Maple syrup urine disease in mennonites: Evidence that the Y393N mutation in E1α impedes assembly of the E1 component of branched-chain α-keto acid dehydrogenase complex

Maple Syrup Urine Disease (MSUD) in Mennonites is associated with homozygosity for a T to A transversion in the E1α gene of the branched-chain α-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 E1α-deficient MSUD lymphoblasts (Lo) as a host. The level of E1β 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 E1α subunit precursors. Transfection with normal E1α cDNA into Lo cells restored decarboxylation activity of intact cells. Western blotting showed that both E1α and E1β subunits were markedly increased. Introduction of Y393N mutant E1αa cDNA failed to produce any measurable decarboxylation activity. Mutant E1α subunit was expressed at a normal level, however, the E1β subunit was undetectable. These results provide the first evidence that Y393N mutation is the cause of MSUD. Moreover, this mutation impedes the assembly of E1α with E1β into a stable α₂β₂ structure, resulting in the degradation of the free E1β subunit.