Larisa H. Cavallari

Warfarin pharmacogenetics: a single VKORC1 polymorphism is predictive of dose across 3 racial groups

Warfarin-dosing algorithms incorporating CYP2C9 and VKORC1 -1639G>A improve dose prediction compared with algorithms based solely on clinical and demographic factors. However, these algorithms better capture dose variability among whites than Asians or blacks. Herein, we evaluate whether other VKORC1 polymorphisms and haplotypes explain additional variation in warfarin dose beyond that explained by VKORC1 -1639G>A among Asians (n = 1103), blacks (n = 670), and whites (n = 3113). Participants were recruited from 11 countries as part of the International Warfarin Pharmacogenetics Consortium effort. Evaluation of the effects of individual VKORC1 single nucleotide polymorphisms (SNPs) and haplotypes on warfarin dose used both univariate and multi variable linear regression. VKORC1 -1639G>A and 1173C>T individually explained the greatest variance in dose in all 3 racial groups. Incorporation of additional VKORC1 SNPs or haplotypes did not further improve dose prediction. VKORC1 explained greater variability in dose among whites than blacks and Asians. Differences in the percentage of variance in dose explained by VKORC1 across race were largely accounted for by the frequency of the -1639A (or 1173T) allele. Thus, clinicians should recognize that, although at a population level, the contribution of VKORC1 toward dose requirements is higher in whites than in nonwhites; genotype predicts similar dose requirements across racial groups.

Regulatory polymorphism in vitamin K epoxide reductase complex subunit 1 (VKORC1) affects gene expression and warfarin dose requirement

Warfarin dose requirements have been associated with 2 main haplotypes in VKORC1, but the responsible polymorphisms remain unknown. To search for regulatory polymorphisms, we measured allelic mRNA expression of VKORC1 in human liver, heart, and B lymphocytes. The observed 2-fold allelic mRNA expression imbalance narrowed possible candidate SNPs to -1639G>A and 1173C<T. This genotype effect was observed selectively in the liver but not in heart or lymphocytes. In vitro expression of VKORC1 gene constructs, including coding and promoter regions, failed to reveal any genotype effect on transcription and mRNA processing. Chromatin immunoprecipitation with antibodies against acetyl-histone3 and K4-trimethyl-histone3 revealed preferential association of the promoter -1639 G allele with active chromatin, consistent with enhanced mRNA expression. The minor -1639 A allele generates a suppressor E-box binding site, apparently regulating gene expression by a mechanism undetectable with reporter gene assays. A clinical association study demonstrated that promoter SNP -1639G>A, and the tightly linked intron1 SNP 1173C>T, predict warfarin dose more accurately than intron 2 SNP 1542G>C in blacks. Increased warfarin dose requirement in blacks was accounted for by lower frequency of the -1639 A allele. Therefore, -1639G>A is a suitable biomarker for warfarin dosing across ethnic populations.

Genetic and Clinical Predictors of Warfarin Dose Requirements in African Americans

The objective of this study was to determine whether, in African‐American patients, additional vitamin K oxidoreductase complex subunit 1 (VKORC1), cytochrome P450 2C9 (CYP2C9), CYP4F2, or apolipoprotein E (APOE) polymorphisms contribute to variability in the warfarin maintenance dose beyond what is attributable to the CYP2C9*2 and *3 alleles and the VKORC1 −1639G>A genotype. In a cohort of 226 African‐American patients, weekly warfarin dose requirements were lower in those with the CYP2C9*8 allele (34 (30–47) mg; P = 0.023) and the CYP2C9 *2, *3, *5, *6, or *11 allele (33(28–40 mg); P < 0.001) as compared with those with the CYP2C9*1/*1 genotype (43 (35–56) mg). The combination of CYP2C9 alleles, VKORC1 −1639G>A genotype, and clinical variables explained 36% of the interpatient variability in warfarin dose requirements. By comparison, a model without the CYP2C9*5, *6, *8, and *11 alleles explained 30% of the variability in dose. No other VKORC1, CYP4F2, or APOE polymorphism contributed to the variance. The inclusion of additional CYP2C9 variants may improve the predictive ability of warfarin dosing algorithms for African Americans.

