An entire exon 3 germ-line rearrangement in the BRCA2 gene: pathogenic relevance of exon 3 deletion in breast cancer predisposition

Muller, Danièle; Rouleau, Étienne; Schultz, Inès; Caputo, Sandrine M.; Lefol, Cédrick; Bièche, Ivan; Caron, Olivier; Noguès, Catherine; Limacher, Jean Marc; Demange, L.; Lidereau, Rosette; Fricker, Jean Pierre; Abecassis, Joseph

doi:10.1186/1471-2350-12-121

Cited by 24 publications

(27 citation statements)

References 36 publications

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“…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 . Conversely, in a recent publication, variant c.68‐7T>A induced 20% of transcript Δ3 in lymphoblastoid cell lines of carrier patients versus 2.8% of Δ3 and 7.2% of Δ(6q 39 ,7) found in our minigene study in MCF‐7 cells .…”

Section: Discussionsupporting

confidence: 75%

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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%

“…Hence, changes in this region may cause severe alterations in protein structure and functionality. Likewise, variants c.316+2T>C, c.316+3delA, c.316+5G>C and c.316+6T>C, which also induced Δ3, had been already catalogued as pathogenic . Consequently, all the Δ3‐inducing variants should be considered deleterious.…”

Section: Discussionmentioning

confidence: 99%

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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“…Several BRCA2 alterations causing the complete loss of exon 3 and the exclusive synthesis of ∆3 transcripts have been ascertained, including c.316 + 5G > C (Bonnet et al., ), c.316 + 3delA and c.68‐925_316 + 2889del (Muller et al., ) and c.156_157insAlu, a variant reported as a founder Portuguese mutation (Peixoto et al., ).…”

Section: Discussionmentioning

confidence: 99%

“…In particular, using semiquantitative approaches, it has been documented that the variant leads to an increase of the naturally occurring transcripts lacking exon 3 (∆3) (Houdayer et al., ; Jarhelle, Riise Stensland, Maehle, & Van Ghelue, ; Sanz et al., ; Thery et al., ; Vreeswijk et al., ). A competitive quantitative PCR (qPCR) analysis estimated that the proportion of the ∆3 transcript compared to full length was approximately 25% in variant samples versus 4% in normal samples (Muller et al., ). More recently, segregation analyses in two families indicated that the variant did not segregate in the affected branches (Santos et al., ).…”

Section: Introductionmentioning

confidence: 99%

The BRCA2 c.68-7T > A variant is not pathogenic: A model for clinical calibration of spliceogenicity

Investigators

2018

Human Mutation

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Although the spliceogenic nature of the BRCA2 c.68‐7T > A variant has been demonstrated, its association with cancer risk remains controversial. In this study, we accurately quantified by real‐time PCR and digital PCR (dPCR), the BRCA2 isoforms retaining or missing exon 3. In addition, the combined odds ratio for causality of the variant was estimated using genetic and clinical data, and its associated cancer risk was estimated by case‐control analysis in 83,636 individuals. Co‐occurrence in trans with pathogenic BRCA2 variants was assessed in 5,382 families. Exon 3 exclusion rate was 4.5‐fold higher in variant carriers (13%) than controls (3%), indicating an exclusion rate for the c.68‐7T > A allele of approximately 20%. The posterior probability of pathogenicity was 7.44 × 10−115. There was neither evidence for increased risk of breast cancer (OR 1.03; 95% CI 0.86–1.24) nor for a deleterious effect of the variant when co‐occurring with pathogenic variants. Our data provide for the first time robust evidence of the nonpathogenicity of the BRCA2 c.68‐7T > A. Genetic and quantitative transcript analyses together inform the threshold for the ratio between functional and altered BRCA2 isoforms compatible with normal cell function. These findings might be exploited to assess the relevance for cancer risk of other BRCA2 spliceogenic variants.

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“…LGRs fully characterized at the molecular level [4,6,9,[15][16][17][18][19][20][21][22][23][24]. Here, we describe the molecular characterization of four additional LGRs indentified in Spanish HBOC families.…”

Section: Introductionmentioning

confidence: 99%

Characterization of four novel BRCA2 large genomic rearrangements in Spanish breast/ovarian cancer families: review of the literature, and reevaluation of the genetic mechanisms involved in their origin

Garibay

Gutiérrez‐Enríquez²,

Garré

et al. 2012

Breast Cancer Res Treat

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Large genomic rearrangements (LGRs) at the BRCA2 locus explain a non-negligible proportion of hereditary breast and ovarian cancer (HBOC) syndromes. The multiplex ligation and probe amplification (MLPA) assay has permitted in recent years to identify several families carrying LGRs at this locus, but very few such alterations have been fully characterized at the molecular level. Yet, molecular characterization is essential to identify recurrent alterations, to analyze the genetic mechanisms underlying such alterations, or to investigate potential genotype/phenotype relationships. We have used MLPA to identify BRCA2 LGRs in 7 out of 813 Spanish HBOC families previously tested negative for BRCA1 and BRCA2 small genomic alterations (substitutions and indels) and BRCA1 LGRs. We used a combination of long-range PCR, restriction mapping, and cDNA analysis to characterize the alterations at the molecular level. We found that Del Exon1-Exon2, Del Exon12-Exon16 and Del Exon22-Exon24 explain one family each, while Del Exon2 appears to be a Spanish founder mutation explaining four independent families. Finally, we have combined our data with a comprehensive review of the literature to reevaluate the genetic mechanisms underlying LGRs at the BRCA2 locus. Our study substantially increases the spectrum of BRCA2 LGRs fully characterized at the molecular level. Further on, we provide data to suggest that non-allelic homologous recombination has been overestimated as a mechanism underlying these alterations, while the opposite might be true for microhomology-mediated events.

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An entire exon 3 germ-line rearrangement in the BRCA2 gene: pathogenic relevance of exon 3 deletion in breast cancer predisposition

Cited by 24 publications

References 36 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

The BRCA2 c.68-7T > A variant is not pathogenic: A model for clinical calibration of spliceogenicity

Characterization of four novel BRCA2 large genomic rearrangements in Spanish breast/ovarian cancer families: review of the literature, and reevaluation of the genetic mechanisms involved in their origin

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