Accumulation of sequence variants in genes of Wnt signaling and focal adhesion pathways in human corneas further explains their involvement in keratoconus

Introduction

Keratoconus (KTCN) is an eye disease characterized by progressive thinning and conical protrusion of the cornea (Karolak & Gajecka, 2017). The structural abnormalities in different layers of corneal tissue result in altered refractive powers and a loss of visual function (Rabinowitz, 1998). The first symptoms of KTCN usually appear during puberty or early in the third decade of life (Rabinowitz, 1998). The management of this condition depends on the disease stage and includes visual correction by contact lenses, corneal collagen cross-linking, or corneal transplant surgery (Sarezky et al., 2017; Vazirani & Basu, 2013). The incidence of KTCN is one in 2,000 individuals in the general population (Rabinowitz, 1998). However, the incidence may vary depending on the geographic location and ethnicity of the studied population. The estimated incidence of KTCN is higher in Indians, Chinese, Pacific, and Maori ethnicities compared with Caucasians (Pearson et al., 2000; Georgiou et al., 2004; Kok, Tan & Loon, 2012; Patel & McGhee, 2013; Gokhale, 2013; Godefrooij et al., 2016). The environmental factors, such as eye rubbing or contact lens wear, influence disease development (Hashemi et al., 2019). However, genetic triggers also play an important role in KTCN (Karolak & Gajecka, 2017; Abu-Amero, Al-Muammar & Kondkar, 2014). Several genomic strategies have been implemented for finding candidate genes, including both simple molecular techniques and high-throughput technologies (Karolak & Gajecka, 2017; Abu-Amero, Al-Muammar & Kondkar, 2014; Bykhovskaya, Margines & Rabinowitz, 2016; Mas Tur et al., 2017; Valgaeren, Koppen & Van Camp, 2018; Loukovitis et al., 2018).

Linkage studies, performed in KTCN families, have led to the identification of multiple chromosomal regions linked to KTCN, including two replicated loci at 5q (Tang et al., 2005; Li et al., 2006; Bisceglia et al., 2009; Rosenfeld et al., 2011; Bykhovskaya et al., 2016). The genome-wide association studies have detected variants mapped near HGF, COL5A1, FOXO1, RAB3GAP1, or ZNF469, associated with KTCN risk (Li et al., 2012; Burdon et al., 2011; Hoehn et al., 2012; Lu et al., 2013). However, their contribution to KTCN needs to be clarified. Several candidate genes for KTCN have also been identified using Sanger sequencing or next-generation sequencing including VSX1, SOD1 and DOCK9 or SKP1, MPDZ, FLG, PPIP5K2, and PCSK1, respectively (Héon et al., 2002; Karolak et al., 2015, 2017; Udar et al., 2006; Lucas et al., 2018; Khaled et al., 2019; Magalhães et al., 2019). However, variants detected in those genes were present in a small fraction of KTCN patients or particular populations only.

Analysis of transcriptome profiles of human KTCN and non-KTCN corneas by a high-throughput RNA sequencing (RNA-Seq) showed the deregulation of numerous genes in KTCN corneas (Kabza et al., 2017). The significant downregulation was observed among genes in collagen synthesis and maturation pathways, as well as in the TGF-β, Hippo and Wnt signaling pathways (Kabza et al., 2017). The results of subsequent RNA studies further support the potential role of genes involved in the extracellular matrix, TGF-β and Wnt molecular cascades in KTCN pathogenesis (Khaled et al., 2018; You et al., 2018; Sharif et al., 2019). Since these signaling pathways influence the corneal organization and play a role in the regulation of extracellular matrix components, the genes encoding the core elements of the mentioned pathways were proposed as novel candidate genes for KTCN.

To assess the involvement of new genetic variants in KTCN etiology, we performed a further molecular investigation of corneas of Polish patients with KTCN, previously tested by RNA-Seq, using exome sequencing (ES) approach.

