DEFICIENCY OF THE E1-BETA SUBUNIT IN THE BRANCHED-CHAIN ALPHA-KETO ACID DEHYDROGENASE COMPLEX DUE TO A SINGLE-BASE SUBSTITUTION OF THE INTRON-5, RESULTING IN 2 ALTERNATIVELY SPLICED MESSENGER-RNAS IN A PATIENTWITH MAPLE-SYRUP-URINE-DISEASE

A patient with maple syrup urine disease (MSUD) associated with a E1 b eta subunit deficiency of the branched-chain cu-keto acid dehydrogenas e (BCKDH) complex was investigated at the molecular level. The defect responsible for the deficiency of the E1 beta subunit protein was iden tified by analysis of cDNA and genomic DNA by polymerase chain reactio n. Total RNA isolated from lymphoblastoid cells was transcribed into c DNA and amplified using a set of primers located within exon 3 and exo n 9 of the E1 beta gene. Agarose gel electrophoresis of cDNA amplifica tion products revealed two shortened bands as well as a faint band of normal size. Nucleotide sequencing of the shortened cDNA amplification products showed that sequences corresponding to exon 5 and both exons 5 and 6 were absent. Nucleotide sequencing of the proband's amplified genomic DNA corresponding to this region of the E1 beta gene revealed a single base substitution from G to T of the invariant GT dinucleoti des at 5' splice site of the intron 5. Analysis of family members usin g primer-specified restriction map modification showed that the patien t is homozygous for this mutation. We postulate that this mutation lea ds to the skipping of either exon 5 or both exons 5 and 6, thus produc ing two shortened E1 beta mRNA. The percentage of normal and two short ened transcripts was estimated to be 9, 71 and 20%, respectively. To o ur best knowledge, this is the first documented example of exon skippi ng in the E1 beta gene as the cause of MSUD and the novel mutation of the invariant G at the 5' splice site which results in two alternative ly spliced mRNA due to the skipping of the preceding exon as well as b oth preceding and following exon.