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To determine the individualized dosage of acenocoumarol, we investigated the contribution of CYP2C9 and VKORC1 genetic polymorphisms on daily acenocoumarol maintenance dose in the patients of south Indian origin.
Materials and methods: The study was conducted in 170 patients attending out- patient clinics. The single nucleotide polymorphisms of CYP2C9*2 (rs1799853), CYP2C9*3 (rs1057910) and VKORC1 (rs9923231) were identified by Real-Time polymerase chain reaction (RT-PCR) method.
Results: The study reveals that, the CYP2C9*1*1, *1*2, *1*3, *2*3 and *3*3 genotype frequencies were found to be 80.3%, 4.3% 13.5%, 0.6% and 1.2%, respectively. The VKORC1 GG, GA and AA genotype frequencies were found to be 84.7%, 14.7% and 0.6%, respectively. Any one variant allele carriers of CYP2C9 (*1*2 and *1*3) required 44% and 28.2% lower daily acenocoumarol dose (2.0± 0.8 (SD) mg and 2.5± 1.2 (SD) mg and 2.0 mg respectively) than the normal CYP2C9*1*1 genotype group (3.4± 1.3(SD) mg). The VKORC1GG genotype carriers required higher dose [3.3±1.3 (SD) mg] as compared to VKORC1GA [2.3± 0.8 (SD) mg] and VKORC1AA [1.0 mg]. A combination of both the genes indicated that patients with CYP2C9*1*2 and VKORC1 GA genotypes required 38% lower dose (2.3±0.9 (SD) mg) than patients with CYP2C9*1*1 and VKORC1 GG genotype (3.5± 1.3(SD) mg). The clinical and genetic variables together contribute to predict 30.6% of the required maintenance dose.
Conclusion: The effect of genetic polymorphisms in the two genes CYP2C9 and VKORC1 were significantly affects the acenocoumarol daily maintenance dose requirement in south Indian population.
Key words: Acenocoumarol, Oral anticoagulants, pharmacogenomics, polymorphisms, south Indians
Oral anticoagulants, including warfarin and acenocoumarol are most widely prescribed for prevention of thromboembolic disorders in patients undergoing for cardiac surgery valvular replacement and for prevention of venous thromboembolism, pulmonary embolism, atrial fibrillation, valvular heart diseases and coronary heart diseases [1-5]. The complications of oral anticoagulant therapy may due to their narrow therapeutic index, the excessive dosage of these drugs leads to bleeding complications (sensitivity) and the lower dosage leads to therapeutic failure (resistance), so careful monitoring is needed for optimal anticoagulation therapy. The effective anticoagulation is measured by monitoring the prothrombin time (PT) expressed as the international normalized ratio (INR) within the desired therapeutic range (2.0- 3.5) . Inter- individual variation is the major problem for determining the oral anticoagulant dose. This variability may dependents on environmental factors but genetic variability also explained and plays role in the variable dose requirements [7, 8].
Oral anticoagulants are primarily metabolized by cytochrome P450 2C9 enzyme (CYP2C9) , and the target enzyme for these drugs was found to be vitamin K epoxide reductase complex subunit 1(VKORC1) . The CYP2C9 has been identified to be polymorphic, two defective alleles (CYP2C9*2 and CYP2C9*3) has been found strongly associated with dose requirement and bleeding complications . The presence of the defective allele leads to lower enzymatic activity, thus leads to reduced metabolism of these drugs and the individuals with defective allele's required lower dose. On the other hand the VKORC1 is involved in the pharmacodynamics action of the oral anticoagulants. The single nucleotide polymorphism in the promoter region at the nucleotide position -1639 G>A, was found to be strongly associated with the lower dose requirement . It has been found that the genetic variation in CYP2C9 and VKORC1 genes together accounts for 35- 50% variability in the dose requirement for initiation and maintenance dose of oral anticoagulants [13, 14]. Recently, the United States Food and Drug Administration (US FDA) updated the label of Coumadin, in 2010 added a table and provided the information with the range of predictive therapeutic doses based on CYP2C9 and VKORC1 genotypes . In our hospital acenocoumarol is widely prescribed for the patients to maintain the anticoagulation in patients after the rheumatic valve replacement surgery. The pharmacogenomics of warfarin was well studied and acenocoumarol was less studied in the world population. In our population there was no such study explaining the association of acenocoumarol and genetic polymorphisms. So, in the present study we aimed to find out the influence of the genetic variability of CYP2C9 and VKORC1 genes on acenocoumarol maintenance dose requirement in the south Indian patients after the heart valve replacement surgery.
