Identification of hub genes and molecular pathways in keratoconus by integrating bioinformatics and literature mining at the RNA level.

OBJECTIVE: Keratoconus (KC) is a condition characterized by progressive corneal steepening and thinning. However, its pathophysiological mechanism remains vague. We mainly performed literature mining to extract bioinformatic and related data on KC at the RNA level. The objective of this study was to explore the potential pathological mechanisms of KC by identifying hub genes and key molecular pathways at the RNA level. METHODS: We performed an exhaustive search of the PubMed database and identified studies that pertained to gene transcripts derived from diverse corneal layers in patients with KC. The identified differentially expressed genes were intersected, and overlapping genes were extracted for further analyses. Significantly enriched genes were screened using "Gene Ontology" (GO) and "Kyoto Encyclopedia of Genes and Genomes" (KEGG) analysis with the "Database for Annotation, Visualization, and Integrated Discovery" (DAVID) database. A protein-protein interaction (PPI) network was constructed for the significantly enriched genes using the STRING database. The PPI network was visualized using the Cytoscape software, and hub genes were screened via betweenness centrality values. Pathways that play a critical role in the pathophysiology of KC were discovered using the GO and KEGG analyses of the hub genes. RESULTS: 68 overlapping genes were obtained. Fifty genes were significantly enriched in 67 biological processes, and 16 genes were identified in 7 KEGG pathways. Moreover, 14 nodes and 32 edges were identified via the PPI network constructed using the STRING database. Multiple analyses identified 4 hub genes, 12 enriched biological processes, and 6 KEGG pathways. GO enrichment analysis showed that the hub genes are mainly involved in the positive regulation of apoptotic process, and KEGG analysis showed that the hub genes are primarily associated with the interleukin-17 (IL-17) and tumor necrosis factor (TNF) pathways. Overall, the matrix metalloproteinase 9, IL-6, estrogen receptor 1, and prostaglandin-endoperoxide synthase 2 were the potential important genes associated with KC. CONCLUSION: Four genes, matrix metalloproteinase 9, IL-6, estrogen receptor 1, and prostaglandin endoperoxide synthase 2, as well as IL-17 and TNF pathways, are critical in the development of KC. Inflammation and apoptosis may contribute to the pathogenesis of KC.

The candidate proteins associated with keratoconus: A meta-analysis and bioinformatic analysis.

PURPOSE: Keratoconus (KC) is a multifactorial disorder. This study aimed to conduct a systematic meta-analysis to exclusively explore the candidate proteins associated with KC pathogenesis. METHODS: Relevant literature published in the last ten years in Pubmed, Web of Science, Cochrane, and Embase databases were searched. Protein expression data were presented as the standard mean difference (SMD) and 95% confidence intervals (CI). The meta-analysis is registered on PROSPERO, registration number CRD42022332442 and was conducted in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analyses statement (PRISMA). GO and KEGG enrichment analysis were performed, as well as the miRNAs and chemicals targeting the candidate proteins were predicted. PPI was analyzed to screen the hub proteins, and their expression was verified by RT-qPCR. RESULTS: A total of 21 studies were included in the meta-analysis, involving 346 normal eyes and 493 KC eyes. 18 deregulated proteins with significant SMD values were subjected to further analysis. In which, 7 proteins were up-regulated in KC compared with normal controls, including IL6 (SMD 1.54, 95%CI [0.85, 2.24]), IL1B (SMD 2.07, 95%CI [0.98, 3.16]), TNF (SMD 2.1, 95%CI [0.24, 3.96]), and MMP9 (SMD 1.96, 95%CI [0.68, 3.24]). While 11 proteins were down-regulated in KC including LOX (SMD 2.54, 95%CI [-4.51, -0.57]). GO and KEGG analysis showed that the deregulated proteins were involved in inflammation, extracellular matrix (ECM) remodeling, and apoptosis. MMP9, IL6, LOX, TNF, and IL1B were regarded as hub proteins according to the PPI analysis, and their transcription changes in stromal fibroblasts of KC were consistent with the results of the meta-analysis. Moreover, 10 miRNAs and two natural polyphenols interacting with hub proteins were identified. CONCLUSION: This study obtained 18 candidate proteins and demonstrated altered cytokine profiles, ECM remodeling, and apoptosis in KC patients through meta-analysis and bioinformatic analysis. It will provide biomarkers for further understanding of KC pathogenesis, and potential therapeutic targets for the drug treatment of KC.

