Folate‐mediated one‐carbon metabolism and neural tube defects: Balancing genome synthesis and gene expression

Bringing clarity to the role of MTHFR variants in neural tube defect prevention

Neural tube defects (NTDs) are the second most prevalent structural birth defect affecting approximately 324,000 births worldwide (Christianson et al., 2006), including 3000 pregnancies in the United States (Mersereau et al., 2004) annually. NTDs result from abnormalities in the development of the spinal cord and brain as well as the tissues that surround these structures such as the bones of the skull and vertebrae. The most common NTDs are anencephaly and myelomeningocele (commonly referred to as spina bifida). Anencephaly results from abnormal development of the portion of the neural tube that will develop into the brain, is uniformly lethal in the pre- or early postnatal period, and has a prevalence of ∼2.6/10,000 births in the United States (Canfield et al., 2006). Spina bifida results from abnormal development of the portion of the neural tube that will develop into the spinal cord, is associated with increased mortality and significant morbidity through the lifespan of survivors (Ouyang et al., 2007), and has a prevalence of ∼3.7/10,000 births in the United States (Canfield et al., 2006). The basis of NTDs in humans (in particular anencephaly and spina bifida) has been extensively studied yet the definitive causes are identified in only a few instances. A small proportion of NTDs occurs as part of chromosomal, single-gene, or teratogenic syndromes. Similarly, NTDs segregate in a Mendelian manner in only a minority of families. The majorities of NTDs are not associated with syndromes and have complex etiologies involving the combinatorial effects of multiple genetic and environmental factors. For instance, both maternal obesity and inadequate maternal folate status/intake of folic acid are associated with an increased risk of nonsyndromic NTDs (Waller et al., 2007; Correa et al., 2008). However, these and other known risk factors only account for a small proportion of NTDs, providing limited avenues for prevention. For instance, implementation of mandatory folic acid fortification of the food supply in the United States was followed by only a 24% reduction in the incidence of spina bifida (Canfield et al., 2006). Thus, additional efforts to understand the underlying genetic and environmental factors contributing to NTDs is imperative to achieve further reductions in the incidence of this birth defect. To accelerate discoveries in NTD research, the International Neural Tube Defects Conference was initiated in 1999 to bring together researchers from a wide variety of disciplines with the common goal of understanding the causes and ultimately preventing NTDs. The Ninth International Neural Tube Defects Conference was held October 26 to 29, 2015, at the AT&T Executive Conference Center in Austin, Texas. The conference was organized by Richard Finnell, Laura Mitchell, Philip Lupo, and John Wallingford and was supported by generous contributions from the National Institutes of Health (NICHD, NINDS, NIEHS); the School of Human Ecology, The University of Texas at Austin; Fibich, Leebron, Copeland, Briggs & Josephson; Rosenblum, Ronan, Kessler & Sarachan; and Blizzard & Nabers. Over 80 attendees from 15 countries throughout the world gathered to discuss recent advances in the field. Since its inception, the NTD Conference has provided a forum for the exchange of information across disparate disciplines of clinical medicine, public health, and the basic sciences. Following in this tradition, the Ninth International NTD Conference included oral and poster presentations covering a diverse spectrum of investigation, from clinical, epidemiological, computational toxicology, developmental biology, and genomics. This special issue of Birth Defects Research highlights a sampling of the interdisciplinary research presented at the Ninth International conference on NTD as well as providing a glimpse of the broad spectrum of research conducted by the vigorous NTD research community. Dr. Edward R.B. McCabe from the March of Dimes Foundation and Dr. Sergiu P. Pasca of Department of Psychiatry at Stanford University (Stanford, CA) gave keynote presentations. Dr. McCabe provided an update on folic acid fortification and a discussion of March of Dimes funding opportunities for research related to NTDs. Dr. Pasca presented his work on the development of novel three-dimensional culture systems to differentiate human induced pluripotent stem cells (IPSCs) into laminated cerebral cortical-like structures for the interrogation of cortical development and disease. The potential for these technologies to be used in NTD research to examine cell polarity and fate in IPSCs from NTD patients was discussed. Because folic acid supplementation does not prevent all NTDs, there are significant efforts in the field to identify additional preventative treatments. One new and exciting approach originates from studies in model systems where it was found that supplementation with inositol prevents NTDs in mouse models. This and other findings prompted initiation of clinical trials to determine if combined inositol and folate supplementation would prevent NTDs in subjects with previous NTD affected pregnancies despite having taken folate supplements. Nicholas Greene (University College London, UK) presented an update on the PONTI (Prevention of Neural Tube Defects by Inositol) study. Their promising results support the efficacy and safety of combined inositol and folate supplementation for the prevention of NTDs in high-risk populations. Furthermore, their study provides strong support for the feasibility of a large double blind clinical trial. Another nutrient of interest for prevention of NTDs is iron, and Irene Zohn (Children's National Medical Center, Washington, DC) presented work demonstrating that iron and folate supplementation prevented NTDs in a mouse model with severe iron deficiency. Unexpectedly, high levels of iron