Comparative transcriptome analysis of hESC- and iPSC-derived corneal endothelial cells.

The corneal endothelium (CE), a monolayer of hexagonal cells constitutes the innermost layer of the cornea that is critical in maintaining clarity by mediating hydration through barrier and pump functions. Corneal endothelial cells (CECs) have limited proliferative potential and therefore generation of CECs has been undertaken by many groups. We previously reported generation of CECs from peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cells (iPSCs). In here, we extend our analysis through next-generation seqeuncing based transcriptome profiling of H9 human embryonic stem cell (hESC)- and human PBMC-originated, iPSC-derived CECs. The differentiating CECs on day 20 (D20) exhibited a tightly packed hexagonal/polygonal shape expressing zona occludens-1 (ZO-1) and N-cadherin at the cell boundaries. Next-generation RNA sequencing of hESC- and iPSC-derived CECs detected expression (≥0.659 RPKM) of 13,546 and 13,536 genes, respectively. Comparative transcriptome analysis of hESC- and iPSC-derived CECs revealed 13,208 (>96%) genes common in both transcriptomes. Among the 13,208 genes common in these transcriptomes, 12,580 (>95%) exhibited a quantitatively similar expression. To the best of our knowledge, this is the first report presenting comparative transcriptome analysis of hESC- and iPSC-derived CECs.

Mutations in AGBL1 cause dominant late-onset Fuchs corneal dystrophy and alter protein-protein interaction with TCF4.

Fuchs corneal dystrophy (FCD) is a hereditary dystrophy of the corneal endothelium and is responsible for majority of the corneal transplantation performed in the United States. Here, we describe three generations of a family with 12 individuals affected by late-onset FCD and in which three individuals are unaffected. Genome-wide mapping provided suggestive linkage at two loci on chromosomal arms 3p and 15q. Alleles at either locus alone were not sufficient to explain FCD; however, considered together, both loci could explain the disorder in this pedigree. Subsequent next-generation sequencing identified a nonsense mutation in AGBL1 in the 15q locus; this mutation would result in a premature termination of AGBL1. Consistent with a causal role for this transcript, further sequencing of our cohort of late-onset-FCD-affected individuals identified two cases harboring the same nonsense mutation and a further three unrelated individuals bearing a second missense allele. AGBL1 encodes a glutamate decarboxylase previously identified in serial analysis of gene expression of corneal endothelium, a finding confirmed by immunohistochemical staining. Wild-type AGBL1 localizes predominantly to the cytoplasm; in sharp contrast, the truncated protein showed distinct nuclear localization. Finally, we show that AGBL1 interacts biochemically with the FCD-associated protein TCF4 and that the mutations found in our cohort of FCD individuals diminish this interaction. Taken together, our data identify a locus for FCD, extend the complex genetic architecture of the disorder, provide direct evidence for the involvement of TCF4 in FCD pathogenesis, and begin to explain how causal FCD mutations affect discrete biochemical complexes.

Mutations in LOXHD1, a recessive-deafness locus, cause dominant late-onset Fuchs corneal dystrophy.

Fuchs corneal dystrophy (FCD) is a genetic disorder of the corneal endothelium and is the most common cause of corneal transplantation in the United States. Previously, we mapped a late-onset FCD locus, FCD2, on chromosome 18q. Here, we present next-generation sequencing of all coding exons in the FCD2 critical interval in a multigenerational pedigree in which FCD segregates as an autosomal-dominant trait. We identified a missense change in LOXHD1, a gene causing progressive hearing loss in humans, as the sole variant capable of explaining the phenotype in this pedigree. We observed LOXHD1 mRNA in cultured human corneal endothelial cells, whereas antibody staining of both human and mouse corneas showed staining in the corneal epithelium and endothelium. Corneal sections of the original proband were stained for LOXHD1 and demonstrated a distinct increase in antibody punctate staining in the endothelium and Descemet membrane; punctate staining was absent from both normal corneas and FCD corneas negative for causal LOXHD1 mutations. Subsequent interrogation of a cohort of >200 sporadic affected individuals identified another 15 heterozygous missense mutations that were absent from >800 control chromosomes. Furthermore, in silico analyses predicted that these mutations reside on the surface of the protein and are likely to affect the protein's interface and protein-protein interactions. Finally, expression of the familial LOXHD1 mutant allele as well as two sporadic mutations in cells revealed prominent cytoplasmic aggregates reminiscent of the corneal phenotype. All together, our data implicate rare alleles in LOXHD1 in the pathogenesis of FCD and highlight how different mutations in the same locus can potentially produce diverse phenotypes.

