Searching for vitamin B12 genes

Abstract

Vitamin B₁₂ (cobalamin, Cbl) is an essential vitamin in humans. Genetic disorders linked to Cbl metabolism have been defined through complementation analysis, and have been assigned to metabolic steps through biochemical analysis. Eight complementation groups have been identified, cblA through cblH. These genetic defects produce functional deficiency in one or both of methionine synthase (MS) or methylmalonyl CoA mutase (MCM), depending on the nature of the block. My quest was to identify missing pieces of the vitamin B12 pathway. Upon examining prokaryotic operon structures using NCBI's "Cluster of Orthologous Genes" (COG) database, I observed a gene flanking MCM in several genomes and another gene in one genome with similarity to human genes. These studies led to the identification of two genes, MMAA and MMAB, responsible for the cblA and cblB complementation groups, respectively. Biochemical examination of the MMAA protein was conducted on Y gfD (the E. coli ortholog). Y gfD was purified, the GTPase activity examined and the interaction with MCM studied. Celis defined the role of YgfD as a lysine-omithine-arginine (LOA) transporter, while Korotkova demonstrated its role in preventing the inactivation of MCM. While the case for LOA transporter role is severely flawed, the role as a protein preventing inactivation of MCM is in conflict with the information known. My studies suggest that YgfD (and the orthologous MMAA protein) may play a role in a larger complex thereby affecting both MCM activity and AdoCbl levels. Three patients were examined for mutations in methylmalonyl CoA epimerase (MCEE). Unlike most cblA patients, these three lacked mutations in the MMAA gene, had high AdoCbl levels and were not responsive to vitamin B₁₂ treatment. One patient, WG2278, is homozygous to R49X in MCEE. The family was also found to be homozygous for R49X. A knockdown of the epimerase expression, using siRNA, revealed that 14C­propionate uptake was affected. A region of chromosome lp34 was identified by Atkinson to be critical to cblC patients. Sixteen candidate genes belonging to that region were tested for mutation in cblC cell lines. Several single nucleotide polymorphisms were identified in these genes, including, a two base pair (AT) deletion. No gene was identified as the gene responsible for the cblC disorder. To identify and test additional genes in vitamin B₁₂ metabolism, I developed a method of "genome subtraction" in which all the genes found in microbial vitamin B₁₂ users are subtracted from non-users leaving only vitamin B₁₂ genes. High, medium and low stringency tests were performed using empirically defined rules. Three genes were identified that were not part of the selection criteria that are involved in vitamin B₁₂ -dependent pathways and represent a validation of the enrichment method. Four novel candidate genes were screened by gene silencing technology to determine if any affected the vitamin B₁₂ pathway. One candidate gene was found to have reduce the metabolism of ¹⁴C-propionate, a test routinely used to evaluate vitamin B₁₂ cofactor function in human cells. To maximize the number of candidates, the genome subtraction queries were combined with an in-depth operon, i.e. custom-designed COG, search. The results of the combined queries yielded 18 candidate genes, six of which are good candidates. It is expected that gene silencing and mutation detection can be performed to assess the remaining candidate genes. Overall, genome subtraction, operon analysis and siRNA testing should be effective tools for the identification of novel vitamin B₁₂ genes. This approach should be generalizable to other ancient pathways that that have been retained during evolution. These methods are also useful even when the protein has a misidentified function.