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Background: Methylation quantitative trait loci (mQTLs) are the genetic variants that may affect the DNA methylation patterns of CpG sites. However, their roles in influencing the disturbances of smoking-related epigenetic changes have not been well established. This study was conducted to address whether mQTLs exist in the vicinity of smoking-related CpG sites (± 50 kb) and to examine their associations with smoking exposure and all-cause mortality in older adults., Results: We obtained DNA methylation profiles in whole blood samples by Illumina Infinium Human Methylation 450 BeadChip array of two independent subsamples of the ESTHER study (discovery set, n  = 581; validation set, n  = 368) and their corresponding genotyping data using the Illumina Infinium OncoArray BeadChip. After correction for multiple testing (FDR), we successfully identified that 70 out of 151 previously reported smoking-related CpG sites were significantly associated with 192 SNPs within the 50 kb search window of each locus. The 192 mQTLs significantly influenced the active smoking-related DNA methylation changes, with percentage changes ranging from 0.01 to 18.96%, especially for the weakly/moderately smoking-related CpG sites. However, these identified mQTLs were not directly associated with active smoking exposure or all-cause mortality., Conclusions: Our findings clearly demonstrated that if not dealt with properly, the mQTLs might impair the power of epigenetic-based models of smoking exposure to a certain extent. In addition, such genetic variants could be the key factor to distinguish between the heritable and smoking-induced impact on epigenome disparities. These mQTLs are of special importance when DNA methylation markers measured by Illumina Infinium assay are used for any comparative population studies related to smoking-related cancers and chronic diseases.

Tobacco smoking and oxidative stress (OS) are both related to a wide spectrum of adverse age-related health outcomes, but their association is not yet well-established. We examined the associations of self-reported smoking indicators, serum cotinine levels and smoking-related DNA methylation biomarkers with two urinary proxy markers of OS, 8-isoprostane (8-iso) and 8-hydroxy-2'-deoxyguanosine (8-oxodG), in two independent subsets of older adults recruited in Germany (discovery set: n = 978, validation set: n = 531). We obtained DNA methylation profiles in whole blood samples by Illumina Human Methylation450K Beadchip and measured the urinary levels of both OS markers using commercial ELISA kits. After controlling for potential confounders, current smoking, cumulative smoking exposure (pack-years) and serum cotinine levels (ng/ml) were strongly associated with 8-iso levels (p values <0.0001, 0.004 and 0.001, respectively). Of 151 previously identified smoking-related CpG sites, 71 loci were associated with 8-iso levels after correction for multiple testing (FDR < 0.05) in the validation phase and were designated as loci related to 8-iso levels defined OS. In addition, serum cotinine levels, cumulative smoking exposure and a smoking index (SI) based on the 71 identified loci manifested monotonic associations with 8-iso levels. However, we did not observe any associations between these smoking indicators and 8-oxodG levels. In conclusion, this study suggests that smoking-related epigenetic alterations are closely correlated with smoking-induced OS. The identified CpG sites could potentially be prognostic epigenetic markers of OS and OS-related health outcomes. Our findings and the underlying mechanisms should be followed up in further, preferably longitudinal studies.

DNA methylation (DNAm) has been revealed to play a role in various diseases. Here we performed epigenome-wide screening and validation to identify mortality-related DNAm signatures in a general population-based cohort with up to 14 years follow-up. In the discovery panel in a case-cohort approach, 11,063 CpGs reach genome-wide significance (FDR<0.05). 58 CpGs, mapping to 38 well-known disease-related genes and 14 intergenic regions, are confirmed in a validation panel. A mortality risk score based on ten selected CpGs exhibits strong association with all-cause mortality, showing hazard ratios (95% CI) of 2.16 (1.10-4.24), 3.42 (1.81-6.46) and 7.36 (3.69-14.68), respectively, for participants with scores of 1, 2-5 and 5+ compared with a score of 0. These associations are confirmed in an independent cohort and are independent from the 'epigenetic clock'. In conclusion, DNAm of multiple disease-related genes are strongly linked to mortality outcomes. The DNAm-based risk score might be informative for risk assessment and stratification.

