Rearrangements In BRCA1 Research Articles

The landscape of BRCA1 and BRCA2 large rearrangements in an international cohort of over 20 000 ovarian tumors identified using next-generation sequencing.

Approximately half of ovarian tumors have defects within the homologous recombination repair pathway. Tumors carrying pathogenic variants (PVs) in BRCA1/BRCA2 are more likely to respond to poly-ADP ribose polymerase (PARP) inhibitor treatment. Large rearrangements (LRs) are a challenging class of variants to identify and characterize in tumor specimens and may therefore be underreported. This study describes the prevalence of pathogenic BRCA1/BRCA2 LRs in ovarian tumors and discusses the importance of their identification using a comprehensive testing strategy. Sequencing and LR analyses of BRCA1/BRCA2 were conducted in 20 692 ovarian tumors received between March 18, 2016 and February 14, 2023 for MyChoice CDx testing. MyChoice CDx uses NGS dosage analysis to detect LRs in BRCA1/BRCA2 genes using dense tiling throughout the coding regions and limited flanking regions. Of the 2217 PVs detected, 6.3% (N = 140) were LRs. Overall, 0.67% of tumors analyzed carried a pathogenic LR. The majority of detected LRs were deletions (89.3%), followed by complex LRs (5.7%), duplications (4.3%), and retroelement insertions (0.7%). Notably, 25% of detected LRs encompassed a single or partial single exon. This study identified 84 unique LRs, 2 samples each carried 2 unique LRs in the same gene. We identified 17 LRs that occurred in multiple samples, some of which were specific to certain ancestries. Several cases presented here illustrate the intricacies involved in characterizing LRs, particularly when multiple events occur within the same gene. Over 6% of PVs detected in the ovarian tumors analyzed were LRs. It is imperative for laboratories to utilize testing methodologies that will accurately detect LRs at a single exon resolution to optimize the identification of patients who may benefit from PARP inhibitor treatment.

15 years of experience of an oncogenetics service in the northeast of Brazil.

e22522 Background: The Oncogenetics service at the Federal University of Bahia was implemented in 2007 and since then has been offering pre- and post-test genetic counseling for cancer susceptibility genes, mainly tumors associated with King's syndrome in the population of the state of Bahia. This is a mixed population of about 80% of African ancestry. Methods: Between the years 2007 to 2021, 462 probands and their families have already been evaluated by NGS analysis, Sanger sequencing. Large rearrangements were detected by multiplex ligation-dependent probe amplification (MLPA) for the BRCA1 and BRCA2 genes. Results: The mean age at diagnosis was 41 years. Pathogenic/probably pathogenic mutations were founded in 118 patients: 67 had breast cancer, 09 with ovarian cancer, 26 with more than one tumor, 15 without cancer, and 01 with another tumor type. Most of the pathogenic variants were located in the BRCA1 gene (27 variants), the two most frequent variants being: c.211G > A (13 probands) and c.3331_3334delCAAG (18 probands), both of Iberian origin. Of patients with mutation, 9.8% had large rearrangements in BRCA1 and 11.5% in BRCA2. Bilateral breast cancer appears predominantly in patients with BRCA1 (4) and BRCA2 (1) variants. In this study, only two mutations in BRCA described as of African origin were found in two patients. Other pathogenic mutations appear in ATM, BARD1, BRIP1, CDH1, CHEK2, FAM175A, FANCM, MSH6, NBN, NF2, PALB2, RAD51, SDHB, SLX4, and TP53 genes. Variants of uncertain significance were identified in 71 patients. Conclusions: The population of the state of Bahia has a peculiar mutational profile, containing mutations that are not very frequent in other regions of the country. Despite being a population with high African ancestry, most mutations are of European origin.

Contribution of large genomic rearrangements in PALB2 to familial breast cancer: implications for genetic testing

BackgroundPALB2 is the most important contributor to familial breast cancer after BRCA1 and BRCA2. Large genomic rearrangements (LGRs) in BRCA1 and BRCA2 are routinely assessed in clinical testing and are...

5' Region Large Genomic Rearrangements in the BRCA1 Gene in French Families: Identification of a Tandem Triplication and Nine Distinct Deletions with Five Recurrent Breakpoints.

