Copy number variants in BRCA1 and BRCA2 genes in Polish patients with breast and ovarian cancer

Copy number variants in BRCA1 and BRCA2 genes in Polish patients with breast and ovarian cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Copy number variants in BRCA1 and BRCA2 genes in Polish patients with breast and ovarian cancer Anna Doraczynska-Kowalik, Rafal Matkowski, Dagmara Michalowska, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9336936/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Purpose: BRCA1 and BRCA2 are key susceptibility genes in hereditary breast and ovarian cancer (HBOC), with mutational status guiding PARP inhibitor therapy. While single-nucleotide variants (SNVs) predominate, the prevalence of copy number variants (CNVs) varies significantly across different populations. This study aims to determine the incidence of BRCA1/2 CNVs in the Polish population, where data remain scarce due to non-mandatory CNV testing. Methods: We retrospectively analysed the results of genetic tests assessing the presence of BRCA1/2 CNVs performed in 2720 Polish probands tested at the Lower Silesian Oncology Centre (2021–2024), including 2702 breast/ovarian cancer patients and 18 relatives. The mean age was 54.7±15.15 years. Genetic testing involved DNA extraction, NGS, and MLPA for CNV confirmation. Variants were classified according to ACMG-AMP guidelines and verified through independent testing. Results: In this study, no BRCA2 CNVs were identified, consistent with previous Central European findings. Pathogenic BRCA1 CNVs were found in 0.85% of probands, affecting 23 individuals from 13 families. Eight distinct BRCA1 CNVs were detected, the most common being exon 21 deletion. Affected families exhibited a high incidence of HBOC-related cancers, with early-onset breast cancer and a notable proportion of triple-negative breast cancer cases. Conclusions: This study highlights the clinical significance of BRCA1 CNVs in Polish patients with HBOC-spectrum cancers and their families. Although rare, these variants were associated with aggressive cancer phenotypes and early onset. Given their diagnostic and therapeutic implications, BRCA1 CNVs should be routinely analysed in high-risk families to ensure accurate detection and personalised treatment planning. breast and ovarian cancer BRCA1 BRCA2 CNVs MLPA NGS Figures Figure 1 Figure 2 Introduction Hereditary predisposition to breast and ovarian cancer (HBOC) is one of the most commonly examined predispositions in Poland. Public funding ensures that many patients with breast, ovarian, pancreatic and prostate cancer and their families have access to genetic diagnostics ranging from testing for a single familial pathogenic variant, testing for a few selected pathogenic variants (most common in the Polish population), and also sequencing of the whole coding part of selected genes (Doraczynska-Kowalik et al. 2022). It is estimated that even about 10-15% of breast cancer patients, 15% of ovarian cancer patients, 5-10% of pancreatic cancer patients, 5% of localized prostate cancer patients and 12% of metastatic prostate cancer patients are likely to be carriers of a germline dominant susceptibility pathogenic variant responsible for a specific hereditary cancer predisposition (Kasuga et al 2022; Kurian et al. 2018; Pritchard et al. 2016; Stoffel et al. 2023; Walsh et al. 2011). Among many susceptibility genes for malignancies mentioned above, BRCA1 and BRCA2 are the two most often tested as their pathogenic or likely pathogenic variants (PVs/LPVs) are characterized by high penetrance (cumulative cancer risks to age 80 are breast cancer risk of 72% for BRCA1 and 69% for BRCA2 mutation carriers and ovarian cancer risk of 44% and 17% respectively) and account for up to 80% of all deleterious alterations recognized in hereditary breast and ovarian cancer (HBOC) families (Kuchenbaecker et al. 2017; McAlarnen etl. 2021). Moreover, BRCA1/2 mutational status together with homologous recombination repair deficiency (HRD) rank are the most important biomarkers for PARPi (poly ADP-ribose polymerase inhibitors) response in ovarian, breast, prostate and pancreatic cancer patients (de Bono et al. 2020; Kindler et al. 2022; Menezes et al. 2022; Tew et al. 2020; Vergote et al. 2022). Worldwide observations show that single-nucleotide variants (SNVs) (previously known as point mutations) dominate germline PVs/LPVs found in BRCA1 and BRCA2 genes. The share of intragenic copy number variants (CNVs), mainly deletions and duplications, in BRCA1 and BRCA2 genes is therefore relatively small; however, it varies greatly depending on the examined populations (Germani et al.; Schmidt et al. 2017). The highest occurrence of BRCA1/2 CNVs is found in the Dutch population, in which 27-36% of all germline PVs/LPVs in the BRCA1 gene are CNVs, making BRCA1 deletions major founder mutations in the Netherlands (Hogervorst et al. 2003; Petrij-Bosch et al. 1997). Surprising results were obtained in the Portuguese population, in which, due to the large share of BRCA2 founder mutation c.156_157insAlu in exon 3 (short interspersed elements (SINEs), Alu elements), CNVs were more often found in the BRCA2 than BRCA1 gene, which is in contrary with observations in other nationalities where CNVs are most often identified in the BRCA1 gene. Overall, in Portugal, CNVs constitute approximately 6% of all BRCA1 germline PVs/LPVs and about 58% of all BRCA2 germline Pvs/LPVs (Peixoto et al. 2009). Moreover, researchers of various nationalities have also shown the important share of CNVs among all germline PVs/LPVs in BRCA1 and BRCA2 genes in their reports on European (Hogervorst et al. 2003; Petrij-Bosch et al. 1997; Peixoto et al. 2009; Concolino et al. 2018; Gad et al. 2002; Hansen et al. 2009; De La Hoya et al. 2006; Engert et al. 2008; Hartmann et al. 2004), Asian (Kwong et al. 2015; Seong et al. 2014; Akin Duman et al. 2023), African (Sluiter et al. 2011), and American (LaDuca et al. 2020) populations [Table 1]. Table 1. The proportion of CNVs reported in patients with germline PVs/LPVs in BRCA1 and BRCA2 depending on the studied population. Country Percentage of CNVs among BRCA1 and BRCA2 PVs/LPVs Year of publication Authors of the publication Netherlands 27-36% 1997; 2003 Petrij-Bosch, A. et al. Hogervorst, F. B. L. et al. Portugal 25% 2009 Peixoto, A. et al. Czech Republic 12.3% 2010 Ticha, I. et al. Italy 12% 2018 Concolino, P. et al. Hungary 10% 2020 Bozsik, A. et al. France 9.5% 2002 Gad, S. et al. Denmark 9.2% 2009 Hansen, T. V. O. et al. Spain 8% 2006 De La Hoya, M. et al. Germany 8% 2004; 2008 Hartmann, C. et al Engert, S. et al. USA 8% 2020 LaDuca, H. et al. China 6.7% 2015 Kwong, A. et al. South Korea 3.7% 2014 Seong, M. W. et al. Türkiye 3.4% 2023 Akin Duman, T. et al. Republic of South Africa 3% 2011 Sluiter, M. D. et al. Regarding the Central and Eastern European population, data are limited due to the small number of studies conducted in this area so far. However, the available information mentions a significant share of BRCA1 CNVs in the Czech and Hungarian populations, where they accounted for about 12.3% and 10% of all germline PVs/LPVs identified in BRCA1, respectively (Bozsik et al. 2020; Ticha et al. 2010). The prevalence of CNVs in the German population in cancer-affected 450 families was estimated to be 2.1%. The most common deletions involved exon 17 of the BRCA1, which, together with the deletion of exon 22 and duplication of exon 13, represent more than 50% of all BRCA1 CNVs. In addition to recurrent and unique minor exon rearrangements, large deletions were detected in two families: deletion of the entire BRCA1 gene and deletion of exons 1-7 (Engert et al. 2008). Data regarding the Polish population is incomplete due to the current lack of mandatory CNVs verification of the BRCA1 and BRCA2 genes in every patient referred for germline and somatic mutations testing of these genes. According to the publication by Rudnicka H. et al. (2013), CNVs of the BRCA1 gene constitute approximately 3.7% of all BRCA1 germline PVs/LPVs in the Polish population. Patients The study group consisted of 2720 probands: 2702 cancer patients and 18 relatives. The probands had a mean age of 54.7±15.15 years (ranging from 18 to 96 years old). The median age was 55. 