Cytokine Gene Polymorphisms in the Susceptibility to Acute Coronary Syndrome

Abstract

Aim: Acute coronary syndrome (ACS) is an inflammatory disease. Cytokines are the central regulators of inflammation and may be a cause or marker of atherosclerosis. Accumulating evidence suggests that polymorphisms at promoter regions of various cytokine genes are known to be associated with their expression levels. In the present study we investigated whether variants at −1082G→A (rs1800896) and −592C→A (rs1800872) of interleukin-10 (IL-10), −1188A→C (rs3212227) of IL-12 p40, −308G→A of tumor necrosis factor-α (TNF-α) (rs1800629), −174G→C of IL-6 (rs1800795) and +874A→T of interferon-γ (IFN-γ) genes (rs2430561) are associated with ACS. Materials and Methods: DNA samples were collected from 1083 subjects and IL-10-1082G→A, −592A→C, TNF-α-308G→A, IL-12 p40-1188 A→C, and IFN-γ+874A→T polymorphisms were identified by amplified refractory mutation system polymerase chain reaction and IL-6-174 G/C, restriction fragment length polymorphism based on standard methods. Results: Six hundred and fifty one ACS patients along with 432 age and sex matched controls were analyzed for various gene polymorphisms. The “low-producer” IL-10-1082 AA (χ²=9.45; p=0.0021; odds ratio [OR]=1.472; 95% confidence interval [CI]=1.15-1.884), “high producer” IL-10-592 CC (χ²=39.42; p=0.001, OR=2.26; 95% CI=1.748-2.292), “low producer”IFN-γ+874AA (χ²=28; p<0.00154; OR=2.36 & 95% CI=1.713-3.251), and “high producer” TNF-α −308AA (χ²=3.213, p=0.073; OR=1.515) genotypes may be responsible for the regulation of immune response leading to inflammation in ACS patients. However, −1188 of the IL-12 gene was not associated with the disease. Conclusion: The polymorphisms at −308G→A of TNF-α, −174G→C of IL-6, +874A→T of IFN-γ and −1082G→A, and −592C→A of IL-10 genes evaluated in the present study are important risk factors for the development of ACS in the South Indian population from Andhra Pradesh. The better understanding of these variants conferring susceptibility to ACS may aid in early diagnosis and development of new methods to create personalized medicine.

Introduction

Inflammation plays an important role in the initiation and development of coronary atherosclerotic diseases such as acute coronary syndrome (ACS). Inflammatory markers such as cytokines play an important role in the pathogenesis of atherosclerosis and mediate the stages of atheroma development from initial leukocyte recruitment to eventual rupture of the atherosclerotic plaque (Libby et al., 2002; Tedgui and Mallat, 2001).

The alteration of cytokine expression and secretion might be due to the disease progression. Previous studies have indicated that genetic polymorphisms within the coding and promoter regions of cytokine genes might influence their production (Morse et al., 1999). Many of the cytokine polymorphisms, specifically expressing high levels of cytokines have been reported to influence susceptibility to various diseases (Bidwell et al., 1999). Presence of variant alleles such as ‘G’ at −174 of interleukin-6 (IL-6), ‘C’ at −592 and ‘G’ at −1082 of IL-10, ‘A’ at −308 of tumor necrosis factor-alpha (TNF-α), ‘A’ at −1188 of IL-12, and ‘T’ at +874 of interferon-gamma (IFN-γ) in the promoter regions have been reported to be involved in the regulation of gene expression (Turner et al., 1997; Wilson et al., 1997; Fishman et al., 1998; Pravica et al., 2000; Seegers et al.,2002).

Very few studies have been carried out to evaluate the role of variants of cytokine genes like IL-6-174 G/C, IL-10-1082G/A, −592A/C, TNF-α −308G/A, IL-12 p40-1188, and IFN-γ +874A/T in the development of ACS. Hence, the present study was carried out with the aim of evaluating the variants of these cytokine genes in association with susceptibility to ACS. These genes were chosen based on their functions in inflammation regulation that may be involved in ACS pathogenesis and their polymorphisms based on their potential regulation of gene expression.

Materials and Methods

Six hundred and fifty one ACS patients, who had ischemic chest pain at rest within the preceding 48 h (i.e., Braunwald class IIIB); and with transient ST-T-segment depression and/or T-wave inversion, were included in the study. The patients with thrombolytic therapy within the previous month and concomitant disease likely to be associated with inflammation (infections and autoimmune disorders) were excluded from the study. Age and sex matched healthy individuals (n=432) of same geographic background and socioeconomic status similar to that of ACS patients were included as control group in the study. Information pertaining to age, gender, habits, health status, family history and the like was recorded in a case proforma and the study was approved by the Institutional ethics committee.

