RESUMEN
The poor correlation of mutational landscapes with phenotypes limits our understanding of the pathogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here we show that oncogenic dosage-variation has a critical role in PDAC biology and phenotypic diversification. We find an increase in gene dosage of mutant KRAS in human PDAC precursors, which drives both early tumorigenesis and metastasis and thus rationalizes early PDAC dissemination. To overcome the limitations posed to gene dosage studies by the stromal richness of PDAC, we have developed large cell culture resources of metastatic mouse PDAC. Integration of cell culture genomes, transcriptomes and tumour phenotypes with functional studies and human data reveals additional widespread effects of oncogenic dosage variation on cell morphology and plasticity, histopathology and clinical outcome, with the highest KrasMUT levels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous KrasMUT in driving tumorigenesis, but have lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumour suppressor alterations (Cdkn2a, Trp53, Tgfß-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive the early progression of PDAC and shape its downstream biology. Our study uncovers universal principles of Ras-driven oncogenesis that have potential relevance beyond pancreatic cancer.
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Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Evolución Molecular , Dosificación de Gen , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Alelos , Animales , Carcinogénesis/genética , Proteínas de Ciclo Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Progresión de la Enfermedad , Femenino , Genes myc , Genes p53 , Humanos , Masculino , Ratones , Mutación , Subunidad p52 de NF-kappa B/genética , Metástasis de la Neoplasia/genética , Proteínas Nucleares/genética , Fenotipo , Fosfoproteínas/genética , Factores de Transcripción/genética , Transcriptoma/genética , Factor de Crecimiento Transformador beta1/genética , Proteínas Señalizadoras YAPRESUMEN
BACKGROUND: Liquid biopsy and Integrative Genomic Profiling (IGP) are yet to be implemented into routine Radiation Oncology. Here we assess the utility of germline, tumour and circulating cell-free DNA-based genomic analyses for the clinical management of early-stage and oligometastatic cancer patients treated by precision radiotherapy. METHODS: We performed germline, tissue- and liquid biopsy NGS panels on 50 early-stage/oligometastatic cancer patients undergoing radiotherapy. We also monitored ctDNA variants in serial liquid biopsies collected during radiotherapy and follow-up and evaluated the clinical utility of such comprehensive approach. RESULTS: The integration of different genomic studies revealed that only 1/3 of the liquid biopsy variants are of tumour origin. Altogether, 55 tumour variants (affecting 3/4 of the patients) were considered potentially actionable (for treatment and prognosis), whereas potential follow-up biomarkers were identified in all cases. Germline cancer-predisposing variants were present in three patients, which would have not been eligible for hereditary cancer testing according to clinical guidelines. The presence of detectable ctDNA variants before radiotherapy was associated with progression-free survival both in oligometastatic patients and in those with early-stage. CONCLUSIONS: IGP provides both valuable and actionable information for personalised decision-making in Radiation Oncology.
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ADN Tumoral Circulante , Neoplasias , Oncología por Radiación , Humanos , ADN Tumoral Circulante/genética , Biomarcadores de Tumor/genética , Biopsia Líquida , Genómica , MutaciónRESUMEN
Here, we show CRISPR/Cas9-based targeted somatic multiplex-mutagenesis and its application for high-throughput analysis of gene function in mice. Using hepatic single guide RNA (sgRNA) delivery, we targeted large gene sets to induce hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). We observed Darwinian selection of target genes, which suppress tumorigenesis in the respective cellular/tissue context, such as Pten or Cdkn2a, and conversely found low frequency of Brca1/2 alterations, explaining mutational spectra in human ICC/HCC. Our studies show that multiplexed CRISPR/Cas9 can be used for recessive genetic screening or high-throughput cancer gene validation in mice. The analysis of CRISPR/Cas9-induced tumors provided support for a major role of chromatin modifiers in hepatobiliary tumorigenesis, including that of ARID family proteins, which have recently been reported to be mutated in ICC/HCC. We have also comprehensively characterized the frequency and size of chromosomal alterations induced by combinatorial sgRNA delivery and describe related limitations of CRISPR/Cas9 multiplexing, as well as opportunities for chromosome engineering in the context of hepatobiliary tumorigenesis. Our study describes novel approaches to model and study cancer in a high-throughput multiplexed format that will facilitate the functional annotation of cancer genomes.