96.3% of probands were women and 3.7% were men. The testing of germline pathogenic or likely pathogenic SNVs and CNVs in BRCA1 and BRCA2 genes was performed on a group of patients diagnosed and treated in the Lower Silesian Oncology, Pulmonology and Haematology Centre (Wroclaw, Poland) in the years 2021-2024, who met at least one of the following criteria: 1) patients with breast cancer to whom PARPi treatment might be considered, 2) patients with breast cancer with significantly affected clinical and/or family history such as: breast cancer before or at the age of 45, bilateral breast cancer (synchronous or metachronous), TNBC (triple negative breast cancer), breast cancer and ovarian cancer in the same patient, male breast cancer, three cases of breast cancer among first- or second-degree relatives (regardless of age at diagnosis), two cases of breast cancer among first- or second-degree relatives (including at least one diagnosis before the age of 50) 3) patients with ovarian cancer in whom the NGS test performed on tDNA (isolated from the malignant clone) for BRCA1 and BRCA2 mutations showed no presence of pathogenic or likely pathogenic SNVs; however, the family and/or clinical history indicated a high probability of HBOC, 4) patients and healthy probands with relatives previously identified as having a germline CNV in BRCA1 or BRCA2. Methods DNA extraction Genomic DNA (gDNA) was extracted from 300 µL of fresh whole blood using a Maxwell RSC Blood DNA Kit (Promega Corporation) and a Maxwell RSC isolator (AS4500, Promega Corporation) according to the manufacturer's instructions. Following extraction, Quantus Fluorometer (Promega) and QuantiFluor ONE dsDNA System (Promega) were used to quantify the DNA using the fluorometric approach. DNA purity was assessed using the Implant NanoPhotometer N60 (Implen). NGS The BRCA1 and BRCA2 exons and exon-intron boundaries were sequenced using the Devyser BRCA kit (Devyser AB, Sweden) according to the manufacturer's protocol. The sequencing process was performed with the MiSeqDx and MiSeq Reagent Micro Kit v2 (300 cycles) or v3 (600 cycles) (Illumina, California, USA). Primary data analysis (cluster density, cluster passing filter, estimated yield, and Q30 score) was carried out directly in the MiSeqDx instrument. Secondary data analysis was carried out using Amplicon Suite Software version 3.7.0 (SmartSeq, Italy). To analyse SNVs, indels, and CNVs, a 200× amplicon coverage was recommended. CNVs were computed using the ratio of the number of reads with both intra-sample and inter-sample normalisation within each run. Variants were classified according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) system (Richards et al. 2015). MLPA Exon deletions and duplications in BRCA1 and BRCA2 were analysed by MLPA using SALSA MLPA Probemix P002 BRCA1, SALSA MLPA Probemix P087 BRCA1 Confirmation and SALSA MLPA Probemix P045 BRCA2/CHEK2 (MRC-Holland, Amsterdam, The Netherlands), respectively. The process was carried out precisely according to the manufacturer's protocol. The PCR products were separated using the 3500xl Genetic Analyser (Applied Biosystems). Data were analysed with the Coffalyser.NET software, version v.240129.0000 (MRC-Holland, Amsterdam, The Netherlands). The MLPA technique was used to diagnose probands with a known familial exon loss, to confirm pathogenic or potentially pathogenic CNVs identified using NGS or in patients with ovarian cancer with highly suggestive family and/or clinical history in whom the NGS test performed on tDNA showed no presence of pathogenic or likely pathogenic SNV in BRCA1 and BRCA2 . Every patient who had either a pathogenic or likely pathogenic copy number variant, or a normal result but a first-degree relative with a pathogenic or likely pathogenic CNV, underwent two separate tests using two different blood samples and two different MLPA kits, or two different techniques (NGS vs. MLPA). Results In our research, no CNV in BRCA2 was found, which is consistent with the results of the previous Polish study, as well as German, Czech and Hungarian studies (Engert et al. 2008; Bozsik et al. 2020; Ticha et al. 2010; Rudnicka et al. 2013). Pathogenic CNVs in BRCA1 were identified in 23 patients from 13 distinct families. Among eight different BRCA1 CNVs with a prevalence of 0.85% in the examined population, four were unique, three were present in two families and one (exon 21 deletion) was detected in three families. Four of the revealed CNVs were one-exon deletions (exons 3, 16, 19, and 21), while four spanned several exons (upstream exon 2, upstream exons 13 and 23, and exons 12-18). The most frequently involved was exon 2 (present in 3 different variants) [Fig.1, Table 3]. Table 2. NGS and MLPA of BRCA1 and BRCA2 performed in cancer patients and their family members. Number of probands Number of tests NGS tests Pathogenic CNVs identified by NGS NGS tests with non-informative CNV MLPA tests Pathogenic CNVs identified by MLPA Total number of pathogenic CNVs Total 2720 3031 2638 9 (0.34% of NGS tests) 583 393 (311 due to non-informative NGS, 82 as a first test) 14 (3.56% od MLPA tests) 23 (0.85% of probands) Cancer patients 2702 3013 2638 9 583 375 (311 due to non-informative NGS, 64 as a first test) 5 14 (0.52% of cancer patients) Family members 18 18 0 0 0 18 (18 as a first test) 9 9 (50% of family members) Families Within 13 families with identified pathogenic CNVs in BRCA1 , 39 BRCA-related cancer cases were noted, including 27 breast cancer, 10 ovarian cancer and two pancreatic cancer cases. All families were severely affected by malignancies typical of HBOC [Table 3, Fig. 2]. What is particularly noteworthy is the young age of breast cancer development in examined families, as 13 out of 27 cases (48%) were diagnosed before or at the age of 45. The mean age of breast cancer diagnosis was 47.4. There was also a significant proportion of TNBC among breast cancer cases, which in our study was 30% (8 out of 27 cases), and only 1 case of TNBC was diagnosed after the age of 45. The mean age of ovarian cancer diagnosis was 54.3, which is also noticeably younger than in the general population. Moreover, four individuals developed two independent BRCA-related cancers, including: 1 individual with bilateral, metachronous breast cancer (first diagnosed at age 39, then TNBC at age 51), one individual diagnosed with ovarian cancer and then pancreatic cancer, and two individuals diagnosed with both breast and ovarian cancer. Within the examined families, a genetic test for CNVs in BRCA1 and/or BRCA2 was performed in 32 individuals, including 14 individuals diagnosed with cancer (8 patients with unilateral breast cancer with predominance of TNBC cases, 1 patient with bilateral breast cancer, 4 patients with ovarian cancer, and one patient with both ovarian and pancreatic cancer) and 18 individuals referred due to the detection of a mutation in a relative. Thanks to the genetic tests performed, the carriage of the pathogenic CNV in BRCA1 was detected in 50% of healthy relatives referred due to the previous identification of such mutation in the family. Table 3. Families with identified BRCA1 CNVs. Family BRCA1 CNVs NM_007294.4 [U] unique variants No. of members tested No. of members with BRCA1 CNVs Significantly affected clinical and/or family history Family 1 BRCA1 upstream-exon 2 deletion 1 1 Yes (TNBC at age 44) Family 2 BRCA1 exon 21 deletion 4 3 Yes (TNBC at age 28) Family 3 BRCA1 exon 19 deletion [U] 3 3 Yes (bilateral BC, first at age 39, then TNBC at age 51) Family 4 BRCA1 upstream-exon 23 deletion [U] 3 1 Yes (TNBC at age 39, BC in a relative) Family 5 BRCA1 upstream – exon 13 deletion [U] 1 1 Yes (OC, 2 BC in relatives) Family 6 BRCA1 exon 12-18 deletion 1 1 Yes (TNBC at age 34, BC and 2 OC in relatives), Ukrainian Family 7 BRCA1 exon 21 deletion 8 3 Yes (OC and PC, OC and BC in a relative) Family 8 BRCA1 exon 16 deletion 2 2 Yes (OC and BC in a relative, OC in another relative) Family 9 BRCA1 upstream-exon 2 deletion 1 1 Yes (TNBC at age 43, BC and PC in relatives) Family 10 BRCA1 exon 3 deletion [U] 1 1 Yes (OC, BC and OC in relatives) Family 11 BRCA1 exon 12-18 deletion 1 1 Yes, (OC, 3 BC in relatives) Family 12 BRCA1 exon 21 deletion 2 1 