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The Cystic Fibrosis Conductance Transmembrane Regulator gene encodes for the CFTR ion channel, which is responsible for the transport of chloride and bicarbonate across the plasma membrane. Mutations in the gene result in impaired ion transport, subsequently leading to perturbed secretion in all exocrine glands and, therefore, the multi-organ disease cystic fibrosis (CF). In recent years, several studies have reported on CFTR expression in immune cells as demonstrated by immunofluorescence, flow cytometry, and immunoblotting. However, these data are mainly restricted to single-cell populations and show significant variation depending on the methodology used. Here, we investigated CFTR transcription and protein expression using standardized protocols in a comprehensive panel of immune cells. Methods: We applied a high-resolution Western blot protocol using a combination of highly specific monoclonal CFTR antibodies that have been optimized for the detection of CFTR in epithelial cells and healthy primary immune cell subpopulations sorted by flow cytometry and used immortalized cell lines as controls. The specificity of CFTR protein detection was controlled by peptide competition and enzymatic Peptide-N-Glycosidase-F (PNGase) digest. CFTR transcripts were analyzed using quantitative real-time PCR and normalized to the level of epithelial T84 cells as a reference. Results: CFTR mRNA expression could be shown for primary CD4+ T cells, NK cells, as well as differentiated THP-1 and Jurkat T cells. In contrast, we failed to detect CFTR transcripts for CD14+ monocytes and undifferentiated THP-1 cells, as well as for B cells and CD8+ T cells. Prominent immunoreactive bands were detectable by immunoblotting with the combination of four CFTR antibodies targeting different epitopes of the CFTR protein. However, in biosamples of non-epithelial origin, these CFTR-like protein bands could be unmasked as false positives through peptide competition or PNGase digest, meaning that the observed mRNA transcripts were not necessarily translated into CFTR proteins, which could be detected via immunoblotting. Our results confirm that mRNA expression in immune cells is many times lower than in that cells of epithelial origin. The immunoreactive signals in immune cells turned out to be false positives, and may be provoked by the presence of a high-affinity protein with a similar epitope. Non-specific binding (e.g., Fab-interaction with glycosyl branches) might also contribute to false positive signals. Our findings highlight the necessity of accurate controls, such as CFTR-negative cells, as well as peptide competition and glycolytic digest in order to identify genuine CFTR protein by immunoblotting. Our data suggest, furthermore, that CFTR protein expression data from techniques such as histology, for which the absence of a molecular weight or other independent control prevents the unmasking of false positive immunoreactive signals, must be interpreted carefully as well.
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Regulador de Condutância Transmembrana em Fibrose Cística , Leucócitos Mononucleares , RNA Mensageiro , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Leucócitos Mononucleares/metabolismo , Western Blotting , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fibrose Cística/metabolismo , Fibrose Cística/genética , Células Matadoras Naturais/metabolismo , Citometria de Fluxo/métodos , Linfócitos T CD4-Positivos/metabolismoRESUMO
BACKGROUND: In recent years, increasingly complex ALI protocols involving specialized, albeit laboratory-specific media have been established, while at the same time, many studies compile the data of only a few ALI donors in spite of site-, protocol- and donor-specific differentiation. METHODS: We describe a simple morphology scoring protocol using histology material derived from epithelia grown on ALI inserts in parallel to other, more complex readouts. RESULTS: Among more than 100 ALI inserts derived from different donors, significant differences in layer score (p = 0.001) and goblet cell score (p = 0.002) were observed when ALI epithelia derived from explanted lung material were compared to trachea-derived ALI cultures. Cortisol withdrawal for the final 2 days of ALI cultures influenced goblet cell density (p = 0.001). CONCLUSIONS: While the histology score provides less resolution than FACS- or OMICs- based single cell analyses, the use of a subportion of the ALI epithelia grown on inserts makes it feasible to combine morphology assessment and other readouts of the same insert. This allows us to control for basic ALI morphology in research and personalized medicine settings in order to assess and, if desired, control for the impact of ALI culture protocols, site- and donor-specific influences on outcome of studies of ALI-derived epithelia.
