Research Article - Biomedical Research (2017) Volume 28, Issue 18

Association between IL-4 genetic polymorphism and risk of coronary artery disease in a Chinese population

Jiangyong Yuan¹, Hui Li^2*, Guizhi Bai³, Jinping Wen¹, Wentao Du¹

¹Department of Cardiology, Affiliated Hospital of Hebei University of Engineering, Handan, Hebei, PR China

²Department of Cardiology, Handan Central Hospital, Handan, Hebei, PR China

³Health Examination Center, the First Hospital of Handan, Handan, Hebei, PR China

*Corresponding Author:

Hui Li
Department of Cardiology
Handan Central Hospital
Handan, Hebei, PR China

Accepted on August 23, 2017

Abstract

We aimed to investigate the association between IL-4 genetic polymorphisms (IL-4 rs2243250, rs2227284, rs2243267, rs2243270) and risk of CAD in a Han Chinese individual. A total of 366 patients with coronary artery disease patients and 366 controls were collected from July 2014 to July 2016. SNP genotyping of IL-4 rs2243250, rs2227284, rs2243267 and rs2243270 was done in a 384-well plate format on the sequenom Mass ARRAY platform. Conditional logistic regression analysis was performed to analyze the relationship between the four SNPs and risk of CAD. We found that those with BMI ≥ 24 (adjusted OR=2.71, 95% CI=1.70-4.30), T2DM (adjusted OR=2.59, 95% CI=1.61-4.17) or hypertension (adjusted OR=2.10, 95% CI=1.47-3.00) were associated with an increased risk of CAD compared with the reference group. The TT (adjusted OR=2.86, 95% CI=1.75-4.68) and CT+TT (adjusted OR=1.39, 95% CI=1.02-1.90) genotypes of rs2243250 were correlated with an elevated risk of CAD when compared with the CC genotype. The GG genotype of rs2243270 was associated with a higher risk of CAD in comparison to the AA genotype (adjusted OR=1.97, 95% CI=1.22-3.18). Our study suggests that IL-4 may be a useful biomarker for prediction of the susceptibility of this study.

Keywords

IL-4, Polymorphism, Coronary artery disease (CAD).

Introduction

Coronary Artery Disease (CAD) is the main cause of death worldwide and the prevalence is increasing in China [1,2]. Coronary artery disease displays a wide range of clinical manifestations, such as subclinical asymptomatic atherosclerosis and some clinical complications [3-5]. Currently, CAD is regarded as one of the most leading cause of death and disability worldwide [6]. The precision in estimating the risk for CAD is crucial for future therapy decision and prevention strategies. Improving the accuracy of risk prediction is particularly important in patients with CAD risk [7]. The prevalence of CAD is usually high in individuals with many lifestyle and dietary factors [8]. Therefore, the use of emergent biomarkers could contribute to identify individuals at high-risk, and pose a critical challenge for CAD prevention.

There is extensive evidence supporting a role of inflammatory response in the pathogenesis of CAD and in the natural history of atherosclerosis [9,10]. Cardiovascular events are more common in patients with high circulating levels of several inflammatory markers, such as hs-C reactive protein (hs-CRP) [11]. The Interleukins (IL) mediate various effects in inflammation. Interleukin 4 (IL-4) is mainly secreted by activated T cells and mononuclear cells [12]. In humans, diminished products of IL-4 are considered to contribute to the autoimmune diseases [13]. As well, polymorphisms of IL-4 gene coding for the cytokine IL-4 have been thought to influence the susceptibility or protection in autoimmune diseases [14-16]. However, few studies reported an association of IL-4 expression and polymorphisms with risk of CAD [17,18]. In the present study, we conducted a case-control study to investigate the association between IL-4 genetic polymorphisms (IL-4 rs2243250, rs2227284, rs2243267 and rs2243270) and risk of CAD in Han Chinese individuals.

Methods

Study subjects

A total of 366 patients with coronary artery disease patients were collected from July 2014 to July 2016 in the department of cardiology of Affiliated Hospital of Hebei University of Engineering. Coronary angiography was performed and all the patients were confirmed to be CAD according to the WHO criteria for diagnosis of patients with CAD in 1979.

