Description
INTRODUCTION
Keratoconus (KC) is a complex, progressive corneal ectasia and one of the most common causes of visual deterioration in young adults. Genetic risk contributes to the development of KC, but we lack understanding of the aetiology of this condition. Here we aim to complement our GWAS approaches with experimental investigation of familial KC, with the potential to identify rare variants not detected in GWAS and variants with larger effect that confer substantial risk of KC.
METHODS
We recruited a KC pedigree with multiple affected individuals across two generations for whole genome sequencing (WGS) and urine sample collection to generate iPSCs. WGS analysis initially focussed on any shared haplotypes that encompass previously identified GWAS loci and rare variants within these loci were assessed for potential pathogenicity. Candidate genes were assessed for biological relevance and corneal expression. Using a newly developed protocol, we differentiated corneal epithelial-like cells (CEpi) from iPSCs and performed immunocytochemistry to test for expression of corneal epithelial cell markers and bulk RNA sequencing.
RESULTS
Two haplotypes encompassing GWAS loci that fully or partially segregated with KC were identified encompassing the candidate genes RORA, DOCK9, COL4A1 and COL4A2. CEpi were differentiated from iPSCs for one KC patient and one control line as a pilot study. Successful differentiation was demonstrated using ICC for CEpi markers (K14, K3, P63 and PAX6). Bulk RNA sequencing confirmed CEpi differentiation and enabled identification of dysregulated genes and variants in candidate loci. COL4A1 and COL4A2 were downregulated in patient CEpi and rare intronic variants were identified in both genes on chr13q32.1-q34.
CONCLUSION
Integrating genome sequencing with iPSC-derived CEpi models for familial KC holds promise for identifying genes, variants and pathways that confer substantial risk of developing KC. We aim to expand this cohort and perform transcriptomics on a larger scale to validate candidate genes and pathways identified in this pilot study.
Lay Abstract
Keratoconus is a common degenerative corneal condition affecting teenagers and young adults. Individuals with keratoconus develop a distorted cornea, the outermost transparent layer of the eye, resulting in deterioration of vision. Currently, our understanding of what causes keratoconus is limited. To help preserve vision, we need to understand what causes keratoconus and who is at risk of developing this condition. In this study we recruited a large family affected by keratoconus with the aim of identifying genetic risk factors. Large families are rarely seen in clinic but provide us with the opportunity to see what DNA changes affected individuals within a family share. This experiment resulted in the identification of several genes as potentially contributing to disease development. Additionally, kidney cells were grown from urine samples collected from the family as part of a pilot study. We then produced stem-like cells from the kidney cells, and grew these in special conditions so that they turned into corneal cells. This allowed us to investigate the effect of the DNA changes in the affected cell type, corneal cells. From this we identified two candidate genes which have DNA changes and are behaving abnormally.
| Lay Title | Investigating the genetic causes of keratoconus using stem cell–derived corneal cells |
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| Role | Postdoctoral Researcher |