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BACKGROUND: MYBPC3 , encoding cardiac myosin binding protein-C (cMyBP-C), is the most mutated gene known to cause hypertrophic cardiomyopathy (HCM). However, since little is known about the underlying etiology, additional in vitro studies are crucial to defining the underlying molecular mechanisms. Accordingly, this study aimed to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with a polymorphic variant (D389V) in MYBPC3 by using human-induced pluripotent stem cell (hiPSC)-derived cardiac organoids (hCOs). METHODS: The hiPSC-derived cardiomyocytes (hiPSC-CMs) and hCOs were generated from human subjects to define the molecular, cellular, and functional changes caused by the MYBPC3 D389V variant. This variant is associated with increased fractional shortening and is highly prevalent in South Asian descendants. Recombinant C0-C2, N'-region of cMyBP-C (wildtype and D389V), and myosin S2 proteins were also utilized to perform binding and motility assays in vitro . RESULTS: Confocal and electron microscopic analyses of hCOs generated from noncarriers (NC) and carriers of the MYBPC3 D389V variant revealed the presence of highly organized sarcomeres. Furthermore, functional experiments showed hypercontractility with increased contraction velocity, faster calcium cycling, and faster contractile kinetics in hCOs expressing MYBPC3 D389V than NC hCOs. Interestingly, significantly increased cMyBP-C phosphorylation in MYBPC3 D389V hCOs was observed, but without changes in total protein levels, in addition to higher oxidative stress and lower mitochondrial membrane potential (ΔΨm). Next, spatial mapping revealed the presence of endothelial cells, fibroblasts, macrophages, immune cells, and cardiomyocytes in the hCOs. The hypercontractile function was significantly improved after treatment with the myosin inhibitor mavacamten (CAMZYOS®) in MYBPC3 D389V hCOs. Lastly, various in vitro binding assays revealed a significant loss of affinity in the presence of MYBPC3 D389V with myosin S2 region as a likely mechanism for hypercontraction. CONCLUSIONS: Conceptually, we showed the feasibility of assessing the functional and molecular mechanisms of HCM using highly translatable hCOs through pragmatic experiments that led to determining the MYBPC3 D389V hypercontractile phenotype, which was rescued by administration of a myosin inhibitor. Novelty and Significance: What Is Known?: MYBPC3 mutations have been implicated in hypertrophic cardiomyopathy. D389V is a polymorphic variant of MYBPC3 predicted to be present in 53000 US South Asians owing to the founder effect. D389V carriers have shown evidence of hyperdynamic heart, and human-induced pluripotent stem cells (hiPSC)-derived cardiomyocytes with D389V show cellular hypertrophy and irregular calcium transients. The molecular mechanism by which the D389V variant develops pathological cardiac dysfunction remains to be conclusively determined.What New Information Does This Article Contribute ?: The authors leveraged a highly translational cardiac organoid model to explore the role of altered cardiac calcium handling and cardiac contractility as a common pathway leading to pathophysiological phenotypes in patients with early HCM. The MYBPC3 D389V -mediated pathological pathway is first studied here by comparing functional properties using three-dimensional cardiac organoids differentiated from hiPSC and determining the presence of hypercontraction. Our data demonstrate that faster sarcomere kinetics resulting from lower binding affinity between D389V-mutated cMyBP-C protein and myosin S2, as evidenced by in vitro studies, could cause hypercontractility which was rescued by administration of mavacamten (CAMZYOS®), a myosin inhibitor. In addition, hypercontractility causes secondary mitochondrial defects such as higher oxidative stress and lower mitochondrial membrane potential (ΔΨm), highlighting a possible early adaptive response to primary sarcomeric changes. Early treatment of MYBPC3 D389V carriers with mavacamten may prevent or reduce early HCM-related pathology. GRAPHICAL ABSTRACT: A graphical abstract is available for this article.
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KIF3A, the gene encoding kinesin family member 3A, is a susceptibility gene locus associated with asthma; however, mechanisms by which KIF3A might influence the pathogenesis of the disorder are unknown. In this study, we deleted the mouse Kif3a gene in airway epithelial cells. Both homozygous and heterozygous Kif3a gene-deleted mice were highly susceptible to aeroallergens from Aspergillus fumigatus and the house dust mite, resulting in an asthma-like pathology characterized by increased goblet cell metaplasia, airway hyperresponsiveness, and Th2-mediated inflammation. Deletion of the Kif3a gene increased the severity of pulmonary eosinophilic inflammation and expression of cytokines (Il-4, Il-13, and Il-17a) and chemokine (Ccl11) RNAs following pulmonary exposure to Aspergillus extract. Inhibition of Kif3a disrupted the structure of motile cilia and impaired mucociliary clearance, barrier function, and epithelial repair, demonstrating additional mechanisms by which deficiency of KIF3A in respiratory epithelial cells contributes to pulmonary pathology. Airway epithelial KIF3A suppresses Th2 pulmonary inflammation and airway hyperresponsiveness following aeroallergen exposure, implicating epithelial microtubular functions in the pathogenesis of Th2-mediated lung pathology.
