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The impact of the CYP2C9 and VKORC1 polymorphisms on acenocoumarol dose requirements in a Romanian population
YBCMD-01670; No. of pages: 5; 4C: Blood Cells, Molecules, and Diseases xxx (2012) xxx–xxx
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The impact of the CYP2C9 and VKORC1 polymorphisms on acenocoumarol dose requirements in a Romanian population Anca Dana Buzoianu a, Florentina Claudia Militaru a, Ştefan Cristian Vesa a,⁎, Adrian Pavel Trifa b, Sorin Crişan c a
Department of Clinical Pharmacology, “Iuliu Haţieganu” University of Medicine and Pharmacy, 6th Pasteur Street, 400349, Cluj-Napoca, Cluj, Romania Department of Medical Genetics, “Iuliu Haţieganu” University of Medicine and Pharmacy, 6th Pasteur Street, 400349, Cluj-Napoca, Cluj, Romania Department of Internal Medicine, 5th Medical Clinic, Municipal Hospital, “Iuliu Haţieganu” University of Medicine and Pharmacy, 11th Tăbăcarilor Street, 400139, Cluj-Napoca, Cluj, Romania
b c
a r t i c l e
i n f o
Article history: Submitted 9 October 2012 Available online xxxx (Communicated by J. Adamson, M.D., 30 October 2012) Keywords: Acenocoumarol Romanian population Genetic variability CYP2C9 VKORC1
a b s t r a c t Aim: To investigate the genotype–phenotype correlation in Romanian patients treated with acenocoumarol. Material and methods: We studied 301 consecutive patients who required treatment with acenocoumarol, admitted within the Municipal Hospital of Cluj-Napoca and the Heart Institute “Niculae Stănciou” in ClujNapoca over a 3-year period. For each patient we recorded clinical parameters which could interfere with the achievement of stable therapeutic international normalized ratio (INR). We performed genetic analysis which consisted of genotyping the CYP2C9 gene and the VKORC1 gene. Patients were divided in three groups according to the acenocoumarol dose needed to reach a stable INR: the low dose group (≤7 mg/week), the medium dose group (>7 mg and b 28 mg/week) and the high acenocoumarol dose group (>28 mg/week). Results: We found that patients' age was significantly different between groups (pb 0.001). No differences existed between groups regarding the pathologies which required anticoagulation therapy or the concomitant treatment. The following parameters increased the odds of receiving a low dose of acenocoumarol: patient's age over 65 years (OR, 3.2; p=0.01; 95%CI: 1.24–8.25), the presence of the CYP2C9*3 allele (OR, 3.4; p =0.006; 95%CI: 1.41–8.34), and the GA or AA genotype of c.-1639G>A polymorphism of VKORC1 (OR, 6.5; p =0.01; 95%CI: 1.38–30.5; respectively OR, 11.6; p =0.003; 95%CI: 2.26–59.58). A high acenocoumarol dose was less likely to be administered to an elderly patient (OR, 0.24; p= 0.001; 95%CI: 0.1–0.56) or to a patient with the GA or AA genotype (OR, 0.2; p b 0.001; 95CI%: 0.09–0.45; respectively OR, 0.05; p=0.006; 95%CI: 0.007–0.43). Conclusion: The stable therapeutic dose of acenocoumarol is dependent of patient's age, the presence of the CYP2C9*3 allele and the c.-1639G>A polymorphism of VKORC1. © 2012 Elsevier Inc. All rights reserved.
Introduction Acenocoumarol is the most widely used oral anticoagulant in Romania and other European countries, indicated for the treatment and prophylaxis of thromboembolic diseases. Anticoagulant treatment is always a challenge for clinicians because of the difficulties to match an appropriate dose. An insufficient dose will lead to a failure to prevent thrombosis, while an overdose will significantly increase the hemorrhagic risk, acenocoumarol being a low therapeutic index drug [1]. The most frequent complications of acenocoumarol treatment are hemorrhagic accidents, including cerebral and digestive hemorrhage, which are the most severe. ⁎ Corresponding author. E-mail addresses: [email protected] (A.D. Buzoianu), [email protected] (F.C. Militaru), [email protected] (Ş.C. Vesa), [email protected] (A.P. Trifa), [email protected] (S. Crişan).
Apart from its toxicity oral anticoagulant treatment is characterized by a great inter-individual variability regarding its efficacy. Treatment monitoring is usually done by INR determination, which has to be kept between specific limits, according to the pathology approached. Variability of the efficacy is manifested through differences in the dose needed to maintain the INR within the therapeutic range and it is determined in a multi-factorial way that involves both genetic and environmental factors. For a long time environmental factors were considered as the main cause for the variations in oral anticoagulant treatment response. These factors include: patient's characteristics (age, sex, and body mass index), vitamin K food intake, co-morbidities (hepatic failure, severe renal failure, heart failure, thyroid diseases, etc.), acute diseases (fever, sepsis, decompensated heart failure, diarrhea, etc.) and concomitant treatments [2,3]. After uncovering the role of the genetic polymorphisms in altering the function of certain specific proteins, the variability of the response to oral anticoagulant treatment was partly attributed to genetic factors. Among these, the CYP P450 system metabolizing enzymes, highly polymorphic in humans, are
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Please cite this article as: A.D. Buzoianu, et al., The impact of the CYP2C9 and VKORC1 polymorphisms on acenocoumarol dose requirements in a Romanian population, Blood Cells Mol. Diseases (2012), http://dx.doi.org/10.1016/j.bcmd.2012.10.010
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very important. The main enzymatic subfamily involved in metabolizing acenocoumarol is CYP2C9. The CYP2C9 polymorphisms identified about 10 years ago vary with race (Caucasian, African or Asian). In the Caucasian population the CYP2C9*1 allele is the most frequent, being considered the wild one. Its most frequent polymorphic variants are CYP2C9*2 (Arg 144-Cys) and CYP2C9*3 (Ile 359-Leu) present in 8–19% and 6–10% of the population respectively, having at least one mutant allele. These SNPs (single nucleotide polymorphisms), CYP2C9*2 and CYP2C9*3, have been associated with a decrease of the enzymatic activity and implicitly with an increase in acenocoumarol toxicity, leading to the need for a lower dose in the carriers of these alleles [4,5]. Acenocoumarol exerts its anticoagulant effect through competitive inhibition of epoxireductase, the enzyme responsible for the gammacarboxylation of the glutamic acid residues during the synthesis of II, VII, IX and X coagulation factors. Thus, the formation of the active form of K vitamin is inhibited, leading to the formation of proteins lacking anticoagulant biological activity (PIVKA). Additionally, oral anticoagulant inhibits the formation of C and S proteins, which have an anticoagulant role. The enzyme K-vitamin-epoxireductase is coded by the subunit 1 of the VKORC gene. The recent discovery of the VKORC1 gene polymorphisms represents a step forward in understanding the inter-individual variability of the necessary oral anticoagulant dose, and recent studies show that the presence of VKORC1 SNPs influences the body's reactivity to oral anticoagulant treatment. Patients with congenital VKORC1 deficits have coagulation disorders or resistance to oral anticoagulant treatment [6]. Twenty-eight polymorphisms of the VKORC1 gene have been described, of which three of the haplotypes are mainly responsible for the VKORC1 genetic variability. VKORC1*2 haplotype has been made responsible for most of the variability of the response to oral anticoagulant [7]. An increased response to acenocoumarol has been observed in healthy subjects carrying c.