Reversal of dual epigenetic repression of non-canonical Wnt-5a normalises diabetic corneal epithelial wound healing and stem cells.

AIMS/HYPOTHESIS: Diabetes is associated with epigenetic modifications including DNA methylation and miRNA changes. Diabetic complications in the cornea can cause persistent epithelial defects and impaired wound healing due to limbal epithelial stem cell (LESC) dysfunction. In this study, we aimed to uncover epigenetic alterations in diabetic vs non-diabetic human limbal epithelial cells (LEC) enriched in LESC and identify new diabetic markers that can be targeted for therapy to normalise corneal epithelial wound healing and stem cell expression. METHODS: Human LEC were isolated, or organ-cultured corneas were obtained, from autopsy eyes from non-diabetic (59.87±20.89 years) and diabetic (71.93±9.29 years) donors. The groups were not statistically different in age. DNA was extracted from LEC for methylation analysis using Illumina Infinium 850K MethylationEPIC BeadChip and protein was extracted for Wnt phospho array analysis. Wound healing was studied using a scratch assay in LEC or 1-heptanol wounds in organ-cultured corneas. Organ-cultured corneas and LEC were transfected with WNT5A siRNA, miR-203a mimic or miR-203a inhibitor or were treated with recombinant Wnt-5a (200 ng/ml), DNA methylation inhibitor zebularine (1-20 µmol/l) or biodegradable nanobioconjugates (NBCs) based on polymalic acid scaffold containing antisense oligonucleotide (AON) to miR-203a or a control scrambled AON (15-20 µmol/l). RESULTS: There was significant differential DNA methylation between diabetic and non-diabetic LEC. WNT5A promoter was hypermethylated in diabetic LEC accompanied with markedly decreased Wnt-5a protein. Treatment of diabetic LEC and organ-cultured corneas with exogenous Wnt-5a accelerated wound healing by 1.4-fold (p<0.05) and 37% (p<0.05), respectively, and increased LESC and diabetic marker expression. Wnt-5a treatment in diabetic LEC increased the phosphorylation of members of the Ca2+-dependent non-canonical pathway (phospholipase Cγ1 and protein kinase Cß; by 1.15-fold [p<0.05] and 1.36-fold [p<0.05], respectively). In diabetic LEC, zebularine treatment increased the levels of Wnt-5a by 1.37-fold (p<0.01)and stimulated wound healing in a dose-dependent manner with a 1.6-fold (p<0.01) increase by 24 h. Moreover, zebularine also improved wound healing by 30% (p<0.01) in diabetic organ-cultured corneas and increased LESC and diabetic marker expression. Transfection of these cells with WNT5A siRNA abrogated wound healing stimulation by zebularine, suggesting that its effect was primarily due to inhibition of WNT5A hypermethylation. Treatment of diabetic LEC and organ-cultured corneas with NBC enhanced wound healing by 1.4-fold (p<0.01) and 23.3% (p<0.05), respectively, with increased expression of LESC and diabetic markers. CONCLUSIONS/INTERPRETATION: We provide the first account of epigenetic changes in diabetic corneas including dual inhibition of WNT5A by DNA methylation and miRNA action. Overall, Wnt-5a is a new corneal epithelial wound healing stimulator that can be targeted to improve wound healing and stem cells in the diabetic cornea. DATA AVAILABILITY: The DNA methylation dataset is available from the public GEO repository under accession no. GSE229328 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229328 ).

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.

Genetics vs chronic corneal mechanical trauma in the etiology of keratoconus.

