Inositol-preventable neural tube defects: understanding the molecular causes and mechanisms of prevention
Lead Research Organisation:
University College London
Department Name: Unlisted
Abstract
During early pregnancy, a crucial event in the developing embryo is the formation of the neural tube , which will later develop into the brain and spinal cord. Failure of the neural tube to form correctly leads to a group of birth defects called neural tube defects (NTDs), in which the brain and/or spinal cord of the fetus become irreversibly damaged, resulting in death before or shortly after birth, or handicap in surviving babies. Overall, NTDs occur in around 1 per 1,000 pregnancies although the rate varies and is significantly higher in some regions (e.g. Northern Ireland and Scotland). Worldwide, approximately 130,000 cases occur every year. The risk of NTDs depends on both inherited genetic factors and non-genetic factors such as diet, but the exact causes are not well understood. However, the risk of an affected pregnancy can be substantially reduced if the mother takes folic acid supplements before and during early pregnancy. Unfortunately, not all cases of NTDs are preventable by folic acid and for this reason, we are seeking additional protective approaches. Using a mouse model that naturally develops folate-resistant NTDs we found that another vitamin, inositol, can prevent NTDs, and we are now carrying out a clinical trial to test whether inositol prevents NTDs in humans. There are two main aims of the present research proposal. First, we will investigate the cause of the inositol-preventable NTDs in the mouse. We have used a technique that lets us separate many proteins and compare which ones are present in normal embryos and those that develop NTDs. We have detected several proteins that seem to be present at lower abundance or in abnormally modified forms in embryos developing NTDs. We will build on these studies by analysing the proteins that seem to be abnormal in the embryos that develop spina bifida. Understanding the role of specific proteins in the mouse may indicate risk factor genes in humans. The second aim of the research is to work out exactly how inositol prevents NTDs, as this may allow improvements to be made in the therapeutic approach. Knowledge of the causes of NTDs in humans may then allow more accurate counselling for affected families who are considering a further pregnancy, and may allow identification of women who will benefit from inositol supplementation.
Technical Summary
Neural tube defects (NTDs), such as spina bifida and anencephaly, are severe congenital malformations caused by failure of closure of the embryonic neural tube. Folic acid supplementation can prevent many cases of NTDs but a significant proportion (at least 30%) are unresponsive, and these defects remain a major health issue in the UK and worldwide. As a system to investigate the mechanisms underlying development of ?folate-resistant? NTDs, and to evaluate potential therapies, the curly tail mutant mouse provides a well-studied model. The incidence of defects in curly tail is influenced by both multiple genetic and environmental factors, as in humans.
We found previously that NTDs in curly tail can be prevented by inositol, acting through a protein kinase C (PKC)-dependent mechanism. This finding suggests that inositol may represent a possible adjunct therapy to folic acid for prevention of NTDs. Indeed, we recently initiated a clinical trial to test this possibility. Our present goals are: (1) to understand the molecular basis of NTDs in the curly tail mouse and (2) to further define the mechanism by which NTDs can be prevented by inositol. In a recent proteomic screen, we compared curly tail and genetically matched wild type embryos, using 2D protein gels and mass spectrometry. We identified a number of protein differences involving changes in protein abundance and post-translational protein modification. Our working hypothesis is that these changes may be associated with predisposition to NTDs.
Here, we will characterise the specific changes in individual candidate proteins by detailed examination of the abundance and spectrum of post-translational variants. We will then evaluate the potential role of the most promising candidate proteins in neural tube closure. Correction of mutant protein profiles by inositol treatment will support such a role, particularly if this is dependent on the PKC isoforms we previously found to be necessary for prevention of NTDs. Tissue expression and subcellular localization will be examined as these may indicate disruption of protein function. Since the curly tail mutation is thought to represent a hypomorphic allele of the transcription factor, Grhl3, we will also examine the protein profile of Grhl3 null mutants, which also exhibit NTDs, in parallel with curly tail. These studies will allow us to prioritise candidate proteins for functional analysis to directly test their requirement in neural tube closure and/or the prevention of NTDs by inositol.
We found previously that NTDs in curly tail can be prevented by inositol, acting through a protein kinase C (PKC)-dependent mechanism. This finding suggests that inositol may represent a possible adjunct therapy to folic acid for prevention of NTDs. Indeed, we recently initiated a clinical trial to test this possibility. Our present goals are: (1) to understand the molecular basis of NTDs in the curly tail mouse and (2) to further define the mechanism by which NTDs can be prevented by inositol. In a recent proteomic screen, we compared curly tail and genetically matched wild type embryos, using 2D protein gels and mass spectrometry. We identified a number of protein differences involving changes in protein abundance and post-translational protein modification. Our working hypothesis is that these changes may be associated with predisposition to NTDs.
Here, we will characterise the specific changes in individual candidate proteins by detailed examination of the abundance and spectrum of post-translational variants. We will then evaluate the potential role of the most promising candidate proteins in neural tube closure. Correction of mutant protein profiles by inositol treatment will support such a role, particularly if this is dependent on the PKC isoforms we previously found to be necessary for prevention of NTDs. Tissue expression and subcellular localization will be examined as these may indicate disruption of protein function. Since the curly tail mutation is thought to represent a hypomorphic allele of the transcription factor, Grhl3, we will also examine the protein profile of Grhl3 null mutants, which also exhibit NTDs, in parallel with curly tail. These studies will allow us to prioritise candidate proteins for functional analysis to directly test their requirement in neural tube closure and/or the prevention of NTDs by inositol.
Organisations
- University College London, United Kingdom (Collaboration, Lead Research Organisation)
- University of Oxford, United Kingdom (Collaboration)
- VU University Medical Center (Collaboration)
- Karolinska Institute, Sweden (Collaboration)
- Imperial College London, United Kingdom (Collaboration)
- Lund University (Collaboration)
- Grants Admin Office (Collaboration)
Publications

