A novel pharmacogenetic testing panel for CYP2C19 genetic polymorphisms

Abstract

Specific drug metabolism rates are defined by the constituency of the cytochrome P450 (CYP) genome, including polymorphic changes in any of 200+ CYP genes. An example is CYP2C19, where associations of gene polymorphisms with variability in certain drug metabolism rates have been linked to inter-individual and inter-ethnic differences in therapeutic outcomes. While pharmacogenomic screening for these variants prior to drug and dosage prescription has well-defined links to better treatment outcomes, current implementation is limited to complex and costly variant-probing and DNA sequencing protocols, which have limited availability in clinical laboratories, leading to slow turnaround times, impacting effective clinical intervention. Here we describe a novel, cost-effective, multiplex genotyping approach to screening CYP2C19 variants. Fluorescence nested allele-specific (FAS) PCR was used with primers to detect CYP2C19 variants of interest in specific hot spots, including the Tier 1 haplotypes identified by the Association for Molecular Pathology (AMP): CYP2C19*2, *3, and *17. The presence/absence of wild-type and mutant alleles were identified independently as haplotypes, and in a multiplex reaction as diplotypes representing the 10 possible genotype combinations/profiles. FAS-PCR achieved the same genotype calls as a pyrosequencing protocol optimized for validating genotypes, but with a simpler and more sensitive interface. The FAS-PCR method correctly identified the genotypes of both synthesized DNA and a human genomic DNA standard. Uniquely, the FAS-PCR protocol generates patterns using one fluorescently-labeled primer irrespective of the number of variants targeted, establishing it as considerably more cost-effective than other allele-specific PCR-based techniques that involve labeling both the forward and reverse primers.