Neurogénesis en el hipocampo: caracterización de un nicho periventricular de células madres neuronales

El presente Trabajo de Fin de Máster aborda un tema de investigación de importancia y actualidad como es el estudio de la neurogénesis en el cerebro adulto. En la introducción empezaremos definiendo la neurogénesis en el cerebro adulto, seguiremos con un recorrido desde las primeras etapas del desarrollo hasta las células madre adultas y finalizaremos con la definición de nicho y su repercusión en este proceso. De esta forma, trataremos de fundamentar la hipótesis y los objetivos a desarrollar en este trabajo.Departamento de Medicina, Dermatología y ToxicologíaMáster en Investigación Biomédic

Functional analyses of a novel splice variant in the CHD7 gene, found by next generation sequencing, Confirm Its pathogenicity in a Spanish patient and diagnose him with CHARGE syndrome

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.This work was funded by Jesús de Gangoiti Barrera Foundation (FJGB15/005). The EAV laboratory is funded by projects of the Spanish Ministry of Economy and Competitiveness, National Plan for R & D 2013–2016, ISCIII (FIS: PI13/01749) co-financed by FEDER from Regional Development European Funds (European Union) and the project CSI090U14 of the Regional ministry of Education (ORDER EDU/122/2014) (Castilla y León, Spain). This study made use of data generated by the UK10K Project. Funding for the UK10K Project was provided by the Wellcome Trust under award WT091310.Peer reviewe

Differential regulation of microRNA-15a by radiation affects angiogenesis and tumor growth via modulation of acid sphingomyelinase

Producción CientíficaActivation of acid sphingomyelinase (SMPD1) and the generation of ceramide is a critical regulator of apoptosis in response to cellular stress including radiation. Endothelial SMPD1 has been shown to regulate tumor responses to radiation therapy. We show here that the SMPD1 gene is regulated by a microRNA (miR), miR-15a, in endothelial cells (ECs). Standard low dose radiation (2 Gy) upregulates miR-15a and decreases SMPD1 levels. In contrast, high dose radiation (10 Gy and above) decreases miR-15a and increases SMPD1. Ectopic expression of miR-15a decreases both mRNA and protein levels of SMPD1. Mimicking the effects of high dose radiation with a miR-15a inhibitor decreases cell proliferation and increases active Caspase-3 & 7. Mechanistically, inhibition of miR-15a increases inflammatory cytokines, activates caspase-1 inflammasome and increases Gasdermin D, an effector of pyroptosis. Importantly, both systemic and vascular-targeted delivery of miR-15a inhibitor decreases angiogenesis and tumor growth in a CT26 murine colorectal carcinoma model. Taken together, our findings highlight a novel role for miR mediated regulation of SMPD1 during radiation responses and establish proof-of-concept that this pathway can be targeted with a miR inhibitor.This work was supported by US NIH (grant R01HL137779 and R01HL143803) to S.AASTRO (Grant ID 534775

Functional classification of DNA variants by hybrid minigenes: identification of 30 spliceogenic variants of BRCA2 exons 17 and 18

