Identification and characterization of variants and a novel 4 bp deletion in the regulatory region of , a risk factor for primary open-angle glaucoma.

Background: Primary open-angle glaucoma (POAG) is a complex disease of multigenic inheritance and the most common subtype of glaucoma. encodes a transcription factor involved in retina, optic nerve, and pituitary development. Previous studies showed a genetic association between the locus and POAG, identifying risk alleles. Whether these alleles are present also in the south Indian population is unclear.

Methods: To address this question, the gene and an already characterized and highly conserved enhancer (Ch14:60974427-60974430) were sequenced in two south Indian cohorts, respectively, composed of 65/65 and 200/200 POAG cases/age-matched controls. We next used Taqman-based allelic discrimination assay to genotype a common variant (rs33912345: c.421A>C) and the rs1048372 SNP in two cohorts, respectively, composed of 557/387 and 590/448 POAG cases/age-matched controls. An additional cohort of 153 POAG cases was subsequently recruited to assess the association of the rs33912345:c.421A>C and rs10483727 variants with more prominent changes in two POAG diagnostic parameters: retinal nerve fiber layer thickness and vertical cup/disc ratio, using spectral domain optical coherence tomography. The activity of the newly identified enhancer variants was assessed by transgenesis in zebrafish and assays.

Results: We identified two known rare and two common variants in the locus and a novel 4 bp deletion in the analyzed enhancer. Contrary to previous studies, we could not establish a significant association between the rs10483727 and rs33912345:c.421A>C variants and PAOG in the south Indian ethnicity but patients carrying the corresponding C or T risk alleles exhibited a dose-dependent reduction of the thickness of the retinal nerve fiber layer and a significant increase in the vertical cup/disc ratio. Transgenesis in zebrafish and assays demonstrated that the newly identified 4 bp deletion significantly reduced reporter expression in cells of the retinal ganglion and amacrine layers, where human is expressed.

Conclusion: Altogether, our data further support the implication of variants as POAG risk factors and implicates haploinsufficiency in POAG pathogenesis.