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Alérgenos/inmunología , Aspergillus fumigatus/inmunología , Asma/inmunología , Células Epiteliales/inmunología , Cinesinas/inmunología , Mucosa Respiratoria/inmunología , Células Th2/inmunología , Animales , Asma/inducido químicamente , Asma/genética , Asma/patología , Citocinas/genética , Citocinas/inmunología , Células Epiteliales/patología , Cinesinas/genética , Pulmón/inmunología , Pulmón/patología , Ratones , Ratones Transgénicos , Mucosa Respiratoria/patología , Células Th2/patologíaRESUMEN
BACKGROUND: There is considerable heterogeneity in asthma treatment response. OBJECTIVE: We sought to identify biomarkers of corticosteroid treatment response in children with asthma and evaluate the utility and mechanistic basis of these biomarkers. METHODS: Children (5-18 years) presenting to the emergency department with an acute asthma exacerbation were recruited and followed during hospitalization. Nasal epithelial cells were collected on presentation to the emergency department (T0) and 18 to 24 hours later (T1), and T1/T0 gene expression ratios were analyzed to identify genes associated with good and poor corticosteroid treatment response phenotypes. The utility of these genes in discriminating between systemic corticosteroid treatment response groups was then tested prospectively in a new cohort of patients. A gene candidate (vanin-1 [VNN1]) that consistently distinguished good versus poor response phenotypes was further studied in an experimental asthma model, and VNN1 promoter methylation was measured by means of bisulfite pyrosequencing in patients. RESULTS: VNN1 mRNA expression changes were associated with systemic corticosteroid treatment response in children with acute asthma, and VNN1 was required for optimal response to corticosteroid treatment in an experimental asthma model. A CpG site within the VNN1 promoter was differentially methylated between good versus poor treatment response groups, and methylation at this site correlated with VNN1 mRNA expression. CONCLUSIONS: We have identified a biological basis for poor corticosteroid treatment response that can be used to distinguish a subgroup of asthmatic children who respond poorly to systemic corticosteroid treatment. VNN1 contributes to corticosteroid responsiveness, and changes in VNN1 nasal epithelial mRNA expression and VNN1 promoter methylation might be clinically useful biomarkers of treatment response in asthmatic children.
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Corticoesteroides/uso terapéutico , Amidohidrolasas/metabolismo , Asma/tratamiento farmacológico , Biomarcadores Farmacológicos/metabolismo , Mucosa Nasal/fisiología , Adolescente , Amidohidrolasas/genética , Animales , Asma/diagnóstico , Células Cultivadas , Niño , Preescolar , Estudios de Cohortes , Metilación de ADN , Progresión de la Enfermedad , Servicios Médicos de Urgencia , Femenino , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Regulación de la Expresión Génica , Hospitalización , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Modelos Animales , Estudios Prospectivos , Resultado del TratamientoRESUMEN
BACKGROUND: Induced pluripotent stem cells (iPSCs) hold tremendous potential, both as a biological tool to uncover the pathophysiology of disease by creating relevant human cell models and as a source of cells for cell-based therapeutic applications. Studying the reprogramming process will also provide significant insight into tissue development. OBJECTIVE: We sought to characterize the derivation of iPSC lines from nasal epithelial cells (NECs) isolated from nasal mucosa samples of children, a highly relevant and easily accessible tissue for pediatric populations. METHODS: We performed detailed comparative analysis on the transcriptomes and methylomes of NECs, iPSCs derived from NECs (NEC-iPSCs), and embryonic stem cells (ESCs). RESULTS: NEC-iPSCs express pluripotent cell markers, can differentiate into all 3 germ layers in vivo and in vitro, and have a transcriptome and methylome remarkably similar to those of ESCs. However, residual DNA methylation marks exist, which are differentially methylated between NEC-iPSCs and ESCs. A subset of these methylation markers related to epithelium development and asthma and specific to NEC-iPSCs persisted after several passages in vitro, suggesting the retention of an epigenetic memory of their tissue of origin. Our analysis also identified novel candidate genes with dynamic gene expression and DNA methylation changes during reprogramming, which are indicative of possible roles in airway epithelium development. CONCLUSION: NECs are an excellent tissue source to generate iPSCs in pediatric asthmatic patients, and detailed characterization of the resulting iPSC lines would help us better understand the reprogramming process and retention of epigenetic memory.
