Department of Clinical and Experimental Medicine, Endocrinology Unit II, University of Pisa and University Hospital of Pisa, Pisa, Italy.
Department of Surgical, Medical and Molecular Pathology, ENT Unit I, University of Pisa and University Hospital of Pisa, Pisa, Italy.
J Clin Endocrinol Metab. 2021 Apr 23;106(5):e2176-e2190. doi: 10.1210/clinem/dgab035.
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.
We aimed to explore potential genetic or epigenetic differences using primary cultures of orbital fibroblasts from GO and control patients.
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.
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.
We propose that, following an autoimmune insult, DNA methylation elicits differential gene expression and sustains the maintenance of GO.
Graves 眼病（GO）是一种自身免疫性疾病，即使在免疫抑制后仍会持续存在。GO 患者的眼眶成纤维细胞即使未暴露于自身免疫中，也会表现出特殊的表型，这可能反映了遗传或表观遗传机制，我们在此进行了研究。
我们旨在使用来自 GO 和对照患者的眼眶成纤维细胞原代培养物来探索潜在的遗传或表观遗传差异。
测量细胞增殖、透明质酸（HA）分泌和 HA 合酶（HAS）。对整个基因组进行下一代测序和基因表达分析，并对来自 6 名 GO 患者和来自转诊中心的 6 名对照患者的眼眶成纤维细胞进行实时 PCR 和全基因组 DNA 甲基化检测。
GO 成纤维细胞的细胞增殖高于对照成纤维细胞。同样，GO 成纤维细胞培养物中的 HA 也较高。GO 和对照成纤维细胞中的 HAS-1 和 HAS-2 没有差异，而 HAS-3 在 GO 成纤维细胞中表达更高。全基因组测序未发现相关基因变异。然而，与对照成纤维细胞相比，GO 中发现了 58 个基因表达差异，并且 RT-PCR 证实了 10 个选定基因的发现。我们推测，差异基因表达与表观遗传机制有关，反映了不同的 DNA 甲基化，因此我们进行了测量。支持我们的假设，GO 成纤维细胞的总体 DNA 甲基化明显更高。
我们提出，在自身免疫攻击后，DNA 甲基化引发差异基因表达并维持 GO 的维持。
