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Under stress, certain eukaryotic proteins and RNA assemble to form membraneless organelles known as stress granules. The most well-studied stress granule components are RNA-binding proteins that undergo liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by intrinsically disordered low-complexity domains (LCDs). Here we show that stress granules include proteasomal shuttle factor UBQLN2, an LCD-containing protein structurally and functionally distinct from RNA-binding proteins. In vitro, UBQLN2 exhibits LLPS at physiological conditions. Deletion studies correlate oligomerization with UBQLN2's ability to phase-separate and form stress-induced cytoplasmic puncta in cells. Using nuclear magnetic resonance (NMR) spectroscopy, we mapped weak, multivalent interactions that promote UBQLN2 oligomerization and LLPS. Ubiquitin or polyubiquitin binding, obligatory for UBQLN2's biological functions, eliminates UBQLN2 LLPS, thus serving as a switch between droplet and disperse phases. We postulate that UBQLN2 LLPS enables its recruitment to stress granules, where its interactions with ubiquitinated substrates reverse LLPS to enable shuttling of clients out of stress granules., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Obesity is associated with higher cardio-metabolic risk even in childhood and adolescence; whether this association is mediated by epigenetic mechanisms remains unclear. We examined the extent to which mid-childhood body mass index (BMI) z-score (median age 7.7 years) was associated with cardio-metabolic risk score in early adolescence (median age 12.9 years) via mid-childhood DNA methylation among 265 children in the Project Viva. We measured DNA methylation in leukocytes using the Infinium Human Methylation450K BeadChip. We assessed mediation CpG-by-CpG using epigenome-wide association analyses, high-dimensional mediation analysis, and natural effect models. We observed mediation by mid-childhood DNA methylation at 6 CpGs for the association between mid-childhood BMI z-score and cardio-metabolic risk score in early adolescence in the high-dimensional mediation analysis (accounting for 10% of the total effect) and in the natural effect model (β = 0.04, P = 3.2e-2, accounting for 13% of the total effect). The natural direct effect of BMI z-score on cardio-metabolic risk score was still evident (β = 0.27, P = 1.1e-25). We also observed mediation by mid-childhood DNA methylation at 5 CpGs that was in the opposite direction from the total effect (natural effect model: β = -0.04, P = 2.0e-2). Mediation in different directions implies a complex role of DNA methylation in the association between BMI and cardio-metabolic risk and needs further investigation. Future studies with larger sample size and greater variability in cardio-metabolic risk will further help elucidate the role of DNA methylation for cardio-metabolic risk.

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, FEV 1 , FEF 25-75% ) 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.

Aim: We tested whether genetic variation in miRNA processing genes modified the association of PM 2.5 with DNA methylation (DNAm) age., Patients & Methods: We conducted a repeated measures study based on 552 participants from the Normative Aging Study with multiple visits between 2000 and 2011 (n = 940 visits). Address-level 1-year PM 2.5 exposures were estimated using the GEOS-chem model. DNAm-age and a panel of 14 SNPs in miRNA processing genes were measured from participant blood samples., Results & Conclusion: In fully adjusted linear mixed-effects models, having at least one copy of the minor rs4961280 [AGO2] allele was associated with a lower DNAm-age (β = -1.13; 95% CI: -2.26 to -0.002). However, the association of PM 2.5 with DNAm-age was significantly (P interaction  = 0.01) weaker in homozygous carriers of the major rs4961280 [AGO2] allele (β = 0.38; 95% CI: -0.20 to 0.96) when compared with all other participants (β = 1.58; 95% CI: 0.76 to 2.39). Our results suggest that miRNA processing impacts DNAm-age relationships. Graphical abstract: miRNA processing AGO2 polymorphism (rs4961280) modifies the association of long-term ambient fine particle exposure with blood DNA methylation age [Formula: see text] The graph depicts lines from a fully adjusted linear regression model with fine particle exposure levels ranging from the tenth to the ninetieth percentile, all other continuous variables held constant at their means, and all other categorical variables held at their most frequent level.

Background: Fine particulate matter (PM2.5) represents a mixture of components with potentially different toxicities. However, little is known about the relative effects of PM2.5 mass and PM2.5 components on mitochondrial DNA (mtDNA) abundance, which may lie on the pathway of PM2.5-associated disease., Methods: We studied 646 elderly male participants in the Normative Aging Study from Greater Boston to investigate associations of long-term exposure to PM2.5 mass and PM2.5 components with mtDNA abundance. We estimated concentrations of pollutants for the 365-day preceding examination at each participant's address using spatial- and temporal-resolved chemical transport models. We measured blood mtDNA abundance using RT-PCR. We applied a shrinkage and selection method (adaptive LASSO) to identify components most predictive of mtDNA abundance, and fit multipollutant linear mixed-effects models with subject-specific intercept to estimate the relative effects of individual PM component., Results: MtDNA abundance was negatively associated with PM2.5 mass in the previous year and-after adjusting for PM2.5 mass-several PM2.5 components, including organic carbon, sulfate (marginally), and nitrate. In multipollutant models including as independent variables PM2.5 mass and PM2.5 components selected by LASSO, nitrate was associated with mtDNA abundance. An SD increase in annual PM2.5-associated nitrate was associated with a 0.12 SD (95% confidence intervals [CI] = -0.18, -0.07) decrease in mtDNA abundance. Analyses restricted to PM2.5 annual concentration below the current 1-year U.S. Environmental Protection Agency standard produced similar results., Conclusions: Long-term exposures to PM2.5-associated nitrate were related to decreased mtDNA abundance independent of PM2.5 mass. Mass alone may not fully capture the potential of PM2.5 to oxidize the mitochondrial genome.See video abstract at, http://links.lww.com/EDE/B274.

