Arq Bras Cardiol abc Arquivos Brasileiros de Cardiologia Arq. Bras. Cardiol. 0066-782X 1678-4170 Sociedade Brasileira de Cardiologia - SBC 7dWQjcdXDRRJJ37nYYrk6Yg S0066-782X2016004100131 10.5935/abc.20160124 Original Articles Epidemiology Hypomethylation of Interleukin-6 Promoter is Associated with the Risk of Coronary Heart Disease Zuo Hong-Peng 1 * Guo Ying-Yu 2 * Che Lin 3 Wu Xian-Zheng 1 Tongji University Tongji Hospital Department of Emergency Shanghai China Department of Emergency, Tongji Hospital, Tongji University, Shanghai, China Tongji University Jilin Oilfield General Hospital Department of Computed Tomography Songyuan Jilin Province China Department of Computed Tomography, Jilin Oilfield General Hospital, Songyuan, Jilin Province, China Tongji University Tongji Hospital Department of Cardiology Shanghai China Department of Cardiology, Tongji Hospital, Tongji University, Shanghai, China Tongji University Mailing Address: Xian-Zheng Wu, No. 389 Xincun Road. Postal Code 200065, Shanghai - China. E-mail: wuxz_tongji@sina.com

Author contributions

Conception and design of the research: Zuo HP, Guo YY, Che L, Wu XZ. Acquisition of data: Zuo HP, Guo YY, Che L. Analysis and interpretation of the data: Zuo HP, Guo YY, Che L, Wu XZ. Statistical analysis: Zuo HP, Wu XZ. Writing of the manuscript: Zuo HP, Guo YY, Che L. Critical revision of the manuscript for intellectual content: Wu XZ. Supervision / as the major investigador: Zuo HP, Guo YY, Wu XZ.

Potential Conflict of Interest

No potential conflict of interest relevant to this article was reported.

 08 2016 107 2 131 136 01 09 2015 10 10 2015 29 02 2016 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background:

Interleukin-6 (IL-6) is implicated in the pathogenesis of coronary heart disease (CHD), and IL-6 expression has associated with reduced DNA methylation of its gene promoter. However, there are no data on IL-6 promoter methylation and the risk of CHD.

 Objective:

To examine whether IL-6 promoter methylation measured in blood leukocyte DNA is associated with CHD risk.

 Methods:

A total of 212 cases with CHD and 218 controls were enrolled. Methylation at two CpG sites in IL-6 promoter was measured by bisulfite pyrosequencing, and the mean IL-6 methylation was calculated by averaging the methylation measures of the two CpGs.

 Results:

Mean methylation level in IL-6 promoter in CHD cases was significantly lower than that in controls (p = 0.023). Logistic regression analysis showed that IL-6 methylation was inversely associated with the risk of CHD. The odds ratios (ORs) of CHD for subjects in the second and first (lowest) tertile of IL-6 methylation were 1.87 (95% CI = 1.10‑3.20) and 2.01 (95% CI = 1.19-3.38) (ptrend = 0.013), respectively, compared to subjects in the third (highest) tertile. The IL-6 hypomethylation-related risk estimates tended to be stronger for acute myocardial infarction (ptrend = 0.006). CpG position-specific analysis showed that hypomethylation of position 1 conferred a more pronounced increase in CHD risk than that of position 2.

 Conclusion:

These findings suggest that DNA hypomethylation of IL-6 promoter is associated with the increased risk for CHD, especially for acute myocardial infarction. The two distinct CpGs in IL-6 may contribute differently to the development of CHD.

 Keywords: Coronary Artery Disease Interleukin-6 DNA Methylation Epigenetic Repression Introduction

DNA methylation is an epigenetic modification that plays a crucial role in controlling gene expression in the genome.1 In mammals, DNA methylation involves addition of methyl groups to cytosine of a CpG dinucleotide to form 5-methylcytosine (5 mC). Epigenetic changes in methylation patterns are increasingly implicated in a number of human diseases.2 Aberrant promoter methylation of several genes has been associated with the development and progression of coronary heart disease (CHD).3-8

The major cause of CHD is atherosclerosis, an inflammatory disease of the arteries associated with lipid and other metabolic alterations. Interleukin-6 (IL-6) is central to inflammatory processes underlying chronic inflammatory diseases including CHD,9-11 and have been shown to induce the expression of other genes that might contribute to the CHD phenotype.12 Genetic variant in IL-6 gene promoter has been associated with its abnormal expression13,14 and increased risk of CHD.15 Methylation modification is an alternative mechanism regulating IL-6 production,16,17 and consistent evidence has shown that IL-6 expression is associated with reduced DNA methylation of its gene promoter.18-24 In addition, DNA methylation in IL-6 promoter has been associated with risk factors for CHD such as air pollution exposure.25 However, to the best of our knowledge, there are no data on IL-6 promoter methylation and the risk of CHD.

Recently, gene silencing by DNA methylation has been suggested to be associated with methylation not only in CpG islands, but also in CpG island shores (i.e., on the island edges).26,27 In the present study on the association between IL-6 promoter methylation and CHD risk, we focused on two individual CpGs within a CpG shore located in the IL-6 gene promoter region, which were described to be associated with lung function and air pollution exposure.25,28

Methods Study participants

A total of 212 patients with CHD, including 120 cases of acute myocardial infarction (AMI), 42 cases of prior myocardial infarction and 50 cases of unstable angina, were recruited from Tongji Hospital, Tongji University between January 2011 and June 2012. The diagnosis of CHD was established by angiographic evidence of ≥ 70% stenosis of 1 major coronary artery, and/or ≥ 50% of the left main coronary artery. As control, 218 CHD-free subjects, determined by history analysis, physical examination, electrocardiography, and echocardiography were recruited from the same hospital during the same period when the case patients were recruited. The controls were frequency-matched to the cases by age (±5 years) and sex. For both CHD and control groups, subjects with hypertension, diabetes, peripheral artery disease, autoimmune-related disease or cancers were excluded. Information on age, sex, height, weight, cigarette smoking, and family history of CHD was obtained using structured questionnaire through in-person interviews. Body mass index (BMI) was calculated using the formula: body weight in kilograms divided by the square of body height in meters (kg/m2). An ever-smoker was defined as a smoker of at least 1 cigarette per day for at least 6 months. Information on serum total cholesterol was collected on the basis of medical records. Written informed consent was obtained from all participants. This study was conducted with approval from the Ethics Committee of Tongji Hospital (E20100401).

