Congenital hereditary endothelial dystrophy 1 (CHED1) and posterior polymorphous corneal dystrophy 1 (PPCD1) are autosomal-dominant corneal endothelial dystrophies that have been genetically mapped to overlapping loci on the short arm of chromosome 20. We combined genetic and genomic approaches to identify the cause of disease in extensive pedigrees comprising over 100 affected individuals. After exclusion of pathogenic coding, splice-site, and copy-number variations, a parallel approach using targeted and whole-genome sequencing facilitated the identification of pathogenic variants in a conserved region of the OVOL2 proximal promoter sequence in the index families (c.-339_361dup for CHED1 and c.-370T>C for PPCD1). Direct sequencing of the OVOL2 promoter in other unrelated affected individuals identified two additional mutations within the conserved proximal promoter sequence (c.-274T>G and c.-307T>C). OVOL2 encodes ovo-like zinc finger 2, a C2H2 zinc-finger transcription factor that regulates mesenchymal-to-epithelial transition and acts as a direct transcriptional repressor of the established PPCD-associated gene ZEB1. Interestingly, we did not detect OVOL2 expression in the normal corneal endothelium. Our in vitro data demonstrate that all four mutated OVOL2 promoters exhibited more transcriptional activity than the corresponding wild-type promoter, and we postulate that the mutations identified create cryptic cis-acting regulatory sequence binding sites that drive aberrant OVOL2 expression during endothelial cell development. Our data establish CHED1 and PPCD1 as allelic conditions and show that CHED1 represents the extreme of what can be considered a disease spectrum. They also implicate transcriptional dysregulation of OVOL2 as a common cause of dominantly inherited corneal endothelial dystrophies.

European Eye Clinic Lexum Antala Staška 1670 80 Prague 140 00 Czech Republic

Institute of Inherited Metabolic Disorders 1st Faculty of Medicine Charles University Prague and General University Hospital Prague Ke Karlovu 2 Prague 128 08 Czech Republic

Institute of Molecular Genetics Vídeňská 1083 Prague 142 20 Czech Republic

Moorfields Eye Hospital London EC1V 2PD UK

UCL Genetics Institute University College London London WC1E 6BT UK

UCL Institute of Ophthalmology University College London London EC1V 9EL UK

UCL Institute of Ophthalmology University College London London EC1V 9EL UK; Institute of Inherited Metabolic Disorders 1st Faculty of Medicine Charles University Prague and General University Hospital Prague Ke Karlovu 2 Prague 128 08 Czech Republic; Department of Ophthalmology 1st Faculty of Medicine Charles University Prague and General University Hospital Prague U Nemocnice 2 Prague 128 08 Czech Republic

UCL Institute of Ophthalmology University College London London EC1V 9EL UK; Moorfields Eye Hospital London EC1V 2PD UK

UCL Institute of Ophthalmology University College London London EC1V 9EL UK; UCL Genetics Institute University College London London WC1E 6BT UK

Weatherall Institute of Molecular Medicine University of Oxford John Radcliffe Hospital Oxford OX3 9DS UK

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Weiss J.S., Møller H.U., Aldave A.J., Seitz B., Bredrup C., Kivelä T., Munier F.L., Rapuano C.J., Nischal K.K., Kim E.K. IC3D classification of corneal dystrophies--edition 2. Cornea. 2015;34:117–159. PubMed

Cibis G.W., Krachmer J.A., Phelps C.D., Weingeist T.A. The clinical spectrum of posterior polymorphous dystrophy. Arch. Ophthalmol. 1977;95:1529–1537. PubMed

Krachmer J.H. Posterior polymorphous corneal dystrophy: a disease characterized by epithelial-like endothelial cells which influence management and prognosis. Trans. Am. Ophthalmol. Soc. 1985;83:413–475. PubMed PMC

Pearce W.G., Tripathi R.C., Morgan G. Congenital endothelial corneal dystrophy. Clinical, pathological, and genetic study. Br. J. Ophthalmol. 1969;53:577–591. PubMed PMC

