Gene-environment interactions in the causation of neural tube defects: folate deficiency increases susceptibility conferred by loss of Pax3 function

Abstract

Risk of neural tube defects (NTDs) is determined by genetic and environmental factors, among which folate status appears to play a key role. However, the precise nature of the link between low folate status and NTDs is poorly understood, and it remains unclear how folic acid prevents NTDs. We investigated the effect of folate level on risk of NTDs in splotch (Sp(2)(H)) mice, which carry a mutation in Pax3. Dietary folate restriction results in reduced maternal blood folate, elevated plasma homocysteine and reduced embryonic folate content. Folate deficiency does not cause NTDs in wild-type mice, but causes a significant increase in cranial NTDs among Sp(2)(H) embryos, demonstrating a gene-environment interaction. Control treatments, in which intermediate levels of folate are supplied, suggest that NTD risk is related to embryonic folate concentration, not maternal blood folate concentration. Notably, the effect of folate deficiency appears more deleterious in female embryos than males, since defects are not prevented by exogenous folic acid. Folate-deficient embryos exhibit developmental delay and growth retardation. However, folate content normalized to protein content is appropriate for developmental stage, suggesting that folate availability places a tight limit on growth and development. Folate-deficient embryos also exhibit a reduced ratio of s-adenosylmethionine (SAM) to s-adenosylhomocysteine (SAH). This could indicate inhibition of the methylation cycle, but we did not detect any diminution in global DNA methylation, in contrast to embryos in which the methylation cycle was specifically inhibited. Hence, folate deficiency increases the risk of NTDs in genetically predisposed splotch embryos, probably via embryonic growth retardation.

Figure 1.

Embryonic folate content shows only partial correlation with maternal blood folate and homocysteine (Hcy) concentration. Whole blood folate (A; n = 10–17) and plasma homocysteine (B; n = 4–6) were quantified in Sp^2H/+ dams after maintenance for at least 6 weeks on the appropriate diet. Embryonic folate content (C; n = 10–14; Sp^2H genotypes pooled) was measured in stage-matched embryos at E10.5 (26–32 somite stage; mean number of somites equal for each treatment). Values are given as mean ± SEM. Maternal blood folate and embryonic folate content were significantly lower on all diets compared with the standard breeding diet (*P < 0.001, **P < 0.05, compared with SD). Embryonic folate content was significantly higher in the FD + DF and FD + BF diet groups than in the folate-deficient group (^†P < 0.001, compared with FD). Maternal homocysteine was elevated in both the FD and FD + BF groups compared with SD and FD + DF (^#P < 0.001). SD, standard breeding diet; FD, folate-deficient diet; FD + DF, folate-deficient but with dietary folate available; FD + BF, folate-deficient but with bacterial folate available.

Figure 2.

Frequency of cranial NTDs among Sp^2H/Sp^2H embryos varies with folate status. (A) Among Sp^2H/Sp^2H embryos that developed under folate-deficient (FD, n = 33) conditions, a significantly higher incidence of exencephaly (at E10.5–11.5) was observed than among embryos developing under conditions of standard diet (SD, n = 34); # indicates significant difference compared with SD (P < 0.05, Fisher exact test). In contrast, incidence of exencephaly under conditions of folate deficiency but with dietary folate available (FD + DF, n = 12) or folate deficiency but with bacterial folate available (FD + BF, n = 14) did not differ from the SD (P > 0.05). Maternal supplementation with folic acid reduced the incidence of exencephaly among both SD embryos (SD + FA, n = 24) and FD embryos (FD + FA, n = 23), although this effect reached statistical significance only for the FD + FA group (*indicates significant difference compared with FD, P < 0.01, Fisher exact test). Thymidine treatment (FD + Thy, n = 7) did not reduce the frequency of exencephaly. (B) The sex of a subset of embryos was determined (indicated as M or F above bar). On the SD the frequency of exencephaly was higher among females (F, n = 7) than males (M, n = 11), and remained higher after folic acid treatment (SD + FA; n = 14 males, 11 females), although these sex differences were not statistically significant. Under folate-deficient (FD) conditions both sexes exhibited a high frequency of exencephaly (n = 19 males, n = 9 females). However, while folic acid significantly reduced the frequency of exencephaly among males (n = 17, *indicates significant difference compared with FD, P < 0.01, Fisher exact test), females were almost completely resistant to folic acid (n = 14) such that the frequency of exencephaly was significantly higher among folic acid-treated FD females than males (**indicates significant difference compared with FD + FA males, P < 0.02).

Figure 3.

Developmental stage and size of embryos after development under varying levels of folate. Splotch embryos (genotypes pooled) developing under folate-deficient (FD) conditions had fewer somites (A) and smaller crown-rump length (B) at E10.5 than embryos in all other dietary groups (*significant difference compared with other groups, P < 0.001, One-way ANOVA). At E11.5, FD embryos still had significantly fewer somites and smaller crown-rump length than embryos from the SD (**P < 0.001). This difference was not rescued by folic acid treatment (FD + FA; **P < 0.001 compared with SD). In contrast, thymidine treatment (FD+THY) resulted in normalization of somite number in folate-deficient embryos (P > 0.05 compared with SD), although FD + THY embryos still had reduced crown-rump length (**P < 0.001 compared with SD). Values are given as mean ± SEM. Numbers of embryos at E10.5: 46 SD; 93 FD; 34 FD + DF; 21 FD + BF; 24 FD+THY. At E11.5: 19 SD; 49 FD; 28 FD + FA; 21 FD+THY. See Figure 1 for dietary group abbreviations.

Figure 4.

Relationship between embryonic growth (protein content per embryo) and developmental stage (somite number) in splotch embryos developing under differing dietary folate conditions. Black circles indicate standard diet (SD), white circles indicate folate-deficient diet (FD). All splotch genotypes were pooled for analysis. Note the exponential increase in protein content with somite stage, with no differences between SD and FD dietary groups. Hence, embryonic growth and developmental progression are not dissociated in folate deficiency.

Figure 5.

Developmental change in embryonic folate status. Folate and protein content were measured for a series of splotch embryos (genotypes pooled for analysis) at different developmental stages (as indicated by somite number). Each data point represents an individual embryo. Embryonic folate content (A) increases with developmental stage for embryos developing under standard dietary conditions (SD, solid line), whereas there is no discernible increase for embryos developing under folate-deficient (FD, dashed line) conditions. Linear regression lines were fitted (FD, solid line, r² = 0.700; SD, dashed line, r² = 0.024) and show significant difference in gradient between dietary groups (P < 0.001, t-test). In contrast, folate concentration (B), normalized to protein content, declines with development. Prior to 20 somites the rate of decline is greater in FD than SD embryos, but after the 20 somite stage there is no apparent difference between SD and FD groups. Thus, at a given somite stage the protein concentration of FD embryos is the same as for SD embryos, although the embryo is gestationally older.

Figure 6.

NTDs in splotch embryos Neural tube closure is completed among all wild-type embryos (SD embryo shown in A), whereas Sp^2H/Sp^2H embryos (B–D) exhibit NTDs, comprising spina bifida (arrowhead in B and D) and/or exencephaly (C and D, region of open neural folds is indicated by arrows). In FD conditions, the region of open neural folds encompasses the midbrain or mid- and hindbrain (C), whereas thymidine-treated embryos exhibit a small region of open neural folds (D).