations at the samelocus and thus lead to the identification of the gene responsible,as occurred with the isolation of the R,B1 gene.

Functionof tumor suppressorgenes Although familial retinoblastoma was classicallyconsidered to be an autosomaldominant trait, demonstration of the action of the retinoblastomageneas a tumor suppressorgeneis consistent with it being, in fact, a recessivetrait, as originally suggestedin the somatic cell hybridization studies carried out by Harris and colleagues.In other words, absenceof the gene product in the homozl,'gousstate leads to the development of this particular

Fis.14.6

'13 Two homotogsof chromosome from a patentw th retinobtastoma s h o wn g a n n t e r s t i t i ad le l e t i o n o f 1 3 q 1 4n t h e r i g h t - h a n dh o m o l o g , asindicated

casesof retinoblastoma suggestedthat it could also be the locus involved in the autosomal dominant familial form of retinoblastoma. Famill' studies using a poll.morphic enzyme, esteraseD, rvhich had prcviously been mapped to that region, rapidl-vconfirmed linkageof the hereditarvform of retinoblastoma to that locus

Lossof heterozygosity Analvsesof thc DNA sequencesin this region of chromosome13 in the peripheralblood and in retinoblastomatumor materialfrom children who had inherited the gene for retinoblastomashowed them to have loss of an allele at the retinoblastomalocus in the tumor material, or what is known as losso.f'hetero4,gosity (LOH), or sometimesas loss of razslitutionalheterozygositv.An example of this is shown in Fig. 14.7A, in which the mother transmits the rctinoblastoma gene along with allele 2 at a closely linked marker locus.The father is homozygousfor allele I at this same locus,with the result that the child is constitutionally an obligate heterozygotc at this locus. Anall,sis of the tumor tissue reveals apparent homozygosity for allele 2. ln fact, there has been loss of the paternallvderived allele 1, i e. LOH in the tumor material. This LOH is consistentwith the 'two-hit' h1'pothesisleading to developmentof the malignancy-as proposedb1,Knudson. LOH can occur through severalmechanisms,which include loss of a chromosomethrough mitotic non-disjunction (p. ,15),a deletion on the chromosome carrying the corresponding allele, or a cross-over between the two homologous genes leading to homozygosity for the mutant allele (Fig. 14.78). Observation

tumor In contrast to oncogenes,tumor suppressor Benesare a class of cellular genes whose normal function is to suppress inappropriate cell proliferation, i.e. the development of a malignanc-vis due to a loss-of-function mutation (p. 25). The tumor suppressoractivity of the retinoblastomagenehas been demonstrated in t:itro in cancer cells. In addition, further support for the RB1 gene acting as a tumor suppressor Bene comesfrom the recognition that individuals rvith the hereditary form of retinoblastoma have an increased risk of developing second new malignancieslater in life, including osteosarcoma, Iibrosarcomaand chondrosarcoma.

protein The RBI gene/pllORB Thc RB I genespecifiesa'1.7-kilobase(kb) transcript that encodes a nuclear protein called pl1ORB,which associateswith DNA and is involved in the regulation of the cell cycle. Fortuitouslv. researchon the mechanism of action of the EIA oncogene of human adenovirus demonstrated that pl10RB forms a complex with E2F-l,which is an EIA oncogene-regulatedinhibitor of the transcription factor E2F The complex so formed interferes rvith the abilit.v of E2F to activate transcription of some key proteins required for DNA synthesis.When p1IORBis in a state it does not interact readily with .62,F-1, h-vperphosphor-vlated so permitting the cell cycle to proceed into the S phase (p. al). Retinoblasts fail to differentiate normally in the presence of mutant pl10Rts. p110Ruinteracts with severalviral oncoproteins,such as the transforming proteins of simian virus (SV) 40 (largeT antigen) and papilloma virus (E7 protein), and is inactivated, thereby liberating cells from normal growth constraints. These findings y-ield insight into the mechanisms of interaction between oncogenesand tumor suppressorgenes As researchcontinues there could well be many other examples whereby oncogenesexert their influence b-vdirectly or indirectly inactivating the function of tumor suppressorgene-encoded proteins or other proteins intimatell, involved in the cell cycle

203

14

CANCER GENETICS A Mother

Father

MCF

Tumour

-:l:l

t:tt:l

lrl

Acquisition of somatic mutation

Loss through Non-disjunction Mitotic Gene non-disjunction andreduplicationrecombination converston

Fig.14.7 (L0H)inthedevelopment A, Diagrammatic representation oftheLoss of heterozygosity ofa tumor. Themother(M)andfather(F)areboth (C)wiLttherefore homozygous for different attetes atthesameLocus, 2-2 and1-1,respectively. ThechiLd be constitutionaLLy heterozygous. 1 - 2l f a n a n a t y s i sDoN f AfromatumoratthattocusreveatsontyasingLeattete,2,thrsisconsistentwithL0H.B,Diagrammatic 'second representations ofthemechanisms leading to the hit'teading tothedevelopment of retinoblastoma,

'C

Tabte14.2 Syndromes andcancersthatshowlossof heterozygosity and their chromosomalLocation Syndromeor cancer Retinob[astoma

Chromosomallocation '13q14

Osteosarcoma

13q,17p

Wilmstumor

11p'13. 11p15, 16q

Renalcarcinoma

3p25.17p13

vonHippet-Lindau disease

3p25

that, although occurring in different codons, are clustered in highly conservedregions in exons 5 to 10. This is in contrast to IP53 mutations in hepatocellular carcinoma, which occur in a 'hot spot' in codon 249.The basechangein this mutated codon, usually G to t could be the result of an interaction with the carcinogenaflatoxin Bl, which is associatedwith liver cancerin China and South Africa, or with the hepatitis B virus that is also implicated as a risk factor in hepatomas. Interestingly, aflatoxin Bl, a ubiquitous food-contaminatingaflatoxinin theseareas,is a mutagen in many animal speciesand induces G to T substitutions in mutagenesisexperiments.If an interaction between hepatitis

Hepatoblastoma

B viral proteins and non-mutated TP53 can be demonstrated, this will further support the role of this virus in the etiology of 9q21.11015.17p13 hepatocellularcarcinoma Cancers frequently have a decreased cell death rate through 3p,13q14,17p and a major factor in the activationof apoptosisis TP53 a?(qt(.tsis, 11p15, 11q, 13q12,13q14.17p13. - p53 has been coined the 'guardian of the genome'. The p53 17q21 protein is a multimeric complex and it functions as a checkpoint '11p15,'17p13 control site in the cell cycle at G1 before the S phase,interacting with other factors, including cyclins and p2l, preventing DNA 'wear and tear' from being replicated' 5q,11p15 damagedthrough normal

G a s t r ci ca n c e r

1 p5, q , 7 q , 1 1 p 3 ,q , 1 7 p , 1 8 p

potyposis Famitiatadenomatous

5q21

carcrnoma Colorectal

1p,5q2'1. Bp,'17p13, 18q21

Neurofibromatosis | (NF1. von Reck[inghausen disease)

17q

Neurofibromatosis Il (NF2)

22q

Meningioma

22q

Muttrpte endocrine neoptasra type| (MENl)

11q

Melanoma

9p21,17q

Ovarian

11q25.16q.17q

Pancreatic

9p21.13q14.17p13

Prostate cancer

1p36.1q.8p.10q. 13q,16q

Btaddercarcinoma Lungcarcinoma Breastcarcinoma

Rhabdomyosarcoma

TP53 The p53 protein was first identified as a host cell protein bound to T antigen, the dominant transforming oncogeneof the DNA tumor virus SV40. After the murine p53 gene rvascloned it was shown to be able to cooperatewith activatedRIS and act as an oncogenetransforming primary rodent cellsln aitro,eventhough the

Mutant p53 protein monomers are more stablethan the normal p53 proteins and can form complexeswith the normal wild-type IP5J, acting in a dominant-negativemanner to inactivateit'

Li-Fraumenisyndrome As mutations rn TP53 appear to be a common event in the genesis of many cancers, an inherited or germline mutation of IP5J would be expected to have serious consequences.This hypothesiswas substantiatedwith the discoveryofsuch a defect in persons with Li-Fraumeni syndrome.Members of families with this rare syndrome (p.212), which is inherited as an autosomal dominant trait, are highly susceptibleto developing a variety of malignanciesat a relatively early age.These include sarcomas' adrenal carcinomas and breast cancer. Point mutations in highly conserved regions of the TP53 gene (codons 245-258) have been identified in the germline of family members, with analysisof the tumor revealineloss of the normal allele.

EPIGEN ICsANDCANCER

rodent cellsexpressedthe wild-type or normal25J. Subsequently, inactivationofp53 was frequently found in murine Friend virusinduced erythroleukemiacells,which led to the proposalthat the IP5J gene was,in fact, a tumor suppressorgene.

Much of this chapter discussesfamilial cancer syndromes that follow mendelian inheritance, characterized by mutations in disease-specificgenes. However, no discussion about cancer geneticsis completewithout consideringepigeneticmechanisms' refers to heritable As discussedin Chapter 6 (p. 98), epigenetics changes to gene expression that are not d:ueto differences in the genetic code. Such gene expression can be transmitted stably through cell divisions,both mitosis and meiosis In cancer,much

The IPJ-l geneis the most frequently mutated of all the known cancergenes.Some 20-25oloof breast and over 50o/oof bladder, colon and lung cancers have been found to have TP53 mutations

is now known about alterations to methylation status of the genome, both hypopmethylation and hLpermethylation' and in this section we also discusstelomere length and cancer.

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14

CANCER GENETICS

D N AM E T H Y L A T I O AN N DG E N O M I C IMPRINTING The methylation of DNA is an epigenerirphenomenon (p. 98), and is the mechanism responsiblefor X-inactivation (p. 98) and genomic imprinting (p 115). Methylation of DNA has the effect of silencing gene expressionand maintaining stabilit-vof the genome,especiall-v in areaslr.herethere is a vast quantity of repetitive DNA (heterochromatin), rvhich might otherwise become erroneousl],'involvedin recombination eventsleading to altered regulation of adjacent genes.Thc relevanceof this for cancer emergedin 1983when studiesshor,redthat the genomesof cancer cells were hl,pometh,vlated compared with those of normal cells, primarilv rvithin repetitive DNA. This /ossa/-imprinting(LOI)may lead to activationofan allelethat is normallv silent, and hencethe high expressionof a product that confers adr.antageous cellular growth. This appearsto be an early er.entin man-vcancersand ma-v correlatewith diseaseseveritv.Chromosomalinstabilitvis stronglv associatedlvith increased tumor frequencv. which has been clearly observedin mouse models, and all thc 'chromosome breakagesvndromes' (p. 277) in humans are associatedwith a signilicantly increasedrisk of canccr, particularlv leukcmia and lvmphoma.LOI and removalof normal genesilencingmav leadto oncogeneactivation,and hencecancerrisk. LOI hasbeenstudied extensivelv at the IGF2/ HI9 loc,ts on chromosome l lpl5 5, previously discussedin Chapter 7 (p. 118). Insulin-like growrh factor 2 (IGF2) and H I9 arenormallv expressedfrom the paternal and maternal alleles, respectivcly (see Fig. 7 26), but relaxed silencing of the maternal allele, i e. hvpomethylation, results in increased1G,F2expression.This has bccn shor.n to be thc most common LOI event acrossa wide rangc of common tumor tvpes (e.9.lung, liver, colon and ovary) aswell asWilms tumor in which it was lirst identified Just as l-ypamethylationma1,-lead to activation of oncogenes, the opposite effect of h.ypennethylation ma-valso give rise ro an

increasedcancer risk, in this case through silencing of tumor suppressorgenesr.hose normal functions include inhibition of cell growth. The aberrant hvpermethylationusuallv affectsCpG islands,which aremostlv unmethylatedin somaticcells.This results in changesin chromatin structure (hypoacetylationof histone) that effcctivelvsilencetranscription. When the genesinvolved in all sorts of cell regulatorv activity are silenced,the cells have a gro\\'th advantage.Earlv hy'permethylationhas been detected in colonic cancer The effects of altered methylation leading to cancer are summarized in Fig. 14.8, although the mechanism(s) that initiate the processesare poorly understood

TELOMER L E N G TAHN DC A N C E R The ends of the chromosomesare knorvnastelomeres(p. 3 I ) and thev are specializedchromatin structures that have a protective function. The sequenceof DNA is specific and consists of multiple double-strandedtandem repeatsas follor,ys:TTAGGG. This sequenceis t1'pically about l0-l5kb long in human cells and is bound by specific proteins. It is also the substrate for telomerase,an enzvme that can lengthen the telomeresin those cclls in which it is expressed.The linal length of DNA at the very tip ofthe telomere is a single-strandedoverhang of 150 to 200 nucleotides.Telomerascrecognizesthe 3' end ofthe overhang, allowing lcngthening to proceed. Everv cell division appearsto result in the loss of TTAGGG repeatsbecauseconvcntionalDNA polvmerasescannot replicate a linear chromosomein its entiret-v,known asthe'end-replication problem' This progessiveloss of telomere length is a form of ccllular clock belier.ed to be linked to both aging and human disease.When telomeres reach a criticalll,' short length there is loss of protcction and a consequenceis chromosomal,and therefore genomic,instabilitl., rvhich meansthe cell is no longer viable. Short telomeres are no\y known to be a feature of the

H y p er m e t h y l a t e d , r ep e t i t i v e h e t er o c hr o m a tni

Fis.14.8 M e t h y t a t roofn D N Aa ^ dc a n c eTr h et o ps c h e m a showsa regionof hypermethylated repetitive DNA L ihne)rt ^ i s. o s e ist s s e q L e ^ c(eh e t e r o c h r o m aW r e L ^ y t aot n i m p r i nct h r o m o s o mi ^es l a b i . rmr ya y rpct rlr rtuh.rh m:\/ ip:n tn :rl ir:trnn nf nnrnnono[<.)

205

In the lowerpa^el hypomeLhytaLed srretcheso' CpGseq.lencebecomemethyLaLed, res;tt,ngirL r a n s cp' t i o n ast L p p r e s s r ool ' t u m o rs u p p - e s s o r :nd

roll ronr

l:tnrrr nonoc

CANCER GENETICS

premature aging syndromes, such as ataxia telangiectasia,and other chromosomebreakagedisorders (p.277), all of which are associatedwith premature onset of various cancers.It appears

colon. An understandingof the developmentof colorectalcancer has shed light on the processof carcinogenesis.

that the rate of telomere shortening is markedly increasedin these conditions, so that cells and tissuesliterally'age'more quickly. It is of great interest that some cancer cells expresshigh levels of telomerase,so that cell viability is maintained. Most metastases havebeen shown to contain telomerase-positivecells,suggesting

Multistageprocessof carcinogenesis

that telomeraseis required to sustain such growth. However, cancer cells generally have shorter telomeresthan the normal cells surrounding them, so telomeraseactivation in cancerrescuesshort telomeresand perpetuatesgenomicallyunstablecells. Telomere length is therefore almost certainly a key concept in many cancers,as well as aging processes,even though the exact mechanismsremain to be elucidated.The relationshipof telomere length to ageand diseaseis displayedgraphically in Fig 14 9.

GENETICS OFCOMMON CANCERS It is estimatedthat about 5oloof colorectaland breastcancersanse as a result of an inherited cancer susceptibility gene.A similar proportion ofmany other cancersaredue to inherited predisposing genetic factors, but there are some notable exceptionsin which onll very low incidencesof dominantly inherited carcinomasare recorded.These include the lung and cervix, as well asleukemias, I.vmphomas and sacrcomas Here external agents or stimuli, and,/or stochastic genetic events, are presumably the main factors.Nevertheless,studiesof the common cancers- bowel or colorectaland breastcancer- haveprovided further insights into the geneticsof cancer

COLORECTAL CANCER Approximately 1 in 40 personsin the developedcountriesof western Europe and North America will develop cancer of the bowel or

The mafority of colorectalcancersare believed to develop from 'benign' adenomas.Conversely only a small proportion of adenomasproceed to invasive cancer Histologically',adenomatous pol.vps less than 1 cm in diameter rarely contain areas of carcinomatouschanges,whereasthe risk ofcarcinomatouschange incrcasesto 5-l0o/o when an adenomareaches2cm in diameter. The transition from a small adenomatouspolyp to an invasive cancer is thought to take between 5 and 10years.Adenomatous polyps lessthan I cm in diameter have mutations in the r4.rgene in less than 10o/oof cases As the size of the polyp increasesto between I and 2 cm, the prevalenceof ras gene mutations is in the region of 10o/c;,rising to approximately 50o/oin full-blown colorectalcancers. Similarly, allele loss of chromosome 5 markers occurs in approximately 40oloof adenomatouspolyps and,70o/oof c a r c i n o m a s .D e l e t i o n s o n c h r o m o s o m e l 7 p i n t h e r e g i o n containing the ZP53 geneoccur in more than 75oloof carcinomas, but this is an uncommon finding in small or intermediatesized pol.vps.A region on l8q is deleted in about l0o/o of small adenomas,rising to almost 50o/owhen the adenoma shows foci of invasir.ecarcinoma, and in more than 70oloof carcinomas ( F i g . 1 4 . 1 0 ) .G e n e s a t t h i s I o c u s i n c l u d e D C C ( d e l e t e d i n colorectal cancer), SMAD2 and SMADI, the latter being part of thc transforming growth factor-B GGF-B) pathway.In some colorectal cancersmutations in the TGF-B receptor gene have been identified. It appears,therefore, that mutations of the RAS and TP53 genesand LOH on 5q and l8q accumulateduring the transition 'benign' adenomato carcinoma.The accumulation from a small ofalterations,rather than the order,appearsto be more important in the development of carcinoma More than one of these four alterationsis seenin only-7o/oof small, earlv adenomas Th'o or

Fi1.14.9 T p l n m e r c l e n n t hn v p r a n e t n n o r m a l G e r m l i ncee l l s

c

u E

Normal cells somatic

a F

Disease

Age

agingsyndromes Life andpremature ceLLs inthebodythatmaintain TheonLy Life, and iengththroughout ieLomere are havehighlevelsoftelomerase, cetts, Somatic thoseofthegermLine a undergo of disease, intheabsence tntelomere decrease slowlyprogressive life,sothatdisease throughout Length a n dc a n c ebr e c o maeni n c r e a s i n g aging riskinthee[dertyln premature oftelomere theprocess syndromes andthertskof isaccelerated. shortening highfromearlyaduLt cancerbecomes Lrfe onwards

207

14 Chromosome 18qloss-800/o tumors DCC, SMD4, SMAD2

APCgenemutation (chromosome 5q loss) >800/0 tumors

K-ras[activation) -500/o tumors TGF-B receptor

Chromosome 17p loss- TP53 >850/o tumors

Fig.14.10 Thedevelopment ofcolorectal cancer process is a muLtistage ofaccumuLating genetic errorsin cells. Theredarrows represent a newcriticaI mutation event, foLlowed byclonaIexpansion Atthe stageofcarcinoma, theprotiferating cetts contain at[thegenetic errorsthathave accumu[ated

The multistage process of the development of cancer is likely, of course, to be an oversimplification. The distinction between oncogenesand tumor suppressor genes(Table 14.3) has not always been clear-cut, e.g. the RE?. oncogene and MEN2 (p. 9l). In addition, the samemutation in some of the inherited cancer syndromes (p. 212) can result in cancers at various sites in different individuals, perhaps as a consequence of the effect of interactions with inherited polymorphic variation in a number of other genesor a variety of environmental agents.

208

vonHippel-Lrndau syndrome

VHL

3p25-26

Muttiple endocrine neoplas a type1l

RET

10q112

Breastcancer

BRCA2

13q12,13

Gastriccancer

CDHI

16q221

Further insight into the processesinvolved in the development of colorectal cancer came from a rare cause of familial colonic cancer known as familial adenomatous polyposis.

polyposis Famitiat adenomatous

more such alterations are seen with increasing frequency when adenomas progress in size and show histological features of malignancy. Over 90olo of carcinomas show two or more such alterations, and approximately 40o/oshow three.

Approximately lolo of persons who develop colorectal cancer do so through inheritance of an autosomal dominant disorder known as familial adenomatous polyposis (FAP). Affected persons develop numerous polyps of the large bowel, which can involve its entirety (Fig. 14.I I ). There is a high risk of carcinomatous change taking place in these polyps, with more than 90oloof persons with FAP eventually developing bowel cancer. The identification of an individual with FAP and an interstitial deletion of a particular region of the long arm of chromosome 5 (5q21) led to the demonstration of linkage of FAP to DNA markers in that region. Subsequent studies led to the isolation of the adenomatous polyposis coli (APQ gene.Analyses of the markers

GENETICS CANCER

colorectaIcancer Hereditarynon-polyposis A proportion of individuals with familial colonic cancermay have a small number of polyps, and the cancersoccur more frequently in the proximal, or right side,of the colon, which is sometimescalled 'site-specific'colonic cancer.The averageageof onset for colonic cancerin this condition is the mid-forties This familial cancerpredisposing svndrome is inherited as an autosomal dominant disorder and is known as hereditary non-polyposis colorectal canccr (HNPCC) - even though polyps may be present (the name helps to distinguish the condition from FAP). There is also a risk of small intestinal cancers,including stomach,endometrial cancerand a varietv of other cancers(seeThble 14.5).

DNAmismotchrepoirgenes Fig.14.11 n i t hp o l y p o sci so l o L a r g eb o w e t f r o m a p e r s ow i p e n eudpt o s h o w s yf M r P F l n a n . m u l t i p lpeo l y ptsh r o u g h otuhtec o t o n( C o u r t e o flon:rlmenl

n[(rrrnorrr 9L

'.

G onor:l vs'

lni rm:nr

I ood<)

linked to the APC genc in cancers from persons rvho have inherited the genefor this disorder haveshown LOH, suggesting a similar mechanism of gcnc action in thc dcvclopment of this tvpe of borvclcanccr Studies in the common, non-hereditary-fbrm of bowel cancer have shorvnsimilar LOH at 5q in the tumor material, with the FAP genebeing deleted in 407o and 70()/t,of sporadicalll' occurring adcnomasand carcinomasof thc colon LOH has also been reportcd at a number of different sites in colonic cancer tumors that include the regionsl8q2l-qter and 17pl2-l3,the lattcr region including the IP5J gene,as well as another geneat 5q21 known 'mutated in asthe colorcctalcancer'(AICC) gene,consistentwith the development of the common form of colonic cancerbeing a multistage process.

When looking for LOH, comparisonof polymorphic microsatellite markers in tumor tissue and constitutional cells in persons with HNPCC somewhat surprisingl-vrevealed the presenceof new rather than fewer allelesin the DNA from tumor tissue.In contrast to the site-specihcchromosomerearrangementsseenwith certain malignancies(p 199), this phenomenon, knorvn as microsatellite instu,bility(MSI) or replicarionrrrar (RER), is generalized, occurring rvith all microsatellite markers analyzed' irrespectir-e of their chrtrmosomallocation. This phenomenonwas recognizedto be similar to that seenin associationrvith mutations in genesknown asmutator genes,such asthe MutHLS genesin yeastand Escherichiurali. In addition, the human homolog of the mutator geneswere locatedin regions of the human chromosomesto which HNPCC had previouslybeen mapped, leading to rapid cloning of the genes responsible for HNPCC in humans (Thble 14 4). The mutator genes code for a slstem of'proof-reading' enzymes and are usually known x mismtttchrepair genes,which detect mismatched base pairs arising through errors in DNA replication or acquired causes, e.g.mutagens Individuals who inherit a mutation in one of the mismatch repair genesresponsiblefor HNPCC are constitutionally heterozygous

'Deletedin colorectaI cancer' Allele loss on chromosome lSq is seen in more than 70o/oof colorectalcarcinomas.The original candidategenefor this region, callcd deletedin colorectulcunter (DCC), has been identified and cloned; it has a high degreeof homolog-vwith the famill- of genes encodingcell adhcsionmolecules.Thc DCC geneis expressedin

r e n e sa s s o c j a t ewdi t h T a b t e1 4 . 4 M r s m a t crhe p a i g h e r e dt a r yn o n - p o l y p o sci so l o r e c t caaLn c e r HNPCC (%)

Humangene

Chromosomal E colihomolog locus

normal colonic mucosabut is cither reducedor absentin colorectal carcinomas.As with IP-iJ, somatic mutations in the DCC gene of the remaining allele occur in some cancers'wheregene

\MSH7

2p15-16

MutS

31

iMSH6

2p15-16

MutS

Rare

expression is absent. The known homology suggeststhat loss of DCC pla.vsa role in cell-cell and ccll basementmembrane intcractions, featuresthat are lost in overt malignancJ'Horvever, mutations in the DCC gene have been found in only a small

hMLHI

3p21

MutL

33

hPMSI

2q31

MutL

Rare

hPM52

7p22

MutL

4

proportion ofcolonic cancers.Other gcnesdeletedin this region in colorectal tumors incl,l.deDPC| (renamed S,l4AD1) andJ[/l I (renamedSMAD2\

Undelermrned [oc

-32

209

14

CANCER GENETICS

for a loss-of-function mutxtion (p. 25) Loss of function of the secondcopv through anl of the mcchanismsdiscussedin relation to LOH (p. 203) results in defectir.emismarch repair leading to

Autosomal dominant transmission is well described for a rare form of juvenile pol1,-posis that mal present in variety of ways,

an rncreasedmutation rate associatcduith an incrcasedrisk of developingmalignancl,.Certain germline mutations, ho\rever,seem to havedominant-negative effects Although HNPCC accountsfor a small proportion of colonic cancers,estimatedas2-4o/ooverall,

including bleedingwith anemir, pain, intussusceptionand failure to thrive. The polyps carry an approximate l3-fold increasedcancer

approximatelv 15o/oof all colorectal cancers exhibit MSI, the proportion being grcater in tumors from personswho developcd colorectalcancerat a vounger age.Some of theseindividuals rvill have inhcrited constitutional mutations in one of the mismatch repair gcnesin the absenccof a famill, history of colonic cancer. In addition, lbr women rvith a constitutional mismatch repair g;encmutation, the lif'etime risk of endomctrial cancer is up to 50o/o. Anall-.sisof tumor DNA for evidence of I,ISI has becomc a routine lirst test in caseswherc a diagnosis of HNPCC is a possibilitr, High levcls of X'ISI are suggiesrivcof the prescnce

the third decade,so that colectomyin adult life may be advisable. Two gcncs have been identilied as causative: SMADI (l8q), previouslv knorvn as DPC|, and BMPR|A (10q22). Both are

of HNPCC-related mutations in the tumor, some of rvhich uill be somatic in origin r,vhcreasin others there will be a germlinc mutation plus a 'sccond hit' in the normal allele.An additional techniquc, immunohistothemistr-1' (IHC), is also pror,ing useful as an invcstigationto discriminate those casessuitable for direct mutation analvsis Thking paraffin-cmbeddedtumor tissue, loss of expression of specilic mismatch repair genes can be tcsted using antibodiesagainstthc proreinshMSH2, hNILHl, hMSH2 and hPNIS2 Where tumor cclls fail to stain (in conrrasr to surrounding normal cells),a lossof expressionof that protein has occurred and direct genemutxtion anal-viscan be justilicd.

O T H E RP O L Y P OSS ISY N D R OME S Although isolated intestinal polvps arc commor, occurrlng even in about lolo of childrcn, there are familial forms of multiple polyposis that are distinct from trAP but shouing heterogeneitv

MYHpotyposis In a recent large study nearly' 2006 of f-amilial polvposis cases shorvedneither dominant inheritance nor evidence of an APC gene mutation Of thesefamilies more than 2Oolcr were found to havemutations in thc MYH gcne,and affectedindividuals werc compound heterozvgotes In contrast to thc other pol-vposis conditions described belor,, NIYH pol-vposisis, therefore, an autosomal recessive trait, thus signilicantly affecting genetic counselingaswell asthe needfor screcningin the rr ider f-amily.The gene,locatcd on chromosomeband 1p33, is thc human homolog

210

Juvenilepolyposissyndrome

of mutY in E toli. This bacterial mismarch repair operaresin ccrnjunction with mutM to correcr A,/G and A,/C base-pair mismatches.In tumors studicd, an excessof G:C toT:A trans\rerslons r.vasobservedin thc APC gcne. Mutations that cffectivell' knock outthe MYH gene,thereforc,lead to defectsin thc baseexcisionrepair path.w.av;this is a form of DNA mismatch repair that. unusualll.,follor,vsautosomalrecessiveinheritance

risk and, oncc diagnosed,regular surveillanceand polvpectomv should bc undertaken.The averageageat diagnosisofcancer is in

components of the TGF-B signaling pathway and SA4AD'| mutations,rvhich accountfor about 600loofcases,appearto carry a higher malignancypotential and the possibilityof largenumbers of gastric polvps

Cowdendisease Also known as multiltle humartomas.yndrome, Cowden diseaseis autosomal dominant but very variable Gastrointestinal polvps are found in about half of the casesand are generally benign hamartomasor adenomas.Multiple lipomas occur with similar frequencJ'and the oral mucosamay havea 'cobblestone'appearance Ntlacrocephaly is common in this condition. Importantlv. however, there is a high incidence (50o/o)of breast cancer in females, usuallv occurring at a ]roungage,and papillary-th-vroidcarcinoma affects about 7olcrof the patients. Mutations in the tumor suppressor PTEN gene on chromosome 10q23, encoding a tvrosine phosphatase, causeCowden disease.A relatedphenotlpe with manl'overlapping features,rvhich glories in the eponl,mous name Bannavan-Rile1--Ruvalcabasyndrome, has also been shown to be due to mutations in PTEN in a large proportion of cases.

Peutz-Jegher syndrome Also autosomaldominant, this condition is characterizedby the prcsenceof dark mclanin spots on the lips, around the mouth, on the palms and plantar areas,and other extremities.These are usuallv prcscnt in childhood and can fade in adult life. Patients often prescnt with colicky abdominal pain from childhood due to the devclopmentof multiple polvps rhar occur throughout the gastrointcstinaltract, although thev aremost common in the small intcstine.These are hamartomasbut there is a significant risk of malignant transformation There is an increasedrisk of cancersat othcr sites,particularlv breast,uterus, ovary and testis,and these tend to occur in earlvadult life. Regularscreeningfor thesecancers throughout life, from carly adulthood, is warranted. Mutations in a novel serine threonine kinase gene, SIK11, located on chromosome 19p,causePJS.

BREAST CANCER Approximately-1 in 12 r,vomenin Western societiesr,villdevelop breast cancer, this being the most common cancer ln women betrveen,f0 and 551,earsof age,rvith approximatell,' I in 3 affected

CANCER GENETICS

women going on to developmctxstaticdisease.Some l5 20o/rtol Nomen who develop breast cancer har,ea familv histor-vof the disorder Famill' studies have shown that thc risk of a \\iomxn developing breast cancer is greater when one or morc of the follorving factors is present in the familv historv: (1) a clustering of casesin close female relatives;(2) earll' age (<50vears) of presentation;(3) the occurrcnce of bilateral disease;(4) the additionaloccurrenceof ovariancanccr Molecular studies of breast cancer tumors have revealed a varietv of different lindings that included amplificationof erb-81, erb-82, tq)tcand int-Z oncogenesas well as LOH at a number of chromosomalsites,including (in descendingorder of frequencl.) 7q, l6q, l3q, l7p, 8p, Zlq, 3p, lSq, 2q and l9p, as rrell as scveral other regions vr,ithknotvn candidate genesor fragile sites.In man1. breasttumors showingLOH, allelelossoccursat t$,o to four of the sites,againsugBestingthat the accumulationof alterations,rather than their order, is important in the evolution of breast cancer One potentiallvkev elementin the developmentof sporadicbreast

O

RIANCANCER

Approximatcl--v1 in 70 women develops ovarian cancer, the incidence increasing with age.The majoritl' arise as a result of gcnctic alterationswithin the ovariansurfaceepithelium and are therelbrc rcferred to as epitheliolovarian cancer.In generalit is a poorlv understood disease,although recent studieshave shown a high frequency-ofLOH at 11q25in tumor tissue.A possible tumor suppressorgeneat this locus is OPCML, which encodesa cell adhesionmoleculethat includesan immunoglobulin domain. Approximatell' 5o/oof rvomen with ovarian cancer have a family historv of thc disorder and it is estimatedthat 1oloof all ovarian cancer fcrllorvsdominant inheritance becauseit is strongl-v prcdisposedb1'single-genemutations. In families with multiple \\iomen affected with ovarian cancer, the age of presentationis l0-1-5years carlier than with non-hereditary ovarian cancer in the generalpopulation. Mutations in BRCAI, BRCA2, and less commonlv the genesresponsiblefor HNPCC, are responsiblein a proportion of thesefamilies,but a susceptibility genelocus for

cancer,and sporadic ovarian cancer,is a neulv discovercdgene named EM.9Y This rras fbund to bc amplilied in 13or'o of breast cancers rnd 17o/oof ovarian cancers,and was ascertaincdrvhen looking for DNA sequencesthat interact with BRCA2.The

site-specificovariancancerhas yet to be identified.

normal function of EMSY mav be to switch off BRCA2; this ma-vpoint to an important pathrvavof control of ccll gror,th in thesetissues.

Prostatecancer is the most common cancer overall after breast clncer, and is thc most common canceraffectingmen; mean have a lif-ctime risk of l0o/o of developing prostate cancer and a 3olo chanccof df ing from it Enquiries into the family history of males presentingwith prostatecancerhaverevealeda significantproportion

BRCAIand BRCA2genes Famil--vstudies of earl-v-onsetor premcnopausal breast cancer shorvedthat it behaved like a dominant trait in manr, families Linkage anal-vsisin these families shorvedthat thc rendencv to develop breast cancer mapped to the long arm of chromosome 17, eventually leading to identification of thc BRCAI gene. A proportion of families rvith earlv-onset breast cancer rhat did not shorvlinkage to this region shorvedlinkage to the long arm of chromosome 13, resulting in the idcntification of the BRCA2 gene. Approximatclv 40-507o of families with earlv-onsetautosomal dominant breastcancerhar.ea mutation in thc BRCAI geneand have been shorvn to have a 60 85o/olifetime risk of developing brcast cancer.Femalestyrth a BRCAI mutation havean increased risk of developingovariancanceq and males an increasedrisk of developing prostate cancer.N{utations in thc BRCA2 geneaccount

PROS

E CANCER

(about 1-5o/o) to have a first-degree male relative with prostate cancer.Familv studieshaveshorvnthat first-degreemale relatir.es of a man prcsenting with prostatecancer have between two and Iivc times the population risk of developingprostatecancer. Anal-vsisof prostatecancer tumor material has revealedLOH of famil.v irt sevcralchromosomallocations.Segregation analy-sis studies of prostate cancer suggestedthat a single dominant susceptibilit),locus could be responsible,accounting for 9o/oof all prostatecancersand up to 40oloof early-onsetprostatecancers (diagnoscdunder age551'ears). Linkage analysisstudiesidentified nr,o major susceptibilit.vloci, hereditary prostatecancer-l and -2 (F{PCI and HPC2), and a number of other more minor susceptibility loci har.ebeenreported. Recentll'mutationsin the ribonucleaseL

der,elopingovariancancer,and malesan increasedrisk ofprostatc cancer In some of the original familial breast cancer families recruited for linkage studies,a number had maleslvho der.eloped breastcancer.Although breastcancerin malesis verl'rare, males

gcnc (RrVlSEl) were identified in trvo families showing linkage to thc HPC1 locus at 1q25. Mutations have been found in the EL.1C2 geneat 17p11,the HPC2 locus,and, rarel1,,mutations in have been identified threc genes - PTEN, ,MXII and KAII in a minoritv of families with familial prostate cancer.A small proportion of familial prostatecanceris associatedwith BRCAI or BRCA2. N{enu'hocarrvmutations inetther BRCAI or BRCA2 har.ean increasedrisk, and in one study; conducted in Ashkenazi Jervs,mcn rvith such mutations had a 160lorisk of prostatecancer b-vage70 years,comparcd with 3.8o/ofor the generalpopulation. Although the majority of prostatecancersoccur in men aged over 65y-ears,individuals with a famill- history of prostate

with mutations in the BRCA2 gene have a 6(% lifetime risk of developingbreastcancer)approximatel-v a 100-foldincreasein the population risk of brcast cancerin males

canccr consistentrvith the possibilit-vof a dominant gene being responsiblehar,ea significantlvincreasedrisk ofdevelopingprostate cancerat a relatively -youngerage (lessthan 55y-ears)Screening

for 30-40o/o of families u'ith early-onset autosomal dominant breast cancer,and the lifetime risk of developingbreastcanceris similar.Although initially mutations inrhe BRCA2 gene\rere not thought to be associatedrvith an increasedrisk of other cancers, \\iomenhcterozvgousfor a mutation alsohavean increasedrisk of

211

14

CANCER GENETICS

bv measuring prostate-specilicantigen (PSA) levcls has been suggested,and is often offered, but problems with specificitl'and sensitir.itvmcan that interpretation of results is diflicult.

