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BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a blistering disease caused by mutations in the gene encoding type VII collagen (C7). RDEB is associated with fibrosis, which is responsible for severe complications. The phenotypic variability observed in siblings with RDEB suggests that epigenetic modifications contribute to disease severity. Identifying epigenetic changes may help to uncover molecular mechanisms underlying RDEB pathogenesis and new therapeutic targets. OBJECTIVES: To investigate histone acetylation in RDEB skin and to explore histone deacetylase inhibitors (HDACi) as therapeutic molecules capable of counteracting fibrosis and disease progression in RDEB mice. METHODS: Acetylated histone levels were detected in human skin by immunofluorescence and in RDEB fibroblasts by enzyme-linked immunosorbent assay (ELISA). The effects of givinostat and valproic acid (VPA) on RDEB fibroblast fibrotic behaviour were assessed by a collagen-gel contraction assay, Western blot and immunocytofluorescence for α-smooth muscle actin, and ELISA for released transforming growth factor (TGF)-ß1. RNA sequencing was performed in HDACi- and vehicle-treated RDEB fibroblasts. VPA was systemically administered to RDEB mice and effects on overt phenotype were monitored. Fibrosis was investigated in the skin using histological and immunofluorescence analyses. Eye and tongue defects were examined microscopically. Mass spectrometry proteomics was performed on skin protein extracts from VPA-treated RDEB and control mice. RESULTS: Histone acetylation decreases in RDEB skin and primary fibroblasts. RDEB fibroblasts treated with HDACi lowered fibrotic traits, including contractility, TGF-ß1 release and proliferation. VPA administration to RDEB mice mitigated severe manifestations affecting the eyes and paws. These effects were associated with fibrosis inhibition. Proteomic analysis of mouse skin revealed that VPA almost normalized protein sets involved in protein synthesis and immune response, processes linked to the increased susceptibility to cancer and bacterial infections seen in people with RDEB. CONCLUSIONS: Dysregulated histone acetylation contributes to RDEB pathogenesis by facilitating the progression of fibrosis. Repurposing of HDACi could be considered for disease-modifying treatments in RDEB.
Recessive dystrophic epidermolysis bullosa (or 'RDEB') is a rare skin disease that affects fewer than 5,000 people in the USA. A similar number of people in Europe are affected. RDEB is caused by mutations in the gene that controls the production of a protein called 'type VII collagen' (or 'C7'). A shortage of C7 causes fragile skin that blisters. In severe forms of RDEB, wounds heal slowly and can even affect a person's life expectancy. Differences in the disease are common in people (even identical twins) with RDEB who have similar levels of C7. This suggests that how severe the disease is could be affected by molecular processes that control other genes. Understanding these processes may help us to find treatments for RDEB. This study was done in Italy, in collaboration with centres in Germany and Switzerland. We wanted to see whether a chemical modification called 'histone acetylation' (which influences gene activity) is different in RDEB and whether it can be targeted by a specific treatment. We found that histone acetylation is reduced in RDEB skin and in skin cells grown in the lab called 'fibroblasts'. When we increased histone acetylation in fibroblasts with two drugs called givinostat and valproic acid, the amount of scar tissue produced decreased. This is important because scar tissue can lead to severe symptoms. We carried out more experiments to study the effects of givinostat and valproic acid in mice with RDEB. We found that valproic acid reduces the severity of RDEB by decreasing the disease's harmful effects and reducing the amount of scar tissue. Our findings suggest that abnormal histone acetylation contributes to the scar tissue seen in RDEB. Our study shows that valproic acid could be useful in treating the scarring seen in RDEB and in reducing the effects of the disease. As this drug is used to treat other diseases, there could be potential for rapid repurposing of it for RDEB.