Both genetic and environmental factors have been considered to play a role in the etiology keratoconus. Eye rubbing, and more recently eye compression due to sleeping position, have been identified to be highly related to the condition, and are present in a high percentage of patients. Today, the predominant model is that these factors can provide the "second hit" necessary to generate the condition in a genetically susceptible individual. In addition, the extremely high prevalence in Arab populations, where endogamy could play a role, the high concordance rate in monozygotic twins, and the presence of family history of the condition between 5 and 23% of cases, support a genetic influence. Segregation analysis studies suggest that keratoconus is a complex non-Mendelian disease. Results from linkage analysis, next generation sequencing studies and genome-wide association studies also have suggested that genetic factors are involved in the condition. Recently, it has been proposed that mechanical trauma (i.e. eye rubbing or eye compression at night), is a sine quanon condition for the onset of keratoconus, and quite possibly its only cause. There are various arguments for and against this hypothesis. Indeed, it is possible, as initially suggested around 55 years ago, that the term "keratoconus" include diverse phenotypically similar conditions, which are actually of different etiology.

Novel nanopolymer RNA therapeutics normalize human diabetic corneal wound healing and epithelial stem cells.

Human diabetic corneas develop delayed wound healing, epithelial stem cell dysfunction, recurrent erosions, and keratitis. Adenoviral gene therapy modulating c-Met, cathepsin F and MMP-10 normalized wound healing and epithelial stem cells in organ-cultured diabetic corneas but showed toxicity in stem cell-enriched cultured limbal epithelial cells (LECs). For a safer treatment, we engineered a novel nanobiopolymer (NBC) that carried antisense oligonucleotide (AON) RNA therapeutics suppressing cathepsin F or MMP-10, and miR-409-3p that inhibits c-Met. NBC was internalized by LECs through transferrin receptor (TfR)-mediated endocytosis, inhibited cathepsin F or MMP-10 and upregulated c-Met. Non-toxic NBC modulating c-Met and cathepsin F accelerated wound healing in diabetic LECs and organ-cultured corneas vs. control NBC. NBC treatment normalized levels of stem cell markers (keratins 15 and 17, ABCG2, and ΔNp63), and signaling mediators (p-EGFR, p-Akt and p-p38). Non-toxic nano RNA therapeutics thus present a safe alternative to viral gene therapy for normalizing diabetic corneal cells.

Persistence of reduced expression of putative stem cell markers and slow wound healing in cultured diabetic limbal epithelial cells.

PURPOSE: To examine the expression of putative limbal epithelial stem cell (LESC) markers and wound healing rates in primary healthy and diabetic human limbal epithelial cells (LECs) cultured on different substrata. METHODS: Primary limbal epithelial cells were isolated from human autopsy corneas and discarded corneoscleral rims with dispase II treatment. LECs were cultured in EpiLife medium on human amniotic membrane (AM) denuded with mild alkali treatment, on plastic dishes and on glass slides coated with a mixture of human fibronectin, collagen type IV, and laminin (FCL). Cultured LECs were fixed in p-formaldehyde or methanol, and the expression of the putative LESC markers ΔNp63α, PAX6, and ABCG2 and keratins K12, K15, and K17 was examined with immunostaining. Wound healing was evaluated in scratch wound assay in LECs cultured on FCL-coated plates 20 h after wounding. RESULTS: LECs cultured on denuded AM expressed ΔNp63α, PAX6 (both showed nuclear staining), K15, K17 (cytoskeleton staining), and ABCG2 (cytoplasmic and/or plasma membrane staining). LECs cultured on FCL-coated slides also expressed these markers, whereas no expression was detected for differentiated corneal epithelial cell marker K12. Decreased expression of LESC markers was observed in diabetic LECs compared to healthy LECs cultured on the FCL-coated slides. This reduction was most prominent for K15 and K17. Diabetic LECs were found to heal scratch wounds slower than healthy cells in accordance with previous results in corneal organ cultures. CONCLUSIONS: Healthy human LECs cultured either on AM or FCL-coated slides preserved LESC marker expression. The observed reduction in LESC marker expression and slower wound healing in cultured diabetic LECs are in line with our earlier reports and may account for diabetic LESC dysfunction and clinically observed impaired corneal epithelial wound healing.

A genome-wide association study identifies a potential novel gene locus for keratoconus, one of the commonest causes for corneal transplantation in developed countries.