-1639G>A polymorphism, the hallmark of VKORC1*2 haplotype [8]. According to several authors [9–11], combined analysis of CYP2C9 and VKORC1 polymorphisms explains 30 to 40% of the inter-individual variability of the equilibrium oral anticoagulant dose and, consequently, the response to treatment and its safety. The study aims to investigate the association between the genetic status in patients treated with acenocoumarol and the phenotypic expression represented by the therapeutic acenocoumarol dose. It also aims to correlate the genetic factors with the clinical ones such as age, sex, and concomitant medication, in order to establish their influence on the acenocoumarol dose. Material and methods The study included 301 consecutive patients who required oral anticoagulant treatment with acenocoumarol, admitted within the Internal Medicine and Cardiology Departments of the Municipal Hospital of Cluj-Napoca and the Cardiology Department of the Heart Institute “Niculae Stăncioiu” in Cluj-Napoca between October 2008 and June 2011. Informed consent was obtained from all the participants prior to their enrolment in the study, including consent on genetic evaluations. The study protocol was submitted for approval to the Ethics Commission of “Iuliu Haţieganu” University of Medicine and Pharmacy in Cluj-Napoca. The following inclusion criteria were used: - age over 18 years - diagnosis of acute deep venous thrombosis of the lower limbs or pulmonary thrombo-embolism, persistent or permanent atrial fibrillation or heart valve prosthesis - no contraindications for anticoagulant treatment (heparin or acenocoumarol allergy, active hemorrhage, active peptic ulcer, ischemic stroke at inclusion or in the week prior to inclusion, hemorrhagic
stroke in the three months prior to inclusion, elevated blood pressure — systolic over 220 mm Hg, diastolic over 120 mm Hg, thrombocytopenia b 100,000/fl, antecedents of heparin-induced thrombocytopenia, and hepatic failure). Pregnant women, patients with a life expectancy of less than 1 year due to co-morbidities and patients who had not given their informed consent were excluded from the study. The following data was recorded for each patient: age, sex, indication for oral anticoagulant treatment, concomitant therapy potentially influencing the metabolization of oral anticoagulants by the enzymatic system of P450 cytochrome (amiodarone, statins, spironolactone, and proton pump inhibitors) and by the oral anticoagulant dose (expressed in mg/week) required to reach a stable target INR (between 2 and 3 for venous thrombosis and atrial fibrillation and between 2.5 and 3.5 for heart valve prostheses) at three consecutive determinations. Patients were divided in three groups according to the acenocoumarol dose needed to reach a stable INR: patients receiving a low dose (≤7 mg/week), patients receiving a medium dose (>7 mg and b28 mg/week) and patients receiving a high acenocoumarol dose(s) (>28 mg/week). The genetic analysis consisted of genotyping the CYP2C9 gene and the VKORC1 gene and assessment of the CYP2C9*2 and CYP2C9*3 polymorphisms. Genotyping for CYP2C9*2 and CYP2C9*3 was carried out using PCR-RFLP technique (polymerase chain reaction-restriction fragment length polymorphism) as described in 1999 by Aynacioglu et al. [12]. Genotyping for polymorphism VKORC1 c.-1639G>A was performed using PCR-RFLP technique as described by Wen et al. [13]. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS, ver. 17, Chicago, IL, USA). Deviations of allelic frequencies from Hardy–Weinberg equilibrium were computed using a Chi-square test. Quantitative variables were tested for normality of distribution using the Kolmogorov–Smirnov test. Descriptive analysis included frequencies for ordinal variables and the median for continuous variables. Differences in the median between groups were analyzed using the Mann–Whitney test or the Kruskal–Wallis test, whichever was appropriate. The correlation between continuous variables was assessed using Spearman's rho. A Chi-square test was used to compare categorical variables. Variables that achieved the criterion of significance at p b 0.25 in univariate analysis were included in multivariate analysis. A multinomial logistic regression was used to assess the influence of certain parameters (age, sex, and concomitant medication such as CYP2C9 inducers or inhibitors, CYP2C9 genotypes and c.-1639G>A polymorphism of the VKORC1) on the probability that a patient would receive a low, medium or a high dose of acenocoumarol. The level of statistical significance was set at p b 0.05. Results A total of 301 patients (146 men and 155 women) who had met the selection criteria were included in this study. The mean age was 64 years, and the median was 66 years. Age had a non-normal distribution (Kolmogorov–Smirnov test; p= 0.02). Acute deep vein thrombosis was the most frequent pathology encountered that required oral anticoagulation (62.1%). Among the medication that can interfere with acenocoumarol metabolization, statins were the most prescribed drugs (42.1%). The lowest acenocoumarol dose was 2 mg/week and the highest was 49 mg/week. The mean dose was 17.3 mg/week and the median was 16 mg/week. The weekly acenocoumarol dose was non-normally distributed. A highly significant negative correlation was found between age and the acenocoumarol weekly dose (Spearman's rho; r =−0.339; pb 0.001). The weekly acenocoumarol dose was not influenced by sex (Mann–Whitney test; p =0.06), by treatment with amiodarone (Mann–Whitney test; p= 0.75), proton pump inhibitors (Mann–Whitney test; p =0.9), spironolactone (Mann–Whitney test; p= 0.43) or statins (Mann–Whitney test; p =0.39).
