人类遗传病人类遗传病 (2).pdf

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1、-I-W.Liu1l2,H.Wang1,S.Zhao3,W.Zhao4,S.Bail,Y.Zhao5,S.Xu5,C.Wu5,W.Huang5,Z.Chen5,G.Feng1,andL.He2*ShanghaiInstitutesforBiologicalSciences,ChineseAcademyofSciences,320YueYangRoad,Shanghai,200031,P.R.China;2Bio-XLifeScienceResearchCenter,POBox501,HaoRanBuilding,ShanghaiJiaoTongUniversity,Shanghai,20003

2、0,P.R.China;3JuniorMiddleSchoolofXunyiCounty,ShananXiprovince,P.R.China;4HospitalofXunyiCounty,ShananXiprovince,P.R.China;and5ChineseNationalHumanGenomeCenteratShanghai,Shanghai,P.R.China;*correspondingauthor,TheNovelGeneLocusforAgenesisofPermanentTeeth(He-Zhaodeficiency)MapstoChromosome1Oq11.2JDent

3、Res80(8):1716-1720,2001ABSTRACTHe-Zhaodeficiencyhasbeenrecentlycharacterizedwithadistinctformofagenesisofpermanentteeththatisdifferentfromotherpreviouslyreporteddisordersoftoothagenesis.Thisinheritedabnormalitysuggeststhatsomegene(s)associatedwiththedevelopmentofpermanentteethmaymutate.Inthisstudy,w

4、emapthegenelocustochromosomelOqi1.2.TheDNApoolingmethodcombinedwithtwo-pointandmulti-pointlinkageanalysishasbeensuccessfullyapplied.ThemaximumLOD(Zmax)scoresfortwo-pointandmulti-pointanalysesare13.29(onmarkerDIOS196)atrecombinationfraction(0)=0and18.09(between markersD1OS1772andD1OS1766),respectivel

5、y.Haplotypeanalysisconfinedthelocuswithinanintervalof5.5cMflankedbymarkersDIOS604andD1OS568.ThisstudyhasdemonstratedanovelgenelocusresponsibleforHe-Zhaodeficiencyandprovidesagoodlikelihoodforthediscoveryofoneofthegenesdeterminingpermanenttoothformationanddevelopment.KEYWORDS:agenesisofpermanentteeth

6、,He-Zhaodeficiency,DNApooling,linkageanalysis,lOql1.2.ReceivedSeptember22,2000;LastrevisionMay29,2001;AcceptedJune14,2001INTRODUCTIONToothagenesis(oneormoreteethmissing)isoneofthemostcommondevelopmentalabnormalitiesinthehuman.Toothagenesismaybeoffamilialorsporadicorigin.Morethan20%ofthepopulationisa

7、ffectedbywisdomtoothdeficiency.Incidencesoftheothertoothagenesisvaryfrom1.6%to9.6%(McKusicketal.,1998).Infact,thereareabout100inheriteddisordersorsyndromesrelatedtothecongenitalabsenceofoneormoreteeth.Themodeofinheritancecanbeautosomal-dominant,-recessive,orX-linked.Thedisorderscanoccurinprimaryorpe

8、rmanentteeth,withvariousabnormalitiesinshape,space,color,location,number,andtoothroot(Graberetal.,1978).SeveralgenesincludingEDA,MSX1,REIG,PAX9,andothergeneticlocirelatingtohumantoothagenesishave beenidentified(Datsonetal.,1996;Kereetal.,1996;Phillipsetal.,1996;Vastardisetal.,1996;Petersetal.,1999;S

9、tocktonetal.,2000).However,thecompletemolecularmechanismofabnormaltoothdevelopmentisstillunclear.He-Zhaodeficiency(collectedinOnlineMendelianInheritanceinManOMIMwithIDnumber604625;http:/www.ncbi.nlm.nih.gov/OMIM)hasbeenrecentlycharacterizedinalargeChinesekindredwithadistinctformofpermanenttoothagene

10、sisthatisdifferentfromotherdisordersoftoothagenesisreportedbefore(Wangetal.,2000).Thisinheritedabnormalitysuggeststhatsomegene(s)associatedwiththedevelopmentofpermanentteethmaymutate.TheaimofthisstudywastolocalizethegenedeterminingHe-Zhaodeficiencytoaidourunderstandingofthemechanism(s)underlyingthef