Simple SummaryLarge genomic rearrangements in BRCA1 consisting of deletions/duplications of one or several exons are complex events, often occurring in the 5′ region. We characterized 10 events in 20 families: one large triplication classified as benign and nine large deletions classified as pathogenic. The breakpoint localization will certainly help to further understand the chromatin structure in regions sensitive to rearrangement.Background: Large genomic rearrangements (LGR) in BRCA1 consisting of deletions/duplications of one or several exons have been found throughout the gene with a large proportion occurring in the 5′ region from the promoter to exon 2. The aim of this study was to better characterize those LGR in French high-risk breast/ovarian cancer families. Methods: DNA from 20 families with one apparent duplication and nine deletions was analyzed with a dedicated comparative genomic hybridization (CGH) array, high-resolution BRCA1 Genomic Morse Codes analysis and Sanger sequencing. Results: The apparent duplication was in fact a tandem triplication of exons 1 and 2 and part of intron 2 of BRCA1, fully characterized here for the first time. We calculated a causality score with the multifactorial model from data obtained from six families, classifying this variant as benign. Among the nine deletions detected in this region, eight have never been identified. The breakpoints fell in six recurrent regions and could confirm some specific conformation of the chromatin. Conclusions: Taken together, our results firmly establish that the BRCA1 5′ region is a frequent site of different LGRs and highlight the importance of the segmental duplication and Alu sequences, particularly the very high homologous region, in the mechanism of a recombination event. This also confirmed that those events are not systematically deleterious.

Abstract 2505: The Oncomine BRCA expanded next-generation sequencing assay: Development and analytical validation of a new panel for detection of SNV, insertions, deletions and copy number variants in a panel of 15 genes involved in homologous recombination repair of double-strand break DNA damage

Abstract Introduction: Many tumors display phenotypic similarity across tissue types, characterized by defects in biological pathways leading to susceptibility to particular therapies. Deficiencies in one such pathway, homologous recombination DNA damage repair (HR DDR), affect the response of breast and ovarian cancers to agents that inhibit PARP function. The Oncomine BRCA Expanded panel targets 15 genes (BRCA1 and 2, ATM, BARD1, BRIP1, CDK12, CHEK2, FANCD2, MRE11, NBN, PALB2, PPP2R2A, RAD51B, RAD54L, and TP53) that play various roles in the HR DDR pathway. The assay includes 1011 amplicons covering both hotspot variants and coding regions of these genes. An additional set of pre-qualified genes is available for assay customization, including a recommended set of 25 genes of particular relevance to HR DDR pathway. Methods: Targeted regions were selected, and amplicons were designed, optimized, and experimentally verified to perform well with semi-conductor based sequencing. Measures used included gene-base and hotspot-base coverage uniformity, variant calling accuracy, and detection of known CNV gains in both FFPE and cell line DNA samples. Large gene rearrangements in BRCA1 and BRCA2, including exon deletions and duplications, could be detected by using a custom exon deletion pipeline. Analysis was performed on an integrated bioinformatics platform, Ion Reporter, which includes variant calling, identification and annotation of the likely deleterious mutations, and production of configurable reports, including visualization of the extent of copy number changes. Results: The assay's performance was evaluated using a variety of sample types, including commercially available controls and FFPE samples for breast, ovarian, prostate, and other tumor types. Sensitivity and positive predictive value for small variant detection was >95% in Acrometrix Hotspot Controls, and >90% of CNV gains greater than 6 were detected in cell line samples. Examples of all detected variant classes were displayed based on the Ion Reporter user interface. Conclusions: We introduce an expanded BRCA panel for researching genetic changes influencing homologous recombination DNA damage repair. The option to customize the gene content of the panel is enabled via a web-based interface to inventoried, pre-qualified targets. This approach allows identifying a broad set of mutation types in a single assay with high accuracy, enabling interrogation of HR DDR genes in FFPE preparations. For research use only. Not for use in diagnostic procedures. Citation Format: Charles Scafe, Yun Zhu, Chenchen Yang, Yu-Ting Tseng, Brooke McKnight, Fernando Farfan, Santoshi Bandla, Seth Sadis, Steven Roman, Fiona C. Hyland. The Oncomine BRCA expanded next-generation sequencing assay: Development and analytical validation of a new panel for detection of SNV, insertions, deletions and copy number variants in a panel of 15 genes involved in homologous recombination repair of double-strand break DNA damage [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2505.

