Mutations in several genes, including BRCA1, BRCA2, TP53, CHEK2, PALB2, and BRIP1, are reported to increase the risk of breast cancer [345678]. Importantly, mutations in BRCA1 and BRCA2 contribute to approximately 5% of breast cancer cases [9]. Moreover, up to 25% of familial breast cancer cases are reported to result from pathogenic mutations in BRCA1 and BRCA2 [10]. Deleterious mutations in BRCA1 or BRCA2 not only lead to breast cancer, but also increase the risk of secondary malignancies, such as prostate cancer, pancreatic cancer, and gastrointestinal cancer [11]. Therefore, genetic testing of BRCA1 and BRCA2 has proven to be a powerful tool to support early prevention and appropriate personalized treatment for breast cancer in the clinic.

In this study, genetics screening of BRCA1 and BRCA2 was performed in patients with breast cancer. All genetic studies were approved by the ethics committee of the Wuhan Red Cross Hospital (approve number: WHSYYY2017003). All patients provided written informed consent in accordance to the Declaration of Helsinki.

Through our screening program, pathogenic mutations were identified in patients with breast cancer using next-generation sequencing methods. However, most of these mutations have been previously reported. Interestingly, a novel heterozygous mutation in BRCA2 (c.849dupT, p.G284Wfs*11) was detected in one family with a history of breast cancer (Figure 1A). This mutation was then confirmed by Sanger sequencing, as shown in Figure 1B. In this family, the female proband (III:6) and her two brothers (III:3, III:7) were carriers, while other members did not carry the mutation (II:1, III:2, IV:3). These results suggest that the BRCA2 mutation in these three members (III:3, III:6, III:7) was likely inherited from their mother (II:2), who was diagnosed with and died from breast cancer.

BRCA2 is localized at 13q13.1 and the length of BRCA2 is 3,418 amino acids. Key functional domains of the BRCA2 protein include the Breast Cancer (BRC) repeats, a DNA binding domain, and the C-terminal testicular receptor 2 (TR2) domain. These domains are critical for the interaction of BRCA2 with DNA or binding partners such as PALB2 and RAD51 [1213]. Moreover, distinct nuclear localization signals (NLSs) in the C-terminal region are essential for its nuclear localization and functions. BRCA2 is essential for DNA repair, DNA replication, telomere homeostasis and cell cycle progression [141516]. The mutation in BRCA2 was located on exon 7. This mutation introduces a stop codon after the 295th amino acid, leading to premature termination of translation. As shown in Figure 1C, the mutant BRCA2 protein lacks nearly all functional domains, including the BRC repeats, the α-helical domain, the oligonucleotide binding domain, and the NLS, and retains only the N-terminal region of BRCA2.

Truncations of large fragments always result in downregulation of genes at the mRNA and protein levels via nonsense mutation-mediated mRNA decay. The whole blood samples of the affected female, her younger brother and three noncarrier controls were collected for BRCA2 mRNA expression testing. As shown in Figure 1D, BRCA2 mRNA expression levels in the blood of the two mutation carriers were reduced by approximately 70% and 90% relative to noncarriers. Furthermore, the mutation-associated downregulation of BRCA2 protein was confirmed by immunofluorescence and western blot assays. As shown in Figure 2A, after overexpression in HEK293T cells, wild-type BRCA2 was expressed in both the nucleus and cytoplasm of cells, as detected by Flag antibody. However, mutant BRCA2 protein could not be detected in the nucleus or cytoplasm. In addition, as shown in Figure 2B, wild-type BRCA2 could be detected with an anti-Flag antibody using western blot. However, mutant BRCA2 protein could not be detected. In summary, these in vivo and in vitro assay results suggested that the mutation (c.849dupT, p. G284Wfs*11) could lead to a significant decrease of BRCA2 expression and therefore be damaging to the function of BRCA2.

By carrying the mutation in BRCA2, the female proband is at a greater risk for right breast cancer and other malignancies, such as ovarian cancer. Therefore, routine examinations of the right breast and other organs are indispensable. Moreover, since cells with BRCA1/2 mutations are more sensitive to DNA cross-linking drugs, such as cisplatin and carboplatin, and less susceptible to taxanes [1718], the effects of this mutation on drug sensitivity should be taken into consideration during treatment. Considering the positive family history and pathogenicity of this mutation, gene testing for other family members was also necessary and meaningful. Another two males were also identified as carriers of the mutation. Although the proband's two brothers had not been diagnosed with any malignancies yet, greater attention should be paid to breast and examinations for other cancers, such as prostate, colon and genitourinary cancer, as 4% to 14% of males with BRCA2 mutations will develop breast cancer at any age [1920], and men under 65 years old with BRCA2 mutations have a 8.6-fold higher risk of developing prostate cancer [21]. Moreover, this mutation was likely to be inherited by the brothers' offspring, who have not yet undergone genetic testing. Therefore, we recommended that these individuals undergo gene screening as soon as possible.

In this study, we identified a novel heterozygous mutation of BRCA2 in a family with a history of breast cancer. Pedigree analysis suggested that the novel mutation could be a highly pathogenic factor in familial breast cancer. In vivo and in vitro studies demonstrated that this frameshift mutation led to BRCA2 mRNA decay and that it could be damaging for BRCA2 function. Our finding not only enriches the Breast Cancer Information Core database (BICD; http://research.nhgri.nih.gov/bic/), but also provides assistance to genetic counseling for this family. More importantly, genetic counseling based on BRCA1/BRCA2 testing was clinically beneficial and significant for this family, as the results contributed to personalized treatments for the patients and prevention and early detection of breast cancer or other malignancies in the family members.