Coexistence of Meesmann Corneal Dystrophy and a Pseudo-Unilateral Lattice Corneal Dystrophy in a Patient With a Novel Pathogenic Variant in the Keratin K3 Gene: A Case Report.

PURPOSE: This study aims to clinically and genetically report a case of coexisting Meesmann corneal dystrophy (MECD) and pseudo-unilateral lattice corneal dystrophy (LCD). METHODS: Clinical characterization was supported by a complete ophthalmological evaluation, including visual acuity measurement and slit-lamp examination. Molecular diagnosis was performed by whole-exome sequencing analyzing the gelsolin, keratin K3 (KRT3), keratin K12, and transforming growth factor-beta-induced genes. RESULTS: A 57-year-old woman presented with recurrent corneal erosions over 17 years and visual impairment in both eyes. Ophthalmological evaluation revealed multiple central tiny cysts in the epithelium of both eyes and lattice linear lesions only in the right cornea. In both eyes, a corneal posterior crocodile shagreen degeneration could also be observed. These findings were compatible with a MECD and a unilateral LCD. Molecular analysis identified the novel heterozygous nucleotide substitution c.1492G>A (amino acid change p.Glu498Lys) in the KRT3 gene, in cosegregation with the MECD familial phenotype. However, no genetic evidence supported the unique LCD phenotype observed in the patient. CONCLUSIONS: To the best of our knowledge, this is the first report of a pseudo-unilateral LCD in a patient with coexistent MECD. Moreover, the genetic analysis showed a novel mutation in the previously MECD-associated gene KRT3.

Xenofree generation of limbal stem cells for ocular surface advanced cell therapy.

BACKGROUND: Limbal stem cells (LSC) sustain the corneal integrity and homeostasis. LSC deficiency (LSCD) leads to loss of corneal transparency and blindness. A clinical approach to treat unilateral LSCD comprises autologous cultured limbal epithelial stem cell transplantation (CLET). CLET uses xenobiotic culture systems with potential zoonotic transmission risks, and regulatory guidelines make necessary to find xenofree alternatives. METHODS: We compared two xenofree clinical grade media and two feeder layers. We used CnT07, a defined commercial medium for keratinocytes, and a modified xenofree supplemented hormonal epithelial medium with human serum (XSHEM). Optimal formulation was used to compare two feeder layers: the gold standard 3T3 murine fibroblasts and human processed lipoaspirate cells (PLA). We tested the expressions of ΔNp63α and cytokeratin 3 and 12 by qPCR and immunofluorescence. Morphology, viability, clonogenicity, proliferation, and cell growth assays were carried out. We also evaluated interleukin 6 (IL-6) and stromal-derived factor 1 (SDF-1) by qPCR and ELISA. RESULTS: XSHEM maintained better LSC culture viability and morphology than CnT07. Irradiated PLA feeder cells improved the undifferentiated state of LSC and enhanced their growth and clonogenicity stimulating IL-6 secretion and SDF-1 expression, as well as increased proliferation and cell growth when compared with irradiated 3T3 feeder cells. CONCLUSIONS: The combination of XSHEM and PLA feeder cells efficiently sustained LSC xenofree cultures for clinical application. Moreover, PLA feeder layers were able to improve the LSC potential characteristics. Our results would have direct clinical application in CLET for advanced therapy.

In-utero epigenetic factors are associated with early-onset myopia in young children.

OBJECTIVES: To assess whether epigenetic mechanisms affecting gene expression may be involved in the pathogenesis of early-onset myopia, we performed genome-wide DNA methylation analyses of umbilical cord tissues, and assessed any associations between CpG site-specific methylation and the development of the disorder when the children were 3 years old. METHODS: Genome-wide DNA methylation profiling of umbilical cord samples from 519 Singaporean infants involved in a prospective birth cohort 'Growing Up in Singapore Towards healthy Outcomes' (GUSTO) was performed using the Illumina Infinium HumanMethylation450K chip microarray. Multivariable logistic regression models were used to assess any associations between site-specific CpG methylation of umbilical cord tissue at birth and myopia risk in 3 year old children, adjusting for potential confounders. Gene expression of genes located near CpG sites that demonstrated statistically significant associations were measured in relevant ocular tissues using human and mouse fetal and adult eye samples. RESULTS: We identified statistically significant associations between DNA methylation levels at five CpG sites and early-onset myopia risk after correcting for multiple comparisons using a false discovery rate of 5%. Two statistically significant CpG sites were identified in intergenic regions: 8p23(p = 1.70×10-7) and 12q23.2(p = 2.53×10-7). The remaining 3 statistically significant CpG sites were identified within the following genes: FGB (4q28, p = 3.60×10-7), PQLC1 (18q23, p = 8.9×10-7) and KRT12 (17q21.2, p = 1.2×10-6). Both PQLC1 and KRT12 were found to be significantly expressed in fetal and adult cornea and sclera tissues in both human and mouse. CONCLUSIONS: We identified five CpG methylation sites that demonstrate a statistically significant association with increased risk of developing early-onset myopia. These findings suggest that variability in the neonatal cord epigenome may influence early-onset myopia risk in children. Further studies of the epigenetic influences on myopia risk in larger study populations, and the associations with adulthood myopia risk are warranted.

