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1.  Ambient particulate air pollution and microRNAs in elderly men 
Background
Ambient particulate matter (PM) has been associated with mortality and morbidity for cardiovascular disease (CVD). MicroRNAs control gene expression at a post-transcriptional level. Altered microRNA expression has been reported in processes related to CVD and PM exposure, e.g. systemic inflammation, endothelial dysfunction and atherosclerosis. Polymorphisms in microRNA-related genes could influence response to PM.
Methods
We investigated the association of exposure to ambient particles in several time windows (4-hours to 28-days moving averages) and blood-leukocyte expression changes in fourteen candidate microRNAs, in 153 elderly males from the Normative Aging Study (examined 2005–2009). Potential effect modification by six single nucleotide polymorphisms (SNPs) in three microRNA-related genes was investigated. Fine PM (PM2.5), black carbon, organic carbon and sulfates were measured at a stationary ambient monitoring site. Linear regression models, adjusted for potential confounders, were used to assess effects of particles and SNP-by-pollutant interaction. An in silico pathways analysis was performed on target genes of miRNAs associated with the pollutants.
Results
We found a negative association for pollutants in all moving averages and miR-1, -126, -135a, -146a, -155, -21, -222 and -9. The strongest associations were observed with the 7-day moving averages for PM2.5 and black carbon and with the 48-hour moving averages for organic carbon. The association with sulfates was stable across the moving averages. The in silico pathway analysis identified 18 pathways related to immune response shared by at least two miRNAs; in particular, the “HMGB1/RAGE signaling pathway” was shared by miR-126, -146a, -155, -21 and -222.
No important associations were observed for miR-125a-5p, -125b, -128, -147, -218 and -96. We found significant SNP-by-pollutant interactions for rs7813, rs910925 and rs1062923 in GEMIN4 and black carbon and PM2.5 for miR-1, -126, -146a, -222 and -9, and for rs1640299 in DGCR8 and SO42− for miR-1 and -135a.
Conclusions
Exposure to ambient particles could cause a downregulation of microRNAs involved in processes related to PM exposure. Polymorphisms in GEMIN4 and DGCR8 could modify these associations.
doi:10.1097/EDE.0000000000000026
PMCID: PMC3977338  PMID: 24257509
2.  Integrative Analysis of miRNA and Inflammatory Gene Expression After Acute Particulate Matter Exposure 
Toxicological Sciences  2013;132(2):307-316.
MicroRNAs (miRNAs) are environmentally sensitive inhibitors of gene expression that may mediate the effects of metal-rich particulate matter (PM) and toxic metals on human individuals. Previous environmental miRNA studies have investigated a limited number of candidate miRNAs and have not yet evaluated the functional effects on gene expression. In this study, we wanted to identify PM-sensitive miRNAs using microarray profiling on matched baseline and postexposure RNA from foundry workers with well-characterized exposure to metal-rich PM and to characterize miRNA relations with expression of candidate inflammatory genes. We applied microarray analysis of 847 human miRNAs and real-time PCR analysis of 18 candidate inflammatory genes on matched blood samples collected from foundry workers at baseline and after 3 days of work (postexposure). We identified differentially expressed miRNAs (fold change [FC] > 2 and p < 0.05) and correlated their expression with the inflammatory associated genes. We performed in silico network analysis in MetaCore v6.9 to characterize the biological pathways connecting miRNA-mRNA pairs. Microarray analysis identified four miRNAs that were differentially expressed in postexposure compared with baseline samples, including miR-421 (FC = 2.81, p < 0.001), miR-146a (FC = 2.62, p = 0.007), miR-29a (FC = 2.91, p < 0.001), and let-7g (FC = 2.73, p = 0.019). Using false discovery date adjustment for multiple comparisons, we found 11 miRNA-mRNA correlated pairs involving the 4 differentially expressed miRNAs and candidate inflammatory genes. In silico network analysis with MetaCore database identified biological interactions for all the 11 miRNA-mRNA pairs, which ranged from direct mRNA targeting to complex interactions with multiple intermediates. Acute PM exposure may affect gene regulation through PM-responsive miRNAs that directly or indirectly control inflammatory gene expression.
doi:10.1093/toxsci/kft013
PMCID: PMC3595525  PMID: 23358196
miRNA expression; integrative analysis; mRNA expression; inflammation; metal-rich particulate matter; microarray.
