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Regulatory T cells (Tregs) control adaptive immunity and restrain type 2 inflammation in allergic disease. Interleukin-33 promotes the expansion of tissue-resident Tregs and group 2 innate lymphoid cells (ILC2s); however, how Tregs locally coordinate their function within the inflammatory niche is not understood. Here, we show that ILC2s are critical orchestrators of Treg function. Using spatial, cellular, and molecular profiling of the type 2 inflamed niche, we found that ILC2s and Tregs engage in a direct (OX40L-OX40) and chemotaxis-dependent (CCL1-CCR8) cellular dialogue that enforces the local accumulation of Gata3high Tregs, which are transcriptionally and functionally adapted to the type 2 environment. Genetic interruption of ILC2-Treg communication resulted in uncontrolled type 2 lung inflammation after allergen exposure. Mechanistically, we found that Gata3high Tregs can modulate the local bioavailability of the costimulatory molecule OX40L, which subsequently controlled effector memory T helper 2 cell numbers. Hence, ILC2-Treg interactions represent a critical feedback mechanism to control adaptive type 2 immunity.
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Imunidade Adaptativa , Fator de Transcrição GATA3 , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores , Animais , Linfócitos T Reguladores/imunologia , Fator de Transcrição GATA3/imunologia , Fator de Transcrição GATA3/metabolismo , Camundongos , Imunidade Adaptativa/imunologia , Linfócitos/imunologia , Imunidade Inata/imunologia , Camundongos Knockout , Células Th2/imunologia , FemininoRESUMO
Single-nuclei RNA sequencing (snRNA-seq) allows for obtaining gene expression profiles from frozen or hard-to-dissociate tissues at the single-nuclei level. Here, we describe a protocol to obtain snRNA-seq data of pancreatic tumors from orthotopically grafted organoid-derived mouse models. We provide details on the establishment of these mouse models, the isolation of single nuclei from pancreatic tumors, and the analysis of the snRNA-seq datasets. For complete details on the use and execution of this protocol, please refer to Mucciolo et al.1.
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The estrogen receptor-α (ER) drives 75% of breast cancers. On activation, the ER recruits and assembles a 1-2 MDa transcriptionally active complex. These complexes can modulate tumour growth, and understanding the roles of individual proteins within these complexes can help identify new therapeutic targets. Here, we present the discovery of ER and ZMIZ1 within the same multi-protein assembly by quantitative proteomics, and validated by proximity ligation assay. We characterise ZMIZ1 function by demonstrating a significant decrease in the proliferation of ER-positive cancer cell lines. To establish a role for the ER-ZMIZ1 interaction, we measured the transcriptional changes in the estrogen response post-ZMIZ1 knockdown using an RNA-seq time-course over 24 h. Gene set enrichment analysis of the ZMIZ1-knockdown data identified a specific delay in the response of estradiol-induced cell cycle genes. Integration of ENCODE data with our RNA-seq results identified that ER and ZMIZ1 both bind the promoter of E2F2. We therefore propose that ER and ZMIZ1 interact to enable the efficient estrogenic response at subset of cell cycle genes via a novel ZMIZ1-ER-E2F2 signalling axis. Finally, we show that high ZMIZ1 expression is predictive of worse patient outcome, ER and ZMIZ1 are co-expressed in breast cancer patients in TCGA and METABRIC, and the proteins are co-localised within the nuclei of tumour cell in patient biopsies. In conclusion, we establish that ZMIZ1 is a regulator of the estrogenic cell cycle response and provide evidence of the biological importance of the ER-ZMIZ1 interaction in ER-positive patient tumours, supporting potential clinical relevance.
