Date Published: March 01, 2018
Author(s): Petra Liskova, Lubica Dudakova, Cerys J. Evans, Karla E. Rojas Lopez, Nikolas Pontikos, Dimitra Athanasiou, Hodan Jama, Josef Sach, Pavlina Skalicka, Viktor Stranecky, Stanislav Kmoch, Caroline Thaung, Martin Filipec, Michael E. Cheetham, Alice E. Davidson, Stephen J. Tuft, Alison J. Hardcastle.
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.
Posterior polymorphous corneal dystrophy (PPCD) is a rare autosomal-dominant disorder, primarily affecting the corneal endothelium and Descemet membrane. The severity and phenotype of PPCD is variable.1 Mild manifestations of the disease include asymptomatic corneal endothelial changes such as vesicular, band-like, and geographic lesions. In severe cases, corneal endothelial failure may occur and corneal transplantation is required to restore vision.1, 2, 3, 4 Aberrant corneal endothelial cells have been shown to proliferate and migrate onto the trabecular meshwork and iris acquiring an epithelial-like morphology.1, 5, 6, 7, 8 Secondary complications are common and include corneal edema, glaucoma, iris adherence to the cornea, and corectopia.1, 2
In this study we identified a locus for autosomal-dominant PPCD, PPCD4, on chromosome 8q22.3–q24.12. WGS revealed three unique non-coding variants within the linked region in the index family (C15), one of which, c.20+544G>T, mapped within a potential regulatory region of GRHL2. Additional recombination events were identified by genotyping in the extended family, that refined the PPCD4 locus (chr8.hg38:101,411,163–109,214,442) and excluded two of the variants, leaving c.20+544G>T as the outstanding candidate disease-causing variant.