Racial discrimination during middle age predicts higher serum phosphorylated tau and neurofilament light chain levels a decade later: A study of aging black Americans.

INTRODUCTION: Recent evidence suggests that exposure to the stress of racism may increase the risk of dementia for Black Americans. METHODS: The present study used 17 years of data from a sample of 255 Black Americans to investigate the extent to which exposure to racial discrimination predicts subsequent changes in serum Alzheimer's Disease Research Center (ADRC) biomarkers: serum phosphorylated tau181(p-tau181), neurofilament light (NfL), and glial fibrillary acidic protein (GFAP). We hypothesized that racial discrimination assessed during middle age would predict increases in these serum biomarkers as the participants aged into their 60s. RESULTS: Our findings indicate that exposure to various forms of racial discrimination during a person's 40s and early 50s predicts an 11-year increase in both serum p-tau181 and NfL. Racial discrimination was not associated with subsequent levels of GFAP. DISCUSSION: These findings suggest that racial discrimination in midlife may contribute to increased AD pathology and neurodegeneration later in life. HIGHLIGHTS: A 17-year longitudinal study of Black Americans. Assessments of change in serum p-tau181, neurofilament light, and glial fibrillary acidic protein. Exposure to racial discrimination during middle age predicted increases in p-tau181 and neurofilament light. Education was positively related to both p-tau181 and exposure to racial discrimination.

Childhood adversity predicts black young adults' DNA methylation-based accelerated aging: A dual pathway model.

We expand upon prior work (Gibbons et al., ) relating childhood stressor effects, particularly harsh childhood environments, to risky behavior and ultimately physical health by adding longer-term outcomes - deoxyribonucleic acid (DNA) methylation-based measures of accelerated aging (DNAm-aging). Further, following work on the effects of early exposure to danger (McLaughlin et al., ), we also identify an additional pathway from harsh childhood environments to DNAm-aging that we label the danger/FKBP5 pathway, which includes early exposure to dangerous community conditions that are thought to impact glucocorticoid regulation and pro-inflammatory mechanisms. Because different DNAm-aging indices provide different windows on accelerated aging, we contrast effects on early indices of DNAm-aging based on chronological age with later indices that focused on predicting biological outcomes. We utilize data from Family and Community Health Study participants (N = 449) from age 10 to 29. We find that harshness influences parenting, which, in turn, influences accelerated DNAm-aging through the risky cognitions and substance use (i.e., behavioral) pathway outlined by Gibbons et al. (). Harshness is also associated with increased exposure to threat/danger, which, in turn, leads to accelerated DNAm-aging through effects on FKBP5 activity and enhanced pro-inflammatory tendencies (i.e., the danger/FKBP5 pathway).

Childhood adversity is linked to adult health among African Americans via adolescent weight gain and effects are genetically moderated.

Identifying the mechanisms linking early experiences, genetic risk factors, and their interaction with later health consequences is central to the development of preventive interventions and identifying potential boundary conditions for their efficacy. In the current investigation of 412 African American adolescents followed across a 20-year period, we examined change in body mass index (BMI) across adolescence as one possible mechanism linking childhood adversity and adult health. We found associations of childhood adversity with objective indicators of young adult health, including a cardiometabolic risk index, a methylomic aging index, and a count of chronic health conditions. Childhood adversities were associated with objective indicators indirectly through their association with gains in BMI across adolescence and early adulthood. We also found evidence of an association of genetic risk with weight gain across adolescence and young adult health, as well as genetic moderation of childhood adversity's effect on gains in BMI, resulting in moderated mediation. These patterns indicated that genetic risk moderated the indirect pathways from childhood adversity to young adult health outcomes and childhood adversity moderated the indirect pathways from genetic risk to young adult health outcomes through effects on weight gain during adolescence and early adulthood.

AHRR methylation predicts smoking status and smoking intensity in both saliva and blood DNA.

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.

AHRR Methylation is a Significant Predictor of Mortality Risk in Framingham Heart Study.

