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The advent of cell culture-based methods for the establishment and expansion of human corneal endothelial cells (CEnC) has provided a source of transplantable corneal endothelium, with a significant potential to challenge the one donor-one recipient paradigm. However, concerns over cell identity remain, and a comprehensive characterization of the cultured CEnC across serial passages has not been performed. To this end, we compared two established CEnC culture methods by assessing the transcriptomic changes that occur during in vitro expansion. In confluent monolayers, low mitogenic culture conditions preserved corneal endothelial cell state identity better than culture in high mitogenic conditions. Expansion by continuous passaging induced replicative cell senescence. Transcriptomic analysis of the senescent phenotype identified a cell senescence signature distinct for CEnC. We identified activation of both classic and new cell signaling pathways that may be targeted to prevent senescence, a significant barrier to realizing the potential clinical utility of in vitro expansion.

PurposeTo report potentially pathogenic mutations in the keratin 3 (KRT3) and keratin 12 (KRT12) genes in two individuals with clinically diagnosed Meesmann corneal dystrophy (MECD).MethodsSlit-lamp examination was performed on the probands and available family members to identify characteristic features of MECD. After informed consent was obtained, saliva samples were obtained as a source of genomic DNA, and screening of KRT3 and KRT12 was performed. Potentially pathogenic variants were screened for in 200 control chromosomes. PolyPhen-2, SIFT, and PANTHER were used to predict the functional impact of identified variants. Short tandem repeat genotyping was performed to confirm paternity.ResultsSlit-lamp examination of the first proband demonstrated bilateral, diffusely distributed, clear epithelial microcysts, consistent with MECD. Screening of KRT3 revealed a heterozygous missense variant in exon 1, c.250C>T (p.(Arg84Trp)), which has a minor allele frequency of 0.0076 and was not identified in 200 control chromosomes. In silico analysis with PolyPhen-2 and PANTHER predicted the variant to be damaging to protein function; however, SIFT analysis predicted tolerance of the variant. The second proband demonstrated bilateral, diffusely distributed epithelial opacities that appeared gray-white on direct illumination and translucent on retroillumination. Neither parent demonstrated corneal opacities. Screening of KRT12 revealed a novel heterozygous insertion/deletion variant in exon 6, c.1288_1293delinsAGCCCT (p.(Arg430_Arg431delinsSerPro)). This variant was not present in either of the proband's parents or in 200 control chromosomes and was predicted to be damaging by PolyPhen-2, PANTHER, and SIFT. Haplotype analysis confirmed paternity of the second proband, indicating that the variant arose de novo.ConclusionsWe present a novel KRT12 mutation, representing the first de novo mutation and the first indel in KRT12 associated with MECD. In addition, we report a variant of uncertain significance in KRT3 in an individual with MECD. Although the potential pathogenicity of this variant is unknown, it is the first variant affecting the head domain of K3 to be reported in an individual with MECD and suggests that disease-causing variants associated with MECD may not be restricted to primary sequence alterations of either the helix-initiation or helix-termination motifs of K3 and K12.

Background/Aims To report the clinical, histopathological and genetic features of a variant of lattice corneal dystrophy (LCD) associated with two pathogenic mutations in the transforming growth factor-B-induced (TGFBI) gene. Methods Clinical characterisation was performed by slit lamp examination and in vivo confocal microscopic imaging (IVCM). Histopathological characterisation was performed with light microscopic examination of an excised corneal button and a peripheral blood samples were collected for TGFBI screening. Results A 42-year-old woman presented with progressive photophobia and decreased visual acuity in both eyes. Slit lamp examination demonstrated punctate and linear branching opacities in the mid and posterior corneal stroma, corresponding to hyper-reflective opacities noted on IVCM and amyloid deposition noted on histopathological examination of an excised corneal button. TGFBI screening revealed two previously reported heterozygous missense mutations: c.337G>A (p.(Val113Ile)) in exon 4 and c.1673T>C (p. (Leu558Pro)) in exon 12. Screening of an affected sibling with a similar phenotype revealed that she was also heterozygous for both mutations, while screening of another sibling with punctate but not linear stromal opacities revealed that she was heterozygous for only the p.(Leu558Pro) mutation. Conclusions The p.(Val113Ile) mutation results in an alteration of the atypical LCD phenotype associated with the p.(Leu558Pro) mutation. This represents only the second report of the alteration of the phenotype of a TGFBI dystrophy by a second, non-homozygous pathogenic mutation, and thus provides insight into the phenotype-genotype correlation of the TGFBI dystrophies. [ABSTRACT FROM AUTHOR]

