Original Article

Clinical and Applied

Thrombosis/Hemostasis
Frequency of Common VKORC1 2018, Vol. 24(2) 323-329
ª The Author(s) 2016

Polymorphisms and Their Impact on Reprints and permission:

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DOI: 10.1177/1076029616680478
Warfarin Dose Requirement in Pakistani journals.sagepub.com/home/cat

Population

Aisha Qayyum, MBBS, MPhil, PhD1, Muzammil Hasan Najmi, MBBS, MPhil, PhD2,
Qaisar Mansoor, BSc, MSc, MPhil3, Muhammad Irfan, MSc, MPhil, PhD4,
Abdul Khaliq Naveed, MBBS, MPhil, FCPS, PhD5, Andleeb Hanif, BSc, MPhil3,
Ali Raza Kazmi, MBBS, FCPS3, and Muhammad Ismail, BSc, MSc, PhD3

Abstract
Polymorphisms in vitamin K epoxide reductase complex subunit 1 (VKORC1) gene lead to interindividual variability in warfarin
dose requirement. The characterization of genotype frequency distribution is required in different populations for construction of
customized dosing algorithms to enhance the efficacy and reduce the toxicity of warfarin therapy. This study was carried out in
Pakistani population to evaluate the contribution of common VKORC1 polymorphisms to warfarin therapy. A total of 550 stable
patients taking warfarin were enrolled after medical history, physical examination, and laboratory investigations. Single blood
sample was collected after informed consent. Genomic DNA was extracted and genotype analysis for VKORC1 1173C>T and
VKORC1–1639G>A polymorphisms was done by polymerase chain reaction–restriction fragment length polymorphism assay. A
number of samples were also analyzed by direct DNA sequencing for validation of results. Data were analyzed using SPSS version
20. Genotype frequency distributions of VKORC1 1173C>T and VKORC1–1639G>A were found to be different from other
populations. Both of these polymorphisms did not demonstrate significant effect on warfarin dose requirement. Although
Cytochrome P450 2C9 (CYP2C9) and VKORC1 polymorphisms together attributed only 3.8% variability in warfarin dose but it
was statistically significant (p value ¼ .004). It is concluded that there is a need to study genotype frequency distribution and their
effect on warfarin dose variability among different populations due to diversity in outcome. At the same time, no effect on warfarin
dose variation explained by VKORC1 polymorphisms and small variability explained by studied genotypes stresses the need for
exploration of more genetic and nongenetic factors in Pakistani population.

Keywords
Warfarin, VKORC1 1173C>T, VKORC1–1639G>A, PCR-RFLP, direct DNA sequencing, CYP2C9 polymorphism