Genetic variants in the FOXO1 and ZNF469 genes are associated with keratoconus in Sweden: a case-control study.

BACKGROUND: Keratoconus (KC) is characterized by pathological thinning and bulging of the cornea that may lead to visual impairment. The etiology of sporadic KC remains enigmatic despite intensive research in recent decades. The purpose of this study was to examine the relationship between previously highlighted genetic variants associated with KC and sporadic KC in a Swedish cohort. METHODS: A total of 176 patients (age 16-70 years) with sporadic KC diagnosed by Scheimpflug-topography (Pentacam) were included. The control group (n = 418; age 70 years) was a subsample originating from the Gothenburg H70 Birth Cohort Studies of ageing. Extraction of DNA from blood samples was performed according to standard procedures, and genotyping was performed using competitive allele specific PCR (KASP) technology. A total of 11 single nucleotide polymorphisms (SNPs) were selected for analysis. RESULTS: Statistically significant associations (p = 0.005) were found between the SNPs rs2721051 and rs9938149 and sporadic KC. These results replicate earlier research that found associations between genetic variants in the FOXO1 and BANP-ZNF469 genes and sporadic KC in other populations. CONCLUSION: Genetic variations in the FOXO1 and BANP-ZNF469 genes may be involved in the pathogenesis of sporadic KC.

Integrated analysis of murine cornea identifies JAK/STAT signaling pathway upregulated specifically in female Vitamin A Deficient mice.

The Keratoconus (KC) is a corneal ectatic disease with unclear etiology. There are increasing studies that reported its association with a variety of inflammatory mechanisms. Vitamin A(VA) is an important nutrient related to inflammation regulation, and its deficiency may cause abnormalities of the ocular surface. However, the proportion of Vitamin A deficiency(VAD) was found surprisingly high among KC patients in our clinic practice. The aim of this study is to explore the effects of VAD on the transcriptome of corneas with the help of the VAD murine model and transcriptomics techniques. Blood samples of KC patients and non-KC controls (NC) were collected and the serum VA concentrations were measured and analyzed. A total of 52 NC and 39 KC were enrolled and the comparison of serum VA showed that the proportion of VAD in KC patients was 48.7% versus 1.9% in NC group. The further analysis of gender differences showed the proportion of VAD in female KC was 88.9% versus 36.7% in KC male patients. To explore the influence of VAD on cornea, the VAD mice fed with VAD diets were used. The RNA sequencing was employed to compare the corneal transcriptomic characteristics between the VAD female mice, NC female mice, VAD male mice and NC male mice. The transcriptome analysis revealed that the upregulated differential genes were mainly enriched in the immune response related pathways in VAD female mice versus NC female mice, especially the genes of JAK-STAT signaling pathway. The downstream molecules of JAK-STAT pathway were also significant after corneal mechanical scratching in female VAD mice. While, the differential genes between VAD male mice and NC male mice were estrogen signaling pathway instead of JAK-STAT pathway. This study indicates that VAD affects the transcriptomics of murine cornea with gender differences, which specifically affects the inflammatory status of the female murine cornea.

Inflammatory profile of keratoconic corneal epithelium.

BACKGROUND: Recent studies have presented inflammatory features on keratoconus (KC) and many inflammatory markers are described in the tears of patients with this disease. The KC pathogenesis is still unknown just like the correlation with inflammatory patterns. However, environmental and genetic issues may be part of the progress of KC. In addition, some systemic features, such as allergy and obesity, seem to be related to the progression of KC. Our purpose was to evaluate the neuropeptides vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), chemokines ligand 2 (CCL-2) and 5 (CCL-5), and interleukins 6 (IL-6) and 8 (IL-8) on corneal epithelial cells and blood of patients with KC and in healthy controls. In addition, the neutrophil-to-lymphocyte ratio (NLR) was evaluated to predict inflammation. METHODS: This including prospective observational study included 32 KC patients who underwent corneal crosslinking (CXL) and 32 control patients who underwent photorefractive keratectomy (PRK). Patients' corneal epithelial cells were removed surgically, and blood (buffy coat) was analyzed. Samples in triplicate were evaluated on rt-PCR for neuropeptides (VIP e NPY), interleukins (IL-6 e IL-8), and chemokines (CCL-2 and CCL-5). RESULTS: Our study showed statistically higher CCL-5 and IL-8 on corneal epithelial cells in patients with KC. Blood cells were statistically higher in VIP and NPY in the KC group. Interleukin-8 on blood cells was statistically significant in KC'S group; for CCL-2 and CCL-5 they were statistically lower in patients with KC compared with controls. NLR showed no difference between the groups. CONCLUSIONS: Our data support the findings of other studies that suggested altering KC status, such as inflammatory corneal disease. The presence of IL-8 in the cornea and blood samples of KC's group suggested systemic disease with a possible local or repercussion action. Further studies are warranted to elucidate KC pathogenesis and its correlation to systemic disease.