Keratoconus is a condition in which the cornea progressively thins over time, and is a major cause for cornea transplantation. To identify keratoconus susceptibility regions, we performed a comprehensive genome-wide association study (GWAS) using a discovery and replication design. A discovery panel of 222 keratoconus Caucasian patients and 3324 Caucasian controls was genotyped using Illumina 370K beadchips. Further associated and fine-mapping single nucleotide polymorphisms (SNPs) (n= 4905) were genotyped in an independent replication case-control panel of 304 cases and 518 controls and a family panel of 307 subjects in 70 families. Logistic regression models implemented in PLINK were performed to test associations in case-control samples with and without principal component (PC) adjustments. Generalized estimation equation models accounting for familial correlations implemented in GWAF were used for association testing in families. No genome-wide associations were identified in the discovery GWAS panel. From the initial testing without adjustments for PCs, the top three SNPs located at 3p26 (rs6442925), 2q21.3 (rs4954218) and 19q13.3 (rs1428642) were identified with unadjusted P-values of 6.5 × 10(-8), 2.4 × 10(-7) and 3.1 × 10(-7), respectively. After adjustments for PCs, rs1428642 became the most significant through the genome with a P-value of 1.4 × 10(-6), while rs6442925 and rs4954218 were less significant (P= 1.9 × 10(-5) and 2.6 × 10(-4)). SNP rs4954218 was confirmed in two independent replication panels with P-values of 0.004 and 0.009, respectively. Meta-analysis revealed a highest association at rs4954218 with adjusted P= 1.6 × 10(-7) (unadjusted P= 1.2 × 10(-9)). These findings suggest SNP rs4954218, located near the RAB3GAP1 gene, previously reported to be associated with corneal malformation, is a potential susceptibility locus for keratoconus.

Optical coherence tomography combined with videokeratography to differentiate mild keratoconus subtypes.

PURPOSE: To develop parameters using a combination of optical coherence tomography (OCT) and videokeratography to detect early keratoconus. METHODS: Videokeratography, wavefront analysis, and measured OCT indices were performed on 180 normal eyes, 46 eyes with moderate keratoconus, 54 eyes with early keratoconus, 7 eyes with forme fruste keratoconus, and 16 eyes with keratoconus "suspect" to determine the most sensitive parameters for separating these groups. RESULTS: A combination of videokeratography and OCT indices (inferior-superior [I-S] value and minimum pachymetry) was statistically the most significant in separating the keratoconus groups from normal eyes (P < .001). Using a newly derived index, the minimum pachymetry divided by the I-S value (pachymetry/asymmetry [PA]/I-S index) with a cut-off of 100, 100% of early and forme fruste keratoconus could be identified as being abnormal with 7 normals misclassified (misclassification rate 2.7%). By adding keratoconus "suspect" to the analysis and an I-S value of 1.2 as a cut-off point, 5 "suspects" were classified as normal and 11 normals as abnormal (misclassification rate 7.8%). The PA/I-S index, with a cut-off point of 100, reduced this misclassification rate to 4.4%. CONCLUSIONS: These results suggest that OCT combined with videokeratography may be more useful for differentiating mild forms of keratoconus than videokeratography alone.

Results of corneal collagen cross-linking in patients with corneal ectasia after laser refractive surgery-A prospective study.

PURPOSE: To evaluate the safety and efficacy of epithelium-off (epi­off) corneal cross-linking (CXL) in patients with post-LASIK corneal ectasia (PLE) SETTING: Private clinical practice DESIGN: Prospective clinical trial METHODS: 82 eyes of adult patients post-LASIK, ages 21-67, with a topography pattern consistent with corneal ectasia, corrected distance visual acuity (CDVA) worse than 20/20, and minimum corneal pachymetry > 400 µm underwent epi­off CXL. Exclusion criteria were patients with corneas that were thinner than 400 µm or demonstrated central corneal scarring, history of herpetic eye disease, pregnancy or nursing. Follow up examinations of spherical equivalent, uncorrected distance visual acuity (UDVA), CDVA, steep keratometry (KSteep) and minimum pachymetry occurred on different but highly overlapping subsets of the operated eyes yearly until 5 years post-CXL. RESULTS: Over the 5 years of follow up, spherical equivalent did not significantly change while UCVA and CDVA stabilized or improved to a non-significant degree. KSteep and minimum pachymetry continued to be decreased to a statistically significant degree (p < 0.05 at 5 years). CONCLUSIONS: CXL in PLE patients is safe and efficacious: it halts progression of PLE and may improve visual function. KSteep and minimum pachymetry decrease post-CXL. Patients with PLE should be encouraged to stop progression of the disease by undergoing epi­off CXL once progression is established.