Aberrant expression of a stabilized ß-catenin mutant in keratocytes inhibits mouse corneal epithelial stratification.

We previously reported that genetic deletion of ß-catenin in mouse corneal keratocytes resulted in precocious corneal epithelial stratification. In this study, to strengthen the notion that corneal keratocyte-derived Wnt/ß-catenin signaling regulates corneal epithelial stratification during mouse development, we examined the consequence of conditional overexpression of a stabilized ß-catenin mutant (Ctnnb1ΔE3) in corneal keratocytes via a doxycycline (Dox)-inducible compound transgenic mouse strain. Histological analysis showed that conditional overexpression of Ctnnb1ΔE3 in keratocytes inhibited corneal epithelial stratification during postnatal development. Unlike the corneal epithelium of the littermate controls, which consisted of 5-6 cell layers at postnatal day 21 (P21), the mutant corneal epithelium contained 1-2 or 2-3 cell layers after Dox induction from embryonic day 0 (E0) to P21 and from E9 to P21, respectively. X-gal staining revealed that Wnt/ß-catenin signaling activity was significantly elevated in the corneal keratocytes of the Dox-induced mutant mice, compared to the littermate controls. Furthermore, RT-qPCR and immunostaining data indicated that the expression of Bmp4 and ΔNp63 was downregulated in the mutant corneas, which was associated with reduced corneal epithelial proliferation in mutant epithelium, as revealed by immunofluorescent staining. However, the expression of Krt12, Krt14 and Pax6 in the mutant corneas was not altered after overexpression of Ctnnb1ΔE3 mutant protein in corneal keratocytes. Overall, mutant ß-catenin accumulation in the corneal keratocytes inhibited corneal epithelial stratification probably through downregulation of Bmp4 and ΔNp63 in the corneal epithelium.

In vivo histology and p.L132V mutation in KRT12 gene in Japanese patients with Meesmann corneal dystrophy.

PURPOSE: To report genetic mutational analysis and in vivo histology of Meesmann corneal dystrophy. STUDY DESIGN: Prospective, case control study. METHODS: Six patients from three independent families with clinically diagnosed Meesmann corneal dystrophy were enrolled in this study. Slit-lamp biomicroscopy with fluorescein vital staining, anterior segment optical coherence tomography (AS-OCT), and in vivo laser confocal microscopy (IVCM) were performed on selected patients. Mutational screening for the keratin genes KRT3 and KRT12 was performed in all six patients and selected unaffected family members. RESULTS: Slit-lamp biomicroscopy revealed numerous intraepithelial microcysts in all affected individuals. AS-OCT revealed hyperreflectivity and high corneal epithelial layer thickness (mean, 64.8µm) in all individuals tested (3/3). By using IVCM, multiple epithelial microcysts and hyperreflective materials (6/6), subepithelial nerve abnormalities (6/6), tiny punctate hyperreflective material (6/6), and needle-like hyperreflective materials (4/6) were observed in the corneal stromal layer. A heterozygous genetic mutation in the KRT12 gene (c.394 C>G, p.L132V) was identified in all six patients. No pathological mutation was observed in the KRT3 gene. CONCLUSION: We identified a heterozygous genetic mutation (c.394 C>G, p.L132V) in the KRT12 gene in six Japanese patients with inherited Meesmann corneal dystrophy. This is the first study to confirm this genetic mutation in Japanese Meesmann corneal dystrophy patients. This mutation has been independently reported in an American Meesmann corneal dystrophy patient, confirming its pathogenicity. AS-OCT and IVCM proved to be useful tools for observing corneal epithelial layer pathology in this dystrophy. Furthermore, IVCM reveals corneal stromal layer pathological changes not previously reported in this dystrophy.