Background: Air particulate matter (PM) is a ubiquitous environmental exposure associated with oxidation, inflammation, and age-related chronic disease. Whether PM is associated with loss of bone mineral density (BMD) and risk of bone fractures is undetermined., Methods: We conducted two complementary studies of: (i) long-term PM <2.5 μm (PM 2.5 ) levels and osteoporosis-related fracture hospital admissions among 9.2 million Medicare enrollees of the Northeast/Mid-Atlantic United States between 2003-2010; (ii) long-term black carbon [BC] and PM 2.5 levels, serum calcium homeostasis biomarkers (parathyroid hormone, calcium, and 25-hydroxyvitamin D), and annualized BMD reduction over a 8-year follow-up of 692 middle-aged (46.7±12.3 yrs), low-income BACH/Bone cohort participants., Findings: In the Medicare analysis, risk of bone fracture admissions at osteoporosis-related sites was greater in areas with higher PM 2.5 levels (Risk ratio [RR] 1.041, 95% Confidence Interval [CI], 1.030, 1.051). This risk was particularly high among low-income communities (RR 1.076; 95% CI, 1.052, 1.100). In the longitudinal BACH/Bone study, baseline BC and PM 2.5 levels were associated with lower serum PTH (Estimate for baseline one interquartile increase in 1-year average BC= -1.16, 95% CI -1.93, -0.38; Estimate for baseline one interquartile increase in 1-year average PM 2.5 = -7.39; 95%CI -14.17, -0.61). BC level was associated with higher BMD loss over time at multiple anatomical sites, including femoral neck (-0.08%/year per one interquartile increase; 95% CI -0.14, -0.02%/year) and ultradistal radius (-0.06%/year per one interquartile increase; 95% CI -0.12, -0.01%/year)., Interpretation: Our results suggest that poor air quality is a modifiable risk factor for bone fractures and osteoporosis, especially in low-income communities., Competing Interests: DECLARATION OF INTEREST We declare that we have no conflicts of interest.

Background: Early-life exposure to lead is associated with deficits in neurodevelopment and with hematopoietic system toxicity. DNA methylation may be one of the underlying mechanisms for the adverse effects of prenatal lead on the offspring, but epigenome-wide methylation data for low levels of prenatal lead exposure are lacking., Objectives: We investigated the association between prenatal maternal lead exposure and epigenome-wide DNA methylation in umbilical cord blood nucleated cells in Project Viva, a prospective U.S.-based prebirth cohort with relatively low levels of lead exposure., Methods: Among 268 mother-infant pairs, we measured lead concentrations in red blood cells (RBC) from prenatal maternal blood samples, and using HumanMethylation450 Bead Chips, we measured genome-wide methylation levels at 482,397 CpG loci in umbilical cord blood and retained 394,460 loci after quality control. After adjustment for batch effects, cell types, and covariates, we used robust linear regression models to examine associations of prenatal lead exposure with DNA methylation in cord blood at epigenome-wide significance level [false discovery rate (FDR)<0.05]., Results: The mean [standard deviation (SD)] maternal RBC lead level was 1.22 (0.63) μg/dL. CpG cg10773601 showed an epigenome-wide significant negative association with prenatal lead exposure (-1.4% per doubling increase in lead exposure; p=2.3×10-7) and was annotated to C-Type Lectin Domain Family 11, Member A ( CLEC11A ), which functions as a growth factor for primitive hematopoietic progenitor cells. In sex-specific analyses, we identified more CpGs with FDR<0.05 among female infants (n=38) than among male infants (n=2). One CpG (cg24637308), which showed a strong negative association with prenatal lead exposure among female infants (-4.3% per doubling increase in lead exposure; p=1.1×10-06), was annotated to Dynein Heavy Chain Domain 1 gene ( DNHD1 ) which is highly expressed in human brain. Interestingly, there were strong correlations between blood and brain methylation for CpG (cg24637308) based on another independent set of samples with a high proportion of female participants., Conclusion: Prenatal low-level lead exposure was associated with newborn DNA methylation, particularly in female infants. https://doi.org/10.1289/EHP1246.

In this article, we consider variable selection for correlated high dimensional DNA methylation markers as multivariate outcomes. A novel weighted square-root LASSO procedure is proposed to estimate the regression coefficient matrix. A key feature of this method is tuning-insensitivity, which greatly simplifies the computation by obviating cross validation for penalty parameter selection. A precision matrix obtained via the constrained ℓ1 minimization method is used to account for the within-subject correlation among multivariate outcomes. Oracle inequalities of the regularized estimators are derived. The performance of our proposed method is illustrated via extensive simulation studies. We apply our method to study the relation between smoking and high dimensional DNA methylation markers in the Normative Aging Study (NAS).

The mitochondrial genome has long been implicated in age-related disease, but no studies have examined its role in the relationship of long-term fine particle (PM 2.5 ) exposure and DNA methylation age (DNAm-age)-a novel measure of biological age. In this analysis based on 940 observations between 2000 and 2011 from 552 Normative Aging Study participants, we determined the roles of mitochondrial DNA haplogroup variation and mitochondrial genome abundance in the relationship of PM 2.5 with DNAm-age. We used the GEOS-chem transport model to estimate address-specific, one-year PM 2.5 levels for each participant. DNAm-age and mitochondrial DNA markers were measured from participant blood samples. Nine haplogroups (H, I, J, K, T, U, V, W, and X) were present in the population. In fully adjusted linear mixed-effects models, the association of PM 2.5 with DNAm-age (in years) was significantly diminished in carriers of haplogroup V (P interaction = 0.01; β = 0.18, 95%CI: -0.41, 0.78) compared to noncarriers (β = 1.25, 95%CI: 0.58, 1.93). Mediation analysis estimated that decreases in mitochondrial DNA copy number, a measure of mitochondrial genome abundance, mediated 12% of the association of PM 2.5 with DNAm-age. Our data suggests that the mitochondrial genome plays a role in DNAm-age relationships particularly in the context of long-term PM 2.5 exposure.

