Effect of the overexpression of BRCA2 unclassified missense variants on spontaneous homologous recombination in human cells

Abstract

Breast Cancer 2 gene (BRCA2) mutation carriers have a 45% chance of developing breast cancer and a 11% risk of developing ovarian cancer by the age of 70. While hundreds of BRCA2-truncating mutations have been associated with an increased cancer risk in carriers, the contribution of unclassified variants (UCVs) to cancer risk remains largely undefined. BRCA2-defective cells show a high degree of chromosome instability. Although a functional assay based on the BRCA2 capability to stimulate DSB-induced homologous recombination (HR) as a way to classify UCVs has been proposed, so far no data are available concerning the effect of BRCA2 UCVs on spontaneous HR. In this study, we proposed a novel functional HR-based assay that determines the effect of the transient overexpression of the BRCA2 variant on spontaneous HR. This assay will help one in the difficult task of classifying UCVs, and it will give more information on how BRCA2 may induce genome instability and on the basic mechanism of BRCA2-induced tumourigenesis. We chose 11 BRCA2 UCVs not previously described or classified in other articles, and distributed along the entire BRCA2-coding region. They are as follows: G173V, D191V, S286P, M927V, T1011R, L1019V, N1878K, S2006R, R2108C, G2353R and V3091I. Basically, because the expression of BRCA2wt and the neutral variants did not increase spontaneous HR, we classified the variants G173V, S286P, M927V, T1011R and L1019V as HR-negative and presumed that they were not pathogenic. The HR-positive variants, D191V, N1878K, S2006R, R2108C, G2353R, and V3091I, which increased HR as much as the cancer-associated variant G2748D, could probably be classified as pathogenic. We observed that all our variants in the C-terminus of the protein behaved differently from the wt, suggesting a role for this protein region in spontaneous HR.

Minimally invasive genomic and transcriptomic profiling of visceral cancers by next-generation sequencing of circulating exosomes

“…Several potentially actionable mutations were identified ( supplementary Table S12, available at Annals of Oncology online ) including an unexpected somatic mutation of BRCA2 , which was not present in the germline DNA. Specifically, the BRCA2 V3091I mutation has previously been reported as conferring a homologous recombination defect in cancer cells [ 23 ]. Three lines of evidence suggest that this BRCA2 mutation is indeed pathogenic: first, the high MAF in exoDNA, underscoring its ‘driver’ status; second, the ‘unstable’ genome phenotype on genome-wide copy number assessment [ 22 ]; and third, the exceptional response to a platinum-containing adjuvant regimen that this patient has had to date (although the overall follow-up period remains limited).…”

“…While cfDNA platforms can certainly elucidate limited panels of genomic abnormalities and even map the emergence of resistance mechanisms during the course of targeted therapies, exosome-based liquid biopsy approaches have the additional benefit of being able to comprehensively profile the cancer transcriptome from the same biosample. In particular, the ability to identify expressed neoantigens (point mutations or fusion transcripts) represents an avenue to interrogate the humoral or cellular responses to such neoantigens in visceral cancers [ 20 , 23 ]. For example, emerging ‘personalized’ adoptive T-cell therapies require elucidation of cancer-specific neoantigens that are expressed and processed in an human leukocyte antigen context [ 26 ].…”

High-Throughput Functional Evaluation ofBRCA2Variants of Unknown Significance

“…However, it should be noted that loss of a certain function does not directly lead to cancer predisposition. For example, an established R3052W pathogenic variant did not show pathogenicity in the centrosome amplification assay 39 , whereas G2353R, once annotated as a benign variant, showed a pathogenic response in the spontaneous homologous recombination assay 40 . The results of each functional assay should be carefully interpreted together with clinical genetic data.…”

Characterization of BRCA1 and BRCA2 variants in multi-ethnic Asian cohort from a Malaysian case-control study

“…This is consistent with a study in Chinese women from Shanghai in which BRCA2 p.Cys315Ser was detected in 1.4% of cases and 0.9% of controls, compared with 0.9% of cases and 1.2% of controls in our study [ 12 ]. Notably, BRCA2 p.Arg2108Cys was evaluated as pathogenic in spontaneous homologous recombination [ 25 ], but our study suggests that this variant is unlikely to be associated with high risk of breast cancer.…”