2014 Progress Report: Epigenetics Project

EPA Grant Number: R834513C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant R834513
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Center for the Health Assessment of Mothers and Children of Salinas - UC Berkeley School of Public Health: CHAMACOS Office, Berkeley, CA
Center Director: Eskenazi, Brenda
Title: Epigenetics Project
Investigators: Eskenazi, Brenda , Arora, Manish , Bradman, Asa , Chevrier, Jonathan , Harley, Kim , Holland, Nina T. , Johnson, Caroline , Lustig, Robert , Sjodin, Andreas , Smith, Donald
Current Investigators: Eskenazi, Brenda , Barcellos, Lisa , Bradman, Asa , Harley, Kim , Holland, Nina T. , Hubbard, Alan , Lustig, Robert
Institution: University of California - Berkeley
Current Institution: University of California - Berkeley , University of California - San Francisco
EPA Project Officer: Louie, Nica
Project Period: August 1, 2009 through July 31, 2014 (Extended to July 31, 2016)
Project Period Covered by this Report: August 1, 2009 through July 31,2014
RFA: Children's Environmental Health and Disease Prevention Research Centers (with NIEHS) (2009) RFA Text |  Recipients Lists
Research Category: Children's Health , Health

Objective:

In Project C, we are investigating the effects of exposure on the epigenome and its relationship with pubertal onset.

Progress Summary:

1. To analyze global DNA methylation in newborn children by three different assays
 
Pyrosequencing assays were used to measure methylation of Alu and LINE-1 repetitive elements in newborns and 9-year-old children. We found significant associations of DNA methylation with host factors. Levels of DNA methylation were lower in 9-year-olds compared to newborns and were higher in boys compared to girls (Figure 1). These findings were recently published in EMM (Huen, et al., 2014). Additionally, we observed no correlation between LINE-1 and Alu methylation levels at either age suggesting that the two assays represent different portions of the methylome.
 
https://cfpub.epa.gov/ncer_abstracts/images/fckimages/index.cfm?imgid=8057
 
Figure 1. Effects of sex and age on LINE-1 and Alu DNA methylation (n = 246 cords and 246 9-year-olds)
 
The 450K assay was performed in blood samples of 250 newborns and 250 9-year-olds from the CHAMACOS cohort. Our newly developed All Sample Mean Normalization (ASMN) procedure for normalization of methylation data performed consistently better than alternative methodologies at reducing batch effects and increasing repeatability among technical replicates. ASMN was submitted as an open source Bioconductor software package in December 2013, and was described in our recent publication in Epigenetics (Yousefi, et al., 2013).
 
2. To determine ontogenetic changes in global DNA methylation in blood of children between birth and 12 years
 
To determine the longitudinal changes in repetitive element DNA methylation (Alu and LINE-1), pyrosequencing assays were performed in > 600 blood samples collected from CHAMACOS children at multiple time points from birth through early adolescence. LINE-1 global DNA methylation was found to decrease 0.3% per month increase in child age (95% CI: 0.2 – 0.6%). A similar decreasing trend was found for Alu methylation, but this association was not statistically significant (p = 0.18). These data were recently published in EMM (Huen, et al., 2014). Analysis of the results of genome-wide methylation by the 450K Beadchip currently are in progress.
 
3. To investigate the relationship of in utero and 9-year-old blood concentrations of DDT/E and PBDEs with global DNA methylation
 
We observed a consistent trend of lower Alu methylation in fetal blood with higher prenatal DDT/E exposure, particularly after adjusting for cell type composition (Figure 2). Furthermore, associations of prenatal exposure with levels of LINE-1 methylation were only identified after examining the co-exposure of DDT/E with PBDEs simultaneously. Our data suggest prenatal exposure to POPs may be linked to hypomethylation in fetal blood and that accounting for co-exposure to cell types may increase sensitivity of epigenetic analyses for epidemiological studies.
 
https://cfpub.epa.gov/ncer_abstracts/images/fckimages/index.cfm?imgid=8058
 
Figure 2. Alu methylation and prenatal o,p’-DDT exposure in newborns (n=94) after adjusting for sex and cell type composition. Higher prenatal exposure to o,p’-DDT was associated with lower levels of methylation (p=0.02, b(95%CI): 20.37(20.69, 20.05)).
 
4. To determine whether global methylation is associated with onset of puberty and hormonal changes
 
In girls there was a suggestive trend of accelerated breast development (Tanner Stage > 1) at age 9 with increased cord LINE-1 methylation (OR(95% CI): 1.23(0.88,1.73)). Furthermore, this relationship was somewhat stronger at age 10.5 (OR(95%CI): 1.76(0.87,3.59). Similarly, odds of pubic hair development at age 9 were positively associated with increased 9 year LINE-1 methylation (OR(95%CI): 1.18(0.84,1.67)) and this relationship was even stronger at age 10.5 (OR(95%CI): 1.3(0.95,1.8)).
 
Hormone levels in 12-year-old CHAMACOS boys (n = 112) were comparable to reference levels for children of this age group. Repeat element methylation was significantly associated with differences in hormone levels in boys. For instance, a one unit increase in Alu methylation (cord blood) was associated with an 80% increase in testosterone levels (p = 0.02; Figure 3). Additionally, both Alu and LINE-1 methylation (9 yrs) were inversely associated with LH at age 12 (p = 0.05 and 0.02, respectively).
 
