Xinping C. Zhao

Comparison of the relative toxicity of travoprost 0.004% without benzalkonium chloride and latanoprost 0.005% in an immortalized human cornea epithelial cell culture system.

The purpose of this study was to compare the relative toxicity of a new topical ophthalmic glaucoma medication, travoprost 0.004% without benzalkonium chloride (BAK), with that of commercially available latanoprost 0.005% (preserved with 0.02% BAK) in immortalized human corneal epithelial cells (HCEs). Tissue culture plates (96 well) containing HCEs were divided into 6 groups. Two groups served as negative controls (70% methanol and gentamicin). Another 2 groups—1 in corneal epithelial culture media and the other in a hydroxypropyl (HP)-Guar gellable lubricant eyedrop—served as live controls. The travoprost 0.004% without BAK and latanoprost 0.005% groups were exposed to 100 μL of the undiluted solutions. Cells were incubated for 25 min at 37°C. A live/dead assay was used to measure the effects of travoprost without BAK and of latanoprost on HCEs compared to 70% methanol and culture medium. Between the 2 glaucoma medications tested, travoprost 0.004% preserved without BAK showed significantly less toxicity on HCEs than did latanoprost 0.005%. This difference may have ramifications in terms of tolerability for patients who use these topical glaucoma drugs on a long-term basis.

CRB1 gene mutations are associated with keratoconus in patients with leber congenital amaurosis.

PURPOSE To present an association of mutations in the CRB1 gene with keratoconus in patients with Leber congenital amaurosis (LCA). METHODS Sixteen patients with genotyped LCA (having the CRB1, CRX, RetGC, RPE65, and AIPL1 mutations) were recruited from one ophthalmology practice and examined for the presence of keratoconus. Corneal topography, visual acuity, and slit lamp biomicroscopic examination were performed in all cases. RESULTS The mean age of the patients was 34.5 years (range, 13-74). Visual acuities ranged from 20/40 to light perception. Corneal topography was successfully collected in 15 of the cases. Five of the 16 cases had slit lamp and/or topographic features consistent with keratoconus. One patient had a clinical picture that was keratoglobus-like. Of these six cases, four had a CRB1 mutation and two had a CRX mutation. Of the three subjects with the CRX mutation, one had keratoconus, one had the keratoglobus-like presentation, and one was normal. Our cohort represents 14 separate, unrelated families. Only one family comprised multiple members with LCA. These were three affected brothers, one with keratoconus, all with CRB1 mutations. CONCLUSIONS Although the results cannot exclude other gene mutations, they suggest that LCA patients with a CRB1 mutation may have a particular susceptibility to keratoconus.

The Role of crumbs Genes in the Vertebrate Cornea

PURPOSE To evaluate the role of crumbs genes and related epithelial polarity loci in the vertebrate cornea. METHODS The authors used histologic analysis and electron microscopy to evaluate the corneas of zebrafish mutant for a crumbs locus oko meduzy (ome) and in mutants of four other loci, nagie oko (nok), heart and soul (has), mosaic eyes (moe), and ncad (formerly glass onion), that function in the same or related genetic pathways. In parallel, they performed an evaluation of corneas in human carriers of a crumbs gene, CRB1, and mutations using topography and biomicroscopy. The expression of the CRB1 gene in the normal human cornea was examined by polymerase chain reaction (PCR) and immunohistochemical staining. RESULTS The corneas of zebrafish mutants display severe abnormalities of the epithelial and stromal layers. The epithelial cells do not properly adhere to each other, and fluid-filled spaces form between them. In addition, the layering of the corneal stroma is poorly formed or absent. The corneas of human carriers of CRB1 mutations display shape deviations compared with what has been observed in normal individuals. A PCR product of the correct size was obtained from normal human corneal samples. Sequence analyses confirmed its identity to be the human CRB1 gene. Immunohistochemical staining using anti-CRB1 yielded positive brown deposits in the human cornea. CONCLUSIONS crumbs genes play a role in the differentiation of the vertebrate cornea. Corneal defects associated with crumbs gene mutations are very severe in the zebrafish model and, in comparison, appear clinically less pronounced in the human eye.

Transgenic Analysis of the Anterior Eye- Specific Enhancers of the Zebrafish gelsolin- like 1 (gsnl1) Gene

The anterior segment of the eye includes such structures as the cornea, lens, iris, and ciliary body and is essential for many visual and physiological functions of the eye. The zebrafish gelsolin‐like 1 (gsnl1) gene encodes an actin regulatory protein and is expressed in the anterior segment of the eye. We report the transgenic analyses of the gsnl1 promoter and enhancer that are required for expression in the anterior segment of the eye. A 6.4‐kb genomic fragment upstream from the translation initiation site (ATG) was capable of driving green fluorescent protein (GFP) expression in transient transgenic embryos and stable transgenic adult fish, which mimics the endogenous gsnl1 expression. The GFP expression was localized in the corneal epithelium (CE) and the annular ligament (AL) at the iridocorneal angle. A unique enhancer for each of these two tissues was identified at 3.7‐kb upstream from the ATG. The 60‐bp AL and 25‐bp CE enhancers were separated by 100‐bp and functioned independently from each other. Deletion analysis indicated that the proximal promoter was located 1.6‐kb upstream from the ATG. Stable GFP transgenic lines were established for future studies of genetic regulation in the anterior segment of the fish eye. Developmental Dynamics 236:1929–1938, 2007.

Individual Phenotypic Variances in a Family With Thiel–behnke Corneal Dystrophy

Purpose: To diagnose Thiel–Behnke dystrophy, an autosomal dominant disease of the anterior basement membrane/Bowman membrane complex and corneal stroma, currently relies primarily on the overall clinical presentation of patient complaints, inheritance pattern, and physical appearance of the corneal findings on slit-lamp examination. Key challenges to accurately identifying the disease are variable and often obscured morphology caused from secondary scarring, creating phenotypic deviation from the “classical” presentation, and mimicry of characteristics typical of other closely related dystrophies. In this report, we demonstrate the high degree of phenotypic variability that can be found in this disease. Methods: A well-characterized family with an established diagnosis of Thiel–Behnke dystrophy mapped to chromosome 10 was evaluated along with the corresponding pedigree. Each individual was examined under slit lamp, and any apparent lesions were photographed. Results: In total, 4 generations were represented with 20 affected members accounted for, ranging from ages 11 to 86 years. We observed 4 phenotypes in this family: (1) the majority displayed “honeycomb” reticular opacities consistent with Thiel–Behnke dystrophy, (2) several subjects showed more granular-like deposits in a geographic distribution, (3) younger subjects with possible early manifestations of the disease possessed small superficial vesicles, and (4) some eyes exhibited an intermediate form with 2 distinct disease presentations at different regions within the same cornea. Taken together, the pedigree demonstrated a wide continuous spectrum of phenotypes from a supposedly singular genetic disorder that may also vary based on the age of patient. Conclusions: These observations show that the clinical phenotypic appearance alone can result in a variety of different and conflicting diagnoses. With such potential for error, improvement in the diagnostic criteria is necessary.