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

Justyna A Karolak; Barbara Ginter-Matuszewska; Katarzyna Tomela; Michal Kabza; Dorota M Nowak-Malczewska; Malgorzata Rydzanicz; Piotr Polakowski; Jacek P Szaflik; Marzena Gajecka

doi:10.7717/peerj.9793

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

PeerJ. 2020 Aug 20:8:e9793. doi: 10.7717/peerj.9793. eCollection 2020.

Authors

Affiliations

¹ Chair and Department of Genetics and Pharmaceutical Microbiology, Poznan University of Medical Sciences, Poznan, Poland.
² Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.
³ Department of Medical Genetics, Medical University of Warsaw, Warsaw, Poland.
⁴ Department of Ophthalmology, Medical University of Warsaw, Warsaw, Poland.

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.

Grants and funding

This study was supported by the National Science Centre in Poland, Grants 2012/05/E/NZ5/02127 and 2018/31/B/NZ5/03280 (Marzena Gajecka). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.