Quantitative Studies of Muscleblind Proteins and Their Interaction With TCF4 RNA Foci Support Involvement in the Mechanism of Fuchs' Dystrophy

PURPOSE: Fuchs' endothelial corneal dystrophy (FECD) is a major cause of vision loss and the most common nucleotide repeat disorder, affecting 4% of United States population greater than 40 years of age. Seventy percent of FECD cases are due to an intronic CTG expansion within the TCF4 gene, resulting in accumulation of CUG repeat RNA nuclear foci in corneal endothelium. Each endothelial cell has approximately two sense foci, and each focus is a single RNA molecule. This study aimed to obtain a better understanding of how rare repeat RNA species lead to disease. METHODS: We quantitatively examined muscleblind-like (MBNL) proteins and their interaction with foci in both patient-derived corneal endothelial cell lines and human corneal endothelial tissue. RESULTS: Using fluorescent in situ hybridization and immunofluorescence, we found that depletion of both MBNL1 and MBNL2 reduces nuclear RNA foci formed by the repeat, suggesting that both are necessary for foci. Quantitative studies of RNA and protein copy number revealed MBNLs to be abundant in the total cellular pool in endothelial cell lines but are much lower in human corneal endothelial tissue. Studies using human tissue nuclear and cytoplasmic fractions indicate that most MBNL proteins are localized to the cytoplasm. CONCLUSIONS: The low levels of MBNL1/2 in corneal tissue, in combination with the small fraction of protein in the nucleus, may make corneal endothelial cells especially susceptible to sequestration of MBNL1/2 by CUG repeat RNA. These observations may explain how a limited number of RNA molecules can cause widespread alteration of splicing and late-onset degenerative FECD.