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Effect of CYP2C9 and VKORC1 genetic
variations on warfarin dose requirements
in Indian patients
Sripriya Natarajan1, Chandrashekhar K. Ponde2, Rajesh M. Rajani2, Farah
Jijina2, Roopkumar Gursahani2, Pradnya P. Dhairyawan1, Tester F. Ashavaid1,3
1Research Laboratories,
2Department of Medicine,
3Department of Lab Medicine, P.D. Hinduja National Hospital
and Medical Research Centre, V. S. Marg, Mahim, Mumbai-400016, India
Correspondence: Tester F. Ashavaid, e-mail: [email protected] or [email protected]
Abstract:
Background: Warfarin, an oral anticoagulant is used in patients who are at increased risk of developing blood clots. The manage-
ment of warfarin therapy is challenging because it shows large inter and intra individual variability in patient response due to factors
like age, gender, diet, concurrent drug interactions and variations in CYP2C9 and VKORC1 genes. Studies implicate that polymor-
phisms in VKORC1 and CYP2C9 genes are associated with reduced doses of warfarin. The aim of our current study was to character-
ize the effects of VKORC1 and CYP2C9 gene variations that contribute to variability in warfarin dosing in Indian patients.
Methods: Genomic DNA was extracted from 103 patients undergoing warfarin therapy. Their mean daily warfarin dose, INR and
demographics were recorded and genotyping of VKORC1 and CYP2C9 gene was performed by PCR-RFLP method.
Results: Individuals with wild type genotypes required highest mean warfarin dosage of 4.72 mg/day while VKORC1 variants re-
quired 3.6 mg/day to maintain their therapeutic INR. CYP2C9*2 genotype was not found to affect the warfarin maintenance dosages.
The odds ratio for developing supra therapeutic INR in patients carrying VKORC1 variant allele when compared to wild types was
13.96 (95% CI; 4.85 – 44.65. Other factors affecting warfarin dosages were age and weight.
Conclusion: Inclusion of pharmacogenetic data along with clinical parameters would help better predict warfarin doses in Indian
patients.
Key words:
CYP2C9, genetic variations, Indians, VKORC1, warfarin
Abbreviations: APOE – apolipoprotein E gene, BMI – body
mass index, bp – base pair, CI – confidence interval, CYP1A1
– cytochrome P450 complex subunit 1A1, CYP1A2 – cyto-
chrome P450 complex subunit 1A2, CYP2C9 – cytochrome
P450 complex subunit 2C9, CYP3A4 – cytochrome P450 com-
plex subunit 3A4, DNA – deoxyribose nucleic acid, DVT –
deep vein thrombosis, df – degrees of freedom, EDTA – ethyle-
nediaminetetraacetic acid, GGCX – g-glutamyl carboxylase
gene, Htz – heterozygous, INR – international normalized ra-
tio, M – 50 base pair DNA ladder, Mut – homozygous mutant,
NC – negative control, OR – odds ratio, PCR-RFLP – po-
lymerase chain reaction-restriction fragment length polymor-
phism, UV – ultraviolet, VKORC1 – vitamin K epoxide reduc-
tase complex subunit 1, WT – wild type
Introduction
Warfarin has been in commercial use for medical pur-
pose since six decades, and till date it is the most popu-
lar oral anticoagulant in use. It is used for prophylaxis
of thromboembolic episodes in deep vein thrombosis
(DVT), pulmonary embolism, mechanical valve re-
placement and stroke due to atrial fibrillation [10].
Warfarin exerts its anticoagulant activity by inhib-
iting the C1 subunit of vitamin K epoxide reductase
enzyme (encoded by VKORC1 gene) and interfering
Pharmacological Reports, 2013, 65, 1375�1382 1375
Pharmacological Reports2013, 65, 1375�1382ISSN 1734-1140
Copyright © 2013by Institute of PharmacologyPolish Academy of Sciences
with cyclic interconversion of vitamin K and its 2,3-
epoxide. This decreases the available vitamin K epox-
ide in liver that is required for carboxylation and in
turn activation of coagulation factors II, VII, IX and X
[2, 8, 10, 18].