Please cite this article as: A.D. Buzoianu, et al., The impact of the CYP2C9 and VKORC1 polymorphisms on acenocoumarol dose requirements in a Romanian population, Blood Cells Mol. Diseases (2012), http://dx.doi.org/10.1016/j.bcmd.2012.10.010
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The characteristics of the participants according to the weekly acenocoumarol therapeutic dose they received are summarized in Table 1. Distribution of CYP2C9 genotypes according to the acenocoumarol therapeutic dose groups is shown in Table 2. The CYP2C9*2 allele was found in 63 cases (20.2%) and the CYP2C9*3 was determined in 51 cases (16.9%). Allelic frequencies reached the Hardy–Weinberg equilibrium. The presence of CYP2C9*2 allele had not been associated with a specific acenocoumarol dose (Chi-square test; p =0.36). In turn, patients with CYP2C9*3 allele were significantly prone to receiving a lower dose of acenocoumarol (Chi-square test; p =0.03). The presence of any CYP2C9 (CYP2C9*2 +CYP2C9*3) allele was associated with the requirement of a low dose of acenocoumarol (Chi-square test; p =0.03). Distribution of genotypes for the c.-1639G>A polymorphism of the VKORC1 according to the acenocoumarol therapeutic dose groups is summarized in Table 3. The frequencies of the VKORC1 GG, VKORC1 GA and VKORC1 AA genotypes were 32.5%, 50.7%, and 16.5%. Allelic frequencies were in Hardy–Weinberg equilibrium. The presence of any of the A allele of c.-1639G>A polymorphism of VKORC1 increased the probability of a patient to be included in the low acenocoumarol dose group (Chi-square test; p b 0.001). In order to find out the independent influence of the parameters on acenocoumarol dose a model has been constructed using a multinomial logistic regression. The reference category was the medium dose of the acenocoumarol group. We dichotomized the age using a cut-off value of 65 years. The suggested model explained about 32% of the acenocoumarol dose variation. We determined that the presence of the CYP2C9*3 allele increases the odds of receiving a low dose of acenocoumarol (OR, 3.4; p =0.006; 95%CI: 1.41–8.34). The GA genotype of c.-1639G>A polymorphism of VKORC1 increased the odds of receiving a low dose of acenocoumarol (OR, 6.5; p =0.01; 95%CI: 1.38–30.5). The presence of AA genotype of c.-1639G>A polymorphism of VKORC1 further increased the odds of receiving a low acenocoumarol dose (OR, 11.6; p= 0.003; 95%CI: 2.26–59.58). Patients aged over 65 years were more likely to receive a low dose of acenocoumarol (OR, 3.2; p =0.01; 95%CI: 1.24–8.25). Elderly patients (>65 years) were less likely to receive a high acenocoumarol dose (OR, 0.24; p= 0.001; 95%CI: 0.1–0.56). Patients with the GA or AA genotype had low odds of being included in the high acenocoumarol group (OR, 0.2; pb 0.001; 95CI%: 0.09–0.45; respectively OR, 0.05; p =0.006; 95%CI: 0.007–0.43).
Discussion Alongside the progress of pharmacogenetics, the hypothesis that genetic variations could either influence drugs' toxicity or determine a suboptimal response to treatment has become more attractive.
Table 1 Characteristics of the study participants divided by acenocoumarol therapeutic dose. Variable
Patients (no, %) Age (years, median) Male (no, %) Female (no, %) Atrial fibrillation (no, %) Deep vein thrombosis (no, %) Prosthetic heart valves (no, %) Amiodarone (no, %) Proton pump inhibitor (no, %) Spironolactone (no, %) Statins (no, %) ⁎ Kruskal–Wallis test. ⁎⁎ Chi-square test.
Acenocoumarol dose
p
Low
Medium
High
31 (10.3%) 73 13 (4.3%) 18 (5.9%) 13 (4.3%) 20 (6.6%) 3 (0.9%) 1 (0.3%) 3 (0.9%) 6 (1.9%) 12 (3.9%)
231 (76.7%) 67 108 (35.8%) 123 (40.8%) 96 (31.8%) 140 (46.5%) 38 (12.6%) 11 (3.6%) 19 (6.3%) 50 (16.6%) 99 (32.8%)
39 (13%) 56 25 (8.3%) 14 (4.6%) 12 (3.3%) 27 (8.9%) 5 (1.6%) 0 2 (0.6%) 7 (2.3%) 16 (5.3%)
– b0.001⁎ 0.09⁎⁎ 0.43⁎⁎ 0.56⁎⁎ 0.55⁎⁎ 0.36⁎⁎ 0.75⁎⁎ 0.84⁎⁎ 0.36⁎⁎
3
Table 2 Genotype distribution of CYP2C9 variants. CYP2C9 genotype
*1/*1 *1/*2 *1/*3 *2/*3 *2/*2 *3/*3
(no, (no, (no, (no, (no, (no,
%) %) %) %) %) %)
Acenocoumarol dose Low
Medium
High
14 (4.6%) 5 (1.6%) 9 (2.9%) 2 (0.6%) 0 1 (0.3%)
152 (50%) 42 (13.9%) 28 (9.3%) 6 (1.9%) 2 (0.6%) 1 (0.3%)
29 (9.3%) 6 (1.9%) 4 (1.3%) 0 0 0
Although acenocoumarol is a frequently used drug, it remains a hard to maneuver one in daily practice. Because of its low therapeutic index and of the inter-individual variability of the therapeutic response, patients under such a treatment require regular medical supervision in order to reach the target INR. Failure to carefully monitor the patient may lead either to excessive anticoagulation with an increased risk for hemorrhagic events, or to the suboptimal anticoagulation, which could in turn lead to an increased stroke risk or to other thrombo-embolic events. The study aims to establish the link between the polymorphisms of the genes involved in the oral anticoagulant metabolism (CYP2C9), the gene VKORC1 (the oral anticoagulant target), and the anticoagulant doses required to maintain the target INR. In addition, the implications of age, sex and co-medication upon the acenocoumarol dose necessary to maintain a stable INR have been studied. The study of the CYP2C9 gene alleles showed a 20.2% frequency for the CYP2C9*2 allele, a result that is consistent with the data in the current literature on the frequency of this allele within the Caucasian population (8–20%) [9]. CYP2C9*3 allele was found in 16.9% of the patients, a value higher than the average reported frequency for this allele within the Caucasian population (6–10%) [9]. GG genotype of the polymorphism c.-1639G>A was present in 98 patients (32.5%). GA genotype was found in 153 patients (50.8%). Tatarunas et al. reported frequencies of 38.6%, 54.4% and 7.2% [14]. Montes et al. found GG genotype in 27.6% of the cases, GA in 40% and AA in 32.3% [15]. Sipeky et al. reported the following percentages in a Hungarian population: GG 35.3%, GA 51.4% and AA 13.3% [16]. The negative correlation between a patient's age and the acenocoumarol dose indicates that patients aged 65 or more have a statistically significant higher probability to receive a lower anticoagulant dose, which is consistent with the data from the relevant literature [17]. This inverse correlation is independent from variations in CYP2C9 and VKORC1 genotypes and from the use of CYP2C9-inducing or inhibiting medication. Patients aged over 65 were 3.2 times more likely to receive a dose of acenocoumarol lower than 7 mg/week, compared to patients younger than 65 years. Furthermore, the probability of older patients to receive an acenocoumarol dose higher than 28 mg/week was 76% lower than that of younger patients. Loebstein et al. initially thought that there were two major determinants of the maintenance dose once therapeutic INR has been reached: age over 65 and CYP2C9 genotype [17]. Montes et al. also find an inverse correlation between age and acenocoumarol dose [15].
