RNAseq analysis of bronchial epithelial cells to identify COPD-associated genes and SNPs.

BACKGROUND: There is a need for more powerful methods to identify low-effect SNPs that contribute to hereditary COPD pathogenesis. We hypothesized that SNPs contributing to COPD risk through cis-regulatory effects are enriched in genes comprised by bronchial epithelial cell (BEC) expression patterns associated with COPD. METHODS: To test this hypothesis, normal BEC specimens were obtained by bronchoscopy from 60 subjects: 30 subjects with COPD defined by spirometry (FEV1/FVC < 0.7, FEV1% < 80%), and 30 non-COPD controls. Targeted next generation sequencing was used to measure total and allele-specific expression of 35 genes in genome maintenance (GM) genes pathways linked to COPD pathogenesis, including seven TP53 and CEBP transcription factor family members. Shrinkage linear discriminant analysis (SLDA) was used to identify COPD-classification models. COPD GWAS were queried for putative cis-regulatory SNPs in the targeted genes. RESULTS: On a network basis, TP53 and CEBP transcription factor pathway gene pair network connections, including key DNA repair gene ERCC5, were significantly different in COPD subjects (e.g., Wilcoxon rank sum test for closeness, p-value = 5.0E-11). ERCC5 SNP rs4150275 association with chronic bronchitis was identified in a set of Lung Health Study (LHS) COPD GWAS SNPs restricted to those in putative regulatory regions within the targeted genes, and this association was validated in the COPDgene non-hispanic white (NHW) GWAS. ERCC5 SNP rs4150275 is linked (D' = 1) to ERCC5 SNP rs17655 which displayed differential allelic expression (DAE) in BEC and is an expression quantitative trait locus (eQTL) in lung tissue (p = 3.2E-7). SNPs in linkage (D' = 1) with rs17655 were predicted to alter miRNA binding (rs873601). A classifier model that comprised gene features CAT, CEBPG, GPX1, KEAP1, TP73, and XPA had pooled 10-fold cross-validation receiver operator characteristic area under the curve of 75.4% (95% CI: 66.3%-89.3%). The prevalence of DAE was higher than expected (p = 0.0023) in the classifier genes. CONCLUSIONS: GM genes comprised by COPD-associated BEC expression patterns were enriched for SNPs with cis-regulatory function, including a putative cis-rSNP in ERCC5 that was associated with COPD risk. These findings support additional total and allele-specific expression analysis of gene pathways with high prior likelihood for involvement in COPD pathogenesis.

Microcystin-LR aerosol induces inflammatory responses in healthy human primary airway epithelium.

Harmful algal blooms plague bodies of freshwater globally. These blooms are often composed of outgrowths of cyanobacteria capable of producing the heptapeptide Microcystin-LR (MC-LR) which is a well-known hepatotoxin. Recently, MC-LR has been detected in aerosols generated from lake water. However, the risk for human health effects due to MC-LR inhalation exposure have not been extensively investigated. In this study, we exposed a fully differentiated 3D human airway epithelium derived from 14 healthy donors to MC-LR-containing aerosol once a day for 3 days. Concentrations of MC-LR ranged from 100 pM to 1 µM. Although there were little to no detrimental alterations in measures of the airway epithelial function (i.e. cell survival, tissue integrity, mucociliary clearance, or cilia beating frequency), a distinct shift in the transcriptional activity was found. Genes related to inflammation were found to be upregulated such as C-C motif chemokine 5 (CCL5; log2FC = 0.57, p = 0.03) and C-C chemokine receptor type 7 (CCR7; log2FC = 0.84, p = 0.03). Functionally, conditioned media from MC-LR exposed airway epithelium was also found to have significant chemo-attractive properties for primary human neutrophils. Additionally, increases were found in the concentration of secreted chemokine proteins in the conditioned media such as CCL1 (log2FC = 5.07, p = 0.0001) and CCL5 (log2FC = 1.02, p = 0.046). These results suggest that MC-LR exposure to the human airway epithelium is capable of inducing an inflammatory response that may potentiate acute or chronic disease.

New-onset acute interstitial lung disease after treatment with erlotinib in a patient with metastatic squamous cell carcinoma of the lung.

Erlotinib is a Human Epidermal Growth Factor Receptor Type 1/tyrosine kinase (EGFR) inhibitor, which is used for non-small-cell lung cancer treatment. Erlotinib usually has a favorable safety profile however; adverse events such as interstitial lung disease (ILD) have been reported in pivotal studies. ILD usually occurs weeks to months after initiating therapy with Erlotinib. We report a case of Erlotinib induced ILD presenting within 5 days of initiating treatment with Erlotinib.

The c-myc x E2F-1/p21 interactive gene expression index augments cytomorphologic diagnosis of lung cancer in fine-needle aspirate specimens.

Morphological analysis of cytologic samples obtained by fine-needle aspirate (FNA) or bronchoscopy is an important method for diagnosing bronchogenic carcinoma. However, this approach has only about 65 to 80% diagnostic sensitivity. Based on previous studies, the c-myc x E2F-1/p21WAF1/CIP1 (p21 hereafter) gene expression index is highly sensitive and specific for distinguishing normal from malignant bronchial epithelial tissues. In an effort to improve sensitivity of diagnosing lung cancer in cytologic specimens, we used Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR) to measure the c-myc x E2F-1/p21 index in cDNA samples from 14 normal lung samples (6 normal lung parenchyma and 8 normal bronchial epithelial cell [NBEC] biopsies), and 16 FNA biopsies from 14 suspected tumors. Based on cytomorphologic criteria, 11 of the 14 suspected tumors were diagnosed as bronchogenic carcinoma and three specimens were non-diagnostic. Subsequent biopsy samples confirmed that the three non-diagnostic samples were derived from lung carcinomas. The index value for each bronchogenic carcinoma was above a cut-off value of 7000 and the index value of all but one normal sample was below 7000. Thus the c-myc x E2F-1/p21 index may augment cytomorphologic diagnosis of bronchogenic carcinoma biopsy samples, particularly those considered non-diagnostic by cytomorphologic criteria.

Disseminated blastomycosis.

A 26-year-old veiled Saudi-Arabian woman presented with hemoptysis, and multiple nodules and abscesses. A skin biopsy specimen revealed yeast forms consistent with Blastomyces dermatitidis. Fungal cultures from bronchoscopy and skin specimens also grew B dermatitidis. She was treated with oral itraconazole (200 mg twice a day). Both lung and skin lesions showed improvement within 6 weeks.