Colocalization analysis of 3' UTR alternative polyadenylation quantitative trait loci reveals novel mechanisms underlying associations with lung function.

While many disease-associated single nucleotide polymorphisms (SNPs) are expression quantitative trait loci (eQTLs), a large proportion of genome-wide association study (GWAS) variants are of unknown function. Alternative polyadenylation (APA) plays an important role in posttranscriptional regulation by allowing genes to shorten or extend 3' untranslated regions (UTRs). We hypothesized that genetic variants that affect APA in lung tissue may lend insight into the function of respiratory associated GWAS loci. We generated alternative polyadenylation (apa) QTLs using RNA sequencing and whole genome sequencing on 1241 subjects from the Lung Tissue Research Consortium (LTRC) as part of the NHLBI TOPMed project. We identified 56 179 APA sites corresponding to 13 582 unique genes after filtering out APA sites with low usage. We found that a total of 8831 APA sites were associated with at least one SNP with q-value < 0.05. The genomic distribution of lead APA SNPs indicated that the majority are intronic variants (33%), followed by downstream gene variants (26%), 3' UTR variants (17%), and upstream gene variants (within 1 kb region upstream of transcriptional start site, 10%). APA sites in 193 genes colocalized with GWAS data for at least one phenotype. Genes containing the top APA sites associated with GWAS variants include membrane associated ring-CH-type finger 2 (MARCHF2), nectin cell adhesion molecule 2 (NECTIN2), and butyrophilin subfamily 3 member A2 (BTN3A2). Overall, these findings suggest that APA may be an important mechanism for genetic variants in lung function and chronic obstructive pulmonary disease (COPD).

Application of the ERS/ATS Spirometry Standards and Race-Neutral Equations in the COPDGene Study.

RATIONALE: The European Respiratory Society (ERS) and the American Thoracic Society (ATS) recommend using z-scores, and the ATS has recommended using Global Lung Initiative (GLI)- "Global" race-neutral reference equations for spirometry interpretation. However, these recommendations have been variably implemented and the impact has not been widely assessed, both in clinical and research settings. OBJECTIVES: We evaluated the ERS/ATS airflow obstruction severity classification. METHODS: In the COPDGene Study (n = 10,108), airflow obstruction has been defined as a forced expiratory volume in one second to forced vital capacity (FEV1/FVC) ratio <0.70, with spirometry severity graded from class 1 to 4 based on race-specific percent predicted (pp) FEV1 cut-points as recommended by the Global Initiative for Chronic Obstructive Lung Disease (GOLD). We compared the GOLD approach, using NHANES III race-specific equations, to the application of GLI-Global equations using the ERS/ATS definition of airflow obstruction as FEV1/FVC ratio < lower limit of normal (LLN) and z-FEV1 cut-points of -1.645, -2.5, and -4 ("zGLI Global"). We tested the four-tier severity scheme for association with COPD outcomes. MEASUREMENTS AND MAIN RESULTS: The lowest agreement between ERS/ATS with zGLI Global and the GOLD classification was observed in individuals with milder disease (56.9% and 42.5% in GOLD 1 and 2) and race was a major determinant of redistribution. After adjustment for relevant covariates, zGLI Global distinguished all-cause mortality risk between normal spirometry and the first grade of COPD (Hazard Ratio 1.23, 95% CI 1.04-1.44, p=0.014), and showed a linear increase in exacerbation rates with increasing disease severity, in comparison to GOLD. CONCLUSIONS: The zGLI Global severity classification outperformed GOLD in the discrimination of survival, exacerbations, and imaging characteristics.

Blood-based Transcriptomic and Proteomic Biomarkers of Emphysema.

