Use of Atropine for Prevention of Childhood Myopia Progression in Clinical Practice.

OBJECTIVE: The most effective strategy to reduce myopia-related complications is to prevent myopia progression during childhood. This review article examines the latest published evidence on the use of atropine in childhood myopia control and discusses practical aspects of applying the findings to clinical practice. Future directions including possible forms of combination therapy are examined. METHODS: A literature search with a focus on randomized controlled trials (RCTs) and meta-analyses on the subject was conducted. Observational studies with control groups were also reviewed to discuss issues regarding feasibility of using atropine for myopia control in clinical practice. RESULTS: Five RCTs and 2 meta-analyses were found. The studies all found beneficial effects of atropine in myopia control, as well as a clear but perhaps clinically insignificant dose-response relationship between atropine and myopia progression rates. Available evidence however is focused on predominantly Chinese populations, and there is a current lack of guidance on timing of therapy initiation, duration of therapy, and treatment cessation. For future directions, combining atropine with other forms of myopia control would be worth considering. CONCLUSIONS: Atropine is robust option for childhood myopia control. Further evidence including RCTs in different populations as well as the upcoming 5-year atropine for the treatment of myopia 2 trial results will provide much needed answers for wider acceptance of its use.

A rabbit model of age-dependant ocular hypertensive response to topical corticosteroids.

OBJECTIVE: To investigate the ocular hypertensive response to topical dexamethasone (DEX), rimexolone (RIM), loteprednol etabonate (LOT) and fluorometholone (FML) in rabbits of different ages. METHODS: Seventy-five rabbits of three age groups (7 weeks, 6 months and 1-year old) received topical administration of 0.1% DEX, 1% RIM, 0.5% LOT, 0.1% FML or balanced salt solution four times daily for 1 month. Intraocular pressure (IOP) was monitored at regular time intervals. After a month, eyes were harvested for histological study with haematoxylin and eosin (H&E), periodic acid Schiff and Masson trichrome staining. Trabecular meshwork changes were graded by masked ocular pathologists. RESULTS: Topical DEX caused the greatest increase in IOP, followed by RIM and FML. LOT caused the least IOP increase. Similar pattern of IOP response to the four corticosteroids was observed in the three studied age groups. Young rabbits (7 week) were the most responsive to corticosteroids among the age groups. Extracellular matrix thickening in the trabecular meshwork region and loss of trabecular meshwork cells were observed after DEX, FML or RIM treatments. CONCLUSION: Young rabbits are more susceptible to steroid induced increase in IOP, even for milder steroids such as fluorometholone and rimexolone.

AC and AG dinucleotide repeats in the PAX6 P1 promoter are associated with high myopia.

PURPOSE: The PAX6 gene, located at the reported myopia locus MYP7 on chromosome 11p13, was postulated to be associated with myopia development. This study investigated the association of PAX6 with high myopia in 379 high myopia patients and 349 controls. METHODS: High myopia patients had refractive errors of -6.00 diopters or greater and axial length longer than 26 mm. Control subjects had refractive errors less than -1.00 diopter and axial length shorter than 24 mm. The P1 promoter, all coding sequences, and adjacent splice-site regions of the PAX6 gene were screened in all study subjects by polymerase chain reaction and direct sequencing. PAX6 P1 promoter-luciferase constructs with variable AC and AG repeat lengths were prepared and transfected into human ARPE-19 cells prior to assaying for their transcriptional activities. RESULTS: No sequence alterations in the coding or splicing regions showed an association with high myopia. Two dinucleotide repeats, (AC)(m) and (AG)(n), in the P1 promoter region were found to be highly polymorphic and significantly associated with high myopia. Higher repeat numbers were observed in high myopia patients for both (AC)(m) (empirical p = 0.013) and (AG)(n) (empirical p = 0.012) dinucleotide polymorphisms, with a 1.327-fold increased risk associated with the (AG)(n) repeat (empirical p = 0.016; 95% confidence interval: 1.059-1.663). Luciferase-reporter analysis showed elevated transcription activity with increasing individual (AC)(m) and (AG)(n) and combined (AC)(m)(AG)(n) repeat lengths. CONCLUSIONS: Our results revealed an association between high myopia and AC and AG dinucleotide repeat lengths in the PAX6 P1 promoter, indicating the involvement of PAX6 in the pathogenesis of high myopia.

