Association between inflammatory gene polymorphismsand coronary artery disease in an Indian population

Indranil Banerjee Æ Umeshwar Pandey ÆOmer M. Hasan Æ Rashmi Parihar ÆVijaya Tripathi Æ Subramaniam Ganesh

Published online: 23 December 2007

� Springer Science+Business Media, LLC 2007

Abstract Background Inflammation is one of the major

components of atherosclerosis which is the underlying

disorder that leads to various diseases including coronary

artery disease (CAD). Genes that are involved in the

inflammatory processes are therefore good candidates for

the risk of CAD. Variations in the genes involved in var-

ious molecular pathways of inflammation have been

implicated to exaggerated atherosclerosis and the risk of

cardiovascular diseases. In this study, we performed a

genetic association study on the single nucleotide poly-

morphisms (SNPs) present in the genes CD14 (-159 C/T),

TNFa (-308 G/A), IL-1a (-889 C/T), IL-6 (-174 G/C),

PSMA6 (-8 C/G), and PDE4D (SNP83 T/C, respectively)

in order to discern their possible role in the susceptibility to

CAD in a North Indian population. Methods Angiograph-

ically proven CAD patients (n = 210) and age, sex and

ethnically matched normal healthy controls (n = 232)

were recruited for this case-control study. Genotypes were

determined by PCR–RFLP method. Chi-square and logistic

regression analyses were performed to compare the geno-

type and allele frequencies between the patient and the

control groups. Results None of the SNPs showed signifi-

cant association with CAD in the study population before

and after adjustment for the confounding risk factors like

age, sex, hypertension, smoking habit, and diabetes. Con-

clusion This study was unable to demonstrate any

association between the six gene variants tested and CAD

in the North Indian population.

Keywords Coronary artery disease � Inflammation �Gene polymorphism � Association

Introduction

Coronary artery disease (CAD) is the most common form

of heart diseases that results due to occlusion in the arteries

supplying blood to the cardiac muscles. According to the

World Health Organization estimates, nearly 7 million

people die of this disease worldwide per year and most of

these deaths occur in the developing countries (http://

www.who.int/cardiovascular_diseases). More than 80% of

sudden cardiac deaths are caused by atherosclerotic CAD,

and the remaining 20% of cases are caused by other dis-

eases [1]. CAD has a multifactorial basis which is

characterized by the interactions between the genetic and

the environmental factors. These interactions largely

determine whether an individual is predisposed to a greater

or lesser extent to CAD [2].

In the pathogenesis of CAD, inflammation plays a cru-

cial role through its contribution to the formation of

atheroma, which eventually leads to the evolution of ath-

eromatous injury, plaque rupture and intraluminal

thrombosis [3]. The lesions of atherosclerosis, the under-

lying cause of CAD and other cardiovascular diseases such

as ischemic stroke, represent a series of highly specific

cellular and molecular responses that could be best

described as inflammatory disease [4]. Due to inflamma-

tion, macrophages and lymphocytes emigrate from the

blood, multiply within the atherosclerotic lesion and lead to

the release of hydrolytic enzymes, cytokines, chemokines,

I. Banerjee � R. Parihar � V. Tripathi � S. Ganesh (&)

Department of Biological Sciences and Bioengineering,

Indian Institute of Technology, Kanpur 208016, India

e-mail: sganesh@iitk.ac.in

U. Pandey � O. M. Hasan

LPS Institute of Cardiology, GSVM Medical College, Kanpur,

India

123

J Thromb Thrombolysis (2009) 27:88–94

DOI 10.1007/s11239-007-0184-8

and growth factors, which induce further damage and lead

to focal necrosis [4]. A greater prevalence of ruptured

atherosclerotic plaques was also found to correspond to an

elevated level of inflammatory markers, indicating a link

between the inflammatory processes and arterial dysfunc-

tions that could lead to various cardiovascular diseases [5].

Recently a number of candidate genes and chromosomal

loci have been identified to be associated with the sus-

ceptibility to myocardial infarction (the most acute form of

CAD) and a majority of these genes have been implicated

in the processes of inflammation [6]. From amongst a

number of genes that have been implicated in the patho-

genesis of CAD and ischemic stroke, we selected for our

study the genes encoding CD14 cell surface glycoprotein

(CD14), tumor necrosis factor-a (TNFa), interleukin-1a(IL-1a), interleukin-6 (IL-6), proteasome subunit a type 6

(PSMA6), and phosphodiesterase 4D (PDE4D), all of

which are involved in the inflammatory mechanisms. CD14

serves as a receptor for bacterial lipopolysaccharide (LPS,

endotoxin), mediating cell activation by LPS, while TNFa,

IL-1, IL-6 contribute to inflammation as proinflammatory

cytokines [7–11]. Enhanced PSMA6 activity has been

suggested to exaggerate inflammation through activation of

nuclear factor jB (NF-jB), a central transcription factor

that regulates the expression of the genes of cytokines and

adhesion molecules involved in atherogenesis [6, 12].