G E N E TC I CO U N S E L IINNG M I L I A L CANCER Recognition of individuals with an inherited susceptibilitr to canccrusuallvrelieson taking a carefulfamil-vhistory to document the presenceor absenceof other family members with similar or relatcd cancers.Thc malignanciesthat develop in susceptible individualsare often the sameasthosethat occur in the population in general Thcre is a number ofother fcaturesthat can suggestan inherited canccr susceptibility svndromc in a famil-v(Bor 1'1l)

I N H E R I T ECDA N C E R . P R E D I S P O S I N G SYNDROMES Although most cancersduc to an inherited cancersyndromeoccur at r specific sitc, families have been described in rvhich canccrs occur lrt morc than one site in an individual or at diffcrcnt sites in vlrious members of thc familr, more commonlv than rvould bc cxpected Thcsc f'amiliesare referred to as having a familial cancer-predisposing svndrome Thc majorit--v of the rare inherited f'amilialcanccr-predisposingsvndromescurrenth. recognizedare dominantlv inherited, rvith offspring of affected individuals having a 50o/i,,chance of inheriting the gene and therefore of being at increasedrisk of developingcancer (Thble 11 5) Thcre is also a number of svndromes, usuallv inherited as autosomal r e c e s s i v cd i s o r d e r s , r v i t h a n i n c r e a s e dr i s k o f d e v e l o p i n g

svndrome at a strikinglv ].oung age.The cancer-predisposing HNPCC used to be separatedclinicall-vinto Lynch tvpe I, in rvhich family' members appear to be at risk onlv for cancer of thc colon, and L1'nch t1'peII (sometimes,confusingly,known ^s the cuncerrttmil1, ;yrlrrrtt), in which family members are also at risk for a number of other cancers,including stomach, cndomctrial, breast and renal transitional cell carclnomas. Hon'ever, progress at the molecular levcl has highlighted the difliculty' of the L1'nch classificationas individuals from families nith both t1'pe I and II characteristicshave been found to har.e mutationsin an1-oneof the mismatchrepair genes(seeThble14.,t). Furthermore, Turcot syndromc is due to mutations in the APC geneand two of the mismatch repair genes,rvhereasMuir-Torre srndrome resultsfrom mutations inthe hMSH2 mismatch repair gene. Individuals at risk in such familics should, however, be screenedfor the appropriatecancers.

RT H E I N H E R I T ESD U S C E P T I B I LFI O TY C O M M OCNA N C E R S The majorit.vof personsat an increasedrisk of developingcancer becauseof their famih. historr, do not have one of the cancerpredisposingsvndromes.The levelof risk for personswith a famill' historr, of one of the common cancerssuch as bowel or breast cancerdcpcndson a numbcr of Ihctors Thcse include the number of persons rvith cancer in the famil-1',horv closelv related the person at risk is to the affectedindir,iduals,and thc age at which

cancer associatedr,vith an increased number of abnormalities in the chromosomeswhcn cultured, or rvhat are knor,vnas thc chromosomalbreakagcsr,,ndromes. These are discussedin dctail in Chapter 18 (p.277).

the affected famil-v-. member(s) dcveloped canccr A few families rvith a largc numbcr of mcmbers affected u.ith one of the

P c r s o n s r ri t h a n i n h e r i t e d f a m i l i a l c a n c c r - p r e d i s p o s i n g svndrome are at risk of developing a second tumor (multifocal or bilatcrrl in the cascofbreast cancer),havean incrcascdrisk of developinga cancerat a relatir.elr-voungeragethan pcrsons rvith the sporadicform, and can developtumors at differcnt sitesin the

individuals rvith cancerin a famil1,,and there is doubt about whether a cancer susceptibilit.v gene is responsible or not. In such an instanceone relieson empirical data gainedfrom epidemiological studiesto provide risk estimates(Tables1,1.6& 1,+.7)

bsdr', although one tvpe of canccr is usuallv predominant.

S C R E E N I NFGO R M I L I A LC A N C E R

B o x 4 . 1 F e a t u r essu g g e svte o f a n i n h e r r t ecda n c e r suscepLibitity synoromer a fa'n Ly

212

A number of different familial cancer-predisposing svndromes havebeendescribed,dependingon the patternsofcanceroccurring in a familli For example,personsr'viththe Li-Fraumeni syndrome (p. 205) are at risk of developingadrcnocorticaltumors, soft-tissue sarcomas,breastcancer,brain tumors and leukemia sometimes

( f r s t o r s e c o n d - d e g r e er)e t a tv e s w r t ha c o m m o n SeveraIclose canceT S e v e r a cI l o s er e t a tv e s w i t h r e t a t e dc a n c e T se g b r e a s ta n d o v a T yo r b o w e Ia n d e n d o m e t r i a l T w of a m t y m e m b e r sw i t h t h e s a m e r a T ec a n c e T A n u n u s u a t t ye a r l ya g e o f o n s e t B r l a t e r a t t u m o rrsn p a i r e do r g a n s S y n c h r o n o uo s Ts u c c e s s l vteu m o T s u ' n o rs i r r , n uooi l l e r e rLo r g a r s y c t e ' r s r o r e , n o i vo u a ,

common cancersare consistentrvith a dominantlv inherited cancer susceptibilitl. gene. In most instances there arc onlv a few

Preventionor earlvdetectionofcancer is the ultimate goalofscreening individuals at risk of familial cancer The mcans of prevention for certain cancerscan includc a changein lifestyle or diet, drug therap).prophllactic surger.\or screening. Screening of persons at risk of familial cancer is usually directed at detecting thc phenotl.pic expressionofthe genotvpe, i.e. surveillancefor a particular canceror its precursor Screening can alsoinclude diagnostic teststhat indirectl.v revealthe genotype, looking for other clinical featuresthat are er-idenceofthc presence or absenceof the gene For example,individuals at risk for trAP can be screenedfor evidenceofthe IPC geneby retinal examination looking for areasof rongenital /zypertrophyof the retinal pigment

C A N C EG RE N E T I C S

Tabte14.5 Syndrome

Modeof inheritance

Gene

Chromosomal site

Main cancer(s)

Breast/ovary famities

AD

BRCAI

11q21

Breast.ovarycoton,prostate

Breastfamilies

AD

BRCA2

13q12

Breast,ovary

Famrtiat potyposis adenomatous

AD

APC

\n?l

duodena[, thyroid Cotorecta[,

Turcotsyndrome

AD

APC hMLHI hMSH2

5q21 3p21

brain Colorecta[,

Hereditary non-potyposis (HNPCC) cotorectaI cancer LynchI

Lynchll

AD

AD

hMSH2 hMSH6 hMLHI hPMSI hPM52

)nl\-16

I Colorecta

3p21 2q31 7n))

hMSH2 hMLHI hPMSI hPMS2

2p15-16 3p21 2q31 7p22

urinarytract.ovarian, Colorectat, endometria[, g a s t r r cs, m a t [b o w e l h e p a t o b i l i a r y

MYHpolyposis

AD

MYH

1p33

Muir-Torre

AD

hMSH2

2p15-16

tumors,laryngeal As Lynchll plussebaceous

potyposrs Juvenite

AD

SMAD4/DPC4 BMPR1A

18q211 10q22

Colorectal

P e rr t z - l c n h e r s

AD

STKll

19p13 3

Gastrointestinal, breast,uterus.ovarytestrs

C o w d e nd i s e a s e

AD

PTEN

10q23

Famitral retinoblastoma

AD

RBl

13q14

Retinoblastoma

Li-Fraumeni

AD

TP53

17o13

adrenalcortex breast,brain,leukemta, Sarcoma,

Multiple endocrine neoptasia (MEN) Typel(MEN1)

AD

MEN1

11q13

T y p el l ( M E N 2 )

AD

RET

"lOq11 2

pancreatic thyroid, anterorpituitary, Parathyroid. istetce[Ls, adrenat (medutlary). pheochromocytoma Thyroid

von Hrppe[-Lindau

AD

VHL

3p25-26

renal,pancreatic, CNShemangioblastoma, pheochromocytoma

basalcetl Gorlin(nevoid carcinoma)

AD

PTCH

9q22

meduttobtastoma carcnomas, syndrome Basatcetl (odontogenrc keratocysts) fibromas, ovarran

Dysptastic nevussyndrome

AD

CMMl

1p

t oleme[anoma, M e t a n o m a( f a m i [ i aaI t y p i c am FAMM)

AD,autosomal domnant,CNScentraL nervous svstem

epithelium, or what is knownas CHRPEs.The finding of CHRPEs increasesthe likelihood ofan individual at risk being heterozygous for the APC gene and therefore developing polyposis and malienancv. We now know that CHRPEs are seenin oersons with

FAP when mutations occur in the first part of the APC gene,an exampleof a genotype-phenotypecorrelation (p. 26). More recently identification of the gene responsible for a number of the cancer-predisposingsyndromes,and determination

213

14

C A N C EG RE N E T I C S

Tabte14.6 Lifetimerrskof colorectal cancerfor an 'rdrrrrdr,: "ulvluuot

: r Lr n ' r In 1 9 lLnU lLh loL F : m i l r r h i c t n l r nC r I l t rLrdnl a L O L -Ur I U uf 6 L6 u lr nu 6r pu lut rr d y n

Poputatron risk

1 in 50

O n ef r r s t - d e g r e er e t a t i v ea f f e c t e d

1in17

O n ef i r s t - d e g r e er e t a t i v ea n d o n e s e c o n d - d e g r e e relattve affected

1in12

Oneretative agedunderage 45years affected

1 in10

Twofirst-degree relatrves affecied

1 in 6

Three or mnre {rrst-dporperelativesaffected

1in2

National Health Service,although it is important to appreciate that this is an evolving area and screening recommendations are subject to change. Furthermore, individualized screening strategies are often devised for women from families rvith BRCAI, BRCA2 and TP53, as well as families with a high risk of colorectalcancer.

Familiatcancer-predisposing syndromes

FromHoutston R S,MurdayVHarocopos C Wrlliams CB,SlackJ 1990Screenrng andgenetrc counsetting forretatives of patients with colorectat cancer in a familyscreening ctlnicBr MedJ 301:366-368

Many familial cancer-predisposingsyndromes are inherited as autosomal dominant traits that are fully penetrant, with the consequent risk for heterozygotesof developing cancer approaching l00o/o.This level of risk means that more invasive means of screeningu'ith more frequent and earlier initiation of screeninBprotocols are justifred than would be acceptablefor the population in general(seeTable 14.9).

Inheritedsusceptibitity for the commoncancers Screening for the common cancers arising from inherited susceptibilitl has only relativelyrecently been establishedand by its very natureinvolr,esa long-term undertakingfor the individual at risk as well as his or her phl.sicianor surgeon It is important

Tabte14.7 Lifetrme lsk of breastcancern females +^ tnnr nf hror
Poputation risk

1in12

Sisterdiagnosed at65-70years of age

1 in 8

Sisterdiagnosedunder4Oyearsofage

1tn4

Twofirst-degreerelatives affected under40yearsof age

1in 3

to emphasizethat the natural enthusiasmfor screeningneeds to be balancedwith the paucity of hard data in many instanceson the relativebenelits and risks. Horvever,recommendedscreening protocols are increasingly evidencebased as more data become available(Box 14.2).

Who shouldbe screened? of the genotypic status,i e. presymptomatic testing (p 306), of an individual at risk allorvsmore efficient deliver-vof surveillance screening for the phenotl'pic expression, e.g. renal cancer, central nervous system tumors and pheochromocvtomasin \on Hippel-Lindau (VHL) disease(Thble 1,t.8).For those who tesr negativefor the family mutation, expensiveand time-consuming screeningis unnecessar\.. As more genesfor cancersusceptibilitlr are discoveredthere rvill be an increasingnumber of conditions for which DNA testingwill enablepresymptomaticdetermination of genotypic status. Although the potential for prevention of cancer through screening persons at high risk is considerable,it is important to remember that the impact on the overall rate of cancer in the population in general will be small as only a minoritv of all common cancers are due to gene mutations that follor,r. straightforwardmendelianinheritance.For manv familial cancers there hasbeen a strong move towardsnationally agreedscreening protocols, especiallvin countries such as the UK where the bulk of health care is provided b-v the state The provision of screeningmust increasinglvbe evidencebasedlr,ith demonstrable cost-benefits In the UK, screeningguidelines produced by the National Institute for Health and Clinical Excellence (NICE)

214

are seenas broadly determining what is availablewithin the UK

In the caseof the rare, dominantly inherited, single-genefamilial cancer-predisposingsvndromessuch as FAP,VHL and multiple endocrine neoplasia(MEN), those who should be screened can be identified on a simple mendelian basis. However, for retinoblastoma,for example,the situation is more complex. If no R-81mutation hasbeenidentified,presvmptomaticgeneticresting cannot be offered. Some individuals with the non-hereditary form havebilateral tumors, u'hereassome with the hereditary form have no tumor (i.e.the condition is non-penetrant)or a unilateraltumor. It ma-vbe impossible to distinguish which form is present, and screeningof second-degree,as well as first-degree,relativesmay be appropriategiven that early detectioncan successfullyprevent blindness.For personswith a family historv of the common cancers, such as bowel or breast cancer,the risk levelsat which screening is recommended,and below which screeningis not likely to be of benefit, will var1,At each extreme of risk the decision is usually straightforward, but with intermediate-levelrisks there can be doubt as to relativebenefitsand risks of screenins.

What ageand how often? Cancer in personsw,ith a familial cancer-predisposingsyndrome tends to occur at a relativel-vearlier age than in the general population and screeningprograms must reflect this. With the

C A N C EG RF N F T I C S

T a b t e1 / + . 8 S r g g e s t esdc r e e r--r gg u r d erIe s ' o - p e - s o n a s t s , g n .cf a r l r s z o ' c a n c e rl a l l c a n L e ' - p r e d i s orogs : 'nia a n 0c 0 m m 0 nc a n c e r s Condition/cancer

Screeningtest

Frequency

Starting age (years)

Familialsusceptibitityfor the common cancers Breast concer Breast

Mammography

Annual

40-50(3yearlyfrom50 untessvery or BRCA2gere h ghr s^.eg BRCAI mutation carrer)

Breast/ovory Breast Ovary

Mammography U5/Doppler: CA125

Annual Annual

40-50(asabove) revrew/ J5 lUn0eT

- LynchI HNPCC riskfamilies Colorectal-high

Colonoscopy

2-3yearty

Cotorectalintermediate rrskfamilies

Cotonoscopy

or Atfirstconsultation age35-4Oyears

25or5yearsbeforetheearlrest inthefamiLy diagnosis 55 Repeatatage

Cotonoscopy US(underevatuation)

for LynchI As above.

US US Gastroscopy

for LynchI As above, Annual AnnuaI AnnuaI 2 yearly

None None Mammography

Annual

40-50

AnnuaI 3 yearty Annuat

12 20 20

HNPCC- Lynch ll Cotorectal EndometrraI Ovary RenaI tract Gastric SmattboweI H cn:tnhilia rrr

Breast Familialcancer-predisposing syndromes Fomili oI odenomotous polyp osis CotorectaI DuodenaI Thyroid(women)

(CHRPE)" RetinaI examination Sinmnid/rnlnnncronvo!

Gasiroscopy None/US?

35-65 35 35 2 5 i d e f i n i t eL y n c hl ' s y n d - o n e

Childhood

Li-Froumeni Breast Sarcoma Brain Leukemia AdrenaIcortex

Mammography None None None None

AnnuaL

40?

Retinoblostomo

RetinaI examination

Frequentty

Frombirth

C a 2 * ,P T H ,p i t u i t a r yh o r m o n e s p a n c r e a l cn o r m o n e s Calcitoninprovocationtesto

AnnuaI

R rrorrc

US Urinary VMA cal. Poo,PTH

? Annual AnnuaI

RetrnaI examinat ono CNSCT/MRI UrinaryVMA Abdominat CT A b d o m l nU a tS

AnnuaI 3 yearly Annual 3 yearty AnnuaI

M u lti ple en docrine neo plasia Type1 Iype2 Medultary thyroid Pheochromocytoma Par:ihrrrnid:dcnnm:

von Hippel-Lindau angioma Retinal Hemangioblastoma Pheochromocytoma Renal

r rn fn :nc

50 vp:rc

'10 '10 10 10

5

\

5 ( 5 y e a r l yr r o m a g e4 u y e a r s /

10 20 2A Toblecontinued

215

14

CANCER GENETICS

T a b t e1 4 . 8 S u g g e s t esdc r e e r i n g J t d er[e s f o -p e r s o r -ast s i g r i ' i c a nr ts k o rc a n c e -' a: r r i L i a l c a n c e r - p f e d i s p os sy inndgr o m e s a l d c o n n . o nc a n c e r(sc o n i d ) Condition/cancer

Screeningtest

Gorlin (nevoid hasol cell carcinomo)syndrome BCCs Ctrnical surveittance Medultobtastoma Clinical surveittance 0dontogenic keratocysts Orthpaniomography

Frequency

Startingage (years)

Annual AnnuaI 6 monthty

10 Infancy 10

'Testto detectheterozygous state olnrndividuals foundto beaffected, pnortocotectomy annuat cotonoscopy andlifelong 4-6-monthly surveitlance oftherectalstump, aftersubtotal cotectom;r U5,ultrasonography; CHRPE, congenitalhypertrophy oftherelinalprgment epthetrum; CT,computed tomography: MRl.magnetic resonance imaging; PTH parathyroid hormone;VMA, vani[[y[ mandelic acid;BCC, basalcetlcarcrnoma

B o x 1 1 . 2 R e q u i r e m e not sf a s c r e e n i ntge s tf o r p e l s o n s a t r i s kf o ra f a m l i a tc a n c e r - p r e d i s pnogss y n d r o m o er a t i n c r e a s erdi s kf o rt h ec o m n f o nc a n c e r s -\e

l e s ts r o u l o d e - e c at - n a [g n a ^ l o r o r e - n a g . n a n rc o r o i to 1 o l q s t s g s p r i o rt o i t s p r o d u c i n gs y m p t o m sw l t h h i g h s e n s i tv t y a n d s p e cf i c r t y T h e t r e a t m e n to f p e r s o n sd e t e c t e db y s c r e e nn g s h o u t di m p r o v et h e pr0gnosrs T h e b e n e f io t f e a r l yd e t e c t i o ns h o u t do u t w e i g hp o t e n ta I h a r m f r o m t h e s c r e e n l n gt e s t T h e t e s ts h o u l do r e f e r a b l b v e n o n - i n v a sv e a s m o s t a t r r s kr n d i v i d u a t s r e q ur e l o n g t e r m s u r v e t t l a n c e A d e q u a t ep r o v i s r o fno r p r e s c r e e n r ncqo u n s e ln g a n d f o l o w u p s h o u t d be availabte

exceptionof FAI in which it is recommendedthat sigmoidoscopv to detect rectal polyps should start in the teenage\€ars)most cancer screeningprograms do not start until 25yearsof age or later. The highest-risk agc band for mosr inherited suscepribiliriesis 35-50vears, but becausecancercan still develop in thosc at risk at a later age,screeningis usually continued thereaftcr.In some families the age of onset of cancer can be especialll.earl-vand it is recommended that screening of at-risk individuals in these families commences 5 vears before the age of onset in the earliest affected member of the familli Again, retinoblastomais an exception to the usual rule because,as it is a cancer of earl-v childhood, screeningstarts in the postnatalperiod with frequcnt o p h t h a l m i ce r a m i n a r i o n . The recommended interval betrveen repeatcd screening proceduresshould be determined from the natural histor.vof the particular cancer.The developmentof colorectalcancerfrom an

215

What sitesshouldbe screened? Having decided who is at risk within a famill', one has to judge which tvpes of cancerare most likel-vto occur and rrhich svstems of the bodr, should be screened

Fomiliol concer- predisposing syndro mes This can be a verv difficult problem with somc of the familv cancer svndromes,such as the old L.vnch tvpe II form of HNPCC, in rvhich a person at risk can developcancerat a number of different sites Screening for ever.vpossiblecancer that can occur rvould mean frequent investigationby a varietv of different specialists and/or investigationsThis would result in an unr,ieldv and u n p l e a s a n tp r o t o c o l . P e r s o n s a t r i s k f o r H N P C C s h o u l d have regular colonoscopl', and females may be offered pelr,ic screening for g-vnecological malignancies,although the eflicacy is ver-vdebatable.Some of the other cancersthat can occur in persons at risk for L1-nch t-rpe II, such as stomach cancer,are not sccn in everv famill', and so screening is usuallv restricted to persons from those families in which these cancers have affected at lcast one famil-v member In persons at risk for Li-Fraumeni syndrome, a llide spectrum of cancerscan occur However, apart from regular mammographv, no satisfactory screening is available for the other malignancies (seeTable 14.8).

lnheritedsusceptibility for thecommonconcers ColorectaI cancer

adenomais believedto take place over a number of vears,and as a result it is thought that 5-vear screeningintervals will sufficc

Colorectal carcinoma holds the greatcstpromise for prer.ention bv scrccning Endoscop-vprovides a sensitiveand specificmeans

If, however, a pol1.p is found, the interval betrveen screening proceduresis usuall;-brought down to 3vears Breast cancer is not detectablein a premalignant stage and earlv diagnosisis critical if there is to bc a good prognosis.Annual mammographv for femalcs at high risk is therefore recommendedfrom the age

of examination of the colorectal mucosa and polvpectomv can be carried out $,ith relatir,eeaseso that screening,diagnosisand treatment can take place concurrentiv Although colonoscopvis the preferred screening method, it requires a skilled operator and, as it is an invasiveprocedure, it has a small but consequent morbiditli Becauseof this, and in order to target screening to

of 35 r,ears.

C A N C EG RE N E T I C S

thosemost likelv to benefit,most geneticcentershaveadoptedthe so-calledAmsterdamcriteriato sclecthigh-risk individuals These minimal criteria suggesta familial form of colonic cancer: I At least three affected relatir.es(related ro one another bv lirst-degree relationships),one a lirst-dcgree relativeofthe other two; FAP excluded. 2 At leasttrvo successivcgenerationsaffected 3 . C a n c e r d i a g n o s e db e f o r e a g e 5 0 v e a r s i n a t l e a s t o n e relative. Failure to visualize the right side of the colon with colonoscopv neccssitatcsa barium enema to vierv this region, particularly in persons at risk for HNPCC, in which proximal right-sided involvement commonlv occurs. For pcrsons r,vith a moderatelv increased risk of developing colorectal cancer, the majoritv of cancersoccur in the distal (left-sided) colon and at a relativelv latcr age Flcxible sigmoidoscopl',u'hich is much less invasir,e providesan adequatescreeningtool for persons than colonoscopr,, in this risk group and can be emploved from the ageof 50years.

Breastcancer In the UK scrccning of rvomen aged 50-vearsand over for breast cancer bv regular mammographv has become establishedas a national program as a result of studies demonstrating improved surr,ival of uomen detected as having carlv breast cancer. For women with an increased risk of developing breast cancer becauseof their family historl', there is conflicting evidence of the relatir.ebenefit of screening rvith respect to the frequencv of mammograph-vand the chanceof developingbreast cancerin thc intcrval bctt,een the screeningprocedures,or rvhatis tcrmcd interval cancer.One reasonis that cancerdetectionratesare lowcr in premenopausalthan in postmenopausalbreast tissue. It is alsoarguedthat the radiation exposureassociatedwith annual mammography could be detrimental if started at an early age, leadingto an increascdrisk ofbreast cancerthrough the screening rvhen carried out o\rera long period of time. This is of particular concern in families s'ith Li Fraumeni svndrome, because mutations in the IP.iJ gene have been shown experimentalll. in r:itro to impair the repair of DNA damagedby X-irradiation. Holvever, most experts currently believe that there is a greater relativebcneht than risk in identifying and treating breastcancer in rvomenfrom this high-risk group, although formal evaluation of such screeningprograms continues. N{ammograph-vis usualll'offered onl-vto women at increased risk of breastcancerafter the ageof 35 years,as interpretation of mammograms is difficult before this age becauseof the densitl, of the breasts.As a consequence,women at increased risk of developingbreastcancershould be taught breastself-examination and undergo regular clinical examination.

Ovariancancer Ovarian cancerin the earll'stagesis frequentl-vasymptomaticand often incurable by the time a woman presentswith symptoms

Earll' diagnosisof ovarian cancer in individuals at high risk is vital, rvith proph-vlacticoophorectomy being the only logical, if radical, alternative The position of the ovarieswithin the pelvis makes screening difficult Ultrasonography provides the most sensitivemeans of screening.tansvaginal scanning is more sensitive than conventional transabdominal scanning, and the usc of color Doppler blood flow imaging further enhances scrccning of rvomen at increased risk If a suspicious feature is seen on scanning and conlirmed on further investigation, laparoscop-vor a lxparotomy is usually required to confirm the diagnosis Screening should be carried out annually as interval cancers can develop if screening is carried out less frcquentlli Nllcasuringthe levels of CAl25, an antigenic determinant of a glvcoprotein that is present in increased levels in the blood of women with ovarian cancer,can be also be used as a screeningtest for women at increasedrisk of developingovarian cancer.CA125 levels are not specific to ovarian cancer,as they arc also increasedin women with a number of other disorders, such as endometriosis In addition, there are problems with sensitivitv (p 309), as CAl25 levels are not necessarilv increasedin all women with ovarian cancer.Becauseof the problemsoutlined with thesevariousscreeningmodalities,many \romen with an increasedrisk of developing ovarlan cancer choose to har.e their ovaries removed prophylactically once their familf is complete. However, this in turn raises the issue o f t h e b e n e f i t s a n d r i s k s a s s o c i a t e dw i t h t a k i n g h o r m o n e replaccmenttherapy'.

Surgical intervention is the treatment of choice for persons at risk for some of the familial cancer-predisposing s-vndromes, e g prophl-lactic thyroidectomy in MEN tvpe 2 (especially IIEN2B) or colectoml,in FAP For personswith a high risk due to an inherited susceptibilit-vfor one of the common cancers (e g colon or breast/ovary), prophylactic surger)ris also an acceptedoption but the decisionis more complex and dependent on the individual patient's choice. The option of prophylactic mastectomvin rvomenat high risk of developingbreastcanceris ver-vappealingto some patients but totall-vabhorrent to others, and alternativemanagementin the form of frequent surveillance, and possibly-drugssuch as the anti-estrogentamoxifen, can be offered For patients at high risk of colonic cancet dietar-v modihcation such as the use of non-digestiblestarch, or the use of drugs such as the aspirin-like non-steroidalanti-inflammatorv sulindac,ma-vhave value (Table 14.9). Pcrsons at an increasedrisk of developing cancer,especially if it is one of the single-genedominant cancer-predisposing svndromes,or one of the single-genecausesof the common cancers,find themseh'esin an unenviable situation concerning both their health and the possibilit.vof transmitting the condition to their children. Unfortunately, they are also likely in future to cxperienceincreasing difficulties in other areasof life, such as insuranceand employment (p. 359)

217

14

CANCER GENETICS

Acceptedtreatment Familialadenomatouspotyposis Totalcotectomy Ovarian cancer families Oophorectomy Breastcancer famIes Bi.arera, n'rastectory MEN2 Totalthyroidectomy Underevaluation Fa'n,la[adeno.natoL]s potyposrs

Li F P, Fraumeni J F 1969Soft tissuesarcomas,breast cancer, and other neoplasms:a familial syndromel Ann Intern Med 7l:747J52 The original descriptionof the Li-Fraumeni ryndrome. Lynch H T 1967'Cancer families': adenocarcinomas(endometrial and colon carcinoma)and multiple primary malignant neoplasms RecentResultsCancerRes 12:125-142 Thedescriptionof the cancerfomily syndromenow knownas Lynch II. Offit K 1998Clinical cancergenetics Wiley-Liss, Chichester Textcoaeringthe clinical aspecxofthe aariousfomilial cancer-gred.isposing syndromes as pell as the commln cancers,alongwith the basiccellularbiologjt and ethital and legalaspecx. VolgelsteinB, Kinzler K W 2002The genetic basisof human cancer. McGraw-Hill, London Verycomprehensiae boobcoaeringin detail the cellularbiologyofcancerand,the clinical aspectsof thefomilial cancer-lredisposing syndromes and,thefamilial c0mm0ncancers

Non-digestibte starch to deLay nn
nf nnhrnncie

S u t , n d a -c t o T e o L c eT e c l aa. n o d u o d e n aaI d e n o m a s B r e a s tc a n c e rf a m r l i e s

- to prevent Tamoxifen j e y c l n p r r e r - lo f h r e a s tc a n c e T A v o i d a n coef o ' a I c o n t ' a c e p r i v e s a n d h o r m o n er e p l a c e m e n t therapy

FURTHER READING CorvellJ K (ed) 1995Moleculargeneticsof cancer.Bios Scientific, Oxford A multiauthor text coteringthe cancerfamil.1,syndromes and the common cantersEelesR A, PonderB AJ, EastonD Il, Horwich A (eds)1996Genetic predispositionto cancer Chapman& Hall, London A goodmultiauthor tefi reriening Ihe tarious cancer-fretlislosingsJ,ndrlmes and the tommonJitmilialtauers, as pell as the acceltedantl controursial areasoJtheir management Harris H, Miller O J, Klein G, Worst ! TachibamT 1969Suppressionof malignancyby'cell fusion Nature 350: 377-3'18 Studrcsthat erentuelhtled I0 the clncepl 0ftumor suppresst)r genes HodgsonS \', Maher E R 1999A practicalguideto human canecr genetics, 2nd edn CambridgeUniversity Press,Cambridge An u!-to-date secondeditionoJ this text touring the derelopingJieltlofhuman cancergenettcs. King R A, RotterJ I, XlotulskyA G (eds)1992The geneticbasisof common diseasesOxford Universitl' Press,Oxford Six chaptersoJ'thistext rct:er the basicbitktgy, epitlemiolog.y andfamilial as?ectsof canter. KnudsonA G 1971Mutation and cancer:sraristicalstudv of retinoblastoma Proc Natl Acad Sci USA 68: 820-823 Proposttlof the 'tmo-hit' hytrothesis for the deulopmentof retinoblastoma.

218

Q

Crtr".r has both genetic and environmental causes.

@ Genetic and environmental factors in the etiology of cancer can be differentiated by epidemiological, family and twin studies, and by analysis of disease,biochemical and viral associations. @ Studies oftumor viruses have revealedgenespresent in humans known as oncogenes that are involved in carcinogenesisby altering cellular control mechanisms. @ Study of rare, dominantly inherited, tumors in humans, such asretinoblastoma,has led to the identification of tumor suppressor genes, consistent with the hypothesis that the development of cancer involves a minimum of two 'hits'. Personsat risk of familial cancer inherit the first 'hit' in the germ cell, the second 'hit' occurring in somatic cells in mitosis. In persons with sporadically occurring cancer,both 'hits' occur in somatic cells. @ So-. 5oloof the common cancers, such as breast and bowel cancer, arise as a result of an inherited cancer susceptibility. Familial susceptibility for cancer can occur as an inherited susceptibility for a single type of cancer or for a number of different types of cancer as part of a familial cancer-predisposingsyndrome. @ P..ron, at risk of an inherited cancer susceptibility can be screenedfor associatedfeatures of a familial cancerpredisposingsyndrome or for particular cancers.

CHAPTER I I

I

Medical genetics usually concentrateson the study of rare unifactorial chromosomal and single-gene disorders. Diseases such as diabetes,cancer,cardiovascularand coronary artery disease,mental health and neurodegenerativedisorders are responsible,horvever,for the majority of the morbidit_vand mortalitl. in developed countries These so-called tommon

If the underl.ving genetic factors were understood, it would be possible to offer genetic testing to identify those persons gcnetically susceptible to a particular disorder. However, the utilit-v of such a test would depend upon the action taken subsequentlyto reduce other risk factors,i.e. lifestyle changes.

diseasesare likely to be of even greater importance in the future, rvith the elderlv accounting for an increasing proportion of the population.

OS FG E N E T I C T Y P EA S N DM E C H A N I S M SUSCEPTIBILITY

The common diseasesdo not usually show a simple pattern of inheritance Instead, the contributing genetic factors are often multiple, interacting 'lvith one another and environmental

Genctic susceptibility for a particular diseasecan occur through single-geneinheritanceof an abnormalgeneproduct involvedin a particular metabolic pathway,for instanceearly coronary artery diseasearising from familial hypercholesterolemia(FH) (p. 167). In an individual with a mutation in the FH gene, genetic

factors in a complex manner. In fact, it is uncommon for eithcr genetic or environmental factors to be entirely responsiblefor a particular common disordcr or diseasein a single individual In most instances both genetic and environmental factors are contributorl.althoughsometimcsone canappearmore important than the other (Fig. 15.1). At one extreme are diseasessuch as Duchenne muscular d]'strophy; these are exclusivelv genetic in origin and the environment plays little or no direct part in the etiology At the other extreme are infectious diseasesthat are almost entirelv the result of environmental factors.Betrveenthesetwo extremes are the common diseasesand disorderssuch as diabetesmellitus, hypertcnsion, cerebrovascularand coronary arterl, disease, schizophrenia,the common cancers)and certain congenital abnormalities,in which both genetic and environmental factors are involvcd

GENETIC SUSCEPTIBILITY TOCOMMON DISEASE For many of the common diseasesa small but significant proportion have single-gene causes,but the major proportion of the genetic basisof common diseasescan be consideredto be the result of an inherited predispositionor geneticsusceptibilit),. Common discascsresult from a complex interaction of the effects of multiple different genes,or what is known asplblgenic inheritance,lr,ith environmental factors and influences, due to what is known as multifuttorial inheritunce(p 136).

susceptibility is the main determinant of the development of coronaryartery diseasebut this can be modified by environmental alteration,such asreductionin dietarycholesteroland avoidanceof other risk factors such as obesity,lack of exerciseand smoking. Inheritance of a single-genesusceptibility does not, however, necessarilylead to development of a disease.For some diseases exposureto specificenvironmentalfactorsis the main determinant in the development of the disease,for instance smoking or occupational dust exposure in the development of pulmonary in personswith o1-antitr-vpsindeficiency(p 182). emph-vsema In other instancesthe mechanismof the geneticsusceptibility is less clear-cut. This can involve inheritance of a single gene pol.vmorphism(p. 128) that leadsto differencesin susceptibility to a disease,for example acetaldehydedehydrogenaseactivity and alcoholism (p. 180). In addition, inherited single-gene poll.morphismsappearto determinethe responseto asyet undefined environmental factors; an example is the antigens of the maior histocompatibility (HLA) complex and specificdiseaseassociations (p. 141), such as t.vpe1 diabetes,rheumatoid arthritis and celiac disease.Lastly, geneticsusceptibilitycan determine differencesin responsesto medical treatment,for instanceisoniazidinactivation statusin the treatment oftuberculosis (p 178).

TODEMONSTRE GENETIC APPROACHES DISEASES TOCOMMON SUSCEPTIBILITY In attemptingto understandthe geneticsof a particular condition, the investigatorcan approach the problem in a number of rvays

219

15

GENETIC FACTORS INCOMMON DISEASES

Hemophilia O s t e o g e n e si im s perfecta

P e p t i cu l c e r D i ab e t e s

Talipes Pyloric stenosis Dislocation of hip

Duchenne m u s c u l adr y s t r o p h y

P h e n ykle t o nur i a G al ac t o s e m ia

I u b e r c ul o s i s

S p i n ab i f i d a l s c h e m i ch e a r td i s e a s e A n k y l o s i n sgp o n d yi tl i s Rore Geneficssimple (Unifoctoriol) High recurrencerisk

Common Geneticscomplex (Multifoctoriol) Lowrecurrencerisk

Fis.15.1 Httm:n

djctr:ctr

rr es nPr occ)ocnl r o d

:c

hoinn

.. u

. ^> C +L hl l. o t t... tn thnrn rh-i a.^ : J< n + ,l ^l n o P aL. Li .Lt utrm .l ronl ^1 9 ^l 1^ 9 fl rl ^U- l l l lt hl l^U O I E . eorlngocl tr ry co n l lrvr Ir nUnr m r lo' lncl lr ll L o l |i n l. -d, u, ->1 O u l l t U L ll u > g L ll d L d l E

entrreLv oenetrc

B o x 1 5 . 1 T y p e so f g e n e t iac p p r o a ctho t h ec o m m o n dr s e a s e s

suggestthat environmental factors are more important. Conversely., maintenance of a low incidence of the diseasein the migrant group would suggestthat geneticfactors are more important.

Poputation/migration studres Famity studies Twn studies Adoption studies

Famitystudies

D^1,,-^-^h -^--^^tt^^r uryrrrut Pt I r tt dJ>uLldLtut r>

BiochemicaIstudies Animatmode[s

(Box 15.1).These can include comparing the prevalenceand incidence in various different population groups, the effects of migration, studying the incidence of the diseaseamong relatives in family studies,comparing the incidence in identical and nonidentical twins, determining the effect of environmenralchanges by adoption studies and studfing the associationof the disease with DNA polymorphisms In addition, stud-vcan be made of the pathologicalcomponentsor biochemicalfactorsof the disease in relatives,such as serum lipids among the relativesof patients with coronary artery disease.Study of diseasesin animalsthat are homologousto diseasesoccurring in humans can also be helpful (p. 73). Before considering the use of these different approaches in a number of the common diseasesin humans, specificaspects of some of theseapproachesrvill be discussedin more detail.

Population/migration studies Differences in the incidence of a particular diseasein different population groups suggestthe possibilitv of geneticfactors being important Thev could, however,alsobe explainedb1,differences

220

in environmental factors. Studies of migrant groups moving from a population group with a low incidence of a diseaseto one rvith a high incidence, in which the incidence of the diseasein the migrant group rises to that of its new population group, would

A genetic susceptibilityto a diseasecan be suggestedby the Iinding of a higher frequency of a family history of the disease in relatir.esthan in the general population. The proportion of affected relativesof a specific relationship (first degree,second degree,etc.)can provide information for empiric recurrencerisks in genetic counseling (p. 253), as well as evidence supporting a genetic contribution (p. 136). Familial aggregation does not, however,prove a genetic susceptibilit]',becausefamilies share a common environment.The frequenc)rof the diseasein spouses,who share the sameenvironment but usuall-vhave a different genetic background, can be used as a control, particularly for possible environmental factors in adult life.

