Abstract

Background: It is critical to see splicing patterns for assessing clinical phenotypes of mutations in the dystrophin gene. However, it is still unclear how to predict alternative splicing pathways from splice site mutations in the dystrophin gene.

Objective: To identify elements determining alternative splicing pathways in intron +1G>A mutations in the dystrophin gene.

Results and conclusion: We discovered that exon 25 is spliced out in +1G>A mutation in intron 25, resulting in mild Becker muscular dystrophy, and that a cryptic splice site within exon 45 is activated in severe Duchenne muscular dystrophy with +1G>A mutation in intron 45. Furthermore, in vitro splicing analysis using a pre-constructed expression vector revealed that mutant intron 25 produced one transcript that lacked exon 25. In contrast, the same splice site mutation in intron 45 produced three splicing products. One product used the same cryptic splice donor site within exon 45 as the in vivo donor site, and another product used a cryptic splice site within the vector sequence. It is remarkable that the available cryptic splice site was not activated by the same G>A mutation of intron 25. We concluded that sequences inserted into the in vitro splicing assay minigene contain cis-elements that determine splicing pathways. By taking other +1G>A mutations in the introns of the dystrophin gene reported in the literature into consideration, we found that cryptic splice site activation is observed only in strong exons. This finding will help to elucidate the molecular pathogenesis of dystrophinopathy and to predict the efficiency of induction of exon skipping with antisense oligonucleotides for Duchenne muscular dystrophy treatment.