Emery's Elements of Medical Genetics, 13th Edition

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Emery's Elements of Medical Genetics, 13th Edition

13t* EDITION EMERY'S E FRcpcH FRcp, MB, chB, Peter D. Turnpenny, BSc, Consuttaht Ctinicat Geneticist RoyalDevonandExet

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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



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


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


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



togetherto grow librariesin developingcountries www.elsevier.com I www.bookaid.org I www.sabre.org

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Cettdivision40 43 Gametogenesis 45 Chromosomeabnormatities

Dedication ii Preface ix Acknowtedgementsx


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


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


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


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



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



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.


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.



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



Purebred Short

x Tt




Secondfilial cross

Hybrid Tall





f2' TT





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.


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


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





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.


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



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


',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.


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




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




FrancisCrick JamesWatson Maurice Wrtkins

Themotecuiar srrucrure of DNA


FrangoisJacob Jacques Monod And16Lwoff

Genetic regulatron



Oncogenic viruses


RobertHotley Gobind Khorana Marshatt Nireberg

Deciphering ofthegenetic code

DavidBattimore Renato Dutbecco HowardTemrn

Interaction between tumor vtruses anonuctear DNA


WernerArber Daniet Nathans Hamrlton Smith

Restrictron endonucteases


BarulBenacerraf JeanDausset George Sne[[

Genetic controtof immunologrc responses


Barbara McClrntock

genes Mobite (transposons)


MichaelBrown JosephGoldstein

Cel[receptors in famrtiat hypercholesterotemia



Genetic aspects oI antibodies


Michaet Bishop Harotd Varmus




Rrchard Roberts Phittip Sharp


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


EdwardLewis Homeotrc andother ChristianeNusstein-Volhard devetopmentatgenes EricWreschaus


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.


Stantey Prusrner






ArvidCarlsson Pau[Greengard EricKandel

Signattransductron Inthe nervoussystem


LelandHartwett Timothy Hunt PauINurse

Regulators ofthece[[cycte


SydneyBrenner RobertHorritz JohnSutston

Genetrc regulation rn development and programmed cettdeath (apoptosrs)

AndrewFrre CraigMetto




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.



Thece[[ular andmolecutar basis of inheritance


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


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


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




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


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


D N Ad o u b l e helix


Chromatin fi ber

Extended section of chromosome

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


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


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^laPdr 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



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


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


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


CAAT box

80bp+: TATA

, TranscriPtion

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

Fig.2.10 Diagrammatic representation geneexpression ofihefactors thatregulate






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


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


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


5ubstrtution Synonymous Non-synonymous



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


Effecton protein product

Sitento Missenseo Nonsenseo Snlirc f

p Gty542X

to Stop Gtycine


c 621+ 1G>T

(1bp) Detetion


p Vat358TyrfsXll

mutation Frameshift

(3bp) Detetion


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


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




Tabte2.5 ExampLes of diseases arisingfromtripletrepeatexpansions Disease

Repeat sequence

Normal range (repeats)

Pathogenic range (repeats)

Repeat location

(HD) Huntington disease





Myotonic dystrophy type1(DM1)


tr ?f








(SBMA) Kennedy disease





Spinocerebeltar 1(5CAl) ata."ia





Spinocerebettar ataxia2 (SCA2)





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





Spinocerebertar ataxra 6 (SCA6)





Spinocerebettar ataxia7 (SCA7)





Sprnocerebeltar ataxra B (SCA8)





Spinocerebe[ar ataxia 12(SCA12)





Spinocerebettar ataxia17(SC417)





(DRPLA) Dentatorubrat-pattidoluysian atrophy





Friedrerch ataxia(FA)









OcutopharyngeaI muscutar dystrophy





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


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.


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.


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


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.




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




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.



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.



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



y radiationo External



Sourceof radiation

Internalyradiation 030


Artificiat radiology Medrcat

0 30


fallout Radioactive

0 01


and Occupationat miscellaneous

0 04





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




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.


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


Typeof DNArepair




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


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


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


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.



Chromosomes andce[[division


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




Examplesof application


Reliabte methods forchromosome preparations

Chromosome numberdetermined as46(1956)


Giemsa chromosome banding

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


(FISH) Ftuorescent rn-sduhybridization

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


(CGH) Comparative genomic hybridization

genomic Mapping imbalances in sotidtumors(1992)



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


(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


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



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




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


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


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 --





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


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.



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



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

A n al y z e


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,



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


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


xtl An




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.








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


f* 19




Fig.3.5 A normaI G-banded malekaryotype







,r s3 19
















,;x ** 2122Y

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


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


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






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,




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



(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



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


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





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




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











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.


Duptrcatron: e g 46XYdup(13Xq1+)




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






Ring,eg 46:XX,r(21)


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


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


cnromosomes cell average slide average

Ratio Profile




L.1Jif I


5 59;




I 1



n=l 5













n=] 4












E 't5


n = 1I

n tr

r--l E

N F -


n=l 7


€ EStt

E L-r

t! 2n








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,


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





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

? os E^ N o





-2 D i s t a n c ei n m i l l i o n so f b a s ep a i r s

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],





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




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






1 1

3 Metaphase


2 ,5




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


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).



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.


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).




Late pachytene

Telophase I

Anaphase ll

Telophase ll

Fis.3.19 Stages of meiosis



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


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


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:


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'.



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




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




Eartyembryonic tife




Numbersof mitoses in gameieformation



Gameteproduction peTmerosrS

4 spermatids

1ovum* 3 potarbodies


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




ff** Fetal life $

Primary spermarocyre

Primary oocyle

$Birth *M

Puberty ffi


Dictyotene E.

ul a t i o n


Secondary oocyte

P o l a rb o d i e s

60-65 days

zysot.f;D Spermatozoa


Fis.3.20 44

Stages ofoogenesis andspermatogenesis n.haploid number



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


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


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



13 Trisomy



18 Trisomy



21 Trrsomy




















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


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


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



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






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




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


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 =





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



222 Atternate




Batanced translocation

(non-homotogous Adjacent-1 centTomeres segregate together)


(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


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


Unbatanced, leading to tnsomyinthezygote



Unbatanced, leading to monosomy inthezygote

c 48



'-+--j =+rr------C€ B llll IJII VV

pachytene qu a dr i v aIen t


yieldsnormal Alternate segregation or balanced haploid complement


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):


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.




H ll

Normal chromosomes






B al an c e d 14121carrier

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



Normal carrler



14121 2 1 Down's syndrome







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


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 )



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




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 complementaryrecombinantchromosc

-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.




A Drosophila


3' Abd-B Abd-A Ubx

Human bein .q

HOXA chr'7p14





HOXB ch.17q21

HOXC chr'12q13

HOXD chr'7q31





























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



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



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



Renal-cotoboma syndrome



Waardenburg syndrome type1






Absentor ectopic thyroidgtand




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




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.



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



GreigsyndromeandPattister-Hall syndrome



Denys-Drash syndrome







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.



l gl l l


]TM :

I a-::::t:





t t t t TD1 P









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


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'



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



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


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



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


in the extracellulardomains of FGFR2.






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


(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



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)


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.



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.




1 28


636 651 726

r 6 0 9 - 6 3 4r







999 1114


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


Developmental anomaly











1l /19^4) +/ ,

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




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


Partial mole


No of chromosomes



Parental originof chTomosomes

23- maternal 46* paternat

At[46 paternal


Yes- butnotviabte


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

V e r yl o w


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:


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.


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.





a + ,/ Testosterone




il"\Q*,^ \


ilt rrellm




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.




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.


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,


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


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.



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


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











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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


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


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



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



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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).



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.


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.




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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.


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


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




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

Fis.7.6 r,y , ^p ^e +--;l -l ^--,,, ! u u -r^ ,'^r.g^ d - L d - - ' rdr r p d r I | dlir;l ^p^o1 ,o, ,y,5- ^l ^- ^o. p ^oyu^e. l^o a- -m L L a L t onn r - e . a - i n , A / Cg e n eT " e p a r i e ^tta c " sa d ; o o s e t s s r e e s p e ca t t yi ^ t ^ e d i s t a tI m b s A w o e v a r i e r yo ' c t r r - i c p ah I e r o t y p e iss a s s o c r a t e c w i t h m u t a t i o n isn t h i so n e q e n e n


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.



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



IV |


nnecteo [J{

matrng consanguineous

Fig.7.7 trait recessive Fami[y treeofanautosomaI



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.



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




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.


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


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


The mode of inheritancewhereby only malesare affectedby a diseasethat is transmitted by normal femaleswas appreciatedby theJews nearly 2000yearsago.They excusedfrom circumcision


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.


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


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





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


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



r9ak,. ) r n t s\





X chromosomes







| | nr..t.o dominanttrait Familytree of an X-Lrnked





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



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.


Y-tinkedinheritance A

ABI^B Normal 6 Xchromosome " N rnactivated


Derivative Xchromosome inactivated

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

C e l ld e a t hd u et o inactivatioo nf a u t o s o m es e g m e n t

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




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).


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


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


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.


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


phenotypes genotypes, andgametes Tabte7.1 Possibte A',Ar,B and0 at theAB0 tocus formedfromthefouratleles Genotype

































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).




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.


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.


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



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


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.) ,


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.





ffi /






'riffi) uu..' y'\

/\ ./-t,

H r e i o sy i' \, "



lHiifli 't''!rj\..!.2' F e r tIi z a t o in



Lossof c hr o m o s o m e

Uniparental isodisomy




+ $;;;'.,... 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.



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)




n,),., A )


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




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



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


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.



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


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






$, ^ - ^ - ,- l1 LrLr

other CDKNlC




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




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



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


S e v e r ed i s e a s e


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)


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).


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.



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


AA Aa Aa


Frequency p2 2pq qt

6. The effectsof medical intervention.



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


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



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


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







AA x Aa




Aa \ Aa




AA x aa



Aa x aa





tpq' n4



p2(p2 + 2pq+ q2)

+ 2pq+ q2) 2pq(p2

q2(p2 + 2pq+ q2)

Relative frequency







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.


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,


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


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


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


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



430 s40 30

490(p2x lo0o) 420(2pqx 1000) 90(q'zx 1000)




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:


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 )



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


(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)







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




1 lze










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.


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



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



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


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


ScLerosteosis Lipoidproteinosis

boneswithcraniaInervepatsies' of craniofactal overgrowth Tallstature, ctyiy synda of skrnandmucousmembranes Thickening

'Ryukyan spinalmuscular atrophy

ctubfoot,scotiosis weakness, Muscte


(offJapan) istands Ryukyan

hypoptasia Cartilage-hair Crevetd syndrome Ellis-van




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



Resistanceor advantage

- c e [ [d i s e a s e Srckle



FaLciparum malaria



South-EastAsiaandtheN4editerranean Fatciparummalaria




Falciparum mataria

Cystic fibrosis



Tubercu[osis? Tho nlrnr ro?





EasternEuropean Jews

Tubercu [osrs?

Congenital adrenal hyperptasia


Yupik Eskrmos

Influenza B

Iype2 diabetes


PimaIndrans andothers

Periodrc starvation




Spontaneous abortion ratelower?

AR:autosomaI recessive: XLR:X-tinked recessive; AD:autosomaI gtucose dominant; G6PD. 6-phosphate dehydrogenase


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/^ -s^o^) c



Hvoertenslon fessentrat)



ofthehip Congenital drslocation



andspinabifida Anencephaty






Cnnoenith ae l artdisease





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


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








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



(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 )


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


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




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








i j I





) trBltli




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









= 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


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


Tabte9.4 Pooulations studied intheInternationa-



l-.1:nMan Prnioet ""H""ts




lbadan. Nigeria


30 trios(adutt andboth parents)



45unretated rndivrduats

Beijing. China


45unrelated individuats


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


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.




'Blood is very a specialjuice.' Johann Wolfgang von Goethe, in


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.




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


Stagein development



GowerI Gowerll PortlandI


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.







A substitutron, m 8356T>C substitution)

Myoctonus, seizures, optrcatrophy, hearing impairment, dementra, myopathy

Erythropoietic porphyrias Congenitalerythropoietic porphyna Erythropoietic protoporphyria

Disordersinvolvingmitochondria MERFF Mt


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.



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



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


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.


'If it rverenot for the great variability among individuals medicine might as well be a scienceand not an art ' Sir Williarn Osler (1892)


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


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).


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




G 5


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


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).



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.


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.




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.



(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



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


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.


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,


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




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



UV tight



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_\.



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.





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


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


'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


D ':'r

. {C l q r s C.. _.-.i, C4


- a',tt"'

C3 + C3a+ C3b-.::ir:-r:1"M a s ct e l d l egranulation Chemotaxis 0psonization


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.


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.


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




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




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


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



Mot.wt (Da)

Serumconcentration Antibodyactivity (ms/mt)

,l n v vl l

1(n nnn

Q 1a

Brndsto m croorganisms andneutratizes bacteriaItoxins +




Produced rnearlyimmuneresponse, especiatty in bacteremia





Guards mucosaI surfaces





Ontymphocyte cellsurface, invotved in control of dclivaLion ard slppress,on





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


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



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 ) '


IT Somatic r e c o m bni a t io n



I Variable DiversityJunctionalConstant regron regron regron regron :, K Chromosome 2.,-






Chromosome 22


Class ing switch



I V 6





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>




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


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


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


Ankytosing spondytitis


C e L i adci s e a s e


defrciency 21-Hydroxyfase




Tabte13.2 Alletesatthe HLALocr



HLA locus

No.of alleles

gravrs Myasthenia








arthritis Rheumatoid




lupuserythematosus Systemic




(Graves disease) Thyrotoxrcosis


testiculirr differentiation and function, its expressiondoes not ncccssarih correlirte rvith thc prcsencc or absenceof testicular





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




Lymphocytes Cett mediated

Ptasma lg celts synthesis

Genetics Treatment

Severe combrned immunodeficiency







DiGeorge/Sed 15d kov6 syndrome

Absent(parathyroids +




Detetion Transplantationof fetaI

Bruton-type agammagtobu [inemia




BMT,enzyme replacement forADAdeficency








AR,autosomal recessive;XR,X-iinked recessive: BMT,bonemarrowtransplant;ADA. adenosine deaminase; N,normal; J, reduced: +. Dresent


0 p s o n i z a t i oonf b a c t e r i a -* fixed to cell surface


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




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


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


EcoRllMspl d o u b l ed i g e s t C

Non-ca rri er


EcoRUHpall double digest NC





--. b+


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




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


(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


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 > drl ,ya sn g or r rl q


accne - -f,1_^_!^_^


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


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



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


humans.These include the locationof oncosenesat chromosomal



,, gag po env i I--f-T__l



.. I gag pol envsrc i il--f-T--f-ll

m os ."gag ,' I-TIT-L r \,/ Oete'tiO


::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


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


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


of both types, of which chronic myeloid leukemia is an erample of the former and Burkitt lymphoma an erample of the latter. 13






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


*!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.




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


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


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


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






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


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''.


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.




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



o oo oo oooo ooo o ooo o /\












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



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


'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










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,


Tabte14.2 Syndromes andcancersthatshowlossof heterozygosity and their chromosomalLocation Syndromeor cancer Retinob[astoma

Chromosomallocation '13q14




11p'13. 11p15, 16q



vonHippet-Lindau disease


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


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


carcrnoma Colorectal

1p,5q2'1. Bp,'17p13, 18q21

Neurofibromatosis | (NF1. von Reck[inghausen disease)


Neurofibromatosis Il (NF2)




Muttrpte endocrine neoptasra type| (MENl)








Prostate cancer

1p36.1q.8p.10q. 13q,16q

Btaddercarcinoma Lungcarcinoma Breastcarcinoma


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.


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.




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[ dPPtrold|Lc llll9llL dl>U Ju,! v,,J, (CBAVD). It is now recognizedthat a small subsetof males have s r c h a s E h L e r s - D a ^ t so ys n d - o m e a verv mild form of CF in which CBAVD is the only significant the others missensemutations were found in the related gene,

clinical problem. Other rare presentationsof CF include chronic pancreatitis, diffuse bronchiectasisand bronchopulmonary


allergic aspergillosis.

CongenitaIcontracturaI arachnodactyly


Also knovvn as Beal syndrome, this is probably the condition originallydescribedby Marfan in 1896 Many'featuresoverlapwith

CF shows autosomal recessiveinheritance. Other autosomal recessivedisorders,such ashemochromatosis,which causestissue




iron overload,show a higher incidence,but CF is by far the most seriousautosomalrecessivedisorder encounteredin children of western European origin Possibleexplanationsthat have been proposedfor this high incidenceinclude multiple CF loci, a high mutation rate, meiotic drive and heterozygote advantage.This latter explanation,possibly mediated by increasedheterozygote resistanceto chloride-secretingbacterially induced diarrhea, is thought to be the most likell-, although absolute proof is

Chromosome 7





Mappingand isolationof the cysticfibrosisgene The CF locus was mapped to chromosome7q31 in 1985 by the demonstration of linkage to the gene for a polymorphic enzyme known as paraoxanase.Shortly afterwards two polymorphic DNA marker loci, known as MET and D7S8, were shown to be closely linked flanking markers.The region between these markers was



250kb CFIR


scrutinized for the presenceof HTF or CpG islands,which are known to be present close to the 5' end of many genes(p. 74). This led to the identification of several new DNA markers that were shown to be verv tightly linked to the CF locus with recombination frequenciesof lessthan lolo. These loci were found to be in linkage disequilibrium (p. 132) with the CF locus, and the CF mutation was found to be associatedwith one particular haplotypein 84o/oofcases.This discoveryoflinkage disequilibrium was consistent with the concept of a single original mutation being responsible for a large proportion of all CF genes.The identification of loci tightly linked to the CF locus narrowed its location down to a region of approximately 500kb. Genes expressedin tissuesinvolvedin CS such aslung and pancreas,and conserved between specieswere identified. The CF gene was eventually cloned by two groups of scientistsin North America in 1989 by a combination of chromosome jumping, physical mapping, isolation of exon sequencesand mutation analysis. It was named the CF transmembrane conductance regulator (CFTR) gene and was shown to span a genomic region of approximatelv250kb and to contain 27 exons.

