Design and Optimization of a CYP2C19*2 Genotyping Assay Based on the SuperSelective Primer Method

Abstract

Introduction: Percutaneous coronary intervention (PCI) is a nonsurgical technique carried out frequently to treat patients suffering obstructive coronary artery disease. These patients are treated with a combination of aspirin and an antiplatelet drug (clopedogril) for one year following the intervention. CYP2C19 enzyme is considered the major hepatic enzyme that contributes in the conversion of clopedogril from inactive prodrug to active drug. Several CYP2C19 genetic variants are associated with a significant decreased enzymatic activity. Accordingly, CYP2C19 genotyping is recommended to test patients for the most frequent poor metabolizer genotype, particularly the CYP2C19*2 variant. The aim of this study is to develop and optimize a new technique for CYP2C19*2 genotyping using the recently invented SuperSelective primer method.

Material and method: BLAST analysis, PCR assay, and Sanger sequencing were used to establish and validate the reference control of the three possible genotypes, namely, the wild-type *1/*1, the heterozygous *1/*2, and the mutant *2/*2. Real-time PCR assay was utilized in the test to optimize the parameters of a SuperSelective primer “CYP2C19*2 24-14/13-6:1:1”. The newly developed assay was used to genotype 81 genomic DNA samples and Sanger sequencing was used for validating accuracy of the new assay.

Result and discussion: The newly developed Real-time PCR assay utilizing “CYP2C19*2 2414/13-6:1:1” SuperSelective primer showed a 100% accuracy in genotyping and distinguishing the heterozygous from the wild-type samples with a minimum discriminatory cutoff of (Ct =7). The assay demonstrated a high genotyping efficiency of all the tested clinical samples across a broad range of their genomic DNA concentrations.

Conclusion: The SuperSelective based CYP2C19*2 genotyping assay, developed in this study, is an accurate and relatively fast genotyping assay that can be used in labs with resource-limited settings for genotype-guided antiplatelet therapy.