The Interleukin-6-572G/C Gene Polymorphism and the Risk of Intracranial Aneurysms in a Chinese Population

Abstract

Interleukin-6 (IL-6) is an important proinflammatory cytokine that plays an important role in the pathogenesis of intracranial aneurysms (IAs). The aim of this study was to investigate the association between the IL-6-572G/C polymorphism and the risk of IAs in a Chinese population. The IL-6-572G/C gene polymorphisms in 220 IA cases and 220 controls were analyzed using the polymerase chain reaction-restriction fragment length polymorphism method. The IL-6-572GG (odds ratio [OR]=3.35, 95% confidence intervals [CIs]=1.65, 6.82; p=0.001) and G allele frequencies (OR=1.48, 95% CIs=1.09, 2.00; p=0.01) in the IA group were higher than those in the control group. The C allele frequencies (OR=0.68, 95% CIs=0.50, 0.92; p=0.01) were significantly lower in patients than in controls. When stratified by the site, shape, size, and the Fisher Grade of IAs, no statistically significant result was observed. This study suggested that the IL-6-572GG genotype was associated with a higher risk of IA in a Chinese population.

Introduction

Rupture of intracranial aneurysm (IA) accounts for more than 90% of subarachnoid hemorrhage (SAH) cases (Inagawa, 2001). SAH is fatal in about 50% of cases, and a significant disability is caused in about 30% of cases, despite the diagnostic and therapeutic developments of the past few decades (Longstreth et al., 1993; Hashikata et al., 2010). IA occurs in about 2% of the general population, which means that over 20 million people in China will have an IA (Liu et al., 2010). IA is about 1.6 times more common in women than in men, and ruptured IA is found mostly between 40 and 60 years of age (Rinkel et al., 1998). Both environmental and genetic factors are associated with IA (Broderick et al., 2003; Ruigrok et al., 2005; Nahed et al., 2007). The concept of genetic factors being involved in the development of aneurysms has led to many studies on genetic determinants for IA in the last decade. Interleukin-6 (IL-6), an important pro-inflammatory cytokine, is a multifunctional protein principally involved in the genesis and maintenance of the inflammatory response (Fragoso et al., 2010). There is evidence that IL-6 plays a major role in the pathogenesis of IA. In addition, there is IL-6 in other arterial aneurysms, such as abdominal aortic aneurysm and thoracic aortic aneurysm. The IL-6 gene is located in the short arm of chromosome 7 and presents polymorphism at position −572 in its promoter region. The IL-6-572G/C promoter polymorphism has been associated with altered plasma secretion of the proinflammatory cytokine in healthy adults. Recently, some studies have indicated that the IL-6-572G/C promoter polymorphism is related to IA (Morgan et al., 2006; Fontanella et al., 2008; Sun et al., 2008; Zhang et al., 2011). The aim of this study was to confirm the association between the IL-6-572G/C polymorphism and the risk of IA in a Chinese population.

Materials and Methods

Study population

The Ethics Committee of the West China Hospital of Sichuan University approved the study protocols, and all the participants gave written informed consent according to the Declaration of Helsinki. Peripheral blood specimens and demographic, medical, and family histories were obtained from 220 sequentially ascertained, unrelated patients at the West China Hospital of Sichuan University, China. Aneurysms in the cases were detected by magnetic resonance imaging or computed tomography and cerebral angiography. The controls were drawn from the same population as the cases and had a similar age and sex distribution. The controls were uncharacterized with respect to vascular disease.

DNA extraction and genotyping

DNA was isolated using a commercial kit (Wizard DNA Purification Kit; Promega). The IL-6-572G/C genotype was determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. On the basis of the GenBank reference sequence, the PCR primers were as follows: forward-5′-GGAGACGCCTTGAAGTAACTGC-3′ and reverse-5′-GAGTTTCCTCTGACTCCATCGCAG-3′. A PCR was initiated by an initial denaturation (5 min at 94°C), followed by amplification for 35 cycles of denaturation (1 min at 94°C), annealing (1 min at 55°C), and extension (1 min at 72°C), and a final extension step (5 min at 72°C). Genotypes were resolved by RFLP using the restriction endonuclease BsrBI (New England Biolabs). Digestion was performed at 37°C in 15 μL using 6 to 10 μL of the PCR product and 3 U of restriction endonuclease for 2 h. The size of the digestion products was determined by the agarose gel electrophoresis.

