Warfarin analogs target disulfide bond-forming enzymes and suggest a residue important for quinone and coumarin binding.

Disulfide bond formation has a central role in protein folding of both eukaryotes and prokaryotes. In bacteria, disulfide bonds are catalyzed by DsbA and DsbB/VKOR enzymes. First, DsbA, a periplasmic disulfide oxidoreductase, introduces disulfide bonds into substrate proteins. Then, the membrane enzyme, either DsbB or VKOR, regenerate DsbA's activity by the formation of de novo disulfide bonds which reduce quinone. We have previously performed a high-throughput chemical screen and identified a family of warfarin analogs that target either bacterial DsbB or VKOR. In this work, we expressed functional human VKORc1 in Escherichia coli and performed a structure-activity-relationship analysis to study drug selectivity between bacterial and mammalian enzymes. We found that human VKORc1 can function in E. coli by removing two positive residues, allowing the search for novel anticoagulants using bacteria. We also found one warfarin analog capable of inhibiting both bacterial DsbB and VKOR and a second one antagonized only the mammalian enzymes when expressed in E. coli. The difference in the warfarin structure suggests that substituents at positions three and six in the coumarin ring can provide selectivity between the bacterial and mammalian enzymes. Finally, we identified the two amino acid residues responsible for drug binding. One of these is also essential for de novo disulfide bond formation in both DsbB and VKOR enzymes. Our studies highlight a conserved role of this residue in de novo disulfide-generating enzymes and enable the design of novel anticoagulants or antibacterials using coumarin as a scaffold.

Precision medicine in cardiovascular therapeutics: Evaluating the role of pharmacogenetic analysis prior to drug treatment.

Pharmacogenomics is the examination of how genetic variation influences drug metabolism and response, in terms of both efficacy and safety. In cardiovascular disease, patient-specific diplotypes determine phenotypes, thereby influencing the efficacy and safety of drug treatments, including statins, antiarrhythmics, anticoagulants and antiplatelets. Notably, polymorphisms in key genes, such as CYP2C9, CYP2C19, VKORC1 and SLCO1B1, significantly impact the outcomes of treatment with clopidogrel, warfarin and simvastatin. Furthermore, the CYP2C19 polymorphism influences the pharmacokinetics and safety of the novel hypertrophic cardiomyopathy inhibitor, mavacamten. In this review, we critically assess the clinical application of pharmacogenomics in cardiovascular disease and delineate present and future utilization of pharmacogenomics. This includes insights into identifying missing heritability, the integration of whole genome sequencing and the application of polygenic risk scores to enhance the precision of personalized drug therapy. Our discussion encompasses health economic analyses that underscore the cost benefits associated with pre-emptive genotyping for warfarin and clopidogrel treatments, albeit acknowledging the need for further research in this area. In summary, we contend that cardiovascular pharmacogenomic analyses are underpinned by a wealth of evidence, and implementation is already occurring for some of these gene-drug pairs, but as with any area of medicine, we need to continually gather more information to optimize the use of pharmacogenomics in clinical practice.

Association between polymorphisms of the VKORC1 and CYP2C9 genes and warfarin maintenance dose in Peruvian patients.

AIMS: The aim of this study was to investigate the association between VKORC1 and CYP2C9 genes polymorphisms and the maintenance dose of warfarin in Peruvian patients. METHODS: An observational study was conducted on outpatients from the Hospital Grau ESSALUD in Lima, Peru. The participants were selected using nonprobabilistic convenience sampling. Inclusion criteria required patients to have been on anticoagulation therapy for >3 months, maintain stable doses of warfarin (consistent dose for at least 3 outpatient visits), and maintain an international normalized ratio within the therapeutic range of 2.5-3.5. DNA samples were obtained from peripheral blood for gene analysis. RESULTS: Seventy patients (mean age of 69.6 ± 13.4 years, 45.7% female) were included in the study. The average weekly warfarin dose was 31.6 ± 15.2 mg. The genotypic frequencies of VKORC1 were as follows: 7.1% (95% confidence interval, 2.4-15.9) for AA; 44.3% (32.4-56.7) for GA; and 48.6% (36.4-60.8) for GG. No deviation from the Hardy-Weinberg equilibrium was observed in the variants studied (P = .56). The mean weekly warfarin doses for AA, GA and GG genotypes were 16.5 ± 2.9, 26.5 ± 9.5 and 37.9 ± 17.1 mg, respectively (P < .001). The genotypic frequencies of CYP2C9 were as follows: 82.8% (72.0-90.8) for CC (*1/*1); 4.3% (1.0-12.0) for CT (*1/*2); and 12.9% (6.1-23.0) for TT (*2/*2). We did not find a significant association between the CYP2C9 gene polymorphism and the dose of warfarin. CONCLUSIONS: The AA genotype of the VKORC1 gene was associated with a lower maintenance dose of warfarin in Peruvian patients.

