Scientific Studies & Publication
Warfarin & CYP2C9 and VKORC1
Title 1 : Warfarin pharmacogenetics meets clinical use.
Blood. 2011 Sep 15;118(11):2938-9.
Schwab M, Schaeffeler E.
The FDA in their revised approval of the warfarin prescribing information of 2007 noted the relevance of genetic testing for VKORC1 and CYP2C9 variants. Yet there is still a lack of prospective clinical trials using genetic testing, underscoring the difficulty of a pharmacogenetics-based initiation dosing strategy. In this issue of Blood, Gong and colleagues provide evidence for the clinical use of a newly established warfarin pharmacogenetics-initiation protocol (WRAPID).
Title 2 : Influence of CYP2C9 and VKORC1 polymorphisms on warfarin dose, anticoagulation attainment and maintenance among European American and African Americans.
Pharmacogenomics. 2008 May;9(5):511-26.
Limdi NA, Arnett DK, Goldstein JA, Beasley TM, McGwin G, Adler BK, Acton RT.
Title 3: Genetic variation of VKORC1 and CYP4F2 genes related to warfarin maintenance dose in patients with myocardial infarction.
J Biomed Biotechnol. 2011;2011:739751.
Kringen MK, Haug KB, Grimholt RM, Stormo C, Narum S, Opdal MS, Fosen JT, Piehler AP, Johansen PW, Seljeflot I, Berg JP, Brørs O.
The aim of this study was to investigate whether the VKORC1*3 (rs7294/9041 G>A), VKORC1*4 (rs17708472/6009 C>T), and CYP4F2 (rs2108622/1347 C>T) polymorphisms were associated with elevated warfarin maintenance dose requirements in patients with myocardial infarction (n = 105) from the Warfarin Aspirin Reinfarction Study (WARIS-II). We found significant associations between elevated warfarin dose requirements and VKORC1*3 and VKORC1*4 polymorphisms (P = .001 and P = .004, resp.), whereas CYP4F2 (1347 C>T) showed a weak association on higher warfarin dose requirements (P = .09). However, analysing these variant alleles in a regression analysis together with our previously reported data on VKORC1*2, CYP2C9*2 and CYP2C9*3 polymorphisms, gave no significant associations for neither VKORC1*3, VKORC1*4 nor CYP4F2 (1347 C>T). In conclusion, in patients with myocardial infarction, the individual contribution to warfarin dose requirements from VKORC1*3, VKORC1*4, and CYP4F2 (1347 C>T) polymorphisms was negligible. Our results indicate that pharmacogenetic testing for VKORC1*2, CYP2C9*2 and CYP2C9*3 is more informative regarding warfarin dose requirements than testing for VKORC1*3, VKORC1*4, and CYP4F2 (1347 C>T) polymorphisms.
Title 4: Prospective evaluation of a pharmacogenetics-guided warfarin loading and maintenance dose regimen for initiation of therapy.
Blood. 2011 Sep 15;118(11):3163-71.
Gong IY, Tirona RG, Schwarz UI, Crown N, Dresser GK, Larue S, Langlois N, Lazo-Langner A, Zou G, Roden DM, Stein CM, Rodger M, Carrier M, Forgie M, Wells PS, Kim RB.
Single-nucleotide polymorphisms in genes that affect warfarin metabolism (cytochrome P450 2C9 gene, CYP2C9) and response (vitamin K epoxide reductase complex 1 gene, VKORC1) have an important influence on warfarin therapy, particularly during initiation; however, there is a lack of consensus regarding the optimal pharmacogenetics-based initiation strategy. We conducted a prospective cohort study in which patients requiring warfarin therapy for atrial fibrillation or venous thromboembolism were initiated with a novel pharmacogenetics-initiation protocol (WRAPID, Warfarin Regimen using A Pharmacogenetics-guided Initiation Dosing) that incorporated loading and maintenance doses based on genetics, clinical variables, and response (n = 167, followed up for 90 days), to assess the influence of genetic variations on anticoagulation responses. Application of the WRAPID algorithm resulted in a negligible influence of genetic variation in VKORC1 or CYP2C9 on time to achievement of first therapeutic response (P = .52, P = .28) and risk of overanticoagulation (P = .64, P = .96). After adjustment for covariates, time to stable anticoagulation was not influenced by VKORC1 or CYP2C9 genotype. Importantly, time spent within or above the therapeutic range did not differ among VKORC1 and CYP2C9 genotype groups. Moreover, the overall time course of the anticoagulation response among the genotype groups was similar and predictable. We demonstrate the clinical utility of genetics-guided warfarin initiation with the WRAPID protocol to provide safe and optimal anticoagulation therapy for patients with atrial fibrillation or venous thromboembolism.
Title 5: Absence of Novel CYP4F2 and VKORC1 Coding Region DNA Variants in Patients Requiring High Warfarin Doses.
Clin Med Res. 2011 Nov;9(3-4):119-24.
Burmester JK, Berg RL, Glurich I, Yale SH, Schmelzer JR, Caldwell MD.
Objective Warfarin is an FDA-approved oral anticoagulant for long-term prevention of thromboembolism. Substantial inter-individual variation in dosing requirements and the narrow therapeutic index of this widely-prescribed drug make safe initiation and dose stabilization challenging. Single nucleotide polymorphisms (SNPs) occurring in CYP2C9, VKORC1, and CYP4F2 genes are known to impact dose, and VKORC1 and CYP4F2 polymorphisms are associated with higher therapeutic dose requirements in our cohort. However, the most advanced regression models using personal, clinical, and genetic factors to predict individual stable dose account for only 50% to 60% of the observed variability in stable theapeutic dose in Caucasians. Design and Methods In this study, we used DNA sequence analysis to determine whether additional variants in CYP4F2 and VKORC1 gene coding regions contribute to variable dosing requirements among individuals for whom the actual dose was the highest relative to regression model- predicted dose. Results and Conclusions No novel DNA variants in the coding regions of these genes were identified among subjects requiring high warfarin doses, suggesting that other factors yet to be defined contribute to variability in warfarin dose requirements in this subset of our cohort.
Title 1 : Review: Will Warfarin Ever Be Replaced?
J Cardiovasc Pharmacol Ther. 2010 Sep;15(3):210-9.
Ruff CT, Braunwald E.
Arterial and venous thromboembolism account for significant morbidity and mortality worldwide. Warfarin, and other vitamin K antagonists (VKAs), have been the only class of oral anticoagulants currently in clinical use and have been so for over 50 years. Although warfarin is effective in preventing thromboembolism, its use is limited by its narrow therapeutic index that necessitates frequent monitoring and dose adjustments resulting in considerable inconvenience to patients and clinicians. There are now several orally administered anticoagulants in late stages of clinical development that may offer effective, safer, and more convenient anticoagulation. This review summarizes and compares data on novel anticoagulants in the prophylaxis and treatment of venous thromboembolism, acute coronary syndromes, and the prevention of stroke in patients with atrial fibrillation.
Title 2 : Patient factors that influence warfarin dose response.
J Pharm Pract. 2010 Jun;23(3):194-204.
White PJ.
Warfarin has long been the mainstay of oral anticoagulation therapy for the treatment and prevention of venous and arterial thrombosis. The narrow therapeutic index of warfarin, and the complex number of factors that influence international normalized ratio (INR) response, makes optimization of warfarin therapy challenging. Determination of the appropriate warfarin dose during initiation and maintenance therapy requires an understanding of patient factors that influence dose response: age, body weight, nutritional status, acute and chronic disease states, and changes in concomitant drug therapy and diet. This review will examine specific clinical factors that can affect the pharmacokinetics and pharmacodynamics of warfarin, as well as the role of pharmacogenetics in optimizing warfarin therapy.
Title 3 : Warfarin pharmacogenetics: a single VKORC1 polymorphism is predictive of dose across 3 racial groups
Blood. 2010 May 6;115(18):3827-34.
Limdi NA, Wadelius M, Cavallari L, Eriksson N, Crawford DC, Lee MT, Chen CH, Motsinger-Reif A, Sagreiya H, Liu N, Wu AH, Gage BF, Jorgensen A, Pirmohamed M, Shin JG, Suarez-Kurtz G, Kimmel SE, Johnson JA, Klein TE, Wagner MJ; International Warfarin Pharmacogenetics Consortium.
