A Polygenic Risk Score for Idiopathic Pulmonary Fibrosis and Interstitial Lung Abnormalities.

Rationale: In addition to rare genetic variants and the MUC5B locus, common genetic variants contribute to idiopathic pulmonary fibrosis (IPF) risk. The predictive power of common variants outside the MUC5B locus for IPF and interstitial lung abnormalities (ILAs) is unknown. Objectives: We tested the predictive value of IPF polygenic risk scores (PRSs) with and without the MUC5B region on IPF, ILA, and ILA progression. Methods: We developed PRSs that included (PRS-M5B) and excluded (PRS-NO-M5B) the MUC5B region (500-kb window around rs35705950-T) using an IPF genome-wide association study. We assessed PRS associations with area under the receiver operating characteristic curve (AUC) metrics for IPF, ILA, and ILA progression. Measurements and Main Results: We included 14,650 participants (1,970 IPF; 1,068 ILA) from six multi-ancestry population-based and case-control cohorts. In cases excluded from genome-wide association study, the PRS-M5B (odds ratio [OR] per SD of the score, 3.1; P = 7.1 × 10-95) and PRS-NO-M5B (OR per SD, 2.8; P = 2.5 × 10-87) were associated with IPF. Participants in the top PRS-NO-M5B quintile had ∼sevenfold odds for IPF compared with those in the first quintile. A clinical model predicted IPF (AUC, 0.61); rs35705950-T and PRS-NO-M5B demonstrated higher AUCs (0.73 and 0.7, respectively), and adding both genetic predictors to a clinical model yielded the highest performance (AUC, 0.81). The PRS-NO-M5B was associated with ILA (OR, 1.25) and ILA progression (OR, 1.16) in European ancestry participants. Conclusions: A common genetic variant risk score complements the MUC5B variant to identify individuals at high risk of interstitial lung abnormalities and pulmonary fibrosis.

Distinct COPD subtypes in former smokers revealed by gene network perturbation analysis.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) varies significantly in symptomatic and physiologic presentation. Identifying disease subtypes from molecular data, collected from easily accessible blood samples, can help stratify patients and guide disease management and treatment. METHODS: Blood gene expression measured by RNA-sequencing in the COPDGene Study was analyzed using a network perturbation analysis method. Each COPD sample was compared against a learned reference gene network to determine the part that is deregulated. Gene deregulation values were used to cluster the disease samples. RESULTS: The discovery set included 617 former smokers from COPDGene. Four distinct gene network subtypes are identified with significant differences in symptoms, exercise capacity and mortality. These clusters do not necessarily correspond with the levels of lung function impairment and are independently validated in two external cohorts: 769 former smokers from COPDGene and 431 former smokers in the Multi-Ethnic Study of Atherosclerosis (MESA). Additionally, we identify several genes that are significantly deregulated across these subtypes, including DSP and GSTM1, which have been previously associated with COPD through genome-wide association study (GWAS). CONCLUSIONS: The identified subtypes differ in mortality and in their clinical and functional characteristics, underlining the need for multi-dimensional assessment potentially supplemented by selected markers of gene expression. The subtypes were consistent across cohorts and could be used for new patient stratification and disease prognosis.

Lung tissue shows divergent gene expression between chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are characterized by shared exposures and clinical features, but distinct genetic and pathologic features exist. These features have not been well-studied using large-scale gene expression datasets. We hypothesized that there are divergent gene, pathway, and cellular signatures between COPD and IPF. METHODS: We performed RNA-sequencing on lung tissues from individuals with IPF (n = 231) and COPD (n = 377) compared to control (n = 267), defined as individuals with normal spirometry. We grouped the overlapping differential expression gene sets based on direction of expression and examined the resultant sets for genes of interest, pathway enrichment, and cell composition. Using gene set variation analysis, we validated the overlap group gene sets in independent COPD and IPF data sets. RESULTS: We found 5010 genes differentially expressed between COPD and control, and 11,454 genes differentially expressed between IPF and control (1% false discovery rate). 3846 genes overlapped between IPF and COPD. Several pathways were enriched for genes upregulated in COPD and downregulated in IPF; however, no pathways were enriched for genes downregulated in COPD and upregulated in IPF. There were many myeloid cell genes with increased expression in COPD but decreased in IPF. We found that the genes upregulated in COPD but downregulated in IPF were associated with lower lung function in the independent validation cohorts. CONCLUSIONS: We identified a divergent gene expression signature between COPD and IPF, with increased expression in COPD and decreased in IPF. This signature is associated with worse lung function in both COPD and IPF.

C FTR variants are associated with chronic bronchitis in smokers.

