Effects of folate supplementation on the risk of spontaneous and induced neural tube defects in Splotch mice

BACKGROUND: Neural tube defects (NTDs) are among the most common human congenital malformations. Although clinical investigations have reported that periconceptional folic acid supplementation can reduce the occurrence of these defects, its mechanism remains unknown. Therefore, the murine mutant Splotch, which has a high incidence of spontaneous NTDs, along with the inbred strains SWV and LM/Bc, were used to investigate the relationship between folate and NTDs. METHODS: To investigate whether folates could reduce spontaneous NTDs, heterozygous Splotch dams (+/Sp) were treated with either folate or folinic acid throughout neurulation, gestational day (GD) 6.5 to 10.5. On GD 18.5 the dams were sacrificed and the fetuses examined for any neural tube defects. Subsequently, Sp/+ dams were treated with arsenic while receiving either a folate or folinic acid supplementation. Similar experiments were performed in the LM/Bc and SWV strains. RESULTS: Neither folate nor folinic acid supplements reduced the frequency of spontaneous NTDs in the embryos from Splotch heterozygote crosses. Arsenic increased the frequency of NTDs and embryonic death in the Splotch, LM/Bc and SWV litters and folinic acid failed to ameliorate the teratogenic effect of this metal. A folate supplement given to arsenic-treated dams proved to be maternally lethal in all three strains. CONCLUSIONS: Splotch embryos were not protected from either spontaneous or arsenic-induced NTDs by folinic or folic acid supplementation. Furthermore, folinic acid supplements did not reduce the incidence of arsenic-induced NTDs in either the LM/Bc or SWV litters.     

碱基切除修复基因Lig3对小鼠胚胎神经发育的影响

摘要 目的：DNA连接酶III（DNA ligase III, Lig3）基因是碱基切除修复通路中的关键基因,在胚胎发育过程中发挥重要作用,通过研究Lig3基因在叶酸代谢障碍状态下的表达情况,探讨其对小鼠胚胎神经发育的影响。方法：采用无特定病原体（specific pathogen free, SPF）级C57BL/6J成年小鼠（8-9周,18-20 g）,雌雄1:1合笼,孕鼠随机分为实验组和对照组,孕7.5天实验组腹腔注射4.5 mg/kg体重甲氨蝶呤（Methotrexate, MTX,二氢叶酸还原酶抑制剂）诱导产生叶酸代谢障碍的小鼠神经管畸形（neural tube defects, NTDs）模型,对照组腹腔注射等体积的生理盐水。孕10.5天体视显微镜下观察胎鼠的发育情况。同时利用200 nM的MTX建立叶酸代谢障碍的小鼠神经干细胞模型。在模型建立成功的基础上,应用实时荧光定量聚合酶链反应(Real time quantitative PCR,RT-qPCR)及免疫印迹（Western blot）等方法研究碱基切除修复通路相关基因Lig3的表达水平。结果：4.5 mg/kg 体重MTX处理孕鼠后胎鼠NTDs的发生率为31.1%（19/61）,而正常对照组未见胎鼠NTDs的发生。在体视显微镜下可见NTDs胎鼠神经管未闭合,而正常胎鼠发育完好。RT-qPCR检测发现叶酸代谢障碍小鼠NTDs 胚胎神经组织中Lig3 mRNA的表达水平明显低于对照组（P<0.05）。Western blot检测发现,与对照组相比,叶酸代谢障碍NTDs胎鼠神经组织中Lig3蛋白水平明显降低（P<0.05）。同时,在MTX处理的神经干细胞中,Lig3的表达水平明显低于对照组（P<0.05）。对凋亡相关蛋白Cleaved caspase-3进行检测发现MTX处理后的NTDs胎鼠神经组织及细胞模型中其表达均明显增加,表明细胞凋亡增加。结论：在叶酸代谢障碍前提下,Lig3表达降低,DNA修复功能减弱,细胞凋亡增加,导致NTDs的发生,为NTDs及出生缺陷的防控提供新思路。     

Similar rhythms of seasonal conceptions in neural tube defects and schizophrenia: a hypothesis of oxidant stress and the photoperiod

