Association of FOXE3-p.Ala170Ala and PITX3-p.Ile95Ile Polymorphisms with Congenital Cataract and Microphthalmia.

PURPOSE: To investigate the association of FOXE3-p.Ala170Ala (rs34082359) and PITX3-p.Ile95Ile (rs2281983) polymorphisms with congenital cataract and microphthalmia in a western Indian population. METHODS: FOXE3-p.Ala170Ala (c.510C>T) and PITX3-p.Ile95Ile (c.285C>T) polymorphisms were genotyped in 561 subjects consisting of 242 cases with congenital cataract, 52 with microphthalmia, and 267 controls using polymerase chain reaction-restriction fragment length polymorphism. Approximately 10% of samples were randomly sequenced for each single nucleotide polymorphism to confirm the genotypes. The prediction of mRNA secondary structure for polymorphism FOXE3-p.Ala170Ala and PITX3-p.Ile95Ile was performed. RESULTS: A significantly high frequency of T allele and a borderline significance in the frequency of TT genotype of FOXE3-p.Ala170Ala was observed in microphthalmia cases, as compared to controls [T allele: OR: [CI] = 1.8 [1.15-2.72], P = 0.0115; TT: OR [CI] = 2.9 [1.14-7.16], P = 0.0291). The frequency of CC genotype was significantly low in microphthalmia cases when compared to controls (CC: OR [CI] = 0.5 [0.24-0.86, P = 0.0150). There was no significant difference in the allele and genotype frequencies of PITX3-p.Ile95Ile between cases and controls. A slight free energy change was observed in the secondary structure of mRNA between the FOXE3-p.Ala170Ala C-allele (-917.60 kcal/mol) and T-allele (-916.80 kcal/mol) and between PITX3-p.Ile95Ile C-allele (-659.80 kcal/mol) and T-allele (-658.40 kcal/mol). CONCLUSION: The present findings indicate that FOXE3-p.Ala170Ala 'T' allele and 'TT' genotype could be predisposing factors for microphthalmia while 'CC' genotype might play a protective role against it. A reduction in the free energy change associated with FOXE3-p.Ala170Ala 'T' allele could further contribute towards disease risk.

Trichostatin A Restores Expression of Adherens and Tight Junction Proteins during Transforming Growth Factor ß-Mediated Epithelial-to-Mesenchymal Transition.

PURPOSE: Adherens junctions and polarity markers play an important role in maintaining epithelial phenotype but get altered during the epithelial-mesenchymal transition (EMT). Alterations of these markers during EMT of lens epithelial cell (LEC) can lead to vision compromising conditions. The aim of this study was to examine if Trichostatin-A (TSA), a histone deacetylase inhibitor, can prevent EMT by restoring the adherens junction complex in LEC. METHODS: Fetal human lens epithelial cell line (FHL124) was used. Cells were treated with 10 ng/ml TGF-ß2 in the presence or absence of TSA. Real time-PCR and western blotting were carried out for HDAC1, HDAC2, CDH1 (E-cad), TJP1 (ZO-1) and CTNNB1 (ß-cat). Level of histone acetylation was analyzed by western blotting. Chromatin Immunoprecipitation was carried out to study the level of acetylated histone H4 and HDAC2 at the promoter regions of CDH1, TJP1, and CTNNB1. E-cad, ZO-1, and ß-cat were localized using immunofluorescence. Kruskal-Wallis test was used for statistical analysis. RESULTS: TSA down-regulated HDAC1 and HDAC2 and led to an increase in global acetylation. The mRNA and protein levels of E-cad, ZO-1, and ß-cat decreased during EMT but were up-regulated by TSA treatment. TSA also helped in stabilizing these proteins at cell-cell junctions during EMT. TSA decreases association of HDAC2 at the promoter regions of adherens junction genes while increasing histone H4 acetylation status. CONCLUSION: TSA increases histone acetylation and restores the adherens junction complex in LECs. TSA helps in preventing EMT and thus shows potential against lens fibrosis and vision compromising conditions.

Genetic investigation of ocular developmental genes in 52 patients with anophthalmia/microphthalmia.

BACKGROUND: Mutation in eye developmental genes has been reported to cause anophthalmia and microphthalmia. However, in India, especially in the Western Indian population, such reports are scarce. Hence, the present study aims to investigate mutations in 15 ocular developmental genes in patients with anophthalmia and microphthalmia in the western region of India. MATERIALS AND METHODS: Genomic DNA was isolated from the blood of 52 individuals affected with microphthalmia and anophthalmia, and 50 healthy normal controls. Polymerase chain reaction (PCR) was carried out for 15 genes including BMP4, CRYBA4, FOXE3, GDF6, GJA3, GJA8, MITF, OTX2, PAX6, PITX3, RAX, SIX3, SIX6, SOX2, and VSX2 using gene-specific primers spanning the exon-intron boundaries and part of a promoter region. The amplified PCR products were purified and then subjected to Sanger's bi-directional sequencing. Nucleotide variations were examined using a basic local alignment search tool (BLAST). RESULTS: Bi-directional sequencing identified 8 novel and 14 known variations. Out of this, the variations GJA3-c.92T>A; p.Ile31Asn, SOX2-c.542C>A; p.Pro181Gln and SOX2-c.541_542delinsGA; p.Pro181Glu were found to be deleterious by in silico analysis. The GJA3-p.Ile31Asn mutation was identified in a patient with bilateral microphthalmia, microcornea, and membranous cataract. The SOX2-p.Pro181Gln and SOX2-p.Pro181Glu mutations were identified in patients with isolated bilateral microphthalmia and microphthalmia with microcornea, respectively. A novel nondeleterious missense variation was identified in the GJA8 gene in a patient with anophthalmia. CONCLUSION: These results support the crucial role of GJA3 and SOX2 in eye development and indicate a detailed functional study to understand the molecular mechanisms underlying the disease pathology.

