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Resumen del póster presentado a la European Human Genetics Conference celebrada en Nuremberg (Alemania) del 23 al 26 de junio de 2012., Mutations in susceptibility breast/ovarian cancer (BOC) genes show different spectra according to geographic origin. To date, few BRCA1 and BRCA2 mutations are described as founder in Spanish population, intriguingly linked to geographic distribution. Other susceptibility genes that share key roles in Fanconi Anemia pathway are now being scanned for mutations. A total of 1275 unrelated families have been tested for BRCA mutations in the IBGM. Samples and written informed consent of BOC cases and relatives were collected at the Genetic Counselling Units of East CyL. DNA was scrutinized for BRCA mutations by HA-CAE and subsequently sequence analysis of abnormal patterns. Additionally, MLPA was performed in high-risk patients with familial history of BOC without point mutations. Moreover, other genes as PALB2 or RAD51C were tested in families with specific cancer features. Seventy-four different deleterious DNA changes in BRCA genes have been identified in our laboratory in 186 unrelated families (69 BRCA1+ and 117 BRCA2+ families). Spanish founder mutations were predominant in our samples: 330A>G, 5242C>A and 5272-1G>A in BRCA1 and 3036_3039delACAA, 5374_5377delTATG and 9254_9258delATCAT in BRCA2. Remarkably, 187_188delAG-BRCA1 mutation was absent in our region. Five different large rearrangements in seven ovarian unrelated patients have been detected during MLPA analysis. No pathological mutations were identified in either RAD51C or PALB2. Our target population shows a particular BRCA1 and BRCA2 mutation spectra where Spanish founder mutations are leading. Although mutations in other susceptibility BOC genes do not yield results so far, comprehensive studies with larger number of samples would be performed.

La presente invención se refiere a un vector génico útil para ensayos funcionales de splicing obtenido a partir del plásmido pSPL3b, que comprende: una deleción del intrón de pSPL3b; una sustitución del MCS de pSPL3b por un casete lacZ sin sitio críptico aceptor de splicing; una mutación de fortalecimiento del sitio aceptor de splicing de pSPL3b; una mutación de inhabilitación de al menos una diana de restricción de pSPL3b; y consiste preferentemente en SEQ ID No: 79. Es asimismo objeto de la invención, un procedimiento de obtención del vector anterior. La invención también se refiere a un minigen que comprende dicho vector con una secuencia nucleotídica clonada en el MCS de lacZ, así como una célula que comprende un vector o minigen de los anteriores. Además, también hace referencia al uso del vector o minigen en ensayos funcionales de splicing, protegiendo también un método para este tipo de ensayos, Consejo Superior de Investigaciones Científicas, Universidad de Valladolid, A1 Solicitud de patente con informe sobre el estado de la técnica

Resumen del póster presentado a la European Human Genetics Conference celebrada en Nuremberg (Alemania) del 23 al 26 de junio de 2012., Splicing disruptions is one key pathogenic mechanism in inherited diseases. We are currently investigating the contribution of aberrant splicing of BRCA1/2 genes to hereditary breast/ovarian cancer. A powerful approach to study the splicing outcomes of DNA variants is a splicing reporter minigene especially when patient RNA is not available. We constructed a single minigene of 8 BRCA2 exons (19 to 26) in a pSPL3-derived plasmid in 5 cloning steps, which is, as far as we know, the largest BRCA2 minigene ever reported. The genomic fragment from exons 19 to 26 is more than 27 kb in length that was reduced to a final insert of 4.7 kb with internal deletions of introns 20, 21, 24 and 25.This construction was transfected in HeLa cells and we observed a main RNA isoform of the expected size of 1.5 Kb that contained the vector constitutive exons and BRCA2 exons 19 to 26. Several splicing variants of each exon were generated in the wild type minigene by PCR mutagenesis and assayed to demonstrate the usefulness and reliability of this large construction. Splicing reporter minigenes are straightforward and robust tools to distinguish between pathogenic mutations and innocuous variants. The use of minigenes with several exons facilitates the analysis of putative splicing variants in a single minigene and emulates the physiological genomic context where the splicing reactions take place., Grants CSI004A10-2 and BIO39/VA27/10 (Junta de Castilla y León) and Grant PI10/2910 (Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Spain).