The contribution of large genomic rearrangements in BRCA1 and BRCA2 to South African familial breast cancer

BackgroundPathogenic variants that occur in the familial breast cancer genes (BRCA1/2) lead to truncated ineffective proteins in the majority of cases. These variants are mostly represented by small deletions/insertions, nonsense- and splice-site variants, although some larger pathogenic rearrangements occur. Currently, their contribution to familial breast cancer (BC) and ovarian cancer (OVC) in South Africa (SA) is unknown.MethodsSeven hundred and forty-four patients affected with BC or OVC were screened for larger genomic rearrangements (LGRs) by means of multiplex ligation-dependent probe amplification or Next Generation Sequencing using the Oncomine™ BRCA research assay.ResultsThe patients represented mostly medium to high-risk families, but also included lower risk patients without a family history of the disease, diagnosed at an early age of onset (< 40 years). Eight LGRs were detected (1.1%); seven in BRCA1 with a single whole gene deletion (WGD) detected for BRCA2. These eight LGRs accounted for 8.7% of the 92 BRCA1/2 pathogenic variants identified in the 744 cases. The pathogenic LGRs ranged from WGDs to the duplication of a single exon.ConclusionsLarger rearrangements in BRCA1/2 contributed to the overall mutational burden of familial BC and OVC in SA. Almost a quarter of all pathogenic variants in BRCA1 were LGRs (7/30, 23%). The spectrum observed included two WGDs, one each for BRCA1 and BRCA2.

2008P - Large genomic rearrangements in BRCA1 and BRCA2 genes in the Portuguese population

BackgroundBRCA1 and BRCA2 are the genes most frequently associated with hereditary breast and ovarian cancer (HBOC). Besides point mutations, BRCA1/2 large genomic rearrangements (LGR) have been observed, with different frequencies in specific populations. This study aims to update the frequency of BRCA1 and BRCA2 LGR in our population, as well as to characterize the gene distribution of these genetic events. MethodsAll high risk pts referred to our multidisciplinary program between 2000-2018 that consented on genetic testing were reviewed for BRCA1 and BRCA2 point pathogenic variants (several methodologies throughout the years including Conformation Sensitive Gel Electrophoresis; Conformation Sensitive Capillary Electrophoresis; Sanger Sequencing and Next Generation Sequencing) and LGR with Multiplex Ligation dependent Probe Amplification (MLPA). Results3763 of 3901 index pts consented on genetic testing and 473 pts had 476 different pathogenic variants (PV). In 3 cases double germline PV were observed: BRCA1 (c.68_69delAG) and CHEK2 [c.(908+1_909-1)_(1095+1_1096-1)del]; ATM (c.8264_8268delATAAG) and PALB2 (c.751C>T); BRCA2 (c.156_157insAlu) and CHEK2 (c.593-1G>T). BRCA1 PV were detected in 151 pts with 24 LGR of these (15,9%) being detected by MLPA. The most frequent BRCA1 LGR was c. (4185+1_4186-1)_(4357+1_4358-1)dup (n=8). BRCA2 PV were detected in 260 pts with 99 LGR: 92 pts with the Portuguese founder mutation c.156_157insAlu (detected by specific PCR or MLPA) and 7 pts with other LGR (detected by MLPA). The second most frequent BRCA2 LGR was c. (8632+1_8633-1)_(8754+1_8755-1)dup (n=5). Nine CHEK2 PV were detected with the SALSA MLPA probemix P045 BRCA2/CHEK2: c.1100delC (8 pts) and c. (908+1_909-1)_(1095+1_1096-1)del (1pt). The total percentage of BRCA1 and BRCA2 LGR in our cohort was 26%. Excluding the Portuguese founder mutation c.156_157insAlu, the prevalence of LGR was 6,5%. ConclusionsThere is a high prevalence of LGR in the Portuguese population, mostly because of the Portuguese founder mutation c.156_157insAlu. Even if excluding this event, other LRG still account for 6,5% of the BRCA1 and BRCA2 pathogenic variants. These results emphasize the importance of testing LGR in all patients with high risk HBOC. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Multiplex ddPCR assay for screening copy number variations in BRCA1 gene.

