RNA toxicity and missplicing identified in patients with Fuchs' endothelial corneal dystrophy

Researchers at Mayo Clinic report the first instance of RNA toxicity in patients with a non-neurological or neuromuscular disease — Fuchs' endothelial corneal dystrophy (FECD).

FECD is a common inherited, degenerative disease that affects the internal endothelial cell monolayer of the cornea. Advanced FECD, treatable only by corneal transplantation, is characterized by guttae, endothelial cell loss and loss of vision due to stromal edema. It affects nearly 5 percent of middle-aged Caucasians in the United States and accounts for more than 14,000 corneal transplantations annually.

Trinucleotide repeat expansion in FECD

In 2010, a genome-wide association study by Keith H. Baratz, M.D., Ophthalmology, at Mayo Clinic's campus in Rochester, Minnesota, and a multidisciplinary research team identified the single nucleotide polymorphism rs613872, located in an intron of the transcription factor 4 (TCF4) gene on chromosome 18, as a marker for FECD. Study results were published by Dr. Baratz and others in a 2010 issue of the New England Journal of Medicine.

This association has now been replicated repeatedly. Subsequent investigation by a research team that included Dr. Baratz and was led by Eric D. Wieben, Ph.D., Biochemistry and Molecular Biology, at Mayo Clinic in Rochester, Minnesota, found an even stronger linkage between a (CTG ∙ CAG)n trinucleotide repeat expansion in a different intron of the TCF4 gene and FECD. Their findings were published in PLOS One in 2012.

"The strongest association among the genes and loci associated with FECD was with an intronic (CTG ∙ CAG)n trinucleotide repeat expansion in the TCF4 gene, which was found in a majority of patients affected with the disease," says Dr. Baratz. A repeat length longer than 150 nucleotides in leukocyte DNA is highly predictive of disease, so this trinucleotide repeat is a prime candidate for being pathogenic in an autosomal dominant, late-onset degenerative disease such as FECD.

The location of the repeat in an intron indicated that an alteration in protein coding was not the cause of the disease but raised the possibility that RNA toxicity might play a role in the pathogenesis of this disorder, as it does in several of the relatively rare neurodegenerative and neuromuscular repeat expansion diseases, such as myotonic dystrophy. They also concluded that the FECD patient population with the (CTG ∙ CAG)n trinucleotide repeat expansion exceeded that of the combined number of patients in all other microsatellite expansion disorders, including Huntington's disease, myotonic dystrophy types 1 and 2, fragile X syndrome, spinocerebellar ataxia, and C9ORF72-associated amyotrophic lateral sclerosis and frontotemporal dementia.

RNA toxicity and missplicing in FECD

A subsequent collaboration between the Mayo team and researchers at The Scripps Research Institute, La Jolla, California, confirmed evidence of RNA toxicity in FECD. "Corneal endothelial cells from patients with FECD harbored a poly(CUG)n RNA that could be visualized as RNA foci containing this condensed RNA and associated proteins," says Dr. Baratz.

The poly(CUG)n RNA colocalized with and sequestered the mRNA splicing factor MBNL1, leading to missplicing of essential MBNL1-regulated mRNAs. Such foci and missplicing were not observed in similar cells from patients with FECD who lack the repeat expansion or in normal corneal endothelial cells.

"RNA-seq splicing data from the corneal endothelium of patients with FECD and controls revealed many differential alternative splicing events," says Dr. Baratz, "including events previously characterized in the context of myotonic dystrophy type 1 as well as splicing changes in genes that could implicate additional biochemical mechanisms involved in FECD pathogenesis."

Repeat-targeting therapies for common genetic diseases

"Our most important finding is that the genetic mechanism of Fuchs' is exactly the same as the genetic mechanism in myotonic dystrophy type 1, which is a severe neuromuscular degeneration," says Dr. Baratz. "It raises the possibility that other common genetic diseases might also be due to unstable repeat expansions.

"Because significant progress has been made in the development of repeat-targeting therapies for myotonic dystrophy, it is likely that these approaches will also be valuable for the treatment of FECD. Therapeutic trials using simple ocular delivery methods such as eyedrops or injections might facilitate rapid advancement of these therapies for FECD and these other debilitating and fatal diseases."

Study results were published by Jintang Du, Ph.D., and others in the Journal of Biological Chemistry in 2015.

For more information

Baratz KH, et al. E2-2 protein and Fuchs's corneal dystrophy. New England Journal of Medicine. 2010;363:1016.

Wieben ED, et al. A common trinucleotide repeat expansion within the transcription factor 4 (TCF4, E2-2) gene predicts Fuchs corneal dystrophy. PLOS One. 2012;7:e49083.

Du J, et al. RNA toxicity and missplicing in the common eye disease Fuchs endothelial corneal dystrophy. Journal of Biological Chemistry. 2015;290:5979.