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Gopalakrishnan P.,Dr G Venkataswamy Eye Research Institute | Haripriya A.,Aravind Eye Hospital | Sundaresan P.,Dr G Venkataswamy Eye Research Institute
International Ophthalmology | Year: 2017

Purpose: Pseudoexfoliation syndrome (PEX) is a late onset disorder of extracellular matrix turnover, associated systemically with cardiovascular and cerebrovascular disease. To evaluate the suggested association of polymorphisms of homocysteine metabolism genes MTHFR (rs1801131, rs1801133) and MTHFD1 (rs8006686) with PEX. Methods: A case–control association study was undertaken, comprising a total of 1472 individuals including 860 unrelated PEX cases and 612 ethnic-matched cataract controls (CC). All the study subjects were genotyped for three SNPs using the TaqMan allelic discrimination assay. Association and statistical analysis were performed with PLINK 1.07 and STATA 11.1. Results: Among the three SNPs genotyped, MTHFR polymorphisms did not exhibit significant association with PEX (rs1801131; p = 0.549, rs1801133; p = 0.408). The intronic SNP rs8006686 showed nearly significant association (p = 0.069), and however did not remain significant after Bonferroni correction. Conclusion: Our study suggests no significant genetic association of MTHFR (rs1801131, rs1801133) and MTHFD1 (rs8006686) polymorphisms in South Indian PEX patients. © 2017 Springer Science+Business Media Dordrecht


Vilas G.L.,University of Alberta | Loganathan S.K.,University of Alberta | Quon A.,University of Alberta | Sundaresan P.,Dr G Venkataswamy Eye Research Institute | And 3 more authors.
Human Mutation | Year: 2012

Mutations in the SLC4A11 gene, which encodes a plasma membrane borate transporter, cause recessive congenital hereditary endothelial corneal dystrophy type 2 (CHED2), corneal dystrophy and perceptive deafness (Harboyan syndrome), and dominant late-onset Fuchs endothelial corneal dystrophy (FECD). We analyzed missense SLC4A11 mutations identified in FECD and CHED2 patients and expressed in transfected HEK 293 cells. Chemical cross-linking and migration in nondenaturing gels showed that SLC4A11 exists as a dimer. Furthermore, co-immunoprecipitation of epitopetagged proteins revealed heteromeric interactions between wild-type (WT) and mutant SLC4A11 proteins. When expressed alone, FECD- and CHED2-causing mutant SLC4A11 proteins are primarily retained intracellularly. Co-expression with WT SLC4A11 partially rescued the cell surface trafficking of CHED2 mutants, but not FECD mutants. CHED2 alleles of SLC4A11 did not affect cell surface processing of WT SLC4A11. In contrast, FECD mutants reduced WT cell surface processing efficiency, consistent with dominant inheritance of FECD. The reduction in movement of WT protein to the cell surface caused by FECD SLC4A11 helps to explain the dominant inheritance of this disorder. Similarly, the failure of CHED2 mutant SLC4A11 to affect the processing of WT protein, explains the lack of symptoms found in CHED2 carriers and the recessive inheritance of the disorder. © 2011 Wiley Periodicals, Inc.


Sundaresan P.,Dr G Venkataswamy Eye Research Institute | Kumar S.M.,Aravind Eye Hospital | Thompson S.,University of Iowa | Fingert J.H.,University of Iowa
Ophthalmic Genetics | Year: 2010

Background: Three mitochondrial mutations account for 95% of Leber's hereditary optic neuropathy (LHON) in the European population: G3640A, G11778A and T14484C. The purpose of the study was to investigate the frequency of these mitochondrial DNA mutations in LHON patients from a South Indian population. Methods: LHON was diagnosed by inheritance pattern, ophthalmologic examination, and by exclusion of non-LHON forms of optic neuropathy. Ninety unrelated LHON patients and 20 at-risk family members (5 with LHON and 15 without LHON) underwent molecular screening for the mitochondrial DNA mutations G3640A, G11778A and T14484C by amplification refractory mutation system (ARMS) polymerase chain reaction (PCR). Positive results were confirmed with bi-directional sequencing. Results: The G11778A mutation was detected in 8 of 90 (8.9%) LHON families. The T14484 mutation was detected in 3 of 90 (3.3%) LHON families. No instances of the G3460A mutation were detected. Other variants were incidentally detected by the DNA sequencing assay. Conclusions: Three mitochondrial mutations (G3640A, G11778A and T14484C) account for the vast majority of LHON cases in Europe. However, these mutations were detected in only 11 (12%) of 90 LHON families from Southern India in our study. These results suggest that a different set of LHON-causing mutations is present in the South Indian population than in the European population. Further study of subjects with LHON from India may lead to the discovery of novel disease-causing mutations and/or genes. © 2010 Informa Healthcare USA, Inc.


