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GeneticdiseasesrevealDNAnucleotideexcisionrepair201005akDrugsAgainstCancer:StoriesofDiscoveryandtheQuestforaCureKurtW.Kohn,MD,PhDScientistEmeritusLaboratoryofMolecularPharmacologyDevelopmentalTherapeuticsBranchNationalCancerInstituteBethesda,Marylandkohnk@nih.govCHAPTER22GeneticdiseasesrevealDNAnucleotideexcisionrepair.RareinheriteddiseasesrevealDNArepairgenesandhowcellscope.By1977,sevengeneticallyinheritedconditionswereknownorsuspectedtohaveDNArepairdefectsandtomaketheunfortunatechildpronetodevelopingcancer(Setlow,1978)(Table22.1):•Xerodermapigmentosum(nucleotideexcisionrepair(NER))(Chapters22and23);•Cockayne’ssyndrome(transcription-coupledNER)(Chapter23);•Fanconi’sanemia(DNAcrosslinkrepairdefect)(Chapter…);•Ataxiatelangiectasia(Chapter…);•Li-Fraumenisyndrome(Chapter…)•Bloom’sprematureaging(progeria)syndrome(unstablegenome)(Chapter…).•Werner’ssyndrome(adultprogeria)(Chapter…)ItisremarkablehowstudiesofcertainrarediseaseshelpedunravelthecomplexitiesofDNAdamagerepair,thefactorsthatpredisposetocancer,andthemoleculartargetsfordrugtreatmentstailoredforthemoleculardefectsinindividualcancers.Table22.1lists7geneticdiseasesthatledtothediscoveryofgenesfortherepairofcertaintypesofDNAdamageorthathelpedcellsinotherwaystosurvivethedamage.Thecurrentchapterrelatesthestoriesofthefirsttwoonthelist(xerodermapigmentosumandCockaynesyndrome);theotherswillbethesubjectoflaterchapters.

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Table22.1.Geneticdisease Defectiveprocess DefectivegenesXerodermapigmentosum(XP) Nucleotideexcisionrepair XPA-XPG;XPVCockaynesyndrome Transcription-coupledrepair CSA,CSBFanconi’sanemia Inter-strandcrosslinkrepair FANCgenesAtaxiatelangiectasia Cellcyclecheckpointactivation ATM,ATRLi-Fraumenisyndrome “Guardianofthegenome” TP53Bloom’ssyndrome Prematureaginginchildhood BLMWerner’ssyndrome Prematureaginginadult WRNTheXerodermapigmentosum(XP)story:acurseandaclue.Theesotericname,xerodermapigmentosum(orsimplyXP),becamecommonparlanceamongresearchersandcliniciansdealingwithskindiseasesandcancer.Likewise,thenamewillappearsomanytimesinthechapterthatthereadermayfinditenterhis/herfamiliarvocabulary.Literally,xerodermapigmentosummeans“pigmenteddryskin,”butthatbenign-soundingnamedoesnotmatchtheseverityofthedisease.Childrencursedwiththisterrible,butfortunatelyveryrareinheriteddisease,cametobecalledchildrenofthedarkandevenvampirechildren,becauseforthemdaylightwasdeadly.Iftheyfailedtoadherestrictlytoadisciplineofthedark,theyhadlittlechanceofsurvivingbeyondtheageof20.Theirquandaryofhowtomeldtheirextremelightsensitivitywiththeirsociallivesbecamethesubjectofseveralfilms(“ChildrenoftheDark”–TVmovie1994.Itmaynothaveconsoledtheseunfortunateyoungpeopleverymuchtoknowthattheirsufferingwouldleadtonewknowledgethatwouldhelpothers,includingmanycursedwithcancer.Childrenwithxerodermapigmentosum(XP)tendtogetfreckle-likepigmentationinsun-exposedskin,severeburnsafterminimalsunexposure,anddamagetosun-exposedpartsoftheeyeswithlossofvisionandocularcancer.Theirgreatestriskofsunexposure,however,ispotentiallydeadlyskincancers.Atleast8subtypesofthediseasewerediscoveredthathavedifferentdiseaseseverities.Themedicalliteratureoftenshowspatientswithadvancedstagesofthedisease.Sinceitaffectsdaylight-exposedskin,itsterribleeffectsareexposedtoview.Theworstofit,however,ishidden:someoftheskinnodulesbecomemalignantcancersthatspreadtoorganswithinthebody.Xerodermapigmentosumisoneoftheworstcancer-proneconditions,anditbecamethemostextensivelystudieddiseaseinallofcancerresearch.Thediseasewassorare,thateveryavailablepatientafflictedwithitwasstudied.Theadvancedcasescommonlyshowninthemedicalliteraturelooksobad,thatIhaveselectedanearlyandrelativelymildcasetoshowinFigure23.1(Anderson,1889).ThediseasewasfirstdescribedbyHebraandMorrisKaposiin1874,whocoinedthename“xerodermapigmentosum”in1882.ThecaseinFigures22.1and22.2datesbackto1889,whenitsinheritednaturewasknown,butitscausewasstillcloudedinmystery(Anderson,

