Extracellular Vesicle-Encapsulated microRNAs as Novel Biomarkers of Lung Health.

Rationale: Early detection of respiratory diseases is critical to facilitate delivery of disease-modifying interventions. Extracellular vesicle-enriched microRNAs (EV-miRNAs) may represent reliable markers of early lung injury. Objectives: Evaluate associations of plasma EV-miRNAs with lung function. Methods: The prospective NAS (Normative Aging Study) collected plasma EV-miRNA measurements from 1996-2015 and spirometry every 3-5 years through 2019. Associations of EV-miRNAs with baseline lung function were modeled using linear regression. To complement the individual miRNA approach, unsupervised machine learning was used to identify clusters of participants with distinct EV-miRNA profiles. Associations of EV-miRNA profiles with multivariate latent longitudinal lung function trajectories were modeled using log binomial regression. Biological functions of significant EV-miRNAs were explored using pathway analyses. Results were replicated in an independent sample of NAS participants and in the HEALS (Health Effects of Arsenic Longitudinal Study). Measurements and Main Results: In the main cohort of 656 participants, 51 plasma EV-miRNAs were associated with baseline lung function (false discovery rate-adjusted P value < 0.05), 28 of which were replicated in the independent NAS sample and/or in the HEALS cohort. A subset of participants with distinct EV-miRNA expression patterns had increased risk of declining lung function over time, which was replicated in the independent NAS sample. Significant EV-miRNAs were shown in pathway analyses to target biological pathways that regulate respiratory cellular immunity, the lung inflammatory response, and airway structural integrity. Conclusions: Plasma EV-miRNAs may represent a robust biomarker of subclinical lung injury and may facilitate early identification and treatment of patients at risk of developing overt lung disease.

Extracellular microRNA and cognitive function in a prospective cohort of older men: The Veterans Affairs Normative Aging Study.

BACKGROUND: Aging-related cognitive decline is an early symptom of Alzheimer's disease and other dementias, and on its own can have substantial consequences on an individual's ability to perform important everyday functions. Despite increasing interest in the potential roles of extracellular microRNAs (miRNAs) in central nervous system (CNS) pathologies, there has been little research on extracellular miRNAs in early stages of cognitive decline. We leverage the longitudinal Normative Aging Study (NAS) cohort to investigate associations between plasma miRNAs and cognitive function among cognitively normal men. METHODS: This study includes data from up to 530 NAS participants (median age: 71.0 years) collected from 1996 to 2013, with a total of 1,331 person-visits (equal to 2,471 years of follow up). Global cognitive function was assessed using the Mini-Mental State Examination (MMSE). Plasma miRNAs were profiled using small RNA sequencing. Associations of expression of 381 miRNAs with current cognitive function and rate of change in cognitive function were assessed using linear regression (N = 457) and linear mixed models (N = 530), respectively. RESULTS: In adjusted models, levels of 2 plasma miRNAs were associated with higher MMSE scores (p < 0.05). Expression of 33 plasma miRNAs was associated with rate of change in MMSE scores over time (p < 0.05). Enriched KEGG pathways for miRNAs associated with concurrent MMSE and MMSE trajectory included Hippo signaling and extracellular matrix-receptor interactions. Gene targets of miRNAs associated with MMSE trajectory were additionally associated with prion diseases and fatty acid biosynthesis. CONCLUSIONS: Circulating miRNAs were associated with both cross-sectional cognitive function and rate of change in cognitive function among cognitively normal men. Further research is needed to elucidate the potential functions of these miRNAs in the CNS and investigate relationships with other neurological outcomes.

Predicting risk of lung function impairment and all-cause mortality using a DNA methylation-based classifier of tobacco smoke exposure.

