遗传学 Genetic Diagnosis-工具与遗传病诊断

Chapter4

ToolsofHumanMolecularGenetics1ThePolymeraseChainReactionMolecularcloningNucleicAcidHybridizationDNAsequenceanalysisWewilldiscuss2AnalysisofindividualDNAand

RNAsequencesPolymeraseChainReaction(PCR)MolecularCloning

3ThePolymeraseChainReaction

聚合酶链式反应PCR:

EnzymaticamplificationofafragmentofDNAlocatedbetweenapairofprimerssensitivenessandspecificity4

PCR_technique5MolecularCloningToisolateaparticulargeneorotherDNAsequenceinlargequantities.TotransfertheDNAsequenceintoasinglecellofamicroorganism,thenreproducestheDNAsequencebygrownofmicroorganism6TheprocessofcloningasegmentofhumanDNAintoaplasmidcloningvector

ToolsofmolecularcloningVector(Plasimd,Virus,Bacterialartificialchromsome,PAC,YAC)RestrictionenzymeTargetgeneHastcell(microorganism)8RestrictionEnzymesEcoRⅠ（palindrome）5’---G^AATTC---3’

5’---G

AATTC---3’

3’---CTTAA^G---5’

3’---CTTAA

G---5’

Vectors

Example:

plasmid(<5000bp),bacteriophagelambda(20kb)，BAC(100-300kb)，YAC(1000-2000kb)

multiplecloningsites,antibioticresistancegene910TheuseofmolecularcloningandthePCRtoisolatelargequantitiesofaparticulardesiredDNAsequenceofinterestinpureformThePolymeraseChainReactionMolecularcloningNucleicAcidHybridizationDNAsequenceanalysisWewilldiscuss11Nucleicacidhybridizati-on:DoublecomplementarystrandsDNAcanbedenaturedbyavarietytreatmentsandcanhybridizetothehomologoussequenceunderconditionsthatfavorformationofdoublestrandedDNA.Probe:aspecificDNAsequencelabeledwithisotopeorfluorescence12Thesouthernblottingprocedure13InSituHybridizationtoChromosomesFluorescenceInSituHybridization(FISH)ComparativeGenomeHybridizationSpectralKaryotyping(SKY)14FluorescenceInSituHybridization(FISH)NucleicacidProbeslabeledwithfluorescencebynicktranslationHybridizedwithinterphasenucleusandmetaphasechromosomeinsitu.AccordingtoBasePaircomplementaryprinciples15FISH16ThePolymeraseChainReactionMolecularcloningNucleicAcidHybridizationDNAsequenceanalysisWewilldiscuss17DNAsequencing

-sangersequencing(末端终止法）PCR(templet)PrimerForprimerRdNTPddNTPwithfluoresent(smallaccountPolymerease18Sangersequencing19Chapter10

ThediagnosisofGeneticDisease20

PrenataldiagnosisThediagnosisofgeneticdisease

Outline21PresymptomaticdiagnosisSymptomaticdiagnosisPrenataldiagnosisThetypesofdiagnosis

-GeneticDisease22SymptomaticdiagnosisClinicaldiagnosisPedigreeanalysisLaboratorytests23Symptomaticdiagnosis(1)ClinicaldiagnosisclinicalsymptomsPhysicalExaminationHistoryTaking(familyhistory,Marritalhistory,Childbearinghistory)BiochemicalexaminationsOtherExaminations(CT,MRIetc)24Symptomaticdiagnosis(2)

Pedigreeanalysis

Apedigreeisadiagramoffamilyrelationshipsthatusessymbolstorepresentpeopleandlinestorepresentgeneticrelationships25Symptomaticdiagnosis(3)LaboratorystudiesCytogenetictestBiochemicaltestGenetest26CytogenetictestDiagnosis:forchromosomesdiseases.Technique:Karyotype-GbandingFluorescenceinsituhybridization(FISH)Material:PeripheralBlood-lymphocyteAmnioticfluid

Chorionicvillus

27Clinicalexample(1)

-Karyotype18-trisomysyndrome

ClinicaldiagnosisCytogenetictestconfirmingclinicaldiagnosisgeneticcounselingG-bandingKARYOTYPE28BiochemicaltestDiagnosis:formetabolicdiseasesDiagnosedbyenzymeassayMaterial:PeripheralBlood-lymphocyteAmnioticfluidChorionicvillus29BiochemicaltestsformetabolicdiseasesUsefulformetabolicdiseasesDiagnosedbyenzymeassay30DNAanalysisMeansofcloselylinkedmarkers-tolinkageanalysis.

