Yang L, Cao Y Y, Qu Y J, Bai J L, Wang H, Jin Y W, Han Y L, Song F
Department of Pediatrics, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China.
Zhonghua Yi Xue Za Zhi. 2017 Feb 14;97(6):418-423. doi: 10.3760/cma.j.issn.0376-2491.2017.06.004.
To detect the subtle variant of survival motor neuron gene 1(SMN1) by Sanger sequencing, and to assess the value of Sanger sequencing for the diagnosis of spinal muscular atrophy(SMA) with compound heterozygous mutation of SMN1. Fifty-two patients suspected SMA were recruited by the Capital Institute of Pediatrics from Jan.2014 to June.2016. PCR was used for amplifying exon7 of SMN1 and SMN2 in 52 patients. Natural different base peaks on the sequencing chromatogram in the SMN1 and SMN2 within the amplified segments were identified with Sanger DNA sequencing to detect the homozygous deletion or heterozygous deletion of SMN1. Then we screened the SMN1 subtle variants in heterozygous deletion patients by genomic Sanger sequencing for the other SMN exons. At last, multiplex ligation-dependent probe amplification(MLPA) was carried out to confirm the results of SMN1 heterozygous deletion, and T-A cloning confirmed the subtle variants were located in SMN1. Forty-seven of 52 cases were homozygous deletion of SMN1, while 5 cases were heterozygous deletion which were confirmed by MLPA.Then, by genomic and T-A cloning sequencing, five SMN1 subtle mutations were separately identified in 5 cases of heterozygous deletion. Sanger sequencing is an effective method for the clinical diagnosis of compound heterozygous mutation of SMN1, and is meaningful for improving genetic diagnosis rate of SMA.
采用桑格测序法检测生存运动神经元基因1（SMN1）的微小变异，并评估桑格测序法对诊断具有SMN1复合杂合突变的脊髓性肌萎缩症（SMA）的价值。2014年1月至2016年6月，首都儿科研究所招募了52例疑似SMA患者。采用聚合酶链反应（PCR）扩增52例患者的SMN1和SMN2基因第7外显子。通过桑格DNA测序法识别扩增片段内SMN1和SMN2测序色谱图上自然出现的不同碱基峰，以检测SMN1的纯合缺失或杂合缺失。然后，我们通过对其他SMN外显子进行基因组桑格测序，在杂合缺失患者中筛选SMN1微小变异。最后，进行多重连接依赖探针扩增（MLPA）以确认SMN1杂合缺失的结果，T-A克隆证实微小变异位于SMN1。52例患者中47例为SMN1纯合缺失，5例为杂合缺失，经MLPA证实。然后，通过基因组和T-A克隆测序，在5例杂合缺失患者中分别鉴定出5个SMN1微小突变。桑格测序是诊断SMN1复合杂合突变的有效临床方法，对提高SMA的基因诊断率具有重要意义。
