Identification of Splicing Defects Caused by Mutations in the Dysferlin Gene


    loading  Checking for direct PDF access through Ovid

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 anin vitroapproach to study the impact ofDYSFmutations on splicing. It was evaluated on a series of 45DYSFmutations, both intronic and exonic. We confirmed splicing alterations for all intronic mutations localized in 5′ or 3′ splice sites. Then, we showed thatDYSFmissense 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% ofDYSFmissense 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.The fifty five exons of DYSF and their phasing are presented. Gradients of blue represent the strength of 5′ and 3′ splice sites, the darker the stronger.SymbolIntronic mutations for which a deleterious effect on splicing is suspected but not confirmedSymbolIntronic mutations for which a deleterious effect on splicing has been confirmed in patientSymbolIntronic mutations for which a deleterious effect on splicing has been shown both in patients RNA and by this studySymbolIntronic mutations for which a deleterious effect on splicing has been shown by this studySymbolMissense mutations for which a deleterious effect on splicing has been confirmed in patients and by this studySymbolMissense mutations for which a deleterious effect on splicing has been shown in our study

    loading  Loading Related Articles