RESUMEN
Neurodegenerative diseases encompass a group of debilitating conditions resulting from progressive nerve cell death. Of these, Alzheimer's disease (AD) occurs most frequently, but is currently incurable and has limited treatment success. Late onset AD, the most common form, is highly heritable but is caused by a combination of non-genetic risk factors and many low-effect genetic variants whose disease-causing mechanisms remain unclear. By mining the FinnGen study database of phenome-wide association studies, we identified a rare variant, rs148726219, enriched in the Finnish population that is associated with AD risk and dementia, and appears to have arisen on a common haplotype with older AD-associated variants such as rs429358. The rs148726219 variant lies in an overlapping intron of the FosB proto-oncogene (FOSB) and ERCC excision repair 1 (ERCC1) genes. To understand the impact of this SNP on disease phenotypes, we performed CRISPR/Cas9 editing in a human induced pluripotent stem cell (hiPSC) line to generate isogenic clones harboring heterozygous and homozygous alleles of rs148726219. hiPSC clones differentiated into induced excitatory neurons (iNs) did not exhibit detectable molecular or morphological variation in differentiation potential compared to isogenic controls. However, global transcriptome analysis showed differential regulation of nearby genes and upregulation of several biological pathways related to neuronal function, particularly synaptogenesis and calcium signaling, specifically in mature iNs harboring rs148726219 homozygous and heterozygous alleles. Functional differences in iN circuit maturation as measured by calcium imaging were observed across genotypes. Edited mature iNs also displayed downregulation of unfolded protein response and cell death pathways. This study implicates a phenotypic impact of rs148726219 in the context of mature neurons, consistent with its identification in late onset AD, and underscores a hiPSC-based experimental model to functionalize GWAS-identified variants.
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Enfermedad de Alzheimer , Células Madre Pluripotentes Inducidas , Humanos , Enfermedad de Alzheimer/metabolismo , Polimorfismo de Nucleótido Simple , Genotipo , NeuronasRESUMEN
BACKGROUND: The remarkable growth of genome-wide association studies (GWAS) has created a critical need to experimentally validate the disease-associated variants, 90% of which involve non-coding variants. METHODS: To determine how the field is addressing this urgent need, we performed a comprehensive literature review identifying 36,676 articles. These were reduced to 1454 articles through a set of filters using natural language processing and ontology-based text-mining. This was followed by manual curation and cross-referencing against the GWAS catalog, yielding a final set of 286 articles. RESULTS: We identified 309 experimentally validated non-coding GWAS variants, regulating 252 genes across 130 human disease traits. These variants covered a variety of regulatory mechanisms. Interestingly, 70% (215/309) acted through cis-regulatory elements, with the remaining through promoters (22%, 70/309) or non-coding RNAs (8%, 24/309). Several validation approaches were utilized in these studies, including gene expression (n = 272), transcription factor binding (n = 175), reporter assays (n = 171), in vivo models (n = 104), genome editing (n = 96) and chromatin interaction (n = 33). CONCLUSIONS: This review of the literature is the first to systematically evaluate the status and the landscape of experimentation being used to validate non-coding GWAS-identified variants. Our results clearly underscore the multifaceted approach needed for experimental validation, have practical implications on variant prioritization and considerations of target gene nomination. While the field has a long way to go to validate the thousands of GWAS associations, we show that progress is being made and provide exemplars of validation studies covering a wide variety of mechanisms, target genes, and disease areas.
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Estudio de Asociación del Genoma Completo , Secuencias Reguladoras de Ácidos Nucleicos , Humanos , Fenotipo , Regiones Promotoras GenéticasRESUMEN
Inhibitors of poly(ADP)-ribose polymerase (PARP) exploit defective DNA repair pathways existing in several forms of cancer, such as those with BRCA mutations, and have proven clinical efficacy as chemosensitizers. However, platinum-based chemopotentiation by PARP inhibitors (PARPi), particularly for non-small cell lung cancer (NSCLC), has only been confirmed in a few preclinical models and the molecular mechanisms that drive PARPi combinatorial synergy with chemotherapeutics remains poorly defined. To better understand these mechanisms, we characterized cisplatin and veliparib efficacy in A549 and Calu6 NSCLC in vivo tumor xenograft models and observed combinatorial synergy in the Calu6 model. Transcriptome-wide analysis of xenografts revealed several differentially expressed genes (DEGs) between untreated and cisplatin + veliparib-treated groups, which were unique from genes identified in either of the single-agent treatment arms. Particularly at 10- and 21-days post-treatment, these DEGs were enriched within pathways involved in DNA damage repair, cell cycle regulation, and senescence. Furthermore, TGF-ß- and integrin-related pathways were enriched in the combination treatment arm, while pathways involved in cholesterol metabolism were identified at earlier time points in both the combination and cisplatin-only groups. These data advance the biological underpinnings of PARPi combined with platinum-based chemotherapy and provides additional insight into the diverse sensitivity of NSCLC models.
