title: In vitro splicing analysis showed that availability of a cryptic splice site is not a determinant for alternative splicing patterns caused by +1G-->A mutations in introns of the dystrophin gene.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pediatrics, Kobe University Graduate School of Medicine, Chuo, Kobe 6500-017, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Habara
authors.forename: Y
authors.initials: Y
authors.lastname: Takeshima
authors.forename: H
authors.initials: H
authors.lastname: Awano
authors.forename: Y
authors.initials: Y
authors.lastname: Okizuka
authors.forename: Z
authors.initials: Z
authors.lastname: Zhang
authors.forename: K
authors.initials: K
authors.lastname: Saiki
authors.forename: M
authors.initials: M
authors.lastname: Yagi
authors.forename: M
authors.initials: M
authors.lastname: Matsuo
mesh_terms: D004252:DNA Mutational Analysis; D016189:Dystrophin / Q000235:genetics*; D005091:Exons; D006801:Humans; D007438:Introns; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics*; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide; D020033:Protein Isoforms; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D015203:Reproducibility of Results
chemical_list: D016189:Dystrophin; D020033:Protein Isoforms; D012333:RNA, Messenger