Two new mutations in the human E1 beta subunit of branched chain alpha-ketoacid dehydrogenase associated with maple syrup urine disease.

Maple syrup urine disease (MSUD) is an autosomal recessive disorder caused by defective function of the mitochondrial branched chain alpha-ketoacid dehydrogenase (BCKD) complex. Mutations in both alleles of any of three genes for component proteins result in the clinical phenotype. Two discrete mutant alleles for the E1 beta subunit of the decarboxylase component in a proband with MSUD are defined and parental origin of each allele identified. The maternal mutation, an A to T transversion at nucleotide 526 in the coding sequence, potentiates an asparagine to tyrosine change at position 126 (N126Y). The paternal mutant allele contains a C to T transition at nucleotide 970 introducing a stop codon (R274 ). Western blot analysis revealed a 75% reduction in the E1 beta-N126Y protein and an absence of the R274* truncated protein in proband cells. Both mutant proteins could be synthesized, imported into mitochondria, and processed in vitro. Functional analysis of the mutant proteins provided new information on the role of E1 beta in the activity of BCKD. In vivo the E1 beta-N126Y protein associated into macromolecular complexes indistinguishable from those formed with the wild type E1 beta protein. However, catalytic activity of these complexes in proband cells was < 1% of wild type activity. Alignment comparisons with other thiamin pyrophosphate-requiring enzymes suggests the N126Y substitution could interfere with interactions of the protein with the cofactor causing inactivity. The truncated E1 beta-R274* protein is unstable and not found in mitochondria from the patient derived cells.