RESUMEN
Pancreatic endocrine cells employ a sophisticated system of paracrine and autocrine signals to synchronize their activities, including glutamate, which controls hormone release and ß-cell viability by acting on glutamate receptors expressed by endocrine cells. We here investigate whether alteration of the excitatory amino acid transporter 2 (EAAT2), the major glutamate clearance system in the islet, may occur in type 2 diabetes mellitus and contribute to ß-cell dysfunction. Increased EAAT2 intracellular localization was evident in islets of Langerhans from T2DM subjects as compared with healthy control subjects, despite similar expression levels. Chronic treatment of islets from healthy donors with high-glucose concentrations led to the transporter internalization in vesicular compartments and reduced [H3]-d-glutamate uptake (65 ± 5% inhibition), phenocopying the findings in T2DM pancreatic sections. The transporter relocalization was associated with decreased Akt phosphorylation protein levels, suggesting an involvement of the phosphoinositide 3-kinase (PI3K)/Akt pathway in the process. In line with this, PI3K inhibition by a 100-µM LY294002 treatment in human and clonal ß-cells caused the transporter relocalization in intracellular compartments and significantly reduced the glutamate uptake compared to control conditions, suggesting that hyperglycemia changes the trafficking of the transporter to the plasma membrane. Upregulation of the glutamate transporter upon treatment with the antibiotic ceftriaxone rescued hyperglycemia-induced ß-cells dysfunction and death. Our data underscore the significance of EAAT2 in regulating islet physiology and provide a rationale for potential therapeutic targeting of this transporter to preserve ß-cell survival and function in diabetes.NEW & NOTEWORTHY The glutamate transporter SLC1A2/excitatory amino acid transporter 2 (EAAT2) is expressed on the plasma membrane of pancreatic ß-cells and controls islet glutamate clearance and ß-cells survival. We found that the EAAT2 membrane expression is lost in the islets of Langerhans from type 2 diabetes mellitus (T2DM) patients due to hyperglycemia-induced downregulation of the phosphoinositide 3-kinase/Akt pathway and modification of its intracellular trafficking. Pharmacological rescue of EAAT2 expression prevents ß-cell dysfunction and death, suggesting EAAT2 as a new potential target of intervention in T2DM.
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Diabetes Mellitus Tipo 2 , Transportador 2 de Aminoácidos Excitadores , Ácido Glutámico , Hiperglucemia , Islotes Pancreáticos , Transportador 2 de Aminoácidos Excitadores/metabolismo , Humanos , Diabetes Mellitus Tipo 2/metabolismo , Ácido Glutámico/metabolismo , Hiperglucemia/metabolismo , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Femenino , Transporte de Proteínas , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Anciano , Adulto , Animales , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
NF1 microdeletion syndrome, accounting for 5-11% of NF1 patients, is caused by a deletion in the NF1 region and it is generally characterized by a severe phenotype. Although 70% of NF1 microdeletion patients presents the same 1.4 Mb type-I deletion, some patients may show additional clinical features. Therefore, the contribution of several pathogenic mechanisms, besides haploinsufficiency of some genes within the deletion interval, is expected and needs to be defined. We investigated an altered expression of deletion flanking genes by qPCR in patients with type-1 NF1 deletion, compared to healthy donors, possibly contributing to the clinical traits of NF1 microdeletion syndrome. In addition, the 1.4-Mb deletion leads to changes in the 3D chromatin structure in the 17q11.2 region. Specifically, this deletion alters DNA-DNA interactions in the regions flanking the breakpoints, as demonstrated by our 4C-seq analysis. This alteration likely causes position effect on the expression of deletion flanking genes.Interestingly, 4C-seq analysis revealed that in microdeletion patients, an interaction was established between the RHOT1 promoter and the SLC6A4 gene, which showed increased expression. We performed NGS on putative modifier genes, and identified two "likely pathogenic" rare variants in RAS pathway, possibly contributing to incidental phenotypic features.This study provides new insights into understanding the pathogenesis of NF1 microdeletion syndrome and suggests a novel pathomechanism that contributes to the expression phenotype in addition to haploinsufficiency of genes located within the deletion.This is a pivotal approach that can be applied to unravel microdeletion syndromes, improving precision medicine, prognosis and patients' follow-up.