Genetic variants associated with warfarin dose in African-American individuals: a genome-wide association study

Summary Background VKORC1 and CYP2C9 are important contributors to warfarin dose variability, but explain less variability for individuals of African descent than for those of European or Asian descent. We aimed to identify additional variants contributing to warfarin dose requirements in African Americans. Methods We did a genome-wide association study of discovery and replication cohorts. Samples from African-American adults (aged ≥18 years) who were taking a stable maintenance dose of warfarin were obtained at International Warfarin Pharmacogenetics Consortium (IWPC) sites and the University of Alabama at Birmingham (Birmingham, AL, USA). Patients enrolled at IWPC sites but who were not used for discovery made up the independent replication cohort. All participants were genotyped. We did a stepwise conditional analysis, conditioning first for VKORC1 −1639G→A, followed by the composite genotype of CYP2C9*2 and CYP2C9*3. We prespecified a genome-wide significance threshold of p<5×10−8 in the discovery cohort and p<0·0038 in the replication cohort. Findings The discovery cohort contained 533 participants and the replication cohort 432 participants. After the prespecified conditioning in the discovery cohort, we identified an association between a novel single nucleotide polymorphism in the CYP2C cluster on chromosome 10 (rs12777823) and warfarin dose requirement that reached genome-wide significance (p=1·51×10−8). This association was confirmed in the replication cohort (p=5·04×10−5); analysis of the two cohorts together produced a p value of 4·5×10−12. Individuals heterozygous for the rs12777823 A allele need a dose reduction of 6·92 mg/week and those homozygous 9·34 mg/week. Regression analysis showed that the inclusion of rs12777823 significantly improves warfarin dose variability explained by the IWPC dosing algorithm (21% relative improvement). Interpretation A novel CYP2C single nucleotide polymorphism exerts a clinically relevant effect on warfarin dose in African Americans, independent of CYP2C9*2 and CYP2C9*3. Incorporation of this variant into pharmacogenetic dosing algorithms could improve warfarin dose prediction in this population. Funding National Institutes of Health, American Heart Association, Howard Hughes Medical Institute, Wisconsin Network for Health Research, and the Wellcome Trust.

Warfarin Dosing in Patients With Impaired Kidney Function

BACKGROUND In patients with kidney impairment, warfarin, a drug metabolized primarily by the cytochrome P-450 system, is initiated at similar doses and managed similarly as in the general medical population. Unfortunately, few data exist to guide dose adjustment in patients with decreased kidney function. Here, we determine the degree of warfarin dose reduction associated with kidney impairment and make recommendations for warfarin dosing. STUDY DESIGN Cross-sectional analysis. SETTING & PARTICIPANTS Long-term warfarin users followed up at anticoagulation clinics (n = 980); 708 participants from the University of Alabama (UAB) and 272 participants from the University of Chicago (UIC). PREDICTOR No/mild (estimated glomerular filtration rate [eGFR] ≥ 60 mL/min/1.73 m(2)), moderate (eGFR, 30-59 mL/min/1.73 m(2)), and severe (eGFR < 30 mL/min/1.73 m(2)) kidney impairment; CYP2C9 and VKORC1 genotype; age; race; sex; body mass; sociodemographic factors; smoking status; alcohol; vitamin K intake; comorbid conditions (eg, congestive heart failure); and drug interactions (eg, amiodarone and statins). OUTCOME & MEASUREMENT Warfarin dose (milligrams per day) was evaluated using linear regression after adjustment for clinical, demographic, and genetic factors. RESULTS Prevalences of moderate (31.8% and 27.6%) and severe kidney impairment (8.9% and 6.6%) were similar in the UAB and UIC cohorts. Warfarin dose requirements were significantly lower in patients with moderate and severe kidney impairment compared with those with no/mild kidney impairment in the UAB (P < 0.001) and UIC (P < 0.001) cohorts. Compared with patients with no/mild kidney impairment, patients with moderate kidney impairment required 9.5% lower doses (P < 0.001) and patients with severe kidney impairment required 19% lower doses (P < 0.001). LIMITATIONS No measurement of warfarin, serum albumin, vitamin K, and coagulation factors; no evaluation of other markers (eg, cystatin). CONCLUSION Moderate and severe kidney impairment were associated with a reduction in warfarin dose requirements.

