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PALB2 loss‐of‐function variants confer high risk of developing breast cancer. Here we present a systematic functional analysis of PALB2 splice‐site variants detected in approximately 113,000 women in the large‐scale sequencing project Breast Cancer After Diagnostic Gene Sequencing (BRIDGES; https://bridges-research.eu/). Eighty‐two PALB2 variants at the intron‐exon boundaries were analyzed with MaxEntScan. Forty‐two variants were selected for the subsequent splicing functional assays. For this purpose, three splicing reporter minigenes comprising exons 1–12 were constructed. The 42 potential spliceogenic variants were introduced into the minigenes by site‐directed mutagenesis and assayed in MCF‐7/MDA‐MB‐231 cells. Splicing anomalies were observed in 35 variants, 23 of which showed no traces or minimal amounts of the expected full‐length transcripts of each minigene. More than 30 different variant‐induced transcripts were characterized, 23 of which were predicted to truncate the PALB2 protein. The pathogenicity of all variants was interpreted according to an in‐house adaptation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG‐AMP) variant classification scheme. Up to 23 variants were classified as pathogenic/likely pathogenic. Remarkably, three ±1,2 variants (c.49‐2A>T, c.108+2T>C, and c.211+1G>A) were classified as variants of unknown significance, as they produced significant amounts of either in‐frame transcripts of unknown impact on the PALB2 protein function or the minigene full‐length transcripts. In conclusion, we have significantly contributed to the ongoing effort of identifying spliceogenic variants in the clinically relevant PALB2 cancer susceptibility gene. Moreover, we suggest some approaches to classify the findings in accordance with the ACMG‐AMP rationale. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Purpose: Promoter mutations may affect transcription and can be associated with human diseases. However, the promoters of the breast cancer (BC) genes are not regularly screened. Our goal was to investigate the BRCA2 promoter in order to study a possible correlation between impaired transcription and disease.Methods: The proximal and core promoter of the BRCA2 gene was sequenced in 95 high-risk BC patients. A BRCA2-promoter insert [− 938 to + 312 from the transcription start site (TSS)] was generated and cloned into the firefly luciferase vector pGL4.10. Promoter variants and deletions were introduced by site-directed mutagenesis and quantified by Dual-Luciferase assays and semi-quantitative RT-PCR.Results: Three different variants were detected in high-risk BC patients: rs3092989, rs206118, and rs563971900. Functional mapping of 13 overlapping deletions revealed four down-regulating segments (TSS positions): −59_−10del/µdel3 (16% of activity of the wild-type construct), −104_−55del/µdel4 (62%), −239_−190del/µdel7 (39%), −464_−415/µdel12 (78%), suggesting the presence therein of putative transcriptional activator motifs. Additionally, six microdeletions rendered luciferase overexpression: +32_+81del/µdel1 (356%), −14_+36del/µdel2 (180%), −194_−145del/µdel6 (154%), −284_−235del/µdel8 (168%), −329_−280del/µdel9 (111%), and −509_−460del/µdel13 (139%), which is indicative of repressor elements. Functional assays of 15 promoter variants (including those detected in patients) showed that ten of them significantly altered expression with seven up-regulating (113-163%) and three down-regulating (rs551887850_G, rs570548398_T, rs55880202_T; 72-83%) SNPs. Eight of them were located in an ENCODE-DNase Hypersensitive Cluster (TSS − 185 to + 105) where most active transcriptional motifs are known to be placed.Conclusions: BRCA2 expression is highly sensitive to promoter variations as most of them induced relevant changes. Moreover, we mapped critical regions of the BRCA2 promoter that may constitute potential targets for regulatory variants. Three SNPs moderately decreased luciferase activity, but confirmation of its potential pathogenicity requires further analysis. These data reinforce the need to screen the promoter regions of breast cancer genes with a view to discovering novel deleterious mutations. [ABSTRACT FROM AUTHOR]

Simple Summary: Genetic variants in more than 10 genes are known to confer moderate to high risks to breast and/or ovarian cancers (BC/OC). In the framework of the international project BRIDGES, a panel of 34 known or suspected BC/OC genes has been sequenced in 60,466 breast cancer patients and 53,461 controls. In this work, we focus on BRIDGES variants detected in the RAD51C gene and their impact on the gene expression step known as splicing (intron removal), whose alteration is a relevant disease mechanism. For this purpose, we bioinformatically analyzed 40 RAD51C variants from the intron/exon boundaries, 20 of which were selected. Then, we developed a biotechnological tool, called splicing reporter minigene, containing RAD51C exons 2 to 8 where any variant can be introduced by site-directed mutagenesis and functionally assayed in MCF-7 cells under the splicing perspective. Nineteen variants impaired splicing, 18 of which induced severe splicing anomalies. Finally, they were clinically interpreted according to strict guidelines whereby 15 variants were classified as Pathogenic/Likely Pathogenic, so they are clinically actionable. Therefore, carrier patients and families may benefit from tailored prevention protocols and therapies. Hereditary breast and/or ovarian cancer is a highly heterogeneous disease with more than 10 known disease-associated genes. In the framework of the BRIDGES project (Breast Cancer Risk after Diagnostic Gene Sequencing), the RAD51C gene has been sequenced in 60,466 breast cancer patients and 53,461 controls. We aimed at functionally characterizing all the identified genetic variants that are predicted to disrupt the splicing process. Forty RAD51C variants of the intron-exon boundaries were bioinformatically analyzed, 20 of which were selected for splicing functional assays. To test them, a splicing reporter minigene with exons 2 to 8 was designed and constructed. This minigene generated a full-length transcript of the expected size (1062 nucleotides), sequence, and structure (Vector exon V1- RAD51C exons_2-8- Vector exon V2). The 20 candidate variants were genetically engineered into the wild type minigene and functionally assayed in MCF-7 cells. Nineteen variants (95%) impaired splicing, while 18 of them produced severe splicing anomalies. At least 35 transcripts were generated by the mutant minigenes: 16 protein-truncating, 6 in-frame, and 13 minor uncharacterized isoforms. According to ACMG/AMP-based standards, 15 variants could be classified as pathogenic or likely pathogenic variants: c.404G > A, c.405-6T > A, c.571 + 4A > G, c.571 + 5G > A, c.572-1G > T, c.705G > T, c.706-2A > C, c.706-2A > G, c.837 + 2T > C, c.905-3C > G, c.905-2A > C, c.905-2_905-1del, c.965 + 5G > A, c.1026 + 5_1026 + 7del, and c.1026 + 5G > T. [ABSTRACT FROM AUTHOR]