Accepted Manuscript

Genetic polymorphisms of eNOS (-786T/C, Intron 4b/4a & 894G/T) and its
association with asymptomatic first degree relatives of coronary heart disease
patients

Galimudi Rajesh Kumar, Kondapalli Mrudula Spurthi, Gundapaneni Kishore Kumar,

Tupurani Mohini Aiyengar, Chiranjeevi Padala, Nivas Shyamala, Cingeetham
Anuradha, Banapuram Swathi, Sanjib Kumar Sahu, Altaf Ali, Hanumanth Surekha
Rani
PII: S1089-8603(16)30110-0
DOI: 10.1016/j.niox.2016.09.001
Reference: YNIOX 1592

To appear in: Nitric Oxide

Received Date: 14 July 2016

Revised Date: 23 August 2016
Accepted Date: 4 September 2016

Please cite this article as: G.R. Kumar, K.M. Spurthi, G.K. Kumar, T.M. Aiyengar, C. Padala, N.
Shyamala, C. Anuradha, B. Swathi, S.K. Sahu, A. Ali, H.S. Rani, Genetic polymorphisms of eNOS
(-786T/C, Intron 4b/4a & 894G/T) and its association with asymptomatic first degree relatives of
coronary heart disease patients, Nitric Oxide (2016), doi: 10.1016/j.niox.2016.09.001.

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 ACCEPTED MANUSCRIPT

Genetic Polymorphisms of eNOS (-786T/C, Intron 4b/4a & 894G/T) and its Association with
Asymptomatic First Degree Relatives of Coronary Heart Disease patients

Galimudi Rajesh Kumar MSc PhD1, Kondapalli Mrudula Spurthi MSc PhD1, Gundapaneni Kishore
Kumar MSc (PhD)1, Tupurani Mohini Aiyengar MSc (PhD)1, PadalaChiranjeevi MSc (PhD)1,
Shyamala Nivas MSc (PhD)1, Cingeetham Anuradha MSc PhD1, Banapuram Swathi MSc (PhD)1,
Sanjib Kumar Sahu MD DNB2, Altaf Ali MSc (PhD)1, Hanumanth Surekha Rani MSc PhD1**

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1
Department of Genetics, Osmania University, Hyderabad
2
Durgabai Deshmukh Hospital and Research Centre, Hyderabad

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** Corresponding Author

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Short/running title: Genetic Polymorphisms of eNOS in FDRS

Name and complete address for the corresponding author

Dr. H. Surekha Rani

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Assistant Professor
Department of Genetics
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Osmania University
Hyderabad-500 007, Telangana State, INDIA
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Abstract

Background
The eNOS gene has been extensively screened for important variations in the promoter (-786T/C),
Intron 4 (4b/4a) and exon 7 (894G/T) regions in multiple diseases. Prevalence of Coronary Heart
Disease (CHD) in the young is much higher in Indians when compared to other ethnic groups and
first-degree relatives (FDRS) of CHD patients are at risk correlating with earlier age-of-onset and
poor prognosis.

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Objective
The objective of this study is to understand the genetic mechanism of eNOS -786T/C, Intron 4b/4a

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and 894G/T polymorphisms/haplotypes in the development of CHD and to identify individuals
having coronary risk factors among the FDRS of CHD patients by comparing them with the general
population.

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Materials &Methods
The study population consisted of 300 CHD patients with angiographically documented, 100 FDRS
and 300 age and sex matched healthy controls. Genotyping of eNOS -786T/C, Intron 4b/4a was

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carried out by Allele specific-Polymerase Chain Reaction (As-PCR) and 894G/T by PCR-restriction
fragment length polymorphism (PCR-RFLP) assays and confirmed by agarose gel electrophoresis.
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Results
The frequency of risk factors for CHD includes age, blood pressure, obesity, diabetes mellitus,
smoking, non-vegetarian diet, family history are higher in CHD patients and FDRS compared to
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controls (p<0.01). The genotype distribution, allele frequencies and haplotypes of the eNOS -
786T/C, 4b/4a and 894G/T were significantly different between CHD patients, FDRS and controls.
eNOS gene haplotypes GCB, TCB (G-allele of 894G/T, C-allele -786T/C, B-allele of Intron 4b/4a
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respectively) were associated with high nitrite/nitrate levels compared to GTB in both FDRS and
CHD patients (p<0.01).
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Conclusion
The study shows the importance of eNOS polymorphism in the pathogenesis of CHD and predicting
the risk of future coronary events in asymptomatic healthy FDRS and suggests the use of new
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therapeutics and initiating the measures for prevention or delaying the progression of the disease by
life style modification or treatment modalities in high-risk individuals.
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Key words: Coronary heart disease (CHD), Endothelial nitric oxide synthase (eNOS), First degree
relatives (FDRS), Haplotypes, Linkage Disequilibrium (LD), RNA-Secondary Structures,
Three-dimensional Modelling.
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Introduction:
Coronary heart disease (CHD) is a multifactorial disease resulting due to the accumulation of
atheromatous plaques within the walls of the coronary arteries that supply the myocardium (the
muscle of the heart) with oxygen and nutrients. CHD is the major cause of morbidity and mortality
in the developed world. The magnitude of this problem is profound, as CHD claims more lives than
all types of cancer combined and the economic costs are also considerable. Although currently a
problem of the developed world, the world health organisation predicted that the acquiring of

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western habits in developing countries results in global economic prosperity and could lead to an
epidemic of atherosclerosis [1].
The Asian Indian population accounts for a fifth of all global deaths from CHD. In the Indian

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subcontinent CHD deaths have doubled since 1990, and are predicted to rise a furthermore 50% by
2030[2].

