【摘要】 目的:采用三重PCR技术检测缺失型杜氏肌营养不良(Duchenne muscular dystrophy,DMD)基因部分外显子和性别鉴定位点amelogenin基因,探讨该技术对DMD家系中致病基因携带者进行植入前遗传学诊断(preimplantation genetic diagnosis,PGD)的可行性。方法:显微操作取正常男性、正常女性和DMD基因8号、47号外显子缺失型DMD患者单个淋巴细胞及无DMD家族史夫妻行体外授精-胚胎移植(in vitro fertilization pre-embryo transfer,IVF-ET)术后废弃的单个卵裂球细胞,采用三重PCR技术扩增DMD基因8号、47号外显子和amelogenin基因。结果:64枚正常人单个淋巴细胞的DMD基因8号、47号外显子和amelogenin基因扩增阳性率分别为93.8%,93.8%和95.3%,假阳性率为2.8%;48枚8号外显子缺失型患者单个淋巴细胞的DMD基因47号外显子和amelogenin基因扩增阳性率均为95.8%,假阳性率为3.3%;48枚47号外显子缺失型患者单个淋巴细胞的DMD基因8号外显子和amelogenin基因扩增阳性率均为95.8%,假阳性率为0;40枚单卵裂球细胞DMD基因8号、47号外显子和amelogenin基因扩增阳性率分别为82.5%,80.0%和77.5%,假阳性率为0。结论:本研究所建立的单细胞三重PCR技术具有较高的扩增阳性率和特异性,有望在缺失型DMD家系中进行PGD的临床应用。更多还原

【Abstract】 ObjectiveTo detect two exons of Duchenne muscular dystrophy (DMD) gene and a gender discrimination locus amelogenin gene by single cell triplex PCR, and to evaluate the possibility of this technique for preimplantation genetic diagnosis (PGD) in DMD family with DMD deletion mutation. MethodsSingle lymphocytes from a normal male, a normal female, two DMD patients (exon 8 and 47 deleted, respectively) and single blastomeres from the couples treated by the in vitro fertilization pre-embryo transfer (IVF-ET) and without family history of DMD were obtained. Exons 8 and 47 of DMD gene were amplified by a triplex PCR assay, the amelogenin gene on X and Y chromosomes were co-amplified to analyze the correlation between embryo gender and deletion status. ResultsIn the normal single lymphocytes, the amplification rate of exons 8 and 47 of DMD and amelogenin gene were 93.8%, 93.8%, and 95.3%,respectively. The false positive rate was 3.3%. In the exon 8 deleted DMD patient, the amplification rate of exon 47 of DMD and amelogenin gene was 95.8%, and the false positive rate was 3.3%. In the exon 47 deleted DMD patient, the amplification rate of exon 8 of DMD and amelogenin gene was 95.8%, and the false positive rate was 0. In the single blastomeres, the amplification rate of exons 8 and 47 of DMD and amelogenin gene was 82.5%, 80.0% and 77.5%, respectively, and the false positive rate was 0. ConclusionThe single cell triplex PCR protocol for the detection of DMD and amelogenin gene is highly sensitive, specific and reliable, and can be used for PGD in those DMD families with DMD deletion mutation.更多还原