Identification of Differentially Expressed Genes in Keratoconus Epithelium Analyzed on Microarrays (original) (raw)
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Genomic strategies to understand causes of keratoconus
Molecular Genetics and Genomics
com thinning, which results in the conical shape of the cornea. These structural changes in the corneal layers induce optical aberrations, leading to a loss of visual acuity due to distorted blurred vision, which is caused by irregular astigmatism, and high myopia (Rabinowitz 1998). Although KTCN is sometimes referred to as a corneal dystrophy, it is not included in International Classification of Corneal Dystrophies (IC3D) (Weiss et al. 2008) and should be distinguished from this group of corneal diseases. However, co-occurrence of KTCN with many types of corneal dystrophies, including Avellino and Fuchs dystrophies (Igarashi et al. 2003; Salouti et al. 2010; Wilson et al. 2014), may indicate that common molecular mechanisms in the pathogenesis of these disorders are involved. Among the general population, the estimated frequency of KTCN is 1 in 2,000 individuals (Rabinowitz 1998), although up-to-date data are not available. The prevalence of KTCN may be different according to patient ethnic origin (Gokhale 2013). The reported prevalence of the disease may also vary depending upon the different diagnostic tests used in the particular studies. The early KTCN or forme fruste KTCN are not detectable at the slit lamp during the anterior segment examination, and in these cases, assessment of the corneal topographic pattern is required to obtain the accurate diagnosis (Saad and Gatinel 2010). The first symptoms of KTCN usually appear during the second decade or early in the third decade of life. The pathogenic features of KTCN may be observed in different layers of the cornea (Fig. 1) (Sherwin and Brookes 2004). These abnormalities include changes in morphology of epithelial cells (Sykakis et al. 2012), deposition of iron particles in the epithelial basement membrane, breaks in Bowman's layer (Rabinowitz 1998), and thinning of stroma correlating with loss of collagen lamellae, altered collagen fibril orientation, and decreased keratocytes density (Patey et al.
Molecular and Histopathological Changes Associated with Keratoconus
BioMed Research International, 2017
Keratoconus (KC) is a corneal thinning disorder that leads to loss of visual acuity through ectasia, opacity, and irregular astigmatism. It is one of the leading indicators for corneal transplantation in the Western countries. KC usually starts at puberty and progresses until the third or fourth decade; however its progression differs among patients. In the keratoconic cornea, all layers except the endothelium have been shown to have histopathological structural changes. Despite numerous studies in the last several decades, the mechanisms of KC development and progression remain unclear. Both genetic and environmental factors may contribute to the pathogenesis of KC. Many previous articles have reviewed the genetic aspects of KC, but in this review we summarize the histopathological features of different layers of cornea and discuss the differentially expressed proteins in the KC-affected cornea. This summary will help emphasize the major molecular defects in KC and identify additional research areas related to KC, potentially opening up possibilities for novel methods of KC prevention and therapeutic intervention.
Further evaluation of differential expression of keratoconus candidate genes in human corneas
PeerJ, 2020
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.
Differential epithelial and stromal protein profiles in keratoconus and normal human corneas
Experimental Eye Research, 2011
The purpose of the study was to identify epithelial and stromal proteins that exhibit up-or down-regulation in keratoconus (KC) vs. normal human corneas. Because previous proteomic studies utilized whole human corneas or epithelium alone, thereby diluted the specificity of the proteome of each tissue, we selectively analyzed the epithelium and stromal proteins. Individual preparations of epithelial and stromal proteins from KC and age-matched normal corneas were analyzed by two independent methods, i.e., a shotgun proteomic using a Nano-Electrospray Ionization Liquid Chromatography Tandem Mass Spectrometry [Nano-ESI-LC-MS (MS) 2 ] and two-dimensional-difference gel electrophoresis (2D-DIGE) coupled with mass spectrometric methods. The label-free Nano-ESI-LC-MS (MS) 2 method identified 104 epithelial and 44 stromal proteins from both normal and KC corneas, and also quantified relative changes in levels of selected proteins, in both the tissues using spectral counts in a proteomic dataset. Relative to normal corneal epithelial proteins, six KC epithelial proteins (lamin-A/C, keratin type I cytoskeletal 14, tubulin beta chain, heat shock cognate 71 kDa protein, keratin type I cytoskeletal 16 and protein S100-A4) exhibited upregulation and five proteins (transketolase, pyruvate kinase, 14-3-3 sigma isoform, phosphoglycerate