Twinstudies If both members of a pair of identical trvins have the samerrait, this could be thought to prove that the trait is hereditary.This is not necessaril.v so.As twins tend to sharethe sameenvironment it is possiblethel'rvill be exposedto the sameenvironmentalfactors. For example,if one of a twin pair contracts a contagiousdisease such as impetigo, it is likely that the other t\,vinwill also become affected.This problem can be partly resolved by comparing differences in the frequencv of a diseaseor disorder between non-identical or diz,'-gotic(DZ) and identical or monozvgoric (MZ) twin pairs. Both members of a pair of twins are said tobe concortLant when either both are affectedor neither is affected.The term discordunt is used when only one member of a pair of twins is affected.Both types of twin havea tendencyto sharethe sameenvironment but, whereas identical tlvins basically have identical genotypes

D}5EA5E5 FACTORS IN COMMON GENETIC

(p. 101),non-identical twins are no more similar geneticalll'than brothers and sisters Ifa diseaseis entirely geneticallvdetermined then, apart from rare eventssuch aschromosomenon-disjunction or a ne\\ mutation occurring in one of a twin pair, both members of a pair of identical twins will be similarll, affected but nonidentical twins can differ. If a diseaseis caused entirely by environmentalfactors,identical and non-identical twins will have similar concordancerates. Although all twins tend to share the same environment, it is probable that this is more likely in identical trvins than in nonidentical twins. Similarities betweenidentical twins can therefore reflect their shared environment as much as their identical genot-\-pes. One way of getting round this difficulty is to studv differences bctrvcen identical twin pairs who, through unusual f'amil.vcircumstances,have been reared apart from an early age.If a particular diseaseis entirely geneticall.vdetermined then, if one identical tr,in is affected,the other will alsobe affectedevenwhen thev have been brought up in different environments It is rare, horvever,for identical twins to be separatedfrom early childhood and so onlv a limited number of studies for any one disorder exist In onc study of identical twins reared separately,the data clearl-vshorvedthat eachpair of twins differed little in height but diffcrcd considerablvin body-weight.These observationssuggest that heredit_rcould play a bigger part in detcrmining staturethan it does in determining body weight.

Adoptionstudies Anothcr approachthat helps to differentiatebetweengeneticand environmental factors is to compare thc frequencl, of a disease in individuals who remain rvith their biological parents with those who are adopted out of their biological family-.Adopted individuals take their genes with thcm to a ner,venvironment If thc frequencl. of a diseasein the individuals adopted out of a famil-vis similar to that seenin thosewho remain with their biological parents, gcnctic factors are likely to be more important. If, conversell-,the frequencvofthe diseasein the adoptedindividuals is similar to that of their adoptive parents,environmental factors are likcll'to be more important.

Polymorphism association studies The rvidespreadexistenceof inherited biochemical,protein, enz-vmeand DNA variants allorvsthe possibility of determining whether particular variantsoccur more commonly in individuals affected with a particular diseasethan in the population in general,or rvhatis known as association. Although demonstration of a pol.vmorphic associationcan suggest that the inherited variation is involved in the etiology of the disorder, for example the demonstration of HLA associationsin the immune response in the causation of the autoimmune disorders (p. 189), it may' reflect only'that a gene nearby in linkage disequilibrium (p. 132) is inr.olvedin causationof the disorder. The human genomecontainsup to l0 million single-nucleotide polymorphisms(SNPs), so the possibilitiesfor associationstudies

are endless!Developmentsin high-throughput microarray SNP genotyping, together with information about SNP haplotypes (from the HapMap Project, p. 142), and the availability of large collectionsof DNA samplesfrom patientswith common diseases, mcans that a new approach is possibleto look for predisposing variants,or to identify loci harboring such variants.Much storeis being placed on thesewhole-genomeassociationstudies (p.1+2) to identifl' genetic variants associatedwith common diseases, rvith thc ultimate goal of reducing the burden of such disease.

B i o c h e m i c as lt u d i e s Anall'-sisof metabolite or enzyme activity levels in biochemical or metabolic pathwayslikely to be involved in the causationof a particular disorder can provide evidenceof genetic contribution to some of the common diseases,such as the hormones involved in thc control ofblood pressurein hypertensionor the regulation of lipid levelsin atherosclerosisHowever, in disorderswhere we have a limited understandingof the biological basis,for example schizophrenia,this approachhas been oflittle use.

AnimaImodels that occurs in humans Recognition of the samedisease,/disorder and in anotherspecies,such asthe mouse,allowsthe possibility of erperimental studies that are often not possible in humans. In man-vinstances,ho'wever,the disorder in the animal model will have a single-gene basis that has been identified or bred for Ner,crtheless,spontaneouslyoccurring or transgenic animal models (p. 75), experimentally'induced for mutations in single gcnes involved in the metabolic processesor diseasepathways of common diseases,will provide vital insights into the genetic contribution to these disorders.

FORMULTI CTORIAL MODELS DISEASE INHERI NCE The searchfor susceptibility loci and polygenes,sometimesalso referred to asquantitatioetrait loci,inhuman multifactorial disorders has met with increasingsuccessin recent years.This is due largell.to knowledgegainedfrom the Human Genome (p' 9) and HapNlap (p. 142) Proiects,together with developmentsin SNP genotl'ping technology. Examples of recent research in some common conditions will be consideredto illustrate the progress to date and the extent ofthe challengesthat lie ahead.

MELLITUS DIABETES There are two main forms of diabetes mellitus (DM) that are clinically distinct. Typ. I (TIDM) is the rarer, iuvenile onset, insulin-dependentform (previouslyabbreviationIDDM), which affects0 4oloofthe population and hasa high incidenceofpotentially seriousrenal, retinal and vascularcomplications.TIDM has a peak

221

15

GENETIC FACTORS INCOMMON DISEASES

ageofonset in adolescenceand can be controlled onlv b-vregular injection of insulin Type 2 DM is the more common, later onset, non-insulin-dependentform that aflectsup to 10o/oofthc population. It usualll,affectsolder personsand mav respond to simple dietar.v restriction of carbohvdrateintake, although manv persons with T2DM require oral hypoglycemicmedication, and some requlre insulin. An additional l-2o/o of persons rvith diabetes have (single gene)forms of diabetes(Table 15.1). monogenic Around l-3o/o of women develop glucoseintolerance during pregnancv.This is known asgestationaldiabetesTheir abnormal glucosetoleranceusually reverts to normal after the pregnanc\', although approximately one-half ro three-quarters of these women go on to develop diabeteslater in life. Diabetescan alsooccur secondaryto a variety ofother rare genetic syndromesand non-geneticdisorders Examplesinclude PraderWilli syndrome (p 266), Bardet Biedl sl.ndrome, Wolfram

Mutations in five additional genes,which encodetranscription factors required for development ofthe B cell, have been reported. The hepatocyte nuclear factor lcr (HNFIA) and hepatocyte nuclearfactor 4a (HNF'IA) genesw€re identified through positional cloning efforts and are associatedwith a more severe,progressive form of diabetesusuall-vdiagnosedduring adolescenceor earl-v adulthood.These patientsaresensitiveto treatmentwith sulfonylurea tablets; this is an example of pharmacogenetics(p. 177). Good glycemic control is important as patients havea long duration of diabetesand may suffer from diabetic complications.Mutations in the HNFIA gene are the most common cause of MODY in most populations(65oloof patients wtih MODY in the UK), and HNFlA mutations are less frequent. Hepatocyte nuclear factor lp (HNF-lB) pla-vsa ke-vrole in kidnev development. Mutations cause renal cysts and diabetes (RCAD), and some female patients also have genital tract

syndrome and Friedreich ataxia DM is therefore etiologically heterogeneous.

malformations Insulin promoter factor I (IPFI), NEURODI and CEL mutations are rare causesof MODY, but highlight the possibility that further genes encoding B-cell transcription

M O N O G E NFI C O R M SO FD I A B E T E S

factors may be mutated in MODY (so called -MODYX genes).

Rare forms of diabetesthat show high penetrancewithin families are usuallydue to mutationsin singlegenes.Nearly 20 monogenic forms of diabetesare known (seeThble1-5.1).

Maturity-onsetdiabetesof the young(M0DY) MODY is an autosomaldominant form of diabetcscharacterized by B-cell dvsfunction. It shorvsclinical heterogeneitr,., r,vhichcan now be explained b1, genetic heterogencitv. Mutations in the glucokinasegene causemild hyperglycemia(usuallv bctween 5.5 and 8mmol/l), which is stablethroughout life and often treated by diet alone Glucokinaseis describedas the pancreaticglucose sensor becauseit catalysesthe rate-limiting step of glucose metabolism in the pancreaticp cell It lvas therefore an obvious candidate gene. Manv patients with glucokinase mutations are asymptomatic and their hyperglvcemia is detected during routine screening,for exampleduring pregnancyor emplovment medicals.The mild phenotype means that finding a glucokinase mutation is 'good news'

NeonataIdiabetes Anall'sis of HLA genotypesin children diagnosedwith diabetes before the age of 6months has shown that these patients have a similar frequcncv of high-risk allelesfor type 1 diabetesas that found in the generalpopulation This suggeststhat type 1 diabetes is rare before 6months of ageand implies a geneticcause. Although definitions of the neonatal period var1,,we know that diabetesis rare before 6 months of ageand the prevalenceis estimatedat lessthan I in 100000.There has been great progress over the past 5 l0-vearsin delining the geneticsof this rare condition Neonatal diabetescan be transient or permanent. N{ore than 70o/oof casesof transient neonatal diabetesresult from the overexpressionof a paternally erpressed gene on chromosome 6q24. These patients are usuallv diagnosedin the first week of life and treated with insulin. Apparent remission occurs by 3 months but there is a tendency for children to developdiabetes in later life

Tabte15.1 Subtypes ofdrabetes

222

Type1 diabetes

Type2 diabetes

MODY

NeonataIdiabetes

Prevalence(%)

<1

<10

<001

<00001

Ageof onset

Chitdhood/adolescence

M i d d t e / o tadq e

Adotescence/early adulthood

Before6 months

Inheritance

Potygenic

Potygenic

Monogenic

Monogenic

No of genes

> 2 0l o c i

NumeTous

At leastB genes

At least10genes

Pathophysiology

Autoimmune

lnsutinsecretion/resistanceB-Cetldvsfunction

MODYmaturrty-onset diabetes oftheyoung(p 181)

dysfunction B-Cet[

}N COMMON DI5EA5E5: GENETIC FACTORS

Permanentneonataldiabctcsdoesnot remit and, until recentll., patients were treated lvith insulin for life. Thc most common causes(more than 50o/o)are mutations in the KC,$1 I or ABCCS genes,rvhich encode the Kir6 2 rnd SURI subunits of the adenosinetriphosphate(ATP)-sensitivepotassium(K-ATP) channel in the pancreaticB cell. Closure ofthcse channelsin responseto ATP generated from glucosc mctabolism is thc kcy' signal for insulin releaseThe effect of activatingmutationsin thesegenesis to preyent channel closureb-vreducing the responseto ATP, and hcnce insulin secretion.The most erciting aspectof this recent discoverv is that most patients r'vith this genetic etiology-can be treated rvith sulfonvlurea drugs, rrhich bind to the channel and causeclosureindependentlyof ATP. Not onl.vhavepatientsbccn ableto stop insulin injectionsand takc sulfonvlureatabletsinstead, but better gl1-ccmiccontrol is achieved, lvhich improves both their qualitl- of life and later risk of diabetic complicattons. Nconatal diabetcsis geneticall.vheterogencousand mutations in the follou,inBgeneshavealsobeen reported in a small number of patients: homozvgous or compound heterozvgous GCK or IPFI mutations (heterozlgous mutations cause N{ODY); heterozl'gousHNFlheta mutations (also cause renal cysts); homozvgous PTF/A or GLIS3 mutations result in diabctcs Neonatal with ccrcbellaraplasiaor h1'pothl,roidism,respectir,ely. diabetesis also a fcaturc of the follol'ing svndromes:\\blcottRallison (homoz-vgousor compound heterozvgous EIF2AK3 mutations) and the X-linked IPEX (immunod-vsregulation, pol-vendocrinopath\i, enteropathl.,X-linkcd) s1'ndrome(F OX P 3 mutations) There are still many patients lvithout a genctic diagnosis, u,hich sugBeststhat therc arc likel-v to be other monogenic etiologies.

TYPE1 DIABETES Initial researchtended to focuson type I diabetes,for rvhich there is greatercvidenceof familial clustering(1,.is l5 for TlDM vcrsus 3.5 for T2DN{ - p 136).The concordanceratesin monozrgotic and dizl.gotic twins are around 50o/oand 12olo,respectivel-v. These observationspoint to a multifactorial etiolog.vwith both cnvironmental and geneticcontributions. Known environmental factorsinclude diet, viral exposurein earlr childhood and certain drugs. The diseaseproccss involves irreversible destruction of insulin producing islet B cells in the pancrcasb-vthe bodl-'s ou''n immunc s.vstem,perhaps as a result of an intcraction between infection and an abnormal geneticall-rprogrammed immune response. The hrst major breakthrough came lvith the recognition of strong associationswith the HLA region on chromosome6p21 The original associationsrvere with the HLA-BS and Bl5 antigens,rvhich are in linkage disequilibrium with the DR3 and DR4 allcles(p 132). It is rvith these that the TIDN{ association is strongest,rvith 95o/oof affectedindividuals having DR3 and/or DR,l, compared with 50o/oof the gcneral population. Follorving for the developmcntof polvmerasechain rexction (PCR) anal-vsis the HLA region, it rvassholr.nthat the HLA contribution to TIDNI susceDtibilitvis determined br: the 57th amino-acid residue

at the DQlocus, where aspartic acid conveys protection' in contrast to othcr allelesthat increasesusceptibility.As this was thc {rrst susceptibilit-ylocus identified for T1DM, it was labeled IDDMI The next locus to be identilied was the insulin geneon chromosome1lpl5, where it rvas shown that variation in the number of tandem repeatsof a 14-basepair (bp) sequenceupstream to thc gene(known as the IN.SVNTR - variablenumber tandem rcpeat) influences diseasesusceptibility-.It is hypothesizedthat long repeatsconvcv protection by increasing expressionof the insulin gene in the fctal thymus gland, thereby reducing the likclihood that insulin-producing B cells will be viewed asforeign bv the mature lmmune s.vstem. Thcse trvo loci contribute l",valuesof approximately3 and 1.3, rcspectivclli However,the total risk ratio forTlDM is around l5 Conhrmation that other loci are involved came first of all from linkagc analvsisusing breeding experimentswith the non-obese diabetic (NOD) strain of mice. These mice shorv a very high incidenceofTlDM rvith immunopathologicalfeaturessimilar to thosc seenin humans.These linkagedata pointed to the existence of ninc or tcn different susceptibility loci in mice. Shortly afterrvards,in 1994, the results of a genome-wide scan in three largc sets of about 100 affected British sibling pairs provided er.idencefor the existcnceof between12 and 20 susceptibilityloci in humans.Thc identitlt of some of thesc loci has been revealed in rccent years and these include the CTLA'|, PTPN2L, IL2RA (CD25) and IFIHI regions T h c c u r r r c n t u n d e r s t a n d i n gi s t h a t T I D M i s i n d e e d a or pol.ygenic multifactorial disorder r,vith an underlying r.tligogenic susceptibilit)' consisting of one maior locrrs (IDDM1 = HLA) and up to 20 minor loci. The products of these loci arc believed to interact in a complex and poorly understood \\ay to confer susceptibilityto environmentaltriggersof autoimmuncpxncreatic P-cell destruction.This phenomenon of geneinteraction is referred to as epistasis.The long-term researchgoalsare firstly to map all rhe IDD,M loci, then to identif.v the relevant genes, and {inally devise new strategiesfor prevention and possibly treatment based on a full understanding of the underlying etiology and pxthogenesis.These objectivesare ambitious, but if successfulh achievedthe rervardsin terms of preventing the health burden posedb1'T1DM rvill be huge.

TYPE2 DIABETES The prevalenceof T2DM is increasingand is predicted to reach 215 million rvorldwideby 2010.Although commonlv believed to be more benign than the earlier-onset,insulin-dependent type I diabetes,patients with T2DM are also prone to both macrovascular and microvascular diabetic complications with corresponding excessmorbidity and mortalitl'. The later age of onset has made family studies difficult, but breakthroughshave been possible through genome-wide scans using hundreds of affectedsibling pairs, and associationstudiesinvolving thousands of casesand control samples. fable 15.2 givesexamplesofvariants in genesthat predispose to T2DM. A number of different approacheshas been utilized to

223

15

GENETIC FACTORS IN COMMON DISEASES

Gene

Variant

CAPNlO

SNP43in intron3 (Gattete)

PPARG

(Proattete) P12A

Adipocyte differentiation Alsobindsinsulinsensitizing drugs

genefromhumanmodetwhere Candidate mutation causes severe insutin resistance

KCN]ll

E23K

c - c e t lK - A T Pc h a n n e l S u b u n i to f p a n c r e a t i B

B r o t o g i c acla n d r d a t e

HNFlA (TCFI)

G319S

Transcription factorin p-cetldeveiopment andfunction

C a n d i d a t fer o m h u m a nm o d e l P r i v a t em i s s e n s ev a r i a n t f o u n di n 0 j i C r e e

IPF 1

D76N

T r a n s c r i p t r of n a c t o r n B - c e t td e v e t o p m e n t andfunction

C a n d i d a tfer o m h u m a n m o d e l

HNF4,4

SNPsin P2 pTomoter

T r a n s c n p t t ofna c t o ri n B - c e l ld e v e l o p m e n t a n df u n c t r o n

P o st i o n a I c l o n i n a gndcandidate f r o m h u m a nm o d e l

TCF7L2

Variants in intron3 (rnc[uding T allele of rs12255372 SNP)

-rarsc'ipL iaclor on t n a l r e g u . a r e 'p . o g t u c a g o . t g e n ee x p T e s s r ornn e n t e r o e n d o c r r ncee l l s

Proteinfunction

PositionaI clonrng in Mexican-American affected sibtng parrs

hnd thesegenesand it has becomeapparentthat, unlike the HLA and INS VNTR loci in TIDM, there are no ma.iorpredisposing Ioci associatedwith T2DM. For example,the odds ratios for the Pl24. (peroxisome proliferaror-activated recepror TPPAR] y) and E23K (Kir6 2) variants are only 1.25 and 1 23; there were

P o s r t i o n acit o nn g i n l c e t a n d rfca m i t i e s

., OHNDISEASE

contradictory reports of their significancein the literature unril sufficiently large-scaleassociationstudies had been performed and confirmed by meta-analysisof all published smaller studies. The contribution of loci varies between populations, with some

Inflammatory bowel disease(IBD) includestwo clinical subrypes: Crohn diseaseand ulcerative colitis. Its prevalencein Western countries is lJo/o and the estimated1,, is 25. Positionalcloning for IBD hasidentified loci at l6q,l2q,6p and 3p.The mosr srriking peak was located at chromosome l6pl2, which was linked to Crohn diseasebut not ulcerativecolitis in the majority of studies. In 200I , two groups working independently and using different

variants being population specific(e.g the G3l95 varianr in rhe HIVFIA gene has been found only in the Oji Cree population in Ontario, Canada) and other loci showing varying contributions depending on allelefrequencies(e.g.there is lessevidencefor the calpain l0 variant at SNP,+3in Europeans,compared with the Mexican American population in which it was identified by linkage) Human models have proven useful as,with the exception

approaches identified disease-predisposingvariants in the CARDIS gene (previously known as NOD2). Crohn diseaseis characterizedby perturbed control of inflammation in the gut and with its interaction with bacteria.One of the groups, Ogura and co-workers, had previously identified a Toll-like receptor, NODZ, which activatesNF-KB, making it responsiveto bacterial lipopolysaccharides.The CARDI5 gene is located within the

of CAPNl0 and TCF7L2, murations in all of these genes cause rare, monogenic forms of diabetes.The identification of TCF7L2 variants shows the power of the genetic, as opposed

16p12region and was therefore a good positional and functional candidate.Sequenceanalysisrevealedthree variants(R702W G908R and 3020insC), which were shown by case-control and TDT studies to be associatedwith Crohn disease.The second group,

to candidate gene, approach, as this gene is not expressedin the pancreas.These variants are common (allele frequency of approximately35o/oin most populations studies to date) and are associatedwith odds ratios of 1.45for heterozygotesand 2 31 for homozygotes.

224

Strategy

Current thinking is thar rype 2 diabetesrisk may be attributed to the additive effects of predisposing variants in many genes. This highlights multiple targets for intervention but the genetic complexity underlying type 2 diabetesmeans that such studies will not be easv.

Hugot et al, Iine-mapped the 16p12region by genotyping SNPs within the 20000000-base(20Mb) interval, and also arrived at the same variants within the CARDI5 gene. These variantsare found in up to l5oloof patientswith Crohn diseasebut in only 5oloof controls.The relativerisk conferred by heterozygousand homozygousgenotypeswas approximately2.5 and 40, respectively. For therapy, drugs that target the NF-KB complex are already the most effective drugs currently available. Perhapsit might be possiblein the future to prevent Crohn disease

by enhancingNOD2 function in the bowel of personswith a genetic predisposition.

Various studies have shown that l0-25o/o of the population is hypertensive, although the prevalence is age dependent, with up to 40o/oof 75-79-year-olds being hypertensive. Hypertension leads to an increased morbidity and mortality through a greater risk of stroke, coronary artery and renal disease. Blood pressure may contribute up to 50oloof the global cardiovascular epidemic. There is substantial evidence that treatment of hypertension prevents the development of these complications. Personswith hypertension fall into two groups. In one group the onset is usually in early adult life and is a consequenceof another disorder, such as kidney disease or abnormalities of certain endocrine glands. This is referred to as secondaryhjtpertension.In the second, more common, group hypertension usually begins in middle age and has no recognized cause.This is known as essentialhypertension.The following discussion is concerned only with essentialhypertension.

CTORS ENVIRONMENTAL IN HYPERTENSION Environmental factors, such as high sodium levels in the diet, obesity, alcohol intake and reduced exercise, are recognized as being associatedwith an increased risk of hypertension. Hypertension is also more prevalent in persons from poorer socioeconomic groups. Studies of adopted children have shown lower correlation of their blood pressure with their biological parents than with children remaining with their biological parents. In addition, migration studies involving persons moving from a population with a low prevalence of hypertension to one with a high prevalence have shown that the immigrant group acquires the frequency of hypertension of their new population group during the course of one to two generations. This suggests that environmental factors are of major importance in the etiology of

ln:KingRA,RotterlI A G Hypertension FromBurkeW Motutsky genetic basisof commondrseases A G (eds)The Motulsky Ch10 Press,Oxford, Unrversity Oxford

EY NES S U S C E P T I B I LGI T To date,thcre is evidencefrom relativelysmall associationstudies for l number of loci that contribute to the prcdisposition to h-vpertensionThese include chromosome 17q (HYTI), another on chromosome l6 that is homologous to the SA locus in rats (HYT3),and chromosome l5q @Yf4. A genome-rvidescanof 2010 affectedsibling pairs (p 74) iailed to confirm these loci but found a maior locus on 6q and minor loci on 2q, 5q and 9q It is hoped that a genome-rvideassociationapproach will .vicld more clues to this common disorder.

hypertension.

S H Y P E R T E N S ION G EN E T I F CAC T ORIN

DISEASE ARTERY CORONARY

Family and twin studies have shown that hypertension is familial (Table 15.3) and that blood pressurecorrelateswith the degree of relationship (Table 15.4).These findings suggestthe importance of genetic factors in the etiology of hypertension. In addition, there are differences in the prevalence of hypertension between populations, hypertension being more

Coronarl, arter-vdiseaseis the most common causeof death in industrialized countries and is rapidly increasing in prevalence in der,clopingcountries.It results from atherosclcrosis,a process that fakes place ovcr many )rearsand involves deposition of lipid in the subendothelial space (intima) of arteries u'ith a

common in persons of Afro-Caribbean origin and less common in Eskimos, Australian Aborigines, and Central and South American Indians.

conscquent narroling of their lumina The first stage involves thc deposition of lipid in the arterial wall that is determined b,v hemodynamic factors. Monoc-vtesadhere to areasof the

225

15

GENETIC FACTORS IN COMN4ON DISEASES

endothelialsurfaceof arterial rvallswith lipid depositsand enter thc vesselwall, proliferatc and diffcrentiate into macrophages The macrophagesscavengethe lipids, producing the classicfattv streaks,and through the action of cytokines,growth factors and adhesionmoleculcsinduce smooth muscle proliferation and the formation of cxtracellular matrix, resulting in the development of thc librous athcroscleroticplaques.The narroting of the coronar\ arteriescompromisesthe metabolic needs of the heart muscle, lcading to mvocardial ischcmia, which, if severe,results in mvocardialinfarction. For the majoritv ofpersons their risk ofcoronary arterl, diseasc is multifactorial or polygenic in origin. A variety of different genetic and environmental risk factors has becn identified that predispose to earlv onset of the atherosclerotic process \\r'ell publicized cnr.ironmental risk factors include lack of exercise.

Tabte15.5 Recurrence lsks for prematurecoronary afterydrsease Proband

Relativerisk

Mate(<55years) Brother Srster

5 25

Female(<65years) Sibtings

7

DatafromStackJ, EvansKA1966Theincreased riskofdeath fromischaemic heartdisease infirstdegree relatives of121men and96womenwithischaemrc heartdisease J MedGenet 3:239-257

dietar-vcholesteroland smoking The advice rvith respect to the potential for prevention ofdeveloping coronarv artery diseasefor thesefactors is obvious.

L I P I DM E

BOLISM

The metabolic path$.aysb-vwhich the bodl'absorbs,synthesizes, transports and catabolizesdietarv and endogenouslipids are compler. Lipids are packagedin intestinal cellsasa complex with variousproteinsknown asapolipoproteins to form triglvceride-rich chvlomicrons.These are sccretedinto the ly.mphand transported to the liver, rvhcre, in associationuith endogenoussvnthesisof triglycerideand cholesterol,thev arepackagedand sccretedinto the circulation as triglyceride-rich, very low-dcnsity lipoproteins (VLDLs).VLDL is degradedro intcrmediate-densitvlipoprotein (IDL), which is further broken dorvn into cholesrerol-richlowdensit-vlipoprotein (LDL). High-density lipoproteins (HDLs) are formed from lipoproteins secretedbv the liver, chvlomicrons andVLDL remnants. High levels of I-DLs are associatedI'ith an increased risk of coronarv artery' disease.Converselr,.,high levels of HDLs are inverselv correlated rvith a risk of coronary arterl disease. Consequentlv.the LDL:HDL ratio has been used as a risk predictor for coronarv arterv disease and as an indicator for therapeutic intervention. Statins are effectivedrugs for lolvering LDl-cholesterol levels

S I N G L E . G ED NIES O R D E O RS FL I P I D ME BOLISM LEADING TOCORONARY ARTERY DISEASE Although there are a number of individually rare inherited disordersof specificlipoproteins,levelsof the variouslipoproteins and the h"vperlipidemiasare determined by a complex interaction of genctic and environmental factors Family studies of some of the hvperlipidemias are, horvever,consistent rvith a single gene being a major factor determining genericsusceptibility.

Familiat hypercholesterolemia The best known disorder of lipid metabolism is familial h1-percholesterolemia (FH) (p. 167). FH is associatedwith a signihcantly increasedrisk of early coronary artery diseaseand is inherited as an autosomal dominant disorder. It has been estimatedthat about 1 person in 500 in the general population, and about I in 20 persons presenting with early coronary artery disease,is heterozygous for a murarion in the ZDZR (lowdensitl,'lipoprotein receptor) gene. Molecular studies in FH have revealedthat it is due to a variety of defectsin the number, function or processingof the LDL receptorson the cell surface (p.168).

MILYAND TWINSTUDIES The familial nature ofcoronary artery'disease hasbeenrecognized since the early part of the twenticth century. The risk to a ltrstdcgreerelativeof a personr,vithprematurccoronarvxrterv discase, defined as occurring before age 55,vearsin males and age65 years in females,is two to seventimes that for the generalpopulation (Thble 15.5). Twin studies of concordance for coronary arter,v-

226

diseasevarv from l5o/oto25o/ofor dizygotic twins and from 39olo to 48o/ofor monozygotic trvins. Although rhese valucs supporr the involvcment of genetic factors, the low concordance rate for monozvgotic t$'ins clearly supports the importance of environmental factors.

Myocyteenhancerfactor2A(MEF2A) In 2003 a positional cloning approach identifed a locus in a large family rvith dominantly inherited coronary artery disease The genetic interval contained 93 genes and analysisof the A4EF2A gene revealed a seven-amino-acid deletion rhat cosegregatedwith the disease.In rilro studiessupporteda functional effect of this deletion upon the transcription factor. Howevel further studies have reported MEF2A murations (including the seven-amino-aciddeletion) present in controls and hence the link between MEF2A variants and coronary artery diseaseis uncertaln.

SC O [ / M OD NI S E A S E S G E N EC T F A C T O RI N

S U S C E P T I B I LGI T EY NES Thus far, the most common strategvhas been to studv candidate genes that encode important rate-limiting proteins in the homeostatic systems involved in maintaining cardiovascular health. Although many putative loci have been implicated, the lack of reproducibility seenin relativell,-small geneticassociation studies has led to uncertaintv about the nature and number of genesinvolved. Genome-wide scanshavehighlighted trvo predominant loci at 2q34-37and3q26-27.The 2q locusencompasses severalcandidate genes,including the uridine diphosphate gll,cosvltransferaseI (UGTIAI) gene A promoter variant in this genecausesincreased bilirubin levels in patients r,ith Gilbert syndrome, and the associationof reduced bilirubin levels t'ith coronarv arterv diseasesupports UGTIAI as a candidate gene.A recent studv of 2658 affectedsib pairs found linkage to a large region (36Mb) on chromosome l7 that includes more than 300 genes,manv of which are possible candidatesbased on knowledge of their function or expressionproliles

EPILEPSIES Epilepsy-is a brain disorder characterizedby recurrent and unprovoked seizures It may be an isolated feature in a patient, part of a disease,or indeed part of a s1,-ndrome. Seizuresmav be partial (focal) or generulized(absencesor tonic clonic seizurcs) Up to l0o/crof the population suffer some sort of seizure in their lifetime and about 3olodevelop epilepsl' by 75 -vearsof age. The causesof epilepsy are extremelv heterogeneous,including acquiredbrain damagefrom head injury, meningitis, encephalitis and birth asphyria.However,overallit is recognizedthat a genetic etiology is present in about 40o/oofcases,and in the absenceofa positive familv historl-the generalrisk of epilepsyto the offspring of those affectedis approximately'4olo Familial clustering occurs in some tvpes, thus providing strong evidence for mendelian forms of epilepsy The classificationof the non-acquired epilepsiesis potentially ver-vconfusing becausethere are manv waysin rvhich a classihcation might be structured. Thc internationall.vagreedclassificationbegins with the t1-peof seizure,rvhetherpartial or generalized,then moves on to whether the causationis idiopathic (no known underlying cause),s.vmptomatic(the consequenceof a known centralnervous s-ystemdisorder), or crvptogenic (presumed to be symptomatic but no causeidentified (Box 15.2).

EPILEPS AY N DC H R O M O S O M E ABERRATIONS Nearll' all chromosome disorders include significant neurodevelopmentaldelay and therefore some form of cerebral even though magnetic resonanceimaging mav show dy-sgenesis, normal featuresThe conditions in which seizuresare particularlv frequent include Angelman syndrome(deletion 15qI 1.2) (p. 117),

B o x 1 5 . 2 A s r m p L i f i ge ed n e r acl i a s s i f i c a t ioofne p i L e p s i e s Partiat(focal) ldiopathic o Auroso-ra. roctu'ral'rorLaL lobeeplepsy don-tna1[ o Familialtemporal lobeeprtepsy Symptomatic Generalized ldiopathic . B e n i g n e o n a t a t f a mci loi anlv u t s i o n s . Juventemyoclonic epilepsy Cryptogenic (lnfantile . Westsyndrome spasms) . Lennox-Gastaut syndrome Symptomatic (tuberous o Neurocutaneous sclerosis) syndromes ([ssencephaly o DevetopmentaI syndromes, syndromes Rettsyndrome) (pyrdoxine . Metabotrc mitochondriaI dependency, disorders leukodystrophies) ureacyctedisorders, disorders, myoclonicepitepsies Progressive . Unverrcht-Lundborg disease . Lafora disease partialor focal undetermined Epilepsies andsyndromes: SpeciaIsyndromes . S tuationrelated seizures) sezures(febrile

ring; chromosome 20, Miller Dieker (lissencephaly)syndrome (dcletion17p13),tetrasomv15q,deletion1p36syndrome(p.269), Wolf Hirschhorn svndrome (deletion 4p) (p.261and PallisterKillian s-vndrome(tertrasomy 12p).

EPILEPSIES SYMPTOMICMENDELIAN This group includcs tuberous sclerosis,an autosomaldominant condition in rvhich a number of different seizure tvpes occur' including infantile spasms,Lennox-Gastaut s1'ndrome(multiple complex scizures) and generalized tonic clonic seizures.The 'tubers' leadsto seizures.In the lissencephalies prescnceofcortical the normal gvrations and folds ofthe cerebralcortex are lost and the surface of the brain is essentiall-vsmooth Miller-Dieker 'classic' lissencephaly,with mental retardation and svndrome is abnormal facial features.It may be due to a hemizvgousdeletion ofl,or mutation in, the Z,IS1 geneat 17p13.Unverricht-Lundborg disease(ULD) and Lafora disease(LD) are both progressir-e following autosomal m1'oclonicepilepsieswith neurodegeneration, reccssir.einheritance They are due to mutations in the cvstatin B gene(ULD) md EPM2A and NHLRCI genes(LD).

Epitepsyand ion channels I o n c h a n n e l s a r e m e m b r a n e - s p a n n i n gp r o t e i n s t h a t f o r m selectivepores for Na*, K*, Cl or Car+ ions.They regulatecell ercitabilit.v,particularl-vin the central nervous system' skeletal and heart muscle;mutations in the genesencodingtheseproteins give rise to the so-called'channelopathies'.

227

15

GENETIC FACTORS IN COMMON DISEASES

An increasing number of idiopathic epilepsl' svndromes belongsto this group. Autosomal dominant benign familial neonatal convulsions are causedbr mutations in the potassium channel genes (KC.\Q2 and KC,\QJ), whereas autosomal dominant

consistent linding is for a susceptibility locus on chromosome 7q, but in addition there is evidencefor linkage to chromosomes 1 7 q , 5 p , 1 1 , 4 a n d 9 . \ r a r i a n t si n t h e s e r o t o n i nr e c e p t o rg e n e (-|-HTT) on chromosome l7q are more common in individuals

nocturnal frontal lobe epilepsf is caused bv mutations in the neuronal nicotinic acetvlcholine receptor genes (C11R,Nll and CHRNA2) that probabl.v reduce pcrmeabilitv for Caz+ ions N,tutationsin sodium channel genes (SCN1A and SCNIB) have been found in generalized epilepsl' with febrile seizures. N{utations in a chloride channel gene(CLCN2) havebeen found in a number of patientswith idiopathic generalizedepilepst'.

rvith autism than in controls, but manv additional loci havebeen implicated in different studies Hence a full understandingof the Beneticcontribution to autism and geneticallybasedtherapeutic strateeiies are likely to be a long way off.

SCHIZOPHRENIA NONM . E N D E L I AENP I L E P S I E S In these large groups of epilepsies,which include .jur.enile mvoclonic epilepsr.,benign childhood epilepsy and the absence epilcpsies,a genetic contribution is well recognizedthrough familv studiesbut, aswith manv complextraits, geneidentilication has proved elusivc To date onl1. two genes, CICNI 1H and GABRD, have been reportcd to have functional yariants that are associatedrvith common epilepsl'.

AUTISM Autism is a severeneurodcvelopmentaldisorder that affects bett'een,l and l0 individualsper 10000, with onset during the first 3 vears of life The incidence is three to four times higher in bo1-sthan in girls, and the diagnostic features are severe impairment in the development of social responsir-eness, r.er\. poor verbal and non-verbal communication, and repetitive, stereotypicbehar,iorand interests Nlanv affected children shou' significant developmentaldelav and, conversell,,manv children r,ith developmentaldelavcan shorvautistic features.For example, autistic behavior is rvell rccognized in children with tuberous sclerosisand various forms of chromosome imbalancc, as well as in bovs nith the fragile X svndrome(p.273). Autism is part of a spectrum that includes Asperger syndrome and, overall, thc 'autistic spectrum disorders'havea much highcr prevalenceof up to 60 per 10000individuals. The causeof autism in isolation (non-svndromal) is not clcar, but thcre is increasing evidence that genetic factors plav an important role. This is basedon rhe resultsof twin studies,which for autistic spectrum disorders vield concordancerates of up to 807o and 20o/oin monozvgotic and dizygotic tr,vins,rcspectiveh., depending on the diagnostic criteria used. On the evidence of familial aggregation,the empiric recurrence risk for siblings is betrveen2o/oand 6olcr,very much greater than the 0.04 0 1o/oin the general population Autism and autistic specrrum disorders are therefore highlv heritable In an attempt to clarifl, the genetic contribution ro autism, large genome-rvidescreeningstudieshavc been performed using

228

severalhundred families u'ith more than one affected member. Using complex computer-basedstatistical anall'sisthe most

Schizophrenia is a serious psychotic illness with an onset usually in late adolescenceor early adult life. It is characterized b y g r o s s l - vd i s o r g a n i z e d t h o u g h t p r o c e s s e sa n d b e h a v i o r , togethcr with a marked deterioration of social and occupational functioning, and can be accompaniedby hallucinations and delusions.

