Lack of association between Glu298Asp polymorphismand coronary artery disease in North Indians

Himanshu Rai • Jacqui Fitt • A. K. Sharma •

Nakul Sinha • Sudeep Kumar • C. M. Pandey •

Suraksha Agrawal • Sarabjit Mastana

Received: 1 August 2011 / Accepted: 19 December 2011 / Published online: 30 December 2011

� Springer Science+Business Media B.V. 2011

Abstract Nitric Oxide (NO) is an important molecule

carrying number of different functions in humans. Published

studies suggest that it may inhibit several key steps involved

in the pathogenesis of atherosclerosis. Inhibition or reduc-

tion of NO due to Glu298Asp polymorphism may accelerate

atherosclerosis. The aim of this study was to determine

whether Glu298Asp polymorphism is implicated in the

pathogenesis of coronary artery disease (CAD) among

North Indian population from the state of Uttar Pradesh,

India. We selected 253 CAD patients and 174 healthy,

normotensive, non-diabetic controls, which were matched

for gender and ethnicity. The Glu298Asp (rs1799983) var-

iant was detected by genotyping subjects, using a poly-

merase chain reaction followed by restriction fragment

length polymorphism. There was no significant difference

found in the genotypic and allelic frequencies between

patients and controls. Our study indicated that Glu298Asp

polymorphism does not play any critical role in the patho-

genesis of CAD, at least in North Indian population.

Keywords Coronary artery disease (CAD) �Glu298Asp polymorphism � Endothelial nitric oxide

synthase (eNOS) � North Indian population

Introduction

Nitric Oxide (NO) is generated by endothelium and is a

potent vasodilator and functions as an important key factor in

the atherosclerotic properties of the endothelium, it is syn-

thesized from L-arginine by at least three isoforms of NO

synthase (NOS) (inducible NOS, constitutive neuronal NOS

and constitutive endothelial NOS [eNOS]) [1]. NO is

responsible for vascular relaxation, it suppresses the adhe-

sion of platelets and leucocytes to the vascular endothelium

[2–5]; it reduces smooth muscle cell proliferation and

migration [5]. It scavenges superoxide radicals and thus has a

vasoprotective effect [2]. Finally it limits the oxidation of

atherogenic low density lipoproteins [3]. These functions of

NO suggest that it may inhibit several key steps involved in

the pathogenesis of atherosclerosis [4].

NO production can be influenced by polymorphisms of

the eNOS gene. The gene is located on the chromosome

7q35–36 and it consists of 26 exons with a total size of

21 kb [6]. The eNOS gene is expressionally and func-

tionally regulated through multiple regulatory steps, and

entails several polymorphisms [3], some of which bear

functional consequences. A point mutation of guanine

(G) to thymine (T) at nucleotide 894 in exon 7 of the eNOS

H. Rai � S. Kumar

Department of Cardiology, Sanjay Gandhi PGIMS,

Lucknow, Uttar Pradesh, India

J. Fitt � S. Mastana

School of Sport Exercise and Health Sciences,

Loughborough University, Leicestershire, UK

A. K. Sharma

Department of Zoology, University of Lucknow,

Lucknow, Uttar Pradesh, India

N. Sinha

Department of Cardiology, Sahara Hospital,

Lucknow, Uttar Pradesh, India

C. M. Pandey

Department of Biostatistics, Sanjay Gandhi PGIMS,

Lucknow, Uttar Pradesh, India

S. Agrawal (&)

Department of Medical Genetics, Sanjay Gandhi PGIMS,

Lucknow 226014, Uttar Pradesh, India

e-mail: sur_ksha_agrawal@yahoo.co.in

123

Mol Biol Rep (2012) 39:5995–6000

DOI 10.1007/s11033-011-1412-z

gene has been first described by Hingorani et al. [7]. This

variant results in the replacement of glutamic acid by

aspartic acid at codon 298 (Glu298Asp). This gene poly-

morphism has been reported to be associated with coronary

spasm [8], essential hypertension [9] and the risk of acute

myocardial infarction (AMI) [3, 10, 11]. Some researchers

recently have reported no association of Glu298Asp poly-

morphism with nitric oxide production [12]. Glu298Asp

polymorphism effects red blood cell (RBC) aggregation as

reported by Bor-Kucukatay et al [13]. Further they dem-

onstrated that the genotypes carrying Asp alleles have

greater RBC aggregability. It has been traditionally proven

that individuals with higher RBC aggregation are more

likely to suffer from acute coronary events [14], especially

the ones involving thrombotic mechanisms.

