Effect of trinucleotide repeat expansion on the expression of TCF4 mRNA in Fuchs' endothelial corneal dystrophy

Purpose: 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. Methods: 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. Results: 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. Conclusions: 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

Gene expression in the corneal endothelium of Fuchs endothelial corneal dystrophy patients with and without expansion of a trinucleotide repeat in TCF4.

Fuchs Endothelial Corneal Dystrophy (FECD) is a late onset, autosomal dominant eye disease that can lead to loss of vision. Expansion of a CTG trinucleotide repeat in the third intron of the transcription factor 4 (TCF4) gene is highly associated with FECD. However, only about 75% of FECD patients in the northern European population possess an expansion of this repeat. The remaining FECD cases appear to be associated with variants in other genes. To better understand the pathophysiology of this disease, we compared gene expression profiles of corneal endothelium from FECD patients with an expanded trinucleotide repeat (RE+) to those that do not have a repeat expansion (RE-). Comparative analysis of these two cohorts showed widespread RNA mis-splicing in RE+, but not in RE- samples. Quantitatively, we identified 39 genes in which expression was significantly different between RE+ and RE- samples. Examination of the mutation profiles in the RE- samples did not find any mutations in genes previously associated with FECD, but did reveal one sample with a rare variant of laminin subunit gamma 1 (LAMC1) and three samples with rare variants in the gene coding for the mitochondrial protein peripheral-type benzodiazepine receptor-associated protein 1 (TSPOAP1)

DNA sequence surrounding the trinucleotide repeat in the intron of the <i>TCF4</i> gene on chromosome 18.

<p>The trinucleotide repeat region is shown in red, and PCR primer sequences used for sizing the repeat region are underlined. This version of the sequence comes from the human reference sequence and contains 25 TGC repeats.</p

STR analysis of PCR amplicons of 3 normal control (A) and 3 FECD patients (B).

<p>The bottom panel is the analysis of a FECD patient with 2 expanded alleles.</p

Genomic Southern Blot of DNA samples from normal control (lanes 1, 4, 7 and 8) and FECD patients (lanes 2, 3, 5, 6, 9–11).

<p>Lanes 12 and 13 are laboratory control samples that have not been evaluated for FECD. Note that the samples in lanes 2, 3 and 6 are from FECD patients that do not have the repeat expansion. The samples in lanes 5, 9, and 10 are samples from FECD cases with repeat expansion over 1500 repeats. Lane L contains sizing standards.</p