EPIDEMIOLOGY Schizophreniais a principal causeof chronic mental illness. There is a 1ololifetime risk for a person to developschizophrenia, and at any one time approximatelv 0.2o/oof the population is affected Schizophreniaoccursmore commonlv in individuals of poorer socioeconomicstatus and has an earlier age of onset and \rorse prognosis in malcs There is an excessof winter births in schizophrenicindividuals,which hassuggestedthat environmental factors, such as certain viral infections or nutritional factors, could be contributort.

EVIDENCE FORGENETICCTORS The natureand extentofthe Beneticcontribution to schizophrenia is not clear.This is partlv becauseofpast and continuing contro\rersy concerningthe definition of schizophreniaand the term schizoid. The latter term refers to the schizophrenia-liketraits often seen in relatir.esof schizophrenics.The problem arisesbecauseclinical criteria to distinguish schizoid from normal personality are lacking.For the sakeof simplicitv we can regard the term schizoid as referring to a person with the fundamental symptoms of schizophreniabut in a milder form. It hasbeenestimatedthat roughly ,[o/oof the general population has schizophrenia or a schizoid personalitv disorder.

Famityand twin studies The rcsults of ser,eralstudies of the prevalenceof schizophrenia and schizoid disorder among the relativesof schizophrenicsare summarized in Thble 15.6. If onlv schizophreniais considered, the concordanceratefor identicaltwins is only 160/o,suggesring the importance of environmental factors If, horvever,schizophrenia and schizoid personalitv disorder are consideredtogether, then almost 90oloof identical co-tt'ins are concordant.

INCOMI/ON DI5EA5E5 GENETlC FACTORS

Tabte15.6 Proportions offrrst-degree relatrves of i n d i v i d r , awt si t i s c h i z o p h r eanw h o a . es , ' n i [ a r lay' f e c t eoor h a v ea s c h i z o iddi s o r d e r Relatives

Proportionof relatives(%) Schizophreniao Schizoid Total schizophrenia + schizoid

ldenticattwins 46

41

87

(ofone Offsprrng schrzoph renrc)

16

33

49

Srblings

14

32

46

Parents

9

35

44

(oftwo Offspring schizophren ics)

34

32

66

3

4

General populatron

'l

oAgecorrected FromHestonL L 1970 Thegenetics of schrzophrenia andschizoid diseaseScience 161 : 249-256

Adoptionstudies Other evidencethat provides compclling support for gcnctic factorsin the causationofschizophreniais pror.idedbv the results of adoption studies. The nature of schizophreniacan result in disruption of the famil1,,l,ith childrcn being placed with adoptir,e parents. Higher frequenciesof schizophrenia in individuals rdoptcd awav from their biological parents and their adoptir,e siblings supports a geneticbasisfor schizophrenia

S U S C E P T I B I L IGTE YN E S 'I'he

obserrationof differencesin concentrationsof ncurotransmittcr suchasdopamineor 5-hrdror-\trrptamine (serotonin), substanccs, in r,ariousparts ofthe brains ofschizophrenicsat autopsl and the pharmacologicalactior.rof antips-vchotic drugs, has suggestedthat genesfor centriri ner\ous s\-stemneurotransmittersubstances and their receptorscould be possiblccandidatcgcncsfor linkage studics No confirmed linkagc has been found, but somc studies halc shou'nevidenceof possiblcassociations rvith particular allelic vrriants, lbr cxamplc thc 5-hvdroxl-tr-vptamincty.pc2a rcccptor The highestrisk factor for schizophreniais for the monozr.gotic trvin of an affected co-tu'in, follorved b1' being the child of trvo schizophrcnic parents.A similar risk also applies to individuals *,ith deletion 22ql I (DiGcorge/Sedlidkovi/velocardiofacial) s-vndrome.This chromosomeregion has therefore been of grcat interest and svstematic approacheshave focused on thc genes cncodingPRODH (proline dehydrogenase) and CONIT (catccholO-methr.l transferase).

A scriesof studies combining linkage and associationanal-Yses hrr,e idcntified promising candidate genes (D71VBPl, NRCI, G72/ G30, TRARI). Thc cvidencefor tu'o of these(DTNBPI and A'RG1) is strong A secondsericsof studiescombining association u,ith lunctional investigationofchanges in the associatedgenein schizophreniahavc identified severaladditional candidategenes (RG.Sl, PPP3CC, ZDHHCS,,1K7',1). Oncc ag;ain,horver.er,promising rcsults from some studies havenot been replicatedin othcrs, and it is clear that there are no simple solutions to thc cause(s)of schizophrenia.

DISEASE ALZHEIMER Dcmentia is characterizedb1-an irreversiblc and progrcssive globll impairmcnt of intcllect) memorvr social skills and control of emotional reactionsin the prcsenceof normal consciousness. to Dcmcntia is ctiologicall-vheterogeneous,occurring secondaril-v and both l larietr,ofnon-genetic causes,such as r-asculardisease int-ectionssuch asacquiredimmune deficicnc-vsvndrome(AIDS), as *'ell rls eieneticcauses.Alzheimer discase(AD) is the most common cause of dcmentia in persons rvith either earl-v-onset clcmcntia(at xgc lessthan 60 1'ears,or presenile)or late onset (ag;c abole 601ears,or senile).Thc classicneuropathologicalfinding in pcrsons lvith AD is the prescnceat post-mortem examination of'am1'loicl dcposits in neurohbrillarl' tanglcs and neuronal or senile plaques. In addition, indir.iduals rvith Dou'n s-vndrome harc an increasedrisk of developingdemcntia(p 261)' which, at autops\:,has identical central nervous svstemfindings to those seenin pcrsonsl ith t1'picalAD

EPIDEMIOLOGY Limited numbers of studies of the incidence and prevalenceof AD are availableorl,ing to problems of ascertainment.Although it hasbcen suggcstedthat cnvironmental factorscould havea role in etiologl.,the risk of der.elopingAD increasesdramaticalll'with agc('lable 15.7).

TWINAND MILYSTUDIES Difl'crcnces in the age of onset of AD in identical trvins are consistcntrvith the importanceof environmentalfactors,but there arc clifficultieswith famill' studiesin AD \'Ianv studiesare based on a clinical diagnosis.Holet'er, a signilicant proportion of persons uith rr clinical diagnosisof AD are found to have other causesat Attempts xutops\ such ascercbrovascularatheroscleroticdisease. to conlirm diagnosesin relatives rvho have died previously are Obviousll',giventhe ageofonset, it is generalll' often unsuccessful. ncither practicalnor possiblcto obtain funding for prospectivestudies of the risk to offspring. Thcrcfore, famill' studies of the risk to siblingsarc the onlv practical trpe of famill'studv to provide reliable data Although there arc numerous retrospcctive reports of llmilies rvith AD that are consistent with autosomal dominant

229

15

GENETIC FACTORS IN COMMON DISEASES

T a b t e1 5 . 7 E s t i - n a t eos{a g e s p e c i f rccL - r u . a L , v e p r e v a t e n coef d e m e n t i a Age interval (years)

Prevalence(%)

<70

13

70-74

23

75-79

04

B0-84

153

85-89

231

90-94

429

>95

509

FromHestonL L 1992 Atzheimer's diseaseIn:KingRA,RotterJ I MotulskyA genetic G (eds)The basisofcommondiseases OxfordUniversity Press,Oxford. Ch 39

inheritance,recurrencerisksin a number ofstudies for first-degrec relativesare lessthan 10o/o.The risks are agerelated and greater the younger the ageofthe diagnosisin the affectedindividual.

B I O C H E M I CSATLU D I E S The am.vloiddepositsin the neurofibrillarl,'tanglesand neuronal plaqueshavebeenshownto consistof the amvloid B A,1precursor protein (APP) The major protein componentof the neurofibrillary tangles has been shown to be derived from a microtubuleassociatedprotein (MAP) calledtau (t). Along rvith other MAPs, it interacts with B-tubulin to stabilizemicrotubulcs.

S I N G L E . G E NDEI S O R D E R S The identificationof APP in the amyloid depositsof rhe neuronal plaques, its mapping in or near to thc critical region of the distal part of chromosome 2lq associatedwith the phenotypic features of Dolr,n sl,ndrome (p. 261) and the increasedrisk of AD in persons rvith Dorn s.vndromelcd to the suggesrion that duplication of the APP genecould be a causeof AD. This mechanism as a causefor familial AD has been found to be the exception rather than the rule, although a small proportion of families with earlv-onsetAD show linkage to the APP locus. Of these families, only a limited number have been found to have mutations in the IPP gene Evidenceof linkagewasfound for anotherlocus for earlv-onset AD mapping to the long arm of chromosomel4q Mutations \\'ere identified in a proportion ofaffected individuals in one ofa novelclass of genes,knorvnaspresenilin-1,norvknown to be a component of

230

the notch signaling pathrvay(p. 8a).A second gene, presenilin2, with homologv to presenilin-l, was mapped to chromosome 1q and has been shorvn to have mutations in a limited number of families r'vithAD. Presenilin-1 and -2 are inteeral membrane

proteinscontainingmultiple transmembranedomainsthat localize to the endoplasmicreticulum and the Golgi complex.A largenumber of mutations in the presenilin-l gene have been identified and appear to account for up to 70o/ctof earlv-onsetAD. All of the preseniledementiasfollorvingautosomaldominant inheritance demonstratehigh penetrance.

SUSCEPTIBILITY GENES In the early 1990s,linkagestudiesidentifieda susceptibilit-v locusto late-onsetAD in a region of the long arm of chromosome 19q, lr,hich lr,askno\\n to contain the apolipoprotein E gene (p. 181). The apolipoprotein E gene is polvmorphic, with three major protein isoforms, e2, e3 and e,+.A number of studies in various populationsand ethnic groups haveshownan increasedfrequency of heterozl'gosity-and homozvgosity for the e4 allele in persons rvith both sporadic and late-onsetfamilial AD. In addition, the e2 allele has been sho\\,nto be associatedrvith a decreasedrisk of developingAD. The finding of apolipoproteinE in senileplaques and neurofibrillar-vtangles,along with its role in lipid transport, possiblf in relation to the nerve injury and regeneration seen in AD, provides further evidence for a possible role in the accelerationof the neurodegenerativeprocessin AD. In addition, there is an associationrvith pol-vmorphismsin the crr-macroglobulin gene and the LDl-related protein, both of rvhich interact with components involved in the clearanceand degradationof lmr loid protein. Although it is clear that the APOE e4 allele is the most significant risk factor for late-onsetAD, found in up ro 40o/oof cases,the strongestassociationis rvith the ageof onsetrather than 'the absoluterisk of developingAD. APOE e,[ allele is therefore neither necessarv nor sufficient for the development of AD, emphasizingthe importance of other geneticand environmental etiological factors. This underlies the recommendation of the US National Institute on Aging and the Alzheimer Association Working Group that IPOE testing is zol used to predict whether an asvmptomaticperson is likelv to der,elopAD. It is evident that other geneticrisk factorscontribute to late-onset Alzheimer disease.NIore than 30 putativeloci havebeenreported, but none has been consistentlvreplicatedin other studies.

HEMOCHROM OSIS Hemochromatosisis a common disorder of iron metabolism that results in accumulationof iron The liver is the most commonlv damagedtissue,r'r'ithiron depositionleadingto cirrhosisand liver failure. Patientsare at increasedrisk ofhepatocellular carcinoma. Other organs that may be affected include the pancreas,heart, pituitar.vgland, skin and joints.The iron overloadis easilytreared by' venesectionand this is very effective at reducing morbidity and mortalitrr The ratio of affectedmales:femalesis 5:1, and the diseaseis underdiagnosedin the general population but overdiagnosedin patients rvith secondaryiron overload.

lN C0lvll'4ON DISEASES GENET CFACTORS

L I N K A GAEN DG E N E I D E N T I F I CI O N In 1996thc HIE gene\\as discoveredcloseto the HLA region on 6p21. T\r'o r,ariantswere described, C282Y and H63D. Between 85()6 and 100orb(depending on thc population) of affected individuals rvere found to be homozvgous for C282Y, and the carrier ftequencv in northern Europe is approximatelv I in 10 H63Dismorc commonin the generaipopulation,and homozvgositr for this variant is associatedr,ith onl1,a modest increased(about fourfold) risk of hcmochromatosis.Compound hetcrozvgositvfor C282 Y andH 63D is associated rvith rcduced penetrance;onlr I o/o are thought likel-vto developsvmptoms.Homozl.gositvfor C282Y was thought to confer a high risk of hemochromatosisand it was suggestedthat population scrcening rvould be useful as the iron overload is casily'treated.Horvever,recent populationbased studies have suggestedthat the penetranceof hereditar-v hemochromatosisdue to homozvgosity for C282Y may be as low as 1(/o

ESTING G E N E TTI C Genetic testing is no\v commonplacein families rvherean index casc is found to bc homoz--vgous for C282Y or compoundlv heterozrgous for C282Y and H63D. Current strategv involves first testing the spouse for carrier status, as there is a I in 10 chancethev s'ill be hetcroz-ygous for C282Y.If the offspring har,e inherited a predisposing genotvpe,annual monitoring of serum

V E N O UT SH R O M O S I S Venousthrombosisrepresentsa maior health problem worldwide, r,ith increasingincidencefrom I in I 00 000 during childhood to 1 in 100in old age.Venousthromboembolism,including deep-vein thrombosis and pulmonarv embolism, is a complex diseasethat resultslrom multiple interactionsbetrveeninherited and acquired risk factors(Box l5 3) Inherited thrombophilias alsoincreasethe risk of fetal loss- both stillbirths and earl-vmiscarriages.

I D E N T I F I CI O NO FG E N E S In the last decadethe identification of two mutations prevalent in European caucasianshas pavcd the way for a number of large cohort studies that have increasedour understanding of venous thrombosis.The factorV Leiden mutation (R506CDrenders the factor\i protein resistantto cleavagebv activatedprotein C, thus increasinggenerationof thrombin. Thc prothrombin variant 20210G>A is located in the 3' untranslatedterminal region (UTR) and is associatedwith increasedprothrombin levels. Thcse variants confer a four- to fir'efold increased risk of state(Table 15.9),but individuals thrombosisin the heteroz-vgous homozl'gousfor one or heterozvgousfor both are at significantly cicvatedrisk (up to S0-fold).

ferritin ler,elsis recommendedin order to dctcct iron ovcrload at an early.stirgeand initiate prompt treatment

G E N E T ITCE S T I N G

G E N E TH I CE T E R O G E N E I T Y

Nlore than 50o/oof casesof venous thromboembolism can be erplainedb.vf-actor\rLciden and the prothrombin variant.Testing is now commonplace,but therc is no evidencethat the detectionof a heritable thrombophilic defect alters managementof the index

H e m o c h r o m a t o s i si s a g e n e t i c a l l vh c t e r o g e n e o u sd i s o r d e r (Tablc 15 8) rvith mutxtions also reported in the transferrin receptor 2 gcne and the SLCl1Al gene, u,hich cncodcs ferroportin In addition to the common recessiveadult-onset form, there is a rare juvenile form rvith iron overloadand organ failure befbrc the rrgeof 30vears, s''hich is fatal if untreated N c o n a t a l h e m o c h r o m a t o s i si s a s e v c r e f o r m o f u n k n o w n etiologl.

case.llol'ever, if an inherited predispositionis identilied, testing can be offered to lirst-degree relatives.Those who test positive mar,be able to reduce their risk of developingthrombosis b1';for example,short-term thromboprophylaxisin periods of increased thrombotic risk, such as surger\. Knorvledge of a genetic predispositionto thrombosiswill alsoinfluencethe choiceof oral contraccDu\res.

T a b t e1 5 . 8 G e n e t ihce t e r o q e n e i nt vh e m o c h r o m a t o s r s Type

lnheritance

Gene

Chromosomelocation

Age of onset(years)

HFE

Autosomalrecessive

HFE

6p213

40-60

HFE2A

Autosomal recessive

HJV

1q21

<30(uvenite)

IFE2B

Autosomalrecessive

HAMP

1 9 n 1l

<30(juvenrLe)

HFE3

Autosomal recessive

TFR?

7q22

40-60

u = =/

Autosomaldomrnant

SLC40A13

2q32

Upto60170"

o J p t o 6 0 y e a r sr r n - a l e sa r d T 0 y e a r si n f e r n a . e s

231

15

GENETICFACTORS IN COMN4ON DISEASES

(FLC), a protein that forms part of the denseprotein-lipid matrix that both preventsepidermal water lossand protectsagainstinvasion

B o x 1 5 . 3 I n h e r i t eadn da c q u i r ecda u s e so f v e n o u s thrombosis Inherited Common FactorV Leden(R506Q) Prothromb n variantG20210A lomozygousC677Tmutationn the methytenetetrahydrofolate reductase gene(MTHFR) Rore Antithrombin defc ency Proten Cdeficiency Proten S deficiency Acquired Surgery andtrauma Prolonged immobrlrzation Previous lhrombosis Pregnancy 0raLcontracept'ves or hor.rorereplacere-rt therdpy O t d ear g e

b1-chemical and infectious agents.Patients with full-blorvn IV were homozygous, or compound heterozygotes, for these mutations, whereasmildly affected patients were heterozvgous for a FLG null allele. Extending the study to two populations in the British Isles, the same group showed a signilicant association between the ^FZG mutations and childhood eczema and/or asthma, with onc or other of the mutations present in approximately 9oloof the populationoverall Whilst this is only part of the explanationfor atopicdisease, mutationsin ,FZGarea very important predisposing factor, and illustrate the point that a very common condition, rvhich would have been thought to be highly multifactorial/ polvgenic,may still be explained,in part, by mendelian genetics. Furthermore, hopes have been raised for novel therapeutic approachesin eczemaif, for example,a pharmaceuticalpreparation of filaggrin can be applied topically to the skin

OPICDIsEASE

AGE-REL EDMACULAR D E G E N E RI O N

Atopic disease, rvhichincludesasthma,eczemaand allergicrhinitis, has shown a genuine increasein frequencl' over the past two to three decadcs,giving rise to much speculation about changing envrronments,atmospheric pollution, altered immunological responseand reduced childhood exposure to infectious agents Up to 20oh of the population in the der.elopedlorld norv has some form of atopic disease. Despite this epidcmiologicalincreasein the frequencyof atopic diseasc,trvin and familv studies have shown that atopic eczema is heritable to a high degree.The natural focus of artenrion in terms of researchhasbeen the immune svstem,but the other kev component is the integritv of the physical barrier in the skin A verv marked skin barrier defect is seenin the condition knorvn as icthvosis vulgaris (IV), which often clusters in families. Atopic dermatitis, or eczema,is frequently observed in individuals rvith mild IV Starting with a number of IV families,Palmer and colleagueshaverecently shorrn that affectedindividuals haveone of tu,o loss-of-function mutations in the geneencoding {ilaggrin

Age-related macular degeneration(AMD) is a leading causeof vision loss and blindness, affecting around 50 million elderly p e o p l e t h r o u g h o u t t h e w o r l d . A M D i s c h a r a c t e r i z e db v a progressive loss of central vision attributable to degenerative and neovascularchangesthat occur at the interface between the neural retina and the underl-r-ingchoroid. E t i o l o g i c a l r e s e a r c h s u g g e s t st h a t A N { D i s a c o m p l e x multifactorial disease.Familial studies have provided strong cvidencefor the heritabilitl.of AMD, with a geneticcontribution suggestedin up to 25o/oof cases.Whole-genome linkage scans haveimplicated nearlv everv chromosomein the human genome, but the strongest linkage evidence \\,as seen at chromosomes l q 2 5 - 3 1a n d 1 0 q 2 6 . In 2005, an associationstud1,of 900 AMD casesand 400 controls was performed to investigatevariants rvithin the 11F1 gene located within the lq25-31 interval Previous studies had implicated the activation of complement in the formation of

T a b t e1 5 . 9 F r e q u e r coyf f a c t o - VL eo e na n dp r o L h r o - n bT;A n 2 l A A v a r i arnnt l . eU Kp o o l t a o t n a n dc o r r e s p o n d ;rni g s ko f v e n 0 u sI n [ 0 m D o S t s

232

Riskfactor

Status

Populationfrequency

Approximateincreasedrisko

F a c t o r VL e i d e n( F V L )

nererozygous rlOmOZygous

4%l1in 25) (1in 625) 015%

10-80

Prothrombin

ileterozygous nomozygous

2%(1in 50) 0 04%(1in 2500)

38 Notknown

F V La n d p r o t h r o m b i n

Heterozygousfor both

0 0B%('1in 1250)

20

o T h e s ev a l u e s a r e i n c r e a s e di n t h e p r e s e n c eo f a n t i t h r o m b i np,r o t e i nC o r p r o t e i nS d e f i c i e n c y

/.4

DISEASFS GENETIC FACTORS INCOMMON

drusen, the hallmark lesion ofAMD. The HFI geneencodesfactor H, the major inhibitor of the alternative complement pathway, which accumulates within drusen. This association study identifed two common missensevariants,I62V and Y402H, that were highly correlated with AMD. Haplotype analysisshowed that multiple HFI variants confer increasedor reduced risk of developingAMD One common at-risk haplotype is present at a frequency of 50o/oin patients with AMD and 29o/oin conrrols (odds ratio 2 46) Homozygotes for this haplotype account for 21o/oof casesand 8o/oof controls (odds ratio 3 51) Several protective haplotypeswere also identilied. The dataobtainedin this study may alsoprovide insight regarding the roles of establishedrisk factors for AMD, such as smoking the most consistentlydocumentedAMD risk factor. Cigarette smoke has been shown to activate the alternative complement pathway through the modification of C3 in aitro. Similar processes acting in xit;o could promote inflammatory events in the eye that hastendrusen formation and exacerbatethe geneticsusceptibiliq/ to AMD that is conferred by the at-risk HFI haplotype. The results of this investigationprovide strong evidencethat a specificcommon haplotype of the complement regulator F1F1 predisposesindividuals to AMD. It is thought that this genetic predisposition, when combined rvith a triggering event, such as infection, may underlie a major proportion of AMD in the human population. Thus, moleculesinvolved in complement activationand its regulation are now prime targetsfor therapeutic intervention in AMD.

FURTHER READING Adams P C 2002Hemochromatosis: clinicalimplications Medscape Gastroenterology eJournal4 A summar.1,0J'tlinital as?ectsoJhemochromatosis and the role ofgenetit testing. Glo.vnA L, \\'eedon M N, OrvenKR et al 2003Large-scaleassociationstudies of variantsin genesencodingthe pancreaticbeta-cellKATP channel subunits Kir6 2 (KCNJ I 1) and SUR1 (ABCC9) confirm that the KCNI1 E23K variantis associated with type 2 diabetesDiabetes52. 568-572 The resultsof a large-scaleassonationstudyand meta-anulysisof pretious studiesto conJirmthat E2.?Kincreases nsb oJ t.ype2 diabetes. Grant S F, ThorleifssonG, ReynisdottirI et al 2006Variantof transcription factor 7-like 2 (f CF7L2) geneconfersrisk of type 2 diabetesNat Genet

38:320-323 The latestgeneticriskfactor identtfedfor tytre2 d,iabetes HagemanG S, AndersonD H, JohnsonLV et al 2005A common haplotype in the complementregulatorvgenefactor H (HFl/CFH) predisposes individualsto age relatedmaculardegenerationProc Natl Acad Sci U S A 1 0 2 : 7 2 2 77 2 3 2 A recentpublicationdescribingthe identtficationrariants in the HFI genethat are assotiatedpith age-relatedmaculardegeneration Heston L L 1966Psvchiatricdisordersin fosterhome rearedchildren of schizophrenicmothers Br J Ps.vchiatr.v 112:819-825 p(Llerdemlnstratinggeneticfactlrs in the etiologyoJschizophreniu. A classic HugotJ P,ChamaillardM, Zouali H et al 2001Associationof NOD2 leucinerich repeatvariantswith susceptibilityto Crohn'sdisease Nature4ll:599-603 Descriptionofthe lositional cllning stra.tegythat led,to the identtJitationofthe NOD2/CARD15 susceptibilitygenefor Crohndisease. JamesonJ L (ed ) 1998Principlesof molecularmedicine HumanaPress, Totorva,Nerv Jersev A largemuhiauthortefi coxeringin detailthemolecularbasisof thecommondiseases as well as many oJ the mlre clmmln single-gene and chromosome disorders

Ogura\ Bonen D K, InoharaN et al 2001A frameshiftmutation in NOD2 with susceptibilityto Crohn'sdiserse Nature 411: 603-606 associated Dexription ofthe positionaltloning strateg.ythat led to the identif.cationofthe genefor Crohn disease. NOD2/CARD15 susceptibiht.y Palmer C N A. Irvine A D Terron-Kwiatkowski A et al 2006 Common lossof-function variantsof the epidermalbarrierprotein filaggrinarea major predisposingfactor for atopicdermatitis.Nature Genet 38: 441446 thegeneticbasisof Kt.y paler lroriding a major breakthroughm understnnding atopicdisease. Plomin R, De FriesJ C, McClearn G E, Rutter M 1997Behavioralgenetics W H Freeman,NewYork Muhiauthor text couring Ihe alprodchesto studling behaxioralgenetirs SeligsohnM D, LubetskyA 2001Geneticsusceptibilityto venous thrombosis N Engl J Med 344: 1222-1231 Comprehensiae r et;ieaof heredit aD) t hromboph lia WeedonM N, McCarthy M I, Hitman G et al 2006Combininginformation the from multiple common susceptibilitypolymorphismsincreases predictivepowerof geneticinformation:a study of replicatedtype Z diabetesvariants PLoS Med 3 to Recente-publitationdescribmgthe fficts oJ'multiplerariants thQ,tPredisqose 4tpe2 diabetes Wicker L S, Clark J, FraserH I et al 2005Type I diabetesgenesand pathways sharedby humansand NOD mice JAutoimmun 25(Suppl):29-33 RexiewoJ'thexalue o/ the NOD mousemodelin identifuingsuveptibiliqygenes Ji)r 4/fe I diabetes

ELEMENTS Q eotn genetic and environmental factors are involved in the etiology of many of the common diseasesaffecting humans.The geneticfactorscan be consideredto be due to an inherited predispositionor genetic susceptibility. @ fne geneticcontribution in a particular condition can by studying the incidenceof diseasein relatives, be assessed comparingconcordanceratesin identical and non-identical trvins, studying differencesbetween populations and the effects of migration, studying the effects of adoption, biochemical studies, evaluating any possible association with other inherited factors and studying animal models. @ nor common disorders, such as diabetesmellitus, hypertension,coronaryartery disease,age-relatedmascular degeneration, schizophrenia, autism, hemochromatosis, \renousthrombosis,the common cancersand the common congenitalabnormalities,a multifactorial mode of inheritance is most likely. However, it is alsobecoming clear that many of theseconditions are heterogeneous,that is, they are caused by a variety of geneticand environmental factors. @ Preventionof the common diseasesinvolvesdetermining causativeenvironmentalagentsaswell as identifying those individuals who are geneticallysusceptibleto such agents. @ Genetic risk factors for diabetes, Crohn disease, Alzheimer disease,hemochromatosis,venous thrombosis and age-relatedmacular degenerationhavebeen identified. Advances in technology together with knowledge of the human genome sequence,promises to expedite the discovery of further genetic variants that predispose to common diseases.

233

235

CHAPTER

Congenitat abnormatities syndromes anddysmorphic t-'

'The.v certainlv give very strangenamesto diseases.' Plato The formation of a human being, a processsometimes known involves an extremely complicated and as yet as morphogenesls, incompletelyunderstoodinteractionof geneticand environmental factors.Holever, our knowledgein this areais increasingrapidly (Ch. 6). Given the extraordinary complexit-vof this processit is not surprising that on occasionit goeswrong. Nor is it surprising that in manl'congenital abnormalitiesgeneticfactors can clearll, be implicated Approximately 2400 dysmorphic syndromes are describedthat are believed to be due to molecular pathology in single genes,and for about 500 the geneshavebeen identihed and a further 200 or more mapped. A further 500 or so sporadicall-v occurring s-vndromesare recognized,for which the precisecause remains elusive In this chapter we shall consider the overall impact of abnormalitiesin morphogenesisby reviewing: l. The incidence of abnormalities at various staqesfrom

Chromosome abnormalities such as trisomy' monosomy or triploidy are found in about 50o/oof all spontaneousabortions' This incidencerisesto 600lowhen a grossstructural abnormality is present and it is very likely that submicroscopic or de notto singlc-gene abnormalities account for a proportion of the rcmainder.

ES ITALABNORM ALITI CONGEN MORTALITY AND PERINATAL Perinatal mortalitl' frgures include all infants who are stillborn after 28 weeks' gestationplus all babieswho die during the first week oflife. Studies undertakenin severalcentersin Europe and North America have shown that 25-30o/o of all perinatal deaths occur as a result of a serious structural abnormality. In 80o/oof thesecasesgenetic factors can be implicated, with a recurrence risk to future pregnanciesof 1oloor more' The relatite contribution of structural abnormalities to perinatal mortality is lower in

conception onwards 2. Their nature and the wavs in which they can be classified 3. Their causes,when known, with particular emphasison the

developing countries, where environmental factors play a much grcater role.

role of genetics.

INFANTS NEWBORN

INCIDENCE IMESTER FIRsT.TR SPONTANEOUS LO55 PREGNANCY It has beenestimatedthat around 50o/oof all human conceptions are lost either before implantation at 5-6 days' postconceptionor shortly afterwards,beforethe woman realizesthat sheis pregnant. Among recognizedpregnanciesat least 157oend in spontaneous miscarriagebefore l2weeks'gestation. Even when material from the abortus can be obtained, it is often very difficult to establish why a pregnancy loss has occurred. Horvever,careful study of large numbers of spontaneouslyaborted embryos has shown that gross structural abnormalitiesare present in 80-85o/o.These abnormalities varv from complete absenceof an embryo in the developingpregnancy sac- a blightedocum to a very distorted bod-vshape,or a specificabnormality in a single body system.

Surve.vsreviewing the incidence of both major and minor anomalies in newborn infants have been undertaken in many parts of the world. A maior anomaly is defined as one that has an adverse outcome on either the function or the social acceptability of the individual (Table 16.1). In contrast, minor abnormalities are of neither medical nor cosmetic importance (Box 16.1). However, the division between major and minor abnormalities is not alway'sstraightforward; for instance, an inguinal hernia occasionallyleads to strangulation of bowel and alwa-vsrequires surgical correction' so there is a small risk of serioussequelae. These surveyshaveconsistentlyshown that 2-3oloofall newborns have at least one major abnormality apparent at birth. The true incidence,taking into account abnormalitiesthat present later in life, such as brain malformations,is probably closeto 5olo Nlinor abnormalitiesarefound in approximatelyl0o/oof all newborn babies' If trvo or more minor abnormalities are present in a newborn infant there is a l0-207o risk that the baby will also havea maior malformation.

237

16

CONGENITALABNORMALITIES ANDDYSMORPHIC SYNDROMES

Systemand abnormality

lncidenceperi000 births

Cardiovascutar

10

Ventricular septaldefect

25

Atrialseptaldefect

1

Patentductusartenosus

1

Teiratogy ofFallot

1

Centrat nervoussystem

10

dying in early infancy, 25olohaving subsequentmental or physical disability, and the remaining 50o/ohaving a fair to good ourlook after treatment

CHILDHOOD MORTALITY Congenitalabnormalitiesmakea significantcontribution to mortality throughout childhood. During the first yearof life approximately 25o/oof all deathsare the result of major structural abnormalities. This value falls to 20olobetweenthe agesof I and l0years and to around 7 .5o/ofor children agedbetween 10 and 15 years. Taking into account the incidence of major and minor abnormalitiesnoted in newborn surveysand the high incidence

Anencephaty

1

Hydrocephaty

j

Microcephaly

1

Lumbosacral sprnabifrda

of defects observed in early spontaneousmiscarriages,it is evident that at least I 5oloof all recognizedhuman conceptionsare

2

Gastrointestinal

4

structurally abnormal (Thble 16.2). It is probable that genetic factors are involved in the causation of at least 50o/oof all structural abnormalities.

Ctefttip/patate

15

Diaphragmatic hernia

05

Esophageat atresia

03

lmperforate anus

02

Limb

2

Transverse amputatron

02

UrogenitaI

4

Bitateral renalagenesis

2

Potycystic kidneys(infantrle)

0 02

So far in this chapter the terms congenitalabnormalit.yand birth defecthave been used in a general senseto describe all types of structural abnormality that can occur in an embrvo, fetus or newborn infant Although these terms are perfectly acceptable for the purpose of lumping togetherall theseabnormalitieswhen studying their overall incidence,they do not provide any insight into possibleunderlying mechanisms.N{ore specific definitions

Bladderexstrophy

0 03

have been devised that have the added advantageof providing a combined clinical and etiologicalclassification.

Box 15.1 Examples of mrnorcongenital structuraI abnormaLities

238

The long-term outlook for a baby with a major abnormality obviously depends on the nature of the specificbirth defect and whether it can be treated successfully.Thken toBether, the overall prognosis for this group of newborns is relatively poor, withZio/o

Preauricutar pitor tag Epicanthic fotds Lacrimat ductstenosrs Brushfield spotsintheiris Lrppits patmarcrease Sing[e Fifthfingerctrnodactyly Syndactyty between second andthirdtoes Supernumerary nipple Umbiticathernia Hydroce[e S a c r a l poi tr d i m p l e

DEFINITION AND CLASSI FICATION OFBIRTHDEFECTS

Tabte16.2 Incidence

(%)

Spontaneousmiscarriages Firsttrimester Secondtrimester

B0-85 25

Att babies Majorabnormality apparent at birth Majorabnormatrty apparent later Minorabnormality period Deathin perinatal Deathin firstyearof tife Deathat1-9years Deathat10-'14years

2-3 2 10 25 25 20 7.5

CONGENTAt ABNORMALITES,ANDDYSMORPHICSYNDROME5

S I N G LAEB N O R M A L I T I E S Single abnormalities ma1''have a genetic or non-gcnctic basis The system of terms used helps us to understand the different mechanismsthat might be implicated,and thesecan be illustrated in schematicform (Fig. 16.1).

n C on c e p t i o

15

Birth

Normad l evelopment

Malformation

Malformation A malformutioz is a primarv structural defect of an organ, or part of an organ, that results from irn inherent abnormality in der.elopment This used to bc knorvn as a primarl- or intrinsic malformation. The prescnceof a malformation implies that thc earlv developmcntof a particular tissucor organ hasbeenarrested or misdirected Common examplesof malformations include congenitalheart abnormalitiessuch as ventricular or atrial septal defects,clefi lip and/or palate,and neural tube defects such as anencephal.v or lumbosacralmvelomeningocele(Fig. 16.2).N4ost malformations inr,olr,ingonlv a single organ sholv multifactorial inhcritancc, imply-ing an interaction of manv gencs r,vith othcr factors(p. 136).Nlultiple malformationsare more likely to be due to chromosomalabnormalities.

Dis r u p t i o n The term disruptionrefers to an abnormal structure of an organ or tissue as a result of cxtcrnal firctors disturbing the normal developmentalprocess.This used to bc knol'n as a sccondar-vor cxtrinsic malformation. Extrinsic factors thltt can disrupt normal developmentinclude ischemia,infection and trauma.An example of a disruption is the effect seenon limb developmentr'r'hcna strand or band of irmnion becomcscntrvined around a bab1.'sforearm or digits (Fig 16 3) 81'delinition a disruption is not glenetic, although occasionallrgeneticlactorscanpredisposeto disruptivc events For

Dysplasia

Fis.16.1 of the differentmechanismsn Schematc reoresentation m o r p h o g e n ess F o rm a l f o r m a t t o dn i.s r u p t i o an n dd y s p l a s i a . t h e b r o k e nLn e s y m b o l z e sd e v e t o p m e n tpaoLt e n at t r a t h e r t h a n of the defect.whlch m ght be Laten timingof the manifestatjon e m b r y o g e n e s i(sA d a p t e fdr o m S p r a n g eer t a t 1 9 B 2E r r o r so f m o r o h o o e n e s icso: n c e p t as n dt e r m s . JP e d l a t r " l 01O6, 0 - 1 6 5 )

examplc, a small proportion of amniotic bands arc causedbY an underlving geneticalll- determined defect in collagen that rvcakcnsthc amnion, making it more liable to tear or rupture spontan(]oush'.

Fig.15.2 r y e . o r e r , n g o c e .ceo n ss t r ^ go ' p r o t r J dn g s o ; n acl o r dc o v e r e db y m e n i n q e s C ^ r Lw d i L na . a r g et h o r a c o l u - b a m

239

15

CONGENITALABNORMALITIES ANDDYSMORPHIC SYNDROMES

Fi9.15.3 Hand(A)andfoot(B)ofa baby (Courtesy wrthdigiiat amputatrons duetoamniotrc bands showing residual strands ofamnron ofDrUna MacFadyen, Leicester Royat Infirmary.)