Yellore V.S., Papp J.C., Sobel E., Khan M.A., Rayner S.A., Farber D.B., Aldave A.J. Replication and refinement of linkage of posterior polymorphous corneal dystrophy to the posterior polymorphous corneal dystrophy 1 locus on chromosome 20. Genet. Med. 2007;9:228–234. PubMed

Gwilliam R., Liskova P., Filipec M., Kmoch S., Jirsova K., Huckle E.J., Stables C.L., Bhattacharya S.S., Hardcastle A.J., Deloukas P., Ebenezer N.D. Posterior polymorphous corneal dystrophy in Czech families maps to chromosome 20 and excludes the VSX1 gene. Invest. Ophthalmol. Vis. Sci. 2005;46:4480–4484. PubMed

Héon E., Mathers W.D., Alward W.L., Weisenthal R.W., Sunden S.L., Fishbaugh J.A., Taylor C.M., Krachmer J.H., Sheffield V.C., Stone E.M. Linkage of posterior polymorphous corneal dystrophy to 20q11. Hum. Mol. Genet. 1995;4:485–488. PubMed

Aldave A.J., Yellore V.S., Yu F., Bourla N., Sonmez B., Salem A.K., Rayner S.A., Sampat K.M., Krafchak C.M., Richards J.E. Posterior polymorphous corneal dystrophy is associated with TCF8 gene mutations and abdominal hernia. Am. J. Med. Genet. A. 2007;143A:2549–2556. PubMed

Krafchak C.M., Pawar H., Moroi S.E., Sugar A., Lichter P.R., Mackey D.A., Mian S., Nairus T., Elner V., Schteingart M.T. Mutations in TCF8 cause posterior polymorphous corneal dystrophy and ectopic expression of COL4A3 by corneal endothelial cells. Am. J. Hum. Genet. 2005;77:694–708. PubMed PMC

Liskova P., Evans C.J., Davidson A.E., Zaliova M., Dudakova L., Trkova M., Stranecky V., Carnt N., Plagnol V., Vincent A.L. Heterozygous deletions at the ZEB1 locus verify haploinsufficiency as the mechanism of disease for posterior polymorphous corneal dystrophy type 3. Eur. J. Hum. Genet. 2015 Published online October 28, 2015. PubMed PMC

Biswas S., Munier F.L., Yardley J., Hart-Holden N., Perveen R., Cousin P., Sutphin J.E., Noble B., Batterbury M., Kielty C. Missense mutations in COL8A2, the gene encoding the alpha2 chain of type VIII collagen, cause two forms of corneal endothelial dystrophy. Hum. Mol. Genet. 2001;10:2415–2423. PubMed

Liskova P., Gwilliam R., Filipec M., Jirsova K., Reinstein Merjava S., Deloukas P., Webb T.R., Bhattacharya S.S., Ebenezer N.D., Morris A.G., Hardcastle A.J. High prevalence of posterior polymorphous corneal dystrophy in the Czech Republic; linkage disequilibrium mapping and dating an ancestral mutation. PLoS ONE. 2012;7:e45495. PubMed PMC

Aldave A.J., Yellore V.S., Vo R.C., Kamal K.M., Rayner S.A., Plaisier C.L., Chen M.C., Damani M.R., Pham M.N., Gorin M.B. Exclusion of positional candidate gene coding region mutations in the common posterior polymorphous corneal dystrophy 1 candidate gene interval. Cornea. 2009;28:801–807. PubMed PMC

Lai I.N., Yellore V.S., Rayner S.A., D’Silva N.C., Nguyen C.K., Aldave A.J. The utility of next-generation sequencing in the evaluation of the posterior polymorphous corneal dystrophy 1 locus. Mol. Vis. 2010;16:2829–2838. PubMed PMC

Kirkness C.M., McCartney A., Rice N.S., Garner A., Steele A.D. Congenital hereditary corneal oedema of Maumenee: its clinical features, management, and pathology. Br. J. Ophthalmol. 1987;71:130–144. PubMed PMC

Toma N.M., Ebenezer N.D., Inglehearn C.F., Plant C., Ficker L.A., Bhattacharya S.S. Linkage of congenital hereditary endothelial dystrophy to chromosome 20. Hum. Mol. Genet. 1995;4:2395–2398. PubMed