Burren KA
(2008)
Gene-environment interactions in the causation of neural tube defects: folate deficiency increases susceptibility conferred by loss of Pax3 function.
in Human molecular genetics

Burren KA
(2010)
The genetic background of the curly tail strain confers susceptibility to folate-deficiency-induced exencephaly.
in Birth defects research. Part A, Clinical and molecular teratology

Cleary JO
(2011)
Magnetic resonance virtual histology for embryos: 3D atlases for automated high-throughput phenotyping.
in NeuroImage

D'Souza SW
(2021)
Maternal Inositol Status and Neural Tube Defects: A Role for the Human Yolk Sac in Embryonic Inositol Delivery?
in Advances in nutrition (Bethesda, Md.)

De Castro SC
(2010)
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.
in Birth defects research. Part A, Clinical and molecular teratology

Dinicola S
(2021)
Inositols: From Established Knowledge to Novel Approaches
in International Journal of Molecular Sciences

Greene ND
(2009)
Genetics of human neural tube defects.
in Human molecular genetics

Greene ND
(2009)
Development of the vertebrate central nervous system: formation of the neural tube.
in Prenatal diagnosis

Greene ND
(2017)
Inositol, neural tube closure and the prevention of neural tube defects.
in Birth defects research

Greene ND
(2016)
Inositol for the prevention of neural tube defects: a pilot randomised controlled trial.
in The British journal of nutrition
Description | Institution Strategic Support Fund |
Amount | £48,000 (GBP) |
Funding ID | ISSF3 |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 07/2017 |
End | 08/2019 |
Description | Prevention of Neural Tube Defects by Inositol and Vitamin B12 (PONTib) - Development Trial |
Amount | £302,630 (GBP) |
Funding ID | T003847 |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2021 |
End | 02/2023 |
Description | Programme grant |
Amount | £1,366,000 (GBP) |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2010 |
End | 03/2016 |
Description | Project Grant |
Amount | £189,516 (GBP) |
Funding ID | GN2656 |
Organisation | Action Medical Research |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2018 |
End | 10/2020 |
Description | SPARKS PhD studentship |
Amount | £89,968 (GBP) |
Funding ID | 17ICH03 |
Organisation | Sparks Charity |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 09/2017 |
End | 09/2020 |
Description | Sparks Project Grant |
Amount | £67,000 (GBP) |
Organisation | Sparks Charity |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 02/2011 |
End | 08/2013 |
Description | Vitamins for the prevention of neural tube defects |
Amount | £89,968 (GBP) |
Funding ID | 17ICH03 |
Organisation | Sparks Charity |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 09/2017 |
End | 09/2020 |
Title | Grainyhead-like 3 transgenic mice |
Description | A mouse line was generated that carries a BAC containing the Grhl3 gene. This results in over-expression of Grhl3 in the embryo. |
Type Of Material | Model of mechanisms or symptoms - mammalian in vivo |
Provided To Others? | No |
Impact | The mice were used in a 'transgenic rescue' experiment which showed that neural tube defects in curly tail mice result from reduced expression of Grhl3. |
Title | Inositol assay - GC-MS |
Description | GC-MS method for inositols |
Type Of Material | Technology assay or reagent |
Year Produced | 2017 |
Provided To Others? | No |
Impact | This method was developed to allow high-throughput analysis of inositol isoforms - the method will be published with the intial research findings |
Description | DNA methylation & folate/inositol deficiency |
Organisation | VU University Medical Center |
Country | Netherlands |
Sector | Academic/University |
PI Contribution | We found that folate and/or inositol deificient dets increase the frequency of neural tube defects in mutant mouse strains. Folate deficiency may impact the methylation cycle, which we previously found to be required for neural tube formation. This raised the possibility that DNA methylation may be altered - the area of this collaboration. |
Collaborator Contribution | Our collaborator has performed mass spectrometry based analysis of DNA methylation on embryo samples from mutant mouse strains maintained under dietary conditions that predispose to neural tube defects. |
Impact | We observed reduced methylation in embryos treated with methylation cycle inhibitors, but not in folate-deficient embryos (Publication 18753144). |
Start Year | 2007 |
Description | Folate assays |