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.
Title 4 : Inhibition of bacterial disulfide bond formation by the anticoagulant warfarin
Proc Natl Acad Sci U S A. 2010 Jan 5;107(1):297-301.
Dutton RJ, Wayman A, Wei JR, Rubin EJ, Beckwith J, Boyd D.
Blood coagulation in humans requires the activity of vitamin K epoxide reductase (VKOR), the target of the anticoagulant warfarin (Coumadin). Bacterial homologs of VKOR were recently found to participate in a pathway leading to disulfide bond formation in secreted proteins of many bacteria. Here we show that the VKOR homolog from the bacterium Mycobacterium tuberculosis, the causative agent of human tuberculosis, is inhibited by warfarin and that warfarin-resistant mutations of mycobacterial VKOR appear in similar locations to mutations found in human patients who require higher doses of warfarin. Deletion of VKOR results in a severe growth defect in mycobacteria, and the growth of M. tuberculosis is inhibited by warfarin. The bacterial VKOR homolog may represent a target for antibiotics and a model for genetic studies of human VKOR. We present a simple assay in Escherichia coli, based on a disulfide-sensitive beta-galactosidase, which can be used to screen for stronger inhibitors of the M. tuberculosis VKOR homolog.
Title 1 : Reconstructing Indian population history.
Nature. 2009 Sep 24;461(7263):489-94.
Reich D, Thangaraj K, Patterson N, Price AL, Singh L.
India has been underrepresented in genome-wide surveys of human variation. We analyse 25 diverse groups in India to provide strong evidence for two ancient populations, genetically divergent, that are ancestral to most Indians today. One, the 'Ancestral North Indians' (ANI), is genetically close to Middle Easterners, Central Asians, and Europeans, whereas the other, the 'Ancestral South Indians' (ASI), is as distinct from ANI and East Asians as they are from each other. By introducing methods that can estimate ancestry without accurate ancestral populations, we show that ANI ancestry ranges from 39-71% in most Indian groups, and is higher in traditionally upper caste and Indo-European speakers. Groups with only ASI ancestry may no longer exist in mainland India. However, the indigenous Andaman Islanders are unique in being ASI-related groups without ANI ancestry. Allele frequency differences between groups in India are larger than in Europe, reflecting strong founder effects whose signatures have been maintained for thousands of years owing to endogamy. We therefore predict that there will be an excess of recessive diseases in India, which should be possible to screen and map genetically.
Title 2 : Warfarin pharmacogenomics.
Curr Opin Mol Ther. 2009 Jun;11(3):243-51.
Cavallari LH, Limdi NA.
Warfarin therapy is complicated by a narrow therapeutic index and substantial interpatient variability in dose response. The cytochrome P450 (CYP)2C9 and vitamin K epoxide reductase complex 1 (VKORC1) genes have recently been determined to be associated with warfarin dose requirements, with reduced doses of this drug being required in patients with the variant CYP2C9*2, CYP2C9*3, or VKORC1 -1639A allele. The combination of genotype and clinical factors explains approximately 50 to 60% of the variance in warfarin dose requirements in Caucasians and Asians, but only 25 to 40% among African Americans. Racial differences in the association between genotype and a patient's response to warfarin treatment may be caused by racial differences in the frequencies of the variant CYP2C9*2, CYP2C9*3, and VKORC1 -1639A alleles or by the influence of non-genetic factors. Genotype also influences the time required to attain therapeutic anticoagulation and the risk for over-anticoagulation and hemorrhage. Although the incorporation of genotype information improves the accuracy of dose prediction, an improvement in anticoagulation control or a reduction in hemorrhagic complications has not been demonstrated. Therefore, the routine use of CYP2C9 and VKORC1 genotyping is not supported by currently available evidence. The results of ongoing or future multicenter clinical trials are expected to clarify the role of pharmacogenomics in the management of warfarin therapy.
Title 3 : Prevalence in the United States of selected candidate gene variants: Third National Health and Nutrition Examination Survey, 1991-1994.
Am J Epidemiol. 2009 Jan 1;169(1):54-66.
Chang MH, Lindegren ML, Butler MA, Chanock SJ, Dowling NF, Gallagher M, Moonesinghe R, Moore CA, Ned RM, Reichler MR, Sanders CL, Welch R, Yesupriya A, Khoury MJ; CDC/NCI NHANES III Genomics Working Group.
Population-based allele frequencies and genotype prevalence are important for measuring the contribution of genetic variation to human disease susceptibility, progression, and outcomes. Population-based prevalence estimates also provide the basis for epidemiologic studies of gene-disease associations, for estimating population attributable risk, and for informing health policy and clinical and public health practice. However, such prevalence estimates for genotypes important to public health remain undetermined for the major racial and ethnic groups in the US population. DNA was collected from 7,159 participants aged 12 years or older in Phase 2 (1991-1994) of the Third National Health and Nutrition Examination Survey (NHANES III). Certain age and minority groups were oversampled in this weighted, population-based US survey. Estimates of allele frequency and genotype prevalence for 90 variants in 50 genes chosen for their potential public health significance were calculated by age, sex, and race/ethnicity among non-Hispanic whites, non-Hispanic blacks, and Mexican Americans. These nationally representative data on allele frequency and genotype prevalence provide a valuable resource for future epidemiologic studies in public health in the United States.
Title 4 : VKORC1 haplotypes in five East-Asian populations and Indians.
Pharmacogenomics. 2009 Oct;10(10):1609-16.
Lee MT, Chen CH, Chuang HP, Lu LS, Chou CH, Chen YT, Liu CY, Wen MS, Lu JJ, Chang CF, Wu JY, Chen YT.
Title 5 : Estimation of the warfarin dose with clinical and pharmacogenetic data.
N Engl J Med. 2009 Feb 19;360(8):753-64.
International Warfarin Pharmacogenetics Consortium, Klein TE, Altman RB, Eriksson N, Gage BF, Kimmel SE, Lee MT, Limdi NA, Page D, Roden DM, Wagner MJ, Caldwell MD, Johnson JA.
Title 6: A genome-wide association study confirms VKORC1, CYP2C9, and CYP4F2 as principal genetic determinants of warfarin dose.
PLoS Genet. 2009 Mar;5(3):e1000433.
Takeuchi F, McGinnis R, Bourgeois S, Barnes C, Eriksson N, Soranzo N, Whittaker P, Ranganath V, Kumanduri V, McLaren W, Holm L, Lindh J, Rane A, Wadelius M, Deloukas P.
We report the first genome-wide association study (GWAS) whose sample size (1,053 Swedish subjects) is sufficiently powered to detect genome-wide significance (p<1.5 x 10(-7)) for polymorphisms that modestly alter therapeutic warfarin dose. The anticoagulant drug warfarin is widely prescribed for reducing the risk of stroke, thrombosis, pulmonary embolism, and coronary malfunction. However, Caucasians vary widely (20-fold) in the dose needed for therapeutic anticoagulation, and hence prescribed doses may be too low (risking serious illness) or too high (risking severe bleeding). Prior work established that approximately 30% of the dose variance is explained by single nucleotide polymorphisms (SNPs) in the warfarin drug target VKORC1 and another approximately 12% by two non-synonymous SNPs (*2, *3) in the cytochrome P450 warfarin-metabolizing gene CYP2C9. We initially tested each of 325,997 GWAS SNPs for association with warfarin dose by univariate regression and found the strongest statistical signals (p<10(-78)) at SNPs clustering near VKORC1 and the second lowest p-values (p<10(-31)) emanating from CYP2C9. No other SNPs approached genome-wide significance. To enhance detection of weaker effects, we conducted multiple regression adjusting for known influences on warfarin dose (VKORC1, CYP2C9, age, gender) and identified a single SNP (rs2108622) with genome-wide significance (p = 8.3 x 10(-10)) that alters protein coding of the CYP4F2 gene. We confirmed this result in 588 additional Swedish patients (p<0.0029) and, during our investigation, a second group provided independent confirmation from a scan of warfarin-metabolizing genes. We also thoroughly investigated copy number variations, haplotypes, and imputed SNPs, but found no additional highly significant warfarin associations. We present power analysis of our GWAS that is generalizable to other studies, and conclude we had 80% power to detect genome-wide significance for common causative variants or markers explaining at least 1.5% of dose variance. These GWAS results provide further impetus for conducting large-scale trials assessing patient benefit from genotype-based forecasting of warfarin dose.