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Neoplasias da Mama , Fator de Transcrição E2F2 , Receptor alfa de Estrogênio , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Receptor alfa de Estrogênio/metabolismo , Receptor alfa de Estrogênio/genética , Feminino , Linhagem Celular Tumoral , Fator de Transcrição E2F2/metabolismo , Fator de Transcrição E2F2/genética , Proliferação de Células/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ligação Proteica , Regiões Promotoras Genéticas/genética , Transdução de Sinais , Ciclo Celular/genética , PrognósticoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. Cancer-associated fibroblasts (CAFs) are recognized potential therapeutic targets, but poor understanding of these heterogeneous cell populations has limited the development of effective treatment strategies. We previously identified transforming growth factor beta (TGF-ß) as a main driver of myofibroblastic CAFs (myCAFs). Here, we show that epidermal growth factor receptor/Erb-B2 receptor (EGFR/ERBB2) signaling is induced by TGF-ß in myCAFs through an autocrine process mediated by amphiregulin. Inhibition of this EGFR/ERBB2-signaling network in PDAC organoid-derived cultures and mouse models differentially impacts distinct CAF subtypes, providing insights into mechanisms underpinning their heterogeneity. Remarkably, EGFR-activated myCAFs promote PDAC metastasis in mice, unmasking functional significance in myCAF heterogeneity. Finally, analyses of other cancer datasets suggest that these processes might operate in other malignancies. These data provide functional relevance to myCAF heterogeneity and identify a candidate target for preventing tumor invasion in PDAC.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Camundongos , Animais , Miofibroblastos/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Carcinoma Ductal Pancreático/tratamento farmacológico , Transdução de Sinais , Fator de Crescimento Transformador beta , Microambiente TumoralRESUMO
High-grade serous ovarian carcinoma (HGSOC) is characterised by poor outcome and extreme chromosome instability (CIN). Therapies targeting centrosome amplification (CA), a key mediator of chromosome missegregation, may have significant clinical utility in HGSOC. However, the prevalence of CA in HGSOC, its relationship to genomic biomarkers of CIN and its potential impact on therapeutic response have not been defined. Using high-throughput multi-regional microscopy on 287 clinical HGSOC tissues and 73 cell lines models, here we show that CA through centriole overduplication is a highly recurrent and heterogeneous feature of HGSOC and strongly associated with CIN and genome subclonality. Cell-based studies showed that high-prevalence CA is phenocopied in ovarian cancer cell lines, and that high CA is associated with increased multi-treatment resistance; most notably to paclitaxel, the commonest treatment used in HGSOC. CA in HGSOC may therefore present a potential driver of tumour evolution and a powerful biomarker for response to standard-of-care treatment.
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Cistadenocarcinoma Seroso , Neoplasias Ovarianas , Humanos , Feminino , Neoplasias Ovarianas/patologia , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Centrossomo/metabolismo , Cistadenocarcinoma Seroso/genéticaRESUMO
Background: Branched-chain aminotransferase 1 (BCAT1) has been proposed to drive proliferation and invasion of isocitrate dehydrogenase (IDH) wild-type glioblastoma cells. However, the Cancer Genome Atlas (TCGA) dataset shows considerable variation in the expression of this enzyme in glioblastoma. The aim of this study was to determine the role of BCAT1 in driving the proliferation and invasion of glioblastoma cells and xenografts that have widely differing levels of BCAT1 expression and the mechanism responsible. Methods: The activity of BCAT1 was modulated in IDH wild-type patient-derived glioblastoma cell lines, and in orthotopically implanted tumors derived from these cells, to examine the effects of BCAT1 expression on tumor phenotype. Results: In cells with constitutively high BCAT1 expression and a glycolytic metabolic phenotype, inducible shRNA knockdown of the enzyme resulted in reduced proliferation and invasion by increasing the concentration of α-ketoglutarate, leading to reduced DNA methylation, HIF-1α destabilization, and reduced expression of the transcription factor Forkhead box protein M1 (FOXM1). Conversely, overexpression of the enzyme increased HIF-1α expression and promoted proliferation and invasion. However, in cells with an oxidative phenotype and very low constitutive expression of BCAT1 increased expression of the enzyme had no effect on invasion and reduced cell proliferation. This occurred despite an increase in HIF-1α levels and could be explained by decreased TCA cycle flux. Conclusions: There is a wide variation in BCAT1 expression in glioblastoma and its role in proliferation and invasion is dependent on tumor subtype.