BACKGROUND: Numerous studies have found that people with schizophrenia tend to be born most often in late winter and least often in late summer. The same rhythm appears in the birth of children with neural tube defects (NTDs). In the northern hemisphere, both disorders thus show a conception peak in May-June and a trough around November-December. The senior author found the same May-June conception peak among left-handed American baseball players and the opposite effect (a November-December peak) among extreme right-handed players. A similar rhythm appeared with respect to characteristics related to artistic as opposed to scientific modes of thought. DISCUSSION: Schizophrenia has been proposed to involve a deficit in the establishment of lateral asymmetry, as does left-handedness. The artist-scientist dichotomy is also believed to involve cerebral dominance. Thus, the similarity of seasonal variation in month of conception between NTDs, schizophrenia, left-handedness, and artistic intuition suggests that these four conditions may share some factor affecting the cellular processes involved in both neural tube closure and asymmetry development during the early-fourth week, neural-fold stages of embryogenesis. We propose that maternal oxidant stress, which can rise with exposure to intense solar radiation, may interfere with both neural tube closure and asymmetry development. The June and December extremes of sunlight would thus explain the peak times of the seasonal fluctuations. Moreover, regardless of mechanisms, the parallel between the two conception rhythms suggests that the same periconceptional folate regimens found effective in preventing NTDs may also lower the risk of schizophrenia. This paper reviews some of the clinical and experimental evidence supporting this hypothesis.     

Folate-mediated one-carbon metabolism and neural tube defects: balancing genome synthesis and gene expression

Neural tube defects (NTDs) refer to a cluster of neurodevelopmental conditions associated with failure of neural tube closure during embryonic development. Worldwide prevalence of NTDs ranges from approximately 0.5 to 60 per 10,000 births, with regional and population-specific variation in prevalence. Numerous environmental and genetic influences contribute to NTD etiology; accumulating evidence from population-based studies has demonstrated that folate status is a significant determinant of NTD risk. Folate-mediated one-carbon metabolism (OCM) is essential for de novo nucleotide biosynthesis, methionine biosynthesis, and cellular methylation reactions. Periconceptional maternal supplementation with folic acid can prevent occurrence of NTDs in the general population by up to 70%; currently several countries fortify their food supply with folic acid for the prevention of NTDs. Despite the unambiguous impact of folate status on NTD risk, the mechanism by which folic acid protects against NTDs remains unknown. Identification of the mechanism by which folate status affects neural tube closure will assist in developing more efficacious and better targeted preventative measures. In this review, we summarize current research on the relationship between folate status and NTDs, with an emphasis on linking genetic variation, folate nutriture, and specific metabolic and/or genomic pathways that intersect to determine NTD outcomes.     

n-6 and n-3 Long-chain polyunsaturated fatty acids in the erythrocyte membrane of Brazilian preterm and term neonates and their mothers at delivery

Placental transfer of the long-chain polyunsaturated fatty acids (LCPUFA) arachidonic (AA) and docosahexaenoic (DHA) acids is selectively high to maintain accretion to fetal tissues, especially the brain. The objectives of the present study were to investigate the essential fatty acid (EFA) and LCPUFA status at birth of preterm and term Brazilian infants and their mothers, from a population of characteristically low intake of n-3 LCPUFA, and to evaluate the association between fetal and maternal status, by the determination of the fatty acid composition of the erythrocyte membrane. Blood samples from umbilical cord of preterm (26-36 weeks of gestation; n = 30) and term (37-42 weeks of gestation; n = 30) infants and the corresponding maternal venous blood were collected at delivery. The LCPUFA composition of the erythrocyte membrane and DHA status were similar for mothers of preterm and term infants. Neonatal AA was higher (P < 0.01) whereas its precursor 18:2n-6 was lower (P < 0.01) than maternal levels, as expected. There was no difference in LCPUFA erythrocyte composition between preterm and term infants, except for DHA. Term infants presented a worse DHA status than preterm infants (P < 0.01) and than their mothers (P < 0.01) at delivery. There was a negative correlation of neonatal DHA with maternal AA and a positive correlation between neonatal AA and maternal AA and 18:2n-6 only at term. These results suggest that the persistent low DHA maternal status, together with the comparatively better AA and 18:2n-6 status, might have affected maternal-fetal transfer of DHA when gestation was completed up to term, and possibly contributed to the worse DHA status of term neonates compared with the preterm neonates.     