3.  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
4.  Storage conditions and stability of global DNA methylation in placental tissue 
Epigenomics  2013;5(3):10.2217/epi.13.29.
Aim
The placenta is an informative and easily available tissue for many epidemiological studies. We analyzed the extent to which storage delay affects DNA methylation.
Material & methods
Biopsies from two placentas were sequentially stored at −80°C after standing at room temperature for 30 min, 1 h, 2 h, 6 h and 24 h. Global DNA methylation was measured by bisulfite pyrosequencing of repetitive elements and the luminometric methylation assay.
Results
Small changes in global DNA methylation in relation to time-to-storage were observed by pyrosequencing, with a coefficient of variation (COV) of 2.49% (placenta 1) and 2.86% (placenta 2), similar to the mean technical variation observed for pyrosequencing (COV: 1.91 and 1.51%, respectively). A luminometric methylation assay yielded more variable results in the two placentas analyzed, both among time points (COV: 9.13 and 10.35%, respectively) and technical replicates (COV: 11.60 and 9.80%, respectively).
Conclusion
Global DNA methylation is stable at room temperature. However, some techniques to measure methylation might be confounded by DNA degradation caused by a delay in storage.
doi:10.2217/epi.13.29
PMCID: PMC3855826  PMID: 23750648
cohort study; DNA methylation stability; luminometric methylation assay; placenta; pyrosequencing; variability
5.  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
6.  ADCYAP1R1 and Asthma in Puerto Rican Children 
Rationale: Epigenetic and/or genetic variation in the gene encoding the receptor for adenylate-cyclase activating polypeptide 1 (ADCYAP1R1) has been linked to post-traumatic stress disorder in adults and anxiety in children. Psychosocial stress has been linked to asthma morbidity in Puerto Rican children.
Objectives: To examine whether epigenetic or genetic variation in ADCYAP1R1 is associated with childhood asthma in Puerto Ricans.
Methods: We conducted a case-control study of 516 children ages 6–14 years living in San Juan, Puerto Rico. We assessed methylation at a CpG site in the promoter of ADCYAP1R1 (cg11218385) using a pyrosequencing assay in DNA from white blood cells. We tested whether cg11218385 methylation (range, 0.4–6.1%) is associated with asthma using logistic regression. We also examined whether exposure to violence (assessed by the Exposure to Violence [ETV] Scale in children 9 yr and older) is associated with cg11218385 methylation (using linear regression) or asthma (using logistic regression). Logistic regression was used to test for association between a single nucleotide polymorphism in ADCYAP1R1 (rs2267735) and asthma under an additive model. All multivariate models were adjusted for age, sex, household income, and principal components.
Measurements and Main Results: Each 1% increment in cg11218385 methylation was associated with increased odds of asthma (adjusted odds ratio, 1.3; 95% confidence interval, 1.0–1.6; P = 0.03). Among children 9 years and older, exposure to violence was associated with cg11218385 methylation. The C allele of single nucleotide polymorphism rs2267735 was significantly associated with increased odds of asthma (adjusted odds ratio, 1.3; 95% confidence interval, 1.02–1.67; P = 0.03).
Conclusions: Epigenetic and genetic variants in ADCYAP1R1 are associated with asthma in Puerto Rican children.
doi:10.1164/rccm.201210-1789OC
PMCID: PMC3733434  PMID: 23328528
methylation; ADCYAP1R1; childhood asthma; Puerto Ricans; violence
7.  Air Pollution and Homocysteine: More Evidence that Oxidative Stress-related Genes Modify Effects of Particulate Air Pollution 
Epidemiology (Cambridge, Mass.)  2010;21(2):198-206.
Background
Ambient particles are associated with cardiovascular events, and recently with total plasma homocysteine. High total plasma homocysteine is a risk for human health. However, the biological mechanisms are not fully understood. One of putative pathways is through oxidative stress. We aimed to examine whether associations of PM2.5 and black carbon with homocysteine were modified by genotypes including HFE H63D, C282Y, CAT (rs480575, rs1001179, rs2284367 and rs2300181), NQO1 (rs1800566), GSTP1 I105V, GSTM1, GSTT1(deletion vs non-deletion) and HMOX-1 (any short vs both long). We attempted to replicate identified genes in an analysis of heart rate variability, and in other outcomes reported in the literature.