Meta-Analysis of Keratoconus Transcriptomic Data Revealed Altered RNA Editing Levels Impacting Keratin Genomic Clusters.

Introduction: Keratoconus (KC) is an ocular disorder with a multifactorial origin. Transcriptomic analyses (RNA-seq) revealed deregulations of coding (mRNA) and non-coding RNAs (ncRNAs) in KC, suggesting that mRNA-ncRNA co-regulations can promote the onset of KC. The present study investigates the modulation of RNA editing mediated by the adenosine deaminase acting on dsRNA (ADAR) enzyme in KC. Materials: The level of ADAR-mediated RNA editing in KC and healthy corneas were determined by two indexes in two different sequencing datasets. REDIportal was used to localize known editing sites, whereas new putative sites were de novo identified in the most extended dataset only and their possible impact was evaluated. Western Blot analysis was used to measure the level of ADAR1 in the cornea from independent samples. Results: KC was characterized by a statistically significant lower RNA-editing level compared to controls, resulting in a lower editing frequency, and less edited bases. The distribution of the editing sites along the human genome showed considerable differences between groups, particularly relevant in the chromosome 12 regions encoding for Keratin type II cluster. A total of 32 recoding sites were characterized, 17 representing novel sites. JUP, KRT17, KRT76, and KRT79 were edited with higher frequencies in KC than in controls, whereas BLCAP, COG3, KRT1, KRT75, and RRNAD1 were less edited. Both gene expression and protein levels of ADAR1 appeared not regulated between diseased and controls. Conclusions: Our findings demonstrated an altered RNA-editing in KC possibly linked to the peculiar cellular conditions. The functional implications should be further investigated.

Evaluation of Ocular Higher-Order Aberrations in First-Degree Relatives of Patients With Keratoconus.

PURPOSE: The aim of this study was to evaluate the corneal higher-order aberrations (HOAs) in first-degree relatives of patients with keratoconus (KCN) and compare with the normal population. METHODS: In this prospective comparative study, 210 eyes from 105 family members of 28 patients with KCN and 210 normal eyes of 105 controls were enrolled. In each eye, corneal topography, tomography, and aberrometry were performed and compared between the 2 groups. RESULTS: This study included 61 female (58.1%) and 44 male participants (41.9%) and 105 age-matched and sex-matched controls with normal topographic cornea. In 14 of 105 first-degree relatives (13.33%) of patients with KCN, KCN was diagnosed with a male preponderance (71.5% male, 28.5% female). Tomographic indices and irregularity indices in 3 and 5 mm zone in Orbscan were significantly higher in the relative group. In addition, other irregularity indices of TMS-4 topography including surface regularity index, surface asymmetry index, difference sector index, SDP, and irregular astigmatism index were significantly higher in family members of patients with KCN. The most prevalent topographic pattern in the control group was the symmetric bowtie (57.1%) and in the relative group was the asymmetric bowtie (39.5%). In addition, significantly thinnest corneal pachymetry was detected in the relative group. Root mean square of all HOAs including vertical trefoil, vertical coma, horizontal coma, horizontal trefoil, quadrifoil, and fourth-order spherical aberrations were significantly greater in the relative group than controls. CONCLUSIONS: Owing to the high prevalence of undiagnosed KCN susceptibility in family members with KCN, keratorefractive surgery should be considered cautiously in these individuals. In addition, comprehensive preoperative examination should be considered to detect subtle topographic and HOAs in these individuals.

Personalized Model to Predict Keratoconus Progression From Demographic, Topographic, and Genetic Data.