Human aniridia limbal epithelial cells lack expression of keratins K3 and K12.

Aniridia is a rare disease of the eye that affects the iris, lens and the cornea. In about 90% of the cases, patients showed a loss of PAX6 function. Patients with aniridia often develop aniridia-related keratopathy (ARK), due to limbal stem cell insufficiency. The aim of this study was to determine the differentiation status of limbal epithelial cells (LECs) in patients with ARK. Epithelial cells were isolated from the limbus region of two patients with aniridia and cultured in KSFM medium supplemented with EGF and BPE. Normal cells were obtained from limbus region of cadaveric control patients. Cells were analyzed with RT-PCR, qPCR and Western blot to evaluate expression of the developmental transcription factor, PAX6, potential stem cell markers, ΔNp63α and ABCG2, and corneal differentiation markers, keratin 12 (K12) and K3. Conjunctival differentiation markers, keratin 13 (K13) and K19 were also investigated. Cells were immunostained to evaluate K3, PAX6, and p63α protein expression. Protein coding sequence of PAX6 from patient LEC-cDNA was cloned and sequenced. RT-PCR showed that K3 and K12 transcripts were absent from patient cells, but present in healthy control preparations. Transcription levels of PAX6, ABCG2, and p63α of aniridia patients show no differences compared to normal control cells. Western blot showed reduced PAX6, protein levels in aniridia-LECs compared to control-LECs. Immunostaining also showed reduced PAX6 and K3 expression in aniridia-LECs compared to control-LECs. One aniridia patient showed a loss of stop codon in half of the cloned transcripts. In the second aniridia patient mRNA degradation through nonsense mediated decay seems to be very likely since we could not identify the mutation c.174C > T (Refseq. NM_000280), or misspliced transcripts in cDNA. We identified decreased PAX6 protein levels in aniridia patients in addition to decreased K12 mRNA levels compared to control cells. This result indicates an altered differentiation of limbal epithelial cells of aniridia patients. Further studies are necessary to evaluate the mechanism of differentiation of limbal epithelial cells in aniridia.

Mutation analysis of TGFBI and KRT12 in a case of concomitant keratoconus and granular corneal dystrophy.

PURPOSE: This study is to summarize the concurrent keratoconus (KC) and granular corneal dystrophy (GCD) phenotype and identify the underlying genetic cause in a 23-year-old male patient. METHODS: A detailed family history and clinical data from the patient and his parents were collected by ophthalmologic examination. The candidate genes were captured and sequenced by targeted next-generation sequencing, and the results were confirmed by Sanger sequencing. RESULTS: The proband was clinically diagnosed as a case of concurrent KC and GCD, which is a very rare presentation. His father and grandmother were diagnosed as GCD in both eyes. There was no character of KC in his father's and grandmother's eyes. A heterozygous TGFBI mutation in exon 4 (c.370G > A) was identified in the proband, which was predicted to generate a missense mutation (p.R124H). The mutation also existed in his father and grandmother. A heterozygous KRT12 mutation in exon 8 (c.1456-1457ins GTA) was identified in the proband, which was predicted to generate an insert mutation and created a premature termination codon. The mutation did not exist in his father and grandmother. The two mutations did not exist in his mother and 200 unrelated normal controls. CONCLUSIONS: KC can co-exist with GCD. The missense mutation (c.370G > A) in the TGFBI gene and insert mutation (c.1456-1457ins GAT) in the KRT12 gene were identified in a 23-year-old male patient with concurrent KC and GCD.

A hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo expansion of human corneal epithelial stem cells.

