VKORC1 Polymorphisms Research Articles

Genetic Determinants of Warfarin Dosing in the Han-Chinese Population

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. In this study, we aimed to identify additional genes influencing warfarin dosing in the Han-Chinese population. In this study, we screened for SNPs in 13 genes (VKORC1, CYP2C9, CYP2C18, PROC, APOE, EPHX1, CALU, GGCX, ORM1, ORM2, factor II, factor VII and CYP4F2) and tested their associations with warfarin dosing with univariate and multiple regression analysis. Polymorphisms in the VKORC1 gene have the strongest effects on warfarin dose, followed by CYP2C9*3. In addition, our results showed that CYP2C18, PROC and EPHX1 have small but significant associations with warfarin dose. In multiple regression analysis, PROC and EPHX1 explained 3% of the dose variation. The incorporation of these two genes into warfarin dosing algorithms could improve the accuracy of prediction in the Han-Chinese population.

Ethnic differences in the population pharmacokinetics and pharmacodynamics of warfarin

Ethnic differences in warfarin maintenance doses have been documented amongst the three major Asian ethnic groups (Chinese, Malay and Indian) in Singapore. Studies have shown that cytochrome P450 2C9 (CYP2C9) polymorphisms alone did not entirely account for these differences. Recent reports suggest that VKORC1 (subunit of vitamin K epoxide reductase) haplotypes are more predictive of warfarin response. Population pharmacokinetic/pharmacodynamic (PK/PD) modelling techniques were employed to characterise the PK and PD of warfarin in a healthy volunteer study of 16 Chinese and Indian subjects following a single 25 mg dose of warfarin. To further investigate the underlying differences in warfarin response, a semi-mechanistic modelling approach (using an indirect response model for PCA activity) incorporating the vitamin K cycle was attempted using population methods with Bayesian inference. All eight Indian subjects had H7H7 VKORC1 haplotypes and three had either *2/wt or *3/wt CYP2C9 genotypes. Six Chinese subjects had H1H1 VKORC1 haplotypes and one had H1H7. All Chinese subjects were homozygous wt/wt for CYP2C9. Simulations to steady state were performed to examine warfarin response in subjects with different CYP2C9 and VKORC1 polymorphisms. The presence of a single *2 or *3 CYP2C9 allele reduced mean [SE (standard error)] S-warfarin clearance by 35% from 0.276 (0.04) to 0.180 (0.11) l/h. Subjects with VKORC1 haplotype groups of H7H7 had increased mean (SE) C (50,S) (concentration of S-warfarin required to achieve 50% of maximum effect) of 479 (7.3) compared to 206 (6.7) ng/ml in subjects with the H1H1 groups. For subjects with the H1H7 haplotype, mean (SE) C (50,S) increased 1.4 times to 288 (1.3) ng/ml compared to subjects with H1H1 haplotypes. Steady state simulations showed that whilst CYP2C9 polymorphisms affect the PK of warfarin, VKORC1 haplotypes may be better predictors of warfarin response. Since 90% of Chinese subjects had the VKORC1 H1 haplotype and 100% of Indian subjects the H7 haplotype in this study, ethnic differences in warfarin response in this study appear to be linked to differences in VKORC1 haplotypes.

VKORC1 Haplotypes in Five East-Asian Populations and Indians

Warfarin, a widely prescribed oral anticoagulant, is used for the prevention of thromboembolism. Several polymorphisms in VKORC1 have been shown to be associated with warfarin dose requirements. The frequencies of these VKORC1 polymorphisms display population differences; however, this has not been examined in many populations. In this study, we examined VKORC1 polymorphisms in five East-Asian populations (Han Chinese, Indonesian, Filipino, Thai and Vietnamese) and Indians. A total of six SNPs in the VKORC1 gene (-1639G>A, 497T>G, 1173C>T, 1542T>G, 2255C>T and 3730G>A) were genotyped. Frequencies, linkage disequilibrium and haplotype structures of these six VKORC1 SNPs were analyzed. Our data showed that 497T>G is only polymorphic in the Indian population. The 497G allele is very rare in the East-Asian populations (frequency < 1%). The remaining SNPs demonstrated high linkage disequilibrium and had similar frequencies and haplotype structures in all but the Indian population. The Indian population is mostly made up of the H7 haplotype (76%) while the rest of the recruited populations consisted of the H1 haplotype (> 80%).