A novel gammaD-crystallin mutation causes mild changes in protein properties but leads to congenital coralliform cataract.

PURPOSE: To identify the genetic lesions for congenital coralliform cataract. METHODS: Two Chinese families with autosomal dominant coralliform cataract, 12 affected and 14 unaffected individuals, were recruited. Fifteen known genes associated with autosomal dominant congenital cataract were screened by two-point linkage analysis with gene based single nucleotide polymorphisms and microsatellite markers. Sequence variations were identified. Recombinant FLAG-tagged wild type or mutant gammaD-crystallin was expressed in human lens epithelial cells and COS-7 cells. Protein solubility and intracellular distribution were analyzed by western blotting and immunofluorescence, respectively. RESULTS: A novel heterozygous change, c.43C>A (R15S) of gammaD-crystallin (CRYGD) co-segregated with coralliform cataract in one family and a known substitution, c.70C>A (P24T), in the other family. Unaffected family members and 103 unrelated control subjects did not carry these mutations. Similar to the wild type protein, R15S gammaD-crystallin was detergent soluble and was located in the cytoplasm. ProtScale and ScanProsite analyses revealed raised local hydrophobicity and the creation of a hypothetical casein kinase II phosphorylation site. CONCLUSIONS: A novel R15S mutation caused congenital coralliform cataract in a Chinese family. R15S possessed similar properties to the wild type gammaD-crystallin, but its predicted increase of hydrophobicity and putative phosphorylation site could lead to protein aggregation, subsequently causing opacification in lens.

An alphaA-crystallin gene mutation, Arg12Cys, causing inherited cataract-microcornea exhibits an altered heat-shock response.

PURPOSE: To investigate the clinical features and molecular basis of inherited cataract-microcornea caused by an alphaA-crystallin gene (CRYAA) mutation in a Chinese family. METHODS: A three-generation Chinese family with members having autosomal dominant cataract and microcornea was recruited. Genomic DNA from peripheral blood or buccal swab samples of five affected and five unaffected members were obtained. Based on 15 genes known to cause autosomal dominant cataract, single nucleotide polymorphisms (SNPs) or microsatellite markers were selected and genotyped for two-point linkage analysis. Direct sequencing was performed to identify the disease-causing mutation. The expression construct coding for recombinant COOH-terminal myc-His-tagged wild type or R12C alphaA-crystallin protein (CRYAA) was expressed in COS-7 cells. Detergent solubility and subcellular distribution of wild type and R12C CRYAA were examined by western blotting and immunofluorescence, respectively. Heat-shock response was monitored by quantitative polymerase chain reaction (qPCR) of heat-shock proteins 70 and 90alpha (HSP70 and HSP90alpha). RESULTS: The five affected family members showed variable lens opacities and microcornea. Clinical features of cataract were asymmetric in two eyes of some affected subjects. A heterozygous missense substitution, c.34C>T, in CRYAA, which is responsible for the R12C amino acid change, segregated with autosomal dominant cataract (ADCC) in this family. This substitution was absent in 103 unrelated controls. When expressed in COS-7 cells, the R12C mutant CRYAA resembled the wild type protein in its solubility when extracted with 0.5% Triton X-100 and with its cytoplasmic localization. However, mutant cells exhibited an altered heat-shock response, evidenced by the delayed expression of HSP70, when compared to cells expressing wild type CRYAA. CONCLUSIONS: The R12C mutation in CRYAA was responsible for a variable type of inherited cataract associated with microcornea in this Chinese family. The altered heat-shock response of mutant cells suggested a change of chaperoning capacity and networking, which could be associated with the pathogenesis of hereditary cataract-microcornea syndrome.

A novel deletion variant of gammaD-crystallin responsible for congenital nuclear cataract.