Table 3 Distribution of genotypes for the c.-1639G>A polymorphism of the VKORC1. c.-1639G>A polymorphism of the VKORC1genotypes
Acenocoumarol dose Low
Medium
High
GG (no, %) GA (no, %) AA (no, %)
2 (0.6%) 19 (6.3%) 10 (3.3%)
69 (22.9%) 123 (40.8%) 39 (12.9%)
27 (8.9%) 11 (3.6%) 1 (0.3%)
Please cite this article as: A.D. Buzoianu, et al., The impact of the CYP2C9 and VKORC1 polymorphisms on acenocoumarol dose requirements in a Romanian population, Blood Cells Mol. Diseases (2012), http://dx.doi.org/10.1016/j.bcmd.2012.10.010
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Although several studies revealed that patients' gender influences the response variability to oral anticoagulant [17,18], our study does not show any significant differences regarding the link between patient gender and the efficient acenocoumarol dose. No statistically significant association has been found between comedication with drugs known to influence oral anticoagulant's metabolism (proton pump inhibitors, spironolactone, statins, and amiodarone) and the daily oral anticoagulant dose. The results of this study do not confirm the data from the literature regarding the influence of the treatment with certain classes of drugs on the acenocoumarol dose. Statins are known to influence the plasma concentration of the oral anticoagulant [19]. Despite an increased number of patients simultaneously receiving statins and acenocoumarol, statins did not influence the oral anticoagulant dose. Proton pump inhibitors (PPI) are drugs known to increase the oral anticoagulant effect. Because of the reduced number of patients treated simultaneously with oral anticoagulant and amiodarone or PPI (24 and 12 patients), no association has been found between these treatments and the oral anticoagulant dose. Apart from the above-mentioned factors (age, sex, and concomitant medication) the influence of genetic factors on the required oral anticoagulant dose for an effective anticoagulation has also been studied. The presence of CYP2C9*2 allele did not influence the assignment of patients to one of the three groups defined by the acenocoumarol dose. Importantly, none of the patients included in the study proved to be CYP2C9*2/*2 homozygous, all the patients presenting CYP2C9*2 allele being heterozygous. Literature results concerning the effect of CYP2C9*2 allele on the daily dose of acenocoumarol are not straightforward. Saraeva et al. [20] showed in a recent study on 96 patients that a high number of carriers of the CYP2C9*2 allele required low acenocoumarol doses, while other authors showed that the presence of the CYP2C9*2 allele has little or no influence at all on the acenocoumarol dose [21–24]. Unlike the CYP2C9*2 heterozygous, carriers of the CYP2C9*3 allele required low acenocoumarol doses, the results being consistent with the relevant literature [14,20]. The patients having the CYP2C9*3 allele were 3.4 times more likely to receive less than 7 mg acenocoumarol per week compared to those not having this allele. This was independent of the presence of other mutations. Studies published so far showed that the patients carrying the CYP2C9*3 allele require a low acenocoumarol dose to reach the anticoagulation target [21,24]. Another important determinant of the oral anticoagulant treatment response variability is represented by the VKORC1 gene polymorphisms. This is why the current study assessed c.-1639G>A polymorphism of the VKORC1 gene and its influence on the acenocoumarol dose. The research on the prevalence of the c.-1639G>A polymorphism of the VKORC1 showed a difference between the three subgroups. The carriers of the AA genotype of c.-1639G>A polymorphism of VKORC1 received lower doses of acenocoumarol. This genotype had the greater impact (OR — 11.6) on the probability of a patient receiving a low acenocoumarol dose. Patients having the AA genotype were 95% less likely to receive a higher acenocoumarol dose than 28 mg/week. The presence of a GA genotype increased by 6 fold the probability of an individual being assigned to the subgroup of patients receiving an acenocoumarol dose lower than 7 mg/week and also lowered the probability to be assigned to the subgroup of patients receiving high acenocoumarol doses by 80%. These results confirm the findings of previous research on the variability of the response to oral anticoagulant treatment [25]. Bodin et al. argued that the VKORC1 genotype accounts for 37% of this variability [8]. The results of this research are consistent with the results of some of the latest relevant literature in the field. Using a complex statistical analysis, Skov et al. showed that the major determinants of the oral anticoagulant dose are: VKORC1 and CYP2C9 gene polymorphisms and age [26]. This study was not a randomized trial. The genetic analysis did not account for mutations which generate resistance to acenocoumarol.
Conclusion Our study demonstrated that the stable therapeutic dose of acenocoumarol is dependent of patient's age, the presence of the CYP2C9*3 allele and c.-1639G>A polymorphism of VKORC1. Acenocoumarol dose was not influenced by the other medication received by patients or the presence of the CYP2C9*2 allele.
Acknowledgments This study was supported by research grant 42-127/2008 “Trombo-Gen”, from the National Center for Programs Management, Romanian Ministry of Education and Research. Contributors Anca D. Buzoianu designed the research, analyzed the data and revised the manuscript; Florentina C. Militaru analyzed the data and wrote the manuscript; Ştefan C. Vesa collected the samples, analyzed the data and wrote the manuscript; Adrian Trifa performed the genetic analyses, analyzed the data and wrote the manuscript; Sorin Crişan collected the samples and analyzed the data. Conflict of interest The authors confirm that there are no conflicts of interest. Disclosure All authors have approved the final form of this article.