Recent studies have reported robust associations of long-term PM2.5 exposure with DNA methylation-based measures of aging; yet, the molecular implications of these relationships remain poorly understood. We evaluated if genetic variation in 3 biological pathways implicated in PM2.5-related disease-oxidative stress, endothelial function, and metal processing-could modify the effect of PM2.5 on DNAm-age, one prominent DNA methylation-based measure of biological age. This analysis was based on 552 individuals from the Normative Aging Study with at least one visit between 2000 and 2011 (n = 940 visits). A genetic-score approach was used to calculate aging-risk variant scores for endothelial function, oxidative stress, and metal processing pathways. One-year PM2.5 and PM2.5 component (sulfate and ammonium) levels at participants' addresses were estimated using the GEOS-chem transport model. Blood DNAm-age was calculated using CpG sites on the Illumina HumanMethylation450 BeadChip. In fully-adjusted linear mixed-effects models, the effects of sulfate on DNAm-age (in years) were greater in individuals with high aging-risk endothelial function variant scores when compared with individuals with low aging-risk endothelial function variant scores (Pinteraction = 0.0007; βHigh = 1.09, 95% CIHigh: 0.70, 1.48; βLow = 0.40, 95% CILow: 0.14, 0.67). Similar trends were observed in fully adjusted models of ammonium and total PM2.5 alone. No effect modification was observed by oxidative stress and metal processing variant scores. Secondary analyses revealed significant associations of serum endothelial markers, intercellular adhesion molecule-1 (β = 0.01, 95% CI: 0.002, 0.012) and vascular cell adhesion molecule-1 (β = 0.002, 95% CI: 0.0005, 0.0026), with DNAm-age. Our results add novel evidence that endothelial physiology may be important to DNAm-age relationships, but further research is required to establish their generalizability., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Long-term PM 2.5 exposure and aging have been implicated in multiple shared diseases; studying their relationship is a promising strategy to further understand the adverse impact of PM 2.5 on human health., Objective: We assessed the relationship of major PM 2.5 component species (ammonium, elemental carbon, organic carbon, nitrate, and sulfate) with Horvath and Hannum DNA methylation (DNAm) age, two DNA methylation-based predictors of chronological age., Methods: This analysis included 552 participants from the Normative Aging Study with multiple visits between 2000 and 2011 (n=940 visits). We estimated 1-year PM 2.5 species levels at participants' addresses using the GEOS-chem transport model. Blood DNAm-age was calculated using CpG sites on the Illumina HumanMethylation450 BeadChip. We fit linear mixed-effects models, controlling for PM 2.5 mass and lifestyle/environmental factors as fixed effects, with the adaptive LASSO penalty to identify PM 2.5 species associated with DNAm-age., Results: Sulfate and ammonium were selected by the LASSO in the Horvath DNAm-age models. In a fully-adjusted multiple-species model, interquartile range increases in both 1-year sulfate (95%CI: 0.28, 0.74, P<0.0001) and ammonium (95%CI: 0.02, 0.70, P=0.04) levels were associated with at least a 0.36-year increase in Horvath DNAm-age. No PM 2.5 species were selected by the LASSO in the Hannum DNAm-age models. Our findings persisted in sensitivity analyses including only visits with 1-year PM 2.5 levels within US EPA national ambient air quality standards., Conclusion: Our results demonstrate that sulfate and ammonium were most associated with Horvath DNAm-age and suggest that DNAm-age measures differ in their sensitivity to ambient particle exposures and potentially disease., (Published by Elsevier Ltd.)

Environmental noise exposure is associated with adverse effects on human health including hearing loss, heart disease, and changes in stress-related hormone levels. Alteration in DNA methylation in response to environmental exposures is a well-known phenomenon and it is implicated in many human diseases. Understanding how environmental noise exposures affect DNA methylation patterns may help to elucidate the link between noise and adverse effects on health. In this pilot study we examined the effects of environmental noise exposure on DNA methylation of genes related to brain function and investigated whether these changes are related with metabolic health. We exposed four groups of male Wistar rats to moderate intensity noise (70-75dB with 20-4000Hz) at night for three days as short-term exposure, and for three weeks as long-term exposure. Noise exposure was limited to 45dB during the daytime. Control groups were exposed to only 45dB, day and night. We measured DNA methylation in the Bdnf, Comt, Crhr1, Mc2r, and Snca genes in tissue from four brain regions of the rats (hippocampus, frontal lobe, medulla oblongata, and inferior colliculus). Further, we measured blood pressure and body weight after long-term noise exposure. We found that environmental noise exposure is associated with gene-specific DNA methylation changes in specific regions of the brain. Changes in DNA methylation are significantly associated with changes in body weight (between Bdnf DNA methylation and Δ body weight: r=0.59, p=0.018; and between LINE-1 ORF DNA methylation and Δ body weight: =-0.80, p=0.0004). We also observed that noise exposure decreased blood pressure (p=0.038 for SBP, p=0.017 for DBP and p 0. 017 for MAP) and decreased body weight (β=-26g, p=0.008). In conclusion, environmental noise exposures can induce changes in DNA methylation in the brain, which may be associated with adverse effects upon metabolic health through modulation of response to stress-related hormones., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Ambient air pollution and temperature have been linked with cardiovascular morbidity and mortality. Metabolic syndrome and its components-abdominal obesity, elevated fasting blood glucose concentration, low high-density lipoprotein cholesterol concentration, hypertension, and hypertriglyceridemia-predict cardiovascular disease, but the environmental causes are understudied. In this study, we prospectively examined the long-term associations of air pollution, defined as particulate matter with an aerodynamic diameter less than or equal to 2.5 µm (PM 2.5 ), and temperature with the development of metabolic syndrome and its components. Using covariate-adjustment Cox proportional hazards models, we estimated associations of mean annual PM 2.5 concentration and temperature with risk of incident metabolic dysfunctions between 1993 and 2011 in 587 elderly (mean = 70 (standard deviation, 7) years of age) male participants in the Normative Aging Study. A 1-μg/m 3 increase in mean annual PM 2.5 concentration was associated with a higher risk of developing metabolic syndrome (hazard ratio (HR) = 1.27, 95% confidence interval (CI): 1.06, 1.52), an elevated fasting blood glucose level (HR = 1.20, 95% CI: 1.03, 1.39), and hypertriglyceridemia (HR = 1.14, 95% CI: 1.00, 1.30). Our findings for metabolic syndrome and high fasting blood glucose remained significant for PM 2.5 levels below the Environmental Protection Agency's health-safety limit (12 μg/m 3 ). A 1°C increase in mean annual temperature was associated with a higher risk of developing elevated fasting blood glucose (HR = 1.33, 95% CI: 1.14, 1.56). Men living in neighborhoods with worse air quality-with higher PM 2.5 levels and/or temperatures than average-showed increased risk of developing metabolic dysfunctions., (© The Author 2016. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Long-term air pollution exposure has been associated with age-related cognitive impairment, possibly because of enhanced inflammation. Leukocytes with longer telomere length (TL) are more responsive to inflammatory stimuli, yet TL has not been evaluated in relation to air pollution and cognition., Objectives: We assessed whether TL modifies the association of 1-year exposure to black carbon (BC), a marker of traffic-related air pollution, with cognitive function in older men, and we examined whether this modification is independent of age and of C-reactive protein (CRP), a marker of inflammation., Methods: Between 1999 and 2007, we conducted 1-3 cognitive examinations of 428 older men in the Veterans Affairs (VA) Normative Aging Study. We used covariate-adjusted repeated-measure logistic regression to estimate associations of 1-year BC exposure with relative odds of being a low scorer (≤ 25) on the Mini-Mental State Examination (MMSE), which is a proxy of poor cognition. Confounders included age, CRP, and lifestyle and sociodemographic factors., Results: Each doubling in BC level was associated with 1.57 (95% CI: 1.20, 2.05) times higher odds of low MMSE scores. The BC-MMSE association was greater only among individuals with longer blood TL (5th quintile) (OR = 3.23; 95% CI: 1.37, 7.59; p = 0.04 for BC-by-TL-interaction). TL and CRP were associated neither with each other nor with MMSE. However, CRP modified the BC-MMSE relationship, with stronger associations only at higher CRP (5th quintile) and reference TL level (1st quintile) (OR = 2.68; 95% CI: 1.06, 6.79; p = 0.04 for BC-by-CRP-interaction)., Conclusions: TL and CRP levels may help predict the impact of BC exposure on cognitive function in older men. Citation: Colicino E, Wilson A, Frisardi MC, Prada D, Power MC, Hoxha M, Dioni L, Spiro A III, Vokonas PS, Weisskopf MG, Schwartz JD, Baccarelli AA. 2017. Telomere length, long-term black carbon exposure, and cognitive function in a cohort of older men: the VA Normative Aging Study. Environ Health Perspect 125:76-81; http://dx.doi.org/10.1289/EHP241., Competing Interests: The views expressed in this paper are those of the authors and do not necessarily represent the views of the U.S. Department of VA. The authors declare they have no actual or potential competing financial interests.

