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Background: Endothelial nitric oxide synthase (eNOS) and potassium voltage-gated channel subfamily J member 11 (KCNJ11) could be the candidate genes for coronary artery disease (CAD). This study investigated the relationship of the eNOS (rs1799983) and KCNJ11 (rs5219) polymorphisms with the presence and severity of CAD in the North Indian population. Methods: This study included 300 subjects, 150 CAD cases and 150 healthy controls. Single nucleotide polymorphism was evaluated by Polymerase chain reaction and Restriction fragment length polymorphism (PCR-RFLP). Analysis was performed by SPSS (version 21.0). Results: We observed that KK genotype of KCNJ11E23K (rs5219) polymorphism (P=0.0001) genotypes and K allele (P=0.0001) was found to be a positive risk factor and strongly associated with CAD. In the case of eNOSG894T (rs1799983) there was no association found with CAD. Conclusion: These results illustrate the probability of associations between SNPs and CAD although specific genetic polymorphisms affecting ion channel function and expression have still to be clarified by further investigations involving larger cohorts.
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Association of eNOS (G894T, rs1799983) and KCNJ11 (E23K, rs5219)
gene polymorphism with coronary artery disease in North Indian population
Syed Tasleem Raza1, Sachendra P Singh1, Saliha Rizvi1, Alina Zaidi1,
Sanchita Srivastava1, Arif Hussain2, Farzana Mahdi1
1. Department of Biochemistry, Era’s Lucknow Medical College and Hospital, Lucknow, India.
2. Department of Molecular biology, Manipal Academy of Higher Education.
Emails:
Syed Tasleem Raza: tasleem24@gmail.com; Sachendra P Singh: singhsachendra189@gmail.com,
singhsachendra189@gmail.com; Saliha Rizvi: rizvi_saliha@rediffmail.com; Alina Zaidi:
zaidialinaa@gmail.com; Sanchita Srivastava: sanchita.1926@gmail.com; Arif Hussain:
arifhussain@manipaldubai.com; Farzana Mahdi: farzana.mahdi@gmail.com
Abstract
Background: Endothelial nitric oxide synthase (eNOS) and potassium voltage-gated channel subfamily J member 11
(KCNJ11) could be the candidate genes for coronary artery disease (CAD). This study investigated the relationship of the
eNOS (rs1799983) and KCNJ11 (rs5219) polymorphisms with the presence and severity of CAD in the North Indian pop-
ulation.
Methods: This study included 300 subjects, 150 CAD cases and 150 healthy controls. Single nucleotide polymorphism
was evaluated by Polymerase chain reaction and Restriction fragment length polymorphism (PCR-RFLP). Analysis was
performed by SPSS (version 21.0).
Results: We observed that KK genotype of KCNJ11E23K (rs5219) polymorphism (P=0.0001) genotypes and K allele
(P=0.0001) was found to be a positive risk factor and strongly associated with CAD. In the case of eNOSG894T (rs1799983)
there was no association found with CAD.
Conclusion: These results illustrate the probability of associations between SNPs and CAD although specic genetic pol-
ymorphisms affecting ion channel function and expression have still to be claried by further investigations involving larger
cohorts.
Keywords: Coronary Artery Disease (CAD); endothelial nitric oxide synthase (eNOS); potassium voltage-gated channel
subfamily J member 11 (KCNJ11); gene polymorphism.
DOI: https://dx.doi.org/10.4314/ahs.v21i3.25
Cite as: Raza ST, Singh SP, Rizvi S, Zaidi A, Srivastava S, Hussain A, et al. Association of eNOS (G894T, rs1799983) and KCNJ11
(E23K, rs5219) gene polymorphism with coronary artery disease in North Indian population. Afri Health Sci. 2021;21(3). 1163-1171.
https://dx.doi.org/10.4314/ahs.v21i3.25
Corresponding author:
Syed Tasleem Raza,
Department of Biochemistry Era’s
Lucknow Medical College and Hospital,
Lucknow, India- 226025.
Ph +91 522 2408122, 2408123
Fax: +91 5222407824
Email: tasleem24@gmail.com
Background
Coronary artery disease (CAD) is an ischemic heart
disease which has been found to be the major cause
of mortality in both developed and developing coun-
tries1. This atherosclerotic disease is inammatory in
nature2 with clinical manifestations which include both
stable and unstable angina, myocardial infarction (MI),
and sudden cardiomyocyte death3. According to ge-
nome-wide association studies (GWAS), numerous ge-
netic mutations have also been found to be robustly al-
lied with CAD4. Other worldwide studies conducted on
the genetic variations in CAD risk among populations
reported that the disease incidence is also attributable
to demographic phenomenon5, 6. Additionally, lifestyle
also plays a profound role in the progression of such
cardiovascular events7. World Health Organization in
the year 2009 documented through several reports that
17.3 million deaths prevailed due to cardiovascular dis-
African Health Sciences, Vol 21 Issue 3, September, 2021
© 2021 Raza ST et al. Licensee African Health Sciences. This is an Open Access article distributed under the terms of the Creative commons Attribution License
(https://creativecommons.org/licenses/BY/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
African
Health Sciences
1163
eases2. Moreover, among Indian population, CAD has
been the chief cause of disability and death8 and has
been the source of mortality leading to increase num-
ber of deaths from the year 1985 which was expected
to be doubled by the year 20159. However, therapeu-
tic measures have signicantly enhanced the prognosis
of CAD patients over the past few decades10. Yet, the
progression of the disease could be halted only in a
few patients via treatments through aspirin, statins, and
β-blockers11. By 1879, it was concluded by pathologists
that CAD was the basis of myocardial infarction (MI)12.
