RNA sequencing of corneas from two keratoconus patient groups identifies potential biomarkers and decreased NRF2-antioxidant responses

Abstract

Keratoconus is a highly prevalent (1 in 2000), genetically complex and multifactorial, degenerative disease of the cornea whose pathogenesis and underlying transcriptomic changes are poorly understood. To identify disease-specific changes and gene expression networks, we performed next generation RNA sequencing from individual corneas of two distinct patient populations-one from the Middle East, as keratoconus is particularly severe in this group, and the second from an African American population in the United States. We conducted a case: control RNA sequencing study of 7 African American, 12 Middle Eastern subjects, and 7 controls. A Principal Component Analysis of all expressed genes was used to ascertain differences between samples. Differentially expressed genes were identified using Cuffdiff and DESeq2 analyses, and identification of over-represented signaling pathways by Ingenuity Pathway Analysis. Although separated by geography and ancestry, key commonalities in the two patient transcriptomes speak of disease-intrinsic gene expression networks. We identified an overwhelming decrease in the expression of anti-oxidant genes regulated by NRF2 and those of the acute phase and tissue injury response pathways, in both patient groups. Concordantly, NRF2 immunofluorescence staining was decreased in patient corneas, while KEAP1, which helps to degrade NRF2, was increased. Diminished NRF2 signaling raises the possibility of NRF2 activators as future treatment strategies in keratoconus. The African American patient group showed increases in extracellular matrix transcripts that may be due to underlying profibrogenic changes in this group. Transcripts increased across all patient samples include Thrombospondin 2 (THBS2), encoding a matricellular protein, and cellular proteins, GAS1, CASR and OTOP2, and are promising biomarker candidates. Our approach of analyzing transcriptomic data from different populations and patient groups will help to develop signatures and biomarkers for keratoconus subtypes. Further, RNA sequence data on individual patients obtained from multiple studies may lead to a core keratoconus signature of deregulated genes and a better understanding of its pathogenesis. Keratoconus (KCN) is a condition where the cornea develops bilateral ectasia, becomes progressively thin and protrudes conically. The patient develops astigmatism, myopia, corneal scarring, with eventual loss of vision 1-5. The major form of keratoconus is asyndromic, where the cornea alone is affected. However, syndromic types of KCN also exist, and are associated with Down, Leber congenital amaurosis, Turner, Marfan and Ehlers-Danlos syndromes 6. Isolated KCN affects individuals in adolescence with an incidence and prevalence of 13.3/100,000

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

“…A recent study reported RNA sequencing of full-thickness corneas from keratoconus patients of African American and Middle Eastern ancestry who had decreased WNT5A and WNT9A expression, as well as the Wnt signaling intermediate secreted frizzled-related protein 1 (SFRP1). 21 In another study of keratoconus, an RNA array was used to compare the corneal epithelium of keratoconus patients undergoing corneal transplant surgery; this study demonstrated Wnt signaling dysregulations, specifically in LEF1, PITX2, and SFRP1. 6 SFRP1 is a Wnt signaling modulator, and in this study, SFRP expression was associated with patient age, and the protein localized mostly in the basal epithelium.…”

“…A recent study reported RNA sequencing of full-thickness corneas from keratoconus patients of African American and Middle Eastern ancestry who had decreased WNT5A and WNT9A expression, as well as the Wnt signaling intermediate secreted frizzled-related protein 1 ( SFRP1 ) . 21 …”

“…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.…”

The Pathomechanism, Antioxidant Biomarkers, and Treatment of Oxidative Stress-Related Eye Diseases

“…However, the use of this mutation as a marker for KC may not be warranted, as the presence of this mutation in KC patients seems rare and varies from study to study [186]. Multiple antioxidant genes regulated by NRF2, including SOD1, GSTM3, and HMOX1, are decreased in patients with KC when compared to those of healthy controls [187]. As for nonenzymatic antioxidants, decreased GSH [121], a reduced glutathione/oxidized glutathione ratio (GSH/GSSG) [188], and stromal serotransferrin [124] have been found in KC corneas when compared with those of healthy corneas.…”

Comprehensive Transcriptome Analysis of Patients With Keratoconus Highlights the Regulation of Immune Responses and Inflammatory Processes

“…Proteomics studies revealed decreased expression of structural proteins (Chaerkady et al, 2013) and increased expression of degradative enzymes. Pathway analyses indicated the involvement of genes related to extracellular matrix organization (Kabza et al, 2017), transforming growth factor β signaling, WNT pathway (You et al, 2018), and nuclear factor erythroid 2-related factor 2-regulated network (Shinde et al, 2020). In recent years, omics studies improved our understanding of the molecular mechanisms of KTCN (McKay et al, 2016;Kabza et al, 2019;Karolak et al, 2020).…”