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Pharmacogenetics of warfarin: current status and future challenges

Abstract

Warfarin is an anticoagulant that is difficult to use because of the wide variation in dose required to achieve a therapeutic effect, and the risk of serious bleeding. Warfarin acts by interfering with the recycling of vitamin K in the liver, which leads to reduced activation of several clotting factors. Thirty genes that may be involved in the biotransformation and mode of action of warfarin are discussed in this review. The most important genes affecting the pharmacokinetic and pharmacodynamic parameters of warfarin are CYP2C9 (cytochrome P450 2C9) and VKORC1 (vitamin K epoxide reductase complex subunit 1). These two genes, together with environmental factors, partly explain the interindividual variation in warfarin dose requirements. Large ongoing studies of genes involved in the actions of warfarin, together with prospective assessment of environmental factors, will undoubtedly increase the capacity to accurately predict warfarin dose. Implementation of pre-prescription genotyping and individualized warfarin therapy represents an opportunity to minimize the risk of haemorrhage without compromising effectiveness.

Introduction

Warfarin is a widely used coumarin anticoagulant prescribed for patients with venous thrombosis and pulmonary embolism, chronic atrial fibrillation and prosthetic heart valves. Interindividual differences in drug response, a narrow therapeutic range and the risk of bleeding, all make warfarin a difficult drug to use clinically. Warfarin dose requirements, the stability of anticoagulation and risk of bleeding are influenced by environmental factors such as the intake of vitamin K, illness, age, gender, concurrent medication and body surface area, and by genetic variation.1, 2, 3, 4, 5, 6, 7, 8 To be able to improve the benefit–harm profile associated with warfarin therapy, all these factors need to be taken into account.

There is increasing interest in whether pharmacogenetics can accurately predict warfarin dose. There have been some recent advances in this area, but much more work needs to be done: at least 30 genes may be involved in the mode of action of warfarin (Table1 and Figure 1). Within these genes, there are thousands of publicly available single nucleotide polymorphisms (SNPs) with unknown function. There are also over 100 genetic variants that are known to change protein function that can be identified through a systematic search of the literature. Well-known examples are polymorphisms that change cytochrome P450 enzyme activity, apolipoprotein E (APOE) variants, factor V Leiden and other mutations in the coagulation system that cause either a bleeding tendency or increase the risk of thrombosis. In this review, we will analyse the different pathways involved in warfarin's action and critically evaluate the likelihood of whether genetic variation in these pathways may truly impact on the safety and ease of use of warfarin in clinical practice.

Table 1 Genes involved in the mechanism of action of warfarin
Figure 1
figure1

An overview of warfarin interactive pathways. This figure illustrates the genes thought to be involved in the action and biotransformation of warfarin and vitamin K.

Variability in the effect of warfarin

The efficacy of warfarin and other vitamin K antagonists in preventing and treating thrombosis has been well demonstrated in numerous randomized controlled trials and meta-analyses.9, 10 The efficacy and safety is, however, contingent on maintaining the anticoagulation within a clinically acceptable ‘therapeutic range’. This may be easier to achieve within the confines of a randomized controlled trial than during everyday real-world clinical practice.

Warfarin has a narrow therapeutic index and thus the dose required to achieve therapeutic anticoagulation is very close to the dose that leads to over-anticoagulation. Furthermore, the maintenance dose varies between different individuals, and ranges from 0.5 mg/day to more than 10 mg/day. This unpredictability leads to difficulties in maintaining patients within a therapeutic anticoagulation range, which usually is an international normalized ratio (INR) of 2.0–3.0. A recent analysis of 2223 patients showed that patients were outside the INR target range one-third of the time, with 15.4% of INR values above 3.0 and 16.7% of INR values below 2.0.11 There was higher mortality, increased risk of stroke and increase in the rate of hospitalization when patients were outside the anticoagulation range.