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Colágeno Tipo VII , Progresión de la Enfermedad , Epidermólisis Ampollosa Distrófica , Fibroblastos , Fibrosis , Inhibidores de Histona Desacetilasas , Piel , Epidermólisis Ampollosa Distrófica/tratamiento farmacológico , Epidermólisis Ampollosa Distrófica/patología , Epidermólisis Ampollosa Distrófica/genética , Animales , Humanos , Inhibidores de Histona Desacetilasas/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Colágeno Tipo VII/genética , Piel/patología , Piel/efectos de los fármacos , Ratones , Ácido Valproico/farmacología , Histonas/metabolismo , Acetilación/efectos de los fármacos , Masculino , Femenino , Modelos Animales de Enfermedad , Factor de Crecimiento Transformador beta1/metabolismo , Células Cultivadas , Niño , CarbamatosRESUMEN
Serine/arginine-rich splicing factors (SRSFs) are a family of proteins involved in RNA metabolism, including pre-mRNA constitutive and alternative splicing. The role of SRSF proteins in regulating mitochondrial activity has already been shown for SRSF6, but SRSF4 altered expression has never been reported as a cause of bone marrow failure. An 8-year-old patient admitted to the hematology unit because of leukopenia, lymphopenia, and neutropenia showed a missense variant of unknown significance of the SRSF4 gene (p.R235W) found via whole genome sequencing analysis and inherited from the mother who suffered from mild leuko-neutropenia. Both patients showed lower SRSF4 protein expression and altered mitochondrial function and energetic metabolism in primary lymphocytes and Epstein-Barr-virus (EBV)-immortalized lymphoblasts compared to healthy donor (HD) cells, which appeared associated with low mTOR phosphorylation and an imbalance in the proteins regulating mitochondrial biogenesis (i.e., CLUH) and dynamics (i.e., DRP1 and OPA1). Transfection with the wtSRSF4 gene restored mitochondrial function. In conclusion, this study shows that the described variant of the SRSF4 gene is pathogenetic and causes reduced SRSF4 protein expression, which leads to mitochondrial dysfunction. Since mitochondrial function is crucial for hematopoietic stem cell maintenance and some genetic bone marrow failure syndromes display mitochondrial defects, the SRSF4 mutation could have substantially contributed to the clinical phenotype of our patient.
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Médula Ósea , Mitocondrias , Neutropenia , Factores de Empalme Serina-Arginina , Niño , Humanos , Empalme Alternativo , Médula Ósea/metabolismo , Médula Ósea/patología , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Dinámicas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Fosfoproteínas/metabolismo , Precursores del ARN/genética , Factores de Empalme Serina-Arginina/genética , Factores de Empalme Serina-Arginina/metabolismoRESUMEN
NAA20 is the catalytic subunit of the NatB complex, which is responsible for N-terminal acetylation of approximately 20% of the human proteome. Recently, pathogenic biallelic variants in NAA20 were associated with a novel neurodevelopmental disorder in five individuals with limited clinical information. We report two sisters harboring compound heterozygous variant (c.100C>T (p.Gln34Ter) and c.11T>C p.(Leu4Pro)) in the NAA20 gene, identified by exome sequencing. In vitro studies showed that the missense variant p.Leu4Pro resulted in a reduction of NAA20 catalytic activity due to weak coupling with the NatB auxiliary subunit. In addition, unpublished data of the previous families were reported, outlining the core phenotype of the NAA20-related disorder mostly characterized by cognitive impairment, microcephaly, ataxia, brain malformations, dysmorphism and variable occurrence of cardiac defect and epilepsy. Remarkably, our two patients featured epilepsy onset in adolescence suggesting this may be a part of syndrome evolution. Functional studies are needed to better understand the complexity of NAA20 variants pathogenesis as well as of other genes linked to N-terminal acetylation.
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Microcefalia , Malformaciones del Sistema Nervioso , Adolescente , Humanos , Dominio Catalítico , Microcefalia/genética , Síndrome , Fenotipo , Acetiltransferasa B N-Terminal/genética , Acetiltransferasa B N-Terminal/metabolismoRESUMEN
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may result in a severe pneumonia associated with elevation of blood inflammatory parameters, reminiscent of cytokine storm syndrome. Steroidal anti-inflammatory therapies have shown efficacy in reducing mortality in critically ill patients; however, the mechanisms by which SARS-CoV-2 triggers such an extensive inflammation remain unexplained. OBJECTIVES: To dissect the mechanisms underlying SARS-CoV-2-associated inflammation in patients with severe coronavirus disease 2019 (COVID-19), we studied the role of IL-1ß, a pivotal cytokine driving inflammatory phenotypes, whose maturation and secretion are regulated by inflammasomes. METHODS: We analyzed nod-like receptor protein 3 pathway activation by means of confocal microscopy, plasma cytokine measurement, cytokine secretion following in vitro stimulation of blood circulating monocytes, and whole-blood RNA sequencing. The role of open reading frame 3a SARS-CoV-2 protein was assessed by confocal microscopy analysis following nucleofection of a monocytic cell line. RESULTS: We found that circulating monocytes from patients with COVID-19 display ASC (adaptor molecule apoptotic speck like protein-containing a CARD) specks that colocalize with nod-like receptor protein 3 inflammasome and spontaneously secrete IL-1ß in vitro. This spontaneous activation reverts following patient's treatment with the IL-1 receptor antagonist anakinra. Transfection of a monocytic cell line with cDNA coding for the ORF3a SARS-CoV-2 protein resulted in ASC speck formation. CONCLUSIONS: These results provide further evidence that IL-1ß targeting could represent an effective strategy in this disease and suggest a mechanistic explanation for the strong inflammatory manifestations associated with COVID-19.