Simultaneously, a total of 366 control group were selected from the outpatient clinics and health examination center of Affiliated Hospital of Hebei University of Engineering, and they matched with patients with age (± 5 y). All the controls received health examination, and they were confirmed to be free of CAD. All respondents voluntary participated in our study and signed informed consent forms before enrolment. This study was approved by the ethics committee of the Affiliated Hospital of Hebei University of Engineering.

All the subjects were unrelated Chinese Han population; and we excluded those with systemic inflammation, rheumatic disease, serious heart failure, malignant tumors, thyroid function derangement and autoimmune diseases, but did not exclude hypertension and diabetes.

Demographic and lifestyle characteristics were collected from the questionnaire survey, including weight, height, alcohol drinking habit and tobacco smoking habit. The clinical information collected from medical records, such as family history of cardiovascular disease, hypertension, Type 2 Diabetes Mellitus (T2DM), Total Cholesterol (TC), Triglyceride (TG), Low Density Lipoprotein cholesterol (LDLc) and High Density Lipoprotein cholesterol (HDL-c). Tobacco smoking status was divided into never smoking and ever smoking. Ever smoking was defined as those who smoked more than 20 cigarettes per week and continued for half a year, and the remaining respondents were considered as never smoking. The alcohol drinking status was divided into never drinking and ever drinking. Ever drinking was defined as those who drank more than 50 g white wine or half a bottle of beer and lasted for half a year. T2DM was defined as those with a fast plasma glucose ≥ 7.0 mmol/l (126 mg/dl) or with a glucose tolerance test (two hours after the oral dose plasma glucose) ≥ 11.1 mol/l (200 mg/dl). Hypertension was defined as those with systolic pressure ≥140 mmHg or diastolic pressure ≥ 90 mmHg.

Three ml of peripheral venous blood was collected from each subject. The blood samples were centrifuged with 1500r/min to separate serum, and stored at -80°C for lipid analysis; the white cell was handled with Blood DNA extraction kit to extract genomic DNA, and then the DNA samples were stored at -20°C preservation for genotype analysis.

SNP genotyping of IL-4 rs2243250, rs2227284, rs2243267 and rs2243270 was done in a 384-well plate format on the sequenom MassARRAY platform (Sequenom, San Diego, USA). Primers for polymerase chain reaction amplification and single base extension assays of the four SNPs were designed by Sequenom Assay Design 3.1 software. The genomic DNA of IL-4 rs2243250, rs2227284, rs2243267 and rs2243270 was amplified with an initial denaturation at 95°C for 2 min; 45 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 60 s; a final extension at 72°C for 5 min. Then the SAP and iPLEX reactions were then carried out. Then the PCR products were desalted, and finally the products were dispensed to a SpectroCHIP and analyzed with MALDI-TOF MS.

Statistical analysis

Data analysis was performed with the software IBM SPSS Statistics for Windows, Version 20.0. (IBM Corp. Armonk, NY, USA). Two tailed P<0.05 was considered to be statistical significant difference. Continuous variables are shown by means ± standard deviations (SD) and categorical variables are displayed by percentages and frequencies (%). First, comparison of demographic, lifestyle and clinical characteristics between the two groups were analyzed by Chisquare test or student t- test. Second, the genotype frequencies of IL-4 rs2243250, rs2227284, rs2243267 and rs2243270 between the two study groups were compared by Chi-square test and the Hardy-Weinberg equilibrium of the four SNPs was assessed with Chi-square (χ²) test with one degree of freedom. Relationship of the four SNPs with risk of CAD was estimated by conditional multivariate logistic regression analysis, and the results were displayed by Odds Ratios (ORs) and 95% Confidence Intervals (CIs). The OR was adjusted for potential confounding factors, such as sex, age, BMI and history of T2DM and hypertension.

Results

Comparison with controls, patients with CAD were more likely to have higher BMI (χ²=43.84, P<0.001), have a history of T2DM (χ²=22.98, P<0.001) and hypertension (χ²=23.18, P<0.001) (Table 1). Moreover, patients with CAD had a higher level of TC (t=2.76, P=0.01), TG (t=3.99, P<0.001) and LDL-c (t=5.80, P<0.001) and had a lower level of HDL-c (t=-6.83, P<0.001).

Table 1. Comparison of the social demographic, lifestyle and clinical characteristics between the two study groups.

