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 melling analysis

作　　者：赵秀丽[1] 刘彦山[1] 黄尚志[1] 孟岩[1] 孙淼[1] 杨威[1] 张学[1] 

机构地区：[1]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,北京100105

出　　处：《中华医学遗传学杂志》2008年第5期515-519,共5页Chinese Journal of Medical Genetics

基　　金：国家“863”项目（2007AA02Z440）;“十五”国家科技攻关计划基金（2004BA720A04）;北京市科学技术委员会研发攻关类基金（D0906005040491）

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ATF7B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，P992L／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。Objective To detect the most prevalent mutations, R778L and P992L of ATP8B 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 eneompassingATP7B 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 Afa 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 heterozygole. Three HRM curve patterns were observed for the fragments encompassingATP7B 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 

分 类 号：R686[医药卫生—骨科学]