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Patients with a substantial number of measurable CHD risk factors may have progression of
atherosclerosis disease at a much faster rate (younger age) compared with those with few or no risk
factors, such as conventional (gender, age), non-modifiable (family history, diabetes mellitus) and
modifiable risk factors (hypertension, obesity, smoking, food habits, physical activity and

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dyslipidemia) [3].
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Family history is a predominant and autonomous CHD risk factor, especially for early onset disease.
It has been reported that having a First Degree Relatives (FDRS) with premature CHD is a
significant risk factor for the development of CHD [4][5].
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Evidence shows that oxidative stress caused due to the disturbance in the balance between the
production of reactive oxygen species (ROS) and antioxidant defence plays a vital role in the
pathogenesis of coronary atherosclerosis and its complications. ROS is produced in cells by oxygen-
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derived species resulting from cellular metabolism and other interactions with cells of exogenous
compounds such as redox-cycling drugs, carcinogenic compounds, and ionising radiations.
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Enhanced formation of ROS may affect four fundamental mechanisms that contribute to
atherogenesis: oxidation of LDL, endothelial dysfunction, vascular smooth muscle cells growth, and
monocytes migration. NO, perhaps the most important endothelium-derived vasorelaxant factor is
scavenged by ROS[6].
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It is evident that physiological and functional alterations in endothelial cells may play a pivotal role
in the evolution & progression of the atherosclerotic lesion. Therefore, polymorphism in
endothelial nitric oxide synthase (eNOS) is considered as one of the major predisposing factors for
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endothelial dysfunction [7][8]. It has been reported that polymorphic variants of 894G/T, -786T/C
and Intron 4b/4a of eNOS may play an important role in atherosclerosis. The genetic variations may
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alter eNOS activity or regulation and possibly leads to endothelial dysfunction and the pathogenesis
of several cardiovascular diseases [9].

Objective:
The objective of this study is to understand the genetic mechanism of eNOS involved in the
development of CHD through analysis of eNOS -786T/C, Intron 4b/4a and 894G/T polymorphisms
and to identify individuals having coronary risk factors among the first-degree relatives (FDRS) of
patients with CHD by comparing them with the general population.
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Materials and methods:

The study population consists of300 patients with angiographically documented CHD, admitted at
the cardiology section of Durgabai Deshmukh Hospital and Research Center, Hyderabad and 100
FDRS of the patients and 300 controls with no known history of any disease. The healthy controls
representing same geographical area without any history of CHD were included after a thorough
interview and clinical examination. Patients with concomitant cardiomyopathy, valvular heart
disease, acute renal failure, acute and chronic bacterial or viral infections, asthma, tumours or

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connective tissue diseases and those who are on dialysis were excluded from the study. This study
was approved by Institutional Ethics Committee for Biomedical Research of Institute of Genetics &
Hospital for Genetic Diseases, Hyderabad.

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Detailed written informed consent was obtained from all the subjects of this study. All the subjects
were examined clinically, and detailed history was recorded with particular reference to the known
risk factors for CHD, including hypertension, family history, diabetes mellitus, smoking, food

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habits, life style, etc.
Following an overnight fast, blood samples were drawn into two tubes, with and without
anticoagulant for biochemical and molecular analysis. Genotyping of eNOS polymorphism was

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carried out in the samples of 300 CHD, 100 FDRS and 300 controls.
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Statistical analysis
Epidemiological and clinical information along with experimental data recorded for all the cases
were coded and tabulated in separate Microsoft Excel sheets for statistical analysis. The different
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statistical tools used to analyse the quantitative and qualitative variables in the present study are
IBM SPSS version 20.0, SNPSTAT(http://bioinfo.iconcologia.net/index.php?module=Snpstats),
HAPLOVIEW(http://www.broad.mit.edu/mpg/haploview),Generalized Multifactor Dimensionality
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Reduction (GMDR) (GMDR Software Beta version 0.7), Multifactor dimensionality reduction
(MDR)and Graph Pad Prism Version 5.0.
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Insilico Analysis
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Secondary structures for eNOS -786T/C, Intron 4b/4a were identified by using RNA Vienna server
tool. Three-dimensional Modelling for eNOS894G/T was identified by I-TASSER online server
tool. Splice site analysis was identified by Human Splice Cite Finder (HSF) tool.
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Results:
Demographic data of Controls, FDRS and CHD patients (Categorized)
The number of CHD patients, FDRS, and controls along with their demographic data is presented in
Table 2 (Categorized). The risk factors for coronary heart disease include gender, history of
hypertension and DM, family history, smoking (male). The ratio of male to females in CHD patients
was about 3.2:1, FDRS 2.3:1 and controls 1.2:1 respectively.

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Demographic data of Controls, FDRS and CHD patients (Continuous)
The continuous demographic data is presented in Table 3. The risk factors for coronary heart

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disease, age and blood pressure, were significantly associated with CHD patients followed by FDRS
compared to controls while BMI was found to be normal among three groups.
Genotype distribution of eNOS -786T/C, Intron 4b/4a and 894G/T polymorphism in Controls,

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CHD patients and FDRS
Genotype distribution, Allele frequency, Chi-square, Odds ratio and 95% confidence interval (CI) of
the eNOS -786T/C, intron 4b/4a and 894G/Tin CHD patients, FDRS and controls are shown in

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Table 4. The study follows the Hardy-Weinberg equilibrium. The frequency of TC genotype in-
786T/C polymorphism is high among CHD patients (55.3%) followed by FDRS (49%) compared to
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controls (34%) while the frequency of 4b/4b genotype was almost similar in the three study groups
and the frequency of GT genotype in894G/T polymorphism is high among CHD patients (44%)
followed by FDRS (40%) compared to controls (30%).
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eNOS -786T/C and Intron 4b/4a Secondary Structure

To find out the stability of SNPs of eNOS we have used the RNA Vienna server and found that -
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786C allele has lesser entropy hence it is more stable than that of -786T allele (Figure 1) and 4b
allele has lesser entropy hence it is more stable than that of 4a allele (Figure 2).
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eNOS 894G/T Three-dimensional Modelling

To find out the Three-dimensional Modelling of eNOS 894G/T we have used I-TASSER online
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server and found that T allele has additional alpha helices when compared to G allele (Figure 3).