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Deleción Cromosómica , Epigénesis Genética , Haploinsuficiencia , Neurofibromatosis 1 , Humanos , Neurofibromatosis 1/genética , Femenino , Masculino , Neurofibromina 1/genética , Cromosomas Humanos Par 17/genética , Fenotipo , Niño , Regiones Promotoras GenéticasRESUMEN
Aberrant activation of the Hh pathway promotes cell proliferation and multi-drug resistance (MDR) in several cancers, including Acute Myeloid Leukemia (AML). Notably, only one Hh inhibitor, glasdegib, has been approved for AML treatment, and most patients eventually relapse, highlighting the urgent need to discover new therapeutic targets. Hh signal is transduced through the membrane of the primary cilium, a structure expressed by non-proliferating mammalian cells, whose stabilization depends on the activity of HDAC6. Here we describe a positive correlation between Hh, HDAC6, and MDR genes in a cohort of adult AML patients, human leukemic cell lines, and a zebrafish model of Hh overexpression. The hyper-activation of Hh or HDAC6 in zebrafish drove the increased proliferation of hematopoietic stem and progenitor cells (HSPCs). Interestingly, this phenotype was rescued by inhibition of HDAC6 but not of Hh. Also, in human leukemic cell lines, a reduction in vitality was obtained through HDAC6, but not Hh inhibition. Our data showed the presence of a cross-talk between Hh and HDAC6 mediated by stabilization of the primary cilium, which we detect for the first time in zebrafish HSPCs. Inhibition of HDAC6 activity alone or in combination therapy with the chemotherapeutic agent cytarabine, efficiently rescued the hematopoietic phenotype. Our results open the possibility to introduce HDAC6 as therapeutic target to reduce proliferation of leukemic blasts in AML patients.
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Proteínas Hedgehog , Inhibidores de Histona Desacetilasas , Leucemia Mieloide Aguda , Adulto , Animales , Proliferación Celular , Proteínas Hedgehog/metabolismo , Células Madre Hematopoyéticas , Histona Desacetilasa 6/metabolismo , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Transducción de Señal , Pez Cebra/metabolismoRESUMEN
Transcriptional changes normally occur during development but also underlie differences between healthy and pathological conditions. Transcription factors or chromatin modifiers are involved in orchestrating gene activity, such as the cohesin genes and their regulator NIPBL. In our previous studies, using a zebrafish model for nipblb knockdown, we described the effect of nipblb loss-of-function in specific contexts, such as central nervous system development and hematopoiesis. However, the genome-wide transcriptional impact of nipblb loss-of-function in zebrafish embryos at diverse developmental stages remains under investigation. By RNA-seq analyses in zebrafish embryos at 24 h post-fertilization, we examined genome-wide effects of nipblb knockdown on transcriptional programs. Differential gene expression analysis revealed that nipblb loss-of-function has an impact on gene expression at 24 h post fertilization, mainly resulting in gene inactivation. A similar transcriptional effect has also been reported in other organisms, supporting the use of zebrafish as a model to understand the role of Nipbl in gene regulation during early vertebrate development. Moreover, we unraveled a connection between nipblb-dependent differential expression and gene expression patterns of hematological cell populations and AML subtypes, enforcing our previous evidence on the involvement of NIPBL-related transcriptional dysregulation in hematological malignancies.
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Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Pez Cebra/metabolismo , Pez Cebra/genética , Animales , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Embrión no Mamífero/citología , Perfilación de la Expresión Génica , Genoma , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genética , CohesinasRESUMEN
The abnormal deposition of proteins in brain tissue is a common feature of neurodegenerative diseases (NDs) often accompanied by the spread of mutated proteins, causing neuronal toxicity. Exosomes play a fundamental role on their releasing in extracellular space after endosomal pathway activation, allowing to remove protein aggregates by lysosomal degradation or their inclusion into multivesicular bodies (MVBs), besides promoting cellular cross-talk. The emerging evidence of pathogenic mutations associated to ND susceptibility, leading to impairment of exosome production and secretion, opens a new perspective on the mechanisms involved in neurodegeneration. Recent findings suggest to investigate the genetic mechanisms regulating the different exosome functions in central nervous system (CNS), to understand their role in the pathogenesis of NDs, addressing the identification of diagnostic and pharmacological targets. This review aims to summarize the mechanisms underlying exosome biogenesis, their molecular composition and functions in CNS, with a specific focus on the recent findings invoking a defective exosome biogenesis as a common biological feature of the major NDs, caused by genetic alterations. Further definition of the consequences of specific genetic mutations on exosome biogenesis and release will improve diagnostic and pharmacological studies in NDs.
