Publications by authors named "Yaron S Rabinowitz"

51 Publications

A multi-ethnic genome-wide association study implicates collagen matrix integrity and cell differentiation pathways in keratoconus.

Commun Biol 2021 Mar 1;4(1):266. Epub 2021 Mar 1.

Section of Ophthalmology, School of Life Course Sciences, King's College London, London, UK.

Keratoconus is characterised by reduced rigidity of the cornea with distortion and focal thinning that causes blurred vision, however, the pathogenetic mechanisms are unknown. It can lead to severe visual morbidity in children and young adults and is a common indication for corneal transplantation worldwide. Here we report the first large scale genome-wide association study of keratoconus including 4,669 cases and 116,547 controls. We have identified significant association with 36 genomic loci that, for the first time, implicate both dysregulation of corneal collagen matrix integrity and cell differentiation pathways as primary disease-causing mechanisms. The results also suggest pleiotropy, with some disease mechanisms shared with other corneal diseases, such as Fuchs endothelial corneal dystrophy. The common variants associated with keratoconus explain 12.5% of the genetic variance, which shows potential for the future development of a diagnostic test to detect susceptibility to disease.
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http://dx.doi.org/10.1038/s42003-021-01784-0DOI Listing
March 2021

Update on the genetics of keratoconus.

Exp Eye Res 2021 Jan 13;202:108398. Epub 2020 Dec 13.

Cornea Genetic Eye Institute, Department of Surgery and Board of the Governors Regenerative Medicine Institute, Beverly Hills, Cedars-Sinai, Los Angeles, CA, United States.

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.
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http://dx.doi.org/10.1016/j.exer.2020.108398DOI Listing
January 2021

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

Nanomedicine 2020 Nov 10;32:102332. Epub 2020 Nov 10.

Eye Program, Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA; Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA; Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA; Neurosurgery, Cedars-Sinai Medical Center, Los Angeles, CA, USA; David Geffen School of Medicine at UCLA, Los Angeles, CA, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.nano.2020.102332DOI Listing
November 2020

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

Exp Eye Res 2021 Jan 24;202:108328. Epub 2020 Oct 24.

Centro Oftalmológico Virgilio Galvis, Floridablanca, Colombia; Universidad de la Sabana, Chía, Colombia.

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.
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http://dx.doi.org/10.1016/j.exer.2020.108328DOI Listing
January 2021

Association of Genetic Variation With Keratoconus.

JAMA Ophthalmol 2020 02;138(2):174-181

Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania, Australia.

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.
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http://dx.doi.org/10.1001/jamaophthalmol.2019.5293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6990728PMC
February 2020

PPIP5K2 and PCSK1 are Candidate Genetic Contributors to Familial Keratoconus.

Sci Rep 2019 12 18;9(1):19406. Epub 2019 Dec 18.

Department of Cellular Biology and Anatomy, Augusta University, Augusta, GA, USA.

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.
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http://dx.doi.org/10.1038/s41598-019-55866-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920454PMC
December 2019

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

Cornea 2019 Aug;38(8):1033-1039

Stein Eye Institute, UCLA, Los Angeles, CA.

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.
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http://dx.doi.org/10.1097/ICO.0000000000002002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612572PMC
August 2019

Author Correction: Cross-ancestry genome-wide association analysis of corneal thickness strengthens link between complex and Mendelian eye diseases.

Nat Commun 2019 01 8;10(1):155. Epub 2019 Jan 8.

Statistical Genetics, QIMR Berghofer Medical Research Institute, Brisbane, QLD, 4029, Australia.

Emmanuelle Souzeau, who contributed to analysis of data, was inadvertently omitted from the author list in the originally published version of this Article. This has now been corrected in both the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41467-018-07819-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325104PMC
January 2019

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

Invest Ophthalmol Vis Sci 2018 06;59(7):2717-2728

Department of Cellular Biology and Anatomy, Augusta University, Augusta, Georgia, United States.

