Genetics & Genomics of Fuchs Endothelial Corneal Dystrophy

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 DESCRIPTION (provided by applicant): Fuchs' endothelial corneal dystrophy (FECD) is an age-related degenerative disorder resulting in corneal edema and loss of vision. FECD occurs in 5% of whites greater than 40 years of age and is the leading indication for corneal transplantation in the U.S. Intronic CTG triplet repeat expansions at CTG18.1 locus of TCF4 are a common genetic cause of FECD in a majority of Caucasians with FECD. Mutant expanded CUG transcripts (CUGexp) accumulate as discrete ribonuclear foci in the endothelium of FECD subjects. Based on other rare neurodegenerative disorders such as Myotonic Dystrophy caused by expanded repeats, these foci exert toxicity by sequestering RNA binding proteins (RBPs) required for normal mRNA regulation. The splicing factor MBNL1 has been shown to co-localize with the CUGexp foci in FECD resulting is missplicing of genes regulated by MBNL1. In this proposal, we will examine intergenerational instability of CTG18.1 in parent-child transmissions using multi-generational FECD families. Expansions and contractions of CTG18.1 will be noted with attention to parent of origin and clinical impact. Somatic instability and expansion of the expanded repeats in FECD endothelium will be examined to account for why this tissue layer is so prone to age-related degeneration. Using slit lamp, confocal, and specular microscopy, we will examine for early corneal findings specific for the expanded repeats in FECD subjects as well as abnormalities in subjects with subclinical disease. We want to critically examine the impact of the CUGexp foci on the transcriptome by performing RNA sequencing of FECD tissue with intermediate and large expansions compared to FECD samples without the expanded repeats. We will examine the transcripts whose splicing is regulated by the MBNL and CELF family of RBPs including genes involved with extracellular matrix proteins and epithelial-mesenchymal transition. The physical interaction of MBNL1 and MBNL2 with the CUGexp transcripts of TCF4 will be examined with RNA immunoprecipitation. We will determine which of these RBPs are critical determinants for foci formation by siRNA mediated down regulation in a FECD cell line. Protein mass spectrometry will be utilized to identify additional RBPs with an affinity for CUGexp. We will test duplex RNA and single-stranded antisense oligonucleotide inhibitors of CUGexp foci formation in a cell culture model systems. We aim to identify lead compounds for therapeutic development.