Systemic Steroid Exposure Is Associated with Differential Methylation in Chronic Obstructive Pulmonary Disease Emily S. Wan1,2, Weiliang Qiu1, Andrea Baccarelli3, Vincent J. Carey1, Helene Bacherman1, Stephen I. Rennard4, Alvar Agustı´5, Wayne H. Anderson6, David A. Lomas7, and Dawn L. DeMeo1,2 1Channing Division of Network Medicine, Brigham and Women’s Hospital, Boston, Massachusetts; 2Division of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts; 3Exposure Epidemiology and Risk Program, Harvard School of Public Health, Boston, Massachusetts; 4Section of Pulmonary and Critical Care, University of Nebraska Medical Center, Omaha, Nebraska; 5Thorax Institute, Hospital Clinic, IDIBAPS, University of Barcelona and CIBER Enfermedades Respiratorias, FISIB, Mallorca, Spain; 6Division of Pulmonary and Critical Care Medicine, University of North Carolina, Chapel Hill, North Carolina; and 7Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom Rationale: Systemic glucocorticoids are used therapeutically to treat a variety of medical conditions. Epigenetic processes such as DNA AT A GLANCE COMMENTARY methylation may reflect exposure to glucocorticoids and may be involved in mediating the responses and side effects associated Scientific Knowledge on the Subject with these medications. Systemic corticosteroids are associated with a wide range of Objectives: To test the hypothesis that differences in DNA methyla- therapeutic responses and side effects that may persist after tion are associated with current systemic steroid use. drug withdrawal. These characteristics suggest a possible Methods: We obtained DNA methylation data at 27,578 CpG sites in role for epigenetic modifications. Previous epigenetic studies 14,475 genes throughout the genome in two large, independent ¼ on steroid exposure have examined histone modifications or cohorts: the International COPD Genetics Network (ndiscovery ¼ DNA methylation of candidate genes involved in steroid 1,085) and the Boston Early Onset COPD study (nreplication 369). Sites were tested for association with current systemic steroid use biosynthesis, metabolism, or receptors. using generalized linear mixed models. Measurements and Main Results: A total of 511 sites demonstrated What This Study Adds to the Field significant differential methylation by systemic corticosteroid use in all three of our primary models. Pyrosequencing validation con- Our study explores the association of differential DNA firmed robust differential methylation at CpG sites annotated to methylation at sites throughout the entire genome. We genes such as SLC22A18, LRP3, HIPK3, SCNN1A, FXYD1, IRF7, AZU1, report robustly associated sites in biologically plausible and SIT1, GPR97, ABHD16B, and RABGEF1. Functional annotation cluster- previously unsuspected pathways. ing demonstrated significant enrichment in intrinsic membrane components, hemostasis and coagulation, cellular ion homeostasis, leukocyte and lymphocyte activation and chemotaxis, protein trans- Keywords: DNA methylation; glucocorticoids; chronic obstructive pul- port, and responses to nutrients. monary disease Conclusions: Our analyses suggest that systemic steroid use is associ- ated with site-specific differential methylation throughout the ge- Systemic glucocorticoids possess potent antiinflammatory, im- nome. Differentially methylated CpG sites were found in biologically munomodulatory, and antineoplastic properties and are integral plausible and previously unsuspected pathways; these genes and in the treatment of chronic obstructive pulmonary disease (COPD) pathways may be relevant in the development of novel targeted and asthma exacerbations, autoimmune diseases, allergic reac- therapies. tions, and certain malignancies. However, despite their clinical utility, systemic glucocorticoid use is often complicated by mul- tiple side effects, including weight gain and redistribution of (Received in original form July 23, 2012; accepted in final form September 27, 2012) body fat, osteoporosis, cardiovascular disease, impaired im- Supported by National Institutes of Health grants R01HL089438, T32 HL007427, mune response and wound healing, and alterations in glucose and K12HL089990; by the Doris Duke Foundation Clinician Scientist Develop- and lipid metabolism. The responses and side effects experienced ment Award (D.L.D.); and by National Institute of Environmental Health Sciences- by individuals exposed to glucocorticoids are highly variable and Harvard School of Public Health New Investigator Fund grant ES00002 (A.B.). may persist for extended periods after drug withdrawal; these phe- Author Contributions: E.S.W. and W.Q. contributed to the concept and design, nomena may be mediated