Methods
Study subjects were 1000 white non-Hispanic men in the Boston area, participating in a cohort study of aging. PM2.5, black carbon, total plasma homocysteine and other covariates were measured at several points in time between 1995 and 2006. We fit mixed models to examine effect modification of genes on associations of pollution with total plasma homocysteine.
Results
Interquartile range (IQR) increases in PM2.5 and black carbon (7-day moving averages) were associated with 1.5% (95% confidence interval = 0.2% to 2.8%) and 2.2% (0.6% to 3.9%) increases in total plasma homocysteine, respectively. GSTT1 and HFE C282Y modified effects of black carbon on total plasma homocysteine, and HFE C282Y and CAT (rs2300181) modified effects of PM2.5 on homocysteine. Several genotypes marginally modified effects of PM2.5 and black carbon on various endpoints. All genes with significant interactions with particulate air pollution had modest main effects on total plasma homocysteine.
Conclusions
Effects of PM2.5 and black carbon on various endpoints appeared to be mediated by genes related to oxidative stress pathways.
doi:10.1097/EDE.0b013e3181cc8bfc
PMCID: PMC3939788  PMID: 20110814
8.  Lipid and endothelial related genes, ambient particulate matter, and heart rate variability --the VA Normative Aging Study 
Background
Many studies have shown that exposures to air pollution are associated with cardiovascular events although the mechanism remains to be clarified. To identify whether exposures to ambient particles act on autonomic function via the lipid/endothelial metabolism pathway, we evaluated whether the effects of particulate matter < 2.5 µm in aerodynamic diameter (PM2.5) on heart rate variability (HRV) were modified by gene polymorphisms related to those pathways.
Methods
We used HRV and gene data from the Normative Aging Study and PM2.5 from a monitor located a kilometer from the examination site. We fitted a mixed effect model to investigate the associations between PM2.5 and repeated measurements of HRV by gene polymorphisms of apolipoprotein E (APOE), lipoprotein lipase (LPL) and vascular endothelial growth factor (VEGF) adjusting for potential confounders chosen a priori.
Results
A 10-µg/m3 increase of PM2.5 in the two days before the examination was associated with 3.8% [95% confidence interval (CI): 0.2%, 7.4%], 7.8% [95 CI: 0.4%, 15.3%] and 10.6% [95% CI: 1.8 %, 19.4%] decreases of the standard deviation of normal-to-normal intervals, low frequency and high frequency, respectively. In general, carriers of wild type APOE, LPL and VEGF genes had stronger effects of particles on HRV compared to those with hetero- or homozygous types. Variations of LPL-N291S, LPL-D9N and APOE-G113C significantly modified effects of PM2.5 on HRV.
Conclusion
Associations between PM2.5 and HRV were modified by gene polymorphisms of APOE, LPL and VEGF and biological metabolism remains to be identified.
doi:10.1136/jech.2008.083295
PMCID: PMC3935361  PMID: 19602472
air pollution; heart rate variability; effect modification; apolipoprotein E; lipoprotein lipase; vascular endothelial growth factor
9.  Maternal OGTT Glucose Levels at 26–30 Gestational Weeks with Offspring Growth and Development in Early Infancy 
BioMed Research International  2014;2014:516980.
Aims. We aim to evaluate the association of maternal gestational oral glucose tolerance test (OGTT) glucose concentrations with anthropometry in the offspring from birth to 12 months in Tianjin, China. Methods. A total of 27,157 pregnant women underwent OGTT during 26–30 weeks gestation, and their children had body weight/length measured from birth to 12 months old. Results. Maternal OGTT glucose concentrations at 26–30 gestational weeks were positively associated with Z-scores for birth length-for-gestational age and birth weight-for-length. Compared with infants born to mothers with normal glucose tolerance, infants born to mothers with gestational diabetes mellitus (impaired glucose tolerance/new diabetes) had higher mean values of Z-scores for birth length-for-gestational age (0.07/0.23; normal group −0.08) and birth weight-for-length (0.27/0.57; normal group −0.001), smaller changes in mean values of Z-scores for length-for-age (0.75/0.62; normal group 0.94) and weight-for-length (0.18/−0.17; normal group 0.37) from birth to month 3, and bigger changes in mean values in Z-scores for weight-for-length (0.07/0.12; normal group 0.02) from month 9 to 12. Conclusions. Abnormal maternal glucose tolerance during pregnancy was associated with higher birth weight and birth length, less weight and length gain in the first 3 months of life, and more weight gain in the months 9–12 of life.
doi:10.1155/2014/516980
PMCID: PMC3943263
10.  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
11.  Predicting DNA methylation level across human tissues 
Nucleic Acids Research  2014;42(6):3515-3528.
