May 01, 2020
Cytosine, thymine and guanine (CTG) trinucleotide repeat (TNR) expansion in an intron of the TCF4 gene is the most common genetic variant associated with Fuchs' endothelial corneal dystrophy (FECD). Several pathogenic mechanisms directly attributable to TNR expansion and common to other TNR diseases have been identified, yet a specific set of mechanisms responsible for pathological progression of FECD is unknown.
To understand events leading from TCF4 TNR expansion to disease phenotype, a team comprising Eric D. Wieben, Ph.D., with Biochemistry and Molecular Biology, Keith H. Baratz, M.D., with Ophthalmology, and Michael P. Fautsch, Ph.D., with the Ophthalmology Research Unit at Mayo Clinic in Rochester, Minnesota, characterized splicing, gene expression and exon sequence changes in a rare cohort of patients with TNR expansions but no phenotypic FECD (RE+/FECD-). Their study was published in Investigative Ophthalmology & Visual Science in 2019.
"One of the challenges of studying TNR expansion diseases is the difficulty in directly associating the varied mechanisms of action from progression to disease, says Dr. Wieben. "At Mayo Clinic, we've developed a large FECD and non-FECD tissue bank where we have characterized samples for TNR expansion within the corneal endothelium. In our database, we identified five patients ranging in age from 68-88 years whose TCF4 genes contain pathological expansions of CTG repeats, but the patients did not have FECD. This enabled us to characterize gene expression in this unique group of samples."
Comparison of RE+/FECD- data to data from patients with FECD and a TNR expansion (RE+/FECD+) revealed significant differences in splicing and gene expression profiles between the two groups. "We found that three genes — MBNL1, KIF13A and AKAP13 — that were previously identified as misspliced in RE+/FECD+ patients were normally spliced in RE+/FECD- samples," says Dr. Fautsch. "Additionally, gene expression analysis of RE+/FECD- patients identified several important signaling pathways that have been implicated in FECD."
Qualitative splicing differences
Previous studies on corneal endothelial tissue obtained from RE+/FECD+ patients have identified a set of 24 genes whose missplicing patterns are a signature of the disease. To determine if these missplicing events were also found in RE+/FECD- samples, researchers performed RNA sequencing and compared the transcriptome of RE+/FECD- samples to previously reported data sets of both RE+/FECD+ and non-FECD patients who did not have a TNR expansion (RE-/FECD-). Analysis of the missplicing events in samples from RE+/FECD- patients showed traits of both RE+/FECD+ and RE-/FECD-.
Analysis of missplicing in RE+/FECD- samples identified four genes whose percent spliced-in (PSI) values were within one standard deviation from the mean of RE+/FECD+ values. For the remaining 17 genes, the PSI values for at least one RE+/FECD- sample fell between those recorded for RE+/FECD+ and the RE-/FECD- samples.
Researchers also examined the impact of the TNR expansion on splicing in the TCF4 gene. "We had previously reported that the intron just upstream of the TNR expansion in TCF4 is preferentially retained in RE+/FECD+ samples, but absent in samples that lacked a TNR expansion," says Dr. Baratz. "Examining this region in the RE+/FECD- samples revealed that this intron is also retained, suggesting that while retention of this intron might be a reliable marker for identifying the presence of a TNR expansion, it is not a reliable marker for FECD status."
Effect of TNR expansion on gene expression
Researchers compared gene expression profiles between RE+/FECD+ and RE+/FECD- patients to identify genes that link TNR expansion to FECD pathophysiology. A total of 810 genes with at least a twofold higher expression in RE+/FECD+ compared with RE+/FECD- samples were identified, including SLC4A11, a gene that has previously been implicated in the pathogenesis of FECD.
Researchers also identified 1,372 genes that have more than a twofold lower expression in RE+/FECD+ samples when compared with RE+/FECD-. Notes Dr. Fautsch: "Analysis of this gene set revealed significant reduction for intracellular signaling pathways, including decreased expression of Toll-like receptor signaling, transforming growth factor-beta (TGFβ) superfamily members, and genes involved in TGFβ2 expression and activation. It also identified a number of downregulated molecules in the RE+/FECD+ group whose expression is associated with cell senescence."
Genetic variants in RE+/FECD-
Exome sequencing performed on leukocyte DNA obtained from the RE+/FECD- patients did not identify any genetic variants common to all five samples, but did identify 94 genes that had uncommon variants in at least two of the RE+/FECD- samples. Comparison of the exome sequencing results with those obtained by RNA sequencing showed that 64 of the 94 genes were expressed in the corneal endothelium.
Dr. Fautsch concludes: "Identification of novel splicing patterns and differential gene expression in RE+/FECD- samples provides new insights and more relevant gene targets that may be protective against FECD disease in vulnerable patients with TCF4 TNR expansions. Insights gained may serve as a platform to develop productive approaches to therapy across the spectrum of TNR diseases."
For more information
Wieben ED, et al. Gene expression and missplicing in the corneal endothelium of patients with a TCF4 trinucleotide repeat expansion without Fuchs' endothelial corneal dystrophy. Investigative Ophthalmology & Visual Science. 2019;60:3636.