Characterization of spliceogenic variants located in regions linked to high levels of alternative splicing:<i>BRCA2</i>c.7976+5G &gt; T as a case study

Montalban, Gemma; Fraile-Bethencourt, Eugenia; López‐Perolio, Irene; Pérez‐Segura, Pedro; Infante, Mar; Durán, Mercedes; Alonso-Cerezo, María Concepción; López‐Fernández, Adrià; Dı́ez, Orland; Hoya, Miguel de la; Velasco, Eladio A.; Gutiérrez‐Enríquez, Sara

doi:10.1002/humu.23583

Cited by 12 publications

(13 citation statements)

References 31 publications

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“…In this context, minigenes represent a simple and robust tool to assay all potential splicing variants, since patient RNA samples are not always available. Actually, 260 BRCA1 / 2 variants have been so far tested by minigenes . Moreover, minigenes present some important advantages such as: (1) analysis of one single allele effect; (2) identification and quantification of all transcripts; (3) study of multiple DNA variants using just one multiple‐exon minigene; (4) assay in many cell types, essential for tissue‐specific alternative splicing; and (5) high reproducibility of physiological and pathological splicing patterns.…”

Section: Discussionsupporting

confidence: 75%

“…Previous results from our group showed that minigene tests of at least 16 different BRCA2 variants from exons 17 to 27 reproduced patient RNA outcomes . Moreover, the reproducibility of MGBR2_2–9 splicing outcomes was supported by previous studies based on patient RNA or minigene data, according to which fourteen variants matched preceding results: c.67+3A>G, c.68‐7T>A, c.316+3del, c.316+5G>C, c.426‐12_426‐8del, c.467A>G, c.516+ 1G>T, c.517G>T, c.572A>G, c.617C>G, c.627C>A/T, c.631G>A, c.631+3A>G and c.681+4A>G .…”

Section: Discussionsupporting

confidence: 75%

“…It is also worthy of mention that 10 ACMG pathogenic/likely pathogenic variants (four of them VUS) affected nucleotides surrounding the ultraconserved positions +1 and +2 of the donor site: c.67G>A, c.67+3A>G, c.316+3delA, c.316+5G>C, c.316+6T>C c.475+3A>T, c.516+4_516+5del, c.631G>A, c.631+3A>G and c.681+4A>G. Given the high conservation of these sequences (http://www.life.umd.edu/labs/mount/RNAinfo/consensus.html; MAGgtaagt), our data confirm the potential ‘spliceogenicity’ of mutations in any of these nucleotides, spanning from the last 3 nt of each exon to the intron +6 position. Importantly, in silico analysis of c.316+5G>C and c.316+6T>C did not detect abrogation of exon 3‐5′SS, like the previously reported variant c.7976+5G>T, which by contrary generated major splicing defects (see supplementary material, Tables S5 and S6) .…”

Section: Discussionmentioning

confidence: 99%

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Mis‐splicing in breast cancer: identification of pathogenic BRCA2 variants by systematic minigene assays