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Asma/genética , Células Madre Embrionarias/metabolismo , Células Epiteliales , Células Madre Pluripotentes Inducidas , Mucosa Nasal/citología , Adolescente , Animales , Línea Celular , Células Cultivadas , Metilación de ADN , Epigenómica , Células Epiteliales/citología , Células Epiteliales/metabolismo , Fibroblastos , Prepucio/citología , Perfilación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , RatonesRESUMEN
Serine proteases are critical for epidermal barrier homeostasis, and their aberrant expression and/or activity is associated with chronic skin diseases. Elevated levels of the serine protease inhibitors SERPINB3 and SERPINB4 are seen in patients with atopic dermatitis and psoriasis. However, their mechanistic role in the skin is unknown. To evaluate the contribution of Serpinb3a (mouse homolog of SERPINB3 and SERPINB4) in atopic dermatitis, we examined the effect of topical Aspergillus fumigatus extract exposure in wild-type and Serpinb3a-null mice on transepidermal water loss (TEWL), sensitization, and inflammation. Allergen exposure induced Serpinb3a expression in the skin, along with increased TEWL, epidermal thickness, and skin inflammation, all of which were attenuated in the absence of Serpinb3a. Attenuated TEWL correlated with decreased expression of the pro-inflammatory marker S100A8. Silencing of SERPINB3/B4 in human keratinocytes decreased S100A8 expression, supporting a role for SERPINB3/B4 in the initiation of the acute inflammatory response. RNA-seq analysis following allergen exposure identified a network of pro-inflammatory genes induced in wild-type mice that was absent in Serpinb3a-null mice. In conclusion, Serpinb3a deficiency attenuates barrier dysfunction and the early inflammatory response following cutaneous allergen exposure, supporting a role for Serpinb3a (mice) and SERPINB3/B4 (humans) early in atopic dermatitis.
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Antígenos de Neoplasias/inmunología , Dermatitis Atópica/inmunología , Serpinas/inmunología , Enfermedad Aguda , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Aspergillus fumigatus/inmunología , Calgranulina A/genética , Calgranulina A/inmunología , Calgranulina A/metabolismo , Enfermedad Crónica , Dermatitis Atópica/genética , Dermatitis Atópica/metabolismo , Modelos Animales de Enfermedad , Epidermis/inmunología , Epidermis/metabolismo , Expresión Génica/inmunología , Humanos , Queratinocitos/inmunología , Queratinocitos/metabolismo , Ratones Endogámicos BALB C , Ratones Noqueados , Serpinas/genética , Serpinas/metabolismo , Pérdida Insensible de Agua/inmunologíaRESUMEN
BACKGROUND: Epithelial genes have previously been associated with asthma but only explain a small fraction of heritability. In part, this might be due to epistasis, which is often not considered. OBJECTIVE: We sought to determine independent and epistatic associations between filaggrin (FLG), serine protease inhibitor Kazal-type 5 (SPINK5), and thymic stromal lymphopoietin (TSLP) gene variants and childhood asthma. METHODS: Using a candidate gene approach, we genotyped 29 variants in FLG, SPINK5, and TSLP in asthmatic, allergic, and nonallergic nonasthmatic white and black children participating in the well-phenotyped Greater Cincinnati Pediatric Clinic Repository. Associations with asthma were also assessed in 6 replication populations. RESULTS: We observed independent associations of variants in SPINK5 (P = .003) and TSLP (P = .006) with childhood asthma; a SPINK5 single nucleotide polymorphism was replicated. In subjects with 1 or more SPINK5 risk alleles, the absence of the TSLP protective minor alleles was associated with a significant increase in asthma (67% vs 53%, P = .0017). In contrast, the presence or absence of TSLP minor alleles did not affect asthma risk in subjects without the SPINK5 risk alleles. The SPINK5 and TSLP epistasis was replicated in a black population (P = .036) who did not display independent association with variants in these genes. CONCLUSIONS: Our results support epistasis between SPINK5 and TSLP, which contributes to childhood asthma. These findings emphasize the importance of using biology to inform analyses to identify genetic susceptibility to complex diseases. The results from our study have clinical relevance and support that the therapeutic effects of anti-TSLP therapy in asthmatic patients might be dependent on SPINK5 genotype.
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Asma/genética , Citocinas/genética , Epistasis Genética , Proteínas de Filamentos Intermediarios/genética , Proteínas Inhibidoras de Proteinasas Secretoras/genética , Adolescente , Población Negra , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Proteínas Filagrina , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Polimorfismo Genético , Inhibidor de Serinpeptidasas Tipo Kazal-5 , Población Blanca , Linfopoyetina del Estroma TímicoRESUMEN
BACKGROUND: IL-13 receptor α2 (IL-13Rα2) binds IL-13 with high affinity and modulates IL-13 responses. There are soluble and membrane forms of IL-13Rα2 generated by alternative splicing in mice, but human subjects express only the membrane form of IL-13Rα2 (memIL-13Rα2). OBJECTIVE: We determined the role of memIL-13Rα2 in the development of allergic inflammation in mouse models of asthma. METHODS: IL-13Rα2-deficient and memIL-13Rα2 lung epithelium-specific transgenic mice were challenged with house dust mite (HDM). Airway hyperresponsiveness (AHR) and inflammation were assessed based on the airway pressure-time index, bronchoalveolar lavage (BAL) cell counts, and lung histology. Mucus production was determined by means of periodic acid-Schiff staining of lung sections, Western blot analysis of chloride channel calcium activated 3 (CLCA3) expression in lung homogenates, and ELISA of Muc5ac in BAL fluid. The expression of cytokines and chemokines was determined by using RT-quantitative PCR. RESULTS: In IL-13Rα2-deficient mice AHR and airway inflammation were attenuated compared with levels seen in wild-type mice after HDM challenge. Lung epithelial overexpression of memIL-13Rα2 in the IL-13Rα2-deficient mice reconstituted AHR and inflammation to levels similar to those observed in HDM-challenged wild-type mice. Mucus production was attenuated in lungs from HDM-treated IL-13Rα2-deficient mice, whereas lung epithelial overexpression of memIL-13Rα2 increased mucus production. Lung epithelial overexpression of memIL-13Rα2 had no effect on levels of the soluble form of IL-13Rα2 in serum or BAL fluid and did not affect IL-13-dependent signal transducer and activator of transcription 6 activation in the lungs. CONCLUSION: These data collectively support a distinct role for memIL-13Rα2 in the lung and suggest that memIL-13Rα2 might contribute to allergic inflammation.
