Bilateral BBSRC-SFI: Deciphering the function of the human Dihydrofolate reductase 2 gene

Folates are a type of B vitamin that consists of a group of small molecules that are needed in nearly every cell in the body to fulfil a number of essential functions including synthesis of DNA for cell division. Like all vitamins, we need to ensure that we consume a sufficient amount of folate to maintain our health. Inadequate folate is implicated in birth defects, inborn errors of metabolism, neurological problems, autism, fatty liver disease, age-related and cognitive impairment and many cancers. We need to understand the pathway that uses folate, known as folate metabolism, and how it is regulated in different tissues and stages of development before and after birth.

The individual steps of folate metabolism are mediated by a specialised set of proteins, called enzymes, one of the most important being Dihydrofolate Reductase (DHFR). Lack of DHFR function suppresses folate metabolism and prevents cells dividing - this is very harmful in normal tissue but can be exploited as the basis of action of some drugs for the treatment of cancer. DHFR is also the route of entry into folate metabolism of folic acid, which is included in fortified foods and vitamin supplements for prevention of birth defects such as spina bifida. Despite public health messages, many women still become pregnant with inadequate folate status. Further research on folate and human health is essential to inform the public health discussion on the introduction of mandatory fortification policies across Europe.
Notably we found that humans and other primates have acquired a second DHFR gene, DHFR2, during evolution whereas other mammals have only one. Little is known about the function of DHFR2 but we find that the gene is active in many tissues and genetic studies show that alteration of DHFR2 may be linked to increased risk of a group of severe birth defects termed neural tube defects (NTDs), in which the early events of brain and spinal cord development fail. These findings suggest that DHFR2 may play a key role during development.

To gain a better understanding of human folate metabolism it is important to investigate the role of DHFR2 and to ask whether it has similar or distinct functions to DHFR. We will study how DHFR2 protein abundance and location changes as stem cells differentiate and become more specialised cell types such as neurons. We will use genetic tools to remove DHFR2 from cells in culture and investigate the consequences for cellular properties such as proliferation and differentiation, as well as activity of folate metabolism. In parallel, we will ask how the relative levels and location of DHFR and DHFR2 differ in varying conditions. This knowledge will all help to understand what it does.

The next step will be to investigate the role of DHFR2 during development and we will find out where DHFR2 is expressed in human embryos at differing stages. In order to move this work to living embryos we will generate new mouse strains in which the mouse DHFR gene is replaced with human DHFR or DHFR2 or both. These mouse models will allow us to ask detailed questions about the function of each protein. Can human DHFR or DHFR2 substitute for the mouse protein? Is the presence of only DHFR2 sufficient for normal development or do these embryos show changes in growth, neural development and/or folate metabolism? In this way, the study of 'humanised' mice that express only DHFR or DHFR2 will tell us about the individual functions of the enzymes that is difficult to address in human cells that have both.

Having established the functions of DHFR2 in human cells and mouse models, the final part of the project will examine the regulation of the protein in more detail. We will test whether it is present in different structural forms, whether it is modified at particular sites and how its production is regulated. Overall, this project will give new insight into a fundamental metabolic pathway that is crucial for human health.

