Pharmacogenomics of warfarin: comprehensive evaluation of important warfarin genomic response factors

Introduction: Warfarin is the most widely prescribed anticoagulant for the prevention and treatment of thromboembolic diseases. However, warfarin use is complicated by its narrow therapeutic range and inter-individual variability in the starting dose required to achieve a stable international normalised ratio (INR). Warfarin is initiated clinically at 5mg/day then subsequent doses are adjusted accordingly to achieve a stable targeted INR. However, inter-individual variability in response to the warfarin starting dose has been observed and this is reported to be attributed to by various genetic and nongenetic factors. Non-genetics factors implicated in the warfarin dose variability include age, gender, body weight, comorbidities and concomitant drugs. Genetic factors affecting warfarin dose variability include variation in genes encoding the warfarin metabolising enzymes and targeted proteins. Genetic variants in CYP2C9 and VKORC1 have been extensively studied on how they affect warfarin dose variability, culminating in several dosing algorithms incorporating genetic (i.e., CYP2C9*2, CYP2C9*3 and VKORC1 g.-1639G>A) and non-genetic factors (i.e., age, body surface area, amiodarone, race, targeted INR, smoking and thromboembolism). However, these studies have often excluded African populations, therefore missing variants that might be important in the prediction of warfarin doses among Africans. Data on variants that specifically affect warfarin dose variability among Africans is lacking, with no dosing algorithms tailored specifically for Africans developed to date. Thus, the main aim of the study is to conduct a comprehensive evaluation of important genetic and non-genetic factors affecting warfarin response, and further make recommendations on variables important for the development of appropriate algorithms for warfarin dosing among black Africans and the Mixed Ancestry population group in Southern Africa. Method: A total of 302 black Africans and 277 Mixed Ancestry adults undergoing warfarin treatment were recruited at INR clinics in the Western Cape Province, South Africa and Harare, Zimbabwe. Their DNA samples were extracted and utilised for downstream analyses. A total of 73 candidate variants involved in either pharmacokinetics or pharmacodynamics of warfarin, were genetically characterised using a combination of allelic discrimination, Sanger sequencing, restriction fragment length polymorphism and iPLEX PGx74 Mass Array platform. Various statistical packages in STATA, R, haploview and plink were employed to determine frequency distribution, linkage disequilibrium and haplotype mapping of the studied genetic variants. Furthermore, genetic and non-genetic variables were correlated with warfarin maintenance dose and their cumulative effect on warfarin dose variability measured through a multivariate step-wise regression analysis in both the black African and Mixed Ancestry cohorts. Whole exome sequencing was done using the ion torrent Sequence ion S5 system in selected black African individuals presenting with extreme phenotypes (i.e., very low dose or very high dose) but who did not harbour variants known to significantly affect warfarin dose requirements. A workflow which applied various bioinformatics tools was employed for the analyses of the resultant raw BAM files, subsequently, population structure and frequency distribution patterns were described among our cohort and individuals in the 1000 Genomes project. Specific variants identified through WES were prioritised according to clinical significance and further genotyped in an enhanced sample size of 252 black Africans, to confirm their effect on warfarin dose requirements

Profiling of warfarin pharmacokinetics‐associated genetic variants: Black Africans portray unique genetic markers important for an African specific warfarin pharmacogenetics‐dosing algorithm

Background: Warfarin dose variability observed in patients is attributed to variation in genes involved in the warfarin metabolic pathway. Genetic variation in CYP2C9 and VKORC1 has been the traditional focus in evaluating warfarin dose variability, with little focus on other genes. Objective: We set out to evaluate 27 single nucleotide polymorphisms (SNPs) in the CYP2C cluster loci and 8 genes (VKORC1, ABCB1, CYP2C9, CYP2C19, CYP2C8, CYP1A2, CYP3A4, and CYP3A5) involved in pharmacokinetics of warfarin. Patients/methods: 503 participants were recruited among black Africans and Mixed Ancestry population groups, from South Africa and Zimbabwe, and a blood sample taken for DNA. Clinical parameters were obtained from patient medical records, and these were correlated with genetic variation. Results: Among black Africans, the SNPs CYP2C rs12777823G>A, CYP2C9 c.449G>A (*8), CYP2C9 c.1003C>T (*11) and CYP2C8 c.805A>T (*2) were significantly associated with warfarin maintenance dose. Conversely, CYP2C9 c.430C>T (*2), CYP2C8 c.792C>G (*4) and VKORC1 g.-1639G>A were significantly associated with maintenance dose among the Mixed Ancestry. The presence of CYP2C8*2 and CYP3A5*6 alleles was associated with increased mean warfarin maintenance dose, whereas CYP2C9*8 allele was associated with reduced warfarin maintenance dose. Conclusion: African populations present with a diversity of variants that are important in predicting pharmacogenetics-based warfarin dosing in addition to those reported in CYP2C9 and VKORC1. It is therefore important, to include African populations in pharmacogenomics studies to be able to identify all possible biomarkers that are potential predictors for drug response