Is a Vitamin K Epoxide Reductase Complex Subunit 1 (VKORC1) Polymorphism a Risk Factor for Nephrolithiasis in Sarcoidosis?

Sarcoidosis is a systemic inflammatory disorder characterized by granuloma formation in various organs. It has been associated with nephrolithiasis. The vitamin K epoxide reductase complex subunit 1 (VKORC1) gene, which plays a crucial role in vitamin K metabolism, has been implicated in the activation of proteins associated with calcification, including in the forming of nephrolithiasis. This study aimed to investigate the VKORC1 C1173T polymorphism (rs9934438) in a Dutch sarcoidosis cohort, comparing individuals with and without a history of nephrolithiasis. Retrospectively, 424 patients with sarcoidosis were divided into three groups: those with a history of nephrolithiasis (Group I: n = 23), those with hypercalcemia without nephrolithiasis (Group II: n = 38), and those without nephrolithiasis or hypercalcemia (Group III: n = 363). Of the 424 sarcoidosis patients studied, 5.4% had a history of nephrolithiasis (Group I), only two of whom possessed no VKORC1 polymorphisms (OR = 7.73; 95% CI 1.79-33.4; p = 0.001). The presence of a VKORC1 C1173T variant allele was found to be a substantial risk factor for the development of nephrolithiasis in sarcoidosis patients. This study provides novel insights into the genetic basis of nephrolithiasis in sarcoidosis patients, identifying VKORC1 C1173T as a potential contributor. Further research is warranted to elucidate the precise mechanisms and explore potential therapeutic interventions based on these genetic findings.

Pharmacogenetics of warfarin dosing in Chinese adults with nonvalvular atrial fibrillation.

BACKGROUND: The guide for the use of genotype-guided warfarin dosing in patients for the treatment of non-valvular atrial fibrillation (AF) is still lacking. AIM: We aimed to evaluate whether genotype-guided warfarin dosing is superior to conventional clinical dosing for the outcomes of interest in Chinese patients. METHOD: Our study consisted of 508 newly recruited and 471 existing Chinese AF patients. Among the total 979 patients, 585 patients received their dose of warfarin determined by a genetic and clinical factor (gene group), while the remaining 394 patients whose dosing was determined empirically in control group. We incorporated CYP2C9 and VKORC1 genotypes into the gene group. The international normalized ratio (INR) measurement and standard protocols were used for further dose adjustment in both groups. The primary outcomes were the percentage of time in the therapeutic range (%TTR) and INR during 12-month follow-up. Secondary safety outcome included bleeding and thrombotic events. RESULTS: Compared with the control group, the average TTR of the gene group was higher [68.4 ± 20.6% vs 48.5 ± 21.6%, P < 0.001]. The average INR monitoring times to reach the therapeutic time in the gene group was lower (P < 0.001). The risk ratios (RR) for cumulative incidence of total bleeding events, minor bleeding events, gastrointestinal bleeding, and intracerebral bleeding events were not significantly different between the two groups (P > 0.05). Comparing to the analysis using existing 471 patients, the analysis using total 979 patients showed that the gene group experienced a lower (RR 0.4 (95% CI 0.2 to 0.8), P = 0.008) incidence of cumulative ischemic stroke. CONCLUSION: Genotype-guided warfarin administration increases the average TTR, reaches higher TTR levels in the early anticoagulant phase, and significantly reduces the risk of ischemic stroke events.

Pharmacogenetics of warfarin and healthcare costs - Real-world data analysis.