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Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Adenosina Difosfato , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Colesterol , Cisplatino , Humanos , Integrinas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Platino (Metal)/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Poli(ADP-Ribosa) Polimerasas/metabolismo , Ribosa/uso terapéutico , Transcriptoma , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Tissue fibrosis, the development of fibrous connective tissue as a result of injury or damage, is associated with many common diseases and cannot be treated effectively. The complex biological processes accompanying fibrosis often involve aberrant signaling through the transforming growth factor beta (TGF-ß) pathway. In the search for mechanisms to repress this signaling, microRNAs have emerged as a novel class of molecules capable of targeting single members of the TGF-ß pathway, or the pathway as a whole. We previously identified miR-1343 as a potent repressor of TGF-ß signaling and fibrosis through the direct attenuation of both canonical TGF-ß receptors. Here, we build upon our previous findings to better characterize the function of endogenous miR-1343 in normal biology and examine the potential role of exogenous miR-1343 as a repressor of TGF-ß signaling. CRISPR/Cas9-mediated deletion of miR-1343 from A549 lung epithelial cells impacts several processes and genes implicated in fibrosis and known to be TGF-ß pathway effectors. Moreover, the responses are opposite to those we observed previously when miR-1343 was overexpressed in the same cell type. We also show that miR-1343 can be shuttled into exosomes, a type of extracellular vesicle that are exported by cells into the surrounding medium and can be absorbed by distant target cells. miR-1343 delivered into primary lung fibroblasts by exosomes has a measurable function in reducing TGF-ß signaling and markers of fibrosis. These results highlight a role for miR-1343 in fine-tuning the TGF-ß pathway and suggest its use as a therapeutic in fibrotic disease.
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Biomarcadores/metabolismo , Micropartículas Derivadas de Células/metabolismo , MicroARNs/administración & dosificación , Células A549 , Exosomas/metabolismo , Fibrosis , Regulación de la Expresión Génica , Células HL-60 , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Chronic respiratory diseases encompass a group of diverse conditions affecting the airways, which all impair lung function over time. They include cystic fibrosis (CF), idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and asthma, which together affect hundreds of millions of people worldwide. MicroRNAs (miRNAs), a class of small non-coding RNAs involved in post-transcriptional gene repression, are now recognized as major regulators in the development and progression of chronic lung disease. Alterations in miRNA abundance occur in lung tissue, inflammatory cells, and freely circulating in blood and are thought to function both as drivers and modifiers of disease. Their importance in lung pathology has prompted the development of miRNA-based therapies and biomarker tools. Here, we review the current literature on miRNA expression and function in chronic respiratory disease and highlight further research that is needed to propel miRNA treatments for lung disorders towards the clinic.
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Asma/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , MicroARNs/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Animales , Asma/tratamiento farmacológico , Biomarcadores/metabolismo , Humanos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , MicroARNs/genética , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológicoRESUMEN
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), but are not good predictors of lung phenotype. Genome-wide association studies (GWAS) previously identified additional genomic sites associated with CF lung disease severity. One of these, at chromosome 11p13, is an intergenic region between Ets homologous factor (EHF) and Apaf-1 interacting protein (APIP). Our goal was to determine the functional significance of this region, which being intergenic is probably regulatory. To identify cis-acting elements, we used DNase-seq and H3K4me1 and H3K27Ac ChIP-seq to map open and active chromatin respectively, in lung epithelial cells. Two elements showed strong enhancer activity for the promoters of EHF and the 5' adjacent gene E47 like ETS transcription factor 5 (ELF5) in reporter gene assays. No enhancers of the APIP promoter were found. Circular chromosome conformation capture (4C-seq) identified direct physical interactions of elements within 11p13. This confirmed the enhancer-promoter associations, identified additional interacting elements and defined topologically associating domain (TAD) boundaries, enriched for CCCTC-binding factor (CTCF). No strong interactions were observed with the APIP promoter, which lies outside the main TAD encompassing the GWAS signal. These results focus attention on the role of EHF in modifying CF lung disease severity.
