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Purpose: To identify inherited or acquired mutations in the VSX1, SOD1, TIMP3 and LOX genes from the combined analysis of corneal and blood samples from patients with Keratoconus., Methods: The casuistry was consisted of samples of peripheral blood and corneal epithelium from 35 unrelated patients with Keratoconus who were submitted to corneal crosslink treatment. Also, blood and corneal epithelium samples from 89 non-keratoconic patients were used to compose the control group. Ophthalmologic evaluations included a clinical examination, topography and tomography. DNA samples were extracted from peripheral blood and from corneal epithelium in both groups and all coding regions of the VSX1, SOD1, TIMP3 and LOX genes were amplified by polymerase chain reaction, denatured and subjected to polyacrylamide gel electrophoresis. Mutational screening was performed by single -s trand conformation polymorphism and direct DNA sequencing., Results: No pathogenic variant was found in all coding regions of VSX1, SOD1, TIMP3 and LOX genes, we detected only few SNPs (single-nucleotide polymorphisms). Among the polymorphisms stand out three of them, corresponding to the synonymous exchange of amino acids: exon 3 of VSX1 Ala182Ala and exon 3 of TIMP3 His83His and Ser87Ser; in patients with Keratoconus and also in control subjects. All the polymorphisms were found in samples of corneal epithelium and corresponding blood., Conclusion: There is absence of KC pathogenic related to mutations in the VSX1, SOD1, TIMP3 and LOX genes in the studied patients.

Keratoconus is a chronic disorder of the cornea, characterized by its progressive thinning, stretching, and conical protrusion. Diagnostics of subclinical keratoconus, as well as its early stages (forme fruste), is a complex problem. The presence of these forms of keratoconus in a patient is one of the reasons for the development of keratectasia after laser refractive surgery. Currently, the role of genetic factors in keratoconus development has been proven. This indicates the possibility of diagnostics of subclinical and forme fruste keratoconus using genetic markers. Knowledge about the patient's genetic susceptibility to keratoconus would allow correcting the tactics of treatment of refractive anomalies and avoiding serious side effects. The studies of causal mutations indicate the genetic heterogeneity of keratoconus, which complicates the development of a diagnostic panel. Selection of candidate variants from the currently known ones based on clear criteria may be one of the approaches for diagnostic markers search. In this review, we have analyzed articles on keratoconus markers in order to form a list of candidate variants for genotyping in the Russian population. The selection criteria took into account the complexes of symptoms in which a marker was found, populations in which a particular marker was investigated, the presence and results of replication studies. The analysis included markers in VSX1, SOD1, ZEB1, LOX, CAST, DOCK9, TGFBI, HGF, MAP3K19, KCND3, COL4A3, COL4A4, COL5A1, FNDC3B, FOXO1, BANP-ZNF469, MPDZ-NF1B, WNT10A genes. Based on the results of the analysis, the following candidate variants were selected for genotyping in the Russian population of patients with keratoconus: rs1536482 and rs7044529 in the COL5A1 gene, rs5745752 and rs2286194 in the HGF gene, rs4954218 in the MAP3K19 gene, rs4839200 near the KCND3 gene, rs2721051 near the FOXO1 gene, rs1324183 between the MPDZ and the NF1B genes, and rs121908120 in the WNT10A gene.

Purpose: Despite numerous studies associating Visual System Homeobox 1 (VSX1), with posterior polymorphous corneal dystrophy and keratoconus, its role in these diseases is unclear. Here we examine the pathogenicity of VSX1 missense mutations in vitro and in a mouse genetic model., Methods: Vsx1 transcriptional repressor activity, protein stability, and subcellular localization activity, was examined using luciferase reporter-based assays, western blotting and immunolabeling, respectively, in transfected human embryonic kidney 293T cells. A genetic model for VSX1 p.P247R was generated to investigate pathogenicity of the mutation, in vivo. A wholemount confocal imaging approach on unfixed intact eyes was developed to examine corneal morphology, curvature, and thickness. Immunolabeling and electroretinography was used to examine retinal phenotype., Results: A mutation corresponding to human VSX1 p.P247R led to enhanced transcriptional repressor activity, in vitro. A mouse model for VSX1 p.P247R did not have any observable corneal defect, but did exhibit an abnormal electroretinogram response characterized by a more prominent ON as opposed to OFF panretinal responsiveness. In vitro analysis of additional VSX1 missense mutations showed that they either enhanced repressor activity or did not alter activity., Conclusions: Our results indicate that although VSX1 sequence variants can alter transcriptional activity, in the context of a mouse genetic model, at least one of these changes does not lead to corneal abnormalities. While we cannot exclude a role for VSX1 as a risk factor for corneal disease, on its own, it does not appear to play a major causative role.

Objective: To investigate the possibility of the visual system homeobox 1 (VSX1) gene as a candidate susceptibility gene for Chinese patients with sporadic keratoconus, and to identify sequence variants of the VSX1 gene in such patients. Methods: Cross-sectional study. Genomic DNA was extracted from the leukocytes in the peripheral venous blood of 50 patients with sporadic keratoconus and 50 control subjects without this ocular disorder. Five exons and the intron-exon splicing of the VSX1 gene were amplified by polymerase chain reaction (PCR). The PCR products were directly sequenced and compared to the GeneBank database to find mutations. Bioinformatics analysis was done to predict the influence of these mutations on proteins. Results: One novel missense heterozygous mutation (p.R131P) was found in exon 1 of the VSX1 gene in one keratoconus patient. Another heterozygous mutation (p.G160V) in exon 2 was found in two keratoconus patients. These mutations were not detected in the control subjects. Bioinformatics analysis predicted that the p.R131P mutation may not cause a pathogenic change, but the p.G160V mutation might be functionally deleterious. In intron 3 of the VSX1 gene, the nucleotide substitution of g.8326G>A was detected to be heterozygous in 3 cases of sporadic keratoconus and 4 cases of control and homozygous in 2 cases of sporadic keratoconus and 1 case of control. The variation of g.8326G>A belonged to a single polymorphism change of the VSX1 gene. Conclusions: The p.R131P detected in this study is a novel mutation of the VSX1 gene. Sequence variants of the VSX1 gene were identified for the first time in Chinese patients with sporadic keratoconus, but their precise role in disease causation requires further investigations. (Chin J Ophthalmol, 2018, 54: 212-217) .