PURPOSE: Variants in CYP2C9 and VKORC1 genes have been associated with individuals' sensitivity to warfarin. The aim of this study was to investigate the differences of healthcare costs of genetically normal and genetically sensitive warfarin responder groups. METHODS: This was a retrospective study linking genotype data from three Finnish biobanks (THL Biobank, Auria Biobank, Helsinki Biobank) with healthcare encounter data of the Finnish Institute of Health and Welfare (THL), drug dispensation data from the Social Insurance Institution of Finland (Kela) and laboratory data from Finnish hospital districts and municipalities. We compared the normal and sensitive warfarin responder groups in terms of healthcare costs related to bleeding and thromboembolic events, INR tests and medication purchases. RESULTS: We found a trend towards increased bleeding-related hospital costs in the sensitive warfarin responder group (881 patients) when compared with the normal responders (1627 patients) with a per patient difference of 150.9 €/year (95% CI: -55.1, 414.6 €/year, p = 0.087). INR test costs were higher in the sensitive responder group with a difference of 7.2 €/year (95% CI: -1.5, 16.4 €/year, p = 0.047). Medication costs were significantly lower in the sensitive responder group with a difference of -14.4 €/year (95% CI: -15.8, -12.9 €/year, p < 0.001). CONCLUSIONS: The difference in the costs of bleeding-related hospitalization between genetically sensitive and normal warfarin responders may justify genotype-guided warfarin dosing. Further studies with larger sample sizes would be needed to verify the result.

Hyper-responsiveness to warfarin in a young patient with the VKORC1 -1639GA/CYP2C9*1*46 genotype: a case report.

BACKGROUND: Warfarin is the most widely used oral anticoagulant; nevertheless, dosing of warfarin is problematic for clinicians worldwide. Inter-individual variability in response to warfarin is attributed to genetic as well as non-genetic factors. Pharmacogenomics studies have identified variants in CYP2C9 and VKORC1 genes as significant predictors of warfarin dose, however, phenotypes of rare variants are not well characterized. CASE PRESENTATION: We report a case of hyper-responsiveness to warfarin in a 22-year-old outpatient with Crohn's disease who presented with a swollen, red, and painful left calf. Deep venous thrombosis (DVT) in the left lower extremity was confirmed via ultrasonography, and hence, anticoagulation therapy of heparin and concomitant warfarin was initiated. Warfarin dose of 7.5 mg/day was estimated by the physician based on clinical factors. Higher than the expected international normalized ratio (INR) value of 4.5 necessitated the reduction of the warfarin dose to 5 and eventually to 2.5 mg/day to reach a therapeutic INR value of 2.6. Pharmacogenetic profiling of the VKORC1 -1639G > A and CYP2C9 *2, *3, *4, *5, *8, *14, *20, *24, *26, *33, *40, *41, *42, *43, *45, *46, *55, *62, *63, *66, *68, *72, *73 and *78 revealed a VKORC1-1639GA/CYP2C9*1*46 genotype. The lower catalytic activity of the CYP2C9*46 (A149T) variant was previously reported in in vitro settings. CONCLUSIONS: This is the first report on a case of warfarin hyper-responsive phenotype of a patient with the heterozygous CYP2C9*1*46 polymorphism.

To establish a model for the prediction of initial standard and maintenance doses of warfarin for the Han Chinese population based on gene polymorphism: a multicenter study.

PURPOSE: The purpose of this paper is to study the correlation between demographic and clinical factors and warfarin dose of patients in Chinese Han population taking warfarin and study gene polymorphisms impact of related gene loci (CYP2C9*3, VKORC1-1639G > A) on warfarin doses, to establish a model to predict initial standard dose and maintenance dose based on CYP2C9*3, VKORC1-1639G > A genotype. METHODS: The study collects the data of patients in our hospital and other subcenters which incorporates 2160 patients to establish the initial dose model and 1698 patients for the stable dose model, and sequences 26 multigene sites in 451 patients. Based on the patient's dosage, clinical data, and demographic characteristics, the genetic and non-genetic effects on the initial dose and stable dose of warfarin are calculated by using statistical methods, and the prediction model of initial standard dose and maintenance dose can be established via multiple linear regression. RESULTS: The initial dose of warfarin (mg/day) was calculated as (1.346 + 0.350 × (VKORC1-1639G > A) - 0.273 × (CYP2C9*3) + 0.245 × (body surface area) - 0.003 × (age) - 0.036 × (amine-iodine) + 0.021 × (sex))2. This model incorporated seven factors and explained 55.3% of the individualization differences of the warfarin drug dose. The maintenance dose of warfarin (mg/day) was calculated as (1.336 + 0.299 × (VKORC1-1639G > A) + 0.480 × (body surface area) - 0.214 × (CYP2C9*3) - 0.074 × (amine-iodine) - 0.003 × (age) - 0.077 × (statins) - 0.002 × (height))2. This model incorporated six factors and explained 42.4% of the individualization differences in the warfarin drug dose. CONCLUSION: The genetic and non-genetic factors affecting warfarin dose in Chinese Han population were studied systematically in this study. The pharmacogenomic dose prediction model constructed in this study can predict anticoagulant efficacy of warfarin and has potential application value in clinical practice.

