Screening and validation of double allele-specific binding F-primers for the measurement of antihypertensive pharmacogenomics

Abstract

Objective: Previous studies have proposed that genetic polymorphisms of CYP2D6^*10, ADRB1, NPPA, CYP3A5^*3, ACE, CYP2C9^*3, and AGTR1 are involved in antihypertensive pharmacogenomics. The purpose of this study is to develop an amplification analysis using double allele-specific (AS) binding primers for accurate measurement of antihypertensive pharmacogenomics.

Methods: To establish a quadruplex quantitative PCR (qPCR) analysis for genotyping of CYP2D6^*10, ADRB1 (1165 G>C), NPPA (2238 T>C) and CYP3A5^*3, and a triplex qPCR analysis for genotyping of ACE (I/D), CYP2C9^*3 and AGTR1 (1166 A>C), mismatch AS F-primers were screened by detection of plasmid/gDNA, and were validated by agreement analysis/reproducibility evaluation, in which the ΔCq (differences in threshold cycles between the wild-type F-primer-based amplification assay and the mutant-type F-primer-based amplification assay) was employed to determine genotypes.

Results: Seven pairs of primers were successfully selected through three rounds of F-primers screening. Except for ADRB1, the robustness assessment showed the amplification efficiency ranging from 0.9 to 1.1. In agreement analysis, two specimens in the training set (n = 203) were defined by the triplex analysis rather than NGS as heterozygotes for ACE, which was evidenced by gel electrophoresis. Reproducibility evaluation demonstrated that the coefficient of variation (CV) was <5%.

Conclusion: Multiplex amplification analysis using screened AS binding primers is a simple, reliable, and accurate tool to guide drug delivery in antihypertensive personalized treatment.

Keywords: F-primers; allele-specific (AS); antihypertensive; genotyping; polymorphism.