甲状腺中的心脏钙结合蛋白基因（CASQ2）在格雷夫斯眼病患者中上调——支持自身免疫针对钙结合蛋白作为触发事件的作用。

The cardiac calsequestrin gene (CASQ2) is up-regulated in the thyroid in patients with Graves' ophthalmopathy--support for a role of autoimmunity against calsequestrin as the triggering event.

机构信息

Department of Medicine, Nepean Clinical School, The University of Sydney, Penrith, NSW, Australia.

出版信息

Clin Endocrinol (Oxf). 2010 Oct;73(4):522-8. doi: 10.1111/j.1365-2265.2009.03753.x.

DOI:10.1111/j.1365-2265.2009.03753.x

PMID:20039900

Abstract

BACKGROUND

Graves' Ophthalmopathy (GO) is a complex eye and orbital disorder that is uniquely linked to Graves' Hyperthyroidism (GH) and has traditionally been considered a cross-reactive immune response against the thyroid stimulating hormone receptor (TSHR) in orbital tissue. However, because there is no direct evidence, such as specific TSHR antibodies or T lymphocytes targeting the orbital tissues in patients with GO compared to those without eye disease, it is important to consider alternative hypotheses for the pathogenesis of GO. The aim of this study was to identify differentially expressed genes within the thyroid of patients with GO and GH as a possible explanation for a thyroid initiated orbital autoimmunity.

METHODS

RNA was extracted from thyroid glands of patients with GO (n = 10) and GH (n = 8) post-total thyroidectomy. RNA samples were arrayed on Illumina® Human Ref-8 Expression BeadChips™ representing 20,589 genes. Microarray results of selected genes were validated by quantitative PCR (qPCR) and levels of protein translation measured by Western blot analysis.

FINDINGS

Two hundred and ninety-five genes were differentially expressed between patients with GO and GH. Of these, the cardiac calsequestrin gene (CASQ2) was the most highly expressed gene in GO (2.2-fold increase, P < 0.05). The succinate dehydrogenase flavoprotein subunit gene (SDHA) was also significantly up-regulated in GO (P < 0.05), 1.4-fold, while genes encoding the thyroid antigens thyroglobulin, thyroid peroxidase and TSHR were not differentially expressed. qPCR verified up-regulation of CASQ2 and down-regulation BMP7, CD80, IGFBP5, and MYD88 genes in GO. Western blot analysis showed that the average CASQ/GAPDH protein expression ratios for GH and GO were 1.04 and 1.03, respectively. t-Test analysis of data generated a P-value of 0.26, therefore no significant difference was found for CASQ protein expression in thyroid tissue between GH and GO.

INTERPRETATION

The skeletal and cardiac calsequestrin proteins share 68.4% amino acid homology. Previous work has shown that RNA levels of skeletal muscle calsequestrin are 4.7 times higher in extraocular muscle (EOM) than in masticatory skeletal muscle (jaw), and cardiac calsequestrin is expressed 2.7 times more in EOM. We postulate that up-regulation of casq2 gene in the thyroid of patients with GH may lead to the production of autoantibodies and sensitized T-lymphocytes, which cross-react with calsequestrin in the EOM of patients who develop ophthalmopathy.

摘要

背景

格雷夫斯眼病（GO）是一种复杂的眼部和眼眶疾病，与格雷夫斯甲状腺功能亢进症（GH）密切相关，传统上被认为是针对眼眶组织中促甲状腺激素受体（TSHR）的交叉反应性免疫反应。然而，由于没有直接证据，例如与没有眼病的患者相比，GO 患者的眼眶组织中存在针对 TSHR 的特异性 TSHR 抗体或 T 淋巴细胞，因此需要考虑 GO 发病机制的替代假说。本研究的目的是鉴定 GO 和 GH 患者甲状腺中的差异表达基因，以解释甲状腺起始的眼眶自身免疫。

方法

从甲状腺全切术后的 GO（n=10）和 GH（n=8）患者的甲状腺中提取 RNA。RNA 样本在 Illumina®Human Ref-8 Expression BeadChips™上进行排列，代表 20,589 个基因。通过定量 PCR（qPCR）验证所选基因的微阵列结果，并通过 Western blot 分析测量蛋白翻译水平。

结果

GO 和 GH 患者之间有 295 个基因表达差异。其中，心脏钙结合蛋白 2 基因（CASQ2）在 GO 中表达最高（增加 2.2 倍，P<0.05）。琥珀酸脱氢酶黄素蛋白亚基基因（SDHA）在 GO 中也明显上调（P<0.05），上调 1.4 倍，而甲状腺抗原甲状腺球蛋白、甲状腺过氧化物酶和 TSHR 的基因表达无差异。qPCR 验证了 GO 中 CASQ2 的上调和 BMP7、CD80、IGFBP5 和 MYD88 基因的下调。Western blot 分析显示，GH 和 GO 的 CASQ/GAPDH 蛋白表达比值分别为 1.04 和 1.03。t 检验分析数据得出 P 值为 0.26，因此 GH 和 GO 甲状腺组织中的 CASQ 蛋白表达无显著差异。

解释

骨骼和心脏钙结合蛋白具有 68.4%的氨基酸同源性。先前的工作表明，眼外肌（EOM）中的骨骼肌钙结合蛋白 RNA 水平比咀嚼骨骼肌（颌）高 4.7 倍，而 EOM 中心脏钙结合蛋白的表达高 2.7 倍。我们假设 GH 患者甲状腺中 casq2 基因的上调可能导致自身抗体和致敏 T 淋巴细胞的产生，这些抗体和致敏 T 淋巴细胞与发生眼病的患者的 EOM 中的钙结合蛋白发生交叉反应。