Yes (BC, TNBC at age 35 in a relative) Family 13 BRCA1 exon 16 deletion 4 4 Yes (TNBC, 6 BC in relatives) Total 32 23 Legend: BC (breast cancer), TNBC (triple negative breast cancer), OC (ovarian cancer), PC (pancreatic cancer) Discussion The widespread use of the NGS technique has made it possible to quickly, accurately and simultaneously analyse whole coding sequences of selected genes for both single-nucleotide variants and copy-number variants. Although pathogenic SNVs are a major cause of BRCA protein loss of function, at least in families suspected with HBOC, the contribution of BRCA CNVs to its aetiology should not be ignored (McDevitt et al. 2024). Previous study on European population of BRCA- associated cancer patients and their family members revealed, that CNVs in BRCA1 accounts for about 1.5-2.0% of all BRCA1 pathogenic variants while for BRCA2 are very rare, or not present in examined groups, except BRCA2 founder pathogenic variant c.156_157insAlu in Portuguese (Engert et al. 2008). Our findings confirmed previous study on 200 unrelated patients of Rudnicka et al. (2013) where exons 13-19 deletion (according to NM_007294.4 Mane Select: exons 12-18), exon 17 deletion (NM_007294.4: exon 16) and exon 22 deletion (NM_007294.4: exon 21) were detected and are consistent with other European studies (Schmidt et al. 2017; Peixoto et al. 2009; Hansen et al. 2009; Engert et al. 2008; Bozsik et al. 2020). Pathogenic CNVs in BRCA1 are most commonly located in three domains/regions: the N-terminal RING domain (exons 2-7, NM_007294.4: 2-6), exons 11-13 (NM_007294.4: 10-12), and the BRCT domain (exons 16-24, NM_007294.4: 15-23) (Wang 2012; Wang et al. 2019). The repeat of regions involved in rearrangements in the BRCA1 gene is caused by recombination of intronic Alu-elements present in this gene and its pseudogene, as well as Alu-Alu homologous recombination in BRCA1 (Engert et al. 2008; Wang et al. 2019). The same Alu-elements may be responsible for developing resistance to PARP inhibitors during therapy in patients with BRCA1 mutations, as they induce rearrangements that allow the return of functional protein expression (Wang et al. 2019). Our observations showed that all families with identified pathogenic CNVs in BRCA1 were characterised by an aggressive phenotype with a predominance of TNBC breast cancers before the age of 45 and frequent cases of two primary BRCA -dependent cancers in the same person. In 8 out of 13 examined families, there were at least three diagnoses of HBOC spectrum malignancies among first- and/or second-degree relatives, which is additional proof of high penetration in carriers. This highlights the importance of a detailed and thorough analysis of the BRCA mutational status, also taking into account CNVs, especially in cases indicating HBOC. What is also important, in as many as 11 out of 13 identified families, the proband who was first detected as a carrier of a pathogenic CNV in BRCA1 was a patient with a current diagnosis of cancer for which personalized PARPi therapy could be considered (7 cases of TNBC, 3 cases of ovarian cancer and 1 case of pancreatic cancer after previously diagnosed ovarian cancer). Moreover, in the families mentioned above, half of the healthy relatives who underwent genetic tests were found to be carriers of the pathogenic marker CNV in BRCA1 , which significantly changed the recommendations regarding oncological prevention. Polish patients and their relatives with detected pathogenic copy number variants in BRCA1 are a small but significant group; therefore, we believe that not only SNVs and indels but also exon rearrangements should be targeted in routine genetic screening diagnostics. As pathogenic BRCA2 gene rearrangements are extremely rare in Poland, their diagnosis should at least be offered to patients with significantly affected clinical and/or family history in which no pathogenic SNVs in the HBOC genes have been detected (Engert et al. 2008; Ticha et al. 2010; McDevitt et al. 2024). Moreover, because of today's high levels of migration (Skorniak et al. 2025), in populations characterized by the presence of frequent changes in selected genes that are considered in national health programs, the patient's ethnicity should also be taken into account, and the diagnostic test adapted accordingly. Conclusion Despite a small share of CNVs among all identified germline PVs/LPVs in BRCA1 and no CNVs in BRCA2 in examined patients, every effort should be made to ensure that these variants are included in the genetic analysis carried out in patients with HBOC spectrum cancers as it can prevent false negative results that may lead to giving up personalized PARPi therapy or losing the chance for individualized cancer prevention for the family. Therefore, despite the relatively low frequency of copy number variants in BRCA1 in Polish patients, in addition to sequencing BRCA1 , BRCA2 , and testing for exon deletions and duplications, these tests should be offered at least to families at high risk of BRCA -associated cancers. Limitations The data obtained on the percentage of BRCA1 and BRCA2 CNVs in the population of Polish patients (except one Ukrainian family) and their family members may be incorrectly estimated because MLPA and NGS tests were not performed in all patients with breast and/or ovarian cancer, but in selected cases, which was related to national prevention and diagnostic programs. MLPA and NGS techniques do not allow assessment of exact breakpoints; therefore, CNVs detected in more than one family could have been in fact different variants. Declarations Funding This research was financed through a statutory subsidy by the Ministry of Health as part of the Department of Oncology Wroclaw Medical University research grant SUBZ.C280.26.041 (record number in the Simple System). Competing interests The authors have no relevant financial or non-financial interests to disclose. Author contributions All authors contributed to the study conception and design. All authors read and approved the final manuscript. Ethics approval This is an observational study. The Wroclaw Medical University Ethics Committee has confirmed that no ethical approval is required (No. 233/2025). Consent to participate Informed consent for genetic diagnostics was obtained from all individual participants included in the study. Data availability The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. References Akin Duman, T., & Ozturk, F. N. (2023). Frequency and distribution of BRCA1/BRCA2 large genomic rearrangements in Turkish population with breast cancer. Journal of Human Genetics, 68 (7), 485–490. https://doi.org/10.1038/S10038-023-01140-6 Bozsik, A., Pócza, T., Papp, J., Vaszkó, T., Butz, H., Patócs, A., et al. (2020). 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Genetics in Medicine, 17 (5), 405–424. https://doi.org/10.1038/gim.2015.30 Rudnicka, H., Debniak, T., Cybulski, C., Huzarski, T., Gronwald, J., Lubinski, J., et al. (2013). Large BRCA1/2 rearrangements in Polish families. Molecular Biology Reports, 40 (12), 6619–6623. https://doi.org/10.1007/s11033-013-2775-0 Schmidt, A. Y., Hansen, T. v. O., Ahlborn, L. B., Jønson, L., Yde, C. W., & Nielsen, F. C. (2017). NGS-based detection of germline CNVs in BRCA1/2. Journal of Molecular Diagnostics, 19 (6), 809–816. https://doi.org/10.1016/j.jmoldx.2017.07.003 Seong, M. W., Cho, S. I., Kim, K. H., Chung, I. Y., Kang, E., Lee, J. W., et al. (2014). Prevalence of BRCA1/2 LGRs in familial breast cancer. BMC Cancer, 14 (1). https://doi.org/10.1186/1471-2407-14-645 Skorniak, J., Rabalski, L., Szynglarewicz, B., Dolega-Kozierowski, B., Kasprzak, P., Zietek, M., et al. (2025). Breast cancer stage among Ukrainian refugees in Poland. JAMA Network Open, 8 (4), e256215. https://doi.org/10.1001/jamanetworkopen.2025.6215 Sluiter, M. D., & Van Rensburg, E. J. (2011). Large genomic rearrangements of BRCA1 and BRCA2: review. Breast Cancer Research and Treatment, 125 (2), 325–349. https://doi.org/10.1007/S10549-010-0817-Z Stoffel, E. M., Brand, R. E., & Goggins, M. (2023). Pancreatic cancer: changing epidemiology and new approaches. Gastroenterology, 164 (5), 752–765. https://doi.org/10.1053/j.gastro.2023.02.012 Tew, W. P., Lacchetti, C., Ellis, A., Maxian, K., Banerjee, S., Bookman, M., et