Identification of Splicing Defects Caused by Mutations in the Dysferlin Gene

Missense, iso-semantic, and intronic muta- tions 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 im- pact 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 muta- tions 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 lead- ing to exonic out of frame deletions. We demonstrated that 20% of DYSF missense mutations have a strong im- pact 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 splic- ing defects due to mutations and could improve prediction tools evaluating splicing defects.