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VKORC1 and CYP2C9 allelic variants influence acenocoumarol dose requirements in Greek patients Aim: To identify the frequencies of the polymorphisms CYP2C9*2, CYP2C9*3 and VKORC1-1639 G>A in the Greek population and investigate whether these polymorphisms and patient demographics (age, sex and comedication) could explain the interindividual variability of acenocoumarol dose requirements for efficient anticoagulation. Materials & methods: CYP2C9*2 (Arg144Cys), CYP2C9*3 (Ile359Leu) and VKORC1–1639G>A allelic variants were analyzed in 98 patients treated with acenocoumarol. Results: Allelic frequencies of CYP2C9*2, CYP2C9*3 and VKORC1A were found to be 0.155, 0.075 and 0.485, respectively. Carriership of at least one CYP2C9*3 allele led to the most pronounced reduction in the required mean dose (p A VKORC1 in the Greek population and investigate whether these polymorphisms and patient demographics (age, sex and comedication) could explain the interindividual variability of acenocoumarol dose. Materials & methods
Participating subjects A retrospective cohort study was conducted in 98 patients receiving chronic oral anticoagulant treatment (acenocoumarol). All the patients signed an informed consent form and fulfiled specific inclusion criteria: Greek origin, a complete history of acenocoumarol exposure, international normalized ratio (INR) within therapeutic range (2.0–3.0) and stable for at least 4 weeks, anti coagulant treatment for ≥2 months and absence of any underlying disease that influences drug metabolism, such as malignancy or hepatic insufficiency. The study was approved by the Ethics Committee of ‘ATTIKON’ University Hospital. Genotyping Genomic DNA was isolated from peripheral blood (2 ml in a ethylenediaminetetraacetic [EDTA] vacutainer) by a QIAamp blood Midi Kit (Qiagen Inc., CA, USA) according to the principles provided from the manufacturer. PCR was performed for genotyping CYP2C9*2, *3 and VKORC1 ‑1639 G>A variants alleles. Cycling procedures applied for all the polymorphisms consisted of 35 cycles at 94˚C for 1 min (denaturation), 59˚C for 1 min (annealing) and 72˚C for 1 min (extension). CYP2C9*2 and VKORC1-1639 G>A polymorphisms were detected using restriction endonucleases (Ava II and NciI, respectively), while the CYP2C9*3 variant allele was genotyped by direct sequencing (Applied Biosystems [ABI], CA, USA; 3730 xl). Table 1 summarizes the SNP positions, primers and amplicons sizes.
Statistical analysis Descriptive statistics, nonparametric tests (Mann–Whitney and Kruskall–Wallis), correlation coefficients (Spearman analysis) and regression multivariable analysis were performed using SPSS software (version 13.0, SPSS, IL, USA). In all cases, 5% was defined as the statistical significant limit. Results Patient characteristics (demographical, clinical and genetic) are summarized in Table 2 .