Tobacco smoking is a preventable environmental factor that contributes to a wide spectrum of age-related health outcomes; however, its association with the development of frailty is not yet well established. We examined the associations of self-reported smoking indicators, serum cotinine levels and smoking-related DNA methylation biomarkers with a quantitative frailty index (FI) in 2 independent subsets of older adults (age 50-75) recruited in Saarland, Germany in 2000 - 2002 (discovery set: n = 978, validation set: n = 531). We obtained DNA methylation profiles in whole blood samples by Illumina HumanMethylation450 BeadChip and calculated the FI according to the method of Mitnitski and Rockwood. Mixed linear regression models were implemented to assess the associations between smoking indicators and the FI. After controlling for potential covariates, current smoking, cumulative smoking exposure (pack-years), and time after smoking cessation (years) were significantly associated with the FI (P-value < 0.05). In the discovery panel, 17 out of 151 previously identified smoking-related CpG sites were associated with the FI after correction for multiple testing (FDR < 0.05). Nine of them survived in the validation phase and were designated as frailty-associated loci. A smoking index (SI) based on the 9 loci manifested a monotonic association with the FI. In conclusion, this study suggested that epigenetic alterations could play a role in smoking-associated development of frailty. The identified CpG sites have the potential to be prognostic biomarkers of frailty and frailty-related health outcomes. Our findings and the underlying mechanisms should be followed up in further, preferably longitudinal studies.

Aim: Whole-blood DNA methylation depends on the underlying leukocyte composition and confounding hereby is a major concern in epigenome-wide association studies. Cell counts are often missing or may not be feasible. Computational approaches estimate leukocyte composition from DNA methylation based on reference datasets of purified leukocytes. We explored the possibility to train such a model on whole-blood DNA methylation and cell counts without the need for purification., Materials & Methods: Using whole-blood DNA methylation and corresponding five-part cell counts from 2445 participants from the London Life Sciences Prospective Population Study, a model was trained on a subset of 175 subjects and evaluated on the remaining., Results: Correlations between cell counts and estimated cell proportions were high (neutrophils 0.85, eosinophils 0.88, basophils 0.02, lymphocytes 0.84, monocytes 0.55) and estimated proportions explained more variance in whole-blood DNA methylation levels than counts., Conclusion: Our model provided precise estimates for the common cell types.

Epigenome-wide association studies have established methylation patterns related to smoking, the major risk factor of lung cancer (LC), which are distinct from methylation profiles disclosed in LC patients. This study simultaneously investigated associations of smoking-associated and LC-related methylation markers with LC mortality. DNA methylation was determined by HM450K assay in baseline blood samples of 1,565 older adults in a population-based case-cohort study. The associations of 151 smoking-associated CpGs (smoCpGs) and 3,806 LC-related CpGs (caCpGs) with LC mortality were assessed by weighted Cox regression models, controlling for potential confounders. Multi-loci methylation scores were separately constructed based on smoCpGs and caCpGs. During a median follow-up of 13.8 years, 60 participants who had a first diagnosis of LC died from LC. The average time between sample collection and LC diagnosis was 5.8 years. Hypomethylation at 77 smoCpGs and 121 caCpGs, and hypermethylation at 4 smoCpGs and 66 caCpGs were associated with LC mortality. The associations were much stronger for smoCpGs than for caCpGs. Hazard ratios (95% CI) were 7.82 (2.91-21.00) and 2.27 (0.75-6.85), respectively, for participants in highest quartile of Score I (based on 81 smoCpGs) and Score II (based on 187 caCpGs), compared with participants in the corresponding lower three quartiles. Score I outperformed Score II, with an optimism-corrected C-index of 0.87 vs. 0.77. In conclusion, although methylation changes of both smoking-associated and LC-related genes are associated with LC mortality, only smoking-associated methylation markers predict LC mortality with high accuracy, and may thus serve as promising candidates to identify high risk populations for LC screening., (© 2016 UICC.)

Telomere length (TL) is associated with an increased risk of aging-related diseases. As a preventable environmental hazard of morbidity and mortality, smoking has been reported to promote TL attrition by producing a variety of oxidants and free radicals. Since DNA methylation has been demonstrated to play an important role in the pathways of smoking and smoking-induced diseases, this study aimed to address whether the smoking-associated DNA methylation changes could be associated with accelerated TL shortening. We obtained DNA methylation profiles in whole blood samples by Illumina Infinium Human Methylation 450 Beadchip array in two independent subsamples of the ESTHER study and measured their relative TL by quantitative PCR. Terminal Restriction Fragment analysis was additionally performed in a subsample to obtain absolute TL in base pairs. TL measurements across panels were standardized by z-transformation. After correction for multiple testing, we successfully confirmed that seven out of 151 smoking-related CpG sites were associated with TL (FDR <0.05). A smoking index based on the seven loci showed monotonic associations with TL, cumulative smoking exposure and time after smoking cessation. In conclusion, our study supports suggestions that epigenetic alterations could play a role in smoking-associated disproportionate aging as reflected by TL. Further research is required to examine whether the identified epigenetic signatures of smoking can be of value in clinical practice to assess individual aging across the lifespan.