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Tratamiento Farmacológico de COVID-19 , Inflamasomas , Antiinflamatorios , Síndrome de Liberación de Citoquinas/tratamiento farmacológico , Citocinas/metabolismo , ADN Complementario , Humanos , Inflamasomas/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Interleucina-1beta/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas NLR , Receptores de Interleucina-1 , SARS-CoV-2RESUMEN
The reinterpretation of variants based on updated annotations is part of the routine work of research laboratories: the more data is collected about a specific variant, the higher the probability to reinterpret its classification. To support this task, we developed VariantAlert, a web-based tool to help researchers and clinicians to be constantly informed about changes in variant annotations extracted from multiple sources. VariantAlert provides daily re-annotation of variants using external resources accessed through application programming interface, such as MyVariant.info providing in turn links to gnomAD, catalogue of somatic mutations In cancer (COSMIC), ClinVar, CIViC, and many others. Researchers and clinicians can submit one or more lists of variants. If a change is detected for the annotation of a variant due to the upgrade of the underlying resource (e.g., change in gnomAD allele frequency, presence in COSMIC database, change in ClinVar classification) the user is notified by email and updated annotations are stored on the web-site. VariantAlert is freely available at https://github.com/next-crs4/VariantAlert. Installation and deployment are easy thanks to the use of the Docker platform. A Makefile allows you to easily bootstrap VariantAlert. VariantAlert is also available as a web service at https://variant-alert.crs4.it/.
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Variación Genética , Programas Informáticos , Humanos , Frecuencia de los Genes , Bases de Datos Factuales , Internet , Bases de Datos GenéticasRESUMEN
Alternative splicing (AS) is crucial for cell-type-specific gene transcription and plays a critical role in neuronal differentiation and synaptic plasticity. De novo frameshift variants in NOVA2, encoding a neuron-specific key splicing factor, have been recently associated with a new neurodevelopmental disorder (NDD) with hypotonia, neurological features, and brain abnormalities. We investigated eight unrelated individuals by exome sequencing (ES) and identified seven novel pathogenic NOVA2 variants, including two with a novel localization at the KH1 and KH3 domains. In addition to a severe NDD phenotype, novel clinical features included psychomotor regression, attention deficit-hyperactivity disorder (ADHD), dyspraxia, and urogenital and endocrinological manifestations. To test the effect of the variants on splicing regulation, we transfected HeLa cells with wildtype and mutant NOVA2 complementary DNA (cDNA). The novel variants NM_002516.4:c.754_756delCTGinsTT p.(Leu252Phefs*144) and c.1329dup p.(Lys444Glnfs*82) all negatively affected AS events. The distal p.(Lys444Glnfs*82) variant, causing a partial removal of the KH3 domain, had a milder functional effect leading to an intermediate phenotype. Our findings expand the molecular and phenotypic spectrum of NOVA2-related NDD, supporting the pathogenic role of AS disruption by truncating variants and suggesting that this is a heterogeneous condition with variable clinical course.
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Discapacidad Intelectual , Trastornos del Neurodesarrollo , Empalme Alternativo , Células HeLa , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Hipotonía Muscular/genética , Proteínas del Tejido Nervioso/genética , Antígeno Ventral Neuro-Oncológico , Trastornos del Neurodesarrollo/genética , Fenotipo , Proteínas de Unión al ARN/genéticaRESUMEN
BACKGROUND: Mutations in the recombinase-activating genes cause severe immunodeficiency, with a spectrum of phenotypes ranging from severe combined immunodeficiency to immune dysregulation. Hematopoietic stem cell transplantation is the only curative option, but a high risk of graft failure and poor immune reconstitution have been observed in the absence of myeloablation. OBJECTIVES: Our aim was to improve multilineage engraftment; we tested nongenotoxic conditioning with anti-CD45 mAbs conjugated with saporin CD45 (CD45-SAP). METHODS: Rag1-KO and Rag1-F971L mice, which represent models of severe combined immune deficiency and combined immune deficiency with immune dysregulation, respectively, were conditioned with CD45-SAP, CD45-SAP plus 2 Gy of total body irradiation (TBI), 2 Gy of TBI, 8 Gy of TBI, or no conditioning and treated by using transplantation with lineage-negative bone marrow cells from wild-type mice. Flow cytometry and immunohistochemistry were used to assess engraftment and immune reconstitution. Antibody responses to 2,4,6-trinitrophenyl-conjugated keyhole limpet hemocyanin were measured by ELISA, and presence of autoantibody was detected by microarray. RESULTS: Conditioning with CD45-SAP enabled high levels of multilineage engraftment in both Rag1 mutant models, allowed overcoming of B- and T-cell differentiation blocks and thymic epithelial cell defects, and induced robust cellular and humoral immunity in the periphery. CONCLUSIONS: Conditioning with CD45-SAP allows multilineage engraftment and robust immune reconstitution in mice with either null or hypomorphic Rag mutations while preserving thymic epithelial cell homeostasis.
