Genome-Wide Association Study: Functional Variant rs2076295 Regulates Desmoplakin Expression in Airway Epithelial Cells

Abstract

<b>Rationale:</b> Genetic association studies have identified rs2076295 in association with idiopathic pulmonary fibrosis (IPF). We hypothesized that rs2076295 is the functional variant regulating <i>DSP</i> (<i>desmoplakin</i>) expression in human bronchial epithelial cells, and <i>DSP</i> regulates extracellular matrix-related gene expression and cell migration, which is relevant to IPF development.<b>Objectives:</b> To determine whether rs2076295 regulates <i>DSP</i> expression and the function of DSP in airway epithelial cells.<b>Methods:</b> Using CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 editing (including regional deletion, indel, CRISPR interference, and single-base editing), we modified rs2076295 and measured <i>DSP</i> expression in edited 16HBE14o- and primary airway epithelial cells. Cellular integrity, migration, and genome-wide gene expression changes were examined in 16HBE14o- single colonies with <i>DSP</i> knockout. The expression of <i>DSP</i> and its relevant matrix genes was measured by quantitative PCR and also analyzed in single-cell RNA-sequencing data from control and IPF lungs.<b>Measurements and Main Results:</b><i>DSP</i> is expressed predominantly in bronchial and alveolar epithelial cells, with reduced expression in alveolar epithelial cells in IPF lungs. The deletion of the DNA region-spanning rs2076295 led to reduced expression of <i>DSP</i>, and the edited rs2076295GG 16HBE14o- line has lower expression of <i>DSP</i> than the rs2076295TT lines. Knockout of <i>DSP</i> in 16HBE14o- cells decreased transepithelial resistance but increased cell migration, with increased expression of extracellular matrix-related genes, including <i>MMP7</i> and <i>MMP9</i>. Silencing of <i>MMP7</i> and <i>MMP9</i> abolished increased migration in <i>DSP</i>-knockout cells.<b>Conclusions:</b> rs2076295 regulates <i>DSP</i> expression in human airway epithelial cells. The loss of <i>DSP</i> enhances extracellular matrix-related gene expression and promotes cell migration, which may contribute to the pathogenesis of IPF.