亨廷顿病的基因诊断

为了简单高效检测HD基因开放阅读框5’端(CAG)ｎ三核苷酸重复序列，建立快速准确的亨廷顿病(Huntington disease, HD)基因诊断方法，应用TaKaRa LA Taq DNA聚合酶配合GC buffer扩增HD基因包含(CAG)ｎ重复序列的目的片段，非变性聚丙烯酰胺凝胶电泳检测后回收(CAG)ｎ拷贝数异常增多的目的片段，再次PCR扩增后将产物连接至T载体，进行DNA测序确定CAG的拷贝数。应用该方法对一个HD家系的3名成员以及20名正常人进行基因诊断，结果显示该HD家系3名成员的一条染色体上的(CAG)ｎ拷贝数在正常范围内，而另一条染色体上的(CAG)ｎ拷贝数异常增多，分别为39、40、41，而20例正常人(CAG)ｎ拷贝数均在正常范围内，正常和HD等位基因之间的(CAG)ｎ拷贝数不相重叠。因此，应用该方法可以对HD进行准确的基因诊断，结果同时也证明HD基因的动态突变是导致中国人亨廷顿病的遗传基础。

Gene diagnosis of Huntington Disease

We report here a simple and effective method to assess the CAG repeat size of HD gene for gene diagnosis of Huntington disease,Genomic DNA sequences in polymorphic CAG repeat HD gene was amplified by PCR with TaKaRa LA Taq DNA polymerase and GC buffer.To distinguish normal alleles from HD alleles,DNA fragments of affected alleles were recovered as templates for secondary PCR.The secondary PCR products were cloned into T vector for sequencing to determine CAG repeat size.A total of 20 normal individuals and 3 members from a HD pedigree were included in this study.Results showed that the CAG repeat numbers in 20 normal individuals and 3 normal alleles from the HD pedigree varied in normal range,while in 3 HD alleles,the copy numbers of CAG repeat were 39,40,41,respectively.There was no overlap between the copy number of the normal and affected alleles.In conclusion,the TaKaRa LA Taq DNA polymerase with GC buffer can be used to effectively amplify CAG repeat of HD gene,which combined DNA sequencing can diagnose HD accurately.In addition,these finding suggest that dynamic mutation in HD gene responsible for the genetic defect in Chinese HD patients.