Download Variability in CYP2C9 allele frequency

Pharmacological Reports
Copyright © 2013
2013, 65, 187–194
by Institute of Pharmacology
ISSN 1734-1140
Polish Academy of Sciences
Variability in CYP2C9 allele frequency: A pilot
study of its predicted impact on warfarin response
among healthy South and North Indians
Risha Nahar1,2, Roumi Deb2, Renu Saxena1, Ratna Dua Puri1,
Ishwar Chander Verma1
1
Centre of Medical Genetics, Sir Ganga Ram Hospital, Rajinder Nagar - 110060, New Delhi, India
2
Amity Institute of Biotechnology, Amity University, Sector-125, Noida - 201301, Uttar Pradesh, India
Correspondence: Risha Nahar, e-mail: [email protected]
Abstract:
Background: Wide variability exists in the frequency of pharmacogenetic markers for anticoagulant response in different populations.
There is insufficient data on the prevalence of these variant genotypes in the Indian population. This study aims to determine the frequency of various genotype combinations of CYP2C9*2, *3 and VKORC1-1639G>A polymorphisms in the South and North Indians.
Methods: Genotyping was carried out by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) technique in 209 North Indians (NI) and 82 South Indians (SI). Warfarin maintenance dose was predicted for all subjects based on FDA
approved genotype-based dose estimates from revised COUMADIN medication guide. Fisher exact test and c2 test were applied to
compare categorical data among the SI and NI groups.
Results: In SI and NI, the allele frequency of CYP2C9*2 was 0.006 and 0.05 (significant variation; p < 0.001); of CYP2C9*3 was 0.09
and 0.11; and of VKORC1-1639A was 0.14 and 0.19 (not significant), respectively. The variation in the frequency of combined
CYP2C9/ VKORC1 genotypes revealed plausible difference in warfarin response among SI and NI. Based on the FDA approved revised dosing guidelines, significantly higher percentage of NI were likely to require intermediate dose (3–4 mg/day; p = 0.015, RR =
2.16) and were also predicted to have an increased risk of bleeding episodes and over anticoagulation (p = 0.012, RR = 1.93).
Conclusions: Genotype frequency of CYP2C9 and VKORC1 SNPs is variable among the two ethno-geographically distinct Indian
populations. This could translate into diverse warfarin response among the Indian population.
Key words:
warfarin, acenocoumarol, India, thrombosis, drug response, adverse effects, CYP2C9, VKORC1
Abbreviations: CYP2C9 – cytochrome P450 subfamily IIC
polypeptide 9, NI – North Indians, SI – South Indians, SNP –
single nucleotide polymorphism, VKORC1 – vitamin K epoxide reductase complex, subunit 1
Introduction
Coumarin anticoagulant drugs are used in the treatment and prophylaxis of venous and arterial thromboembolic events; warfarin (Coumadin) being the
most widely prescribed worldwide. Acenocoumarol
and phenprocoumon are administered in Europe [3]
while in India, acenocoumarol (Acitrom) and warfarin
are the most commonly prescribed. The widespread
use of these drugs is limited by the wide interindividual variability (20 fold) in the dose required to
achieve therapeutic anticoagulation [24]. Current dosage protocols (based on demographic or clinical information) run the risk of severe bleeding and hemorrhage (when on higher dose) or slow and inadequate
anticoagulation (when on lower dose).
Pharmacological Reports, 2013, 65, 167–194
187
Warfarin and acenocoumarol are metabolized primarily in the liver by cytochrome P-450 2C9 enzyme (encoded by CYP2C9), and exert their anticoagulant effect
by inhibiting vitamin K epoxide reductase complex,
subunit 1 protein (encoded by VKORC1). In western
countries, pharmacogenomic studies on these two genes
have clearly shown their value in individualizing treatment. These have helped the physicians to reduce the
risk of bleeding and hemorrhage without compromising
the effectiveness of therapy [26]. Indeed, in year 2007,
the US Food and Drug Administration (FDA) considered evidence of the benefits of genotyping to be sufficiently strong for it to revise warfarin label in year 2007,
recommending lowering of initial doses in patients having the CYP2C9 *2 or *3 or VKORC1-1639G>A alleles
[8]. In 2010, the Coumadin® label was revised again
with specific pharmacogenetic guided dosing ranges derived from multiple published clinical studies [6].