Warfarin is a racemic mixture of R- and S-warfarin
and they both differ in their potency and metabolism. S-
warfarin is 5 times more potent than R-warfarin and me-
tabolized mainly by CYP2C9 enzyme in liver to S-7-
hydroxywarfarin whereas R-warfarin is metabolized to
minor end products like R- 6-, 8- and 10-hydroxywarfarin
by CYP1A1, CYP1A2 and CYP3A4 [2, 8, 10].
Currently, the anticoagulation status of an individual
is monitored by blood prothrombin time using interna-
tional normalized ratio (INR). Warfarin has a narrow
range for INR [8] and maintaining this therapeutic
range is very important as a sub therapeutic INR
caused due to underdosing leads to thrombosis and
a supra therapeutic INR due to overdosing increases
the risk of bleeding. Physicians find it extremely prob-
lematic to adjust warfarin dosage as this treatment
shows a large inter- and intraindividual variability. The
cause of this is the complexity of coagulation cascade,
and numerous clinical factors like age, gender, BMI,
vitamin K intake and concomitant drugs [13, 15].
Evidence accumulated in the last decade attributes
almost 40% variability in warfarin dosage to genetic
variants in genes involved in warfarin metabolism and
activity. Aithal and his associates were the first to re-
port direct associations between warfarin dosage and
CYP2C9 gene variants in 1999 [1], while the informa-
tion on VKORC1 gene variants on warfarin doses be-
came available in 2004 [23].
The most reported and studied single nucleotide
polymorphisms in CYP2C9 gene are
CYP2C9*2(430C>T) and CYP2C9*3(1075A>C).
Both *2 and *3 alleles have impaired hydroxylation
of S-warfarin with 12% and less than 5% of wild type
activity, respectively. These variations decrease the
degradation and clearance of warfarin and therefore, a
low dosage is needed to maintain therapeutic INR
[16].
VKORC1-1639G>A, a promoter polymorphism,
present in linkage disequilibrium with VKORC
11173C>T is also an important factor in affecting
warfarin dosage. This polymorphism alters the bind-
ing site for VKORC1 transcription factor and leads to
lower VKORC1 mRNA expression in human liver. As
a result, the steady-state concentration of tissue
VKOR decreases making a person with this variation
more susceptible to inhibition by warfarin and caus-
ing warfarin sensitivity [7, 16].
African-Americans require the highest doses of war-
farin followed by Caucasians, and Asians are most sen-
sitive to warfarin [7]. Amongst Asians too, Indians re-
quire higher doses than Chinese, Japanese and Malays.
A part of this variability can be contributed to differ-
ences in frequency and effect of these genetic varia-
tions. The inhabitants of North India and South India
are ethnically and also genetically very different with
huge difference in the allele frequencies of VKORC1
and CYP2C9 gene variations [12, 19, 22]. Very few
data and that too especially from the South Indian
population is available on the effect of these variations
on warfarin dosage. Hence, due to lack of sufficient
data in a heterogeneous Indian population, the aim of
our study was to identify the magnitude of effect of
CYP2C9*2, CYP2C9*3, VKORC1-1639G>A and
VKORC11173C>T genetic variations on warfarin dos-
age in subjects residing in the metropolitan city of
Mumbai that includes inhabitants who are diverse eth-
nically, culturally and genetically and can thereby rep-
resent the whole of Indian population.
Materials and Methods
Patients
The study included 103 patients who were receiving
warfarin therapy at P. D. Hinduja National Hospital
and Medical Research Centre, Mumbai, India. The
clinical indications of warfarin therapy were mechani-
cal valve replacement, DVT, pulmonary embolism,
atrial fibrillation and stroke. Patients with liver, kid-
ney or gastrointestinal diseases were excluded from
the study. Clinical information including age, gender,
weight, concomitant drugs, mean daily warfarin dos-
age and mean INRs were noted down. The study was
approved by the institutional review board and re-
search ethics committee and informed consent was
obtained from all participants.
DNA extraction and genotyping
Whole blood samples were collected in EDTA tubes.