Controversial results have been obtained with respect to

its association with coronary artery disease (CAD).

Glu298Asp polymorphism has been implicated as a risk

factor for myocardial infarction [10, 11, 15], coronary

artery spasm [10] and CAD [2, 4, 12, 16, 17]. Hingorani

et al. [3] identified the Glu298Asp variant as a major risk

factor for heart disease in population from United King-

dom. Colombo et al. [4] also reported similar association in

population from Italy; they reported a threefold higher

chance of developing coronary artery disease for Asp298

homozygotes compared to the individuals having Glu298

allele. On the contrary, some other studies have also shown

lack of its association with CAD [18–23]. Geo-ethnic dif-

ferences in the populations may have imparted the

observed diversity.

Our understanding of the host’s genetic contribution to

CAD risk and its progression may be improved by the

detection of genetic polymorphism that regulates suscep-

tibility to CAD. Elucidation of genetic susceptibility profile

may also allow implementation of specific prophylaxis

implementation strategies in high risk patients and devel-

opment of potentially novel therapies. Our study is an

attempt in this direction.

Materials and methods

Subjects

253 proven CAD patients and 174 healthy, sex matched,

controls were prospectively included in the study after

obtaining a written informed consent. CAD status was

determined by the detection of at least 50% or more ste-

nosis in one or more native coronary arteries of the patient,

verified through coronary angiography. The healthy case

controls had no known history of ischemic heart disease,

hypertension, diabetes, endocrine or metabolic disorders.

They were selected after administering a treadmill test to

negate the possibility of the patients having an underlying

CAD. Patients who experienced even a single episode of

acute coronary syndrome during last 6 weeks were exclu-

ded. All selected cases and controls were ‘‘North Indians’’

and were residents of Uttar Pradesh, a densely populated

state in northern India. Data pertaining to demographics,

anthropometrics and clinical history was collected using a

uniform clinical proforma. 3 ml of EDTA whole blood was

collected for DNA extraction. DNA was extracted from

blood by salting out method using phenol–chloroform as

described by Comey et al [25] and was purified by ethanol

precipitation. This DNA was used as a template for eNOS

Glu298Asp polymorphism analysis.

An additional 3 ml of blood was drawn for lipid estima-

tion. Methodology used for total cholesterol (TC), Triglyc-

erides (TG) and high density lipoprotein cholesterol (HDL-

c) estimation was done using Bi-directionally interfaced

fully automated analyser. Low and very low density lipo-

protein cholesterols (LDL-c and VLDL-c) were calculated

for subjects with fasting serum TG levels (\400 mg/dl)

using the Friedewald formula [24]; i.e. LDL-c = TC-HDL-

c–(TG/5) and VLDL-c = 0.20(TG) respectively.

DNA genotyping

The Glu298Asp (rs1799983) variant was detected by

genotyping the cases and controls by a polymerase chain

reaction-restriction fragment length polymorphism (PCR-

RFLP) as described by Salimi et al. [16]. All samples were

genotyped after blinding the disease/control status. The

457 bp PCR product was cleaved into the following

genotypes: TT: one band at 457 bp, GT: 3 bands, 457, 320

and 137 bp, and GG: 2 of bands 320 and 137 bp (Fig. 1).

Statistical analysis

Genotypes were determined by viewing the presence or

absence of bands on the gel photographs. The genotypes

were scored, allele frequencies were calculated by allele

Fig. 1 Gel photograph of the eNOS Glu298Asp polymorphism

showing, lane 1 homozygous for mutation (TT) band of 457 bp,

lanes 2, 3, 4, 7, 10 wild type (GG) band of 320 and 137 bp lanes 5, 6,

8, 9 heterozygous for mutation (GT) band of 457, 320 and 137, lane11 DNA Ladder of 100 bp

5996 Mol Biol Rep (2012) 39:5995–6000

123

counting. Statistical analysis was carried out using the

computer packages EXCEL and SPSS (version 16.0). Inde-

pendent t test using SPSS 16.0 software was used to analyse

differences between the means of continuous variables.

Two-tailed P values of\0.05 were considered to be statis-

tically significant. Discrete variables, genotype distribution

and Hardy–Weinberg equilibrium (HWE) were tested using

v2 test. To assess the association of risk between variables,

Odds Ratios (OR’s) were calculated using a contingency

programme with 95% confidence intervals (CI).

