RESUMO
Phospholipase C isozymes (PLCs) hydrolyze phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and diacylglycerol, important signaling molecules involved in many cellular processes. PLCG1 encodes the PLCγ1 isozyme that is broadly expressed. Hyperactive somatic mutations of PLCG1 are observed in multiple cancers, but only one germline variant has been reported. Here we describe three unrelated individuals with de novo heterozygous missense variants in PLCG1 (p.Asp1019Gly, p.His380Arg, and p.Asp1165Gly) who exhibit variable phenotypes including hearing loss, ocular pathology and cardiac septal defects. To model these variants in vivo, we generated the analogous variants in the Drosophila ortholog, small wing (sl). We created a null allele slT2A and assessed the expression pattern. sl is broadly expressed, including in wing discs, eye discs, and a subset of neurons and glia. Loss of sl causes wing size reductions, ectopic wing veins and supernumerary photoreceptors. We document that mutant flies exhibit a reduced lifespan and age-dependent locomotor defects. Expressing wild-type sl in slT2A mutant rescues the loss-of-function phenotypes whereas expressing the variants causes lethality. Ubiquitous overexpression of the variants also reduces viability, suggesting that the variants are toxic. Ectopic expression of an established hyperactive PLCG1 variant (p.Asp1165His) in the wing pouch causes severe wing phenotypes, resembling those observed with overexpression of the p.Asp1019Gly or p.Asp1165Gly variants, further arguing that these two are gain-of-function variants. However, the wing phenotypes associated with p.His380Arg overexpression are mild. Our data suggest that the PLCG1 de novo heterozygous missense variants are pathogenic and contribute to the features observed in the probands.
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Neuroendocrine neoplasms (NENs) are highly vascularized malignancies arising from cells of the diffuse neuroendocrine system. An intricated cross-talk exists between NEN cells and the tumor microenvironment, and three main molecular circuits (VEGF/VEGFR pathway, FGF-dependent signaling and PDGF/PDGFR axis) have been shown to regulate angiogenesis in these neoplasms. Multiple randomized trials have investigated antiangiogenic agents over the past two decades, and sunitinib is currently approved for the treatment of advanced, progressive, G1/G2 pancreatic NENs. In recent years, two phase III clinical trials have demonstrated the efficacy and safety of surufatinib, a multi-tyrosine kinase angioimmune inhibitor, in patients with well-differentiated pancreatic and extrapancreatic NENs, and two studies of this agent are currently underway in Europe and US. The HIF-2α inhibitor belzutifan has recently received regulatory approval for the treatment of tumors arising in the context of Von-Hippel Lindau syndrome including pancreatic NENs, and a study of this drug in patients with sporadic tumors is presently ongoing. Combinations of antiangiogenic agents with chemotherapeutics and targeted drugs have been tested, with accumulating toxicities being a matter of concern. The potential of antiangiogenic agents in fine-tuning the immune microenvironment of NENs to enhance the activity of immune checkpoint inhibitors has been only partially elucidated, and further research should be carried out at this regard. Here, we review the current understanding of the biology of angiogenesis in NENs and provide a summary of the latest clinical investigations on antiangiogenic drugs in this malignancy.