INTRODUCTION: Loss-of-function variants in both copies of the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF); however, there is evidence that reduction in CFTR function due to the presence of one deleterious variant can have clinical consequences. Here, we hypothesise that CFTR variants in individuals with a history of smoking are associated with chronic obstructive pulmonary disease (COPD) and related phenotypes. METHODS: Whole-genome sequencing was performed through the National Heart, Lung, and Blood Institute TOPMed (TransOmics in Precision Medicine) programme in 8597 subjects from the COPDGene (Genetic Epidemiology of COPD) study, an observational study of current and former smokers. We extracted clinically annotated CFTR variants and performed single-variant and variant-set testing for COPD and related phenotypes. Replication was performed in 2118 subjects from the ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints) study. RESULTS: We identified 301 coding variants within the CFTR gene boundary: 147 of these have been reported in individuals with CF, including 36 CF-causing variants. We found that CF-causing variants were associated with chronic bronchitis in variant-set testing in COPDGene (one-sided p=0.0025; OR 1.53) and in meta-analysis of COPDGene and ECLIPSE (one-sided p=0.0060; OR 1.52). Single-variant testing revealed that the F508del variant was associated with chronic bronchitis in COPDGene (one-sided p=0.015; OR 1.47). In addition, we identified 32 subjects with two or more CFTR variants on separate alleles and these subjects were enriched for COPD cases (p=0.010). CONCLUSIONS: Cigarette smokers who carry one deleterious CFTR variant have higher rates of chronic bronchitis, while presence of two CFTR variants may be associated with COPD. These results indicate that genetically mediated reduction in CFTR function contributes to COPD related phenotypes, in particular chronic bronchitis.

Development of a Blood-based Transcriptional Risk Score for Chronic Obstructive Pulmonary Disease.

Rationale: The ability of peripheral blood biomarkers to assess chronic obstructive pulmonary disease (COPD) risk and progression is unknown. Genetics and gene expression may capture important aspects of COPD-related biology that predict disease activity. Objectives: Develop a transcriptional risk score (TRS) for COPD and assess the contribution of the TRS and a polygenic risk score (PRS) for disease susceptibility and progression. Methods: We randomly split 2,569 COPDGene (Genetic Epidemiology of COPD) participants with whole-blood RNA sequencing into training (n = 1,945) and testing (n = 624) samples and used 468 ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points) COPD cases with microarray data for replication. We developed a TRS using penalized regression (least absolute shrinkage and selection operator) to model FEV1/FVC and studied the predictive value of TRS for COPD (Global Initiative for Chronic Obstructive Lung Disease 2-4), prospective FEV1 change (ml/yr), and additional COPD-related traits. We adjusted for potential confounders, including age and smoking. We evaluated the predictive performance of the TRS in the context of a previously derived PRS and clinical factors. Measurements and Main Results: The TRS included 147 transcripts and was associated with COPD (odds ratio, 3.3; 95% confidence interval [CI], 2.4-4.5; P < 0.001), FEV1 change (ß, -17 ml/yr; 95% CI, -28 to -6.6; P = 0.002), and other COPD-related traits. In ECLIPSE cases, we replicated the association with FEV1 change (ß, -8.2; 95% CI, -15 to -1; P = 0.025) and the majority of other COPD-related traits. Models including PRS, TRS, and clinical factors were more predictive of COPD (area under the receiver operator characteristic curve, 0.84) and annualized FEV1 change compared with models with one risk score or clinical factors alone. Conclusions: Blood transcriptomics can improve prediction of COPD and lung function decline when added to a PRS and clinical risk factors.

MALT1-Dependent Cleavage of HOIL1 Modulates Canonical NF-κB Signaling and Inflammatory Responsiveness.

Nuclear factor kappa B (NF-κB) is a critical transcription factor involved in regulating cell activation, inflammation, and survival. The linear ubiquitin chain assembly complex (LUBAC) which consists of HOIL1, HOIP, and SHARPIN, catalyzes the linear ubiquitination of target proteins-a post-translational modification that is essential for NF-κB activation. Human germline pathogenic variants that dysregulate linear ubiquitination and NF-κB signaling are associated with immunodeficiency and/or autoinflammation including dermatitis, recurrent fevers, systemic inflammation and enteropathy. We previously identified MALT1 paracaspase as a novel negative regulator of LUBAC by proteolytic cleavage of HOIL1. To directly investigate the impact of HOIL1 cleavage activity on the inflammatory response, we employed a stable transduction system to express and directly compare non-cleavable HOIL1 with wild-type HOIL1 in primary HOIL1-deficient patient skin fibroblasts. We discovered that non-cleavable HOIL1 resulted in enhanced NF-κB signaling in response to innate stimuli. Transcriptomics revealed enrichment of inflammation and proinflammatory cytokine-related pathways after stimulation. Multiplexed cytokine assays confirmed a 'hyperinflammatory' phenotype in these cells. This work highlights the physiological importance of MALT1-dependent cleavage and modulation of HOIL1 on NF-κB signaling and inflammation, provides a mechanism for the autoinflammation observed in MALT1-deficient patients, and will inform the development of therapeutics that target MALT1 paracaspase and LUBAC function in treating autoinflammatory skin diseases.