Results

It is evident from Table 1 that the subjects in CAD group

were significantly older than control group with a mean age

of 51.29 ± 11.78 versus 44.82 ± 13.57 years respectively

(P \ 0.001). The effect of age on CAD risk was analysed by

creating age brackets. We classified the subjects into three

different groups; \40 years, 40–54 years and C55 years

respectively. Table 1 shows the OR for CAD associated with

each of the three age brackets. An individual falling under

40 years of age bracket is shown to be protective against

CAD (OR = 0.25, CI = 0.16–0.40, P \ 0.005). The age

group 40–54 years showed a statistically increased risk of

CAD (OR = 1.59, CI = 1.05–2.41, P = 0.038). The age

group of over 55 years showed a further increased risk of

CAD (OR = 1.90, CI = 1.27–2.84, P = 0.002).

All serum lipid parameters i.e. TC, TG, HDL-c, LDL-c

and VLDL-c were significantly higher into subjects in

CAD group when compared to those of control group,

P \ 0.0005. There was a significantly higher percentage of

smokers in the CAD group; 62.06% compared with 51.15%

(P = 0.032) in controls. Smokers were found to be at a

higher risk to develop CAD, OR = 1.56 (CI = 1.06–2.31),

P = 0.032. Therefore for a non smoker the risk of devel-

oping CAD was significantly reduced (OR = 0.64,

CI = 0.43–0.95, P = 0.032). No significant difference

were found for gender (P = 0.150) or dietary habit

(P = 0.080) between the two groups.

eNOS Glu298Asp genotypes and allele frequencies

The distributions of the Glu298Asp genotypes in both CAD

cases and controls satisfied the Hardy–Weinberg equilib-

rium. Table 2 shows the distribution of the genotypes and

alleles frequency among the CAD and control groups

which were consistent. Among CAD group the genotypic

frequency was 62.8, 33.2 and 4% for GG, GT and TT

respectively, and in those in controls were 68.4, 28.7 and

2.9% for GG, GT and TT. We found no significant dif-

ference between any of the genotype or allele frequencies

between the CAD and control groups (P [ 0.05).

Due to the positive association of smoking with CAD,

the three genotypes were analysed with respect to smoking

status. Our results demonstrated higher frequency of T

allele in patients who were smokers whilst lower frequency

of G allele in patients which suggests that G allele may be

protective against CAD, while T allele may confer risk of

CAD. Consequently the TT genotype showed the highest

OR of 1.54 compared to an OR of 0.72 for the GG geno-

type. However, we must add that none of these results

reached statistical significance, P [ 0.05 (Table 3).

Independent t test between lipid levels and different

genotypes for the entire group (patients ? controls) was

performed. Significant difference in mean LDL-c levels

Table 1 Demographic and biochemical characteristics of CAD patient and control groups

CAD patients (n = 253) Controls (n = 174) OR (95% CI) v2 P value

Age (years) Mean ± SD (SE) 51.29 ± 11.78 (0.74) 44.82 ± 13.57 (1.03) 0.000*

\40 years n (%) 36 (14.23) 69 (39.65) 0.25 (0.16–0.40) 34.58 0.000*

40–54 years n (%) 97 (38.34) 49 (28.16) 1.59 (1.05–2.41) 4.306 0.038*

C55 years n (%) 120 (47.43) 56 (32.18) 1.90 (1.27–2.84) 9.273 0.002*

% male n (%) 218 (86.17) 140 (80.46) 1.51 (0.90–2.54) 2.075 0.150

TC (mg/dl) Mean ± SD (SE) 175.50 ± 87.43 (5.50) 135.05 ± 31.27 (2.37) 0.000*

TG (mg/dl) Mean ± SD (SE) 200.49 ± 104.88 (6.59) 138.07 ± 60.65 (4.60) 0.000*

HDL-c (mg/dl) Mean ± SD (SE) 32.08 ± 12.96 (0.81) 27.63 ± 9.89 (0.75) 0.000*

LDL-c (mg/dl) Mean ± SD (SE) 101.56 ± 44.78 (2.84) 81.57 ± 24.24 (1.84) 0.000*

VLDL-c (mg/dl) Mean ± SD (SE) 39.81 ± 17.34 (1.10) 28.59 ± 12.40 (0.94) 0.000*

Smokers n (%) 157 (62.06) 89 (51.15) 1.56 (1.06–2.31) 4.585 0.032*

% Non vegetarian n (%) 144 (56.92) 84 (48.27) 1.44 (0.98–2.13) 3.062 0.080

TC total cholesterol, TG triglycerides, HDL-c high density lipoprotein cholesterol, LDL-c low density lipoprotein cholesterol, VLDL-c very low