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-β, 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.
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http://dx.doi.org/10.1167/iovs.18-24267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984031PMC
June 2018

Cross-ancestry genome-wide association analysis of corneal thickness strengthens link between complex and Mendelian eye diseases.

Nat Commun 2018 05 14;9(1):1864. Epub 2018 May 14.

Statistical Genetics, QIMR Berghofer Medical Research Institute, QLD 4029, Brisbane, Australia.

Central corneal thickness (CCT) is a highly heritable trait associated with complex eye diseases such as keratoconus and glaucoma. We perform a genome-wide association meta-analysis of CCT and identify 19 novel regions. In addition to adding support for known connective tissue-related pathways, pathway analyses uncover previously unreported gene sets. Remarkably, >20% of the CCT-loci are near or within Mendelian disorder genes. These included FBN1, ADAMTS2 and TGFB2 which associate with connective tissue disorders (Marfan, Ehlers-Danlos and Loeys-Dietz syndromes), and the LUM-DCN-KERA gene complex involved in myopia, corneal dystrophies and cornea plana. Using index CCT-increasing variants, we find a significant inverse correlation in effect sizes between CCT and keratoconus (r = -0.62, P = 5.30 × 10) but not between CCT and primary open-angle glaucoma (r = -0.17, P = 0.2). Our findings provide evidence for shared genetic influences between CCT and keratoconus, and implicate candidate genes acting in collagen and extracellular matrix regulation.
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http://dx.doi.org/10.1038/s41467-018-03646-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5951816PMC
May 2018

Corneal Cross-Linking for Pediatric Keratcoconus Review.

Cornea 2018 Jun;37(6):802-809

Cornea Eye Institute, Beverly Hills, CA.

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.
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http://dx.doi.org/10.1097/ICO.0000000000001579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5938128PMC
June 2018

TSC1 Mutations in Keratoconus Patients With or Without Tuberous Sclerosis.

Invest Ophthalmol Vis Sci 2017 12;58(14):6462-6469

Department of Surgery and Board of the Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States.

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.
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http://dx.doi.org/10.1167/iovs.17-22819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760196PMC
December 2017

Comparison of the Effect of Epithelial Removal by Transepithelial Phototherapeutic Keratectomy or Manual Debridement on Cross-linking Procedures for Progressive Keratoconus.

J Refract Surg 2016 Oct;32(10):699-704

Purpose: To compare the visual, refractive, keratometric, topographic, and pachymetric outcomes of corneal collagen cross-linking (CXL) for progressive keratoconus following epithelial removal by transepithelial phototherapeutic keratectomy (PTK) or manual debridement.

Methods: In this analysis, 339 eyes (78% male, 22% female) that had undergone CXL following manual epithelial debridement (n = 180) or ablation via PTK (n = 159) were evaluated preoperatively and at 6, 12, and 24 months postoperatively for uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), maximum corneal keratometry, pachymetry, and spherical equivalent. The data were analyzed in a t test to evaluate the relative efficacy of each epithelial removal procedure.

Results: Manual epithelial debridement and ablation via PTK produce equivalent changes for all variables at each time interval with the exception of maximum corneal keratometry at 6 months postoperatively, for which PTK exhibited a significantly improved (flatter) result. This difference was present but not statistically significant at 12 and 24 months postoperatively.

Conclusions: Prior to CXL, both manual epithelial debridement and ablation via PTK result in equivalent visual, refractive, and keratometric outcomes up to 24 months postoperatively. [J Refract Surg. 2016;32(10):699-704.].
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http://dx.doi.org/10.3928/1081597X-20160712-01DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5963884PMC
October 2016

Genetics in Keratoconus: where are we?

Eye Vis (Lond) 2016 27;3:16. Epub 2016 Jun 27.

Regenerative Medicine Institute and Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, USA ; Cornea Genetic Eye Institute, 50 N. La Cienega Blvd. Suite #340, Beverly Hills, CA 90211 USA ; The Jules Stein Eye Institute, University of California Los Angeles, Los Angeles, USA.