Rationale: Emphysema is a chronic obstructive pulmonary disease phenotype with important prognostic implications. Identifying blood-based biomarkers of emphysema will facilitate early diagnosis and development of targeted therapies. Objectives: To discover blood omics biomarkers for chest computed tomography-quantified emphysema and develop predictive biomarker panels. Methods: Emphysema blood biomarker discovery was performed using differential gene expression, alternative splicing, and protein association analyses in a training sample of 2,370 COPDGene participants with available blood RNA sequencing, plasma proteomics, and clinical data. Internal validation was conducted in a COPDGene testing sample (n = 1,016), and external validation was done in the ECLIPSE study (n = 526). Because low body mass index (BMI) and emphysema often co-occur, we performed a mediation analysis to quantify the effect of BMI on gene and protein associations with emphysema. Elastic net models with bootstrapping were also developed in the training sample sequentially using clinical, blood cell proportions, RNA-sequencing, and proteomic biomarkers to predict quantitative emphysema. Model accuracy was assessed by the area under the receiver operating characteristic curves for subjects stratified into tertiles of emphysema severity. Measurements and Main Results: Totals of 3,829 genes, 942 isoforms, 260 exons, and 714 proteins were significantly associated with emphysema (false discovery rate, 5%) and yielded 11 biological pathways. Seventy-four percent of these genes and 62% of these proteins showed mediation by BMI. Our prediction models demonstrated reasonable predictive performance in both COPDGene and ECLIPSE. The highest-performing model used clinical, blood cell, and protein data (area under the receiver operating characteristic curve in COPDGene testing, 0.90; 95% confidence interval, 0.85-0.90). Conclusions: Blood transcriptome and proteome-wide analyses revealed key biological pathways of emphysema and enhanced the prediction of emphysema.

Lung Transcriptomics Links Emphysema to Barrier Dysfunction and Macrophage Subpopulations.

RATIONALE: While many studies have examined gene expression in lung tissue, the gene regulatory processes underlying emphysema are still not well understood. Finding efficient non-imaging screening methods and disease-modifying therapies has been challenging, but knowledge of the transcriptomic features of emphysema may help in this effort. OBJECTIVES: Our goals were to identify emphysema-associated biological pathways through transcriptomic analysis of bulk lung tissue, to determine the lung cell types in which these emphysema-associated pathways are altered, and to detect unique and overlapping transcriptomic signatures in blood and lung samples. METHODS: Using RNA-sequencing data from 446 samples in the Lung Tissue Research Consortium (LTRC) and 3,606 blood samples from the COPDGene study, we examined the transcriptomic features of chest computed tomography-quantified emphysema. We also leveraged publicly available lung single-cell RNA-sequencing data to identify cell types showing COPD-associated differential expression of the emphysema pathways found in the bulk analyses. MEASUREMENTS AND MAIN RESULTS: In the bulk lung RNA-seq analysis, 1,087 differentially expressed genes and 34 dysregulated pathways were significantly associated with emphysema. We observed alternative splicing of several genes and increased activity in pluripotency and cell barrier function pathways. Lung tissue and blood samples shared differentially expressed genes and biological pathways. Multiple lung cell types displayed dysregulation of epithelial barrier function pathways, and distinct pathway activities were observed among various macrophage subpopulations. CONCLUSIONS: This study identified emphysema-related changes in gene expression and alternative splicing, cell-type specific dysregulated pathways, and instances of shared pathway dysregulation between blood and lung.

Sex-biased Regulation of Extracellular Matrix Genes in COPD.

Compared to men, women often develop COPD at an earlier age with worse respiratory symptoms despite lower smoking exposure. However, most preventive, and therapeutic strategies ignore biological sex differences in COPD. Our goal was to better understand sex-specific gene regulatory processes in lung tissue and the molecular basis for sex differences in COPD onset and severity. We analyzed lung tissue gene expression and DNA methylation data from 747 individuals in the Lung Tissue Research Consortium (LTRC), and 85 individuals in an independent dataset. We identified sex differences in COPD-associated gene regulation using gene regulatory networks. We used linear regression to test for sex-biased associations of methylation with lung function, emphysema, smoking, and age. Analyzing gene regulatory networks in the control group, we identified that genes involved in the extracellular matrix (ECM) have higher transcriptional factor targeting in females than in males. However, this pattern is reversed in COPD, with males showing stronger regulatory targeting of ECM-related genes than females. Smoking exposure, age, lung function, and emphysema were all associated with sex-specific differential methylation of ECM-related genes. We identified sex-based gene regulatory patterns of ECM-related genes associated with lung function and emphysema. Multiple factors including epigenetics, smoking, aging, and cell heterogeneity influence sex-specific gene regulation in COPD. Our findings underscore the importance of considering sex as a key factor in disease susceptibility and severity.

Activation of CD8+ T Cells in Chronic Obstructive Pulmonary Disease Lung.