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Please cite this article as: A.D. Buzoianu, et al., The impact of the CYP2C9 and VKORC1 polymorphisms on acenocoumarol dose requirements in a Romanian population, Blood Cells Mol. Diseases (2012), http://dx.doi.org/10.1016/j.bcmd.2012.10.010
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The impact of the CYP2C9 and VKORC1 polymorphisms on acenocoumarol dose requirements in a Romanian population Anca Dana Buzoianu a, Florentina Claudia Militaru a, Ştefan Cristian Vesa a,⁎, Adrian Pavel Trifa b, Sorin Crişan c a
Department of Clinical Pharmacology, “Iuliu Haţieganu” University of Medicine and Pharmacy, 6th Pasteur Street, 400349, Cluj-Napoca, Cluj, Romania Department of Medical Genetics, “Iuliu Haţieganu” University of Medicine and Pharmacy, 6th Pasteur Street, 400349, Cluj-Napoca, Cluj, Romania Department of Internal Medicine, 5th Medical Clinic, Municipal Hospital, “Iuliu Haţieganu” University of Medicine and Pharmacy, 11th Tăbăcarilor Street, 400139, Cluj-Napoca, Cluj, Romania
b c
a r t i c l e
i n f o
Article history: Submitted 9 October 2012 Available online xxxx (Communicated by J. Adamson, M.D., 30 October 2012) Keywords: Acenocoumarol Romanian population Genetic variability CYP2C9 VKORC1
a b s t r a c t Aim: To investigate the genotype–phenotype correlation in Romanian patients treated with acenocoumarol. Material and methods: We studied 301 consecutive patients who required treatment with acenocoumarol, admitted within the Municipal Hospital of Cluj-Napoca and the Heart Institute “Niculae Stănciou” in ClujNapoca over a 3-year period. For each patient we recorded clinical parameters which could interfere with the achievement of stable therapeutic international normalized ratio (INR). We performed genetic analysis which consisted of genotyping the CYP2C9 gene and the VKORC1 gene. Patients were divided in three groups according to the acenocoumarol dose needed to reach a stable INR: the low dose group (≤7 mg/week), the medium dose group (>7 mg and b 28 mg/week) and the high acenocoumarol dose group (>28 mg/week). Results: We found that patients' age was significantly different between groups (pb 0.001). No differences existed between groups regarding the pathologies which required anticoagulation therapy or the concomitant treatment. The following parameters increased the odds of receiving a low dose of acenocoumarol: patient's age over 65 years (OR, 3.2; p=0.01; 95%CI: 1.24–8.25), the presence of the CYP2C9*3 allele (OR, 3.4; p =0.006; 95%CI: 1.41–8.34), and the GA or AA genotype of c.-1639G>A polymorphism of VKORC1 (OR, 6.5; p =0.01; 95%CI: 1.38–30.5; respectively OR, 11.6; p =0.003; 95%CI: 2.26–59.58). A high acenocoumarol dose was less likely to be administered to an elderly patient (OR, 0.24; p= 0.001; 95%CI: 0.1–0.56) or to a patient with the GA or AA genotype (OR, 0.2; p b 0.001; 95CI%: 0.09–0.45; respectively OR, 0.05; p=0.006; 95%CI: 0.007–0.43). Conclusion: The stable therapeutic dose of acenocoumarol is dependent of patient's age, the presence of the CYP2C9*3 allele and the c.-1639G>A polymorphism of VKORC1. © 2012 Elsevier Inc. All rights reserved.
Introduction Acenocoumarol is the most widely used oral anticoagulant in Romania and other European countries, indicated for the treatment and prophylaxis of thromboembolic diseases. Anticoagulant treatment is always a challenge for clinicians because of the difficulties to match an appropriate dose. An insufficient dose will lead to a failure to prevent thrombosis, while an overdose will significantly increase the hemorrhagic risk, acenocoumarol being a low therapeutic index drug [1]. The most frequent complications of acenocoumarol treatment are hemorrhagic accidents, including cerebral and digestive hemorrhage, which are the most severe. ⁎ Corresponding author. E-mail addresses: [email protected] (A.D. Buzoianu), [email protected] (F.C. Militaru), [email protected] (Ş.C. Vesa), [email protected] (A.P. Trifa), [email protected] (S. Crişan).
Apart from its toxicity oral anticoagulant treatment is characterized by a great inter-individual variability regarding its efficacy. Treatment monitoring is usually done by INR determination, which has to be kept between specific limits, according to the pathology approached. Variability of the efficacy is manifested through differences in the dose needed to maintain the INR within the therapeutic range and it is determined in a multi-factorial way that involves both genetic and environmental factors. For a long time environmental factors were considered as the main cause for the variations in oral anticoagulant treatment response. These factors include: patient's characteristics (age, sex, and body mass index), vitamin K food intake, co-morbidities (hepatic failure, severe renal failure, heart failure, thyroid diseases, etc.), acute diseases (fever, sepsis, decompensated heart failure, diarrhea, etc.) and concomitant treatments [2,3]. After uncovering the role of the genetic polymorphisms in altering the function of certain specific proteins, the variability of the response to oral anticoagulant treatment was partly attributed to genetic factors. Among these, the CYP P450 system metabolizing enzymes, highly polymorphic in humans, are
1079-9796/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcmd.2012.10.010
Please cite this article as: A.D. Buzoianu, et al., The impact of the CYP2C9 and VKORC1 polymorphisms on acenocoumarol dose requirements in a Romanian population, Blood Cells Mol. Diseases (2012), http://dx.doi.org/10.1016/j.bcmd.2012.10.010
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very important. The main enzymatic subfamily involved in metabolizing acenocoumarol is CYP2C9. The CYP2C9 polymorphisms identified about 10 years ago vary with race (Caucasian, African or Asian). In the Caucasian population the CYP2C9*1 allele is the most frequent, being considered the wild one. Its most frequent polymorphic variants are CYP2C9*2 (Arg 144-Cys) and CYP2C9*3 (Ile 359-Leu) present in 8–19% and 6–10% of the population respectively, having at least one mutant allele. These SNPs (single nucleotide polymorphisms), CYP2C9*2 and CYP2C9*3, have been associated with a decrease of the enzymatic activity and implicitly with an increase in acenocoumarol toxicity, leading to the need for a lower dose in the carriers of these alleles [4,5]. Acenocoumarol exerts its anticoagulant effect through competitive inhibition of epoxireductase, the enzyme responsible for the gammacarboxylation of the glutamic acid residues during the synthesis of II, VII, IX and X coagulation factors. Thus, the formation of the active form of K vitamin is inhibited, leading to the formation