Gene identification for the cblD defect of vitamin B12 metabolism.

BACKGROUND Vitamin B12 (cobalamin) is an essential cofactor in several metabolic pathways. Intracellular conversion of cobalamin to its two coenzymes, adenosylcobalamin in mitochondria and methylcobalamin in the cytoplasm, is necessary for the homeostasis of methylmalonic acid and homocysteine. Nine defects of intracellular cobalamin metabolism have been defined by means of somatic complementation analysis. One of these defects, the cblD defect, can cause isolated methylmalonic aciduria, isolated homocystinuria, or both. Affected persons present with multisystem clinical abnormalities, including developmental, hematologic, neurologic, and metabolic findings. The gene responsible for the cblD defect has not been identified. METHODS We studied seven patients with the cblD defect, and skin fibroblasts from each were investigated in cell culture. Microcell-mediated chromosome transfer and refined genetic mapping were used to localize the responsible gene. This gene was transfected into cblD fibroblasts to test for the rescue of adenosylcobalamin and methylcobalamin synthesis. RESULTS The cblD gene was localized to human chromosome 2q23.2, and a candidate gene, designated MMADHC (methylmalonic aciduria, cblD type, and homocystinuria), was identified in this region. Transfection of wild-type MMADHC rescued the cellular phenotype, and the functional importance of mutant alleles was shown by means of transfection with mutant constructs. The predicted MMADHC protein has sequence homology with a bacterial ATP-binding cassette transporter and contains a putative cobalamin binding motif and a putative mitochondrial targeting sequence. CONCLUSIONS Mutations in a gene we designated MMADHC are responsible for the cblD defect in vitamin B12 metabolism. Various mutations are associated with each of the three biochemical phenotypes of the disorder.