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1.  Association between birth weight and DNA methylation of IGF2, glucocorticoid receptor and repetitive elements LINE-1 and Alu 
Epigenomics  2013;5(3):271-281.
Aim
We examined the association between birth weight and methylation in the imprinted IGF/H19 loci, the nonimprinted gene NR3C1 and repetitive element DNA (LINE-1 and Alu).
Materials & methods
We collected umbilical cord venous blood from 219 infants born in Mexico City (Mexico) as part of a prospective birth cohort study and analyzed DNA methylation using pyrosequencing.
Results
Birth weight was not associated with DNA methylation of the regions studied. One of the CpG dinucleotides in the IGF2 imprinting control region (ICR)1 includes a potential C–T SNP. Among individuals with an absence of methylation at this site, probably due to a paternally inherited T allele, birth weight was associated with mean methylation status of both IGF2 ICR1 and ICR2. However, this association would not have survived adjustment for multiple testing.
Conclusion
While we did not detect an association between DNA methylation and birth weight, our study suggests a potential gene–epigene interaction between a T allele in the IGF2 ICR1 and methylation of ICRs of IGF2, and fetal growth.
doi:10.2217/epi.13.24
PMCID: PMC3787720  PMID: 23750643
Alu; birth weight; DNA methylation; fetal growth; glucocorticoid receptor; IGF2; imprinting; LINE-1; NR3C1; SNP
2.  Blood Hypomethylation of Inflammatory Genes Mediates the Effects of Metal-rich Airborne Pollutants on Blood Coagulation 
Objectives
Recent investigations have associated airborne Particulate Matter (PM) with increased coagulation and thrombosis, but underlying biological mechanisms are still incompletely characterized. DNA methylation is an environmentally-sensitive mechanism of gene regulation that could potentially contribute to PM-induced hypercoagulability. We aimed to test whether altered methylation mediates environmental effects on coagulation.
Methods
We investigated 63 steel workers exposed to a wide range of PM levels, as a work-related condition with well-characterized prothrombotic exposure. We measured personal PM10 (PM≤10 μm in aerodynamic diameter), PM1 (≤1 μm), and air metal components. We determined leukocyte DNA methylation of NOS3 (nitric-oxide-synthase-3) and EDN1 (endothelin-1) through bisulfite-pyrosequencing and we measured Endogenous Thrombin Potential (ETP), as a global coagulation-activation test after standardized triggers.
Results
ETP increased in association with PM10 (β=20.0, 95%CI: 3.0, 37.0), PM1 (β=80.8 95%CI: 14.9, 146.7), and zinc (β=51.3, 95%CI: 0.01, 111.1) exposures. NOS3 methylation was negatively associated with PM10 (β=−0.2, 95%CI: −0.4, −0.03), PM1 (β=−0.8, 95%CI: −1.4, −0.1), zinc (β=−0.9, 95%CI: −1.4, −0.3) and iron (β=−0.7, 95%CI: −1.4, −0.01) exposures. Zinc exposure was negatively associated with EDN1 (β=−0.3, 95%CI: −0.8, −0.1) methylation. Lower NOS3 (β=−42.3; p<0.001) and EDN1 (β=−14.5; p=0.05) were associated with higher ETP. Statistical mediation analysis formally confirmed NOS3 and EDN1 hypomethylation as intermediate mechanisms for PM-related coagulation effects.
Conclusions
Our study showed for the first time, that gene hypomethylation contributes to environmentally-induced hypercoagulability.
doi:10.1136/oemed-2012-101079
PMCID: PMC3963398  PMID: 23476046
Air pollution; DNA methylation; coagulation
3.  Association between blood pressure and DNA methylation of retrotransposons and pro-inflammatory genes 
Background Methylation of deoxyribonucleic acid (DNA) is an epigenetic regulator of gene expression that changes with age, but its contribution to aging-related disorders, including high blood pressure (BP), is still largely unknown. We examined the relation of BP to the methylation of retrotransposon sequences of DNA and of selected candidate genes.
Methods This investigation included 789 elderly participants in the Normative Aging Study, ranging in age from 55 to 100 years, who had longitudinal measurements of DNA methylation. In these subjects’ DNA we measured the proportion of methylated sites in retrotransposable sequences and in pro-inflammatory genes, expressed as the percent of 5-methylated cytosines (%5mC) among all cytosines. From one to four methylation measurements were made for each subject between 1999 and 2009. We fit mixed-effects models, using repeated measures of BP as the outcome and DNA methylation as the explanatory variable, adjusting for confounding variables. We also fit a Bayesian mixed-effects structural equation model to account for heterogeneity in the effects of methylation sites within each gene.
Results An increase in inter-quartile range (IQR) in the methylation of Alu elements was associated with an increase of 0.97 mm Hg in diastolic blood pressure (DBP) (95% CI 0.32–1.57), but no such association was observed for long interspersed nuclear element-1 (LINE-1). We also found positive associations between DBP and methylation of the genes for toll-like receptor 2 (TLR2) and inducible nitric oxide synthase (iNOS), and a negative association between DBP and methylation of the gene for interferon-γ (IFN-γ). Associations between methylation and systolic blood pressure (SBP) were weaker than those between methylation and DBP. Bayesian mixed-effects structural equation model results were similar for both DBP and SBP models.
