Download Pharmacogenetics of warfarin: current status and future

The Pharmacogenomics Journal (2007) 7, 99–111
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REVIEW
Pharmacogenetics of warfarin: current status and
future challenges
M Wadelius1 and
M Pirmohamed2
1
Department of Medical Sciences, Clinical
Pharmacology, Uppsala University Hospital,
Uppsala, Sweden and 2Department of
Pharmacology and Therapeutics, University of
Liverpool, Liverpool, UK
Correspondence:
Dr M Wadelius, Department of Medical
Sciences, Clinical Pharmacology, Uppsala
University Hospital, Entrance 61 3rd floor,
SE-751 85 Uppsala, Sweden.
E-mail: [email protected]
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.
The Pharmacogenomics Journal (2007) 7, 99–111. doi:10.1038/sj.tpj.6500417;
published online 19 September 2006
Keywords: warfarin; vitamin K epoxide reductase complex subunit 1; VKORC1; cytochrome
P450 enzyme; CYP2C9; vitamin K-dependent protein
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.1–8 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
Received 24 April 2006; revised 13 July 2006;
accepted 24 July 2006; published online 19 literature. Well-known examples are polymorphisms that change cytochrome
P450 enzyme activity, apolipoprotein E (APOE) variants, factor V Leiden and
September 2006
Pharmacogenetics of warfarin
M Wadelius and M Pirmohamed
100
Table 1
Genes involved in the mechanism of action of warfarin
Protein name
Gene
Location
ORM1
Chr 9: 114 083 890–
114 087 309 bp
6
802
201
Alpha-1-acid glycoprotein 2,
Orosomucoid 2
ORM2
Chr 9: 114 171 703–
114 175 086 bp
6
760
201
P-glycoprotein, Multidrug
resistance protein 1
ABCB1
(MDR1)
Chr 7: 85 668 428–
85 877 818 bp
29
4643
1279
Metabolism
Cytochrome P450 2C9
CYP2C9
Chr 10: 96 688 405–
96 739 137 bp
9
1847
490
Cytochrome P450 1A1
CYP1A1
7
2601
512
Cytochrome P450 1A2
CYP1A2
7
1618
516
Cytochrome P450 2A6
CYP2A6
Chr 15: 72 798 943–
72 804 930 bp
Chr 15: 72 828 257–
72 834 505 bp
Chr 19: 46 041 284–
46 048 180 bp
9
1751
494
Cytochrome P450 2C8
CYP2C8
Chr 10: 96 786 520–
96 819 244 bp
9
1923
490
Cytochrome P450 2C18
CYP2C18
9
2418
490
Cytochrome P450 2C19
CYP2C19
Chr 10: 96 432 700–
96 485 937 bp
Chr 10: 96 437 901–
96 603 007 bp
9
1901
490
Cytochrome P450 3A4
CYP3A4
13
2768
503
Cytochrome P450 3A5
CYP3A5
Chr 7: 97 889 181–
97 916 385 bp
Chr 7: 97 780 394–
97 812 183 bp
13
1707
502
NR1I2
Chr 3: 120 982 021–
121 020 021 bp
9
2753
473
NR1I3
Chr 1: 158 012 528–
158 021 028 bp
9
1337
348
APOE
Chr 19: 50 100 879–
50 104 489 bp
4
1179
317
Apolipoprotein E serves as a ligand
for receptors that mediate the
uptake of vitamin K61–64
Chr 16: 31 009 677–
31 013 777 bp
3
997
163
A hepatic epoxide hydrolase that
catalyses the reduction of vitamin
K. The target of warfarin56,69,70
A hepatic epoxide hydrolase in the
endoplasmic reticulum that may be
complexed with VKOR86,87,89
A detoxifying enzyme that has the
potential to reduce the quinine
form of vitamin K62,90,91
Biotransformation of warfarin
Transport
Alpha-1-acid glycoprotein 1,
Orosomucoid 1
Cytochrome P450 inducibility
Pregnane X receptor (PXR)
Constitutive androstane
receptor (CAR)
Biotransformation of vitamin K
Transport
Apolipoprotein E
Vitamin K cycle
Vitamin K epoxide reductase VKORC1
Exons
Transcript Protein
(bp)
(aa)
Epoxide hydrolase 1,
microsomal
EPHX1
Chr 1: 222 304 587–
222 339 995 bp
9
1605
455
NAD(P)H dehydrogenase,
quinone 1
NQO1
Chr 16: 68 300 807–
68 317 893 bp
6
2448
274
The Pharmacogenomics Journal
Function of protein
A plasma glycoprotein that
functions as a carrier of warfarin in
the blood16,17
A plasma glycoprotein that
functions as a carrier of warfarin in
the blood16,17
A cellular efflux pump for
xenobiotics.20 Warfarin is a weak
inhibitor and maybe a substrate18
Polymorphic hepatic drug
metabolizing enzyme. Metabolism
of S-warfarin26,28
Extrahepatic oxidation, inducible.