Purpose. To report the identification of the first de novo UBIAD1 missense mutation in an individual with Schnyder corneal dystrophy (SCD). Methods. A slit lamp examination was performed on a 47-year-old woman without a family history of corneal disorders. The proband’s parents, two sisters, and son were also examined and genomic DNA from all six individuals was collected. The exons and exon-intron boundaries of UBIAD1 were screened using Sanger sequencing. Identified mutations were screened for in 200 control chromosomes. In silico analysis predicted the impact of identified mutations on protein function and structure. Results. Slit lamp examination of the proband revealed findings consistent with SCD. Corneas of the family members appeared unaffected. Screening of UBIAD1 in the proband identified a novel heterozygous c.308C>T mutation, predicted to encode the missense amino acid substitution p.(Thr103Ile). This mutation was not identified in any of the family members or in 200 control chromosomes and was predicted to be damaging to normal protein function and structure. Conclusions. We present a novel heterozygous de novo missense mutation in UBIAD1, p.(Thr103Ile), identified in a patient with classic clinical features of SCD. This highlights the value of genetic testing in clinical diagnostic settings, even in the absence of a positive family history. [ABSTRACT FROM AUTHOR]

Background: Hereditary benign intraepithelial dyskeratosis (HBID) is a rare autosomal-dominant disorder of the conjunctiva and oral mucosa first described in and predominantly affecting descendents of Haliwa-Saponi Native Americans. We report a spontaneous case of histopathologically-confirmed HBID affecting an individual not of Native American ancestry. Materials and Methods: Report of a case with histopathologic examination of an excised conjunctival specimen as well as molecular and cytogenetic analysis. Results: A Caucasian boy with a history of oral lesions and conjunctival injection from birth developed bilateral corneal opacities at age 5 and underwent penetrating keratoplasty, with recurrence of the corneal opacification shortly after surgery. Examination of a conjunctival biopsy specimen revealed features consistent with HBID. Copy number variant (CNV) analysis revealed a de novo 4q35 duplication that overlapped the duplication previously associated with HBID, although no genes were identified in the common interval.NLRP1gene sequencing failed to reveal a presumed pathogenic variant. Conclusions: HBID may develop de novo in individuals who are not of Native American ancestry. The absence of coding regions in a duplicated region of 4q35 common to both the individual that we report and previously associated with HBID raises questions regarding the significance of this CNV in the pathogenesis of HBID. [ABSTRACT FROM PUBLISHER]

Background: The genetic basis of X-linked megalocornea (MGC1) was reported in 2012 to be caused by mutations in the CHRDL1 gene. We sought to confirm that mutations in CHRDL1 are associated with MGC1 in a previously unreported pedigree. Materials and Methods: Slit lamp examination, corneal pachymetry, corneal topography and DNA collection for screening of the CHRDL1 gene were performed for members of an affected family. Results: Examination of a woman and her four sons, ranging in age between 3 and 15 years, demonstrated horizontal corneal diameters of 14 mm in three of the four sons and a normal corneal diameter of 12 mm in the mother and other son. Central corneal thickness in the individuals with enlarged corneal diameters averaged 474 microns, compared to 604 microns in their unaffected brother. Corneal topographic imaging demonstrated an average K value of 44.4 D in the affected individuals compared with 41.6 D in their unaffected sibling. Screening of the CHRDL1 gene demonstrated the novel hemizygous frameshift mutation c.167delC (p.(Pro56Leu*8)) in exon 3 in the affected individuals and in the heterozygous state in their mother. This mutation was not present in the unaffected brother or in unrelated controls. Conclusion: We provide the initial confirmation that X-linked megalocornea is associated with mutations in the CHRDL1 gene. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the functional role that the zinc e-box binding homeobox 1 ( ZEB1 ) gene, which underlies the genetic basis of posterior polymorphous corneal dystrophy 3 (PPCD3), plays in corneal endothelial cell proliferation, apoptosis, migration, and barrier function., Methods: A human corneal endothelial cell line (HCEnC-21T) was transfected with siRNA targeting ZEB1 mRNA. Cell proliferation, apoptosis, migration, and barrier assays were performed: Cell proliferation was assessed with cell counting using a hemocytometer; cell apoptosis, induced by either ultraviolet C (UVC) radiation or doxorubicin treatment, was quantified by measuring cleaved caspase 3 (cCASP3) protein levels; and cell migration and barrier function were monitored with electric cell-substrate impedance sensing (ECIS)., Results: ZEB1 knockdown in HCEnC-21T cells transfected with siRNA targeting ZEB1 did not result in a significant difference in cell proliferation when compared with control. Although knockdown of ZEB1 in HCEnC-21T cells sensitized the cells to UV-induced apoptosis, ZEB1 knockdown did not alter the cells' susceptibility to doxorubicin-induced apoptosis, as measured with cCASP3 protein levels, compared with controls. Similarly, no difference was observed in cell migration following ZEB1 knockdown. However, cell barrier function increased significantly following ZEB1 knockdown., Conclusions: The corneal endothelium in PPCD3 is characterized by morphologic, anatomic, and molecular features that are more consistent with an epithelial-like rather than an endothelial-like phenotype. Although these characteristics have been well documented, we demonstrate for the first time that susceptibility to UV-induced apoptosis and cell barrier function are significantly altered in the setting of reduced ZEB1 . The significance of an altered cellular response to apoptotic stimuli and increased cell barrier function in the pathobiology of PPCD remains to be fully elucidated.