Conclusions The results of our study suggest that changes in DNA methylation of some pro-inflammatory genes and retrotransposable elements are related to small changes in BP.
doi:10.1093/ije/dys220
PMCID: PMC3600626  PMID: 23508416
Epigenetics; DNA methylation; blood pressure; inflammation; Bayesian model
4.  Allergen sensitization is associated with increased DNA methylation in older men 
Background
Variation in epigenetic modifications, arising from either environmental exposures or internal physiological changes, can influence gene expression, and may ultimately contribute to complex diseases such as asthma and allergies. We examined the association of asthma and allergic phenotypes with DNA methylation levels of retrotransposon-derived elements.
Methods
We used data from 704 men (mean age 73) in the longitudinal Normative Aging Study to assess the relationship between asthma, allergic phenotypes and DNA methylation levels of the retrotransposon derived elements Alu and LINE-1. Retrotransposons represent a large fraction of the genome (> 30%), and are heavily methylated to prevent expression. Percent methylation of Alu and LINE-1 elements in peripheral white blood cells was quantified using PCR pyrosequencing. Data on sensitization to common allergens by skin prick testing, asthma, and methacholine responsiveness was gathered approximately 8 years prior to DNA methylation analysis.
Results
Prior allergen sensitization was associated with increased methylation of Alu (β=0.32 [sensitized vs. non-sensitized], p value 0.003), in models adjusted for pack-years, BMI, smoking, air pollutants, percent eosinophils, white blood cell count and age. Of the men interviewed, 5 % of subjects reported diagnosis of asthma. Neither Alu, nor LINE-1 methylation was associated with asthma.
Conclusions
These data suggest that increased DNA methylation of repetitive elements may be associated with allergen sensitization, but does not appear to be associated with asthma. Future work is needed to identify potential underlying mechanisms for these relationships.
doi:10.1159/000343004
PMCID: PMC3730837  PMID: 23257623
allergen sensitization; DNA methylation; Alu; and LINE-1
5.  Repetitive element hypomethylation in blood leukocyte DNA and cancer incidence, prevalence and mortality in elderly individuals: the Normative Aging Study 
Cancer causes & control : CCC  2010;22(3):437-447.
Background
Global genomic hypomethylation is a common epigenetic event in cancer that mostly results from hypomethylation of repetitive DNA elements. Case-control studies have associated blood leukocyte DNA hypomethylation with several cancers. Because samples in case-control studies are collected after disease development, whether DNA hypomethylation is causal or just associated with cancer development is still unclear.
Methods
In 722 elderly subjects from the Normative Aging Study cohort, we examined whether DNA methylation in repetitive elements (Alu, LINE-1) was associated with cancer incidence (30 new cases, median follow-up: 89 months), prevalence (205 baseline cases), and mortality (28 deaths, median follow-up: 85 months). DNA methylation was measured by bisulfite pyrosequencing.
Results
Individuals with low LINE-1 methylation (
Conclusion
These findings suggest that individuals with lower repetitive element methylation are at high risk of developing and dying from cancer.
doi:10.1007/s10552-010-9715-2
PMCID: PMC3752839  PMID: 21188491
Repetitive elements; DNA methylation; Epigenetics; Blood; Cancer risk
Epidemiology (Cambridge, Mass.)  2010;21(6):819-828.
Background
Epigenetic features such as DNA hypomethylation have been associated with conditions related to cardiovascular risk. We evaluated whether lower blood DNA methylation in heavily methylated repetitive sequences predicts the risk of ischemic heart disease and stroke.
Methods
We quantified blood DNA methylation of LINE-1 repetitive elements through PCR-pyrosequencing in 712 elderly individuals from the Boston-area Normative Aging Study. We estimated risk-factor adjusted relative risks (RRs) for ischemic heart disease and stroke at baseline (242 prevalent cases); as well as in incidence (44 new cases; median follow-up, 63 months); and subsequent mortality from ischemic heart disease (86 deaths; median follow-up, 75 months).
Results
Blood LINE-1 hypomethylation was associated with baseline ischemic heart disease (RR=2.1 [95% confidence interval = 1.2 to 4.0] for lowest vs. highest methylation quartile) and for stroke (2.5 [0.9 to 7.5]). Among participants free of baseline disease, individuals with methylation below the median also had higher risk of developing ischemic heart disease (4.0 [1.8 to 8.9]) or stroke (5.7 [0.8 to 39.5]). In the entire cohort, persons with methylation below the median had higher mortality from ischemic heart disease (3.3 [1.3 to 8.4]) and stroke (2.8 [0.6 to 14.3]). Total mortality was also increased (2.0 [1.2 to 3.3]). These results were confirmed in additional regression models using LINE-1 methylation as a continuous variable.
Conclusions
Subjects with prevalent IHD and stroke exhibited lower LINE-1 methylation. In longitudinal analyses, persons with lower LINE-1 methylation were at higher risk for incident ischemic heart disease and stroke, and for total mortality.
doi:10.1097/EDE.0b013e3181f20457
PMCID: PMC3690659  PMID: 20805753
Environmental Health  2013;12:47.
Background
Exposure to pollutants including metals and particulate air pollution can alter DNA methylation. Yet little is known about intra-individual changes in DNA methylation over time in relationship to environmental exposures. Therefore, we evaluated the effects of acute- and chronic metal-rich PM2.5 exposures on DNA methylation.
Methods
Thirty-eight male boilermaker welders participated in a panel study for a total of 54 person days. Whole blood was collected prior to any welding activities (pre-shift) and immediately after the exposure period (post-shift). The percentage of methylated cytosines (%mC) in LINE-1, Alu, and inducible nitric oxide synthase gene (iNOS) were quantified using pyrosequencing. Personal PM2.5 (particulate matter with an aerodynamic diameter ≤ 2.5 μm) was measured over the work-shift. A questionnaire assessed job history and years worked as a boilermaker. Linear mixed models with repeated measures evaluated associations between DNA methylation, PM2.5 concentration (acute exposure), and years worked as a boilermaker (chronic exposure).