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Susceptibilidad a Enfermedades , Exosomas/metabolismo , Variación Genética , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Animales , Biomarcadores , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/fisiopatología , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Enfermedades Neurodegenerativas/patologíaRESUMEN
BACKGROUND: Emissions from diesel vehicles and biomass burning are the principal sources of primary ultrafine particles (UFP). The exposure to UFP has been associated to cardiovascular and pulmonary diseases, including lung cancer. Although many aspects of the toxicology of ambient particulate matter (PM) have been unraveled, the molecular mechanisms activated in human cells by the exposure to UFP are still poorly understood. Here, we present an RNA-seq time-course experiment (five time point after single dose exposure) used to investigate the differential and temporal changes induced in the gene expression of human bronchial epithelial cells (BEAS-2B) by the exposure to UFP generated from diesel and biomass combustion. A combination of different bioinformatics tools (EdgeR, next-maSigPro and reactome FI app-Cytoscape and prioritization strategies) facilitated the analyses the temporal transcriptional pattern, functional gene set enrichment and gene networks related to cellular response to UFP particles. RESULTS: The bioinformatics analysis of transcriptional data reveals that the two different UFP induce, since the earliest time points, different transcriptional dynamics resulting in the activation of specific genes. The functional enrichment of differentially expressed genes indicates that the exposure to diesel UFP induces the activation of genes involved in TNFα signaling via NF-kB and inflammatory response, and hypoxia. Conversely, the exposure to ultrafine particles from biomass determines less distinct modifications of the gene expression profiles. Diesel UFP exposure induces the secretion of biomarkers associated to inflammation (CCXL2, EPGN, GREM1, IL1A, IL1B, IL6, IL24, EREG, VEGF) and transcription factors (as NFE2L2, MAFF, HES1, FOSL1, TGIF1) relevant for cardiovascular and lung disease. By means of network reconstruction, four genes (STAT3, HIF1a, NFKB1, KRAS) have emerged as major regulators of transcriptional response of bronchial epithelial cells exposed to diesel exhaust. CONCLUSIONS: Overall, this work highlights modifications of the transcriptional landscape in human bronchial cells exposed to UFP and sheds new lights on possible mechanisms by means of which UFP acts as a carcinogen and harmful factor for human health.
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Biomasa , Bronquios/metabolismo , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Material Particulado/efectos adversos , Emisiones de Vehículos/envenenamiento , Bronquios/citología , Bronquios/efectos de los fármacos , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Humanos , TranscriptomaRESUMEN
Human clear cell renal cell carcinoma (ccRCC) is therapy resistant; therefore, it is worthwhile studying in depth the molecular aspects of its progression. In ccRCC the biallelic inactivation of the VHL gene leads to stabilization of hypoxia-inducible factors (HIFs). Among the targets of HIF-1α transcriptional activity is the LOX gene, which codes for the inactive proenzyme (Pro-Lox) from which, after extracellular secretion and proteolysis, derives the active enzyme (Lox) and the propeptide (Lox-PP). By increasing stiffness of extracellular matrix by collagen crosslinking, Lox promotes tumor progression and metastasis. Lox and Lox-PP can reenter the cells where Lox promotes cell proliferation and invasion, whereas Lox-PP acts as tumor suppressor because of its Ras recision and apoptotic activity. Few data are available concerning LOX in ccRCC. Using an in vitro model of ccRCC primary cell cultures, we performed, for the first time in ccRCC, a detailed study of endogenous LOX and also investigated their transcriptomic profile. We found that endogenous LOX is overexpressed in ccRCC, is involved in a positive-regulative loop with HIF-1α, and has a major action on ccRCC progression through cellular adhesion, migration, and collagen matrix stiffness increment; however, the oncosuppressive action of Lox-PP was not found to prevail. These findings may suggest translational approaches for new therapeutic strategies in ccRCC.