We observed that the genotype frequencies of IL-4 rs2243250 (χ²=19.78, P<0.001) and rs2243270 (χ²=8.53, P=0.01) showed significant differences between patients and controls. However, the genotype distributions of the other four SNPs did not show significant differences in the study groups. All the four SNPs of IL-4 were not deviated from the Hardy-Weinberg equilibrium in controls (P>0.05) (Table 2).

By conditional logistic regression analysis, we found that those with BMI ≥ 24 (adjusted OR=2.71, 95% CI=1.70-4.30), T2DM (adjusted OR=2.59, 95% CI=1.61-4.17) or hypertension (adjusted OR=2.10, 95% CI=1.47-3.00) were associated with an increased risk of CAD compared with the reference group (Table 3). Moreover, the TT (adjusted OR=2.86, 95% CI=1.75-4.68) and CT+TT (adjusted OR=1.39, 95% CI=1.02-1.90) genotypes of rs2243250 were correlated with an elevated risk of CAD when compared with the CC genotype. The GG genotype of rs2243270 was associated with a higher risk of CAD in comparison to the AA genotype (adjusted OR=1.97, 95% CI=1.22-3.18).

Table 2. Genotype frequencies of IL-4 rs2243250, rs2227284, rs2243267 and rs2243270 between the two study groups.

Table 3. Logistic regression analysis for the risk factors of CAD.

Discussion

Genetic polymorphisms may affect the expression and activity of IL-4 and influence the development and progression of disease. In this study, we found that IL-4 rs2243250 and rs2243270 were associated with risk of CAD.

It is reported that inflammation is of major importance for the development of coronary artery disease [19]. Inflammatory cells and signaling molecules play an important role in the pathogenesis of this disease through modulating the arterial wall, promoting lipoprotein retention and plaque formation [19]. Previous studies have indicated that several inflammatory biomarkers could forecast cardiovascular outcome [20,21]. In our study, we found that rs2243250 and rs2243270 were positively associated with the risk of CAD. With regard to rs2243250 being located in the translation start site, we found that individuals with rare TT genotype and CT genotype showed a higher tendency to develop CAD than those with CC homozygous. The variant allele of rs2243250 has been identified as a functional polymorphism being relevant with increased IL-4 transcriptional and translational activity. Rosenwasser reported that minor T-allele of rs2243250 was related to an elevated IL-4 activity by greater binding to nuclear transcriptional factor than C allele [22]. Therefore, we hypothesized cautiously according to allele frequency distribution in Chinese that the rare T allele of rs2243250 may influence genetic susceptibility to CAD or severity of the disease via modifying the IL-4 activity in Chinese Han population.

Many previous studies have reported the association between rs2243250 polymorphism and risk of cardiovascular disease. However, they failed to reach consistent results [17,23-29]. Mahmoudi et al. reported that the IL-4 rs2243250 polymorphism and IL-4 haplotypes were significantly associated with risk of ischemic heart failure in Iranian [25]. However, Chou et al. revealed lack of association between IL-4 polymorphisms and rheumatic heart disease [23]. The possible explanation for the inconsistent results arises from these following aspects: rs2243250 polymorphism may exert diverse effects in the pathogenesis of cardiovascular disease in different populations; cardiovascular diseases are multi- factorial and gene-environment and gene-gene interactions may influence the disease susceptibility.

We also found that rs2243270 is in the promoter region of IL-4 and we observed that the GG genotype was associated with an increased risk of CAD. Only two previous studies reported the association between rs2243270 and disease susceptibility [29,30]. Kim et al. performed a study in Korean population on the association between IL-4 SNPs and asthmatics, and they found an association of IL-4 rs2243250 and rs2070874 with aspirin intolerance, but rs2243270 was not associated with aspirin hypersensitivity [29]. Rong et al. found that rs2243270 was associated with RCC and had a significant decreased risk of RCC [30].

Two limitations should be mentioned in the present study. First, since patients and controls were selected from only one hospital of China, the selection samples may not represent Han Chinese in other places, and the selection bias may be occurred. Second, the small sample size was quite small, resulting in a low statistical power to find differences between the patients and controls.

In conclusion, this study provides a new insight in CAD that rs2242250 and rs2243270 in the promoter region of IL-4 are positively associated with the risk of CAD in Han Chinese individuals. Our study suggests that IL-4 may be a useful biomarker for prediction of the susceptibility of this study.

