Haplotype analysis reveals that the recurrent BRCA1 deletion of exons 23 and 24 is a Greek founder mutation

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1 Clin Genet 2017: 91: Printed in Singapore. All rights reserved Short Report 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: /cge Haplotype analysis reveals that the recurrent BRCA1 deletion of exons 23 and 24 is a Greek founder mutation Apostolou P., Pertesi M., Aleporou-Marinou V., Dimitrakakis C., Papadimitriou C., Razis E., Christodoulou C., Fountzilas G., Yannoukakos D., Konstantopoulou I., Fostira F. Haplotype analysis reveals that the recurrent BRCA1 deletion of exons 23 and 24 is a Greek founder mutation. Clin Genet 2017: 91: John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2016 A recurrent large genomic rearrangement (LGR) encompassing exons 23 and 24 of the BRCA1 gene has been identified in breast-ovarian cancer families of Greek origin. Its breakpoints have been determined as c _*8273del11052 (RefSeq: NM_ ) and a diagnostic polymerase chain reaction (PCR) has been set up for rapid screening. In a series of 2,092 high-risk families completely screened for BRCA1 and BRCA2 germline mutations, we have found the deletion in 35 families (1.68%), representing 7.83% of the mutations identified in both genes and 10.3% of the total BRCA1 mutations. In order to characterize this deletion as a founder mutation, haplotype analysis was conducted in 60 carriers from 35 families, using three BRCA1 intragenic microsatellite markers and four markers surrounding the BRCA1 locus. Our results demonstrate a common shared core disease-associated haplotype of 2.89Mb. Our calculations estimate that the deletion has originated from a common ancestor 1450 years ago, which most probably inhabited the Asia Minor area. The particular (LGR) is the third mutation of such type that is proven to have a Greek founder effect in the Greek population, illustrating the necessity for LGRs testing in individuals of Greek descent. Conflict of interest The authors declare that they have no conflict of interest. P. Apostolou a,b, M. Pertesi a,c, V. Aleporou-Marinou b, C. Dimitrakakis d, C. Papadimitriou e,e.razis f, C. Christodoulou g, G. Fountzilas h, D. Yannoukakos a, I. Konstantopoulou a and F. Fostira a a Molecular Diagnostics Laboratory, INRaSTES, National Center for Scientific Research Demokritos, Athens, Greece, b Department of Genetics & Biotechnology, Faculty of Biology, National and Kapodistrian University of Athens, Athens, Greece, c Division of Hematology and Transfusion Medicine, Faculty of Medicine, Department of Laboratory Medicine, Lund University, Lund, Sweden, d Breast Unit of the 1st Department of Obstetrics and Gynecology, Athens University Medical School, Athens, Greece, e Department of Clinical Therapeutics, "Alexandra" Hospital, School of Medicine, University of Athens, Athens, Greece, f Third Medical Oncology Department, Hygeia Hospital, Athens, Greece, g Second Department of Medical Oncology, Metropolitan Hospital, Piraeus, Greece, and h School of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece Key words: BRCA breast cancer founder mutation hereditary large genomic rearrangements ovarian cancer Corresponding author: Dr Florentia Fostira, Molecular Diagnostics Laboratory, INRaSTES, National Center for Scientific Research Demokritos, Patriarchou Gregoriou E & Neapoleos Str., Aghia Paraskevi, Athens, Greece. Tel.: ; fax: ; florentia_fostira@hotmail.com; florentia@rrp.demokritos.gr 482

2 Haplotype analysis reveals that the recurrent BRCA1 deletion of exons 23 and 24 Received 26 April 2016, revised and accepted for publication 24 June 2016 The identification of BRCA1 and BRCA2 as breast and ovarian cancer predisposing genes, which took place more than two decades ago (1, 2), along with the implementation of genetic testing into clinical practice, which counts more than a decade, has led to effective management of mutation carriers which has subsequently increased their survival. The majority of reported germline deleterious mutations are point or small insertions/deletions that cause premature termination of translation. Furthermore, many large genomic rearrangements (LGRs), which lead to a peptide of abnormal function, have been