Differences in methylation across tissues are critical to cell differentiation and are key to understanding the role of epigenetics in complex diseases. In this investigation, we found that locus-specific methylation differences between tissues are highly consistent across individuals. We developed a novel statistical model to predict locus-specific methylation in target tissue based on methylation in surrogate tissue. The method was evaluated in publicly available data and in two studies using the latest IlluminaBeadChips: a childhood asthma study with methylation measured in both peripheral blood leukocytes (PBL) and lymphoblastoid cell lines; and a study of postoperative atrial fibrillation with methylation in PBL, atrium and artery. We found that our method can greatly improve accuracy of cross-tissue prediction at CpG sites that are variable in the target tissue [R2 increases from 0.38 (original R2 between tissues) to 0.89 for PBL-to-artery prediction; from 0.39 to 0.95 for PBL-to-atrium; and from 0.81 to 0.98 for lymphoblastoid cell line-to-PBL based on cross-validation, and confirmed using cross-study prediction]. An extended model with multiple CpGs further improved performance. Our results suggest that large-scale epidemiology studies using easy-to-access surrogate tissues (e.g. blood) could be recalibrated to improve understanding of epigenetics in hard-to-access tissues (e.g. atrium) and might enable non-invasive disease screening using epigenetic profiles.
doi:10.1093/nar/gkt1380
PMCID: PMC3973306  PMID: 24445802
13.  Cross-sectional association between polyfluoroalkyl chemicals and cognitive limitation in the National Health and Nutrition Examination Survey (NHANES) 
Neuroepidemiology  2012;40(2):125-132.
Background/Aims
Our limited understanding of how polyflouoroalkyl chemicals (PFCs) may impact human health suggests the potential for a protective impact on brain health. This study was designed to explore the association between PFCs and cognitive ability in older adults.
Methods
We assessed the association between four PFCs, perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), perfluorononanoic acid (PFNA), and perfluorohexane sulfonic acid (PFHxS), and self-reported limitation due to difficulty remembering or periods of confusion using data from participants aged 60–85 from the 1999–2000 and 2003–2008 National Health and Nutrition Examination Surveys. We also considered whether diabetic status or diabetic medication use modifies this association in light of in vitro evidence that PFCs may act on the same receptors as some diabetic medications.
Results
In multivariable adjusted models, point estimates suggest a protective association between PFCs and self-reported cognitive limitation (OR (95% CI) for a doubling in PFOS, 0.90 (0.78, 1.03); PFOA, 0.92 (0.78, 1.09); PFNA, 0.91 (0.79, 1.04); PFHxS, 0.93 (0.82, 1.06)). The protective association was concentrated in diabetics, with strong, significant protective associations in non-medicated diabetics.
Conclusions
This cross-sectional study suggests that there may be a protective association between exposure to PFCs and cognition in older adults, particularly diabetics.
doi:10.1159/000342310
PMCID: PMC3658136  PMID: 23095808
NHANES; National Health and Nutrition Examination Survey; CDC; Centers for Disease Control; cognitive function; polyfluoroalkyl chemicals; PFCs; epidemiology; risk factor
14.  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
15.  Maternal Prepregnancy Body Mass Index and Gestational Weight Gain on Pregnancy Outcomes 
PLoS ONE  2013;8(12):e82310.
Objective
The aim of the present study was to evaluate the single and joint associations of maternal prepregnancy body mass index (BMI) and gestational weight gain (GWG) with pregnancy outcomes in Tianjin, China.
Methods
Between June 2009 and May 2011, health care records of 33,973 pregnant women were collected and their children were measured for birth weight and birth length. The independent and joint associations of prepregnancy BMI and GWG based on the Institute of Medicine (IOM) guidelines with the risks of pregnancy and neonatal outcomes were examined by using Logistic Regression.
