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12 páginas, 4 tablas.-- El pdf del artículo es el manuscrito de autor.-- et al. ; Environmental or lifestyle factors are likely to explain part of the heterogeneity in breast and ovarian cancer risk among BRCA1 and BRCA2 mutation carriers. We assessed parity as a risk modifier in 515 and 503 Spanish female carriers of mutations in BRCA1 and BRCA2, respectively. Hazard ratios (HR) and their corresponding 95% confidence intervals (CI) were estimated using weighted Cox proportional hazards regression, adjusted for year of birth and study centre. The results for ever being parous and number of live-births were very similar for carriers of mutations in both genes. For all mutation carriers combined, the estimated HR associated with ever having had a live-birth was 0.74 (95% confidence interval [CI] = 0.55–1.01, P = 0.06), and that associated with each live-birth was 0.87 (95%CI = 0.77–0.98, P = 0.02). The latter association was observed only in women aged 40 and above (HR = 0.81, 95%CI = 0.70–0.94, P = 0.004 vs. HR = 0.99, 95%CI = 0.83–1.18, P = 0.9 for women under age 40), and this trend was highly consistently observed for carriers of mutations in each gene. There was no evidence of an association between breast cancer risk and age at first birth for parous BRCA1 or BRCA2 mutation carriers (P-trend ≥ 0.3). The power to detect associations with ovarian cancer risk was much lower, especially for BRCA2 mutation carriers. Nevertheless, having a live-birth was associated with protection for BRCA1 mutation carriers (HR = 0.41, 95%CI = 0.18–0.94, P = 0.03), and a strong and consistent protective effect of age at first birth was observed for parous carriers of mutations in both genes (HR = 0.65, 95%CI = 0.52–0.83, P < 0.001). This is the third independent study to find that, as in the general population, parity appears to be associated with protection from breast cancer in women with mutations in BRCA1 and BRCA2. Parity appears to be protective for ovarian cancer in BRCA1 mutation carriers, but its role in BRCA2 mutation carriers remains unclear. Whether later age at first birth is also protective for ovarian cancer in mutation carriers requires further confirmation. ; This work was partly supported by a grant from the Fondo de Investigación Sanitario [PI081120]. TC, MdH and ED-R were supported by the RTICC (RD06/0020/0021), Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation and a grant from the Fundación de Investigación Médica Mutua Madrileña (FMMA/06). The work at the Instituto Catalán de Oncología was supported by grants ISCIII-RETIC RD06/0020/1051 and 2005SGR00018. The Fundación Pública Galega de Medicina Xenómica -SERGAS component of this work was partially supported by grants from the Ministerio de Sanidad y Consumo (PI052275) and the Xunta de Galicia (PGIDIT06BTF910101PR) to AV. AB has a fellowship from the Instituto de Salud Carlos III. The work at the Instituto de Biologia y Genetica Molecular was partly supported by the Hereditary Cancer Prevention Programme of the Regional Government of Castilla y León, and grant PI06/1102 (Fondo de Investigación Sanitaria, Instituto de Salud Carlos III). The work at the Hospital de Cruces was funded by the grant BIO07/CA/006. ; Peer reviewed

et al. ; Rare sequence variants in >high-risk> disease genes, often referred as unclassified variants (UVs), pose a serious challenge to genetic testing. However, UVs resulting in splicing alterations can be readily assessed by in vitro assays. Unfortunately, analytical and clinical interpretation of these assays is often challenging. Here, we explore this issue by conducting splicing assays in 31 BRCA2 genetic variants. All variants were assessed by RT-PCR followed by capillary electrophoresis and direct sequencing. If assays did not produce clear-cut outputs (Class-2 or Class-5 according to analytical International Agency for Research on Cancer guidelines), we performed qPCR and/or minigene assays. The latter were performed with a new splicing vector (pSAD) developed by authors of the present manuscript (patent #P201231427 