The most feared adverse effect associated with anticoagulation is bleeding. Major and fatal bleeding events occur at a rate of 7.2 and 1.3/100 patient years, respectively, according to a meta-analysis of 33 studies.12 Bleeding rates may be lower in specialized anticoagulation clinics,13 and when monitoring is more frequent.11 Bleeding events are most likely to occur within the first 90 days of therapy, but the incidence never falls to zero. The risk of bleeding is higher when INR is over 3, but bleeding can also occur when the INR is within the therapeutic range.13 Apart from the mortality and morbidity associated with warfarin-related bleeds, there is also a cost element: a recent analysis showed that the average cost per patient of a bleeding episode was $15 988 (range $2707–$64 446) with a mean length of stay of 6 days.13

Warfarin interactive pathways

At least 30 genes may be involved in the mechanism by which warfarin exerts its anticoagulant effect (Table 1 and Figure 1). The most important gene in the pharmacokinetics of warfarin is CYP2C9 (cytochrome P450 2C9 gene), whereas the central gene in the pharmacodynamics of warfarin is VKORC1 (vitamin K epoxide reductase complex subunit 1 gene).

Transportation of warfarin

The molecular basis of the pharmacokinetics of warfarin has been extensively studied.14 Warfarin is rapidly absorbed from the stomach and the upper gastrointestinal tract, with a bioavailability of 100%.15 In the circulating blood, warfarin is 99% protein bound largely to albumin and alpha-1-acid glycoproteins. The latter are encoded by ORM1 (orosomucoid 1 gene or alpha-1-acid glycoprotein 1 gene) and ORM2 (orosomucoid 2 gene or alpha-1-acid glycoprotein 2 gene).16, 17 It has been shown that warfarin binds preferentially to certain genetic variants of alpha-1-acid glycoproteins that can be separated by chromatography.17 Whether this has any effect on warfarin dose requirement seems rather unlikely given the binding to albumin.

Based on an inhibition assay, there is some evidence that the transport of warfarin across plasma membranes of cells, for example in the liver, may be mediated by P-glycoprotein (multidrug resistance protein 1), which is encoded by ABCB1 (MDR1; ATP-binding cassette transporter B1 gene).18 However, the evidence is scant and seems less probable given that warfarin has a very good bioavailability. Polymorphisms in ABCB1 have been linked to changes in mRNA and protein expression, and to the pharmacokinetic profiles of various drugs.19 The widely studied synonymous exon 26 C3435T variant has been the subject of numerous studies with conflicting results.19, 20, 21, 22 Interestingly, it has been shown that a haplotype containing the exon 26 C3435T variant (which could be expected to reduce drug efflux) was overrepresented among patients requiring a low dose of warfarin to maintain therapeutic anticoagulation.1 This needs to be replicated in another cohort, but is unlikely to be of major importance.

Biotransformation of warfarin

The influence of genetic variation on warfarin pharmacokinetics has been the focus of several review articles.4, 14, 23, 24, 25 Warfarin is administered as a racemate comprising R- and S-enantiomers: the S-form being 3–5 times more active than the R-form.26, 27 Once warfarin has entered the liver, S-warfarin is metabolized by cytochrome P450 2C9 (CYP2C9) to 7-hydroxywarfarin.26, 28 Many different polymorphisms in CYP2C9 that vary according to ethnicity and in terms of their functional effects have been described (http://www.imm.ki.se/CYPalleles/cyp2c9.htm). Most of the warfarin studies have so far concentrated on CYP2C9*2 and CYP2C9*3 variants. Compared with extensive metabolizers, who are homozygous for the wild-type *1 allele, homozygosity for *2 reduces CYP2C9 enzyme activity to 12% whereas homozygosity for *3 reduces enzyme activity to 5%.29, 30, 31 In accordance with this, many studies have shown that patients with the CYP2C9*2 and CYP2C9*3 variant alleles require lower mean daily warfarin doses (Table 2).1, 2, 3, 5, 6, 7, 8, 32, 33, 34, 35, 36, 37, 38 A systematic review and meta-analysis of nine studies has established that the CYP2C9*2 and CYP2C9*3 alleles lead to 17 and 37% reduction in the daily warfarin dose, respectively.24 In all studies, the overall variance in warfarin dose accounted for by CYP2C9*2 and CYP2C9*3 was below 20%. Moreover, CYP2C9 alleles *4 (identified in the Japanese), *5 and *6 (found in Afro-Americans) and *11 (rare in both Europeans and Afro-Americans) all lead to a reduction in warfarin dose requirement.39, 40

