经DNA测序证实的肝豆状核变性基因突变热区的研究

目的研究我国肝豆状核变性（ＷＤ）基因突变的特征。方法应用聚合酶链反应－单链构像多态（ＰＣＲ－ＳＳＣＰ）技术，结合ＤＮＡ测序技术，筛查４０个ＷＤ家系的５６例患者及无亲缘关系的６０名正常人的ＷＤ基因第５、８、１４号外显子（ｅｘｏｎ５，ｅｘｏｎ８，ｅｘｏｎ１４）的突变及多态。结果２１例患者（来自１５个ＷＤ家系）在ｅｘｏｎ８检出２种错义突变，占３７．５％（１５／４０），其中２例（来自同一家系）发生Ａｒｇ７７８Ｇｌｎ纯合子突变（２．５％），８例（分别来自８个ＷＤ家系）发生Ａｒｇ７７８Ｌｅｕ纯合子突变（２０．０％），余１１例（来自６个ＷＤ家系）发生Ａｒｇ７７８Ｌｅｕ杂合子突变（１５．０％，６／４０）。此外，在第８号外显子区域发现了两种多态；在ｅｘｏｎ５和ｅｘｏｎ１４侧翼的内含子区域各发现了１种新多态。这是我国首次经ＤＮＡ测序证实的ＷＤ基因突变热区。结论ｅｘｏｎ８为我国ＷＤ病人基因突变的热区之一，这对于建立准确快速的ＷＤ直接基因诊断方法具有重要意义，该测序方法对明确基因变异的具体部位和内容的具有一定的重要性。

Hot point mutations of Wilson disease gene in Chinese with DNA sequencing

Objective To study the prevalence of mutations of Wilson disease (WD) gene in Chinese. Methods The subjects included 60 unrelated normal Chinese and 56 patients of 40 WD families. Genomic DNA was prepared from peripheral blood leukocytes by a salt out method. Polymerase chain reaction single strand conformation polymorphism(PCR SSCP) and subsequent direct sequencing were used to identify the mutations and polymorphisms of exon5,8,14 of WD in these subjects. Results We identified two missene mutations in 21 patients from 15 unrelated WD families(37.5%): 2 patients in 1 family were homozygous for Arg778Gln mutation,8 patients in 8 families were homozygous for Arg778Leu mutation and 11 patients in 6 families were heterozygous for Arg778Leu mutation. In the same time, we found two polymorphisms in exon8 and a new polymorphism in intronic sequence of exon5 and exon14,respectively. Conclusion Exon8 is a hot point mutation of WD in Chinese. It is important to use direct DNA sequencing for determining the mutations or polymorphisms of WD gene.