Results
After adjustment for all confounding factors, maternal prepregnancy BMI was positively associated with risks of gestational diabetes mellitus (GDM), pregnancy-induced hypertension, caesarean delivery, preterm delivery, large-for-gestational age infant (LGA), and macrosomia, and inversely associated with risks of small-for-gestational age infant (SGA) and low birth weight. Maternal excessive GWG was associated with increased risks of pregnancy-induced hypertension, caesarean delivery, LGA, and macrosomia, and decreased risks of preterm delivery, SGA, and low birth weight. Maternal inadequate GWG was associated with increased risks of preterm delivery and SGA, and decreased risks of LGA and macrosomia, compared with maternal adequate GWG. Women with both prepregnancy obesity and excessive GWG had 2.2–5.9 folds higher risks of GDM, pregnancy-induced hypertension, caesarean delivery, LGA, and macrosomia compared with women with normal prepregnancy BMI and adequate GWG.
Conclusions
Maternal prepregnancy obesity and excessive GWG were associated with greater risks of pregnancy-induced hypertension, caesarean delivery, and greater infant size at birth. Health care providers should inform women to start the pregnancy with a BMI in the normal weight category and limit their GWG to the range specified for their prepregnancy BMI.
doi:10.1371/journal.pone.0082310
PMCID: PMC3869661  PMID: 24376527
16.  Systemic Steroid Exposure Is Associated with Differential Methylation in Chronic Obstructive Pulmonary Disease 
Rationale: Systemic glucocorticoids are used therapeutically to treat a variety of medical conditions. Epigenetic processes such as DNA methylation may reflect exposure to glucocorticoids and may be involved in mediating the responses and side effects associated with these medications.
Objectives: To test the hypothesis that differences in DNA methylation are associated with current systemic steroid use.
Methods: We obtained DNA methylation data at 27,578 CpG sites in 14,475 genes throughout the genome in two large, independent cohorts: the International COPD Genetics Network (ndiscovery = 1,085) and the Boston Early Onset COPD study (nreplication = 369). Sites were tested for association with current systemic steroid use using generalized linear mixed models.
Measurements and Main Results: A total of 511 sites demonstrated significant differential methylation by systemic corticosteroid use in all three of our primary models. Pyrosequencing validation confirmed robust differential methylation at CpG sites annotated to genes such as SLC22A18, LRP3, HIPK3, SCNN1A, FXYD1, IRF7, AZU1, SIT1, GPR97, ABHD16B, and RABGEF1. Functional annotation clustering demonstrated significant enrichment in intrinsic membrane components, hemostasis and coagulation, cellular ion homeostasis, leukocyte and lymphocyte activation and chemotaxis, protein transport, and responses to nutrients.
Conclusions: Our analyses suggest that systemic steroid use is associated with site-specific differential methylation throughout the genome. Differentially methylated CpG sites were found in biologically plausible and previously unsuspected pathways; these genes and pathways may be relevant in the development of novel targeted therapies.
doi:10.1164/rccm.201207-1280OC
PMCID: PMC3622442  PMID: 23065012
DNA methylation; glucocorticoids; chronic obstructive pulmonary disease
17.  DNA methylation differences in exposed workers and nearby residents of the Ma Ta Phut industrial estate, Rayong, Thailand 
Background Adverse biological effects from airborne pollutants are a primary environmental concern in highly industrialized areas. Recent studies linked air pollution exposures with altered blood Deoxyribo-nucleic acid (DNA) methylation, but effects from industrial sources and underlying biological mechanisms are still largely unexplored.
Methods The Ma Ta Phut industrial estate (MIE) in Rayong, Thailand hosts one of the largest steel, oil refinery and petrochemical complexes in south-eastern Asia. We measured a panel of blood DNA methylation markers previously associated with air pollution exposures, including repeated elements [long interspersed nuclear element-1 (LINE-1) and Alu] and genes [p53, hypermethylated-in-cancer-1 (HIC1), p16 and interleukin-6 (IL-6)], in 67 MIE workers, 65 Ma Ta Phut residents and 45 rural controls. To evaluate the role of DNA damage and oxidation, we correlated DNA methylation measures with bulky DNA and 3-(2-deoxy-β-D-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG) adducts.