Immunomodulatory function of the cystic fibrosis modifier gene BPIFA1.

BACKGROUND: Cystic fibrosis (CF) is characterized by a progressive decline in lung function due to airway obstruction, infection, and inflammation. CF patients are particularly susceptible to respiratory infection by a variety of pathogens, and the inflammatory response in CF is dysregulated and prolonged. BPI fold containing family A, member 1 (BPIFA1) and BPIFB1 are proteins expressed in the upper airways that may have innate immune activity. We previously identified polymorphisms in the BPIFA1/BPIFB1 region associated with CF lung disease severity. METHODS: We evaluated whether the BPIFA1/BPIFB1 associations with lung disease severity replicated in individuals with CF participating in the International CF Gene Modifier Consortium (n = 6,365). Furthermore, we investigated mechanisms by which the BPIFA1 and BPIFB1 proteins may modify lung disease in CF. RESULTS: The association of the G allele of rs1078761 with reduced lung function was replicated in an independent cohort of CF patients (p = 0.001, n = 2,921) and in a meta-analysis of the full consortium (p = 2.39x10-5, n = 6,365). Furthermore, we found that rs1078761G which is associated with reduced lung function was also associated with reduced BPIFA1, but not BPIFB1, protein levels in saliva from CF patients. Functional assays indicated that BPIFA1 and BPIFB1 do not have an anti-bacterial role against P. aeruginosa but may have an immunomodulatory function in CF airway epithelial cells. Gene expression profiling using RNAseq identified Rho GTPase signaling pathways to be altered in CF airway epithelial cells in response to treatment with recombinant BPIFA1 and BPIFB1 proteins. CONCLUSIONS: BPIFA1 and BPIFB1 have immunomodulatory activity and genetic variation associated with low levels of these proteins may increase CF lung disease severity.

Correction to: RNA sequencing identifies novel non-coding RNA and exon-specific effects associated with cigarette smoking.

Following publication of the original article [1], the authors provided an updated accession number in the "Availability of data and materials" section of the declarations.

RNA sequencing identifies novel non-coding RNA and exon-specific effects associated with cigarette smoking.

BACKGROUND: Cigarette smoking is the leading modifiable risk factor for disease and death worldwide. Previous studies quantifying gene-level expression have documented the effect of smoking on mRNA levels. Using RNA sequencing, it is possible to analyze the impact of smoking on complex regulatory phenomena (e.g. alternative splicing, differential isoform usage) leading to a more detailed understanding of the biology underlying smoking-related disease. METHODS: We used whole-blood RNA sequencing to describe gene and exon-level expression differences between 229 current and 286 former smokers in the COPDGene study. We performed differential gene expression and differential exon usage analyses using the voom/limma and DEXseq R packages. Samples from current and former smokers were compared while controlling for age, gender, race, lifetime smoke exposure, cell counts, and technical covariates. RESULTS: At an adjusted p-value <0.05, 171 genes were differentially expressed between current and former smokers. Differentially expressed genes included 7 long non-coding RNAs that have not been previously associated with smoking: LINC00599, LINC01362, LINC00824, LINC01624, RP11-563D10.1, RP11-98G13.1, AC004791.2. Secondary analysis of acute smoking (having smoked within 2-h) revealed 5 of the 171 smoking genes demonstrated an acute response above the baseline effect of chronic smoking. Exon-level analyses identified 9 exons from 8 genes with significant differential usage by smoking status, suggesting smoking-induced changes in isoform expression. CONCLUSIONS: Transcriptomic changes at the gene and exon levels from whole blood can refine our understanding of the molecular mechanisms underlying the response to smoking.

Endoplasmic Reticulum Stress and Chemokine Production in Cystic Fibrosis Airway Cells: Regulation by STAT3 Modulation.