PurposeTo develop a hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo cultivation of human corneal epithelial stem cells (CESCs).Patients and MethodsCESCs were cultivated from donor limbal explants on the HyStem-C Hydrogel bio-scaffold in 12-well plates for 3 weeks. Group A used the traditional supplemented hormonal epidermal medium (SHEM) and group B used the defined SHEM (without fetal bovine serum and toxin A, adding 20% serum replacement). The growth and morphology of the cultured cells were assessed by phase contrast microscope. The expressions of specific cell markers were assessed by immunofluorescence staining and quantitative real-time PCR (qRT-PCR).ResultsSuccessful cultures of CESCs were obtained in both groups, resulting in multilayered stratified epithelia. Comparing to group A, the cells in group B was grown slightly slower and formed less cellular layers at the end of culture. The corneal specific cytokeratin (K) 12 and differentiation markers, involucrin, and connexin 43, were mainly expressed in the superficial cellular layers in both groups. Interestingly, certain basal cells were immune-positive to proposed stem cell markers such as K19, ABCG2, and integrin ß1 in both groups. There was no significant difference between the two groups with regard to the gene expression levels of all these selected corneal markers (all P>0.05).ConclusionsThe hyaluronan hydrogel scaffold-based xeno-free culture system may support the expansion of regenerative CESCs without the risk of xeno component contamination. The regenerated epithelium maintains similar characteristics of native corneal epithelium.

PAX6 Isoforms, along with Reprogramming Factors, Differentially Regulate the Induction of Cornea-specific Genes.

PAX6 is the key transcription factor involved in eye development in humans, but the differential functions of the two PAX6 isoforms, isoform-a and isoform-b, are largely unknown. To reveal their function in the corneal epithelium, PAX6 isoforms, along with reprogramming factors, were transduced into human non-ocular epithelial cells. Herein, we show that the two PAX6 isoforms differentially and cooperatively regulate the expression of genes specific to the structure and functions of the corneal epithelium, particularly keratin 3 (KRT3) and keratin 12 (KRT12). PAX6 isoform-a induced KRT3 expression by targeting its upstream region. KLF4 enhanced this induction. A combination of PAX6 isoform-b, KLF4, and OCT4 induced KRT12 expression. These new findings will contribute to furthering the understanding of the molecular basis of the corneal epithelium specific phenotype.

Keratin 12 missense mutation induces the unfolded protein response and apoptosis in Meesmann epithelial corneal dystrophy.

Meesmann epithelial corneal dystrophy (MECD) is a rare autosomal dominant disorder caused by dominant-negative mutations within the KRT3 or KRT12 genes, which encode the cytoskeletal protein keratins K3 and K12, respectively. To investigate the pathomechanism of this disease, we generated and phenotypically characterized a novel knock-in humanized mouse model carrying the severe, MECD-associated, K12-Leu132Pro mutation. Although no overt changes in corneal opacity were detected by slit-lamp examination, the corneas of homozygous mutant mice exhibited histological and ultrastructural epithelial cell fragility phenotypes. An altered keratin expression profile was observed in the cornea of mutant mice, confirmed by western blot, RNA-seq and quantitative real-time polymerase chain reaction. Mass spectrometry (MS) and immunohistochemistry demonstrated a similarly altered keratin profile in corneal tissue from a K12-Leu132Pro MECD patient. The K12-Leu132Pro mutation results in cytoplasmic keratin aggregates. RNA-seq analysis revealed increased chaperone gene expression, and apoptotic unfolded protein response (UPR) markers, CHOP and Caspase 12, were also increased in the MECD mice. Corneal epithelial cell apoptosis was increased 17-fold in the mutant cornea, compared with the wild-type (P < 0.001). This elevation of UPR marker expression was also observed in the human MECD cornea. This is the first reporting of a mouse model for MECD that recapitulates the human disease and is a valuable resource in understanding the pathomechanism of the disease. Although the most severe phenotype is observed in the homozygous mice, this model will still provide a test-bed for therapies not only for corneal dystrophies but also for other keratinopathies caused by similar mutations.

CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting.

CRISPR/Cas9-based therapeutics hold the possibility for permanent treatment of genetic disease. The potency and specificity of this system has been used to target dominantly inherited conditions caused by heterozygous missense mutations through inclusion of the mutated base in the short-guide RNA (sgRNA) sequence. This research evaluates a novel approach for targeting heterozygous single-nucleotide polymorphisms (SNPs) using CRISPR/Cas9. We determined that a mutation within KRT12, which causes Meesmann's epithelial corneal dystrophy (MECD), leads to the occurrence of a novel protospacer adjacent motif (PAM). We designed an sgRNA complementary to the sequence adjacent to this SNP-derived PAM and evaluated its potency and allele specificity both in vitro and in vivo. This sgRNA was found to be highly effective at reducing the expression of mutant KRT12 mRNA and protein in vitro. To assess its activity in vivo we injected a combined Cas9/sgRNA expression construct into the corneal stroma of a humanized MECD mouse model. Sequence analysis of corneal genomic DNA revealed non-homologous end-joining repair resulting in frame-shifting deletions within the mutant KRT12 allele. This study is the first to demonstrate in vivo gene editing of a heterozygous disease-causing SNP that results in a novel PAM, further highlighting the potential for CRISPR/Cas9-based therapeutics.

