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The RE1-silencing transcription factor (REST) acts either as a repressor or activator of transcription depending on the genomic and cellular context. REST is a key player in brain cell differentiation by inducing chromatin modifications, including DNA methylation, in a proximity of its binding sites. Its dysfunction may contribute to oncogenesis. Mutations in IDH1/2 significantly change the epigenome contributing to blockade of cell differentiation and glioma development. We aimed at defining how REST modulates gene activation and repression in the context of the IDH mutation-related phenotype in gliomas. We studied the effects of REST knockdown, genome wide occurrence of REST binding sites, and DNA methylation of REST motifs in IDH wild type and IDH mutant gliomas. We found that REST target genes, REST binding patterns, and TF motif occurrence proximal to REST binding sites differed in IDH wild-type and mutant gliomas. Among differentially expressed REST targets were genes involved in glial cell differentiation and extracellular matrix organization, some of which were differentially methylated at promoters or gene bodies. REST knockdown differently impacted invasion of the parental or IDH1 mutant glioma cells. The canonical REST-repressed gene targets showed significant correlation with the GBM NPC-like cellular state. Interestingly, results of REST or KAISO silencing suggested the interplay between these TFs in regulation of REST-activated and repressed targets. The identified gene regulatory networks and putative REST cooperativity with other TFs, such as KAISO, show distinct REST target regulatory networks in IDH-WT and IDH-MUT gliomas, without concomitant DNA methylation changes. We conclude that REST could be an important therapeutic target in gliomas.
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Neoplasias Encefálicas , Metilación de ADN , Redes Reguladoras de Genes , Glioma , Isocitrato Deshidrogenasa , Mutación , Isocitrato Deshidrogenasa/genética , Glioma/genética , Glioma/patología , Glioma/metabolismo , Humanos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Regulación Neoplásica de la Expresión Génica/genéticaRESUMEN
OBJECTIVE: To analyze the correlation between the miRNA expression profile in vestibular schwannoma (VS) tumor tissue and preoperative patient's hearing status, using the RNA-seq technique. METHODS: Nineteen tumor samples were collected from patients operated for VS in a Tertiary Academic Center. Samples were classified into "good hearing" and "poor hearing" study group based on the results of audiometric studies. Tumor miRNA expression was analyzed using high-throughput RNA sequencing (RNA-seq) technique, using NovaSeq 6000 Illumina system. Functional analysis was performed with the use of DIANA miRpath v. 4.0 online tool. RESULTS: The most overexpressed miRNAs in VS samples derived from poor hearing patients belonged to miR 449a/b, miR 15/16-1, and hypoxamiR families. Functional analysis showed that the differentially expressed miRNAs regulate cellular pathways associated with hypoxia, adherence junction functions, and signaling pathways such as Hippo, FOXO, MAPK, and Wnt signaling pathway. CONCLUSION: Our study identified a specific miRNA expression profile in VS tumor tissues that correlates with hearing impairment. These results suggest potential new molecular mechanisms related to hearing loss in the course of VS. LEVEL OF EVIDENCE: 3 (cohort study) Laryngoscope, 134:3778-3785, 2024.
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Pérdida Auditiva , Secuenciación de Nucleótidos de Alto Rendimiento , MicroARNs , Neuroma Acústico , Humanos , Neuroma Acústico/genética , MicroARNs/genética , Masculino , Femenino , Persona de Mediana Edad , Pérdida Auditiva/genética , Pérdida Auditiva/etiología , Adulto , Análisis de Secuencia de ARN/métodos , AncianoRESUMEN
PURPOSE: To assess the differences in the miRNA expression profile between small (stage I Koos classification) and large solid vestibular schwannoma (VS) tumors, using the RNA-seq technique. METHODS: Twenty tumor samples (10 small and 10 large tumors) were collected from patients operated for VS in a Tertiary Academic Center. Tumor miRNA expression was analyzed using high-throughput RNA sequencing (RNA-seq) technique, with NovaSeq 6000 Illumina system. Bioinformatics analysis was done using statistical software R. Gene enrichment and functional analysis was performed using miRTargetLink 2.0 and DIANA miRpath 3.0 online tools. RESULTS: We identified 9 differentially expressed miRNAs in large VS samples: miR-7, miR-142 (-3p and -5p), miR-155, miR-342, miR-1269, miR-4664, and miR-6503 were upregulated, whereas miR-204 was significantly down-regulated in comparison to small VS samples. Gene enrichment analysis showed that the most enriched target genes were SCD, TMEM43, LMNB2, JARID2, and CCND1. The most enriched functional pathways were associated with lipid metabolism, along with signaling pathways such as Hippo and FOXO signaling pathway. CONCLUSION: We identified a set of 9 miRNAs that are significantly deregulated in large VS in comparison to small, intracanalicular tumors. The functional enrichment analysis of these miRNAs suggests novel mechanisms, such as that lipid metabolism, as well as Hippo and FOxO signaling pathways that may play an important role in VS growth regulation.