Congenital hereditary endothelial dystrophy (CHED), Harboyan syndrome (CHED with progressive sensorineural deafness), and potentially a subset of individuals with late-onset Fuchs' endothelial corneal dystrophy are caused by mutations in the SLC4A11 gene that results in corneal endothelial cell abnormalities. Originally classified as a borate transporter, the function of SLC4A11 as a transport protein remains poorly understood. Elucidating the transport function(s) of SLC4A11 is needed to better understand how its loss results in the aforementioned posterior corneal dystrophic disease processes. Quantitative PCR experiments demonstrated that, of the three known human NH 2 -terminal variants, SLC4A11-C is the major transcript expressed in human corneal endothelium. We studied the expression pattern of the three variants in mammalian HEK-293 cells and demonstrated that the SLC4A11-B and SLC4A11-C variants are plasma membrane proteins, whereas SLC4A11-A is localized intracellularly. SLC4A11-B and SLC4A11-C were shown to be multifunctional ion transporters capable of transporting H + equivalents in both a Na + -independent and Na + -coupled mode. In both transport modes, SLC4A11-C H + flux was significantly greater than SLC4A11-B. In the presence of ammonia, SLC4A11-B and SLC4A11-C generated inward currents that were comparable in magnitude. Chimera SLC4A11-C-NH 2 -terminus-SLC4A11-B experiments demonstrated that the SLC4A11-C NH 2 -terminus functions as an autoactivating domain, enhancing Na + -independent and Na + -coupled H + flux without significantly affecting the electrogenic NH 3 -H (n) + cotransport mode. All three modes of transport were significantly impaired in the presence of the CHED causing p.R109H (SLC4A11-C numbering) mutation. These complex ion transport properties need to be addressed in the context of corneal endothelial disease processes caused by mutations in SLC4A11., (Copyright © 2016 the American Physiological Society.)

Purpose: To report potentially pathogenic mutations in the keratin 3 (KRT3) and keratin 12 (KRT12) genes in two individuals with clinically diagnosed Meesmann corneal dystrophy (MECD)., Methods: Slit-lamp examination was performed on the probands and available family members to identify characteristic features of MECD. After informed consent was obtained, saliva samples were obtained as a source of genomic DNA, and screening of KRT3 and KRT12 was performed. Potentially pathogenic variants were screened for in 200 control chromosomes. PolyPhen-2, SIFT, and PANTHER were used to predict the functional impact of identified variants. Short tandem repeat genotyping was performed to confirm paternity., Results: Slit-lamp examination of the first proband demonstrated bilateral, diffusely distributed, clear epithelial microcysts, consistent with MECD. Screening of KRT3 revealed a heterozygous missense variant in exon 1, c.250C>T (p.(Arg84Trp)), which has a minor allele frequency of 0.0076 and was not identified in 200 control chromosomes. In silico analysis with PolyPhen-2 and PANTHER predicted the variant to be damaging to protein function; however, SIFT analysis predicted tolerance of the variant. The second proband demonstrated bilateral, diffusely distributed epithelial opacities that appeared gray-white on direct illumination and translucent on retroillumination. Neither parent demonstrated corneal opacities. Screening of KRT12 revealed a novel heterozygous insertion/deletion variant in exon 6, c.1288_1293delinsAGCCCT (p.(Arg430_Arg431delinsSerPro)). This variant was not present in either of the proband's parents or in 200 control chromosomes and was predicted to be damaging by PolyPhen-2, PANTHER, and SIFT. Haplotype analysis confirmed paternity of the second proband, indicating that the variant arose de novo., Conclusions: We present a novel KRT12 mutation, representing the first de novo mutation and the first indel in KRT12 associated with MECD. In addition, we report a variant of uncertain significance in KRT3 in an individual with MECD. Although the potential pathogenicity of this variant is unknown, it is the first variant affecting the head domain of K3 to be reported in an individual with MECD and suggests that disease-causing variants associated with MECD may not be restricted to primary sequence alterations of either the helix-initiation or helix-termination motifs of K3 and K12.

Purpose: To report the identification of a novel frameshift mutation and copy number variation (CNV) in PIKFYVE in two probands with fleck corneal dystrophy (FCD)., Methods: Slit-lamp examination was performed to identify characteristic features of FCD. After genomic DNA was collected, PCR amplification and automated sequencing of all 41 exons of PIKFYVE was performed. Using genomic DNA, quantitative PCR (qPCR) was performed to detect CNVs within PIKFYVE., Results: In the first FCD proband, numerous panstromal punctate opacities were observed in each of the proband's corneas, consistent with the diagnosis of FCD. Screening of PIKFYVE demonstrated a novel heterozygous frameshift mutation in exon 19, c.3151dupA, which is predicted to encode for a truncated PIKFYVE protein, p.(Asp1052Argfs*18). This variant was identified in an affected sister but not in the proband's unaffected mother or brother or 200 control chromosomes. The second FCD proband presented with bilateral, discrete, punctate, grayish-white stromal opacities. Exonic screening of PIKFYVE revealed no causative variant. However, CNV analysis demonstrated the hemizygous deletion of exons 15 and 16., Conclusions: We report a novel heterozygous frameshift mutation (c.3151dupA) and a CNV in PIKFYVE, representing the first CNV and the fifth frameshift mutation associated with FCD.