Missense mutations in TCF8 cause late-onset Fuchs corneal dystrophy and interact with FCD4 on chromosome 9p.

Fuchs corneal dystrophy (FCD) is a degenerative genetic disorder of the corneal endothelium that represents one of the most common causes of corneal transplantation in the United States. Despite its high prevalence (4% over the age of 40), the underlying genetic basis of FCD is largely unknown. Here we report missense mutations in TCF8, a transcription factor whose haploinsufficiency causes posterior polymorphous corneal dystrophy (PPCD), in a cohort of late-onset FCD patients. In contrast to PPCD-causing mutations, all of which are null, FCD-associated mutations encode rare missense changes suggested to cause loss of function by an in vivo complementation assay. Importantly, segregation of a recurring p.Q840P mutation in a large, multigenerational FCD pedigree showed this allele to be sufficient but not necessary for pathogenesis. Execution of a genome-wide scan conditioned for the presence of the 840P allele identified an additional late-onset FCD locus on chromosome 9p, whereas haplotype analysis indicated that the presence of the TCF8 allele and the disease haplotype on 9p leads to a severe FCD manifestation with poor prognosis. Our data suggest that PPCD and FCD are allelic variants of the same disease continuum and that genetic interaction between genes that cause corneal dystrophies can modulate the expressivity of the phenotype.

Descemet membrane endothelial keratoplasty in eyes with COL8A2-associated corneal dystrophy.

Purpose: The p.(Leu450Trp) COL8A2 mutation, associated with an early-onset corneal endothelial dystrophy, can result in bullous keratopathy within the first few decades of life. People with this condition frequently experience anterior corneal changes in keratometry as the disease worsens, which may potentially affect refractive error after endothelial keratoplasty. We describe outcomes of the first cases of Descemet Membrane Endothelial Keratoplasty (DMEK) for patients with known mutations in this gene. Observations: Four eyes from two patients with COL8A2-associated corneal dystrophy underwent DMEK for this condition at a tertiary academic center. Preoperative and postoperative Scheimpflug imaging and manifest refraction was conducted. Mean central corneal thickness decreased from 713 µm preoperatively to 529 µm at one month. Despite long-standing corneal haze, all eyes reached between 20/20 and 20/30 best corrected visual acuity, and minimum postoperative central corneal thickness reached 482, 479, 479 and 533 µm. Refractive changes frequently occurred during the first postoperative year, with 3.6 D, 3.3 D, 3 D, and 0.8 D shifts in spherical equivalent taking place within this time period in the four eyes. Conclusions and Importance: In two patients with the p.(Leu450Trp) mutation in COL8A2 who underwent DMEK, resolution of corneal edema resulted in centrally thin corneas and refractive shifts postoperatively. Despite chronic edema, excellent visual acuity was achieved in all eyes.

Cigarette Smoke Triggers Loss of Corneal Endothelial Cells and Disruption of Descemet's Membrane Proteins in Mice.

Purpose: To investigate changes at a molecular level in the mouse corneal endothelium (CE) exposed to chronic cigarette smoke (CS). Methods: Pregnant mice (gestation days 18-20) were placed in a whole-body exposure smoking chamber, and a few days later pups were born. After 3.5 months of CS exposure, a ConfoScan4 scanning microscope was used to examine the corneal endothelial cells (CECs) of CS-exposed and control (Ct) mice. The CE was peeled under a microscope and maintained as four biological replicates (two male and two female) for CS-exposed and Ct mice; each replicate consisted of 16 CEs. The proteome of the CE was investigated through mass spectrometry. Results: The CE images of CS-exposed and Ct mice revealed a difference in the shape of CECs accompanied by a nearly 10% decrease in CEC density (P < 0.00003) following CS exposure. Proteome profiling identified a total of 524 proteins exhibiting statistically significant changes in CE from CS-exposed mice. Importantly, proteins associated with Descemet's membrane (DM), including COL4α1, COL4α2, COL4α3, COL4α4, COL4α5, COL4α6, COL8α1, COL8α2, and FN1, among others, exhibited diminished protein levels in the CE of CS-exposed mice. Conclusions: Our data confirm that exposure to CS results in reduced CEC density accompanied by diminished levels of multiple collagen and extracellular matrix proteins associated with DM.

