摘要 植入前遗传学诊断(Prei娜lantationGeneticDiagnosis,pGD)是 与辅助生殖技术相结合进行的一种遗传学诊断技术PGD在遗传诊 断后将无遗传缺陷的胚胎植入母体子宫,诊断在妊娠发生之前,因此 防止了遗传病的发生杜氏肌营养不良(Duchennemusculardystrophs, DMI))是神经肌肉系统中最常见的X一连锁隐性遗传病,具有较高的 发病率和死亡率,目前尚无有效治疗方法,主要通过产前诊断防止 DMI〕患儿的出生对DMD进行PGD不仅可以防止DMD患儿的出 生,而且能够避免反复流产!引产对孕妇造成的身心伤害,是一种优 于产前诊断的孕前诊断由于PGD的分析材料极其有限,通常只有 1一2个细胞,限制了DMD一PGD的开展,而常用的FISH性别鉴定又 导致正常男性胚胎的销毁,因此开展DMD一PGD,需建立有效的单细 胞扩增方法和更安全的诊断技术 目的:本文旨在通过多重置换扩增(Multipledisplacement amplifieation,MDA)及单倍体分析(haplot0ing)的联合运用,探 讨MDA和单倍体分析在D入ID一PGD中应用的可行性,拟建立一种更 为高效!安全的DMD一PGD方法 方法:选择高杂合度的8个STR位点及3个性别鉴定位点,对 12对无DMD家族史的ICSI/IVF一ET夫妇的胚胎行MDA后的植入前 单体型分析(prei娜lantationgenetich即lotyping,pGH)(其中3对仅 用4个STR及1个性别鉴定位点)首先采用MDA对单卵裂球细胞 进行全基因组扩增,然后对胚胎及其亲本进行单倍体分析在随机假 设母本某一单体型为致病单体型的前提下,对胚胎进行遗传分析 结果:共收集胚胎19枚,其中双原核胚胎n枚,单原核胚胎1 枚,三原核胚胎2枚,不明受精胚胎5枚共取卵裂球51枚,1枚 MDA发生污染,6枚MDA失败,单卵裂球MDA成功率863% (44/51)44枚MDA成功的卵裂球单体型均获成功分析,单倍体分 析成功率100%(44/44),MDA一PGH成功率86.3%(44/51)33枚双 原核胚胎卵裂球中,28枚MDA成功,MDA一PGH成功率84.9% (28召3),平均ADo(alleledropout)和好(amplifieationfailure) 分别为25.3%和3.2%在假设母亲某一单体型携带D胭D3一11号和 44一45号外显子缺失突变的前提下,25枚卵裂球单倍体分析结果示胚 胎为正常男性或DMD女性携带者,另外3枚卵裂球为单倍体,示胚 介硕士中文摘要 胎异常取自异常受精胚胎的18枚卵裂球中,16枚MDA成功, MDA一PGH成功率88.9%(16/18) 结论:1.本实验MDA成功率86.3%,比文献报道稍低,单倍体 分析成功率达100%,可联合用于临床PGD2.对DMD行植入前单 倍体分析,各单体型针对突变选择的STRS中,)2个为有效位点时, 选择4个和选择8个STRS的单倍体分析成功率均可达100%3.单 倍体分析联合性别鉴定,不仅可鉴定DMD女性携带者,还可鉴定正 常男性胚胎,避免FISH性别鉴定造成正常男性胚胎的销毁 关键词植入前遗传学诊断,DMD,多重置换扩增,单倍体分析,STR硕士英文摘要 ABSTRACT PreimPlantationgenetiediagnosis(PGD)15anewgenetiediagnosis technology,develoPingwithassistedreProduetivetechnology.PGD15 PerformedbeforePregnancy,andthenembryoswithoutgeneticdisorders aretransferredtomother.5uterus.50itPreventstheoccurreneeof inheriteddiseases.DuchermemusculardystroP坤(DMD)15anX一linked recessiVeandneuromuscularinheriteddiseasewhiehhasahighfrequency, withhighattackrateandmortalityrate.There15noutilitytherapyProeess uPtonow,50themainmethodtoPreventbirthoftheDMDPatients15 Prenataldiagnosis.PGDofDMDeannotonlyPreventbirthoftheDMI) Patierits,butalsogreatlyhelPtoreducetheP坤sicalandmentalPressure causedbyelectiveabortionandterminationofgravidityoftheabnormal fetusamongPregnantwomen.50PGD15verysignificantinthe PrecautionofDMD.ButthestuffinPGDusuallyonlyhasoneortwo cells,whieh15limitedandPreventstheapPlieationofPGDofDMD,and sexingusingFISHwouldinducediscardingofnormalmaleembryos.50 theestablishnlentofafeasiblesingleeellamPlificationanddiagnosis technology15akeystePinearryingoutPGDofDMDinclinicalPractice. O衍ective:TostudythefeasibilityofmultiPledisPlacement amPlifieation(MDA)eombinedhaplotyPinginDMD一PGD,andto