Rationale: Despite the importance of inflammation in chronic obstructive pulmonary disease (COPD), the immune cell landscape in the lung tissue of patients with mild-moderate disease has not been well characterized at the single-cell and molecular level. Objectives: To define the immune cell landscape in lung tissue from patients with mild-moderate COPD at single-cell resolution. Methods: We performed single-cell transcriptomic, proteomic, and T-cell receptor repertoire analyses on lung tissue from patients with mild-moderate COPD (n = 5, Global Initiative for Chronic Obstructive Lung Disease I or II), emphysema without airflow obstruction (n = 5), end-stage COPD (n = 2), control (n = 6), or donors (n = 4). We validated in an independent patient cohort (N = 929) and integrated with the Hhip+/- murine model of COPD. Measurements and Main Results: Mild-moderate COPD lungs have increased abundance of two CD8+ T cell subpopulations: cytotoxic KLRG1+TIGIT+CX3CR1+ TEMRA (T effector memory CD45RA+) cells, and DNAM-1+CCR5+ T resident memory (TRM) cells. These CD8+ T cells interact with myeloid and alveolar type II cells via IFNG and have hyperexpanded T-cell receptor clonotypes. In an independent cohort, the CD8+KLRG1+ TEMRA cells are increased in mild-moderate COPD lung compared with control or end-stage COPD lung. Human CD8+KLRG1+ TEMRA cells are similar to CD8+ T cells driving inflammation in an aging-related murine model of COPD. Conclusions: CD8+ TEMRA cells are increased in mild-moderate COPD lung and may contribute to inflammation that precedes severe disease. Further study of these CD8+ T cells may have therapeutic implications for preventing severe COPD.

Clonal Somatic Mutations in Chronic Lung Diseases Are Associated with Reduced Lung Function.

Rationale: Constantly exposed to the external environment and mutagens such as tobacco smoke, human lungs have one of the highest somatic mutation rates among all human organs. However, the relationship of these mutations to lung disease and function is not known. Objectives: To identify the prevalence and significance of clonal somatic mutations in chronic lung diseases. Methods: We analyzed the clonal somatic mutations from 1,251 samples of normal and diseased noncancerous lung tissue RNA sequencing with paired whole-genome sequencing from the Lung Tissue Research Consortium. We examined the associations of somatic mutations with lung function, disease status, and computationally deconvoluted cell types in two of the most common diseases represented in our dataset, chronic obstructive pulmonary disease (COPD; 29%) and idiopathic pulmonary fibrosis (IPF; 13%). Measurements and Main Results: Clonal somatic mutational burden was associated with reduced lung function in both COPD and IPF. We identified an increased prevalence of clonal somatic mutations in individuals with IPF compared with normal control subjects and individuals with COPD independent of age and smoking status. IPF clonal somatic mutations were enriched in disease-related and airway epithelial-expressed genes such as MUC5B in IPF. Patients who were MUC5B risk variant carriers had increased odds of developing somatic mutations of MUC5B that were explained by increased expression of MUC5B. Conclusions: Our identification of an increased prevalence of clonal somatic mutation in diseased lung that correlates with airway epithelial gene expression and disease severity highlights for the first time the role of somatic mutational processes in lung disease genetics.

Blood Gene Expression and Immune Cell Subtypes Associated with Chronic Obstructive Pulmonary Disease Exacerbations.

Rationale: Acute exacerbations of chronic obstructive pulmonary disease (AE-COPDs) are associated with a significant disease burden. Blood immune phenotyping may improve our understanding of a COPD endotype at increased risk of exacerbations. Objective: To determine the relationship between the transcriptome of circulating leukocytes and COPD exacerbations. Methods: Blood RNA sequencing data (n = 3,618) from the COPDGene (Genetic Epidemiology of COPD) study were analyzed. Blood microarray data (n = 646) from the ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints) study were used for validation. We tested the association between blood gene expression and AE-COPDs. We imputed the abundance of leukocyte subtypes and tested their association with prospective AE-COPDs. Flow cytometry was performed on blood in SPIROMICS (Subpopulations and Intermediate Outcomes in COPD Study) (n = 127), and activation markers for T cells were tested for association with prospective AE-COPDs. Measurements and Main Results: Exacerbations were reported 4,030 and 2,368 times during follow-up in COPDGene (5.3 ± 1.7 yr) and ECLIPSE (3 yr), respectively. We identified 890, 675, and 3,217 genes associated with a history of AE-COPDs, persistent exacerbations (at least one exacerbation per year), and prospective exacerbation rate, respectively. In COPDGene, the number of prospective exacerbations in patients with COPD (Global Initiative for Chronic Obstructive Lung Disease stage ⩾2) was negatively associated with circulating CD8+ T cells, CD4+ T cells, and resting natural killer cells. The negative association with naive CD4+ T cells was replicated in ECLIPSE. In the flow-cytometry study, an increase in CTLA4 on CD4+ T cells was positively associated with AE-COPDs. Conclusions: Individuals with COPD with lower circulating lymphocyte counts, particularly decreased CD4+ T cells, are more susceptible to AE-COPDs, including persistent exacerbations.