Table 2 A selection of studies on CYP2C9 polymorphisms in warfarin-treated patients

S-warfarin may also be metabolized by CYP2C8, CYP2C18 and CYP2C19 to form 4-hydroxywarfarin, although these are minor pathways.28 The genes encoding these P450 isoforms contain many functional polymorphisms. Two studies have so far found no effect of the CYP2C19*2 variant allele on warfarin therapy.2, 41 The role of other CYP2C isoforms has not been adequately evaluated, but would be predicted to be small. Furthermore, the coumarin hydroxylase variant CYP2A6*2 has been suspected to cause warfarin sensitivity.35, 42 However, these reports from one laboratory have not been replicated and there is no good evidence that CYP2A6 actually metabolizes warfarin.

R-warfarin, which is the less active enantiomer, is mainly metabolized by cytochrome P450 enzymes CYP1A2 (to 6- and 8-hydroxywarfarin) and CYP3A4 (to 10-hydroxywarfarin).26, 28, 43 In addition, CYP1A1, CYP2C8, CYP2C18, CYP2C19 and CYP3A5 may be involved in the metabolism of R-warfarin.26, 28, 43, 44, 45, 46, 47 There are as yet no published studies indicating that polymorphisms in these enzymes influence warfarin dosing.1, 48

Many of the P450 isoforms involved in the metabolism of warfarin are inducible; indeed, this is the mechanism of the well-known interactions that occur when warfarin is co-prescribed with drugs, for instance the aromatic anticonvulsants and herbal medicines such as St John's Wort.49, 50 The mechanism of induction of the P450 isoforms is dependent on the nuclear hormone receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR), but nothing has yet been published on whether warfarin dose requirement is affected by variation in the genes encoding these receptors, NR1I2 (pregnane X receptor gene) and NR1I3 (constitutive androstane receptor gene).50, 51, 52, 53, 54

Distribution and hepatic uptake of vitamin K

Warfarin targets the vitamin K epoxide reductase complex in the liver, thereby interfering with the recycling of vitamin K (Figure 1).25, 55, 56, 57, 58, 59 A high intake of fat-soluble vitamin K can reverse the action of warfarin, and a low or erratic intake of dietary vitamin K may be partly responsible for unstable control of anticoagulation in warfarin patients.60 Vitamin K1 is absorbed from the small intestine along with dietary fat, transported by chylomicrons in the blood and subsequently cleared by the liver through an APOE receptor-specific uptake.61, 62, 63 Uptake of chylomicrons and thus vitamin K1 into the liver varies between different APOE alleles, the rank order being *E4>*E3>*E2.61, 64 Consistent with this, patients with the APOE*E2 allele, who allegedly have the least efficient uptake of vitamin K1, have an increased risk of warfarin-associated intracerebral haemorrhage.65 In a Swedish cohort, it was shown that CYP2C9*1/*1 individuals (extensive metabolizers) who were homozygous for APOE*E4 were given significantly higher warfarin doses than other CYP2C9 extensive metabolizers.66 In agreement with this, a Dutch study showed that APOE*E4 carriers required slightly higher maintenance doses of the anticoagulant phenprocoumon, but surprisingly carriers of APOE*E4 required lower maintenance doses of acenocoumarol.67 In Italian patients, where the E4 allele is rare, no association was found between warfarin dose requirements and APOE genotype.68 The contradictory results of these candidate gene association studies reflect the lack of a clear description of the exact role of different APOE genotypes in vitamin K uptake and intracellular handling.