al. (2020). PARP inhibitors in ovarian cancer: ASCO guideline. Journal of Clinical Oncology, 38 , 3468–3493. https://doi.org/10.1200/JCO.20 Ticha, I., Kleibl, Z., Stribrna, J., Kotlas, J., Zimovjanova, M., Mateju, M., et al. (2010). Screening for genomic rearrangements in Czech patients. Breast Cancer Research and Treatment, 124 (2), 337–347. https://doi.org/10.1007/s10549-010-0745-y Vergote, I., González-Martín, A., Ray-Coquard, I., Harter, P., Colombo, N., Pujol, P., et al. (2022). European consensus on BRCA/HRD testing in ovarian cancer. Annals of Oncology, 33 (3), 276–287. https://doi.org/10.1016/j.annonc.2021.11.013 Walsh, T., Casadei, S., Lee, M. K., Pennil, C. C., Nord, A. S., Thornton, A. M., et al. (2011). Mutations in 12 genes for inherited ovarian carcinoma. PNAS, 108 (44), 18032–18037. https://doi.org/10.1073/pnas.1115052108 Wang, B. (2012). BRCA1 tumor suppressor network. Cell and Bioscience, 2 (1). https://doi.org/10.1186/2045-3701-2-6 Wang, Y., Bernhardy, A. J., Nacson, J., Krais, J. J., Tan, Y. F., Nicolas, E., et al. (2019). BRCA1 intronic Alu elements drive rearrangements. Nature Communications, 10 (1). https://doi.org/10.1038/s41467-019-13530-6 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9336936","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":626839970,"identity":"36afc0b6-a061-4c7e-861d-4bbf97c6baff","order_by":0,"name":"Anna 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Technology","correspondingAuthor":false,"prefix":"","firstName":"Gabriela","middleName":"","lastName":"Janus-Szymanska","suffix":""},{"id":626839981,"identity":"8b4855da-04c7-4e74-b446-7c18734671ee","order_by":11,"name":"Ireneusz Pawlak","email":"","orcid":"","institution":"Lower Silesian Center of Oncology, Pulmonology and Hematology","correspondingAuthor":false,"prefix":"","firstName":"Ireneusz","middleName":"","lastName":"Pawlak","suffix":""},{"id":626839982,"identity":"15346c38-cc11-479e-b349-6b8d937ca60c","order_by":12,"name":"Adam Maciejczyk","email":"","orcid":"","institution":"Wroclaw Medical University","correspondingAuthor":false,"prefix":"","firstName":"Adam","middleName":"","lastName":"Maciejczyk","suffix":""},{"id":626839983,"identity":"149e9b0a-e914-4f57-b642-c3b904af1877","order_by":13,"name":"Jolanta Szelachowska","email":"","orcid":"","institution":"Wroclaw Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jolanta","middleName":"","lastName":"Szelachowska","suffix":""},{"id":626839984,"identity":"87c7f544-0637-4051-81a9-b5c4243a0605","order_by":14,"name":"Izabela Laczmanska","email":"","orcid":"","institution":"Wroclaw Medical University","correspondingAuthor":false,"prefix":"","firstName":"Izabela","middleName":"","lastName":"Laczmanska","suffix":""}],"badges":[],"createdAt":"2026-04-06 19:53:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9336936/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9336936/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107896143,"identity":"92e142fa-eeac-495c-90c8-017d437caef2","added_by":"auto","created_at":"2026-04-27 10:52:11","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":48510,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBRCA1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(NM_007294.4) deletions specific to the examined families.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9336936/v1/692b7eb841af9fc791c3e887.jpg"},{"id":107896144,"identity":"f060d1cb-7fdd-4d18-bdda-0e838ea5057d","added_by":"auto","created_at":"2026-04-27 10:52:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":781486,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFamilies with identified \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBRCA1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e CNVs\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9336936/v1/1204022ab856e7a043339991.jpg"},{"id":108006561,"identity":"7426720b-014f-4467-836c-d88f67eb585a","added_by":"auto","created_at":"2026-04-28 12:56:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1217183,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9336936/v1/0c8d7c98-701c-4ac8-917f-2a75e78f7b53.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Copy number variants in BRCA1 and BRCA2 genes in Polish patients with breast and ovarian cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHereditary predisposition to breast and ovarian cancer (HBOC) is one of the most commonly examined predispositions in Poland. Public funding ensures that many patients with breast, ovarian, pancreatic and prostate cancer and their families have access to genetic diagnostics ranging from testing for a single familial pathogenic variant, testing for a few selected pathogenic variants (most common in the Polish population), and also sequencing of the whole coding part of selected genes (Doraczynska-Kowalik et al. 2022).\u003c/p\u003e\n\u003cp\u003eIt is estimated that even about 10-15% of breast cancer patients, 15% of ovarian cancer patients, 5-10% of pancreatic cancer patients, 5% of localized prostate cancer patients and 12% of metastatic prostate cancer patients are likely to be carriers of a germline dominant susceptibility pathogenic variant responsible for a specific hereditary cancer predisposition (Kasuga et al 2022; Kurian et al. 2018; Pritchard et al. 2016; Stoffel et al. 2023; Walsh et al. 2011). Among many susceptibility genes for malignancies mentioned above, \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u0026nbsp;\u003c/em\u003eare the two most often tested as their pathogenic or likely pathogenic variants (PVs/LPVs) are characterized by high penetrance (cumulative cancer risks to age 80 are breast cancer risk of 72% for \u003cem\u003eBRCA1\u003c/em\u003e and 69% for \u003cem\u003eBRCA2\u003c/em\u003e mutation carriers and ovarian cancer risk of 44% and 17% respectively) and account for up to 80% of all deleterious alterations recognized in hereditary breast and ovarian cancer (HBOC) families (Kuchenbaecker et al. 2017; McAlarnen etl. 2021). Moreover, \u003cem\u003eBRCA1/2\u003c/em\u003e mutational status together with homologous recombination repair deficiency (HRD) rank are the most important biomarkers for PARPi (poly ADP-ribose polymerase inhibitors) response in ovarian, breast, prostate and pancreatic cancer patients (de Bono et al. 2020; Kindler et al. 2022; Menezes et al. 2022; Tew et al. 2020; Vergote et al. 2022).\u003c/p\u003e\n\u003cp\u003eWorldwide observations show that single-nucleotide variants (SNVs) (previously known as point mutations) dominate germline PVs/LPVs found in \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e genes. The share of intragenic copy number variants (CNVs), mainly deletions and duplications, in \u003cem\u003eBRCA1\u0026nbsp;\u003c/em\u003eand \u003cem\u003eBRCA2\u0026nbsp;\u003c/em\u003egenes is therefore relatively small; however, it varies greatly depending on the examined populations (Germani et al.; Schmidt et al. 2017).\u003c/p\u003e\n\u003cp\u003eThe highest occurrence of \u003cem\u003eBRCA1/2\u003c/em\u003e CNVs is found in the Dutch population, in which 27-36% of all germline PVs/LPVs in the \u003cem\u003eBRCA1\u003c/em\u003e gene are CNVs, making \u003cem\u003eBRCA1\u003c/em\u003e deletions major founder mutations in the Netherlands (Hogervorst et al. 2003; Petrij-Bosch et al. 1997). Surprising results were obtained in the Portuguese population, in which, due to the large share of \u003cem\u003eBRCA2\u003c/em\u003e founder mutation c.156_157insAlu in exon 3 (short interspersed elements (SINEs), Alu elements), CNVs were more often found in the \u003cem\u003eBRCA2\u003c/em\u003e than \u003cem\u003eBRCA1\u003c/em\u003e gene, which is in contrary with observations in other nationalities where CNVs are most often identified in the \u003cem\u003eBRCA1\u003c/em\u003e gene. Overall, in Portugal, CNVs constitute approximately 6% of all \u003cem\u003eBRCA1\u003c/em\u003e germline PVs/LPVs and about 58% of all \u003cem\u003eBRCA2\u003c/em\u003e germline Pvs/LPVs (Peixoto et al. 2009). Moreover, researchers of various nationalities have also shown the important share of CNVs among all germline PVs/LPVs in \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e genes in their reports on European (Hogervorst et al. 2003; Petrij-Bosch et al. 1997; Peixoto et al. 2009; Concolino et al. 2018; Gad et al. 2002; Hansen et al. 2009; De La Hoya et al. 2006; Engert et al. 2008; Hartmann et al. 2004), Asian (Kwong et al. 2015; Seong et al. 2014; Akin Duman et al. 2023), African (Sluiter et al. 2011), and American (LaDuca et al. 2020) populations [Table 1].