The Interleukin 6 - 572 G>C (rs1800796) Polymorphism Is Associated with the Risk of Developing Acute Coronary Syndrome

Abstract

Background: Inflammation plays an essential role in the development and progression of atherosclerotic lesions and plaque disruption. Cytokines are central regulators in immunoinflammatory mechanisms. The aim of the present study was to evaluate the role of INF-γ and IL-6 gene polymorphisms as susceptibility markers for acute coronary syndromes (ACS) in a group of Mexican patients. Methods: Four polymorphisms (INF-γ -155 G>A, INF-γ -179 T>G, IL-6 -572 G>C, and IL-6 -1426 T>G) of the INF-γ and IL-6 genes were analyzed by 5′ exonuclease TaqMan genotyping assays in a group of 284 patients with ACS (mean age = 58.88 ± 11.7 years) and 247 healthy unrelated controls (mean age = 56.03 ± 4.13 years). Results: Distribution of INF-γ -179 G>T (rs2069709) and INF-γ -155 G>A (rs2069710) polymorphisms was similar in patients and healthy controls. A significant difference was observed in the distribution of IL-6 -572 G>C (rs1800796) genotypes between patients with ACS and healthy controls (p = 0.019). Estimated odds ratio and 95% confidence interval (CI) showed a protective role of the IL-6 -572 (rs1800796) CC when compared with the GG genotype (wild type) (odds ratio = 0.6, 95% CI = 0.33-1.08). In this case, heterozygous individuals with GC genotype had 2.19-fold increased risk of developing ACS than CC homozygous (p = 0.007, 95% CI = 1.24-3.96). Conclusions: The results suggest that IL-6 -572 G>C (rs1800796) polymorphism could be involved in the risk of developing ACS in Mexican individuals.

Introduction

Coronary atherosclerosis is a complex multifactorial process resulting from an excessive inflammatory response to various forms of injurious stimuli to the arterial wall (Ross, 1999; Garcia-Moll, 2005; Fragoso et al., 2009). The inflammatory processes, coupled with dyslipidemia, advanced glycation, and infectious agents play an important role in the progression of atherosclerotic plaque (Libby, 2002; Virmani et al., 2005). The transition of a stable coronary atherosclerotic lesion into a ruptured plaque, with subsequent thrombus formation, results in the clinical manifestation of an acute coronary syndrome (ACS) (Achar et al., 2005; Mouco et al., 2005; Mälarstig et al., 2006; Fragoso et al., 2009). Several cytokines have been associated with the development and severity of coronary diseases (Aukrust et al., 2008). Interferon-gamma (INF-γ) plays a central role in the immune and inflammatory response to a wide range of stimuli (Angerio, 2009). Also, it is a cytokine essential in the development and propagation of T helper 1 type immune response and is an important mediator of innate immunity. In addition, it activates monocytes and macrophages, which when taking part in inflammatory responses produce free radicals and pro-inflammatory cytokines such as interleukin-6 (IL-6). IL-6 is a pleiotropic cytokine with a broad range of humoral and cellular immune effects relating to inflammation, host defense, and tissue injury (Abeywardena et al., 2009; Dienz and Rincon, 2009; Fonseca et al., 2009). Several studies correlate the plasma levels of INF-γ and IL-6 with the instability of the atherosclerotic plaque (Ridker et al., 2000; Wang et al., 2004; Heinisch et al., 2005; Tanaka et al., 2005; Pasqui et al., 2006). INF-γ presents two single-nucleotide polymorphisms (SNPs) in the promoter region -179 G>T (rs2069709) and -155 G>A (rs2069710), which are associated with an increased expression of INF-γ in several diseases (Bream et al., 2002; Koch et al., 2005; Qi et al., 2005; Huang et al., 2007). On the other hand, two SNPs -572 G>C (rs1800796) and -1426 T>G (rs2069827) of IL-6 gene have been associated with an increased expression of IL-6 gene in atherosclerosis, myocardial infarction, blood pressure, type 2 diabetes, and coronary artery disease (Humphries et al., 2001; Tanaka et al., 2005; Qi et al., 2006). The IL-6 -572 G allele and GG genotype have been associated with high levels of IL-6, C-reactive protein, and fibrinogen in patients with coronary artery disease, ACS, hypertension, and diabetes (Mälarstig et al., 2007; Wong et al., 2007; Jang et al., 2008) and have been associated with the risk of developing several diseases including coronary artery disease (Fu et al., 2006; Koh et al., 2009).

