title: Exon first nucleotide mutations in splicing: evaluation of in silico prediction tools.
abstract: Mutations in the first nucleotide of exons (E(+1)) mostly affect pre-mRNA splicing when found in AG-dependent 3' splice sites, whereas AG-independent splice sites are more resistant. The AG-dependency, however, may be difficult to assess just from primary sequence data as it depends on the quality of the polypyrimidine tract. For this reason, in silico prediction tools are commonly used to score 3' splice sites. In this study, we have assessed the ability of sequence features and in silico prediction tools to discriminate between the splicing-affecting and non-affecting E(+1) variants. For this purpose, we newly tested 16 substitutions in vitro and derived other variants from literature. Surprisingly, we found that in the presence of the substituting nucleotide, the quality of the polypyrimidine tract alone was not conclusive about its splicing fate. Rather, it was the identity of the substituting nucleotide that markedly influenced it. Among the computational tools tested, the best performance was achieved using the Maximum Entropy Model and Position-Specific Scoring Matrix. As a result of this study, we have now established preliminary discriminative cut-off values showing sensitivity up to 95% and specificity up to 90%. This is expected to improve our ability to detect splicing-affecting variants in a clinical genetic setting.
authors.affiliation: Molecular Genetics Laboratory, Centre for Cardiovascular Surgery and Transplantation, Brno, Czech Republic ; Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
authors.forename: Lucie
authors.initials: L
authors.lastname: Grodecká
authors.affiliation: Molecular Genetics Laboratory, Centre for Cardiovascular Surgery and Transplantation, Brno, Czech Republic.
authors.forename: Pavla
authors.initials: P
authors.lastname: Lockerová
authors.affiliation: Molecular Genetics Laboratory, Centre for Cardiovascular Surgery and Transplantation, Brno, Czech Republic.
authors.forename: Barbora
authors.initials: B
authors.lastname: Ravčuková
authors.affiliation: International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
authors.forename: Emanuele
authors.initials: E
authors.lastname: Buratti
authors.affiliation: International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
authors.forename: Francisco E
authors.initials: FE
authors.lastname: Baralle
authors.affiliation: Institute of Biostatistics and Analyses, Masaryk University, Brno, Czech Republic.
authors.forename: Ladislav
authors.initials: L
authors.lastname: Dušek
authors.affiliation: Molecular Genetics Laboratory, Centre for Cardiovascular Surgery and Transplantation, Brno, Czech Republic ; Central European Institute of Technology, Masaryk University, Brno, Czech Republic ; Institute of Clinical Immunology and Allergology, St. Anne's University Hospital and Masaryk University, Brno, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Freiberger
mesh_terms: D000077329:Agammaglobulinaemia Tyrosine Kinase; D000361:Agammaglobulinemia / Q000235:genetics; D003198:Computer Simulation; D005091:Exons; D040181:Genetic Diseases, X-Linked / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D017354:Point Mutation; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D022821:RNA Splice Sites; D012326:RNA Splicing; D017422:Sequence Analysis, DNA; D012984:Software
chemical_list: D022821:RNA Splice Sites; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase