Ectopic GRHL2 Expression Due to Non-coding Mutations Promotes Cell State Transition and Causes Posterior Polymorphous Corneal Dystrophy 4

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

doi:10.1016/j.ajhg.2018.02.002

Abstract

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.

Highlights

Posterior polymorphous corneal dystrophy (PPCD) is a rare autosomal-dominant disorder, primarily affecting the corneal endothelium and Descemet membrane
We considered that an additional PPCD locus and/or a variant not captured by Whole-Exome Sequencing (WES) might be causative in this family
GRHL2 encodes a transcription factor that is a direct transcriptional repressor of ZEB1.49 Given this role, in addition to the lack of GRHL2 expression in the corneal endothelium, we hypothesized that the putative regulatory mutations could lead to inappropriate transcriptional activation and ectopic expression of GRHL2 in the corneal endothelium, similar to the mechanism we proposed for the variants in OVOL2.2 To explore this hypothesis further, a full-thickness corneal sample from individual identified in the proband (II):[1] from family C23, with the GRHL2 c.20þ544G>T variant, was analyzed by IHC and compared to control tissue

Summary

Introduction

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. 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]

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