Evaluation of the OPTC gene in primary open angle glaucoma: functional significance of a silent change

BACKGROUND: We investigated the molecular basis of primary open-angle glaucoma (POAG) using Opticin (OPTC) as a candidate gene on the basis of its expression in the trabecular meshwork cells involved in the disease pathogenesis. Two hundred POAG patients and 100 controls were enrolled in this study. The coding sequence of OPTC was amplified by PCR from genomic DNA of POAG patients, followed by SSCP, DHPLC and DNA sequencing. Subsequent bioinformatic analysis, site-directed mutagenesis, quantitative RT-PCR and western blot experiments were performed to address the functional significance of a 'silent' change in the OPTC coding region while screening for mutations in POAG patients. RESULTS: We detected two missense (p.Glu66Gly & p.Ile89Thr) and one silent change (p.Phe162Phe; c.602 C>T) that was present in 3 different patients but in none of the 100 controls screened. The mutant (c.602T) mRNA was predicted to have remarkably different secondary structure compared to the wild-type transcript by in silico approaches. Subsequent wet-lab experiments showed lower expression of the gene both at the mRNA and protein levels. CONCLUSION: Our study suggests OPTC as a candidate gene for POAG. Further, it highlights the importance of investigating the 'silent' variations for functional implication that might not be apparent from only in silico analysis

Molecular Characterization of Primary Open Angel Glucoma In Indian Patients.

Glaucoma, a heterogeneous group of neurodegenerative disorders of the eye, affects about 67 million people worldwide. In India about 1.5 million people are blind due to glaucoma. Among different subtypes, Primary Open Angle Glaucoma (POAG) is the most common form of the disease where loss of vision is progressive and often silent. To find the genetic basis of the disease among Indians, I have been involved in screening for mutation in multiple candidate genes, both implicated and projected based on the expression in trabecular meshwork (TM) cells and evaluation of the allelic variants including functional analysis where feasible and warranted. In addition, potential functional implication for the digenic mode of inheritance of MYOC and CYP1B1 defects has been examined, and potential association of p53 polymorphism with POAG has been investigated to explore the complex nature of the disease. In most cases, the study was conducted using a pool of 200 unrelated POAG patients comprising 155 sporadic and 45 familial cases and 100 unrelated ethnically matched controls. A brief overview of the findings from the studies is presented below

Distribution of p53 codon 72 polymorphism in Indian primary open angle glaucoma patients

Purpose: Glaucoma is a complex neurodegenerative disorder of the eye. Primary Open Angle Glaucoma (POAG) is the most common type, accounting for over half of the total cases. Recently, a significant difference in the distribution of the codon 72 polymorphism of the tumor suppressor gene p53 between control subjects and POAG patients of Chinese origin (p=0.00782) was demonstrated. The proline residue at codon 72 of the p53 gene was significantly over represented in the POAG patients relative to healthy controls. The purpose of this study was to investigate whether the reported association between the p53 polymorphism and POAG is a common phenomenon irrespective of geographical location or ethnicity of the population. Methods: Sixty seven unrelated POAG patients, ranging from 10-65 years of age (mean±SD of 41.16±18.52 years), and 112 control subjects having a similar age range of 18-63 years (mean±SD of 36.64±14.65 years) were enrolled in this study. A region of the p53 gene encompassing two polymorphic sites, a 16 bp duplication in intron 3 and a BstU I RFLP in exon 4, were amplified by polymerase chain reaction from Indian POAG patients and normal healthy controls. A single base change (G to C) in codon 72 alters the amino acid residue from arginine to proline and removes the polymorphic BstU I site mentioned above. The amplified DNA fragments were digested with the restriction enzyme and the digestion patterns of the fragments were used to identify the alleles for both the polymorphic sites. Results: No significant association between p53 alleles and Indian POAG patients were observed by analyzing either codon 72 polymorphism (p=0.5627) or the intronic 16 bp duplication polymorphism (p=0.059). Haplotype analysis, reported to be a better predictor of association of the p53 gene with different types of cancer, was also performed and no association of any haplotype was detected with POAG (p=0.1831). Conclusions: Association between the p53 gene encoding for proline at codon 72 and POAG presumably exists in some ethnic populations but cannot be used as a predictor for the role of the gene as a common regulator of cell death of retinal ganglions leading to POAG

Molecular Basis for Involvement of CYP1B1 in MYOC Upregulation and Its Potential Implication in Glaucoma Pathogenesis

<div><p><em>CYP1B1</em> has been implicated in primary congenital glaucoma with autosomal recessive mode of inheritance. Mutations in <em>CYP1B1</em> have also been reported in primary open angle glaucoma (POAG) cases and suggested to act as a modifier of the disease along with <em>Myocilin</em> (<em>MYOC</em>). Earlier reports suggest that over-expression of myocilin leads to POAG pathogenesis. Taken together, we propose a functional interaction between CYP1B1 and myocilin where 17β estradiol acts as a mediator. Therefore, we hypothesize that 17β estradiol can induce <em>MYOC</em> expression through the putative estrogen responsive elements (EREs) located in its promoter and CYP1B1 could manipulate <em>MYOC</em> expression by metabolizing 17β estradiol to 4-hydroxy estradiol, thus preventing it from binding to <em>MYOC</em> promoter. Hence any mutation in <em>CYP1B1</em> that reduces its 17β estradiol metabolizing activity might lead to <em>MYOC</em> upregulation, which in turn might play a role in glaucoma pathogenesis. It was observed that 17β estradiol is present in Human Trabecular Meshwork cells (HTM) and Retinal Pigment Epithelial cells (RPE) by immunoflouresence and ELISA. Also, the expression of enzymes related to estrogen biosynthesis pathway was observed in both cell lines by RT-PCR. Subsequent evaluation of the EREs in the <em>MYOC</em> promoter by luciferase assay, with dose and time dependent treatment of 17β estradiol, showed that the EREs are indeed active. This observation was further validated by direct binding of estrogen receptors (ER) on EREs in <em>MYOC</em> promoter and subsequent upregulation in <em>MYOC</em> level in HTM cells on 17β estradiol treatment. Interestingly, <em>CYP1B1</em> mutants with less than 10% enzymatic activity were found to increase the level of endogenous myocilin in HTM cells. Thus the experimental observations are consistent with our proposed hypothesis that mutant CYP1B1, lacking the 17β estradiol metabolizing activity, can cause MYOC upregulation, which might have a potential implication in glaucoma pathogenesis.</p> </div

