TCF4 Triplet Repeat Expansion and Nuclear RNA Foci in Fuchs' Endothelial Corneal Dystrophy

Abstract

Purpose: 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.

Keywords: CTG18.1; Fuchs' endothelial corneal dystrophy; RNA nuclear foci; TCF4; triplet repeat expansion.

Figure 1

Nuclear RNA foci accumulate in corneal endothelial cells with CTG18.1 triplet repeat expansion in TCF4. (A) Fluorescence in situ hybridization with a (CAG)₆CA-5′ Texas red-labeled 2-O-methyl RNA 20-mers probe (Integrated DNA Technologies) on endothelial keratoplasty samples of FECD subjects with CTG18.1 triplet repeat expansion revealed punctate, nuclear CUG repeat RNA foci (red). These foci were absent in control tissue without the expansion. Affected FECD subjects shown here had CTG18.1 expansion with 1300 CTG repeats or 91 repeats, whereas the control endothelial sample had two short alleles with 12 and 14 CTG repeats at the locus. DNA was stained with DAPI (blue). Scale bar: 25 μm. (B) Preoperative in vivo specular microscopy of the central endothelium of the FECD subjects corresponding to endothelial samples examined by FISH in (A) revealed typical findings for the disorder, including increased cellular polymorphism (with loss of normal hexagonal pattern) and polymegathism (variation in cell size) with focal dark spots corresponding to corneal guttae (arrows) (left) and large dark areas corresponding to confluence of the corneal guttae and marked loss of endothelial cell density and grotesque morphology of remaining cells (right). Scale bar: 100 μm. (C) Fluorescence in situ hybridization on endothelial samples from two control corneas revealed punctate, nuclear RNA foci (red) and subsequent genotyping of the donor corneal tissue revealed expansion of CTG18.1 triplet repeat. The tissue harbored expanded alleles with either 100 CTG repeats or 87 CTG repeats. DNA was stained with DAPI (blue). Scale bar: 25 μm. (D) Postmortem specular microscopy of the central endothelium of corresponding donor corneas with nuclear RNA foci revealed either moderate diffuse polymorphism and polymegathism (left) or severe diffuse polymorphism and polymegathism (right). The presence of foci seen in these endothelial samples with morphological changes typical for the early stages of FECD indicate that the foci are present and may play a critical role early in the disease course. Scale bar: 100 μm. (E) Quantification of the percentage of endothelial cells with foci detected by RNA-FISH as in (A). Gray bars indicate eight endothelial keratoplasty samples from FECD subjects with CTG18.1 expansion and the two white bars indicate the donor cornea endothelial samples with the triplet repeat expansion. Bars represent SE. The number of CTG repeats at this locus is shown for each sample below the corresponding bar.

Figure 2

The RNA nuclear foci are specific for CTG18.1 expansion-mediated endothelial dysfunction. The RNA foci were absent in the endothelial sample from a subject with endothelial dysfunction without FECD that had two short alleles of 12 CTG repeats (left). Note the paucicellularity of the endothelium that is characteristic of endothelial specimens with corneal decompensation. The RNA foci were absent in an endothelial sample from an FECD subject without CTG18.1 repeat expansion with 12 and 17 repeats at locus (right).

Figure 3

Foci are sensitive to degradation with RNase. The nuclear foci were sensitive to degradation with RNase I but not DNase I treatment. Note chromatin digestion with DNase and loss of nuclear borders in middle panel. The DNA was stained with DAPI (blue). These results are consistent with these foci being composed primarily of RNA. Scale bar: 10 μm.

Figure 4

Expression of TCF4. (A) Gene schematic diagram of TCF4 showing 20 exons and relevant elements including intronic CTG18.1 locus and single nucleotide polymorphism rs613872 (NCBI Accession # NM_001083962.1). The CTG18.1 repeat locus reaches the threshold for instability with more than 37 CTG repeats. The gene regulatory basic helix-loop-helix is present in constitutive exon 18. (B) Semiquantitative RT-PCR expression of all exons of TCF4 in control corneal endothelial tissue. Control endothelial tissue was from an 18-year-old donor with two short alleles with 14 and 17 CTG repeats at locus. Similar expression results were seen using control endothelial samples from a 60-year-old donor as well as a 66-year-old donor (data not shown). (C) Bar diagram depicting qPCR analysis of total TCF4 mRNA (control, n = 5; FECD, n = 5). Demographic information of the FECD subjects and control corneas is shown in Table 2. The mean age of the FECD subjects was 63.0 years and the mean age of the donors was 60.6 years (P = 0.70). The qPCR analysis of endothelial samples from patients with FECD compared with control endothelial samples reveals no significant fold change in total TCF4 mRNA level. Bars represent SE. Details of primers are included in Supplementary Table S1.