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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

FOXE3 contributes to Peters anomaly through transcriptional regulation of an autophagy-associated protein termed DNAJB1.

FOXE3 is a lens-specific transcription factor that has been associated with anterior segment ocular dysgenesis. To determine the transcriptional target(s) of FOXE3 that are indispensable for the anterior segment development, we examined the transcriptome and the proteome of cells expressing truncated FOXE3 responsible for Peters anomaly identified through linkage-coupled next-generation whole-exome sequencing. We found that DNAJB1, an autophagy-associated protein, was the only candidate exhibiting differential expression in both screens. We confirmed the candidacy of DNAJB1 through chromatin immunoprecipitation and luciferase assays while knockdown of DNAJB1 in human lens epithelial cells resulted in a mitotic arrest. Subsequently, we targeted dnajb1a in zebrafish through injection of a splice-blocking morpholino. The dnajb1a morphants exhibited underdeveloped cataractous lenses with persistent apoptotic nuclei. In conclusion, here we report DNAJB1 is a transcriptional target of FOXE3 in a novel pathway that is crucial for the development of the anterior segment of the eye.

Distinct Clinical Phenotype of Corneal Dystrophy Predicts the p.(Leu450Trp) Substitution in COL8A2.

PURPOSE: The p.(Leu450Trp) substitution in Collagen, Type VIII, Alpha 2 (COL8A2) is associated with an early-onset corneal dystrophy. Here we identify distinct anterior corneal and keratorefractive changes associated with this disease and replicate its distinguishing endothelial characteristics in a new family. METHODS: We reviewed clinical data from a large family associated with the p.(Leu450Trp) COL8A2 mutation. We compared clinical photographs and keratometry over an 11-year period. We sought to replicate these findings, and after a 40-year-old male subject presented similarly, we obtained a peripheral blood sample and sequenced COL8A2. RESULTS: Of 10 individuals with the p.(Leu450Trp) substitution, clinical records noted corneal edema in 6, of which 4 showed epithelial microcystic edema. Eleven-year progression data reveal a marked increase in subepithelial corneal edema and gradual, profound increase in anterior corneal astigmatism. Sequencing of genomic DNA from the unrelated individual predictably identified a c.1349T>G [p.(Leu450Trp)] heterozygous variation in COL8A2. Confocal microscopy confirmed attenuated endothelium, and histopathology revealed no guttae, consistent with findings from a previously identified family. CONCLUSIONS: Peripheral, anterior microcystic corneal edema represents a characteristic aspect of the phenotype associated with the p.(Leu450Trp) substitution in COL8A2, in at least 2 of 3 known affected families worldwide. We describe long-term progression and Descemet stripping endothelial keratoplasty for this disease for the first time.

Retroillumination Photography Analysis Enhances Clinical Definition of Severe Fuchs Corneal Dystrophy.

PURPOSE: Retroillumination photography analysis (RPA) provides an objective assessment of the number and distribution of guttae in Fuchs corneal dystrophy. Here, we assess its correlation with clinical grading using slit-lamp biomicroscopy across varying levels of severity. METHODS: Retroillumination photographs were conducted for 95 affected corneas with slit-lamp flash photography after pupillary dilation. Individual guttae were counted manually and the position of individual points recorded. Clinical grading using the Krachmer scale was documented for each eye during examination, and regression analyses were performed to identify the strength of association with number of guttae. We assessed range at each stage of clinical grading and used the Mann-Whitney U test to assess whether clinical grading levels demonstrated successively higher numbers of guttae. RESULTS: Krachmer score ranged from 1 to 5, with mean of 2.6. Mean numbers of guttae at each level of severity were 289 (1+), 999 (2+), 2669 (3+), 5474 (4+), and 7133 (5+). Each stage demonstrated significantly higher numbers of guttae than its preceding level except from 4+ to 5+ (P = 0.30), consistent with the definition of 4+ as the highest level defined by the presence of guttae. Higher levels of clinical grading were associated with larger ranges of guttae (P < 0.01). A linear regression model resulted in a strong fit between RPA and Krachmer score (r = 0.81). CONCLUSIONS: In this largest study of RPA data and comparison with subjective clinical grading of Fuchs dystrophy severity, RPA correlates strongly and demonstrates enhanced definition of severity at advanced stages of disease.

Overview of the Cornea: Structure, Function, and Development.

The cornea is a transparent tissue with significant refractive and barrier functions. The epithelium serves as the principal barrier to fluid and pathogens, a function performed through production of tight junctions, and constant repopulation through differentiation and maturation of dividing cells in its basal cell layer. It is supported posteriorly by basement membrane and Bowman's layer and assists in maintenance of stromal dehydration. The stroma composes the majority of corneal volume, provides support and clarity, and assists in ocular immunity. The posterior cornea, composed of Descemet membrane and endothelium, is essential for stromal dehydration, maintained through tight junctions and endothelial pumps. Corneal development begins with primitive formation of epithelium and lens, followed by waves of migration from cells of neural crest origin between these two structures to produce the stroma and endothelium. Descemet membrane is secreted by the latter and gradually thickens.

Fuchs Corneal Dystrophy.

Fuchs corneal dystrophy (FCD) is a hereditary, progressive disease of the posterior cornea which results in excrescences of Descemet membrane, endothelial cell loss, corneal edema, and, in late stages, bullous keratopathy. Structural changes are noted principally in Descemet membrane and the endothelium, with thickening of Descemet membrane, loss of barrier function, and increased corneal hydration, although secondary effects occur throughout all layers. Multiple chromosomal loci and, more recently, causal genetic mutations have been identified for this complex disorder, including in TCF8, SLC4A11, LOXHD1, and AGBL1. A trinucleotide repeat in TCF4 correlates strongly with disease status and interacts in common pathways with previously identified genes. Dysregulation of pathways involving oxidative stress and apoptosis, epithelial-to-mesenchymal transition, microRNA, mitochondrial genes, and unfolded protein response has been implicated in FCD pathogenesis.

Expansion of CTG18.1 Trinucleotide Repeat in TCF4 Is a Potent Driver of Fuchs' Corneal Dystrophy.

PURPOSE: To analyze the expansion of CTG18.1 allele associated with Fuchs' corneal dystrophy (FCD) in our large cohort of late-onset FCD cases. METHODS: CTG repeats within the CTG18.1 allele were estimated by short tandem repeat (STR) and triplet primed PCR (TP-PCR) assays in our large cohort of 574 late-onset FCD cases and 354 controls and large multigeneration familial cases. The age versus severity relationships were analyzed in FCD genotypes, namely, nonexpanded (N/N), monoallelic expansion (N/X), and biallelic expansion (X/X) with N ≤ 40 CTG monomers. The threshold for causality conferred by an expansion of CTG18.1 was identified by excluding the population of FCD cases who harbored an allele length equivalent to the maximum CTG monomers observed in the controls. RESULTS: The expanded CTG18.1 for (CTG)n>40 showed a strong association (P = 1.56 × 10(-82)) with FCD. Importantly, we delineated the threshold of expansion to 103 CTG repeats above which the allele confers causality in 17.8% of FCD cases. Regression analyses demonstrated a significant correlation between disease severity and age in individuals who harbor either a monoallelic expansion or a biallelic expansion at (CTG) n > 40. These analyses helped predict FCD in two previously unaffected individuals based on their CTG18.1 expansion genotype. CONCLUSIONS: A monoallelic expansion of CTG18.1 contributes to increased disease severity and is causal at (CTG)n>103, whereas a biallelic expansion is sufficient to be causal for FCD at (CTG)n>40. This study highlights the largest contributory causal allele for FCD.

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.