of proteins lacking anticoagulant biological activity (PIVKA). Additionally, oral anticoagulant inhibits the formation of C and S proteins, which have an anticoagulant role. The enzyme K-vitamin-epoxireductase is coded by the subunit 1 of the VKORC gene. The recent discovery of the VKORC1 gene polymorphisms represents a step forward in understanding the inter-individual variability of the necessary oral anticoagulant dose, and recent studies show that the presence of VKORC1 SNPs influences the body's reactivity to oral anticoagulant treatment. Patients with congenital VKORC1 deficits have coagulation disorders or resistance to oral anticoagulant treatment [6]. Twenty-eight polymorphisms of the VKORC1 gene have been described, of which three of the haplotypes are mainly responsible for the VKORC1 genetic variability. VKORC1*2 haplotype has been made responsible for most of the variability of the response to oral anticoagulant [7]. An increased response to acenocoumarol has been observed in healthy subjects carrying c.-1639G>A polymorphism, the hallmark of VKORC1*2 haplotype [8]. According to several authors [9–11], combined analysis of CYP2C9 and VKORC1 polymorphisms explains 30 to 40% of the inter-individual variability of the equilibrium oral anticoagulant dose and, consequently, the response to treatment and its safety. The study aims to investigate the association between the genetic status in patients treated with acenocoumarol and the phenotypic expression represented by the therapeutic acenocoumarol dose. It also aims to correlate the genetic factors with the clinical ones such as age, sex, and concomitant medication, in order to establish their influence on the acenocoumarol dose. Material and methods The study included 301 consecutive patients who required oral anticoagulant treatment with acenocoumarol, admitted within the Internal Medicine and Cardiology Departments of the Municipal Hospital of Cluj-Napoca and the Cardiology Department of the Heart Institute “Niculae Stăncioiu” in Cluj-Napoca between October 2008 and June 2011. Informed consent was obtained from all the participants prior to their enrolment in the study, including consent on genetic evaluations. The study protocol was submitted for approval to the Ethics Commission of “Iuliu Haţieganu” University of Medicine and Pharmacy in Cluj-Napoca. The following inclusion criteria were used: - age over 18 years - diagnosis of acute deep venous thrombosis of the lower limbs or pulmonary thrombo-embolism, persistent or permanent atrial fibrillation or heart valve prosthesis - no contraindications for anticoagulant treatment (heparin or acenocoumarol allergy, active hemorrhage, active peptic ulcer, ischemic stroke at inclusion or in the week prior to inclusion, hemorrhagic
stroke in the three months prior to inclusion, elevated blood pressure — systolic over 220 mm Hg, diastolic over 120 mm Hg, thrombocytopenia b 100,000/fl, antecedents of heparin-induced thrombocytopenia, and hepatic failure). Pregnant women, patients with a life expectancy of less than 1 year due to co-morbidities and patients who had not given their informed consent were excluded from the study. The following data was recorded for each patient: age, sex, indication for oral anticoagulant treatment, concomitant therapy potentially influencing the metabolization of oral anticoagulants by the enzymatic system of P450 cytochrome (amiodarone, statins, spironolactone, and proton pump inhibitors) and by the oral anticoagulant dose (expressed in mg/week) required to reach a stable target INR (between 2 and 3 for venous thrombosis and atrial fibrillation and between 2.5 and 3.5 for heart valve prostheses) at three consecutive determinations. Patients were divided in three groups according to the acenocoumarol dose needed to reach a stable INR: patients receiving a low dose (≤7 mg/week), patients receiving a medium dose (>7 mg and b28 mg/week) and patients receiving a high acenocoumarol dose(s) (>28 mg/week). The genetic analysis consisted of genotyping the CYP2C9 gene and the VKORC1 gene and assessment of the CYP2C9*2 and CYP2C9*3 polymorphisms. Genotyping for CYP2C9*2 and CYP2C9*3 was carried out using PCR-RFLP technique (polymerase chain reaction-restriction fragment length polymorphism) as described in 1999 by Aynacioglu et al. [12]. Genotyping for polymorphism VKORC1 c.-1639G>A was performed using PCR-RFLP technique as described by Wen et al. [13]. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS, ver. 17, Chicago, IL, USA). Deviations of allelic frequencies from Hardy–Weinberg equilibrium were computed using a Chi-square test. Quantitative variables were tested for normality of distribution using the Kolmogorov–Smirnov test. Descriptive analysis included frequencies for ordinal variables and the median for continuous variables. Differences in the median between groups were analyzed using the Mann–Whitney test or the Kruskal–Wallis test, whichever was appropriate. The correlation between continuous variables was assessed using Spearman's rho. A Chi-square test was used to compare categorical variables. Variables that achieved the criterion of significance at p b 0.25 in univariate analysis were included in multivariate analysis. A multinomial logistic regression was used to assess the influence of certain parameters (age, sex, and concomitant medication such as CYP2C9 inducers or inhibitors, CYP2C9 genotypes and c.-1639G>A polymorphism of the VKORC1) on the probability that a patient would receive a low, medium or a high dose of acenocoumarol. The level of statistical significance was set at p b 0.05. Results A total of 301 patients (146 men and 155 women) who had met the selection criteria were included in this study. The mean age was 64 years, and the median was 66 years. Age had a non-normal distribution (Kolmogorov–Smirnov test; p= 0.02). Acute deep vein thrombosis was the most frequent pathology encountered that required oral anticoagulation (62.1%). Among the medication that can interfere with acenocoumarol metabolization, statins were the most prescribed drugs (42.1%). The lowest acenocoumarol dose was 2 mg/week and the highest was 49 mg/week. The mean dose was 17.3 mg/week and the median was 16 mg/week. The weekly acenocoumarol dose was non-normally distributed. A highly significant negative correlation was found between age and the acenocoumarol weekly dose (Spearman's rho; r =−0.339; pb 0.001). The weekly acenocoumarol dose was not influenced by sex (Mann–Whitney test; p =0.06), by treatment with amiodarone (Mann–Whitney test; p= 0.75), proton pump inhibitors (Mann–Whitney test; p =0.9), spironolactone (Mann–Whitney test; p= 0.43) or statins (Mann–Whitney test; p =0.39).
