Folic acid: investigation of its relevance for DNA methylation and the enzymes that reduce it.

DNA methylation is a biochemical process where a DNA base, usually cytosine, is enzymatically methylated at the 5-carbon position. An epigenetic modification associated with gene regulation, DNA methylation plays a key role in a wide variety of biological processes. Changes in DNA methylation patterns have also been implicated in the pathogenesis of many human diseases. Following up on a randomised control trial (RCT) studying the effects of prolonged folic acid supplementation during pregnancy, we sought to identify regions of the genome that altered their methylation status in response to intervention, considering the methyl-donor for DNA methyltransferase reactions is a product of folate metabolism. Using a genome-wide, array-based method of analysis on a subset of the cohort (n=6) from the RCT, we identified 5 novel folate-sensitive differentially methylated regions (FS-DMRs): Inosital hexakisphosphate kinase 1 (IP6K1), Chromosome 9 Open Reading Frame 44 (Chr9ORF44), RAS p21 protein activator 4 (RASA4), Sphingosine-1-phosphate lyase 1 (SPGL1) and Chromosome 19 Open Reading Frame 75 (Chr19ORF75). A confirmation analysis was carried out using a loci-specific method of analysis across the rest of the cohort (n = 238) on three of these FS-DMRs (IP6K1, RASA4, and Chr9ORF44), but they did not reach statistical significance. In a cell culture model, changes in methylation across selected regions were confirmed to have a significant impact on gene expression (IP6K1, RASA4 and GPS2). A final analysis of dihydrofolate reductase (DHFR), responsible for reducing folic acid to its active form, investigated whether specific post-translational modifications was a feature of this enzyme. Following up on a SUMOylation analysis of DHFR previously published in the literature, our own analysis did not yield the same results, exposing a potential source of false positive results from a commercially available kit wildly used in this field