Pos1030 dna-Methylation Profiling in the Blood of Patients With Rheumatoid Arthritis-Associated Interstitial Lung Disease

Abstract

BackgroundRheumatoid Arthritis-associated Interstitial Lung Disease (RA-ILD) is an extraarticular manifestation with clear clinical relevance because it impairs quality of life and survival. Despite this, RA-ILD etiology and pathogenesis are only partially understood. However, we know that fibrosis and collagen deposition, aging and telomere shortening, the expression of the mucin MUC5B gene, the RA autoantibodies and smoking are contributing elements. A better knowledge of RA-ILD pathogenesis will lead to improved outcomes for the patients.ObjectivesThis study aims to identify CpG sites that are differentially methylated in the blood of RA-ILD patients compared to matched RA patients without ILD. These findings will point to genes and pathways of specific relevance to RA-ILD pathogenesis.MethodsWe compared two matched groups of 32 patients with RA: RA patients diagnosed with ILD less than one year before sample collection; RA patients without ILD. The two groups were assessed by High-Resolution Computed Tomography (HRCT) and matched by sex, age, smoking (ever/never), and anti-CCP status. Blood samples were used for DNA extraction. Methylation of 850000 CpG was measured with the Infinium Methylation Epic BeadChip (Illumina). Identification of the differentially methylated positions (DMP) and differentially methylated regions (DMR) was performed using the R application ShinyÉPICO [1]. Gene set enrichment analysis (GSEA) was done with the R package methylGSA [2].ResultsA total of 6679 DMP with ≥ 2 % difference and FDR < 0.05 were identified in the autosomal chromosomes. In addition, 119 DMR (72 in gene bodies, 32 in promoters, and 15 in CpG Islands) were discovered. Some of these DMP y DMR are associated with genes of known relevance (Figure 1): genes of mucins (MUC6, MUC13) and collagens (COL9A3, COL21A1), genes involved in telomere maintenance (TERF, TERT1, POT1), and some genes related to immunological processes (HLA-DRA, VCAM1, TGFBR2…). A systematic analysis of the associated genes with GSEA showed significant enrichment of 62 gene ontology (GO) terms and 49 Reactome pathways. Several of the top enriched GO terms referred to the detection of chemical stimuli involved in sensory perception, either by smell or taste. This finding was replicated in the enriched pathways. Other top GO terms identified chromosome changes during mitosis: centromeric duplication, chromosome segregation, and others. They corresponded to top-enriched pathways involved in the M phase of mitosis and cell cycle checkpoints. Another group of GO terms referred to post-translational modifications of proteins: deacylation of proteins, which coincided with top pathways involved in chromatin modifications, including acetylation and deacetylation of histones; and ubiquitination of proteins, also reflected in several top enriched pathways. Besides, enriched GO terms that referred to the catabolic process of nuclear-transcribed mRNA correlated with top pathways of mRNA metabolism. In addition, there were other significantly enriched gene sets, such as the involved in the transport of intracellular vesicles and the location in the cell of proteins, mitochondria, RNA, and chromosomes, the host interaction with viral infections, gene silencing by small RNA, SUMOylation, and the RHO GTPase cycle.ConclusionWe observed significant differences in blood DNA methylation between matched RA-ILD and RA-control patients. Some of these differences were related to potentially interesting genes (mucins, collagen, telomere maintenance), and biological processes and pathways (detection of chemical stimuli, post-translational modifications of proteins, chromosome and intracellular vesicle localization, and transport) that will help us to better understand RA-ILD pathogenesis.References[1] Morante-Palacios O, Ballestar E. Bioinformatics 2021; 2: 257-259[2] Ren X, Kuan PF. Bioinformatics; 2019. 11: 1958-1959AcknowledgementsWe would like to thank other MARILD network authors: Yolanda Lopez-Golan, Ángeles Villaronga-Torres, Carmen Pena, Silvia Gomez-Sabater, Rocío Caño, María Martín López, Raúl Castellanos, Raquel López Mejías, Arkaitz Mucientes Ruiz and Susana Holgado.Funding for this study was provided by the Instituto de Salud Carlos III through grants PI20/01268, RD16/0012/0014 and RD21/0002/0003, co-funded by the European Union.Disclosure of InterestsNone Declared.