Collagen synthesis disruption and downregulation of core elements of TGF-β, Hippo, and Wnt pathways in keratoconus corneas

Abstract

To understand better the factors contributing to keratoconus (KTCN), we performed comprehensive transcriptome profiling of human KTCN corneas for the first time using an RNA-Seq approach. Twenty-five KTCN and 25 non-KTCN corneas were enrolled in this study. After RNA extraction, total RNA libraries were prepared and sequenced. The discovery RNA-Seq analysis (in eight KTCN and eight non-KTCN corneas) was conducted first, after which the replication RNA-Seq experiment was performed on a second set of samples (17 KTCN and 17 non-KTCN corneas). Over 82% of the genes and almost 75% of the transcripts detected as differentially expressed in KTCN and non-KTCN corneas were confirmed in the replication study using another set of samples. We used these differentially expressed genes to generate a network of KTCN-deregulated genes. We found an extensive disruption of collagen synthesis and maturation pathways, as well as downregulation of the core elements of the TGF-β, Hippo, and Wnt signaling pathways influencing corneal organization. This first comprehensive transcriptome profiling of human KTCN corneas points further to a complex etiology of KTCN.

Figure 1

Hierarchical clustering of KTCN and non-KTCN samples and PCA plots in the discovery study. Heat maps indicate hierarchical clustering of KTCN and non-KTCN samples based on the expression values of differentially expressed genes before removing the samples (a) and after removing the samples (b). PCA plots are based on the expression values of all genes before removing the samples (c) and after removing the samples (d).

Figure 2

Relative quantification and comparison of the expression of TGFB3, CTGF, and COL21A1 genes in KTCN and non-KTCN individuals. Bars indicate mean expression (white in KTCN, black in non-KTCN); the error bar show SD, P-value <0.01.

Figure 3

Dysregulated TGF-β, Hippo, and Wnt pathways in KTCN corneas. Yellow rectangle indicates gene with upregulated expression in KTCN corneas; genes with downregulated expression are shown in blue rectangles. Genes without detectable expression alterations but that are core elements of the pathways are also included and shown in gray rectangles. Downregulated genes, important for functioning of the cornea, are shown as rectangles with additional frames. Statistically significant downregulation of gene expression in KTCN corneas is visible in the presented pathways, whereas only one upregulated gene is exposed.