Project Summary Effective treatments for COPD are disappointingly limited because the subtypes of this complex disease are not well defined. COPD may be initiated by susceptibility of different cell types to pollutants, including epithelial cells, endothelial cells, fibroblasts, dendritic cells, T cells, or any other cell type. We have shown that primary human airway epithelial cells (HAECs) with an arginine (HAECArg ) compared with a proline ( HAECPro) at locus 72 of the p53 protein, express higher mucin levels and that the same is found in nasal brushings from humans. Single- cell RNA sequencing showed that differentiated HAECArg show increased numbers of mucous cells while HAECPro cultures show squamous cells. Therefore, genetic variants may cause susceptibility depending on the cell type and the type of injury that initiates the chronic inflammatory condition. To begin investigating this possibility, we generated whole genome genotyping and bulk RNA sequence data from HAECs of 185 individuals cultured as basal cells. We identified 1,014 genes that harbored at least one expression quantitative trait locus (eQTL) and 16 distinct loci containing known COPD-associated genetic variants including a region near MUTYH. Based on these findings, we propose to test the hypotheses that multiple genetic loci associated with COPD harbor eQTLs or protein QTLs that are active in airway epithelia exposed to cigarette smoke (CS). Colocalization analysis of these QTLs with COPD genome-wide association study (GWAS) results will identify airway-specific genes that initiate COPD development. Aim 1 will identify CS-responsive genes and pathways and link eQTLs and protein QTLs to COPD-associated genetic variants, with MUTYH being the first identified gene to investigate. Aim 2 will determine the role of the Tp53 variants in airway epithelial differentiation. Isogenic HAECArg and HAECPro cultures will be compared for cell types by single cell RNA sequencing and investigate whether the p53Arg or p53Pro variants facilitate a mucous or squamous differentiation process depending on culture conditions. Mechanisms underlying the p53Arg-mediated expression of mucin genes will be elucidated. These studies will help define pathways that characterize a specific COPD subtype and provide biomarkers for early diagnosis, patient stratification. The data from HAEC185 will also provide a resource for future studies on any chronic disease that originates from susceptible airway epithelial cells.