11、ormationanddevelopmentofhumanteeth.SUBJECTS&METHODSSampleCollectionandClinicalEvaluationThesamplesofthekindredwerecollectedfromasmallvillageinShaanxiProvinceinnorthwestChina.Anovelformofagenesisofpermanentteethinthispedigreewasobserved:(1)Therewerenosignificantclinicalmanifestationsotherthantheoligo

12、dontiainthispedigree.(2)Theoligodontiarangedfromafewteethtotheentiresetofteeth.Onlythepermanentteethwereinvolvedandusuallyappearedatage7or8years,whenprimaryteetharenormallyreplacedbypermanentteeth.Someofaffectedindividualsdevelopedthefirstandsecondmolars.(3)Insomecases,itappearedthattheremainingteet

13、hwerenotpermanentteeth;instead,theywereprimaryandsometimesremaineduntilsubjectswereintheirforties(Wangetal.,2000).Toconfirmthediagnosis,wetookradiographsofallthepartiallyaffected1716He-ZhaoDeficiencyLocusMapsto1OqI1.2membersdescribedinourpreviousreport.Wefoundfromtheradiographsthat:(1)somesubjects,s

14、uchasIV-30,V-19,andV-22,hadretainedtheirpermanentteethalthoughtheyhadoneortwoteethmissing;(2)exceptforlossesofacoupleofteeth,subjectsV-3,V-8,V-9,V-18,andV-21hadmoreorlessretainedtheirprimaryteeth,eventhoughtheywereover15yearsofage,whichindicatedthatthesemembersshouldbeaffected;and(3)subjects111-11,I

15、V-3,IV-25,IV-31,IV-33,andIV-48,whohadbeenpreviouslydiagnosedbyvisualinspectionasaffectedmembers,hadalltheirremainingteeth,whichwerenormalpermanentteethbutwithsomelargegapsbetweenteethwhichlookedliketheteethweremissing(Fig.1).Astandardinformedconsenttotheprotocolwassignedbyalltheparticipantsortheirre

16、latives.DNAPreparationandPoolingDNAwasextractedfromvenousbloodbyastandardmethodasdescribedpreviously(Yangetal.,2000).TheconcentrationwasmeasuredbyHoeferDyNAQuant200Fluorometer(AmershamPharmaciaBiotechAB,Uppsala,Sweden).Weconstructed3DNApanelsbypooling10iLof20ngDNAfromeachof20affectedmembers(panelA,1

17、11-2,111-7,III-10,111-13,111-16,111-22,IV-4,IV-14,IV-17,IV-20,IV-26,IV-28,IV-29,IV-34,IV-43,IV-47,IV-49,V-2,V-18,V-21),19unaffectedsiblings(panelU,111-17,111-19,IV-6,IV-18,IV-19,IV-21,IV-25,IV-30,IV-31,IV-33,IV-45,IV-48,V-5,V-6,V-7,V-10,V-1,V-17,V-24),and18normalsubjects(panelC,111-5,111-8,111-12,11

18、1-14,111-18,111-20,111-21,IV-5,IV-9,IV-15,IV-16,IV-24,IV-27,IV-36,IV-37,IV-42,IV-44,IV-46),actingascontrols,respectively.DeterminationofCandidateMarkersExceptformarkersonchromosomeXandY,382dinucleotide-repeat-basedmicrosatellitemarkerswithaverage10-cMintervalswererecruitedformultiplexpolymerasechain

19、-reaction(PCR):From10to20pairsofprimerswereaddedintoonetubeaccordingtoclassificationbythecompany(Perkin-ElmerAppliedBiosystems,Foster,CA,USA).Oneofeachpairofprimerswaslabeledatthe5endbyFAM,NED,orHEX,respectively.PCRwasperformedinatotalvolumeof5pLcontaining40ngDNA,0.0002nmolofeachprimer,and0.2UofHotS

20、tarTaqDNApolymerase(QIAGENGmbH,Hilden,Germany)onathermalcycler9700(Perkin-ElmerAppliedBiosystems,Foster,CA,USA)understandardconditions:1cycleat95Cfor12mintodenaturetheDNA,followedby14touchdowncycleswithdenaturingconditionsat95Cfor30sec,annealingtemperaturefrom63Cto56Cfor1min,andFigure1.Thepedigreewi