Background.-The ability to predict mortality is useful to clinicians, policy makers and insurers. At the current time, prediction of future mortality is still an inexact process with some proposing that epigenetic assessments could play a role in improving prognostics. In past work, we and others have shown that DNA methylation status at cg05575921, a well-studied measure of smoking intensity, is also a predictor of mortality. However, the exact extent of that predictive capacity and its independence of other commonly measured mortality risk factors are unknown. Objective.-To determine the capacity of methylation to predict mortality. Method.-We analyzed the relationship of methylation at cg05575921 and cg04987734, a recently described quantitative marker of heavy alcohol consumption, to mortality in the Offspring Cohort of the Framingham Heart Study using proportional hazards survival analysis. Results.-In this group of participants (n = 2278) whose average age was 66 ± 9 years, we found that the inclusion of both cg05575921 and cg04987734 methylation to a base model consisting of age and sex only, or to a model containing 11 commonly used mortality risk factors, improved risk prediction. What is more, prediction accuracy for the base model plus methylation data was increased compared to the base model plus known predictors of mortality (CHD, COPD, or stroke). Conclusion.-Cg05575921, and to a smaller extent cg04987734, are strong predictors of mortality risk in older Americans and that incorporation of DNA methylation assessments to these and other loci may be useful to population scientists, actuaries and policymakers to better understand the relationship of environmental risk factors, such as smoking and drinking, to mortality.

A Four Marker Digital PCR Toolkit for Detecting Heavy Alcohol Consumption and the Effectiveness of Its Treatment.

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.

Prevention of Early Substance Use Mediates, and Variation at SLC6A4 Moderates, SAAF Intervention Effects on OXTR Methylation.

The Strong African American Family (SAAF) program has been shown to have a variety of short and long-term benefits for participating youth and families. However, biological mechanisms potentially influencing long-term effects on resilience in young adulthood have not been examined. In the current investigation, we examine the effects of SAAF on methylation of the OXTR gene in young adulthood, focusing on a regulatory region previously identified to be both responsive to stress and implicated in resilience. Using the subsample of participants from the original study for whom methylation data was available (N = 388), we replicated the previously reported G × E effect on prevention of early substance use and then examined whether there would also be a moderated effect on OXTR methylation in early adulthood, with "s" allele carriers, but not "LL" participants, showing a significant indirect effect of SAAF on OXTR methylation. Results suggest that for susceptible youth (i.e., "s" allele carriers), preventive intervention may "get under the skin," in a manner potentially beneficial for long-term outcomes. Implications for examination of OXTR methylation in future prevention research are discussed.

Sharing the Burden of the Transition to Adulthood: African American Young Adults' Transition Challenges and Their Mothers' Health Risk.

For many African American youth, the joint influences of economic and racial marginalization render the transition to stable adult roles challenging. We have gained much insight into how these challenges affect future life chances, yet we lack an understanding of what these challenges mean in the context of linked lives. Drawing on a life course framework, this study examines how young African Americans' experiences across a variety of salient domains during the transition to adulthood affect their mothers' health. Results suggest that stressors experienced by African Americans during the transition to adulthood (e.g., unemployment, troubled romantic relationships, arrest) heighten their mothers' cumulative biological risk for chronic diseases, or allostatic load, and reduce subjective health. These results suggest that the toll of an increasingly tenuous and uncertain transition to adulthood extends beyond young people to their parents. Hence, increased public investments during this transition may not only reduce inequality and improve life chances for young people themselves, but may also enhance healthy aging by relieving the heavy burden on parents to help their children navigate this transition.

A Droplet Digital PCR Assay for Smoking Predicts All-Cause Mortality.

OBJECTIVES: -Determine whether an epigenetic assay for smoking predicts all-cause mortality in adults participating in a longitudinal study of Iowa adoptees. BACKGROUND: -Improved biomarkers for smoking are needed given its large public health impact and significant limitations of both self-report and current biomarkers, such as cotinine in detecting smoking. In the past 5 years, multiple epigenome-wide association studies of smoking have identified loci suitable for translation as epigenetic biomarkers for smoking, in particular the CpG cg05575921. Digital polymerase chain reaction methods hold promise for the development of this and other epigenetic biomarkers. METHODS: -Participants in the Iowa Adoption Studies were interviewed regarding their smoking habits. DNA was prepared from whole blood and bisulfite-converted for methylation analysis and digital droplet polymerase chain reaction assay of methylation at cg05575921 was performed. National Death Index records were requested for 584 study participants, resulting in 24 complete matches, 210 partial matches and 350 non-matching records. Complete matches were coded as deceased while the remainder were coded as alive (ie, censored). In total, methylation data and vital status information were available for a total of N = 193 subjects, including 15 deceased and 178 non-deceased. Cox regression was used to examine the ability of cg05575921 methylation as a continuous value to predict the timing of mortality with and without the inclusion of age, sex, race, BMI, marital status, educational status, socioeconomic status, cardiovascular risk factors, and a history of cancer as covariates. RESULTS: -Methylation at cg05575921 predicted the hazard of mortality as the sole predictor and after accounting for major demographic and clinical risk factors. The fitted model showed the hazard ratio increased by 3.5% for every 1% decrease in methylation. CONCLUSIONS: -Decreased methylation at cg05575921, an emerging epigenetic biomarker for smoking, was associated with early mortality in a longitudinal study of adults after accounting for the impact of major demographic and clinical risk factors for all-cause mortality. This approach may be useful in clinical research or actuarial assessments.

