遗传 ›› 2005, Vol. 27 ›› Issue (6): 861-864.

• 研究报告 •    下一篇

亨廷顿病的基因诊断

莫亚勤1;2;李麓芸2;卢光琇2   

  1. 中山大学附属第二医院妇产科,广州 510120
  • 收稿日期:2005-01-28 修回日期:2005-03-29 出版日期:2005-12-10 发布日期:2005-12-10
  • 通讯作者: 卢光琇

Gene diagnosis of Huntington Disease

MO Ya-Qin1,2,LI Lu-Yun2,LU Guang-Xiu 2   

  1. The Second Affiliated Hospital of Sun Yat-sen University, Guangzhou 510120,China
  • Received:2005-01-28 Revised:2005-03-29 Online:2005-12-10 Published:2005-12-10
  • Contact: LU Guang-Xiu

摘要: 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。

关键词: 动态突变, 三核苷酸重复, HD基因, 亨廷顿病

Abstract: To investigate the methods to effectively and simply assess the CAG repeat size of HD gene which was necessary for gene diagnosis of Huntington disease, the sequence including polymorphic CAG repeat of HD gene was amplified by PCR with TaKaRa LA Taq DNA polymerase and GC buffer. PCR products were analyzed on polyacrylamide gel to distinguish normal alleles from HD alleles. The DNA fragments of affected alleles were recovered from polyacrylamide gel as templets for secondary PCR. The secondary PCR products were cloned into T vector for sequencing analysis to determine CAG repeat size. A total of 20 normal individuals and 3 members from a HD pedigree were included in this study. The results showed that the CAG repeat numbers in 20 normal individuals and 3 normal alleles from a HD pedigree varied in normal range, while in 3 HD alleles, the copy number 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 amplified CAG repeat of HD gene, which combined polyacrylamide gel electrophoresis and DNA sequencing analysis can diagnose HD accurately. In addition, these finding suggested that the dynamic mutation in HD gene be responsible for the genetic defect in Chinese HD patients.

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