BACKGROUND: The Epigenetic Smoking Status Estimator (EpiSmokEr) predicts smoking phenotypes based on DNA methylation at 121 CpG sites. OBJECTIVE: Evaluate associations of EpiSmokEr-predicted versus self-reported smoking phenotypes with lung function and all-cause mortality in a cohort of older adults. METHODS: The prospective Normative Aging Study collected DNA methylation measurements from 1999 to 2012 with follow-up through 2016. The R package EpiSmokEr derived predicted smoking phenotypes based on DNA methylation levels assayed by the Illumina HumanMethylation450 Beadchip. Spirometry was collected every 3-5 years. Airflow limitation was defined as forced expiratory volume in 1 s/forced vital capacity <0.7. Vital status was monitored through periodic mailings. RESULTS: Among 784 participants contributing 5414 person-years of follow-up, the EpiSmokEr-predicted smoking phenotypes matched the self-reported phenotypes for 228 (97%) never smokers and 22 (71%) current smokers. In contrast, EpiSmokEr classified 407 (79%) self-reported former smokers as never smokers. Nonetheless, the EpiSmokEr-predicted former smoking phenotype was more strongly associated with incident airflow limitation (hazard ratio [HR] = 3.15, 95% confidence interval [CI] = 1.50-6.59) and mortality (HR = 2.11, 95% CI = 1.56-2.85) compared to the self-reported former smoking phenotype (airflow limitation: HR = 2.21, 95% CI = 1.13-4.33; mortality: HR = 1.08, 95% CI = 0.86-1.36). Risk of airflow limitation and death did not differ among self-reported never smokers and former smokers who were classified as never smokers. The discriminative accuracy of EpiSmokEr-predicted phenotypes for incident airflow limitation and mortality was improved compared to self-reported phenotypes. CONCLUSIONS: The DNA methylation-based EpiSmokEr classifier may be a useful surrogate of smoking-induced lung damage and may identify former smokers most at risk of adverse smoking-related health effects.

Gaseous air pollutants and DNA methylation in a methylome-wide association study of an ethnically and environmentally diverse population of U.S. adults.

Epigenetic mechanisms may underlie air pollution-health outcome associations. We estimated gaseous air pollutant-DNA methylation (DNAm) associations using twelve subpopulations within Women's Health Initiative (WHI) and Atherosclerosis Risk in Communities (ARIC) cohorts (n = 8397; mean age 61.3 years; 83% female; 46% African-American, 46% European-American, 8% Hispanic/Latino). We used geocoded participant address-specific mean ambient carbon monoxide (CO), nitrogen oxides (NO2; NOx), ozone (O3), and sulfur dioxide (SO2) concentrations estimated over the 2-, 7-, 28-, and 365-day periods before collection of blood samples used to generate Illumina 450 k array leukocyte DNAm measurements. We estimated methylome-wide, subpopulation- and race/ethnicity-stratified pollutant-DNAm associations in multi-level, linear mixed-effects models adjusted for sociodemographic, behavioral, meteorological, and technical covariates. We combined stratum-specific estimates in inverse variance-weighted meta-analyses and characterized significant associations (false discovery rate; FDR<0.05) at Cytosine-phosphate-Guanine (CpG) sites without among-strata heterogeneity (PCochran's Q > 0.05). We attempted replication in the Cooperative Health Research in Region of Augsburg (KORA) study and Normative Aging Study (NAS). We observed a -0.3 (95% CI: -0.4, -0.2) unit decrease in percent DNAm per interquartile range (IQR, 7.3 ppb) increase in 28-day mean NO2 concentration at cg01885635 (chromosome 3; regulatory region 290 bp upstream from ZNF621; FDR = 0.03). At intragenic sites cg21849932 (chromosome 20; LIME1; intron 3) and cg05353869 (chromosome 11; KLHL35; exon 2), we observed a -0.3 (95% CI: -0.4, -0.2) unit decrease (FDR = 0.04) and a 1.2 (95% CI: 0.7, 1.7) unit increase (FDR = 0.04), respectively, in percent DNAm per IQR (17.6 ppb) increase in 7-day mean ozone concentration. Results were not fully replicated in KORA and NAS. We identified three CpG sites potentially susceptible to gaseous air pollution-induced DNAm changes near genes relevant for cardiovascular and lung disease. Further harmonized investigations with a range of gaseous pollutants and averaging durations are needed to determine the effect of gaseous air pollutants on DNA methylation and ultimately gene expression.

Pulmonary Function and Blood DNA Methylation: A Multiancestry Epigenome-Wide Association Meta-analysis.