Results
PM2.5 exposure was associated with increased methylation in the promoter region of the iNOS gene (β = 0.25, SE: 0.11, p-value = 0.04). Additionally, the number of years worked as a boilermaker was associated with increased iNOS methylation (β = 0.03, SE: 0.01, p-value = 0.03). No associations were observed for Alu or LINE-1.
Conclusions
Acute and chronic exposure to PM2.5 generated from welding activities was associated with a modest change in DNA methylation of the iNOS gene. Future studies are needed to confirm this association and determine if the observed small increase in iNOS methylation are associated with changes in NO production or any adverse health effect.
doi:10.1186/1476-069X-12-47
PMCID: PMC3700827  PMID: 23758843
DNA methylation; PM2.5; iNOS; Welders; LINE-1; Alu; Boilermakers
Epidemiology (Cambridge, Mass.)  2012;23(2):332-340.
BACKGROUND
Previous studies suggest that air pollution is related to thrombosis, inflammation, and endothelial dysfunction. Mechanisms and sources of susceptibility are still unclear. One possibility is that these associations can be modified by DNA methylation states.
METHODS
We conducted a cohort study with repeated measurements of fibrinogen, C-reactive protein, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in 704 elderly men participating in the Veterans Administration Normative Aging Study (2000-2009). We investigated short- and intermediate-term air pollution effects on these blood markers, and epigene-environment interactions by DNA methylation of Alu, LINE-1, tissue factor (F3), Toll-Like Receptor 2 (TLR-2), and ICAM-1.
RESULTS
We found effects of particle number, black carbon, nitrogen dioxide (NO2), and carbon monoxide (CO) on fibrinogen. Ozone was a significant predictor of C-reactive protein and ICAM-1. Particle number, black carbon, NO2, CO, PM2.5, and sulfates were associated with ICAM-1and VCAM-1. An interquartile range increase in 24-hour exposure for NO2; was associated with a 1.7% (95% confidence interval = 0.2% to 3.3%) increase in fibrinogen for ozone a 10.8% (2.2% to 20.0%) increase in C-reactive protein for particle number, a 5.9% (3.6% to 8.3%) increase in ICAM-1; and for PM2.5 a 3.7% (1.7% to 5.8%) increase in VCAM-1. The air pollution effect was stronger among subjects having higher Alu, lower LINE-1, tissue factor, or TLR-2 methylation status.
CONCLUSION
We observed associations of traffic-related pollutants on fibrinogen, and both traffic and secondary particles on C-reactive protein, ICAM-1, and VCAM-1. There was effect modification by DNA methylation status, indicating that epigenetic states can convey susceptibility to air pollution.
doi:10.1097/EDE.0b013e31824523f0
PMCID: PMC3285258  PMID: 22237295
Epigenetics  2012;7(3):253-260.
Maternal diet affects offspring DNA methylation in animal models, but evidence from humans is limited. We investigated the extent to which gestational intake of methyl donor nutrients affects global DNA methylation in maternal and umbilical cord blood. Among mother-infant pairs in Project Viva, a folate-replete US population, we estimated maternal intakes of vitamin B12, betaine, choline, folate, cadmium, zinc and iron periconceptionally and during the second trimester. We examined associations of these nutrients with DNA methylation, measured as %5-methyl cytosines (%5mC) in Long Interspersed Nuclear Element-1 (LINE-1), in first trimester (n = 830) and second trimester (n = 671) maternal blood and in cord blood at delivery (n = 516). Cord blood methylation was higher for male than female infants {mean [standard deviation (SD)] 84.8 [0.6] vs. 84.4 [0.7]%}. In the multivariable-adjusted model, maternal intake of methyl donor nutrients periconceptionally and during the second trimester of pregnancy was not positively associated with first trimester, second trimester or cord blood LINE-1 methylation. Periconceptional betaine intake was inversely associated with cord blood methylation [regression coefficient = −0.08% (95% confidence interval (CI): −0.14, −0.01)] but this association was attenuated after adjustment for dietary cadmium, which itself was directly associated with first trimester methylation and inversely associated with cord blood methylation. We also found an inverse association between periconceptional choline [−0.10%, 95% CI: −0.17, −0.03 for each SD (∼63 mg/day)] and cord blood methylation in males only. In this folate-replete population, we did not find positive associations between intake of methyl donor nutrients during pregnancy and DNA methylation overall, but among males, higher early pregnancy intakes of choline were associated with lower cord blood methylation.
doi:10.4161/epi.7.3.19082
PMCID: PMC3335948  PMID: 22430801
DNA methylation; pregnancy; cord blood; maternal diet; cadmium
Epigenetics  2012;7(3):261-269.