Purpose: Common polymorphic variants upstream of Zinc finger protein gene 469 (ZNF469) have been associated with central corneal thickness. Rare ZNF469 variants have been shown in keratoconus patients. The aim of the current study was to investigate the frequency of ZNF 469 gene variants in rapidly progressive advance keratoconus patients who underwent corneal transplant surgery by the age of 30, compared to their frequency in the normal Turkish population., Methods: A search in a patient database was performed to identify patients with a rapidly progressive keratoconus requiring corneal transplant surgery by the age of 30 in at least one eye. Twenty-six advance keratoconus patients (study group) and 109 health subjects (control group) were included in the study. Blood samples were donated, and genomic DNA was extracted. The entire coding sequence of the ZNF469 gene including the 84 bp of the putative intron was amplified using PCR primers and analyzed using next generation sequencing (NGS)., Results: Fifteen single nucleotide polymorphisms previously reported and registered to the dbSNP database were detected in the study group. The allele frequencies of these polymorphisms were higher in the keratoconus group compared to the control group and to the ExAC genome database. Three new missense heterozygote variants and one new synonym variant were detected in keratoconus group. According to prediction software, the P873T and Q2188H variants were shown to be non-tolerated, whereas G3424S could be tolerated. The synonymous variant R1060R is not predicted to lead to abnormal splicing by Human Splicing Finder in silico analysis., Conclusion: New detected ZNF 469 P873T and Q2188H heterozygote coding variants in isolated advance keratoconus patients may be associated with the disease pathogenesis.

Background: Keratoconus normally presents as a sporadic disease. Although different studies have found sequence variants of the visual system homeobox 1 (VSX1) gene associated with keratoconus in humans, no research has detected such variants in sporadic keratoconus patients from China. To investigate the possibility of VSX1 being a candidate susceptibility gene for Chinese patients with sporadic keratoconus, we performed sequence screening of this gene in such patients., Methods: Whole DNA was obtained from the leukocytes in the peripheral venous blood of 50 patients with sporadic keratoconus and 50 control subjects without this ocular disorder. Polymerase chain reaction single-strand conformation polymorphism analysis and direct DNA sequencing technology were used to detect sequence variation in the five exons and splicing regions of the introns of the VSX1 gene. The sequencing results were analyzed using DNAstar software., Results: One novel missense heterozygous sequence variant (p.Arg131Pro) was found in the first exon of the VSX1 gene in one keratoconus patient. Another heterozygous sequence variant (p.Gly160Val) in the second exon was found in two keratoconus patients. These variants were not detected in the control subjects. In the third intron of the VSX1 gene, c.8326G > A nucleotide substitution (including heterozygous and homozygous change) was also discovered. The frequency of this variation did not differ significantly between patients and controls, it should belong to single-nucleotide polymorphism of the VSX1 gene. Bioinformatic analysis also predicted that one missense sequence variation (p.Arg131Pro) may not cause a pathogenic change., Conclusions: In this study, we added one novel missense sequence variation (p.Arg131Pro) in the coding region of the VSX1 gene to the range of VSX1 coding region variations observed in patients with sporadic keratoconus from China. Our work suggests that VSX1 sequence variants might be involved in the pathogenesis of sporadic keratoconus, but their precise role in disease causation requires further investigation.

The aim of this study was to screen the visual system homeobox 1 (VSX1) gene in Turkish patients with keratoconus (KC). The patient group consisted of 44 patients who had undergone corneal transplant surgery before the age of 30, for advanced and rapidly progressive KC. The control group comprised 250 healthy individuals. We detected two missense mutations, D144N and D295Y, in exon 2 and exon 5 of the VSX1 gene, respectively, using next-generation sequencing analysis. The pathologic effects of the D144N and D295Y missense mutations on protein function were determined with bioinformatic analysis tools, SIFT, PolyPhen, and MutationTaster. Aspartic acid at the 144th position was more preserved among species than aspartic acid at the 295th position of the VSX1 protein. In the control group, five different genetic variations were detected, two of which (rs8123716 and rs12480307) were synonymous with variations in the patient group. Our results suggested that the D144N and D295Y mutations might have a role in the pathogenesis of KC disease.

Background: Keratoconus is an ocular degeneration characterized by the thinning of corneal stroma that may lead to varying degrees of myopia and visual impairment. Genetic factors have been reported in the pathology of keratoconus where Asians have a higher incidence, earlier onset, and undergo earlier corneal grafts compared to Caucasians. The visual system homeobox 1 (VSX1) gene forms part of a paired-like homeodomain transcription factor which is responsible for ocular development. The gene was marked as a candidate in genetic studies of keratoconus in various populations. Single nucleotide polymorphisms (SNPs) in the VSX1 gene have been reported to be associated with keratoconus. The detection of the SNPs involves DNA amplification of the VSX1 gene followed by genomic sequencing. Thus, the objective of this study aims to establish sensitive and accurate screening protocols for the molecular characterization of VSX1 polymorphisms., Methods: Keratoconic (n = 74) and control subjects (n = 96) were recruited based on clinical diagnostic tests and selection criteria. DNA extracted from the blood samples was used to genotype VSX1 polymorphisms. In-house designed primers and optimization of PCR conditions were carried out to amplify exons 1 and 3 of the VSX1 gene. PCR conditions including percentage GC content, melting temperatures, and differences in melting temperatures of primers were evaluated to produce sensitive and specific DNA amplifications., Results: Genotyping was successfully carried out in 4 exons of the VSX1 gene. Primer annealing temperatures were observed to be crucial in enhancing PCR sensitivity and specificity. Annealing temperatures were carefully evaluated to produce increased specificity, yet not allowing sensitivity to be compromised. In addition, exon 1 of the VSX1 gene was amplified using 2 different sets of primers to produce 2 smaller amplified products with absence of non-specific bands. DNA amplification of exons 1 and 3 consistently showed single band products which were successfully sequenced to yield reproducible data., Conclusions: The use of in-house designed primers and optimized PCR conditions allowed sensitive and specific DNA amplifications that produced distinct single bands. The in-house designed primers and DNA amplification protocols established in this study provide an addition to the current repertoire of primers for accurate molecular characterization of VSX1 gene polymorphisms in keratoconus research.