Molecular analyses

Genomic DNA was isolated from peripheral blood by the salting out method (Lahiri and Nurnberger, 1991). Polymorphisms of IL-10 −1082G/A, −592A/C, TNF-α −308G/A, IL-12 p40-1188, and IFN-γ +874A/T were evaluated by the amplified refractory mutation system polymerase chain reaction (PCR) using the specific primers and PCR conditions summed up in Table 1 (Olomolaiye et al., 1998; Perrey et al., 1999; Louis et al., 2000; Pravica et al.,2000; Latsi et al., 2003; Lopez-Maderuelo et al., 2004). However, IL-6-174 G/C polymorphism was detected by PCR-restriction fragment length polymorphism technique using the restriction enzyme Nla III (MBI Fermentas, Inc.). Primers and PCR conditions have also been summed up in Table 1 (Boiardi et al., 2006; Belluco et al., 2003). The PCR was carried out with specific primers in a 30 μL volume with 10× buffer (500 mM KCl, 100 mM Tris-HCl [pH 8.3], 15 mM MgCl₂); 10 pM of each specific oligonucleotide primers; 200 μM dNTPs; 100 ng of genomic DNA; and 1.0 U Taq DNA polymerase (Banglore Genei). The PCR products were 780, 161, 319, 183, and 263 bp for IL-12 p40, IL-10-1082, IL-10-592, TNF-α −308, and IFN-γ +874 genes, respectively. In case of IL-6 PCR product size was 190 bp that was digested with Nla III restriction enzyme at 37°C overnight and electrophoresed on a 2% agarose gel with ethidium bromide staining. After digestion 140/58 bp, 198/140/58 bp, and 198 bp represented GG homozygotes, GC heterozygotes, and CC homozygotes, respectively. The details have been given in Table 1.

Table 1.

Brief Description of Polymerase Chain Reaction of the Genes Under Study

Cytokine	SNP/RS number	Annealing temp. Amplicon size method reference	Primer sequences	Expression (reference)
IL-12 p40	-1188 A/C rs3212227	60°C −1 min 780 bp ARMS PCR (Latsi et al., 2003)	A reverse primer 5′-ttgtttcaatgagcatttagcatct3′ C reverse primer 5′-gtttcaatgagcatttagcatcg3′ Forward primer 5′-atcttggagcga atgggcat3′	C high levels (Seegers et al., 2002)
IL-10	-1082 G/A rs1800896	60°C −1 min 161 bp ARMS PCR (Lopez et al., 2004)	A reverse primer 5′-aacactactaaggcttctttgggta3′ G reverse primer 5′-aacactactaaggcttctttgggtg3′	G high levels (Turner et al., 1997)
IL-10	-592 C/A rs1800872	60°C −1 min 319 bp ARMS PCR (Lopez et al., 2004)	A reverse primer 5′-atcctgtgaccccgcctgta-3′ C reverse primer 5′-atcctgtgaccccgcctgtc3′ Forward primer 5′-tgagaaataattgggtcccc3′	C high levels (Turner et al., 1997)
TNF-α	-308 G/A rs1800629	60°C-30s 183 bp ARMS PCR (Louis et al., 2000)	Common primer 5′-tctcggtttcttctccatcg-3′ primer G 5′-ataggttttgaggggcatgg-3′ primer A 5′-ataggttttgaggggcatga-3′ Internal Control 5′-gagtctccgggtcagaatga-3′	A high levels (Wilson et al., 1997)
IFN-γ	+874A/T rs2430561	60°C −1 min 263 bp ARMS PCR (Pravica et al., 2000)	Consensus primer 5′-tcaacaaagctgatactcca-3′ A allele specific 5′-ttcttacaacacaaaatcaaatca-3 T allele specific 5′-ttcttacaacacaaaatcaaatct-3′ Control forward 5′-gccttcccaaccattccctta-3′ Control reverse 5′ tcacggatttctgttgtgtttc-3′	T high levels (Pravica et al., 2000)
IL-6	-174 G/C rs1800795	60°C −1 min 198 bp (198/140/58 bp) PCR RFLP (Boiardi et al., 2006)	Forward primer 5′-ttgtcaagacatgccaagtgct− 3′ Reverse primer 5′-gcctcagagacatctccagtcc-3′	G high levels (Fishman et al., 1998)

ARMS, amplified refractory mutation system; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; IL, interleukin; TNF, tumor necrosis factor; IFN, Interferon.