Genotype-Guided Dosing of Coumarin Derivatives: The European Pharmacogenetics of Anticoagulant Therapy (EU-PACT) Trial Design

The narrow therapeutic range and wide interpatient variability in dose requirement make anticoagulation response to coumarin derivatives unpredictable. As a result, patients require frequent monitoring to avert adverse effects and maintain therapeutic efficacy. Polymorphisms in VKORC1 and CYP2C9 jointly account for about 40% of the interindividual variability in dose requirements. To date, several pharmacogenetic-guided dosing algorithms for coumarin derivatives, predominately for warfarin, have been developed. However, the potential benefit of these dosing algorithms in terms of their safety and clinical utility has not been adequately investigated in randomized settings. The European Pharmacogenetics of Anticoagulant Therapy (EU-PACT) trial will assess, in a single-blinded and randomized controlled trial with a follow-up period of 3 months, the safety and clinical utility of genotype-guided dosing in daily practice for the three main coumarin derivatives used in Europe. The primary outcome measure is the percentage time in the therapeutic range for international normalized ratio. This report describes the design and protocol for the trial.

Pharmacogenetic Impact of VKORC1 and CYP2C9 Allelic Variants on Warfarin Dose Requirements in a Hispanic Population Isolate

Warfarin is the most prescribed oral anticoagulant worldwide. Because of the complexity of warfarin therapy, we attempted to dissect genetic from bioenvironmental factors influencing warfarin dose responses in individuals of a genetic isolate of Hispanic ancestry. A total of 191 patients with standard values of international normalized ratio were recruited. Three groups with a significantly different warfarin dose response were identified, that is, sensitive (2.28 +/- 0.50 mg/d), intermediate (4.2 +/- 0.76 mg/d), and resistant (7.40 +/- 1.54 mg/d; Tukey test, P < .001). Age had a significant inverse correlation with warfarin dose (P < .001; effective dose diminished 0.56 mg/d/decade). Required doses were higher for individuals with CYP2C9 variants containing the allele *1 compared to those individuals with variants composed of other alleles (P = .006). Similarly, individuals with VKORC1-1639GG and VKORC1-1639GA genotypes also required higher doses compared to the AA genotype (P < .001). Evaluation of potential gene-gene interactions between CYP2C9 and VKORC1 polymorphisms showed significant differences in dosing for CYP2C9 genotypes within the VKORC1-1639G/A subgroup (P = .013). A stepwise multivariate linear regression analysis showed that 38.2% of the warfarin dose response variance was accounted for by a model involving age (20.9%), VKORC1-1639G/A (11.3%), and CYP2C9*1, *2, and *3 variants (7.1%). These results corroborate previous findings on warfarin pharmacogenetics and define a contrastable gene-bioenvironment interaction model suited to be used in Hispanic populations.

Thrombotic genetic risk factors and warfarin pharmacogenetic variants in São Miguel's healthy population (Azores).

