Molecular Genetics and Molecular Biology of Cataract

Author: Alpana Dave

Dave, Alpana, 2015 Molecular Genetics and Molecular Biology of Cataract, Flinders University, School of Medicine

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Cataract is an opacification of the ocular lens and is classified as congenital cataract (CC) or as age-related cataract (ARC) depending on the age-of-onset. Causative mutations and SNPs in EPHA2 have been implicated in congenital and age-related cataract, respectively. EPHA2 encodes a tyrosine kinase receptor which is expressed in lens cells and plays an important role in lens development. The main objective of this project was to investigate the role of EPHA2 in congenital and age-related cataract development.

CC causing mutations in EPHA2 were previously reported through linkage and candidate-gene analysis including a mutation reported in an Australian family. However, the genetic contribution of mutations in EPHA2 to CC cases in Australia is unknown. To determine this, EPHA2 gene was screened for mutations in a South-Eastern Australian cohort of CC cases. We found two families, each with a novel mutation and two new families with a previously reported causative mutation in EPHA2. The mutations in EPHA2 accounted for 4.7% of CC cases in South-Eastern Australia.

Further, we investigated the effect of two previously reported and three mutations found in this study on EPHA2 localisation. EPHA2 is highly expressed in epithelial cells and interacts with cell-junction proteins. Therefore we analysed the effect of these mutations in epithelial cell lines. Two mutant proteins localised in the perinuclear region and co-localised with a cis-golgi marker. These mutations possibly delay recruitment of mutant EPHA2 to the cell membrane affecting its function. The other three mutations localised to the cell periphery as the wild-type protein.

Environmental factors namely, age, gender and ultraviolet-B (UV-B) radiation are associated with increased risk of ARC development and also affect EPHA2 expression. We investigated if these environmental factors interact with EPHA2 and consequently affect ARC development using an Epha2-/- mice. These mice, on FVB/NJ background, were reported to develop ARC between 5-8 months of age while Epha2+/- mice had clear lenses until 14 months of age. However, we found that Epha2-/- and Epha2+/- mice on C57BL/6J background developed cataract by 4 months and 8 months of age, respectively, likely attributable to differences in the background strain compared to the previous report. Consistent with previous reports, diffused localisation of cell-junction proteins in Epha2 knockout lenses suggested that lack of Epha2 may affect lens cellular junctions. Accumulation of a partial Epha2 fusion protein, generated as a result of the knockout, may contribute to the difference in phenotype observed in Epha2 knockout mouse strains. Additionally, gender had a small effect on cataract progression in Epha2 knockout mice. UV-B exposure of Epha2+/- and Epha2+/+ mice demonstrated a dose-dependent difference in cataract development in both the genotypes and no difference in UV-B-induced cataract between the two genotypes.

Overall, mutations in EPHA2 contribute to a significant proportion of CC in South-Eastern Australia. Analysis of causative mutations and studies in Epha2-/- mouse suggest a role of Epha2 at cell-junctions in the lens, disruption of which results in cataract. Additionally, this study demonstrates an effect of age and gender on Epha2 mediated ARC development.

Keywords: Cataract, EPHA2, Genetics, Congenital cataract, Age-related cataract, cell culture, Knockout mouse model, Ultraviolet light

Subject: Ophthalmology thesis

Thesis type: Doctor of Philosophy
Completed: 2015
School: School of Medicine
Supervisor: Dr. Shiwani Sharma