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Sistemas CRISPR-Cas/genética , Carcinoma Hepatocelular/genética , Modelos Animales de Enfermedad , Genómica/métodos , Ensayos Analíticos de Alto Rendimiento , Neoplasias Hepáticas/genética , Mutagénesis/genética , Animales , Secuencia de Bases , Marcación de Gen , Técnicas Histológicas , Hígado/metabolismo , Ratones , Datos de Secuencia Molecular , Selección Genética/genéticaRESUMEN
An ageing world population has fuelled interest in regenerative remedies that may stem declining organ function and maintain fitness. Unanswered is whether elimination of intrinsic instigators driving age-associated degeneration can reverse, as opposed to simply arrest, various afflictions of the aged. Such instigators include progressively damaged genomes. Telomerase-deficient mice have served as a model system to study the adverse cellular and organismal consequences of wide-spread endogenous DNA damage signalling activation in vivo. Telomere loss and uncapping provokes progressive tissue atrophy, stem cell depletion, organ system failure and impaired tissue injury responses. Here, we sought to determine whether entrenched multi-system degeneration in adult mice with severe telomere dysfunction can be halted or possibly reversed by reactivation of endogenous telomerase activity. To this end, we engineered a knock-in allele encoding a 4-hydroxytamoxifen (4-OHT)-inducible telomerase reverse transcriptase-oestrogen receptor (TERT-ER) under transcriptional control of the endogenous TERT promoter. Homozygous TERT-ER mice have short dysfunctional telomeres and sustain increased DNA damage signalling and classical degenerative phenotypes upon successive generational matings and advancing age. Telomerase reactivation in such late generation TERT-ER mice extends telomeres, reduces DNA damage signalling and associated cellular checkpoint responses, allows resumption of proliferation in quiescent cultures, and eliminates degenerative phenotypes across multiple organs including testes, spleens and intestines. Notably, somatic telomerase reactivation reversed neurodegeneration with restoration of proliferating Sox2(+) neural progenitors, Dcx(+) newborn neurons, and Olig2(+) oligodendrocyte populations. Consistent with the integral role of subventricular zone neural progenitors in generation and maintenance of olfactory bulb interneurons, this wave of telomerase-dependent neurogenesis resulted in alleviation of hyposmia and recovery of innate olfactory avoidance responses. Accumulating evidence implicating telomere damage as a driver of age-associated organ decline and disease risk and the marked reversal of systemic degenerative phenotypes in adult mice observed here support the development of regenerative strategies designed to restore telomere integrity.
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Envejecimiento/metabolismo , Envejecimiento/patología , Telomerasa/deficiencia , Telomerasa/metabolismo , Envejecimiento/efectos de los fármacos , Animales , Reacción de Prevención/efectos de los fármacos , Encéfalo/anatomía & histología , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/patología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Daño del ADN/efectos de los fármacos , Proteína Doblecortina , Activación Enzimática/efectos de los fármacos , Reactivadores Enzimáticos/farmacología , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Vaina de Mielina/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/enzimología , Células-Madre Neurales/patología , Tamaño de los Órganos/efectos de los fármacos , Fenotipo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Medicina Regenerativa , Olfato/efectos de los fármacos , Olfato/fisiología , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Telomerasa/genética , Telómero/efectos de los fármacos , Telómero/metabolismo , Telómero/patologíaRESUMEN
Aging is a multifactorial process that affects most of the biological functions of the organism and increases susceptibility to disease and death. Recent studies with animal models of accelerated aging have unveiled some mechanisms that also operate in physiological aging. However, little is known about the role of microRNAs (miRNAs) in this process. To address this question, we have analysed miRNA levels in Zmpste24-deficient mice, a model of Hutchinson-Gilford progeria syndrome. We have found that expression of the miR-29 family of miRNAs is markedly upregulated in Zmpste24(-/-) progeroid mice as well as during normal aging in mouse. Functional analysis revealed that this transcriptional activation of miR-29 is triggered in response to DNA damage and occurs in a p53-dependent manner since p53(-/-) murine fibroblasts do not increase miR-29 expression upon doxorubicin treatment. We have also found that miR-29 represses Ppm1d phosphatase, which in turn enhances p53 activity. Based on these results, we propose the existence of a novel regulatory circuitry involving miR-29, Ppm1d and p53, which is activated in aging and in response to DNA damage.