Sundaresan P.,Dr G Venkataswamy Eye Research Institute | Simpson D.A.,Queen's University of Belfast | Sambare C.,Queen's University of Belfast | Sambare C.,H V Desai Eye Hospital | And 10 more authors.
Genetics in Medicine | Year: 2015

Purpose:The aim of this study was to determine whether mutations in mitochondrial DNA play a role in high-pressure primary open-angle glaucoma (OMIM 137760) by analyzing new data from massively parallel sequencing of mitochondrial DNA.Methods:Glaucoma patients with high-tension primary open-angle glaucoma and ethnically matched and age-matched control subjects without glaucoma were recruited. The entire human mitochondrial genome was amplified in two overlapping fragments by long-range polymerase chain reaction and used as a template for massively parallel sequencing on an Ion Torrent Personal Genome Machine. All variants were confirmed by conventional Sanger sequencing.Results:Whole-mitochondrial genome sequencing was performed in 32 patients with primary open-angle glaucoma from India (n = 16) and Ireland (n = 16). In 16 of the 32 patients with primary open-angle glaucoma (50% of cases), there were 22 mitochondrial DNA mutations consisting of 7 novel mutations and 8 previously reported disease-associated sequence variants. Eight of 22 (36.4%) of the mitochondrial DNA mutations were in complex I mitochondrial genes.Conclusion:Massively parallel sequencing using the Ion Torrent Personal Genome Machine with confirmation by Sanger sequencing detected a pathogenic mitochondrial DNA mutation in 50% of the primary open-angle glaucoma cohort. Our findings support the emerging concept that mitochondrial dysfunction results in the development of glaucoma and, more specifically, that complex I defects play a significant role in primary open-angle glaucoma pathogenesis.Genet Med 17 4, 279-284.


Sureshkumar J.,Dr G Venkataswamy Eye Research Institute | Haripriya A.,Aravind Eye Hospital | Muthukkaruppan V.,Dr G Venkataswamy Eye Research Institute | Kaufman P.L.,University of Wisconsin - Madison | Tian B.,Aravind Eye Hospital
Graefe's Archive for Clinical and Experimental Ophthalmology | Year: 2012

Background To determine whether the cytoskeletal drugs H-7 and Latrunculin B (LAT-B) inhibit posterior capsular opacification (PCO) in the cultured human lens capsular bag. Methods Following extracapsular cataract (lens) extraction in human donor eyes, the capsular bag was prepared and cultured by standard techniques. Forty-eight capsular bags were studied, of which 13 were treated with H-7 (50, 100 or 300 μM), 12 with 1% BSS (vehicle of H-7), 11 with LAT-B (2, 5 or 10 ?M), and 12 with 0.25% DMSO (vehicle of LATB). Forty out of the 48 capsular bags were from paired eyes of 20 donors, with one bag being treated withH-7/LAT-B and the other with BSS/DMSO for each pair, including 20 for the H-7-BSS protocol and 20 for the LAT-B-DMSO protocol. The medium with the cytoskeletal drug/vehicle was replaced every 3-4 days for 4 weeks. PCO was assessed daily using inverted phase-contrast microscopy, and scored on a 4-point scale. Results In all cultures with BSS or DMSO, residual lens epithelial cells (LECs) on the anterior capsule migrated to and proliferated on the posterior capsule by 3-7 days, and apparent LEC growth on the posterior capsule with severe capsular wrinkling (PCO Grade 3) was seen by 2-3 weeks. When treated continuously with H-7 or LAT-B, the migration and proliferation of LECs and the capsular wrinkling were inhibited in a dose-dependent manner, with the inhibition being complete (PCO Grade 0) in the 300 μM H-7 (n08, p<0.001) or 10 μM LAT-B culture (n03, p00.002). Conclusion H-7 and LAT-B dose-dependently inhibited PCO formation in the cultured human lens capsular bags, suggesting that cytoskeletal drugs might prevent PCO formation after surgery in the human eye. © 2011 Springer-Verlag.