BackgroundThe Azorean population presents the highest standardized mortality rate for cardiovascular diseases (CVD) when compared to mainland Portugal and other populations. Since thrombosis is a common cause of CVD, we assessed four polymorphisms in three thrombotic risk genes – F5 (G1691A), F2 (G20210A) and MTHFR (C677T, A1298C), in 469 healthy blood donors from São Miguel Island (Azores). We also analysed the CYP2C9 (C430T, A1075C) and VKORC1 (G1639A) variants in fifty-eight individuals with predisposition to thrombosis (possessing at least one variation in F5 or F2 genes and one in MTHFR) to evaluate their warfarin drug response genetic profiles.ResultsAmong the 469 individuals, the data showed that thrombotic risk allele frequencies – 1691A (4.9%), 20210A (1.8%), 677T (41.7%) and 1298C (24.8%) – were similar to other Caucasians, but significantly different from mainland Portuguese (χ2, p < 0.001). The combined analysis of these variants identified twenty-two different genetic profiles (genotype order: F5, F2, MTHFR C677T and A1298C). Complete homozygosity for all wild-type alleles (GG GG CC AA) was present in 11.7%, being GG GG CT AA (22.4%) the most frequent profile. The results also demonstrated that 12.4% (58 out of 469) of São Miguel islanders have increased genetic predisposition to thrombosis. Subsequently, we evaluated these individuals for their warfarin response genetic profiles. The data showed that seven out of fifty-eight individuals are poor metabolizers (two with CYP2C9*2/*2 and five with CYP2C9*2/*3 genotypes). VKORC1 polymorphism analysis identified twelve individuals (20.7%) with AA genotype, who probably will require lower doses of warfarin. The joint analysis of CYP2C9 and VKORC1 revealed that 79.3% (46 out of 58) of the individuals carry at least one polymorphism in these genes. Within these, twenty-five individuals (43.1%) need intermediate and/or low doses of warfarin, if treatment is started.ConclusionThe present study demonstrated, for the first time, that São Miguel, and possibly the Azores population, shows significant differences on allele frequencies of thrombotic risk factors when compared to mainland Portugal. This research constitutes a primary approach for future studies on CVD, as well as for the implementation of warfarin dosing protocols using the patient's genotypic information.

Personalized Therapy and Pharmacogenomics: Future Perspective

expression of the gene (regulation, inhibition and induction) and, of course, at the protein level. A systems biology approach using all the different ‘omics’ is well adapted. The pathways that could be involved should be screened systematically [4]. The latest developments in the genome-wide search have considerably promoted the emergence of new biomarkers that could represent future drug targets. Current genome-wide studies are focusing on cardiovascular diseases including hypertension and related metabolic disturbances, diabetes, lipids, inflammation and also cancer, as well as various other psychological traits. These recent results represent a major shift from classical linkage and associationbased genetic studies, moving the focus towards deciphering the genomic variations network, thus identifying novel therapeutic targets and genetic biomarkers that will be useful for drug discovery. Bernhard Winkelmann (Cardiology Group Frankfurt-Sachsenhausen, Frankfurt, Germany) [5] and Laurent Becquemont (Hopital Bicetre, France) [6] both presented good examples of retrospective genome-wide association studies that confirmed that the most significant independent effect was associated with the VKORC1 polymorphisms (p = 6.2 × 10) and in the CYP2C9 polymorphism (rs1057910 CYP2C9*3 and rs4917639), with warfarin at moderate significance levels (p = 10) [7]. A more recent polymorphism found in the CYP4F2 gene has just been described but has not yet been clinically confirmed [8]. Recently, a new biomarker of statin effect was also highlighted by a genome-wide scan approach of the OATP1B1 rs4363657 SNP and was found to have a high degree of confidence (p = 4 × 10) [9]. Therefore, these three genes could explain a large fraction of the dose variance, which could help future clinical trials to produce meaningful and definitive results. A goal of pharmacogenomics is to guide drug development and selection in order to optimize therapeutic benefits and minimize the potential risk of toxicity. Genetic-based differences in drug metabolism have been recognized for 50 years, but these are better understood since two accidents occured 30 years ago with anti-anginal drug perhexiline and the antidiabetic phenylbiguanide. Accordingly, there is a growing need for ways to better identify individuals who have the highest probability of benefiting from pharmacological interventions, and those who have the lowest risk of developing side effects when exposed to drugs. Since the first Santorini meeting in 2002 with Klaus Lindpaintner (Roche Center for Medical Genomics, Basel, Switzerland), each Biologie Prospective conference has helped shape the field of pharmaco genomics, particularly, the 2006 and 2008 conferences [1,2] following the publication of recommendations by David Gurwitz (Tel Aviv University, Israel) [3], which were published after the second Santorini meeting in 2004.