Genomic DNA was extracted using modified Miller et
al. method [17]. The extracted DNA was genotyped
for VKORC1-1639G>A, VKORC11173C>T, CYP2C9
1376 Pharmacological Reports, 2013, 65, 1375�1382
*2(430C>T) and CYP2C9*3(1075A>C) polymor-
phisms using PCR-RFLP technique. The restriction
enzyme digested products were separated on 3% aga-
rose gel and visualized with ethidium bromide under
UV illumination. The 50 µl reaction mixture for
VKORC1-1639G>A variation consisted of 1X PCR
buffer (Fermentas), 200 µM dNTPs, 3 mM MgCl2, 17
pmol of both forward and reverse primers, 1 unit of
Taq DNA polymerase (Fermentas) and 2 µl of tem-
plate DNA. The 50 µl reaction mixture for each
VKORC11173C>T, CYP2C9*2 and CYP2C9*3 varia-
tion consisted of 1X PCR buffer, 200 µM dNTPs, 1.5
mM MgCl2, 20 pmol of both forward and reverse
primers, 1 unit of Taq DNA polymerase and 2 µl of
template DNA. The primer sequences, PCR condi-
tions and restriction enzymes (Fermentas) used are
summarized in Table 1. The results were further vali-
dated by outsourcing representative samples of all the
variations for automated DNA sequencing by Sanger’s
method to Chromous Biotech, Bangalore. A represen-
tative gel picture and chromatogram for VKORC1 and
CYP2C9 genotypes are given in Figure 1.
Pharmacological Reports, 2013, 65, 1375�1382 1377
Gene variants and warfarin dosing in IndiansSripriya Natarajan et al.
Tab. 1. PCR conditions for genetic analysis of CYP2C9 and VKORC1 variations. a The PCR conditions were repeated for 34 cycles. WT – wildtype; Htz – heterozygous; Mut – homozygous mutant; bp – base pairs
Variation PrimersPCR
conditionsaPCR
product sizeRestrictionenzyme (RE)
REdigestion product size
VKORC1-
1639G>A
F-5’GAGCCAGCAGGAGAGGGAAATAT3’R-5’GTTTGGACTACAGGTGCCTGCC 3’
94°C/1 min68°C/1 min72°C/1 min
291 bp Msp I WT-167 + 124 bpHtz-291 + 167 + 124 bp
Mut-291 bp
VKORC1
1173C>T
F-5’CTAAGATGAAAAGCAGGGCCTAC3’R-5’CTGCCCGAGAAAGGTGATTTCC3’
94°C/1 min60°C/1 min72°C/1 min
201 bp Sty I WT-127 + 74 bpHtz-201 + 127 + 74 bp
Mut-201 bp
CYP2C9*2(430C>T)
F-5’TCCTAGTTTCGTTTCTCTTCCTGT3’R-5’ATAGTAGTCCAGTAAGGTCAGTGA3’
94°C/1 min60°C/1 min72°C/1 min
221 bp Ava II WT-122 + 99 bpHtz-221 + 122 + 99 bp
Mut-221 bp
CYP2C9*3(1075A>C)
F-5’CACGAGGTCCAGAGATGCATTG3’R-5’CTTCGAAAACATGGAGTTGCAGT3’
94°C/1 min60°C/1 min72°C/1 min
135 bp Nsi I WT-116 + 19 bpHtz-135 + 116 + 19 bp
Mut-135 bp
Fig. 1. Representative gel picture andchromatogram showing VKORC1 andCYP2C9 genotypes. 1a: Agarose gel(3%) showing VKORC1 genotypes[Lane 1, 4 – Wild type for VKORC1-1639G>A and VKORC11173C>T, re-spectively; Lane 2, 5 – Heterozygousfor VKORC1-1639G>A and VKORC11173C>T, respectively; Lane 3, 6 –Homozygous mutant for VKORC1-1639G>A and VKORC11173C>T, res-pectively]; 1b-1c: DNA chromatogramfor VKORC1-1639G>A and VKORC11173CT, respectively; 2a: Agarose gel(3%) showing CYP2C9 genotypes[Lane 1, 4 – Wild type for CYP2C9*2and CYP2C9*3, respectively; Lane 2 –Heterozygous for CYP2C9*2; Lane 3 –Homozygous mutant for CYP2C9*2];2b-2c: DNA chromatogram for CYP-2C9*2(430C>T) and CYP2C9*3 (1075-A>C), respectively (Sequencing forCYP2C9*3 variation with reverse pri-mer). ¶ L – 50 base pair DNA ladder,NC – negative control, bp – base pair
Statistical analysis
The distribution of alleles for their accordance with
Hardy Weinberg equilibrium was calculated by c2
test. Comparisons of mean daily warfarin dosage be-
tween different genotypes was calculated by Mann-
Whitney test and odds ratio at 95% CI was also calcu-
lated where necessary. The effect of age, gender,
weight, medications and genotypes on the mean war-
farin dosage was calculated by multivariate logistic
regression analysis. All analysis was carried out using
statistical software Epi Info 3.5.3.
