Splicing analysis disclosed a determinant single nucleotide for exon skipping caused by a novel intraexonic four-nucleotide deletion in the dystrophin gene

Background: Mutations in exonic splicing enhancer sequences are known to cause splicing errors. Although exonic splicing enhancers have been identified as a stretch of purine-rich sequences, it has been difficult to precisely pinpoint the determinant nucleotides in these sequences. This article reports that a 4-bp deletion in exon 38 of the dystrophin gene induced complete exon 38 skipping in vivo. Moreover, the third nucleotide of the deletion was shown to be determinant for the exonic splicing enhancer activity in in vivo splicing analysis of hybrid minigenes encoding mutant exons. Method: Genomic DNA analysis of a 2-year-old boy with a raised level of serum creatine kinase yielded a 4-bp deletion 11 bp upstream of the 39 end of exon 38 of the dystrophin gene (c. 5434-5437del TTCA), disrupting a predicted SC35-binding site. Result: Interestingly, his dystrophin mRNA was shown to completely lack exon 38 (exon 382 transcript). As the exon 382 transcript coded for a truncated dystrophin protein, this exon skipping was determined to be a modifying factor of his phenotype. In an in vivo splicing assay, a hybrid minigene encoding exon 38 with the 4-bp deletion was shown to induce complete exon 38 skipping, confirming the deleted region as a splicing enhancer sequence. Site-directed mutagenesis of the deleted sequence showed that the complete exon 38 skipping was caused by mutation of the third nucleotide position of the deletion (C5436), whereas mutations at the other three nucleotide positions induced partial exon skipping. Conclusion: Our results underline the potential of understanding the regulation of exonic splicing enhancer sequences and exon skipping therapy for treatment of Duchenne's muscular dystrophy.