2013 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 , Barcellos, Lisa , Bradman, Asa , Harley, Kim , Holland, Nina T. , Lustig, Robert
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: June 1, 2012 through May 31,2013
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 (R834513C003) we are investigating the effects of exposure to environmental chemicals on the epigenome and its relationship with pubertal onset.

The specific aims are:

  1. To analyze global DNA methylation in newborn children by three different assays.
  2. To determine ontogenetic changes in global DNA methylation in blood of children between birth and 12 years.
  3. To investigate the relationship of in utero and 9-year-old blood concentrations of DDT/E and PBDEs with global DNA methylation.
  4. To determine whether global methylation is associated with onset of puberty and hormonal changes.
  5. To examine site-specific methylation in relation to age, sex, exposure to DDT/E and PBDEs and puberty onset.

Progress Summary:

Progress on Specific Aims:

  1. To analyze global DNA methylation in newborn children by three different assays.

    When we were funded in 2009, 27K Infinium arrays were considered “state-of the art”, and we initially have done analyses of CHAMACOS samples using these BeadChips. However, by the end of 2011, it became apparent that there were significant technical issues with these chips, and we could not reliably use 27K data that were already generated for the majority of the subjects. Fortunately, new 450K arrays that were issued in 2012, appear to be off a much better quality and provide significantly more information (genome-wide, 485,577 CpG sites) to address our Specific Aims. Still, this year we determined that the normalization methodologies provided by Illumina’s Genome Studio software for 450K assay data were inadequate for reducing technical variability, potentially biasing results. To overcome this obstacle, we conducted a comparative assessment of 10 normalization methods including the GenomeStudio(R) Illumina method, the lumi smooth quantile approach, and the newly developed by us All Sample Mean Normalization (ASMN) method. We also examined the performance of normalization methods in combination with correction for the two types of Infinium chemistry utilized on the 450K BeadChip®. The proposed ASMN method performed consistently well both at reducing batch effects and improving replicate comparability. It will be applied to conduct the final analyses of 450K CHAMACOS data. A manuscript describing these results has been submitted to Epigenetics. Currently, we have 450K data generated for a subset of newborns (Discovery dataset, N=70 and Validation N=72). However, we may not have sufficient resources remaining on this grant to do all 250 newborn analyses using 450K considering high costs. We did complete the analyses of global methylation by Alu and LINE-1 assays for all 250 newborns as initially proposed (paper submitted to EMM). Additional comparisons between DNA methylation between three assays will be completed in Year 5.

    We performed differential cell count on blood samples from CHAMACOS 113 newborns (using banked blood smears) to address potential confounding by the variation in DNA methylation in different white blood cell types. No significant relationship between levels of LINE-1 DNA methylation and percentages of white blood cells in newborns has been found (P>0.05). The lack of effect of differential cell counts on global DNA methylation is an additional confirmation that blood is an appropriate tissue to use for epigenetic analyses in population studies.

  2. To determine ontogenetic changes in global DNA methylation in blood of children between birth and 12 years.

    We partially completed analyses of samples at 2 year old (N=45) and 5 year old (N=43), and we plan to finalize the analyses of ontogenetic changes in DNA methylation in Year 5, including 12 year old samples from the same children (currently being collected). Increasing the number of newborns and 9 year old children (n=250 each time-point) for two assays, we confirmed our preliminary results showing lower global methylation levels in older children. Among those with measures at both time points (n=134) levels were lower at 9 years of age compared to birth (LINE-1, p=0.04; Alu, p=0.07). We found similar results using GEE to model population differences among all subjects. On average, mean LINE-1 DNA methylation in 9 year old was 0.38% 5mC lower than that in newborns (p=0.001). Although these differences are not large, previous studies have shown that even small differences in methylation can be biologically meaningful. For 450K assay, however, the data will be limited to a subset of 25 subjects at all 4 proposed time points due to funding limitations as described above.

  3. To investigate the relationship of in utero and 9-year-old blood concentrations of DDT/E and PBDEs with global DNA methylation.

    Confirming observations in our preliminary data, we found small but consistent decreases in global methylation measured by both LINE-1 and Alu assays in response to prenatal OC and PBDE exposures in cord blood DNA. We did not observe any effect of prenatal exposures on global methylation in 9-year-old children. Furthermore, since postnatal exposure to PBDEs is generally much higher in our cohort, we also looked at PBDE measurements during childhood. Although we have not completed measurement of 9 year old DDT/E and PBDE, we did analyze the relationship between 7 year blood concentrations and global methylation at age 9. We did not find a significant relationship between exposure and methylation for those time points. A manuscript describing the relationship of age, sex, and prenatal POPs exposure with global methylation in newborns and 9 year olds was submitted to Environmental and Molecular Mutagenesis. The power to analyze the relationship between prenatal exposures and 450K data will be limited as explained above. These analyses will be conducted in Year 5 after new ASMN normalization has been implemented.

  4. To determine whether global methylation is associated with onset of puberty and hormonal changes.

    We analyzed the association of LINE-1 and Alu global methylation at birth and 9 years of age with onset of puberty as determined by Tanner staging. Among girls, we found no evidence of a relationship between global methylation and increased odds of being at a Tanner Stage>1. However, among boys, we found that increased LINE-1 methylation at birth was associated with lower odds (OR(95%CI):0.49(0.31-0.79) of being at a Tanner Stage>1 at age 9 suggesting that global hypomethylation may be related to later pubertal onset in boys. Furthermore, we observed a similar trend for Alu methylation, however it was not statistically significant (OR(95%CI): 0.68(0.21-2.19). Next, we plan to analyze the relationship of DNA methylation with hormonal levels as soon as the FSH/LH/T measurements are completed (by Esoterix in 2013).

