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

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.

ZEB1 insufficiency causes corneal endothelial cell state transition and altered cellular processing

“…Approximately 30% of affected individuals demonstrate a monoallelic mutation of the ZEB1 gene, resulting in ZEB1 insufficiency [12]. A smaller percentage of affected individuals demonstrate non-coding mutations in OVOL2 and GRHL2 , presumably as a result of ectopic expression of either gene in the corneal endothelium, with subsequent repression of ZEB1 transcription [13–16]. As a consequence of ZEB1 insufficiency, various epithelial-like features are observed in PPCD corneal endothelium, including a stratified organization, desmosomal intracellular junctions, and expression of an epithelial-like transcriptomic profile, including increased/ectopic expression of epithelial-associated keratins and cadherins (e.g., CDH1 ), and decreased expression of CDH2 [12, 17, 18].…”

The Right Gene, Expressed at the Wrong Time, or at the Wrong Place

“…Also a specific form of autosomal-dominant corneal endothelial dystrophy, known as posterior polymorphous corneal dystrophy (PPCD), may be due to ectopic gene expression [Liskova et al, 2018]. The clinical course of this disorder is highly variable, ranging from asymptomatic changes of the corneal endothelium, up to blurred and even complete loss of vision.…”

“…Changes in sequence, or structural genome variations, not affecting the coding sequence of a gene are generally considered not to be clinically relevant. Nevertheless, chromosomal breakpoints, copy number variations (CNVs), or single nucleotide variations (SNVs) outside the coding sequence of plausible candidate genes have been observed in some patients with dominant developmental disorders [Krebs et al, 1997;Klopocki et al, 2011;Liskova et al, 2018]. Such variations do not alter the functional properties of the encoded proteins.…”

“…Disturbances of the delicate balance between ZEB1, OVOL2, and GRHL2 levels will thus result in either of 3 forms of PPCD [Liskova et al, 2018]. OVOL2 ectopic expression in corneal endothelial cells leads to high OVOL2/ low ZEB1 PPCD1.…”

“…While mutations within the coding sequence or loss of 1 allele of GRHL2 are linked to hearing loss, upregulation and ectoptic expression of GRHL2 are associated with PPCD without hearing impairment [Liskova et al, 2018]. Hence, a specific and novel mechanism for noncoding GRHL2 SNVs in causing PPCD has to be conceived.…”