The vitamin K cycle

Genes involved in the vitamin K cycle have recently been shown to be crucial determinants of the response to warfarin. Warfarin and other vitamin K antagonists exert their anticoagulant effects by preventing the regeneration of vitamin K from vitamin K epoxide by inhibiting the enzyme vitamin K epoxide reductase (Figure 1).57 In 2004, the gene encoding this enzyme was identified as VKORC1.69, 70 Rare mutations in the human VKORC1 gene that convey resistance to warfarin have been identified.69, 71 Furthermore, a number of studies have shown that common SNPs in VKORC1 are strongly associated with warfarin maintenance dose in several populations (Table 3).72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 Additionally, a similar relationship has been demonstrated with the other vitamin K antagonists acenocoumarol and phenprocoumon,84, 85 with one study suggesting an association with coumarin-related bleeding.85 The associated VKORC1 SNPs are within a region of strong linkage disequilibrium, and a combination of several SNPs does not contribute greater information than one individual SNP.73, 74, 81 The molecular mechanism by which VKORC1 polymorphisms lead to variation in response to warfarin has not been resolved. It has been suggested that VKORC1 is regulated at a transcriptional level, as at least one of five correlated SNPs is a promoter polymorphism (−1639G>A, rs9923231) and is associated with low mRNA levels in liver specimens.74 In addition, a study has shown that a VKORC1 promoter cloned into a human hepatoma cell line was 44% more active if it contained the wild-type −1639G than the A allelic variant.75 This is biologically plausible given that the SNP resides within an E-box site, which can be important in determining tissue-specific transcription.75 However, no study has yet demonstrated that the change in mRNA levels is associated with a change in protein levels or indeed in functional activity in vitro. The VKORC1 gene is located in a large haplotype block where there are numerous SNPs that potentially could mediate the functional effect, and even though the evidence suggests transcriptional regulation of VKORC1, no single causative SNP or set of SNPs has been identified. In addition, there is no definite proof that transcriptional regulation is involved, as another study utilizing transfected HepG2 cells failed to show that a construct containing the G variant had activity that was different from that containing the A variant.84

Table 3 A selection of studies on VKORC1 polymorphisms in warfarin-treated patients

Although the gene for VKORC1 has been identified, the mechanism by which it functions as a reductase is unclear. The protein resides in the endoplasmic reticulum, and may be complexed with microsomal epoxide hydrolase (encoded by EPHX1), to produce a multiprotein complex that is responsible for vitamin K epoxide reduction.86, 87 Microsomal epoxide hydrolase by itself does not possess vitamin K epoxide reductase activity.88 Interestingly, a recent study in an Israeli population has shown an association between high doses of warfarin and a coding EPHX1 polymorphism (rs1051740) in CYP2C9 extensive metabolizers.89 However, the authors’ contention that this EPHX1 polymorphism leads to high-dose requirements beyond the effect of CYP2C9 needs to be replicated in another population. Moreover, it has been suggested that the antioxidant enzyme nicotine adenine dinucleotide phosphate (NAD(P)H) dehydrogenase, also called flavoprotein DT-diaphorase, has the potential to reduce dietary vitamin K.62, 90, 91 Its gene, NQO1 (NAD(P)H dehydrogenase, quinone 1 gene), has not been studied with respect to warfarin treatment.

Reduced vitamin K is an essential cofactor for the activation of vitamin K-dependent proteins by gamma-glutamyl carboxylase.92, 93, 94 Gamma-glutamyl carboxylase is an integral endoplasmic reticulum protein that localizes in close proximity to the vitamin K epoxide reductase complex. A very rare autosomal recessive bleeding disorder due to combined deficiency of the vitamin K-dependent coagulation factors II , VII, IX and X, and proteins C, S and Z is caused by mutations in the gamma-glutamyl carboxylase gene (GGCX).92, 95 SNPs as well as microsatellite markers that might affect warfarin dosing have recently been identified in the GGCX gene. For example, an intronic polymorphism that increases warfarin dose requirements was identified in a Swedish population.73 A microsatellite in intron 6 has been associated with warfarin dose in the Japanese;96 a similar analysis in a Swedish population showed that warfarin dose requirements increase with the number of microsatellite repeats.97 On the other hand, a coding polymorphism (rs699664) that leads to a change from arginine to glutamine at residue 325 is not associated with warfarin sensitivity or resistance.73, 89 Taken as a whole, the effect of GGCX seems to be rather modest.96, 97

