Altered gene expression in dry age-related macular degeneration suggests early loss of choroidal endothelial cells

Abstract

Purpose: Age-related macular degeneration (AMD) is a major cause of blindness in developed countries. The molecular pathogenesis of early events in AMD is poorly understood. We investigated differential gene expression in samples of human retinal pigment epithelium (RPE) and choroid from early AMD and control maculas with exon-based arrays.

Methods: Gene expression levels in nine human donor eyes with early AMD and nine control human donor eyes were assessed using Affymetrix Human Exon ST 1.0 arrays. Two controls did not pass quality control and were removed. Differentially expressed genes were annotated using the Database for Annotation, Visualization and Integrated Discovery (DAVID), and gene set enrichment analysis (GSEA) was performed on RPE-specific and endothelium-associated gene sets. The complement factor H (CFH) genotype was also assessed, and differential expression was analyzed regarding high AMD risk (YH/HH) and low AMD risk (YY) genotypes.

Results: Seventy-five genes were identified as differentially expressed (raw p value <0.01; ≥50% fold change, mean log2 expression level in AMD or control ≥ median of all average gene expression values); however, no genes were significant (adj. p value <0.01) after correction for multiple hypothesis testing. Of 52 genes with decreased expression in AMD (fold change <0.5; raw p value <0.01), 18 genes were identified by DAVID analysis as associated with vision or neurologic processes. The GSEA of the RPE-associated and endothelium-associated genes revealed a significant decrease in genes typically expressed by endothelial cells in the early AMD group compared to controls, consistent with previous histologic and proteomic studies. Analysis of the CFH genotype indicated decreased expression of ADAMTS9 in eyes with high-risk genotypes (fold change = -2.61; raw p value=0.0008).

Conclusions: GSEA results suggest that RPE transcripts are preserved or elevated in early AMD, concomitant with loss of endothelial cell marker expression. These results are consistent with the notion that choroidal endothelial cell dropout or dedifferentiation occurs early in the pathogenesis of AMD.

Figure 1

Overview of bioinformatics pipeline. Software is shown in the upper left corner of the boxes while the analytic process is indicated in the center of the boxes.

Figure 2

Quality control plots generated by arrayQualityMetrics before and after removal of CTRL3 and CTRL9. A and B are a false color heatmaps indicating the distances between arrays, computed as the absolute mean distance between the data. C and D indicate the sum of distances computed for A and B. For C the outlier threshold was 7.14 (vertical bar), and for D the outlier threshold was 5.79 (vertical bar). When analyzing all 18 samples, both CTRL3 and CTRL9 were flagged as exceeding the outlier threshold (A and C). When CTRL3 and CTRL9 were omitted, only ARM7 exceeded the threshold (D). E and F are boxplots of the gene-level signal intensity distribution across the arrays. For E and F, the Kolmogorov-Smirnov statistic K_a was calculated based on the distanced between each individual array and the pooled distribution of all arrays. G and H show K_a for each array with outlier thresholds (vertical bars) of 0.0509 and 0.049, respectively. Only CTRL3 was flagged by this metric (G).

Figure 3

Distribution of mean expression before median thresholding. Mean expression is log₂ of gene-level signal intensity. The median indicated by the red line.

Figure 4

Heatmap of differentially expressed genes shown in Table 2. Dark shading indicates low expression; light shading indicates high expression.

Figure 5

Heatmap of genes previously associated with or possibly implicated in AMD. Dark shading indicates low expression; light shading indicates high expression.

Figure 6

Heatmap of genes highly expressed in the RPE. Dark shading indicates low expression; light shading indicates high expression.

Figure 7

Heatmap of genes associated with endothelial cells. Note the trend toward decreased expression in the AMD samples (ARM 1–9). Dark shading indicates low expression; light shading indicates high expression.