Deformation

Dysplasia

A deformationis a defect resulting from an abnormal mechanical force that distorts an otherwisenormal structure.Well recognized examples include dislocation of the hip and mild 'positional'

A dysplasia is an abnormal organization of cells into tissue. Usually the effectsare seenin all parts of the body in which that particular tissue is present. For example, in a skeletaldysplasia such as thanatophoricdysplasia,which is causedby murations in FGFR3 (p. 9 I ), almost all parts of the skeletonare affected(Fig. 16.5). Similarly, in an ectodermal dysplasia, widely dispersed

talipes ('club foot') (Fig. l6 4), both of which can be causedby Iack of amniotic fluid (oligohydramnios)or inrrauterine crowding due to twinning or a structurally abnormal uterus. Deformations usually occur late in pregnancyand conveya good prognosiswith appropriatetreatment, for instancegentle splinting for talipes,as the underlying organ is fundamentally normal in structure.

tissuesofectodermal origin, such ashair, teeth, skin and nails,are involved (Fig. 16.6). Most dysplasiasare causedby single-gene defectsand are associatedwith high recurrencerisks for siblings and,/or offspring.

MULTIPLEABNO RMALITIES Sequence The most logical and easily understood pattern of multiple abnormalities is the concept of a sequence. This describes the findings that occur as a consequenceof a cascadeof eventsinitiated by a single primary factor. This can often be a single organ malformation.In the'Potter'sequence,chronic leakageof amniotic fluid or defective urinary output results in oligohydramnios (Fig. 16.7).This, in turn, leads to fetal compression,resulting in squashedfacial features,dislocation of the hips, talipes and pulmonary hypoplasia (Fig. 16.8), usually resulting in early neonatal death from respiratory failure.

Syndrome

Fis.15.4 240

LowerLimbs ofa babywithtalipesequrnovarus

In practice the term synrlromeis used very loosely (e.g.the amniotic band 'syndrome'), but in theory it should be reserved for consistentand recognizablepatterns of abnormalities for which there will often be a known underlying cause.These underlying

SYNDROMES CONGENITALABNORI'4ALTIESAND DYSMORPHIC

16

Fis.15.5 bodiesandcurvedfemora shortribs,ftatvertebraI A. Infantwith thanatophoric dyspLasia B, Radrograph oftheinfantshowing

Fis.16.6 Hair (A)and teeth(B) of a malewith ectodermaldvsplasia

causescan include chromosome abnormalities, as in Down syndrome,or single-genedefects,asin theVan der Woude syndrome, in which cleft lip and/ or palateoccurs in associationwith pits in

diagnosisof individual syndromeshasbeen greatly helped by the development of computerized databases(see Appendix). It is possibleto obtain a list of differential diagnosesby providing the

the lower lip (Fig. 16.9). Several thousand multiple malformation syndromes are now recognized.This field of study is known as dysmorphology.The

searchfacility of the databasewith detailsof key abnormal clinical features.Even with the help of this extremely valuablediagnostic tool. there are, unfortunately, many dysmorphic children for

211

16

CONGENITALABNORN4ALIT] E5 AND DYSMORPHICSYNDROMES

R e du c e d u n n a r y0 u r p u r

D i s l o c a t i oonf h i p sa n dt a l i p e s

Fig.15.9 Posteriorcleftpatateand lower lip pits n a ch td with Vander W o u d es y n d r o m e

Fis.16.7 T r e ' P o l t e - s e q J e n c es n o w i n gt h e c a s u a o o e f e v e - t s. e a dn g r o a n d r e s u l t i n fgr o m o t i g o h y d r a m n i (orse d u c e vdo l u m eo f a m n i o t i c flurd) rvhom no diagnosiscan be reached,so that it can be very-difficult to provide accurate information about the likely prognosis and recurrcnccrisk (p. 330).

Association Thc term u.ssociution has been introduced in recognition of the fact that certain malformations tend to occur together more often than rvould be expected by chance, let this non-random occurrcnceof abnormalitiescannot be explainedon the basisof a sequenceor a syndrome.The main differencesfrom a svndrome are the lack of consistencvof abnormalities from one affected individual to another and the absenceof a satisfactorl,' underlying explanation. The names of associationsare often acron)rms devisedby juggling the hrst letters of the organsor svsremsmost commonlr involved; for example,theVATER associationfeatures -oertebral,anal, rracheo-esophageal and renal abnormalities. Associationsconvev a low risk of recurrence and are generally thought not to be genctic in origin even rhough the underlying causcis usuallv unknor,vn It is recognizedthat this classificationof birth defects is not perfect It till be readilv apparentthat it is far from being either fullv comprehensiveor mutuallv exclusive.For example,bladder outflou obstruction causedbv a primarl' malformation such as a urethral valve will result in the oligoh.v-'dramnios or Potter scquence,leading to secondarvdeformations such as dislocation of'the hip and talipes.To complicatematters further, the absence of both kidnevs, lhich rlill result in the same sequenceof

Fis.15.8 242

F a ca l a p p e a r a n coef a b a b yw t h P o t t e rs e q u e n c e d u et o o t r g o h y d ' a m r - iaossa c o n s e q u e ^ coe' b r l a r e r ar eI n a la g e n e s i s N o t et h e s q u a s h e d a p p e a r a n ccea u s e db y t n u t e r ac o m p r e s so n

cvents,is usualll'erroneouslvreferred to as Potter syndrome. Despite this scmantic confusion, adhercnceto this classification is important becauseit scnes as an aid to understandingand recurrcncerisks (Ch. l7).

SlYCN D R O M E S A INEDSD Y S M O R P H C O N G ETNA L A B N O R M A L I T

GENETIC CAUSES OFMALFORMATIONS Therc are mlnv recognized causesof congcnital abnormalitr,, although it is notablc that in up to -50(lirof all cirsesno cleu crplanation can be established(Trblc 16 3)

AB CHROMOSOM EN O R M A L I T I E S 'I'hcse

accountfbr approximatclr'6(lirof all recognizedcongcnital a b n o r m a l i t i e s .A s a g e n e r a l r u l c l n r p c r c e p t i b l e d e g r e c o f rntosomll imbalancc, such as duplication, delction, trisomr- or monosom\r l'ill result in severestructural and developmcntal abnorm:rlitr If vcrv ser.erethis can lead to carlv spontaneous miscarriag;e.Common chromosomc svndromes are dcscribed in detail in Chapter lu. It is not kno\rn whether malformations causedb1 a signihcantchromosomeabnormalitl; such asa trisomr', arc thc result of dosagecffects of the indir,idual gencs involr,ed ('additivc' model) or generaldevelopmcntalinstabilitl' caused br a large number of abnormal developmentalgcne products ('interlctir c' model).

S I N G L E - G E NDEE F E C T S T h e s e l c c o u n t f o r a p p r o r i m a t e l r 7 . 5 o , i ro f a l l c o n g e n i t a l abnormalitics Somc of these irre isol:rtcd,that is, thef involve onlr.one orgirnor slstcm (Table16.'l).Othcr single-genedcfccts lesult in multiple congenitll :rbnormalitr, sr-ndromcsinvolving mail orllxns or svstemsthat do not har,canr:obr,iousundcrlf ing embrlological reltrtionship Iror examplc, cctrodact-vlv(Fig 16.10) in isolation can be inherited rrs irn autosomtll dominant trlit, occrsionallv autosomal recessivc,and ralelr X-linked. It can also occur as onc mlnifcstation of thc EL,C sr-nclromc (cctoderm:rldrsplasia,cctrodxctlh and rleft lip/palate), uhich '..Llso shors alrtosomaldominant inhcritance. There is abundant

1

T a b t e1 5 . 4 C o n g e n i t a l a b n o r m a ltiht iaetcs a nb e c a u s e d b v s i n q l e - c e ndee f e c t s lnheritance lsolated CentrolnervoussYstem Hydrocephalus Megalencephaty Microcephaly

XR AD AD/AR

Oculor Anrridia Cataracts Microphthalmia

AD AD/AR AD/AR

Limb Brachydactyty Ectrodactyly Polydactyty

AD AD/ARiXR AD

Other polycystic nfantite

ARkidneYs

Syndromes Anerr

AD

EEC

AD

Meckel

AR

Roberts

AR

V a nd e r W o u d e

AD

Abnormalities

CraniosYnostosts s Y r o a ct Y t Y EctodermaldYsPlasta cteftliP/ eclrodactYtY, Patate EncePhatocete. p o . y o a c t T loyo, l Y c Y sct kidneYs CleftliP/Paiate phocomelia C t e f t i p / p a l a t el i.p p i t s

X-tinked recessive;XR AR,autosomaI dominant; AD,autosomal IVE TECCSS

cr iclenccfron.rthe stucll'of dl smorphologl thrt scveraldilfcrcnt mutrrtions,lllclic ol non-allelic, can causcsimilar if not identical rnllfbrmrrtions.

abnormaLities Tabte16.3 Causesof conqenitaI Cause

Multrfactorial

30-40 6 7.5 20 30

Environmental D r u g sa n dc h e m i c a t s lnfections illness Maternat agents Physical Unknown Total

5-10 2 2 2 1 50 100

Genetic Chromosomal S i n g l eg e n e

Fis.16.10 Appearanceof the feetin a childwith ectrodactyly

243

15

CONGENITALABNORN4ALITI ESAND DYS[/ORPHICSYNDROMES

The importance of identifving birth defectsthat show singlegcne inheritance is twofold From the famil.v'spoint of view it is essentialthat correct counseling is availableso that close relativescan be alerted to possiblerisks. From the academic point of viet! the identification of a molecular defcct causing

X{any examplesof progressin identifl,'ingthe genesthat cause congenital abnormalities could be gir.cn. Holvever, progress has been particularlv impressive in relation to well defined svndromes,and trvo recent examplcsfrom the field of pediatric geneticsare highlighted In both casesrhe function of the gene

In a threc-generationDutch family NS was mapped to 12q22 in 199,t,but it was not until 2001 that mutations were identified in the protein tyrosine phosphatase,non-receptor-type, I 1 (PTPNI I) gene.Attcntion has rurned rapidly ro phenorypegenotvpe correlation, and mutation-positive caseshave a much higher frequenc,vof PS than mutation-negativecases,and very few mutations have been found in patients with cardiomyopathy. However, facial featurcsare simila( whether or not a mutation is found N{utations in PIPN11 account for about half of all cases of NS. Recentlv mutations in the KRl.l gene,and subsequently IIAPZKI and SOS,I genes,havebeen found in a small proporrion of PTPNI I-negativc NS casesThese genesbelong to the same pathwav, known as the RAS-MAPK pathway The protein product of PTPNII is SHP-2 and this, togerher with SOSI

in rclation to r,videsprcad expressionin many tissueshas vet to be dctermined.

positivclv transducessignalsto Ras-GTP, a downstreameffector (Fig 16.12).Thc KR4.S murarionsin NS appearto lead to K-ras

an isolated single-geneabnormalitv in a particular organ can provide a clue as to the locus of a susceptibilitl' gene for similar malformations affecting the same organ that appear to shovr, m u l t i l a c t o r i ai ln h e r i r a n c t

Noonansyndrome First described bv Noonan and Ehmke in 1963, this condition is rvell known to pcdiatricians.The incidcnce may be as high as I in 2000 births, affecting malesand femalesequall1,. Thc clinical features bear a resemblancc to those of Turner svndrome in females - short str.ture, neck r,vcbbing,increased carrying angle at the clbow and congenital heart disease pulmonary s t e n o s i s( P S ) i s t h e m o s t c o m m o n l c s i o n b u t a t r i a l s e p t a l defect (ASD), vcntricular septal defcct (VSD) and occasionallv hypertrophic cardiomvopathvoccur. A characteristicpcctus deformitl,' mav be seen, as rvcll as thc face, which includes hypertelorism, dorvn-slantingpalpebral fissuresand low-set ears (Fig. 16.11) Some patienrshave a mild bleedingdiathesis,and learning difficulties are prescnt in about one-quarter

proteins with impaired responsivenessto GTPase activating (GAP) proteins (see also p. 201) Neurolibromarosis,by far the most common of this group, is dealt rvith later in chapter 18 (p. 289). For manv years clinical dysmorphologists have recognized overlappingf'eaturesbetweenNS and the rarer conditions known a s c a r d i o - f a c i o - c u t a n e o u s( C F C ) s v n d r o m e a n d C o s t e l l o svndrome. Thcse conditions are noru recognized to form part of a spectrum of disorders explained b1' mutations in different components of the RAS-MAPK pathrvay,with each syndrome displafing considerablegenerichererogeneiry(Table 16.5).Many of the mutations are gain-of-function missensemutations, which mav explain the increasein solid tumors in Costello syndrome as well asccllular proliferation in sometissuesin CFC svndrome,e g. h1-perkcratosis. The effect is for RAS to bind GTP, which results in activation of the pathrval.(gain of function). Neurofibromatin is a GAP protein, and functions as a rumor-supprcssor

l

:::rrrr:

Fis.16.11 244

NoonansyndromeA. In a babypresentingwith cardiomyopathy at birth (whichlaterresotved)B, In a ch td C,In a 57-vear-oLd man

ICSYNDROMES TIESAND DYSMORPH CONGENITALABNORN4AL

16

CellMembrane-1

pathwayand Tabte15.5 Genesof the RAS-IVAPK svndromes associated Gene

Noonan syndrome

PTPNll

Commonupto 50%

KRAS

Rare

CFC syndrome

Costetlo sYndrome

Rare

Rare - >50% Common

HRAS S0Sl N u c l e a rm e m b r a n e

Rare

BRAF MAP2Kl

Rare

MAP2K2

Fig.16.12 HRASandKRASareactivated TheRAS MAPKpathway. bySPH-2 - inhrbition (redarrows)Orange andSOS1 arTows Thepathway s d y s ' e q u l a t be ydm u t a t i o n' nsk e yc o m p o n e n.tes s. u l t i nrgr phenotypes thed stinctbutrelated of Noonan syndrome, CFC (seeTabte Costelto syndrome, syndrome, andNeurofibromatosis .- -\ ^' protein rsa GAP(GTPase that 165) Neurofrbromrn actvating) f u n c t ' o nassa T U - o r s u p p r e s s o Mru. r a rR - tA Sp r o t e r ndsr s p . a y mpairedGTPase activity andareresistant to GAPsTheeffect rsfor RASto bindGTPwhlchresuitsin activation ofthepathwav (garo n ff u n c t i o n )

Sotossyndrome First describedin 1964,this is one of the'overgrowth'syndromes, previously knorvn as cerebral gigantism. Birth weight is usually increasedand macrocephall,-notcd.Early feeding difficulties and hvpotonia mav prompt manv investigations and there is often motor delay and ataxia Height progressesalong the top of, or above, the normal centile lines but final adult height is not necessarilv increased. Advanced bone age may be present, large hands and feet, and the cerebral ventricles may be mildll. dilated on magnetic resonanceimaging or computed t o m o g r a p h v .T h e f a c e i s c h a r a c t e r i s t i c( F i g l 6 1 3 ) w i t h a

Common upto 50%

Some

Some

Some

Rare

associatedco-regulator with 23 exons The Japanesefound a small number of frameshift mutations in their patients but, intcrestingll', a stud-vof European casesfound that mutations rvercfar more common than deletions For the large majority of casesthe mutations and deletions occur de noao.

CE RIALINHERITAN MULTIFACTO This accounts for the maioritl' of congenital abnormalities in rvhich genetic factors can clearly be implicated. These include most isolated ('non-s1'ndromal') malformations involving the heart, central nervous svstem and kidneys (Box 16.2).For many of these conditions empirical risks have been derived (p. 337) basedon large epidemiologicalfamily studies,so that it is usually possibleto provide the parents of an affected child with a clear indication of the likelihood that a future child will be similarly affected.Risks to the offspring of patients who were themselves trcated successfullyin childhood are becoming available.These are usually similar to the risks that apply to siblings,as would be predicted b-r-the multifactorial model (p. 136)

HETEROGENEITY GENETIC

high prominent forehead, hypertelorism with dorvn-slanting palpebral fissures,a characteristicnose in early childhood, and a long pointed chin. Scoliosis develops in some casesduring adolescence.Parent child transmissionis rare, probably because most patients have learning difficulties. However, the author has recentll,encountereda family with individuals affectedin at least

It haslong beenrecognizedthat speci{rccongenitalmalformations can have manv different causes(p. 243), hence the importance of tr.ving to distinguish between syndromal and isolated cases This causal diversity has become increasingly apparent as devclopmentsin molecular biology have led to the identification of highly conservedfamilies of genes that plal' crucial roles in

three generations. Among patients with Sotos syndrome (SS) reported to have balancedchromosometranslocationswere two with breakpoints at 5q35 From these crucial patients a Japanesegroup in 2002 went on to identify a 22-Mb deletion in a seriesof SS cases.The

carlv embryogenesis. This subjectis discussedat length in Chapter 6. In this chapter trvo specific malformations, holoprosencephalyand neural tube defects.will be consideredto demonstratethe rate of progressin this field and the extent of the challengethat lies aheadbeforefull

deletion takes out a gene called NSD/, an androgen receptor-

understandineis achieved.

245

16

C O N G E N I TAABLN O R M A L I TAI E NSDD Y S M O R P H SIYCN D R O M E S

Fis.16.13 S o t o s s y n d r o mAe, l n a y o u n g c h i l d w h o h a s t h e t y p ihci ag th f o r e h e al da ,r g e h e a d a n d c h a r a c t e n s t i c t p t o t h e nBo. T se h e s a m en d i v i d u a t a g e dl 8 y e a r s w , i t h l e a r n i n qd i f f i c u e[ ts a n da s p i n a cI u r v a t u T(es c o l i o s i s )

B o x 1 5 . 2 l s o l a t e(dn o n - s y n d r o m a mLa)l f o r m a t i o n t hsa t s h o wm u l t i f a c t o r i n ah l eriLance Cardiac A t r i a ls e p t a ld e f e c t Tetralogyof Fallot Patend t u c t u sa r t e r i o s u s V e n t rc u l a rs e p t a ld e f e c t CentraInervous system A n en c e p ha l y Encepha[ocele 5p na bifrda Genitourinary Hypospadias R e n aal g e n e s r s R e n a ld y s g e n e ss 0ther Cleftlip/patate C o n g e nt a l d i s t o c aotn o f h i p s Talrpes

Hotoprosencepha ly This severcand often fatal malformation is crruscdbr. a failure of cleavageof the embrronic forebrain or prosenccphalon\ormallr, this divides transverseir into the tclcncephalon and the

216

diencephalon The telencephalondivides in thc sagittalplane to form the cercbral hemisphercs and thc olfactor_vtracts and bulbs Thc dienccphalon der.elopsro form thc thalamic nuclei,

thc pineal gland, the optic chiasm and the opric nervcs. In holoprosenccphah-there is incomplete or parrial failure of these devclopmentalprocesses,and in the ser,erealobar form this rcsults ir.ran abnormal facial appearance(Fig. 6 6, p. 87) and profound ncuroder.elopmcntalimpairment. E t i o l o g i c a l l l . , h o l o p r o s e n c e p h a l l .c a n b e c l a s s i f i e d a s chromosomal, syndromal or isolated Chromosomal causes account for around 30-409/oof all cases,rvith the most common trbnormalitr.bcingtrisomv 13 (p. 264). Other chromosomalcauscs include dclctionsof 18p,2p21,7q36and 21q22.3,duplicationof 3p2.l-pter, duplication or deletion of l3q, and triploidl. $ 269). Svndromal causesof holoprosenccphalr are numerous and inciudc relatir,elvrvcll knorvn conditions such as the DiGeorge/ velocaldiofacial sr.ndromc (pp. 267) and a host of much rarer m u l t i p l e m a l f o r m a t i o n s r . ' n d r o m e s s, o m c o f r v h i c h s h o w autosomal reccssiveinheritance One of these. Smith-LemliOpitz slndrome (SLOS) (p.2i6), is associatcd rvith lou,levelsof cholestcrol;this is relo.ant in that it is knou,n that cholesterolis necessarvfor normal functioning of the hedgehogdcvelopmcnt genefamill'(p 85). This leads conr,enientlvto the third group consisting of isolatcd holoproscncephah',u,hich until recently r,vaslargelv uncxplained. Howcver, o\.er the past fe\\rvears three geneshar.e bccn identified. Heterozlgous mutations in these gcnes can cause verv varilble degrees of holoprosencephal).,ranging from mild 'microforms' rvith minimal fcatures such as anosmia to thc full-blou'n lethal alobar form Thev are sonic hedgehog (SHI, on chromosomc 7q36, ZIC2 on chromosome 13q32, and 5'1XJ on chromosomc 2p21. Of these .911F1is thought

sYNDROMES ESANDDYSMORPHIC CONGENITAtABNOR|\/ALITI

to make the greatestcontribution, accounting for up to 20o/oof all familial casesand betrveen lo/o and l0o/o of isolated cases S o m e s i b l i n g r e c u r r e n c e s o f h o l o p l o s e n c e p h a l v ,n o t d u c to SLOS, have been shown to be due to germline mutations in thesegenes.

at thc k)wer end of the developing neural tube leads to a spinal lesion such as a lumbosacral myelocele or meningomyelocele (seeFig 16.2),and a defect involving the head plus cervical and These different entities thoracic spine leadsto craniorachischisis. rclate to the different embrvologicalclosure points of the neural

The fact that so many familial casesremain unexplained indicatesthat more holoprosencephalvgenesarvaitidentification. The extent of causal heterogeneityshown by this condition is further illustrated by its associationrvith poorlv controlled maternal type I diabetesmellitus (p. 250).

tube. N{ost NTDs have seriousconsequencesAnencephaly and craniorachischisisare not compatiblewith survival for more than a few hours after birth Large lumbosacrallesionsusuallv cause partial or complete paralysisof the lower limbs with impaired

NeuraItube defects

16

bladder and bowel continence As with manv malformations NTDs can be classified etiologicallyunder the headingsof chromosomal,syndromal and isolated. Chromosomal causesinclude trisomy l3 and trisomy

Neural tube defects(NTDs), such asspinabifida and anencephaly, illustrate many of the underll,ing principles of multifactorial inheritance and emphasizethe importance of try-ing to identify possibleadverseenvironmental factors These conditions result

18, in both of which NTDs show an incidenceof around 5-10o/o. S-vndromalcausesinclude the relativelyrare autosomalrecessive disorder Mcckel syndrome in which an encephaloceleoccurs in associationrvith polycystic kidneys and polydactyly. However,

from defectiveclosure of the der.elopingneural tube during the first month of embrl'onic life. A defect occurring at the upper end of the developingneural tube results in either exencephal-v/ anenccphalyor an enccphalocele(Fig. 16.14).A defect occurring

most NTDs represent isolated malformations in otherwise normal infants and most isolated NTDs are believed to show multilactorial inheritance. Thc empirical recurrence risks to first-degree relatives (siblings and offspring) vary according to the local population incidence and arc as high as 4-5olo in areaswhere NTDs are common. The incidence in the UK is highest in people of Celtic origin. If such indir-iduals move from their country of origin to another part of the world, the incidence of NTDs in thcir offspring declines but remains higher than amongst the indigcnous population These observationssuggestthat there is a relativcly high incidence of adversesusceptibility genesin the Celtic populations. No single NTD susceptibility genes have been identified in humans, although there is some evidence that the common 677C>-t poll-morphism in the meth-vlenetetrahydrofolate reductase(MTHFR) gene can be a susceptibility factor in some populations.Reduction in MTHFR activity results in decreased plasma folate levels, which are known to be causally associated rvith NTDs (see below). Researchefforts have also focused on developmentalgenes,such as the PAX family (p. 89), which are crpressedin the embryonic neural tube and vertebral column' In mouse models about 80 geneshave been linked to exencephaly, about 20 to lumbosacral meningomyelocele,and about 5 to craniorachischisis.One example is an interaction between mutations of PAXI and the platelet-derived growth factor ct gene(PDCFRA) that resultsin severeNTDs in l00o/oof doublemutant embryos.This rare exampleof digenic inheritanceserves as a useful illustration of the difficulties posed bv a search for susceptibility-genesin a multifactorial disorder.However, to date thcre have been no equivalent breakthroughs in understanding the processesin human NTDs Environmental factors involved in the etiologl' of NTDs that have been identified include poor socioeconomicstatus' multiparitl, and r.alproic acid embryopathy. Firm evidence has

Ftg.16.14 -- ^,'rl ^ L- ^.. ..:.L hrLnrelc H U d U y V V r Ir rd r d rg t r U L L P r r c .o- .n.e- p_ n r. .-.- - -.-

alsoemergedthat periconceptionalmultivitamin supplementation reduces the risk of recurrence bv a factor of 70-75o/owhen a u.omanhas had one affected child. Severalstudies haveshown that

247

16

CONGENITALABNORMALITIES ANDDYSMORPHIC SYNDROM ES

folic acid is likely to be the effectiveconstituent in multivitamin preparations.In both the UK and the USA it is recommended that all women who havehad a previouschild with a NTD should take,f-5 mg folic acid daily both beforeand during the earlv srages of all subsequentpregnancies Similarly, in the UK it has been recommendedthat all women who are trying to conceiveshould take 0.4m9 folic acid daily. In the USA, where bread is fortified rvith folic acid, this recommendation applies to all women of reproductive age throughout their reproductive years. In the UK this recommendationhas not as yet resulted in a noticeable decline in the incidence of NTDs, although a longer period ma-v be necessaryto show anv such effect.

Drug

Effects

ACEinhrbitors

Renaldysplasia

Atcohol

Cardiac defects, mrcrocephaty, characteristic facies

Ch[oroqurne

Chorioretinitis, deafness

DiethylstilbestroI Uterinematformations,vaginaladenocarcinoma Lithium

Cardiac defects(Ebstein anomatv)

Phenytorn

Cardiac defects, cteftpatate, digitaIhypoptasia

Retinoids

Earandeyedefects. hydrocephalus

Strcninmvrin

Dcafncag

An agentthat can causea birth defect by interfering with normal embryonic or fetal der.elopment is known as a teratogen Manv

Tetracyctrne

DentaIenamel hypoptasia

teratogens have been identilied and exhaustive tests are now undertakenbefore any new drug is approved for use by pregnant women. The potential effectsof any particular teratogenusually depend on the dosageand timing of administration during pregnanc)',along with the susceptibility of both the mother and

Thatidomide

Phocomelia, cardiac andearabnormalities

Valproic acid

Neurattube defects, clefting. [imbdefects, characteristic facies

Warfarin

Nasalhypoplasia, stippted epiphyses

fetus. An agent that conveys a high risk of teratogenesis,such as the rubella virus or thalidomide, can usually be identilied relatively quickl1..Unfortunately, it is much more difficult to detect a low-grade teratogen that causesan abnormality in only a small proportion of cases.This is becauseof the relativel_v high background incidence of congenital abnormalities,and

ACE,angiotensrn-converting enzyme

ENVIRONMENTAL AGENTS (TERATOGENS)

also becausemany pregnant women take medication at some time in pregnancy,often for an ill-defined 'flu-like' illness. Despite extensivestudy, controversy still surrounds the use of a number of drugs in pregnancy.The antinauseadrug Debendox has been the subject of successfullitigation in the USA despite the absenceof firm evidenceto support a definite teratogeniceffect.

D R U GASN DC H E M I C A L S Drugs and chemicals that have a proven teratogenic effect in humans are listed in Thble 16.6. Overall, rhese account for approximately2o/oof all congenitalabnormalities. N{any other drugs have been proposed as possibleteratogens but the relativelysmall numbers of reported casesmakeit difficult to confirm that they are definitely teratogenic.This applies to manv anticancerdrugs, such as methotrexateand chlorambucil, and to anticonvulsants,such as sodium valproate,carbamazepine and primadone Exposure to environmental chemicals is

248

Tabte16.6 Drugswithd proventeratogenic effectin humans

also an area of widespread concern. Organic mercurials ingested in contaminated fish in Minamata, Japan, as a result of industrial pollution caused a 'cerebral palsy-like' syndrome in babies who had been exposed in utero. Controversl-

surrounds the use of agents used in warfare such as dioxin (Agent Orange) in Vietnam and various nerve gasesin the Gulf War.

The thatidomidetragedy Thalidomide was used widely in Europe during 1958-1962 as a sedative In 1961 an associationwith severelimb anomaliesin babieswhosemothers had taken the drug during the first trimester was recognizedand the drug was subsequentlywithdrawn from use.It hasbeenestimatedthat during this short period more than 10000 babies were damaged by thalidomide. Review of these babies'records indicated that the critical period for fetal damage wasbetween20 and 35 daysfrom conceprion,i.e. 34-50 daysafter the beginning of the last menstrual period. The most characteristic abnormality caused by thalidomide was phocomelia(Fig. 16.15).This is the name given to a limb that is malformed due to absenceof some or all of the long bones, with retention of digits giving a'flipper' or'seal-like' appearance. Other external abnormalities included ear defects,microphthalmia and cleft lip,/palate.In addition, approximately,l0o/oof 'thalidomide babies' died in early infancy as a result of severeinternal abnormalities affecting the heart, kidneys or gastrointestinal tract. Some 'thalidomide babies' have grown up and had children of their orvn,and in somecasestheseoffspring havealsohad similar defects. It is therefore most likely, not surprisingly, that

SIYCN D R O M E S ES CONGENITALABNORM AALNI TDD Y S M O R P H

16

Fig.16.15 embryopathy. A chitdwiththaLidomide oftheuppertimbs(ametia) Thereisabsence a inad T h eL o w e[ ri m b s h o wp h o c o m e L (Couriesy R polydactyly. of EmeritusProfessor of Leeds) Unrversity W SmitheLLs,

thalidomide was wrongly blamed in a proportion of casesthat were in fact due to single-gene defects following autosomal dominant inheritance(e.9.SALL4 mtt*ions in Okihiro syndrome). The thalidomide tragedy focusedattention on the importance of avoiding all drugs in pregnancy as far as is possible,unless absolute safety has been established. Drug manufacturers undertake extensive research trials before releasing a drug for general use, and invariably urge caution about the use of any new drug in pregnancy.Monitoring systems,in the form of congenital abnormality registers,havebeen set up in most Westerncountries 'epidemic' on the scaleof the thalidomide so that it is unlikely that an tragedycould ever happenagain.

FetaIalcoholsyndrome Children born to mothers who have consistently consumed Iarge quantities of alcohol during pregnancytend to show a distinctive facial appearance,with short palpebral fissures (eye apertures) and a long smooth philtrum (upper lip). They also show mild developmental delay and are often hyperactive and clumsy in later childhood. This condition is referred to as the fetal alcohol syndrome. There is uncertainty about the level of alcohol consumption that is'safe'in pregnancyand there is evidencethat even mild-to-moderate ingestion can be harmful. Generally,it is advised that all women should try to abstain from alcohol intake completely throughout pregnancy.

MATERNAL INFECTIONS Several infectious agents can interfere with embryogenesis and fetal development (Table 16.7).The developing brain, eyes and ears are particularllr susceptibleto damageby infection.

Rubella The rubella virus, which damages between l5o/o and 25o/o of all babies infected during the first trimester, also causes

cardiovascularmalformations such as patent ductus arteriosus and peripheral pulmonary artery stenosis.Congenital rubella infection can be prevented by the widespread use of immunization programs basedon administration of either the measles,mumps' rubella (MMR) vaccine in early childhood or the rubella vaccine alone to young adult women.

Cytomegalovirus At present no immunization is availableagainst cytomegalovirus (CMV) and naturally occurring infection doesnot alwaysproduce long-term immunity. The risk of abnormality is greatest when infection occurs during the Iirst trimester. Overall this virus causesdamagein only 5oloofinfected pregnancies.

Toxoplasmosis Maternal infection with the parasite causing toxoplasmosis conveys a risk of 20o/othat the fetus will be infected during the first trimestet rising to 75o/oin the second and third trimesters. Vaccines against toxoplasmosis are not available.

Table16.7 lnfection

Effects

Viral Cytomegalovirus Chororetinitis,deafness,microcephaty Herpessimptex MicrocephaLy,microphthatmia retinitis, cardiacdefects cataracts, Microcephaly, Rubetta Varicettazoster MicrocephalV.chorioretinitis,skindefects BacteriaI Syphilis Parasitic Toxoptasmosis

rhinitis ostertis, Hydrocephatus,

Hydrocephalus.microcephaly,cataracts. deafness chorioretinitis.

249

16

C O N G E N I T A L A B N O R M AALNI TDIDEYSS M O R P H SIYCN D R O M E S

If a woman is exposedto any of theseinfectious agentsduring pregnancythen an attempt can be made to establishwhether the fetus hasbeen infected by sampling fetal blood to look for specihc immunoglobulin M antibodies Fetal blood analvsiscan also reveal generalizedevidenceof infection, such as abnormal liver function and thrombocvtopenia. There is some evidence to suggest that maternal infection with listeria can cause a miscarriage,and a dehnite association has been establishedbetweenmaternal infection rvith this agent and neonatalmeningitis. Maternal infection with 1tar.^0,-irus can causese\rereanemiain the fetus, resulting in hydrops fetalis and pregnancy-loss.

P H Y S I C AALGE N T S Women who have had babies with congenital abnormalities arc often understandably anxious that exposure to agents such as radiolvaves,ultrasound or magnetic fields could have caused their babies' problems. Unfortunatel-v,it is difficult to prove or disprove anl, of these possibilitiesas exposureto these physical agentsis almost universal However, there is evidence that two speciltc physical agents, ionizing radiation and prolonged hyperthermia, can have teratogeniceffects.

l o n i z i n gr a d i a t i o n Heavy doses of ionizing radiation, far in excessof those used in routine diagnostic radiography; can causemicrocephaly and ocular defectsin the developingfetus.The most sensitivetime of exposureis from 2 to 5 weeksafter conception Ionizing radiation can also have mutagenic (p. 26) and carcinogenic effects, and although the risks associatedwith low-dosediagnosticprocedures are minimal radiographv should be avoided during pregnancy if at all possible.

assessed by regular monitoring of blood glucoseand glycosylated hemoglobin levels.

Phenylketonuria The other main maternal metabolic condition that convevsa risk to offspring is untreated phenylketonuria(p. 158).A high serum levcl of phenl'lalaninein a pregnant woman with phenylketonuria who is not on a specialdiet will almost invariablyresult in serious damage to the fetus, r,r.iththe incidence of mental retardation in the offspring close to l00o/o.Structural abnormalitiescan include microcephaly and congenital heart defects.All women u'ith phenylketonuriashould be strongly advised to adhere to a strict and closelv monitored low phenvlalaninediet both before and during pregnancv.

MaternaIepitepsy There is a large body of literature devoted to the question of maternal epilepsr.,the link uith congenital abnormalities, and the teratogenic effects of antiepileptic drugs (AEDs) The largestand best controlled studiessuggesrthat maternal epilepsy itself is not associatedwith an increased risk of congenital abnormalities.Hotever, all studies have shown an increased incidence of birth defects in babies exposed to AEDs. The risks are in the region of 5-10o/o,rvhich is two to four times the background population risk. Thcse figures applv mainly to single drug therapv.and may be doubled if the fetus is exposed to more than one AED. Some drugs are more teratogenic than others, and data suggest that, of the AEDs currently most commonly prcscribed, the highest risks apply to sodium valproate.The range of abnormalitiesoccurring in the 'fetal

Prolonged hyperthermia

anticonvulsant svndromes' (FACS) are rvide, including neural tube defect (about 2olo),oral clefting, genitourinaryabnormalities

There is evidencethat prolongedhi,perthermiain earlypregnancy can causemicrocephalv and microphthalmia as rvell as neuronal

such as h-vpospadias, congenital heart diseaseand limb defects. The abnormalitiesthemselvesare not specificto FACS; making

migration defects.Consequentlyit is recommendedthat careshould be taken to avoid excessiveuse of hot baths and saunasdurine the lirst trimester

a diagnosis in an individual case can therefore be difficult. Sometimescharacteristicfacial featuresare seen,particularly in fetal valproatesyndrome (trig. 16.16). The most controversialaspectof AEDs and FACS is the risk of learning difficulties and behavioralproblems.Controlled studies in this areaare difficult to setup, but many reports, and much clinical

M A T ER N AIL L LNESS A number of maternal illnessesare associatedwith an increased risk of an untoward pregnancv outcome.

