title: A novel splicing silencer generated by DMD exon 45 deletion junction could explain upstream exon 44 skipping that modifies dystrophinopathy.
abstract: Duchenne muscular dystrophy (DMD), a progressive muscle-wasting disease, is mostly caused by exon deletion mutations in the DMD gene. The reading frame rule explains that out-of-frame deletions lead to muscle dystrophin deficiency in DMD. In outliers to this rule, deletion junction sequences have never previously been explored as splicing modulators. In a Japanese case, we identified a single exon 45 deletion in the patient's DMD gene, indicating out-of-frame mutation. However, immunohistochemical examination disclosed weak dystrophin signals in his muscle. Reverse transcription-PCR amplification of DMD exons 42 to 47 revealed a major normally spliced product with exon 45 deletion and an additional in-frame product with deletion of both exons 44 and 45, indicating upstream exon 44 skipping. We considered the latter to underlie the observed dystrophin expression. Remarkably, the junction sequence cloned by PCR walking abolished the splicing enhancer activity of the upstream intron in a chimeric doublesex gene pre-mRNA in vitro splicing. Furthermore, antisense oligonucleotides directed against the junction site counteracted this effect. These indicated that the junction sequence was a splicing silencer that induced upstream exon 44 skipping. It was strongly suggested that creation of splicing regulator is a modifier of dystrophinopathy.
authors.affiliation: Department of Pediatrics, Graduate School of Medicine, Kobe University, Kobe, Japan.
authors.forename: Ery Kus
authors.initials: EK
authors.lastname: Dwianingsih
authors.affiliation: Department of Medical Rehabilitation, Faculty of Rehabilitation, Kobegakuin University, Kobe, Japan.
authors.forename: Rusdy Ghazali
authors.initials: RG
authors.lastname: Malueka
authors.affiliation: Department of Medical Rehabilitation, Faculty of Rehabilitation, Kobegakuin University, Kobe, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nishida
authors.affiliation: Department of Pathology and Applied Neurobiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Itoh
authors.affiliation: Department of Pediatrics, Graduate School of Medicine, Kobe University, Kobe, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Lee
authors.affiliation: Department of Pediatrics, Graduate School of Medicine, Kobe University, Kobe, Japan.
authors.forename: Mariko
authors.initials: M
authors.lastname: Yagi
authors.affiliation: Department of Pediatrics, Graduate School of Medicine, Kobe University, Kobe, Japan.
authors.forename: Kazumoto
authors.initials: K
authors.lastname: Iijima
authors.affiliation: Department of Pediatrics, Graduate School of Medicine, Kobe University, Kobe, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takeshima
authors.affiliation: Department of Medical Rehabilitation, Faculty of Rehabilitation, Kobegakuin University, Kobe, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Matsuo
mesh_terms: D001483:Base Sequence; D002648:Child; D016189:Dystrophin / Q000235:genetics* / Q000378:metabolism; D005091:Exons / Q000235:genetics; D016368:Frameshift Mutation; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics*; D059372:Nucleotide Motifs; D016376:Oligonucleotides, Antisense; D010641:Phenotype; D012322:RNA Precursors / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D037941:Silencer Elements, Transcriptional / Q000235:genetics*
chemical_list: D016189:Dystrophin; D016376:Oligonucleotides, Antisense; D012322:RNA Precursors; D012333:RNA, Messenger