CSIC). We have identified three clinically relevant Class-5 variants (c.682-2A>G, c.7617+1G>A, and c.8954-5A>G), and 27 analytical Class-2 variants (not inducing splicing alterations). In addition, we demonstrate that rs9534262 (c.7806-14T>C) is a BRCA2 splicing quantitative trait locus. Splicing analyses are widely used in the clinical setting to address the significance of rare genetic variants. However, interpretation of results is often challenging. Here we show that incorporating capillary electrophoresis (EP) and direct sequencing to the analysis of RT-PCR splicing assays often produces clear-cut analytical outputs. Indeed, we have produced clear-cut outputs in 29 out of 31 BRCA2 variants investigated, including the identified of clinically relevant splicing aberrations, and one splicing quantitative trait locus. ; Contract grant sponsors: EAV was supported by research grants PI10/2910 (Instituto de Salud Carlos III), CSI004A10-2 (Consejería de Educación, Regional Government of Castilla y León) and BIO39/VA27/10 (Consejería de Sanidad, Regional Government of Castilla y León). Alberto Acedo is supported by the European Social Fund and Regional Government of Castilla y León (P.O. 2007-2013). ZGC is supported by the Genetic Counseling Program (Regional Government of Valencia). SGE is funded by a Miguel Servet contract (CP10/00617, Instituto de Salud Carlos III). HUVH-VHIO research was supported by a Miguel Servet research grant CP10/00617, Instituto de Salud Carlos III). MH was supported by research grants PI 09/00859 and PI 12/00539 (Instituto de Salud Carlos III). Instituto de Salud Carlos III research grants are partially supported by European Regional Development FEDER Funds. ; Peer Reviewed

7 páginas, 3 figuras, 2 tablas.-- El pdf del artículo es la versión pre-print.-- et al. ; Lynch syndrome is caused by germline mutations in MSH2, MLH1, MSH6, and PMS2 mismatch repair genes and leads to a high risk of colorectal and endometrial cancer. It was recently shown that constitutional 3′ end deletions of EPCAM could cause Lynch syndrome in tissues with MSH2 deficiency. We aim to establish the spectrum of mutations in MSH2-associated Lynch syndrome cases and their clinical implications. Probands from 159 families suspected of having Lynch syndrome were enrolled in the study. Immunohistochemistry and microsatellite instability (MSI) analyses were used on the probands of all families. Eighteen cases with MSH2 loss were identified: eight had point mutations in MSH2. In 10 Lynch syndrome families without MSH2 mutations, EPCAM-MSH2genomic rearrangement screening was carried out with the use of multiplex ligation–dependent probe amplification and reverse transcriptase PCR. We report that large germline deletions, encompassing one or more exons of the MSH2 gene, cosegregate with the Lynch syndrome phenotype in 23% (8 of 35) of MSI families tested. A new combined deletion EPCAM-MSH2 was identified and characterized by break point analysis, encompassing from the 3′ end region of EPCAM to the 5′ initial sequences of the MSH2 (c.859-1860_MSH2:646-254del). EPCAM-MSH2 fusion transcript was isolated. The tumors of the carriers show high-level MSI and MSH2 protein loss. The clinical correlation provided evidence that the type of mutation and the extension of the deletions involving the MSH2 gene could have different implications in cancer predisposition. Thus, the identification of EPCAM-MSH2 rearrangements and their comprehensive characterization should be included in the routine mutation screening protocols for Lynch syndrome. ; This work was supported by the Regional Government of Castilla y Le on (Spain). L. Perez-Cabornero and A. Acedo were supported by a predoctoral FPI fellowship from the Government of Castilla y León (Spain). ; Peer reviewed

