525 Epigenome-wide Association Study in the European Prospective Investigation Into Cancer and Nutrition (EPIC-Turin) Identifies Novel Genes Associated With Smoking

Epigenetics Unit, Department of Surgery and Cancer, Imperial College London, W12 0NN, UK.
Human Molecular Genetics (Impact Factor: 6.39). 11/2012; 22(5). DOI: 10.1093/hmg/dds488
Source: PubMed


A single cytosine–guanine dinucleotide (CpG) site within coagulation factor II (thrombin) receptor-like 3 (F2RL3) was recently found to be hypomethylated in peripheral blood genomic DNA from smokers compared with former and non-smokers.
We performed two epigenome-wide association studies (EWAS) nested in a prospective healthy cohort using the Illumina 450K
Methylation Beadchip. The two populations consisted of matched pairs of healthy individuals (n = 374), of which half went on to develop breast or colon cancer. The association was analysed between methylation and smoking
status, as well as cancer risk. In addition to the same locus in F2RL3, we report several loci that are hypomethylated in smokers compared with former and non-smokers, including an intragenic
region of the aryl hydrocarbon receptor repressor gene (AHRR; cg05575921, P = 2.31 × 10−15; effect size = 14–17%), an intergenic CpG island on 2q37.1 (cg21566642, P = 3.73 × 10−13; effect size = 12%) and a further intergenic region at 6p21.33 (cg06126421, P = 4.96 × 10−11, effect size = 7–8%). Bisulphite pyrosequencing validated six loci in a further independent population of healthy individuals
(n = 180). Methylation levels in AHRR were also significantly decreased (P < 0.001) and expression increased (P = 0.0047) in the lung tissue of current smokers compared with non-smokers. This was further validated in a mouse model of
smoke exposure. We observed an association with breast cancer risk for the 2q37.1 locus (P = 0.003, adjusted for the smoking status), but not for the other loci associated with smoking. These data show that smoking
has a direct effect on the epigenome in lung tissue, which is also detectable in peripheral blood DNA and may contribute to
cancer risk.

