目的:脊髓性肌萎缩症(SMA)是由存活运动神经元1(SMN1)的纯合子缺失和复合杂合突变引起的神经肌肉疾病,严重程度与存活运动神经元2(SMN2)的拷贝数有关。本研究旨在开发一种快速、全面的SMA诊断方法。
方法:采用扩增难治性突变系统聚合酶链反应-毛细管电泳(ARMS-PCR-CE)方法,对292例临床疑似SMA患儿和394名家族成员进行检测,针对19个报告的突变,并将结果与多重连接依赖性探针扩增(MLPA)的结果进行了比较。通过SMN1远程PCR和Sanger测序进一步证实了具有鉴定的点突变的个体。
结果:共有202名SMA儿童,272个运营商,在这项研究中确定了212名正常人。SMN1和SMN2中外显子7和8的R值分布在这些队列中没有发现差异,变异系数始终低于0.08。为了检测SMN1和SMN2中的外显子7和8拷贝数,ARMS-PCR-CE结果与MLPA的结果一致。约4.95%(10/202)的研究患者具有复合杂合突变。
结论:ARMS-PCR-CE检测是一个全面、快速,同时检测SMN1/SMN2中外显子7和8的拷贝数,以及SMN1中的19点突变和SMN2中的2个增强子的SMA的准确诊断方法。这种方法可以有效地减少诊断的时间范围,促进早期干预和预防出生缺陷。
OBJECTIVE: Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by homozygous deletion and compound heterozygous mutations in survival motor neuron 1 (SMN1), with severity tied to the copy number of survival motor neuron 2 (SMN2). This study aimed to develop a rapid and comprehensive method for the diagnosis of SMA.
METHODS: A total of 292 children with clinically suspected SMA and 394 family members were detected by the amplification refractory mutation system polymerase chain reaction-capillary electrophoresis (ARMS-PCR-CE) method, which targeted 19 reported mutations, and the results were compared with those in multiplex ligation-dependent probe amplification (MLPA). Individuals with identified point mutations were further confirmed by SMN1 long-range PCR and Sanger sequencing.
RESULTS: A total of 202 children with SMA, 272 carriers, and 212 normal individuals were identified in this study. No difference was found in the R-value distribution of exons 7 and 8 in SMN1 and SMN2 among these cohorts, with coefficients of variation consistently below 0.08. To detect exon 7 and 8 copy numbers in SMN1 and SMN2, the ARMS-PCR-CE results were concordant with those of MLPA. Approximately 4.95 % (10/202) of the study patients had compound heterozygous mutations.
CONCLUSIONS: The ARMS-PCR-CE assay is a comprehensive, rapid, and accurate diagnostic method for SMA that simultaneously detects copy numbers of exons 7 and 8 in SMN1/SMN2, as well as 19 point mutations in SMN1 and 2 enhancers in SMN2. This approach can effectively reduce the time frame for diagnosis, facilitating early intervention and preventing birth defects.