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Cromosomas Humanos Par 11/genética , Fibrosis Quística/genética , Fibrosis Quística/patología , Regulación de la Expresión Génica , Factores de Transcripción/fisiología , Células CACO-2 , Células Cultivadas , Cromatina/metabolismo , Elementos de Facilitación Genéticos , Sitios Genéticos , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Células K562 , Polimorfismo de Nucleótido Simple , Índice de Severidad de la Enfermedad , Factores de Transcripción/genéticaRESUMEN
Irreversible respiratory obstruction resulting from progressive airway damage, inflammation and fibrosis is a feature of several chronic respiratory diseases, including cystic fibrosis (CF), idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). The cytokine transforming growth factor ß (TGF-ß) has a pivotal role in promoting lung fibrosis and is implicated in respiratory disease severity. In the present study, we show that a previously uncharacterized miRNA, miR-1343, reduces the expression of both TGF-ß receptor 1 and 2 by directly targeting their 3'-UTRs. After TGF-ß exposure, elevated intracellular miR-1343 significantly decreases levels of activated TGF-ß effector molecules, pSMAD2 (phosphorylated SMAD2) and pSMAD3 (phosphorylated SMAD3), when compared with a non-targeting control miRNA. As a result, the abundance of fibrotic markers is reduced, cell migration into a scratch wound impaired and epithelial-to-mesenchymal transition (EMT) repressed. Mature miR-1343 is readily detected in human neutrophils and HL-60 cells and is activated in response to stress in A549 lung epithelial cells. miR-1343 may have direct therapeutic applications in fibrotic lung disease.
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MicroARNs/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Fibrosis Pulmonar/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Regiones no Traducidas 3' , Transición Epitelial-Mesenquimal , Humanos , Pulmón/metabolismo , MicroARNs/genética , Proteínas Serina-Treonina Quinasas/genética , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/fisiopatología , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Transducción de Señal , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína smad3/genética , Proteína smad3/metabolismoRESUMEN
The Myc family of transcription factors regulates a variety of biological processes, including the cell cycle, growth, proliferation, metabolism, and apoptosis. In Caenorhabditis elegans, the "Myc interaction network" consists of two opposing heterodimeric complexes with antagonistic functions in transcriptional control: the Myc-Mondo:Mlx transcriptional activation complex and the Mad:Max transcriptional repression complex. In C. elegans, Mondo, Mlx, Mad, and Max are encoded by mml-1, mxl-2, mdl-1, and mxl-1, respectively. Here we show a similar antagonistic role for the C. elegans Myc-Mondo and Mad complexes in longevity control. Loss of mml-1 or mxl-2 shortens C. elegans lifespan. In contrast, loss of mdl-1 or mxl-1 increases longevity, dependent upon MML-1:MXL-2. The MML-1:MXL-2 and MDL-1:MXL-1 complexes function in both the insulin signaling and dietary restriction pathways. Furthermore, decreased insulin-like/IGF-1 signaling (ILS) or conditions of dietary restriction increase the accumulation of MML-1, consistent with the notion that the Myc family members function as sensors of metabolic status. Additionally, we find that Myc family members are regulated by distinct mechanisms, which would allow for integrated control of gene expression from diverse signals of metabolic status. We compared putative target genes based on ChIP-sequencing data in the modENCODE project and found significant overlap in genomic DNA binding between the major effectors of ILS (DAF-16/FoxO), DR (PHA-4/FoxA), and Myc family (MDL-1/Mad/Mxd) at common target genes, which suggests that diverse signals of metabolic status converge on overlapping transcriptional programs that influence aging. Consistent with this, there is over-enrichment at these common targets for genes that function in lifespan, stress response, and carbohydrate metabolism. Additionally, we find that Myc family members are also involved in stress response and the maintenance of protein homeostasis. Collectively, these findings indicate that Myc family members integrate diverse signals of metabolic status, to coordinate overlapping metabolic and cytoprotective transcriptional programs that determine the progression of aging.
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Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Unión al ADN/genética , Longevidad/genética , Transactivadores/genética , Animales , Regulación de la Expresión Génica/genética , Factor I del Crecimiento Similar a la Insulina/genética , Transducción de Señal/genética , Activación Transcripcional/genéticaRESUMEN
Myosin 1e (myo1e) is an actin-dependent molecular motor that plays an important role in kidney functions. Complete knockout of myo1e in mice and Myo1E mutations in humans are associated with nephrotic syndrome and focal segmental glomerulosclerosis. In this paper, we tested the hypothesis that myo1e is necessary for normal functions of glomerular visceral epithelial cells (podocytes) using podocyte-targeted knockout of myo1e. Myo1e was selectively knocked out in podocytes using Cre-mediated recombination controlled by the podocin promoter. Myo1e loss from podocytes resulted in proteinuria, podocyte foot process effacement, and glomerular basement membrane disorganization. Our findings indicate that myo1e expression in podocytes is necessary for normal glomerular filtration and that podocyte defects are likely to represent the primary pathway leading to glomerular disease associated with Myo1E mutations.