Metabolism of R-warfarin28,43,45
Hepatic oxidation, inducible.
Metabolism of R-warfarin28,43
Polymorphic hepatic drug
metabolizing enzyme. Metabolism
of S-warfarin?35
Polymorphic hepatic drug
metabolizing enzyme. Minor
pathway for R- and S-warfarin26,28
Found in the liver and lung. Minor
pathway for R- and S-warfarin28,44
Polymorphic hepatic drug
metabolizing enzyme. Minor
pathway for R- and S-warfarin28,44
Hepatic oxidation, inducible.
Metabolism of R-warfarin28
Polymorphic hepatic and
extrahepatic oxidation.
Metabolism of R-warfarin?46
Mediates induction of CYP2C9,
CYP3A4, other CYP enzymes and
ABCB151–53
Transcriptional regulation of a
number of genes including CYP2C9
and CYP3A454
Pharmacogenetics of warfarin
M Wadelius and M Pirmohamed
101
Table 1 Continued
Protein name
Calumenin
Gene
Location
CALU
Chr 7: 127 973 368–
128 005 478 bp
7
3316
315
Chr 2: 85 687 865–
85 700 237 bp
15
3155
758
Chr 11: 46 697 331–
46 717 631 bp
14
1997
622
Gamma-glutamyl carboxylase GGCX
Vitamin K-dependent proteins
Coagulation factor II,
F2
prothrombin
Exons
Transcript Protein
(bp)
(aa)
Coagulation factor VII
F7
Chr 13: 112 808 124–
112 822 348 bp
9
2459
466
Coagulation factor IX
F9
Chr X: 138 340 437–
138 373 137 bp
8
2780
461
Coagulation factor X
F10
8
1524
488
Protein C
PROC
Chr 13: 112 825 128–
112 851 846 bp
Chr 2: 127 892 246–
127 903 048 bp
9
1756
461
Protein S
PROS1
Chr 3: 95 074 647–
95 175 395 bp
15
3275
676
Protein Z
PROZ
Chr 13: 112 860 971–
112 874 700 bp
8
1488
400
Growth-arrest-specific
protein 6
GAS6
Chr 13: 113 546 903–
113 590 421 bp
15
2499
678
SERPINC1
Chr 1: 170 604 596–
170 618 130 bp
7
1559
464
F5
Chr 1: 166 215 067–
166 287 379 bp
25
6914
2224
Other coagulation proteins
Anti-thrombin III
Coagulation factor V
Function of protein
Binds to the vitamin K epoxide
reductase complex and inhibits the
effect of warfarin98,99
Carboxylates vitamin K-dependent
coagulation factors and proteins in
the vitamin K cycle92–94
Converts fibrinogen to fibrin,
activates FV, FVIII, FXI, FXIII,
protein C93,108
Is converted to FVIIa and then
converts FIX to FIXa and FX to
FXa93,108
Makes a complex with FVIIIa and
then converts FX to its active
form93,108
Converts FII to FIIa in the presence
of factor Va93,108
Activated protein C counteracts
coagulation together with protein
S by inactivating FVa and VIIIa93,108
Cofactor to protein C that
degrades coagulation factors Va
and VIIIa93,108
Is together with protein Zdependent protease inhibitor,
a cofactor for the inactivation
of FXa93,100
Participates in many processes, for
example, potentiation of agonistinduced platelet aggregation62
Inhibits FIIa, FIXa, Xa, XIa and XIIa.
Anti-thrombin deficiency increases
risk of thrombosis108
A cofactor that activates FII
together with FXa. An F5 mutation
leads to risk of thrombosis108
Protein and gene names, location in NCBI build 35, number of exons, size of transcript and protein and function of the proteins are included in the table.
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.
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
The Pharmacogenomics Journal
Pharmacogenetics of warfarin
M Wadelius and M Pirmohamed
102
Figure 1 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.