Statistical methods

Base line characteristics between the groups were compared by t test. The polymorphisms were tested for Hardy Weinberg equilibrium analysis using the SNPStats and SNP Analyzer web-based tool (Yoo et al., 2005; Sole et al., 2006). The genotype associations between ACS patients and controls were examined by using odds ratio (OR) with 95% confidence interval (CI) and chi-square (χ²) analysis. A p-value of <0.05 was considered statistically significant.

Results

Six hundred and fifty one ACS patients and 432 controls were employed in the study. Demographic features of the study group have been presented in Table 2. The mean age of patients and controls was 53.56±11.72; 52.62±8.45 (mean±SD; years), respectively. In the present study 76.65% ACS patients were males, indicating that the prevalence of ACS is greater in males in comparison with females.

Table 2.

Demographic Characteristics in the Study Population

Parameters	Controls	ACS	p-value
Number	432	651	NA
Males, n (%)	265 (61.34)	499 (76.65)	NA
Females, n (%)	167 (38.66)	152 (23.34)	NA
Age (mean±SD)	52.62±8.45	53.56±11.72	0.1359
BMI, kg/m2	22.1±2.6	26.95±2.46	<0.001
Smokers, n (%)	29 (6.71%)	241 (37.02)	NA
Alcoholics, n (%)	15 (3.47%)	207 (31.79)	NA
Diabetes mellitus, n (%)	6 (1.39)	101 (15.51)	NA
Hypertension, n (%)	16 (3.71)	308 (47.31)	NA
History of CHD, n (%)	10 (2.31)	202 (31.02)	NA
LVEF (% mean±SD)	54.56±7.64	46.75±6.36	<0.001
SBP, mm Hg (mean±SD)	120±12	134±28	<0.001
DBP, mm Hg (mean±SD)	80±6	85±20	0.001
Hemoglobin (grams) (mean±SD)	15.7±3.2	15.0±2.4	0.005
TCHOL (mg/dL) (mean±SD)	162.9±16.2	210.02±22.7	<0.001
TGL (mg/dL) (mean±SD)	139.1±10.5	166.1±14.5	<0.001
HDL (mg/dL) (mean±SD)	41.30±5.4	33.1±5.2	<0.001
LDL (mg/dL) (mean±SD)	93.70±17.9	142.44±13.2	<0.001

ACS, acute coronary syndrome; BMI, body mass index.

The body mass index (BMI) values in ACS patients ranged between19.1-34.6 (mean±SD: 26.95±2.46) whereas in the control group it ranged between17.8-26.4 (22.1±2.6). Out of 651 ACS patients 241 (37.02%) were smokers and 207 (31.79%) were alcoholics. 15.51%, 47.31%, and 31.02% of ACS patients had a previous history of diabetes, hypertension, and a family history of CHD, respectively. In the control group 6.71% were smokers, 3.47% were alcoholics; 1.39%, 3.71%, and 2.31% had a previous history of diabetes, hypertension, and the family history of CHD, respectively.

The estimated mean levels of cholesterol, triglycerides, and LDL cholesterol were significantly higher in patients in comparison with controls; whereas the HDL cholesterol in ACS patients was significantly lower in comparison with controls (Table 2). The distribution of genotypes and allelic frequency of various cytokine SNPs in ACS patients and controls have been presented in Table 3 and their association with the disease has been given in Table 4.

Table 3.

Distribution of Genotypes and Alleles of Various Cytokine Gene Polymorphisms in Acute Coronary Syndrome Patients and Controls

	ACS 651		Control 432
Genotype/Allele	No.	Frequency	No.	Frequency
IL-10-1082 G/A
GG	73	0.11	74	0.17
GA	260	0.39	188	0.43
AA	318	0.48	170	0.39
G	406	0.31	336	0.38
A	896	0.68	528	0.61
IL-10-592 C/A
CC	321	0.49	130	0.30
CA	253	0.38	228	0.52
AA	77	0.11	74	0.17
C	895	0.68	488	0.56
A	407	0.31	376	0.43
TNF-α A−308 G/A
GG	280	0.43	247	0.57
GA	307	0.47	156	0.36
AA	64	0.9	29	0.6
G	867	0.66	650	0.75
A	435	0.33	214	0.24
IL-12 p40-1188 A/C
AA	224	0.34	153	0.35
CA	336	0.51	222	0.51
CC	91	0.13	57	0.13
A	636	0.48	528	0.61
C	664	0.51	336	0.38
IFN-γ +874T/A
TT	168	0.25	192	0.44
AT	301	0.46	179	0.41
AA	182	0.27	61	0.14
T	637	0.48	563	0.65
A	665	0.51	301	0.34
IL-6-174 G/C
GG	134	0.20	135	0.31
GC	294	0.45	206	0.47
CC	223	0.34	91	0.21
G	562	0.43	476	0.55
C	740	0.56	388	0.44

Table 4.

