Abstract
Purpose :
Fuchs endothelial corneal dystrophy (FECD) is highly associated with an expansion of a CTG trinucleotide repeat in the transcription factor 4 (TCF4) gene. However, mutations in a variety of other genes (including COL8A2, SLC4A11, and ZEB1) have been found to cause FECD in some patients, and additional loci have been associated with the disease (LAMC1, KANK4 and others). To better understand how different mutations can lead to a common clinical phenotype, we compared gene expression profiles from the corneal endothelium of FECD patients with a repeat expansion (RE+) in TCF4 to those that did not have a repeat expansion (RE-).
Methods :
Corneal endothelium was removed at the time of keratoplasty from patients with FECD. Total RNA was extracted, and RNA libraries were prepared using TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold. Sequencing was performed on Illumina HiSeq instruments. Pairwise comparisons were performed between 18 RE+ FECD samples and 6 RE- samples using computational pipeline (MAP-RSeq) and Mixture of Isoforms (MISO) software packages to identify quantitative and qualitative changes in gene expression. TCF4 trinucleotide repeat length from leukocyte DNA was determined using a combination of GeneScan analysis and Southern blotting.
Results :
RE+ FECD samples demonstrated increased expression of 28 genes and decreased expression of 11 genes when compared to RE- samples. Pathway analysis of this set of genes did not reveal enrichment for any particular pathway. Splicing differences in MBNL1, NUMA1 and PPFIBP1 transcripts were identified in every pairwise comparison, and transcripts of 17 additional genes showed differential splicing in the majority of comparisons. Exome sequencing of the RE- samples did not identify likely causative mutations in SLC4A11, COL8A2, ZEB1, or TCF4, but one RE- sample did have a rare variant in the LAMC1 gene.
Conclusions :
Splicing patterns and differential expression of a number of genes differ between corneal endothelium isolated from FECD patients with and without a CTG trinucleotide repeat expansion in the TCF4 gene. This reinforces the premise that mis-splicing events seen in the RE+ samples are a direct consequence of the CTG repeat expansion at the TCF4 locus and that there are important biological differences among FECD genotypes.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.