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

Studies on Glucoma Genes: Molecular Defects in Primary Open Angel Glucoma Patients and Single Nuclotide Polymorphism in Indian Population.

Glaucoma, the second leading cause of blindness, is a heterogeneous group of optic neuropathies having complex genetic bases. Primary Open Angle glaucoma (POAG) is the most prevalent among glaucoma subtypes. Among the 14 reported loci implicated for the disease, only three underlying candidate genes have been characterized till date viz. Myocilin (MYOC), Optineurin (OPTN) and WDR36. Among them MYOC has been reported to be responsible for 2-4% of the adult onset cases of the disease whereas there has been relatively less evidence for any major involvement of OPTN and WDR36 with the disease till date. While digenic cases involving mutations in MYOC and CYP1B1 have been associated with the disease, SNPs in genes like APOE and p53 have also been reported to contribute to the apparent complexity of the disease. Hence analysis of the role of other genetic variation like intragenic SNPs as potential predisposing factors for the disease would be a natural strategy to decipher the complex genetic etiology of 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

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