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目的 筛选中国人肝豆状核变性 (Wilsondisease,WD)的高频突变点 ,并建立酶切诊断方法。方法 对中国人WD39个家系 45例患者的 3～ 2 0号外显子进行聚合酶链反应 (PCR) 单链构象多态性 (SSCP)分析 ,对有异常者进行序列分析 ,并通过对该突变点的酶切再次筛选。结果 ① 8号外显子存在 13个泳动异常 ,测序表明 778号密码子由精氨酸错义突变为亮氨酸 (Arg778Leu) ,针对该位点的MspI酶切表明该位点的突变率为 2 8.8% ,对 3个患者家系进行了酶切分析 ;②对 12号外显子的935号密码子进行PCR SSCP及TaiI酶切分析 ,结果均无异常 ;③ 18号外显子发现 16例泳动异常 ,自动测序序列均正常 ;④ 4、5、13、19号等外显子均存在 1～ 2例SSCP的泳动异常。结论 研究结果支持Arg778Leu作为中国人WD的高频突变点。本研究建立的针对 8号外显子突变热点的MspI酶切方法可作为中国人WD患者外显子突变的初步筛选方法。
Objective To screen the high frequency mutation point of Wilsondisease (WD) in Chinese and establish the diagnostic method of digestion. Methods Single nucleotide polymorphism (SSCP) analysis of exon 3 ~ 20 of 45 Chinese patients with WD39 pedigree was performed. Sequence analysis of abnormalities was performed. The point of digestion again screening. Results ① There were 13 abnormalities of migration in exon 8, and sequencing showed that the codon 778 was changed from arginine to leucine (Arg778Leu). The MspI digestion of this site showed that the mutation rate was 2 8.8% of the three patients were analyzed by restriction enzyme digestion; ② Exon 12 of exon 935 codons PCR SSCP and TaiI digestion analysis, the results were no abnormalities; exon 18 found 16 cases of migration Abnormal, automatic sequencing sequences are normal; ④ 4,5,13,19 and other exons there are 1 to 2 cases of SSCP migration abnormalities. Conclusion The results support Arg778Leu as a high frequency mutation in Chinese WD. The MspI digestion method for the exon 8 mutation hot spot established in this study can be used as a preliminary screening method for exon mutations in Chinese WD patients.