Comparison of TCF4 repeat expansion length in corneal endothelium and leukocytes of patients with Fuchs endothelial corneal dystrophy.

Eric D Wieben,Shubham Basu,Sanjay V Patel,Michael P Fautsch,Leo J Maguire,Tommy A Rinkoski,Ross A Aleff,Keith H Baratz,Alfred S Lewin

doi:10.1371/journal.pone.0260837

Eric D Wieben, Shubham Basu + Show 7 more

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https://doi.org/10.1371/journal.pone.0260837

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Journal: PloS one	Publication Date: Dec 2, 2021
Citations: 6	License type: CC BY 4.0

Affiliation: Mayo Clinic

Abstract

Expansion of CTG trinucleotide repeats (TNR) in the transcription factor 4 (TCF4) gene is highly associated with Fuchs Endothelial Corneal Dystrophy (FECD). Due to limitations in the availability of DNA from diseased corneal endothelium, sizing of CTG repeats in FECD patients has typically been determined using DNA samples isolated from peripheral blood leukocytes. However, it is non-feasible to extract enough DNA from surgically isolated FECD corneal endothelial tissue to determine repeat length based on current technology. To circumvent this issue, total RNA was isolated from FECD corneal endothelium and sequenced using long-read sequencing. Southern blotting of DNA samples isolated from primary cultures of corneal endothelium from these same affected individuals was also assessed. Both long read sequencing and Southern blot analysis showed significantly longer CTG TNR expansion (>1000 repeats) in the corneal endothelium from FECD patients than those characterized in leukocytes from the same individuals (<90 repeats). Our findings suggest that the TCF4 CTG repeat expansions in the FECD corneal endothelium are much longer than those found in leukocytes.

Highlights

Fuchs Endothelial Corneal Dystrophy (FECD) is a late onset eye disease associated with an expanded trinucleotide repeat (TNR) in the transcription factor 4 (TCF4) gene and a lack of other systemic findings
Using surgical samples and corresponding blood obtained from the same patients, we identified TCF4 CTG repeat expansions longer in FECD corneal endothelial RNA than in leukocyte DNA
FECD(1) and FECD(2) have bands that are approximately 15 kb, consistent with expansions of approximately 4500 repeats. These data are consistent with the large size of the Iso-Seq corneal endothelial transcripts found in RNA samples from the same patient. To determine if this is a characteristic of FECD Human corneal endothelial cell (HCEC), we isolated genomic DNA from 8 additional primary HCEC lines we had previously established from FECD patients and assessed TCF4 repeat length by Southern blot (Fig 4b)

Summary

Introduction

Fuchs Endothelial Corneal Dystrophy (FECD) is a late onset eye disease associated with an expanded trinucleotide repeat (TNR) in the TCF4 gene and a lack of other systemic findings. This work revealed that intron sequences upstream of the CTG repeats in TCF4 accumulate in corneal endothelial tissue obtained from FECD patients with repeat expansions. The ability to identify the repeat length in corneal endothelial cells is not feasible due to the inability to isolate sufficient amounts of DNA from FECD corneal surgical samples. The corneal endothelium obtained from a single, pauci-cellular surgical specimen limits the ability to extract the necessary amount of DNA required for determination of repeat length with current technology. Using surgical samples and corresponding blood obtained from the same patients, we identified TCF4 CTG repeat expansions longer in FECD corneal endothelial RNA than in leukocyte DNA. Analysis of repeat length in genomic DNA from primary cultures of corneal endothelial cells revealed DNA expansions comparable to those found in corneal endothelial tissue from the same individuals

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