Purpose: Keratoconus (KTCN) is a degenerative disorder of the eye that results in the conical shape and thinning of the cornea and is a leading cause for corneal transplantations. A number of studies suggest that genetic factors play a role in KTCN etiology. Some candidate gene variants have recently been shown to be associated with KTCN. The purpose of our study was to verify the role of VSX1, TGFBI, DOCK9, IPO5, and STK24 sequence variants in Polish KTCN patients., Methods: Forty-two Polish patients with sporadic KTCN and 50 control individuals were enrolled into this study. Both affected and unaffected individuals underwent detailed ophthalmic examination. The mutations screening in the candidate genes was performed by the direct sequencing method., Results: Analysis of VSX1, TGFBI, DOCK9, IPO5, and STK24 genes identified numerous sequence variants. Variants c.-264_-255delGGGGTGGGGT, c.627 + 23G > A, c.809-6_809-5insT, and c.*200G > T in the VSX1 gene, and heterozygous c.1598G > A mutation (Arg533Gln) in exon 12 of TGFBI were detected for the first time in KTCN patients. Two known sequence variants of TGFBI c.1620T > C (Phe540Phe) and c.1678 + 23G > A were observed in KTCN patients and control individuals. The newly reported c.717 + 43A > G substitution in intron 7 of DOCK9 was identified in both KTCN patients and healthy individuals., Conclusions: 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.

Background: Visual system homeobox gene (VSX1) plays a major role in the early development of craniofacial and ocular tissues including cone opsin gene in the human retina. To date, few disease-causing mutations of VSX1 have been linked to familial and sporadic keratoconus (KC) in humans. In this study, we describe the clinical features and screening for VSX1 gene in families with KC from India., Methods: Clinical data and genomic DNA were collected from patients with clinically diagnosed KC and their family members. The study was conducted on 20 subjects of eight families from India. The coding exons of VSX1 gene were amplified using PCR and amplicons were analyzed by direct sequencing. Predictive effect of the mutations was performed using Polyphen-2, SIFT and mutation assessor algorithms. Additionally, haplotypes of VSX1 gene were constructed for affected and unaffected individuals using SNPs., Results: In the coding region of VSX1, one novel missense heterozygous change (p.Leu268His) was identified in five KC patients from two unrelated families. Another family of three members had a novel heterozygous change (p.Ser251Thr). These variants co-segregated with the disease phenotype in all affected individuals but not in the unaffected family members and 105 normal controls. In silico analysis suggested that p.Leu268His could have a deleterious effect on the protein coded by VSX1, while p.Ser251Thr has a neutral effect on the functional properties of VSX1. Haplotype examination revealed common SNPs around the missense change (p.Leu268His) in two unrelated KC families., Conclusions: In this study, we add p.Leu268His, a novel missense variation in the coding region of VSX1 to the existing repertoire of VSX1 coding variations observed in Indian patients with the characteristic phenotype of KC. The variant p.Ser251Thr might be a benign polymorphism, but further biophysical studies are necessary to evaluate its molecular mechanism. The shared haplotype by two families with the same variant suggests the possibility of a founder effect, which requires further elucidation. We suggest that p.Leu268His might be involved in the pathogenesis of KC, which may help in the genetic counselling of patients and their family.

Background: A number of mutations in the VSX1 and SOD1 genes have been reported to be associated with keratoconus (KC), however the results from different studies are controversial. In this study, we conducted the genotyping of common polymorphisms [VSX1: D144E, H244R, R166W, G160D; SOD1: intronic 7-base deletion (c.169 + 50 delTAAACAG)], in a case-control sample panel of the Greek population., Materials and Methods: A case-control panel, with 33 KC patients and 78 healthy controls, were surveyed. DNA from each individual was tested for the VSX1: D144E, H244R, R166W, G160D and SOD1: intronic 7-base deletion (c.169 + 50 delTAAACAG) polymorphisms by direct sequencing., Results: We observed no polymorphisms of the VSX1 gene in the case-control panel. Concerning the SOD1 intronic 7-base deletion (c.169 + 50 delTAAACAG), our findings suggest that heterozygous carriers are over-represented among KC cases compared to healthy controls (p = 0.002)., Conclusions: We cannot confirm the previously reported association of the polymorphism in the VSX1 gene with KC. Our results suggest a possible causative role of SOD1 in the pathogenesis of KC. Further studies are required to identify other important genetic factors involved in the pathogenesis and progression of KC.

Purpose: A recent genome-wide association study (GWAS) identified six loci associated with central corneal thickness that also conferred associated risk of keratoconus (KC). We aimed to assess whether genetic associations existed for these loci with KC or corneal curvature in an independent cohort of European ancestry., Methods: In total, 157 patients with KC were recruited from public and private clinics in Melbourne, Australia, and 673 individuals without KC were identified through the Genes in Myopia study from Australia. The following six single-nucleotide polymorphisms (SNPs) that showed a statistically significant association with KC in a recent GWAS study were selected for genotyping in our cohort: rs4894535 (FNDC3B), rs1324183 (MPDZ-NF1B), rs1536482 (RXRA-COL5A1), rs7044529 (COL5A), rs2721051 (FOXO1), and rs9938149 (BANP-ZNF469). The SNPs were assessed for their association with KC or corneal curvature using logistic or linear regression methods, with age and sex included as covariates. Bonferroni corrections were applied to account for multiple testing., Results: Genotyping data were available for five of the SNPs. Statistically significant associations with KC were found for the SNPs rs1324183 (P = 0.001; odds ratio [OR], 1.68) and rs9938149 (P = 0.010; OR, 1.47). Meta-analysis of previous studies yielded genome-wide significant evidence of an association for rs1324183, firmly establishing it as a KC risk variant. None of the SNPs were significantly associated with corneal curvature., Conclusions: The SNPs rs1324183 in the MPDZ-NF1B gene and rs9938149 (between BANP and ZNF4659) were associated with KC in this independent cohort, but their association was via a non-corneal curvature route.

