Folate deficiency increases the incidence of dolutegravir-associated foetal defects in a mouse pregnancy model.

BACKGROUND: Dolutegravir (DTG) is a recommended first-line regimen for all people with Human Immunodeficiency Virus (HIV) infection. Initial findings from Botswana, a country with no folate fortification program, showed an elevated prevalence of neural tube defects (NTDs) with peri-conceptional exposure to DTG. Here we explore whether a low folate diet influences the risk of DTG-associated foetal anomalies in a mouse model. METHODS: C57BL/6 mice fed a folate-deficient diet for 2 weeks, were mated and then randomly allocated to control (water), or 1xDTG (2.5 mg/kg), or 5xDTG (12.5 mg/kg) both administered orally with 50 mg/kg tenofovir disoproxil fumarate 33.3 mg/kg emtricitabine. Treatment was administered once daily from gestational day (GD) 0.5 to sacrifice (GD15.5). Foetuses were assessed for gross anomalies. Maternal and foetal folate levels were quantified. FINDINGS: 313 litters (103 control, 106 1xDTG, 104 5xDTG) were assessed. Viability, placental weight, and foetal weight did not differ between groups. NTDs were only observed in the DTG groups (litter rate: 0% control; 1.0% 1xDTG; 1.3% 5xDTG). Tail, abdominal wall, limb, craniofacial, and bleeding defects all occurred at higher rates in the DTG groups versus control. Compared with our previous findings on DTG usage in folate-replete mouse pregnancies, folate deficiency was associated with higher rates of several defects, including NTDs, but in the DTG groups only. We observed a severe left-right asymmetry phenotype that was more frequent in DTG groups than controls. INTERPRETATION: Maternal folate deficiency may increase the risk for DTG-associated foetal defects. Periconceptional folic acid supplementation could be considered for women with HIV taking DTG during pregnancy, particularly in countries lacking folate fortification programs. FUNDING: This project has been funded by Federal funds from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN275201800001I and award #R01HD104553. LS is supported by a Tier 1 Canada Research Chair in Maternal-Child Health and HIV. HM is supported by a Junior Investigator award from the Ontario HIV Treatment Network.

Investigating Genetic Determinants of Plasma Inositol Status in Adult Humans.

BACKGROUND: Myo-inositol (MI) is incorporated into numerous biomolecules, including phosphoinositides and inositol phosphates. Disturbance of inositol availability or metabolism is associated with various disorders, including neurological conditions and cancers, whereas supplemental MI has therapeutic potential in conditions such as depression, polycystic ovary syndrome, and congenital anomalies. Inositol status can be influenced by diet, synthesis, transport, utilization, and catabolism. OBJECTIVES: We aimed to investigate potential genetic regulation of circulating MI status and to evaluate correlation of MI concentration with other metabolites. METHODS: GC-MS was used to determine plasma MI concentration of >2000 healthy, young adults (aged 18-28 y) from the Trinity Student Study. Genotyping data were used to test association of plasma MI with single nucleotide polymorphisms (SNPs) in candidate genes, encoding inositol transporters and synthesizing enzymes, and test for genome-wide association. We evaluated potential correlation of plasma MI with d-chiro-inositol (DCI), glucose, and other metabolites by Spearman rank correlation. RESULTS: Mean plasma MI showed a small but significant difference between males and females (28.5 and 26.9 µM, respectively). Candidate gene analysis revealed several nominally significant associations with plasma MI, most notably for SLC5A11 (solute carrier family 5 member 11), encoding a sodium-coupled inositol transporter, also known as SMIT2 (sodium-dependent myo-inositol transporter 2). However, these did not survive correction for multiple testing. Subsequent testing for genome-wide association with plasma MI did not identify associations of genome-wide significance (P < 5 × 10-8). However, 8 SNPs exceeded the threshold for suggestive significant association with plasma MI concentration (P < 1 × 10-5), 3 of which were located within or close to genes: MTDH (metadherin), LAPTM4B (lysosomal protein transmembrane 4 ß), and ZP2 (zona pellucida 2). We found significant positive correlation of plasma MI concentration with concentration of dci and several other biochemicals including glucose, methionine, betaine, sarcosine, and tryptophan. CONCLUSIONS: Our findings suggest potential for modulation of plasma MI in young adults by variation in SLC5A11, which is worthy of further investigation.

