SAGE Expression Analysis of Trabecular Meshwork to Identify POAG Susceptibility Genes

Abstract

Abstract: : Purpose: Elevated intraocular pressure is the leading risk factor for primary open angle glaucoma. We hypothesize that gene expression changes in the trabecular meshwork (TM) will reveal the mechanism of this IOP elevation. We have profiled gene expression in the TM to shed light on the metabolic activity of this tissue and to identify candidate susceptibility genes for POAG. Methods: Serial Analysis of Gene Expression (SAGE) was performed on 5µg total RNA from primary cultures of human TM cells, which had been isolated from two POAG patients and two controls. Candidate genes were sequenced in 16 POAG patients and controls. Taqman was used to genotype single nucleotide polymorphisms for association analysis. Results: Among the ten most abundantly expressed genes in the TM are matrix Gla protein (MGP) and Cohen syndrome 1 (COH1). Variants in MGP increase calcification of soft tissue, which could increase resistance to fluid flow in the TM. Variants in COH1 cause multiple ocular phenotypes. The SAGE data also reveal alternative splicing of transcripts. For example, there are two common splice forms of the serine arginine–rich pre–mRNA splicing factor (SR–A1), but only one of these forms is expressed in the TM.We have also combined these SAGE expression profiles with our existing genomic linkage data for POAG (HMG 9: 1109). Those four linkage regions contain 1065 UNIGENE clusters, but only 118 of these genes are expressed in the TM, and even fewer are also differentially expressed in patients and controls. Because they are both positional and functional candidates, these genes are prioritized for analysis in POAG patients and controls. High throughput SNP association and gene sequencing is underway for these candidates. To date no variants associated with POAG have been found in NDN (Necdin homolog), NDNL2 (Necdin–like 2), and the receptors GABRα5, ß3, and γ3. Conclusions: SAGE expression profiles of the TM shed light on the biological activity of that tissue, and provide a rich source of prioritized candidate susceptibility genes for POAG.

Keywords: trabecular meshwork • gene/expression • metabolism