Folic acid and neural tube defects
In 1976, Smithells et al. (
1976) demonstrated decreased levels of several micronutrients, especially folate, in serum of women pregnant with a neural tube defect (NTD)-affected child during the first trimester. Subsequent small trials in women who had a NTD-affected pregnancy indicated that periconceptional supplementation with 0.4–5 mg of folic acid per day decreased the recurrence risk of NTDs about fourfold (Blom
2009). In 1991, the MRC Vitamin Study Research group conducted a large international double-blind placebo-controlled randomized trial in 1,817 women who had a previous NTD-affected pregnancy and demonstrated a 72% reduction in NTD recurrence risk for periconceptional folic acid supplementation (Vitamin Study Research Group
1991). The protective effect of periconceptional folic acid supplementation on NTD occurrence risk has mainly been demonstrated in retrospective case-control studies (Blom
2009). However, a large randomized controlled trial in Hungarian women showed that supplementation with multivitamins including 0.8 mg folic acid reduced the first occurrence of NTDs by 93% (Czeizel and Dudas
1992).
The results of these studies led to the general advice for women planning a pregnancy to take 0.4 mg of folic acid per day at least 1 month before conception and during the first trimester of pregnancy. Women who are at high risk because of a previous NTD-affected pregnancy are advised to take 4 mg of folic acid daily in the periconceptional period (Pitkin
2007). In the years after the endorsement of these recommendations, the expected decline in NTD prevalence failed to occur. In 1998, the United States mandated folic acid fortification of enriched cereal grain products was followed by a decline of NTD rates of 20–30%.
The observation that women who use folic acid periconceptional are at reduced risk for NTD-affected pregnancies is one of the most promising clues to the etiology of NTDs. In the search for mechanisms underlying the beneficial effect of folic acid in relation to NTDs, several mechanisms have been suggested. One mechanism playing a possible a role in NTD etiology is methylation, as discussed in more detail below.
Methylation
It is becoming more and more evident that, besides genetic variation and environmental factors, epigenetic mechanisms also play an important role in the etiology of complex diseases (Blom et al.
2006). Since NTDs are multifactorial in origin, epigenetic mechanisms may well be involved in their etiology. DNA methylation, which is reprogrammed during early embryogenesis (Reik et al.
2001), is part of the epigenetic code and is a chief regulator of gene expression (Dean et al.
2005), has been hypothesized to be associated with NTDs (Blom et al.
2006).
The methylenetetrahydrofolate reductase (MTHFR) enzyme has a unique function in that it regulates the availability of methyl groups for methylation reactions at the cost of purines and pyrimidine synthesis. The 677C>T variant in the
MTHFR gene renders a less active thermolabile MTHFR enzyme (Frosst et al.
1995), resulting in elevated levels of 10-formylTHF and a decreased availability of 5-methylTHF in red blood cells (Bagley and Selhub
1998). The observations that the
MTHFR 677TT genotype decreases the availability of 5-methylTHF for methylation (Frosst et al.
1995), results in a reduction of global DNA methylation (Friso et al.
2002; Castro et al.
2004), and is a genetic risk factor for NTDs (van der Put et al.
1995; Blom et al.
2006) support the concept that DNA methylation plays an important role in NTD etiology. The observation that inactivation of the DNA methyltransferase
DNMT3B disrupts de novo DNA methylation, and causes multiple developmental defects including NTDs in mice, further supports the importance of DNA methylation (Okano et al.
1999). In addition, culturing rat embryos on low methionine containing media-induced NTDs resulted in protein hypomethylation that was specific for those proteins located in the neural tube, indicating that protein methylation may also play a role in proper neural tube closure (Coelho and Klein
1990; Moephuli et al.
1997).
Using a chick embryo model, our group tested the hypothesis that methylation is crucial for proper neural tube closure (Afman et al.
2005). Inhibition of SAHH in our in vitro chick embryo model resulted in inhibition of methylation, as judged from the increased AdoHcy concentration and a decreased AdoMet/AdoHcy ratio, and a widening of the anterior neuropore (ANP). Comparable results were obtained by inhibition of MAT. Inhibition of MAT in cultured mouse embryos also decreased the AdoMet/AdoHcy ratio, and these embryos also presented with cranial closure defects (Dunlevy et al.
2006). The AdoMet/AdoHcy ratio in mothers of a NTD-affected child has been demonstrated to be decreased (Zhao et al.
2006). Only one study, however, has actually examined DNA methylation status in relation to NTDs (Al-Gazali et al.
2001). In that study, the AdoMet/AdoHcy ratio and DNA methylation were both decreased in a single case with trisomy 21 and spina bifida (Al-Gazali et al.
2001). Taken together, these studies have lead to the hypothesis that methylation is hampered in women that are pregnant with a NTD-affected child and that folic acid supplementation prevents NTDs by donating methyl groups, hereby improving methylation (Blom et al.
2006).
In the future, large-scale studies in which DNA methylation patterns of healthy newborns and those with a NTD will be characterized and compared using array-based approaches, which will shed more light on the association between an impaired DNA methylation and NTD etiology in humans and may identify new genes essential for neural tube closure. If an impaired DNA methylation is indeed associated with NTD etiology, the next step will be to examine whether folic acid supplementation of the mother can affect DNA methylation patterns in her child.
The ultimate goal of identifying genes that are under epigenetic control and involved in NTD etiology is the identification of molecular pathways that are essential for proper neural tube closure and can be manipulated and in this way prevent NTDs.