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1889).Uptothattime,only44casesofthediseasewereknown.ThiscasewasreportedbyT.McCallAndersonoftheUniversityofGlasgowintheBritishMedicalJournal.Itwasofa9-yearoldboy,who,despitehisskinlesionsandthelossofhislefteye--whichwasremovedbecauseithadatumorgrowingonit--stilllookedtobeotherwiseingenerallygoodhealth.YoucanseefromFigure22.1thathisskinlesionsweremainlyontheface,neck,uppershoulders,andlowerarms:thepartsofhisbodythatwouldhavebeenmostexposedtothesun.Microscopicexaminationofhistumorswasnotedtobetypicalofepithelioma(epithelialcancer)(Figure22.2).Hisparentswerebothingoodhealth,buthisonlysisterwassimilarlyaffectedandhaddiedattheageof9.Thiswasconsistentwiththeknownrecessiveinheritanceofthedisease.Theexactgeneticshoweverwereconfusing;wenowknowthereasonfortheconfusion:itwasbecausethedisease,withclinicalvariation,canbecausedbymutationofanyoneofseveralgenes,locatedondifferentchromosomes(Table22.1).

Figure22.1.Woodcutofa9-yearoldboywithxerodermapigmentosum(Anderson,1889).Thechildhasmultiplecancersonhisfaceandhislefteyewasremovedbecausetherewasacancerinit.Unlessprotectedagainstsunlightorevenagainstdaylight,hisconditioncouldbecomemuchworse.Atthetimethispicturewasmade(circa1889),littlewasknownaboutthedisease.Pigmentation(darkening)isalsoshowninthesun-exposedareasaroundhisneckandthelowerpartsofhisarms.

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Figure22.2.Amicrograph(x250)ofaskintumorfromthepatientinFigure22.1(Anderson,1889).Thecentralportion(labeledAinthemicrograph)wastypicalforasquamouscarcinomawherethecancerretainsabizarrememoryofthearrangementofthemultilayeredcellsoftheouterskin.Thecancercellsshowedtheusuallargeirregularnuclei,comparedwiththefibroblastsinthestroma(labeledB)withinwhichthetumorsaregrowing.Abloodvesselisseenontheleft.Acaseofxerodermapigmentosumina10-yearold,alsopublishedin1889intheBritishMedicalJournal,notedtheroleofsunexposurebytheunusualinvolvementofthischild’sfeetandlowerlegs(Hunter,1889):

Itmayberemarkedherethatthesechildrenwenttoschoolduringthesummermonthswithoutshoesorstockingsandthetrousersoftenrolledup.Thelatterisdonesoasnottointerferewithrunningorjumping,andinwetweathertokeepthem(thetrousers)clean.Thisisacommonhabitinthecountrydistricts,andthus,inthesecases, the feetand legswhichareaffectedwerealsoexposed (Hunter,1889).