 
5. To examine site-specific methylation in relation to age, sex, exposure to DDT/E and PBDEs and puberty onset
 
We assessed the association of prenatal DDT/E exposures on DNA methylation measured by 450K Beadchip assay in cords (n = 308) and 9-year-old children (n = 213) but did not find any associations after adjusting for multiple comparisons (using false discovery rate [FDR]). For prenatal PBDE exposures, we identified eight significantly associated CpG sites (three were significant after Bonferonni correction and eight by FDR), several of which were consistently associated across different BDE-congeners, in CHAMACOS fetal blood DNA (Figure 4). We did not find any significant association in 9-year-old blood DNA. We currently are preparing a manuscript on these results (and those with DDT/E exposure).
 
https://cfpub.epa.gov/ncer_abstracts/images/fckimages/index.cfm?imgid=8060
 
Figure 4. Manhattan plot for association between prenatal log BDE-100 and 450K methylation at birth (red line Bonferonni cutoff for multiple testing, significant hits by FDR in blue rectangle)
 
We did not find significant association of site-specific methylation (cord blood and 9 yrs) with puberty status (Tanner Stage > 1) at age 10.5 in girls or boys. However, methylation at age 9 was significantly associated with testosterone levels in 12-year-old boys for five different CpG sites after adjusting for multiple testing by FDR. These CpG sites were located in the following genes: NBAS, MIR574, TTC40, FBRSL1, and THEG. Using structurally enhanced pathway enrichment analysis (SEPEA) of this methylation data, we found that several important pathways were significantly enriched including glutathione metabolism, amino sugar and nucleotide sugar metabolism, and the PPAR and insulin signaling pathways.
 

Future Activities:

In the next year, we plan to finalize the analyses of complex multi-dimensional data from 450K Beadchips, and to focus on validation of the found “hits” by assessment of expression of these genes by Nanostring and qPCR, as well as by alternative platform such as targeted bisulfite pyrosequencing or next generation sequencing. Several manuscripts currently in preparation will be submitted for publication.


Journal Articles on this Report : 8 Displayed | Download in RIS Format

Other subproject views: All 60 publications 22 publications in selected types All 22 journal articles
Other center views: All 666 publications 138 publications in selected types All 137 journal articles
Type Citation Sub Project Document Sources
Journal Article Chadwick LH, Sawa A, Yang IV, Baccarelli A, Breakefield XO, Deng H-W, Dolinoy DC, Fallin MD, Holland NT, Houseman EA, Lomvardas S, Rao M, Satterlee JS, Tyson FL, Vijayanand P, Greally JM. New insights and updated guidelines for epigenome-wide association studies. Neuroepigenetics 2015;1:14-19. R834513 (2014)
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  • Journal Article Dave V, Yousefi P, Huen K, Volberg V, Holland N. Relationship between expression and methylation of obesity-related genes in children. Mutagenesis 2015;30(3):411-420. R834513 (2013)
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  • Journal Article Holland N, Lizarraga D, Huen K. Recent progress in the genetics and epigenetics of paraoxonase: why it is relevant to children's environmental health. Current Opinion in Pediatrics 2015;27(2):240-247. R834513 (2013)
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  • Journal Article Huen K, Harley K, Beckman K, Eskenazi B, Holland N. Associations of PON1 and genetic ancestry with obesity in early childhood. PLoS One 2013;8(5):e62565. R834513 (2013)
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  • Journal Article Huen K, Yousefi P, Bradman A, Yan L, Harley KG, Kogut K, Eskenazi B, Holland N. Effects of age, sex, and persistent organic pollutants on DNA methylation in children. Environmental and Molecular Mutagenesis 2014;55(3):209-222. R834513 (2012)
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  • Journal Article Volberg V, Harley KG, Aguilar RS, Rosas LG, Huen K, Yousefi P, Dave V, Phan N, Lustig RH, Eskenazi B, Holland N. Associations between perinatal factors and adiponectin and leptin in 9-year-old Mexican-American children. Pediatric Obesity 2013;8(6):454-463. R834513 (2013)
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  • Journal Article Volberg V, Heggeseth B, Harley K, Huen K, Yousefi P, Dave V, Tyler K, Vedar M, Eskenazi B, Holland N. Adiponectin and leptin trajectories in Mexican-American children from birth to 9 years of age. PLoS One 2013;8(10):e77964 (8 pp.). R834513 (2014)
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  • Journal Article Yousefi P, Huen K, Schall RA, Decker A, Elboudwarej E, Quach H, Barcellos L, Holland N. Considerations for normalization of DNA methylation data by Illumina 450K BeadChip assay in population studies. Epigenetics 2013;8(11):1141-1152. R834513 (2012)
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  • Supplemental Keywords:

    epigenetics, methylation, DDT, DDE, PBDEs, flame retardants, manganese, maneb, puberty, RFA, Health, Scientific Discipline, INTERNATIONAL COOPERATION, Health Risk Assessment, Biochemistry, Children's Health, Environmental Policy, Biology, farmworkers, pesticide exposure, flame retardants, PBDE, children's vulnerablity, neurochemical effects, harmful environmental agents, biological markers, agricultural community

    Relevant Websites:

    Center for Environmental Research and Children's Health Exit

    Progress and Final Reports:

    Original Abstract
  • 2010 Progress Report
  • 2011 Progress Report
  • 2012 Progress Report
  • 2013 Progress Report
  • 2015 Progress Report
  • Final

  • Main Center Abstract and Reports:

    R834513    Center for the Health Assessment of Mothers and Children of Salinas - UC Berkeley School of Public Health: CHAMACOS Office, Berkeley, CA

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R834513C001 CHAMACOS Cohort Project: Pesticides and PBDE on Neurobehavior and Puberty
    R834513C002 Project B: Exposure Project: Mn, DDT/E and PBDE Exposure to Farmworker Children
    R834513C003 Epigenetics Project
    R834513C004 Community Outreach and Translation Core