Rationale: Methylation integrates factors present at birth and modifiable across the lifespan that can influence pulmonary function. Studies are limited in scope and replication. Objectives: To conduct large-scale epigenome-wide meta-analyses of blood DNA methylation and pulmonary function. Methods: Twelve cohorts analyzed associations of methylation at cytosine-phosphate-guanine probes (CpGs), using Illumina 450K or EPIC/850K arrays, with FEV1, FVC, and FEV1/FVC. We performed multiancestry epigenome-wide meta-analyses (total of 17,503 individuals; 14,761 European, 2,549 African, and 193 Hispanic/Latino ancestries) and interpreted results using integrative epigenomics. Measurements and Main Results: We identified 1,267 CpGs (1,042 genes) differentially methylated (false discovery rate, <0.025) in relation to FEV1, FVC, or FEV1/FVC, including 1,240 novel and 73 also related to chronic obstructive pulmonary disease (1,787 cases). We found 294 CpGs unique to European or African ancestry and 395 CpGs unique to never or ever smokers. The majority of significant CpGs correlated with nearby gene expression in blood. Findings were enriched in key regulatory elements for gene function, including accessible chromatin elements, in both blood and lung. Sixty-nine implicated genes are targets of investigational or approved drugs. One example novel gene highlighted by integrative epigenomic and druggable target analysis is TNFRSF4. Mendelian randomization and colocalization analyses suggest that epigenome-wide association study signals capture causal regulatory genomic loci. Conclusions: We identified numerous novel loci differentially methylated in relation to pulmonary function; few were detected in large genome-wide association studies. Integrative analyses highlight functional relevance and potential therapeutic targets. This comprehensive discovery of potentially modifiable, novel lung function loci expands knowledge gained from genetic studies, providing insights into lung pathogenesis.

Metabolomic signatures of the short-term exposure to air pollution and temperature.

BACKGROUND: Short-term exposures to air pollution and temperature have been reported to be associated with inflammation and oxidative stress. However, mechanistic understanding of the affected metabolic pathways is still lacking and literature on the short-term exposure of air-pollution on the metabolome is limited. OBJECTIVES: We aimed to determine changes in the plasma metabolome and associated metabolic pathways related to short-term exposure to outdoor air pollution and temperature. METHODS: We performed mass-spectrometry based untargeted metabolomic profiling of plasma samples from a large and well-characterized cohort of men (Normative Aging Study) to identify metabolic pathways associated with short-term exposure to PM2.5, NO2, O3, and temperature (one, seven-, and thirty-day average of address-specific predicted estimates). We used multivariable linear mixed-effect regression and independent component analysis (ICA) while simultaneously adjusting for all exposures and correcting for multiple testing. RESULTS: Overall, 456 white men provided 648 blood samples, in which 1158 metabolites were quantified, between 2000 and 2016. Average age and body mass index were 75.0 years and 27.7 kg/m2, respectively. Only 3% were current smokers. In the adjusted models, NO2, and temperature showed statistically significant associations with several metabolites (19 metabolites for NO2 and 5 metabolites for temperature). We identified six metabolic pathways (sphingolipid, butanoate, pyrimidine, glycolysis/gluconeogenesis, propanoate, and pyruvate metabolisms) perturbed with short-term exposure to air pollution and temperature. These pathways were involved in inflammation and oxidative stress, immunity, and nucleic acid damage and repair. CONCLUSIONS: This is the first study to report an untargeted metabolomic signature of temperature exposure, the largest to report an untargeted metabolomic signature of air pollution, and the first to use ICA. We identified several significant plasma metabolites and metabolic pathways associated with short-term exposure to air pollution and temperature; using an untargeted approach. Those pathways were involved in inflammation and oxidative stress, immunity, and nucleic acid damage and repair. These results need to be confirmed by future research.

Ambient PM2.5 species and ultrafine particle exposure and their differential metabolomic signatures.

