NTDs stand out as one of few birth defects for which primary prevention strategies are available. Research spanning decades, including randomized and community-based trials demonstrate that maternal, periconceptional supplementation with FA alone, or multivitamins containing FA can reduce risk of NTDs in offspring (1
). How FA acts to prevent NTDs is a major outstanding question, and the answer will be complex because folate is central to numerous cellular reactions. These include production of purines and thymidylate, the building blocks for DNA and RNA biosynthesis, and production of the universal methyl donor S-adenosyl- methionine (SAM), utilized in methylation of DNA, histones, proteins, and lipids. Therefore, deficits in FA metabolism could affect cell proliferation, cell survival, transcriptional regulation, or a host of other cellular reactions; defects in any of these processes could disrupt NTC. To bring insight into the mechanisms by which FA acts during NTC, studies have turned to animal models, in particular mouse NTD models, which are thought to be representative of human neurulation anatomically and molecularly and in which folate levels can be altered prior to or during pregnancy.
Curiously, even though targeted mutations have been made in numerous mouse genes required for FA metabolism or utilization, only three (Folr1, RFC, Mthfd1l) have overt NTD phenotypes (24
). Even under conditions of folate deficiency, there is relatively little evidence of altered NTD incidence for mouse FA pathway mutations (27
). Moreover little compelling association has been established between Folr1 or RFC and human NTDs and despite a long-standing focus on this pathway, only a few polymorphisms have been identified as possible risk factors for human NTDs, such as the 677C > T SNP in the MTHFR gene (11
). Currently, in the folate-replete population in the US, the majority of human pregnancies are within the normal range of FA levels and recent studies found little association between NTD risk and maternal FA intake, perhaps suggesting that FA-sensitive NTDs have largely been prevented (28
). Therefore, data to date suggest that deficits in the FA pathway likely represent only a modest fraction of NTD risk.
Looking beyond the folate pathway to elucidate gene–environment interactions
A lack of evidence between FA pathway mutants and NTD risk indicates the need for novel approaches to elucidate how FA impacts NTC. The large number of mouse NTD models with no apparent link to the FA pathway provide enormous potential to explore how genetics impact responsiveness to FA and to define mechanisms by which FA influences NTC. This potential has been only minimally realized, as only 23 of the >200 mouse NTD mutants have been tested for FA responsiveness (27
). FA treatment has some preventive effect in 11 mouse NTD models and in a few cases this correlates with compromised FA utilization. Splotch2H
) and Mthfd2l
mutants show a deficit in FA metabolism, but Cited2, Fkbp8, Fuz
, and curly tail
) mutants do not, indicating that disrupted FA metabolism is unlikely to be the full explanation for FA-mediated effects. The requirement for FA in DNA synthesis might be expected to impact cell proliferation or survival, but to date there is no evidence that these processes are normalized in FA-preventable mouse NTD models. We therefore currently have little that ties together NTD models that are FA-responsive or FA-resistant at a mechanistic level. Testing a much larger set of NTD models will expand this dataset and may reveal common pathways or targets and should lead to better predictions as to whether FA, or perhaps another treatment, would be most effective in preventing NTDs.
Contrary to expectations, in a few mouse NTD models FA treatment resulted in detrimental effects, including an increased risk for NTDs and embryo loss prior to the time of NTC (29
). Although it is largely assumed that FA prevents NTDs by correcting the embryological defect, these findings of early embryo loss are consistent with the possibility raised back in 1997 based on miscarriage risk that embryo loss may explain some of the decrease in human NTD occurrence upon FA supplementation (31
). If these unexpected findings are relevant to human NTDs, it could suggest that, for certain mutations, FA may not be protective or even neutral in its action, although the consensus is that in humans, at a population-scale, FA has a preventive effect. Additional studies in animal models will be required to determine the basis for the observed detrimental effect and whether there are particular gene or pathway mutations that are more susceptible, either positively or negatively to FA effects.
Possible epigenetic changes induced by FA
A striking but understudied aspect of FA is the potential to cause epigenomic changes. Changes in SAM levels could impact DNA methylation and histone modification, both of which can influence gene transcription. Indeed, there is evidence that methyl donor-enriched diets can induce alterations in gene expression and long-term generational exposure can result in increasing variation in DNA methylation even in wildtype mice (32
). Moreover, questions have been raised as to whether the increase in FA intake acting through the methylation cycle may predispose to allergic airway disease, although the current evidence is conflicting (33
). With respect to NTD risk, some mutant mice showed a beneficial response to increased FA over a single gestation period but a detrimental response over multiple generations (30
). This contradictory response depending on the length of FA exposure highlights the difficulties in considering how best to model human exposure to FA as currently implemented. Moreover, it raises the important but little studied question of whether there may be unexpected effects of long-term FA fortification and supplementation in humans or potential effects due to increased levels of metabolized and unmetabolized FA.
The variation in NTD risk depending on the length of FA exposure (30
) points toward the possibility of epigenetic changes. Consistent with this idea, mutations in genes that affect DNA methylation, histone modification (in particular acetylation) or chromatin remodeling result in NTDs in mice (4
). Furthermore, the anti-epileptic drug valproic acid is a histone deacetylase inhibitor and it is a well-known risk factor for NTDs in humans (18
). Interestingly, NTDs in mice bearing mutations in the histone acetyltransferases Gcn5 or Cited2 can be prevented with FA supplementation (38
). Epigenetic influence has also been suggested to help explain the predominance of cranial NTDs in females versus males. X chromosome inactivation is maintained by DNA methylation and hence there is more demand on the methylation cycle in female cells after every division relative to male cells (40
). Epigenomic studies as outlined below should bring new insights into how FA may affect transcriptional programs during NTC.
In summary, we have a relatively poor understanding of FA action on NTC and little insight into why some genetic mutants respond, positively or negatively, to this environmental factor. As described below, efforts to understand the cellular mechanisms governing NTC in animal models are providing important new tools for NTD research. Investigation of FA action in a much larger set of mouse NTD models will help to reveal whether particular developmental processes or molecular pathways can be related in terms of FA responsiveness and to gain molecular insights into optimal interventions to prevent NTDs.