Lung function is a strong predictor of mortality. While inflammatory markers have been associated with lung function decrease, pathways are still poorly understood and epigenetic changes may participate in lung function decline mechanisms. We studied the cross-sectional association between DNA methylation in nine inflammatory genes and lung function in a cohort of 756 elderly men living in the metropolitan area of Boston. Participants donated a blood sample for DNA methylation analysis and underwent spirometry at each visit every 3 to 5 y from 1999–2006. We used separate multivariate mixed effects regression models to study the association between each lung function measurement and DNA methylation within each gene. Decreased CRAT, F3 and TLR2 methylation was significantly associated with lower lung function. One interquartile range (IQR) decrease in DNA methylation was associated with lower forced vital capacity (FVC) and forced expiratory volume in one second (FEV1), respectively by 2.94% (p < 10−4) and 2.47% (p < 10−3) for F3 and by 2.10% (p < 10−2) and 2.42% (p < 10−3) for TLR2. Decreased IFNγ and IL6 methylation was significantly associated with better lung function. One IQR decrease in DNA methylation was associated with higher FEV1 by 1.75% (p = 0.02) and 1.67% (p = 0.05) for IFNγ and IL6, respectively. These data demonstrate that DNA methylation may be part of the biological processes underlying the lung function decline and that IFNγ and IL6 may have ambivalent roles through activation of negative feedback.
doi:10.4161/epi.7.3.19216
PMCID: PMC3335949  PMID: 22430802
DNA methylation; genes; spirometry; FEV1; lungs; TLR2; F3; INOS; GCR; OGG1
Background Estimates of global DNA methylation from repetitive DNA elements, such as Alu and LINE-1, have been increasingly used in epidemiological investigations because of their relative low-cost, high-throughput and quantitative results. Nevertheless, determinants of these methylation measures in healthy individuals are still largely unknown. The aim of this study was to examine whether age, gender, smoking habits, alcohol drinking and body mass index (BMI) are associated with Alu or LINE-1 methylation levels in blood leucocyte DNA of healthy individuals.
Methods Individual data from five studies including a total of 1465 healthy subjects were combined. DNA methylation was quantified by PCR-pyrosequencing.
Results Age [β = −0.011% of 5-methyl-cytosine (%5mC)/year, 95% confidence interval (CI) −0.020 to −0.001%5mC/year] and alcohol drinking (β = −0.214, 95% CI −0.415 to −0.013) were inversely associated with Alu methylation. Compared with females, males had lower Alu methylation (β = −0.385, 95% CI −0.665 to −0.104) and higher LINE-1 methylation (β = 0.796, 95% CI 0.261 to 1.330). No associations were found with smoking or BMI. Percent neutrophils and lymphocytes in blood counts exhibited a positive (β = 0.036, 95% CI 0.010 to 0.061) and negative (β = −0.038, 95% CI −0.065 to −0.012) association with LINE-1 methylation, respectively.
Conclusions Global methylation measures in blood DNA vary in relation with certain host and lifestyle characteristics, including age, gender, alcohol drinking and white blood cell counts. These findings need to be considered in designing epidemiological investigations aimed at identifying associations between DNA methylation and health outcomes.
doi:10.1093/ije/dyq154
PMCID: PMC3304518  PMID: 20846947
Blood; DNA methylation; epigenetics; meta-analysis; repetitive elements
Epigenetics  2012;7(1):63-70.
DNA methylation has been associated with age-related disease. Intra-individual changes in gene-specific DNA methylation over time in a community-based cohort has not been well described. We estimated the change in DNA methylation due to aging for nine genes in an elderly, community-dwelling cohort of men. Seven hundred and eighty four men from the Veterans Administration Normative Aging Study who were living in metropolitan Boston from 1999–2009 donated a blood sample for DNA methylation analysis at clinical examinations repeated at approximately 3-5 year intervals. We used mixed effects regression models. Aging was significantly associated with decreased methylation of GCR, iNOS and TLR2 and with increased methylation of IFNγ, F3, CRAT and OGG. Obstructive pulmonary disease at baseline modified the effect of aging on methylation of IFNγ (interaction p = 0.04). For participants who had obstructive pulmonary disease at their baseline visit, the rate of change of methylation of IFNγ was -0.05% 5-methyl-cytosine (5-mC) per year (95% CI: -0.22, 0.13), but was 0.14% 5-mC per year (95% CI: 0.05, 0.24) for those without this condition. Models with random slopes indicated significant heterogeneity in the effect of aging on methylation of GCR, iNOS and OGG. These findings suggest that DNA methylation may reflect differential biological aging.
doi:10.4161/epi.7.1.18749
PMCID: PMC3329504  PMID: 22207354
aging; DNA Mmthylation; epigenesis; genetic
BMJ Open  2012;2(5):e001231.
Objectives
To investigate the association between methylation of transposable elements Alu and long-interspersed nuclear elements (LINE-1) and lung function.
Design
Cohort study.
Setting
Outpatient Veterans Administration facilities in greater Boston, Massachusetts, USA.
Participants
Individuals from the Veterans Administration Normative Aging Study, a longitudinal study of aging in men, evaluated between 1999 and 2007. The majority (97%) were white.
Primary and secondary outcome measures
Primary predictor was methylation, assessed using PCR-pyrosequencing after bisulphite treatment. Primary outcome was lung function as assessed by spirometry, performed according to American Thoracic Society/European Respiratory Society guidelines at the same visit as the blood draws.
Results
In multivariable models adjusted for age, height, body mass index (BMI), pack-years of smoking, current smoking and race, Alu hypomethylation was associated with lower forced expiratory volume in 1 s (FEV1) (β=28 ml per 1% change in Alu methylation, p=0.017) and showed a trend towards association with a lower forced vital capacity (FVC) (β=27 ml, p=0.06) and lower FEV1/FVC (β=0.3%, p=0.058). In multivariable models adjusted for age, height, BMI, pack-years of smoking, current smoking, per cent lymphocytes, race and baseline lung function, LINE-1 hypomethylation was associated with more rapid decline of FEV1 (β=6.9 ml/year per 1% change in LINE-1 methylation, p=0.005) and of FVC (β=9.6 ml/year, p=0.002).