Deficient or Excess Folic Acid Supply During Pregnancy Alter Cortical Neurodevelopment in Mouse Offspring.

Folate is an essential micronutrient required for both cellular proliferation through de novo nucleotide synthesis and epigenetic regulation of gene expression through methylation. This dual requirement places a particular demand on folate availability during pregnancy when both rapid cell generation and programmed differentiation of maternal, extraembryonic, and embryonic/fetal tissues are required. Accordingly, prenatal neurodevelopment is particularly susceptible to folate deficiency, which can predispose to neural tube defects, or when effective transport into the brain is impaired, cerebral folate deficiency. Consequently, adequate folate consumption, in the form of folic acid (FA) fortification and supplement use, is widely recommended and has led to a substantial increase in the amount of FA intake during pregnancy in some populations. Here, we show that either maternal folate deficiency or FA excess in mice results in disruptions in folate metabolism of the offspring, suggesting diversion of the folate cycle from methylation to DNA synthesis. Paradoxically, either intervention causes comparable neurodevelopmental changes by delaying prenatal cerebral cortical neurogenesis in favor of late-born neurons. These cytoarchitectural and biochemical alterations are accompanied by behavioral abnormalities in FA test groups compared with controls. Our findings point to overlooked potential neurodevelopmental risks associated with excessively high levels of prenatal FA intake.

Impaired folate 1-carbon metabolism causes formate-preventable hydrocephalus in glycine decarboxylase-deficient mice.

Ventriculomegaly and hydrocephalus are associated with loss of function of glycine decarboxylase (Gldc) in mice and in humans suffering from non-ketotic hyperglycinemia (NKH), a neurometabolic disorder characterized by accumulation of excess glycine. Here, we showed that ventriculomegaly in Gldc-deficient mice is preceded by stenosis of the Sylvian aqueduct and malformation or absence of the subcommissural organ and pineal gland. Gldc functions in the glycine cleavage system, a mitochondrial component of folate metabolism, whose malfunction results in accumulation of glycine and diminished supply of glycine-derived 1-carbon units to the folate cycle. We showed that inadequate 1-carbon supply, as opposed to excess glycine, is the cause of hydrocephalus associated with loss of function of the glycine cleavage system. Maternal supplementation with formate prevented both ventriculomegaly, as assessed at prenatal stages, and postnatal development of hydrocephalus in Gldc-deficient mice. Furthermore, ventriculomegaly was rescued by genetic ablation of 5,10-methylene tetrahydrofolate reductase (Mthfr), which results in retention of 1-carbon groups in the folate cycle at the expense of transfer to the methylation cycle. In conclusion, a defect in folate metabolism can lead to prenatal aqueduct stenosis and resultant hydrocephalus. These defects are preventable by maternal supplementation with formate, which acts as a 1-carbon donor.

Cellular mechanisms underlying Pax3-related neural tube defects and their prevention by folic acid.

Neural tube defects (NTDs), including spina bifida and anencephaly, are among the most common birth defects worldwide, but their underlying genetic and cellular causes are not well understood. Some NTDs are preventable by supplemental folic acid. However, despite widespread use of folic acid supplements and implementation of food fortification in many countries, the protective mechanism is unclear. Pax3 mutant (splotch; Sp2H ) mice provide a model in which NTDs are preventable by folic acid and exacerbated by maternal folate deficiency. Here, we found that cell proliferation was diminished in the dorsal neuroepithelium of mutant embryos, corresponding to the region of abolished Pax3 function. This was accompanied by premature neuronal differentiation in the prospective midbrain. Contrary to previous reports, we did not find evidence that increased apoptosis could underlie failed neural tube closure in Pax3 mutant embryos, nor that inhibition of apoptosis could prevent NTDs. These findings suggest that Pax3 functions to maintain the neuroepithelium in a proliferative, undifferentiated state, allowing neurulation to proceed. NTDs in Pax3 mutants were not associated with abnormal abundance of specific folates and were not prevented by formate, a one-carbon donor to folate metabolism. Supplemental folic acid restored proliferation in the cranial neuroepithelium. This effect was mediated by enhanced progression of the cell cycle from S to G2 phase, specifically in the Pax3 mutant dorsal neuroepithelium. We propose that the cell-cycle-promoting effect of folic acid compensates for the loss of Pax3 and thereby prevents cranial NTDs.