Aldave A.J., Han J., Frausto R.F. Genetics of the corneal endothelial dystrophies: an evidence-based review. Clin. Genet. 2013;84:109–119. PubMed PMC

Stránecký V., Hoischen A., Hartmannová H., Zaki M.S., Chaudhary A., Zudaire E., Nosková L., Barešová V., Přistoupilová A., Hodaňová K. Mutations in ANTXR1 cause GAPO syndrome. Am. J. Hum. Genet. 2013;92:792–799. PubMed PMC

van de Steeg E., Stránecký V., Hartmannová H., Nosková L., Hřebíček M., Wagenaar E., van Esch A., de Waart D.R., Oude Elferink R.P., Kenworthy K.E. Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver. J. Clin. Invest. 2012;122:519–528. PubMed PMC

Plagnol V., Curtis J., Epstein M., Mok K.Y., Stebbings E., Grigoriadou S., Wood N.W., Hambleton S., Burns S.O., Thrasher A.J. A robust model for read count data in exome sequencing experiments and implications for copy number variant calling. Bioinformatics. 2012;28:2747–2754. PubMed PMC

Cingolani P., Platts A., Wang L., Coon M., Nguyen T., Wang L., Land S.J., Lu X., Ruden D.M. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin) 2012;6:80–92. PubMed PMC

Paila U., Chapman B.A., Kirchner R., Quinlan A.R. GEMINI: integrative exploration of genetic variation and genome annotations. PLoS Comput. Biol. 2013;9:e1003153. PubMed PMC

Li H., Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010;26:589–595. PubMed PMC

Kumar P., Henikoff S., Ng P.C. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat. Protoc. 2009;4:1073–1081. PubMed

Cartharius K., Frech K., Grote K., Klocke B., Haltmeier M., Klingenhoff A., Frisch M., Bayerlein M., Werner T. MatInspector and beyond: promoter analysis based on transcription factor binding sites. Bioinformatics. 2005;21:2933–2942. PubMed

Peh G.S., Beuerman R.W., Colman A., Tan D.T., Mehta J.S. Human corneal endothelial cell expansion for corneal endothelium transplantation: an overview. Transplantation. 2011;91:811–819. PubMed

Valtink M., Gruschwitz R., Funk R.H., Engelmann K. Two clonal cell lines of immortalized human corneal endothelial cells show either differentiated or precursor cell characteristics. Cells Tissues Organs (Print) 2008;187:286–294. PubMed

Dudakova L., Liskova P., Trojek T., Palos M., Kalasova S., Jirsova K. Changes in lysyl oxidase (LOX) distribution and its decreased activity in keratoconus corneas. Exp. Eye Res. 2012;104:74–81. PubMed

Massie I., Dziasko M., Kureshi A., Levis H.J., Morgan L., Neale M., Sheth R., Tovell V.E., Vernon A.J., Funderburgh J.L., Daniels J.T. Advanced imaging and tissue engineering of the human limbal epithelial stem cell niche. Methods Mol. Biol. 2015;1235:179–202. PubMed PMC

Merjava S., Malinova E., Liskova P., Filipec M., Zemanova Z., Michalova K., Jirsova K. Recurrence of posterior polymorphous corneal dystrophy is caused by the overgrowth of the original diseased host endothelium. Histochem. Cell Biol. 2011;136:93–101. PubMed

Studeny P., Jirsova K., Kuchynka P., Liskova P. Descemet membrane endothelial keratoplasty with a stromal rim in the treatment of posterior polymorphous corneal dystrophy. Indian J. Ophthalmol. 2012;60:59–60. PubMed PMC

Sánchez-Tilló E., Lázaro A., Torrent R., Cuatrecasas M., Vaquero E.C., Castells A., Engel P., Postigo A. ZEB1 represses E-cadherin and induces an EMT by recruiting the SWI/SNF chromatin-remodeling protein BRG1. Oncogene. 2010;29:3490–3500. PubMed

