BACKGROUND: Fuchs endothelial corneal dystrophy (FECD) is the most common repeat-mediated disease in humans. It exclusively affects corneal endothelial cells (CECs), with ≤81% of cases associated with an intronic TCF4 triplet repeat (CTG18.1). Here, we utilise optical genome mapping (OGM) to investigate CTG18.1 tissue-specific instability to gain mechanistic insights. METHODS: We applied OGM to a diverse range of genomic DNAs (gDNAs) from patients with FECD and controls (n = 43); CECs, leukocytes and fibroblasts. A bioinformatics pipeline was developed to robustly interrogate CTG18.1-spanning DNA molecules. All results were compared with conventional polymerase chain reaction-based fragment analysis. FINDINGS: Analysis of bio-samples revealed that expanded CTG18.1 alleles behave dynamically, regardless of cell-type origin. However, clusters of CTG18.1 molecules, encompassing ∼1800-11,900 repeats, were exclusively detected in diseased CECs from expansion-positive cases. Additionally, both progenitor allele size and age were found to influence the level of leukocyte-specific CTG18.1 instability. INTERPRETATION: OGM is a powerful tool for analysing somatic instability of repeat loci and reveals here the extreme levels of CTG18.1 instability occurring within diseased CECs underpinning FECD pathophysiology, opening up new therapeutic avenues for FECD. Furthermore, these findings highlight the broader translational utility of FECD as a model for developing therapeutic strategies for rarer diseases similarly attributed to somatically unstable repeats. FUNDING: UK Research and Innovation, Moorfields Eye Charity, Fight for Sight, Medical Research Council, NIHR BRC at Moorfields Eye Hospital and UCL Institute of Ophthalmology, Grantová Agentura České Republiky, Univerzita Karlova v Praze, the National Brain Appeal's Innovation Fund and Rosetrees Trust.

Cornea Cataract and External Disease Division Wilmer Eye Institute Johns Hopkins Medicine Baltimore USA

Department of Ophthalmology 1st Faculty of Medicine Charles University and General University Hospital Prague Prague Czech Republic; Department of Paediatrics and Inherited Metabolic Disorders 1st Faculty of Medicine Charles University and General University Hospital Prague Prague Czech Republic

Department of Paediatrics and Inherited Metabolic Disorders 1st Faculty of Medicine Charles University and General University Hospital Prague Prague Czech Republic

Moorfields Eye Hospital London UK

UCL Institute of Ophthalmology London UK

UCL Institute of Ophthalmology London UK; Moorfields Eye Hospital London UK

UCL Queen Square Institute of Neurology Department of Neuromuscular Diseases London UK

Zobrazit více v PubMed

Matthaei M., Hribek A., Clahsen T., Bachmann B., Cursiefen C., Jun A.S. Fuchs endothelial corneal dystrophy: clinical, genetic, pathophysiologic, and therapeutic aspects. Annu Rev Vis Sci. 2019;5:151–175. doi: 10.1146/annurev-vision-091718-014852. PubMed DOI

Mathews P., Benbow A., Corcoran K., DeMatteo J., Philippy B., Van Meter W. 2022 Eye banking statistical report—executive summary. Eye Bank Corneal Transplant. 2023;2 doi: 10.1097/ebct.0000000000000008. DOI

NHS organ and tissue donation and transplantation activity report 2023/2024. Section 10 - Cornea Activity. 2023. https://www.odt.nhs.uk/statistics-and-reports/annual-activity-report/

Joyce N.C. Proliferative capacity of corneal endothelial cells. Exp Eye Res. 2012;95:16–23. doi: 10.1016/j.exer.2011.08.014. PubMed DOI PMC

Price M.O., Mehta J.S., Jurkunas U.V., Price F.W. Corneal endothelial dysfunction: evolving understanding and treatment options. Prog Retin Eye Res. 2021;82 doi: 10.1016/j.preteyeres.2020.100904. PubMed DOI

Fautsch M.P., Wieben E.D., Baratz K.H., et al. TCF4-mediated Fuchs endothelial corneal dystrophy: insights into a common trinucleotide repeat-associated disease. Prog Retin Eye Res. 2021;81 doi: 10.1016/j.preteyeres.2020.100883. PubMed DOI PMC

Zarouchlioti C., Sanchez-Pintado B., Tear N.J.H., et al. Antisense therapy for a common corneal dystrophy ameliorates TCF4 repeat expansion-mediated toxicity. Am J Hum Genet. 2018;102:528. doi: 10.1016/j.ajhg.2018.02.010. PubMed DOI PMC

