Analysis of exonic mutations leading to exon skipping in patients with pyruvate dehydrogenase E1 alpha deficiency

The pyruvate dehydrogenase (PDH) complex is situated at a key position in energy metabolism and is responsible for the conversion of pyruvate to acetyl CoA. In the literature, two unrelated patients with a PDH complex deficiency and splicing out of exon 6 of the PDH E1 alpha gene have been described, although intronic/exonic boundaries on either side of exon 6 were completely normal. Analysis of exon 6 in genomic DNA of these patients revealed two exonic mutations, a silent and a missense mutation. Although not experimentally demonstrated, the authors in both publications suggested that the exonic mutations were responsible for the exon skipping. In this work, we were able to demonstrate, by performing splicing experiments, that the two exonic mutations described in the PDH E1 alpha gene lead to aberrant splicing. We observed a disruption of the predicted wild-type pre-mRNA secondary structure of exon 6 by the mutated sequences described. However, when we constructed mutations that either reverted or disrupted the wild-type predicted pre-mRNA secondary structure of exon 6, we were unable to establish a correlation between the aberrant splicing and disruption of the predicted structure. The mutagenic experiments described here and the silent mutation found in one of the patients suggest the presence of an exonic splicing enhancer in the middle region of exon 6 of the PDH E1alpha gene.