 DNA methylation analysis of IL-6 promoter

Peripheral blood leukocytes were isolated by Ficoll-Hypaque density gradient centrifugation. DNA was extracted from leukocytes using the QIAamp DNA Blood kit (Qiagen, Shanghai, China), and then bisulfite-converted with the Zymo EZ DNA Methylation kit (Zymo, CA, USA). PCR-based pyrosequencing was performed to quantitate methylation of the IL-6 promoter. PCR to amplify the target region covering the two CpGs [Genbank Accession no.M18403, chromosome 7: 22733847 (position1) and chromosome 7: 22733841 (position 2)] was carried out in a 50 µl reaction volume containing 25 µl of GoTaq Master mix (Promega, WI, USA), 10 pmol of biotinylated forward primer (biotin-TAT TTT AGT TTT GAG AAA GGA GGT G), 10 pmol of reverse primer (CAA TAC TCT AAA ACC CAA CAA AAA C), and 50 ng of bisulfite-treated genomic DNA. The cycling program was 5 min at 95°C followed by 45 cycles of each 95°C for 1 min, 57°C for 1 min and 72°C for 1 min and a final elongation for 5 min at 72 °C. Then, sequencing was performed on the PSQ HS 96 Pyrosequencing System using 0.3 µM pyrosequencing primer (TCC TAA TAC AAA CAA CCC C). Non-CpG cytosine residue was used as built-in control to verify bisulfite conversion. The degree of methylation was expressed for each CpG as %5 mC (%5 mC) over the sum of methylated and unmethylated cytosines. %5 mC levels of the two CpGs were averaged to obtain a mean methylation measure of the IL-6 promoter.

 Statistical analysis

Differences in age (< 65, ≥ 65 years), sex, BMI (< 24, 24-27.9, ≥ 28 kg/m2), ever-smoker (no, yes), serum total cholesterol (< 4.67, 4.67-5.47, > 5.47 mmol/L) and family history of CHD (no, yes) between CHD cases and controls were evaluated using χ2-test. Tertile cut-points of mean, position 1 and position 2 methylation measures in IL-6 promoter were based on the values among controls. The associations of IL-6 methylation with CHD risk were estimated by computing the odds ratios (ORs) and 95% confidence intervals (CIs) from multivariate logistic regression analyses with adjustment for BMI (< 24, 24-27.9, ≥ 28 kg/m2), serum total cholesterol (< 4.67, 4.67-5.47, > 5.47 mmol/L) and family history of CHD (no, yes). Further adjustment by age (< 65, ≥ 65 years), sex and ever-smoker (no, yes) did not materially alter the risk estimates and thus these variables were not included in the final models. All tests were two-sided and a p value of less than 0.05 was considered significant. Data was analyzed with Stata 10.1 software (Stata Corporation, College Station, TX).

 Results Characteristics of the study subjects

The characteristics of the CHD cases and controls are shown in Table 1. No significant differences between cases and controls were observed in the distributions of age, sex, or ever-smoker. Cases with CHD were more likely to have higher BMI (p = 0.029), higher serum total cholesterol (p < 0.001) and frequent family history of CHD (p < 0.001) than controls.

Characteristics of coronary heart disease (CHD) cases and control subjects.
Characteristic Controls, n (%) CHD cases, n (%) p value*
Age (years)
< 65 102 (46.8) 108 (50.9)
≥ 65 116 (53.2) 104 (49.1) 0.389
Sex
Female 46 (21.1) 48 (22.6)
Male 172 (78.9) 164 (77.4) 0.699
Body mass index (kg/m2)
< 24 125 (57.3) 96 (45.3)
24-27.9 61 (28.0) 69 (32.5)
≥ 28 32 (14.7) 47 (22.2) 0.029
Ever-smoker
No 100 (45.9) 79 (37.3)
Yes 118 (54.1) 133 (62.7) 0.070
Serum total cholesterol (mmol/L)
< 4.67 73 (33.5) 34 (16.1)
4.67-5.47 75 (34.4) 52 (24.5)
> 5.47 70 (32.1) 126 (59.4) < 0.001
Family history of CHD
No 214 (98.2) 185 (87.3)
Yes 4 (1.8) 27 (12.7) < 0.001

p value obtained from a χ2-test comparing cases and Controls.

 Association between mean IL-6 methylation and CHD risk

CHD cases had significantly reduced mean IL-6 methylation level than controls (mean (standard deviation, SD): 41.2 (0.7) versus 43.4 (0.6), p = 0.023, Figure 1). Logistic regression analysis showed that IL-6 promoter methylation was inversely associated with the risk of CHD (Table 2). The ORs of CHD for subjects in the second and first (lowest) tertile of mean IL-6 methylation were 1.87 (95% CI = 1.10-3.20) and 2.01 (95% CI = 1.19-3.38) (ptrend = 0.013), respectively, compared to individuals in the third (highest) tertile. When evaluated by clinical types of CHD, the IL-6 hypomethylation-related risk estimates tended to be stronger for AMI, with OR of 2.00 (95% CI = 3.2-5.2) for the second tertile and 2.57 (95% CI = 1.33-4.95) for the first tertile (ptrend = 0.006, Table 2).

Comparison of IL-6 methylation levels measured in blood leukocyte in coronary heart disease (CHD) cases and controls (p = 0.023).