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MicroARNs , Neuroma Acústico , Humanos , Neuroma Acústico/genética , MicroARNs/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ARNRESUMEN
Diffuse intrinsic pontine gliomas (DIPGs) are deadly pediatric brain tumors, non-resectable due to brainstem localization and diffusive growth. Over 80% of DIPGs harbor a mutation in histone 3 (H3.3 or H3.1) resulting in a lysine-to-methionine substitution (H3K27M). Patients with DIPG have a dismal prognosis with no effective therapy. We show that histone deacetylase (HDAC) inhibitors lead to a significant reduction in the H3.3K27M protein (up to 80%) in multiple glioma cell lines. We discover that the SB939-mediated H3.3K27M loss is partially blocked by a lysosomal inhibitor, chloroquine. The H3.3K27M loss is facilitated by co-occurrence of H2A.Z, as evidenced by the knockdown of H2A.Z isoforms. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis confirms the occupancy of H3.3K27M and H2A.Z at the same SB939-inducible genes. We discover a mechanism showing that HDAC inhibition in DIPG leads to pharmacological modulation of the oncogenic H3.3K27M protein levels. These findings show the possibility of directly targeting the H3.3K27M oncohistone.
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Neoplasias Encefálicas , Glioma Pontino Intrínseco Difuso , Glioma , Humanos , Niño , Histonas , Proteínas Mutantes , Glioma/genética , Neoplasias Encefálicas/genética , Inhibidores de Histona Desacetilasas/farmacologíaRESUMEN
Loricrin keratoderma (LK) is a rare autosomal dominant genodermatosis caused by LORICRIN gene mutations. The pathogenesis of the disease is not yet fully understood. So far, only 10 pathogenic variants in LORICRIN have been described, with all of them but one being deletions or insertions. The significance of rare nonsense variants remains unclear. Furthermore, no data regarding the RNA expression in affected patients are available. The aim of this study is to describe the two variants in the LORICRIN gene found in two distinct families: the novel pathogenic variant c.639_642dup and a rare c.10C > T (p.Gln4Ter) of unknown significance. We also present the results of the transcriptome analysis of the lesional loricrin keratoderma epidermis of a patient with c.639_642dup. We show that in the LK lesion, the genes associated with epidermis development and keratocyte differentiation are upregulated, while genes engaged in cell adhesion, differentiation developmental processes, ion homeostasis and transport, signaling and cell communication are downregulated. In the context of the p.Gln4Ter clinical significance evaluation, we provide data indicating that LORICRIN haploinsufficiency has no skin consequences. Our results give further insight into the pathogenesis of LK, which may have therapeutic implications in the future and important significance in the context of genetic counseling.