Distribution of non-synonymous Vkorc1 mutations in roof rats (Rattus rattus) in France and in Spain - consequences for management.

Rodent control is mainly done using anticoagulant rodenticides leading to the death of rodents through internal bleeding by targeting the VKORC1 protein. However, mutations in VKORC1 can lead to resistance to anticoagulant rodenticides that can cause treatment failure in the field. This study provides the first insight into the distribution, frequency and characterization of Vkorc1 mutations in roof rats (Rattus rattus) in France and in three administrative areas of Spain. The roof rat is present in France while it was thought to have almost disappeared with the expansion of the brown rat. Nevertheless, it has been found mainly in maritime areas. 151 roof rats out of 219 tested presented at least one missense mutation in the coding sequences of Vkorc1 gene (i.e. 69.0% of the rat). Nine Vkorc1 genotypes were detected (Y25F, A26P, R40G, S57F, W59C, W59R, H68N, Y25F/K152T and Y25F/W59R. Biochemical characterization of the consequences of these different genotypes proved that these various genotypes did not induce severe resistance to anticoagulant rodenticides. Even if many mutations of the Vkorc1 gene are present in roof rat populations in France, their management may be based in a first approach, considering the low levels of resistance induced, on the use of first-generation anticoagulants less dangerous for wildlife. The use of second-generation may be considered when treatment failure is observed or when bait consumption is limited.

Vitamin K-Dependent Protein Activation: Normal Gamma-Glutamyl Carboxylation and Disruption in Disease.

Vitamin K-dependent (VKD) proteins undergo an unusual post-translational modification, which is the conversion of specific Glu residues to carboxylated Glu (Gla). Gla generation is required for the activation of VKD proteins, and occurs in the endoplasmic reticulum during their secretion to either the cell surface or from the cell. The gamma-glutamyl carboxylase produces Gla using reduced vitamin K, which becomes oxygenated to vitamin K epoxide. Reduced vitamin K is then regenerated by a vitamin K oxidoreductase (VKORC1), and this interconversion of oxygenated and reduced vitamin K is referred to as the vitamin K cycle. Many of the VKD proteins support hemostasis, which is suppressed during therapy with warfarin that inhibits VKORC1 activity. VKD proteins also impact a broad range of physiologies beyond hemostasis, which includes regulation of calcification, apoptosis, complement, growth control, signal transduction and angiogenesis. The review covers the roles of VKD proteins, how they become activated, and how disruption of carboxylation can lead to disease. VKD proteins contain clusters of Gla residues that form a calcium-binding module important for activity, and carboxylase processivity allows the generation of multiple Glas. The review discusses how impaired carboxylase processivity results in the pseudoxanthoma elasticum-like disease.

VKORC1 gene polymorphism (-1639G>A) in warfarin therapy patients of Pakistani population.

