天津医药
天津醫藥
천진의약
TIANJIN MEDICAL JOURNAL
2014年
7期
697-700
,共4页
脊髓性肌萎缩,儿童%序列分析, DNA%多态性,限制性片段长度%基因诊断%SMN基因
脊髓性肌萎縮,兒童%序列分析, DNA%多態性,限製性片段長度%基因診斷%SMN基因
척수성기위축,인동%서렬분석, DNA%다태성,한제성편단장도%기인진단%SMN기인
spinal muscular atrophies of childhood%sequence analysis,DNA%polymorphism,restriction fragment length%genetic testing%SMN gene
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501 bp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189 bp，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。
目的：探索將測序技術應用于缺失型脊髓性肌萎縮癥（SMA）基因診斷的可行性。方法設計2對引物，PCR擴增SMA緻病基因運動神經元生存基因（SMN1）與其同源基因SMN2之間5箇不同堿基所在區域，第1對引物正嚮擴增SMN1內含子6至7間長度為501 bp片段，包含4箇不同堿基位點g.31957、32006、32154及32269；第2對引物反嚮擴增SMN1外顯子8區域，長度為189 bp，包含1箇不同堿基位點g.32734。根據測序圖譜區分SMA患者與攜帶者和（或）正常人。將此方法應用于7箇臨床疑似SMA傢繫的診斷，併與聚閤酶鏈反應-限製性片段長度多態性分析（PCR-RFLP）法進行比較。結果6例測序圖譜顯示SMN內含子6至外顯子8之間5箇SMN1和SMN2差異位點g.31957、32006、32154、32269及32734均隻有SMN2特有堿基a、T、g、g和A，患兒父母（攜帶者）相同位點顯示為a/g、T/C、g/a、g/a和A/G。說明患者缺失SMN1基因，缺失範圍包括內含子6至外顯子8；攜帶者則既有SMN1基因，又有SMN2基因，與患者能區分開。1例檢測結果為a、T、g、g和A/G，說明其缺失範圍不含外顯子8。測序法與PCR-RFLP方法結果一緻。結論測序技術在缺失型SMA患者的基因診斷上優于經典PCR-RFLP法，更方便快捷，結果更明確，建議替代常用的PCR-RFLP法。
목적：탐색장측서기술응용우결실형척수성기위축증（SMA）기인진단적가행성。방법설계2대인물，PCR확증SMA치병기인운동신경원생존기인（SMN1）여기동원기인SMN2지간5개불동감기소재구역，제1대인물정향확증SMN1내함자6지7간장도위501 bp편단，포함4개불동감기위점g.31957、32006、32154급32269；제2대인물반향확증SMN1외현자8구역，장도위189 bp，포함1개불동감기위점g.32734。근거측서도보구분SMA환자여휴대자화（혹）정상인。장차방법응용우7개림상의사SMA가계적진단，병여취합매련반응-한제성편단장도다태성분석（PCR-RFLP）법진행비교。결과6례측서도보현시SMN내함자6지외현자8지간5개SMN1화SMN2차이위점g.31957、32006、32154、32269급32734균지유SMN2특유감기a、T、g、g화A，환인부모（휴대자）상동위점현시위a/g、T/C、g/a、g/a화A/G。설명환자결실SMN1기인，결실범위포괄내함자6지외현자8；휴대자칙기유SMN1기인，우유SMN2기인，여환자능구분개。1례검측결과위a、T、g、g화A/G，설명기결실범위불함외현자8。측서법여PCR-RFLP방법결과일치。결론측서기술재결실형SMA환자적기인진단상우우경전PCR-RFLP법，경방편쾌첩，결과경명학，건의체대상용적PCR-RFLP법。
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 the region including 5 different bases in SMA-causative gene SMN1 and its homologue copy SMN2 by polymerase chain reaction (PCR). The first primer amplified a fragment 501 bp long spanning from SMN intron 6 to intron 7 targeting four different bases (g.31957, 32006, 32154 and 32269). The second primer reversely amplified a 189 bp long fragment within SMN exon 8 including one base-pair differ-ence (g.32734). PCR procedure was followed by Sanger sequencing technique to identify the 5 different bases. SMA patients caused by SMN1 homozygous deletion were distinguished from carriers or normal controls by absence of SMN1 specific bas-es in sequence chromatograms. This assay was performed in 7 SMA suspected patients and their parents. The specimens were also detected by PCR- restriction fragment length polymorphism (RFLP) method. Results It was found that 6 of 7 SMA suspected patients showed only SMN2 specific bases at the 5 different base positions among the region from intron 6 to exon 8, which meant the patient displaying only SMN2-specific nucleotide a, T, g, g and A at g.31957, 32006, 32154, 32269 and 32734, while their parents (carriers) showed a/g, T/C, g/a, g/a and A/G at the same sites. SMN1 gene was deleted in the patient, and the deletion region was inferred from intron 6 to exon 8. Because carriers had both SMN1 and SMN2 genes, they can be discriminated from the SMN1 deleted patient. One of 7 patients yield an unique sequence chromatogram of a, T, g, g and A/G, indicating that exon 8 of SMN1 was not deleted in this patient. Conclusion DNA sequencing analysis is an alter-native simple method for detecting SMA caused by homozygous deletion of SMN1. We recommend to replace the widely used PCR-RFLP method with DNA sequencing assay.
