Folic acid intervention during pregnancy alters DNA methylation, affecting neural target genes through two distinct mechanisms

Background: We previously showed that continued folic acid (FA) supplementation beyond the first trimester of pregnancy appears to have beneficial effects on neurocognitive performance in children followed for up to 11 years, but the biological mechanism for this effect has remained unclear. Using samples from our randomized controlled trial of folic acid supplementation in second and third trimester (FASSTT), where significant improvements in cognitive and psychosocial performance were demonstrated in children from mothers supplemented in pregnancy with 400 mu g/day FA compared with placebo, we examined methylation patterns from cord blood (CB) using the EPIC array which covers approximately 850,000 cytosine-guanine (CG) sites across the genome. Genes showing significant differences were verified using pyrosequencing and mechanistic approaches used in vitro to determine effects on transcription. Results: FA supplementation resulted in significant differences in methylation, particularly at brain-related genes. Further analysis showed these genes split into two groups. In one group, which included the CES1 gene, methylation changes at the promoters were important for regulating transcription. We also identified a second group which had a characteristic bimodal profile, with low promoter and high gene body (GB) methylation. In the latter, loss of methylation in the GB is linked to decreases in transcription: this group included the PRKAR1B/HEATR2 genes and the dopamine receptor regulator PDE4C. Overall, methylation in CB also showed good correlation with methylation profiles seen in a published data set of late gestation foetal brain samples. Conclusion: We show here clear alterations in DNA methylation at specific classes of neurodevelopmental genes in the same cohort of children, born to FA-supplemented mothers, who previously showed improved cognitive and psychosocial performance. Our results show measurable differences at neural genes which are important for transcriptional regulation and add to the supporting evidence for continued FA supplementation throughout later gestation.