Objective: To investigate whether the tag single nucleotide polymorphisms (tSNPs) in VSX1, COL4A3, COL4A4, IL1A and IL1B genes were associated with keratoconus (KTCN) in the Han Chinese population., Methods: Ninety-seven KTCN patients and 101 healthy controls were enrolled in this study. All cases were diagnosed on the basis of clinical examination. Twenty-one tSNPs were selected for association study in five genes. SNP genotyping was performed by Sequenom MassARRAY RS1000. Sequenom Typer 4.0 Software, PLINK, Haploview and SHEsis software platform were used to perform data management and analyses., Results: Three tSNPs in the VSX1 gene were observed to be associated with KTCN risk at a 5% level by χ(2) test (rs56157240 and rs12480307, p = 0.0499, OR: 6.42, 95% CI: 0.77-53.78; rs6050307, p = 1.22 × 10(-7), OR: 0.05, 95% CI: 0.01-0.23). Rs2071376 in the IL1A gene was also associated with KTCN (p = 0.0487, OR: 1.51, 95% CI: 1.00-2.26). Three haplotypes in the VSX1 gene were found to be associated with risk of KTCN (p < 0.05)., Conclusions: Our findings confirmed previous reports that polymorphisms of VSX1 and IL1A genes were associated with risk of KTCN in the Chinese population, suggesting an important determinant of KTCN development by VSX1 and IL1A genes.

Keratoconus is a progressive corneal thinning disease associated with significant tissue remodeling activities and activation of a variety of signaling networks. However, it is not understood how differential gene and protein expression direct function in keratoconus corneas to drive the underlying pathology, ectasia. Research in the field has focused on discovering differentially expressed genes and proteins and quantifying their levels and activities in keratoconus patient samples. In this study, both microarray analysis of total ribonucleic acid (RNA) and whole proteome analyses are carried out using corneal epithelium and tears from keratoconus patients and compared to healthy controls. A number of structural proteins, signaling molecules, cytokines, proteases, and enzymes have been found to be deregulated in keratoconus corneas. Together, the data provide clues to the complex process of corneal degradation which suggest novel ways to clinically diagnose and manage the disease. This review will focus on discussing these recent advances in the knowledge of keratoconus biology from a gene expression and function point-of-view.

Purpose: Mutations in the visual system homeobox 1 (VSX1) gene have been described at a low frequency in keratoconus and posterior polymorphous corneal dystrophy (PPCD). The putative role is controversial for several reasons, including a lack of mutations detected in other population cohorts. This study aims to determine whether VSX1 contributes to the genetic pathogenesis of keratoconus and PPCD in a New Zealand population, and includes analysis of a Polynesian population., Methods: Recruitment of patients with keratoconus and PPCD, comprehensive clinical examination including corneal topography and pachymetry, and collection of biologic samples for DNA extraction were undertaken. Mutational analysis of VSX1 (exons 1-7) with PCR and sequencing with bioinformatic assessment of variants was performed. Probable pathogenic variants were screened for in a control population using high-resolution melting analysis., Results: Forty-seven patients with keratoconus, including 15 familial cases, and ten unrelated patients with PPCD were recruited. Two pathogenic changes were detected; a novel change c.173C>T (p.Pro58Leu) was found in a patient with PPCD, predicted to be pathogenic, and not seen in 200 ethnically matched control alleles. The previously reported c.731A>G (p.His244Arg) was detected in a patient with sporadic keratoconus, and not present in the controls. No family members were available for segregation analysis., Conclusions: This study reports the presence of pathogenic mutations in VSX1 in PPCD and keratoconus, including a novel disease-causing variant. The affected numbers are small, but given the growing body of evidence of pathogenic segregating changes in VSX1 in disease cohorts, the expression in keratocytes as part of wound healing, and the documented association of PPCD and keratoconus, it seems likely that the role of VSX1 as a genetic factor contributing to disease is real.

Background: The involvement of VSX1 gene for the genetic basis of keratoconus is unclear and controversial. The genetic screening of VSX1 from different ethnic populations can enlighten this subject. The aim of the present study is to investigate the role of VSX1 gene in patients with sporadic cases of keratoconus from South India., Methods: The VSX1 gene coding regions, including exon-intron boundaries were screened by direct sequencing analysis in 117 sporadic cases of keratoconus. The identified variations were also analyzed in 108 ethnic matched healthy blood donors., Results: In the VSX1 gene screening, no pathogenic mutation was identified, whereas we could find the presence of four reported single nucleotide polymorphisms; c.546A>G (rs12480307), c.627+23G>A (rs6138482), c.627+84T>A (rs56157240) and c.504-24C>T (IVS3-24C). These variations were observed in similar frequency between cases and controls., Conclusions: The lack of VSX1 pathogenic variations in a large number of unrelated sporadic keratoconus patients tend to omit its role, and corroborate the involvement of other genetic, environmental or behavioural factors in the development of this complex disorder.

Objective: Keratoconus (KC) is an eye disorder in which the cornea is swollen, thinned and deformed. Despite extensive studies, the pathophysiological processes and genetic etiology of KC are unknown. The disease incidence is approximately 1 in 2,000, and it is the most common cause of corneal transplantation in the USA. Many genes are involved in the disease, but evidence suggests a major role for VSX1 in the etiology of KC. This study aimed to determine the frequency of mutations in exons 2, 3 and 4 of the VSX1 gene in Chaharmahal va Bakhtiari province in the southwest of Iran., Study Design: In this experimental study, mutations in 3 exons, namely exons 2, 3 and 4, of VSX1 were investigated in 50 patients with KC and 50 healthy control subjects. DNA was extracted using a standard phenol-chloroform method. PCR-single-strand conformational polymorphism/heteroduplex analysis was performed, followed by DNA sequencing to confirm the identified motility shifts., Results: H244R mutations were found in 1 patient and also in 1 healthy control subject. Furthermore, 12 polymorphisms were identified in patients with KC and 7 in healthy control subjects [rs6138482 and c.546A>G (rs12480307)]., Conclusion: Our investigation showed that KC-related VSX1 mutations were found in a very small proportion of the studied patients from Iran. Further investigations on other genes are needed to clarify their roles in KC pathogenesis.

Purpose: The visual system homeobox 1 (VSX1) gene variants have recently been shown to be associated with keratoconus. To replicate this finding, we performed a genetic analysis of the VSX1 gene in a Korean case-control sample., Methods: Patients with keratoconus and healthy control subjects were recruited from Seoul National University Hospital. A diagnosis of keratoconus was made based on clinical examinations and the presence of characteristic topographic features. For all patients and controls, the whole coding region and the exon-intron junctions of the VSX1 gene were analyzed by direct sequencing., Results: Fifty-three patients with keratoconus and 100 healthy volunteers were included. We observed 2 novel missense substitutions (Leu17Val and Val199Leu) and 1 previously reported substitution (Gly160Val) in 6 of the 53 affected probands. Because these substitutions have been identified in unaffected individuals, they were not considered to be pathogenic. No intragenic polymorphism was associated with a significantly increased risk of keratoconus., Conclusions: We cannot confirm the previously reported association of the VSX1 gene variants with keratoconus. Our results suggest that the VSX1 gene and its mutations with amino acid changes do not play a major role in the pathogenesis of keratoconus.