Causes of and Clinical Features Associated with Death in Tobacco Cigarette Users by Lung Function Impairment.

Rationale: Cigarette smoking contributes to the risk of death through different mechanisms. Objectives: To determine how causes of and clinical features associated with death vary in tobacco cigarette users by lung function impairment. Methods: We stratified current and former tobacco cigarette users enrolled in Genetic Epidemiology of Chronic Obstructive Pulmonary Disease (COPDGene) into normal spirometry, PRISm (Preserved Ratio Impaired Spirometry), Global Initiative for Chronic Obstructive Lung Disease (GOLD) 1-2 COPD, and GOLD 3-4 COPD. Deaths were identified via longitudinal follow-up and Social Security Death Index search. Causes of death were adjudicated after a review of death certificates, medical records, and next-of-kin interviews. We tested associations between baseline clinical variables and all-cause mortality using multivariable Cox proportional hazards models. Measurements and Main Results: Over a 10.1-year median follow-up, 2,200 deaths occurred among 10,132 participants (age 59.5 ± 9.0 yr; 46.6% women). Death from cardiovascular disease was most frequent in PRISm (31% of deaths). Lung cancer deaths were most frequent in GOLD 1-2 (18% of deaths vs. 9-11% in other groups). Respiratory deaths outpaced competing causes of death in GOLD 3-4, particularly when BODE index ⩾7. St. George's Respiratory Questionnaire score ⩾25 was associated with higher mortality in all groups: Hazard ratio (HR), 1.48 (1.20-1.84) normal spirometry; HR, 1.40 (1.05-1.87) PRISm; HR, 1.80 (1.49-2.17) GOLD 1-2; HR, 1.65 (1.26-2.17) GOLD 3-4. History of respiratory exacerbations was associated with higher mortality in GOLD 1-2 and GOLD 3-4, quantitative emphysema in GOLD 1-2, and airway wall thickness in PRISm and GOLD 3-4. Conclusions: Leading causes of death vary by lung function impairment in tobacco cigarette users. Worse respiratory-related quality of life is associated with all-cause mortality regardless of lung function.

Alternative poly-adenylation modulates α1-antitrypsin expression in chronic obstructive pulmonary disease.

α1-anti-trypsin (A1AT), encoded by SERPINA1, is a neutrophil elastase inhibitor that controls the inflammatory response in the lung. Severe A1AT deficiency increases risk for Chronic Obstructive Pulmonary Disease (COPD), however, the role of A1AT in COPD in non-deficient individuals is not well known. We identify a 2.1-fold increase (p = 2.5x10-6) in the use of a distal poly-adenylation site in primary lung tissue RNA-seq in 82 COPD cases when compared to 64 controls and replicate this in an independent study of 376 COPD and 267 controls. This alternative polyadenylation event involves two sites, a proximal and distal site, 61 and 1683 nucleotides downstream of the A1AT stop codon. To characterize this event, we measured the distal ratio in human primary tissue short read RNA-seq data and corroborated our results with long read RNA-seq data. Integrating these results with 3' end RNA-seq and nanoluciferase reporter assay experiments we show that use of the distal site yields mRNA transcripts with over 50-fold decreased translation efficiency and A1AT expression. We identified seven RNA binding proteins using enhanced CrossLinking and ImmunoPrecipitation precipitation (eCLIP) with one or more binding sites in the SERPINA1 3' UTR. We combined these data with measurements of the distal ratio in shRNA knockdown experiments, nuclear and cytoplasmic fractionation, and chemical RNA structure probing. We identify Quaking Homolog (QKI) as a modulator of SERPINA1 mRNA translation and confirm the role of QKI in SERPINA1 translation with luciferase reporter assays. Analysis of single-cell RNA-seq showed differences in the distribution of the SERPINA1 distal ratio among hepatocytes, macrophages, αß-Tcells and plasma cells in the liver. Alveolar Type 1,2, dendritic cells and macrophages also vary in their distal ratio in the lung. Our work reveals a complex post-transcriptional mechanism that regulates alternative polyadenylation and A1AT expression in COPD.

