引物延伸变性高效液相色谱法诊断遗传性非综合征性耳聋  

Rapid,accurate genotyping of the deafness genes mutations using a novel multiplex PE and DHPLC assay

作　　者：门美超[1] 薛晋杰[2] 潘乾[2] 田湘娥[1] 冯永[1] 

机构地区：[1]中南大学湘雅医院耳鼻咽喉头颈外科,湖南长沙410008 [2]中南大学中国医学遗传学国家重点实验室,湖南长沙410078

出　　处：《中国现代医学杂志》2011年第10期1143-1146,共4页China Journal of Modern Medicine

基　　金：国家自然科学基金(No:30470954No:30671150);"十一五"国家攻关计划(No:2007BA118B13)

摘　　要：目的建立针对中国人常见的遗传性非综合征性耳聋基因诊断的新方法。方法 PCR扩增的靶序列,经引物延伸,得到中国人遗传性非综合征性耳聋的6个常见突变的特异性延伸片段,用全变性高效液相色谱分析延伸片段混合物,分离图谱可鉴定被检样本的基因型。结果盲法分析显示,120例样品的引物延伸变性高效液相色谱与直接测序检测结果符合率为100%。其中84例耳聋患者诊断为上述6个常见突变中的突变。该法的突变检出率为70%(84/120)。结论该法是一种准确、高效的检测非综合征性遗传性耳聋突变的分析方法,值得推广。【Objective】 NSHL（Nonsyndromic hearing loss） is a common inherited disorder of nervous system worldwide and rapid,accurate genotyping methodologies for specific,causative mutations of NSHL are needed for diagnosis of this disease in different ethnic populations.【Methods】 In this study,we performed a novel multiplex PE（primer extension） reaction in combination with DHPLC（denaturing high-performance liquid chromatography） for simultaneously detecting and genotyping the six most common mutations（GJB2-235delC,MT-DNA-A1555G,PDSIVS7-2ag,GJB2-299delAT,MT-DNA-C1494T and PDS-C1229T mutation） in the NSHL in Chinese populations.This method involved the amplification of target sequence followed by a purification step,a multiplex PE reaction and a fully-denaturing DHPLC analysis on the Transgenomic Wave DNA fragment analysis system.【Results】 In a blinded study,this technique accurately genotyped 100%（120/120） of samples simultaneously characterized by direct sequencing successfully.【Conclusion】 This combined PE/DHPLC approach is simple,rapid,highly accurate and cost-effective for use in genotyping common disease-causing mutations,and strongly suggests that this technique may be used widely.

关 键 词：遗传性耳聋 变性高效液相色谱 基因突变 引物延伸 

分 类 号：R394[医药卫生—医学遗传学] R446[医药卫生—基础医学]