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Genetic association and stress mediated down-regulation in trabecular meshwork implicates MPP7 as a novel candidate gene in primary open angle glaucoma.

ABSTRACT: BACKGROUND:Glaucoma is the largest cause of irreversible blindness affecting more than 60 million people globally. The disease is defined as a gradual loss of peripheral vision due to death of Retinal Ganglion Cells (RGC). The RGC death is largely influenced by the rate of aqueous humor production by ciliary processes and its passage through the trabecular meshwork (TM) in the anterior part of the eye. Primary open angle glaucoma (POAG), the most common subtype, is a genetically complex disease. Multiple genes and many loci have been reported to be involved in POAG but taken together they explain less than 10 % of the patients from a genetic perspective warranting more studies in different world populations. The purpose of this study was to perform genome-wide search for common variants associated with POAG in an east-Indian population. METHODS:The study recruited 746 POAG cases and 697 controls distributed into discovery and validation cohorts. In the discovery phase, genome-wide genotype data was generated on Illumina Infinium 660 W-Quad platform and the significant SNPs were genotyped using Illumina GGGT assay in the second phase. Logistic regression was used to test association in the discovery phase to adjust for population sub-structure and chi-square test was used for association analysis in validation phase. Publicly available expression dataset for trabecular meshwork was used to check for expression of the candidate gene under cyclic mechanical stress. Western blot and immunofluorescence experiments were performed in human TM cells and murine eye, respectively to check for expression of the candidate gene. RESULTS:Meta-analysis of discovery and validation phase data revealed the association of rs7916852 in MPP7 gene (p = 5.7x10(-7)) with POAG. We have shown abundant expression of MPP7 in the HTM cells. Expression analysis shows that upon cyclic mechanical stress MPP7 was significantly down-regulated in HTM (Fold change: 2.6; p = 0.018). MPP7 protein expression was also found to be enriched in the ciliary processes of the murine eye. CONCLUSION:Using a genome-wide approach we have identified MPP7 as a novel candidate gene for POAG with evidence of its expression in relevant ocular tissues and dysregulation under mechanical stress possibly mimicking the disease scenario.

SUBMITTER: Vishal M

PROVIDER: S-EPMC4802647 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Genetic association and stress mediated down-regulation in trabecular meshwork implicates MPP7 as a novel candidate gene in primary open angle glaucoma.

Vishal Mansi M Sharma Anchal A Kaurani Lalit L Alfano Giovanna G Mookherjee Suddhasil S Narta Kiran K Agrawal Jyoti J Bhattacharya Iman I Roychoudhury Susanta S Ray Jharna J Waseem Naushin H NH Bhattacharya Shomi S SS Basu Analabha A Sen Abhijit A Ray Kunal K Mukhopadhyay Arijit A

BMC medical genomics 20160322

<h4>Background</h4>Glaucoma is the largest cause of irreversible blindness affecting more than 60 million people globally. The disease is defined as a gradual loss of peripheral vision due to death of Retinal Ganglion Cells (RGC). The RGC death is largely influenced by the rate of aqueous humor production by ciliary processes and its passage through the trabecular meshwork (TM) in the anterior part of the eye. Primary open angle glaucoma (POAG), the most common subtype, is a genetically complex ...[more]

