Identification of a BRCA2 mutation in a Turkish family with early‐onset breast cancer

Key Clinical Message We used a multi‐gene panel testing to identify the germline variants in a mother‐daughter pair with early‐onset breast cancer, and detected one pathogenic protein‐truncating variant in BRCA2. Our results highlight the importance of genetic testing in identifying the pathogenic mutation running in cancer families.

up to 25% of all familial breast cancers. 3 Additional cancer predisposition genes (e.g. ATM, PALB2, TP53, BARD1, CHEK2) are also implied in hereditary breast cancer. 3 Most breast cancer predisposition genes have functions in maintaining genome integrity and cell cycle control. ATM, a serine/threonine protein kinase, functions as a transducer of DNA damage signals, and activates downstream proteins, including BRCA1 and BRCA2, by phosphorylation. 4 BRCA1 is an E3 ubiquitin protein ligase and a transcriptional activator. It plays a central role in coordinating cellular pathways in response to DNA damage. Most notably, BRCA1 stimulates DNA repair mechanisms, and arrests cell cycle progression to ensure that DNA is repaired before division. 5 BRCA2 is an ssDNA binding protein, and has a vital role in DNA damage response by regulating homologous recombination.
Genetic tests are recommended for individuals suspected to have germline variants. The results of these tests might be important for personalizing the management of the disease and take preventive measures for the subjects and their families. With multi-gene panel testing, germline variants in the exons of many cancer predisposing genes can be screened in a run in a cost-effective manner. In this case report, we studied a Turkish mother-daughter pair with early-onset breast cancers using a multi-gene panel and identified two variants of uncertain significance (VUS) in ATM, BRCA1 and a pathogenic variant in BRCA2.

| MATERIALS AND METHODS
The index patient (the daughter) was recruited in the Surgical Oncology Department of Umraniye Teaching and Research Hospital (UEAH), Istanbul in 2015. Blood samples from the index and her mother, who was also found to have had early-onset breast cancer, were collected and subsequent genetic tests were performed in the joint Genomic Laboratory (GLAB) of UEAH and Istanbul Technical University. 6 Genomic DNAs were isolated using PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific) from blood samples of the index patient and her mother. DNA libraries were prepared using TruSight Cancer Kit (Illumina), and sequenced in MiSeq sequencer (Illumina), using 2 × 150-bp paired-end reads. Sequence assembling and variant calling were done using Sophia DDM software (Sophia Genetics). SIFT, PolyPhen-2, MutationTaster, Provean, and Mutation Assessor tools were also used to predict the pathogenicity of missense variants. [7][8][9][10] The family history of cancer was interrogated by medical geneticists at UEAH (Figure 1). The study was carried out with the given consent of the patients F I G U R E 1 Pedigree of the index patient with early-onset breast cancer

| RESULTS
The mother-daughter pair studied in this case report developed breast cancers at young age, suggesting an underlying germline mutation. The daughter was diagnosed with bilateral triple negative invasive ductal carcinoma at the age of 33 in 2015, and had bilateral modified radical mastectomy. Cancer has metastasized to the brain; and the patient is currently receiving chemotherapy. The mother was diagnosed with breast cancer at the age of 40 in 2006, had unilateral mastectomy, and is currently alive. Both patients were referred for genetic testing. Pathological information of the index is described in Table 1. Three germline variants, common to both patients, were detected using a multi-gene panel. The variants in ATM (NM_000051.3c.8965C>G:p.Gln2989Glu) and BRCA1 (NM_007294.3c.3424G>C:p.Ala1142Pro) cause nonsynonymous amino acid changes. The clinical significance of these variants is not known. Further analysis is needed to assess their pathogenicity. On the other hand, the variant in BRCA2 (NM_000059.3c.7655_7658delTTAA:p.Ile2552Thrfs) causes a premature stop codon, leading to the truncation of the C-terminal 866 amino acids in BRCA2 protein. Truncating variants in BRCA2 are highly pathogenic for breast cancer. Variants in BRCA1 and BRCA2 genes were also confirmed by Sanger sequencing.