Dubey S.K.,Dr G Venkataswamy Eye Research Institute | Mahalaxmi N.,Dr G Venkataswamy Eye Research Institute | Vijayalakshmi P.,Aravind Eye Hospital | Sundaresan P.,Dr G Venkataswamy Eye Research Institute
Molecular Vision | Year: 2015

Purpose: Aniridia is a rare panocular disorder characterized by iris hypoplasia and other associated eye anomalies. Heterozygous null mutations in paired box gene 6 (PAX6) are the major cause of the classic aniridia phenotype. This study aims to detect the mutational spectrum of PAX6 and associated phenotypes in southern Indian patients with sporadic and familial aniridia. Methods: Genomic DNA was isolated from peripheral blood from all participants. The coding regions and flanking intronic sequences of PAX6 were screened with Sanger sequencing in 30 probands with aniridia. The identified variations were further evaluated in available family members and 150 healthy controls. The pathogenic potential of the mutations were assessed using bioinformatics tools. Results: Thirteen different mutations were detected in eight sporadic and five familial cases. Eleven novel mutations, including five insertions (c.7_10dupAACA, c.567dupC, c.704dupC, c.868dupA and c.753_754insTA), two deletions (c.242delC and c.249delT), and four splicing variants (c.10+1G>A, c.141G>A, c.141+4A>G and c.764A>G) were identified in this study. Clinical findings of the patients revealed phenotypic heterogeneity with the same or different mutations. Conclusions: This study reported 11 novel mutations and thus expanded the spectrum of PAX6 mutations. Interestingly, all mutations reported in this study were truncations, which confirms the hypothesis that haploinsufficiency of PAX6 causes the aniridia phenotype. Our observations revealed inter- and intrafamilial phenotypic variability with PAX6 mutations. The common ocular findings associated with PAX6 mutations were iris hypoplasia, nystagmus, and foveal hypoplasia reported in almost all cases, with cataract, glaucoma, and keratopathy reported in approximately 50% of the patients. © 2015 Molecular Vision.


Priya C.G.,Dr G Venkataswamy Eye Research Institute | Prasad T.,Dr G Venkataswamy Eye Research Institute | Prajna N.V.,Aravind Eye Hospital | Muthukkaruppan V.,Dr G Venkataswamy Eye Research Institute
Microscopy Research and Technique | Year: 2013

Purpose: Till date there is no exclusive marker for human corneal epithelial stem cells (CESCs). In this study, our strategy is to combine high expression of ABCG2, a putative SC marker with high N/C ratio to develop a specific method for identification of CESCs. Methods: Limbal/corneal epithelial cells (LECs/CECs) were isolated from cadaver eyes by enzymatic treatment and the cytospin smears/cryosections were immunostained for ABCG2, p63, or connexin-43 (Cx-43) and counterstained with propidium iodide/DAPI. Analysis was carried out for (1) ABCG2 expression, N/C ratio, (2) p63, ABCG2, N/C ratio, and (3) ABCG2, Cx-43, N/C ratio using confocal/fluorescence microscopy. Results: ABCG2 was highly expressed by cells in the basal and suprabasal layers of limbus. The SCs identified on the basis of our earlier method using high levels of p63 expression along with high (>0.7) N/C ratio were strongly positive for ABCG2 and negative for differentiation marker Cx-43. High expression of ABCG2 was also observed in cells with high p63 expression and low (<0.7) N/C ratio; however, they were positive for Cx-43. Combining high ABCG2 expression with large N/C ratio in fluorescence microscopy identified 5.3 ± 3.5% LECs as putative SCs and this was confirmed by confocal microscopic analysis. Conclusions: We demonstrate the importance of combining ABCG2 expression with N/C ratio for identification and quantification of human CESCs. This method will have application in evaluating the role of niche cells/factors in the maintenance of stemness. © 2012 Wiley Periodicals, Inc.


