A genome-wide association study of plasma concentrations of warfarin enantiomers and metabolites in sub-Saharan black-African patients

Abstract

Diversity in pharmacogenomic studies is poor, especially in relation to the inclusion of black African patients. Lack of funding and difficulties in recruitment, together with the requirement for large sample sizes because of the extensive genetic diversity in Africa, are amongst the factors which have hampered pharmacogenomic studies in Africa. Warfarin is widely used in sub-Saharan Africa, but as in other populations, dosing is highly variable due to genetic and non-genetic factors. In order to identify genetic factors determining warfarin response variability, we have conducted a genome-wide association study (GWAS) of plasma concentrations of warfarin enantiomers/metabolites in sub-Saharan black-Africans. This overcomes the issue of non-adherence and may have greater sensitivity at genome-wide level, to identify pharmacokinetic gene variants than focusing on mean weekly dose, the usual end-point used in previous studies. Participants recruited at 12 outpatient sites in Uganda and South Africa on stable warfarin dose were genotyped using the Illumina Infinium H3Africa Consortium Array v2. Imputation was conducted using the 1,000 Genomes Project phase III reference panel. Warfarin/metabolite plasma concentrations were determined by high-performance liquid chromatography with tandem mass spectrometry. Multivariable linear regression was undertaken, with adjustment made for five non-genetic covariates and ten principal components of genetic ancestry. After quality control procedures, 548 participants and 17,268,054 SNPs were retained. CYP2C9*8, CYP2C9*9, CYP2C9*11, and the CYP2C cluster SNP rs12777823 passed the Bonferroni-adjusted replication significance threshold (p < 3.21E-04) for warfarin/metabolite ratios. In an exploratory GWAS analysis, 373 unique SNPs in 13 genes, including CYP2C9*8, passed the Bonferroni-adjusted genome-wide significance threshold (p < 3.846E-9), with 325 (87%, all located on chromosome 10) SNPs being associated with the S-warfarin/R-warfarin outcome (top SNP rs11188082, CYP2C19 intron variant, p = 1.55E-17). Approximately 69% of these SNPs were in linkage disequilibrium (r ² > 0.8) with CYP2C9*8 (n = 216) and rs12777823 (n = 8). Using a pharmacokinetic approach, we have shown that variants other than CYP2C9*2 and CYP2C9*3 are more important in sub-Saharan black-Africans, mainly due to the allele frequencies. In exploratory work, we conducted the first warfarin pharmacokinetics-related GWAS in sub-Saharan Africans and identified novel SNPs that will require external replication and functional characterization before they can be considered for inclusion in warfarin dosing algorithms.

Keywords: black-African; genome-wide association study; personalized medicine; pharmacokinetics; warfarin.

FIGURE 1

Flow chart for included participants. ^aOnly patients who were within the dynamic range included in analysis. IBD = identity-by-descent.

FIGURE 2

Manhattan plots of warfarin enantiomers and metabolites. Genome-wide association analyses were carried out using natural logarithm transformed analyte concentrations, adjusted for age, sex, weight, simvastatin/amiodarone and efavirenz statuses, and ten principal components by frequentist association testing assuming an additive model of inheritance.

FIGURE 3

Manhattan plots of analyte (warfarin enantiomers, metabolites) ratios. Genome-wide association analyses were carried out using natural logarithm transformed analyte concentration ratios, adjusted for age, sex, weight, simvastatin/amiodarone status and efavirenz statuses, and ten principal components by frequentist association testing assuming an additive model of inheritance. The top SNPs already known to significantly influence warfarin pharmacokinetics are annotated.

FIGURE 4

Regional LocusZoom plot of the established CYP2C9 SNP rs7900194 (CYP2C9*8). The linkage disequilibrium (LD) pattern is based on the 1,000 genomes African populations (Genomes Project et al., 2010). In this study, most of the SNPs shown above to have an r ² between 0.6 and 0.8 (orange circles) were in LD with rs7900194 (r ² > 0.8).