Results of corneal crosslinking in adolescents with progressive keratoconus: prospective study.

PURPOSE: To evaluate the safety and efficacy of epithelium-off (epi-off) corneal crosslinking (CXL) in adolescents with progressive keratoconus (KC). SETTING: Private clinical practice. DESIGN: Nonrandomized prospective clinical trial. METHODS: 230 adolescent patients aged 10 to 19 years with progressive KC (increasing maximum keratometry [Kmax] or astigmatism of 1.00 diopter or greater associated with decreased corrected distance visual acuity [CDVA]) underwent CXL. Exclusion criteria were age at time of CXL younger than 10 years or older than 19 years, corneas that were thinner than 400 µm or demonstrated central corneal scarring, history of herpetic eye disease, or pregnancy or nursing. Follow-up examinations of uncorrected distance visual acuity (UDVA), CDVA, Kmax, and minimum pachymetry occurred on 130 eyes at 1 year, 77 eyes at 2 years, and 55 eyes at 3 years post-CXL. RESULTS: In this study, 230 eyes of adolescent patients were evaluated. UDVA significantly improved from preoperatively to 1 year, 2 years, and 3 years post-CXL. CDVA values significantly improved from preoperatively to 1 year, 2 years, and 3 years post-CXL. Kmax values significantly reduced (improved) from preoperatively to 1 year and 3 years post-CXL and reduced (improved) (P = .22) from preoperatively to 2 years post-CXL. Minimum pachymetry decreased significantly from preoperatively to 1 year, 2 years, and 3 years post-CXL. CONCLUSIONS: CXL in patients aged 10 to 19 years was safe and efficacious, halted progression of KC and could improve UCVA, CDVA, and Kmax. Minimum pachymetry decreased and stabilized post-CXL. Ophthalmologists should encourage adolescent patients with KC to obtain prompt evaluation and possible CXL to halt progression of the disease.

Association of Genetic Variation With Keratoconus.

Importance: Keratoconus is a condition in which the cornea progressively thins and protrudes in a conical shape, severely affecting refraction and vision. It is a major indication for corneal transplant. To discover new genetic loci associated with keratoconus and better understand the causative mechanism of this disease, we performed a genome-wide association study on patients with keratoconus. Objective: To identify genetic susceptibility regions for keratoconus in the human genome. Design, Setting, and Participants: This study was conducted with data from eye clinics in Australia, the United States, and Northern Ireland. The discovery cohort of individuals with keratoconus and control participants from Australia was genotyped using the Illumina HumanCoreExome single-nucleotide polymorphism array. After quality control and data cleaning, genotypes were imputed against the 1000 Genomes Project reference panel (phase III; version 5), and association analyses were completed using PLINK version 1.90. Single-nucleotide polymorphisms with P < 1.00 × 10-6 were assessed for replication in 3 additional cohorts. Control participants were drawn from the cohorts of the Blue Mountains Eye Study and a previous study of glaucoma. Replication cohorts were from a previous keratoconus genome-wide association study data set from the United States, a cohort of affected and control participants from Australia and Northern Ireland, and a case-control cohort from Victoria, Australia. Data were collected from January 2006 to March 2019. Main Outcomes and Measures: Associations between keratoconus and 6 252 612 genetic variants were estimated using logistic regression after adjusting for ancestry using the first 3 principal components. Results: The discovery cohort included 522 affected individuals and 655 control participants, while the replication cohorts included 818 affected individuals (222 from the United States, 331 from Australia and Northern Ireland, and 265 from Victoria, Australia) and 3858 control participants (2927 from the United States, 229 from Australia and Northern Ireland, and 702 from Victoria, Australia). Two novel loci reached genome-wide significance (defined as P < 5.00 × 10-8), with a P value of 7.46 × 10-9 at rs61876744 in patatin-like phospholipase domain-containing 2 gene (PNPLA2) on chromosome 11 and a P value of 6.35 × 10-12 at rs138380, 2.2 kb upstream of casein kinase I isoform epsilon gene (CSNK1E) on chromosome 22. One additional locus was identified with a P value less than 1.00 × 10-6 in mastermind-like transcriptional coactivator 2 (MAML2) on chromosome 11 (P = 3.91 × 10-7). The novel locus in PNPLA2 reached genome-wide significance in an analysis of all 4 cohorts (P = 2.45 × 10-8). Conclusions and Relevance: In this relatively large keratoconus genome-wide association study, we identified a genome-wide significant locus for keratoconus in the region of PNPLA2 on chromosome 11.

