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

Investigation published in Frontiers in genetics

May 28th, 2019

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: (E14q5), Δ (E14p10), Δ(E14p246), Δ(E14q256), Δ(E14), Δ(E15p12), Δ(E15p13), Δ(E15p83), Δ(E15) and a 942-nt fragment of unknown structure. All transcripts, except for Δ(E14q256) and Δ(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




Fraile-Bethencourt E., Valenzuela-Palomo A., Diez-Gomez B., Caloca MJ., Gomez S. and Velasco EA.




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

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

Participants:

Consejo Superior de Investigaciones CientíficasGrupo de Splicing y Cáncer. Instituto de Biología y Genética Molecular (IBGM). Consejo Superior de Investigaciones Científicas (CSIC).

Universidad ComplutenseCentro de Vigilancia Sanitaria Veterinaria (VISAVET). Universidad Complutense (UCM).







FACTOR YEAR Q
3.517 2018

NLMID: 101560321

PMID: 31191615

ISSN: 1664-8021



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


JOURNAL: Front Genet


NUMERACIÓN: 10:503


AÑO: 2019


PUBLISHER: Lausanne : Frontiers Research Foundation


AUTHORS: Fraile-Bethencourt E., Valenzuela-Palomo A., Diez-Gomez B., Caloca MJ., Gomez S. and Velasco EA.


VISAVET PARTICIPANTS


Susana Gómez Barrero

DOI: https://doi.org/ 10.3389/fgene.2019.00503


CITE THIS PUBLICATION:

Fraile-Bethencourt E., Valenzuela-Palomo A., Diez-Gomez B., Caloca MJ., Gomez S. and Velasco EA. Minigene Splicing Assays Identify 12 Spliceogenic Variants of BRCA2 Exons 14 and 15. Frontiers in genetics. 10:503. 2019. (A). ISSN: 1664-8021. DOI: 10.3389/fgene.2019.00503