Keratoconus (KC) is a non-inflammatory thinning and protrusion of the cornea in which the cornea assumes a conical shape. Complex etiology of this condition at present remains an enigma. Although environmental factors have been involved in KC pathogenesis, strong underlining genetic susceptibility has been proven. The lack of consistent findings among early genetic studies suggested a heterogeneity and complex nature of the genetic contribution to the development of KC. Recently, genome-wide linkage studies (GWLS) and genome-wide association studies (GWAS) were undertaken. Next-generation sequencing (NGS)-based genomic screens are also currently being carried out. Application of these recently developed comprehensive genetic tools led to a much greater success and increased reproducibility of genetic findings in KC. Involvement of the LOX gene identified through GWLS has been confirmed in multiple cohorts of KC patients around the world. KC susceptibility region located at the 2q21.3 chromosomal region near the RAB3GAP1 gene identified through GWAS was independently replicated. Rare variants in the ZNF469 gene (mutated in corneal dystrophy Brittle Cornea Syndrome) and in the TGFBI gene (mutated in multiple corneal epithelial-stromal TGFBI dystrophies) have been repeatedly identified in familial and sporadic KC patients of different ethnicities. Additional comprehensive strategies using quantitative endophenotypes have been successfully employed to bring further understanding to the genetics of KC. Additional genetic determinants including the COL5A1 gene have been identified in the GWAS of KC-related trait central corneal thickness. These recent discoveries confirmed the importance of the endophenotype approach for studying complex genetic diseases such as KC and showed that different connective tissue disorders may have the same genetic determinants.
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http://dx.doi.org/10.1186/s40662-016-0047-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4922054PMC
June 2016

Abnormal regulation of extracellular matrix and adhesion molecules in corneas of patients with keratoconus.

Int J Keratoconus Ectatic Corneal Dis 2016 May-Aug;5(2):63-70

Regenerative Medicine Institute and Department of Surgery, Cedars-Sinai, USA.

Aim: To identify changes in the expression of genes coding for extracellular matrix (ECM) proteins in patients with non-inflammatory corneal disorder keratoconus (KC), patients with corneal scarring, and normal controls.

Materials And Methods: Total RNA extracted from corneal tissue of 13 KC patients, 2 patients with corneal scaring and 4 normal controls was analyzed using Human Extracellular Matrix & Adhesion Molecules Profiler PCR Array. Statistically significant changes in gene expression were identified using the Data Analysis software.

Results: Comparison of KC and control corneas with thresholds of 1.5 or greater fold change and a p-value of 0.05 or lower, revealed 21 differentially expressed genes, 16 genes were downregulated and 5 were upregulated. Among transcripts downregulated in KC patients we identified THBS1, ADAMTS1, SPP1, several collagens and integrins. We found TGFBI (BIGH3) gene was the most significantly upregulated transcript.

Conclusion: Development of keratoconus results in deregulation of gene expression of extracellular matrix and adhesion molecules.

Clinical Significance: Downregulation of collagens and upregulation of TGFBI repeatedly identified in KC patients may be used as clinical markers of the disease.
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http://dx.doi.org/10.5005/jp-journals-10025-1123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627975PMC
October 2017

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

Mol Vis 2015 30;21:1357-67. Epub 2015 Dec 30.

Eye Program, Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA; David Geffen School of Medicine at UCLA, Los Angeles, CA.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4704770PMC
September 2016

Screening for Keratoconus and Related Ectatic Corneal Disorders.