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.

PPIP5K2 and PCSK1 are Candidate Genetic Contributors to Familial Keratoconus.

Keratoconus (KC) is the most common corneal ectatic disorder affecting >300,000 people in the US. KC normally has its onset in adolescence, progressively worsening through the third to fourth decades of life. KC patients report significant impaired vision-related quality of life. Genetic factors play an important role in KC pathogenesis. To identify novel genes in familial KC patients, we performed whole exome and genome sequencing in a four-generation family. We identified potential variants in the PPIP5K2 and PCSK1 genes. Using in vitro cellular model and in vivo gene-trap mouse model, we found critical evidence to support the role of PPIP5K2 in normal corneal function and KC pathogenesis. The gene-trap mouse showed irregular corneal surfaces and pathological corneal thinning resembling KC. For the first time, we have integrated corneal tomography and pachymetry mapping into characterization of mouse corneal phenotypes which could be widely implemented in basic and translational research for KC diagnosis and therapy in the future.

Three VSX1 gene mutations, L159M, R166W, and H244R, are not associated with keratoconus.

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.

Corneal Cross-Linking for Pediatric Keratcoconus Review.

PURPOSE: To comprehensively review the available published literature for cross-linking in the pediatric population. METHODS: Review of the literature published in English in PubMed. RESULTS: Two hundred ten publications were considered. One hundred fifteen were considered relevant to this review. CONCLUSIONS: Studies of cross-linking in pediatric patients are sparse, with relatively short follow-up times, and mostly on small groups of patients. Treatment with cross-linking halts progression of keratoconus in the pediatric population, and early treatment seems to be cost-effective compared with later penetrating keratoplasty. Long-term effects and regression rates remain unclear, and further studies are needed in this population.

Differential Expression of Coding and Long Noncoding RNAs in Keratoconus-Affected Corneas.

Purpose: Keratoconus (KC) is the most common corneal ectasia. We aimed to determine the differential expression of coding and long noncoding RNAs (lncRNAs) in human corneas affected with KC. Methods: From the corneas of 10 KC patients and 8 non-KC healthy controls, 200 ng total RNA was used to prepare sequencing libraries with the SMARTer Stranded RNA-Seq kit after ribosomal RNA depletion, followed by paired-end 50-bp sequencing with Illumina Sequencer. Differential analysis was done using TopHat/Cufflinks with a gene file from Ensembl and a lncRNA file from NONCODE. Pathway analysis was performed using WebGestalt. Using the expression level of differentially expressed coding and noncoding RNAs in each sample, we correlated their expression levels in KC and controls separately and identified significantly different correlations in KC against controls followed by visualization using Cytoscape. Results: Using |fold change| ≥ 2 and a false discovery rate ≤ 0.05, we identified 436 coding RNAs and 584 lncRNAs with differential expression in the KC-affected corneas. Pathway analysis indicated the enrichment of genes involved in extracellular matrix, protein binding, glycosaminoglycan binding, and cell migration. Our correlation analysis identified 296 pairs of significant KC-specific correlations containing 117 coding genes enriched in functions related to cell migration/motility, extracellular space, cytokine response, and cell adhesion. Our study highlighted the potential roles of several genes (CTGF, SFRP1, AQP5, lnc-WNT4-2:1, and lnc-ALDH3A2-2:1) and pathways (TGF-ß, WNT signaling, and PI3K/AKT pathways) in KC pathogenesis. Conclusions: Our RNA-Seq-based differential expression and correlation analyses have identified many potential KC contributing coding and noncoding RNAs.