Please cite this article as: A.D. Buzoianu, et al., The impact of the CYP2C9 and VKORC1 polymorphisms on acenocoumarol dose requirements in a Romanian population, Blood Cells Mol. Diseases (2012), http://dx.doi.org/10.1016/j.bcmd.2012.10.010
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The characteristics of the participants according to the weekly acenocoumarol therapeutic dose they received are summarized in Table 1. Distribution of CYP2C9 genotypes according to the acenocoumarol therapeutic dose groups is shown in Table 2. The CYP2C9*2 allele was found in 63 cases (20.2%) and the CYP2C9*3 was determined in 51 cases (16.9%). Allelic frequencies reached the Hardy–Weinberg equilibrium. The presence of CYP2C9*2 allele had not been associated with a specific acenocoumarol dose (Chi-square test; p =0.36). In turn, patients with CYP2C9*3 allele were significantly prone to receiving a lower dose of acenocoumarol (Chi-square test; p =0.03). The presence of any CYP2C9 (CYP2C9*2 +CYP2C9*3) allele was associated with the requirement of a low dose of acenocoumarol (Chi-square test; p =0.03). Distribution of genotypes for the c.-1639G>A polymorphism of the VKORC1 according to the acenocoumarol therapeutic dose groups is summarized in Table 3. The frequencies of the VKORC1 GG, VKORC1 GA and VKORC1 AA genotypes were 32.5%, 50.7%, and 16.5%. Allelic frequencies were in Hardy–Weinberg equilibrium. The presence of any of the A allele of c.-1639G>A polymorphism of VKORC1 increased the probability of a patient to be included in the low acenocoumarol dose group (Chi-square test; p b 0.001). In order to find out the independent influence of the parameters on acenocoumarol dose a model has been constructed using a multinomial logistic regression. The reference category was the medium dose of the acenocoumarol group. We dichotomized the age using a cut-off value of 65 years. The suggested model explained about 32% of the acenocoumarol dose variation. We determined that the presence of the CYP2C9*3 allele increases the odds of receiving a low dose of acenocoumarol (OR, 3.4; p =0.006; 95%CI: 1.41–8.34). The GA genotype of c.-1639G>A polymorphism of VKORC1 increased the odds of receiving a low dose of acenocoumarol (OR, 6.5; p =0.01; 95%CI: 1.38–30.5). The presence of AA genotype of c.-1639G>A polymorphism of VKORC1 further increased the odds of receiving a low acenocoumarol dose (OR, 11.6; p= 0.003; 95%CI: 2.26–59.58). Patients aged over 65 years were more likely to receive a low dose of acenocoumarol (OR, 3.2; p =0.01; 95%CI: 1.24–8.25). Elderly patients (>65 years) were less likely to receive a high acenocoumarol dose (OR, 0.24; p= 0.001; 95%CI: 0.1–0.56). Patients with the GA or AA genotype had low odds of being included in the high acenocoumarol group (OR, 0.2; pb 0.001; 95CI%: 0.09–0.45; respectively OR, 0.05; p =0.006; 95%CI: 0.007–0.43).
Discussion Alongside the progress of pharmacogenetics, the hypothesis that genetic variations could either influence drugs' toxicity or determine a suboptimal response to treatment has become more attractive.
Table 1 Characteristics of the study participants divided by acenocoumarol therapeutic dose. Variable
Patients (no, %) Age (years, median) Male (no, %) Female (no, %) Atrial fibrillation (no, %) Deep vein thrombosis (no, %) Prosthetic heart valves (no, %) Amiodarone (no, %) Proton pump inhibitor (no, %) Spironolactone (no, %) Statins (no, %) ⁎ Kruskal–Wallis test. ⁎⁎ Chi-square test.
Acenocoumarol dose
p
Low
Medium
High
31 (10.3%) 73 13 (4.3%) 18 (5.9%) 13 (4.3%) 20 (6.6%) 3 (0.9%) 1 (0.3%) 3 (0.9%) 6 (1.9%) 12 (3.9%)
231 (76.7%) 67 108 (35.8%) 123 (40.8%) 96 (31.8%) 140 (46.5%) 38 (12.6%) 11 (3.6%) 19 (6.3%) 50 (16.6%) 99 (32.8%)
39 (13%) 56 25 (8.3%) 14 (4.6%) 12 (3.3%) 27 (8.9%) 5 (1.6%) 0 2 (0.6%) 7 (2.3%) 16 (5.3%)
– b0.001⁎ 0.09⁎⁎ 0.43⁎⁎ 0.56⁎⁎ 0.55⁎⁎ 0.36⁎⁎ 0.75⁎⁎ 0.84⁎⁎ 0.36⁎⁎
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Table 2 Genotype distribution of CYP2C9 variants. CYP2C9 genotype
*1/*1 *1/*2 *1/*3 *2/*3 *2/*2 *3/*3
(no, (no, (no, (no, (no, (no,
%) %) %) %) %) %)
Acenocoumarol dose Low
Medium
High
14 (4.6%) 5 (1.6%) 9 (2.9%) 2 (0.6%) 0 1 (0.3%)
152 (50%) 42 (13.9%) 28 (9.3%) 6 (1.9%) 2 (0.6%) 1 (0.3%)
29 (9.3%) 6 (1.9%) 4 (1.3%) 0 0 0
Although acenocoumarol is a frequently used drug, it remains a hard to maneuver one in daily practice. Because of its low therapeutic index and of the inter-individual variability of the therapeutic response, patients under such a treatment require regular medical supervision in order to reach the target INR. Failure to carefully monitor the patient may lead either to excessive anticoagulation with an increased risk for hemorrhagic events, or to the suboptimal anticoagulation, which could in turn lead to an increased stroke risk or to other thrombo-embolic events. The study aims to establish the link between the polymorphisms of the genes involved in the oral anticoagulant metabolism (CYP2C9), the gene VKORC1 (the oral anticoagulant target), and the anticoagulant doses required to maintain the target INR. In addition, the implications of age, sex and co-medication upon the acenocoumarol dose necessary to maintain a stable INR have been studied. The study of the CYP2C9 gene alleles showed a 20.2% frequency for the CYP2C9*2 allele, a result that is consistent with the data in the current literature on the frequency of this allele within the Caucasian population (8–20%) [9]. CYP2C9*3 allele was found in 16.9% of the patients, a value higher than the average reported frequency for this allele within the Caucasian population (6–10%) [9]. GG genotype of the polymorphism c.-1639G>A was present in 98 patients (32.5%). GA genotype was found in 153 patients (50.8%). Tatarunas et al. reported frequencies of 38.6%, 54.4% and 7.2% [14]. Montes et al. found GG genotype in 27.6% of the cases, GA in 40% and AA in 32.3% [15]. Sipeky et al. reported the following percentages in a Hungarian population: GG 35.3%, GA 51.4% and AA 13.3% [16]. The negative correlation between a patient's age and the acenocoumarol dose indicates that patients aged 65 or more have a statistically significant higher probability to receive a lower anticoagulant dose, which is consistent with the data from the relevant literature [17]. This inverse correlation is independent from variations in CYP2C9 and VKORC1 genotypes and from the use of CYP2C9-inducing or inhibiting medication. Patients aged over 65 were 3.2 times more likely to receive a dose of acenocoumarol lower than 7 mg/week, compared to patients younger than 65 years. Furthermore, the probability of older patients to receive an acenocoumarol dose higher than 28 mg/week was 76% lower than that of younger patients. Loebstein et al. initially thought that there were two major determinants of the maintenance dose once therapeutic INR has been reached: age over 65 and CYP2C9 genotype [17]. Montes et al. also find an inverse correlation between age and acenocoumarol dose [15].