Statistical analysis

SPSS version 13.0 software for Windows (SPSS Inc.) was used for the statistical analysis. Differences between continuous variables were assessed by the Student's t-test, whereas those between categorical variables were evaluated using the Pearson χ² test. The existence of differences in allelic and genotypic frequencies between different groups was assessed by means of the Pearson χ² test and calculating the odds ratio (OR) with the 95% confidence intervals (CIs). Two-sided p-values were used, with p<0.05 considered statistically significant. The genotype distribution and allele frequencies were compared to the Hardy-Weinberg equilibrium model using the Pearson χ² test.

Results

Characteristics of patients with IAs and controls are listed in Table 1. As to clinical characteristics, there was no statistical significant difference in gender, age, or the prevalence of risk factors, including hypertension, smoking, and drinking, between the patient with IAs and control groups, as shown in Table 1. The distribution of all genotypes was in the Hardy-Weinberg equilibrium.

Table 1.

Characteristics of Patients with Intracranial Aneurysms and Controls

	Aneurysms	Controls	p
Total No.	220	220
Gender (%)			0.44
Male	95 (43.2)	103 (46.8)
Female	125 (56.8)	117 (53.2)
Age at diagnosis (years), Mean±SD	47.4±11.3	45.6±10.7	0.09
Hypertension (%)			0.49
Yes	82 (37.3)	75 (34.1)
No	138 (62.7)	145 (65.9)
Smoking (%)			0.83
Yes	56 (25.5)	54 (24.5)
No	164 (74.5)	166 (75.5)
Drinking (%)			0.82
Yes	47 (21.4)	45 (20.5)
No	173 (78.6)	175 (79.5)
Family history of IA (%)
Yes	5 (2.3)	/
No	215 (97.7)	/
Site of aneurysm (%)
ACA	98 (44.6)	/
ICA	76 (34.5)	/
MCA	46 (20.9)	/
Shape of aneurysm (%)
Saccular	178 (80.9)	/
Fusiform	33 (15.0)	/
Dissecting	9 (4.1)	/
Size of aneurysm (%)
Small (<15 mm)	155 (70.5)	/
Larger (15-25 mm)	56 (25.4)	/
Giant (>25 mm)	9 (4.1)	/
The Fisher Grade
Grade 1	24 (10.9)	/
Grade 2	101 (45.9)	/
Grade 3	65 (29.6)	/
Grade 4	30 (13.6)	/

IA, intracranial aneurysms; ACA, anterior cerebral artery; ICA, internal carotid artery; MCA, middle cerebral artery.

IL-6-572GG (OR=3.35, 95% CIs=1.65, 6.82; p=0.001) and G allele frequencies (OR=1.48, 95% CIs=1.09, 2.00; p=0.01) in the IA group were higher than those in the control group (Table 2). The C allele frequencies (OR=0.68, 95% CIs=0.50, 0.92; p=0.01) were significantly lower in patients than in controls (Table 2). When stratified by the site, shape, size, and the Fisher Grade of IAs, no statistically significant result was observed (Table 3).

Table 2.

Genotype Frequencies of Interleukin-6-572G/C Polymorphisms Among Intracranial Aneurysm Patients and Controls

Genotype	Aneurysms, n (%)	Controls, n (%)	OR (95% CIs)	p-Value
GG	33 (15.0)	11 (5.0)	3.35 (1.65,6.82)	0.001
GC	66 (30.0)	77 (35.0)	0.80 (0.53,1.19)	0.26
CC	121 (55.0)	132 (60.0)	0.82 (0.56,1.19)	0.29
G allele frequency	132 (30.0)	99 (22.5)	1.48 (1.09,2.00)	0.01
C allele frequency	308 (70.0)	341 (77.5)	0.68 (0.50,0.92)	0.01

OR, odds ratio; CI, confidence interval.

Table 3.