High dietary folate in pregnant mice leads to pseudo-MTHFR deficiency and altered methyl metabolism, with embryonic growth delay and short-term memory impairment in offspring.

Methylenetetrahydrofolate reductase (MTHFR) generates methyltetrahydrofolate for methylation reactions. Severe MTHFR deficiency results in homocystinuria and neurologic impairment. Mild MTHFR deficiency (677C > T polymorphism) increases risk for complex traits, including neuropsychiatric disorders. Although low dietary folate impacts brain development, recent concerns have focused on high folate intake following food fortification and increased vitamin use. Our goal was to determine whether high dietary folate during pregnancy affects brain development in murine offspring. Female mice were placed on control diet (CD) or folic acid-supplemented diet (FASD) throughout mating, pregnancy and lactation. Three-week-old male pups were evaluated for motor and cognitive function. Tissues from E17.5 embryos, pups and dams were collected for choline/methyl metabolite measurements, immunoblotting or gene expression of relevant enzymes. Brains were examined for morphology of hippocampus and cortex. Pups of FASD mothers displayed short-term memory impairment, decreased hippocampal size and decreased thickness of the dentate gyrus. MTHFR protein levels were reduced in FASD pup livers, with lower concentrations of phosphocholine and glycerophosphocholine in liver and hippocampus, respectively. FASD pup brains showed evidence of altered acetylcholine availability and Dnmt3a mRNA was reduced in cortex and hippocampus. E17.5 embryos and placentas from FASD dams were smaller. MTHFR protein and mRNA were reduced in embryonic liver, with lower concentrations of choline, betaine and phosphocholine. Embryonic brain displayed altered development of cortical layers. In summary, high folate intake during pregnancy leads to pseudo-MTHFR deficiency, disturbed choline/methyl metabolism, embryonic growth delay and memory impairment in offspring. These findings highlight the unintended negative consequences of supplemental folic acid.

Inositol, neural tube closure and the prevention of neural tube defects.

Susceptibility to neural tube defects (NTDs), such as anencephaly and spina bifida is influenced by genetic and environmental factors including maternal nutrition. Maternal periconceptional supplementation with folic acid significantly reduces the risk of an NTD-affected pregnancy, but does not prevent all NTDs, and "folic acid non-responsive" NTDs continue to occur. Similarly, among mouse models of NTDs, some are responsive to folic acid but others are not. Among nutritional factors, inositol deficiency causes cranial NTDs in mice while supplemental inositol prevents spinal and cranial NTDs in the curly tail (Grhl3 hypomorph) mouse, rodent models of hyperglycemia or induced diabetes, and in a folate-deficiency induced NTD model. NTDs also occur in mice lacking expression of certain inositol kinases. Inositol-containing phospholipids (phosphoinositides) and soluble inositol phosphates mediate a range of functions, including intracellular signaling, interaction with cytoskeletal proteins, and regulation of membrane identity in trafficking and cell division. Myo-inositol has been trialed in humans for a range of conditions and appears safe for use in human pregnancy. In pilot studies in Italy and the United Kingdom, women took inositol together with folic acid preconceptionally, after one or more previous NTD-affected pregnancies. In nonrandomized cohorts and a randomized double-blind study in the United Kingdom, no recurrent NTDs were observed among 52 pregnancies reported to date. Larger-scale fully powered trials are needed to determine whether supplementation with inositol and folic acid would more effectively prevent NTDs than folic acid alone. Birth Defects Research 109:68-80, 2017. © 2016 The Authors Birth Defects Research Published by Wiley Periodicals, Inc.

Formate supplementation enhances folate-dependent nucleotide biosynthesis and prevents spina bifida in a mouse model of folic acid-resistant neural tube defects.