Human Splice Cite Finder (HSF) Analysis

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eNOS Intron 4a/4b allele variants were analysed by human splicing finder and found that Intron 4a
polymorphic variant created a new intron-exon splicing enhancer site (ESE) and doesnt have any
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impact on splicing.

HapMap Analysis of eNOS -786T/C, Intron 4b/4a and 894G/T polymorphisms

To find out the allele frequencies of eNOS -786T/C, Intron 4b/4a and 894G/T in different ethnic
groups along with Indians we have used HapMap and found that the T allele of -786, the b allele of
Intron 4b/4a and G allele of 894 were higher in our population (Figure 4).
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eNOS haplotypes (-786T/C, 894G/T& 4b/4a) and their association with CHD susceptibility in
Controls, CHD patients, and FDRS
Based on the genotype distributions, we estimated and compared four haplotypes between the
Controls, CHD patients, and FDRS groups. We found that GCB, TCB haplotypes (894G/T, -786T/C
& 4b/4a) were significantly associated with CHD patients and FDRS (Table 5).

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Plots Representing Linkage Disequilibrium (LD) of alleles at different positions of eNOS
(-786T/C, Intron 4b/4a and 894G/T) in Controls, CHD patients and FDRS

The SNP combination of eNOS -786T/C, Intron 4b/4a and 894G/T was found to be in complete LD

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among CHD patients (D'=1.0) and strong LD in both Controls and FDRS (D'=0.75-0.95)which
indicates influence of above combination on disease susceptibility (Figure 5).

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MDR gene-gene interaction

MDR has been performed to find out SNPs (Single nucleotide polymorphisms) interaction and its
relation to disease. It has been found that eNOS-786T/C is the best model, and it is strongly

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interacting with 4b/4a and 894G/T based on Cross consistency value (CV) and p-value between
three study groups Controls vsCHD patients (Figure 6), Controls vs FDRS (Figure 7).
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Fruchterman-Rheingold analysis between Controls, FDRS and CHD Patients
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It has been found that eNOS-786T/C and 4b/4a are having a strong synergetic interaction in CHD
patients with entropy of 10.21% and FDRS with 6.07 %, while 894G/T and 4b/4a having moderate
synergistic interaction with entropy of 2.86% and 4.98% respectively. The interaction between -
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786T/C and 894G/T is found to be lower (Figure 8).

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eNOS haplotypes vs. nitrite/nitrate levels in Controls, FDRS and CHD patients
The presence of the GCB and TCB haplotypes of the eNOS gene polymorphism was associated
with the higher serum nitrite/nitrate levels compared to GTB Haplotype in both FDRS and CHD
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patients(p = 0.01) (Figure 9). There is no significant difference in the levels of serum nitrite/nitrate
levels in controls.
Discussion:
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As compared to other ethnic groups, CHD rates among Indians are 2 to 4 fold higher overall and 5
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to 10 fold higher in those younger than 40 years of age, irrespective of gender, region, or social
class. The manifestation of CHD in a more extensive form at a younger age despite a relatively low
longitudinal burden of conventional risk factor points to some other risk factors, presumably
genetically determined, which predispose our population to an increased risk at a much younger
age[10].
Several reports have been revealed that having an FDR with premature CHD is a significant risk
factor for the development of CHD. It has also been reported that the relativity of developing early-
onset CHD in a first-degree relative is between 3.8 to 12.1 and depends on the age of onset (AOO)
of the proband and higher risk correlates with earlier AOO [11][12].
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The potency of a positive family history as a risk factor for the disease may well derive from its
integrated reflection of multiple environmental and genetic factors shared by members of a family.
The risk of CHD that arises from genetic cause should result on average from the same risk factor
within a single family [13].
Among the several gene polymorphic variants common polymorphic variations in the promoter,
coding region, and introns of the eNOS gene have been the focus of interest as potential sources of
inter-individual variation and its role in the pathobiology of cardiovascular disease. Many studies

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have addressed and reported possible relationships between polymorphisms of eNOS and vascular
disease states such as coronary artery disease [14][15], myocardial infarction [16][17][18],
hypertension[19], stroke[20] or coronary artery spasm[21].But, in the present study, we have
evaluated the role of 3 polymorphisms in CHD patients, FDRs and controls, and also analysed the

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haplotypes, LD, MDR, substantiated by insilico analysis of secondary structures and three-
dimensional modelling to understand the molecular mechanism of eNOS in CHD development.

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We have analysed the role of eNOS-786T/C polymorphism in the development of CHD and found
that there was a significant association between eNOS-786C allele and CHD. The study satisfied the
Hardy-Weinberg equilibrium. The genotype distribution, allele frequencies and odds ratios of
eNOS-786T/C polymorphism among controls, CHD patients and FDRS are presented in Table 4,

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and we found that the C allele might increase susceptibility to develop CHD. To know the stability
of the eNOS -786T/C polymorphism insilico analysis was performed using RNA Vienna server tool
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for secondary structure prediction and it was found that -786 C allele has lesser free energy
compared to -786T allele, which shows its stability (Figure 1) and may enhance the activity of gene
expression.
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Another widely studied polymorphism of eNOS is a VNTR (Variable Number Tandem Repeats) in
intron 4 (27-bp repeats). Recent findings indicate that this polymorphism regulates eNOS
expression through small interference RNA (siRNA). Endothelial cells with carriers of the allele
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with five repeats express higher quantities of siRNA, thus leading to lower eNOS expression when
compared with those carrying the allele with four repeats [22].
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There are studies, which report that the eNOS-Intron 4b/4a polymorphism of the eNOS gene has an
association with CHD in several populations [23][24][25]. In the present study, we have also
evaluated the eNOS-VNTR 4b/4a polymorphism in the development of CHD and found that there is
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a significant increase in the frequency of 4b4b-genotype when compared to 4b4a and 4a4a
genotypes in CHD patients & FDRS, indicating the role of eNOS-Intron 4b/4a polymorphism in the
development of CHD and its complications. The study satisfied the Hardy-Weinberg equilibrium.
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The genotype distribution, allele frequencies and Odds ratios of eNOS-Intron 4b/4a polymorphism
among controls, CHD patients, and FDRS are presented in Table 4 and we found that the 4b allele
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might increase susceptibility for developing CHD.