The cysticfibrosistransmembraneconductance regulatorprotein The structure of CFTR is consistent with a protein product containing 1480amino acidswith a molecular weight of 168kDa. It is believed to consist of two rransmembrane(TM) domains that anchor it to the cell membrane, two nucleotide binding folds (NBFs) that bind ATP, and a regulatory (R) domain, which is phosphorylatedby protein kinase-A (Fig. 19.8). The primary role of the C,EIR protein is to act as a chloride channel Activation by phosphorylationof the regulatorydomain, followed by binding of ATP to the NBF domains, opens the outwardly rectifying chloride channeland exertsa negativeeffect on intracellular sodium absorption by closure of the epithelial


sodium channel. The net effect is to reduce the Ievel of intracellular sodium chloride, which improves the quality of cellular mucous secretions

O u t w a r d l yr e c t i f y i n g c h l o r i d ce h a n n e l

Extracellular fluid

Fi9.19.8 geneandprotein Thecystic product, fibrosis locus. whichinftuences g ch[oride close[y adjacent epithetial sodium andoutwa rdLyrectifyin c h a n n e lR s ,r e g u t a t odr yo m a i nN; B En u c t e o t i b d ien d i nfqo l d ; T M ,t r a n s m e m b r adnoem a r n

Mutationsin the cysticfibrosistransmembrane conductance regulatorgene The first mutation to be identified in CFTR was a deletion of three adjacentbasepairs at the 508th codon which results in the lossof a phenylalanineresidue.This mutation is known asAF508 (A for deletion and F for phenl,lalanine)and it has been shown to account for approximately70o/oof all mutations in CFZR, with the highest incidence of 88o/obeing in Denmark (Table 19.3). The AF508 mutation can be demonstrated relatively simply by polymerasechain reaction (PCR) (p. 58) using primers that flank the 508th codon (Fig. 19.9). More than 1500 other mutations in the CIIR gene have been identifi ed. These include missense,frameshift, splice-site,nonsense and deletion mutations. N4ostof theseare extremely uncommon, although a few can account for a small but significant proportion of mutations in a particular population. For example,the G542X and G55lD mutations account for 12o/oand 3o/oof all CF mutations in the AshkenaziJewish and North American caucasian populations, respectively.Commercial multiplex PCR-based kits have been developed that detect approximately 90o/oof all



Tabte19.3 Contribution ofAF50Bmutation toaltCF mutations Country Denmark





















(EWGCFG) DatafromEuropean Working Groupon CFGenetrcs gradient ofdistribution rnEurope ofthemajorCFmutation andof its associated haptotype HumGenet1990:85:436-441.and worldwide - reportfromtheCystic surveyoftheAF5O8mutation Fibrosrs Genetic Analysis Consortium AmJ HumGenet1990; 47 354-359

l-letqroduplex 0 an 0 s

.-98bp L u L c r L y






Expressedas odds







RISKS EMPIRIC Up to this point risks have been calculated for single-gene disorders using knowledge of basic mendelian genetics and applied probability theory. In many counseling situations it is not possibleto arrive at an accuraterisk figure in this way,either becausethe disorder in question does not show single-gene inheritance or becausethe clinical diagnosiswith which the family has been referred shows causal heterogeneity (p. 338). In these situations it is usually necessaryto resort to the use of observedor empiricrfuPs.These are basedon observationsderived from family and population studies rather than theoretical calculations.

DISORDERS MULTIFACTORIAL One of the basic principles of multifactorial inheritance is that the risk of recurrence in first-degree relatives,siblings and offspring, equals the square root of the incidence of the diseasein the general population (p. 136), i.e. P/2, where P equals the general population incidence. For example, if the r/100s, then the theoretical general population incidence equals r/1666' risk to a first-degree relative equals the square root of which approximatesto 1 in 32 or 3o/o.The theoretical risks for second- and third-degree relatives can be shown to approximate to P3/+and Ptls, ,.rp."tiuely. Therefore, if there is stronEisupport for multifactorial inheritance, it is reasonable to use these theoretical risks when counseling close family relatives. However, when using this approach it is important to remembcr that the confirmation of multifactorial inheritance will often havebeen basedon the study of observedrecurrencerisks. Consequently it is generally more appropriate to refer back to the original family studies and counsel on the basis of the risks derived in these(Table 22.7) Ideally,referenceshould be made to local studiesasrecurrence risks can differ quite substantially in different communities, ethnic groups and geographicallocations. For example, the recurrence risk for neural tube defects in siblings is quoted as '1olo.This, essentially,is an averagerisk. The actual risk varies from 2-3o/o in south-east England up to 8o/oin Northern Ireland, and also shows an inverserelationship with the family's socioeconomicstatus, being greatest for mothers in poorest clrcumstances. Unfortunately, empiric risks are rarely availablefor families in which there are several affected family members, or for disorders with variable severity or different sex incidences.For example, in a family where several members have been affected by cleft lip,/palate, the empiric risks based on population data may not apply - the condition may appear to be segregatingas an autosomaldominant trait with a high penetrance.In the absence of a s1'ndromediagnosisbeing made and genetic testing being possible,the clinical geneticist has to make the best judgement about recurrencerisk.





lncidence (per 1000)

Sex ratio (M:F)

Unaffectedparentshavinga second affected child (%)

Affected parents having an effected child (%)

Cteft[iP+ s[sftp3b1s










CongenitaI heartdefect




a It-+A^-


6 [mother affected)

Congenital dislocation ofthehip


(inmates) Hypospadias




NeuraItubedefect Anencephaty Spinabifida

15 25

Pytoricstenosis Mateindex Femateindex

25 05











1:2 2:3




2 10

4 17




Incidence (per1000)

Sex ratio (M:F)

Unaffectedparentshaving a secondaffectedchitd(%)





(idiopathic) Epitepsy


'l :1






(idiopathic) MentaIretardation





Profound childhood sensorineural deafness





CONDITIONS SHOWING CAUSAL HETEROGENEITY Many referrals to genetic clinics relate to a clinical phenotype rather than to a preciseunderlying diagnosis(Thble22.8).In these situations great care must be taken to ensure that all appropriate



diagnosticinvestigationshave been undertaken before resorting to the use of empiric risk data (p. 337). It is worth emphasizing that the use of empiric risks for conditions such as sensorineuralhearing loss in childhood is at best a compromise, as the figure quoted to an individual family

Affected parents having an affected chitd (%)


will rarely be the correct one for their particular diagnosis. Severe sensorineural hearing loss in a young child is usually caused either by single-gene inheritance, most commonly autosomal recessive but occasionally autosomal dominant or sex-linked recessive,or by an environmental condition such as rubella embryopathy.Therefore, for most families the correct risk of recurrence will be either 25o/o or 0o/o. In practice it is often not possibleto establishthe precisecause,so that the only option availableis to offer the family an empiric or 'average' risk.

BayesT 1958An essaytowards solving a problem in the doctrine of chances. Biometrika 45: 296-315 A reproductionof t he ReaerendB ayi original esay 0n lrobahiliu thelrJl that wasfrst published,posthumousfu, in 1763. Emery A E H 1986Methodology in medical genetics,2nd edn. Churchill Livingstone, Edinburgh An introductionto statisticalmethod;of analysisin humanand medical genettcs. Murphy E A, ChaseG A 1975Principles of genetic counseling.YearBook Medical Publications,Chicago A aery thorough explanation of the useof Bayes' theoremin genetic counseling. Young I D 1999Introduction to risk calculation in genetic counselling, 2nd edn. Oxford University Press,Oxford A short introductory guide to all aspectsof rish cahulation in geneticcounseling Highly recommended.

O nirt calculationin geneticcounselingrequires a knowledgeand understandingofbasic probabilitytheory. 'prior' risks to Bayes'theoremenablesinitial background be modifiedby'conditional'information to givean overall probability or risk for a particular event such as carrier status. dominantinheritance @ For disordersshowingautosomal it is often necessaryto considerfactors such as reduced penetrance anddelayedageofonset.For disordersshowing autosomalrecessiveinheritance,risks to offspring are determinedby calculatingthe probabilitythat eachparent is a carrier and then multiplying the product of these probabilitiesby r/a. @ In sex-linkedrecessiveinheritancea particular problemariseswhenonly onemalein a family is affected. The results of carrier tests that show overlap between carriersandnon-carrierscanbe incorporatedin a bayesian calculation. @ Poly-orphic DNA markerslinked to the disease disordersfor carrier locuscanbe usedin manysingle-gene detection,preclinicaldiagnosisand prenataldiagnosis. for multifactorial risksareavailable @ Empiric (observed) heterogeneous conditions disordersand for etiologically hearing loss. suchasnon-syndromalsensorineural



'So little

Treatment ofgenetic disease

done So much to do.' Alexander Graham Bell

Many geneticdisordersare characterizedby progressivedisability or chronic ill-health for which there is, at present, no effective treatment. Consequentlvone of the most exciting aspectsof the developmentsin biotechnologyis the prospectof new treatments mediated through gene transfer, RNA modification or stem cell therapl'. It is important, horlever, to keep a perspectiveon the limitations of these approachesfor the immediate future and to consider, in the first instancc, conventional approachesto the treatment of geneticdisease.

CONVENTIONAL APPROACH ESTO TREATMENT OFGENETIC DISEASE Most genetic disorders cannot be cured or even ameliorated using conventionalmethods of treatment. Sometimes this is becausethe underlying gene and gene product have not been identified so that there is little, if any,understandingof the basic metabolicor moleculardefect.If. however.this is understoodthen dietary restriction, as in phenylketonuria (p. 158), or hormone replacement, as in congenital adrenal hyperplasia (p. 165), can be used very successfullyin the treatment of the disorder. In a few disorders,such as homocystinuria (p. 163) and some of the organic acidurias (p. 175), supplementation with a r-itamin or co-enzymccan increasethe activitv of the defectiveenzvme with benehcialeffect (Table 23.l).

PROTEI N/ENZYM E REPLACEM ENT If a genetic disorder is found to be the result of a deficiencv of or an abnormality in a specific enzJrmeor protein, treatment could, in theory, involve replacementofthe deficient or defective enzyme or protcin. An obviously successfulexampleof this is the use of factor VIII concentratein the treatment of hemoohilia A



For most of the inborn errors of metabolism in which an enzyme deficiencr, has been identified, recombinant DNA techniquesmay be used to biosynthesizethe missing or defectir.e gene product; hor,vever, injection of the enzyme or protein mav

not be successfulif the metabolic processesinvolved are carried out within cells and the protein or enzyme is not normally transported into the cell. Artilicial delivery svstems, such as liposomes,allow proteins to crossthe cell membrane.Liposomes are artilicially preparedcell-like structuresin which one or more bimolecular layersof phospholipid encloseone or more aqueous compartments,which can include specificproteins.Although, in theory it was thought that liposomeswould work, they havemet rvith limited successin the treatment of genetic disorders such as the mucopoll'saccharidosesIn some instances,however, biochemicalmodification of the protein or enzyme allowsutilization of normal cellular transport mechanismsto target the enzyme to its normal location within the cell. For example,modilications in asusedin the treatmentof Gaucherdiseaseenableit B-glucosidase to enter the lysosomes,resulting in an effectiveform of treatment (p. 170). Another example is the modification of adenosine deaminase(ADA) by an inert polymer,pol-vethylene glycol(PEG), to generxtea replacementenzyme that is lessimmunogenic and has an extendedhalf-life.

DRUG TREATMENT In some genetic disorders drug therapy is possible; for example, statins can help to lower cholesterol levels in familial h y p e r c h o l e s t e r o l e m i a( p . 1 6 7 ) . S t a t i n s f u n c t i o n i n d i r e c t l y through the lorv-densitvlipoprotein (LDL) receptorby inhibiting endogenouscholesterolbiosvnthesisat the rate-limiting step that is mediated by hy-droxymethyl glutary'l co-enzyme A (HMGCoA) reductase.This leadsto upregulation of the LDL receptor and incrcasedLDL clearancefrom plasma In others, avoidance of certain drugs or foods can prevent the manifestation of the disorder, for example sulfonamides and fava beans in glucose-6-phosphatedehydrogenase(G6PD) deficiencv (p. 179). Drug therapy might also be directed at a subset of patients according to their molecular defect. A recent example is a trial r.here gentamicin was administered via nasal drops to patients with cvstic fibrosis.Aminogll,cosideanribiotics such as gentamicinor amikacin causeread-through of premature stop codons in aitro and onlv patients with nonsensemutations (p 25) showedevidenceofexpressionoffull-length cvstic fibrosis transmembrane conductance regulator (CFTR) protein in the nasalepithelium



T a b t e2 3 . 1 E x a m p L eosfv a r i o u sm e t h o d fso r t r e a t r ngge n e t idc i s e a s e Treatment


Enzymeinductionby drugs Phenobarbitone

jaundice Congenital non-hemolytic

Replacementof deficientenzyme/protein Btoodtransfusion

deficiencv deaminase SCIDdueto adenosine

Bone marrow transplantation


Enzyme/proteinpreparations Trypsin q-Antitrypsin Cryoprecipitate/factorVlll B-Glucosidase

deficrency Trypsinogen q-Antitrypsin deficiency Hemophitia A Gaucher disease

Replacementof deficientvitamin or coenzyme a u6 a u12

Homocystinuria acidemta Methylmalonic

Brotin D

rtckets Vitamrn D-resistant

Replacementof deficientproduct Cortisone Thyroxine

adrenaI hyperptasia CongenitaI hypothyroidism CongenitaI

Substraterestrictionin diet Amino ocids Phenytatanine Leucine. isoteucine. va[ine

Phenylketonuria Maptesyrupurinedisease

Corbohydrate Ga|.actose


Lipid Chotesterol

Famitial hypercholesterolemia



Drug therapy Aminocaproic acid Dantrotene Chotestyramine Pancreatic enzymes Peniciltamine

Angioneurotic edema hyperthermia Matignant Famitial hyperchotesterolemia fibrosrs Cystic Witsondisease, cystinuna

Drug/dietaryavoidance Sulfonamides rates Barbrtu

G6PDdeficiency Porphyria

Replacementof diseasedtissue Kidneytransptantation Bonemarrowtransptantation

potycystic Fabrydisease kidneydisease, Adult-onset syndrome Wiskott-Atdrich X-tinkedSCID.

RemovaIof diseasedtissue Cotectomy 5p[enectomy

potyposts FamitiaI adenomatous spherocytosis Hereditary






TISSUETRANSPLANTATION Replacement of diseased tissue has been a further option since the advent of tissue t-vping (p. 377). An example is renal transplantation in adult polycystic kidney disease or lung transplantationin patients with cystic fibrosis. An exciting new treatment for type I diabetesmellitus is islet transplantation.Islet cells are prepared from donated pancreases (usually two per patient) and injected into the liver ofthe recipient. The 'Edmonton' protocol has proved very successful:at 3 years post-transplant more than 80o/oof patients are still producing their own insulin.


biosynthetica[y using Tabte23.2 Proteinsproduced r e c o m b i n aD n tN At e c h n o L o a v Protein



Diabetes meltitus


Shortstature dueto growthhormone defrciency


Hemophitia A

Factor lX

Hemophitia B



(X-tinked A Fabrydisease Lysosomal Cx-Ga[actosidase storage disorder) B-lnterferon

Muttipte scterosis

The adventof recombinantDNA technologvhas alsoled to rapid progressin the availability of biosynthetic gene products for the treatment of certain inherited diseases

GENETHERAPY B I O S Y N T H E SOI S FG E N EP R O D U C T S Insulin used in the treatment of diabetesmellitus was previously obtained from pig pancreases.This had to be purified for use very carefully,and even then it occasionallyproduced sensitivity reactionsin patients.However, with recombinant DNA technolog-v. microorganisms can be used to synthesize insulin from the human insulin gene. This is inserted, along with appropriate sequencesto ensure efficient transcription and translation, into a recombinant DNA vector such as a plasmid and cloned in a microorganism, such as Escherichiacoli In this way large quantities of insulin can be made An artificial gene that is not identical with the natural gene needs to be constructed for this purpose. However, synthetically produced genescannot contaln the non-coding intervening sequences,or introns (p. 16), found in the majority of structural genesin eukaryotic organisms, as microorganismssuch asd. coli do not possessa meansfor splicing of the messengerRNA (mRNA) after transcription. Recombinant DNA technology is being employed in the production of a number of other biosynthetic products (Thble 23.2).The biosynthesisof medically important peptides in this way is usually more expensivethan obtaining the product from conventional sourcesbecauseof the researchand development involved. For example,the cost oftreating one patient can exceed d50 000 per year.However, biosynthetically derived products have the dual advantagesof providing a pure product that is unlikely to induce a sensitivity reaction and one that is free of the risk of chemicalor biologicalcontamination.In the past,the useof growth hormone from human cadaver pituitaries has been associated with the transmission of Creutzfeldt-Jakob disease,and human immunodeficiency virus (HIV) has been a contaminant in cryoprecipitate containing factor VIII used in the treatment of hemophilia A (p. 300).