Stratification Analysis of Interleukin-6-572G/C Genotype Frequency in Intracranial Aneurysms

		GG			GC			CC
Variable	Aneurysm	n (%)	OR (95% CIs)	p	n (%)	OR (95% CIs)	p	n (%)	OR (95% CIs)	p
Site of aneurysm (%)	220	33 (15.0)	1 (Reference)		66 (30.0)	1 (Reference)		121 (55.0)	1 (Reference)
ACA	98	13 (13.3)	0.88 (0.45,1.75)	0.73	30 (30.6)	1.02 (0.62,1.67)	0.94	55 (56.1)	1.02 (0.69,1.52)	0.92
ICA	76	12 (15.8)	1.05 (0.52,2.14)	0.89	23 (30.3)	1.01 (0.59,1.73)	0.98	41 (53.9)	0.98 (0.63,1.52)	0.93
MCA	46	8 (17.4)	1.16 (0.50,2.67)	0.73	13 (28.3)	0.94 (0.48,1.85)	0.86	25 (54.3)	0.99 (0.58,1.69)	0.97
Shape of aneurysm (%)	220	33 (15.0)	1 (Reference)		66 (30.0)	1 (Reference)		121 (55.0)	1 (Reference)
Saccular	178	27 (15.2)	1.01 (0.59,1.75)	0.97	54 (30.3)	1.01 (0.67,1.52)	0.96	97 (54.5)	0.99 (0.71,1.38)	0.96
Fusiform	33	5 (15.2)	1.01 (0.37,2.77)	0.98	10 (30.3)	1.01 (0.47,2.16)	0.98	18 (54.5)	0.99 (0.54,1.84)	0.98
Dissecting	9	1 (11.1)	0.74 (0.09,6.04)	0.78	2 (22.2)	0.74 (0.16,3.51)	0.71	6 (66.7)	1.21 (0.42,3.49)	0.72
Size of aneurysm (%)	220	33 (15.0)	1 (Reference)		66 (30.0)	1 (Reference)		121 (55.0)	1 (Reference)
Small (<15 mm)	155	23 (14.8)	0.99 (0.56,1.75)	0.97	47 (30.3)	1.01 (0.66,1.55)	0.96	85 (54.8)	1.00 (0.71,1.41)	0.99
Larger (15-25 mm)	56	9 (16.1)	1.07 (0.49,2.37)	0.87	17 (30.4)	1.01 (0.55,1.86)	0.97	30 (53.5)	0.97 (0.59,1.60)	0.92
Giant (>25 mm)	9	1 (11.1)	0.74 (0.09,6.04)	0.78	2 (22.2)	0.74 (0.16,3.51)	0.71	6 (66.7)	1.21 (0.42,3.49)	0.72
The Fisher Grade	220	33 (15.0)	1 (Reference)		66 (30.0)	1 (Reference)		121 (55.0)	1 (Reference)
Grade 1	24	4 (16.7)	1.11 (0.36,3.41)	0.85	7 (29.2)	0.97 (0.40,2.36)	0.95	13 (54.1)	0.99 (0.48,2.00)	0.97
Grade 2	101	15 (14.9)	0.99 (0.52,1.91)	0.98	31 (30.7)	1.02 (0.63,1.67)	0.93	55 (54.4)	0.99 (0.67,1.47)	0.96
Grade 3	65	9 (13.8)	0.92 (0.42,2.03)	0.84	20 (30.8)	1.03 (0.58,1.82)	0.93	36 (55.4)	1.01 (0.63,1.60)	0.98
Grade 4	30	5 (16.7)	1.11 (0.40,3.07)	0.84	8 (26.7)	0.89 (0.39,2.03)	0.78	17 (56.6)	1.03 (0.55,1.94)	0.93

Discussion

The concept of genetic factors being involved in the development of aneurysms has led to many studies on genetic determinants for IA in the last decade. A genetic meta-analysis of 8 genes and 13 polymorphisms in ∼20,000 individuals showed that there was a likely genetic basis to sporadic IA. However, the evidence base was small when compared against other complex disorders (McColgan et al., 2010). Hashikata et al. (2010) found that a region between introns 7 and 15 of CDKN2BAS carrying the rs1333040-T allele might confer the risk for IA in Japanese patients. The rs2621215 single-nucleotide polymorphism (SNP) in intron 46 of the COL1A2 gene was found to be marginally associated with an increased risk of IA development in the Korean population examined. The rs42524 polymorphism of COL1A2 could be a genetic risk factor for sporadic IA among individuals of the Chinese Han ethnicity (Zhu et al., 2008). Polymorphisms of homocysteine metabolism are possible risk factors for the formation of IA (Semmler et al., 2008). Polymorphisms within the kallikrein gene cluster are associated with IA, suggesting that the kallikreins are important candidate genes for IA (Weinsheimer et al., 2007). The study by Pannu et al. (2006) and Peters et al. (1999) supported a role for MMP-9 in the pathogenesis of IA. Endothelial nitric oxide synthase T-786C SNP can be a putative genetic marker differentiating small versus large ruptured IA (Khurana et al., 2003). The data by Takenaka et al. (1999) provided evidence of an association between aneurysm development and a polymorphism at a genetic variant of endoglin in patients with these lesions.