Materials and methods
The patients were recruited for the study from out-patient department of cardio thoracic and vascular surgery and department of cardiology in the Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER) hospital, Pondicherry. All patients received anticoagulation treatment with acenocoumarol to achieve an INR in the target range of 2.0 to 3.5. Patients of age group 18-65 years and of either gender were recruited for the study. Their nativity as south Indian was assessed by their status based on family history of three generations living in Tamil Nadu, Pondicherry, Kerala, Karnataka and Andhra Pradesh and speaking any of the south Indian language as their mother tongue. Written informed consent was obtained from all the patients participated in this study. Institutional ethics committee approved this study.
Patients with liver or renal dysfunction or on treatment with drugs potentially interacting with acenocoumarol, pregnant and lactating women, smokers and alcoholics were excluded. Patients' demographic details were obtained from patients' case records. Patients with three consecutive measurements of INR value between 2 and 3.5, three months after the initiation of acenocoumarol therapy were included for the study.
Genotyping for CYP2C9 and VKORC1 -1639G>A
Five milliliters of venous blood were collected from the patients for genotyping. DNA was extracted by phenol-chloroform extraction procedure. Genotyping of CYP2C9 and VKORC1 were carried out in real-time thermo cycler (7300 Applied Biosystems; Life Technologies Corporation, Carlsbad, CA, USA) using TaqMan SNP genotyping assays (VKORC1 (rs9923231) assay ID: C__30996661_30, CYP2C9*2 (rs1799853) (assay by design), CYP2C9*3 (rs1057910) assay ID: C_27104892_10). The PCR was carried out in triplicate in a 25-µL ï¬nal volume that contained 12.5 µL of TaqMan universal PCR master mix (2x), 1.25 µL of 20x working stock of TaqMan SNP genotyping assay and 5.0 µL of genomic DNA diluted in DNAase free water and 6.25 µL of MilliQ water (Millipore Corporate Headquarters, Billerica, MA, USA). The thermo cycler conditions included one cycle at 95°C for 10 min to activate the AmpliTaq gold DNA polymerase followed by 40 cycles of denaturation at 92°C for 15 sec and annealing/extension at 60°C for 1 min. The genotype and allele calls were analyzed using 7300 SDS software version 1.4.0.
GraphPad Instat® version 3.06 (San Diego, USA) and IBM® SPSS® Statistics version 19.0 (SPSS Inc., Chicago, IL, USA) were used for statistical analysis. The genotype frequencies were analyzed for Hardy- Weinberg equilibrium by chi-square test. The average daily maintenance dose between the genotype groups were compared by Kruskal Wallis test and Mann- Whitney U-test. The genotype and dose relationship was evaluated using linear regression analysis. Stepwise multivariate regression analysis was used to find the influence of the independent variables (age, BMI, concomitant medications, comorbid conditions and genetic polymorphisms) on the dependent variable (logarithmic transferred daily maintenance dose). p<0.05 was considered statistically significant.