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Carcinoma de Células Renales/patología , Colágeno/metabolismo , Neoplasias Renales/patología , Proteína-Lisina 6-Oxidasa/metabolismo , Anciano , Anciano de 80 o más Años , Western Blotting , Carcinoma de Células Renales/enzimología , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Progresión de la Enfermedad , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Neoplasias Renales/enzimología , Masculino , Microscopía de Fuerza Atómica , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Cultivo Primario de Células , Reacción en Cadena en Tiempo Real de la Polimerasa , Transfección , Células Tumorales CultivadasRESUMEN
BACKGROUND: Exposure to particulate matter (PM) is associated with increased incidence of cardiovascular disease and increased coagulation, but the molecular mechanisms underlying these associations remain unknown. Obesity may increase susceptibility to the adverse effects of PM exposure, exacerbating the effects on cardiovascular diseases. Extracellular vesicles (EVs), which travel in body fluids and transfer microRNAs (miRNAs) between tissues, might play an important role in PM-induced cardiovascular risk. We sought to determine whether the levels of PM with an aerodynamic diameter ≤ 10 µm (PM10) are associated with changes in fibrinogen levels, EV release, and the miRNA content of EVs (EV-miRNAs), investigating 1630 overweight/obese subjects from the SPHERE Study. RESULTS: Short-term exposure to PM10 (Day before blood drawing) was associated with an increased release of EVs quantified by nanoparticle tracking analysis, especially EVs derived from monocyte/macrophage components (CD14+) and platelets (CD61+) which were characterized by flow cytometry. We first profiled miRNAs of 883 subjects by the QuantStudio™ 12 K Flex Real Time PCR System and the top 40 EV-miRNAs were validated through custom miRNA plates. Nine EV-miRNAs (let-7c-5p; miR-106a-5p; miR-143-3p; miR-185-5p; miR-218-5p; miR-331-3p; miR-642-5p; miR-652-3p; miR-99b-5p) were downregulated in response to PM10 exposure and exhibited putative roles in cardiovascular disease, as highlighted by integrated network analysis. PM10 exposure was significantly associated with elevated fibrinogen levels, and five of the nine downregulated EV-miRNAs were mediators between PM10 exposure and fibrinogen levels. CONCLUSIONS: Research on EVs opens a new path to the investigation of the adverse health effects of air pollution exposure. EVs have the potential to act both as markers of PM susceptibility and as potential molecular mechanism in the chain of events connecting PM exposure to increased coagulation, which is frequently linked to exposure and CVD development.
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Coagulación Sanguínea/efectos de los fármacos , Enfermedades Cardiovasculares/sangre , Vesículas Extracelulares/efectos de los fármacos , MicroARNs/sangre , Obesidad/sangre , Material Particulado/toxicidad , Índice de Masa Corporal , Enfermedades Cardiovasculares/inducido químicamente , Estudios Transversales , Vesículas Extracelulares/metabolismo , Femenino , Citometría de Flujo , Humanos , Exposición por Inhalación/análisis , Modelos Lineales , Masculino , MicroARNs/genética , Persona de Mediana Edad , Análisis Multivariante , Obesidad/complicaciones , Tamaño de la PartículaRESUMEN
AIMS: Exposure to particulate air pollution is associated with increased blood pressure (BP), a well-established risk factor for cardiovascular disease. To elucidate the mechanisms underlying this relationship, we investigated whether the effects of particulate matter of less than 10µm in aerodynamic diameter (PM10) on BP are mediated by microRNAs. METHODS AND RESULTS: We recruited 90 obese individuals and we assessed their PM10 exposure 24 and 48h before the recruitment day. We performed multivariate linear regression models to investigate the effects of PM10 on BP. Using the TaqMan® Low-Density Array, we experimentally evaluated and technically validated the expression levels of 377 human miRNAs in peripheral blood. We developed a mediated moderation analysis to estimate the proportion of PM10 effects on BP that was mediated by miRNA expression. PM10 exposure 24 and 48h before the recruitment day was associated with increased systolic BP (ß=1.22mmHg, P=0.019; ß=1.24mmHg, P=0.019, respectively) and diastolic BP (ß=0.67mmHg, P=0.044; ß=0.91mmHg, P=0.007, respectively). We identified nine miRNAs associated with PM10 levels 48h after exposure. A conditional indirect effect (CIE=-0.1431) of PM10 on diastolic BP, which was mediated by microRNA-101, was found in individuals with lower values of mean body mass index. CONCLUSIONS: Our data provide evidence that miRNAs are a molecular mechanism underlying the BP-related effects of air pollution exposure, and indicate miR-101 as epigenetic mechanism to be further investigated.