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Enfermedades Cutáneas Genéticas , Humanos , Enfermedades Cutáneas Genéticas/metabolismo , Epidermis/metabolismo , Perfilación de la Expresión GénicaRESUMEN
High-grade gliomas are aggressive, deadly primary brain tumors. Median survival of patients with glioblastoma (GBM, WHO grade 4) is 14 months and <10% of patients survive 2 years. Despite improved surgical strategies and forceful radiotherapy and chemotherapy, the prognosis of GBM patients is poor and did not improve over decades. We performed targeted next-generation sequencing with a custom panel of 664 cancer- and epigenetics-related genes, and searched for somatic and germline variants in 180 gliomas of different WHO grades. Herein, we focus on 135 GBM IDH-wild type samples. In parallel, mRNA sequencing was accomplished to detect transcriptomic abnormalities. We present the genomic alterations in high-grade gliomas and the associated transcriptomic patterns. Computational analyses and biochemical assays showed the influence of TOP2A variants on enzyme activities. In 4/135 IDH-wild type GBMs we found a novel, recurrent mutation in the TOP2A gene encoding topoisomerase 2A (allele frequency [AF] = 0.03, 4/135 samples). Biochemical assays with recombinant, wild type (WT) and variant proteins demonstrated stronger DNA binding and relaxation activity of the variant protein. GBM patients carrying the altered TOP2A had shorter overall survival (median OS 150 vs 500 days, P = .0018). In the GBMs with the TOP2A variant we found transcriptomic alterations consistent with splicing dysregulation. luA novel, recurrent TOP2A mutation, which was found exclusively in four GBMs, results in the TOP2A E948Q variant with altered DNA binding and relaxation activities. The deleterious TOP2A mutation resulting in transcription deregulation in GBMs may contribute to disease pathology.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Glioblastoma/patología , Neoplasias Encefálicas/metabolismo , Glioma/genética , Pronóstico , ADN , Isocitrato Deshidrogenasa/genética , MutaciónRESUMEN
Studies conducted on large populations show a lack of connection between vaccination and serious neurological symptoms. However, there are isolated cases that indicate such a relationship. These reports on adverse effects following immunization (AEFI) reduce social confidence in vaccination; however, their background may be rare genetic defects. The aim of the presented study was to examine if neurological AEFI in children may be associated with variants in genes related to neurodevelopment. To identify such possible associations, a descriptive study of the Polish case series was conducted. We performed next-generation sequencing in patients who, up to 4 weeks of injection of any vaccine, manifested neurological AEFI. We included 23 previously normally developing children with first seizures that occurred after vaccination. We identified pathogenic/likely pathogenic variants in genes engaged in neurodevelopment in nine patients and variants of uncertain significance in another nine patients. The mutated genes belonged to the group of genes related to epilepsy syndromes/epileptic encephalopathy. We showed that AEFI might have a genetic background. We hypothesized that in some AEFI patients, the vaccine might only trigger neurological symptoms that would have been manifested anyway as a result of a pathogenic variant in a gene engaged in neurodevelopment.
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Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Vacunas , Humanos , Niño , Polonia , Inmunización , Vacunación/efectos adversos , Vacunas/efectos adversos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/etiología , Convulsiones/genética , Convulsiones/inducido químicamente , Factores de Riesgo , Sistemas de Registro de Reacción Adversa a MedicamentosRESUMEN
Heat Shock Factor 1 (HSF1), a transcription factor frequently overexpressed in cancer, is activated by proteotoxic agents and participates in the regulation of cellular stress response. To investigate how HSF1 level affects the response to proteotoxic stress, we integrated data from functional genomics analyses performed in MCF7 breast adenocarcinoma cells. Although the general transcriptional response to heat shock was impaired due to HSF1 deficiency (mainly chaperone expression was inhibited), a set of genes was identified, including ATF3 and certain FOS and JUN family members, whose stress-induced activation was stronger and persisted longer than in cells with normal HSF1 levels. These genes were direct HSF1 targets, suggesting a dual (activatory/suppressory) role for HSF1. Moreover, we found that heat shock-induced inflammatory response could be stronger in HSF1-deficient cells. Analyses of The Cancer Genome Atlas data indicated that higher ATF3, FOS, and FOSB expression levels correlated with low HSF1 levels in estrogen receptor-positive breast cancer, reflecting higher heat shock-induced expression of these genes in HSF1-deficient MCF7 cells observed in vitro. However, differences between the analyzed cancer types were noted in the regulation of HSF1-dependent genes, indicating the presence of cell-type-specific mechanisms. Nevertheless, our data indicate the existence of the heat shock-induced network of transcription factors (associated with the activation of TNFα signaling) which includes HSF1. Independent of its chaperone-mediated cytoprotective function, HSF1 may be involved in the regulation of this network but prevents its overactivation in some cells during stress.