Organisation | Trinity College Dublin |
Country | Ireland |
Sector | Academic/University |
PI Contribution | We found that folate deficiency increases suceptibility to neural tube defects in various mouse strains that are genetically predisposed to neural tube defects. In addition, we showed that in some cases these defects were preventable by inositol. We performed biochemical analysis of folate content in different genetic models under varying dietary conditions. |
Collaborator Contribution | Our collaborator contributed reagents and expertise to enable us to establish microbiological folate assays, and contributed expertise in data analysis. |
Impact | This collaboration enabled measurement of folate in embryos that developed under differing conditions, to correlate folate status with neural tube defect risk. Publications: 18753144 and 19824061 |
Start Year | 2006 |
Description | Gene-nutrient interactions - Trinity Cohort Study |
Organisation | Trinity College Dublin |
Country | Ireland |
Sector | Academic/University |
PI Contribution | Our research team analysed samples from the cohort and produced quantitative data on levels of myo-inositol, chiro-inositol and glucose |
Collaborator Contribution | Our partners provided data on additional nutrients/biomarkers in the cohort. They generated GWAS data on the cohort to allow analysis of gene-nutrient interactions. |
Impact | Data is under analysis |
Start Year | 2017 |
Description | Gene-nutrient interactions: microbes to mammals |
Organisation | Imperial College London |
Department | MRC London Institute of Medical Sciences |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | In this project we are seeking to carry our large-scale screens to identify gene-nutrient interactions. We seek to identify nutritional factors which ameliorate or exacerbate specific both defects and post-natal disease mammals. Screens are carried out using bacteria and C.elegans models carrying mutations in the orthologous genes to those responsible for human disease. Our group provides the mammalian models for testing of candidate molecules and carries out sensitive mass spectrometry analysis to examine mechanisms by which nutritional status alters microb/host response. |
Collaborator Contribution | Our partners carry our high throughout screens of nutrients in panels of E.coli and C.elegans genetic mutants. |
Impact | Manuscriipts; Cabreiro et al. Cell (2013) PMID: 23540700 Leung et al. Mol Cell Biochem (2013) PMID: 23483428 Scott et al. Cell (2017) PMID: 28431245 Joint funding: 2017-2019: Wellcome Institutional Strategic Support Fund (ISSF3) (£48,000) Gene-nutrient interactions: from Microbes to Mammals. Greene, Cabreiro |
Start Year | 2012 |
Description | Gene-nutrient interactions: microbes to mammals |
Organisation | University College London |
Department | School of Life and Medical Sciences |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | In this project we are seeking to carry our large-scale screens to identify gene-nutrient interactions. We seek to identify nutritional factors which ameliorate or exacerbate specific both defects and post-natal disease mammals. Screens are carried out using bacteria and C.elegans models carrying mutations in the orthologous genes to those responsible for human disease. Our group provides the mammalian models for testing of candidate molecules and carries out sensitive mass spectrometry analysis to examine mechanisms by which nutritional status alters microb/host response. |
Collaborator Contribution | Our partners carry our high throughout screens of nutrients in panels of E.coli and C.elegans genetic mutants. |
Impact | Manuscriipts; Cabreiro et al. Cell (2013) PMID: 23540700 Leung et al. Mol Cell Biochem (2013) PMID: 23483428 Scott et al. Cell (2017) PMID: 28431245 Joint funding: 2017-2019: Wellcome Institutional Strategic Support Fund (ISSF3) (£48,000) Gene-nutrient interactions: from Microbes to Mammals. Greene, Cabreiro |
Start Year | 2012 |
Description | Inositol assay |
Organisation | Lund University |
Department | Department of Engineering, Division of Fluid Mechanics |
Country | Sweden |
Sector | Academic/University |
PI Contribution | Our research group has developed methodology for analysis of inositol in biological samples. We have also analysed the effect of inositol supplementation in animal models. This work has led to a clinical trial of inositol for prevention of neural tube defects. These projects require analysis of inositol in large sets of samples. |
