Epigenome-wide association study in the European Prospective Investigation into Cancer and Nutrition (EPIC-Turin) identifies novel genetic loci associated with smoking
TL;DR: The 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.
Abstract: 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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TL;DR: The results of this study confirm the broad effect of tobacco smoking on the human organism, but also show that quitting tobacco smoking presumably allows regaining the DNA methylation state of never smokers.
Abstract: Environmental factors such as tobacco smoking may have long-lasting effects on DNA methylation patterns, which might lead to changes in gene expression and in a broader context to the development or progression of various diseases. We conducted an epigenome-wide association study (EWAs) comparing current, former and never smokers from 1793 participants of the population-based KORA F4 panel, with replication in 479 participants from the KORA F3 panel, carried out by the 450K BeadChip with genomic DNA obtained from whole blood. We observed wide-spread differences in the degree of site-specific methylation (with p-values ranging from 9.31E-08 to 2.54E-182) as a function of tobacco smoking in each of the 22 autosomes, with the percent of variance explained by smoking ranging from 1.31 to 41.02. Depending on cessation time and pack-years, methylation levels in former smokers were found to be close to the ones seen in never smokers. In addition, methylation-specific protein binding patterns were observed for cg05575921 within AHRR, which had the highest level of detectable changes in DNA methylation associated with tobacco smoking (-24.40% methylation; p = 2.54E-182), suggesting a regulatory role for gene expression. The results of our study confirm the broad effect of tobacco smoking on the human organism, but also show that quitting tobacco smoking presumably allows regaining the DNA methylation state of never smokers.
679 citations
Cites background or result from "Epigenome-wide association study in..."
...Furthermore, AHRR was also found to be differentiallymethylated in the very recent study of Shenker et al. carried out in whole blood [17]....
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...who subsequently developed breast or colon cancer and matched controls [17]....
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...Replication of sites found within Shenker and co-workers, accompanied by additional findings for the corresponding regions, could also be achieved for the genes GNG12 (cg25189904), GFI1 (cg09935388), CNTNAP2 (cg25949550) and LRP5 (cg21611682) (please see Table S2 for additional sites found within these genes) [17]....
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...CpG sites in this region were also found to be differentially-methylated in pulmonary macrophages within the study of Monick et al. [12] and in whole blood within the study of Shenker [17]....
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...However, Shenker et al. analyzed the relationship between different blood cell fractions and whole blood DNA from the same individual by the 450K....
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United States Department of Health and Human Services1, Erasmus University Rotterdam2, University of California, Berkeley3, Johns Hopkins University4, Icahn School of Medicine at Mount Sinai5, University of Southern California6, Duke University7, University of Bristol8, University Medical Center Groningen9, University of California, San Francisco10, North Carolina State University11, Karolinska Institutet12, Pompeu Fabra University13, University of Paris14, University of Memphis15, Centre Hospitalier Universitaire de Grenoble16, University of Bergen17, Isfahan University of Medical Sciences18, Brigham and Women's Hospital19, Oslo University Hospital20, Utrecht University21, French Institute of Health and Medical Research22, Norwegian Institute of Public Health23, Johns Hopkins University School of Medicine24, Harvard University25, International Agency for Research on Cancer26, Paris Descartes University27, Michigan State University28, Centre national de la recherche scientifique29, Fred Hutchinson Cancer Research Center30, Swiss Tropical and Public Health Institute31, University of Basel32, Stockholm County Council33, University of Southampton34
TL;DR: This large scale meta-analysis of methylation data identified numerous loci involved in response to maternal smoking in pregnancy with persistence into later childhood and provide insights into mechanisms underlying effects of this important exposure.