BACKGROUND: The metabolomic signatures of short- and long-term exposure to PM2.5 have been reported and linked to inflammation and oxidative stress. However, little is known about the relative contribution of the specific PM2.5 species (hence sources) that drive these metabolomic signatures. OBJECTIVES: We aimed to determine the relative contribution of the different species of PM2.5 exposure to the perturbed metabolic pathways related to changes in the plasma metabolome. METHODS: We performed mass-spectrometry based metabolomic profiling of plasma samples among men from the Normative Aging Study to identify metabolic pathways associated with PM2.5 species. The exposure windows included short-term (one, seven-, and thirty-day moving average) and long-term (one year moving average). We used linear mixed-effect regression with subject-specific intercepts while simultaneously adjusting for PM2.5, NO2, O3, temperature, relative humidity, and covariates and correcting for multiple testing. We also used independent component analysis (ICA) to examine the relative contribution of patterns of PM2.5 species. RESULTS: Between 2000 and 2016, 456 men provided 648 blood samples, in which 1158 metabolites were quantified. We chose 305 metabolites for the short-term and 288 metabolites for the long-term exposure in this analysis that were significantly associated (p-value < 0.01) with PM2.5 to include in our PM2.5 species analysis. On average, men were 75.0 years old and their body mass index was 27.7 kg/m2. Only 3% were current smokers. In the adjusted models, ultrafine particles (UFPs) were the most significant species of short-term PM2.5 exposure followed by nickel, vanadium, potassium, silicon, and aluminum. Black carbon, vanadium, zinc, nickel, iron, copper, and selenium were the significant species of long-term PM2.5 exposure. We identified several metabolic pathways perturbed with PM2.5 species including glycerophospholipid, sphingolipid, and glutathione. These pathways are involved in inflammation, oxidative stress, immunity, and nucleic acid damage and repair. Results were overlapped with the ICA. CONCLUSIONS: We identified several significant perturbed plasma metabolites and metabolic pathways associated with exposure to PM2.5 species. These species are associated with traffic, fuel oil, and wood smoke. This is the largest study to report a metabolomic signature of PM2.5 species' exposure and the first to use ICA.

Metabolomic signatures of the long-term exposure to air pollution and temperature.

BACKGROUND: Long-term exposures to air pollution has been reported to be associated with inflammation and oxidative stress. However, the underlying metabolic mechanisms remain poorly understood. OBJECTIVES: We aimed to determine the changes in the blood metabolome and thus the metabolic pathways associated with long-term exposure to outdoor air pollution and ambient temperature. METHODS: We quantified metabolites using mass-spectrometry based global untargeted metabolomic profiling of plasma samples among men from the Normative Aging Study (NAS). We estimated the association between long-term exposure to PM2.5, NO2, O3, and temperature (annual average of central site monitors) with metabolites and their associated metabolic pathways. We used multivariable linear mixed-effect regression models (LMEM) while simultaneously adjusting for the four exposures and potential confounding and correcting for multiple testing. As a reduction method for the intercorrelated metabolites (outcome), we further used an independent component analysis (ICA) and conducted LMEM with the same exposures. RESULTS: Men (N = 456) provided 648 blood samples between 2000 and 2016 in which 1158 metabolites were quantified. On average, men were 75.0 years and had an average body mass index of 27.7 kg/m2. Almost all men (97%) were not current smokers. The adjusted analysis showed statistically significant associations with several metabolites (58 metabolites with PM2.5, 15 metabolites with NO2, and 6 metabolites with temperature) while no metabolites were associated with O3. One out of five ICA factors (factor 2) was significantly associated with PM2.5. We identified eight perturbed metabolic pathways with long-term exposure to PM2.5 and temperature: glycerophospholipid, sphingolipid, glutathione, beta-alanine, propanoate, and purine metabolism, biosynthesis of unsaturated fatty acids, and taurine and hypotaurine metabolism. These pathways are related to inflammation, oxidative stress, immunity, and nucleic acid damage and repair. CONCLUSIONS: Using a global untargeted metabolomic approach, we identified several significant metabolites and metabolic pathways associated with long-term exposure to PM2.5, NO2 and temperature. This study is the largest metabolomics study of long-term air pollution, to date, the first study to report a metabolomic signature of long-term temperature exposure, and the first to use ICA in the analysis of both.

Biomarkers of aging and lung function in the normative aging study.

Elderly individuals who are never smokers but have the same height and chronological age can have substantial differences in lung function. The underlying biological mechanisms are unclear. To evaluate the associations of different biomarkers of aging (BoA) and lung function, we performed a repeated-measures analysis in the Normative Aging Study using linear mixed-effect models. We generated GrimAgeAccel, PhenoAgeAccel, extrinsic and intrinsic epigenetic age acceleration using a publically available online calculator. We calculated Zhang's DNAmRiskScore based on 10 CpGs. We measured telomere length (TL) and mitochondrial DNA copy number (mtDNA-CN) using quantitative real-time polymerase chain reaction. A pulmonary function test was performed measuring forced expiratory volume in 1 second / forced vital capacity (FEV1/FVC), FEV1, and maximum mid-expiratory flow (MMEF). Epigenetic-based BoA were associated with lower lung function. For example, a one-year increase in GrimAgeAccel was associated with a 13.64 mL [95% confidence interval (CI), 5.11 to 22.16] decline in FEV1; a 0.2 increase in Zhang's DNAmRiskScore was associated with a 0.009 L/s (0.005 to 0.013) reduction in MMEF. No association was found between TL/mtDNA-CN and lung function. Overall, this paper shows that epigenetics might be a potential mechanism underlying pulmonary dysfunction in the elderly.

