Original Article

Clinical and Applied

Thrombosis/Hemostasis
Frequency of Common CYP2C9 2017, Vol. 23(7) 800-806
ª The Author(s) 2016

Polymorphisms and Their Impact Reprints and permission:

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DOI: 10.1177/1076029616654264
on Warfarin Dose Requirement journals.sagepub.com/home/cat

in Pakistani Population

Aisha Qayyum, MBBS, MPhil, PhD1,

Muzammil Hasan Najmi, MBBS, MPhil, PhD2,
Qaisar Mansoor, BSc, MSc, MPhil3, Zia-ur-Rehman Farooqi, BSc, MSc, MPhil4,
Abdul Khaliq Naveed, MBBS, MPhil, FCPS, PhD5, Andleeb Hanif, BSc, MPhil3,
Syed Ali Raza Kazmi, MBBS, FCPS3, and Muhammad Ismail, BSc, MSc, PhD3

Abstract
Polymorphisms in cytochrome P450 (CYP) 2C9 (CYP2C9) gene result in interindividual variability in warfarin dose requirement.
There is a need for characterization of genotype frequency distribution 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 CYP2C9 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 CYP2C9*2 and CYP2C9*3 poly-
morphisms 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 the results. Data were analyzed using SPSS version 20. Genotype
frequency distribution of CYP2C9*2 and CYP2C9*3 was found to be different from other populations. Of these 2 polymorphisms,
CYP2C9*2 did not demonstrate significant effect on warfarin dose requirement, whereas CYP2C9*3 did show significant effect (P
value ¼ .012). 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.

Keywords
warfarin, CYP2C9 polymorphism, CYP2C9*2, CYP2C9*3, direct DNA sequencing, PCR-RFLP

