A novel BRCA1 duplication and new insights on the spectrum and frequency of germline large genomic rearrangements in BRCA1/BRCA2

Heritable breast cancers account for 5% to 10% of all breast cancers, and monogenic, highly penetrant genes cause them. Around 90% of pathogenic variants in BRCA1 and BRCA2 are observed using gene sequencing, with another 10% identified through gene duplication/deletion analysis, which differs across various communities. In this study, we performed a next-generation sequencing panel and MLPA on 1484 patients to explain the importance of recurrent germline duplications/deletions of BRCA1-2 and their clinical results and determine how often BRCA gene LGRs were seen in people suspected of hereditary breast and ovarian cancer syndrome. The large genomic rearrangements (LGRs) frequency was approximately 1% (14/1484). All 14 mutations were heterozygous and detected in patients with breast cancer. BRCA1 mutations were more predominant (n = 8, 57.1%) than BRCA2 mutations (6, 42.9%). The most common recurrent mutations were BRCA2 exon three and BRCA1 exon 24 (23) deletions. To the best of our knowledge, BRCA1 5’UTR-exon11 duplication has never been reported before. Testing with MLPA is essential to identify patients at high risk. Our data demonstrate that BRCA1-2 LGRs should be considered when ordering genetic testing for individuals with a personal or family history of cancer, particularly breast cancer. Further research could shed light on BRCA1-2 LGRs’ unique carcinogenesis roles.


Introduction
Hereditary breast and ovarian cancer syndrome (HBOC) is characterized by an increased incidence of male and female breast cancer, ovarian cancer (Fallopian tube and peritoneal cancers), and, to a lesser extent, other cancers such as prostate cancer, pancreatic cancer, and melanoma. For breast and ovarian cancers, germline genetic testing has long been used to assess the likelihood of hereditary cancers. Heritable breast cancers account for 5 to 10% of all breast cancers, and they are caused by monogenic, highly penetrant genes [1].
HBOC is caused by germline BRCA1 and BRCA2 mutations [2]. BRCA1 and BRCA2 germline mutations are responsible for up to 30% of all inheritable breast cancers [3]. More than 2000 pathogenic variants in the BRCA1 and BRCA2 genes have been identified [4]. Carriers of the BRCA1 mutation have a 60-65% risk of developing breast cancer (BC) until the age of 70 and a 40-60% risk of developing ovarian cancer (OC). The corresponding percentages for BRCA2 mutation carriers are 45-55% for BC and 11-16.5% for OC [5]. The most prevalent malignancy in individuals with a germline BRCA1 or BRCA2 pathogenic variant is breast cancer, which varies from 46% to 87%. BRCA germline pathogenic variants raise the incidence of ovarian cancer from 16.5% to 63%. BRCA1 and BRCA2 germline pathogenic forms are inherited in an autosomal dominant pattern. Each person with a BRCA1 or BRCA2 germline pathogenic variant has a 50% risk of transmitting the variant to their offspring. Around 90% of pathogenic variants in BRCA1 and BRCA2 are observed using gene sequencing, with another 10% identified through gene duplication/deletion analysis, which differs across various communities [6]. The effects of BRCA mutations have also been discovered to be connected to prostate, pancreatic, stomach, and colorectal cancers, although it is difficult to determine the level of harm [7].

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The majority of BRCA1 and BRCA2 mutations are small deletions, insertions, nonsense mutations, or splice variants that result in a truncated protein. Despite this, a number of significant large genomic rearrangements (LGRs) involving these genes have been reported [8]. These modifications are generally pathogenic since deletions or insertions of broad genomic sequences inside a coding area result in out-offrame translation, resulting in a nonfunctional mutant protein. BRCA1 LGRs may account for a sizable proportion of all disease-causing mutations in various populations, while BRCA2 LGRs are less commonly found [9].
Various techniques were used to identify large deletions and duplications, including southern blotting, semiquantitative multiplex PCR, real-time PCR, restriction analysis, long-range PCR, and sequencing [10]. Multiplex Ligationdependent Probe Amplification (MLPA) has been the most frequently used strategy for detecting these BRCA1/2 gene mutations [8]. The most successful technique for routine BRCA1/2 molecular screening was a diagnostic molecular algorithm focused on NGS (as the first step) and MLPA (as the second step). Large genomic rearrangements (LGRs) have been extensively studied in breast and ovarian cancer patients from various countries during the last several years. Numerous experiments demonstrate their presence in the genetic predisposition to gynecological tumors, and a large number of novel BRCA1/2 gene rearrangements have been recorded [11].
In this study, we performed a next-generation sequencing panel and MLPA on 1484 patients to explain the importance of recurrent germline duplications/deletions of BRCA1-2 and their clinical results and determine how often BRCA gene LGRs were seen in people suspected of HBOC syndrome. Our data broadens the spectrum of BRCA gene LGRs and provides insights for genotype-phenotype correlations for hereditary breast and ovarian cancer syndrome.

Patients
Consent for the publication and any additional related information was taken from the patients or their parents involved in the study. Most of the patients came to our clinic in their first few years of diagnosis. Clinical histories and molecular results were reviewed for all unrelated patients who examined at the Department of Medical Genetics, University of Health Sciences, Dışkapı Yıldırım Beyazıt Training and Research Hospital, Ankara, Turkey. According to National Comprehensive Cancer Network (NCCN) guidelines for breast-ovarian cancer, patients were evaluated. Patients underwent BRCA1-2 NGS panel test between January 2017 and December 2020 at Ankara Central Genetic Laboratory (Ankara, Turkey). All have a strong family history with at least three cancers in relatives (1st, 2nd, 3rd degree). Patients with uncertain/missing data have been filtered. Participants who underwent the MLPA test for the BRCA1/BRCA2 deletions or duplications after NGS screening have been chosen for the study.

