多重置换扩增(MDA)结合荧光PCR对植入前胚胎进行性别诊断的研究

Multiple Displacement Amplification and Subsequent Fluorescent-PCR for Gender Determination in Preimplantation Genetic Diagnosis(PGD)

目的:建立多重置换扩增(MDA)联合荧光PCR对植入前胚胎进行性别诊断的方法,为开展性连锁遗传病的性别筛选提供实验依据。方法:在行IVF-ET助孕的患者中,选取废弃的第3日新鲜或冷冻后的2PN受精胚胎。通过显微操作活检得到单个卵裂球细胞,采用MDA方法对单个/2个卵裂球细胞行全基因组扩增,然后采用荧光PCR方法对AMEL、X22、SRY和XHPRT 4个性染色体相关基因或STR位点进行扩增,对其扩增产物行毛细管电泳检测,分析电泳结果确定植入前胚胎的性别。以父母的外周血单个淋巴细胞作为对照进行分析。结果:①采用蛋白酶K法(n=21)和碱法(n=17)对卵裂球细胞进行裂解行MDA,扩增成功率未见统计学差异(85.7%vs 82.4%,P>0.05);单个卵裂球组(n=15)和2个卵裂球组(n=23)MDA扩增成功率亦未见统计学差异(80.0%vs87.0%,P>0.05)。②利用MDA方法对15个胚胎的单个/2个卵裂球进行扩增,扩增成功率为86.7%(n=13)。13个胚胎中7个为男性,6个为女性,性别检测成功率100%;等位基因脱扣(alleledropout,ADO)发生率为7.7%。结论:PGD中MDA是有效且可靠的全基因组扩增方法,MDA结合荧光PCR可以用于性连锁遗传病的性别筛选、致病基因检测和对胚胎进行HLA分型。

Objective： To establish a preimplantation genetic gender diagnosis method that combined multiple displacement amplification（MDA） with fluorescent PCR so that to provide experimental basis for the sex-linked genetic disease screening of embryos.Methods： The discarded 2 PN derivate embryos of day 3 were collected after embryo transfer and cryopreservation from the patients conducted IVF-ET in our center from Oct.2009 to Apr.2010.One or two blastomeres were biopsied by micromanipulation and whole genome of the blastomere was amplified by multiple displacement amplification（MDA） method,then 4 sex chromosome related genes or STR loci markers（AMEL,X22,SRY and XHPRT） were amplified using fluorescence PCR method and the amplified products and the amplification products were sized using Capillary electrophoretogram to determine the numbers of sex chromosomes.The embryo sex was determined using parent lymphocyte＇s results as positive control.Results： 1） There was no statistical differences of amplification success rate between the proteinase K（n=21） or Alkaline（n=17） lysing methods（85.7% vs.82.4%,P0.05） and also between single blastomere（n=15） and the two blastomeres（n=23） groups（80.0% vs 87.0%,P0.05）.2） The MDA amplification success rate was 86.7%（n=13） in 15 samples of 1 or 2 blastomeres.There were 7 male embryos and 6 female embryos in the 13 embryos.The fluorescent PCR efficiency was 100% and ADO rate was 7.7%.Conclusion： MDA is an efficient and valid whole genome amplification method for PGD.MDA combined with fluorescent PCR can be used for gender determination in the sex-linked genetic disease screening,monogenic inheritance disorder and HLA-typing of embryos.