Collaborator Contribution | Our collaborator has developed novel methodology for high throughput analysis of inositol. Together we are testing the method and applying to biological samples. |
Impact | Manuscript in preparation |
Start Year | 2016 |
Description | Karolinska - Genetics of NTDs |
Organisation | Karolinska Institute |
Country | Sweden |
Sector | Academic/University |
PI Contribution | We have been investigating the development and prevention of neural tube defects in grainyhead-like3 mutant mice. These studies have involved generation of novel transgenic mouse strains. Proteomic analysis has identified a modifier gene, which is a candidate to be involved in human neural tube defects. |
Collaborator Contribution | Our collaborator has contributed to design and analysis of microarray and transgenic investigations of grainyhead-like mutant mice. In addition Dr Gustavsson has collected patient DNA samples for analysis of candidate genes for neural tube defects. |
Impact | We showed that reduced expression of grainyhead-like3 is the cause of neural tube defects in the curly tail mouse model. We identified a polymorphism as a modifier of neural tube defects risk in curly tail mice and began analysis in human patients. Publications: 18683893 and 17720888 |
Start Year | 2006 |
Description | Lamin B1 Cell Biology |
Organisation | University of Oxford |
Department | Sir William Dunn School of Pathology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We identified a variant form of lamin B1 in mice that are predisposed to spina bifida. We carried otu genetic experiments that showed that the lamin B1 variant strongly influences risk of spina bifida. We generated constructs and primary cell lines for analysis by our collaborators. |
Collaborator Contribution | Our collaborators carried out FLIP assay and cell morphometric analysis to test the functional effect of a variant of lamin B1 that we found to affect susceptibility to spina bifida |
Impact | This data forms part of 2 published manuscripts. |
Start Year | 2007 |
Title | Steps towards a clinical trial of inositol |
Description | Ths intervention aims to prevent neural tube defects (NTDs) by maternal supplelemtation with vitamins, inositol and/or B12. NTDs are common birth defects with a high prevalence in many low and middle income countries. NTDs can be lethal at birth (anencephaly) or cause severe disability (spina bifida). Primary NTD prevention is based on peri-conceptional supplementation with folic acid (FA), but considerable numbers of NTDs still occur, indicating frequent FA-resistance. Research in mice, and our pilot clinical trial in high-risk women, both show a potential preventive effect of inositol (vitamin B8) for FA-resistant NTDs. Vitamin B12 supplements could also enhance FA prevention of NTDs, although this is untested. This Trial Development grant will prepare for a future large scale, randomised controlled trial of inositol and B12, as a means of preventing a greater proportion of NTDs than with FA alone. China offers a unique opportunity for the study, given the large size of the reproductive population, high NTD rate, and well organised birth defects surveillance. Study particpantswill be women with a history of NTD-affected pregnancy who wish to become pregnant again. They will be randomised to four intervention arms: (i) inositol, (ii) vitamin B12, (iii) inositol + vitamin B12, (iv) no treatment. All women will receive FA (5 mg/day). We will test key requirements for a subsequent, fully-powered phase III trial in China: effectiveness of the NTD surveillance mechanisms, proportions of potential subjects who are eligible to enter the study, consent to randomisation, comply with the study protocol, and have an informative pregnancy outcome. This study will be a vital step towards providing an evidence base for enhancing pregnancy supplementation worldwide, to further reduce the global burden of NTDs. |
Type | Preventative Intervention - Nutrition and Chemoprevention |
Current Stage Of Development | Late clinical evaluation |
Year Development Stage Completed | 2021 |
Development Status | Under active development/distribution |
Impact | The pilot study in the UK led to uptake of inositol supplments by some women plannng pregnancy. The PONTib-Development study has broadened the research and is aimed to lead to a full-scale clinical trial |
Description | Anomalies of the Fetal CNS, Neural Tube Defects and the PONTI Symposium |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Health professionals |