Higher order wavefront aberrations and topography in early and suspected keratoconus.

PURPOSE: To determine whether higher order wavefront aberrations or a combination of topography and wavefront variables distinguishes between early and suspected keratoconus. METHODS: This prospective comparative study evaluated 70 eyes using videokeratography (Tomey TMS-1) and aberrometry (Alcon LADARWave). Videokeratography and clinical evaluation were used to divide the eyes into three groups: 50 normal eyes, 10 eyes with early keratoconus, and 10 eyes with suspected keratoconus. Data were analyzed to determine whether higher order aberrations could separate eyes with early and suspected keratoconus from normal eyes. The product of the Inferior-Superior (I-S) topographic value in combination with the wavefront vertical coma also was evaluated to determine whether this could distinguish normal eyes from eyes with early and suspected keratoconus. RESULTS: Differences in vertical coma, root-mean-square coma, and secondary astigmatism for the three groups were statistically significant. Vertical coma was -0.03 +/- 0.28 for normal eyes, -0.525 +/- 0.253 for eyes with suspected keratoconus, and -1.949 +/- 1.416 for eyes with early keratoconus. Root-mean-square coma was 0.229 +/- 0.149 for normal eyes, 0.639 +/- 0.250 for eyes with suspected keratoconus, and 2.034 +/- 1.532 for eyes with early keratoconus. The product of vertical coma and the topographic I-S value separated the three groups better than aberrometry alone (P < .0001). CONCLUSIONS: Although both vertical coma and the I-S topographic value were useful for distinguishing among the three study groups, a combination of wavefront aberrometry and videokeratography appears to be the most sensitive way for distinguishing among normal eyes, eyes with suspected keratoconus, and eyes with early keratoconus.

TSC1 Mutations in Keratoconus Patients With or Without Tuberous Sclerosis.

Purpose: To test candidate genes TSC1 and TSC2 in a family affected by tuberous sclerosis complex (TSC) where proband was also diagnosed with bilateral keratoconus (KC) and to test the hypothesis that defects in the same gene may lead to a nonsyndromic KC. Methods: Next-generation sequencing of TSC1 and TSC2 genes was performed in a proband affected by TSC and KC. Identified mutation was confirmed by Sanger DNA sequencing. Whole exome sequencing (WES) was performed in patients with nonsyndromic KC. Sanger DNA sequencing was used to confirm WES results and to screen additional patients. RT-PCR was used to investigate TSC1 expression in seven normal human corneas and eight corneas from patients with KC. Various in silico tools were employed to model functional consequences of identified mutations. Results: A heterozygous nonsense TSC1 mutation g.132902703C>T (c.2293C>T, p.Gln765Ter) was identified in a patient with TSC and KC. Two heterozygous missense TSC1 variants g.132896322A>T (c.3408A>T, p.Asp1136Glu) and g.132896452G>A (c.3278G>A, p.Arg1093Gln) were identified in three patients with nonsyndromic KC. Two mutations were not present in The Genome Aggregation (GnomAD), The Exome Aggregation (ExAC), and 1000 Genomes (1000G) databases, while the third one was present in GnomAD and 1000G with minor allele frequencies (MAF) of 0.00001 and 0.0002, respectively. We found TSC1 expressed in normal corneas and KC corneas, albeit with various levels. Conclusions: Here for the first time we found TSC1 gene to be involved in bilateral KC and TSC as well as with nonsyndromic KC, supporting the hypothesis that diverse germline mutations of the same gene can cause genetic disorders with overlapping clinical features.