kinase 1, and NADPH dehydrogenase (quinone) 1) showed down-regulation. A similar relative analysis showed that three KC stromal proteins (decorin, vimentin and keratocan) were up-regulated and five stromal proteins (TGF-betaig h3 (Bigh3), serotransferrin, MAM domain-containing protein 2 and isoforms 2C2A of collagen alpha-2[VI] chain) were down-regulated. The 2D-DIGE-mass spectrometry followed by Decyder software analysis showed that relative to normal corneas, the KC corneal epithelium exhibited upregulation of four proteins (serum albumin, keratin 5, L-lactate dehydrogenase and annexin A8) and downregulation of four proteins (FTH1 [Ferritin heavy chain protein 1], calpain small subunit 1, heat shock protein beta 1 and annexin A2). A similar relative analysis of stroma by this method also showed upregulation of aldehyde dehydrogenase 3A1 (ALDH3A1), keratin 12, apolipoprotein A-IV precursor, haptoglobin precursor, prolipoprotein and lipoprotein Gln in KC corneas. Together, the results suggested that the Nano-ESI-LC-MS(MS) 2 method was superior than the 2D-DIGE method as it identified a greater number of proteins with altered levels in KC corneas. Further, the epithelial and stromal structural proteins of KC corneas exhibited altered levels compared to normal corneas, suggesting that they are affected due to structural remodeling during KC development and progression. Additionally, because several epithelial and stromal enzymes exhibited up-or down-regulation in the KC corneas relative to normal corneas, the two layers of KC corneas were under metabolic stress to adjust their remodeling.
Investigative Opthalmology & Visual Science, 2005
PURPOSE. To increase the database of genes expressed in human cornea and to gain insights into the molecular basis of keratoconus (KC). METHODS. A cDNA library was constructed from KC corneas harvested at keratoplasty and used for expressed sequence tag (EST) analysis. Data were analyzed using grouping and identification of sequence tags (GRIST). Expression of selected clones was examined by RT-PCR. RESULTS. A total of 7680 clones was sequenced from the 5Ј end. After bioinformatics analysis, 4090 clusters of clones, each potentially representing individual genes, were identified. Of these, 887 genes were represented by more than one clone. The five most abundant transcripts, represented by Ͼ60 clones each, were for keratin-12, TGFBI (BIGH3), decorin, ALDH3, and enolase 1, all known markers for cornea. Many other markers for epithelial, stromal, and endothelial genes were also present. One cluster of six clones came from an apparently novel gene (designated KC6) located on chromosome 18 at p12.3. RT-PCR of RNA from several human tissues detected KC6 transcripts only in cornea. In addition, no clones were observed for the usually prominent corneal epithelial cell marker aquaporin 5 (AQP5), a water channel protein. Semiquantitative RT-PCR confirmed that expression of AQP5 is much lower in KC cornea than in non-KC cornea. CONCLUSIONS. This analysis increases the database of genes expressed in the human cornea and provides insights into KC. KC6 is a novel gene of unknown function that shows corneapreferred expression, whereas the suppression of transcripts for AQP5 provides the first clear evidence of a molecular defect identified in KC.
Characterization of Corneal Epithelial Cells in Keratoconus
Translational Vision Science & Technology, 2019
Purpose: We studied the cellular characteristics of epithelial cells in the cone and extraconal periphery of corneas in keratoconus eyes. Methods: This prospective observational study was conducted at Narayana Nethralaya Eye Institute. A total of 83 and 42 eyes with keratoconus and normal topography, respectively, were included in the study. Corneal epithelial cells were collected and analyzed for apoptosis, proliferation, epithelial-mesenchymal transition, and differentiation status using molecular and biochemical tools. Statistical analysis was performed using the Student's t-test. Results: Corneal epithelial cells from the cone showed significantly higher expression of proapoptotic marker BAX (P , 0.005) compared to controls. Significantly elevated expression of cell cycle markers CYCLIN D1 (P , 0.005) and Ki67 (P , 0.005) were noted in the extraconal region compared to controls. Cells of the cone showed significantly higher ZO-1 (P , 0.005) and lower vimentin (P , 0.005) compared to controls. Significantly lower expression of the differentiation marker CK3/12 (P , 0.05) was observed in cones compared to controls. Conclusions: Cones of keratoconic corneas show enhanced cell death, poor differentiation, proliferation and epithelial-mesenchymal transition. The cellular changes of the corneal epithelial cells in the cone and extraconal region differ significantly in a keratoconus corneas. Translational Relevance: Characterization of patient-specific corneal epithelial cellular status in keratoconus has the potential to determine the optimal treatment and therapeutic outcomes paving the way towards personalized treatment in the future.