Mouro Pinto R., Arning L., Giordano J.V., et al. Patterns of CAG repeat instability in the central nervous system and periphery in Huntington's disease and in spinocerebellar ataxia type 1. Hum Mol Genet. 2020;29:2551–2567. doi: 10.1093/hmg/ddaa139. PubMed DOI PMC

Wheeler V.C., Dion V. Modifiers of CAG/CTG repeat instability: insights from mammalian models. J Huntingt Dis. 2021;10:123–148. doi: 10.3233/JHD-200426. PubMed DOI PMC

Monckton D.G. The contribution of somatic expansion of the CAG repeat to symptomatic development in huntington's disease: a historical perspective. J Huntingt Dis. 2021;10:7–33. doi: 10.3233/JHD-200429. PubMed DOI PMC

Hafford-Tear N.J., Tsai Y.-C., Sadan A.N., et al. CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy-associated TCF4 triplet repeat. Genet Med. 2019;21:2092–2102. doi: 10.1038/s41436-019-0453-x. PubMed DOI PMC

Wieben E.D., Aleff R.A., Rinkoski T.A., et al. Comparison of TCF4 repeat expansion length in corneal endothelium and leukocytes of patients with Fuchs endothelial corneal dystrophy. PLoS One. 2021;16 doi: 10.1371/journal.pone.0260837. PubMed DOI PMC

Depienne C., Mandel J.-L. 30 years of repeat expansion disorders: what have we learned and what are the remaining challenges? Am J Hum Genet. 2021;108:764–785. doi: 10.1016/j.ajhg.2021.03.011. PubMed DOI PMC

Nakamori M., Sobczak K., Puwanant A., et al. Splicing biomarkers of disease severity in myotonic dystrophy. Ann Neurol. 2013;74:862–872. doi: 10.1002/ana.23992. PubMed DOI PMC

Handsaker R.E., Kashin S., Reed N.M., et al. Long somatic DNA-repeat expansion drives neurodegeneration in Huntington disease 2. bioRxiv. 2024 doi: 10.1101/2024.05.17.592722. DOI

Benn C.L., Gibson K.R., Reynolds D.S. Drugging DNA damage repair pathways for trinucleotide repeat expansion diseases. J Huntingt Dis. 2021;10:203–220. doi: 10.3233/JHD-200421. PubMed DOI PMC

Ciosi M., Cumming S.A., Chatzi A., et al. Approaches to sequence the HTT CAG repeat expansion and quantify repeat length variation. J Huntingt Dis. 2021;10:53–74. doi: 10.3233/JHD-200433. PubMed DOI PMC

Ciosi M., Maxwell A., Cumming S.A., et al. A genetic association study of glutamine-encoding DNA sequence structures, somatic CAG expansion, and DNA repair gene variants, with Huntington disease clinical outcomes. EBioMedicine. 2019;48:568–580. doi: 10.1016/j.ebiom.2019.09.020. PubMed DOI PMC

Erdmann H., Schöberl F., Giurgiu M., et al. Parallel in-depth analysis of repeat expansions in ataxia patients by long-read sequencing. Brain J Neurol. 2023;146:1831–1843. doi: 10.1093/brain/awac377. PubMed DOI

Höijer I., Tsai Y.-C., Clark T.A., et al. Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing. Hum Mutat. 2018;39:1262–1272. doi: 10.1002/humu.23580. PubMed DOI PMC

Yuan Y., Chung C.Y.-L., Chan T.-F. Advances in optical mapping for genomic research. Comput Struct Biotechnol J. 2020;18:2051–2062. doi: 10.1016/j.csbj.2020.07.018. PubMed DOI PMC

Facchini S., Dominik N., Manini A., et al. Optical genome mapping enables detection and accurate sizing of RFC1 repeat expansions. Biomolecules. 2023;13:1546. doi: 10.3390/biom13101546. PubMed DOI PMC

Morato Torres C.A., Zafar F., Tsai Y.-C., et al. ATTCT and ATTCC repeat expansions in the ATXN10 gene affect disease penetrance of spinocerebellar ataxia type 10. HGG Adv. 2022;3 doi: 10.1016/j.xhgg.2022.100137. PubMed DOI PMC

Maroilley T., Tsai M.-H., Mascarenhas R., et al. A novel FAME1 repeat configuration in a European family identified using a combined genomics approach. Epilepsia Open. 2023;8:659–665. doi: 10.1002/epi4.12702. PubMed DOI PMC

Efthymiou S., Lemmers R.J.L.F., Vishnu V.Y., et al. Optical genome mapping for the molecular diagnosis of facioscapulohumeral muscular dystrophy: advancement and challenges. Biomolecules. 2023;13:1567. doi: 10.3390/biom13111567. PubMed DOI PMC