Cellular Response of Limbal Stem Cells on Polycaprolactone Nanofibrous Scaffolds for Ocular Epithelial Regeneration.

PURPOSE: The aim of this study was to develop nanofibrous polycaprolactone (PCL) substrate for limbal stem cell (LSC) expansion that can serve as a potential alternative substrate to replace human amniotic membrane (AM). MATERIALS AND METHODS: The human limbus stem cell was used to evaluate the biocompatibility of substrates (nanofibrous scaffold and, human AM) based on their phenotypic profile, viability, proliferation and attachment ability. RESULTS: Biocompatibility results indicated that the all substrates were highly biocompatible, as LSCs could favorably attach and proliferate on the nanofibrous surface. Microscopic figures showed that the human LSCs were firmly anchored to the substrates and were able to retain a normal corneal stem cell phenotype. Microscopic analyses illustrated that cells infiltrated the nanofibers and successfully formed a three-dimensional corneal epithelium, which was viable for two weeks. Immunocytochemistry (ICC) and real time-PCR results revealed no change in the expression profile of LECs grown on nanofibrous substrate when compared to those grown on human AM. CONCLUSION: In addition, electrospun nanofibrous PCL substrate provides not only a milieu supporting LSCs expansion, but also serve as a useful alternative carrier for ocular surface tissue engineering and could be used as an alternative substrate to AM.

Identification of presumed pathogenic KRT3 and KRT12 gene mutations associated with Meesmann corneal dystrophy.

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.

siRNA silencing of the mutant keratin 12 allele in corneal limbal epithelial cells grown from patients with Meesmann's epithelial corneal dystrophy.

PURPOSE: The aim of this study is to further assess our previously reported keratin 12 (K12)-Leu132Pro specific siRNA in silencing the mutant allele in Meesmann's Epithelial Corneal Dystrophy (MECD) in experimental systems more akin to the in vivo situation through simultaneous expression of both wild-type and mutant alleles. METHODS: Using KRT12 exogenous expression constructs transfected into cells, mutant allele specific knockdown was quantified using pyrosequencing and infrared Western blot analysis, while the silencing mechanism was assessed by a modified rapid amplification of cDNA ends (5'RACE) method. Corneal limbal biopsies taken from patients suffering from MECD were used to establish cultures of MECD corneal limbal epithelial stem cells and the ability of the siRNA to silence the endogenous mutant KRT12 allele was assessed by a combination of pyrosequencing, qPCR, ELISA, and quantitative-fluorescent immunohistochemistry (Q-FIHC). RESULTS: The siRNA displayed a potent and specific knockdown of K12-Leu132Pro at both the mRNA and protein levels with exogenous expression constructs. Analysis by the 5'RACE method confirmed siRNA-mediated cleavage. In the MECD cells, an allele-specific knockdown of 63% of the endogenous mutant allele was observed without effect on wild-type allele expression. CONCLUSIONS: Combined with an effective delivery vehicle this siRNA approach represents a viable treatment option for prevention of the MECD pathology observed in K12-Leu132Pro heterozygous individuals.

KRT12 mutations and in vivo confocal microscopy in two Japanese families with Meesmann corneal dystrophy.

