Background: Systemic sclerosis (SSc) is a heterogeneous autoimmune disorder characterized by immune imbalance, fibrosis and vasculopathy. SSc etiology is complex, and requires the interplay of genetics and environmental factors, which is reflected in the epigenome. Accordingly, DNA methylation emerges as one of the most studied epigenetic marks, as gene expression levels are sensitive to changes in the methylome.

Objectives: We aimed to analyze DNA methylation changes in the largest SSc cohort to date employing a comprehensive and integrative approach in whole-blood samples.

Methods: An epigenome-wide association study was conducted in a cohort of 179 SSc patients and 241 healthy donors (HD). Transcription factor binding sites and GO term enrichment analysis was carried out with the results of the differential methylation analysis. Transcriptome analysis was performed with further integration with the methylation data.

Results: 525 differentially methylated positions (DMPs) were identified, 255 of them were hypomethylated and 270 hypermethylated. These DMPs were enriched within transcription factor binding sites (TFBSs) of immune related transcription factors, such as those belonging to the Runt family and CEBP (p-value=1x10 ^-12 ), a myeloid transcription factor involved in interleukin 6 signaling molecular network. GO Terms analysis also revealed an enrichment in T cell development molecular routes, as well as in pathways related with cell mobility. Gene expression analysis identified 2,530 genes as differentially expressed genes (DEGs). Upon integrating these results with methylation data, we uncovered 842 expression quantitative trait methylation (eQTM) associations, involving 361 DMPs and 553 DEGs. These eQTMs represent disease specific CpG-gene interactions and underscored the involvement of genes in the interferon pathway, along with other processes relevant to SSc, including fibrotic and vascular pathways. Furthermore, our results highlighted the significance of neutrophils and T cells in SSc pathogenesis. To illustrate this approach, the gene RUNX3 , which is a relevant transcription factor in plasmacytoid dendritic cell biology and SSc related fibrosis [1]. A DMP within its first exon was hypermethylated (FDR=3.21x10 ^-3 , Δβ=0.032), correlating with decreased levels of the RUNX3 gene (r=-0.32, FDR=2.23x10 ^-6 ). Furthermore, our analysis also reported an enrichment of DMPs within RUNX3 TFBS.

Conclusion: Our results have unveiled differences in DNA methylation between SSc patients and HD. Additionally, through an integrative approach we were able to report a significant association between DNA methylation impairment and gene expression levels, uncovering novel genes and molecular mechanisms involved in SSc inflammatory, vasculopathy and fibrotic pathways.

REFERENCES: [1] Affandi AJ, Carvalheiro T, Ottria A, et al (2019) Low RUNX3 expression alters dendritic cell function in patients with systemic sclerosis and contributes to enhanced fibrosis. Ann Rheum Dis 78:1249–1259.

Acknowledgements: We thank Sofia Vargas and Gema Robledo for their excellent technical assistance and all the patients and control donors for their essential collaboration. The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under the Grant Agreement Number 115565 (PRECISESADS project), resources of which are composed of financial contribution from the European Union’s Seventh Framework Program (FP7/2007–2013) and EFPIA companies’ in-kind contribution. This research was also funded by Redes de Investigación Cooperativa Orientadas a Resultados en Salud (RICORS) (RD21/0002/0039). M.A.H. is a recipient of a Miguel Servet fellowship (CP21/00132) from Instituto de Salud Carlos III. L.O.F. work was supported by a Juan de la Cierva Incorporacion fellowship (IJC2019-040746-I) funded by MCIN/AEI/10.13039/501100011033. J.M.L. is recipient of a PFIS fellowship (FI23/00231) from Instituto de Salud Carlos III. This research is part of the doctoral degree awarded to J.M.L., within the Biomedicine program from the University of Granada.

Disclosure of Interests: None declared.