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测序技术在缺失型脊髓性肌萎缩症基因诊断中的应用

孟英韬,舒剑波   

  1. 天津市儿童医院
  • 收稿日期:2013-11-08 修回日期:2014-02-21 出版日期:2014-07-15 发布日期:2014-07-15
  • 通讯作者: 孟英韬

Sequencing Technology in Molecular Diagnosis of Spinal Muscular Atrophy Caused by SMN1 Deletion

Ying-tao MENG 2   

  • Received:2013-11-08 Revised:2014-02-21 Published:2014-07-15 Online:2014-07-15
  • Contact: Ying-tao MENG

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摘要：

【摘要】目的 探索将测序技术应用于缺失型脊髓性肌萎缩症（SMA）基因诊断的可行性。方法设计2对引物，PCR扩增SMA致病基因运动神经元生存基因（SMN1）与其同源基因SMN2之间5个不同碱基所在区域，第1对引物正向扩增SMN1内含子6至7间长度为501bp片段，包含4个不同碱基位点g.31957、32006、32154及32269；第2对引物反向扩增SMN1外显子8区域，长度为189bp，包含1个不同碱基位点g.32734。根据测序图谱区分SMA患者与携带者和（或）正常人。将此方法应用于7个临床疑似SMA家系的诊断，并与聚合酶链反应-限制性片段长度多态性分析（PCR-RFLP）法进行比较。结果6例测序图谱显示SMN内含子6至外显子8之间5个SMN1和SMN2差异位点 g.31957、32006、32154、32269及32734均只有SMN2特有碱基a、T、g、g和A，患儿父母（携带者）相同位点显示为a/g、 T/C、g/a、g/a和A/G。说明患者缺失SMN1基因，缺失范围包括内含子6至外显子8；携带者则既有SMN1基因，又有 SMN2基因，与患者能区分开。1例检测结果为a、T、g、g和A/G，说明其缺失范围不含外显子8。测序法与PCR-RFLP 方法结果一致。结论测序技术在缺失型SMA患者的基因诊断上优于经典PCR-RFLP法，更方便快捷，结果更明确，建议替代常用的PCR-RFLP法。

关键词: 脊髓性肌萎缩, 儿童, 序列分析, DNA, 基因诊断

Abstract:

[Abstract] Objective To investigate the feasibility of DNA sequencing analysis in molecular diagnosis for spinal muscular atrophy (SMA).Methods Two pairs of primers were utilized to amplify theregion including5different bases in SMA-causative geneSMN1and its homologue copySMN2bypolymerase chain reaction (PCR). The first primer amplified a fragment 501bp long spanning fromSMNintron 6to intron 7targeting four different bases (g.31957, 32006, 32154and 32269). The second primer reversely amplified a 189bp long fragment withinSMNexon8including one base-pair differ? ence (g.32734). PCR procedure was followed by Sanger sequencing technique to identify the 5different bases. SMA patients caused bySMN1homozygous deletion were distinguished from carriers or normal controls by absence ofSMN1specific bas? es in sequence chromatograms. This assay was performed in7SMA suspected patients and their parents. The specimens were also detected by PCR- restriction fragment length polymorphism (RFLP) method.Results It was found that 6of7 SMA suspected patients showed onlySMN2specific bases at the5different base positionsamong the region from intron6to exon8, which meant the patient displaying only SMN2-specific nucleotide a, T, g, g and A at g.31957, 32006, 32154, 32269 and32734, while their parents (carriers) showed a/g, T/C, g/a, g/a and A/G at the same sites. SMN1gene was deleted in the patient, and the deletion region was inferred from intron6to exon 8. Because carriers had bothSMN1andSMN2genes, they can be discriminated from theSMN1deleted patient. One of7patients yield an unique sequence chromatogram of a, T, g, g and A/G, indicating thatexon8ofSMN1was not deleted in this patient.Conclusion DNA sequencing analysis is an alter? native simple method for detecting SMA caused by homozygous deletion ofSMN1. We recommend to replace the widely used PCR-RFLP method with DNA sequencing assay.

Key words: spinal muscular atrophies of childhood, sequence analysis, DNA, genetic testing