Exploration of molecular factors impairing superoxide dismutase isoforms activity in human senile cataractous lenses.

PURPOSE: To explore different molecular factors impairing the activities of superoxide dismutase (SOD) isoforms in senile cataractous lenses. METHODS: Enzyme activity of SOD isoforms, levels of their corresponding cofactors copper (Cu), manganese (Mn), zinc (Zn), and expression of mRNA transcripts and proteins were determined in the lenses of human subjects with and without cataract. DNA from lens epithelium (LE) and peripheral blood was isolated. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) followed by sequencing was carried out to screen somatic mutations. The impact of intronic insertion/deletion (INDEL) variations on the splicing process and on the resultant transcript was evaluated. Genotyping of IVS4+42delG polymorphism of SOD1 gene was done by PCR-restriction fragment length polymorphism (RFLP). RESULTS: A significant decrease in Cu/Zn- and Mn-SOD activity (P < 0.001) and in Cu/Zn-SOD transcript (P < 0.001) and its protein (P < 0.05) were found in cataractous lenses. No significant change in the level of copper (P = 0.36) and an increase in the level of manganese (P = 0.01) and zinc (P = 0.02) were observed in cataractous lenses. A significant positive correlation between the level of Cu/Zn-SOD activity and the levels of Cu (P = 0.003) and Zn (P = 0.005) was found in the cataractous lenses. DNA sequencing revealed three intronic INDEL variations in exon4 of SOD1 gene. Splice-junction analysis showed the potential of IVS4+42delG in creating a new cryptic acceptor site. If it is involved in alternate splicing, it could result in generation of SOD1 mRNA transcripts lacking exon4 region. Transcript analysis revealed the presence of complete SOD1 mRNA transcripts. Genotyping revealed the presence of IVS4+42delG polymorphism in all subjects. CONCLUSIONS: The decrease in the activity of SOD1 isoform in cataractous lenses was associated with the decreased level of mRNA transcripts and their protein expression and was not associated with either modulation in the level of enzyme cofactors or with INDEL variations.

Matrix metalloproteinase-9 activity in human lens epithelial cells of cortical, posterior subcapsular, and nuclear cataracts.

PURPOSE: To evaluate the level of matrix metalloproteinase-9 (MMP-9) activity in lens epithelial cells (LECs) derived from different types of cataract in patients having phacoemulsification. SETTING: Iladevi Cataract & IOL Research Centre, Memnagar, Ahmedabad, India. METHODS: This observational study of 275 patients having phacoemulsification was performed to evaluate the level of MMP-9 activity in LECs. All anterior lens capsules harboring the LECs and derived from the surgical curvilinear capsulorhexis were obtained during phacoemulsification. The anterior lens capsule samples were processed to analyze MMP-9 activity using a succinylated gelatin assay. The samples were grouped based on age and on pure cataract type. RESULTS: The level of MMP-9 activity in LECs was highest in eyes with cortical cataract. A significant difference in the level of MMP-9 activity was found in different types of cataract (P<.001). The highest level of MMP-9 activity was in patients older than 60 years. The MMP-9 activity increased gradually with age irrespective of cataract type (P<.001). CONCLUSIONS: A significant difference was observed in the level of MMP-9 activity between different types of cataract. The activity of MMP-9 was highest in eyes with cortical cataract. The level of MMP-9 activity increased with age in the LECs of patients with age-related cataract.

Activity of superoxide dismutase isoenzymes in lens epithelial cells derived from different types of age-related cataract.

PURPOSE: To evaluate the level of superoxide dismutase (SOD) and SOD isoenzyme activity in lens epithelial cells (LECs) derived from different types of cataract in patients having phacoemulsification. SETTING: Iladevi Cataract & IOL Research Centre, Memnagar, Ahmedabad, India. METHODS: This observational study of 109 patients having phacoemulsification was performed to evaluate the level of activity in LECs of total superoxide dismutase (TSOD) and 2 superoxide dismutase isoenzymes: copper- (Cu) and zinc (Zn)-dependent SOD (Cu/Zn-SOD) and manganese (Mn)-dependent SOD (Mn-SOD). The curvilinear capsulorhexis (lens capsule) harboring LECs was obtained during phacoemulsification. The anterior lens capsule samples were processed for assaying SOD activity using the nitro blue tetrazolium reduction assay. The samples were grouped based on age and on pure cataract types. RESULTS: The highest level of TSOD, Cu/Zn-SOD, and Mn-SOD activity was in patients 50 years or younger. The activity declined gradually with age (P<.001). The level of TSOD activity increased in cortical cataract. The level of Cu/Zn-SOD and Mn-SOD isoenzyme activity in LECs was higher in cortical cataracts. A significant difference in the level of TSOD and Cu/Zn-SOD activity was found with different types of cataract (P<.001). CONCLUSIONS: The level of TSOD, Cu/Zn-SOD, and Mn SOD isoenzyme activity decreased with age in LECs of patients with age-related cataract. A significant difference in the level of TSOD and Cu/Zn-SOD isoenzyme activity between different types of cataract was observed. The activity of all 3 SOD isoenzymes was highest in cortical cataracts.