EFFECTS OF MATERNAL SOCIAL FACTORS ON CHILD’S EPIGENETICS. STUDIES IN PREGNANT WOMEN AND IN OBESE CHILDREN. – EPICHILD

We recruited 150 obese and healthy children at Bicêtre Hospital (Le Kremlin Bicêtre) ; and 500 pregnant women at Antoine Béclère Hospital (Clamart). Patients and controls were included in the study according to the French bioethics law with families being carefully informed and having signed a detailed informed consent agreed by the French ethics board.
We have studied DNA methylation using the Illumina 450K array.

Our first challenge is that the DNA samples available for methylation analysis are generally derived from heterogeneous cell populations. For example, the DNA samples most readily available from large numbers of individuals are from whole blood, which consists of many distinct populations in varying proportions. It has been shown that these functionally distinct populations have unique DNA methylation signatures, thus cell heterogeneity may act as a potential confounder when investigating DNA methylation differences between cases and controls, if cell distribution itself differs by disease status. To address we adjusted for cell proportion our DNA methylation analysis.
We found 47 differentially methylated CGs between obese (BMI +2.5DS) and controls and 547 CGs between super-obese (BMI +4DS) and controls. We are replicating these results by pyrosequencing.

We recruited 500 pregnant women and the last baby will born in February, 2016. After that, we will measure DNA methylation in cord blood, try to correlate in utero social stress and epigenetic marks and determine whether obesity marks and stress marks overlap.

We are working on an article about DNA methylation marks in obese and healthy children.

This study will investigate the biological underpinnings by which early life experiences are able to shape the regulatory systems underlying the development of childhood obesity. For reasons that are explained in the full version of the project, childhood obesity is our paradigm for approaching the biological link between early life experiences and later phenotype, as a phenotype sensitive to maternal social factors. We hypothesize that epigenetics (here the CpG methylation of genomic DNA), because it can keep a durable biological memory of early social events, could be this missing biological link.
Our protocol includes two separate studies, performed in parallel that will bring complementary answers to the question.
i) We will search for a relationship between social factors in the pregnant mother and offspring’s epigenetic marks generated during embryogenesis and fetal development: inherited methylation marks in cord blood DNA that will be found associated with maternal factors will be called Eg1. We will determine whether Eg1 are linked with each studied category of social or socially dependent factors in the mothers (acute stress, socioeconomic and educational level, smoking, obesity, alcohol, folic acid, nutritional factors).
ii) We will also study the relationship between childhood obesity, maternal social factors and epigenetic marks: marks that will be found associated in obese child DNA with childhood obesity will be called Eg2. We will also study potential correlations of Eg2 with maternal factors including those having their effects during the postnatal life of the babies.
iii) By combining the two sets of methylation data, we will determine whether Eg1 and Eg2 overlap, indicating that some loci are both sensitive to prenatal maternal factors and associated with obesity, tracing a possible epigenetic path between mother’s acquired social phenotype and child inherited obesity phenotype. If not, it will suggest that social maternal factors acting during pregnancy are not predisposing to child obesity, a negative result that remains important. In this case, the non-overlapping Eg1 and Eg2 will be the new findings of our study.
iv) The stability of Eg1 and Eg2 will bet tested by re-sampling studied individuals.

Methods for studying epigenetic marks: To determine Eg1 and Eg2, we will study i) selected genomic loci where methylation imprints are candidates; ii) whole genome DNA methylation with the CHARM methylation array. Given the high cost of the methylation arrays, we will perform genome wide studies of CpG methylation only in a small number of representative subjects, calculated though to be able to give significant statistical results. If we find that methylation at some CpGs associates either with maternal factors or child obesity, we will study these CpGs specifically in blood cells DNA from a large number of i) pregnant women and their neonates ii) obese and non obese children. Finally, to know if blood cells reflect the methylation levels at these loci in the tissues involved in obesity, CpG methylation will be determined in several tissues from autopsied fetuses, neonates or children. This will allow to evaluate the tissue specific duration of the methylation patterns.