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This dataset corresponds to a comprehensive study of splice-site variants of the breast cancer susceptibility gene CHEK2. Variant data have been obtained from the large-scale sequencing project BRIDGES that sequenced 34 genes in 113,000 women. A set of 128 CHEK2 variants at the intron-exon boundaries were identified, 52 of which were predicted spliceogenic. These were introduced into the three minigene constructs by site-directed mutagenesis and tested in MCF-7 cells. Forty-six variants impaired splicing, 26 of which were classified as pathogenic/likely pathogenic variants according to ACMG/AMP-based guidelines, so carrier patients and families may benefit from tailored prevention protocols and personalized therapies.

This dataset corresponds to a comprehensive splicing analysis of splice-site variants of exons 1 to 3 of the breast cancer susceptibility gene PALB2. These variants were reported at the ClinVar database. Loss-of-function variants at the PALB2 gene are known to confer risk to breast and ovarian cancers. A total of 31 PALB2 variants at the intron/exon boundaries were analyzed with MaxEntScan. Sixteen variants were selected and genetically engineered into a PALB2 splicing reporter minigene. We found that 12 variants disrupted splicing and six of them could be classified as likely pathogenic. Hence, they are clinically actionable findings so variant-carriers may benefit from tailored prevention protocols and therapies.

This dataset corresponds to a systematic splicing analysis of splice-site variants of the breast cancer susceptibility gene ATM, which had been sequenced in 113,000 women who took part of the large-scale sequencing project BRIDGES. A set of 381 variants at the intron-exon boundaries were identified, 128 of which were predicted spliceogenic. After further filtering, we ended up selecting 56 variants for splicing analysis in four minigene constructs. Forty-eight variants impaired splicing, 29 of which were classified as pathogenic/likely pathogenic variants according to ACMG/AMP-based guidelines, so that carrier patients and families may benefit from tailored prevention protocols and personalized therapies.

[Introducción]: La disrupción del splicing del pre-ARNm es un mecanismo deletéreo frecuente en el cáncer hereditario. Nuestro objetivo en este estudio fue analizar funcionalmente mediante minigenes híbridos las variantes potencialmente espliceogénicas reportadas en el proyecto europeo BRIDGES (https://cordis.europa.eu/project/id/634935) en el gen de susceptibilidad al cáncer de mama CHEK2., [Material y Métodos]: Se identificaron un total de 128 variantes BRIDGES en las fronteras exón/intrón del gen CHEK2, de las cuales nos quedamos 52 tras el filtrado bioinformático con la herramienta MaxEntScan. Para el testado funcional de dichas variantes candidatas se construyeron 3 minigenes (mgChk2_ex1-7, mgChk2_ex6-10 y mgChk2_ex11-15), los cuales abarcaban los 15 exones de CHEK2. Finalmente, las 52 variantes seleccionadas se incorporaron en el minigén correspondiente mediante mutagénesis dirigida y se ensayaron funcionalmente en células MCF-7., [Resultados]: Cuarenta y seis variantes (88,5%) alteraban el splicing, 34 de las cuales indujeron graves anomalías de splicing sin dejar rastro o cantidades insignificantes del transcrito full-length del minigén. Un total de 89 transcritos diferentes fueron identificados, 82 de los cuales pudieron caracterizarse (59 PTC, 7 in-frame, 5 eliminaban el codón de inicio, 6 afectaban a la 5'UTR, 2 missense y 3 full-length). Curiosamente, el análisis de la variante c.684-2A>G reveló el uso de un sitio aceptor TG creado de novo 1 nucleótido upstream del canónico, cuyo reconocimiento depende de una serie de secuencias críticas ubicadas en el exón 6., [Conclusiones]: La incorporación de las lecturas generadas por los minigenes a un esquema de clasificación basado en las directrices ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) nos permitió clasificar 27 variantes de CHEK2 como patogénicas/probablemente patogénicas y 5 como probablemente benignas. Sin embargo, 20 variantes (38%) seguían siendo de significado clínico incierto, lo que refleja en parte los complejos patrones de splicing de este gen.

This dataset corresponds to a systematic splicing analysis of PALB2 splice-site variants that had been detected in over 113,000 women who took part of the large scale sequencing project BRIDGES. Eighty-two variants of the intron-exon boundaries were bioinformatically analyzed, and 42 of them were selected for subsequent splicing assays in three minigene constructs. Thirty-five variants impaired splicing, 23 of which were classified as pathogenic/likely pathogenic variants according to ACMG/AMP-based guidelines, so that carrier patients and families may benefit from tailored prevention protocols and personalized therapies.

