Non-communicable diseases including cardiovascular diseases, metabolic diseases and cancer are the leading causes of death in developed countries. These diseases are predicted to also be the leading causes of death in developing countries by 2020. Stemming the increase in the prevalence of these diseases will require a more detailed understanding of their etiology using a life course approach. Existing data linking early chemical and non-chemical stressors to these adult-onset diseases derives either from well-powered cross- section or retrospective cohort studies, or under-powered prospective cohorts with short follow-up. Epigenetic alterations - a mechanism by which genes respond to the environment - have been hypothesized to link observed associations between early stressors and multiple common diseases. However, prior cross-sectional and retrospective studies have lacked early life covariate data and specimens that would help to examine the links between early life stressors and later life disease. To address these knowledge gaps, in 2005-2011, we developed the pre-birth Newborn Epigenetics STudy (NEST) cohort comprising more than 2000 women, and followed their offspring until age 3-5 years. The NEST cohort has become a resource used by our group to identify novel associations between chemical and non-chemical stressors, and early signs of these non- communicable diseases, including cardiometabolic dysfunction. This resource has also been used to replicate novel findings by other groups, to pool with other cohorts to enhance statistical power, and to test new hypotheses by others. Our overarching goal for this application is to maintain this resource. This will be accomplished through the retention of trained staff with the critical skills to, (i) maintain and enrich the cohort by collecting additional data and specimens, (ii) develop and implement quality control and quality assurance protocols on existing and to-be-collected data and specimens, and, (iii) establish a comprehensive web-based database to increase our capability for data re-use and pooling with other cohorts to enhance statistical power. Direct web access will also simplify the process of data sharing with other birth cohorts and prepare our data for linkage. We will follow the cohort until age 11-17 years. This age range coincides with peri-puberty and puberty?developmental windows of heightened susceptibility to the non-communicable diseases under investigation. We also will link NEST data with identifiable Health System- and State-run medical records. Completing the proposed study will result in an enriched specimen repository with quality control and quality assurance, and annotated epidemiologic and clinical data. These data and specimens will facilitate rapid hypothesis testing by our group as well as data sharing and linkage with other cohorts to enhance statistical power. Data contributing to our understanding of the developmental origins of adult-onset non-communicable diseases are critical for guiding public health intervention efforts.

The alteration of gene expression mediated by epigenetic modifications, specifically DNA methylation, has been proposed as a mechanism by which chemical and biological factors during gestation and childhood may influence health and adult disease onset. Epigenetics of imprinted genes, which exhibit expression of one parental allele, represent a promising area of fetal programming research, as many imprinted genes are involved in early growth and development. The aim of the proposed study is to characterize the relationship of prenatal exposure to common endocrine disruptors, phthalates, with DNA methylation of imprinted genes in newborns. The analysis will include DNA samples from the well-characterized CHAMACOS birth cohort study of hundreds of Mexican-American children and their mothers followed from early pregnancy through adolescence. In order to discern relationships between epigenetic profiles of newborns and early life exposure parameters, DNA methylation of imprinted genes in newborns will be assessed using targeted pyrosequencing, followed by validation of the hits by next generation sequencing. DNA methylation of imprinted genes will also be interrogated for associations with biological factors (sex, gestational age, and birth weight), phthalate exposure during pregnancy, and parental obesity. The CHAMACOS study provides a unique opportunity to assess biological and environmental interactions with imprinted gene epigenetics in a cohort with substantial environmental stressors and a high prevalence of parental pre-pregnancy obesity. The research outlined in this proposal will contribute to the limited knowledge regarding the effects of in utero phthalate exposure on methylation of imprinted genes, which thus far includes limited data on only two imprinted genes.7,8 We will expand this analysis to include several additional imprinted genes with high biological significance for fetal development. We will also validate findings of the association between methylation marks and parental obesity from another birth cohort (NEST).9,10 Data generated by the state-of-the art epigenetic and bioinformatic methodologies will provide insights into imprinted gene epigenetics and may identify potential new targets for intervention in pregnant women.

