联合应用PCR扩增和Sanger测序对脊髓小脑型共济失调进行基因诊断的临床意义

Genetic diagnosis of spinocerebellar ataxia by PCR amplification and Sanger sequencing

目的分析1个遗传性脊髓小脑型共济失调(SCA)家系的基因突变情况,对该家系进行分型,为遗传咨询提供依据。方法采集1个SCA家系5例个体(含3例患者)的外周血,提取基因组DNA。设计9对引物,采用聚合酶链反应(PCR)分别扩增SCA1、SCA2、SCA3、SCA6、SCA7、SCA8、SCA12、SCA17、齿状肌麻痹性萎缩(DRPLA)基因,筛选该家系的致病基因。对筛选出的SCA致病基因通过Sanger测序进行验证。结果 3例患者SCA3基因中CAG重复序列数分别为76、78和75,超出文献报道的12~44重复数的正常范围,确诊为SCA3家系。通过PCR扩增及Sanger测序,家系中1例携带CAG重复序列数为74的个体被确诊为SCA3致病基因携带者。结论联合应用PCR扩增和Sanger测序是一种简单易行的SCA基因诊断途径,可应用于携带者诊断和产前诊断。

Objective To investigate the causative mutations in a spinocerebellar ataxia(SCA) pedigree and the types of the SCA pedigree,and to provide a reference for genetic counseling. Methods Genomic DNA was extracted from peripheral blood of 5 subjects,including 3 SCA patients,in the SCA pedigree. Totally,9 sets of primers were designed for the polymerase chain reaction(PCR) amplification of 9 fragments,including SCA1,SCA2,SCA3,SCA6,SCA7,SCA8,SCA12,SCA17 and dentatorubral-pallidoluysian atrophy(DRPLA) gene,respectively. Causative mutations were screened and were validated by Sanger sequencing. Results Repeat lengths of 76,78 and 75,above that of 12-44 CAG triplets reported in healthy people from literatures,were observed in the 3 patients from the SCA3 pedigree. Another member carrying a repeat length of 74 in the pedigree was diagnosed as a causative mutation of the SCA3 gene carrier by Sanger sequencing. Conclusions PCR amplification combined with Sanger sequencing is a convenient and efficient method to identify causative mutations for SCA gene,which can be used for carrier screening and prenatal diagnosis.