Comparison of Genotypes and Alleles of Various Cytokine Gene Polymorphisms in Acute Coronary Syndrome Patients and Controls

Genotype/Allele	F value	Odds ratio (95% CI)	p-value
IL-10-1082 G/A
GG vs. GA+AA	7.741	0.611 (0.430-0.866)	0.0053
GA vs. GG+AA	1.371	0.863 (0.674-1.104)	0.2423
AA vs. GG+GA	9.45	1.472 (1.150-1.884)	0.0021
GG vs. AA	10.83	0.527 (0.363-0.765)	0.0009
G vs. A	13.69	0.712 (0.594-0.852)	0.0002
A vs. G		1.404 (1.173-1.682)
IL-10-592 C/A
CC vs. CA+AA	39.42	2.260 (1.748-2.921)	<0.001
CA vs. CC+AA	20.35	0.568 (0.444-0.727)	<0.001
AA vs. CC+CA	6.077	0.649 (0.459-0.916)	0.0136
CC vs. AA	19.68	2.373 (1.625-3.465)	<0.001
C vs. A	33.8	1.694 (1.418-2.025)	<0.001
A vs. C		0.590 (0.493-0.705)
TNF-α −308 G/A
GG vs. GA+AA	20.84	0.565 (0.442-0.722)	<0.001
GA vs. GG+AA	12.94	1.579 (1.23-2.026)	<0.001
AA vs. GG+GA	3.213	1.515 (0.959-2.392)	0.073
GG vs. AA	7.253	0.513 (0.320-0.822)	0.0070
G vs. A	18.47	0.656 (0.541-0.795)	<0.001
A vs. G		1.524 (1.257-1.848)
IL-12 P40-1188 A/C
AA vs. CA+CC	0.1162	0.956 (0.741-1.234)	0.7332
CA vs. AA+CC	0.0052	1.009 (0.791-1.287)	0.9424
CC vs. AA+CA	0.1352	1.069 (0.748-1.526)	0.7131
AA vs. CC	0.1133	0.917 (0.621-1.354)	0.7364
A vs. C	0.1745	0.963 (0.807-1.149)	0.6761
C vs. A		1.038 (0.870-1.238)
IFN-γ +874T/A
TT vs. AA+AT	40.61	0.434 (0.335-0.563)	<0.001
AT vs. AA+TT	2.424	1.216 (0.950-1.554)	0.1196
AA vs. AT+TT	28.54	2.360 (1.713-3.251)	<0.001
TT vs. AA	46.32	0.293 (0.205-0.418)	<0.001
T vs. A	55.39	0.512 (0.429-0.611)	<0.001
A vs. T		1.953 (1.636-2.331)
IL-6-174 G/C
GG vs. GC+CC	15.81	0.570 (0.431-0.753)	<0.001
GC vs. GG+CC	0.665	0.903 (0.708-1.153)	0.4148
CC vs. GG+GC	21.92	1.952 (1.472-2.589)	<0.001
GG vs. CC	27.4	0.405 (0.287-0.569)	<0.001
G vs. C	29.6	0.619 (0.520-0.736)	<0.001
C vs. G		1.615 (1.358-1.921)

CI, confidence interval.

In case of IL-10-1082 G→A polymorphism the AA genotype and A allele significantly associated with ACS (χ²=9.45; p=0.0021; OR=1.472; 95% CI=1.15-1.884 & χ²=13.69; p=0.00021, OR=1.404, 95% CI=1.173-1.682, respectively). As far as IL −10-592 C→A polymorphism is concerned, the C allele significantly associated with ACS (χ²=33.8; p<0.0001, OR=1.694, 95% CI=1.418-2.025).The frequency of homozygous CC and heterozygous CA genotypes in ACS were significantly high in ACS patients (χ²=39.42; p=0.001, OR=2.26; 95% CI=1.748-2.292 & χ²=20.35; p=0.001,OR=0.568; 95% CI=0.4447-0.7274, respectively).

There was a statistically significant difference in the AA genotypic distribution and A allelic frequency in case of TNF-α 308 G→A polymorphism between the patients and healthy controls (χ²=3.213, p=0.073; OR=1.515; 95% CI=0.9597-2.392, and χ² =18.47, p=0.0001; OR=1.524; 95% CI=1.257-1.848, respectively). The intermediate producer GA genotype at this position also showed a significant association with the ACS (χ²=12.94; p=0.00032; OR=1.579; 95% CI=1.23-2.026).