The CAD occurs due to atherosclerosis or atheroscle-
rotic occlusions of the coronary arteries13. Endothelial
dysfunction of the arterial wall due to the accumula-
tion of modied low-density lipoprotein (LDL) in the
intima of the coronary vessels gives rise to atheroscle-
rosis14. High concentration of LDL permeates the dis-
rupted endothelium and undergoes oxidation15 which
draws leukocytes which are further scavenged by mac-
rophages, leading to the development of foamy cells.
These foamy-texture cells undergo replication and form
lesions which aid in early detection of atherosclerosis.
This cascade triggers signals which attract smooth mus-
cle cells (SMCs) to this site and provoke their prolif-
eration and production of extracellular matrix (ECM),
including collagen and proteoglycans which further ini-
tiates atherosclerotic brous plaque and encroaches on
the lumen of the coronary vessel and small blood ves-
sels subsequently calcifying the plaques thus forming
nal lesion that may be highly thrombogenic16. This is
followed by obstruction of blood ow leading impaired
myocardial oxygen demand and supply17. These events
give rise to the symptoms related to CAD.
Nitric oxide synthase (NOS) is one of the chief can-
didate genes in CAD. It synthesizes NO in a catabol-
ic reaction in presence of L-arginine18. The gene is
located on chromosome 7q36. The three isoforms of
NOS are neuronal isoforms (nNOS), inducible isoform
(iNOS) and endothelial NOS (eNOS). The capability
of a blood vessel to dilate is largely dependent upon the
activity of eNOS hence the present article will focus on
this isoform as one of the candidate gene. Endotheli-
al NOS (eNOS), also known as nitric oxide synthase 3
(NOS3) or constitutive NOS (eNOS), is an enzyme in
humans which is encoded by the NOS3 gene located in
the 7q35-7q36 region of chromosome 7,19. eNOS also
acts as potent regulator of blood pressure and blood
ow20. Elevated levels of iron could lead to free hydrox-
yl radicals synthesis resulting in LDL oxidation21 which
is considered as one of the prime factor in pathogenesis
of atherosclerosis and cardiovascular diseases22 due to a
lipid accumulation in macrophages and foam cells, thus
conferring toxicity to cells.
Another gene that has been focused in this article
is KCNJ11 gene as it also guides the formation of
ATP-sensitive potassium (K-ATP) channel in cardio-
myocytes. These ATP sensitive channel play important
role in CAD23. Usually, the K-ATP channels in cardiac
tissue are closed when the intracellular ATP concentra-
tion is high24, but get activated in cardiovascular patho-
logical states which include ischemia, reperfusion, cell
stress and apoptosis25,26 due to which the cellular mem-
brane hyperpolarization occurs which promotes te syn-
thesis of NO thus increasing the permeability of the
vascular wall. This leads to the pathologic development
of coronary atherosclerosis. Any mild mutations or pol-
ymorphisms modifying the KATP channel current or
activity will be correlated with diseases. It was reported
that the common polymorphism E23K of this gene is
associated with higher susceptibility to CAD in many
population.
The purpose of this study is to disclose the relation-
ship between G894T (rs1799983) polymorphisms of
the eNOS gene and KCNJ11 E23K (rs5219) with the
presence of CAD in North Indian population. Single
nucleotide polymorphisms (SNPs) could serve as prog-
nostic factor as they inuence disease incidence and can
be employed in everyday clinical practice and decision
making; hence, it is essential to identify those SNPs that
have strongest predisposition. Location of SNPs play
inuential role in incidence of diseases. Any variation
of the SNP could be directly connected to the disease if
it is located within or close to the translated region. As a
result, various research studies envisaged the identica-
tion of candidate and sensitive genes for CAD and their
polymorphisms in relation to the risk of this disease.
Methods
Subject’s enrollment
This case-control study was performed on 300 subjects
including 150 CAD cases (who underwent coronary
angiography) and 150 controls were recruited. Blood
samples were collected in anti-coagulation EDTA vi-
als from the Cardiology Unit (Department of Medi-
cine), Era’s Lucknow Medical College & Hospital of
Lucknow, Uttar Pradesh. A written informed consent
was obtained from all participants prior to sampling. All
the baseline clinical prolic collection for each patient
was done that included clinical variable such as age, sex,
blood pressure, body mass index, height, weight, lipid
African Health Sciences, Vol 21 Issue 3, September, 20211164
prole etc. The diagnosis of CAD was dened as >
50% reduction of coronary artery diameter. Cases were
classied according to the number of signicant sten-
otic vessels as follows: angigraphically-normal vessel
(n = 19), 1-vessel (SVD) (n = 44), 2-vessel (DVD) (n
= 58) and 3- vessel (TVD) (n = 64). Control subjects
(n=160) were dened as those individuals who had no
personal or family history of cardiovascular disease or
diabetes. Protocol and procedures were in accordance
with the standards of the Institutional Ethical/ Review
Committee.