Genome-wide and digital polymerase chain reaction epigenetic assessments of alcohol consumption.

The lack of readily employable biomarkers of alcohol consumption is a problem for clinicians and researchers. In 2014, we published a preliminary DNA methylation signature of heavy alcohol consumption that remits as a function of abstinence. Herein, we present new genome-wide methylation findings from a cohort of additional subjects and a meta-analysis of the data. Using DNA from 47 consecutive heavy drinkers admitted for alcohol detoxification in the context of alcohol treatment and 47 abstinent controls, we replicate the 2014 results and show that 21,221 CpG residues are differentially methylated in active heavy drinkers. Meta-analysis of all data from the 448,058 probes common to the two methylation platforms shows a similarly profound signature with confirmation of findings from other groups. Principal components analyses show that genome-wide methylation changes in response to alcohol consumption load on two major factors with one component accounting at least 50% of the total variance in both smokers and nonsmoking alcoholics. Using data from the arrays, we derive a panel of five methylation probes that classifies use status with a receiver operator characteristic area under the curve (AUC) of 0.97. Finally, using droplet digital polymerase chain reaction (PCR), we convert these array-based findings to two marker assays with an AUC of 0.95 and a four marker set AUC of 0.98. We conclude that DNA methylation assessments are capable of quantifying alcohol use status and suggest that readily employable digital PCR approaches for substance consumption may find widespread use in alcohol-related research and patient care.

Methylation of the oxytocin receptor gene mediates the effect of adversity on negative schemas and depression.

Building upon various lines of research, we posited that methylation of the oxytocin receptor gene (OXTR) would mediate the effect of adult adversity on increased commitment to negative schemas and in turn the development of depression. We tested our model using structural equation modeling and longitudinal data from a sample of 100 middle-aged, African American women. The results provided strong support for the model. Analysis of the 12 CpG sites available for the promoter region of the OXTR gene identified four factors. One of these factors was related to the study variables, whereas the others were not. This factor mediated the effect of adult adversity on schemas relating to pessimism and distrust, and these schemas, in turn, mediated the impact of OXTR methylation on depression. All indirect effects were statistically significant, and they remained significant after controlling for childhood trauma, age, romantic relationship status, individual differences in cell types, and average level of genome-wide methylation. These finding suggest that epigenetic regulation of the oxytocin system may be a mechanism whereby the negative cognitions central to depression become biologically embedded.

When inflammation and depression go together: The longitudinal effects of parent-child relationships.

Parent-child relationships have long-term effects on health, particularly later inflammation and depression. We hypothesized that these effects would be mediated by later romantic partner relationships and elevated stressors in young adulthood, helping promote chronic, low grade, inflammation as well as depressive symptoms, and driving their covariation. It has been proposed recently that youth experiencing harsher parenting may also develop a stronger association between inflammation and depressive symptoms in adulthood and altered effects of stressors on outcomes. In the current investigation, we test these ideas using an 18-year longitudinal study of N = 413 African American youth that provides assessment of the parent-child relationship (at age 10), pro-inflammatory cytokine profile and depressive symptoms (at age 28), and potential mediators in early young adulthood (assessed at ages 21 and 24). As predicted, the effect of harsher parent-child relationships (age 10) on pro-inflammatory state and increased depressive symptoms at age 28 were fully mediated through young adult stress and romantic partner relationships. In addition, beyond these mediated effects, parent-child relationships at age 10 moderated the concurrent association between inflammation and depressive symptoms, as well as the prospective association between romantic partner relationships and inflammation, and resulted in substantially different patterns of indirect effects from young adult mediators to outcomes. The results support theorizing that the association of depression and inflammation in young adulthood is conditional on earlier parenting, and suggest incorporating this perspective into models predicting long-term health outcomes.

Smoking in young adulthood among African Americans: Interconnected effects of supportive parenting in early adolescence, proinflammatory epitype, and young adult stress.