OBJECTIVE: To observe vitamin K epoxide reductase complex subunit 1-1639 G>A polymorphism in patients resistant to warfarin therapy, and to calculate the allele frequency of the polymorphism in local patients. Methods: The cross-sectional case-control study was conducted at the Punjab Institute of Cardiology, Lahore, from 2013 to 2014 and comprised patients with heart valve replacement. Thy were divided into warfarin-resistant group 1 taking 10mg/day, 70mg/week and control group 2 taking a standard dose of 5mg/day, 35mg/week. The vitamin K epoxide reductase complex subunit 1-1639 G>A polymorphism analysis was done by polymerase chain reaction, followed by restriction fragment length polymorphism technique. Data was analysed using SPSS 20. RESULTS: Of the 146 patients, there were 73(50%) in each of the two groups. In group 1, there were 37(50.68%) males and 36(49.32%) were females with an overall mean age of 33±12 years, while group 2 had 36(49.32%) males and 37(50.68%) females with an overall mean age of 37±13 years. There were no significant differences in mean values of age, serum cholesterol, triglycerides and albumin levels between the groups (p>0.05). The G allele was the most frequently found in both groups, with 140(96%) in group-1 and 137(94%) in group-2. Overall, the homozygous GG genotype was significantly higher in the sample 132(90.4%) (p<0.05). CONCLUSIONS: There was evidence found that vitamin K epoxide reductase complex subunit 1-1639 G>A polymorphism alone may not be the dominant genetic factor associated with warfarin response variability.

Seasonal diet-based resistance to anticoagulant rodenticides in the fossorial water vole (Arvicola amphibius).

Anticoagulant rodenticides (AR) resistance has been defined as "a major loss of efficacy due to the presence of a strain of rodent with a heritable and commensurately reduced sensitivity to the anticoagulant". The mechanism that supports this resistance has been identified as based on mutations in the Vkorc1 gene leading to severe resistance in rats and mice. This study evaluates the validity of this definition in the fossorial water vole and explores the possibility of a non-genetic diet-based resistance in a strict herbivorous rodent species. Genetic support was explored by sequencing the Vkorc1 gene and the diet-based resistance was explored by the dosing of vitamins K in liver of voles according to seasons. From a sample of 300 voles, only 2 coding mutations, G71R and S149I, were detected in the Vkorc1 gene in the heterozygous state with low allele frequencies (0.5-1%). These mutations did not modify the sensitivity to AR, suggesting an absence of genetic Vkorc1-based resistance in the water vole. On the contrary, vitamin K1 was shown to be 5 times more abundant in the liver of the water vole compared to rats. This liver concentration was shown to seasonally vary, with a trough in late winter and a peak in late spring/early summer related to the growth profile of grass. This increase in concentration might be responsible for the increased resistance of water voles to AR. This study highlights a non-genetic, diet-related resistance mechanism in rodents to AR. This diet-based resistance might explain the different evolution of the Vkorc1 gene in the fossorial water vole compared to rats and mice.

Non-genetic factors and polymorphisms in genes CYP2C9 and VKORC1: predictive algorithms for TTR in Brazilian patients on warfarin.

PURPOSE: This study was designed to evaluate the association of non-genetic factors and polymorphisms CYP2C9*2 (rs1799853), CYP2C9*3 (rs1075910), and VKORC1-G1639A (rs9923231) with time in therapeutic range (TTR), and to build a regression model to predict the quality of oral anticoagulation control in a sample of Brazilian patients. METHODS: This is a retrospective cohort study developed at an anticoagulation clinic of a university hospital. Overall, 312 patients were included. The quality of oral anticoagulation control was evaluated by TTR. TTR was dichotomized for analysis, using two cutoff points for classification as inadequate (TTR ≤ 60.0%) and optimal (TTR ≥ 75.0%) control. RESULTS: The average age was 60.4 ± 13.5 years, with a predominance of women (187; 59.9%). The -G1639A polymorphism of the VKORC1 gene, when evaluated, based on the recessive inheritance pattern [AA × (GA + GG)], patients with AA genotype exhibited a higher TTR (68.2% versus 62.8%, p = 0.017). TTR ≤ 60.0% was associated with number of drugs in chronic use, assistance for warfarin administration, reports of not taking warfarin, absenteeism, sex (female), and target INR (International Normalized Ratio; 2.00-3.00). TTR ≥ 75.0% was associated with sex (male), target INR (2.00-3.00), assistance for warfarin administration, reports of not taking warfarin, and absenteeism. The two algorithms proposed showed adequate ability to predict TTR presenting good sensitivity and specificity. CONCLUSIONS: Our findings provided useful information for risk stratification depending on TTR level and for future investigations on the quality of oral anticoagulation control in Brazilian anticoagulation clinics.