Gene therapy hasbeen defined by the UK GeneTherapyAdvisory Committee (GTAC) as 'the deliberate introduction of genetic material into human somatic cells for therapeutic,prophylactic or diagnostic purposes' It includes techniques for delivering synthetic or recombinant nucleic acids into humans; genetically modified biological vectors (such as viruses or plasmids), geneticallymodified stem cells, oncolytic viruses, nucieic acids associatedwith delivery vehicles,naked nucleic acids, antisense techniques (e.9. gene silencing, gene correction or gene modification), geneticvaccines,DNA or RNA technologiessuch as RNA interference, and xenotransplantation of animal cells (but not solid organs). Advancesin molecular biology leading to the identification of many important human diseasegenesand their protein products have raised the prospect of gene therapy for manl' genetic and non-genetic disorders.The first human gene therapy trial began in 1990,but it is important to emphasizethat, although it is often presentedas the new panaceain medicine, progress to date has been limited and there are many practicaldifficulties to overcome before genetherapy can deliver its promise.

REGULATO RYREOUI REMENTS There has been much publicity about the potential usesand abuses of gene therapy. Regulatory bodies have been established in severalcountries to overseethe technical, therapeutic and safety aspectsof gene therapy programs (p. 360). There is universal agreement that germline gene therapy, in which genetic changes could be distributed to both somaticand germ cells, and thereby be transmitted to future generations,is morally and ethically unacceptable. Therefore all programs are focusingonly on somatic cell genetherap.y,in which the alteration in genetic information is


targeted to specificcells, tissuesor organs in which the disorder is manifest In the USA the Human Gene Therapv Subcommittee of thc National Institutes of Health has produced guidelinesfor protocols oftrials ofgcne therapythat musr be submittedfor approvalto both the trood and Drug Administration and the Recombinant DNA Advisory.Committee, along rvith their institutional rer-iervboards (IRBs) In the UK the GTAC adr.iseson the ethical accepribilir]. of proposalsfor genetherapy researchin humans,taking account of the scientific merits, and the potcntial benefitsand risks. More than 800 clinical trials of genetherapv havebeen approved

product, for instanceb1-producing antibodiesto the protein product. Lastl-1', it is esscntialto demonstratethat introduction of thc foreign geneor DNA sequcncehasno deleteriouseffects,such as inad\,ertentll lcading to a malignanc-Yor a mutagenic effect on either the somaticor the germ-cell lines, for examplethrough mistakesarising as a result of the insertion of the gene or DNA sequenceinto the host DNA, or what is known as insertional mutagencsis In tr'vo patients who developed leukemia after gene therap.vfor XL-SCID, the retrovirus used to deliver the 1-c (IL2RG) genc \\,'asshown to have inserted into the LMO-2 oncogenc,rr hich plays a role in some forms of childhood leukemia,

for childrcn and adults fbr a varietv of genetic and non-genetic disorders. For the most part these appear to be proceeding

on chromosome1l.

without event, although the unexpectcddeath of a patient in one trial in 7999 and,the development of leukemia in three of 11 children uho receivedgenctherapyfor X-linked severecombined


immunodeficiencl'(XL-SCID) (p. 191)has highlighted the risks of gcnc thcrapy

TEC H N I C AAS L PECTS Bcforc a gcne therapv trial is possible, there are a number of technical aspectsthat must be addressed


Onc of the basic prerequisites for assessingthe suitability of gene thcrap-ytrials in humans is the existenceof an animal model. Although there are naturalll'' occurring animal models for some inherited human diseases,for most there is no animal countcrpart. Thc techniquesused to generateanimal models for human diseasearc outside the scopeof this book, but much effort has focuscd on the production of animal models that faithfully recrcate diseascphenotypes Animal models flor cystic librosis, Duchenne muscular dystroph-v(DMD), Huntington disease irnd Fiiedreich ataxiahavc been generatedand provide iust a ferv eramples that mav be used to evaluategenetherapy before trials

One of thc basic prerequisitesof gcne therap]' is that the gene involved should have been cloned This should include not onlr,

in humans.

the structural genc but also the DNA sequencesinvolved in the control and regulation of expressionof that gene.

In-uterofetal genetherapy

Targetceils,tissueand organ The specificcells, tissueor organ affectedby the diseaseprocess must be identi{ied and accessiblebefore treatmcnt options can be considered.Again, this seemsobvious. Some of the early attempts at treatinB the inherited disordersof hemoglobin, such as B-thalassemia,involved removing bone marrorv from affected indir.iduals,treating itin ritro, and then returning it to the patient b-vtransfusion.Although in principle this could have rvorked,to har.ean)' likelihood of successthe particular cells that neededto be targetedrvere the small numbcr of bone-marrow stem cells from rvhich the immature rcd blood cells,or reticuloc-ytes,develop.


The report of successfuladenovirusvector-mediatedin-uterogene therapr in a cystic fibrosis mouse model in 1997meansthat fetal gcne therap,vin tLteroma.vbe possiblein humans.At present it is considcredunacceptablebecauseof the possibilitv of inadvertent gcrm-cell modification The useof stem cells geneticallymodified ex tiro should reduce this risk. However, in-utero stem-cell trlnsplantation rvithout genetic modification currentl!- offers the best prospects for the successful treatment of serious ncurodegencratir.edisorderswith a very earl-vonset,such asKrabbe discaseor Hurler syndrome (p. 169).

ORGANS TARGET In manv instancesgene therap.vr,villneed to be, and should be, dirccted or limited to a particular organ, tissueor bodl- system.

The meansb1'which a foreign geneis introduced need to be both eflicient and safe.If genetherapy is to be consideredas a realistic


alternativeto conventionaltreatments,there shouldbe unequivocal evidencefrom trials of genetherapy carried out in animal models that the inserted gene functions adequatel,-v rvith appropriatc regulatorv,promoter and enhancersequences. In addition, it needs

\i'iral vectorsfor genetherapy ofinherited hepatic disordershave bcen of limited use owing to the lack of vectors that specifically t a r g e t h e p a t o c v t e s .A l t h o u g h l i v e r c e l l s a r e r e f r a c t o r y t o retrovirusesin t;iao, thev ate, somewhatsurprisingly,susceptible to transfection b-v retroviruses lz uitro. Cells removed from the liver b1'partial hepatectomy can be teated in oltra and then reinjectedvia the portal venoussystem,from which they seedin the

to be sholn that the treated tissue or cell population has a reasonablclifespan, that the gene product continues to bc expressed,and that the bod-r-doesnot react adverselvto the gene





liver The effectiveness ofthis approachhasbeendcmonstratedby the lowering of cholesterollevelsin a rabbit animal model with a defect in the LDL receptor.The injection of the hepatocytesinto the portal venoussystemis, however,associatedwith a signilicant

granulocytecolony-stimulatingfactor (G-CSF) and the cytotoxic agent 5-fluorouracil. Reliable identification of specific stem-cell types would enablethem to be enriched for, thereby increasing the likelihood of success.

risk of thrombosisof the portal venoussystemthat can lead to the complication of portal hvpertension. Nevertheless,becauseof the seriousoutlook for homozygoteswith mutations in the LDL receptor (p.226), genetherapyby this meanshas been attempted


in a woman homozygous for a LDL receptor defect. This led, in the short term, to a reduction of LDL levels, although the long-term benefit is, as yet, undetermined. Other disorders affecting or involving the liver in which a similar approachcould be consideredare phenylketonuria,o,-antitrypsin deficiencyand hemophiliaA.

Centralnervoussystem CNS-directed vector systems are being developed in rvhich replication-defective neurotropic adenoviruseslacking the socalled El region can bc produced and then be made infective by growing them in cells engineeredto expressthe El genes.In addition, lentiviruses could be used for the treatment of CNS disorders, such as Parkinson and Alzheimer diseases,because they integrate into the host genome of non-dividing cells and could, therefore,act as a delivery system for stableexpression. Another approachthat has beensuggestedin geneticdisorders affecting the CNS is to transplant cells that havebeen genetically modified in oitro into specific regions of the brain, such as the caudate nucleus in persons with or at risk of Huntington disease.

Gene transfer can be carried out either ex oiao by treatment of cells or tissue from an affected individual in culture, with reintroduction into the affectedindividual, or in aiuo if cellscannot be cultured or be replacedin the affectedindividual (Fig 23.1). The ex-tito approachis limited to disordersin which the relevant cell population can be removed from the affected individual, modified geneticalll,,and then replaced The in-aiao approach is the most direct strategyfor genetransfer and can theoreticallybe used to treat many hereditary disorders.Although severalstudies in animal modelshavedemonstratedthat it is feasibleat leastpartially to target viral gene-transfervectorsto different organs,targeting strategieshavenot been used clinically for hereditary disorders. There are two main methods for delivering genetransfer,viral and non-r'iral

ViraIagents A number of different viruses can be used to transport foreign genetic material into cells. Each of these has its particular (Thble 23 3). advantagesand disadvantages

Muscle Unlike other tissues,direct injection of foreign DNA into muscle has met with some successin terms of retention and expression of the foreign genein the treated muscle.Alternatively-,injection of myoblastsinto muscle results in their incorporation into recipient muscle bundles.Although animal model work showed some promise of efficacy this approachhas met with difliculties in humans. Direct DNA injection has, however, been used to expressthe protein products of genes,transferred in t,itnt into myoblasts,that are unrelated to muscle function, such as human growth hormone and factorVIIL Other primary cell types, such as fibroblaststreated in oitro, could also be transplantedback as skin grafts to deliver circulating geneproducts.



In the treatment of disordersaffectingthe bone marroq problems arise due to the small numbers of srem cells, which need to be transducedif there is to be more than a transient responsewith gene therapl Stem cells often constitute less than lolo of the total cells present. Pretreatment of the bone marrow to expand the number of stem cells has been tried for certain inherited immunological disordersby the use of growth factorssuch as the

Fis.23.1 ln-vrvoandex-vivogenetherapy. ln-vivogenetherapydetivers genetically modf edceltsdirectly totheparienr A^ examples CFTR g e n et h e r a puys i n g[ i p o s o m eosr a d e n o v r rvuisan a s asI p r a y s lrrm lho nriiont .nOdifieS Fx-Vivn nene the-a _m " n\/o< rellc " rn/v r F

the- in vrtroandthen relu.^s them [o LhepatrentAn exampteis the treatmentof fibroblasts from patientswith hemophiliaB by the additionof the factorlX gene Modifiedfibrobtasts are then iniected intothe stomachcavity



s f q e r et r a n s f e r T a b t e2 3 . 3 M e t h o o o Feature



Adeno-associated Lentivirus vtrus




Maximum insert size(kb)








Chromosomal IntegTatlon








Duratron of expressron








Hostimmune response









Possrbitity of insertionaI mutagenesis






Possibility of non- specific events

In S e r l o n a l




These are RNA viruses that can integrate into the host DNA b-v making a copl''of their RNA molecule using the enzvme reverse transcriptase(p. 375).The provirus so formed is the template for the production of the mRNAs for the various viral gene products

Adcnoviruses can be used as vectors in gene therapy as they infect a rvide variety of cell t1'pes.They have advantagesover oncoretrovirusesin that they are stableand can easilybe purified to produce high titers for infection. Unlike retroviruses,they can

and the ns11,genomic RNA of the virus. If the provirus is stabll' integrated into dividing stem cells, all subsequentprogenv cells rvill inherit a copv of thc viral genome Onc of thc disadvantages associated with the useof retroviruscs as a vector system in gene therapy is that only a relatively small

infect non-dividing cells and carry up to 36kb of foreign DNA. In addition, the.varesuitablefor targetedtreatment of specifictissues such as the rcspiratort tract, and have been extensivelyused in genetherap-\trials for the treatment of cvstic fibrosts. Adenovirusesdo not integrate into the host genome,thereby avoiding the possibility of insertional mutagenesisbut having the disadvantagethat expressionof the introduced gene is usually unstable and often transient. They also contain genes known to be involved in the process of malignant transformation' so thcre is a potential risk that they could inadvertently induce malignancy.Bv virtue of their infectivity, they can produce advcrseefl'ectssecondaryto infection and by stimulating the host immune response.This was demonstrated by a vector-related death follorving intravascular administration of high doses

DNA sequencecan be introduced into thc target cells usually lessthan 7 kb which limits their use For example,even if all of the introns were removed from the dystrophin gene (p 298) for use in gene therapy of DMD, the gene would still be much too large to be incorporated into a retroviral vector Attempts have been made to overcomethis by inserting a modified dystrophin gene in which a large amount of the gene has been deleted, but which still has relativell' normal function. This is known as a mini-d-vstrophingene. A seconddisadvantageofusing retrovirusesas vectorsin genc therap.vis that they can onlv integrateinto cellsthat divide shortllafter infection This limits their potential use,as few cell types are dividing continualll.,although retrovirusesmav be beneficialfor targeting cancersrvithin non-dividing cells.

Lentiviruses The lentivirus family includes HIV. Lentiviruses are complex viruscs that infect macrophagesand lymphocl'tes, but, unlike oncoretroviruses,thev can be integrated into non-dividing cells The.v ma1.,therefore, be useful in the treatment of neurological conditions

(3.8 x l0tr) of adenovirus particles to a patient with ornithine deficiencl'. transcarbamylase

Adeno- ossocioted viru ses Adeno-associatedviruses are non-pathogenic parvoviruses in humansthat require co-infection with helper adenovirusesor certain members of the herpes virus family to achieveinfection. In the absenceof the helper virus, the adeno-associatedvirus DNA intcgratesinto chromosomalDNA at a specificsiteon the Iong arm of chromosome19 (19q13.3-qter) Subsequentinfection with an viral DNAadenovirusactivatesthe integrated adeno-associated producing virions The-vhavethe advantagesof being able to infect




a wide varietv of cell tvpes, exhibiting long-term geneexpression and not generatingan immune responseto transducedcells.The safety of adeno-associatedviruses as vectors occurs bv virtue of their site-specificintegration but, unfortunatell., this is often impaired with the inclusion of foreign DNA in the virus. The disadvantagesof adeno-associated viruses include the fact that they can be actirated b-vany adenovirusinfection and that, although 95oloof the vector genome is removed, thev can take inserts of foreign DNA of onlv up to 5 kb in size Someof the more recentdevelopmentsof the adeno-associated viruses as vectors for gene therapy allow the prospect of the introduction of pharmacologically controlled induction of the erpressionof transducedgenes

Herpesvirus Herpesviruses are neurotropic (i e. they infect nervous tissue) and, if suitably modified, could be used ro target gene therapl. to the CNS for the treatment of neurological disorders such as Parkinson disease.An immediate disadvantageof using herpesvirusesas a vector svstem is their directly-toxic effectson nerve cells as rvell as the consequentimmune response,although recentmodilicationsofthis potentialneurotropic vector havebeen produced that are devoid of viral expressionand neurotoxicitr-. Herpes viruses,however,do not integrate into the host genome, and therefore it is likelv that the expressionof introduced genes lvould be temporary and unstable.

NokedDNA Direct injection of DNA into cells hasbeen used in genetherap1,, such asthe mini-dystrophin geneinto myoblastsin the mousemodel for DMD (p.299). Although successhas been reported in terms of evidence of localized gene expression,this approach clearly has a limited place except for the possibility of the expressionof hormones or proteins for which small amounts will result in a significantclinical effect (e g. erythropoietin or factorVIII).

Liposome- m ediotedDNA tronsfer Liposomes are lipid bilayerssurrounding an aqueousvesiclethat can facilitate the introduction of foreign DNA into a target cell (trig. 23.2).A disadvantageof liposomesis that they are not verv efficient in genetransfer and the expressionof the foreign geneis transient, so that the treatment has to be repeated.An advantage of liposome-mediatedgene transfer is that a much larger DNA sequencecan be introduced into the target cells or tissuesthan with r.iral vector systems.This can be as large as an artificially constructed mini-chromosome which, in addition to a specific structural Bene,can include elementsinvolved in the regulation of geneexpressionin a physiologicallycontrolled fashion, as well ascentromericand telomeric sequencesthat will allow replication of the foreign DNA in mitotic divisions.Recent modilications of cationic lipid DNA complexeshavebeendevelopedthat enhance the efficacvof,genetransduction.