References

1. Pragani MA, Desai KP, Morrone D, Sidhu MS, Boden WE. The role of nitrates in the management of stable ischemic heart disease: A review of the current evidence and guidelines. Rev Cardiovasc Med 2017; 18: 14-20.
2. Lingel JM, Srivastava MC, Gupta A. Management of coronary artery disease and acute coronary syndrome in the chronic kidney disease population: A review of the current literature. Hemodial Int 2017.
3. Urowitz MB, Gladman D, Ibanez D, Fortin P, Sanchez-Guerrero J, Bae S, Clarke A, Bernatsky S, Gordon C, Hanly J, Wallace D, Isenberg D, Ginzler E, Merrill J, Alarcon G, Steinsson K, Petri M, Dooley MA, Bruce I, Manzi S, Khamashta M, Ramsey-Goldman R, Zoma A, Sturfelt G, Nived O, Maddison P, Font J, van Vollenhoven R, Aranow C, Kalunian K, Stoll T, Buyon J. Clinical manifestations and coronary artery disease risk factors at diagnosis of systemic lupus erythematosus: data from an international inception cohort. Lupus 2007; 16: 731-735.
4. Tresch DD, Aronow WS. Clinical manifestations and diagnosis of coronary artery disease. Clin Geriatr Med 1996; 12: 89-100.
5. Kuznetsov VA, Yaroslavskaya EI, Bessonova MI, Bessonov IS, Zyrianov IP, Kolunin GV, Krinochkin DV, Maryinskih LV, Bektasheva AS. Clinical manifestations and risk factors of coronary artery disease in patients with diabetes mellitus in western Siberia. Int J Circumpolar Health 2010; 69: 278-284.
6. Brown RA, Shantsila E, Varma C, Lip GY. Epidemiology and pathogenesis of diffuse obstructive coronary artery disease: the role of arterial stiffness, shear stress, monocyte subsets and circulating micro particles. Ann Med 2016; 9: 1-12.
7. Allan GM, Garrison S, McCormack J. Comparison of cardiovascular disease risk calculators. Curr Opin Lipidol 2014; 25: 254-265.
8. Abou Sherif S, Ozden Tok O, Taskoylu O, Goktekin O, Kilic ID. Coronary artery aneurysms: A review of the epidemiology, pathophysiology, diagnosis, and treatment. Front Cardiovasc Med 2017; 4: 24.
9. Luc G, Bard JM, Juhan-Vague I, Ferrieres J, Evans A, Amouyel P, Arveiler D, Fruchart JC, Ducimetiere PS. C-reactive protein, interleukin-6, and fibrinogen as predictors of coronary heart disease: the PRIME study. Arterioscler Thromb Vasc Biol 2003; 23: 1255-1261.
10. Christiansen MK, Larsen SB, Nyegaard M, Neergaard-Petersen S, Ajjan R, Wurtz M, Grove EL, Hvas AM, Jensen HK, Kristensen SD. Coronary artery disease-associated genetic variants and biomarkers of inflammation. PLoS One 2017; 12: e0180365.
11. Liao JK. Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein. Curr Atheroscler Rep 2009; 11: 243-244.
12. Tay SS, Plain KM, Bishop GA. Role of IL-4 and Th2 responses in allograft rejection and tolerance. Curr  Opin Organ Transplant 2009; 14: 16-22.
13. Wallis SK, Cooney LA, Endres JL, Lee MJ, Ryu J, Somers EC, Fox DA. A polymorphism in the interleukin-4 receptor affects the ability of interleukin-4 to regulate Th17 cells: a possible immunoregulatory mechanism for genetic control of the severity of rheumatoid arthritis. Arthritis Res Ther 2011; 13: 15.
14. Zaaber I, Mestiri S, Hammedi H, Marmouch H, Mahjoub S, Tensaout BB, Said K. Association of Interleukin-1B and Interleukin-4 gene variants with autoimmune thyroid diseases in Tunisian population. Immunol Invest 2016; 45: 284-297.
15. Yousefi A, Mahmoudi E, Zare Bidoki A, Najmi Varzaneh F, Baradaran Noveiry B, Sadr M, Motamed F, Najafi M, Farahmand F, Rezaei N. IL4 gene polymorphisms in Iranian patients with autoimmune hepatitis. Expert Rev Gastroenterol Hepatol 2016; 10: 659-663.