Resumen del póster presentado a la European Human Genetics Conference celebrada en Nuremberg (Alemania) del 23 al 26 de junio de 2012., MUTYH-associated polyposis (MAP) is an autosomal recessive cancer predisposition syndrome associated with the development of colorectal tumors and colonic polyps at an early age. MAP syndrome is associated to germline biallelic mutations in the MUTYH gene which lead to deficient DNA repair through the base-excision repair system and accumulation of G:C-T:A transversions. Occurrence of such mutations in oncogenes and tumor suppressor genes drives colorectal carcinogenesis and is associated with the development of colonic polyps. The aim of this study was to assess the frequency of germline MUTYH mutations in patients with MAP and other hereditary colorectal cancer (CRC) phenotypes. A total of 30 patients were included. Samples were screened for the MUTYH germline mutations by capillary array electrophoresis (HA_CAE) method. In all mutation-positive cases, results were confirmed by sequencing. Biallelic germline MUTYH mutations were identified in 2 of 30 (6.6%) patients with a phenotype of hereditary colorectal cancer. Besides, four monoallelic variants were detected in different samples that are described in LOVD database (MUTYH_00078, MUTYH_00005, MUTYH_00063, MUTYH_00086). A genotype-phenotype correlation has found in these patientns. Germline MUTYH mutation screening should be considered in the differential diagnosis of hereditary colorectal syndromes, we are considering the validity and applicability of MUTYH mutation screening in our population.

Póster presentado al 4th Familial Cancer Conference celebrado en Madrid del 7 al 8 de Junio del 2010.

Póster presentado al 4th Familial Cancer Conference celebrado en Madrid del 7 al 8 de Junio del 2010.-- et al., This work was partially supported by FMM, FIS09/00859 and Consejería de Educación, Junta de Castilla y León, grant CSI004A10-2. A.B. is a founded of the “Instituto de Salud Carlos III” (FI05/00026).

International audience; The mutation spectrum of and presents a wide range of unique mutations in breast/ovarian cancer patients but recurrent mutations with founder effects have also been described. 5344delAATA and 9538delAA are recurrent mutations in Castilla-León (Spain) representing 10.6% of 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-León. Furthermore, both mutations are associated with earlier onset of breast cancer (5344delAATA: 37.4 years, = 0.033; 9538delAA: 39.4 years, = 0.008). The identification of founder effects improves the genetic screening strategy to be followed and facilitates the clinical management of asymptomatic carriers

5 páginas.-- et al., CYP2C9 is a major liver enzyme responsible of the metabolism of many clinically important drugs. The presence of CYP2C9 genetic polymorphisms has been associated with marked interindividual variability in its catalytic activity that could result in drug toxicity. Here we present frequencies of the most common CYP2C9 coding variants CYP2C9*2 (C430T) and CYP2C9*3 (A1075C) in representative samples of four regions from Spain (Basque Country, n=358; Catalonia, n=240; Central Spain, n=190 and Galicia, n=288) and one northern Italian region, (Verona, n=164), which range between 0.125 and 0.165 in the case of CYP2C9*2 and between 0.071 and 0.085 for CYP2C9*3. No significant differences between CYP2C9 allele frequencies were found comparing all the sampled populations. A more extensive comparative analysis using allele frequency data of populations widely spread over Europe was performed, showing significant differences in the CYP2C9*2 allele frequencies distribution between some of the regions, being quite homogeneous in the case of CYP2C9*3 variant. The results obtained show that above 40% of our samples carry a mutate allele, which can result in a poor metabolization of low therapeutic index drugs as oral anticoagulants (warfarin, acenocoumarol), oral antidiabetic drugs and some non-steroidal anti-inflammatory drugs. Our study constitutes both a large (n=1240) and robust allele frequency database on CYP2C9 polymorphisms, which represents one of the most numerous CYP2C9*2 and *3 database existing to date.

International audience; Environmental or lifestyle factors are likely to explain part of the heterogeneity in breast and ovarian cancer risk among and mutation carriers. We assessed parity as a risk modifier in 515 and 503 Spanish female carriers of mutations in and , 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, = 0.06), and that associated with each live-birth was 0.87 (95%CI = 0.77–0.98, = 0.02). The latter association was observed only in women aged 40 and above (HR = 0.81, 95%CI = 0.70–0.94, = 0.004 vs. HR = 0.99, 95%CI = 0.83–1.18, = 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 or mutation carriers (-trend ≥ 0.3). The power to detect associations with ovarian cancer risk was much lower, especially for mutation carriers. Nevertheless, having a live-birth was associated with protection for mutation carriers (HR = 0.41, 95%CI = 0.18–0.94, = 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