Introduction from single-nucleotide base substitution from C to T at codon 430

Warfarin has been the most commonly prescribed oral antic-
oagulant for prophylaxis and treatment of various thromboem-
bolic conditions. It is administered as a racemic preparation of
R- and S-enantiomers. Two enantiomers not only differ in
potency but also in their half-lives and metabolism. R-
warfarin is mainly metabolized by cytochrome P450 (CYP)
1A2, CYP3A4, CYP2C19, and CYP1A1, whereas S-warfarin
mainly by CYP2C9. As S-warfarin is 3 to 5 times more potent
than R-warfarin, so changes in blood levels of S-warfarin affect
the anticoagulant response significantly.1,2
The CYP2C9 enzyme is encoded by CYP2C9 gene that has
been found to contain at least 57 CYP2C9 variant alleles defined
by the human CYP Allele Nomenclature Committee (http://
www.cypalleles.ki.se/cyp2c9.htm), but of these CYP2C9*3 and
CYP2C9*2 have been well studied because of their significant
effect on S-warfarin metabolism. The CYP2C9*2 allele results
located in exon 3 (rs1799853). This single-nucleotide polymorph-
ism (SNP) causes a change in amino acid residue from arginine to
cysteine at codon position 144 (Arg144Cys) on the surface of
1
Department of Pharmacology, Fazaia Medical College, Air University,
Islamabad, Pakistan
2
Department of Pharmacology, Foundation University Medical College,
Islamabad, Pakistan
3
Institute of Biomedical and Genetic Engineering, Islamabad, Pakistan
4
Department of Medical Technology, ShifaTameer-e-Millat University,
Islamabad, Pakistan
5
Department of Biochemistry, Islamic International Medical College, Riphah
International University, Rawalpindi, Pakistan
Corresponding Author:
Aisha Qayyum, Department of Pharmacology & Therapeutics, Fazaia Medical
College, Air University, E-9 Islamabad, Pakistan.
Email: aisha30102000@yahoo.com
Qayyum et al
CYP2C9 enzyme. The CYP2C9*3 allele results from substitution
from A to C at codon 1075 located at exon 7 (rs1057910). This
SNP leads to a change in amino acid residue from isoleucine to
leucine at codon position 359 (Ile359Leu) inside the CYP2C9
enzyme. The most commonly present allele is CYP2C9*1, which
is regarded as wild type and produces CYP2C9 enzyme with
normal activity, whereas the presence of these 2 SNPs results in
decrease in the activity of CYP2C9 enzyme that is more with
CYP2C9*3 than with CYP2C9*2 allele.3-8
The presence of polymorphic alleles of CYP2C9 decreases the
S-warfarin metabolic rate resulting in increased levels of S-
warfarin which in turn leads to lower doses of warfarin required
to produce the therapeutic response without any bleeding risk.6,9,10
The reported studies have demonstrated that VKORC1 and
CYP2C9 gene variants together with demographic factors
account for 50% to 60% variance in warfarin dose require-
ment.10-13 Different dosing algorithms have been constructed
from data including demographic factors along with the fre-
quencies of common CYP2C9 and VKORC1 genotypes in their
populations. The use of pharmacogenetic-based dosing algo-
rithms has shown better outcome in the form of improved
efficacy and less adverse effects such as bleeding.10,14-17
A comprehensive dosing model that is applicable regardless
of ethnicity can be developed by characterizing the frequency of
common polymorphisms in different ethnic populations. This
will further help in drafting worldwide applicable clinical guide-
lines for warfarin prescription. 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 CYP2C9*2 and
CYP2C9*3 polymorphisms in Pakistani population and to deter-
mine its effect on warfarin dose requirement.
Materials and Methods
Clinical Settings and Protocol
The clinical data collection and laboratory investigations were
carried out at 2 major clinical setups in Pakistan providing
anticoagulation therapy, Armed Forces Institute of Cardiology,
Rawalpindi, 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 Patients
The study was conducted in accordance with the current Good
Clinical Practices18 and the Declaration of Helsinki.19 Study
patients were adults of either gender between the age of 18
and 65 years who were receiving warfarin therapy. All parti-
cipants were Pakistani citizens belonging to different regions
of Pakistan to provide representation from all areas. Each
patient was evaluated by detailed medical history, physical
examination, and laboratory tests. Stable patients taking
801
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 normal-
ized ratio of the prothrombin time within the range of 1.5 to
3.5.14,20-22 The patients with hepatic and renal disease, any
comorbid disease, or taking any concurrent medication or
diet, which could have affected warfarin therapy, were
excluded.
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.23 The protocol was slightly
modified per requirement of the working laboratory. DNA
isolation kit was used (QIAamp DNA Mini; Qiagen Inc,
Valencia, CA, USA) to extract genomic DNA from some of
the blood samples that were either less in quantity or some-
what clotted.
Polymerase Chain Reaction-Restriction Fragment Length
Polymorphism Assay
Genotyping of the CYP2C9*2 and CYP2C9*3 polymorphisms
was done by polymerase chain reaction-restriction fragment
length polymorphism (PCR-RFLP) assays. The sequences of
forward (50 -GGAGGATGGAAAACAGAGACTTA-30 ) and
reverse (50 -TGAGCTAACAACCAGGACTCAT-30 ) primers
used for CYP2C9*2 and forward (50 -GCTGTGGTGCAC-
GACGTCCAGAGATGC-3 0 ) and reverse (5 0 -ACACA-
CACTGCCAGACACTAGG-30 ) primers for CYP2C9*3 were
obtained from a reported study.24 The optimization of PCR
with reproducible results was performed. The PCR was carried
out for each sample in a final volume of 20 mL containing
initial and final concentrations of the reagents as given in
Table 1. The thermal stages with their temperatures and num-
ber of cycles are given in Table 2. The amplified DNA frag-
ment harboring the CYP2C9*2 was digested with AVAII
restriction enzyme, whereas for CYP2C9*3 restriction enzyme
NsiI was used. The digestion was carried out by adding 1 mL of
respective enzyme (10 U/mL) and 3mL 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.
DNA Sequencing
In order to validate the PCR-RFLP genotypes; direct
sequencing of 100 samples was done through automated
capillary sequencing method. Both SNPs were amplified
using the same primers as mentioned in PCR. The sequen-
cing reaction product was purified and loaded to ABI
802 Clinical and Applied Thrombosis/Hemostasis 23(7)

Table 1. Polymerase Chain Reaction (PCR) Reagents With Their Concentrations Used for PCR.

Serial No. Reagents With Initial Concentration Reagents With Final Concentration Quantity Used in mL

1 dH2O – 11.8
2 10 PCR buffer 1X PCR buffer 2
3 25 mmol/L MgCl2 1.25 mmol/L MgCl2 1
4 2 mmol/L dNTPs 0.1 mmol/L dNTPs 1
5 5 U/mL Taq DNA polymerase 0.05 U Taq DNA polymerase 0.2
6 20 nmol/L forward primer 1.0 nmol/L 1
7 20 nmol/L reverse primer 1.0 nmol/L 1
8 40 ng/mL sample genomic DNA 4 ng 2
– Total reaction volume – 20
Abbreviations: dNTPs, deoxynucleotides; PCR, polymerase chain reaction.

