Neural tube defects induced by folate deficiency in mutant curly tail (Grhl3) embryos are associated with alteration in folate one-carbon metabolism but are unlikely to result from diminished methylation

Abstract

Background: Folate one-carbon metabolism has been implicated as a determinant of susceptibility to neural tube defects (NTDs), owing to the preventive effect of maternal folic acid supplementation and the higher risk associated with markers of diminished folate status.

Methods: Folate one-carbon metabolism was compared in curly tail (ct/ct) and genetically matched congenic (+(ct)/+(ct)) mouse strains using the deoxyuridine suppression test in embryonic fibroblast cells and by quantifying s-adenosylmethionine (SAM) and s-adenosylhomocysteine (SAH) in embryos using liquid chromatography tandem mass spectrometry. A possible genetic interaction between curly tail and a null allele of 5,10-methylenetetrahydrofolate reductase (MTHFR) was investigated by generation of compound mutant embryos.

Results: There was no deficit in thymidylate biosynthesis in ct/ct cells, but incorporation of exogenous thymidine was lower than in +(ct)/+(ct) cells. In +(ct)/+(ct) embryos the SAM/SAH ratio was diminished by dietary folate deficiency and normalized by folic acid or myo-inositol treatment, in association with prevention of NTDs. In contrast, folate deficiency caused a significant increase in the SAM/SAH ratio in ct/ct embryos. Loss of MTHFR function in curly tail embryos significantly reduced the SAM/SAH ratio but did not cause cranial NTDs or alter the frequency of caudal NTDs.

Conclusions: Curly tail fibroblasts and embryos, in which Grhl3 expression is reduced, display alterations in one-carbon metabolism, particularly in the response to folate deficiency, compared to genetically matched congenic controls in which Grhl3 is unaffected. However, unlike folate deficiency, diminished methylation potential appears to be insufficient to cause cranial NTDs in the curly tail strain, nor does it increase the frequency of caudal NTDs.

Figure 1. Analysis of folate-dependent thymidylate biosynthesis using the deoxyuridine (dU) suppression test in curly tail (ct/ct) and congenic (+^ct/+^ct) cells

(A) Incorporation of [³H]thymidine into dTMP (and thence to DNA), via the thymidine kinase-mediated salvage pathway (white bars), is suppressed by addition of dU which stimulates de novo synthesis of dTMP by thymidylate synthase (grey bars). Reduction of folate availability (culture in low folate medium) increases the incorporation of [³H]thymidine into cells of both genotypes (# indicates significant difference compared with equivalent genotype/condition for standard medium, p<0.001). Under ‘low folate’ conditions the incorporation is diminished in ct/ct cells compared with +^ct/+^ct (* indicates significant difference compared with +^ct/+^ct cells under the same conditions, p<0.02). (B) The % suppression of thymidine incorporation by dU is higher in ct/ct cells than in +^ct/+^ct cells under standard culture conditions (* indicates significant difference compared with +^ct/+^ct, p<0.05), but not under ‘low folate’ conditions.