Interpretations, descriptive statistics and graphics
The LRGs were named using Human Genome Variation Society (http:// www. HGVS. org/ varno men) guidelines and classified using the American Society of Medical Genetics and Genomics (ACMG) criteria for the interpretation and reporting of single gene copy number variants [12]. Descriptive statistical calculations have been made, and graphics have been prepared with Python (version 3.9.2).

Results
Females (1442, 97.17%) were more than males (42, 2.83%). The mean age was 49 (in females 48.6 and males 57.8) with a minimum age of 19 and a maximum of 88. Most of the patients were between 40 and 60 ages (Fig. 1, Table 1).

Discussion
This is the first study mentioning the importance of recurrent BRCA large genomic rearrangements (LGRs) in patients with breast cancer phenotype in the Turkish population. Recurrent and other LGRs were observed only in the patients with breast cancer. The principal finding of our study is that BRCA1 LGRs were more predominant in the Turkish population, but BRCA2 mutation was more recurrent. The mean ages in males and females were 57.8 and 48.6, respectively. Most of the cancers observed between 40 and 60 years of age. Currently, the MLPA procedure is the gold standard for establishing a conclusive molecular diagnosis. The BRCA Tumor (Multiplicom, Niel, Belgium) Panel and Sophia DDM framework (Sophia Genetics SA, Saint Sulpice, Switzerland) were found to be the most appropriate combination for concomitant and optimal CNV, SNV, and indel detection in this situation [13]. In this study, it was also observed that analyzing LGRs with Sophia DDM was very helpful and informative. Researchers have also shown that BRCA1 gene mutations occur more often in high-risk cancer databases than BRCA2 gene mutations [14]. The present study's findings corroborate current information in the literature [15].
CNVs are more prevalent in the BRCA1 gene because of the proliferation of intronic Alu repeat sequences [16].
The most recurrent rearrangement detected in this study was BRCA2 exon three deletions, which were found to be associated with a high risk of breast and ovarian cancer [17] (Fig. 2). This mutation and BRCA1 exon 24 deletion were detected in both female and male breast cancers. Both deletions and BRCA1 exons 18-19 deletion reported many times across Europe [18][19][20]. We observed high BRCA LGRs frequency in triple-negative breast cancer patients (9/14), as mentioned in the literature [21]. LGRs were not detected in patients with ovarian, pancreas, or other cancers except for breast cancer. Because the respective exons are not in the frame, the altered transcript will degrade through nonsensemediated decay and prevent DNA repair.
To the best of our knowledge, BRCA1 5'UTR-exon11 (5'UTR_ex10) [LRG_292t1: c.(?_-232)_ (4096 + 1_4097-1) dup] duplication has never been reported before (Fig. 3, Table 1). It was observed in a 57-year-old female patient with bilateral invasive ductal carcinoma. Bilateral tumor status was ER + , PR + , and c-erbB2 scores were 2 + . Bilateral axillary lymph node metastases were detected. Her deceased father was diagnosed with gastric cancer at the age of 86. She has two female cousins with breast cancer connected with her mother. No cancer was detected in her mother, her two brothers, and her two sisters.
Ten percent of the pathogenic variants in BRCA1 and BRCA2 were established through deletion/duplication tests, which may differ between populations [6,22]. The frequency of the LGRs was approximately 1% in this study. Among all the BRCA mutations detected in the center, the frequency A B LGR rate of 7.9 percent in the Myriad data [6]. According to Smith et al., the overall CNV rate for BRCA1 and BRCA2 was marginally higher, with an average of 11.9 percent in European families [23]. The results of this study help provide current information about the relationship between BRCA1 and BRCA2 LGRs status and the development of breast cancer. The main strength of our study is that it involved a diverse, welldefined community of participants, many of whom had symptoms that manifested in the clinical sense, allowing us to generalize our findings to patients. We used a specialized diagnostic center with extensive cancer gene testing experience to do an exhaustive and functional cancer gene analysis.

Conclusion
Our study gives a novel insight into the diagnosis of patients suspected of having breast cancer. Testing with MLPA is essential to identify patients at high risk. Our data demonstrate that BRCA1-2 LGRs should be considered when ordering genetic testing for individuals with a personal or family history of cancer, particularly breast cancer. Further research could shed light on BRCA1-2 LGRs' unique carcinogenesis roles. To detect and explain the critical mutations, large cohorts are needed. A more detailed examination of cancer genetics research is needed to improve patient risk management, prognosis, and treatment decisions.
Author contributions IS designed the research, analyzed and interpreted the results (including the coding part with Python version 3.9.2), wrote the manuscript, and approved the final manuscript. HS collected the data, analyzed and interpreted the results, reviewed the manuscript, and approved the final manuscript.
Funding No financial assistance was received.

Data availability
The data (including patient demographic information and mutations) and the code of the current study are available from the corresponding author on reasonable request.

Conflict of interest
The authors declare that there is no conflict of interest.
Ethical approval We confirm that we have read the journal's position on issues involved in the ethical publication, and we affirm that this report is consistent with those guidelines. The Ethics Committee of the University of Health Sciences, Dışkapı Yıldırım Beyazıt Training and Research Hospital approved the study (08.03.2021-106/27).
Informed consent Informed consent was obtained from the patients or their parents (mentioned within "Patients").