论文部分内容阅读
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 and apply the High Performance Liquid Chromatography (DHPLC) to detect the exon 8 mutation of ATP7B gene in Wilson disease (WD). Methods The exon 8 fragment of WD gene was amplified by polymerase chain reaction (PCR). The amplified product was directly analyzed by DHPLC. Sequencing samples were determined according to different chromatographic peaks. The mutation types were determined after sequencing. Results The positive rate of WD gene exon 8 gene mutation was 55.2% in 203 probands, 22 in Arg778Leu homozygous mutation and 90 in heterozygous mutation. The frequency of gene mutation was 33.0%. Three missense mutations, one frameshift insertion mutation and one polymorphic site (C2310G) were found, and the polymorphic site C2310G was completely linked with the Arg778Leu mutation. Among them, 76 cases had probable Arg778Leu missense mutation, 22 cases Arg778Leu homozygous missense mutation, 10 cases Arg778Gln missense mutation, 2 cases Tyr713Silent missense mutation (C2139G) and 2 cases heterozygous 2304C insert mutation. Conclusion WD exon 8 is the first mutation hot region of ATP7B gene. DHPLC technology is a highly efficient, sensitive, rapid and easy method of gene mutation detection. Can be used for WD clinical gene diagnosis.