Genetic burden of dysregulated cytoskeletal organisation in the pathogenesis of pulmonary fibrosis

Background Pulmonary fibrosis (PF) is a shared characteristic of chronic interstitial lung diseases of mixed aetiology. Previous studies on PF highlight a pathogenic role for common and rare genetic variants. This study aimed to identify rare pathogenic variants that are enriched in distinct biological pathways and dysregulated gene expression. Methods Rare variants were identified using whole genome sequencing (WGS) from two independent PF cohorts, the PROFILE study and the Genomics England 100K (GE100KGP) cohort, with the gnomAD database as a reference. Four pathogenic variant categories were defined: loss of function variants, missense variants, protein altering variants, and protein truncating variants. Gene burden testing was performed for rare variants defined as having a minor allele frequency <0.1%. Overrepresentation analysis of gene ontology terms and gene concept network analysis were used to interpret functional pathways. Integration of publicly available transcriptomic datasets was performed using weighted gene co-expression network analysis of idiopathic pulmonary fibrosis (IPF) lung tissue compared with healthy controls. Results Burden testing was performed on 507 patients from the PROFILE study and 451 PF patients from GE100KGP cohort, compared with 76,156 control participants from the gnomAD database. Ninety genes containing significantly more pathogenic rare variants in cases than in controls were observed in both cohorts. Fifty-six genes included missense variants and 87 genes included protein altering variants. For missense variants, HMCN1 , encoding hemicentin-1, and RGPD1 , encoding a protein with a RanBD1 domain, were highly associated with PF in both PROFILE (p=5.70E-22 and p=4.48E-51, respectively) and GE100KGP cohorts (p=2.27E-24 and p=1.59E-36, respectively). 56 of 90 genes with significant burden were observed within modules correlated with disease in transcriptomic analysis, including HMCN1 and RGPD1 . Enriched functional categories from genetic and transcriptomic analyses included pathways involving extracellular matrix constituents, cell adhesion properties and microtubule organisation. Conclusions Rare pathogenic variant burden testing and weighted gene co-expression network analysis of transcriptomic data provided complementary evidence for pathways regulating cytoskeletal dynamics in PF pathogenesis. Functional validation of candidates could provide novel targets for intervention strategies. ### Competing Interest Statement LVW receives research funding from Orion Pharma, GSK, and Genentech, consulting fees from Galapagos, Boehringer Ingelheim, and GSK, and travel support from Genentech, has research collaboration with AstraZeneca, Nordic Bioscience, and Sysmex (OGT), and serves on advisory board for Galapagos. TMM, via his institution, has received industry-academic funding from Astra Zeneca and GlaxoSmithKline R and D; and consultancy or speaker fees from Abbvie, Amgen, Astra Zeneca, Bayer, Boehringer Ingelheim, BMS, CSL Behring, Endeavor, Fibrogen, Galapagos, Galecto, GlaxoSmithKline, IQVIA, Merck, Pliant, Pfizer, Qureight, Roche, Sanofi-Aventis, Structure Therapeutics, Trevi and Vicore. He is supported by an NIHR Clinician Scientist Fellowship (NIHR Ref: CS-2013-13-017) British Lung Foundation Chair in Respiratory Research (C17-3). RGJ received grant funding to institution from AstraZeneca, Biogen, Galecto, GlaxoSmithKline, Nordic Bioscience, RedX, and Pliant Therapeutics, consulting fees from AstraZeneca, Brainomix, Bristol Myers Squibb, Chiesi, Cohbar, Daewoong, GlaxoSmithKline, Veracyte, Resolution Therapeutics, and Pliant Therapeutics, payments or honoraria from Boehringer Ingelheim, Chiesi, Roche, PatientMPower, and AstraZeneca, payment for expert testimony from Pinsent Masons LLP, is the president of Action for Pulmonary Fibrosis and in the leadership of NuMedii.