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GCNA proteins are expressed across eukarya in pluripotent cells and have conserved functions in fertility. GCNA homologs Spartan (DVC-1) and Wss1 resolve DNA-protein crosslinks (DPCs), including Topoisomerase-DNA adducts, during DNA replication. Here, we show that GCNA mutants in mouse and C. elegans display defects in genome maintenance including DNA damage, aberrant chromosome condensation, and crossover defects in mouse spermatocytes and spontaneous genomic rearrangements in C. elegans. We show that GCNA and topoisomerase II (TOP2) physically interact in both mice and worms and colocalize on condensed chromosomes during mitosis in C. elegans embryos. Moreover, C. elegans gcna-1 mutants are hypersensitive to TOP2 poison. Together, our findings support a model in which GCNA provides genome maintenance functions in the germline and may do so, in part, by promoting the resolution of TOP2 DPCs.
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Replicação do DNA , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Mitose , Proteínas Nucleares/metabolismo , Espermatócitos/citologia , Animais , Caenorhabditis elegans , Dano ao DNA , Reparo do DNA , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Genoma , Células Germinativas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas Nucleares/genética , Espermatócitos/metabolismo , EspermatogêneseRESUMO
The Piwi-interacting RNA (piRNA) pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. piRNA biogenesis requires a specialized machinery that converts long single-stranded precursors into small RNAs of â¼25-nucleotides in length. This process involves factors that operate in two different subcellular compartments: the nuage/Yb body and mitochondria. How these two sites communicate to achieve accurate substrate selection and efficient processing remains unclear. Here, we investigate a previously uncharacterized piRNA biogenesis factor, Daedalus (Daed), that is located on the outer mitochondrial membrane. Daed is essential for Zucchini-mediated piRNA production and the correct localization of the indispensable piRNA biogenesis factor Armitage (Armi). We found that Gasz and Daed interact with each other and likely provide a mitochondrial "anchoring platform" to ensure that Armi is held in place, proximal to Zucchini, during piRNA processing. Our data suggest that Armi initially identifies piRNA precursors in nuage/Yb bodies in a manner that depends on Piwi and then moves to mitochondria to present precursors to the mitochondrial biogenesis machinery. These results represent a significant step in understanding a critical aspect of transposon silencing; namely, how RNAs are chosen to instruct the piRNA machinery in the nature of its silencing targets.
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Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , RNA Helicases/metabolismo , RNA Interferente Pequeno/biossíntese , Animais , Linhagem Celular , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Técnicas de Silenciamento de Genes , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/metabolismoRESUMO
Estrogen Receptor alpha (ERα) plays a major role in most breast cancers, and it is the target of endocrine therapies used in the clinic as standard of care for women with breast cancer expressing this receptor. The two methods ChIP-seq (chromatin immunoprecipitation coupled with deep sequencing) and RIME (Rapid Immunoprecipitation of Endogenous Proteins) have greatly improved our understanding of ERα function during breast cancer progression and in response to anti-estrogens. A critical component of both ChIP-seq and RIME protocols is the antibody that is used against the bait protein. To date, most of the ChIP-seq and RIME experiments for the study of ERα have been performed using the sc-543 antibody from Santa Cruz Biotechnology. However, this antibody has been discontinued, thereby severely impacting the study of ERα in normal physiology as well as diseases such as breast cancer and ovarian cancer. Here, we compare the sc-543 antibody with other commercially available antibodies, and we show that 06-935 (EMD Millipore) and ab3575 (Abcam) antibodies can successfully replace the sc-543 antibody for ChIP-seq and RIME experiments.
