Multigene Panel Sequencing Identifies a Novel Germline Mutation Profile in Male Breast Cancer Patients.

Even though male breast cancer (MBC) risk encompasses both genetic and environmental aetiologies, the primary risk factor is a germline pathogenic variant (PV) or likely pathogenic variant (LPV) in BRCA2, BRCA1 and/or PALB2 genes. To identify new potential MBC-specific predisposition genes, we sequenced a panel of 585 carcinogenesis genes in an MBC cohort without BRCA1/BRCA2/PALB2 PV/LPV. We identified 14 genes carrying rare PVs/LPVs in the MBC population versus noncancer non-Finnish European men, predominantly coding for DNA repair and maintenance of genomic stability proteins. We identified for the first time PVs/LPVs in PRCC (pre-mRNA processing), HOXA9 (transcription regulation), RECQL4 and WRN (maintenance of genomic stability) as well as in genes involved in other cellular processes. To study the specificity of this MBC PV/LPV profile, we examined whether variants in the same genes could be detected in a female breast cancer (FBC) cohort without BRCA1/BRCA2/PALB2 PV/LPV. Only 5/109 women (4.6%) carried a PV/LPV versus 18/85 men (21.2%) on these genes. FBC did not carry any PV/LPV on 11 of these genes. Although 5.9% of the MBC cohort carried PVs/LPVs in PALLD and ERCC2, neither of these genes were altered in our FBC cohort. Our data suggest that in addition to BRCA1/BRCA2/PALB2, other genes involved in DNA repair/maintenance or genomic stability as well as cell adhesion may form a specific MBC PV/LPV signature.

5' Region Large Genomic Rearrangements in the BRCA1 Gene in French Families: Identification of a Tandem Triplication and Nine Distinct Deletions with Five Recurrent Breakpoints.

BACKGROUND: Large genomic rearrangements (LGR) in BRCA1 consisting of deletions/duplications of one or several exons have been found throughout the gene with a large proportion occurring in the 5' region from the promoter to exon 2. The aim of this study was to better characterize those LGR in French high-risk breast/ovarian cancer families. METHODS: DNA from 20 families with one apparent duplication and nine deletions was analyzed with a dedicated comparative genomic hybridization (CGH) array, high-resolution BRCA1 Genomic Morse Codes analysis and Sanger sequencing. RESULTS: The apparent duplication was in fact a tandem triplication of exons 1 and 2 and part of intron 2 of BRCA1, fully characterized here for the first time. We calculated a causality score with the multifactorial model from data obtained from six families, classifying this variant as benign. Among the nine deletions detected in this region, eight have never been identified. The breakpoints fell in six recurrent regions and could confirm some specific conformation of the chromatin. CONCLUSIONS: Taken together, our results firmly establish that the BRCA1 5' region is a frequent site of different LGRs and highlight the importance of the segmental duplication and Alu sequences, particularly the very high homologous region, in the mechanism of a recombination event. This also confirmed that those events are not systematically deleterious.

The c.5242C>A BRCA1 missense variant induces exon skipping by increasing splicing repressors binding.

Several unclassified variants (UV) of BRCA1 can be deleterious by affecting normal pre-mRNA splicing. Here, we investigated the consequences at the mRNA level of the frequently encountered c.5242C>A UV in BRCA1 exon 18. We show that the c.5242C>A variant induces skipping of exon 18 in UV carriers and in vitro. This alteration predicted to disrupt the first BRCT domain of BRCA1. We show that two splicing repressors, hnRNP A1 and hnRNP H/F, display a significant preference toward binding with the mutated exon 18 and assemble into a protein complex. Sequence analysis of the region surrounding the c.5242C>A change reveals the presence of hnRNP A1 and hnRNP H/F binding sites, which are modified by several UVs. Mutation of these sites alters the RNA binding ability of both splicing regulators. In conclusion, our work supports the model of the pathogenicity of the c.5242C>A BRCA1 variant that induces exon skipping by creating a sequence with silencer properties. We propose that other UVs in exon 18 interfere with splicing complex assembly by perturbing the binding of hnRNP A1 and hnRNP H/F to their respective cis-elements. RNA analysis is therefore necessary for the assessment of the consequences of UVs on splicing of disease-associated genes and to enable adequate genetic counseling for breast/ovarian cancer families.