Mutation screening of the breast cancer genes BRCA1 and BRCA2 identifies a large fraction of variants of uncertain clinical significance (VUS) whose functional and clinical interpretations pose a challenge for genomic medicine. Likewise, an increasing amount of evidence indicates that genetic variants can have deleterious effects on pre-mRNA splicing. Our goal was to investigate the impact on splicing of a set of reported variants of BRCA2 exons 17 and 18 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. Fiftytwo candidate variants were selected with splicing prediction programs, introduced in MGBR2_ ex14-20 by site-directed mutagenesis and assayed in triplicate in MCF-7 cells. Wild type MGBR2_ ex14-20 produced a stable transcript of the expected size (1,802 nucleotides) and structure (V1-[BRCA2_ exons_ 14-20]-V2). Functional mapping by microdeletions revealed essential sequences for exon recognition on the 3 ' end of exon 17 (c. 79447973) and the 5 ' end of exon 18 (c. 7979-7988, c. 7999-8013). 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. A wide range of splicing motifs were affected including the canonical splice sites (15 variants), novel alternative sites (3 variants), the polypyrimidine tract (3 variants) and enhancers/silencers (9 variants). According to the guidelines of the American College of Medical Genetics and Genomics (ACMG), 20 variants could be classified as pathogenic (c. 7806-2A > G, c. 7806-1G > A, c. 7806-1G > T, c. 7806-1_ 7806-2dup, c. 7976+ 1G > A, c. 7977-3_ 7978del, c. 7977-2A > T, c. 7977-1G > T, c. 7977-1G > C, c. 8009C > A, c. 8331+ 1G > T and c. 8331+ 2T > C) or likely pathogenic (c. 78069T > G, c. 7976G > C, c. 7976G > A, c. 7977-7C > G, c. 7985C > G, c. 8023A > G, c. 8035G > T and c. 8331G > A), accounting for 30.8% of all pathogenic/likely pathogenic variants of exons 17-18 at the BRCA Share database. The remaining 8 variants (c. 7975A > G, c. 7977-6T > G, c. 7988A > T, c. 7992T > A, c. 8007A > G, c. 8009C > T, c. 8009C > G, and c. 8072C > T) induced partial splicing anomalies with important ratios of the full-length transcript (>= 70%), so that they remained classified as VUS. Aberrant splicing is therefore especially prevalent in BRCA2 exons 17 and 18 due to the presence of active ESEs involved in exon recognition. Splicing functional assays with minigenes are a valuable strategy for the initial characterization of the splicing outcomes and the subsequent clinical interpretation of variants of any disease-gene, although these results should be checked, whenever possible, against patient RNA

Minigene Splicing Assays Identify 12 Spliceogenic Variants of BRCA2 Exons 14 and 15

A relevant fraction of BRCA2 variants is associated with splicing alterations and with an increased risk of hereditary breast and ovarian cancer (HBOC). In this work, we have carried out a thorough study of variants from BRCA2 exons 14 and 15 reported at mutation databases. A total of 294 variants from exons 14 and 15 and flanking intronic sequences were analyzed with the online splicing tools NNSplice and Human Splicing Finder. Fifty-three out of these 294 variants were selected as candidate splicing variants. All variants but one, were introduced into the minigene MGBR2_ex14-20 (with exons 14–20) by site-directed mutagenesis and assayed in MCF-7 cells. Twelve of the remaining 52 variants (23.1%) impaired splicing at different degrees, yielding from 5 to 100% of aberrant transcripts. Nine variants affected the natural acceptor or donor sites of both exons and three affected putative enhancers or silencers. Fluorescent capillary electrophoresis revealed at least 10 different anomalous transcripts: (E14q5), Δ (E14p10), Δ(E14p246), Δ(E14q256), Δ(E14), Δ(E15p12), Δ(E15p13), Δ(E15p83), Δ(E15) and a 942-nt fragment of unknown structure. All transcripts, except for Δ(E14q256) and Δ(E15p12), are expected to truncate the BRCA2 protein. Nine variants induced severe splicing aberrations with more than 90% of abnormal transcripts. Thus, according to the guidelines of the American College of Medical Genetics and Genomics, eight variants should be classified as pathogenic (c.7008-2A > T, c.7008-1G > A, c.7435+1G > C, c.7436-2A > T, c.7436-2A > G, c.7617+1G > A, c.7617+1G > T, and c.7617+2T > G), one as likely pathogenic (c.7008-3C > G) and three remain as variants of uncertain clinical significance or VUS (c.7177A > G, c.7447A > G and c.7501C > T). In conclusion, functional assays by minigenes constitute a valuable strategy to primarily check the splicing impact of DNA variants and their clinical interpretation. While bioinformatics predictions of splice site variants were accurate, those of enhancer or silencer variants were poor (only 3/23 spliceogenic variants) which showed weak impacts on splicing (∼5–16% of aberrant isoforms). So, the Exonic Splicing Enhancer and Silencer (ESE and ESS, respectively) prediction algorithms require further improvement

Comprehensive Functional Characterization and Clinical Interpretation of 20 Splice-Site Variants of the RAD51C Gene

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 predictions, minigene analyses, and ACMG-AMP clinical classification of 42 germline PALB2 splice-site variants.