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Anticorpos , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Receptor alfa de Estrogênio/imunologia , Imunoprecipitação/métodos , Especificidade de Anticorpos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Células MCF-7RESUMO
BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare disease characterized by pulmonary arteriole remodeling, elevated arterial pressure and resistance, and subsequent heart failure. Compared with adult-onset disease, pediatric-onset PAH is more heterogeneous and often associated with worse prognosis. Although BMPR2 mutations underlie ≈70% of adult familial PAH (FPAH) cases, the genetic basis of PAH in children is less understood. METHODS: We performed genetic analysis of 155 pediatric- and 257 adult-onset PAH patients, including both FPAH and sporadic, idiopathic PAH (IPAH). After screening for 2 common PAH risk genes, mutation-negative FPAH and all IPAH cases were evaluated by exome sequencing. RESULTS: We observed similar frequencies of rare, deleterious BMPR2 mutations in pediatric- and adult-onset patients: ≈55% in FPAH and 10% in IPAH patients in both age groups. However, there was significant enrichment of TBX4 mutations in pediatric- compared with adult-onset patients (IPAH: 10/130 pediatric versus 0/178 adult-onset), and TBX4 carriers had younger mean age-of-onset compared with BMPR2 carriers. Mutations in other known PAH risk genes were infrequent in both age groups. Notably, among pediatric IPAH patients without mutations in known risk genes, exome sequencing revealed a 2-fold enrichment of de novo likely gene-damaging and predicted deleterious missense variants. CONCLUSIONS: Mutations in known PAH risk genes accounted for ≈70% to 80% of FPAH in both age groups, 21% of pediatric-onset IPAH, and 11% of adult-onset IPAH. Rare, predicted deleterious variants in TBX4 are enriched in pediatric patients and de novo variants in novel genes may explain ≈19% of pediatric-onset IPAH cases.
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Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Análise Mutacional de DNA/métodos , Sequenciamento do Exoma/métodos , Exoma , Hipertensão Pulmonar Primária Familiar/genética , Mutação , Proteínas com Domínio T/genética , Adolescente , Adulto , Idade de Início , Criança , Hipertensão Pulmonar Primária Familiar/diagnóstico , Hipertensão Pulmonar Primária Familiar/fisiopatologia , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Fenótipo , Valor Preditivo dos Testes , Fatores de Risco , Adulto JovemRESUMO
BACKGROUND & AIMS: We performed a genome-wide association study (GWAS) to identify genetic risk factors for drug-induced liver injury (DILI) from licensed drugs without previously reported genetic risk factors. METHODS: We performed a GWAS of 862 persons with DILI and 10,588 population-matched controls. The first set of cases was recruited before May 2009 in Europe (n = 137) and the United States (n = 274). The second set of cases were identified from May 2009 through May 2013 from international collaborative studies performed in Europe, the United States, and South America. For the GWAS, we included only cases with patients of European ancestry associated with a particular drug (but not flucloxacillin or amoxicillin-clavulanate). We used DNA samples from all subjects to analyze HLA genes and single nucleotide polymorphisms. After the discovery analysis was concluded, we validated our findings using data from 283 European patients with diagnosis of DILI associated with various drugs. RESULTS: We associated DILI with rs114577328 (a proxy for A*33:01 a HLA class I allele; odds ratio [OR], 2.7; 95% confidence interval [CI], 1.9-3.8; P = 2.4 × 10-8) and with rs72631567 on chromosome 2 (OR, 2.0; 95% CI, 1.6-2.5; P = 9.7 × 10-9). The association with A*33:01 was mediated by large effects for terbinafine-, fenofibrate-, and ticlopidine-related DILI. The variant on chromosome 2 was associated with DILI from a variety of drugs. Further phenotypic analysis indicated that the association between DILI and A*33:01 was significant genome wide for cholestatic and mixed DILI, but not for hepatocellular DILI; the polymorphism on chromosome 2 was associated with cholestatic and mixed DILI as well as hepatocellular DILI. We identified an association between rs28521457 (within the lipopolysaccharide-responsive vesicle trafficking, beach and anchor containing gene) and only hepatocellular DILI (OR, 2.1; 95% CI, 1.6-2.7; P = 4.8 × 10-9). We did not associate any specific drug classes with genetic polymorphisms, except for statin-associated DILI, which was associated with rs116561224 on chromosome 18 (OR, 5.4; 95% CI, 3.0-9.5; P = 7.1 × 10-9). We validated the association between A*33:01 terbinafine- and sertraline-induced DILI. We could not validate the association between DILI and rs72631567, rs28521457, or rs116561224. CONCLUSIONS: In a GWAS of persons of European descent with DILI, we associated HLA-A*33:01 with DILI due to terbinafine and possibly fenofibrate and ticlopidine. We identified polymorphisms that appear to be associated with DILI from statins, as well as 2 non-drug-specific risk factors.