Purpose: To decipher the biological pathways involved in keratoconus pathophysiology by determining the patterns of differential gene expression between keratoconus and control corneas., Methods: RNA was extracted from surgically removed corneas of 10 keratoconus patients and from normal corneas of 10 control patients who had undergone enucleation of an eye for ocular melanoma. Several hundred thousand RNA transcripts were assessed using exon microarrays. Statistical comparison and identification of differentially regulated and differentially spliced RNA transcripts was performed by comparing keratoconus cases and controls. In addition, relevant biological pathways were identified by information extraction using network biology., Results: Eighty-seven genes showed significant differences in expression levels. Among these, 69 were downregulated in keratoconus patients, particularly partners of the transcription factor AP-1. The 18 overexpressed genes include mucins, keratins, and genes involved in fibroblast proliferation. In addition, 36 genes were shown to be differentially spliced, including 9 among those that were differentially expressed. Network biology and analysis using Gene Ontology descriptors suggest that many members of both groups belong to pathways of apoptosis and regulation of the balance between cellular differentiation and proliferation., Conclusions: This work constitutes the first genome-wide transcriptome analysis of keratoconus patient corneas that include all currently known genes and exons. Differential expression suggests that mechanisms of cell loss resulting from antiproliferative and hyperapoptotic phenotypes may be responsible for the pathogenesis of keratoconus. Array information, experimental design, raw intensities, and processed log(2) ratios were deposited at the European Bioinformatic Institute's ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/). The accession number is E-MEXP-2777.

Purpose: To screen the visual system homebox 1 (VSX1) gene in Saudi Arabian keratoconus patients., Methods: We sequenced the entire coding region, exon-intron boundaries in clinically confirmed keratoconus patients (n=55) and 50 ethnically matched healthy controls. All cases and controls were unrelated., Results: Sequencing VSX1 revealed the presence of five nucleotide changes, 3 of which were non-coding (g.8326 G>A, g.10945 G>T, and g.11059 A>C) and 2 were synonymous-coding sequence changes (g.5053 G>T and g.8222 A>G). All five sequence changes were benign polymorphisms with no apparent clinical significance., Conclusions: In our keratoconus cohort, no pathogenic VSX1 mutation(s) were identified.

Purpose: To look for segregation of Visual System Homeobox 1 (VSX1) mutations in family members of a patient with keratoconus., Methods: Our initial molecular genetic studies conducted to identify the role of VSX1 in the causation of keratoconus had identified a novel mutation in one patient. He later presented to the clinic affected with vernal kerato conjunctivitis (VKC) accompanied by his brother, also similarly affected. All the family members were called and detailed clinical evaluations were undertaken. DNA from the blood samples of all family members was amplified using primers specific for VSX1 and analyzed by direct sequencing to look for segregation of the mutation in the family members. Protein modeling studies were done to assess the effect of the mutation on protein structure and function., Results: Clinical examination of the family revealed bilateral keratoconus and VKC in the proband and his brother. One of his sisters had VKC without keratoconus and his parents and another sister were normal. Molecular analysis identified the VSX1 mutation Q175H in the affected brother and in the mother who had neither VKC nor keratoconus but only the VSX1 Q175H sequence change., Conclusions: The VSX1 Q175H mutation may be a pathogenic variant with incomplete penetrance. Protein modeling studies show that the mutation affects the DNA binding properties of the protein. This VSX1 variant exhibiting low penetrance may require the presence of some modifier genes or environmental factors for disease presentation. VSX1 may have an important role in the pathogenesis of keratoconus which needs further investigation.

Purpose: To evaluate mutations in the visual system homeobox gene 1 (VSX1) and superoxide dismutase 1 (SOD1) genes with keratoconus (KTCN), direct sequencing was performed in an Iranian population., Methods: One hundred and twelve autosomal dominant KTCN patients and fifty-two unaffected individuals from twenty-six Iranian families, as well as one hundred healthy people as controls were enrolled. Genomic DNA was extracted from whole blood sample. Then to study the possible linkage between KTCN and six known loci linkage analysis was performed using 12 short tandem repeat (STR) markers. Also, the entire coding region and intron-exon boundaries of VSX1 and SOD1 were amplified by the PCR technique in each proband. Subsequently, PCR products were subjected to direct sequencing. Co-segregation analysis of the identified mutation was conducted in the family members. An Amplification Refractory Mutation System PCR (ARMS-PCR) was additionally employed for detection of the identified mutation in healthy controls., Results: Linkage analysis of aforementioned loci did not detect evidence for linkage to KTCN. Direct PCR sequencing revealed two single nucleotide polymorphisms (SNPs; g.1502T>G and g.9683C>T), as well as two missense mutations that have been previously reported (R166W and H244R) in VSX1. We also found three undescribed SNPs (g.4886G>A, g.4990C>G, and g.9061T>A) in SOD1. The R166W and H244R mutations were co-segregated in affected family members but not in those that were unaffected. Moreover, the ARMS-PCR strategy did not detect the identified mutations in controls., Conclusions: Our data suggest a significant association between KTCN patients and VSX1 genetic alterations (p.R166W and p.H244R). Although our findings support VSX1 as a plausible candidate gene responsible for keratoconus, other chromosomal loci and genes could be involved in KTCN development. Taken together, our results suggest that p.R166W and p.H244R could have possible pathogenic influences on KTCN.

Purpose: To evaluate the involvement of Visual System Homeobox 1 (VSX1), Secreted Protein Acidic and Rich in Cysteine (SPARC), Superoxide Dismutase 1 (SOD1), Lysyl Oxidase (LOX), and Tissue Inhibitor of Metalloproteinase 3 (TIMP3) in sporadic and familial keratoconus., Methods: Mutational analysis of the five genes was performed by sequencing and fragment analysis in a large cohort of 302 Italian patients, with a diagnosis of keratoconus based on clinical examination and corneal topography. The variants identified in VSX1 and SPARC were also assessed in the available relatives of the probands., Results: A novel mutation p.G239R and previously reported mutations were found in VSX1. Novel and already reported variants were identified in SPARC and SOD1, whose pathogenic significance has not been established. No pathogenic variants have been identified in LOX and TIMP3., Conclusions: Molecular analysis of the five genes in a cohort of 225 sporadic and 77 familial keratoconus cases confirms the possible pathogenic role of VSX1 though in a small number of patients; a possible involvement of LOX and TIMP3 could be excluded; and the role played by SOD1 and SPARC in determining the disease as not been definitively clarified. Further studies are required to identify other important genetic factors involved in the pathogenesis and progression of the disease that in the authors' opinion, and according with several authors, should be considered as a complex disease.

