Deficiency of the E1β subunit in the branched-chain α-keto acid dehydrogenase complex due to a single base substitution to the intron 5, resulting in two alternatively spliced mRNAs in patient with maple syrup urine disease

Abstract A patient with maple syrup urine disease (MSUD) associated with a E1β subunit deficiency of the branced-chain α-keto acid dehydrogenase (BCKDH) complex was investigated at the molecular level. The defect responsible for the deficiency of the E1β subunit protein was identified by analysis of cDNA and genomic DNA by polymerase chain reaction. Total RNA isolated from lymphoblastoid cells was transcribed into cDNA and amplified usign a set of primers located within exon 3 and exon 9 of the E1β gene. Agarose gel electrophoresis of cDNA amplification products revealed two shortened bands as well as a faint band or 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 E1β gene revealed a single base substitution from G to T of the invariant GT dinucleotides at 5′ splice site of the intron 5. Analysis of family members using primer-specified restriction map modification showed that the patient is homozygous for this mutation. We postulate that this mutation leads to the skipping of either exon 5 or both exons 5 and 6, thus producing two shortened E1β mRNA. The percentage of normal and two shortened transcripts was estimated to be 9, 71 and 20%, respectively. To our best knowledge, this is the first documented example of exon skipping in the E1β gene as the cause of MSUD and the novel mutation of the invariant G at the 5′ splice site which results in two alternatively spliced mRNA due to the skipping of the preceding exon as well as both preceding and following exon.