In 2006, Takahashi et al. [22] showed that the dosing
requirement for a similar intensity of anticoagulation
varies across different populations. Asians (Chinese,
Japanese, Malays) were reported to require lower maintenance dose than Caucasians and Indians, while African Americans require higher dose [22]. Before introducing pharmacogenetic-based dosing of coumarin anticoagulants in Indians, it would be useful to determine
the prevalence of the different genotype combinations of
CYP2C9 (*2, *3) and VKORC1 (-1639G>A) in the
South and North Indian population. The people of
Northern India are known to be descendents of Aryans
(Indo-European-speaking group) while the South Indian
population are considered to be the Dravidian ethnic
group [2]. Dermatoglyphic and genomic marker studies
have confirmed the diversity between these two ethnogeographical Indian ethnic populations [2, 17]. This
study was aimed to identify and compare the proportion
of South and North Indians who are carriers of variant
CYP2C9 and VKORC1 polymorphisms. As this is a pilot study in health subjects, a more systematic prospective study including larger cohort of patients with well
defined clinical and extended candidate gene-SNP panel
are needed to get a finer picture of variability in warfarin
response among South and North Indians.
Materials and Methods
The study was carried out at the Centre of Medical
Genetics, Sir Ganga Ram Hospital, New Delhi, India.
188
Pharmacological Reports, 2013, 65, 187–194
The study protocol was approved by the hospital’s
Ethics Review Board and is in accordance with the
ethical guidelines for biomedical research on human
participants (Indian Council of Medical Research).
Study population
Healthy volunteers (n = 291) were recruited from
a period of February to November 2011. The ‘healthy’
status was based on the information they had provided
themselves during their visit to the genetic clinic for
routine antenatal screening. Prospective organ donors
who visited the clinic for HLA matching tests were
assumed to be healthy. Of these, 209 (102 males; 107
females) belonged to the Northern states of India
[Punjab (n = 65), Haryana (n = 38), Uttar Pradesh (n =
35), New Delhi (n = 40), Uttarakhand (n = 31) – IndoAryan ethnic group] and 82 (40 males; 42 females)
were from Southern India [Tamil Nadu (n = 25),
Andhra Pradesh (n = 32), Kerala (n = 25) – Dravidian
ethnic group]. The study was planned with a 5% precision level (95% confidence interval) and sample
sizes were calculated accordingly. Ethnicity was established by inquiring about the individuals’ maternal
and paternal grand-parental place of origin and language. Informed consent was obtained from all subjects prior to their inclusion in the study.
DNA extraction
DNA was isolated from two ml of whole blood (with
EDTA as anticoagulant) using standard salt-extraction
method [14] and stored at 4°C prior to genotyping
analysis. The DNA purity and quantity was measured
using UV visible spectrophotometer (NanoDrop
2000, Thermo Scientific, USA).
Genotyping of CYP2C9*2 (430C>T); *3 (1075A>C)
and VKORC1-1639G>A polymorphisms
Polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) was used
for genotyping VKORC1–1639G>A, CYP2C9*2 and
CYP2C9*3 as described previously [7, 21]. Amplification of DNA samples was carried out by three separate PCR reactions, each with 20 picomoles of the
specific primer pair, 0.1 mM dNTP, 100 to 200 ng genomic DNA, 0.6 U of Taq DNA polymerase (Genei)
in 10 mM Tris–HCl (pH 9.0), 1.5 mM MgCl2, 50 µM
KCl. PCR amplification was accomplished with
Predicted coumarin response in healthy Indians
Risha Nahar et al.