The endoplasmic reticulum chaperone protein calumenin, encoded by CALU (calumenin gene), can bind to the vitamin K cycle and inhibit its activity.98, 99 It has been shown that silencing of the CALU gene with small interfering RNA results in a fivefold increase in gamma-carboxylase.99 Furthermore, overexpression of calumenin in the liver produces warfarin resistance in rats by protecting vitamin K epoxide reductase from inhibition by warfarin.98 Whether this is a mechanism of warfarin resistance in man is unknown at present, particularly as calumenin is expressed at low levels in the human liver. Only one coding polymorphism in the human CALU gene (rs2290228) has so far been related to warfarin dose requirements.79

Vitamin K-dependent proteins

Many vitamin K-dependent proteins have been implicated in warfarin sensitivity. The main vitamin K-dependent proteins are clotting factors II (prothrombin), VII, IX and X, proteins C, S and Z and growth-arrest-specific protein 6, encoded by F2, F7, F9, F10, PROC, PROS1, PROZ and GAS6.62, 93, 100 Two independent studies have shown that a polymorphism in F2 causing a change from threonine to methionine at residue 165 leads to increased sensitivity to warfarin,96, 101 whereas a third study did not show this.83 It has also been shown that promoter polymorphisms in F7 have an effect on warfarin sensitivity.83, 96, 101 Mutations in the propeptide of F9, causing a change from alanine to valine or threonine at residue −10, lead to a rapid drop in factor IX during warfarin treatment and are the reason for bleeding in rare cases.102, 103 Promoter polymorphisms and a synonymous coding polymorphism in exon 7 of F10 have also been studied, but no effect on warfarin sensitivity was seen.83, 96

Unlike other vitamin K-dependent factors, protein C and S work as natural anticoagulants. After administration of warfarin, protein C and S decline more rapidly than other vitamin K-dependent proteins, and this may contribute to the poor antithrombotic efficacy during the first day of anticoagulant therapy.104, 105 The temporary imbalance is exaggerated in patients with a hereditary deficiency of protein C or S, which leads to a relative hypercoagulable state at the start of warfarin treatment.106, 107 Rare genetic variants of PROC and PROS1 did not, however, affect warfarin dose requirement in a Japanese population.96 Two vitamin K-dependent proteins, encoded by PROZ and GAS6, have not been studied with respect to warfarin sensitivity. Two non-vitamin K-dependent clotting proteins of interest for warfarin pharmacogenetics are anti-thrombin III and factor V. Anti-thrombin III inhibits factors II, IX, X, XI and XII, and anti-thrombin III deficiency, both the congenital form caused by mutations in SERPINC1 (anti-thrombin III gene) and the acquired form, may create a hypercoagulable state during warfarin induction.107, 108 A point mutation in the factor V gene (Arg506Gln or FV Leiden), which commonly causes thromboembolism and warfarin treatment, is not known to affect dose requirement.109

Alternative approaches

Although we understand a lot about the pharmacokinetics and dynamics of warfarin (Figure 1), it is possible, and indeed likely, that other genes are involved in the outcome of treatment. Such genes may act in trans (e.g. transcription factors) and may therefore not be identified by the candidate gene approach. Owing to recent advances in genotyping technologies, it is now feasible to find these other genes through genome-wide association studies. Compared with targeted analysis of candidate genes based on the known actions and metabolism of warfarin, a genome-wide approach is advantageous because (a) it has a better chance of identifying previously unknown genes that influence warfarin therapy and (b) the cost and effort per genotype produced is significantly lower than for the analysis of a limited number of candidate genes. However, there is a need for large sample sizes to ensure adequate statistical power (which in effect renders these studies expensive), and better statistical approaches need to be developed.