PURPOSE: To generate a prognostic model to predict keratoconus progression to corneal crosslinking (CXL). DESIGN: Retrospective cohort study. METHODS: We recruited 5025 patients (9341 eyes) with early keratoconus between January 2011 and November 2020. Genetic data from 926 patients were available. We investigated both keratometry or CXL as end points for progression and used the Royston-Parmar method on the proportional hazards scale to generate a prognostic model. We calculated hazard ratios (HRs) for each significant covariate, with explained variation and discrimination, and performed internal-external cross validation by geographic regions. RESULTS: After exclusions, model fitting comprised 8701 eyes, of which 3232 underwent CXL. For early keratoconus, CXL provided a more robust prognostic model than keratometric progression. The final model explained 33% of the variation in time to event: age HR (95% CI) 0.9 (0.90-0.91), maximum anterior keratometry 1.08 (1.07-1.09), and minimum corneal thickness 0.95 (0.93-0.96) as significant covariates. Single-nucleotide polymorphisms (SNPs) associated with keratoconus (n=28) did not significantly contribute to the model. The predicted time-to-event curves closely followed the observed curves during internal-external validation. Differences in discrimination between geographic regions was low, suggesting the model maintained its predictive ability. CONCLUSIONS: A prognostic model to predict keratoconus progression could aid patient empowerment, triage, and service provision. Age at presentation is the most significant predictor of progression risk. Candidate SNPs associated with keratoconus do not contribute to progression risk.

A novel variant in TGFBI causes keratoconus in a two-generation Chinese family.

BACKGROUND: This study aims to investigate the genetic abnormalities in a two-generation Chinese family affected by keratoconus (KC). A two-generation Chinese family affected by KC was studied. MATERIALS AND METHODS: A total of 118 unrelated healthy individuals without KC were recruited as controls. The family history, clinical data, and peripheral blood leukocytes were collected from all subjects. Whole exome sequencing was performed using the genomic DNA of the proband (II.2) and the other two affected family members (I.1 and II.3). Afterwards, polymerase chain reaction was performed for the other enrolled subjects to verify the variants identified in family members with KC. The PolyPhen2, SIFT, PROVEIN and Mutation Taster software programs were applied to analyze the functional consequences of the variants. RESULTS: A single nucleotide polymorphism (VARIANT) (c.1406 G > A [rs759370852]) in the transforming growth factor beta-induced (TGFBI) gene was identified in all affected family members, which resulted in a p.R469H amino acid change. This variant was not detected in the controls. The variant c.1406 G > A in TGFBI was predicted as probably damaging with software programs. CONCLUSION: A novel variant c.1406 G > A in TGFBI has been identified, and probably contributes to the pathogenesis of KC.

Is there a genetic link between Keratoconus and Fuch's endothelial corneal dystrophy?

Keratoconus (KC) is a noninflammatory, bilateral, progressive corneal stromal thinning disorder that leads to corneal ectasia, central anterior corneal stromal scarring, irregular myopic astigmatism, and reduced visual acuity. Fuchs endothelial corneal dystrophy (FECD) is bilateral, sporadic, or autosomal dominant, non-inflammatory, dystrophy involving the corneal endothelium. As the dystrophy evolves, the major complaints include glare, halos, and reduced visual acuity. Very rarely, both the pathologies can co-exist in the same patient. After the initial report of KC with FECD, there have been nearly 70 cases of coexistence reported in the literature till now. The reported number of cases varies from region to region and the majority of cases have no documented family history, hence it is difficult to approximate the incidence and prevalence based on the available literature. Hence, we hypothesize that both pathologies have probably the same genetic link. Available literature supporting the hypothesis has been discussed. We have also discussed various probable mechanisms of coexistence and proposed a management algorithm while assessing and attempting surgery in these patients. We also propose that all these patients must undergo genetic screening and counselling to prevent the spread of these pathologies in future generations.

USH2A gene variants cause Keratoconus and Usher syndrome phenotypes in Pakistani families.