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Asma/inmunología , Hipersensibilidad/inmunología , Subunidad alfa2 del Receptor de Interleucina-13/metabolismo , Pulmón/inmunología , Pulmón/patología , Animales , Asma/complicaciones , Asma/metabolismo , Asma/patología , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/metabolismo , Hiperreactividad Bronquial/patología , Líquido del Lavado Bronquioalveolar , Membrana Celular/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Hipersensibilidad/complicaciones , Hipersensibilidad/metabolismo , Hipersensibilidad/patología , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Subunidad alfa2 del Receptor de Interleucina-13/genética , Subunidad alfa2 del Receptor de Interleucina-13/inmunología , Masculino , Ratones , PyroglyphidaeRESUMEN
BACKGROUND: Allergic disorders, including asthma, allergic rhinitis, atopic dermatitis, eosinophilic esophagitis, and food allergy, are a major global health burden. The study and management of allergic disorders is complicated by the considerable heterogeneity in both the presentation and natural history of these disorders. Biorepositories serve as an excellent source of data and biospecimens for delineating subphenotypes of allergic disorders, but such resources are lacking. METHODS: In order to define subphenotypes of allergic disease accurately, we established an infrastructure to link and efficiently utilize clinical and epidemiologic data with biospecimens into a single biorepository called the Greater Cincinnati Pediatric Clinic Repository (GCPCR). Children with allergic disorders as well as healthy controls are followed longitudinally at hospital clinic, emergency department, and inpatient visits. Subjects' asthma, allergy, and skin symptoms; past medical, family, social, diet, and environmental histories; physical activity; medication adherence; perceived quality of life; and demographics are ascertained. DNA is collected from all participants, and other biospecimens such as blood, hair, and nasal epithelial cells are collected on a subset. RESULTS: To date, the GCPCR has 6,317 predominantly Caucasian and African American participants, and 93% have banked DNA. This large sample size supports adequately powered genetic, epidemiologic, environmental, and health disparities studies of childhood allergic diseases. CONCLUSIONS: The GCPCR is a unique biorepository that is continuously evaluated and refined to achieve and maintain rigorous clinical phenotype and biological data. Development of similar disease-specific repositories using common data elements is necessary to enable studies across multiple populations of comprehensively phenotyped patients.
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RATIONALE AND OBJECTIVE: Autophagy is a cellular process directed at eliminating or recycling cellular proteins. Recently, the autophagy pathway has been implicated in immune dysfunction, the pathogenesis of inflammatory disorders, and response to viral infection. Associations between two genes in the autophagy pathway, ATG5 and ATG7, with childhood asthma were investigated. METHODS: Using genetic and experimental approaches, we examined the association of 13 HapMap-derived tagging SNPs in ATG5 and ATG7 with childhood asthma in 312 asthmatic and 246 non-allergic control children. We confirmed our findings by using independent cohorts and imputation analysis. Finally, we evaluated the functional relevance of a disease associated SNP. MEASUREMENTS AND MAIN RESULTS: We demonstrated that ATG5 single nucleotide polymorphisms rs12201458 and rs510432 were associated with asthma (pâ=â0.00085 and 0.0025, respectively). In three independent cohorts, additional variants in ATG5 in the same LD block were associated with asthma (p<0.05). We found that rs510432 was functionally relevant and conferred significantly increased promotor activity. Furthermore, Atg5 expression was increased in nasal epithelium of acute asthmatics compared to stable asthmatics and non-asthmatic controls. CONCLUSION: Genetic variants in ATG5, including a functional promotor variant, are associated with childhood asthma. These results provide novel evidence for a role for ATG5 in childhood asthma.
