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DNA Methylation and Smoking in Korean Adults: Epigenome-Wide Association Study Mi Kyeong Lee1,2,3, Yoonki Hong3, Sun-Young Kim4, Stephanie J

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DNA Methylation and Smoking in Korean Adults: Epigenome-Wide Association Study Mi Kyeong Lee1,2,3, Yoonki Hong3, Sun-Young Kim4, Stephanie J Lee et al. Clinical Epigenetics (2016) 8:103 DOI 10.1186/s13148-016-0266-6 RESEARCH Open Access DNA methylation and smoking in Korean adults: epigenome-wide association study Mi Kyeong Lee1,2,3, Yoonki Hong3, Sun-Young Kim4, Stephanie J. London1*† and Woo Jin Kim3*† Abstract Background: Exposure to cigarette smoking can increase the risk of cancers and cardiovascular and pulmonary diseases. However, the underlying mechanisms of how smoking contributes to disease risks are not completely understood. Epigenome-wide association studies (EWASs), mostly in non-Asian populations, have been conducted to identify smoking-associated methylation alterations at individual probes. There are few data on regional methylation changes in relation to smoking. Few data link differential methylation in blood to differential gene expression in lung tissue. Results: We identified 108 significant (false discovery rate (FDR) < 0.05) differentially methylated probes (DMPs) and 87 significant differentially methylated regions (DMRs) (multiple-testing corrected p < 0.01) in current compared to never smokers from our EWAS of cotinine-validated smoking in blood DNA from a Korean chronic obstructive pulmonary disease cohort (n = 100 including 31 current, 30 former, and 39 never smokers) using Illumina HumanMethylation450 BeadChip. Of the 108 DMPs (FDR < 0.05), nine CpGs were statistically significant based on Bonferroni correction and 93 were novel including five that mapped to loci previously associated with smoking. Of the 87 DMRs, 66 were mapped to novel loci. Methylation correlated with urine cotinine levels in current smokers at six DMPs, with pack-years in current smokers at six DMPs, and with duration of smoking cessation in former smokers at eight DMPs. Of the 143 genes to which our significant DMPs or DMRs annotated, gene expression levels at 20 genes were associated with pack-years in lung tissue transcriptome data of smokers (Asan Biobank, n = 188). Conclusions: Our study of differential methylation in Koreans confirmed previous findings from non-Asian populations and revealed novel loci in relation to smoking. Smoking-related differential methylation in blood is associated with gene expression in lung tissue, an important target of adverse health effects of smoking, supporting the potential functional importance of methylation in smoking-related disease. Keywords: DNA methylation, Smoking, Epigenome-wide association study, Cotinine, Duration of smoking cessation, Gene expression Background cardiovascular diseases [5], the underlying mechanisms Smoking is well-known for its adverse health effects [1]; are not completely understood. One proposed mechan- however, between 10 and 35 % of people still smoke ism is through DNA methylation. daily worldwide [2]. Despite established evidence of the DNA methylation, a type of epigenetic modification, causal relationships between smoking and elevated risk plays a key role in regulating gene expression [6]. Unlike of diseases including cancers [3] and pulmonary [4] and DNA sequence, methylation has cell-type and tissue- specific characteristics. DNA methylation can be impacted * Correspondence: [email protected]; [email protected] by age [7], gender [8], and exposures such as obesity [9] †Equal contributors and smoking [10]. 1 Epidemiology Branch, National Institute of Environmental Health Sciences, At least 16 epigenome-wide association studies (EWASs) Department of Health and Human Services, National Institutes of Health, Research Triangle Park, Durham, NC 27709, USA of the association between smoking and blood DNA 3Department of Internal Medicine and Environmental Health Center, methylation in adults have been published [11–26]. Only Kangwon National University Hospital, School of Medicine, Kangwon one study was conducted in an East Asian population [26]; National University, Chuncheon-si, Gangwon-do 19300, South Korea Full list of author information is available at the end of the article most have been conducted in populations of European © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lee et al. Clinical Epigenetics (2016) 8:103 Page 2 of 17 ancestry with others in African American, Arab, and South and weight (kg) were measured twice for each partici- Asian populations. There is no study in Koreans. There are pant using a body composition