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. The proportion of CNVs reported in patients with germline PVs/LPVs in \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e depending on the studied population.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCountry\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePercentage of CNVs among \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e PVs/LPVs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eYear of publication\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAuthors of the publication\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNetherlands\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e27-36%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1997; 2003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePetrij-Bosch, A. et al.\u003c/p\u003e\n \u003cp\u003eHogervorst, F. B. L. et al.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePortugal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e25%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePeixoto, A. et al.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCzech Republic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e12.3%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2010\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTicha, I. et al.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eItaly\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e12%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eConcolino, P. et al.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHungary\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBozsik, A. et al.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFrance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGad, S. et al.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDenmark\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9.2%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHansen, T. V. O. et al.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSpain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDe La Hoya, M. et al.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGermany\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2004; 2008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHartmann, C. et al\u003c/p\u003e\n \u003cp\u003eEngert, S. et al.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eUSA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLaDuca, H. et al.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eChina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6.7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKwong, A. et al.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSouth Korea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3.7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSeong, M. W. et al.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eTürkiye\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3.4%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAkin Duman, T. \u0026nbsp;et al.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;Republic of South Africa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSluiter, M. D. et al.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eRegarding the Central and Eastern European population, data are limited due to the small number of studies conducted in this area so far. However, the available information mentions a significant share of \u003cem\u003eBRCA1\u003c/em\u003e CNVs in the Czech and Hungarian populations, where they accounted for about 12.3% and 10% of all germline PVs/LPVs identified in \u003cem\u003eBRCA1,\u003c/em\u003e respectively (Bozsik et al. 2020; Ticha et al. 2010). The prevalence of CNVs in the German population in cancer-affected 450 families was estimated to be 2.1%. The most common deletions involved exon 17 of the BRCA1, which, together with the deletion of exon 22 and duplication of exon 13, represent more than 50% of all \u003cem\u003eBRCA1\u0026nbsp;\u003c/em\u003eCNVs. In addition to recurrent and unique minor exon rearrangements, large deletions were detected in two families: deletion of the entire \u003cem\u003eBRCA1\u003c/em\u003e gene and deletion of exons 1-7 (Engert et al. 2008).\u003c/p\u003e\n\u003cp\u003eData regarding the Polish population is incomplete due to the current lack of mandatory CNVs verification of the\u003cem\u003e\u0026nbsp;BRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u0026nbsp;\u003c/em\u003egenes in every patient referred for germline and somatic mutations testing of these genes. According to the publication by Rudnicka H. et al. (2013), CNVs of the \u003cem\u003eBRCA1\u003c/em\u003e gene constitute approximately 3.7% of all \u003cem\u003eBRCA1\u003c/em\u003e germline PVs/LPVs in the Polish population.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study group consisted of \u003cstrong\u003e2720\u003c/strong\u003e probands: 2702 cancer patients and 18 relatives. The probands had a mean age of 54.7±15.15 years (ranging from 18 to 96 years old). The median age was 55. 96.3% of probands were women and 3.7% were men.\u003c/p\u003e\n\u003cp\u003eThe testing of germline pathogenic or likely pathogenic SNVs and CNVs in \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e genes was performed on a group of patients diagnosed and treated in the Lower Silesian Oncology, Pulmonology and Haematology Centre (Wroclaw, Poland) in the years 2021-2024, who met at least one of the following criteria:\u003c/p\u003e\n\u003cp\u003e1) patients with breast cancer to whom PARPi treatment might be considered,\u003c/p\u003e\n\u003cp\u003e2) patients with breast cancer with significantly affected clinical and/or family history such as: breast cancer before or at the age of 45, bilateral breast cancer (synchronous or metachronous), TNBC (triple negative breast cancer), breast cancer and ovarian cancer in the same patient, male breast cancer, three cases of breast cancer among first- or second-degree relatives (regardless of age at diagnosis), two cases of breast cancer among first- or second-degree relatives (including at least one diagnosis before the age of 50)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3) patients with ovarian cancer in whom the NGS test performed on tDNA (isolated from the malignant clone) for \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u0026nbsp;\u003c/em\u003emutations showed no presence of pathogenic or likely pathogenic SNVs; however, the family and/or clinical history indicated a high probability of HBOC,\u003c/p\u003e\n\u003cp\u003e4) patients and healthy probands with relatives previously identified as having a germline CNV in \u003cem\u003eBRCA1\u003c/em\u003e or \u003cem\u003eBRCA2.\u003c/em\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eDNA extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA (gDNA) was extracted from 300 µL of fresh whole blood using a Maxwell RSC Blood DNA Kit (Promega Corporation) and a Maxwell RSC isolator (AS4500, Promega Corporation) according to the manufacturer's instructions. Following extraction, Quantus Fluorometer (Promega) and QuantiFluor ONE dsDNA System (Promega) were used to quantify the DNA using the fluorometric approach. DNA purity was assessed using the Implant NanoPhotometer N60 (Implen).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNGS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e exons and exon-intron boundaries were sequenced using the Devyser BRCA kit (Devyser AB, Sweden) according to the manufacturer's protocol. The sequencing process was performed with the MiSeqDx and MiSeq Reagent Micro Kit v2 (300 cycles) or v3 (600 cycles) (Illumina, California, USA).