Introduction carrying G allele. Patients with genotype AA are at risk of

bleeding if same dose given as required by those possessing
Warfarin has been the most commonly prescribed oral antic-
GG genotype. The other SNP significantly affecting warfarin
oagulant for various thromboembolic disorders. Warfarin
results in anticoagulation by inhibiting vitamin K epoxide
reductase (VKOR) enzyme. The gene coding for VKOR is 1
Department of Pharmacology, Fazaia Medical College, Air University,
VKOR complex subunit 1 (VKORC1).1,2 Since VKORC1 Islamabad, Pakistan
2
breakthrough in 2004, several single-nucleotide polymorph- Department of Pharmacology, Foundation University Medical College,
isms (SNPs) have been identified in VKORC1 but very few Islamabad, Pakistan
3
Institute of Biomedical and Genetic Engineering, Islamabad, Pakistan
of them have been found to be affecting the warfarin dose 4
Department of Zoology, Pir Mehr Ali Shah Arid Agriculture University,
requirement. Out of these SNPs, 2 have shown significant Rawalpindi, Pakistan
5
effect on warfarin anticoagulant response. First one is a pro- Department of Biochemistry, Islamic International Medical College, Riphah
moter polymorphism, VKORC1–1639G>A suggested to cause International University, Rawalpindi, Pakistan
changes in VKORC1 transcription-binding site leading to
Corresponding Author:
decreased VKORC1 mRNA expression in human liver cells Aisha Qayyum, Department of Pharmacology, Fazaia Medical College, Air
and decreased VKOR protein synthesis. Patients possessing University, E-9 Islamabad, Pakistan.
A allele require less doses of warfarin as compared to those Email: aisha30102000@yahoo.com
324
anticoagulant response is VKORC1 1173C>T. The carriers of
TT genotype require less dose of warfarin as compared to those
possessing CC genotype to produce same anticoagulation
response. Both VKORC1–1639G>A and VKORC1 1173C>T
have been found to be in linkage disequilibrium (LD).3-7
Different dosing algorithms have been constructed by
researchers from data comprising of demographic factors along
with the frequencies of common VKORC1 and CYP2C9 poly-
morphisms in their populations. The use of genotype-based dos-
ing algorithms has shown better results in terms of improved
efficacy, less adverse effects such as bleeding, and less frequent
laboratory monitoring.4,8-12 We have already studied CYP2C9
polymorphisms and their effect on warfarin dose in Pakistani
population.13 That study data were used to assess the overall
influence of both CYP2C9 and VKORC1 polymorphisms on
warfarin dose requirement in present study. To expedite the use
of VKORC1 genotyping along with CYP2C9 genotypes routi-
nely in anticoagulation practice, it is imperative to determine the
allelic frequency in different populations. A comprehensive dos-
ing model that is applicable regardless of ethnicity can be devel-
oped by identifying the allelic frequency of common alleles in
different ethnic populations. Pakistan, although among the top
10 populous countries in the world, has been underrepresented in
pharmacogenetic studies.
The present study was carried out to determine the genotype
frequencies of VKORC1–1639G>A and VKORC1 1173C>T
polymorphisms in Pakistani population and to determine its
effect on warfarin dose requirement. Furthermore, the com-
bined effect of both CYP2C9 and VKORC1 polymorphisms
on warfarin dose variance was assessed.
Materials and Methods
Study Settings and Protocol
The clinical data collection and laboratory investigations were
done at 2 major clinical setups in Pakistan providing anticoa-
gulation therapy—Armed Forces Institute of Cardiology Raw-
alpindi, and National Institute of Cardiovascular Diseases,
Karachi. The analytical procedures were carried out at Centre
for Research in Experimental and Applied Medicine, Army
Medical College Rawalpindi in collaboration with Institute of
Biomedical and Genetic Engineering Islamabad. The study
protocol was approved by ethical committees of institutes.
Study Participants
The study was conducted in accordance with the current
Good Clinical Practices14 and the Declaration of Helsinki.15
Study participants were adults of either gender between 18
and 65 years of age who were receiving warfarin therapy.
Stable patients taking warfarin were recruited in the study
after informed consent. A stable patient was defined as the
one whose warfarin dose had been constant for at least 3
previous clinic visits over a minimum period of 3 months
and had an international normalized ratio of the prothrombin
time within the range of 1.5 to 3.5.11,16-18 The patients having
Clinical and Applied Thrombosis/Hemostasis 24(2)
hepatic and renal disease, any comorbid disease, or taking
any concurrent medication or diet which would have affected
warfarin therapy were excluded. All participants were Pakis-
tani citizens belonging to different regions of Pakistan to
provide representation from all areas. Each participant was
evaluated by detailed medical history, physical examination,
and laboratory tests.
Genotyping
A total of 550 patients were recruited in the study. A 2 mL of
blood sample was collected in EDTA containing tube and
stored at 4 C for genotyping. The genomic DNA from all of
the samples was isolated mainly by standard organic method
involving chloroform and phenol.19 The protocol was slightly
modified as per requirement of the laboratory working. DNA
isolation kit was used (QIAamp DNA Mini; Qiagen Inc Valen-
cia, CA, USA) to extract genomic DNA from some of the
blood samples that were either less in quantity or somewhat
clotted.
Polymerase Chain Reaction–Restriction Fragment Length
Polymorphism Assay
Genotyping of the VKORC1–1639G>A and VKORC1
1173C>T polymorphisms were done by polymerase chain reac-
tion–restriction fragment length polymorphism (PCR-RFLP)
assays. The sequences of forward (50 -GCCAGCAGGAGAGG-
GAAATA-3 0 ) and reverse (5 0 -AGTTTGGACTA-
0
CAGGTGCCT-3 ) primers used for VKORC1–1639G>A
were obtained from a reported study.20 The sequences of for-
ward (50 -AGAGACTTACTTAAGGTCTA-30 ) and reverse
(50 -TTCCAAGAAGCCACCTGGGC-30 ) for VKORC1 1173C>T
were kindly provided by Professor Pieter H. Reitsma.21 The
optimization of PCR with reproducible results was carried out.
The PCR was carried out for each sample in a final volume of
20 mL containing, 40 ng genomic DNA (2 mL), 2 mL 10 PCR
buffer without Mg2þ, 1 mL of 25 mM MgCl2, 0.2 mL of 5 U/mL
Taq DNA polymerase, 1 mL of 2 mM dNTPs, and 1 mL of each
20 nM forward and reverse primers. The final volume was
adjusted to 20 mL with deionized water. The thermal stages
included initial denaturation at 94 C for 5 minutes followed
by 35 cycles of 45 seconds each of the following—94 C,
55 C, and 72  C. Final extension was carried out at 72  C
for 10 minutes, and the product was subjected to digestion.
The amplified DNA fragment harboring the VKORC1–
1639G>A was digested with Msp1 restriction enzyme (New
England Biolabs (NEB) Inc Ipswich, MA, USA), whereas for
VKORC1 1173C>T, restriction enzyme Sty1 (NEB) was used.
The digestion was carried out by adding 1 mL of respective
enzyme (10 U/mL) and 3 mL 10 digestion buffer to 10 mL of
the PCR product and adjusting the final volume to 30 mL with
dH2O. The mixture was incubated at 37 C for 16 hours. The size
of the RFLP bands was depicted with DNA size reference
ladder.
Qayyum et al 325

