Understanding the role of the Glycine Cleavage System in Neural Tube Defects

Lead Research Organisation: University College London
Department Name: Institute of Child Health

Abstract

Neural tube defects (NTDs) are common birth defects that arise in early pregnancy caused by incomplete formation of the neural tube, which will later develop into the brain and spinal cord. As a result, the brain and/or spinal cord of the fetus become irreversibly damaged, resulting in death around birth, or long term disability in surviving children. The most common forms of NTDs are anencephaly, affecting the brain, and spina bifida, which affects the lower spinal cord. They occur in approximately 1-2 per 1,000 pregnancies and total at least 170,000 new cases per year worldwide.
The risk of NTDs depends on both inherited factors and environmental influences such as maternal diet, diabetes or exposure to certain chemicals. Because of the many possible contributory factors, the exact causes of NTDs in any affected individual are usually unknown. 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 NTDs are prevented by folic acid - perhaps up to 50% of all cases fail to respond - and so additional therapies are needed. In order to make further progress towards prevention of all NTDs we need a better understanding of their causes, in particular the genes that increase a person's risk of NTD. Moreover, it will be important to identify new preventive therapies for NTDs which may be used individually or in combination with folic acid. In families where genetic risk factors have been identified this also means that family-specific therapies may be offered.
All cells require efficient handling of small molecules called folates, which are related to folic acid, for many different functions. It appears that some NTDs are caused by an inherited abnormality in the way cells in the embryo handle folates. We studied a group of proteins called the "glycine cleavage system" (GCS), that are involved in folate handling. Some patients with NTDs had defects in these proteins, whereas unaffected people did not. This finding suggests that problems with the GCS may directly cause NTDs. In support of this idea, mouse embryos that have GCS defects also develop NTDs.
This project will make use of mouse models lacking function of glycine decarboxylase (Gldc), part of the GCS. The mouse models provide an opportunity to study the role of these folate handling proteins in the embryo and how the associated NTDs may be prevented. We have found that handling of folates is altered in Gldc-deficient embryos and we will now use detailed metabolic studies to work out exactly how these changes come about. We will use genetic approaches to turn off Gldc function or to restore function only in the neural tube. This will tell us which tissues in the developing embryo need Gldc function, to ensure normal development.
The next step is to understand which of the outputs of folate metabolism are disrupted in Gldc-deficient embryos and to test which of the changes are responsible for NTDs. This will be achieved by detailed biochemical analysis and embryo imaging using markers of particular cellular changes, followed by use of supplements to correct these defects. Folate metabolism is essential for synthesis of DNA, which is needed for cells to divide, and methylation reactions that modify gene expression and other functions. Folate metabolism may also be needed for regulation of the level of reactive oxygen species (free radicals) which can be damaging within cells. An imbalance in production and removal of these molecules, termed oxidative stress, is important in a number of diseases, including diabetes. We will examine whether Gldc-deficiency causes oxidative stress in developing embryos, that may contribute to NTDs.
Understanding the molecular and cellular causes of NTDs in Gldc-deficient embryos is an important step towards developing new therapies to prevent more NTDs in humans than is currently possible using folic acid alone.

Technical Summary

This project aims to understand how abnormal folate one-carbon metabolism (FOCM) causes neural tube defects (NTDs). We focus on a mitochondrial-specific component of FOCM, the glycine cleavage system (GCS), which has been implicated in human NTDs. Mice lacking expression of the GCS component glycine decarboxylase (Gldc) exhibit abnormal FOCM, elevated glycine levels and NTDs. Main goals are:
1. To understand the metabolic basis of NTDs in Gldc-deficient embryos, we will adopt complementary approaches: (i) metabolic labelling to track one-carbon units; (ii) mass spectrometry-based quantification of folate profiles in differing subcellular compartments and cell types; (iii) gene expression/pathway analysis.
2. Genetic approaches, involving conditional knockout and rescue of Gldc expression, will be used to determine whether there is tissue heterogeneity in the requirement for GCS activity in FOCM and to test whether Gldc function in the neuroepithelium is necessary and sufficient for neural tube closure.
3. Outputs of FOCM include provision of one-carbon units for nucleotide biosynthesis and/or methylation reactions. We will determine which of these outputs are compromised in Gldc-deficient embryos and whether they mechanistically contribute to NTDs. Downstream effects on DNA copy number/integrity and DNA/protein methylation will be evaluated. The relative importance of diminished methylation and nucleotide biosynthesis in Gldc-mediated NTDs will be tested by supplementation strategies and genetic approaches that channel one-carbon units towards either output.
4. Mitochondrial FOCM contributes to production of NADPH, required for redox balance. We will ask whether Gldc-deficiency results in redox imbalance and oxidative stress using mass spectrometry based read-outs of reactive oxygen species, as well as imaging and reporter assays in embryos. The functional contribution of oxidative stress to NTDs will be assessed using cellular markers and rescue experiments.