Cornea 2015 Aug;34(8):e20-2

*Department of Ophthalmology, Emory University, Atlanta, GA †Emory Vision, Emory Eye Center, Atlanta, GA ‡Cleveland Clinic Cole Eye Institute, Cleveland, OH §Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, OH ¶Transplant Center, Surgery Institute, Cleveland Clinic, OH ‖Department of Biomedical Engineering, Case Western Reserve University, OH **Department of Ophthalmology, University of Sao Paulo, Sao Paulo, Brazil ††Department of Ophthalmology, Federal University of Rio de Janeiro, Sao Paulo, Brazil ‡‡Cornea Genetic Eye Institute, Beverly Hills, CA §§Institute for Translational Genomics and Population Sciences, Los Angeles BioMedical Research Institute, Harbor-UCLA Medical Center, Torrance, CA ¶¶The Jules Stein Eye Institute, University of California-Los Angeles, Los Angeles, CA ‖‖Cullen Eye Institute, Baylor College of Medicine, Houston, TX ***Stulting Research Center, Woolfson Eye Institute, Atlanta, GA †††Department of Ophthalmology, Mount Sinai School of Medicine, New York, NY.

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http://dx.doi.org/10.1097/ICO.0000000000000500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6392063PMC
August 2015

Differentiation of human limbal-derived induced pluripotent stem cells into limbal-like epithelium.

Stem Cells Transl Med 2014 Sep 28;3(9):1002-12. Epub 2014 Jul 28.

Regenerative Medicine Institute, Eye Program, and Departments of Biomedical Sciences, Neurosurgery, Genomics Core, and Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA; Norris Comprehensive Cancer Center Bioinformatics Core and Division of Hematology, University of Southern California, Los Angeles, California, USA; David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA

Limbal epithelial stem cell (LESC) deficiency (LSCD) leads to corneal abnormalities resulting in compromised vision and blindness. LSCD can be potentially treated by transplantation of appropriate cells, which should be easily expandable and bankable. Induced pluripotent stem cells (iPSCs) are a promising source of transplantable LESCs. The purpose of this study was to generate human iPSCs and direct them to limbal differentiation by maintaining them on natural substrata mimicking the native LESC niche, including feederless denuded human amniotic membrane (HAM) and de-epithelialized corneas. These iPSCs were generated with nonintegrating vectors from human primary limbal epithelial cells. This choice of parent cells was supposed to enhance limbal cell differentiation from iPSCs by partial retention of parental epigenetic signatures in iPSCs. When the gene methylation patterns were compared in iPSCs to parental LESCs using Illumina global methylation arrays, limbal-derived iPSCs had fewer unique methylation changes than fibroblast-derived iPSCs, suggesting retention of epigenetic memory during reprogramming. Limbal iPSCs cultured for 2 weeks on HAM developed markedly higher expression of putative LESC markers ABCG2, ΔNp63α, keratins 14, 15, and 17, N-cadherin, and TrkA than did fibroblast iPSCs. On HAM culture, the methylation profiles of select limbal iPSC genes (including NTRK1, coding for TrkA protein) became closer to the parental cells, but fibroblast iPSCs remained closer to parental fibroblasts. On denuded air-lifted corneas, limbal iPSCs even upregulated differentiated corneal keratins 3 and 12. These data emphasize the importance of the natural niche and limbal tissue of origin in generating iPSCs as a LESC source with translational potential for LSCD treatment.
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http://dx.doi.org/10.5966/sctm.2014-0076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4149305PMC
September 2014

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

J Refract Surg 2014 Feb;30(2):80-7

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.
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http://dx.doi.org/10.3928/1081597X-20140120-02DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4000528PMC
February 2014

A simple alkaline method for decellularizing human amniotic membrane for cell culture.

PLoS One 2013 13;8(11):e79632. Epub 2013 Nov 13.

Eye Program, Cedars-Sinai Medical Center, Los Angeles, California, United States of America ; Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States of America ; Departments of Biomedical Sciences, Surgery, and Neurosurgery, Cedars-Sinai Medical Center, Los Angeles, California, United States of America.

Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for transplantation to patients with limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane, because denuded membrane is poorly immunogenic and better supports repopulation by dissociated limbal epithelial cells. Amniotic membrane denuding usually involves treatment with EDTA and/or proteolytic enzymes; in many cases additional mechanical scraping is required. Although ensuring limbal cell proliferation, these methods are not standardized, require relatively long treatment times and can result in membrane damage. We propose to use 0.5 M NaOH to reliably remove amniotic cells from the membrane. This method was used before to lyse cells for DNA isolation and radioactivity counting. Gently rubbing a cotton swab soaked in NaOH over the epithelial side of amniotic membrane leads to nearly complete and easy removal of adherent cells in less than a minute. The denuded membrane is subsequently washed in a neutral buffer. Cell removal was more thorough and uniform than with EDTA, or EDTA plus mechanical scraping with an electric toothbrush, or n-heptanol plus EDTA treatment. NaOH-denuded amniotic membrane did not show any perforations compared with mechanical or thermolysin denuding, and showed excellent preservation of immunoreactivity for major basement membrane components including laminin α2, γ1-γ3 chains, α1/α2 and α6 type IV collagen chains, fibronectin, nidogen-2, and perlecan. Sodium hydroxide treatment was efficient with fresh or cryopreserved (10% dimethyl sulfoxide or 50% glycerol) amniotic membrane. The latter method is a common way of membrane storage for subsequent grafting in the European Union. NaOH-denuded amniotic membrane supported growth of human limbal epithelial cells, immortalized corneal epithelial cells, and induced pluripotent stem cells. This simple, fast and reliable method can be used to standardize decellularized amniotic membrane preparations for expansion of limbal stem cells in vitro before transplantation to patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0079632PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3827346PMC
July 2014

C.57 C > T Mutation in MIR 184 is Responsible for Congenital Cataracts and Corneal Abnormalities in a Five-generation Family from Galicia, Spain.

Ophthalmic Genet 2015 ;36(3):244-7

a Cornea Genetic Eye Institute , Beverly Hills , CA , USA .

A c.57 C > T mutation in the seed region of MIR184 located at the 15q25.1 chromosomal region has been independently associated with autosomal dominant keratoconus with early-onset anterior polar cataract in the Northern Irish family and with autosomal dominant EDICT (Endothelial Dystrophy, Iris hypoplasia, Congenital cataracts, and stromal Thinning) syndrome. In this study we report a five-generation family originating in Galicia, Spain with early onset cataracts and variable corneal abnormalities which include non-ectatic corneal thinning and severe early-onset keratoconus. We identified a heterozygous c.57 C > T mutation in miR-184 in the proband and two additional affected relatives on the maternal side. This finding represents a third independent occurrence of this mutation in familiar ocular disease thus strengthening the link between miR-184 abnormalities and inherited eye defects.
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http://dx.doi.org/10.3109/13816810.2013.848908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3991767PMC
April 2016

Genetic association of COL5A1 variants in keratoconus patients suggests a complex connection between corneal thinning and keratoconus.

Invest Ophthalmol Vis Sci 2013 Apr 12;54(4):2696-704. Epub 2013 Apr 12.

Cornea Genetic Eye Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA.

Purpose: Single nucleotide polymorphisms (SNPs) located near or within the COL5A1 gene, at 9q34.2-q34.3 chromosomal region have been reported in association with central corneal thickness (CCT). Using family linkage analysis, we identified a keratoconus susceptibility locus at 9q34. These findings led us to perform an association study between COL5A1 variation and keratoconus susceptibility.

Methods: A Caucasian case-control cohort of 222 keratoconus patients and 3324 controls was selected as the discovery panel. An independent case-control panel of 304 cases and 518 controls and a family panel of 186 subjects were replicated for genotyping and association. Forty-four SNPs (21 for discovery and 23 for fine-mapping) spanning 300 kilobases in and around COL5A1 were genotyped and tested for genetic association. Logistic regression models implemented in PLINK were used to test for association in case controls. Generalized estimating equation models accounting for familial correlations implemented in genome-wide interaction analyses with family data were used for association testing in families.