Abstract: Epigenetic modifications, including DNA methylation, represent a potential mechanism for environmental impacts on human disease. Maternal smoking in pregnancy remains an important public health problem that impacts child health in a myriad of ways and has potential lifelong consequences. The mechanisms are largely unknown, but epigenetics most likely plays a role. We formed the Pregnancy And Childhood Epigenetics (PACE) consortium and meta-analyzed, across 13 cohorts (n = 6,685), the association between maternal smoking in pregnancy and newborn blood DNA methylation at over 450,000 CpG sites (CpGs) by using the Illumina 450K BeadChip. Over 6,000 CpGs were differentially methylated in relation to maternal smoking at genome-wide statistical significance (false discovery rate, 5%), including 2,965 CpGs corresponding to 2,017 genes not previously related to smoking and methylation in either newborns or adults. Several genes are relevant to diseases that can be caused by maternal smoking (e.g., orofacial clefts and asthma) or adult smoking (e.g., certain cancers). A number of differentially methylated CpGs were associated with gene expression. We observed enrichment in pathways and processes critical to development. In older children (5 cohorts, n = 3,187), 100% of CpGs gave at least nominal levels of significance, far more than expected by chance (p value < 2.2 × 10(-16)). Results were robust to different normalization methods used across studies and cell type adjustment. In this large scale meta-analysis of methylation data, we identified numerous loci involved in response to maternal smoking in pregnancy with persistence into later childhood and provide insights into mechanisms underlying effects of this important exposure.
646 citations
Cites result from "Epigenome-wide association study in..."
...64 3 10 (193)), which has been observed as differentially methylated in relation to smoking in many studies of adults and children.(7,8,10,28,35,53) Our enrichment testing of the genome-wide results is in line with previous findings showing that island shores, enhancers, and DNase I hypersensitive sites are more dynamic (susceptible tomethylation changes) than promoter regions(54) and imprinted loci....
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Harvard University1, Brigham and Women's Hospital2, National Institutes of Health3, Wake Forest University4, Icahn School of Medicine at Mount Sinai5, University of Edinburgh6, University of Queensland7, University of California, Los Angeles8, University of Exeter9, University of Alabama at Birmingham10, Boston University11, Erasmus University Rotterdam12, University of Minnesota13, University of Cambridge14, Universidad Autónoma Metropolitana15, International Agency for Research on Cancer16, University of Michigan17, University of Washington18, Emory University19, University of California, Berkeley20, German Cancer Research Center21, Imperial College London22, Northwestern University23, Mayo Clinic24, Max Planck Society25, Group Health Cooperative26
TL;DR: Cigarette smoking has a broad impact on genome-wide methylation that, at many loci, persists many years aftersmoking cessation, indicating a pattern of persistent altered methylation, with attenuation, after smoking cessation.
Abstract: Background —DNA methylation leaves a long-term signature of smoking exposure and is one potential mechanism by which tobacco exposure predisposes to adverse health outcomes, such as cancers, osteoporosis, lung, and cardiovascular disorders.
Methods and Results —To comprehensively determine the association between cigarette smoking and DNA methylation, we conducted a meta-analysis of genome-wide DNA methylation assessed using the Illumina BeadChip 450K array on 15,907 blood derived DNA samples from participants in 16 cohorts (including 2,433 current, 6,518 former, and 6,956 never smokers). Comparing current versus never smokers, 2,623 CpG sites (CpGs), annotated to 1,405 genes, were statistically significantly differentially methylated at Bonferroni threshold of p<1×10-7 (18,760 CpGs at False Discovery Rate (FDR)<0.05). Genes annotated to these CpGs were enriched for associations with several smoking-related traits in genome-wide studies including pulmonary function, cancers, inflammatory diseases and heart disease. Comparing former versus never smokers, 185 of the CpGs that differed between current and never smokers were significant p<1×10-7 (2,623 CpGs at FDR<0.05), indicating a pattern of persistent altered methylation, with attenuation, after smoking cessation. Transcriptomic integration identified effects on gene expression at many differentially methylated CpGs.
Conclusions —Cigarette smoking has a broad impact on genome-wide methylation that, at many loci, persists many years after smoking cessation. Many of the differentially methylated genes were novel genes with respect to biologic effects of smoking, and might represent therapeutic targets for prevention or treatment of tobacco-related diseases. Methylation at these sites could also serve as sensitive and stable biomarkers of lifetime exposure to tobacco smoke.