The curly tail mouse provides a model for neural tube defects (spina bifida and exencephaly) that are resistant to prevention by folic acid. The major ct gene, responsible for spina bifida, corresponds to a hypomorphic allele of grainyhead-like 3 (Grhl3) but the frequency of NTDs is strongly influenced by modifiers in the genetic background. Moreover, exencephaly in the curly tail strain is not prevented by reinstatement of Grhl3 expression. In the current study we found that expression of Mthfd1L, encoding a key component of mitochondrial folate one-carbon metabolism (FOCM), is significantly reduced in ct/ct embryos compared to a partially congenic wild-type strain. This expression change is not attributable to regulation by Grhl3 or the genetic background at the Mthfd1L locus. Mitochondrial FOCM provides one-carbon units as formate for FOCM reactions in the cytosol. We found that maternal supplementation with formate prevented NTDs in curly tail embryos and also resulted in increased litter size. Analysis of the folate profile of neurulation-stage embryos showed that formate supplementation resulted in an increased proportion of formyl-THF and THF but a reduction in proportion of 5-methyl THF. In contrast, THF decreased and 5-methyl THF was relatively more abundant in the liver of supplemented dams than in controls. In embryos cultured through the period of spinal neurulation, incorporation of labelled thymidine and adenine into genomic DNA was suppressed by supplemental formate, suggesting that de novo folate-dependent biosynthesis of nucleotides (thymidylate and purines) was enhanced. We hypothesise that reduced Mthfd1L expression may contribute to susceptibility to NTDs in the curly tail strain and that formate acts as a one-carbon donor to prevent NTDs.

Inositol for the prevention of neural tube defects: a pilot randomised controlled trial.

Although peri-conceptional folic acid (FA) supplementation can prevent a proportion of neural tube defects (NTD), there is increasing evidence that many NTD are FA non-responsive. The vitamin-like molecule inositol may offer a novel approach to preventing FA-non-responsive NTD. Inositol prevented NTD in a genetic mouse model, and was well tolerated by women in a small study of NTD recurrence. In the present study, we report the Prevention of Neural Tube Defects by Inositol (PONTI) pilot study designed to gain further experience of inositol usage in human pregnancy as a preliminary trial to a future large-scale controlled trial to evaluate efficacy of inositol in NTD prevention. Study subjects were UK women with a previous NTD pregnancy who planned to become pregnant again. Of 117 women who made contact, ninety-nine proved eligible and forty-seven agreed to be randomised (double-blind) to peri-conceptional supplementation with inositol plus FA or placebo plus FA. In total, thirty-three randomised pregnancies produced one NTD recurrence in the placebo plus FA group (n 19) and no recurrences in the inositol plus FA group (n 14). Of fifty-two women who declined randomisation, the peri-conceptional supplementation regimen and outcomes of twenty-two further pregnancies were documented. Two NTD recurred, both in women who took only FA in their next pregnancy. No adverse pregnancy events were associated with inositol supplementation. The findings of the PONTI pilot study encourage a large-scale controlled trial of inositol for NTD prevention, but indicate the need for a careful study design in view of the unwillingness of many high-risk women to be randomised.

High folic acid consumption leads to pseudo-MTHFR deficiency, altered lipid metabolism, and liver injury in mice.

BACKGROUND: Increased consumption of folic acid is prevalent, leading to concerns about negative consequences. The effects of folic acid on the liver, the primary organ for folate metabolism, are largely unknown. Methylenetetrahydrofolate reductase (MTHFR) provides methyl donors for S-adenosylmethionine (SAM) synthesis and methylation reactions. OBJECTIVE: Our goal was to investigate the impact of high folic acid intake on liver disease and methyl metabolism. DESIGN: Folic acid-supplemented diet (FASD, 10-fold higher than recommended) and control diet were fed to male Mthfr(+/+) and Mthfr(+/-) mice for 6 mo to assess gene-nutrient interactions. Liver pathology, folate and choline metabolites, and gene expression in folate and lipid pathways were examined. RESULTS: Liver and spleen weights were higher and hematologic profiles were altered in FASD-fed mice. Liver histology revealed unusually large, degenerating cells in FASD Mthfr(+/-) mice, consistent with nonalcoholic fatty liver disease. High folic acid inhibited MTHFR activity in vitro, and MTHFR protein was reduced in FASD-fed mice. 5-Methyltetrahydrofolate, SAM, and SAM/S-adenosylhomocysteine ratios were lower in FASD and Mthfr(+/-) livers. Choline metabolites, including phosphatidylcholine, were reduced due to genotype and/or diet in an attempt to restore methylation capacity through choline/betaine-dependent SAM synthesis. Expression changes in genes of one-carbon and lipid metabolism were particularly significant in FASD Mthfr(+/-) mice. The latter changes, which included higher nuclear sterol regulatory element-binding protein 1, higher Srepb2 messenger RNA (mRNA), lower farnesoid X receptor (Nr1h4) mRNA, and lower Cyp7a1 mRNA, would lead to greater lipogenesis and reduced cholesterol catabolism into bile. CONCLUSIONS: We suggest that high folic acid consumption reduces MTHFR protein and activity levels, creating a pseudo-MTHFR deficiency. This deficiency results in hepatocyte degeneration, suggesting a 2-hit mechanism whereby mutant hepatocytes cannot accommodate the lipid disturbances and altered membrane integrity arising from changes in phospholipid/lipid metabolism. These preliminary findings may have clinical implications for individuals consuming high-dose folic acid supplements, particularly those who are MTHFR deficient.

Nucleotide precursors prevent folic acid-resistant neural tube defects in the mouse.

Closure of the neural tube during embryogenesis is a crucial step in development of the central nervous system. Failure of this process results in neural tube defects, including spina bifida and anencephaly, which are among the most common birth defects worldwide. Maternal use of folic acid supplements reduces risk of neural tube defects but a proportion of cases are not preventable. Folic acid is thought to act through folate one-carbon metabolism, which transfers one-carbon units for methylation reactions and nucleotide biosynthesis. Hence suboptimal performance of the intervening reactions could limit the efficacy of folic acid. We hypothesized that direct supplementation with nucleotides, downstream of folate metabolism, has the potential to support neural tube closure. Therefore, in a mouse model that exhibits folic acid-resistant neural tube defects, we tested the effect of specific combinations of pyrimidine and purine nucleotide precursors and observed a significant protective effect. Labelling in whole embryo culture showed that nucleotides are taken up by the neurulating embryo and incorporated into genomic DNA. Furthermore, the mitotic index was elevated in neural folds and hindgut of treated embryos, consistent with a proposed mechanism of neural tube defect prevention through stimulation of cellular proliferation. These findings may provide an impetus for future investigations of supplemental nucleotides as a means to prevent a greater proportion of human neural tube defects than can be achieved by folic acid alone.

Mutations in genes encoding the glycine cleavage system predispose to neural tube defects in mice and humans.

Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects of the central nervous system. The complex multigenic causation of human NTDs, together with the large number of possible candidate genes, has hampered efforts to delineate their molecular basis. Function of folate one-carbon metabolism (FOCM) has been implicated as a key determinant of susceptibility to NTDs. The glycine cleavage system (GCS) is a multi-enzyme component of mitochondrial folate metabolism, and GCS-encoding genes therefore represent candidates for involvement in NTDs. To investigate this possibility, we sequenced the coding regions of the GCS genes: AMT, GCSH and GLDC in NTD patients and controls. Two unique non-synonymous changes were identified in the AMT gene that were absent from controls. We also identified a splice acceptor site mutation and five different non-synonymous variants in GLDC, which were found to significantly impair enzymatic activity and represent putative causative mutations. In order to functionally test the requirement for GCS activity in neural tube closure, we generated mice that lack GCS activity, through mutation of AMT. Homozygous Amt(-/-) mice developed NTDs at high frequency. Although these NTDs were not preventable by supplemental folic acid, there was a partial rescue by methionine. Overall, our findings suggest that loss-of-function mutations in GCS genes predispose to NTDs in mice and humans. These data highlight the importance of adequate function of mitochondrial folate metabolism in neural tube closure.

Quantitative analysis of myo-inositol in urine, blood and nutritional supplements by high-performance liquid chromatography tandem mass spectrometry.

Myo-inositol plays key physiological functions, necessitating development of methodology for quantification in biological matrices. Limitations of current mass spectrometry-based approaches include the need for a derivatisation step and/or sample clean-up. In addition, co-elution of glucose may cause ion suppression of myo-inositol signals, for example in blood or urine samples. We describe an HPLC-MS/MS method using a lead-form resin based column online to a triple quadrupole tandem mass spectrometer, which requires minimum sample preparation and no derivatisation. This method allows separation and selective detection of myo-inositol from other inositol stereoisomers. Importantly, inositol was also separated from hexose monosaccharides of the same molecular weight, including glucose, galactose, mannose and fructose. The inter- and intra-assay variability was determined for standard solutions and urine with inter-assay coefficient of variation (CV) of 1.1% and 3.5% respectively, while intra-assay CV was 2.3% and 3.6%. Urine and blood samples from normal individuals were analysed.

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.

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.