Results
A total of 103 patients (56 males and 47 females) who
were on warfarin therapy were included into the
study. The other baseline demographics are given in
Table 2.
The mean INR for majority of the patients (n = 54;
53%) was 2.0–3.5 irrespective of their clinical indica-
tion, whereas 49 patients (47%) had their mean INRs
lower than that required by clinical indication but
were considered stable by their physicians.
VKORC1-1639G>A and 1173C>T variation was
found to be present in complete linkage disequilib-
rium in our study sample. The genotypic frequencies
of VKORC1 and CYP2C9 genetic variants are given
in Table 3. The frequency of VKORC1 variant allele
was found to be 13.11% and its wild type allele was
found to be 86.89%. The allelic frequency of CYP2C9
wild type allele *1 was found to be 94.6%, whereas
it’s variant allele *2 was found to be present in 5.4%
of our study samples. The variant allele *3 of
CYP2C9 was not found in our study sample. All the
variations were found to be in accordance with Hardy
Weinberg equilibrium (p = 0.04 for VKORC1 and p =
1.86 for CYP2C9*2; df = 1).
1378 Pharmacological Reports, 2013, 65, 1375�1382
Fig. 2. Mean warfarin dosage among VKORC1 and CYP2C9*2 genotypes. § – statistically significant
Tab. 2. Baseline characteristics of study population (n = 103). a Themost common concurrent medications interacting with warfarin andtheir frequency in the study sample. Many patients had more thanone concurrent interacting drugs
Mean age (years): 52 ± 14
Mean weight (kg): 66.5 ± 13.6
Gender:
Male 56 (54%)
Female 47 (46%)
Clinical indication:
Mechanical valve replacement 55 (53%)
Deep vein thrombosis 24 (23%)
Pulmonary embolism 7 (7%)
Atrial fibrillation 6 (6%)
Stroke 6 (6%)
Others 5 (5%)
Concurrent interacting medicationsa:Aspirin 25
Amiodarone 8
Thyroxine 7
Clopidogrel 9
Fenofibrate 2
Phenytoin 3
The mean daily warfarin dosage among different
genotypes is given in Figure 2. No significant differ-
ence (p = 0.67) was seen in the mean daily warfarin
requirements between CYP2C9*2 heterozygotes and
the wild types. Only one patient was found to be ho-
mozygous mutant for CYP2C9*2 variation and re-
quired 2 mg less warfarin dose per day than the wild
types. The mean daily warfarin dosage in patients
with VKORC1 variant allele was, however, found to
be significantly (p = 0.02) lower than those with the
presence of wild type alleles. Patients who were ho-
mozygous mutant for the VKORC1 variant allele re-
quired almost 2 mg/day less and those who were het-
erozygous for the VKORC1 variant allele required
1 mg/day less than their wild type counterparts. Also
one patient was found to be compound heterozygote
for VKORC1 and CYP2C9*2 variations and required
4.5 mg/day warfarin dose.
Along with the genotypic variation, certain clinical
parameters are also found to affect warfarin dosages.
A multiple logistic regression analysis was carried out
with weight, age and VKORC1 genotype with mean
daily warfarin dosage. The results of regression analy-
ses are presented in Table 4. The R2 for final multiple
regression analysis model is 21%.
