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

LPL杂合子缺乏的受试者(携带者)在合并环境因素(如高胰岛素状态和/或高酒精摄入量，刺激肝脏甘油三酯合成)时，容易发生轻度高甘油三酯血症(IV型高脂蛋白血症)，而携带者如果没有环境因素，则是正常血脂。因此，识别LPL基因杂合子突变作为早期诊断，对于防止高甘油三酯血症的发展和随后的动脉粥样硬化的发展，使患者改变到更健康的生活方式是重要的。我们建立了检测LPL基因杂合性突变的电化学杂交方法，用于检测LPL基因的杂合性突变。该杂合性突变是指LPL基因第818位(G188E：818G正常等位基因和818A突变等位基因)发生G-A转换的杂合子，以及LPL基因外显子5第916位G缺失(916G正常等位基因和916G-del突变等位基因)。区分野生型(Wt)等位基因和突变型(Mt)等位基因是基于固定在金电极上的Wt探针和mt探针(13-15mer)与外显子5(350bp)的聚合酶链式反应产物的杂交。以FND为嵌入剂，在460 mV下用差示脉冲伏安法对杂交后的双链DNA进行定量，FND电化学杂交检测可以快速区分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。对Wt(916G)等位基因和Mt(919G-del)等位基因的区分也得到了相同的结果。该方法也适用于其他LPL基因突变的检测。

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.