Investigating the effect of folic acid on the neural tube defect methylome

Neural tube defects (NTDs) are common birth defects, which arise when the embryonic precursor of the brain and spinal cord fail to close during embryonic development. NTDs affecting the brain (anencephaly) are lethal before or at birth, whereas lower spinal defects (spina bifida), which comprises around half of all NTDs, lead to life-long neurological handicap. They occur in approximately 1-2 per 1000 pregnancies and total around 170,000 new cases per year worldwide.

One of the most striking things about NTDs is the fact that many cases are preventable by the mother taking periconceptional supplements of folic acid. Despite this, the worldwide prevalence has not dramatically reduced. This is largely because it has not been possible to ensure that adequate levels of folic acid are available to the mother in every case. Voluntary supplementation does not work for at least the estimated 'half of all pregnancies' that are unplanned, while existing food fortification programmes do not appear to increase folate intake to a sufficient level.

In addition to this, the mechanism of action of folate supplementation is simply not well understood. In this study, we plan to use a model system that will allow us to investigate the effect of different amounts of dietary folate. We will use a method to accurately measure DNA methylation, which is a downstream effect thought to be directly affected by available folate levels. We will investigate DNA methylation in conditions of low, normal and high folate levels in a normal mouse strain to see what affect it has. We will then use a mouse model that is known to be sensitive to folate-preventable NTDs.

This investigation will confirm the role of folate levels in DNA methylation and in particular, whether this is an important mechanism in the development of this birth defect. By pinpointing specific DNA targets, we will be able to direct our research towards better NTD prevention in the future.

This project sets out to investigate the effect of embryonic folate status on DNA methylation in wild type and mutant mouse embryos susceptible to NTDs. To do this, we will use the recently developed high throughput, bisulphite-sequencing (BS-Seq) method. We will use both diet and supplementation methods to restrict or increase intake of folic acid. Neurulation stage embryos from wild type or NTD mutant animals will be investigated for locus specific, genome wide effects on DNA methylation (epigenome). Male and female animals will be analysed independently in order to investigate sex specific differences. Candidate regions undergoing treatment specific epigenetic effects will be independently verified and investigated in additional mouse strains with different NTD causing mutations (e.g. Curlytail) or down regulation of the folate cycle (Mthfrnull). Global gene expression changes will be evaluated using RNA-seq and gene specific changes of interest confirmed using RT-PCR and in situ hybridization.

Our experiments have been designed to generate the most reliable data possible by ensuring we use appropriate intra-experimental controls, e.g. wild-type animals versus mutant animals and using an appropriate number of samples in each arm of the experiment, e.g. 2 wild-type DNA samples and 2 mutant DNA samples. Experiments will be performed according to protocols we have already standardized. We have developed the appropriate expertise in our labs at ICH and KCL to be able to effectively carry out each of the techniques described for this project.

The use of animals to achieve the objectives is necessary as there is no alternative way to reproducibly study the developmental time points in known affected and unaffected human samples. The mouse is a validated and widely used model for the study of human embryology. We aim to use as many animals as needed to achieve a robust data output and will consider in vitro alternatives where appropriate.

The key long-term aims of our research are to better understand the causes of NTDs and to develop the most effective therapeutic approaches to prevent NTDs. These are likely to involve strategies that will both complement folic acid and better understand its application to allow the 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 understanding the causes and progress on prevention; (c) healthcare professionals whose aim is to offer counselling for future pregnancy and 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. NTDs are also amongst the most common human birth defects. In the UK more than 1,200 babies are affected by NTDs annually and around 170,000 per year worldwide. 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 to be in excess of $500,000. If failure of closure occurs, the only treatments available are palliative; e.g. 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.

Peri-conceptional folic acid supplementation is one of the few truly effective preventative treatments available. The current proposal is designed to generate a better understanding of the processes of normal and abnormal neural tube development and how folic acid interacts with this. Moreover, our findings may indicate the importance of DNA methylation and expression of particular genes, suggesting a potential role for additional pro-methylation regimes in therapeutic approaches. The ultimate goal will be to reduce the clinical, financial and psychological burden on the individual, family and health service.