Pluripotent stem cell-derived corneal endothelial cells as an alternative to donor corneal endothelium in keratoplasty.

Here, we evaluate the efficacy of cryopreserved human embryonic stem cell (hESC)-derived corneal endothelial cells (CECs) to form a functional monolayer of corneal endothelium (CE) in rabbits and monkeys. We injected cryopreserved hESC-derived CECs into the anterior chamber of rabbits and monkeys either immediately after mechanical scraping of the central CE or a few days later when corneal edema developed. All preclinical models developed deturgesced and clear corneas following the injection of cryopreserved hESC-derived CECs and remained comparable to the corneas of the untreated eye. Confocal scanning microscopy confirmed an intact structure of hexagonal/polygonal cells and immunohistochemical analysis illustrated a monolayer expressing barrier and pump function proteins in the regenerated CE. The necropsy examination confirmed no remarkable change in multiple tissues assessed for teratoma formation. In conclusion, our data demonstrate the efficacy of cryopreserved hESC-derived CECs to form a functional CE on the denuded Descemet's membrane.

Missense mutations in the sodium borate cotransporter SLC4A11 cause late-onset Fuchs corneal dystrophy.

Homozygous mutations in the Borate Cotransporter SLC4A11 cause two early-onset corneal dystrophies: congenital hereditary endothelial dystrophy (CHED) and Harboyan syndrome. More recently, four sporadic patients with late-onset Fuchs corneal dystrophy (FCD), a common age-related disorder, were also reported to harbor heterozygous mutations at this locus. We therefore tested the hypothesis that SLC4A11 contributes to FCD and asked whether mutations in SLC4A11 are responsible for familial cases of late-onset FCD. We sequenced SLC4A11 in 192 sporadic and small nuclear late-onset FCD families and found seven heterozygous missense novel variations that were absent from ethnically matched controls. Familial data available for one of these mutations showed segregation under a dominant model in a three-generational family. In silico analyses suggested that most of these substitutions are intolerant, whereas biochemical studies of the mutant protein indicated that these alleles impact the localization and/or posttranslational modification of the protein. These results suggest that heterozygous mutations in SLC4A11 are modest contributors to the pathogenesis of adult FCD, suggesting a causality continuum between FCD and CHED. Taken together with a recent model between FCD and yet another early onset corneal dystrophy, PPCD, our data suggest a shared pathomechanism and genetic overlap across several corneal dystrophies.

Cataract surgery in Fuchs corneal dystrophy.

PURPOSE OF REVIEW: Patients with decreased vision due to Fuchs corneal dystrophy and cataract can present with a number of challenges to determine the best surgical option for restoring sight. The ophthalmologist must first distinguish how much of the vision loss is due to the cataract versus the Fuchs corneal dystrophy (FCD) before determining the best surgical course, either cataract surgery alone or cataract surgery combined with full thickness or lamellar keratoplasty. This review will discuss the various techniques and clinical signs to ascertain the source of vision loss and how to use this clinical information to help guide the surgical decision making process in order to optimize the best possible outcome. RECENT FINDINGS: Over the last several years, the technique of endothelial keratoplasty has evolved to become an important alternative in the treatment of FCD. Anticipating the correct intraocular lens power for a patient undergoing cataract surgery alone followed by Descemet stripping endothelial keratoplasty (DSEK) or combined cataract surgery with DSEK requires understanding the hyperopic shift that can occur with DSEK and incorporating this correction preoperatively in the intraocular lens power selection. SUMMARY: New developments in the surgical treatment of Fuchs corneal dystrophy have greatly enhanced our ability to restore vision and reduce the dependence on contact lenses in this genetic disease.

Monoclonal gammopathy of "ocular" significance.