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BACKGROUND: Neuroendocrine tumors (NETs) overexpress somatostatin receptors (SSTRs). METHODS: We developed a second-generation, ligand-based, anti-SSTR chimeric antigen receptor (CAR) incorporating the somatostatin analog octreotide in its extracellular moiety. RESULTS: Anti-SSTR CAR T cells exerted antitumor activity against SSTR+NET cell linesin vitro. The killing activity was highly specific, as demonstrated by the lack of CAR T cell reactivity against NET cells engineered to express mutated variants of SSTR2/5 by CRISPR/Cas9. When adoptively transferred in NSG mice, anti-SSTR CAR T cells induced significant antitumor activity against human NET xenografts. Although anti-SSTR CAR T cells could recognize the murine SSTRs as shown by their killing ability against murine NET cells, no obvious deleterious effects on SSTR-expressing organs such as the brain or the pancreas were observed in mice. CONCLUSIONS: Taken together, our results establish anti-SSTR CAR T cells as a potential candidate for early phase clinical investigations in patients with NETs. More broadly, the demonstration that a known peptide drug can direct CAR T cell targeting may streamline the potential utility of multiple peptide motifs and provide a blueprint for therapeutic applications in a variety of cancers.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Tumores Neuroendócrinos , Animais , Humanos , Ligantes , Camundongos , Tumores Neuroendócrinos/tratamento farmacológico , Octreotida , Somatostatina/uso terapêuticoRESUMO
The COVID-19 pandemic has added another layer of complexity to the fears of patients with neuroendocrine tumors (NETs). Little is known regarding the psychological impact of the COVID-19 outbreak on patients with gastroenteropancreatic or bronchopulmonary (BP) NETs. We longitudinally surveyed the mental symptoms and concerns of NET patients during the plateau phase of the first (W1) and second epidemic waves (W2) in Italy. Seven specific constructs (depression, anxiety, stress, health-related quality of life, NET-related quality of life, patient-physician relationship, psychological distress) were investigated using validated screening instruments, including DASS-21, EORTC QLQ-C30, EORTC QLQ GI.NET21, PDRQ9 and IES-R. We enrolled 197 patients (98 males) with a median age of 62 years. The majority of the patients had G1/G2 neoplasms. Some 38% of the patients were on active treatment. At W1, the prevalence of depression, anxiety and stress was 32%, 36% and 26% respectively. The frequency of depression and anxiety increased to 38% and 41% at W2, whereas no modifications were recorded in the frequency of stress. Poor educational status was associated with higher levels of anxiety at both W1 (odds ratio [OR] = 1.33 ± 0.22; p = .07) and W2 (OR = 1.45 ± 0.26; p = .03). Notably, post-traumatic stress symptoms were observed in the 58% of the patients, and both single marital status (OR = 0.16, 95% confidence interval [CI] = 0.06-0.48; p = .0009) and low levels of formal education (OR = 0.47, 95% CI = 0.23-0.99; p = .05) predicted their occurrence. No significant deteriorations of health-related quality of life domains were observed from W1 to W2. High patient care satisfaction was documented despite the changes in health systems resource allocation. NET patients have an increased risk of developing post-traumatic stress symptoms as result of the COVID-19 pandemic. Specific screening measures and psychological interventions should be implemented in NET clinics to prevent, recognize and treat mental distress in this vulnerable population.
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Multiple systemic treatments are currently available for advanced cancers of the digestive tract, but none of them is curative. Adoptive T-cell immunotherapy refers to the extraction, modification and re-infusion of autologous or allogenic T lymphocytes for therapeutic purposes. A number of clinical trials have investigated either non-engineered T cells (i.e., lymphokine-activated killer cells, cytokine induced killer cells, or tumor-infiltrating lymphocytes) or engineered T cells (T cell receptor-redirected T cells or chimeric antigen receptor T cells) in patients with digestive tract malignancies over the past two decades, with variable degrees of success. While the majority of completed trials have been primarily aimed at assessing the safety of T-cell transfer strategies, a new generation of studies is being designed to formally evaluate the antitumor potential of adoptive T-cell immunotherapy in both the metastatic and adjuvant settings. In this review, we provide an overview of completed and ongoing clinical trials of passive T-cell immunotherapy in patients with cancers of the digestive tract, focusing on present obstacles and future strategies for achieving potential success.
