Molecular Phenotypes in Cultured Maple Syrup Urine Disease Cells: Complete E1 α cDNA sequence and mRNA and subunit contents of the human branched chain α-keto acid dehydrogenase complex

Abstract

The activity of the branched-chain α-keto acid dehydrogenase complex is deficient in patients with the inherited maple syrup urine disease (MSUD). To elucidate the molecular basis of this metabolic disorder, we have isolated three overlapping cDNA clones encoding the E1 α subunit of the human enzyme complex. The composite human E1 α cDNA consists of 1783 base pairs encoding the entire human E1 α subunit of 400 amino acids with calculated Mr = 45,552. The human E1 α and the previously isolated human E2 cDNAs were used as probes in Northern blot analysis with cultured fibroblasts and lymphoblasts from seven unrelated MSUD patients. The results along with those of Western blotting have revealed five distinct molecular phenotypes according to mRNA and protein-subunit contents. These consist of type I, where the levels of E1 α mRNA and E1 α and E1 β subunits are normal in cells, but E1 activity is deficient; Type II, where the E1 α mRNA is present in normal quantity, whereas the contents of E1 α and E1 β subunits are reduced; Type III, where the level of E1 α mRNA is markedly reduced with a concomitant loss of E1 α and E1 β subunits; Type IV, where the contents of both E2 mRNA and E2 subunits are markedly reduced; and Type V, where the E2 mRNA is normally expressed, but the E2 subunit is markedly reduced or completely absent. Type V includes thiamin-responsive (WG-34) and certain classical MSUD cells. These molecular phenotypes have demonstrated the complexity of MSUD and identified the affected gene in different patients for further characterization.