Tan Jianxin, Zhang Jingjing, Sun Ruihong, Jiang Zhu, Wang Yuguo, Ma Dingyuan, Jiao Jiao, Chen Hao, Lin Yingchun, Zhang Qinxin, Xu Zhengfeng, Hu Ping
Department of Prenatal Diagnosis, Women's Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing 210004, People's Republic of China.
Department of Laboratory Medicine, The First School of Clinical Medicine, Nanjing Medical University, Nanjing 210029, People's Republic of China.
Clin Chim Acta. 2023 Aug 1;548:117496. doi: 10.1016/j.cca.2023.117496. Epub 2023 Jul 20.
Spinal muscular atrophy (SMA) is an autosomal recessive inherited neuromuscular condition caused by biallelic mutations in the survival of motor neuron 1 (SMN1) gene. A homozygous deletion of the SMN1 gene accounts for approximately 95-98% of SMA patients. A highly homologous gene survival motor neuron 2 (SMN2) can partially compensate for SMN1 deletion, and its copy number is associated with disease severity. Population-based carrier screening by simultaneous quantification of SMN1 and SMN2 copy numbers is the best method to prevent SMA.
In this study, a total of 516 samples were re-tested for the SMN1 copy number by using quantitative polymerase chain reaction (qPCR), multiplex ligation probe amplification (MLPA), droplet digital PCR (ddPCR), high-resolution melting (HRM) analysis, and PCR-based capillary electrophoresis (PCR/CE) simultaneously. Then, the performance of these methods was compared by using MLPA results as the reference.
The results of qPCR, ddPCR, HRM, and PCR/CE in detecting heterozygous deletion of SMN1 exon 7 and the results of ddPCR, HRM, and PCR/CE in detecting ≥2 copies of SMN1 exon7 are totally consistent with those of MLPA. The sensitivity and specificity of qPCR for detection of 2 copies of SMN1 exon 7 were 99.7% and 98.8%, respectively. The sensitivity and specificity of qPCR for detection of >2 copies of SMN1 exon 7 were 96.3% and 99.8%, respectively. Compared with the MLPA results, the sensitivity and specificity of qPCR and HRM for detection of heterozygous deletion of SMN1 exon 8 were 100% and 100%, respectively. They were 99.4% and 100%, respectively for detection of 2 copies, and 100% and 100%, respectively for detection of >2 copies. The results of PCR/CE in detecting SMN1 exon 8 were consistent with those of MLPA.
All these four methods show excellent performance in detecting heterozygous deletion of SMN1 exon 7. All PCR/CE results are totally concordant with those of MLPA. As the most cost-effective method, qPCR also shows high sensitivity and specificity in detecting SMN1. Taken together, our study provides useful information to select appropriate methods for SMA carrier screening.
脊髓性肌萎缩症（SMA）是一种常染色体隐性遗传性神经肌肉疾病，由运动神经元存活基因1（SMN1）的双等位基因突变引起。SMN1基因的纯合缺失约占SMA患者的95 - 98%。高度同源的运动神经元存活基因2（SMN2）可部分补偿SMN1的缺失，其拷贝数与疾病严重程度相关。通过同时定量SMN1和SMN2拷贝数进行基于人群的携带者筛查是预防SMA的最佳方法。
在本研究中，共516份样本同时采用定量聚合酶链反应（qPCR）、多重连接探针扩增技术（MLPA）、微滴数字PCR（ddPCR）、高分辨率熔解曲线分析（HRM）以及基于PCR的毛细管电泳（PCR/CE）对SMN1拷贝数进行重新检测。然后，以MLPA结果为参照比较这些方法的性能。
qPCR、ddPCR、HRM以及PCR/CE检测SMN1外显子7杂合缺失的结果，以及ddPCR、HRM和PCR/CE检测≥2个拷贝SMN1外显子7的结果与MLPA结果完全一致。qPCR检测2个拷贝SMN1外显子7的灵敏度和特异性分别为99.7%和98.8%。qPCR检测>2个拷贝SMN1外显子7的灵敏度和特异性分别为96.3%和99.8%。与MLPA结果相比，qPCR和HRM检测SMN1外显子8杂合缺失的灵敏度和特异性分别为100%和100%。检测2个拷贝时分别为99.4%和100%，检测>2个拷贝时分别为100%和100%。PCR/CE检测SMN1外显子8的结果与MLPA结果一致。
这四种方法在检测SMN1外显子7杂合缺失方面均表现出色。所有PCR/CE结果与MLPA结果完全一致。作为最具成本效益的方法，qPCR在检测SMN1时也显示出高灵敏度和特异性。综上所述，我们的研究为选择合适的SMA携带者筛查方法提供了有用信息。