supplementation reduced folate levels in both pregnant dams and embryos, suggesting that high levels of iron supplementation might be counterproductive during pregnancy. Other presentations emphasized the need to carefully consider the role of environmental factors and gene–environment interactions in human studies. As summarized in this issue, Maitreyi Mazumdar (Harvard University, Cambridge, MA) presented findings from a new case-control study of NTDs in Bangladesh. This study explored how arsenic might influence NTD risk through disruption of maternal glucose and folate metabolism. Margaret Nguyen (The University of Texas Health Science Center at Houston, Texas) examined the role of maternal gene–micronutrient interactions on the risk of myelomeningocele among offspring using a case–parent trio approach. While they found no significant interactions, it is apparent that future studies should not only account for gene–environment interactions, but also account for maternal genotypes. Studies in mouse models paralleled this human work. Stephen Gross (Weill Medical College of Cornell University, New York, NY) presented results from a collaborative study with Richard Finnell (The University of Texas at Austin, Austin, TX) and Margaret Ross (Weill Medical College of Cornell University, New York, NY) using a novel metabolite profiling technique to define the metabolic perturbations that occur in both pregnant dams and their offspring in response to valproate exposure, a known teratogen that increases NTD risk. Several presentations focused on the identification of inherited genetic risk factors that increase susceptibility to NTDs. Included in this issue is an extensive review by Margaret Ross (Weill Medical College of Cornell University, New York, NY) that documents the various genomic approaches used in evaluating the risk of spina bifida in humans. This important review reminds us that the goal of human genomic studies is to find strategies for optimizing conditions to promote healthy birth outcomes. Furthermore, compared with other relatively common structural birth defects, large-scale genome-wide studies to identify the genes responsible for NTDs are currently missing from the literature. Animal model systems provide an essential complement to human genomic studies by identifying novel candidate genes and pathways required for neural tube closure. Several presentations on the genes and pathways involved in NTDs in these model systems were given. The plethora of genes discussed illustrates the diverse cellular functions required to generate the mechanical forces and tissue movements that drive neural tube closure. As an example, in this issue an extensive review by Saikat Mukhopadhyay (University of Texas Southwestern Medical Center, Dallas, TX) summarizes our current knowledge of the involvement of G-protein coupled receptor signaling and neural tube closure. The complex genetic landscape underlying NTDs is also echoed in mouse genetic studies. Dr. Heather McDermid (University of Alberta, Canada) presented an update on the analysis of genetic modifiers present in various inbred mouse strains and the opportunity the identification of these modifiers presents toward providing a greater understanding the complex genetics of NTDs. One of the goals of the conference is to provide opportunities for trainees to learn about the opportunities in the field of NTD research and present their research. At each meeting, the Marcy Speer Memorial Awards are bestowed to the predoctoral and postdoctoral level trainees judged to have given the most meritorious platform presentations. This award is in honor of Marcy Speer (1959–2007), one of the initial founders of the International NTD Conference. The awardees for 2015 were Deepthi Vijayraghavan (predoctoral) and Youssef Kousa (postdoctoral). Deepthi Vijayraghavan is a graduate student at the University of Pittsburgh, Pittsburgh, PA (mentor Dr. Lance Davidson). Ms. Vijayraghavan presented her research on the physical mechanics that shape, fold, and form the neural tube. Dr. Youssef Kousa is currently a Medical Resident (Children's National Medical Center in Washington DC) and presented research from his Ph.D. thesis with Brian Schutte (Michigan State University, Lansing, MI) on shared pathways in orofacial and neural tube development. At the conclusion of the meeting, the possibility that the NTD community might contribute to a white paper representing interests of the greater birth defects research community was discussed. This white paper calls for strategies and investments to tackle significant challenge involved in the study of birth defects with the goal of understanding the genetic basis of these complex disorders. The product of these discussions titled “White paper on the study of birth defects” is published in this special issue of Birth Defects Research. This white paper is authored by Mustafa Khokha (Yale University School of Medicine, New Haven, CN), Laura Mitchell (University of Texas Health Science Center at Houston, Houston, TX), and John Wallingford (University of Texas at Austin, Austin, TX) and signed by representatives of multiple scientific societies with vested interests in understanding and preventing birth defects. The International Conference on NTDs provides an interdisciplinary forum for sharing current research on the causes of NTDs. This meeting helps to foster existing and new collaborations as well as providing opportunities in career development for trainees. The breadth of subjects covered by the papers in this issue illustrates the complexities faced by researchers in understanding the underlying factors that contribute to NTDs and the challenges in developing new strategies to prevent their occurrence. More information about this and future international conferences on NTDs can be found at http://ntdconference.com Philip J. Lupo1 Irene Zohn2 1Department of Pediatrics, Section of Hematology--Oncology, Baylor College of Medicine, Houston, Texas 2Neuroscience Research, Children's National Medical Center, Washington, DC