Impact of methylenetetrahydrofolate reductase deficiency and low dietary folate on the development of neural tube defects in splotch mice

BACKGROUND: The etiology of neural tube defects (NTDs) is multifactorial, with environmental and genetic determinants. Folate supplementation prevents the majority of NTDs, and a polymorphism in methylenetetrahydrofolate reductase (MTHFR) has become recognized as a genetic risk factor. The mechanisms by which folate affects NTD development are unclear. The Splotch (Sp) mouse is a well-characterized mouse model for studying spontaneous NTDs. To assess the potential interaction between folate metabolism and the Sp mutant in NTD development, we studied mice with both Sp and Mthfr mutations, as well as the interaction between Sp and low dietary folate. METHODS: Wild-type, single Mthfr+/-mutant, single Sp/+mutant, and double mutant (Mthfr+/-, Sp/+) female mice were mated with males of the same genotype. Embryos were examined for NTDs on gestational day (GD) 13.5. To investigate the effects of folate deficiency on Sp mice, Sp/+female mice were fed a control diet (CD), a moderately folic acid-deficient diet (MFADD), or a severely folic acid-deficient diet (SFADD). They were mated with Sp/+males and the embryos were examined. RESULTS: There were no differences in the incidence or severity of NTDs in embryos from double-mutant mating pairs compared to those from single Sp mutants. Embryos from Mthfr+/-dams did not exhibit NTDs. Diets deficient in folate did not influence the incidence or severity of NTDs in embryos from Sp/+mice. CONCLUSIONS: We did not observe an interaction between Sp and Mthfr mutations, or between the Sp mutation and low dietary folate, in NTD development in Splotch mice.     

Reduced folate carrier gene is a risk factor for neural tube defects in a Chinese population

BACKGROUND: There is a considerable body of data demonstrating that periconceptional supplementation of folic acid can prevent a significant proportion of neural tube defects (NTDs). At present, the mechanism by which folic acid exerts its beneficial effect remains unknown. Folate transporter genes, including the reduced folate carrier gene (RFC1), have been proposed as NTD risk factors. METHODS: The study population included 104 nuclear families with NTDs and 100 nonmalformed control families. We investigated the possible association between a common RFC1 polymorphism (A80G) and NTD risk among offspring, as well as potential gene-environment interactions between the infant RFC1 genotype and maternal periconceptional use of folic acid through a population-based case-control study. RESULTS: We observed that the infants of the GG genotype were associated with a 2.56-fold increased risk of NTDs when compared to the AA genotype (odds ratio [OR], 2.56; 95% confidence interval [CI], 1.04-6.36) in our study population. Among mothers who did not utilize folic acid supplements, the risk for having a child with an NTD was 3.30 (95% CI, 1.15-9.65) for offspring with the GG genotype, compared to the reference (AA) genotype. Children who had the GG genotype and whose mothers did not take folic acid had an elevated risk for NTDs (OR, 8.80; 95% CI, 2.83-28.69), compared to offspring with the AA and GA genotypes whose mothers utilized folic acid supplements. CONCLUSIONS: Our findings suggest that the RFC1 G allele is likely to be an important genetic factor in determining folate transport and subsequently may be a risk factor for NTDs in this Chinese population.     

Neural tube defects,folate, and immune modulation

Periconceptional supplementation with folic acid has led to a significant worldwide reduction in the incidence of neural tube defects (NTDs). However, despite increasing awareness of the benefits of folic acid supplementation and the implementation of food fortification programs in many countries, NTDs continue to be a leading cause of perinatal morbidity and mortality worldwide. Furthermore, there exists a significant subgroup of women who appear to be resistant to the protective effects of folic acid supplementation. The following review addresses emerging clinical and experimental evidence for a role of the immune system in the etiopathogenesis of NTDs, with the aim of developing novel preventative strategies to further reduce the incidence of NTD‐affected pregnancies. In particular, recent studies demonstrating novel roles and interactions between innate immune factors such as the complement cascade, neurulation, and folate metabolism are explored. Birth Defects Research (Part A) 97:602–609, 2013. © 2013 Wiley Periodicals, Inc.     

