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

Wieben, Eric D.; Aleff, Ross A.; Rinkoski, Tommy A.; Baratz, Keith H.; Basu, Shubham; Patel, Sanjay; Maguire, Leo J.; Fautsch, Michael P.

doi:10.1371/journal.pone.0260837

Cited by 9 publications

(6 citation statements)

References 19 publications

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“…In this study, we examined ungenotyped FECD patient surgical samples and utilized a variety of primary and immortalized cell culture models from FECD patients with pathological expansions of trinucleotide repeats in intron 3 of the TCF4 gene (the most common genotype that causes a late onset of disease) to characterize lipid peroxidation and key iron metabolites in FECD and determine whether ferroptosis plays a role in its pathogenesis (46)(47)(48).…”

Section: Introductionmentioning

confidence: 99%

“…We hypothesized that both FECD genetic background and UVA increase CEC susceptibility to nonapoptotic oxidative cell death and lipid peroxidation through the accumulation of toxic intracellular concentrations of ferrous iron. To test this hypothesis, we studied ungenotyped FECD patient surgical samples and immortalized and primary cell culture models from FECD patients with pathological expansions of trinucleotide repeats in intron 3 of the TCF4 gene (the most common genotype associated with FECD (49)(50)(51)) and evaluated for increases in lipid peroxidation, cytosolic Fe 2+ , and susceptibility to ferroptosis attributable to genetic background and UVA exposure.…”

Section: Introductionmentioning

confidence: 99%

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TCF4trinucleotide repeat expansions and UV irradiation increase susceptibility to ferroptosis in Fuchs endothelial corneal dystrophy

Saha

Skeie

Schmidt

et al. 2022

Preprint

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Fuchs endothelial corneal dystrophy (FECD), a progressive polygenic disease that causes degeneration of the corneal endothelium, is the leading indication for corneal transplantation in the U.S. Characteristically, loss of corneal endothelial cells and formation of degenerative extracellular guttae result in failure to maintain appropriate corneal hydration through active ion pumping to counter the passive leakage of aqueous humor. FECD pathogenesis is linked to oxidative stress and environmental exposure to ultraviolet A (UVA), and impaired endogenous response to oxidative stress is common to the disease across multiple genotypes. UV irradiation is also known to cause cellular damage by ferroptosis, a nonapoptotic oxidative cell death resulting from iron-mediated lipid peroxidation. Although a possible role for ferroptosis in FECD has been postulated to account for the increased susceptibility to oxidative damage and lipid peroxidation, this has not been explored systematically. In this study, we investigated the roles of genetic background and UVA exposure in causing lipid membrane damage and endothelial cell degeneration in FECD. We first sought clinical evidence of iron-mediated lipid peroxidation in surgical samples obtained from FECD patients undergoing endothelial keratoplasty, and found increased levels of cytosolic ferrous iron (Fe2+) and evidence of lipid peroxidation were present in end-stage diseased tissues compared with healthy human donor corneas. Next, using immortalized (F35T) and primary cell cultures modeling the TCF4 intronic trinucleotide repeat expansion genotype, we found that alterations in gene and protein expression involved in ferroptosis were conserved in both cell culture and tissue models of FECD disease compared to controls, including elevated levels of the ferroptosis-specific marker transferrin receptor 1. F35T immortalized cells showed significantly higher basal lipid peroxidation than control HCEC-B4G12 cells, indicating higher susceptibility to ferroptosis attributable to genetic background. Then, we tested the impact of physiologically relevant doses of UVA irradiation on F35T cell cultures and found increased cytosolic Fe2+ iron levels indicating a role for ferroptosis in FECD disease progression. Finally, we tested the effects of inhibitory ferroptosis molecules. We found that F35T cells were more prone to RSL3 induced ferroptosis than healthy controls with deferoxamine chelation which indicated increased susceptibility in FECD cells, and cell death could be prevented after experimentally-induced ferroptosis using solubilized antioxidant ubiquinol indicating a role for anti-ferroptosis therapies. This investigation demonstrates that FECD genetic background and UVA exposure contribute to basal and incidental iron-mediated lipid peroxidation and cell death in FECD, and provides the basis for future investigations of ferroptosis-mediated oxidative damage and disease progression in FECD.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

TCF4trinucleotide repeat expansions and UV irradiation increase susceptibility to ferroptosis in Fuchs endothelial corneal dystrophy

Saha

Skeie

Schmidt

et al. 2022

Preprint

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“…We have previously utilised a long-read amplification-free sequencing method to determine that expanded copies of CTG18.1 (defined as ≥50 repeats) are somatically unstable in peripheral blood leukocytes, with inherited repeat lengths positively correlating with increased CTG18.1 instability (Hafford-Tear et al 2019). Southern blot data of FECD patient-derived corneal endothelial cell cultures have also suggested that CTG18.1 repeat length is greater in affected cells compared to leukocytes, in keeping with the dogma that STRs typically display high levels of instability within affected cell types (Wieben et al 2021;Depienne and Mandel 2021). For example, in myotonic dystrophy type 1, the diseaseassociated repeat has been reported to expand up to 4,000 repeats in skeletal muscle, representing up to a 25-fold increase in length compared to blood (Nakamori et al 2013).…”