Diabetesmeltitus

250

sacral agenesis,femoral h-vpoplasia,holoprosencephaly'and sirenomelia ('mermaidism'). The likelihood of an abnormality is inverselv related to the quality of the control of the mother's b l o o d g l u c o s el e v e l s d u r i n g e a r l v p r e g n a n c y T h i s c a n b e

Maternal diabetesmellitus is associatedwith a rwo- to threefold i n c r e a s ei n t h e i n c i d e n c e o f c o n g e n i t a l a b n o r m a l i t i e s i n offspring. Malformations that occur most commonly in such infants include congenital heart disease,neural tube defects,

experience,stronglv suggestthat the incidenceofthese problems is significantlyhigher than in the generalpopulation.For practical purposes,potential risks to the fetus have to be weighed against the dangersof stoppingAED treatmentand risking seizuresduring pregnancv.If the patient has been seizure-freefor at least2 years she can be offered withdrawal of anticonvulsanttreatment before proceedingwith a pregnancy..If therap-vis essential,then singledrug treatment is much preferred and sodium valproateshould be avoidedif possible

SIYCN D R O M E S NS DD Y S M O R P H C O N G E N I TAABIN O R M A T I TA] E

15

CAUSE OFUNKNOWN MALFORMATIONS In up to 50o/oofall congenitalabnormalitiesno clearcausecan be cstablished This applies to manv relatively common conditions such as isolated diaphragmatic hernia, tracheo-esophageal listula, anal atresia and single limb reduction defects. For an isolated Iimb defect, such as absenceof a hand, it is reasonable to postulatethat loss of vascularsupply at a critical time during thc developmentof the limb bud leads to developmentalarrest, with the formation of only vestigial digits. It is much more difficult to envisagehow vascular occlusion could result in an abnormalit-v such as esophagealatresia with an associated fistula. t rachco-esophageal

ANDASYMMETRY SYMMETRY

Fis1 . 6.16

A c - r . dw r r l f e t a v. a t p r o a rsey n d ' o - e S ^ e h a sa o ' o a d^ a s a .r o o l , b t r - t n a s arI: pa n da t h n u p p e rI p

Whcn tr1-ingto assesswhether a birth defect is genetic or nongenetic it ma-vbe helpful to consider aspectsof symmetry. As a broad generalization,symmetrical and midline abnormalities frequently har.e a genetic basis.Asymmetrical defects are less likel-r-to have a genetic basis In the examples shown in Fig. l 6 1 7 , t h e c h i l d w i t h c l e i d o c r a n i a ld y s p l a s i a( F i g ' l 6 . l 7 A )

Fig.16.17 A , A b o y w i t h c t e i d o c - a ^d iyasl p t a s a i n w h o - t ^ e c . a v ' c t e s ^ a v e f a i t e d r o o e v e l o o . ^ e ^ c e t - e - e m a r k a b t e ' n o o t t t y o [ ^ iHs es a " ol s- o lders - c o ^ d - c t i v ed e a ' n e s si s a r e c o g -z e d h a sa r e , a L i v elrayr g e ( ^ y p e . r e t o r i s m ) p r o b [ e s p r e s e n t e w d , t h e a T H e w , L h w i d e l y s p a c e d e y e s "eao r o r r rr r o e L o o - : t ^ , , c n t : c r c , , . - i l . . - - - ; + ^ - r lm r r dt ri Lr rrr. Lr ro r c.r r, 9. f9l up Jq.i,nr r. :ynup9nLprtriLr rbra!Ls S B . A C h r t d w i t ^ C o ^ g e n t a l l e d t u l c J K c l t r t d t u y > p t d > d >u 5 u d t t y r r d r r c > ri l r u r c r r d ^I r Li -r ,>, >, ^u -cd^ r. u] d- ., t^r. >. ,ym

e c ko fs y - m e l r ys u g g e s las^ o n - g e n e lci ca u s e l i m bo e r o r m , r ideuser oa r n i o l c b a n d sT ^ ec o m p l e rl a

251

16

CONGENITALABNORMALITIES ANDDYSMORPHIC SYNDROMES

has symmetrical defects (absent or hypoplastic clavicles) and other features indicating a generalized tissue disorder that is overwhelmingly likely to have a genetic basis.The striking asymmetry of the limb deformities in Fig. 16.17Bis likely ro have a non-senetic basis

COUNSELING In caseswhere the precise diagnosisis uncertain an assessment of symmetry and midline involvement may be helpful for genetic counseling.Although it is obviously frustrating for the parentsof a child with one of theseabnormalitiesthat no factualexplanation can be given, in many casesthey can at leastbe reassuredthat the empirical risk of recurrence for siblings is very low. It is worth n o t i n g t h a t t h i s d o e s n o t n e c e s s a r i l ym e a n r h a t g e n e t i c factors are irrelevant. Some 'unexplained' malformations and syndromes could well be due to new dominant mutations (p. 106), submicroscopicmicrodeletions or uniparental disomy

(p.1ls). AII of these would convey negligible recurrence risks to future siblings, although those casesdue to nerv mutations or microdeletionswould be associatedwith a significant risk to the offspring of affected individuals. There is oprimism rhat molecular techniqueswill provide at leastsome of the answersto thesemany unresolvedissues.

FURTHER READING AaseJ 1990Diagnosticdysmorphology.Plenum,London A detailedtext oJ'theart and scienceofdysmorphology. HansonJ W 1997Human teratologl In: Rimoin D L, ConnorJ M, PyeritzR E (eds)Principlesand practiceof medicalgenetics,3rd edn Churchill Livingstone, New York, pp 697-724 A comprehensiae, balancedoxeraiemoJknonn anrl suslectedhumunrcrurugtns. patternsof human malformation, JonesK L 1996Smith's recognizable 5th edn Saunders,Philadelphia Thestandardpediatrictextbookguideto syndromes. Smithells R W, Newman C G H 1992Recognition of thalidomide defects. J Med Genet 29:716-723 A comprehensite accountof the spectrumoJ-abnormalitiescausedby thalidomide. SprangerJ,BenirschkeK, HallJ G et al 1982Errors ofmorphogenesis: conceptsand terms Recommendationsof an international u'orking group J Pediatr100:160 165 A shlrt ?afer prlxirling n rlassificationand iariJication ofthe termsusedto describebirth defec ts. StevensonR E, Hall J G, GoodmanR M 1993Human malformations and related anomalies Oxford Unir.ersity Press,New York Thedefnitiue guide,in tpo tolumes,to humanmalformntions

252

ELEMENTS Q Congenital abnormalitiesare apparentat birth in I in 40 ofall newborn infants.They account for 20-25o/oof alI deathsoccurring in the perinatal period and in childhood up to the ageof l0years. @ A singleabnormality can be classifiedasa malformation, a deformation, a dysplasiaor a disruption. Multiple abnormalties can be classifiedas a sequence,a syndrome or an assocratlon. @ Congenital abnormalitiescan be causedby chromosome imbalance,single-genedefects,multifactorial inheritance or non-genetic factors. Most isolated malformations, including isolatedcongenitalheart defectsand neural tube defects, show multifactorial inheritance, whereas most dysplasiashave a single-geneetiology-. @ l,tuny congenital malformations, including cleft lipl palate,congenital heart defectsand neural tube defects,show etiological heterogeneity, so that when counseling it is important to establish whether these malformations are isolatedor are associatedwith other abnormalities @ nt"ny environmental agentshavebeen shown to have a teratogeniceffect and, ifat all possible,great careshould be taken to avoid exposureto theseagentsduring pregnancy.

CHAPTER

counseting Genetic

'What's the differencebetween... a doctor ... and God?' Q 'God A doesn't think He's a doctor.' Anon

2 The mode of inheritance of the disorder and the risk of d e re l o p i n ga n d / o r t r a n s m i t t i n gi t 3. The choicesor options availablefor dealing with the risks.

Anv couple that has had a child with a seriousabnormality must inevitably reflect on why this happened and whether anlchild(ren) thev choose to have in future might be similarly affected. Similarll', individuals with a family history of a serious disorder are likell, to be concernedthat the1,could either develop the disorder or transmit it to future generations The.v are also very concerned about the risk that their normal children might

It is also agreedthat genetic counselingshould include a strong communicative and supportive element, so that those who seek information are able to reach their own fully informed decisions

transmit the condition to their offspring. For all those affected by a genetic condition that is seriousto them, great sensitivity is neededin communication.Just a few words spokenwith genuine caring concern can put patients at caseand allow a meaningful sessionto proceed; just a few carelessrvords that make light of a serious situation can damagecommunication irrevocabh'.The importance of confidenceand trust in the relationship between patient and health professionalcan never be underestimated. Realization of the needs of such individuals and couples, together rvith awarenessof the importance of providing them with accurate and appropriate information, has led to the widespreadintroduction of genetic counseling clinics in parallelwith the establishmentof clinical geneticsasa recognized medical specialtl'.

DEFINITION Since the first introduction of genetic counseling services approximately 40years ago many attempts have been made to devisea satisfactoryand all-embracing definition. A theme common to all is the concept of genetic counseling being a process of communicationand educationthat addressesconcernsrelating to the developmentandlor transmissionof a hereditary disorder. An individual who seeks genetic counseling is known as a consuhand.During the genetic counselingprocessit is widely agreedthat the counselorshould try to ensurethat the consultand is provided rvith information that enableshim or her to understand: 1. The medical diagnosisand its implications in terms of prognosisand possibletreatment

without undue pressureor stress(Box 17 l).

NGTHEDIAGNOSIS ESTABLISHI The most crucial step in anlr genetic consultation is that of establishingthe diagnosis.If this is incorrect, then inappropriate and totally misleadinginformation could be given,with potentially traglc consequences Rcaching a diagnosisin clinical geneticsusually involves the three fundamental steps of any medical consultation: taking a historl',carrying out an examination,and undertakingappropriate investigations.Often, detailedinformation about the consultand's famil-vhistory will have been obtained by a skilled genetic nurse counsclor as part of a preclinic home visit. A full and accurate famil.v history is a cornerstonein the whole genetic assessment and counseling process Further information about the family and pcrsonal medical history often emergesat the clinic visit, rvhen a full examination can be undertaken and appropriate investigationsinitiated. These can include chromosome and molecular studies as well as referral on to specialistsin other Iields, such as neurology and ophthalmology' It cannot be overemphasizedthat the quality of genetic counseling is dependent upon the availability of facilities that ensurean accuratediagnosiscan be made. Even when a firm diagnosis has been made, problems can arise if the disorder in question shows etiologicalheterogeneity. Common examplesinclude hearing loss and non-specificmental retardation,both of which can be causedby either environmental or geneticfactors In thesesituationsempirical risks can be used (p. 337), although theseare not as satisfactorvas risks basedon a prcciseand specificdiagnosis. A disorder is said to shorv genetic heterlgeneityif it can be caused by more than one genetic mechanism (p 370). Many

253

17

GENETC I CO U N S E L I N G

B o x 1 7 . 1 S l e p si n g e n e t i c o u n s erIg D a g n o s i s b a s e do n a c c u r a t ef a m i t yh i s t o r ym , ed cathstory, e x a m r n a t r oann d i n v e s tg a t i o n s R i s ka s s e s s m e n t Communication D s c u s s i o no f o p t r o n s l o n g - t e r m c o n l a c ta n d s u p p o r t

such disorders are rccognized,and counselingcan be extremelv difficult if the hetcrogeneitv extends to diffcrent modes of inheritance. Commonly encountered cxamples include thc vanous forms of Ehlers-Danlos svndrome (Fig. 17.1),Charcot-

Tabte17.1 Hereditary drsorders thatcanshowdifferent p a t t e r nosf r n h e r i t a n c e Disorder

patterns Inheritance

Cerebeltar ataxia (HMSN) Charcot-Marie-Tooth drsease Congenital cataract Ehters-Dantos syndro'ne lchthyosis Microcephaly Polycystic kidney disease pigmentosa Retrnitis Sensorineural rearingloss

AD.AR AD,AR,XR AD,AR,XR AD.AR.XR AD AR,XR AD,AR AD,AR AD,ARXR,M AD AR.XR,M

AD,autosomal domrnant;AR, autosomal recessive;XR,X*ltnKeo Tecess ve;M,mitochondriaI

N'Iarie-Toothdisease(p 286) and retinitis pigmentosa,all of rvhich can sho\\ autosomal dominant, autosomal reccssiveand X-linked recessiveinheritance(Thble 17.1). Fortunatclr,,progrcss in molecular geneticsis providing solutions to some of these problems. For example, mutations in the gene that codes for rhodopsin, a retinal pigment protein, are found in approximatelv 30o/crof families shorving autosomal dominant inheritance of retinitis pigmentosa(Fig. 17.2)and rhe molecularbasisof rhe most common forms of Charcot-N,Iarie-Tooth disease(t-vpe l), also

Fis.17.2 F u n d ussh o w i ntgy p i c a l p i q m e n tcahray n g eosfr e t r n i t r s prgmentosa

known as hereditarv motor and sensorvneuropathJr(HMSN), is now understood (p. 286).

CALCULATING AND PRESENTI NG T H ER I S K Fi1.17.1

254

E h l e r s - D a n t ossy n d r o m eT h e i n h e r i t a n cpea t t e r ni n t h r sc a s ei s a u t o s o m a l d o m i n abnet c a u s e f a t h e ra n d s o n a r e a f f e c t e d

In some counseling situations,calculation of thc recurrence risk is rclirtivelv straightforrvardand requires littlc morc than a reirsonableknor'vledgeof mcndelian inheritance.Horvever,many flactors,such as delal'ed age of onset, reduced penetranceand

GENETIC COUNSETING

the use of linked DNA markers, can result in the calculation becoming much more complex. The theoretical aspectsof risk calculationare consideredin more dctail in Chapter 22 The provision of a recurrence risk does not simply involve conyeying a stark risk figure in isolation. It is verr important that the information provided is understood, and that parents are given as much background information as possible to help thcm reach their own decision As a working rule of thumb, recurrence risks should be quantified, qualifred and placed in context.

17

RISKSIN CONTEXT PLACING Prospectiveparentsseenat a geneticcounselingclinic should be provided rvith information that enablesthem to put their risks in context so as to be able to decide for themselveswhether a risk is'high' or'low'. For example,it can be helpful (but alsoalarming) to point out that approximately 1 in 40 of all babieshas a congenital malformation or handicappingdisorder.Therefore, an additional quoted risk of I in 50, although initially alarming, might on reflection be perceived as relatively lour As an arbitrary guide, risks of 1 in l0 or greater tend to be regardedas high, I in 20 or lessas low, and intermediate valuesas moderate.

. T H EN U ME R IC AL VALUE Q U A N T I F I C A T ION O F AR I S K Most prospectiveparentswill be familiar to somedegreewith the concept of risk, but not everyoneis comfortablewith probability' theory and the alternative ways of expressingrisk, such as in the form of odds or as a percentage.Thus, for example, a risk of 1 in ,1 can be presented as an odds ratio of 3 to I against, or numericalll- as 25o/o.Consistency and clarity are important if

D I S C U S S I N G TOHPET I O N S

confusion is to be avoided. It is also essentialto emphasizethat a risk appliesto eachpregnancl,-andthat chancedoes not have a memor\i For example,the fact that parcnts have just had a child rvith an autosomalrecessivedisorder (recurrencerisk of 1 in 4) doesnot mean that their next three children will be unaffected A

Har.ing establishedthe diagnosis and discussedthe risk of occurrence/recurrence,the counselor is then obliged to ensure that the consultands are provided with all of the information neccssaryfor them to make their own informed decisions.This should include detailsof all the choicesopen to them. For example, if relevant,the availabilit.vof prenataldiagnosisshould be discussed, togcther rvith details of the techniques, limitations and risks associatedwith the variousmethods employed(Ch 21). Mention will sometimesbe made of other reproductive options These can

useful analog-vis that of the tossedcoin that cannot be expected to remember rvhcthcr it landed heads or tails at the last throrv and cannot therefbrc bc cxocctcd to know what it should do at the ncxt throx,. It is also important that genetic counselorsshould not be seen exclusivell' as prophets of doom Continuing the pcnn-v

include alternative approachesto conception, such as artificial inseminationusing donor sperm (AID), the use of donor ova and preimplantation geneticdiagnosis(p. 325)' These techniquescan bc used when one partner is infertile, as in the caseof Klinefelter syndrome and Turner syndrome (Ch l8), or simply to bypass the possibility that one or other partner will transmit his or her

analogl',the good side of the coin should also be emphasized, particularl-yif the odds stronglv favor a successfuloutcome. For example,a couple faced with a probability of I in 25 that their

gene(s)to the baby. disadvantageous These are issuesthat should be broached with great care and sensitivitli For some couples the prospect of prenatal diagnosis

next babv rvill havea neural tube defect should be reminded that thcre are 2,1chancesout of 25 that their next babv will not be

follorvedby selectivetermination of pregnancy is unacceptable, rvhereasothers view this as their only means of ensuring that an-vchildren they do have rvill be healthy.Whatever the personal viervsof the counselor,the consultandsare entitled to knowledge of prenataldiagnosticproceduresthat are technicall-vfeasibleand

affected.

O U A L I F I C A T-ITOHNEN A T U ROEF AR I S K Severalstudieshaveindicatedthat the factor that most influenccs parentsrvhendeciding rvhetheror not to haveanotherchild is the nature of thc long-term burden, or sevcrifi,,associatedrvith a risk 'high' risk rather than its prccisc numerical value. Therefore a of 1 in 2 for a trir.ial problem such as an extra digit (polydactyl.v) rvill deter verv few parents.In contrast a'low' risk of I in 25 for a disablingcondition such as a neural tube defect can havea very signi{rcantdeterrent effect. A woman rvho grew up watching her brother developDuchenne musculardystrophy and subsequently die from thc condition aged2l years,may'not risk having children er,enif there is only a I o/ochancethat she is a carrier. Other factors, such as rvhethera condition can be treated successfulll',whether it is associatedwith pain and suffering, and lvhether prenatal diagnosisis available,will all be relevant to the decision-making process

legalll' permissible.

ANDSUPPORT COMMUNICATION The ability to communicate is essentialin genetic counseling. Communicationis a trvo-wa.vprocessNot only doesthe counselor provide information, he or shealsohas to be receptiveto the fears and aspirations,expressedor unexpressed,of the consultand A rcadinessto listen is a key attribute for anyoneinvolved in genetic counseling, as is an ability to present information in a clear, sympatheticand appropriatemanner Often an indir,idual or couple will be extremely upset when {irst made arvareof a geneticdiagnosis,and it is very common for guilt feelings to set in. The individual or couple may look back

255

17

GENETIC COUNSELING

and scrutinize everv event and happening, for example during a pregnanc\i The delivery of potentially distressing information cannotbe carried out in isolation.Genetic counselorsneed to take rnto account the complex psychological and emotional factors that can influence the counseling dialog The setting should be agreeable,private and quiet, with ample time for discussionand questions.When possible,technical terms should be avoided or, if used, full1, explained. Qrestions should be answered openll, and honestlv, and if information is lacking it is certainlv not a fault or sign of weaknessto admit that this is so. Most couples respect and recognize the truth, and some parents of children whose condition cannot be diagnosedderive a curious pleasure from knowing that their child appearsto be unique and has bamboozled the medical profession (unfortunately, this is not particularlv difficult!). Despite all of these measures,a counselingsessioncan be so intense and intimidating that the amount and accuracy of information retained on follow-up at a later date can be ver-v limited For this reasona letter summarizing the topics discussed at a counseling sessionis usuallv sent to the famill,-afterwards. In addition, thev are sometimescontacted at a later date by a member of the counseling team, mostly bv telephone but sometimesby a home visit, thereby providing an opportunitv for clarification of anv confusing issuesand for further questionsto be answered. It rvould be rvrongsimplv to conveyinformation of a distressing nature without offering an opportunity for furthcr discussionand long-term support. Most genetic counseling centers maintain informal contact with relevant families through a network of genetic nurse counselorswho are familiar with the family and

- DIRECTIVE GENETIC COUNSELING ORNON-DIRECTIVE? It has alreadvbeen emphasizedthat geneticcounselingshould be viewed as a communication processthat provides information. The ultimate goal is to ensurethar an individual or couple can reach their own decisions based on full information about risks and options. There is universal agreement that genetic counseling should be non-directive, with no attempt being made to steerthe consultandalong a particular courseof action. In the samespirit the genetic counselorshould alsostrive to be non-judgmenral, even ifa decisionis reachedthat seemsill advisedor is contrary ro the counselor'sown beliefs.Thus the role of the geneticcounseloris to facilitateand enhanceindividual autonomy rather than to give adviceor recommend a particular courseof action. Genetic counselorsare sometimesaskedwhat thel'themselves would do if placed in the consultand's position. Generally it is preferable to avoid being drawn into expressingan opinion, opting instead to suggestthat the consultand try to imagine how he or she might feel in the future having pursued each ofthe available options.This approach,sometimesreferred to as 'scenario-based decision counseling', provides individuals with an opportunity for careful reflection This is particularly important if one of the options under considerationinvolvesa potentially irreversible reproductive decision such as sterilization. There is a well establishedmaxim that it is the consultandsand not the counselors who haveto live rvith the consequences oftheir decisionsand, indeed, consultandsshould be encouragedto make the decisionthat they can best live with the one that they are leastlikely to regret.

their particular circumstances.This is especially valuable for prospective parents who subsequentlyrequest specific prenatal diagnostic investigations, and for presvmptomatic adults who are shown to be at high risk of developing late-onsetautosomal dominant disorders such as Alzheimer diseaseand Huntington disease(p.282). Genctic registers(p. 313) provide a useful means of ensuring that effectivecontact can be maintained with all such relevant familv members

PATIENT SUPPORT GROUPS Finally, mention should be made of the widespreadnetrvork of support groups that now exists.These organizationshave usually been establishedby highl-vmorivaredand well informed parents

256

OUTCOMES IN GENETIC COUNSELING The issue of defining outcomesin genetic counselingis difficult and contentious, but also topical in today's climate of health economy where everything has to be justified. The difficulty arisesbecauseof the rather nebulousnature of geneticcounseling, which, in contrast to most medical activities, does not have any easilyquantifred end points, such as rate of infection or survival after surgerv.The issue is topical becauseof the increasing pressure being applied by funding authorities, with emphasis on demonstrable quality and effectiveness.Finally; the issue is contentiousbecauseit raisesseriousquestionsabout the purpose of geneticcounselingand whether this should be viewed assimply the provision ofinformation or rvhetherthere should be identified

or affected families who can provide enormous support and companionship for others affectedbv a particular geneticcondition or syndrome. When confronted by a new diagnosis of a rare disorder many families feel very isolated; if at all possiblethey should be given contact information so that rhey havethe option

benefits for society in terms of a reduction in the incidence of geneticdisease. In practice the three main outcome measuresthat have been

of communicating with other affectedfamilies who havehad similar experiences Referral ro an appropriate support group would now be viewed as an essentialintegral component of the genetic counselingprocess.As well as providing support and information for affected families, these groups have often been very successful in fosterine research.

assessed are recall, impact on subsequentreproductive behavior, and patientsatisfaction.Mosr studieshaveshownthat the majority ofindividuals rvho haveatrendeda geneticcounselingclinic havea reasonablerecall of the information given, particularly if this was reinforced bv a personal letter or follow-up visit. Nevertheless, confusion can arise, and as many as 30o/oof counseleeshave

G E N E T ICCO U N S E L I N G

difficultl- in remembering a preciserisk figure. Studies that have focused on the subsequentreproductive behavior of couples that have attended a genetic counseling clinic have shorvn that approximatelv 50o/ohave been influenced to some extent The factors that have been shown to be influential are the severitv of the disorder, the desire of the parents to have children, and whether prenatal diagnosis and/or treatment are available. Finally; studies that have attempted to assesspatient satisfaction have strugglcd to addressthe problem of how this should best be defined. For example,an individual could be very satisliedwith the way in which they rverecounseledbut remain very dissatislied b-v lack of a precise diagnosis or the availability of subsequent prenatal diagnostictests. In an increasingly cost-conscioussociety it is not surprising that the purchasers of health care, lvhether privately or statc funded, are keen to identify quantifiabletargets rvith which thel' 'effectiveness'of genctics services,and genctic can assessthe counselingin particular. Outcomes such asthe number of abnormal pregnanciesterminatedor individualsscreenedcan seemattractive to administrators and politicians who are preoccupied rvith balancingbudgetsand cost bcneht analyses.In contrast, clinical geneticistsand non-medical counselor colleaguesuniversalll' reject the use of theseoutcomc criteria on the grounds that theyhint at a eugenics philosophl. that is totally unacceptablein a society in r,vhichpatient autonomy-is a guiding ethical principle Instead thel' emphasize the benefits of an educatcd informed communit,v rvith enhanced indir.idual autonomy. The goals of satisfaction as cxpressed b,y the users of genctic services and their ability to make informed decisionsare seenas much more acceptablethan a reduction in the financialand personalburdens causedb1'genetic discase In the future it is possiblc that an outcome measure such as 'perceivcd personal control' will be developed.This rvill almost certainly incorporate both information and satisfactioncriteria, together with an assessmentof whether individuals have been able to understand the information and come to terms with their situation,thereb.venablingthem to makeappropriatelife decisions s,ith rvhich they are comfortable The idea is alsoconsistentwith gcneralgovernmcnt polic-vto encourageindividuals to take more personalcontrol of their own health agenda.

PROBLEMS IN GENETIC SPECIAL COUNSELING There are a number of specialproblems that can arise in genetic counseling.

CO N SA N G U IN IT Y rclationshipis one betweenblood relativesrvho A consanguineous havc at leastone common ancestorno more remote than a greatgrcat-grandparent. Consanguineousmarriage is widespread in many parts of the rvorld (Thble 17.2). In Arab populations the

17

T a b t e1 7 . 2 W o r l d w i dien c i d e n coef c o n s a n g u i n e o u s marrage Country

Incidence(%)

Kuwatt SaudiArabia Joroan Pakistan lndia Syria Egypt LeDanon Algeria Japan UK.USA France,

54 54 50 40-50 5*60 33 28 25 23 2-4 2

GJ' JaberL.Halpern inciuding sources fromvarious Dataadapted wortdwide of consanguinity ShohaiM 1998Thetmpact Genet 1,12-17 Community

most common t.vpeof consanguineousmarriage occurs between lirst cousinsrvho are the children of trvo brothers, whereasin the Indian subcontinent uncle-niece marriagesare the most commonly encountercd form of consanguineousrelationship.Although there is in these communities some recognition of the potential genetic effects of consanguinity,there is also a disadvantageous strongly held vierv that these are greatlv outweighed b-v social irdvantagessuch as greaterfamil-vand marital stability. \'{anv studies have sholvn that among the offspring of consanguineousmarriages there is an increasedincidence of both congenital malformations and other conditions that will present latcr,such ashearinglossand mentalretardation For the offspring of hrst cousins the incidence of congenital malformations is increasedto approximateh trvice that seen in the offspring of unrelated parcnts. Almost all of this increasein morbidity and for autosomal recessive mortalit.v is attributed to homozl--gositY disorders,a finding consistentwith Garrod's original observation 'the mating of {rrst cousinsgivesexactlvthe conditions most that character to show likely to enable a rare, and usually-recessive, itself'(p.107). On the basis of studies of children born to consanguineous parcnts it has been estimated that the averagc human carries betlvcen one and two genes for a harmful autosomal recessive disorder, together with severalmutations for conditions that result in lethalit.vbefore birth. Most prospectiveconsangurneous parents are concerned primaril-v with the risk that they will have a handicapped child, and fortunately the overall risks are usually relativell,'small. When estimating a risk for a particular consanguineousrelationship it is generally assumed that each common ancestorcarried one deleteriousrecessivemutatlon Therefore' for hrst cousins' the probabilitlr that their first child rvill be homozl-gous for their common grandfather's dcleteriousgenervill be 1 in 64 (Fig. 17.3).Similarly, the risk that

257

17

GENETIC COUNSELING

this child will be homozygous for the common grandmother's recessivegene will also be I in 64. This gives a rotal probability that the child will be homozygous for one of the grandparent,s deleterious genes of I in 32. This risk should be added to the general population risk of I in 40 thar any baby will have a maior congenital abnormality (p.237), to give an overall risk of approximately I in 20 that a child born to first-cousin parents will be either malformed or handicapped in some way. Risks arising from consanguinity for more distant relatives are much lower. For consanguineousmarriagesthere is alsoa slightly increased risk that a child will have a multifactorial disorder. In practice this risk is usually very small. In contrast, a closefamily history of an autosomalrecessivedisorder can conveya relativelyhigh risk that a consanguineouscouple will have an affected child. For example, if the sibling of someonewith an autosomal recessivedisorder marries a first cousin, the risk that their first baby will be affected

Geneticrelationship

Proportionof sharedgenes

Riskof abnormality in offspring(%)

First degree Parent-chr [d Brother-sister

112

50

Seconddegree Uncte-n iece Aunt-nephew Double frrstcousins

114

5 10

Third degree Firstcousins

1/B

3-5

equalsI in24(p.126).

INCEST Incestuous relationships are those that occur between first-degree relatives - in other words, brother-sister or parent-child (Thble 17.3).Marriage betweenfirst-degree relarivesis forbidden, both on religious grounds and by legislation,in almost every culture. Incestuousrelationships are associatedwith a very high risk of abnormality in offspring, with lessthan half the children of such unions being entirely healthy (Table 17.4).

,F

lntettectual impairment Severe Mitd

25 35

A D O P T I OAN N DG E N E T D I CI S O R D E R S

tlz

1t"

1l+

tl+

1l8x1l8 =1le+

The issue of adoption can arise in severalsituations relating to genetics.Firstly, parents at high risk of having a child with a serious abnormality often expressinterest in adopting rather than running the risk of having an affected baby. In generic terms this is a perfectly reasonableoption, although in practice the number of couples wishing to adopt usually far exceedsthe number of babiesand children availablefor adoption. The physician with a knowledgeof geneticscan also be called upon to try to determine whether a child who is being placed for adoption will develop a genetic disorder. For the offspring of consanguineous or incestuous matings, risks can be given as outlined previously (seeTables 17.3 & 17.4).Adoption socieries sometimesalso wish to place a child with a family history of a particular hereditary disorder. This raises the difficult ethical

dilemma of predictive testing in childhood for conditions showing onset in adult life (p. 357). Increasinglyit is felt that such testing Fis.17.3 should not be undertaken unless this will be of direct medical Proba bi[tythatthefirstchitdoffirstcousins wiI be homozygous for thedeteterious allele*carriedbythecommongreat-grandfather: benefit to the child. In practice, even when a child is actually A simitarriskof1in 64wittapptyto thedeLeterious attelebetonging affected by a genetic disorder, suitable adoptive parenrs can tothecommongreat-grandmother, grvrng a totatriskof1 in32 usually be found.

258

17 Concernaboutthe possiblemisuseof genetictestingin neonares and young children who are up for adoption has prompted the American Society of Human Geneticsand the American College of Medical Genetics to issue joint recommendations.These are

M12 A

F

M12

F

B

basedon the best interestsof the child. They can be summarized as supporting genetic testing in such children only when the testing would be appropriate for all children of that age and when the tests are undertaken for disorders that manifest during childhood and for which preventivemeasurescan be undertaken during childhood. The joint statementdoes not support testing for untreatabledisorders of adult onset or for detecting predispositions to 'physical, mental or behavioral traits within the normal range'.

DISPUTED PATERNITY This presentsa difficult problem for which the help of a clinical geneticistis sometimessought. Until recently paternity could never be proved with absolutecertainty,although it could be disproved or excluded in two ways.If a child was found to possessa blood group or other polymorphism not presentin either the mother or the putative father, then paternity could be confidently excluded For example,if the mother and putative father both lackedblood group B, but this was present in the child, the putative father could be excluded. Similarly; if a child lacked a marker that the putative father would have had to transmit to all of his children, then once again paternity could be excluded. As an example, a putative father with blood group AB could not have a child with blood group O. Earlv attempts to establishpaternity were based on analysis of severaldifferent polymorphic systems,such as blood groups, isoenzymes and human Ieukocyte antigen (HLA) haplotypes. The results of these studies can be consistent with paternity but cannot give absolute proof of it. Depending on the number of polymorphic systems analyzed and their frequenciesin the general population, it is possibleto calculate the relative probability that a particular male is the father compared with any male taken at random from the general population. The limitations of these approacheshave been overcomeby the development of genetic fingerprinting using minisatellite repeat sequence probes (pp. 17, 67) and single nucleotide polymorphisms (SNPs). The pattern of DNA fragments generated by' these probes, and SNP variants, is so highly polymorphic that the restriction map obtained is unique to each individual, with the exception of identical twins (Fig 17.,t). If DNA from the child and the mother is analyzed, then the bands inherited from the biological father can be analyzedand comparedwith thosepresentin DNA from the putative father(s). If these match, this gives an extremely high mathematical probability that the putative and biological fathers are the same individual.

Fis.17.4 probeswith ustngtwominrsatettite nt obtained fingerpr Genetic DNAfroma mother(M),father(F)andtheirtwrns(1and2) s n de a c hb a n di nt h e T h et w i n sh a v ea n i d e n t i csael to fb a n d a of Dr Raymond fromoneoftheparents(Courtesy twinsoriginates of Leicester University SirAtecJeffreys, andProfessor Daig[ersh E H.MacFadyen R,Mackay fromYoungI D,Dalgleish Reproduced in monozygoLic oftheGsyndrome expression U l'41988Discorda^t lromthe twinsAmJ MedGenet29:863-869withpermission Americon Journotof MedicolGenetics)

READING FURTHER ASHG/ACMG Statement2000 Genetictestingin adoption Am J Hum Gcnet 66: 761-767 of Human Ceneticsand the of theAmerimn Societ.y Thejoint recommendatitns AmericanCollegerf Medical Ceneticson genehctestingin Toungchiklren nhu are beingplaredfor adoPtion ClarkeA (ed ) 1994Geneticcounselling.Practiceand principles Routledge, London

259

17

GENETIC COUNSELING

A thoughtfuland prlr0catne muhi-authlr text that atldresses tlfficult issues such aslredictixe testing,ureening,prenataldiagnosisand confdentialit.y. ClarkeA, ParsonsE, WilliamsA 1996Outcomesand processin genetic counselingClin Genet 50:462469 A critical retiep of preaiousstudiesoJ'theoutcomes of geneticcounseling. Frets P G, Niermeijer M F 1990 Reproductive planning after genetic counselling:a perspectivefrom the last decade Clin Genet 38: 295-306 A reaiep of studiesundertakenbetpeen1980 antl 1989 to determinephich factorsare m0stimp0rtantin infiuencingreprotluctiaedecisions Harper P S 1998Practical genetic counselling, 5th edn ButterworthHeinemann, Oxford An extremelyusefulpracticalguid,eto all aspecxof geneticcounseling. JaberL, Halpern G J, ShohatM 1998The impact of consanguiniry worldwide Community Genet l: 12-17 A reoiewof the incidenceand consequences of rcnsanguinityin aariouspurts of the porld. JeffreysA J, Brookfield J F I Semeonoff R 1985Positive identification of an immigration test-case using human DNA fingerprints Nature 317: 818-819 A cletterdemonstratilnof the aalueof geneticfngerprinting in analyzing alleged famifu relationships Tirrnpenny P (ed ) 1995Secretsin the genes:adoption, inheritance and genetic diseaseBritish Agenciesfor Adoption and Fostering, London A multi-author basicten coxeringaspects0fgeneticsrelet)antt0 the adoption l)roIess.

iiFtE[fENT5 Q Genetic counseling may be defined asa communication processthat dealswith the risk of developing or transmitting a genetic disorder. @ fft. most important steps in genetic counseling are the establishment of a diagnosis,estimation of a recurrence risk, communication of relevantinformation and provision of long-term support. @ Genetic counseling should be non-directive and the genetic counselor should be non-iudgmental. The goal of genetic counseling is to provide accurate information that enables counselees to make their own fully informed decisions. @ Marriage between blood relativesconveys an increased risk for an autosomal recessivedisorder in future offspring. The probability that first cousinswill have a child with an autosomal recessive condition is approximately 3o/o, although this risk can be greater if there is a family history of a specificgenetic disorder. @ fn. most sensitivetechnique for paternity tesring is genetic fingerprinting Other polymorphic systems can disprove paternity but cannot establishwith such a high statistical probability that a particular male is the biological father.

260

CHAPTER

Chromosome disorders

The developmentof a reliabletechniquefor chromosomeanalysis in 1956soon led to the discoverythat severalpreviouslydescribed conditions were due to an abnormality in chromosomenumber. Within 3 yearsthe causesof Down syndrome (47,XX/XY, +21), Klinefelter syndrome (47,XXY) and Tirrner syndrome (45,X) had beenestablished.Shortll'afterwards other autosomaltrisom-v svndromeswere recognized,and graduallyover the ensuing1,ears many other multiple malformation syndromeswere describedin which there was loss or gain of chromosomematerial To date, at least 20000 chromosomalabnormalitieshavebeen registeredon laboratorydatabases. On an individual basismost of these are very rare, but together the-vmake a major contribution to human morbidit-v and mortalitv Chromosome abnormalities account for a large proportion of spontaneouspregnancv loss and childhood disabilitl', and also contribute to the genesisof a significant proportion of malignancv in both childhood and adult life as a consequenceof acquired somatic chromosome aberrations. In Chapter 3 the basicprinciples of chromosomestructure and function were described.Details of chromosomebehaviorduring cell division were also provided, along with a theoreticalaccount of the nature of chromosome abnormalities and how thesecan both arise and be transmitted in families. In this chapter the medical aspectsof chromosomeabnormalitiesare consideredand specihc chromosomeabnormalitv svndromesdescribed.

OE FC H R O M O S O M E INCIDENC ABNORMALITIES

From conception onwards the incidence of chromosome abnormalities falls rapidly. By birth it has declined to a level of 0 5-1o/o,although the total is much higher (5olo)in stillborn infants. Table 18.2 lists the incidence figures for chromosome abnormalitiesbasedon newborn surveys.It is notablethat among the commonly recognized aneuploidy syndromes there is also r high proportion of spontaneouspregnancy loss (Thble l8 3). This is illustrated by comparison of the incidence of conditions such as Dorvn syndrome at the time of chorionic villus sampling (ll-l2weeks), amniocentesis (16weeks)and term (Fig. 18.1).