Thus, the aim of the present study was to establish the role of the INF-γ and IL-6 gene polymorphisms in the risk of developing ACS in a clinically well characterized cohort of Mexican Mestizo patients.

Materials and Methods

Patients and controls

The study included 284 Mexican Mestizo patients with ACS (225 men and 59 women, mean age 58.88 ± 11.7). One hundred eighty-four of them presented myocardial infarction and 100 presented unstable angina. ACS were diagnosed on the basis of clinical history, physical examination with electrocardiography, chest radiography, echocardiography, and coronary angiography. The diagnosis of ACS was made according to the World Health Organization and the American Heart Association/American College of Cardiology (Richardson et al., 1996). Also, a group of 247 healthy unrelated individuals (195 men and 52 women, mean age 56.03 ± 4.13) with neither symptoms nor previous diagnosis of cardiovascular problem and systemic disease was studied as control group. This group was selected from the first wave of the “Integrated study of depression among elderly” that included 2600 individuals. The population base consists of persons 50 years or older insured by Mexican Institute of Social Security and residing in Mexico City. According to the medical records, none of these individuals presented diabetes, hypertension, or dyslipidemia. All included subjects (patients and controls) were ethnically matched, and we considered as Mexican Mestizos only those individuals who had been born in Mexico for three generations, including their own. A Mexican Mestizo is defined as someone born in Mexico who is a descendant of the original autochthonous inhabitants of the region and of individuals, mainly Spaniards of Caucasian and/or Black origin, who came to America during the 16th century. The Bioethics and Research Committee approved the present study, and all study subjects signed an informed consent letter.

DNA extraction

Genomic DNA from whole blood containing ethylenediaminetetraacetic acid was isolated by standard technique (Lahiri and Nurnberger, 1991).

Determination of the IL-6 and INF-γ genotypes

The IL-6 -572 G>C (rs1800796), IL-6 -1426 T>G (rs2069827), INF-γ -179 G>T (rs2069709), and INF-γ -155 G>A (rs2069710) SNPs were genotyped using 5′ exonuclease TaqMan genotyping assays on a 7900HT Fast real-time PCR system according to the manufacturer's instructions (Applied Biosystems, Foster City, CA).

Statistical analysis

Gene frequencies of IL-6 and INF-γ polymorphisms of patients and controls were obtained by direct counting. Further, Hardy-Weinberg equilibrium (HWE) was evaluated by the chi-square test. The compute values of empirical power of the association tests, provided by the sample for all SNPs, were estimated using two different methods: With the power and sample size calculations software version 3.0.14 and the formula for power calculation for the difference in the proportions, estimations were similar. Mean and standard deviation were estimated for continuous variables and percentages for discrete ones. Differences in alleles and genotype distribution between patients and healthy controls were assessed with χ² or Fisher's exact test as appropriate. Odds ratios (OR) with 95% confidence intervals (CI) were estimated for each polymorphism between patients with ACS and healthy controls; homozygous wild type genotypes were used as references. For the IL-6 -572 (rs1800796) polymorphism, the OR with 95% CI was estimated between the GC and CC genotypes. To assess the association between the presence of a particular genotype and the presence of ACS, and comorbidity, OR and 95% CI were estimated between patients with ACS with and without diabetes mellitus, hypertension, hypercholesterolemia, prior acute myocardial infarction, and Major Adverse Cardiac Events. Body mass index and smoking status were also considered. The analysis was performed with the Stata 10.0 for windows software. Statistical significance was accepted at p < 0.05. Pair wise linkage disequilibrium (D') estimations between polymorphisms and haplotype reconstruction were performed with Haploview version 4:1 (Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA).

Results

Characteristics of the study sample

Baseline characteristics are shown in Table 1. No differences were identified in relation to age and sex between patients with ACS and healthy controls. Comorbidity is only described in the group of patients with ACS; hypertension, dyslipidemia, and myocardial infarction were the most frequent in this group.

Table 1.