Assuntos
Neoplasias do Sistema Digestório/terapia , Imunoterapia Adotiva/métodos , Humanos , Receptores de Antígenos QuiméricosRESUMO
Management of localized well-differentiated pancreatic neuroendocrine tumors (panNETs) is controversial and primarily dependent on tumor size. Upfront surgery is usually recommended for tumors larger than 2 cm in diameter since they frequently show metastatic potential, whereas smaller panNETs are generally characterized by an indolent clinical course, with a rate of relapse or metastasis below 15%. To explore whether increased tumor size is paralleled by genomic variations, we compared the rate and the mutational patterns of putative driver genes that are recurrently altered in these tumors by investigating differential cohorts of panNET surgical specimens smaller (n = 27) or larger than 2 cm (n = 29). We found that the cumulative number of mutations detected in panNETs >2 cm was significantly higher (p = 0.03) relative to smaller tumors, while mutations of DAXX were significantly more frequent in the cohort of larger tumors (p = 0.05). Moreover, mutations of DAXX were associated with features of malignancy including increased grade, nodal involvement and lymphovascular invasion, and independently predicted both relapse after surgery (p = 0.05) and reduced DFS in multivariable analysis (p = 0.02). Our data suggest that alterations of the DAXX/ATRX molecular machinery increase the malignant potential of panNETs, and that identification of mutations of DAXX/ATRX in small, nonfunctioning tumors can predict the malignant progression observed in a minority of them.
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Proteínas Correpressoras/genética , Regulação Neoplásica da Expressão Gênica , Chaperonas Moleculares/genética , Mutação , Tumores Neuroendócrinos/genética , Neoplasias Pancreáticas/genética , Adulto , Idoso , Análise Mutacional de DNA , Feminino , Variação Genética , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Recidiva Local de Neoplasia , Prognóstico , Estudos Retrospectivos , RiscoRESUMO
OPINION STATEMENT: The treatment landscape of well-differentiated neuroendocrine tumors (NETs) has considerably expanded in recent years, and both somatostatin analogs, radiolabeled somatostatin analogs, everolimus, and sunitinib have been incorporated within the therapeutic armamentarium against these malignancies. Even in the context of multiple treatment options available, cytotoxic chemotherapy plays a pivotal role in the management of pancreatic NETs (panNETs), while its activity in midgut carcinoids and lung NETs is still debated. High response rates, ranging from 30 to 70%, have been consistently reported in studies of panNETs investigating streptozotocin-, temozolomide-, or platinum-based regimens, and an unprecedented prolongation of progression-free survival has been recently demonstrated in a prospective, randomized trial of capecitabine and temozolomide in patients with progressive panNETs. As a general principle, cytotoxic chemotherapy appears particularly appropriate in patients with bulky, symptomatic, or rapidly progressing tumors, especially of pancreatic origin, or in the salvage setting of NET patients who have failed alternative therapeutic options. Emerging evidence has also shown the potential efficacy of induction chemotherapy in patients with locally advanced or oligometastatic panNET, but prospective validation is needed before implementation of this approach in routine clinical practice. At present, there is no consensus on adjuvant therapy in pulmonary NETs, and differences between guideline recommendations at this regard mainly stem from the lack of high-level evidence. In the future, the identification of molecular biomarkers of response to chemotherapy might allow better patient preselection, thus leading to improved outcomes.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Tumores Neuroendócrinos/tratamento farmacológico , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Terapia Combinada , Humanos , Gradação de Tumores , Seleção de Pacientes , Medicina de Precisão/métodos , Resultado do TratamentoRESUMO
Kabuki syndrome is a rare autosomal dominant condition characterized by facial features, various organs malformations, postnatal growth deficiency and intellectual disability. The discovery of frequent germline mutations in the histone methyltransferase KMT2D and the demethylase KDM6A revealed a causative role for histone modifiers in this disease. However, the role of missense mutations has remained unexplored. Here, we expanded the mutation spectrum of KMT2D and KDM6A in KS by identifying 37 new KMT2D sequence variants. Moreover, we functionally dissected 14 KMT2D missense variants, by investigating their impact on the protein enzymatic activity and the binding to members of the WRAD complex. We demonstrate impaired H3K4 methyltransferase activity in 9 of the 14 mutant alleles and show that this reduced activity is due in part to disruption of protein complex formation. These findings have relevant implications for diagnostic and counseling purposes in this disease.