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Proteínas de Unión al ADN , Genes fos , Factor de Transcripción Activador 3/genética , Factor de Transcripción Activador 3/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción del Choque Térmico/genética , Respuesta al Choque Térmico/genética , Humanos , Inflamación/genética , Factores de Transcripción/metabolismoRESUMEN
The formation of the synovial joint begins with the visible emergence of a stripe of densely packed mesenchymal cells located between distal ends of the developing skeletal anlagen called the interzone. Recently the transcriptome of the early synovial joint was reported. Knowledge about enhancers would complement these data and lead to a better understanding of the control of gene transcription at the onset of joint development. Using ChIP-sequencing we have mapped the H3-signatures H3K27ac and H3K4me1 to locate regulatory elements specific for the interzone and adjacent phalange, respectively. This one-stage atlas of candidate enhancers (CEs) was used to map the association between these respective joint tissue specific CEs and biological processes. Subsequently, integrative analysis of transcriptomic data and CEs identified new putative regulatory elements of genes expressed in interzone (e.g., GDF5, BMP2 and DACT2) and phalange (e.g., MATN1, HAPLN1 and SNAI1). We also linked such CEs to genes known as crucial in synovial joint hypermobility and osteoarthritis, as well as phalange malformations. These analyses show that the CE atlas can serve as resource for identifying, and as starting point for experimentally validating, putative disease-causing genomic regulatory regions in patients with synovial joint dysfunctions and/or phalange disorders, and enhancer-controlled synovial joint and phalange formation.
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Osteoartritis , Transcriptoma , Proteínas Adaptadoras Transductoras de Señales , Extremidades , Humanos , Articulaciones , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Spatial chromatin organization is crucial for transcriptional regulation and might be particularly important in neurons since they dramatically change their transcriptome in response to external stimuli. We show that stimulation of neurons causes condensation of large chromatin domains. This phenomenon can be observed in vitro in cultured rat hippocampal neurons as well as in vivo in the amygdala and hippocampal neurons. Activity-induced chromatin condensation is an active, rapid, energy-dependent, and reversible process. It involves calcium-dependent pathways but is independent of active transcription. It is accompanied by the redistribution of posttranslational histone modifications and rearrangements in the spatial organization of chromosome territories. Moreover, it leads to the reorganization of nuclear speckles and active domains located in their proximity. Finally, we find that the histone deacetylase HDAC1 is the key regulator of this process. Our results suggest that HDAC1-dependent chromatin reorganization constitutes an important level of transcriptional regulation in neurons.
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Cromatina/metabolismo , Histona Desacetilasa 1/metabolismo , Neuronas/metabolismo , Animales , Señalización del Calcio , Cromatina/ultraestructura , Cromosomas de los Mamíferos/metabolismo , Metabolismo Energético , Hipocampo/citología , Potenciación a Largo Plazo , Ratones Endogámicos C57BL , Ratas Wistar , Transcripción GenéticaRESUMEN
Heat shock factor 1 (HSF1), a key regulator of transcriptional responses to proteotoxic stress, was linked to estrogen (E2) signaling through estrogen receptor α (ERα). We found that an HSF1 deficiency may decrease ERα level, attenuate the mitogenic action of E2, counteract E2-stimulated cell scattering, and reduce adhesion to collagens and cell motility in ER-positive breast cancer cells. The stimulatory effect of E2 on the transcriptome is largely weaker in HSF1-deficient cells, in part due to the higher basal expression of E2-dependent genes, which correlates with the enhanced binding of unliganded ERα to chromatin in such cells. HSF1 and ERα can cooperate directly in E2-stimulated regulation of transcription, and HSF1 potentiates the action of ERα through a mechanism involving chromatin reorganization. Furthermore, HSF1 deficiency may increase the sensitivity to hormonal therapy (4-hydroxytamoxifen) or CDK4/6 inhibitors (palbociclib). Analyses of data from The Cancer Genome Atlas database indicate that HSF1 increases the transcriptome disparity in ER-positive breast cancer and can enhance the genomic action of ERα. Moreover, only in ER-positive cancers an elevated HSF1 level is associated with metastatic disease.
