Cooper, C.B.

Abstract Introduction Resilient smokers, who maintain respiratory health despite smoking, remain poorly characterized. Airway epithelial cell gene expression in resilient smokers can provide insight into mechanisms and pathways that may protect smokers from developing smoking-related lung disease. The objective of this study is to identify genes and potential pathways associated with lung-related resilience. Methods Resilient smokers were defined based on a previously published multi-dimensional definition which includes the absence of: 1) spirometric airflow limitation, 2) symptoms (cough/sputum, dyspnea), 3) radiographic (HRCT) abnormalities, 4) exacerbations, and 5) an accelerated FEV1 decline. “Non-resilient” smokers had preserved spirometry but showed abnormalities in at least one domain of resilience. We analyzed airway epithelial brush samples from 21 resilient and 34 non-resilient smokers from SPIROMICS. Differential gene expression was performed using DESeq2 package in R, which estimates differential expression using generalized linear models. We adjusted for sequencing batch, age (continuous), race, gender, and current smoking status. While controlling for these covariates we evaluated differential gene expression between resilient and non-resilient smokers, the interaction between resilience and smoking status, and in each sub-category of resilient (absence of symptoms, radiographic abnormalities, exacerbations, and accelerated decline in FEV1). Results We identified 98 genes that were differentially expressed (FDR ≤0.1) when comparing resilient to non-resilient smokers, 20 of those genes were up regulated in resilient smokers (Figure 1). Notable genes included cadherin-12 (CDH12), aspartate beta-hydroxylase (ASPH), and protein kinase AMP-activated alpha 2 (PRKAA2). CDH12 is involved in cell adhesion and epithelial integrity. ASPH influences cell adhesion and signaling. PRKAA2 plays role in cellular homeostasis and stress response, which may be altered in non-resilient smokers, affecting cellular metabolism and inflammation. BPI folding containing family A member (BPIFA1) was significantly associated with smoking status in the interaction analysis, suggesting its role in modulating the impact of smoking on resilience. BPIFA1 has anti-microbial properties, regulates immune responses, and contributes mucosal surface protection. Sub-category analysis identified 17 genes associated with resilience to symptoms and 2 genes related to physiologic resilience (FEV1 decline), though no significant genes were associated with radiographic or exacerbation resilience. Conclusion Differential gene expression of airway epithelial cells identified distinct expression patterns between resilient and non-resilient smokers. Specifically, genes associated with epithelial cell adhesion, signaling, and cellular homeostasis were up regulated in resilient smokers. These findings suggest that resilient smokers may have transcriptomic profiles and potential mechanisms that mitigate the effects of cigarette smoke.

Abstract Rationale: MUC5B and MUC5AC are neighboring genes on chromosome 11p15 that encode the two predominant mucins secreted in the respiratory airways. Increased airway mucin concentrations and an imbalance between these two mucins drive COPD symptoms and the development of airflow obstruction or COPD, but the epigenomic regulation of this loci has not been characterized in the airways. Methods: We isolated DNA from bronchial airway brushings of 72 participants with >20 pack-years smoking history from the Subpopulations and Intermediate Outcomes Measures in COPD Study (SPIROMICS). 36 participants had GOLD-based moderate-to-severe COPD (FEV1/FVC<0.70, FEV1<80%) and 36 were without or with mild COPD. We used the Illumina Infinium Methylation EPIC BeadChip Array to assess proportion of DNA methylation (β) spanning 689,937 CpG sites. We evaluated proportional DNA methylation (β) at 96 CpG loci within a 103kB region using matrixQTL for cis-expression quantitative trait methylation (eQTM: 10kB flanks relative to MUC5AC/MUC5B) integrating bulk RNA-seq data and mQTL models integrating gene sequencing data to identify DNA variation associated with proportion eQTM methylation. Models included age, sex, BMI, clinical site, current smoking vs past smoking, total pack-years, three ancestral components of ancestry, chipset, and position. Results: We identified a top eQTM, cg00524486, marginally associated with MUC5B expression (coefficient estimate=8.75, punadjusted=0.0014, pFDR=0.065, Figure 1B). This eQTM was associated with CT scan parametric response mapping-based functional small airways disease (PRM-fSAD, beta coefficient[β]=3.64, p=0.011). We tested for DNA variation associated with methylation of this top eQTM which identified two methylation quantitative trait loci (mQTL: rs885454 [β=0.044, minor allele frequency[MAF]=0.09, pFDR=0.01], rs868902 [β=0.020, MAF=0.43, pFDR=0.08, Figure 1A) associated with cg00524486 methylation, of which the more frequent mQTL, rs868902, was marginally associated with PRM-fSAD (p=0.07) in the larger SPIROMICS cohort with MUC5B DNA sequencing data. Conclusions: This preliminary multi-omic study of the lower airway epithelium demonstrates that the expression of MUC5B is epigenetically regulated by neighboring CpG probe methylation (eQTM) represented by DNA variation (mQTL). These studies require further confirmation in a larger dataset of airway samples and support a role for epigenetic variation as a biologic contributor and biomarker of early COPD pathogenesis.