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Envejecimiento , Daño del ADN , Regulación de la Expresión Génica , MicroARNs/biosíntesis , Fosfoproteínas Fosfatasas/biosíntesis , Proteína p53 Supresora de Tumor/biosíntesis , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Fibroblastos/fisiología , Proteínas de la Membrana/deficiencia , Metaloendopeptidasas/deficiencia , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Proteína Fosfatasa 2C , Análisis de Secuencia de ADNRESUMEN
Accelerated aging syndromes represent a valuable source of information about the molecular mechanisms involved in normal aging. Here, we describe a progeroid syndrome that partially phenocopies Hutchinson-Gilford progeria syndrome (HGPS) but also exhibits distinctive features, including the absence of cardiovascular deficiencies characteristic of HGPS, the lack of mutations in LMNA and ZMPSTE24, and a relatively long lifespan of affected individuals. Exome sequencing and molecular analysis in two unrelated families allowed us to identify a homozygous mutation in BANF1 (c.34G>A [p.Ala12Thr]), encoding barrier-to-autointegration factor 1 (BAF), as the molecular abnormality responsible for this Mendelian disorder. Functional analysis showed that fibroblasts from both patients have a dramatic reduction in BAF protein levels, indicating that the p.Ala12Thr mutation impairs protein stability. Furthermore, progeroid fibroblasts display profound abnormalities in the nuclear lamina, including blebs and abnormal distribution of emerin, an interaction partner of BAF. These nuclear abnormalities are rescued by ectopic expression of wild-type BANF1, providing evidence for the causal role of this mutation. These data demonstrate the utility of exome sequencing for identifying the cause of rare Mendelian disorders and underscore the importance of nuclear envelope alterations in human aging.
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Proteínas de Unión al ADN/genética , Proteínas Nucleares/genética , Adulto , Núcleo Celular , Células Cultivadas , Preescolar , Análisis Mutacional de ADN , Femenino , Fibroblastos/metabolismo , Ligamiento Genético , Homocigoto , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Mutación , Proteínas Nucleares/metabolismo , Linaje , Fenotipo , Progeria/genética , Progeria/metabolismo , Progeria/patología , Conformación Proteica , Alineación de SecuenciaRESUMEN
BACKGROUND AND AIM: The majority of mismatch repair (MMR) gene mutations causing Lynch syndrome (LS) occur either in MLH1 or MSH2. However, the relative contribution of PMS2 is less well defined. The aim of this study was to evaluate the role of PMS2 in LS by assessing the pathogenicity of variants of unknown significance (VUS) detected in the mutational analysis of PMS2 in a series of Spanish patients. METHODS: From a cohort of 202 LS suspected patients, 13 patients showing loss of PMS2 expression in tumours were screened for germline mutations in PMS2, using a long range PCR based strategy and multiplex ligation dependent probe amplification (MLPA). Pathogenicity assessment of PMS2 VUS was performed evaluating clinicopathological data, frequency in control population and in silico and in vitro analyses at the RNA and protein level. RESULTS: Overall 25 different PMS2 DNA variants were detected. Fourteen were classified as polymorphisms. Nine variants were classified as pathogenic: seven alterations based on their molecular nature and two after demonstrating a functional defect (c.538-3C>G affected mRNA processing and c.137G>T impaired MMR activity). The c.1569C>G variant was classified as likely neutral while the c.384G>A remained as a VUS. We have also shown that the polymorphic variant c.59G>A is MMR proficient. CONCLUSIONS: Pathogenic PMS2 mutations were detected in 69% of patients harbouring LS associated tumours with loss of PMS2 expression. In all, PMS2 mutations account for 6% of the LS cases identified. The comprehensive functional analysis shown here has been useful in the classification of PMS2 VUS and contributes to refining the role of PMS2 in LS.