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Doença Hepática Induzida por Substâncias e Drogas/genética , Cromossomos Humanos Par 2/genética , Antígenos HLA-A/genética , Alelos , Antidepressivos/efeitos adversos , Antifúngicos/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Feminino , Fenofibrato/efeitos adversos , Genes MHC Classe I/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Hipolipemiantes/efeitos adversos , Masculino , Pessoa de Meia-Idade , Naftalenos/efeitos adversos , Razão de Chances , Fenótipo , Inibidores da Agregação Plaquetária/efeitos adversos , Polimorfismo de Nucleotídeo Único , Sertralina/efeitos adversos , Terbinafina , Ticlopidina/efeitos adversos , População Branca/genéticaRESUMO
BACKGROUND: The Trial of Early Aggressive Therapy in Juvenile Idiopathic Arthritis (TREAT trial) was accompanied by a once-in-a-generation sample collection for translational research. In this paper, we report the results of whole blood gene expression analyses and genomic data-mining designed to cast light on the immunopathogenesis of polyarticular juvenile idiopathic arthritis (JIA). METHODS: TREAT samples and samples from an independent cohort were analyzed on Affymetrix microarrays and compared to healthy controls. Data from the independent cohort were used to validate the TREAT data. Pathways analysis was used to characterize gene expression profiles. Furthermore, we correlated differential gene expression with new information about functional regulatory elements within the genome to develop models of aberrant gene expression in JIA. RESULTS: There was a strong concordance in gene expression between TREAT samples and the independent cohort. In addition, rheumatoid factor (RF)-positive and RF-negative patients showed only small differences on whole blood expression profiles. Analysis of the combined samples showed 158 genes represented by 176 probes that showed differential expression between TREAT subjects at baseline and healthy controls. None of the differentially expressed genes were encoded within linkage disequilibrium blocks containing single nucleotide polymorphisms known to be associated with risk for JIA. Functional analysis of these genes showed functional associations with multiple processes associated with innate and adaptive immunity, and appeared to reflect overall suppression of STAT1-3/interferon response factor-mediated pathways. CONCLUSIONS: Despite their limitations, whole blood expression profiles clearly distinguish children with polyarticular JIA from healthy controls. Whole blood expression profiles identify several immunologic pathways of biologic relevance that will need to be pursued in homogeneous cell populations in order to clarify mechanisms of pathogenesis. TRIAL REGISTRATION: ClinicalTrials.gov registry #NCT00443430 , originally registered 2 March 2007 and last updated 30 May 2013.
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Artrite Juvenil/genética , Artrite Juvenil/imunologia , Perfilação da Expressão Gênica/métodos , Adolescente , Antirreumáticos/uso terapêutico , Artrite Juvenil/tratamento farmacológico , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , TranscriptomaRESUMO
OBJECTIVE: Flupirtine is a nonopioid analgesic with regulatory approval in a number of European countries. Because of the risk of serious liver injury, its use is now limited to short-term pain management. We aimed to identify genetic risk factors for flupirtine-related drug-induced liver injury (DILI) as these are unknown. MATERIALS AND METHODS: Six flupirtine-related DILI patients from Germany were included in a genome-wide association study (GWAS) involving a further 614 European cases of DILI because of other drugs and 10,588 population controls. DILI was diagnosed by causality assessment and expert review. Human leucocyte antigen (HLA) and single nucleotide polymorphism genotypes were imputed from the GWAS data, with direct HLA typing performed on selected cases to validate HLA predictions. Four replication cases that were unavailable for the GWAS were genotyped by direct HLA typing, yielding an overall total of 10 flupirtine DILI cases. RESULTS: In the six flupirtine DILI cases included in the GWAS, we found a significant enrichment of the DRB1*16:01-DQB1*05:02 haplotype compared with the controls (minor allele frequency cases 0.25 and minor allele frequency controls 0.013; P=1.4 × 10(-5)). We estimated an odds ratio for haplotype carriers of 18.7 (95% confidence interval 2.5-140.5, P=0.002) using population-specific HLA control data. The result was replicated in four additional cases, also with a haplotype frequency of 0.25. In the combined cohort (six GWAS plus four replication cases), the haplotype was also significant (odds ratio 18.7, 95% confidence interval 4.31-81.42, P=6.7 × 10(-5)). CONCLUSION: We identified a novel HLA class II association for DILI, confirming the important contribution of HLA genotype towards the risk of DILI generally.