Conclusions
In multiple regression analysis, Alu hypomethylation was associated with lower lung function, and LINE-1 hypomethylation was associated with more rapid lung function decline in a cohort of older and primarily white men from North America. Future studies should aim to replicate these findings and determine if Alu or LINE-1 hypomethylation may be due to specific and modifiable environmental exposures.
doi:10.1136/bmjopen-2012-001231
PMCID: PMC3488751  PMID: 23075571
Epidemiology; Genetics
Environmental Health Perspectives  2012;120(7):1061-1066.
Background: Arsenic is an epigenetic toxicant and could influence fetal developmental programming.
Objectives: We evaluated the association between arsenic exposure and DNA methylation in maternal and umbilical cord leukocytes.
Methods: Drinking-water and urine samples were collected when women were at ≤ 28 weeks gestation; the samples were analyzed for arsenic using inductively coupled plasma mass spectrometry. DNA methylation at CpG sites in p16 (n = 7) and p53 (n = 4), and in LINE-1 and Alu repetitive elements (3 CpG sites in each), was quantified using pyrosequencing in 113 pairs of maternal and umbilical blood samples. We used general linear models to evaluate the relationship between DNA methylation and tertiles of arsenic exposure.
Results: Mean (± SD) drinking-water arsenic concentration was 14.8 ± 36.2 μg/L (range: < 1–230 μg/L). Methylation in LINE-1 increased by 1.36% [95% confidence interval (CI): 0.52, 2.21%] and 1.08% (95% CI: 0.07, 2.10%) in umbilical cord and maternal leukocytes, respectively, in association with the highest versus lowest tertile of total urinary arsenic per gram creatinine. Arsenic exposure was also associated with higher methylation of some of the tested CpG sites in the promoter region of p16 in umbilical cord and maternal leukocytes. No associations were observed for Alu or p53 methylation.
Conclusions: Exposure to higher levels of arsenic was positively associated with DNA methylation in LINE-1 repeated elements, and to a lesser degree at CpG sites within the promoter region of the tumor suppressor gene p16. Associations were observed in both maternal and fetal leukocytes. Future research is needed to confirm these results and determine if these small increases in methylation are associated with any health effects.
doi:10.1289/ehp.1104173
PMCID: PMC3404653  PMID: 22466225
Alu; arsenic; developmental programming; DNA methylation; environmental exposures; epigenetics; in utero exposure; LINE-1; p16; p53
PLoS ONE  2012;7(6):e39220.
Background
DNA methylation is an epigenetic mechanism that has been increasingly investigated in observational human studies, particularly on blood leukocyte DNA. Characterizing the degree and determinants of DNA methylation stability can provide critical information for the design and conduction of human epigenetic studies.
Methods
We measured DNA methylation in 12 gene-promoter regions (APC, p16, p53, RASSF1A, CDH13, eNOS, ET-1, IFNγ, IL-6, TNFα, iNOS, and hTERT) and 2 of non-long terminal repeat elements, i.e., L1 and Alu in blood samples obtained from 63 healthy individuals at baseline (Day 1) and after three days (Day 4). DNA methylation was measured by bisulfite-PCR-Pyrosequencing. We calculated intraclass correlation coefficients (ICCs) to measure the within-individual stability of DNA methylation between Day 1 and 4, subtracted of pyrosequencing error and adjusted for multiple covariates.
Results
Methylation markers showed different temporal behaviors ranging from high (IL-6, ICC = 0.89) to low stability (APC, ICC = 0.08) between Day 1 and 4. Multiple sequence and marker characteristics were associated with the degree of variation. Density of CpG dinucleotides nearby the sequence analyzed (measured as CpG(o/e) or G+C content within ±200bp) was positively associated with DNA methylation stability. The 3′ proximity to repeat elements and range of DNA methylation on Day 1 were also positively associated with methylation stability. An inverted U-shaped correlation was observed between mean DNA methylation on Day 1 and stability.
Conclusions
The degree of short-term DNA methylation stability is marker-dependent and associated with sequence characteristics and methylation levels.
doi:10.1371/journal.pone.0039220
PMCID: PMC3379987  PMID: 22745719
Preterm birth affects over 12% of all infants born in the US yet the biology of early delivery remains unclear, including whether epigenetic mechanisms are involved. We examined associations of maternal and umbilical cord blood long interspersed nuclear element-1 (LINE-1) DNA methylation with length of gestation and odds of preterm birth in singleton pregnancies in Project Viva. In white blood cells from maternal blood during 1st trimester (n=914) and 2nd trimester (n=922), and from venous cord blood at delivery (n=557), we measured LINE-1 by pyrosequencing (expressed as %5 methyl cytosines within the LINE-1 region analyzed [%5mC]). We ran linear regression models to analyze differences in gestation length, and logistic models for odds of preterm birth (<37 v. ≥37 weeks gestation), across quartiles of LINE-1. Mean(SD) LINE-1 levels were 84.3(0.6), 84.5(0.4), and 84.6(0.7) %5mC for 1st trimester, 2nd trimester and cord blood, respectively. Mean(SD) gestational age was 39.5(1.8) weeks, and 6.5% of infants were born preterm. After adjustment for maternal age, race/ethnicity, BMI, education, smoking status, and fetal sex, women with the highest vs. lowest quartile of 1st trimester LINE-1 had longer gestations (0.45 weeks [95% CI 0.12, 0.78]) and lower odds of preterm birth (OR 0.40 [0.17, 0.94]), whereas associations with cord blood LINE-1 were in the opposite direction (−0.45 weeks, −0.83, −0.08) and (OR 4.55 [1.18, 17.5]). In conclusion, higher early pregnancy LINE-1 predicts lower risk of preterm birth. In contrast, preterm birth is associated with lower LINE-1 in cord blood.