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Adenosina Trifosfatasas/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Mutación de Línea Germinal , Estudios de Cohortes , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Reparación de la Incompatibilidad de ADN , Variación Genética , Células HEK293 , Humanos , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto , Polimorfismo Genético , TransfecciónRESUMEN
The molecular diagnosis of mismatch repair-deficient cancer syndromes is hampered by difficulties in sequencing the PMS2 gene, mainly owing to the PMS2CL pseudogene. Next-generation sequencing short reads cannot be mapped unambiguously by standard pipelines, compromising variant calling accuracy. This study aimed to provide a refined bioinformatic pipeline for PMS2 mutational analysis and explore PMS2 germline pathogenic variant prevalence in an unselected hereditary cancer (HC) cohort. PMS2 mutational analysis was optimized using two cohorts: 192 unselected HC patients for assessing the allelic ratio of paralogous sequence variants, and 13 samples enriched with PMS2 (likely) pathogenic variants screened previously by long-range genomic DNA PCR amplification. Reads were forced to align with the PMS2 reference sequence, except those corresponding to exon 11, where only those intersecting gene-specific invariant positions were considered. Afterward, the refined pipeline's accuracy was validated in a cohort of 40 patients and used to screen 5619 HC patients. Compared with our routine diagnostic pipeline, the PMS2_vaR pipeline showed increased technical sensitivity (0.853 to 0.956, respectively) in the validation cohort, identifying all previously PMS2 pathogenic variants found by long-range genomic DNA PCR amplification. Fifteen HC cohort samples carried a pathogenic PMS2 variant (15 of 5619; 0.285%), doubling the estimated prevalence in the general population. The refined open-source approach improved PMS2 mutational analysis accuracy, allowing its inclusion in the routine next-generation sequencing pipeline streamlining PMS2 screening.
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Biología Computacional , Pruebas Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto , Humanos , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Biología Computacional/métodos , Pruebas Genéticas/métodos , Análisis Mutacional de ADN/métodos , Mutación de Línea Germinal , Síndromes Neoplásicos Hereditarios/genética , Síndromes Neoplásicos Hereditarios/diagnósticoRESUMEN
The translation elongation factor eEF1A promotes protein synthesis. Its methylation by METTL13 increases its activity, supporting tumor growth. However, in some cancers, a high abundance of eEF1A isoforms is associated with a good prognosis. Here, we found that eEF1A2 exhibited oncogenic or tumor-suppressor functions depending on its interaction with METTL13 or the phosphatase PTEN, respectively. METTL13 and PTEN competed for interaction with eEF1A2 in the same structural domain. PTEN-bound eEF1A2 promoted the ubiquitination and degradation of the mitosis-promoting Aurora kinase A in the S and G2 phases of the cell cycle. eEF1A2 bridged the interactions between the SKP1-CUL1-FBXW7 (SCF) ubiquitin ligase complex, the kinase GSK3ß, and Aurora-A, thereby facilitating the phosphorylation of Aurora-A in a degron site that was recognized by FBXW7. Genetic ablation of Eef1a2 or Pten in mice resulted in a greater abundance of Aurora-A and increased cell cycling in mammary tumors, which was corroborated in breast cancer tissues from patients. Reactivating this pathway using fimepinostat, which relieves inhibitory signaling directed at PTEN and increases FBXW7 expression, combined with inhibiting Aurora-A with alisertib, suppressed breast cancer cell proliferation in culture and tumor growth in vivo. The findings demonstrate a therapeutically exploitable, tumor-suppressive role for eEF1A2 in breast cancer.
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Aurora Quinasa A , Neoplasias de la Mama , Neoplasias Mamarias Animales , Fosfohidrolasa PTEN , Factor 1 de Elongación Peptídica , Animales , Femenino , Humanos , Ratones , Aurora Quinasa A/genética , Aurora Quinasa A/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Glucógeno Sintasa Quinasa 3 beta , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Factor 1 de Elongación Peptídica/genética , Factor 1 de Elongación Peptídica/metabolismoRESUMEN
Accurate classification of genetic variants is crucial for clinical decision-making in hereditary cancer. In Spain, genetic diagnostic laboratories have traditionally approached this task independently due to the lack of a dedicated resource. Here we present SpadaHC, a web-based database for sharing variants in hereditary cancer genes in the Spanish population. SpadaHC is implemented using a three-tier architecture consisting of a relational database, a web tool and a bioinformatics pipeline. Contributing laboratories can share variant classifications and variants from individuals in Variant Calling Format (VCF) format. The platform supports open and restricted access, flexible dataset submissions, automatic pseudo-anonymization, VCF quality control, variant normalization and liftover between genome builds. Users can flexibly explore and search data, receive automatic discrepancy notifications and access SpadaHC population frequencies based on many criteria. In February 2024, SpadaHC included 18 laboratory members, storing 1.17 million variants from 4306 patients and 16 343 laboratory classifications. In the first analysis of the shared data, we identified 84 genetic variants with clinically relevant discrepancies in their classifications and addressed them through a three-phase resolution strategy. This work highlights the importance of data sharing to promote consistency in variant classifications among laboratories, so patients and family members can benefit from more accurate clinical management. Database URL: https://spadahc.ciberisciii.es/.