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Asma/genética , Proteínas Asociadas a Microtúbulos/genética , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Adolescente , Asma/metabolismo , Asma/patología , Proteína 5 Relacionada con la Autofagia , Proteína 7 Relacionada con la Autofagia , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Frecuencia de los Genes , Genes Reporteros , Estudios de Asociación Genética , Células HEK293 , Haplotipos , Humanos , Desequilibrio de Ligamiento , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Mucosa Nasal/metabolismo , Análisis de Secuencia de ADN , Transcripción Genética , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismoRESUMEN
BACKGROUND: Small proline rich protein 2B (SPRR2B) is a skin and lung epithelial protein associated with allergic inflammation in mice that has not been evaluated in human atopic diseases. OBJECTIVE: To determine whether single-nucleotide polymorphisms (SNPs) in SPRR2B are associated with childhood eczema and with the phenotype of childhood eczema combined with asthma. METHODS: Genotyping for SPRR2B and filaggrin (FLG) was performed in 2 independent populations: the Cincinnati Childhood Allergy & Air Pollution Study (CCAAPS; N = 762; birth-age, 4 years) and the Greater Cincinnati Pediatric Clinical Repository (GCPCR; N = 1152; ages 5-10 years). Eczema and eczema plus asthma were clinical outcomes based on parental report and clinician's diagnosis. Genetic analyses were restricted to whites and adjusted for sex in both cohorts and adjusted for environmental covariates in CCAAPS. RESULTS: Variants in SPRR2B were not significantly associated with eczema in either cohort after Bonferroni adjustment. Children from both cohorts with the CC genotype of the SPRR2B rs6693927 SNP were at 4 times the risk for eczema plus asthma (adjusted odds ratio, 4.1; 95% confidence interval, 1.5-10.9; P = .005 in CCAAPS; and adjusted odds ratio, 4.0; 95% confidence interval, 1.8-9.1; P < .001 in the GCPCR), however. SNPs in SPRR2B were not in strong linkage disequilibrium with the R501X and del2282 FLG mutations, and these findings were independent of FLG. CONCLUSIONS: An SNP in SPRR2B was predictive of asthma among white children with eczema from 2 independent populations. SPRR2B polymorphisms may serve as important predictive markers for the combined eczema plus asthma phenotype.
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Asma/genética , Proteínas Ricas en Prolina del Estrato Córneo/genética , Eccema/genética , Polimorfismo de Nucleótido Simple , Asma/complicaciones , Asma/diagnóstico , Biomarcadores , Niño , Preescolar , Estudios de Cohortes , Eccema/complicaciones , Eccema/diagnóstico , Femenino , Proteínas Filagrina , Predisposición Genética a la Enfermedad , Variación Genética , Genotipo , Humanos , Lactante , Recién Nacido , Proteínas de Filamentos Intermediarios/genética , Masculino , Eliminación de SecuenciaRESUMEN
BACKGROUND: Asthma is a chronic inflammatory disease with a strong genetic predisposition. A major challenge for candidate gene association studies in asthma is the selection of biologically relevant genes. METHODOLOGY/PRINCIPAL FINDINGS: Using epithelial RNA expression arrays, HapMap allele frequency variation, and the literature, we identified six possible candidate susceptibility genes for childhood asthma including ADCY2, DNAH5, KIF3A, PDE4B, PLAU, SPRR2B. To evaluate these genes, we compared the genotypes of 194 predominantly tagging SNPs in 790 asthmatic, allergic and non-allergic children. We found that SNPs in all six genes were nominally associated with asthma (p<0.05) in our discovery cohort and in three independent cohorts at either the SNP or gene level (p<0.05). Further, we determined that our selection approach was superior to random selection of genes either differentially expressed in asthmatics compared to controls (pâ=â0.0049) or selected based on the literature alone (pâ=â0.0049), substantiating the validity of our gene selection approach. Importantly, we observed that 7 of 9 SNPs in the KIF3A gene more than doubled the odds of asthma (ORâ=â2.3, p<0.0001) and increased the odds of allergic disease (ORâ=â1.8, p<0.008). Our data indicate that KIF3A rs7737031 (T-allele) has an asthma population attributable risk of 18.5%. The association between KIF3A rs7737031 and asthma was validated in 3 independent populations, further substantiating the validity of our gene selection approach. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that KIF3A, a member of the kinesin superfamily of microtubule associated motors that are important in the transport of protein complexes within cilia, is a novel candidate gene for childhood asthma. Polymorphisms in KIF3A may in part be responsible for poor mucus and/or allergen clearance from the airways. Furthermore, our study provides a promising framework for the identification and evaluation of novel candidate susceptibility genes.
