Rationale and objectives Self-reported smoking underestimates disease risk. Smoking affects DNA methylation, in particular the cg05575921 site in the aryl hydrocarbon receptor repressor (AHRR ) gene. We tested the hypothesis that AHRR cg05575921 hypomethylation is associated with risk of smoking-related morbidity and mortality. Methods From the Copenhagen City Heart Study representing the Danish general population, we studied 9234 individuals. Using bisulphite treated leucocyte DNA, AHRR (cg05575921) methylation was measured. Rs1051730 ( CHRN3A ) genotype was used to evaluate smoking heaviness. Participants were followed for up to 22 years for exacerbations of COPD, event of lung cancer and all-cause mortality. Six-year lung cancer risk was calculated according to the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial (PLCO M2012 ). Measurements and main results AHRR (cg05575921) hypomethylation was associated with former and current smoking status, high daily and cumulative smoking, short time since smoking cessation (all p values –31 ), and the smoking-related CHRN3A genotype (−0.48% per T-allele, p=0.002). The multifactorially adjusted HRs for the lowest versus highest methylation quintiles were 4.58 (95% CI 2.83 to 7.42) for COPD exacerbations, 4.87 (2.31 to 10.3) for lung cancer and 1.67 (1.48 to 1.88) for all-cause mortality. Finally, among 2576 high-risk smokers eligible for lung cancer screening by CT, observed cumulative incidences of lung cancer after 6 years for individuals in the lowest and highest methylation quintiles were 3.7% and 0.0% (p=2×10 –7 ), whereas predicted PLCO M2012 6-year risks were similar (4.3% and 4.4%, p=0.77). Conclusion AHRR (cg05575921) hypomethylation, a marker of smoking behaviour, provides potentially clinical relevant predictions of future smoking-related morbidity and mortality.

https://thorax.bmj.com/content/thoraxjnl/72/7/646.full.pdf

AHRR (cg05575921) hypomethylation marks smoking behaviour, morbidity and mortality

The evidence for epigenome-wide associations between smoking and DNA methylation continues to grow through cross-sectional studies. However, few large-scale investigations have explored the associations using observations for individuals at multiple time-points. Here, through the use of the Illumina 450K BeadChip and data collected at two time-points separated by approximately 7 years, we investigate changes in methylation over time associated with quitting smoking or remaining a former smoker, and those associated with continued smoking. Our results indicate that after quitting smoking the most rapid reversion of altered methylation occurs within the first two decades, with reversion rates related to the initial differences in methylation. For 52 CpG sites, the change in methylation from baseline to follow-up is significantly different for former smokers relative to the change for never smokers (lowest p-value 3.61 x 10-39 for cg26703534, gene AHRR). Most of these sites’ respective regions have been previously implicated in smoking-associated diseases. Despite the early rapid change, dynamism of methylation appears greater in former smokers vs never smokers even four decades after cessation. Furthermore, our study reveals the heterogeneous effect of continued smoking: the methylation levels of some loci further diverge between smokers and non-smokers, while others re-approach. Though intensity of smoking habit appears more significant than duration, results remain inconclusive. This study improves the understanding of the dynamic link between cigarette smoking and methylation, revealing the continued fluctuation of methylation levels decades after smoking cessation and demonstrating that continuing smoking can have an array of effects. The results can facilitate insights into the molecular mechanisms behind smoking-induced disturbed methylation, improving the possibility for development of biomarkers of past smoking behavior and increasing the understanding of the molecular path from exposure to disease.

/pdf/the-dynamics-of-smoking-related-disturbed-methylation-a-two-1gbr4nuoi2.pdf

The dynamics of smoking-related disturbed methylation: a two time-point study of methylation change in smokers, non-smokers and former smokers.

Epigenetic signatures of starting and stopping smoking.