The missing association: sequencing-based discovery of novel SNPs in VKORC1 and CYP2C9 that affect warfarin dose in African Americans.

It is well recognized that the genetic variants VKORC–‐1–39, CYP2C9*2, and CYP2C9*3 contribute to warfarin dose response. This has led to warfarin dosing algorithms that include these polymorphisms and explains between 47% and 56% of variability in dose in Caucasians. However, these polymorphisms explain significantly less of the variance in dose among African Americans. In order to identify novel variations that affect warfarin dose in African Americans, we used a targeted resequencing strategy that examined evolutionarily conserved sequences and regions of putative transcriptional binding. Through ethnicity‐specific warfarin dose model building in 330 African Americans, we identified two novel genetic associations with higher warfarin dose, namely, VKORC–‐8–91 (rs61162043, P = 0.0041) and 18786 in CYP2C9 (rs7089580, P = 0.035). These novel finds are independent of the previous associations with these genes. Our regression model, encompassing both genetic and clinical variables, explained 40% of the variability in warfarin dose in African‐American subjects, significantly more than any model thus far.

A Polymorphism in the VKORC1 Regulator Calumenin Predicts Higher Warfarin Dose Requirements in African Americans

Warfarin demonstrates a wide interindividual variability in response that is mediated partly by variants in cytochrome P450 2C9 (CYP2C9) and vitamin K 2,3‐epoxide reductase complex subunit 1 (VKORC1). It is not known whether variants in calumenin (CALU) (vitamin K reductase regulator) have an influence on warfarin dose requirements. We resequenced CALU regions in a discovery cohort of dose outliers: patients with high (>90th percentile, n = 55) or low (<10th percentile, n = 53) warfarin dose requirements (after accounting for known genetic and nongenetic variables). One CALU variant, rs339097, was associated with high doses (P = 0.01). We validated this variant as a predictor of higher warfarin doses in two replication cohorts: (i) 496 patients of mixed ethnicity and (ii) 194 African‐American patients. The G allele of rs339097 (the allele frequency was 0.14 in African Americans and 0.002 in Caucasians) was associated with the requirement for a 14.5% (SD ± 7%) higher therapeutic dose (P = 0.03) in the first replication cohort and a higher‐than‐predicted dose in the second replication cohort (allele frequency 0.14, one‐sided P = 0.03). CALU rs339097 A>G is associated with higher warfarin dose requirements, independent of known genetic and nongenetic predictors of warfarin dose in African Americans.

The IGNITE network: a model for genomic medicine implementation and research

BackgroundPatients, clinicians, researchers and payers are seeking to understand the value of using genomic information (as reflected by genotyping, sequencing, family history or other data) to inform clinical decision-making. However, challenges exist to widespread clinical implementation of genomic medicine, a prerequisite for developing evidence of its real-world utility.MethodsTo address these challenges, the National Institutes of Health-funded IGNITE (Implementing GeNomics In pracTicE; www.ignite-genomics.org) Network, comprised of six projects and a coordinating center, was established in 2013 to support the development, investigation and dissemination of genomic medicine practice models that seamlessly integrate genomic data into the electronic health record and that deploy tools for point of care decision making. IGNITE site projects are aligned in their purpose of testing these models, but individual projects vary in scope and design, including exploring genetic markers for disease risk prediction and prevention, developing tools for using family history data, incorporating pharmacogenomic data into clinical care, refining disease diagnosis using sequence-based mutation discovery, and creating novel educational approaches.ResultsThis paper describes the IGNITE Network and member projects, including network structure, collaborative initiatives, clinical decision support strategies, methods for return of genomic test results, and educational initiatives for patients and providers. Clinical and outcomes data from individual sites and network-wide projects are anticipated to begin being published over the next few years.ConclusionsThe IGNITE Network is an innovative series of projects and pilot demonstrations aiming to enhance translation of validated actionable genomic information into clinical settings and develop and use measures of outcome in response to genome-based clinical interventions using a pragmatic framework to provide early data and proofs of concept on the utility of these interventions. Through these efforts and collaboration with other stakeholders, IGNITE is poised to have a significant impact on the acceleration of genomic information into medical practice.