Behavioral effects of prenatal folate deficiency in mice

BACKGROUND: Folate supplementation decreases the incidence of birth defects such as neural tube defects (NTDs). We and others have shown that gestational dietary folate deficiency that does not produce overt NTDs can alter fetal neural histology. Accordingly, murine offspring were examined for the possible functional consequences of prenatal folate deficiency. METHODS: CD-1 mice were fed a diet of chow containing 400, 600, or 1200 nmol of folic acid/kg of chow for eight weeks prior to breeding and until GD18, at which time all dams were placed on folate-replete chow. Behavioral tests of male and female offspring included righting reflex, negative geotaxis, forelimb hanging, motor coordination, open field activity, and elevated plus maze activity. RESULTS: Of greatest significance, the adult offspring that were prenatally folate-deficient exhibited more anxiety-related behavior in the elevated plus maze. Offspring of the 400 nmol of folic acid/kg of chow diet group exhibited significantly shorter durations in the open arms and longer durations in the closed arms. Further, these two behaviors were dose-related. There was also a trend for the prenatally folate-deficient adult mice to exhibit more thigmotaxis (wall-hugging) behavior in the open field, entering the central area less frequently than controls. There were few other differences in tested behaviors between folate-deficient and folate-replete mice. CONCLUSIONS: Prenatal folate deficiency that is repleted at birth can manifest later with increased anxiety 9-12 weeks after birth.     

Inositol- and folate-resistant neural tube defects in mice lacking the epithelial-specific factor Grhl-3

The neural tube defects (NTDs) spina bifida and anencephaly are widely prevalent severe birth defects. The mouse mutant curly tail (ct/ct) has served as a model of NTDs for 50 years, even though the responsible genetic defect remained unrecognized. Here we show by gene targeting, mapping and genetic complementation studies that a mouse homolog of the Drosophila grainyhead (grh) gene, grainyhead-like-3 (Grhl3), is a compelling candidate for the gene underlying the curly tail phenotype. The NTDs in Grhl3-null mice are more severe than those in the curly tail strain, as the Grhl3 alleles in ct/ct mice are hypomorphic. Spina bifida in ct/ct mice is folate resistant, but its incidence can be markedly reduced by maternal inositol supplementation periconceptually. The NTDs in Grhl3-/- embryos are also folate resistant, but unlike those in ct/ct mice, they are resistant to inositol. These findings suggest that residual Grhl3 expression in ct/ct mice may be required for inositol rescue of folate-resistant NTDs.     

Eicosapentaenoic acid in the treatment of schizophrenia and depression: rationale and preliminary double-blind clinical trial results

It has been hypothesised that polyunsaturated fatty acids (PUFA) play an important role in the aetiology of schizophrenia and depression. Evidence supporting this hypothesis for schizophrenia includes abnormal brain phospholipid turnover shown by 31P Magnetic Resonance Spectroscopy, increased levels of phospholipase A2, reduced niacin skin flush response, abnormal electroretinogram, and reduced cell membrane levels of n-3 and n-6 PUFA. In depression, there is strong epidemiological evidence that fish consumption reduces risk of becoming depressed and evidence that cell membrane levels of n-3 PUFA are reduced. Four out of five placebo-controlled double- blind trials of eicosapentaenoic acid (EPA) in the treatment of schizophrenia have given positive findings. In depression, two placebo-controlled trials have shown a strong therapeutic effect of ethyl-EPA added to existing medication. The mode of action of EPA is currently not known, but recent evidence suggests that arachidonic acid (AA) if of particular importance in schizophrenia and that clinical improvement in schizophrenic patients using EPA treatment correlates with changes in AA.     