About 70% of breast cancers rely on supplies of a hormone called estrogen which is the main hormone responsible for female physical characteristics to grow. Breast cancer cells that are sensitive to estrogen possess proteins known as estrogen receptors and are classified as estrogen-receptor positive. When estrogen interacts with its receptor in a cancer cell, it stimulates the cell to grow and migrate to other parts of the body. Therefore, therapies that decrease the amount of estrogen the body produces, or inhibit the receptor itself, are widely used to treat patients with estrogen receptor-positive breast cancers. When estrogen interacts with an estrogen receptor known as ERα it can also activate a protein called HSF1, which helps cells to survive under stress. In turn, HSF1 regulates several other proteins that are necessary for ERα and other estrogen receptors to work properly. Previous studies have suggested that high levels of HSF1 may worsen the outcomes for patients with estrogen receptor-positive breast cancers, but it remains unclear how HSF1 acts in breast cancer cells. Vydra, Janus, Kus et al. used genetics and bioinformatics approaches to study HSF1 in human breast cancer cells. The experiments revealed that breast cancer cells with lower levels of HSF1 also had lower levels of ERα and responded less well to estrogen than cells with higher levels of HSF1. Further experiments suggested that in the absence of estrogen, HSF1 helps to keep ERα inactive. However, when estrogen is present, HSF1 cooperates with ERα and enhances its activity to help cells grow and migrate. Vydra, Janus, Kus et al. also found that cells with higher levels of HSF1 were less sensitive to two drug therapies that are commonly used to treat estrogen receptor-positive breast cancers. These findings reveal that the effect HSF1 has on ERα activity depends on the presence of estrogen. Therefore, cancer therapies that decrease the amount of estrogen a patient produces may have a different effect on estrogen receptor-positive tumors with high HSF1 levels than tumors with low HSF1 levels.
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Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/genética , Estrógenos/metabolismo , Factores de Transcripción del Choque Térmico/genética , Transducción de Señal , Adulto , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Receptor alfa de Estrógeno/metabolismo , Femenino , Factores de Transcripción del Choque Térmico/metabolismo , Humanos , Persona de Mediana Edad , Adulto JovenRESUMEN
Glioblastomas (GBM) are the common and aggressive primary brain tumors that are incurable by conventional therapies. Immunotherapy with immune checkpoint inhibitors is not effective in GBM patients due to the highly immunosuppressive tumor microenvironment (TME) restraining the infiltration and activation of cytotoxic T cells. Clinical and experimental studies showed the upregulation of expression of the arginase 1 and 2 (ARG1 and ARG2, respectively) in murine and human GBMs. The elevated arginase activity leads to the depletion of L-arginine, an amino-acid required for the proliferation of T lymphocytes and natural killer cells. Inhibition of ARG1/2 in the TME may unblock T cell proliferation and activate effective antitumor responses. To explore the antitumor potential of ARG1/2 inhibition, we analyzed bulk and single-cell RNA sequencing (scRNA-seq) data from human and murine gliomas. We found the upregulation of ARG1/2 expression in GBMs, both in tumor cells and in tumor infiltrating microglia and monocytes/macrophages. We employed selective arginase inhibitors to evaluate if ARG1/2 inhibition in vitro and in vivo exerts the antitumor effects. A novel, selective ARG1/2 inhibitor - OAT-1746 blocked microglia-dependent invasion of U87-MG and LN18 glioma cells in a Matrigel invasion assay better than reference compounds, without affecting the cell viability. OAT-1746 effectively crossed the blood brain barrier in mice and increased arginine levels in the brains of GL261 glioma bearing mice. We evaluated its antitumor efficacy against GL261 intracranial gliomas as a monotherapy and in combination with the PD-1 inhibition. The oral treatment with OAT-1746 did not affect the immune composition of TME, it induced profound transcriptomic changes in CD11b+ cells immunosorted from tumor-bearing brains as demonstrated by RNA sequencing analyses. Treatment with OAT-1746 modified the TME resulting in reduced glioma growth and increased antitumor effects of the anti-PD-1 antibody. Our findings provide the evidence that inhibition of ARG1/2 activity in tumor cells and myeloid cells in the TME unblocks antitumor responses in myeloid cells and NK cells, and improves the efficacy of the PD-1 inhibition.