Recent studies have identified biomarkers of chronological age based on DNA methylation levels. Since active smoking contributes to a wide spectrum of aging-related diseases in adults, this study intended to examine whether active smoking exposure could accelerate the DNA methylation age in forms of age acceleration (AA, residuals of the DNA methylation age estimate regressed on chronological age). We obtained the DNA methylation profiles in whole blood samples by Illumina Infinium Human Methylation450 Beadchip array in two independent subsamples of the ESTHER study and calculated their DNA methylation ages by two recently proposed algorithms. None of the self-reported smoking indicators (smoking status, cumulative exposure and smoking cessation time) or serum cotinine levels was significantly associated with AA. On the contrary, we successfully confirmed that 66 out of 150 smoking-related CpG sites were associated with AA, even after correction for multiple testing (FDR <0.05). We further built a smoking index (SI) based on these loci and demonstrated a monotonic dose-response relationship of this index with AA. In conclusion, DNA methylation-based biological indicators for current and past smoking exposure, but not self-reported smoking information or serum cotinine levels, were found to be related to DNA methylation defined AA. Further research should address potential mechanisms underlying the observed patterns, such as potential reflections of susceptibility to environmental hazards in both smoking related methylation changes and methylation defined AA., Competing Interests: The authors declare that they have no competing interests.

Background: Smoking is a major cause of morbidity and mortality. Smoking-related epigenetic biomarkers may open new avenues to better quantify the adverse health effects of smoking, and to better understanding of the underlying mechanisms. We aimed to evaluate the clinical implications of F2RL3 methylation, a novel epigenetic biomarker of smoking exposure disclosed by recent genome-wide methylation studies., Methods: Blood DNA methylation at F2RL3 (also known as PAR-4) was quantified in baseline samples of 3588 participants aged 50-75 years in a large population-based prospective cohort study by MALDI-TOF mass spectrometry. Deaths were recorded during a median follow-up of 10.1 years. The associations of methylation intensity and of smoking with all-cause, cardiovascular, cancer and other mortality were assessed by Cox's proportional hazards regression, controlling for potential confounding factors., Results: Lower methylation intensity at F2RL3 was strongly associated with mortality. After adjustment for multiple covariates including smoking, hazard ratios [95% confidence interval (CI)] for death from any cause, cardiovascular disease, cancer or other causes were 2.60 (95% CI, 1.81-3.74), 2.45 (95% CI, 1.28-4.68), 2.94 (95% CI, 1.68-5.14) and 2.39 (95% CI, 1.11-5.16), respectively, in subjects in the lowest quartile of methylation intensity compared with subjects in the highest quartile. The associations with mortality outcomes were much stronger among men than among women. In addition, strong positive associations of smoking with each of the outcomes were substantially weakened, and almost disappeared when controlling for F2RL3 methylation intensity., Conclusions: F2RL3 methylation is a strong predictor of mortality, including all-cause, cardiovascular, cancer and other mortality. Systemic adverse effects of smoking may be mediated by pathways associated with F2RL3 methylation., (© The Author 2014; all rights reserved. Published by Oxford University Press on behalf of the International Epidemiological Association.)

Background: Recent genome-wide DNA methylation studies have found a pronounced difference in methylation of the F2RL3 gene (also known as PAR-4) in blood DNA according to smoking exposure. Knowledge on the variation of F2RL3 methylation by various degrees of smoking exposure is still very sparse., Objectives: We aimed to assess dose-response relationships of current and lifetime active smoking exposure with F2RL3 methylation., Methods: In a large population-based study, we quantified blood DNA methylation at F2RL3 for 3,588 participants using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Associations of smoking exposure with methylation intensity were examined by multiple linear regression, controlling for potential confounding factors and paying particular attention to dose-response patterns with respect to current and lifetime smoking exposure as well as time since cessation of smoking., Results: F2RL3 methylation intensity showed a strong association with smoking status (p < 0.0001), which persisted after controlling for potential confounding factors. Clear inverse dose-response relationships with F2RL3 methylation intensity were seen for both current intensity and lifetime pack-years of smoking. Among former smokers, F2RL3 methylation intensity increased gradually from levels close to those of current smokers for recent quitters to levels close to never smokers for long-term (> 20 years) quitters., Conclusions: F2RL3 methylation is a promising biomarker for both current and long-term past tobacco exposure, and its predictive value for smoking-related diseases warrants further exploration.

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., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)