Endoplasmic reticulum (ER) stress has been recognized to play an important role in chronic inflammatory diseases such as cystic fibrosis (CF), and targeting ER stress may be useful for alleviating damaging neutrophilic inflammation in CF airways. Cellular models were used in conjunction with data from a recent CF genome-wide association study (GWAS) meta-analysis to determine modulators of ER stress-mediated inflammation. Surprisingly, cells undergoing ER stress during inflammatory stimulation showed reduced interleukin 8 (IL-8) and CXCL1 secretion (P < .001). Neutralization of CXCL1 and IL-8 reduced neutrophil chemotaxis >50% to supernatants from IL-1ß-stimulated CF airway epithelial cells (P < .01). The clinical importance of these chemokines was validated by association of CXCL1 and IL8 polymorphisms with changes in lung disease severity in patients with CF (n = 6365; IL8, P = .001; CXCL1, P = .001), confirming that targeting these chemokine pathways could help improve lung disease. We determined that production of these chemokines was partially controlled by ER stress in a signal transducer and activator of transcription 3 (STAT3)-dependent manner, whereby ER stress inhibited STAT3 activation. Our findings support a role for CXCL1 and IL-8 in CF lung disease severity and identify STAT3 as a modulating pathway. Targeting these pathways may help improve health outcomes in CF.

Endosomal pH modulation by peptide-gold nanoparticle hybrids enables potent anti-inflammatory activity in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays a central role in the pathophysiology of many acute and chronic human inflammatory diseases, and pharmacological regulation of TLR responses is anticipated to be beneficial in many inflammatory conditions. Currently there are no specific TLR inhibitors in clinical use. To overcome this challenge, we have developed a nano-based TLR inhibitor (peptide-gold nanoparticle hybrids) that inhibits a broad spectrum of TLR responses. Through mechanistic studies, we established that specific peptide decorated-gold nanoparticles that display high cellular uptake in phagocytic immune cells modulate endosomal pH, leading to significant attenuation of signaling through multiple TLRs. Using a global transcriptomic approach, we defined the broad anti-inflammatory activity of the nanoparticle in human peripheral blood mononuclear cells. In vivo studies confirmed the beneficial immunomodulatory activity since treatment with the nanoparticle significantly reduced weight loss, improved the disease activity index, and ameliorated colonic inflammation in a murine model of intestinal inflammation. This work enhances our fundamental understanding of the role of peptide coatings on the nanoparticle surface in regulating innate immune signaling, and identifies specific peptide decorated nanoparticles that may represent a novel class of anti-inflammatory therapeutics for human inflammatory diseases.

Polymorphisms associated with expression of BPIFA1/BPIFB1 and lung disease severity in cystic fibrosis.

BPI fold containing family A, member 1 (BPIFA1) and BPIFB1 are putative innate immune molecules expressed in the upper airways. Because of their hypothesized roles in airway defense, these molecules may contribute to lung disease severity in cystic fibrosis (CF). We interrogated BPIFA1/BPIFB1 single-nucleotide polymorphisms in data from an association study of CF modifier genes and found an association of the G allele of rs1078761 with increased lung disease severity (P = 2.71 × 10(-4)). We hypothesized that the G allele of rs1078761 is associated with decreased expression of BPIFA1 and/or BPIFB1. Genome-wide lung gene expression and genotyping data from 1,111 individuals with lung disease, including 51 patients with CF, were tested for associations between genotype and BPIFA1 and BPIFB1 gene expression levels. Findings were validated by quantitative PCR in a subset of 77 individuals. Western blotting was used to measure BPIFA1 and BPIFB1 protein levels in 93 lung and 101 saliva samples. The G allele of rs1078761 was significantly associated with decreased mRNA levels of BPIFA1 (P = 4.08 × 10(-15)) and BPIFB1 (P = 0.0314). These findings were confirmed with quantitative PCR and Western blotting. We conclude that the G allele of rs1078761 may be detrimental to lung function in CF owing to decreased levels of BPIFA1 and BPIFB1.

Cell culture-induced aberrant methylation of the imprinted IG DMR in human lymphoblastoid cell lines.

DNA methylation patterns are often poorly conserved through cell culturing. To determine the effect of cell immortalization and culture on DNA methylation profiles, we analyzed methylation in the differentially methylated regions (DMR) of five imprinted domains: the intergenic (IG) DMR on chromosome 14q32; potassium voltage-gated channel, KQT-like subfamily, member 1, (KCNQ1); small nuclear ribonucleoprotein polypeptide N (SNRPN), mesoderm specific transcript homolog (MEST); and H19 in lymphoblastoid cell lines (LCLs). In the IG DMR we found an aberrant methylation pattern that was consistent through all the cell lines tested and significantly different from that of noncultured peripheral blood cells. Using a generalized linear mixed model to compare methylation profiles, we show that recently derived LCLs significantly differ from the CEPH LCLs. This implies a gradual cell-culture related deterioration of DNA methylation in the IG DMR with at least two steps that may be identified: loss of methylation at CG sites 1 and 8; and loss of allelic differences in DNA methylation. The IG DMR methylation profile also confirms the high level of clonality of the CEPH LCLs. We conclude that non-transformed primary cells may be less susceptible to epigenetic anomalies and therefore may provide a more accurate reflection of gene expression in vivo.