Socioeconomic position, lifestyle habits and biomarkers of epigenetic aging: a multi-cohort analysis.

Differences in health status by socioeconomic position (SEP) tend to be more evident at older ages, suggesting the involvement of a biological mechanism responsive to the accumulation of deleterious exposures across the lifespan. DNA methylation (DNAm) has been proposed as a biomarker of biological aging that conserves memory of endogenous and exogenous stress during life.We examined the association of education level, as an indicator of SEP, and lifestyle-related variables with four biomarkers of age-dependent DNAm dysregulation: the total number of stochastic epigenetic mutations (SEMs) and three epigenetic clocks (Horvath, Hannum and Levine), in 18 cohorts spanning 12 countries.The four biological aging biomarkers were associated with education and different sets of risk factors independently, and the magnitude of the effects differed depending on the biomarker and the predictor. On average, the effect of low education on epigenetic aging was comparable with those of other lifestyle-related risk factors (obesity, alcohol intake), with the exception of smoking, which had a significantly stronger effect.Our study shows that low education is an independent predictor of accelerated biological (epigenetic) aging and that epigenetic clocks appear to be good candidates for disentangling the biological pathways underlying social inequalities in healthy aging and longevity.

Analysis of repeated leukocyte DNA methylation assessments reveals persistent epigenetic alterations after an incident myocardial infarction.

BACKGROUND: Most research into myocardial infarctions (MIs) have focused on preventative efforts. For survivors, the occurrence of an MI represents a major clinical event that can have long-lasting consequences. There has been little to no research into the molecular changes that can occur as a result of an incident MI. Here, we use three cohorts to identify epigenetic changes that are indicative of an incident MI and their association with gene expression and metabolomics. RESULTS: Using paired samples from the KORA cohort, we screened for DNA methylation loci (CpGs) whose change in methylation is potentially indicative of the occurrence of an incident MI between the baseline and follow-up exams. We used paired samples from the NAS cohort to identify 11 CpGs which were predictive in an independent cohort. After removing two CpGs associated with medication usage, we were left with an "epigenetic fingerprint" of MI composed of nine CpGs. We tested this fingerprint in the InCHIANTI cohort where it moderately discriminated incident MI occurrence (AUC = 0.61, P = 6.5 × 10-3). Returning to KORA, we associated the epigenetic fingerprint loci with cis-gene expression and integrated it into a gene expression-metabolomic network, which revealed links between the epigenetic fingerprint CpGs and branched chain amino acid (BCAA) metabolism. CONCLUSIONS: There are significant changes in DNA methylation after an incident MI. Nine of these CpGs show consistent changes in multiple cohorts, significantly discriminate MI in independent cohorts, and were independent of medication usage. Integration with gene expression and metabolomics data indicates a link between MI-associated epigenetic changes and BCAA metabolism.

Metastable DNA methylation sites associated with longitudinal lung function decline and aging in humans: an epigenome-wide study in the NAS and KORA cohorts.

DNA methylation is an epigenetic regulator of gene transcription, which has been found to be both metastable and variable within human cohort studies. Currently, few studies have been done to identify metastable DNA methylation biomarkers associated with longitudinal lung function decline in humans. The identification of such biomarkers is important for screening vulnerable populations. We hypothesized that quantifiable blood-based DNA methylation alterations would serve as metastable biomarkers of lung function decline and aging, which may help to discover new pathways and/or mechanisms related to pulmonary pathogenesis. Using linear mixed models, we performed an Epigenome Wide Association Study (EWAS) between DNA methylation at CpG dinucleotides and longitudinal lung function (FVC, FEV1, FEF25-75%) decline and aging with initial discovery in the Normative Aging Study, and replication in the Cooperative Health Research in the Region of Augsburg cohort. We identified two metastable epigenetic loci associated with either poor lung function and aging, cg05575921 (AHRR gene), or lung function independently of aging, cg06126421 (IER3 gene). These loci may inform basic mechanisms associated with pulmonary function, pathogenesis, and aging. Human epigenomic variation, may help explain features of lung function decline and related pathophysiology not attributable to DNA sequence alone, such as accelerated pulmonary decline in smokers, former smokers, and perhaps non-smokers. Our EWAS across two cohorts, therefore, will likely have implications for the human population, not just the elderly.