Background: Chronic low-grade inflammation reflects a subclinical immune response implicated in the pathogenesis of complex diseases. Identifying genetic loci where DNA methylation is associated with chronic low-grade inflammation may reveal novel pathways or therapeutic targets for inflammation., Results: We performed a meta-analysis of epigenome-wide association studies (EWAS) of serum C-reactive protein (CRP), which is a sensitive marker of low-grade inflammation, in a large European population (n = 8863) and trans-ethnic replication in African Americans (n = 4111). We found differential methylation at 218 CpG sites to be associated with CRP (P < 1.15 × 10 -7 ) in the discovery panel of European ancestry and replicated (P < 2.29 × 10 -4 ) 58 CpG sites (45 unique loci) among African Americans. To further characterize the molecular and clinical relevance of the findings, we examined the association with gene expression, genetic sequence variants, and clinical outcomes. DNA methylation at nine (16%) CpG sites was associated with whole blood gene expression in cis (P < 8.47 × 10 -5 ), ten (17%) CpG sites were associated with a nearby genetic variant (P < 2.50 × 10 -3 ), and 51 (88%) were also associated with at least one related cardiometabolic entity (P < 9.58 × 10 -5 ). An additive weighted score of replicated CpG sites accounted for up to 6% inter-individual variation (R2) of age-adjusted and sex-adjusted CRP, independent of known CRP-related genetic variants., Conclusion: We have completed an EWAS of chronic low-grade inflammation and identified many novel genetic loci underlying inflammation that may serve as targets for the development of novel therapeutic interventions for inflammation.

Long-term exposure to air pollution is associated with age-related diseases. We explored the association between accelerated biological aging and air pollution, a potential mechanism linking air pollution and health. We estimated long-term exposure to PM10, PM2.5, PM2.5 absorbance/black carbon (BC), and NOx via land-use regression models in individuals from the KORA F4 cohort. Accelerated biological aging was assessed using telomere length (TeloAA) and three epigenetic measures: DNA methylation age acceleration (DNAmAA), extrinsic epigenetic age acceleration (correlated with immune cell counts, EEAA), and intrinsic epigenetic age acceleration (independent of immune cell counts, IEAA). We also investigated sex-specific associations between air pollution and biological aging, given the published association between sex and aging measures. In KORA an interquartile range (0.97 µg/m3) increase in PM2.5 was associated with a 0.33 y increase in EEAA (CI = 0.01, 0.64; P = 0.04). BC and NOx (indicators or traffic exposure) were associated with DNAmAA and IEAA in women, while TeloAA was inversely associated with BC in men. We replicated this inverse BC-TeloAA association in the Normative Aging Study, a male cohort based in the USA. A multiple phenotype analysis in KORA F4 combining all aging measures showed that BC and PM10 were broadly associated with biological aging in men. Thus, we conclude that long-term exposure to air pollution is associated with biological aging measures, potentially in a sex-specific manner. However, many of the associations were relatively weak and further replication of overall and sex-specific associations is warranted.

Background: Among nondiabetic individuals, higher fasting blood glucose (FBG) independently predicts diabetes risk, cardiovascular disease, and dementia. Ambient PM2.5 (particulate matter with aerodynamic diameter ≤ 2.5 μm) is an emerging determinant of glucose dysregulation. PM2.5 effects and mechanisms are understudied among nondiabetic individuals., Objectives: Our goals were to investigate whether PM2.5 is associated with an increase in FBG and to explore potential mediating roles of epigenetic gene regulation., Methods: In 551 nondiabetic participants in the Normative Aging Study, we measured FBG, and DNA methylation of four inflammatory genes (IFN-γ, IL-6, ICAM-1, and TLR-2), up to four times between 2000 and 2011 (median = 2). We estimated short- and medium-term (1-, 7-, and 28-day preceding each clinical visit) ambient PM2.5 at each participant's address using a validated hybrid land-use regression satellite-based model. We fitted covariate-adjusted regression models accounting for repeated measures., Results: Mean FBG was 99.8 mg/dL (SD = 10.7), 18% of the participants had impaired fasting glucose (IFG; i.e., 100-125 mg/dL FBG) at first visit. Interquartile increases in 1-, 7-, and 28-day PM2.5 were associated with 0.57 mg/dL (95% CI: 0.02, 1.11, p = 0.04), 1.02 mg/dL (95% CI: 0.41, 1.63, p = 0.001), and 0.89 mg/dL (95% CI: 0.32, 1.47, p = 0.003) higher FBG, respectively. The same PM2.5 metrics were associated with 13% (95% CI: -3%, 33%, p = 0.12), 27% (95% CI: 6%, 52%, p = 0.01) and 32% (95% CI: 10%, 58%, p = 0.003) higher odds of IFG, respectively. PM2.5 was negatively correlated with ICAM-1 methylation (p = 0.01), but not with other genes. Mediation analysis estimated that ICAM-1 methylation mediated 9% of the association of 28-day PM2.5 with FBG., Conclusions: Among nondiabetics, short- and medium-term PM2.5 were associated with higher FBG. Mediation analysis indicated that part of this association was mediated by ICAM-1 promoter methylation. Citation: Peng C, Bind MA, Colicino E, Kloog I, Byun HM, Cantone L, Trevisi L, Zhong J, Brennan K, Dereix AE, Vokonas PS, Coull BA, Schwartz JD, Baccarelli AA. 2016. Particulate air pollution and fasting blood glucose in nondiabetic individuals: associations and epigenetic mediation in the Normative Aging Study, 2000-2011. Environ Health Perspect 124:1715-1721; http://dx.doi.org/10.1289/EHP183., Competing Interests: The views expressed in this paper are those of the authors and do not necessarily represent the views of the U.S. Department of Veterans Affairs. The authors declare they have no actual or potential competing financial interests.

