The contribution folate and vitamin B12 genes to disease.

National Human Genome Research Institute

Investigators

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Abstract

Research in the Gene and Environment Interaction Section is focused on defining changes in the genes that underlie inherited susceptibilities to common diseases such as cancer and birth defects. Changes in folate and vitamin B12 metabolism are associated with tumor formation, birth defects and cognitive decline. Folate and vitamin B12 genes are also involved in the methylation of DNA and normal brain function. We are searching for genetic variants in genes related to folate, methionine and homocysteine metabolism. Individuals affected with spina bifida (one form of neural tube defects) will be tested for these variants. Variants found at higher frequency in individuals with disease will help us identify genes associated with risk. We will also apply genetic and genomic technologies to assess the role of genetics in a subset of other rare birth defects. The field of neural tube defects has historically focused on evaluating variation in genes in the folate/vitamin B12 metabolic pathway (e.g., DHFR2, published in 2021). As genotyping technology has increasingly offered more information at lower cost, the next logical step in this research is to screen the entire genome for additional genes associated with NTDs. This type of experiment requires a very large sample size. Although we have one of the world's largest samples of NTDs that are available for genetic research, our sample is too small to carry out a genome wide association study (GWAS). In collaboration with Anne Molloy, Trinity College Dublin, we have organized an international collaboration with the goal of pooling samples for a GWAS. Over ten groups have joined this collaborative effort. The total number of samples collected by all groups exceeds 12,000 and includes nearly 3000 cases. We have obtained external funding to coordinate this study and collect the samples at a central location. We have collected DNA samples from these collaborators, and in the past year we have continued recruitment of additional investigator participation in replication studies. All samples have been submitted to the genotyping center and we have recently received the genome-wide genotyping data for the cohort. We are still awaiting the data release of a collaborator’s control cohort that will be included in analyses. Our plan for analyses includes case-control tests to detect the effects of both case and maternal genotypes on the risk of having an NTD or carrying an NTD pregnancy, respectively. We also plant to perform family-based tests to look for genotypic contribution to risk of an NTD. We plan to divide our participants into a discovery and replication cohort. We additionally have the commitment of six other investigative teams who plan to contribute their existing genotypic and/or sequencing data on their NTD participants (n>2000) to perform a completely independent replication. Other groups have measured the impact of genetic variants on the level of total vitamin B12 in blood. We have measured vitamin B12 in the blood of over 2,000 individuals using standard methods. We then subjected these samples to assays that resolve circulating vitamin B12 into pools that correspond to its two major carrier proteins; transcobalamin, the bioactive carrier that is taken up by all cells, or haptocorrin, which is taken up by the liver for eventual recirculation or elimination. We previously published our work describing our insight that the variant repeatedly reported to influence circulating vitamin B12 is actually influencing the subset of vitamin B12 bound to haptocorrin (Velkova 2017). In a previous study of this cohort (Molloy 2016), we published that the common genetic variant most associated with a circulating marker of vitamin B12 deficiency was in a gene unrelated to vitamin B12 transport or metabolism. These findings could be relevant to clinical measures of vitamin B12 testing where it is known that individuals on either side of the normal range are given false positive and negative results. It may be possible to use individual genetic variation to refine the interpretation of clinical testing. In addition to vitamin B12, we have recently published on genetic influence on other circulating metabolites related to the folate one-carbon metabolic pathway. Such variants may be part of the normal population variation and still, in combination with other genetic and environmental factors, contribute to disease states. We have collaborated to publish on genetic variants influencing formate (Brosnan 2018), glycine (OReilly 2018), folate and homocysteine (Shane 2018), vitamin B6 (Stevelink 2019), and most recently inositol (Weston 2022). Our recent work seeking to determine whether a variant we identified