Bone Lead Levels and Risk of Incident Primary Open-Angle Glaucoma: The VA Normative Aging Study.

BACKGROUND: Oxidative stress may play an important role in the etiology of primary open-angle glaucoma (POAG). The association between risk of POAG and lead exposure, which is an environmental source of oxidative stress, has not been fully investigated yet. OBJECTIVE: Our objective was to determine the association between bone lead­a biomarker of cumulative lead dose (tibia lead) or an endogenous source of stored lead (patella lead)­and incident POAG. METHODS: We examined a prospective cohort of 634 POAG-free men [mean baseline age=66.8 y of age (SD=6.7)] from the Normative Aging Study (NAS) who had tibia and patella K X-ray fluorescence lead measurements between 1 January 1991 and 31 December 1999. They also had standard ocular evaluations by NAS optometrists until 31 December 2014. POAG cases were identified by consistent reports of enlarged or asymmetric cup-to-disc ratio together with visual field defect or existence of disc hemorrhage. We used Cox proportional hazards regressions to estimate hazard ratios (HRs) of incident POAG and adjusted survival curves to examine changes in the risk of POAG during follow-up according to bone lead quartiles. RESULTS: We identified 44 incident cases of POAG by the end of follow-up (incidence rate=74 per 10,000 person-years; median follow-up=10.6 y). In fully adjusted models, 10-fold increases in patella lead and tibia lead were associated with HRs of 5.06 (95% CI: 1.61, 15.88, p=0.005) and 3.07 (95% CI: 0.94, 10.0, p=0.06), respectively. The HRs comparing participants in the third and fourth quartiles with the lowest quartile were 3.41 (95% CI: 1.34, 8.66) and 3.24 (95% CI: 1.22, 8.62) for patella lead (p-for-trend=0.01), and 3.84 (95% CI: 1.54, 9.55) and 2.61 (95% CI: 0.95, 7.21) for tibia lead (p-for-trend=0.02). CONCLUSIONS: Our study provides longitudinal evidence that bone lead may be an important risk factor for POAG in the U.S. population. https://doi.org/10.1289/EHP3442.

Lung function association with outdoor temperature and relative humidity and its interaction with air pollution in the elderly.

While the effects of weather variability on cardio-respiratory mortality are well described, research examining the effects on morbidity, especially for vulnerable populations, is warranted. We investigated the associations between lung function and outdoor temperature (T in Celsius degrees (°C)) and relative humidity (RH), in a cohort of elderly men, the Normative Aging Study. Our study included 1103 participants whose forced vital capacity (FVC), forced expiratory volume in one second (FEV1), and weather exposures were assessed one to five times during the period 1995-2011 (i.e. 3162 observations). Temperature and relative humidity were measured at one location 4 h to 7 days before lung function tests. We used linear mixed-effects models to examine the associations with outdoor T and RH. A 5-degree increase in the 3-day moving average T was associated with a significant 0.7% decrease (95%CI: -1.24, -0.20) in FVC and a 5% increase in the 7-day moving average RH was associated with a significant 0.2% decrease (95%CI: -0.40, -0.02) in FVC and FEV1. The associations with T were greater when combined with higher exposures of black carbon with a 1.6% decrease (95%CI -2.2; -0.9) in FVC and a 1% decrease (95%CI -1.7; -0.4) in FEV1. The relationships between T and RH and lung function were linear. No synergistic effect of T and RH was found. Heat and lung function are two predictors of mortality. Our findings suggest that increases in temperature and relative humidity are related to decreases in lung function, and such observations might be amplified by high black carbon levels.

Improving estimation and prediction in linear regression incorporating external information from an established reduced model.

