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Establishment of an early diagnostic system for the detection of heart disease-related gene mutations with a novel electrochemical array chip

新型电化学阵列芯片建立心脏病相关基因突变早期诊断系统

基本信息

批准号：
14570376
负责人：
IKEDA Yasuyuki
金额：
$ 1.86万
依托单位：
NATIONAL CARDIOVASCULAR CENTER RESEARCH INSTITUTE
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

Subjects (carriers) with heterozygous LPL deficiency are prone to develop mild hypertriglyceridemia (type IV hyperlipoproteinemia) when complicated with environmental factors such as a hyperinsulinemic state and/or a high alcohol intake, which stimulate triglyceride synthesis in the liver, while carriers are normolipidemic provided that they do not have the environmental factors. Identification of heterozygous LPL gene mutations as an early diagnosis, therefore, is important for preventing the development of hypertriglyceridemia and subsequent development of atherosclerosis by getting the patient to change to a more healthful lifestyle. We aimed to develop and evaluate an electrochemical hybridization assay with ferrocenylnaphthalene diimide (FND) for the detection of heterozygous LPL gene mutations.We developed the electrochemical hybridization assay for the detection of heterozygous LPL mutations, which are heterozygotes for a G-to-A transition at the 818th position (G188E : 818G normal allele and 818A mutant allele) and for a deletion of G at the 916th of the LPL gene-exon 5 (916G normal allele and 916G-del mutant allele). Discriminating between a wild type (Wt) allele and mutant type (Mt) allele is based on the hybridization between two probes such as a Wt probe and a Mt probe (13-15 mer) immobilized on a gold electrode and the PCR product od exon 5 (350 bp). The hybridized double-stranded DNA was quantified by a differential pulse voltammetry at 460 mV using FND as an intercalator.The electrochemical hybridization assay with FND allowed quick discrimination among a Wt (818G)/Wt (818G) homozygote, a Wt (818G)/Mt (818A) heterozygote and a Mt (818A)/Mt (818A) homozygote of the LPL gene. The same results were obtained for the discrimination of a Wt (916g) allele and a Mt (919G-del) allele. This method is also applicable for the detection of other LPL gene mutations.

杂合子LPL缺乏症的受试者（携带者）在合并高胰岛素状态和/或高酒精摄入等刺激肝脏中甘油三酯合成的环境因素时，容易发生轻度高甘油三酯血症（IV型高脂蛋白血症），而携带者在没有环境因素的情况下，则是正常血脂。因此，鉴别杂合LPL基因突变作为早期诊断对于通过让患者改变更健康的生活方式来预防高甘油三酯血症的发展和随后的动脉粥样硬化的发展是重要的。我们的目的是建立和评估与二茂铁萘二亚胺（FND）的电化学杂交检测杂合LPL基因突变。我们开发了用于检测LPL杂合突变的电化学杂交试验，这些杂合突变是LPL基因第818位的G到a过渡（G188E: 818G正常等位基因和818A突变等位基因）和LPL基因第916位-外显子5的G缺失（916G正常等位基因和916G-del突变等位基因）。野生型（Wt）和突变型（Mt）等位基因的区分是基于固定在金电极上的两个探针(Wt探针和Mt探针（13-15 mer）和PCR产物5外显子（350 bp）之间的杂交。用FND作为插层剂，在460 mV下用差分脉冲伏安法定量杂交双链DNA。利用FND进行电化学杂交，可以快速区分LPL基因的Wt (818G)/Wt （818G）纯合子、Wt (818G)/Mt （818A）杂合子和Mt (818A)/Mt （818A）纯合子。Wt （916g）和Mt （919G-del）等位基因的区分结果相同。该方法同样适用于其他LPL基因突变的检测。