Tejasvita Gaikwad

Influence of CYP2C9 and VKORC1 gene polymorphisms on warfarin dosage, over anticoagulation and other adverse outcomes in Indian population.

The aim of this study was to determine the frequencies of SNPs in the vitamin K epoxide reductase complex subunit 1 (VKORC1) and cytochrome P450 2C9 (CYP2C9) genes and their effect on warfarin dose requirement, over anticoagulation and other adverse outcomes in Indian population. A total of 145 warfarin treated patients for various clinical conditions were screened for VKORC1 and CYP2C9 gene polymorphisms by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. We found that homozygous VKORC1-1639 AA and CYP2C9 (*)3/(*)3 polymorphisms showed 100% association with risk of over anticoagulation and other adverse events. Carriers of two heterozygous variant genotypes also showed significant association with risk of over anticoagulation and bleeding. Single variant carrier patients were found to require low warfarin dose as compared to wild type (CYP2C9(*)1/(*)1 and VKORC1- 1639 GG) patients. The major impact of VKORC1 and CYP2C9 genotypes was observed in the first month of anticoagulation. A drastic variation from other Asian countries was observed in Indian population with regard to the distribution of different VKORC1 -1639 genotypes. Our results suggest that both VKORC1 and CYP2C9 genotypes showed significant impact on warfarin dose requirement, over anticoagulation in the first month of anticoagulation and number of bleeding episodes. The variation in therapeutic dosage of warfarin and the associated adverse events across different populations is due to the wide differences in the frequency of these warfarin sensitive alleles.

Warfarin Dose Model for the Prediction of Stable Maintenance Dose in Indian Patients

The main aim of this study was to screen various genetic and nongenetic factors that are known to alter warfarin response and to generate a model to predict stable warfarin maintenance dose for Indian patients. The study comprised of 300 warfarin-treated patients. Followed by extensive literature review, 10 single-nucleotide polymorphisms, that is, VKORC1-1639 G>A (rs9923231), CYP2C9*2 (rs1799853), CYP2C9*3 (rs1057910), FVII R353Q (rs6046), GGCX 12970 C>G (rs11676382), CALU c.*4A>G (rs1043550), EPHX1 c.337T>C (rs1051740), GGCX: c.214+597G>A (rs12714145), GGCX: 8016G>A (rs699664), and CYP4F2 V433M (rs2108622), and 5 nongenetic factors, that is, age, gender, smoking, alcoholism, and diet, were selected to find their association with warfarin response. The univariate analysis was carried out for 15 variables (10 genetic and 5 nongenetic). Five variables, that is, VKORC1-1639 G>A, CYP2C9*2, CYP2C9*3, age, and diet, were found to be significantly associated with warfarin response in univariate analysis. These 5 variables were entered in stepwise and multiple regression analysis to generate a prediction model for stable warfarin maintenance dose. The generated model scored R 2 of .67, which indicates that this model can explain 67% of warfarin dose variability. The generated model will help in prescribing more accurate warfarin maintenance dosing in Indian patients and will also help in minimizing warfarin-induced adverse drug reactions and a better quality of life in these patients.

Delayed warfarin induced skin necrosis in a patient with poor warfarin metabolizing activity due to interrupted warfarin therapy