Warfarin Pharmacogenomics for Precision Medicine in Real-Life Clinical Practice in Southern Africa: Harnessing 73 Variants in 29 Pharmacogenes

Pharmacogenomics is universally relevant for worldwide modern therapeutics and yet needs further development in resource-limited countries. While there is an abundance of genetic association studies in controlled medical settings, there is a paucity of studies with a naturalistic design in real-life clinical practice in patients with comorbidities and under multiple drug treatment regimens. African patients are often burdened with communicable and noncommunicable comorbidities, yet the application of pharmacogenomics in African clinical settings remains limited. Using warfarin as a model, this study aims at minimizing gaps in precision/personalized medicine research in African clinical practice. We present, therefore, pharmacogenomic profiles of a cohort of 503 black Africans (n = 252) and Mixed Ancestry (n = 251) patients from Southern Africa, on warfarin and co-prescribed drugs in a naturalized noncontrolled environment. Seventy-three (n = 73) single nucleotide polymorphisms (SNPs) in 29 pharmacogenes were characterized using a combination of allelic discrimination, Sanger sequencing, restriction fragment length polymorphism, and Sequenom Mass Array. The common comorbidities were hypertension (43-46%), heart failure (39-45%), diabetes mellitus (18%), arrhythmia (25%), and HIV infection (15%). Accordingly, the most common co-prescribed drugs were antihypertensives, antiarrhythmic drugs, antidiabetics, and antiretroviral therapy. We observed marked variation in major pharmacogenes both at interethnic levels and within African subpopulations. The Mixed Ancestry group presented a profile of genetic variants reflecting their European, Asian, and African admixture. Precision medicine requires that African populations begin to capture their own pharmacogenetic SNPs as they cannot always infer with absolute certainty from Asian and European populations. In the current historical moment of the COVID-19 pandemic, we also underscore that the spectrum of drugs interacting with warfarin will likely increase, given the systemic and cardiovascular effects of COVID-19, and the anticipated influx of COVID-19 medicines in the near future. This observational clinical pharmacogenomics study of warfarin, together with past precision medicine research, collectively, lends strong support for incorporation of pharmacogenetic profiling in clinical settings in African patients for effective and safe administration of therapeutics

Meta-analysis of genome-wide association studies of stable warfarin dose in patients of African ancestry.

Warfarin dose requirements are highly variable due to clinical and genetic factors. While genetic variants influencing warfarin dose have been identified in European and East Asian populations, more work is needed to identify African-specific genetic variants to help optimize warfarin dosing. We performed genome-wide association studies (GWAS) in four African cohorts from Uganda, South Africa, and Zimbabwe, totalling 989 warfarin-treated participants who reached stable dose and had international normalized ratios within therapeutic ranges. We also included two African American cohorts recruited by the International Warfarin Pharmacogenetics Consortium (n=316) and the University of Alabama at Birmingham (n=199). Following the GWAS, we performed standard error-weighted meta-analyses and then conducted stepwise conditional analyses to account for known loci (the CYP2C cluster SNP rs12777823 and CYP2C9 in chromosome 10; VKORC1 in chromosome 16). The genome-wide significance threshold was set at PA revealed an additional locus on chromosome 2 (top SNPs rs116057875/rs115254730/rs115240773, P=3.64×10-8), implicating the MALL gene, that could indirectly influence warfarin response through interactions with caveolin-1. In conclusion, our meta-analysis of six cohorts of warfarin-treated patients of African ancestry reaffirmed the importance of CYP2C9 and VKORC1 in influencing warfarin dose requirements. We also identified a new locus (MALL), that still requires direct evidence of biological plausibility