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Contaminantes Atmosféricos/toxicidad , Presión Sanguínea/efectos de los fármacos , Exposición a Riesgos Ambientales , MicroARNs/genética , Tamaño de la Partícula , Material Particulado/toxicidad , Adulto , Femenino , Humanos , Italia , Masculino , MicroARNs/metabolismo , Persona de Mediana Edad , Obesidad/etiología , Sobrepeso/etiologíaRESUMEN
Synaptic abnormalities are a hallmark of several neurological diseases, and clarification of the underlying mechanisms represents a crucial step toward the development of therapeutic strategies. Rett syndrome (RTT) is a rare neurodevelopmental disorder, mainly affecting females, caused by mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene, leading to a deep derangement of synaptic connectivity. Although initial studies supported the exclusive involvement of neurons, recent data have highlighted the pivotal contribution of astrocytes in RTT pathogenesis through non-cell autonomous mechanisms. Since astrocytes regulate synapse formation and functionality by releasing multiple molecules, we investigated the influence of soluble factors secreted by Mecp2 knock-out (KO) astrocytes on synapses. We found that Mecp2 deficiency in astrocytes negatively affects their ability to support synaptogenesis by releasing synaptotoxic molecules. Notably, neuronal inputs from a dysfunctional astrocyte-neuron crosstalk lead KO astrocytes to aberrantly express IL-6, and blocking IL-6 activity prevents synaptic alterations.
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miRNA target genes prediction represents a crucial step in miRNAs functional characterization. In this context, the challenging issue remains predictions accuracy and recognition of false positive results. In this article myMIR, a web based system for increasing reliability of miRNAs predicted targets lists, is presented. myMIR implements an integrated pipeline for computing ranked miRNA::target lists and provides annotations for narrowing them down. The system relies on knowledge base data, suitably integrated in order to extend the functional characterization of targeted genes to miRNAs, by highlighting the search on over-represented annotation terms. Validation results show a dramatic reduction in the quantity of predictions and an increase in the sensitivity, when compared to other methods. This improves the predictions accuracy and allows the formulation of novel hypotheses on miRNAs functional involvement.
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Biología Computacional/métodos , Genoma , MicroARNs/química , Anotación de Secuencia Molecular/métodos , Reproducibilidad de los Resultados , Programas InformáticosRESUMEN
Cellular senescence is characterized by proliferation and migration exhaustion, senescence-associated secretory phenotype (SASP), and oxidative stress. Senescent vascular smooth muscle cells (VSMCs) contribute to cardiovascular diseases and atherosclerotic plaque instability. Since there are no unanimously agreed senescence markers in human VSMCs, to improve our knowledge, we looked for new possible senescence markers. To this end, we first established and characterized a model of replicative senescence (RS) in human aortic VSMCs. Old cells displayed several established senescence-associated markers. They stained positive for the senescence-associated ß-galactosidase, showed a deranged proliferation rate, a dramatically reduced expression of PCNA, an altered migratory activity, increased levels of TP53 and cell-cycle inhibitors p21/p16, and accumulated in the G1 phase. Old cells showed an altered cellular and nuclear morphology, downregulation of the expression of LMNB1 and HMGB1, and increased expression of SASP molecules (IL1ß, IL6, IL8, and MMP3). In these senescent VSMCs, among a set of 12 manually selected long non-coding RNAs (lncRNAs), we detected significant upregulation of PURPL and NEAT1. We observed also, for the first time, increased levels of RRAD mRNA. The detection of modulated levels of RRAD, PURPL, and NEAT1 during VSMC senescence could be helpful for future studies on potential anti-aging factors.