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Bases de Datos Genéticas , Humanos , España , Variación Genética , Neoplasias/genética , Genes Relacionados con las Neoplasias , Predisposición Genética a la EnfermedadRESUMEN
The mouse ortholog of human FACE-1, Zmpste24, is a multispanning membrane protein widely distributed in mammalian tissues and structurally related to Afc1p/ste24p, a yeast metalloproteinase involved in the maturation of fungal pheromones. Disruption of the gene Zmpste24 caused severe growth retardation and premature death in homozygous-null mice. Histopathological analysis of the mutant mice revealed several abnormalities, including dilated cardiomyopathy, muscular dystrophy and lipodystrophy. These alterations are similar to those developed by mice deficient in A-type lamin, a major component of the nuclear lamina, and phenocopy most defects observed in humans with diverse congenital laminopathies. In agreement with this finding, Zmpste24-null mice are defective in the proteolytic processing of prelamin A. This deficiency in prelamin A maturation leads to the generation of abnormalities in nuclear architecture that probably underlie the many phenotypes observed in both mice and humans with mutations in the lamin A gene. These results indicate that prelamin A is a specific substrate for Zmpste24 and demonstrate the usefulness of genetic approaches for identifying the in vivo substrates of proteolytic enzymes.
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Adipocitos/patología , Proteínas de la Membrana/deficiencia , Metaloendopeptidasas/deficiencia , Músculos/patología , Proteínas Nucleares/metabolismo , Precursores de Proteínas/metabolismo , Adipocitos/metabolismo , Animales , Núcleo Celular/patología , Femenino , Humanos , Lamina Tipo A , Masculino , Proteínas de la Membrana/genética , Metaloendopeptidasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculos/metabolismo , Miocardio/patología , Fenotipo , Procesamiento Proteico-PostraduccionalRESUMEN
In contrast to mono- or biallelic loss of tumor-suppressor function, effects of discrete gene dysregulations, as caused by non-coding (epi)genome alterations, are poorly understood. Here, by perturbing the regulatory genome in mice, we uncover pervasive roles of subtle gene expression variation in cancer evolution. Genome-wide screens characterizing 1,450 tumors revealed that such quasi-insufficiency is extensive across entities and displays diverse context dependencies, such as distinct cell-of-origin associations in T-ALL subtypes. We compile catalogs of non-coding regions linked to quasi-insufficiency, show their enrichment with human cancer risk variants, and provide functional insights by engineering regulatory alterations in mice. As such, kilo-/megabase deletions in a Bcl11b-linked non-coding region triggered aggressive malignancies, with allele-specific tumor spectra reflecting gradual gene dysregulations through modular and cell-type-specific enhancer activities. Our study constitutes a first survey toward a systems-level understanding of quasi-insufficiency in cancer and gives multifaceted insights into tumor evolution and the tissue-specific effects of non-coding mutations.
RESUMEN
Premature aging syndromes often result from mutations in nuclear proteins involved in the maintenance of genomic integrity. Lamin A is a major component of the nuclear lamina and nuclear skeleton. Truncation in lamin A causes Hutchinson-Gilford progerial syndrome (HGPS), a severe form of early-onset premature aging. Lack of functional Zmpste24, a metalloproteinase responsible for the maturation of prelamin A, also results in progeroid phenotypes in mice and humans. We found that Zmpste24-deficient mouse embryonic fibroblasts (MEFs) show increased DNA damage and chromosome aberrations and are more sensitive to DNA-damaging agents. Bone marrow cells isolated from Zmpste24-/- mice show increased aneuploidy and the mice are more sensitive to DNA-damaging agents. Recruitment of p53 binding protein 1 (53BP1) and Rad51 to sites of DNA lesion is impaired in Zmpste24-/- MEFs and in HGPS fibroblasts, resulting in delayed checkpoint response and defective DNA repair. Wild-type MEFs ectopically expressing unprocessible prelamin A show similar defects in checkpoint response and DNA repair. Our results indicate that unprocessed prelamin A and truncated lamin A act dominant negatively to perturb DNA damage response and repair, resulting in genomic instability which might contribute to laminopathy-based premature aging.
