Grantee Research Project Results
2011 Progress Report: Epigenetics Project
EPA Grant Number: R834513C003Subproject: 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 Integrative Research on Childhood Leukemia and the Environment - 2015
Center Director: Metayer, Catherine
Title: Epigenetics Project
Investigators: Eskenazi, Brenda , Holland, Nina T. , Bradman, Asa , Harley, Kim , Barcellos, Lisa , Lustig, Robert
Current Investigators: Eskenazi, Brenda
Institution: University of California - Berkeley
EPA Project Officer: Hahn, Intaek
Project Period: August 1, 2009 through July 31, 2014 (Extended to July 31, 2017)
Project Period Covered by this Report: August 1, 2010 through May 31,2011
RFA: Children's Environmental Health and Disease Prevention Research Centers (with NIEHS) (2009) RFA Text | Recipients Lists
Research Category: Children's Health , Human Health
Objective:
In Project C, we are investigating the effects of exposure on the epigenome and its relationship with pubertal onset. This year, we have continued to assess global methylation in DNA isolated from banked clot specimens of CHAMACOS children using three different assay methods: pyrosequencing (Alu and LINE-1 repeats) and Illumina Infinium. Preliminary analyses suggest that global methylation patterns are likely fluctuating throughout early childhood. If verified through expanded analysis, the opposite directions of the change in global DNA methylation by LINE-1 and Illumina 27k assays may highlight the importance of using site specific methylation data. Furthermore, we’ve found that global DNA methylation may be modified by prenatal exposure to PBDEs, providing a potential mechanism by which PBDEs and possibly other toxicants affect health.
The specific aims of this project are:
- To analyze global DNA methylation in newborn children by three different assays.
- To determine ontogenetic changes in global DNA methylation in blood of children between birth and 12 years.
- To investigate the relationship of in utero and 9-year-old blood concentrations of DDT/E and PBDEs with global DNA methylation.
- To determine whether global methylation is associated with onset of puberty and hormonal changes.
- To examine site-specific methylation in relation to age, sex, exposure to DDT/E and PBDEs and puberty onset.
Progress Summary:
1. To analyze global DNA methylation in newborn children by three different assays.
To date, we have isolated DNA, bisulfite-converted and analyzed global methylation by pyrosequencing in 280 banked clot specimens from CHAMACOS children collected at birth to assess DNA methylation, or CpG sites, as measured by three different assays. We found that, on average, the LINE-1 assay repeats are more than twice as likely to be methylated than Alu assay repeats or CpG sites covered by the Illumina Methylation 27k array (i.e., of approximately 27,000 CpG sites) (77.8 vs. 28.8 or 27.5 %5mC).
The recently released Illumina Infinium Methylation 450K Assay greatly expands the number of CpG sites examined from the earlier 27k assay (from 25,450 to 485,577 CpG sites), which we previously ran for 72 newborns. The expanded assay retains ~90% of the sites that the 27k array investigated. We conducted Infinium Methylation 450k (Illumina) analyses on DNA isolated for 140 newborns.
We also analyzed LINE-1 and Alu methylation in DNA isolated from serum samples for 25 children at birth that had previously been analyzed for clot DNA LINE-1 methylation in order to look at methylation differences by cell composition. We found that LINE-1 methylation was significantly higher in DNA isolated from clots compared to serum (71.1 vs. 69.6 %5mC, p=0.01).
2. To determine ontogenetic changes in global DNA methylation in blood of children between birth and 12 years.
Global methylation via pyrosequencing of Alu and Line-1 repeats was performed for 376 additional blood samples since 5/7/2010, for a total of 486 samples analyzed at delivery, 2, 5, 7, and 9 years of age.
Using mixed effects models analysis we observed that mean LINE-1 DNA methylation increased with age (1.59 percentage points) from birth to 9 years of age (p<0.0005). However, we observed the opposite trend in global methylation as measured by the Illumina 27k assay over time, where methylation decreased by 0.32 percentage points from 0 to 9 years of age (p<0.005). While the trend of global methylation percentage over time was the opposite for the two assays, LINE-1 and Illumina 27k, this may be explained by the different CpG sites examined by these assays which may behave differently over time.
3. To investigate the relationship of in utero and 9-year-old blood concentrations of DDT/E and PBDEs with global DNA methylation.
We expanded our analysis on the effect of prenatal organochlorine (p,p’-DDT, o,p’ -DDT, and p,p’-DDE) exposure on LINE-1 and Alu methylation at birth (n=129) and 9 years of age (n=184). At birth, we found a borderline significant inverse relationship between OC’s (p,p’-DDT and o,p’ -DDT) and LINE-1 methylation (P=0.10 and p=0.11, respectively). This effect was not observed in our Alu global methylation data. Furthermore, we did not find such a relationship between prenatal OC levels and global methylation (LINE-1 & Alu) at 9 years of age.