Fraile-Bethencourt

Valenzuela-Palomo

Díez-Gómez

et al. 2019

The Journal of Pathology

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Splicing disruption is a common mechanism of gene inactivation associated with germline variants of susceptibility genes. To study the role of BRCA2 mis-splicing in hereditary breast/ovarian cancer (HBOC), we performed a comprehensive analysis of variants from BRCA2 exons 2-9, as well as the initial characterization of the regulatory mechanisms of such exons. A pSAD-based minigene with exons 2-9 was constructed and validated in MCF-7 cells, producing the expected transcript (1016-nt/V1-BRCA2_exons_2-9-V2). DNA variants from mutational databases were analyzed by NNSplice and Human Splicing Finder softwares. To refine ESE-variant prediction, we mapped the regulatory regions through a functional strategy whereby 26 exonic microdeletions were introduced into the minigene and tested in MCF-7 cells. Thus, we identified nine spliceogenic ESE-rich intervals where ESE-variants may be located. Combining bioinformatics and microdeletion assays, 83 variants were selected and genetically engineered in the minigene. Fifty-three changes impaired splicing: 28 variants disrupted the canonical sites, four created new ones, 10 abrogated enhancers, eight created silencers and three caused a double-effect. Notably, nine spliceogenic-ESE variants were located within ESE-containing intervals. Capillary electrophoresis and sequencing revealed more than 23 aberrant transcripts, where exon skipping was the most common event. Interestingly, variant c.67G>A triggered the usage of a noncanonical GC-donor 4-nt upstream. Thirty-six variants that induced severe anomalies (>60% aberrant transcripts) were analyzed according to the ACMG guidelines. Thus, 28 variants were classified as pathogenic, five as likely pathogenic and three as variants of uncertain significance. Interestingly, 13 VUS were reclassified as pathogenic or likely pathogenic variants. In conclusion, a large fraction of BRCA2 variants (∼64%) provoked splicing anomalies lending further support to the high prevalence of this disease-mechanism. The low accuracy of ESE-prediction algorithms may be circumvented by functional ESE-mapping that represents an optimal strategy to identify spliceogenic ESE-variants. Finally, systematic functional assays by minigenes depict a valuable tool for the initial characterization of splicing anomalies and the clinical interpretation of variants.

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Section: Discussionsupporting

confidence: 75%

Section: Discussionsupporting

confidence: 75%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Mis‐splicing in breast cancer: identification of pathogenic BRCA2 variants by systematic minigene assays

Fraile-Bethencourt

Valenzuela-Palomo

Díez-Gómez

et al. 2019

The Journal of Pathology

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“…The minigene MGBR2_14-20 had previously been used to study variants of exons 16, 17, and 18, proving that it is a reliable and robust tool to functionally assay splicing variants (Fraile-Bethencourt et al, 2017, 2018; Montalban et al, 2018). The MGBR2_14-20 is a 10.7 Kb construct which, after transfection in MCF-7 cells, produces a transcript with the following structure: V1-BRCA2_exons from 14 to 20-V2 (1,806 nt) (Figure 1).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2019

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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.

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“…A recent study reported that variants affecting splicing, namely spliceogenic, are the third most common type occurring in BRCA1 and BRCA2 genes, accounting for 10.1% and 7.6% of the total, respectively (Rebbeck et al, ). Although multiple spliceogenic variants producing abnormal transcripts have been identified in HBOC families, the clinical relevance of the splicing outcomes can be sometimes difficult to determine (de Caputo et al, ; Colombo et al, ; la Hoya et al, ; Montalban et al, ). Furthermore, numerous BRCA1/2 alternative splicing events have been described in several tissues and cell lines, adding complexity to the interpretation of splicing outcomes generated from variant alleles (Colombo et al, ; Fackenthal et al, ).…”

Section: Introductionmentioning

confidence: 99%

Incorporation of semi‐quantitative analysis of splicing alterations for the clinical interpretation of variants inBRCA1andBRCA2genes

Montalban¹,

Bonache²,

Moles‐Fernández³

et al. 2019

Human Mutation

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BRCA1 and BRCA2 (BRCA1/2) genetic variants that disrupt messenger RNA splicing are commonly associated with increased risks of developing breast/ovarian cancer.The majority of splicing studies published to date rely on qualitative methodologies (i.e., Sanger sequencing), but it is necessary to incorporate semi-quantitative or quantitative approaches to accurately interpret the clinical significance of spliceogenic variants. Here, we characterize the splicing impact of 31 BRCA1/2 variants using semi-quantitative capillary electrophoresis of fluorescent amplicons (CE), Sanger sequencing and allele-specific assays. A total of 14 variants were found to disrupt splicing. Allelic-specific assays could be performed for BRCA1 c.302−1G>A and BRCA2 c.

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Characterization of spliceogenic variants located in regions linked to high levels of alternative splicing:BRCA2c.7976+5G > T as a case study

Cited by 12 publications

References 31 publications

Mis‐splicing in breast cancer: identification of pathogenic BRCA2 variants by systematic minigene assays

Mis‐splicing in breast cancer: identification of pathogenic BRCA2 variants by systematic minigene assays

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

Incorporation of semi‐quantitative analysis of splicing alterations for the clinical interpretation of variants inBRCA1andBRCA2genes

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