Thesetworeports(Anderson,1889;Hunter,1889),bothpublishedintheBritishMedicalJournalin1889andavailableontheinternet,giveadetaileddescriptionoftheskinlesionsandthecourseofthediseasein4patients.Theseearlyreports,whilelittlewasasyetknownaboutthedisease,showwhatphysiciansof~130yearsagosawandexperienced.Next,wemoveaheadabout78years.Severaldifferentgeneswerebythenknownthatcausedifferentclinicalmanifestationofthedisease,althoughallofthementailedskinlesioncausedbysunlight.Thecellsfrompatientshavingmutationsinaparticulargenewerereferredtoasa“complementationgroup”becausethenormalversionofthe

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respectivegenecouldcurethedefectinthecorrespondingmutantcells.Thedefectmeasuredwas“unscheduledDNAsynthesis”whichwillbeexplainedinamoment.Acomplementationgroup,incommonparlance,becamenearlythesameasreferringtoaparticularmutantgene(Table22.2).Itwas1967Ithink,inasmallconferenceintheNIHClinicalCenter;wehadaguestspeaker.ItwasJamesE.CleaverfromtheUniversityofCalifornia.HeclaimedtohavedetectedadefectinunscheduledDNAsynthesisincellsfrompatientswithxerodermapigmentosum.Someofuswereskeptical,becausewehadexpectedthatphenomenon,butnoonehadyetbeenabletodemonstrateit.Moreover,hisdataseemedtobebarelyabovethenoiselevel.Withinayearorso,however,hepublishedmoreconvincingdata(Cleaver,1968)andtheskepticsweresoononthebandwagonastheirinvestigationscorroboratedandextendedCleaver’sclaims(KraemerandDiGiovanna,2015).Sonow,whatis“unscheduledDNAsynthesis”andhowisitrelatedtoDNArepair?Well,ourconceptwasthatcellswhoseDNAwasdamagedbyultravioletlightwouldbeundergoingasmallamountofDNAsynthesisforrepairafterthedamagewascutout.Thiswouldbeoccurringevenincellsthatwereresting,i.e.,notcyclingincelldivisionandhencenotundergoingthenormalDNAsynthesisphase.Normally,thosecellswouldnotbesynthesizingDNA.ButtorepairDNAdamage,eventhosecellswouldbeundergoingasmallamountofunscheduledDNAsynthesis:itis“unscheduled”becauseitwouldbeoccurringevenifthecellsarenotinthecelldivisioncycle.WehadreasontobelievethatcellsfromxerodermapigmentosumcellsweredefectiveintheirabilitytorepairDNAdamagecausedbyultravioletlight–orbysunshine.Therefore,theymightnotexhibitunscheduledDNAsynthesis,asCleaverhasshowntobethecase.Toputitinanotherway:exposingnormalskintoultravioletlightinducesDNAdamage,whichispromptlyrepairedbyaprocessthatinvolvesasmallamountofDNAsynthesistoreplacethedamagedDNAregionsthatwerecutout.ThisDNAsynthesiswouldbeoccurringevenincellsthatwerenotindivisioncycleandhencewouldnotordinarilyundergoanyDNAsynthesis.SincethatDNArepairsynthesiswouldbeoccurringinquiescent(i.e.non-dividing,non-cycling)cells,itwastermed“unscheduled.”XerodermapigmentosumpatientswerepresumedtobedefectiveinsuchDNA“excision”repairandthereforewouldbeunabletocarryoutunscheduledDNAsynthesis.TheinabilityofskincellsfromxerodermapigmentosumpatientstoexhibitunscheduledDNAsynthesisinresponsetoultravioletlightwaslaterconfirmeddirectlyinpatients(Figure22.4)(Epsteinetal.,1970).Asmallregionofthepatient’sskinwasirradiatedwithultravioletlight;then,tritium-labeledthymidinewasinjectedundertheirradiatedportionofskin,whichwasthenbiopsiedandradiographedtoshowthecellsthathadincorporatedtheradioactivethymidineintoDNA(Figure22.4).Idonotknowwhetherthepatientshadgiveninformedconsentforthisprocedureofinjectingradioactivematerialintotheskin–itmightnotyethavebeenroutinelyrequiredtobedocumented.