Comparative biological properties of the four stereoisomers of difethialone, a second-generation anticoagulant rodenticide, in rats: development of a model allowing to choose the appropriate stereoisomeric ratio.

The current management of rodent pest populations is based on second-generation anticoagulant rodenticides (SGAR). These molecules, of which difethialone is part, are much more efficient than the first generation. Nevertheless, this efficiency comes with a major drawback, SGARs are tissue persistent that increases the exposure of rodent predators to them. According to its chemical structure, difethialone has four stereoisomers, whose specific inhibition potency and pharmacokinetic have never been described and might be useful to design new eco-friendly rodenticides. The study aimed to investigate the ability to inhibit anticoagulant target enzyme (VKORC1) and the pharmacokinetics in rats of the four difethialone stereoisomers in rats. We show that stereoisomers are all highly efficient to inhibit VKORC1 activity, but they have distinct initial half-life with 6.0 h, 25.4 h, 69.3 h, and 82.3 h for, respectively, E4-trans, E2-cis, E1-trans, and E3-cis stereoisomer. These results open the way of the development of eco-friendly and efficient rodenticide by mixing some of these stereoisomers. Preferential incorporation of the E4-trans stereoisomer (high inhibitory VKORC1 potency, relatively shorter liver half-life) into difethialone rodenticides baits might result in a more eco-friendly product than current commercially available difethialone formulations. In addition, we put forward modelling to help design bait according to the circumstance of use (presence of non-target species, food competition, etc.) by modulating the theorical AUC and and the theorical concentration of the product at the death of the rodent pest. Thus, this modeling might allow to diminish the use of laboratory animal in assay.

Genotype-guided warfarin therapy: Still of only questionable value two decades on.

WHAT IS KNOWN AND OBJECTIVE: Despite an apparently sound pharmacological basis, clinical studies of genotype-guided warfarin dosing have yielded mixed and conflicting results, leading to reluctance in its clinical implementation. The objective of this critique is to re-evaluate key warfarin pharmacogenetic studies with a view to explaining why this may be so. METHODS: Major widely-cited warfarin pharmacogenetic studies as well as recent meta-analyses were identified and a critical analysis of these was undertaken to identify factors that may account for poor clinical implementation of pre-treatment genotyping. RESULTS AND DISCUSSION: Critical examination of major warfarin pharmacogenetic studies identified a number of methodological concerns such as marked variations in study designs with different variously-defined measures of outcome. Genotype testing involved only a limited number of CYP2C9/VKORC1 alleles. Claims of benefits of genotyping are based almost exclusively on INR-related parameters which are known to be highly time-labile and of limited value in predicting clinical risk or benefit. This is evidenced by lack of any significant effect of genotyping on rates of bleeding or thromboembolic events. Neither have the effects of potential phenoconversion or medication non-adherence in study populations been adequately investigated. Although the effect of ethnicity/race is now better characterised, studies lack the power to determine whether any benefits claimed are indication-sensitive. WHAT IS NEW AND CONCLUSION: Since 60% of inter-individual variability in warfarin dose/response is due to other factors (many of which are non-genetic), expectations of eliminating this variability simply by CYP2C9/VKORC1 genotyping are over-optimistic and efforts cost-ineffective. Real-world studies have not always corroborated trials-based claims of clinical benefit. It is time to consider redirecting scarce resources away from the study of warfarin pharmacogenetics to pharmacogenetic research of potentially greater clinical relevance.

An analysis of allele, genotype and phenotype frequencies, actionable pharmacogenomic (PGx) variants and phenoconversion in 5408 Australian patients genotyped for CYP2D6, CYP2C19, CYP2C9 and VKORC1 genes.

