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

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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.