This is an open access article distributed under the terms of the Creative Commons Attribution License. ; [Introduction]: The underlying pathogenic mechanism of a large fraction of DNA variants of disease-causing genes is the disruption of the splicing process. We aimed to investigate the effect on splicing of the BRCA2 variants c.8488-1G > A (exon 20) and c.9026_9030del (exon 23), as well as 41 BRCA2 variants reported in the Breast Cancer Information Core (BIC) mutation database. [Methods]: DNA variants were analyzed with the splicing prediction programs NNSPLICE and Human Splicing Finder. Functional analyses of candidate variants were performed by lymphocyte RT-PCR and/or hybrid minigene assays. Forty-one BIC variants of exons 19, 20, 23 and 24 were bioinformatically selected and generated by PCR-mutagenesis of the wild type minigenes. [Results]: Lymphocyte RT-PCR of c.8488-1G > A showed intron 19 retention and a 12-nucleotide deletion in exon 20, whereas c.9026_9030del did not show any splicing anomaly. Minigene analysis of c.8488-1G > A displayed the aforementioned aberrant isoforms but also exon 20 skipping. We further evaluated the splicing outcomes of 41 variants of four BRCA2 exons by minigene analysis. Eighteen variants presented splicing aberrations. Most variants (78.9%) disrupted the natural splice sites, whereas four altered putative enhancers/silencers and had a weak effect. Fluorescent RT-PCR of minigenes accurately detected 14 RNA isoforms generated by cryptic site usage, exon skipping and intron retention events. Fourteen variants showed total splicing disruptions and were predicted to truncate or eliminate essential domains of BRCA2. [Conclusions]: A relevant proportion of BRCA2 variants are correlated with splicing disruptions, indicating that RNA analysis is a valuable tool to assess the pathogenicity of a particular DNA change. The minigene system is a straightforward and robust approach to detect variants with an impact on splicing and contributes to a better knowledge of this gene expression step. ; This work was supported by grants PI10/2910 of the Instituto de Salud Carlos III (Spanish Ministry of Science and Innovation), and CSI004A10-2 (Consejería de Educación) and BIO39/VA27/10 (Consejería de Sanidad) of the Regional Government of Castilla y León. Alberto Acedo is supported by the European Social Fund and Consejeria de Educación de la Junta de Castilla y León under the P.O. Castilla y León 2007-2013. ; Support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). ; Peer reviewed

11 páginas, 3 figuras, 2 tablas.-- et al.-- El pdf del artículo es la versión post-print. ; [Purpose]: Most BRCA1/2 mutations are of unknown clinical relevance. An increasing amount of evidence indicates that there can be deleterious effects through the disruption of the splicing process. We have investigated the effect of aberrant splicing of BRCA1/2 on hereditary breast/ovarian cancer (HBOC). [Experimental Design]: DNA variants were analyzed with splicing prediction programs to select putative splicing mutations. Splicing assays of 57 genetic variants were done by lymphocyte reverse transcription-PCR and/or hybrid minigenes in HeLa and nontumor breast epithelial cells. [Results]: Twenty-four BRCA1/2 variants of Spanish HBOC patients were bioinformatically preselected. Functional assays showed that 12 variants induced anomalous splicing patterns, 6 of which accounted for 58.5% of BRCA1 families. To further evaluate the defective splicing of BRCA1/2, we analyzed 31 Breast Cancer Information Core Database (BIC) and two artificial variants that were generated by mutagenesis. Sixteen variants induced different degrees of aberrant splicing. Altogether, anomalous splicing was caused by 28 BRCA1/2 variants of all types, indicating that any DNA change can disrupt pre-mRNA processing. We show that a wide range of regulatory elements can be involved, including the canonical and cryptic splice sites, the polypyrimidine tract, and splicing enhancers/silencers. Twenty mutations were predicted to truncate the BRCA proteins and/or to delete essential domains, thus supporting a role in HBOC. [Conclusions]: An important fraction of DNA variants of BRCA1/2 presents splicing aberrations that may represent a relevant disease-causing mechanism in HBOC. The identification of splicing disruptions by functional assays is a valuable tool to discriminate between benign polymorphisms and pathogenic mutations. ; PI061102, (Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Spain) and 200820I135 (Consejo Superior de Investigaciones Científicas, Ministerio de Ciencia e Innovación, Spain), the Hereditary Cancer Prevention Programme of the Regional Government of Castilla y León, and a grant from the Associazione Italiana Ricerca sul Cancro, Italy (F. Pagani). ; Peer reviewed