Keywords Warfarin induced skin necrosis. Poor metabolizers of warfarin. Vitamin K. Heparin Warfarin-induced skin necrosis (WISN) is a rare but potentially devastating complication of oral anticoagulation occurring within 3-10 days of warfarin therapy. Potential causes of WISN include overdose of warfarin, drugs which affect liver function, acquired or hereditary protein C, protein S or antithrombin deficiency, factor V Leiden mutation, and lupus anticoagulants [1]. The dermatologic complications occur in 0.01 to 0.1 % of warfarin-treated patients [2]. A 31 year old man presented with severe pain and increased swelling in left lower limb in September 2011. Venous Doppler showed thrombosis in external iliac, common femoral vein extending to popliteal and proximal tibial vein. The patient was put on LMWH (CLEXANE®) 50 mg daily, which was overlapped with 5 mg warfarin. After achieving the therapeutic range by day 4, enoxaparin was stopped and warfarin was continued without any other medication. His thrombophilia profile was normal and he had no other known precipitating conditions. Patient presented 2 months later with elevated INR of 5.84, but without any bleeding symptoms. Warfarin was immediately stopped and a single parenteral dose of vitamin K1, 10 mg, was given. After 48 hours of vitamin K1 administration, INR dropped to 1.3 and the patient was started on low dose warfarin at 2 mg/day but without overlapping with heparin. On day 4 after reinitiation of warfarin, multiple erythematous lesions, petechiae and swelling were noted on the affected foot followed by skin necrosis. Complete blood count (eosinophil count 245/uL), autoimmune profile, alkaline phosphatise, SGOT, SGPT and bilirubin were within normal limits; both protein C and protein S were reduced to <20 % of the normal control. The sample was screened for the common warfarin sensitive gene polymorphisms, i.e. CYP2C9*2, CYP2C9*3 and VKORC1-1639G/A, and was found to be homozygous for CYP2C9*3 polymorphism. The histopathology of the lesions could not be done in our case, but considering the time of onset and other clinical and laboratory findings, a diagnosis of WISN was made. Ultrasonography (USG) showed partial canalisation of all the veins in the affected limb. The patient was restarted on enoxaparin 50 mg subcutane-ous daily, along with 2 mg warfarin. After 6 days of heparin bridging treatment, heparin was stopped and only warfarin, 2 mg, was continued under observation. The patients INR reached 2.4 after two weeks of warfarin therapy and he has been maintained on that dose. Local wound …

Dosing algorithms for vitamin K antagonists across VKORC1 and CYP2C9 genotypes: comment

We read with interest the recent report by Baranova et al. [1] in which the authors have carried out secondary analysis of European Pharmacogenetics of Anticoagulant Therapy (EU-PACT) data by evaluating the performance of different dosing algorithms and time intervals of outcome assessment across VKORC1CYP2C9 genetic subgroups. When administering warfarin, it is important to have knowledge of polymorphisms other than VKORC1CYP2C9, which are known to affect warfarin dosage. Identifying both low and high-dose variants of these polymorphisms is important to draw a genomic guided algorithm. Although their contribution is not substantial, they may cumulatively impact the laboratory and clinical outcomes. The authors have shown that the subgroup with the VKORC1 AA-CYP2C9 *1/*1 genotype is associated with a higher risk of sub-therapeutic INR with the genotypeguided dose, but the sample size in this group is too small (PG algorithm, n = 29; non-PG algorithm, n = 34) to draw a definitive conclusion. We have previously analyzed the data from Asian countries and have shown that East Asian and South-East Asian populations predominately carry these variant genotypes (CYP2C9*1/*1 > 90%; VKORC1 AA 55–85%) [2]. Studies in these populations may give an opportunity to analyze larger sample sizes in this subgroup of patients and will help in confirming these findings. It is also known that interindividual variation in vitamin K antagonist dosage has been mainly attributed to genetic variables and also to some extent to environmental, demographic and clinical factors. Other non-genetic factors, including ethnicity, age, body mass index (BMI), weight and gender, drug and food interaction and existing pathology, are also known to alter the response to vitamin K antagonists. Although the novel oral anticoagulants are overall safe and effective alternatives to vitamin K antagonists, several factors still limit their use in developing countries such as India.

Possible impact of factor V Leiden genotype on warfarin induced bleeding

We read with interest the report, recently published by Nahar et al.[1] on the prevalence of warfarin sensitive alleles in factor V Leiden (FVL) mutation carriers. The study provides preliminary evidence for the need of pre‐prescription genotyping of warfarin sensitive polymorphisms (CYP2C9*2, *3 and Vitamin‐K epoxide reductase complex subunit1 [VKORC1]‐1639G/A) in patients who are at risk of thrombosis (carriers of thrombophilic marker) and require anticoagulation therapy.

Warfarin pharmacogenetics: How close are we to clinical practice?