Baseline Clinical Characteristics of the Studied Individuals

	Patients with ACS	Healthy controls
	(n = 284)	(n = 247)
Age years mean (±SD)	58.88 (±11.7)	56.03 (±4.13)
Male n (%)	225 (79)	195 (79)
Body mass index (media [SD] kg/m²)	27.1 (±4.41)	25.4 (±3.1)
Arterial hypertension n (%)	168 (59)	—
Diabetes n (%)	125 (44)	—
Dyslipidemia n (%)	178 (63)	—
Smoking n (%)	114 (55)	—
Alcohol use n (%)	22 (8)	—
MACE, n (%)	99 (34)	—
Myocardial infarction n (%)	184 (65)	—
Unstable angina n (%)	100 (35)	—
Death n (%)	1 (0.35)	—

ACS, acute coronary syndrome; MACE, Major Adverse Cardiac Events; SD, standard deviation.

Allele and genotype frequencies

The HWE hypothesis was tested on the combined case-control sample, and all the studied SNPs were in HWE. Allele and genotype frequencies of the studied polymorphisms in the whole group of patients with ACS and healthy controls are shown in Table 2. A significant difference was observed in the distribution of IL-6 -572 G>C (rs1800796) genotypes between patients with ACS and healthy controls (p = 0.019). The distribution of INF-γ polymorphisms (INF-γ -155 G>A and INF-γ -179 T>G) was similar in both groups.

Table 2.

Allele and Genotype Distribution of IL-6 and INF-γ Gene Polymorphisms in Patients with Acute Coronary Syndrome and Healthy Controls

		Patients with ACS	Healthy controls
		n = 284	n = 247
		(n [%])	(n [%])	p-Value
IL-6 -572 (rs1800796)
Alleles	C	192 (34)	175 (35)	NS
	G	376 (66)	319 (65)
Genotypes	GG	115 (41)	108 (44)	0.019
	GC	146 (51)	103 (42)
	CC	23 (8)	36 (15)
IL-6 -1426 (rs2069827)
Alleles	G	551 (97)	485 (98)	NS
	T	17 (3)	9 (2)
Genotypes	GG	267 (94)	238 (96)	NS
	GT	17 (6)	9 (4)
INF-γ -179 (rs2069709)
Alleles	G	568 (100)	492 (99.6)	NS
	T	0	2 (0.4)
Genotypes	GG	284 (100)	245 (99)	NS
	GT	0	2 (1)
INF-γ -155 (rs2069710)
Alleles	A	568 (100)	491 (99)	NS
	G	0	3 (1)
Genotypes	AA	284 (100)	244 (99)	NS
	AG	0	3 (1)

NS, not significant.

Estimated OR and 95% CI showed a protective role of the IL-6 -572 (rs1800796) CC when compared with the GG genotype (wild type) (OR = 0.6, 95% CI = 0.33-1.08). In this case, heterozygous individuals with GC genotype had a 2.19-fold increased risk of developing ACS than CC homozygous individuals (p = 0.007, 95% CI = 1.24-3.96). No association was found between GG homozygous and GC heterozygous (OR = 1.33, 95% CI = 0.92-1.91) (Table 3). The estimated power for the comparison between the GC and CC genotype for SNP IL-6 -572 (rs1800796) was 91 and 88 with the two different methods used. In addition, the possible association of clinical characteristics of patients with ACS with the IL-6 and INF-γ polymorphisms was analyzed. Patients with ACS with and without some clinical features showed similar distribution of the studied polymorphisms (data not shown).

Table 3.

Estimated Risk Between Patients and Healthy Controls According to IL-6 -572 Polymorphism

	OR	95% CI	p-Value
IL-6 -572 (rs1800796)
GG	1
GC	1.33	0.92-1.91	NS
CC	0.60	0.33-1.08	NS
GC vs. CC	2.19	1.24-3.96	0.007

CI, confidence interval; OR, odds ratio.

On the other hand, the IL-6 and INF-γ polymorphisms analyzed in the present study showed a strong linkage disequilibrium with a D′ = 0.89 for IL-6 and D′ = 1 for INF-γ polymorphisms. However, the distribution of the different haplotypes was similar in patients and healthy controls (data not shown).

