Dataset Information

Identification of Eight Spliceogenic Variants in BRCA2 Exon 16 by Minigene Assays.

ABSTRACT: Genetic testing of BRCA1 and BRCA2 identifies a large number of variants of uncertain clinical significance whose functional and clinical interpretations pose a challenge for genetic counseling. Interestingly, a relevant fraction of DNA variants can disrupt the splicing process in cancer susceptibility genes. We have tested more than 200 variants throughout 19 BRCA2 exons mostly by minigene assays, 54% of which displayed aberrant splicing, thus confirming the utility of this assay to check genetic variants in the absence of patient RNA. Our goal was to investigate BRCA2 exon 16 with a view to characterizing spliceogenic variants recorded at the mutational databases. Seventy-two different BIC and UMD variants were analyzed with NNSplice and Human Splicing Finder, 12 of which were selected because they were predicted to disrupt essential splice motifs: canonical splice sites (ss; eight variants) and exonic/intronic splicing enhancers (four variants). These 12 candidate variants were introduced into the BRCA2 minigene with seven exons (14-20) by site-directed mutagenesis and then transfected into MCF-7 cells. Seven variants (six intronic and one missense) induced complete abnormal splicing patterns: c.7618-2A>T, c.7618-2A>G, c.7618-1G>C, c.7618-1G>A, c.7805G>C, c.7805+1G>A, and c.7805+3A>C, as well as a partial anomalous outcome by c.7802A>G. They generated at least 10 different transcripts: ?16p44 (alternative 3'ss 44-nt downstream; acceptor variants), ?16 (exon 16-skipping; donor variants), ?16p55 (alternative 3'ss 55-nt downstream), ?16q4 (alternative 5'ss 4-nt upstream), ?16q100 (alternative 5'ss 4-nt upstream), ?16q20 (alternative 5'ss 20-nt downstream), as well as minor (?16p93 and ?16,17p69) and uncharacterized transcripts of 893 and 954 nucleotides. Isoforms ?16p44, ?16, ?16p55, ?16q4, ?16q100, and ?16q20 introduced premature termination codons which presumably inactivate BRCA2. According to the guidelines the American College of Medical Genetics and Genomics these eight variants could be classified as pathogenic or likely pathogenic whereas the Evidence-based Network for the Interpretation of Germline Mutant Alleles rules suggested seven class 4 and one class 3 variants. In conclusion, our study highlights the relevance of splicing functional assays by hybrid minigenes for the clinical classification of genetic variations. Hence, we provide new data about spliceogenic variants of BRCA2 exon 16 that are directly correlated with breast cancer susceptibility.

SUBMITTER: Fraile-Bethencourt E

PROVIDER: S-EPMC5977032 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Identification of Eight Spliceogenic Variants in BRCA2 Exon 16 by Minigene Assays.

Fraile-Bethencourt Eugenia E Valenzuela-Palomo Alberto A Díez-Gómez Beatriz B Acedo Alberto A Velasco Eladio A EA

Frontiers in genetics 20180524

Genetic testing of <i>BRCA1</i> and <i>BRCA2</i> identifies a large number of variants of uncertain clinical significance whose functional and clinical interpretations pose a challenge for genetic counseling. Interestingly, a relevant fraction of DNA variants can disrupt the splicing process in cancer susceptibility genes. We have tested more than 200 variants throughout 19 BRCA2 exons mostly by minigene assays, 54% of which displayed aberrant splicing, thus confirming the utility of this assay ...[more]

PMID: 29881398

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Project description: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.

Project description:Mutation 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. Fifty-two 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 (1,806 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.7944-7973) 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.7806-9T>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.

Dataset Information

Identification of Eight Spliceogenic Variants in BRCA2 Exon 16 by Minigene Assays.

Publications

Identification of Eight Spliceogenic Variants in BRCA2 Exon 16 by Minigene Assays.

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