Computational protocol: Comparative In Vitro and In Silico Analyses of Variants in Splicing Regions of BRCA1 and BRCA2 Genes and Characterization of Novel Pathogenic Mutations

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Protocol publication

[…] Nine computational programs were investigated to verify their accuracy in correctly predicting the effect on mRNA splicing of the variants analyzed in vitro. These included 5 tools integrated in the Alamut application (Interactive Biosoftware, Version 2.1, Roven, France) , namely: Splice Site Finder (SSF) , MaxEntScan(MES) , Splice Site Prediction by Neural Network (NNSPLICE) , GeneSplicer (GS) , and Human Splicing Finder (HSF) , plus the following additional tools: NetGene2 (NG2) , , SpliceView (SV) , SplicePredictor (SP) , and Automated Splice Site Analyses (ASSA) .Gene regions addressed by the variants under analyses were submitted to bioinformatics analyses using the human default parameter settings of the different programs. For all programs except ASSA, the splice site prediction scores (SSPSs) in the wild-type and the mutated sequences were compared and the relative percent difference was calculated as follows: [(SSPSmut-SSPSwt)/SSPSwt ]x100. For ASSA, which measures the binding affinity of the spliceosome to wild-type and mutated splice sites using information theory-based values (Ri) measured in bits (where a 1 bit change represents a 2-fold change ), the percent difference of binding affinity in the mutated compared to the wild-type sequences was calculated as follows: [2(Rimut-Riwt)−1]×100.In addition, we verified the ability of bioinformatics programs to identify the alternative splice sites that were observed in in vitro analyses to be activated following the destruction of the natural splice sites. For programs that were able to identify all such alternative splice sites, the sequence encompassing 500 bp upstream and downstream the natural splice site affected by the alteration was submitted to bioinformatics analyses and the SSPS and Ri patterns in the mutated sequences were analyzed. […]

Pipeline specifications

Software tools MaxEntScan, NNSplice, GeneSplicer, HSF, NetGene2, SpliceView, SplicePredictor
Application WGS analysis
Organisms Homo sapiens