Receptor- m edi ated endocytosis N o n - v i r am I ethods There is a number of different non-viral methodsof genetherapr,-. These have the theoretical advantageof not eliciting an immune responseand of being saferand simpler to use,aswell asallowing large-scaleproduction, but their efficacyis limited

L i p o s o m-eD N A complex

Liposome /'kr


Foreign gene


Plasm id


Fis.23.2 346

D i a g - a m m a t irce o ' e s e n t a t i oo^f I , p o s o - e - m e d i a L egde . e L n e ' a p y

A variation of liposome-mediatedgene transfer is to target the DNA to specific receptors on the surfacesof cells. A complex is made betrveenplasmid DNA containing the foreign gene or DNA sequenceand specilicpolypeptideligandsfor which the cell has a receptor on its surface.For example,DNA complexedto a

H u m a nc e l l


glycoproteincontaininBgalactosewill be recognizedbv receptors on the surfacc of liver cells that are specific to gl.vcoproteins rvith a terminal galactoseThis rcsults in internalization of thc complex into endocytic vcsiclcs,which are then transported to the lysosomesvr,herethe complex is degraded. In order for the foreign geneto be expressed,it has to cscapefrom thc lysosome Thc rate at which it escapcsfrom the lvsosomescan be increased b-vinclusion of adenovirusor influcnza gcnc products.

cancer,viral infections including HId and polyglutamine repeat disorders.RNA interferencehas scquencesin neurodegeneratir,e been shown to be successfulin mice; the next step is a human pilot studl'. One possible application is to target SCNI gene duplications and triplications in patients with Parkinson disease rvho hil,e abcrrant 0-synuclein dosageThere areconcerns,however, regarding recent reports that RNA interference may induce an interferon rcsponse



RNA modification therapv targetsmRNA, either bv suppressing mRNA levels or b1. correcting/adding function to thc nRNA. There are three main approachesto modifying mRNA to treat monogenic disorders:usc of antisenseoligonucleotides,RNA

Ribozl'mes arc RNA moleculcs rvith enzymatic activity that recognize specific RNA sequencesand catal.vzea site-specific phosphodiesterbond cleavagervithin the target molecule. This method has potential for replacingmutant sequencesor reducing mutant mRNA levels in loss-of-function dominant disorders. The structure of ribozvmes consistsof two regions of antisense RNA (referred to as the flanking complementarit.vregions) that

interferenceand riboz-vmes.

Antisenseoligonucleotides Antisense therapy ma1-be used to modulate the expressionof gencs associatedlvith malignanciesand other genetic disorders The principle of antiscnsc technologv is the sequence-specific binding of an antisenseoligonucleotidc (t,vpicall1.18 to 30 bases in length) to a target mRNA that results in inhibition of gene expressionat the protein lcvcl Antisense oligonucleotidescan be delivered to the cell by liposomes,but the folding of mRNAs or interaction rvith proteins mav pre\rent them binding to the targct. Nevertheless,one compound has already been approved for treatmentof cytomegalovirus-inducedretinitis, and a number of other trials are ongoing. The identification of exon-splicingcnhancer(ESE) sequences rvithin the past decadehas increasedour understanding of the processof exon splicing. If an ESE is mutated, the exon is morc likel-vto be spliccd out. Somc protcins rvith in-frame rvhole-exon deletions retain some residual activit)', for example d-vstrophin mutations in Becker muscular dystrophy (p. 297).ln an in-ritro experiment using muscle cells from trvo patients rvith DMD, blocking an ESE with an antisenseoligonucleotiderestoredthe reading framc The detectionof significantlevelsof dystrophin protein in the musclc cclls confirmed the therapeuticpotential of this approach.

RNAinterference This tcchnique also has broad therapeutic application, as an-v interference genemx),bc a potential target for silencingb"v'.'RNA In contrast to antisenseoligonucleotidctherapy where the target mRNA is bound, asa result of RNA interfercnce the target mRNA is cleavedand it is estimatedto be up to 1000-fold more active. RNA interference works through thc targeted degradation of mRNAs containing homologous sequenccsto synthetic doublcstranded RNA molecules knorvn as small interfering RNAs (siRNAs) (Fig 23.3).The siRNAs ma.vbe deliveredin drug form using strategiesdevelopedto stabilizeantisenseoligonucleotides, or from plasmids or viral vectors. In u^itro,siRNAs have been sho$,n to reducc the expressionof Bcr-Abl and Bcl2 targets in


flank thc nucleolytic motif and provide the target specificity. ';itro to correct hereditar.v Ribozl'me constructshavebeentestedin There have poll.neuropathy disordcrssuch asfamilial amyloidotic on the is focused been no clinical trials to date,but one strategy The approach pigmentosa autosomaldominant form of retinitis is sclectivclyto txrget the dominant version of the Benetranscrlpt' as successfullyachievedin rodent and large mammalian models

CORRECTION GENE TARGETED A promising nelr' approach is to repair genesin sirz through the ccllular DNA repair machinery (p. 28) Proof of principle has been demonstratedin an animal model of Pompe disease.The point mutation was targetedby chimeric double-strandedDNA-RNA oligonucleotidcscontaining the correct nucleotide sequence. Repair rvasdemonstratedat the DNA level and normal enzyme actir,it]'was restorcd. Thc lateststrategyusesengineeredzinc-finger nucleases(ZFNs) to stimulate homologous recombination. Targeted cleavageof DNA is achievedby zinc-finger proteins designedto recognize unique chromosomal sites and fused to the non-specific DNA clear,agedomain of a restriction enzyme' A double-strand break induced by thc resulting ZFNs can createspecificchangesin the gcnome by' stimulating homology-directedDNA repair between the locus of interest and an extrachromosomalmolecule' There arc many potential problems to overcome' such as the possible immunogenicit-vof ZFNs, but this technique mal'-be particularly promising for er-t;ixo geneticmanipulation.

THERAPY STEM.CELL SOMATIC Stcm cellsareunspecializedcellsthat are definedby their capacity lbr self-rcneu'aland the ability to differentiate into specialized cclls along manv lineages.Somatic stem cells can differentiate into the cell t1'pesfound in the tissuefrom which they are derived (Fig. 23..t).The-vare usuallv describedb-vreferenceto the organ of origin (such as hematopoieticstem cells).







il|l ||tl

Il Il



In a c t i v e RISC

Mechan ismofRNAinterference Doubte-stranded (ds)RNAsareprocessed byDicer. in anATPp'ocess. dependent to produce smaLL Interfering RNAs(siRNA) ofabout21to 23 nucleotides in lengthwrthtwo-nucteotrde overhangs ateach e n d S h o rht a i r p i(ns . )R N A se, i t h epr r o d u c e d endogenously or expressed fromvrraL vectors,



:rp :lsn n'n.trsctrd

hv f)ircp rnto siRNA

A n A T P - d- -pTn- p n d' p_ n t

hplie:cp ic reor rirp.ltn

unwind r h ed s R N Aa,t t o w i nogn es t r a n d lo b i n dt ot h eR N A - r n d u cseidt e n c i ncgo m p t e x ( R I S CB) r n d i nogft h ea n t i s e n sReN A s t r a n d activates theRISCto cleave mRNAscontaining a homologou s q u e n c(eF r o mL i e b e r m a n se etaL2OO3Trends MotMed9 397-403. w i t hp e r m i s s i o n )

Target mRNA

llllll||tl Cleaved t a r g e tm R N A






' )


Embryonic stemcells


D i f f e r e n t i a t ecde l l so f e n d o d e r m a l , m e s o d e r m aolr e c t o d e r m aol r i g i n





Somatic s t e mc e l l s


C n r :Unp: h l or u J U, nr rmr o: rLir.L c) L+ga m rLag lt lt c>. -dol .tlr. d t>U L ou

D i f f e r e n t i a t ecde lI t y p e s f r o m t h e t i s s u eo f o r i g i n

Bone-marrow transplantation is a form of somatic stem cell therapy that has been used for more than 40vears.Although it can be an effective treatment for a number of genetic disorders, including ADA deficiency, SCID, lvsosomal srorage diseases and Fanconi anemia, the associatedrisks of infection due to lmmunosuppressionand graft-versus-hostdiseaseare high. The

Generation of embryonic andsomatic sler-cettsThefusiono{thespermand eggduringfertilization estab[ishes a d;ptoid zygote thatdivrdes to create the blastocyst Embryonrc stemcetts(ESCs) arederived fromtheinnerce[[massofthe btastocyst ESCsin cutture arecapabte of self-renewaI withoutdifferentiatron and rntoarlcet[typesof a.eabteto d,fferentiate t h ee n d o d e r m m,e s o d e r m a n de c t o d e r m [ i n e a g euss i n ga p p r o p r i astieg n a t s n lf u

seLf-renewaI and,withappropriate signaLs. differentrate intovarrous ce[[typesfromthe tissuefromwhichtheyarederived

main limitation is the lack of a suitablebone-marrow donor, but it is hoped that the use of stem cells derived from cord blood may overcomethis problem in the future. Tiansplantation of stem cells (e.g. pluripotent hematopoietic stem cells) in utero offers the prospect of a novel mode of treatment for genetic disorderswith a congenital onset.The immaturity of


the fetal immune systemmeans that the fetus will be tolcrant of foreign cells so that there is no nced to match the donor cells with thosc of the fetus.Tiials of in-utero stem cell transplantationare under rvay fbr a number of disorders,including SCIQ chronic granulomatousdiseaseand hemoglobinopathies.

E M BR Y O N ISCT E MC E L LT H E R A P Y Teratomas(bcnign) and teratocarcinomas(malignant)arc tumors that are found most commonly in the gonads. Their name is 'teratos'(monster) and it dcscribes derived from the Greek word thcir appearancewell, as these tumors contain teeth, pieces of bone, musclcs,skin and hair A key experimentdemonstratedthat if a single cell is removed from one of thesetumors and injectcd intrapcritoneally,it acts as a stem cell b_r-producing all the cell t-vpesfound in a tcratocarcrnoma. Mouse embryonic stem cells rvere first isolated and cultured 25 yearsago.Studies of human embry-onicstem cells havelagged behind, but the pace of research has increascd exponentialll' in recent 1-ears,following the achievementin 1998 of the first stem cells.Embr-vonicstem cclls are cultured human embr_r-onic derived from the inner cell mass of embryos at the blastoc-yst stage (Fig. 23.4). They are pluripotent, w'hich means thcy can give rise to derivativesof all three germ la-vers,i c. all cell types that arc ftrund in the adult oreanism

Embryonicstem ce[[sfor transptantation

thc right conditions. If this differentiation of adult stem cells can be controlled in the laboratory,these cells may becomethe basis of thcrapics for many diseases.

Genetherapyusingembryonicstem cells An alternatc strategv is to use ESCs as delivery vehiclesfor genesthat mcdiate phenotype correction through gene-transfer tcchnologl'.One potential barrier to using human ESCs to treat genetic disorders is immunorejection of the transplanted cells b1' the host. This obstaclemight be overcomeby using gene transfcr with the relevant normal gene to autologouscells (such as cultured skin fibroblasts), transfer of the corrected nucleus to an enucleatcdegg from an unrelated donor, development of 'corrccted' ESCs and, {inalll', differentiation and transplantation of the corrected relevantcells to the samepatient (Fig'.23.5). A crucial component of future clinical applications of this 'personalized'human ESC lines is the ability to derive stratcei-v using the nuclear transfer technique. Although researchon this tcchnology has been controversial,the efficient transfer of somatic cell nuclei to enucleatedoocytesfrom unrelated donors, and the subsequent derivation of human ESC lines from the is a technical hurdle that seemslikely to be resulting blastoc-vsts, overcomeduring the next few Jrears.


The ability of an embryonic stem cell (ESC) to differentiateinto an1't1'peof cell meansthat the potential applicationsof ESC therapl' arc vast. One approach involvcs the differentiation of ESCs iz pro\ide specializedcells for transplantation.For example, "^itr0to it is possibleto culture mouse ESCs to generatedopamine-

The disorders that are possiblecandidatesfor genetherapy include both genctic and non-genetic diseases(Table 23 4).

producing neurons. When these neural cells wcre transplanted into a mouse model for Parkinson disease,the dopamineproducing neurons showed long-term survival and ultimately corrected thc phenotype.This'therapeutic cloning' strategyhas been proposed as a future therapy for other brain disordcrs such

Thcre are a number of single-gene diseasesthat are obvious candidatesfor genetheraPl'.

as stroke and neurodegencrativediseases. It ma1'alsobe possibleto geneticallyengineerESCs in order to

One of the first diseasesfor which gene therapy has been attcmpted in humans is the inherited immunodeficiencydisorder causedb-vadenosinedeaminase(ADA) deficiency (p. 171).The most successfulconventional treatment for ADA deficiency is bone-marrow transplantationbut, in the absenceof a compatible donor, patientsmav be treated rvith PEG-conjugatedADA. In 1990 the lirst gene-therapytrial enrolled l0 patients with ADA-SCID. Although no adverseeventswere reported, none of 'cured', probably becauseof the lorv efficiency thc patients was of gcne transfer from the retroviral vector Although transduced 'l'cells havc been shown to persist for more than l0,vearsand still expresstransgenicADA, the therapeutic effect of gene therap-vremained difficult to assessbecauseof the concomitant trcatment rvith bovine ADA coniugatedto PEG-ADA. A recent report showed that discontinuation of PEG-ADA resulted in a

improve their utilit.v for transplantation.For example,although bone marror,v-derivedmesenchymal stem cells can develop into cardiac muscle in aio|, their potential as a treatment for cardiac discaseis limited, in part, by'their poor viability aftcr transplantation.Tiansduction with a vector containing the mouse Akt-l gene reduced cell death of the stcm cells. When these modihed stem cellswereinjectedinto the heart of a rat 60min after suffering a heart attack,myoclte regenerationwas observed,with subsequentnormalization of cardiacfunction. There has been much debate as to whether ESCs arc an essentialprerequisite,asadult stem cellshavebeen lbund in manv more tissues than was once thought possible.This hnding has led scientiststo ask whether adult stem cells could be used for trxnsplantation.Certain kinds ofadult stem cell seemto havethe abilit-vto differentiateinto a number of diffe rent cell types,given



Aden osine deomin osedeficiencY

strong selectiveadvantagefor gene-correctedT cells associated $,ith restoration of T:cell functions and antibody responses




T r a n s f enr o r m a lg e n eo r c o r r e c tm u t a t i o ni n v i t r o





P a t i e n tc e l l s

G e n e t i clal y corrected cell

t D o n o re g gw i t h p a t i e n t ' s corrected c e l ln u c l e u s


G e n e t i c a l lcyo r r e c t e da u t o l o g o u s s t e mc e l l sf o r t r a n s p l a n t a t i o n


Fis.23.5 E m b r y o n i c s t e m c e l l s f o r g e n e t h e r a p y . T h e s t r a t e g y d e p i c t e d s t a r t s w i t h T ef im s (aepga t i e n t w i t h a m o n o g e n i c b roovbl nt agscftersot)tm d t s o r d e r a n d t h e n t r a n s f e r r i n g t h e n o r m a t g e n e u s i n g a v e c t o r ( o r p e r h a p s b y c o r r en c tviint rgot h ) Tehmeuntuact il oe nu s f r o m a c o i r e c t e d cellisthentransferred to an enucteated eggobtained froman unretated donorbysomatic cellnuctear transfer: Theegg,nowcontanrng thegenetically corrected genomeofthepatient. is activated to devetop intoa blastocyst in vitro,and corrected autotogous stemcellsare derived fromtheinnerceLL massThestemcetlsarethendirected to differentiate rntoa snecrfic celL tvneenrltransferred tothepatient, thereby correcting thedisorder:

to neoantigen, but incomplete correction of the metabolic defect. Recentll',an improved genetransfer protocol in bone-marrow

However, progress to date has been slow and, although gene therapy can correct the primary and secondarydefectsassociated with cysticfibrosis,the extent and duration of geneexpressionhas

CD34+ cells combined with lorv-dosechemorherapy resulted in multilineage,stableengraftment of transduced progenitors at substantial levels, restoration of immune functions, correction of the ADA metabolic defect, and proven clinical benefit, in the absenceof PEG-ADA.

been inadequate,orving to the rapid turnover oflung epithelial cells There have also been concernsabout the safety of some current

Hemoglobinopothies Attempts at treating B-thalassemiaand sickle-celldiseaseby gene therapy havenot beeneffectiveasyet, primarilv becausethe numbers of cx- and B-globin chains must be equal (p. la9). Gene therapy must, therefore, be dose specific,and this is not possibleat the presenttrme

delivery systems,especiallyfollowing the adenovirus-triggered death of one patient. For viral vecrors,the main challengesare accessto target cells and host immunity, which preventsefficient re-administration. Non-viral vectors haveimproved greatly over the past 5 years,but improvements in efficacyare needed.In the lung, delivery of nakedDNA has been inefficient and lipid-based vectors have achieved efficient gene transfer only at dosesthat elicit limiting inflammarory responses.Molecular conjugatesor polymer-baseddelivery overcomessome limitations, with good ability to transfectnon-dividing cells.Improvementsof viral and non-viral vectors continue to advancethe construction of stable, safeand eflicaciousvectorsthat can be re-administered.

Cysticfibrosis In contrast to the hemoglobinopathies,cystic fibrosis should be more amenableto genetherapy as the level of functional protein sufficient to produce a clinical responsemay be as low as 5 l0o/o


and the lung is a relativelyaccessibletissue.

HemophilioA ond B Hemophilia A and B are excellentcandidatesfor genetherapy as a modest increasein the level of factor VIII or IX, respectively, will be of major clinical benefit Recent trials have used direct



Duchenne muscuIor dystrophy T a b t e2 3 . 4 G e n e t iacn dn o n -g e n e r ,dci s e a s ersh a Ic a n potentralty be trealedby generherapy Geneticdisorder


l m m u n ed e f i c i e n c y

A d e n o s i n ed e a m i n a s ed e f i c r e n c y P u r i n en u c l e o s i d e phosphorytase d e f i ce n c y C h ' o n i cg ' a n l l o n ' a t o u sd i s e a s e


Low-densrty [ipoprotern receptor abnormatities


(A) Facto r V ll defioency (B) Factor lXdeflcrency

Gaucher disease

Glucocerebrosidase defrciencv

^/,,-^^^..-^--L--ir^-:-\/r r v r u L u p u r y s d L Lr d r u u 5 t S v t

R _ c l , r e r , r n n. _i d_ :_c- o -n- a, f r- ,. : n c y


q-Antitrypsin deficency

Cystic fibrosis



Phenytalanine hydroxytase defici ency


Ornithine transcarbamytase dofirionnr

Citrutt nemra

Argininosuccinate synthetase deficiency

Muscutar dystrophy

Dystrophin mutations

T h a t a s s e m i a / s i c kc leet l a n e m a


M a l r g n am ne t tanoma n.,--;-^ --^-^-

Thc main difficultylvith genetherapv for DN{D is the sheersize of the drstrophin gcne the complementary DNA (cDNA) is l'lkb. A trial is in progressusing plasmid vectors that can accommodatc the large cDNA, but delivery to the muscle i s i n c f f i c i c n t . \ n a l t c r n a t i r es t r a t e g r i s t o u s e x n t i s e n s e oligonucleotidesto force cxon skipping and convert out-offrame deletions that causeDMD to in-frame deletions usually associated uith thc milder Beckermusculard.vstrophyphenotypc This approach ma-vbc successfulfor up to 75o/oof patients rvith DNID. A recent report has describcd the intravenous infusion of a 31-mer phosphorothioateoligonucleotidc against thc splicing enhancer sequcnceof exon 19 in a patient with an out-of'-frame,exon 20 deletion of thc dystrophin gene.This antisenseconstruct rvas administered at 1-week intervals for ,1l'ccks with no evidence of side-effects.Exon 19 skipping appeared in a portion of the dystrophin mRNA in peripheral lvmphocvtcs after the infusion and a muscle biops,vtaken I week after the linal infusion sholled that thc novel in-frame mRNA, lrrcking both exons l9 and 20, represented approximately 601c, of' the total reverse transcription polymerasechain-reaction (RLPCR) product Dl-strophin was idcntified histochemicalll, in the sarcolcmma of muscle cells aftcr oligonucleotide trcatment. Thesc hndings demonstrate that phosphorothioate oligonucleotidesmxy be administeredsafelv to children with D\{D, and thrt a simplc intravenous infusion is an cffective dclir,er]. mechanism for oligonucleotidcs that lead to exon skipping in DN'ID skeletalmuscles There is also the possibilit-rof upregulating a d]'strophin homolog, utrophin. Immune reiection is not a problem and studics in rhe mdx mousc have shown significant improvement in musclc function. Researchis norv under way to find a pharmacologicalcompound that will upregulate utrophin cxprcsslon.

Braintumors Neuroblastoma

Hutchi nson-Gilford progeroi d syndrome

R e n a lc a n c e r

A c q u i r e di m m u n e d e f i c i e n c y s y n d r o m e( A 1 D S ) a --i,^.,.--,,|.-


Rheumatoid arthrltis

Thc great majorit-v of casesof the premature aging condition, H u t c h i n s o n G i l f o r d p r o g e r o i d s - v n d r o m e( H G P S ; p 1 0 5 ) arc caused b1-a single nucleotide mutation (c.1824 C>T) in thc LMNA gene. This mutation activates an aberrant cryptic splicc site in LMNA pre-mRNA, leading to svnthesis of a truncatcd lamin A protein and concomitant rcduction in wildtvpc lamin A. Fibroblasts from individuals with HGPS havc scvcrc morphological abnormalities in the structure of the nuclcar envelope RNA interfercnce technologv has been used to suppressthc erprcssion of this mutant protein, lvith the long-term goal of

intramuscular injection of adeno-associated r,irus expressing factorVIII or e x-rir^otreatment of libroblastswith plasmid-borne factor IX follorvcdby injection into the sromachcavitl: Although initial rcsults \\ere encouraging,the transient rise in factor\rIII levels rvas modcst (0.5-.1oloof normal) and these clinical trials havebcen halted.

halting thc pathogenesisof the coronar-yartery atherosclerosis thar tl.pically leads to the death of patients with HGPS. S h o r t h a i r p i n R N A ( s h R N A ) c o n s t r u c t s w e r e d e s i g n e dt o trrgct the mutated pre-spliced or mature LMNA mRNAs, and exprcssed in HGPS fibroblasts carrying the c. 1824 C>T mutations using lcntiviruses. One of the shRNAs targctcd to the mutatcd mRNA rcduced the erpression levelsof the mutant




protein more than fourfold and also ameliorated the abnormal nuclear morphologl':

Commonmultifactorialdiseases In the majority of human diseasesin which there is a genctic etiology both genes and environmental factors are involved (p 219). Gene therapy will have a much more widespread impact in medicine if it can be used in this group of disorders It is important, however, to remember that, for most of the common multifactorial diseasesin humans, the identification and subsequentavoidanceof causativeenvironmental factors is likely to be much more effectivethan gene therapy at present or in the near future.

Concer In contrast to the limited number of gene therapy trials for single-gene disorders, numerous cancer-gene therapy trials have been initiated. Gene therapy for cancer aims to kill cancer cells selectively,either directly bv the use of toxins targeted at cancer cells, or by enhancing the bodv's immune response. Supply turnor suppressor genes It has beenproposedthat the targetedintroduction of recognized tumor suppressorgenes (p. 201), such as TpS3, to cancer cells could result in control of their growth. More detailed knowledge of the biology of cancer is needed before this approach can be undertaken reliably and safely'. Inhibit oncogenic proteins Imatinib (alsoknown as STI-571 or Glivec) is a protein tr rosine kinase inhibitor used to treat chronic myeloid leukemia. It is a very effective treatment that works b.v binding the Bcr-Abl fusion protein resulting from the t(9;22) translocation.This is an exampleof effectivedrug designresulting from knowledgeof the molecular etiolog]'. Stimulate natural killing of turnor cells Mitogens, such as interleukin-2, introduced in t,itro into melanosomes that have been removed from a patient with malignant melanoma and then reintroduced into the patient, could be used to activate the patient's immune response.The use of liposome-bound plasmid DNA containing foreign histocompatibility genes to transduce tumor cells to enhance the immune responsehas also been proposed as a possibleform of gene therapy in cancer.Again, a better understanding of the malignantprocessand the bodv's immune responseto malignancv is necessarybefore this form of genetherapl'-can be effective Introduce genes that selectively darnage cancer cells The introduction of the tumor necrosisfactor gene into tumorinfiltrating lymphocvtes, which can then be returned to the patient,


has been promoted as another approach for gene therapy in cancer. More recently, a proposal has been made to introduce what have been called conditionally toxic or suicide genes into cancer cells An example is the thymidine kinase gene of the herpes simplex virus, which allows the metabolism of the drug ganciclovir by cellular kinasesinto the triphosphate form that inhibits DNA polymerase,resulting in the death of the cancer 'bystander' effect. It cells as well as surrounding cells through a has alsobeen proposedthat'antiangiogenic' genescould be used to compromise the circulatory supply of tumors An example is the inhibition of the angiogenic vascular endothelial growth factor (VEGF).

Coronory o rtery diseose VEGF is alsoa target for genetherapy in coronary artery disease rvhere patients cannot be treated by angioplasty or coronaryarter-ybypass grafting The aim is to overexpressVEGF and lncreaseanglogenesrs.

PeripheroI voscuIor diseose A number of different trials of gene therapy in persons with peripheral arterial vasculardiseaseare under way, involving the introduction of genesthat lead to proliferation of new vessels (e g.VEGF) and the production of anticlotting agents.

Autoimmunedisorders The use of gene therapy in autoimmune disorders to restore immune homeostasishas been suggested.Interleukin-1 (IL1) is an immune system signal that triggers inflammation. For example, it has been proposed that gene therapy could be used to introduce genes for the IL-l receptor antagonist protein into svnovial cells in persons with rheumatoid arthritis, in which the inflammatory process is believed to plav a key etiolosicalrole.

FURTHER READING AndersonW F 1992Human genctherap1,. Science256: 808 813 A considerution o.fgenetherapyby oneof its main pro2onents Graw J, BrackmannH H, OldenburgJ, SchneppenheimR, SpannaglM, SchwaabR 2006HaemophiliaA: from mutationanalysisto nerv therapies.Nat Rcv Genet 6: 488 501 Rexien ol'hemophiliaA geneticsand genetherap.y. LiebermanJ, Song E, Lee S-K et al 2003Interferingwith disease: opportunitiesand roadblocksto harnessingRNA interference. tends Mol Med 9: 397 403 Article devribing thepotenttalapllicttions of RNA mterJirence. O'Connor T P, Crl stalR G 2006Geneticmedicines:treatmenrstrategies for hereditarydisordersNat Rev GenetT:261-76 A recentreuep article that summarizeslhe detelopmentof'geneticmedicines'. SolterD 2006From teratocarcinomas to embryonicstemcellsand beyond:a history of embr.vonicstemcell researchNat Rev Genet 7:319-27

Thefavinating history of stem-cellresearchpith insightsinto thefuture applications of embryonicstemcelk. Van Deutekom J C ! van Ommen G-J B 2003Advancesin Duchenne muscular dystrophy gene therapy.Nature Rev Genet 4: 774-783 A reoieo of genetherapyfor DMD that includesa goodozterzsieo of d.ffirent str&te[tes,

O teatment of genetic diseaseby conventional means requires identification of the gene product and an understanding of the pathophysiology of the disease process.Therapeutic options can include dietary restriction or supplementation, drug therapy, replacement of an abnormal or deficient protein or enzyme, and replacement or removal of an abnormal tissue. @ Recombinant DNA technology has enabled humanderived biosynthetic gene products such as human insulin and growth hormone to be produced for the treatment of human disease. @ B"fote a trial of gene therapy is carried out in humans, the gene involved must be characterized, the particular cell type or tissue to be targeted must be identified, an efficient, reliable and safevector system that results in stable continued expressionofthe introduced gene has to be developed, and the safety and effectiveness of the particular modality of gene therapy has to be demonstrated in an animal model. @ Germline gene therapy is universally viewed as ethically unacceptable, whereas somatic cell gene therapy is generally viewed as being acceptable, as this is seen as similar to existing treatments such as organ transplantation. @ Ernbtyonic stem cells might be used therapeutically in a regenerative approach in which they are differentiated lz aitro to specialized cell types (or progenitors of the target specialized cells), and then transplanted in oioo to replace diseasedcells or tissues.Alternatively they could be used as delivery vehicles for gene-transfer technology.





'The mere existenceof the complete referencemap and DNA sequencedown to the last nucleotide mav lead to thc absurditv of reductionism - the misconceptionthat r,r'eknor, everl'thing it meansto be human or to thc absurdity of determinism - that what we are is a dircct and inevitable consequenceof w-hatour genomeis ' Victor McKusick (1991) Ethics is the branch of knowledgethat dealslr.ith moral principlcs, rvhich in turn relate to principles of right, \rrong, justicc and standardsof behavior.Traditionalll; the referencepoints arebased on a synthesis of the philosophical and religious views of rvell informed, respected,thinking members of society.In this tvay a codc of practice evolvesthat is seenas reasonableand acceptable by a majority and that often forms the basis for professional guidelines or regulations. It might be argued that there arc no 'absolutes'in ethical and moral debates.In complex scenarios, rvherethcre may be competing and conflicting claimsto an ethical principle, practical decisionsand actions often have to be based on a balancing of duties, responsibilitiesand rights. Ethics, like science,is not static but moves on, and in I'actthe development of the two disciplinesis closelyintertwined Ethical issues arisc in all branches of medicine but human geneticsposesparticularchallcngesbecausegeneticidentity impinges not just on an individual but also on rhe exrendedfamil1.,and on societv in general In the minds of the general public, clinical geneticsand genctic counseling can easill,be confused with eugenics- delined asthe scienceof improving' a specicsthrough breeding. It is important to stressthat the modern specialty of clinical gcneticshasabsolutelynothing in common with the appalling eugenic philosophiesthat were practiced in Nazi Germany and, to a much lesserextent,elsewherein Europe and the USA bett een the two world tvars. Emphasis has alrcadv been placed on the fundamental principle that genetic counseling is a non-directbe and n on-j udgmenta I communication proccss lvherebl. factual knowledgeis impartcd to facilitateinformed personalchoice(Ch. l7) Indeed, clinical geneticistshavebeenpioneersin recenrrrmes in practicing and promoting non-parcrnalism in medicine, and


5oloof the original budget for the Human Genome Project \\ xs set asidefor funding studies into the ethical and socialimplications of the knowledgegainedfrom the projcct Coercion and eugenics certainlv haveno placein modern medical genctics.

Nevertheless,this subjectlends itselfto ethicaldebate,not least becauseof the new challengesand opportunities provided bv discoveriesin molecular genetics.In this chapter some of the more controversial and difficult areas are considered It soon becomesapparentthat for many of theseissuesthere is no clearly right or wrong approach,and individual views will vary widel1,. Sometimesin a clinical setting the best that can be hoped for is to arrive at a mutuall.vacceptablecompromise, with an explicit agreementthat opposingviewsarerespectedand, personalconscience permitting, a patient's expressedrvishesare carried out. As genetictestingand DNA technologiesenter the mainstream of medicine,and awareness of the ethicalissuesgrowsand impacts on societr,,so there is a need for somerestrictionsand protections to be enshrined in law.This chapter therefore touches on some dcvelopments in this area. The Western rvorld is becoming increasingly familiar with courts of law making final decisions, for examplein relation to contentiousend-of-life issues,and this trend is likelv to continue.

PRINCIPLES GENERAL The time-honoredfour principlesof medicalethicsthat command wide consensusare listed in Box21.L Developedand championed by the American ethicistsTom Beauchampand JamesChildress, theseprinciples provide an acceptableframer,vork,although close scrutiny of many difficult dilemmas highlights limitations in theseprinciples and apparent conflicts betrveenthem Evervone involved in clinical geneticswill sooneror later be confronted by complex and challenging ethical situations,some of which pose particularly difficult problems u'ith no obvious solution, and certainlv no perfect one. Just as patients need to balance risks when making a decisionabouta treatmentoption, so the clinician/ counselor mav need to balancethese principles one against the other.A particular difficulty in medical geneticscan be the principle of autonomy,given that we all shareour geneswith our biological relativcs Individual autonomy needs sometimesto be weighed againstthe principle of doing good, and doing no harm, to close familv membcrs. The Beauchampand Childressframework of ethicalprinciples is, unsurprisingll', not the only one in useand others havedeveloped


Box24.1 Fundamental ethicalorincioles A u t o n o m y i n c o r p o r a t i nrge s p e c ft o r t h e n d i v i d u a tp r r v a c yt h ,e i m p o r t a n c eo f i n f o r m e dc o n s e n a t ndconftdentiality B e n e f i c e n c e- t h e p r i n cp t eo f s e e k i n gt o d o g o o da n d t h e r e f o r e a c t i n g n r h e b e s l i r t e r e s t so f t h e p d L r e n r N o n - m a l e f i c e n c e- t h e p r i n cp t eo f s e e k i n go, v e r a l ln, o tt o h a r m , r e n o t t o l e a v et h e p a t i e n it n a w o r s e c o n d i t i o nt h a n b e f o r et r e a t m e n t J u s t i c e - i n c o r p o r a t i nfga i r n e s sf o r t h e p a t i e n t n t h e c o n t e x o t fthe Lt r > U u l

LU> dvdlldutg.

^ ^ :' r lr r r r o c < eL]UlLy U

: n dt u u n pn rn' Jn tr

r- J rt n rI 'l y' ,

Framework Box24.3 The EthoxCentreClinicaLEthrcs (MikeParker) andotherfacts(eg famitydynamrcs, clinical 1 Whataretherelevant eelo-al nrart

by Mike Parkerof Oxford's Ethox Centre (Box 24 3), which builds on previous proposals.Taken together,these provide a practical approach to clinical ethics, which is an expanding discipline in health care. In practice,the issuesthat commonlv arisein the geneticsclinic during anv patient contact are outlined below.