\u003c/p\u003e\n\u003cp\u003ePrimary data analysis (cluster density, cluster passing filter, estimated yield, and Q30 score) was carried out directly in the MiSeqDx instrument. Secondary data analysis was carried out using Amplicon Suite Software version 3.7.0 (SmartSeq, Italy). To analyse SNVs, indels, and CNVs, a 200× amplicon coverage was recommended. CNVs were computed using the ratio of the number of reads with both intra-sample and inter-sample normalisation within each run. Variants were classified according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) system (Richards et al. 2015).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMLPA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExon deletions and duplications in \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u0026nbsp;\u003c/em\u003ewere analysed by MLPA using SALSA MLPA Probemix P002 \u003cem\u003eBRCA1,\u003c/em\u003e SALSA MLPA Probemix P087 \u003cem\u003eBRCA1\u0026nbsp;\u003c/em\u003eConfirmation and SALSA MLPA Probemix P045 \u003cem\u003eBRCA2/CHEK2\u0026nbsp;\u003c/em\u003e(MRC-Holland, Amsterdam, The Netherlands), respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe process was carried out precisely according to the manufacturer's protocol. The PCR products were separated using the 3500xl Genetic Analyser (Applied Biosystems). Data were analysed with the Coffalyser.NET software, version v.240129.0000 (MRC-Holland, Amsterdam, The Netherlands).\u003c/p\u003e\n\u003cp\u003eThe MLPA technique was used to diagnose probands with a known familial exon loss, to confirm pathogenic or potentially pathogenic CNVs identified using NGS or in patients with ovarian cancer with highly suggestive family and/or clinical history in whom the NGS test performed on tDNA showed no presence of pathogenic or likely pathogenic SNV in \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e. Every patient who had either a pathogenic or likely pathogenic copy number variant, or a normal result but a first-degree relative with a pathogenic or likely pathogenic CNV, underwent two separate tests using two different blood samples and two different MLPA kits, or two different techniques (NGS \u003cem\u003evs.\u0026nbsp;\u003c/em\u003eMLPA).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn our research, no CNV in \u003cem\u003eBRCA2\u003c/em\u003e was found, which is consistent with the results of the previous Polish study, as well as German, Czech and Hungarian studies (Engert et al. 2008; Bozsik et al. 2020; Ticha et al. 2010; Rudnicka et al. 2013). Pathogenic CNVs in \u003cem\u003eBRCA1\u003c/em\u003e were identified in 23 patients from 13 distinct families. Among eight different \u003cem\u003eBRCA1\u003c/em\u003e CNVs with a prevalence of 0.85% in the examined population, four were unique, three were present in two families and one (exon 21 deletion) was detected in three families. Four of the revealed CNVs were one-exon deletions (exons 3, 16, 19, and 21), while four spanned several exons (upstream exon 2, upstream exons 13 and 23, and exons 12-18). The most frequently involved was exon 2 (present in 3 different variants) [Fig.1, Table 3].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. NGS and MLPA of \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e performed in cancer patients and their family members.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"726\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of probands\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of tests\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNGS tests\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePathogenic CNVs identified by NGS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNGS tests with non-informative CNV\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMLPA tests\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePathogenic CNVs identified by MLPA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal number of\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003epathogenic\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCNVs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e2720\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3031\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2638\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9 (0.34% of NGS tests)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e583\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e393 (311 due to non-informative NGS, 82 as a first test)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e14 (3.56% od MLPA tests)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e23 (0.85% of probands)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCancer patients\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e2702\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2638\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e583\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e375 (311 due to non-informative NGS, 64 as a first test)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e14 (0.52% of cancer patients)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFamily members\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e18 (18 as a first test)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9 (50% of family members)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eFamilies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWithin 13 families with identified pathogenic CNVs in \u003cem\u003eBRCA1\u003c/em\u003e, 39 BRCA-related cancer cases were noted, including 27 breast cancer, 10 ovarian cancer and two pancreatic cancer cases. All families were severely affected by malignancies typical of HBOC [Table 3, Fig. 2]. What is particularly noteworthy is the young age of breast cancer development in examined families, as 13 out of 27 cases (48%) were diagnosed before or at the age of 45. The mean age of breast cancer diagnosis was 47.4. There was also a significant proportion of TNBC among breast cancer cases, which in our study was 30% (8 out of 27 cases), and only 1 case of TNBC was diagnosed after the age of 45. The mean age of ovarian cancer diagnosis was 54.3, which is also noticeably younger than in the general population. Moreover, four individuals developed two independent BRCA-related cancers, including: 1 individual with bilateral, metachronous breast cancer (first diagnosed at age 39, then TNBC at age 51), one individual diagnosed with ovarian cancer and then pancreatic cancer, and two individuals diagnosed with both breast and ovarian cancer. Within the examined families, a genetic test for CNVs in \u003cem\u003eBRCA1\u003c/em\u003e and/or \u003cem\u003eBRCA2\u003c/em\u003e was performed in 32 individuals, including 14 individuals diagnosed with cancer (8 patients with unilateral breast cancer with predominance of TNBC cases, 1 patient with bilateral breast cancer, 4 patients with ovarian cancer, and one patient with both ovarian and pancreatic cancer) and 18 individuals referred due to the detection of a mutation in a relative. Thanks to the genetic tests performed, the carriage of the pathogenic CNV in \u003cem\u003eBRCA1\u003c/em\u003e was detected in 50% of healthy relatives referred due to the previous identification of such mutation in the family.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Families with identified \u003cem\u003eBRCA1\u003c/em\u003e CNVs.