We examined two potentially interacting, connected pathways by which parental supportiveness during early adolescence (ages 1-13) may come to be associated with later African American young adult smoking. The first pathway is between parental supportiveness and young adult stress (age 19), with stress, in turn, predicting increased smoking at age 20. The second pathway is between supportive parenting and tumor necrosis factor (TNF) gene methylation (i.e., TNFm), a proinflammatory epitype, with low levels indicating greater inflammatory potential and forecasting increased risk for smoking in response to young adult stress. In a sample of 382 African American youth residing in rural Georgia, followed from early adolescence (age 10-11) to young adulthood (age 20), supportive parenting indirectly predicted smoking via associations with young adult stress, IE = -0.071, 95% confidence interval [-0.132, -0.010]. In addition, supportive parenting was associated with TNFm measured at age 20 (r = .177, p = .001). Further, lower TNFm was associated with a significantly steeper slope (b = 0.583, p = .003) of increased smoking in response to young adult stress compared to those with higher TNFm (b = 0.155, p = .291), indicating an indirect, amplifying role for supportive parenting via TNFm. The results suggest that supportive parenting in early adolescence may play a role in understanding the emergence of smoking in young adulthood.

A pilot investigation of the impact of smoking cessation on biological age.

BACKGROUND AND OBJECTIVES: Smoking is known to increase biological age. However, whether this process is reversible through smoking cessation is not known. In this pilot study, we attempt to determine whether smoking cessation reduces biological age. METHODS: We conducted regression analyses of methylation data from 22 subjects, as they entered and exited inpatient substance use treatment, to determine change in biological age, as indicated by the deviation of their methylomic age from chronological age across two time points. RESULTS: We found that, as compared to those subjects who did not stop smoking, subjects who significantly decreased their smoking consumption over a 1 month time period exhibited a marked reduction in methylomic age. CONCLUSION: The rapid and substantial reversal of accelerated aging associated with successful smoking cessation suggests that it can reverse well-known smoking effects on methylomic aging. This preliminary finding can be readily examined in other, larger data sets, and if replicated, this observation may provide smokers with yet another good reason to quit smoking. SCIENTIFIC SIGNIFICANCE: Successful smoking cessation makes patients appear biologically younger than they were at baseline, and to do so quite rapidly. In today's youth driven society, our observations may serve as a powerful impetus for some to quit smoking. (Am J Addict 2017;26:129-135).

Optimizing the chances of success in the search for epigenetic biomarkers: Embracing genetic variation.

The emphasis on clinical translation in biomedical research continues to grow. This focus has been particularly notable in those investigators using epigenetic approaches to decipher the biology of complex behavioral disorders. As a result of these efforts, reproducible findings for several disorders, such as smoking, have been generated, giving rise to hopes that biomarkers for other behavioral illnesses would be forthcoming. Unfortunately, that biomedical cornucopia has not yet materialized. In this editorial, we review progress to date and discuss barriers to generating epigenetic biomarkers for complex behavioral disorders. We highlight the need to incorporate information on genetic variation and develop more powerful bioinformatics tools in order to optimize the likelihood of success. We emphasize that searches should focus on clearly defined, readily distinguishable behavioral constructs and suggest that some well-intentioned methods, such as correction for cellular heterogeneity, may actually impede the identification of clinically relevant biomarkers in peripheral blood. Finally, we describe how the understanding created by the development of these biomarkers may lead to more valid animal models of neuropsychiatric illness. We conclude that the prospects for epigenetic biomarkers for complex disorders are bright, but emphasize that the journey to the clinical implementation of these findings will be a slow, iterative process.

Genetically contextual effects of smoking on genome wide DNA methylation.

Smoking is the leading cause of death in the United States. It exerts its effects by increasing susceptibility to a variety of complex disorders among those who smoke, and if pregnant, to their unborn children. In prior efforts to understand the epigenetic mechanisms through which this increased vulnerability is conveyed, a number of investigators have conducted genome wide methylation analyses. Unfortunately, secondary to methodological limitations, these studies were unable to examine methylation in gene regions with significant amounts of genetic variation. Using genome wide genetic and epigenetic data from the Framingham Heart Study, we re-examined the relationship of smoking status to genome wide methylation status. When only methylation status is considered, smoking was significantly associated with differential methylation in 310 genes that map to a variety of biological process and cellular differentiation pathways. However, when SNP effects on the magnitude of smoking associated methylation changes are also considered, cis and trans-interaction effects were noted at a total of 266 and 4353 genes with no marked enrichment for any biological pathways. Furthermore, the SNP variation participating in the significant interaction effects is enriched for loci previously associated with complex medical illnesses. The enlarged scope of the methylome shown to be affected by smoking may better explicate the mediational pathways linking smoking with a myriad of smoking related complex syndromes. Additionally, these results strongly suggest that combined epigenetic and genetic data analyses may be critical for a more complete understanding of the relationship between environmental variables, such as smoking, and pathophysiological outcomes.