Genomic DNA isolation
Genomic DNA isolation from peripheral blood sam-
ples was done using the standard phenol-chloroform
extraction method and was checked on 1% agarose gel
stained with ethidium bromide. Quantication of the
extracted DNA was done using Nanodrop (TM) 1000
UV/VIS Spectrophotometer. Finally the isolated DNA
was stored at -20°C until further analysis was done.
PCR Amplication
Endothelial nitric oxide synthase (eNOS) (G894T,
rs1799983) Gene Polymorphism
Forward Primer: 5’ CATGAGGCT-
CAGCCCCAGAAC 3’
Reverese Primer: 5’ AGTCAATCCCTTTG-
GTGCTCAC 3’
Approximately 100 ng of genomic DNA was ampli-
ed in a total volume of 25 μL containing 2.5 μL of
thermophilic DNA Polymerase Buffer (supplied with
Taq polymerase; Promega), 3.0 mmol/L MgCl2, 200
μmol/L deoxynucleotide triphosphates, 10 pmol/L of
each primer/span> and 1 U of Taq polymerase.
The PCR amplication conditions were 95 °C for 5
mins followed by 40 cycles of denaturation at 94 °C
for 30 s, annealing at 66 °C for 30 s, extension at 72 °C
for 30 s. The nal extension step was carried out at 72
°C for 7 min. The 206-bp PCR product (10 µl) was di-
gested with MboI restriction endonuclease (NEB, UK)
overnight and the digested products were analyzed on
3% agarose gel and visualized by ultraviolet transillu-
mination after ethidium bromide staining. The 206 bp
amplicon containing a thymine at nucleotide position
894 (corresponding to an aspartic acid at amino acid
position 298, TT genotype) was cleaved into two frag-
ments of 119 bp and 87 bp in length by MboI diges-
tion but not for a guanine in this position (206 bp, GG
genotype).
Potassium voltage-gated channel subfamily J member
11(KCNJ11) E23K (rs5219) Gene Polymorphism
Forward Primer: 5’ GACTCTGCAGTGAGGCCCTA
3’
Reverse Primer: 5’ ACGTTGCAGTTGCCTTTCTT
3’
Approximately 100 ng of genomic DNA was ampli-
ed in a total volume of 25 μL containing 2.5 μL of
Thermophilic DNA Polymerase Buffer (supplied with
Taq polymerase; Promega), 3.0 mmol/L MgCl2, 200
μmol/L deoxynucleotide triphosphates, 10 pmol/L of
each primer and 1 U of Taq polymerase.
The PCR amplication conditions were 95 °C for 5
mins followed by 40 cycles of denaturation at 94 °C for
30 s, annealing at 66 °C for 30 s, extension at 72 °C for
30 s. The nal extension step was performed at 72 °C
for 7 min. The 210-bp PCR product (10 µl) was digest-
ed with Ban II restriction endonuclease (NEB, UK) for
1 hr at 37°C and the digested products were analyzed
on 3% agarose gel and visualized by ultraviolet transil-
luminatior after ethidium bromide staining.
Statistical analysis
QUANTO (v.online) software for each single nucleotide
polymorphism (SNP) was used to calculate the sample
size. Minor allelic frequency (MAF) and distribution
were used for evaluation. The Hardy-Weinberg equi-
librium for each locus is evaluated by the Chi-square
test (X2). The continuous variables for each group
were expressed as mean ± SD and compared with the
Student's t test after determining the normality of the
Kolmogorov-Smirnov Z test. In both groups, the al-
lele frequencies were compared by comparing the 2 x 2
matrix. P <0.05 was considered statistically signicant.
The 95% condence interval (CI) ratio was determined
in the logistic regression model to describe the strength
of the two SNPs. All analyzes were performed by SPSS
(version 21.0).
Results
Restriction fragment length polymorphism analy-
sis for determination of genotype
For eNOS gene polymorphisms, the GT genotype
shows two bands of base pairs 206 bp and 119 bp. GG
genotype shows single band of 206 bp and TT geno-
type shows two bands of 119 bp and 87bp. In case of
KCNJ11 gene polymorphisms, UD shows undigested
band of 210bp, EK genotype shows three bands of 210
bp, 178 bp and 150 bp; EE genotype shows one band
of 150 bp and KK genotype shows two bands of 178
bp and 150 bp.
African Health Sciences, Vol 21 Issue 3, September, 2021 1165
Demographic and clinical characteristics
Demographic, clinical and biochemical characteristics
of the studied population were recorded from both
cases and controls. The mean age of cases and con-
trols were 52.68±5.440 and 52.00±5.188 respectively
(P=0.269). Clinical parameters such as BMI (P=0.005),
LDL(0) and total cholesterol (0) was signicantly high-
er in cases as compared to controls whereas, HDL
(p<0.001), Triglyceride (p=0.003)and VLDL(p=0.003)
level was found to be signicantly lower in cases as
compared to controls (Table 1).