Germinal and somatic rearrangements in BRCA1 gene play a significant role in carcinogenesis of breast and ovarian cancer. The present study is dedicated to the development of multiplex droplet digital PCR (ddPCR) assay for detecting large deletions and duplications in the BRCA1 gene. In-house tetraplex ddPCR assay for BRCA1 gene analysis was used for testing of DNA samples with BRCA1 status. DNA specimens were purified from 24 individuals. The presence of BRCA1 rearrangements in samples was confirmed by a commercial MLPA-based kit. An amplitude-based multiplex ddPCR assay was developed: 8 multiplexes, each containing primers and probes to amplify 3 BRCA1 exons and 1 reference gene (ALB or RPP30). A novel assay demonstrated 100% concordance with the commercial MLPA-based kit, identifying 9 specimens with different deletions in BRCA1, 1 with duplication, and 14 with the wild-type BRCA1. We have designed a simple, precise, and cost-effective assay for BRCA1 rearrangement testing, based on ddPCR. The developed assay is the first multiplex ddPCR-based test that provides results in accordance with MLPA and can be used for routine clinical screening.

Frequency of Rearrangements Versus Small Indels Mutations in BRCA1 and BRCA2 Genes in Turkish Patients with High Risk Breast and Ovarian Cancer.

The current rearrangement ratio of BRCA1 and BRCA2 genes is not known in the Turkish population. Rearrangements are not routinely investigated in many Turkish laboratories. This creates problems and contradictions between clinics. Therefore, the aim of this study was to evaluate the distribution and frequency of rearrangements in BRCA1 and BRCA2 genes in high-risk families and to clarify the limits of BRCA1 and BRCA2 testing in Turkey. The study included 1809 patients at high risk of breast cancer or ovarian cancer. All patients were investigated for both small indels and rearrangements of BRCA genes using DNA sequencing and multiplex ligation-dependent probe amplification (MLPA) analysis. The overall frequency of rearrangements was 2% (25/1262). The frequency of rearrangements was 1.7% (18/1086) and 4% (9/206) in patients with breast cancer and ovarian cancer, respectively. The frequency of rearrangements was 3.7% (8/215) in patients with triple-negative breast cancer. The rearrangement rate was 7.7% (2/26) in patients with both breast and ovarian cancer. Rearrangements were found with high rates and were strongly associated with bilateral and triple-negative status of patients with breast cancer, which are signs of high risk for breast and ovarian cancer. Analysis of rearrangements should definitely be included in routine clinical practice in Turkey for high-risk families and also for improved cancer risk prediction for families.

A comprehensive BRCA1/2 NGS pipeline for an immediate Copy Number Variation (CNV) detection in breast and ovarian cancer molecular diagnosis

BackgroundThe aim of this study was to assess the ability of a Next Generation Sequencing (NGS)-based approach in the detection of BRCA1/2 Large Genomic Rearrangements (LGRs). MethodsWe investigated 287 consecutive unrelated Italian women affected by breast or ovarian cancer. A NGS pipeline with a reliable Copy number Variation (CNV) prediction algorithm was applied. In addition, all samples were investigated using MAQ (Multiplex Amplicon Quantification) assay, while the MLPA (Multiplex Ligation-dependent Probe Amplification) technique was used as alternative method if necessary. ResultsSmall pathogenic variants were identified in 80 samples. During NGS analysis, 11 samples were identified by Amplicon Suite Software (SmartSeq, Novara, Italy) as positive for large deletions or duplications. However, MAQ assay provided positive results in 19 patients. In particular, BRCA2 exon 25 deletion, not revealed by NGS CNV prediction algorithm, was detected in 8 unrelated women as false positive result. Sequencing analysis showed IVS24-113T/G (c.9257-113T>G) variant in 6 of these 8 patients, while the novel IVS24-129G>A (c.9257-129G>A) variant was detected in only two samples. DiscussionThe prevalence of large rearrangements in BRCA1/2 genes was the 12% of all disease-causing mutations detected in our patients. In particular, BRCA1 rearrangements were the 14.5% of all BRCA1 causing variants identified. Differently, BRCA2 large deletions were only the 6.9% of all mutations occurring in this gene. While MAQ assay showed 2,8% of false positive results, our integrative NGS-based approach fully satisfied the sensitivity and specificity parameters required on the BRCA1/2 LGRs detection. The workflow represents a robust and easy-to-use method for full BRCA1/2 screening, which can be easily implemented in routine diagnostic testing.