Materials and Methods

Results

This pilot ES screening of KTCN corneas revealed 117 potentially relevant variants with MAF ≤ 0.01 in our internal DMG cohort and public databases and fulfilling all of the remaining filtering criteria (Table S2). Genes with identified rare nucleotide variants were enriched in 14 molecular pathways, including Wnt signaling (VANGL1, WNT1, PPP3CC, LRP6, FZD2) and focal adhesion (BIRC2, PAK6, COL4A4, PPP1R12A, PTK6) pathways (Table 1). The analysis of functional interactions between genes from overrepresented molecular pathways allowed for further narrowing the number of candidate KTCN variants (Table 2).

Sanger sequencing performed in blood samples derived from the five studied individuals confirmed all variants identified with the use of ES in matched corneas.

The overrepresentation analysis performed among genes filtered in a step-wise manner in control datasets revealed an enrichment in 16 (i.e., O-linked glycosylation, C-type lectin receptors, termination of O-glycan biosynthesis, nucleotide-binding oligomerization domain) and nine (i.e., cargo recognition for clathrin-mediated endocytosis VLDL clearance, glyoxylate and dicarboxylate metabolism, statin pathway DNA damage recognition in GG-NER) pathways in HM patients and individuals without ocular disease, respectively. No genes involved in pathways enriched in KTCN corneas were overrepresented in our control sample sets containing randomly selected Polish individuals without ocular abnormalities (n = 5) and Polish patients with HM (n = 5).

The TPM values of genes from overrepresented pathways in corneas of patients carrying particular gene variants and in KTCN individuals without the analyzed gene variation were calculated based on our previous RNA-Seq study data (Kabza et al., 2017). The expression values of the genes varied between patients with particular variants and the mean expression values of the same genes in the KTCN individuals without these variants. The expression profile of genes from overrepresented pathways in KTCN corneas is presented in Table 3.

The data were deposited in the freely accessible ClinVar database (submission ID: SUB3758236 and SUB4926771).

Discussion

The rapid development of next-generation DNA sequencing methods has greatly improved the ability to detect genetic variations (Koboldt et al., 2013). However, KTCN, like many other ophthalmic diseases, displays genetic heterogeneity hindering the identification of a factor unambiguously influencing its development (Karolak & Gajecka, 2017; Stone et al., 2004).

The results of this pilot ES study of corneas obtained from five KTCN patients undergoing penetrating keratoplasty revealed various rare possibly pathogenic variants in genes that were overrepresented in several molecular pathways, including the Wnt signaling and focal adhesion. Interestingly, these pathways have been proposed as involved in KTCN etiology based on our previous RNA-Seq study, performed in experimental material derived from the same KTCN patients, as well as other experiments (Kabza et al., 2017; Khaled et al., 2018; You et al., 2018; Sharif et al., 2019). In addition, each of five patients had at least one variant in genes from these particular pathways. Among genes from overrepresentation gene sets were LRP6, FZD2, COL4A4, and WNT1.

Ocular cells, including corneal epithelial stem cells, express components of the Wnt/β-catenin signaling pathway during eye development (Nakatsu et al., 2011). The LRP6 gene encodes the low-density lipoprotein receptor-related protein, which is a component of a Wnt receptor complex. This complex is involved in Wnt ligands binding resulting in the nuclear translocation of β-catenin and regulation of the transcription of target genes (Zhang et al., 2015). Knock-out of β-catenin or its both co-receptors (Lrp5 and Lrp6) in mouse corneal stromal cells resulted in premature stratification of the corneal epithelium, suggesting these genes play a role in the regulation of corneal morphogenesis (Zhang et al., 2015). In this study, the expression of LRP6 in the cornea of the carrier of variant c.4822C>T (KC17) was unchanged compared to the other four KTCN individuals. However, since this variant was identified in KC17 patient in combination with another variant in the gene of Wnt signaling pathway (PPP3CC), we suggest that this variant might be a part of the specific combination of KTCN variants and its identification is not incidental.