PCR-STR(PCR)RFLP（Southernblotting)

Directdetectionofthemutation.

PCR-sequencingtodetectpointmutationPCR-SSCPtodetectpointmutationMLPA(MultiplexLigationProbesAssay)todetectduplication/deletionSouthernblottingtodetectduplication/deletion315.7kb3.4kb2.4kbRFLP-Linkageanalysis32ClinicalExample（1）

-DMD/BMD(XR)ToconfirmwhetheritisgeneticdiseaseToanalyzeitsinheritedmannersToestimatetheriskofthediseaseinthisfamilyprobandmotherDuplicationmutationdetectedbyMLPASummaryClinicaldiagnosisPredigreeanalysisgenetestingConfirmingclinicaldiagnosisGeneticcounseling33PCR-SequencingtodetectpointmutationFatherprobandMotherClinicalExample（2）

-Methylmalonicacidemia(AR)DevelopmentretardationMentalretardationVomiting,etc.Vitb12diagnosistherapy34Prenataldiagnosis35PrenataldiagnosisPrenataldiagnosisdeterminesthehealthandconditionofanunbornfetus.36ThehistoryofprenataldiagnosisIn1966,SteeleandBregdetectedDownsyndrome.Cellcultured

Karyotypeanalysis37TherequiresofprenataldiagnosisClinicalgenetic(includinggeneticcounseling)ObstetricsUltrasonographyLaboratoryservice38Thegoalsofprenataldiagnosis①Toprovidearangeofinformedchoice.②Toprovidereassuranceandtoreduceanxiety.especiallyamonghigh-riskgroups.③haveahealthone.④theoptionofappropriatemanagement.⑤Toenableprenataltreatmentoftheaffectedfetus.39TheindicationforprenataldiagnosisTheageofpregnantwoman(>35yearsold)Positiveprenatalscreening.Highdangerfamily.Otherwell-definedriskfactors.4041ThecontentofprenataldiagnosisNoninvasivetesting

Maternalserumscreening(MSS),UltrasonographyInvasivetesting

Amniocentesis,Chorionicvillussampling,CordocentesisLaboratoryservice

Cytogenetictesting,Biochemicaltesting,Genetesting42Noninvasivetesting43NoninvasivetestingMaternalserumscreeningUltrasonography44Maternalserumscreening(MSS1)MSS:alsocalledtriplescreenmeasuresthreebloodmarkers:①Alphafetoprotein(MS-AFP),②unconjugatedestriol(uE3),③humanchorionicgonadotrophin(HCG).Thesemarkersareproducedbythefetusand/orplacenta.Performedbetween15-20+6weeksatgestationalage.45Maternalserumscreening(MSS2)Thesetriplesmarkersareusedtoscreenthreediseases:①Downsyndrome②Trisomy18③neuraltubedefect(NTD)46Downsyndrome47Trisomy18syndrome48Neuraltubedefect(NTD)49ConditionMSAFPuE3HCGNeuraltubedefect↑

NormalNormalTrisomy21↓

↓↑Trisomy18↓↓↓5051ThecautionofMSSMSSisonlyscreeningtest,notdiagnostictest.FurthercounselinganddiagnostictestingshouldbeofferedtowomenwhoseMSSscreeningtestresultispositive.NegativeresultofMSSTheriskiszero≠52UltrasonographyinprenataldiagnosisItisimportanttodetectfetalassessment:fetalage,multiplepregnancies,fetalviability.Itisimportantforthedetectionofmorphologicalanomalies,suchas,anencephaly,cystichygrome(囊性水瘤).53Ultrasonographyingeneticdisorderchromosomeaneuploidy.single-genedisorders(Holt-Oramsyndrome).multifactorialdisorders.Determinationoffetalsex.54Detectingchromosomeaneuploidy

①Theaorticisthmus(主动脉峡)issignificantly

narrowerintrisomy21,18,13,and

Turnersyndromethaninnormal

fetuses.

②Measurmentofnuchaltranslucency

thickness(NTT)at10-14weeksmay

provetobeauseful

methodfor

screeningchromosomaldefects.55Nuchaltranslucencythickness(NTT)Nuchaltranslucencythickness:betweentheskinandthesofttissueoverlyingthecervicalspine.0.12cminanormal11-weekfetus.TheaccumulationoffluidbehindthefetalneckNT↑56Ultrasoundforsingle-genedisorderNormalfetusHolt-OramsyndromeADdiseaseTBX5transcriptionfactorgenemutation57Invasivetesting58TheprincipalindicationsforInvasivetesting①Advancedmaternalage.②Previouschildwithadenovochromosomeabnormality.③Presenceofstructuralchromosomeabnormalityinoneoftheparents.④FamilyhistoryofageneticdisorderthatmaybediagnosedorruledoutbybiochemicalorDNAanalysis.