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Background: Defects in expression, maturation or function of the epithelial membrane glycoprotein CFTR are causative for the progressive disease cystic fibrosis. Recently, molecular therapeutics that improve CFTR maturation and functional defects have been approved. We aimed to verify whether we could detect an improvement of CFTR protein expression and maturation by triple therapy with elexacaftor-tezacaftor-ivacaftor (ELX/TEZ/IVA). Methods: Rectal suction biopsies of 21 p.Phe508del homozygous or compound heterozygous CF patients obtained pre- and during treatment with ELX/TEZ/IVA were analyzed by CFTR Western blot that was optimized to distinguish CFTR glycoisoforms. Findings: CFTR western immunoblot analysis revealed that-compared to baseline-the levels of CFTR protein increased by at least twofold in eight out of 12 patients upon treatment with ELX/TEZ/IVA compared to baseline (p < 0.02). However, polydispersity of the mutant CFTR protein was lower than that of the fully glycosylated wild type CFTR Golgi isoform, indicating an incompletely glycosylated p.Phe508el CFTR protein isoform C* in patients with CF which persists after ELX/TEZ/IVA treatment. Interpretation: Treatment with ELX/TEZ/IVA increased protein expression by facilitating the posttranslational processing of mutant CFTR but apparently did not succeed in generating the polydisperse spectrum of N-linked oligosaccharides that is characteristic for the wild type CFTR band C glycoisoform. Our results caution that the lower amounts or immature glycosylation of the C* glycoisoform observed in patients' biomaterial might not translate to fully restored function of mutant CFTR necessary for long-term provision of clinical benefit.
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INTRODUCTION AND AIM: The signal transducer and activator of transcription 3 (STAT3) has been identified as one of the cystic fibrosis (CF) modifying genes. In this study, we aimed to assess the association between STAT3 genotype and CF patient survival over several decades and to investigate the effect of STAT3 inhibition on epithelial CFTR expression. METHODS: We analyzed the informative genetic marker STAT3Sat for its association with survival in 174 p.Phe508del-CFTR homozygous CF patients treated at the CF center in Hannover spanning birth cohorts from >3 decades (1959-1994). Furthermore, we treated two epithelial cell lines with STAT3 inhibitors and monitored changes of CFTR protein expression by western blot. RESULTS: Only for p.Phe508del-CFTR homozygous patients born prior to 1975, survival was significantly influenced by STAT3sat genotype (P = 0.023). The expression levels of STAT3 and CFTR positively correlated in epithelial cell lines (P = 0.01). CONCLUSIONS: Our results in different birth cohorts identified a time-dependent impact of STAT3 genotype on CF patients' survival and found that improved symptomatic treatment of later-born CF patients obviates STAT3's modifying influence. Consistent with our previous results, STAT3-specific inhibition resulted in increased CFTR expression in the epithelial cell line 16HBE14o-. Thus, care should be taken when CF-modifying genes are studied in cross-sectional cohorts as the impact of modifying genes might not be invariant in the light of changing therapeutic regimens.
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Fibrose Cística , Humanos , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Estudos Transversais , GenótipoRESUMO
Cystic fibrosis is a genetic disease caused by mutation of the CFTR gene, which encodes a chloride and bicarbonate transporter in epithelial cells. Due to the vast range of geno- and phenotypes, it is difficult to find causative treatments; however, small-molecule therapeutics have been clinically approved in the last decade. Still, the search for novel therapeutics is ongoing, and thousands of compounds are being tested in different assays, often leaving their mechanism of action unknown. Here, we bring together a CFTR-specific compound database (CandActCFTR) and systems biology model (CFTR Lifecycle Map) to identify the targets of the most promising compounds. We use a dual inverse screening approach, where we employ target- and ligand-based methods to suggest targets of 309 active compounds in the database amongst 90 protein targets from the systems biology model. Overall, we identified 1038 potential target-compound pairings and were able to suggest targets for all 309 active compounds in the database.
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Fibrose Cística , Humanos , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Cloretos/metabolismo , Ligantes , Bicarbonatos/metabolismo , Mutação , Preparações FarmacêuticasRESUMO
An adequate visualization form is required to gain an overview and ultimately understand the complex and diverse biological mechanisms of diseases. Recently, disease maps have been introduced for this purpose. A disease map is defined as a systems biological map or model that combines metabolic, signaling, and physiological pathways to create a comprehensive overview of known disease mechanisms. With the increase in publications describing biological interactions, efforts in creating and curating comprehensive disease maps is growing accordingly. Therefore, new computational approaches are needed to reduce the time that manual curation takes. Test mining algorithms can be used to analyse the natural language of scientific publications. These types of algorithms can take humanly readable text passages and convert them into a more ordered, machine-usable data structure. To support the creation of disease maps by text mining, we developed an interactive, user-friendly disease map viewer. The disease map viewer displays text mining results in a systems biology map, where the user can review them and either validate or reject identified interactions. Ultimately, the viewer brings together the time-saving advantages of text mining with the accuracy of manual data curation.