In case of −1188A→C polymorphism of IL-12 p40 genotypes and alleles were almost equally distributed in the ACS patients when compared with healthy controls. The polymorphism did not show significant association with the disease.

There was a statistically significant difference in the genotypic distribution and allelic frequency in case of IFN-γ +874T→A polymorphism between patients and healthy controls. The AA genotype and A allele significantly associated with the disease (χ²=28.54; p<0.001; OR=2.36; 95% CI=1.713-3.251& χ²=55.39; p<0.0001OR=1.953; 95% CI=1.636-2.331, respectively).

As far as IL-6-174G→C polymorphism was concerned, the C allele (χ² =29.6; p<0.001, OR=1.615; 95% CI=1.358-1.921) and CC genotype (χ² =21.92; p<0.0001; OR=1.952; 95% CI=1.472-2.589) significantly associated with the disease.

Discussion

Coronary artery disease is the leading cause of mortality worldwide and its acute manifestations are often unstable angina and non-ST-segment-elevation myocardial infarction. Epidemiologists in India and international agencies, such as the World Health Organization, have been sounding an alarm on the rapidly rising burdens of CVD for the past 15 years. (Reddy, 2007) South Asians have high rates of acute myocardial infarction at younger ages compared with individuals from other countries but the reasons for this are unclear (Joshi et al., 2007). Inflammation within the atherosclerotic plaque contributes to its destabilization and subsequent disruption. The systemic inflammation in ACS originates from inflammatory processes within the arterial wall after plaque disruption (Libby et al., 2002).

Various studies divulged that polymorphisms located within critical promoter or regulatory regions of genes coding for cytokines may affect gene transcription resulting in expression of cytokines (Bidwell et al., 1999). In the present study we observed a significant association of AA genotype of −1082G→A and CC genotype of −592 of IL-10 C→A polymorphism, AA genotype at −308 of TNF-γ G→A polymorphism, AA genotype at +874 of IFN-γ A→T polymorphism, and CC genotype at −174 of IL-6 G→C polymorphism with the disease whereas IL-12 p40-1188 C→A polymorphism did not significantly associate with the disease. To our knowledge, this is the first study reporting association of these cytokine polymorphism associations with development of ACS in the South Indian population from Andhra Pradesh.

IL-10, a multifunctional cytokine inhibits monocyte/macrophage function during inflammation by downregulating the production of proinflammatory cytokines. The IL-10 gene promoter polymorphisms at −1082 (G/A), −819 (C/T) and −592 (C/A) independently influence IL-10 expression (Turner et al., 1997). Various studies suggest that IL-10 genotypes strongly influence the CRP secretions and have been associated with a lower risk of cardiovascular disease by limiting the inflammatory activation (Girnd et al., 2003). In the present study we observed that IL10-1082 AA genotype, IL-10-592 CC, and GC genotypes significantly associated with ACS which is in accordance with our previous studies in stroke and diabetes (Karunakar et al.,2009; Munshi et al., 2010).

TNF-α is the most important proinflammatory cytokine. It has been localized in atheromatous plaques and contributes to the progression of atheroma by augmenting the local inflammatory response (Barath et al., 1990; Vaddi et al., 1994). Further, high levels of TNF-α systemically affect a number of mediators of atherosclerotic process, altering lipid homeostasis and promoting endothelial dysfunction (Fernandez-Real and Ricart, 2003). The TNF-α gene is situated in the HLA class III region (6p21) and is highly polymorphic (Wilson et al., 1997). The substitution of guanine by adenine at −308 in the promoter region of TNF-α gene generates less common A allele, resulting in enhanced transcriptional activity of TNF and thereby higher TNF secretion (Kroeger et al., 1997). Presence of AG+AA genotypes displayed significant enhanced levels of the most relevant biochemical myocardial ischaemia markers like troponin I, creatine kinase, lactate dehydrogenase, myoglobin and others in ACS patients (Antonicelli et al., 2005). Various studies suggest that individuals with ‘A’ allele may develop left ventricular dysfunction through a TNF-α dependent pathway, leading to ACS progression (Densem et al., 2002). Similarly in the present study AA and GA genotypes significantly associated with the disease (p=0.0001).

IL-12 is a proinflammatory cytokine that promotes atherosclerosis (Lee et al., 1999). Uyemura et al. (1996) demonstrated that IL-12 mRNA gets strongly expressed in atherosclerotic lesions as compared with normal arteries. The high levels of IL-12 protein induce the production of IFN-γ (Trinchieri et al., 2003). The 3′UTR region of the IL-12 gene influences the amount of translated protein by several mechanisms, including effects on mRNA stability and on transcriptional activity (Grzybowska et al., 2001; Morahan et al., 2002). The AA genotype of IL-12-1188C→A polymorphism has been shown to be associated with increased IL-12 expression in vitro (Seegers et al., 2002), but recent studies failed to show such an association between this polymorphism and CAD (Momiyama et al., 2005). In the current study also we found that the IL-12 p40-1188 polymorphism had no association with ACS.