Background: Continuing chronic and sporadic high-level of lead exposure in some regions in the U.S. has directed public attention to the effects of lead on human health. Long-term lead exposure has been associated with faster cognitive decline in older individuals; however, genetic susceptibility to lead-related cognitive decline during aging has been poorly studied., Methods: We determined the interaction of APOE-epsilon variants and environmental lead exposure in relation to age-related cognitive decline. We measured tibia bone lead by K-shell-x-ray fluorescence, APOE-epsilon variants by multiplex PCR and global cognitive z-scores in 489 men from the VA-Normative Aging Study. To determine global cognitive z-scores we incorporated multiple cognitive assessments, including word list memory task, digit span backwards, verbal fluency test, sum of drawings, and pattern comparison task, which were assessed at multiple visits. We used linear mixed-effect models with random intercepts for individual and for cognitive test., Results: An interquartile range (IQR:14.23μg/g) increase in tibia lead concentration was associated with a 0.06 (95% confidence interval [95%CI]: -0.11 to -0.01) lower global cognition z-score. In the presence of both ε4 alleles, one IQR increase in tibia lead was associated with 0.57 (95%CI: -0.97 to -0.16; p-value for interaction: 0.03) lower total cognition z-score. A borderline association was observed in presence of one ε4 allele (Estimate-effect per 1-IQR increase: -0.11, 95%CI: -0.22, 0.01) as well as lack of association in individuals without APOE ε4 allele., Conclusions: Our findings suggest that individuals carrying both ε4 alleles are more susceptible to lead impact on global cognitive decline during aging., Competing Interests: The authors have no conflict of interest to disclose., (Copyright © 2016. Published by Elsevier Inc.)

Motivation: High-dimensional DNA methylation markers may mediate pathways linking environmental exposures with health outcomes. However, there is a lack of analytical methods to identify significant mediators for high-dimensional mediation analysis., Results: Based on sure independent screening and minimax concave penalty techniques, we use a joint significance test for mediation effect. We demonstrate its practical performance using Monte Carlo simulation studies and apply this method to investigate the extent to which DNA methylation markers mediate the causal pathway from smoking to reduced lung function in the Normative Aging Study. We identify 2 CpGs with significant mediation effects., Availability and Implementation: R package, source code, and simulation study are available at https://github.com/YinanZheng/HIMA CONTACT: lei.liu@northwestern.edu., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: DNA methylation leaves a long-term signature of smoking exposure and is one potential mechanism by which tobacco exposure predisposes to adverse health outcomes, such as cancers, osteoporosis, lung, and cardiovascular disorders., Methods and Results: To comprehensively determine the association between cigarette smoking and DNA methylation, we conducted a meta-analysis of genome-wide DNA methylation assessed using the Illumina BeadChip 450K array on 15 907 blood-derived DNA samples from participants in 16 cohorts (including 2433 current, 6518 former, and 6956 never smokers). Comparing current versus never smokers, 2623 cytosine-phosphate-guanine sites (CpGs), annotated to 1405 genes, were statistically significantly differentially methylated at Bonferroni threshold of P<1×10 -7 (18 760 CpGs at false discovery rate <0.05). Genes annotated to these CpGs were enriched for associations with several smoking-related traits in genome-wide studies including pulmonary function, cancers, inflammatory diseases, and heart disease. Comparing former versus never smokers, 185 of the CpGs that differed between current and never smokers were significant P<1×10 -7 (2623 CpGs at false discovery rate <0.05), indicating a pattern of persistent altered methylation, with attenuation, after smoking cessation. Transcriptomic integration identified effects on gene expression at many differentially methylated CpGs., Conclusions: Cigarette smoking has a broad impact on genome-wide methylation that, at many loci, persists many years after smoking cessation. Many of the differentially methylated genes were novel genes with respect to biological effects of smoking and might represent therapeutic targets for prevention or treatment of tobacco-related diseases. Methylation at these sites could also serve as sensitive and stable biomarkers of lifetime exposure to tobacco smoke., Competing Interests: B. M. P. serves on Data Safety Monitoring Board (DSMB) of a clinical trial of a device funded by the manufacturer (Zoll LifeCor) and on the Steering Committee of the Yale Open Data Access Project funded by Johnson & Johnson. C.E.E. is currently employed by Astra Zeneca, although the work was completed prior to the employment. All other authors declare no conflicts of interest., (© 2016 American Heart Association, Inc.)

Estimates of biological age based on DNA methylation patterns, often referred to as "epigenetic age", "DNAm age", have been shown to be robust biomarkers of age in humans. We previously demonstrated that independent of chronological age, epigenetic age assessed in blood predicted all-cause mortality in four human cohorts. Here, we expanded our original observation to 13 different cohorts for a total sample size of 13,089 individuals, including three racial/ethnic groups. In addition, we examined whether incorporating information on blood cell composition into the epigenetic age metrics improves their predictive power for mortality. All considered measures of epigenetic age acceleration were predictive of mortality (p≤8.2x10 -9 ) , independent of chronological age, even after adjusting for additional risk factors (p<5.4x10 -4 ) , and within the racial/ethnic groups that we examined (non-Hispanic whites, Hispanics, African Americans). Epigenetic age estimates that incorporated information on blood cell composition led to the smallest p-values for time to death (p=7.5x10 -43 ). Overall, this study a) strengthens the evidence that epigenetic age predicts all-cause mortality above and beyond chronological age and traditional risk factors, and b) demonstrates that epigenetic age estimates that incorporate information on blood cell counts lead to highly significant associations with all-cause mortality., Competing Interests: The Regents of the University of California is the sole owner of a provisional patent application directed at the invention of measures of epigenetic age acceleration for which SH is a named inventor. The other authors declare no conflicts of interest.