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"603\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFamily\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eBRCA1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;CNVs\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eNM_007294.4\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e[U] unique variants\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of members tested\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of members with \u003cem\u003eBRCA1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;CNVs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSignificantly affected clinical and/or family history\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFamily 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1\u003c/em\u003e upstream-exon 2 deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes (TNBC at age 44)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFamily 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1\u003c/em\u003e exon 21 deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes (TNBC at age 28)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFamily 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1\u003c/em\u003e exon 19 deletion [U]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes (bilateral BC,\u0026nbsp;first at age 39, then TNBC at age 51)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFamily 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1\u003c/em\u003e upstream-exon 23 deletion [U]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes (TNBC at age 39, BC in a relative)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFamily 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1\u003c/em\u003e upstream – exon 13 deletion [U]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes (OC, 2 BC in relatives)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFamily 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1\u003c/em\u003e exon 12-18 deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes (TNBC at age 34, BC and 2 OC in relatives), Ukrainian\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFamily 7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1\u003c/em\u003e exon 21 deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes (OC and PC, OC and BC in a relative)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFamily 8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1\u003c/em\u003e exon 16 deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes (OC and BC in a relative, OC in another relative)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFamily 9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1\u003c/em\u003e upstream-exon 2 deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes (TNBC at age 43, BC and PC in \u0026nbsp;relatives)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFamily 10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1\u003c/em\u003e exon 3 deletion [U]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes (OC, BC and OC in relatives)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFamily 11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1\u003c/em\u003e exon 12-18 deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes, \u0026nbsp;(OC, 3 BC in relatives)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFamily 12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1\u003c/em\u003e exon 21 deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes (BC, TNBC at age 35 in a relative)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFamily 13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBRCA1\u003c/em\u003e exon 16 deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes (TNBC, 6 BC in relatives)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eLegend:\u003c/strong\u003e BC (breast cancer), TNBC (triple negative breast cancer), OC (ovarian cancer), PC (pancreatic cancer)\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe widespread use of the NGS technique has made it possible to quickly, accurately and simultaneously analyse whole coding sequences of selected genes for both single-nucleotide variants and copy-number variants. Although pathogenic SNVs are a major cause of BRCA protein loss of function, at least in families suspected with HBOC, the contribution of \u003cem\u003eBRCA\u0026nbsp;\u003c/em\u003eCNVs to its aetiology should not be ignored (McDevitt et al. 2024).\u003c/p\u003e\n\u003cp\u003ePrevious study on European population of \u003cem\u003eBRCA-\u003c/em\u003eassociated cancer patients and their family members revealed, that CNVs in \u003cem\u003eBRCA1\u003c/em\u003e accounts for about 1.5-2.0% of all \u003cem\u003eBRCA1\u003c/em\u003e pathogenic variants while for \u003cem\u003eBRCA2\u003c/em\u003e are very rare, or not present in examined groups, except \u003cem\u003eBRCA2\u003c/em\u003e founder pathogenic variant c.156_157insAlu in Portuguese (Engert et al. 2008). Our findings confirmed previous study on 200 unrelated patients of Rudnicka et al. (2013) where exons 13-19 deletion (according to NM_007294.4 Mane Select: exons 12-18), exon 17 deletion (NM_007294.4: exon 16) and exon 22 deletion (NM_007294.4: exon 21) were detected and are consistent with other European studies (Schmidt et al. 2017; Peixoto et al. 2009; Hansen et al. 2009; Engert et al. 2008; Bozsik et al. 2020).\u003c/p\u003e\n\u003cp\u003ePathogenic CNVs in \u003cem\u003eBRCA1\u0026nbsp;\u003c/em\u003eare most commonly located in three domains/regions: the N-terminal RING domain (exons 2-7, NM_007294.4: 2-6), exons 11-13 (NM_007294.4: 10-12), and the BRCT domain (exons 16-24, NM_007294.4: 15-23) (Wang 2012; Wang et al. 2019). The repeat of regions involved in rearrangements in the\u003cem\u003e\u0026nbsp;BRCA1\u003c/em\u003e gene is caused by recombination of intronic Alu-elements present in this gene and its pseudogene, as well as Alu-Alu homologous recombination in \u003cem\u003eBRCA1\u003c/em\u003e (Engert et al. 2008; Wang et al. 2019). The same Alu-elements may be responsible for developing resistance to PARP inhibitors during therapy in patients with \u003cem\u003eBRCA1\u003c/em\u003e mutations, as they induce rearrangements that allow the return of functional protein expression (Wang et al. 2019).\u003c/p\u003e\n\u003cp\u003eOur observations showed that all families with identified pathogenic CNVs in \u003cem\u003eBRCA1\u003c/em\u003e were characterised by an\u0026nbsp;aggressive phenotype with a predominance of TNBC breast cancers before the age of 45 and frequent cases of two primary \u003cem\u003eBRCA\u003c/em\u003e-dependent cancers in the same person. In 8 out of 13 examined families, there were at least three diagnoses of HBOC spectrum malignancies among first- and/or second-degree relatives, which is additional proof of high penetration in carriers. This highlights the importance of a detailed and thorough analysis of the \u003cem\u003eBRCA\u0026nbsp;\u003c/em\u003emutational status, also taking into account CNVs, especially in cases indicating HBOC. What is also important, in as many as 11 out of 13 identified families, the proband who was first detected as a carrier of a pathogenic CNV in \u003cem\u003eBRCA1\u003c/em\u003e was a patient with a current diagnosis of cancer for which personalized PARPi therapy could be considered (7 cases of TNBC, 3 cases of ovarian cancer and 1 case of pancreatic cancer after previously diagnosed ovarian cancer). Moreover, in the families mentioned above, half of the healthy relatives who underwent genetic tests were found to be carriers of the pathogenic marker CNV in \u003cem\u003eBRCA1\u003c/em\u003e, which significantly changed the recommendations regarding oncological prevention.\u003c/p\u003e\n\u003cp\u003ePolish patients and their relatives with detected pathogenic copy number variants in \u003cem\u003eBRCA1\u003c/em\u003e are a small but significant group; therefore, we believe that not only SNVs and indels but also exon rearrangements should be targeted in routine genetic screening diagnostics. As pathogenic \u003cem\u003eBRCA2\u003c/em\u003e gene rearrangements are extremely rare in Poland, their diagnosis should at least be offered to patients with significantly affected clinical and/or family history in which no pathogenic SNVs in the HBOC genes have been detected (Engert et al. 2008; Ticha et al. 2010; McDevitt et al. 2024).\u003c/p\u003e\n\u003cp\u003eMoreover, because of today\u0026apos;s high levels of migration (Skorniak et al. 2025), in populations characterized by the presence of frequent changes in selected genes that are considered in national health programs, the patient\u0026apos;s ethnicity should also be taken into account, and the diagnostic test adapted accordingly.