density lipoprotein cholesterol, CI confidence interval

* P \ 0.05 was considered to be statistically significant

Mol Biol Rep (2012) 39:5995–6000 5997

123

between GG and TT genotypes were seen (90.07 ± 36.69

vs. 113.15 ± 33.18 mg/dl respectively for GG and TT,

95% CI = 4.02–42.14, P = 0.018). No other association

of serum lipid levels with any of the genotypes was

observed.

Discussion

It has been reported that a point mutation of guanine to

thymine at nucleotide 894 in exon 7 of the NOS3 gene,

results in replacement of glutamic acid by aspartic acid at

codon 298 [8]. Philip et.al. [26] suggested lesser production

of NO in eNOS T allele carriers, which may be responsible

for its association with CAD. Glu298Asp gene polymor-

phism has been considered to be a major risk factor for

CAD and several recent studies have shown a possible

association between CAD and Glu298Asp gene polymor-

phism in Italian [4], German [2], British [3], Iranian [16]

and Arabian [17] populations, however on the contrary

some studies reported no association of this polymorphism

with CAD in Austrian [20], Chilian [21], Korean [22],

Turkish [23], Canadian [19] and white Australian popula-

tion [18]. Hibi et al. [11] showed that homozygous Japa-

nese subjects carrying Glu298Asp polymorphism may be

genetically predisposed to acute MI; however, no relation

of this mutation was shown with the severity of coronary

atherosclerosis. Hingorani et al. [3] observed a larger

number of homozygotes for Asp298 variant among patients

with CAD when compared to controls and suggested it as a

risk factor for CAD. Gardemann et al. [2] concluded that

younger patients with T allele show increased risk of CAD

and/or MI. We have also observed such a trend but without

statistical significance. In the present study we found

higher odds ratios for T allele (OR = 1.24) than G allele

(OR = 0.81) but the difference did not reach statistical

significance. Our data revealed a trend that the individuals

with T (Asp) allele could be at a higher risk of developing

the disease. It is possible the presence of Asp allele disturbs

the intricacies of gene–gene interactions, causing the dis-

ease in the mutant individuals. One possible mechanism

has been recently proposed by Bor-Kucukatay et al. [13],

which suggests that genotypes carrying Asp alleles have

higher RBC aggregations. RBC aggregation has tradition-

ally been proven to be increased in patients with CAD as

compared to healthy controls [14].

Contrary to our results, a recent study in Indian Tamilian

population by Angeline et al. [12] have reported a signif-

icantly higher frequency of TT genotype and T allele in

acute MI patients as opposed to those in CAD free controls,

suggesting Glu298Asp polymorphism as a risk factor for

AMI. This report further establishes the presence of geo-

ethnic differences present between north and south Indian

populations and their different susceptibility to diseases

and thus reinforces the need to study these populations

individually.

CAD is largely dependent on the precise orchestration of

numerous factors. The INTERHEART study demonstrated

that traditional risk factors viz. diabetes mellitus, hyper-

tension, raised Apo B/A1 ratio, raised waist to hip ratio,

psychosocial stress, tobacco use, reduced rate of moderate

alcohol consumption, reduced intake of fruits and vegeta-

bles, and reduced physical activity accounted for over 90%

of the composite cardiovascular risk to an individual [27].

However all the individuals exposed to above mentioned

risk factors do not develop CAD indicating that there may

be genetic factors causing predisposition to CAD. In a

normal individual, NO pathway is activated leading

increased NO availability which is further responsible for

normal vasodilation, resulting no rise in the blood pressure.