Abstract 4278: Screening for BRCA1 founder mutation in Mexican population: an accessible approach

Abstract Aim: To implement an accessible strategy for detection of the Mexican founder mutation NG_005905.2:g.118973_133629del, located in BRCA1, which deletes exons 9-12 (BRCA1 ex9-12del), as part of the health care setting of patients with hereditary breast and ovarian cancer (HBOC), from the National Cancer Institute (Mexico). Material and methods: We designed a polymerase chain reaction (PCR) based method, for the targeted detection of the deletion of exons 9 through 12, of the BRCA1 gene. The method was validated with previously verified results of the deletion by multiplex ligation-dependent probe amplification assay (MLPA), or deep sequencing of the region. Finally, the technique was used for the detection of BRCA1 ex9-12del mutation in a group of 302 HBOC patients, in whom the existence of BRCA1 and BRCA2 point mutations had been ruled by pyrosequencing. Results: The concordance of our method, with previously reported BRCA1 ex9-12del mutation results, was 100%. Also, we provided a profile of the clinical and familial characteristics of 302 Mexican patients with clinical suspicion of HBOC, in whom the detection of BRCA1 ex9-12del mutation was performed. In this way, 21 patients (6.95%) were identified as BRCA1 ex9-12del mutation carriers. Among whom, a higher proportion of triple negative breast cancer was found (73% versus 21%, p=0.0005; two sided Fisher's exact test) . Conclusions: A feasible molecular diagnostic strategy for the detection of BRCA1 ex9-12del mutation was established in Mexican patients, with HBOC. In this way, we identified 21 (21/302) carriers of the deletion, who represented the 6.95%. Related to the clinical features, triple negative breast cancer carriers stood out. It had been reported that large genomic rearrangements in BRCA1 are associated with an increased frequency of high risk features. These type of approach is appropriate in populations where a founder effect is recognized. For its simplicity and effectiveness, it can be transferred to other laboratories, for the benefit of a greater number of Mexican patients and with the aim of minimize costs of testing. Because of the frequency of BRCA1 ex9-12del mutation, this method has been implemented as the first line of BRCA1 clinical genetic analysis in patients with suspected HBOC, at National Cancer Institute (Mexico ). Citation Format: Rosa M. Alvarez, Jose Velazquez, Veronica Fragoso, Julieta Dominguez, Paulina Nuñez, Talia Wegman, Yuliana Sanchez, Silvia Vidal, Enrique Bargallo, Dolores Gallardo, Abelardo Meneses, Luis A. Herrera. Screening for BRCA1 founder mutation in Mexican population: an accessible approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4278. doi:10.1158/1538-7445.AM2017-4278

Identification and Characterization of a New BRCA2 Rearrangement in an Italian Family with Hereditary Breast and Ovarian Cancer Syndrome.

Many studies document the involvement of BRCA1/2 gene rearrangements in genetic predisposition to breast and ovarian cancer. Large genomic rearrangements (LGRs) of BRCA1 account for 0-27% of all disease-causing mutations in various populations, while LGRs in BRCA2 are rarer. Here, we describe a novel BRCA2 LGR, involving the duplication of exons 4-26, in an Italian family with hereditary breast and ovarian cancer (HBOC) syndrome. Our purpose was to provide an effective characterization of this variant using a combination of different methods able to establish the exact breakpoints of the duplication. A multiplex amplicon quantification (MAQ) assay was used as the primary screening method in the detection of LGRs. Array comparative genomic hybridization (CGH), reverse transcriptase polymerase chain reaction (RT-PCR) and long-range PCR were used for the careful characterization of the rearrangement and breakpoint regions. The Repeat Masker program was employed to identify Alu sequences at breakpoint junctions. Array CGH and long-range PCR strategies revealed that the BRCA2 exons 4-26 duplication (g.12016_87170dup) involved exactly 75,154bp nucleotides between intron 3 and intron 26 of the gene. Given that no Alu repeats were found at the junction sites, we support the hypothesis that the new duplication could be the result of a microhomology-mediated event (MH) involving very short homologous sequences at an upstream breakpoint. LGR investigation is mandatory in BRCA1/2 routine testing in order to provide a complete result for a targeted therapeutic decision. Nevertheless, the characterization and classification of novel BRCA1/2 variants represents a crucial step in the support of genetic counselling. Our results, including a comprehensive co-segregation analysis, indicate that the novel duplication identifed has a pathogenic role and would be considered a causing-disease variant in genetic and oncologic counselling.

Characterization of a new BRCA1 rearrangement in an Italian woman with hereditary breast and ovarian cancer syndrome.