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Algoritmos , Mineração de Dados , Mineração de Dados/métodos , PublicaçõesRESUMO
The Transmembrane member 16A (TMEM16A), also known as anoctamin-1 (ANO1), is a calcium-activated chloride channel present in the airway epithelium. It is known to be involved in the apical chloride secretion indicating that TMEM16A could be addressed for the treatment of chloride secretion defects like in Cystic- Fibrosis (CF). In this paper we generated knockout cell lines using CRISPR/Cas9-mediated ablation in a healthy human iPSC line (MHHi001-A), in a CF patient iPSC line (MHHi002-A) and in its corrected counterpart (MHHi002-A-1). These lines can be used for gaining information about the role of TMEM16A for mucus secretion and/or production and evaluating its therapeutic potential.
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Fibrose Cística , Células-Tronco Pluripotentes Induzidas , Humanos , Anoctamina-1/genética , Anoctamina-1/metabolismo , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Cloretos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Mutação , Células Clonais/metabolismoRESUMO
Cystic fibrosis (CF) is a monogenic disease, characterized by massive chronic lung inflammation. The observed variability in clinical phenotypes in monozygotic CF twins is likely associated with the extent of inflammation. This study sought to investigate inflammation-related aberrant DNA methylation in CF twins and to determine to what extent acquired methylation changes may be associated with lung cancer.Blood-based genome-wide DNA methylation analysis was performed to compare the DNA methylomes of monozygotic twins, from the European CF Twin and Sibling Study with various degrees of disease severity. Putatively inflammation-related and differentially methylated positions were selected from a large lung cancer case-control study and investigated in blood by targeted bisulphite next-generation-sequencing. An inflammation-related locus located in the Plakophilin-3 (PKP3) gene was functionally analysed regarding promoter and enhancer activity in presence and absence of methylation using luciferase reporter assays.We confirmed in a unique cohort that monozygotic twins, even if clinically discordant, have only minor differences in global DNA methylation patterns and blood cell composition. Further, we determined the most differentially methylated positions, a high proportion of which are blood cell-type-specific, whereas others may be acquired and thus have potential relevance in the context of inflammation as lung cancer risk factors. We identified a sequence in the gene body of PKP3 which is hypermethylated in blood from CF twins with severe phenotype and highly variably methylated in lung cancer patients and controls, independent of known clinical parameters, and showed that this region exhibits methylation-dependent promoter activity in lung epithelial cells.
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Fibrose Cística , Neoplasias Pulmonares , Estudos de Casos e Controles , Fibrose Cística/genética , Metilação de DNA , Epigênese Genética , Células Epiteliais , Humanos , Inflamação/genética , Pulmão , Neoplasias Pulmonares/genética , Placofilinas/genética , Gêmeos Monozigóticos/genéticaRESUMO
Background: Cystic fibrosis (CF) is a genetic disease caused by mutations in CFTR, which encodes a chloride and bicarbonate transporter expressed in exocrine epithelia throughout the body. Recently, some therapeutics became available that directly target dysfunctional CFTR, yet research for more effective substances is ongoing. The database CandActCFTR aims to provide detailed and comprehensive information on candidate therapeutics for the activation of CFTR-mediated ion conductance aiding systems-biology approaches to identify substances that will synergistically activate CFTR-mediated ion conductance based on published data. Results: Until 10/2020, we derived data from 108 publications on 3,109 CFTR-relevant substances via the literature database PubMed and further 666 substances via ChEMBL; only 19 substances were shared between these sources. One hundred and forty-five molecules do not have a corresponding entry in PubChem or ChemSpider, which indicates that there currently is no single comprehensive database on chemical substances in the public domain. Apart from basic data on all compounds, we have visualized the chemical space derived from their chemical descriptors via a principal component analysis annotated for CFTR-relevant biological categories. Our online query tools enable the search for most similar compounds and provide the relevant annotations in a structured way. The integration of the KNIME software environment in the back-end facilitates a fast and user-friendly maintenance of the provided data sets and a quick extension with new functionalities, e.g., new analysis routines. CandActBase automatically integrates information from other online sources, such as synonyms from PubChem and provides links to other resources like ChEMBL or the source publications. Conclusion: CandActCFTR aims to establish a database model of candidate cystic fibrosis therapeutics for the activation of CFTR-mediated ion conductance to merge data from publicly available sources. Using CandActBase, our strategy to represent data from several internet resources in a merged and organized form can also be applied to other use cases. For substances tested as CFTR activating compounds, the search function allows users to check if a specific compound or a closely related substance was already tested in the CF field. The acquired information on tested substances will assist in the identification of the most promising candidates for future therapeutics.
