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.

Changes in Nuclear Gene Expression Related to Mitochondrial Function Affect Extracellular Matrix, Collagens, and Focal Adhesion in Keratoconus

“…Of the 1223 nuclear-encoded genes related to mitochondrial function, 13 – 19 56 and 41 were significantly down- and upregulated, respectively, in KTCN corneas compared with non-KTCN corneas based on our previous RNA-seq study 20 , 21 , 37 ( Supplementary Table S2 ). Seven of these 97 differentially expressed genes overlapped those from the top overrepresented molecular pathways that were previously described as deregulated in KTCN, including TGFB1 , P4HB , and BCL2 , which are involved in the extracellular matrix (ECM) organization, collagen formation, focal adhesion, non-integrin membrane–ECM interactions, and elastic fiber formation ( Table 1 ).…”

Transcriptomic and Immunohistochemical Analysis of Progressive Keratoconus Reveal Altered WNT10A in Epithelium and Bowman's Layer

“… 8 A Wnt ligand— WNT5A —was initially shown to be dysregulated in this study of full-thickness corneal tissue, but a follow-up study showed similar expression in keratoconus patients and controls. 19 Nevertheless, DNA methylation profiles were shown to be different for WNT3 and WNT5A in keratoconus full-thickness corneal samples. 40 Of note, the control group used in these studies comprised full-thickness abnormal corneas that underwent surgery for reasons other than keratoconus (e.g., ulcers and bullous keratopathy), so the results may have been skewed by comparison to abnormal as opposed to healthy controls.…”

“…All corneal epithelial patient samples used in the RNA-seq study originated from patients with mild to moderate, progressive disease, whereas some previous studies used full-thickness corneas tissue obtained during keratoplasty, which is typically reserved for end-stage disease. 8 , 9 , 19 , 20 Therefore, these samples may provide a more precise representation of the transcriptomic profile of cells with early yet active keratoconus. In addition, a large number of keratoconus samples were obtained for both the large qPCR validation cohort and immunohistochemistry studies.…”

“…To identify key keratoconus regulators, transcriptomic studies of keratoconus corneas have been performed [7][8][9][10][19][20][21] ; however, most were conducted using whole corneas containing all layers, rather than focusing on epithelium. 8,9,[19][20][21] One study did compare epithelial gene expression in keratoconus and control cases but used cases for which disease severity and progression status were not clearly defined. 7 Using samples from progressing cases is important as end-stage keratoconus corneas may show secondary changes, which mask early molecular drivers of disease activity.…”

More than meets the eye: Expanding and reviewing the clinical and mutational spectrum of brittle cornea syndrome

“…Little is known of the function of the ZNF469 protein, but the ZNF469 locus has been associated with central corneal thickness in several genome-wide association studies (Hoehn et al, 2012;Lu et al, 2010;Ulmer et al, 2012;Vitart et al, 2010;Vithana et al, 2011). Some studies describe a link between ZNF469 and keratoconus, but others could not confirm this finding (Davidson et al, 2015;Karolak et al, 2020;Lechner et al, 2014;Lucas et al, 2017;Vincent et al, 2014;Yu et al, 2017). A specific novel heterozygous PRDM5 missense variant also segregated within a Pakistani family with Axenfeld-Rieger syndrome, a disorder mainly affecting the anterior segment of the eye and sometimes extending to systemic manifestations .…”