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Anormalidades Múltiplas/genética , Proteínas de Ligação a DNA/genética , Face/anormalidades , Doenças Hematológicas/genética , Mutação de Sentido Incorreto , Proteínas de Neoplasias/genética , Doenças Vestibulares/genética , Anormalidades Múltiplas/enzimologia , Simulação por Computador , Proteínas de Ligação a DNA/metabolismo , Doenças Hematológicas/enzimologia , Histona Desmetilases/genética , Humanos , Modelos Moleculares , Mutação , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Conformação Proteica , Análise de Sequência de Proteína , Doenças Vestibulares/enzimologiaRESUMO
Autophagy is a catabolic process needed for maintaining cell viability and homeostasis in response to numerous stress conditions. Emerging evidence indicates that the ubiquitin system has a major role in this process. TRIMs, an E3 ligase protein family, contribute to selective autophagy acting as receptors and regulators of the autophagy proteins recognizing endogenous or exogenous targets through intermediary autophagic tags, such as ubiquitin. Here we report that TRIM50 fosters the initiation phase of starvation-induced autophagy and associates with Beclin1, a central component of autophagy initiation complex. We show that TRIM50, via the RING domain, ubiquitinates Beclin 1 in a K63-dependent manner enhancing its binding with ULK1 and autophagy activity. Finally, we found that the Lys-372 residue of TRIM50, critical for its own acetylation, is necessary for its E3 ligase activity that governs Beclin1 ubiquitination. Our study expands the roles of TRIMs in regulating selective autophagy, revealing an acetylation-ubiquitination dependent control for autophagy modulation.
Assuntos
Proteína Beclina-1/metabolismo , Proteínas de Membrana/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Acetilação , Animais , Autofagia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/genética , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Camundongos , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Motivation: Drug repositioning has been proposed as an effective shortcut to drug discovery. The availability of large collections of transcriptional responses to drugs enables computational approaches to drug repositioning directly based on measured molecular effects. Results: We introduce a novel computational methodology for rational drug repositioning, which exploits the transcriptional responses following treatment with small molecule. Specifically, given a therapeutic target gene, a prioritization of potential effective drugs is obtained by assessing their impact on the transcription of genes in the pathway(s) including the target. We performed in silico validation and comparison with a state-of-art technique based on similar principles. We next performed experimental validation in two different real-case drug repositioning scenarios: (i) upregulation of the glutamate-pyruvate transaminase (GPT), which has been shown to induce reduction of oxalate levels in a mouse model of primary hyperoxaluria, and (ii) activation of the transcription factor TFEB, a master regulator of lysosomal biogenesis and autophagy, whose modulation may be beneficial in neurodegenerative disorders. Availability and implementation: A web tool for Gene2drug is freely available at http://gene2drug.tigem.it. An R package is under development and can be obtained from https://github.com/franapoli/gep2pep. Contact: dibernardo@tigem.it. Supplementary information: Supplementary data are available at Bioinformatics online.
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Biologia Computacional/métodos , Simulação por Computador , Reposicionamento de Medicamentos/métodos , Software , Animais , Linhagem Celular , Descoberta de Drogas/métodos , Humanos , CamundongosRESUMO
To orchestrate the genomic response to cellular stress signals, p53 recognizes and binds to DNA containing specific and well-characterized p53-responsive elements (REs). Differences in RE sequences can strongly affect the p53 transactivation capacity and occur even between closely related species. Therefore, the identification and characterization of a species-specific p53 Binding sistes (BS) consensus sequence and of the associated target genes may help to provide new insights into the evolution of the p53 regulatory networks across different species. Although p53 functions were studied in a wide range of species, little is known about the p53-mediated transcriptional signature in Danio rerio. Here, we designed and biochemically validated a computational approach to identify novel p53 target genes in Danio rerio genome. Screening all the Danio rerio genome by pattern-matching-based analysis, we found p53 RE-like patterns proximal to 979 annotated Danio rerio genes. Prioritization analysis identified a subset of 134 candidate pattern-related genes, 31 of which have been investigated in further biochemical assays. Our study identified runx1, axin1, traf4a, hspa8, col4a5, necab2, and dnajc9 genes as novel direct p53 targets and 12 additional p53-controlled genes in Danio rerio genome. The proposed combinatorial approach resulted to be highly sensitive and robust for identifying new p53 target genes also in additional animal species.