Valproic acid increases expression of methylenetetrahydrofolate reductase (MTHFR) and induces lower teratogenicity in MTHFR deficiency

Valproate (VPA) treatment in pregnancy leads to congenital anomalies, possibly by disrupting folate or homocysteine metabolism. Since methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of folate interconversion and homocysteine metabolism, we addressed the possibility that VPA might have different teratogenicity in Mthfr(+/+) and Mthfr(+/-) mice and that VPA might interfere with folate metabolism through MTHFR modulation. Mthfr(+/+) and Mthfr(+/-) pregnant mice were injected with VPA on gestational day 8.5; resorption rates and occurrence of neural tube defects (NTDs) were examined on gestational day 14.5. We also examined the effects of VPA on MTHFR expression in HepG2 cells and on MTHFR activity and homocysteine levels in mice. Mthfr(+/+) mice had increased resorption rates (36%) after VPA treatment, compared to saline treatment (10%), whereas resorption rates were similar in Mthfr(+/-) mice with the two treatments (25-27%). NTDs were only observed in one group (VPA-treated Mthfr(+/+)). In HepG2 cells, VPA increased MTHFR promoter activity and MTHFR mRNA and protein (2.5- and 3.7-fold, respectively). Consistent with cellular MTHFR upregulation by VPA, brain MTHFR enzyme activity was increased and plasma homocysteine was decreased in VPA-treated pregnant mice compared to saline-treated animals. These results underscore the importance of folate interconversion in VPA-induced teratogenicity, since VPA increases MTHFR expression and has lower teratogenic potential in MTHFR deficiency.     

High rates of neural tube defects in Ukraine

BACKGROUND: Oral consumption of synthetic folic acid can prevent neural tube defects (NTDs), which are some of the most severe congenital anomalies. The prevalence of NTDs in Ukraine and other countries of the former U.S.S.R. has not been well studied. We determined the prevalence of NTD-affected pregnancies in Northwestern Ukraine as background for policy decisions related to flour fortification in this country. METHODS: The Ukrainian-American Birth Defects Program was established in 1999 and conducts population- based surveillance of birth defects in several oblasts (states) of Ukraine. We determined the prevalence of NTDs in the Volyn and Rivne oblasts of Northwestern Ukraine for three years, 2000-2002. RESULTS: There were 75,928 births in the two oblasts in 2000-2002. There were 159 cases of NTDs among live births, stillbirths, and induced abortions. The prevalence of NTDs in the two oblasts in Northwestern Ukraine is 2.1 per 1000 births. CONCLUSIONS: The prevalence of NTD-affected pregnancies we found in Northwestern Ukraine is almost four times what it should be. This prevalence suggests that population folate deficiency is widespread in Ukraine. Universal folic acid fortification of flour milled in Ukraine is urgently needed to end this epidemic of birth defects. Such fortification would be expected to prevent folate deficiency anemia, heart attacks, and strokes.     

Folate status and neural tube defects

Periconceptional folic acid supplementation prevents approximately 70% of neural tube defects (NTDs). While most women carrying affected fetuses do not have deficient blood folate levels, the risk of having an NTD affected child is inversely correlated with pregnancy red cell folate levels. Current research is focused on the discovery of genetic abnormalities in folate related enzymes which might explain the role of folate in NTD prevention. The first candidate gene to emerge was the C677T variant of 5,10-methylenetetrahydrofolate reductase. Normal subjects who are homozygous for the mutation (TT) have red cell folate status some 20% lower than expected. It is now established that the prevalence of the TT genotype is significantly higher among spina bifida cases and their parents. Nevertheless, our studies show that the variant does not account for the reduced blood folate levels in many NTD affected mothers. We conclude that low maternal folate status may in itself be the most important risk factor for NTDs and that food fortification may be the only population strategy of benefit in the effort to eliminate NTDs.     

Biochemical effects of supplemented long-chain polyunsaturated fatty acids in hyperphenylalaninemia

Hyperphenylalaninemic (HPA) children display low levels of long-chain polyunsaturated fatty acids (LCPUFA), particularly docosahexaenoic acid (DHA), in circulating lipids and erythrocytes. We have investigated the effects on the blood fatty acid status and lipid picture of a balanced supplementation with LCPUFA in HPA children through a double-blind, placebo-controlled trial. A total of 20 well-controlled HPA, school-age children were randomized to receive through a 12-month trial fat capsules supplying either 26% fatty acid as LCPUFA (including 4.6%gamma -linolenic acid, 7.4% arachidonic acid, AA, 5.5% eicosapentaenoic acid and 8% DHA) or placebo (olive oil). The study supplementation was administered in order to provide 0.3-0.5% of the individual daily energy requirements as LCPUFA. Reference data were obtained from healthy children of comparable age. Among HPA children (whose DHA status was poor at baseline), those supplemented with LCPUFA showed an increase of around 100% in the baseline DHA levels in plasma phospholipids and erythrocytes. No changes of AA levels were observed. Blood lipid levels did not significantly change. A balanced supplementation with LCPUFA in treated HPA children may improve the DHA status without adversely affecting the AA status.     

