Comparison of DNA Methylation Profiles in Sibships Discordant for Intrauterine Exposure to Maternal Gestational Diabetes Mellitus

Abstract

Introduction: In utero diabetic exposure is reported to be associated with predisposition to metabolic diseases in later life, supporting the hypothesis of Developmental Origins of Health and Disease (DOHaD). One of the underlying mechanisms proposed so far is by alterations in DNA methylation levels, which are associated with gene expression. To assess epigenetic modifications at DNA methylation levels associated with prenatal exposure to diabetes, we conducted a discordant sibship study, in which a total of 38 siblings showing discordance for intrauterine exposure to maternal gestational diabetes mellitus were recruited. Methods: We collected data on birth outcomes and data of anthropometric, physiological, and biochemical measurements after recruitment. DNA methylation levels of peripheral leukocytes were examined at over 485,000 CpG sites using Illumina Infinium HumanMethylation 450 BeadChip assays. To obtain an overview of methylation profiles across samples, intersample distance was measured using unsupervised hierarchical clustering methods. Within-pair differential methylation analysis was performed on genomic region levels including CpG islands, genes, and promoters, as well as on CpG site levels. Pathway and function analysis was conducted to identify biological implications associated with differentially methylated sites and regions. Results: In a Manhattan distance-based unsupervised hierarchical clustering analysis of methylation levels across samples, three of the sibling pairs were closest to each other, while most of the rest were closely related to each other based on prenatal GDM exposure status, independent of siblings. A within-pair differential analysis on CpG site levels showed that 18 sites were differentially methylated at p-values < 10-5. Among those significantly differential CpG sites was cg08407434, which is associated with HNF4A loci, with the mean pairwise difference of methylation levels of 1.3% (p-value = 9.1×10-7). In the region-level differential methylated analysis, 23 genes and 24 promoter regions were differentially methylated at p-value < 10-3. In the pathway analysis, the main pathway overrepresented by hypermethylated gene regions was immune responses. Discussion: To the best of our knowledge, this is the first epigenome-wide study investigating methylation profiles in discordant sibships for their maternal GDM. Our findings suggest that in utero exposure to diabetic environment has epigenetic effects, particularly altering DNA methylation levels, and thus reinforce the evidence for the hypothesis of DOHaD.