Whole Exome Sequencing Reveals Novel Candidate Genes in Familial Forms of Glaucomatous Neurodegeneration.

Glaucoma is the largest cause of irreversible blindness with a multifactorial genetic etiology. This study explores novel genes and gene networks in familial forms of primary open angle glaucoma (POAG) and primary angle closure glaucoma (PACG) to identify rare mutations with high penetrance. Thirty-one samples from nine MYOC-negative families (five POAG and four PACG) underwent whole-exome sequencing and analysis. A set of prioritized genes and variations were screened in an independent validation cohort of 1536 samples and the whole-exome data from 20 sporadic patients. The expression profiles of the candidate genes were analyzed in 17 publicly available expression datasets from ocular tissues and single cells. Rare, deleterious SNVs in AQP5, SRFBP1, CDH6 and FOXM1 from POAG families and in ACACB, RGL3 and LAMA2 from PACG families were found exclusively in glaucoma cases. AQP5, SRFBP1 and CDH6 also revealed significant altered expression in glaucoma in expression datasets. Single-cell expression analysis revealed enrichment of identified candidate genes in retinal ganglion cells and corneal epithelial cells in POAG; whereas for PACG families, retinal ganglion cells and Schwalbe's Line showed enriched expression. Through an unbiased exome-wide search followed by validation, we identified novel candidate genes for familial cases of POAG and PACG. The SRFBP1 gene found in a POAG family is located within the GLC1M locus on Chr5q. Pathway analysis of candidate genes revealed enrichment of extracellular matrix organization in both POAG and PACG.

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

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.

Increased Epithelial Expression of CTGF and S100A7 with Elevated Subepithelial Expression of IL-1ß in Trachomatous Trichiasis.

PURPOSE: To characterize the histological appearance and expression of pro-inflammatory mediators, growth factors, matrix metalloproteinases and biomarkers of epithelial-mesenchymal transition (EMT) in healthy control and trachomatous trichiasis (TT) conjunctival tissue. METHODS: Conjunctival biopsies were taken from 20 individuals with TT and from 16 individuals with healthy conjunctiva, which served as controls. Study participants were of varying ethnicity and were living in a trachoma-endemic region of northern Tanzania. Formalin-fixed paraffin-embedded tissue sections were stained using hematoxylin and eosin or by immunohistochemistry using antibodies against: IL-1ß, IL-6, IL-17A, IL-22, CXCL5, S100A7, cleaved caspase 1 (CC1), PDGF, CTGF, TGFß2, MMP7, MMP9, E-cadherin, vimentin, and αSMA. RESULTS: Tissue from TT cases had a greater inflammatory cell infiltrate relative to controls and greater disruption of collagen structure. CTGF and S100A7 were more highly expressed in the epithelium and IL-1ß was more highly expressed in the substantia propria of TT cases relative to controls. Latent TGFß2 was slightly more abundant in the substantia propria of control tissue. No differences were detected between TT cases and controls in the degree of epithelial atrophy, the number of myofibroblasts or expression of EMT biomarkers. CONCLUSIONS: These data indicate that the innate immune system is active in the immunopathology of trachoma, even in the absence of clinical inflammation. CTGF might provide a direct link between inflammation and fibrosis and could be a suitable target for therapeutic treatment to halt the progression of trachomatous scarring.