12 páginas, 7 tablas.-- et al., We screened index cases from 410 Spanish breast/ovarian cancer families and 214 patients (19 of them males) with breast cancer for germ-line mutations in the BRCA1 and BRCA2 genes, using SSCP, PTT, CSGE, DGGE, and direct sequencing. We identified 60 mutations in BRCA1 and 53 in BRCA2. Of the 53 distinct mutations observed, 11 are novel and 12 have been reported only in Spanish families (41.5%). The prevalence of mutations in this set of families was 26.3%, but the percentage was higher in the families with breast and ovarian cancer (52.1%). The lowest proportion of mutations was found in the site-specific female breast cancer families (15.4%). Of the families with male breast cancer cases, 59.1% presented mutations in the BRCA2 gene. We found a higher frequency of ovarian cancer associated with mutations localized in the 5′ end of the BRCA1 gene, but there was no association between the prevalence of this type of cancer and mutations situated in the ovarian cancer cluster region (OCCR) region of exon 11 of the BRCA2 gene. The mutations 187underscore;188delAG, 330A>G, 5236G>A, 5242C>A, and 589underscore;590del (numbered after GenBank U14680) account for 46.6% of BRCA1 detected mutations whereas 3036underscore;3039del, 6857underscore;6858del, 9254underscore;9258del, and 9538underscore;9539del (numbered after GenBank U43746) account for 56.6% of the BRCA2 mutations. The BRCA1 330A>G has a Galician origin (northwest Spain), and BRCA2 6857underscore;6858del and 9254underscore;9258del probably originated in Catalonia (northeast Spain). Knowledge of the spectrum of mutations and their geographical distribution in Spain will allow a more effective detection strategy in countries with large Spanish populations.

5 páginas, 1 tabla., Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Furthermore, the involvement of this gene in other pathologies „associated with CF”, like Congenital Absence of the Vas Deferens (bilateral or unilateral, CBAVD or CUAVD, resp.) is known, though the mutations causing these phenotypes within a defined population are different from those observed in CF patients. While cystic fibrosis patients have CFTR mutations in both alleles, most of the patients suffering from CF-related pathologies have mutations in only one allele. Frequently, the second CFTR allele is not mutant but polyvariant carrying a combination of alleles of polymorphic loci, called Polyvariant Mutant Genes (PMG) which, as a whole haplotype, contribute to lowering the level of CFTR transcripts. The loci involved in these PMG are Tn, TGm (both in intron 8), and M470V (exon 10) of the CFTR gene. We have carried out an exhaustive analysis of the CFTR gene in order to determine its possible role in 15 azoospermic Spanish patients. We have found that all CBAVD cases with a CF mutation carry the haplotype 5T-12(TG)-V470 on the second allele which is different from that found in other related pathologies causing azoospermia, like Congenital Bilateral Absence of Seminal Vesicles (CBASV) or CUAVD, where we have found the haplotype 5T-11(TG)-M470 which is more efficient producing CFTR protein. We believe that the latter phenotypes represent incomplete forms of CBAVD. Given the high frequency of severe CF mutations an exhaustive screening of CFTR mutations should be offered when assisted reproduction technologies are available. Moreover, when two mutations (or one mutations and a PMG) are detected, their phase should be established, since the existence of double mutants could modify the phenotype and it may suggest that the alteration in the other CFTR gene remains unidentified.