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Anticuerpos Monoclonales/farmacología , Trasplante de Médula Ósea , Proteínas de Homeodominio/genética , Inmunoconjugados/farmacología , Antígenos Comunes de Leucocito/antagonistas & inhibidores , Saporinas/farmacología , Inmunodeficiencia Combinada Grave/terapia , Acondicionamiento Pretrasplante , Aloinjertos , Animales , Anticuerpos Monoclonales/efectos adversos , Proteínas de Homeodominio/inmunología , Inmunoconjugados/efectos adversos , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/inmunología , Ratones , Ratones Noqueados , Saporinas/efectos adversos , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/inmunologíaRESUMEN
The advent of Whole Genome Sequencing (WGS) broadened the genetic variation detection range, revealing the presence of variants even in non-coding regions of the genome, which would have been missed using targeted approaches. One of the most challenging issues in WGS analysis regards the interpretation of annotated variants. This review focuses on tools suitable for the functional annotation of variants falling into non-coding regions. It couples the description of non-coding genomic areas with the results and performance of existing tools for a functional interpretation of the effect of variants in these regions. Tools were tested in a controlled genomic scenario, representing the ground-truth and allowing us to determine software performance.
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Genómica , Programas Informáticos , Humanos , Genómica/métodos , Secuenciación Completa del Genoma/métodos , Genoma , Genoma HumanoRESUMEN
BACKGROUND: Improving the availability and usability of data and analytical tools is a critical precondition for further advancing modern biological and biomedical research. For instance, one of the many ramifications of the COVID-19 global pandemic has been to make even more evident the importance of having bioinformatics tools and data readily actionable by researchers through convenient access points and supported by adequate IT infrastructures. One of the most successful efforts in improving the availability and usability of bioinformatics tools and data is represented by the Galaxy workflow manager and its thriving community. In 2020 we introduced Laniakea, a software platform conceived to streamline the configuration and deployment of "on-demand" Galaxy instances over the cloud. By facilitating the set-up and configuration of Galaxy web servers, Laniakea provides researchers with a powerful and highly customisable platform for executing complex bioinformatics analyses. The system can be accessed through a dedicated and user-friendly web interface that allows the Galaxy web server's initial configuration and deployment. RESULTS: "Laniakea@ReCaS", the first instance of a Laniakea-based service, is managed by ELIXIR-IT and was officially launched in February 2020, after about one year of development and testing that involved several users. Researchers can request access to Laniakea@ReCaS through an open-ended call for use-cases. Ten project proposals have been accepted since then, totalling 18 Galaxy on-demand virtual servers that employ ~ 100 CPUs, ~ 250 GB of RAM and ~ 5 TB of storage and serve several different communities and purposes. Herein, we present eight use cases demonstrating the versatility of the platform. CONCLUSIONS: During this first year of activity, the Laniakea-based service emerged as a flexible platform that facilitated the rapid development of bioinformatics tools, the efficient delivery of training activities, and the provision of public bioinformatics services in different settings, including food safety and clinical research. Laniakea@ReCaS provides a proof of concept of how enabling access to appropriate, reliable IT resources and ready-to-use bioinformatics tools can considerably streamline researchers' work.
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COVID-19 , Nube Computacional , Biología Computacional , Humanos , SARS-CoV-2 , Programas InformáticosRESUMEN
Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, a relevant number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NSG recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPCs coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by bone marrow fibrosis.