Allele frequencies Among the 98 patients of the study group, CYP2C9 allele frequencies were estimated to be 0.77 for the wild-type (wt) 2C9*1, 0.155 for 2C9*2 and 0.075 for 2C9*3 variant allele. VKORC1 -1639G>A allele frequencies were 0.515 and 0.485 for G and A alleles, respectively. Allele frequencies in both genes followed Hardy–Weinberg equilibrium. Patients were stratified in terms of acenocoumarol dose. Those receiving less than 1 mg/day were classified as low dosers (n = 7), moderate, more than 1 mg/day and 4 mg/day or less (n = 74) and high dosers, more than 4 mg/day (n = 17). Table 3 shows the distribution of CYP2C9 and VKORC1 genotypes among the dosing groups. Impact of CYP2C9 polymorphisms on acenocoumarol dose requirements The mean maintenance acenocoumarol dose was clearly related to CYP2C9 haplotype (F igur e 1) . Wild-type patients required 2.91 mg (±1.32), while *1/*3 and *2/*3 compound heterozygotes required 1.73 mg (± 1.02) and 1.28 mg (± 0.57), respectively (Kruskall– Wallis test, p = 0.004). Carriers of CYP2C9*2 allele required a lower acenocoumarol dosage (2.51 ± 1.51 mg) than the wild-type patients (2.91 ± 1.32 mg) to maintain the target INR, but this difference was not statistically significant (Mann–Whitney test, p = 0.3). By contrast, the carriership of at least one CYP2C9*3 allele reduced the mean acenocoumarol dose (1.64 ± 0.95 mg) in a statistically significant way (Mann–Whitney test, p T) 416 5’-GGA-GGA-TGG-AAA-ACA-GAG-ACT-TA-3’ 5’-TGA-GCT-AAC-AAC-CAG-GAC-TCA-T-3’ CYP2C9*3 (A>C) 1075 5’-CTC-CTT-TTC-CAT-CAG-TTT-TTA-CT-3’ 5’-ACA-CAC-ACT-GCC-AGA-CAC-TAG-G-3’ VKORC1 (G>A) 3673 (-1639) 5’-ATC-CCT-CTG-GGA-AGT-CAA-GC-3’ 5’-CAC-CTT-CAA-CCT-CTC-CAT-CC-3’
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VKORC1 & CYP2C9 allelic variants influence acenocoumarol dose requirements
Influence of the VKORC1 -1639A allele on acenocoumarol dose requirements The presence of the VKORC1-1639G>A poly morphism affected the acenocoumarol dose regime. Wild-type patients needed higher amounts (3.54 ± 1.48 mg) in comparison with heterozygous G/A (2.85 ± 1.17 mg) and especially homozygous A/A (1.3 ± 0.34 mg) patients (Kruskall–Wallis test, p A genotype
Patients (%)
G/G G/A A/A
26.53 50 23.47
DVT: Deep venous thrombosis; PE: Pulmonary embolism.
including patient age, CYP2C9*2, *3 and VKORC1 A carriership. The partial R² for age, CYP2C9*2, CYP2C9*3 and VKORC1 A were estimated as 0.08, 0.05, 0.13 and 0.135, respectively. Discussion Acenocoumarol exerts a significant anticoagulant effect on patients with thrombotic disorders. Due to the high interindividual variability of the dose response and its narrow therapeutic range, Table 3. Distribution of CYP2C9 and VKORC1 genotypes among the dosing groups. Genotype
Dosing groups Low (≤1 mg/day) n=7
Median (>1 and ≤4 mg/day) n = 74
High (>4 mg/day) n = 17
2 0 4 0 1
44 20 7 1 2
11 5 1 0 0
0 0 7
16 42 16
10 7 0
CYP2C9 1/1 1/2 1/3 2/2 2/3 VKORC1 G/G G/A A/A
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8.00
mdose (mg)
2.91 6.00 2.51
1.73
4.00
1.28
2.00
0.00 1/1 n = 57
1/2 n = 25
1/3 n = 12
2/2 n=1
2/3 n=3
CYP2C9 genotype
Figure 1. Boxplots exhibiting the association of CYP2C9 genotype and acenocoumarol dose. Boxes show the median (black box) and interquartile range and contain 25–75 percentiles, while circles indicate outliers. The mean value is noted above each boxplot.
serious adverse outcomes are often observed. Until now, empirical determination of dosage was the unique choice for treatment. After the recent clarification of coumarin pharmaco genetics, several clinical dosing algorithms have been proposed, although not yet established, depending on individual hereditary, environmental and clinical factors [19] . CYP2C9 and VKORC1 polymorphisms are considered to be the principle hereditary
mdose (mg)
8.00 6.00 4.00 2.00 0
3.54
2.85
G/G n = 26
G/A n = 49 VKORC1 GEN
1.30 A/A n = 23
Figure 2. Boxplots showing the association of VKORC1-1639G>A genotype and acenocoumarol dose. Boxes show the median (black box) and interquartile range and contain 25–75 percentiles, while circles indicate outliers. The mean value is noted above each boxplot.