AUTONOMY It is the patient who should be empoweredand in charge when it comesto decisionsthat have to be made.The degreeto which this is possibleis a function of the quality of information given. Sometimes patients are still seeking some form of guidance in order to givethem confidencein the decisionthey reach,and it will require the judgment of the clinician/counselor as to how much guidance is appropriate in a given situation. The patient should feel comfortable to proceed no further, and opt out if they wish at anv stageofthe process;this appliesparticularly in the context

'inre's' ! i v , v


v v T r - ,

d e c i s i o n - m a k i npgr o c e s s ? 2 W h a t w o u t dc o n s t i t u t a en appropriate . W h o l s t o b e h e t dr e s p o n s i b t e ? . W h e n d o e st h e d e c i s i o nh a v et o b e m a d e ? . W h o s h o u [ db e i n v o l v e d ? . W h a t a r e l h e p r o c e d u r a l r u l e(se g c o n f i d e n t i a t i t y ) ? 1 J

them into practical approaches.These include the Jonsen Framework (Box24.2) and the more detailed schemedeveloped



^! rL^ ^.,^ll^Ll^ >L U tC dvdttdutc

^^r:^^upUUt >

4 W h a t a r e t h e m o r a t l ys i g n i f i c a nf et a t u r e so f e a c ho p t i o nf;o r e x a m p t e o W h a t d o e st h e p a t e n t w a n t t oh a p p e n ? o l s t h e p a t i e n ct o m p e t e n t ? . l f t h e p a t l e n it s n o t c o m p e t e n tw h a t i s i n h i s o r h e r ' b e s L interests? . W h a t a r e t h e f o r e s e e a b tceo n s e q u e n c eosf e a c ho p t i o n ? 5 W h a t d o e st h e l a w / g u i d a n csea ya b o u te a c ho f t h e s eo p t i o n s ? b F o r e a c hr e a t r s t iocp t i o n r, d e n t i f tyh e m o r a Ia r g u m e n t si n f a v o ra n d a g an s t 7 C h o o s ea n o p t , o nb a s e do n , u d g n e n t o [ t h e r e l a t ' v em e r i L so f t h e s e arguments; . H o w d o e st h i s c a s ec o m p a r ew i t h o t h e r s ? o A r e t h e r ea n y k e yt e r m s f o r w h i c ht h e m e a n i n gn e e d st o b e a g r e e de , q b e s ti r t e r e s t ,p e r s o r ' ? 'va[id . Arethearguments ? o C o n s i d etrh e f o r e s e e a b lceo n s e q u e n c e(st o c aaI n d m o r e b r o a d ) . D o t h e o p l i o n sr e s p e c tp e T s o n ? s . W h a t w o u t db e t h e i m p l i c a t i o nos f t h i s d e c i s i o na p p te d a s a g e n e r a lr u l e ? B d e n t i f yt h e s t r o n g e s t c o u n t e r - a r g u m e n ttthoe o p t i o ny o u h a v e cnosen 9 C a ny o u r e b u tt h i s a r g u m e n t ?W h a t a r e y o u r r e a s o n s ? 1 0 M a k ea d e c i so n '11 R e v i e wt h i s d e c i s i o ni n t h e l i g h to f w h a t a c t u a t l yh a p p e n sa, n d l e a r n from t

of predictive genetictesting.

I N F O R M EC DH O I C E The patient is entitled to full information about all options available in a given situation, including the option of not participating. Potentialconsequences ofeachdecisionoption shouldbe discussed. No duressshould be applied and the clinician/counselor should not have a vested interest in the patient pursuing any particular courseof action

Box24.2 TheJonsonFramework: a practicaL approach to clinicalethics Indications for medicalintervention Estab[ish a diagnosis fortreatment Determrne theoptrons andtheprognoses foreach oftheopions lf so.whatdoeshe Preferences of patient- lsthepatient competent? n r < h c ' a n n i 7 l f n n r . n m n' o' rt' ce Ln!t

u , h : t i cJ i rn| r. h' a! n Aricni< hcql intara.t? /u.'L

'1, vvr,u-

Ouatityof tife- Wilttheproposed treatment improve thepatient s qualityof tife? Contextual features- Doreliqious, cultural or leqalfactors havean imn:et nn iho dorr e d r L r - +r f r, ^e ^| d- +L, U, -t^e u t Ule sLU0v -^-^---l

. W h o r s d o n g t h e s l u d y a n dw h e r e s i t b e r n gc a r r i e do u t ? o A v a i l a bLt y o f r e s u t t sa n d t h e i r m p t c a t r o n sf o r t h e i n d i v r d u a l a n d e x t e n d e df a m i l yr e g a r d t n gh e a l t h e , m p t o y m e nat n d l n s u r a n c e . A n o n y m i t yo f l e s t i n ga n d c o n fd e n t r a l i toyf r e s u l t s . n 1 n l o - r < ' o r , : n e nl f- l N Ar . d - n r s . r l e r . e n n t h p r - . o 5 e d , C - t prolects . P o t e n l i a l c o m m e r c i a l a p p loi cn as at n d p r o f i t

to ensurethat all aspectsof informed consentare addressedrvhen samplcsarc collectedfor geneticresearch.Just as signedconsent for genetictesting and storageof DNA has becomeroutine in the servicesetting (although not a legal requirement under the UK Human Tissue Act 200.1),similar proceduresshould bc adhered to in a researchsetting.

ETHICAL DILEMMAS INAWIDER CONTEXT Rcccnt progressin genetics,most notabl].in the areaof molecular tcsting, has brought thc ethical debateinto a much uider public arena.Topics such as insurance and DNA databases,patenting, gencthcrap\; populationscreening,cloning and stem-cellresearch are now rightlv vicwed asbeing of major societal,commercialand political importance, and perhaps not surprisinglv the.vfeature prominentlv in media discussion.All of thesesubtectsimpact on the specialtv of medical geneticsand each of these will now be consideredin turn.

GENETIC AS N DI N S U R A N C E The availabilitl of prcdictive tests for disordcrs of adult onset that conver a risk for chronic ill-health, r'viththe possibilit.vof a reduction in lifc cxpectancl,,hasgeneratedwidespreadconcernabout the extent to which the results of these tests should be revealed to outsidc agencies.Chief among these are the life insurance companies. In countries rvith only limited welfare medical servicesthis is also an issue for private health-care insurance, including critical illnessand disabilityincome.In addition,if either life or health insuranceis arrangedthrough an emplo-ver,then in


predictivc tcst rrill take out large policies without revealingtheir 'antiselection'or'adverse true risk status This is referredto as selection'.On the other hand, there is a ver-vreal fear among the gcnctics communitv that individuals rvho test positive will find themselvesr.ictims of discrimination, possiblyto the extent that they bccomeuninsurable This concernalsoextendsto thosewho have a famill' historv of a late-onsetdisorder, who might be refused insurancc unlessthel' undcrgo predictive testing. Cloncernsabout this ver-vrcal possibility that DNA testing 'genetic underclass' have led to the *'ill create an uninsurable introduction of legislation in manl' parts of the USA aimed at limiting the use of genetic information b1' health insurcrs This culminated in 1996 in President Clinton signing The Health Insurance Portabilit,-vand Accountabilitt' Act, r'vhich expresslv preyentscmplover-basedhealth plans from refusing coverageon geneticgrounds rvhena person changesemployment. In the UK this u'holearea\\'asconsideredin 1995b-vthe House of Commons S c i e n c ea n d T e c h n o l o g l ' C o m m i t t e e , w h i c h r e c o m m e n d e d thrrt a Human Genetics Advisor-v Commission be established to ovcrvier, dcvclopments in human genetics. In 1997 this Advisorl'Commission (non subsumcdinto the Human Genetics Commission) recommcnded that applicants for life insurance should not har,cto disclosethe results of anr genetic test to ir prospcctive insurer and that a moratorium on disclosure of genetic tcst results should last for at least 2years until genetic testing had been carefulll' cvaluated. the Associationof British Insurers (ABI) had a vieur Iner-itabl1., In thc 1999 revision of its Code of Practice. the ABI rciterated its r,iervthat applicantsshould not be askedto undergo genetic tcsting and that cxisting genetictest results need not be disclosed in applicationsfor mortgage-relatedlife assuranceup to a total of d100000 In 2005 the UK government negotiatedan agreement u'ith the ABI to extend restriction on the use of predictive genetic testsbf insurersto November 201I . The nelv document, entitled 'Concordat and Moratorium on Geneticsand Insurance',set out that no one r,ill be required to disclosethe result of a predictive gcnctictestunlesslirst approvedby-thegovernment'sGeneticsand InsuranceCommittee(GAIC) (seebelow).Disclosureis permitted on lif'einsurancegrcaterthan {500 000 and critical illness/income protection insuranceof more than {300000, which accountsfor less than 3%r of all policies. Genetic results generatedthrough researchstudiesdo not haveto be disclosedto insurers(Box 24.5).

theorv there could alsobe implicationsfor long-term careerprospects asa positive predictivc genetictest could lead to rvithdrawalofan

In the UK, the GAIC has been establishedwith a broad mcmbership draln from the insurance industrl., the medical communitv and intcrested public bodies. One of the chief remits of this committee is to evaluategenetic tests as they are developed, and the ABI has stated in its code of practice that

offcr of cmployment. The lifc insurance industrv is competitive and profit driven Privateinsuranceis basedon the concept of 'mutualitv', whereb-v risksarepooledfor individualsin similarcircumstances. In contrast, public health servicesare based on thc principle of'solidaritv',

gcnctic test rcsults should not be taken into account until each spccilic test has been fullv r.alidated. Thc issuesinvolved in genetictesting and the insuranceindustrv are not likely to be resolved easily.If'polygenes' conve-ving susceptibilitl' to common disorders of adult life are identihed,

rvhereb-vhealth provision for evervone is funded from general taxation. It is understandablethat the life insurance industry is concerncd that individuals rvho receivea oositive result from a

thcn almost thc entire population could find itself at the mercv of a prolit-driven, commerciallv focused, insurance industrr'. Among the g;eneticscommunit-vthere is a strong view that




Box24.5 Keypointsrnthe'Concordat and Moratorium o n G e n e t i casn dI n s u r a n cnee g o t i a t ebde t w e e tnh e U K Governmea nn t dt h eA s s o c i a t i oonf B r i t i s hI n s u r e r(sA B l ) /rnntr\ r A o o Lc a r l s n L r s t- l o ro e a s k e dl o l r d e ' g o p r e d r r iv e g e n e r r tce s t r r g o R e s l l t so f n p n p t. t p s t st h a t h a v eb e e nu n d e r t a k e ns h o u l db e d i s r n s p dr r n- n r r o ' l a n i n , l | l r 0 n l v t h e r e s u t t so f t e s t st h a t h a v eb e e nv a l i d a t e ds h o u t db e r a x e n ntoaccount o E x i s tn 9 g e n e l i ct e s t r e s u l t sn e e dn o t b e d l s c l o s e di n a p p tc a t i o n s f o r l r f ei n s L r r a n cneo L i c i ersr nt o a t o t a to f f 5 0 0 0 0 0 , a n d f o r c r i t i c a l i t l n e s s / i n c o mper o t e c t i o n l n s u r a n c eu p t o 1 3 0 0 0 0 0 r T h e r e s u t t so f g e n e t i ct e s t su n d e r t a k e nb y a n a p p t i c a nwt i t [ n o t b e l a k e ni n t oa c c o u nw t h e n a s s e s sn g a n a p p l r c aot n f r o m a n o l h e r i n d i vd u a t ,a n d v i c ev e r s a o C , e n p l ilce s t st r l . o n a s r t r t o i a p c c e q r r h< - .r l r re n n o t h : v e t Ob e d i s c l o s e dt o i n s u r e T s ' A g e n e t i ct e s t i s d e fn e d a s a n e x a m i n a t i o o nfthe chromosome, D N A o r R N A l o f i n d o u t i f t h e r e i s a n o t h e r w i s eu n d e t e c t a b lree l a t e d g e n o t y p ew, h i c h m a y i n d c a t ea n i n c r e a s e dc h a n c eo f t h a t i n d v i d u a l d e v e t o p i na g s p e c r f idc i s e a s ei n t h e f u t u r e

legislation is urgentll, needed to ensure that those tvho are geneticall)rdisadvantaged,through no fault of their own, do not face discrimination when seeking health-care or long-term life insurance.These are powerful argumcnts favoring retention of the principles of the UK National Health Service The possibility of government-controlled DNA databases for the population has also brought the insurance debate into focus. The use of DNA matching (fingerprinting) in criminal investigationsis now so sophisticatedthat there is a natural desireon the part of law enforcersto be ableto identify the DNA fingerprint for anyone in the general population. The existing databasein the UK, rvhich once contained material solely from sentenced offenders, is being expanded. For certain types of crime, whole sectionsof a communitl, are invited to come forward to give a sample of DNA so that the-vcan be eliminated from enquiries Bv the summer of 2003 one in 30 people in the UK alreadyfeaturedon the database.Large numbers of sampleshave been, or will be, collected as part of big population studies such asALSPAC (Avon Longitudinal Studv of Parentsand Children) or the UK Biobank project. Clearly,there is a need for safeguards to be built into the use that is made of DNA collectionslike these. and accessto them Debate will certainly continue on the use and misuseof personalgeneticdata.

GENE PATENTING ANDTHEHUMAN GENOME PROJECT The controversysurrounding the patentingofnaturally occurring human DNA sequences,be they complete genes or expressed sequencetags (ESTS), neatly encapsulatesthe conflict between


harsh commercial realism and altruistic academicidealism. On the one hand, biotechnologycompaniesthat haveinvestedheavill'

in molccularresearchcan arBuewith convictionthat both they and their shareholders are entitled to benefit from the fruits of their labors Biotechnology researchis indeed expensive,and it can reasonabl.v be argued that any commercial companyis entitled to a fair return on its investments Those who embracea more idealistic view havearguedthat the human genomerepresentshumankind's 'common heritage' and that information gained through the Human Genome Project, or other molecular research, should be freell' availablefor the benelit of everyone.Proponentsof this latter vierv frequentl-vcite alleged exploitation of patients and communities who havedonated their blood samplesfor research, little realizingthat their generositycould be exploitedfor financial gain.This is amply illustratedby the furore surrounding the proposed use (or abuse) of a centralized medical databaseof the entire Icelandic population to help identify 'polygenes' for potential commercialgain (p. l4l). There havealsobeen somehigh-profile court casesover the issuein the USA Given the complexit]' of the legal issues involved, it is not surprising that the international community has struggled to identifl, satisfactor.vsolutions. With the exception of the USA, most national regulatorl- bodies prohibit pa-vment for the procurement of human genetic material, but their r-iewson patenting are much lesswell defined. In the new millennium, the realitv is that many important human geneshave been patented, and companiessuch as Myriad Genetics in the USA sought to impose their exclusivelicencefor genetictesting for BRCAI and BRCA2 (p. 2ll). In fact, in 200,1the European Patent Oflice revoked the patent, denying Myriad a licence fee from ever1, BRCA test undertakenin Europe This test casehas highlighted translatlantic differencesover thesecontentious issues.In the short term the impact of gene patents will be mainly on single-gene disorders,and clearl-vif excessivecostsare imposed this will raise a direct challengeto the fundamentalethicalprinciple ofequity of access.In the longer term genepatenting could impinge directly on testing for common polygenic disordersand for treatment of both genetic and non-genetic problems, such as baldness and obesit1,,using gene therapy-.As illustrations of the prevailing levelsof investment,it is worth noting that the rights to one gene associatedwith obesitywere sold in 1995for $70 million, whereas in 1997DeCODE, the Icelandic genomicscompanyat the center ofthe controversyregardingnational assent,sold the potential rights to l2 genes,possibly associatedwith common complex diseases, to Hoffman-La Roche for 9200 million

THERAPY GENE One of the most exciting aspectsof recent progressin molecular biology is the prospect of successfulgene therapy (p. 3,12), although it is obviously disappointing that this potential has not yet been realized. It is understandablethat both the general public and the health-careprofessionsshould be concernedabout the possible side-effects and abuse of gene therapy. Frequent referencehasbeenmadeto the'slippery slope'argument,whereby it is claimed that one innocent concessionwill lead inevitably to uncontrolled experimentation.To addressthese anxieties,


adr,isorl or regulator-vcommitteeshavcbeenestablishedin several countries to assessthe practical and ethical aspectsof gene therapy researchprograms. Concern centers around nr,o fundamcntal issucs.The first rclatesto the practicalaspectsofensuring informed consenton the part of patientswho lvish to participatein genetherapv research Adult paticnts and parcnts of alfected children could wcll be despcrateto participate in gcnc therapy research,particularl-vif their diseascis otherwise incurablc Consequentlvthev could be temptcd to disregard the possiblehazardsof what is essentially a nerv,untricd and unproven therapcutic approach In the UK, the Committee on the Ethics of Gcne Therapv recommended that, until shorvn to be safe, all gene therapy programs should be subjectedto careful scrutinv bv local hospital researchethics committees. In addition, a national supervisorv bod1.,the Gene Therapl'Advisory Committee, has bccn establishedto reviervall proposalsto conduct genetherapv in humans and to monitor and record complications in ongoing gcnc therapv trials In this r,va1. it is anticipatedthat the rights of individual paricnrsin rerms of both consentand confidentialitv u,ill be safeguarded The second aspectof gene therapv that gencratesconcern is the possibilitl*that it could be used for cugenic purposes On this point thc British committee has recommended that genetic modification inr,olving the germline should not be attempted. Therefore, bv limiting gene therapy to somaric cells, it should not be possible for ncrvly modilicd genes to be transmitted to future generations.This committee has also rccommended that somaticccll gcnetherapvshould be usedonlv to try to treat serious diseases,and should not be used to alter human characteristics such asintclligenceor athletic prowess)nor usedin any sensethat could be cunslruedas cosmctic. Thc potential benchts of gene therapv are enormous and, althoughit is disappointingthat initial success hasbeenverv limited, it is ino'itrble that both somatic and germline gene therap-v$,ill continue to be the focus of intense researchactivit),.Thc degree of caution erercisedbv national committeesoverseeingthc ethics of gene therapr.illustratesthe care that is being taken b-r medical and gor.erningbodies to ensure that human gene manipulation rvill not be abuscd This is dcmonstrated by the halting of gene thcrxp\ trials at an American institute follorving the death of a ].oung man rvho rvas bcing treated for ornithine decarboxylasc deficienoi The US Food and Drug Administration promptlv suspcndcd this particular studv and halted all others pending further inr.estigations


Pilot studics assessingthe responsesto cystic fibrosis carrier screening in caucasianpopulations have yielded conflicting results(p. 74) The differing reccptions to thesevarious screeningprograms illustrate the importancc of informed consentand the difficulties and informed choice.For example,it of cnsuring both autonom.vis nolr,UK government polic.vfor cvstic {ibrosis (CF) screening to be introduced into the neonatalscreeningprogram, and this is currcnth being implemented Whilst aimed at identiff ing babies rvith CE, the scrccningwill inevitably detect a proportion who are simplv carriers.It has also been suggestedthat screeningfor CF carricr statusbe introduccd as an option for all schoolchildren at age 16]cars. Clearl-vnewborn infants cannot make an informed choice and it is doubtful whether all 16-year-oldsare sufliciently mature to make a fulll'reasoned decisionfor themselves. Consequentl,qmanl' pilot studies have fbcused on adults and their responsesto the offer of carrier testing, either from their generalpractitioner or at the antenatalclinic This has raised the vcxcdquestionofwhcther an offer from a respectedfamily doctor could bc interpreted asan implicit recommendationto participate thirt cannoteasily'be refused A personal'opportunistic' invitation to participate from a general practitioner -vieldsa much higher rcceptanceratc than a casualuritten invitation to attend for screening at a future date.This differencein ratesof uptakecould simply be a rcflcction of inertia, but could also indicate that individuals feel pressurizcdto agreeto a test that they do not necessarilywant. It is, therefbre,important that eventhe most well intended offer ofcarrier detectionshould be worded carefullvso asto ensurethat participation is entirclv voluntary Full counselingin the event of :r positiveresult is alsocssentialto minimize the risk of any feeling of stigmatizationor geneticinferiorit-v. In population scrcening confidentialitl'-is also important. \4an1-t'ill not rvish their carrier statusto be known b,vclassmates or collcaguesat \{ork The issue of confidentiality will become prrticularlr difficult for individuals who are found to be geneticall-v susccptibleto a potcntial industrial hazardsuch assmokeor dust. There is concern that such individuals will be discriminated againstbv potential emplovers (p. 359), and doctors could find themsclvcsin the invidious position of having to provide information that jeopardizestheir patient's emplovment prospects 'l'hese anricties led in 1995 to the Equal Emplol'ment Opportunitv Commission in the USA issuing a guideline that allorvs for an-vonedenied emplovment becauseof disease susceptibility to claim protection under the Americans with DisrrbilitiesAct

P O P U L A T I OSNC R E E N I N G Population screening programs offering carrier detection for common autosomal recessivedisorders have been in operation for manv 1.ears(p. 308). These have been well received for thalassemiaand Tay-Sachs disease,for rvhich screeninghas been carefull,v planned using well informed and highl-v motivated targct populations.In contrast, earlv efforts to introduce sicklccell carrier detcction in North America rverelargelv unsuccessful becauseof misinformation, discrimination and stigmatization.

