Abstract
Aims
To investigate the influence of APOE genotypes and VKORC1 haplotypes on warfarin dose requirements in Asian patients.
Methods
A total of 174 Asian patients (Chinese, n = 96; Malays, n = 50; Indians, n = 28) who had stable daily warfarin doses for at least 1 month were recruited. Following genomic DNA extraction from venous blood, pharmacogenetic analysis of APOE and VKORC1 genes was done by DNA sequencing.
Results
The majority of the Asian patients (78%) harboured the APOE ε3/ε3 genotype. Different APOE genotypes were found not to have any significant influence on mean daily warfarin dose requirements. Warfarin dose requirements in the pooled Asian patients homozygous for the VKORC1 H1 haplotype were significantly lower compared with patients homozygous for the H7 haplotype (H1-H1 vs. H7-H7: 2.79 ± 1.06 mg day−1vs. 5.45 ± 2.3 mg day−1, P < 0.001).
Conclusions
The present study suggests that APOE variants have minimal impact on warfarin dose requirements in Asian patients, probably due to the low frequency of ε4 allele containing genotypes.
What is already known about this subject
Recent studies on pharmacogenetics of warfarin have implicated apolipoproteinE (APOE) polymorphisms to influence the vitamin K dependent coagulation cascade and hence the efficacy of warfarin.
Studies among Caucasian and African Americans showed a significant but conflicting role of apolipoproteinE (APOE) isoforms in warfarin pharmacogenetics.
The contribution of APOE isoforms in influencing variations in warfarin requirements in Asian subjects remains to be investigated.
What this study adds
This is the first report of a population study in Asians exploring the role of isoforms encoded by three APOE alleles (ε2, ε3, ε4) in influencing warfarin dose requirements.
The present study showed that the APOE ε3/ε3 isoform is the predominant genotype in the Asian population.
The study also showed that APOE isoforms may not be important in affecting warfarin pharmacodynamics in Asian patients. It also suggested that the impact of different APOE isoforms depended on the frequency of APOE genotypes in the population, in particular the ε4 allele containing genotypes.
Keywords: apolipoproteinE, Asian, VKORC1 haplotypes, warfarin dose
Introduction
Warfarin is a commonly prescribed oral anticoagulant with wide interindividual variations in therapeutic response. Risk of haemorrhage frequently complicates warfarin therapy and occurs at a rate of 7.6–16.5 per 100 patients per year [1]. Warfarin is administered as a racemate of R- and S-enantiomers with S-warfarin being 3–5 fold more active than R-warfarin and metabolized mainly by the cytochrome P450 2C9 (CYP2C9) enzyme [2]. The CYP2C9 gene is polymorphic and the reference CYP2C9*1 allele yields a more active enzyme than the CYP2C9*2 and *3 alleles. Warfarin exerts its pharmacodynamic effects by inhibiting VKORC1 (vitamin K epoxide reductase complex 1), a multicomponent lipid-protein enzyme system [3]. Major warfarin dose related haplotypes [H1(CCATTG), H7(TCGTCA) and H9(TAGTCG)] have been assigned based on the rs7196161, rs17880887, rs9923231, rs2884737, rs9934438 and rs17880624 polymorphisms in the VKORC1 gene [4].