Table 1: Comparison of Biochemical parameters in Cases and Controls
Patients
Cases (N=150)
Controls
(N=150)
t or χ2
P value
Age (years)
52.68±5.440
52.00±5.188
1.108
0.269
Gender
Male (n,%)
116 (77.3%)
70 (46.7%)
29.938
<0.0001
Female (n,%)
34 (22.7%)
80 (53.3%)
BMI (Kg/m2)
24.80±4.595
23.71±1.233
2.797
0.005
HDL (mg/dL)
42.98±3.488
56.11±9.848
15.388
<0.0001
LDL (mg/dL)
150.35±30.270
71.43±28.916
23.09
0
Total cholesterol (mg/dL)
216.11±30.922
153.59±22.173
20.126
0
Triglyceride (mg/dL)
113.89±14.683
129.48±62.532
2.972
0.003
VLDL (mg/dL)
22.80±3.061
25.92±12.449
2.981
0.003
Table 1: Comparison of Biochemical parameters in Cases and Controls
Patients
Controls
(N=150)
t or χ2
P value
Age (years)
52.00±5.188
1.108
0.269
Gender
Male (n,%)
70 (46.7%)
29.938
<0.0001
Female (n,%)
80 (53.3%)
BMI (Kg/m2)
23.71±1.233
2.797
0.005
HDL (mg/dL)
56.11±9.848
15.388
<0.0001
LDL (mg/dL)
71.43±28.916
23.09
0
Total cholesterol (mg/dL)
153.59±22.173
20.126
0
Triglyceride (mg/dL)
129.48±62.532
2.972
0.003
VLDL (mg/dL)
25.92±12.449
2.981
0.003
Genotyping
Genotypes & alleles frequencies of KCNJ11 and eNOS
genes in cases and healthy controls
The results were analyzed as allelic and genotypic fre-
quencies of the KCNJ11E23K and eNOSG894T pol-
ymorphisms (Table 2). The frequency of KK geno-
type of KCNJ11E23K polymorphism was found to be
highly signicant in cases with 25 fold higher risk of
CAD (p≤0.0001) in comparison to control. Similarly
the frequency of K* genotype of KCNJ11E23K pol-
ymorphism was also found to be highly signicant in
cases with 3.45 fold higher risk of CAD (p≤0.0001).
EK in combination with KK (EK+KK) was also found
highly signicant in cases with 6.30 fold higher risk of
CAD. However the frequency of EK genotype of KC-
NJ11E23K polymorphism was moderately signicant
in control in comparison to CAD cases (p=0.001). No
signicant association was found in EE polymorphisms
between cases and control. In case of eNOSG894T
polymorphisms no signicant association was found
between cases and control.
Association of genotype eNOS and KCNJ11 in
male and female cases & controls
The results were analyzed as association of genotype
KCNJ11E23K and eNOSG894T in male and female
cases (Table 3). In case of males the frequency of EK
genotype of KCNJ11E23K polymorphism was found
to be moderately signicant in cases with 0.23 fold high-
er risk of CAD (p=0.004) in comparison to control.
The frequency of KK genotype was found to be highly
signicant in cases with 0.02 fold higher risk of CAD
(p≤0.0001) Similarly, the frequency of K* genotype
of KCNJ11E23K polymorphism was also found to be
highly signicant in cases with 0.25 fold higher risk of
CAD (p≤0.0001). Whereas no signicant association
was found in EE and E* genotype between cases and
control. In case of females, the frequency of KK geno-
type of KCNJ11E23K polymorphism was found to be
moderately signicant in cases with 0.09 fold higher risk
of CAD (p=0.001) in comparison to control. Similarly
the frequency of K* genotype of KCNJ11E23K poly-
morphism was also found to be moderately signicant
in cases with 0.35 fold higher risk of CAD (p=0.001).
However no signicant association was found in EE,
EK and E* genotype betweecases and control. Again in
case of eNOSG894T polymorphisms no signicant as-
sociation was found between cases and control in both
males and females.
African Health Sciences, Vol 21 Issue 3, September, 20211166
Table 2: Genotypes & alleles frequencies of KCNJ11 and eNOS genes in cases and healthy controls.
Genotypes
Cases
Controls
OR (95%CI)
P value
Number
(N=150)
Frequency
(%)
Number
(N=150)
Frequency (%)
eNOS G894T
GG
95
63.3
101
67.3
1(Ref)
GT
48
32
42
28
1.215 (0.737-2.003)
0.445
TT
7
4.7
7
4.7
1.063 (0.359-3.145)
0.912
GT+TT
55
36.7
49
32.7
1.19 (0.741-1.921)
0.467
Allele
G*
238
79.3
244
81.33
1(Ref)
T*
62
20.7
56
18.67
1.14 (0.759-1.698)
0.538
KCNJ11 E23K
EE
9
6
43
28.7
1(Ref)
EK
73
48.7
94
62.7
3.710 (1.700-8.101)
0.001
KK
68
45.3
13
8.7
24.991 (9.843-63.452)
<0.0001
EK+KK
141
94
107
71.3
6.30 (2.941-13.478)
<0.0001
Allele
E*
91
30.33
180
60
1(Ref)
K*
209
69.67
120
40
3.45 (2.458-4.828)
<0.0001
