title: Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools.
abstract: The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient's RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants), including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs). We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZEI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases.
authors.affiliation: Inserm U1079-IRIB, University of Rouen, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.
authors.forename: Omar
authors.initials: O
authors.lastname: Soukarieh
authors.affiliation: Inserm U1079-IRIB, University of Rouen, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.
authors.forename: Pascaline
authors.initials: P
authors.lastname: Gaildrat
authors.affiliation: Inserm U1079-IRIB, University of Rouen, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.
authors.forename: Mohamad
authors.initials: M
authors.lastname: Hamieh
authors.affiliation: Inserm U1079-IRIB, University of Rouen, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Drouet
authors.affiliation: Inserm U1079-IRIB, University of Rouen, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.|Department of Genetics, University Hospital, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Baert-Desurmont
authors.affiliation: Inserm U1079-IRIB, University of Rouen, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.|Department of Genetics, University Hospital, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Frébourg
authors.affiliation: Inserm U1079-IRIB, University of Rouen, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.
authors.forename: Mario
authors.initials: M
authors.lastname: Tosi
authors.affiliation: Inserm U1079-IRIB, University of Rouen, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Martins
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D019313:BRCA1 Protein / Q000235:genetics; D024682:BRCA2 Protein / Q000235:genetics; D003198:Computer Simulation; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D005091:Exons / Q000235:genetics*; D005260:Female; D006801:Humans; D000070957:MutL Protein Homolog 1; D025542:Neurofibromin 1 / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics*; D010051:Ovarian Neoplasms / Q000235:genetics* / Q000473:pathology; D022821:RNA Splice Sites / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D024682:BRCA2 Protein; C551750:BRCA2 protein, human; C505032:CFTR protein, human; C501486:MLH1 protein, human; D025542:Neurofibromin 1; D009687:Nuclear Proteins; D022821:RNA Splice Sites; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000070957:MutL Protein Homolog 1