BACKGROUND: Retinitis pigmentosa (RP) is the most common inherited retinal dystrophy, affecting approximately 1 in 4000 individuals worldwide. The most common form of syndromic RP is Usher syndrome (USH) accounting for approximately 20-30 % of RP cases. Mutations in the USH2A gene cause a significant proportion of recessive non-syndromic RP and USH type II (USH2). This study aimed to determine the causative role of the USH2A gene in autosomal recessive inherited ocular diseases and to establish genotype-phenotype correlation associated with USH2A variants. METHODS: We performed direct Sanger sequencing and co-segregation analysis of the USH2A gene to identify disease causing variants in a non-syndromic RP family, two USH2 families and two Keratoconus (KC) families. RESULTS: Disease causing variants in the USH2A gene were identified in two families displayed KC and USH2 phenotypes. A novel variant c.4029T > G, p.Asn1343Lys in the USH2A gene was detected in a Pakistani family with KC phenotype. In addition, a missense variant (c.7334 C > T, p. Ser2445Phe) in the USH2A gene was found segregating in another Pakistani family with USH2 phenotype. Homozygosity of identified missense USH2A variants was found associated with autosomal recessive inherited KC and USH2 phenotypes in investigated families. These variants were not detected in ethnically matched healthy controls. Moreover, the USH2A variants were predicted to be deleterious or potentially disease causing by PolyPhen-2, PROVEAN and SIFT. CONCLUSIONS: This study provided first evidence for association of a novel USH2A variant with KC phenotype in a Pakistani family as well as established the phenotype-genotype correlation of a USH2A variant (c.7334 C > T, p. Ser2445Phe) with USH2 phenotype in another Pakistani family. The phenotype-genotype correlations established in present study may improve clinical diagnosis of affected individuals for better management and counseling.

Gemelarity-based diagnosis for subclinical keratoconus / Diagnóstico baseado em gemelaridade para ceratocone subclínico

ABSTRACT We report a case of two twins for whom advanced keratoconus is present in one of the siblings and no clear sign of the disease could be found for the other.

RESUMO Relatamos um caso de dois gêmeos em que o ceratocone avançado está presente em um dos irmãos e nenhum sinal da doença foi encontrado no outro.

Association with Corneal Remodeling Related Genes, ALDH3A1, LOX, and SPARC Genes Variations in Korean Keratoconus Patients.

PURPOSE: To determine whether the cornea remodeling-related genes aldehyde dehydrogenase 3A1 (ALDH3A1), lysyl oxidase (LOX), and secreted protein acidic and rich in cysteine (SPARC) were potential susceptibility candidate genes for keratoconus in Korean patients, we investigated the associations of single nucleotide polymorphisms (SNPs) in these three genes in Korean patients with keratoconus. METHODS: Genomic DNA was extracted from blood samples of unrelated patients with keratoconus and healthy control individuals. For screening of genetic variations, all exons from the entire coding regions of the ALDH3A1, LOX, and SPARC genes were directly sequenced to determine the presence of mutations. Control individuals were selected from the general population without keratoconus. RESULTS: In this study, we detected nine SNPs in ALDH3A1, four SNPs in LOX, and 18 SNPs in SPARC. rs116992290, IVS3-62c>t, rs116962241, and rs2228100 in ALDH3A1 and rs2956540 and rs1800449 in LOX were significantly different between patient and control groups. In the SPARC gene, the distribution of the *G allele of EX10+225 T>G (p = 0.018; odds ratio, 1.869) was strongly associated with the risk of keratoconus in the Korean population. In haplotype analysis, C-G of rs2956540-rs2288393 in LOX(p = 0.046) and C-C-G and G-G-G of rs60610024-rs2228100-rs57555435 (p = 0.021 and p < 0.001), G-A of IVS3-62 a>g - rs116962241 in ALDH3A1(p = 0.048) predisposed significantly to keratoconus. After cross-validation consistency and permutation tests, two locus model was the best SNP variations interaction pattern. CONCLUSIONS: Our results suggested that genetic variations in ALDH3A1, LOX, and SPARC genes were associated with a predisposition for keratoconus in Korean individuals. Moreover, variations in ALDH3A1 and LOX may serve as strong biomarkers for keratoconus.

Association of Collagen Gene (COL4A3) rs55703767 Variant With Response to Riboflavin/Ultraviolet A-Induced Collagen Cross-Linking in Female Patients With Keratoconus.