Table 3: Association of genotype eNOS and KCNJ11 in male and female cases & controls
Genotypes
Male
OR (95%CI)
P value
Female
OR (95%CI)
P value
Cases
(n=116)
Controls
(70)
Cases
(n=34)
Controls
(n=80)
eNOS G894T
GG
77 (66.4)
48 (68.6)
1(Ref)
18 (52.9)
53 (66.2)
1(Ref)
GT
34 (29.3)
18 (25.7)
0.85 (0.432-1.669)
0.635
14 (41.2)
24 (30.0)
0.58 (0.249-1.360)
0.212
TT
5 (4.3)
4 (5.7)
1.28 (0.328-5.017)
0.72
2 (5.9)
3 (3.8)
0.51 (0.079-3.297)
0.479
Allele
G*
188
(81.0)
114 (81.4)
1(Ref)
50 (73.5)
130
(81.2)
1(Ref)
T*
44 (19.0)
26 (18.6)
0.97 (0.569-1.668)
0.925
18 (26.5)
30 (18.8)
0.64 (0.328-1.252)
0.193
KCNJ11 E23K
EE
6 (5.2)
21 (30.0)
1(Ref)
3 (8.8)
22 (27.5)
1(Ref)
EK
57 (49.1)
46 (65.7)
0.23 (0.086-0.619)
0.004
16 (47.1)
48 (60.0)
0.41 (0.108-1.550)
0.189
KK
53 (45.7)
3 (4.3)
0.02 (0.004-0.071)
<0.0001
15 (44.1)
10 (12.5)
0.09 (0.021-0.387)
0.001
Allele
E*
69 (29.7)
88 (62.9)
1(Ref)
22 (32.4)
92 (57.5)
1(Ref)
K*
163
(70.3)
52 (37.1)
0.25 (0.161-0.390)
<0.0001
46 (67.6)
68 (42.5)
0.35 (0.195-0.642)
0.001
African Health Sciences, Vol 21 Issue 3, September, 2021 1167
Fig: 1 Restriction fragment length polymorphism analysis for determination of genotype. (a) For eNOS gene
polymorphisms, The GT genotype shows two bands of 206 bp and 119 bp; The GG genotype shows single band of 206 bp, The
TT genotype shows two bands of 119 bp and 87bp. (b) For KCNJ11 gene polymorphisms, UD shows Undigested band of
210bp, the EK genotype shows three bands of 210 bp, 178 bp and 150 bp; the EE genotype shows one band of 150 bp; The KK
genotype shows two bands of 178 bp and 150 bp. M1 AND M2 are 100 bp molecular ladder.
Fig: 1 Restriction fragment length polymorphism analysis for determination of genotype. (a) For eNOS gene
polymorphisms, The GT genotype shows two bands of 206 bp and 119 bp; The GG genotype shows single band of 206 bp, The
TT genotype shows two bands of 119 bp and 87bp. (b) For KCNJ11 gene polymorphisms, UD shows Undigested band of
210bp, the EK genotype shows three bands of 210 bp, 178 bp and 150 bp; the EE genotype shows one band of 150 bp; The KK
genotype shows two bands of 178 bp and 150 bp. M1 AND M2 are 100 bp molecular ladder.
Discussion
CAD has been found to be the major cause of mor-
tality and morbidity in human. Widespread studies pe-
rused on the epidemiology of CAD have reported the
incidence of CAD in almost all parts of the world. In
the developing countries, these cardiovascular diseases
(CVD) are categorized as epidemic. Several case–con-
trol studies, consortia, genome-wide association analy-
sis and epidemiological studies have been conducted to
understand the different aspects of this disease. Con-
ventional threats include type 2 diabetes (T2D), arterial
hypertension, dyslipidemia and cigarette smoking27.
Single nucleotide polymorphisms (SNPs) could serve
as prognostic factor as they inuence disease incidence
and can be employed in everyday clinical practice and
decision making; hence, it is essential to identify those
SNPs that have strongest predisposition. Hence, this
article reveals the allelic and genotypic frequencies of
the KCNJ11E23K and eNOSG894T polymorphisms
as they are related to atherosclerotic consequences
which lead to CAD.
A point mutation found at nucleotide 894 in exon 7
where guanine (G) shifts to thymine (T) in the eNOS
gene transforms the coding sequence from glutamic
acid to aspartic acid in codon 298 (Glu298Asp, also
known as the G894T, rs1799983)28. Recently through
several reports it was concluded that eNOSG894T
(Glu-298→Asp) mutation at exon 7 of the eNOS gene
is related to coronary spasm28, myocardial infarction
(MI)29, 30, and hypertension31. Previous reports, illustrate
that the Glu2983Asp polymorphism is signicantly as-
sociated with the presence of CAD32. However our re-
sults did not show any signicant association in eNOS
genotype between cases and control. Our results were
also not discordant with the outcomes of Chang et al
and Kerkeni et al33,34 who found a signicant correla-
tion between Glu298Asp polymorphism and CAD. Al-
though Abolhalaj et al also revealed that the difference
in eNOS gene expression was not statistically signi-
cant between patients and control35.