Effect of supplementation of arachidonic acid (AA) or a combination of AA plus docosahexaenoic acid on breastmilk fatty acid composition

We investigated whether supplementation with arachidonic acid (20:4 omega 6; AA), or a combination of AA and docosahexaenoic acid (22:6 omega 3; DHA) would affect human milk polyunsaturated fatty acid (PUFA) composition. Ten women were daily supplemented with 300 mg AA, eight with 300 mg AA, 110 mg eicosapentaenoic acid (20:5 omega 3; EPA) and 400 mg DHA, for one week and eight women served as unsupplemented controls. Milk samples were collected on days 0, 1 and 7. The fatty acid composition of the milk was analyzed by capillary gas chromatography with flame ionisation detection. Supplementation with AA alone had no effect on breastmilk AA, but tended to reduce EPA and DHA levels. Administration of a combination of AA, EPA and DHA tended to increase both milk AA and long chain PUFA (LCPUFA)omega 3 content. A larger simultaneous increase of milk AA, DHA and EPA than observed in the present study can probably be accomplished by the use of a combination of a lower LCPUFA omega 6/LCPUFA omega 3 ratio and higher AA, EPA and DHA dosages.     

The genetic background of the curly tail strain confers susceptibility to folate‐deficiency‐induced exencephaly

BACKGROUND : Suboptimal maternal folate status is considered a risk factor for neural tube defects (NTDs). However, the relationship between dietary folate status and risk of NTDs appears complex, as experimentally induced folate deficiency is insufficient to cause NTDs in nonmutant mice. In contrast, folate deficiency can exacerbate the effect of an NTD‐causing mutation, as in splotch mice. The purpose of the present study was to determine whether folate deficiency can induce NTDs in mice with a permissive genetic background which do not normally exhibit defects. METHODS : Folate deficiency was induced in curly tail and genetically matched wild‐type mice, and we analyzed the effect on maternal folate status, embryonic growth and development, and frequency of NTDs. RESULTS : Folate‐deficient diets resulted in reduced maternal blood folate, elevated homocysteine, and a diminished embryonic folate content. Folate deficiency had a deleterious effect on reproductive success, resulting in smaller litter sizes and an increased rate of resorption. Notably, folate deficiency caused a similar‐sized, statistically significant increase in the frequency of cranial NTDs among both curly tail (Grhl3 mutant) embryos and background‐matched embryos that are wild type for Grhl3. The latter do not exhibit NTDs under normal dietary conditions. Maternal supplementation with myo‐inositol reduced the incidence of NTDs in the folate‐deficient wild‐type strain. CONCLUSIONS : Dietary folate deficiency can induce cranial NTDs in nonmutant mice with a permissive genetic background, a situation that likely parallels gene‐nutrient interactions in human NTDs. Our findings suggest that inositol supplementation may ameliorate NTDs resulting from insufficient dietary folate. Birth Defects Research (Part A), 2010. © 2009 Wiley‐Liss, Inc.     

No indications for altered essential fatty acid metabolism in two murine models for cystic fibrosis