Fig. 1. (i) Agarose gel band pattern for VKORC1-1639A: The variant abolishes Mspl restriction site. Lane 1 is the undigested PCR product. Lanes
3 and 7 show heterozygosity (AG) while lanes 2, 4, 5 and 6 show pattern of the wild type genotype (GG). (ii) Agarose gel band pattern for
CYP2C9*2 (430T): The variant abolishes AvaII restriction site. Lane 1 is the undigested PCR product. Lanes 2, 3, 4 and 6 show homozygosity
for the wild type genotype (CC) while lanes 5, 7 and 8 are heterozygous for the polymorphism (CT). (iii) Polyacrylamide gel band pattern for
CYP2C9*3 (1075C): The variant abolishes NsiI restriction site: Lane 1 is the undigested PCR product. Lanes 3, 4 and 13 show heterozygosity
(AC) while the rest show the wild type genotype (AA)
4 min of initial denaturation at 94°C; followed by 30
cycles of 94°C, 60°C, and 72°C for 1 min each and
a final extension at 72°C for 7 min. Controls of
known genotype as well as no template controls were
included in each batch of PCR. Restriction fragment
size analysis was performed. The Nsi1 digested products were then separated on 10% polyacrylamide gel
followed by silver staining while the AvaII and MspI
digested products were separated on 2% agarose and
visualized by ethidium bromide staining. The resulting band patterns for the restriction fragment analysis
for the three SNPs are illustrated in Figure 1. The
genotyping results were validated by gene resequencing. Since the cleavage recognition site for NsiI restriction enzyme used for detection of CYP2C9*3
(1075A>C) may be affected by the presence of
CYP2C9*4 (1076T>C), 50 samples observed to be
heterozygous or homozygous for the *3 variant were
resequenced to detect *4 variant in these samples.
Although other variants of VKORC1 and CYP2C9
may be present, the alleles tested in the current study
are the most important for determining dose requirement and are the only valid biomarkers for warfarin
sensitivity and dosing considered by the FDA.
Prediction of warfarin response based on FDA
approved dose estimates and recommendations
Based on the 2010 revised Coumadin dosing recommendations with pharmacogenetic data [6], the expected warfarin maintenance dose range was esti-
mated independently in the two ethno-geographic
groups in the present study. The FDA approved classification of genotype-guided warfarin dose estimates
have been “derived from multiple published clinical
studies” and are as given below; s with *1/*1, AA;
*1/*3, GG; *1/*3, AG; *1/*2, AG; *1/*2, AA; *2/*2,
GG; *2/*2, AG and *2/*3, GG are recommended 3.0
to 4.0 mg/day and those with genotypes *1/*1, GG;
*1/*1, AG and *1/*2, GG have been suggested
a higher dose range of 5.0 to 7.0 mg/day. The revised
dosing recommendations also highlight the prolonged
time to achieve therapeutic INR effect for a given
dosage regimen in carriers of CYP2C9 *1/*3, *2/*2,
*2/*3, and *3/*3 as compared to non-carriers of the
above CYP2C9 genotypes (*1/*1; *1/*2). The guidelines, based on several published studies, also emphasize the increased risk of over anticoagulation
(INR>3) or bleeding in CYP2C9 *2 and *3 carriers [6].
Statistical analysis
Genotype frequencies were checked for HardyWeinberg equilibrium using c2 test. Fisher exact probability test and c2 test (with Yates correction) were applied to compare categorical data among the SI and
NI groups. Relative risks were calculated where applicable. All statistical analysis was done using the SPSS
package (version 15.0). A p value < 0.05 was considered to be statistically significant.
Pharmacological Reports, 2013, 65, 167–194
189
Results
Tab. 1. Comparison of CYP2C9 and VKORC1 genotype and allele
frequencies in the study cohorts
South Indians
(SI, n = 82)
The subjects were of the age range 19 to 43 years
(mean age = 28 ± 8.8 years). The genotype and allele
frequencies of CYP2C9 (*1, *2, *3) and VKORC11639G>A in the South and North Indian population
are shown in Table 1. The genotypic frequencies were
found to be in Hardy-Weinberg equilibrium (p = 0.1).
The allele and genotype frequency did not differ significantly within subjects of different states within
North India or South India and hence these are represented as two groups; the North Indians and South Indians, respectively.
Genotype$ frequencies n (%)
CYP2C9 (430C>T, 1075A>C)
*1/*1
69 (84.1) P
*1/*2
1 (1.2)
*1/*3
9 (11)
*2/*3
0
*3/*3
3 (3.7)
Among the SI and NI, no significant difference was
observed in the VKORC1-1639 A minor allele frequency (Tab. 1) and neither in the percent of carriers
of the polymorphism (AA and GA; 28.1% vs. 35.9%
in SI and NI, respectively).