We report a novel BRCA1 LGR, involving the complete duplication of exon 3, in an Italian patient with a strong family history of breast and ovarian cancer. Our purpose is to provide an effective characterization of this LGR using a combination of different methods able to establish the exact breakpoints of the duplication. MAQ assay was used as primary screening method in LGRs detection. Array CGH, RT-PCR, and Long-PCR were used for a careful characterization of rearrangement and breakpoint regions. The Repeat Masker program was employed to identify Alu sequences at breakpoint junctions. RNA analysis showed that this in tandem duplication of exon 3 causes an in frame insertion of 18 amino acids within the protein. Array CGH and Long-PCR strategies revealed that the duplication (g.100411_102863dup) involves exactly 2.452 nucleotides between intron 2 and intron 3 of the gene. In addition, while an Alu Sx sequence was identified at upstream breakpoint, no Alu repeats were found at downstream junction. This supports the hypothesis that the new duplication was the result of a non-homologous recombination event between Alu and Non-Alu sequences. Our strategy, which combines a comprehensive set of methodologies, has been able to characterize the new BRCA1 duplication confirming, as previously reported, that MAQ assay represents a reliable and effective method for a primary screening of BRCA rearrangements. We underline the relevance of incorporating quantitative methods for BRCA genes dosage testing into routine diagnostic practice.

Recurrent large genomic rearrangements in BRCA1 and BRCA2 in an Irish case series

Mutations in BRCA1 and BRCA2 confer a highly increased risk of cancers, mainly of the breast and ovary. Most variants are point mutations or small insertions/deletions detectable by Sanger sequencing. Large genomic rearrangements, including deletions/duplications of multiple exons, are not routinely detectable by Sanger sequencing, but can be reliably identified by Multiplex Ligation-dependent Probe Amplification (MLPA), and account for 5-17% mutations in different populations. Comprehensive mutation testing using these two methods has been facilitated via our centre since 2005. The aim of this study was to investigate the incidence of and phenotype associated with large genomic rearrangements in BRCA1 and BRCA2 in an Irish cohort. An observational cohort study was undertaken. Patients with large genomic rearrangements in BRCA1/BRCA2 were identified from a prospectively maintained database of MLPA test results. Phenotypic and genotypic data were retrieved by chart review. Large genomic rearrangements in BRCA1 were identified in 49 families; and in BRCA2 in 7 families, representing ~11% of mutations in BRCA1/BRCA2 in Ireland. The most common large genomic rearrangement in BRCA1 was deletion of exons 1-23 (11 families, 7 from Co. Galway). Other common mutations included deletions of exon 3 (8 families) and exons 1-2 (6 families). Deletion of exons 19-20 in BRCA2 represented the familial mutation in five families, all from East Ireland (Wexford/Wicklow/Dublin). It is evident that a significant proportion of highly penetrant pathogenic variants in BRCA1 and BRCA2 will be missed if testing is limited to PCR-based Sanger sequencing alone. Screening for large genomic rearrangements in BRCA1 and BRCA2 in the routine diagnostic workflow is critical to avoid false negative results.

Architectural plasticity of human BRCA2-RAD51 complexes in DNA break repair.

The tumor suppressor BRCA2 is a large multifunctional protein mutated in 50–60% of familial breast cancers. BRCA2 interacts with many partners and includes multiple regions with potentially disordered structure. In homology directed DNA repair BRCA2 delivers RAD51 to DNA resulting in removal of RPA and assembly of a RAD51 nucleoprotein filament. Dynamic rearrangements of BRCA2 likely drive this molecular hand-off initiating DNA strand exchange. We show human BRCA2 forms oligomers which can have an extended shape. Scanning force microscopy and quantitative single molecule fluorescence define the variety of BRCA2 complexes, reveal dramatic rearrangements upon RAD51 binding and the loading of RAD51 patches on single strand DNA. At sites of repair in cell nuclei, super-resolution microscopy shows BRCA2 and RAD51 arranged in largely separate locations. We identified dynamic structural transitions in BRCA2 complexes suggested to facilitate loading of RAD51 onto RPA coated single strand DNA and subsequent release of BRCA2.

