Wan Lei, Tsai Chang-Hai, Tsai Yuhsin, Hsu Chin-Moo, Lee Cheng-Chun, Tsai Fuu-Jen
Department of Medical Research, China Medical University Hospital, Taichung, Taiwan.
Biochem Biophys Res Commun. 2006 Jun 30;345(2):734-8. doi: 10.1016/j.bbrc.2006.04.136. Epub 2006 May 3.
Wilson disease (WD) is an autosomal recessive disorder of copper metabolism, which is caused by mutation in copper-transporting ATPase (ATP7B). In the present study, we report a molecular diagnosis method to screen the WD chromosome in patients or in heterozygotic carriers in Taiwan. Exons 8, 11, 12, 13, 16, 17, and 18 of ATP7B are selected for the screening of mutations. The most common mutation, Arg778Leu or Arg778Gln, was first screened by PCR-RFLP then we combined single-stranded conformation polymorphism (SSCP) analysis followed by direct DNA sequencing on the DNA fragments with mobility shift on SSCP analysis. The diagnostic rate was compared with standard ATP7B whole gene sequencing analysis. Ten different mutations were identified among 29 WD patients; among them four were novel (Ala1168Pro, Thr1178Ala, Ala1193Pro, and Pro1273Gln). The false positive rates were tested against 100 normal individuals and listed as follows: exon 8: 5%; exon 11: 4%; exon 12: 6%; exon 13: 5%; exon 16: 5%; exon 17: 3%; exon 18: 4%. The Arg778Leu mutation exhibited the highest allelic frequency (43.1%). The detection rate of WD chromosomes is 65.52%, which is as sensitive as whole gene sequencing scanning. According to our results, WD chromosomes in Taiwan are predominantely located at exons 8, 11, 12, 13, 16, 17, and 18. The standard sequencing analysis on the entire gene is time consuming. We recommend screening these 7 exons first on those individuals who have a higher risk in having WD, before whole gene and promoter sequencing analysis in Taiwan.
威尔逊病（WD）是一种常染色体隐性铜代谢紊乱疾病，由铜转运ATP酶（ATP7B）突变引起。在本研究中，我们报告了一种分子诊断方法，用于筛查台湾患者或杂合子携带者的WD染色体。选择ATP7B的第8、11、12、13、16、17和18外显子进行突变筛查。首先通过PCR-RFLP筛查最常见的突变Arg778Leu或Arg778Gln，然后我们结合单链构象多态性（SSCP）分析，随后对在SSCP分析中出现迁移率变化的DNA片段进行直接DNA测序。将诊断率与标准的ATP7B全基因测序分析进行比较。在29例WD患者中鉴定出10种不同的突变；其中4种是新突变（Ala1168Pro、Thr1178Ala、Ala1193Pro和Pro1273Gln）。针对100名正常个体测试了假阳性率，结果如下：第8外显子：5%；第11外显子：4%；第12外显子：6%；第13外显子：5%；第16外显子：5%；第17外显子：3%；第18外显子：4%。Arg778Leu突变表现出最高的等位基因频率（43.1%）。WD染色体的检测率为65.52%，与全基因测序扫描的灵敏度相同。根据我们的结果，台湾的WD染色体主要位于第8、11、12、13、16、17和18外显子。对整个基因进行标准测序分析耗时较长。我们建议在台湾对那些患WD风险较高的个体首先筛查这7个外显子，然后再进行全基因和启动子测序分析。