Polygenic risk scores identify heterogeneity in asthma and chronic obstructive pulmonary disease.

BACKGROUND: Asthma and chronic obstructive pulmonary disease (COPD) have distinct and overlapping genetic and clinical features. OBJECTIVE: We sought to test the hypothesis that polygenic risk scores (PRSs) for asthma (PRSAsthma) and spirometry (FEV1 and FEV1/forced vital capacity; PRSspiro) would demonstrate differential associations with asthma, COPD, and asthma-COPD overlap (ACO). METHODS: We developed and tested 2 asthma PRSs and applied the higher performing PRSAsthma and a previously published PRSspiro to research (Genetic Epidemiology of COPD study and Childhood Asthma Management Program, with spirometry) and electronic health record-based (Mass General Brigham Biobank and Genetic Epidemiology Research on Adult Health and Aging [GERA]) studies. We assessed the association of PRSs with COPD and asthma using modified random-effects and binary-effects meta-analyses, and ACO and asthma exacerbations in specific cohorts. Models were adjusted for confounders and genetic ancestry. RESULTS: In meta-analyses of 102,477 participants, the PRSAsthma (odds ratio [OR] per SD, 1.16 [95% CI, 1.14-1.19]) and PRSspiro (OR per SD, 1.19 [95% CI, 1.17-1.22]) both predicted asthma, whereas the PRSspiro predicted COPD (OR per SD, 1.25 [95% CI, 1.21-1.30]). However, results differed by cohort. The PRSspiro was not associated with COPD in GERA and Mass General Brigham Biobank. In the Genetic Epidemiology of COPD study, the PRSAsthma (OR per SD: Whites, 1.3; African Americans, 1.2) and PRSspiro (OR per SD: Whites, 2.2; African Americans, 1.6) were both associated with ACO. In GERA, the PRSAsthma was associated with asthma exacerbations (OR, 1.18) in Whites; the PRSspiro was associated with asthma exacerbations in White, LatinX, and East Asian participants. CONCLUSIONS: PRSs for asthma and spirometry are both associated with ACO and asthma exacerbations. Genetic prediction performance differs in research versus electronic health record-based cohorts.

Integrating Genetics, Transcriptomics, and Proteomics in Lung Tissue to Investigate Chronic Obstructive Pulmonary Disease.

The integration of transcriptomic and proteomic data from lung tissue with chronic obstructive pulmonary disease (COPD)-associated genetic variants could provide insight into the biological mechanisms of COPD. Here, we assessed associations between lung transcriptomics and proteomics with COPD in 98 subjects from the Lung Tissue Research Consortium. Low correlations between transcriptomics and proteomics were generally observed, but higher correlations were found for COPD-associated proteins. We integrated COPD risk SNPs or SNPs near COPD-associated proteins with lung transcripts and proteins to identify regulatory cis-quantitative trait loci (QTLs). Significant expression QTLs (eQTLs) and protein QTLs (pQTLs) were found regulating multiple COPD-associated biomarkers. We investigated mediated associations from significant pQTLs through transcripts to protein levels of COPD-associated proteins. We also attempted to identify colocalized effects between COPD genome-wide association studies and eQTL and pQTL signals. Evidence was found for colocalization between COPD genome-wide association study signals and a pQTL for RHOB and an eQTL for DSP. We applied weighted gene co-expression network analysis to find consensus COPD-associated network modules. Two network modules generated by consensus weighted gene co-expression network analysis were associated with COPD with a false discovery rate lower than 0.05. One network module is related to the catenin complex, and the other module is related to plasma membrane components. In summary, multiple cis-acting determinants of transcripts and proteins associated with COPD were identified. Colocalization analysis, mediation analysis, and correlation-based network analysis of multiple omics data may identify key genes and proteins that work together to influence COPD pathogenesis.