Results In covariate-adjusted models, MIE workers, compared with rural residents, showed lower LINE-1 (74.8% vs 78.0%; P < 0.001), p53 (8.0% vs 15.7%; P < 0.001) and IL-6 methylation (39.2% vs 45.0%; P = 0.027) and higher HIC1 methylation (22.2% vs 15.3%, P < 0.001). For all four markers, Ma Ta Phut residents exhibited methylation levels intermediate between MIE workers and rural controls (LINE-1, 75.7%, P < 0.001; p53, 9.0%, P < 0.001; IL-6, 39.8%, P = 0.041; HIC1, 17.8%, P = 0.05; all P-values vs rural controls). Bulky DNA adducts showed negative correlation with p53 methylation (P = 0.01). M1dG showed negative correlations with LINE-1 (P = 0.003) and IL-6 methylation (P = 0.05).
Conclusions Our findings indicate that industrial exposures may induce alterations of DNA methylation patterns detectable in blood leucocyte DNA. Correlation of DNA adducts with DNA hypomethylation suggests potential mediation by DNA damage.
doi:10.1093/ije/dys129
PMCID: PMC3535756  PMID: 23064502
Air pollution; Ma Ta Phut; blood leucocytes; DNA methylation; DNA damage
18.  AIR POLLUTION EXPOSURE AND TELOMERE LENGTH IN HIGHLY EXPOSED SUBJECTS IN BEIJING, CHINA: A REPEATED-MEASURE STUDY 
Environment international  2012;48:10.1016/j.envint.2012.06.020.
Background
Ambient particular matter (PM) exposure has been associated with short- and long-term effects on cardiovascular disease (CVD). Telomere length (TL) is a biomarker of CVD risk that is modified by inflammation and oxidative stress, two key pathways for PM effects. Whether PM exposure modifies TL is largely unexplored.
Objectives
To investigate effects of PM on blood TL in a highly-exposed population.
Methods
We measured blood TL in 120 blood samples from truck drivers and 120 blood samples from office workers in Beijing, China. We measured personal PM2.5 and Elemental Carbon (EC, a tracer of traffic particles) using light-weight monitors. Ambient PM10 was obtained from local monitoring stations. We used covariate-adjusted regression models to estimate percent changes in TL per an interquartile-range increase in exposure.
Results
Covariate-adjusted TL was higher in drivers (mean=0.87, 95%CI: 0.74; 1.03) than in office workers (mean=0.79, 95%CI: 0.67; 0.93; p=0.001). In all participants combined, TL increased in association with personal PM2.5 (+5.2%, 95%CI: 1.5; 9.1; p=0.007), personal EC (+4.9%, 95%CI: 1.2; 8.8; p=0.01), and ambient PM10 (+7.7%, 95%CI: 3.7; 11.9; p<0.001) on examination days. In contrast, average ambient PM10 over the 14 days before the examinations was significantly associated with shorter TL (−9.9%, 95%CI: −17.6; −1.5; p=0.02).
Conclusions
Short-term exposure to ambient PM is associated with increased blood TL, consistent with TL roles during acute inflammatory responses. Longer exposures may shorten TL as expected after prolonged pro-oxidant exposures. The observed TL alterations may participate in the biological pathways of short- and long-term PM effects.
doi:10.1016/j.envint.2012.06.020
PMCID: PMC3821920  PMID: 22871507
Particulate Matter; Personal Monitoring; Telomere length; Traffic pollution; China
19.  Genome-wide study of DNA methylation alterations in response to Diazinon exposure in vitro 
Pesticide exposure has repeatedly been associated with cancers. However, molecular mechanisms are largely undetermined. In this study, we examined whether exposure to diazinon, a common organophosphate that has been associated with cancers, could induce DNA methylation alterations. We conducted genome-wide DNA methylation analyses on DNA samples obtained from human hematopoietic K562 cell exposed to diazinon and ethanol using the Illumina Infinium HumanMethylation27 BeadChip. Bayesian-adjusted t-tests were used to identify differentially methylated gene promoter CpG sites. We identified 1069 CpG sites in 984 genes with significant methylation changes in diazinon-treated cells. Gene ontology analysis demonstrated that some genes are tumor suppressor genes, such as TP53INP1 (3.0-fold, q-value<0.001) and PTEN (2.6-fold, q-value<0.001), some genes are in cancer-related pathways, such as HDAC3 (2.2-fold, q-value=0.002), and some remain functionally unknown. Our results provided direct experimental evidence that diazinon may modify gene promoter DNA methylation levels, which may play a pathological role in cancer development.
