Folate metabolism and development of Neural Tube Defects

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

Abstract

During early pregnancy, a crucial event in the developing embryo is the formation of a structure called 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 long term disability in surviving babies. Spina bifida is the best known type of NTD. 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, so additional therapies are needed. In order to make further progress towards prevention of all NTDs we need a better understanding of their causes. At the present time the genetic cause of an NTD cannot be explained in most patients, but considerable progress is being made towards unravelling the complexity of these diseases. Our long-term goal is to identify preventive therapies for NTDs which may be used individually or in combination. In families where genetic risk factors have been identified this also means that family-specific therapies may be offered.

In all cells, efficient handling of small molecules called folates, which are related to folic acid, is needed for a number of different functions including making DNA for cells to divide. An important question is whether some NTDs are caused by an inborn abnormality in the way that the cells in the developing baby handle folate. We recently studied a group of proteins that are involved in folate handling and found that some patients with NTDs have defects in these proteins, whereas unaffected people did not. This finding suggests that problems with these problems may directly cause NTDs. In support of this idea, mouse embryos that have defects in the same group of proteins also develop NTDs. These mouse models provide an opportunity to study what job these folate handling proteins are doing in the embryo and how the associated NTDs may be prevented.

Technical Summary

Neural tube defects (NTDs), such as spina bifida and anencephaly, are severe congenital abnormalities caused by failure of closure of the embryonic neural tube. The causes of human NTDs are still largely unknown, but appear to involve multiple genetic and environmental factors. Folic acid supplementation can prevent some NTDs but a significant proportion (at least 30%) are unresponsive, and these defects remain a major health issue in the UK and worldwide. Abnormalities in folate one-carbon metabolism (FOCM) have been implicated in the possible causation of NTDs but the genetic basis of this relationship remains elusive. Key outstanding questions include which components of FOCM may be involved in NTDs, the mechanism by which impaired FOCM influences the morphological process of neural tube closure and whether FOCM-related NTDs can be prevented by folic acid and/or other exogenous agents. Emerging evidence suggests that neural tube closure depends on activity of the glycine cleavage system (GCS), a component of mitochondrial FOCM. We have identified functional mutations in GLDC and AMT in patients with NTDs, while loss-of-function of the corresponding genes has generated NTDs in novel mouse models. In the current project we will build on these preliminary findings by, (1) determining the developmental and cellular basis of NTDs in GCS mutant embryos. Next, (2) the metabolic consequence of impaired GCS activity in mouse embryos and patient cell lines will be analysed by mass spectrometry-based quantification of FOCM components, measurement of flux through FOCM and assay of FOCM outputs. Finally, (3) we will test potential therapies for prevention of GCS-related NTDs. We will follow up pilot data suggesting an ameliorating effect of methionine and additional approaches will be informed by data from metabolic assays and in silico modelling. Therapies will be optimised by an iterative process of testing and evaluation of metabolic and developmental outcome

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 randomised, double blind trial is underway to test the efficacy of inositol for prevention of NTDs. 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. Two elements will be required:

(i) Identification of genetic risk factors for NTDs. Although complicated by the complex, multifactorial nature of NTDs, progress is being made towards understanding which genes may be involved. A large international consortium has been established in the NTD research community in order to make progress towards large-scale genome wide association and copy number variant studies of NTDs. The applicants have committed to contribute DNA samples from NTD patients to this consortium.

(ii) 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 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 pathog

Publications

10 25 50
 
Description Action Medical Research Project
Amount £177,915 (GBP)
Funding ID GN2403 
Organisation Action Medical Research 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2016 
End 04/2018
 
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 MRC Project Grant
Amount £858,719 (GBP)
Funding ID MR/N003713/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 03/2016 
End 02/2020
 
Description Newlife Project Grant
Amount £119,841 (GBP)
Organisation Newlife 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2014 
End 06/2016
 
Description Royal Society International Exchange Scheme
Amount £11,500 (GBP)
Funding ID 000021750 
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 03/2015 
End 02/2017
 
Description UCL/MRC Confidence in Concept
Amount £83,470 (GBP)
Organisation University College London 
Sector Academic/University
Country United Kingdom
Start 10/2015 
End 09/2016
 
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 LC-MS/MS method - folates 
Description This method allows simultaneous quantification of mutiple folate species (6 major and their polyglutamated forms). Folate metabolism is crucial to cellular function and implicated in several diseases. 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact This methodology has been applied to models of human genetic disease, aging, diabetes and birth defects. 
 
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-environment interactions in NTDs 
Organisation Peking University
Country China 
Sector Academic/University 
PI Contribution The aim is to develop a collaborative project focusing on neural tube defects (NTDs), a group of common birth defects, that includes spina bifida and anencephaly. Understanding the causes of NTDs is hindered by their complex, multifactorial etiology which involves both genetic and environmental factors and in most individuals the causes are not well understood. The UK team contributes expertise in experimental analysis of the mechanisms, both genetic and environmental, underlying NTDs.
Collaborator Contribution The China Team provides expertise in the epidemiology and population surveillance of NTDs in a high-risk area. Together we aim to establish projects (i) for analysis of genetic risk variants in this population and (ii) for experimental analysis of environmental factors identified through population.
Impact No outputs yet. the project is multi-disciplinary involving molecular biology/genetics, developmental biology, epidemiology and clinical trials.
Start Year 2015
 
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 University of Malaya - NTDs 
Organisation University of Malaya
Country Malaysia 
Sector Academic/University 
PI Contribution Providing expertise and advice on experimental design, technology and data interpretation.
Collaborator Contribution Provision of funding for research project in collaborator laboratory at University of Malaya.
Impact Collaborative projects with funding from HIR (University of Malaya) Visit as High Impact Research Icon to University of Malaya (2015).
Start Year 2013
 
Description Newspaper Coverage (Nucleotides paper) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Press release describing findings from publication PMID: 23935126.
Interview with The Times

Press coverage in:
Evening Standard (9/8/2013), The Times, Daily Telegraph, Daily Mail, Mirror, Yorkshire Post, Scottish Herald and Scotsman (10/82013)
Year(s) Of Engagement Activity 2013