through epigenetic processes. statistical support, data analysis, and interpretation of the data. A.B. contributed Epigenetic changes, such as histone modifications (1, 2), to the concept of the study and the interpretation of the data. V.J.C. contributed DNA methylation, and alterations in chromatin structure, vary to the statistical support and analysis of the data. H.B. participated in the design between individuals (3) and can affect glucocorticoid receptor and performance of the experiments. S.I.R., A.A., W.H.A., and D.A.L. contributed to the concept of the study and to the acquisition of the data. D.L.D. contributed expression and binding (3, 4) as well as the subsequent re- to the funding, concept and design, data collection, statistical support, analysis, sponses to endogenous (5) or exogenously administered (1, 2, and interpretation of the data. All authors contributed substantially to drafting 6) steroids. Conversely, glucocorticoid exposure can cause epi- and editing the manuscript. genetic changes and may be mediated in part through changes Correspondence and requests for reprints should be addressed to Emily S. Wan, in the expression or activity of DNA methyltransferases (7–9). M.D., M.P.H., 181 Longwood Ave, 4th floor, Boston, MA 02115. E-mail: Dynamic, site-specific changes in DNA methylation have been [email protected] observed after glucocorticoid exposure in vitro (4) and in vivo This article has an online supplement, which is accessible from this issue’s table of (10). However, investigations into the associations between glu- contents at www.atsjournals.org cocorticoids and DNA methylation remain limited to a small Am J Respir Crit Care Med Vol 186, Iss. 12, pp 1248–1255, Dec 15, 2012 number of CpG sites within selected candidate genes (10–13). Copyright ª 2012 by the American Thoracic Society Originally Published in Press as DOI: 10.1164/rccm.201207-1280OC on October 11, 2012 We hypothesized that, given the extensive systemic effects of Internet address: www.atsjournals.org therapeutic glucocorticoids, exposure to these medications would Wan, Qiu, Baccarelli, et al.: DNA Methylation and Systemic Steroids in COPD 1249 be associated with differential methylation in peripheral blood Statistical Analysis DNA at multiple sites throughout the genome. Some of these results The ComBat (19) function as implemented in the sva package (20, 21) have been previously reported in the form of an abstract (14). in R (release 2.12) was used to account for batch effects. To account for familial correlations in the data, generalized linear mixed models (22) were used to analyze the methylation data. The Illumina “b” value was METHODS modeled as the dependent variable, with oral steroid use included as the independent variable. Models were adjusted for age, sex, and FEV1 Subjects and Cohorts % predicted (Model 1); pack-years of smoking (Model 2); and current The discovery cohort consisted of 1,085 subjects participating in the In- smoking status (Model 3). CpG sites with a P value below the 2 ternational COPD Genetics Network (15, 16) (ICGN), a family-based Bonferroni-adjusted threshold for significance (,1.89 3 10 6)in study with probands between the ages of 45 and 65 years, 5 or more ICGN and a P value of less than 0.05 with the same direction of effect pack-years of cigarette smoking, FEV1 less than 60% predicted, and in EOCOPD were considered significant. a FEV1/FVC ratio of less than 90% predicted. Siblings of probands with at least 5 pack-years of cigarette smoking were also enrolled. The replication cohort consisted of 369 subjects from the Boston Severe Subgroup Analysis: Matched Cohort Early-Onset COPD Study (17) (EOCOPD), a family-based study Subjects in ICGN who reported current systemic steroid use were where probands have an FEV1 less than 40% predicted by age 53 in matched (1:1) by sex, age (62 yr), and FEV1 % predicted (610%) with a the absence of severe -1 antitrypsin deficiency. Although extended subjects who did not report current systemic steroid use (ntotal ¼ 160). pedigrees were recruited, DNA methylation data were obtained only Differences in the mean Illumina b values were tested using an unpaired on probands and their siblings. The protocols were approved by the Student’s t test. appropriate Internal Review Boards; all subjects provided written in- formed consent. RESULTS The characteristics of the subjects in each cohort are listed in Variable Definitions Table 1, and the ranges for continuous variables by current Subjects in ICGN were asked to list all current medications. Current systemic steroid use are shown in Table E1 in the online sup- systemic steroid use was present if they answered affirmatively to the plement.