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Genoma , Elementos de Resposta , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Peixe-Zebra/genética , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Evolução Molecular , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSC70/genética , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Alinhamento de Sequência , Transdução de Sinais , Fator 4 Associado a Receptor de TNF/genética , Fator 4 Associado a Receptor de TNF/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
GNB5 encodes the G protein ß subunit 5 and is involved in inhibitory G protein signaling. Here, we report mutations in GNB5 that are associated with heart-rate disturbance, eye disease, intellectual disability, gastric problems, hypotonia, and seizures in nine individuals from six families. We observed an association between the nature of the variants and clinical severity; individuals with loss-of-function alleles had more severe symptoms, including substantial developmental delay, speech defects, severe hypotonia, pathological gastro-esophageal reflux, retinal disease, and sinus-node dysfunction, whereas related heterozygotes harboring missense variants presented with a clinically milder phenotype. Zebrafish gnb5 knockouts recapitulated the phenotypic spectrum of affected individuals, including cardiac, neurological, and ophthalmological abnormalities, supporting a direct role of GNB5 in the control of heart rate, hypotonia, and vision.
Assuntos
Bradicardia/genética , Bradicardia/fisiopatologia , Deficiências do Desenvolvimento/genética , Subunidades beta da Proteína de Ligação ao GTP/genética , Genes Recessivos/genética , Mutação/genética , Nó Sinoatrial/fisiopatologia , Adolescente , Animais , Criança , Deficiências do Desenvolvimento/fisiopatologia , Feminino , Subunidades beta da Proteína de Ligação ao GTP/deficiência , Refluxo Gastroesofágico/genética , Refluxo Gastroesofágico/fisiopatologia , Deleção de Genes , Frequência Cardíaca/genética , Heterozigoto , Humanos , Masculino , Hipotonia Muscular/genética , Mutação de Sentido Incorreto/genética , Linhagem , Fenótipo , Doenças Retinianas/genética , Doenças Retinianas/fisiopatologia , Convulsões/genética , Síndrome , Adulto Jovem , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Proteínas de Peixe-ZebraRESUMO
BACKGROUND: Human gliomas are a heterogeneous group of primary malignant brain tumors whose molecular pathogenesis is not yet solved. In this regard, a major research effort has been directed at identifying novel specific glioma-associated genes. Here, we investigated the effect of TRIM8 gene in glioma. METHODS: TRIM8 transcriptional level was profiled in our own glioma cases collection by qPCR and confirmed in the independent TCGA glioma cohort. The association between TRIM8 expression and Overall Survival and Progression-free Survival in TCGA cohort was determined by using uni-multivariable Cox regression analysis. The effect of TRIM8 on patient glioma cell proliferation was evaluated by performing MTT and clonogenic assays. The mechanisms causing the reduction of TRIM8 expression were explored by using qPCR and in vitro assays. RESULTS: We showed that TRIM8 expression correlates with unfavorable clinical outcome in glioma patients. We found that a restored TRIM8 expression induced a significant reduction of clonogenic potential in U87MG and patient's glioblastoma cells. Finally we provide experimental evidences showing that miR-17 directly targets the 3' UTR of TRIM8 and post-transcriptionally represses the expression of TRIM8. CONCLUSIONS: Our study provides evidences that TRIM8 may participate in the carcinogenesis and progression of glioma and that the transcriptional repression of TRIM8 might have potential value for predicting poor prognosis in glioma patients.
