Abstract

Background: Splicing patterns are critical for assessing clinical phenotype of mutations in the dystrophin gene. However, it is still unclear how to predict alternative splicing pathways in such cases of splice-site mutation in the dystrophin gene.

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

Results: We found that exon 25 is spliced out in the +1G→A mutation in intron 25, resulting in mild Becker muscular dystrophy, and that a cryptic splice site within exon 45 was activated in severe Duchenne muscular dystrophy with a mutation of +1G→A mutation in 45. Furthermore, in vitro splicing analysis using a pre-constructed expression vector showed that the 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 donor splice site within exon 45 as the in vivo donor site and another product used a cryptic splice site within the vector sequence. Notably, the available cryptic splice site was not activated by the same G→A mutation of intron 25.

Conclusion: It was 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, it seems that cryptic splice-site activation is seen only in strong exons. This finding will help to elucidate the molecular pathogenesis of dystrophinopathy and to predict efficiency of induction of exon skipping with antisense oligonucleotides for treatment of Duchenne muscular dystrophy.