PURPOSE: Diagnostic criteria for monoclonal gammopathy of undetermined significance (MGUS) do not currently include ocular phenotypic changes. Here, we offer a new diagnostic approach that is useful in patients with posteriorly located corneal depositions and present evidence to support the theory that the aqueous humor is a source for monoclonal proteins accumulated in the cornea. OBSERVATIONS: A 77-year-old woman presented to the clinic with a gradual decrease in visual acuity over 6 months. Slit lamp examination revealed bilateral central guttae consistent with Fuchs corneal dystrophy, peripheral circular band-like corneal opacities in the deep stroma, and bilateral nuclear sclerotic and cortical cataracts. Anterior segment optical coherence tomography confirmed corneal opacities in the posterior stroma and Descemet membrane. Immunological studies revealed increased serum IgG levels of 3220 mg/dL and serum electrophoresis showed an abnormal monoclonal band of 2.4 g/dL identified as IgG lambda by immunofixation electrophoresis. The patient was referred to the hematology clinic where she underwent further systemic workup and was diagnosed with MGUS. Immunofixation electrophoresis of aqueous sampling, which was performed at the time of cataract surgery, confirmed the presence of the IgG lambda gammopathy in the anterior chamber. CONCLUSIONS AND IMPORTANCE: Monoclonal gammopathy, although rare, should be included in the differential diagnosis of corneal opacities, as the ocular finding can be the initial manifestation of a systemic disease that can potentially be life-threatening. When corneal biopsy is not feasible due to the location of corneal pathology, aqueous sampling may be an alternative approach towards a clinical diagnosis. We propose a new terminology, "monoclonal gammopathy of ocular significance," for patients diagnosed with MGUS, however, their only significant clinical finding is ocular manifestation.

Identification of a Novel TCF4 Isoform in the Human Corneal Endothelium.

PURPOSE: Alternative splice isoforms of TCF4, a gene implicated in Fuchs corneal dystrophy, have been identified in multiple human tissues outside of the eye. The aim of this study was to identify the transcriptional profile of TCF4 in the corneal endothelium. METHODS: We extracted RNA from the donor corneal endothelium and performed rapid amplification of cDNA ends. We tested the expression pattern of 1 newly identified isoform (7b) in a panel of cDNA derived from multiple human tissues and included cDNA from corneal endothelial (CE) and retinal pigment epithelial cell lines. To further delineate differential expression of TCF4 splice variants that span CTG18.1, we analyzed expression of 6 alternative splice isoforms that are transcribed from either exon 2 or 3 in RNA extracted from the corneal endothelium of 3 normal donors and a CE cell line. RESULTS: We identified 11 different isoforms in control CE tissue, including 1 isoform (7b) not reported previously. This isoform is enriched specifically in the corneal endothelium and placenta compared with other tissues in a panel of human cDNA. CONCLUSIONS: We demonstrate the complex expression profile of TCF4 in the human corneal endothelium and reveal expression of alternative splice variants of TCF4.

Pilot Study of Audiometric Patterns in Fuchs Corneal Dystrophy.

Purpose: Although Fuchs corneal dystrophy (FCD) is considered an eye disease, a small number of studies have identified genes related to both FCD and hearing loss. Whether FCD is related to hearing loss is unknown. Method: This is a case-control study comparing pure-tone audiometry hearing thresholds in 180 patients with FCD from a hospital-based ophthalmology clinic with 2,575 population-based controls from a nationally representative survey, the National Health and Nutrition Examination Survey (from cycles 2005-06 and 2009-10). Generalized estimating equations were used to compare mean better-hearing ear thresholds in the 2 groups adjusted for age, sex, race, and noise exposure. Results: Patients with FCD had higher hearing thresholds (worse hearing) in lower frequencies (mean difference at 0.5 kHz = 3.49 dB HL) and lower hearing thresholds (better hearing) in higher frequencies (difference at 4 kHz = -4.25 dB HL) compared with population-based controls. Conclusion: In the first study to use objectively measured hearing, FCD was associated with poorer low-frequency and better high-frequency audiometric thresholds than population controls. Further studies are needed to characterize this relationship.

Generation and Proteome Profiling of PBMC-Originated, iPSC-Derived Corneal Endothelial Cells.

