基因组杂交技术在诊断和产前诊断不平衡染色体畸变中

广州医学院硕士学位论文微阵列比较基因组杂交技术在诊断和产前诊断不平衡染色体畸变中的应用姓名：符芳申请学位级别：硕士专业：妇产科学指导教师：廖灿201005182G5MbfluorescenceinsituhybridizationFISHspectralkaryotypinSKY24array-basedcomparativegenomichybridizationarray-CGHaCGHDNAArray-CGHDNAcopynumbervariantsCNVsDNADNAArray-CGH-array-CGH31.array-basedcomparativegenomichybridizationarray-CGHoraCGHarray-CGH2.array-CGH3.aCGHGFISHSKYC1.2008120091221191121417G2121SNP6.02PCRFISHSKYarray-CGH3C24array-CGHFISHSKYGC34121array-CGHG17DNADNA4DNA2.1G13[G46XYr(13)(p10q34)?]array-CGH138.5Mb46XYdel(13)(q33.2→q34)3.246G46218DNA34Mb[G46XYadd18],aCGH46,XY,dup(18)q12.3→q22.3)46G9aCGH9DNA30MbaCGH46,XX,dup(9)(9p13→q13)pat4.35DNADNA3G8[G46,XY,add(8)]aCGH8DNA46,XY,dup(8)(p21.3-p11.3),de1(8)(p23.3→p23.1)5GX[G46,X,der(X)],aCGHXDNA63.45Mb52.7Mb46,X,dup(X)(q21.31→q28),del(X)(p22.33→p11.22)5.7—10Garray-CGH17380kb46XXdel(17)(q21.31-q31.32)6.11—15Garray-CGHX112kb46XXdel(X)(p22.33)7.16—1716G5array-CGH22700kb46XYdel(22)(q11.2),BDiGeorge17array-CGHDNA8.aCGHPCRFISHaCGH1.Array-CGH2.Array-CGHDNADNADNAFISHSKY3.Array-CGH-4.Array-CGHDNAcopynumbervariations,CNVDNAGFISHaCGHCNV5.Garray-CGHDNAFISHPCR6CCNV-7Theapplicationofarray-basedcomparativegenomichybridizationindiagnosisandprenataldiagnosisofunbalancedchromosomeaberrationAbstractPartialdeletionandduplicationarecommonunbalancedchromosomalaberrationsandoftenassociatedwithmultiplecongenitalanomalies,developmentaldelayandmentalretardation.AlthoughaconventionalcytogeneticanalysisbyG-bandingcandetectnumericalaberrationsandsomeapparentstructuralaberrations,itcannotdetectdeletionsorduplicationssmallerthan5Mb.Itispossibletoconfirmordetectthesuspectmicro-deletionsorduplicationsbyfluorescenceinsituhybridization(FISH).However,withoutanyoftheindicativechromosomalaberrationinformation,FISHisnotfeasible.Eventhoughthe24fluoresencetlylabeledchromosomalpaintingprobesutilizedbyspectralkaryotypin(SKY)candetecttheoriginoftheapparenttranslocationsandmarkerchromosomes,SKYisimpossibletodetecttheaberrationswithinachromosome.Array-basedcomparativegenomichybridization(array-CGHoraCGH)isarecentlydevelopedmolecularkaryotypingtechnologypermitingthesimultaneousrapidhighresolutiongenome-widecopynumbervariants(CNVs)analysisandmappingofDNAsequencesbyahybridizationexperiment.Sincedeveloped,itisadmittedtobethemosteffectivetoolfordetectingchromosomemicrodeletionsandmicroduplications.Array-CGHtechnologycanimprovethediagnosticdetectionrateofthesesmallchromosomalabnormalitieswithnovelhigh-resolution,whole-genomescreeningefficiencyandassistclinicalreferringdoctorstopreciseevaluatepatients'conditionandtheirprognosisbaseonthegenotype-phenotyperelationanalysis.OurcurrentstudyisthefirstinvestigationofapplicationaCGHtechnologyindiagnosisandprenataldiagnosisofunbalancedchromosomeaberrationinmainlandChina.8Objection6.Establishingthetechnicalsystemofarray-basedcomparativegenomichybridization(array-CGH),andtostudytheapplicationofarray-CGHintheunbalancedchromosomalaberration.7.Tostudythesensibilityandacccuracyofarray-CGHtodiagnosistheunbalancedchromosomalaberration.8.Toinvestigatehowtocombinationthetechnicalofarray-CGH,G-banding,FISH,C-bandingandsilver-staininginthediagnosisofunbalancedchromosomalaberration,andtobuildupanexercisablestandardclinicalprocedure.SubjectsandMethods1.SubjectsDuringJan.2008toDec.2009,itselected21casesofchromosomeanalysisthatcamefromthegeneticclinicofGuangzhouwomenandchildrenmedicalcentreorotherhospitals,including19casesofperipheralbloodsample,1amnionicfluidand1fetalcordbloodsample.Among21cases,therewere4casesofnormalcontrolledgenome,theremainingcasesweresuspectedofunbalancedchromosomeabnormalities,buttheconventionalG-bandingcouldnotconfirmthediagnosis.2.Method(1)Allof21DNAsampleswereperformedaccordingtothestandardoperationprocedureofSNP6.0chip(Affimetrix),andwereanalyzedbythematchingscannerandsoftwareofcomputer.9(2)UsingPCRorFISHtoconfirmthearray-CGHresultstoassesstheaccuracyandsensibilityofarray-CGHindiagnosistheunbalancedchromosomeaberration.(3)UsingC-bandingandsilverstainingtechnologytoconfirmtheduplicationoccurredinheterochromatinandsatelliteregiontoevaluatetheprognosis.(4)Tostudytheeffectinallkindsofbandingtechniquesandanalysistechniquesofchromosomes,andtobuildupanstandardclinicalprocedure.3.follow-upWemaderoutinepostnatalfollow-upfortheprenatalcases.Results1.Allof21samplesweresuccessfuldetectedbyarray-CGHtechnology,includingwhich17patientcasesthatcannotdiagnosedbytheconventionalGbandingwereallpreciseidentifiedatmolecularlevel.2.Case1:Therewassomeabnormalatthelongarmofchromosome13[thekaryotypeofG-bandingis46,XY,13q-?].Theresultofarray-CGHis46,XY,del(13)(q33.2→q34).Thesizeofthedeletionis8.5Mb.3.Case2,4and6:Theywereallfoundtohavesomepartofextramaterialonchromosome18andchromosome9,respectively,whichthesourcewerenotdeterminedbyroutineG-banding.Case6isthefatherofcase4.Thearray-CGHresultofcase2is46,XY,dup(18)q12.3→q22.3),thesizeofduplicationis34Mb.Therewasainheritedduplicationonthelongarmofchromosome9atcase4transmittedfromherfather.Thearray-CGHresultis46,XX,dup(9)(9p13→q13)pat.Thesizeoftheduplicationi