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A Novel Human Congenital Cataract Mutation in EPHA2 Kinase Domain (p.G668D) Alters Receptor Stability and Function
Yi Zhai1,2, Sha Zhu2, Jinyu Li2
1Department of Ophthalmology and Visual Sciences, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
Purpose:
To identify the genetic defect in a four-generation Chinese family that causes autosomal dominant congenital posterior subcapsular cataracts, and to understand how this EPHA2 kinase domain mutation affects EPHA2 activity.
Methods:
Variants in 54 cataract-associated genes were screened by targeted next generation sequencing (NGS) and then validated by Sanger sequencing. EPHA2 wild-type cDNA was synthesized in vitro, and EPHA2 p.G668D mutant was constructed by PCR site-directed mutagenesis. Western blotting and fluorescence microscopy were used to analyze the expression level of protein and its subcellular localization, respectively. A wound-healing assay was performed to analyze changes to cell migration.
Results:
A novel heterozygous missense mutation was identified in the kinase domain of the EPHA2 gene (c.2003G>A, p.G668D). This is the third congenital cataract mutation being reported in this domain. Functional study revealed that the kinase domain mutation (p.G668D) decreased EphA2 protein level (P = 0.036) via a proteasome-dependent pathway, altered its subcellular localization of the EphA2 from cell-cell contacts to a diffuse perimembranous distribution, and changed the distribution of β-catenin as well. The expression of mutant EphA2 significantly promoted the migration of human lens epithelial cells (P = 0.002).
Conclusions:
Our study presented the evidence for a novel EPHA2 kinase domain mutation that causes congenital posterior subcapsular cataracts. The first functional study on an EPHA2 kinase domain mutation that causes a congenital cataract revealed that the G668D mutation destabilized the receptor, changed its subcellular localization, and altered the activation of EphA2 with its ligand ephrin. The mutant EphA2 resulted in a reduced inhibition of cell migration. As a consequence, the c.G668D mutation promoted cell migration and caused the formation of cataracts.
Insights
A novel mutation in the EPHA2 gene causes congenital cataracts by affecting protein levels and cell migration. This discovery sheds light on the genetic basis of posterior subcapsular cataracts.
Area of Science:
- Genetics
- Molecular Biology
- Ophthalmology
Background:
- Congenital cataracts are a leading cause of childhood blindness.
- Genetic factors play a significant role in the development of cataracts.
- Mutations in the EPHA2 gene have been implicated in cataract formation.
Purpose of the Study:
- Identify the genetic cause of autosomal dominant congenital posterior subcapsular cataracts in a Chinese family.
- Investigate the functional impact of an EPHA2 kinase domain mutation on EPHA2 activity and lens cell behavior.
Main Methods:
- Targeted next-generation sequencing (NGS) and Sanger sequencing to identify gene variants.
- In vitro synthesis and mutagenesis of EPHA2 cDNA.
- Western blotting and fluorescence microscopy to analyze protein expression and localization.
- Wound-healing assays to assess cell migration.
Main Results:
- A novel heterozygous missense mutation (c.2003G>A, p.G668D) was identified in the EPHA2 kinase domain.
- The G668D mutation decreased EphA2 protein levels via a proteasome-dependent pathway.
- The mutation altered EphA2 subcellular localization and affected beta-catenin distribution.
- Mutant EphA2 significantly promoted human lens epithelial cell migration.
Conclusions:
- A novel EPHA2 kinase domain mutation is causative for congenital posterior subcapsular cataracts.
- The G668D mutation destabilizes EphA2, alters its localization, and affects ligand binding.
- Reduced inhibition of cell migration by mutant EphA2 leads to cataract formation.
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