The IL-6-572G/C gene polymorphisms studied in this article have been associated with susceptibility to several other diseases. Results of the study by Godarzi et al. (2011) showed that the IL-6-572G/C polymorphism plays an important role in susceptibility to systemic lupus erythematosus in Iranian patients. The study by Wang and Jia (2010) showed that the IL-6-572G/C promoter polymorphism modifies the Alzheimer's risk in APOE epsilon four carriers. The study by Lee et al. (2010) suggested that the IL-6-572G/C polymorphism was associated with the bone mineral density in the lumbar spine in Korean girls with adolescent idiopathic scoliosis. The study by Jingjin et al. (2010) suggested that the IL-6-572G/C polymorphism may contribute to the susceptibility to chronic periodontitis in the Chinese Han population. A family study by Guzman-Guzman et al. (2010) suggested that IL-6-572GG might confer susceptibility for the development of subclinical inflammation and type 2 diabetes in Mexican families. The study by Fragoso et al. (2010) suggested that the IL-6-572G/C polymorphism was associated with the risk of developing acute coronary syndrome in Mexican individuals. The study by Xiao et al. (2009) suggested that the IL-6-572 genotype and allele distributions are unique to subjects with type 2 diabetes mellitus and chronic periodontitis in a Chinese population. A systematic review and a meta-analysis indicated that the IL-6-572G/C polymorphism was associated with the pathogenesis of periodontitis, including chronic periodontitis or aggressive periodontitis (Shao et al., 2009). The study by Reiman et al. (2008) suggested that the IL-6-174 GG genotypes were associated with a higher incidence of histologic chorioamnionitis. The study by Gu et al. (2008) suggested that the IL-6-572G/C polymorphism might be used as a relevant risk estimate for sepsis in trauma patients in the Chinese Han population.

The molecular mechanism of the association between the IL-6-572G/C polymorphism and the risk of IA is still relatively unclear. An important part is played by inflammation in IA formation. IL-6, an important pro-inflammatory cytokine, is a multifunctional protein principally involved in the genesis and maintenance of the inflammatory response (Fragoso et al., 2010). There is evidence that IL-6 plays a major role in the pathogenesis of IA. The IL-6-572G/C promoter polymorphism has been associated with altered plasma secretion of the proinflammatory cytokine in healthy adults. Morgan et al. (2006) did the first study to show that IL-6 promoter polymorphisms are associated with intracranial aneurysmal disease. Zhang et al. (2011) speculated about the following possible mechanisms: (1) the increased C allele frequency of IL-6-572G/C may worsen the inflammatory and immune responses of the hemal wall in IA patients by increasing the serum concentration of IL-6, resulting in the injury of blood vessel endothelial cells of the hemal wall, the inhibition of collagen expression and the increase of the hemal wall fragility to increase the incidence of aneurysms (Zhang et al., 2011); (2) IL-6 may enhance the incidence of IAs by increasing intracranial atherosclerosis (Skirgaudas et al., 1996); and (3) the increasing allele frequency of IL-6-572G/C may aggravate the effect of neurotoxicity after ruptured IAs to enhance brain injury after SAH (Zhang et al., 2011).

In conclusion, this study suggested that the IL-6-572GG genotype was associated with a higher risk of IA in the Chinese population. To confirm this finding, an additional analysis might need to be performed using a larger sample size.

Footnotes

Acknowledgments

The research of this article was supported by the Youth Project No. 30801185 (to Y.L.) and the General Program No.30872673 (to C.Y.) from the National Natural Science Foundation of China.

Author Disclosure Statement

No competing financial interests exist.

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