Another gene that has been focused in this article
is KCNJ11 gene as it also guides the formation of
ATP-sensitive potassium (K-ATP) channel in cardio-
myocytes. These ATP sensitive channels play important
role in CAD20. Our result stated that the frequency of
KK genotype and K* genotype of KCNJ11E23K pol-
ymorphism was found to be highly signicant in cases
with 25 and 3.45 fold higher risk respectively in CAD
(p≤0.0001) in comparison to control. EK in combina-
tion with KK (EK+KK) was also found highly signi-
cant in cases with 6.30 fold higher risk of CAD. How-
ever the frequency of EK genotype of KCNJ11E23K
polymorphism was moderately signicant in control in
comparison to CAD cases (p=0.001). No signicant
association was found in EE polymorphisms between
cases and control. In case of males the frequency of EK
genotype of KCNJ11E23K polymorphism was found
to be moderately signicant in cases with 0.23 fold high-
er risk of CAD (p=0.004) in comparison to control and
the frequency of KK genotype was found to be highly
African Health Sciences, Vol 21 Issue 3, September, 20211168
signicant in cases with 0.02 fold higher risk of CAD
(p≤0.0001) Similarly, the frequency of K* genotype
of KCNJ11E23K polymorphism was also found to be
highly signicant in cases with 0.25 fold higher risk of
CAD (p≤0.0001). Whereas no signicant association
was found in EE and E* genotype between cases and
control. In case of females, the frequency of KK geno-
type of KCNJ11E23K polymorphism was found to be
moderately signicant in cases with 0.09 fold higher risk
of CAD (p=0.001) in comparison to control. Similarly
the frequency of K* genotype of KCNJ11E23K poly-
morphism was also found to be moderately signicant
in cases with 0.35 fold higher risk of CAD (p=0.001).
However no signicant association was found in EE,
EK and E* genotype between cases and control. Fedele
et al aimed to evaluate the clinical aspects of SNPs in
genes associated with ischemic heart disease in which
KCNJ11 was one of the candidate genes. Through this
study he observed a signicant difference (p<0.05) for
SNP rs 5219 of KCNJ11 gene. However, he also de-
duced that SNP 5219 of KCNJ11 has inverse corre-
lation with cardiovascular dysfunction as its frequency
was higher in control group36. Their results suggest that
genetic polymorphism could also be used to identify
patients with low risk of CAD regardless of presence
T2DM and dyslipidemia. Whereas, our results showed
correspondence with the results of Xiong et al who
demonstrated that the E23K gene polymorphism in
KCNJ11 gene is related to susceptibility to CAD37. Al-
though, on the other hand, Samadikuchaksaraei et al as
did not nd any association between CAD and E23K
polymorphism in Iranian patients38. The sample size of
this polymorphism was small; therefore we cannot rule
out the possibility of the results being affected. Hence,
genome-wide association study on distinct population
with large sample size is suggested as a more wide-
spread approach answering many more questions.
Conclusion
Polymorphism study can serve as prognostic factor as it
inuences disease occurrence and could aid in everyday
clinical practice hence, it is essential to identify those
polymorphisms that have strongest predisposition.
Further studies on these genes in specic and large
populations are required to improve our understand-
ing regarding the role of gene polymorphisms and also
to achieve any possible clinical signicance in terms of
prognosis or therapeutic intervention for CAD patients.
Acknowledgements
We are thankful to the Secretary, Era’s educational Trust
for providing us support to conduct the research.
Funding
Not applicable.
Conict of interest
The authors declare that they have no conict of in-
terest.
Ethical approval
The study was approved by Institutional ethical Com-
mittee and all procedures performed in the study were
in accordance with the laid ethical standards of the in-
stitution and with the 1964 Helsinki declaration and its
later amendments or comparable ethical standards.
Informed consent
Written informed consent was obtained from the pa-
tients prior to their participation in the research.
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... Six Asian [12,22,35],P. [43,45] and present study, three European [4,33,36], and one studies African [11] were included for meta-analysis of NOS3 (Fig. 3). These results indicate that NOS3 rs1799983 variant is the risk factor for cardiovascular diseases among different populations, and it follows recessive genetic contrast model of disease. ...
... Our meta-analysis indicates a significant association of NOS3 rs1799983 with all four genetic model's understudy; however, no heterogeneity was found under recessive model only. The studies from diverse populations that include Asian [12,22,35],P. [43] and present case-control study, European [4,33,36], and African [11] showed CVD risk causing impact of NOS3 rs1799983 variant under recessive model; however, only one study from Asian population [45] showed protective impact. To understand the impact of missense rs1799983 variant on structure and functional role of NOS3 protein, in silico studies were conducted that also showed change in the structure due to substitution of amino acids (GLN > ARG, 557) that behaves as pathogenic variant. ...
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Background Cardiovascular diseases (CVDs) are complex diseases determined by various environmental risk factors and genetic susceptibility, and NOS3 and PON1 are considered one of the susceptible genes for CVD. Our study aims to evaluate the association of NOS3 rs1799983 and PON1 rs662 variants with CVD. Methods A case–control study was conducted among equal number (252) of cases and controls in the Pakistani population to investigate the significance of NOS3 (rs1799983) and PON1 (rs662) variants in causing CVD risk. Genotyping was performed using Tetra-ARMS PCR to evaluate the genotype–phenotype correlation. For meta-analysis, the case–control studies of NOS3 rs1799983 and PON1 (rs662) variants and CVD were included by searching various databases according to PRISMA guideline. Eligible data were extracted and pooled and were analyzed using Review Manager version 5.4 based on four different genetic models. Results Our case–control study showed that both NOS3 rs1799983 (OR = 2.39, p = < 0.0001, AIC value = 710.50) and PON1 rs662 (OR = 7.30, p = < 0.0001, AIC value = 680.10) variants significantly increase the risk of CVD under recessive genetic contrast model. The meta-analysis of NOS3 rs1799983 showed association with CVD under all four genetic model's understudy, however, no heterogeneity was found under recessive model only. Meta-analysis for PON1 rs662 showed association with homozygous genetic contrast model only, whereas heterogeneity was observed under all the genetic model's understudy. Conclusions We found NOS3 rs1799983 was associated with the increased risk of CVD under four genetic contrast models, while PON1 rs662 polymorphisms associated with homozygous genetic contrast model in different populations only. These results can be utilized to identify individuals at high risk of CVDs and for disease management.
