Simple SummaryHereditary Breast and Ovarian Cancer syndrome (HBOC) is an inherited trait that predisposes adults to an earlier onset of cancer than the general population. HBOC is an autosomal dominant condition caused by heterozygous mutations in one of the HBOC genes. Pathogenic variants inBRCA1andBRCA2are considered to be the most prevalent causes of HBOC, though mutations on other less common genes have also been described. In 2017, the French Genetic and Cancer Group recommended the screening of 13 genes in individuals with a strong suspicion of HBOC. Here, we report and discuss the results of a retrospective analysis of genetic data related to HBOC gene sequences in a large cohort of 4630 French cases. This work also demonstrated the importance of retesting BRCA1/2 negative cases, as well as the relevance of functional splicing tests in variant classification.AbstractThe use of multigene panel testing for patients with a predisposition to Hereditary Breast and Ovarian Cancer syndrome (HBOC) is increasing as the identification of mutations is useful for diagnosis and disease management. Here, we conducted a retrospective analysis of BRCA1/2 and non-BRCA gene sequencing in 4630 French HBOC suspected patients. Patients were investigated using a germline cancer panel including the 13 genes defined by The French Genetic and Cancer Group (GGC)—Unicancer. In the patients analyzed, 528 pathogenic and likely pathogenic variants (P/LP) were identified, includingBRCA1(n = 203, 38%),BRCA2(n = 198, 37%),PALB2(n = 46, 9%),RAD51C(n = 36, 7%),TP53(n = 16, 3%), andRAD51D(n = 13, 2%). In addition, 35 novel (P/LP) variants, according to our knowledge, were identified, and double mutations in two distinct genes were found in five patients. Interestingly, retesting a subset of BRCA1/2-negative individuals with an expanded panel produced clinically relevant results in 5% of cases. Additionally, combining in silico (splicing impact prediction tools) and in vitro analyses (RT-PCR and Sanger sequencing) highlighted the deleterious impact of four candidate variants on splicing and translation. Our results present an overview of pathogenic variations of HBOC genes in the southeast of France, emphasizing the clinical relevance of cDNA analysis and the importance of retesting BRCA-negative individuals with an expanded panel.