Pharmacogenetic relevance of CYP4F2 V433M polymorphism on acenocoumarol therapy

VKORC1 and CYP2C9 polymorphisms are used to predict the safe dose of oral anticoagulant therapy. A new variant of CYP4F2 (V433M) has recently been related to the required warfarin dose. We evaluated its influence in earliest response to acenocoumarol in 100 selected men who started anticoagulation (3 mg for 3 consecutive days). V433M genotype exerted a gene dosage-dependent effect on the decrease of factors II, VII, IX, and X in the earliest response to acenocoumarol, with homozygous 433V subjects being the most sensitive. Similarly, after the initiation of therapy, international normalized ratio also experienced a gene dosage-dependent effect (P = .015), and 433V subjects needed 4 mg/week less than 433M carriers to achieve a steady anticoagulation (P = .043). Multivariate linear regression analysis revealed a significant contribution of V433M polymorphism to variability of both early international normalized ratio value (R(2) = 0.14) and dose requirements (R(2) = 0.19). Our data underline the relevant role of CYP4F2 V433M polymorphism in the pharmacogenetics of coumarin anticoagulants.

Validation of Clinical Testing for Warfarin Sensitivity: Comparison of CYP2C9-VKORC1 Genotyping Assays and Warfarin-Dosing Algorithms

Responses to warfarin (Coumadin) anticoagulation therapy are affected by genetic variability in both the CYP2C9 and VKORC1 genes. Validation of pharmacogenetic testing for warfarin responses includes demonstration of analytical validity of testing platforms and of the clinical validity of testing. We compared four platforms for determining the relevant single nucleotide polymorphisms (SNPs) in both CYP2C9 and VKORC1 that are associated with warfarin sensitivity (Third Wave Invader Plus, ParagonDx/Cepheid Smart Cycler, Idaho Technology LightCycler, and AutoGenomics Infiniti). Each method was examined for accuracy, cost, and turnaround time. All genotyping methods demonstrated greater than 95% accuracy for identifying the relevant SNPs (CYP2C9 *2 and *3; VKORC1 -1639 or 1173). The ParagonDx and Idaho Technology assays had the shortest turnaround and hands-on times. The Third Wave assay was readily scalable to higher test volumes but had the longest hands-on time. The AutoGenomics assay interrogated the largest number of SNPs but had the longest turnaround time. Four published warfarin-dosing algorithms (Washington University, UCSF, Louisville, and Newcastle) were compared for accuracy for predicting warfarin dose in a retrospective analysis of a local patient population on long-term, stable warfarin therapy. The predicted doses from both the Washington University and UCSF algorithms demonstrated the best correlation with actual warfarin doses.

Pharmacogenetic characteristics of patients with complicated phenprocoumon dosing

Anticoagulation therapy with coumarins necessitates a strict individualization of dosing. Whereas the impacts of the cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase (VKORC1) polymorphisms on warfarin dosing are clearly established, the role of these genetic variants on dosing and the safe use of phenprocoumon are less well investigated and, to a certain degree, controversial. We studied the most frequent functional polymorphisms of VKORC1, CYP2C9, and CYP3A5 in 60 consecutive patients demonstrating complicated phenprocoumon-mediated anticoagulation and in 120 controls. The frequencies of the less active VKORC1 haplotype A-group alleles (p < 0.0001) and of CYP2C9 genotypes with two variant alleles (p = 0.035) were higher in the patient cohort than in the control group, while the frequency of patients carrying only one variant CYP2C9 allele was unchanged relative to the control subjects (RR 1.2; p = 0.49). The data suggest a fundamental role of VKORC1 haplotypes and a minor role of CYP2C9 variants in the anticoagulation property of phenprocoumon.

A Genome-Wide Association Study Confirms VKORC1, CYP2C9, and CYP4F2 as Principal Genetic Determinants of Warfarin Dose

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<1.5×10−7) 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 ∼30% of the dose variance is explained by single nucleotide polymorphisms (SNPs) in the warfarin drug target VKORC1 and another ∼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 = 8.3×10−10) 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.