628 citations
TL;DR: Healthy human aging throughout the lifetime is focused on and the dynamics of DNA methylation is discussed, as well as how interactions between the genome, environment, and the epigenome influence aging rates are discussed.
Abstract: The process of aging results in a host of changes at the cellular and molecular levels, which include senescence, telomere shortening, and changes in gene expression. Epigenetic patterns also change over the lifespan, suggesting that epigenetic changes may constitute an important component of the aging process. The epigenetic mark that has been most highly studied is DNA methylation, the presence of methyl groups at CpG dinucleotides. These dinucleotides are often located near gene promoters and associate with gene expression levels. Early studies indicated that global levels of DNA methylation increase over the first few years of life and then decrease beginning in late adulthood. Recently, with the advent of microarray and next‐generation sequencing technologies, increases in variability of DNA methylation with age have been observed, and a number of site‐specific patterns have been identified. It has also been shown that certain CpG sites are highly associated with age, to the extent that prediction models using a small number of these sites can accurately predict the chronological age of the donor. Together, these observations point to the existence of two phenomena that both contribute to age‐related DNA methylation changes: epigenetic drift and the epigenetic clock. In this review, we focus on healthy human aging throughout the lifetime and discuss the dynamics of DNA methylation as well as how interactions between the genome, environment, and the epigenome influence aging rates. We also discuss the impact of determining ‘epigenetic age’ for human health and outline some important caveats to existing and future studies.
622 citations
Cites background from "Epigenome-wide association study in..."
...Interestingly, this negative correlation is not upheld when comparing expression and DNA methylation for a specific gene across individuals (van Eijk et al., 2012; Lam et al., 2012; Gutierrez Arcelus et al., 2013; Wagner et al., 2014)....
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...Conversely, DNA methylation in the gene body is often positively associated with levels of gene expression (Lister et al., 2009; Gutierrez Arcelus et al., 2013)....
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...…signature, cigarette smoke has been linked to changes in DNA methylation at the AHRR locus both in smokers and in children of smokers (Saxonov et al., 2006; Monick et al., 2012; Joubert et al., 2012; Shenker et al., 2013; Sun et al., 2013 Elliott et al., 2014; Lee et al., 2014; Shah et al., 2014)....
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...Clear relationships also exist between DNA methylation and genetic variability, and recent studies have identified genetic loci whose variation is strongly associated with DNA methylation at nearby sites (Fraser et al., 2012; Gamazon et al., 2013; Gutierrez Arcelus et al., 2013; Moen et al., 2013)....
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...Levels of DNA methylation at a promoter-associated CpG island are generally negatively associated with gene expression, although some specific genes show the opposite effect (Weber et al., 2007; Lam et al., 2012; Gutierrez Arcelus et al., 2013)....
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TL;DR: Blood DNA methylation patterns as biomarkers of smoking exposure provides a reservoir for constructing a smoking exposure index score which could be used to more precisely quantify long-term smoking exposure and evaluate the risks of smoking-induced diseases.
Abstract: Active smoking is a major preventable public health problem and an established critical factor for epigenetic modification. In this systematic review, we identified 17 studies addressing the association of active smoking exposure with methylation modifications in blood DNA, including 14 recent epigenome-wide association studies (EWASs) and 3 gene-specific methylation studies (GSMSs) on the gene regions identified by EWASs. Overall, 1460 smoking-associated CpG sites were identified in the EWASs, of which 62 sites were detected in multiple (≥3) studies. The three most frequently reported CpG sites (genes) in whole blood samples were cg05575921 (AHRR), cg03636183 (F2RL3), and cg19859270 (GPR15), followed by other loci within intergenic regions 2q37.1 and 6p21.33. These significant smoking-related genes were further assessed by specific methylation assays in three GSMSs and reflected not only current but also lifetime or long-term exposure to active smoking. In conclusion, this review summarizes the evidences for the use of blood DNA methylation patterns as biomarkers of smoking exposure for research and clinical practice. In particular, it provides a reservoir for constructing a smoking exposure index score which could be used to more precisely quantify long-term smoking exposure and evaluate the risks of smoking-induced diseases.