Title 7 : The largest prospective warfarin-treated cohort supports genetic forecasting.
Blood. 2009 Jan 22;113(4):784-92.
Wadelius M, Chen LY, Lindh JD, Eriksson N, Ghori MJ, Bumpstead S, Holm L, McGinnis R, Rane A, Deloukas P.
Genetic variants of cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase (VKORC1) are known to influence warfarin dose, but the effect of other genes has not been fully elucidated. We genotyped 183 polymorphisms in 29 candidate genes in 1496 Swedish patients starting warfarin treatment, and tested for association with response. CYP2C9*2 and *3 explained 12% (P = 6.63 x 10(-34)) of the variation in warfarin dose, while a single VKORC1 SNP explained 30% (P = 9.82 x 10(-100)). No SNP outside the CYP2C gene cluster and VKORC1 regions was significantly associated with dose after correction for multiple testing. During initiation of therapy, homozygosity for CYP2C9 and VKORC1 variant alleles increased the risk of over-anticoagulation, hazard ratios 21.84 (95% CI 9.46; 50.42) and 4.56 (95% CI 2.85; 7.30), respectively. One of 8 patients with CYP2C9*3/*3 (12.5%) experienced severe bleeding during the first month compared with 0.27% of other patients (P = .066). A multiple regression model using the predictors CYP2C9, VKORC1, age, sex, and druginteractions explained 59% of the variance in warfarin dose, and 53% in an independent sample of 181 Swedish individuals. In conclusion, CYP2C9 and VKORC1 significantly influenced warfarin dose and predicted individuals predisposed to unstable anticoagulation. Our results strongly support that initiation of warfarin guided by pharmacogenetics would improve clinical outcome.
Title 8 : VKORC1 haplotypes in five East-Asian populations and Indians.
Pharmacogenomics. 2009 Oct;10(10):1609-16.
Lee MT, Chen CH, Chuang HP, Lu LS, Chou CH, Chen YT, Liu CY, Wen MS, Lu JJ, Chang CF, Wu JY, Chen YT.
Title 9 : Genetic determinants of warfarin dosing in the Han-Chinese population.
Pharmacogenomics. 2009 Dec;10(12):1905-13.
Lee MT, Chen CH, Chou CH, Lu LS, Chuang HP, Chen YT, Saleem AN, Wen MS, Chen JJ, Wu JY, Chen YT.
Warfarin, a widely prescribed oral anticoagulant, is used for the prevention of thromboembolism. Polymorphisms in CYP2C9 and VKORC1 have been shown to be associated with warfarin dose requirements. However, it is likely that other genes could also affect warfarin dose.
Title 1 : Apolipoprotein E genotype and warfarin dosing among Caucasians and African Americans.
Pharmacogenomics J. 2008 Feb;8(1):53-60.
Kimmel SE, Christie J, Kealey C, Chen Z, Price M, Thorn CF, Brensinger CM, Newcomb CW, Whitehead AS.
Warfarin sodium is a vitamin K antagonist that is plagued by large variability in patient response, including higher dose requirements among African Americans. Polymorphisms in the gene encoding apolipoprotein E (APOE) may partly explain this variability by altering transport of vitamin K to the liver. In a prospective cohort study of 232 individuals (52.2% Caucasian and 47.8% African American) initiating warfarin therapy, the weekly maintenance dose was significantly higher for African Americans than for Caucasians (mean 42.9 versus mean 36.9 mg, P=0.018), and the epsilon4 allele was more common among African Americans (37.8 versus 26.4% for Caucasians). In multivariable analyses, the presence of the epsilon4 allele was associated with a statistically significantly higher warfarin dose among African Americans (median 45.0 mg in epsilon4 carriers versus 35.0 mg in non-epsilon4 carriers, P=0.014) but not Caucasians (38.1 versus 35.0 mg, P=0.60). In addition, warfarin maintenance dose increased among African Americans according to genotype previously associated with differential hepatic chylomicron clearance (epsilon2/epsilon2 or epsilon2/epsilon3: 30.0 mg; epsilon3/epsilon3: 35.0 mg; epsilon3/epsilon4 or epsilon4/epsilon4: 45.0 mg; P=0.012), although the epsilon4/epsilon4 genotype was rare and not clearly associated with higher doses. The association of APOE with warfarin dosing was independent of CYP2C9 and VKORC1 polymorphisms. APOE polymorphisms thus may be important determinants of warfarin maintenance dose and could explain at least some of the observed racial differences in dose requirements.
Title 2 : VKORC1 Asp36Tyr warfarin resistance marker is common in Ethiopian individuals.
Blood. 2008 Apr 1;111(7):3903-4.
Aklillu E, Leong C, Loebstein R, Halkin H, Gak E.
Warfarin is the mainstay of oral anticoagulation worldwide. However, its use is complicated by a narrow therapeutic index with potentially severe adverse effects and high interindividual variability in dose requirements. The explanation for this variability is multifactorial and determined by genetic factors, concomitant drugs, age, diet, and race. African-American patients are more warfarin resistant than whites, while Chinese and Japanese are mostly warfarin sensitive.1,2 Extensive research in this field yielded several important genetic markers of warfarin dose response, predominantly including markers of warfarin sensitivity caused by common polymorphisms in the major warfarin target gene VKORC1 and its metabolizing enzyme CYP2C9.
Title 3 : African genetic diversity: implications for human demographic history, modern human origins, and complex disease mapping.
Annu Rev Genomics Hum Genet. 2008;9:403-33.
Campbell MC, Tishkoff SA.
Comparative studies of ethnically diverse human populations, particularly in Africa, are important for reconstructing human evolutionary history and for understanding the genetic basis of phenotypic adaptation and complex disease. African populations are characterized by greater levels of genetic diversity, extensive population substructure, and less linkage disequilibrium (LD) among loci compared to non-African populations. Africans also possess a number of genetic adaptations that have evolved in response to diverse climates and diets, as well as exposure to infectious disease. This review summarizes patterns and the evolutionary origins of genetic diversity present in African populations, as well as their implications for the mapping of complex traits, including disease susceptibility.
Title 4 : Dosing algorithms to predict warfarin maintenance dose in Caucasians and African Americans.
Clin Pharmacol Ther. 2008 Sep;84(3):332-9.
Schelleman H, Chen J, Chen Z, Christie J, Newcomb CW, Brensinger CM, Price M, Whitehead AS, Kealey C, Thorn CF, Samaha FF, Kimmel SE.
The objective of this study was to determine whether warfarin dosing algorithms developed for Caucasians and African Americans on the basis of clinical, environmental, and genetic factors will perform better than an empirical starting dose of 5 mg/day. From April 2002 through December 2005, 259 subjects (Caucasians and African Americans) who started using warfarin were prospectively followed until they reached maintenance dose. The Caucasian algorithm included 11 variables (R(2) = 0.43). This model (which predicted 51% of the doses to within 1 mg of the observed dose) performed better than 5 mg/day (which predicted 29% of the doses to within 5 +/- 1 mg). The African-American algorithm included 10 variables (R(2) = 0.28). This model predicted 37% of the doses to within 1 mg of the observed dose, representing a small improvement compared with 5 mg/day (which predicted 34% of the doses to within 1 mg of 5 mg/day). These results were similar to the results we obtained from testing other published algorithms. The dosing algorithms explained <45% of the observed variability in Caucasians, and the algorithms performed only marginally better for African Americans when compared with giving 5 mg empirically.
Title 5 : VKORC1 polymorphisms, haplotypes and haplotype groups on warfarin dose among African- Americans and European-Americans.
Pharmacogenomics. 2008 Oct;9(10):1445-58.