Purpose: To screen the visual system homeobox 1 (VSX1) gene in keratoconus patients., Methods: The enntire coding region of VSX1, including intron-exon boundaries were amplified in keratoconus cases (n=50) and controls (n=50). All sequences were analyzed against the ensemble sequence (ENSG00000100987) for VXS1., Results: Sequencing analysis showed four alterations (p.A182A, p.R217H, p.P237P, and g.25059612C>T) in VSX1 of which g.25059612C>T (in intron 2) was found to be novel. Of these four, p.A182A and p.P237P were present in both cases as well as controls while p.R217H and g.25059612C>T were limited to cases only. All these changes were non-pathogenic., Conclusions: In our study no pathogenic VSX1 mutation was identified. The role of VSX1 in the pathogenesis of keratoconus is still controversial. VSX1 mutations are responsible for a very small fraction of all observed keratoconus cases. The absence of pathogenic mutations in VSX1 in our patients indicates that other genetic loci like 13q32 as suggested by a recent study may be involved in the pathogenesis of this disorder.

Purpose: To perform mutational screening of the visual system homeobox gene 1 (VSX1; MIM#605020) in patients with sporadic and familial keratoconus (MIM#148300) in a European population and, for the first time, report the mutational analysis of the two newly identified VSX1exons., Methods: VSX1sequence variants in patients with keratoconus were evaluated by direct sequencing of the entire coding region, including two novel exons. In familial keratoconus cases, segregation of potentially pathogenic VSX1variants was assessed to determine pathogenicity. Transcript analysis was carried out on splice site and synonymous sequence variants not detected in controls., Results: A total of 66 unrelated patients with keratoconus from the European population (27 with familial keratoconus; 39 with sporadic keratoconus) were analysed for VSX1 mutations. Four sequence variants were not observed in 100 healthy control individuals: c.432C>G (p.D144E), c.479G>A (p.G160D), c.789C>T (p.S263S), and an intronic change c.844-13T>A (numbered with respect to NM&#95;014588). Segregation was not detected for p.D144E and c.844-13T>A. The change in p.G160D was observed in two patients with sporadic keratoconus. Although predicted to alter VSX1 splicing, p.S263S had no effect on transcript processing. Four known SNPs were detected and the following polymorphic variants were observed in keratoconus patients and controls: c.711T>A (NM&#95;199425; p.P237P), c.844-5&#95;-6insT (NM&#95;014588), c.*28G>T (DQ854811/DQ854812), and c.*50G>A (DQ854809/DQ854810)., Conclusions: VSX1has a minor role in keratoconus pathogenesis. The pathogenicity of p.G160D remains controversial and this change may represent a rare polymorphism or genetic modifier. Further evidence is provided that the previously reported variant, p.D144E, is a polymorphism.

Purpose: Keratoconus (KC) is a bilateral, noninflammatory, and progressive corneal ectasia that occurs mostly as a sporadic disorder, but it has long been recognized that a significant minority of patients also exhibit a family history. In recent years, several candidate genes, including VSX1 and SOD1, have been proposed and some disease-causing mutations have been identified., Methods: To investigate the role of the 2 genes in 113 Slovenian patients with sporadic and familial KC, the complete coding region with corresponding intronic sequences was analyzed. The same regions of both genes were also checked in 100 healthy blood donors. We also checked the relation of 627+23G>A polymorphism in the VSX1 gene with the hereditary form of the disease., Results: No disease-causing mutations were identified in either gene. We did discover a significant association of 627+23G>A polymorphism distribution (VSX1) with unrelated patients diagnosed with the hereditary form of KC., Conclusion: The absence of pathogenic mutations in our large number of unrelated patients with KC indicates that other genetic factors are involved in the development of this disorder.

Purpose: To evaluate the possible role of the VSX1 gene in a group of patients from the Indian subcontinent with keratoconus., Methods: Molecular analysis of 66 patients with a diagnosis of keratoconus, based on clinical examination and corneal topography, was carried out. DNA extraction from peripheral blood followed by Polymerase Chain Reaction (PCR) amplification of the VSX1 gene was performed. The entire coding region and the exon-intron junctions of the VSX1 gene were analyzed by direct sequencing., Results: A novel change at c.525G>C, replacing amino acid glutamine at position 175 with histidine, was found in one affected individual. One of the previously reported SNPs (rs12480307) was found with equal frequency in both patients and controls., Conclusions: This is the first report from the Indian subcontinent exploring the role of VSX1 in the causation of keratoconus. One novel mutation (Q175H) predicted to be a potentially damaging change was seen in an affected individual; this substantiates the importance of this gene but its precise role in disease causation needs further investigation.

Purpose: To present an association of mutations in the CRB1 gene with keratoconus in patients with Leber congenital amaurosis (LCA)., Methods: Sixteen patients with genotyped LCA (having the CRB1, CRX, RetGC, RPE65, and AIPL1 mutations) were recruited from one ophthalmology practice and examined for the presence of keratoconus. Corneal topography, visual acuity, and slit lamp biomicroscopic examination were performed in all cases., Results: The mean age of the patients was 34.5 years (range, 13-74). Visual acuities ranged from 20/40 to light perception. Corneal topography was successfully collected in 15 of the cases. Five of the 16 cases had slit lamp and/or topographic features consistent with keratoconus. One patient had a clinical picture that was keratoglobus-like. Of these six cases, four had a CRB1 mutation and two had a CRX mutation. Of the three subjects with the CRX mutation, one had keratoconus, one had the keratoglobus-like presentation, and one was normal. Our cohort represents 14 separate, unrelated families. Only one family comprised multiple members with LCA. These were three affected brothers, one with keratoconus, all with CRB1 mutations., Conclusions: Although the results cannot exclude other gene mutations, they suggest that LCA patients with a CRB1 mutation may have a particular susceptibility to keratoconus.