Variation in eNOS activity can be important in modu-

lating mechanisms influenced by environmental conditions

(especially those which can influence the NOS activity,

such as smoking and alcohol consumption), which may

indirectly increase the risk of CAD. In our study we have

found higher odds ratio for Asp allele (T allele)

(OR = 1.33), suggesting a possible association with CAD

Table 2 Genotype and allele

frequencies in CAD patients

versus controls

* P \ 0.05 was considered to

be statistically significant

Genotypes/Alleles CAD Patients

(n = 253)

Controls

(n = 174)

OR (95% CI) v2 P value

GG 159 (62.8%) 119 (68.4%) 0.78 (0.52–1.18) 1.162 0.281

GT 84 (33.2%) 50 (28.7%) 1.23 (0.81–1.88) 0.759 0.384

TT 10 (4%) 5 (2.9%) 1.39 (0.47–4.41) 0.107 0.743

G allele (frequency) 0.79 0.83 0.81 (0.57–1.14) 1.252 0.263

T Allele (frequency) 0.21 0.17 1.24 (0.87–1.77)

Table 3 Odds ratio for genotypes with CAD risk in smokers

Genotypes/Alleles Odds ratio (95% CI) v2 P value

GG 0.72 (0.42–1.25) 1.077 0.299

GT 1.31 (0.74–2.29) 0.627 0.428

TT 1.54 (0.40–5.96) 0.095 0.758

G allele 0.75 (0.47–1.19) 1.207 0.272

T allele 1.33 (0.84–2.12) 1.207 0.272

* P \ 0.05 was considered to be statistically significant

5998 Mol Biol Rep (2012) 39:5995–6000

123

in smokers, however our results did not reach statistical

significance (P = 0.272).The n-3 fatty acid levels are

possibly related to flow mediated dilation in Asp298 car-

riers but not Glu298 homozygotes. Thus the Glu298Asp

polymorphism may be associated with differences in the

response of the endothelium to both smoking and n-3 fatty

acid status. These early findings are suggestive of gene-

environment interactions with the different NOS3 poly-

morphisms and warrants cautious interpretations. However

in our study we selected CAD patients using very stringent

conditions and observed no association. Significant asso-

ciation of Asp allele with the risk of MI in smokers has also

been reported recently by Dafni et al. [15] in Greek

population. These data, possibly suggest, that presence

of established underlying endothelial dysfunction, as

observed among cigarette smokers, may be necessary for

this polymorphism to attenuate endothelial function and

predispose patients to increased cardiovascular risk. The

extent of the interaction of this polymorphism in smokers,

affecting incidence of CAD in North Indians, however

seems to be limited.

Some studies have shown Asp allele to be associated

with higher LDL-cholesterol. This association has already

been established in hypertensive African–Americans [28]

and Venezuelans [29]. However, the increases were mod-

est, but for the 298Asp genotypes carrying two rather than

one allele did increase the lipid abnormalities significantly,

thus implicating Asp allele as a factor responsible for

raised LDL values. Ours is the first study to report the

association of higher levels of LDL cholesterol with

Asp298 variant among north Indians, the exact mechanism

of this association although needs functional assays.

Due to the lack of functional studies, the exact mecha-

nism pertaining to the relation of Glu298Asp polymor-

phism with enzyme activity, NO production, serum lipids

or CAD susceptibility remains unclear. However, few

published reports explain the mechanism that how

Glu298Asp polymorphism effects the enzyme activity and

NO production. Tesauro et al. [28] demonstrated that eNOS

isoforms are processed differently depending on the pres-

ence of aspartate or glutamate at position 298. This affects

the gene product of eNOS. If aspartate, is present at posi-

tion 298 instead of glutamate, at position 298 it results into

the cleavage in normal tissue and in cells over expressing

eNOS. Since eNOS Asp298 is subjected to selective pro-

teolytic cleavage in endothelial cells and vascular tissues it

might account for reduced vascular NO generation. [29] On

the contrary a study by Mc Donald et al. [30] demonstrated

that the Asp substitution at 298 does not have a major

effect in modulating eNOS activity in vivo. His findings

were further validated by Angeline et al. [12], who reported

no significant difference in NO levels in different

eNOS Glu298Asp genotypes in Tamilian (South Indian)

population. Thus it would be safe to say that due to the lack

of decisive functional studies, especially involving Asian-

Indian population, the exact mechanism pertaining to the

relation of Glu298Asp polymorphism with enzyme activ-

ity, NO production, serum lipids or CAD susceptibility

remains to be largely unclear and warrants further

investigation.

Conclusion

To conclude, our results show that genotype frequencies for

Glu/Glu, Asp/Glu, and Asp/Asp, and Asp allele, are not

significantly different between individuals with and without

CAD in North Indian population. Our data also suggests that

the Asp allele of NOS3 Glu298Asp polymorphism may

increase the risk of CAD among smokers.

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