In a subset of 191 newborn children who had polybrominated diphenylether ether (PBDE) exposure data available, we observed an inverse relationship between the prenatal serum concentrations of two PBDEs (congeners PBDE-47 & -100, and the sum of 5 PBDE congeners with greater than 50% detection) and Alu global methylation. These data suggest a potential pathway by which early exposures (e.g.,. PBDEs) may affect long term health. In the future we will also determine whether similar effects persist at age 9.
4. To determine whether global methylation is associated with onset of puberty and hormonal changes.
Assessment of pubertal onset at age 9 was completed in 314 children. These data will be available for analysis following data entry and cleaning. However, preliminary analysis (n=100) showed that at age 9, 37% of girls had signs of breast development and 41% of boys had begun gonadal development. In girls, BMI was slightly higher for those in Tanner stage 2, but no such trend was seen in boys.
5. To examine site-specific methylation in relation to age, sex, exposure to DDT/E and PBDEs and puberty onset.
As part of our preliminary analysis of data generated by the Illumina 450k platform, we analyzed genome-wide and site-specific DNA methylation in 42 CHAMACOS children for subjects that had matched data both at birth and 9 years of age and found meaningful variation in methylome by sex and age. After adjusting for multiple testing by controlling for the False Discovery Rate (FDR), over 2500 CpG sites were differentially methylated by age. Furthermore, several CpG sites in the HDAC4, FRMD6, TAP1, and FGR genes identified were also found to be differentially methylated in buccal DNA among newborns and 18-month-old children from a different study in Australia (personal communication, Dr. Jeff Craig). While it was not surprising that many CpG sites in sex chromosomes were differentially methylated by gender, among newborns, we also found sex- specific differences in 73 CpG sites located in autosomes (38 genes). Fewer sites were differentially methylated by gender in 9-year-olds, however 14 of the 15 sites were also identified in newborns. Further, three of these loci also had gender-specific differences in methylation in other studies of DNA from cord blood (Adkins 2011) and adult saliva specimens (Liu 2010).
We examined the relationship of genome-wide site specific CpG methylation (measured as β: proportion of methylated cytosines per specific CpG site) in newborn DNA with obesity parameters at age 9, including obesity status and waist circumference in CHAMACOS children stratified by sex. Preliminary pathway analysis of the 200 sites (approximately 130 genes) that differed most by obesity parameters, using the Benjamini and Hochberg adjustment for multiple testing, identified clusters of genes that were involved in relevant biological pathways. In 9-year-old girls, top gene hits were significantly enriched with genes related to adipogenesis for both waist circumference (NR2F1, CNTFR, LPIN1, p = 0.02) and overweight status (GATA2, GATA3, CNTFR, UCP1, p =0.02). Among boys, top candidate genes for overweight status (MEF2A, NCOR2, SMAD3, RB1, p = 0.003) were significantly enriched in clusters related to insulin sensitivity and Type II Diabetes. We found no overlap between genes identified in boys and girls, suggesting sex-specific methylation differences.
Future Activities:
Next year, we will expand pyrosequencing and Infinium analyses to additional samples from the same children at other ages (2, 5, 9 years old), and confirm our preliminary finds for Alu, LINE-1 and Illumina for a larger sample size. Additionally, as CHAMACOS puberty data become available at the end of 2011, we will analyze how global and site-specific methylation changes through pubertal development.
Journal Articles on this Report : 6 Displayed | Download in RIS Format
| Other subproject views: | All 77 publications | 38 publications in selected types | All 38 journal articles |
|---|---|---|---|
| Other center views: | All 697 publications | 170 publications in selected types | All 169 journal articles |
Supplemental Keywords:
Exposure assessment, environmental, disease, toxin, health effects, child health, biomarkers, epigenetic, DNA methylation, reproductive health, neurobehavioral health, pubertal onset, metabolic syndrome, obesity, cognition, attention, memory, motor, executive function, social cognition, infants, children, Latino, farmworker, agriculture, pesticide, fungicide, PBDE, DDT, Mn, California, CA, Salinas Valley, community-based, deciduous teeth, back-extrapolation, GIS, maternal, international cooperation, scientific discipline, health, RFA, biology, health risk assessment, children's health, biochemistry, environmental policy, neurological development, farmworkers, harmful environmental agents, flame retardants, agricultural community, pesticide exposure, neurochemical effects, biological markers, children's vulnerability, 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 communityRelevant Websites:
Progress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R834513 Center for Integrative Research on Childhood Leukemia and the Environment - 2015 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
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
- Final Report
- 2016 Progress Report
- 2015 Progress Report
- 2014 Progress Report
- 2013 Progress Report
- 2012 Progress Report
- 2010 Progress Report
- Original Abstract
38 journal articles for this subproject
Main Center: R834513
697 publications for this center
169 journal articles for this center