Background: The mitochondrion is the primary target of oxidative stress in response to exogenous environments. Mitochondrial DNA (mtDNA) is independent from nuclear DNA and uses separate epigenetic machinery to regulate mtDNA methylation. The mtDNA damage induced by oxidative stress can cause mitochondrial dysfunction and is implicated in human diseases; however, mtDNA methylation has been largely overlooked in environmental studies relating to human disease. The purpose of this study was to examine the association between exposure to fine metal-rich particulates (particulate matter <2.5 µm in diameter [PM2.5]) from welding in a boilermaker union and blood mtDNA methylation in relation to heart rate variability., Methods and Results: Forty-eight healthy men were recruited on multiple sampling cycles at the Boilermaker Union Local 29, located in Quincy, Massachusetts. We measured personal PM2.5 in the background ambient environment. We measured blood mtDNA methylation in the mtDNA promoter (D-loop) and genes essential for ATP synthesis (MT-TF and MT-RNR1) by bisulfite pyrosequencing. All analyses were adjusted for demographics, type of job, season, welding-work day, and mtDNA methylation experimental batch effect. The participants' PM2.5 exposure was significantly higher after a welding-work day (mean 0.38 mg/m(3)) than the background personal level (mean 0.15 mg/m(3), P<0.001). Blood mtDNA methylation in the D-loop promoter was associated with PM2.5 levels (β=-0.99%, SE=0.41, P=0.02). MT-TF and MT-RNR1 methylation was not associated with PM2.5 exposure (β=0.10%, SE=0.45, P=0.82). Interaction of PM2.5 exposure levels and D-loop promoter methylation was significantly associated with markers of heart rate variability., Conclusions: Blood mtDNA methylation levels were negatively associated with PM2.5 exposure and modified the adverse relationships between PM2.5 exposure and heart rate variability outcomes., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Background: Ambient particles have been shown to exacerbate measures of biological aging; yet, no studies have examined their relationships with DNA methylation age (DNAm-age), an epigenome-wide DNA methylation based predictor of chronological age., Objective: We examined the relationship of DNAm-age with fine particulate matter (PM 2.5 ), a measure of total inhalable particle mass, and black carbon (BC), a measure of particles from vehicular traffic., Methods: We used validated spatiotemporal models to generate 1-year PM 2.5 and BC exposure levels at the addresses of 589 older men participating in the VA Normative Aging Study with 1-3 visits between 2000 and 2011 ( n = 1032 observations). Blood DNAm-age was calculated using 353 CpG sites from the Illumina HumanMethylation450 BeadChip. We estimated associations of PM 2.5 and BC with DNAm-age using linear mixed effects models adjusted for age, lifestyle/environmental factors, and aging-related diseases., Results: After adjusting for covariates, a 1-µg/m 3 increase in PM 2.5 (95% CI: 0.30, 0.75, P <0.0001) was significantly associated with a 0.52-year increase in DNAm-age. Adjusted BC models showed similar patterns of association (β = 3.02, 95% CI: 0.48, 5.57, P = 0.02). Only PM 2.5 (β = 0.54, 95% CI: 0.24, 0.84, P = 0.0004) remained significantly associated with DNAm-age in two-particle models. Methylation levels from 20 of the 353 CpGs contributing to DNAm-age were significantly associated with PM 2.5 levels in our two-particle models. Several of these CpGs mapped to genes implicated in lung pathologies including LZTFL1, PDLIM5 , and ATPAF1 ., Conclusion: Our results support an association of long-termambient particle levels with DNAm-age and suggest that DNAm-age is a biomarker of particle-related physiological processes.

Introduction: Air pollution exposure has been linked to impaired cognitive aging, but little is known about biomarkers modifying this association. MicroRNAs (miRNAs) control gene expression and neuronal programming. miRNA levels vary due to single nucleotide polymorphisms (SNPs) in genes processing miRNAs from precursor molecules., Objectives: To investigate whether SNPs in miRNA-processing genes are associated with cognition and modify the relationship between black carbon (BC), marker of traffic-related pollution, and cognitive functions., Methods: 533 Normative Aging Study men (mean±SD 72±7years) were tested ≤4 times (mean=1.7 times) using seven cognitive tests between 1995 and 2007. We tested interactions of 16 miRNA-related SNPs with 1-year average BC from a validated land-use-regression model. We used covariate-adjusted logistic regression for low (≤25) Mini-Mental tate Examination (MMSE) and mixed-effect regression for a global cognitive score combining six other tests., Results: Global cognition was negatively associated with the homozygous minor variant of rs595961 AGO1 (-0.42SD; 95%CI: (-0.71, -0.13)) relative to the major variant. BC-MMSE association was stronger in heterozygous carriers of rs11077 XPO5 (OR=1.99; 95%CI: (1.39, 2.85)) and minor variant carriers of GEMIN4 rs2740348 (OR=1.34; 95%CI: (1.05, 1.7)), compared to their major variant. The BC-global-cognition association was stronger in heterozygous carriers of GEMIN4 rs4968104 (-0.10SD; 95%CI: (-0.18, -0.02)), and GEMIN4 rs910924 (-0.09SD; 95%CI: (-0.17, -0.02)) relative to the major variant. Blood miRNA expression analyses showed associations only of XPO5 rs11077 with miR-9 and miR-96., Conclusions: Carriers of particular miRNA-processing SNPs had higher susceptibility to BC in BC-cognition associations, possibly due to influences on miRNA expression., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Biological measures of aging are important for understanding the health of an aging population, with epigenetics particularly promising. Previous studies found that tumor tissue is epigenetically older than its donors are chronologically. We examined whether blood Δage (the discrepancy between epigenetic and chronological ages) can predict cancer incidence or mortality, thus assessing its potential as a cancer biomarker. In a prospective cohort, Δage and its rate of change over time were calculated in 834 blood leukocyte samples collected from 442 participants free of cancer at blood draw. About 3-5 years before cancer onset or death, Δage was associated with cancer risks in a dose-responsive manner (P = 0.02) and a one-year increase in Δage was associated with cancer incidence (HR: 1.06, 95% CI: 1.02-1.10) and mortality (HR: 1.17, 95% CI: 1.07-1.28). Participants with smaller Δage and decelerated epigenetic aging over time had the lowest risks of cancer incidence (P = 0.003) and mortality (P = 0.02). Δage was associated with cancer incidence in a 'J-shaped' manner for subjects examined pre-2003, and with cancer mortality in a time-varying manner. We conclude that blood epigenetic age may mirror epigenetic abnormalities related to cancer development, potentially serving as a minimally invasive biomarker for cancer early detection.

