A Robust and Fast/Multiplex Pharmacogenetics Assay to Simultaneously Analyze 17 Clinically Relevant Genetic Polymorphisms in CYP3A4, CYP3A5, CYP1A2, CYP2C9, CYP2C19, CYP2D6, ABCB1, and VKORC1 Genes

Abstract

In the field of pharmacogenetics, the trend is to analyze a panel of several actionable genetic polymorphisms. It may require the use of high-throughput sequencing which demands expensive reagents/instruments and specific skills to interpret results. As an alternative, the aim of this work was to validate an easy, fast, and inexpensive multiplex pharmacogenetics assay to simultaneously genotype a panel of 17 clinically actionable variants involved in drug pharmacokinetics/pharmacodynamics. We designed primers to perform a multiplex PCR assay using a single mix. Primers were labeled by two fluorescent dye markers to discriminate alleles, while the size of the PCR fragments analyzed by electrophoresis allowed identifying amplicon. Polymorphisms of interest were CYP3A4*22, CYP3A5*3, CYP1A2*1F, CYP2C9*2-*3, CYP2C19*2-*3-*17, VKORC1-1639G > A, ABCB1 rs1045642-rs1128503-rs2229109-rs2032582, and CYP2D6*3-*4-*6-*9. The assay was repeatable and a minimum quantity of 10 ng of DNA/ sample was needed to obtain accurate results. The method was applied to a validation cohort of 121 samples and genotyping results were consistent with those obtained with reference methods. The assay was fast and cost-effective with results being available within one working-day. This robust assay can easily be implemented in laboratories as an alternative to cumbersome simplex assays or expensive multiplex approaches. Together it should widespread access to pharmacogenetics in clinical routine practice.

Keywords: CYP450; multiplex; panel; personalized medicine; pharmacogenetics.

Figure 1

Resolution of the 17 amplicons corresponding to the clinically relevant genetic polymorphisms. Example of an electrophoregram obtained for one DNA sample. The green peaks correspond to the fluorescence of the HEX probe. The blue peaks correspond to the fluorescence of the FAM probe.

Figure 2

Resolution of a bi-allelic polymorphisms. Primer design for CYP3A5 rs776746 (A). Primer design for CYP2D6 rs5030655 (B). Representative electropherogram results of the migration of fragments according to genotype (C). CYP3A5 rs776746 illustrates results of bi-allelic discrimination for a single-nucleotide polymorphism. CYP2D6 rs5030655 illustrates results for a polymorphism based on the deletion of 1 nucleotide. The green peak corresponds to the fluorescence of the HEX probe (WT) and the blue peak corresponds to the fluorescence of the FAM probe (Variant).

Figure 3

Resolution of tri-allelic SNP. Primer design for ABCB1 rs2032582 (A). Representative electrophoregram results of the migration of fragments according to genotype (B). ABCB1 rs2032582 illustrates results of tri-allelic discrimination for a single-nucleotide polymorphism. The green peak corresponds to the fluorescence of the HEX probe and the blue peak corresponds to the fluorescence of the FAM probe.