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文献汇报赖方秾2016.1.18借助二代测序，以及关联分析，使后代避免携带单基因遗传病以及染色体异常背景介绍：IVF：ICSI：背景介绍：WHATISPGD?PGD(PreimpantationGeneticDiagnosis)isusedtodetectknowngeneticmutationsbeforeimplantation.GeneticDiseaseDetectionSinglegenetestingisusedforpatientsthathaveaknowngenemutationandcanhelpidentifythosegeneticdiseasesineachembryobeforeimplantation.TestablegeneticdiseasesincludeCysticFibrosis,TaySachsDisease,FragileXSyndrome.Byidentifyingembryoswiththesegeneabnormalities,wecanavoidimplantingthembeforepregnancyoccurs.背景介绍：WHATISPGS?PGS(PreimpantationGeneticScreening)isacompletescreeningofanembryochromosomes.ChromosomeScreeningThemostcommonerrorinembryosiscalledaneuploidy.Thismeanstherearetoomanyortoofewchromosomes.ScreeningembryoswithPGSpriortotransferhelpstoachievehigherimplantationrates,fewerpregnancylossesandlowerstheriskofhavingtoconsiderpregnancytermination.背景介绍：背景介绍：背景介绍：DNAMutationandChromosomalVariationBasesubstitutiontransitiontransversionDeletionmutationInsertionmutationDNAMutationA-G,T-CA-T,A-CG-T,G-CChromosomalVariationStructuralVariationinversiontranslocationDuplicationDeletionNumberVariationEuploidyPolyploidyAneuploidyTriploidyTeraploidy23x323x4MonosomyTrisomyaneuploidyChromosomenumberVariationaneuploidymultiploidyHutchinson-GilfordProgeriaMutationChromosomalVariationPrader-WillisyndromesDown'ssyndromeStructuralVariationChromosomalNumberVariationLinkageanalysisPGD方法：1.PCR2.FISH3.array-CGH4.SNP-arrayBiopsy方法：1.PolarBody(Day1/Day2)2.EarlyStageBlastomere(Day3)3.Blastocyst(Day5/Day6)1.PCR-PGD(Pointmutation,Lowthroughput,Amplification,Primerdesigning…)1.FISH-PGD(Aneuploidy,Falsepositive,Falsenegative,Timecost…)3.arrayCGH(comparativegenomichybridization)-PGD(Aneuploidy,Knownsequence,Lowresolution…)4.SNParray-PGD(Aneuploidy&Mutation,KnownSNP,Linkageanalysisratherthantargetmutation…)技术路线结论通过测序，染色体非整倍性以及关联分析检测突变等位基因的方法家族患病史及囊胚活检Hereditarymultipleexostoses（HME）HereditarydominantdisorderFrame-shiftpointmutationc.233delCatEXT2X-linkedchromosomerecessivehereditydisorderPointmutationc.T1085GatEDA1Hypohidroticectodermaldysplasia0.1X1000通过MARSALA的方法同时检测SNV和CNVE05,E06,E09,E10,E11,E12carriedmutatedSNVmonosomydeletiontrisomyE06,E07,E08,E09,E12,E13,E14,E16,E18havechromosomeabnormality1.PCRLowdepth结论通过MARSALA的方法同时检测SNV和CNVE01,E03carriedmutatedSNVE03haveaneuploidy通过MARSALA进行关联分析通过MARSALA进行关联分析E05,E06,E09,E10,E11,E12have~10concordantSNPsclosedtothetargetmutation通过MARSALA进行关联分析通过MARSALA进行关联分析E01,E03have~10concordantSNPsclosedtothetargetmutation囊胚PGD/PGS结果与极体活检结果进行比较囊胚PGD/PGS结果与极体活检结果进行比较E01,E03have~10concordantSNPsclosedtothetargetmutationE03haveaneuploidyNormalPaternal?Spermaneuploidy?囊胚PGD/PGS结果与极体活检结果进行比较通过MARSALA进行关联分析Transferred桑格测序，CGHarray，STR分析进行验证E09?AllelicDropout?E05,E06,E09,E10,E11,E12carriedSTRlinkedwithmutatedallele(consistent)E12(monosomy8),E14(deletionorduplication3),E18(trisomy22)monosomydeletiontrisomyE06,E07,E08,E09,E12,E13,E14,E16,E18havechromosomeabnormality胎儿验证MARSALA花费普通测序检测点突变：30x左右测序深度MARSALA：0.1x（人大约0.3G数据,30$/sample），IlluminaHiSeq2500测序平台根据作者以往经验，MALBAC做关联分析2X测序深度足以MARSALA的局限性分析过程因事先需要知道父母双方携带致病突变的信息，所以新生突变（denovomutation）不能检测。需要使用特异性的引物，也是局限性的潜在可能，虽然花费不高，但可能耗时间，但在PGD诊断以前引物信息可能已经确定，也可能不需要额外时间。个人认为：如应用于临床，还应提高单细胞或少量细胞扩增的成功率制定标准化的数据评估方法，如对CNV异常进行定义收集东亚人遗传背景清楚的遗传病SNV信息，整合建立数据库，对确切机理的遗传病新发突变进行避免标准化的扩增方法，在测序成本下调的情况下，合理增加测序深度谢谢!