Department of Ophthalmology, Flinders University, Bedford Park, SA, 5042, Australia.
Adelaide Medical School and Robinson Research Institute, University of Adelaide, Adelaide, SA, 5042, Australia.
Exp Eye Res. 2021 Sep;210:108692. doi: 10.1016/j.exer.2021.108692. Epub 2021 Jul 3.
Fuchs' endothelial corneal dystrophy (FECD) is a progressive vision impairing disease caused by thickening of Descemet's membrane and gradual degeneration and loss of corneal endothelial cells. The aim of this study was to identify differentially expressed genes between FECD-affected and unaffected corneal endothelium to gain insight into the pathophysiological mechanisms underlying this disease. Microarray gene expression analysis was performed on total RNA from FECD-affected and unaffected corneal endothelium-Descemet's membrane (CE-DM) specimens using the Illumina HumanHT-12 v4.0 expression array. RNA from pools of FECD-affected (n = 3 per pool) and individual unaffected (n = 3) specimens was used for comparison. Altered expression of a sub-set of differentially expressed genes was validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in independent specimens. Bioinformatics analysis was performed using InnateDB to reveal functional relationships among the differentially expressed genes and molecular pathways involved in the disease. A total of 16,513 genes were found expressed in the corneal endothelium of which 142 genes were differentially expressed between FECD-affected and unaffected endothelium (log fold-change ≥1.5, corrected p-value ≤0.05). Most of the genes were up-regulated (126) and a small proportion down-regulated (16) in affected corneal endothelium. Of the twelve genes prioritised for validation, differential expression of 10 genes, including those ranked 57th and 81st by significance validated by qRT-PCR (8 up-regulated and 2 downregulated, corrected p ≤ 0.05), one gene showed a trend for up-regulation in affected endothelium, consistent with the microarray analysis and another was up-regulated in an independent study indicating robustness of the differential expression dataset. Bioinformatic analysis revealed significant over-representation of differentially expressed genes in extracellular matrix reorganisation, cellular remodelling, immune response, and inflammation. Network analysis showed functional inter-relatedness of the majority of the dysregulated genes and revealed known direct functional relationships between 20 of the genes; many of these genes have roles in macrophage differentiation, phagocytosis and inflammation. This is the second report of microarray gene expression analysis in FECD. This study revealed a set of highly dysregulated genes in the corneal endothelium in FECD. More than a third of the dysregulated genes in the disease have been discovered for the first time and thus are novel. The dysregulated genes strongly suggest the presence of phagocytic cells, most likely immune cells, and inflammation in corneal endothelium in the disease. This study provides a molecular framework for delineating the mechanisms underlying these cellular processes in FECD.
福斯曼角膜内皮营养不良症（FECD）是一种进行性视力损害疾病，由 Descemet 膜增厚和角膜内皮细胞逐渐变性和丧失引起。本研究的目的是鉴定 FECD 相关和不相关角膜内皮之间差异表达的基因，以深入了解该疾病的病理生理机制。使用 Illumina HumanHT-12 v4.0 表达谱对来自 FECD 相关和不相关角膜内皮-Descemet 膜（CE-DM）标本的总 RNA 进行了微阵列基因表达分析。使用来自 FECD 相关（每组 3 个样本）和个体不相关（每组 3 个样本）的 RNA 进行比较。在独立的标本中通过定量逆转录聚合酶链反应（qRT-PCR）验证了一组差异表达基因的表达变化。使用 InnateDB 进行生物信息学分析，以揭示差异表达基因之间的功能关系和疾病中涉及的分子途径。在角膜内皮中发现了总共 16513 个表达的基因，其中 142 个基因在 FECD 相关和不相关的内皮之间表达差异（对数倍数变化≥1.5，校正后的 p 值≤0.05）。大多数基因在受影响的角膜内皮中上调（126 个），一小部分下调（16 个）。在为验证而确定的 12 个基因中，10 个基因的差异表达通过 qRT-PCR 验证（8 个上调和 2 个下调，校正的 p 值≤0.05），其中包括排名第 57 位和第 81 位的基因，一个基因在受影响的内皮中表现出上调趋势，与微阵列分析一致，另一个基因在独立研究中上调，表明差异表达数据集的稳健性。生物信息学分析显示，差异表达基因在细胞外基质重组、细胞重塑、免疫反应和炎症中显著过表达。网络分析显示，大多数失调基因之间存在功能相关性，并揭示了 20 个基因之间的已知直接功能关系；这些基因中的许多基因在巨噬细胞分化、吞噬和炎症中发挥作用。这是 FECD 中第二次进行微阵列基因表达分析。本研究揭示了 FECD 中角膜内皮中一组高度失调的基因。疾病中超过三分之一的失调基因是首次发现，因此是新的。失调基因强烈表明疾病中存在吞噬细胞，很可能是免疫细胞和炎症。本研究为阐明 FECD 中这些细胞过程的机制提供了一个分子框架。