We consider a situation where there is rich historical data available for the coefficients and their standard errors in a linear regression model describing the association between a continuous outcome variable Y and a set of predicting factors X, from a large study. We would like to use this summary information for improving inference in an expanded model of interest, Y given X,B. The additional variable B is a new biomarker, measured on a small number of subjects in a new dataset. We formulate the problem in an inferential framework where the historical information is translated in terms of nonlinear constraints on the parameter space and propose both frequentist and Bayes solutions to this problem. We show that a Bayesian transformation approach proposed by Gunn and Dunson is a simple and effective computational method to conduct approximate Bayesian inference for this constrained parameter problem. The simulation results comparing these methods indicate that historical information on E(Y|X) can improve the efficiency of estimation and enhance the predictive power in the regression model of interest E(Y|X,B). We illustrate our methodology by enhancing a published prediction model for bone lead levels in terms of blood lead and other covariates, with a new biomarker defined through a genetic risk score.

The Inflammatory Potential of Dietary Manganese in a Cohort of Elderly Men.

Manganese is an essential nutrient that may play a role in the production of inflammatory biomarkers. We examined associations between estimated dietary manganese intake from food/beverages and supplements with circulating biomarkers of inflammation. We further explored whether estimated dietary manganese intake affects DNA methylation of selected genes involved in the production of these biomarkers. We analyzed 1023 repeated measures of estimated dietary manganese intakes and circulating blood inflammatory biomarkers from 633 participants in the Normative Aging Study. Using mixed-effect linear regression models adjusted for covariates, we observed positive linear trends between estimated dietary manganese intakes and three circulating interleukin proteins. Relative to the lowest quartile of estimated intake, concentrations of IL-1ß were 46% greater (95% CI - 5, 126), IL-6 52% greater (95% CI - 9, 156). and IL-8 32% greater (95% CI 2, 71) in the highest quartiles of estimated intake. Estimated dietary manganese intake was additionally associated with changes in DNA methylation of inflammatory biomarker-producing genes. Higher estimated intake was associated with higher methylation of NF-κß member activator NKAP (Q4 vs Q1: ß = 3.32, 95% CI - 0.6, 7.3). When stratified by regulatory function, higher manganese intake was associated with higher gene body methylation of NF-κß member activators NKAP (Q4 vs Q1: ß = 10.10, 95% CI - 0.8, 21) and NKAPP1 (Q4 vs Q1: ß = 8.14, 95% CI 1.1, 15). While needed at trace amounts for various physiologic functions, our results suggest estimated dietary intakes of manganese at levels slightly above nutritional adequacy contribute to inflammatory biomarker production.

Low-Level Cumulative Lead and Resistant Hypertension: A Prospective Study of Men Participating in the Veterans Affairs Normative Aging Study.

Background Bone lead offers a better method over blood lead measurement to discern long-term lead exposure and accumulation. We examined the risk of resistant hypertension based on bone lead levels in a prospective cohort study of NAS (Normative Aging Study). Methods and Results Participants had clinic data on hypertension (systolic blood pressure, diastolic blood pressure, and antihypertension medication), lead (blood, bone-patella, bone-tibia), and demographic and confounding variables. Cases of resistant hypertension were identified by meeting criteria for: (1) inadequate systolic blood pressure (>140 mm Hg) or diastolic blood pressure (>90 mm Hg) while taking 3 medications or (2) requiring >4 medications for blood pressure control. A modified Poisson regression was used for model analysis. Of the 475 participants, 97 cases of resistant hypertension (20.4%) were identified. Among the cases of resistant hypertension, the median tibia and patella lead levels were 20 µg/g and 25 µg/g, respectively, while median tibia and patella lead levels were 20 µg/g and 27.5 µg/g, respectively, in participants without resistant hypertension. Tibia lead demonstrated a significant association with resistant hypertension (relative risk, 1.19; 95% confidence interval, 1.01-1.41 [ P=0.04]) per interquartile range increase in tibia lead (13-28.5 µg/g). Patella lead was not associated with resistant hypertension (relative risk, 1.10; 95% confidence interval, 0.92-1.31 [ P=0.31]) per interquartile range increase in patella lead (18-40 µg/g). Blood lead levels were not significantly associated with resistant hypertension (relative risk, 1.11; 95% confidence interval, 0.88-1.40 [ P=0.38]). Conclusions Tibia lead represents a novel risk factor for resistant hypertension. Our study demonstrates an increased association between tibia lead and resistant hypertension status, with an increased risk of 19% per 1 interquartile range increase in tibia lead.