Limdi NA, Beasley TM, Crowley MR, Goldstein JA, Rieder MJ, Flockhart DA, Arnett DK, Acton RT, Liu N.
Title 6 : Regulatory polymorphism in vitamin K epoxide reductase complex subunit 1 (VKORC1) affects gene expression and warfarin dose requirement.
Blood. 2008 Aug 15;112(4):1013-21
Wang D, Chen H, Momary KM, Cavallari LH, Johnson JA, Sadée W.
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.
Title 7 : A genome-wide scan for common genetic variants with a large influence on warfarin maintenance dose.
Blood. 2008 Aug 15;112(4):1022-7.
Cooper GM, Johnson JA, Langaee TY, Feng H, Stanaway IB, Schwarz UI, Ritchie MD, Stein CM, Roden DM, Smith JD, Veenstra DL, Rettie AE, Rieder MJ.
Warfarin dosing is correlated with polymorphisms in vitamin K epoxide reductase complex 1 (VKORC1) and the cytochrome P450 2C9 (CYP2C9) genes. Recently, the FDA revised warfarin labeling to raise physician awareness about these genetic effects. Randomized clinical trials are underway to test genetically based dosing algorithms. It is thus important to determine whether common single nucleotide polymorphisms (SNPs) in other gene(s) have a large effect on warfarin dosing. A retrospective genome-wide association study was designed to identify polymorphisms that could explain a large fraction of the dose variance. White patients from an index warfarin population (n = 181) and 2 independent replication patient populations (n = 374) were studied. From the approximately 550 000 polymorphisms tested, the most significant independent effect was associated with VKORC1 polymorphisms (P = 6.2 x 10(-13)) in the index patients. CYP2C9 (rs1057910 CYP2C9*3) and rs4917639) was associated with dose at moderate significance levels (P approximately 10(-4)). Replication polymorphisms (355 SNPs) from the index study did not show any significant effects in the replication patient sets. We conclude that common SNPs with large effects on warfarin dose are unlikely to be discovered outside of the CYP2C9 and VKORC1 genes. Randomized clinical trials that account for these 2 genes should therefore produce results that are definitive and broadly applicable.
Title 8 : Warfarin pharmacogenetics: CYP2C9 and VKORC1 genotypes predict different sensitivity and resistance frequencies in the Ashkenazi and Sephardi Jewish populations.
Am J Hum Genet. 2008 Feb;82(2):495-500.
Scott SA, Edelmann L, Kornreich R, Desnick RJ.
Warfarin is a widely used anticoagulant that has a narrow therapeutic range because of both genetic and environmental factors. CYP2C9( *)2 (p.R144C), CYP2C9( *)3 (p.I359L), and the VKORC1 promoter (g.-1639G-->A) polymorphisms occur frequently in patients who are warfarin "sensitive" and require lower doses, whereas patients with VKORC1 missense mutations are warfarin "resistant" and require higher doses. To compare the CYP2C9 and VKORC1 allele and genotype frequencies among 260 Ashkenazi (AJ) and 80 Sephardi Jewish (SJ) individuals, we genotyped six CYP2C9 and eight VKORC1 alleles by using the Tag-It Mutation Detection Kit and PCR-RFLP assays. The "sensitive"CYP2C9( *)2 and ( *)3 alleles had significantly higher frequencies in SJ than in AJ individuals, 0.194 and 0.144 versus 0.127 and 0.081, respectively (p <or= 0.001). In contrast, the VKORC1 p.D36Y mutation, which predicts warfarin "resistance," had a significantly higher frequency in AJ than in SJ individuals, 0.043 versus 0.006, respectively (p <or= 0.025). Of note, 11.3% of AJ individuals predicted to be CYP2C9 extensive metabolizers and 8.7% of those predicted to be intermediate and poor metabolizers were VKORC1 p.D36Y carriers who require markedly higher warfarin doses. Thus, approximately 10% of all AJ individuals would be misclassified when only genotyping CYP2C9( *)2, ( *)3, and VKORC1 g.-1639G-->A, underscoring the importance of screening for p.D36Y prior to initiating warfarin anticoagulation in AJ individuals. Taken together, our findings show that approximately 85% of AJ and approximately 90% of SJ individuals have at least one "sensitive" (CYP2C9( *)2, ( *)3, VKORC1 g.-1639G-->A) or "resistant" (VKORC1 p.D36Y) allele, indicating that each group has different warfarin pharmacogenetics and would benefit from genotype-based dose predictions.
Title 9 : Use of pharmacogenetic and clinical factors to predict the therapeutic dose of warfarin.
Clinical Pharmacology & Therapeutics 84, 326-331 (September 2008)
BF Gage, C Eby, JA Johnson, E Deych, MJ Rieder, PM Ridker, PE Milligan, G Grice, P Lenzini, AE Rettie, CL Aquilante, L Grosso, S Marsh, T Langaee, LE Farnett, D Voora, DL Veenstra, RJ Glynn, A Barrett and HL McLeod
Initiation of warfarin therapy using trial-and-error dosing is problematic. Our goal was to develop and validate a pharmacogenetic algorithm. In the derivation cohort of 1,015 participants, the independent predictors of therapeutic dose were: VKORC1 polymorphism −1639/3673 G>A (−28% per allele), body surface area (BSA) (+11% per 0.25 m2), CYP2C9*3 (−33% per allele), CYP2C9*2 (−19% per allele), age (−7% per decade), target international normalized ratio (INR) (+11% per 0.5 unit increase), amiodarone use (−22%), smoker status (+10%), race (−9%), and current thrombosis (+7%). This pharmacogenetic equation explained 53–54% of the variability in the warfarin dose in the derivation and validation (N= 292) cohorts. For comparison, a clinical equation explained only 17–22% of the dose variability (P < 0.001). In the validation cohort, we prospectively used the pharmacogenetic-dosing algorithm in patients initiating warfarin therapy, two of whom had a major hemorrhage. To facilitate use of these pharmacogenetic and clinical algorithms, we developed a nonprofit website, http://www.WarfarinDosing.org.
Title 10 : Prospective study of warfarin dosage requirements based on CYP2C9 and VKORC1 genotypes.
Clin Pharmacol Ther. 2008 Jul;84(1):83-9.
Wen MS, Lee M, Chen JJ, Chuang HP, Lu LS, Chen CH, Lee TH, Kuo CT, Sun FM, Chang YJ, Kuan PL, Chen YF, Charng MJ, Ray CY, Wu JY, Chen YT.
Polymorphisms in CYP2C9 and VKORC1 have been shown to be associated with warfarin dose requirements and could be used to predict warfarin dose. We conducted a prospective study in which warfarin dose was prescribed based on CYP2C9 and VKORC1 polymorphisms in 108 Han-Chinese patients without prior warfarin treatments. Using the genotype-based dosing, 83% of patients reached stable, therapeutic international normalized ratio (INR) within 2 weeks of treatment initiation and none of the patients developed clinical bleeding or thromboembolic event. Ten percent (11) of patients with INR > 4 and no clinical bleeding were detected during this study. At 12 weeks, 69% of the patients' maintenance doses matched the prediction. Dosing algorithms incorporating genetic factors, age, and body surface area were developed, which could explain up to 62% of the total variation (R(2) of 0.62). This study demonstrated that pharmacogenetics-based dosing could improve time to stable, therapeutic INR, reduce adverse events, and achieve high sensitivity.
Title 1 : Gammaglutamyl carboxylase (GGCX) tagSNPs have limited utility for predicting warfarin maintenance dose.
J Thromb Haemost. 2007 Nov;5(11):2227-34.
Rieder MJ, Reiner AP, Rettie AE.
Title 2 : Rapid and accurate haplotype phasing and missing-data inference for whole-genome association studies by use of localized haplotype clustering.
Am J Hum Genet. 2007 Nov;81(5):1084-97.
Browning SR, Browning BL.