Prevalence of BRCA1 and BRCA2 large genomic rearrangements in Tunisian high risk breast/ovarian cancer families: Implications for genetic testing

Germline mutations in the BRCA tumor suppressor genes account for a substantial proportion of hereditary breast/ovarian cancer. However, this contribution is lower than expected. This underestimation can partly be explained by the BRCA alterations missed by using Sanger sequencing methods. Thus, large genomic rearrangements (LGRs) in BRCA1 and BRCA2 are responsible for 4-28% of all inherited BRCA mutations. In this study, Multiplex ligation-dependent probe amplification (MLPA) assay was used for detection of large rearrangements of BRCA1 and BRCA2 genes in 36 unrelated high-risk breast/ovarian cancer patients negative for BRCA1/2 point mutations. MLPA assay for all exons of both genes and for 1100delC variant of CHEK2 gene were performed. Positive MLPA results were confirmed by real-time quantitative PCR (qPCR). Two different rearrangements in the BRCA1 gene were identified consisting of exon 5 deletion and exon 20 duplication. MLPA analysis did not reveal any large genomic rearrangements in BRCA2 gene. Overall BRCA1/2 LGRs prevalence among high-risk Tunisian patients was 5.5%. Quantitative real-time PCR confirmed MPLA findings. Our results suggest the usefulness of screening for LGRs in BRCA genes in the Tunisian population. To avoid false-negative results, we suggest that MLPA should be used in genetic testing programs. These results are important for guidance counseling and clinical management of Tunisian high-risk patients.

BRCA1 and BRCA2 rearrangements in Brazilian individuals with Hereditary Breast and Ovarian Cancer Syndrome.

Approximately 5-10% of breast cancers are caused by germline mutations in high penetrance predisposition genes. Among these, BRCA1 and BRCA2, which are associated with the Hereditary Breast and Ovarian Cancer (HBOC) syndrome, are the most frequently affected genes. Recent studies confirm that gene rearrangements, especially in BRCA1, are responsible for a significant proportion of mutations in certain populations. In this study we determined the prevalence of BRCA rearrangements in 145 unrelated Brazilian individuals at risk for HBOC syndrome who had not been previously tested for BRCA mutations. Using Multiplex Ligation-dependent Probe Amplification (MLPA) and a specific PCR-based protocol to identify a Portuguese founder BRCA2 mutation, we identified two (1,4%) individuals with germline BRCA1 rearrangements (c.547+240_5193+178del and c.4675+467_5075-990del) and three probands with the c.156_157insAlu founder BRCA2 rearrangement. Furthermore, two families with false positive MLPA results were shown to carry a deleterious point mutation at the probe binding site. This study comprises the largest Brazilian series of HBOC families tested for BRCA1 and BRCA2 rearrangements to date and includes patients from three regions of the country. The overall observed rearrangement frequency of 3.44% indicates that rearrangements are relatively uncommon in the admixed population of Brazil.

Abstract P2-09-17: Frequency of large rearrangements in BRCA1, BRCA2, ATM, CHEK2, and PALB2 in hereditary cancer testing

Abstract Objectives Large deletions and duplications are recognized as a cause of many inherited cancer syndromes; however, few studies have examined the frequency. In one study, large rearrangements accounted for 11-14% of pathogenic variants in BRCA1 and 1-3% of pathogenic variants in BRCA2 (Judkins 2012). Additionally, in a study of invasive ovarian cancer patients, large deletions and duplications accounted for 6% of BRCA1 and 3% of BRCA2 pathogenic variants (Zhang 2011). The frequency of large deletions and duplications in moderate penetrance genes, such as ATM, CHEK2, and PALB2, has not been carefully studied. We therefore sought to determine the frequency of pathogenic and likely pathogenic (P/LP) large deletions and duplications in BRCA1, BRCA2, ATM, CHEK2, and PALB2. Methods We analyzed a consecutive case series of patients who had BRCA1/2 and cancer panel testing. The data were analyzed to determine the percentage of deletions or duplications of greater than 250 bp identified on array comparative genome hybridization as a percentage of all pathogenic variants (PVs)/likely pathogenic variants (LPVs) in each of BRCA1, BRCA2, ATM, CHEK2, and PALB2. Results Large deletions/duplications accounted for 10.1% (55/545) of cases with a BRCA1 PV/LPV and 1.8% (10/556) of cases with a BRCA2 PV/LPV. By ethnicity, BRCA1/2 large deletions/duplications accounted for a higher fraction of PV/LPVs in Asians (11.3%) and Hispanics (8.1%). In Hispanic individuals, all of the large deletions/duplications were identified in BRCA1, accounting for 14.6% of all BRCA1 PV/LPV in this group. In the moderate penetrance genes, large deletions/duplications accounted for 4.6% (9/195), 5.1% (22/436), and 9.9% (13/132) of cases with a PV/LPV in ATM, CHEK2, and PALB2, respectively. There were two recurrent deletions in our cohort: the CHEK2 founder Slavic deletion of exons 9-10 observed in 16 of our cases, and a deletion of exon 11 in PALB2 observed in 6 cases. Conclusions Large deletions/duplications account for a substantial proportion of clinically significant variants in hereditary cancer genes. The proportion of large deletions/duplications in BRCA1 and BRCA2 reported here is similar to previous data. However, our cohort suggests that Asian individuals may have a higher proportion of mutations due to large deletions/duplications than previously reported. Among moderate risk cancer genes, PALB2 has the highest proportion of large deletions/duplications. Citation Format: Bissonnette J, Doonanco K, Xu Z, Klein RT, Hruska KS. Frequency of large rearrangements in BRCA1, BRCA2, ATM, CHEK2, and PALB2 in hereditary cancer testing. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-09-17.