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CFTR encodes for a chloride and bicarbonate channel expressed at the apical membrane of polarized epithelial cells. Transepithelial sodium transport mediated by the amiloride-sensitive sodium channel ENaC is thought to contribute to the manifestation of CF disease. Thus, ENaC is a therapeutic target in CF and a valid cystic fibrosis modifier gene. We have characterized SCNN1B as a genetic modifier in the three independent patient cohorts of F508del-CFTR homozygotes. We could identify a regulatory element at SCNN1B to the genomic segment rs168748-rs2303153-rs4968000 by fine-mapping (Pbest = 0.0177), consistently observing the risk allele rs2303153-C and the contrasting benign allele rs2303153-G in all three patient cohorts. Furthermore, our results show that expression levels of SCNN1B are associated with rs2303153 genotype in intestinal epithelia (p = 0.003). Our data confirm that the well-established biological role of SCNN1B can be recognized by an association study on informative endophenotypes in the rare disease cystic fibrosis and calls attention to reproducible results in association studies obtained from small, albeit carefully characterized patient populations.
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Fibrose Cística/genética , Canais Epiteliais de Sódio/genética , Genes Modificadores , Polimorfismo de Nucleotídeo Único , Alelos , Homozigoto , HumanosRESUMO
Different causative therapeutics for CF patients have been developed. There are still no mutation-specific therapeutics for some patients, especially those with rare CFTR mutations. For this purpose, high-throughput screens have been performed which result in various candidate compounds, with mostly unclear modes of action. In order to elucidate the mechanism of action for promising candidate substances and to be able to predict possible synergistic effects of substance combinations, we used a systems biology approach to create a model of the CFTR maturation pathway in cells in a standardized, human- and machine-readable format. It is composed of a core map, manually curated from small-scale experiments in human cells, and a coarse map including interactors identified in large-scale efforts. The manually curated core map includes 170 different molecular entities and 156 reactions from 221 publications. The coarse map encompasses 1384 unique proteins from four publications. The overlap between the two data sources amounts to 46 proteins. The CFTR Lifecycle Map can be used to support the identification of potential targets inside the cell and elucidate the mode of action for candidate substances. It thereby provides a backbone to structure available data as well as a tool to develop hypotheses regarding novel therapeutics.
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Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Combinação de Medicamentos , Humanos , Mutação , Mapas de Interação de Proteínas , Análise de SistemasRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) gene is influenced by the fundamental cellular processes like epithelial differentiation/polarization, regeneration and epithelial-mesenchymal transition. Defects in CFTR protein levels and/or function lead to decreased airway surface liquid layer facilitating microbial colonization and inflammation. The SERPINA1 gene, encoding alpha1-antitrypsin (AAT) protein, is one of the genes implicated in CF, however it remains unknown whether AAT has any influence on CFTR levels. In this study we assessed CFTR protein levels in primary human lung epithelial cells grown at the air-liquid-interface (ALI) alone or pre-incubated with AAT by Western blots and immunohistochemistry. Histological analysis of ALI inserts revealed CFTR- and AAT-positive cells but no AAT-CFTR co-localization. When 0.5 mg/mL of AAT was added to apical or basolateral compartments of pro-inflammatory activated ALI cultures, CFTR levels increased relative to activated ALIs. This finding suggests that AAT is CFTR-modulating protein, albeit its effects may depend on the concentration and the route of administration. Human lung epithelial ALI cultures provide a useful tool for studies in detail how AAT or other pharmaceuticals affect the levels and activity of CFTR.