IFN-γ is a principal mediator of innate and adaptive immunity. IFN-γ has been shown to influence many features of atherosclerosis such as foam cell formation and plaque development (McLaren and Ramji, 2009). The human IFN-γ gene is located on chromosome 12q24.1. A polymorphism at +874T→A position of the first intron of IFN-γ gene can putatively influence the secretion of IFN-γ; in particular the T allele correlates with increased levels of the cytokine expression (Pravica et al., 2000). In the present study we found an association of AA genotype and A allele with the disease.

IL-6 is a pleiotropic inflammatory cytokine which has been associated with CVD (Ridker et al., 2000). Fishman et al. (1998) first described a polymorphism in the 5’ flanking region (IL-6 174G→C) that alters in vitro secretion of IL-6. Various studies have shown an association of the IL-6 174 C allele with increased levels of IL-6 in vivo (Jenny et al., 2002) and CRP (Humphries et al., 2001; Vickers et al., 2002). Atherosclerosis process is generally the pathologic background for ACS (Ross, 1999). Old atherosclerotic patients with IL-6-174 GG genotype showed a higher degree of inflammation (increased IL-6 and TNF-α and decreased IL-10), enhanced stress related protein production, and a major risk for plaque rupture, and therefore possible appearance of MI, when compared with patients with the CC or CG (C+) genotypes (Girndt et al., 2003; Giacconi et al., 2004). The present observations in ACS patients are plausible and in line with the previous positive associations of IL6 −174G→C as a risk factor for ACS. The CC genotype of IL6-174 polymorphism was more frequently observed in ACS patients. (Rauramaa et al., 2000; Jones et al., 2001; Basso et al., 2002; Jerrard-Dunne et al., 2003; Antonicelli et al., 2005; Weger et al., 2005).

Previous studies have revealed contradictory results as far as the association of these polymorphisms with ACS is concerned. Some studies have found an association with the normal allele whereas others have shown an association with the mutant allele (Kroeger et al., 1997; Rauramaa et al., 2000; Jones et al., 2001; Basso et al., 2002; Densem et al., 2002; Georges et al., 2001; Jerrard-Dunne et al., 2003; Giacconi et al., 2004; Antonicelli et al., 2005; Momiyama et al., 2005; Weger et al., 2005). These discrepancies might be on account of ethnic variation.

Based on the current evaluation of ACS, the “low-producer” IL-10-1082 AA genotype, “high producer”IL-10-592 CC genotype, ‘low producer’ IFN-γ +874AA genotype, and “high producer” TNF-α −308AA genotype may be responsible for the regulation of an immune response leading to inflammation in ACS patients. The polymorphisms at −308G→A of TNF-α, −174G→C of IL-6, +874A→T of IFN-γ and −1082G→A, and −592C→A of IL-10 genes evaluated in the present study are important risk factors for the development of ACS in the South Indian population from Andhra Pradesh. However, −1188 of IL-12 gene is not associated with the disease.

In summary, if the findings of this study are replicated, these cytokine polymorphisms, along with traditional risk factors, would help clinicians in early identification of ACS patients at higher risk for their better management.

Conclusion

The results from our study support that the cytokine polymorphisms; −308G→A of TNF-α, −174G→C of IL-6, +874A→T of IFN-γ and −1082G→A, and −592C→A of IL-10 are associated with ACS susceptibility whereas IL-12 p40 +118A→C polymorphism is not associated with the disease. The better understanding of the various genotypes conferring the susceptibility of the diseases may aid in early diagnosis and development of new methods to create personalized medicine.

Footnotes

Acknowledgments

We are grateful to the department of Biotechnology India for their financial support in carrying out the study. We thank Dr. Shubho Das Gupta for the clinical and technical help rendered during the study.

Disclosure Statement

No competing financial interests exist.

References

Antonicelli

, Olivieri

, Cavallone

et al. 2005. Tumor necrosis factor alpha gene −308G>A polymorphism is associated with ST-elevation myocardial infarction and with high plasma levels of biochemical ischemia markers. Coron Artery Dis, 16:489-493.

Barath

, Fishbein

, Cao

et al. 1990. Detection and localization of tumor necrosis factor in human atheroma. Am J Cardiol, 65:297-302.