Future challenges for clinical practice

The studies discussed above clearly show that genetic variation, especially in CYP2C9 and VKORC1, is extremely important for the variability in the response to warfarin. Polymorphisms in the VKORC1 and CYP2C9 genes and a limited subset of environmental determinants account for around 50–60% of the variance in warfarin dose requirement.73, 76, 77, 79, 81, 82, 83 In six studies, the relative contribution of VKORC1 is greater than that of CYP2C9,73, 74, 78, 81, 82, 83 in two, CYP2C9 has a greater quantitative contribution,72, 76 whereas VKORC1 and CYP2C9 contribute equally in one study.79 Sconce et al.76 have gone on to develop a dosing table based on a regression equation combining age, height and CYP2C9*2 and CYP2C9*3, and the VKORC1 SNP −1639G>A.

The data from various pharmacogenetic studies worldwide have been considered by the FDA in an open hearing (http://www.fda.gov/ohrms/dockets/ac/05/slides/2005-4194S1_Slide-Index.htm).73, 74, 110, 111 The interesting questions are whether this will lead to a recommendation for genotyping in the label for warfarin, and if this would change clinical practice and, more importantly, improve the use and safety of warfarin. Before these questions can be answered several important issues need to be considered.

First, the estimates for the variance in warfarin dosing have been derived from retrospective studies in homogeneous populations. Thus, it is unclear how a combined variance of 55–60% will translate into predictive values in diverse populations. Furthermore, the retrospective nature of the studies undertaken so far is likely to underestimate the environmental contribution and overestimate the genetic contribution. This is a consistent feature of genetic association studies, which is perhaps best exemplified by the association of ACE gene polymorphisms and risk of myocardial infarction.112

Second, it could be argued that the maintenance dose of warfarin could be achieved rapidly by more intensive monitoring particularly in specialist anticoagulant clinics. However, this has not been studied in comparison to genetic individualization. The crucial issue to assess here is how closely the induction dose predicts the maintenance dose. Encouragingly, a randomized study of 5 mg of warfarin as a starting dose versus an initial dose calculated on the basis of weight, age, serum albumin and presence of malignancy resulted in the latter regimen leading to a slightly but significantly quicker time to onset of anticoagulation (5 versus 4.2 days).113 Genetic individualization of dose might further speed up this process.

Third, the ultimate aim of individualizing warfarin dosing is not only to improve the stability of anticoagulation control, but also to reduce the risk of bleeding with warfarin. Some,5, 8, 34, 114 but not all,1, 33, 115 studies have shown an association between bleeding and genetic factors such as CYP2C9 polymorphisms (Table 2). Prospective and retrospective data have shown that the intensity of anticoagulation and deviation in anticoagulation control are the strongest predictors for the risk of bleeding.116 It is likely to be more difficult to consistently show an association between genetic factors and warfarin-related bleeding because (a) it is relatively uncommon and therefore most of the studies are under-powered with respect to bleeding as an end point, (b) there are differences between studies in the definitions used for the severity of bleeding, (c) some patients bleed at normal INR values,13 and in these patients in particular, there may be underlying causes such as tumours,117, 118 and (d) there may be other co-incidental genetic polymorphisms that contribute to the risk of bleeding, for example those involved in platelet aggregation.119 Nevertheless, a meta-analysis of CYP2C9 genetic polymorphisms showed that the relative bleeding risk for CYP2C9*2 was 1.91 (95% CI 1.16–3.17) and for CYP2C9*3 1.77 (95% CI 1.07–2.91).24 For either variant, the relative risk was 2.26 (95% CI 1.36–3.75).