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Asma/genética , Perfilación de la Expresión Génica , Frecuencia de los Genes/genética , Cinesinas/genética , ARN/genética , Adolescente , Niño , Preescolar , Femenino , Humanos , Hipersensibilidad/genética , Masculino , Polimorfismo de Nucleótido Simple/genética , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Glutathione S-transferase pi (GSTPi) is the predominant redox regulator in the lung. Although evidence implicates an important role for GSTPi in asthma, the mechanism for this has remained elusive. OBJECTIVES: We sought to determine how GSTPi is regulated in asthma and to elucidate its role in maintaining redox homeostasis. METHODS: We elucidated the regulation of GSTPi in children with asthma and used murine models of asthma to determine the role of GSTPi in redox homeostasis. RESULTS: Our findings demonstrate that GSTPi transcript levels are markedly downregulated in allergen- and IL-13-treated murine models of asthma through signal transducer and activator of transcription 6-dependent and independent pathways. Nuclear factor erythroid 2-related factor 2 was also downregulated in these models. The decrease in GSTPi expression was associated with decreased total glutathione S-transferase activity in the lungs of mice. Examination of cystine intermediates uncovered a functional role for GSTPi in regulating cysteine oxidation, whereby GSTPi-deficient mice exhibited increased oxidative stress (increase in percentage cystine) compared with wild-type mice after allergen challenge. GSTPi expression was similarly downregulated in children with asthma. CONCLUSIONS: These data collectively suggest that downregulation of GSTPi after allergen challenge might contribute to the asthma phenotype because of disruption of redox homeostasis and increased oxidative stress. Furthermore, GSTPi might be an important therapeutic target for asthma, and evaluation of GSTPi expression might prove beneficial in identifying patients who would benefit from therapy targeting this pathway.
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Asma/fisiopatología , Regulación hacia Abajo , Gutatión-S-Transferasa pi/metabolismo , Estrés Oxidativo/fisiología , Adolescente , Alérgenos/inmunología , Animales , Asma/metabolismo , Niño , Preescolar , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Gutatión-S-Transferasa pi/genética , Homeostasis , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Oxidación-Reducción , Transducción de Señal , Pruebas CutáneasRESUMEN
BACKGROUND: Candidate gene case-control studies have identified several single nucleotide polymorphisms (SNPs) that are associated with asthma susceptibility. Most of these studies have been restricted to evaluations of specific SNPs within a single gene and within populations from European ancestry. Recently, there is increasing interest in understanding racial differences in genetic risk associated with childhood asthma. Our aim was to compare association patterns of asthma candidate genes between children of European and African ancestry. METHODOLOGY/PRINCIPAL FINDINGS: Using a custom-designed Illumina SNP array, we genotyped 1,485 children within the Greater Cincinnati Pediatric Clinic Repository and Cincinnati Genomic Control Cohort for 259 SNPs in 28 genes and evaluated their associations with asthma. We identified 14 SNPs located in 6 genes that were significantly associated (p-values <0.05) with childhood asthma in African Americans. Among Caucasians, 13 SNPs in 5 genes were associated with childhood asthma. Two SNPs in IL4 were associated with asthma in both races (p-values <0.05). Gene-gene interaction studies identified race specific sets of genes that best discriminate between asthmatic children and non-allergic controls. CONCLUSIONS/SIGNIFICANCE: We identified IL4 as having a role in asthma susceptibility in both African American and Caucasian children. However, while IL4 SNPs were associated with asthma in asthmatic children with European and African ancestry, the relative contributions of the most replicated asthma-associated SNPs varied by ancestry. These data provides valuable insights into the pathways that may predispose to asthma in individuals with European vs. African ancestry.
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Asma/etnología , Asma/genética , Negro o Afroamericano/genética , Estudios de Asociación Genética , Población Blanca/genética , Adolescente , Estudios de Casos y Controles , Niño , Preescolar , Estudios de Cohortes , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética/estadística & datos numéricos , Predisposición Genética a la Enfermedad/etnología , Genotipo , Humanos , Masculino , Polimorfismo de Nucleótido SimpleRESUMEN
OBJECTIVES: To determine the relationship between single nucleotide polymorphisms in candidate genes associated with multiple asthma phenotypes and health-related quality of life (HRQOL). STUDY DESIGN: A cross-sectional study was conducted at a pediatric hospital in 275 school-aged children diagnosed with asthma and their caregivers. Genomic DNA was obtained from children, and caregivers completed a measure of their child's HRQOL. Analysis of variance was used to investigate the association between single nucleotide polymorphisms and HRQOL. RESULTS: Children homozygous for the major variant at IL-4RA rs 1805010 had significantly better HRQOL than their counterparts. Significant associations with pulmonary function were not observed. CONCLUSIONS: Genes associated with asthma phenotype can be associated with HRQOL at least partly independent of pulmonary function.
