Project Summary/Abstract Section Little is known regarding how conditions during pregnancy and early life may impact epigenetic alterations and aging processes that could subsequently manifest in midlife as atherosclerosis, type 2 diabetes, adiposity and cognitive decline. In particular, there are sizeable and important knowledge gaps regarding associations of prenatal conditions (e.g. intrauterine growth restriction, maternal serum cortisol levels, maternal smoking and socioeconomic adversity) with epigenetic processes (often measured as DNA methylation) that could alter gene expression and disease outcomes. Our goal is to assess aging processes in 500 of the Collaborative Perinatal Project (CPP) participants born in 1959-1966 who had detailed prenatal assessments, biosample collection and phenotypic assessments during the first 8 years of life. Specific analytic objectives include: (a) to evaluate whether DNA methylation can be accurately measured in umbilical cord serum that has been stored frozen for 44-52 years; (b) to perform pilot analyses to investigate if DNA methylation in subcutaneous adipocytes and umbilical cord serum is associated with measures of adiposity derived from DEXA scans, CT lumbar region scans (to assess visceral fat), waist circumference and BMI; (c) to perform pilot analyses to evaluate whether prenatal conditions are associated with adiposity and adiposity-related DNA methylation in subcutaneous adipocytes and umbilical cord serum. The study will recruit approximately 500 CPP participants from Rhode Island. Clinical assessments include adiposity, atherosclerosis (coronary artery calcium CT scans), cognitive function, diabetes (fasting glucose), and blood biobanking for future analyses of cardiovascular risk factors (e.g. lipids). Subcutaneous adipocyte biopsies and buccal cell collection will be performed. Umbilical cord serum DNA and adipocyte DNA will be obtained using established protocols, and 100 samples will be run through deep sequencing DNA methylation assays as biomarkers of epigenetic characteristics. This will provide pilot data for future proposed work that will propose DNA methylation analyses of all collected samples. The analytic approach will include a locus-by-locus analysis for each 26,486 autosomal CpG, assessing associations of methylation with a) adiposity, and b) perinatal conditions, correcting for false discovery rate. Within-sibling analyses will be performed to account for shared family and environment as potential confounders. This study offers to provide innovative information to advance understanding of how epigenetic processes are involved in aging, and how perinatal and early life factors may be important determinants of aging. It is one of very few data sources available worldwide regarding prenatal, early life and middle-age determinants of health.

Specific Aims Section There is great interest in understanding how prenatal and early life factors influence aging processes, including the role of epigenetics and aging-related phenotypes of adiposity, atherosclerosis, type 2 diabetes and cognitive function. In recent years a number of discoveries suggested that the fetal environment and early life conditions are important in affecting these phenotypes. However, much of this research has been limited by fairly crude measures, both of key biological conditions (such as birth weight) and early life circumstances (such as father’s occupation as a measure of childhood socioeconomic position). Advances from animal and clinical literature have helped to identify key biological and social processes during fetal and early postnatal development that are hypothesized to result in epigenetic alterations which, in part, may alter normative aging processes. Most of these specific early risk conditions have not been investigated in a large prospective fashion, with high quality epigenetic data. As a result, there is much to learn regarding health-altering factors such as fetal hazards including maternal smoking, maternal cortisol levels during pregnancy, placental insufficiency and intrauterine growth restriction, and early life conditions such as child BMI, blood pressure and growth parameters, as well as family socioeconomic position and maternal cognitive function. In part due to the lengthy follow-up time (e.g. 40 years) required between early life factors and the expression of later life chronic conditions, there is a dearth of information in this area. The Collaborative Perinatal Project (CPP) assessed approximately 17,000 mother-fetus dyads in Rhode Island and Massachusetts during 1959–1966. This cohort includes a wealth of perinatal and early life information (up to age 8 years), as well as substantial data on mothers and fathers. Placental tissue has been assessed for pathology. Umbilical cord serum and maternal serum during pregnancy have been collected and remain in storage. Child participants are now middle-aged, and offer the opportunity to understand how perinatal and early life factors may influence aging processes. During the proposed study, our goal is to perform an assessment of 500 of these participants, collect high quality clinical data and conduct initial analyses. This unique scientific database will then be of service to the scientific community for years to come. Specific aims are: 1. To begin establishing an epigenetic database on middle aged participants, with DNA from subcutaneous adipocytes, blood, buccal cells and umbilical cord serum. High quality exposure data have already been directly collected prenatally and during the first 8 years of life. New biological materials will be collected including aforementioned DNA as well as a number of variables at midlife such as adiposity, atherosclerosis (via coronary artery calcification), type 2 diabetes, biological risk factors for cardiovascular disease, cognitive function, depressive symptomatology and health behaviors. Deep sequencing DNA methylation assays will be performed on the umbilical cord serum and subcutaneous adipocytes of 100 participants. This approach will provide high quality outcomes and biobanked materials to set the stage for a larger future database that will be used both during the initial two years of support and for multiple future epigenetic studies with this cohort. Within this database, we broadly aim to evaluate associations between social and environmental risks across the life course, measures of DNA methylation and midlife health outcomes. Specific analytic aims to be achieved within the funding period for this grant are outlined below. 2. To evaluate whether DNA methylation can be accurately measured in umbilical cord serum that has been stored frozen for 46-52 years. 3. To perform pilot analyses to investigate if DNA methylation in subcutaneous adipocytes and umbilical cord serum is associated with measures of adiposity derived from DEXA scans, CT lumbar region scans (to assess visceral fat), waist circumference and BMI. 4. To perform pilot analyses to evaluate whether prenatal conditions are associated with adiposity and adiposityrelated DNA methylation in subcutaneous adipocytes and umbilical cord serum. This study offers to provide innovative information to advance understanding of how epigenetic processes are involved in aging, and how perinatal and early life factors may be important determinants of aging. It is one of the very few data sources available worldwide regarding prenatal, early life and middle-age determinants of health. This study offers the potential to make major inroads towards understanding modifiable determinants of aging across the life course, beginning during prenatal and early years of life. This could serve to expand the complement of public health strategies that may enhance healthy aging and prevent disease outcomes.

Public Health Relevance Section The search for modifiable influences on healthy aging and aging-related disease has led to considerable interest in the potential health impact of conditions during pregnancy and early childhood (e.g. intrauterine grown restriction, maternal smoking, maternal stress during pregnancy, economic distress, maternal cognitive function, childhood cognitive function, childhood blood pressure, childhood body growth and obesity, childhood neglect and maternal mental health). Such information may contribute to critical and effective new alternatives to enhance healthy aging and prevent later disease, by public health interventions early in life. This project will add to the scientific understanding of biological mechanisms by which prenatal and early life factors may influence health, including the potential discovery of biomarkers as targets for treatment and prevention efforts. Overall, this study is one of the very few available sources of prenatal, early life and middle-aged health determinants and health outcomes worldwide, and offers the potential to make inroads in more fully understanding prenatal and early life determinants of health.