PMCID: PMC3377352  PMID: 22720130
Preterm; epigenetics; LINE-1; DNA methylation
17.  Erratum to 
Epigenetics  2012;7(6):667.
It has come to the attention of the authors that Table 5 was not included in Epigenetics Volume 7, Issue 3 in the manuscript: Lepeule J, Baccarelli A, Tarantini L, Motta V, Cantone L, Litonjua AA, et al. Gene promoter methylation is associated with lung function in the elderly: The normative aging study. Epigenetics 2012; 7:261-9.
The citation for Table 5 should have appeared on p. 264, “Sensitivity analyses. The sensitivity analyses including par­ticipants with chronic lung diseases showed similar associations between lung function and DNA methylation as the main analy­ses, with only slight variations in significance (Table 5).”
doi:10.4161/epi.20942
PMCID: PMC3398994
BACKGROUND
Lower blood DNA methylation has been associated with atherosclerosis and high cardiovascular risk. Mechanisms linking DNA hypomethylation to increased cardiovascular risk are still largely unknown.
In a population of community-dwelling elderly individuals, we evaluated whether DNA methylation in LINE-1 repetitive element, heavily methylated sequences dispersed throughout the human genome, was associated with circulating Vascular Cell Adhesion Molecule-1 (VCAM-1), Inter-Cellular Adhesion Molecule-1 (ICAM-1), and C-reactive protein (CRP).
METHODS AND RESULTS
We measured LINE-1 methylation by bisulfite PCR-Pyrosequencing on 742 blood DNA samples from male participants in the Boston area Normative Aging Study (mean age=74.8 years). Mean serum VCAM-1 increased progressively in association with LINE-1 hypomethylation (from 975.2 to 1063.4 ng/ml in the highest vs. lowest methylation quintiles; p-trend=0.004). The association between VCAM-1 and LINE-1 hypomethylation was significant in individuals without ischemic heart disease or stroke (n=480; p=0.001), but not in those with prevalent disease (n=262; p=0.57). Serum ICAM-1 and CRP were not associated with LINE-1 methylation (p-trend=>0.25). All results were confirmed by multivariable analyses adjusting for age, BMI, smoking, pack-years, and ischemic heart disease/stroke.
CONCLUSIONS
LINE-1 element hypomethylation is associated with higher serum VCAM-1. Our data provide new insights into epigenetic events that may accompany the development of cardiovascular disease.
PMCID: PMC3155741  PMID: 20305373
cell adhesion molecules; epidemiology; cardiovascular diseases; risk factors; LINE-1; VCAM-1
Exposure to ambient air particles matter (PM) has been associated with increased risk of lung cancer. Aberrant tumor suppressor gene promoter methylation has emerged as a promising biomarker for cancers, including lung cancer. Whether exposure to PM is associated with peripheral blood leukocyte (PBL) DNA methylation in tumor suppressor genes has not been evaluated. In 63 male healthy steel workers with well-characterized exposure to metal-rich particles nearby Brescia, Italy, we evaluated whether exposure to PM and metal components was associated with PBL DNA methylation in 4 tumor suppressor genes (i.e., APC, p16, p53 and RASSF1A). Blood samples were obtained on the 1st (baseline) and 4th day (post-exposure) of the same work week and DNA methylation was measured using pyrosequencing. A linear mixed model was used to examine the correlations of the exposure with promoter methylation levels. Mean promoter DNA methylation levels of APC or p16 were significantly higher in post-exposure samples compared to that in baseline samples (p-values = 0.005 for APC, and p-value = 0.006 for p16). By contrast, the mean levels of p53 or RASSF1A promoter methylation was decreased in post-exposure samples compared to that in baseline samples (p-value = 0.015 for p53; and p-value < 0.001 for RASSF1A). In post-exposure samples, APC methylation was positively associated with PM10 (β = 0.27, 95% CI: 0.13-0.40), and PM1 (β = 0.23, 95% CI: 0.09-0.38). In summary, ambient PM exposure was associated with PBL DNA methylation levels of tumor suppressor genes of APC, p16, p53 and RASSF1A, suggesting that such methylation alterations may reflect processes related to PM-induced lung carcinogenesis.
doi:10.1186/1743-8977-8-25
PMCID: PMC3180673  PMID: 21878113
Environmental Health Perspectives  2011;119(7):977-982.
Background: DNA methylation is a potential pathway linking environmental exposures to disease. Exposure to particulate air pollution has been associated with increased cardiovascular morbidity and mortality, and lower blood DNA methylation has been found in processes related to cardiovascular morbidity.
Objective: We hypothesized that prolonged exposure to particulate pollution would be associated with hypomethylation of repetitive DNA elements and that this association would be modified by genes involved in glutathione metabolism and other host characteristics.
Methods: DNA methylation of the long interspersed nucleotide element–1 (LINE-1) and the short interspersed nucleotide element Alu were measured by quantitative polymerase chain reaction pyrosequencing in 1,406 blood samples from 706 elderly participants in the Normative Aging Study. We estimated changes in repetitive element DNA methylation associated with ambient particles (particulate matter ≤ 2.5 µm in aerodynamic diameter), black carbon (BC), and sulfates (SO4), with mixed models. We examined multiple exposure windows (1–6 months) before DNA methylation measurement. We investigated whether this association was modified by genotype and phenotype.