CYP4F2 Is a Vitamin K1 Oxidase: An Explanation for Altered Warfarin Dose in Carriers of the V433M Variant

Genetic polymorphisms in VKORC1 and CYP2C9, genes controlling vitamin K(1) (VK1) epoxide reduction and (S)-warfarin metabolism, respectively, are major contributors to interindividual variability in warfarin dose. The V433M polymorphism (rs2108622) in CYP4F2 has also been associated with warfarin dose and speculatively linked to altered VK1 metabolism. Therefore, the purpose of the present study was to determine the role of CYP4F2 and the V433M polymorphism in the metabolism of VK1 by human liver. In vitro metabolic experiments with accompanying liquid chromatography-tandem mass spectrometry analysis demonstrated that recombinant CYP4F2 (Supersomes) and human liver microsomes supplemented with NADPH converted VK1 to a single product. A screen of all commercially available P450 Supersomes showed that only CYP4F2 was capable of metabolizing VK1 to this product. Steady-state kinetic analysis with recombinant CYP4F2 and with human liver microsomes revealed a substrate K(m) of 8 to 10 microM. Moreover, anti-CYP4F2 IgG, as well as several CYP4F2-selective chemical inhibitors, substantially attenuated the microsomal reaction. Finally, human liver microsomes genotyped for rs2108622 demonstrated reduced vitamin K(1) oxidation and lower CYP4F2 protein concentrations in carriers of the 433M minor allele. These data demonstrate that CYP4F2 is a vitamin K(1) oxidase and that carriers of the CYP4F2 V433M allele have a reduced capacity to metabolize VK1, secondary to an rs2108622-dependent decrease in steady-state hepatic concentrations of the enzyme. Therefore, patients with the rs2108622 polymorphism are likely to have elevated hepatic levels of VK1, necessitating a higher warfarin dose to elicit the same anticoagulant response.

Polymorphisms in VKORC1 and GGCX are not major genetic determinants of vitamin K-dependent coagulation factor activity in Western Germans

Polymorphisms in VKORC1 and GGCX are not major genetic determinants of vitamin K-dependent coagulation factor activity in Western Germans -

Association of Sequence Variations in Vitamin K Epoxide Reductase and γ-Glutamyl Carboxylase Genes with Biochemical Measures of Vitamin K Status

Genetic factors, specifically the VKORC1 and GGCX genes, have been shown to contribute to the interindividual variability in response to the vitamin K-antagonist, warfarin, which influences the dose required to achieve the desired anticoagulation response. These differences in warfarin sensitivity may be explained by differences in vitamin K status. Men and women (n=416, 60-80 y), primarily of European descent, were genotyped for common polymorphisms in VKORC1 and GGCX. Cross-sectional associations exist between polymorphisms and biochemical markers of vitamin K [plasma phylloquinone, percent undercarboxylated osteocalcin (%ucOC)]. VKORC1 rs8050894 GG homozygotes had significantly higher cross-sectional measures of plasma phylloquinone than carriers of the CG or CC genotypes (plasma phylloquinone geometric means: GG 0.874+/-0.092 versus CG/CC 0.598+/- 0.044; p=0.020), whereas carriers of VKORC1 rs7294 AA or AG had significantly lower plasma phylloquinone concentrations compared to GG homozygotes (plasma phylloquinone geometric means: 0.579+/-0.045 versus 0.762+/-0.057; p=0.035). Cross-sectional analyses also revealed that heterozygous carriers of GGCX rs10187424 and rs7568458 had significantly lower %ucOC relative to either homozygous group. Polymorphisms in genes encoding enzymes involved in vitamin K metabolism may modulate plasma concentrations of phylloquionone and percent carboxylation of osteocalcin.

Serum Undercarboxylated Osteocalcin as Biomarker of Vitamin K Intake and Risk of Prostate Cancer: A Nested Case-Control Study in the Heidelberg Cohort of the European Prospective Investigation into Cancer and Nutrition