Table 1. Allele and Genotype Frequency Distributions of VKORC1–1639G>A and VKORC1 1173C>T Polymorphisms Along With Their
Relationship With Warfarin Dose.

Number of Observed 95% Confi- Warfarin Dose Warfarin Dose

Participants Frequency, dence Expected H-W (mg/wk), Mean + (mg/d), Mean +
Genotypes (%) % Interval Frequency, % P Value SD SD P Value

VKORC1–1639G>A 527 (100) – – – .0003 – – .984

GG 88 (16.7) 16.7 13.52-19.88 26.8 38.84 + 14.68 5.55 + 2.1
GA 370 (70.2) 70.2 66.3-74.1 50 38.88 + 13.3 5.55 + 1.9
AA 69 (13.1) 13.1 10.22-15.98 23.2 38.57 + 9.6 5.51 + 1.37
VKORC1 1173C>T 516 (100) – – – .582 – – .946
CC 115 (22.3) 22.3 18.71-25.89 25 38.57 + 13.36 5.51 + 1.91
CT 285 (55.2) 55.2 50.91-59.49 50 38.47 + 12.67 5.49 + 1.81
TT 116 (22.5) 22.5 18.9-26.1 25 38.94 + 13.0 5.56 + 1.86
Abbreviations: H-W, Hardy-Weinberg; SD, standard deviation; VKORC1, vitamin K epoxide reductase complex subunit 1.

DNA Sequencing VKORC1–1639G>A polymorphism. The allele and genotype

frequency distribution along with expected H-W frequencies
In order to validate the PCR-RFLP genotypes, direct sequen-
for both SNPs are given in Table 1. A p value of more than .05
cing of 100 samples was done through automated capillary
in case of VKORC1 1173C>T implies that observed frequen-
sequencing method. Both SNPs were amplified using the same
cies did not deviate from H-W equilibrium, whereas p value of
primers as mentioned in PCR. The sequencing reaction product
less than .05 in case of VKORC1–1639G>A implies that
was purified and loaded to ABI Genetic Analyzer 3130
observed frequencies are deviated from H-W equilibrium. The
(Applied Biosystem; Life Technologies Waltham, MA, USA)
effect of VKORC1–1639G>A and VKORC1 1173C>T poly-
for sequencing.
morphism on warfarin dose was determined, and the compar-
The amplified PCR product was resolved at 200 bp in case
ison has been summarized in Table 1. There was no statistically
of VKORC1 1173C>T polymorphism. The PCR fragment con-
significant effect of different genotypes on warfarin dose
taining C allele was digested into 2 fragments of 144 and 56 bp,
requirement (p value >.05). Multiple linear regression was per-
whereas T allele was not digested and resolved at 200 bp. The
formed incorporating 4 common SNPs, that is, VKORC1
amplified PCR product was resolved at 290 bp for VKORC1–
1173C>T, VKORC1–1639G>A, CYP2C9*2, and CYP2C9*3.
1639G>A. The PCR fragment containing G allele was digested
The regression modeling revealed that total variation in war-
into 2 fragments of 124 and 166 bp, whereas A allele was not
farin dose explained by these SNPs was 3.8% (R2 ¼ .038) and
digested and resolved at 290 bp.
was statistically significant (p Value ¼ .004).

