Characterizing DNA Methylation and Hydroxymethylation in Cord Blood and Identifying Sex-Specific Differences using the Illumina EPIC Array.

DNA methylation, an epigenetic mark, has become a common outcome in epidemiological studies with the aid of affordable and reliable technologies. Yet the most widespread technique used to assess methylation, bisulfite conversion, does not allow for the differentiation of regular DNA methylation (5-mC) and other cytosine modifications, like that of hydroxymethylation (5-hmC). As both 5-mC and 5-hmC have distinct biological roles, sometimes with opposing effects, it is crucial to understand the difference between these marks. To characterize 5-mC and 5-hmC in cord blood and expand on previously published results in smaller cohorts, 73 samples from infants in the Michigan Mother Infant Pairs cohort were paired bisulfite and oxidative bisulfite converted. 5-mC and 5-hmC were assessed on the Illumina Infinium EPIC array, using maximum likelihood methods, and sex-specific differences of these marks were analyzed. 5-mC and 5-hmC were both broadly distributed across the genome, and 5-hmC was prevalent, with proportions of 0.01-0.55. Sex-specific analysis revealed total methylation was different on 17,000 sites (q<0.05), but only different at 1,866 and 5 sites of 5-mC and 5-hmC specifically. These results add additional support to the literature and demonstrate the importance of differentiating between 5-mC and 5-hmC in epidemiological studies going forward.