RESUMEN
Long non-coding RNAs (lncRNAs) have emerged as key regulators of cellular senescence by transcriptionally and post-transcriptionally modulating the expression of many important genes involved in senescence-associated pathways and processes. Among the different lncRNAs associated to senescence, Senescence Associated Long Non-coding RNA (SALNR) was found to be down-regulated in different cellular models of senescence. Since its release in 2015, SALNR has not been annotated in any database or public repository, and no other experimental data have been published. The SALNR sequence is located on the long arm of chromosome 10, at band 10q23.33, and it overlaps the 3' end of the HELLS gene. This investigation helped to unravel the mystery of the existence of SALNR by analyzing publicly available short- and long-read RNA sequencing data sets and RT-PCR analysis in human tissues and cell lines. Additionally, the expression of HELLS has been studied in cellular models of replicative senescence, both in silico and in vitro. Our findings, while not supporting the actual existence of SALNR as an independent transcript in the analyzed experimental models, demonstrate the expression of a predicted HELLS isoform entirely covering the SALNR genomic region. Furthermore, we observed a strong down-regulation of HELLS in senescent cells versus proliferating cells, supporting its role in the senescence and aging process.
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ARN Largo no Codificante , Humanos , ARN Largo no Codificante/genética , Senescencia Celular/genética , Regulación hacia Abajo , Línea Celular , Fibroblastos/fisiología , ADN Helicasas/genéticaRESUMEN
Spinal neurofibromatosis (SNF) is a form of neurofibromatosis type 1 (NF1) characterized by bilateral neurofibromas involving all spinal roots. The pathogenic mechanisms determining the SNF form are currently unknown. To verify the presence of genetic variants possibly related to SNF or classic NF1, we studied 106 sporadic NF1 and 75 SNF patients using an NGS panel of 286 genes encoding RAS pathway effectors and neurofibromin interactors and evaluated the expression of syndecans (SDC1, SDC2, SDC3, SDC4), the NF1 3' tertile interactors, by quantitative real-time PCR. We previously identified 75 and 106 NF1 variants in SNF and NF1 cohorts, respectively. The analysis of the distribution of pathogenic NF1 variants in the three NF1 tertiles showed a significantly higher prevalence of NF1 3' tertile mutations in SNF than in the NF1 cohort. We hypothesized a potential pathogenic significance of the 3' tertile NF1 variants in SNF. The analysis of syndecan expression on PBMCs RNAs from 16 SNF, 16 classic NF1 patients and 16 healthy controls showed that the expression levels of SDC2 and SDC3 were higher in SNF and NF1 patients than in controls; moreover, SDC2, SDC3 and SDC4 were significantly over expressed in patients mutated in the 3' tertile compared to controls. Two different mutational NF1 spectra seem to characterize SNF and classic NF1, suggesting a pathogenic role of NF1 3' tertile and its interactors, syndecans, in SNF. Our study, providing new insights on a possible role of neurofibromin C-terminal in SNF, could address effective personalized patient management and treatments.
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Neurofibromatosis , Neurofibromatosis 1 , Humanos , Neurofibromatosis 1/genética , Neurofibromina 1/genética , Mutación , Sindecanos/genética , Genes de Neurofibromatosis 1RESUMEN
SUMMARY: Chromosomal patterns of genomic signals represent molecular fingerprints that may reveal how the local structural organization of a genome impacts the functional control mechanisms. Thus, the integrative analysis of multiple sources of genomic data and information deepens the resolution and enhances the interpretation of stand-alone high-throughput data. In this note, we present PREDA (Position RElated Data Analysis), an R package for detecting regional variations in genomics data. PREDA identifies relevant chromosomal patterns in high-throughput data using a smoothing approach that accounts for distance and density variability of genomics features. Custom-designed data structures allow efficiently managing diverse signals in different genomes. A variety of smoothing functions and statistics empower flexible and robust workflows. The modularity of package design allows an easy deployment of custom analytical pipelines. Tabular and graphical representations facilitate downstream biological interpretation of results. AVAILABILITY: PREDA is available in Bioconductor and at http://www.xlab.unimo.it/PREDA. CONTACT: silvio.bicciato@unimore.it SUPPLEMENTARY INFORMATION: Supplementary information is available at Bioinformatics online.