Data Analysis
Data were analyzed using SPSS version 20.0. The genotypes
and allele frequencies were estimated from the observed num-
bers of each specific allele, and 95% confidence interval was
calculated. The expected Hardy-Weinberg (H-W) frequencies
for genotypes were calculated using H-W Equilibrium equa-
tion. Genetic data for deviation from H-W equilibrium were
tested using w2 test. Analysis of variance (ANOVA) was
applied for comparison of different VKORC1 genotypes with
warfarin dose requirement. A p value of less than .05 was taken
as statistically significant. The ANOVA was followed by post
hoc Tukey test for pair-wise comparison if ANOVA gave p
value of less than .05. Multiple linear regression was carried
out to study the overall influence of both CYP2C9 and
VKORC1 polymorphisms on warfarin dose requirement. A p
value of less than .05 was taken as statistically significant.
Results
A total of 253 male and 297 female participated in the study
with the mean age of 36.9 + 12.1 years. A total of 516 samples
gave successful results for VKORC1 1173C>T and 527 for
Discussion
This study was the first large scale study in Pakistan to report the
population-based frequencies of VKORC1 1173C>T and
VKORC1–1639G>A genotypes and their impact on warfarin
dose requirement. The genotypes frequency distribution in
Pakistani population was different from other international stud-
ies conducted in different populations as shown in Table 2. It is
evident that different genotypes have diverse prevalence and
effect in different populations and regions. These observations
imply that every population has to have its own local frequency
data. Even the population of geographically close countries may
not be having same genotype frequency distribution as obvious
from the results obtained from Pakistani population and those
countries surrounding our geographical boundary. It is also evi-
dent from the comparison of results from studies conducted in
different Asian countries that the word ‘‘Asian population’’ can-
not be reflective of one homogenous population. Different coun-
tries in Asia have different genotypes frequency distribution so
each region or country should be treated as an individual entity.
It has been confirmed by some multiethnic studies conducted in
Asia including participants belonging to different Asian
326 Clinical and Applied Thrombosis/Hemostasis 24(2)

Table 2. Frequency Distribution of VKORC1–1639G>A and VKORC1 1173C>T Genotypes in Different Populations.

VKORC1–1639G>A VKORC1 1173C>T

Population GG GA AA CC CT TT References

Pakistani 16.7 70.2 13.1 22.3 55.2 22.5 Present study

3,6,9,10,22-29
Caucasian 19-59.7 27-60 12-32.5 33.7-43 43.9-50.7 12.7-24
18,30-33
Japanese 0-0.8 15-16 83-86.6 0-1.2 13.8-15.5 83.7-85
11,17,34-37
Chinese 0-1.3 11.5-15.7 80.7-88 1-6 14-25 69-85
38-40
Indian 64-81 17-34 0-2.1 75.9-82.4 16.7-22.9 1-1.6
41,42
Iranian 15 58 27 34.5 48.3 17.2
43-45
Egyptian 23.8-25.5 51-56.6 18-25 17.5 20.5 62
46
Turkish 25 51.5 23.5 – – –
47
Malaysian 1.6 26.4 72 1.7 32.3 66
48
Indonesian 4 29.5 66.5 – – –
49
Korean – – – 1.5 23.1 75.4
Abbreviation: VKORC1, vitamin K epoxide reductase complex subunit 1.