identified in the aforementioned genes (3, 4). To date, more than 100 LGRs have been characterized in BRCA1 (5) and approximately one third of those in BRCA2 (5). Most of these LGRs result from unequal homologous recombination between Alu repeats accumulated at the BRCA1 and BRCA2 genetic loci (6). The distribution of BRCA1 and BRCA2 LGRs shows ethnical variation and more specifically, they are more prevalent in the Netherlands (7) and Italy (8) but less frequent in Germany (3). Up to date, two BRCA1 LGRs involving a 3.2 kb deletion of exon 20, along with a 4.4 kb deletion of exon 24, have been identified as founder mutations for the Greek population (9, 10). Our previous work has shown that these two, along with the mutations p.g1738r and c.5266dupc, account for 54% of all mutations detected in both BRCA1 and BRCA2 in individuals of Greek descent (11). The aim of this study was to characterize the BRCA1 LGR which deletes BRCA1 exons 23 and 24 and to further elucidate its contribution towards breast and/or ovarian cancer susceptibility, thus expanding the number of Greek founder alleles. Patients and methods Study group The current study involved 2,092 high-risk breast/ ovarian cancer families that were recruited from several hospitals in Greece in collaboration with the Hellenic Cooperative Oncology Group (HeCOG). Eligibility criteria were previously described (12). The study was approved by the Bioethics Committees of NCSR Demokritos (240/EHΔ/11.3, updated on February 14, 2014) and Papageorgiou Hospital (193rd Decision of Bioethics Committee, January 15, 2014). Written informed consent was provided from all individuals before genetic analysis was performed. Genomic DNA was isolated from peripheral blood lymphocytes using the salt extraction protocol proposed by Miller et al. (13). Multiplex ligation-dependent probe amplification (MLPA) analysis MLPA analysis was performed using the SALSA P002-D1 BRCA1 MLPA kit according to the manufacturer s instructions, while analysis was performed with the Coffalyser software (MRC Holland, Amsterdam, the Netherlands). Breakpoint analysis of the BRCA1 LGR deleting exons 23 and 24 The genomic breakpoints of the BRCA1 LGR were determined by long range PCR using the TaKaRa LA Taq (Takara Bio Inc., Kyoto, Japan), according to the manufacturer s instructions. A subsequent nested PCR gave rise to smaller DNA fragments, which were gel extracted using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) and were sequenced using the v3.1 BigDye Terminator Cycle Sequencing kit on an ABI 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA). Custom-designed PCR for the BRCA1 LGR Genotyping for the BRCA1 deletion was performed by a customized diagnostic PCR assay, where the PCR products 501 and 418 bp were produced for the mutant and the wild-type BRCA1 allele, respectively. All mutation-positive samples were confirmed by MLPA. Primers and protocols are available upon request. Haplotype analysis Haplotype analysis was carried out in all 60 mutation carriers, 14 family relatives that were non-mutation carriers and 66 cancer-free, age-matched women. Three BRCA1 intragenic polymorphic microsatellite markers (D17S1323, D17S1322, D17S855) and four extragenic markers (D17S951, D17S1801, D17S1299, D17S800), spanning a 2.89 Mb region around the BRCA1 gene were studied. Estimation of the BRCA1 mutation age The DMLE2.2 software method was used to estimate the age of the BRCA1 mutation. The program uses the Markov Chain Monte Carlo algorithm to allow Bayesian estimation of the mutation age (14). Results Molecular characterization of the BRCA1 genomic rearrangement The BRCA1 deletion of exons 23 and 24 was initially identified by MLPA in two unrelated patients diagnosed with very early-onset breast cancer at 28 and 26 years, previously tested negative for BRCA1 and BRCA2 point mutations (Fig. 1a). In order to characterize the large 483