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Figure22.3.JamesEdwardCleaver(1938-),discovererofthemolecularprocessthatisdefectiveinthegeneticdisease,xerodermapigmentosum.ThiswastheseminaldiscoveryofahumanDNArepairdeficientdisease.

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Figure22.4.SkinfromaxerodermapigmentosumpatientisunabletorepairDNAdamagecausedbyultravioletlight.TherepairrequiresasmallamountofDNAsynthesistoreplacethedamagedDNAregions.ThesestudiesdemonstrateddefectiveDNArepairsynthesis(“unscheduledDNAsynthesis”)inskinofxerodermapigmentosumpatients.Left,normalskin:noradioactivitygrains.Middle,irradiatednormalskin:manycellsshowradioactivitygrains,indicativeofunscheduledDNAsynthesis.Right,irradiatedxerodermaskin:nounscheduledDNAsynthesis(aheavilylabelledcellisundergoingreplicativeDNAsynthesis)(Epsteinetal.,1970).AnunusualvariantofXPisdiscovered.Asoftenhappensinscience,anewdiscovery,whenfurtherinvestigated,becomeschallengedbyfindingsthatdon’tfittheoriginalconcept.AndsoitwasthatthefourthNIHXPpatienthadthehighlightsensitivityoftheskin,butshowednodefectinunscheduledDNAsynthesis.Aboutthesametime,JimCleaverattheUniversityofCaliforniainSanFranciscoreported3moreXPpatientswhosecellshadnormalunscheduledDNAsynthesisafterbeingexposedtoultravioletlight(Cleaver,1972).ThosepatientsdefinedavariantofXPthathadnormalnucleotideexcisionrepair(NER);thisvariantofXPbecameknownasXPV.ThedefectinXPVturnedouttobeamutationinaspecialDNApolymerase,knownasPol-etaorPolH,whichisneededtocompletethesmallamountofDNAsynthesisacrossthedefectleftbehindafterNERremovedtheoffendingthyminedimer(or,moregenerally,pyrimidinedimer)(DiGiovannaandKraemer,2012).Cleaverlaterremarkedthat,ifhisfirstpatienthadbeenoftheXPVtype,hemighthaveerroneouslyconcludedthatXPhadnormalDNArepair(KraemerandDiGiovanna,2015).Subtypesofxerodermapigmentosum:complementationgroupsEvenamongXPpatientswhosecellsweredefectiveinunscheduledDNAsynthesis,therewasconsiderablevariabilityinhowseverethediseasewas,howsensitivethecellswereto

