Genetic Profiling of Orbital Fibroblasts from Patients with Graves' Orbitopathy

Abstract

Context: Graves' orbitopathy (GO) is an autoimmune disease that persists when immunosuppression is achieved. Orbital fibroblasts from GO patients display peculiar phenotypes even if not exposed to autoimmunity, possibly reflecting genetic or epigenetic mechanisms, which we investigated here.

Objective: We aimed to explore potential genetic or epigenetic differences using primary cultures of orbital fibroblasts from GO and control patients.

Methods: Cell proliferation, hyaluronic acid (HA) secretion, and HA synthases (HAS) were measured. Next-generation sequencing and gene expression analysis of the whole genome were performed, as well as real-time-PCR of selected genes and global DNA methylation assay on orbital fibroblasts from 6 patients with GO and 6 control patients from a referral center.

Results: Cell proliferation was higher in GO than in control fibroblasts. Likewise, HA in the cell medium was higher in GO fibroblasts. HAS-1 and HAS-2 did not differ between GO and control fibroblasts, whereas HAS-3 was more expressed in GO fibroblasts. No relevant gene variants were detected by whole-genome sequencing. However, 58 genes were found to be differentially expressed in GO compared with control fibroblasts, and RT-PCR confirmed the findings in 10 selected genes. We postulated that the differential gene expression was related to an epigenetic mechanism, reflecting diverse DNA methylation, which we therefore measured. In support of our hypothesis, global DNA methylation was significantly higher in GO fibroblasts.

Conclusions: We propose that, following an autoimmune insult, DNA methylation elicits differential gene expression and sustains the maintenance of GO.

Trial registration: ClinicalTrials.gov NCT04488406.

Keywords: Graves’ orbitopathy; genetics; thyroid eye disease.