Recent pharmacogenetic studies of warfarin have implicated apolipoproteinE (APOE) polymorphisms to influence the vitamin K dependent coagulation cascade and hence the efficacy of warfarin [5, 6]. Plasma vitamin K concentrations are dependent on the APOE receptor mediated clearance of chylomicron particles and their remnants where it serves as a ligand mediating the uptake of vitamin K into hepatocytes [7]. APOE is a glycoprotein consisting of 299 amino acids encoded by the human APOE gene on chromosome 19q13.2 which is 3.7 kb and consists of 4 exons. Two nucleotide variations on exon 4 in the APOE gene give rise to three isoforms encoded by three APOE alleles, namely, ε2 (Cys112/Cys158), ε3 (Cys112/Arg158) and ε4 (Arg112/Arg158) [8]. The receptor affinities of the proteins encoded by the three allelic isoforms of APOE towards chylomicron remnants varies in the order ε2 < ε3 < ε4[9]. Further investigations to elucidate the relationships between the different APOE genotypes, vitamin K concnetrations and warfarin dose requirements showed that patients homozygous for the APOE ε4 allele required higher warfarin doses and that the allele accounted for 6% of the variance in warfarin dose requirements among the CYP2C9 extensive metabolizers. However, Visser et al.[10] reported that patients homozygous for the APOE ε2 allele required higher doses of acenocoumarol than their counterparts harbouring the APOE ε3/ε4 or ε4/ε4, implying that rapid uptake of vitamin K among subjects with an APOE ε2 allele could increase its hepatic availability, raising warfarin requirements.
The objective of the present study was to determine the influence of APOE ε2, ε3 and ε4 allelic isoforms on warfarin dose requirements in Asian patients of known VKORC1 genotypes. To the best of our knowledge, this is the first study exploring the relation of APOE pharmacogenetics to warfarin dose requirements in Asians.
Methods
Study subjects and genotyping
A total of 174 Asians comprising of 96 Chinese, 50 Malays and 28 Indians were enrolled from the Anticoagulation Clinic at the Singapore General Hospital and the National University Hospital, Singapore. Malay and Indian subjects were also enrolled in the study by Lee et al.[11]. Ethnicity was assigned by verification against National Registry Identification Cards (NRIC). All patients were required to have their daily warfarin doses stable for at least 1 month and stabilized INR values (range 2–3) within the desired therapeutic range over the same duration. The indications for anticoagulant therapy were mainly for prevention or treatment of thromboembolic diseases such as atrial fibrillation, deep vein thrombosis or prosthetic valve replacement. Patients with congestive heart failure (NYHA class 3 or greater), liver cirrhosis or thyroid disease and patients on medications known to interact with warfarin were excluded from the study. All patients gave written informed consent to participate and the protocol was approved by the institution ethics committee.
Genomic DNA was extracted from venous blood using the phenol-chloroform method and APOE genotypes were determined by PCR (F: 5′-TCAAGGTTGCAGTGAACCATGTTCAGGC-3′, R: 5′-AGAGCTAGGGAAGGACAGA GACAGAGC-3′) and fragments analyzed by direct DNA sequencing (Beckman Coulter Inc., CA, USA) with 5′-TGATGGACGAGACCATGAAGGAGTTG-3′ as the sequencing primer. Genotyping for the VKORC1 polymorphisms was also performed in all the subjects using the technique previously described [11]. VKORC1 haplotypes were assigned according to Rieder et al.[4].
Statistical analysis
The chi-squared and Fisher's exact test was used to analyze allele and genotype frequencies. Individual haplotypes were derived using the expectation maximization algorithm. The nonparametric Kruskal–Wallis test was used to measure the variability in warfarin dose requirements in relation to APOE genotype and VKORC1 haplotype pairs. For the analysis of covariance, covariates included APOE and VKORC1 genotype status, age, weight, height, gender, ethnic group status and the INR. Bonferroni correction was applied for multiple testings. All statistical analyses including estimation of confidence intervals were done using Stata (STATA statistical software release 7.0, USA).
Results
The median age of the patients was 58 years (range 29–87 years). Univariate and multivariate analysis showed that age (P < 0.001), ethnicity (Chinese, P = 0.017; Malays, P < 0.001; Indians, P < 0.001) and VKORC1 haplotype (H1, P < 0.001; H7, P < 0.001) correlated significantly with daily warfarin dose requirements. Patients older than 60 years of age required significantly lower daily doses of warfarin (n = 82; 3.08 ± 1.49 mg day−1) compared with younger patients (n = 92; 4.71 ± 2.18 mg day−1; P < 0.0001). The daily warfarin doses were significantly different among the three ethnic groups (P = 0.0001) and were significantly lower among Chinese (3.68 ± 1.68 mg day−1) and Malays (3.28 ± 1.39 mg day−1) than in Indians (6.21 ± 2.94 mg day−1, P < 0.001 in each case).
