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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 RXR? (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.

SUBMITTER: Pasutto F

PROVIDER: S-EPMC5457519 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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

Pasutto Francesca F Zenkel Matthias M Hoja Ursula U Berner Daniel D Uebe Steffen S Ferrazzi Fulvia F Schödel Johannes J Liravi Panah P Ozaki Mineo M Paoli Daniela D Frezzotti Paolo P Mizoguchi Takanori T Nakano Satoko S Kubota Toshiaki T Manabe Shinichi S Salvi Erika E Manunta Paolo P Cusi Daniele D Gieger Christian C Wichmann Heinz-Erich HE Aung Tin T Khor Chiea Chuen CC Kruse Friedrich E FE Reis André A Reis André A Schlötzer-Schrehardt Ursula U

Nature communications 20170523

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 intron ...[more]

PMID: 28534485

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Project description:BACKGROUND: LOXL1 gene is the most important genetic risk factor known so far for pseudoexfoliation glaucoma (XFG). Our purpose was to evaluate the potential association of individual genetic variants of the lysyl oxidase-like 1 (LOXL1) gene and haplotypes with XFG in Spanish patients. METHODS: Blood samples were collected from a total of 105 Spanish patients with XFG and 200 healthy controls. The entire LOXL1 gene along with the promoter, coding and non-coding regions including the 5'- and 3'-untranslated regions, were sequenced using next-generation sequencing in 99 XFG patients. SNPs rs16958477 (promoter), rs1048661 (exon 1), rs3825942 (exon 1), rs2165241 (intron 1) and rs3522 (exon 7) in LOXL1 were genotyped by restriction fragment-length polymorphism (RFLP) in all Spanish control participants and in six additional XFG patients, and a case-control association study was performed. Comparisons of the allelic and genotypic frequencies were performed using standard ?(2) test with Bonferroni and Pearson corrections. Logistic regression analyses were permormed using Sigmaplot v11. Haplotypes frequencies were performed using HaploView 4.0. RESULTS: Sequencing of the LOXL1 gene in XFG participants identified a total of 212 SNPs, of which 49 exhibited allelic frequencies with significant differences between cases and controls, and 66 were not previously described. The allele frequencies of SNPs rs16958477, rs1048661, rs3825942, rs2165241, were significantly associated with an increased risk for XFG, however the SNP rs3522 was not. The haplotype frequencies of SNPs rs16958477, rs1048661, rs3825942 and rs2165241 and their association with XFG indicated that the CGGT haplotype, containing all four risk alleles, and the AGGT haplotype, which carries the protective allele of rs16958477 and three risk alleles of the other three SNPs, were significantly associated with XFG (p?=?4.5×10(-6), and p?=?8.8×10(-6)), conferring more than 2-fold increased disease susceptibility. CONCLUSIONS: SNPs of the LOXL1 gene are associated with XFG in the Spanish population. This information adds new support to the distinct risk association frequencies of LOXL1 alleles with XFG in Western European and Asian populations. Identification and validation of additional SNPs along the entire LOXL1 gene of XFG cases may provide insightful information on their potential role in the pathogenesis of this disease.

Project description:PurposeTo study lysyl oxidase-like 1 (LOXL1) expression in freshly collected lens capsules from pseudoexfoliation syndrome (XFS), pseudoexfoliation glaucoma (XFG), and normal cataract control individuals. We also investigated the effects of four glaucoma drug medications on LOXL1 expression in primary human lens epithelial cell cultures to see if they could affect LOXL1 expression.MethodsLens capsules were collected at the time of cataract surgery. Controls were matched to age, sex, and ethnicity. Total RNA was isolated from individual lens capsule samples and real-time PCR was performed on each sample using primers flanking the sixth exon of the LOXL1 gene. Cell cultures were grown to confluence in four separate six-well plates at 37 °C in 5% CO2. Each plate was then treated with one of four different glaucoma drugs (brinzolamide 1%, brimonidine tartrate 0.1%, timolol maleate 0.5%, and latanoprost 0.005%) once daily for seven days (at both 1:1,000 and 1:100 concentrations relative to media). Controls were not treated with any drug but media was changed in the same manner. After one week of treatment, cells were harvested and total RNA isolated. Real-time PCR was performed on each group of cells.ResultsSeven XFS, seven XFG, and ten cataract control specimens were analyzed. LOXL1 expression was detected in the lens capsule specimens from each of the four groups. Significant expression differences were found between the control and XFG groups and XFS and XFG groups. No significant difference was observed between the control and XFS group. No significant decrease in LOXL1 expression was seen with drug incubation of the four medications (Brinzolamide, Timolol, Latanoprost, and Brimonidine) at the 1:1,000 drug:media concentrations versus controls. At 10-fold higher concentrations (1:100 drug:media), brinzolamide, timolol maleate, and latanoprost showed small increases in LOXL1 expression relative to controls. This effect was not observed with brimonidine tartrate.ConclusionsThese results establish that LOXL1 expression is reduced in lens capsule specimens from XFG individuals but not XFS. The drug treatment incubation studies suggest that the change in LOXL1 expression observed in XFG is not attributable to glaucoma drug therapy. If a causative functional relationship can be validated, modification of LOXL1 expression in affected tissues may represent a novel treatment strategy for this disorder.

Project description:PURPOSE: Single nucleotide polymorphisms (SNPs) within the lysyl oxidase like-1 gene (LOXL1; rs1048661 and rs3825942) were found to confer risk to pseudoexfoliation glaucoma (XFG) through the pseudoexfoliation syndrome (XFS) in Nordic, Caucasian, and two Asiatic populations (Indian and Japanese). The prevalence (0.2%-0.7%) of XFS in the Chinese is considerably lower compared to Nordic populations. The aim of this study was to determine the association of LOXL1 in Chinese subjects with XFS/XFG. METHODS: Chinese subjects with clinically diagnosed XFS/XFG and normal controls were recruited. Genomic DNA was extracted, and the two LOXL1 SNPs (rs1048661 and rs3825942) were genotyped by bidirectional sequencing. Allele and genotype frequencies were compared between cases and unrelated controls using PLINK. Linkage disequilibrium (LD) calculations and haplotype association analysis were done using the Haploview package and WHAP package, respectively. RESULTS: Sixty-two Chinese patients (17 XFG and 45 XFS) and 171 Chinese controls were studied. The G allele of LOXL1 SNP rs3825942 was moderately associated (OR=10.97, p=0.0018) with pseudoxfoliation in the Chinese. The frequency of the G allele of rs1048661 was not significantly different in cases compared to controls (p=0.142) in the allelic association test. However, the genotype test showed marginal association for rs1048661 (p=0.030). Only three haplotypes were observed (T-G, G-G, and G-A) with G-G as a risk haplotype (p=0.0034) and G-A as a protective haplotype (p=0.00039). T-G, which was a risk haplotype in the Japanese, was not associated with XFG in the Chinese (p=0.124). CONCLUSIONS: Polymorphisms in LOXL1 confer risk to XFS/XFG in the Chinese. The lower incidence of XFS compared to other populations suggests additional genetic or environmental factors to have a major influence on the phenotypic expression of XFS in the Chinese. The G allele of rs3825942 has been shown to be associated with XFS/XFG in all populations studied to date.

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

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

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