Purpose: Corneal endothelial cells (CECs) are critical in maintaining clarity of the cornea. This study was initiated to develop peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cell (iPSC)-derived CECs. Methods: We isolated PBMCs and programmed the mononuclear cells to generate iPSCs, which were differentiated to CECs through the neural crest cells (NCCs). The morphology of differentiating iPSCs was examined at regular intervals by phase contrast microscopy. In parallel, the expression of pluripotent and corneal endothelium (CE)-associated markers was investigated by quantitative real-time PCR (qRT-PCR). The molecular architecture of the iPSC-derived CECs and human corneal endothelium (hCE) was examined by mass spectrometry-based proteome sequencing. Results: The PBMC-originated, iPSC-derived CECs were tightly adherent, exhibiting a hexagonal-like shape, one of the cardinal characteristics of CECs. The CE-associated markers expressed at significantly higher levels in iPSC-derived CECs at days 13, 20, and 30 compared with their respective levels in iPSCs. It is of importance that only residual expression levels of pluripotency markers were detected in iPSC-derived CECs. Cryopreservation of iPSC-derived CECs did not affect the tight adherence of CECs and their hexagonal-like shape while expressing high levels of CE-associated markers. Mass spectrometry-based proteome sequencing identified 10,575 proteins in the iPSC-derived CEC proteome. In parallel, we completed proteome profiling of the hCE identifying 6345 proteins. Of these, 5763 proteins were identified in the iPSC-derived CECs, suggesting that 90.82% of the hCE proteome overlaps with the iPSC-derived CEC proteome. Conclusions: We have successfully developed a personalized approach to generate CECs that closely mimic the molecular architecture of the hCE. To the best of our knowledge, this is the first report describing the development of PBMC-originated, iPSC-derived CECs.

CTG18.1 Expansion in TCF4 Among African Americans With Fuchs' Corneal Dystrophy.

Purpose: Studies of Fuchs' dystrophy have largely focused on individuals of European origin. Characterization of disease among African Americans is required to ensure prognostic factors and therapeutic approaches are applicable across diverse patient populations. Methods: We assessed all self-reported black and white patients aged older than 40 years at a tertiary care institution with a diagnosis of cataract over a 3-year period for concurrent diagnosis of Fuchs' dystrophy. Affected patients in a longitudinal cohort were invited to provide a blood sample from which we extracted genomic DNA. The CTG18.1 trinucleotide repeat length was determined using a two-step, triplet repeat primed PCR protocol. Expansion was defined as >40 CTG repeats. Demographic information, including race, was documented. Results: Of 59,365 self-reported black and white adults who presented for cataract evaluation, the odds ratio of presenting with Fuchs' dystrophy among black compared to white patients was 0.6992 (95% confidence interval [CI], 0.6210-0.7872). A total of 60 black and 549 white patients with Fuchs' corneal dystrophy enrolled in the longitudinal study, of which 21 (35.0%) black and 343 (62.5%) white patients demonstrated trinucleotide repeat expansion, a significant difference (P = 7.7 × 10-5). In a multivariable linear regression model, repeat expansion but not race was significantly associated with mean clinical grading of severity. Conclusions: Black patients with Fuchs' dystrophy were less likely than white patients to demonstrate CTG18.1 allele expansion. The data contribute to our understanding of population differences in clinical presentation, and highlight the need for considering diversity of patient populations in clinical research.

CTG18.1 Expansion in TCF4 Increases Likelihood of Transplantation in Fuchs Corneal Dystrophy.

PURPOSE: Fuchs dystrophy is the leading indication of corneal transplantation in the United States. A CTG18.1 trinucleotide repeat in TCF4 correlates with increased severity in Fuchs dystrophy; however, quantitative estimates of increased transplantation risk, including effects of age and sex, are unclear. METHODS: In a tertiary institution clinical practice, 574 participants were enrolled in a longitudinal study of Fuchs dystrophy after slit-lamp biomicroscopy confirmed significant central guttae and/or corneal transplantation in both eyes. We documented clinical history, examination findings, and demographic information. We acquired blood samples, extracted DNA, and sequenced the CTG18.1 trinucleotide repeat in TCF4. In this retrospective case-control study, the number of participants with triplet expansion, defined as greater than 40 CTG repeats, and transplantation status were assessed. Kaplan-Meier estimates of timing and transplantation events were produced. The Cox proportional hazard regression model was used to assess the relationship between age, sex, triplet expansion, and surgery. RESULTS: A total of 106 participants (18.5%) previously underwent corneal transplantation in at least 1 eye at the time of initial evaluation. A higher proportion of individuals harboring allele expansion had undergone transplantation (78/357, 21.8%) compared with those without the expanded allele (28/217, 12.9%), a significant association (P = 0.007). The log-rank test demonstrates a significant difference in survival function over time (P = 0.027), with a hazard ratio of 1.64 (95% confidence interval, 1.05-2.55). CONCLUSIONS: Expansion of the TCF4 CTG trinucleotide repeat was associated with 1.64 times higher likelihood of corneal transplantation at a given age in patients with Fuchs dystrophy.