The demographic characteristic of the study cohort were obtained from patient case records (Table 1). Among the 170 patients, a total of 163 patients were included for the analysis, seven patients data were excluded for analysis due to loss of samples during processing of DNA. The mean average daily dose of acenocoumarol was determined to be 3.15± 1.3(SD) mg. In the present study, the allele frequencies of CYP2C9*1 (89.2%), CYP2C9*2 (2.5%), CYP2C9*3 (8.3%), were consistent with those reported for the study population. The VKORC1 G and A allele frequencies were found to be 92.1% and 7.9%, respectively. The genotype frequencies of these variants were found to be in Hardy- Weinberg equilibrium.
The daily maintenance dose in the patients with variant genotype group was significantly lower than the normal genotype group (Table 2). The normal genotypes carriers of CYP2C9 and VKORC1 were required nearly the same maintenance dose of acenocoumarol (3.4± 1.3(SD) mg for CYP2C9*1*1 and 3.3± 1.3(SD) mg for VKORC1 GG). Patients having two defective alleles in CYP2C9 gene required significantly lower dose (2.0 mg/day) than other genotype groups. The homozygote variant in VKORC1 (AA) was found in only one patient and the daily maintenance dose was lower (1.0 mg/day) than the other genotype group.
The effect of combination of variant genotypes on maintenance dose was compared between the other genotype combinations (Table 3). The patients carrying both the variant genotypes in (CYP2C9 and VKORC1) genes and any one variant in any one gene (CYP2C9 or VKORC1) were required lower dose as compared to the normal genotype carriers. Due to skewed distribution the daily dose was converted into logarithmic transformed dose and was taken into univariate and multivariate analysis, univariate analysis revealed age (p<0.05), body weight (p<0.0001), height (p<0.05) and body mass index (BMI) (p<0.0001), genetic polymorphism in the two genes CYP2C9*2 (p<0.0001), CYP2C9*3 (p<0.0001), VKORC1-1639G>A (p<0.0001) significantly influenced the daily dose. Multivariate stepwise regression analysis was performed by adding all the significant factors from univariate analysis (Table 4). The multivariate analysis revealed that the combined effect of age, BMI and genotype contributes 30.6% dose variation. The clinical factors age and BMI together contributes 11.2% of the dose variation. The genetic factors alone contribute for 19.4% dose variation. In our study 5 (3.1%) patients were reported to have bleeding risk. But there was no significant association found with the genetic polymorphisms and other factors.
This study was designed to analyze the influence of CYP2C9*2, *3 variant allele and VKORC1 -1639A variant allele on acenocoumarol dose requirement in south Indian patients. The clinical factors also considered and were taken into account as well. Over the past few years the influence of CYP2C9 and VKORC1 polymorphisms on oral anticoagulants has been extensively studied in various populations [16-30]. The allele and genotype frequencies reported in the present study were similar to the previously reported frequency in our population . According to the genotype groups the CYP2C9*1*1 carriers required higher maintenance dose than the other variant genotype groups (CYP2C9*1*2, *1*3, *2*3 and *3*3). Similarly according to the VKORC1 genotypes the GG genotype carriers required higher dose than the variant genotype (GA, AA) groups. The combined effect of these genes reveals that the carriers of normative genotype in both the genes required higher dose (3.5± 1.3(SD) mg) than other genotype groups. The presence of CYP2C9*1*2 variant shows 45% lower dose requirement and CYP2C9*1*3 variant shows that 29% dose reduction, the presence of both variant alleles (CYP2C9*2*3 and CYP2C*3*3) required 45% dose reduction than the CYP2C9*1*1 carriers. According to the VKORC1 genotype groups VKORC1 GA carriers required 30% lower dose and VKORC1 AA carriers required 73% lower dose than the VKORC1 GG carriers. Therefore, the carriers of wild type genotype in CYP2C9 and VKORC1 of our study population were treated with higher acenocoumarol dose than the heterozygous and homozygous variant genotype groups. Other than the genetic factors the clinical factors such as age, body mass index were also significantly associated with the dose requirements.