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Trasplante de Células Madre Hematopoyéticas , Osteopetrosis , ATPasas de Translocación de Protón Vacuolares , Antígenos CD34 , Terapia Genética , Células Madre Hematopoyéticas/metabolismo , Humanos , Osteoclastos/metabolismo , Osteopetrosis/genética , Osteopetrosis/terapia , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Recent data suggest that imbalances in the composition of the gut microbiota (GM) could exacerbate the progression of Parkinson disease (PD). The effects of levodopa (LD) have been poorly assessed, and those of LD-carbidopa intestinal gel (LCIG) have not been evaluated so far. The aim of this study was to identify the effect of LD and LCIG, in particular, on the GM and metabolome. METHODS: Fecal DNA samples from 107 patients with a clinical diagnosis of PD were analyzed by next-generation sequencing of the V3 and V4 regions of the 16S rRNA gene. PD patients were classified in different groups: patients on LCIG (LCIG group, n = 38) and on LD (LD group, n = 46). We also included a group of patients (n = 23) without antiparkinsonian medicaments (Naïve group). Fecal metabolic extracts were evaluated by gas chromatography mass spectrometry. RESULTS: The multivariate analysis showed a significantly higher abundance in the LCIG group of Enterobacteriaceae, Escherichia, and Serratia compared to the LD group. Compared to the Naïve group, the univariate analysis showed a reduction of Blautia and Lachnospirae in the LD group. Moreover, an increase of Proteobacteria, Enterobacteriaceae, and a reduction of Firmicutes, Lachnospiraceae, and Blautia was found in the LCIG group. No significant difference was found in the multivariate analysis of these comparisons. The LD group and LCIG group were associated with a metabolic profile linked to gut inflammation. CONCLUSIONS: Our results suggest that LD, and mostly LCIG, might significantly influence the microbiota composition and host/bacteria metabolism, acting as stressors in precipitating a specific inflammatory intestinal microenvironment, potentially related to the PD state and progression.
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Microbioma Gastrointestinal , Enfermedad de Parkinson , Antiparkinsonianos , Carbidopa , Combinación de Medicamentos , Geles , Humanos , Levodopa , Metaboloma , Enfermedad de Parkinson/tratamiento farmacológico , ARN Ribosómico 16S/genéticaRESUMEN
Colorectal cancer (CRC) is a leading cause of cancer death worldwide, and about 20% is metastatic at diagnosis and untreatable. Increasing evidence suggests that the heterogeneous nature of CRC is related to colorectal cancer stem cells (CCSCs), a small cells population with stemness behaviors and responsible for tumor progression, recurrence, and therapy resistance. Growing knowledge of stem cells (SCs) biology has rapidly improved uncovering the molecular mechanisms and possible crosstalk/feedback loops between signaling pathways that directly influence intestinal homeostasis and tumorigenesis. The generation of CCSCs is probably connected to genetic changes in members of signaling pathways, which control self-renewal and pluripotency in SCs and then establish function and phenotype of CCSCs. Particularly, various deregulated CCSC-related miRNAs have been reported to modulate stemness features, controlling CCSCs functions such as regulation of cell cycle genes expression, epithelial-mesenchymal transition, metastasization, and drug-resistance mechanisms. Primarily, CCSC-related miRNAs work by regulating mainly signal pathways known to be involved in CCSCs biology. This review intends to summarize the epigenetic findings linked to miRNAome in the maintenance and regulation of CCSCs, including their relationships with different signaling pathways, which should help to identify specific diagnostic, prognostic, and predictive biomarkers for CRC, but also develop innovative CCSCs-targeted therapies.
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Neoplasias Colorrectales/metabolismo , Células Madre Neoplásicas/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal/genética , Transición Epitelial-Mesenquimal/fisiología , Retroalimentación Fisiológica , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Modelos Biológicos , Células Madre Neoplásicas/patología , Transducción de SeñalRESUMEN
Glycogen storage disease type Ia (GSDIa) is an inherited metabolic disorder caused by mutations in the enzyme glucose-6-phosphatase-α (G6Pase-α). Affected individuals develop renal and liver complications, including the development of hepatocellular adenoma/carcinoma and kidney failure. The purpose of this study was to identify potential biomarkers of the evolution of the disease in GSDIa patients. To this end, we analyzed the expression of exosomal microRNAs (Exo-miRs) in the plasma exosomes of 45 patients aged 6 to 63 years. Plasma from age-matched normal individuals were used as controls. We found that the altered expression of several Exo-miRs correlates with the pathologic state of the patients and might help to monitor the progression of the disease and the development of late GSDIa-associated complications.