  5. To examine site-specific methylation in relation to age, sex, exposure to DDT/E and PBDEs and puberty onset.

    Expanding upon our preliminary analysis of PPARγ site-specific methylation (determined by Illumina 450K) with health outcomes and biomarkers of obesity in our discovery set, we confirmed similar results in our validation set. The same pattern of methylation across the sites was found in both sets at birth and 9 year old time points. CpG sites located in the north and south shore tended to be highly methylated, while those in the CpG island had lower methylation. Using the discovery set, we found that methylation at birth in sites 1 and 18 was associated with birth weight. Methylation at 9 years in sites 1, 20, and 22 was associated with 9-year BMI. Additionally, at 9-year time point, methylation in sites 1 and 20 was significantly and positively associated with adiponectin and negatively associated with leptin, biomarkers of obesity. PPARγ site 1 methylation at birth had statistically significant inverse associations with child birth weight (β=-2.5, P=0.04; β=-3.4, P=0.03) in both the discovery and validation sets. Site 1 methylation at 9 years was associated with 9-year BMI in the discovery set (β=-4.8, P<0.001) and borderline significant in the validation set (β=-2.7, P=0.08). Further, using pooled data from both batches, we found that PPARγ site 1 methylation remained related to child size at birth and 9-years, even after Bonferroni adjustment for multiple testing. Our results suggest that PPARγ methylation may play an important role in obesity development in children, reflecting child birth weight and BMI. Next steps of the analyses will included the relationship with puberty onset, as well as validation of DNA methylation finding for PPARγ and other sites by alternative technologies (deep pyrosequencing and Nanostring).

    Significance: We expanded analyses of DNA methylation in CHAMACOS children using three different state-of the art assays. We developed improved approach to analysis of complex Illumina 450K data to decrease technical variability. This is of particular importance in large population studies, where batch variability can bias results. We also found the blood cell heterogeneity, which has been thought to bias methylation results as well, was not associated with differences in global methylation in CHAMACOS newborns. Dr. Holland has contributed to the Special NIH Panel on “Surrogate Tissues for Epigenetic Studies."

Future Activities:

In the next year we will finalize analyses of available Illumina 450K data using newly developed ASMN methodology, both genome-wide and site-specific. Recent publications also have suggested filtering for potentially biased probes (i.e., sex linked probes and those containing SNP). Therefore, we will re-examine sites differentially methylated by sex, age, puberty, and environmental exposures. We will complete the analyses of ontogenetic changes in DNA methylation as soon as 12 year old samples are collected. We also will expand on the analyses of the relationship of epigenetic changes with hormones and puberty onset using a complete global methylation data for Alu and LINE-1 assays.


Journal Articles on this Report : 5 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 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)
R834513 (2014)
R834513 (2015)
R834513 (Final)
R834513C003 (2013)
R834513C003 (2014)
R834513C003 (2015)
  • Abstract from PubMed
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  • Abstract: Oxford Journals-Abstract
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  • Other: ResearchGate-Abstract & Full Text-PDF
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  • Journal Article Eskenazi B, Chevrier J, Rauch SA, Kogut K, Harley KG, Johnson C, Trujillo C, Sjodin A, Bradman A. In utero and childhood polybrominated diphenyl ether (PBDE) exposures and neurodevelopment in the CHAMACOS study. Environmental Health Perspectives 2013;121(2):257-262. R834513 (2012)
    R834513 (2013)
    R834513 (2014)
    R834513 (Final)
    R834513C001 (2012)
    R834513C001 (2013)
    R834513C003 (2013)
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  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: EHP-Full Text-HTML
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  • Other: EHP-Full Text-PDF
  • 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)
    R834513 (2014)
    R834513 (2015)
    R834513 (Final)
    R834513C003 (2013)
    R834513C003 (2014)
    R834513C003 (2015)
    R834513C004 (2013)
  • Full-text from PubMed
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  • Abstract: LWW-Abstract
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  • Other: University of California, Berkeley-Full Text-PDF
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  • Journal Article Warner M, Schall RA, Harley KG, Bradman A, Barr D, Eskenazi B. In utero DDT and DDE exposure and obesity status of 7-year-old Mexican-American children in the CHAMACOS cohort. Environmental Health Perspectives 2013;121(5):631-636. R834513 (2013)
    R834513 (2014)
    R834513 (Final)
    R834513C001 (2013)
    R834513C001 (2014)
    R834513C003 (2013)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: EHP-Full Text-HTML
  • Abstract: EHP-Abstract
  • Other: EHP-Full Text-PDF
  • 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)
    R834513 (2013)
    R834513 (2014)
    R834513 (2015)
    R834513 (Final)
    R834513C003 (2012)
    R834513C003 (2013)
    R834513C003 (2014)
    R834513C003 (2015)
  • Abstract from PubMed
  • Full-text: Taylor & Francis-Full Text-HTML
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  • Abstract: Taylor & Francis-Abstract
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  • Other: Taylor & Francis-Full Text-PDF
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  • Supplemental Keywords:

    epigenetics, methylation, DDT, DDE, PBDEs, flame retardants, manganese, maneb, puberty, RFA, Scientific Discipline, Health, 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 EPA Disclaimer

    Progress and Final Reports:

    Original Abstract
  • 2010 Progress Report
  • 2011 Progress Report
  • 2012 Progress Report
  • 2014 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