title: In vitro correction of a pseudoexon-generating deep intronic mutation in LGMD2A by antisense oligonucleotides and modified small nuclear RNAs.
abstract: Limb-girdle muscular dystrophy type 2A (LGMD2A) is the most frequent autosomal recessive muscular dystrophy. It is caused by mutations in the calpain-3 (CAPN3) gene. The majority of the mutations described to date are located in the coding sequence of the gene. However, it is estimated that 25% of the mutations are present at exon-intron boundaries and modify the pre-mRNA splicing of the CAPN3 transcript. We have previously described the first deep intronic mutation in the CAPN3 gene: c.1782+1072G>C mutation. This mutation causes the pseudoexonization of an intronic sequence of the CAPN3 gene in the mature mRNA. In the present work, we show that the point mutation generates the inclusion of the pseudoexon in the mRNA using a minigene assay. In search of a treatment that restores normal splicing, splicing modulation was induced by RNA-based strategies, which included antisense oligonucleotides and modified small-nuclear RNAs. The best effect was observed with antisense sequences, which induced pseudoexon skipping in both HeLa cells cotransfected with mutant minigene and in fibroblasts from patients. Finally, transfection of antisense sequences and siRNA downregulation of serine/arginine-rich splicing factor 1 (SRSF1) indicate that binding of this factor to splicing enhancer sequences is involved in pseudoexon activation.
authors.affiliation: Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain; Neuroscience Area, Health Research Institute Biodonostia, San Sebastian, Spain.
authors.forename: Lorea
authors.initials: L
authors.lastname: Blázquez
authors.forename: Ana
authors.initials: A
authors.lastname: Aiastui
authors.forename: Maria
authors.initials: M
authors.lastname: Goicoechea
authors.forename: Mafalda
authors.initials: M
authors.lastname: Martins de Araujo
authors.forename: Aurélie
authors.initials: A
authors.lastname: Avril
authors.forename: Cyriaque
authors.initials: C
authors.lastname: Beley
authors.forename: Luis
authors.initials: L
authors.lastname: García
authors.forename: Juan
authors.initials: J
authors.lastname: Valcárcel
authors.forename: Puri
authors.initials: P
authors.lastname: Fortes
authors.forename: Adolfo
authors.initials: A
authors.lastname: López de Munain
mesh_terms: D017398:Alternative Splicing; D002154:Calpain / Q000235:genetics; D002460:Cell Line; D005091:Exons; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D005786:Gene Expression Regulation; D023061:Gene Order; D006801:Humans; D007438:Introns; D008875:Middle Aged; D009124:Muscle Proteins / Q000235:genetics; D049288:Muscular Dystrophies, Limb-Girdle / Q000235:genetics*; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000235:genetics* / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D000068103:Serine-Arginine Splicing Factors
chemical_list: D009124:Muscle Proteins; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; C105884:CAPN3 protein, human; D002154:Calpain
keywords: AON; LGMD2A; antisense oligonucleotide; exon skipping; limb-girdle muscular dystrophy; pseudoexon; usnRNA