The difference-in-differences (DID) approach is a well known strategy for estimating the effect of an exposure in the presence of unobserved confounding. The approach is most commonly used when pre-and post-exposure outcome measurements are available, and one can assume that the association of the unobserved confounder with the outcome is equal in the two exposure groups, and constant over time. Then, one recovers the treatment effect by regressing the change in outcome over time on the exposure. In this paper, we interpret the difference-in-differences as a negative outcome control (NOC) approach. We show that the pre-exposure outcome is a negative control outcome, as it cannot be influenced by the subsequent exposure, and it is affected by both observed and unobserved confounders of the exposure-outcome association of interest. The relation between DID and NOC provides simple conditions under which negative control outcomes can be used to detect and correct for confounding bias. However, for general negative control outcomes, the DID-like assumption may be overly restrictive and rarely credible, because it requires that both the outcome of interest and the control outcome are measured on the same scale. Thus, we present a scale-invariant generalization of the DID that may be used in broader NOC contexts. The proposed approach is demonstrated in simulations and on a Normative Aging Study data set, in which Body Mass Index is used for NOC of the relationship between air pollution and inflammatory outcomes.

The shape of the non-linear relationship between temperature and mortality varies among cities with different climatic conditions. There has been little examination of how these curves change over space and time. We evaluated the short-term effects of hot and cold temperatures on daily mortality over six 7-year periods in 211 US cities, comprising over 42 million deaths. Cluster analysis was used to group the cities according to similar temperatures and relative humidity. Temperature-mortality functions were calculated using B-splines to model the heat effect (lag 0) and the cold effect on mortality (moving average lags 1-5). The functions were then combined through meta-smoothing and subsequently analyzed by meta-regression. We identified eight clusters. At lag 0, Cluster 5 (West Coast) had a RR of 1.14 (95% CI: 1.11,1.17) for temperatures of 27 °C vs 15.6 °C, and Cluster 6 (Gulf Coast) has a RR of 1.04 (95% CI: 1.03,1.05), suggesting that people are acclimated to their respective climates. Controlling for cluster effect in the multivariate-meta regression we found that across the US, the excess mortality from a 24-h temperature of 27 °C decreased over time from 10.6% to 0.9%. We found that the overall risk due to the heat effect is significantly affected by summer temperature mean and air condition usage, which could be a potential predictor in building climate-change scenarios., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Polybrominated diphenyl ethers (PBDEs) are known endocrine disrupting chemicals used commonly as flame retardants in everything from electronics to furniture. Exposure to PBDEs during early development has been linked to neurodevelopmental delays. Despite mounting evidence of neurological harm from PBDE exposure, the molecular mechanisms underlying these effects on brain function remain unknown. We examined the effects of perinatal exposure to BDE-47, the most biologically active and prevalent BDE congener in North America, on epigenetic patterns in the frontal lobe of Wistar rats. Dams were gavaged with BDE-47 (0.002 and 0.2mg/kg body weight) at gestation days 9 and 16, and postnatal days 1, 8, and 15. Frontal lobes from offspring at postnatal day 41 were collected to measure 5-methylcytosine (5mC) in mitochondrial cytochrome c oxidase genes (Mt-co1, Mt-co2, and Mt-co3), global nuclear 5-hydroxymethylcytosine (5hmC) content, 5mC in repetitive elements L1Rn, and 5mC in nuclear genes (Bdnf, Crhr1, Mc2r, Nr3c1, and Snca) related to behavioral and brain functions in the nuclear genome. We observed a significant decrease in %5mC in Mt-co2 (difference from control=-0.68%, p=0.01 at the 0.2mg/kg BDE-47). 5mC in repetitive elements L1Rn decreased at 0.002 mg/kg BDE-47 (difference=-1.23%, p=0.02). Decreased nuclear 5mC was observed in Bdnf and Nr3c1 in BDE-47 exposed rats. However, we did not observe significant effects of PBDE toxicity on DNA methylation patterns for the majority of genes in the brain., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: DNA methylation levels change with age. Recent studies have identified biomarkers of chronological age based on DNA methylation levels. It is not yet known whether DNA methylation age captures aspects of biological age., Results: Here we test whether differences between people's chronological ages and estimated ages, DNA methylation age, predict all-cause mortality in later life. The difference between DNA methylation age and chronological age (Δage) was calculated in four longitudinal cohorts of older people. Meta-analysis of proportional hazards models from the four cohorts was used to determine the association between Δage and mortality. A 5-year higher Δage is associated with a 21% higher mortality risk, adjusting for age and sex. After further adjustments for childhood IQ, education, social class, hypertension, diabetes, cardiovascular disease, and APOE e4 status, there is a 16% increased mortality risk for those with a 5-year higher Δage. A pedigree-based heritability analysis of Δage was conducted in a separate cohort. The heritability of Δage was 0.43., Conclusions: DNA methylation-derived measures of accelerated aging are heritable traits that predict mortality independently of health status, lifestyle factors, and known genetic factors.