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Aminopiridinas/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/genética , Cadeias beta de HLA-DQ/genética , Cadeias HLA-DRB1/genética , Adulto , Idoso , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Essential tremor (ET) is one of the most common causes of tremor in humans. Despite its high heritability and prevalence, few susceptibility genes for ET have been identified. To identify ET genes, whole-exome sequencing was performed in 37 early-onset ET families with an autosomal-dominant inheritance pattern. We identified candidate genes for follow-up functional studies in five ET families. In two independent families, we identified variants predicted to affect function in the nitric oxide (NO) synthase 3 gene (NOS3) that cosegregated with disease. NOS3 is highly expressed in the central nervous system (including cerebellum), neurons and endothelial cells, and is one of three enzymes that converts l-arginine to the neurotransmitter NO. In one family, a heterozygous variant, c.46G>A (p.(Gly16Ser)), in NOS3, was identified in three affected ET cases and was absent in an unaffected family member; and in a second family, a heterozygous variant, c.164C>T (p.(Pro55Leu)), was identified in three affected ET cases (dizygotic twins and their mother). Both variants result in amino-acid substitutions of highly conserved amino-acid residues that are predicted to be deleterious and damaging by in silico analysis. In three independent families, variants predicted to affect function were also identified in other genes, including KCNS2 (KV9.2), HAPLN4 (BRAL2) and USP46. These genes are highly expressed in the cerebellum and Purkinje cells, and influence function of the gamma-amino butyric acid (GABA)-ergic system. This is in concordance with recent evidence that the pathophysiological process in ET involves cerebellar dysfunction and possibly cerebellar degeneration with a reduction in Purkinje cells, and a decrease in GABA-ergic tone.
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Tremor Essencial/genética , Mutação de Sentido Incorreto , Óxido Nítrico Sintase Tipo III/genética , Adulto , Idade de Início , Endopeptidases/genética , Tremor Essencial/patologia , Proteínas da Matriz Extracelular/genética , Feminino , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Linhagem , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genéticaRESUMO
BACKGROUND: We have previously shown that childhood-onset rheumatic diseases show aberrant patterns of gene expression that reflect pathology-associated co-expression networks. In this study, we used novel computational approaches to examine how disease-associated networks are altered in one of the most common rheumatic diseases of childhood, juvenile idiopathic arthritis (JIA). METHODS: Using whole blood gene expression profiles derived from children in a pediatric rheumatology clinical trial, we used a network approach to understanding the impact of therapy and the underlying biology of response/non-response to therapy. RESULTS: We demonstrate that therapy for JIA is associated with extensive re-ordering of gene expression networks, even in children who respond inadequately to therapy. Furthermore, we observe distinct differences in the evolution of specific network properties when we compare children who have been treated successfully with those who have inadequate treatment response. CONCLUSIONS: Despite the inherent noisiness of whole blood gene expression data, our findings demonstrate how therapeutic response might be mapped and understood in pathologically informative cells in a broad range of human inflammatory diseases.