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Envejecimiento Prematuro/genética , Daño del ADN/genética , Reparación del ADN/fisiología , Inestabilidad Genómica , Lamina Tipo A/genética , Proteínas de la Membrana/genética , Metaloendopeptidasas/genética , Animales , Células de la Médula Ósea/fisiología , Células de la Médula Ósea/efectos de la radiación , Senescencia Celular/genética , Proteínas Cromosómicas no Histona , Aberraciones Cromosómicas , ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Fibroblastos/patología , Fibroblastos/efectos de la radiación , Rayos gamma , Histonas/genética , Histonas/metabolismo , Histonas/efectos de la radiación , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lamina Tipo A/metabolismo , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Ratones , Ratones Mutantes , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Recombinasa Rad51 , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
HYPOTHESIS: Adult genetic sensorineural hearing loss (SNHL) may be underestimated. BACKGROUND: The diagnosis of genetic hearing loss is challenging, given its extreme genetic and phenotypic heterogeneity, particularly in adulthood. This study evaluated the utility of next-generation sequencing (NGS) in the etiological diagnosis of adult-onset SNHL. MATERIALS AND METHODS: Adults (>16 yr old) with SNHL were recruited at the Otolaryngology Department at Marqués de Valdecilla University Hospital (Spain). Environmental factors, acoustic trauma, endolymphatic hydrops, and age-related hearing loss were excluding criteria. An NGS gene panel was used, including 196 genes (OTOgenics v3) or 229 genes (OTOgenics v4) related to syndromic and nonsyndromic hearing loss. RESULTS: Sixty-five patients were included in the study (average age at the onset of SNHL, 41 yr). Fifteen pathogenic/likely pathogenic variants considered to be causative were found in 15 patients (23% diagnostic yield) in TECTA (4), KCNQ4 (3), GJB2 (2), ACTG1 (1), COL2A1 (1), COCH (1), COCH/COL2A1 (1), STRC (1), and ABHD12 (1). Three patients had syndromic associations (20% of patients with genetic diagnosis) that had not been previously diagnosed (two Stickler type I and one polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, cataract syndrome). Seven variants of unknown significance were found in COL11A1 (1), GSMDE (2), DNTM1 (1), SOX10 (1), EYA4 (1), and TECTA (1). CONCLUSION: NGS gene panels can provide diagnostic yields greater than 20% for adult SNHL, with a significant proportion of variant of unknown significance that could potentially contribute to increasing diagnostic output. Identifying a genetic cause enables genetic counseling, provides prognostic information and can reveal unrecognized syndromes contributing to an accurate management of their associated manifestations.
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Catarata , Sordera , Pérdida Auditiva Sensorineural , Pérdida Auditiva , Adulto , Humanos , Pérdida Auditiva Sensorineural/diagnóstico , Pérdida Auditiva Sensorineural/genética , Sordera/complicaciones , Secuenciación de Nucleótidos de Alto Rendimiento , Pérdida Auditiva/complicaciones , Mutación , Transactivadores , Péptidos y Proteínas de Señalización Intercelular/genética , Monoacilglicerol Lipasas/genéticaRESUMEN
OBJECTIVE: The biallelic inheritance of an expanded intronic pentamer (AAGGG)exp in the gene encoding replication factor C subunit 1 (RFC1) has been found to be a cause of cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS). This study describes clinical and genetic features of our patients with clinical suspicion of the syndrome. STUDY DESIGN: A retrospective descriptive study from an ataxia database comprising 500 patients. SETTING: The study was performed at the Otorhinolaryngology Department of a hospital in the north of Spain. METHODS: Specific genetic testing for CANVAS was performed in 13 patients with clinical suspicion of complete or incomplete syndrome. The clinical diagnosis was supported by quantitative vestibular hypofunction, cerebellar atrophy, and abnormal sensory nerve conduction testing. RESULTS: Nine of 13 (69%) patients met clinical diagnostic criteria for definite CANVAS disease. The first manifestation of the syndrome was lower limb dysesthesia in 8 of 13 patients and gait imbalance in 5 of 13. Eleven of 13 (85%) patients were carriers of the biallelic (AAGGG)exp in RFC1. CONCLUSION: A genetic cause of CANVAS has recently been discovered. We propose genetic screening for biallelic expansions of the AAGGG pentamer of RFC1 in all patients with clinical suspicion of CANVAS, since accurate early diagnosis could improve the quality of life of these patients.