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Variación Genética , Genómica/métodos , Programas InformáticosRESUMEN
BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is characterized by recurrent copy number alterations (CNAs) and loss of heterozygosity (LOH), which may have potential diagnostic and prognostic applications. Here, we explored whether ccRCC primary cultures, established from surgical tumor specimens, maintain the DNA profile of parental tumor tissues allowing a more confident CNAs and LOH discrimination with respect to the original tissues. METHODS: We established a collection of 9 phenotypically well-characterized ccRCC primary cell cultures. Using the Affymetrix SNP array technology, we performed the genome-wide copy number (CN) profiling of both cultures and corresponding tumor tissues. Global concordance for each culture/tissue pair was assayed evaluating the correlations between whole-genome CN profiles and SNP allelic calls. CN analysis was performed using the two CNAG v3.0 and Partek software, and comparing results returned by two different algorithms (Hidden Markov Model and Genomic Segmentation). RESULTS: A very good overlap between the CNAs of each culture and corresponding tissue was observed. The finding, reinforced by high whole-genome CN correlations and SNP call concordances, provided evidence that each culture was derived from its corresponding tissue and maintained the genomic alterations of parental tumor. In addition, primary culture DNA profile remained stable for at least 3 weeks, till to third passage. These cultures showed a greater cell homogeneity and enrichment in tumor component than original tissues, thus enabling a better discrimination of CNAs and LOH. Especially for hemizygous deletions, primary cultures presented more evident CN losses, typically accompanied by LOH; differently, in original tissues the intensity of these deletions was weaken by normal cell contamination and LOH calls were missed. CONCLUSIONS: ccRCC primary cultures are a reliable in vitro model, well-reproducing original tumor genetics and phenotype, potentially useful for future functional approaches aimed to study genes or pathways involved in ccRCC etiopathogenesis and to identify novel clinical markers or therapeutic targets. Moreover, SNP array technology proved to be a powerful tool to better define the cell composition and homogeneity of RCC primary cultures.
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Carcinoma de Células Renales/patología , Línea Celular Tumoral/citología , Neoplasias Renales/patología , Carcinoma de Células Renales/química , Carcinoma de Células Renales/genética , Línea Celular Tumoral/química , Línea Celular Tumoral/ultraestructura , Forma de la Célula , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Dosificación de Gen , Perfilación de la Expresión Génica , Inestabilidad Genómica , Genotipo , Humanos , Inmunofenotipificación , Neoplasias Renales/química , Neoplasias Renales/genética , Pérdida de Heterocigocidad , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Polimorfismo de Nucleótido Simple , Eliminación de SecuenciaRESUMEN
The integration of high-throughput genomic data represents an opportunity for deciphering the interplay between structural and functional organization of genomes and for discovering novel biomarkers. However, the development of integrative approaches to complement gene expression (GE) data with other types of gene information, such as copy number (CN) and chromosomal localization, still represents a computational challenge in the genomic arena. This work presents a computational procedure that directly integrates CN and GE profiles at genome-wide level. When applied to DNA/RNA paired data, this approach leads to the identification of Significant Overlaps of Differentially Expressed and Genomic Imbalanced Regions (SODEGIR). This goal is accomplished in three steps. The first step extends to CN a method for detecting regional imbalances in GE. The second part provides the integration of CN and GE data and identifies chromosomal regions with concordantly altered genomic and transcriptional status in a tumor sample. The last step elevates the single-sample analysis to an entire dataset of tumor specimens. When applied to study chromosomal aberrations in a collection of astrocytoma and renal carcinoma samples, the procedure proved to be effective in identifying discrete chromosomal regions of coordinated CN alterations and changes in transcriptional levels.
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Perfilación de la Expresión Génica , Genómica/métodos , Neoplasias/genética , Astrocitoma/genética , Carcinoma/genética , Aberraciones Cromosómicas , Interpretación Estadística de Datos , Dosificación de Gen , Humanos , Neoplasias Renales/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido SimpleRESUMEN
Multiple myeloma (MM) is characterized by marked genomic heterogeneity. Beyond structural rearrangements, a relevant role in its biology is represented by allelic imbalances leading to significant variations in ploidy status. To elucidate better the genomic complexity of MM, we analyzed a panel of 45 patients using combined FISH and microarray approaches. We firstly generated genome-wide profiles of 41 MMs and four plasma cell leukemias, using a self-developed procedure to infer exact local copy numbers (CNs) for each sample. Our analysis allowed the identification of a significant fraction of patients showing near-tetraploidy. Furthermore, a conventional hierarchical clustering analysis showed that near-tetraploidy, 1q gain, hyperdiploidy, and recursive deletions at 1p and chromosomes 13, 14, and 22 were the main aberrations driving samples grouping. Moreover, mapping information was integrated with gene expression profiles of the tumor samples. A multiclass analysis of transcriptional profiles characterizing the different clusters showed marked gene-dosage effects, particularly concerning 1q transcripts; this finding was also confirmed by a nonparametric analysis between normalized gene expression levels and local CN variations (1027 highly-significant correlated genes). Finally, we identified several loci in which gene expression correlated with the occurrence of loss of heterozygosity. Our results provide insights into the composite network linking genome structure and transcriptional features in MM.
