A bronchial gene signature specific for severe COPD that is retained in the nose.

Introduction: A subset of COPD patients develops advanced disease with severe airflow obstruction, hyperinflation and extensive emphysema. We propose that the pathogenesis in these patients differs from mild-moderate COPD and is reflected by bronchial gene expression. The aim of the present study was to identify a unique bronchial epithelial gene signature for severe COPD patients. Methods: We obtained RNA sequencing data from bronchial brushes from 123 ex-smokers with severe COPD, 23 with mild-moderate COPD and 23 non-COPD controls. We identified genes specific to severe COPD by comparing severe COPD to non-COPD controls, followed by removing genes that were also differentially expressed between mild-moderate COPD and non-COPD controls. Next, we performed a pathway analysis on these genes and evaluated whether this signature is retained in matched nasal brushings. Results: We identified 219 genes uniquely differentially expressed in severe COPD. Interaction network analysis identified VEGFA and FN1 as the key genes with the most interactions. Genes were involved in extracellular matrix regulation, collagen binding and the immune response. Of interest were 10 genes (VEGFA, DCN, SPARC, COL6A2, MGP, CYR61, ANXA6, LGALS1, C1QA and C1QB) directly connected to fibronectin 1 (FN1). Most of these genes were lower expressed in severe COPD and showed the same effect in nasal brushings. Conclusions: We found a unique severe COPD bronchial gene signature with key roles for VEGFA and FN1, which was retained in the upper airways. This supports the hypothesis that severe COPD, at least partly, comprises a different pathology and supports the potential for biomarker development based on nasal brushes in COPD.

Smoking induces shifts in cellular composition and transcriptome within the bronchial mucus barrier.

BACKGROUND AND OBJECTIVE: Smoking disturbs the bronchial-mucus-barrier. This study assesses the cellular composition and gene expression shifts of the bronchial-mucus-barrier with smoking to understand the mechanism of mucosal damage by cigarette smoke exposure. We explore whether single-cell-RNA-sequencing (scRNA-seq) based cellular deconvolution (CD) can predict cell-type composition in RNA-seq data. METHODS: RNA-seq data of bronchial biopsies from three cohorts were analysed using CD. The cohorts included 56 participants with chronic obstructive pulmonary disease [COPD] (38 smokers; 18 ex-smokers), 77 participants without COPD (40 never-smokers; 37 smokers) and 16 participants who stopped smoking for 1 year (11 COPD and 5 non-COPD-smokers). Differential gene expression was used to investigate gene expression shifts. The CD-derived goblet cell ratios were validated by correlating with staining-derived goblet cell ratios from the COPD cohort. Statistics were done in the R software (false discovery rate p-value < 0.05). RESULTS: Both CD methods indicate a shift in bronchial-mucus-barrier cell composition towards goblet cells in COPD and non-COPD-smokers compared to ex- and never-smokers. It shows that the effect was reversible within a year of smoking cessation. A reduction of ciliated and basal cells was observed with current smoking, which resolved following smoking cessation. The expression of mucin and sodium channel (ENaC) genes, but not chloride channel genes, were altered in COPD and current smokers compared to never smokers or ex-smokers. The goblet cell-derived staining scores correlate with CD-derived goblet cell ratios. CONCLUSION: Smoking alters bronchial-mucus-barrier cell composition, transcriptome and increases mucus production. This effect is partly reversible within a year of smoking cessation. CD methodology can predict goblet-cell percentages from RNA-seq.

High miR203a-3p and miR-375 expression in the airways of smokers with and without COPD.

