Naturally occurring<i>BRCA2</i>alternative mRNA splicing events in clinically relevant samples

Fackenthal, James D.; Yoshimatsu, Toshio F.; Zhang, Bifeng; Garibay, Gorka Ruíz de; Colombo, Mara; Vecchi, Giovanna De; Ayoub, Samantha; Lal, Kumar; Olopade, Olufunmilayo I.; Vega, Ana; Santamariña, Marta; Blanco, Ana; Wappenschmidt, Barbara; Becker, Alexandra; Houdayer, Claude; Walker, Logan C.; López‐Perolio, Irene; Thomassen, Mads; Parsons, Michael T.; Whiley, Phillip J.; Blok, Marinus J.; Brandão, Rita D.; Tserpelis, Demis; Baralle, Diana; Montalban, Gemma; Gutiérrez‐Enríquez, Sara; Dı́ez, Orland; Lázaro, Conxi; Investigators, kConFab; Spurdle, Amanda B.; Radice, Paolo; Hoya, Miguel de la

doi:10.1136/jmedgenet-2015-103570

Cited by 45 publications

(80 citation statements)

References 47 publications

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“…The NNSplice, MaxEnt and HSF algorithms accurately anticipated the splice site disruptions and the generation of novel active sites, but the splicing outcomes were absolutely unpredictable reinforcing the current need of functional assays. The characterization of the physiological alternative splicing events of BRCA1 and BRCA2 [14,47] and improved computer tools will help to estimate the aberrant transcripts that a particular DNA variant may generate. It is also worthy to mention that three variants of the polypyrimidine tract, c.7806-9T>G, c.7977-7C>G and c.7977-6T>G, produced defective splicing.…”

Section: Discussionmentioning

confidence: 99%

“…Certainly, exons 17 and 18 undergo naturally-occurring alternative splicing producing minor transcripts Δ18 and Δ17+18 [28], although in our study Δ18 was only detected at even lower levels in the wild type minigene (<1%; S3 Table), together with the full-length transcript (≥99%). This may probably be due to: i) the genomic context that influences exon recognition [15]; ii) tissue-dependent alternative splicing as we used different host cells (MCF-7 vs. HeLa); and iii) RNA preparation and storage conditions, primer design, PCR conditions, and PCR product detection methodology can introduce small variations in splicing isoform ratios as previously reported [14,42]. …”

Section: Discussionmentioning

confidence: 99%

“…According to GENCODE (V.24, http://www.gencodegenes.org/stats/current.html), the average number of protein coding transcripts per gene is ~4. In this regard, it has been recently reported the existence of 24 naturally occurring alternative splicing events of the BRCA2 gene [14]. Alternative splicing not only allows transcriptome and proteome diversity, but it also regulates important processes such as embryonic development or cell differentiation.…”

Section: Introductionmentioning

confidence: 99%

“…Recognition of a GC donor site at the BTK gene was associated with splicing enhancers for SR proteins 9G8, Tra2β and SC35 [27]. The BRCA2 intron 17 has also a 5’ GC motif and there have been identified minor natural alternative splicing isoforms Δ18 (exon 18 skipping) and Δ17,18 (exons 17+18 skipping) [14,28]. To study the regulatory mechanisms of both exons and how DNA variants affect this process we constructed a large minigene with exons 14 to 20 in the pSAD plasmid (MGBR2_ex14-20) to keep the genomic context.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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“…This is adding to a growing body of data inferring clinical relevance of BRCA2 splicing. While many BRCA2 splice variants have been linked to distinct BRCA2 mutations, 12, 13, 17, 23, 24, 25, 26 our findings of the splice variant BRCA2 ΔE5 , which previously has only been described associated with the c.475+1G>A variant, 17 and the novel BRCA2 ΔE5+7 in the revertant SBRes cells infers a more general role of BRCA2 splicing in normal and malignant tissue, of which regulation is only partially understood. However, an integral role of splicing in double strand repair and chemo-resistance is increasingly recognized.…”

Section: Discussionmentioning

confidence: 55%

Acquired cross-linker resistance associated with a novel spliced BRCA2 protein variant for molecular phenotyping of BRCA2 disruption

et al. 2017

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BRCA2 encodes a protein with a fundamental role in homologous recombination that is essential for normal development. Carrier status of mutations in BRCA2 is associated with familial breast and ovarian cancer, while bi-allelic BRCA2 mutations can cause Fanconi anemia (FA), a cancer predisposition syndrome with cellular cross-linker hypersensitivity. Cancers associated with BRCA2 mutations can acquire chemo-resistance on relapse. We modeled acquired cross-linker resistance with an FA-derived BRCA2-mutated acute myeloid leukemia (AML) platform. Associated with acquired cross-linker resistance was the expression of a functional BRCA2 protein variant lacking exon 5 and exon 7 (BRCA2ΔE5+7), implying a role for BRCA2 splicing for acquired chemo-resistance. Integrated network analysis of transcriptomic and proteomic differences for phenotyping of BRCA2 disruption infers impact on transcription and chromatin remodeling in addition to the DNA damage response. The striking overlap with transcriptional profiles of FA patient hematopoiesis and BRCA mutation associated ovarian cancer helps define and explicate the ‘BRCAness’ profile.

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Thorough in silico and in vitro cDNA analysis of 21 putativeBRCA1andBRCA2splice variants and a complex tandem duplication inBRCA2allowing the identification of activated cryptic splice donor sites inBRCA2exon 11

et al. 2018

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For 21 putative BRCA1 and BRCA2 splice site variants, the concordance between mRNA analysis and predictions by in silico programs was evaluated. Aberrant splicing was confirmed for 12 alterations. In silico prediction tools were helpful to determine for which variants cDNA analysis is warranted, however, predictions for variants in the Cartegni consensus region but outside the canonical sites, were less reliable. Learning algorithms like Adaboost and Random Forest outperformed the classical tools. Further validations are warranted prior to implementation of these novel tools in clinical settings. Additionally, we report here for the first time activated cryptic donor sites in the large exon 11 of BRCA2 by evaluating the effect at the cDNA level of a novel tandem duplication (5' breakpoint in intron 4; 3' breakpoint in exon 11) and of a variant disrupting the splice donor site of exon 11 (c.6841+1G > C). Additional sites were predicted, but not activated. These sites warrant further research to increase our knowledge on cis and trans acting factors involved in the conservation of correct transcription of this large exon. This may contribute to adequate design of ASOs (antisense oligonucleotides), an emerging therapy to render cancer cells sensitive to PARP inhibitor and platinum therapies.

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Naturally occurringBRCA2alternative mRNA splicing events in clinically relevant samples

Cited by 45 publications

References 47 publications

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

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

Acquired cross-linker resistance associated with a novel spliced BRCA2 protein variant for molecular phenotyping of BRCA2 disruption

Thorough in silico and in vitro cDNA analysis of 21 putativeBRCA1andBRCA2splice variants and a complex tandem duplication inBRCA2allowing the identification of activated cryptic splice donor sites inBRCA2exon 11

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