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

Transcripción y splicing de brca2 y su relación con la susceptibilidad a cáncer de mama y ovario hereditario

Programa de Doctorando en Investigación Biomédica.[Introducción]: El Cáncer de Mama y Ovario Hereditario (CMOH) es una enfermedad con herencia autosómica dominante caracterizada por la aparición temprana del tumor. Actualmente, aunque se han asociado 26 genes a la aparición de la enfermedad, sólo dos de ellos están considerados como genes principales de cáncer de mama: BRCA1 y BRCA2. El rastreo de los genes BRCA en pacientes de alto riesgo ha dado lugar a la aparición de miles de variantes, muchas de ellas clasificadas como variantes de significado clínico desconocido (VUS). Éstas suelen ser variantes missense, sinónimas, deleciones o inserciones in-frame y variantes en regiones no codificantes como los intrones o las UTRs (Untranslated Region). En este contexto, los ensayos funcionales juegan un papel esencial para conocer el impacto de este tipo de variantes y su asociación con la enfermedad. El objetivo de esta tesis doctoral se centra en el estudio de la transcripción y el splicing de BRCA2. Se pretende estudiar los mecanismos reguladores de la expresión génica y evaluar las variantes que puedan alterar dichos procesos y estar asociadas a la susceptibilidad a CMOH.[Métodos]: Las variantes, recogidas de las bases de las principales bases de datos (BIC, UMD, ClinVar y Ensembl) y encontradas en pacientes del sistema de prevención de cáncer de Castilla y León (España), fueron analizadas in silico. Se seleccionaron aquellas variantes que potencialmente alteraban los mecanismos de transcripción o de splicing. El estudio de la transcripción se realizó a través de un vector reportero con el gen de luciferasa bajo el promotor de BRCA2. Los resultados fueron analizados mediante Dual-Luciferase Assays y RT-PCR semi-cuantitativa. Para estudiar el splicing, se construyeron dos minigenes (MGBR2_2-9 y MGBR2_14-20) basados en el vector pSAD (Patente P201231427,CSIC). Las variantes fueron introducidas mediante mutagénesis dirigida y ensayadas en células MCF-7. El estudio de regiones reguladoras se realizó a través de microdeleciones. Los factores implicados en el splicing se estudiaron mediante ensayos de interacción RNA-proteína (pull-down) y ensayos de pérdida de función (siRNA). Los transcritos generados fueron analizados mediante gel de agarosa, secuenciación SANGER y electroforesis capilar.[Resultados]: Las secuencias reguladoras de la transcripción se estudiaron a través de 13 microdeleciones a lo largo del promotor de BRCA2. Los resultados mostraron que seis disminuían significativamente la transcripción y tres la aumentaban. Una vez mapeado el promotor, se ensayaron 15 variantes que podrían alterar elementos reguladores, tres de ellas encontradas en pacientes del sistema de prevención de Cáncer de Castilla y León (rs3092989_A, rs206118_G y rs563971900_T). Diez variantes alteraron significativamente la expresión génica, de las cuales, tres provocaron la subexpresión del gen (rs551887850_G, rs570548398_T y rs55880202_T). Cabe destacar que ocho de las 10 variantes reguladoras mapeaban en el cluster 121 de hipersensibilidad a DNAsa, una región crítica para el inicio de la transcripción. Por otro lado, el estudio del splicing reveló que exones 3 a 8, 15, 17 y 18 de BRCA2 requieren de elementos reguladores de splicing (SREs) para su correcto reconocimiento. Además, los resultados indican que los factores SRSF3 y SRSF2 juegan un papel importante en el splicing de los exones 3 y 18, respectivamente. En total, se analizaron mediante minigenes 200 variantes repartidas a lo largo de los exones 2-9 y 14-18 de BRCA2. La mayoría de las variantes produjeron alteraciones en el splicing (104), entre ellas 49 eran exónicas (missense, sinónimas, nonsense y frameshift). Aproximadamente el 70% de las variantes espliceogénicas (73/104) provocaron graves alteraciones (>60% de transcritos aberrantes). En total, 74 variantes fueron clasificadas como patogénicas o probablemente patogénicas según los criterios propuestos por ACMG (American College of Medical Genetic and Genomics), 30 de las cuales se encontraban previamente catalogadas como VUS.[Conclusiones]: La transcripción y el splicing son mecanismos fundamentales en la expresión génica que pueden verse alterados debido a cambios en la secuencia de DNA. El promotor de BRCA2 es sensible a cambios de nucleótido y su rastreo debería incluirse en los estudios genéticos de pacientes de CMOH. Por otro lado, cualquier variación en la secuencia podría alterar el splicing. En este contexto, los estudios con minigenes proporcionan datos fiables para evaluar el impacto de las variantes cuando no se dispone de RNA de pacientes.Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (proyecto ID PI13/01749; 2014-2016). Consejería de Educación de la Junta de Castilla y León (proyecto ID CSI90U14). Beca predoctoral de la Universidad de Valladolid cofinanciada por el Banco Santander (2015-2019), la beca EMBO Short-Fellowship (ASTF 517-2016), la beca ERASMUS+ y la ayuda para estancias brteves en el desarrollo de tesis doctorales de la Universidad de Valladolid 2018.Peer reviewe

