Pseudoexfoliation syndrome-associated genetic variants affect transcription factor binding and alternative splicing of LOXL1

Abstract

Although lysyl oxidase-like 1 (LOXL1) is known as the principal genetic risk factor for pseudoexfoliation (PEX) syndrome, a major cause of glaucoma and cardiovascular complications, no functional variants have been identified to date. Here, we conduct a genome-wide association scan on 771 German PEX patients and 1,350 controls, followed by independent testing of associated variants in Italian and Japanese data sets. We focus on a 3.5-kb four-component polymorphic locus positioned spanning introns 1 and 2 of LOXL1 with enhancer-like chromatin features. We find that the rs11638944:C>G transversion exerts a cis-acting effect on the expression levels of LOXL1, mediated by differential binding of the transcription factor RXRα (retinoid X receptor alpha) and by modulating alternative splicing of LOXL1, eventually leading to reduced levels of LOXL1 mRNA in cells and tissues of risk allele carriers. These findings uncover a functional mechanism by which common noncoding variants influence LOXL1 expression.

Posttranscriptional Regulation of LOXL1 Expression Via Alternative Splicing and Nonsense-Mediated mRNA Decay as an Adaptive Stress Response

“…39 On the other hand, LOXL1 expression was found to be significantly downregulated below homeostatic levels in elastin-rich intraand extraocular connective tissues, such as the lamina cribrosa and aortic wall. 19,26 Reduced LOXL1 expression levels were found to be associated with pronounced elastotic and biomechanical alterations, which have been suggested to predispose to the development of glaucoma and cardiovascular FIGURE 2. Effects of NMD suppression on expression levels of LOXL1 and LOXL1-a.…”

“…24 All hTCF were homozygous for the risk alleles of the two coding SNPs, rs1048661T>G and rs3825942A>G, as well as the four intronic SNPs, rs12905253G>A, r s11638944C>G, rs12441130T>C, rs11631579A>G, which had been previously shown to influence alternative splicing of LOXL1. 26 Primary human optic nerve head astrocyte (hONHA) cultures were generated from lamina cribrosa tissue of five healthy donor eyes (74 6 9 years) with appropriate research content as previously described. 19 Primary human trabecular meshwork cells (hTMC) were obtained from Provitro (Berlin, Germany) and grown in Dulbecco's modified Eagle's medium (DMEM; Pan Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (FBS; Pan Biotech) and 1% antibiotic (penicillin/streptomycin/amphotericin B; Pan Biotech).…”

“…One of these SNPs, rs11638944:C>G, was found to act as an expression quantitative trait locus affecting transcriptional output of LOXL1, mediated by differential transcription factor binding and alternative pre-mRNA splicing. 26 We showed that increased transcriptional activity at this locus is associated with reduced binding of retinoid X receptor alpha and with enhanced alternative splicing coupled with nonsense-mediated decay (NMD), which altogether reduces the levels of LOXL1 mRNA in cells and tissues with the alternative allele. The alternatively spliced transcript, LOXL1-a, includes an additional exon (Ex1A) in the first intron of LOXL1, introducing a premature termination codon (PTC) in exon 2 via frameshift, subjecting this unproductive isoform to NMD.…”

“…33 An essential intermediary step, by which stress factors inhibit the NMD pathway to downregulate gene expression, is the phosphorylation of the eukaryotic translation initiation factor 2-a (eIF2-a), known to be a critical factor for the initiation of many stress pathways. 34 Based on our previous finding that constitutive NMD-coupled alternative splicing is influenced by PEXassociated genetic variants in LOXL1, 26 we hypothesized that this pathway represents a means to regulate steady-state levels of LOXL1 mRNA and protein as an adaptive response to PEX-associated cues and stress factors.…”

Recent advances in risk factors associated with ocular exfoliation syndrome

“…It has been suggested that the risk sequence had an increased activity at the transcriptional level that was associated with decreased binding affinity of transcription factor (RXR α ) and enhanced differential splicing of LOXL1 pre‐mRNA along with nonsense‐mediated mRNA decay (Pasutto et al. ). Differential splicing coupled to nonsense‐mediated mRNA decay was later shown to regulate expression of LOXL1 at the post‐transcriptional level in response to XFS‐associated environmental and nutritional factors (Berner et al.…”