Diagnosis of Spinal Muscular Atrophy: A Simple Method for Quantifying the Relative Amount of Survival Motor Neuron Gene 1/2 Using Sanger DNA Sequencing
Diagnosis of Spinal Muscular Atrophy: A Simple Method for Quantifying the Relative Amount of Survival Motor Neuron Gene 1/2 Using Sanger DNA Sequencing
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脊髓性肌萎缩症的诊断：使用桑格 DNA 测序定量运动神经元存活基因 1/2 相对量的简单方法
DOI：
10.4103/0366-6999.247198
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发表时间：
2018-12
影响因子：
6.1
通讯作者：
Fang Song
中科院分区：
文献类型：
--
作者：
Yan‑Yan Cao;Wen‑Hui Zhang;Yu‑Jin Qu;Jin‑Li Bai;Yu‑Wei Jin;Hong Wang;Fang Song
背景：脊髓性肌萎缩症（SMA）是由存活运动神经元基因1 （SMN1）纯合缺失或复合杂合突变引起的，是诊断SMA的关键。本研究旨在建立并评价一种新的SMA诊断方法。方法：共纳入1494例疑似SMA患儿。本研究于2003 - 2014年对1364例疑似SMA患儿采用传统的MLPA和TA克隆方法，2015 - 2016年对130例疑似SMA患儿采用新策略进行检测，并采用MLPA和TA克隆相结合的方法进行验证。采用聚合酶链反应，用相同的引物同时扩增SMN1和SMN2。突变测量软件通过计算桑格测序中的等位基因比例来检测和量化SMN1变异。最后，比较了两种策略的周转时间和成本。结果：1364例疑似SMA患儿中，SMN1纯合缺失576例，SMN1复合杂合突变27例。130例中，SMN1纯合子缺失59例，杂合子缺失8例，SMN1在第7外显子上的特异性峰值比例分别为34.6±1.0%和25.5±0.5%，分别为1:2和1:3。在6/8的杂合缺失病例中，检测到p.Ser8Lysfs *23（2例）、p.Leu228*、p.Pro218Hisfs *26、p.Ser143Phefs*5、p.Tyr276His等5个变异，突变等位基因比例分别为31.9%、23.9%、37.6%、32.8%、24.5%、23.6%，与SMN1外显子7特异位点相似，提示这些细微突变位于SMN1。这些结果与MLPA和TA克隆结果一致。两种策略的周转时间分别为7.5 h和266.5 h。新战略的成本仅为传统战略的28.5%。结论：Sanger测序联合突变测量分析在SMA诊断中具有潜在的应用价值。
Background: Spinal muscular atrophy (SMA) is caused by homozygous deletion or compound heterozygous mutation of survival motor neuron gene 1 (SMN1), which is the key to diagnose SMA. The study was to establish and evaluate a new diagnostic method for SMA. Methods: A total of 1494 children suspected with SMA were enrolled in this study. Traditional strategy, including multiplexed ligation-dependent probe amplification (MLPA) and TA cloning, was used in 1364 suspected SMA children from 2003 to 2014, and the 130 suspected SMA children were tested by a new strategy from 2015 to 2016, who were also verified by MLPA combined with TA cloning. The SMN1 and SMN2 were simultaneously amplified by polymerase chain reaction using the same primers. Mutation Surveyor software was used to detect and quantify the SMN1 variants by calculating allelic proportions in Sanger sequencing. Finally, turnaround time and cost of these two strategies were compared. Results: Among 1364 suspected SMA children, 576 children had SMN1 homozygous deletion and 27 children had SMN1 compound heterozygous mutation. Among the 130 cases, 59 had SMN1 homozygous deletion and 8 had heterozygous deletion: the SMN1-specific peak proportion on exon 7 was 34.6 ± 1.0% and 25.5 ± 0.5%, representing SMN1:SMN2 to be 1:2 and 1:3, respectively. Moreover, five variations, including p.Ser8Lysfs *23 (in two cases), p.Leu228*, p.Pro218Hisfs *26, p.Ser143Phefs*5, and p.Tyr276His, were detected in 6/8 cases with heterozygous deletion, the mutant allele proportion was 31.9%, 23.9%, 37.6%, 32.8%, 24.5%, and 23.6%, which was similar to that of the SMN1-specific site on exon 7, suggesting that those subtle mutations were located in SMN1. All these results were consistent with MLPA and TA cloning. The turnaround times of two strategies were 7.5 h and 266.5 h, respectively. Cost of a new strategy was only 28.5% of the traditional strategy. Conclusion: Sanger sequencing combined with Mutation Surveyor analysis has potential application in SMA diagnosis.
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影响因子：
3.5
作者：
Xiufeng Song;Jian‐Hong Deng;C. J. Liu;Yidong Bai
通讯作者：
Xiufeng Song;Jian‐Hong Deng;C. J. Liu;Yidong Bai
影响因子：
5.3
作者：
Dong C;Vincent K;Sharp P
通讯作者：
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影响因子：
20.3
作者：
Schmidt, Janine;Enric Ramis-Zaldivar, Joan;Quintanilla-Martinez, Leticia
通讯作者：
Quintanilla-Martinez, Leticia
影响因子：
5.3
作者：
Alias, Laura;Bernal, Sara;Tizzano, Eduardo F.
通讯作者：
Tizzano, Eduardo F.
影响因子：
4.5
作者：
Elango T;Sun J;Zhu C;Zhou F;Zhang Y;Sun L;Yang S;Zhang X
通讯作者：
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