BackgroundRegular smoking is associated with a wide variety of syndromes with prominent inflammatory components such as cancer, obesity and type 2 diabetes. Heavy regular smoking is also associated with changes in the DNA methylation of peripheral mononuclear cells. However, in younger smokers, inflammatory epigenetic findings are largely absent which suggests the inflammatory response(s) to smoking may be dose dependent. To help understand whether peripheral mononuclear cells have a role in mediating these responses in older smokers with higher cumulative smoke exposure, we examined genome-wide DNA methylation in a group of well characterized adult African American subjects informative for smoking, as well as serum C-reactive protein (CRP) and interleukin-6 receptor (IL6R) levels. In addition, complementary bioinformatic analyses were conducted to delineate possible pathways affected by long-term smoking.ResultsGenome-wide DNA methylation analysis with respect to smoking status yielded 910 significant loci after Benjamini-Hochberg correction. In particular, two loci from the AHRR gene (cg05575921 and cg23576855) and one locus from the GPR15 gene (cg19859270) were identified as highly significantly differentially methylated between smokers and non-smokers. The bioinformatic analyses showed that long-term chronic smoking is associated with altered promoter DNA methylation of genes coding for proteins mapping to critical sub-networks moderating inflammation, immune function, and coagulation.ConclusionsWe conclude that chronic regular smoking is associated with changes in peripheral mononuclear cell methylation signature which perturb inflammatory and immune function pathways and may contribute to increased vulnerability for complex illnesses with inflammatory components.

Many existing DNA repositories do not have robust characterizations of smoking, while for many currently ongoing studies, the advent of vaping has rendered traditional cotinine‐based methods of determining smoking status unreliable. Previously, we have shown that methylation status at cg05575921 in whole blood DNA can reliably predict cigarette consumption. However, whether methylation status in saliva can be used similarly has yet to be established. Herein, we use DNA from 418 biochemically confirmed smokers or nonsmokers to compare and contrast the utility of cg05575921 in classifying and quantifying cigarette smoking. Using whole blood DNA, a model incorporating age, gender, and methylation status had a receiver operating characteristic (ROC) area under the curve (AUC) for predicting smoking status of 0.995 with a nonlinear demethylation response to smoking. Using saliva DNA, the ROC AUC for predicting smoking was 0.971 with the plot of the relationship of DNA methylation to daily cigarette consumption being very similar to that seen for whole blood DNA. The addition of information from another methylation marker designed to correct for cellular heterogeneity improved the AUC for saliva DNA to 0.981. Finally, in 31 subjects who reported quitting smoking 10 or more years previously, cg05575921 methylation was nonsignificantly different from controls. We conclude that DNA methylation status at cg05575921 in DNA from whole blood or saliva predicts smoking status and daily cigarette consumption. We suggest these epigenetic assessments for objectively ascertaining smoking status will find utility in research, clinical, and civil applications.

Substance abuse has an enormous impact on economic and quality of life measures throughout the world. In more developed countries, overutilization of the most common forms of substances of abuse, alcohol and tobacco, is addressed primarily through prevention of substance use initiation and secondarily through the treatment of those with substance abuse or dependence. In general, these therapeutic approaches to substance abuse are deemed effective. However, there is a broad consensus that the development of additional tools to aid diagnosis, prioritize treatment selection and monitor treatment response could have substantial impact on the effectiveness of both substance use prevention and treatment. The recent demonstrations by a number of groups that substance use exposure is associated with robust changes in DNA methylation signatures of peripheral blood cells suggests the possibility that methylation assessments of blood or saliva could find broad clinical applications. In this article, we review recent progress in epigenetic approaches to substance use assessment with a particular emphasis on smoking (and alcohol) related applications. In addition, we highlight areas, such as the epigenetics of psychostimulant, opioid and cannabis abuse, which are markedly understudied and could benefit from intensified collaborative efforts to define epigenetic biomarkers of abuse and dependence.

Objectives To examine the effect of the relationship between alcohol and cigarette consumption on biological aging using deoxyribonucleic acid (DNA) methylation-based indices. Design We examined the association between DNA methylation indices of smoking and alcohol to those for biological aging in two independent cohorts using the epigenetic “clock” provided by Hannum and colleagues. Setting Longitudinal studies of aging and the effect of psychosocial stress. Participants Publicly available genome-wide methylation data from participants in two ethnically informative cohorts (n=656 white, n=180 black). Measurements Deviation of biological age from chronological age as a result of smoking and alcohol consumption. Results Greater cigarette consumption was associated with accelerated biological aging, with strong effects evident at even low levels of exposure. In contrast, alcohol consumption was associated with a mixed effect on biological aging and pronounced nonlinear effects. At low and heavy levels of alcohol consumption, there was accelerated biological aging, whereas at intermediate levels of consumption there was a relative decelerating effect. The decelerating effects of alcohol were particularly notable at loci for which methylation increased with age. Conclusion These data support prior epidemiological studies indicating that moderate alcohol use is associated with healthy aging, but we urge caution in interpreting these results. Conversely, smoking has strong negative effects at all levels of consumption. These results also support the use of methylomic indices as a tool for assessing the impact of lifestyle on aging.

