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.

Keratoconus (KTCN), a non-inflammatory corneal disorder characterized by stromal thinning, represents a major cause of corneal transplantations. Genetic and environmental factors have a role in the etiology of this complex disease. Previously reported linkage analysis revealed that chromosomal region 13q32 is likely to contain causative gene(s) for familial KTCN. Consequently, we have chosen eight positional candidate genes in this region: MBNL1, IPO5, FARP1, RNF113B, STK24, DOCK9, ZIC5 and ZIC2, and sequenced all of them in 51 individuals from Ecuadorian KTCN families and 105 matching controls. The mutation screening identified one mutation and three sequence variants showing 100% segregation under a dominant model with KTCN phenotype in one large Ecuadorian family. These substitutions were found in three different genes: c.2262A4C (p.Gln754His) and c.720+43A4G in DOCK9; c.2377-132A4C in IPO5 and c.1053+29G4C in STK24. PolyPhen analyses predicted that c.2262A4C (Gln754His) is possibly damaging for the protein function and structure. Our results suggest that c.2262A4C (p.Gln754His) mutation in DOCK9 may contribute to the KTCN phenotype in the large KTCN-014 family.

ABSTRACT.Purpose: Keratoconus (KTCN) is a degenerative disorder characterized by stromal thinning and protrusion of the cornea, resulting in severe impairment of visual function. A recent study proposed that rare heterozygous mutations in ZNF469 determine KTCN aetiology. Methods: To investigate the contribution of ZNF469 to KTCN, we Sanger sequenced ZNF469 in 42 unrelated Polish patients with KTCN and 49 Polish individuals with high myopia (HM) and compared the results with whole-exome sequencing (WES) data performed in 268 Polish individuals without ocular abnormalities. Results: The average number of ZNF469 non-synonymous variants was 16.31 and 16.0 for individuals with KTCN and HM, respectively (p = 0.3724). All identified variants were previously reported. Alternative allele frequency (AAF) was determined based on the WES results. Among missense variants, only one (rs528085780) has AAF ≤ 0.001 and was identified in one patient with sporadic KTCN. However, the resulting Arg1864Lys substitution was not predicted to be deleterious. Conclusion: In summary, we have not found a significant enrichment of sequence variants in ZNF469 in Polish patients with KTCN. High prevalence of ZNF469 variants identified in our KTCN group is typical for a common genetic variation observed in general population. Our findings indicate that variation in ZNF469 is not responsible for KTCN and other genetic variants are involved in the development and progression of this disease in Polish patients.

Our investigation showed that KTCN-related sequence variants of analyzed genes were found in a very small proportion of the studied patients indicating that genes other than VSX1, TGFBI, DOCK9, IPO5, and STK24 are involved in the development and progression of KTCN in Polish patients. Our results support the hypothesis about the genetic heterogeneity of KTCN.

Oxidative stress may play a role in the pathogenesis of keratoconus (KC) and Fuchs endothelial corneal dystrophy (FECD). Iron may promote the stress by the Fenton reaction, so its homeostasis should be strictly controlled. Transferrin is essential for iron homeostasis because it transports iron from plasma into cells. The malfunction of transferrin, which may be caused by variation in its gene (TF) variation, may contribute to oxidative stress and change KC and FECD risk. To verify this hypothesis we investigated the association between three polymorphisms of the TF gene, g.3296G>A (rs8177178), g.3481A>G (rs8177179), and c.–2G>A (rs1130459), and KC and FECD occurrence. Genotyping was performed in blood lymphocytes in 216 patients with KC, 130 patients with FECD and 228 controls by PCR-RFLP. We studied also the influence of other risk factors. The A/A genotype and the A allele of the g.3296G>A polymorphism were associated with KC occurrence, while the G allele was negatively correlated with it. We observed a decrease in KC occurrence associated with the A/G genotype of the g.3481A>G polymorphism. We did not find any association between the c.–2G>A polymorphism and KC. No association was found between all three polymorphisms and FECD occurrence.

Background Keratoconus (KTCN) is a protrusion and thinning of the cornea, resulting in loss of visual acuity. The etiology of KTCN remains unclear. The purpose of this study was to assess the potential involvement of new genetic variants in KTCN etiology based on both the genomic and transcriptomic findings recognized in the same corneal tissues. Methods Corneal tissues derived from five unrelated Polish individuals with KTCN were examined using exome sequencing (ES), followed by enrichment analyses. For comparison purposes, the datasets comprising ES data of five randomly selected Polish individuals without ocular abnormalities and five Polish patients with high myopia were used. Expression levels of selected genes from the overrepresented pathways were obtained from the previous RNA-Seq study. Results Exome capture discovered 117 potentially relevant variants that were further narrowed by gene overrepresentation analyses. In each of five patients, the assessment of functional interactions revealed rare (MAF ≤ 0.01) DNA variants in at least one gene from Wnt signaling (VANGL1, WNT1, PPP3CC, LRP6, FZD2) and focal adhesion (BIRC2, PAK6, COL4A4, PPP1R12A, PTK6) pathways. No genes involved in pathways enriched in KTCN corneas were overrepresented in our control sample sets. Conclusions The results of this first pilot ES profiling of human KTCN corneas emphasized that accumulation of sequence variants in several genes from Wnt signaling and/or focal adhesion pathways might cause the phenotypic effect and further points to a complex etiology of KTCN.

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.

Oxidative stress is implicated in the pathogenesis of many diseases, including serious ocular diseases, keratoconus (KC) and Fuchs endothelial corneal dystrophy (FECD). Flap endonuclease 1 (FEN1) plays an important role in the repair of oxidative DNA damage in the base excision repair pathway. We determined the association between two single nucleotide polymorphisms (SNPs), c.–441G>A (rs174538) and g.61564299G>T (rs4246215), in the FEN1 gene and the occurrence of KC and FECD. This study involved 279 patients with KC, 225 patients with FECD and 322 control individuals. Polymerase chain reaction (PCR) and length polymorphism restriction fragment analysis (RFLP) were applied. The T/T genotype of the g.61564299G>T polymorphism was associated with an increased occurrence of KC and FECD. There was no association between the c.–441G>A polymorphism and either disease. However, the GG haplotype of both polymorphisms was observed more frequently and the GT haplotype less frequently in the KC group than the control. The AG haplotype was associated with increased FECD occurrence. Our findings suggest that the g.61564299G>T and c.–441G>A polymorphisms in the FEN1 gene may modulate the risk of keratoconus and Fuchs endothelial corneal dystrophy.