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Exosomas/genética , Enfermedad del Almacenamiento de Glucógeno Tipo I/genética , Enfermedades Renales/genética , Hígado/lesiones , Hígado/metabolismo , MicroARNs/genética , Adolescente , Adulto , Factores de Edad , Animales , Biomarcadores/metabolismo , Estudios de Casos y Controles , Niño , Preescolar , Estudios de Cohortes , Exosomas/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ontología de Genes , Glucosa-6-Fosfatasa/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo I/sangre , Enfermedad del Almacenamiento de Glucógeno Tipo I/patología , Humanos , Enfermedades Renales/sangre , Enfermedades Renales/patología , Masculino , Ratones , MicroARNs/metabolismo , Persona de Mediana Edad , Factores de Tiempo , Adulto JovenRESUMEN
Crisponi syndrome (CS)/cold-induced sweating syndrome type 1 (CISS1) is a very rare autosomal-recessive disorder characterized by a complex phenotype with high neonatal lethality, associated with the following main clinical features: hyperthermia and feeding difficulties in the neonatal period, scoliosis, and paradoxical sweating induced by cold since early childhood. CS/CISS1 can be caused by mutations in cytokine receptor-like factor 1 (CRLF1). However, the physiopathological role of CRLF1 is still poorly understood. A subset of CS/CISS1 cases remain yet genetically unexplained after CRLF1 sequencing. In five of them, exome sequencing and targeted Sanger sequencing identified four homozygous disease-causing mutations in kelch-like family member 7 (KLHL7), affecting the Kelch domains of the protein. KLHL7 encodes a BTB-Kelch-related protein involved in the ubiquitination of target proteins for proteasome-mediated degradation. Mono-allelic substitutions in other domains of KLHL7 have been reported in three families affected by a late-onset form of autosomal-dominant retinitis pigmentosa. Retinitis pigmentosa was also present in two surviving children reported here carrying bi-allelic KLHL7 mutations. KLHL7 mutations are thus associated with a more severe phenotype in recessive than in dominant cases. Although these data further support the pathogenic role of KLHL7 mutations in a CS/CISS1-like phenotype, they do not explain all their clinical manifestations and highlight the high phenotypic heterogeneity associated with mutations in KLHL7.
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Alelos , Autoantígenos/genética , Deformidades Congénitas de la Mano/complicaciones , Deformidades Congénitas de la Mano/genética , Hiperhidrosis/complicaciones , Hiperhidrosis/genética , Mutación , Retinitis Pigmentosa/complicaciones , Retinitis Pigmentosa/genética , Trismo/congénito , Secuencia de Aminoácidos , Autoantígenos/química , Niño , Preescolar , Muerte Súbita , Facies , Femenino , Humanos , Lactante , Masculino , Modelos Moleculares , Linaje , Fenotipo , Síndrome , Trismo/complicaciones , Trismo/genéticaRESUMEN
BACKGROUND: KBG syndrome is a very rare autosomal dominant disorder, characterized by macrodontia, distinctive craniofacial findings, skeletal findings, post-natal short stature, and developmental delays, sometimes associated with seizures and EEG abnormalities. So far, there have been over 100 cases of KBG syndrome reported. CASE PRESENTATION: Here, we describe two sisters of a non-consanguineous family, both presenting generalized epilepsy with febrile seizures (GEFS+), and one with a more complex phenotype associated with mild intellectual disability, skeletal and dental anomalies. Whole exome sequencing (WES) analysis in all the family members revealed a heterozygous SCN9A mutation, p.(Lys655Arg), shared among the father and the two probands, and a novel de novo loss of function mutation in the ANKRD11 gene, p.(Tyr1715*), in the proband with the more complex phenotype. The reassessment of the phenotypic features confirmed that the patient fulfilled the proposed diagnostic criteria for KBG syndrome, although complicated by early-onset isolated febrile seizures. EEG abnormalities with or without seizures have been reported previously in some KBG cases. The shared variant, occurring in SCN9A, has been previously found in several individuals with GEFS+ and Dravet syndrome. CONCLUSIONS: This report describe a novel de novo variant in ANKRD11 causing a mild phenotype of KGB syndrome and further supports the association of monogenic pattern of SCN9A mutations with GEFS+. Our data expand the allelic spectrum of ANKRD11 mutations, providing the first Brazilian case of KBG syndrome. Furthermore, this study offers an example of how WES has been instrumental allowing us to better dissect the clinical phenotype under study, which is a multilocus variation aggregating in one proband, rather than a phenotypic expansion associated with a single genomic locus, underscoring the role of multiple rare variants at different loci in the etiology of clinical phenotypes making problematic the diagnostic path. The successful identification of the causal variant in a gene may not be sufficient, making it necessary to identify other variants that fully explain the clinical picture. The prevalence of blended phenotypes from multiple monogenic disorders is currently unknown and will require a systematic re-analysis of large WES datasets for proper diagnosis in daily practice.