RAD51C loss-of-function variants are associated with an increased risk of breast and ovarian cancers. Likewise, splicing disruptions are a frequent mechanism of gene inactivation. Taking advantage of a previous splicing-reporter minigene with exons 2-8 (mgR51C_ex2-8), we proceeded to check its impact on the splicing of candidate ClinVar variants. A total of 141 RAD51C variants at the intron/exon boundaries were analyzed with MaxEntScan. Twenty variants were selected and genetically engineered into the wild-type minigene. All the variants disrupted splicing, and 18 induced major splicing anomalies without any trace or minimal amounts (<2.4%) of the minigene full-length (FL) transcript. Twenty-seven transcripts (including the wild-type and r.904A FL transcripts) were identified by fluorescent fragment electrophoresis; of these, 14 were predicted to truncate the RAD51C protein, 3 kept the reading frame, and 8 minor isoforms (1.1-4.7% of the overall expression) could not be characterized. Finally, we performed a tentative interpretation of the variants according to an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme, classifying 16 variants as likely pathogenic. Minigene assays have been proven as valuable tools for the initial characterization of potential spliceogenic variants. Hence, minigene mgR51C_ex2-8 provided useful splicing data for 40 RAD51C variants.

The ataxia telangiectasia-mutated (ATM) protein is a major coordinator of the DNA damage response pathway. ATM loss-of-function variants are associated with 2-fold increased breast cancer risk. We aimed at identifying and classifying spliceogenic ATM variants detected in subjects of the large-scale sequencing project BRIDGES. A total of 381 variants at the intron-exon boundaries were identified, 128 of which were predicted to be spliceogenic. After further filtering, we ended up selecting 56 variants for splicing analysis. Four functional minigenes (mgATM) spanning exons 4-9, 11-17, 25-29, and 49-52 were constructed in the splicing plasmid pSAD. Selected variants were genetically engineered into the four constructs and assayed in MCF-7/HeLa cells. Forty-eight variants (85.7%) impaired splicing, 32 of which did not show any trace of the full-length (FL) transcript. A total of 43 transcripts were identified where the most prevalent event was exon/multi-exon skipping. Twenty-seven transcripts were predicted to truncate the ATM protein. A tentative ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme that integrates mgATM data allowed us to classify 29 ATM variants as pathogenic/likely pathogenic and seven variants as likely benign. Interestingly, the likely pathogenic variant c.1898+2T>G generated 13% of the minigene FL-transcript due to the use of a noncanonical GG-5'-splice-site (0.014% of human donor sites). Circumstantial evidence in three ATM variants (leakiness uncovered by our mgATM analysis together with clinical data) provides some support for a dosage-sensitive expression model in which variants producing ¿30% of FL-transcripts would be predicted benign, while variants producing ¿13% of FL-transcripts might be pathogenic.

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.

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.

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.

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-m

[Background] PALB2 monoallelic loss-of-function germ-line variants confer a breast cancer risk comparable to the average BRCA2 pathogenic variant. Recommendations for risk reduction strategies in carriers are similar. Elaborating robust criteria to identify loss-of-function variants in PALB2—without incurring overprediction—is thus of paramount clinical relevance. Towards this aim, we have performed a comprehensive characterisation of alternative splicing in PALB2, analysing its relevance for the classification of truncating and splice site variants according to the 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines., [Methods] Alternative splicing was characterised in RNAs extracted from blood, breast and fimbriae/ovary-related human specimens (n=112). RNAseq, RT-PCR/CE and CloneSeq experiments were performed by five contributing laboratories. Centralised revision/curation was performed to assure high-quality annotations. Additional splicing analyses were performed in PALB2 c.212–1G>A, c.1684+1G>A, c.2748+2T>G, c.3113+5G>A, c.3350+1G>A, c.3350+4A>C and c.3350+5G>A carriers. The impact of the findings on PVS1 status was evaluated for truncating and splice site variant., [Results] We identified 88 naturally occurring alternative splicing events (81 newly described), including 4 in-frame events predicted relevant to evaluate PVS1 status of splice site variants. We did not identify tissue-specific alternate gene transcripts in breast or ovarian-related samples, supporting the clinical relevance of blood-based splicing studies., [Conclusions] PVS1 is not necessarily warranted for splice site variants targeting four PALB2 acceptor sites (exons 2, 5, 7 and 10). As a result, rare variants at these splice sites cannot be assumed pathogenic/likely pathogenic without further evidences. Our study puts a warning in up to five PALB2 genetic variants that are currently reported as pathogenic/likely pathogenic in ClinVar.