5 páginas, 1 figura, 1 tabla.-- et al.-- El pdf del artículo es el manuscrito de autor. ; The mutation spectrum of BRCA1 and BRCA2 presents a wide range of unique mutations in breast/ovarian cancer patients but recurrent mutations with founder effects have also been described. BRCA2 5344delAATA and 9538delAA are recurrent mutations in Castilla-Leo´n (Spain) representing 10.6% of BRCA2 positive families. By genotyping eleven chromosome 13 markers (4.3 Mb) we demonstrate that each mutation shows core haplotypes of 1.66 and 0.87 Mb, respectively, supporting a common ancestor in Castilla-Leo´n. Furthermore, both mutations are associated with earlier onset of breast cancer (5344delAATA: 37.4 years, P = 0.033; 9538delAA: 39.4 years, P = 0.008). The identification of founder effects improves the genetic screening strategy to be followed and facilitates the clinical management of asymptomatic carriers. ; This work has been supported by the Regional Government of Castilla y León, and grants PI061102 (Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación) and 200820I135 (Consejo Superior de Investigaciones Científicas, Ministerio de Ciencia e Innovación). ; Peer reviewed

10 páginas, 3 figuras, 3 tablas.-- et al.-- El pdf del artículo es la versión post-print. ; The distribution of BRCA1 and BRCA2 germ line mutations in breast/ovarian cancer families varies among different populations, which typically present a wide spectrum of unique mutations. Splicing mutation 5272-1G>A of BRCA1 and frameshift mutation 5374delTATG of BRCA2 are highly prevalent mutations in Castilla-León (Spain), accounting for 18.4% and 13.6% of BRCA1 and BRCA2 positive families, respectively. To test the presence of founder effects, 9 Spanish 5272-1G>A and 13 5374delTATG families were genotyped with polymorphic markers linked to BRCA1 or BRCA2. All the 5272-1G>A families shared a common haplotype in eight markers (1.1 Mb region) and the mutation age was estimated in 15 generations (∼380 years). A conserved haplotype associated to 5374delTATG was observed in four markers (0.82 Mb). The mutation occurred approximately 48 generations ago (∼1200 years). Each mutation likely arose from a common ancestor that could be traced to a small area of Castilla-León and expanded to other Spanish regions. They can have a significant impact on the clinical management of asymptomatic carriers as well as on the genetic screening strategy to be followed in populations with Spanish ancestries. ; This work has been supported by the Regional Government of Castilla y León, and grants PI061102, PI052275, PI050864) (Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación), 200820I135 (Consejo Superior de Investigaciones Científicas, Ministerio de Ciencia e Innovación), INT07/191 (Instituto de Salud Carlos III), PGDIT06BTF910101PR (Xunta de Galicia). EB was supported by The Health Department of The Basque Country Government (BOPV, 17 Jul 2008, p.16983). ; Peer reviewed

et al. ; It has been demonstrated that monoallelic PALB2 (Partner and Localizer of BRCA2) gene mutations predispose to familial breast cancer. Some of the families reported with germline PALB2 mutations presented male breast cancer as a characteristic clinical feature. Therefore, we wanted to investigate the contribution of germline PALB2 mutations in a set of 131 Spanish BRCA1/BRCA2-negative breast/ovarian cancer families with at least one male breast cancer case. The analysis included direct sequencing of all coding exons and intron/exon boundaries as well as a Multiplex Ligation-dependent Probe Amplification-based analysis of genomic rearrangements. For the first time we have identified a genomic rearrangement of PALB2 gene involving a large deletion from exon 7 to 11 in a breast cancer