Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
Department of Genomic Medical Sciences, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Invest Ophthalmol Vis Sci. 2019 Feb 1;60(2):779-786. doi: 10.1167/iovs.18-25760.
CTG trinucleotide repeat (TNR) expansion is frequently found in transcription factor 4 (TCF4) in Fuchs' endothelial corneal dystrophy (FECD), though the effect of TNR expansion on FECD pathophysiology remains unclear. The purpose of this study was to evaluate the effect of TNR expansion on TCF4 expression in corneal endothelium of patients with FECD.
Peripheral blood DNA and Descemet membrane with corneal endothelium were obtained from 203 German patients with FECD. The CTG TNR repeat length in TCF4 was determined by short tandem repeat (STR) assays and Southern blotting using genomic DNA. Genotyping of rs613872 in TCF4 was performed by PCR. TCF4 mRNA levels in corneal endothelium were evaluated by quantitative PCR using three different probes. Control corneal endothelial samples were obtained from 35 non-FECD subjects.
The STR assay and Southern blotting showed that 162 of the 203 patients with FECD (80%) harbored CTG trinucleotide repeat lengths larger than 50. Quantitative PCR using all three probes demonstrated that TCF4 mRNA is significantly upregulated in the corneal endothelium of patients with FECD, regardless of the presence of TNR expansion. However, the length of the TNR tended to show a positive correlation with TCF4 expression level. No correlation was shown between the genotype of TCF4 SNP, rs613872, and the level of TCF4 expression.
Our findings showed that TCF4 mRNA is upregulated in the corneal endothelium of patients with FECD. Further studies on the effects of TCF4 upregulation on corneal endothelial cell function will aid in understanding the pathophysiology of FECD.
在 Fuchs 内皮角膜营养不良（FECD）中，经常在转录因子 4（TCF4）中发现 CTG 三核苷酸重复（TNR）扩展，尽管 TNR 扩展对 FECD 病理生理学的影响尚不清楚。本研究的目的是评估 TNR 扩展对 FECD 患者角膜内皮中 TCF4 表达的影响。
从 203 名德国 FECD 患者中获得外周血 DNA 和带有角膜内皮的 Descemet 膜。使用基因组 DNA 通过短串联重复（STR）分析和 Southern 印迹测定 TCF4 中的 CTG TNR 重复长度。通过 PCR 对 TCF4 中的 rs613872 进行基因分型。使用三种不同的探针通过定量 PCR 评估角膜内皮中的 TCF4 mRNA 水平。从 35 名非 FECD 受试者中获得对照角膜内皮样本。
STR 分析和 Southern 印迹表明，203 名 FECD 患者中有 162 名（80%）患者的 CTG 三核苷酸重复长度大于 50。使用所有三种探针的定量 PCR 表明，无论 TNR 扩展是否存在，TCF4 mRNA 在 FECD 患者的角膜内皮中均显著上调。然而，TNR 的长度与 TCF4 表达水平呈正相关趋势。TCF4 SNP，rs613872 的基因型与 TCF4 表达水平之间没有相关性。
我们的发现表明，在 FECD 患者的角膜内皮中 TCF4 mRNA 上调。进一步研究 TCF4 上调对角膜内皮细胞功能的影响将有助于了解 FECD 的病理生理学。