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Desequilibrio Alélico , Dosificación de Gen , Hibridación Fluorescente in Situ/métodos , Mieloma Múltiple/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Polimorfismo de Nucleótido Simple , Anciano , Anciano de 80 o más Años , Aberraciones Cromosómicas , Mapeo Cromosómico , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Genoma Humano , Humanos , Pérdida de Heterocigocidad , Masculino , Persona de Mediana Edad , Mieloma Múltiple/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Oxidorreductasa que Contiene Dominios WWRESUMEN
Noonan syndrome (NS) is an autosomal-dominant disorder with variable expressivity and locus heterogeneity. Despite several RAS pathway genes were implicated in NS, 20-30% of patients remain without molecular diagnosis, suggesting the involvement of further genes or multiple mechanisms. Eight patients out of 60, negative for conventional NS mutation analysis, with heterogeneous NS phenotype were investigated by means of target resequencing of 26 RAS/MAPK pathway genes. A trio was further characterized by means of whole-exome sequencing. Protein modeling and in silico prediction of protein stability allowed to identify possible pathogenic RAS pathway variants in four NS patients. A new c.355T>C variant in LZTR1 was found in patient 43. Two patients co-inherited variants in LRP1 and LZTR1 (patient 53), or LRP1 and SOS1 genes (patient 67). The forth patient (56) carried a compound heterozygote of RASAL3 gene variants and also an A2ML1 variant. While these subclinical variants are singularly present in healthy parents, they co-segregate in patients, suggesting their addictive effect and supporting a digenic inheritance, as alternative model to a more common monogenic transmission. The ERK1/2 and SAPK/JNK activation state, assessed on immortalized lymphocytes from patients 53 and 67 showed highest phosphorylation levels compared to their asymptomatic parents. These findings together with the lack of their co-occurrence in the 1000Genomes database strengthen the hypothesis of digenic inheritance in a subset of NS patients. This study suggests caution in the exclusion of subclinical variants that might play a pathogenic role providing new insights for alternative hereditary mechanisms.
Asunto(s)
Exoma , Herencia Multifactorial , Mutación , Síndrome de Noonan/genética , Fenotipo , Adulto , Anciano , Femenino , Proteínas Activadoras de GTPasa/genética , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Sistema de Señalización de MAP Quinasas/genética , Masculino , Persona de Mediana Edad , Síndrome de Noonan/patología , Proteína SOS1/genética , Factores de Transcripción/genética , alfa-Macroglobulinas/genéticaRESUMEN
Histone deacetylase 8 (HDAC8), a class I HDAC that modifies non-histone proteins such as p53, is highly expressed in different hematological neoplasms including a subtype of acute myeloid leukemia (AML) bearing inversion of chromosome 16 [inv(16)]. To investigate HDAC8 contribution to hematopoietic stem cell maintenance and myeloid leukemic transformation, we generated a zebrafish model with Hdac8 overexpression and observed an increase in hematopoietic stem/progenitor cells, a phenotype that could be reverted using a specific HDAC8 inhibitor, PCI-34051 (PCI). In addition, we demonstrated that AML cell lines respond differently to PCI treatment: HDAC8 inhibition elicits cytotoxic effect with cell cycle arrest followed by apoptosis in THP-1 cells, and cytostatic effect in HL60 cells that lack p53. A combination of cytarabine, a standard anti-AML chemotherapeutic, with PCI resulted in a synergistic effect in all the cell lines tested. We, then, searched for a mechanism behind cell cycle arrest caused by HDAC8 inhibition in the absence of functional p53 and demonstrated an involvement of the canonical WNT signaling in zebrafish and in cell lines. Together, we provide the evidence for the role of HDAC8 in hematopoietic stem cell differentiation in zebrafish and AML cell lines, suggesting HDAC8 inhibition as a therapeutic target in hematological malignancies. Accordingly, we demonstrated the utility of a highly specific HDAC8 inhibition as a therapeutic strategy in combination with standard chemotherapy.