Heterogeneity and Progression of Chronic Obstructive Pulmonary Disease: Emphysema-Predominant and Non-Emphysema-Predominant Disease.

While variation in emphysema severity between patients with chronic obstructive pulmonary disease (COPD) is well-recognized, clinically applicable definitions of the emphysema-predominant disease (EPD) and non-emphysema-predominant disease (NEPD) subtypes have not been established. To study the clinical relevance of the EPD and NEPD subtypes, we tested the association of these subtypes with prospective decline in forced expiratory volume in 1 second (FEV1) and mortality among 3,427 subjects with Global Initiative for Chronic Obstructive Lung Disease (GOLD) spirometric grade 2-4 COPD at baseline in the Genetic Epidemiology of COPD (COPDGene) Study, an ongoing national multicenter study that started in 2007. NEPD was defined as airflow obstruction with less than 5% computed tomography (CT) quantitative densitometric emphysema at -950 Hounsfield units, and EPD was defined as airflow obstruction with 10% or greater CT emphysema. Mixed-effects models for FEV1 demonstrated larger average annual FEV1 loss in EPD subjects than in NEPD subjects (-10.2 mL/year; P < 0.001), and subtype-specific associations with FEV1 decline were identified. Cox proportional hazards models showed higher risk of mortality among EPD patients versus NEPD patients (hazard ratio = 1.46, 95% confidence interval: 1.34, 1.60; P < 0.001). To determine whether the NEPD/EPD dichotomy is captured by previously described COPDGene subtypes, we used logistic regression and receiver operating characteristic (ROC) curve analysis to predict NEPD/EPD membership using these previous subtype definitions. The analysis generally showed excellent discrimination, with areas under the ROC curve greater than 0.9. The NEPD and EPD COPD subtypes capture important aspects of COPD heterogeneity and are associated with different rates of disease progression and mortality.

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.

Plasma metabolomics and quantitative interstitial abnormalities in ever-smokers.

BACKGROUND: Quantitative interstitial abnormalities (QIA) are an automated computed tomography (CT) finding of early parenchymal lung disease, associated with worse lung function, reduced exercise capacity, increased respiratory symptoms, and death. The metabolomic perturbations associated with QIA are not well known. We sought to identify plasma metabolites associated with QIA in smokers. We also sought to identify shared and differentiating metabolomics features between QIA and emphysema, another smoking-related advanced radiographic abnormality. METHODS: In 928 former and current smokers in the Genetic Epidemiology of COPD cohort, we measured QIA and emphysema using an automated local density histogram method and generated metabolite profiles from plasma samples using liquid chromatography-mass spectrometry (Metabolon). We assessed the associations between metabolite levels and QIA using multivariable linear regression models adjusted for age, sex, body mass index, smoking status, pack-years, and inhaled corticosteroid use, at a Benjamini-Hochberg False Discovery Rate p-value of ≤ 0.05. Using multinomial regression models adjusted for these covariates, we assessed the associations between metabolite levels and the following CT phenotypes: QIA-predominant, emphysema-predominant, combined-predominant, and neither- predominant. Pathway enrichment analyses were performed using MetaboAnalyst. RESULTS: We found 85 metabolites significantly associated with QIA, with overrepresentation of the nicotinate and nicotinamide, histidine, starch and sucrose, pyrimidine, phosphatidylcholine, lysophospholipid, and sphingomyelin pathways. These included metabolites involved in inflammation and immune response, extracellular matrix remodeling, surfactant, and muscle cachexia. There were 75 metabolites significantly different between QIA-predominant and emphysema-predominant phenotypes, with overrepresentation of the phosphatidylethanolamine, nicotinate and nicotinamide, aminoacyl-tRNA, arginine, proline, alanine, aspartate, and glutamate pathways. CONCLUSIONS: Metabolomic correlates may lend insight to the biologic perturbations and pathways that underlie clinically meaningful quantitative CT measurements like QIA in smokers.

Alpha-1 Antitrypsin MZ Heterozygosity Is an Endotype of Chronic Obstructive Pulmonary Disease.