16. Mahmoudi M, Tahghighi F, Ziaee V, Harsini S, Rezaei A, Soltani S, Sadr M, Moradinejad MH, Aghighi Y, Rezaei N. Interleukin-4 single nucleotide polymorphisms in juvenile systemic lupus erythematosus. Int J Immunogenet 2014; 41: 512-517.
17. Basol N, Celik A, Karakus N, Ozturk SD, Yigit S. The evaluation of angiotensin-converting enzyme (ACE) gene I/D and IL-4 gene intron 3 VNTR polymorphisms in coronary artery disease. In Vivo 2014; 28: 983-987.
18. Mamta PS. An association of serum vitamin D, IL-4 level and VDR gene polymorphism in CAD with and without T2DM. J Clin Exp Cardiolog 2016; 7: 54.
19. Libby P, Ridker PM, Hansson GK. Progress and challenges in translating the biology of atherosclerosis. Nature 2011; 473: 317-325.
20. Kaptoge S, Di Angelantonio E, Pennells L, Wood AM, White IR. C-reactive protein, fibrinogen and cardiovascular disease prediction. N Engl J Med 2012; 367: 1310-1320.
21. Kaptoge S, Seshasai SR, Gao P, Freitag DF, Butterworth AS, Borglykke A, Di Angelantonio E, Gudnason V, Rumley A, Lowe GD, Jorgensen T, Danesh J. Inflammatory cytokines and risk of coronary heart disease: new prospective study and updated meta-analysis. Eur Heart J 2014; 35: 578-589.
22. Rosenwasser LJ, Klemm DJ, Dresback JK, Inamura H, Mascali JJ, Klinnert M, Borish L. Promoter polymorphisms in the chromosome 5 gene cluster in asthma and atopy. Clin Exp Allergy 1995; 2: 74-78.
23. Chou HT, Tsai CH, Chen WC, Tsai FJ. Lack of association of genetic polymorphisms in the interleukin-1beta, interleukin-1 receptor antagonist, interleukin-4 and interleukin-10 genes with risk of rheumatic heart disease in Taiwan Chinese. Int Heart J 2005; 46: 397-406.
24. Sobti RC, Maithil N, Thakur H, Sharma Y, Talwar KK. VEGF and IL-4 gene variability and its association with the risk of coronary heart disease in north Indian population. Mol Cell Biochem 2010; 341: 139-148.
25. Konenkov VI, Shevchenko AV, Prokof'ev VF, Maksimov VN. Complex of genotypes of cytokines as a genetic factor of risk of development of myocardial infarction of in European population of Russia men. Kardiologiia 2012; 52: 22-29.
26. Mahmoudi MJ, Hedayat M, Taghvaei M, Nematipour E, Farhadi E, Esfahanian N, Sadr M, Mahmoudi M, Nourijelyani K, Amirzargar AA, Rezaei N. Association of interleukin-4 gene polymorphisms with ischemic heart failure. Cardiol J 2014; 21: 24-28.
27. Tong YQ, Ye JJ, Wang ZH, Zhang YW, Zhan FX, Guan XH, Geng YJ, Hou SY, Li Y, Cheng JQ, Lu ZX, Liu JF. Association of variable number of tandem repeat polymorphism in the IL-4 gene with ischemic stroke in the Chinese Uyghur population. Genet Mol Res 2013; 12: 2423-2431.
28. Yousry SM, Sedky Y, Sobieh A. Association of IL-4 (intron 3) and IL-10 (-1082) gene polymorphisms with risk of mitral valve disease in children with rheumatic heart disease. Cardiol Young 2016; 26: 1290-1296.
29. Kim BS, Park SM, Uhm TG, Kang JH, Park JS, Jang AS, Uh ST, Kim MK, Choi IS, Cho SH, Hong CS, Lee YW, Lee JY, Choi BW, Park HS, Park BL, Shin HD, Chung IY, Park CS. Effect of single nucleotide polymorphisms within the interleukin-4 promoter on aspirin intolerance in asthmatics and interleukin-4 promoter activity. Oncotarget 2017; 20: 748-758.
30. Rong H, He X, Wang L, He Y, Kang L, Jin T. Associations between polymorphisms in the IL-4 gene and renal cell carcinoma in Chinese Han population. Oncotarget 2017.