Discussion

It is widely accepted that both pro- and anti-inflammatory mediators produced by activated immunocompetent cells, endothelial, and vascular smooth muscle cells are important for the development of the atherosclerosis and its progress to ACS (Wang et al., 2004; Heinisch et al., 2005; Tanaka et al., 2005; Pasqui et al., 2006). Several polymorphisms located in genes that code pro- and anti-inflammatory molecules have been associated with the risk of developing cardiovascular diseases. Elevated levels of IL-6 have been associated with the development and severity of coronary disease as well as with the transition to plaque instability (Gabriel et al., 2000; Ridker et al., 2000). Regarding INF-γ, Heinisch et al. (2005) reported increased levels of this cytokine in patients with ACS. Considering that INF-γ is produced by T-lymphocytes, which, among their functions inhibit collagen production by smooth muscle cells (Amento et al., 1991), INF-γ could have a fundamental role in the stabilization of atherosclerotic plaques. In the present work, we studied two polymorphisms located in the IL-6 and two located in the INF-γ genes in patients with ACS. It has been reported that −155 SNP is located upstream of the INF-γ gene promoter area, is close to the nuclear factors-activated T-cell site (-168 taaacgcgaaa-160), and may affect the stability of this region (Barbulescu et al., 1997). Also, Bream et al. (2002) reported an important effect of the INF-γ -179 polymorphism in the expression of the INF-γ in T cell lines in response to tumor necrosis factor alpha. These results suggest that this polymorphism could be related with the mRNA expression of the INF-γ gene. However, the effect of these two polymorphisms was not observed in our study showing similar distribution of INF-γ polymorphisms (INF-γ -155 G>A and INF-γ -179 T>G) in patients with ACS and healthy controls. Regarding the IL-6 gene, our study showed a different distribution of the -572 G>C polymorphism in patients with ACS and healthy controls. This polymorphism appears to be associated with the risk of developing ACS showing a protective role for the CC genotype. Individuals with GC genotype showed a 2.19-fold increased risk of developing ACS when compared with individuals with the CC genotype. This polymorphism (which is identical to -634 G>C) has been associated with the progression of diabetic nephropathy and increased plasma levels of IL-6 after coronary artery bypass graft surgery (Brull et al., 2001; Kitamura et al., 2002). Also, the effect of the −572 G>C polymorphism on risk of coronary heart disease and on intermediate risk traits including fibrinogen and systolic blood pressure was previously studied (Humphries et al., 2001). This study reported no association of this polymorphism with levels of any trait or risk of developing coronary heart disease. After reviewing several articles in which IL-6 -572 polymorphism was used for different purposes (Holla et al., 2004; Komatsu et al., 2005; Gordon et al., 2008; Shao et al., 2009), we found that the distribution of genotypes among the populations in each study was different from the Mexicans. The Caucasian population showed a larger proportion of GG genotype, a small proportion of GC, and practically nonexistent or a very low proportion of CC genotype (Gordon et al., 2008). On the other hand, Oriental populations had a large proportion of CC genotype followed by the GC and a small proportion of GG genotype (Komatsu et al., 2005). The Mexican population from the present study had a similar proportion of GG and GC genotype with a proportion of CC genotype larger than Caucasians but smaller than Orientals. This distribution of genotypes is different from the other two populations and could have been a determinant factor to find the association between the GC and CC genotype.

In summary, our data suggest that the IL-6 -572 G>C (rs1800796) polymorphism has an important role in the risk of developing ACS. However, our data are preliminary due to the study sample size and additional studies in a larger number of individuals and in other populations that could help define the true role of this marker as a risk factor for developing ACS.

Footnotes

Acknowledgments

This work was submitted in partial fulfillment of the requirements for the D.Sc. Degree for José Manuel Fragoso Lona at Universidad Nacional Autónoma de México. José Manuel Fragoso Lona was supported by a fellowship from the Consejo Nacional de Ciencia y Tecnología Number CVU: 49158. This work was supported in part by grants from the Consejo Nacional de Ciencia y Tecnología (50352-M/24147) and Fundación Gonzalo Rio Arronte, Mexico City, Mexico. The authors are grateful to the study participants. Institutional Review Board approval was obtained for all sample collections.

Disclosure Statement

No competing financial interests exist.

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