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
The Pharmacogenomics Journal
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
Pharmacogenetics of warfarin
M Wadelius and M Pirmohamed
103
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 alpha1-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.19–22 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.4,14,23–25
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%.29–31 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).1–3,5–8,32–38 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
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.26,28,43–47 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 coprescribed 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).50–54
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).25,55–59 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 receptorspecific uptake.61–63 Uptake of chylomicrons and thus
vitamin K1 into the liver varies between different APOE
alleles, the rank order being *E44*E34*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
The Pharmacogenomics Journal
Pharmacogenetics of warfarin
M Wadelius and M Pirmohamed
104
Table 2
A selection of studies on CYP2C9 polymorphisms in warfarin-treated patients
Reference
Patient populations
n
Warfarin dose requirement
Risk of adverse event
Furuya et al. (1995)6
Steward et al. (1997)7
Aithal et al. (1999)114
British Caucasian
American Caucasian
British Caucasian
94
1
88
CYP2C9*2 associated with low dose
CYP2C9*3 associated with low dose
CYP2C9*2 and *3 associated with
low dose
Ogg et al. (1999)8
British
233
CYP2C9*3 associated with low dose
Taube et al. (2000)33
British
561
Margaglione et al.
(2000)34
Freeman et al.
(2000)35
Loebstien et al.
(2001)3
Leung et al. (2001)127
Italian Caucasian
180
Tabrizi et al. (2002)36
American Caucasian 81%
Afro-American 19%
American Caucasian 91%
Asian 4%
Afro-American 3%
Hispanic 2%
Italian Caucasian
153
CYP2C9*2 and *3 associated with
low dose
CYP2C9*2 and *3 associated with
low dose
CYP2C9*2 and *3 associated with
low dose
CYP2C9*2 and *3 associated with
low dose
CYP2C9 208Val carriers require a
lower dose
CYP2C9*2 and *3 associated with
low dose
CYP2C9*2 and *3 associated with
low dose
Not studied
High initial INR
Low-dose requirement associated with
raised INR during induction and risk of
major bleeding
CYP2C9*3 associated with risk of early
bleeding
No increased risk of severe overanticoagulation or bleeding
Variant alleles associated with risk of
bleeding
Not studied
Wadelius et al.
(2004)1
Peyvandi et al.
(2004)37
Joffe et al. (2004)115
Swedish Caucasian
225
Italian
125
Lindh et al. (2005)38
Swedish
Higashi et al. (2002)5
Scordo et al. (2002)2
American Caucasian 78%
Afro-American 22%
Israeli
Hong Kong Chinese
American Caucasian
38
156
89
185
93
73
219
CYP2C9*2 and *3 associated with
low dose and S-warfarin-clearance
CYP2C9*2 and *3 associated with
low dose
CYP2C9*2 and *3 associated with
low dose
CYP2C9*2 and *3 associated with
low dose
CYP2C9*2 and *3 associated with
low dose
Not studied
Not studied
Not studied
Variant alleles associated with high
initial INR, longer time to stable INR
and risk of serious bleeding
Not studied
Variant alleles not associated with
bleeding
Variant alleles associated with INR43
during induction
A tendency to an association between
variant alleles and INR46, but no
association with bleeding
Variant alleles associated with INR43
during induction
INR, international normalized ratio.
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
The Pharmacogenomics Journal
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).72–83 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
Pharmacogenetics of warfarin
M Wadelius and M Pirmohamed
105
promoter polymorphism (1639G4A, 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
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
Table 3
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 DTdiaphorase, 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 gammaglutamyl carboxylase.92–94 Gamma-glutamyl carboxylase is
an integral endoplasmic reticulum protein that localizes in
close proximity to the vitamin K epoxide reductase
A selection of studies on VKORC1 polymorphisms in warfarin-treated patients
Reference
Patient populations
Rost et al. (2004)69
Warfarin resistant
6
D’Andrea et al. (2005)72
Harrington et al. (2005)71
Italian Caucasian
Warfarin resistant
147
4
Wadelius et al. (2005)73
Swedish Caucasian