Planned Impact

A key long-term aim of this research will be to develop therapeutic approaches for NTDs that will complement folic acid to allow prevention of more NTDs than is currently possible. The major beneficiaries will therefore be (a) children who would otherwise have been born with a birth defect, (b) families who have encountered NTDs and are motivated towards progress on prevention; (c) healthcare professionals whose aim is to improve health status of mother and fetus during pregnancy; (d) the pharmaceutical industry, whose nutritional supplement sector has a growing interest in prevention of common birth defects such as NTDs.

Neural tube defects are clinically devastating to affected individuals: anencephaly is lethal at birth and spina bifida may result in lifelong disabilities. There are major implications for health care provision - the estimated cost of life time medical care for a spina bifida patient in the USA is estimated at in excess of $500,000. If failure of closure occurs, the only treatments available are palliative; eg. in utero surgery may prevent further degeneration but does not result in recovery of damaged tissue. The optimum approach is primary prevention as exemplified by population-wide use of folic acid supplements when planning pregnancy.

At the present time the recurrence risk for NTDs is more than 10-fold higher than for a first affected pregnancy. Prospective mothers are advised to take folic acid and, while this has proven a very successful strategy, risk is only reduced by up to 70%. In fact, following fortification of the food supply with folic acid in the USA, only a 26% reduction in NTD prevalence has been detected. The applicants previously identified inositol as a preventive therapy for genetically-determined mouse NTDs that are unresponsive to folic acid. This finding has been taken forward to clinical testing and a pilot randomised, double blind trial, to test the efficacy of inositol for prevention of NTDs , has been completed. The experience of the applicants in taking a therapy from the laboratory to the clinic will facilitate the exploitation of findings from the current project towards clinical impact. This could be achieved on a timescale of 5-10 years.

In the longer term, genetic analysis could be offered to allow identification of parental risk factors, which would then indicate the most effective preventive therapy. This strategy would also address the possibility that therapies which are effective in some individuals may be inactive or even harmful in others. Such a 'pharmacogenomics' type of model is currently unrealistic for NTDs. However, in an era when personalised exome sequencing will become a reality, it is time to begin development of therapies that may be appropriate for a subset of NTDs with a particular underlying pathology.

Identification of genetic risk factors is the aim of large-scale studies in the NTD research community. The next step will involve development of preventive strategies based on measurable metabolic and/or cellular defects in NTD models/patients. In the current project this approach will be used to address NTDs associated with a specific group of genes (encoding the glycine cleavage system) that appear to play a key role in neural tube development in both mice and humans. This offers the possibility of: (a) refining the likely range of influence of existing treatments, particularly folic acid (i.e. which NTDs are likely to respond to folic acid and which are not?), and (b) identifying promising novel therapies which may be more effective than folic acid in NTDs with particular patterns of molecular pathology.

Publications

10 25 50

 
Description MGDW
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
Impact Organiser of the Mammalian Genetics and Development Workshop. This is a meeting of the Genetics Society held annually at UCL Institute of Child Health with 60-70 attendees from throughout the UK. In particular, this meeting is focused on training and education of PhD students and Post-Doctoral researchers.
 
Description Innovator Award
Amount £365,837 (GBP)
Funding ID 210774 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2018 
End 09/2020
 
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 Leadership Award
Amount £106,994 (GBP)
Funding ID V2117 
Organisation Great Ormond Street Hospital Children's Charity (GOSHCC) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2016 
End 09/2019
 
Description NIHR BRC Project Grant
Amount £50,000 (GBP)
Organisation Great Ormond Street Hospital (GOSH) 
Department NIHR Great Ormond Street Biomedical Research Centre
Sector Public
Country United Kingdom
Start 04/2017 
End 03/2018
 
Description Newlife Start-Up Grant
Amount £13,259 (GBP)
Funding ID SG/15-16/04 
Organisation Newlife 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2016 
End 04/2017
 
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 Sea and Currents Fund
Amount £650 (GBP)
Organisation University College London 
Sector Academic/University
Country United Kingdom
Start 08/2016 
End 08/2016
 