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Antirreumáticos/uso terapêutico , Artrite Juvenil/tratamento farmacológico , Artrite Juvenil/genética , Resistência a Medicamentos/genética , Adolescente , Criança , Pré-Escolar , Etanercepte/uso terapêutico , Feminino , Humanos , Masculino , Metotrexato/uso terapêutico , Prednisona/uso terapêutico , Análise de Sequência de RNA , TranscriptomaRESUMO
Uveal melanoma (UM) is an aggressive intraocular malignancy with limited therapeutic options. Both primary and metastatic UM are characterized by oncogenic mutations in the G-protein alpha subunit q and 11. Furthermore, nearly 40% of UM has amplification of the chromosomal arm 8q and monosomy of chromosome 3, with consequent anomalies of MYC copy number. Chromatin regulators have become attractive targets for cancer therapy. In particular, the bromodomain and extra-terminal (BET) inhibitor JQ1 has shown selective inhibition of c-Myc expression with antiproliferative activity in hematopoietic and solid tumors. Here we provide evidence that JQ1 had cytotoxic activity in UM cell lines carrying Gnaq/11 mutations, while in cells without the mutations had little effects. Using microarray analysis, we identified a large subset of genes modulated by JQ1 involved in the regulation of cell cycle, apoptosis and DNA repair. Further analysis of selected genes determined that the concomitant silencing of Bcl-xL and Rad51 represented the minimal requirement to mimic the apoptotic effects of JQ1 in the mutant cells, independently of c-Myc. In addition, administration of JQ1 to mouse xenograft models of Gnaq-mutant UM resulted in significant inhibition of tumor growth.Collectively, our results define BRD4 targeting as a novel therapeutic intervention against UM with Gnaq/Gna11 mutations.
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Azepinas/uso terapêutico , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Triazóis/uso terapêutico , Neoplasias Uveais/tratamento farmacológico , Neoplasias Uveais/genética , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Genes myc/fisiologia , Humanos , Melanoma/patologia , Camundongos , Camundongos SCID , Terapia de Alvo Molecular , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Neoplasias Uveais/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Congenital diaphragmatic hernia (CDH) is a serious birth defect that accounts for 8% of all major birth anomalies. Approximately 40% of cases occur in association with other anomalies. As sporadic complex CDH likely has a significant impact on reproductive fitness, we hypothesized that de novo variants would account for the etiology in a significant fraction of cases. We performed exome sequencing in 39 CDH trios and compared the frequency of de novo variants with 787 unaffected controls from the Simons Simplex Collection. We found no significant difference in overall frequency of de novo variants between cases and controls. However, among genes that are highly expressed during diaphragm development, there was a significant burden of likely gene disrupting (LGD) and predicted deleterious missense variants in cases (fold enrichment = 3.2, P-value = 0.003), and these genes are more likely to be haploinsufficient (P-value = 0.01) than the ones with benign missense or synonymous de novo variants in cases. After accounting for the frequency of de novo variants in the control population, we estimate that 15% of sporadic complex CDH patients are attributable to de novo LGD or deleterious missense variants. We identified several genes with predicted deleterious de novo variants that fall into common categories of genes related to transcription factors and cell migration that we believe are related to the pathogenesis of CDH. These data provide supportive evidence for novel genes in the pathogenesis of CDH associated with other anomalies and suggest that de novo variants play a significant role in complex CDH cases.
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Anormalidades Congênitas/genética , Hérnia Diafragmática/genética , Mutação de Sentido Incorreto , Feminino , Humanos , MasculinoRESUMO
OBJECTIVE: To determine whether gene expression profiles identified in peripheral whole blood samples could be used to determine therapeutic outcome in a cohort of children with newly diagnosed polyarticular juvenile idiopathic arthritis (JIA). METHODS: Whole blood samples from the Trial of Early Aggressive Therapy (TREAT) in JIA patients were analyzed on Illumina microarrays, and differential gene expression was compared to expression in healthy controls. Microarray results were validated by real-time quantitative polymerase chain reaction in an independent cohort of samples. Pathway analysis software was used to characterize gene expression profiles. Support vector machines were used to develop predictive models for different patient classes. RESULTS: Differential gene expression profiles for rheumatoid factor (RF)-positive and RF-negative patients were remarkably similar. Pathway analysis revealed a broad range of affected pathways, consistent with current mechanistic theories. Modeling showed that the prognosis at 6 months was strongly linked to gene expression at presentation, irrespective of treatment. CONCLUSION: Gene expression is linked to therapeutic outcome, and gene expression in the peripheral blood may be a suitable target for a prognostic test.
