Non-invasive Analysis of the Airway Transcriptome Discriminates Clinical Phenotypes of Asthma
ABSTRACT: BACKGROUND: Evaluation of the airway transcriptome may reveal patterns of gene expression that are associated with clinical phenotypes of asthma. To define transcriptomic endotypes of asthma (TEA) we analyzed gene expression in induced sputum that correlate with phenotypes of disease. METHODS: Gene expression was measured in sputum of subjects with asthma using Affymetrix HuGene ST 1.0 microarrays. Unsupervised clustering analysis of genes in pathways selected from the Kyoto Encyclopedia of Genes and Genomes (KEGG) identified TEA clusters. Clinical characteristics were compared and logistic regression analysis of matched blood samples defined an expression profile to determine the TEA cluster assignment in a cohort of children with asthma for validation. RESULTS: Three TEA clusters were identified. TEA cluster 1 had the most subjects with a history of intubation (P = 0.05), a lower pre-bronchodilator FEV1 (P = 0.006), a higher bronchodilator response (P = 0.03), and higher exhaled nitric oxide levels (P = 0.04), compared to the other TEA clusters. TEA cluster 2, the smallest cluster had the most subjects that were hospitalized for asthma (P = 0.04). Subjects in TEA cluster 3, the largest cluster, had normal lung function, low exhaled nitric oxide levels, and lower inhaled steroid requirements. Evaluation of TEA clusters in children confirmed that TEA clusters 1 and 2 are associated with a history of intubation (P = 5.58 x 10-06) and hospitalization (P = 0.01), respectively. CONCLUSIONS: Patterns of gene expression in the sputum and blood reveal TEA clusters that are associated with severe asthma phenotypes in children and adults. Gene expression was measured in sputum of subjects with asthma using Affymetrix HuGene ST 1.0 microarrays. Unsupervised clustering analysis of genes in pathways selected from the Kyoto Encyclopedia of Genes and Genomes (KEGG) identified TEA clusters. Clinical characteristics were compared and logistic regression analysis of matched blood samples defined an expression profile to determine the TEA cluster assignment in a cohort of children with asthma for validation.
Project description:This study identifies differentially expression genes in the sputum of people with eosinophilic, neutrophilic and paucigranulocytic asthma. A selection of markers identified using this microarray were further validated using qPCR on a wider sample set. Gene expression profiles were generated from induced sputum samples from 47 asthma patients and were grouped by the inflammatory phenotype assigned using sputum cell counts into neutrophilic asthma (n=12), eosinophilic asthma (n=17) and paucigranulocytic asthma (n=18). RNA was extracted, amplified and hybridised to Illumina Sentrix HumanRef-8 Version 2 Expression BeadChips, and genes that were differentially expressed between asthma inflammatory phenotypes were compared.
Project description:Sputum cells collected before (visit 2) and after (visit 4) allergen challenge in asthma patients were isolated and RNA purified for analysis on gene expression arrays. Human subject recruitment part of NIH sponsored protocol as part of the Eosinophil Program Project Grant (PI: Dr. Nizar Jarjour) Sputum cell RNA collected from induced sputum cells before and 48 hours after whole-lung allergen challenge.
Project description:Severe asthma is a collection of disease entities with varying pathophysiological characteristics that result in symptoms of cough, wheeze and breathlessness, with frequent exacerbations. To address the problem of phenotypic difference and heterogeneity, the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) project was set up as a public-private partnership within the framework of the Innovative Medicines Initiative (IMI), engaging academia, the pharmaceutical industry and patient groups. The goal of this investigation was to identify proteomic fingerprints in induced sputum that characterize patients with severe asthma and to determine whether subgroups of severe asthmatics can be identified. Furthermore, we were interested in elucidating the biological pathways that showed differences between subgroups. This dataset is a description of the normal sputum proteome.
Project description:Systemic inflammation is reported to be associated with neutrophilic airway inflammation in asthma, this study aimed to examine the molecular mechanisms of the neutrophilia that is associated with systemic inflammation, and hypothesized that asthma patients with systemic inflammation have a group of genes that are differentially expressed and are assciated with airway inflammation. 50 asthma patients were recruited and grouped as asthmatics with systemic inflammation (n=18) and asthamtics without systemic inflammation (n=16) accroding to the levels of serum CRP and IL-6. RNA was extracted from induced sputum and was reverse-transcribed into cDNA. Gene profiling was performed using Illumina Sentrix HumanRef-8 Version 2 Expression BeadChips, and genes that were differentially expressed between asthmatics with systemic inflammation and asthmatics without systemic inflammation were compared and valided using qPCR.