... Structure: The human eNOS gene is located in the 7q35-7q36 loci on chromosome 7 [110]; similar to the iNOS gene, it is comprised of 26 exons [111] and 25 introns that span 22 kb of the DNA, approximately [112]. Exon 26 ranging from between 68 bp (exon 14) and 579 bp (exon 26). ...
... In certain physiological conditions, nNOS could produce H 2 O 2 and superoxide (O 2 ) [139], which are key mediators in non-neuronal cell signaling. Besides, an antioxidant cytoprotection function has been suggested for nNOS, which is mediated via endoplasmic reticulum chaperone Grp94/gp96 and Ca 2+ homeostasis [110]. ...
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Nitric oxide (NO) is a small molecule, produced by nitric oxide synthase (NOS), with various physio-pathological functions in the body. There are three main NOS isoforms, including the endothelial (eNOS), inducible (iNOS), and neuronal NOS (nNOS), that existing in the peripheral organs and nervous systems of humans and rodents. Moreover, NOS includes other identified NOS isoforms, such as retinal Muller glial cells (mNOS), mitochondrial (mtNOS), penile (PnNOS), testis-specific (TnNOS), and invertebrate Drosophila NOS (dNOS) are the lesser-known types. It is proposed that the versatile functions of NOS isoforms depended on various NOS splice variant subtypes and their expression in the neural (e.g., brain, and spinal cord) and non-neuronal tissues (e.g., lung, kidney, liver and GI tract). Therefore, this review summarizes the NOS subtypes, splice variants, targeted splicing expression in the body, and their proposed physio-pathological functions. At last, alternative NOS subtypes and isoforms, which have previously received scant attention, will be addressed in this article.
... The protective role of the rs5215_G/G genotype has been already demonstrated [18,19], while the role of rs1799983_T/T genotype is controversial in the literature. The presence of the T allele may associate with CV protection and reduced vascular stiffness [55], even if other evidence has demonstrated a predisposing role to vascular stiffness [56] and IHD [57] through the reduced production of NO [58]. Other evidence showed no relationship between this SNP and IHD [59]. ...
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Genetic susceptibility may influence ischemic heart disease (IHD) predisposition and affect coronary blood flow (CBF) regulation mechanisms. The aim of this study was to investigate the association among single nucleotide polymorphisms (SNPs) of genes encoding for proteins involved in CBF regulation and IHD. A total of 468 consecutive patients were enrolled and divided into three groups according to coronary angiography and intracoronary functional tests results: G1, patients with coronary artery disease (CAD); G2, patients with coronary microvascular dysfunction (CMD); and G3, patients with angiographic and functionally normal coronary arteries. A genetic analysis of the SNPs rs5215 of the potassium inwardly rectifying channel subfamily J member 11 (KCNJ11) gene and rs1799983 of the nitric oxide synthase 3 (NOS3) gene, respectively encoding for the Kir6.2 subunit of ATP sensitive potassium (KATP) channels and nitric oxide synthase (eNOS), was performed on peripheral whole blood samples. A significant association of rs5215_G/G of KCNJ11 and rs1799983_T/T of NOS3 genes was detected in healthy controls compared with CAD and CMD patients. Based on univariable and multivariable analyses, the co-presence of rs5215_G/G of KCNJ11 and rs1799983_T/T of NOS3 may represent an independent protective factor against IHD, regardless of cardiovascular risk factors. This study supports the hypothesis that SNP association may influence the crosstalk between eNOS and the KATP channel that provides a potential protective effect against IHD.
... The associations of 163 genetic loci with CAD have been revealed so far by GWAS [21]. Our previous studies have also shown the association of gene polymorphism with CAD [22,23]. Due to the presence in noncoding regions, most of the gene variants are associated with CAD resulting into the development of disease by regulating gene expression [24]. ...