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determinants. Our study, which is the first on acenocoumarol pharmacogenomics in Greeks, showed that the frequencies of VKORC1 A, CYP2C9*2 and CYP2C9*3 allele (48.5, 15.5 and 7.5%, respectively) are within the expected range which is already reported for the Caucasians (32–50%, 8–20% and 6–11%) [3,15,20] . Our results regarding the CYP2C9*2 and CYP2C9*3 alleles frequencies are consistent with that already reported for the Greek population (12.9 and 8.1%, respectively) [21] . VKORC1 has been proven to be the major congenital influence factor of the high inter individual variability in coumarin dose adjustment. Oldenburg et al. found that SNPs located at the coding region of VKORC1 led to coumarin resistance, while those in the untranslated region to coumarin sensitivity [16] . One of these polymorphisms is the ‑1639 G>A in the flanking region of this gene. Our data reinforce this conclusion, as VKORC1 A allele presence leads undoubtedly to a reduced dosage. In patients receiving low dose (7 mg/day) the frequency was only 0.42 (7/17). Acenocoumarol is mainly metabolized by the CYP2C9 isoform of cytochrome P450. Carriers of CYP2C9 variants *2 and *3 are poor metabolizers, and therefore coumarin sensitive [10,22] . CYP2C9*3 allele has a significant impact on acenocoumarol sensitivity in Greek individuals, as five of the seven low-dose patients (71.4%) were CYP2C9*3 carriers. By contrast, the CYP2C9*2 variant was detected in only one of the low dosers, indicating its minor role. As it is already known, the impact of CYP2C9*2 allele is less significant on acenocoumarol dose than on warfarin dose determination [23] . Pharmacokinetic data may provide an explanation, since S‑warfarin is thoroughly metabolized by CYP2C9, while R‑acenocoumarol is partially metabolized by CYP2C9 (50%) [9] . From genotyping of the patients receiving acenocoumarol in high dosage (>7 mg/day) no-one was found to be either a VKORC1 AA or CYP2C9*3 carrier. It is also essential to correlate the impact of both VKORC1 and CYP2C9 SNPs on acenocoumarol dose variability. Patients carrying both VKORC1‑1639 A/A and CYP2C9*3 received the lowest maintenance dose (0.94 mg/day) of the cohort, while wt patients (VKORC1 GG and CYP2C9*1) required the highest dose (3.67 mg/day). From investigating the importance of CYP2C9*3 allele separately, it was found that patients wt future science group
VKORC1 & CYP2C9 allelic variants influence acenocoumarol dose requirements
90.00
74.6
62
48.8
Low
33 34 85 Moderate
High
80.00 Age (years)
for both VKORC1 and CYP2C9 received a significantly higher maintenance dose (3.67 mg/ day) than wt VKORC1 and CYP2C9*3 carriers (2.45 mg/day). During the anticoagulant treatment, bleeding complications were observed in two patients. A male aged 48 years presented with subconjuctival hemorrhage, and a female aged 63 years with nose bleeding. The genetic backgrounds of these patients were CYP2C9*1/*3, VKORC1-1639 A /A and CY P2C9*1/ *1, VKORC1-1639 G/A, respectively. In both cases, the bleeding event was detected far from the induction phase of acenocoumarol therapy (5.5 and 22 months, respectively). Among the nongenetic factors, patient sex, age, statin and azole treatment were associated with acenocoumarol dose [20] . Patient sex was not related to the dosage regime. By contrast, patients age was correlated to acenocoumarol dose. Older patients received a statistically significant lower acenocoumarol dose. Hepatic
Research Article
70.00 60.00 50.00 40.00 30.00 20.00
Acenocoumarol dose
Figure 3. Boxplots showing the association between patient age and acenocoumarol dose. Boxes show the median (black box) and interquartile range and contain 25–75 percentiles, while circles indicate outliers. The mean value is noted above each boxplot.