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bekilledbyultravioletlight,andtheclinicalpictureingeneral.Particularlypuzzlingwasthatthediseaseofsomepatientshadneurologicalsymptoms,sometimesquitesevere.Anextremecaseofthelatterwasasyndromethatdeserveditsownname:DeSanctis–Cacchionesyndrome.Itisoneoftherarest,mostsevereformsofxerodermapigmentosum(XP).Inadditionbeinghighlysensitivetodaylight,theseXPpatientsareofshortstatureanddevelopprogressiveneurologicdegeneration.ThesyndromewasfirstrecognizedbydeSanctisandCacchionein1932,whodescribedthreebrotherswithXPwhohadmicrocephaly,mentaldeficiency,dwarfism,gonadalhypoplasia,progressiveneurologicdeterioration,deafness,andataxiabeginningattheageof2years.Thisnewsyndrome,thattheseunfortunatechildrenhad,wasdescribedin1932inanobscureItalianjournal:deSanctisC.,CacchioneA.L’idioziaxerodermica.RivSperFreniatrMedLegAlienMent.1932;56:269–292.WhatcausedthesedifferenttypesofXPcouldonlybeinvestigatedafternewmethodsandconceptsweredeveloped,whichtookseveraldecades.Thestorybegantounfoldin1971whenåDirkBootsma(Figure22.5),aProfessorofGeneticsatErasmusUniversityinRotterdam,thoughtthatthelargedifferencesintheclinicalpictureofXPpatientsmightbeduetodifferentgenes,eachofwhich,whenmutated,wouldcauseaparticularclinicalformofthedisease.Hereasonedthat,iftwoofthosedefectivegenes,eachonadifferentchromosome,wereputintothesamecell,theymightcomplementeachotherandrestorenormalunscheduledDNAsynthesis.HewantedtotestthatideausingcellsfromtwoverydifferentformsofXP:theclassicvarietyandtheDeSanctis–Cacchionetype.Buthowcouldoneputthepresumeddifferentdefectivegenesintothesamecell?Bootsmaandhiscolleaguesdevelopedamethodthatusedtheabilityofcertainviruses(inactivatedSendaivirus)tofusecellstogethertoproducecellsthatsometimeshadtwonuclei.However,althoughsomeofthebinucleatecellshadanucleusfromeachoftheXPtypes,oftentimestheyhadnucleifromthesametype.Inordertodistinguishwhosedifferentcases,theyusedaclevertrick:theycombinedcellsfromamalepatientwhohadoneXPtypewithcellsfromafemalepatientwhohadtheotherXPtypeofthedisease.TheythenexposedthecellstoUVtoproduceDNAdamage.Bycarefullyexaminingthechromatinineachnucleusofabinucleatecell,theycouldtheycouldtellwhetheritcamefromamaleorfemalecell.Theyindeedfoundthatthoseandonlythosebinucleatecellsthathadbothamaleandafemalenucleus–onenucleusfromeachXPtype--complementedeachothertorestoreunscheduledDNAsynthesis(Figure22.6)(DeWeerd-Kasteleinetal.,1972).Otherinvestigators,particularlyKenKraemeratNIH(Figure22.7),thenjumpedinand,usingamodifiedmethod,foundthattherewereinfactseveraldifferentcomplementationgroupsamongtheXPpatients,theircells,andtheirmutatedgenes(Table22.2).Thegenethatwasmutatedineachcomplementationgroupwasclonedanditsfunctionsdetermined.Quiteremarkably,theproteinsproducedbyalloftheseXPgenewerefoundtoworktogethertorepairDNAdamagebyaveryimportantmechanism:DNAnucleotideexcisionrepair(NER).Howthismechanismworksisthesubjectofthenextchapter.

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Figure22.5.DirkBootsma(1936-),aProfessorofGeneticsatErasmusUniversityRotterdam,Netherlands,developedacell-fusionmethodbywhichhediscoveredxerodermapigmentosumcomplementationgroups.(FromNedTijdschrGeneeskd200212oktober;146(41).)

Figure22.6.HowBootsmaandhiscolleaguesshowedthatageneresponsibleforwhatwasthencalledtheclassictypeofXPandageneresponsiblefortheDeSanctis–CacchionetypeofXPcomplementedeachothertorestoretheunscheduledDNAsynthesisprocessofDNArepair(DeWeerd-Kasteleinetal.,1972).TheyfusedcellsfromamalechildwhohadonetypeofXPwithcellsfromafemalechildwhohadtheothertypeofXP.Eachpanelshowsacellwithtwonuclei.ThecellshadbeenexposedtoUVtoproduceDNAdamageandthenincubatedwithradioactivethymidine.ThecellontherightshowsradioactivespotsscatteredinbothnucleiwhereunscheduledDNAsynthesiswasoccurring.ThecellontheleftshowsnounscheduledDNAsynthesis.Thenucleiinthecellontheleftbothcamefrommaledonors.Thesamewastrueifbothnucleicamefromfemales.Onlycellsthathadonemaleandonfemalenucleus–therefore,amixtureoftheXPtypes–showedunscheduledDNAsynthesis:thecellhadagoodcopyofbothgenes,onefromeachnucleus.