Folate one-carbon metabolism (FOCM) is a complex interlinked network of reactions that provides one-carbon units for a range of cellular functions including DNA synthesis and methylation reactions. Intergrity of FOCM is therefore essential throughout life and dysfunction is associated with a range of disorders including birth defects, inborn errors of metabolism and cancer. Dihydrofolate reductase (DHFR) mediates reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), which is needed for recycling of folates following DHF production by thymidylate synthase. DHFR is also the only enzyme that provides an entry route for folic acid, the form of folate found in supplements and fortified foods to prevent birth defects, into FOCM. DHFR's essential role in cell proliferation has made it a well-established drug target for the treatment of cancer and rheumatoid arthritis. We found that humans and other primates have a second DHFR enzyme, DHFR2. The amino acid differences between DHFR and DHFR2 cause variation in their enzymatic properties and their differential regulation affects their relative abundance in different cells and tissue types. Our genetic association study showed that common polymorphisms in DHFR2 (and not DHFR) increases risk of the common birth defect, Neural Tube Defects (NTD) and we confirmed that DHFR2 mRNA is expressed in a series of human embryos at Carnegie stages 16-22 and 10 weeks post-conception. We propose that DHFR2 plays a role in FOCM that is distinct to DHFR and that it is specifically required during early embryonic development and during periods of rapid cellular proliferation. This project will elucidate the cellular function of DHFR2 by assessing the interplay between human DHFR and DHFR2 in FOCM during cellular differentiation and early embryonic development in genome edited cell lines and humanised mouse models. Elucidation of DHFR2 function will give new insight into FOCM and its role in human health.

The impact of our research is particularly relevant in terms of knowledge exchange between the research team and various stakeholder and end-user groups, as detailed below.

Academic folate one-carbon metabolism research community. The project will have relevance to researchers focussing on FOCM in a wide variety contexts from biochemistry and cellular biology to human physiology and disease.

Pharmaceutical industry. This project may have medium-term impact on disease treatment by providing data on the efficacy of DHFR-targeted drugs such as Methotrexate, used in treatment of severe psoriasis, rheumatid arthritis and cancers. Clinical trials to date have had no knowledge of the role of DHFR2 and we showed that pre-clinical animal models lack an equivalent gene. The functional characterisation of DHFR2 will inform future drug development.

Public health policy advisors. Dihydrofolate reductase is the only enzyme that can reduce folic acid to its biologically active form so investigation of this human gene family will inform public health policy on mandatory/voluntary fortification of foods with folic acid Europe and globally. There is currently discussions on introducing mandatory folic acid fortification across Europe to drive NTD prevention with definitive health economic arguments to support this. This project can inform this discussion by providing novel data on the only gene family that can metabolise this form of folate. The 2015 WHO guidelines recognise the need for more intense investigation of the folate pathway.

Genetics research community. Human genetics: This project aims to understand the function of a human gene (DHFR2) that we have already found to be associated with neural tube defects and which may have other, as yet unrecognised, roles in disease. An improved understanding of the function of DHFR2 and its potential influence on susceptibility to environmental factors, such as dietary folate status, may in the future pave the way for 'personalised' preventive therapy based on genetic risk factors.

Mouse genetics: Work in the current project, to generate humanised models in which human genes (DHFR, DHFR2) are inserted into the mouse locus, will complement ongoing international efforts by the International Mouse Phenotyping Consortium (IMPC) to generate loss of function mouse models for every gene. The MRC's Mouse Network aims to expedite the entry of mice from research. AC leads the Developmental Anomalies (DevAn) Consortium in the MRC's Mouse Network within IMPC. Considerable progress has been made in making searchable data from mouse models available to the research community and we will seek to link our findings to these efforts.

Human fetal development stakeholders. The Human Developmental Biology Resource (HDBR), funded by the MRC and Wellcome Trust since 1999, is a national and international service co-led by AC (London HDBR Director), that provides difficult-to-access late human embryonic and early fetal tissue for studies of gene and protein expression in relation to development. We previously used this tissue for both mRNA and metabolite studies and will perform mRNA and proteomic studies in the current project. The project focuses on a primate-specific gene whose expression pattern and protein abundance cannot be assessed in rodent models. This project will make use of the human fetal tissue bank and by carrying out the mouse research in parallel we will ensure that our findings closely link to the national human development agenda.

Information and education for the public. The applicants have close links with organisations that have a vested interest in folate metabolism including patients groups relating to spina bifida and hydrocephalus as well as inborn errors of metabolism.

Training and education. Team members are involved in outreach activities to local schools, and in organising visits to the Institute's labs by sixth-form science students each year.