Association of IL-6 promoter methylation with risk of coronary heart disease (CHD).
Methylation (%5 mC) Controls CHD cases
All AMI Prior MI Angina
n (%) n (%) OR (95% CI)* n (%) OR (95% CI)* n (%) OR (95% CI)* n (%) OR (95% CI)*
Mean (Tertile†)
T3 (> 47.7) 73 (33.5) 42 (19.8) 1.0 (reference) 21 (17.5) 1.0 (reference) 9 (21.4) 1.0 (reference) 12 (24.0) 1.0 (reference)
T2 (40.1-47.7) 71 (32.6) 77 (36.3) 1.87 (1.10-3.20) 41 (34.2) 2.00 (1.01-3.94) 18 (42.9) 2.02 (0.84-4.86) 18 (36.0) 1.76 (0.73-4.24)
T1 (< 40.1) 74 (33.9) 93 (43.9) 2.01 (1.19-3.38) 58 (48.3) 2.57 (1.33-4.95) 15 (35.7) 1.63 (0.66-4.01) 20 (40.0) 1.83 (0.77-4.33)
p-trend=0.013 p-trend=0.006 p-trend=0.325 p-trend=0.182
Position 1 (Tertile†)
T3 (> 51.7) 73 (33.5) 44 (20.8) 1.0 (reference) 25 (20.8) 1.0 (reference) 8 (19.1) 1.0 (reference) 11 (22.0) 1.0 (reference)
T2 (43.5-51.7) 71 (32.6) 77 (36.3) 2.04 (1.20-3.49) 38 (31.7) 1.71 (0.88-3.32) 19 (45.2) 2.37 (0.96-5.84) 20 (44.0) 2.50 (1.01-6.17)
T1 (< 43.5) 74 (33.9) 91 (42.9) 2.17 (1.29-3.66) 57 (47.5) 2.44 (1.29-4.62) 15 (35.7) 1.94 (0.77-4.92) 19 (38.0) 2.16 (0.88-5.27)
p-trend=0.05 p-trend=0.006 p-trend=0.196 p-trend=0.114
Position 2 (Tertile†)
T3 (> 44.0) 73 (33.5) 51 (24.1) 1.0 (reference) 30 (25.0) 1.0 (reference) 9 (21.4) 1.0 (reference) 12 (24.0) 1.0 (reference)
T2 (35.9-44.0) 71 (32.6) 63 (29.7) 1.25 (0.74-2.12) 28 (23.3) 0.93 (0.48-1.81) 17 (40.5) 2.06 (0.84-5.02) 18 (36.0) 1.74 (0.72-4.20)
T1 (< 35.9) 74 (33.9) 98 (46.2) 1.73 (1.05-2.86) 62 (51.7) 1.81 (0.99-3.29) 16 (38.1) 1.82 (0.74-4.47) 20 (40.0) 1.79 (0.76-4.23)
p-trend=0.030 p-trend=0.038 p-trend=0.220 p-trend=0.197

%5mC: percentage of 5-methylcytosine; AMI: acute myocardial infarction; CI: confidence interval; MI: myocardial infarction; OR: odds ratio.

Adjusted by body mass index (<24, 24-27.9, ≥28 kg/m2), serum total cholesterol (<4.67, 4.67-5.47, >5.47 mmol/L) and CHD family history (no, yes).

The tertiles of IL-6 methylation measures were based on values among control subjects.

CpG position-specific association between IL-6 methylation and CHD risk

Our main analysis considered the mean methylation of the two CpGs in IL-6 promoter. However, methylation at specific positions within a gene's promoter may affect gene expression differently. Therefore, we further examined the associations between position-specific methylation in IL-6 and CHD risk. As shown in Table 2, although significant ptrend values for CHD and AMI were observed for both CpG positions, hypomethylation of position 1 conferred a more pronounced increase in CHD risk when compared to that of position 2.

 Discussion

Promoter methylation is an essential epigenetic mechanism for the regulation of the IL-6 expression.16,17 It has been reported that hypomethylation of IL-6 promoter was associated with the pathogenesis of systemic lupus erythematosus, rheumatoid arthritis and chronic periodontitis.18,24,29 In the present study, we demonstrated for the first time that DNA hypomethylation in IL-6 promoter was associated with increased risk for CHD, especially for AMI. These data suggest that demethylation of the IL-6 promoter might be a common epigenetic basis for the development of a variety of inflammation-associated diseases. In this context, the associations between IL-6 promoter methylation and the risk of other inflammatory diseases merit further investigations.

Methylation level of IL-6 promoter has been associated with air pollution exposure,25 which has been known to increase cardiovascular morbidity and mortality.30 In addition, serum IL-6 level has been associated with increased risk of mortality in patients with CHD.31 In the present study, we observed that hypomethylation in IL-6 promoter was associated with a stronger risk estimate for AMI, a clinical type of CHD contributing to large cases of mortality worldwide.32 Based on these preliminary results and the current literature, it is tempting to speculate that demethylation of the IL-6 promoter may play a role not only in the development, but also in the prognosis of CHD. In line with this hypothesis, the methylation level of the IL-6 promoter has been reported to be significantly correlated to the development and the severity of systemic lupus erythematosus.18,24

There is increasing evidence that methylation at specific positions within a gene's promoter may be more important for gene expression than the mean methylation of CpG sites in the promoter region.33,34 In the present study, we therefore further examined whether the CHD associations we observed in the main analysis using mean methylation were specific to certain positions in the IL-6 promoter. We observed that hypomethylation of the CpG at position 1 in IL-6 promoter conferred a more pronounced increase in CHD and AMI risk estimates than that at position 2. Previously, methylation levels of these two CpGs in IL-6 have been differently associated with air pollution exposure.25 These data suggest that differential DNA hypomethylation of the two distinct CpGs in IL-6 may reflect different cumulative effects from endogenous and exogenous exposure factors, and then contribute differently to the susceptibility to human diseases including CHD. Transcription factor binding sites (BAF155, Inil, c-Myc, BAF170, Max, NRSF and Nrf1) were present for position 1, whereas position 2 was free of the binding sites,28 which may partly explain the different results we and others observed for these two positions. Further studies are required on whether the different CpGs in IL-6 promoter have differential effects on IL-6 expression.

