Analysis of genetically determined gene expression suggests role of inflammatory processes in exfoliation syndrome

Abstract

Background: Exfoliation syndrome (XFS) is an age-related systemic disorder characterized by excessive production and progressive accumulation of abnormal extracellular material, with pathognomonic ocular manifestations. It is the most common cause of secondary glaucoma, resulting in widespread global blindness. The largest global meta-analysis of XFS in 123,457 multi-ethnic individuals from 24 countries identified seven loci with the strongest association signal in chr15q22-25 region near LOXL1. Expression analysis have so far correlated coding and a few non-coding variants in the region with LOXL1 expression levels, but functional effects of these variants is unclear. We hypothesize that analysis of the contribution of the genetically determined component of gene expression to XFS risk can provide a powerful method to elucidate potential roles of additional genes and clarify biology that underlie XFS.

Results: Transcriptomic Wide Association Studies (TWAS) using PrediXcan models trained in 48 GTEx tissues leveraging on results from the multi-ethnic and European ancestry GWAS were performed. To eliminate the possibility of false-positive results due to Linkage Disequilibrium (LD) contamination, we i) performed PrediXcan analysis in reduced models removing variants in LD with LOXL1 missense variants associated with XFS, and variants in LOXL1 models in both multiethnic and European ancestry individuals, ii) conducted conditional analysis of the significant signals in European ancestry individuals, and iii) filtered signals based on correlated gene expression, LD and shared eQTLs, iv) conducted expression validation analysis in human iris tissues. We observed twenty-eight genes in chr15q22-25 region that showed statistically significant associations, which were whittled down to ten genes after statistical validations. In experimental analysis, mRNA transcript levels for ARID3B, CD276, LOXL1, NEO1, SCAMP2, and UBL7 were significantly decreased in iris tissues from XFS patients compared to control samples. TWAS genes for XFS were significantly enriched for genes associated with inflammatory conditions. We also observed a higher incidence of XFS comorbidity with inflammatory and connective tissue diseases.

Conclusion: Our results implicate a role for connective tissues and inflammation pathways in the etiology of XFS. Targeting the inflammatory pathway may be a potential therapeutic option to reduce progression in XFS.

Keywords: Exfoliation syndrome; GTEx; GWAS; TWAS; predicted expressions; transcriptomics.

Fig. 1

Manhattan plot for GWAS meta-analysis and PrediXcan analysis of the genotyping data for XFS. a The lower half of the plot is for the XFS meta-analysis summary statistics data Aung et al., 2017, while the upper half of the plot shows results from PrediXcan analysis for 48 GTEx tissues. On the X axis is plot of variant/gene associations along the chromosomes, while Y axis represent the significance levels for the associations. The legend for PrediXcan analysis on the 48 GTEx tissues, a color for each tissue, is on the right. For both plots the blue dotted line is the “suggestive” genome-wide significant threshold (p < 1e-4), while the red line is the genome-wide significant threshold. On the lower plot, the gene labels are for genes reported/mapped to genome-wide significant signals in GWAS result, while in the upper plot is for genes that are associated at genome-wide significant threshold. For genes associated with XFS at genome-wide threshold in more than one tissues, only the tissue with lowest p-value is labeled. The GWAS plot has been truncated to p < 1e-220 for clarity. b genes in the region in chromosome 15 that show significant association. The size of the balloon for each gene-tissue association is proportional to -log10_pvalue and color corresponds to the predicted direction of expression changes: dark-red and blue for increased and decreased expression changes, respectively. Only four genes (EDC3, ULK3, HCN4 & FAM219B) in the whole region were not associated with XFS

Fig. 2

Conditional analysis to prioritize XFS associated genes: a) Manhattan plot for PrediXcan analysis of European ancestry individuals in tissues with predicted gene expression for a) LOXL1 and b) conditioned on LOXL1 predicted gene expressions c) Manhattan plot for PrediXcan analysis of European ancestry individuals in tissues with predicted gene expression for STOML1 and d) conditioned on STOML1 predicted gene expressions e) correlation in gene expression in for genes in chr15q22–25 in lung tissue for i) reference GTEx data f) predicted gene expression in BioVU cohort. In each case on the X axis is plot of variant/gene associations along the chromosomes, while Y axis represent the significance levels for the associations. The legend for PrediXcan analysis on the GTEx tissues, a color for each tissue, is on the right. For both plots the blue dotted line is the “suggestive” genome-wide significant threshold (p < 1e-4), while the red line is the genome-wide significant threshold. On the lower plot, the gene labels are for genes reported/mapped to genome-wide significant signals in GWAS result, while in the upper plot is for genes that are associated at genome-wide significant threshold. For genes associated with XFS at genome-wide threshold in more than one tissues, only the tissue with lowest p-value is labeled. g linkage disequilibrium between variants in prediction models for LOXL1 and other chr15q22–25 genes associated with XFS in lung tissue based on pairwise r² and D′ parameters. Relative genome location for variants in each gene models are roughly demarcated by diagonal lines next to gene symbols. Proximate location for the variant shared between LOXL1 and STOML1, rs12102019 is labelled

Fig. 3

Expression of NEO1, CD276, INSYN1, LOXL1, STOML1, UBL7, ARID3B and SCAMP2 mRNA in iris tissues derived from normal human donors (control) (n = 19) and donors with XFS syndrome (n = 12) using real-time PCR technology. Expression levels were reduced in XFS specimens compared to control specimens, with significant differences for NEO1, CD276, LOXL1, UBL7, ARID3B and SCAMP2. The relative expression levels were normalized relative to GAPDH and are represented as mean values ± SD (*p < 0.05; **p < 0.01, ***p < 0.001)