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Figure22.7.KennethKraemer(left)andVilhelmBohr(right)in2005,commemoratingfourdecadesofresearchonDNArepairatNIH.KraemerreceivedanMDdegreeatTuftsMedicalSchoolandbecameboardcertifiedinDermatologyandInternalMedicine.HecametoNIHin1971asaclinicalassociateintheDermatologyBranchandhasbeenleadingground-breakingresearchonxerodermapigmentosumandrelateddiseases.In1980,heestablishedandhassinceledanNIHSpecialInterestGrouponDNARepairinwhichhepioneeredinternetconferencingtobringtogetherresearchersfromseveralinstitutionsindifferentcities.Bohr,adescendentofNielsBohr,receivedanMDdegreein1978,followedbyPhDandD.Sc.degreesin1987fromtheUniversityofCopenhageninDenmark.TogetherwithPhilipHanawaltatStanfordUniversity,hepioneeredinvestigationsoftranscription-coupledDNArepair,whichhecontinuedinmyLaboratory.In1992,hebecameChiefoftheLaboratoryofMolecularGeneticsintheNationalInstituteofAgingwherehehadbeenleadingstudiesofDNArepairandcancer.Table22.2.Xerodermapigmentosumgenes(complementationgroups)Gene Synonyms ChromosomeXPA XP1 9q22.33XPB ERCC3(excisionrepair3,helicasesubunit) 2q14.3XPC RAD4 3p15.1XPD ERCC2(excisionrepair2,helicasesubunit) 19q13.32XPE DDB2(damage-specificDNAbinding2) 11p11.2XPF FANCQ,RAD1,ERCC4(excisionrepair4,endonuclease) 16p13.12XPG ERCC5(excisionrepair5,endonuclease) 13q33.1XPV XPvariant,DNApolymeraseeta 6q21.1(Informationfromthehumangenenomenclaturecommittee(HGNC)website.)

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TheCockaynesyndromestoryButtheXPstoryhadyetanothertwistinadifferent,butcloselyrelated,geneticdisease.In1936,EdwardAlfredCockayne,apediatricianattheGreatOrmondStreetHospitalforSickChildren(whichstillstands(Figure22.8))inBloomsbury,London,describedtwochildrenwithapreviouslyunkownsyndromethatwastobearhisname(Figure22.9)(Cockayne,1936).Hecharacterizedthesyndromeas“dwarfismwithretinalatrophyanddeafness.”AlthoughCockayne’ssyndromebecamecloselylinkedwithxerodermapigmentosum,thesechildrendidnothavesunsensitivityandtheirskinwasclear(Figure22.9).That85-yearoldpaperwasnoteasytofindandmaybecomeincreasinglydifficulttofindastimegoesby;therefore,IamreproducinghereCockayne’soriginaldescriptionofthesyndrome,aswellasimagesheincludedinhispaper(Figures22.10-22.12):Thetwochildrenwiththisdystrophy,agirlagedsevenyearsandelevenmonthsandaboyagedsixyearsandthreemonthswereadmittedtotheHospitalforSickChildren,GreatOrmondStreetinJune,1935.Theparents,whoarenativesofnorthHampshire,areofEnglishrace,normalandnotblood-relations,andtheyhavebeenunabletotracetheoccurrenceoftheconditionintheirascendantsorcollaterals.....The dwarfs are so much alike in facial appearance, build and disposition, that the same generaldescriptionwillsuffice.Bothhavesmallheads,thatofthegirlbeingthesmaller,but,althoughthevaultoftheskullisflattenedandthecircumferencesmall,thegeneralshapeisnormal,andneitherchildhasthe receding forehead characteristic of microcephaly. Their faces are small with sunken eyes andprominentsuperiormaxillae.Theyareslightlybuiltwithshort,slendertrunksandundulylonglegs,andtheirfeetandhandsaretoolargeinproportion.Thethirdandfourthfingersoftheirhandsaredeviatedalittletowardsthemesialline.Bothareactiveandtheirmovementsarequickandbird-like.Theyarefriendly and playful, invariably good tempered, and laugh with obvious enjoyment at the slightestprovocation.Althoughtheyareimitative,theyhaveacertainamountofinitiativeandinplayingwithtoysarenomoredestructivethanmostchildrenoftheirageandclass.Theyfrequentlymakenoiseswhichatfirstsoundlikespeech,butactualwordscanseldomberecognized,althoughthegirlhasbeenheardtosay'mother'and'doitagain'andtheboyhassaid'doctor'severaltimes.Theydonotanswertotheirnamesorobeyspokenwords,nordotheytakeanynoticeofasoundmadebehindtheirheads,buttheyarequicktoobeysigns.Mr.JamesCrooks,F.R.C.S.,whosawthem,saysthatalthoughnottotallydeaf,theirhearingisgreatlyimpaired.Itisdifficulttotellhowmuchoftheirbackwardnessisduetodeafnessandhowmuchtomentaldeficiency.Theirbehaviourisnottheusualbehaviourofdeafchildren.Theyappeartobealittlebelowtheaverageinintelligenceandarefarmoreexcitableandlaughmuchmorereadilythanchildrenofnormalmentalitywhetherdeafornot.ChildrenwithCockayne’ssyndromerarelysurvivedtoadulthood.