We also analysed the secondary structure of eNOS-Intron 4b/4a and found that 4b repeat has lesser
free energy than that of 4a repeat indicating its stability (Figure 2) further confirming its role in
CHD patients. As 4b/4a SNP was mapped at the intronic position, therefore we analysed the role of
SNP in splicing mechanism by HSF analysis, and it has been found that 4b/4a is creating an intronic
ESE site and do not have any impact on splicing mechanism of the gene.
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The third most studied eNOS polymorphism is 894G/T, corresponds to a guanine to thymine
substitution at position 894. In particular, the G to T polymorphism at position 894, resulting in a
change from Glu to Asp at codon 298, is the only polymorphism identified to date that changes the
eNOS protein sequence, leading to speculation that genetic variation at this site may alter eNOS
activity or regulation. The 298Asp variant has been associated with myocardial infarction
[26][27][24] and reported as a risk factor for coronary artery disease in a Caucasian population [28],
while other studies do not support these findings [20][18]. A paucity of quantitative functional data
has fueled controversy as to whether the Glu298Asp variant directly alters eNOS activity or

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regulation, rather than being a possible genetic marker of association with the disease phenotype in
some populations.
It was also evident that substitution of amino acid alters the function of protein Glu298Asp

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(G894T). Further, bioinformatic analysis revealed the conformational change from helix to tight
turn in the eNOS suggesting that homozygosity for the Asp298 variant may result in a reduction in
eNOS activity [29].

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Earlier to this study we have analysed the 894G/T polymorphism in 150 controls,150 CHD cases
[30]and found significant association in development of CHD while in the present study we have
further confirmed our observations by including 300 controls, 300 CHD cases and 100 FDRS and

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found that there was a significant association between eNOS 894T-allele and CHD. The study
satisfied the Hardy-Weinberg equilibrium. The genotype distribution, allele frequencies and Odds
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ratios of eNOS-894G/T polymorphism among controls, CHD patients, and FDRS are presented in
Table 4, and FDRS with T allele carriers may have the susceptibility to develop CHD.
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Further, we have analysed the three-dimensional modelling of eNOS 894G/T by I-TASSER online
server [31] and found that the T allele showed additional alpha helices when compared to the wild-
type (G' allele) (Figure 3). Based on the above observation, we propose a hypothesis, wherein the
addition of the alpha helices could act as compounding factor for the reduced function of the
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catalytic substrate encoded by eNOS, thus reducing the vasodilatory effect of the final product
(NO).
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To evaluate the combined effect of eNOS3 polymorphisms (eNOS -786T/C, Intron 4b/4a &
894G/T) with CHD, we have performed haplotype analysis using SNPSTAT programme and found
that G-T-B haplotype was the most frequently found among controls compared to CHD patients,
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and FDRS, thus considered as a reference group and G-C-B and T-C-B haplotypes were found to be
significantly associated with 19.85(8.41-46.84) and 20.92(7.25-60.34) increased risk respectively in
CHD patients (Table 5), while in FDRS it has been found that17.94 (6.40-5.028) and 5.78 (1.91-
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17.47) (Table 5) folds of risk for CHD development. Further, we have also performed global
haplotype association test and confirmed that the eNOS 3 haplotypes are strongly associated with
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CHD development.
In addition to above haplotype analysis we have also analysed the allele frequency distribution of
eNOS-786T/C, Inton 4b/4a & 894G/T in various populations along with Indians by HapMap and
found that T allele of -786, b allele of Intron 4b/4a and G allele of 894 were significantly high
indicating the association between eNOS polymorphism and CHD (Figure 4).
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The SNP combination of eNOS -786T/C, 4b/4a and 894G/T was found to be in complete LD among
CHD patients (D'=1.0) and strong LD in both controls and FDRS(D'=0.75-0.95) which indicates the
influence of above combination on disease susceptibility due to increased minor allele frequencies
in CHD patients and FDRS (Figure 5).
In the present study MDR gene-gene interaction has been shown between controls, CHD patients
and FDRS in figure 6 and 7 respectively and the best single loci model for predicting CHD risk was
found to be eNOS-786T/C (TC and CC genotypes of eNOS-786T/C polymorphism were found to

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be the highest risk genotype for development of CHD compared to TT genotype). The best two
(eNOS-786T/C and 4b/4a), three (eNOS 894G/T, -786T/C and 4b/4a) loci models had shown
significantly increased sign test p-value with CVC values indicating the risk towards CHD
development.

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The MDR has also revealed Fruchterman-Rheingold interaction analysis between Controls, FDRS
and CHD Patients and it has been found that there is an strong synergetic interaction between -

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786T/C & 4b/4a and 4b/4a &894G/T when compared to the -786T/C & 894G/T between three study
groups.
It is also explained that the carriers of the eNOS 298Asp allele is associated with the lower

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endogenous NO formation. It is possible that VNTR 4b/4a gene polymorphisms could contribute to
CHD risk by decreasing NO production, which subsequently affects the development of CHD.
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However, because this variant is located in the intronic region, it is possible that it is in linkage
disequilibrium with other functional variants in regulatory regions of the NOS gene. In 2002, Wang
et al. demonstrated that the eNOS 4a allele coordinated with the T-786C variant in the promoter
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region to regulate the transcriptional efficiency of eNOS in a haplotype-specific fashion while the
carriers of the Asp allele is associated with the lower endogenous NO formation. Indeed, recent
studies showed that this polymorphism affects the amounts of eNOS located within the caveolae,
thus affecting eNOS activity and NO production [22][32].
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In the present study, the NO end products(nitrite/nitrate) levels were higher in FDRS and CHD
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patients compared to controls. We further examined the nitrite/nitrate levels among the subjects with
respect to eNOS haplotypes. Interestingly, we found higher nitrite/nitrate levels among GCB, TCB
haplotypes carriers in FDRS and CHD patients compared to GTB haplotype (Figure ). The high
levels of NO products could be due to increased oxidative stress and inflammatory conditions
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among the CHD patients and FDRs.