PURPOSE: To investigate a novel deletion variant of gammaD-crystallin (CRYGD) identified in a Chinese family with nuclear congenital cataract. METHODS: A Chinese family with five affected members diagnosed with nuclear cataract and four unaffected members were recruited for the mutational screening of 15 known candidate genes for autosomal dominant congenital cataract. Two-point linkage analysis with single nucleotide polymorphism markers and microsatellite markers flanking these genes together with direct sequencing was applied to identify the disease-causing mutation. Recombinant NH(2)-terminal FLAG-tagged wildtype or mutant gammaD-crystallin was expressed in COS-7 cells. The expression pattern, protein solubility and intracellular distribution were analyzed by western blotting and confocal double immunofluorescence. RESULTS: Linkage analysis located the candidate region in the gammaC-crystallin and gammaD-crystallin gene cluster. Direct sequencing identified a c.494delG in CRYGD, which cosegregated with the disease in all affected members. Neither the unaffected family members nor the 103 unrelated controls carried this deletion mutation, which causes a frameshift and an early termination of polypeptide to become G165fs. A significantly reduced solubility was observed for this mutant. Unlike wildtype gammaD-crystallin, which existed in both the nucleus and cytoplasm, G165fs was colocalized with lamin A/C on the nuclear envelope. CONCLUSIONS: We have identified a novel mutation, c.494delG, in CRYGD, which was associated with nuclear cataract. This is the first deletion mutation of CRYGD found to cause autosomal dominant congenital cataract. The mutant protein with loss of solubility and localization to the nuclear envelope is hypothesized to impair nuclear transfiguration and degradation in lens fiber cell differentiation, leading to opacity formation during lens development.

SNPs and interaction analyses of myocilin, optineurin, and apolipoprotein E in primary open angle glaucoma patients.

PURPOSE: To evaluate the association of myocilin (MYOC), optineurin (OPTN), and apolipoprotein E (APOE) genes and their interactions in primary open angle glaucoma (POAG). METHODS: A cohort of 400 unrelated POAG patients (294 high tension glaucoma, HTG, and 106 normal tension glaucoma, NTG) and 281 unrelated control subjects were recruited. All coding exons and splicing junctions in MYOC and OPTN were screened for sequence alterations. Common polymorphisms in APOE were genotyped. Single genes were investigated by univariate and haplotype analysis, and gene-gene interactions by logistic regression and stratified analysis. Multiple comparisons were corrected by the Bonferroni method. Bioinformatics analysis was performed to assess the conservation of mutation sites across species and to predict putative motifs and secondary structures in mutated proteins. RESULTS: Disease-causing mutations in MYOC and OPTN were identified in 1.75% and 1% of POAG patients, respectively. Most of these mutations were highly conserved across species, many predicted to create new motifs or change protein secondary structures. No individual MYOC polymorphisms significantly contributed to HTG or NTG. A haplotype containing the minor allele of the MYOC IVS2+35A>G increased NTG risk (p=0.0001). Three OPTN polymorphisms, T34T, IVS5+38T>G, and IVS8-53T>C increased NTG risk (p<0.0008), while IVS5+38T>G increased HTG risk (p=0.0006). One haplotype that contains the minor alleles of 3 OPTN polymorphisms, T34T, IVS5+38T>G, and IVS7+24G>A, increased NTG risk (p=0.0002). APOE epsilon4 carriers had a decreased NTG risk (p=0.007). Possible gene-gene interactions were found between MYOC, OPTN, and APOE. CONCLUSIONS: Disease-causing mutations in MYOC and OPTN accounted for only a small proportion of Chinese POAG patients. Common polymorphisms in MYOC, OPTN, and APOE might interactively contribute to POAG, indicating a polygenic etiology.

Growth factor changes in ex vivo expansion of human limbal epithelial cells on human amniotic membrane.

PURPOSE: Human limbal epithelial cells cultured on human amniotic membrane have been used for transplantation to treat corneal surface injuries. We determined whether the amniotic basement membrane affects the growth of human limbal epithelial cells through the production of growth factors. METHODS: The epithelial cells grown out from limbal basal epithelium were placed on conventional culture plastic or on the epithelial side of denuded amniotic membrane under serum-free conditions. Culture supernatant was assayed for growth factor release at 24, 48, and 96 hours. RESULTS: The cells grown on both substrata produced similar levels of epidermal growth factor (EGF). Cells grown on amniotic membrane showed enhanced secretion of tissue inhibitor of metalloproteinase type 1 (TIMP1) and reduced production of transforming growth factor beta1 and beta2. Depletion of EGF and TIMPI in cell culture slowed down cell growth and reduced EGF receptor expression, respectively. CONCLUSION: Increased TIMPI influences the proteolytic system in the cell and extracellular matrix interaction, and decreased transforming growth factor beta1 and beta2 may stimulate corneal cell proliferation. We show that the amniotic membrane leads to differential expression of cytokines of limbal epithelial cells cultured on its surface. Such effects may be favorable to the growth and differentiation of the cells when used for ocular surface reconstruction.