doi:10.1016/j.etap.2012.07.012
PMCID: PMC3514648  PMID: 22964155
Diazinon exposure; DNA methylation alteration; carcinogenesis
20.  Multivariate Gene Selection and Testing in Studying the Exposure Effects on a Gene Set 
Statistics in biosciences  2012;4(2):319-338.
Studying the association between a gene set (e.g., pathway) and exposures using multivariate regression methods is of increasing importance in genomic studies. Such an analysis is often more powerful and interpretable than individual gene analysis. Since many genes in a gene set are likely not affected by exposures, one is often interested in identifying a subset of genes in the gene set that are affected by exposures. This allows for better understanding of the underlying biological mechanism and for pursuing further biological investigation of these genes. The selected subset of “signal” genes also provides an attractive vehicle for a more powerful test for the association between the gene set and exposures. We propose two computationally simple Canonical Correlation Analysis (CCA) based variable selection methods: Sparse Outcome Selection (SOS) CCA and step CCA, to jointly select a subset of genes in a gene set that are associated with exposures. Several model selection criteria, such as BIC and the new Correlation Information Criterion (CIC), are proposed and compared. We also develop a global test procedure for testing the exposure effects on the whole gene set, accounting for gene selection. Through simulation studies, we show that the proposed methods improve upon an existing method when the genes are correlated and are more computationally efficient. We apply the proposed methods to the analysis of the Normative Aging DNA methylation Study to examine the effects of airborne particular matter exposures on DNA methylations in a genetic pathway.
doi:10.1007/s12561-012-9072-7
PMCID: PMC3524591  PMID: 23264831
Canonical Correlation Analysis; Epigenetics; Global test; Sparsity; Variable Selection; Tuning Parameter
21.  DNA methylation in repetitive elements and post-traumatic stress disorder: a case–control study of US military service members 
Epigenomics  2012;4(1):10.2217/epi.11.116.
Aim
We investigated serum DNA methylation patterns in genomic repetitive elements, LINE-1 and Alu, for post-traumatic stress disorder (PTSD) cases and controls who were US military service members recently deployed to Afghanistan or Iraq.
Methods
Cases (n = 75) had a postdeployment diagnosis of PTSD. Controls (n = 75) were randomly selected service members with no postdeployment PTSD diagnosis. Pre- and post-deployment sera were accessed, DNA was extracted and DNA methylation (percentage 5-methyl cytosine) was quantifed via pyrosequencing. Conditional and unconditional logistic regressions were used to compare: cases post- to pre-deployment; controls post- to pre-deployment; cases to controls predeployment; cases to controls postdeployment.
Results
LINE-1 was hypermethylated in controls post- versus pre-deployment (odds ratio [OR]: 1.33; 95% CI: 1.06–1.65) and hypomethylated in cases versus controls postdeployment (OR: 0.82; 95% CI: 0.67–1.01). Alu was hypermethylated for cases versus controls predeployment (OR: 1.46; 95% CI: 1.08–1.97).
Conclusion
Patterns of hypermethylation of LINE-1 in controls postdeployment and of Alu in cases postdeployment are intriguing and may suggest resilience or vulnerability factors.
doi:10.2217/epi.11.116
PMCID: PMC3809831  PMID: 22332656
Alu; combat; DNA methylation; epigenetics; LINE-1; Operation Enduring Freedom; Operation Iraqi Freedom; post-traumatic stress disorder; repetitive element
22.  Exposure to airborne particulate matter is associated with methylation pattern in the asthma pathway 
Epigenomics  2013;5(2):147-154.
Background
Asthma exacerbation and other respiratory symptoms are associated with exposure to air pollution. Since environment affects gene methylation, it is hypothesized that asthmatic responses to pollution are mediated through methylation.