Title Gldc conditional targeted mice 
Description Two lines of Gldc mutant mice have been generated. In order to examine the development of related diseases in humans (Non-Ketotic Hyperglycinemia, neural tube defects, hydrocephalus), conditional knockout and conditional rescue lines have been generated using cre-lox technology 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2016 
Provided To Others? No  
Impact Conditional rescue demonstrated proof of principle for genetic rescue of NKH - leading to initiation of gene therapy projects 
 
Title Gldc mutant mice 
Description We generated novel mouse lines carrying loss of function alleles of Gldc. These mice provide a model for neural tube defects (NTDs) and Non-Ketotic Hyerglycinemia (NKH). 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2015 
Provided To Others? Yes  
Impact The Gldc mouse lines provide important models of human birth defects and childhood disease: NTDs - these mice carry the same mutation as some NTD patients. The identification of preventive approaches in this model highlighted possible new therapies for human NTDs. NKH - these mice represent a unique mouse model for NKH. Hence, new projects investigating the pathogenesis and possible therapy for this disease have been initiated. 
URL http://www.nature.com/ncomms/2015/150304/ncomms7388/full/ncomms7388.html
 
Title Gldc RNA-Seq 
Description RNA-seq data on brain tissue affected by GLDC mutation - not yet published 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? No  
Impact Stimulated new lines of research 
 
Description Amino acid analysis in Non-Ketotic Hyperglycinemia and defects of the Glycine Cleavage System 
Organisation Technion - Israel Institute of Technology
Country Israel 
Sector Academic/University 
PI Contribution Our research team has developed a mouse model for Non-Ketotic Hyperglycinemia, a rare autosomal recessive neurometabolic disease. NKH patients develop intractable epilepsy and this model provides a system to better understand the pathogenic mechanisms and to test therapy.
Collaborator Contribution Analysis of D-serine levels in genetic mutant tissue.
Impact Still in progress
Start Year 2016
 
Description Causes of NTDs in China 
Organisation Peking University
Department Peking University Academic Health Science Centre
Country China 
Sector Academic/University 
PI Contribution We have engaged in collaborative laboratory research including reciprocal research visits. We have participated in epidemiological studies especially contributing to study design and data analysis.
Collaborator Contribution Our partners have carried our laboratory research on environmental factors that may increase risk of birth defects.Our partners have carried out epidemiological studies using birth defect surveillance data in North China.
Impact We have co-authored three publications. We have developed plans for a large-scale clinical trial for which a funding application has been submitted. We have initiated a study on genetics of NTDs.
Start Year 2017
 
Description Dictyostelium - Cannabidiol 
Organisation Royal Holloway, University of London
Country United Kingdom 
Sector Academic/University 
PI Contribution Our team used mass spectrometry approaches to analyse folate metabolism in the model system for the analysis of cannabidiol mechanism of action. The study involved analysis of gene knockouts of the glycine cleavage system.
Collaborator Contribution Our collaborators carried out a large-scale genetic screen to identify proteins that participate in cannabidiol action.
Impact Manuscriput submitted for publication.
Start Year 2018
 
Description Folate mass spectrometry 
Organisation McGill University
Department Montreal Children's Hospital Research Institute
Country Canada 
Sector Academic/University 
PI Contribution As part of this project we developed novel mass spectrometry methodology for analysis of multiple folates. This method can be used to analyse folate metabolism in a wide range of experimental systems.
Collaborator Contribution UCL Ageing - developed a model of delayed aging in C.elegans, exploring the mechanism by which metformin increases lifespan. Continued projects investigate the role of the microbiome in drug metabolism. McGill University - developed a novel mouse model targeting MTHFD1s to model a variant associated with human disease. We analysed this model. Ottawa - developed models for reduced function of BHMT in mouse blastocysts Prague - developed a model of hypertension in the rat.
Impact Publications: Cabreiro et al., 2013 PMID: 23540700 Leung et al, 2013 PMID: 23483428 Christensen et al., 2013 PMID: 23704330 Zhang et al. FASEB Journal. 2015 Pravenec et al. PMID: 30036071
Start Year 2012
 
