Validation of methodology for efficient genotyping of CYP2D6 and CYP2C19

Abstract There are some data associating variants in the CYP2D6 and/or CYP2C19 genes with concentration-to-dose ratios, efficacy, and retention in treatments. However, much of the above arises from relatively small studies or large datasets with limited genotyping methodologies. Our aim was to develop and validate comprehensive and accurate genotyping methodology for these two genes to facilitate regenotyping in large datasets and hence the generation of more accurate clinical associations. TaqMan copy number variant (CNV) assays for CYP2D6 were used to identify samples from a relevant large dataset (GENDEP study, N = 853) with particularly challenging genotypes to call. These and those representing as broad a range of CYP2D6 and CYP2C19 genotypes as possible by prior available data (AmpliChip CYP450 and TaqMan CYP2C19*17) were chosen for further analysis (N = 96). Genotyping techniques employed were: Luminex CYP2D6 xTAGv3 and Luminex CYP2C19 xTAGv3, PharmacoScan, the Ion S5 AmpliSeq Pharmacogenomics Panel, TaqMan single nucleotide variant (SNV) assays, and, for the CYP2D6 hybrids, long-range polymerase chain reactions (L-PCRs) with Sanger sequencing. Agena was also used for CYP2C19. The TaqMan SNV assays were able to assist with identifying which gene was duplicated or in tandem for multiple copy variants. A multiplex assay was adaptable for analysis of CYP2D6 hybrid genes, with Sanger sequencing data being consistent with the data arising; we provide these data for efficient genotyping of such CYP2D6 hybrid genes with adaptable multiplex methods. Consensus genotypes generated to date resulted in revision of assigned enzyme activity score for 28/96(29%) and 2/93 samples (2.2%) for CYP2D6 and CYP2C19, respectively.