When the patients carrying VKORC1 and CYP2C9
variations were compared with those having wild type
genotypes, it was found that the likelihood of devel-
oping a supra therapeutic INR within four standard
5 mg/day warfarin dose was 13.96 times higher
among those who carry a VKORC1 variant allele (OR
= 13.96; 95% CI; 4.85 – 44.65). None of the CYP2C9
heterozygote developed supra therapeutic INR, but
CYP2C9*2/*2 homozygote developed supra thera-
peutic INR within four doses of standard 5 mg/day
warfarin dosage.
Discussion
In this study, we characterized the effect of CYP2C9
and VKORC1 genetic variations and clinical parame-
ters on warfarin dose requirements in patients under-
going warfarin therapy.
Although a majority of patients (53%) had their
INR within therapeutic range, 47% of patients had
their INR in the sub-therapeutic range. These indi-
viduals have been on long term warfarin therapy. It is
reported that the risk of thrombosis in such patients is
markedly reduced by long term anticoagulation but is
associated with higher rate of major bleeding. This
risk is further increased with addition of antiplatelet
therapy [5, 14]. Hence, even though these patients’
INRs were less than what their clinical indication de-
manded, they were included into the study because
the physicians considered them stable.
About 90% of the Chinese population carries
VKORC1 variant allele, whereas only 20% of the
Caucasians have these variations [6, 20, 24, 30]. In In-
dians, VKORC1 variant allele is present in 12-14% of
population as given by earlier published articles [12,
19, 22]. The importance of the functional promoter
polymorphism, –1639G>A in VKORC1 gene on the
warfarin dose requirements has also been studied by
many [1, 6, 16, 20, 21, 24, 27, 30, 31]. Our results
were also consistent with their findings with patients
who were identified as harboring VKORC1 variant al-
lele (even as a single copy), requiring lower than stan-
dard warfarin doses to maintain their INR in the thera-
peutic range. In fact, even the presence of a single
copy of the variant allele made the individual sensi-
tive to warfarin therapy. This variant allele was found
Pharmacological Reports, 2013, 65, 1375�1382 1379
Gene variants and warfarin dosing in IndiansSripriya Natarajan et al.
Tab. 3. Distribution of VKORC1 and CYP2C9 genotypes (n = 103)
Gene Genotype Frequency (%)
VKORC1 VKORC1-1639GG and 1173CC(wild type)
78 (76)
VKORC1-1639GA and 1173CT(heterozygous)
23 (22)
VKORC1-1639AA and 1173TT(homozygous mutant)
2 (2)
CYP2C9 CYP2C9*1/*1 (wild type) 93 (90)
CYP2C9*1/*2 (heterozygous) 9 (9)
CYP2C9*2/*2 (homozygousmutant)
1 (1)
Tab. 4. Multiple regression analysis between weight, age andVKORC1 genotype with warfarin dosage. Model R2 = 21.0%
Predictor Coefficient p value
Intercept 3.291 –
Weight 0.048 0.008
Age –0.034 0.01
VKORC1 genotype –1.16 0.003
in 13.11% of our study population while the GA and
AA genotype was found in 22 and 2% of the study
population, respectively.
Seventy six percent patients with VKORC1 variant
allele developed supra therapeutic INR (> 4) within
four standard warfarin doses of 5 mg/day. Their INR
was, however, brought down to therapeutic range and
was maintained within it when their warfarin dose
was decreased to 2–3 mg/day. Thus, the likelihood of
developing a supra therapeutic INR for an individual
carrying VKORC1 variation was found to be 13.96
times higher than the wild types (OR = 13.96; 95%
CI: 4.85 – 44.65).
In Caucasians, CYP2C9*2 variation is found in
16% and CYP2C9*3 variation in 6.5% of the popula-
tion. It is very rare or absent in Chinese and Japanese
population, whereas it is found to be present in 3–8%
in specific Indian cohorts [6, 12, 20, 22, 24, 30]. In
our study sample, we found *2 variant allele to be
present in 5.4% of our study population whereas *3
variant allele was completely absent. This could be
due to the fact that even though this variant is not
completely absent in our population, it is present in
very low frequencies and therefore, may have been
undetected in our small sample size. The results were
also further validated by outsourcing 10% of the rep-
resentative samples in form of PCR products for each
of the variations, for automated DNA sequencing as
also mentioned in the methods section. These varia-
tions due to its rarity have not been studied and re-
ported much in Asians but have been associated with
reduced warfarin doses in Caucasian populations [21,
26, 27, 31]. However, in our study sample, individuals
having *2 variation did not have significant difference
in warfarin dosage over the wild types.
