Variants associated with exfoliation glaucoma affect promoter activity of the LOXL1 antisense gene

Abstract

Purpose: Exfoliation glaucoma (XFG) is the most common identifiable form of open-angle glaucoma in the world. Coding variants in LOXL1 have been significantly associated with increased risk of XFG in populations worldwide. However, none are shared across all populations, suggesting that XFG may be caused by alterations in the regulatory regions of LOXL1. In a South African exfoliation dataset, the most significantly associated variants are located within the first intron of the LOXL1 gene. Interestingly, this region is located upstream of the LOX1 antisense RNA (LOXL1-AS1), which encodes a regulatory RNA for LOXL1 on the antisense strand of LOXL1. Here, we evaluate whether these highly associated intronic variants alter the promoter activity of LOXL1-AS1.

Methods: To study the promoter activity, we used a dual-luciferase reporter assay to characterize a 7kb fragment of intron 1, and to determine the effects of 5 selected SNPs within that region, all of which are highly associated with XFG in the South African dataset. These SNPs include rs1550437, rs6495085, rs6495086, rs8034403, and rs8034017. The 7 kb risk haplotype was cloned, promoter activity was tested in a HEK293 cell line (embryonic kidney cells), and the effect of substituting non-risk alleles was studied for each SNP individually.

Results: Data from the dual-luciferase reporter assay indicated that the 7kb-region in intron 1 contains a promoter for LOXL1-AS1, with the strongest activity localized in a 1600bp fragment immediately proximal to intron 1. Substitution with the non-risk alleles for SNPs rs8034403 and rs8034017 resulted in reduced luciferase activity (p<0.01). However, substitution with the non-risk alleles for SNPs rs1550437, rs6495085, and rs6495086 resulted in increased luciferase activity (p<0.01). Interestingly, the full 7 kb fragment containing risk alleles of these 5 SNPs eliminated all promoter activity.

Conclusions: Our dual-luciferase assay data suggests that XFG-associated variants could contribute to XFG risk by altering promoter activity for LOXL1-AS1. Altered LOXL1-AS1 expression may regulate the expression of LOXL1, which serves as a reasonable mechanism for disease risk. Future directions include testing of full risk and non-risk haplotypes, and determining the impact of overexpression or knockdown of LOXL1-AS1 on LOXL1 expression and activity.

Keywords: 539 genetics