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.

Progress and challenges in diagnosis of dysferlinopathy

“…While the pathogenic effect of nonsense and frame‐shift deletion/insertion mutations is obvious, missense mutations may be pathogenic when they lead to deleterious effects on mRNA splicing (in 15–20% of cases) or to mislocalization of the protein. Two missense mutations (c.463G>A and c.2641A>C) were proven to cause partial exon skipping …”

“…Some mutations that cause exon skipping still preserve the reading frame, but the corresponding deleted region could be important for protein function, thus leading to its degradation and deleterious effect . One such example is the in‐frame skipping of exon 32, which produces a functional truncated protein .…”

Diagnostic use of Massively Parallel Sequencing in Neuromuscular Diseases: Towards an Integrated Diagnosis

“…To increase the benefit of MPS in a diagnosis setting, improvement in bioinformatics, storage of data and interpretation of variants are areas that require further resource development, as well as expanded and multidisciplinary databases and analysis pipelines and tools, and increased training for biologists and clinicians. High throughput assay to assess RNA integrity [55] and protein function have been described [43] and should be further developed for functional validation of genetic variants.…”

Splicing impact of deep exonic missense variants in CAPN3 explored systematically by minigene functional assay

“…Since this assay is not adapted for U12 type introns, The Intron Annotation and Orthology Database (IAOD)(Gault et al 2017; Turunen et al 2013) was used to identify this type of introns, leading to exclusion of exons 19 and 20 from the analysis. As described previously (Kergourlay et al 2014; Puppo et al 2015), exons and approximately 150 bp of flanking introns were amplified using the Expand high fidelity PCR system (Roche, Basel, Switzerland). Amplicons were subsequently cloned into the pCAS2 vector.…”

“…Assigning the clinical significance to a newly identified exonic single nucleotide variant (SNV, either synonymous or missense) is much more difficult, despite help from ACMG guidelines that take into account several lines of pathogenicity evidence (Richards et al 2015). It is important to note that synonymous variants or missense variants with low impact on protein function can still be pathogenic by disrupting splicing (Duguez, Bartoli, and Richard 2006; Kergourlay et al 2014). There is a great risk of misinterpreting the clinical significance of these variants using standard approaches, which could potentially lead to incorrect diagnosis.…”