Table 2. Thermal Profile for the PCR for CYP2C9 Alleles. bp. The amplified PCR product was resolved at 298 bp for
CYP2C9*3. The PCR fragment containing A allele was
Number of
Thermal Stages Temperature Time Cycles
digested into 2 fragments of 274 bp and 24 bp, whereas C allele
was not digested and resolved at 298 bp. The results obtained
Stage 1 Initial denaturation 94 C 5 minutes 1 on gel electrophoresis were confirmed on DNA sequencing
Stage 2 Denaturation 94 C 45 seconds 35 electropherogram. The results obtained from both methods
Annealing 60 C 45 seconds were in 100% concordance.
Extension 72 C 45 seconds
A total of 509 samples gave successful results for
Stage 3 Final extension 72 C 10 minutes 1
Storage 4 C CYP2C9*2 and 527 for CYP2C9*3 polymorphism. The allele
and genotype frequency distribution along with expected H-W
Abbreviation: CYP2C9, cytochrome P450 (CYP) 2C9; PCR, polymerase chain frequencies for both SNPs are given in Table 3. A P value of
reaction.
more than .05 in both SNPs implies that observed frequencies
did not deviate from H-W equilibrium. The effect of
CYP2C9*2 and CYP2C9*3 polymorphisms on warfarin dose
was determined, and the comparison has been summarized in
Genetic Analyzer 3130 (Applied Biosystem, Life Technol- Table 4. There was statistically significant effect of different
ogies, Carlsbad, California) for sequencing. The samples for CYP2C9*3 genotypes on warfarin dose requirement (P value
DNA sequencing were selected randomly among all samples <.05), whereas CYP2C9*2 polymorphism did not have any
giving positive results for PCR-RFLP so that the results can significant effect on warfarin dose (P value >.05).On the basis
be validated. of pairwise comparison of different CYP2C9*3 genotypes
(Table 5), it has been inferred that patients possessing homo-
Data Analysis zygous polymorphic CYP2C9*3/*3 genotype required lesser
warfarin dose when compared to homozygous wild-type
Data were analyzed using SPSS version 20.0. The genotypes
CYP2C9*1/*1 genotype (AA), as the difference in dose
and allele frequencies were estimated from the observed num-
requirement was statistically significant (P value <.05).
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- Discussion
tion. Genetic data for deviation from H-W equilibrium were
This study was the first large-scale study in Pakistan to report
tested using w2test. Analysis of variance (ANOVA) was applied
the population-based frequencies of CYP2C9*2 and
for comparison of different CYP2C9 genotypes with warfarin
CYP2C9*3 genotypes and their impact on warfarin dose
dose requirement. A P value of <.05 was taken as statistically
requirement. This study has pointed to the predominance of
significant. Analysis of variance was followed by post hoc
wild-type genotype that is more evident in CYP2C9*2 when
Tukey test for pairwise comparison if ANOVA gave a P value
compared to CYP2C9*3 in Pakistani population. The geno-
of <.05.
types frequency distribution in Pakistani population was differ-
ent from other reported studies conducted in different
populations as shown in Table 6. It is evident that these 2 poly-
Results morphisms have diverse prevalence and effect in different
The amplified PCR product was resolved at 396 base pair (bp) populations.24,36,38,43,50-52 In view of these observations, every
in case of CYP2C9*2 polymorphism. The PCR fragment con- population has to have its own local frequency data. Even if we
taining C allele was digested into 2 fragments of 223 bp and compare our results with those populations surrounding our
173 bp, whereas T allele was not digested and resolved at 396 geographical boundary, the diversity is obvious. It is also
Qayyum et al 803

Table 3. Allele and Genotype Frequency Distributions of CYP2C9*2 and CYP2C9*3 Polymorphisms.

Genotypes Number of Patients, n Observed Frequency, % 95% Confidence Interval Expected H-W Frequency, % P Value

CYP2C9*2 509 - - - .066

CC (CYP2C9*1/*1) 470 92.3 89.98-94.62 91.4
CT (CYP2C9*1/*2) 33 6.5 4.36-8.64 8.4
TT (CYP2C9*2/*2) 6 1.2 0.25-2.15 0.2
CYP2C9*3 527 - - - .47
AA (CYP2C9*1/*1) 325 61.7 57.55-65.85 59.3
AC (CYP2C9*1/*3) 163 30.9 26.95-34.85 35.4
CC (CYP2C9*3/*3) 39 7.4 5.17-9.63 5.3
Abbreviation: H-W, Hardy-Weinberg.

Table 4. Relationship of Warfarin Dose With CYP2C9 Genotypes.

Genotypes Number of Patients, n (%) Warfarin Dose, mg/week, Mean (SD) Warfarin Dose, mg/day, Mean (SD) P Value

CYP2C*2 509 (100) .173a

CC (CYP2C9*1/*1) 470 (92.3) 38.84 + 13.52 5.55 + 1.93
CT (CYP2C9*1/*2) 33 (6.5) 38.56 + 11.56 5.51 + 1.65
TT (CYP2C9*2/*2) 6 (1.2) 28.55 + 7.43 4.08 + 1.06
CYP2C*3 527 (100) .012b
AA (CYP2C9*1/*1) 325 (61.7) 40.53 + 13.53 5.79 + 1.93
AC (CYP2C9*1/*3) 163 (30.9) 37.64 + 13.69 5.38 + 1.96
CC (CYP2C9*3/*3) 39 (7.4) 35.02 + 14.35 5.0 + 2.05
Abbreviation: SD, standard deviation.
a
Nonsignificant.
b
Significant.