Basso

, Lowe

, Rumley

et al. 2002. Interleukin-6-174G>C polymorphism and the risk of coronary heart disease in West of Scotland coronary prevention study (WOSCOPS) Arterioscler Thromb Vasc Biol, 22:599-604.

Belluco

, Olivieri

, Bonafe

et al. 2003. −174G>C polymorphism of interleukin 6 gene promoter affects interleukin 6 serum level in patients with colorectal cancer. Clin Cancer Res, 9:2173-2176.

Bidwell

, Keen

, Gallagher

et al. 1999. Cytokine gene polymorphism in human disease: on-line database. Genes Immun, 1:3-19.

Boiardi

, Casali

, Farnetti

et al. 2006. Relationship between interleukin 6 promoter polymorphism at position 174, IL-6 serum levels, and the risk of relapse/recurrence in polymyalgia rheumatica. J Rheumatol, 33:703-708.

Densem

, Hutchinson

, Yonan

et al. 2002. Tumour necrosis factor α gene polymorphism: a predisposing factor to non-ischaemic myocardial dysfunction? Heart, 87:153-155.

Fernandez-Real

, Ricart

. 2003. Insulin resistance and chronic cardiovascular inflammatory syndrome. Endocr. Rev, 24:278-301.

Fishman

, Faulds

, Jeffery

et al. 1998. The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemiconset juvenile chronic arthritis. J Clin Invest, 102:1369-1376.

10.

Georges

, Loukaci

, Poirier

et al. 2001. Interleukin-6 gene polymorphisms and susceptibility to myocardial infarction. The ECTIM study. J Mol Med, 79:303-305.

11.

Giacconi

, Cipriano

, Albanese

et al. 2004. The −174G/C polymorphism of IL-6 is useful to screen old subjects at risk for atherosclerosis or to reach successful ageing. Exp Gerontol, 39:621-628.

12.

Girndt

, Ulrich

, Kaul

et al. 2003. Uremia-associated immune defect: the IL-10-CRP axis. Kidney Int, 63:S76-S79.

13.

Grzybowska

, Wilczynska

, Siedlecki

. 2001. Regulatory functions of 3′UTRs. Biochem Biophys Res Commun, 288:291-295.

14.

Humphries

, Luong

, Ogg

et al. 2001. The interleukin-6-174 G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in healthy men. Eur Heart J, 22:2243-2252.

15.

Jenny

, Tracy

, Ogg

et al. 2002. In the elderly, interleukin-6 plasma levels and the −174G>C polymorphism are associated with the development of cardiovascular disease. Arterioscler Thromb Vasc Biol, 22:2066-2071.

16.

Jerrard-Dunne

, Sitzer

, Risley

et al. 2003. Interleukin-6 promoter polymorphism modulates the effects of heavy alcohol consumption on early carotid artery atherosclerosis. Stroke, 34:402-407.

17.

Jones

, Brull

, Brown

et al. 2001. Interleukin-6 (IL-6) and the prognosis of abdominal aortic aneurysms. Circulation, 103:2260-2265.

18.

Joshi

, Islam

, Pais

et al. 2007. Risk factors for early myocardial infarction in South Asians compared with individuals in other countries. JAMA, 297:286-294.

19.

Karunakar

, Madhavi

, Srikanth Babu

BMV

et al. 2009. Association of tumor necrosis factor alpha, interferon gamma and interleukin10 gene polymorphisms with peripheral neuropathy in South Indian patients with type 2 diabetes. Cytokine, 47:173-177.

20.

Kroeger

, Carville

, Abraham

. 1997. The −308 tumor necrosis factor alpha promoter polymorphism effects transcription. Mol Immunol, 34:391-399.

21.

Lahiri

, Nurnberger

. 1991. A rapid non-enzymatic method for the preparation of HMW DNA from blood RFLP studies. Nucleic Acid Res, 19:5444.

22.

Latsi

, Pantelidis

, Vassilakis

et al. 2003. Analysis of IL-12 p40 subunit gene and IFN-γ G5644A polymorphisms in idiopathic pulmonary fibrosis. Respir Res, 4:6.

23.

Lee

, Yen

, Pan

et al. 1999. The role of interleukin 12 in the development of atherosclerosis in ApoE-deficient mice. Arterioscler Thromb Vasc Biol, 19:734-742.

24.

Libby

, Ridker

, Maseri

. 2002. Inflammation and atherosclerosis. Circulation, 105:1135-1143.

25.

Lopez-Maderuelo

, Arnalich

, Serantes

et al. 2004. Interferon-γ and interleukin-10 gene polymorphisms in pulmonary tuberculosis. Am J Respir Crit Care Med, 167:970-975.

26.

