warfarin has an established role in secondary prevention of atherothrombotic disease , reducing new thromboembolic events . however , warfarin dosage is a challenge to clinicians because of its narrow therapeutic range and patient variability in obtained inr levels . an important issue is to improve individual warfarin dose prediction , especially in the initial phase of treatment , in order to avoid thrombosis and treatment - induced bleeding . an increasing number of genetic variations affecting warfarin pharmacokinetics and/or pharmacodynamics have recently been reported to have major impact on dosage requirements , that is , polymorphisms in the cyp2c9 , vkorc1 , and cyp4f2 genes     [ 27 ] . in caucasians , the vkorc1 gene is represented by three main haplotypes : vkorc1 * 2 ( rs9934438/6484 c  >  t ) , vkorc1 * 3 ( rs7294/9041 g  >  a ) , and vkorc1 * 4 ( rs17708472/6009 c  >  t )     . previously we have found vkorc1 * 2 to account for 24.5% , and the combination of cyp2c9 * 2 and cyp2c9 * 3 ( rs1799853 and rs1057910 ) to account for 7.2% of the warfarin maintenance dose variation in patients from the warfarin aspirin reinfarction study ( waris - ii ) . in the present investigation , we aimed to study whether the high - dose polymorphisms vkorc1 * 3 , vkorc1 * 4 , and cyp4f2 ( rs2108622/1347 c  >  t ) were associated with elevation in warfarin maintenance dose requirements in the same patient group . the patients in this study were a subgroup of the waris - ii trial who had been assigned to three different antithrombotic regimens after acute myocardial infarction     . the patients were randomly assigned to treatment with either a daily dose of 160  mg aspirin , warfarin ( target inr 2.84.2 ) , or 75  mg of aspirin combined with warfarin ( target inr 2.02.5 ) and followed for 4 years . blood samples for genotyping were available for 212 patients from oslo university hospital , ullevl . of the 140 patients on warfarin treatment , from both treatment groups combined , 105 patients were informative for weekly warfarin maintenance dose and used for genetic association analyses ( warfarin total group , table 1 ) . the vkorc1 * 3 ( rs7294/9041 g  >  a ) polymorphism was pcr amplified on a lightcycler ( roche.com ) real - time instrument under standard conditions essentially as reported   with an annealing temperature of 60c . forward and reverse primers were 5'-gagccttgcctaaggg and 5'-tggaaagagctttggagac , respectively , whereas the lc red640-labelled sensor probe and the fluorescence - labelled anchor probe were 5'-tgtgcgggtatggcagga and 5'-cgtgtggcacatttggtccattgt , respectively . cyp4f2 ( rs2108622/1347 c  >  t ) and vkorc1 * 4 ( rs17708472 ) were genotyped by taqman assays on abi 7900 ht fast pcr system ( applied biosystems , ca , usa ) . t ) assay were commercially available at abi ( cat no c_16179493_40 ) , whereas primers and probes for vkorc1 * 4 ( rs17708472/6009 c  >  t ) were custom made by abi ( forward primer : 5'gcccggcccttaagtaattctt , reverse primer : 5'tcccagtctctgatgcaaaacc , wild - type probe : vic - tgaaccgttatactagccttg , mutant probe : fam - ccgttataccagccttg ) . genotyping of vkorc1 * 2 ( rs9934438/6484 c  >  t ) , cyp2c9 * 2 ( rs1799853 ) , and cyp2c9 * 3 ( rs1057910 ) was done previously . the mann - whitney u - test was used to test the difference in warfarin dosage between two groups . multivariate regression analyses were performed to evaluate the individual impact of vkorc1 * 3 , * 4 , and cyp4f2 ( 1347 c  > t ) polymorphisms on mean warfarin maintenance dose , after adjustment for the covariates age and treatment group . the significance level of p  .05 was set for entry into the final model . genotype distributions of the vkorc1 * 2 , vkorc1 * 3 , vkorc1 * 4 , and cyp4f2 ( 1347 c  >  t ) polymorphisms in 212 patients from the oslo subset of the waris - ii trial are summarized in table 1 . none of the single nucleotide polymorphisms ( snps ) deviated from the hardy - weinberg equilibrium . combined with our previous waris - ii data , three polymorphisms in the vkorc1 gene have been analysed representing three distinct haplotypes : vkorc1 * 2 , vkorc1 * 3 , and vkorc1 * 4 . the vkorc1 * 1 haplotype was found in one patient only ( table 1 ) . boxplot of mean weekly warfarin dose for combinations of different vkorc1 haplotypes is shown ( figure 1 ) . patients homozygous for the vkorc1 * 2 haplotype had significantly lower mean warfarin maintenance dose compared to patients homozygous for either vkorc1 * 3 or * 4 haplotypes ( p < .001 and p = .003 , resp . , mann - whitney ) , whereas there was no significant difference in warfarin dosage between homozygotes of vkorc1 * 3 and * 4 ( p = .80 , mann - whitney ) . heterozygotes with combinations of vkorc1 * 2 and vkorc1 * 3 or * 4 haplotypes had significantly lowered maintenance dose compared to heterozygotes of vkorc1 * 3 and * 4 ( p = .002 and p = .023 , resp . , mann - whitney ) . there was a slight increase in mean weekly warfarin dose in patients who were heterozygous ( ct ) , but not for patients who were homozygous ( tt ) for the cyp4f2 ( 1347 c  > t ) t allele compared to patients with the normal cc - genotype ( p = .14 and p = .51 , resp . , mann - whitney ) , indicating a lack of trend between the three cyp4f2 genotypes ( figure 2 ) . in three separate multivariate regression analyses , vkorc1 * 3 and vkorc1 * 4 were associated with higher dose requirements when adjusted for age and treatment group only ( p = .001 and p = .004 , resp . ) , while cyp4f2 ( 1347 c  > t ) showed a weak association on higher warfarin dose requirement ( p = .09 ) . however , a stepwise multivariate regression analysis of vkorc1 * 3 , * 4 , and cyp4f2 ( 1347 c  > t ) together with our earlier reported data on vkorc1 * 2 , cyp2c9 * 2 , and cyp2c9 * 3 , age and treatment group gave no significant associations for neither vkorc1 * 3 , * 4 nor cyp4f2 ( 1347 c  > in our earlier study of 212 patients with myocardial infarction ( the waris - ii study ) , up to 32% of the variability in warfarin dose requirements could be explained by the vkorc1 * 2 , cyp2c9 * 2 , and cyp2c9 * 3 polymorphisms . this is in accordance with results by wadelius et al .     , where 201 swedish patients with mixed indications ( mainly atrial fibrillation and heart valve prosthesis ) were studied . whereas all these three snps contributed to reduced warfarin dosage requirements , the vkorc1 * 3 and * 4 haplotypes and recently the cyp4f2 ( rs2108622/1347 c  >  t)-polymorphism have been associated with increased dose requirement     [ 26 ] . carriers of any of these six genetic variants may be at increased risk of being outside the narrow therapeutic range for inr , especially during the initial phase of the anticoagulation treatment . the combined effect of different genetic variants upon warfarin dose requirement may therefore be difficult to foresee and needed to be clarified by further studies . the frequencies of the three vkorc1 haplotypes ( * 2 , * 3 , and * 4 ) in this study were in concordance with previous reports     [ 8 , 1214 ] . , we found that all carriers of the vkorc1 * 2 haplotype needed lower doses of warfarin compared to vkorc1 * 3 and * 4 carriers , irrespective of homozygosity or heterozygosity of the * 2 allele . in separate multivariate regression analysis , vkorc1 * 3 and vkorc1 * 4 were associated with higher dose requirements , however , inclusion of both vkorc1 * 3 and * 4 into our previously described regression model , did not explain more of