as influencing circulating levels of bioavailable vitamin B12 might have clinical consequence has been recently published (Pangilinan 2021). We have additionally been evaluating which genetic variants most influence mitochondrial metabolites related to the one-carbon metabolic pathway. This has led to a new collaboration to determine whether a gene harboring a variant that influences choline might in fact be an as-yet unidentified transporter. These experiments established for the first time that SLC25A48 was located in the inner mitochondrial membrane as an excellent candidate to the heretofore uncharacterized choline transporter (Bernard 2024). Work by others confirmed that this protein is responsible for choline transport. Additionally, we have recently published work on improved methods for measuring mitochrondrial DNA (mtDNA) heteroplasmy (Walsh 2022), and have reported use of a mouse model to examine alterations in mtDNA heteroplasmy in the context of dietary changes related to one-carbon metabolism (Walsh 2024). We are also using animal models to understand the biology of genes involved in vitamin B12 metabolism. We have developed strains of zebrafish and mice in which we have disrupted vitamin B12 transport genes. In our zebrafish model, we targeted the only known circulatory carrier of vitamin B12, Tcn2. Although these fish should not be able to deliver vitamin B12 to their cells and tissue, they appear to develop normally. This led to a search for an alternate vitamin B12 transport protein in zebrafish. A bioinformatics approach revealed two coding regions that are highly similar to the known carrier protein. We have shown that in an artificial system these partial proteins, encoded by Tcnba and Tcnbb, can be expressed and bind vitamin B12 with affinities comparable to known carrier proteins. These proteins may have a biological role in vitamin B12 transport in these fish. This work has been published (Benoit 2018). Since then, we have additionally characterized the effect of knocking out the Tcn2 gene in zebrafish (Benoit 2021). Without this vitamin B12 transporter, zebrafish exhibit a shorter body length but otherwise appear normal. However, female fish without Tcn2 give rise to offspring with gross developmental and metabolic defects. Since this is observed regardless of the paternal genotype, this seems to be a maternal effect. We speculate that these female fish without Tcn2 may not be able to deposit sufficient vitamin B12 into the yolk for normal development. Despite the lack of maternal care, these animals could serve as a model maternal-fetal nutrition. More recently we have been similarly characterizing knockout lines of Tcnba and Tcnbb to determine whether they share similar phenotypes. We have also been working on combinations of double knockouts as well as a triple knockout to delete all three vitamin B12-binding transport proteins. Our preliminary results indicate the absence of Tcnba has a similar effect as the absence of Tcn2 in terms of reduced length and maternal effects on the offspring. Double knockout lines exhibit more severe phenotypes, supporting a model for these genes having similar vitamin B12-binding functions. We anticipate completion of this work by 2026 and the submission of a manuscript. Our other model of vitamin B12 deficiency is in mice, where we have targeted the cellular receptor for vitamin B12 uptake. These animals appear to mimic a number of aspects of vitamin B12 deficiency in humans, especially when placed on a diet lacking vitamin B12. First, these mice exhibit the metabolic hallmarks of vitamin B12 deficiency observed in humans (elevated circulating homocysteine and methylmalonic acid). They are also prone to developing anemia as they age, which can be temporarily rescued with an injection of vitamin B12. Last, we have been investigating female-specific infertility in these mice. These dams appear to ovulate normally, and we have shown their embryos can develop for a few days but implantation is generally unsuccessful. Maternal injections of vitamin B12 restore the ability of these dams to sustain a pregnancy. This work has been published (Bernard 2018). Future work is needed to determine whether vitamin B12 deficiency in the offspring also contributes to the apparent infertility of their dams. In the past year we have been exploring the impact of vitamin B12 deficiency on neurological function (gene expression in the brain, ability to sense heat, balance, and anxious behaviors) and retinal health. Future work with this animal model may include interrogating the effect of vitamin B12 deficiency on cell division (e.g., melanocyte activity as it relates to hair color). The literature contains a variety of strength of evidence of the impact of vitamin B12 on these aspects of human health, and our mouse model provides a way to more fully interrogate these processes.