21、thHe-Zhaodeficiencyandhaplotypesofaffectedmembers.Sharedhaplotypeisindicatedbyblackbar.Thewhitespaceinabarindicatesarecombination.AkeyrecombinationcanbefoundinIV-20.Theblanksquares,blacksquares,blankcircles,andblackcirclesrefertounaffectedmales,affectedmales,unaffectedfemales,andaffectedfemales,resp

22、ectively.N00NO0NO0-sNNoN,-NO000N0NN0NNNNONO0OO0NNNNOOONMrCNeNNonnvv-T.0kstooN NNNO0N0NNka-.00-cos4waN.-tDOONNrU.tNO,N000Dwt-O0NN08NON,Oeb0NW9coV-MMtl0dMCnCoCaOM00N-00srNtoCl0NNCO0OO0OOO-aONO-NNNNbN|O-NNeeN0NtC-00tcooOno-NNNC-NN,NOON|0-NraNN0NNN0Nro-7NNOOt.NN00Nr7t)etOs-C-N NC-NtNt-NOO0OtroNNNNONNNNC

23、OOaO0tOO0OoOOOoooOoaoOIJDentRes80(8)20011717tNNoNOtNNNNNNNNNONNC-NcNo-NNNNO0tN.NN0ItW-NNN0N-91-)mC4toto-.NNWnW0C-NNNNNNNONO-NN-NNNN,-0-V.NNONO-NNNN-NOC-NgtWC,0NNON&ON0-0NNNNNn0NNNNtOgo.new2-Mraa0toco-tc-MMWN-ov2orNNtNoONOONN-ON0-ONOC-NN0NONOOOC-NNNNNN 0N0NNNr)VIncovW&4asNetNe0etcmvNt;nN43vL,NNWtSWP-

24、ot1stMMtenNWe-W)VcmK0SN-4041sDY*eStnMWNV2VWNNtoNNN.7-V-ewsN74Nt,-NO0NN-ONrN-X-NN-ONN-NsO0O0NNNNNO.CO0ONN-NNNOCO0-7rntC-NN0NNOONNtst-nH7tterONON-KNeN-ONNO0NNnN*-ZNONNNNON-N0N4rN*-tOFWNNNNONONO0rNNNNNIINstNNabNONO4-O0NNONNNNONONO0er-1-0NN-0etNON00eeKN.NONNNNONNN0NNONC-NN0-NN=NNOO7NOlKFutNN-000-Liuetal

25、.Chromosome10Markerinterval(cM)AU0a:DlOS196AUyCb:D1S218Figure2.GenotypesofthepositivecandidatemarkersgeneratedfromtheDNApoolings.ThepeakprofilesbasedontheresultsanalyzedbysoftwareGenotyper2.0forpositivemarkerswereformedbytransformationfromthegelelectrophoresisimageswithsoftwareGenescanAnalysis2.1.(a

26、)Arareallelesparkingintheaffectedpooledpanel.(b)Acommonallelewhichresultedinthelowersignificantdifferenceamong3panels.(a)Arrowsindicatethesparkingpeaks.Notes:A=panelA;U=panelU;C=panelC;0=Overlaid.Figure3.ThelinkageregiothegenefortheHe-Zhaodediagramshowsthecytoge1Oql1.2ofchromosome10genelocusfortheHe

27、-Zhawasmappedwithin5.5ctvmarkersDlOS604andD1OSextensionat72Cfor1.5min.Inthetouchdowncycles,annealingtemperaturewasdecreased0.5Caftereachcycle.Afterthetouchdowncycles,anadditional25cycleswithanannealingtemperatureat56Cwereadded.Finally,theproductswereincubatedat72Cfor10min.PCRproducts1.5,uLeachwerede

28、naturedandloadedonto5%denaturedsequencinggelonanABI377sequencer(Perkin-Ehner,Foster,CA,USA)under3000V,60mA,and200W.ThesoftwareGenescanAnalysis2.1andGenotyper2.0(Perkin-ElmerAppliedBiosystems,Foster,CA,USA)wasusedforthegenotypingofmarkers.Thepeakprofilesofeachmarkerfromthe3panelswereoverlaidandcompar

29、edbysimplevisualinspection.AnymarkerthatshowedauniqueorahigherpeakinpanelAcomparedwithpanelUandpanelC(whichcouldberegardedasareferenceforthealleliccontributioninthepopulation)wasselectedasacandidatemarker(Fig.2).Toavoidthefalse-positiveresultandanon-specificproductofPCR,weamplifiedallthemarkersaddsc

