• epigenetic
• genome
• graves orbitopathy
• methyolome

Context. Graves’ orbitopathy (GO) is an autoimmune disease persisting when immunosuppression is achieved. Orbital fibroblasts from GO patients display peculiar phenotypes even if not exposed to autoimmunity, possibly reflecting genetic or epigenetic mechanisms, furthermore the extent to which mononuclear cells and TSH-receptor autoantibodies (TRAb) contribute to Graves’ orbitopathy is not completely defined. Design. The design entailed the use of primary cultures of orbital fibroblasts from GO and control patients in order to understand any genetic or epigenetic mechanism involved in the development of the disease, and the investigation about the relationship between the immunohistochemical phenotype of orbital infiltrating cells and GO features in a large number of patients.
Methods To understand the genetic and/or epigenetic mechanism underlying the GO peculiar phenotypes of fibroblasts, 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 (RT-PCR) of selected genes and global DNA methylation assay.
To assess the relationship between the immunohistochemical phenotype and GO feature, we conducted an observational cohort study in 76 consecutive patients with GO (16 men and 60 women) who underwent orbital decompression over a period of 18 consecutive months. An ophthalmological evaluation was performed in all patients, as well as immunohistochemistry for CD3, CD4, CD8, CD56 (T-cell markers), CD25 (T and B-cell marker), CD20, CD19 (B-cell markers), and CD138 (plasmacell marker) in specimens collected at decompressive surgery.
Main Outcome Measure. Exome sequencing, gene expression of the whole genome, methylome analysis and immunohistochemical assay. 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 ten 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.
Regarding the immunohistochemical assay, having established cutoff values for each marker, cell infiltrates were found in 60 patients (78.9%; CD3: 39.4%, CD4 55.2%, CD8 50%, CD56: 0%, CD25: 28.9%, CD20: 51.3%, CD19: 25%, CD138: 26.3%). Eleven (14.4%) stained exclusively for CD138 (plasmacells). Patients with CD4-positive mononuclear cells had a significantly greater GO clinical activity score (CAS) (mean difference 1.07, 95% CI − 0.33 to − 1.82, P = 0.004 by univariate, P = 0.05 by multivariate analysis). CAS as well as the remaining GO features were not affected significantly by the mononuclear cell subpopulations in multivariate analyses.
Conclusions. We propose that, following an autoimmune insult, DNA methylation elicits differential gene expression and sustains the maintenance of GO.
From the immunohistochemical point of view mononuclear cell infiltrates are present in the majority of GO patients, with a small percentage represented exclusively by plasmacells. CD4 cells exert a major role on GO activity. These findings may represent a further advancement in the comprehension of GO pathogenesis.