Kazemi Mohammad, Salehi Mansoor, Kheirollahi Majid
Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
Medical Genetic Center of Genome, Isfahan, Iran.
Int J Mol Cell Med. 2017 Winter;6(1):13-21. Epub 2017 Feb 15.
This study aimed to verify the reliability of the 7 tissue differentially methylated regions used in the methylated DNA immunoprecipitation (MeDIP) real- time quantitative polymerase chain reaction (real-time qPCR) based approach of fetal DNA in maternal blood to diagnosis of fetal trisomy 21. Forty pregnant women with high risk pregnancy who were referred after first or second trimester screening tests, were selected randomly. For each sample whole DNA extraction (mother and fetus), fragmentation of DNA, immunoprecipitation of methylated DNA and real- time qPCR using 7 primer pairs was performed. D-value for each sample was calculated using the following formula D = -4.908+ 0.254 X+ 0.409 X+ 0.793 X+ 0.324 X+ 0.505 X+ 0.508 X+ 0.691 X. In all normal cases, D value was negative, while it was positive in all trisomy cases. Therefore, all normal and trisomy 21 cases were classified correctly which correspond to 100% specificity and 100% sensitivity for this method. The MeDIP real-time qPCR method has provided the opportunity for noninvasive prenatal diagnosis of fetal trisomy 21 to be potentially employed into the routine practice of diagnostic laboratories.
本研究旨在验证基于甲基化DNA免疫沉淀（MeDIP）实时定量聚合酶链反应（实时qPCR）方法检测孕妇血液中胎儿DNA以诊断胎儿21三体综合征时所使用的7个组织差异甲基化区域的可靠性。随机选取40名在孕早期或孕中期筛查试验后被转诊的高危孕妇。对每个样本进行全DNA提取（母亲和胎儿）、DNA片段化、甲基化DNA免疫沉淀以及使用7对引物进行实时qPCR。每个样本的D值使用以下公式计算：D = -4.908 + 0.254X + 0.409X + 0.793X + 0.324X + 0.505X + 0.508X + 0.691X。在所有正常病例中，D值为阴性，而在所有三体病例中D值为阳性。因此，所有正常和21三体病例均被正确分类，该方法的特异性和敏感性均为100%。MeDIP实时qPCR方法为胎儿21三体综合征的非侵入性产前诊断提供了机会，有望应用于诊断实验室的常规实践中。