Fumonisin, Folate, and Neural Tube Defects

5.13 - Fumonisin, Folate and other Methyl Donors and Neural Tube Defects

Focused Revision: Policy statement on folic acid and neural tube defects

Neural tube defects (NTDs) are malformations of the brain and the spinal cord, resulting from lack of closure of the developing neural tube during embryological development. NTDs are complex birth disorders with a worldwide prevalence from 0.5 to 2.0 per 1000 births. The two most common types of NTD are anencephaly and spina bifida, which involve failure of neural tube closure in the cranial and in the back regions respectively. NTDs are hypothesised to be a result of genetic abnormalities and environmental factors; yet, the aetiology remains undetermined. Research studies over the past decade have indicated that maternal folate deficiency is an extremely important risk factor for an NTD birth, with maternal folate supplementation decreasing risk. However, the role that folate plays in NTD risk is not well defined. Key Concepts: Neural tube defects (NTDs) are congenital malformations of the brain and spinal cord. The prevalence rates of NTDs vary worldwide. NTDs are complex birth defects composed of both genetic and environmental components. The underlying mechanism remains undetermined. NTDs are classified by whether they are open or closed and by the location of the lesion. Folic acid supplementation ameliorates NTD risk. The majority of NTDs are sporadic and nonsyndromic. To date, association studies in relatively small patient cohorts have been used in an attempt to find causative genes. Future additional approaches will include Genome‐Wide Association Study (GWAS) and high‐throughput sequencing.