Whole-genome association studies present many new statistical and computational challenges due to the large quantity of data obtained. One of these challenges is haplotype inference; methods for haplotype inference designed for small data sets from candidate-gene studies do not scale well to the large number of individuals genotyped in whole-genome association studies. We present a new method and software for inference of haplotype phase and missing data that can accurately phase data from whole-genome association studies, and we present the first comparison of haplotype-inference methods for real and simulated data sets with thousands of genotyped individuals. We find that our method outperforms existing methods in terms of both speed and accuracy for large data sets with thousands of individuals and densely spaced genetic markers, and we use our method to phase a real data set of 3,002 individuals genotyped for 490,032 markers in 3.1 days of computing time, with 99% of masked alleles imputed correctly. Our method is implemented in the Beagle software package, which is freely available.
Title 3 : A second generation human haplotype map of over 3.1 million SNPs.
Nature. 2007 Oct 18;449(7164):851-61.
International HapMap Consortium
We describe the Phase II HapMap, which characterizes over 3.1 million human single nucleotide polymorphisms (SNPs) genotyped in 270 individuals from four geographically diverse populations and includes 25-35% of common SNP variation in the populations surveyed. The map is estimated to capture untyped common variation with an average maximum r2 of between 0.9 and 0.96 depending on population. We demonstrate that the current generation of commercial genome-wide genotyping products captures common Phase II SNPs with an average maximum r2 of up to 0.8 in African and up to 0.95 in non-African populations, and that potential gains in power in association studies can be obtained through imputation. These data also reveal novel aspects of the structure of linkage disequilibrium. We show that 10-30% of pairs of individuals within a population share at least one region of extended genetic identity arising from recent ancestry and that up to 1% of all common variants are untaggable, primarily because they lie within recombination hotspots. We show that recombination rates vary systematically around genes and between genes of different function. Finally, we demonstrate increased differentiation at non-synonymous, compared to synonymous, SNPs, resulting from systematic differences in the strength or efficacy of natural selection between populations.
Title 4 : Warfarin response and vitamin K epoxide reductase complex 1 in African Americans and Caucasians.
Clin Pharmacol Ther. 2007 May;81(5):742-7.
Schelleman H, Chen Z, Kealey C, Whitehead AS, Christie J, Price M, Brensinger CM, Newcomb CW, Thorn CF, Samaha FF, Kimmel SE.
The objective of this study was to determine whether two vitamin K epoxide reductase complex 1 (VKORC1) polymorphisms contribute to the variability in warfarin response, particularly in African Americans. The effect of the VKORC1 1173C/T and -1639G/A polymorphisms was examined in a prospective cohort study of 338 warfarin users. Subjects carrying an 1173T allele had a lower warfarin maintenance dose compared with subjects with the CC genotype in African Americans (-12.10 mg/week+/-4.93; P=0.02) and Caucasians (-14.41 mg/week+/-3.28; P<0.001). Before reaching maintenance dose, only Caucasians with the T allele had significantly increased risk of international normalized ratio >3 (odds ratio=3.10; 95% confidence interval: 1.73-5.55) compared with Caucasians with the CC genotype. Polymorphisms in the VKORC1 gene are associated with warfarin maintenance dose requirements among both African Americans and Caucasians. However, these polymorphisms may not be as useful in predicting over-anticoagulation among African Americans.
Title 5 : Factors affecting the interindividual variability of warfarin dose requirement in adult Korean patients.
Pharmacogenomics. 2007 Apr;8(4):329-37.
Cho HJ, Sohn KH, Park HM, Lee KH, Choi B, Kim S, Kim JS, On YK, Chun MR, Kim HJ, Kim JW, Lee SY.
Title 6 : The largest prospective warfarin-treated cohort supports genetic forecasting.
Blood. 2009 Jan 22;113(4):784-92.
Wadelius M, Chen LY, Lindh JD, Eriksson N, Ghori MJ, Bumpstead S, Holm L, McGinnis R, Rane A, Deloukas P.
Genetic variants of cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase (VKORC1) are known to influence warfarin dose, but the effect of other genes has not been fully elucidated. We genotyped 183 polymorphisms in 29 candidate genes in 1496 Swedish patients starting warfarin treatment, and tested for association with response. CYP2C9*2 and *3 explained 12% (P = 6.63 x 10(-34)) of the variation in warfarin dose, while a single VKORC1 SNP explained 30% (P = 9.82 x 10(-100)). No SNP outside the CYP2C gene cluster and VKORC1 regions was significantly associated with dose after correction for multiple testing. During initiation of therapy, homozygosity for CYP2C9 and VKORC1 variant alleles increased the risk of over-anticoagulation, hazard ratios 21.84 (95% CI 9.46; 50.42) and 4.56 (95% CI 2.85; 7.30), respectively. One of 8 patients with CYP2C9*3/*3 (12.5%) experienced severe bleeding during the first month compared with 0.27% of other patients (P = .066). A multiple regression model using the predictors CYP2C9, VKORC1, age, sex, and druginteractions explained 59% of the variance in warfarin dose, and 53% in an independent sample of 181 Swedish individuals. In conclusion, CYP2C9 and VKORC1 significantly influenced warfarin dose and predicted individuals predisposed to unstable anticoagulation. Our results strongly support that initiation of warfarin guided by pharmacogenetics would improve clinical outcome.
Title 7 : Association of warfarin dose with genes involved in its action and metabolism.
Hum Genet. 2007 Mar;121(1):23-34.
Wadelius M, Chen LY, Eriksson N, Bumpstead S, Ghori J, Wadelius C, Bentley D, McGinnis R, Deloukas P.
We report an extensive study of variability in genes encoding proteins that are believed to be involved in the action and biotransformation of warfarin. Warfarin is a commonly prescribed anticoagulant that is difficult to use because of the wide interindividual variation in dose requirements, the narrow therapeutic range and the risk of serious bleeding. We genotyped 201 patients for polymorphisms in 29 genes in the warfarin interactive pathways and tested them for association with dose requirement. In our study, polymorphisms in or flanking the genes VKORC1, CYP2C9, CYP2C18, CYP2C19, PROC, APOE, EPHX1, CALU, GGCX and ORM1-ORM2 and haplotypes of VKORC1, CYP2C9, CYP2C8, CYP2C19, PROC, F7, GGCX, PROZ, F9, NR1I2 and ORM1-ORM2 were associated with dose (P < 0.05). VKORC1, CYP2C9, CYP2C18 and CYP2C19 were significant after experiment-wise correction for multiple testing (P < 0.000175), however, the association of CYP2C18 and CYP2C19 was fully explained by linkage disequilibrium with CYP2C9*2 and/or *3. PROC and APOE were both significantly associated with dose after correction within each gene. A multiple regression model with VKORC1, CYP2C9, PROC and the non-genetic predictors age, bodyweight, drug interactions and indication for treatment jointly accounted for 62% of variance in warfarin dose. Weaker associations observed for other genes could explain up to approximately 10% additional dose variance, but require testing and validation in an independent and larger data set. Translation of this knowledge into clinical guidelines for warfarin prescription will be likely to have a major impact on the safety and efficacy of warfarin.
Title 8 : Allelic variants in the CYP2C9 and VKORC1 loci and interindividual variability in the anticoagulant dose effect of warfarin in Italians.
Pharmacogenomics. 2007 Nov;8(11):1545-50.
Borgiani P, Ciccacci C, Forte V, Romano S, Federici G, Novelli G.
Title 9 : Pharmacogenetics of warfarin: current status and future challenges.
Pharmacogenomics J. 2007 Apr;7(2):99-111.
Wadelius M, Pirmohamed M.
Warfarin is an anticoagulant that is difficult to use because of the wide variation in dose required to achieve a therapeutic effect, and the risk of serious bleeding. Warfarin acts by interfering with the recycling of vitamin K in the liver, which leads to reduced activation of several clotting factors. Thirty genes that may be involved in the biotransformation and mode of action of warfarin are discussed in this review. The most important genes affecting the pharmacokinetic and pharmacodynamic parameters of warfarin are CYP2C9 (cytochrome P(450) 2C9) and VKORC1 (vitamin K epoxide reductase complex subunit 1). These two genes, together with environmental factors, partly explain the interindividual variation in warfarin dose requirements. Large ongoing studies of genes involved in the actions of warfarin, together with prospective assessment of environmental factors, will undoubtedly increase the capacity to accurately predict warfarin dose. Implementation of pre-prescription genotyping and individualized warfarin therapy represents an opportunity to minimize the risk of haemorrhage without compromising effectiveness.
