LOXL1 genetic variants are significantly associated with exfoliation syndrome (XFS); however, the impact of the associated variants on disease development is not yet understood. Initially the associated common missense variants, R141L (Arginine 141 Leucine) and G153D (Glycine 153 Asparate), were considered to be pathogenic alleles.1 “Flipping” of the risk allele in certain populations for both missense variants has provided strong evidence that these missense changes are not biologically significant (Fig. 1).2,3 In addition, in an in vitro assay, the associated missense alleles do not affect LOXL1 enzymatic activity.4 Together, these results suggest that other LOXL1 variant(s), in linkage disequilibrium with the common missense variants, predispose to XFS by affecting protein function and/or gene expression. Several lines of evidence suggest that a reduction in LOXL1 gene expression contributes to disease development and each are discussed in the following sections.
Using ocular tissue samples (iris, lens, and ciliary body) from 25 XFS/exfoliation glaucoma cases and 25 controls, the expression level and localization of LOXL1, LOXL2, and lysyl oxidase (LOX) were investigated and correlated with the LOXL1 risk genotype and also disease stages (early vs. late).5 LOXL1 ocular expression was reduced by approximately 20% per risk allele of rs1048661(R141).5 An effect on gene expression was not observed for G153D. In addition, LOXL1 was observed to have higher levels of expression in early disease stages compared with late and this was not correlated with risk genotypes. The expression of LOXL2 and LOX was not altered in XFS-derived tissue samples compared with controls.
The fact that common LOXL1 missense changes (R141L and G153D) are not likely to have biologically relevant effects suggests that other variants in the LOXL1 genomic region must underlie the significant association observed with disease. Haplotype analysis can be used to identify functional variants within a genomic region. Functional variants could include those in coding and/or noncoding regions. To identify variants associated with disease risk, haplotype analysis using variants distributed throughout the LOXL1 regulatory regions and gene coding regions identified a risk haplotype in a white population from the United States that included a LOXL1 promoter region variant previously shown to reduce gene expression (rs16958477).6,7 These variants and other regulatory elements that could influence LOXL1 expression are shown in Figure 2. Of particular interest are the LOXL1-AS1 (antisense RNA) and its promoter, the LOXL1 promoter elements, and a highly conserved region in the distal 5′ LOXL1 regulatory region. Further work will be necessary to determine whether these elements impact disease pathogenesis.
In addition to LOXL1, several other factors may contribute to XFS pathogenesis, including TGF-β1 (transforming growth factor beta 1), oxidative stress, UV light, and hypoxia. Factors potentially influencing XFS pathogenesis were tested for their effect on LOXL1 expression using cultured fibroblasts from Tenon capsule biopsies from 5 patients with cataract and XFS and 3 patients with cataract without XFS.8 Changes in LOXL1 expression were also investigated for high and low LOXL1 risk haplotypes (based on the R141L and G153D polymorphisms).8 Each of these disease-associated factors (TGF-β1, oxidative stress, UV light, and hypoxia) induced significant increases in expression of LOXL1 and elastic proteins in cells with the low-risk haplotype but had comparatively lower expression in cells with the high-risk haplotype. These results suggest that the high-risk LOXL1 haplotype is associated with reduced gene expression at baseline and also under conditions in which LOXL1 gene expression increases in response to factors impacting disease pathogenesis.
To investigate the impact of loss of LOXL1 function on disease development, the phenotypic features of a LOXL1 null mouse were characterized.9 The LOXL1 null mouse was found to have some features of XFS, including lens abnormalities consistent with cataract and disruption of the blood-aqueous barrier.10 The LOXL1 null mouse did not have deposition of exfoliation fibrillar material or elevated intraocular pressure and glaucoma. These results suggest that loss of LOXL1 enzyme activity contributes to disease predisposition, but that other factors, which may be genetic and/or environmental, are necessary for the disease to be fully manifest.
Collectively, these results support the hypothesis that dysregulation of LOXL1 expression is a contributing factor to exfoliation disease development. Further study defining the regulatory elements necessary for regulation of LOXL1 expression could point to possible therapeutic targets.
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