⑤FamilyhistoryofanX-linkeddisorderforwhichthereisnospecificprenataldiagnostictest⑥Riskofaneuraltubedefect（suchas,highMSAFPofMSS)⑦ThepositiveresultofMSSandultrasound59ThemethodsofinvasivetestingAmniocentesisChorionicvillussamplingCordocentsis60Amniocentesis(羊膜腔穿刺）Amniocentesisreferstotheprocedureofremovingasampleofamnioticfluidtransabdominallybysyringe.time:15-16thweekafterthefirstdayofthelastmenstrualperiod.Method:transabdominally,locatedintheamnioticcavitybyultrasound.Howtoavoidpollutingwithmother’sblood.Culturingamnioticfluidcells61ThepurposeofamniocentesisTheconcentrationofAFP(AFAFP):NTDandAnencephaly(无脑畸形）.Chromosomeanalysis-KaryotypeandFISHBiochemicaldetection-enzymeanalysisDNAanalysis62Clinicalexample

-prenataldiagnosisofNTDRiskofaneuraltubedefectpregnancyMSSDetectingMSAFPIfMSAFPisatnormallevelItshouldbedetectedbyultrasoundtoexcludeNTDIfMSAFPishigherthannormallevelItshouldbegivenAmniocentesistodetectAFAFP.IfAFAFPishigherthannormallevel,itshouldbeexcludedothercausesofelevatedamnioticfluid–AFAFP,suchasfetalbloodcontamination,Fetaldeath,twinpregnancy,etc.63Thecomplicationofamniocentesismiscarriageinthemidtrimester.talipesequinovarus（马蹄型内翻足）LeakageofamnioticfluidInfectionInjurytothefetusbyneedlepuncture.64ChorionicVillusSampling65Thevilliarederivedfromthetrophoblast,theextra-embryonicpartoftheblastocyst66Thetimeofcvs:10-12thweeksThevillisampled:tertiaryvilli67ThepurposeofCVSChromosomeanalysis-KaryotypeandFISHBiochemicaldetection-enzymeanalysisDNAanalysis68ThefeaturesofCVSTheadvantagesofCVS:thestageofsamplingisearlierthanamniotensis.ThedisadvantagesofCVS:①AFPcannotbeassayedatthisstage.②Therateoffetallossincreasesapproximately1%③about2%ofCVSsamplingsyieldambiguousresultsduetochromosomal

mosaicism(truemosaicismandpseudomosaicism).69Cordocentensis（脐血穿刺）Cordocentensisisaprocedureusedtoobtainasampleoffetalblooddirectlyfromtheumbilicalcordwithultrasonographicguidance.Time:19-21thweekofpregnancy.70cordocentensis71Laboratorytest72LaboratorytestCytogeneticsBiochemicaltestDNAanalysis73PrenataldiagnosisbycytogeneticSummaryClinicaldiagnsisGeneticcounselingLabortarytestingKaryotype-GbandingGeneticcounselingCVSoramniocentesisKaryotype-Gbanding74？probandmotherFemalefetusPrenataldiagnosisbyPCR-sequencing75ALD-XRFeaturesoflaboratorytesting76Problemsinchromosomeanalysis

forprenataldiagnosisMosaicism:referstothepresenceoftwoormorecelllinesinanindividualortissuesample.CulturefailureUnexpectedadversefindings.无法预测表型的染色体畸形，如染色体数目正常但为常见变异体，罕见的重排或标记染色体,应确定双亲的核型，判断是遗传的还是新发的，才能评估这种变异对胎儿的影响。77MosaicismTruemosaicism：isdetectedinmultiplecoloniesfromseveraldifferentprimarycultures.Mosaicismistrulypresentinthefetus，PseudomosaicismAnartifactoccurringintissuecultureMaternalcellcontamination,especiallyCVS.Confinedplacentalmosaicism,inCVSstudies,2%ofpregnancies.Mosaicismispresentintheplacentalbutnotinthefetus.78ThefeaturesofBiochemicaltests

forprenataldiagnosisAdvantages:Biochemicalassayscanbedetecteddirectlythegeneproduct.Isnoteasytobecontaminatedinthebiochemi