The aim of this study is to provide a systematic review of the known epigenetic alterations caused by cigarette smoke; establish an evidence-based perspective of their clinical value for screening, diagnosis, and treatment of smoke-related disorders; and discuss the challenges and ethical concerns associated with epigenetic studies. A well-defined, reproducible search strategy was employed to identify relevant literature (clinical, cellular, and animal-based) between 2000 and 2019 based on AMSTAR guidelines. A total of 80 studies were identified that reported alterations in DNA methylation, histone modifications, and miRNA expression following exposure to cigarette smoke. Changes in DNA methylation were most extensively documented for genes including AHRR, F2RL3, DAPK, and p16 after exposure to cigarette smoke. Likewise, miR16, miR21, miR146, and miR222 were identified to be differentially expressed in smokers and exhibit potential as biomarkers for determining susceptibility to COPD. We also identified 22 studies highlighting the transgenerational effects of maternal and paternal smoking on offspring. This systematic review lists the epigenetic events/alterations known to occur in response to cigarette smoke exposure and identifies the major genes and miRNAs that are potential targets for translational research in associated pathologies. Importantly, the limitations and ethical concerns related to epigenetic studies are also highlighted, as are the effects on the ability to address specific questions associated with exposure to tobacco/cigarette smoke. In the future, improved interpretation of epigenetic signatures will lead to their increased use as biomarkers and/or in drug development.

A systematic review of smoking-related epigenetic alterations.

Large-scale epigenome-wide association meta-analyses have identified multiple ‘signatures’’ of smoking. Drawing on these findings, we describe the construction of a polyepigenetic DNA methylation score that indexes smoking behavior and that can be utilized for multiple purposes in population health research. To validate the score, we use data from two birth cohort studies: The Dunedin Longitudinal Study, followed to age-38 years, and the Environmental Risk Study, followed to age-18 years. Longitudinal data show that changes in DNA methylation accumulate with increased exposure to tobacco smoking and attenuate with quitting. Data from twins discordant for smoking behavior show that smoking influences DNA methylation independently of genetic and environmental risk factors. Physiological data show that changes in DNA methylation track smoking-related changes in lung function and gum health over time. Moreover, DNA methylation changes predict corresponding changes in gene expression in pathways related to inflammation, immune response, and cellular trafficking. Finally, we present prospective data about the link between adverse childhood experiences (ACEs) and epigenetic modifications; these findings document the importance of controlling for smoking-related DNA methylation changes when studying biological embedding of stress in life-course research. We introduce the polyepigenetic DNA methylation score as a tool both for discovery and theory-guided research in epigenetic epidemiology.

/pdf/establishing-a-generalized-polyepigenetic-biomarker-for-1pbi2ufxmi.pdf

Establishing a generalized polyepigenetic biomarker for tobacco smoking.

Smoking is the largest preventable cause of morbidity and mortality in the world. Although there are effective pharmacologic and behavioral treatments for smoking cessation, our inability to objectively quantify smokers’ progress in decreasing smoking has been a barrier to both clinical and research efforts. In prior work, we and others have shown that DNA methylation at cg05575921, a CpG residue in the aryl hydrocarbon receptor repressor (AHRR), can be used to determine smoking status and infer cigarette consumption history. In this study, we serially assessed self-report and existing objective markers of cigarette consumption in 35 subjects undergoing smoking cessation therapy, then quantified DNA methylation at cg05575921 at study entry and three subsequent time points. Five subjects who reported serum cotinine and exhaled carbon monoxide verified smoking abstinence for the three months prior to study exit averaged a 5.9% increase in DNA methylation at cg05575921 (p<0.004) over the six month study. Although the other 30 subjects did not achieve smoking cessation at the six-month time point, their self-reported reduction of cigarette consumption (mean = 6 cigarettes per day) was associated with a 2.8% increase DNA methylation at cg05575921 (p<0.05). Finally, a survey of subjects as they exited the study demonstrated strong support for the clinical use of epigenetic biomarkers. We conclude that AHRR methylation status is a quantifiable biomarker for progress in smoking cessation that could have substantial impact on both smoking cessation treatment and research.

/pdf/reversion-of-ahrr-demethylation-is-a-quantitative-biomarker-cuv7u9am0z.pdf

Reversion of AHRR Demethylation Is a Quantitative Biomarker of Smoking Cessation

Objectives: Early identification of smoking, essential for the successful implementation of interventions, arrests the escalation of smoking and smoking-associated risk behaviors in adoles...

Kyra Vercande

Papers

Reversion of AHRR Demethylation Is a Quantitative Biomarker of Smoking Cessation

Saliva DNA Methylation Detects Nascent Smoking in Adolescents