Accuracy and utility of an epigenetic biomarker for smoking in populations with varying rates of false self-report.

Better biomarkers to detect smoking are needed given the tremendous public health burden caused by smoking. Current biomarkers to detect smoking have significant limitations, notably a short half-life for detection and lack of sensitivity for light smokers. These limitations may be particularly problematic in populations with less accurate self-reporting. Prior epigenome-wide association studies indicate that methylation status at cg05575921, a CpG residue located in the aryl hydrocarbon receptor repressor (AHRR) gene, may be a robust indicator of smoking status in individuals with as little as half of a pack-year of smoking. In this study, we show that a novel droplet digital PCR assay for measuring methylation at cg05575921 can reliably detect smoking status, as confirmed by serum cotinine, in populations with different demographic characteristics, smoking histories, and rates of false-negative self-report of smoking behavior. Using logistic regression models, we show that obtaining maximum accuracy in predicting smoking status depends on appropriately weighting self-report and cg05575921 methylation according to the characteristics of the sample being tested. Furthermore, models using only cg05575921 methylation to predict smoking perform nearly as well as those also including self-report across populations. In conclusion, cg05575921 has significant potential as a clinical biomarker to detect smoking in populations with varying rates of accuracy in self-report of smoking behavior.

MTHFR methylation moderates the impact of smoking on DNA methylation at AHRR for African American young adults.

Smoking has been shown to have a large, reliable, and rapid effect on demethylation of AHRR, particularly at cg05575921, suggesting that methylation may be used as an index of cigarette consumption. Because the availability of methyl donors may also influence the degree of demethylation in response to smoking, factors that affect the activity of methylene tetrahydrofolate reductase (MTHFR), a key regulator of methyl group availability, may be of interest. In the current investigation, we examined the extent to which individual differences in methylation of MTHFR moderated the association between smoking and demethylation at cg05575921 as well as at other loci on AHRR associated with a main effect of smoking. Using a discovery sample (AIM, N = 293), and a confirmatory sample (SHAPE, N = 368) of young adult African Americans, degree of methylation of loci in the first exon of MTHFR was associated with amplification of the association between smoking and AHRR demethylation at cg05575921. However, genetic variation at a commonly studied MTHFR variant, C677T, did not influence cg05575921 methylation. The significant interaction between MTHFR methylation and the smoking-induced response at cg05575921 suggests a role for individual differences in methyl cycle regulation in understanding the effects of cigarette consumption on genome wide DNA methylation.

Epigenetic silencing of the human NOS2 gene: rethinking the role of nitric oxide in human macrophage inflammatory responses.

Macrophages, including alveolar macrophages, are primary phagocytic cells of the innate immune system. Many studies of macrophages and inflammation have been done in mouse models, in which inducible NO synthase (NOS2) and NO are important components of the inflammatory response. Human macrophages, in contrast to mouse macrophages, express little detectable NOS2 and generate little NO in response to potent inflammatory stimuli. The human NOS2 gene is highly methylated around the NOS2 transcription start site. In contrast, mouse macrophages contain unmethylated cytosine-phosphate-guanine (CpG) dinucleotides proximal to the NOS2 transcription start site. Further analysis of chromatin accessibility and histone modifications demonstrated a closed conformation at the human NOS2 locus and an open conformation at the murine NOS2 locus. In examining the potential for CpG demethylation at the NOS2 locus, we found that the human NOS2 gene was resistant to the effects of demethylation agents both in vitro and in vivo. Our data demonstrate that epigenetic modifications in human macrophages are associated with CpG methylation, chromatin compaction, and histone modifications that effectively silence the NOS2 gene. Taken together, our findings suggest there are significant and underappreciated differences in how murine and human macrophages respond to inflammatory stimuli.

Parenting, Socioeconomic Status Risk, and Later Young Adult Health: Exploration of Opposing Indirect Effects via DNA Methylation.

A sample of 398 African American youth, residing in rural counties with high poverty and unemployment, were followed from ages 11 to 19. Protective parenting was associated with better health, whereas elevated socioeconomic status (SES) risk was associated with poorer health at age 19. Genome-wide epigenetic variation assessed in young adulthood (age 19), was associated with both SES risk and protective parenting. Three categories of genes were identified whose methylation was associated with parenting, SES risk, and young adult health. Methylation was a significant mediator of the impact of parenting and SES risk on young adult health. Variation in mononuclear white blood cell types was also examined and controlled, showing that it did not account for observed effects of parenting and SES risk on health.