insufficiency and concurrent medication in older patients could lead to inadequate acenocoumarol metabolism by the liver microsomes. GENCYP
1/1
1/2
1/3
2/3
5.00
4.05 4.00
Mean mdose (mg)
3.67
3.04
3.01
3.00
2.51
1.92
2.00
1.56
1.52
1.36
1.12 1.00
0.71
0.00 G/G n = 14
G/A n = 34
VKORC1
A/A n=9
G/G n=7
G/A n = 12
A/A n=6
VKORC1
G/G n=3
G/A n=2
VKORC1
A/A n=7
G/G n=2
G/A
A/A n=1
VKORC1
Figure 4. Bar charts showing the relationship between CYP2C9 genotypes, VKORC1 genotypes and acenocoumarol dose. Mean values are noted above each column.
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Box 1. Acenocoumarol dosing algorithm.
Dose = anti log 71.083 - 0.004 x age - 0.188 x VKORC1 genotype - 0.073 x CYP2C9 genotype A
VKORC1 genotype is 1 for G/G, 2 for G/A, 3 for A/A and CYP2C9 genotype is 1 for CYP2C9 1/1, 2 for CYP2C9 1/2, 3 for CYP2C9 1/3, 4 for CYP2C9 2/2 and 5 for CYP2C9 2/3.
Furthermore, exposure to statins and azoles, which are considered to decrease CYP2C9 activity [20] , was not found to affect acenocoumarol dose requirements in our cohort patients. A possible explanation might be that statins and azoles are weak CYP2C9 inhibitors, and the CYP2C9*2 variant allele has a minor role in acenocoumarol metabolism. After the correlation of each factor with acenocoumarol dose separately, we constructed a regression multivariable model, in order to investigate the interactions of the studied factors and their impact on the dosage regime. In the model, CYP2C9 and VKORC1-1639 G/A genotypes provided a significant 49% explanation of the dose variability, which reached 55% when age was incorporated in the model. Sex, statin and azole comedication were excluded from the model. Therefore, the genetic influence of CYP2C9 and VKORC1 could explain 49% in acenocoumarol dose variability, a finding similar to the one observed in Caucasian patients treated with warfarin [22] . This is very interesting as, although warfarin and acenocoumarol have the same pharmacodynamics (VKORC1 is the molecular target of both coumarins), they do not share the exact pharmacokinetic properties (R‑acenocoumarol is partially metabolized by CYP2C9). Potential limitations of our study, such as its retrospective character, the small sample size of cases and their heterogeneity concerning age and cause of anticoagulation should be considered.
Nonetheless, our findings clearly suggest that the VKORC1 genotype produces the highest single contribution to acenocoumarol interindividual dose variability (R² = 0.4). This is in agreement with findings from other studies, which have demonstrated that the larger part of variability in acenocoumarol dose requirements is explained by the VKORC1-1639 G>A genotype [3,6,23] . Furthermore, our results confirm those of Hermida et al. that the effect of CYP2C9*2 on acenocoumarol requirements is much smaller than the effect observed by the CYP2C9*3 variant allele (R² = 0.05 and 0.13, respectively, in our study) [4] . Conclusion In conclusion, among the parameters that we investigated in our study, age, CYP2C9 genotype, and, mainly, VKORC1-1639 G>A genotype were found to play a major role in the interindividual variability of acenocoumarol dose requirements in Greek. Nevertheless, a significant 45% of the dose variability still remains unexplained. The aforementioned determinants must be co-estimated with other nongenetic and hereditary factors (SNPs in clotting factors and in the vitamin K cycle) to accomplish a safer and more effective anticoagulant outcome. Future perspective It is important to co-estimate the polymorphisms mentioned above with others of genes encoding for several proteins such as GGCX, calumenin,
Table 4. Association of CYP2C9 and VKORC1 genotypes and demographics with acenocoumarol maintenance dose (dependent variable). Variable
Standardized coefficient
All Sex Age Statins‡ Azoles CYP2C9 genotype
0.052 -0.350** -0.151 -0.016 -0.319**
VKORC1 genotype CYP2C9 and VKORC1 genotype
-0.625
**
R²
p-value
0.55
0.12