Predicted warfarin response based on combined CYP2C9 and VKORC1 genotypes
The majority of both North and South Indians were envisaged to require higher (5–7 mg/day) warfarin maintenance dose to achieve therapeutic anticoagulation
[74.6% (156), 85.4% (70), respectively] due to the
overall high prevalence of CYP2C9 *1/*1 and
190
Pharmacological Reports, 2013, 65, 187–194
145 (69.4)
20 (9.6)
42 (20)
2 (1)
P
0
GG
59 (71.9)
134 (64.1)
GA
23 (28.1)
72 (34.4)
0
3 (1.4)
AA
VKORC1 allele and genotypes
P
VKORC1-1639G>A
CYP2C9 allele and genotypes
The CYP2C9*2 allele frequency showed highly significant variation in the SI and NI (p = 0.007). Five
CYP2C9 genotypes were detected in these two cohorts (*1/*1, *1/*2, *1/*3, *2/*3, *3/*3). Of these,
genotypes *1/*1 and *3/*3 were observed to be in
significantly higher frequency in the SI (n = 82) as
compared to NI (n = 209; p = 0.01 and 0.02, respectively). Conversely, the SI had a much lower frequency of *1/*2 genotype as compared to the NI (p =
0.01). A total of 26.5% were carriers of the *2 and/or
*3 variant alleles with significant deviation in frequency observed between the SI and NI groups (15.9
vs. 30.6%, respectively; p = 0.01). Resequencing of
the 50 DNA samples with observed *3 variant (genotyped using NsiI digestion) did not reveal presence of
*4 allele in any sample.
North Indians
(NI, n = 209)
$$
Allele frequencies
CYP2C9*1
0.9
0.84
CYP2C9*2
0.006 P
0.05
CYP2C9*3
0.09
0.11
VKORC1-1639G
0.86
0.81
VKORC1-1639A
0.14
0.19
$
Genotype *1/*1 = 430CC/1075AA; *1/*2 = 430CT/1075AA; *1/*3 =
430CC/1075AC; *2/*2 = 430TT/1075AA; *2/*3 = 430CT/1075AC;
*3/*3 = 430CC/1075CC, CYP2C9 *1 is the wild type allele and refers
to CYP2C9 alleles other than *2 and *3. P Statistically significant
(p value < 0.05) variation in frequency between SI and NI, $$ Allele
*1 = 430C/1075A; *2 = 430T; *3 = 1075C
VKORC1 GG wild-type genotypes. The percentage of
NI likely to require intermediate (3–4 mg/day) dose
was significantly greater than that of SI [24.4% (51) vs.
11% (9), respectively; p = 0.015, relative risk (RR) =
2.16; 95% confidence interval (CI) = 1.12–4.18]. The
subjects who were likely to require low dose (0.5–2.0
mg/day) formed the minority group [0.9% (2) NI and
3.6% (3) SI] showing no significant variation. Also,
considerably larger proportion of NI were genetically
predisposed to warfarin-induced bleeding episodes or
over anticoagulation (INR > 3) due to increased frequency of CYP2C9*2 and *3 variants as compared to
SI [30.6% (64) vs. 15.9% (13), respectively; p = 0.012;
RR = 1.93; 95% CI = 1.13–3.31]. Prolonged time to
achieve stable therapeutic INRs was expected in 14.6%
(12) of South Indians and 21% (44) of North Indians
with no significant deviation (p = 0.249).
Predicted coumarin response in healthy Indians
Risha Nahar et al.