BRCA1, BRCA2 and PALB2 mutations and CHEK2 c.1100delC in different South African ethnic groups diagnosed with premenopausal and/or triple negative breast cancer.

BackgroundCurrent knowledge of the aetiology of hereditary breast cancer in the four main South African population groups (black, coloured, Indian and white) is limited. Risk assessments in the black, coloured and Indian population groups are challenging because of restricted information regarding the underlying genetic contributions to inherited breast cancer in these populations. We focused this study on premenopausal patients (diagnosed with breast cancer before the age of 50; n = 78) and triple negative breast cancer (TNBC) patients (n = 30) from the four South African ethnic groups. The aim of this study was to determine the frequency and spectrum of germline mutations in BRCA1, BRCA2 and PALB2 and to evaluate the presence of the CHEK2 c.1100delC allele in these patients.MethodsIn total, 108 South African breast cancer patients underwent mutation screening using a Next-Generation Sequencing (NGS) approach in combination with Multiplex Ligation-dependent Probe Amplification (MLPA) to detect large rearrangements in BRCA1 and BRCA2.ResultsIn 13 (12 %) patients a deleterious mutation in BRCA1/2 was detected, three of which were novel mutations in black patients. None of the study participants was found to have an unequivocal pathogenic mutation in PALB2. Two (white) patients tested positive for the CHEK2 c.1100delC mutation, however, one of these also carried a deleterious BRCA2 mutation. Additionally, six variants of unknown clinical significance were identified (4 in BRCA2, 2 in PALB2), all in black patients. Within the group of TNBC patients, a higher mutation frequency was obtained (23.3 %; 7/30) than in the group of patients diagnosed before the age of 50 (7.7 %; 6/78).ConclusionThis study highlights the importance of evaluating germline mutations in major breast cancer genes in all of the South African population groups. This NGS study shows that mutation analysis is warranted in South African patients with triple negative and/or in premenopausal breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1913-6) contains supplementary material, which is available to authorized users.

BRCA1 and BRCA2 mutations in ethnic Lebanese Arab women with high hereditary risk breast cancer.

Breast cancer is the most common malignancy among women in Lebanon and in Arab countries, with 50% of cases presenting before the age of 50 years. Between 2009 and 2012, 250 Lebanese women with breast cancer who were considered to be at high risk of carrying BRCA1 or BRCA2 mutations because of presentation at young age and/or positive family history (FH) of breast or ovarian cancer were recruited. Clinical data were analyzed statistically. Coding exons and intron-exon boundaries of BRCA1 and BRCA2 were sequenced from peripheral blood DNA. All patients were tested for BRCA1 rearrangements using multiplex ligation-dependent probe amplification (MLPA). BRCA2 MLPA was done in selected cases. Overall, 14 of 250 patients (5.6%) carried a deleterious BRCA mutation (7 BRCA1, 7 BRCA2) and 31 (12.4%) carried a variant of uncertain significance. Eight of 74 patients (10.8%) aged ≤40 years with positive FH and only 1 of 74 patients (1.4%) aged ≤40 years without FH had a mutated BRCA. Four of 75 patients (5.3%) aged 41-50 years with FH had a deleterious mutation. Only 1 of 27 patients aged >50 years at diagnosis had a BRCA mutation. All seven patients with BRCA1 mutations had grade 3 infiltrating ductal carcinoma and triple-negative breast cancer. Nine BRCA1 and 17 BRCA2 common haplotypes were observed. Prevalence of deleterious BRCA mutations is lower than expected and does not support the hypothesis that BRCA mutations alone cause the observed high percentage of breast cancer in young women of Lebanese and Arab descent. Studies to search for other genetic mutations are recommended.