Project description:We set out to test the hypothesis that ozone inhalation exposure significantly alters miRNA expression profiles within the airway of humans. Adult human volunteers were exposed to 0.4 ppm ozone for two hours. Induced sputum samples were collected from each subject 48 hours pre-exposure and 6 hours post-exposure. Genome-wide miRNA expression profiles were evaluated using microarray analysis. Twenty healthy non-asthmatic adult subjects aged 18 – 37 years, with no history of smoking in the past 10 years, completed the study. The ozone exposures were conducted in an exposure chamber. Each subject was exposed to 0.4 ppm ozone for two hours while performing four 15-minute sessions of intermittent moderate exercise (expiratory minute ventilation, 30–40 L/min) on a treadmill, separated by 15 minutes of seated rest. Induced sputum samples were collected from each subject 48 hours pre-exposure and 6 hours post-exposure. RNA samples were homogenized in TRIzol, and extracted according to standard TRIzol protocol. RNA was labeled and hybridized to the Agilent Human miRNA Microarray (v1.0).
Project description:RNA sequencing (RNA-seq) of Mycobacterium abscessus in four infection-relevant culture conditions: hypoxic stress, artificial sputum medium, kanamycin-treated medium, and erythromycin-treated medium. Triplicate cultures of M. abscessus were grown in (1) Artificial Sputum media, (2) hypoxic conditions, (3) the presence of kanamycin, and (4) the presence of erythromycin. Triplicate controls were prepared for sample (1) and samples (2-4).
Project description:Arrays comparing Pseudomonas aeruginosa growth in a defined synthetic cystic fibrosis sputum medium with and without aromatic amino acids. Additional arrays comparing wild-type Pseudomonas aeruginosa and phhR mutant P. aeruginosa in defined synthetic cystic fibrosis sputum medium.
Project description:Asthma is a heterogeneous disease. Exercise-induced bronchoconstriction (EIB) is a distinct syndrome that occurs in 30-50% of asthmatics and is characterized by high levels of pro-inflammatory eicosanoids. We identified genes differentially expressed in the airways of asthmatics with EIB relative to asthmatics without EIB. Genes related to epithelial repair and mast cell infiltration including beta-tryptase and carboxypeptidase A3 were upregulated by exercise challenge in the asthma group with EIB. We confirmed that two novel mediators trefoil factor 3 (TFF3) and transglutaminase 2 (TGM2) have increased expression in airways cells and secreted product in the airways. In vitro studies indicate that 1) TFF3 induces nitric oxide synthase in airway epithelial cells from asthmatics and 2) TGM2 augments the enzymatic activity of secreted phospholipase A2 (sPLA2) group X, an enzyme recently been implicated in asthma pathogenesis. Since PLA2 serves as the first rate-limiting step leading to eicosanoid generation, these results suggest that TGM2 may be a key initiator of the airway inflammatory cascade in asthma. EIB positive and EIB negative subjects sampled pre- and post-exercise
Project description:The overall goal of the Severe Asthma Research Program (SARP) is to identify and characterize subjects with severe asthma to understand pathophysiologic mechanisms in severe asthma. Subjects with mild and moderate asthma were recruited for comparison but the program was enriched for subjects with severe asthma from multiple centers. Subjects were comprehensively phenotyped for asthma related traits including lung function, atopy, questionnaires on medical and family history, exhaled nitric oxide and health care utilization including exacerbations and symptoms. Asthma is a heterogenous disease cluster analysis in SARP has shown multiple subphenotypes and endotypes.
Project description:Asthma is caused by a combination of poorly understood genetic and environmental factors. We found multiple markers on chromosome 17q21 to be strongly and reproducibly associated with childhood onset asthma in family and case-referent panels with a combined P < 10-12. In independent replication studies the 17q21 locus showed strong association with diagnosis of childhood asthma in 2,320 subjects from a cohort of German children (P = 0.0003) and in 3,301 subjects from the British 1958 Birth Cohort (P = 0.0005). We systematically evaluated the relationships between markers of the 17q21 locus and transcript levels of genes in EBV-transformed lymphoblastoid cell lines from children in the asthma family panel used in our association study. The SNPs associated with childhood asthma were consistently and strongly associated (P <10-22) in cis with transcript levels of ORMDL3, a member of a gene family that encode transmembrane proteins anchored in the endoplasmic reticulum. The results indicate that genetic variants regulating ORMDL3 expression are determinants of susceptibility to childhood asthma. Experiment Overall Design: Gene expression levels were evaluated in 404 children. We then evaluated the relationship between SNPs in the 17q21 region (which show association to asthma in the same children) with gene expression levels. See http://www.sph.umich.edu/csg/liang/asthma/