Shrivastav, A.K.

Abstract RATIONALE. Significant sex-differences exist in asthma across the lifespan. Asthma prevalence and severity are greater in pre-pubescent boys compared to girls but greater in women after puberty. In women, asthma may improve after menopause when sex hormones wane. To address potential mechanisms underlying these changes, we compared the airway transcriptomics signatures of post-menopausal vs. premenopausal women. METHODS. We used bulk RNA seq data from bronchial epithelial cells from three different cohorts (SARP III, GEO-GSE85567, GEO-GSE201955) to perform a comprehensive analysis of differential gene expression and regulation. Data were combined into a meta-analysis, to compare pre-menopausal (n=114 cases with asthma, 48 controls) to post-menopausal women (n=52 cases with asthma, 13 controls). Differential gene expression between pre- and postmenopausal women was analyzed separately for asthma (Figure 1A) and control groups (not shown). Significant differentially expressed genes (DEGs) were identified by contrasting effect sizes (log fold-changes) between asthma and control groups using a Z-score interaction test. Accordingly, a scatter plot (Figure 1B) was created to represent whether differential gene expression between pre- and post-menopausal conditions is modified by disease status (asthma vs. control). The scatter plot also highlights that these findings are related to asthma per. se. and not to normal aging and menopausal transition. Pathway enrichment analysis was conducted using the differential expression data, followed by differential regulation analysis of the identified pathways. Statistical significance was assessed using a false discovery rate (FDR) threshold of 5%. RESULTS. Out of 15,468 genes that passed quality control, 19 genes were differentially expressed in bronchial epithelial cells from pre- and post-menopausal women with asthma (as compared to controls) (Figure 1A). Of these, 5 genes were down-regulated (ITLN1, POSTN, FCRL3, CCL22, PCSK6), and 14 genes were up-regulated (SPP1, S100A9, A2ML1, TNFRSF10C, CSF3R, OLFM1, C1QL2, NLRP3, HAL, PI3, RNASE1, TMEM178B, TNNI3, RNF175) when comparing pre- and post-menopausal women with asthma (Figure 1A). Of these 19 DEGs, 14 were differentially expressed in pre- as compared to post-menopausal women with asthma, but not in controls (Figure 1B). Comparing pre- to post-menopausal women with asthma, biologic pathways related to the innate and adaptive immune system activation and mitochondrial function (Electron transport Chain oxidative phosphorylation and ATP production) were both differentially regulated and expressed. CONCLUSIONS. This analysis highlights several DEGs and pathways that characterize asthma pathogenesis when comparing pre- and post-menopausal women, and emphasize the role of female sex hormones on the immune system response and mitochondrial function in asthma pathogenesis.

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.