The past decade has seen substantial advances in our understanding of the genetic factors influencing response to a variety of drugs including warfarin. Though there has been an increase in the clinical use of pharmacogenetic information to individualize treatment regimens, yet there generally has not been broad acceptance of pharmacogenetic testing. Warfarin remains the mainstay therapy for oral anticoagulation; it also remains one of the most challenging medications to manage despite over 60 years of experience with the drug. It is one of the top 10 drugs related to adverse drug events and hospitalization. Clinical management of warfarin therapy mainly is complicated by a narrow therapeutic index and high inter‐ and intra‐individual variability in drug disposition and response. Supratherapeutic international normalized ratio (INRs) can lead to fatal hemorrhage while subtherapeutic INRs can lead to thrombosis. Growing evidence indicates that up to 60% of the individual pharmacological response to warfarin might be due to genetic variables and affected by polymorphisms in the genes mainly, vitamin K epoxide reductase complex subunit 1 (VKORC1), the target enzyme of warfarin and cytochrome P450 2C9 (CYP2C9), the main enzyme involved in warfarin metabolism. Although the genotypes of VKORC1 and CYP2C9 are clearly the most important genetic factors for warfarin response, genetic variations in other genes for instance, CYP4F2 and GGCX also show significant association with warfarin response. Non‐genetic factors such as age (elderly patients), diet (vitamin K rich diet), disease states (thyroid activity, liver or renal dysfunction, fever), other drugs (e.g., amiodarone, propafnone, metronidazole, tamoxifen, etc.), life‐style (alcohol intake, smoking, exercise) can also substantially modulate the response to warfarin. VKORC1 polymorphisms have been reported to be more potent modifiers of warfarin response than the CYP2C9 polymorphisms. Recent work by Rieder et al.[1] has also shown that individuals with VKORC1 A haplotype (H1 and H2) require low warfarin dose as a result of a decreased expression of messenger ribonucleic acid when compared to individuals with VKORC1 B haplotype (H7, H8 and H9). In the present issue of the journal, Kumar et al.[2] have studied genotype, allele and haplotype frequencies of VKORC1 and CYP4F2 in South Indian population. The authors have used 5 VKORC1 single nucleotide polymorphisms (SNPs) (similar to the report by Rieder et al.) to draw the VKORC1 haplotypes and the haplotype frequency has been compared with that of other populations. Except two SNPs, a strong LD pattern (D’ > 0.8) has been observed in this study for all the remaining studied SNPs in VKORC1 gene. In addition, the genotype frequencies of CYP4F2 were also found to be distinct in Indian population when compared to the rest of the populations. Thus, the study further

Polymorphisms of warfarin metabolizing enzymes in an Indian population

We read with interest the report recently published by Buzoianu et al. [1] on the effect of older age apart from CYP2C9*3 (Cytochrome P450 2C9*3) and c.-1639 G>A polymorphism of VKORC1 (Vitamin K epoxide reductase complex subunit 1). These polymorphisms are associated with lower dose requirement of acenocoumarol in the Romanian population. The pharmacokinetic parameters of bothwarfarin andacenocoumarol are slightly different and the importance of CYP2C9 for the clearance of each anticoagulant differs. CYP2C9 isoenzyme appears to be the major enzyme responsible for the clearance of warfarin and to a little lesser extent for acenocoumarol [2]. Thus the effect of the CY2PC9 variants should not differ much for these two anticoagulants. Different authors have shown different degrees of association of cytochrome and VKORC1 polymorphismswith both over anticoagulation and warfarin dosage. In a similar study from our center in Mumbai, in 145 patients on warfarin, we found that, VKORC1-1639 AA, CYP2C9*3/*3 and two heterozygous variant genotype carrier patients were found to be significantly associated with risk of over anticoagulation and reduced stable warfarin dose for achieving INR values of 2–3 (P b 0.0001). Carriers of VKORC1-1639 GA, CYP2C9*1/*2 and CYP2C9*1/*3 were not found to be significantly associated with the risk of over anticoagulation with bleeding but were found to be associated with reduced warfarin dose requirement for obtaining an INR of 2–3. Wild allele carrier patients required 6.25 mg stable average daily warfarin dose, while patients who were carriers of VKORC1-1639 GA, CYP2C9*1/*2 and CYP2C9*1/*3 required 3.75 mg, 4.4 mg, and 3.75 mg stable average daily warfarin dose respectively [3]. The main difference between the two studies is that, no association of CYP2C9*2 allele was observed related to dose requirement in the study cohort of Buzoianu et al., while the same was significant in our study. The effect of age on warfarin dosage has been reported by several authors and this has been one of the important clinical parameter included in the pharmacogenomic algorithms of both acenocoumarol and warfarin [4–6]. In our series only 5 out 145 patients were above