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eDespite a small share of CNVs among all identified germline PVs/LPVs in \u003cem\u003eBRCA1\u003c/em\u003e and no CNVs in \u003cem\u003eBRCA2\u003c/em\u003e in examined patients, every effort should be made to ensure that these variants are included in the genetic analysis carried out in patients with HBOC spectrum cancers as it can prevent false negative results that may lead to giving up personalized PARPi therapy or losing the chance for individualized cancer prevention for the family. Therefore, despite the relatively low frequency of copy number variants in \u003cem\u003eBRCA1\u003c/em\u003e in Polish patients, in addition to sequencing \u003cem\u003eBRCA1\u003c/em\u003e, \u003cem\u003eBRCA2\u003c/em\u003e, and testing for exon deletions and duplications, these tests should be offered at least to families at high risk of \u003cem\u003eBRCA\u003c/em\u003e-associated cancers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimitations\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data obtained on the percentage of \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u0026nbsp;\u003c/em\u003eCNVs in the population of Polish patients (except one Ukrainian family) and their family members may be incorrectly estimated because MLPA and NGS tests were not performed in all patients with breast and/or ovarian cancer, but in selected cases, which was related to national prevention and diagnostic programs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMLPA and NGS techniques do not allow assessment of exact breakpoints; therefore, CNVs detected in more than one family could have been in fact different variants.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was financed through a statutory subsidy by the Ministry of Health as part of the Department of Oncology Wroclaw Medical University research grant\u0026nbsp;SUBZ.C280.26.041\u0026nbsp;(record number in the Simple System).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAll authors contributed to the study conception and design. All authors read and approved the final manuscript.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThis is an observational study.\u0026nbsp;\u003c/em\u003e\u003cem\u003eThe Wroclaw Medical University Ethics Committee\u003c/em\u003e\u003cem\u003e\u0026nbsp;has confirmed that no ethical approval is required\u0026nbsp;\u003c/em\u003e\u003cem\u003e(No. 233/2025).\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eInformed consent\u0026nbsp;\u003c/em\u003e\u003cem\u003efor genetic diagnostics\u0026nbsp;\u003c/em\u003e\u003cem\u003ewas obtained from all individual participants included in the study.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u003cstrong\u003eAkin Duman, T., \u0026amp; Ozturk, F. 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Prevalence of BRCA1/2 LGRs in familial breast cancer. \u003cem\u003eBMC Cancer, 14\u003c/em\u003e(1). https://doi.org/10.1186/1471-2407-14-645\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eSkorniak, J., Rabalski, L., Szynglarewicz, B., Dolega-Kozierowski, B., Kasprzak, P., Zietek, M., et al.\u003c/strong\u003e (2025). Breast cancer stage among Ukrainian refugees in Poland. \u003cem\u003eJAMA Network Open, 8\u003c/em\u003e(4), e256215. https://doi.org/10.1001/jamanetworkopen.2025.6215\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eSluiter, M. D., \u0026amp; Van Rensburg, E. J.\u003c/strong\u003e (2011). Large genomic rearrangements of BRCA1 and BRCA2: review. \u003cem\u003eBreast Cancer Research and Treatment, 125\u003c/em\u003e(2), 325\u0026ndash;349. https://doi.org/10.1007/S10549-010-0817-Z\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eStoffel, E. M., Brand, R. E., \u0026amp; Goggins, M.\u003c/strong\u003e (2023). 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BRCA1 tumor suppressor network. \u003cem\u003eCell and Bioscience, 2\u003c/em\u003e(1). https://doi.org/10.1186/2045-3701-2-6\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eWang, Y., Bernhardy, A. J., Nacson, J., Krais, J. J., Tan, Y. F., Nicolas, E., et al.\u003c/strong\u003e (2019). BRCA1 intronic Alu elements drive rearrangements. \u003cem\u003eNature Communications, 10\u003c/em\u003e(1). https://doi.org/10.1038/s41467-019-13530-6\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-cancer-research-and-clinical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jocr","sideBox":"Learn more about [Journal of Cancer Research and Clinical Oncology](https://www.springer.com/journal/432)","snPcode":"432","submissionUrl":"https://submission.nature.com/new-submission/432/3","title":"Journal of Cancer Research and Clinical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"breast and ovarian cancer, BRCA1, BRCA2, CNVs, MLPA, NGS","lastPublishedDoi":"10.21203/rs.3.rs-9336936/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9336936/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePurpose: \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e are key susceptibility genes in hereditary breast and ovarian cancer (HBOC), with mutational status guiding PARP inhibitor therapy. While single-nucleotide variants (SNVs) predominate, the prevalence of copy number variants (CNVs) varies significantly across different populations. This study aims to determine the incidence of BRCA1/2 CNVs in the Polish population, where data remain scarce due to non-mandatory CNV testing.\u003c/p\u003e\n\u003cp\u003eMethods: We retrospectively analysed the results of genetic tests assessing the presence of \u003cem\u003eBRCA1/2\u003c/em\u003e CNVs performed in 2720 Polish probands tested at the Lower Silesian Oncology Centre (2021–2024), including 2702 breast/ovarian cancer patients and 18 relatives. The mean age was 54.7±15.15 years. Genetic testing involved DNA extraction, NGS, and MLPA for CNV confirmation. Variants were classified according to ACMG-AMP guidelines and verified through independent testing.\u003c/p\u003e\n\u003cp\u003eResults: In this study, no \u003cem\u003eBRCA2\u003c/em\u003e CNVs were identified, consistent with previous Central European findings. Pathogenic \u003cem\u003eBRCA1\u003c/em\u003e CNVs were found in 0.85% of probands, affecting 23 individuals from 13 families. Eight distinct \u003cem\u003eBRCA1\u003c/em\u003e CNVs were detected, the most common being exon 21 deletion. Affected families exhibited a high incidence of HBOC-related cancers, with early-onset breast cancer and a notable proportion of triple-negative breast cancer cases.\u003c/p\u003e\n\u003cp\u003eConclusions: This study highlights the clinical significance of \u003cem\u003eBRCA1\u003c/em\u003e CNVs in Polish patients with HBOC-spectrum cancers and their families. Although rare, these variants were associated with aggressive cancer phenotypes and early onset. Given their diagnostic and therapeutic implications, \u003cem\u003eBRCA1\u003c/em\u003e CNVs should be routinely analysed in high-risk families to ensure accurate detection and personalised treatment planning.\u003c/p\u003e","manuscriptTitle":"Copy number variants in BRCA1 and BRCA2 genes in Polish patients with breast and ovarian cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-27 10:52:07","doi":"10.21203/rs.3.rs-9336936/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-01T13:58:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-01T10:20:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"135561080393100969390055052216618572518","date":"2026-04-21T07:46:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-18T19:48:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-17T11:41:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-17T05:34:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Cancer Research and Clinical Oncology","date":"2026-04-15T20:15:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-cancer-research-and-clinical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jocr","sideBox":"Learn more about [Journal of Cancer Research and Clinical Oncology](https://www.springer.com/journal/432)","snPcode":"432","submissionUrl":"https://submission.nature.com/new-submission/432/3","title":"Journal of Cancer Research and Clinical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4339d8a1-adb1-4ad5-97e8-89404add548d","owner":[],"postedDate":"April 27th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-01T13:58:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-01T10:20:35+00:00","index":39,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-17T11:38:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-27 10:52:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9336936","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9336936","identity":"rs-9336936","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}