(TRISOMY 21) DOWNSYNDROME This condition derives its name from Dr Langdon Down, who first dcscribed it in the Clinical Lecture Reports of the London Hospital in 1866.The chromosomal basis of Down syndrome was not establisheduntil 1959by Leieune and his colleaguesin Paris.

lncidence The overall birth incidence, when adiusted for the increasingly rvidespreadimpact of antenatalscreening,is approximately I in 1000 in the UK, which has a national register.In the USA the birth incidence has recently been estimated at approximately I in 800. In the UK approximately600loof Down syndrome cases are detected prenatally. There is a strong associationbetween the incidence of Down syndrome and advancing maternal age (tble 18.4).

features Ctinicat

survival be-vondthe first few days or weeks after fertilization is not possible.Approximately 50o/oof all spontaneousmiscarriages havea chromosomeabnormality (Table 18.1)and the incidenceof

These are summarized in Box 18.1.The most common finding in the newborn period is severehypotonia. Usually the facial characteristicsof upward sloping palpebral lissures' small ears and protrudingtongue(Figs 18.2& 18.3)prompt rapid suspicion of the diagnosis,although this can be delayed in very small or prcmature babies. Single palmar creasesare found in 50o/oof children with Down syndrome (Fig. 18.4), in contrast to 2-3o/o

chromosomalabnormalitiesin morphologically normal embryos is around 20olo.These observationsindicate that chromosome abnormalitiesaccountfor the lossof a very high proportion of all

of the general population. Congenital cardiac abnormalities are present in 40-45olo of babies with Down syndrome, rvith the three most common lesionsbeing atrioventricular canal defects,

human conceptions.

ventricular septal defectsand patent ductus arteriosus.

Chromosome abnormalities are present in at least l07o of all spermatozoaand 25oloof mature oocytes.Between l5o/oand20o/o of all recognized pregnanciesend in spontaneousmiscarriage, and many more zygotes and embrvos are so abnormal that

261

18

CHROMOSOME DISORDERS

O

Abnormatity

Incidence(%)

Trisomy 13

2

Trisomy'16

15

Trisomy'18

3

Trisomy 21

5

Tnsomyother

25

MonosomyX

20

o c

34

-'ip

o oy

5

0ther

10

38 40 42 M a t e r n a al g e( y e a r s )

44

46

Fis.18.1 A p p r o x i m ai tnec i d e n coeft r i s o m y 2al t t h e t i m e o fc h o r r o n r c (CVS) (11-12weeks). (16weeks) villussampling amniocentesis and (DatafromHookE B,CrossP K.Jackson delrvery L,Pergament E.Brambati B 1988Maternatage-specific ratesof4Z +21and othercytogenetic abnormatities diagnosed inthefrrsttrrmester o fp r e g n a n ci nyc h o r i o nvi ci l l u sb i o p ssyp e c i m e nAsmJ H u m Genet42:797-807, andCuckle H S WaLd NJ,Thompson S G1987 Estimating a womans rrskof having a pregnancy associated with Downsyndrome usingherageandserumalpha-fetoprotein Level BrJ Obstet Gynaeco[ 94:387-402)

15

Tetraptoidy

36

Abnormatity

Incidenceper10000births

Autosomes Trsomy13 Trisomy 1B Trrsomy 21

2 3 15

Naturalhistory Affected children show a broad range of intellectual ability with IQscores ranging from 25 to 75. The averageIQof young adults with Down syndrome is around 40-45. Social skills are relatively well advancedand most children with Down syndrome are happy

5ex chromosomes Femalehirths 45,X 41.XXX

1-2 10

Molebirths 47.XXY 47.XW

10 10

Otherunbalancedrearrangements10 Batancedrearrangements 30 Total 90

and very affectionate.Adult height is usually around l50cm. In the absenceofa severecardiacanomaly,which leadsto early death in l5-20o/o of cases,averagelife expectancyis 50-60 years.Most affected adults develop Alzheimer diseasein later life, possibly because of a gene dosage effect as the locus for the amyloid precursor protein geneis on chromosome2l . This geneis known to be implicated in some familial casesof Alzheimer disease

(p.229). Chromosome findings

262

Disorder

Proportionundergoingspontaneous pregnancytoss(%)

Trisomy'13

95

Trisomy 18

95

Trisomy 21

80

MonosomyX

98

These are listed in Thble 18.5. In casesresulting from trisomy 21, the additional chromosome is maternal in origin in more than 90o/oof cases,and DNA studies have shown that this arises most commonly as a result of non-disjunction in maternal meiosis I (p. a3). Robertsonian translocations (p. 49) account for approximately4o/oof all cases,in roughly one-third of which one of the parents is found to be a carrier of the translocation. Children with mosaicism are often less severely affected than those with the full syndrome. Efforts have been made to correlate the various clinical features in Down syndrome with trisomy for specific regions of chromosome 21, by studying children with partial trisomy for

SORDERS C H R O M O S OD MIE

18

Tabte18.4 Incidence of Downsvndromein relationto maternalage Maternalage at delivery(years)

Incidenceof Downsyndrome

20

1i n1 5 0 0

25

1in1350

30

'1 in900

35

1in400

36

1in 300

31

1in250

38

1 in200

39

1in150

40

1in100

41

1in85

42

1in65 'l

43

Fi9.18.2 A ch[dwrthDownsyndrome

in 50

a+

1in40

45

1in30

a fromCuckte H S.WaldN .J.ThompsonS G1987Estimating Adapted a pregnancy assocrated withDownsyndrome womans riskof having Gynaecol leveiBrJ Obstet usingherageandserumatpha-fetoprotern 94:387-442

Fig.18.3 B o x 1 8 . 1 C o m m o nf i n dn c si n D o w ns v n d r o m e

o fa c h i l dw i t h C l o s e - uvpr e wo ft h ee y e sa n dn a s abl r i d g e D o w ns y n d r o mseh o w i nugp w a r sdt o p i npga l p e b r a l f i s s u r e s icfolds eprcanth spotsandbilateral Brushfield

Newbornperiod f l r e n n n . : < ' c p n \ / c y r p q q r . r c h a ls k , r

Craniofacial protruding epicanthic fotds, tonguesma[[ears,upward BrachycephaLy, clonrnn ne nchre

f ccr rrpc

Limbs palr-arcrease. --nat[.r odteora.anx off'flhrr'qerwidegap S,ngte f rst and second toes between Cardiac ^+-.-r-^!t u ..^^+-i-, r-- -^^r- ,rfectscommonatrlOventrrCular canat, vtrr tt rLUrdr 5trPrdr uc

HLt tdt d

^-+^^+ f, ,-+ pdtcL L uutLu>

o

Ltr |UJUJ

0ther shortstature alresia, lirschsprung d sease. AnaIatresla, duodenal strabsmus

different regions There is some support for a Dorvn syndrome 'critical region'at the distalend ofthe long arm (21q22)aschildren with trisomy for this region usually-havetypical Down syndrome facialfeatures Chromosome2l is a'gene-poor'chromosomewith

Fi9.18.4 thesingle Note syndrome wrthDown ofanadult Thehands shortcurvedftfth palmarcreasein the lefthandplusbrLateral fingers(clinodactyly)

253

18

C H R O M O S OD MIE SORDERS

T a b t e1 8 . 5 C h r o m o s o maeb n o r m a l i t i e t nsD o w n syndrome Abnormality

(%) Frequency

Trisomy

95

Transtocation

4

Mosaicism

1

a high ratio of AT to GC sequences (p 35).Ar presentthe onlv reasonablyrvell establishedgenotype-phenotvpe correlation in trisomy 2l is the high incidence of Alzheimer diseasc,attributcd to the amyloid precursor protein genedosageeffect

Recurrence risk For straightforrvardtrisomy 21 the recurrence risk is relatcd to maternal age and is usuallv of the order of I in 200 to 1 in 100 In translocationcasessimilar figures apply if neither parenr is x carrier In familial translocationcases,the recurrence risks vart' from around 1-3o/ofor male carriers up to 10 1.5olo for fcmale carriers,with the exception of verl' rare carriers of a 21q2lq translocation,for whom the recurrencerisk is l00o/o(p. 51). Prenataldiagnosiscan be offered basedon analysisofchorionic villi or cultured amniotic cclls.Prenatalscreeningprograms have becn introduced basedon the so-called'triple'or'quadruple' testsofmaternal serum at 16 rveeks'gestation (p 318)

(TRISOMY PATAU SYNDROME 13) (TRISOMYlS) ANDEDWARDS SYNDROME

conditions there are usuallv severelearning difficulties, often in association'w.ithfailure to thrive. Horvever,there is considerable

These verv severeconditions \\,erefirst describedin 1960and share manv features in common (Figs 18 5 & 18.6).The,,-both shorv incidence figures of approximatelr,I in 5000 and convev a verv poor prognosis,rvith mosr affccted infants d1,ingduring the first days or weeksof life In the unusual event of long-term survival there are severelearning difliculties. Cardiacabnormalitiesoccur in at least90o/oof all cases. Usualll' chromosomeanalysisrer.ealsstraightforward trisoml.. Both of these disorders show an associationbctrveenincreasing incidence and advancedmaternal age,and in both the additional

variabilin.,particularlv in Wolf-Hirschhorn svndrome,and a poor corrclation of the phenotvpewith the preciselossof chromosomal material as determined by molecular analy-sis. The cri-du-chat svndrome derives its name from thc characteristiccat-like crv of affected neonatesthat results from underdevelopmentof the larvnx. Both conditions are rare, with estimated incidences of approximatelv I in 50000 births. In some children with clinical f'eaturesof one of these svndromes, but apparentlv normal chromosomes,the presenceof a verv subtle deletion can be demonstratedbv fluorescentin-situ h.vbrrdization(FISH, p. 34)

c h r o m o s o m e i s u s u a l h o f m a t e r n a l o r i g i n ( s e eT a b l e 3 . 5 , p. 48) Approximatelv l0o/o of casesare caused br mosaicism

using locus-specihcprobes for 4p or 5p (trig. 18.9).

o r u n b a l a n c e d r e a r r a n g e m e n t s ,p a r t i c u l a r l l ' r o b e r t s o n i a n translocationsin casesof Patau sYndrome.

C H R O M O S ODMEEL E T I O N A N DM I C R O D E L E TSI O YNDROMES Microscopicall-vvisible deletions of the terminal portions of chromosomes4 and 5 causethe \Volf Hirschhorn (4p-) (Fig. 18.7)

264

Fis.18.5 Facialview ofa ch tdwithtrisomy13showing severeb Lateralcleft Irpandpatate

and cri-du-chat (5p ) (Fig. l8 8) syndromes,respeciveh'.In both

Microdeletions Through a combination of high-resolution prometaphase banding (p 33) and FISH (p 3,1),severalpreviouslv unexplained syndromeshave been shown to be due to submicroscopicor 'micro' deletions Some of these microdelellazsinvolve loss of onlv a ferv genes at closelv adjacent loci, resulting in what are knou,n as contiguottsgenesyntlromes.For example, several bo1'srvith Duchenne muscular dystrophy (DMD) have been

C H R O M O S OD NI4SEO R D E R S

18

Fig.18.8 F a ca I v e w o f a 2 - y e a r o l d b o y w t h c r i - d u - c h a t s y n d r o m e

Fi9.18.6 o c c i o uat n d L i g L t l y A b a o yw r r - t - s o - y 1 8 N o r et h e p - o m i n e r L c t e n c h eh da n d s

Fi9.18.9 F I S Hs h o w i nfga i t u roefa c h r o m o s o m4ep l o c u s - s p eccpi fr o b e i nea c h i l dw t t ht h e ( r e dt)o h y b rd i z et o o n en u m b e4r c h T o m o s o m Theyellowprobeactsasa markerfor syndrome Wotf-Hirschhorn s yf N r g e l t h ec e n t r o m eor efe a c hn u m b e r 4c h r o m o s o m( eC o u r t e o N o t t i n g h a m H o s p i t a L , C t y ) Smith

Fig.18.7 - Wotf-Hrrschhorn syndrome w th deletion4psyndrome A chiLd

described rvho also havc other X-linked disorders, such as kinase de{icienc-vThe loci for rctinitis pigmentosaand gl.'-'cerol thesc disorders are knorvn to bc verv close to the DMD locus on Xp21.

265

18

C H R O M O S OD MI E SORDERS

In severalother microdeletion syndromesit is likely thar more than a few loci are involved. Examples are given in Table 18.6. Even when considered together these conditions are rare, but developmentsin microarray comparativegenomic hybridization (CGH) (p. 37) are beginning to reveal that microdeletion syndromes, and possibly also microduplication syndromes, are more common than is realized, especially'in idiopathic mental retardation. Recently reported series using microarral. CGH suggest that a submicroscopic chromosome abnormality is the cause of at least l0o/o of casesof idiopathic mental retardation. Many malformations and svndromes have been describedfor which there is as yet no recognizedcause(p. 251), but some of these will turn out to be due to microdeletions or duplications.

Lessonsfrom microdeletion syndromes Retinoblostoma The first clue to the location of the gene for retinoblastomawas provided by the discovery thar approximatelv 5o/oof children presentingwith the condition had other abnormalities,including learning difficulties. In severalof these children a constitutional interstitial deletion of a region of the long arm of chromosome13 was identified. The smallestregion of overlap was l3ql4, which was subsequentlyshown to be the position of the locus for the autosomal dominant form of retinoblastoma.This in turn led to the cloning of the gene and identification of the gene product

(p.202).

Wilms'tumor A proportion of children who develop the rare renal embryonal neoplasm known as l44lms' tumor (or hypernephroma) also h a v e a n i r i d i a ( = a b s e n ti r i s ; s e e F i g . 1 8 1 l ) , g e n i t o u r i n a r y abnormalities and /etardation of growth and development This combination of findings is referred to as the WAGR syndrome. Chromosome analysis in these children reveals a n i n t e r s t i t i a l d e l e t i o n o f c h r o m o s o m e1 1 p 1 3 ( F i g . 1 8 . 1 0 ) . Molecular studies have identified several genes within this deletion Loss of one of these, PAX6, is responsible for t h e a n i r i d i a ( F i g . l 8 . l l ) a n d c a n b e e s t a b l i s h e db y F I S H probe analysis. Loss of another, known as WTI, catses the development of Wilms' tumor (see also loss of imprinting and Wilms' tumor, p 206). This knowledge can now be used to predict whether a newly diagnosedchild with an 1lpl3 deletion is at high risk of developing a Wilms' tumor, and this can also be achievedby a separateFISH analysiswtng WTI as a locus, specific probe. Failure of WTI to hybridize to the deletion site indicatesthat the risk for developingWilms' tumor is high. It is important to note that, in contrast to retinoblastoma, the genetic aspectsof Wilms' tumor have proved to be extremely complicated, with several different autosomal loci involved. Nevertheless,thesediscoveriesbasedon the study of microdeletion syndromes have been extremely valuable in leading to the isolation of genesthat causethesetwo embryonal tumors.

AngeImon ond Proder-W i lli syndromes Recentdevelopmentsin thesedisordershavegeneratedparticular interest. Children with Angelman syndrome (see Fig. 7.23, p. ll8) show inappropriate laughter, with convulsions,poor coordination (ataxia) and learning difliculties. Children with

266

T a b t e1 8 . 5 M i c r o d e l e t i soynn d r o m e s

Prader-Willi syndrome (seeFig. 7 .21, p. 117) are extremely floppy (hypotonic) in early infancy and develop quite marked obesity

Syndrome

Chromosome

and mild-to-moderate learning difficulties in later years.A large proportion of children with thesedisordershavea microdeletion

Detetion 1p36

1

involving the proximal part of the long arm of chromosome 15q

Witliams

l

Langer-Giedion

8

WAGR

11

Angetman

15

Prader-Witti

15

Rubinstein-Taybi

16

Milter-Dieker

17

Smith-Magenis

17

DiGeorge/Sedt5dkov5/VCFS

22

(lsql -13).

WAGR, l4ltmstumononrridia, genitourinary malformations and retardation of growthanddevelopment

It is now known that if the deletion occurs de noao on the paternally inherited number l5 chromosomethe child will have Prader-Willi syndrome. In contrast, a deletion occurring ar the same region on the maternally inherited number l5 chromosome causesAngelman syndrome. Non-deletion cases also exist and are often due to uniparenral disomy (p. ll5), with both number l5 chromosomesbeing paternal in origin in Angelman syndrome, and maternal in origin in Prader-Willi syndrome.Thus loss of a critical region from a paternal number l5 chromosome causesthe Prader-Willi syndrome. Loss of an identical or similar region from a maternallyinherited number l5 chromosomecausesAngelman syndrome.These observationsare fundamental to the concept of imprinting (seeFig. 7 .22, p. ll7) and illustrate how new technologicaldevelopmentscoupled with clinical observationhave helped identify new underlying genetic mechanisms.

C H R O M O S OD Nl/SEO R D E R S

18

I

f

Fis.18.10 A ,M e t a p h a s e p r e a sdh o w i ntgh en u m b e1r 1c h r o m o s o m (easr r o w e d T)h ec h r o m o s o mi ned i c a t ebdyt h es i n g t ae r r o wh a sa ni n t e r s t i t i a I f 6 d e l e t r o n i n t h e s h o r tS ae rm e F i g s l S&l ' 'l1 8 1(2C o u r t e s yMoef g H e a t h , C r t y H o s p i t a t , N o t t i nFgl hSaHms )hBo w t n g f a t l uarPeAo X (A) green (red) The syndrome with WAGR probe from a child rn locusspecifrc to hybrrdize to thedeteted number11chromosome shown p r o b e a c t s a s a m a r k e r f o r i h e c e n t r oema ec rhenoufm b e r l l c h r o m o s(oCmoeu r t e s yDorfJ o h n C r o l l a . S a l i s b u r y a n d D r V e r o n i c a v a n H e y n i n g eEnd, i n b u r g) h invoh-ing the cardiac outflow tract, along with thymic and parath),'roidhypoplasia. Molecular and FISH studies have shown that most, if not all, casesare due to a microdeletion invol'r,ingthe proximal long arm of chromosome 22 (22q11'2) Dr Eva Sedlidkovi from Praguereported a largeseriesofchildren with a congenitallyshort palate in 1955, l0years earlier than DiGeorge, and these patients clearl-vhad the same condition. A similar phenotype was described by Shprintzen, with cardiac malformations, cleft palate and a recognizablefacial appearance - now usually referred to as velocardiofacialsyndrome (VCFS). Becauseof the confusion of eponyms and other terms given to 'Deletion 22q11 syndrome' now this condition over the years, has the most widespreadacceptance(although at the molecular

Fis.18.11 presenting 1'1p13 withaniridia on routine A babywithdeletion neonataiexamrnatron

level the deleted DNA segment is referred to as the DiGeorge Critical Region - DGCR). Figure 18.12 shorvsindividuals with deletion 22q11.2at different ages.Becauseit is the most common of the microdeletion syndromesit has been intenselyresearched. It is variableand many affectedindividuals are able to reproduce, so that the condition follows autosomaldominant inheritance in

somefamilies.A 3-Mb deletionoccursbecausethis small region of gcnome is flanked by two identical sequencesof DNA, known as low-copy repeats (LCRs), of the type that occur frequently throughout the genome. At meiosis the chromosomes can be Di George/ Sed16ikov6/VeIocardiofocioI syndrome confused when they align, such that the downstream DNA I is approximately DiGeorgesvndrome a disorderaffecting sequencealigns with the upstream. If recombination occurs occurringsporadically, characterized betrveenthesetwo flanking regions a deletion of 3 Mb results on in 4000births,usuall-v particularly those one chromosome 22. lt has recently emerged that many of the by a high incidenceof heart malformations,

267

18

C H R O I V O S ODMI S EO R D E R S

Fis.18.12 D e l e t i o2n2 q 1(1DG e o r g e / S e d t j d k o v S / v eot o f accarrasdly) n d r o mA e , A y o u n iqn f a nB t , A y o u n cgh i l dC A n o t d e r c h t d D , T h es a m e r n d i v i d u a l(aCs)a sa na d u lat q e d4 9 v e a r s

268

phenotvpic {'eaturesmar.be duc hrgelr to hlploinsufhcicncr. forthe 7BX1 gene I n d i r . i c l u r r l sd i a g n o s e c ll ' i t h t h i s c o n c l i t i o n s h o u l d b e invcstigatedlbr cardilc mallbrmirtions, calcium and parathr.roid s t a t u s ,i m m u n e f u n c t i o n , a n d p o s s i b l er c n l l a n o m a l i c s b v ultrasonog;raph-v. About half hare short stature and a small proportion of thesc have partial gro\\ th hormone deficicncr

A signilicant proportion, at least 25olo,have schizophrcnia-like episodesin adult lif'e.

Duplicotion22q11.2 In considerltion of thc mechanismthat gives rise to dcletion 22q11 2 srndrome (i.e. misalignedpairing at meiosisof the LCRs

CHROMO5OME DISORDERS

that flank thc 3-Mb region), one rvould predict that gametes duplicated for this submicroscopicsegment of DNA llould bc present in equal numbers, and therefore perhaps gir ing rise

hugging'. Somedegreeof learningdifficulty is the norm. The sleep pattern can often be managedb1'-fudicious use of melatonin.

to individuals duplicated for band 22q11.2. Interestingh,,onl1, recentlv has information been accumulating on the clinical featurespresent in individuals rvith this duplication. It ma-vwell

Deletion1p36syndrome

bc a relativelvcommon occurrcncebut will not be detectedunless a clinician asksthc laboratorv to perform the22ql1.2 FISH test. This will not happen,of course,unlessthe clinician suspectsthis region to be implicated,so at presentduplication 22qll.2 is almost certainly-underdiagnosed. It is cmerging that therc is no clearly consistent phenotype associatedwith this duplication. Some casesbear similarity to the tleletion 22qll.2 phenotype,but reports have highlighted marked variability'.The problems range from isolated mild learning diflicultics to multiple abnormalities lvith non-specilic dvsmorphic features,congenital heart disease,cleft palate,hearing loss and postnatalgrowth dehciencli

Williomssyndrome

Improvemcnts in cvtogenetictechniquesand the use of FISH is leadingto the identilicationand characterizationofadditional rare microdcletion s-vndromes.One of these, which emerged during thc 1990s,is deletion lp36 syndrome.The featuresare hypotonia' microccphah, growth delal-,severelearning difficulties, epilepsy (including infantile spasms),characteristicallystraight eyebrows rvith slightl.vdeep-seteyes,and midface hypoplasia(Fig 18.15).

ANDLEARNING PROBES MULTITELOMERIC DIFFICULTIES A ke-vdcvelopment in the use of FISH technology has been a set of subtclomeric probes for all chromosomes.Their use has begun to be routine in the investigationof non-speci{iclearning difficulties with or without dysmorphic features, especiall-vrvhen thcre is a famill' history of similar problems that follows a

This svndrome is due to a microdeletion at chromosome Tqll and diagnosiscan be confirmed by FISH. The clinical phenot-vpe lr,as lirst reported by Williams in 1961 and later expanded b1. Beuren (it is sometimesknou'n as Williams-Beuren svndrome). H-vpercalcemiais a variablefeature in childhood and sometimcs p e r s i s t s , r v h i l s t s u p r a v a l v u l a ra o r t i c s t e n o s i s( S V A S ) a n d

pattcrn that could be explained by the segregationof balanced and unbalanced forms of a reciprocal translocation (p. 47). Thc rationale is based on the observationthat the most viable

periphcral pulmonarl. artery stcnosisarc congenitalabnormalities

in about 5o/oof casesanaly-zed,although the figure is slightl-v higher for casesof severelearning diffrculties.About half of the positive casesare tJenoxoand the remainder are familial. Positive findings are especiallyimportant for familial casesin order to

of thc great vessels Haploinsufficiencv at 7q1l leads to loss of one copv of the genethat encodeselastin,a component of connective tissue This is probably the ke,'-factor causing SIAS and the vascular problems that are more common in later life Individuals havc a characteristicappearance(Fig. 18.13)with mild short stature,a full lower lip and sloping shoulders.Equall-v charactcristicis their behavior They are typicalll- ven outgoing in childhood - having rvhatis known as a 'cocktail party manner' - but become rather withdrawn and sensitive as adults All are intcllcctuallf impaired to the extent that they cannot lcad independent lives, and the vast majoritl' do not reproduce, although parent-child transmissionhas bcen reported.

Smith-Mogenis syndrom e This microdeletion sl,ndrome is due to loss of chromosome material at 17p 11.2;this is often visible cytogeneticallyon a good qualit-vkarl'ot-vpe.As with DiGeorgc svndrome the mechanism of deletion in many casesinvolves homologous recombination betrveen flanking LCRs Thc physical characteristrcs are not convincinglv distinctive (Fig. 18.14),but congenitalheart disease occurs in one-third, scoliosisdevelopsin late childhood in more than one-half, and hearing impairment in about trvo-thirds The s1'ndromeis most likel.vto be recognizcdby' the behavioral characteristics:aschildren, thesepatientsexhibit self-harming(headbanging, pulling out nails and inserting objects into orifices), 'selfa persistcntlv disturbed sleep pattern, and characteristic

18

unbalancedtranslocationsare those that are small and thereforc involvc verv tcrminal (telomeric)chromosomesegmentsA revierv of the use of the svstem has shorvnthat abnormalitiesare found

identif-vbalancedcarriers.Telomeric FISH probesare now being replacedbv a polymerasechain reaction (PCR)-basedtechnique calledmultiplex ligation-dependentprobe amplification (MLPA) (p. 67) This is an easierand lesstime-consuming analysisin the laboratorvbut doesnot detect balancedtranslocations.

TRIPLOIDY (69,XXX, 69,XXY, 69,XYY) is a relatively common finding in material cultured from spontaneousabortions, but is seenonlv rarelv in a liveborn infant. Such a child almost always shorvssevere intrauterine gro\vth retardation with relative preservationof head growth at the expenseof a small thin trunk

tiploidl'

involving the third and fourth hngers and/or the second Syndact.vl.v and third toes is a common finding Casesof triploidy due to a doublc paternal contribution usually miscarry in early to midprcgnancy and are associatedrvith partial hydatidiform changes in the placenta(p 96) Caseswith a double maternal contribution surr,ivefor longer but rarel-Ybeyond the early neonatalperiod.

of lto Hypomelanosis Several children rvith mosaicism for diploidy/triploidy have been identified. These can demonstrate the clinical picture

269

18

CHROMOSOME DISORDERS

Fi9.18.13 A person withWiLliams (B)anolderchiLd (C)andrnhiseartv syndrome asa baby(A),ayoung (D) chiLd forties

seen in full triploidy but in a milder form. An alternarive presentationoccurs as the condition knolvn as hypomelanosisof

270

Ito. In this curious disorder the skin shows alternating patterns of normallv pigmented and depigmentedstreaksthat correspond to the embryological developmentallines of the skin known as B l a s c h k o ' sl i n e s ( s e e F i g . 7 . 1 7 ; 1 8 . 1 6 ) .M o s t c h i l d r e n w i t h hypomelanosis of Ito have moderate learning difficulties and convulsions that can be particularly diflicult to treat. There is

increasing evidence that this clinical picture representsa nonspecific embryological response to cell or tissue mosaicism. A similar pattern of skin pigmentation is sometimesseenin women with one of the rare X-linked dominanr disorders (p. 110) with skin involvement, such as incontinentia pigmenti (seeFig. 7.17). Such women can be considered as being mosaic, as some cells expressthe normal genewhereasothers expressonly the mutant gene.

DM l SEO R D E R S CHROl\4OSO

18

Fis.18.14 A y o u n gp e r s o nw r t hS m i t h - M a g e n sr sy n d r o m et :h ef a c i a I f e a t u r ea s ' e n o rr i g ^ t yd s t i n c t i v eb r t t h e p l i l t r - m s u s u a l t ys h o r L A s b a b i e sc. h r o m o s o m es t u d i e sa r e o f t e nr e q u e s t e b de c a u s e t h e o o s s r btrv. o f D o w - s v n d r o m ei s - a r s e d

Fig.18.15 - verystraight eyebrows, 1p36 syndrome withdeletion A child difficutties e p i l e p s ay n d l e a r n t n g

D I S O R D EO RS FTHE S E XC H R O M O S O M E Scanbe rather self-obsesscdin their behavior Adults'rvith Klinefelter SYNDROME (4ZXXY) KLINEFELTER First describcdin7912, this rclativcl.vcommon condition with an incidence of I in 1000 male live births was in 1959 shown to be due to the presenceof an additional X chromosome.

features Ctinicat In childhood the diagnosiscan be suspectedin a male presenting with clumsinessor mild learning difficulties, particularlf in relation to verbal skills.The overallverbalIQis reducedb1,l0 to 20 points below that of unaffectedsiblinesand controls. and thesechildren

svndrome tend to be slightly taller than averagervith long lorverlimbs. Approximately 30o/oof adult malcs with Klinefelter svndrome show moderatel.vsevcregynecomastia(enlargementof the breasts)and all are infertile, with small soft testes.There is an incrcasedincidence of leg ulcers, osteoporosisand carcinomaof the breastin adult life Tieatment with testosteronefrom pubert-v onrvards is benelicial for the devclopment of secondary sexual characteristics and the long-term prevention of osteoporosis. Nfalesrvith Klinefelter svndrome arc usually infertile due to the althortghfertility absenccof sperm in their semen(azoospermiu), has been achievedfor a small number of affectedmales using the techniques of testicular sperm aspiration and intracytoplasmic sperm injection(ISCI)

Fi9.18.16 M o s a ipca t t e ronfs k i np i g m e n t a t ioonn t h ea r mo fa c h i t dw r t hh y p o m e l a n o s t s e dr t hp e r m , sosn o ' l t o ( R e p r o d r cw fromJenkrD n s.M a r t i nK , Y o u nI gD 1 9 9 3 with of ltoassociated Hypomelanosis o rt r i s o m7y a n da p p a r e n t m o s a i c i sfm 'pseudomosaicism' a ta m n i o c e n tse s J MedGenet3]783-184)

271

18

C H R O M O S OD MI E SORDERS

Chromosome findings Usually the karyotype shows an additional X chromosome. Molecular studies have shown that there is a roughly equal chance that this will have been inherited from the mother or from the father. The maternally derived casesare associated with advancingmaternal age A small proportion of casesshow mosaicism(i.e. 46,XY / 47,XXY) Rarely,a male with more than two X chromosomescan be encountered,for example48,XXXY or 49,XXXXY. These individuals are usually quite severely retarded and also share physical characteristicswith Klinefelter men, often to a more marked degree

(45,X) TURNER SYNDROME This condition was lirst describedin 1938.The absenceof a Barr body consistent with the presenceof only one X chromosome, wasnoted in 1954and cytogeneticconfirmation was forthcoming in 1959. Although common at conception and in spontaneous abortions(seeTable 18.1), the incidencein liveborn femaleinfants is loq with estimatesranging from I in 5000 to I in 10000.

Ctinicatfeatures Presentation can be at any time from pregnancy to adult life. Increasingly,Turner svndrome is being detected during the second trimester as a result of routine detailed ultrasonography,which can revealeither generalizededema(hydrops)or swellinglocalized to the neck (nuchal cysr or thickened nuchal pad) (Fig. I 8. I 7). At birth many babies with Turner syndrome look entirely normal. Others show the residue of intrauterine edema with puffy extremities (Fig. 18 18) and neck webbing. Other findings can include a low posterior hair-line, increased carrying angles at

Fis.18.18 Thefootofan infant wrthTurnersyndrome showng edemaand s m a tnLa i t s

the elbows,short fourth metacarpals,widely spacednipples and coarctationofthe aorta, which is presentin 15oloofcases. Intelligence in Turner syndrome is normal. However, studies haveshown somedifferencesin socialcognition and higher-order executivefunction skills accordingto whether the X chromosome was paternalor maternal in origin (Ch. 6; p. 100).The rwo main medical problems are short stature and ovarian failure. The short stature becomesapparent by mid-childhood, and without growth hormone treatment the averageadult height is 145cm. This short stature is due, at least in part, to haploinsufliciency for the SHOX gene, which is located in the pseudoautosomal region (p. 112). Ovarian failure commencesduring the second half of intrauterine life and almost invariably leads to primary amenorrheaand infertility. Estrogen replacementtherapy should be initiated at adolescencefor the development of secondary sexualcharacteristicsand long-term prevention of osteoporosis. In-t:itro fertilization using donor eggs offers the prospect of pregnancyfor women with Tirrner syndrome.

Fis.18.17 272

Ultrasono graph icscanat]8 weeksgestation g nyorops showin fetatisNotethehatooffluidsurroundrng thefetus(Courtesy of Dr D Rose, Cty HospitaL, Nottrngham )

Chromosome findings These are summarized in Thble 18.7.The most common finding is 45,X, sometimeserroneouslyreferred to as 45,XO It has been

SORDERS C H R O M O S OD MIE

18

F R A G I LXE S Y N D R O M E Tabte18.7 Chromosome findinqsrnTurnersvndrome Karyotype

Frequency(%)

MonosonyX-45,X

50

g 45.X146.XX Mosaicism-e

2A

- 46.Xi(Xq) lsochromosome

15

This condition, which could equalll' rvellbe classiliedasa singlegenedisorder rather than a chromosomeabnormalit-v'hasthe unique distinction of being both the most common inherited causeof learning difliculties and the first disordcr in which a d1--namic mutation was identilied (p. 23) Martin and Bell described the condition in the 1940sbefore the chromosome era, and it has

Rrng- 46,X.r(X)

5

also becn known as Martin-Bell svndrome The chromosomal abnormality rvasfirst describedin 1969 but the signilicancenot fullv realizeduntil 1977.In 1991the underlying molecular defect

- 46,X,det(Xp) Detetion

5

lvas discovered.

0ther

5

lncidence Fragile X svndrome affects approximatell' 1 in 5000 males and accountsfor 'f-So/oof all males with learning difficulties

shown that Turner syndrome arisesin 807o of instancesthrough a sex chromosome (X or Y) being lost in paternal meiosis.In a signilicant proportion of casestherc is chromosome mosaicism and those with a normal cell line (16,XX) have a chanceof being fertile. Those r,vithsomeY-chromosomematerial in thcir second cell line must be investigated for possible gonadal dvsgencsis; intracellular malc gonadscan occasionallvbecomemalignant and need to be removed surgicalll'.

XX X F EM A L ES Birth survc,vshave shown that approrimatcll' 0 lob of all femalcs har,ca'[7,XXX karyot-vpeThcsc rvomenusually haveno ph1-sical abnormalitiesbut can sho'w'a mild reduction of betrveen10 and 20 points in intellectual skills and sometimesquite oppositional

Ctinicatfeatures Older boys and adult males usualll'' have a recognizablefacial appcarancervith high forehead,large ears,long face and prominent jaw (trig. 18.19).After puberty most affectedmaleshavelargetestes (macro-orchidism). There may also be evidence of connective tissue rveakness,with hyperextensibleioints' stretch marks on the skin (striac) and mitral valve prolapse.The learning diflicultics are moderate to severeand man-vaffected bo.vsshow autistic features and/or hyperactive behavior. Speech tends to be halting and repetitive Female carriers can sholv some of the facial features,and approximately 50o/oof women with the full mutation shorvmild-to-moderate learning diffi culties.

behavior This is rarely of sufficient severity to rcquire special education.Studieshaveshorvnthat thc additional X chromosome is of maternal origin in 95o/oof casesand usually arisesfrom an error in meiosisI. Women rvith a 47,XXX karyotypeusually show normal fertility and havechildren rvith normal karyot-vpes. As rvith malesrvho havemore than two X chromosomes,women rvith more than three X chromosomesshow a high incidence of learning difhculties,the severityof which is directl-vrelatedto the number of X chromosomesDresent.

XYYMALES This condition shows an incidence of about 1 in 1000 in malcs in newborn surveys but is found in 2 3o/oof males who are in institutions becauseof learning difficulties or antisocialcriminal behavior Hower.er,it is important to stress that most '[7,XYY men have neither learning difficulty nor a criminal record, although they can show emotional immaturitv and impulsive behavior Fertility is normal. Ph1'sicalappearanceis normal and stature is usuallv above average.Intelligence is mildly impaircd, rvith an overall IQscorc of 10 to 20 points below a control sample.The additional Y chromosomemust ariseas a rcsult of non-disjunction in paternal meiosisII or as a posl-z]gotic cvent.

Fis.18.19 A famrlyaffectedby frag teX syndromeTwo sistersbothcarriers of a smatlFR4XAmutationinheritedfrom theirfathelhavehad affectedsonswith differentdegreesof learningdifficuLty.

273

18

C H R O M O S OD MIE SORDERS

The fragiteX chromosome The fragile X syndrometakesits name from the appearanceof the X chromosome, which showsafi,agile site close to the telomere at the end of the long arm atXq}7.3 (Fig. 18.20).A fragile site is a non-staining gap usually involving both chromatids at a point at which the chromosome is liable to break. In this condition, detection of the fragile site involves the use of special culture techniques such as folate or thymidine depletion, which can result in the fragile site being detectablein up ro 50o/oof cells from affected males.Demonstrarion of the fragile site in female carriers is much more difficult and cvtogeneticstudiesalonearenot a reliable means of carrier detection, in that, although a positive result confirms carrier status, the absenceof the fragile site does not excludea woman from beine a carrier.

