基于DHPLC技术的Wilson病基因诊断  

Denaturing High Performance Liquid Chromatography for the Gene Diagnosis of Chinese Wilson's Disease

作　　者：杨文明[1] 汪美霞[1] 杨静芳[2] 鲍远程[1] 李瑞娟[3] 董婷[1] 汪瀚[1] 杨兴涛[1] 陈彪[2,4] 

机构地区：[1]安徽中医学院第一附属医院 [2]首都医科大学宣武医院 [3]安徽中医学院 [4]国家人类基因组北京研究中心

出　　处：《中西医结合心脑血管病杂志》2008年第12期1430-1432,共3页Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease

基　　金：安徽省科技攻关计划基金项目(No.06013057A);国家中医药管理局科研项目(No.06-07LP23);国家科技攻关计划863项目(No.2002BA711A07);安徽省学科拔尖人才基金及重点学科基金资助项目

摘　　要：目的建立并应用高效液相色谱(DHPLC)技术检测Wilson病(WD)ATP7B基因第8外显子突变。方法采用聚合酶链式反应(PCR)扩增WD基因第8外显子片段,扩增产物直接进行DHPLC检测分析,根据色谱峰型不同确定测序样品,经测序后确定突变类型。结果203例先证者中WD基因外显子8基因突变阳性检出率为55.2%,Arg778Leu纯合突变22例,杂合突变90例,基因突变频率为33.0%。共发现3种错义突变、1种移码插入突变和1种多态性位点(C2310G),且多态位点C2310G与Arg778Leu突变完全连锁。其中76例先证者带有Arg778Leu杂合错义突变,22例为Arg778Leu纯合错义突变,10例为Arg778Gln杂合错义突变,2例Tyr713Silent错义突变(C2139G),2例为杂合2304C插入突变。结论WD基因外显子8是ATP7B基因的第一突变热区。DHPLC技术是一种高效、灵敏、快速简便的基因突变检测方法。可用于WD的临床基因诊断。Objective To establish the technique platform of denaturing high performance liquid chromatography （DHPLC） to detect the distribution of exon 8 mutation of Chinese Wilson＇s disease（WD） gene. Methods The exon 8 fragments of WD gene were amplified using the polymerase chain reaction and amplification productions were directly screened by DHPLC. Sequencing samples were selected by different type of chromatographic peak. Results Two .hundreds and three Chinese WD probands were detected in the study. The positive detection rate of exon 8 mutation of WD gene was 55. 2%, 22 probands were homozygous mutation for the Arg778Len, and 90 were heterozygous mutation. The mutation frequency was 33.0%. Three missense mutations, 1 frameshift insertion mutation and 1 polymorphism（C2310G） had been identified in 203 Chinese WD probands. The complete linkage between the polymorphic sitc C2310G and mutation site Arg778Leu had been confirmed. The total 76 were found to be heterozygous missense mutations, 22 homozygous missense mutations for the Arg778Leu, 10 heterozygous missense mutations for the Arg778Gln, 2 missense mutations for the Tyr713Silent and 2 heterozygous mutations for the Ins2304C. Conclusion The exon 8 was first pathological mutation spot for Chinese WD, DHPI.C was a effective, sensitive, rapid and convenient detection method of gene mutation, and it could be used as the method for gene diagnosis.

关 键 词：高效液相色谱 WILSON病 基因突变 

分 类 号：R742.4[医药卫生—神经病学与精神病学]