Presence of 17β estradiol in ocular cells.

<p>Confocal images are shown for HTM (<i>Panel A</i>), RPE (<i>Panel B</i>) and HEK 293 (<i>Panel C</i>) cells using anti-17β estradiol antibody and counterstained with FITC labeled secondary antibody. DAPI was used to stain the nucleus. In each panel control cells were treated only with FITC labeled secondary antibody, but not primary antibody, to assess the background noise. The scale of magnification is shown in each panel. The level of 17β estradiol in HTM and RPE cell lines were estimated by ELISA (<i>Panel D</i>). Similar estimation in low glucose (LG) and high glucose (HG) media containing 10% charcoal treated FBS did not show presence of 17β estradiol. The experiments were done in triplicate.</p

CYP1B1 mutants cause upregulation of MYOC in HTM cells.

<p><i>A</i>: <i>Increased MYOC expression with mutant CYP1B1</i>. Western blot analysis of mutant CYP1B1 and myocilin showed increased expression of MYOC in the presence of mutant CYP1B1 clones with reduced (<10%) 17β estradiol metabolizing activity. <b><i>B</i></b><b>: </b><i>Quantitative analysis of MYOC expression</i>. The histogram shows levels of expression of endogenous MYOC in HTM cells transfected with wild type and mutant CYP1B1 clones. All the three mutants of CYP1B1 (i.e. E229K, R368H and R523T) considerably over-expressed MYOC compared to the normal CYP1B1. The R368H and R523T showed statistically significant over expression of myocilin with a p-value of 0.023 and 0.014, respectively. However, the effect of E229K mutant was not found to be statistically significant. This experiment was repeated three times [*p-value- <0.05].</p

Expression of 17β estradiol synthesizing enzymes in HTM and RPE cells.

<p><i>A</i>: 17β estradiol synthesis pathway. The key enzymes are highlighted by red squares. <b><i>B</i></b><b>:</b> Semi-quantitative RT-PCR showing the presence of key 17β estradiol synthesizing enzymes in HTM and RPE cells. Three independent experiments were done for each enzyme in both cell lines. The identity of each product was confirmed by sequencing (data not shown). NTC: No cDNA template control.</p

Functional evaluation of putative EREs in <i>MYOC</i> promoter.

<p><i>A</i>: Serial constructs of <i>MYOC</i>-promoter region containing ERE and AP1 sites cloned in promoter less PGL3 basic vector. Black solid arrows indicate the forward and reverse primers used to amplify the inserts for subcloning. Also, the alphanumeric nomenclature of the constructs corresponds to the first initial of <i>myocilin</i> (M) followed by the size of the insert in base pairs. <b><i>B</i></b><b>:</b> Luciferase activity in extracts from RPE cells transfected with the clones containing <i>MYOC</i> constructs and treated with 17β estradiol (250 nM or 1000 nM). <b><i>C</i></b><b>:</b> Ratio of luciferase activity in cell extracts between induced and uninduced RPE cells for all 4 serial constructs upon dose (250 nM and 1000 nM) and time (4 hrs & 8 hrs) dependent treatment of 17β estradiol. The time points were taken based on the previous experiment in <i>Panel B</i>. <b><i>D</i></b><b>:</b> The M3194 construct was transfected in RPE cells and subjected to increasing amount of 4-hydroxy tamoxifen (4-OHT; 17β estradiol competitor) treatment followed by luciferase assay. A gradual decrease in <i>MYOC</i> promoter activity was observed with increasing amount of 4-OHT. <b><i>E</i></b><b>:</b> Significant upregulation of endogenous myocilin with 17β estradiol treatment in HTM cell. (**p-value<0.001, ***p-value<0.0001). Three independent replicates were performed for all the experiments described here.</p

Nuclear localization of ERα upon 17β estradiol treatment in human RPE cells.

<p><i>A</i>: Confocal images of human RPE cells upon dose (250 mM & 1000 mM) and time (4 hr and 8 hr) dependent treatment with 17β estradiol. Cells were stained with human specific ERα-antibody followed by Alexa Fluor® 488 labeled anti-rabbit secondary antibody (<i>Upper panel</i>). For all conditions, corresponding superimposed image with DAPI are given (<i>Lower panel</i>). Arrows point to the cells where nuclear localization of ERα was observed. <b><i>B</i></b><b>:</b> Histogram showing the percentage of RPE cells with ERα localized in the nucleus upon treatment with 17β estradiol in a dose and time dependent manner. Each experiment was done in triplicate. <b><i>C</i></b><b>:</b> Cross sectional 3D view of nuclear localization of ERα in RPE cell is shown [Scale bar: 10 µm].</p