PDE4D is a key regulator of cAMP signal transduction and

is implicated in inflammation for its role in upregulating

the processes of cell proliferation and migration [13]. The

single nucleotide polymorphisms (SNPs) present in the

genes CD14 (-159 C/T), TNFa (-308 G/A), IL-1a (-889

C/T), IL-6 (-174 G/C), PSMA6 (-8 C/G), and PDE4D

(SNP83 T/C, respectively), were found to be associated

with increased risk of cardiovascular diseases in different

populations [6, 14–19].

In this case-control association study, we sought to

examine whether the above six SNPs in the genes impli-

cated in the inflammatory processes were also associated

with CAD in a population belonging to North India.

Methods

Study design and population

The study population was comprised of 210 sporadic CAD

cases and 232 unrelated, healthy control individuals

(Table 1). The cases (male 166, female 44), diagnosed with

either myocardial infarction or unstable angina, were

recruited for this study from the LPS Institute of Cardiol-

ogy, GSVM Medical College, Kanpur, India, between

February 2006 and December 2006. The patients, with age

ranging from 31 to 84 years (average age = 56.3 ± 12.1

years), primarily diagnosed with cardiac abnormality by

electrocardiography (ECG), were subjected to coronary

angiography for confirmation of CAD. Angiographic evi-

dence showing[50% stenosis of at least one segment of a

major coronary artery was defined as CAD. Control sub-

jects (male 166, female 66) were recruited from the same

hospital as the cases, and their clinical histories were

reviewed in an interview. They were unrelated individuals

(average age = 56.0 ± 9.5 years) representing the same

geographic location and ethnic population (northern Indian

population). All the control subjects were sampled to match

the CAD patients for age and sex. Control subjects had the

vascular risk factors such as hypertension, diabetes melli-

tus, and smoking habit but had no history of cardiac

diseases, no symptoms of other atherosclerotic vascular

diseases and had normal electrocardiogram.

Arterial hypertension was defined as having a previous

diagnosis of hypertension or if systolic or diastolic blood

pressure was [140 mmHg or [90 mmHg, respectively, or

both on at least two different occasions. Subjects were

classified as having diabetes mellitus if fasting blood glu-

cose level was [126 mg/dL or if they had any history of

being diagnosed with the disease. Subjects were defined as

smokers who were previously or currently addicted to

tobacco smoking, whereas nonsmokers were the subjects

having no history of previous or current smoking.

Genotype determination

Blood samples were drawn from cases and controls, and

genomic DNA was extracted from blood samples by

‘salting out’ method. Using the isolated genomic DNA as

template, polymerase chain reactions (PCR) were carried

out, followed by restriction fragment length polymorphism

(RFLP) analyses to determine the SNPs. The detailed

information on the PCR conditions, primer sequences,

restriction enzymes etc are summarized in Table 2.

Statistical analysis

Categorical data of the study population was reported as

frequencies and percentages, and the continuous data as

means with standard deviations. Comparisons of the cate-

gorical variables between cases and controls were done using

v2 test using 2 9 2 contingency tables. To compare the age

and the levels of cholesterol, triglyceride, LDL, HDL, and

VLDL across the two groups, t-tests were performed.

Shapiro–Wilk tests were performed to examine whether the

levels of cholesterol, triglyceride, LDL, HDL, and VLDL

follow normal distribution in the case and the control groups.

Deviation from Hardy–Weinberg equilibrium proportions

Association between inflammatory gene polymorphisms and coronary artery disease 89

123

was tested for each genetic marker. To assess the indepen-

dent contribution of genotype to CAD, multivariate logistic

regression analyses were performed adjusting for age, sex,

hypertension, smoking habit, and diabetes mellitus. For each

odds ratio (OR), 95% confidence intervals (CIs) were cal-

culated. A probability value \0.05 was considered a

statistically significant result and two-tailed P value was

considered. All analyses were carried out using the software

StatsDirect (Version 2.5.7).