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Chromatin structure and accessibility, and combinatorial binding of transcription factors to regulatory elements in genomic DNA control transcription. Genetic variations in genes encoding histones, epigenetics-related enzymes or modifiers affect chromatin structure/dynamics and result in alterations in gene expression contributing to cancer development or progression. Gliomas are brain tumors frequently associated with epigenetics-related gene deregulation. We perform whole-genome mapping of chromatin accessibility, histone modifications, DNA methylation patterns and transcriptome analysis simultaneously in multiple tumor samples to unravel epigenetic dysfunctions driving gliomagenesis. Based on the results of the integrative analysis of the acquired profiles, we create an atlas of active enhancers and promoters in benign and malignant gliomas. We explore these elements and intersect with Hi-C data to uncover molecular mechanisms instructing gene expression in gliomas.
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Cromatina , Glioma/genética , Secuencias Reguladoras de Ácidos Nucleicos , Sitios de Unión , Neoplasias Encefálicas/genética , Inmunoprecipitación de Cromatina , ADN/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Epigénesis Genética , Epigenómica , Proteína Forkhead Box M1 , Expresión Génica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioblastoma , Código de Histonas , Histonas , Humanos , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismoRESUMEN
LMNA-related congenital muscular dystrophy (L-CMD) is the most severe phenotypic form of skeletal muscle laminopathies. This paper reports clinical presentation of the disease in 15 Polish patients from 13 families with genetically confirmed skeletal muscle laminopathy. In all these patients floppy infant syndrome was the first manifestation of the disease. The genetic diagnosis was established by next generation sequencing (targeted panel or exome; 11 patients) or classic Sanger sequencing (4 patients). In addition to known pathogenic LMNA variants: c.116A > G (p.Asn39Ser), c.745C > T (p.Arg249Trp), c.746G > A (p.Arg249Gln), c.1072G > A (p.Glu358Lys), c.1147G > A (p.Glu383Lys), c.1163G > C (p.Arg388Pro), c.1357C > T (p.Arg453Trp), c.1583C > G (p.Thr528Arg), we have identified three novel ones: c.121C > G (p.Arg41Gly), c.1127A > G (p.Tyr376Cys) and c.1160T > C (p.Leu387Pro). Eleven patients had de novo mutations, 4 - familial. In one family we observed intrafamilial variability of clinical course: severe L-CMD in the male proband, intermediate form in his sister and asymptomatic in their mother. One asymptomatic father had somatic mosaicism. L-CMD should be suspected in children with hypotonia in infancy and delayed motor development, who have poor head control, severe hyperlordosis and unstable and awkward gait. Serum creatine kinase may be high (~1000IU/l). Progression of muscle weakness is fast, leading to early immobilization. In some patients with L-CMD joint contractures can develop with time. MRI shows that the most frequently affected muscles are the serratus anterior, lumbar paraspinal, gluteus, vastus, adductor magnus, hamstrings, medial head of gastrocnemius and soleus. Ultra-rare laminopathies can be a relatively common cause of generalized hypotonia in children. Introduction of wide genome sequencing methods was a breakthrough in diagnostics of diseases with great clinical and genetic variability and allowed approach "from genotype do phenotype". However target sequencing of LMNA gene could be considered in selected patients with clinical picture suggestive for laminopathy.