The clinical classification of BRCA2 variants of uncertain significance (VUS) poses a challenge in human genetics. Historically, variants have been catalogued from the protein outlook, however, upstream gene-expression mechanisms, such as splicing, could be impaired by changes in the DNA sequence. Disrupted splicing variants could alter the ORF provoking the BRCA2 truncation and be involved in cancer development. Here, we have evaluated the splicing effect of 83 BRCA2 exons 2-9 variants by functional assays using the minigene MGBR2_2-9. The MGBR2_2-9 was built based on the splicing vector pSAD (Patent-P201231427, CSIC). In silico selected BRCA2 variants, from BIC and UMD databases, were introduced into MGBR2_2-9 and assayed in MCF-7 cells. Transcripts were analyzed by capillary electrophoresis and sequenced. After the bioinformatic analysis of 302 BRCA2 variants, 83 were functionally tested. Results showed that 53 variants impaired splicing (26 intronic and 27 exonic). Among them, 36 provoked ¿2/3 of aberrant transcripts. According to the ACMG and ENIGMA criteria, our results support the re-classification of 12 variants from VUS to pathogenic (c.67+1G>T, c.67+3A>G, c.426-12del5, c.441A>G, c.451G>A, c.475+3A>T, c.516+4delAA, c.517-1G>A, c.517G>T, c.631+1G>A, c.632+3A>G and c.632-3G>C) and 3 from VUS to likely pathogenic (c.97G>A, c.100G>A, c.476-3C>A). The reproducibility of this technique was supported by previous studies based on minigenes or/and patient RNA. MGBR2_2-9 is a robust tool which provides RNA data for the clinical interpretation of splicing variants.

Mutations in CHD7 have been shown to be a major cause of CHARGE syndrome, which presents many symptoms and features common to other syndromes making its diagnosis difficult. Next generation sequencing (NGS) of a panel of intellectual disability related genes was performed in an adult patient without molecular diagnosis. A splice donor variant in CHD7 (c.5665 + 1G > T) was identified. To study its potential pathogenicity, exons and flanking intronic sequences were amplified from patient DNA and cloned into the pSAD® splicing vector. HeLa cells were transfected with this construct and a wild-type minigene and functional analysis were performed. The construct with the c.5665 + 1G > T variant produced an aberrant transcript with an insert of 63 nucleotides of intron 28 creating a premature termination codon (TAG) 25 nucleotides downstream. This would lead to the insertion of 8 new amino acids and therefore a truncated 1896 amino acid protein. As a result of this, the patient was diagnosed with CHARGE syndrome. Functional analyses underline their usefulness for studying the pathogenicity of variants found by NGS and therefore its application to accurately diagnose patients.

[Purpose]: Promoter mutations may affect transcription and can be associated with human diseases. However, the promoters of the breast cancer (BC) genes are not regularly screened. Our goal was to investigate the BRCA2 promoter in order to study a possible correlation between impaired transcription and disease. [Methods]: The proximal and core promoter of the BRCA2 gene was sequenced in 95 high-risk BC patients. A BRCA2-promoter insert [- 938 to + 312 from the transcription start site (TSS)] was generated and cloned into the firefly luciferase vector pGL4.10. Promoter variants and deletions were introduced by site-directed mutagenesis and quantified by Dual-Luciferase assays and semi-quantitative RT-PCR. [Results]: Three different variants were detected in high-risk BC patients: rs3092989, rs206118, and rs563971900. Functional mapping of 13 overlapping deletions revealed four down-regulating segments (TSS positions): -59_-10del/µdel3 (16% of activity of the wild-type construct), -104_-55del/µdel4 (62%), -239_-190del/µdel7 (39%), -464_-415/µdel12 (78%), suggesting the presence therein of putative transcriptional activator motifs. Additionally, six microdeletions rendered luciferase overexpression: +32_+81del/µdel1 (356%), -14_+36del/µdel2 (180%), -194_-145del/µdel6 (154%), -284_-235del/µdel8 (168%), -329_-280del/µdel9 (111%), and -509_-460del/µdel13 (139%), which is indicative of repressor elements. Functional assays of 15 promoter variants (including those detected in patients) showed that ten of them significantly altered expression with seven up-regulating (113-163%) and three down-regulating (rs551887850_G, rs570548398_T, rs55880202_T; 72-83%) SNPs. Eight of them were located in an ENCODE-DNase Hypersensitive Cluster (TSS - 185 to + 105) where most active transcriptional motifs are known to be placed. [Conclusions]: BRCA2 expression is highly sensitive to promoter variations as most of them induced relevant changes. Moreover, we mapped critical regions of the BRC