11 páginas.-- et al., [ES]: La enfermedad de Darier es un trastorno cutáneo cuya expresividad clínica es variable y diversa, y está caracterizada por la presencia de pápulas queratósicas, lesiones en mucosas, fundamentalmente en boca, alteraciones ungueales y queratodermia palmoplantar, pudiendo afectarse otros órganos de la economía. Se hereda de una forma autosómica dominante y su prevalencia es aproximadamente de 1/50.000. El gen causante de la enfermedad de Darier fue localizado en el brazo largo del cromosoma 12, bandas q23-q24.1. Desde un punto de vista clínico, hemos estudiado tres familias de nuestro entorno, encontrando que las alteraciones ungueales y la queratodermia palmoplantar son las más frecuentes. La afectación cutaneo-mucosa variaba ampliamente en intensidad y extensión, de unos pacientes a otros, tanto ínter como intrafamiliarmente. Por otro lado, hemos efectuado un análisis de ligamiento genético con nueve microsatélites del cromosoma 12q23-q24.1. El conjunto de nuestras tres familias muestran un ligamiento significativo con todos estos marcadores. El análisis de recombinantes nos ha permitido establecer un intervalo crítico donde se localiza el gen Darier, delimitado por el marcador centromérico o proximal D12S78 y el marcador telomérico o distal D12S1646., [EN]: Darier's disease is a disorder of the skin whose clinical expressivity is widely variable and diverse. It is mainly characterized by keratotic papules, oral mucosal alterations, nail distrophy and palmoplantar keratodermia, though other organs may be also affected. Darier's disease is inherited in an autosomal dominant pattern with a prevalence of about 1 in 50,000. The causative gene of Darier's disease was localized on the long arm of chromosome 12, bands q23-q24.1. We have clinically studied three families from our region. The nail distrophy and the palmoplantar keratodermia have been the most frequent signs found in our study. The cutaneous-mucosal affectation showed, in a large degree, an inter and intrafamilial variation. In addition, we have carried out a genetic linkage analysis with nine microsatellites of chromosome 12q23-q24.1 Our set of families gave significant lod scores with all these markers. Our recombinant analysis allowed us to establish a critical interval for the Darier's disease gene delimited, proximally by marker D12S78 and distally by marker D12S1646.

Spinal muscular atrophy is an autosomal recessive disorder which affects about 1 in 10 000 individuals. The three clinical forms of SMA were mapped to the 5q13 region. Three candidate genes have been isolated and shown to be deleted in SMA patients: the Survival Motor Neuron gene (SMN), the Neuronal Apoptosis Inhibitory Protein gene (NAIP) and the XS2G3 cDNA. In this report we present the molecular analysis of the SMN exons 7 and 8 and NAIP exon 5 in 65 Spanish SMA families. NAIP was mostly deleted in type I patients (67.9%) and SMN was deleted in 92.3% of patients with severe and milder forms. Most patients who lacked the NAIP gene also lacked the SMN gene, but we identified one type II patient deleted for NAIP exon 5 but not for SMN exons 7 and 8. Two other patients carried deletions of NAIP exon 5 and SMN exon 7 but retained the SMN exon 8. Three polymorphic variants from the SMN gene, showing changes on the sequence of the centromeric (cBCD541) and telomeric copies of the SMN gene, were found. In addition, we show several genetic rearrangements of the telomeric SMN gene, which include duplication of this gene in one normal chromosome, and putative gene conversion events in affected and normal chromosomes. Altogether these results corroborate the high genetic variability of the SMA region. Finally, we have determined the ratio between the number of centromeric and telomeric copies of the SMN gene in parents of SMA patients, showing that the majority of parents of types II and III patients carried three or more copies of the cBCD541 gene; we suggest a relationship between the number of copies of cBCD541 and the disease phenotype., Fondo de Investigación Sanitaria 93/0027–01

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.

PALB2 loss-of-function variants confer high risk of developing breast cancer. Here we present a systematic functional analysis of PALB2 splice-site variants detected in approximately 113,000 women in the large-scale sequencing project Breast Cancer After Diagnostic Gene Sequencing (BRIDGES; https://bridges-research.eu/). Eighty-two PALB2 variants at the intron-exon boundaries were analyzed with MaxEntScan. Forty-two variants were selected for the subsequent splicing functional assays. For this purpose, three splicing reporter minigenes comprising exons 1-12 were constructed. The 42 potential spliceogenic variants were introduced into the minigenes by site-directed mutagenesis and assayed in MCF-7/MDA-MB-231 cells. Splicing anomalies were observed in 35 variants, 23 of which showed no traces or minimal amounts of the expected full-length transcripts of each minigene. More than 30 different variant-induced transcripts were characterized, 23 of which were predicted to truncate the PALB2 protein. The pathogenicity of all variants was interpreted according to an in-house adaptation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) variant classification scheme. Up to 23 variants were classified as pathogenic/likely pathogenic. Remarkably, three ±1,2 variants (c.49-2A>T, c.108+2T>C, and c.211+1G>A) were classified as variants of unknown significance, as they produced significant amounts of either in-frame transcripts of unknown impact on the PALB2 protein function or the minigene full-length transcripts. In conclusion, we have significantly contributed to the ongoing effort of identifying spliceogenic variants in the clinically relevant PALB2 cancer susceptibility gene. Moreover, we suggest some approaches to classify the findings in accordance with the ACMG-AMP rationale. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.