Results: An interquartile range (IQR) increase in BC over a 90-day period was associated with a decrease of 0.31% 5-methylcytosine (5mC) (95% confidence interval, 0.12–0.50%) in Alu. An IQR increase in SO4 over a 90-day period was associated with a decrease of 0.27% 5mC (0.02–0.52%) in LINE-1. The glutathione S-transferase mu-1–null genotype strengthened the association between BC and Alu hypomethylation.
Conclusion: Prolonged exposure to BC and SO4 particles was associated with hypomethylation of two types of repetitive elements.
doi:10.1289/ehp.1002773
PMCID: PMC3222977  PMID: 21385671
air pollution; DNA methylation; epigenetics; gene–environment. Environ Health Perspect 119:977–982 (2011). doi:10.1289/ehp.1002773 [Online 8 March 2011]
Environmental Health Perspectives  2011;119(7):964-969.
Background: Epidemiology investigations have linked exposure to ambient and occupational air particulate matter (PM) with increased risk of lung cancer. PM contains carcinogenic and toxic metals, including arsenic and nickel, which have been shown in in vitro studies to induce histone modifications that activate gene expression by inducing open-chromatin states. Whether inhalation of metal components of PM induces histone modifications in human subjects is undetermined.
Objectives: We investigated whether the metal components of PM determined activating histone modifications in 63 steel workers with well-characterized exposure to metal-rich PM.
Methods: We determined histone 3 lysine 4 dimethylation (H3K4me2) and histone 3 lysine 9 acetylation (H3K9ac) on histones from blood leukocytes. Exposure to inhalable metal components (aluminum, manganese, nickel, zinc, arsenic, lead, iron) and to total PM was estimated for each study subject.
Results: Both H3K4me2 and H3K9ac increased in association with years of employment in the plant (p-trend = 0.04 and 0.006, respectively). H3K4me2 increased in association with air levels of nickel [β = 0.16; 95% confidence interval (CI), 0.03–0.3], arsenic (β = 0.16; 95% CI, 0.02–0.3), and iron (β = 0.14; 95% CI, 0.01–0.26). H3K9ac showed nonsignificant positive associations with air levels of nickel (β = 0.24; 95% CI, –0.02 to 0.51), arsenic (β = 0.21; 95% CI, –0.06 to 0.48), and iron (β = 0.22; 95% CI, –0.03 to 0.47). Cumulative exposures to nickel and arsenic, defined as the product of years of employment by metal air levels, were positively correlated with both H3K4me2 (nickel: β = 0.16; 95% CI, 0.01–0.3; arsenic: β = 0.16; 95% CI, 0.03–0.29) and H3K9ac (nickel: β = 0.27; 95% CI, 0.01–0.54; arsenic: β = 0.28; 95% CI, 0.04–0.51).
Conclusions: Our results indicate histone modifications as a novel epigenetic mechanism induced in human subjects by long-term exposure to inhalable nickel and arsenic.
doi:10.1289/ehp.1002955
PMCID: PMC3222996  PMID: 21385672
environmental carcinogens; epigenetics; histone modifications; metals; particulate matter
Background
Particulate matter (PM) exposure has been linked to increased risk of cardiovascular disease, possibly resulting from hypercoagulability and thrombosis. Lung and systemic inflammation from PM inhalation may activate blood coagulation, but mechanisms for PM-related hypercoagulability are still largely unknown.
Objectives
To identify coagulation mechanisms activated by PM in a population with well-characterized exposure.
Methods
We measured prothrombin time [PT], activated-partial-thromboplastin time [aPTT], Endogenous Thrombin Potentials [ETP] with/without exogenous triggers and with/without soluble thrombomodulin, tissue-plasminogen activator antigen [t-PA], D-dimer, and C-reactive protein [CRP] in 37 workers in a steel-production plant with well-characterized exposure to PM with aerodynamic diameter <1μm (PM1) and coarse PM (PM10-PM1). Blood samples were collected from each subject on the first (baseline) and last (post-exposure) day of a four-day workweek. We analysed differences between baseline and post-exposure levels using paired Student’s t-test. We fitted multivariate mixed-regression models to estimate the associations of inter-quartile range PM1 and coarse PM exposure with parameter levels.
Results
None of the parameters showed any significant changes in post-exposure samples, compared to baseline. However, exposure levels were associated with shorter PT (β[PM1]=−0.33 sec, p=0.08; β[PMcoarse]=−0.33 sec, p=0.01), and higher ETP without exogenous triggers and with thrombomodulin (β[PM1]=+99 nM*min, p=0.02; β[PMcoarse]=+66 nM*min, p=0.05), t-PA (β[PM1]=+0.72 ng/mL, p=0.01; β[PMcoarse]=+0.88 ng/mL, p=0.04), and CRP (β[PM1]=+0.59 mg/L, p=0.03; β[PMcoarse]=+0.48 mg/L, p=0.01).
Conclusions
PM exposure did not show any short-term effect within the week of the study. The association of PM exposure with PT, ETP, CRP provides some evidence of long-term effects on inflammation and coagulation.
doi:10.1111/j.1538-7836.2009.03694.x
PMCID: PMC3093960  PMID: 19922434
Coagulation; Endogenous Thrombin Potential; Environmental Risk Factors; Occupational Health; Particulate Matter
Environmental Health Perspectives  2010;119(5):622-627.