PURPOSE: To investigate for the first time the association of collagen COL4A3 (rs55703767), COL5A1 (rs7044529), and COL4A4 (rs2229813) variants with response to corneal collagen cross-linking (CXL) with riboflavin and ultraviolet A in patients with keratoconus (KC). METHODS: A total of 147 eligible patients with KC were genotyped for the specified collagen variants using real-time TaqMan-based polymerase chain reaction. Adjusted odds ratio (OR) with 95% confidence interval (CI) was applied to assess the strength of the association with response to CXL for a decrease in maximum keratometry and/or an increase in corneal thickness. RESULTS: Eighty-two patients (55.8%) had post-CXL successful outcomes. The overall analysis revealed that minor allele frequencies of COL4A3, COL5A1, and COL4A4 variants were 0.22, 0.22, and 0.38, respectively. The G/T genotype of the COL4A3 variant was more prevalent in the successful group (43%) compared with the failure group (23%) (P < 0.001). COL4A3 (rs55703767) was associated with a good response under heterozygote (OR: 2.19, 95% CI, 1.04-4.59, P < 0.001) and overdominant (OR: 2.59, 95% CI, 1.25-5.38, P = 0.008) models. By contrast, COL5A1 and COL4A4 variants were not associated with the effective response after CXL treatment. Interestingly, stratification analysis by sex revealed that CXL was more successful in female patients with KC under heterozygote (OR: 4.71, 95% CI, 1.74-12.75), dominant (OR: 3.16, 95% CI, 1.29-7.78), and overdominant (OR: 5.18, 95% CI, 1.92-13.95) models for COL4A3 (rs55703767) variant. CONCLUSIONS: The COL4A3 (rs55703767) variant, among other study variants, could be implicated in CXL riboflavin/ultraviolet A treatment response in patients with KC in the study population. Large-scale replication and follow-up studies in different ethnic groups are warranted.

Update on the genetics of keratoconus.

In the past few years we have seen a great acceleration of discoveries in the field of keratoconus including new treatments, diagnostic tools, genomic and molecular determinants of disease risk. Recent genome-wide association studies (GWAS) of keratoconus cases and population wide studies of variation in central corneal thickness and in corneal biomechanical properties confirmed already identified genes and found many new susceptibility variants and biological pathways. Recent findings in genetic determinants of familial keratoconus revealed functionally important variants and established first mouse model of keratoconus. Latest transcriptomic and expression studies started assessing novel non-coding RNA targets in addition to identifying tissue specific effects of coding genes. First genomic insights into better prediction of treatment outcomes are bringing the advent of genomic medicine into keratoconus clinical practice.

Multi-level consistent changes of the ECM pathway identified in a typical keratoconus twin's family by multi-omics analysis.

BACKGROUND: Keratoconus (KC) is a common, degenerative disorder of the cornea, and genetic factors play a key role in its development. However, the genetic etiology of KC is still unclear. This study used the family of twins as material, using, for the first time, multi-omics analysis, to systematically display the changes in KC candidate factors in patients at the DNA, RNA, and protein levels. These can evaluate candidate pathogenic factors in depth and lock onto pathogenic targets. RESULTS: The twins in this study presented classic phenotypes, clear diagnoses, complete case data, and clinical samples, which are excellent materials for genetically studying KC. Whole-exome sequencing was conducted on both the twins and their parents. Transcriptome sequencing was conducted on proband's and health individual's primary human corneal fibroblast cells. Quantitative Real-time PCR and western blot were used to validate the differential gene expressions between the proband and controls. By integrating genomics, transcriptome, and protein level data, multiple consecutive events of KC were systematically analyzed to help better understand the molecular mechanism and genetic basis of KC. The results showed that the accumulation of rare, micro-effect risk variants was the pathogenic factor in this Chinese KC family. Consistent changes in extracellular matrices (ECMs) at the DNA and RNA levels suggested that ECM related changes play a key role in KC pathogenesis. The major gene variants (WNT16, CD248, COL6A2, COL4A3 and ADAMTS3) may affect the expression of related collagens or ECM proteins, thus reducing the amount of ECM in corneas and resulting in KC. CONCLUSIONS: This study, the first to explore the genetic etiology of KC via multi-omics analysis under the polygenetic model, has provided new insights into the genetic mechanisms underlying KC and an effective strategy for studying KC pathogenesis in the future.

The association between genetic polymorphism of glutathione peroxidase 1 (rs1050450) and keratoconus in a Turkish population / A associação entre o polimorfismo genético da glutationa-peroxidase 1 (rs1050450) e pacientes com ceratocone em uma população turca

ABSTRACT Purpose: To investigate the potential associations between keratoconus and catalase rs1001179, superoxide dismutase 2 rs4880, and glutathione peroxidase 1 rs1050450 gene polymorphisms in a Turkish population. Methods: The study group included 121 unrelated keratoconus patients and 94 unrelated healthy controls. Blood samples (200 ml) were collected from all patients and controls to isolate genomic DNA. Genotyping was performed to identify rs1001179, rs4880, and rs1050450 using real-time polymerase chain reaction (PCR). Genotype and allele frequencies were calculated; their associations with keratoconus risk were assayed, and the association with keratoconus risk and demographic factors was examined. Results: Glutathione peroxidase 1 rs1050450 polymorphism was present in 41% cases compared with 29% controls (OR=1.66; 95% CI=1.11-2.50; p=0.014). No association was observed between catalase rs1001179 and SOD2 rs4880 polymorphisms and keratoconus (for all, p>0.05). Conclusions: This study evaluated possible relationships between rs1050450, rs1001179, and rs4880 polymorphisms and keratoconus susceptibility. We found a possible association between glutathione peroxidase 1 rs1050450 polymorphism and an increased risk of keratoconus. However, the genotype and allele frequencies were identical in the catalase rs1001179 and superoxide dismutase 2 rs4880 polymorphisms. Further studies are needed to analyze the effect of such variations in identifying keratoconus susceptibility.