In conclusion, the results of the present study demonstrate that the eNOS -786T/C, intron 4b/4a and
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894G/T genotypes and haplotypes (G-C-B and T-C-B) are significantly associated with the
development of CHD in combination with potential risk factors. Therefore, it is necessary to
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consider the haplotypes while examining the role of eNOS in CHD. Further research in this area
need to be given high priority for understanding mechanisms responsible for the decreased
expression of eNOS that is associated with the onset/development of cardiovascular dysfunction and
may contribute to the prevention and treatment of cardiovascular diseases.
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Acknowledgments

We thank all the participants of this study, ICMR-SRF for providing fellowship, and UGC-MRP for

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providing infrastructure and chemicals.

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Funding sources: ICMR-SRF, UGC-MRP, UGC-CPEPA, DST-FIST, UPE-FAR New Delhi, India

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Author Disclosure Statement

No competing financial interests exist

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References

[1] A. Sawant, R. Mankeshwar, S. Shah, R. Raghavan, G. Dhongde, H. Raje, S. DSouza, A.

Subramanium, P. Dhairyawan, S. Todur, T.F. Ashavaid, Prevalence of metabolic syndrome
in Urban India, Cholesterol. 2011 (2011). doi:10.1155/2011/920983.
[2] S.-T. Tan, W. Scott, V. Panoulas, J. Sehmi, W. Zhang, J. Scott, P. Elliott, J. Chambers, J.S.
Kooner, Coronary heart disease in Indian Asians., Glob. Cardiol. Sci. Pract. 2014 (2014) 13

PT
23. doi:10.5339/gcsp.2014.4.
[3] J.G. Canto, C.I. Kiefe, W.J. Rogers, E.D. Peterson, P.D. Frederick, W.J. French, C.M.
Gibson, C. V Pollack, J.P. Ornato, R.J. Zalenski, J. Penney, A.J. Tiefenbrunn, P. Greenland,

RI
Number of coronary heart disease risk factors and mortality in patients with first myocardial
infarction., JAMA. 306 (2011) 21207. doi:10.1001/jama.2011.1654.

SC
[4] K.G. Rajesh, S.K. Mrudula, K.G. Kishore, K. Mohanalatha, M. Saraswati, A.T. Mohini, P.
Chiranjeevi, R.G. Srilatha, S. Nivas, P. Kaushik, S.K. Sanjib, R.H. Surekha, Evaluation of
Hs-CRP levels and interleukin 18 (-137G/C) promoter polymorphism in risk prediction of
Coronary Artery Disease in First Degree Relatives, PLoS One. 10 (2015) 113.

U
doi:10.1371/journal.pone.0120359.
AN
[5] M.T. Scheuner, W.C. Whitworth, H. McGruder, P.W. Yoon, M.J. Khoury, Familial risk
assessment for early-onset coronary heart disease., Genet. Med. 8 (2006) 525531.
doi:10.1097/01.gim.0000232480.00293.00.
M

[6] K.K. Griendling, G.A. FitzGerald, Oxidative Stress and Cardiovascular Injury Part I: Basic
Mechanisms and In Vivo Monitoring of ROS, Circulation. 108 (2003) 19121916.
doi:10.1161/01.CIR.0000093660.86242.BB.
D

[7] R. De Caterina, Endothelial dysfunctions: common denominators in vascular disease., Curr.

Opin. Clin. Nutr. Metab. Care. 3 (2000) 45367. doi:10.1097/00041433-200002000-00003.
TE

[8] P. a Marsden, H.H. Heng, S.W. Scherer, R.J. Stewart, a V Hall, X.M. Shi, L.C. Tsui, K.T.
Schappert, Structure and chromosomal localization of the human constitutive endothelial
nitric oxide synthase gene., J. Biol. Chem. 268 (1993) 1747817488.
EP

[9] C. Dafni, N. Drakoulis, O. Landt, D. Panidis, M. Reczko, D. V Cokkinos, Association of the

eNOS E298D polymorphism and the risk of myocardial infarction in the Greek population,
C

BMC Med. Genet. 11 (2010) 133. doi:10.1186/1471-2350-11-133.

[10] K. R.R., K. A., A. S., B. M., T. N., Prevalence of cardiovascular risk factors in Indian
AC

patients undergoing coronary artery bypass surgery, J. Assoc. Physicians India. 54 (2006)
371375. http://www.japi.org/may2006/O-
371.pdf\nhttp://ovidsp.ovid.com/ovidweb.cgi?T=JS&PAGE=reference&D=emed7&NEWS=
N&AN=16909733.
[11] S.H. Shah, N.J. Freedman, L. Zhang, D.R. Crosslin, D.H. Stone, C. Haynes, J. Johnson, S.
Nelson, L. Wang, J.J. Connelly, M. Muehlbauer, G.S. Ginsburg, D.C. Crossman, C.J.H.
Jones, J. Vance, M.H. Sketch, C.B. Granger, C.B. Newgard, S.G. Gregory, P.J. Goldschmidt-
Clermont, W.E. Kraus, E.R. Hauser, Neuropeptide Y gene polymorphisms confer risk of
 ACCEPTED MANUSCRIPT

early-onset atherosclerosis, PLoS Genet. 5 (2009).