Fourth, genetic individualization of warfarin therapy needs to be shown to be cost-effective. If it greatly adds to the cost of treating patients, and given the huge usage of warfarin in the general population, it may be difficult to persuade health-care organizations to fund genetic testing. Hopefully, the rapid development of genetic technology will lead to more sophisticated assays at a lower cost, and this is likely to facilitate incorporation of genetic analyses in clinical practice. A small retrospective study has already suggested that CYP2C9 genotyping is potentially effective in preventing bleeding with a marginal cost.120 However, this needs to be performed in a larger study, and is currently being assessed as part of the UK prospective study (see below).

Fifth, it has been stated that regulation is likely to be the key factor that will drive the implementation of pharmacogenetics into clinical practice. This is true to an extent. The possibility that pharmacogenetic information is going to be incorporated into the warfarin label is an important development. A similar example is azathioprine, which for a long time has been known to be metabolized by thiopurine methyltransferase (TPMT), which is polymorphically expressed, with low expressers being at higher risk of leukopenia. Although the polymorphic metabolism is mentioned in the label for azathioprine, there is no mandatory statement regarding dose individualization according to genotype or phenotype. A Europe-wide survey has shown that TPMT testing before azathioprine use occurs in only about 12% of cases,121 and in Australasia, pharmacogenetic testing for drug metabolizing enzymes are performed rarely in clinical practice.122 Pharmacogenetic labelling is in these cases for information only and not mandatory, and the absence of clear guidelines may lessen the probability that the test is used. Many factors are needed for regulators to change the nature of the warnings in the product label, the most important of which are a strong research base and good evidence of clinical relevance.122 Other factors may also be important including overcoming financial and perception barriers, education regarding pharmacogenetics and adequate information on the benefits of pre-prescription testing.121 Such a multi-pronged approach is going to be needed to incorporate pharmacogenetics into the prescribing of warfarin.

Finally, various other strategies have been suggested to improve the safety of anticoagulation therapy including computer decision support systems,123 the use of patient self-monitoring devices124 and the use of drugs that inhibit other targets in the anticoagulation pathway, for example the oral thrombin inhibitors.125 Whether we should use pharmacogenetic-based warfarin therapy in competition or in conjunction with these other approaches is not clear.

To address these issues, as clinicians, we feel that it will be necessary to undertake large prospective studies of variation in response to warfarin therapy. It has clearly been shown that prospective randomized controlled trials based on CYP2C9 genotyping are feasible.111, 126 It should therefore be possible to conduct randomized controlled trials based on CYP2C9 and VKORC1 polymorphisms. Concerning other genes outlined in this review, it is important to note that two studies in Swedish patients (one in 200 patients and another in 1500 patients) are examining polymorphisms in all these pathways in collaboration with the Sanger Institute, UK, and are due to report soon. These studies are retrospective and will not be able to determine the relative contributions of genetic and environmental factors and the interaction between them. These questions are, however, likely to be answered in a prospective study of up to 2000 patients that is currently ongoing in the UK (http://www.genres.org.uk/prp/projectsliverpool2.htm). This study is not only looking at all the genes mentioned here, but it is assessing environmental factors including the clinical (age, gender, ethnicity, disease, concurrent medication, adherence to treatment), pharmacological (R- and S-warfarin levels), biochemical (vitamin K and epoxide levels) and haematological (clotting factor levels) phenotypes. The study will be able to assess the cost-effectiveness of pre-prescription genotyping, and provide values for positive and negative prediction, and numbers needed to screen. These developments will provide the necessary framework to undertake prospective randomized controlled trials to assess the clinical utility of pre-prescription genotyping for warfarin.