Background
Shortened leukocyte telomere length (LTL) is a marker of cardiovascular risk that has been recently associated with long-term exposure to ambient particulate matter (PM). However, LTL is increased during acute inflammation and allows for rapid proliferation of inflammatory cells. Whether short-term exposure to proinflammatory exposures such as PM increases LTL has never been evaluated.
Objectives
We investigated the effects of acute exposure to metal-rich PM on blood LTL, as well as molecular mechanisms contributing to LTL regulation in a group of steel workers with high PM exposure.
Methods
We measured LTL, as well as mRNA expression and promoter DNA methylation of the telomerase catalytic enzyme gene [human telomerase reverse transcriptase (hTERT)] in blood samples obtained from 63 steel workers on the first day of a workweek (baseline) and after 3 days of work (postexposure).
Results
LTL was significantly increased in postexposure (mean ± SD, 1.43 ± 0.51) compared with baseline samples (1.23 ± 0.28, p-value < 0.001). Postexposure LTL was positively associated with PM10 (β = 0.30, p-value = 0.002 for 90th vs. 10th percentile exposure) and PM1 (β = 0.29, p-value = 0.042) exposure levels in regression models adjusting for multiple covariates. hTERT expression was lower in postexposure samples (1.31 ± 0.75) than at baseline (1.68 ± 0.86, p-value < 0.001), but the decrease in hTERT expression did not show a dose–response relationship with PM. We found no exposure-related differences in the methylation of any of the CpG sites investigated in the hTERT promoter.
Conclusions
Short-term exposure to PM caused a rapid increase in blood LTL. The LTL increase did not appear to be mediated by PM-related changes in hTERT expression and methylation.
doi:10.1289/ehp.1002486
PMCID: PMC3094411  PMID: 21169126
epigenetics; particulate matter; telomerase; telomere length
Background
Lung cancer kills more than 1 million people worldwide each year. Whereas several human papillomavirus (HPV)–associated cancers have been identified, the role of HPV in lung carcinogenesis remains controversial.
Methods
We selected 450 lung cancer patients from an Italian population–based case–control study, the Environment and Genetics in Lung Cancer Etiology. These patients were selected from those with an adequate number of unstained tissue sections and included all those who had never smoked and a random sample of the remaining patients. We used real-time polymerase chain reaction (PCR) to test specimens from these patients for HPV DNA, specifically for E6 gene sequences from HPV16 and E7 gene sequences from HPV18. We also tested a subset of 92 specimens from all never-smokers and a random selection of smokers for additional HPV types by a PCR-based test for at least 54 mucosal HPV genotypes. DNA was extracted from ethanol- or formalin-fixed paraffin-embedded tumor tissue under strict PCR clean conditions. The prevalence of HPV in tumor tissue was investigated.
Results
Specimens from 399 of 450 patients had adequate DNA for analysis. Most patients were current (220 patients or 48.9%) smokers, and 92 patients (20.4%) were women. When HPV16 and HPV18 type–specific primers were used, two specimens were positive for HPV16 at low copy number but were negative on additional type-specific HPV16 testing. Neither these specimens nor the others examined for a broad range of HPV types were positive for any HPV type.
Conclusions
When DNA contamination was avoided and state-of-the-art highly sensitive HPV DNA detection assays were used, we found no evidence that HPV was associated with lung cancer in a representative Western population. Our results provide the strongest evidence to date to rule out a role for HPV in lung carcinogenesis in Western populations.
doi:10.1093/jnci/djr003
PMCID: PMC3057981  PMID: 21293027
Global hypomethylation has been shown to increase genome instability potentially leading to increased cancer risk. We determined whether global methylation in blood leukocyte DNA was associated with gastric cancer in a population-based study on 302 gastric cancer cases and 421 age- and sex-matched controls in Warsaw, Poland, between 1994 and 1996. Using PCR-pyrosequencing, we analyzed methylation levels of Alu and LINE-1, 2 CG-rich repetitive elements, to measure global methylation levels. Gastric cancer risk was highest among those with lowest level of methylation in either Alu (OR = 1.3, 95% CI = 0.9–1.9) or LINE-1 (OR = 1.4, 95% CI = 0.9–2.0) relative to those with the highest levels, although the trends were not statistically significant. For Alu, the association was stronger among those aged 70 or older (OR = 2.6, 95% CI = 1.3–5.5, p for interaction = 0.02). We did not observe meaningful differences in the associations by other risk factors and polymorphisms examined. For LINE-1, the association tended to be stronger among individuals with a family history of cancer (OR = 3.1, 95% CI = 1.4–7.0, p for interaction = 0.01), current alcohol drinkers (OR = 1.9, 95% CI = 1.0–3.6, p for interaction = 0.05), current smokers (OR = 2.3, 95% CI = 1.1–4.6, p for interaction = 0.02), those who rarely or never consumed fruit (OR = 3.1, 95% CI = 1.2–8.1, p for interaction = 0.03), CC carriers for the MTRR Ex5+123C>T polymorphism (OR = 2.3, 95% CI = 1.2–4.4, p for interaction = 0.01) and TT carriers for the MTRR Ex15+572T>C polymorphism (OR = 1.7, 95% CI = 1.0–2.8, p for interaction = 0.06). The association was not different by sex, Helicobacter pylori infection, intake of folate, vitamin B6 and total protein and the remaining polymorphisms examined. Our results indicate that interactions between blood leukocyte DNA hypomethylation and host characteristics may determine gastric cancer risk.
doi:10.1002/ijc.25190
PMCID: PMC3009461  PMID: 20099281
gastric cancer; methylation; global hypomethylation; gastric cancer risk

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