MAPLE SYRUP URINE DISEASE IN MENNONITES - EVIDENCE THAT THE Y393N MUTATION IN E1-ALPHA IMPEDES ASSEMBLY OF THE E1-COMPONENT OF BRANCHED-CHAIN ALPHA-KETO ACID DEHYDROGENASE COMPLEX

Maple Syrup Urine Disease (MSUD) in Mennonites is associated with homozygosity for a T to A transversion in the E1-alpha-gene of the branched-chain alpha-keto acid dehydrogenase complex. This causes a tyrosine to asparagine substitution at position 393 (Y393N). To assess the functional significance of this missense mutation, we have carried out transfection studies using E1-alpha-deficient MSUD lymphoblasts (Lo) as a host. The level of E1-beta-subunit is also greatly reduced in Lo cells. Efficient episomal expression in lymphoblasts was achieved using the EBO vector. The inserts employed were chimeric bovine-human cDNAs which encode mitochondrial import competent E1-alpha-subunit precursors. Transfection with normal E1-alpha cDNA into Lo cells restored decarboxylation activity of intact cells. Western blotting showed that both E1-alpha and E1-beta-subunits were markedly increased. Introduction of Y393N mutant E1-alpha cDNA failed to produce any measurable decarboxylation activity. Mutant E1-alpha-subunit was expressed at a normal level, however, the E1-beta-subunit was undetectable. These results provide the first evidence that Y393N mutation is the cause of MSUD. Moreover, this mutation impedes the assembly of E1-alpha with E1-beta into a stable alpha-2-beta-2 structure, resulting in the degradation of the free E1-beta-subunit.