Fuchs' Endothelial Corneal Dystrophy and RNA Foci in Patients With Myotonic Dystrophy

Abstract

Purpose: The most common cause of Fuchs' endothelial corneal dystrophy (FECD) is an intronic CTG repeat expansion in TCF4. Expanded CUG repeat RNA colocalize with splicing factor, muscleblind-like 1 (MBNL1), in nuclear foci in endothelium as a molecular hallmark. Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a CTG repeat expansion in the 3'-untranslated region (UTR) of DMPK. In this study, we examine for RNA-MBNL1 foci in endothelial cells of FECD subjects with DM1, test the hypothesis that DM1 patients are at risk for FECD, and determine prevalence of TCF4 and DMPK expansions in a FECD cohort.

Methods: Using FISH, we examined for nuclear RNA-MBNL1 foci in endothelial cells from FECD subjects with DM1. We examined 13 consecutive unrelated DM1 patients for FECD using slit-lamp and specular microscopy. We genotyped TCF4 and DMPK repeat polymorphisms in a FECD cohort of 317 probands using short-tandem repeat and triplet repeat-primed PCR assays.

Results: We detected abundant nuclear RNA foci colocalizing with MBNL1 in endothelial cells of FECD subjects with DM1. Six of thirteen DM1 patients (46%) had slit-lamp and specular microscopic findings of FECD, compared to 4% disease prevalence (P = 5.5 × 10-6). As expected, 222 out of 317 (70%) FECD probands harbored TCF4 expansion, while one subject harbored DMPK expansion without prior diagnosis of DM1.

Conclusions: Our work suggests that DM1 patients are at risk for FECD. DMPK mutations contribute to the genetic burden of FECD but are uncommon. We establish a connection between two repeat expansion disorders converging upon RNA-MBNL1 foci and FECD.

Figure 1

Nuclear RNA foci accumulate and colocalize with MBNL1 in corneal endothelial cells with triplet repeat expansion in DMPK gene. (A) FISH with a (CAG)₆CA-5′ Texas red-labeled 2-O-methyl RNA 20-mers probe (Integrated DNA Technologies) on endothelial cells of FECD/DM1 subject (16-1348) with an expanded DMPK allele with 300 CTG repeats revealed punctate, nuclear RNA foci (red). RNA foci were present in endothelial cells from a FECD subject (16-3407) with an expanded TCF4 allele with 71 CTG repeats and absent in cells from unaffected subject (16-0729) without the DMPK and TCF4 repeat expansions. DNA was stained with DAPI (blue). The scale bar represents 25 μm. (B) Colocalization of MBNL1 with the expanded CUG repeat nuclear foci. After hybridization with RNA probe (red), cells were stained with anti-MBNL1 antibody (green) and DAPI (blue). The scale bar represents 10 μm.

Figure 2

Specular microscopy of corneal endothelium in myotonic dystrophy type 1 patients. (A) Specular microscopy of the central endothelium of DM1 subject with FECD Krachmer grade of 5 OD (oculus dexter, right eye) and 4 OS (oculus sinister, left eye) revealed typical FECD findings including numerous focal dark spots corresponding to corneal guttae, increased cellular polymorphism (loss of normal hexagonal pattern), and polymegathism (variation in cell size). Photomicrograph images represent a surface area of 400 by 220 μm. Bar represents 100 μm. (B) Specular imaging of endothelium of DM1 subject harboring both DMPK1 and TCF4 triplet repeat expansions with FECD Krachmer grade of 3 in each eye. Numerous focal dark spots corresponding to corneal guttae are shown in OD. Large dark areas corresponding to confluence of the corneal guttae and marked loss of endothelial cell density and grotesque morphology of remaining cells are shown in OS. (C) Specular images of DM1 subject without FECD showing absence of guttae and normal endothelial cell density and morphology in both eyes.