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Figure22.8.TheGreatOrmondStreetHospitalforSickChildreninBloomsbury,London,whereEdwardAlfredCockaynesawtwochildrenin1935whohadanewsyndrome,whichcametobearhisname(Cockayne,1936).(CreativeCommons,Wikipedia)

Figure22.9.A7-yearoldgirlwiththesyndromedescribedbyCockaynein1936(right)standingnexttoanormalgirlofthesameage(left)(Cockayne,1936).Theaffectedgirl,althoughmuchshorterthannormal,hadrelativelylonglegsandlargehands.Herhead,however,wasrelativelysmall.Herskinwasclearandhadnosignofthesun-induceddamagethatischaracteristicinxerodermapigmentosum.

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Figure22.10.Cockaynesaidtheaffectedchildhadanabnormallysmallheadwithsunkeneyesandprominentfrontupperjaw(Cockayne,1936).

Figure22.11.Cockaynedescribedherskullasbeingsmallofsmallcircumferencewiththickenedbonesandprominentupperjaw(Cockayne,1936).

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Figure22.12.TheretinalatrophyCockaynenotedintheaffectedchildren(Cockayne,1936).Henotedmarkedlynarrowedretinalarteriesandatrophicchanges,particularlyinthecentralregionoftheretina.

Figure22.13.FibroblastcellsfromtheskinofCockayne’ssyndrome(CS)childrenwereunusuallysensitivetobeingkilledbyultravioletlight(UV)(left)buthadnormalsensitivitytox-rays(right)(Schmickeletal.,1977).Xerodermapigmentosumcellshadgiventhesamepattern:highsensitivitytoUVbutnottox-rays.ItwasfourdecadesafterCockayne’sdescriptionbeforethefirstcluetothecauseofthesyndromearrived.ItcamefromthelaboratoryofSchmickelandcoworkersattheUniversityofMichigan.Theyshowedthatfibroblastcellsderivedfromtheskinofaffectedchildrenwereunusuallysensitivetoultravioletlight(UV),whereastheirsensitivitytox-rayswasnormal(Schmickeletal.,1977)(Figure22.13).ThiscuriousdifferenceofbeinghighlysensitivetoDNAdamagecausedbyUVbutnottoDNAdamagecausedbyx-raywasexactlythesameasinxerodermapigmentosum(XP).However,despitethecellsofmostCockayne’ssyndrome(CS)childrenbeinghighlysensitivetoUV,thechildrenwerenot