PALB2 loss-of-function variants confer high risk of developing breast cancer. Here we present a systematic functional analysis of PALB2 splice-site variants detected in approximately 113,000 women in the large-scale sequencing project Breast Cancer After Diagnostic Gene Sequencing (BRIDGES; https://bridges-research.eu/). Eighty-two PALB2 variants at the intron-exon boundaries were analyzed with MaxEntScan. Forty-two variants were selected for the subsequent splicing functional assays. For this purpose, three splicing reporter minigenes comprising exons 1-12 were constructed. The 42 potential spliceogenic variants were introduced into the minigenes by site-directed mutagenesis and assayed in MCF-7/MDA-MB-231 cells. Splicing anomalies were observed in 35 variants, 23 of which showed no traces or minimal amounts of the expected full-length transcripts of each minigene. More than 30 different variant-induced transcripts were characterized, 23 of which were predicted to truncate the PALB2 protein. The pathogenicity of all variants was interpreted according to an in-house adaptation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) variant classification scheme. Up to 23 variants were classified as pathogenic/likely pathogenic. Remarkably, three ��1,2 variants (c.49-2A>T, c.108+2T>C, and c.211+1G>A) were classified as variants of unknown significance, as they produced significant amounts of either in-frame transcripts of unknown impact on the PALB2 protein function or the minigene full-length transcripts. In conclusion, we have significantly contributed to the ongoing effort of identifying spliceogenic variants in the clinically relevant PALB2 cancer susceptibility gene. Moreover, we suggest some approaches to classify the findings in accordance with the ACMG-AMP rationale. �� 2021 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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.

The c.156_157insAlu BRCA2 mutation has so far only been reported in hereditary breast/ovarian cancer (HBOC) families of Portuguese origin. Since this mutation is not detectable using the commonly used screening methodologies and must be specifically sought, we screened for this rearrangement in a total of 5,443 suspected HBOC families from several countries. Whereas the c.156_157insAlu BRCA2 mutation was detected in 11 of 149 suspected HBOC families from Portugal, representing 37.9% of all deleterious mutations, in other countries it was detected only in one proband living in France and in four individuals requesting predictive testing living in France and in the USA, all being Portuguese immigrants. After performing an extensive haplotype study in carrier families, we estimate that this founder mutation occurred 558 ± 215 years ago. We further demonstrate significant quantitative differences regarding the production of the BRCA2 full length RNA and the transcript lacking exon 3 in c.156_157insAlu BRCA2 mutation carriers and in controls. The cumulative incidence of breast cancer in carriers did not differ from that of other BRCA2 and BRCA1 pathogenic mutations. We recommend that all suspected HBOC families from Portugal or with Portuguese ancestry are specifically tested for this rearrangement.

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.

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 (3.4%) of the full-length (FL) transcript. In addition, ESE elements of the alternative exon 3 were mapped by testing four overlapping exonic microdeletions (≥30-bp), revealing an ESE-rich interval (c.202_235del) with critical sequences for exon 3 recognition that might have been affected by germline variants. Next, 26 BRIDGES variants and 16 artificial exon 3 single-nucleotide substitutions were also assayed. Thirty variants impaired splicing with variable amounts (0–65.1%) of the FL transcript, although only c.202G>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.

