Large Genomic Rearrangements of BRCA1 and BRCA2 among Patients Referred for Genetic Analysis in Galicia (NW Spain): Delimitation and Mechanism of Three Novel BRCA1 Rearrangements Laura Fachal1, Ana Blanco1, Marta Santamarin˜ a2, Angel Carracedo1, Ana Vega1* 1 Fundacio´nPu´blica Galega de Medicina Xeno´mica-SERGAS. Grupo de Medicina Xeno´mica, CIBERER, IDIS, Santiago de Compostela, Spain, 2 Grupo de Medicina Xeno´mica -USC, University of Santiago de Compostela, CIBERER, IDIS, Santiago de Compostela, Spain Abstract In the Iberian Peninsula, which includes mainly Spain and Portugal, large genomic rearrangements (LGRs) of BRCA1 and BRCA2 have respectively been found in up to 2.33% and 8.4% of families with hereditary breast and/or ovarian cancer (HBOC) that lack point mutations and small indels. In Galicia (Northwest Spain), the spectrum and frequency of BRCA1/ BRCA2 point mutations differs from the rest of the Iberian populations. However, to date there are no Galician frequency reports of BRCA1/BRCA2 LGRs. Here we used multiplex ligation-dependent probe amplification (MLPA) to screen 651 Galician index cases (out of the 830 individuals referred for genetic analysis) without point mutations or small indels. We identified three different BRCA1 LGRs in four families. Two of them have been previously classified as pathogenic LGRs: the complete deletion of BRCA1 (identified in two unrelated families) and the deletion of exons 1 to 13. We also identified the duplication of exons 1 and 2 that is a LGR with unknown pathogenicity. Determination of the breakpoints of the BRCA1 LGRs using CNV/SNP arrays and sequencing identified them as NG_005905.2:g.70536_180359del, NG_005905.2:g.90012_97270dup, and NC_000017.10:g.41230935_41399840delinsAluSx1, respectively; previous observa- tions of BRCA1 exon1-24del, exon1-2dup, and exon1-13del LGRs have not characterized them in such detail. All the BRCA1 LGRs arose from unequal homologous recombination events involving Alu elements. We also detected, by sequencing, one BRCA2 LGR, the Portuguese founder mutation c.156_157insAluYa5. The low frequency of BRCA1 LGRs within BRCA1 mutation carriers in Galicia (2.34%, 95% CI: 0.61–7.22) seems to differ from the Spanish population (9.93%, 95% CI: 6.76– 14.27, P-value = 0.013) and from the rest of the Iberian population (9.76%, 95% CI: 6.69–13.94, P-value = 0.014). Citation: Fachal L, Blanco A, Santamarin˜a M, Carracedo A, Vega A (2014) Large Genomic Rearrangements of BRCA1 and BRCA2 among Patients Referred for Genetic Analysis in Galicia (NW Spain): Delimitation and Mechanism of Three Novel BRCA1 Rearrangements. PLoS ONE 9(3): e93306. doi:10.1371/ journal.pone.0093306 Editor: Amanda Ewart Toland, Ohio State University Medical Center, United States of America Received December 30, 2013; Accepted February 28, 2014; Published March 31, 2014 Copyright: ß 2014 Fachal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants to AV from the Xunta de Galicia (10 PXIB 9101 297 PR) and the Fundacio´n Mutua Madrilen˜a. LF is supported by the Isabel Barreto programme of the Xunta de Galicia and the European Social Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction the frequency of BRCA2 LGRs is the highest reported to date (57.89% of BRCA2 mutations) [6–8]. The two major high-penetrance breast cancer susceptibility Of the published studies of the frequency of BRCA1/2 LGRs in genes, BRCA1 [1] and BRCA2 [2], account for approximately 26% the Iberian Peninsula or regions thereof [6–17], none has of all cases of hereditary breast and/or ovarian cancer (HBOC) specifically examined the population of Galicia (NW Spain), a [3]. To date, some 1,700 BRCA1 variants and 1,900 BRCA2 region with a distinct genetic identity attributable to its historical variants have been reported (Breast Cancer Information Core relative isolation, its cultural identity, and the occurrence of a database, http://research.nhgri.nih.gov/bic/). However, only 81 marked population bottleneck around 1000 years ago [18]. This BRCA1 variants and 17 BRCA2 variants are large genomic population features a number of founder mutations [18–20], rearrangements (LGRs) [4], and the prevalence of BRCA1/2 including a BRCA1 mutation, c.211A.G (referred to LGRs varies widely among different populations, mainly due to NM_007294.3; BIC 330A.G), which is present in more than the existence of founder rearrangements. For example, in the 50% of Galician HBOC families with BRCA1/2 mutations [21]. Netherlands, BRCA1 LGRs constitute up to 27% of all BRCA1 In view of to date there are no Galician frequency reports of mutations (see Sluiter et al. [4] and references therein), whereas to BRCA1/BRCA2 LGRs and the observed differences between date, only one BRCA2 LGR has been reported in a proband of Galicia and the rest of