Two-stage genome-wide linkage scan in keratoconus sib pair families.

PURPOSE: To identify susceptibility gene loci for keratoconus. METHODS: A genome-wide linkage analysis was performed with data from 67 keratoconus sib pair families with 110 affected sib pairs of white or Hispanic origin. A total of 351 subjects were genotyped for 380 microsatellite markers along the genome at approximately 10-cM density. An additional 58 microsatellite markers at approximately 2-cM density in the identified linkage regions on chromosomes 4, 5, 9, 12, and 14 were also genotyped. Multipoint linkage analysis was performed in all pedigrees by nonparametric methods and maximum likelihood estimates of identity by descent sharing as implemented in GeneHunter (http://linkage.rockefeller.edu/soft/gh/ provided in the public domain by Rockefeller University, New York, NY). RESULTS: The strongest evidence of linkage was observed at the telomere (159 cM) of chromosome 9 (lod = 4.5) in all pedigrees. Other regions suggestive of linkage were identified at 176 cM of chromosome 4 (lod = 2.7), 143 cM of chromosome 5 (lod = 2.0), 7 cM of chromosome 9 (lod = 2.8), 12 cM of chromosome 11 (lod = 2.3), 27 cM of chromosome 12 (lod = 2.3), and 14 cM of chromosome 14 (lod = 2.9). Two significant linkage regions were also observed on chromosomes 17 at 86 cM (lod = 3.9) and 9 at 34 cM (lod = 3.8) in the Hispanic subjects only. After fine mapping these regions (with the exception of chromosomes 11 and 17), most linkage peaks remained similar (lod = 2.2 at 176 cM on chromosome 4; lod = 1.7 at 146 cM on chromosome 5; lod = 3.5 at 160 cM on chromosome 9; lod = 2.5 at 7 cM on chromosome 12; and lod = 2.6 at 19 cM on chromosome 14). CONCLUSIONS: These results indicate that one or more loci may contribute to keratoconus susceptibility.

No VSX1 gene mutations associated with keratoconus.

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.

INTACS inserts using the femtosecond laser compared to the mechanical spreader in the treatment of keratoconus.

PURPOSE: To determine the efficacy of INTACS insertion using a femtosecond laser in the treatment of keratoconus and to compare it to the technique using a mechanical spreader. METHODS: INTACS were inserted in 10 eyes using the mechanical spreader to create the channels and subsequently on another 20 eyes using the femtosecond laser. Uncorrected (UCVA) and best spectacle-corrected visual acuity (BSCVA), manifest refraction, and corneal topography were measured prior to surgery, at 6 months (femtosecond group), and 1 year (mechanical group). Pre- and postoperative data were analyzed to determine changes in the above parameters. RESULTS: Both groups showed significant reduction in average keratometry (K), spherical equivalent refraction, BSCVA, UCVA, surface regularity index (SRI), and surface asymmetry index (SAI). The laser group performed better in all parameters except change in SRI. Results of the laser versus the mechanical spreader were as follows: reduction in spherical equivalent refraction (3.98 vs 2.96), change in average K (2.91 vs 2.52), improvement in UCVA (4.13 vs 3.63), improvement in BSCVA (3.92 vs 1.63), change in SRI (0.37 vs 0.64), and change in SAI (1.00 vs 0.70). Statistical analysis, however, did not reveal any statistically significant differences between the two groups for any single parameter studied. The biggest improvement in the laser group versus the mechanical group was BSCVA (P=.09). Overall success, defined as contact lens or spectacles tolerance, was 85% in the laser group and 70% in the mechanical group. CONCLUSIONS: Inserting INTACS using the femtosecond laser to create the channels is as effective as using the mechanical spreader.