SUMMARY Heritable epigenetic factors can contribute to complex disease etiology. Here we examine the contribution of DNA methylation to complex traits that are precursors to heart disease, diabetes and osteoporosis. We profiled DNA methylation in the liver using bisulfite sequencing in 90 mouse inbred strains, genome-wide expression levels, proteomics, metabolomics and sixty-eight clinical traits, and performed epigenome-wide association studies (EWAS). We found associations with numerous clinical traits including bone density, insulin resistance, expression, protein and metabolite levels. A large proportion of associations were unique to EWAS and were not identified using GWAS. Methylation levels were regulated by genetics largely in cis, but we also found evidence of trans regulation, and we demonstrate that genetic variation in the methionine synthase reductase gene Mtrr affects methylation of hundreds of CpGs throughout the genome. Our results indicate that natural variation in methylation levels contributes to the etiology of complex clinical traits.

Methylenetetrahydrofolate reductase (MTHFR) generates methyltetrahydrofolate for methylation reactions. Severe MTHFR deficiency results in homocystinuria and neurologic impairment. Mild MTHFR deficiency (677C > T polymorphism) increases risk for complex traits, including neuropsychiatric disorders. Although low dietary folate impacts brain development, recent concerns have focused on high folate intake following food fortification and increased vitamin use. Our goal was to determine whether high dietary folate during pregnancy affects brain development in murine offspring. Female mice were placed on control diet (CD) or folic acid-supplemented diet (FASD) throughout mating, pregnancy and lactation. Three-week-old male pups were evaluated for motor and cognitive function. Tissues from E17.5 embryos, pups and dams were collected for choline/methyl metabolite measurements, immunoblotting or gene expression of relevant enzymes. Brains were examined for morphology of hippocampus and cortex. Pups of FASD mothers displayed short-term memory impairment, decreased hippocampal size and decreased thickness of the dentate gyrus. MTHFR protein levels were reduced in FASD pup livers, with lower concentrations of phosphocholine and glycerophosphocholine in liver and hippocampus, respectively. FASD pup brains showed evidence of altered acetylcholine availability and Dnmt3a mRNA was reduced in cortex and hippocampus. E17.5 embryos and placentas from FASD dams were smaller. MTHFR protein and mRNA were reduced in embryonic liver, with lower concentrations of choline, betaine and phosphocholine. Embryonic brain displayed altered development of cortical layers. In summary, high folate intake during pregnancy leads to pseudo-MTHFR deficiency, disturbed choline/methyl metabolism, embryonic growth delay and memory impairment in offspring. These findings highlight the unintended negative consequences of supplemental folic acid.

Fifteen to 20% of pregnant women may exceed the recommended intake of folic acid (FA) by more than four-fold. This excess could compromise neurocognitive and motor development in offspring. Here, we explored the impact of an FA-supplemented diet (5× FASD, containing five-fold higher FA than recommended) during pregnancy on brain function in murine offspring, and elucidated mechanistic changes. We placed female C57BL/6 mice for one month on control diets or 5× FASD before mating. Diets were maintained throughout pregnancy and lactation. Behavioural tests were conducted on 3-week-old pups. Pups and mothers were sacrificed at weaning. Brains and livers were collected to examine choline/methyl metabolites and immunoreactive methylenetetrahydrofolate reductase (MTHFR). 5× FASD led to hyperactivity-like behavior and memory impairment in 3-week-old pups of both sexes. Reduced MTHFR protein in the livers of FASD mothers and male pups resulted in choline/methyl metabolite disruptions in offspring liver (decreased betaine) and brain (decreased glycerophosphocholine and sphingomyelin in male pups, and decreased phosphatidylcholine in both sexes). These results indicate that moderate folate supplementation downregulates MTHFR and alters choline/methyl metabolism, contributing to neurobehavioral alterations. Our findings support the negative impact of high FA on brain development, and may lead to improved guidelines on optimal folate levels during pregnancy.

Background: Moderately high folic acid intake in pregnant women has led to concerns about deleterious effects on the mother and fetus. Common polymorphisms in folate genes, such as methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolaseformyltetrahydrofolate synthetase (MTHFD1) R653Q, may modulate the effects of elevated folic acid intake. Objectives: We investigated the effects of moderate folic acid supplementation on reproductive outcomes and assessed the potential interaction of the supplemented diet with MTHFD1-synthetase (Mthfd1S) deficiency in mice, which is a model for the R653Q variant. Design: Female Mthfd1S +/+ and Mthfd1S +/2 mice were fed a folic acid-supplemented diet (FASD) (5-fold higher than recommended) or control diets before mating and during pregnancy. Embryos and placentas were assessed for developmental defects at embryonic day 10.5 (E10.5). Maternal folate and choline metabolites and gene expression in folate-related pathways were examined. Results: The combination of FASD and maternal MTHFD1-synthetase deficiency led to a greater incidence of defects in E10.5 embryos (diet 3 maternal genotype, P = 0.0016; diet 3 embryonic genotype, P = 0.054). The methylenetetrahydrofolate reductase (MTHFR) protein and methylation potential [ratio of S-adenosylmethionine (major methyl donor):S-adenosylhomocysteine) were reduced in maternal liver. Although 5-methyltetrahydrofolate (methylTHF) was higher in maternal circulation, the methylation potential was lower in embryos. The presence of developmental delays and defects in Mthfd1S +/2 embryos was associated with placental defects (P = 0.003). The labyrinth layer failed to form properly in the majority of abnormal placentas, which compromised the integration of the maternal and fetal circulation and presumably the transfer of methylTHF and other nutrients. Conclusions: Moderately higher folate intake and MTHFD1-synthetase deficiency in pregnant mice result in a lower methylation potential in maternal liver and embryos and a greater incidence of defects in embryos. Although maternal circulating methylTHF was higher, it may not have reached the embryos because of abnormal placental development; abnormal placentas were observed predominantly in abnormally developed embryos. These findings have implications for women with high folate intakes, particularly if they are polymorphic for MTHFD1 R653Q.