应用高分辨熔解曲线技术检测中国人Wilson病的常见基因突变
Rapid detection of common ATP7B mutations in Wilson disease by high resolution melting analysis
赵秀丽 1刘彦山 1黄尚志 1孟岩 1孙淼 1杨威 1张学1
作者信息
- 1. 100005,北京,中国医学科学院基础医学研究所北京协和医学院基础学院,麦库西克-张孝骞遗传医学中心
- 折叠
摘要
目的 应用PCR-高分辨熔解曲线分析技术检测中国人Wilson病(Wilson disease,WD)患者中ATP7B基因内高频突变区第8和13外显子.方法 酚-氯仿法提取外周全血基因组DNA;PCR扩增患者ATP7B基因第8和13外显子及两个外显子中的突变热点区域,PCR产物经HR-1进行高分辨熔解曲线分析;进一步以限制性内切酶或(和)DNA测序法对高分辨熔解曲线检测结果进行验证.结果 在30例WD患者中,检测到R778L纯合子3例,R778L杂合子6例,P992L杂合子6例,P992D/S975Y的复合杂合子1例,R778L/P992L复合杂合子2例和R778L/752.33delG复合杂合子1例.本组样本中,R778L、P992L和S975Y的基因频率分别为25%、15%和1.67%.测序及限制性内切酶分析结果完全与高分辨熔解曲线分析结果一致.结论 高分辨熔解曲线分析检测ATP7B基因突变具有简便、快速、特异和灵敏等优点,可作为Wilson病患者及携带者突变筛查的优选方法.
Abstract
Objective To detect the most prevalent mutations,R778L and P992L of A TP8B gene,in Chinese Wilson disease(WD)patients by high resolution melting(HRM)analysis after polymerase chain reaction(PCR).Methods Genomic DNA Was extracted from peripheral blood samples obtained from 30 cases of WD by the standard phenol/chloroform method.DNA fragments encompassing ATP7B exons 8 and 13 were produced by PCR amplification.The amplicons containing the R778L or P992L mutations were then generated by nested PCR.The nested PCR products were subjected to HRM analysis using the HR-1 instrument.Mutations detected in HRM analysis were verified by restriction analysis using restriction enzyme(Msp I or Alu I or Afs I)or DNA sequencing.Results HRM analysis of the fragments encompassing ATP7B exon 8 showed four curve patterns.Subsequent restriction analysis and DNA sequencing proved that the four different curves represent four different genotypes:the wild type,the R778L/R778L homozygote,the R778L heterozygote,and the R778L/752.33delG compound heterozygote.Three HRM curve patterns were observed for the fragments encompassing ATP7B exon 13,representing the wild type,the P992L heterozygote,and the P992L/S975Y compound heterozygote.In our studied samples,allele frequencies of the R778L,P992L and S975Y mutations were 25%,15% and 1.67%, respectively.Conclusion HRM analysis is a simple.accurate and sensitive approach for rapid detection of the ATP7B mutations and could be used as an optimized method for genetic testing in WD.
关键词
Wilson病/ATP7B基因/高分辨熔解曲线分析/突变检测Key words
Wilson disease/ATP7B gene/high resolution melting/mutation detection引用本文复制引用
基金项目
国家高技术研究发展计划(863计划)(2007AA022440)
国家科技攻关项目(2004BA720A04)
北京市科学技术委员会研发攻关类基金(0906005040491)
出版年
2008
NETL
NSTL
维普
万方数据