332 citations
Cites background or methods from "Epigenome-wide association study in..."
...studies carried out pyrosequencing [12, 14, 19], one used MALDI-TOF [5], and one used quantitative-PCR [17] as additional supplementary measurement in their validation phases (Table 1)....
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...The population sizes varied from 107 to 1793 in discovery panels, two studies only included males [13, 18], and four only included females [14, 17, 19, 21]....
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...Shenker, (2013) [14] UK 184 0 NA 190 71....
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References
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TL;DR: DNA methylation is mapped in the entire Arabidopsis thaliana genome at high resolution, indicating that genic transcription and DNA methylation are closely interwoven processes.
Abstract: Cytosine methylation, a common form of DNA modification that antagonizes transcription, is found at transposons and repeats in vertebrates, plants and fungi. Here we have mapped DNA methylation in the entire Arabidopsis thaliana genome at high resolution. DNA methylation covers transposons and is present within a large fraction of A. thaliana genes. Methylation within genes is conspicuously biased away from gene ends, suggesting a dependence on RNA polymerase transit. Genic methylation is strongly influenced by transcription: moderately transcribed genes are most likely to be methylated, whereas genes at either extreme are least likely. In turn, transcription is influenced by methylation: short methylated genes are poorly expressed, and loss of methylation in the body of a gene leads to enhanced transcription. Our results indicate that genic transcription and DNA methylation are closely interwoven processes.
1,321 citations
"Epigenome-wide association study in..." refers background in this paper
...vice versa.(6-8) According to this rationale, we predicted that decreased levels of methylation in...
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TL;DR: EPIC is a multi-centre prospective cohort study designed to investigate the relation between diet, nutritional and metabolic characteristics, various lifestyle factors and the risk of cancer in middle-aged men and women.
Abstract: Background The most consistent result of epidemiological studies on diet and cancer is that a diet rich in vegetables, fruit and, more generally, in plant foods is associated with a reduced risk of cancer at several anatomical sites. Epidemiological studies have been less consistent regarding the putative increase in risk related to consumption of fat or meat. In addition it has not been possible to identify clearly the biological role of specific nutrients or non-nutrient food components in the prevention or causation of cancer. Limitations in the precision and validity of traditional dietary intake measurements and limited use of biomarkers combined with narrow ranges of variations in dietary habits within single populations, have been the main reasons for the limited success in identifying more specific diet and cancer links. Methods EPIC is a multi-centre prospective cohort study designed to investigate the relation between diet, nutritional and metabolic characteristics, various lifestyle factors and the risk of cancer. The study is based in 22 collaborating centres in nine European countries and includes populations characterized by large variations in dietary habits and cancer risk. Data are collected on diet, physical activity, sexual maturation and reproductive history, lifetime consumption of alcohol and tobacco, previous and current illnesses and current medication. Following a common protocol and using identical equipment, blood samples are collected, aliquoted into plasma, serum, white blood cells and erythrocytes, and stored in liquid nitrogen at -196 degrees C for future laboratory analyses on cancer cases and matched healthy controls. Anthropometric measurements are taken according to a standard protocol. It is planned to include around 400,000 middle-aged men and women. Results and conclusions The collection of questionnaire data, anthropometric measurements and blood samples is under way. Almost 340,000 subjects had been included in the study by mid-1996, and recruitment is expected to be almost complete by 1997. Follow-up for cancer incidence and total mortality has started and it is expected that about 23000 cancer cases will be identified during the first 10 years of follow-up.
841 citations
"Epigenome-wide association study in..." refers methods in this paper
...Study participants were drawn from the Italian component of the European Prospective Investigation into Cancer and Nutrition (EPIC-Turin) cohort, a large general population cohort consisting of 520 000 individuals with standardized lifestyle and personal history questionnaires, anthropometric data and blood samples collected for DNA extraction (24,25)....
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TL;DR: This report summarises the epidemiological evidence on the association between tobacco smoking and cancer, which was reviewed by an international group of scientists convened by IARC, and shows a consistent and statistically significant association between exposure to environmental tobacco smoke and lung cancer risk.