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Neoplasias Encefálicas/genética , Proteínas de Transporte/biossíntese , Glioma/genética , Proteínas do Tecido Nervoso/biossíntese , Prognóstico , Neoplasias Encefálicas/patologia , Proteínas de Transporte/genética , Proliferação de Células/genética , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Gradação de Tumores , Proteínas do Tecido Nervoso/genéticaRESUMO
RNA polymerase III (Pol III) synthesizes tRNAs and other small noncoding RNAs to regulate protein synthesis. Dysregulation of Pol III transcription has been linked to cancer, and germline mutations in genes encoding Pol III subunits or tRNA processing factors cause neurogenetic disorders in humans, such as hypomyelinating leukodystrophies and pontocerebellar hypoplasia. Here we describe an autosomal recessive disorder characterized by cerebellar hypoplasia and intellectual disability, as well as facial dysmorphic features, short stature, microcephaly, and dental anomalies. Whole-exome sequencing revealed biallelic missense alterations of BRF1 in three families. In support of the pathogenic potential of the discovered alleles, suppression or CRISPR-mediated deletion of brf1 in zebrafish embryos recapitulated key neurodevelopmental phenotypes; in vivo complementation showed all four candidate mutations to be pathogenic in an apparent isoform-specific context. BRF1 associates with BDP1 and TBP to form the transcription factor IIIB (TFIIIB), which recruits Pol III to target genes. We show that disease-causing mutations reduce Brf1 occupancy at tRNA target genes in Saccharomyces cerevisiae and impair cell growth. Moreover, BRF1 mutations reduce Pol III-related transcription activity in vitro. Taken together, our data show that BRF1 mutations that reduce protein activity cause neurodevelopmental anomalies, suggesting that BRF1-mediated Pol III transcription is required for normal cerebellar and cognitive development.
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Anormalidades Múltiplas/genética , Deficiência Intelectual/genética , Mutação , RNA Polimerase III/metabolismo , Fatores Associados à Proteína de Ligação a TATA/genética , Transcrição Gênica , Anormalidades Múltiplas/diagnóstico , Adolescente , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Encéfalo/patologia , Proliferação de Células , Criança , Pré-Escolar , Exoma , Fácies , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Deficiência Intelectual/diagnóstico , Imageamento por Ressonância Magnética , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Linhagem , Fenótipo , Conformação Proteica , Isoformas de Proteínas , Irmãos , Síndrome , Fatores Associados à Proteína de Ligação a TATA/química , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Peixe-ZebraRESUMO
Kabuki syndrome (KS) is a multiple congenital anomalies syndrome characterized by characteristic facial features and varying degrees of mental retardation, caused by mutations in KMT2D/MLL2 and KDM6A/UTX genes. In this study, we performed a mutational screening on 303 Kabuki patients by direct sequencing, MLPA, and quantitative PCR identifying 133 KMT2D, 62 never described before, and four KDM6A mutations, three of them are novel. We found that a number of KMT2D truncating mutations result in mRNA degradation through the nonsense-mediated mRNA decay, contributing to protein haploinsufficiency. Furthermore, we demonstrated that the reduction of KMT2D protein level in patients' lymphoblastoid and skin fibroblast cell lines carrying KMT2D-truncating mutations affects the expression levels of known KMT2D target genes. Finally, we hypothesized that the KS patients may benefit from a readthrough therapy to restore physiological levels of KMT2D and KDM6A proteins. To assess this, we performed a proof-of-principle study on 14 KMT2D and two KDM6A nonsense mutations using specific compounds that mediate translational readthrough and thereby stimulate the re-expression of full-length functional proteins. Our experimental data showed that both KMT2D and KDM6A nonsense mutations displayed high levels of readthrough in response to gentamicin treatment, paving the way to further studies aimed at eventually treating some Kabuki patients with readthrough inducers.
