变性高效液相色谱法在家族性阿尔茨海默病突变位点检测中的应用

The Application of Denaturing High Performance Liquid Chromatography for Detection of Point Mutation of Familial Alzheimer Disease

目的 通过对一个阿尔茨海默病 (Alzheimer disease,AD)大家系突变位点的检测 ,探讨中国人家族性 AD(familial Alzheimer disease,FAD)的发病机制 ,同时观察变性高效液相色谱法 (denaturing high performance liq-uid chromatography,DHPLC)的实用价值。方法 应用 DHPLC方法及 DNA直接测序技术 ,对一个有 1 3 0人的AD大家系和 5 0名正常对照者进行了淀粉样蛋白前体 (amyloid precursor protein,APP)基因、早老素 1 (prese-nilin1 ,PS1 )基因的突变位点检测。结果 (1 ) 5例 AD患者及 4例家系中的无痴呆症状者 ( -3 0、3 7、41、43 )经DHPLC检测有 PS1基因的第 5外显子的突变峰 ,家系中其他成员无突变峰。 PS1基因其他外显子检测及 APP基因检测 ,家系中所有成员和正常对照组均无突变峰。 (2 )上述 9例经 DNA直接测序在 PS1基因第 5外显子的1 3 6号密码子发生了 GCT→ GGT错义突变 ,使丙氨酸变为甘氨酸 (Ala1 3 6Gly) ,无突变峰者 DNA测序均未发现异常。结论 (1 ) DHPLC检测显示出这一 AD大家系 PS1基因第 5外显子存在突变 ,该突变位点可能为中国人FAD患者早老素基因突变点之一。 (2 ) DHPLC技术可作为大样本筛查突变位点的一种便捷可靠手段。

Objective To observe the value of denaturing high performance liquid chromatography (DHPLC) in detecting the point mutation of familial Alzheimer diease(FAD).Methods DHPLC and DNA sequencing were used to examine the amyloid precursor protein and presenilin 1(PS1) gene of a 130 member family and a 50 member control group.Results (1)The result of DHPLC tests proved double peaks in 5 members with AD symptoms and in 4 members without AD symptoms(Ⅳ 30,37,41,43),Which indicated the possibility of mutation in PS1 exon 5. There were no double peaks in other exons of PS1 gene and amyloid precursor protein (APP) gene in all family members as well as normal control group.(2) DNA sequencing revealed that there was a missense mutation of GCT to GGT in code 136 of PS1 exon 5 in 9 members with double peak, and with a substitution of Ala by Gly (Ala136Gly).Conclusions (1) The proved mutation in exon 5 of PS1 in the present study may be a responsible PS1 gene mutation for Chinese familial Alzheimer disease. (2)DHPLC techique hasproved to be a rapid and reliable method for screening mutation site in large samples.