Infants and young children spend more than 95% of their time indoors where they receive chronic exposures to semi-volatile organic compounds (SVOCs) from contact with indoor dust due to their increased hand to mouth activity and crawling behavior. Several SVOCs commonly detected in indoor dust are considered "chemical obesogens", which are defined as chemicals that alter lipid homeostasis and fat storage, alter metabolic set points, or disrupt energy balance, resulting in fat accumulation and obesity. Several recent studies have suggested that perinatal exposure to obesogens may result in increased odds of obesity in children. Current research also suggests that many of these chemicals compounds act via a mechanism that includes activation of peroxisome proliferator-activated nuclear receptors (PPARs), leading to adipogenesis. Our preliminary studies demonstrate that rats prenatally exposed to an environmentally relevant dust mixture were heavier at birth, and throughout their life. Current approaches to measuring children's exposure to chemicals present in dust have typically relied on simplistic and crude dust ingestion rates, or measurements in serum or urine, the latter being expensive and typically isolated to one or few specific chemicals. Therefore, there is a critical need to provide better estimates of integrated exposure to SVOC mixtures and validate appropriate exposure markers. Given the chronic and sometimes high SVOC exposure that occurs in some homes, we hypothesize that early life exposure to mixtures of SVOCs in indoor dust leads to increased odds of obesity in children, and that these effects are associated primarily with chemicals in dust having high PPARγ and adipogenic activity. To address this hypothesis we have recruited a multi-disciplinary team that will leverage the resources of, and collaborate with, an ongoing NIH funded birth cohort examining associations between prenatal nutrition, secondhand tobacco smoke, and epigenetics modifications on obesity in children. We will conduct a nested case control study with 100 overweight/obese and 100 non-obese toddlers and quantify their prenatal and postnatal exposure to mixtures of SVOCs using targeted and non-targeted (e.g. screening) approaches. House dust samples will be characterized for both adipogenic and PPAR activity using in vitro assays, which will provide greater insight into effects related to actual exposure t dust particles. Associations between obesity (and other health outcomes available in the study), contaminant exposure (individual chemicals and mixtures) and adipogenic/PPARγ activity in dust samples will be examined. This work will contribute new data regarding the potential health impacts from pre- and postnatal exposure to contaminant mixtures present in house dust, and help identify mitigating factors.

Prenatal and postnatal environmental tobacco smoke (ETS) exposure have been associated with a range of adverse cognitive and neurobehavioral outcomes in children, including higher rates of Attention-Deficit / Hyperactivity Disorder (ADHD). However, our understanding of both the developmental timing of ETS-induced effects on cognitive outcomes, as well as the possible mechanisms underlying such effects is limited. This study will take advantage of a perinatal birth cohort that has obtained prospectively collected data from the first trimester through infancy inclusive of surveys regarding smoking history, data from medical records, maternal blood at the first trimester, and cord blood and buccal cells at delivery. Preliminary data indicates prenatal exposure and cord blood DNA methylation are related to externalizing behavioral problems at one year. The proposed study will conduct detailed assessments of childhood cognitive, neurobehavioral function, and ADHD symptoms among a subcohort of children (n=400) at ages 3-5 years and two-years later at 5-7 years. Using maternal blood specimens collected during the first trimester, cord blood at birth, and blood specimens from the children at the first postnatal assessments we will characterize cotinine levels. DNA methylation for select regulatory control regions for genes that have been associated with ADHD symptoms or similar neurodevelopmental phenotypes will be characterized from the child's cord blood and their peripheral blood collected at 3-5 and 5-7 years of age. We hypothesize that prenatal and postnatal exposure to ETS will be associated with cognitive deficits in executive functioning, that DNA methylation will be associated with deficits, and that the association between ETS and cognitive and neurobehavioral outcomes will be partially mediated by DNA methylation. The study will be the first of its kind to help disentangle the associations between ETS and childhood cognitive outcomes by exploring potential epigenetic factors that may help explain these associations. Because DNA methylation is malleable, the findings may inform novel methods for improving cognitive deficits resulting from ETS.

Early life environmental tobacco smoke (ETS) exposure is associated with detrimental outcomes on neurodevelopment, including Attention-Deficit / Hyperactivity Disorder (ADHD), but the mechanisms are unclear. Epigenetic changes induced by ETS exposure may underlie these associations. Our long-term goal is to utilize DNA methylation profiles to identify children at risk for developing adverse neurodevelopmental outcomes. The objective is to uncover the epigenetic mechanism linking ETS exposure in early life to phenotypes associated with ADHD. We hypothesize that early life ETS exposure induces persistent and targeted functional changes in DNA methylation that associate with neurodevelopmental phenotypes. The rationale is that defining the repertoire of ETS-induced functional DNA methylation changes will offer exciting new opportunities for risk stratification and early interventions to reduce and prevent adverse behavioral and cognitive outcomes of ETS exposure. Our aims are as follows: 1) Identify ETS-related methylation targets. We will test the hypothesis that early life ETS exposure in rats will induce measurable shifts in methylation at loci relevant to neurobehavioral outcomes that are detectable in both brain and blood. Whole genome bisulfite sequencing will identify ETS-vulnerable loci with validation by pyrosequencing. 2) Identify ETS-altered methylation-expression relationships in frontal cortex. We hypothesize that early life ETS exposure in rats induces changes in frontal cortex gene expression at ADHD-relevant genes. Whole transcriptome profiling will identify these changes. We will also evaluate methylation expression relationships from the in vivo results and in an in vitro model of neuronal differentiation. 3) Determine if DNA methylation varies with ETS dose in humans. Prenatal and peri-natal cotinine levels and DNA methylation in children, from bisulfite pyrosequencing at ADHD-related genes and candidate loci from the above aims, will determine if methylation varies with exposure. The proposed research is expected to reveal ETS-related epigenetically vulnerable genes that mechanistically explain the link between ADHD and exposure to ETS and that may serve as biomarkers of past exposure.

