Further evaluation of differential expression of keratoconus candidate genes in human corneas

Abstract

Background: Keratoconus (KTCN) is a progressive eye disease, characterized by changes in the shape and thickness of the cornea that results in loss of visual acuity. While numerous KTCN candidate genes have been identified, the genetic etiology of the disease remains undetermined. To further investigate and verify the contribution of particular genetic factors to KTCN, we assessed 45 candidate genes previously indicated as involved in KTCN etiology based on transcriptomic and genomic data.

Methods: The RealTime ready Custom Panel, covering 45 KTCN candidate genes and two reference transcripts, has been designed. Then, the expression profiles have been assessed using the RT-qPCR assay in six KTCN and six non-KTCN human corneas, obtained from individuals undergoing a penetrating keratoplasty procedure.

Results: In total, 35 genes exhibiting differential expression between KTCN and non-KTCN corneas have been identified. Among these genes were ones linked to the extracellular matrix formation, including collagen synthesis or the TGF-β, Hippo, and Wnt signaling pathways. The most downregulated transcripts in KTCN corneas were CTGF, TGFB3, ZNF469, COL5A2, SMAD7, and SPARC, while TGFBI and SLC4A11 were the most upregulated ones. Hierarchical clustering of expression profiles demonstrated almost clear separation between KTCN and non-KTCN corneas. The gene expression levels determined using RT-qPCR showed a strong correlation with previous RNA sequencing (RNA-Seq) results.

Conclusions: A strong correlation between RT-qPCR and earlier RNA-Seq data confirms the possible involvement of genes from collagen synthesis and the TGF-β, Hippo, and Wnt signaling pathways in KTCN etiology. Our data also revealed altered expression of several genes, such as LOX, SPARC, and ZNF469, in which single nucleotide variants have been frequently identified in KTCN. These findings further highlight the heterogeneous nature of KTCN.

Keywords: CTGF; Gene expression; Hippo signaling; Keratoconic cornea; Keratoconus genetics; TGF-β pathway; TGFB3; TGFBI; Wnt signaling; ZNF469.

Figure 1. Hierarchical clustering analysis of gene expression.

A heat map indicates hierarchical clustering (Ward linkage) of keratoconus (KTCN) and non-KTCN samples (KR) based on the expression values of 36 analyzed genes. Data was clustered as groups of genes (vertical line) and groups of individuals (horizontal line). Color scale encodes Z-score of gene expression; results indicated in green and red point to expression values above and below the median, respectively. Upper color labeling shows KTCN samples in red and non-KTCN samples in blue.

Figure 2. Gene expression correlations between RT-qPCR and RNA-Seq data.

The diagram shows the relationship between binary logarithms (log2) of the fold change values obtained from RT-qPCR (vertical axis) and RNA-Seq (horizontal axis) experiments for 36 analyzed genes. The Pearson correlation and linear model coefficients, as well as linear regression line are indicated.