Tab. 2. Allele frequency of the three SNPs in different world-wide populations
Ethnic Population
Reference study
Caucasian
Takahashi et al. 2006
Swedish
Yasar et al. 1999 (i);
Wadelius et al. 2007 (ii)
French
Yang et al. 2003 (i);
Bodin et al. 2005 (ii)
Netherlands
Schalekamp et al. 2004
(i); 2006 (ii)
n
CYP2C9*2 (%)
CYP2C9*3 (%)
VKORC1-1639A (%)
115
14.3
10.9
42.2
430 (i),
181 (ii)
10.7
7.4
39
151 (i);
222 (ii)
15
8.0
42
231
(i & ii)
16.5
19.9
64.9
Greek
Markatos et al. 2008
98
15.5
7.5
48.5
German
Burian et al. 2002 (i);
Geisen et al. 2005 (ii)
118 (i);
200 (ii)
14
5.0
42
Russian
Gaikovitch et al. 2003
290
10.5
6.7
NA
Japanese
Takahashi et al. 2006
64
0
1.6
89.1
Chinese
Yang et al. 2003 (i);
Yuan et al. 2005 (ii)
394 (i);
104 (ii)
0.1
3.6
91
Korean
Yoon et al. 2001
574
0
1.1
NA
Takahashi et al. 2006
66
0
0.8
8.6
African-American
$
Lee et al. 2006
43
4
18
NA
South India$$
Jose et al. 2004
346
4
8
NA
South India$$$
Adithan et al. 2003
135
2.6
6.7
NA
Indians
#
Rathore et al. 2010
102
4.9
3.92
14.22
South Indians (SI)
Present study
82
0.6
9
14
North Indians (NI)
Present study
209
5
11
19
Total cohort (SI + NI)
Present study
291
4
10
17
North India
NA = Not available; CYP2C9*2 (430C>T; rs1799853); CYP2C9*3 (1075A>C; rs1057910); VKORC1 -1639G>A (G3673A; rs9923231); $ from
Singapore); $$ from states of Andhra Pradesh, Karnataka or Kerala; $$$ only from Tamil Nadu state; # mainly from Uttar Pradesh state
Discussion
The present study has analyzed genotypes of three
SNPs in a total of 582 chromosomes (n = 291) of
which 164 chromosomes (n = 82) were of individuals
of SI (Dravidian ethnic origin) and 418 of NI origin (n
= 209). This study is the largest of its kind in the Indian subcontinent to compare the genotype-based predicted warfarin response in two ethno-geographically
distinct groups in India. The current study, reports the
allele and genotype frequency of VKORC1-1639G>A
SNP in the SI population and reports no significant
difference from that in NI. However, the VKORC11639 minor allele frequency in the current population
was observed to be smaller in contrast to Caucasians
[22], and other world populations [4, 10, 19, 20, 25,
27, 28] except the African Americans [22] (Tab. 2).
The CYP2C9*2 allele frequency was observed to
be significantly different between the SI and NI
groups. More specifically, the relatively smaller *2 allele frequency in SI was similar to that reported in
other Asian populations (Japanese [22], Chinese [27,
30] and Korean [29]) and African Americans [22],
while the *2 allele frequency in NI is similar to the
Caucasians [22] and Russians [9]. In two other studies
in Southern India, the CYP2C9*2 allele occurred with
a frequency of 2.6% (includes subjects originating
from Tamil Nadu) [1] and 4% (includes subjects from
Kerala, Andhra Pradesh and Karnataka) [11] while in
the 82 SI analyzed in the present study, it was comparatively much lesser (0.6%). This discrepancy may
Pharmacological Reports, 2013, 65, 167–194
191
be due to the smaller sample size of SI in the present
study and variation in the ethno-geographic origin of
subjects in the above studies.
The CYP2C9*3 allele frequency in the present Indian
cohort corresponds to that observed in some Caucasian
populations [4, 13, 22, 25, 27, 28], but comparatively
lesser than that reported in the Netherlands [19, 20]. A
recent study [16] which analyzed 102 NI reported
smaller frequencies of CYP2C9*3 and VKORC1-1639A
than that reported in the 209 NI in the current study. This
discrepancy could be because most of the study cohort
in the above study originated from the state of Uttar
Pradesh while the present study included individuals
originating from the states of Punjab, Haryana, New
Delhi, Uttarakhand and Uttar Pradesh.
There is strong evidence that patients who are homozygous or heterozygous for CYP2C9*2, *3 or/and
VKORC1-1639G>A are sensitive to warfarin and acenocoumarol. They require low maintenance dose
(CYP2C9*2, *3 carriers requiring reduction in dose
by 16% and 41% for heterozygous and homozygous
carriers, respectively), take a longer time to achieve
stable dose with international normalized ratio (INR)
values within the therapeutic range (2.0 to 3.0–3.5) [3,
5, 18, 22]. Multiple variants of CYP2C9 or VKORC1
increase the risk of over anticoagulation (INR ³ 4.0)
and bleeding episodes [5]. Individuals heterozygous
for the variants (CYP2C9*1/*2, *1/*3, VKORC1 AG)
are partially sensitive to coumarin derivatives requiring intermediate doses [5, 22]. Carriers of CYP2C9
variants also have delayed stabilization of anticoagulation with both drugs [3]. Individuals with CYP2C9
*1/*1, VKORC1 -1639GG genotype are known to require the highest amount of weekly dose and are
sometimes known as extensive or ultra metabolizers
[5, 18, 22].
