oa Genome-Wide Analysis of Biobanked Blood, Saliva and Cord Blood Identifies DNA Methylation Marks Related to Environmental Programming of Respiratory Allergy

The Developmental Origins of Health and Disease (DOHaD) concept describes how the environment impinges on intra-uterine development and early childhood and how it induces changes in the development that have long term impact on later health and disease risk. Environmental exposures including parental lifestyle and diet, obesity and chemical exposure have been shown to modulate disease risk. The effects do not simply disrupt development or induce disease themselves, but can affect how rapidly disease develops. Epigenetic changes are plausible molecular mechanisms that can explain disease development trajectories. We hypothesize that early life exposures can alter DNA methylation patterns, thereby predisposing the child to develop respiratory allergy (RA) later in life. We have used two longitudinal birth cohorts and biobanked samples in Belgium to discover and confirm DNA methylation signatures that can differentiate between RA cases and controls. Blood and saliva samples of 11-year old children from the discovery cohort were analyzed using Illumina Methylation 450K BeadChips (20 RA cases and 20 controls). Saliva is proposed as a DNA source that can be easily collected in a decentralized manner in children. In addition, we analyzed biobanked cord blood samples of the same children in order to detect if altered DNA methylation marks could be detected at birth, as a proxy for intra-uterine changes. The discovered methylation signatures were studied in blood and saliva samples in a second cohort of 5-year old children (N = 78). We installed an analytical pipeline for sample processing and data analysis. R-based software was used for data normalization and Comb-p tool was used to identify differentially methylated regions (DMR). The Illumina Methylation 450K BeadChips showed that the methylation status was comparable between blood and saliva, with ± 11% of the probes having a differential methylation pattern (Padj < 0.05 and |Δβ| >0.1). The results suggest that saliva is a suitable biofluid for genome-wide DNA methylation studies and that there is a substantial overlap with blood profiles. A comparison between RA cases and controls revealed 83 DMR in blood of the 11-year old children; CB 26 DMR in cord blood and 5 DMR in saliva. Two DMR were identified as being in common between blood, saliva and cord blood samples using a Venn diagram analysis. The DMR were located in genes involved in IL4 signalling, Th2-response and phagocytosis; pathways that are implicated in RA disease. These 2 DMR were confirmed by iPLEX MassArray analysis in the same birth cohort (N = 40) as well as in a second cohort (N = 78). This project provided novel insights in the molecular mechanisms that may predispose children to chronic disease development, such as RA. We have identified DNA methylation marks in saliva and blood of children that are relevant for RA. We have also observed these methylation marks in the children's biobanked cord blood taken many years before RA development. We are among the first to show the utility of saliva to study DNA methylation changes in children. Or results contribute to the DOHaD concept that has very important implication for many societies and for global health policy.