Results

Characteristics of cases and control subjects

The characteristics of the patients with CAD and those of

control subjects are summarized in Table 1. The genotype

frequencies for the CD14, TNFa, IL-1a, IL-6, PSMA6, and

PDE4D polymorphisms in the control groups were in Hardy-

Weinberg equilibrium. The patients with CAD had higher

Table 1 Clinical

characteristics of the study

populationa

* Values shown in bold font

indicate statistical significancea n, number; SD, standard

deviation

Continuous and categorical

variables were tested by t-test

and v2 analysis, respectively

Case (n = 210) Control (n = 232) P value*

Age in years ± SD 56.3 ± 12.1 56.0 ± 9.5 0.79

Male gender, n (%) 166 (79) 166 (71) 0.07

Hypertension, n (%) 104 (50) 110 (47) 0.66

Diabetes, n (%) 66 (31) 34 (20) \0.0001

Smoking, n (%) 53 (25) 42 (18) 0.06

Cholesterol ± SD (mg/dL) 174.1 ± 36.1 188.8 ± 39.2 \0.0001

Triglycerides ± SD (mg/dL) 167.2 ± 54.5 178.3 ± 66.7 0.05

LDL ± SD (mg/dL) 100.3 ± 31.6 113.7 ± 29.8 \0.0001

HDL ± SD (mg/dL) 41.7 ± 11.5 40.3 ± 8.0 0.14

VLDL ± SD (mg/dL) 34.0 ± 11.1 36.3 ± 16.9 0.10

Table 2 Primers, PCR conditions and genotyping

Genes and

polymorphisms

Primers sequence

(fragment size obtained after PCR)

Annealing temperature

�C/extension time in

seconds/number

of cycles

Restriction

enzyme

Fragments in

bp obtained

after digestion

Reference

CD14

159 C/T

50-GTGCCAACAGATGAGGTTCAC-30 (forward)

50-CCTCTGTGAACCCTGATCAC-30 (reverse)

(453 bp)

57/60/40 Eco471

(AvaII)

T/T: 353, 100

C/C: 453

C/T: 453, 353, 100

Present

studya

IL6

-174 G/C

50-TGACTTCAGCTTTACTCTTTGT-30 (forward)

50-CTGATTGGAAACCTTATTAAG-30 (reverse)

(198 bp)

55/60/40 SfaNI C/C: 198

G/G: 140, 58

G/C: 198, 140, 58

[20]

IL1a

-889 C/T

50-GGGGGCTTCACTATGTTGC

CCACACTGGACTAA-30 (forward)

50-GAAGGCATGGATTTTTACATATG

ACCTTCCATG-30 (reverse)

(300 bp)

57/60/40 NcoI T/T: 300

C/C: 266, 43

C/T: 300, 266, 43

[21]

TNFa

-308 G/A

50-AGGCAATAGGTTTTGAGGGCCAT-30

(forward)

50-TCCTCCCTGCTCCGATTCCG-30 (reverse)

(107 bp)

55/60/40 NcoI A/A: 107

G/G: 87, 20

G/A: 107, 87, 20

[22]

PSMA6

-8 C/G

50-CCAGATGAAAGCCTGAAAGC-30 (forward)

50-GGTAATGTGGCGGTCAAAAC-30 (reverse)

(343 bp)

60/60/40 RsaI G/G: 201, 94, 48

C/C: 201, 142

G/C: 201, 142, 94, 48

Present

studya

PDE4D

SNP83 T/C

50-TTGTTTCTAGTGTTAGCCTTG-30 (forward)

5-ATTTGGCCTTGCAATATAC-30 (reverse)

(492 bp)

60/60/40 TaiI C/C: 288, 204

T/T: 492

C/T: 492, 288, 204

[23]

a The PCR–RFLP method to detect the SNP was developed in the present study

90 I. Banerjee et al.

123

prevalence of diabetes mellitus than the asymptomatic

controls, but no statistically significant difference in smok-

ing habit and the number of hypertensive individuals were

found to exist between the two groups. Although the mean

levels of total cholesterol, triglycerides, and low-density

lipoproteins (LDL) were significantly higher in the controls

as compared to the cases, the values in each group showed

significant departure from normality (data not shown).

Genotype frequencies

We calculated the genotype and allele frequencies of the

CD14, TNFa, IL-1a, IL-6, PSMA6, and PDE4D gene

polymorphisms of the CAD group of the study population

and compared them with that of the control group

(Table 3). No evidence of association was found between

the polymorphisms of the candidate genes studied and the

risk of CAD in the study population. Also in the multi-

variate logistic regression analysis, no significant

association was detected (Table 4).