PURPOSE: To identify genetic mutations and study the corneal epithelium in Japanese patients with Meesmann corneal dystrophy. DESIGN: Laboratory investigation and prospective observational case series. METHODS: Slit-lamp biomicroscopy with fluorescein vital staining and in vivo confocal microscopy were performed. Mutation screening of the KRT3 and KRT12 genes was performed via polymerase chain reaction and direct sequencing for 5 patients in 2 families. RESULTS: Slit-lamp biomicroscopy revealed multiple corneal intraepithelial microcysts in all patients. A clear zone was seen in the younger generation, whereas mild subepithelial opacity was seen in the older generation. In the in vivo confocal microscopy, numerous corneal intraepithelial microcysts and hyperreflective materials, which were believed to be degenerative cells, were detected closer to the basal layer of the corneal epithelium in older patients. The superficial layer contained more enlarged microcysts, and the hyperreflective materials showed atrophic changes, as compared to the basal layer. The demarcation line between the microcysts and normal epithelial cells was clearly visualized by in vivo confocal microscopy and corresponded to the demarcation line of the clear zone observed by the slit-lamp examination. Two heterozygous mutations (Q130P, L140Q) in the KRT12 gene, one of which (L140Q) was novel, were identified only in the affected patients of the families. CONCLUSIONS: We identified a novel missense mutation of the KRT12 gene in Meesmann corneal dystrophy. The in vivo confocal microscopy examinations revealed previously unreported depth-dependent ultrastructural changes in the living cornea of Meesmann corneal dystrophy patients.

Autosomal-dominant Meesmann epithelial corneal dystrophy without an exon mutation in the keratin-3 or keratin-12 gene in a Chinese family.

Meesmann epithelial corneal dystrophy (MECD) is a dominantly inherited disorder, characterized by fragility of the anterior corneal epithelium and formation of intraepithelial microcysts. It has been described in a number of different ancestral groups. To date, all reported cases of MECD have been associated with either a single mutation in one exon of the keratin-3 gene (KRT3) or a single mutation in one of two exons of the keratin-12 gene (KRT12). Each mutation leads to a predicted amino acid change in the respective keratin-3 or keratin-12 proteins that combine to form the corneal-specific heterodimeric intermediate filament protein. This case report describes a four-generation Chinese kindred with typical autosomal-dominant MECD. Exon sequencing of KRT3 and KRT12 in six affected and eight unaffected individuals (including two spouses) did not detect any mutations or nucleotide sequence variants. This kindred demonstrates that single mis-sense mutations may be sufficient but are not required in all individuals with the MECD phenotype. It provides a unique opportunity to investigate further genomic and functional heterogeneity in MECD.

Allele-specific siRNA silencing for the common keratin 12 founder mutation in Meesmann epithelial corneal dystrophy.

PURPOSE: To identify an allele-specific short interfering RNA (siRNA), against the common KRT12 mutation Arg135Thr in Meesmann epithelial corneal dystrophy (MECD) as a personalized approach to treatment. METHODS: siRNAs against the K12 Arg135Thr mutation were evaluated using a dual luciferase reporter gene assay and the most potent and specific siRNAs were further screened by Western blot. Off-target effects on related keratins were assessed and immunological stimulation of TLR3 was evaluated by RT-PCR. A modified 5' rapid amplification of cDNA ends method was used to confirm siRNA-mediated mutant knockdown. Allele discrimination was confirmed by quantitative infrared immunoblotting. RESULTS: The lead siRNA, with an IC(50) of thirty picomolar, showed no keratin off-target effects or activation of TLR3 in the concentration ranges tested. We confirmed siRNA-mediated knockdown by the presence of K12 mRNA fragments cleaved at the predicted site. A dual tag infrared immunoblot showed knockdown to be allele-specific, with 70% to 80% silencing of the mutant protein. CONCLUSIONS: A potent allele-specific siRNA against the K12 Arg135Thr mutation was identified. In combination with efficient eyedrop formulation delivery, this would represent a personalized medicine approach, aimed at preventing the pathology associated with MECD and other ocular surface pathologies with dominant-negative or gain-of-function pathomechanisms.

Severe Meesmann's epithelial corneal dystrophy phenotype due to a missense mutation in the helix-initiation motif of keratin 12.

PURPOSE: To describe a severe phenotype of Meesmann's epithelial corneal dystrophy (MECD) and to determine the underlying molecular cause. METHODS: We identified a 30-member family affected by MECD and examined 11 of the 14 affected individuals. Excised corneal tissue from one affected individual was examined histologically. We used PCR and direct sequencing to identify mutation of the coding regions of the KRT3 and KRT12 genes. RESULTS: Cases had an unusually severe phenotype with large numbers of intraepithelial cysts present from infancy and they developed subepithelial fibrosis in the second to third decade. In some individuals, the cornea became superficially vascularized, a change accompanied by the loss of clinically obvious epithelial cysts. Visual loss from amblyopia or corneal opacity was common and four individuals were visually impaired (≤6/24 bilaterally) and one was blind (<6/60 bilaterally). In all affected family members, there was a heterozygous missense mutation c. 395T>C (p. L132P) in exon 1 of the KRT12 gene, which codes for the helix-initiation motif of the K12 polypeptide. This sequence change was not found in unaffected family members or in 100 unaffected controls. CONCLUSIONS: The Leu132Pro missense mutation is within the helix-initiation motif of the keratin and is predicted to result in a significant structural change of the K12 protein. The clinical effects are markedly more severe than the phenotype usually associated with the Arg135Thr mutation within this motif, most frequently seen in European patients with MECD.

