The molecular characterization of inborn errors of vitamin Bââ metabolism : cblA, cblB and cblC

This work investigates the molecular basis of three genetic diseases of vitamin B12 metabolism: cblA, cblB and cblC. Two genes responsible for isolated forms of methylmalonic aciduria types cblA and cblB, called MMAA and MMAB respectively, were recently identified. We sequenced the coding sequence and flanking regions of the MMAA and MMAB genes from the gDNA of 37 cblA and 35 cblB patient cell lines and identified 31 novel mutations in total. The biochemical properties of these cell lines were examined in cell culture. Haplotype analysis was used to investigate the history of mutations. The occurrence of both rare and common mutations were identified. Attempts were made to make genotype-phenotype correlations and to understand the effects of mutations on protein function. In the MMAA gene 18 novel mutations were identified, eight of which were common to two or more individuals. One mutation, c.433C>T represented 43% of pathogenic alleles and a common haplotype was identified. Diagnostic tests were designed for every mutation identified. In the MMAB gene, 13 novel mutations were identified. Most mutations were clustered in exon 7. One mutation, c.556C>T accounted for 33% of pathogenic alleles, associated with disease presentation in the first year of life, was observed on a common haplotype and seen almost exclusively in European individuals. We used a combination of linkage, sibling pair, homozygosity mapping and haplotype analyses to refine the disease locus and identify the gene responsible for cblC disease on chromosome 1p called MMACHC. We examined the gDNA of 244 cblC patient cell lines and identified 42 different mutations. The large number of patient samples allowed for the identification of specific genotype phenotype correlations. Of the mutations with elevated frequency in the patients examined, the c.271dupA and c.331C>T mutations were associated with early onset disease whereas c.394C>T was associated with late onset disease. Other missense mutations were also associated with onset of disease later in life. Seven mutations showed clustering by ethnicity. Eight SNPs were identified spanning the gDNA of the MMACHC gene and allowing for the identification of specific haplotypes and the determination of recurrent vs common mutations. Infection of the wild-type MMACHC gene into cblC patient fibroblast cell lines showed correction of the cellular phenotype. Examination of EST databases and northern blot analysis demonstrated MMACHC is ubiquitously expressed in humans with higher levels in fetal liver. Multiple sequence alignment of genomic DNA in eukaryotes and of the polypeptide sequence demonstrated that MMACHC is well conserved in eukaryotes. Two functional domains were identified in the MMACHC gene product by comparison with bacterial genes involved in vitamin B12 related functions: a putative vitamin B 12 binding domain and a TonB-like domain. Molecular modeling demonstrated that the C-terminal region of the gene product folds similarly to TonB from E. coli and suggesting that the C-terminal region of MMACHC may function in a similar manner.

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