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

Several unclassified variants (UVs) have been identified in splicing regions of disease-associated genes and their characterization as pathogenic mutations or benign polymorphisms is crucial for the understanding of their role in disease development. In this study, 24 UVs located at BRCA1 and BRCA2...

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Autores principales: Colombo, Mara, De Vecchi, Giovanna, Caleca, Laura, Foglia, Claudia, Ripamonti, Carla B., Ficarazzi, Filomena, Barile, Monica, Varesco, Liliana, Peissel, Bernard, Manoukian, Siranoush, Radice, Paolo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3579815/
https://www.ncbi.nlm.nih.gov/pubmed/23451180
http://dx.doi.org/10.1371/journal.pone.0057173
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author Colombo, Mara
De Vecchi, Giovanna
Caleca, Laura
Foglia, Claudia
Ripamonti, Carla B.
Ficarazzi, Filomena
Barile, Monica
Varesco, Liliana
Peissel, Bernard
Manoukian, Siranoush
Radice, Paolo
author_facet Colombo, Mara
De Vecchi, Giovanna
Caleca, Laura
Foglia, Claudia
Ripamonti, Carla B.
Ficarazzi, Filomena
Barile, Monica
Varesco, Liliana
Peissel, Bernard
Manoukian, Siranoush
Radice, Paolo
author_sort Colombo, Mara
collection PubMed
description Several unclassified variants (UVs) have been identified in splicing regions of disease-associated genes and their characterization as pathogenic mutations or benign polymorphisms is crucial for the understanding of their role in disease development. In this study, 24 UVs located at BRCA1 and BRCA2 splice sites were characterized by transcripts analysis. These results were used to evaluate the ability of nine bioinformatics programs in predicting genetic variants causing aberrant splicing (spliceogenic variants) and the nature of aberrant transcripts. Eleven variants in BRCA1 and 8 in BRCA2, including 8 not previously characterized at transcript level, were ascertained to affect mRNA splicing. Of these, 16 led to the synthesis of aberrant transcripts containing premature termination codons (PTCs), 2 to the up-regulation of naturally occurring alternative transcripts containing PTCs, and one to an in-frame deletion within the region coding for the DNA binding domain of BRCA2, causing the loss of the ability to bind the partner protein DSS1 and ssDNA. For each computational program, we evaluated the rate of non-informative analyses, i.e. those that did not recognize the natural splice sites in the wild-type sequence, and the rate of false positive predictions, i.e., variants incorrectly classified as spliceogenic, as a measure of their specificity, under conditions setting sensitivity of predictions to 100%. The programs that performed better were Human Splicing Finder and Automated Splice Site Analyses, both exhibiting 100% informativeness and specificity. For 10 mutations the activation of cryptic splice sites was observed, but we were unable to derive simple criteria to select, among the different cryptic sites predicted by the bioinformatics analyses, those actually used. Consistent with previous reports, our study provides evidences that in silico tools can be used for selecting splice site variants for in vitro analyses. However, the latter remain mandatory for the characterization of the nature of aberrant transcripts.
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spelling pubmed-35798152013-02-28 Comparative In Vitro and In Silico Analyses of Variants in Splicing Regions of BRCA1 and BRCA2 Genes and Characterization of Novel Pathogenic Mutations Colombo, Mara De Vecchi, Giovanna Caleca, Laura Foglia, Claudia Ripamonti, Carla B. Ficarazzi, Filomena Barile, Monica Varesco, Liliana Peissel, Bernard Manoukian, Siranoush Radice, Paolo PLoS One Research Article Several unclassified variants (UVs) have been identified in splicing regions of disease-associated genes and their characterization as pathogenic mutations or benign polymorphisms is crucial for the understanding of their role in disease development. In this study, 24 UVs located at BRCA1 and BRCA2 splice sites were characterized by transcripts analysis. These results were used to evaluate the ability of nine bioinformatics programs in predicting genetic variants causing aberrant splicing (spliceogenic variants) and the nature of aberrant transcripts. Eleven variants in BRCA1 and 8 in BRCA2, including 8 not previously characterized at transcript level, were ascertained to affect mRNA splicing. Of these, 16 led to the synthesis of aberrant transcripts containing premature termination codons (PTCs), 2 to the up-regulation of naturally occurring alternative transcripts containing PTCs, and one to an in-frame deletion within the region coding for the DNA binding domain of BRCA2, causing the loss of the ability to bind the partner protein DSS1 and ssDNA. For each computational program, we evaluated the rate of non-informative analyses, i.e. those that did not recognize the natural splice sites in the wild-type sequence, and the rate of false positive predictions, i.e., variants incorrectly classified as spliceogenic, as a measure of their specificity, under conditions setting sensitivity of predictions to 100%. The programs that performed better were Human Splicing Finder and Automated Splice Site Analyses, both exhibiting 100% informativeness and specificity. For 10 mutations the activation of cryptic splice sites was observed, but we were unable to derive simple criteria to select, among the different cryptic sites predicted by the bioinformatics analyses, those actually used. Consistent with previous reports, our study provides evidences that in silico tools can be used for selecting splice site variants for in vitro analyses. However, the latter remain mandatory for the characterization of the nature of aberrant transcripts. Public Library of Science 2013-02-22 /pmc/articles/PMC3579815/ /pubmed/23451180 http://dx.doi.org/10.1371/journal.pone.0057173 Text en © 2013 Colombo et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Colombo, Mara
De Vecchi, Giovanna
Caleca, Laura
Foglia, Claudia
Ripamonti, Carla B.
Ficarazzi, Filomena
Barile, Monica
Varesco, Liliana
Peissel, Bernard
Manoukian, Siranoush
Radice, Paolo
Comparative In Vitro and In Silico Analyses of Variants in Splicing Regions of BRCA1 and BRCA2 Genes and Characterization of Novel Pathogenic Mutations
title Comparative In Vitro and In Silico Analyses of Variants in Splicing Regions of BRCA1 and BRCA2 Genes and Characterization of Novel Pathogenic Mutations
title_full Comparative In Vitro and In Silico Analyses of Variants in Splicing Regions of BRCA1 and BRCA2 Genes and Characterization of Novel Pathogenic Mutations
title_fullStr Comparative In Vitro and In Silico Analyses of Variants in Splicing Regions of BRCA1 and BRCA2 Genes and Characterization of Novel Pathogenic Mutations
title_full_unstemmed Comparative In Vitro and In Silico Analyses of Variants in Splicing Regions of BRCA1 and BRCA2 Genes and Characterization of Novel Pathogenic Mutations
title_short Comparative In Vitro and In Silico Analyses of Variants in Splicing Regions of BRCA1 and BRCA2 Genes and Characterization of Novel Pathogenic Mutations
title_sort comparative in vitro and in silico analyses of variants in splicing regions of brca1 and brca2 genes and characterization of novel pathogenic mutations
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3579815/
https://www.ncbi.nlm.nih.gov/pubmed/23451180
http://dx.doi.org/10.1371/journal.pone.0057173
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