Keratin 13 is a more specific marker of conjunctival epithelium than keratin 19
Molecular Vision, 2011
Purpose To evaluate the expression patterns of cytokeratin (K) 12, 13, and 19 in normal epithelium of the human ocular surface to determine whether K13 could be used as a marker for conjunctival epithelium. Methods Total RNA was isolated from the human conjunctiva and central cornea. Those transcripts that had threefolds or higher expression levels in the conjunctiva than the cornea were identified using microarray technique. Expression levels of three known signature genes and of two conjunctival genes, K13 and K19 were confirmed by using quantitative real-time PCR (qRT–PCR). Protein expression of K12, K13, and K19 was confirmed by immunostaining with specific antibodies on histologic sections of human sclerocornea that contained the conjunctiva, limbus, and cornea and on impression cytology (IC) specimens of the cornea and conjunctiva from normal donors. Double staining of K13/K12 and K19/K12 on histologic sections and IC specimens was performed. Results There were 337 transcripts...
Advances in Therapy
Introduction: Keratoconus (KC) is a complex, genetically heterogeneous multifactorial degenerative disorder characterized by corneal ectasia and thinning. Its incidence is approximately 1/2000-1/50,000 in the general popula-factor(s),'' ''genetics,'' ''genes,'' ''genetic association(s),'' ''proteins'', ''collagen'' and ''cornea'' were used. In total, 272 articles were retrieved, reviewed and selected, with greater weight placed on more recently published evidence. Based on the reviewed literature, an attempt was made to tabulate the up-and down-regulation of genes involved in KC and their protein products and to delineate the mechanisms involved in CXL. Results: A total of 117 proteins and protein classes have been implicated in the pathogenesis and pathophysiology of KC. These have been tabulated in seven distinct tables according to their gene coding, their biochemistry and their metabolic control. Conclusion: The pathogenesis and pathophysiology of KC remain enigmatic. Emerging evidence has improved our understanding of the molecular characteristics of KC and could further improve the success rate of CXL therapies.
Altered KSPG expression by keratocytes following corneal injury
Mol Vis, 2003
The corneal surface consists of a multi-layered (5-7 cell layers) non-keratinized, stratified squamous epithelium. In part the strength of the corneal epithelium is maintained by its tissue-specific expression of intermediate filaments consisting of paired K3/K12 keratins, which is a characteristic of corneal-type epithelial differentiation [1]. The next layer is the stroma that comprises 90% of the corneal thickness in humans and accounts for the major contribution towards corneal curvature and clarity, and refractive power. Collagen I, V, VI, and XII make up the majority of the stroma [2-5] along with the dermatan sulfate proteoglycan decorin [6], and the keratan sulfate proteoglycans (KSPG), e.g., lumican [7], keratocan [8], and osteoglycin/mimecan [9]. Large epithelial wounds and alkali burns often lead to poor visual outcome, including conjunctivalization, stromal scarring, recurrent epithelium erosions, vascularization, and limbal deficiency [10-17]. The resultant corneal stroma after wound healing is one of disorganized collagen fibrils and opacity. This outcome is significantly different from the uniform collagen fibril diameter and interfibrillar spacing allowing virtually unobscured light resonance resulting from corneal development [18]. The divergence of these two mechanisms suggests the involvement of other factors and/or a difference in gene expression during wound healing. Keratocytes are the major cell-type in the corneal stroma expressing the KSPGs and collagen [19-21] and MMPs for tissue remodeling during wound healing. One early event well documented is a population of keratocytes undergo apoptosis following a corneal wound [22-28]. It remains elusive whether the stromal cells repopulate the injured corneas maintain keratocyte phenotype. Previous reports demonstrate differences (inflammation, necrosis, ulceration, and scarring) in the wound healing process of various wound types, specifically noting a unique healing process for the alkali burn wound [29-33]. One possible explanation is the variation in cytokines and growth factors expressed after a specific wound type [31,34]. Potential source for the initial factors are the tear, the remaining keratocytes and epithelial cells [31-37]. These signaling molecules have the potential to recruit inflammatory cells, promote apoptosis, and contribute to changes of gene expression patterns accounting for opaque scar tissue formation [38-45]. The purpose of this study was to examine the expression of corneal KSPGs as an indication of keratocyte phenotype