[12] J.J. Connelly, T. Wang, J.E. Cox, C. Haynes, L. Wang, S.H. Shah, D.R. Crosslin, A.B. Hale,
S. Nelson, D.C. Crossman, C.B. Granger, J.L. Haines, C.J.H. Jones, J.M. Vance, P.J.
Goldschmidt-Clermont, W.E. Kraus, E.R. Hauser, S.G. Gregory, GATA2 is associated with
familial early-onset coronary artery disease, PLoS Genet. 2 (2006) 12651273.
[13] R.R. Williams, S.C. Hunt, G. Heiss, M.A. Province, J.T. Bensen, M. Higgins, R.M.

PT
Chamberlain, J. Ware, P.N. Hopkins, Usefulness of cardiovascular family history data for
population-based preventive medicine and medical research (The Health Family Tree Study
and the NHLBI Family Heart Study), Am. J. Cardiol. 87 (2001) 129135.
doi:10.1016/S0002-9149(00)01303-5.

RI
[14] K.M. Alkharfy, N.M. Al-Daghri, O.S. Al-Attas, M.S. Alokail, H.M. Draz, T. Hussain,
Endothelial Nitric Oxide Synthase Gene Polymorphisms (894G > T and-786T > C) and Risk

SC
of Coronary Artery Disease in a Saudi Population, Arch. Med. Res. 41 (2010) 134141.
doi:DOI 10.1016/j.arcmed.2010.02.004.
[15] X.L. Wang, A.S. Sim, R.F. Badenhop, R.M. McCredie, D.E. Wilcken, A smoking-dependent

U
risk of coronary artery disease associated with a polymorphism of the endothelial nitric oxide
synthase gene, Nat Med. 2 (1996) 4145. http://www.ncbi.nlm.nih.gov/pubmed/8564837.
AN
[16] K. Hibi, T. Ishigami, K. Tamura, S. Mizushima, N. Nyui, T. Fujita, H. Ochiai, M. Kosuge, Y.
Watanabe, Y. Yoshii, M. Kihara, K. Kimura, M. Ishii, S. Umemura, Endothelial nitric oxide
synthase gene polymorphism and acute myocardial infarction., Hypertension. 32 (1998) 521
M

6. doi:10.1161/01.HYP.32.3.521.
[17] Y. Shimasaki, H. Yasue, M. Yoshimura, M. Nakayama, K. Kugiyama, H. Ogawa, E. Harada,
D

T. Masuda, W. Koyama, Y. Saito, Y. Miyamoto, Y. Ogawa, K. Nakao, Association of the

missense Glu298Asp variant of the endothelial nitric oxide synthase gene with myocardial
infarction, J. Am. Coll. Cardiol. 31 (1998) 15061510. doi:10.1016/S0735-1097(98)00167-3.
TE

[18] Poirier, Mao, Mallet, Nicaud, Herrmann, Evans, Ruidavets, Arveiler, Luc, Tiret, Soubrier,
Cambien, Polymorphisms of the endothelial nitric oxide synthase gene no consistent
EP

association with myocardial infarction in the ECTIM study, Eur. J. Clin. Invest. 29 (1999)
284290. doi:10.1046/j.1365-2362.1999.00451.x.
[19] T. Nakayama, M. Soma, Y. Takahashi, Y. Izumi, K. Kanmatsuse, M. Esumi, Association
C

analysis of CA repeat polymorphism of the endothelial nitric oxide synthase gene with
essential hypertension in Japanese, Clin Genet. 51 (1997) 2630.
AC

[20] H.S. Markus, Y. Ruigrok, N. Ali, J.F. Powell, Endothelial nitric oxide synthase exon 7
polymorphism, ischemic cerebrovascular disease, and carotid atheroma., Stroke. 29 (1998)
19081911. doi:10.1161/01.STR.29.9.1908.
[21] M. Nakayama, H. Yasue, M. Yoshimura, Y. Shimasaki, K. Kugiyama, H. Ogawa, T.
Motoyama, Y. Saito, Y. Ogawa, Y. Miyamoto, K. Nakao, T-786-->C mutation in the 5-
flanking region of the endothelial nitric oxide synthase gene is associated with coronary
spasm., Circulation. 99 (1999) 28642870. doi:10.1161/01.CIR.99.22.2864.
 ACCEPTED MANUSCRIPT

[22] R. Lacchini, P.S. Silva, J.E. Tanus-Santos, A pharmacogenetics-based approach to reduce

cardiovascular mortality with the prophylactic use of statins., Basic Clin. Pharmacol. Toxicol.
106 (2010) 35761. doi:10.1111/j.1742-7843.2010.00551.x.
[23] P. Narne, K.C. Ponnaluri, S. Singh, M. Siraj, M. Ishaq, Association of the genetic variants of
endothelial nitric oxide synthase gene with angiographically defined coronary artery disease
and myocardial infarction in South Indian patients with type 2 diabetes mellitus., J. Diabetes
Complications. 27 (2013) 25561. doi:10.1016/j.jdiacomp.2012.10.009.

PT
[24] J. da Costa Escobar Piccoli, V. Manfredini, F.I. Hamester, J.B. Bandinelli, I.M. Turkienicz,
J.A.B. Chies, A. Peres, L.C. Bodanese, M.R. Bogo, Interaction between endothelial nitric
oxide synthase gene polymorphisms (-786T>C, 894G>T and intron 4 a/b) and cardiovascular

RI
risk factors in acute coronary syndromes., Arch. Med. Res. 43 (2012) 20511.
doi:10.1016/j.arcmed.2012.03.011.

SC
[25] T. Angeline, W. Isabel, G.J. Tsongalis, Endothelial nitric oxide gene polymorphisms, nitric
oxide production and coronary artery disease risk in a South Indian population, Exp. Mol.
Pathol. 89 (2010) 205208. doi:10.1016/j.yexmp.2010.08.009.