Background: Short-term fine particles (PM(2.5)) exposure is associated with reduced heart rate variability, a strong predictor of cardiac mortality among older people. Identifying modifiable factors that confer susceptibility is essential for intervention. We evaluated whether Toll-like receptor 2 (TLR2) methylation, a reversible immune-epigenetic process, and its dietary modulation by flavonoids and methyl nutrients, modify susceptibility to heart rate variability effects following PM(2.5) exposure., Methods and Results: We measured heart rate variability and PM(2.5) repeatedly over 11 years (1275 total observations) among 573 elderly men from the Normative Aging Study. Blood TLR2 methylation was analyzed using pyrosequencing. Daily flavonoid and methyl nutrients intakes were assessed through the Food Frequency Questionnaire (FFQ). Every 10 μg/m(3) increase in 48-hour PM(2.5) moving average was associated with 7.74% (95% CI: -1.21% to 15.90%; P=0.09), 7.46% (95% CI: 0.99% to 13.50%; P=0.02), 14.18% (95% CI: 1.14% to 25.49%; P=0.03), and 12.94% (95% CI: -2.36% to 25.96%; P=0.09) reductions in root mean square of successive differences, standard deviation of normal-to-normal intervals, low-frequency power, and high-frequency power, respectively. Higher TLR2 methylation exacerbated the root mean square of successive differences, standard deviation of normal-to-normal intervals, low-frequency, and high-frequency reductions associated with heightened PM2.5 (P(interaction)=0.006, 0.03, 0.05, 0.04, respectively). Every interquartile-range increase in flavonoid intake was associated with 5.09% reduction in mean TLR2 methylation (95% CI: 0.12% to 10.06%; P=0.05) and counteracted the effects of PM2.5 on low frequency (P(interaction)=0.05). No significant effect of methyl nutrients on TLR2 methylation was observed., Conclusions: Higher TLR2 methylation may confer susceptibility to adverse cardiac autonomic effects of PM2.5 exposure in older individuals. Higher flavonoid intake may attenuate these effects, possibly by decreasing TLR2 methylation., (© 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Background: APOE is the biomarker with the greatest known influence on cognitive function; however, the effect of complex haplotypes involving polymorphisms rs449647, rs405509, rs440446, rs429358 and rs7412 has never been studied in older populations., Methods: We evaluated APOE polymorphisms using multiplex PCR for genotyping and Mini-Mental State Examination (MMSE) to evaluate cognitive function in 819 individuals from VA Normative Aging Study., Results: Combinatorial analysis of all polymorphisms and individual analysis of polymorphisms rs449647, rs405509, rs440446 and rs7412 did not show any association with cognitive performance. Polymorphism rs429358 was associated with better cognitive performance (odds of MMSE ≤ 25 = 0.63, 95% CI 0.42-0.95; p = 0.03) in the oldest subsample (5th quintile of age) (odds of MMSE ≤ 25 = 0.34; 95% CI 0.13-0.86; p = 0.02). APOE allele ε4 was also associated with better cognitive performance (odds of MMSE ≤ 25 = 0.61, 95% CI 0.40-0.94; p = 0.02), also in the oldest subsample (odds of MMSE ≤ 25 = 0.35, 95% CI 0.14-0.90; p = 0.03)., Conclusions: These results suggest a beneficial effect of polymorphism rs429358 in the oldest men.

Background: Traffic-related air pollution has been linked with impaired cognition in older adults, possibly due to effects of oxidative stress on the brain. Mitochondria are the main source of cellular oxidation. Haplogroups in mitochondrial DNA (mtDNA) mark individual differences in oxidative potential and are possible determinants of neurodegeneration. The aim of this study was to investigate whether mtDNA haplogroups determined differential susceptibility to cognitive effects of long-term exposure to black carbon (BC), a marker of traffic-related air pollution., Methods: We investigated 582 older men (72 ± 7 years) in the VA Normative Aging Study cohort with ≤4 visits per participant (1.8 in average) between 1995-2007. Low (≤25) Mini Mental State Examination (MMSE) was used to assess impaired cognition in multiple domains. We fitted repeated-measure logistic regression using validated-LUR BC estimated in the year before their first visit at the participant's address., Results: Mitochondrial haplotyping identified nine haplogroups phylogenetically categorized in four clusters. BC showed larger effect on MMSE in Cluster 4 carriers, including I, W and X haplogroups, [OR = 2.7; 95% CI (1.3-5.6)], moderate effect in Cluster 1, including J and T haplogroups [OR = 1.6; 95% CI: (0.9-2.9)], and no effect in Cluster 2 (H and V haplogroups) [OR = 1.1; 95% CI: (0.8-1.5)] or Cluster 3 (K and U haplogroups) [OR = 1.0; 95% CI: (0.6-1.6)]. BC effect varied only moderately across the I, X, and W haplogroups or across the J and T haplogroups., Conclusions: The association of BC with impaired cognition was worsened in carriers of phylogenetically-related mtDNA haplogroups in Cluster 4. No BC effects were detected in Cluster 2 and 3 carriers. MtDNA haplotypes may modify individual susceptibility to the particle cognitive effects.

Background: The prognostic value of evoked potentials (EPs) in multiple sclerosis (MS) has not been fully established. The correlations between the Expanded Disability Status Scale (EDSS) at First Neurological Evaluation (FNE) and the duration of the disease, as well as between EDSS and EPs, have influenced the outcome of most previous studies. To overcome this confounding relations, we propose to test the prognostic value of EPs within an appropriate patient population which should be based on patients with low EDSS at FNE and short disease duration., Methods: We retrospectively selected a sample of 143 early relapsing remitting MS (RRMS) patients with an EDSS < 3.5 from a larger database spanning 20 years. By means of bivariate logistic regressions, the best predictors of worsening were selected among several demographic and clinical variables. The best multivariate logistic model was statistically validated and prospectively applied to 50 patients examined during 2009-2011., Results: The Evoked Potentials score (EP score) and the Time to EDSS 2.0 (TT2) were the best predictors of worsening in our sample (Odds Ratio 1.10 and 0.82 respectively, p=0.001). Low EP score (below 15-20 points), short TT2 (lower than 3-5 years) and their interaction resulted to be the most useful for the identification of worsening patterns. Moreover, in patients with an EP score at FNE below 6 points and a TT2 greater than 3 years the probability of worsening was 10% after 4-5 years and rapidly decreased thereafter., Conclusions: In an appropriate population of early RRMS patients, the EP score at FNE is a good predictor of disability at low values as well as in combination with a rapid buildup of disability. Interestingly, an EP score at FNE under the median together with a clinical stability lasting more than 3 years turned out to be a protective pattern. This finding may contribute to an early identification of benign patients, well before the term required to diagnose Benign MS (BMS).