Mutation screening of BRCA2 identifies a large fraction of variants of uncertain clinical significance (VUS). Our goal was to investigate the impact of reported variants of BRCA2 exons 17 and 18 on splicing to assess their role in hereditary breast cancer and to identify critical regulatory elements that may constitute hotspots for spliceogenic variants. A splicing reporter minigene with BRCA2 exons 14 to-20 (MGBR2_ex14-20) was constructed in the pSAD vector. Fifty-two candidate variants were selected with splicing prediction programs, introduced in MGBR2_ex14-20 by site-directed mutagenesis and assayed in MCF-7 cells. Functional mapping by microdeletions revealed essential sequences for exon recognition on the 3' end of exon 17 and the 5' end of exon 18. Thirty out of the 52 selected variants induced anomalous splicing in minigene assays with >16 different aberrant transcripts, where exon skipping was the most common event. According to the biological indicators of pathogenicity, 18 variants could be classified as disease-causing, accounting for 28.6% of all pathogenic variants of exons 17-18 at the BRCA Share database. Aberrant splicing is especially prevalent in BRCA2 exons 17 and 18 due to the presence of active ESEs, which are recognized by splicing factor as SC35 and Tra2ß that play an important role in exon recognition. Splicing functional assays with minigenes are a valuable strategy for the interpretation of VUS of any disease-gene.

Las moléculas sensoras del inflamasoma pueden detectar una amplia gama de señales moleculares para responder a diferentes microorganismos o al estrés del tejido, desencadenando una repuesta inflamatoria a través de la activación de las caspasas inflamatorias. Estudios previos del laboratorio han demostrado que la sPLA2- IIA aumenta la proliferación y la capacidad fagocítica de la microglía (ref de nuestro paper). En estos estudios se observo que las acciones de la sPLA2-IIA se producen a través de la transactivacion del EGFR que da lugar a la fosforilacíon de proteínas como ER, p70S6K y rS6 Asimismo, en células de microglía, el LPS también es capaz de inducir la transactivación del EGFR (resultados sometidos a publicación) y este mecanismo esta implicado en la modulación de la fagocitosis inducida por el LPS. En base a estos estudios, en esta memoria se ha continuado con la caracterización de la señalización inducida por estos estimulos proinflamatorios en las celulas de microglía BV-2. La microglía constituye la principal fuente de mediadores inflamatorios en el sistema nervioso central, actuando además como diana de alguno de ellos. Estas células contribuyen tanto a la neurodegeneración como a la neuroprotección, por lo tanto es fundamental identificar los mecanismos de activación del inflamasoma NLRP3 y caracterizar las vías de señalización implicadas en la regulación de este. La expresión del inflamasoma NLRP3 y del factor de transcripción nuclear (NF-kB) inducida por sPLA2-IIA es independiente de la transactivacion del EGFR Sin embargo sPLA2-IIA utiliza la via de señalización de la PKc para inducir la producción de NLRP3 y del factor de transcripción nuclear (NF-kB) (figura 6). Por otro lado, el LPS favorece la expresión de NLRP3, y del factor de transcripción nuclear (NF-kB) independientemente de la transactivación del EGFR siendo sensible a inhibidores de la ruta de la PKc ( Además, hemos comprobado que es indispensable la producción de ROS para inducir la ex, Máster en Investigación Biomédica

Las moléculas sensoras del inflamasoma pueden detectar una amplia gama de señales moleculares para responder a diferentes microorganismos o al estrés del tejido, desencadenando una repuesta inflamatoria a través de la activación de las caspasas inflamatorias. Estudios previos del laboratorio han demostrado que la sPLA2- IIA aumenta la proliferación y la capacidad fagocítica de la microglía (ref de nuestro paper). En estos estudios se observo que las acciones de la sPLA2-IIA se producen a través de la transactivacion del EGFR que da lugar a la fosforilacíon de proteínas como ER, p70S6K y rS6. Asimismo, en células de microglía, el LPS también es capaz de inducir la transactivación del EGFR (resultados sometidos a publicación) y este mecanismo esta implicado en la modulación de la fagocitosis inducida por el LPS. En base a estos estudios, en esta memoria se ha continuado con la caracterización de la señalización inducida por estos estimulos proinflamatorios en las celulas de microglía BV-2. La microglía constituye la principal fuente de mediadores inflamatorios en el sistema nervioso central, actuando además como diana de alguno de ellos. Estas células contribuyen tanto a la neurodegeneración como a la neuroprotección, por lo tanto es fundamental identificar los mecanismos de activación del inflamasoma NLRP3 y caracterizar las vías de señalización implicadas en la regulación de este. La expresión del inflamasoma NLRP3 y del factor de transcripción nuclear (NF-kB) inducida por sPLA2-IIA es independiente de la transactivacion del EGFR. Sin embargo sPLA2-IIA utiliza la via de señalización de la PKc para inducir la producción de NLRP3 y del factor de transcripción nuclear (NF-kB) (figura 6). Por otro lado, el LPS favorece la expresión de NLRP3, y del factor de transcripción nuclear (NF-kB) independientemente de la transactivación del EGFR siendo sensible a inhibidores de la ruta de la PKc. Además, hemos comprobado que es indispensable la producción de ROS para inducir la e