Abstract Rationale Chronic obstructive pulmonary disease (COPD) manifests later in life and is a leading cause of death worldwide. While smoking-related lung pathology is a long-recognized COPD etiotype, emerging evidence suggests that early-life growth conditions also play an important role. A recently-developed index of early-life growth conditions – the difference between attained height and genotype-predicted height (height-GaP) – has been validated against prospectevely ascertained adverse early-life growth conditions. This study used height-GaP to evaluate the association of early-life growth conditions with COPD phenotype and prognosis later in life. Methods Among current and former smokers with spirometry-defined COPD in the SubPopulations and Intermediate Outcomes Measures in COPD Study (SPIROMICS), we computed height-GaP as the difference between attained height and genotype-predicted height. Regression models were fit for respiratory mortality (defined by primary diagnostic code), dyspnea prevalence (Medical Research Council dyspnea rating >1), chronic bronchitis prevalence, airflow obstruction severity as quantified by FEV1Q, and functional capacity (six-minute walk distance), adjusting for age, age2, sex, race-ethnicity, genotype-predicted height, cigarette smoking status and pack-years. Sensitivity analyses accounted for competing risks of death (Fine-Gray subdistribution hazard model) and accounted for later-life height loss (restricting sample to participants under 50 years). Results Among 1,258 participants with COPD (mean±SD age: 65.4±8.0 years, 42.4% female, mean±SD pack-years: 53.6±28.2, mean±SD FEV1%: 63.3±23.9%), there were 202 respiratory deaths over a median 7.5 years of follow-up (Figure). Larger height-GaP deficit was associated with higher respiratory mortality (adjusted hazard ratio [aHR]: 1.30 [95%CI: 1.12 to 1.50] per 1-SD increment in height-GaP deficit). This association remained significant after accounting for competing events (p=0.001) and when restricted to participants under 50 years (p=0.032). Larger height-GaP deficit was associated with higher dyspnea prevalence (adjusted prevalence ratio: 1.13 [95%CI: 1.02 to 1.25] per 1-SD increment in height-GaP deficit), lower FEV1Q (adjusted mean difference: -0.37 [95%CI: -0.45 to -0.29] per 1-SD height-GaP deficit), and shorter 6-minute walk (adjusted mean difference: -16.6 m [95%CI: -23.1 m to -10.0 m] per 1-SD increment in height-GaP deficit), but not chronic bronchitis or CAT score ≥10 (p>0.05 for each). Conclusions Among current and former smokers with COPD, a quantitative index of early-life conditions was associated with higher respiratory mortality, in addition to dyspnea, airflow obstruction severity and reduced functional capacity. These results suggest that early-life growth conditions may influence COPD phenotype and prognosis later in life.