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Lamina Tipo A/genética , Hipotonía Muscular/genética , Distrofias Musculares/complicaciones , Distrofias Musculares/genética , Adolescente , Niño , Preescolar , Femenino , Genotipo , Humanos , Imagen por Resonancia Magnética , Masculino , Mutación , Linaje , FenotipoRESUMEN
High-grade gliomas (HGGs), the most common and aggressive primary brain tumors in adults, inevitably recur due to incomplete surgery or resistance to therapy. Intratumoral genomic and cellular heterogeneity of HGGs contributes to therapeutic resistance, recurrence, and poor clinical outcomes. Transcriptomic profiles of HGGs at recurrence have not been investigated in detail. Using targeted sequencing of cancer-related genes and transcriptomics, we identified single nucleotide variations, small insertions and deletions, copy number aberrations (CNAs), as well as gene expression changes and pathway deregulation in 16 pairs of primary and recurrent HGGs. Most of the somatic mutations identified in primary HGGs were not detected after relapse, suggesting a subclone substitution during the tumor progression. We found a novel frameshift insertion in the ZNF384 gene which may contribute to extracellular matrix remodeling. An inverse correlation of focal CNAs in EGFR and PTEN genes was detected. Transcriptomic analysis revealed downregulation of genes involved in messenger RNA splicing, cell cycle, and DNA repair, while genes related to interferon signaling and phosphatidylinositol (PI) metabolism are upregulated in secondary HGGs when compared to primary HGGs. In silico analysis of the tumor microenvironment identified M2 macrophages and immature dendritic cells as enriched in recurrent HGGs, suggesting a prominent immunosuppressive signature. Accumulation of those cells in recurrent HGGs was validated by immunostaining. Our findings point to a substantial transcriptomic deregulation and a pronounced infiltration of immature dendritic cells in recurrent HGG, which may impact the effectiveness of frontline immunotherapies in the GBM management. KEY MESSAGES: Most of the somatic mutations identified in primary HGGs were not detected after relapse. Focal CNAs in EGFR and PTEN genes are inversely correlated in primary and recurrent HGGs. Transcriptomic changes and distinct immune-related signatures characterize HGG recurrence. Recurrent HGGs are characterized by a prominent infiltration of immature dendritic and M2 macrophages.
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Neoplasias Encefálicas/genética , Neoplasias Encefálicas/inmunología , Glioma/genética , Glioma/inmunología , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/inmunología , Adulto , Anciano , Variaciones en el Número de Copia de ADN , Células Dendríticas/inmunología , Receptores ErbB/genética , Femenino , Humanos , Macrófagos/inmunología , Masculino , Persona de Mediana Edad , Mutación , Fosfohidrolasa PTEN/genética , Transactivadores/genética , Transcriptoma , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
Anti-tumour therapies eliminate proliferating tumour cells by induction of DNA damage, but genomic aberrations or transcriptional deregulation may limit responses to therapy. Glioblastoma (GBM) is a malignant brain tumour, which recurs inevitably due to chemo- and radio-resistance. Human RecQ helicases participate in DNA repair, responses to DNA damage and replication stress. We explored if a helicase RECQL4 contributes to gliomagenesis and responses to chemotherapy. We found upregulated RECQL4 expression in GBMs associated with poor survival of GBM patients. Increased levels of nuclear and cytosolic RECQL4 proteins were detected in GBMs on tissue arrays and in six glioma cell lines. RECQL4 was detected both in cytoplasm and mitochondria by Western blotting and immunofluorescence. RECQL4 depletion in glioma cells with siRNAs and CRISPR/Cas9 did not affect basal cell viability, slightly impaired DNA replication, but induced profound transcriptomic changes and increased chemosensitivity of glioma cells. Sphere cultures originated from RECQL4-depleted cells had reduced sphere forming capacity, stronger responded to temozolomide upregulating cell cycle inhibitors and pro-apoptotic proteins. RECQL4 deficiency affected mitochondrial network and reduced mitochondrial membrane polarization in LN18 glioblastoma cells. We demonstrate that targeting RECQL4 overexpressed in glioblastoma could be a new strategy to sensitize glioma cells to chemotherapeutics.
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Accumulating evidence suggests that glioma stem cells (GSCs), which are rare cells characterized by pluripotency and self-renewal ability, are responsible for glioblastoma (GBM) propagation, recurrence and resistance to therapies. Bone morphogenic proteins (BMPs) induce GSC differentiation, which leads to elimination of GSCs and sensitization of glioma to chemotherapeutics. Alterations in the epidermal growth factor receptor (EGFR) gene are detected in more than half of GBMs; however, the role of EGFR in the chemoresistance of GSCs remains unknown. Here, we examined whether EGFR signaling affects BMP4-induced differentiation of GSCs and their response to the alkylating drug temozolomide (TMZ). We show that BMP4 triggers the SMAD signaling cascade in GSCs independent of the EGFR level. BMP4 downregulated the levels of pluripotency markers (SOX2 and OLIG2) with a concomitant induction of an astrocytic marker (GFAP) and a neuronal marker (ß-Tubulin III). However, GSCs with different EGFR levels responded differently to treatments. BMP4-induced differentiation did not enhance sensitivity to TMZ in EGFRlow GSCs, in contrast to EGFRhigh GSCs, which underwent apoptosis. We then identified differences in cell cycle regulation. In EGFRlow cells, BMP4-triggered G1 cell cycle arrest which was not detected in EGFRhigh cells. RNA-seq profiles further highlighted transcriptomic alterations and distinct processes characterizing EGFR-dependent responses in the course of BMP4-induced differentiation. We found that the control of BIM (the pro-apoptotic BCL-2 family protein) by the AKT/FOXO3a axis only operated in BMP4-differentiated EGFRhigh cells upon TMZ treatment.