Spain in regard to the spectrum and Dutch and German ancestry [5]. On the contrary, in Portugal prevalence of point mutations of BRCA1 and BRCA2, we decided BRCA1 LGRs represents the ,6% of BRCA1 mutations while, due to investigate whether a similar situation holds for BRCA1/2 to the Portuguese BRCA2 founder mutation c.156_157insAluYa5, LGRs. We accordingly screened for BRCA1/2 LGRs among PLOS ONE | www.plosone.org 1 March 2014 | Volume 9 | Issue 3 | e93306 Large Genomic Rearrangements of BRCA1 and BRCA2 Galician families referred for genetic examination of BRCA1/2. (.0.15 standard deviations from the mean) were removed, the Here we describe three novel BRCA1 LGRs, propose likely whole normalization process was repeated, and so on until the originating mechanisms, and compare the frequency of LGRs in standard deviations of all probe signals were ,0.15. Galicia with published results for the remainder of the Iberian Peninsula. SNP arrays and analysis of breakpoint regions LGRs were characterized using the Cytogenetics Whole- Materials and Methods Genome 2.7 M Array in combination with the Genome-Wide Human SNP Array 6.0, or alternatively the CytoScan HD Array Participants (all from Affymetrix). The results were analyzed with the Our reference laboratory handles essentially all Galician Chromosome Analysis Suite (Affymetrix), the breakpoint regions patients referred for evaluation of the possibility of HBOC by delimited by the array markers were examined in the UCSC means of DNA analysis. Between 1997 and 2012 we examined Genome Browser (http://genome.ucsc.edu/; assembly NCBI37/ BRCA1/2 in 830 patients referred to us in accordance with the hg19), and repetitive sequences in these regions were identified criteria established in Galician oncological guidelines [22], which using RepeatMasker [24] within the Genome Browser. Breaks currently recommend referral if patients have (i) three or more first were pinpointed by sequencing as next described. degree relatives who have suffered breast or ovarian cancer, (ii) two affected first degree relatives if one was aged ,40 years at Sequencing diagnosis, (iii) two affected first or second degree relatives if both PCR primers were designed using Primer3 software (http:// suffered breast cancer at age ,50 years, or if one suffered bilateral frodo.wi.mit.edu/primer3/); primer sequences and PCR condi- breast cancer and one was aged ,50 years at diagnosis, or if at tions are described in the Table S1. Sequencing was performed least one of these cancers was ovarian or a male breast cancer, (iv) using BigDye Terminator v3.1 sequencing kits (Applied Biosys- age ,30 years at diagnosis of a breast cancer, (v) both breast and tems, USA). Electrophoresis was carried out on an ABI 3730 xl ovarian cancer, (vi) bilateral breast cancer diagnosed before the DNA analyzer (Applied Biosystems, USA). age of 40 years, or (vii) a family history of deleterious mutation of a Statistical analyses breast cancer susceptibility gene. These patients were first screened Association tests were performed using two-degrees of freedom for the founder mutations BRCA2 c.156_157insAluYa5 and Pearson’s chi-square test with Yates correction. Statistical analyses BRCA1 c.211A.G (BIC 330A.G) using protocols respectively were performed using the statistical package stats with the software described by Peixoto et al. [7] and Vega et al. [21], after which R v3.0.2. A nominal P-value of 0.05 was considered significant. other BRCA1/2 mutations were sought by bi-directional sequenc- ing of exons and flanking intronic splice sites. Of the 830 referred Results patients, 125 were found to have point mutations or small indels in BRCA1, and 54 point mutations or small indels in BRCA2. The 651 Among the 651 apparently unrelated index cases studied we with no point mutations or small indels were included in the found four different LGRs, three in BRCA1 by MLPA and one in present study, as were members of their families when this was BRCA2 by sequencing (Table 1). appropriate and possible. Relationships between index cases were investigated through Exon1-2dup the genealogical tree, which include at least three generations, and A duplication of BRCA1 exons 1-2 was identified in a 66-year- the family name that in Spain included the surname from the old woman in whom breast cancer was diagnosed at age 64 years. father and also from the mother. Previously, her three sisters had developed breast cancer (at ages 35, 49 and 59 years), as had her two maternal aunts (according to Ethics Statement the family, though this was not confirmed) (Family I, Fig. 1). The The study conformed to Spanish biomedical research legislation SNP array bracketed the downstream breakpoint but not the (Ley 14/2007) and was approved by the Galician Ethical upstream one (Fig.