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Asma/genética , Polimorfismo de Nucleótido Simple , Calidad de Vida , Análisis de Varianza , Niño , Estudios Transversales , Femenino , Marcadores Genéticos , Genotipo , Gutatión-S-Transferasa pi/genética , Homocigoto , Hospitales Pediátricos , Humanos , Interleucina-13/genética , Interleucina-4/genética , Receptores de Lipopolisacáridos/genética , Masculino , Receptores de Interleucina-4/genética , Encuestas y CuestionariosRESUMEN
Increases in the epidermal growth factor receptor (EGFR) have been associated with the severity of airway thickening in chronic asthmatic subjects, and EGFR signaling is induced by asthma-related cytokines and inflammation. The goal of this study was to determine the role of EGFR signaling in a chronic allergic model of asthma and specifically in epithelial cells, which are increasingly recognized as playing an important role in asthma. EGFR activation was assessed in mice treated with intranasal house dust mite (HDM) for 3 wk. EGFR signaling was inhibited in mice treated with HDM for 6 wk, by using either the drug erlotinib or a genetic approach that utilizes transgenic mice expressing a mutant dominant negative epidermal growth factor receptor in the lung epithelium (EGFR-M mice). Airway hyperreactivity (AHR) was assessed by use of a flexiVent system after increasing doses of nebulized methacholine. Airway smooth muscle (ASM) thickening was measured by morphometric analysis. Sensitization to HDM (IgG and IgE), inflammatory cells, and goblet cell changes were also assessed. Increased EGFR activation was detected in HDM-treated mice, including in bronchiolar epithelial cells. In mice exposed to HDM for 6 wk, AHR and ASM thickening were reduced after erlotinib treatment and in EGFR-M mice. Sensitization to HDM and inflammatory cell counts were similar in all groups, except neutrophil counts, which were lower in the EGFR-M mice. Goblet cell metaplasia with HDM treatment was reduced by erlotinib, but not in EGFR-M transgenic mice. This study demonstrates that EGFR signaling, especially in the airway epithelium, plays an important role in mediating AHR and remodeling in a chronic allergic asthma model.
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Remodelación de las Vías Aéreas (Respiratorias)/fisiología , Asma/fisiopatología , Hiperreactividad Bronquial/complicaciones , Células Epiteliales/enzimología , Receptores ErbB/metabolismo , Transducción de Señal , Animales , Asma/complicaciones , Asma/parasitología , Asma/patología , Hiperreactividad Bronquial/parasitología , Hiperreactividad Bronquial/patología , Hiperreactividad Bronquial/fisiopatología , Enfermedad Crónica , Modelos Animales de Enfermedad , Activación Enzimática , Células Epiteliales/patología , Receptores ErbB/antagonistas & inhibidores , Células Caliciformes/patología , Inflamación/complicaciones , Inflamación/patología , Pulmón/parasitología , Pulmón/patología , Pulmón/fisiopatología , Metaplasia , Ratones , Músculo Liso/patología , Pyroglyphidae/fisiologíaRESUMEN
BACKGROUND: Asthma is a major public health burden worldwide. Studies from our group and others have demonstrated that SERPINB3 and SERPINB4 are induced in patients with asthma; however, their mechanistic role in asthma has yet to be determined. OBJECTIVE: To evaluate the role of Serpin3a, the murine homolog of SERPINB3 and SERPINB4, in asthma. METHODS: We studied wild-type Balb/c and Serpinb3a-null mice in house dust mite or IL-13-induced asthma models and evaluated airway hyperresponsiveness, inflammation, and goblet cell hyperplasia. RESULTS: Airway hyperresponsiveness and goblet cell hyperplasia were markedly attenuated in the Serpinb3a-null mice compared with the wild-type mice after allergen challenge, with minimal effects on inflammation. Expression of sterile alpha motif pointed domain containing v-ets avian erythroblastosis virus E26 oncogene homolog transcription factor (SPDEF), a transcription factor that mediates goblet cell hyperplasia, was decreased in the absence of Serpinb3a. IL-13-treated Serpinb3a-null mice showed attenuated airway hyperresponsiveness, inflammation, and mucus production. CONCLUSION: Excessive mucus production and mucus plugging are key pathologic features of asthma, yet the mechanisms responsible for mucus production are not well understood. Our data reveal a novel nonredundant role for Serpinb3a in mediating mucus production through regulation of SPDEF expression. This pathway may be used to target mucus hypersecretion effectively.
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Asma/inmunología , Moco/inmunología , Proteínas Proto-Oncogénicas c-ets/inmunología , Serpinas/inmunología , Animales , Asma/metabolismo , Asma/patología , Líquido del Lavado Bronquioalveolar , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Expresión Génica , Regulación de la Expresión Génica/inmunología , Células Caliciformes/inmunología , Células Caliciformes/metabolismo , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Moco/metabolismo , Proteínas Proto-Oncogénicas c-ets/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serpinas/metabolismoRESUMEN
Eczema is very common and increasing in prevalence. Prospective studies investigating environmental and genetic risk factors for eczema in a birth cohort are lacking. We evaluated risk factors that may promote development of childhood eczema in the Cincinnati Childhood Allergy and Air Pollution Study (CCAAPS) birth cohort (n=762) of infants with at least one atopic parent. Objective environmental exposure data were available for each participant. At annual physical examinations, children underwent skin prick tests (SPTs), eczema was diagnosed by a clinician, and DNA was collected. Among Caucasian children, 39% developed eczema by age 3. Children with a pet dog were significantly less likely to have eczema at age one (odds ratio (OR)=0.62, 95% confidence interval (CI): 0.40-0.97) or at both ages 2 and 3 (OR=0.54, 95% CI: 0.30-0.97). This finding was most significant among children carrying the CD14-159C/T CC genotype. Carriers of the CD14-159C/T and IL4Ralpha I75V single-nucleotide polymorphisms (SNPs) had an increased risk of eczema at both ages 2 and 3 (OR=3.44, 95% CI: 1.56-7.57), especially among children who were SPT+. These results provide new insights into the pathogenesis of eczema in high-risk children and support a protective role for early exposure to dog, especially among those carrying the CD14-159C/T SNP. The results also demonstrate a susceptibility effect of the combination of CD14 and IL4Ralpha SNPs with eczema.
