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.

Early flap displacement after LASIK.

PURPOSE: To evaluate the risks of flap displacement after LASIK. DESIGN: Retrospective case series. PARTICIPANTS: We included 41 845 consecutive adults who underwent LASIK surgery at Optical Express in the United Kingdom, including 81 238 eyes, of which 14 555 were hyperopic and 66 681 myopic or mixed astigmatic. We treated 57 241 eyes with the IntraLase FS-60 femtosecond laser and 23 997 with the Moria S.A. ONE Use-Plus automated microkeratome. METHODS: We calculated the incidence of all flap displacements in the study population during an observational time period of ≥12 months after surgery. Independent variables were entered into logistic regression models to identify risk factors. Postoperative outcomes were assessed. MAIN OUTCOME MEASURES: The incidence and odds ratios (OR) of flap displacement in the study population and in categories of refractive error and flap surgery technique. RESULTS: The incidence of flap displacements was 10 in 81 238 LASIK procedures (0.012%), including 8 hyperopic eyes (0.055%) and 2 myopic eyes (0.003%). All flap displacements occurred within 48 hours of surgery and none were preceded by ocular trauma. They were classified as "early flap displacements" (EFD). The incidence of EFD after microkeratome surgery was 0.033% (n = 8), and after femtosecond laser it was 0.003% (n = 2). In hyperopic eyes having microkeratome surgery, the incidence was 0.179% (n = 7). In a logistic regression model, the strongest predictor of EFD after LASIK was hyperopia, recording an OR of 19.29 (P<0.001). The OR of developing an EFD after microkeratomy was 10.53 times higher than after femtosecond laser (P<0.005). In hyperopes, the OR of an EFD was 18.87 times higher after microkeratomy than after femtosecond treatment. Four of 10 displaced flaps needed secondary surgery, and 1 eye lost 2 lines of best-corrected visual acuity. CONCLUSIONS: The incidence of flap displacements during a 12-month period after LASIK was extremely low (0.012%). Although the small number of displacements with the femtosecond laser limits conclusions, the risk of EFD was higher after microkeratome surgery than femtosecond laser.

Excimer laser surface ablation - a review.

Corneal surface laser ablation procedures for the correction of refractive error have enjoyed a resurgence of interest, especially in patients with a possible increased risk of complications after lamellar surgery. Improvements in the understanding of corneal biomechanical changes, the modulation of wound healing, laser technology including ablation profiles and different methods for epithelial removal have widened the scope for surface ablation. This article discusses photorefractive keratectomy, trans-epithelial photorefractive keratectomy, laser-assisted sub-epithelial keratomileusis and epithelial-laser-assisted in situ keratomileusis.

Documentation of corneal epithelial defects with fluorescein-enhanced digital fundus camera photography.

The advent of digital photography in the ophthalmic setting has provided not only a means of documenting pathology, but with instantaneous results, it is possible to aid clinical diagnosis and management. This study was designed to demonstrate the ability to image corneal epithelial lesions stained with fluorescein, with a digital fundus camera set on fluorescein angiography settings. The contrast of this technique demonstrated both gross and subtle corneal epithelial lesions better than traditional methods. The results obtained demonstrated the high sensitivity and high contrast images this technique can facilitate in every ophthalmic practice equipped with a fundus camera with digital fluorescein angiography capability.

Potential Effects of Corneal Cross-Linking upon the Limbus.

Corneal cross-linking is nowadays the most used strategy for the treatment of keratoconus and recently it has been exploited for an increasing number of different corneal pathologies, from other ectatic disorders to keratitis. The safety of this technique has been widely assessed, but clinical complications still occur. The potential effects of cross-linking treatment upon the limbus are incompletely understood; it is important therefore to investigate the effect of UV exposure upon the limbal niche, particularly as UV is known to be mutagenic to cellular DNA and the limbus is where ocular surface tumors can develop. The risk of early induction of ocular surface cancer is undoubtedly rare and has to date not been published other than in one case after cross-linking. Nevertheless it is important to further assess, understand, and reduce where possible any potential risk. The aim of this review is to summarize all the reported cases of a pathological consequence for the limbal cells, possibly induced by cross-linking UV exposure, the studies done in vitro or ex vivo, the theoretical bases for the risks due to UV exposure, and which aspects of the clinical treatment may produce higher risk, along with what possible mechanisms could be utilized to protect the limbus and the delicate stem cells present within it.