From cell studies, Vitamin K is known to exert anticancer effects on a variety of cancer cell lines, including prostate cancer cells. Recently, we reported an inverse association between dietary intake of menaquinones (vitamin K(2)), but not phylloquinone (vitamin K(1)), and risk of prostate cancer. In this nested case-control study including 250 prostate cancer cases and 494 matched controls, we aimed to confirm this cancer-protective effect using serum undercarboxylated osteocalcin (ucOC), a biomarker of vitamin K status inversely associated with vitamin K intake. In addition, effect modification by a functionally relevant polymorphism in the vitamin K epoxide reductase gene (VKORC1) was assessed. Serum ucOC and intact total osteocalcin (iOC) were analyzed with the use of ELISA tests. Serum ucOC was expressed relative to iOC (i.e., as ucOC/iOC ratio). Conditional logistic regression was used to calculate multivariate adjusted odds ratios (OR) and 95% confidence intervals (95% CI). Serum ucOC/iOC ratio was positively associated with advanced-stage (OR per 0.1 increment, 1.38; 95% CI, 1.03-1.86) and high-grade prostate cancer (OR, 1.21; 95% CI, 1.00-1.46) but not with total prostate cancer. The significant association with advanced-stage prostate cancer was confirmed when serum ucOC/iOC ratio was jointly modeled with menaquinone intake data. There was indication of a lower prostate cancer risk in carriers of the A allele (compared with GG carriers) of the +2255 VKORC1 polymorphism with increasing menaquinone intake (P(interaction) = 0.14) whereas no distinct effect modification was observed for the ucOC/iOC ratio (P(interaction) = 0.37). The increased risks of advanced-stage and high-grade prostate cancer with higher serum ucOC/iOC ratio strengthen the findings for dietary menaquinone intake.

Use of Pharmacogenetic Factors to Predict Warfarin Dose in Children - a Retrospective Cohort Study

Background: Warfarin is the most commonly used oral anticoagulant, but its use is limited by the narrow therapeutic index. Single nucleotide polymorphisms in cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase (VKORC1) have been shown to impact warfarin dose requirements. Use of such pharmacogenetic data to improve warfarin dosing has been widely studied in adult populations and is being integrated into clinical practice. However, there remains a paucity of data on the appropriate use of this genetic information in the pediatric population. We developed a retrospective review of pediatric patients on warfarin therapy to evaluate associations between CYP2C9 and VKORC1 polymorphisms and warfarin dose requirements in children.Methods: Patients were recruited from outpatient clinics at Texas Children's Hospital and Stollery Children's Hospital. Any patient less than 18 years of age that had taken warfarin and achieved maintenance dose was eligible. Blood sample or buccal swab was collected for genotyping and clinical data was collected from the medical record. Genotyping was performed by melting curve analysis to detect CYP2C9*2, CYP2C9*3, or VKORC1 −1639 mutations. Warfarin genotyping reagents were obtained from Idaho Technology and assays were run using the Roche LightCyler instrument.Results: This study was initiated in March 2008 and is ongoing. To date 47 children (23M: 24F) have been enrolled. Indications for warfarin therapy include: arrhythmia (n=2), artificial heart valve (n=10), pulmonary hypertension (n=10), deep vein thrombosis (n=9), pulmonary embolism (n=2), Fontan (n=10), stroke (n=2), other (n=2). The mean age is 9.3 years (range 1–18); median target International Normalized Ratio (INR) range is 2–3 (with similar target INRs across genotypes). Twenty-nine patients have fully evaluable data at the time of this report, of whom 21 (72%) are wild type CYP2C9, 6 (21%) carry at least 1 mutant allele, and 2 (7%) are homozygous or compound heterozygote. As compared with wild type patients (mean dose 0.14mg/ kg/day), patients with heterozygous (0.09mg/kg/day; p=0.04) or homozygous (0.05mg/ kg/day; p=0.001) genotype required significantly lower maintenance daily warfarin dose. For VKORC1 −1639 genotype 6 (21%) are AA, 11 (38%) are GA, and 12 (41%) are GG. As compared with GG patients (mean dose 0.16mg/kg/day), patients with 1 (0.11mg/kg/ day; p=0.12) or 2 (0.09mg/kg/day; p=0.02) A allele required lower doses.Discussion: We present the first pediatric series to evaluate the influence of CYP2C9 and VKORC1 genotype on warfarin dosing. These data demonstrate genotype frequency similar to adults, and suggests that genotype influences the maintenance warfarin dose in children. Enrollment to this study is ongoing, and will allow a more precise evaluation of the impact of genotype and additional demographic data on pediatric dosing. These data will provide for development of a pediatric dosing algorithm to predict maintenance warfarin dose a priori.