analyzer IOI 353 (Aarna few data where reported smoking has been biochemically Systems., Udaipur, India); the average value of two mea- validated [11, 21, 25] or where methylation has been surements was used for further analyses. Body mass evaluated in relation to quantitative biomarkers of smoking index (BMI, kg/m2) was calculated by dividing the [21, 27], pack-years, or duration of smoking cessation weight (kg) by the square of the height (m2). [22–25]. Only one EWAS correlated differential methyla- Self-reported smoking status—current, former, and tion in blood with gene expression in lung tissue, and only never smoking—was obtained from the questionnaire, and one locus was examined in 10 individuals [19]. the current status of non-smoking versus smoking was The published EWASs of smoking have identified indi- confirmed by urine cotinine levels (nmol/L) measured by vidual differentially methylated probes (DMPs) rather than immunoassay (Immulite 2000 Xpi; Siemens, NY, USA). differentially methylated regions (DMRs). Identification of One self-reported never smoker was re-assigned to DMRs associated with an exposure can provide stronger current smoker based on a urine cotinine level of evidence for causality than single DMPs [28]. In addition, 16,909 nmol/L, higher than our cut-point for current DMR analysis is statistically more powerful for detection smoking status of 283 nmol/L [32]. Smokers provided the of association with disease traits or exposures [29]. duration (years) and amount (cigarette packs) of cigarette To identify both DMPs and DMRs in relation to smoking. Pack-years were calculated by multiplying the smoking, we conducted an EWAS in 100 adults from a number of smoked cigarette packs per day by the number Korean chronic obstructive pulmonary disease (COPD) of years smoked. Duration of smoking cessation (years) cohort using the Infinium HumanMethylation450 Bead- was reported by former smokers. Chip (450k). For the DMPs of genome-wide significance, we investigated their relationship with smoking intensity Genomic DNA preparation and DNA methylation profiling (urine cotinine) and cumulative smoking (pack-years) in We used blood DNA samples from participants’ baseline current smokers and duration of smoking cessation in visits for methylation profiling. The DNA quality was former smokers. As a replication look-up, we also evalu- checked with a spectrophotometer (NanoDrop® ND- ated association between methylation and smoking at 1000 UV-vis), and genomic DNA was diluted to 50 ng/ previously published probes in our data. For the loci to μl using Quant-iT PicoGreen (Invitrogen, Carlsbad, CA, which significant DMPs or DMRs mapped, we examined USA). Bisulfite-conversion using EZ DNA methylation differential transcriptome profiles in relation to pack- kit (Zymo Research, Irvine, CA, USA) was carried out years in lung tissue from a separate population—188 according to the manufacturer’s protocols. smokers from the Asan Biobank [30]. The Infinium HumanMethylation450 BeadChip (Illu- mina, Inc., San Diego, CA, USA) was used for our genome- Methods wide methylation profiling. The methylation value (β)—a Study participants and exposure to cigarette smoking: ratio between methylated probe intensity and total probe the Korean COPD cohort intensity—is interpreted as the proportion of methylation We aimed to compare methylation in current and and ranges between 0 (unmethylated) and 1 (methylated). former smokers separately to never smokers. For this The signal extraction and normalization using Beta MIx- purpose, we measured DNA methylation in 100 of 190 ture Quantile dilation (BMIQ) [33] were conducted in participants in a Korean COPD cohort [31]. Of the 100 ChAMP [34]. The ComBat [35] method was applied to ad- participants, 60 had COPD and 40 were without COPD. just for batch effects. Cell-type composition was estimated The breakdown by smoking was 39 never, 30 former, by Houseman’s algorithm [36] in minfi [37]. Cytosine- and 31 current smokers. Subjects were recruited from a phosphate-guanine (CpG) probe filtering criteria [38] were rural area in Korea. Having available clinical informa- applied to eliminate sources of possible false positive re- tion, computed tomography (CT) data, survey question- sults, excluding probes that had a detection p value above naire, and blood/urine samples were used for sample 0.01 in any sample; had a bead-count less than 3 in 5 % or selections of methylation profiling. Additional approxi- more of samples; were non-CpG probes; or were non- mate frequency matching on age and smoking status specific probes [39]. To minimize the effects of extreme was applied. Details of the
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