Roca H., Hernandez J., Weidner S., McEachin R.C., Fuller D., Sud S., Schumann T., Wilkinson J.E., Zaslavsky A., Li H. Transcription factors OVOL1 and OVOL2 induce the mesenchymal to epithelial transition in human cancer. PLoS ONE. 2013;8:e76773. PubMed PMC

Chen Y., Huang K., Nakatsu M.N., Xue Z., Deng S.X., Fan G. Identification of novel molecular markers through transcriptomic analysis in human fetal and adult corneal endothelial cells. Hum. Mol. Genet. 2013;22:1271–1279. PubMed PMC

Li B., Dai Q., Li L., Nair M., Mackay D.R., Dai X. Ovol2, a mammalian homolog of Drosophila ovo: gene structure, chromosomal mapping, and aberrant expression in blind-sterile mice. Genomics. 2002;80:319–325. PubMed PMC

Notara M., Daniels J.T. Characterisation and functional features of a spontaneously immortalised human corneal epithelial cell line with progenitor-like characteristics. Brain Res. Bull. 2010;81:279–286. PubMed

Chung D.W., Frausto R.F., Ann L.B., Jang M.S., Aldave A.J. Functional impact of ZEB1 mutations associated with posterior polymorphous and Fuchs’ endothelial corneal dystrophies. Invest. Ophthalmol. Vis. Sci. 2014;55:6159–6166. PubMed PMC

Lee B., Villarreal-Ponce A., Fallahi M., Ovadia J., Sun P., Yu Q.C., Ito S., Sinha S., Nie Q., Dai X. Transcriptional mechanisms link epithelial plasticity to adhesion and differentiation of epidermal progenitor cells. Dev. Cell. 2014;29:47–58. PubMed PMC

de Vooght K.M., van Wijk R., van Solinge W.W. Management of gene promoter mutations in molecular diagnostics. Clin. Chem. 2009;55:698–708. PubMed

Teng A., Nair M., Wells J., Segre J.A., Dai X. Strain-dependent perinatal lethality of Ovol1-deficient mice and identification of Ovol2 as a downstream target of Ovol1 in skin epidermis. Biochim. Biophys. Acta. 2007;1772:89–95. PubMed PMC

Lin Y.C., Boone M., Meuris L., Lemmens I., Van Roy N., Soete A., Reumers J., Moisse M., Plaisance S., Drmanac R. Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations. Nat. Commun. 2014;5:4767. PubMed PMC

Shaw G., Morse S., Ararat M., Graham F.L. Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells. FASEB J. 2002;16:869–871. PubMed

Murphy C., Alvarado J., Juster R., Maglio M. Prenatal and postnatal cellularity of the human corneal endothelium. A quantitative histologic study. Invest. Ophthalmol. Vis. Sci. 1984;25:312–322. PubMed

Sherrard E.S., Novakovic P., Speedwell L. Age-related changes of the corneal endothelium and stroma as seen in vivo by specular microscopy. Eye (Lond.) 1987;1:197–203. PubMed

Mackay D.R., Hu M., Li B., Rhéaume C., Dai X. The mouse Ovol2 gene is required for cranial neural tube development. Dev. Biol. 2006;291:38–52. PubMed PMC

Menendez L., Kulik M.J., Page A.T., Park S.S., Lauderdale J.D., Cunningham M.L., Dalton S. Directed differentiation of human pluripotent cells to neural crest stem cells. Nat. Protoc. 2013;8:203–212. PubMed

Duband J.L., Delannet M., Monier F., Garret S., Desban N. Modulations of cellular interactions during development of the neural crest: role of growth factors and adhesion molecules. Curr. Top. Microbiol. Immunol. 1996;212:207–227. PubMed

Wulle K.G., Lerche W. Electron microscopic observations of the early development of the human corneal endothelium and Descemet’s membrane. Ophthalmologica. 1969;157:451–461. PubMed

Jirsova K., Merjava S., Martincova R., Gwilliam R., Ebenezer N.D., Liskova P., Filipec M. Immunohistochemical characterization of cytokeratins in the abnormal corneal endothelium of posterior polymorphous corneal dystrophy patients. Exp. Eye Res. 2007;84:680–686. PubMed

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