Automated Retroillumination Photography Analysis for Objective Assessment of Fuchs Corneal Dystrophy.

PURPOSE: Retroillumination photography analysis is an objective tool for the assessment of the number and distribution of guttae in eyes affected with Fuchs corneal dystrophy (FCD). Current protocols include manual processing of images; here, we assess validity and interrater reliability of automated analysis across various levels of FCD severity. METHODS: Retroillumination photographs of 97 FCD-affected corneas were acquired, and total counts of guttae were previously summated manually. For each cornea, a single image was loaded into ImageJ software. We reduced color variability and subtracted background noise. Reflection of light from each gutta was identified as a local area of maximum intensity and counted automatically. Noise tolerance level was titrated for each cornea by examining a small region of each image with automated overlay to ensure appropriate coverage of individual guttae. We tested interrater reliability of automated counts of guttae across a spectrum of clinical and educational experience. RESULTS: A set of 97 retroillumination photographs was analyzed. Clinical severity as measured by a modified Krachmer scale ranged from a severity level of 1 to 5 in the set of analyzed corneas. Automated counts by an ophthalmologist correlated strongly with Krachmer grading (R = 0.79) and manual counts (R = 0.88). Intraclass correlation coefficients demonstrated strong correlation at 0.924 (95% CI, 0.870-0.958) among cases analyzed by 3 students, and 0.869 (95% CI, 0.797-0.918) among cases for which images were analyzed by an ophthalmologist and 2 students. CONCLUSIONS: Automated retroillumination photography analysis allows for grading of FCD severity with high resolution across a spectrum of disease severity.

Genome-wide association study identifies three novel loci in Fuchs endothelial corneal dystrophy.

The structure of the cornea is vital to its transparency, and dystrophies that disrupt corneal organization are highly heritable. To understand the genetic aetiology of Fuchs endothelial corneal dystrophy (FECD), the most prevalent corneal disorder requiring transplantation, we conducted a genome-wide association study (GWAS) on 1,404 FECD cases and 2,564 controls of European ancestry, followed by replication and meta-analysis, for a total of 2,075 cases and 3,342 controls. We identify three novel loci meeting genome-wide significance (P<5 × 10-8): KANK4 rs79742895, LAMC1 rs3768617 and LINC00970/ATP1B1 rs1200114. We also observe an overwhelming effect of the established TCF4 locus. Interestingly, we detect differential sex-specific association at LAMC1, with greater risk in women, and TCF4, with greater risk in men. Combining GWAS results with biological evidence we expand the knowledge of common FECD loci from one to four, and provide a deeper understanding of the underlying pathogenic basis of FECD.

Linkage of late-onset Fuchs corneal dystrophy to a novel locus at 13pTel-13q12.13.

PURPOSE: To identify the gene locus underlying the inheritance of late-onset Fuchs corneal dystrophy (FCD) in a large white kindred. METHODS: Genotypes of small tandem repeat polymorphisms were obtained from 17 affected and 3 unaffected family members, followed by genetic linkage analysis. RESULTS: In this family, classic late-onset FCD appeared to be inherited as a single, dominant Mendelian trait. In two exceptional sibships, however, children aged 10 and 13 years had FCD. In each sibship, both parents were found to be affected, opening the possibility that this unusually early age of onset was the result of homozygosity for an FCD mutation. Genotype results, however, were not consistent with consanguinity of the parents, who appear to have independent cases of FCD. A whole-genome linkage scan mapped FCD to a single locus at 13pTel-13q12.13, with significant two-point LOD scores of 3.91 at D13S1236 and 3.80 at D13S1304. The 26.4-Mb disease interval contains the chromosome 13 nucleolus organizer (RNR1), the centromere, and 44 annotated protein-encoding genes. So far, exons of 10 of these genes have been screened, but no mutations have been found. CONCLUSIONS: FCD1 is the first genetic locus to be identified for late-onset FCD, a common disease of the aging cornea. The exceptional early onset of the disease observed in two children is unusual and might be the result of digenic interaction between FCD1 and an independent late-onset FCD mutation.