ANDSTEMCELLRESEARCH CLONING 'Doll-v' the shcep,born in Jull' 1996 at Roslin, near Edinburgh, \\iasthe first mammal to be cloned from an adult cell, and when her existenccwasannouncedabout 6months later the world suddenly becameintenselv interestedin cloning. Doll-v was'conceived'b1'fusing individual mammarv gland cells rvith unfertilized eggs from rvhich the nucleus had been removed, and 277 attempts failed bcfore a succcssful pregnancy ensued. It rvas immediatelv




assumed that the technolog-ywould sooner or later lead to a cloned human being and there have been some unsubstantiated, almost certainlv bogus, claims to this effect. In fact, there has been rvidcspreadrejection of an1,desirability for human raprotluctiae

with a number of successfultreatmentshaving been pioneered If thesecould be harvestedand manipulated to the s me pltential as embryonic stem cells, those objecting to embryo researchr,vould be satisfied.Clearl1.,this remainsa contentiousand hotly debated

cloning, \rith strong statementsemanatingfrom politicians,religious leadersand scientists.Experiments with animals have continucd to havea ver]'poor successrate, and for this reasonaloneno rational person is advocating'experiments' in humans. In some cloned

ethical issue

animals the featureshave suggestedpossibledefectsin genomic imprinting. Dolly died prematurelyfrom lung diseasein Februarv 2003 and she had a number ofcharacteristicssuggestingshe rvas not biologically normal Lessons havc been learned from Dolly in relation to cell nuclear replacement(CNR) technologl.and this has,potentialll', openedthe door to understandingmore about cell differentiation The focus has thereforeshifted to therapeuticcloning using stem cells,and to the prospectsthis holds with respectto human disease. If stem cells were subjected to nuclear transfer from a patient in need, they might be stimulated to grow into an1'tissue tr-pe, perhaps in unlimited quantities and geneticallyidentical to the patient, thus avoidingrejection.The potential possibilitiesarelegion, and one can envisagenovel treatmcnt for Parkinsonor Alzheimer disease,mvocardial infarction, osteoporosisor se\-ereburns, to name but a felr,. The main ethical diflicultv ariscsin relation to thc source of stem cells Currentll,, the best sourceis believedto be emhr.yonic stem cells, and the UK Parliament moved swiftl-vto approve an extensionto rcsearchon early human embrvos for this purpose. Researchon human embryos up to 14day-sof age was alread--v permitted under the Human Fertilization and Embryology Act 1990 The UK has therefore become one of the most attractive placesto rvork in the stem-cell researchfield because,although regulated,it is legal.Publicly funded researchofthis kind cannotbc undertakenin either the USA or mainland Europe at the present time. Although legal, progresshas been painfulll. slow for those engagedin this work, and the whole held attractedverv negative publicity when the allegedbreakthrough of a Korean group, who claimed in earl-v2004 to have created cloned human embrvos for research,was found to be fraudulent. One major problem is the supply and quality ofoocytes for usein nuclearcell transfer.Research Broupsmust usually make do with relativelypoor-quality oocytes left over from infertility treatment. One group in the UK, Newcastle, has been granted a licence to collect frcsh eggs for stem-cell researchfrom egg donors, in return for a reduction in the cost of IVF treatment,a decisionthat rvasgreetedwith alarm and concern in some quarters.This group \ras also the first, in 2005, to createa human blastocystafter nuclear transfer. Those rvho obiect to the use of embr1,'onicstem cells believe it could be the lirst step towards reproductive cloning, and some consider it unethical becauseit treatsthe embryo rvith disrcspect and as a means to an end. In fact, the Human Fertilization and


Embryologv Act 1990permits thc creationof human embryosfor research,but only about 100 havebeen createdsince thc HFEA began granting licences.Those who object have further pointed out that significant advancesare being made on adult stem cclls,

CONCLUSION It is clear that cthical issuesare of major importance in medical genetics.Each new discover-vbrings new challengesand raises new dilemmas for u'hich there are usually no easyansrversOn a global scalethe computerizationof medicalrecords,togetherwith the rvidespreadintroduction ofgenetic testing, makesit essential that safeguardsare introduced to ensure that fundamental principles such as privacv and confidentiality are maintained. l,{embers of the medical genetics community will continue to play a pivotal role in trying to balancethe needsof their patients and families lr ith the demandsof an increasingl-vcost-conscious societyand a commerciallv driven biotechnologyindustry. Costbenefrt arguments can be persuasivein cold financial terms but take no account of the fundamental human and socialissuesthat are oftcn involved Increasingly-, it will fall upon the shouldcrs of the medical gcneticscommunity to trv to ensurethat the interests of their patients and families take precedence,and to$,ardsthat end it is hoped that this chapter,and indeed the rest of this book, can make a positir.econtribution.

READING FURTHER AmericanSocietyof Human GeneticsReport 1996Statementon informed conscntfor gcncticrcscarchAmJ Hum Genet 59: 171471 Thestatementof theAmericanSodet.yoJ'HumanCenetit:sBoard o.fDirettors relatingto int'irmetlconsentin geneticresearth on the issues AssociationofBritish Insurers1999Genetictesting ABI codeofpractice ABI. London A.fbrmil statementof thepnnciplesandpracttceadoptetl b.ythe British insuranceindustr.ynith regardto genetictesting. British N{edicalAssociation1998Human geneticsChoiceand responsibilityOxford University Press,Oxford A comprehenutenile-runging reportproducedby a BMA medicalethics nmmittee sleennggrouf on the ethttal issues raisel b.y;4enetitsin clinirul pracnte. Br.vantJ, Baggottla VelleL, SearleJ (eds)2002Bioethicsfor scicntists John Wiley,Chichester A multi aulhor tetl oJ nde scopemith many nntribtttions releaantto medical genellts

ClarkeA (ed ) 1997'Ihe genetic testing of children Bios Scientific, Oxford A comprehensixe multi-uuthor tett dealing mth thr important subjett Clothier Committee 1992 Rcport of the Committee on the Ethics of Gene Therapy HMSO, London Recommendations oJ the comminee charel b.y Sir Cecil Clothrcr on the ethical asfetts 0.f slmatic cell and germline gene thera!.y. Collins F S 1999 Shattuck lecture medical and socictal consequences of thc human gcnome project N EnglJ Med 3,11:28-37 A nntemplrary {)terrrew oJ the Human Genome Pro.jett ntth emqhasis on its 2ossibleethical and social tmplicunons l{arper P S, ClarkcAJ 1997 Genetics society and clinical practice Bios Scientific. Oxford

24 A thoughtfuluccountof the importantethicaland socialaspects0.frecent detelupments rn iiniral genetircHuman GeneticsCommission2002Inside information:balancinginterests in the useofpersonalgeneticdata DepartmentofHealth, London A detailedworhingparty reportby the Human GenehcsClmmhsion;oz'ering the useand abuseof personalgeneticinformation JonsenA R, SieglerM, WinsladeW J 1992Clinicalethics:a practical approachto ethicaldecisionsin clinicalmedicine(3rd edn) McGraw Hill, NewYork The keyre.ference that outlinestheJonsenJiameworbfor decisionmahingin clinical ethics Knoppers B M 1999Status,saleand patentingof human geneticmaterial: an international survey.Nature Genet 22: 23-26 An article pritten in the light of a laruJmarblegaland socialpolicy document, 'Du'ectit:e the 0n the Legal Prltection of BiotechnologyInxentions',Ji"omthe EuropeanParliament, 1998 KnoppersB M, ChadwickR 2005Human geneticresearch:emerging trends in ethics Nature Rev Genet 6: 75 79 An ourtien oJ'currentinternationalpolicies0n genepa,tenting. Mclnnis M G 1999The assentof a nation:genethicsand Iceland Clin Genet 55:234-239 raisedby the decisionoJ the A criticul reaiewofthe tomplexethical issues Icelandicgoxernmentt0 c0llablratein geneticresearchnith a biotechnolog.y nmpany. Nuffield Council on Bioethics1993Geneticscreening:ethicalissues Nuffield Council on Bioethics.London A ur1 helpful dotumentfor prortssionalguidance. Nuffield Council on Bioethics1998Mental disordersand genetics: the ethicalcontext Nuffield Council on Bioethics.London in the tontext 0J ment&l A Jurther detailedtlocumentdealingwith geneticissues health PokorskiRJ 1997Insuranceunderwriting in the geneticera.Am J Hum Genet 60: 205-216 testsbJtIhe surroundingthe useo.l'genehc A detailedaccountofthe issues insuranteindustry. Ro.valCollege of Physicians,Ro.valCollege of Pathologists,British Society of Human Genetics2006Consentand confidentialityin geneticpractice

guidanceon genetictestingand sharinggeneticinformation.Report of theJoint Committee on Medical Genetics.RCI RCPath, BSHG' London especiallyin conJidentialityissues, 4 detailedaorking 1)art.yre?ortth0t consitlers the contextof the Human TissueAct 2001

ELEMENTS Q ntUcal considerationsimpinge on almost every aspect of clinical genetics.In a wider context developments in molecular biology have important ethical implications for societyat large. problems @ Subjectsthat can generateparticularly difficult in clinical geneticsinclude prenataldiagnosis,predictivetesting in childhood and genetictesting in the extendedfamily. @ Possible applications of molecular genetics of importanceon a wider scaleinclude the use of genetictest results by the insurance industry, gene patenting' gene therapyand populationscreening. the @ Th.t. are no easyor correct solutions for many of clifficult ethical problemsthat arisein clinical genetics.It is important that guidelines and regulations are established that recognize each individual's fundamental entitlement to respectfor autonomy, informed choice and consent' privacy and confidentiality.



Websites andCtinical Databases

The rate of generationof inlormation about human, medical and clinical geneticsmeansthat accessto current information is \,ital to both the student and the doctot particularlvas patientsand familiesoften come ro the clinic armed with the sameinformation! There is a numbcr of generallvebsitesthat studentsrvill find uscful as entrv points, u.ith a wealthof links to other sitesfor ,surfing' A number of educationalwebsitcsis now available;manv include animateddiagramsto assistthe student Clinical geneticistsregularly use a number of expert databases to assist in the diagnosisof geneticdisordersand diseases, someof which are listed Other specializedwebsitesinclude mutation databascs,information on nucleotide and protein sequencesand current projects such as HapN{ap (p.112). Lastll, studentsmav find it ofinterest to look at rhc professionalsocieties' rvebsitcsas thev containmany usefullinks

UCSC GenorneBioinformatics http://genome ucsc.edu,/ Unit:ersit.yoJ Caltfornr,ant Santa Cruz genomebropser. Human Genome Organization http: //wrvw.hugo-international.orgl The websiteof the internationalorganizationof scientistsinvolvedin the Human GenomeProject InternationalHapX,IapProject http://wlvwhapmap org/ The pehste o.ftheprojectt0 map czmmln DNA xariants

MOLECULAR GENETICS WEBSITES GENERAL GENETIC WEBSITES Online MendelianInherirancein Man (OMIX,{) http://rvwu,.ncbinlm nih gov/omim/ Onlineatcessto MtKusick's mtalogue,an inxiluubb resourtefitr tlinical generic informationpith u peahh of links to man.yotherresottres. GeneticInterestGroup http://wwwgig orguk/ Website aJJittedwith genetic Jir allianceoJorgamzationssulforting 1)e0ple r|isorders. Gene tests http://www: genetests org/ IncludesuseJulretiens ofgeneticdisorders Orphanet http://www orpha net/ A pebsfiepith informationaboutrttre diseases, incluling manygeneliciisorders

HUMANGENOME WEBSITES Research Program on Ethical, Legal and Social Implications ofHuman Genome Project http: / /u'rvu,.nhgri nih gor: /PolicyEthics/ Site about ethrcal, legal and sociul implicutions oJ the Human Genome projett Genome Database http://rvwr*gdb orgl

Human GeneMutation Database http://wwlrhgmd cf.acuk/aclindex php A databaseoJ'thereportedmutationsxnhumangenes BROAD Institute http:,/,/rvlvu:broad mit edu/ Human genema!, sequencing and soJiwareltrogra,ms The RockefellerUniversity Genetic Linkage Analysis Resources http: //linkage rockefeller.edu/ Linkageanafusisand marhermappingpr0grdms MammalianGeneticsUnit and Mouse GenomeCentre http://nwu:mgu.har mrc ac uk,/ Mousegenomesite Drosophi Ia meI anogus ter Genome Database http://flybase bio indianaedu/ A comprehensixe database Jbr informationon thegeneticsand molecularbiology a/D. melanogaster,imluding thegenomesequente. Caenorhahditis elegazsGenetics and Genomics http: / /eleganssx.med.edu/genomeshtml C. elegansgenomeprojectinfurnation YeastGenomeProject http://mips gsf.delgenre/projlpast/index jsp Yeastgenomeprojectinformiltion


An entl,clopedm of the .urrent stole oJ'knonledg of lhe hunun genome


Ensembl Genomc Brorvser http: /,/wwu:enscmbl org/ Joint proyct betneen the Eurofeqn Bioinfitrmatits Societ.ynnd the Sanger Institute to prodde ann|tated euharyott( genomes

Dolan DNA Learning Centerat Cold Spring Harbor Laboratorv http://lvu.urdnalc orgl InJirmttion nhoulgtnts ittduratinn

George Mason University http: //www.ncc. gmu.edu/dna/ Tiutorial on DNA structure, replication, transcription arul translation. University of KansasMedical Center http:/ /www.kumc.edu/ gec/ For educatorsinterestedin humangeneticsand,the Human GenomeProject,

American Society of Human Genetics http://www.ashg.orgl

London Medical Databases http: / /www.lmdatabases.com/ Includ.esthe Winter-Baraitser Dysmorpholog Databasq the Baraitser-lllinter NeurogeneticsDatabaseond the London Ophthalmic GenetiesDatabase.

Human Cytogenetic Databaseand Catalogueof ChromosomeAberrations in Man http:/ /www.forschungsportal.ch/ unizh/ p772.hnn A diagnosticdatabaseoith features and rcferenceson nore than I 200 chr omosomaI abnormalities.

