High- and Moderate-Risk Variants Among Breast Cancer Patients and Healthy Donors Enrolled in Multigene Panel Testing in a Population of Central Russia

Abstract

Assessments of breast cancer (BC) risk in carriers of pathogenic variants identified by gene panel testing in different populations are highly in demand worldwide. We performed target sequencing of 78 genes involved in DNA repair in 860 females with BC and 520 age- and family history-matched controls from Central Russia. Among BC patients, 562/860 (65.3%) were aged 50 years or less at the time of diagnosis. In total, 190/860 (22%) BC patients were carriers of 198 pathogenic/likely pathogenic (P/LP) variants in 30 genes, while among controls, 32/520 (6.2%) carriers of P/LP variants in 17 genes were identified. The odds ratio [95% confidence interval] was 16.3 [4.0-66.7] for BRCA1; 12.0 [2.9-45.9] for BRCA2; and 7.3 [0.9-56.7] for ATM (p < 0.05). Previously undescribed BRCA1/2, ATM, and PALB2 variants, as well as novel recurrent mutations, were identified. The contribution to BC susceptibility of truncating variants in the genes BARD1, RAD50, RAD51C, NBEAL1 (p. E1155*), and XRCC2 (p. P32fs) was evaluated. The BLM, NBN, and MUTYH genes did not demonstrate associations with BC risk. Finding deleterious mutations in BC patients is important for diagnosis and management; in controls, it opens up the possibility of prevention and early diagnostics.

Keywords: ATM; BRCA1/2; breast cancer risk; healthy controls; multigene panel testing; pathogenic variant; patients.

Figure 1

Distribution of P/LP variants in different genes in BC patient carriers (a) and healthy control carriers (b). The lower part of the figure shows a ranking of genes by decreasing frequencies of mutations.

Figure 2

P/LP variants and breast cancer risk. ORs (large dots) and corresponding 95% confidence intervals (horizontal lines) for the association between breast cancer and pathogenic variants in various genes, sorted by OR.

Figure 3

Location of P/LP variants found in the study along the BRCA1 gene. Domain structure of the BRCA1 gene: N-terminal RING (really interesting new gene) domain, serine-rich region, coiled-coil domain, SCD (serine-containing domain), and BRCT (BRCA1 C-terminal) repeats. BARD1, BAP1, CHEK2, PALB2, BRCA2, ATM, ATR, BACH1, CtIP, p53, ABRAXAS1, and BRIP1 bind to the BRCA1 protein at specific sites (domains and proteins binding to them are marked in blue).

Figure 4

Location of P/LP variants found in the study along the BRCA2 gene. Domain structure of the BRCA2 gene: RING—Really Interesting New Gene, BRC repeats—region consisting of approximately 1000 amino acids that bind to the protein Bacterial RecA homolog DNA recombinase (RAD51), Helical—Helical domain that comprises 190 amino acids, OB—Oligonucleotide/oligosaccharide-binding fold, Tower—Tower domain, TR2—C-terminal RAD51 binding site, NLS—Nuclear localization signal, P—Phosphorylation site. PALB2, BRCA1, EMSY, RAD51, PIk1, p53, DMC1, DSS1, CDK2 proteins, and ssDNA (single-stranded DNA) bind to BRCA2 at specific sites.

Figure 5

Association between P/LP variants in different genes and clinical features of patients with breast cancer: (a) estrogen receptor status, (b) bilaterality of breast cancer, (c) family history of breast or ovarian cancer.

Figure 6

Schematic representation of protein interactions involved in DNA DSB repair pathways. DSBs in DNA can be repaired by non-homologous end-joining (NHEJ), alternative end-joining (A-EJ), single-strand annealing (SSA), or HR. Multigene panel included 48/78 genes coding the key proteins involved in these DNA damage repair pathways. (1) Repair begins with recognition of DSB sites by the MRN complexes (MRE11-RAD50-NBN), which recruit Ataxia telangiectasia mutated (ATM) kinase. Depending on cell cycle and cyclin-dependent kinase (CDK) activity, ATM promotes different repair pathways. In the G0 and G1 phases, CDK activity is low and ATM phosphorylates 53BP1, favoring EJ pathways with almost no processing of DSB ends, which results in short nucleotide insertions and deletions at the sites of repair. In the S and G2 phases, CDK activity increases and ATM phosphorylates BRCA1, allowing DSB end resection. (2) C-terminal binding protein-interacting protein (CtIP) and the MRN complex lyse DNA strands from 5′ to 3′, generating stretches of single-stranded DNA (ssDNA) at DNA ends for A-EJ, SSA, and HR. Ablation of the BRCA1–Abraxas interaction with these complexes promotes extensive resection. (3) Bloom syndrome RecQ-like helicase (BLM), WRN RecQ-like helicase (WRN), and exonuclease 1 (EXO1) account for additional resection, forming overhanging ssDNA ends that can invade the homologous template. Replication protein A (RPA) binds to ssDNA, activating the ATM RAD3-related (ATR) kinase, ATR phosphorylates checkpoint kinase 1 (CHK1), which arrests the cell cycle, allowing time for DSB repair. In the M phase, both HR and EJ are blocked and DSBs arising during mitosis are repaired by SSA, which results in large-scale chromosomal rearrangements. (4) ATM-phosphorylated checkpoint kinase 2 (CHK2) recruits the BRCA complex, consisting of BRCA1/2, PALB2, BRIP1, BARD1, and SLX4. The BRCA complex regulates the interaction of RAD51 paralog complexes BCDX2 (RAD51B–RAD51C–RAD51D-XRCC2) and CX3 (RAD51C–XRCC3) with RAD51 recombinase. RAD51 is loaded on ssDNA to form the RAD51–ssDNA nucleofilament. (5) RAD51 catalyzes the homology search and synthesizes DNA using sister chromatids as a template, which ensures a low error likelihood. The Fanconi anemia (FA) Core Complex (FANCA, B, C, E, F, G, L, M, and T) activates FANCD2 and FANCI by mono-ubiquitinating the proteins as a response to DNA damage. The activated FANCD2–I heterodimeric proteins are subsequently transported to the sites of DNA repair, which contain FA downstream proteins of the BRCA complex. Polδ, coded by the POLD1 gene, is involved in replication-coupled DNA events associated with repair, including NHEJ and break-induced recombination (blocks highlighted in light blue illustrate the main steps of HR, key participating proteins are marked in other colors).