Purpose: To identify the genetic defect associated with keratoconus (KC) in an Ashkenazi Jewish family and to evaluate its nature and its phenotypic expression within carriers., Methods: A three generation Ashkenazi Jewish family with KC was ascertained. Diagnosis was based on clinical examination and corneal topography. Segregation analysis was performed using micro-satellite polymorphic markers in close proximity to 7 previously associated KC loci and genes. Mutation analysis of the VSX1 gene was performed by direct sequencing of PCR-amplified exons, and a BseR1 restriction assay. In selected cases, where the genotype was consistent with KC, additional effort to detect subtle corneal changes was made by computerized Orbscan measurements., Results: We found co-segregation between the KC phenotype and a polymorphic marker close to the VSX1. Sequencing revealed a previously described missense mutation (D144E). All of the mutation carriers manifested pathologic corneal findings; some had overt KC while others had subtle corneal alterations identifiable only by Orbscan., Conclusions: These findings support the pathogenic role of VSX1 gene in KC. The variable expression among the carriers, suggests the involvement of other factors in determining the final phenotype.

Purpose: Three mutations, L159M, R166W, and H244R, in the VSX1 gene have been recently reported to be associated with keratoconus by direct sequencing in familial panels. In an attempt to confirm this observation, we surveyed the same mutations of the VSX1 gene for a white sporadic keratoconus case-control panel and a larger familial panel to test its association with keratoconus., Methods: A case-control panel, with 77 keratoconus patients and 71 healthy controls, and a keratoconus familial panel, with 444 individuals from 75 families, were surveyed. DNA from each individual was tested for the previously reported mutations by ABI allelic discrimination technology (L159M and R166W) and restriction fragment length polymorphism assay (H244R)., Results: We observed no mutations of R166W and H244R and 1 heterozygous mutation of L159M in a healthy individual in the case-control panel. For the familial panel, we observed no polymorphism of R166W; 3 heterozygous for H244R, with 2 affected and 1 unaffected; and 5 heterozygous for L159M, with 3 affected and 2 unaffected., Conclusions: We cannot confirm the previously reported association of the polymorphism in the VSX1 gene with keratoconus. In our case-control sample panel and the larger familial sample panel, we did not observe the reported polymorphism of the VSX1 gene, and the distribution of these 3 polymorphisms was not significant enough to support a pathogenetic role in keratoconus.

Keratoconus is a bilateral ectatic disorder characterized by the central thinning of corneal tissue leading to visual impairment. To investigate the possibility of visual system homeobox 1 (VSXI) as a candidate susceptibility gene for Korean patients with keratoconus, we performed a mutation screening of the VSXI gene in 249 unrelated patients with keratoconus and 208 control subjects without the ocular disorder. We found two heterozygous novel missense mutations in exon 2: N151S and G160V. The G160V mutation was identified in 13 keratoconus patients (5.3%), and the N151S mutation was found in only one keratoconus patient (0.4%). We also detected three synonymous polymorphisms and four intragenic polymorphisms. The IVS1-11*a allele was associated with a significantly increased risk of keratoconus in Korean patients [3.6 vs. 0.5%, p = 0.001, odds ratio (OR) = 7.76, 95% confidence interval (CI) 1.989-30.241). Other polymorphisms did not show an association with keratoconus risk. Our data is the first reported VSX1 mutation screening in Korean keratoconus patients. We detected two novel missense mutations and one intragenic polymorphism in the VSX1 gene, which show a strong statistical association with unrelated keratoconus patients. Consequently, our study suggests that VSX1 gene variants seem to be significant genetic variants for keratoconus predisposition in unrelated Korean patients.

Purpose: To evaluate the role of the visual system homeobox gene 1 (VSX1) in the pathogenesis of familial keratoconus., Methods: Families with two or more individuals with keratoconus were recruited and their members examined. The coding region and intron-exon junctions of the VSX1 gene were sequenced in affected individuals. In cases where there were possible pathogenic changes, segregation within the pedigree was analyzed. Meta analysis of reports on an association of p.D144E change with keratoconus phenotype was performed., Results: Probands from a panel of 85 apparently unrelated keratoconus families were included. Eleven sequence variants were observed, including the previously reported c.432C>G (p.D144E) change and two novel intronic single nucleotide polymorphisms. However, these three changes did not cosegregate with the disease phenotype., Conclusions: We excluded the c.432C>G sequence alteration as the direct cause of the disease. Lack of possibly pathogenic VSX1 sequence variants in the familial panel suggests that involvement of this gene in the pathogenesis of keratoconus is likely to be confined to a small number of pedigrees, at least in the population studied.

Purpose: To report the development of corneal ectasia and hydrops in a patient with autosomal recessive cornea plana., Methods: Retrospective observational case report., Results: A 16-year-old male with a prior diagnosis of autosomal recessive cornea plana who complained of unilateral visual loss of one month's duration was found to have corneal edema consistent with resolving hydrops in the affected eye. The edema resolved over time, and keratometry revealed high astigmatism in both eyes despite documentation of no significant corneal astigmatism 11 years before. Slit-lamp examination confirmed corneal thinning in both eyes corresponding to the meridian of the astigmatism. The prior diagnosis of cornea plana was confirmed by molecular genetic testing., Conclusions: Although not a characteristic finding of cornea plana, corneal ectasia can rarely occur and be associated with corneal hydrops.

Purpose: To determine whether mutations of the VSX1 gene play a pathogenetic role in the development of keratoconus (KTCN)., Methods: DNA extraction, PCR amplification, and direct sequencing of the VSX1 gene were performed in 100 unrelated patients with diagnoses of clinical and topographic features of KTCN., Results: Of the four previously identified presumed pathogenic mutations in the VSX1 gene (Leu17Pro, Asp144Glu, Leu159Met, and Arg166Trp), only Asp144Glu was identified in a single affected patient. Two novel single nucleotide polymorphisms (SNPs), both resulting in synonymous substitutions, were identified: c.53G>T (Ser6Ser) in four affected patients and c.209G>T (Pro58Pro) in two affected patients. Two previously reported SNPs were also identified: c.426C>A (Arg131Ser) in one affected patient and c.581A>G (Ala182Ala) in 51 of the 100 affected patients., Conclusions: Only one of the presumed pathogenic mutations in the VSX1 gene, Asp144Glu, was identified in a single member of the cohort of affected patients. However, as previously demonstrated, Asp144Glu is a non-disease-causing polymorphism. The absence of pathogenic mutations in the VSX1 gene in a large number of unrelated KTCN patients indicates that other genetic factors are involved in the development of this disorder.