The moleculardefect In gene mapping the fragile X locus is knorvn as FRAXA.T\e FRAXA mutation consistsof an increasein the size of a region in the 5'-untranslatedregion of the fragile X learning difficulties (F,MR-I) gene This region contains a long CGG trinucleotidc repeat sequence.In the DNA of a normal person there are betr,veen 10 and 50 copiesofthis triplet repeatand theseare inherited in a stablefashion. However, a small increaseto between 59 and 200 renders this repeat sequenceunstable,a condition in uhich it is referred to as ^ l)remutation.Allelesof 51-58 are referred to as intermediate. A man rvho carries a premutation is knorvn as a ,normal transmitting male', although it has been recognizedrecentlv that these premutation carriers are at increasedrisk of a lateonset neurologicalcondition named 'fragile X tremor/ataxia syndrome'. All of his daughtersrvill inherit the premutation and will be of normal intelligence,bur when thesedaughterscome to have sons there is a high risk that the premutation will undergo a further increasein size during meiosis Ifthis reachesa critical sizeof greaterthan 200 CGG triplets, it becomesa full mutation. This processis sometimesreferred to as expansionof the triplet repeatsequence. The full mutation is unstable not onlv during female meiosis but alsoin somaticmitotic divisions Consequentll',in an affected male gel electrophoresisshowsa 'smear' of DNA consistinqof manv

different-sizedallelesrather than a singleband (Fig 18.21).Note that a normal allele and premutation can be identified by PCR, whereasSouthern blotting is necessaryto detect full mutations as the long GCC expansionis often refractory to PCR amplification. At the molecular level a full mutation suppressestranscription of the FMR-I geneby hypermethylation, and this in turn is thought to be responsiblefor the clinical featuresseenin males,and in some females with a large expansion (Thble 18.8).The FMR-I gene contains l7 exons encoding a cyroplasmic protein that plays a crucial role in the developmentand function ofcerebral neurons The FMR-I protein can be detected in blood using specihc monoclonal antibodies. Another fragile site closely adjacent to FRAXA has been identilred at Xq28. This is known as FRAXE. The expansion mutations at FRAXE also involve CGG triplet repeats and occur at approximately one-quarter the frequencv of FRAXA mutations, or less. Some males with these mutations have mild learning difficulties, whereas others are just as severely affectedasmen with FRAXA. These men are usually ascertained through the discovery of a fragile sire on chromosome analysis rvith a normal result on FRAXA mutation analysis. A third fragile site, FRAXF, has recently been identified close to FRAXA and,FRAXE. This does nor seem ro cause anv clinical abnormalitl:

Geneticcounseling and the fragileX syndrome This common causeof learning difficulties presenrsa major counselingproblem. Inheritance can be regardedas modihed or atypical Xlinked. All of the daughtersof a normal transmitting male will carry the premutation. Their male offspring are at risk of inheriting either the premutation or a full mutation This risk is dependent on the size of the premutation in the mother, r,vithmutations greater than 100 CGG repeatsalmost invariably increasingin size to becomefull mutations. For a woman r,vhocarries a full mutation there is a 50o/orisk that eachof her sons will be affectedwith the full syndrome and that each of her daughters will inherit the full mutation. As approrimately 50o/oof females with the full mutation have mild learning difliculties, the risk that a female carrier of a full mutation r'villhave a daughter with learning difficulties equals 1/, x t/2,t e.

Fis.18.20 274

X chromosome fromseveraI maLes withfragile X syndrome(Courtesy ofAshleyWitkinson, CttyHospitat, Nottingham )

CHROMOsOME D15ORDERS

18

Thc fragile X syndrome is a condition for which population screening could be offered, either among selectedhigh-risk groups such asmaleswith learning difficulties or on a widespread gencral population basis.Such programs will have to surmount major cthical, {rnancialand logistical concerns if they are to rchieve widespreadacceptance(p. 308).

SORDERS C H R O M O S ODMI E PHENOTYPES ANDBEHAVIORAL The distinctive behavior of children with Williams syndrome 'cocktail party manner' - has been recognized their outgoing as part of the condition for a long time. As the microdeletion conditions have emerged it has been increasingly clear that patternsofbehavior can reliabl-vbe attributed to certain disorders. This is verv striking in Smith-Magenis s-vndrome,but also apparent to a lesser extent in deletion 22qll, cri-du-chat, Angelman and Prader-Willi syndromes. It is also apparent rn the aneuploidies(Dorvn and Klinefelter syndromes),as well as in 47,XXX and '|7,XYY and fragile X syndromes. Behavioral

Fis.18.21 S o u t h e rbnt o o t fD N A f r o ma f a m r lsyh o wn ge x p a n s i oonft h e CGGtripletrepeatbeng passed froma normaltransmitt ng male +L-^,,^hL;- ^f, i^-+^ ^---;^-d:rrnhlerln

h c r c n n r n , i l hf r : n . l p X

L ri l u u g

Ir>uuugoLtrLdr

'l _p : r n r n 'nY "' "'

d i [ [ i er r l t r p c ( f n r r r r p c r i n f f l r G T : v , n r S t ' : m p c c

IrEt

-_-f,

Hncn t:l

Leeds)

genotype-phenotype Tabte18.8 Fragr[e X syndrome: correl'aiio ns No. oftriptet repeats (normal range10-50)

Fragilesite

phenotvpes have therefore become an area of considerable i n t e r e s t t o c l i n i c a l s c i e n t i s t s a n d t h e o b s e r v a t i o n sl e n d support to the belief that behavior, to some extent at least, is gcnetically determined. In studl-ing chromosome disorders rvc are of course looking at genetically abnormal situations, and lrom this we cannot necessarilyextrapolate directl-vto 'normal' situations. For the latter, twin studies have provided substantialand valuableinformation This field of study remains complex and understandably controversial However, most now accept that behavior is a complex interaction of genetic background, ph.vsicalinfluencesduring early development (e.9. fetal u'ell-being),nurturing experiences,family size, culture and belief svstems.

Intetligence detectabte

OFSEXUAL DISORDERS DIFFERENTIATION

Males (intermediate alleles) 5'1-58 59-200(premutation)

No

Normal(normai transmitting maLe)

200-2000(futlmutation)

Yes(inupto

Moderate-to-severe

\ r l / ^n t . c t t c t

tc:rnrnn nrtfiCUlties

The process of sexual differentiation has been described in Chapter 6 (p 96). Given the complexity of the sequentialcascade of cvents that takesplace between6 and 14weeksof embrvonic life, it is not surprisingthat errors can occur Many of theseerrors can lead to sexual ambiguity or to discordancebetween the chromosomal sex and the appearanceof the external genitalia

59-200(premutation)

No

Normal

These disordersare also sometimesreferred to as various forms of intersex(Box 18.2).

200-2000(fu[tmutatron)

Yes(usualty <10% of cetts)

50%normat,50%mitd learningdifficutties

Females atletes) 51-58(intermediate

r/u.Prenatal diagnosiscan be offered basedon analysisof DNA from chorionic villi, but in the event of a female fetus with a full mutation accurateprediction of phenotype cannot be made

T R U EH E R M A P H R O D I T I S M In this extremely rare condition an individual has both tcsticular and ovariantissue,often in associationwith ambiguous genitalia. When an exploratorv operation is carried out in

275

18

C H R O M O S OD MIE SORDERS

Box 18.2 Disordersof sexuaIdifferentration a n dd e v e l o p m e n t (Klinefeltersyndrome) Seminiferous tubuledysgenesis 4ZXXY48 XXXY48,XXYY49,XXXXY (Turnersyndrome) Ovariandysgenesis 45,X,46,X,i(Xq), 46,X,det(Xp), 46.X(X) True hermaphroditism 46,XX withY dervedsequences 46,XX/46,XY ch merism Malepseudohermaphroditism Androgen insensitivity - teslicula' fe'nn zatror Corrptete - Reifenstein Incomplete syndrome lnhorn errors of testosterone biosynthesis e g 5cr-Reductase defciency 45,X/46,XY mosaicism Femalepseudohermaphroditism CongenitaL adrenal hyperptasia Maternal androgen ingestonor androgen-secreting tumor

Fis.18.22 these patients an ovary can be found on one side and a testis on the other. Alternatively, there can be a mixture of ovarian and testicular tissue in the gonad, which is known as an ovotestis. Most patients with true hermaphroditism have a 46,XX karyotype, and in many of these individuals the paternally derived X chromosome carries Y chromosome-specific DNA sequencesas a result of illegitimate crossing over between the X and Y chromosomesduring meiosis I in spermatogenesis

(Fis.18.22). A small proportion of patientswith true hermaphroditism are found to be chimeras with both 46,XX and 46,XY cell lines, a situation analogousto freemartins in cattle (p. 5,1).

M A L EP S E U D O H E R M A P H R O D I T I S M In pseudohermaphroditism there is gonadal tissue of only one sex. The external genitalia can be ambiguous or of the sex opposite to that of the chromosomes. Thus in male pseudohermaphroditism there is a 46,XY karyotype with ambiguous or female genitalia. The most widely recognized causeof male pseudohermaphroditism is androgen insensitivity (p. 166). In this condition, which is also known as testicularfeminization sjtndlome, the karyotvpe is normal male and the external phenotype rs essen-

276

tially that of a normal female. Internally the vaginaends blindly and the uterus and fallopian tubes are absent.Testesare located in the abdomen or in the inguinal canal, where they can be mistakenfor inguinal herniae.This condition is causedby the absence of androgenreceptorsin the target organs,so that, although testosterone is formed normally, its peripheral masculinizing effects are blocked.These androgen receptors are coded for bv a geneon the X chromosomein which both deletions and point

F I S Hs h o w i nhgy b r r d i z a toi ofanY c h r o m o s o mpea i nttot h es h o r t arm ofanX chromosome in a 46,XXmale(Courtesy of N gel S m r t hC. i t yH o s p r t aN[ o, t t i n g h a)m

mutations have been identified. Curiously, expansion of a CAG repeat in the hrst exon of this androgen receptor gene causes a neurological disorder known as Kennedy disease,or spinobulbar muscular atrophy (SBMA). This is a rare example of the phenomenon referred to as a'gene within a gene'. Other causesof male pseudohermaDhroditisminclude: l. An incomplete form of androgen insensitivity known as Reifenstein syndrome in which affected males have hypospadias, small testesand gynecomastia. 2. Enzyme defects in testosterone synthesis such as 5cxreductasedeliciency(seeFig. I 1.5),in which the externalgenitalia are ambiguousat birth but undergo masculinization(virilization) at puberty. 3. Chromosome mosaicism (45,X/46,XY), in which most individuals are normal males but a small proportion have ambiguousor female external genitalia. 4. Campomelic dysplasia, which is caused by mutations in the SOX9 gene on chromosome 17. SOX? is believed to be an important gene in the regulatory parhway by which SRY causesmasculinization of the undifferentiated fetal eonads

(p.eo). 5. The Smith-Lemli-Opitz syndrome, which is caused by deficiency of 7-dehydrocholesterol reductase,an enzyme involved in cholesterolbiosynthesis.Some severelyaffected male infants have female external genitalia.

SORDERS C H R O M O S OD MIE

18

F E M A LP ES E U D O H E R M A P H R O D I T I S M In female pseudohermaphroditism,the karyotype is female and the external genitalia are virilized so that they either resemble those of a normal male or are ambiguous. Congenital adrenal hyperplasia (CAH) is by far rhe most important cause of female pseudohermaphroditism(p. 165). This can be caused by several different enzyme defects in the adrenal cortex, all of which show autosomal recessive inheritance.Reduced cortisol production leads to an increasein adrenocorticotrophichormone (ACTH) secretion,which in turn causeshyperplasiaof the adrenalglands.In the most common form of CAH, due to 2l-hydroxylase deficiency,hormone synthesis switches from the manufacture of cortisol and aldosterone to the androgen pathway (seeFig. 11.5, p. 166), leading to striking virilization of a female fetus (seeFig. 11.6, p. 167).The lack of cortisol and aldosteroneusually leads to rapid collapseshortly after birth, which can prove fatal unlessappropriatehormone and electrolytesupplementationis initiated. Rarer causesof female pseudohermaphroditism include an androgen-secretingtumor and maternal androgen ingestion during pregnancv.

CHROMOSOMAL BREAKAGE SYNDROMES Constitutional and acquired chromosome abnormalitiesthat predisposeto malignancy are consideredin Chapter 14. In addition to theseconditions, it is recognizedthat a small number of hereditary disordersis characterizedby an excessofchromosome breaksand gapsas well as an increasedsusceptibility to neoplasia.

ATAXIATELANGIECTASIA This is an autosomal recessivedisorder that presents in early childhood with ataxia,oculocutaneoustelangiectasia (Fig. 18.23), radiation sensitivit-v,and susceptibility to sinus and pulmonary infection (p 192). There is a l0-20o/o risk of leukemia or lymphoma. Cells from patients show an increasein spontaneous chromosome abnormalities,such as chromatid gaps and breaks, rvhich are enhanced by radiation. The gene for ataxia telangiectasiais called ATA4 (M = mutation) and maps to chromosome llq23. The protein product is thought to act as a 'checkpoint' protein kinase,which interacts with the TP53 and BRCAI gene products to arrest cell division and thereby allow repair of radiation-induced chromosome breaks before the S phasein the cell cycle.

BLOOM SYNDROME Children with this autosomal recessivedisorder are small with a light-sensitive facial rash and reduced immunoglobulin levels (IgA and IgM) The risk of lymphoreticular malignancy is approximately20olo.Cultured cells show an increasedfrequency

Fis.18.23 in a chitdwrthataxrate[angiectasia Ocutartetangrectasia

of chromosome breaks,particularly if they are exposedin oitro to ultraviolet light. The gene for Bloom syndrome maps to chromosome 15q26,where it encodesone member of a group of enzvmescalled the DNA helicases(p. l4). These are responsible for unwinding double-strandedDNA prior to replication, repair and recombination. Normally the Bloom syndrome gene plays a major role in maintaining genome stability. When defective in the homozygous state, DNA repair is impaired and the rate of recombination between sister chromatids is increased dramatically This can be demonstrated by looking for sister chromatid exchanges(seebelow).

FANCONIANEMIA This autosomalrecessivedisorder is associatedwith upper limb abnormalities involving the radius and thumb (Fig 18.24), increasedpigmentation and bone marrow failure leading to deficiency of all types of blood cells (i.e. pancytopenia).There is also an increased risk of neoplasia, particularly leukemia, lymphoma and hepaticcarcinoma.Multiple chromosomalbreaks are observedin cultured cells(Fig. 18.25)and the basicdefect lies in the repair of DNA strand cross-links. There are five known subtypesof Fanconi anemia,each causedby recessivemutations at different autosomal loci. The commonest, type A, maps to chromosome 16q24. lt is not known how any of the Fanconi anemia genes is involved in maintaining the integrity of DNA cross-links.

P IAG M E N T O S A XERODERM This exists in at least seven different forms, all of which show autosomal recessiveinheritance. Patients present with a lightsensitivepigmented rash and usually die from skin malignancy in sun-exposed areas before the age of 2}years. Cells cultured from these patients show chromosome abnormalities only after

277

18

C H R O M O S OD MIE SORDERS

exposureto ultraviolet light. These disordersare due to defectsin the nucleotide excision repair pathway'.This involves endonuclease cleavage5' and 3' to each damaged nucleotide, excision of the damaged nucleotide(s),and finally restoration of the damaged strand using the intact opposite strand as a template.

AE N DS I S T E R C H R O M O S OBMREE A K A G EXCHANGE CHROMATID instability is provided Strong evidenceof increasedchromosome by the demonstration of an increasednumber of sisterchromatid (SCEs) in cultured cells. An SCE is an exchange exchanges (crossing over) of genetic material between the two chromatids

Fie.18.24 B i t a t e rraaLd i aaIp t a s rwar t ha b s e nt th u m b sn a n i n f a nwt r t h Fancoa nn i emia

of a chromosome in mitosis, in contrast to recombination in meiosisI, which is betweenhomologouschromatids.SCEs can be demonstratedby differencesin the uptakeof certain stainsby the two chromatidsof eachmetaphasechromosomeafter two rounds of cell division in the presence of the thymidine analogue 5bromodeoxyuridine(BUdR), which becomesincorporatedin the newly synthesizedDNA (Fig. 18.26).There are normally about l0 SCEs per cell, but the number is greatlyincreasedin cellsfrom

Fis.18.25 M - t t ; p t ec f , T o m o s o l eb r e a k sa r d g a p s n a m e t a p h a s se p r e a dp r e p a r e d f r o m a c h t d w t h F a n c o narn e m t a

278

DM I SEO R D E R S CHRON4OSO

18

anaLvsis for chromosome Box 18.3 Indications es abnormatit congenitaI Muttrp[e mentaIretardatton Unexptained development in sexual Sexual ambguityor abnormality nfertitity miscarrtage Recurrent stttlbirth Unexp[ained syndromes breakage andchromosome Ma[gnancy

t 2. Information about the prognosis can be provided, along with detailsof the relevantsupport group and an offer of contact

Fis.18.26 preparation showing sisterchromatid exchanges Chromosome (arrowed)

with other families. 3. A chromosomal diagnosis should facilitate the provision of accurate information about the recurrence risk for future siblings. Although it can be very distressingfor parents to be told that their child has a chromosome abnormality, they will often be relieved that an explanation for their child's problems has been found

patients with Bloom syndrome and xeroderma pigmentosa. In the latter condition this is apparentonly after the cells havebeen exposedto ultraviolet light At present it is not clear horv SCEs relate to the increased chromosome breakageobservedin these two disorders,but it is thought that the explanation could involve one of the steps in DNA replication. It is also of interest that the number of SCEs in normal cells is increasedon exposureto certain carcinogens and chemical mutagens.For this reasonthe frequency of SCEs in cells in culture has been suggestedas a useful in-ritro test of the carcinogenicitl, and mutagenicity of chemical compounds (p 28).

FORCHROMOSOMAL INDICATIONS ANALYSIS It should be apparent from the contents of this chapter that chromosome abnormalities can present in many different ways. Consequently it is appropriate to consider the indications for chromosome analysisunder a number of different headings (Box18.3).

ITALABNORMALITIES MULTIPLECONGEN Every child with multiple congenital abnormalitiesshould have studies undertaken. This is important for several ;|;ff::"-. l. Establishinga chromosomaldiagnosiswill prevent further potentially unpleasantinvestigationsbeing undertaken.

DIFFICULTIES LEARNING UNEXPLAINED Chromosome abnormalitiescauseat least one-third of the 50o/o of learning difficulties that are attributable to genetic factors. Although most children with a chromosome abnormality have other features such as growth retardation and physical anomalies, this is not always so. If the fragile X syndrome is a possibility, it is important that the cytogeneticslaboratory is informed so that the correct culture conditions are used (p.274), although in most centers the fragile X syndrome is now diagnosed by molecular methods rather than chromosomeanalysis.If standard karyotyping and fragile X syndrome testing is negative' many geneticistsnow frequently requestmultitelomeric probe analysis, especiallyif there is a positive family history of learning difficulties (p.269) In the future it is hoped that microarray CGH (p. 8) will resolve many diagnostic problems in individuals and families with learning difficulties, when the technique becomesroutinely availablewithin clinical serrice testing.

AMBIGUITY SEXUAL The birth of a child with ambiguousgenitaliashould be regarded as a medical emergency, not only because of the inevitable parentalanxiety,but alsobecauseof the importance of ruling out the potentially life-threateningdiagnosisof salt-losingcongenital adrenal hyperplasia(p. 165). A chromosome analysisshould be among the first investigationsundertaken. Disorders of sexual development presenting in later life with problems such as delayed puberty, primary amenorrhea or male gynecomastiaare also strong indications for chromosome

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CHROMOSOME DISORDERs

analysisas a first-line investigation.This can provide a diagnosis such as Turner syndrome (45,X) or Klinefelter syndrome (47,XXY). Alternatively a normal karyotype will stimulate a search for other possible explanations, such as an endocrine abnormality.

INFERTILITY ANDRECURRENT MISCARRIAGE Unexplained involuntary infertility should prompt a request for chromosomestudies,particularly if investigationsrevealevidence

M A L I G N A NACNYDC H R O M O S O M E BREAKAGE SYNDROMES Certain types of leukemia and many solid tumors, such as retinoblastoma (p. 202) and Wilms' tumor (p. 266), are associated with specific chromosomal abnormalities that can be of both diagnostic and prognostic value. Clinical features suggestiveof a chromosomebreakagesyndrome(p.278), such asa combination of photosensitivity and short stature, should also lead to appropriate chromosome fragility studies, such as analysis of sister chromatid exchanges

of azoospermiain the male partner. At least 5oloof such men are found to have Klinefelter syndrome. More rarely a complex chromosome rearrangement such asa translocation can causesuch severemechanicaldisruption in meiosis that complete failure of gametogenesls ensues. At least 15oloofall recognizedpregnanciesend in spontaneous miscarriage;in 50o/oof casesthis is becauseof a chromosome abnormality (p. 261). Unfortunately, some couples experience recurrent pregnancy loss, which is usually defined as three or more spontaneous miscarriages.Often no explanation is ever found and many such couples go on to havesuccessfulpregnancres. However, in 3-60/o of such couples one partner is found to carry a chromosomerearrangementthat predisposesto severeimbalance through malsegregationat meiosis(p. 4l). Consequentlyit is now standardpracticeto offer chromosomeanalysisto all coupleswho haveexperiencedthree or more spontaneouspregnancylosses.

UNEXPLAINEDSTILLBIRTH/NEONATAL DEATH The presenceof growth retardation and at least one congenital abnormality in a stillbirth or neonatal death would be an indication for chromosome studies based on analvsis of blood or skin collected from the baby before or as soon after death as possible. Skin fibroblasts continue to be viable for several days after death. Chromosome abnormalities account for 5olo of all stillbirths and neonaraldeaths,and nor all of these babies have multiple abnormalities that would immediately suggest a chromosomalcause.

280

FURTHER READING De Grouchy J, Turleau C 1984Clinical atlasof human chromosomes, 2nd edn.John Wiley, Chichester A lao^ish11, illustratedatlas of hnownchromosomal syndromes Donnai D, Karmiloff-Smith A 2000 Williams syndrome: from genotype through to the cognitive phenotype Am J Med Genet (Semin Med Genet)97: 164-171 A retien dealingmith onemicrodeletionsyndromein detail and the elforts to unrlerstandhomthephenotypecan beeqlained by the molecular findmgs GardnerRJ M, SutherlandG R 1996Chromosomeabnormalitiesand geneticcounselling,2nd edn Oxford University Press,Oxford A useJitlupdatedguideto geneticcounseling in Ji,miliespith a chromosome disorderHagermanRJ, SilvermanA C (eds)1991FragileX syndrome. Diagnosis,treatmentand researchJohnsHopkins University Press, Baltimore A detailedaccountof the clinical and geneticas?ectsoJ'thefragile X syndrome JacobsP A, Browne C, GregsonN, JoyceC, White H 1992Estimates of the lrequency of chromosomeabnormalitiesdetectablein unselectednewbornsusingmoderatelevelsof banding J Med Genet 2 9 : 1 0 31 0 8 A rexiep ofthe resultsofmore than 14000 prenatal tliugnoses with estimates of the incidenceof chromosome abnormalitiesin term infants RatcliffeS 1999Long term outcomeof children of sexchromosome abnormalities. Arch Dis Child 80: 192-195 A aer-yusefuland cleardescnptnn uJthe cognitite und socialoutcomes oJ long-termfollow-up studiesoJ'setchrumosome aneuphtidies. SchinzelA 1994Human cvtogeneticsdatabaseOxford University Press. Oxford A regularl-yuldated clmputerized,rlatabaseof all hnownchromosome abnormalitiesThis r an inxaluableaid to diagnosisand counseling.

C H R O M O S OD I /IES O R D E R S

18

ELEMENTS Q Ch.o-osome abnormalities account for 50o/oof all spontaneousmiscarriagesand are present in 0.5-l 0oloof all newborn infants. @ Do*n syndrome is the most common autosomal chromosomal syndrome and shows a strong association between increasing incidence and advancing maternal age. Some 95o/oof all casesare causedby trisomy 21. Chromosome studies are necessary in all casesso that the rare but important casesdue to unbalanced familial robertsoniantranslocationscan be identified. (0 n" increasingnumber of chromosomemicrodeletion syndromes are being recognized.These havehelped in gene mapping and in enhancing understanding of underlying genetic mechanisms such as imprinting. Microdeletions of chromosome 15 are found in both Angelman and Prader-Willi syndromes, and are maternally and paternally derived, respectively. @ friptoiay is a common finding in spontaneouslyaborted products ofconception but rare in a live-born infant. Some children with diploidy/triploidy mosaicism present with learning difficulties and areasof depigmentation, a condition known as hypomelanosisof Ito. @ Sex chromosome abnormalities include Klinefelter syndrome (47,XXY), Turner syndrome (45,X), the XYY syndrome(47,XYY) and the triple X syndrome(47,XXX).

In all of theseconditions intelligence is either normal or only mildly impaired. Infertility is the rule in Klinefelter and Turner syndromes.Fertility is normal in the XYY and the triple X syndrome. @ fn. fragile X syndrome is the most common inherited causeoflearning difficulties. It is associatedwith a fragile site on the long arm of the X chromosomeand showsmodified Xlinked inheritance. Affected males have moderate-toseverelearning difficulties; carrier femalescan show mild learning difficulties. At the molecular level there is expansion of a CGG triplet repeat, which can exist as a premutation or a full mutation. @ Disorders of sexual differentiation include true and pseudohermaphroditism. True hermaphroditism is extremely rare.Male pseudohermaphroditismis causedmost commonly by androgeninsensitivity'an X-linked disorder involving the formation of androgen receptors'The most common cause of female pseudohermaphroditism is congenital adrenalhyperplasia,in which virilized infants can collapsewith adrenalfailure during the first week of life. rLte @ fn. chromosome breakage syndromes Lre increased by characterized disorders recessive autosomal chromosome breakage in cultured cells and an increased tendency to neoplasia,such as leukemia and lymphoma. They are causedby underlying defectsin DNA repatr.

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CHAPTER

Singte-gene disorders

To date, more than 10000 single-genetraits and disorders have been identified. Most of theseare individualll, rare, but together thev affect betrveen 1oloand 2o/c;of the general population at any one time. The management of these disorders in affected individuals and in their extended families presenrs the major workload challengein clinical genetics A wide varietl' of single-genedisorders has been mentioned throughout this book In this chapter some of the more common and important single-gene disorders are described, as well as a small number that hold particular interest for clinicians, with particular emphasis on their basic molecular defects. Each of these disorders illustrates important genetic principles and, for manr,;the identihcation of the mutational basis and isolation of the associatedprotein product representsomeof the most notable scientihc achievementsof the last decadeor two.

HUNTINGTON DISEASE

CLINICAL FEATURES In the common adult-onset form of HD, the drseasecourse is characterizedby a slowlv progressivemovement disorder and an insidious impairment of intellectual function with psychiatric disturbance and eventual dementia. The average age of onset is around 40vears and the mean duration of the illness is approximately 15years. Chorea is the most common movement abnormalitv.This takesthe form of subtle involuntary movements such as facial grimacing, twitching of the face and limbs, folding of the arms and crossing of the legs As the diseaseprogressesthe gait becomes very unsteady and speech unclear I n t e l l e c t u a l c h a n g e si n t h e e a r l y s t a g e so f H D i n c l u d e memory impairment and poor concentration span. Anxiety and panic attacks,mood changesand depression,aggressive behavior, paranoia, irrationality, increased libido and alcohol abusecan also occur. There is a gradual deterioration in intellectual function, leading eventuallv to total incapacitation and dementia.

Huntington disease(HD), also knor,vnas Huntington chorea, derives its eponymous title from Dr George Huntington, who describedmultiple affectedindividuals in a largeNorth American kindred in 1872.GeorgeHunrington's paper,which rvaspublished in the Philadelphia journal The Medical and Surgital Reforter, gave a graphic description of the progressir.eneurological disabilitv that has endowedHD with the unenviablerepurarionof being one of the most fearedand unpleasanthereditary disorders encountered in humans. The natural historl- is characterized b.v slowly progressiveselectivecell death in the central nervous system, for which there is no effective treatment or cure. The prevalencein most parts of the world is approximately 1 in 10000, although in some areas,such as Thsmaniaand the Lake Maracaibo region of Venezuela,much higher figures have been noted.

282

JuvenileHuntingtondisease In recent studies (since 1970) up to 5oloof HD caseshave presentedbefore the age of 20years; in some earlier studies the proportion was up to l0o/o. Instead of chorea there is rigidity:, with slowingof voluntarv movement and clumsinessA decline in school performanceheralds the onset of a severeprogressive dementia, often in associationwith epileptic seizures.The averageduration ofthe iliness is around 10-15r.ears.

GENETICS Traditionalll' HD has been said to show autosomal dominant inheritance with a variable age of onset, close to complete penetrance, and a very low mutation rate In addition, it has

Generallr,;HD is a disorder of middle to late adult life However,it can start at virtuallv anv age,including a rare jur.enile form that presents much younger and with different clinical features.This is one of severalenigmaticaspectsof HD that have

been noted that the disorder often shows anticipation, whereby t h e o n s e t i s a t a v o u n g e r a g e i n s u c c e e d i n gg e n e r a t i o n s , particularly when transmitted by a male. The discovery of the

beenexplained,at leastin part, bv the discoveryof the underlying molecular defect

HD gene in 1993 provided an explanation for some of these observations.

SORDERS S I N G L E - G EDN] E

M a p p i n ga n di s o l a t i o no f t h e H u n t i n g t o d ni s e a s e 9ene HD rvasonc of the Iirst disordersto be mappedb-vlinkageanal-vsis using polvmorphic DNA markers lr,hen, in 1983, the disorder rvasfound to show close linkage rvith a probe knorvn as G8 on the short arm of chromosome4. This initial scarchfor the HD locus u'ashelped enormouslv b-vthe collection of blood samplcsfrom a huge pedigreecontaining over 100affectedsubjectsliving on the shoresof Lake Nlaracaiboin\-enezuela As rvcll as providing the first mcansof predictivetestingfor HD, this lvork alsorer,caledthat HD homozi,-gotcsare no more severel-vaffectedthan heterozvgotes This is in contrast to man-yother autosomaldominant disorders (p. 103) When thc gcne itsclf rvasisolatedin 1993 it was found to contain a highl.v poll'morphic CAG (polyglutamine) repeat sequencelocatedin the 5' region. The messengerRNA (nRNA) 350kDa, knorvnashuntingtin codesfor a protein of approximatel-'-(also IT15) Huntingtin is expressedin manr different cells throughout the ccntral ncrvous svstem, as rvell as other tissues, although its function remains unclear One proposed rolc, as -vet unproven, is that it is involved in apoptosis(cell death).

19

T a b l e1 9 . 1 C o m p a r i s oonf g e n e t racs p e c tos f H u n t i n g t o n d i s e a sa e n dm y o t o n idcy s t r o P h Y Huntingtondisease

Myotonicdystrophy

nherrtance

domlnant Autosomal

dominant Autosomal

Chromosome tocus

4p163

19q13 3

Trinucleotide repeat

CAGn 5'transtated regton

CTGin3'untranstated reglon

Repeat sizes

Normal<26 Mutable27-35 Reduced Penetrance 36-39 >40 Fullypenetrant

Normal<37

Huntingtin

kinase MDprotein

product Protein (DMPK)

Earty-onsetform Juvenite paternatV Usuatly transmitted

Fut[mutatton 50-2000+

Congenltal Usuallymaternatly transmltted

nisease T h e m u t a t i o ni n H u n t i n g t o d Almost all individuals rvith HD possessan cxpansionof a CAG p o l l ' g l u t a m i n e r e p e a t s c q u c n c cl o c a t e d i n t h e 5 ' r c g i o n o f the HD gcnc HD is thus an example of a disorder causedbv expansionof a triplet repeat (p. 23), a mutational mechanism that u'aslirst identihed in humans in contrast to almost all othcr tvpes of mutation that ucrc first reported in othcr speciessuch 'asDrusophildand mice. A joint rvorking partv of thc American Collegcof 14edicalGeneticsand thcAmerican Societl'of Human Geneticsrecommcndedthat HD genesshould be categorizedundcr four hcadingson the basisof CAG repeat length (Thble l9 1).

Normololleles Alleles containing 26 or ferver CAG rcpeats are not associated uith diseasemanif'estationsand are stablein mciosis

diseaseor complete absenceof diseaseexpresslon'l.e. nonpcnetrlnce.

Diseose olleles Thc final group of HD gcnes contains '10 or more CAG repcats These are invariabl-rassociatedwith disease'although sometimesthis ma1'not develop until the seventh or eighth dccade.There is a direct relationshipbetrveenlength of repeatand diseaseexpression,rvith the averageage of onset for repeat sizes Most of ,10,,15and 50 being 57,37 and 26 years,respectively. affccted adults have repeat sizesof betrvecn36 and 50, rvhereas jur,enilecasesoften have an expansiongreaterthan 55 repeats.

Parentof origineffectin diseasetransmission Mutobleolleles Allele sizesof 27 to 35 CAG repeatsdo not causcdiseasebut mav shorvmeiotic instabilitl'rvith a potential to increascor decrease 'mutable' allelesthus constitute a reservoirfor nerv in sizc These mutations When an af-fcctcdindividual prescnts with rvhat appearsto be a ner'r,mutation,it usuall.vemergesthat the fbther carriesa mutableallele.Furthcrmore, thereis evidencethat mutable allelesthat expand are associatedwith a particular haplotvpe, as identilicd b-vintragenic and flanking DNA markers.This implies that certain haplot.vpesare more mutable than others.

olleles Reduced Denetronce This third cateeiorvconsists of alleles containing 36 to 39 CAG repeats These are associatedrvith either late-onset

Autosomal dominant inheritance is well established,rvith an offspring risk of 1 in 2 regardlessof uhether the affectedparent is malc or female Horvever,for reasonsthat are not understood' mciotic instabilit-vappearsto be much greaterin spermatogenesis than in oogenesis This is reflected in anticipation' occurring mainl.v*'hen the mutant alleleis transmitted b.va male.Juveniles with the rigid form of HD have almost ahvays inherited the mutant allele from their more mildly affectedfather. Ser,eralpossibleexplanationshave been suggestedfor this pref'ercntialtransmission of expanded alleles b1' the male One possibilitv is that expansion causedby slippage(p 24) of DNA poll.merase simpll, reflects the numbcr of mitoses undergone during gametogenesis.In Chapter 3 it was pointed out that spcrmatogenesisinvolvcs a much larger number of mitotic ciivisionsthan oogenesis(p. 43).An alternativepossibilitf is based

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S I N G L E _ G EDNIE SORDERS

on the observationthat huntingtin is expressedin oocytes,so that there could be selectionagainstoocyteswith large cxpansronsas a consequenceof preferential apoptosis

CLINICAL APPLICATIONS ANDFUTURE PROSPECTS The discoveryof the HD genehasmeant that accuratepredictive testing is possible,although there is universalagreementthat this should be offered only as part of a well planned and carefully monitored counselingpackage.Experienceto date indicatesthat more women than men take up the offer of predictive testing, with the degree of psychologicaldisturbance in those given positive results being less than was expected. Some 600loof candidates test negative(i.e. they receivegood news),and the reasonsfor this departure from the expected50o/oarenot clear. Prenatal diagnosisis also possiblefor those couples who lind this acceptable,although onlv about 25 such tests are performed in the UK annually.Obviously there are considerableemotional and ethicalissuesassociated with termination ofpregnancy,on the grounds that a child could go on to develop a neurodegenerative diseasein middle age.This will be particularlv pertinenr when effectivetherapeuticstrategieshavebeen devised.One appealing approach is based on the observation that large CAG repeats result in intracellular accumulation of huntingtin ,aggregates', which are cleavedby a proteaseknown as caspaseto form a toxic product that causescell death (apoptosis). Caspaseinhibitors have been shown to have a benelicial effect in a HD mouse model. Another therapeutic approach undergoing trial is fetal neuronal cell transfer into regions ofthe brain, such asthe caudatenucleus and putamen, which become atrophic in the earlv stagesof the disease.This approach carries ethical considerationsthat will be difficult for some couples.

to tonic muscle spasm with prolonged relaxation, which can manifest as a delay in releasing the grip on shaking hands Other clinical abnormalitiescan include cataracts(Fig. 19.1), cardiacconduction defects,disturbed gastrointestinalperistalsis (dysphagia,constipation,diarrhea),weaksphincters,increasedrisk of diabetesmellitus and gallstones,somnolence,frontal balding and testicular atrophr,'. The ageof onset is very variableand in its mildest form usually runs a relatively benign course. However, as the age of onset becomes earlier, so the clinical symptoms increasein severity and more body systemsare involved. In the 'congenital' form, affectedbabiespresentat birth with hypotonia, talipes and respiratory distress that can prove life threatening (seeFig 7 18). Children who survive tend to show a lack offacial expression('mvopathic facies')with delayedmotor development and learning difficulties (Fig. 19.2). The diagnosis of MD used to be based on rhe finding o f m y o t o n i c d i s c h a r g e s s e e n o n e l e c t r o m y o g r a p h y .T h i s unpleasantinvestigation has now been supersededby mutation analysis.

GENETICS It has long been recognizedthat MD showsautosomaldominant inheritance, with increasing severity in succeedinggenerarions. At one time it was thought that this phenomenon of anticipation (p. lla) was a reflection of ascertainment bias, caused by the greater likelihood of detecting a mildly affected