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BackgroundGraves’ orbitopathy (GO) is an eye disease occurring in patients with autoimmune thyroid disorders (AITD), most commonly Graves´ disease. It is characterized by inflammation affecting soft tissues of the orbit. A recent study demonstrated an association between fibroblast hypomethylation and disease activity in GO (Virakul et al., Front Endocrinol, 2021). Because procurement of fibroblast from GO patients require an invasive sampling, we wondered whether analysis of global DNA methylation in circulating immune cells obtained from peripheral blood could contribute to early detection of GO from patients with AITD.ObjectivesTo compare global DNA methylation pattern in circulating immune cells obtained from AITD patients with GO and without GO history and healthy controls.MethodsGlobal DNA methylation was quantified in circulating immune cell populations by flow cytometry using 5-methylcytosine antibody in patients with GO (n=10), AITD without GO history (n=9) and healthy controls (n=8). Immune populations (CD4+ and CD8+ T cells, B cells, monocytes and CD56dim/bright NK cells) and their activation status were identified using CD3/4/8/14/16/19/25/45/56/69 antibodies.ResultsIn patients with GO, global DNA methylation was reduced by ~50% in the activated (CD25+) CD8+ T cells and by ~35% in the whole CD8+ T cell population compared to patients with AITD (p=0.006). Moreover, percentage of CD8+ T cells, but not activated subpopulation, was higher in GO when compared to AITD (p=0.04). Hypomethylation by ~20% was detected in monocytes as well as in CD56dim NK cells and their activated (CD69+) subpopulation when GO was compared with AITD (p≤0.02). Of these cell populations, percentage of monocytes was also higher in GO when compared to AITD (p=0.04). Global methylation in B cells, CD4+ T cells and CD56bright NK cells did not differ between patients with GO and patients with AITD (p>0.05). Of these populations, higher percentage of B cells was detected in GO when compared to AITD group (p=0.02). Analysis of larger patient cohorts is in progress with particular emphasis on the relationship of methylation patterns to GO disease activity.ConclusionThis is the first study identifying the different global methylation profile of circulating immune cells in patients with GO characterized by DNA hypomethylation in CD8+ T cells, CD56dim NK cells and monocytes compared to patients with AITD. Our study nominates hypomethylation as a non-invasive biomarker of GO and should be validated in a larger cohort of patients.AcknowledgementsMH CZ NU21J-01-00017, IGA_LF_2022_11, MH CZ – DRO (FNOL, 00098892), CZ.02.2.69/0.0/0.0/19_073/0016713Disclosure of InterestsNone declared