J Breast Cancer.  2018 Sep;21(3):330-333. 10.4048/jbc.2018.21.e33.

The Novel Pathogenic Mutation c.849dupT in BRCA2 Contributes to the Nonsense-Mediated mRNA Decay of BRCA2 in Familial Breast Cancer

• Li S
• Ma J
• Hu C
• Zhang X
• Xiao D
• Hao L
• Xia W
• Yang J
• Hu L
• Liu X
• Dong M
• Ma D
• Liu R

• KMID: 2421374
• DOI: http://doi.org/10.4048/jbc.2018.21.e33

Abstract

In this study, we used next-generation sequencing methods to screen 300 individuals for BRCA1 and BRCA2. A novel mutation (c.849dupT) in BRCA2 was identified in a female patient and her unaffected brothers. This mutation leads to the truncation of BRCA2 functional domains. Moreover, BRCA2 mRNA expression levels in mutation carriers are significantly reduced compared to noncarriers. Immunofluorescence and western blot assays showed that this mutation resulted in reduced BRCA2 protein expression. Thus, we identified a novel mutation that damaged the function and expression of BRCA2 in a family with breast cancer history. The pedigree analysis suggested that this mutation is strongly associated with familial breast cancer. Genetic counsellors suggest that mutation carriers in this family undergo routine screening for breast cancer, as well as other malignancies, such as prostate and ovarian cancer. The effects of this BRCA2 mutation on drug resistance should be taken into consideration during treatment.

Keyword

BRCA2 genes; Breast neoplasms; High-throughput nucleotide sequencing; Mutation; Nonsense mediated mRNA decay

MeSH Terms

Blotting, Western
BRCA2 Protein
Breast Neoplasms*
Breast*
Drug Resistance
Female
Fluorescent Antibody Technique
Genes, BRCA2
High-Throughput Nucleotide Sequencing
Humans
Mass Screening
Nonsense Mediated mRNA Decay*
Ovarian Neoplasms
Pedigree
Prostate
RNA, Messenger
Siblings
BRCA2 Protein
RNA, Messenger

Figure

• 

  Figure 1 A novel mutation in BRCA2. (A) Pedigree of the family with BRCA2 mutation. The family member II:1, III:2, III:3, III:6, III:7, and IV:3 were screened for the BRCA1 and BRCA2 gene, and III:3, III:6, III:7 carry the novel mutation. (B) The mutation (c.849dupT, p.G284Wfs*11) in BRCA2 identified in this family was confirmed by Sanger sequencing. (C) The diagram of human BRCA2 protein with the location of its variant identified. The PALB2 binding domain in the N-terminal is represented by green box. The blue boxes represent the Breast Cancer (BRC) repeats, which form the RAD51 binding domain. The α-helix domain (HD) is indicated by a red box. The gray boxes denote the oligonucleotide binding (OB) domain. HD and OB domains are important for single strand DNA (ssDNA) binding. The yellow boxes indicate the nuclear localization signal (NLS). NLS is also essential for RAD51 binding. The mutation (G284Wfs*11) contributed to the truncation of BRC repeats, HD domain, OB domain and NLS. (D) The real-time quantitative polymerase chain reaction demonstrated that BRCA2 mRNA expression in female and male carriers was significantly lower than that in noncarriers. LC=lung cancer; BC=breast cancer; WT=wild-type; Mut=mutant; PALB2=partner and localizer of BRCA2; GAPDH=glyceraldehyde-3-phosphate dehydrogenase.

• 

  Figure 2 The abolished expression of the mutant BRCA2 in vitro. (A) The immunofluorescent demonstrated that wild-type (WT) of BRCA2 was overexpressed in both nucleus and cytoplasm of HEK293T cells which could be detected by anti-Flag antibody. However, mutant BRCA2 protein could not be detected in HEK293T cells, which were transiently transfected with the Flag-tagged mutant BRCA2 plasmids. (B) Western blotting also showed that wild-type of BRCA2 was successfully detected by anti-Flag antibody, and that mutant BRCA2 protein could not be detected in HEK293T cells. DAPI=4´,6-diamidino-2-phenylindole; NC=negative control; GAPDH=glyceraldehyde-3-phosphate dehydrogenase.

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