Neural tube defects and folic acid in Japan: Prologue introduction ‐ Understanding of the current status of Japan and the proposal from Japanese Teratology Society

Relation between hypomethylation of long interspersed nucleotide elements and risk of neural tube defects

Obesity is associated with twofold increased risk of neural tube defects (NTD). Research has repeatedly shown that about 70% of NTD are folic-acid dependent. Yet, there is controversy whether folic acid status is the main determinant of the increased risk of obesity-induced NTD. The rational for this review is to update and discuss the evidence on the link between obesity, folic acid and NTD, in an attempt to shed light on the question whether optimal folic acid dose schedule can mitigate this risk. During pregnancy maternal folate requirements increase by 5–10-fold, as folate is diverted towards the placenta and fetus, as well as supporting different maternal organs. Correspondingly, low maternal folate status has been associated with birth defects in fetal anatomical regions particularly sensitive to reduced folate intake including oral cleft, cardiovascular defects and NTD. A recent study has documented decreased placental folate transporter expression and activity in the first and second trimesters among obese mothers. This may explain the higher incidence on NTD in infants of obese women, as less folate may find its way to the developing fetus during the sensitive periods for creating NTD. Recent pharmacokinetic results indicate that steady state levels of folate are almost perfectly defined by the dose per lean body weight (LBW). The mean dose per kg LBW that would be expected to result in steady state serum folate level of > 15.9 nmol/L was identified as 0.0073 mg/kg LBW. A large study found no differences in dietary supplementations of folic acid, yet obese women exhibited lower median serum folate as well as lower mean serum B12 levels, but no differences in mean RBC folate levels. There was a negative correlation between increasing BMI and both serum folate and plasma B12. Future research will be needed to incorporate more fully, in addition to evidence of NTD, obesity and folic acid intake, also direct measurements of serum and RBC folate, as well as other confounders, in order to create a model that will shed light on these complex interactions.

Head morphogenesis is a complex process that is controlled by multiple signaling centers. The most common defects of cranial development are craniofacial defects, such as cleft lip and cleft palate, and neural tube defects, such as anencephaly and encephalocoele in humans. More than 400 genes that contribute to proper neural tube closure have been identified in experimental animals, but only very few causative gene mutations have been identified in humans, supporting the notion that environmental influences are critical. The intrauterine environment is influenced by maternal nutrition, and hence, maternal diet can modulate the risk for cranial and neural tube defects. This article reviews recent progress toward a better understanding of nutrients during pregnancy, with particular focus on mouse models for defective neural tube closure. At least four major patterns of nutrient responses are apparent, suggesting that multiple pathways are involved in the response, and likely in the underlying pathogenesis of the defects. Folic acid has been the most widely studied nutrient, and the diverse responses of the mouse models to folic acid supplementation indicate that folic acid is not universally beneficial, but that the effect is dependent on genetic configuration. If this is the case for other nutrients as well, efforts to prevent neural tube defects with nutritional supplementation may need to become more specifically targeted than previously appreciated. Mouse models are indispensable for a better understanding of nutrient-gene interactions in normal pregnancies, as well as in those affected by metabolic diseases, such as diabetes and obesity.

Intersection of complex genetic traits affecting maternal metabolism, fetal metabolism, and neural tube defect risk: Looking for needles in multiple haystacks

In 2018, the Botswana Tsepamo Study reported a nine-fold increased risk of neural tube defects in infants whose mothers were treated with dolutegravir (DTG) from the time of conception. As maternal folate supplementation and status is a well known modifier of neural tube defect (NTD) risk, we sought to evaluate birth outcomes in mice fed normal and low folic acid diets treated with DTG during pregnancy. DTG was evaluated for developmental toxicity using pregnant mice fed normal or low folic acid diet. CD-1 mice were provided diet with normal (3 mg/kg) or low (0.3 mg/kg) folic acid. They were treated with water, a human therapeutic-equivalent dose, or supratherapeutic dose of DTG from mouse embryonic day E6.5 to E12.5. Pregnant dams were sacrificed at term (E18.5) and fetuses were inspected for gross, internal, and skeletal defects. Fetuses with exencephaly, an NTD, were present in both therapeutic human equivalent and supratherapeutic exposures in dams fed low folic acid diet. Cleft palates were also found under both folate conditions. Recommended dietary folic acid levels during mouse pregnancy ameliorate developmental defects that arise from DTG exposure. Since low folate status in mice exposed to DTG increases the risk for NTDs, it is possible that DTG exposures in people living with HIV with low folate status during pregnancy may explain, at least in part, the elevated NTD risk signal observed in Botswana. Based on these results, future studies should consider folate status as a modifier for DTG-associated NTD risk.