Results: Two CCT associated SNPs (rs1536482 and rs7044529 near and within COL5A1) were identified in the keratoconus discovery cohort (P values of 6.5 × 10(-3) and 7.4 × 10(-3)). SNP rs1536482 was replicated in the second case-control sample (P = 0.02), and SNP rs7044529 was replicated in a keratoconus family panel (P = 0.03). Meta P values of rs1536482 and rs7044529 in the keratoconus cohorts were 1.5 × 10(-4) (odds ratio [OR] = 1.30) and 2.9 × 10(-3) (OR = 1.39). After Bonferroni correction, the association of SNP rs1536482 remained significant (P = 6.5 × 10(-3)).

Conclusions: SNPs in the COL5A1 region, which regulate normal variation in CCT, may play a role in the thinning associated with keratoconus.
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http://dx.doi.org/10.1167/iovs.13-11601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3630822PMC
April 2013

An association between the calpastatin (CAST) gene and keratoconus.

Cornea 2013 May;32(5):696-701

Cornea Genetic Eye Institute, Los Angeles, CA, USA.

Purpose: Keratoconus (KC) is a genetically heterogeneous corneal dystrophy. Previously, we performed 2 genome-wide linkage scans in a 4-generation autosomal dominant pedigree and repeatedly mapped a KC locus to a genomic region located on chromosome 5q overlapping the gene encoding the inhibitor of calpains, calpastatin (CAST). To test whether variants in CAST gene are involved in genetic susceptibility to KC, we performed genetic testing of polymorphic markers in CAST gene in family and case-control panels of patients with KC.

Methods: We genotyped single-nucleotide polymorphisms (SNPs) located in CAST gene in 262 patients in 40 white KC families and in a white case-control panel with 304 cases and 518 controls. Generalized estimating equation models accounting for familial correlations implemented in GWAF program were used for association testing in families. Logistic regression models implemented in PLINK were performed to test the associations in case-control samples.

Results: Genetic testing of the first set of 7 SNPs in familial samples revealed 2 tentative nominally significant markers (rs4869307, P = 0.03; rs27654, P = 0.07). Additional genotyping of 12 tightly spaced SNPs identified CAST SNP rs4434401 to be associated with KC in both familial and case-control panels with P values of 0.005 and 0.05, respectively, and with combined meta P value of familial and case-control cohorts of 0.002 or after Bonferroni correction of 0.04.

Conclusions: Linkage analysis and genetic association support involvement of CAST gene in the genetic susceptibility to KC. In silico analysis of CAST expression suggests differential regulation of calpain/calpastatin system in cornea as a potential mechanism of functional defect.
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http://dx.doi.org/10.1097/ICO.0b013e3182821c1cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3653445PMC
May 2013

Genome-wide association analyses identify multiple loci associated with central corneal thickness and keratoconus.

Nat Genet 2013 Feb 6;45(2):155-63. Epub 2013 Jan 6.

Queensland Institute of Medical Research, Statistical Genetics, Herston, Brisbane, Queensland, Australia.

Central corneal thickness (CCT) is associated with eye conditions including keratoconus and glaucoma. We performed a meta-analysis on >20,000 individuals in European and Asian populations that identified 16 new loci associated with CCT at genome-wide significance (P < 5 × 10(-8)). We further showed that 2 CCT-associated loci, FOXO1 and FNDC3B, conferred relatively large risks for keratoconus in 2 cohorts with 874 cases and 6,085 controls (rs2721051 near FOXO1 had odds ratio (OR) = 1.62, 95% confidence interval (CI) = 1.4-1.88, P = 2.7 × 10(-10), and rs4894535 in FNDC3B had OR = 1.47, 95% CI = 1.29-1.68, P = 4.9 × 10(-9)). FNDC3B was also associated with primary open-angle glaucoma (P = 5.6 × 10(-4); tested in 3 cohorts with 2,979 cases and 7,399 controls). Further analyses implicate the collagen and extracellular matrix pathways in the regulation of CCT.
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http://dx.doi.org/10.1038/ng.2506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3720123PMC
February 2013

Penetrating keratoplasty using femtosecond laser-enabled keratoplasty with zig-zag incisions versus a mechanical trephine in patients with keratoconus.