A deficiency of essential fatty acids (EFA) is frequently described in cystic fibrosis (CF), but whether this is a primary consequence of altered EFA metabolism or a secondary phenomenon is unclear. It was suggested that defective long-chain polyunsaturated fatty acid (LCPUFA) synthesis contributes to the CF phenotype. To establish whether cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction affects LCPUFA synthesis, we quantified EFA metabolism in cftr-/-CAM and cftr+/+CAM mice. Effects of intestinal phenotype, diet, age, and genetic background on EFA status were evaluated in cftr-/-CAM mice, DeltaF508/DeltaF508 mice, and littermate controls. EFA metabolism was measured by 13C stable isotope methodology in vivo. EFA status was determined by gas chromatography in tissues of cftr-/-CAM mice, DeltaF508/DeltaF508 mice, littermate controls, and C57Bl/6 wild types fed chow or liquid diet. After enteral administration of [13C]EFA, arachidonic acid (AA) and docosahexaenoic acid (DHA) were equally 13C-enriched in cftr-/-CAM and cftr+/+CAM mice, indicating similar EFA elongation/desaturation rates. LA, ALA, AA, and DHA concentrations were equal in pancreas, lung, and jejunum of chow-fed cftr-/-CAM and DeltaF508/DeltaF508 mice and controls. LCPUFA levels were also equal in liquid diet-weaned cftr-/-CAM mice and littermate controls, but consistently higher than in age- and diet-matched C57Bl/6 wild types. We conclude that cftr-/-CAM mice adequately absorb and metabolize EFA, indicating that CFTR dysfunction does not impair LCPUFA synthesis. A membrane EFA imbalance is not inextricably linked to the CF genotype. EFA status in murine CF models is strongly determined by genetic background.     

Effects of inositol supplementation in a cohort of mothers at risk of producing an NTD pregnancy

Neural tube defects (NTDs), most commonly spina bifida and anencephaly, can be prevented with periconceptional intake of folic acid in about 70% of cases. Recurrence of NTDs despite supplementation of high dose of folic acid further suggests that a proportion of NTD cases might be resistant to folic acid. Moreover, heterogeneity of NTDs has been suggested in animal studies, indicating that only some sub-type of NTDs should be considered sensitive to folate intake. Inositol isomers (particularly myo- and chiro-inositol) can prevent folate-resistant NTDs in the curly-tail mutant mouse, suggesting that some cases of human NTDs might benefit from inositol supplementation. In humans, lower inositol blood concentration was found in pregnant women carrying NTD fetuses, whereas a periconceptional combination therapy with folic acid associated with inositol has been linked to normal live births, despite high NTD recurrence risk. Fifteen pregnancies from 12 Caucasian women from different parts of Italy with at least one previous NTD-affected pregnancy underwent periconceptional combined myo-inositol and folic acid supplementation. Maternal serum α-feto-protein levels were found in the normal range, and normal results on ultrasound examination were found in all the pregnancies that followed. No collateral effects or intense uterine contractions were demonstrated in this pilot study in any of the pregnancies after inositol supplementation, and seventeen babies were born without any type of NTD.     

Analyses of copy number variation reveal putative susceptibility loci in MTX‐induced mouse neural tube defects

Copy number variations (CNVs) are thought to act as an important genetic mechanism underlying phenotypic heterogeneity. Impaired folate metabolism can result in neural tube defects (NTDs). However, the precise nature of the relationship between low folate status and NTDs remains unclear. Using an array‐comparative genomic hybridization (aCGH) assay, we investigated whether CNVs could be detected in the NTD embryonic neural tissues of methotrexate (MTX)‐induced folate dysmetabolism pregnant C57BL/6 mice and confirmed the findings with quantitative real‐time PCR (qPCR). The CNVs were then comprehensively investigated using bioinformatics methods to prioritize candidate genes. We measured dihydrofolate reductase (DHFR) activity and concentrations of folate and relevant metabolites in maternal serum using enzymologic method and liquid chromatography/tandem mass spectrometry (LC/MS/MS). Three high confidence CNVs on XqA1.1, XqA1.1‐qA2, and XqE3 were found in the NTD embryonic neural tissues. Twelve putative genes and three microRNAs were identified as potential susceptibility candidates in MTX‐induced NTDs and possible roles in NTD pathogenesis. DHFR activity and 5‐methyltetrahydrofolate (5‐MeTHF), 5‐formyltetrahydrofolate (5‐FoTHF), and S‐adenosylmethionine (SAM) concentrations of maternal serum decreased significantly after MTX injection. These findings suggest that CNVs caused by defects in folate metabolism lead to NTD, and further support the hypothesis that folate dysmetabolism is a direct cause for CNVs in MTX‐induced NTDs. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 877–893, 2014