The FZD2 gene encodes frizzled class receptor 2, another protein functioning as Wnt ligands receptor and regulating the eye development through Wnt/β-catenin signaling. Xenopus frizzled-2, a homolog of the human frizzled receptor, is highly expressed in the embryo, including the developing eye (Deardorff & Klein, 1999). Interestingly, a significant increase of mRNA and protein expression of secreted frizzled-related protein 1 (SFRP1), which is a Wnt antagonist, has been detected in KTCN corneal epithelium and corneal buttons (Sutton et al., 2010; You et al., 2013a). In contrast, tear SFRP1 level has been significantly decreased in KTCN patients compared to control individuals (You et al., 2013b). The c.1238G>A variant in FZD2 was identified in the cornea of KC16 patient in combination with c.41C>T variant in VANGL1 (Wnt signaling) and c.2347G>A variant in COL4A4 (focal adhesion). It is known that collagens are major components of human corneas and the thinning of corneal stroma in KTCN may be the final effect of a disorganized collagen lamellae arrangement (Meek et al., 2005; Mathew et al., 2015). The COL4A4 gene is expressed in human cornea (Kabza et al., 2017) and based on several studies it has been reported as a candidate gene for KTCN (Stabuc-Silih et al., 2009; Wang et al., 2013; Kokolakis et al., 2014; Saravani et al., 2015). However, genetic analyses of this gene in different populations have given ambiguous results about the role of COL4A4 in KTCN development (Stabuc-Silih et al., 2009; Wang et al., 2013; Kokolakis et al., 2014; Saravani et al., 2015). While there was no difference in expression of COL4A4 and VANGL1 in the patient’s cornea, the expression value of FZD2 in the patient KC16 was lower compared to the expression observed in the other four KTCN individuals. However, further research should be performed to interpret the obtained data.

The combination of variants in WNT1 and PTK6 was revealed in the patient KC15. PTK6 encodes tyrosine kinase 6 protein, which is involved in focal adhesion, as well as in GTPases and MAP kinases regulation. Signaling by PTK6 is implicated in controlling the differentiation of normal epithelium and tumor growth (Brauer & Tyner, 2010). However, there is no report about the role of PTK6 in the maintenance of corneal epithelium.

Also, in KC18 and KC19 patients, variants in other elements (BIRC2 and PAK6 with PPP1R12A, respectively) of Wnt signaling and/or focal adhesion pathways were observed. These results further suggest that the alteration of genes regulating these signaling pathways might be an important risk factor for KTCN.

To determine the likelihood of seeing the overrepresentation of genes from Wnt signaling and focal adhesion in KTCN samples by chance, we performed analyses of exome data of five randomly selected Polish individuals without eye disease, as well as five Polish individuals with HM. Variants from control ES data were selected using the same filtering criteria as we used for the KTCN study, followed by enrichment analyses. No genes involved in Wnt signaling or focal adhesion pathways were overrepresented in our control sample sets, confirming that enrichment of variants in genes from these pathways in KTCN individuals was not incidental.

The small sample size disabled a reliable statistical analysis and due to this limitation, the general conclusions could not be made. However, the results of this pilot study gave additional insight into the role of the Wnt signaling and/or focal adhesion pathways in KTCN development and showed the possible indications for further KTCN research. The identification of rare variants in different genes further supports the heterogeneity of KTCN with multiple genes underlying its pathogenesis (Karolak & Gajecka, 2017; Abu-Amero, Al-Muammar & Kondkar, 2014; Bykhovskaya, Margines & Rabinowitz, 2016; Mas Tur et al., 2017; Valgaeren, Koppen & Van Camp, 2018).

Conclusions

Summarizing, this first pilot ES profiling of human KTCN corneas indicates that the accumulation of variants in several genes from Wnt signaling and/or focal adhesion pathways might cause the phenotypic effect and further explain the involvement of these pathways in KTCN. Moreover, it also supports the hypothesis about the complex basis of KTCN. Since five patients were evaluated, the role of variants in genes of these overrepresented pathways in KTCN etiology should be further elucidated in a larger group of patients using high throughput methods.

Supplemental Information