Title 10 : Estimation of warfarin maintenance dose based on VKORC1 (-1639 G>A) and CYP2C9 genotypes.
Clin Chem. 2007 Jul;53(7):1199-205.
Zhu Y, Shennan M, Reynolds KK, Johnson NA, Herrnberger MR, Valdes R Jr, Linder MW.
Title 1 : Global pharmacogenetics: giving the genome to the masses.
Pharmacogenomics. 2006 Jun;7(4):625-31.
Marsh S, Van Booven DJ, McLeod HL.
With pharmacogenetics comes the promise of individualized therapy selection for many common diseases where multiple treatment options are available. Recent advances including the Human Genome Project, the International HapMap project, advances in throughput and reduction in cost of genetic testing, and the inclusion of genotype-related dosing recommendations into package inserts all point to the integration of pharmacogenetics into clinical practice. However, many countries will not have access to pharmacogenetics resources in the near future. Generation of global genotype profiles will provide a useful, but not perfect resource for incorporating pharmacogenetics into national drug formularies in the form of prioritization or surveillance where individual genotype data would not be attainable. The PharmacoGenetics for Every Nation Initiative is a first step to making pharmacogenetics applicable on a global level.
Title 2 : Genotypes of the cytochrome p450 isoform, CYP2C9, and the vitamin K epoxide reductase complex subunit 1 conjointly determine stable warfarin dose: a prospective study.
J Thromb Thrombolysis. 2006 Dec;22(3):191-7.
Carlquist JF, Horne BD, Muhlestein JB, Lappé DL, Whiting BM, Kolek MJ, Clarke JL, James BC, Anderson JL.
Title 3 : VKORC1 gene variations are the major contributors of variation in warfarin dose in Japanese patients.
Clin Pharmacol Ther. 2006 Aug;80(2):169-78.
Obayashi K, Nakamura K, Kawana J, Ogata H, Hanada K, Kurabayashi M, Hasegawa A, Yamamoto K, Horiuchi R.
Title 4 : Different contributions of polymorphisms in VKORC1 and CYP2C9 to intra- and interpopulation differences in maintenance dose of warfarin in Japanese, Caucasians and African- Americans.
Pharmacogenet Genomics. 2006 Feb;16(2):101-10.
Takahashi H, Wilkinson GR, Nutescu EA, Morita T, Ritchie MD, Scordo MG, Pengo V, Barban M, Padrini R, Ieiri I, Otsubo K, Kashima T, Kimura S, Kijima S, Echizen H.
Title 1 : Haploview: analysis and visualization of LD and haplotype maps.
Bioinformatics. 2005 Jan 15;21(2):263-5.
Barrett JC, Fry B, Maller J, Daly MJ.
Research over the last few years has revealed significant haplotype structure in the human genome. The characterization of these patterns, particularly in the context of medical genetic association studies, is becoming a routine research activity. Haploview is a software package that provides computation of linkage disequilibrium statistics and population haplotype patterns from primary genotype data in a visually appealing and interactive interface.
Title 2 : VKORC1 haplotypes and their impact on the inter-individual and inter-ethnical variability of oral anticoagulation.
Thromb Haemost. 2005 Oct;94(4):773-9.
Geisen C, Watzka M, Sittinger K, Steffens M, Daugela L, Seifried E, Müller CR, Wienker TF, Oldenburg J.
In order to elucidate the role of VCORC1 sequence variants in warfarin sensitivity, we established a complete SNP map of the VKORC1 gene locus in 200 blood donors from Western Germany. Nearly all of the genetic variability of the VKORC1 gene in Europeans is reflected by three main haplotypes. Recently described polymorphisms associated with low warfarin dose requirement (dbSNP:rs9934438; dbSNP:rs17878363) were found in complete linkage disequilibrium with the VKORC1*2 haplotype. In two patient cohorts of European origin with either increased coumarin sensitivity (n= 14) or partial coumarin resistance (n=36) the VKORC1*2 frequency varied highly significant between the two groups and also when compared to 200 blood donor controls (coumarin sensitive 96%, coumarin resistant 7%, controls 42%) thus demonstrating a strong association between these two phenotypes and the VKORC1 haplotype (p = 1.6 x 10(-8) for coumarin sensitive and p = 1.9 x 10(-8) for coumarin resistant). Analysis of database derived VKORC1 genotypes of African Americans and Chinese revealed that haplotype frequencies in these populations differ significantly from the European sample (for VKORC1*2: Europeans 42%, Chinese 95%, African Americans 14%). These observations suggest VKORC1 as principal genetic modulator of the ethnic differences in warfarin response. Since hereditary pharmacodynamic (VKORC1) and pharmacokinetic (CYP2C9) factors account for up to 50% of the inter-individual variability of the warfarin response, these genetic markers may serve as clinically relevant predictors of warfarin dosing in future studies.
Title 3 : Effect of VKORC1 haplotypes on transcriptional regulation and warfarin dose.
N Engl J Med. 2005 Jun 2;352(22):2285-93.
Rieder MJ, Reiner AP, Gage BF, Nickerson DA, Eby CS, McLeod HL, Blough DK, Thummel KE, Veenstra DL, Rettie AE.
Title 4 : Common VKORC1 and GGCX polymorphisms ssociated with warfarin dose.
Pharmacogenomics J. 2005;5(4):262-70.
Wadelius M, Chen LY, Downes K, Ghori J, Hunt S, Eriksson N, Wallerman O, Melhus H, Wadelius C, Bentley D, Deloukas P.
We report a novel combination of factors that explains almost 60% of variable response to warfarin. Warfarin is a widely used anticoagulant, which acts through interference with vitamin K epoxide reductase that is encoded by VKORC1. In the next step of the vitamin K cycle, gamma-glutamyl carboxylase encoded by GGCX uses reduced vitamin K to activate clotting factors. We genotyped 201 warfarin-treated patients for common polymorphisms in VKORC1 and GGCX. All the five VKORC1 single-nucleotide polymorphisms covary significantly with warfarin dose, and explain 29-30% of variance in dose. Thus, VKORC1 has a larger impact than cytochrome P450 2C9, which explains 12% of variance in dose. In addition, one GGCX SNP showed a small but significant effect on warfarin dose. Incorrect dosage, especially during the initial phase of treatment, carries a high risk of either severe bleeding or failure to prevent thromboembolism. Genotype-based dose predictions may in future enable personalised drug treatment from the start of warfarin therapy.
Title 5 : Association of Vitamin K epoxide reductase complex 1 (VKORC1) variants with warfarin dose in a Hong Kong Chinese patient population.
Pharmacogenet Genomics. 2005 Oct;15(10):687-91.
Veenstra DL, You JH, Rieder MJ, Farin FM, Wilkerson HW, Blough DK, Cheng G, Rettie AE.
Title 6 : The impact of CYP2C9 and VKORC1 genetic polymorphism and patient characteristics upon warfarin dose requirements: proposal for a new dosing regimen.
Blood. 2005 Oct 1;106(7):2329-33.
Sconce EA, Khan TI, Wynne HA, Avery P, Monkhouse L, King BP, Wood P, Kesteven P, Daly AK, Kamali F.
Current dosing algorithms do not account for genetic and environmental factors for warfarin dose determinations. This study investigated the contribution of age, CYP2C9 and VKORC1 genotype, and body size to warfarin-dose requirements. Studied were 297 patients with stable anticoagulation with a target international normalized ratio (INR) of 2.0 to 3.0. Genetic analyses for CYP2C9 (*2 and *3 alleles) and VKORC1 (-1639 polymorphism) were performed and venous INR and plasma R- and S-warfarin concentrations determined. The mean warfarin daily dose requirement was highest in CYP2C9 homozygous wild-type patients, compared with those with the variant *2 and *3 alleles (P < .001) and highest in patients with the VKORC1 (position -1639) GG genotype compared with those with the GA genotype and the AA genotype (P < .001). Mean warfarin daily dose requirements fell by 0.5 to 0.7 mg per decade between the ages of 20 to 90 years. Age, height, and CYP2C9 genotype significantly contributed to S-warfarin and total warfarin clearance, whereas only age and body size significantly contributed to R-warfarin clearance. The multivariate regression model including the variables of age, CYP2C9 and VKORC1 genotype, and height produced the best model for estimating warfarin dose (R2 = 55%). Based upon the data, a new warfarin dosing regimen has been developed. The validity of the dosing regimen was confirmed in a second cohort of patients on warfarin therapy.