Conclusion and summary

Despite the fact that warfarin is an old drug, there is currently unprecedented interest in the pharmacology and effectiveness of warfarin, which is partly due to a general interest in whether pharmacogenetics can improve the use of common medicines. This research has led to the identification of striking genetic predisposing factors in two genes, CYP2C9 and VKORC1, explaining a large part of the interindividual variation in warfarin dose requirement. To what extent variability in other genes in the warfarin interactive pathways influences warfarin therapy remains to be resolved. Most studies to date have had an inadequate sample size to be able to detect small genetic effects in these other genes, and the findings highlighted in some of the genetic association studies may thus be due to pure chance. For the intraindividual variation in warfarin dose, environmental factors such as the intake of vitamin K and interacting medications will be more important than genetic factors. Whether the identified genetic and environmental factors will improve the use and safety of warfarin in clinical practice is unclear, but is likely to be resolved in the next couple of years with ongoing and newly planned studies of all known warfarin interactive pathways. Irrespective of whether pre-prescription genotyping impacts directly on the use of warfarin, we are learning much more about the pharmacology of warfarin because of the current interest in warfarin pharmacogenetics. An indirect benefit of this will be an increase in the knowledge of how to prescribe warfarin, and translation of this knowledge into clinical guidelines, is likely to have a major impact on the safety of warfarin.

Abbreviations

ABCB1 :

ATP-binding cassette transporter B1 gene, P-glycoprotein gene or MDR1

APOE:

apolipoprotein E gene

CALU :

calumenin gene

CAR:

constitutive androstane receptor

CYP1A1:

cytochrome P450 1A1 gene

CYP1A2:

cytochrome P450 1A2 gene

CYP2A6:

cytochrome P450 2A6 gene

CYP2C18:

cytochrome P450 2C18 gene

CYP2C19:

cytochrome P450 2C19 gene

CYP2C8:

cytochrome P450 2C8 gene

CYP2C9:

cytochrome P450 2C9 gene

CYP3A4:

cytochrome P450 3A4 gene

CYP3A5:

cytochrome P450 3A5 gene

EPHX1 :

epoxide hydrolase 1, microsomal gene

F2 :

coagulation factor II gene or prothrombin gene

F5 :

coagulation factor V gene

F7 :

coagulation factor VII gene

F9 :

coagulation factor IX gene

F10 :

coagulation factor X gene

FII:

coagulation factor II or prothrombin

FIIa:

coagulation factor II activated or thrombin

FIX:

coagulation factor IX

FIXa:

coagulation factor IX activated

FV:

coagulation factor V

FVII:

coagulation factor VII

FVIIa:

coagulation factor VII activated

FX:

coagulation factor X

FXa:

coagulation factor X activated

GAS6 :

growth-arrest-specific 6 gene

GGCX :

gamma-glutamyl carboxylase gene

MDR1 :

multidrug resistance gene 1, P-glycoprotein gene or ABCB1

NQO1 :

NAD(P)H dehydrogenase, quinone 1 gene

NR1I2 :

pregnane X receptor gene

NR1I3 :

constitutive androstane receptor gene

ORM1 :

orosomucoid 1 gene or alpha-1-acid glycoprotein 1 gene

ORM2 :

orosomucoid 2 gene or alpha-1-acid glycoprotein 2 gene

PROC :

protein C gene

PROS1 :

protein S gene

PROZ :

protein Z gene

PT INR:

prothrombin time international normalized ratio

PXR:

pregnane X receptor

SERPINC1 :

anti-thrombin III gene

SNP:

single nucleotide polymorphism

VKORC1 :

vitamin K epoxide reductase complex subunit 1 gene

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Acknowledgements

The support of the UK Department of Health, which is funding the prospective UK warfarin pharmacogenetics study, is gratefully acknowledged. The Uppsala warfarin study is supported by the Swedish Society of Medicine, Foundation for Strategic Research, Heart and Lung Foundation and the Clinical Research Support (ALF) at Uppsala University. The support of David Bentley and the Wellcome Trust Sanger Institute is acknowledged. The sponsors had no role in the writing of this review.

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Correspondence to M Wadelius.

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Wadelius, M., Pirmohamed, M. Pharmacogenetics of warfarin: current status and future challenges. Pharmacogenomics J 7, 99–111 (2007). https://doi.org/10.1038/sj.tpj.6500417

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Keywords

  • warfarin
  • vitamin K epoxide reductase complex subunit 1
  • VKORC1
  • cytochrome P450 enzyme
  • CYP2C9
  • vitamin K-dependent protein

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