第四届遗传咨询师培训-讲义cytogenetics-richard choy shenyan

CHOYKwongWaiM(Med)Sc,E-mail:概-如何提高产前核型分析质-临床-微阵列比较组杂交技术WhydowestudyhumanMechanismsinGenetic 异常的产前

（绒毛活检Diagnosis-Diagnosis-Green:1qterRed:1p36FluorescenceinsituLocus-specificDNA ysisofcertain edMMSUsedwhenaspecificMMSis orforverificationofaCGHdataVisualizebalancedtranslocationinComplexrearrangementsina如何提高产 核型分析质PartIProcessingofChorionicVillusCleaningofCVSculturesetProcessingofGbandingHighresolutionbandinginInterpretationofRisktofetusofaninvasiveprocedure?1in1inIt1in1in如何提高产 核型分析质ThetissuecultureSampleSampletransportoptimaltransportcondition_avoidextremeReducingthetransportSamplesetCleaningtheCVSCultureandHarvestGDirtyPrecleanedSemi-DirtyAnotherexampleofCleaned1020253010202530ABARotatethedishandSharpcutofthevillionaglasscoverCsameasDRotateCsameasDRotatethedishandcut Cutvilliunderthescopebeforecollagenasetreatment.Singlediscretepc,notearingapartvilli30minin 40minin如何提高产 核型分析质CulturesetTypically,20mgofCVSAftercleaningandremovingthedecidualandmaternaltissueincludingbloodclotsTrypsinfor30minutes,spindownandaddcollegenaseforanother40minutesAfterspindownthesuspension,thecellpelletscanbesetupbytwoindependenttechnologistsasAandBculturesFor10mgstartingmaterial,itcanbesetupintwocoverslipsuchasA1andA2;B1andB2Changemediaon3rdOnday4,If30%confluentwithmultipledividingcellsHarvestonday4orday5,changemediaReadyforCover-SlipDryinginGenieinsituautomatedharvestingThe‘GoldStandard’forprenatalinsitucultureharvesting:overInstallationsExampleCVSBandingBanding AFCulturesetAFCulturesetTypically20mloffluidat>16weeksofgestationin2tubesandsetupbytwotechnologistsAfterspindownandexaminethepelletsize,ingeneral,itcouldberesuspendedin1mlofculturemediaandplace0.5mloneachofthecoverslipandlabelasA1andA2andplaceinincubator;becarefulnottoletthecellsflowoutsideofthecoverslipinthepetridishNextday,Add1mlofmediatotheexistingChangemedia yonday3andeveryotherIf4coloniesandeachcolonyhasmorethan50cellswithshiningdividingcells,proceedtoharvestisExampleofA如何提高产 核型分析质GbandingtechniquecriticalPreparationofSlidesorAgingofslides95oCfor20Trypsin150mLHanksbalancedsaltsolution(1x)(HBSS),usingthepHmeter,adjusttopH7.0-7.2withNaHCO3150mLHBSS+3ml2.5%Trypsin,usingthepHmeter,adjusttopH7.0-7.2withNaHCO3.GiemsaBandingChromosomeBanding GeneralRuleofLevelI_Singlecellabnormality(Singlecellabnormalitiesshouldnotbetakenasanindicationoftruefetalmosaicism)LevelII_Mosaicisminvolvingmultiplecellsconfinedtoasingleflaskshouldnotberegardedasanindicationoftruefetalmosaicism.LevelIII_Mosaicisminvolvingmultiplecellsdistributedovermorethanoneflaskshouldberegardedasastrongindicationoftruefetalmosaicism.MosaicismoftheXX/XYtypeisusuallyduetomaternalcellcontamination.OccasionallyitcanbeafemalefetuswithXYcellsfromanunknownsource.FlaskvsinsitucoverslipIngeneral,ourroutinecytogenetic ysisiscounting15cellswith5cells yzed.Incaseswhereclinicallyrelevantmosaicismised,countingatleast30cellsisperformedtoexcludemosaicismof10%at95%confidencelevel.Ifoneabnormalcellisfoundin30cellscounted,wewillscaleupthenumberofcellscountingto100cellsif临床应FactorsaffectNIPTMolecular

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Oligo+SNP探外(核型分析)vs(微阵列WhatisMicroarrayused用DetecttheCopyNumberVariationinthehumangenome:deletion，duplication. Enablestheidentificationofcriticalgene(s)thathavealteredcopynumberandmayberesponsibleforthedevelopmentandprogressionofaparticulardisease.PrenatalCMAonculturedCVSshowinglossofchromosome8pandgainofPrenatalCMAonculturedCVSshowinglossofchromosome8pandgainofPrenatalCMAonculturedCVSshowinglossofchromosome8pandgainofPrenatalCMAonculturedCVSshowinglossofchromosome8pandgainof CMA Genetic/ChromosomalMicrodeletions/- omericrearrangement:5-15%of->200wellrecognized-CommonCri-du-Chat1q21.2,2q22.3,5q21.2etcmicrodeletion什么是微缺失／微扩增综合征Asyndromecausedbyachromosomaldeletion/duplicationspanningseveralgenesthatistoosmalltobedetectedunderthemicroscopeusingconventionalcytogeneticmethods.Dependingonthesizeofthedeletion,othertechniques,suchasFISHorothermethodsofDNA ysisarerequiredtoidentifythedeletion>200microdeletionsyndromesin>80microduplicationRatioaboutPartofGenomicdisorders 267differentgenomicloci,21179什么是微缺失／微扩增综合征LupskiJRand iewiczP.PLoSGenetCopynumbervariation组拷贝数变 Angelman:Alagille:Rett:Sotos:CHARGE:AreTheyCommonclinicalGrowthDevelopmentaldelay(DD)/In lectualdisability(ID) Seizures癫痫Hypotoniahypertonia肌张高FunctionalPhysicalPhysical ComingdowntoonesinglediagnosisPrenataldiagnosisorsuspicionbysonographicassessmentat20weekisalmostimpossibleinthemajorityofcases.HighIncidenceIncidenceoutof100,000Live1Nussbaumetal.2007.ThompsonandThompsonGeneticsinMedicine(7thedn).OxfordSaunders:Philadelphia 3

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Del(6)(q24toDel(6)(q24to34OSCAR-high34OSCAR-highrisk,NT8.49mm,cystichygromawithSCedema.CVS:aCGH:USG917-NFUSG921-normalmo