Common polymorphisms in the genes encoding CYP2D6, CYP2C19, CYP2C9 and VKORC1 enzymes have an important role in predicting the occurrence of adverse effects and the efficacy of substrate medications. Drug-induced changes to the enzyme's phenotype, a process called phenoconversion, comprise another important factor contributing to interindividual variability in drug response. To date, there is lack of data on the frequency of these common polymorphisms and phenoconversion in the pan-ethnic Australian population. The aim of this study was to (1) describe allele, genotype and phenotype frequencies for CYP2D6, CYP2C19, CYP2C9 and VKORC1 enzymes in the pan-ethnic Australian population and (2) evaluate the frequency of actionable pharmacogenomic (PGx) variants and phenoconversion. Frequencies were calculated using the records of 5408 Australian patients (obtained from myDNA's propriety database), who were consecutively tested with the DNAdose PGx test which included the CYP2D6, CYP2C19, CYP2C9 and VKORC1 genes. In 2509 patients with listed medications at the time of testing, phenoconversion frequencies were calculated for CYP2D6, CYP2C19 and CYP2C9 enzymes. Allele, genotype and phenotype frequencies in our Australian patients correlated with a Caucasian population. Approximately 96% of patients had at least one actionable PGx variant. A five-fold increase in the frequency of poor metabolisers (PMs) for CYP2D6 and CYP2C19 was predicted by phenoconversion. Our study results indicate a high frequency of actionable PGx variants in our Australian population. With the addition of drug-induced phenoconversion, our results provide further support for the utilisation of PGx testing in clinical practice as another tool assisting prescribers in the application of personalised medicine.

Warfarin dose requirement in patients having severe thrombosis or thrombophilia.

AIMS: Warfarin dose requirement varies significantly. We compared the clinically established doses based on international normalized ratio (INR) among patients with severe thrombosis and/or thrombophilia with estimates from genetic dosing algorithms. METHODS: Fifty patients with severe thrombosis and/or thrombophilia requiring permanent anticoagulation, referred to the Helsinki University Hospital Coagulation Center, were screened for thrombophilias and genotyped for CYP2C9*2 (c.430C>T, rs1799853), CYP2C9*3 (c.1075A>C, rs1057910) and VKORC1 c.-1639G>A (rs9923231) variants. The warfarin maintenance doses (target INR 2.0-3.0 in 94%, 2.5-3.5 in 6%) were estimated by the Gage and the International Warfarin Pharmacogenetics Consortium (IWPC) algorithms. The individual warfarin maintenance dose was tailored, supplementing estimates with comprehensive clinical evaluation and INR data. RESULTS: Mean patient age was 47 years (range 20-76), and BMI 27 (SD 6), 68% being women. Forty-six (92%) had previous venous or arterial thrombosis, and 26 (52%) had a thrombophilia, with 22% having concurrent aspirin. A total of 40% carried the CYP2C9*2 or *3 allele and 54% carried the VKORC1-1639A allele. The daily mean maintenance dose of warfarin estimated by the Gage algorithm was 5.4 mg (95% CI 4.9-5.9 mg), and by the IWPC algorithm was 5.2 mg (95% CI 4.7-5.7 mg). The daily warfarin maintenance dose after clinical visits and follow-up was higher than the estimates, mean 6.9 mg (95% CI 5.6-8.2 mg, P < 0.006), with highest dose in patients having multiple thrombophilic factors (P < 0.03). CONCLUSIONS: In severe thrombosis and/or thrombophilia, variation in thrombin generation and pharmacodynamics influences warfarin response. Pharmacogenetic dosing algorithms seem to underestimate dose requirement.

Impact of genetic and clinical factors on warfarin therapy in patients early after heart valve replacement surgery.

PURPOSE: Factors influencing responsiveness to warfarin at treatment onset time were not well identified in Chinese patients undergoing heart valve replacement. We sought to select the most relevant factors that associated with patient response to warfarin early after heart valve surgery. METHODS: In this observational study, 289 patients starting warfarin therapy early after heart valve replacement surgery were enrolled. CYP2C9 *1, *2, *3, and *5; VKORC1-1639 G>A, CYP4F2 V433M, and GGCX rs11676382 genotypes; clinical characteristics, response to therapy, and bleeding and thrombosis events were collected. The primary outcomes were the time to the first INR equal to or more than lower limit of therapeutic range and the warfarin dose requirements. Stepwise multiple linear regression was performed to develop a dosing algorithm to predict the warfarin dose requirements. RESULTS: The results of univariate analysis showed lone VKORC1-1639 G>A, CYP2C9 *1/*3, cefazolin, cefoperazone-sulbactam, increased BMI, Δhemoglobin, and white blood cell count could significantly affect patient responsiveness to warfarin in the initial period of anticoagulation. Multivariate analysis resulted in an equation: Accumulated warfarin doses (mg) = 17.068 VKORC1-1639 G>A - 4.261 hypertension + 0.593 BMI - 0.115 age - 4.852 CYP2C9 *1/*3 - 2.617 cefazolin - 4.902 cefoperazone-sulbactam - 4.537, which could explain 40.2% of the variability in warfarin dose needed to reach the first INR equal to or more than lower limit of therapeutic range. CONCLUSIONS: Both genetic and clinical factors contributed to anticoagulation effect of warfarin in the initial period of treatment. Our findings could provide a basis for the personalized management of warfarin use in the early stage of anticoagulation in northern Chinese patients.