Materials & methods
We study the possibility that airborne particulate matter affects gene methylation in the asthma pathway. We measured methylation array data in clinic visits of 141 subjects from the Normative Aging Study. Black carbon and sulfate measures from a central monitoring site were recorded and 30-day averages were calculated for each clinic visit. Genespecific methylation scores were calculated for the genes in the asthma pathway, and the association between the methylation in the asthma pathway and the pollution measures was analyzed using sparse Canonical Correlation Analysis.
Results
The analysis found that exposures to black carbon and sulfate were significantly associated with the methylation pattern in the asthma pathway (p-values 0.05 and 0.02, accordingly). Specific genes that contributed to this association were identified.
Conclusion
These results suggest that the effect of air pollution on asthmatic and respiratory responses may be mediated through gene methylation.
doi:10.2217/epi.13.16
PMCID: PMC3767408  PMID: 23566092
sulfate; black carbon; epigenetics; gene-specific methylation scores; pathway analysis; sulfate
23.  Arsenic exposure and DNA methylation among elderly men 
Epidemiology (Cambridge, Mass.)  2012;23(5):668-676.
BACKGROUND
Arsenic exposure has been linked to epigenetic modifications such as DNA methylation in in vitro and animal studies. This association has also been explored in highly exposed human populations, but studies among populations environmentally exposed to low arsenic levels are lacking.
METHODS
We evaluated the association between exposure to arsenic, measured in toenails, and blood DNA methylation in Alu and Long Interspersed Nucleotide Element-1 (LINE-1) repetitive elements in elderly men environmentally exposed to low levels of arsenic. We also explored potential effect modification by plasma folate, cobalamin (vitamin B12), and pyridoxine (vitamin B6). The study population was 581 participants from the Normative Aging Study in Boston, of whom 434, 140, and 7 had 1, 2, and 3 visits, respectively, between 1999-2002 and 2006-2007. We used mixed-effects models and included interaction terms to assess potential effect modification by nutritional factors.
RESULTS
There was a trend of increasing Alu and decreasing LINE-1 DNA methylation as arsenic exposure increased. In subjects with plasma folate below the median (< 14.1 ng/ml), arsenic was positively associated with Alu DNA methylation (β=0.08 [95% confidence interval = 0.03 to 0.13] for one interquartile range [0.06μg/g] increase in arsenic) while a negative association was observed in subjects with plasma folate above the median (β=-0.08 [-0.17 to 0.01]).
CONCLUSIONS
We found an association between arsenic exposure and DNA methylation in Alu repetitive elements that varied by folate level. This suggests a potential role for nutritional factors in arsenic toxicity.
doi:10.1097/EDE.0b013e31825afb0b
PMCID: PMC3448132  PMID: 22833016
24.  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
Although pesticides are subject to extensive carcinogenicity testing before regulatory approval, pesticide exposure has repeatedly been associated with various cancers. This suggests that pesticides may cause cancer via non-mutagenicity mechanisms. The present study provides evidence to support the hypothesis that pesticide-induced cancer may be mediated in part by epigenetic mechanisms. We examined whether exposure to 7 commonly used pesticides (i.e., fonofos, parathion, terbufos, chlorpyrifos, diazinon, malathion, and phorate) induces DNA methylation alterations in vitro. We conducted genome-wide DNA methylation analyses on DNA samples obtained from the human hematopoietic K562 cell line exposed to ethanol (control) and several OPs using the Illumina Infinium HumanMethylation27 BeadChip. Bayesian-adjusted t-tests were used to identify differentially methylated gene promoter CpG sites. In this report, we present our results on three pesticides (fonofos, parathion, and terbufos) that clustered together based on principle component analysis and hierarchical clustering. These three pesticides induced similar methylation changes in the promoter regions of 712 genes, while also exhibiting their own OP-specific methylation alterations. Functional analysis of methylation changes specific to each OP, or common to all three OPs, revealed that differential methylation was associated with numerous genes that are involved in carcinogenesis-related processes. Our results provide experimental evidence that pesticides may modify gene promoter DNA methylation levels, suggesting that epigenetic mechanisms may contribute to pesticide-induced carcinogenesis. Further studies in other cell types and human samples are required, as well as determining the impact of these methylation changes on gene expression.
doi:10.1002/em.21718
PMCID: PMC3753688  PMID: 22847954
Pesticide exposure; DNA methylation alteration; Carcinogenesis

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