Factors influencing warfarin dose requirements in African–Americans

INTRODUCTION African-Americans are under-represented in studies assessing contributors to warfarin response. Our primary objective was to determine whether the genes for cytochrome P450 (CYP) 2C9, nicotinamide adenine dinucleotide phosphate, reduced, quinone oxidoreductase (NQO1) and vitamin K epoxide reductase complex subunit 1 (VKORC1) are associated with warfarin dose requirements in African-Americans. PATIENTS AND METHODS The following factors were assessed: demographics; clinical data; the CYP2C9 Arg144Cys (*2), Ile358Leu (*3) and Asp360Glu (*5); NQO1 Pro187Ser (*1/*2); and VKORC1 G6853C genotypes were analyzed in 115 African-Americans on stable warfarin doses. RESULTS Allele frequencies were 0.05 for the CYP2C9 *2, *3 or *5 alleles; 0.20 for NQO1 *2; and 0.25 for VKORC1 6853C. Possession of a CYP2C9*2, *3 or *5 allele was associated with a 38% lower warfarin dose compared with the *1/*1 genotype (30 +/- 13 vs 48 +/- 18 mg/week; p = 0.003). Neither the NQO1 *1/*2 nor VKORC1 G6853C genotype was associated with warfarin dose requirements in the population as a whole or in CYP2C9*1 allele homozygotes. Multiple regression analysis revealed that CYP2C9 genotype (p = 0.015), age (p < 0.001) and body surface area (p < 0.001) were jointly associated with warfarin dose requirements, and together explained 33% of the variability in warfarin dose requirements among African-Americans. DISCUSSION Our data suggest that CYP2C9 genotype, age and body size are important determinants of warfarin dose requirements in African-Americans. Our data further suggest that the VKORC1 G6853C polymorphism alone may not be useful for predicting warfarin dose requirements in this racial group.

Pharmacogenetics and Cardiovascular Disease—Implications for Personalized Medicine

The past decade has seen tremendous advances in our understanding of the genetic factors influencing response to a variety of drugs, including those targeted at treatment of cardiovascular diseases. In the case of clopidogrel, warfarin, and statins, the literature has become sufficiently strong that guidelines are now available describing the use of genetic information to guide treatment with these therapies, and some health centers are using this information in the care of their patients. There are many challenges in moving from research data to translation to practice; we discuss some of these barriers and the approaches some health systems are taking to overcome them. The body of literature that has led to the clinical implementation of CYP2C19 genotyping for clopidogrel, VKORC1, CYP2C9; and CYP4F2 for warfarin; and SLCO1B1 for statins is comprehensively described. We also provide clarity for other genes that have been extensively studied relative to these drugs, but for which the data are conflicting. Finally, we comment briefly on pharmacogenetics of other cardiovascular drugs and highlight β-blockers as the drug class with strong data that has not yet seen clinical implementation. It is anticipated that genetic information will increasingly be available on patients, and it is important to identify those examples where the evidence is sufficiently robust and predictive to use genetic information to guide clinical decisions. The review herein provides several examples of the accumulation of evidence and eventual clinical translation in cardiovascular pharmacogenetics.