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Fuchs endothelial corneal dystrophy (FECD) is a common disease for which corneal transplantation is the only treatment option in advanced stages, and alternative treatment strategies are urgently required. Expansion (≥50 copies) of a non-coding trinucleotide repeat in TCF4 confers >76-fold risk for FECD in our large cohort of affected individuals. An FECD subject-derived corneal endothelial cell (CEC) model was developed to probe disease mechanism and investigate therapeutic approaches. The CEC model demonstrated that the repeat expansion leads to nuclear RNA foci, with the sequestration of splicing factor proteins (MBNL1 and MBNL2) to the foci and altered mRNA processing. Antisense oligonucleotide (ASO) treatment led to a significant reduction in the incidence of nuclear foci, MBNL1 recruitment to the foci, and downstream aberrant splicing events, suggesting functional rescue. This proof-of-concept study highlights the potential of a targeted ASO therapy to treat the accessible and tractable corneal tissue affected by this repeat expansion-mediated disease.

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.

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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.

BackgroundAxenfeld-Rieger syndrome (ARS) is characterised by typical anterior segment anomalies, with or without systemic features. The discovery of causative genes identified ARS subtypes with distinct phenotypes, but our understanding is incomplete, complicated by the rarity of the condition.MethodsGenetic and phenotypic characterisation of the largest reported ARS cohort through comprehensive genetic and clinical data analyses.Results128 individuals with causative variants in PITX2 or FOXC1, including 81 new cases, were investigated. Ocular anomalies showed significant overlap but with broader variability and earlier onset of glaucoma for FOXC1-related ARS. Systemic anomalies were seen in all individuals with PITX2-related ARS and the majority of those with FOXC1-related ARS. PITX2-related ARS demonstrated typical umbilical anomalies and dental microdontia/hypodontia/oligodontia, along with a novel high rate of Meckel diverticulum. FOXC1-related ARS exhibited characteristic hearing loss and congenital heart defects as well as previously unrecognised phenotypes of dental enamel hypoplasia and/or crowding, a range of skeletal and joint anomalies, hypotonia/early delay and feeding disorders with structural oesophageal anomalies in some. Brain imaging revealed highly penetrant white matter hyperintensities, colpocephaly/ventriculomegaly and frequent arachnoid cysts. The expanded phenotype of FOXC1-related ARS identified here was found to fully overlap features of De Hauwere syndrome. The results were used to generate gene-specific management plans for the two types of ARS.ConclusionSince clinical features of ARS vary significantly based on the affected gene, it is critical that families are provided with a gene-specific diagnosis, PITX2-related ARS or FOXC1-related ARS. De Hauwere syndrome is proposed to be a FOXC1opathy.

Keratoconus is characterised by reduced rigidity of the cornea with distortion and focal thinning that causes blurred vision, however, the pathogenetic mechanisms are unknown. It can lead to severe visual morbidity in children and young adults and is a common indication for corneal transplantation worldwide. Here we report the first large scale genome-wide association study of keratoconus including 4,669 cases and 116,547 controls. We have identified significant association with 36 genomic loci that, for the first time, implicate both dysregulation of corneal collagen matrix integrity and cell differentiation pathways as primary disease-causing mechanisms. The results also suggest pleiotropy, with some disease mechanisms shared with other corneal diseases, such as Fuchs endothelial corneal dystrophy. The common variants associated with keratoconus explain 12.5% of the genetic variance, which shows potential for the future development of a diagnostic test to detect susceptibility to disease.

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.