An improved method for detecting coronary heart disease (CHD) could have substantial clinical impact. Building on the idea that systemic effects of CHD risk factors are a conglomeration of genetic and environmental factors, we use machine learning techniques and integrate genetic, epigenetic and phenotype data from the Framingham Heart Study to build and test a Random Forest classification model for symptomatic CHD. Our classifier was trained on n = 1,545 individuals and consisted of four DNA methylation sites, two SNPs, age and gender. The methylation sites and SNPs were selected during the training phase. The final trained model was then tested on n = 142 individuals. The test data comprised of individuals removed based on relatedness to those in the training dataset. This integrated classifier was capable of classifying symptomatic CHD status of those in the test set with an accuracy, sensitivity and specificity of 78%, 0.75 and 0.80, respectively. In contrast, a model using only conventional CHD risk factors as predictors had an accuracy and sensitivity of only 65% and 0.42, respectively, but with a specificity of 0.89 in the test set. Regression analyses of the methylation signatures illustrate our ability to map these signatures to known risk factors in CHD pathogenesis. These results demonstrate the capability of an integrated approach to effectively model symptomatic CHD status. These results also suggest that future studies of biomaterial collected from longitudinally informative cohorts that are specifically characterized for cardiac disease at follow-up could lead to the introduction of sensitive, readily employable integrated genetic-epigenetic algorithms for predicting onset of future symptomatic CHD.

The current investigation was designed to examine the association of parenting during late childhood and early adolescence, a time of rapid physical development, with biological propensity for inflammation. Based on life course theory, it was hypothesized that parenting during this period of rapid growth and development would be associated with biological outcomes and self-reported health assessed in young adulthood. It was expected that association of parenting with health would be mediated either by effects on methylation of a key inflammatory factor, Tumor necrosis factor (TNF), or else by association with a pro-inflammatory shift in the distribution of mononuclear blood cells. Supporting expectations, in a sample of 398 African American youth residing in rural Georgia, followed from age 11 to age 19, parenting at ages 11–13 was associated with youth reports of better health at age 19. We found that parenting was associated with changes in TNF methylation as well as with changes in cell-type composition. However, whereas methylation of TNF was a significant mediator of the association of parenting with young adult health, variation in mononuclear white blood cell types was not a significant mediator of the association of parenting with young adult health. The current research suggests the potential value of examining the health-related effects of parenting in late childhood and early adolescence. Further examination of protection against pro-inflammatory tendencies conferred by parenting appears warranted.

Background.—Heavy alcohol consumption (HAC) is a shared concern of the forensic, medical and insurance underwriting communities. Unfortunately, there is a relative lack of clinically employable tools for detecting HAC and monitoring treatment response. Building on the results of 3 genome wide methylation studies, we have previously shown in a small group of samples that methylation sensitive digital PCR assays (MSdPCR) have the potential to accurately classify individuals with respect to HAC in a small set of individuals. Objective.—We now expand on those earlier findings using data and biomaterials from 143 participants with current HAC and 200 abstinent controls. Results.—We show that a set of 4 digital PCR assays that have a receiver operating characteristic (ROC) area under the curve (AUC) of 0.96 for detecting those with HAC. After a mean of 21 days of inpatient enforced abstinence, methylation status at one of these markers, cg04987734, began to revert to baseline values. Re-examination of methylation data from our smaller 2014 study with respect to this locus demonstrated a similarly significant reversion pattern at cg04987734 in association with treatment enforced abstinence. Conclusions.—We conclude that clinically implementable dPCR tools can sensitively detect the presence of HAC and that they show promise for monitoring alcohol treatment results. These dPCR tools could be useful to clinicians and researchers in monitoring those enrolled in substance use disorder treatment, employee wellness programs and insurance underwriting.

Smoking is associated with poorer health outcomes for both African and European Americans. In order to better understand whether ethnic-specific genetic variation may underlie some of these differences, we compared the smoking-associated genome-wide methylation signatures of African Americans with those of European Americans, and followed up this analysis with a focused examination of the most ethnically divergent locus, cg19859270, at the GPR15 gene. We examined the association of methylation at this locus to the rs2230344 SNP and GPR15 gene and protein expression. Consistent with prior analyses, AHRR residue cg05575921 was the most differentially methylated residue in both African Americans and European Americans. However, the second most differentially methylated locus in African Americans, cg19859270, was only modestly differentially methylated in European Americans. Interrogation of the methylation status of this CpG residue found in GPR15, a chemokine receptor involved in HIV pathogenesis, showed a significant interaction of ethnicity with smoking as well as a marginal effect of genotype at rs2230344, a neighboring non-synonymous SNP, but only among African Americans. Gene and protein expression analyses showed that demethylation at cg19859270 was associated with an increase in both mRNA and protein levels. Since GPR15 is involved in the early stages of viral replication for some HIV-1 and HIV-2 isolates, and the prevalence of HIV is increased in African Americans and smokers, these data support a possible role for GPR15 in the ethnically dependent differential prevalence of HIV.