Influence of Genetic Polymorphisms of VKORC1 and CYP2C9 in Patients on Phenprocoumon Steady-State Dose Requirements

The variants of the (Vitamin-K epoxid reductase subcomplex 1) VKORC1 and of cytochrome P450 2C9 (CYP2C9) genes are know to influence the steady state dose of the vitamin-K antagonist (VKA) phenprocoumon during oral anticoagulant therapy. We have analysed in detail the influence of the VCORC1 1173CC and 1173CT variants in combination with the CYP2C9 variants on the steady state dose of phenprocoumon. The weekly phenprocoumon dosage was calculated after a 3 to 6 months of stable anticoagulation by the international normalized ratio (INR) which had to be in a range from 2 to 3 in 114 patients. VKORC1 1173CC, CT, TT and CYP2C9*1/*1, *1/*2, *1/*3, *2/*2, *2/*3 and *3/*3 genotypes were determined using direct DNA sequencing and realtime PCR. Patients carrying VKORC1 1173CT and CYP2C9*1/*1 genotypes required a 33% lower steady state dose of phenprocoumon compared to VKORC1 1173CC and CYP2C9*1/*1 genotypes (p=0.0001). Patients with VKORC1 1173CC in combination with CYP2C9*1/*2, *1/*3 needed 10% and 34% lower steady state dose of phenprocoumon compared to the combination with CYP2C9*1/*1, respectively (p=0.0001). Patients carrying VKORC1 1173CT needed lower doses of phenprocoumon in combination with CYP2C9*1/*1 (−32%), *1/*2 (−29%), or *1/*3 (−33%), and VKORC1 1173TT in combination with CYP2C9*1/*3 (−44%) compared to VKORC1 1173CC combined with CYP2C9*1/*1 (all p<0.001). CYP2C9 variants did not influence the dose of carriers of VKORC1 1173CT gene. Both genotypes together influenced the phenprocoumon maintenance dosage in this naturalistic study design. Genotyping for CYP2C9 and VCORC1 therefore might be a suitable tool for dose individualization in patients at oral anticoagulant drug therapy.

VKORC1 Polymorphisms in Amerindian Populations of Brazil

Noncoding polymorphisms in the VKORC1 gene associate with variation of interindividual dosing requirements of warfarin and other coumarin anticoagulants. The frequency of VKORC1 polymorphisms displays distinct interpopulation differences. Here, we report the distribution of the VKORC1 3673G>A, 5808T>G, 6853G>C and 9041G>A SNPs in three endogamous Amerindian (Native American) populations, namely, Guarani-Kaiowá, Guarani-Nandeva and Kaingang. Individual DNA from 180 healthy adults was genotyped for the VKORC1 polymorphisms using TaqMan Detection System assays. The ARLEQUIN 3.1 software package was used to estimate haplotype frequency and linkage disequilibrium. The VKORC1 3673G>A, 5808T>G, 6853G>C and 9041G>A polymorphisms were in Hardy-Weinberg equilibrium in each population. The 5808G allele was absent or rare (<3%), whereas 3673A, 6853C and 9041A were frequent (34-63%) in the three Amerindian populations. No difference was detected in allele or genotype frequency bewteen the two Guarani populations, whereas significant differences were observed between Kaingang and Guarani. Polymorphisms 3673G>A, 6853G>C and 9041G>A were in significant linkage disequilibrium in both Guarani and Kaingang (pairwise r2 values: 0.77-1.0). Haplotypes ATCG and GTGA accounted for more than 94% of the haplotypes in both populations, ATCG being the most common in Guarani (49.5%) and GTGA in Kaingang (54%). These data disclose the uniqueness of the frequency distribution of the VKORC1 SNPs in the Amerindians, compared with Asian, African and European populations. In view of the vast interpopulational diversity among Amerindians, the present data should not be interpreted as representative of other extant Amerindian peoples. Our estimates that 40% of Kaingang and 60% of Guarani have haplotypes including the variant 3673A allele suggest that these two Amerindian populations comprise high proportions of individuals requiring reduced warfarin doses.