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    • "Nevertheless, given the relatively small sample size, replication of our newly identified results in larger samples is needed. The difference we observed in AHRR mean methylation levels (14%, range 1–32%) between smokers and non-smokers was comparable with those reported in larger non-twin independent populations studied by other authors (17% in [3]; 24.4% in [29]; 7.4% in [9]; 15% in [8]; 22% in [7]). However, large-scale epigenetic studies are still ongoing, and a threshold to define the minimum DNA methylation changes able to exert functional significance at the population level has not yet been firmly established. "
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    ABSTRACT: Exposure to cigarette smoking affects the epigenome and could increase the risk of developing diseases such as cancer and cardiovascular disorders. Changes in DNA methylation associated with smoking may help to identify molecular pathways that contribute to disease etiology. Previous studies are not completely concordant in the identification of differentially methylated regions in the DNA of smokers. We performed an epigenome-wide DNA methylation study in a group of monozygotic (MZ) twins discordant for smoking habits to determine the effect of smoking on DNA methylation. As MZ twins are considered genetically identical, this model allowed us to identify smoking-related DNA methylation changes independent from genetic components. We investigated the whole blood genome-wide DNA methylation profiles in 20 MZ twin pairs discordant for smoking habits by using the Illumina Human-Methylation450 BeadChip. We identified 22 CpG sites that were differentially methylated between smoker and non-smoker MZ twins by intra-pair analysis. We confirmed eight loci already described by other groups, located in AHRR, F2RL3, MYOG1 genes, at 2q37.1 and 6p21.33 regions, and also identified several new loci. Moreover, pathway analysis showed an enrichment of genes involved in GTPase regulatory activity. Our study confirmed the evidence of smoking-related DNA methylation changes, emphasizing that well-designed MZ twin models can aid the discovery of novel DNA methylation signals, even in a limited sample population. © 2015 Allione et al. 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 author and source are credited.
    Full-text · Article · Jun 2015 · PLoS ONE
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    • "Hypomethylation of cg05575921 at AHRR has been reported to be associated with increasing lymphoblast AHRR gene expression in vivo (Monick et al. 2012). It has also been observed that AHRR expression in human lung tissues was inversly correlated with methylation levels of cg23576855 and cg21161138 at AHRR, with 5.7-fold increased expression in five current smokers compared with five non smokers (Shenker et al. 2013a). AHRR and the aryl hydrocarbon receptor (AHR) constitute a feedback loop in which the AHR hetero dimer activates the expression of the AHRR gene, and the expressed AHRR inhibits the function of AHR in oncogenesis (Mimura et al. 1999). "
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    ABSTRACT: With epigenome-wide mapping of DNA methylation, a number of novel smoking-associated loci have been identified. We aimed to assess dose-response relationships of methylation at the top hits from the epigenome-wide methylation studies with smoking exposure as well as with total and cause-specific mortality. In a population-based prospective cohort study in Germany, methylation was quantified in baseline blood DNA of 1000 older adults by the Illumina 450K assay. Deaths were recorded during a median follow-up of 10.3 years. Dose-response relationships of smoking exposure with methylation at 9 CpGs were modeled by restricted cubic spline regression. Associations of individual and aggregate methylation patterns with all-cause, cardiovascular and cancer mortality were assessed by multiple Cox regression. Clear dose-response relationships with respect to current and lifetime smoking intensity were consistently observed for methylation at 6 of the 9 CpGs. Seven of the 9 CpGs were also associated with mortality outcomes to various extents. A methylation score based on the top 2 CpGs (cg05575921 and cg06126421) showed the strongest associations with all-cause, cardiovascular and cancer mortality, with adjusted hazard ratios (95% CI) of 3.59 (2.10, 6.16), 7.41 (2.81, 19.54), 2.48 (1.01, 6.08), respectively, for participants with methylation levels in the lowest quartile at both CpGs. Adding methylation at those 2 CpGs into a model that included the variables of the Systematic Coronary Risk Evaluation chart for fatal cardiovascular risk prediction improved the predictive discrimination. The novel methylation biomarkers are highly informative for both smoking exposure and smoking-related mortality outcomes. In particular, these biomarkers may substantially improve cardiovascular risk prediction. Nevertheless, the findings of the present study need to be further validated in additional large longitudinal studies.
    Full-text · Article · May 2015 · Environmental Health Perspectives
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    • "Altered methylation of cancer-related genes is, in fact, frequently observed in lung tumors of smokers [8] [9] [10] [11], and a progressive accumulation of epigenetic alterations is also observed in the respiratory epithelium of cancer-free heavy smokers [12] [13] [14] and in exfoliated cells of smokers sputum [15]. Moreover, changes in the methylation profile of cancer related genes have been observed in plasma DNA from cancer-free heavy smokers [16] [17], and smoking-related changes in methylation at a number of CpG sites have been identified in epigenome-wide investigations in blood cells of subjects with different smoking habits [18] [19] [20] [21]. The mechanism(s) by which tobacco smoke could affect DNA methylation is not elucidated. "
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    ABSTRACT: The influence of DNA repair capacity, plasma nutrients and tobacco smoke exposure on DNA methylation was investigated in blood cells of twenty-one couples of monozygotic twins with discordant smoking habits. All study subjects had previously been characterized for mutagen sensitivity with challenge assays with ionizing radiation in peripheral blood lymphocytes. Plasma levels of folic acid, vitamin B12 and homocysteine were also available from a previous investigation. In this work DNA methylation in the promoter region of a panel of ten genes involved in cell cycle control, differentiation, apoptosis and DNA repair (p16, FHIT, RAR, CDH1, DAPK1, hTERT, RASSF1A, MGMT, BRCA1 and PALB2) was assessed in the same batches of cells isolated for previous studies, using the methylation-sensitive high-resolution melting technique. Fairly similar profiles of gene promoter methylation were observed within co-twins compared to unrelated subjects (p=1.23×10(-7)), with no significant difference related to smoking habits (p=0.23). In a regression analysis the methylation index of study subjects, used as synthetic descriptor of overall promoter methylation, displayed a significant inverse correlation with radiation-induced micronuclei (p=0.021) and plasma folic acid level (p=0.007) both in smokers and in non-smokers. The observed association between repair of radiation-induced DNA damage and promoter methylation suggests the involvement of the DNA repair machinery in DNA modification. Data also highlight the possible modulating effect of folate deficiency on DNA methylation and the strong influence of familiarity on the individual epigenetic profile. Copyright © 2015 Elsevier B.V. All rights reserved.
    Full-text · Article · Jan 2015 · Mutation Research/Genetic Toxicology and Environmental Mutagenesis
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