MOLECULAR-BASIS OF INTERMITTENT MAPLE-SYRUP-URINE-DISEASE - NOVEL MUTATIONS IN THE E2 GENE OF THE BRANCHED-CHAIN ALPHA-KETO ACID DEHYDROGENASE COMPLEX

The E2 gene of the branched-chain alpha-keto acid dehydrogenase (BCKDH ) complex was studied at the molecular level in three patients with in termittent maple syrup urine disease (MSUD). All three patients had hi gher BCKDH activity than did those with the classical phenotype. In th e first patient, a single base substitution from A to G in intron 8 cr eated a new 5' splice site and caused an insertion of 126 nucleotides between exons 8 and 9 by activating an upstream cryptic 3' splice site in the same intron. The predicted mRNA encoded a truncated protein wi th 282 amino acids including 4 novel ones at the carboxyl terminus, co mpared with the normal protein with 421 amino acids. In vitro, the reg ion from the patient but not from a normal control was recognized and was recovered as a novel exon, indicating that the single substitution was responsible for incorporation of the region into mRNA. This mutat ion probably supports an exon definition model in which the spliceosom e recognizes a 3' splice site and then scans downstream for an accepta ble 5' splice site, thereby defining an exon. The second patient was h omozygous for a G to T transversion at nucleotide 1463 in exon 11, whi ch predicted a substitution of the termination codon by a leucine resi due and the addition of 7 extra amino acids at the carboxyl terminus. For each mutation, these two patients were homozygous and their parent s were heterozygous. The third patient was a compound heterozygote for a C to G transversion at nucleotide 309 in exon 4 and a G to A transi tion at nucleotide 1165 in exon 9, causing an Ile-to-Met substitution at amino acid 37 and a Gly-to-Ser substitution at amino acid 323, resp ectively. Taken together, these results indicate that the molecular ba sis of intermittent phenotype MSUD in some patients can be due to muta tions in the E2 gene, giving rise to a low but significant residual ac tivity of the BCKDH complex.