Table 3 Distribution of genotypes for the c.-1639G>A polymorphism of the VKORC1. c.-1639G>A polymorphism of the VKORC1genotypes
Acenocoumarol dose Low
Medium
High
GG (no, %) GA (no, %) AA (no, %)
2 (0.6%) 19 (6.3%) 10 (3.3%)
69 (22.9%) 123 (40.8%) 39 (12.9%)
27 (8.9%) 11 (3.6%) 1 (0.3%)
Please cite this article as: A.D. Buzoianu, et al., The impact of the CYP2C9 and VKORC1 polymorphisms on acenocoumarol dose requirements in a Romanian population, Blood Cells Mol. Diseases (2012), http://dx.doi.org/10.1016/j.bcmd.2012.10.010
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Although several studies revealed that patients' gender influences the response variability to oral anticoagulant [17,18], our study does not show any significant differences regarding the link between patient gender and the efficient acenocoumarol dose. No statistically significant association has been found between comedication with drugs known to influence oral anticoagulant's metabolism (proton pump inhibitors, spironolactone, statins, and amiodarone) and the daily oral anticoagulant dose. The results of this study do not confirm the data from the literature regarding the influence of the treatment with certain classes of drugs on the acenocoumarol dose. Statins are known to influence the plasma concentration of the oral anticoagulant [19]. Despite an increased number of patients simultaneously receiving statins and acenocoumarol, statins did not influence the oral anticoagulant dose. Proton pump inhibitors (PPI) are drugs known to increase the oral anticoagulant effect. Because of the reduced number of patients treated simultaneously with oral anticoagulant and amiodarone or PPI (24 and 12 patients), no association has been found between these treatments and the oral anticoagulant dose. Apart from the above-mentioned factors (age, sex, and concomitant medication) the influence of genetic factors on the required oral anticoagulant dose for an effective anticoagulation has also been studied. The presence of CYP2C9*2 allele did not influence the assignment of patients to one of the three groups defined by the acenocoumarol dose. Importantly, none of the patients included in the study proved to be CYP2C9*2/*2 homozygous, all the patients presenting CYP2C9*2 allele being heterozygous. Literature results concerning the effect of CYP2C9*2 allele on the daily dose of acenocoumarol are not straightforward. Saraeva et al. [20] showed in a recent study on 96 patients that a high number of carriers of the CYP2C9*2 allele required low acenocoumarol doses, while other authors showed that the presence of the CYP2C9*2 allele has little or no influence at all on the acenocoumarol dose [21–24]. Unlike the CYP2C9*2 heterozygous, carriers of the CYP2C9*3 allele required low acenocoumarol doses, the results being consistent with the relevant literature [14,20]. The patients having the CYP2C9*3 allele were 3.4 times more likely to receive less than 7 mg acenocoumarol per week compared to those not having this allele. This was independent of the presence of other mutations. Studies published so far showed that the patients carrying the CYP2C9*3 allele require a low acenocoumarol dose to reach the anticoagulation target [21,24]. Another important determinant of the oral anticoagulant treatment response variability is represented by the VKORC1 gene polymorphisms. This is why the current study assessed c.-1639G>A polymorphism of the VKORC1 gene and its influence on the acenocoumarol dose. The research on the prevalence of the c.-1639G>A polymorphism of the VKORC1 showed a difference between the three subgroups. The carriers of the AA genotype of c.-1639G>A polymorphism of VKORC1 received lower doses of acenocoumarol. This genotype had the greater impact (OR — 11.6) on the probability of a patient receiving a low acenocoumarol dose. Patients having the AA genotype were 95% less likely to receive a higher acenocoumarol dose than 28 mg/week. The presence of a GA genotype increased by 6 fold the probability of an individual being assigned to the subgroup of patients receiving an acenocoumarol dose lower than 7 mg/week and also lowered the probability to be assigned to the subgroup of patients receiving high acenocoumarol doses by 80%. These results confirm the findings of previous research on the variability of the response to oral anticoagulant treatment [25]. Bodin et al. argued that the VKORC1 genotype accounts for 37% of this variability [8]. The results of this research are consistent with the results of some of the latest relevant literature in the field. Using a complex statistical analysis, Skov et al. showed that the major determinants of the oral anticoagulant dose are: VKORC1 and CYP2C9 gene polymorphisms and age [26]. This study was not a randomized trial. The genetic analysis did not account for mutations which generate resistance to acenocoumarol.
Conclusion Our study demonstrated that the stable therapeutic dose of acenocoumarol is dependent of patient's age, the presence of the CYP2C9*3 allele and c.-1639G>A polymorphism of VKORC1. Acenocoumarol dose was not influenced by the other medication received by patients or the presence of the CYP2C9*2 allele.
Acknowledgments This study was supported by research grant 42-127/2008 “Trombo-Gen”, from the National Center for Programs Management, Romanian Ministry of Education and Research. Contributors Anca D. Buzoianu designed the research, analyzed the data and revised the manuscript; Florentina C. Militaru analyzed the data and wrote the manuscript; Ştefan C. Vesa collected the samples, analyzed the data and wrote the manuscript; Adrian Trifa performed the genetic analyses, analyzed the data and wrote the manuscript; Sorin Crişan collected the samples and analyzed the data. Conflict of interest The authors confirm that there are no conflicts of interest. Disclosure All authors have approved the final form of this article.
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Please cite this article as: A.D. Buzoianu, et al., The impact of the CYP2C9 and VKORC1 polymorphisms on acenocoumarol dose requirements in a Romanian population, Blood Cells Mol. Diseases (2012), http://dx.doi.org/10.1016/j.bcmd.2012.10.010