Our study was based on accurate quantitative analysis using pyrosequencing methodology, which is suitable for measuring subtle changes in DNA methylation and can produce individual measures of methylation at more than one CpG site, thus reflecting more accurately DNA methylation in the region.35,36 However, several limitations of the present study should be noted. Firstly, while our data demonstrate that IL-6 promoter hypomethylation was associated with CHD, whether the methylation pattern is a cause or a consequence of the development of CHD cannot be determined in our case-control study design. Prospective studies are required to elucidate the temporal nature of this association. However, given the implication of IL-6 in the pathogenesis of CHD and the inverse correlation of IL-6 promoter methylation with CHD risk factors,25,37 our results suggest that demethylation of the IL-6 promoter may contribute to the risk of developing CHD. Secondly, the functional effect of the IL-6 promoter hypomethylation was not further investigated in the present study. Finally, several studies have observed correlations of DNA methylation in IL-6 promoter with diet and environmental exposures,25,38-40 which would confound the associations between IL-6 promoter hypomethylation and risk of CHD that we observed.

 Conclusion

In summary, although limited by relatively small sample size, the present study suggest that DNA hypomethylation in IL-6 promoter is associated with the increased risk for CHD, especially for AMI. Demethylation of the two CpGs in IL-6 may contribute differently to the susceptibility to CHD. Because of the exploratory nature of the present study, future studies will be needed to verify our findings.

The authors Hong-Peng Zuo and Ying-Yu Guo have contributed equally to the manuscript

Sources of Funding

There were no external funding sources for this study.

Study Association

This study is not associated with any thesis or dissertation work.

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Gene promoter methylation is associated with lung function in the elderly: the Normative Aging Study. Epigenetics. 2012;7(3):261-9. Erratum in: Epigenetics. 2012;7(6):667. Lepeule J Baccarelli A Motta V Cantone L Litonjua AA Sparrow D Gene promoter methylation is associated with lung function in the elderly: the Normative Aging Study Erratum in Epigenetics. 2012;7(6):667 Epigenetics 2012 7 3 261 269 29 Nile CJ, Read RC, Akil M, Duff GW, Wilson AG. Methylation status of a single CpG site in the IL6 promoter is related to IL6 messenger RNA levels and rheumatoid arthritis. Arthritis Rheum. 2008;58(9):2686-93. Nile CJ Read RC Akil M Duff GW Wilson AG Methylation status of a single CpG site in the IL6 promoter is related to IL6 messenger RNA levels and rheumatoid arthritis Arthritis Rheum 2008 58 9 2686 2693 30 Brook RD, Rajagopalan S, Pope CA 3rd, Brook JR, Bhatnagar A, Diez-Roux AV, et al; American Heart Association Council on Epidemiology and Prevention, Council on the Kidney in Cardiovascular Disease, and Council on Nutrition, Physical Activity and Metabolism. Particulate matter air pollution and cardiovascular disease: an update to the scientific statement from the American Heart Association. Circulation. 2010;121(21):2331-78. Brook RD Rajagopalan S Pope 3rd CA Brook JR Bhatnagar A Diez-Roux AV American Heart Association Council on Epidemiology and Prevention Council on the Kidney in Cardiovascular Disease Council on Nutrition, Physical Activity and Metabolism Particulate matter air pollution and cardiovascular disease: an update to the scientific statement from the American Heart Association Circulation 2010 121 21 2331 2378 31 Su D, Li Z, Li X, Chen Y, Zhang Y, Ding D, et al. Association between serum interleukin-6 concentration and mortality in patients with coronary artery disease. Mediators Inflamm. 2013;2013:726178. Su D Li Z Li X Chen Y Zhang Y Ding D Association between serum interleukin-6 concentration and mortality in patients with coronary artery disease Mediators Inflamm 2013 2013 726178 726178 32 Rahim MA, Rahim ZH, Ahmad WA, Hashim OH. Can saliva proteins be used to predict the onset of acute myocardial infarction among high-risk patients? Int J Med Sci. 2015;12(4):329-35. Rahim MA Rahim ZH Ahmad WA Hashim OH Can saliva proteins be used to predict the onset of acute myocardial infarction among high-risk patients Int J Med Sci 2015 12 4 329 335 33 Murayama A, Sakura K, Nakama M, Yasuzawa-Tanaka K, Fujita E, Tateishi Y, et al. A specific CpG site demethylation in the human interleukin 2 gene promoter is an epigenetic memory. EMBO J. 2006;25(5):1081-92. Murayama A Sakura K Nakama M Yasuzawa-Tanaka K Fujita E Tateishi Y A specific CpG site demethylation in the human interleukin 2 gene promoter is an epigenetic memory EMBO J 2006 25 5 1081 1092 34 Zou B, Chim CS, Zeng H, Leung SY, Yang Y, Tu SP, et al. Correlation between the single-site CpG methylation and expression silencing of the XAF1 gene in human gastric and colon cancers. Gastroenterology. 2006;131(6):1835-43. Zou B Chim CS Zeng H Leung SY Yang Y Tu SP Correlation between the single-site CpG methylation and expression silencing of the XAF1 gene in human gastric and colon cancers Gastroenterology 2006 131 6 1835 1843 35 Yang AS, Estecio MR, Doshi K, Kondo Y, Tajara EH, Issa JP. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res. 2004;32(3):e38. Yang AS Estecio MR Doshi K Kondo Y Tajara EH Issa JP A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements Nucleic Acids Res 2004 32 3 e38 36 Tost J, Dunker J, Gut IG. Analysis and quantification of multiple methylation variable positions in CpG islands by Pyrosequencing. Biotechniques. 2003;35(1):152-6. Tost J Dunker J Gut IG Analysis and quantification of multiple methylation variable positions in CpG islands by Pyrosequencing Biotechniques 2003 35 1 152 156 37 Aumueller E, Remely M, Baeck H, Hippe B, Brath H, Haslberger AG. Interleukin-6 CpG methylation and body weight correlate differently in type 2 Diabetes patients compared to obese and lean controls. J Nutrigenet Nutrigenomics. 2015;8(1):26-35. Aumueller E Remely M Baeck H Hippe B Brath H Haslberger AG Interleukin-6 CpG methylation and body weight correlate differently in type 2 Diabetes patients compared to obese and lean controls J Nutrigenet Nutrigenomics 2015 8 1 26 35 38 Zhang FF, Santella RM, Wolff M, Kappil MA, Markowitz SB, Morabia A. White blood cell global methylation and IL-6 promoter methylation in association with diet and lifestyle risk factors in a cancer-free population. Epigenetics. 2012;7(6):606-14. Zhang FF Santella RM Wolff M Kappil MA Markowitz SB Morabia A White blood cell global methylation and IL-6 promoter methylation in association with diet and lifestyle risk factors in a cancer-free population Epigenetics 2012 7 6 606 614 39 Kirchner H, Nylen C, Laber S, Barres R, Yan J, Krook A, et al. Altered promoter methylation of PDK4, IL1 B, IL6, and TNF after Roux-en Y gastric bypass. Surg Obes Relat Dis. 2014;10(4):671-8. Erratum in: Surg Obes Relat Dis. 2015;11(3):735. Kirchner H Nylen C Laber S Barres R Yan J Krook A Altered promoter methylation of PDK4, IL1 B, IL6, and TNF after Roux-en Y gastric bypass Erratum in Surg Obes Relat Dis. 2015;11(3):735 Surg Obes Relat Dis 2014 10 4 671 678 40 Peluso M, Bollati V, Munnia A, Srivatanakul P, Jedpiyawongse A, Sangrajrang S, et al. DNA methylation differences in exposed workers and nearby residents of the Ma Ta Phut industrial estate, Rayong, Thailand. Int J Epidemiol. 2012;41(6):1753-60. Peluso M Bollati V Munnia A Srivatanakul P Jedpiyawongse A Sangrajrang S DNA methylation differences in exposed workers and nearby residents of the Ma Ta Phut industrial estate, Rayong, Thailand Int J Epidemiol 2012 41 6 1753 1760 Artigo Original Hipometilação do Promotor de Interleucina-6 Associada com Risco de Doença Arterial Coronariana Zuo Hong-Peng 1 * Guo Ying-Yu 2 * Che Lin 3 Wu Xian-Zheng 1 Department of Emergency, Tongji Hospital, Tongji University, Shanghai, China Department of Computed Tomography, Jilin Oilfield General Hospital, Songyuan, Jilin Province, China Department of Cardiology, Tongji Hospital, Tongji University, Shanghai, China Correspondência: Xian-Zheng Wu, No. 389 Xincun Road. Postal Code 200065, Shanghai - China. E-mail: wuxz_tongji@sina.com