Title 7 : A novel functional VKORC1 promoter polymorphism is associated with inter-individual and inter-ethnic differences in warfarin sensitivity.
Hum Mol Genet. 2005 Jul 1;14(13):1745-51.
Yuan HY, Chen JJ, Lee MT, Wung JC, Chen YF, Charng MJ, Lu MJ, Hung CR, Wei CY, Chen CH, Wu JY, Chen YT.
Warfarin, a commonly prescribed anticoagulant, exhibited large inter-individual and inter-ethnic differences in the dose required for its anticoagulation effect. Asian populations, including Chinese, require a much lower maintenance dose than Caucasians, for which the mechanisms still remain unknown. We determined DNA sequence variants in CYP2C9 and VKORC1 in 16 Chinese patients having warfarin sensitivity (< or = 1.5 mg/day, n = 11) or resistance (> or = 6.0 mg/day, n = 5), 104 randomly selected Chinese patients receiving warfarin, 95 normal Chinese controls and 92 normal Caucasians. We identified three CYP2C9 variants, CYP2C9*3, T299A and P382L, in four warfarin-sensitive patients. A novel VKORC1 promoter polymorphism (-1639 G > A) presented in the homozygous form (genotype AA) was found in all warfarin-sensitive patients. The resistant patients were either AG or GG. Among the 104 randomly selected Chinese patients receiving warfarin, AA genotype also had lower dose than the AG/GG genotype (P < 0.0001). Frequencies of AA, AG and GG genotypes were comparable in Chinese patients receiving warfarin (79.7, 17.6 and 2.7%) and normal Chinese controls (82, 18 and 0%), but differed significantly from Caucasians (14, 47 and 39%) (P < 0.0001). The promoter polymorphism abolished the E-box consensus sequences and dual luciferase assay revealed that VOKRC1 promoter with the G allele had a 44% increase of activity when compared with the A allele. The differences in allele frequencies of A/G allele and its levels of VKORC1 promoter activity may underscore the inter-individual differences in warfarin dosage as well as inter-ethnic differences between Chinese and Caucasians.
Title 8 : A polymorphism in the VKORC1 gene is associated with an interindividual variability in the dose-anticoagulant effect of warfarin.
Blood. 2005; 105(2): 645-649.
Giovanna D'Andrea, Rosa Lucia D'Ambrosio, Pasquale Di Perna, Massimiliano Chetta, Rosa Santacroce, Vincenzo Brancaccio, Elvira Grandone, and Maurizio Margaglione
Patients require different warfarin dosages to achieve the target therapeutic anticoagulation. The variability is largely genetically determined, and it can be only partly explained by genetic variability in the cytochrome CYP2C9 locus. In 147 patients followed from the start of anticoagulation with warfarin, we have investigated whether VKORC1 gene mutations have affected doses of drug prescribed to acquire the target anticoagulation intensity. Two synonymous mutations, 129C>T at Cys43 and 3462C>T at Leu120, and 2 missense mutations, Asp38Tyr and Arg151Gln, were identified. None of these mutations was found to affect the interindividual variability of warfarin prescribed. Finally, 2 common polymorphisms were found, 1173C>T in the intron 1 and 3730G>A transition in the 3' untranslated region (UTR). Regardless of the presence of confounding variables, the mean adjusted dose required of warfarin was higher (6.2 mg) among patients with the VKORC1 1173CC genotype than those of patients carrying the CT (4.8 mg; P = .002) or the TT genotype (3.5 mg; P < .001). In the present setting, VKORC1 and CYP2C9 genetic variants investigated accounted for about a third (r2, 0.353) of the interindividual variability. Genetic variants of the VKORC1 gene locus modulate the mean daily dose of drug prescribed to acquire the target anticoagulation intensity.
Title 1 : Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2.
Nature. 2004 Feb 5;427(6974):537-41.
Rost S, Fregin A, Ivaskevicius V, Conzelmann E, Hörtnagel K, Pelz HJ, Lappegard K, Seifried E, Scharrer I, Tuddenham EG, Müller CR, Strom TM, Oldenburg J.
Coumarin derivatives such as warfarin represent the therapy of choice for the long-term treatment and prevention of thromboembolic events. Coumarins target blood coagulation by inhibiting the vitamin K epoxide reductase multiprotein complex (VKOR). This complex recycles vitamin K 2,3-epoxide to vitamin K hydroquinone, a cofactor that is essential for the post-translational gamma-carboxylation of several blood coagulation factors. Despite extensive efforts, the components of the VKOR complex have not been identified. The complex has been proposed to be involved in two heritable human diseases: combined deficiency of vitamin-K-dependent clotting factors type 2 (VKCFD2; Online Mendelian Inheritance in Man (OMIM) 607473), and resistance to coumarin-type anticoagulant drugs (warfarin resistance, WR; OMIM 122700). Here we identify, by using linkage information from three species, the gene vitamin K epoxide reductase complex subunit 1 (VKORC1), which encodes a small transmembrane protein of the endoplasmic reticulum. VKORC1 contains missense mutations in both human disorders and in a warfarin-resistant rat strain. Overexpression of wild-type VKORC1, but not VKORC1 carrying the VKCFD2 mutation, leads to a marked increase in VKOR activity, which is sensitive to warfarin inhibition.
Title 2 : Identification of the gene for vitamin K epoxide reductase.
Nature. 2004 Feb 5;427(6974):541-4.
Li T, Chang CY, Jin DY, Lin PJ, Khvorova A, Stafford DW.
Vitamin K epoxide reductase (VKOR) is the target of warfarin, the most widely prescribed anticoagulant for thromboembolic disorders. Although estimated to prevent twenty strokes per induced bleeding episode, warfarin is under-used because of the difficulty of controlling dosage and the fear of inducing bleeding. Although identified in 1974 (ref. 2), the enzyme has yet to be purified or its gene identified. A positional cloning approach has become possible after the mapping of warfarin resistance to rat chromosome 1 (ref. 3) and of vitamin K-dependent protein deficiencies to the syntenic region of human chromosome 16 (ref. 4). Localization of VKOR to 190 genes within human chromosome 16p12-q21 narrowed the search to 13 genes encoding candidate transmembrane proteins, and we used short interfering RNA (siRNA) pools against individual genes to test their ability to inhibit VKOR activity in human cells. Here, we report the identification of the gene for VKOR based on specific inhibition of VKOR activity by a single siRNA pool. We confirmed that MGC11276 messenger RNA encodes VKOR through its expression in insect cells and sensitivity to warfarin. The expressed enzyme is 163 amino acids long, with at least one transmembrane domain. Identification of the VKOR gene extends our understanding of blood clotting, and should facilitate development of new anticoagulant drugs.
Title 3 : Use of pharmacogenetics and clinical factors to predict the maintenance dose of warfarin.
Thromb Haemost. 2004 Jan;91(1):87-94.
Gage BF, Eby C, Milligan PE, Banet GA, Duncan JR, McLeod HL.