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Vestibulopatía Bilateral/diagnóstico , Vestibulopatía Bilateral/genética , Ataxia Cerebelosa/diagnóstico , Ataxia Cerebelosa/genética , Expansión de las Repeticiones de ADN/genética , Proteína de Replicación C/genética , Anciano , Bases de Datos Factuales , Diagnóstico Diferencial , Femenino , Pruebas Genéticas , Humanos , Intrones/genética , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , España , Evaluación de Síntomas , SíndromeRESUMEN
Progeria syndromes are rare disorders that involve premature aging. Mutations in BANF1 have been recently reported to cause a new hereditary progeroid syndrome that we now propose to call the Néstor-Guillermo progeria syndrome (NGPS). We describe herein the clinical features of the first two NGPS patients, who phenocopy features of classic progerias (i.e., Hutchinson-Gilford progeria syndrome or mandibuloacral dysplasia), such as aged appearance, growth retardation, decreased subcutaneous fat, thin limbs, and stiff joints. However, these NGPS patients have a distinctive phenotype. In their early adulthood (32 and 24 years of age), they have no signs of cardiovascular impairment, diabetes mellitus, or hypertriglyceridemia. In contrast, they suffer profound skeletal abnormalities that affect their quality of life. The observed differences are of utmost importance to patients and their families and palliation of osseous manifestations is a priority, given their relatively long lifespan. We define NGPS as a chronic progeria because of its slow clinical course and relatively long survival, despite its early onset. Understanding the differences between progeria syndromes might contribute to the development of treatment strategies for common skeletal conditions, as well as aging itself.
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Enfermedades del Desarrollo Óseo/patología , Proteínas de Unión al ADN/genética , Proteínas Nucleares/genética , Progeria/genética , Adolescente , Adulto , Enfermedades del Desarrollo Óseo/genética , Niño , Preescolar , Enfermedad Crónica , Análisis Mutacional de ADN , Pruebas Genéticas , Trastornos del Crecimiento/genética , Trastornos del Crecimiento/patología , Humanos , Masculino , Mutación , Fenotipo , Progeria/diagnóstico , Progeria/patología , Adulto JovenRESUMEN
Zmpste24 (also called FACE-1) is a metalloproteinase involved in the maturation of lamin A (Lmna), an essential component of the nuclear envelope. Both Zmpste24- and Lmna-deficient mice exhibit profound nuclear architecture abnormalities and multiple histopathological defects that phenocopy an accelerated ageing process. Similarly, diverse human progeroid syndromes are caused by mutations in ZMPSTE24 or LMNA genes. To elucidate the molecular mechanisms underlying these devastating diseases, we have analysed the transcriptional alterations occurring in tissues from Zmpste24-deficient mice. We demonstrate that Zmpste24 deficiency elicits a stress signalling pathway that is evidenced by a marked upregulation of p53 target genes, and accompanied by a senescence phenotype at the cellular level and accelerated ageing at the organismal level. These phenotypes are largely rescued in Zmpste24-/-Lmna+/- mice and partially reversed in Zmpste24-/-p53-/- mice. These findings provide evidence for the existence of a checkpoint response activated by the nuclear abnormalities caused by prelamin A accumulation, and support the concept that hyperactivation of the tumour suppressor p53 may cause accelerated ageing.
Asunto(s)
Envejecimiento/fisiología , Proteínas de la Membrana/deficiencia , Metaloendopeptidasas/deficiencia , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Envejecimiento/genética , Animales , Células Cultivadas , Eliminación de Gen , Heterocigoto , Lamina Tipo A/deficiencia , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Ratones , Ratones Noqueados , Fenotipo , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Autophagy is a highly regulated intracellular process involved in the turnover of most cellular constituents and in the maintenance of cellular homeostasis. It is well-established that the basal autophagic activity of living cells decreases with age, thus contributing to the accumulation of damaged macromolecules during aging. Conversely, the activity of this catabolic pathway is required for lifespan extension in animal models such as Caenorhabditis elegans and Drosophila melanogaster. In this work, we describe the unexpected finding that Zmpste24-null mice, which show accelerated aging and are a reliable model of human Hutchinson-Gilford progeria, exhibit an extensive basal activation of autophagy instead of the characteristic decline in this process occurring during normal aging. We also show that this autophagic increase is associated with a series of changes in lipid and glucose metabolic pathways, which resemble those occurring in diverse situations reported to prolong lifespan. These Zmpste24(-/-) mice metabolic alterations are also linked to substantial changes in circulating blood parameters, such as leptin, glucose, insulin or adiponectin which in turn lead to peripheral LKB1-AMPK activation and mTOR inhibition. On the basis of these results, we propose that nuclear abnormalities causing premature aging in Zmpste24(-/-) mice trigger a metabolic response involving the activation of autophagy. However, the chronic activation of this catabolic pathway may turn an originally intended pro-survival strategy into a pro-aging mechanism and could contribute to the systemic degeneration and weakening observed in these progeroid mice.
