天津医药

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运用MLPA进行脊肌萎缩症的基因诊断

陈红苓1,孟英韬2,舒剑波2,宋力3   

  1. 1. 济南市儿童医院
    2. 天津市儿童医院
    3. 天津市儿童医院儿研所
  • 收稿日期:2012-01-28 修回日期:2012-06-15 出版日期:2012-11-15 发布日期:2012-11-15
  • 通讯作者: 宋力

Gene Diagnosis of Spinal Muscular Atrophy Using MLPA

1,Ying-tao MENG   

  • Received:2012-01-28 Revised:2012-06-15 Published:2012-11-15 Online:2012-11-15

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摘要： 目的 应用多重连接依赖式探针扩增法( MLPA)和PCR-RFLP技术对30例临床疑似SMA的病例进行基因诊断，并对两种方法进行比较。方法 盐析方法提取30例家系成员外周血DNA，常规PCR方法扩增SMN 7、8外显子，用DralⅠ和DdelⅠ酶切PCR产物，琼脂糖凝胶电泳检测PCR及酶切产物。同时利用MLPA试剂盒P021方法进行验证比较。结果 经PCR-RFLP方法判断22例SMN1第7+8号外显子纯合缺失患儿和2例Ex7纯合缺失患儿，与MLPA方法一致；其余6例PCR-RFLP方法未见异常家系经MLPA方法分析发现1例患儿和2例母亲为SMN1外显子7、8杂合缺失携带者；MLPA方法还在SMA家系中发现3例“2+0”型携带者。SMN2拷贝数在SMA患者中以4、5为主、携带者以2、3为主、正常人以1、2多见，其各拷贝数的频率分布在病人组与携带者组（x2=30.694）、病人组与正常人组（x2=21.997）中p<0.001，而在携带者与正常人组（x2=3.5）p>0.05。结论 与PCR-RFLP相比，MLPA更加简单、准确、高效，还能够准确定量SMN1、SMN2，是一种高效的遗传病基因诊断方法。

关键词: 脊肌萎缩症, MLPA, RFLP, SMN基因, 基因诊断

Abstract: Abstract Objective To perform gene diagnosis for 30 suspected patients of spinal muscular atrophy (SMA) using multiplex ligation dependent probe amplification (MLPA) and PCR-restriction fragment length polymorphism (PCR-RFLP), and compare the results of the two methods. Method Genomic DNA was isolated from peripheral blood of each subject from 30 families using salting-out method．DNA concentration was determined by nucleic acid quantitative instrument. Exon 7 and 8 of SMN gene was amplified by allele specific PCR.The PCR products were digested with DraⅠand DdeⅠ, detected by agarose gel electrophoresis. Simultaneously,the DNA samples were analyzed by SALSA MLPA KIT P021. Results Both PCR-RFLP and MLPA analysis showed the same that 22 patients with exon 7 and 8 homozygous deletion, and 2 patients with only exon 7 homozygous deletion of SMN1. The other 6 cases and parents presented no homozygous deletion by PCR-RFLP, but one child and two morthers of them were detected heterozygous by MLPA. Also MLPA analysis found three “2+0”carriers from 3 families. The data also showed that the SMN2 copy numbers were mainly 4 or 5 in SMA patients, while the carriers and the normal individuals were 2 or 3 and 1 or 2 copies respectively. There were clear statistical significance in the groups of patient -carrier and patient-normal individuals ( p<0.001). The carrier -normal group appeared no statistical significance( p>0.05). Conclution Compared with PCR-RFLP, MLPA is more convenient, precise, high-effective, and it can accuratly quantitative SMN1 and SMN2, so it is a kind of technique of gene diagnosis for common genetic disease .

Key words: Spinal muscular atrophy, MLPA, PFLP, SMN gene, Gene diagnosis