Contribuição dos autores

Concepção e desenho da pesquisa: Zuo HP, Guo YY, Che L, Wu XZ. Obtenção de dados: Zuo HP, Guo YY, Che L. Análise e interpretação dos dados: Zuo HP, Guo YY, Che L, Wu XZ. Análise estatística: Zuo HP, Wu XZ. Redação do manuscrito: Zuo HP, Guo YY, Che L. Revisão crítica do manuscrito quanto ao conteúdo intelectual importante: Wu XZ. Supervisão / como investigador principal: Zuo HP, Guo YY, Wu XZ.

Potencial Conflito de Interesse

Declaro não haver conflito de interesses pertinentes.

 Resumo Fundamento:

Interleucina-6 (IL-6) está implicada na patogênese de doença arterial coronariana (DAC), sendo sua expressão associada com redução da metilação de DNA do promotor do seu gene. Entretanto, não há dados sobre metilação do promotor de IL-6 e risco de DAC.

 Objetivo:

Verificar se a metilação do promotor de IL-6 medida no DNA de leucócitos sanguíneos acha-se associada com risco de DAC.

 Métodos:

este estudo arrolou 212 casos com DAC e 218 controles. Metilação em dois sítios de CpG no promotor de IL-6 foi medida por pirosequenciamento de bissulfito, sendo a metilação média de IL-6 calculada pela média das medidas de metilação dos dois CpGs.

 Resultados:

A média do nível de metilação no promotor de IL-6 nos casos de DAC foi significativamente mais baixa do que nos controles (p = 0,023). Análise de regressão logística mostrou associação inversa entre metilação de IL-6 e risco de DAC. As razões de chance (OR) de DAC para indivíduos no segundo e no primeiro (mais baixo) tercis de metilação de IL-6 foram 1,87 (IC 95%: 1,10-3,20) e 2,01 (IC 95%: 1,19-3,38) (ptrend = 0,013), respectivamente, comparadas à de indivíduos no terceiro (mais alto) tercil. As estimativas de risco relacionado à hipometilação de IL-6 tenderam a ser mais fortes para infarto agudo do miocárdio (ptrend = 0,006). Análise com especificidade de posição de CpG mostrou que hipometilação na posição 1 conferiu maior elevação no risco de DAC do que na posição 2.

 Conclusão:

Tais achados sugerem que a hipometilação de DNA do promotor de IL-6 está associada com elevado risco de DAC, especialmente para infarto agudo do miocárdio. Os dois CpGs distintos no promotor de IL-6 podem contribuir de modo diferente para o desenvolvimento de DAC.

