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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    • "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.
    Mutation Research/Genetic Toxicology and Environmental Mutagenesis 01/2015; 779. DOI:10.1016/j.mrgentox.2015.01.006 · 2.42 Impact Factor
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    • "There is evidence that the leading risk factors for lung cancer, tobacco smoke and air pollution, can influence DNA methylation in the lungs and in blood. A study nested within the EPIC cohort reported hypomethylation of F2RL3, AHRR and two intergenic regions in the blood of smokers in comparison to non-smokers [92]. A follow-up study from the same group demonstrated that four loci, including AHRR, may serve as blood-based markers of tobacco smoke exposure [93]. "
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    ABSTRACT: Epigenetic epidemiology includes the study of variation in epigenetic traits and the risk of disease in populations. Its application to the field of cancer has provided insight into how lifestyle and environmental factors influence the epigenome and how epigenetic events may be involved in carcinogenesis. Furthermore, it has the potential to bring benefit to patients through the identification of diagnostic markers that enable the early detection of disease and prognostic markers that can inform upon appropriate treatment strategies. However, there are a number of challenges associated with the conduct of such studies, and with the identification of biomarkers that can be applied to the clinical setting. In this review, we delineate the challenges faced in the design of epigenetic epidemiology studies in cancer, including the suitability of blood as a surrogate tissue and the capture of genome-wide DNA methylation. We describe how epigenetic epidemiology has brought insight into risk factors associated with lung, breast, colorectal and bladder cancer and review relevant research. We discuss recent findings on the identification of epigenetic diagnostic and prognostic biomarkers for these cancers.
    Biochemical and Biophysical Research Communications 08/2014; 455(1-2). DOI:10.1016/j.bbrc.2014.08.002 · 2.30 Impact Factor
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    • "In the recent years, association mapping of molecular phenotypes such as gene expression, DNA methylation, or chromatin accessibility as quantitative traits (eQTL, mQTL, dsQTL) has revealed how genetic variants contribute to inter-individual variability and provided additional insights into the modulation of disease susceptibility [1], [20], [31], [32], [33], [34]. The recent technical advances in low-cost genome-wide DNA methylation assays (such as the Illumina 450 k methylation array [35], RRBS [36], and BSPP [16]) have catalyzed a new wave of epigenome-wide association studies aiming to characterize the contribution of both genetic and environmental factors to disease susceptibility [4], [37], with encouraging progress already in sight [18], [38], [39], [40]. However, while new analysis techniques have connected genetic variants, CpG methylation, and disease phenotypes, it remains unclear to what extent we should expect interaction to occur between genetic variation and the variability of DNA methylation, what fraction of interactions are able to be captured with current approaches, and what strategy we should use to efficiently capture these interactions. "
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    ABSTRACT: Genetic polymorphisms can shape the global landscape of DNA methylation, by either changing substrates for DNA methyltransferases or altering the DNA binding affinity of cis-regulatory proteins. The interactions between CpG methylation and genetic polymorphisms have been previously investigated by methylation quantitative trait loci (mQTL) and allele-specific methylation (ASM) analysis. However, it remains unclear whether these approaches can effectively and comprehensively identify all genetic variants that contribute to the inter-individual variation of DNA methylation levels. Here we used three independent approaches to systematically investigate the influence of genetic polymorphisms on variability in DNA methylation by characterizing the methylation state of 96 whole blood samples in 52 parent-child trios from 22 nuclear pedigrees. We performed targeted bisulfite sequencing with padlock probes to quantify the absolute DNA methylation levels at a set of 411,800 CpG sites in the human genome. With mid-parent offspring analysis (MPO), we identified 10,593 CpG sites that exhibited heritable methylation patterns, among which 70.1% were SNPs directly present in methylated CpG dinucleotides. We determined the mQTL analysis identified 49.9% of heritable CpG sites for which regulation occurred in a distal cis-regulatory manner, and that ASM analysis was only able to identify 5%. Finally, we identified hundreds of clusters in the human genome for which the degree of variation of CpG methylation, as opposed to whether or not CpG sites were methylated, was associated with genetic polymorphisms, supporting a recent hypothesis on the genetic influence of phenotypic plasticity. These results show that cis-regulatory SNPs identified by mQTL do not comprise the full extent of heritable CpG methylation, and that ASM appears overall unreliable. Overall, the extent of genome-methylome interactions is well beyond what is detectible with the commonly used mQTL and ASM approaches, and is likely to include effects on plasticity.
    PLoS ONE 07/2014; 9(7):e99313. DOI:10.1371/journal.pone.0099313 · 3.23 Impact Factor
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