Abstract 3531: A novel germline mutation in the non-coding region of BRCA2, c.-40+1 G&gt;A, is associated with hereditary breast cancer
Authors: Hakozaki Yumi , Fujimoto Yumi , Kobayashi Susumu S. , Ushiama Mineko , Hiraoka Yumie , Harano Kenichi , Kogawa Takahiro , Fujii Satoshi , Kuwata Takeshi , Yoshida Teruhiko , Ohashi Akihiro , Mukohara Toru 
Published: September 2020

Abstract
Background: BRCA2 plays an important role in DNA double-strand break repair, especially homologous recombination repair, to maintain genomic stability across generations. Germline mutations of BRCA2 increase a lifetime risk of developing breast and ovarian cancer, which is termed hereditary breast-ovarian cancer syndrome (HBOC). In this study, we identified a novel pathogenic mutation at a splicing site in the non-coding region of BRCA2, which is not covered by commercially-available BRCA2 genetic tests, in a breast cancer patient with a familial history of HBOC-related cancers. This germline mutation was previously reported in a Fanconi anemia family as well (Bakker J.L. et al., 2014).
Methods: Genomic DNA and total RNA were isolated from peripheral blood mononuclear cells (PBMCs) of the breast cancer patient. The oncogenic mutation screening with genomic DNA was conducted using the next generation sequence (NGS)-based panel analysis of 147 familial cancer (FC)-associated genes, termed the National Cancer Center (NCC) Oncopanel FC test. Immunohistochemistry of BRCA2 was conducted using the surgical breast tumors as well as adjacent normal breast tissues of the patient. The sporadic breast tumors without BRCA2 mutation was used as a control.
Results: The NCC Oncopanel FC test detected the novel germline mutation at c.-40+1 G&gt;A in the non-coding region of BRCA2 in a breast cancer patient, who has a familial history of HBOC-related cancers. The germline mutation was a mono-allelic mutation found in the maternal allele and not in the paternal allele. Since the patient has a single nucleotide polymorphism (SNP) at c.-26 G&gt;A in the paternal allele, this SNP site was used to identify the paternal-derived BRCA2 mRNA. RT-PCR analysis in the patient's PBMCs only detected the paternal-derived transcripts harboring c.-26 G&gt;A, suggesting that the transcripts of the maternal-derived BRCA2 allele with c.-40+1 G&gt;A mutation would not be expressed, or not be stabilized, in the patient's PBMCs. Immunohistochemical analysis revealed that BRCA2 protein was detected in normal mammary epithelial cells of the breast tissues. In the malignant tissues, however, BRCA2 protein was potently downregulated in the BRCA2-mutant breast cancer cells but not in the BRCA2 wild-type breast cancer cells, assuming that the mutant cancer cells may have the second hit in the paternal allele of BRCA2. These findings suggest that this novel BRCA2 germline mutation could be involved in loss of function of BRCA2 leading to HBOC syndrome.
Conclusions: We identified the novel germline mutation in the non-coding region of BRCA2, c.-40+1 G&gt;A, which appears to be involved in hereditary breast cancer.
Citation Format: Yumi Hakozaki, Yumi Fujimoto, Susumu S. Kobayashi, Mineko Ushiama, Yumie Hiraoka, Kenichi Harano, Takahiro Kogawa, Satoshi Fujii, Takeshi Kuwata, Teruhiko Yoshida, Akihiro Ohashi, Toru Mukohara. A novel germline mutation in the non-coding region of BRCA2, c.-40+1 G&gt;A, is associated with hereditary breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3531.