 Palavras-chave: Doença Arterial Coronariana Interleucina-6 Metilação de DNA Repressão Epigenética Introdução

Metilação de DNA é uma modificação epigenética que desempenha papel crucial no controle da expressão de genes no genoma.1 Nos mamíferos, a metilação de DNA envolve a adição de grupos metila à citosina do dinucleotídeo CpG para formar 5-metilcitosina (5 mC). Alterações epigenéticas nos padrões de metilação são cada vez mais implicadas em inúmeras doenças humanas.2 Metilação aberrante do promotor de vários genes foi associada com desenvolvimento e progressão de doença arterial coronariana (DAC).3-8

A principal causa de DAC é aterosclerose, uma doença inflamatória das artérias associada com alterações metabólicas, inclusive lipídicas. Interleucina-6 (IL-6) ocupa posição central nos processos inflamatórios subjacentes de doenças inflamatórias crônicas, incluindo DAC,9-11 e mostrou induzir a expressão de outros genes que podem contribuir para o fenótipo de DAC.12 Uma variante genética no promotor do gene IL-6 foi associada com sua expressão anormal13,14 e elevado risco de DAC.15 Modificação da metilação é um mecanismo alternativo que regula a produção de IL-6.16,17 Evidência consistente mostrou que a expressão de IL-6 acha-se associada com reduzida metilação de DNA do seu promotor de gene.18-24 Além disso, metilação de DNA no promotor de IL-6 foi associada com fatores de risco para DAC, como exposição à poluição do ar.25 Entretanto, até onde se sabe, não há dados sobre a metilação do promotor de IL-6 e o risco de DAC.

Recentemente, foi sugerido que o silenciamento gênico através de metilação de DNA acha-se associado com metilação não apenas nas ilhas de CpG, mas também nas bordas das ilhas de CpG.26,27 No presente estudo sobre a associação entre metilação do promotor de IL-6 e risco de DAC, focamos em dois CpGs individuais em uma borda de CpG localizada na região do promotor de gene IL-6, descritos como associados com função pulmonar e exposição à poluição do ar.25,28

Métodos Participantes do estudo

Um total de 212 pacientes com DAC, incluindo 120 casos de infarto agudo do miocárdio (IAM), 42 casos de infarto do miocárdio prévio e 50 casos de angina instável, foi recrutado do Hospital Tongji, Universidade Tongji, entre janeiro de 2011 e junho de 2012. O diagnóstico de DAC foi estabelecido por evidência angiográfica de estenose ≥ 70% de uma artéria coronária maior, e/ou ≥ 50% do tronco da coronária esquerda. Como controle, 218 indivíduos sem DAC, determinado por análise da história, exame físico, eletrocardiografia e ecocardiografia, foram recrutados do mesmo hospital e durante o mesmo período. Os controles foram pareados com os casos por idade (±5 anos) e sexo. Excluíram-se dos grupos DAC e controle os indivíduos com hipertensão, diabetes, doença arterial periférica, doenças autoimunes e cânceres. Informação sobre idade, sexo, altura, peso, tabagismo e história familiar de DAC foi obtida usando-se questionário estruturado em entrevistas pessoais. Índice de massa corporal (IMC) foi calculado através da fórmula: peso corporal em quilogramas dividido pelo quadrado da altura em metros (kg/m2). Definiu-se tabagismo como o consumo de pelo menos um cigarro por dia por pelo menos seis meses. A informação sobre nível sérico de colesterol total foi obtida dos prontuários médicos. Todos os participantes assinaram o termo de consentimento livre e informado. Este estudo foi conduzido com a aprovação do Comitê de Ética do Hospital Tongji (E20100401).

 Análise da metilação de DNA do promotor de IL-6

Os leucócitos do sangue periférico foram isolados por centrifugação de gradiente de densidade (Ficoll-Hypaque). O DNA foi extraído dos leucócitos usando-se o kit QIAamp DNA Blood (Qiidaden, Xangai, China), e então convertidos com bissulfito com o kit Zymo EZ DNA Methylation (Zymo, CA, EUA). O pirosequenciamento baseado em PCR foi realizado para quantificar a metilação do promotor de IL-6. Realizou-se PCR para amplificar a região-alvo cobrindo os dois CpGs [Genbank Accession no.M18403, cromossomo 7: 22733847 (posição 1) e cromossomo 7: 22733841 (posição 2)] em um volume de reação de 50 µl contendo 25 µl da mistura GoTaq Master (Promega, WI, EUA), 10 pmol de primer forward biotinilado (biotina-TAT TTT AGT TTT GAG AAA GGA GGT G), 10 pmol de primer reverse (CAA TAC TCT AAA ACC CAA CAA AAA C), e 50 ng de DNA genômico tratado com bissulfito. O programa de ciclagem consistiu em 5 min a 95 °C seguidos de 45 ciclos, cada um de 95°C por 1 min, 57 °C por 1 min e 72°C por 1 min, com um alongamento final por 5 min a 72°C. Realizou-se, então, sequenciamento no sistema PSQ HS 96 Pyrosequencing System usando 0,3 µM de primer de pirosequenciamento (TCC TAA TAC AAA CAA CCC C). Utilizou-se resíduo de citosina não CpG como controle para verificar a conversão de bissulfito. O grau de metilação foi expresso para cada CpG como %5 mC em relação à soma de citosinas metiladas e não metiladas. Calculou-se a média dos níveis de %5 mC dos dois CpGs para se obter uma medida média de metilação do promotor de IL-6.

 Análise estatística

Diferenças de idade (< 65, ≥ 65 anos), sexo, IMC (< 24; 24-27,9; ≥ 28 kg/m2), tabagismo (não, sim), nível sérico de colesterol total (< 4,67; 4,67-5,47; > 5,47 mmol/L) e história familiar de DAC (não, sim) entre os casos de DAC e os controles foram avaliadas usando-se o teste do qui-quadrado. Os tercis dos pontos de corte das medidas de metilação do promotor de IL-6 (média, posição 1 e posição 2) foram baseados em valores dos controles. As associações de metilação de IL-6 com risco de DAC foram estimadas calculando-se as razões de chance (OR) e intervalos de confiança (IC) a 95% das análises de regressão logística multivariada com ajuste para IMC (< 24; 24-27,9; ≥ 28 kg/m2), nível sérico de colesterol total (< 4,67; 4,67-5,47; > 5,47 mmol/L) e história familiar de DAC (não, sim). Ajuste adicional para idade (< 65, ≥ 65 anos), sexo e tabagismo (não, sim) não alterou as estimativas de risco e, consequentemente, tais variáveis não foram incluídas nos modelos finais. Todos os testes foram bicaudais, sendo um valor de p menor do que 0,05 considerado significativo. Os dados foram analisados com o programa Stata 10.1 (Stata Corporation, College Station, TX, EUA).