establishaneffieielltandsafePGDofDMD. Method:8STRswithhighheteroZygosityand3sexingmarkers wereehosen,andMl)A一PGHwasPreformedat12couPleswithoutDMD familyhistory.AfterwholegenomeamPlifieationofsingleblastomeres byMDA,haplotyPingwastaken.High一andlow一riskhaplotyPewere suPPosedrandomly,andthenPerformedgenetieanalysisontheembryos. Result:19embryoswereobtained,ineluding11embryoswithZPN, 1embryowith1PN,Zembryoswith3PN,andsembryoswith0PN.sl blastomereswereobtained,inwhich1bastomerewascontaminatedin MDA,6MDAfailed,MDAfromsingleblastomeressueeessratewas 86.3%(44/51).All44blastomereswithsuccessMDAwerehaplotyPed, with100%(44/44)successrateofhaplotyPingand86.3%(44/51)success rateofMDA一PGH.In33blastomeresfromembryoswithZPN,28MDA 111丫硕士英文摘要 succeeded,with84.9%(28/33)sueeessrateofMDA一PGH,theaverage ADOandAFwere25.3%and3.2%,resPectively.OnthehyPothesisthat theeertainhaplotyPeofmothershadthemutationofdeletionofexon3一11 andexon44一45ofDMD,thehaplotyPingresultsof25blastomeres showedthatembryoswerenormalmaleorfemalecarriersandtheother3 werehaploid.In18blastomeresfromembryoswithIPN,3PNandOPN, 16MDAsucceeded,with88.9%(16/18)successrateofMDA一PGH. Conelusion:1.Inthisresearehwegot86.3%successrateofMDA, and100%suecessrateofhaplotyPing,whiehconfirmedthefeasibilityof MDAcombinedhaplotyPinginelinicalPraeticeofPGD.2.ForPGHof DMD,100%suceessrateofhaplotyPingcouldbegotwllen4or8STRs WerechosenonthebasiSofmutation,butatleast2sTRSshouldbefully informativeineveryhaplotyPe.3.HaplotyPingeouldidentifyDMD femaleearrierandnormalmale,byPassingthediseardingofnormalmale embryosfollowingsexingbyFISH. KEYWORDSPreimPlantationgeneticdiagnosis,D入们〕,multiPle disPlacementamPlifieation,haplotyPing,STR IV硕士目录 目录 摘要...,...............................................工 ABSTRACT....................................................工I工 主要实验仪器!试剂与耗材.....................................VI 前言.........................................................l 1.研究对象..............................................3 2.方法......................................................5 2.1多重置换扩增(MDA).....................................5 2.1.1使用R即11一g试剂盒行MDA前的准备....................5 2.1.2多重置换扩增........................................6 2.2单倍体分析...............................#6 2.2.1PCR扩增.............................................7 2.2,2基因扫描及单倍体分析................................9 3.结果.....................................................10 4.讨论.....................................................25 5.结论.....................................................28