family. We have also identified several PALB2 variants, but no other obvious deleterious PALB2 mutation has been found. Thus, our study does not support an enrichment of PALB2 germline mutations in the subset of breast cancer families with male breast cancer cases. The identification of intronic and exonic variants indicates the necessity of assessing the implications of variants that do not lead to PALB2 truncation in the pathoghenicity of the PALB2 gene. ; This study was supported by grants from the Xunta de Galicia (10PXIB 9101297PR) and FMM Foundation given to AV. TC, M de H, and PPS were supported by PS09/00859, RD06/0020/0021 from RTICC, Instituto de Salud Carlos III. JB and AO were supports by the AECC, and RD06/0020/1060 from RTICC ICO: Contract grant sponsor: Asociación Española Contra el Cáncer, Spanish Health Research Fund; Carlos III Health Institute; Catalan Health Institute and Autonomous Government of Catalonia. Contract grant numbers: ISCIIIRETIC RD06/0020/1051, PI10/01422, PI10/ 31488, and 2009SGR290. E.V. grants, CSI004A10-2 (Consejería de Educación, Junta de Castilla y León) and BIO39/VA27/10 (Consejería de Sanidad, Junta de Castilla y León). ; Peer Reviewed

Supplementary Tables S1–S3 and figures S1 and S2 can be found at: http://carcin.oxfordjournals.org/.-- et al. ; BRCA2-c.2808_2811del (3036delACAA) is one of the most reported germline mutations in non-Ashkenazi breast cancer patients. We investigated its genetic origin in 51 Spanish carrier families that were genotyped with eleven 13q polymorphic markers. Three independent associated haplotypes were clearly distinguished accounting for 23 (WCL), 20 (ECL) and 6 families (SoS). Mutation age was estimated with the DMLE software in a range of 45-68 and 45-71 generations for WCL and ECL haplotypes, respectively. The most prevalent variants, c.2808_2811del and c.2803G>A, were located in a double-hairpin loop structure (c.2794-c.2825) predicted by Quikfold that was proposed as a mutational hotspot. To check this hypothesis, random mutagenesis was performed over a 923-bp fragment of BRCA2 and 86 DNA variants were characterized. Interestingly, three mutations reported in the mutation databases (c.2680G>A, c.2944del and c.2957dup) were replicated and 20 affected the same position with different nucleotide changes. Moreover, five variants were placed in the same hairpin-loop of c.2808_2811del, and one affected the same position (c.2808A>G). In conclusion, our results support that at least three different mutational events occurred to generate c.2808_2811del. Other highly prevalent DNA variants, such as BRCA1-c.68_69delAG and BRCA2-c.5946delT and c.8537delAG, are concentrated in hairpin-loops suggesting that these structures may represent mutational hotspots. ; Instituto de Salud Carlos III (PI10/2910 to E.A.V., ISCIIIRETIC: RD06/0020/1051 and PI10/01422 to L.F.); Junta de Castilla y León, Consejería de Educación (CSI004A10-2 to E.A.V.); Consejería de Sanidad (BIO39/VA27/10 to E.A.V.); Cancer Prevention Program of the Regional Government of Castilla y León; The European Social Fund and Consejería de Educación de la Junta de Castilla y León under the P.O. Castilla y León 2007–13 to A.A.; Spanish Health Research Fund; Carlos III Health Institute; Catalan Health Institute, Government of Catalonia (2009SGR290 to L.F.); Spanish Association Against Cancer (to L.F.). ; Peer Reviewed

© 2021 by the authors. ; RAD51D loss-of-function variants increase lifetime risk of breast and ovarian cancer. Splicing disruption is a frequent pathogenic mechanism associated with variants in susceptibility genes. Herein, we have assessed the splicing and clinical impact of splice-site and exonic splicing enhancer (ESE) variants identified through the study of ~113,000 women of the BRIDGES cohort. A RAD51D minigene with exons 2–9 was constructed in splicing vector pSAD. Eleven BRIDGES splice-site variants (selected by MaxEntScan) were introduced into the minigene by site-directed mutagenesis and tested in MCF-7 cells. The 11 variants disrupted splicing, collectively generating 25 different aberrant transcripts. All variants