In a large family of Czech origin, we mapped a locus for an autosomal-dominant corneal endothelial dystrophy, posterior polymorphous corneal dystrophy 4 (PPCD4), to 8q22.3-q24.12. Whole-genome sequencing identified a unique variant (c.20+544G>T) in this locus, within an intronic regulatory region of GRHL2. Targeted sequencing identified the same variant in three additional previously unsolved PPCD-affected families, including a de novo occurrence that suggests this is a recurrent mutation. Two further unique variants were identified in intron 1 of GRHL2 (c.20+257delT and c.20+133delA) in unrelated PPCD-affected families. GRHL2 is a transcription factor that suppresses epithelial-to-mesenchymal transition (EMT) and is a direct transcriptional repressor of ZEB1. ZEB1 mutations leading to haploinsufficiency cause PPCD3. We previously identified promoter mutations in OVOL2, a gene not normally expressed in the corneal endothelium, as the cause of PPCD1. OVOL2 drives mesenchymal-to-epithelial transition (MET) by directly inhibiting EMT-inducing transcription factors, such as ZEB1. Here, we demonstrate that the GRHL2 regulatory variants identified in PPCD4-affected individuals induce increased transcriptional activity in vitro. Furthermore, although GRHL2 is not expressed in corneal endothelial cells in control tissue, we detected GRHL2 in the corneal "endothelium" in PPCD4 tissue. These cells were also positive for epithelial markers E-Cadherin and Cytokeratin 7, indicating they have transitioned to an epithelial-like cell type. We suggest that mutations inducing MET within the corneal endothelium are a convergent pathogenic mechanism leading to dysfunction of the endothelial barrier and disease.

• Keywords
• GRHL2, PPCD, corneal dystrophy, corneal edema, corneal endothelium, ectopic expression, epithelial-to-mesenchymal transition, mesenchymal-to-epithelial transition, non-coding mutation, regulatory region,
• MeSH
• Corneal Dystrophies, Hereditary genetics   MeSH
• DNA-Binding Proteins genetics   MeSH
• Transcription, Genetic MeSH
• Genetic Loci MeSH
• HEK293 Cells MeSH
• DNA, Intergenic genetics   MeSH
• Introns genetics   MeSH
• Humans MeSH
• Models, Genetic MeSH
• Mutation genetics   MeSH
• Promoter Regions, Genetic genetics   MeSH
• Family MeSH
• Pedigree MeSH
• Endothelium, Corneal pathology   MeSH
• Base Sequence MeSH
• Whole Genome Sequencing MeSH
• Transcription Factors genetics   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• GRHL2 protein, human MeSH Browser
• DNA, Intergenic MeSH
• Transcription Factors MeSH

PURPOSE: Posterior polymorphous corneal dystrophy (PPCD) is characterized by abnormal proliferation of corneal endothelial cells. It was shown that TGF-β2 present in aqueous humor (AH) could help maintaining the corneal endothelium in a G1-phase-arrest state. We wanted to determine whether the levels of this protein are changed in AH of PPCD patients. METHODS: We determined the concentrations of active TGF-β2 in the AH of 29 PPCD patients (42 samples) and 40 cadaver controls (44 samples) by ELISA. For data analysis the PPCD patients were divided based on either the molecular genetic cause of their disease as PPCD1 (37 samples), PPCD3 (1 sample) and PPCDx (not linked to a known PPCD loci, 4 samples) or on the presence (17 samples) or absence (25 samples) of secondary glaucoma or on whether they had undergone penetrating keratoplasty (PK, 32 samples) or repeated PK (rePK, 7 samples). RESULTS: The level of active TGF-β2 in the AH of all PPCD patients (mean ± SD; 386.98 ± 114.88 pg/ml) in comparison to the control group (260.95 ± 112.43 pg/ml) was significantly higher (P = 0.0001). Compared to the control group, a significantly higher level of active TGF-β2 was found in the PPCD1 (P = 0.0005) and PPCDx (P = 0.0022) groups. Among patients the levels of active TGF-β2 were not significantly affected by gender, age, secondary glaucoma or by the progression of dystrophy when one or repeated PK were performed. CONCLUSION: The levels of active TGF-β2 in the AH of PPCD patients are significantly higher than control values, and thus the increased levels of TGF-β2 could be a consequence of the PPCD phenotype and can be considered as another feature characterizing this disease.

• MeSH
• Corneal Dystrophies, Hereditary metabolism   MeSH
• Glaucoma metabolism   MeSH
• Keratoplasty, Penetrating methods   MeSH
• Aqueous Humor metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Cornea metabolism   MeSH
• Endothelium, Corneal metabolism   MeSH
• Transforming Growth Factor beta2 metabolism   MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Transforming Growth Factor beta2 MeSH

A substantial proportion of patients with posterior polymorphous corneal dystrophy (PPCD) lack a molecular diagnosis. We evaluated 14 unrelated probands who had a clinical diagnosis of PPCD who were previously determined to be negative for mutations in ZEB1 by direct sequencing. A combination of techniques was used including whole-exome sequencing (WES), single-nucleotide polymorphism (SNP) array copy number variation (CNV) analysis, quantitative real-time PCR, and long-range PCR. Segregation of potentially pathogenic changes with disease was confirmed, where possible, in family members. A putative run of homozygosity on chromosome 10 was identified by WES in a three-generation PPCD family, suggestive of a heterozygous deletion. SNP array genotyping followed by long-range PCR and direct sequencing to define the breakpoints confirmed the presence of a large deletion that encompassed multiple genes, including ZEB1. Identification of a heterozygous deletion spanning ZEB1 prompted us to further investigate potential CNVs at this locus in the remaining probands, leading to detection of two additional heterozygous ZEB1 gene deletions. This study demonstrates that ZEB1 mutations account for a larger proportion of PPCD than previously estimated, and supports the hypothesis that haploinsufficiency of ZEB1 is the underlying molecular mechanism of disease for PPCD3.

• MeSH
• Corneal Dystrophies, Hereditary genetics   pathology   MeSH
• Gene Deletion * MeSH
• Child MeSH
• Adult MeSH
• Haploinsufficiency * MeSH
• Heterozygote MeSH
• Polymorphism, Single Nucleotide MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Pedigree MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Zinc Finger E-box-Binding Homeobox 1 genetics   MeSH
• DNA Copy Number Variations MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Zinc Finger E-box-Binding Homeobox 1 MeSH
• ZEB1 protein, human MeSH Browser

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.

• MeSH
• Alleles * MeSH
• Corneal Dystrophies, Hereditary genetics   MeSH
• DNA MeSH
• Humans MeSH
• Mutation * MeSH
• Promoter Regions, Genetic * MeSH
• Pedigree MeSH
• Base Sequence MeSH
• Sequence Homology, Nucleic Acid MeSH
• Transcription Factors genetics   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Ovol2 protein, human MeSH Browser
• Transcription Factors MeSH