Peh G.S.L., Chng Z., Ang H.-P., et al. Propagation of human corneal endothelial cells: a novel dual media approach. Cell Transplant. 2015;24:287–304. doi: 10.3727/096368913X675719. PubMed DOI

Saha S., Skeie J.M., Schmidt G.A., et al. 2022. TCF4 trinucleotide repeat expansions and UV irradiation increase susceptibility to ferroptosis in Fuchs endothelial corneal dystrophy 2022. DOI

Angelbello A.J., Benhamou R.I., Rzuczek S.G., et al. A small molecule that binds an RNA repeat expansion stimulates its decay via the exosome complex. Cell Chem Biol. 2021;28:34–45.e6. doi: 10.1016/j.chembiol.2020.10.007. PubMed DOI PMC

Wieben E.D., Aleff R.A., Tosakulwong N., et al. A common trinucleotide repeat expansion within the transcription factor 4 (TCF4, E2-2) gene predicts Fuchs corneal dystrophy. PLoS One. 2012;7 doi: 10.1371/journal.pone.0049083. PubMed DOI PMC

Mootha V.V., Gong X., Ku H.-C., Xing C. Association and familial segregation of CTG18.1 trinucleotide repeat expansion of TCF4 gene in Fuchs' endothelial corneal dystrophy. Invest Ophthalmol Vis Sci. 2014;55:33–42. doi: 10.1167/iovs.13-12611. PubMed DOI PMC

Mantere T., Neveling K., Pebrel-Richard C., et al. Optical genome mapping enables constitutional chromosomal aberration detection. Am J Hum Genet. 2021;108:1409–1422. doi: 10.1016/j.ajhg.2021.05.012. PubMed DOI PMC

Bhattacharyya N., Chai N., Hafford-Tear N.J., et al. Deciphering novel TCF4-driven mechanisms underlying a common triplet repeat expansion-mediated disease. PLoS Genet. 2024;20 doi: 10.1371/journal.pgen.1011230. PubMed DOI PMC

Malik I., Kelley C.P., Wang E.T., Todd P.K. Molecular mechanisms underlying nucleotide repeat expansion disorders. Nat Rev Mol Cell Biol. 2021;22:589–607. doi: 10.1038/s41580-021-00382-6. PubMed DOI PMC

Hu J., Rong Z., Gong X., et al. Oligonucleotides targeting TCF4 triplet repeat expansion inhibit RNA foci and mis-splicing in Fuchs' dystrophy. Hum Mol Genet. 2018;27:1015–1026. doi: 10.1093/hmg/ddy018. PubMed DOI PMC

Gomes-Pereira M., Fortune M.T., Monckton D.G. Mouse tissue culture models of unstable triplet repeats: in vitro selection for larger alleles, mutational expansion bias and tissue specificity, but no association with cell division rates. Hum Mol Genet. 2001;10:845–854. doi: 10.1093/hmg/10.8.845. PubMed DOI

Goold R., Flower M., Moss D.H., et al. FAN1 modifies Huntington's disease progression by stabilizing the expanded HTT CAG repeat. Hum Mol Genet. 2019;28:650–661. doi: 10.1093/hmg/ddy375. PubMed DOI PMC

Liu H., Cheng J., Zhuang X., Qi B., Li F., Zhang B. Genomic instability and eye diseases. Adv Ophthalmol Pract Res. 2023;3:103–111. doi: 10.1016/j.aopr.2023.03.002. PubMed DOI PMC

Liu C., Miyajima T., Melangath G., et al. Ultraviolet A light induces DNA damage and estrogen-DNA adducts in Fuchs endothelial corneal dystrophy causing females to be more affected. Proc Natl Acad Sci U S A. 2020;117:573–583. doi: 10.1073/pnas.1912546116. PubMed DOI PMC

Rajagopal S., Donaldson J., Flower M., Hensman Moss D.J., Tabrizi S.J. Genetic modifiers of repeat expansion disorders. Emerg Top Life Sci. 2023;7:325–337. doi: 10.1042/ETLS20230015. PubMed DOI PMC

Zhang Q., Wang Y., Xu Y., et al. Optical genome mapping for detection of chromosomal aberrations in prenatal diagnosis. Acta Obstet Gynecol Scand. 2023;102:1053–1062. doi: 10.1111/aogs.14613. PubMed DOI PMC

Ghorbani F., de Boer-Bergsma J., Verschuuren-Bemelmans C.C., et al. Prevalence of intronic repeat expansions in RFC1 in Dutch patients with CANVAS and adult-onset ataxia. J Neurol. 2022;269:6086–6093. doi: 10.1007/s00415-022-11275-9. PubMed DOI PMC

Genetic and Demographic Determinants of Fuchs Endothelial Corneal Dystrophy Risk and Severity