Ananthi S.,Dr G Venkataswamy Eye Research Institute | Venkatesh Prajna N.,Aravind Eye Hospital | Lalitha P.,Aravind Eye Hospital | Valarnila M.,Dr G Venkataswamy Eye Research Institute | Dharmalingam K.,Madurai Kamaraj University
PLoS ONE | Year: 2013

Fusarium is the major causative agent of fungal infections leading to corneal ulcer (keratitis) in Southern India and other tropical countries. Keratitis caused by Fusarium is a difficult disease to treat unless antifungal therapy is initiated during the early stages of infection. In this study tear proteins were prepared from keratitis patients classified based on the duration of infection. Among the patients recruited, early infection (n = 35), intermediate (n = 20), late (n = 11), samples from five patients in each group were pooled for analysis. Control samples were a pool of samples from 20 patients. Proteins were separated on difference gel electrophoresis (DIGE) and the differentially expressed proteins were quantified using DeCyder software analysis. The following differentially expressed proteins namely alpha-1-antitrypsin, haptoglobin α2 chain, zinc-alpha-2-glycoprotein, apolipoprotein, albumin, haptoglobin precursor - β chain, lactoferrin, lacrimal lipocalin precursor, cystatin SA III precursor, lacritin precursor were identified using mass spectrometry. Variation in the expression level of some of the proteins was confirmed using western blot analysis. This is the first report to show stage specific tear protein profile in fungal keratitis patients. Validation of this data using a much larger sample set could lead to clinical application of these findings. © 2013 Ananthi et al.


Ananthi S.,Dr G Venkataswamy Eye Research Institute | Santhosh R.S.,Dr G Venkataswamy Eye Research Institute | Nila M.V.,Dr G Venkataswamy Eye Research Institute | Prajna N.V.,Aravind Eye Hospital | And 2 more authors.
Experimental Eye Research | Year: 2011

The proteomic profile of tear fluid is of fundamental interest in eye research. In this study we optimized the tear sample preparation method for two-dimensional (2D) analysis and determined the protein profile of tear fluid from healthy males and females. To find the most efficient method for tear sample preparation, four widely applied precipitation methods and ultrafiltration were compared. Of these, TCA precipitation & ultrafiltration resulted in efficient sample concentration and desalting. Use of a nonionic wetting agent, Tergitol NP7, in rehydration solution during isoelectric focusing improves protein separation in 2D gel electrophoresis considerably. Using this optimized method, tear protein profile was analyzed from healthy males and females. Of the thirty six tear proteins identified by LC-MS/MS, seven tear proteins were found to be significantly up regulated in the healthy female tear samples when compared to the male tear samples. These results indicate that the tear protein profile differs with respect to the sex. Mostly, the up regulated proteins are involved in the local immune defense; implying that there may be a sex difference in the ability to defend against infection at the anterior segment of the eyes of normal individuals. © 2011 Elsevier Ltd.


Balasubbu S.,Dr G Venkataswamy Eye Research Institute
Investigative ophthalmology & visual science | Year: 2012

Glaucoma comprises a heterogeneous group of optic neuropathies with a complex genetic basis. It is the second leading cause of irreversible blindness in the world. This study investigates the association of SNPs on chromosome 2p with primary open angle glaucoma (POAG) in a Southern Indian population. Case-control analysis was performed using 220 unrelated POAG cases and 220 age-matched unaffected controls recruited through the Aravind Eye Hospital and its outlying clinics. Five SNPs (rs1533428, rs12994401, rs10202118, rs11125375, and rs11889995) on chromosome 2p were evaluated in these two groups and genotyped using Taq Man SNP genotyping assay. Statistical analysis was performed using the SVS program package by Golden Helix to identify the distributions of allele and genotype frequencies, Fisher exact test P values, and odds ratios and to check Hardy-Weinberg equilibrium. Among the five SNPs screened, SNP rs10202118, showed a P = 0.026 for the basic allelic test, P = 0.004 for the genotypic test, and P = 0.0014 for the recessive model. The second suggestive marker was rs11125375, which also showed P = 0.033 for the recessive model. The associated SNPs formed a common disease haplotype. The remaining three SNPs showed insignificant association in this study population. This was the first study to demonstrate the association of SNPs on chromosome 2p in patients with POAG in the Indian population. The two tagging SNPs (rs10202118 and rs11125375) on chromosome 2p are the most likely sites underlying the significant association with POAG in this study population.

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