PMID: 27001270

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Project description:We previously identified and characterized human trabecular meshwork stem cells (TMSCs) based on high expression of ABCG2/p75 positivity and high nucleus to cytoplasmic ratio. These TMSCs expressing high ABCG2 and p75 were located to the insert region of the human TM. Additionally, we demonstrated an age-related reduction in the TMSC content which was significantly associated with TM cell loss. In continuation, this study was aimed to determine the TMSC content in glaucomatous donor eyes wherein a drastic reduction in TM cellularity has already been reported. Anterior segments from known glaucomatous (n = 6) and age-matched normal (n = 8) donors were dissected into four quadrants. A minimum of three sections from each quadrant were used for histopathological analysis as well as immunostaining. Analysis of hematoxylin and eosin-stained sections from glaucomatous tissues revealed a decrease in total TM cellularity, thickening of trabecular beams, fusion of trabeculae, absence of patent Schlemm's canal compared to age-matched controls. In addition, the TM thickness at various positions of the meshwork and the coronal as well as the meridional diameters of the Schlemm's canal were observed to be significantly reduced in glaucomatous eyes. Further, sections from both the groups were immunostained for universal stem cell marker ABCG2 and neural crest derived stem cell marker p75. The images were acquired using Leica SP8 confocal microscope. Quantification of total TM cellularity based on nuclear counterstain (mean ± SD) using ImageJ identified 69.33 ± 12.77 cells/section in control eyes. In glaucomatous donors, the TM cellularity was found to be reduced significantly to 41.83 ± 9.0 (p = 0.0007). In addition, a reduction in the percentage of TMSCs (cells with high ABCG2 expression and p75 positivity) was evident in glaucomatous donors (0.14 ± 0.17%) compared to age-matched controls (4.73 ± 5.46%) (p = 0.064). Thus, the present study confirmed the significant decline in TM cellularity and a reducing trend in the TMSC content, though this reduction was non-significant in glaucomatous donor eyes. Further studies are essential to elucidate the role of TMSCs in the pathogenesis of primary open angle glaucoma.

Project description:PURPOSE: Primary open-angle glaucoma (POAG), which is the most common form of glaucoma, has been associated with a heterogeneous genetic component. A genome-wide association study has identified a common sequence variant at 7q31 (rs4236601 [A]) near the caveolin genes in patients with POAG. Caveolins are a family of integral membrane proteins which participate in many cellular processes, including vesicular transport, cholesterol homeostasis, signal transduction, cell adhesion and migration. The goal of this study was to investigate the expression and regulation of caveolin 1 (CAV-1) and caveolin 2 (CAV-2) in normal and glaucoma trabecular meshwork (TM) cells. METHODS: CAV-1 and CAV-2 protein expression was quantified by immunoblot analysis using lysates isolated from primary and immortalized TM cells or TM tissue dissected from normal and POAG eyes. The localization of caveolins in TM cells was assessed by immunofluorescent microscopy. CAV-1 and CAV-2 protein expression was also investigated in TM cells at various time points after subjecting the cells to known glaucomatous insults like dexamethasone (DEX) and tumor growth factor beta2 (TGF-?2) treatment. Phosphorylation of CAV-1 at tyrosine 14 in normal and glaucoma TM cell lines was evaluated using a specific monoclonal antibody (Ab). The 5' upstream region of the CAV-1 gene was amplified and the sequence variant rs4236601 (A/G polymorphic site) and several putative transcription factor-binding sites were modified by in vitro mutagenesis. The effect of nucleotide sequence modifications in the CAV-1 upstream region on gene expression was assayed in a luciferase-based system in TM and non-TM cells. RESULTS: CAV-1 and CAV-2 are expressed in TM cells, with localization to the cytoplasm and perinuclear region. DEX increased CAV-1 expression in immortalized glaucoma TM cells by 2.8±0.1 (n=3) fold at 24 h and 2.5±0.1 (n=3) fold at 48 h, compared to 1.3±0.06 (n=3) fold at 24 and 48 h in immortalized normal TM cells. Phosphorylation of CAV-1 at Tyr14 was reduced by 3.2±0.15 (n=3) fold in glaucomatous TM cells when compared to normal TM cells. In POAG and normal TM tissue, CAV-1 expression was found to be uniform. CAV-2, on the other hand, was variable in independent normal and glaucoma TM tissue. Substitution of a G for an A at base pair -2,388 upstream of the start codon of CAV-1, corresponding to the minor allele rs4236601 [A], increased transcriptional activity in TM and non-TM cells when compared to the native sequence. Deletion analysis of putative transcription factor binding sites in the CAV-1 promoter region caused cell-specific effects on gene expression. CONCLUSIONS: CAV-1 and CAV-2 are expressed in normal and glaucoma tissue and TM cell lines. Phosphorylation of Tyr14 in CAV-1 and transcriptional regulation of CAV-1 expression may have a role in glaucomatous alterations in TM cells.

Dataset Information

Genetic association and stress mediated down-regulation in trabecular meshwork implicates MPP7 as a novel candidate gene in primary open angle glaucoma.

Publications

Genetic association and stress mediated down-regulation in trabecular meshwork implicates MPP7 as a novel candidate gene in primary open angle glaucoma.

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