Section: Introductionmentioning

confidence: 74%

Tissue-Specific Dynamics ofTCF4Triplet Repeat Instability Revealed by Optical Genome Mapping

Zarouchlioti,

Efthymiou,

Fracchini

et al. 2024

Preprint

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Here, we demonstrate the utility of optical genome mapping (OGM) to interrogate the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (termed CTG18.1) and gain novel insights into the tissue-specific nature of the disease. Genomic DNA (gDNA) samples derived from peripheral blood leukocytes and primary corneal endothelial cells (CECs) were analysed by OGM. Concurrently, all samples were genotyped by standard PCR-based methods to classify their expansion status. Individuals with one or more CTG18.1-expanded alleles (≥50 CTG repeats) detected in their leukocyte-derived gDNA were classified as expansion-positive. A customised bioinformatics pipeline was developed to perform CTG18.1-targeted OGM analysis. All linearised gDNA molecules containing labels flanking CTG18.1 were extracted, corrected for the repeats on the reference human genome and sized. Analysis of paired bio-samples revealed that expanded CTG18.1 alleles behave dynamically, regardless of cell-type origin, but displayed significantly higher levels of instability within the diseased corneal endothelium. Clusters of CTG18.1 molecules of approximately 1,800-11,900 repeats, beyond the ranges observed in individual-matched leukocyte samples, were detected in all CEC gDNA samples from expansion-positive cases. In conclusion, OGM is a powerful method to analyse the somatically unstable CTG18.1 locus. More generally, this work exemplifies the broader utility of OGM in exploring somatically unstable short tandem repeat loci. Furthermore, this study has highlighted the extreme levels of tissue-specific CTG18.1 somatic instability occurring within the diseased corneal endothelium, which we hypothesise plays a pivotal role in driving downstream pathogenic mechanisms of CTG18.1-mediated FECD.

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“…However, results from one recent study that used long read sequencing of the RNA isolated from FECD corneal endothelium suggest that the TCF4 repeat expansion in corneal endothelium is much larger than in peripheral leukocytes of patients. 42 We hypothesize that cells with larger repeat lengths may be more prone to foci formation.…”

Section: Discussionmentioning

confidence: 98%

The TCF4 Trinucleotide Repeat Expansion of Fuchs’ Endothelial Corneal Dystrophy: Implications for the Anterior Segment of the Eye

Gong

Johnson

et al. 2023

Invest. Ophthalmol. Vis. Sci.

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Purpose In the United States, 70% of Fuchs’ endothelial corneal dystrophy (FECD) cases are caused by an intronic trinucleotide repeat expansion in the TCF4 gene. CUG repeat RNA transcripts from this expansion accumulate as nuclear foci in the corneal endothelium. In this study, we sought to detect foci in other anterior segment cell types and assess their molecular impact. Methods We examined CUG repeat RNA foci appearance, expression of downstream affected genes, gene splicing, and TCF4 RNA expression in corneal endothelium, corneal stromal keratocytes, corneal epithelium, trabecular meshwork cells, and lens epithelium. Results CUG repeat RNA foci, the hallmark of FECD in corneal endothelium (found in 84% of endothelial cells), are less detectable in trabecular meshwork cells (41%), much less prevalent in stromal keratocytes (11%) or corneal epithelium (4%), and absent in lens epithelium. With few exceptions including mis-splicing in the trabecular meshwork, differential gene expression and splicing changes associated with the expanded repeat in corneal endothelial cells are not observed in other cell types. Expression of the TCF4 transcripts including full-length isoforms containing the repeat sequence at the 5′ end is much higher in the corneal endothelium or trabecular meshwork than in the corneal stroma or corneal epithelium. Conclusions Expression of the CUG repeat containing TCF4 transcripts is higher in the corneal endothelium, likely contributing to foci formation and the large molecular and pathologic impact on those cells. Further studies are warranted to examine any glaucoma risk and impact of the observed foci in the trabecular meshwork of these patients.

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

Cited by 9 publications

References 19 publications

TCF4trinucleotide repeat expansions and UV irradiation increase susceptibility to ferroptosis in Fuchs endothelial corneal dystrophy

TCF4trinucleotide repeat expansions and UV irradiation increase susceptibility to ferroptosis in Fuchs endothelial corneal dystrophy

Tissue-Specific Dynamics ofTCF4Triplet Repeat Instability Revealed by Optical Genome Mapping

The TCF4 Trinucleotide Repeat Expansion of Fuchs’ Endothelial Corneal Dystrophy: Implications for the Anterior Segment of the Eye

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