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.

Publications

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

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Gustavsson P (2008) Grainyhead genes and mammalian neural tube closure. in Birth defects research. Part A, Clinical and molecular teratology

 
Description Institution Strategic Support Fund
Amount £48,000 (GBP)
Funding ID ISSF3 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2017 
End 08/2019
 
Description Programme grant
Amount £1,366,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2010 
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 04/2018 
End 10/2020
 
Description SPARKS PhD studentship
Amount £89,968 (GBP)
Funding ID 17ICH03 
Organisation Sparks 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2017 
End 09/2020
 
Description Sparks Project Grant
Amount £67,000 (GBP)
Organisation Sparks 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2011 
End 08/2013
 
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
 
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