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.


CONCLUSIONS
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.
Acharya
Moulinath
M
Molecular & Human Genetics Division, Indian Institute of Chemical Biology, Kolkata, India. moulinath@gmail.com <moulinath@gmail.com>
Mookherjee
Suddhasil
S
Bhattacharjee
Ashima
A
Thakur
Sanjay K D
SK
Bandyopadhyay
Arun K
AK
Sen
Abhijit
A
Chakrabarti
Subhabrata
S
Ray
Kunal
K
D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D001483:Base Sequence; D016022:Case-Control Studies; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis* / Q000235:genetics*; D005901:Glaucoma / Q000235:genetics*; D006801:Humans; D007194:India; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011509:Proteoglycans / Q000096:biosynthesis* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D016326:Extracellular Matrix Proteins; C404108:OPTC protein, human; D011509:Proteoglycans