Abstract
BackgroundWe 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.ResultsWe 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.ConclusionOur 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.
Highlights
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
Three different genes, Myocilin (MYOC) and Optineurin (OPTN) and WDR36 [4,5,6] and eleven additional chromosomal loci have been reported to be linked to POAG [5,6,7,8,9,10,11,12,13,14]
We examined Opticin (OPTC) as a candidate for POAG since it is expressed in the trabecular meshwork – the ocular site where the POAG related pathogenesis occurs [18]
Summary
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. 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, sitedirected 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. Primary Open Angle Glaucoma (POAG) is the most common form of the disease [3] where the vision is lost in a progressive manner and, if untreated, results in bilateral blindness. About 33 million people are affected with POAG [3]. Digenic form of the disease has been reported based on mutations in MYOC and CYP1B1 genes [15]
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