3 Apostolou et al. Fig. 1. (a) Ratio plot for a large genomic rearrangement (LGR) carrier showing the deletion of BRCA1 exons 23 and 24. The exon dosage is reported on the y-axis (normal values spanning are indicated with red and blue lines). The low BRCA1 gene dosage at exons 23 and 24 is indicated by the red arrow. (b) Long range polymerase chain reaction (PCR), where the 12,325 and 1,200 bp bands correspond to wild-type and mutant allele, respectively. (c) Physical map showing the microsatellite markers that were used for the haplotype analysis. The distances between the markers on the chromosome 17 are indicated. The markers span a 2.89 Mb region on chromosome 17. deletion, a unique forward primer, located upstream of exon 23, was combined with several reverse primers located downstream of BRCA1 exon 24. Subsequently, two PCR fragments of 12,325 bp and 1200 bp were amplified, which represent the wild type and mutant allele, respectively (Fig. 1b), whereas the deletion size was confirmed as 11,053 bp using Sanger sequencing and is defined as: c _*8273del11052 (RefSeq NM_ ) or g _180579del11052 (GenBank NG_ ). 484

4 Haplotype analysis reveals that the recurrent BRCA1 deletion of exons 23 and 24 Frequency of the BRCA1 genomic rearrangement The frequency of this BRCA1 LGR was determined as 1.68% (35/2,092) in the study group of Greek, high-risk families by a custom-designed PCR, while all positive samples were confirmed using MLPA. Age of disease onset and family history of the deletion carriers are summarized in Table S1, Supporting Information. Clinicopathological characteristics of carriers Among the 35 families with the BRCA1 LGR, 60 mutation carriers were identified, the vast majority of which (76.6%; 46/60) were diagnosed with breast and/or ovarian cancer. Interestingly, seven individuals were diagnosed with both breast and ovarian cancer. The mean age of breast cancer diagnosis was 41.1 years (range: years), whereas the mean age of ovarian cancer diagnosis was 47.8 years (range: years). It is noteworthy that 79.4% (27/34) were diagnosed before the age of 50. Of all breast cancers diagnosed among the mutation carriers, 73.9% (17/23) were ductal, 13% (3/23) were medullary, whereas 8.7% (2/23) were lobular carcinomas. Breast tumors were more frequently associated with higher nuclear grade and more specifically, 85.7% (18/21) were grade III, while none was grade I. According to the available information 72.2% (13/18), of the breast cancers were triple negative (Table 1, panel a), while almost half of the tumors (47%; 8/17) had nodal invasion at diagnosis. In total, 19 of the 60 mutation carriers were diagnosed with ovarian cancer, most frequently being serous carcinoma (66.6%; 10/15), of high grade (75%; 9/12). Histopathology data are summarized in Table 1, panel b. Interestingly, 30.4% (14/46) of the mutation carriers developed a second BRCA-related tumor. More specifically, 26% (12/46) and 4.4% (2/46) were diagnosed with breast cancer and ovarian cancer, respectively, whereas 10.8% (5/46) were diagnosed with both breast and ovarian cancer after initial diagnosis. The mean time for second breast and ovarian cancer diagnosis was 7.25 years (range: 1 18 years) and 11.5 years (range: 1 20 years), respectively. Haplotype analysis Haplotype analysis of seven microsatellite markers has revealed the common haplotype along 2.89 Mb, indicating a common ancestral origin for the BRCA1 LGR deleting BRCA1 exons 23 and 24 (Fig. 1c), indicating the founder effect of this mutation in the Greek population (Table S2a and b) (12). Moreover, haplotype analysis was also performed in 132 chromosomes of 66 healthy age-matched Greek women in order to determine the population allele frequencies of these loci (Table S3).The pedigrees of two typical families, members of which carry the BRCA1 LGR are illustrated in Fig. 2. Mutation age estimation The mutation was estimated as originating between 47 and 78 generations, which involves 1,175 to 1,950 years Table 1. Histopathological characteristics of BRCA1 LGR carriers diagnosed with (a) breast and (b) ovarian cancers. a (a) Histology Ductal 73.9% (17/23) Medullary 13% (3/23) Lobular 8.7% (2/23) Mixed 4.4% (1/23) Grade III 85.7% (18/21) II 14.3% (3/21) ER Positive 27.2% (6/22) Negative 72.8% (16/22) PR Positive 18.2% (4/22) Negative 81.8% (18/22) HER2 Positive 11.1% (2/18) Negative 88.9% (16/18) Lymph node involvement Yes 47% (8/17) No 53% (9/17) (b) Histology Serous 75% (12/16) Endometrioid 6.2% (1/16) Undifferentiated 6.2% (1/16) Other/mixed histology 12.5% (2/16) Grade III 84.6% (11/13) II 15.4% (2/13) LGR, large genomic