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Minigene Splicing Assays Identify 12 Spliceogenic Variants of BRCA2 Exons 14 and 15

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Fraile Bethencourt, Eugenia & Valenzuela Palomo, Alberto & Díez Gómez, Beatriz & Caloca, María José & Gómez Barrero, Susana & Velasco Sampedro, Eladio Andrés (2019-05 ) .Minigene Splicing Assays Identify 12 Spliceogenic Variants of BRCA2 Exons 14 and 15.

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Fraile Bethencourt, Eugenia & Valenzuela Palomo, Alberto & Díez Gómez, Beatriz & Caloca, María José & Gómez Barrero, Susana & Velasco Sampedro, Eladio Andrés. 2019-05 .Minigene Splicing Assays Identify 12 Spliceogenic Variants of BRCA2 Exons 14 and 15.

https://hdl.handle.net/20.500.12080/39628
dc.contributor.author Fraile Bethencourt, Eugenia
dc.contributor.author Valenzuela Palomo, Alberto
dc.contributor.author Díez Gómez, Beatriz
dc.contributor.author Caloca, María José
dc.contributor.author Gómez Barrero, Susana
dc.contributor.author Velasco Sampedro, Eladio Andrés
dc.date.accessioned 2024-02-09T09:10:15Z
dc.date.available 2024-02-09T09:10:15Z
dc.date.created 2019-05
dc.date.issued 2019-05
dc.identifier.uri https://hdl.handle.net/20.500.12080/39628
dc.description.abstract A relevant fraction of BRCA2 variants is associated with splicing alterations and with an increased risk of hereditary breast and ovarian cancer (HBOC). In this work, we have carried out a thorough study of variants from BRCA2 exons 14 and 15 reported at mutation databases. A total of 294 variants from exons 14 and 15 and flanking intronic sequences were analyzed with the online splicing tools NNSplice and Human Splicing Finder. Fifty-three out of these 294 variants were selected as candidate splicing variants. All variants but one, were introduced into the minigene MGBR2_ex14-20 (with exons 14¿20) by site-directed mutagenesis and assayed in MCF-7 cells. Twelve of the remaining 52 variants (23.1%) impaired splicing at different degrees, yielding from 5 to 100% of aberrant transcripts. Nine variants affected the natural acceptor or donor sites of both exons and three affected putative enhancers or silencers. Fluorescent capillary electrophoresis revealed at least 10 different anomalous transcripts: H(E14q5), 1 (E14p10), 1(E14p246), 1(E14q256), 1(E14), 1(E15p12), 1(E15p13), 1(E15p83), 1(E15) and a 942-nt fragment of unknown structure. All transcripts, except for 1(E14q256) and 1(E15p12), are expected to truncate the BRCA2 protein. Nine variants induced severe splicing aberrations with more than 90% of abnormal transcripts. Thus, according to the guidelines of the American College of Medical Genetics and Genomics, eight variants should be classified as pathogenic (c.7008-2A > T, c.7008-1G > A, c.7435+1G > C, c.7436-2A > T, c.7436-2A > G, c.7617+1G > A, c.7617+1G > T, and c.7617+2T > G), one as likely pathogenic (c.7008-3C > G) and three remain as variants of uncertain clinical significance or VUS (c.7177A > G, c.7447A > G and c.7501C > T). In conclusion, functional assays by minigenes constitute a valuable strategy to primarily check the splicing impact of DNA variants and their clinical interpretation. While bioinformatics predictions of splice site variants were accurate, those of enhancer or silencer variants were poor (only 3/23 spliceogenic variants) which showed weak impacts on splicing (¿5¿16% of aberrant isoforms). So, the Exonic Splicing Enhancer and Silencer (ESE and ESS, respectively) prediction algorithms require further improvement. es_ES
dc.format application/pdf es_ES
dc.language eng es_ES
dc.rights CC-BY es_ES
dc.rights.uri http://creativecommons.org/licenses/by/4.0/deed.es es_ES
dc.title Minigene Splicing Assays Identify 12 Spliceogenic Variants of BRCA2 Exons 14 and 15 es_ES
dc.type info:eu-repo/semantics/article es_ES
dc.rights.accessrights info:eu-repo/semantics/openAccess es_ES
dc.identifier.location N/A es_ES


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