Neural tube defects (NTDs) are a common congenital disorder resulting from failed neural tube formation, the precursor of the brain and spinal cord. The complex etiology of NTDs involve both genetic and environmental factors, thus investigating gene‐environment interactions is critical to understanding how NTDs occur or how NTDs may be prevented. For example, iron deficiency is among the most prevalent micronutrient deficiencies in pregnancy and there is evidence that iron deficiency can increase the risk of NTDs. Folic acid (FA) fortification in grain was implemented in the US in 1998 and now in many countries for the purpose of prevention of neural tube defects (NTDs). In 2017, the US Preventive Services Task Force further recommended the consumption of FA‐containing multivitamin/minerals (MVM) for women of childbearing age. Despite the great benefit of FA on NTD prevention, NTDs remain a serious risk.Our studies focus on mouse models of NTDs and I will discuss two projects. First, the benefits of MVM supplementation are emphasized, but a comparison between MVM supplementation and FA alone on neural tube closure and organ development has not been evaluated, particularly in the context of genetic mutations that lead to NTDs. Using a set of NTD models we have compared MMV to FA supplementation. MVM supplementation shows better improvement than FA alone in the penetrance of spinal and cranial NTDs. We are also using human iPSCs to generate organoids that assume a shape similar to the human spinal neural tube, and display neural tube characteristics, for example apical constriction and interkinetic nuclear migration. Subjecting the organoids to various molecular perturbations, such as Rho‐kinase inhibitor‐induced defects in apical constriction, we have found that both MVM and FA largely prevents this molecular deficit. The goal is to use the mouse NTD model and organoid system to better understand how FA/MVM act to prevent NTDs.Second, we are striving to move beyond the relatively non‐specific recommendations of FA/MVM supplementation to more personalized therapies that take into account the molecular underpinning of the NTD. For example, Iron homeostasis is not only controlled by the concentration of iron in diet, but by proteins responsible for its cellular uptake and metabolism. Sorting nexin 3 (Snx3) is known to regulate recycling of the transferrin receptor (TFRC), a mechanism necessary for cellular uptake of iron, and when Snx3 is knocked out in mice, embryos develop cranial NTDs. We hypothesize that in Snx3 mutants, mistrafficking of TFRC results in disrupted iron homeostasis which contributes to the failure of NT closure. Our current studies are evaluating whether intracellular iron levels are altered in Snx3 mutants and whether maternal diets supplemented with iron can prevent NTDs in the Snx3 background. The hope is that by taking into account the biological process that is disrupted, more tailored therapies can be envisioned to better prevent NTDs.

Folic acid and prevention of neural-tube defects

Neural tube defects (NTDs), a common birth defect in humans, result from the failure of the embryonic neural tube (NT) to close properly. NT closure is a complex, poorly understood morphogenetic process influenced by genes and environment. The most effective environmental influence in decreasing the risk for NTDs is folic acid (FA) fortification and supplementation, and these findings led to the recommendation of periconceptual FA intake and mandatory fortification of the US grain supply in 1998. To explore the relationship between genetics and responsiveness to FA supplementation, we used five mouse NTDs models-Zic2, Shroom3, Frem2, Grhl2 (Grainyhead-like 2) and L3P (Line3P)-and a long-term generational FA supplementation scheme. Contrary to expectations, we find that three genetic mutants respond adversely to FA supplementation with increased incidence of NTDs in homozygous mutants, occurrence of NTDs in heterozygous embryos and embryonic lethality prior to NT closure. Because of these unexpected responses, we examined NTD risk after short-term FA supplementation. Our results indicate that, for the same genetic allele, NTD risk can depend on the length of FA exposure. Our data indicate that, depending on the gene mutation, FA supplementation may adversely influence embryonic development and NT closure.