Br J Ophthalmol 2012 Sep 11;96(9):1195-9. Epub 2012 Jul 11.

Cornea Eye Institute, Beverly Hills, CA 90211, USA.

Background/aims: This paper will compare the visual outcomes of two different penetrating keratoplasty (PKP) techniques in patients with keratoconus. It is a retrospective comparative surgical case series of 116 keratoconus patients (137 eyes) who had PKP at the Cornea Eye Institute, Beverly Hills, California, USA.

Methods: 56 keratoconus patients (66 eyes) underwent femtosecond laser-enabled keratoplasty (FLEK) with a zig-zag incision configuration. Their visual parameters were compared with those of 60 patients (71 eyes) who had traditional blade mechanical trephination PKP. The range of follow-up was between 3 and 6 months. The main outcome measures included uncorrected visual acuity and best spectacle-corrected visual acuity (BSCVA), manifest refractive spherical equivalent and topographically determined astigmatism.

Results: BSCVA was significantly better as early as 3 months postoperatively (p=0.001) in the FLEK group. Visual recovery to 20/40 after 3 months was significantly better in the FLEK group (p<0.001). Topographic astigmatism was lower in the FLEK group, but the difference between the two groups reached significance only at 3 months of follow-up (p=0.001). Postoperative complications noted were not different between the two groups.

Conclusions: Faster visual recovery and better long-term outcomes were observed in keratoconus patients who had FLEK compared with those who had the mechanical PKP procedure with 6 months of postoperative follow-up.
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http://dx.doi.org/10.1136/bjophthalmol-2012-301662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3598602PMC
September 2012

Variation in the lysyl oxidase (LOX) gene is associated with keratoconus in family-based and case-control studies.

Invest Ophthalmol Vis Sci 2012 Jun 28;53(7):4152-7. Epub 2012 Jun 28.

Regenerative Medicine Institute, Ophthalmology Research, Department of Surgery, Division of Surgical Research, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA.

Purpose: Keratoconus is a bilateral noninflammatory progressive corneal disorder with complex genetic inheritance and a common cause for cornea transplantation in young adults. A genomewide linkage scan in keratoconus families identified a locus at 5q23.2, overlapping the gene coding for the lysyl oxidase (LOX). LOX encodes an enzyme responsible for collagen cross-linking in a variety of tissues including the cornea. Corneal collagen cross-linking with long-wave ultraviolet light and riboflavin is a promising new treatment for keratoconus. To determine whether LOX is a genetic determinant of the pathogenesis of keratoconus, we analyzed association results of LOX polymorphisms in two independent case-control samples and in keratoconus families.

Methods: Association results were analyzed of single-nucleotide polymorphisms (SNPs) in the LOX gene from a Genome-Wide Association Study (GWAS) investigation in two independent panels of patients with keratoconus and controls and in keratoconus families.

Results: Evidence of association was found at SNPs rs10519694 and rs2956540 located in intron 4 of LOX in the GWAS discovery case-control panel with P values of 2.3×10(-3) and 7×10(-3), respectively. The same two SNPs were found to be associated with keratoconus by family-based association testing with P values of 2.7×10(-3) and 7.7×10(-4), respectively. Meta P values of 4.0×10(-5) and 4.0×10(-7) were calculated for SNPs rs10519694 and rs2956540 by analyzing case-control and family samples simultaneously. Sequencing of LOX exons in a subset of keratoconus patients identified two polymorphisms, rs1800449 and rs2288393, located in LOX transcripts I and II, associated with keratoconus in case-control and family samples with a meta P value of 0.02.

Conclusions: Results provided strong genetic evidence that LOX variants lead to increased susceptibility to developing of keratoconus.
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http://dx.doi.org/10.1167/iovs.11-9268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3760233PMC
June 2012