应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.

Combining approach with multiplex PCR and MLPA to detect deletion and duplication in DMD patients,carriers, and prenatal diagnosis

Objective Applying multiplex PCR and multiplex ligation-dependent probe amplification (MLPA) in a clinical setting to detect deletions and duplications in the Duchenne/Becker muscular dystrophy (DMD/BMD) gene not only for patients, but also for identification of possible carriers and prenatal diagnosis. Methods Multiplex PCR was used first in patients clinically diagnosed with DMD/BMD to examine 26 exons for a large deletion in the two hot regions of the dystrophin gene. For patients without a deletion detected in the aforementioned regions, MLPA was used to further examine all 79 exons to determine whether a deletion in the remaining non-hot regions or any duplication was present. A similar approach was applied to suspected carriers. In requested prenatal diagnosis cases, specific PCR was used to detect deletions, while MLPA was applied to detect duplications. Results Multiplex PCR was used to examine 26 exons within the two hot regions in the Dystrophin gene for 22 patients with DMD;13 (13/22) had multi-exon deletions. For the 9 patients without deletions in the 26 exons, MLPA was used to examine 79 exons. 3 patients had duplications, 1 patient had a single deletion in exon 18, and no deletions or duplications could be detected in the remaining 5 patients. Of the 16 carriers, 2 out of the 3 that had family history had deletions, while the other 13 carriers were mothers of affected children who were sporadic patients without family history. Of them, 8 mothers were carriers for either deletions or duplications. For prenatal diagnosis, 9 fetuses were examined (one case was twins). Of them, 2 fetuses had familial deletions or duplications detected. These results were verified after induced abortion. In 7 fetuses, no deletions or duplications were detected and all developed into children. Conclusion Multiplex PCR can detect 92.86% of deletions and is useful for prenatal diagnosis of deletions because it is simple, reliable and inexpensive. It can be the first choice in DMD/BMD gene diagnosis. MLPA is important for detecting deletions in non-hot regions/exons and duplications in the DMD/BMD gene, as well as for carrier detection.