Research in the Molecular Pathogenesis 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 cancer or 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. In the past we found that variants in two genes, TCN2 and TCN2R, appear to affect the levels of vitamin B12 in the blood during pregnancy. In addition, variants in TCN2R are associated the risk of neural tube defects. These findings may be related to birth defects and also may help to explain why some elderly individuals become anemic and suffer neurological symptoms from vitamin B12 deficiency. We also found that mothers carrying a specific variant in a second gene, MTHFD1, have a 50% increased risk bearing a child with a neural tube defect. This previously un-described variant may be responsible for up to 25% of all neural tube defects. Approximately one in five individuals in the population carry one of these risk factors. We recently determined that this particular variant was also an risk factor for placental abruption a common cause of miscarriage and for miscarriages that occur in the second trimester. We have synthesized copies of these genes in the laboratory and are currently using an experimental system to determine exactly how these variants alter the function of these proteins. We have tested more than 64 additional genes for variants that might perturb folate metabolism and therefore be associated with an increased risk of having a child with an neural tube defect. This was carried out by genotyping more than 1,200 single nucleotide polymorphisms in a large number of families affected with neural tube defects and unaffected controls. This large experiment has allowed us to exclude most of the genes on this list. Results for approximately a dozen genes suggest that they are associated with neural tube defects. Over the past year we have carried out second series of experiments to determine if the genes identified in the first stage of these are definitively associated with neural tube defects. These data reveal that the candidate gene approach did not identify the additional gene variants associated with neural tube defects. 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 worlds 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 and 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 3,000. We have obtained external funding to coordinate this study and collect the samples at a central location. Sample collection began toward the end of this year. We have also continued to work on the biology of the genes we have found to be associated with NTDs. One of these is a gene that produces a protein that binds vitamin B12 and transports it from the blood into the tissues. We have published data demonstrating that several variants in this transporter are associated with a risk of having a child with an NTD. While we now know which variants are associated with risk, we do not yet know if they are actually causing the risk or are linked to additional variants that change the function of the protein. To screen for additional variants, we sequenced the DNA containing this transporter gene in a large number of individuals. This sequencing experiment uncovered a number of previously unidentified variants in this gene. We measured the impact of these variants on the function of the transporter. The majority of variants tested do not appear to have an adverse effect of the function of the receptor. However, we have found that several of the variants are associated with changes in vitamin B12 levels in a large sample of healthy individuals. We tested these variants in a large sample of elderly individuals to determine if specific variants are associated with changes in vitamin B12 levels and disease conditions. We have confirmed that these variants strongly influence vitamin B12 levels in plasma.The results of the disease association have yet to be analyzed. We have also carried out experiments aimed at determining the relationship between folate, vitamin B12 and DNA methylation. These experiments are difficult to carry out in humans. We have used zebrafish as a model organism for these studies and have produced a whole genome methylation map of the zebrafish genome covering important developmental stages. We have also characterized the genes in the folate/ vitamin B12 pathway in zebrafish. During this work we were able to assign a predicted function to a number of genes that had not been characterized. We also discovered that the disruption of folate metabolism produces developmental defects in zebrafish. By using a number of methods, we conclude that the defects are due to a lack of cell division not a change in developmental signaling. This work was published in PLOS Developmental Biology. To follow up on this work we have produced a genome wide maps of DNA methylation in the zebrafish genome. Several maps were produced corresponding to important developmental stages and from fish in which we have interfered with normal methylation metabolism. These data shed new light the importance of examining DNA methylation outside of the promoter regions of genes. We also connected these methylation maps with published maps of gene expression. These analyses have allowed us to examine the role of DNA methylation on alternatively spliced mRNAs. A benefit of our work is that a detailed knowledge of the function of the the genes in the folate vitamin B12 metabolic pathways will add to our understanding of neural tube defects and potentially help guide public health policy in the area of nutritional supplementation.

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Dimopoulos, Aggeliki; Sicko, Robert J; Kay, Denise M et al. (2017) Rare copy number variants in a population-based investigation of hypoplastic right heart syndrome. Birth Defects Res 109:8-15
Carter, Tonia C; Sicko, Robert J; Kay, Denise M et al. (2017) Copy-number variants and candidate gene mutations in isolated split hand/foot malformation. J Hum Genet 62:877-884
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