| DISCUSSION
The germline variant in ATM (8965C>G) results in the change of glutamine to glutamic acid at position 2989, which is located in the FATC domain of ATM. 11 FATC domain is the binding region for Tip60. The interaction between ATM and Tip60, is important for the activation of ATM. 12 In silico prediction tools (SIFT, PolyPhen, MutationTaster, Provean, Mutation assessor) showed conflicting interpretation for this variant.
The variant in BRCA1 results in the change of alanine to proline at position 1142, which is not located in any known functional domain. Proline leads to rigid turns in protein secondary structures, hence missense variants including proline might affect protein folding and structure. However, in silico prediction tools showed conflicting interpretation for this variant.
TTAA deletion in exon 16 of BRCA2 (NM_000059.3c. 7655_7658delTTAA:p.Ile2552Thrfs) causes a frameshift starting from codon 2552 and leading to a stop codon after adding 95 amino acids. The wild-type BRCA2 protein is 3418 amino acid long. Thus, this variant is predicted to be highly pathogenic. The resulting truncated protein would be devoid of very important functional domains, including the SEM1-binding site, DNA-binding site, nuclear localization signal (NLS), and the CDK phosphorylation site at S3291, which also binds RAD51. 2,13 SEM1 stabilizes BRCA2 by preventing its degradation. In vitro studies showed that loss of SEM1 binding to BRCA2, or depletion of either protein, led to hypersensitivity to DNA damage. 13 DNA-binding site is responsible for binding of BRCA2 to single-stranded DNA and acting as a junction between single-strand and double-strand DNA to manage Rad51-mediated homolog recombination. 14 Deletion of NLS site causes aberrant localization of BRCA2, preventing its function in maintaining the integrity of DNA and leading to carcinogenesis. 15 RAD51 directly binds ssDNA and recruitment is provided by BRCA2. RAD51 binding to BRCA2 at the C-terminus is dependent on the phosphorylation of serine at 3291 by CDK. 2 Premature termination codons (PTCs) introduced by BRCA2 mutations also causes degradation of the BRCA2 mRNA by nonsense-mediated mRNA decay (NMD), a protective mechanism that prevents the expression of truncated proteins. PTC-containing BRCA2 transcripts are significantly less prevalent than their counterparts. Therefore, NMD mechanism recognizes PTCs in BRCA2 transcripts and leads to their degradation. 16  Zealander breast cancer patients. 17,18 Now, we report that this pathogenic variant is also found in a Turkish breast cancer family. Deleterious BRCA2 variants also predispose to ovarian cancer, and might occur in families with Fanconi anemia. However, we did not find any member of the family with these diseases. Furthermore, no other breast cancer case was found in the maternal side of the family, suggesting that the pathogenic BRCA2 variant might be a de novo germline mutation in the mother of the index. However, we could not receive the consent of the maternal aunts of the index to test this. Apart from the pathogenic BRCA2 variant, both the index and her mother carry the same two VUS in ATM and BRCA1. Hence, we cannot infer the clinical significance of these VUS on the basis of the limited data we have. Strikingly, the consanguineous family presented here has an aggregation of lung cancers (Figure 1). Two paternal uncles of the index had lung cancer; and the paternal cousin and the maternal uncle had early-onset lung cancer. Despite that the uncles of the index were found to be heavy smokers, frequent presentation of lung cancer in the family might also indicate a genetic predisposition. A germline mutation spectrum study in 555 lung adenocarcinoma cases found deleterious variants in ATM and BRCA2 genes, suggesting that inherited risk factors might lead to lung adenocarcinoma. 19 Three of the lung cancer patients in the family have already died, and the paternal cousin was not reached. Therefore, we were not able to examine the cosegregation pattern of lung cancer with the two VUS in ATM and BRCA1 genes, and the pathogenic variant in BRCA2 to test the link between these genes and lung cancer.
In conclusion, in this case report we provide another evidence for the pathogenicity of truncating germline BRCA2 variants in breast cancer.

ACKNOWLEDGMENT
This study was funded by Istanbul Development Agency and Istanbul Technical University Internal Research Funds.