title: Identification of splicing defects caused by mutations in the dysferlin gene.
abstract: Missense, iso-semantic, and intronic mutations are challenging for interpretation, in particular for their impact in mRNA. Various tools such as the Human Splicing Finder (HSF) system could be used to predict the impact on splicing; however, no diagnosis result could rely on predictions alone, but requires functional testing. Here, we report an in vitro approach to study the impact of DYSF mutations on splicing. It was evaluated on a series of 45 DYSF mutations, both intronic and exonic. We confirmed splicing alterations for all intronic mutations localized in 5' or 3' splice sites. Then, we showed that DYSF missense mutations could also result in splicing defects: mutations c.463G>A and c.2641A>C abolished ESEs and led to exon skipping; mutations c.565C>G and c.1555G>A disrupted Exonic Splicing Enhancer (ESE), while concomitantly creating new 5' or 3' splice site leading to exonic out of frame deletions. We demonstrated that 20% of DYSF missense mutations have a strong impact on splicing. This minigene strategy is an efficient tool for the detection of splicing defects in dysferlinopathies, which could allow for a better comprehension of splicing defects due to mutations and could improve prediction tools evaluating splicing defects.
authors.affiliation: Aix Marseille Université, GMGF, Marseille, 13385, France; Inserm, UMR_S 910, Marseille, 13385, France.
authors.forename: Virginie
authors.initials: V
authors.lastname: Kergourlay
authors.forename: Ghadi
authors.initials: G
authors.lastname: Raï
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Blandin
authors.forename: David
authors.initials: D
authors.lastname: Salgado
authors.forename: Christophe
authors.initials: C
authors.lastname: Béroud
authors.forename: Nicolas
authors.initials: N
authors.lastname: Lévy
authors.forename: Martin
authors.initials: M
authors.lastname: Krahn
authors.forename: Marc
authors.initials: M
authors.lastname: Bartoli
mesh_terms: D000073939:Dysferlin; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D009124:Muscle Proteins / Q000235:genetics*; D009154:Mutation; D012326:RNA Splicing / Q000235:genetics*
chemical_list: C114839:DYSF protein, human; D000073939:Dysferlin; D008565:Membrane Proteins; D009124:Muscle Proteins
keywords: DYSF; diagnostics tests; dysferlin; neuromuscular disease; splicing