In the present study, extrapolation of FDA approved genotype-guided warfarin dose estimates revealed that a minority of North and South Indians
were predicted to require low (0.5–2 mg/day) dose
to achieve therapeutic anticoagulation. Intra-ethnic
variation in warfarin response was observed where in
a significantly higher percentage of NI would require
intermediate (3–4 mg/day) dose and would be at an increased risk of bleeding episodes and over anticoagulation (INR > 3) as compared to SI. This estimation is
based on reports from multiple published studies [3,
5, 18, 22]. However, since the SI sample group is
smaller than that of NI, extrapolation of these dose estimates in rest of the Indian population must be done
192
Pharmacological Reports, 2013, 65, 187–194
with caution. Large prospective studies are warranted
to extend our findings and truly explore the variation
in coumarin response in the South and North Indians.
Several studies [15, 23] have established that
VKORC1 SNP (-1639 A allele) accounts for 19% to
30% of variance in the dose of warfarin, while
CYP2C9 SNPs (*2, *3) have a contribution of 3.2 to
12%. Prediction of warfarin response solely dependent on genotype, as attempted in the current study, is
limited in explaining only a part of the inter-individual variability. As the present study was carried out
in a healthy population (who were not on anticoagulation therapy), it is limited in not taking into account
the non-genetic and clinical factors such as age, sex,
ethnicity, body weight, concomitant medications,
comorbidities and dietary vitamin K intake that are responsible for more than 50% variability in oral anticoagulation therapy [6]. There is a lack of genome wide
association studies of oral anticoagulant response in the
South and North Indian populations. Hence, the plausible role of rare variations in CYP2C9 and VKORC1
genes and variation in other genes (APOE, CALU,
EPHX1, ABCB1, GGCX, CYP4F2) reported to be associated with warfarin response in other ethnic groups
[15, 23–25], remains uncertain in the Indian population, and hence were not included in the current pilot
study. Clearly, this may result in reduced accuracy of
predicting warfarin dose in clinical settings.
Variation in coumarin response and its genetic contribution in different populations vary mostly due to
differences in the frequencies of the common
CYP2C9 and VKORC1 polymorphisms or prevalence
of specific polymorphisms in certain populations. The
present study demonstrated that the North and South
Indian populations are predicted to require variable
warfarin dose due to their varied genotype frequency
and may necessitate different pharmacogenetic dosing
algorithms for the two Indian populations. However,
a more systematic study including larger cohorts with
well defined demographic/ethnicity profiles, extended
candidate gene and SNP panel and comparison of predictive algorithms are needed to get a finer picture of
variability in warfarin response among Indians. The
identification of coumarin-sensitive patients by pharmacogenetic testing, along with appropriate clinical
evaluation will allow the informed patient and physician to work together for optimizing long term anticoagulant therapy while avoiding adverse effects of
the drug.
Predicted coumarin response in healthy Indians
Risha Nahar et al.
Conclusions
Studies have proved that prospective pharmacogenetic-based dosing for oral anticoagulants can
help better optimize the dose and reduce drug associated bleeding events in patients. Before pharmacogenetic testing can be introduced in the Indian population, knowledge about the frequency of the variant
genotypes is important to predict its usefulness and
general applicability.
The two ethno-geographically distinct populations,
the South and North Indians, are observed to be diverse with respect to frequency of CYP2C9 *2 allele
and *1/*1, *1/*2 and *3/*3 genotypes.
The CYP2C9*3 allele frequency (10%) was similar
to the Caucasians but higher than other Asian populations. The VKORC1-1639A allele frequency (17%)
was observed to be much lower than the Caucasian
and Asian populations.
Prediction of warfarin dose requirement revealed
variability among SI and NI. Significantly higher percentage of NI were estimated to require intermediate
dose (3–4 mg/day) and were also predicted to have an
increased risk of bleeding episodes and over anticoagulation as compared to SI.
Conflict of interest statement:
All authors declare: no financial relationships with any organizations
that might have an interest in the submitted work in the previous
three years, no other relationships or activities that could appear
to have influenced the submitted work.
Acknowledgments:
We would like to thank Sir Ganga Ram Hospital for the research
grant to conduct the study and acknowledge Ms. Parul Takkar
for her help with statistical analyses.
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Received: September 2, 2011; in the revised form: August 8, 2012;
accepted: October 8, 2012.