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Barreira Alveolocapilar/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Mucosa Respiratória/metabolismo , alfa 1-Antitripsina/metabolismo , Biomarcadores , Linhagem Celular , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/metabolismo , Imunofluorescência , Humanos , Imuno-Histoquímica , alfa 1-Antitripsina/genéticaRESUMO
Sixteen monozygotic cystic fibrosis (CF) twin pairs of whom 14 pairs were homozygous for the most common p.Phe508del CFTR mutation were selected from the European Cystic Fibrosis Twin and Sibling Study Cohort. The monozygotic twins were examined in their T cell receptor (TCR) repertoire in peripheral blood by amplicon sequencing of the CDR3 variable region of the ß-chain. The recruitment of TCR J and V genes for recombination and selection in the thymus showed a strong genetic influence in the CF twin cohort as indicated by the shortest Jensen-Shannon distance to the twin individual. Exceptions were the clinically most discordant and/or most severely affected twin pairs where clonal expansion probably caused by recurrent pulmonary infections overshadowed the impact of the identical genomic blueprint. In general the Simpson clonality was low indicating that the population of TCRß clonotypes of the CF twins was dominated by the naïve T-cell repertoire. Intrapair sharing of clonotypes was significantly more frequent among monozygotic CF twins than among pairs of unrelated CF patients. Complete nucleotide sequence identity was observed in about 0.11% of CDR3 sequences which partially should represent persisting fetal clones derived from the same progenitor T cells. Complete amino acid sequence identity was noted in 0.59% of clonotypes. Of the nearly 40,000 frequent amino acid clonotypes shared by at least two twin siblings 99.8% were already known within the immuneACCESS database and only 73 had yet not been detected indicating that the CDR3ß repertoire of CF children and adolescents does not carry a disease-specific signature but rather shares public clones with that of the non-CF community. Clonotypes shared within twin pairs and between unrelated CF siblings were highly abundant among healthy non-CF people, less represented in individuals with infectious disease and uncommon in patients with cancer. This subset of shared CF clonotypes defines CDR3 amino acid sequences that are more common in health than in disease.
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Regiões Determinantes de Complementaridade , Fibrose Cística , Receptores de Antígenos de Linfócitos T alfa-beta , Gêmeos Monozigóticos/genética , Adolescente , Adulto , Criança , Pré-Escolar , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Fibrose Cística/genética , Fibrose Cística/imunologia , Feminino , Humanos , Masculino , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologiaRESUMO
Blood of the three clinically most concordant and most discordant p.Phe508del homozygous monozygous twin pairs of the European Cystic Fibrosis Twin and Sibling Study was examined in two postzygotic attributes that generate diversity between monozygous twins, i.e. the repertoire of the CDR3 region of the T-cell receptor ß chains and the DNA methylation at 450,000 genomic CpG sites. Methylation patterns in peripheral blood of twins changed at selected cell-type-independent positions and the immune cells of the twins showed individual profiles of the T cell receptor repertoire reflecting the plasticity of the immune system of genetically identical humans with cystic fibrosis to cope with the environment.
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Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Doenças em Gêmeos/genética , Gêmeos Monozigóticos/genética , Adolescente , Feminino , Homozigoto , Humanos , Lactente , Recém-Nascido , MasculinoRESUMO
BACKGROUND: Nasal potential difference (NPD) and intestinal current measurements (ICM) are cystic fibrosis transmembrane conductance regulator (CFTR) biomarkers recommended to make a diagnosis in individuals with inconclusive sweat test and CFTR genetics and a clinical suspicion for cystic fibrosis (CF) or CFTR-related disorder (CFTR-RD). METHODS: NPD and ICM were measured according to standard operating procedures of the European Cystic Fibrosis Society Diagnostic Network Working Group. RESULTS: We assessed 219 individuals by NPD or ICM who had been referred to our laboratory due to clinical symptoms suggestive of CF, but inconclusive sweat test and CFTR genetics (median age: 16.3 years, range 0.4 to 76 years). CF or CFTR-related disorder was diagnosed in 22 of 29 patients (76%) with a CFTR genotype of unknown or variable clinical significance and in 51 of 190 carriers (27%) of one (35/42) or no (16/148) identified CFTR mutation. If two CFTR sequence variants had been identified, the outcome of NPD and ICM was consistent with the classification of the CFTR2 database. Moreover, a suspected false-positive diagnosis of CF was confirmed in seven and withdrawn in eight patients. Of 26 individuals assessed by both NPD and ICM, eleven individuals exhibited discordant tracings of ICM and NPD, with one measurement being in the CF range and the other in the normal range. CONCLUSION: The majority of patients whom we diagnosed with CF or CFTR-RD by extended electrophysiology are carriers of the wild-type CFTR coding sequence on at least one of their CF alleles. The disease-causing genetic lesions should reside in the non-coding region of CFTR or elsewhere in the genome, affecting the regulation of CFTR expression in a tissue-depending fashion which may explain the large within-group variability of CFTR activity in the respiratory and intestinal epithelium seen in this group.
