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Matthias Zenkel, Francesca Pasutto, Panah Liravi, Steffen Uebe, Friedrich E Kruse, Ursula Schlotzer-Schrehardt; Functional regulatory variants of LOXL1 contribute to dysregulated expression of lysyl oxidase-like 1 in pseudoexfoliation syndrome/glaucoma. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4381.
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© ARVO (1962-2015); The Authors (2016-present)
To identify and functionally characterize regulatory variants in the lysyl oxidase-like 1 (LOXL1) gene, which is known to be the main genetic risk factor for pseudoexfoliation (PEX) syndrome/glaucoma.
In addition to the non-synonymous PEX-associated exonic SNPs (rs1048661, rs3825942) we selected 16 SNPs distributed throughout the first intronic region of the LOXL1 gene (chr15:74220326-74235290, GRCh37/hg19) which showed the highest association (p<10-30) with PEX in our German cohorts of 771 PEX patients and 1365 healthy subjects. These SNPs were cloned into a luciferase reporter construct under the control of a 1.5 kb LOXL1 promoter fragment. Dual-luciferase reporter assays were carried out to determine the effects of 7 different risk- and nonrisk-haplotype combinations on LOXL1 promoter activity in HEK293T cells and in Tenon’s capsule fibroblasts and trabecular meshwork cells obtained from PEX and control patients. Total RNA and genomic DNA was isolated from ocular tissue specimens obtained from donor eyes with PEX syndrome/glaucoma (n=40) and age-matched normal donor eyes (n=40). The mRNA expression of LOXL1 was measured by quantitative real-time PCR assays and correlated with the patients’ individual SNP genotypes.
Dual-luciferase reporter assays showed that a risk-haplotype including 14 intronic SNPs (rs8023330, rs1550436, rs2165241, rs28588430, rs28617339, rs4886778, rs8027022, rs2028386, rs4337252, rs12440667, rs12905253, rs11638944, rs12441130, rs11631579) consistently reduced LOXL1 promoter activity by 30% (p<0.01) in Tenon’s capsule fibroblasts and trabecular meshwork cells. Several deletion constructs indicated that the reduction in promoter activity was achieved by 4 SNPs. Further risk-haplotype combinations with the 2 exonic SNPs had no differential effect on promoter activity. In ocular tissues of both PEX and control eyes, expression of LOXL1 was significantly reduced (20%, p<0.01) in correlation with the presence of the risk-conferring haplotype.
These findings point to a functional link between PEX-associated noncoding SNPs of the LOXL1 gene and the dysregulation of LOXL1 expression in PEX tissues, which appears to be a key pathomechanism in glaucoma development in PEX patients.
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