British Society for Human Genetics http: / /www.bshg.org.uk/ European Society of Human Genetics http://www.eshg.orgl Human Genetics Society of Australasia http: / /www.hgsa.com.aul




A. Abbreviationfor adenine Acentric. Lacking a ccntromere Acetylation. The introductionof an acetvlgroup into a moleculc;often usedbv thc bod1.to help eliminatesubsrances bt the lir,cr. Acrocentric.'ltrm usedto describca chromosomervhcrethc centromercls ncar onc cnd and the short arm usullly'consistsof satellitematcrial Acute-phase proteins. A number of proteinsinr,olvedin innatc immunitr, produccdin reactionto infection,including C-rcactir.eprotein,mrnnosc.binding protein and serum amvloidP component Adeninc A purinc basein DNA and RNA Adcnomatous polyposis coli (APC) SceFamilialadcnomatous pol.vposis AIDS. Acquired immune dcficiencl svndrome Allcle (= allelomorph). Altcr natir.efor m of a genefbund ar the samelocus on homokrgouschromosomcs. Allograft. A tissuegraft betrvccnnon-idcnticalindividuals Allotypes. Gencticalll'determincdvarianrsof antibodies Alpha (cl)-thalassemia. Inherited disorclerof hcmoglobininvolving underproductionof the cr-globinchainsoccurringmost cornmonrt.rn personsfrom South-EastAsra Alternative pathwa)'. Onc of the two pathwa\,sof the activationof complementrvhich,in this instance,inr.olvescell mcmbrirnesof mlcroorganrsms Alu repeat. Short repeatcdDNA scquences that appearto havchomologr. rvith transposablc clementsin other organisms Am.'lhc group of genetic\.arinnrsassociatcd rvith the immunoglobulin(Ig) A heavl-chain Amino acid. An organiccompoundcontainingboth carborvl ( COOH) and amino ( NHl) groups Amniocentesis. Procedurefbr obtainingamnioticfluid and cellsfor prenataldiagnosis Amorph. A mutationthat lcadsto completelossof lunction Anaphase. Thc stagcofcell divisionrrhcn the chromosomesleavcthe equatorialplateand migratc to oppositcpolesof thc spindle Anaphase lag. Loss of a chromosomcasit nor,csto the polc of the cell during anaphase; can leadto monosom\,. Aneuploid. A chromosomcnumber that is not an exactmultiple of the h a p l o i dn u m b e r ,i e 2 N - I o r 2 N i 1 r v h e r e N i st h e h a p l o i dn u m b e r o f chromosomes Anterior information. Information previouslrknolvn that lerrdsto the prior probabilitr Antibody (= immunoglobulin). A serum protein that is lormed in responscto an antigenicstimulusand renctsspccificalll.rviththat antigcn. Anticipation The tendenc_v for someautosomaldominrnt diseascs to manif'cstat an earlierageand/or to incrcascin ser.eritlwith each s u c c e e d i nggc n e r J t i o n Anticodon The complementarytriplct of the transfcrRNA (IRNA) molcculethat binds to it rvirh a parricularamino acid Antigen A substrncethat elicitsthc svnthesisof antibodl,rvith rvhichit spccificalhreacts Antigen binding fragment (Fab). Thc fragmcnrof the antibody.molccule producedbv papaindigestionresponsiblcfor antigcnbinding Antiparallel. Oppositeoricntationof the nro strandsof a DNA duplcr: o n er u n s i n t h e 3 ' t o 5 ' d i r c c t i o n t. h c o t h e ri n t h c 5 ' t o 3 ' d i r c c t i o n Antisense oligonucleotide. A short oligonucleotidcst'nrhesized to bind to a particularRNA or DNA sequenccto block its crpression

Antisense strand The templatestrandof DNA Apical ectodermal ridge. Area of ectodermin thc developinglimb bud that produccsgrowth frctors Apolipoproteins. A numbcr of differentprotcinsthat are involvedin lipid l r a n s p o r t r t i o ni n t h c c i r c u l a t i o n Apoptosis. Programmedinr.olutionor cell deathof a dcvelopingrissueor organof the bod1,. Artificial insemination by donor (AID). Use of semcnfrom a male donor asa reproductivcoption for couplcsat high risk of transmittinga geneticdisorder Ascertainment. Thc finding and selectionof familiesrvith a hereditary disordcr, Association. The occurrenceof a particularallelein a group of patients more often than can be accountedfor br, chance Assortative mating (= non-random mating). The preferentialselection of a spousewith a particularphenotirpe Atherosclerosis.The fattJ'degenerative plaquethat accumulates in the i n ti m e l u a l l o f b l o o dr c s s c l : Autoimmune diseases.Diseasesthat are thought to be causedby the b o d r n o t r e c o g n i z i nigt s o u n l n l i g c n s Autonomous replication sequences.DNA scquences that are necessarv f o r r e c u r r t cr c p l i c a t i o nu i t h i n r c a s t . Autoradiographl-. Dctectionof radioactivell.labeledmoleculeson an X-ray film. Autosomal dominant. A gcncon one of the non-ser chromosomesthat manifcstsin the heterozvgous state Autosornal inheritance. Thc patternof inheriranceshorvnby'a disorder or trait determincdby a geneon one ofthe non-scxchromosomes Autosomal recessive A genelocatedon one of the non-sexchromosomes that manifestsin the homozygousstate Autosome. Anv of the 22 non sexchromosomes Autozygositl.. Homozl'gosity.as a rcsult of identitl,b.vdescentfrom a common ancestor A z o o s p e r m i a . \ b s e n e co f s p e r mi n s c m c n B lymphocytes. Antibodl- producinglymphocltesinvolr.edin humoral lmmunlt\.. Bacterial artificial chromosome (BAC). An artificialchromosomc creatcdfrom modificationof the fertilitv factorof plasmidsthar allows incorporationofup to 330kb offoreign DNA Bacteriophage (= phage). A virus rhat infectsbactcria. Balanced polymorphism. Tr.o different genericvarianrsthat are stabli, presentin a population,i c. selectiveadvantages and disadr.antages cancel cachother out Balanced translocation SeeReciprocaltranslocation Bare lymphocyte syndrome. A rare autosomalrecessive ftrrm of severe combinedimmunodeficiencydue to abscnceof the classII moleculesof thc major histocompatibilitycomplex Barr body. The condensationof the inactiveX chromosomeseenin the nucleusof ccrtaint1-pesof cellsfrom femalesSecSexchromarin Base. Short for the nitrogenousbasesin nuclcicacid molecules(A, adenine; I thlmine; U, uracil; C, cvtosine,G, guanine) Base pair (bp). A pair of complcmentar\.bascsin DNA (A rvith T, G rvith C) Bayes' theorem. Combiningthe prior and conditionalprobabilitiesof certaincventsor the resultsof specificteststo givea joint probabilitvto dcrive the posterioror relativeprobabilitl'.

' .',,ri 6IO55ARY

Bence-Jonesprotein. The antibodi ofa singlcspeciesproducedin largc amountsb1 a personrvith multiple mvekrma,r tumor of antibodyproducingplasmacells Beta (B)-thalassemia. Inherited disorderof hemoglobininvolving underproductionof the B-globinchain,occurringmost commonlyin personsfrom thc mediterraneanrcgion and Indian subcontincnt Bias of ascertainment. An artifactthat must bc takcninto accountin family'studiesrvhenlookingat segregationratios,causcdbJ'families coming to attentionbecausethcv hare affectedindividual(s) Biochemical disorder. An inheritcd disorderinvoh.inga metabolic pathwa],i e an inborn error ofmetabolism Biological or genetic determinism.'l'hc prcmiscthat our generic make up is the only factor dctcrmining all aspcctsofour healthand discasc Biosynthesis. Use of recombinantDNA techniquesto producemolecules of biologicaland medicalimportancein thc laboratorvor commercirlll,. Bipolar illness. Affectivemanic-dcpressive illness Bivalent A pair ofsynapsedhomologouschromosomes Blastocyst. L,arl)'embrvoconsistingof embrvoblastand trophoblast Blastomere. A singlccell of the earlr'fertilizcd conceptus Blood chimera A mixture of cellsof diff'crcntgcncticorigin present in twins as a result of an cxchangeof cellsr.iathe placentabetwccn non-idcnticaltwtns in utero Boundary elemcnts. Short sequences of DNA, usuallyfrom 500bp to 3kb in size,that block or inhibit the influenceofregulatory elementsof adjaccntgenes Break-point cluster (bcr). Regionof chromosome22 involvedin the translocationsccnin the majority of personsrvith chronic mveloid leukemia C. Abbreviationfor cvtosine CAAT box A conserved,non-coding,so-called'promoter'sequenceabout 80bp upstreamfrom the start of transcription Cancer family syndromc A tcrm usedto describethe clustcringin certainfamiliesof particulartvpcs of cancers,in which it has been proposedthat the differentti'pesofmalignancl.couldbe due to a single dominantgcnc,specificallrLvnch tvpe IL Cancer genetics.The studv of the geneticcausesof cancer Candidate gene.A genelrhosefunction or locationsuggeststhat it is likelr to bc rcsponsiblcfor a particulargeneticdiseaseor disorder 5' Cap \lodification of thc nascentmRNA b-rthe additionof a methl'lated gutrninenucleotideto the 5' cnd of the moleculebJ'an unusual5' to 5' triphosphatelinkage CA repeat. A short dinucleotidescquenceprcscntas tandemrepeats at multiple sitesin the human genomc,producingmicrosatellitc polr morphisms p;ene;maleor femalefor Carrier. Personheterozvgousfor a recessive autosomalgenesor femalelbr X-linkcd gcncs Cascade screening. Identificationwithin a family of carriersfor an autosomalrecessive disorderor pcrsonsrvith an autosomaldominant genefollouing ascertainment of an indcx case Cell-mediated immunity'. Immunit-vthat involvesthe T l.vmphoc.vtes in hghting intracellulirrinfection;is alsoinvolvedin transplantation rcjceti,rn rn.l .lchi e.l hr pcrscnsitirit1. Cellular oncogene. SeeProto-oncogene Centimorgan (cM). Unit usedto measuremap distances, equivalentto a l0.16 chanccof rccombination(crossingover) 'fhe conccptthat gcneticinformationis usuallv Central dogma transmittedonlv from DNA to RNA to protein Centric fusion. The fusion of the centromeresof tu.oacrocentrie chromosomesto form x robertsoniantranslocation Centriole. The cellularstructurefrom which microtubulesradiatein the mitotic spindleinvolvedin the separationof chromosomes ln mltosls Centromere (= kinetochore). The point at which the trvo chromatidsof a chromosomeare joined,and the region of the chromosomethat becomes attachcdto thc spindlc during ccll division Chemotaxis The attractionof phagocvtesto thc sitc of infectionb.v componentsof complement Chiasmata. Cross olers betu'eenchromosomes in meiosis Chimera An individual composedof two populationsof cellsrvith diff'erentgenotl'pes Chorion. Lalcr of cellscoveringa fertilizedovum, someof u'hich (the chorion frondosum)rvill later form the placenta

guidance Chorionic villus sampling. Procedureusing ultrasonographic to obtainchorionicvilli from the chorion frondosumfor prenatal diagnosis Chromatid. During cell dir.isioneachchromosomedivideslongitudinally into two strands,or chromatids,which areheld togctherby the centromcrc of the chromosomcs Chromatin. The tertiarl coiling of the nucleosomes u'irh rssociatedproteins Chromatin fiber FISH. Usc of extendedchromatinor DNA fibcrsrvith fluoresccnt in-situh,v6ridizarion(FISH) to order physically DNA clones or sequcnces Chromosomal analysis. The processof countingand analyzingthc of an individual bandingpatternof the chromosomes Chromosomal fragments. Acentricchromosomesthat can ariscasa resultof scgregationof a paracentricinversionand that areusuall.v incapableof replication Chromosome instability. The presenccof breaksand gapsin the with chromosomesfrom personsrvith a number of disordersassociated an increasedrisk of neoplasia. Chromosome mapping. Assigninga geneor DNA sequenceto a specific chromosomeor a particularregion of a chromosome Chromosome-mediated gene transfer. The techniqueof transferring to somaticcell hybrids to enable or parts of chromosomes chromosomes more detailedchromosomemapping. Chromosome painting. The hybridizationiz srlz of fluoresccnt-labeled probesto a chromosomepreparationto allowidentificationof a particular chromosomc(s) Chromosome walking. Using an orderedassembli,of clonesto cxtend from a knrxvnstart point Chromosomes. Thread likc, darkly stainingbodiesrvithin the nucleuscomposedof DNA and chromatinthat carrv the genetic infcrrmation elementsin the promoterregion that act on gencs C/-s-acting.Regulatory' on the samcchromosomc Class switching. Term usedfor the normal changcin antibodyclassfrom lg\{ to IgG in the immune response Classic gene families. Multigene familiesthat shorva high degreeof sequencchomology Classic pathway. One of the two wa.vsof activationof complement,in this complexes instanceinvolr.ingantigcn-antibodJ' Clone. A group of cells,all of which arederivedfrom a singleccll by repeatedmitoses,all havingthe samegeneticinformation of clonesthat havebeenmappedand orderedto Clone contigs. Asscmbl-v producean ovcrlappingarray. Cloning in silico. The useof a number of computerprogramsthat for sequencehomology can searchgenomicDNA sequenccdatabases specificto all genessuch to known genes)as rvellasDNA sequences rs the conscrvedintron/cxon splicejunctions,promotersequencesr poll'adcn1''lation sitesand stretchesof open-readingframes(C)RFs)to idcntif.vnovelgenes cM. Abbrer.iationfor centimorgan Co-dominance. When both allelesare expressedin the heterozygote Codon. A sequenceof threeadjacentnucleotidesthat codesfor one amtno acid or chain termination Common cancers.The cancersthat occur commonlyin humans,such as boweland breastcancer that occur commonlvin humans,e g Common diseases.The diseases diabetes,etc crnccr,coronar)'artery disease, Community genetics.The branchof medicalgcneticsconccrnedwith on a populationbasis. screeningand the prcventionof geneticdiseases Comparative genomics. The identificationof orthologousgencsin differentspecies Compctent. Making bacterialcell membranepermeableto DNA by a variety'of differentmethods,including exposureto certainsaltsor high voltagc Complement. A seriesof at least10 serum proteinsin humans(and other 'classic'or the 'alternative' vcrtebrates)that can be activatedb.vcither the aboutthe destructionof to bring pathlvay'and that interactin sequence cellularantigens Complementary DNA (cDNA). DNA svnthesizedfrom mRNA by the enzvmcreversetranscrlptase Complementary strands. The specificpairing of the basesin the DNA of the purinesadenineand guaninewith thymine and cvtosine




Complcte ascertainment. A term uscdin segregationanalysisfor a tvpc of study that identifiesall affcctcdindividualsin a population Compound heterozygote. An indir.idual rvho is affected with an autosomalrcccssivedisordcrhavingtwo dilfercnt mutationsin homologousgencs Concordance. When both membersof a pair of twins exhibit thc samc trait they are saidto bc concordant If onlv one tivin hasthe trait they arc saidto bc discordant Conditional knockout. A mutation that is expressedonly undcr certain conditions,e g raiscdtemperature Conditional probability. C)bservations or resrsthat can be usedto modif.v prior probabiliticsusing ba1'esian calculationin risk estimations Conditionally toxic or suicide gene. Genesthat are introduccdin gene therap-rand that, under certainconditionsor after the introductionofa certainsubstance, rvill kill the cell Confined placcntal mosaicism. The occurrenceof a chromosomal abnormalitv in chorionic villus samplesobtainedfor first trimester prcnatal diagnosisin rvhichthe fetushasa normal chromosomalcomplement Congenital. Anv abnormalitl l.hethcr gcneticor not, thlt is prcscntat birth Congenital hypertrophy of the retinal pigment epithelium (CHRPE). Abnormal retinal pigmentationthat, rvhenpresentin persons at risk for familial adenomatous polvposis,is evidenccof the heterozvgous state Coniugation A chemicalprocessin rvhichtwo moleculesare joined, often usedto describethe processbl lvhich certaindrugs or chemicals can then be excretedb-vthe bodl; e g acctvlationof isoniazidbv thc liver Consangrrineousmarriage A marriagebetlvecn'blood relatives', that is, betweenpcrsonswho havcone or more ancestorsin common, most frcquentl)'bctweenfirst cousins Consanguinitl'. Relationshipberrveenblood relatives Consensus scquence.A GGGCGGG sequcnccpromoterelementto thc 5'side ofgenesin eukarvotesinvoh.cdin the control of'gcnccxpression Conservative substitution. Singlebasc-pairsubstitutionthat, although resultingin thc replacemcntbv a differcnt amino acid, if chemicall-r. similar,hasno funcrionaleflect Constant region. The portion of thc light and heavychainsof antibodies in which the amino-acidsequenceis rclativelyconstantfrom moleculeto molecule Constitutional. Prcsentin the fcrtilized samete Constitutional heterozygositv.'l'h. prcicncein an intlividualat the rime of conccptionof obligatehetcrozvgositvat a locusrvhcnthe parentsare homozvgousat that locusfor diflerent alleles Consultand. Thc personprcscntingfor geneticadvice Contigs. Contiguousor overlappingDNA clones Contiguous gene syndrome. Disorderresultingfrom dcletionof adjacent genes Continuous trait. A trait, suchas height,for which thereis a rangc of observations or 6ndings,in contrastto traits that areall or none (cf. discontinuous),suchas cleft lip and palatc Control gene.A genethat can turn other gcneson or off, i e rcgulate Cordocentesis The procedurcofobtaining fctal blood samplesfbr prenataldiagnosis Corona radiata. Ccllularlaversurroundingthe matureoocvte Cor pulmonale. Right sidedheart failurethilt can occur after seriouslung discase,such as in personsrvith cvsticIibrosis Correlation. Statisticalmeasureof the degreeof association or resemblance betweent\yo parameters Cosmid. A plasmidthat hashad rhe maximum DNA removcdto allorv the largestpossibleinsert for cloningbut srill hasthe DNA scquences neccssarvftr in-tin'o packaginginto an infectivephageparticlc Co-twins. Both mcmbersof a twin pair,rvhethcrdizvgoticor mr)noz\gotic Counselee.Personreceivinggeneticcounseling Coupling. \\ihen a certainallcleat a particularlocusis on the same chromosomcrvith a spccificalleleat a closelvlinked locus CpG dinucleoti