Interrogation of CYP2D6 Structural Variant Alleles Improves the Correlation Between CYP2D6 Genotype and CYP2D6-Mediated Metabolic Activity

Abstract

The cytochrome P450 2D6 (CYP2D6) gene locus is challenging to accurately genotype due to numerous single nucleotide variants and complex structural variation. Our goal was to determine whether the CYP2D6 genotype-phenotype correlation is improved when diplotype assignments incorporate structural variation, identified by the bioinformatics tool Stargazer, with next-generation sequencing data. Using CYP2D6 activity measured with substrates dextromethorphan and metoprolol, activity score explained 40% and 34% of variability in metabolite formation rates, respectively, when diplotype calls incorporated structural variation, increasing from 36% and 31%, respectively, when diplotypes did not incorporate structural variation. We also investigated whether the revised Clinical Pharmacogenetics Implementation Consortium (CPIC) recommendations for translating genotype to phenotype improve CYP2D6 activity predictions over the current system. Although the revised recommendations do not improve the correlation between activity score and CYP2D6 activity, perhaps because of low frequency of the CYP2D6*10 allele, the correlation with metabolizer phenotype group was significantly improved for both substrates. We also measured the function of seven rare coding variants: one (A449D) exhibited decreased (44%) and another (R474Q) increased (127%) activity compared with reference CYP2D6.1 protein. Allele-specific analysis found that A449D is part of a novel CYP2D6*4 suballele, CYP2D6*4.028. The novel haplotype containing R474Q was designated CYP2D6*138 by PharmVar; another novel haplotype containing R365H was designated CYP2D6*139. Accuracy of CYP2D6 phenotype prediction is improved when the CYP2D6 gene locus is interrogated using next-generation sequencing coupled with structural variation analysis. Additionally, revised CPIC genotype to phenotype translation recommendations provides an improvement in assigning CYP2D6 activity.

Figure 1

Association among CYP2D6 metabolite formation rate and CYP2D6 messenger RNA (mRNA) and protein content. CYP2D6 metabolite formation rate was correlated with CYP2D6 mRNA content quantitated by RNA‐seq: dextrorphan formation rate (a) and alpha‐hydroxymetoprolol formation rate (b); with CYP2D6 protein content quantitated by liquid‐chromatography tandem mass spectrometry: dextrorphan formation rate (c) and alpha‐hydroxymetoprolol formation rate (d). FPKM, fragments per kilobase of transcript per million mapped reads

Figure 2

Diplotypes and activity scores assigned with single nucleotide variation (SNV) data alone and with Stargazer structural variation data. Columns on the left show diplotypes and activity scores (AS) assigned using allele calls from SNV data alone. Corrected diplotypes and AS, based on Stargazer allele assignments, are displayed in the columns on the right. AS are color‐coded as follows: 3 (dark blue); 2.5 (medium blue); 2 (light blue); 1.5 (light yellow); 1 (dark yellow); 0.5 (orange); and 0 (red). Arrows indicate the direction of the change in AS assignments with the incorporation of structural data: decrease (↓), increase (↑), and no change (→).

Figure 3

Association between CYP2D6 metabolite formation rate and CYP2D6 activity score (AS). Dextrorphan formation rate by AS assigned with single nucleotide variation (SNV) data alone (a), with Stargazer (c), and with revised Clinical Pharmacogenetics Implementation Consortium (CPIC) definitions (e). Alpha‐hydroxymetoprolol formation rate by activity score assigned with SNV data alone (b), with Stargazer (d), and with revised CPIC definitions (f). Boxes represent interquartile range with interior line representing the median. Error bars represent 1.5× the interquartile range. Number of samples in each AS category is given in parentheses.

Figure 4

Comparison between current and revised Clinical Pharmacogenetics Implementation Consortium (CPIC) genotype to phenotype translation tables for dextromethorphan and metoprolol. Dextrorphan formation rate by phenotype (IM, intermediate metabolizer; NM, normal metabolizer; PM, poor metabolizer; UM, ultrarapid metabolizer) assigned with current (a) and revised (c) CPIC definitions; alpha‐hydroxymetoprolol formation rate by phenotype assigned with current (b) and revised (d) CPIC definitions. Boxes represent interquartile range with interior line representing the median. Error bars represent 1.5× the interquartile range. Number of samples in each phenotype category is given in parentheses.

Figure 5

Functional characterization of rare CYP2D6 coding variants with CYP2D6 probe tic‐ABP1P. Each CYP2D6 variant was induced in an isogenic yeast strain and function was characterized with a tic‐ABP1P CYP2D6 probe. Fluorescence was normalized to that of CYP2D6 wildtype (medium grey; horizontal dashed line represents 100% activity). Control strains (light grey): empty vector, inactive variant (C443H), and decreased function variant (P34S). Rare coding variant strains (dark gray): P8S, S168A, D198N, P267L, V338M, A449D, and R474Q. Error bars indicate SEs from at least two independent replicates.