Splicing disruption is a common mechanism of gene inactivation associated with germline variants of susceptibility genes. To study the role of BRCA2 mis-splicing in hereditary breast/ovarian cancer (HBOC), we performed a comprehensive analysis of variants from BRCA2 exons 2-9, as well as the initial characterization of the regulatory mechanisms of such exons. A pSAD-based minigene with exons 2-9 was constructed and validated in MCF-7 cells, producing the expected transcript (1016-nt/V1-BRCA2_exons_2-9-V2). DNA variants from mutational databases were analyzed by NNSplice and Human Splicing Finder softwares. To refine ESE-variant prediction, we mapped the regulatory regions through a functional strategy whereby 26 exonic microdeletions were introduced into the minigene and tested in MCF-7 cells. Thus, we identified nine spliceogenic ESE-rich intervals where ESE-variants may be located. Combining bioinformatics and microdeletion assays, 83 variants were selected and genetically engineered in the minigene. Fifty-three changes impaired splicing: 28 variants disrupted the canonical sites, four created new ones, 10 abrogated enhancers, eight created silencers and three caused a double-effect. Notably, nine spliceogenic-ESE variants were located within ESE-containing intervals. Capillary electrophoresis and sequencing revealed more than 23 aberrant transcripts, where exon skipping was the most common event. Interestingly, variant c.67G>A triggered the usage of a noncanonical GC-donor 4-nt upstream. Thirty-six variants that induced severe anomalies (>60% aberrant transcripts) were analyzed according to the ACMG guidelines. Thus, 28 variants were classified as pathogenic, five as likely pathogenic and three as variants of uncertain significance. Interestingly, 13 VUS were reclassified as pathogenic or likely pathogenic variants. In conclusion, a large fraction of BRCA2 variants (∼64%) provoked splicing anomalies lending further support to the high prevalence of this disease-mechanism. The low accuracy of ESE-prediction algorithms may be circumvented by functional ESE-mapping that represents an optimal strategy to identify spliceogenic ESE-variants. Finally, systematic functional assays by minigenes depict a valuable tool for the initial characterization of splicing anomalies and the clinical interpretation of variants., EAV’s lab was supported by grants from the Spanish Ministry of Economy and Competitivity, Plan Nacional de I+D+I 2013-2016, ISCIII (PI13/01749 and PI17/00227) co-funded by FEDER from Regional Development European Funds (European Union), and grants CSI090U14 (ORDEN EDU/122/2014) and CSI242P18 (actuación cofinanciada P.O. FEDER 2014-2020 de Castilla y León) from the Consejería de Educación, Junta de Castilla y León. EFB is supported by a predoctoral fellowship from the University of Valladolid and Banco de Santander (2015–2019). AVP is supported by a predoctoral fellowship from Consejería de Educación, Junta de Castilla y León (2018–2022).

PALB2 loss-of-function variants confer high risk of developing breast cancer. Here, we present a systematic functional analysis of PALB2 splice-site variants detected in ~113,000 women of the large-scale sequencing project BRIDGES (Breast Cancer After Diagnostic Gene Sequencing; https://bridges-research.eu/). Eighty-two PALB2 variants at the intron-exon boundaries were analyzed with MaxEntScan. Forty-two variants were selected for the subsequent splicing functional assays. For this purpose, three splicing reporter minigenes comprising exons 1-12 were constructed. The 42 potential spliceogenic variants were introduced into the minigenes by site-directed mutagenesis and assayed in MCF-7/MDA-MB-231 cells. Splicing anomalies were observed in 35 variants, 23 of which showed no traces or minimal amounts of the expected full-length transcripts of each minigene. More than 30 different variant-induced transcripts were characterized, 23 of which were predicted to truncate the PALB2 protein. The pathogenicity of all variants was interpreted according to an in-house adaptation of the ACMG/AMP (American College of Medical Genetics and Genomics and the Association for Molecular Pathology) variant classification scheme. Up to 23 variants were classified as Pathogenic/Likely Pathogenic. Remarkably, three ±1,2 variants (c.49-2A>T, c.108+2T>C and c.211+1G>A) were classified as variants of unknown significance since they produced significant amounts of either in-frame transcripts of unknown impact on the PALB2 protein function or the minigene full-length transcripts. In conclusion, we have significantly contributed to the ongoing effort of identifying spliceogenic variants in the clinically relevant PALB2 cancer susceptibility gene. Moreover, we suggest some approaches to classify the findings in accordance with the ACMG/AMP rationale., PD, MPGV, DFE, MdlH, and EAV have received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 634935. The laboratory of EAV 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 Educacion, Junta de Castilla y Leon, ref. CSI242P18 (actuacion cofinanciada P.O. FEDER 2014-2020 de Castilla y Leon). The laboratory of MdlH is supported by a grant from the Spanish Ministry of Science and Innovation, Plan Nacional de I+D+I 2013-2016, ISCIII (PI20/00110) co-funded by FEDER from Regional Development European Funds (European Union). Programa Estratégico Instituto de Biología y Genética Molecular (IBGM), Escalera de Excelencia, Junta de Castilla y León (ref. CLU-2019-02). AV-P is supported by a predoctoral fellowship from the Consejería de Educación, Junta de Castilla y León (2018–2022). LS-M is supported by a predoctoral fellowship from the AECC-Scientific Foundation, Sede Provincial de Valladolid (2019–2023). AE-S is supported through the Operational Program for Youth Employment and Youth Employment Initiative (YEI), called by the Community of Madrid in 2020, and co-financed by the European Social Fund.