U
[26] A.M. Zigra, L.S. Rallidis, G. Anastasiou, E. Merkouri, A. Gialeraki, ENOS gene variants and
the risk of premature myocardial infarction, Dis. Markers. 34 (2013) 431436.
AN
doi:10.3233/DMA-130987.
[27] A.S. Arun Kumar, G. Umamaheswaran, R. Padmapriya, J. Balachandar, C. Adithan,
Endothelial nitric oxide synthase gene polymorphisms and the risk of acute myocardial
M

infarction in a South Indian population, Mol Biol Rep. 40 (2013) 12751281.

doi:10.1007/s11033-012-2170-2.
D

[28] M.Z. Gad, M.F. Abdel Rahman, I.M. Hashad, S.M. Abdel-Maksoud, N.M. Farag, K. Abou-
Aisha, Endothelial nitric oxide synthase (G894T) gene polymorphism in a random sample of
the Egyptian population: comparison with myocardial infarction patients., Genet. Test. Mol.
TE

Biomarkers. 16 (2012) 695700. doi:10.1089/gtmb.2011.0342.

[29] G. Lembo, N. De Luca, C. Battagli, G. Iovino, a Aretini, M. Musicco, G. Frati, F. Pompeo,
EP

C. Vecchione, B. Trimarco, A common variant of endothelial nitric oxide synthase

(Glu298Asp) is an independent risk factor for carotid atherosclerosis., Stroke. 32 (2001) 735
40. http://www.ncbi.nlm.nih.gov/pubmed/11239195.
C

[30] R.G. Kumar, M.K. Spurthi, K.G. Kumar, S.K. Sahu, S.H. Rani, Endothelial nitric oxide
synthase polymorphism G298T in association with oxidative DNA damage in coronary
AC

atherosclerosis, J. Genet. 91 (2012) 349352. doi:10.1007/s12041-012-0183-1.

[31] J. Yang, R. Yan, A. Roy, D. Xu, J. Poisson, Y. Zhang, The I-TASSER Suite: protein structure
and function prediction, Nat Meth. 12 (2015) 78. http://dx.doi.org/10.1038/nmeth.3213.
[32] V. Godfrey, S.-L. Chan, A. Cassidy, R. Butler, A. Choy, T. Fardon, A. Struthers, C. Lang,
The functional consequence of the Glu298Asp polymorphism of the endothelial nitric oxide
synthase gene in young healthy volunteers., Cardiovasc. Drug Rev. 25 (2007) 280288.
doi:10.1111/j.1527-3466.2007.00017.
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Figure Legends
Figure 1: eNOS -786 T/C Secondary Structure
Figure 2: eNOS 4b/4a Secondary Structure
Figure 3: eNOS 894G/T Three dimensional modeling
Figure 4: HapMap analysis of eNOS -786T/C, INTRON 4b/4a and 894 G/T polymorphisms
Figure 5: Plots Representing Linkage Disequilibrium (LD) of alleles at different positions of

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eNOS (-786T/C, Intron 4b/4a and 894 G/T) in Controls, CHD patients and FDRS
Figure 6: MDR gene-gene interaction in Controls vs. CHD Patients

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Figure 7: MDR gene-gene interaction in Controls vs. FDRS
Figure 8: Fruchterman-Rheingold analysis between Controls, FDRS and CHD Patients

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Figure 9: eNOS haplotypes vs. Nitrite/Nitrate levels in Controls, FDRS and CHD patients

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Results:

Table 1

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Conditions of genotyping assays for the selected polymorphisms of eNOS gene

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Genotyping Band pattern (bp) in
SNP Primers Tm (o C)
method AGE
CF:5'TTTCTCCAGCCCCTCAGATG3'

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TT-250+176,250
eNOS -786T/C RP: 5' AGG CCC AGC AAG GAT GTA GT 3'
AS-PCR 57.1 TC-250+176,250+176

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rs2070744 CP:5'CAT CAA GCT CTT CCC TGG CC 3'
CC-250,250+176
TP:5'CAT CAA GCT CTT CCC TGG CT 3'
4b/4b-420

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eNOS 4b/4a FP:5'AGGCCCTATGGTAGTGCCTTG 3'
PCR 60 4b/4a-420,393
Intron RP:5'TCTCTTAGTGCTGTGGTCAC 3'
4a/4a-393

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GG-206
eNOS 894G/T PCR-RFLP FP:5'CATGAGGCTCAGCCCCAGAAC 3'
61 GT-206,119, 87

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rs1799983 MboI RP:5'AGTCAATCCCTTTGGTGCTCAC 3'
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Table 2
Demographic data of Controls, FDRS and CHD patients (Categorised)

CHD
Controls Chi Square FDRS Chi Square

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patients
Pearson Fisher Pearson Fisher Exact
N (%) N (%) Df N (%) Df

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value Exact Test vale Test
Male 168 (56) 229 (76.3) 70 (70)
27.70 1 0.001* 6.10 1 0.01*

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Female 132 (44) 71 (23.7) 30 (30)
Veg 82 (27.3) 86 (28.7) 26 (26)
0.132 1 0.785 0.068 1 0.89

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Non veg 218 (72.7) 214 (71.3) 74 (74)

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Smoker 105 (35) 127 (42.3) 30 (30)
3.401 1 0.07* 0.839 1 0.39
Non Smoker 195 (65) 173 (57.7) 70 (70)

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DM 0 87 (29) 13 (13)
101.7 1 0.001* 40.31 1 0.001*
No DM 300 213 (71) 87 (87)

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With Family History 0 20 (6.7) 100 (100)
20.690 1 0.001* 363.5 1 0.001*

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Without Family
300 280 (93.3) NIL
History
With HTN 0 116 (38.7) 51 (51)
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143.8 1 0.001* 175.35 1 0.001*
Without HTN 300 184 (61.3) 49 (49)
*Significant at p<0.05
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Table 3

Demographic data of Controls, FDRS and CHD patients (Continuous)

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Controls FDRS CHD

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N=300 N=100 N=300

Mean Mean Mean

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Mean SD Mean SD Mean SD
Error Error Error

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Age(Years) 52.018.08 0.46 42.6015.3* 1.5 54.2612.06* 0.697

BMI(Kgm2)