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Background: Cardiometabolic adversities constitute an enormous public health challenge. Along with lifestyle/socio-cultural determinants, disease outcome is modulated by genetic variants as well. Genetic polymorphisms like eNOS Glu298Asp (rs1799983) and AGT Met235Thr (rs699) have been identified as significant contributors to cardiometabolic phenotypes (obesity, hypertension and dyslipidemia) due to their role in vascular homeostasis, endothelial function, cardiac performance, etc. However, the association of these two genetic polymorphisms with cardiometabolic phenotypes among Indian Bhil tribal population have not yet been determined. Methods: This was a cross-sectional study conducted among Bhil tribal population from India. The study recruited 297 participants aged 25–75 years through door-to-door household survey. Data on demographic and lifestyle variables were collected using a pre-tested modified interview schedule. Anthropometric (height, weight, waist circumference and hip circumference) and physiological (systolic and diastolic blood pressure) parameters were measured to define obesity and hypertension. Fasting blood samples were collected and serum was separated for lipid profiling (TC, TG, HDL-C, LDL-C and VLDL-C) and DNA was extracted from whole blood for genetic analysis (PCR-RFLP). Multivariable logistic regression was employed to identify association of genetic polymorphisms with cardiometabolic phenotypes. Results: The frequency of GG, GT and TT genotypes of eNOS rs1799983 were 52.86%, 45.45% and 1.68%, respectively and 0.24 minor allele frequency (MAF). The frequency of TT, TC and CC genotypes of AGT rs699 were 37.71%, 41.41% and 20.88%, respectively and 0.42 MAF. GT genotype and T allele (GT + TT genotypes) of rs1799983 were inversely associated with high WC, prehypertension, low HDL-C, and high LDL-C (all adjusted OR values <1 and p < 0.05). Conversely, TC and C allele (TC + CC genotypes) of rs699 were significantly associated with dyslipidemia, specifically high TC, TG and VLDL-C (all adjusted OR values >1.6 and p < 0.05). Also, individuals with the combination of high TC and TC genotype or C allele of rs699 appeared to have survival disadvantage at older age. Conclusion: With high frequency of AGT rs699 mutant C allele and significant association with dyslipidemia, our study suggests screening of candidate gene polymorphisms for cardiometabolic diseases among tribal populations to identify risk genotypes or alleles, which may pave way for future pharmacogenetic therapy and precision medicine.
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Conventionally, ischemic heart disease (IHD) is equated with large vessel coronary disease. However, recent evidence has suggested a role of compromised microvascular regulation in the etiology of IHD. Because regulation of coronary blood flow likely involves activity of specific ion channels, and key factors involved in endothelium-dependent dilation, we proposed that genetic anomalies of ion channels or specific endothelial regulators may underlie coronary microvascular disease. We aimed to evaluate the clinical impact of single-nucleotide polymorphisms in genes encoding for ion channels expressed in the coronary vasculature and the possible correlation with IHD resulting from microvascular dysfunction. 242 consecutive patients who were candidates for coronary angiography were enrolled. A prospective, observational, single-center study was conducted, analyzing genetic polymorphisms relative to (1) NOS3 encoding for endothelial nitric oxide synthase (eNOS); (2) ATP2A2 encoding for the Ca(2+)/H(+)-ATPase pump (SERCA); (3) SCN5A encoding for the voltage-dependent Na(+) channel (Nav1.5); (4) KCNJ8 and KCNJ11 encoding for the Kir6.1 and Kir6.2 subunits of K-ATP channels, respectively; and (5) KCN5A encoding for the voltage-gated K(+) channel (Kv1.5). No significant associations between clinical IHD manifestations and polymorphisms for SERCA, Kir6.1, and Kv1.5 were observed (p > 0.05), whereas specific polymorphisms detected in eNOS, as well as in Kir6.2 and Nav1.5 were found to be correlated with IHD and microvascular dysfunction. Interestingly, genetic polymorphisms for ion channels seem to have an important clinical impact influencing the susceptibility for microvascular dysfunction and IHD, independent of the presence of classic cardiovascular risk factors.
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This study tries to find detectable signals of gene flow of Sub-Saharan origin into the Mediterranean in four genomic regions previously associated with coronary artery disease. A total of 366 single-nucleotide polymorphisms were genotyped in 772 individuals from 10 Mediterranean countries. Population structure analyses were performed, in which a noticeable Sub-Saharan component was found in the studied samples. The overall percentage of this Sub-Saharan component presents differences between the two Mediterranean coasts. D-statistics suggest possible Sub-Saharan introgression into one of the studied genomic regions (10q11). We also found differences in linkage disequilibrium patterns between the two Mediterranean coasts, possibly attributable to differential Sub-Saharan admixture. Our results confirm the potentially important role of human demographic history when performing epidemiological studies.European Journal of Human Genetics advance online publication, 18 January 2017; doi:10.1038/ejhg.2016.200.
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Coronary heart disease affects ≈15.4 million individuals in the United States1 and is one of the main causes of mortality and morbidity.1 A familial component contributes to cardiovascular disease (CVD) susceptibility,2 but it was not until the emergence of genomewide association studies (GWAS) that genetic loci were identified that displayed consistent associations with coronary artery disease (CAD) across multiple cohorts. The present article describes recent advances in the understanding of the genetic basis of CAD. Family2 and twin3 studies provided convincing evidence that CAD clusters in families and has a heritable component2–5; however, the delineation of the specific genetic architecture that predisposes to CAD has been challenging, with initial candidate gene approaches yielding inconsistent results.6 In contrast to candidate gene approaches that focus on genetic variation in genes whose gene products are known to play an important role in cardiovascular physiology, GWAS simultaneously assess the association with CAD of hundreds of thousands of genetic variants distributed across the whole genome. Therefore, GWAS represent an essentially unbiased approach that is not limited by the current (patho)physiological understanding of CVD and bears the potential of discovering completely new molecular mechanisms that predispose to CAD. In 2007, the first GWAS for CAD were published.7–9 The main finding was a locus on chromosome 9p21, which is still the most consistently associated CAD locus to date.10 Subsequent studies revealed that this locus is related to a broad spectrum of vascular phenotypes, including CAD and myocardial infarction,7–9 coronary artery calcification,11 peripheral artery disease,12,13 and abdominal aortic aneurysm.14 To increase power for the detection of genetic variants with smaller effect sizes, large consortia have been built that combine genetic-epidemiological data from multiple cohorts and tens …