countries.50-52 The results of present study and the other reported
studies in different parts of world in different ethnicities show
how much genetic diversity is encountered all over the world.
Although major part of human genome has been found to be
similar among humans but still the small part is playing its part in
this diversity. Different evolutionary changes have taken their
toll over the period of centuries that have led to population
heterogeneity across the world. Many genetic studies have been
carried out to know the origin of their population to trace their
ancestory.53-57
There are some notable findings regarding VKORC1 geno-
typing and their effect on warfarin anticoagulant therapy in
present study. One is that there was no significant effect of
VKORC1–1639G>A and VKORC1 1173C>T polymorphisms
on warfarin dose requirement. The other one was that no LD
between VKORC1 1173C>T and VKORC1–1639G>A geno-
types was found in the present study. A number of studies have
shown the presence of LD between VKORC1–1639G>A and
VKORC1 1173C>T genotypes.17,38,47 On the basis of this LD,
some studies have grouped different VKORC1 SNPs into cer-
tain haplotypes, and further effect on warfarin dose requirement
was then studied on the basis of these haplotypes.12,52,58 But in
our study, we did not find LD existing between these 2 SNPs. It
may be due to large number of patients with heterozygous var-
iant genotype (GA) which also made VKORC1–1639G>A not
following H-W equilibrium. It is suggested that existence of
either too less or too many heterozygous genotypes in data can
cause deviation from Hardy-Weinberg Equilibrium (HWE). We
have excluded the genotyping technique error by carrying out
direct DNA sequencing as well. Pakistani population is hetero-
geneous and this could lead to changes in genotype presentation
and number.59-61 A number of other studies have also shown
such deviation from HWE without affecting the results.6,12,62-64
As far as the status of LD among these SNPs is concerned, a
recent Italian study also reported nonexistence of LD between
VKORC1 1173C>T and VKORC1–1639G>A genotypes.10 A
number of studies have revealed that different VKORC1 SNPs
group differently to form haplotypes because of diverse LD
relationship in different populations and their distribution varies
in different populations.8,38,52,58,65 In light of our results, it is
imperative that frequency and LD studies for commonly
reported VKORC1 SNPs65,66 ought to be carried out in different
populations including Pakistan to identify the haplotype struc-
ture in these particular populations.
The other noteworthy finding in present study is the effect of
VKORC1 SNPs on warfarin dose requirement. In Pakistani pop-
ulation, both VKORC1–1639G>A and VKORC1 1173C>T poly-
morphic alleles did not have any significant effect on warfarin
dose requirement. Majority of studies have demonstrated that the
presence of variant alleles in VKORC1 1173C>T and VKORC1–
1639G>A polymorphisms results in significant reduction in war-
farin dose requirement.6,10,22,23,43 Few other studies also reported
similar results as ours, in which polymorphism did not show
significant effect on warfarin anticoagulant response.41,51,67 One
aspect of this finding is that we have only analyzed the effect of 2
of the commonly reported VKORC1 SNPs but there are many
more SNPs and mutations in other genes that have been recently
studied in different populations and found to be significantly
affecting the warfarin dose.38,65,66,68,69 These polymorphisms
have not only been found to be responsible for increased sensi-
tivity to the warfarin anticoagulant response but some have been
responsible for warfarin resistance which means patients posses-
sing variant allele require more dose as compared to wild-type
genotype.4,9,10,24,70 It has also been suggested that the presence of
VKORC1 Asp36Tyr polymorphic allele that leads to increased
warfarin dose requirement even overrides the dose reducing
effect of VKORC1–1639A allele.71,72 This implies that there may
be other VKORC1 SNPs prevalent in Pakistani population having
more significant effect on warfarin dose requirement as compared
to these 2 studied SNPs. There may be a possibility of existence of
currently unidentified environmental factors or unknown genetic
variants linked to VKORC1 genotype expression, which could
influence vitamin K metabolic pathway. Another explanation
comes from a recent study that has pointed out the existence of
mechanisms and factors other than VKOR playing significant
role in recycling of vitamin K.73 At the same time, only 3.8%
Qayyum et al
variance in dose was explained by the commonly studied SNPs
(VKORC1 1173C>T, VKORC1–1639G>A, CYP2C9*2, and
CYP2C9*3). In our study on CYP2C9 genotypes, for CYP2C9*2
polymorphism, there was statistically no significant difference in
dose requirement among different genotypes. For CYP2C9*3,
patients with homozygous variant genotype (*3/*3) warfarin dose
was significantly lower than homozygous wild type (*1/*1), but
the dose requirement of heterozygous variant genotypes was not
significantly different.13 That is why the overall variability caused
by all 4 SNPs to the warfarin dose is small (3.8%). There is a wide
range of dose variability explained by these genotypes, reported
in other international studies.10,11,24,30,31,34-35,42,46,49,72 This
implies that the studied SNPs are not the significant contribu-
tor to warfarin dose variability in Pakistani population.
Other SNPs and their regulators may have more contribu-
tion to warfarin dose variance in our population. Another
aspect supporting our finding is that there are more than 30
gene products regulating warfarin pharmacokinetics or phar-
macodynamics, and genetic polymorphisms in these genes
affect warfarin anticoagulant response. Polymorphisms in
some of these genes have been studied in some populations.
These studies have reported substantial prevalence and sig-
nificant effect on warfarin dose by these gene products.24,34-
36,44,46,62,66,69,72
These polymorphisms may be considered to
be prevalent and substantial contributors to warfarin dose
variability in Pakistani population. Further studies on such
polymorphisms are recommended to identify the major
genetic factors in Pakistani population.
Conclusion
From this study, it is concluded that the frequency distribution
of VKORC1–1639G>A and VKORC1 1173C>T genotypes in
Pakistani population is different from that reported in other
world populations. As both VKORC1 SNPs were not found
to be affecting warfarin dose requirement and also the overall
effect of common CYP2C9 and VKORC1 polymorphisms on
dose variance was not significant, so there is need for analysis
of other SNPs in VKORC1 and CYP2C9 as well as polymorph-
isms in other genes reported to affect warfarin therapy.
Acknowledgements
The authors are thankful to National University of Sciences and Tech-
nology (NUST) for facilitating to carry out this project.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for
the research, authorship, and/or publication of this article: The authors
are grateful to Higher Education Commission of Pakistan for funding
this project.
327
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