 Resultados Características dos indivíduos do estudo

As características dos casos com DAC e dos controles são mostradas na Tabela 1. Não houve significativa diferença entre os casos e os controles quanto à distribuição de idade, sexo e tabagismo. Os casos com DAC tiveram maior probabilidade de apresentar maior IMC (p = 0,029), maior nível sérico de colesterol total (p < 0,001) e história familiar de DAC mais frequente (p < 0,001) do que os controles.

Características dos casos com doença arterial coronariana (DAC) e controles
Característica Controles, n (%) Casos com DAC, n (%) Valor de p*
Idade (anos)
< 65 102 (46,8) 108 (50,9)
≥ 65 116 (53,2) 104 (49,1) 0,389
Sexo
Feminino 46 (21,1) 48 (22,6)
Masculino 172 (78,9) 164 (77,4) 0,699
Índice de massa corporal (kg/m2)
< 24 125 (57,3) 96 (45,3)
24-27.9 61 (28,0) 69 (32,5)
≥ 28 32 (14,7) 47 (22,2) 0,029
Tabagista
Não 100 (45,9) 79 (37,3)
Sim 118 (54,1) 133 (62,7) 0,070
Colesterol total sérico (mmol/l)
< 4.67 73 (33,5) 34 (16,1)
4.67-5.47 75 (34,4) 52 (24,5)
> 5.47 70 (32,1) 126 (59,4) < 0,001
História familiar de DAC
Não 214 (98,2) 185 (87,3)
Sim 4 (1,8) 27 (12,7) < 0,001

p valor obtido do teste do qui-quadrado comparando casos e controles.

 Associação entre metilação média de IL-6 e risco de DAC

Os casos de DAC apresentaram significativa redução do nível médio de metilação de IL-6 em relação aos controles [média (desvio-padrão, DP): 41,2 (0,7) versus 43,4 (0,6), p = 0,023 (Figura 1)]. Análise de regressão logística mostrou associação inversa entre a metilação do promotor de IL-6 e o risco de DAC (Tabela 2). As OR de DAC para indivíduos no segundo e primeiro tercis (mais baixos) da metilação média de IL-6 foram 1,87 (IC 95%: 1,10-3,20) e 2,01 (IC 95%: 1,19-3,38) (prend = 0,013), respectivamente, em comparação às dos indivíduos do terceiro tercil (mais alto). Quando avaliados quanto aos tipos clínicos de DAC, as estimativas de risco relacionado à hipometilação de IL-6 tenderam a ser mais fortes para IAM, com OR de 2,00 (IC 95%: 3,2-5,2) para o segundo tercil e 2,57 (IC 95%: 1,33-4,95) para o primeiro tercil (ptrend = 0,006, Tabela 2).

Comparações dos níveis de metilação de IL-6 medidos em leucócitos sanguíneos em casos com doença arterial coronariana (DAC) e em controles (p = 0,023).

Associação de metilação no promotor de IL-6 com risco de doença arterial coronariana (DAC)
Metilação (%5 mC) Controles Casos com DAC
Todos IAM IM prévio Angina
n (%) n (%) OR (IC 95%)* n (%) OR (IC 95%)* n (%) OR (IC 95%)* n (%) OR (IC 95%)*
Média (Tercil†)
T3 (> 47,7) 73 (33,5) 42 (19,8) 1,0 (referência) 21 (17,5) 1,0 (referência) 9 (21,4) 1,0 (referência) 12 (24,0) 1,0 (referência)
T2 (40,1-47,7) 71 (32,6) 77 (36,3) 1,87 (1,10-3,20) 41 (34,2) 2,00 (1,01-3,94) 18 (42,9) 2,02 (0,84-4,86) 18 (36,0) 1,76 (0,73-4,24)
T1 (< 40,1) 74 (33,9) 93 (43,9) 2,01 (1,19-3,38) 58 (48,3) 2,57 (1,33-4,95) 15 (35,7) 1,63 (0,66-4,01) 20 (40,0) 1,83 (0,77-4,33)
p-trend=0,013 p-trend=0,006 p-trend=0,325 p-trend=0,182
Posição 1 (Tercil†)
T3 (> 51,7) 73 (33,5) 44 (20,8) 1,0 (referência) 25 (20,8) 1,0 (referência) 8 (19,1) 1,0 (referência) 11 (22,0) 1,0 (referência)
T2 (43,5-51,7) 71 (32,6) 77 (36,3) 2,04 (1,20-3,49) 38 (31,7) 1,71 (0,88-3,32) 19 (45,2) 2,37 (0,96-5,84) 20 (44,0) 2,50 (1,01-6,17)
T1 (< 43,5) 74 (33,9) 91 (42,9) 2,17 (1,29-3,66) 57 (47,5) 2,44 (1,29-4,62) 15 (35,7) 1,94 (0,77-4,92) 19 (38,0) 2,16 (0,88-5,27)
p-trend=0,05 p-trend=0,006 p-trend=0,196 p-trend=0,114
Posição 2 (Tercil†)
T3 (> 44,0) 73 (33,5) 51 (24,1) 1,0 (referência) 30 (25,0) 1,0 (referência) 9 (21,4) 1,0 (referência) 12 (24,0) 1,0 (referência)
T2 (35,9-44,0) 71 (32,6) 63 (29,7) 1,25 (0,74-2,12) 28 (23,3) 0,93 (0,48-1,81) 17 (40,5) 2,06 (0,84-5,02) 18 (36,0) 1,74 (0,72-4,20)
T1 (< 35,9) 74 (33,9) 98 (46,2) 1,73 (1,05-2,86) 62 (51,7) 1,81 (0,99-3,29) 16 (38,1) 1,82 (0,74-4,47) 20 (40,0) 1,79 (0,76-4,23)
p-trend=0,030 p-trend=0,038 p-trend=0,220 p-trend=0,197

%5mC: porcentagem de 5-metilcitosina; IAM: infarto agudo do miocárdio; IC: intervalo de confiança; IM: infarto do miocárdio; OR: razão de chance.

