title: Cryptic splice activation but not exon skipping is observed in minigene assays of dystrophin c.9361+1G>A mutation identified by NGS.
abstract: Next-generation sequencing (NGS) discloses nucleotide changes in the genome. Mutations at splicing regulatory elements are expected to cause splicing errors, such as exon skipping, cryptic splice site activation, partial exon loss or intron retention. In dystrophinopathy patients, prediction of splicing outcomes is essential to determine the phenotype: either severe Duchenne or mild Becker muscular dystrophy, based on the reading frame rule. In a Vietnamese patient, NGS identified a c.9361+1G>A mutation in the dystrophin gene and an additional DNA variation of A>G at +117 bases in intron 64. To ascertain the consequences of these DNA changes on dystrophin splicing, minigene constructs were prepared inserting dystrophin exon 64 plus various lengths of intron 64. Exon 64 skipping was observed in the minigene construct with 160 nucleotide (nt) of intron 64 sequence with both c.9361+1A and +117G. In contrast, minigene constructs with larger flanking intronic domains resulted in cryptic splice site activation rather than exon skipping. Meanwhile, the cryptic splice site activation was induced even in +117G when intron 64 was elongated to 272 nt and longer. It was expected that cryptic splice site activation is an in vivo splicing outcome.
authors.affiliation: Department of Physical Therapy, Faculty of Rehabilitation, Kobe Gakuin University, Kobe, Japan.
authors.forename: Emma Tabe Eko
authors.initials: ETE
authors.lastname: Niba
authors.affiliation: Department of Physical Therapy, Faculty of Rehabilitation, Kobe Gakuin University, Kobe, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nishida
authors.affiliation: Center for Gene and Protein Research, Hanoi Medical University, Hanoi, Vietnam.
authors.forename: Van Khanh
authors.initials: VK
authors.lastname: Tran
authors.affiliation: Department of Medical Genetics, Metabolism and Endocrinology and Clinical Research, Research Institute for Child Health, Vietnam National Hospital of Pediatrics, Hanoi, Vietnam.
authors.forename: Dung Chi
authors.initials: DC
authors.lastname: Vu
authors.affiliation: Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Matsumoto
authors.affiliation: Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Awano
authors.affiliation: Department of Pediatrics, Hyogo College of Medicine, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Lee
authors.affiliation: Department of Pediatrics, Hyogo College of Medicine, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takeshima
authors.affiliation: Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Hisahide
authors.initials: H
authors.lastname: Nishio
authors.affiliation: Department of Physical Therapy, Faculty of Rehabilitation, Kobe Gakuin University, Kobe, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Matsuo
mesh_terms: D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D016189:Dystrophin / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007438:Introns / Q000235:genetics; D008297:Male; D009154:Mutation / Q000235:genetics*; D022821:RNA Splice Sites / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics
chemical_list: D016189:Dystrophin; D022821:RNA Splice Sites