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Proteína 11 Similar a Bcl2/metabolismo , Proteína Morfogenética Ósea 4/farmacología , Neoplasias Encefálicas/metabolismo , Diferenciación Celular , Receptores ErbB/metabolismo , Proteína Forkhead Box O3/metabolismo , Glioma/metabolismo , Temozolomida/farmacología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioma/genética , Glioma/patología , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patología , Transcriptoma/genéticaRESUMEN
In the light of the increasing occurrence of antifungal resistance, there is an urgent need to search for new therapeutic strategies to overcome this phenomenon. One of the applied approaches is the synthesis of small-molecule compounds showing antifungal properties. Here we present a continuation of the research on the recently discovered anti-Candida albicans agent 4-AN. Using next generation sequencing and transcriptional analysis, we revealed that the treatment of C. albicans with 4-AN can change the expression profile of a large number of genes. The highest upregulation was observed in the case of genes involved in cell stress, while the highest downregulation was shown for genes coding sugar transporters. Real-time PCR analysis revealed 4-AN mediated reduction of the relative expression of genes engaged in fungal virulence (ALS1, ALS3, BCR1, CPH1, ECE1, EFG1, HWP1, HYR1 and SAP1). The determination of the fractional inhibitory concentration index (FICI) showed that the combination of 4-AN with amphotericin B is synergistic. Finally, flow cytometry analysis revealed that the compound induces mainly necrosis in C. albicans cells.
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Antifúngicos/farmacología , Candida albicans/crecimiento & desarrollo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Genoma Fúngico , Necrosis , Virulencia/genética , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Candida albicans/efectos de los fármacos , Candida albicans/genética , Proteínas Fúngicas/genética , Perfilación de la Expresión GénicaRESUMEN
Central nervous system (CNS)-border associated macrophages (BAMs) maintain their steady-state population during adulthood and are not replaced by circulating monocytes under physiological conditions. Their roles in CNS integrity and functions under pathological conditions remain largely unknown. Until recently, BAMs and microglia could not be unequivocally distinguished due to expression of common macrophage markers. We investigated the transcriptional profiles of immunosorted BAMs from rat sham-operated and ischemic brains using RNA sequencing. We found that BAMs express the distinct transcriptional signature than microglia and infiltrating macrophages. The enrichment of functional groups associated with the cell cycle in CD163+ cells isolated 3 days after the ischemic injury indicates the proliferative capacity of these cells. The increased number of CD163+ cells 3 days post-ischemia was corroborated by flow cytometry and detecting the increased number of CD163+ cells positive for a proliferation marker Ki67 at perivascular spaces. CD163+ cells in the ischemic brains up-regulated many inflammatory genes and parenchymal CD163+ cells expressed iNOS, which indicates acquisition of a pro-inflammatory phenotype. In mice, BAMs typically express CD206 and we found a subset of these cells expressing CD169. Chimeric mice generated by transplanting bone marrow of donor Cx3cr1gfpCCR2rfp mice to wild type hosts showed an increased number of CX3CR1+CD169+ perivascular macrophages 3 days post-ischemia. Furthermore, these cells accumulated in the brain parenchyma and we detected replacement of perivascular macrophages by peripheral monocytic cells in the sub-acute phase of stroke. In line with the animal results, post-mortem brain samples from human ischemic stroke cases showed time-dependent accumulation of CD163+ cells in the ischemic parenchyma. Our findings indicate a unique transcriptional signature of BAMs, their local proliferation and migration of inflammatory BAMs to the brain parenchyma after stroke in animal models and humans.