Description Folate mass spectrometry 
Organisation Ottawa Hospital Research Institute
Country Canada 
Sector Academic/University 
PI Contribution As part of this project we developed novel mass spectrometry methodology for analysis of multiple folates. This method can be used to analyse folate metabolism in a wide range of experimental systems.
Collaborator Contribution UCL Ageing - developed a model of delayed aging in C.elegans, exploring the mechanism by which metformin increases lifespan. Continued projects investigate the role of the microbiome in drug metabolism. McGill University - developed a novel mouse model targeting MTHFD1s to model a variant associated with human disease. We analysed this model. Ottawa - developed models for reduced function of BHMT in mouse blastocysts Prague - developed a model of hypertension in the rat.
Impact Publications: Cabreiro et al., 2013 PMID: 23540700 Leung et al, 2013 PMID: 23483428 Christensen et al., 2013 PMID: 23704330 Zhang et al. FASEB Journal. 2015 Pravenec et al. PMID: 30036071
Start Year 2012
 
Description Folate mass spectrometry 
Organisation University College London
Department Institute of Healthy Ageing
Country United Kingdom 
Sector Academic/University 
PI Contribution As part of this project we developed novel mass spectrometry methodology for analysis of multiple folates. This method can be used to analyse folate metabolism in a wide range of experimental systems.
Collaborator Contribution UCL Ageing - developed a model of delayed aging in C.elegans, exploring the mechanism by which metformin increases lifespan. Continued projects investigate the role of the microbiome in drug metabolism. McGill University - developed a novel mouse model targeting MTHFD1s to model a variant associated with human disease. We analysed this model. Ottawa - developed models for reduced function of BHMT in mouse blastocysts Prague - developed a model of hypertension in the rat.
Impact Publications: Cabreiro et al., 2013 PMID: 23540700 Leung et al, 2013 PMID: 23483428 Christensen et al., 2013 PMID: 23704330 Zhang et al. FASEB Journal. 2015 Pravenec et al. PMID: 30036071
Start Year 2012
 
Description Formate assays 
Organisation Memorial University of Newfoundland
Country Canada 
Sector Academic/University 
PI Contribution Our team has developed mouse models with genetic defects in folate metabolism and/or diets with altered folate content. These provide experimental models for examining gene-environment interactions in the causation of neural tube defects.
Collaborator Contribution The team at university of Newfoundland have developed methodology for assay of formate which is applied to these models.
Impact Manuscripts published: PMID: 26924399 Sudiwala et al. PMID: 25736695 Pai et al.
Start Year 2013
 
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 Metabolic tracing 
Organisation Cornell University
Department Weill Cornell Medicine
Country United States 
Sector Academic/University 
PI Contribution We generated metabolically labelled tissue for analysis - focussing disease of the glycine cleavage system
Collaborator Contribution Collaborators performed unbiased metabolic tracing
Impact Publication in Cell Reports
Start Year 2016
 
Description Ultrasound scanning of NTDs and hydrocephalus 
Organisation University College London
Department Institute for Women's Health
Country United Kingdom 
Sector Academic/University 
PI Contribution We have a mouse model for Gldc deficiency in which embryos develop neural tube defects or hydrocephalus.
Collaborator Contribution In order to monitor developmental progression of defects we have made use of high resolution ultrasound scanning. This enables analysis of disease progression and monitoring of treatment efficacy.
Impact Presentation at conferences. Manuscript published: PMID: 28056489
Start Year 2014
 
Description Ultrasound scanning of NTDs and hydrocephalus 
Organisation University of Milan
Country Italy 
Sector Academic/University 
PI Contribution We have a mouse model for Gldc deficiency in which embryos develop neural tube defects or hydrocephalus.
Collaborator Contribution In order to monitor developmental progression of defects we have made use of high resolution ultrasound scanning. This enables analysis of disease progression and monitoring of treatment efficacy.
Impact Presentation at conferences. Manuscript published: PMID: 28056489
Start Year 2014
 
Description NKH Family Conference 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Patients, carers and/or patient groups
Results and Impact The NKH Family Conference brought together patient families, carers, health professionals and researchers to raise awareness and discuss current research and treatment of Non-Ketotic Hyperglycinemia
Year(s) Of Engagement Activity 2018
 
Description The NKH Family Conference 2017 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Patients, carers and/or patient groups
Results and Impact We hosted the Family Conference for Non-Ketotic Hyperglycinemia - attended by families and patients, clinicians and researchers. The conference stimulated discussion between families and researchers, provided a two-way learning experience and led to initiation of new research activity led by patient priorities.
Year(s) Of Engagement Activity 2017
URL http://www.nkhconference.com/
 
Description Twitter 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Twitter feed. Sharing f news from the lab and comment on relevant issues
Year(s) Of Engagement Activity 2013,2014,2015,2016,2017
URL https://twitter.com/NTDs_research