Discussion

A number of case-control association studies revealed

variants in many genes are implicated in increasing or

decreasing the susceptibility to CAD or myocardial

infarction. However, true association of some genes with

the disease could not be determined with certainty due to

inconsistency in the results among different studies and

lack of replication in most of the associations with CAD

and myocardial infarction [24]. In this study, we evaluated

the association between some of the SNPs in the CD14,

TNFa, IL-1a, IL-6, PSMA6, and PDE4D genes and CAD

in a northern Indian population. These six gene variants

were previously found to be associated with cardiovascular

diseases in different populations. The rationale for this

study was to examine whether these gene variants are also

independent risk factors for CAD in our study population.

CD14 is a glycoprotein receptor expressed by mature

monocytes, macrophages, and neutrophils. Upon bacterial

LPS binding, CD14 triggers the production of proinflam-

matory cytokines such as TNFa, IL-1, IL-6, and growth

Table 3 Genotype and allele distribution

CD14 -159 C/T IL6 -174 G/C IL1a -889 C/T

Genotypes Cases Controls P value Genotypes Cases Controls P value Genotypes Cases Controls P value

TT 49 68 0.16 CC 8 4 0.24 TT 21 19 0.51

TC 116 126 CG 43 57 TC 92 99

CC 45 38 GG 159 171 CC 97 114

Alleles Cases Controls P value Alleles Cases Controls P value Alleles Cases Controls P value

T 214 262 0.10 C 59 65 0.99 T 134 137 0.44

C 206 202 G 361 399 C 286 327

TNFa -308 G/A PSMA6 -8 C/G PDE4D SNP83 T/C

Genotypes Cases Controls P value Genotypes Cases Controls P value Genotypes Cases Controls P value

AA 1 0 0.47 GG 15 10 0.21 CC 48 41 0.18

AG 28 31 GC 72 79 CT 111 121

GG 181 201 CC 123 143 TT 51 70

Alleles Cases Controls P value Alleles Cases Controls P value Alleles Cases Controls P value

A 30 31 0.79 G 102 99 0.30 C 207 203 0.10

G 390 433 C 318 365 T 213 261

Table 4 Multivariate logistic regression analysisa

Genes and

polymorphisms

Genotype

contrasts

OR 95% CI P value

CD14 -159 C/T TT vs. TC + CC 0.76 0.49–1.17 0.21

IL6 -174 G/C CC vs. CG + GG 1.95 0.56–6.78 0.29

IL1a -889 C/T TT vs. TC + CC 1.20 0.61–2.37 0.59

TNFa -308 G/A AA vs. AG + GG – – 0.98

PSMA6 -8 C/G GG vs. GC + CC 1.68 0.71–3.93 0.24

PDE4D SNP83 T/C CC vs. CT + TT 1.34 0.83–2.16 0.24

a Adjusted for age, sex, hypertension, smoking habit, and diabetes

Association between inflammatory gene polymorphisms and coronary artery disease 91

123

factors [4, 25]. These cytokines contribute to inflammation

and atherogenesis through various processes including

vascular endothelial wall injury, expression of adhesion

molecules for monocytes on the arterial wall, etc. [10, 26-

28]. PSMA6 gene encodes for a protein that is one of the

constituents of the 26S proteasome system. When lym-

photoxin a (a member of the TNF family) binds to its

receptor, proteasome activates NF-jB by degrading its

inhibitory partner IjB. NF-jB then expresses a wide

variety of genes like TNFa, IL-1, IL-6, chemokines,

adhesion molecules, etc. The milieu of these molecules in

arterial wall leads to enhanced inflammation and conse-

quently, results in atherogenesis [12, 29]. PDE4D regulates

cAMP signal transduction in various cell types including

inflammatory, endothelial, and smooth muscle cells [18].

Decreased cAMP level enhances vascular smooth muscle

cell proliferation and migration, the events representing

atherosclerosis [18].

Case-control association studies on the -159 C/T

polymorphism in the CD14 gene and myocardial infarc-

tion, and the finding that healthy males with TT genotype

have higher density of CD14 on monocytes suggested this

SNP could be a risk factor for atherosclerosis and myo-

cardial infarction [30]. Higher frequencies of the T allele

and TT genotype were found in patients with CAD than in

healthy control subjects [14, 31, 32]. But, a lack of asso-

ciation of this SNP with CAD was reported from other

studies [30, 33]. We also failed to find any association of

the -159 C/T polymorphism in the CD14 gene with CAD

in this study. A -308 G/A polymorphism in the promoter

region of the TNFa gene was reported to be associated with

cardiovascular diseases [17, 28, 34]. The A allele was

found to be associated with increased expression of TNFaafter in vitro stimulation, suggesting that the SNP could