Knowledge of pharmacogenetics may help clinicians predict their patients' therapeutic dose of warfarin, thereby decreasing the risk of bleeding during warfarin initiation. Our goal was to use pharmacogenetics to develop an algorithm that uses genetic, clinical, and demographic factors to estimate the warfarin dose a priori. We collected a blood sample, demographic variables, laboratory values, smoking status, names of medications, and dietary history from 369 patients who were taking a maintenance dose of warfarin. Using polymerase chain reaction, we genotyped each participant for the presence of 8 polymorphisms in the cytochrome P450 2C9 system. Using multiple regression, we quantified the association between warfarin dose and all factors. Advanced age, lower body surface area (BSA), and the presence of cytochrome P450 2C9 *2 or *3 single nucleotide polymorphisms were strongly associated (P < 0.001) with lower warfarin dose: the maintenance dose decreased by 8% per decade of age, by 13% per standard deviation decrease in BSA, by 19% per 2C9*2 allele, and by 30% per 2C9*3 allele. Warfarin doses were 29% lower in patients who took amiodarone, 12% lower in patients who took simvastatin, 21% lower in patients whose target INR was 2.5 rather than 3.0, and 11% lower in white rather than African-American participants (P < 0.05 for these comparisons). An algorithm that included these factors and one of borderline significance (sex), explained 39% of the variance in the maintenance warfarin dose. Use of this pharmacogenetic model had potential to prevent patients from being overdosed when initiating warfarin: we estimate that only 24 (6.5%) patients would have been over- dosed by >2 mg/day with pharmacogenetic dosing compared to 59 (16%) patients who would have been overdosed if they had been prescribed the empirical dose of 5 mg/day (P < 0.001). In conclusion, the maintenance warfarin dose can be estimated from demographic, clinical, and pharmacogenetic factors that can be obtained at the time of warfarin initiation.
Title 1 : A comparison of bayesian methods for haplotype reconstruction from population genotype data.
Am J Hum Genet. 2003 Nov;73(5):1162-9.
Stephens M, Donnelly P.
In this report, we compare and contrast three previously published Bayesian methods for inferring haplotypes from genotype data in a population sample. We review the methods, emphasizing the differences between them in terms of both the models ("priors") they use and the computational strategies they employ. We introduce a new algorithm that combines the modeling strategy of one method with the computational strategies of another. In comparisons using real and simulated data, this new algorithm outperforms all three existing methods. The new algorithm is included in the software package PHASE, version 2.0, available online (http://www.stat.washington.edu/stephens/software.html).
Title 1 : Human P450 metabolism of warfarin
Pharmacol Ther. 1997;73(1):67-74.
Kaminsky LS, Zhang ZY.
The anticoagulant drug warfarin occurs as a pair of enantiomers that are differentially metabolized by human cytochromes P450 (CYP). R-warfarin is metabolized primarily by CYP1A2 to 6- and 8-hydroxywarfarin, by CYP3A4 to 10-hydroxywarfarin, and by carbonyl reductases to diastereoisomeric alcohols. S-warfarin is metabolized primarily by CYP2C9 to 7-hydroxywarfarin. Potential warfarin-drug interactions could occur with any of a very wide range of drugs that are metabolized by these P450s, and a number of such interactions have been reported. The efficacy of warfarin is affected primarily when metabolism of S-warfarin is altered.
The R144C change in the CYP2C9*2 allele alters interaction of the cytochrome P450 with NADPH:cytochrome P450 oxidoreductase
Pharmacogenetics. 1997 Jun;7(3):203-10.
Crespi CL, Miller VP.
We have examined the kinetics of substrate metabolism by cDNA-expressed human CYP2C9 and the R144C variant. Both enzymes exhibited similar apparent K(m) values for (S)-warfarin 7-hydroxylation, diclofenac 4'-hydroxylation and lauric acid 11-hydroxylation. In contrast, the R144C variant (relative to CYP2C9) had slower rates of metabolism for all three substrates. The difference was most pronounced for (S)-warfarin. Surprisingly, the magnitude of the difference was found to be dependent on the cytochrome P450 to NADPH-cytochrome P450 reductase (OR) ratio in the system (the difference being more pronounced at higher OR to P450 ratios) implying that the R144C change affects interaction of the P450 with OR. The rates of (S)-warfarin 7-hydroxylation by CYP2C9 and the R144C variant also exhibited differential dependence on salt concentration which further supported a difference in interaction with OR. When OR was bypassed and the hydroxylation was supported by cumene hydroperoxide, no difference in the rates of diclofenac 4'-hydroxylation was observed for CYP2C9 and the R144C variant regardless of OR to P450 ratio. However, for (S)-warfarin 7-hydroxylation, some OR-dependence was maintained even when the reaction was supported by cumene hydroperoxide. Finally, we compared CYP2C9 activity and CYP2C9 protein levels for human lymphoblast expressed (high OR to P450 ratio) to human liver microsomes using immunoblotting and enzyme selective substrates. Human liver microsomal CYP2C9 and human lymphoblast-expressed CYP2C9 showed comparable amounts of activity per unit enzyme. This final observation indicates that the high OR to P450 ratio is the preferred model and predicts that the R144C change in human liver microsomal CYP2C9 should markedly reduce the rates of substrate metabolism. The implications of these observations for the interpretation of results with cDNA-expressed enzymes is discussed.
Title 1 : Allelic variants of human cytochrome P450 2C9: baculovirus-mediated expression, purification, structural characterization, substrate stereoselectivity, and prochiral selectivity of the wild-type and I359L mutant forms.
Arch Biochem Biophys. 1996 Sep 15;333(2):447-58
Haining RL, Hunter AP, Veronese ME, Trager WF, Rettie AE.
The purpose of the present studies was to define the role of the I359L allelic variant of CYP2C9 in the metabolism of the low therapeutic index anticoagulant warfarin, by performing in vitro kinetic studies with the two enantiomers of the drug. To obtain sufficient quantities of these variants to perform kinetic studies at physiologically relevant substrate concentrations, methodology was established for the high-level expression, purification, and structural characterization of wild-type CYP2C9 and CYP2C9V1 using the baculovirus system. Both forms were expressed at levels up to 250 nmol/liter and purified in 50-55% yield to specific contents of 13-14 nmol holoenzyme/mg protein. The purified preparations were characterized by Edman degradation and electrospray-mass spectrometry. Both forms of the enzyme metabolized the pharmacologically more potent (S)-enantiomer of warfarin with the same regioselectivity; however, CYP2C9V1 exhibited a fivefold lower Vmax and a fivefold higher Km compared to the wild-type enzyme for this substrate. Neither form of the enzyme formed significant quantities of the (R)-warfarin phenols. Additional studies performed with prochiral arylalkyl sulfides provided confirmation of the low turnover rates catalyzed by CYP2C9V1 and demonstrated further that sulfoxide product stereochemistry did not differ significantly between the two variants. Therefore, decreased catalytic efficiency rather than a gross alteration in substrate orientation appears to be the consequence of this putative active-site mutation. The greatly decreased catalytic efficiency of the I359L variant suggests that leucine homozygotes would eliminate (S)-warfarin, and probably many other CYP2C9 substrates, at much slower rates in vivo than individuals expressing the wild-type enzyme.
Title 1 : Hydroxylation of warfarin by human cDNAexpressed cytochrome P-450: a role for P-4502C9 in the etiology of (S)-warfarin-drug interactions
Chem Res Toxicol. 1992 Jan-Feb;5(1):54-9.
Rettie AE, Korzekwa KR, Kunze KL, Lawrence RF, Eddy AC, Aoyama T, Gelboin HV, Gonzalez FJ, Trager WF.
Previous kinetic studies have identified a high-affinity (S)-warfarin 7-hydroxylase present in human liver microsomes which appears to be responsible for the termination of warfarin's biological activity. Inhibition of the formation of (S)-7-hydroxywarfarin, the inactive, major metabolite of racemic warfarin in humans, is known to be the cause of several of the drug interactions experienced clinically upon coadministration of warfarin with other therapeutic agents. In order to identify the specific form(s) of human liver cytochrome P-450 involved in this particular toxicity, we have determined the metabolic profiles of 11 human cytochrome P-450 forms expressed in HepG2 cells toward both (R)- and (S)-warfarin. Of the 11 forms examined only 2C9 displayed the regioselectivity and stereoselectivity appropriate for the high-affinity human liver microsomal (S)-7-hydroxylase. We further compared Michaelis-Menten and sulfaphenazole inhibition constants for (S)-warfarin 7-hydroxylation catalyzed by cDNA-expressed 2C9 and by human liver microsomes. Similar kinetic constants were obtained for each enzyme source. It is concluded that 2C9 is likely to be a principal form of human liver P-450 which modulates the in vivo anticoagulant activity of the drug. It is further concluded that those drug interactions with warfarin that arise as a result of decreased clearance of the biologically more potent S-enantiomer may have as their common basis the inhibition of P-450 2C9.