【摘要】 目的:应用多重连接依赖式探针扩增法(MLPA)和聚合酶链式限制性片段长度多态性分析(PCR-RFLP)技术对30例临床疑似脊肌萎缩症(SMA)患者进行基因诊断,并对两种方法进行比较。方法:盐析方法提取30例家系成员外周血DNA,常规PCR方法扩增SMN7、8外显子,用DralⅠ和DralⅠ酶切PCR产物,琼脂糖凝胶电泳检测PCR及酶切产物。同时利用MLPA试剂盒P021方法进行验证比较。结果:经PCR-RFLP方法判断22例SMN1第7+8号外显子纯合缺失患儿和2例Ex7纯合缺失患儿,与MLPA方法一致;其余6例PCR-RFLP方法未见异常家系,经MLPA方法分析发现1例患儿和2例母亲为SMN1外显子7、8杂合缺失携带者;MLPA方法还在SMA家系中发现3例"2+0"型携带者。SMN2拷贝数在SMA患者中以4、5为主,携带者以2、3为主,正常人以1、2多见,其各拷贝数的频率分布在患者组与携带者组(χ2=30.694)、患者组与正常人组(χ2=21.997)中差异有统计学意义(P<0.01),而在携带者与正常人组差异无统计学意义(χ2=3.5,P>0.05)。结论:与PCR-RFLP相比,MLPA更加简单、准确、高效,还能够准确定量SMN1、SMN2,是一种高效的遗传病基因诊断方法。更多还原

【Abstract】 Objective: To perform genetic diagnosis for 30 suspected patients with spinal muscular atrophy (SMA) using multiplex ligation dependent probe amplification (MLPA) and PCR-restriction fragment length polymorphism (PCR-RFLP), and compare the results of the two methods. Methods: The genomic DNA was isolated using salting-out method from peripheral blood of each subject from 30 families.Exon 7 and 8 of SMN genes were amplified by allele specific PCR. The PCR products were digested with DralⅠand DralⅠdigested PCR products. Simultaneously, the DNA samples were analyzed by SALSA MLPA Kit P021. Results: Both PCR-RFLP and MLPA analysis showed the same that 22 patients with exon 7 and 8 homozygous deletion, and 2 patients with only exon 7 homozygous deletion of SMN1. The other 6 cases presented no homozygous deletion by PCR-RFLP, but one child and two mothers of them were detected heterozygous by MLPA. Also MLPA analysis found three "2+0"carriers from 3 families. The data also showed that the SMN2 copy numbers were mainly 4 or 5 in SMA patients, while the carriers and the normal individuals were 2 or 3 and 1 or 2 copies respectively. There was clear statistical significance in the group of patient -carrier and group of patient-normal individuals (P < 0.01). The carrier -normal group appeared no statistical significance (P > 0.05). Conclusion: Compared with PCR-RFLP, MLPA is more convenient, precise, high-effective, and it can accurately quantitative analysis of SMN1 and SMN2. MLPA is a kind of technique of genetic diagnosis for common genetic disease.更多还原