The APOE genotype frequencies were found to be in Hardy–Weinberg equilibrium (Table 1) and the allele frequencies were comparable with previous reports [12]. There were no patients carrying the ε2/ε2 or the ε2/ε4 genotype whereas the ε4/ε4 genotype was observed only in the Chinese subjects (1%). The daily warfarin doses in patients harbouring different APOE genotypes were in the similar range and pairwise analysis showed no significant difference (P > 0.05 in each case) (Table 2). Analysis of patients based on ethnicity as well as stratification by warfarin doses (greater than or less than 5 mg day−1) did not reveal any significant influence of the APOE genotypes on warfarin dose requirements.
Table 1.
APOE genotype and allele frequencies and VKORC1 haplotypes in Asian patients on warfarin therapy
| APOE genotype frequencies, n (%) | APOE allele frequencies (95% CI) | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Populations | n | ε2/ε2 | ε2/ε3 | ε3/ε3 | ε3/ε4 | ε4/ε4 | ε2 | ε3 | ε4 |
| Chinese | 96 | 0 | 16 (16.7) | 67 (69.8) | 12 (12.5) | 1 (1.1) | 0.08 (0.04, 0.12) | 0.84 (0.79, 0.89) | 0.07 (0.03, 0.11) |
| Malays | 50 | 0 | 4 (8.0) | 42 (84.0) | 4 (8.0) | 0 | 0.04 (0.001, 0.08) | 0.92 (0.87, 0.97) | 0.04 (0.001, 0.08) |
| Indians | 28 | 0 | 1 (3.6) | 26 (92.9) | 1 (3.6) | 0 | 0.02 (−0.02, 0.06) | 0.96 (0.91, 1.01) | 0.02 (−0.02, 0.06) |
| Pooled Asians | 174 | 0 | 21 (12.1) | 135 (78.2) | 17 (9.8) | 1 (0.6) | 0.06 (0.04, 0.08) | 0.89 (0.86, 0.92) | 0.04 (0.02, 0.06) |
| VKORC1 haplotype frequencies (%) | VKORC1 haplotype pairs, n | |||||||
|---|---|---|---|---|---|---|---|---|
| n | H1 (CCATTG) | H7 (TCGTCA) | H9 (TAGTCG) | H1-H1 | H1-H9 | H7-H7 | H7-H9 | |
| Chinese | 96 | 86.4 | 12.5 | 1.1 | 82 | 1 | 13 | – |
| Malays | 50 | 80.0 | 20.0 | 0 | 40 | – | 10 | – |
| Indians | 28 | 7.1 | 85.7 | 7.2 | 2 | – | 24 | 2 |
| Pooled Asians | 174 | 71.8 | 26.4 | 1.8 | 124 | 1 | 47 | 2 |
n, number of subjects.
Table 2.