Verification of pharmacogenomics-based algorithms to predict warfarin maintenance dose using registered data of Japanese patients.

PURPOSE: Large inter-individual differences in warfarin maintenance dose are mostly due to the effect of genetic polymorphisms in multiple genes, including vitamin K epoxide reductase complex 1 (VKORC1), cytochromes P450 2C9 (CYP2C9), and cytochrome P450 4F2 (CYP4F2). Thus, several algorithms for predicting the warfarin dose based on pharmacogenomics data with clinical characteristics have been proposed. Although these algorithms consider these genetic polymorphisms, the formulas have different coefficient values that are critical in this context. In this study, we assessed the mutual validity among these algorithms by specifically considering racial differences. METHODS: Clinical data including actual warfarin dose (AWD) of 125 Japanese patients from our previous study (Eur J Clin Pharmacol 65(11):1097-1103, 2009) were used as registered data that provided patient characteristics, including age, sex, height, weight, and concomitant medications, as well as the genotypes of CYP2C9 and VKORC1. Genotyping for CYP4F2*3 was performed by the PCR method. Five algorithms that included these factors were selected from peer-reviewed articles. The selection covered four populations, Japanese, Chinese, Caucasian, and African-American, and the International Warfarin Pharmacogenetics Consortium (IWPC). RESULTS: For each algorithm, we calculated individual warfarin doses for 125 subjects and statistically evaluated its performance. The algorithm from the IWPC had the statistically highest correlation with the AWD. Importantly, the calculated warfarin dose (CWD) using the algorithm from African-Americans was less correlated with the AWD as compared to those using the other algorithms. The integration of CYP4F2 data into the algorithm did not improve the prediction accuracy. CONCLUSION: The racial difference is a critical factor for warfarin dose predictions based on pharmacogenomics.

Genetic polymorphisms associated with the prevalence of retinal vein occlusion in a Greek population.

PURPOSE: To investigate possible associations of single-nucleotide polymorphisms (SNPs) from five genes with branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO). METHODS: A total of 69 patients with retinal vein occlusion-RVO (24 with BRVO and 45 with CRVO), and 82 controls, were enrolled in this study. All subjects were screened for hypertension, diabetes mellitus, hyperlipidemia, glaucoma, anticoagulant medication, smoking status and history of stroke. The genotyping of AGTR1-A1166C, adiponectin + 276 G/T, MMP2-1306C/T, Gpla/lla-C807T/G873A and VKORC1-G1639A polymorphisms was performed using restriction fragment length polymorphism or allele-specific polymerase chain reaction. RESULTS: The percentage of the AGTR1-A1166C C allele carriers and Gpla/lla-C807T/G873A T/A carriers was significantly higher in the CRVO patients than in the controls (P = 0.00001 and P = 0.0004, respectively). At the multiple logistic regression analysis, the AGTR1-A1166C C allele carrier status and the Gpla/lla-C807T/G873A T/A allele carrier status were found to be associated with an increased risk of CRVO. Moreover, adiponectin + 276 G/T T allele carriers had a significantly increased risk of RVO in subjects ≥ 75 years old. There was no significant difference between the BRVO patients and controls concerning the genotype or the allele frequency distributions of these SNPs. The genotype distributions or allelic frequencies of the other evaluated polymorphisms did not significantly differ between the patients with RVO and the control subjects. CONCLUSIONS: AGTR1 A1166C and Gpla/lla C807T/G873A polymorphisms are likely to be risk factors for CRVO. Adiponectin + 276 G/T SNP is likely to predispose to RVO in older subjects.