Louis

, Leyder

, Malaise

et al. 2000. Lack of association between adult asthma and the tumour necrosis factor a-308 polymorphism gene. Eur Respir J, 16:604-608.

27.

McLaren

, Ramji

. 2009. Interferon gamma: a master regulator of atherosclerosis. Cytokine Growth Factor Rev, 20:125-135.

28.

Momiyama

, Ohmori

, Nagano

et al. 2005. Polymorphism of the 3′-untranslated region of interleukin-12 p40 gene is not associated with the presence or severity of coronary artery disease. Circ J, 69:793-797.

29.

Morahan

, Huang

, Wu

et al. 2002. Association of IL12B promoter polymorphism with severity of atopic and non atopic asthma in children. Lancet, 360:455-459.

30.

Morse

, Olomolaiye

, Wood

et al. 1999. Induced heteroduplex genotyping of TNF-alpha, IL-1beta, IL-6 and IL-10 polymorphisms associated with transcriptional regulation. Cytokine, 11:789-795.

31.

Munshi

, Rajeshwar

, Kaul

et al. 2010. Interleukin-10-1082 promoter polymorphism and ischemic stroke risk in a South Indian population. Cytokine, 52:221-224.

32.

Olomolaiye

, Wood

, Bidwell

JLA

. 1998. Novel Nla III polymorphism in the human IL-6 promoter. Eur J Immunogenet, 25:267.

33.

Perrey

, Turner

, Pravica

et al. 1999. ARMS-PCR methodologies to determine IL-10, TNF-α, TNF-13 and TGF-131 gene polymorphism. Transpl Immunol, 7:127-128.

34.

Pravica

, Perrey

, Stevens

et al. 2000. A single nucleotide polymorphism in the first intron of the human IFN-g gene: absolute correlation with a polymorphic CA microsatellite marker of high IFN-g production. Hum Immunol, 61:863-866.

35.

Rauramaa

, Väisänen

, Luong

et al. 2000. Stromeolysin-1 and interleukin-6 gene promoter polymorphisms are determinants of asymptomatic carotid artery atherosclerosis. Arterioscler Thromb Vasc Biol, 20:2657-2662.

36.

Reddy

. 2007. From around the world: focus on India. India wakes up to the threat of cardiovascular diseases. J Am Coll Cardiol, 50:1370-1372.

37.

Ridker

, Hennekens

, Buring

et al. 2000. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med, 342:836-843.

38.

Ross

. 1999. Atherosclerosis—an inflammatory disease. N Engl J Med, 340:115-126.

39.

Seegers

, Zwiers

, Strober

et al. 2002. A TaqI polymorphism in the 3’UTR of the IL-12 p40 gene correlates with increased IL-12 secretion. Genes Immun, 3:419-423.

40.

Sole

, Guino

, Valls

et al. 2006. SNPStats: a web tool for the analysis of association studies. Bioinformatics, 22:1928-1929.

41.

Tedgui

, Mallat

. 2001. Interleukin-10: an anti-atherogenic cytokine. Pathol Biol, 49:107-108.

42.

Trinchieri

, Pflanz

, Kastelein

. 2003. The IL-12 family of heterodimeric cytokines. New players in the regulation of T cell responses. Immunity, 19:641-644.

43.

Turner

, Williams

, Sankaran

et al. 1997. An investigation of polymorphism in the interleukin—10 gene promoter. Eur J Immunogenet, 24:1-8.

44.

Uyemura

, Demer

, Castle

et al. 1996. Cross-regulatory roles of interleukin (IL)-12 and IL-10 in atherosclerosis. J Clin Invest, 97:2130-2138.

45.

Vaddi

, Nicolini

, Mehta

et al. 1994. Increased secretion of tumor necrosis factor-alpha and interferon-gamma by mononuclear leukocytes in patients with ischemic heart disease. Relevance in superoxide anion generation. Circulation, 90:694-699.

46.

Vickers

, Green

, Terry

et al. 2002. Genotype at a promoter polymorphism of the interleukin-6 gene is associated with baseline levels of plasma C-reactive protein. Cardiovasc Res, 53:1029-1034.

47.

Weger

, Steinbrugger

, Haas

et al. 2005. Role of the interleukin-6-174 G>C gene polymorphism in retinal artery occlusion. Stroke, 36:249-252.

48.

Wilson

, Symons

, McDowell

et al. 1997. Effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation. Proc Natl Acad Sci U S A, 94:3195-3199.

49.

Yoo

, Seo

, Kim

. 2005. SNPAnalyzer: a web-based integrated workbench for single-nucleotide polymorphism analysis. Nucleic Acids Res, 33:483-488.