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25.35 5.1 0.29 25.414.9 0.49 28.664.797 0.277
Sy-BP

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121.65 7.2 0.419 124.414.3 1.43 135.7321.8 1.260
(mmHg)

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Di-BP
80.975.4 0.312 83.209.8 0.98 90.2911.8 0.685
(mmHg)
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*Significant at p<0.05
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Table 4

Genotype distribution of eNOS -786T/C, Intron 4b/4a and 894G/T polymorphism in Controls, CHD patients and FDRS

Controls CHD CHD Odds Ratio FDRS FDRS Odds Ratio

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Genotype N=300 N=300 Odds ratio N=100 Odds ratio
2 p-Value 2 p-Value
n (%) n (%) (95% CI) n (%) (95% CI)
TT

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186 (62) 102 (34) 1.00 43 (43) 1.00
8.54
Co- 38.06 2.97 <0.0001* 2.08 0.003*

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TC 102 (34) 166(55.3) 49 (49)
eNOS dominant (2.10-4.19) (1.29-3.34)
-786T/C 4.86 2.88
CC 12 (4) 32 (10.7) 1.46 0.22 8 (8) 0.176 0.67
(2.40-9.85) (1.11-7.49)

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Allele
T 474 (79) 370 (62) 2.337 135(68) 1.81

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frequency <0.0001* 10.29 0.0013*
C 126 (21) 230 (38) 42.37 (1.81-3.024) 65 (32) (1.265-2.58)
4b4b
197 (65.7) 201 (67)

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1.00 67(67) 1.00
Co- 0.007 0.97 0.93 0.0001 0.97 0.99
4b4a 91 (30.3) 90 (30) 30(30)

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dominant (0.68-1.38) (0.59-1.59)
Intron
0.74 0.74
4b/4a 4a4a 12 (4) 9 (3) 0.13 0.71 3(3) 0.007 0.92

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(0.30-1.78) (0.20-2.68)
Allele 4b 485(81) 492(82) 0.9258 164(82) 0.92
0.65 0.06 0.79
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frequency 4a 115(19) 108(18) 0.19 (0.692-1.23) 36(18) (0.60-1.39)
GG
192(64) 154 (51) 1.00 51(51) 1.00
Co-
C

dominant 11.5 1.83 0.001* 3.879 1.67 0.07*

GT 90(30) 132(44) 40(40)
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(1.30-2.57) (1.03-2.71)
894G/T
0.97 1.88
TT 18(6) 14(5) 2.21 0.13 9(9) 0.0011 0.97
(0.47-2.01) (0.80-4.44)
Allele G 474(79) 440(73) 1.368 142(71) 1.536
4.99 0.02* 4.978 0.02*
frequency T 126(21) 160(27) (1.047-1.787) 58(29) (1.064-2.205)
*Significant at p<0.05
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Table 5

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eNOS haplotypes (894G/T, -786T/C & 4b/4a) and their association with CHD susceptibility in Controls, CHD patients and FDRS

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Haplotype Frequency Controls vs CHD Controls vs FDRS

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Odds Ratio Odds Ratio
894 -786 4b/4a Controls CHD FDRS pvalue p -value
(95%CI) (95%CI)

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G T B 0.7799 0.6167 0.6698 1.00 (Ref) --- 1.00 (Ref) ---

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T C A 0.1917 0.18 0.18 1.36(0.99-1.87) 0.055* 1.19(0.77-1.85) 0.44

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G C B 0.0101 0.1167 0.0402 19.85(8.41-46.84) <0.0001* 5.78(1.91-17.47) 0.002*

T C B 0.0082 0.0867 0.1048 20.92(7.25-60.34) <0.0001* 17.94(6.40-5028) 0.0001*

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*Significant at p<0.05
Global haplotype association p-value: <0.0001*
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Haplotypes <0.01 frequency were excluded from analysis

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Figure 1: eNOS -786 T/C Secondary Structure

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Figure 2: eNOS 4b/4a Secondary Structure

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Figure 3: eNOS 894G/T Three dimensional modeling

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Figure 4: HapMap analysis of eNOS -786T/C, INTRON 4b/4a and 894 G/T polymorphisms

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Figure 5: Plots Representing Linkage Disequilibrium (LD) of alleles at different positions of eNOS (-786T/C, Intron 4b/4a and
894 G/T) in Controls, CHD patients and FDRS

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Figure 6: MDR gene-gene interaction in Controls vs. CHD Patients

Model Training Balance Testing Balance Sign test (p) Cross Value Consistency

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Accuracy Accuracy (CVC)
786 0.6400 0.6395 9(0.0107) 10/10
786,4b/4a 0.6850 0.6859 10(0.0010) 10/10

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894,786,4b/4a 0.6850 0.6859 10(0.0010) 10/10

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Figure 7: MDR gene-gene interaction in Controls vs. FDRS

Model Training Balance Accuracy Testing Balance Accuracy Sign test (p) Cross Value Consistency

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(CVC)
786 0.5949 0.5937 9(0.0107) 10/10
786,4b/4a 0.6266 0.6204 8(0.0547) 10/10

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894,786,4b/4a 0.6268 0.6155 8(0.0547) 10/10

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Figure 8: Fruchterman-Rheingold analysis between Controls, FDRS and CHD Patients

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Figure 9: eNOS haplotypes vs. Nitrite/Nitrate levels in Controls, FDRS and CHD patients

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Highlights

Having an FDR with premature CHD is a significant risk factor for development of
CHD.
To study the role eNOS polymorphism (eNOS -786T/C, Intron 4b/4a and 894 G/T) in
CHD patients and FDRS.
eNOS -786TC, intron 4b/4a, and 894GT haplotypes (G-C-B and T-C-B) are

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significantly associated with the development of CHD.
It is the first kind of study to investigate eNOS -786TC, Intron 4b/4a, and 894GT
polymorphisms in FDRS.
Higher nitrite/nitrate levels among GCB, TCB haplotypes carriers in FDRS and CHD

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patients.

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