30、annedthemtwicewiththesameprotocol.GenotypingandLinkageAnalysisEighty-sevenmemberswerecarefullyselectedfromthefamilyforindividualgenotypingaccordingtothedistributionoftheaffectedpeople.Two-pointlinkageanalysiswascarriedoutbyLINKAGEpackageversion5.1(Ott,1991),andmulti-pointlinkageanalysiswasdonebyFAST

31、MAPtosimulateamulti-pointLODscoreofthefulldataset(CurtisandGurling,1993).Themarkerswithanaverageresolutionof1cMformulti-pointlinkageanalysisandforhaplotypeconstructionwereorderedintermsofthegeneticmapofGenethon(Dibetal.,1996).Duetothemodeofautosomal-dominantinheritancedemonstratedbythisfamily,wetook

32、intoaccountthepossibilityofuncertaindiagnosisbysettingallelicfrequencyofthediseaselocusandthephenocopyrateat0.00001and0.5%,respectively.Penetranceof0.95wasassumedforallthesamplesselectedintheanalysis,andwasalsosetfrom0.70to0.95toavoidafalse-positiveresult.Theallelefrequencyforthemicrosatellitemarker

33、wasarbitrarilysetequalto1/n,wherenisthenumberofallelesobserved.Toreducetheincorrectdiagnosisthatoccurredinthefirstsurvey,wealsoperformedtheaffected-onlymodelusingtheaffectedmemberswhowerediagnosedasaffectedinthefirstandsecondsurveys.RESULTSTofacilitatethegenemapping,werecruited382)ncontainingmicrosa

34、tellitemarkerscoveringthewholesficiency.Thegenometogenotypethe3pooledDNApanels-neticregionstngo,inwhichtheconsistingofaffectedprobands,unaffectediodeficiencysiblings,andnormalindividualsinthispedigree,Aflankedbyrespectively.Wethenusedlinkageanalysisto;568.confirmthecandidateregionsselectedbytheDNApo

35、olingmethod.Forfurtherfinemapping,weselected17othermarkersfromthepositiveregiontoperformgenotyping,linkageanalysis,andhaplotypeconstruction.DNAPoolingScanInmultiplexPCR,351outof382(91.88%)markersweresuccessfullyamplifiedwithclearpeaktraces.Nosignificantdifferenceintheduplicatescanshasbeenfound.Sixty

36、-five(18.52%)candidatelociwereobtainedfromthecomparisonbetweenthetestgroupandcontrolgroupoftheDNApoolsinthefirst-phasescanning.LinkageAnalysisandHaplotypeConstructionAftergenotypingeachcandidatelocusinindividualsamplesofthepedigree,wefound,fromtwo-pointlinkageanalysis,thattwolociclosetoeachotheronch

37、romosome10,DIOS196andD10S1652,showedLODscore13.29(0=0)and2.64,respectively.Thesedatawerestronginsupportoflinkage.Toconfirmtheresults,weselected17otherinformativemicrosatellitemarkersrangingfromD1OS208toDIOS537fromthesex-averagegeneticmapofGenethon(Dibetal.,1996)forfurtherindividualgenotyping.Eventua

38、lly,thecloselinkagebetweenthesemarkersandthediseaselocuswasclearlydemonstrated.Themaximumtwo-pointLODscoreobtainedfromD10S196was13.29(0=0)(Table).ThemaximumLODscoreof18.09,revealedbysimulatedmulti-pointlinkageanalysis,indicatedthatthecloselinkagefellintotheregionflankedbymarkersD1OS1772andD10S1766.A

39、lltheLODscoresremainedpositivewhenwechangedthepenetrancefrom0.70to0.95.Furthermore,theanalysisbytheaffected-onlymodel,withuseoftheaffectedmembers,alsoshowedpositiveresults(Table).Weconstructedhaplotypesforall87membersofthefamilytocheckthecriticalrecombinantsbetweenmarkersandJDentRes80(8)20011718He-Z

40、haoDeficiencyLocusMapsto1OqI1.2Table.TheLODScoresObtainedfromtheAnalysisoftheAffected-onlyModel(penetrance=0.99)andtheWhole-pedigreeModel(penetrance=0.95)with19MicrosatelliteMarkers(Bothmodelsshowedsignificantpositiveresults.)LODScoreattheta=Marker00.010.050.10.20.30.4ZmaxThetaAffected-onlyanalysis:

