Non-InvasivePre-ImplantationAneuploidyScreeningandDiagnosisofBetaThalassemiaIVSⅡMutationusingSpentEmbryoCultureMedium
ABSTRACTBackground:Cell-freenuclearDNAhasbeenisolatedfromspentembryoculturemedium.WhetherthissmallamountofDNAcanbeamplifiedatthewholegenomelevelandtheconcordancerateofkaryotypesandspecificallelesbetweenbiopsiedcellsandmediahasnotbeenevaluated.
Methods:Sevencoupleswererecruited,88donatedembryosandtheircorrespondingmediawerecollectedforwholegenomeamplification(WGA).TheefficiencyofWGA,theconcordanceofchromosomestatusandtheHBBgeneIVSⅡallelebetweenbiopsiedcellsandmediawereinvestigated.
Results:AfterWGA,theDNAdetectionratewas90.90%withameanconcentrationof26.15ng/μl.Thefullchromosomeconcordanceratebetweenbiopsiedcellsandmediumwas64.52%,anditincreasedto90.00%fordiploidblastocystsamples.AnalysisofthemutatedIVSⅡlocusandSNPlinkageverifiedthattheDNApresentinthemediumoriginatedfromembryoniccells.
Conclusion:WeconfirmedthatnuclearDNAispresentinspentculturemedium,andthatthemajorityofthisDNAcanbeamplifiedforsubsequentanalysis.Ourresultsshowedthatnon-invasiveembryogenetictestingatthechromosomal-levelusingmediumcanconcordanttothebiopsiedcells,butitneedsfurtheroptimizedbeforeuseinclinicalapplications.KeyWords:Pre-implantationgeneticdiagnosisandscreening;blastocyst;spentculturemedium;wholegenomeamplification;concordanceJUSTAC
摘要
背景：先前已经由胚胎培养后的培养液中分离得到细胞游离核酸DNA。是否这些少量的DNA能够被用于全基因组水平的扩增，并且活检细胞和培养液在核型和特异性等位基因的一致率方面尚未得到评估。
方法：获得七对夫妇志愿者，88枚捐献胚胎，收集相应培养液用于全基因组扩增。
方法：研究活检细胞和培养液间的WGA效率，染色体状态一致性，HBB基因IVSⅡ等位基因。
结果：WGA结束后，DNA检测率为90.90%，平均浓度为26.15ng/μl。活检细胞和培养液间全部染色体一致率为64.52%，对于二倍体囊胚样品，可以增加到90%。突变的IVSⅡ基因座和SNP连锁的分析证实存在于培养基中的DNA来源于胚胎细胞。结论：我们证实核算DNA存在于培养过胚胎的培养液，这些DNA大部分能够被扩增用于后续分析。我们的结果显示，使用培养过胚胎的培养液在染色体水平进行的非侵入性胚胎基因检测能够获得与活检细胞一致的结果，但在临床应用前还需要进一步优化。
关键词：植入前遗传学诊断和筛查;囊胚;培养过胚胎的培养液;全基因组扩增;一致性
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