Assuntos
Anormalidades Múltiplas/genética , Face/anormalidades , Doenças Hematológicas/genética , Doenças Vestibulares/genética , Anormalidades Múltiplas/tratamento farmacológico , Linhagem Celular , Códon sem Sentido/efeitos dos fármacos , Estudos de Coortes , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Estudos de Associação Genética , Gentamicinas/farmacologia , Gentamicinas/uso terapêutico , Haploinsuficiência , Doenças Hematológicas/tratamento farmacológico , Histona Desmetilases/genética , Proteínas de Homeodomínio/genética , Humanos , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , Proteínas Nucleares/genética , Sítios de Splice de RNA , Análise de Sequência de DNA , Transcrição Gênica , Doenças Vestibulares/tratamento farmacológicoRESUMO
TBC1D7 forms a complex with TSC1 and TSC2 that inhibits mTORC1 signaling and limits cell growth. Mutations in TBC1D7 were reported in a family with intellectual disability (ID) and macrocrania. Using exome sequencing, we identified two sisters homozygote for the novel c.17_20delAGAG, p.R7TfsX21 TBC1D7 truncating mutation. In addition to the already described macrocephaly and mild ID, they share osteoarticular defects, patella dislocation, behavioral abnormalities, psychosis, learning difficulties, celiac disease, prognathism, myopia, and astigmatism. Consistent with a loss-of-function of TBC1D7, the patient's cell lines show an increase in the phosphorylation of 4EBP1, a direct downstream target of mTORC1 and a delay in the initiation of the autophagy process. This second family allows enlarging the phenotypic spectrum associated with TBC1D7 mutations and defining a TBC1D7 syndrome. Our work reinforces the involvement of TBC1D7 in the regulation of mTORC1 pathways and suggests an altered control of autophagy as possible cause of this disease.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/genética , Doença Celíaca/genética , Deficiência Intelectual/genética , Megalencefalia/genética , Luxação Patelar/genética , Fosfoproteínas/metabolismo , Autofagia , Proteínas de Transporte/metabolismo , Doença Celíaca/patologia , Proteínas de Ciclo Celular , Linhagem Celular , Exoma , Feminino , Predisposição Genética para Doença , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Deficiência Intelectual/patologia , Peptídeos e Proteínas de Sinalização Intracelular , Megalencefalia/patologia , Mutação , Luxação Patelar/patologia , LinhagemRESUMO
The E3 Ubiquitin ligase TRIM50 promotes the formation and clearance of aggresome-associated polyubiquitinated proteins through HDAC6 interaction, a tubulin specific deacetylase that regulates microtubule-dependent aggresome formation. In this report we showed that TRIM50 is a target of HDAC6 with Lys-372 as a critical residue for acetylation. We identified p300 and PCAF as two TRIM50 acetyltransferases and we further showed that a balance between ubiquitination and acetylation regulates TRIM50 degradation.
Assuntos
Histona Desacetilases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Acetilação , Animais , Linhagem Celular , Células HEK293 , Células HeLa , Desacetilase 6 de Histona , Histona Desacetilases/genética , Humanos , Camundongos , Microtúbulos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/química , Ubiquitinação , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Williams Beuren syndrome (WBS) is a multisystemic disorder caused by a hemizygous deletion of 1.5 Mb on chromosome 7q11.23 spanning 28 genes. A few patients with larger and smaller WBS deletion have been reported. They show clinical features that vary between isolated SVAS to the full spectrum of WBS phenotype, associated with epilepsy or autism spectrum behavior. Here we describe four patients with atypical WBS 7q11.23 deletions. Two carry ~3.5 Mb larger deletion towards the telomere that includes Huntingtin-interacting protein 1 (HIP1) and tyrosine 3-monooxygenase/tryptophan 5-monooxigenase activation protein gamma (YWHAG) genes. Other two carry a shorter deletion of ~1.2 Mb at centromeric side that excludes the distal WBS genes BAZ1B and FZD9. Along with previously reported cases, genotype-phenotype correlation in the patients described here further suggests that haploinsufficiency of HIP1 and YWHAG might cause the severe neurological and neuropsychological deficits including epilepsy and autistic traits, and that the preservation of BAZ1B and FZD9 genes may be related to mild facial features and moderate neuropsychological deficits. This report highlights the importance to characterize additional patients with 7q11.23 atypical deletions comparing neuropsychological and clinical features between these individuals to shed light on the pathogenic role of genes within and flanking the WBS region.