41、DIOS208DlOS1768DlOS1780DlOS1746DIOS1791D1OS604DlOS1793DlOS1772DlOSI766DlOS196DlOS538DIOS225DlOS1724DIOS220DIOS568DOS539DIOS546DlOS1652DIOS537Whole-pedigreeanalysis:DIOS208DlOS1768DlOSI780DlOS1746DlOS1791D1OS604DlOSI793DIOS1772DIOS1766DlOS196DIOS538DIOS225DlOSI724DlOS220D1OS568DIOS539DIOS546DlOSI652-

42、6.76-6.79-1.55-0.852.05-1.935.232.633.725.733.83.391.595.9-1.213.453.74-3.13-1.74-7.43-5.281.255.352.82.1310.73.698.0313.295.876.333.6712.952.575.131.48-2.390.840.813.362.0321.155.132.573.645.633.723.321.555.82.483.373.671.262.873.054.428.478.162.745.0110.593.627.8913.145.796.243.612.817.735.151.571

43、.341.881.873.662.381.81.624.732.333.315.193.413.041.385.362.833.043.361.713.214.765.338.58.122.545.2910.023.357.3512.435.435.843.3112.118.075.071.752.312.032.033.452.241.551.614.212.032.894.643.012.681.164.792.692.642.971.683.045.035.27.917.462.284.969.1836.6311.414.945.32.9311.17.694.741.772.621.68

44、1.732.691.651.051.253.121.422.053.472.221.930.763.592.081.822.161.32.364.334.126.275.681.733.827.212.285.089.033.784.082.158.766.233.751.532.421.061.151.780.960.60.771.980.851.242.261.431.170.412.341.331.051.340.81.532.972.654.283.581.162.484.931.493.376.282.452.691.346.044.262.511.091.740.390.530.8

45、50.360.240.320.890.350.531.070.670.50.151.10.60.420.60.330.71.331.172.091.40.591.222.380.661.573.151.051.210.542.961.991.150.560.852.032.033.662.382.051.645.232.633.725.733.83.391.595.92.833.443.741.723.215.035.358.628.2935.3110.73.698.0313.295.876.333.6712.958.085.161.782.640.0940.0980.0440.04900.0

46、7000000000.049000.660.0470.0950.0610.260.2400.039000000000.0410.160.0810.121thediseaselocus(Fig.1).Toavoidtheimpactofincompletepenetrance,wechoseonlythekeyrecombinantsfromtheaffectedmembersinanalyses.Thediseaselocuswasidentifiedwithina5.5-cMregionflankedbymarkersDlOS604andDIOS568onchromosomelOqll.2(

47、Fig.3).ExceptforV-6,whowasdiagnosedasanormalindividualbutsharedonediseasehaplotype,allotherhaplotypes(basedonboththenormalandtheaffectedmembers)showednodifferencesinrecombinationinsegregationofthediseasehaplotype.DISCUSSIONInthiswork,wehavesuccessfullymappedthegenelocusfortheHe-ZhaodeficiencyontolOq

48、l1.2,aswassupportedbytheevidenceoftwo-pointLODscore13.09at0=0andmulti-pointLODscore18.09.Therecombinationdataobtainedfromhaplotypesacrossalltheaffectedindividualsindicatedthatthegenelocuswaslikelytobewithintheregionof5.5cMflankedbymarkersDOS604andDOS568.Inthehaplotypeconstruction,V-6wastheonlyonewho

49、wasdiagnosedasanormalphenotypethatsharedadiseasehaplotype.Weconcludedthatthismemberwasrelativelyyoung(14yearsold)withanunclearphenotypewhichledtoconfusionwiththediagnosis.Severalgenesinthe1Oq11.2regioncanbeconsideredascandidates,suchasDickkopf-1(Dkk-1),PRKG1B,andaKOXzincfingergenecluster.Dkk-1isamem

50、berofanewgeneJDentRes80(8)20011719JDentRes80(8)2001familythatencodessecretiveproteinsandagrowth-factorantagonistoftheSpemannorganizerexpressedintheheadorganizerandrequiredforheadformation(Glinkaetal.,1998).WhenmRNAofDkk-1wasco-injectedwithBMP(BoneMorphogeneticProtein)inhibitor,acompleteheadstructure