Previous studies have been found that the VKORC1 genetic polymorphisms were a better predictor than CYP2C9 genotype . In our study we have observed VKORC1 genotype alone contributes 9.0 % of the dose variation. Previous studies have explained that age and height were the important predictors and shows a greater variability in dosage of Coumadin anticoagulants [29, 30]. In agreement with the previous studies, we found that the dose requirement significantly reduced with age and shows 6.8% variability and associated with significant reduction in daily maintenance dose, but in our study height was not associated with the dose. Other than age, body mass index explains increased in dose requirement, in our study similarly the BMI increased 4.4% of acenocoumarol dose.
Other than the inter-individual variability, inter population variability also shows greater differences in oral anticoagulant dose requirements . Previous studies have been explained the influence of this genetic polymorphisms and bleeding risk [33, 34]. Another study found that patients receiving low doses of warfarin and who were carriers of one or more CYP2C9 variant allele were show four times more likely to develop major bleeding . In our study there were five patients reported bleeding episodes but that were not statistically significant with the CYP2C9 and VKORC1 genotype (p>0.05) and other variables.
Many studies have developed algorithm based on the genotype for determination of warfarin and acenocoumarol dose [35-41]. However few studies were determined the pharmacogenetic algorithm for acenocoumarol for the individual populations [15, 20, 41]. In Caucasian and Asian populations, genotype predicts 25% of the variability in warfarin dose. Recent study  found that the frequencies of VKORC1 AA, AG and GG genotypes in Chinese patients taking warfarin (80, 18 and 2.7%) were significantly different from Caucasians (14, 47 and 39%) and shows that the Chinese require lower dosages of warfarin than Caucasian to achieve the same degree of anticoagulation. In the present study it was observed that the VKORC1 genotype frequency (84.7, 14.7, 0.6%) were different from Caucasians and also from other Asians, so our population may require the intermediate dosage of acenocoumarol.
Previous studies have been explained that, in addition to the VKORC1 promoter region polymorphisms (-1639 G>A), the polymorphism in the non-coding region also contributes majorly to determine the warfarin and acenocoumarol dose [43, 44]. The haplotype analysis in the previous study shows that the haplotype A (SNPs at nucleotide positions 3730G>A, 2255C>T, 1542G>C, 1173 C>T and -1639 G>A) versus wild type alleles in haplotype B can be used to determine whether the person required low, intermediate or high dose of oral anticoagulants .
In Indian population there were very few studies reported the association of oral anticoagulants and the genetic variations. The study in North Indian patients were conducted and explained up to 41.4% variability in acenocoumarol dose requirement . An another study conducted in Andhra Pradesh population explained 61% variability in warfarin dose requirements based on genetic and clinical parameters . Furthermore, a recent study conducted in Malaysian Indians has been shown that the Indians required more warfarin dose than the Chinese and Malaysians patients .
In the previous studies it was explained CYP4F2 genetic variation is the third most predictor and explains 1 to 2% dose variability with oral anticoagulant dose [48- 50]. The previous studies were included this factor in their algorithm and significantly explains the variation in the pharmacogenetic algorithm [40, 46, 50, 51]. In our study we could able to explain only 30.6% of dose variability in the patient cohort, further, adding together the intronic region polymorphisms in the VKORC1 gene and the other genes (GGCX, EPHX1, CYP4F2, Factor V and VI) may significantly improve our predictive model with large sample size. The limitation of the present study was the strict exclusion criteria for not including the patients with interacting drugs, liver and renal dysfunction, alcoholics and smokers. This was to find the exact association of the genetic variability on acenocoumarol dose requirements. Further, additional genetic, clinical and environmental factors may significantly contribute to improve our dosing model. The present study provided only the underlying clinical and genetic variation and their fraction of influence on acenocoumarol daily maintenance dose in our population.
In conclusion, the association between the CYP2C9*2, CYP2C9*3 and VKORC1 (-1639 G>A) genetic polymorphism and the acenocoumarol maintenance dose requirement has been studied. The present study provided the basic information for developing a pharmacogenetic algorithm for predicting the initial dose of acenocoumarol in South Indian patients.