Germline variants in cancer genes in high-risk non-BRCA patients from Puerto Rico

Abstract

Inherited pathogenic variants in genes that confer moderate to high risk of breast cancer may explain up to 50% of familial breast cancer. This study aimed at identifying inherited pathogenic variants in breast cancer cases from Puerto Rico that were not linked to BRCA1 or BRCA2. Forty-eight breast cancer patients that met the clinical criteria for BRCA testing but had received a negative BRCA1/2 result were recruited. Fifty-three genes previously implicated in hereditary cancer predisposition were captured using the BROCA Agilent cancer risk panel followed by massively parallel sequencing. Missense variants of uncertain clinical significance in CHEK2 were evaluated using an in vitro kinase assays to determine their impact on function. Pathogenic variants were identified in CHEK2, MUTYH, and RAD51B in four breast cancer patients, which represented 8.3% of the cohort. We identified three rare missense variants of uncertain significance in CHEK2 and two variants (p.Pro484Leu and p.Glu239Lys) showed markedly decreased kinase activity in vitro comparable to a known pathogenic variant. Interestingly, the local ancestry at the RAD51B locus in the carrier of p.Arg47* was predicted to be of African origin. In this cohort, 12.5% of the BRCA-negative breast cancer patients were found to carry a known pathogenic variant or a variant affecting protein activity. This study reveals an unmet clinical need of genetic testing that could benefit a significant proportion of at-risk Latinas. It also highlights the complexity of Hispanic populations as pathogenic factors may originate from any of the ancestral populations that make up their genetic backgrounds.

Figure 1

(a) Proportion of unrelated BC patients by carrier status and (b) distribution after grouping by age of onset and family history characteristics. Carriers categories are: harboring at least one coding variant classified as likely pathogenic or pathogenic (L/Pathogenic), VUS in NCCN clinically actionable genes, VUS in other genes, or no L/Pathogenic variant or VUS identified. Early onset breast cancer refers to individuals for which diagnosis was at 45 years of age or less. Positive family history includes only individuals for which a first, or second degree relative was diagnosed with an early onset breast cancer. NCCN clinically actionable BC genes are ATM, BRCA1, BRCA2, CDH1, CHEK2, NBN, NF1, PALB2, PTEN, STK11, and TP53 (NCCN Guidelines for Genetic/Familial High-Risk assessment v3.2019). The complete list of genes included in the BROCA panel is available in the methods section. Coding variants included truncating, missense, deletions and duplications. Synonymous variants were excluded from this analysis. BC breast cancer; VUS variant of uncertain significance.

Figure 2

Family history of cancer (site and age of diagnosis, dx) in relatives of carriers of a known or predicted pathogenic variant in CHEK2 (a), MUTYH (b), and RAD51B (c). Sites of carcinoma are Br breast cancer, Col colorectal cancer, Gas gastric cancer, MM multiple myeloma, Pr prostate cancer. Probands are designated by a red arrow.

Figure 3

(A) Diagram human CHK2 protein domains and variants tested in kinase assays in the current study (purple) or known pathogenic variants (red). (B) Ribbon and Surface view of human CHK2 dimer (aa 89–501) with variants tested in this study (purple) and known pathogenic variants (red) (PDB ID: 3I6U). (C) Western blot analysis of CHK2 expression and activation in HEK293T expressing wild-type CHK2 or the indicated missense variants. Cells were exposed to 6 Gy IR and WCL (whole cell lysates) were extracted 1 h post-exposure. (D) Immunoprecipitated FLAG-CHK2 complexes from HEK293T lysates were immunoblotted for CHK2 and phospho-CHK2. E. In vitro kinase assays from HEK293T cell lysates. FLAG-CHK2 immunoprecipitates were incubated with a purified GST-CDC25C (aa 200–256) peptide. Both auto-phosphorylation of CHK2 and phosphorylation of CDC25C were used as a measure of CHK2 kinase activity.