Establishment of the method for prenatal diagnosis of genetic disease using DNA probes and chorionic villus sampling

应用三种基因内探针结合孕早期绒毛取样进行Duchenne型肌营养不良症（DMD）的携带者检测和产前诊断。共分析13个DMD家系，其中5个家系进行携带者检测，8个家系进行产前诊断。从外周白色血细胞中提取DNA进行携带者检测（90例）。对于产前检测，从9个月经周（8个胎儿）的绒毛取样获得的绒毛中提取。用适当的限制酶消化DNA，然后在1%琼脂糖凝胶中电泳过夜。根据碱转移法的方案将DNA从凝胶转移到尼龙膜上。PERT 87探针通过切口平移标记，比活度为0.7 - 1.3 x 10^9cpm/mug。预杂交4小时后，在41 ℃下将膜杂交15小时。清洗后，将半干燥的膜暴露于X射线片以制备用于限制性片段长度多态性分析的放射自显影。在产前诊断的情况下，胎儿性别是通过快速筛查试验确定的Y染色体特异性重复序列带Yq 12。在5个家庭的5名高危女性中，2名被诊断为携带者，3名被诊断为非携带者。产前诊断8个家系8例胎儿，男4例，女4例。确定所有4只雄性胎仔均未受影响。在4例女性胎儿中，3例被诊断为非携带者，其余1例的携带者状态未被嘲笑，因为她的母亲对所有测试都没有提供信息。建立了妊娠早期DMD的产前诊断方法。这种方法也适用于其他遗传性疾病。

Carrier detection and prenatal diagnosis of Duchenne muscular dystrophy(DMD) were performed combined with the use of three intragenic genomic probes and chorionic villus sampling in early pregnancy. Total of 13 families with at least one DMD were analyzed, 5 were for carrier detection, the rest 8 for prenatal diagnosis. DNA was extracted from peripheral white blood cells for carrier testing (90 individuals). For prenatal detection, it was extracted from chorionic villi obtained by chorionic villus sampling at 9 menstrual weeks(8 fetuses). DNA was digested with appropriate restriction enzyme followed by overnight electrophoresis into 1% agarose gels. DNA was transferred from the gel to nylon membrane according to a protocol of alkaline transfer method. The PERT 87 probes were labeled by nick translation to a specific activity of 0.7 to 1.3 x 10^9cpm/mug. The membranes were hybridized 15 hours at 41 ﾟC after 4 hours prehybridization. After washing the semi-dried membranes were exposed to X-ray films to make autoradiograms for restriction fragment length polymorphisms analysis. In instances of prenatal diagnosis fetal sex was determined by a rapid screening test with a Y chromosome-specific repeat sequence in band Yq12. Of the 5 at-risk females in 5 families for scarier detection 2 were diagnosed as carriers, 3 as non-carriers. Out of 8 fetuses from 8 families for prenatal diagnosis 4 were males and 4 were females. All of 4 male fetuses were determined to be unaffected. Of 4 female fetuses, 3 were diagnosed as non- carrier, carrier status of the remaining one was not derided because her mother was not informative for all testings. The method for prenatal diagnosis of DMD in early pregnancy was established. This method is applicable to other genetic diseases.