rearrangement. ER, estrogen receptor. PR, progesterone receptor. a Percentages were calculated with respect to the total number of patients with the known relevant information. (r = 0.135). Therefore, the mutation was probably introduced in the Greek population approximately 58 generations ago (corresponding to 1,450 years). Discussion Several population studies have shown a great variation of the frequency and spectrum of BRCA1 and BRCA2 mutations. We have previously shown that the Greek population, despite its diverse BRCA1 and BRCA2 mutation spectrum, is influenced by strong founder effects. Three LGRs clustered at the 3 end of BRCA1 (exons 20 24) have a significant contribution to the gene s Greek mutational spectrum (12). Such recurrent mutations derive from a unique older mutational event and disperse through the population by founder effects, as we have already proven for both deletion of BRCA1 exon 20 and exon 24 (10). Deletion of both BRCA1 exons 23 and 24 has been previously reported in families from Greece, Italy, Germany, Spain and South-Africa. The 11 kb deletion bearing the exact breakpoints with the one presently investigated has been initially characterized in a Greek family residing in South Africa (5), whereas none of the deletions of the 485

5 Apostolou et al. Fig. 2. Pedigrees of families, members of which carry the BRCA1 large genomic rearrangement (LGR). Probands are represented by arrows, whereas breast cancers are colored in black. The results of the haplotype analysis are shown. The alleles that are associated with the disease are in red. 486

6 Haplotype analysis reveals that the recurrent BRCA1 deletion of exons 23 and 24 same exons reported in other populations bear the same breakpoints. More specifically, the deletions identified in families of Spanish, Italian and German ancestries were 7.6 kb (15), 3.4 Kb (16), 8.1 kb (17), respectively. In all the above cases, albeit concerning distinct individually occurring genomic events, the Alu-repeat dense region stretching from intron 22 to about 30 Kb downstream of exon 24 is involved and therefore favor recombination events. We have carried out haplotype analysis in 60 deletion carriers and have shown that the majority of carriers (57/60) share the same disease-associated 2.9 Mb haplotype, suggesting that it represents a Greek founder mutation. The common ancestor has been estimated to have lived around 1,450 years ago. Recent ancestors have been living most probably the last centuries in the Asia Minor, a cultural and commercial center of the Greek and Byzantine civilization. The migration of the Greek population, because of Greco-Turkish War of , resulted to the dispersion of the mutation through the Aegean islands, where most of the carrier families originate. The frequency of BRCA1 exons 23 and 24 deletion is 1.68% in the group of 2,092 high-risk families studied, whereas the carriers showed similar clinicopathological characteristics with those reported in the numerous studies published. Breast cancer arising in carriers was mainly of the triple-negative subtype (72.2%) (18), although this is bias due to our selection criteria, with the medullary type being overrepresented (13%), while most ovarian cancers were high-grade serous. Moreover, the increased cumulative risk for a second diagnosis of a BRCA-related cancer, estimated as 27 and 13% for breast and ovarian cancer diagnosis, respectively in a 10-year time frame BRCA1 mutation carriers has been also observed in these patient series (19, 20). More specifically, one third of the individuals carrying the BRCA1 LGR were diagnosed with a second primary malignancy. Combined data from our previous and current work showed that in the above study group 340 families carried a BRCA1 mutation and 107 families carried a BRCA2 mutation, respectively. Notably, the deletion of both exons 23 and 24 was identified in 10.3% (35/340) of families carrying a BRCA1 mutation and in 7.83% (35/447) of families carrying a mutation in either BRCA1 or BRCA2. This high frequency has significant screening priority implications. Supporting Information Additional supporting information may be found in the online version of this article at the publisher s web-site. 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