Folate metabolism and development of Neural Tube Defects

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.

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

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