Identification of the most common BRCA alterations through analysis of germline mutation databases: Is droplet digital PCR an additional strategy for the assessment of such alterations in breast and ovarian cancer families?

Abstract

Breast and ovarian cancer represent two of the most common tumor types in females worldwide. Over the years, several non‑modifiable and modifiable risk factors have been associated with the onset and progression of these tumors, including age, reproductive factors, ethnicity, socioeconomic status and lifestyle factors, as well as family history and genetic factors. Of note, BRCA1 and BRCA2 are two tumor suppressor genes with a key role in DNA repair processes, whose mutations may induce genomic instability and increase the risk of cancer development. Specifically, females with a family history of breast or ovarian cancer harboring BRCA1/2 germline mutations have a 60‑70% increased risk of developing breast cancer and a 15‑40% increased risk for ovarian cancer. Different databases have collected the most frequent germline mutations affecting BRCA1/2. Through the analysis of such databases, it is possible to identify frequent hotspot mutations that may be analyzed with next‑generation sequencing (NGS) and novel innovative strategies. In this context, NGS remains the gold standard method for the assessment of BRCA1/2 mutations, while novel techniques, including droplet digital PCR (ddPCR), may improve the sensitivity to identify such mutations in the hereditary forms of breast and ovarian cancer. On these bases, the present study aimed to provide an update of the current knowledge on the frequency of BRCA1/2 mutations and cancer susceptibility, focusing on the diagnostic potential of the most recent methods, such as ddPCR.

Keywords: BRCA1; BRCA2; DNA repair; Sanger sequencing; breast cancer; droplet digital PCR; germline mutations; large genomic rearrangements; next‑generation sequencing; ovarian cancer.

Figure 1

Non-modifiable and modifiable risk factors for breast and ovarian cancer.

Figure 2

BRCA1 structure and functions. aa, amino acids; ATM, ataxia-telangiectasia mutated kinase; ATR, ATM-related kinase; BACH1, BTB domain and CNC homolog 1; BARD1, BRCA1 associated RING domain 1; BRCA1, BReast CAncer 1; BRCT, C-terminal domain; Chk1, checkpoint kinase 1; GADD45, growth arrest and DNA damage-inducible 45; HDAC1, histone deacetylase 1; NLS, nuclear localization sequences; OLA1, Obg-Like ATPase 1; RAP80, receptor-associated protein 80; RMN, RAD50-MRE11-NBS1 complex; PALB2, partner and localizer of BRCA2; RNA pol II, RNA polymerase II; STAT1, signal transducer and activator of transcription 1; SWI-SNF, SWItch/sucrose non-fermentable complex; ZBRK1, zinc finger and BRCA1-interacting protein with KRAB domain-1.

Figure 3

BRCA2 structure and functions. aa, amino acids; BARD1, BRCA1 associated RING domain 1; BOD1L, biorientation of chromosomes in cell division 1-like; BRAF35, BRCA2-associated factor 35; BRCA2, BReast CAncer 2; DSS1, SEM1 26S proteasome subunit; EMSY, BRCA2-interacting transcriptional repressor; FANCD2, Fanconi anemia complementation group D2; OB, oligonucleotide-binding; NLS, nuclear localization sequences; PAI-1, plasminogen activator inhibitor 1; PALB2, partner and localizer of BRCA2; PDS5B, PDS5 cohesin associated factor B; SMAD3, mothers against decapentaplegic homolog 3; TAD, transcription activation domain; TR2, RAD51-binding site.

Figure 4

Schematic timeline of the technologies developed for the detection of BRCA1 and BRCA2 mutations. ddPCR, digital droplet PCR; MLPA, multiplex ligation-dependent probe amplification; NGS, next-generation sequencing.