Assuntos
Artrite Juvenil/tratamento farmacológico , Artrite Juvenil/genética , Perfilação da Expressão Gênica , Imunoglobulina G/uso terapêutico , Metotrexato/uso terapêutico , Farmacogenética , Prednisolona/uso terapêutico , Receptores do Fator de Necrose Tumoral/uso terapêutico , Adolescente , Artrite Juvenil/diagnóstico , Criança , Pré-Escolar , Estudos de Coortes , Quimioterapia Combinada , Etanercepte , Feminino , Testes Genéticos , Humanos , Masculino , Prognóstico , Reprodutibilidade dos Testes , Estudos Retrospectivos , Fator Reumatoide/genética , Método Simples-Cego , Fatores de Tempo , Resultado do TratamentoRESUMO
Current aquatic chemical testing guidelines recognize that solvents can potentially interfere with the organism or environmental conditions of aquatic ecotoxicity tests and therefore recommend concentration limits for their use. These recommendations are based on evidence of adverse solvent effects in apical level tests. The growing importance of subapical and chronic endpoints in future test strategies, however, suggests that the limits may need reassessment. To address this concern, microarrays were used to determine the effects of organic solvents, dimethylformamide (DMF) and dimethylsulfoxide (DMSO), on the transcriptome of zebrafish (Danio rerio) embryos. Embryos were exposed for 48 h to a range of concentrations between 0.025 and 32.0 ml/L. Effects on survival and development after 24 and 48 h were assessed microscopically, with no effects on mortality or morphology up to 2.0 and 16.0 ml/L for DMF and DMSO. However, analysis of 48-h embryonic RNA revealed large numbers of differentially expressed genes at concentrations well below the 0.1 ml/L solvent limit level. The enrichment of differentially expressed genes was found for metabolic, developmental, and other key biological processes, some of which could be linked to observed morphological effects at higher solvent concentrations. These findings emphasize the need to remove or lower as far as possible the concentrations of solvent carriers in ecotoxicology tests.
Assuntos
Dimetil Sulfóxido/toxicidade , Dimetilformamida/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Monitoramento Ambiental/métodos , Solventes/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Dimetil Sulfóxido/metabolismo , Dimetilformamida/metabolismo , Ecotoxicologia , Embrião não Mamífero/metabolismo , Solventes/metabolismo , Testes de Toxicidade , Transcriptoma , Poluentes Químicos da Água/metabolismo , Peixe-Zebra/embriologiaRESUMO
Large-scale toxicogenomic screening approaches offer great promise for generating a bias-free system-wide view of toxicological effects and modes-of-action of chemicals and ecotoxicants. However, early applications of microarray technology have identified relatively small groups of responding genes with which to define new targets for analysis by conventional means. We have trialled a more intensive approach to the design and interpretation of array experiments incorporating a balanced interwoven ANOVA design with higher levels of biological replication, a more thorough analysis of errors and false discovery rates, and an analysis of response patterns using gene network models. Zebrafish embryos were exposed from 1.5 h post-fertilization for 72 h to ecotoxicants representing different classes--2,4-dichlorophenol, 3,4-dichloroaniline, pentachlorophenol, and cadmium chloride--at low concentrations producing a developmental disturbance to 10% of embryos and half of this dose. Extracted whole embryo RNA was then analyzed on microarrays. Analysis revealed responses of 3000-5000 genes, which is 10-1000 times greater than previously reported, with significance at lower levels of fold change. Some gene responses were common to multiple toxicants, and others were restricted to just one or two toxicants. The gene expression profiles for the different toxicants were distinctive, and analysis using network-based models provided a high level of detail of affected processes, some of which were novel. This approach provides a more highly refined view of toxic effects, from which meaningful patterns of response can be discerned and related to functional deficits and from which more reliable indicators of toxicological effect can be predicted.