A growing consensus suggests that epigenetics, i.e., mitotically heritable information that controls gene expression without a change in the DNA sequence, plays an important role in endocrine adjustment associated with the genesis of obesity and obesity-related adult-onset chronic diseases. Because imprinted genes depend on epigenetic mechanisms such as DNA methylation for their monoallelic expression, variation in methyl-group donor nutrients makes them vulnerable to epigenetic deregulation, especially since only one allele is functional. Deregulation of the paternally expressed IGF2 has been associated with increased risk of obesity, overgrowth disorders and obesity-related cancers in animal and epidemiologic studies. DLK1 exhibits many of the same structural and regulatory characteristics of the coordinately regulated IGF2 imprinted domain. Despite the seeming importance of imprinted genes in obesity and adult-onset chronic disease, the population frequency of, and risk factors associated with deregulation (aberrant methylation and loss of imprinting) of these paternally expressed genes is unknown. Exposure to diets with a high methyl group donor content has been associated with aberrant methylation and cancer. Our central hypothesis is that in utero and early ex utero exposure to `methyl-rich' diets increases the risk of aberrant methylation at imprint control centers of imprinted domains, increasing the risk of loss of imprinting and increased circulating concentrations of growth factors they encode, and subsequently, rapid weight gain in the offspring. Our specific aims are: (1) To determine whether maternal intake of B vitamins-- folate, vitamins B12 and B6--are associated with aberrant methylation and loss of imprinting of insulin-like growth factor-2 (IGF2) and Delta, drosophila homolog-like-1 (DLK1) in the offspring; (2) To determine whether aberrant methylation and loss of imprinting of IGF2 and DLK1 is associated with serum concentrations of IGF2 and DLK1 in infants, and (3) To determine whether early childhood exposure to these B vitamins is associated with aberrant methylation and subsequent weight gain during the first three years of life. To address these aims, we will recruit and estimate micronutrient intake in 1,500 women in the first and second/third trimester of pregnancy. At delivery, we will collect cord blood and buccal cell specimens from which we will evaluate the methylation profile and loss of imprinting in IGF2 and DLK1 in relation to maternal nutrition. At ages one, two and three, we will collect buccal cells, infant data on usual diet, weight and height measurements, to determine whether infant nutrition is associated with an aberrant methylation profile and subsequent infant weight gain. Loss of imprinting is mitotically heritable, but, in the absence of mutation in methylation-regulating genes, can be reversed with normal imprinting restored. Thus, identification of risk factors associated with deregulation of imprint control domains may hold therapeutic and focused public health intervention prospects for childhood obesity and adult onset, obesity-related chronic diseases.

Loss of Imprinting in IGF2 has been found in a wide spectrum of adult chronic diseases including diabetes, cardiovascular diseases and malignancies. IGF2 imprint disorders have also been reported in patients with the human overgrowth disorder Beckwith-Wiedemann syndrome. Patients with Wilm's tumor and hepatoblastoma have a higher prevalence of LOI in IGF2. Methylation changes of differentially methylated regions on exon 3 and 9 of IGF2 have also been reported in lymphocytes of patients with colon cancer and leukemia. Factors underlying these epigenetic alterations are unknown, although environmental exposures such as cigarette smoking have been implicated. The timing of this epigenetic event is also unknown, although accumulating evidence suggests that LOI in IGF2 may occur in-utero. We hypothesize that LOI in IGF2 is a response, in part, to aberrant methylation changes in IGF2, and because these methylation patterns are mitotically heritable, contributes to deleterious outcomes inherent in IGF2 deregulation. The specific aims of the study are: 1) To characterize methylation patterns and estimate the prevalence of LOI in IGF2 in-utero; 2) To evaluate whether maternal exposure to cigarette smoking is associated with LOI in IGF2; and 3) To evaluate whether LOI in IGF2 is associated with rapid infant weight gain during the first year of life, characteristic of infants of smoking mothers. The proposed research builds on an existing data collection structure of the Cord Blood Transplantation (COBLT) Project. This project recruits and stores cord blood of participants from all Obstetrics Care facilities in Durham, Orange and Wake Counties. We will prospectively identify 200 smoking and 200 non-smoking mothers from the Project database and obtain up to 5ml of cord blood at delivery to determine IGF2 DNA methylation patterns and biallelic expression of IGF2. We will then examine these patterns according to maternal smoking status and infant weight gain. Because LOI is potentially reversible with imprinting restored since the DMA sequence remains unaltered, (unless mutation is in genes regulating methylation) identifying the timing of IGF2 LOI and factors influencing this epigenetic event has wide ranging intervention prospects on clinically apparent chronic disease incidence. The proposed study also has the potential to provide a foundation for future studies investigating the etiology of chronic diseases, including diabetes, cardiovascular diseases and some cancers.