Transcripción y Splicing de BRCA2 y su relación con la susceptibilidad a cáncer de mama y ovario hereditario

El Cáncer de Mama y Ovario Hereditario (CMOH) es una enfermedad con herencia autosómica dominante caracterizada por la aparición temprana del tumor. Actualmente, aunque se han asociado 26 genes a la aparición de la enfermedad, sólo dos de ellos están considerados como genes principales de cáncer de mama: BRCA1 y BRCA2. El rastreo de los genes BRCA en pacientes de alto riesgo ha dado lugar a la aparición de miles de variantes, muchas de ellas clasificadas como variantes de significado clínico desconocido (VUS). Éstas suelen ser variantes missense, sinónimas, deleciones o inserciones in-frame y variantes en regiones no codificantes como los intrones o las UTRs (Untranslated Region). En este contexto, los ensayos funcionales juegan un papel esencial para conocer el impacto de este tipo de variantes y su asociación con la enfermedad. El objetivo de esta tesis doctoral se centra en el estudio de la transcripción y el splicing de BRCA2. Se pretende estudiar los mecanismos reguladores de la expresión génica y evaluar las variantes que puedan alterar dichos procesos y estar asociadas a la susceptibilidad a CMOH.Departamento de Bioquímica y Biología Molecular y FisiologíaDoctorado en Investigación Biomédic

Nucleic Acid Sensing in the Tumor Vasculature

Endothelial cells form a powerful interface between tissues and immune cells. In fact, one of the underappreciated roles of endothelial cells is to orchestrate immune attention to specific sites. Tumor endothelial cells have a unique ability to dampen immune responses and thereby maintain an immunosuppressive microenvironment. Recent approaches to trigger immune responses in cancers have focused on activating nucleic acid sensors, such as cGAS-STING, in combination with immunotherapies. In this review, we present a case for targeting nucleic acid-sensing pathways within the tumor vasculature to invigorate tumor-immune responses. We introduce two specific nucleic acid sensors—the DNA sensor TREX1 and the RNA sensor RIG-I—and discuss their functional roles in the vasculature. Finally, we present perspectives on how these nucleic acid sensors in the tumor endothelium can be targeted in an antiangiogenic and immune activation context. We believe understanding the role of nucleic acid-sensing in the tumor vasculature can enhance our ability to design more effective therapies targeting the tumor microenvironment by co-opting both vascular and immune cell types