Title : Functional validation of African-specific non-coding variants impacting warfarin metabolism
Abstract:
Background: CYP2C9 is a major cytochrome P450 enzyme responsible for metabolizing approximately 15% of clinically prescribed drugs, including warfarin, losartan, and NSAIDs. Genetic variants influencing CYP2C9 expression and activity contribute to variability in drug metabolism, efficacy, and risk of adverse drug reactions. Despite the critical role of CYP2C9, most pharmacogenomic studies have underrepresented African populations, where unique genetic diversity exists. Asiimwe et al. (2022) conducted a pharmacokinetic GWAS in Sub-Saharan Black African patients treated with warfarin and identified multiple non-coding variants associated with the S-warfarin/R-warfarin plasma concentration ratio, implicating the CYP2C cluster.
Objective: This study aimed to functionally validate selected non-coding variants in the CYP2C cluster, identified in the Sub-Saharan African warfarin GWAS, with a focus on their regulatory impact on CYP2C9 expression.
Methods: Four candidate SNPs were prioritized using in silico tools (FUMA-GWAS, RegulomeDB) to identify variants likely to affect promoter function. Wild-type and variant alleles were cloned into luciferase reporter constructs containing the CYP2C9 promoter. Constructs were co-transfected with a renilla luciferase control into HepG2 cells. Promoter activity was quantified by dual-luciferase assay 48 hours post-transfection. Statistical differences were evaluated using the Mann-Whitney U test.
Results: Among the variants tested (rs74150717 (A>G), rs114439291 (A>G), rs60204155 (A>G) and chr10:96537727-96537728 (CA > C)), rs60204155 significantly decreased CYP2C9 promoter activity (p = 0.0317), consistent with GWAS findings where this variant was associated with increased plasma S-warfarin levels (Beta = 0.51852 ± 0.07907). This suggests that rs60204155 reduces CYP2C9 expression, potentially leading to altered metabolism of S-warfarin, the more potent warfarin enantiomer.
Conclusion: By providing functional validation of an African-specific CYP2C9 regulatory variant, this study advances the pharmacogenomic understanding of warfarin metabolism in underrepresented populations and can help refine warfarin dosing algorithms specifically for African patients. They also support the integration of genomic data with functional assays to improve the accuracy of drug response predictions, thereby enhancing the safety and effectiveness of therapy.
