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Title: Gene expression profiling in Keratoconus
Author: Muszynska , Dorota
Awarding Body: Queen's University Belfast
Current Institution: Queen's University Belfast
Date of Award: 2013
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Keratoconus (MIM#148300), a common bilateral, progressive corneal thinning disorder, is the leading indication for corneal transplantation in the developed world. Keratoconus usually arises in the teenage years and presents a significant health burden in work-age adults. Despite the visual and social impact of keratoconus, our lack of understanding of the molecular pathology of keratoconus is a major obstacle to the development of new therapeutic approaches. This study represents the first reported application of massively parallel sequencing of mRNA (RNA-Seq) to perform whole genome transcriptome analysis of keratoconic keratocytes. Genes-enrichment analysis identified several pathway maps that are disrupted in keratoconic keratocytes and associated with the pathogenesis of keratoconus. Microarray gene expression was used to validate differentially expressed genes identified by RNA-Seq in a global manner, whereas quantitative reverse transcriptase polymerase chain reaction was performed on selected candidate genes. Wnt signalling, TGF-beta signalling. ECM-matrix interactions, oxidative stress and inflammatory- related genes were specifically identified in keratoconic keratocytes and implicated in the pathogenesis of keratoconus. Analysis of individual target genes identified altered expression of both known and novel keratoconus-related genes, in particular, SFRP1, BMP4, CBS, POSTN, C0L11Al, COL4Al, SOD1, IL6, and SP3. Functional analyses and expression profiling of keratoconic keratocytes harbouring a novel heterozygous missense mutation (c.l920G>T; p.Gln640His) in the zinc finger E-box binding gene 1 (ZEB1) was also performed. The mutant ZEB1 protein was stable and localised to the nucleus resulting in an enhanced transcriptional repressor of known ZEB1 targets, involved in epithelialmesenchymal transition and collagen synthesis. This ZEB1 mutation results in a gain-In-function with enhanced transcriptional repression of a number of gene targets associated with keratoconus, corneal thickness and Fuchs endothelial corneal dystrophy. This study has identified a number of molecular targets for keratoconus and provides a significant insight into the gene pathways involved in keratoconus pathogenesis. Further functional studies can build on this evidence to interrogate disease pathogenesis, identify novel genes and develop molecular therapies for keratoconus.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available