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Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Adolescente , Adulto , Idoso , Biomarcadores , Criança , Pré-Escolar , Fibrose Cística/diagnóstico , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Lactente , Pessoa de Meia-Idade , Valores de Referência , Suor , Adulto JovemRESUMO
SCNN1B encodes the beta subunit of the epithelial sodium channel ENaC. Previously, we reported an association between SNP markers of SCNN1B gene and disease severity in cystic fibrosis-affected sibling pairs. We hypothesized that factors interacting with the SCNN1B genomic sequence are responsible for intrapair discordance. Concordant and discordant pairs differed at six SCNN1B markers (Praw = 0.0075, Pcorr = 0.0397 corrected for multiple testing). To identify the factors binding to these six SCNN1B SNPs, we performed an electrophoretic mobility shift assay and captured the DNA-protein complexes. Based on protein mass spectrometry data, the epithelial splicing regulatory protein ESRP2 was identified when using SCNN1B-derived probes and the ESRP2-SCNN1B interaction was independently confirmed by coimmunoprecipitation assays. We observed an alternative SCNN1B transcript and demonstrated in 16HBE14o- cells that levels of this transcript are decreased upon ESRP2 silencing by siRNA. Furthermore, we confirmed that mildly and severely affected siblings have different ESPR2 genetic backgrounds and that ESRP2 markers are linked to the response of CF patients' nasal epithelium to amiloride, indicating ENaC involvement (Pbest = 0.0131, Pcorr = 0.068 for multiple testing). Our findings demonstrate that sibling pairs clinically discordant for CF can be used to identify meaningful DNA regulatory elements and interacting factors.
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Fibrose Cística/genética , Suscetibilidade a Doenças , Canais Epiteliais de Sódio/genética , Genes Modificadores , Proteínas de Ligação a RNA/metabolismo , Irmãos , Alelos , Processamento Alternativo , Sítios de Ligação , Mapeamento Cromossômico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Técnicas de Silenciamento de Genes , Estudos de Associação Genética , Patrimônio Genético , Genótipo , Haplótipos , Humanos , Íntrons , Masculino , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Proteínas de Ligação a RNA/genética , Análise de Sequência de DNARESUMO
Developing safe and efficient non-viral delivery systems remains a major challenge for in vivo applications of gene therapy, especially in cystic fibrosis. Unlike conventional cationic polymers or lipids, the emerging poloxamine-based copolymers display promising in vivo gene delivery capabilities. However, poloxamines are invalid for in vitro applications and their in vivo transfection efficiency is still low compared with viral vectors. Here, we show that peptides developed by modular design approaches can spontaneously form compact and monodisperse nanoparticles with poloxamines and nucleic acids via self-assembly. Both messenger RNA and plasmid DNA expression mediated by peptide-poloxamine nanoparticles are greatly boosted in vitro and in the lungs of cystic fibrosis mice with negligible toxicity. Peptide-poloxamine nanoparticles containing integrating vectors enable successful in vitro and in vivo long-term restoration of cystic fibrosis transmembrane conductance regulator deficiency with a safe integration profile. Our dataset provides a new framework for designing non-viral gene delivery systems qualified for in vivo genetic modifications.
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Fibrose Cística/genética , Fibrose Cística/terapia , Etilenodiaminas/química , Genoma , Nanopartículas/química , Peptídeos/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Camundongos , Nanopartículas/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TransgenesRESUMO
BACKGROUND: The impact of complex alleles on CFTR processing and function has yet not been investigated in native human tissue. METHODS: Intestinal current measurements (ICM) followed by CFTR immunoblot were performed on rectal biopsies taken from two siblings who are compound heterozygous for the CFTR mutations p.Phe508del and the complex allele p.[Arg74Trp;Val201Met;Asp1270Asn]. RESULTS: Normal and subnormal chloride secretory responses in the ICM were associated with normal and fourfold reduced amounts of the mature glycoform band C CFTR, respectively, consistent with the unequal clinical phenotype of the siblings. CONCLUSION: The combined use of bioassay and protein analysis is particularly meaningful to resolve the CFTR phenotype of "indeterminate" borderline CFTR genotypes on a case-to-case basis.