RESUMO Objetivo: Investigar as possíveis associações entre o ceratocone e os polimorfismos rs1001179 da catalase, rs4880 da superóxido-dismutase 2 e rs1050450 da glutationa-peroxidase 1 rs1050450 em uma população turca. Métodos: O grupo de estudo incluiu 121 pacientes com ceratocone não relacionados e 94 controles saudáveis também sem pa rentesco. Amostra de sangue (200 mL) foram coletadas de todos os pacientes e controle para isolar o DNA genômico. A genotipagem foi realizada para identificar rs1001179, rs4880 e rs1050450 utilizando a reação em cadeia da polimerase (PCR) em tempo real. As frequências de genótipos e alelos foram calculadas, suas associações com o risco de ceratocone foram avaliadas, e a associação com risco de ceratocone e fatores demográficos foi examinada. Resultados: O polimorfismo da glutationa-peroxidase 1 rs1050450 estava presente em 41% dos casos, comparado com 29% dos controles (OR=1,66, IC 95%=1,11-2,50; p=0,014). Não foi observada associação entre o ceratocone e os polimorfismos rs1001179 e SOD2 rs4880 da catalase (para todos, p>0,05). Conclusões: Este estudo avaliou possíveis relações entre os polimorfismos rs1001179, rs4880 e suscetibilidade a cerato cone. Encontramos uma possível associação entre po limorfis mo da glutationa-peroxidase 1 rs1050450 e um risco aumentado de ceratocone. No entanto, o genótipo e as frequências alélicas foram idênticas nos polimorfismos rs1001179 da catalase e superóxido-dismutase 2 rs4880. Mais estudos são necessários para esclarecer o efeito dessas va riações na detecção da sus cetibilidade ao ceratocone.

Whole-exome sequencing in familial keratoconus: the challenges of a genetically complex disorder / Sequenciamento do exoma no ceratocone familiar: os desafios de uma doença genética complexa

ABSTRACT Purpose: The underlying genetic causes of keratoconus are essentially unknown. Here, we conducted whole-exome sequencing in 2 Brazilian families with keratoconus. Methods: Whole-exome sequencing was performed on 6 keratoconus-affected individuals of 2 unrelated pedigrees from Southern Brazil. Pathogenic variants were identified in a modified Trio analysis (1 parent and 2 children) using candidate gene filtering. All the affected subjects underwent detailed corneal tomographic evaluation. Clinically relevant variants that were present in affected individuals at minor allele frequencies <1% were examined in the 1000 Genomes Project single nucleotide polymorphism ABraOM and transcription gene (RefSeq and Ensembl) databases. Results: In family 1, a sequence variant in chromosome 1 (q21.3) was observed within the filaggrin gene. All the tested family members shared a heterozygous missense pathogenic variant in the c.4678C>T position. In family 2, exome analysis demonstrated a sequence variant in chromosome 16 (q24.2) within the gene encoding zinc finger protein 469 (ZNF469). Members of family 2 shared a heterozygous missense variant in the c.1489G>A position. In addition, the exomes of the 2 families were examined for shared genetic variants among all affected individuals. Filtering criteria did not identify any rare sequence variants in a single gene segregated in both families. Conclusion: Our findings show that a complete genotype-phenotype correlation could not be identified, suggesting that keratoconus is a genetically heterogeneous disease. In addition, we believe that whole-exome sequencing-based segregation analysis is probably not the best strategy for identifying variants in families with isolated keratoconus.