but one produced negligible levels (A demonstrated a complete aberrant splicing pattern without the FL transcript. On the other hand, c.214T>C increased efficiency of exon 3 recognition, so only the FL transcript was detected (100%). In conclusion, 41 RAD51D spliceogenic variants (28 of which were from the BRIDGES cohort) were identified by minigene assays. We show that minigene-based mapping of ESEs is a powerful approach for identifying ESE hotspots and ESE-disrupting variants. Finally, we have classified nine variants as likely pathogenic according to ACMG/AMP-based guidelines, highlighting the complex relationship between splicing alterations and variant interpretation. ; P.D., M.P.G.V., D.F.E., M.d.l.H. and E.A.V. have received funding from the European Union's Horizon 2020 research and innovation program under grant agreement no. 634935. E.A.V.'s lab is supported by grants from the Spanish Ministry of Science and Innovation, Plan Nacional de I + D + I 2013–2016, ISCIII (PI17/00227 and PI20/00225), co-funded by FEDER from Regional Development European Funds (European Union) and from the Consejería de Educación, Junta de Castilla y León, ref. CSI242P18 (actuación cofinanciada P.O. FEDER 2014–2020 de Castilla y León). M.d.l.H.'s lab is supported by grants from the Spanish Ministry of Science and Innovation, Plan Nacional de I + D + I 2013–2016, ISCIII (PI15/00059 and PI20/00110) and co-funded by FEDER from Regional Development European Funds (European Union). L.S.-M. is supported by a predoctoral fellowship from the AECC-Scientific Foundation, Sede Provincial de Valladolid (2019–2023). A.V.-P. is supported by a predoctoral fellowship from the Consejería de Educación, Junta de Castilla y León (2018–2022). Programa Estratégico Instituto de Biología y Genética Molecular (IBGM), Escalera de Excelencia, Junta de Castilla y León (Ref. CLU-2019-02).

© 2020 by the authors. ; Hereditary breast and/or ovarian cancer is a highly heterogeneous disease with more than 10 known disease-associated genes. In the framework of the BRIDGES project (Breast Cancer Risk after Diagnostic Gene Sequencing), the RAD51C gene has been sequenced in 60,466 breast cancer patients and 53,461 controls. We aimed at functionally characterizing all the identified genetic variants that are predicted to disrupt the splicing process. Forty RAD51C variants of the intron-exon boundaries were bioinformatically analyzed, 20 of which were selected for splicing functional assays. To test them, a splicing reporter minigene with exons 2 to 8 was designed and constructed. This minigene generated a full-length transcript of the expected size (1062 nucleotides), sequence, and structure (Vector exon V1- RAD51C exons_2-8- Vector exon V2). The 20 candidate variants were genetically engineered into the wild type minigene and functionally assayed in MCF-7 cells. Nineteen variants (95%) impaired splicing, while 18 of them produced severe splicing anomalies. At least 35 transcripts were generated by the mutant minigenes: 16 protein-truncating, 6 in-frame, and 13 minor uncharacterized isoforms. According to ACMG/AMP-based standards, 15 variants could be classified as pathogenic or likely pathogenic variants: c.404G > A, c.405-6T > A, c.571 + 4A > G, c.571 + 5G > A, c.572-1G > T, c.705G > T, c.706-2A > C, c.706-2A > G, c.837 + 2T > C, c.905-3C > G, c.905-2A > C, c.905-2_905-1del, c.965 + 5G > A, c.1026 + 5_1026 + 7del, and c.1026 + 5G > T. ; PD: MPGV: DFE: MdlH and EAV have received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement no. 634935. EAV lab is supported by grants from the Spanish Ministry of Science and Innovation, Plan Nacional de I + D+I 2013–2016, ISCIII (PI17/00227) co-funded by FEDER from Regional Development European Funds (European Union) and from the Consejería de Educación, Junta de Castilla y León, ref. CSI242P18 (actuación cofinanciada P.O. FEDER 2014–2020 de Castilla y León). MdlH is supported by grants from the Spanish Ministry of Science and Innovation, Plan Nacional de I + D+I 2013–2016, ISCIII (PI15/00059) co-funded by FEDER from Regional Development European Funds (European Union).