Purpose: Keratoconus is a noninflammatory corneal disorder that is clinically and genetically heterogeneous. Mutations in the VSX1 (visual system homeobox 1) gene have been identified for two distinct, inherited corneal dystrophies: posterior polymorphous corneal dystrophy and keratoconus. To evaluate the possible role of the VSX1 gene in a series of Italian patients, 80 keratoconus-affected subjects were screened for mutations., Methods: The diagnosis of keratoconus was made on the basis of clinical examination and corneal topography. The whole coding region and the exon-intron junctions of the VSX1 gene were analyzed by direct sequencing., Results: Three already-described changes, D144E, G160D, and P247R, and a novel L17P mutation were found in 7 of 80 unrelated patients (8.7%). Two undescribed intronic polymorphisms are also reported., Conclusions: Mutational analysis of the VSX1 gene in a series of Italian patients revealed one novel mutation and confirmed an important role played by this gene in a significant proportion of patients affected by keratoconus, when it is inherited as an autosomal dominant trait with variable expressivity and incomplete penetrance.

Introduction: Keratoconus is a non-inflammatory ocular disease characterised by conical deformation, progressive thinning and scarring of the central cornea. Despite intensive investigations, the exact cause of the disease still remains unclear. Clinical studies provide strong indications of a major genetic role in the aetiology. We set out to examine the involvement in the manifestation of keratoconus of any of the 5,600 gene specificities available on the Affymetrix GeneChip HuGeneFL., Methods: After examination of two corneas they were stored in RNAlater, RNA was extracted and hybridised on the chips. Using a combination of dyes it was possible to read the chips with laser detection and to visualise the gene expression pattern., Results: We found an upregulation of collagens, versican, metalloproteinases and cell adhesion proteins. A downregulation was observed for TIGR protein, cytokeratins, eyes absent homologue (Eab1) and the proteins for radical treatment selenoprotein P and monooxygenase., Conclusions: Our results indicate that keratoconus is a process in which repair and scar-formation mechanisms operate at the same time. As candidate genes for this mechanism, collagen IV and related proteoglycans were favoured.

Purpose: The present study was conducted to investigate differential gene expression in individual samples derived from fresh-frozen human keratoconus and normal corneal epithelium, using gene microarrays., Methods: Total RNA was extracted (11 keratoconus and 8 normal samples), labeled, and hybridized to microarrays (GeneChip; Affymetrix, Inc., Santa Clara, CA). GeneChip data were validated by verifying the expression profiles of 10 genes by real-time PCR and by recalculation using dChip software (Wong Laboratory, Department of Biostatistics, Harvard School of Public Health, Boston, MA). Furthermore, 3 of the 10 encoded proteins were stained by immunohistochemistry., Results: In comparison with normal cornea, the expression of 471 of the 5600 genes on the microarrays was changed in the keratoconus samples. This number was reduced to 47 with increased expression and 9 with decreased expression when more stringent selection parameters were applied. These genes are believed to be involved in keratoconus. Two of the candidate genes, lysyl oxidase and tissue inhibitor of metalloproteinase 3, are known to be involved in other eye diseases. Expression profiles were reproduced with the software dChip (Wong Laboratory) and real-time PCR. Increases in keratin 6 and 13 were also detected at the protein level., Conclusions: Keratoconus epithelium appears to be characterized by massive changes of the cytoskeleton, reduced extracellular matrix remodeling, altered transmembrane signaling, and modified cell-to-cell and cell-to-matrix interactions. Validation of gene expression with dChip analysis and real-time PCR indicates GeneChip to be a valid technique for investigation of epithelium from single dissected corneal samples. Association between alterations at RNA and protein levels was observed for some of the tested candidates.

We identified mutations in the VSX1 homeobox gene for two distinct inherited corneal dystrophies; posterior polymorphous dystrophy (PPD) and keratoconus. One of the mutation (R166W) responsible for keratoconus altered the homeodomain and impaired DNA binding. Two other sequence changes (L159M and G160D) were associated with keratoconus and PPD, respectively, and involved a region adjacent to the homeodomain. The G160D substitution, and a fourth defect affecting the highly conserved CVC domain (P247R), occurred in a child with very severe PPD who required a corneal transplant at 3 months of age. In this family, relatives with the G160D change alone had mild to moderate PPD, while P247R alone caused no corneal abnormalities. However, with either the G160D or P247R mutation, electroretinography detected abnormal function of the inner retina, where VSX1 is expressed. These data define the molecular basis of two important corneal dystrophies and reveal the importance of the CVC domain in the human retina.

Purpose: A two-generation consanguineous Pakistani family with autosomal recessive Leber congenital amaurosis (LCA, MIM 204,000) and keratoconus was identified. All affected individuals have bilateral keratoconus and congenital pigmentary retinopathy. The goal of this study was to link the disease phenotype in this family., Methods: Genomic DNA was amplified across the polymorphic microsatellite poly-CA regions identified by markers. Polymerase chain reaction (PCR) products were separated by nondenaturing polyacrylamide gel electrophoresis. Alleles were assigned to individuals, which allowed calculation of LOD scores using the Cyrillic and MLINK software program. The retinal guanylate cyclase (RETGC-1, GDB symbol GUC2D) and pigment epithelium-derived factor (PEDF) genes were analyzed by heteroduplex analysis and direct sequencing for mutations in diseased individuals., Results: Based on a whole genome linkage analysis the first locus for this combined phenotype has been mapped to chromosome 17p13. Linkage analysis gave a two point LOD score of 3.21 for marker D17S829. Surrounding this marker is a region of homozygosity of 15.77 cM, between the markers D17S1866 and D17S960; however, the crossover for the marker D17S1529 refines the region to 10.77 cM within which the disease gene is predicted to lie. Mutation screening of the nearby RETGC-1 gene, which has been shown to be associated with LCA1, revealed no mutations in the affected individuals of this family. Similarly, another prime candidate in the region PEDF was also screened for mutations. The factor has been shown to be involved in the photoreceptor differentiation and neuronal survival. No mutations were found in this gene either. Furthermore, RETGC-1 was physically excluded from the critical disease region based on the existing physical map., Conclusions: It is therefore suggested that this combined phenotype maps to a new locus and is due to an as yet uncharacterized gene within the 17p13 chromosomal region.