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Anomalías Múltiples/genética , Enfermedades del Desarrollo Óseo/genética , Epilepsia Generalizada/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Discapacidad Intelectual/genética , Mutación , Fenotipo , Proteínas Represoras/genética , Convulsiones Febriles/genética , Anomalías Dentarias/genética , Anomalías Múltiples/diagnóstico por imagen , Anomalías Múltiples/etiología , Anomalías Múltiples/fisiopatología , Adolescente , Alelos , Enfermedades del Desarrollo Óseo/diagnóstico por imagen , Enfermedades del Desarrollo Óseo/etiología , Enfermedades del Desarrollo Óseo/fisiopatología , Brasil , Electroencefalografía , Epilepsia Generalizada/fisiopatología , Facies , Femenino , Sitios Genéticos , Heterocigoto , Humanos , Discapacidad Intelectual/diagnóstico por imagen , Discapacidad Intelectual/etiología , Discapacidad Intelectual/fisiopatología , Canal de Sodio Activado por Voltaje NAV1.7/genética , Linaje , Convulsiones Febriles/fisiopatología , Anomalías Dentarias/diagnóstico por imagen , Anomalías Dentarias/etiología , Anomalías Dentarias/fisiopatología , Secuenciación del ExomaRESUMEN
Crisponi/cold-induced sweating syndrome (CS/CISS) is a rare autosomal recessive disorder characterized by a complex phenotype (hyperthermia and feeding difficulties in the neonatal period, followed by scoliosis and paradoxical sweating induced by cold since early childhood) and a high neonatal lethality. CS/CISS is a genetically heterogeneous disorder caused by mutations in CRLF1 (CS/CISS1), CLCF1 (CS/CISS2) and KLHL7 (CS/CISS-like). Here, a whole exome sequencing approach in individuals with CS/CISS-like phenotype with unknown molecular defect revealed unpredicted alternative diagnoses. This approach identified putative pathogenic variations in NALCN, MAGEL2 and SCN2A. They were already found implicated in the pathogenesis of other syndromes, respectively the congenital contractures of the limbs and face, hypotonia, and developmental delay syndrome, the Schaaf-Yang syndrome, and the early infantile epileptic encephalopathy-11 syndrome. These results suggest a high neonatal phenotypic overlap among these disorders and will be very helpful for clinicians. Genetic analysis of these genes should be considered for those cases with a suspected CS/CISS during neonatal period who were tested as mutation negative in the known CS/CISS genes, because an expedited and corrected diagnosis can improve patient management and can provide a specific clinical follow-up.
Asunto(s)
Secuenciación del Exoma , Deformidades Congénitas de la Mano/diagnóstico , Deformidades Congénitas de la Mano/genética , Hiperhidrosis/diagnóstico , Hiperhidrosis/genética , Trismo/congénito , Muerte Súbita , Facies , Femenino , Humanos , Lactante , Masculino , Linaje , Fenotipo , Trismo/diagnóstico , Trismo/genéticaRESUMEN
The balance between self-renewal and differentiation is crucial to ensure the homeostasis of the hematopoietic system, and is a hallmark of hematopoietic stem cells. However, the underlying molecular pathways, including the role of micro-RNA, are not completely understood. To assess the contribution of micro-RNA, we performed micro-RNA profiling of hematopoietic stem cells and their immediate downstream progeny multi-potent progenitors from wild-type control and Pbx1-conditional knockout mice, whose stem cells display a profound self-renewal defect. Unsupervised hierarchical cluster analysis separated stem cells from multi-potent progenitors, suggesting that micro-RNA might regulate the first transition step in the adult hematopoietic development. Notably, Pbx1-deficient and wild-type cells clustered separately, linking micro-RNAs to self-renewal impairment. Differential expression analysis of micro-RNA in the physiological stem cell-to-multi-potent progenitor transition and in Pbx1-deficient stem cells compared to control stem cells revealed miR-127-3p as the most differentially expressed. Furthermore, miR-127-3p was strongly stem cell-specific, being quickly down-regulated upon differentiation and not re-expressed further downstream in the bone marrow hematopoietic hierarchy. Inhibition of miR-127-3p function in Lineage-negative cells, achieved through a lentiviral-sponge vector, led to severe stem cell depletion, as assessed with serial transplantation assays. miR-127-3p-sponged stem cells displayed accelerated differentiation, which was uncoupled from proliferation, accounting for the observed stem cell reduction. miR-127-3p overexpression in Lineage-negative cells did not alter stem cell pool size, but gave rise to lymphopenia, likely due to lack of miR-127-3p physiological downregulation beyond the stem cell stage. Thus, tight regulation of miR-127-3p is crucial to preserve the self-renewing stem cell pool and homeostasis of the hematopoietic system.
Asunto(s)
Diferenciación Celular , Células Madre Hematopoyéticas/citología , MicroARNs/fisiología , Animales , Linaje de la Célula/genética , Análisis por Conglomerados , Cruzamientos Genéticos , Perfilación de la Expresión Génica , Hematopoyesis , Homeostasis , Humanos , Células K562 , Lentivirus/genética , Ratones , Ratones Noqueados , Estrés Oxidativo , Factor de Transcripción 1 de la Leucemia de Células Pre-B/metabolismoRESUMEN
We report here a novel de novo missense variant affecting the last amino acid of exon 30 of CREBBP [NM_004380, c.5170G>A; p.(Glu1724Lys)] in a 17-year-old boy presenting mild intellectual disability and dysmorphisms but not resembling the phenotype of classical Rubinstein-Taybi syndrome. The patient showed a marked overweight from early infancy on and had cortical heterotopias. Recently, 22 individuals have been reported with missense mutations in the last part of exon 30 and the beginning of exon 31 of CREBBP, showing this new phenotype. This additional case further delineates the genotype-phenotype correlations within the molecular and phenotypic spectrum of variants in CREBBP and EP300.