201
Rieder et al. (2005)74
American Caucasian
554
Yuan et al. (2005)75
Sconce et al. (2005)76
Veenstra et al. (2005)77
Taiwan Chinese
British Caucasian
Hong Kong Chinese
120
335
69
Geisen et al. (2005)78
European warfarin resistant
Vecsler et al. (2006)79
Mushiroda et al. (2006)80
Israeli
Japanese
100
828
Takahashi et al. (2006)81
American Caucasian 47%
Japanese 26%
Afro-American 26%
Singapore Chinese 53%
Malay 31%
Indian 16%
American Caucasian 91%
Afro-American 7%
Hispanic 1% Asian 0.3%
243
Lee et al. (2006)82
Aquilante et al. (2006)83
n
12
275
350
Warfarin dose requirement
Risk of adverse
event
Coding polymorphisms increase dose
requirement in warfarin resistant
1173C4T associated with dose
Coding polymorphisms increase dose
requirement in warfarin resistant
1639G4A, 1173C4T and 2255C4T
associated with dose
Haplotypes defined by 4451C4A, 497T4G,
1542G4C and 3730G4A associated with dose
1639G4A associated with dose
1639G4A associated with dose
Haplotypes defined by 4451C4A, 497T4G,
1542G4C and 3730G4A associated with dose
1639G4A and 1173C4T associated with
dose in warfarin resistant
1542G4C associated with dose
1639G4A, 1173C4T, 1542G4C, 2255C4T
and 3730G4A associated with dose
1173C4T associated with dose
Not studied
Haplotypes defined by 4931T4C,
1639G4A, 1173C4T, 1542G4C and
2255C4T associated with dose
1639G4A is associated with dose
Not studied
Not studied
Not studied
Not studied
Not studied
Not studied
Not studied
Not studied
Not studied
Not studied
Not studied
Not studied
Not studied
Translation into rs numbers: 4451C4A ¼ rs17880887, 1639G4A ¼ rs9923231, 497T4G ¼ rs2884737, 1173C4T ¼ rs9934438, 1542G4C ¼ rs8050894,
2255C4T ¼ rs2359612, 3730G4A ¼ rs7294.
The Pharmacogenomics Journal
Pharmacogenetics of warfarin
M Wadelius and M Pirmohamed
106
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 gammacarboxylase.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
The Pharmacogenomics Journal
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.73,76,77,79,81–83 In six studies, the relative contribution
of VKORC1 is greater than that of CYP2C9,73,74,78,81–83 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
1639G4A.
Pharmacogenetics of warfarin
M Wadelius and M Pirmohamed
107
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
The Pharmacogenomics Journal
Pharmacogenetics of warfarin
M Wadelius and M Pirmohamed
108
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 preprescription 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
The Pharmacogenomics Journal
warfarin, and translation of this knowledge into clinical
guidelines, is likely to have a major impact on the safety of
warfarin.
Abbreviations
ABCB1
APOE
CALU
CAR
CYP1A1
CYP1A2
CYP2A6
CYP2C18
CYP2C19
CYP2C8
CYP2C9
CYP3A4
CYP3A5
EPHX1
F2
F5
F7
F9
F10
FII
FIIa
FIX
FIXa
FV
FVII
FVIIa
FX
FXa
GAS6
GGCX
MDR1
NQO1
NR1I2
NR1I3
ORM1
ORM2
PROC
PROS1
PROZ
PT INR
PXR
SERPINC1
SNP
VKORC1
ATP-binding cassette transporter B1 gene,
P-glycoprotein gene or MDR1
apolipoprotein E gene
calumenin gene
constitutive androstane receptor
cytochrome P450 1A1 gene
cytochrome P450 1A2 gene
cytochrome P450 2A6 gene
cytochrome P450 2C18 gene
cytochrome P450 2C19 gene
cytochrome P450 2C8 gene
cytochrome P450 2C9 gene
cytochrome P450 3A4 gene
cytochrome P450 3A5 gene
epoxide hydrolase 1, microsomal gene
coagulation factor II gene or prothrombin gene
coagulation factor V gene
coagulation factor VII gene
coagulation factor IX gene
coagulation factor X gene
coagulation factor II or prothrombin
coagulation factor II activated or thrombin
coagulation factor IX
coagulation factor IX activated
coagulation factor V
coagulation factor VII
coagulation factor VII activated
coagulation factor X
coagulation factor X activated
growth-arrest-specific 6 gene
gamma-glutamyl carboxylase gene
multidrug resistance gene 1, P-glycoprotein gene or ABCB1
NAD(P)H dehydrogenase, quinone 1 gene
pregnane X receptor gene
constitutive androstane receptor gene
orosomucoid 1 gene or alpha-1-acid glycoprotein 1 gene
orosomucoid 2 gene or alpha-1-acid glycoprotein 2 gene
protein C gene
protein S gene
protein Z gene
prothrombin time international normalized ratio
pregnane X receptor
anti-thrombin III gene
single nucleotide polymorphism
vitamin K epoxide reductase complex subunit 1 gene
Acknowledgments
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.
Duality of Interest
None.
Pharmacogenetics of warfarin
M Wadelius and M Pirmohamed
109
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