Association Of EphA2Polymorphisms With Age-related Cataract And Pathogenic Mechanisms In A Han Chinese Population

BackgroundAge-related cataract (ARC) is the leading cause of visual impairment worldwide. Thegrowing percentage of aging people leads to the increase of the incidence of ARC,therefore,it is becoming more and more important to elucidate the pathogenesis of ARC. The etiologyand pathogenesis of ARC are still unknown up to date. It has been well established thatARC is a complex polygenic disease which is correlated with genetic and environmentfactors. Genetic studies have demonstrated that Eph-receptor tyrosine kinase-type A2(EphA2) was involved in the pathogenesis of ARC. Polymorphisms of EphA2have beenfound to be associated with cortical ARC in Caucasian population. The association ofEphA2polymorphisms with ARC in Chinese population remains to be unraveled.Abnormal apoptosis of lens epithelial cells (LECs) play an important role on theoccurrence and development of non-congenital cataract. EphA2receptor is widelyexpressed in cells derived from epithelium. It has been known that the signal pathwaymediated by EphA2kinase plays an important role in proliferation, cell adhesion, migrationand apoptosis of cells by activating MAPK/ERK signal transduction. Aging is a knownimportant risk factor for ARC. Therefore, we hypothesize that variations of EphA2genecould lead to ARC by affecting expression and phosphorylation of EphA2, inhibitingMAPK/ERK signal transduction and inducing lens epithelial cells apoptosis, and that agingcould accelerate the progress of ARC formation by enhancing the pathogenicity ofvariations of EphA2gene.ObjectivesTo investigate the association of EphA2polymorphisms and ARC in Han Chinesepopulation, elucidate the pathogenic mechanisms of ARC by observing the effects of EphA2polymorphisms and aging on apoptosis of human lens epithelial cells and evaluating the role of MAPK/ERK signal transduction pathway on the formation of ARC.Methods1. We carried out a population-based genetic association study. Fivesingle-nucleotide polymorphisms (SNPs) in EphA2gene, including rs3768293, rs3754334,rs477558, rs707455, and rs7548209, were genotyped in422Han Chinese patients withage-related cortical cataract and317age, sex and ethnically matched healthy controls usinga PCR restriction fragment length polymorphism (PCR-RFLP) assay. We identified thedifferences in the frequencies of the genotype and allele of EphA2gene between corticalARC groups and controls, explored the association of genetic polymorphisms of EphA2with susceptibility to cortical ARC and searched for single nucleotide polymorphisms(SNPs) correlated with ARC.2. The anterior capsule epithelium sample of ARC patients were collected duringcataract surgery and that of healthy controls were collected during keratoplasty. The mRNAexpressions of EphA2and ERK1/2were determined with quantitative real-time PCR, theprotein expressions of EphA2, phospho-EphA2, ERK1/2and phospho-ERK1/2weredetected with Western-blot. The expression and clinical significance of EphA2andERK1/2in the lens anterior capsule epithelium of ARC patients were investigated.3. The mRNA expressions of caspase-3, caspase-9and bcl-2were determined withquantitative real-time PCR. The protein expressions of caspase-3and active caspase-3inlens anterior capsule epithelium of ARC patients were determined with Western-blot. Theeffect of apoptosis-related genes in the occurrence and progress of ARC was evaluated.Results1. Five SNPs in EphA2gene were genotyped in422Han Chinese patients withcortical ARC and317healthy controls. There were significant differences in frequencies ofgenotype and allele of rs477558and rs7548209in EphA2gene in cortical ARC patients.The frequencies of AA genotype of rs477558, GG genotype and G allele of rs7548209were significantly increased in ARC patients compared with controls (Pc <0.05).Significantly decreased frequencies of rs477558AG genotype, rs7548209CG genotypeand C allele were observed in ARC patients (Pc <0.05). There was no difference in thefrequencies of the genotype and allele of rs3768293, rs3754334, and rs707455SNPsbetween patients with ARC and controls(Pc＞0.05). In addition, when stratified for aged patients with ARC, both of the frequencies of GG genotype and G allele of rs7548209significantly increased with age (Pc <0.05).2. The mRNA expressions of EphA2and ERK1/2, the protein expressions of EphA2,phospho-EphA2, ERK1/2and phospho-ERK1/2were lower in ARC patients than inhealthy controls (P <0.01); The mRNA expressions of EphA2and ERK1/2, the proteinexpressions of EphA2, phospho-EphA2, ERK1/2and phospho-ERK1/2were lower in theARC patients with age of over75than that of less than75(P <0.05); There were nodifferences of the mRNA expressions of EphA2and ERK1/2and protein expression ofEphA2and p-EphA2, ERK1/2and p-ERK1/2among the ARC patients of differentgenotypes of SNP rs7548209.3. The mRNA level of caspase-3and caspase-9and the protein expression ofcaspase-3and active caspase-3were increased in the ARC patients (P <0.01). The mRNAlevel of caspase-3and caspase-9and the protein expression of caspase-3and activecaspase-3were higher in the ARC patients with age of over75than that of less than75(P <0.05). The mRNA level of bcl-2was also detected by real-time qPCR assay and was lowerin ARC patients than in healthy controls (P <0.01), lower in ARC patients with age ofover75than that of less than75(P <0.05). There were no differences of the mRNA levelof caspase-3, caspase-9and bcl-2and the protein expression of caspase-3and activecaspase-3among the ARC patients of different genotype groups of SNP rs7548209.Conclusion1. EphA2polymorphisms are associated with age-related cortical cataract in a HanChinese population. In addition, when stratified for age of the patients with ARC, both ofthe frequencies of GG genotype and G allele of rs7548209are significantly increased withage.2. Aging can affect the apoptosis of LECs related to cataract formation. Themechanism might be the increase of apoptosis of LECs resulting from age-dependentreduction of protein level and the activity of EphA2in LECs and inhibition of ERK1/2signal pathway during aging.3. The rs7548209polymorphism did not modify the steady-state level of EphA2, theapoptosis and the apoptosis-related signaling pathway in LECs, which suggests thatrs7548209in EphA2, might not be a disease-causing locus for cortical ARC, but a marker of this disease.4. The association of rs7548209polymorphism in EphA2with ARC might be theresult of combined interactions of the apoptosis of LECs during aging and other unknownpathogenic mechanisms leading to ARC.