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

对Duchenne型肌营养不良症(DMD)孕妇进行携带者检测和产前诊断，并结合三种基因内探针和早孕绒毛标本进行分析。总共分析了13个至少有1个DMD的家庭，其中5个用于携带者检测，其余8个用于产前诊断。从外周血白细胞中提取DNA进行携带者检测(90人)。产前检测取自9个月经周的绒毛标本(8例胎儿)。用适当的限制性内切酶消化DNA，过夜后用1%琼脂糖凝胶进行电泳。按碱性转移法将DNA从凝胶中转移到尼龙膜上。PERT 87探针通过尼克翻译标记为比活度为0.7~1.3×10^9cpm/mug。预杂交4小时后，在41ﾟC下杂交15小时。清洗后的半干膜用X光胶片摄取放射自显影进行限制性片段长度多态性分析。在产前诊断中，胎儿性别是通过Yq12带中Y染色体特定重复序列的快速筛查试验来确定的。在5个家庭的5名高危女性中，2人被诊断为携带者，3人被诊断为非携带者。产前诊断的8个家系的8个胎儿中，男性4例，女性4例。4例男性胎儿均未受影响。在4名女性胎儿中，3名被诊断为非携带者，其余1名的携带者身份没有被嘲笑，因为她的母亲没有提供所有检测的信息。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.