DNA Methylation Analysis Identifies Loci for Blood Pressure Regulation.

Genome-wide association studies have identified hundreds of genetic variants associated with blood pressure (BP), but sequence variation accounts for a small fraction of the phenotypic variance. Epigenetic changes may alter the expression of genes involved in BP regulation and explain part of the missing heritability. We therefore conducted a two-stage meta-analysis of the cross-sectional associations of systolic and diastolic BP with blood-derived genome-wide DNA methylation measured on the Infinium HumanMethylation450 BeadChip in 17,010 individuals of European, African American, and Hispanic ancestry. Of 31 discovery-stage cytosine-phosphate-guanine (CpG) dinucleotides, 13 replicated after Bonferroni correction (discovery: N = 9,828, p < 1.0 × 10-7; replication: N = 7,182, p < 1.6 × 10-3). The replicated methylation sites are heritable (h2 > 30%) and independent of known BP genetic variants, explaining an additional 1.4% and 2.0% of the interindividual variation in systolic and diastolic BP, respectively. Bidirectional Mendelian randomization among up to 4,513 individuals of European ancestry from 4 cohorts suggested that methylation at cg08035323 (TAF1B-YWHAQ) influences BP, while BP influences methylation at cg00533891 (ZMIZ1), cg00574958 (CPT1A), and cg02711608 (SLC1A5). Gene expression analyses further identified six genes (TSPAN2, SLC7A11, UNC93B1, CPT1A, PTMS, and LPCAT3) with evidence of triangular associations between methylation, gene expression, and BP. Additional integrative Mendelian randomization analyses of gene expression and DNA methylation suggested that the expression of TSPAN2 is a putative mediator of association between DNA methylation at cg23999170 and BP. These findings suggest that heritable DNA methylation plays a role in regulating BP independently of previously known genetic variants.

Empirical comparison of reduced representation bisulfite sequencing and Infinium BeadChip reproducibility and coverage of DNA methylation in humans.

We empirically examined the strengths and weaknesses of two human genome-wide DNA methylation platforms: rapid multiplexed reduced representation bisulfite sequencing and Illumina's Infinium BeadChip. Rapid multiplexed reduced representation bisulfite sequencing required less input DNA, offered more flexibility in coverage, and interrogated more CpG loci at a higher regional density. The Infinium covered slightly more protein coding, cancer-associated and mitochondrial-related genes, both platforms covered all known imprinting clusters, and rapid multiplexed reduced representation bisulfite sequencing covered more microRNA genes than the HumanMethylation450, but fewer than the MethylationEPIC. Rapid multiplexed reduced representation bisulfite sequencing did not always interrogate exactly the same CpG loci, but genomic tiling improved overlap between different libraries. Reproducibility of rapid multiplexed reduced representation bisulfite sequencing and concordance between the platforms increased with CpG density. Only rapid multiplexed reduced representation bisulfite sequencing could genotype samples and measure allele-specific methylation, and we confirmed that Infinium measurements are influenced by nearby single-nucleotide polymorphisms. The respective strengths and weaknesses of these two genome-wide DNA methylation platforms need to be considered when conducting human epigenetic studies.

Testing for the indirect effect under the null for genome-wide mediation analyses.

Mediation analysis helps researchers assess whether part or all of an exposure's effect on an outcome is due to an intermediate variable. The indirect effect can help in designing interventions on the mediator as opposed to the exposure and better understanding the outcome's mechanisms. Mediation analysis has seen increased use in genome-wide epidemiological studies to test for an exposure of interest being mediated through a genomic measure such as gene expression or DNA methylation (DNAm). Testing for the indirect effect is challenged by the fact that the null hypothesis is composite. We examined the performance of commonly used mediation testing methods for the indirect effect in genome-wide mediation studies. When there is no association between the exposure and the mediator and no association between the mediator and the outcome, we show that these common tests are overly conservative. This is a case that will arise frequently in genome-wide mediation studies. Caution is hence needed when applying the commonly used mediation tests in genome-wide mediation studies. We evaluated the performance of these methods using simulation studies, and performed an epigenome-wide mediation association study in the Normative Aging Study, analyzing DNAm as a mediator of the effect of pack-years on FEV1 .