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Envejecimiento Prematuro/fisiopatología , Autofagia , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Progeria/fisiopatología , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP , Envejecimiento Prematuro/genética , Animales , Modelos Animales de Enfermedad , Glucosa/metabolismo , Hormonas/sangre , Humanos , Metabolismo de los Lípidos , Proteínas de la Membrana/genética , Metaloendopeptidasas/genética , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Progeria/genética , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Transducción de SeñalRESUMEN
The stereocilia of the inner ear sensory cells contain the actin-binding protein radixin, encoded by RDX. Radixin is important for hearing but remains functionally obscure. To determine how radixin influences hearing sensitivity, we used a custom rapid imaging technique to visualize stereocilia motion while measuring electrical potential amplitudes during acoustic stimulation. Radixin inhibition decreased sound-evoked electrical potentials. Other functional measures, including electrically induced sensory cell motility and sound-evoked stereocilia deflections, showed a minor amplitude increase. These unique functional alterations demonstrate radixin as necessary for conversion of sound into electrical signals at acoustic rates. We identified patients with RDX variants with normal hearing at birth who showed rapidly deteriorating hearing during the first months of life. This may be overlooked by newborn hearing screening and explained by multiple disturbances in postnatal sensory cells. We conclude radixin is necessary for ensuring normal conversion of sound to electrical signals in the inner ear.
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Proteínas del Citoesqueleto/metabolismo , Células Ciliadas Auditivas Externas/metabolismo , Proteínas de la Membrana/metabolismo , Estereocilios/metabolismo , Estimulación Acústica , Alelos , Animales , Arsenicales/farmacología , Preescolar , Proteínas del Citoesqueleto/genética , Modelos Animales de Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Expresión Génica , Variación Genética , Genotipo , Cobayas , Células Ciliadas Auditivas Externas/efectos de los fármacos , Pérdida Auditiva/diagnóstico , Pérdida Auditiva/genética , Humanos , Mecanotransducción Celular/genética , Proteínas de la Membrana/genética , Modelos Biológicos , Linaje , Estereocilios/efectos de los fármacosRESUMEN
INTRODUCTION: Sensorineural hearing loss (SNL) is the most prevalent sensory deficit in our environment. Next generation genomic sequencing (NGS) enables an aetiological diagnosis in a high percentage of patients. Our pilot study shows the results of the systematic application of NGS in a Childhood Hearing Loss Unit, as well as its implications for the clinical management of patients and their families. MATERIAL AND METHOD: We included 27 patients diagnosed with SNL between 2014 and 2017, in which an environmental cause was ruled out. The genetic test consisted of a panel of genes analyzed by NGS (OTOgenicsTM panel). This panel has been designed to include genes associated with sensorineural or mixed hearing loss, early onset or late, syndromic and non-syndromic, regardless of their inheritance pattern. RESULTS: A genetic diagnosis was obtained in 56% (15/27) of the patients (62% in the case of bilateral SNL). Of the patients, 5/27 (19%) presented pathogenic variants in the GJB2 gene and the rest pathogenic and / or probably pathogenic variants in other genes associated with isolated SNL (PR2X2, TECTA and STRC), with syndromic SNL (CHD7, GATA3, COL4A5, MITF and SOX10) or with syndromic and non-syndromic SNL (BSND, ACTG1 and CDH23). DISCUSSION: The aetiological diagnosis of SNL is a challenge in clinical practice. Our series demonstrates that it is possible to implement genetic diagnosis in the care routine and that this information has prognostic and therapeutic implications.