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MOHD HUSSAIN SHAH, Noemi Tabanera, Subbiah R Krishnadas, Manju Pillai, Paola Bovolenta, Periasamy Sundaresan; Identification and characterization of variants and a novel 4bp deletion in the regulatory region of SIX6, a risk factor for Primary Open Angle Glaucoma . Invest. Ophthalmol. Vis. Sci. 2017;58(8):4900.
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© ARVO (1962-2015); The Authors (2016-present)
The SIX6 gene has been associated with POAG in different populations. Here, we studied if there exists a genetic association between SIX6 with POAG and/or its related quantitative endophenotypes in the south Indian population.
The cohort of individuals analyzed included 265 POAG and 265 age matched controls from the south Indian population. All individuals received a comprehensive ocular examination before the collection of blood samples, to extract genomic DNA. PCR amplification, taqman based allelic discrimination assay, automated sequencing of the SIX6 and its enhancer region were performed on the purified DNA samples. A novel 4 base pair deletion identified in the SIX6 enhancer region was functionally tested using transgenesis in zebrafish and luciferase assay in HEK293 cell line
We sequenced the SIX6 gene and its enhancer region and identified two known rare, two common variants and a novel 4bp deletion in an already characterized putative enhancer region (Chr14:60974427-60974430). In contrast to previous studies, we could not establish a significant genetic association between the rs33912345 (Odds ratio (OR) =0.919, P=0.4) and rs10483727 SNPs at the SIX1-SIX6 locus OR=0.95, P=0.58) with POAG . However, patients carrying either the rs33912345 ‘C’ or the rs10483727 “T” risk alleles presented a significant and dose-dependent reduction of the peripapillary retinal nerve fiber layer thickness (RNFL), which was more evident in the superior and temporal-inferior quadrants, well in agreement with the knowledge that RNFL become thinner in glaucoma patients. We also established that patients carrying two copies of the ‘‘C’’ or ‘‘T’’ risk allele presented a statistically significant increase of the vertical cup disc ratio (p=0.012; p=0.009).Using transgenesis in zebrafish and luciferase assay, we determined that the newly identified 4bp deletion in the enhancer region significantly reduces reporter expression in retinal ganglion and amacrine cell layers, where human SIX6 is expressed, suggesting that SIX6 activity might be reduced in the retina of patients carrying this deletion.
Our data provides additional support to the previously proposed implication of SIX6 variants as POAG risk factors and further suggests that the reduced levels of SIX6 expression might be implicated in POAG pathogenesis.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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