Smoking is a leading cause of chronic obstructive pulmonary disease (COPD). It is known to have a significant impact on gene expression and (inflammatory) cell populations in the airways involved in COPD pathogenesis. In this study, we investigated the impact of smoking on the expression of miRNAs in healthy and COPD individuals. We aimed to elucidate the overall smoking-induced miRNA changes and those specific to COPD. In addition, we investigated the downstream effects on regulatory gene expression and the correlation to cellular composition. We performed a genome-wide miRNA expression analysis on a dataset of 40 current- and 22 ex-smoking COPD patients and a dataset of 35 current- and 38 non-smoking respiratory healthy controls and validated the results in an independent dataset. miRNA expression was then correlated with mRNA expression in the same patients to assess potential regulatory effects of the miRNAs. Finally, cellular deconvolution analysis was used to relate miRNAs changes to specific cell populations. Current smoking was associated with increased expression of three miRNAs in the COPD patients and 18 miRNAs in the asymptomatic smokers compared to respiratory healthy controls. In comparison, four miRNAs were lower expressed with current smoking in asymptomatic controls. Two of the three smoking-related miRNAs in COPD, miR-203a-3p and miR-375, were also higher expressed with current smoking in COPD patients and the asymptomatic controls. The other smoking-related miRNA in COPD patients, i.e. miR-31-3p, was not present in the respiratory healthy control dataset. miRNA-mRNA correlations demonstrated that miR-203a-3p, miR-375 and also miR-31-3p expression were negatively associated with genes involved in pro-inflammatory pathways and positively associated with genes involved in the xenobiotic pathway. Cellular deconvolution showed that higher levels of miR-203a-3p were associated with higher proportions of proliferating-basal cells and secretory (club and goblet) cells and lower levels of fibroblasts, luminal macrophages, endothelial cells, B-cells, amongst other cell types. MiR-375 expression was associated with lower levels of secretory cells, ionocytes and submucosal cells, but higher levels of endothelial cells, smooth muscle cells, and mast cells, amongst other cell types. In conclusion, we identified two smoking-induced miRNAs (miR-375 and miR-203a-3p) that play a role in regulating inflammation and detoxification pathways, regardless of the presence or absence of COPD. Additionally, in patients with COPD, we identified miR-31-3p as a miRNA induced by smoking. Our identified miRNAs should be studied further to unravel which smoking-induced inflammatory mechanisms are reactive and which are involved in COPD pathogenesis.

Identification of asthma-associated microRNAs in bronchial biopsies.

BACKGROUND: Changes in microRNA (miRNA) expression can contribute to the pathogenesis of many diseases, including asthma. We aimed to identify miRNAs that are differentially expressed between asthma patients and healthy controls, and explore their association with clinical and inflammatory parameters of asthma. METHODS: Differentially expressed miRNAs were determined by small RNA sequencing on bronchial biopsies of 79 asthma patients and 82 healthy controls using linear regression models. Differentially expressed miRNAs were associated with clinical and inflammatory asthma features. Potential miRNA-mRNA interactions were analysed using mRNA data available from the same bronchial biopsies, and enrichment of pathways was identified with Enrichr and g:Profiler. RESULTS: In total, 78 differentially expressed miRNAs were identified in bronchial biopsies of asthma patients compared with controls, of which 60 remained differentially expressed after controlling for smoking and inhaled corticosteroid treatment. We identified several asthma-associated miRNAs, including miR-125b-5p and miR-223-3p, based on a significant association with multiple clinical and inflammatory asthma features and their negative correlation with genes associated with the presence of asthma. The most enriched biological pathway(s) affected by miR-125b-5p and miR-223-3p were inflammatory response and cilium assembly/organisation. Of interest, we identified that lower expression of miR-26a-5p was linked to more severe eosinophilic inflammation as measured in blood, sputum as well as bronchial biopsies. CONCLUSION: Collectively, we identified miR-125b-5p, miR-223-3p and miR-26a-5p as potential regulators that could contribute to the pathogenesis of asthma.

iFUSE: integrated fusion gene explorer.

UNLABELLED: We present iFUSE (integrated fusion gene explorer), an online visualization tool that provides a fast and informative view of structural variation data and prioritizes those breaks likely representing fusion genes. This application uses calculated break points to determine fusion genes based on the latest annotation for genomic sequence information, and where relevant the structural variation (SV) events are annotated with predicted RNA and protein sequences. iFUSE takes as input a Complete Genomics (CG) junction file, a FusionMap fusion detection report file or a file already analysed and annotated by the iFUSE application on a previous occasion. RESULTS: We demonstrate the use of iFUSE with case studies from tumour-normal SV detection derived from Complete Genomics whole-genome sequencing results. AVAILABILITY: iFUSE is available as a web service at http://ifuse.erasmusmc.nl.