655 citations
"Epigenome-wide association study in..." refers background in this paper
...There was no strong association (P , 1 × 1025) between methylation levels at any of these loci and disease status, despite smoking being a weak risk factor for colon cancer (Supplementary Material, Table S2) (2,3)....
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...There was no strong association (P , 1 × 10(25)) between methylation levels at any of these loci and disease status, despite smoking being a weak risk factor for colon cancer (Supplementary Material, Table S2) (2,3)....
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...A secondary analysis was performed that also adjusted for the case–control status, which did not significantly alter the results (Supplementary Material, Table S2)....
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TL;DR: The results of the present study provide direct evidence that AhR is involved in carcinogenesis, a transcriptional regulator of the gene for CYP1A1, a widely distributed environmental carcinogen.
Abstract: The contribution of the aryl hydrocarbon receptor (AhR) in induction of a battery of xenobiotic-metabolizing enzymes has been studied extensively. However, no direct proof has been obtained that it plays a role in modulating carcinogenesis. To address the question of whether AhR is required for tumor induction, we have investigated the response of AhR-deficient mice to benzo[a]pyrene (B[a]P), a widely distributed environmental carcinogen. B[a]P treatment induced expression of the cytochrome P450 gene Cyp1a1 in the skin and liver of AhR-positive mice bearing +/+ and +/− genotypes and did not induce expression of the cytochrome P450 gene Cyp1a1 in AhR-null mice in either skin or liver. In contrast, Cyp1a2 gene expression was positive in liver irrespective of the presence or absence of the AhR gene, or B[a]P treatment, although its inducibility was lost in the AhR(−/−) mouse. All AhR-positive male mice of both +/+ and +/− genotypes that received subcutaneous injection of B[a]P (2 mg) on the first and the eighth days had developed subcutaneous tumors at the site of injection at the end of the 18-week experiment. In contrast, no tumors were apparent in any of the AhR-deficient mice. Likewise, topical application of B[a]P (200 μg) at weekly intervals to the skin of female mice for 25 weeks produced skin tumors only in the AhR-positive mice. Thus the carcinogenic action of B[a]P may be determined primarily by AhR, a transcriptional regulator of the gene for CYP1A1. The results of the present study provide direct evidence that AhR is involved in carcinogenesis.
630 citations
"Epigenome-wide association study in..." refers background in this paper
...These compounds are metabolized by AhR (10,11), releasing further carcinogenic metabolites that have been implicated in lung cancer development (12)....
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TL;DR: The results, which were based on a rigorous replication approach, show that the gene coding for a potential drug target of cardiovascular importance features altered methylation patterns in smokers.
Abstract: Tobacco smoking is responsible for substantial morbidity and mortality worldwide, in particular through cardiovascular, pulmonary, and malignant pathology. CpG methylation might plausibly play a role in a variety of smoking-related phenomena, as suggested by candidate gene promoter or global methylation studies. Arrays allowing hypothesis-free searches on a scale resembling genome-wide studies of SNPs have become available only very recently. Methylation extents in peripheral-blood DNA were assessed at 27,578 sites in more than 14,000 gene promoter regions in 177 current smokers, former smokers, and those who had never smoked, with the use of the Illumina HumanMethylation 27K BeadChip. This revealed a single locus, cg03636183, located in F2RL3, with genome-wide significance for lower methylation in smokers (p = 2.68 × 10−31). This was similarly significant in 316 independent replication samples analyzed by mass spectrometry and Sequenom EpiTyper (p = 6.33 × 10−34). Our results, which were based on a rigorous replication approach, show that the gene coding for a potential drug target of cardiovascular importance features altered methylation patterns in smokers. To date, this gene had not attracted attention in the literature on smoking.
605 citations
"Epigenome-wide association study in..." refers background or methods in this paper
...methylated in current smokers (n = 65) compared to former (n = 56) and non-smokers (n = 56).(1)...
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...and have identified further AHRR probes that were significantly associated with smoking.(1, 5)...
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...compared to former and non-smokers.(1) For the present analysis, we used multivariate linear...
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