Rationale: Multiple studies have demonstrated an increased risk of chronic obstructive pulmonary disease (COPD) in heterozygous carriers of the AAT (alpha-1 antitrypsin) Z allele. However, it is not known if MZ subjects with COPD are phenotypically different from noncarriers (MM genotype) with COPD. Objectives: To assess if MZ subjects with COPD have different clinical features compared with MM subjects with COPD. Methods: Genotypes of SERPINA1 were ascertained by using whole-genome sequencing data in three independent studies. We compared outcomes between MM subjects with COPD and MZ subjects with COPD in each study and combined the results in a meta-analysis. We performed longitudinal and survival analyses to compare outcomes in MM and MZ subjects with COPD over time. Measurements and Main Results: We included 290 MZ subjects with COPD and 6,184 MM subjects with COPD across the three studies. MZ subjects had a lower FEV1% predicted and greater quantitative emphysema on chest computed tomography scans compared with MM subjects. In a meta-analysis, the FEV1 was 3.9% lower (95% confidence interval [CI], -6.55% to -1.26%) and emphysema (the percentage of lung attenuation areas <-950 HU) was 4.14% greater (95% CI, 1.44% to 6.84%) in MZ subjects. We found one gene, PGF (placental growth factor), to be differentially expressed in lung tissue from one study between MZ subjects and MM subjects. Conclusions: Carriers of the AAT Z allele (those who were MZ heterozygous) with COPD had lower lung function and more emphysema than MM subjects with COPD. Taken with the subtle differences in gene expression between the two groups, our findings suggest that MZ subjects represent an endotype of COPD.

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.

Identifying chronic obstructive pulmonary disease from integrative omics and clustering in lung tissue.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a highly morbid and heterogenous disease. While COPD is defined by spirometry, many COPD characteristics are seen in cigarette smokers with normal spirometry. The extent to which COPD and COPD heterogeneity is captured in omics of lung tissue is not known. METHODS: We clustered gene expression and methylation data in 78 lung tissue samples from former smokers with normal lung function or severe COPD. We applied two integrative omics clustering methods: (1) Similarity Network Fusion (SNF) and (2) Entropy-Based Consensus Clustering (ECC). RESULTS: SNF clusters were not significantly different by the percentage of COPD cases (48.8% vs. 68.6%, p = 0.13), though were different according to median forced expiratory volume in one second (FEV1) % predicted (82 vs. 31, p = 0.017). In contrast, the ECC clusters showed stronger evidence of separation by COPD case status (48.2% vs. 81.8%, p = 0.013) and similar stratification by median FEV1% predicted (82 vs. 30.5, p = 0.0059). ECC clusters using both gene expression and methylation were identical to the ECC clustering solution generated using methylation data alone. Both methods selected clusters with differentially expressed transcripts enriched for interleukin signaling and immunoregulatory interactions between lymphoid and non-lymphoid cells. CONCLUSIONS: Unsupervised clustering analysis from integrated gene expression and methylation data in lung tissue resulted in clusters with modest concordance with COPD, though were enriched in pathways potentially contributing to COPD-related pathology and heterogeneity.

Blood RNA sequencing shows overlapping gene expression across COPD phenotype domains.

RATIONALE: COPD can be assessed using multidimensional grading systems with components from three domains: pulmonary function tests, symptoms and systemic features. Clinically, measures may be used interchangeably, though it is not known if they share similar pathobiology. OBJECTIVE: To use RNA sequencing (RNA-seq) to determine if there is an overlap in the underlying biological mechanisms and consequences driving different components of the multidimensional grading systems. METHODS: Whole blood was collected for RNA-seq from current and former smokers in the Genetic Epidemiology of COPD study. We tested the overlap in gene expression and biological pathways associated with case-control status and quantitative COPD phenotypes within and between the three domains. RESULTS: In 2647 subjects, there were 3030 genes differentially expressed in any of the three domains or case-control status. There were five genes that overlapped between the three domains and case-control status, including G protein-coupled receptor 15(GPR15), sestrin 1 (SESN1) and interferon-induced guanylate-binding protein 1 (GBP1), which were associated with longitudinal decline in FEV1. The overlap between the three domains was enriched for pathways related to cellular components. CONCLUSIONS: We identified gene sets and pathways that overlap between 12 COPD-related phenotypes and case-control status. There were no pathways represented in the overlap between the three domains and case-control status, but we identified multiple genes that demonstrated a consistent pattern of expression across several of the phenotypes. Patterns of gene expression correlation were generally similar to the correlation of clinical phenotypes in the PFT and symptom domains but not the systemic features.

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.