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Eccema/genética , Hipersensibilidad/genética , Animales , Animales Domésticos , Gatos , Niño , Preescolar , Estudios de Cohortes , Perros , Exposición a Riesgos Ambientales , Femenino , Predisposición Genética a la Enfermedad , Humanos , Lactante , Masculino , Polimorfismo de Nucleótido Simple , Factores de RiesgoRESUMEN
BACKGROUND: Established indicators of central obesity include waist circumference, waist/height ratio, and the conicity index. Studies using such measures (as opposed to body mass index [BMI] percentiles) to characterize the association between obesity and asthma are lacking, despite the fact that these measures have been shown to be most relevant for many other chronic diseases. OBJECTIVES: We sought to examine measures assessing the distribution of obesity in the context of childhood allergic rhinitis and asthma and to elucidate the association of obesity, including central obesity, with allergic asthma in children. METHODS: Children with allergic rhinitis with (cases) or without (control subjects) asthma were recruited. BMI percentiles were derived by using national growth charts. Waist circumference, waist/height ratio, and conicity index values were obtained. RESULTS: Central obesity was associated with asthma, asthma severity, lower lung function, and reduced atopy in asthmatic subjects. CONCLUSION: Measures of central obesity are more associated with the presence of asthma and asthma severity in children with allergic rhinitis when compared with standard BMI measures.
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Antropometría , Asma/epidemiología , Asma/fisiopatología , Obesidad/diagnóstico , Adolescente , Índice de Masa Corporal , Niño , Preescolar , Femenino , Humanos , Modelos Logísticos , Masculino , Prevalencia , Rinitis Alérgica Perenne/epidemiología , Rinitis Alérgica Estacional/epidemiología , Pruebas Cutáneas , Estados Unidos/epidemiología , Circunferencia de la CinturaRESUMEN
OBJECTIVES: To determine whether infants exposed to environmental tobacco smoke (ETS) having the interleukin 4 (IL-4) or interleukin 13 (IL-13) gene polymorphisms were at increased risk of wheezing. STUDY DESIGN: A birth cohort of 758 infants was evaluated annually by a questionnaire, physical examination, and skin prick testing. DNA samples from 560 children were genotyped for IL-4 C-589T and IL-13 C-1112T. The relationship of ETS exposure and genotype with the outcome of wheezing was analyzed. RESULTS: At the time of evaluation, mean age was 13.4 +/- 2.2 months. The prevalence of sensitization was 29%, and wheezing without a cold was 26.2%. The interaction of ETS exposure and the CT/TT genotypes for IL-4 C-589T showed a significant association with wheezing (odds ratio: 10.84; 95% confidence interval: 1.12-104.64, P = .04) in African-American infants. CONCLUSIONS: In African-American infants with a family history of atopy, the interaction of ETS and IL-4 C-589T demonstrated a 10-fold risk associated with wheezing without a cold.
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Negro o Afroamericano , Exposición a Riesgos Ambientales/efectos adversos , Interleucina-4/genética , Polimorfismo de Nucleótido Simple/genética , Ruidos Respiratorios/etiología , Contaminación por Humo de Tabaco/efectos adversos , Estudios de Cohortes , Femenino , Predisposición Genética a la Enfermedad/etnología , Humanos , Lactante , Interleucina-13/genética , Masculino , Factores SocioeconómicosRESUMEN
BACKGROUND: Recent genetic evidence supports that an underlying defect in skin barrier function contributes to the pathogenesis of atopic dermatitis (AD). The integrity of the skin barrier can be assessed objectively by measuring transepidermal water loss (TEWL). Previous investigations of TEWL as a biomarker of skin barrier function have been limited by small sample size, and studies including African American subjects are lacking. OBJECTIVE: We sought to determine whether children with AD have inherently altered skin barrier function by comparing TEWL as a measure of skin barrier function in African American and white children with AD with that in control subjects without AD. METHODS: TEWL was measured on nonlesional normal-appearing skin at 4 sites (the volar forearm, dorsal arm, lower leg, and cheek) in (1) children with AD (cases), (2) children with asthma or allergic rhinitis but without AD (allergic control subjects), and (3) nonatopic control subjects. AD severity was assessed by using the objective SCORAD index. RESULTS: TEWL was increased in children with AD compared with that seen in both control groups at most of the anatomic sites tested (P < .05). TEWL also correlated with objective SCORAD score. The presence of allergic sensitization or other allergic conditions did not affect TEWL among children with AD. TEWL was higher in white than in African American children. CONCLUSION: Skin barrier function as assessed by TEWL is intrinsically compromised in children with AD but not in children with other allergic conditions. The magnitude of skin barrier dysfunction correlates with AD disease severity.