Protein Composition of TGFBI-R124C- and TGFBI-R555W-Associated Aggregates Suggests Multiple Mechanisms Leading to Lattice and Granular Corneal Dystrophy.

PURPOSE: Transforming growth factor beta-induced (TGFBI)-related dystrophies constitute the most common heritable forms of corneal dystrophy worldwide. However, other than the underlying genotypes of these conditions, a limited knowledge exists of the exact pathomechanisms of these disorders. This study expands on our previous research investigating dystrophic stromal aggregates, with the aim of better elucidating the pathomechanism of two conditions arising from the most common TGFBI mutations: granular corneal dystrophy type 1 (GCD1; R555W) and lattice corneal dystrophy type 1 (LCD1; R124C). METHODS: Patient corneas with GCD1 and LCD1 were stained with hematoxylin and eosin and Congo red to visualize stromal nonamyloid and amyloid deposits, respectively. Laser capture microdissection was used to isolate aggregates and extracted protein was analyzed by mass spectrometry. Proteins were identified and their approximate abundances were determined. Spectra of TGFBIp peptides were also recorded and quantified. RESULTS: In total, three proteins were found within GCD1 aggregates that were absent in the healthy control corneal tissue. In comparison, an additional 18 and 24 proteins within stromal LCD1 and Bowman's LCD1 deposits, respectively, were identified. Variances surrounding the endogenous cleavage sites of TGFBIp were also noted. An increase in the number of residues experiencing cleavage was observed in both GCD1 aggregates and LCD1 deposits. CONCLUSIONS: The study reveals previously unknown differences between the protein composition of GCD1 and LCD1 aggregates, and confirms the presence of the HtrA1 protease in LCD1-amyloid aggregates. In addition, we find mutation-specific differences in the processing of mutant TGFBIp species, which may contribute to the variable phenotypes noted in TGFBI-related dystrophies.

Development of allele-specific gene-silencing siRNAs for TGFBI Arg124Cys in lattice corneal dystrophy type I.

PURPOSE: This study aimed to investigate the potency and specificity of short-interfering RNA (siRNA) treatment for TGFBI-Arg124Cys lattice corneal dystrophy type I (LCDI) using exogenous expression constructs in model systems and endogenous gene targeting in an ex vivo model using corneal epithelial cell cultures. METHODS: A panel of 19 TGFBI-Arg124Cys-specific siRNAs were assessed by a dual-luciferase reporter assay. Further assessment using pyrosequencing and qPCR was used to identify the lead siRNA; suppression of mutant TGFBIp expression was confirmed by Western blot and Congo red aggregation assays. An ex vivo model of LCDI was established using limbal biopsies from corneal dystrophy patients harboring the Arg124Cys mutation. Treatment efficiency of the siRNA was assessed for the inhibition of the mutant allele in the primary patient's corneal epithelial cells using pyrosequencing, quantitative PCR (qPCR), and an ELISA. RESULTS: A lead siRNA was identified, and demonstrated to be potent and specific in inhibiting the TGFBI-Arg124Cys mutant allele at the mRNA and protein levels. Besides high allele specificity, siRNA treatment achieved a 44% reduction of the endogenous Arg124Cys allele in an ex vivo model of LCDI. CONCLUSIONS: We have identified a lead siRNA specific to the TGFBI-Arg124Cys mutant allele associated with LCDI. Silencing of exogenous TGFBI was observed at mRNA and protein levels, and in an ex vivo model of LCDI with an efficient suppression of the endogenous mutant allele. This result indicates the potential of siRNA treatment as a personalized medicine approach for the management of heritable TGFBI-associated corneal dystrophies.

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.