contribute to the susceptibility and severity of inflamma-

tory diseases [28]. In our study population, there was no

significant association between this SNP with CAD. The

-889 C/T polymorphisms in the IL-1a genes was found to

confer risk for stroke, while conflicting results were

obtained from the association studies on the -174 G/C

polymorphism in the IL-6 gene and the risk of myocardial

infarction and CAD [15, 16, 19]. IL-1a mRNA and protein

levels increase in the carriers of TT genotype as compared

to those having CC variant, indicating a potential role of

this promoter polymorphism in inflammation [35]. In this

study, we could not find any association between the SNPs

in IL-6 and IL-1a genes and the risk of CAD. Recently, the

-8 C/G polymorphism in the in the 50-untranslated region

of exon 1 of PSMA6 gene was found to be strongly asso-

ciated with the risk of myocardial infarction in the Japanese

population [6]. The G allele showed significantly higher

expression of PSMA6 than the C allele and was suspected

to confer risk for myocardial infarction through the

exaggeration of inflammation [6]. However, another recent

study in the Japanese population has shown this SNP in the

PSMA6 gene does not contribute appreciably to myocar-

dial infarction, but contribute slightly to atherosclerosis

[36]. The SNP83 (a T/C transition in the intron 1) in

PDE4D gene was found to be associated with ischemic

stroke in the Icelandic population [18]. Subsequent studies

in different populations on SNP83 showed conflicting

results, making it difficult to determine whether this

polymorphism is an independent risk factor for ischemic

stroke. However, the overall association of this SNP with

the pathogenesis of ischemic stroke was confirmed by a

recent meta-analysis [37]. Since PDE4D contributes to

atherosclerosis through its participation in the inflamma-

tory processes, its role could be validated in other vascular

diseases like CAD. We, therefore, tested whether this gene

variant confers risk for CAD in the study population, and

failed to find any association.

In the present study, it was surprising to find that the

mean levels of total cholesterol, triglycerides, and low-

density lipoproteins (LDL) were significantly higher in the

controls as compared to the cases. Similar results on total

cholesterol levels were reported in some studies on stroke

in other Asian populations [23, 38]. But, in the present

study, the values of these parameters in either group were

found to be departed significantly from normality. There-

fore, the apparently paradoxical results that we observed on

the mean levels of total cholesterol, triglycerides, and LDL

in relation to CAD pathogenesis, could be attributable to

the low statistical power of this study, and should be

considered with caution.

Since the most common and major pathological changes

in ischemic heart diseases and cerebral infarction are ath-

erosclerosis and thrombogenesis, several studies have so

far investigated whether the sequence variations in the

inflammatory genes increase the risk for these diseases

[28]. But no study on Indian population has so far evalu-

ated the risk of CAD associated with the inflammatory

gene polymorphisms included in this study. We, therefore,

endeavored to investigate the role of the inflammatory gene

variants, previously found to be associated with cardio-

vascular diseases in other populations. However, our data

did not support the hypotheses that the polymorphisms in

the CD14, TNFa, IL-1a, IL-6, PSMA6, and PDE4D genes

might be associated with the development of CAD. These

negative results should be considered with caution, owing

to some potential limitations of the study. First, the sample

size of this study was relatively small. Low statistical

power could have resulted in negative findings. Therefore,

to confirm our results, larger study population is needed.

Second, the possibility of some selection-bias in hospital-

based control subjects could not be ruled out. Nevertheless,

the genotype distributions of the controls were found to

92 I. Banerjee et al.

123

conform to the Hardy–Weinberg equilibrium. Third, the

Indian subcontinent is comprised of large number of eth-

nically varied populations. Thus, the study population (the

population in the city of Kanpur) could represent only a

fraction of the populations belonging to the northern India.

So, the extrapolation of the data to other populations of the

country or to the northern Indian population as a whole

should be carried out with caution.

The gene polymorphisms studied here showed associa-

tion with CAD pathogenesis in populations belonging to

different countries, but India. Thus it is likely that the

observed lack of association may relate in part to the dif-

ferences in allele frequencies across populations due to

variations in ethnicity. Nonetheless, the present study

provides insights about the genetic markers of inflamma-

tion in CAD pathogenesis in a population-specific manner.

The results of this study may contribute to discern the role

of these inflammatory gene polymorphisms in the devel-

opment of CAD in robust statistical approaches like meta-

analysis. Studies with larger population size are warranted

to investigate the relationship of these SNPs in inflamma-

tory genes with CAD in order to provide more definitive

conclusion.

Acknowledgments This study was supported by an intramural

research funding from the Indian Institute of Technology, Kanpur

(IITK). IB was supported by a research studentship from the IITK.

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