RESUMO Objetivos: As causas genéticas subjacentes do ceratocone são essencialmente desconhecidas. Aqui, realizamos o sequenciamento de todo exoma de duas famílias brasileiras com ceratocone. Métodos: O sequenciamento total do exoma foi realizado em 6 indivíduos com ceratocone de duas famílias distintas do sul do Brasil. Variantes patogênicas foram identificadas em uma análise no formato de trio-modificada (um dos pais e dois filhos) usando a filtragem de genes candidatos. Todos os indivíduos afetados passaram por avaliação de tomografia de córnea. Variantes clinicamente relevantes que estavam presentes em indivíduos afetados em menores frequências alélicas <1% foram avaliadas na base de dados de polimorfismo de nucleotídeo único do 1000 Genomes Project ABraOM e do gene de transcrição (RefSeq e Ensembl). Resultados: Na família 1, uma variante de sequência no cromossomo 1 (q21.3) foi observada dentro do gene da filagrina. Todos os membros dessa família compartilhavam uma mutação missense na posição c.4678C>T. Na família 2, a análise do exoma demonstrou uma variante alélica no cromossomo 16 (q24.2) dentro do gene que codifica a proteína de dedo de zinco 469 (ZNF469). Os membros dessa família compartilham uma mutação missense heterozigota na posição c.1489G>A. Além disso, os exomas das duas famílias foram avaliados para variantes genéticas compartilhadas entre todos os indivíduos afetados. Os critérios de filtragem não identificaram variantes de sequência rara em um único gene segregado em ambas as famílias. Conclusão: Nossos achados indicam que uma completa correlação genótipo-fenótipo não pode ser identificada, sugerindo que o ceratocone é uma doença geneticamente heterogênea. Além disso, acreditamos que análises de segregação baseadas no sequenciamento de todo exoma provavelmente não é a melhor estratégia para identificar variantes em famílias isoladas com ceratocone.

Autosomal Recessive Non-syndromic Keratoconus: Homozygous Frameshift Variant in the Candidate Novel Gene GALNT14.

BACKGROUND: Keratoconus (KC) is usually bilateral, noninflammatory progressive corneal ectasia in which the cornea becomes progressively thin and conical. Despite the strong evidence of genetic contribution in KC, the etiology of KC is not understood in most cases. METHODS: In this study, we used whole-exome sequencing to identify the genetic cause of KC in two sibs in a consanguineous family. The Homozygous frameshift variant NM_001253826.1:c.60delC;p.Leu21Cysfs*6 was identified in the gene Nacetylgalactosaminyltransferase 14 (GALNT14). The variant does not exist in all public databases neither in our internal exome database. Moreover, no database harbours homozygous loss of function variants in the candidate gene. RESULT: GALNT14 catalyses the initial reaction in O-linked oligosaccharide biosynthesis, the transfer of an N-acetyl-D- galactosamine residue to a serine or threonine residue on target proteins especially Mucins. CONCLUSION: As alterations of mucin's glycosylation are linked to a number of eye diseases, we demonstrate in this study an association between the truncated protein GALNT14 and KC.

Corneal Perforation After Corneal Cross-Linking in Keratoconus Associated With Potentially Pathogenic ZNF469 Mutations.

PURPOSE: To report a case of bilateral and repetitive corneal perforations after corneal cross-linking (CXL) for keratoconus in a woman harboring potentially pathogenic variants in the ZNF469 gene and to characterize the keratoconus phenotype in this woman and her daughter who shared the same ZNF469 mutations. METHODS: Clinical characterization of the proband and her daughter followed by sequencing of the genes associated with brittle cornea syndrome, ZNF469 and PRDM5, in both individuals. RESULTS: An Ashkenazi Jewish woman in her sixth decade presented with diffuse corneal thinning and progressive steepening consistent with keratoconus. After CXL, epithelium-off in the first eye and epithelium-on in the second, she developed spontaneous corneal perforations in each eye. Her daughter in her fourth decade demonstrated a similar pattern of diffuse corneal thinning and progressive corneal steepening but did not undergo CXL and did not develop corneal perforation. Screening of the ZNF469 and PRDM5 genes revealed 3 missense ZNF469 variants (c.2035G>A, c.10244G>C, and c.11119A>G) in cis arrangement on 1 allele of ZNF469 in both proband and her daughter. Although the 3 variants share low (<0.01) global minor allele frequencies, each has significantly higher minor allele frequencies (0.01-0.03) in the Ashkenazi Jewish population, leading to uncertainty regarding a pathogenic role for the identified variants. CONCLUSIONS: CXL may be associated with the development of corneal perforation in particular at-risk individuals with keratoconus. Identifying clinical and genetic risk factors, including screening of ZNF469 and PRDM5, may be useful in the prevention of significant complications after CXL.