Identification and Functional Clustering of Global Gene Expression Differences between Age-related Cataract and Clear Human Lenses

Abstract

Abstract: : Purpose: To identify the global gene expression differences between lens epithelia isolated from age-related cataract relative to clear human lenses and to functionally cluster the differentially expressed genes.Methods: RNA was extracted from 156 epithelial tags recovered from cataract surgery and from 20 approximately age-matched epithelial tags microdissected from clear lenses. Gene expression changes between these samples were detected by DNA microarray hybridization using Affymetrix U133A chips containing about 20,000 genes. Genes were clustered according to their known functions using the EASE statistical/functional algorhythm. Secondary confirmations of identified gene expression differences were conducted on separately prepared RNA samples by semi-quantitative RT-PCR. Results: Analysis of over 20,000 genes revealed that 143 transcripts were increased and 363 transcripts were decreased at levels of 4-fold or greater between epithelia isolated from cataract versus age-matched clear lenses. Of the genes increased in cataract, the majority have known functions involving transcription, nucleic acid binding and processing, protein processing, ion transport and cell growth/maintenance. Of the genes decreased in cataract, the majority have known functions involving heat-shock/chaperone activity, protein synthesis/degradation, oxidative stress and metal binding. The predominant category of altered genes was found to be chaperones which included HSP90, HSP40 sub-families A, B and C, HSP70 1A and 1B, chaperonin containing protein subunits 3,4 and 7, HSP27, peptidyl-prolyl isomerase B, chaperonin 10, prefoldins 4 and 5 and calnexin. The expression levels of 10 out of 13 differentially expressed genes were confirmed using separately prepared populations of RNA indicating that the microarray data is about 77% accurate. Conclusions: These data provide evidence that cataract is associated with multiple previously identified and novel changes in lens epithelial gene expression. Although not all of the detected gene expression differences were confirmable and spatial/dissection differences between samples cannot be excluded from the present study, the identified pathways provide insight into those mechanisms likely to be important for cataract. Individual genes, their associated pathways and ongoing functional analysis of these will be described.

Keywords: cataract • gene/expression • gene microarray