目的:TCF4内含子CTG18.1三联体重复位点的扩展对欧亚人群中Fuchs内皮角膜营养不良(FECD)的发展具有重要的风险,但是扩大的重复导致内皮退化的机制迄今尚不清楚。这项研究的目的是检查FECD内皮样本是否存在RNA核灶,有毒RNA的标志,以及TCF4单倍体不足的证据。
方法:使用荧光原位杂交,我们检测了CTG18.1扩增的FECD受试者角膜内皮样本中是否存在含有扩增的CUG转录本的核RNA灶.我们还通过定量实时PCR检查了TCF4表达水平的任何变化。
结果:在包含CTG18.1扩增的FECD受试者(n=8)的内皮样本中发现了许多离散的核RNA病灶,但在缺乏扩张的对照组中没有(n=5)(P=7.8×10(-4))。扩增阳性内皮样品中具有病灶的细胞的百分比范围为33%至88%。来自没有CTG18.1扩增的FECD受试者和没有FECD的具有内皮功能障碍的受试者的内皮样品中不存在RNA病灶。编码基本螺旋-环-螺旋结构域的组成型TCF4外显子的表达随着CTG18.1扩增而未改变。
结论:我们的研究结果表明,RNA核病灶是CTG18.1扩增介导的内皮疾病的病理标志。以前仅在由重复扩增引起的罕见神经退行性疾病中发现了RNA核病灶。我们在FECD中检测到丰富的核糖核酸灶,这暗示了毒性RNA在这种常见疾病中的作用。
OBJECTIVE: Expansion of the intronic
CTG18.1 triplet repeat locus within TCF4 contributes significant risk to the development of Fuchs\' endothelial corneal dystrophy (FECD) in Eurasian populations, but the mechanisms by which the expanded repeats result in degeneration of the endothelium have been hitherto unknown. The purpose of this study was to examine FECD endothelial samples for the presence of RNA nuclear foci, the hallmark of toxic RNA, as well as evidence of haploinsufficiency of TCF4.
METHODS: Using fluorescence in situ hybridization, we examined for the presence of nuclear RNA foci containing expanded CUG transcripts in corneal endothelial samples from FECD subjects with
CTG18.1 expansion. We also examined for any changes in expression levels of TCF4 by quantitative real-time PCR.
RESULTS: Numerous discrete nuclear RNA foci were identified in endothelial samples of FECD subjects (n = 8) harboring the
CTG18.1 expansion, but not in controls lacking the expansion (n = 5) (P = 7.8 × 10(-4)). Percentage of cells with foci in expansion-positive endothelial samples ranged from 33% to 88%. RNA foci were absent in endothelial samples from an FECD subject without
CTG18.1 expansion and a subject with endothelial dysfunction without FECD. Expression of the constitutive TCF4 exon encoding the basic helix-loop-helix domain was unaltered with CTG18.1 expansion.
CONCLUSIONS: Our findings suggest that the RNA nuclear foci are pathognomonic for
CTG18.1 expansion-mediated endothelial disease. The RNA nuclear foci have been previously found only in rare neurodegenerative disorders caused by repeat expansions. Our detection of abundant ribonuclear foci in FECD implicates a role for toxic RNA in this common disease.