Ex vivo expanded autologous limbal epithelial cells on amniotic membrane using a culture medium with human serum as single supplement.

In patients with limbal stem cell deficiency (LSCD), transplantation of ex vivo expanded human limbal epithelial cells (HLECs) can restore the structural and functional integrity of the corneal surface. However, the protocol for cultivation and transplantation of HLECs differ significantly, and in most protocols growth additives such as cholera toxins, exogenous growth factors, hormones and fetal calf serum are used. In the present article, we compare for the first time human limbal epithelial cells (HLECs) cultivated on human amniotic membrane (HAM) in a complex medium (COM) including fetal bovine serum to a medium with human serum as single growth supplement (HSM), and report on our first examinations of HLECs expanded in autologous HSM and used for transplant procedures in patients with LSCD. Expanded HLECs were examined by genome-wide microarray, RT-PCR, Western blotting, and for cell viability, morphology, expression of immunohistochemical markers and colony forming efficiency. Cultivation of HLECs in HSM produced a multilayered epithelium where cells with markers associated with LESCs were detected in the basal layers. There were few transcriptional differences and comparable cell viability between cells cultivated in HSM and COM. The p63 gene associated with LESCs were expressed 3.5 fold more in HSM compared to COM, and Western blotting confirmed a stronger p63α band in HSM cultures. The cornea-specific keratin CK12 was equally found in both culture conditions, while there were significantly more CK3 positive cells in HSM. Cells in epithelial sheets on HAM remaining after transplant surgery of patients with LSCD expressed central epithelial characteristics, and dissociated cells cultured at low density on growth-arrested fibroblasts produced clones containing 21 ± 12% cells positive for p63α (n = 3). In conclusion, a culture medium without growth additives derived from animals or from animal cell cultures and with human serum as single growth supplement may serve as an equivalent replacement for the commonly used complex medium for ex vivo expansion of HLECs on HAM.

Keratin 13 is a more specific marker of conjunctival epithelium than keratin 19.

PURPOSE: To evaluate the expression patterns of cytokeratin (K) 12, 13, and 19 in normal epithelium of the human ocular surface to determine whether K13 could be used as a marker for conjunctival epithelium. METHODS: Total RNA was isolated from the human conjunctiva and central cornea. Those transcripts that had threefolds or higher expression levels in the conjunctiva than the cornea were identified using microarray technique. Expression levels of three known signature genes and of two conjunctival genes, K13 and K19 were confirmed by using quantitative real-time PCR (qRT-PCR). Protein expression of K12, K13, and K19 was confirmed by immunostaining with specific antibodies on histologic sections of human sclerocornea that contained the conjunctiva, limbus, and cornea and on impression cytology (IC) specimens of the cornea and conjunctiva from normal donors. Double staining of K13/K12 and K19/K12 on histologic sections and IC specimens was performed. RESULTS: There were 337 transcripts that were preferentially expressed in the conjunctiva. K13 and K19 were among the top twenty transcripts in the conjunctiva and this preferential expression pattern of K13 and K19 was confirmed by qRT-PCR. Immunohistochemical studies showed that K13 was expressed at the posterior limbal epithelium and conjunctival epithelium but was totally absent in the cornea. K12 was expressed in the corneal and anterior limbal epithelia except for the basal layer and was absent from the conjunctiva. In contrast, K19 was detected in the corneal, limbal and conjunctival epithelia. Immunostaining of the IC specimens showed K12(+) epithelial cells in the corneal region, K13(+) cells in the conjunctival area, and K19(+) cells in the corneal and conjunctival specimens. Expression of K13 and K12 on the ocular surface was mutually exclusive on both the histologic and IC samples using double immunostaining. CONCLUSIONS: K13 is more specific to the conjunctival epithelial cells than K19 and potentially could be used as a marker to identify conjunctival epithelial cells in limbal stem cell deficiency.