Functional classification of DNA variants by hybrid minigenes: identification of 30 spliceogenic variants of BRCA2 exons 17 and 18

dc.contributor.authorFraile-Bethencourt, Eugenia
dc.contributor.authorDíez-Gómez, Beatriz
dc.contributor.authorVelásquez-Zapata, Valeria
dc.contributor.authorAcedo, Alberto
dc.contributor.authorSanz, David J.
dc.contributor.authorVelasco, Eladio A.
dc.contributor.funderBanco Santander
dc.contributor.funderUniversidad de Valladolid
dc.contributor.funderMinisterio de Economía y Competitividad
dc.contributor.funderEuropean Regional Development Fund
dc.contributor.funderConsejería de Educación, Junta de Castilla y León
dc.date.accessioned2018-06-15T11:47:20Z
dc.date.available2018-06-15T11:47:20Z
dc.date.issued2017
dc.description.abstractMutation screening of the breast cancer genes BRCA1 and BRCA2 identifies a large fraction of variants of uncertain clinical significance (VUS) whose functional and clinical interpretations pose a challenge for genomic medicine. Likewise, an increasing amount of evidence indicates that genetic variants can have deleterious effects on pre-mRNA splicing. Our goal was to investigate the impact on splicing of a set of reported variants of BRCA2 exons 17 and 18 to assess their role in hereditary breast cancer and to identify critical regulatory elements that may constitute hotspots for spliceogenic variants. A splicing reporter minigene with BRCA2 exons 14 to-20 (MGBR2_ ex14-20) was constructed in the pSAD vector. Fiftytwo candidate variants were selected with splicing prediction programs, introduced in MGBR2_ ex14-20 by site-directed mutagenesis and assayed in triplicate in MCF-7 cells. Wild type MGBR2_ ex14-20 produced a stable transcript of the expected size (1,802 nucleotides) and structure (V1-[BRCA2_ exons_ 14-20]-V2). Functional mapping by microdeletions revealed essential sequences for exon recognition on the 3 ' end of exon 17 (c. 79447973) and the 5 ' end of exon 18 (c. 7979-7988, c. 7999-8013). Thirty out of the 52 selected variants induced anomalous splicing in minigene assays with > 16 different aberrant transcripts, where exon skipping was the most common event. A wide range of splicing motifs were affected including the canonical splice sites (15 variants), novel alternative sites (3 variants), the polypyrimidine tract (3 variants) and enhancers/silencers (9 variants). According to the guidelines of the American College of Medical Genetics and Genomics (ACMG), 20 variants could be classified as pathogenic (c. 7806-2A > G, c. 7806-1G > A, c. 7806-1G > T, c. 7806-1_ 7806-2dup, c. 7976+ 1G > A, c. 7977-3_ 7978del, c. 7977-2A > T, c. 7977-1G > T, c. 7977-1G > C, c. 8009C > A, c. 8331+ 1G > T and c. 8331+ 2T > C) or likely pathogenic (c. 78069T > G, c. 7976G > C, c. 7976G > A, c. 7977-7C > G, c. 7985C > G, c. 8023A > G, c. 8035G > T and c. 8331G > A), accounting for 30.8% of all pathogenic/likely pathogenic variants of exons 17-18 at the BRCA Share database. The remaining 8 variants (c. 7975A > G, c. 7977-6T > G, c. 7988A > T, c. 7992T > A, c. 8007A > G, c. 8009C > T, c. 8009C > G, and c. 8072C > T) induced partial splicing anomalies with important ratios of the full-length transcript (>= 70%), so that they remained classified as VUS. Aberrant splicing is therefore especially prevalent in BRCA2 exons 17 and 18 due to the presence of active ESEs involved in exon recognition. Splicing functional assays with minigenes are a valuable strategy for the initial characterization of the splicing outcomes and the subsequent clinical interpretation of variants of any disease-gene, although these results should be checked, whenever possible, against patient RNA.en
dc.description.sponsorshipMinisterio de Economía y Competitividad (Plan Nacional de I+D+I 2013-2016, ISCIII (Fis: PI13/01749)); European Regional Development Fund (FEDER; Centre of Excellence in Genomics EXCEGEN Grant 3.2.0304.11-0312); Consejería de Educación, Junta de Castilla y León (Grant CSI090U14 ORDEN EDU/122/2014); Universidad de Valladolid and Banco de Santander (Predoctoral Fellowship)en
dc.description.statusPeer reviewed
dc.description.versionPublished Versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.articleide1006691
dc.identifier.citationFraile-Bethencourt, E., Díez-Gómez, B., Velásquez-Zapata, V., Acedo, A., Sanz, D. J. and Velasco, E. A. (2017) 'Functional classification of DNA variants by hybrid minigenes: identification of 30 spliceogenic variants of BRCA2 exons 17 and 18', PLOS Genetics, 13(3), e1006691 (21pp). doi: 10.1371/journal.pgen.1006691en
dc.identifier.doi10.1371/journal.pgen.1006691
dc.identifier.endpage21
dc.identifier.issn1553-7404
dc.identifier.issued3
dc.identifier.journaltitlePLOS Geneticsen
dc.identifier.startpage1
dc.identifier.urihttps://hdl.handle.net/10468/6358
dc.identifier.volume13
dc.language.isoenen
dc.publisherPublic Library of Scienceen
dc.relation.urihttp://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1006691
dc.rights© 2017, Fraile-Bethencourt et al. 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 credited.en
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subjectBreast cancer susceptibilityen
dc.subjectUnclassified variantsen
dc.subjectSplicing assaysen
dc.subjectOvarian canceren
dc.subjectSynonymous mutationsen
dc.subjectSequence variantsen
dc.subjectHuman genesen
dc.subjectRnaen
dc.subjectDiseaseen
dc.subjectGuidelinesen
dc.titleFunctional classification of DNA variants by hybrid minigenes: identification of 30 spliceogenic variants of BRCA2 exons 17 and 18en
dc.typeArticle (peer-reviewed)en
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