Expression Profile of Optic Nerve Head (ONH) Astrocytes From Caucasian and African American Using Oligonucleotide Microarrays

Abstract

Abstract: : Purpose: To compare gene expression profiles of normal human astrocytes cultured from ONH of Caucasian (CA) and African American (AA) using oligonucleotide microarrays. Methods:We used oligonucleotide Affymetrix microarray (Human 133A genechips) to compare gene expression levels in cultured ONH astrocytes from 7 CA (age 61±11) and 7 AA (age 58±10). Replicates of both individual and pooled RNAs were used to gauge technical errors, and data from a total of 34 genechips were normalized with RMA, dChip and Lowess. Genes showing significant differential expression levels were detected by SAM and empirical Bayes (Smyth, 2004) analysis. Clustering analysis was applied to the selected genes to discover co–regulated gene sets in both ethnic groups. Differential gene expression was confirmed by quantitative (q) RT–PCR and protein levels were measured by Western blots. Proliferation, adhesion and migration assays tested physiological responses. Results: A total of 97 genes were differentially expressed between AA and CA (false positive rate 5%). Multiple analyses selected 8 known genes and 2 ESTs as upregulated, and 6 genes and 1 EST down regulated in AA compared to CA. A significant upregulation of elastin (ELN) mRNA (4.6–fold) was confirmed by qRT–PCR and immunoblot in AA astrocytes. ELN clustered with TGFß1, a positive regulator of ELN synthesis and astrocyte differentiation, and with DDX17, an ATP–dependent helicase that regulates pre–mRNA processing. The signal transduction genes GPR56, a G–protein coupled receptor (2.7–fold), and RAB3B, a Ras GTP–binding protein (1.7–fold), were upregulated in AA. Cell adhesion was 20% lower in AA astrocytes compared to CA (P<0.001), consistent with significant down regulation of expression of integrin α6 (–1.9–fold) and basement membrane collagens types XIII (–1.6–fold) and XV (–3.7–fold). Conclusions: This initial study revealed significant differences in gene expression of ELN, a major structural component of the lamina cribrosa, between AA and CA ONH astrocytes. Down regulation of cell adhesion genes and adhesive properties suggest functional differences in AA astrocytes. Our data suggest gene expression differences in ONH astrocytes that may underlie higher susceptibility to glaucomatous optic neuropathy in AA.

Keywords: gene/expression • astrocytes: optic nerve head • gene microarray