Ajustado para índice de massa corporal (<24; 24-27,9; ≥28 kg/m2), colesterol total sérico (<4,67; 4,67-5,47; >5,47 mmol/l) e história familiar de DAC (não, sim).

Os tercis das medidas de metilação de IL-6 foram baseados em valores dos controles.

Associação entre metilação de IL-6 em posição específica de CpG e risco de DAC

Nossa análise principal considerou a metilação média de dois CpGs no promotor de IL-6. Entretanto, metilação em posições específicas dentro de um promotor de gene pode afetar a expressão gênica de forma diferente. Assim, examinamos ainda as associações entre metilação de IL-6 em posição específica e risco de DAC. Como mostra a Tabela 2, embora tenham sido observados significativos valores de ptrend para DAC e IAM para as duas posições de CpG, a hipometilação de posição 1 conferiu um risco de DAC mais elevado se comparada à da posição 2.

 Discussão

Metilação de promotor é um mecanismo epigenético essencial para a regulação da expressão de IL-6.16,17 Foi relatada associação entre a hipometilação do promotor de IL-6 e a patogênese de lúpus eritematoso sistêmico, artrite reumatoide e periodontite crônica.18,24,29 No presente estudo, demonstramos pela primeira vez a associação entre hipometilação de DNA no promotor de IL-6 e elevado risco para DAC, especialmente IAM. Tais dados sugerem que a desmetilação do promotor de IL-6 constitua uma base epigenética comum para o desenvolvimento de várias doenças associadas à inflamação. Nesse contexto, a associação entre metilação do promotor de IL-6 e risco de outras doenças inflamatórias merece investigação adicional.

O nível de metilação do promotor de IL-6 foi associado com exposição à poluição do ar,25 conhecida por aumentar a morbimortalidade cardiovascular.30 Além disso, o nível sérico de IL-6 foi associado com elevado risco de mortalidade em pacientes com DAC.31 No presente estudo, observamos que a hipometilação no promotor de IL-6 foi associada com uma estimativa mais forte de risco para IAM, um tipo clínico de DAC que contribui para muitos casos de mortalidade mundo afora.32 Com base nesses resultados preliminares e na literatura atual, somos tentados a especular que a desmetilação do promotor de IL-6 possa desempenhar papel não apenas no desenvolvimento, mas também no prognóstico da DAC. Em linha com essa hipótese, relatou-se que o nível de metilação do promotor de IL-6 está significativamente correlacionado com o desenvolvimento e a gravidade de lúpus eritematoso sistêmico.18,24

Há evidência crescente de que a metilação em posições específicas dentro do promotor de um gene possa ser mais importante para a expressão gênica do que a metilação média de sítios de CpG na região do promotor.33,34 No presente estudo, portanto, examinamos ainda se as associações de DAC observadas na análise principal usando metilação média foram específicas para certas posições no promotor de IL-6. Notamos que hipometilação de CpG na posição 1 no promotor de IL-6 conferiu um aumento mais pronunciado nas estimativas de risco de DAC e de IAM do que aquela na posição 2. Previamente, níveis de metilação desses dois CpGs na IL-6 foram associados de modo diferente com exposição à poluição do ar.25 Tais dados sugerem que hipometilação diferencial de DNA nos dois CpGs distintos na IL-6 possa refletir diferentes efeitos cumulativos de exposição a fatores endógenos e exógenos, contribuindo diferentemente para a suscetibilidade a doenças humanas, incluindo DAC. Sítios de ligação de fatores de transcrição (BAF155, Inil, c-Myc, BAF170, Max, NRSF e Nrf1) estavam presentes para a posição 1, enquanto a posição 2 não apresentava sítios de ligação,28 podendo explicar em parte os diferentes resultados observados por nós e outros autores para essas duas posições. Estudos adicionais são necessários para esclarecer se os diferentes CpGs no promotor de IL-6 têm efeitos diferentes na expressão de IL-6.

Nosso estudo foi baseado em análise quantitativa precisa usando pirosequenciamento, que é adequado para medir alterações sutis na metilação de DNA e pode produzir medidas individuais de metilação em mais de um sítio de CpG, refletindo assim, de maneira mais acurada, a metilação de DNA na região.35,36

Entretanto, várias limitações do presente estudo devem ser apontadas. Primeiro, enquanto nossos dados demonstram que hipometilação do promotor de IL-6 associou-se com DAC, se tal padrão de metilação é causa ou consequência do desenvolvimento de DAC não pôde ser determinado no nosso estudo de design caso-controle. Estudos prospectivos são necessários para elucidar a natureza temporal dessa associação. Entretanto, dada a implicação da IL-6 na patogênese de DAC e a correlação inversa da metilação do promotor de IL-6 com fatores de risco de DAC,25,37 nossos resultados sugerem que desmetilação do promotor de IL-6 possa contribuir para o risco de desenvolvimento de DAC. Segundo, o efeito funcional do promotor de hipometilação de IL-6 não foi investigado mais profundamente no presente estudo. Por fim, vários estudos relataram correlações de metilação de DNA no promotor de IL-6 com dieta e exposições ambientais,25,38-40 podendo confundir as associações entre hipometilação do promotor de IL-6 e risco de DAC por nós observadas.

 Conclusão

Em resumo, embora limitado pelo relativamente pequeno tamanho amostral, o presente estudo sugere que hipometilação de DNA no promotor de IL-6 esteja associada com elevado risco para DAC, em especial IAM. Desmetilação dos dois CpGs na IL-6 pode contribuir de modo diferente para a suscetibilidade para DAC. Devido à natureza exploratória do presente estudo, investigação adicional se faz necessária para verificar seus achados.

Os autores Hong-Peng Zuo and Ying-Yu Guo contribuiram igualmente para o manuscrito.

Fontes de Financiamento

O presente estudo não teve fontes de financiamento externas.

Vinculação Acadêmica

Não há vinculação deste estudo a programas de pós-graduação.