APOE genotypes and VKORC1 haplotype pairs in relation to daily warfarin dose requirements (mean ± SD) in Asian patients
| Warfarin dose (mg day−1; mean ± SD) | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Gene | Genotype/ Haplotype pairs | n | Pooled Asian | n | Chinese | n | Malays | n | Indians |
| APOE | ε2/ε2 | 0 | NA | 0 | NA | 0 | NA | 0 | NA |
| ε2/ε3 | 21 | 3.26 ± 1.21 | 16 | 3.29 ± 1.40 | 4 | 2.82 ± 1.08 | 1 | 5 | |
| ε3/ε3 | 135 | 4.06 ± 2.15 | 67 | 3.71 ± 1.68 | 42 | 3.34 ± 1.44 | 26 | 6.13 ± 2.9 | |
| ε3/ε4 | 17 | 3.91 ± 1.88 | 12 | 4.02 ± 2.10 | 4 | 3.06 ± 1.19 | 1 | 6 | |
| ε4/ε4 | 1 | 3.75 | 1 | 3.75 | 0 | NA | 0 | NA | |
| VKORC1 | H1-H1 | 124 | 2.79 ± 1.06 | 82 | 2.82 ± 1.05 | 40 | 2.92 ± 1.11 | 2 | 1.75 ± 0.50 |
| H1-H9 | 1 | 7.5 | 1 | 7.5 | – | – | – | – | |
| H7-H7 | 47 | 5.45 ± 2.3 | 13 | 5.71 ± 1.40 | 10 | 4.71 ± 1.39 | 24 | 6.41 ± 2.53 | |
| H7-H9 | 1 | 3 | – | – | – | – | 1 | 3 | |
| 1 | 9 | – | – | – | – | 1 | 9 | ||
n, number of subjects; NA, not appropriate.
The majority of the patients carried the low-dose (H1; 71.8%) VKORC1 haplotype, followed by the high-dose (H7; 26.4% and H9; 1.8%) haplotypes (Table 1). The warfarin dose requirements in the pooled Asian patients homozygous for the H1 haplotype were significantly lower compared with patients homozygous for the H7 haplotype (H1-H1 vs. H7-H7: 2.79 ± 1.06 mg day−1vs. 5.45 ± 2.3 mg day−1, P < 0.001) (Table 2). The warfarin dose requirements were also significantly lower among subjects homozygous for the H1 haplotype compared with patients homozygous for the H7 haplotype when examined in individual ethnic groups (P < 0.02 in each case).
Discussion
The majority of the Asian patients in the present study carried the ε3/ε3 genotype (78.2%), which is associated with intermediate vitamin K clearance. The ε4/ε4 genotype which is associated with faster clearance of vitamin K-containing chylomicrons was detected in only one patient, suggesting minimal phenotypic impact of this rare isoform on warfarin dosage requirement in Asians. Similar to the study by Visser et al.[10], a recent study by Sconce et al.[13] showed a small (2%) but significant impact of the ε4 allele on low dose warfarin requirement in Caucasians. This genotypic-phenotypic effect, however, was absent in the Italian population, probably due to the low frequency of the ε4 allele in this population [14]. In another report, the presence of the ε4 allele associated significantly with higher warfarin dose requirements among African-Americans but not among Caucasians, who incidentally had a lower frequency of the ε4 allele [15]. Taken collectively, these studies suggest that the impact of different APOE isoforms depend on the frequency of APOE genotypes in the population, in particular the ε4 allele containing genotypes which range from 37.8% in African Americans to 3.6% among Indians in the present study [15]. The influence of APOE genotypes may also vary with different coumarin anticoagulants such as acenocoumarol and phenprocoumon which have different pharmacokinetic profiles compared with warfarin [16].
Recent studies suggest that the VKORC1 haplotypes are a major determinant of warfarin dose requirements in patients of different ethnic background [4]. Similar to previous reports among Asians [17], this study showed that subjects harbouring the H7 haplotypes tend to require higher doses of warfarin in comparison with the H1 haplotype, independent of individual ethnic status. The contributions of CYP2C9 genetic polymorphisms have been reported to have minimal impact on warfarin dosing in Asian patients as the frequency of the low-activity CYP2C9*2 and *3 alleles is low in Asian ethnic groups [18].
In conclusion, the present study suggests that the reported influence of APOE variants on warfarin dose requirements may not be important in affecting warfarin pharmacodynamics in Asian patients, probably due to the low frequency of the ε4 allele containing genotypes.
Acknowledgments
The authors would like to acknowledge the Singapore General Hospital Research Fund (SRF #96/03) for funding this study.
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