Normal cells

CS cells

Normal and CS cells

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highlysensitivetosunlightandtheircellsremovedthyminedimersfromtheirDNAatanearnormalrate,contrarytotheinabilityofxerodermapigmentosumcellstoremovethosedimers(Schmickeletal.,1977).IttookalmostanotherdecadetoresolvethepuzzleofwhyCScellsresembledXPcellsinbeingunusuallysensitivetoUV,eventhoughthey(theCScells)removedthyminedimersfromtheirDNAnormally,asopposedtotheinabilityofXPcellstodoso.TheconfusingfindingsabouttherelationshipbetweenCockayne’ssyndromeandxerodermapigmentosumwereatlastclarifiedbyVilhelmBohr(Figure22.7),workingatStanfordwithPhilipHanawaltandlateratNCIinmyLaboratory,pinneddownaspecialnucleotideexcisionrepair(NER)mechanismdesignedspecificallyandexclusivelytorepairDNAdamageinregionsofthegenomethatwerebeingactivelybeingtranscribedatthetimethatthedamagewaspresent.Itwasadistincttypeofrepairandwasnamed“transcription-couplednucleotideexcisionrepair”(TCNER)(Bohretal.,1985).ResearchersinTheNetherlandsandtheUKthenshowedthatthegeneticdefectinCockayne’ssyndromewasinfactduetoadefectinTCNER(Venemaetal.,1990)(Chapter23).ButwhydidadefectinTCNERmakecellssensitivetobeingkilledbyUV(Figure22.13)?Thereasonwasthattranscription(RNAsynthesis),asitprogressedalongtheDNA,occasionallycollidedwithUV-inducedthyminedimer(orotherpyrimidinedimer).ThecollisionproducedapeculiarDNAdamageconfigurationthatwasapttoleadtodeathofthecell.ThisdisasterwasavoidedbyaspecialNERmachinery(TCNER)thatwasattachedtotranscriptionmachinery.WhentranscriptionencounteredaUV-induceddimer,itwaspromptlyexcisedbyTCNER,allowingthetranscriptionmachinerytocontinuemerelyonitsway.TheTCNERmachineryinCockaynesyndrome(CS)cellshoweverwasdefective,whichputthecellsatriskwhenevertranscriptioncollidedwithaUV-induceddimer.Thus,whilemostofthedimersscatteredinthegenomewereefficientlyremovedbyNER,thesmallfractionofdimersinvolvedintranscription-collisionneededthespecialTCNERtoberemoved.Consequently,CScellswerekilledbyUVeventhoughthelargemajorityofdimerswereremovedfromtheirDNA.However,itremainedpuzzlingwhydifferentcasesofCockayne’ssyndromesometimeshaddifferentpatternsandseveritiesofthesymptoms,andtherewerecasesthathadbothCockaynesyndromeandxerodermapigmentosumsymptoms(NanceandBerry,1992).Assooftenhappensinresearch,therealworld,asopposedtosimplerworldsoftheory,hidescomplicationsthatchallengeresearchers,asinatreasurehunt.Findingsinthathuntarethesubjectofthenextchapter.ReferencesAnderson, T.M. (1889). Note of a Rare Form of Skin Disease: Xeroderma Pigmentosum. British

medical journal 1, 1284-1285.

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Bohr, V.A., Smith, C.A., Okumoto, D.S., and Hanawalt, P.C. (1985). DNA repair in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall. Cell 40, 359-369.

Cleaver, J.E. (1968). Defective repair replication of DNA in xeroderma pigmentosum. Nature 218, 652-656.

Cleaver, J.E. (1972). Xeroderma pigmentosum: variants with normal DNA repair and normal sensitivity to ultraviolet light. J Invest Dermatol 58, 124-128.

Cockayne, E.A. (1936). Dwarfism with retinal atrophy and deafness. Arch Dis Child 11, 1-8. De Weerd-Kastelein, E.A., Keijzer, W., and Bootsma, D. (1972). Genetic heterogeneity of

xeroderma pigmentosum demonstrated by somatic cell hybridization. Nature: New biology 238, 80-83.

DiGiovanna, J.J., and Kraemer, K.H. (2012). Shining a light on xeroderma pigmentosum. J Invest Dermatol 132, 785-796.

Epstein, J.H., Fukuyama, K., Reed, W.B., and Epstein, W.L. (1970). Defect in DNA synthesis in skin of patients with xeroderma pigmentosum demonstrated in vivo. Science 168, 1477-1478.

Hunter, W.B. (1889). Notes of Three Cases of Xeroderma Pigmentosum, or Dermatosis Kaposi. British medical journal 2, 69-71.

Kraemer, K.H., and DiGiovanna, J.J. (2015). Forty years of research on xeroderma pigmentosum at the US National Institutes of Health. Photochem Photobiol 91, 452-459.

Nance, M.A., and Berry, S.A. (1992). Cockayne syndrome: review of 140 cases. Am J Med Genet 42, 68-84.

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