Results and Impact | Two presentations of 15 minutes duration entitled 'Developmental Biology of the CNS' and 'Prevention of Human NTDs'. The audience comprised healthcare professionals in the field of obstetrics and fetal medicine. Enthusiastic feedback resulted in greater participation in current clinical trial. Improved participation in clinical trial. |
Year(s) Of Engagement Activity | 2008 |
Description | BBC News 24, BBC Online, BBC Regional News |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | I was interviewed for a television piece which focussed on spina bifida, its possible prevention by folic acid and the need for additional therapies as folic acid does not prevent all cases. The interview also introduced our current clinical trial. Increased recruitment into clinical trial. |
Year(s) Of Engagement Activity | 2010 |
Description | BBC Radio - Five Live |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Live radio interview discussing spina bifida, the need for women to take folic acid and the possibility that additional therapies are required. Resulted in increased awareness of clinical trial for prevention of neural tube defects by inositol - additional women entered trial after hearing radio interview. Additional media interest in clinical trial. Contact from potantial trial participants and clnicians |
Year(s) Of Engagement Activity | 2010 |
Description | BBC TV - Breakfast |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Live TV interview discussing spina bifida, the need for women to take folic acid and the possibility that additional therapies are required. Resulted in increased awareness of clinical trial for prevention of neural tube defects by inositol - additional women entered trial after seeing TV interview. Increased public awareness of neural tube defects, the need for folic acid supplementation and the possible benefit of additional therapies, such the as one being tested in the clinical trial run by this resaerch group. |
Year(s) Of Engagement Activity | 2010 |
Description | Charity/Donors visit |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Supporters |
Results and Impact | Visit by fundraisers from Medical Research Charity and from their supporters/donors |
Year(s) Of Engagement Activity | 2017 |
Description | Congenital Anomaly Register meeting |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Health professionals |
Results and Impact | Scientific contact and invited speaker for the Congenital Anomaly Register & Information Service, Wales. Increased awareness of inositol as possible preventive agent for neural tube defects and the PONTI clinical trial. |
Year(s) Of Engagement Activity | 2007 |
Description | Newspaper/online coverage (Inositol Study) |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Media (as a channel to the public) |
Results and Impact | Press release and media interviews resulted in coverage on the BBC online website and in newspapers (eg. Evening Standard) which sparked online discussion ( 3 international news outlets) and further dissemination of findings (eg. via social media). |
Year(s) Of Engagement Activity | 2016 |
URL | http://www.bbc.co.uk/news/uk-scotland-35503529 |
Description | Otto Wolff Lecture. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Health professionals |
Results and Impact | Presentation of a 1 hour lecture entitled 'Inositol a novel preventive therapy for neural tube defects alongside folic acid?'. This is an invited lecture to the combined academic and medical staff of UCL Institute of Child Health and Great Ormond Street Hospital NHS Trust. Improved knowledge of inositol as a possible therapy for NTDs |
Year(s) Of Engagement Activity | 2007 |
Description | PONTI Study Outcomes reported |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Media (as a channel to the public) |
Results and Impact | Publicity surrounding publication of findings from the PONTI study for prevention of neural tube defects. Media outlets included London Evening Standard and BBC online as well as newsletter of Shine. |
Year(s) Of Engagement Activity | 2014,2016 |
URL | https://www.ucl.ac.uk/ich/research/developmental-biology-cancer/developmental-biology-birth-defects/... |
Description | Parliamentary Committee |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Policymakers/politicians |
Results and Impact | Participation in All Party Parliamentary Group discussion on impact of folic acid fortification |
Year(s) Of Engagement Activity | 2018 |