the variability in warfarin dose requirements , possibly because the vkorc1 activities associated with vkorc1 * 3 and vkorc1 * 4 were similar . since the contribution of differentiating between * 3 and * 4 ( high dose variants ) to warfarin dosage was negligible compared to the considerable contribution of   * 2 ( low dose variant ) , genotyping for vkorc1 * 2 instead of vkorc1 * 2 , * 3 , and * 4 may be sufficient for routine purposes in caucasians . t ) polymorphism in our study were similar to results from others     [ 3 , 15 ] . this cyp4f2 variant , first discovered by caldwell and coauthors     , has been found to explain from 1.5%7% of warfarin dosing variability     [ 3 , 1517 ] . in our study of 105 patients we had 80% power to detect significance for cyp4f2 explaining 7% of the dose variance and 24% power to detect significance explaining a modest dosing variability of 1.5% . the lack of association between the cyp4f2 ( 1347 c  > t ) polymorphism and warfarin dosage in this study indicates that the impact of this polymorphism on warfarin dosage is likely to be small in caucasians . however , four of the seven patients who were homozygous for the cyp4f2 tt - genotype were carriers of either vkorc1 * 2 or cyp2c9 * 3 ( 2 were homozygous for vkorc1 * 2 and 2 were heterozygous for both vkorc1 * 2 and cyp2c9 * 3 ) which may have confounded the cyp4f2 effect . our findings based on myocardial infarction patients confirm that among the hitherto known genetic variants suspected to influence warfarin maintenance dosage , vkorc1 * 2 , vkorc1 * 3 , and vkorc1 * 4 are of major importance in addition to cyp2c9 variants , whereas the cyp4f2 ( 1347 c  > t ) polymorphism in caucasians is likely to be of relatively little importance . since the low dose variant vkorc1 * 2 in particular , as well as the cyp2c9 * 2 and cyp2c9 * 3 variants explain up to 32% of the variability , these variant alleles would probably be sufficient comprehensive genetic markers in caucasians for individual warfarin dose prediction in clinical routine . in order to make a more complete survey of the genetic background for enzymes affecting individual warfarin pharmacokinetics and/or pharmacodynamics , populations with higher carrier frequency of the vkorc*1 haplotype ( ethnic africans ) would probably also benefit from a more detailed genotyping program to elucidate individual warfarin dosage requirements .
the aim of this study was to investigate whether the vkorc1 * 3 ( rs7294/9041 g  >  a ) , vkorc1 * 4 ( rs17708472/6009 c  >  t ) , and cyp4f2 ( rs2108622/1347 c  >  t ) polymorphisms were associated with elevated warfarin maintenance dose requirements in patients with myocardial infarction ( n = 105 ) from the warfarin aspirin reinfarction study ( waris - ii ) . 
 we found significant associations between elevated warfarin dose requirements and vkorc1 * 3 and vkorc1 * 4 polymorphisms ( p = .001 and p = .004 , resp . ) , whereas cyp4f2 ( 1347 c  > 
 t ) showed a weak association on higher warfarin dose requirements ( p = .09 ) . 
 however , analysing these variant alleles in a regression analysis together with our previously reported data on vkorc1 * 2 , cyp2c9 * 2 and cyp2c9 * 3 polymorphisms , gave no significant associations for neither vkorc1 * 3 , vkorc1 * 4 nor cyp4f2 ( 1347 c  > 
 t ) . in conclusion , in patients with myocardial infarction , the individual contribution to warfarin dose requirements from vkorc1 * 3 , vkorc1 * 4 , and cyp4f2 ( 1347 c  > 
 t ) polymorphisms was negligible . 
 our results indicate that pharmacogenetic testing for vkorc1 * 2 , cyp2c9 * 2 and cyp2c9 * 3 is more informative regarding warfarin dose requirements than testing for vkorc1 * 3 , vkorc1 * 4 , and cyp4f2 ( 1347 c  > 
 t ) polymorphisms .