When considering family history, hereditary breast cancer (HBC) syndromes correspond to nearly 5-10% of all breast cancer (BC) cases. However, pathogenic variants in known moderate- and high-risk BC genes explain only ~30% of familial BC cases. Recent advances in sequencing technology resulted in an increasing number of BC genes being revealed in previously negative families by using extended gene panels and whole-exome sequencing (WES). WES offers the opportunity to concomitantly investigate several rare risk genes as well as to identify new BC-predisposing genes. Thus, in this study we used WES to disclose variants contributing to BC increased risk in patients that were negative for mutations in three major BC genes (BRCA1/2 and TP53) and met stringent clinical criteria indicating a genetic predisposition to BC. We selected 17 women (two of them sisters) who fulfilled one or more of the following criteria: early onset BC (<36 years); bilateral BC; breast and other primary related tumor (ovary, fallopian tube, or primary peritoneal tumors). Firstly, we used WES data to search for rare variants in 27 well-established and emerging HBC predisposing genes. This analysis identified two patients harboring truncating mutations, one in ATM (p.Tyr2334Glnfs*4 – pathogenic in ClinVar database) and one in BARD1 (Tyr739Leufs*2 – not described, probably pathogenic according to ACMG guidelines). Additionally, we identified nine variants of unknown clinical significance in these genes, occurring in seven patients. For the remaining 15 patients with no putative pathogenic mutations in HBC genes, we first applied quality filters to exclude false-positive results (included variants: variant base coverage ≥5X, variant frequency ≥25%, QD>2, FS<6). Remaining variants were excluded if present in population databases with a minor allele frequency (MAF) > 0.01 or present in five BRCA1 mutated patients sequenced in the same platforms. Next, using a functional-based variant prioritization, we selected candidate variants according to the predicted impact in the protein function, the affected gene, and segregation with the phenotype (in the family-based study of the two sisters). Candidate variants included all loss-of-function variants as well as missense and in-frame indels occurring in a specific list of 651 genes of interest (DNA repair and cancer-related genes) and if predicted to be damaging by at least 3/6 prediction software. The resulting 244 selected variants were submitted for technical validation by targeted NGS and, of these, 220 were validated (89%). Using the same custom panel, we evaluated 25 control samples of healthy Brazilian women for filtering common polymorphisms in our population, resulting in a final 139 variants in 129 genes (89 LOF and 50 missense variants). For two families---one with WES of two affected sisters and target NGS of one affected aunt; one with WES of the affected daughter and target NGS of the affected mother---we were able to perform cosegregation analysis in other family members and 11 out of 23 variants were found to be segregating with the disease. These 11 distinct genes as well as 82 genes harboring LOF variants were evaluated in an independent cohort of 34 WES of high-risk BC patients. Several rare, possibly damaging variants were identified in this cohort, providing additional evidence of the potential role in BC predisposition of some new genes. Of those, we highlight ATRX, FANCC, TET2, ERCC1, PTPRD, and ROS1 genes, which are involved in DNA repair and other tumorigenic pathways. Overall, our results provide a set of putative BC-predisposing genes and underpin WES as a useful tool for assessing the complex landscape of HBC predisposition. The discovery and validation of new HBC genes in different populations will continue to provide insights into disease mechanisms, eventually leading to the development of more effective therapies and improved management of affected families.

Citation Format: Giovana T. Torrezan, Fernanda GSR Almeida, Marcia CP Figueiredo, Bruna DF Barros, Claudia A. Paula, Renan Valieris, Jorge ES Souza, Elisa N. Ferreira, Felipe CC Silva, Amanda F. Nobrega, Maria Isabel Achatz, Edenir I. Palmero, Rodrigo F. Ramalho, Sandro J. Souza, Dirce M. Carraro. Complex landscape of germline variants in hereditary and early-onset breast cancer ascertained through whole exome sequencing [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A37.