Table 5. Pairwise Comparison of Warfarin Dose Among CYP2C9*3 Table 6. Minor Allele Frequency (MAF) Distribution of CYP2C9*2
Genotypes. and CYP2C9*3 Genotypes in Different Populations.

CYP2C9*3 Genotypes P Value MAF (%)

AA (CYP2C9*1/*1) AC .071a Population CYP2C9*2 CYP2C9*3 References

CC .046b
AC (CYP2C9*1/*3) AA .071a Pakistani 4.45 22.85 Present study
CC .528a Caucasian 7.1-14 4.7-13.6 16,24–30
CC (CYP2C9*3/*3) AA .046b African 0 0.5-1 31
AC .528a Indian 0.6-4.8 1.7-13.2 32–35
Iranian 7.3-28 0-4.5 31,36
a
Nonsignificant. Chinese 0 3-5.7 15,21,37
b
Significant. Japanese 0 1.8-5.9 22,38–42
Israeli 9-13.6 7.4-8.5 43,44
noticeable from studies conducted in different Asian countries Egyptian 3.3-14 3.7-12.2 45–48
Indonasian 0 2.5 49
that the word ‘‘Asian population’’ cannot be reflective of one
homogenous population. This observation has been confirmed
by some multiethnic studies conducted in Asia including
patients belonging to different Asian countries.38,53,54 So each variant allele on warfarin dose requirement is concerned, the
region should be treated as an individual entity. A small part of results are not based on the diversity of genotype frequency
human genome is playing its part in this genetic diversity, as distribution but on the basis of the relative contribution of this
the major part has been found to be similar among humans. variant allele on warfarin anticoagulant response. Some of the
Different evolutionary changes have played their part over the studies have demonstrated significant effect on warfarin dose
period of centuries that have led to population heterogeneity that is reduced in patients possessing variant alleles.16,25,45,58,59
across the globe.55-57 Other studies have reported similar results as ours, where
Although warfarin dose was less in patients with homozy- CYP2C9*2 variant alleles did not show any significant effect
gous variant genotype for CYP2C9*2 polymorphism, there was on warfarin dose requirement.10,17,32,43,46,50,60,61 Even within
no statistically significant difference in dose requirement the same population, different studies presented different
among different genotypes. As regards the effect of CYP2C9*2 results, some exhibiting effect, others not.17,45,46,61,62 This
804
implies that although CYP2C9*2 is a contributor to warfarin
dose variance because it decreases CYP2C9 enzymatic activity
to some extent,50,51,63 but if this effect is not that strong, at
times it is weighed down or modified by other genetic and
nongenetic factors that act as more observable contributors.
In our study, warfarin dose requirement for patients with
homozygous variant genotype for CYP2C9*3 (*3/*3) poly-
morphism was significantly lower than homozygous wild type
(*1/*1). But the dose requirement of heterozygous variant gen-
otypes was not significantly different from either homozygous
wild type or homozygous variant genotypes. Apart from the fact
that different genotype frequency distributions are reported in
different populations, the effect of CYP2C9*3 variant allele on
warfarin dose was found to be significant in almost all the stud-
ies with the exception of few.39,64 Same significant effect was
seen in patients in the present study as well. The patients with
variant allele achieved therapeutic anticoagulant response with
significantly less dose of warfarin when compared to those hav-
ing wild-type genotype. This implies that CYP2C9*3 variant
allele is an effectual contributor to warfarin dose variability by
decreasing the CYP2C9 enzymatic activity leading to reduced S-
warfarin clearance. This aspect can be further elucidated in the
future studies by analyzing S- and R-warfarin levels in plasma
and their relationship with different CYP2C9 genotypes.
Conclusion
The frequency distributions of CYP2C9*2 and CYP2C9*3
genotypes in Pakistani population were different from those
reported in other world populations. CYP2C9*2 polymorphism
was not found to be affecting warfarin dose, whereas presence
of CYP2C9*3 polymorphic alleles reduced the dose require-
ment. This implies that CYP2C9*3 variant allele is an effectual
contributor to warfarin dose variability.
Acknowledgment
The authors are grateful to Higher Education Commission (HEC) of
Pakistan for funding this project. The authors are thankful to National
University of Sciences and Technology (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: Project was
funded by Higher Education Commission (HEC) Pakistan.
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