Asunto(s)
Proteína de Unión a CREB/genética , Exones/genética , Mutación , Síndrome de Rubinstein-Taybi/genética , Síndrome de Rubinstein-Taybi/patología , Adolescente , Femenino , Estudios de Asociación Genética , Humanos , Masculino , Linaje , PronósticoRESUMEN
Colorectal cancer (CRC) is a leading cause of cancer death worldwide and about 20% is metastatic at diagnosis and untreatable. The anti-EGFR therapy in metastatic patients is led by the presence of KRAS-mutations in tumor tissue. KRAS-wild-type CRC patients showed a positive response rate of about 70% to cetuximab or panitumumab combined with chemotherapy. MiRNAs are promising markers in oncology and could improve our knowledge on pathogenesis and drug resistance in CRC patients. This class of molecules represents an opportunity for the development of miRNA-based strategies to overcome the ineffectiveness of anti-EGFR therapy. We performed an integrative analysis of miRNA expression profile between KRAS-mutated CRC and KRAS-wildtype CRC and paired normal colic tissue (NCT). We revealed an overexpression of miR-425-5p in KRAS-mutated CRC compared to KRAS-wild type CRC and NCT and demonstrated that miR-425-5p exerts regulatory effects on target genes involved in cellular proliferation, migration, invasion, apoptosis molecular networks. These epigenetic mechanisms could be responsible of the strong aggressiveness of KRAS-mutated CRC compared to KRAS-wildtype CRC. We proved that some miR-425-5p targeted genes are involved in EGFR tyrosine kinase inhibitor resistance pathway, suggesting that therapies based on miR-425-5p may have strong potential in targeting KRAS-driven CRC. Moreover, we demonstrated a role in the oncogenesis of miR-31-5p, miR-625-5p and miR-579 by comparing CRC versus NCT. Our results underlined that miR-425-5p might act as an oncogene to participate in the pathogenesis of KRAS-mutated CRC and contribute to increase the aggressiveness of this subcategory of CRC, controlling a complex molecular network.
Asunto(s)
Neoplasias Colorrectales/genética , Epigénesis Genética/genética , MicroARNs/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Apoptosis/efectos de los fármacos , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cetuximab/administración & dosificación , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Mutación/genética , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Proteínas de Neoplasias/genética , Panitumumab/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Proto-Oncogénicas B-raf/genéticaRESUMEN
BACKGROUND: Wiskott-Aldrich syndrome (WAS) is a rare primary immunodeficiency caused by mutations in Wiskott-Aldrich syndrome protein (WASp), a key regulator of cytoskeletal dynamics in hematopoietic cells. A high proportion of patients experience autoimmunity caused by a breakdown in T- and B-cell tolerance. Moreover, excessive production of type I interferon (IFN-I) by plasmacytoid dendritic cells (pDCs) contributes to autoimmune signs; however, the factors that trigger excessive innate activation have not been defined. OBJECTIVE: Neutrophil extracellular traps (NETs) emerged as major initiating factors in patients with diseases such as systemic lupus erythematosus and rheumatoid arthritis. In this study we explored the possible involvement of aberrant neutrophil functions in patients with WAS. METHODS: We evaluated the expression of a set of granulocyte genes associated with NETs in a cohort of patients with WAS and the presence of NET inducers in sera. Using a mouse model of WAS, we analyzed NET release by WASp-null neutrophils and evaluated the composition and homeostasis of neutrophils in vivo. By using depletion experiments, we assessed the effect of neutrophils in promoting inflammation and reactivity against autoantigens. RESULTS: Transcripts of genes encoding neutrophil enzymes and antimicrobial peptides were increased in granulocytes of patients with WAS, and serum-soluble factors triggered NET release. WASp-null neutrophils showed increased spontaneous NETosis, induced IFN-I production by pDCs, and activated B cells through B-cell activating factor. Consistently, their depletion abolished constitutive pDC activation, normalized circulating IFN-I levels, and, importantly, abolished production of autoantibodies directed against double-stranded DNA, nucleosomes, and myeloperoxidase. CONCLUSIONS: These findings reveal that neutrophils are involved in the pathogenic loop that causes excessive activation of innate cells and autoreactive B cells, thus identifying novel mechanisms that contribute to the autoimmunity of WAS.