Abstract 4415: The Utility of Genotyping in the First Evaluation of a Novel Vitamin K Antagonist: ATI-5923

(S)-Warfarin, the most active enantiomer of warfarin, is metabolized by CYP2C9. Genetic variations of the CYP2C9 gene result in slower metabolism and are major contributors to inter-patient dose variability with resultant INR instability, especially in patients on multiple medications. This leads to higher hemorrhagic and thrombotic complications. ATI-5923 (ARYx therapeutics Inc.) is a novel VKOR inhibitor that undergoes metabolism by human carboxyles-terase and not by CYP2C9. We hypothesize that time in therapeutic range (TTR) based on Rosendaal method is better for ATI-5923 than for historic warfarin and that maintenance dose is independent of CYP2C9 polymorphism, but dependent on VKOR haplotype. This was a multi-center, open-label, 3 month study of 66 patients with AF treated with ATI-5923. All except two patients were on prior warfarin. Periodic INR testing occurred twice weekly for the first 3 weeks and then weekly during the maintenance phase. Genetic testing for the CYP2C9 and VKORC1 polymorphism was performed on all patients. Drug dosing was performed using a standardized nomogram. Pre-study INRs and warfarin dose data were collected for up to one year prior to study entry. 77 % of patients were in therapeutic range (INR 2–3) after 7 days and 80% had stable INR (3 consecutive INR values within range without dose adjustment) by 3 months. For the maintenance phase the mean TTR was 71.5 and 59.3% for ATI-5923 and warfarin respectively (12.2% improvement; p=0.0009). Patients with the VKOR AA haplotype required lower maintenance doses than VKOR GA or GG haplotype (8.6 vs 15.8 and 18.7mg; p &lt; 0.0001). CYP2C9 genotype had no influence on maintenance dose. CYP2C9 variants (n=20), showed a trend toward better control compared to warfarin; 16% improvement vs. 11% for wild type CYP2C9 (n =42). ATI-5923 has better INR control than prior warfarin therapy. VKOR AA patients require a significantly lower maintenance dose. CYP2C9 variants show a trend to added improvement compared to the CYP2C9 wild type, suggesting that, unlike warfarin, ATI-5923 is not influenced by CYP2C9 genetic variations.

VKORC1 Polymorphisms, Haplotypes and Haplotype Groups on Warfarin Dose Among African–Americans and European–Americans

Although the influence of VKORC1 and CYP2C9 polymorphisms on warfarin response has been studied, variability in dose explained by CYP2C9 and VKORC1 is lower among African-Americans compared with European-Americans. This has lead investigators to hypothesize that assessment of VKORC1 haplotypes may help capture a greater proportion of the variability in dose for this under-represented group. However, the inadequate representation of African-Americans and the assessment of a few VKORC1 polymorphisms have hindered this effort. To determine if VKORC1 haplotypes or haplotype groups explain a higher variability in warfarin dose, we comprehensively assessed VKORC1 polymorphisms in 273 African-Americans and 302 European-Americans. The influence of VKORC1 polymorphisms, race-specific haplotypes and haplotype groups on warfarin dose was evaluated in race-stratified multivariable analyses after accounting for CYP2C9 (*2, *3, *5, *6 and *11) and clinical covariates. VKORC1 explained 18% (30% with CYP2C9) variability in warfarin dose among European-Americans and 5% (8% with CYP2C9) among African-Americans. Four common haplotypes in European-Americans and twelve in African-Americans were identified. In each race VKORC1 haplotypes emerged into two groups: low-dose (Group A) and high-dose (Group B). African-Americans had a lower frequency of Group A haplotype (10.6%) compared with European-Americans (35%, p < 0.0001).The variability in dose explained by VKORC1 haplotype or haplotype groups was similar to that of a single informative polymorphism. Our findings support the use of CYP2C9, VKORC1 polymorphisms (rs9934438 or rs9923231) and clinical covariates to predict warfarin dose in both African- and European-Americans. A uniform set of common polymorphisms in CYP2C9 and VKORC1, and limited clinical covariates can be used to improve warfarin dose prediction for a racially diverse population.