Individuals harboring the CYP2C9*3 variation are
said to have increased risk of supra therapeutic INR
and bleeding complications within 3 months of start
of warfarin therapy [26]. In our study sample
CYP2C9*3 allele was absent and none of the indi-
viduals having CYP2C9*2 variation in heterozygous
form developed supra therapeutic INR. In contrast,
patients harboring VKORC1 variation developed su-
pra therapeutic INR, and thus it can be suggested that
in our study sample, CYP2C9 gene variants had very
little or no effect as compared to VKORC1 variation,
on the warfarin dosage or the risk of developing supra
therapeutic INR and bleeding complications. This is
consistent with the earlier findings which state that re-
duced warfarin dosage by Asians may be contributed
mainly by VKORC1 genetic variations because
CYP2C9 gene variants are very rare or absent in them
[20, 30].
According to the literature, an individual, carrying the
*3 allele of CYP2C9 gene is more important than carry-
ing VKORC1 variant allele, since VKORC1 variant al-
lele only contributes to 25–30% reduction in warfarin
dosage as opposed to almost 95% reduction explained
by CYP2C9*3 variant [26–28]. But on a population
level, VKORC1 is more important simply because it is
found in higher frequency in our population.
Clinical parameters like age, weight, gender have
already been shown to affect warfarin dosage [8].
Hence, a univariate logistic regression analysis of
weight, age, gender, concurrent medications and
VKORC1 and CYP2C9 genotypes individually with
mean daily warfarin dosage was carried out. Of these,
only weight, age and VKORC1 genotype were found
to have p < 0.1. Thus, a multiple logistic regression
analysis was carried out with weight, age and
VKORC1 genotype with mean daily warfarin dosage.
Although it has been seen that females require lower
warfarin doses than males [8], in our present study it
was seen that gender had no effect on warfarin dos-
age. Also warfarin dose requirement increases propor-
tionally with weight, while with increase in age, war-
farin dose requirement decreases. Thus the warfarin
dosage required by a patient needs to be carefully esti-
mated.
Three individuals, in spite of being VKORC1 het-
erozygote, required higher warfarin doses compared
to other VKORC1 heterozygotes and likewise, few in-
dividuals having wild type genotypes required much
lower maintenance dose of warfarin. This can be at-
tributed to certain other variations in same genes or
other genes like CYP4F2, GGCX, APOE, clotting fac-
tor genes, etc. [3, 4, 11, 25, 29]. Hence, further studies
are required to identify these variations and their ef-
fect on warfarin doses.
As cited by Titus, earlier this year, several smaller
studies have drawn attention to the benefit of imple-
menting pharmacogenetic testing for warfarin ther-
apy. This idea is also being backed by various ongo-
ing trails – GIFT, COAG, EU-PACT, etc. [28].
In conclusion, we report that clinical parameters like
age and weight along with VKORC1 gene variants af-
fect the warfarin dosage in our study population. Pa-
tients who are having VKORC1 variations required
lower than 5 mg/day of warfarin dose to maintain INR
within therapeutic range, while majority of CYP2C9*2
1380 Pharmacological Reports, 2013, 65, 1375�1382
heterozygotes and wild types maintained their thera-
peutic INR with the standard 5 mg/day of warfarin
dosage. Thus, along with clinical parameters, a prior
knowledge of the patient’s genotype can be used to de-
cide about initial warfarin dose required by an individ-
ual to maintain therapeutic INR and can thereby help in
reducing the risk of developing supra therapeutic INR
and bleeding complications in Indian patients.
Conflict of interest:
There is no actual or potential conflict of interest capable
of influencing the judgment on the part of any authors.
Acknowledgment:
This study was funded by National Health and Education Society
(NHES) of P. D. Hinduja National Hospital and Medical Research
Centre.
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Received: November 21, 2012; in the revised form: April 12,
2013; accepted: April 18, 2013.
1382 Pharmacological Reports, 2013, 65, 1375�1382