Genetic association study of exfoliation syndrome identifies a protective rare variant at LOXL1 and five new susceptibility loci

Abstract

Exfoliation syndrome (XFS) is the most common known risk factor for secondary glaucoma and a major cause of blindness worldwide. Variants in two genes, LOXL1 and CACNA1A, have previously been associated with XFS. To further elucidate the genetic basis of XFS, we collected a global sample of XFS cases to refine the association at LOXL1, which previously showed inconsistent results across populations, and to identify new variants associated with XFS. We identified a rare protective allele at LOXL1 (p.Phe407, odds ratio (OR) = 25, P = 2.9 × 10^-14) through deep resequencing of XFS cases and controls from nine countries. A genome-wide association study (GWAS) of XFS cases and controls from 24 countries followed by replication in 18 countries identified seven genome-wide significant loci (P < 5 × 10^-8). We identified association signals at 13q12 (POMP), 11q23.3 (TMEM136), 6p21 (AGPAT1), 3p24 (RBMS3) and 5q23 (near SEMA6A). These findings provide biological insights into the pathology of XFS and highlight a potential role for naturally occurring rare LOXL1 variants in disease biology.

Figure 1

LOXL1 p.Y407F regulates ECM synthesis and improves cellular adhesion. (a) Schematic diagram of LOXL1 indicating the protein domain positions for the variants evaluated in this study. b) Immunofluorescent staining of HA-tagged LOXL1 variants overexpressed in HLEC cells labelled with anti-HA for detection of overexpressed forms of LOXL1 (red) and elastin (green). Cell nuclei are stained in blue. The heat map for elastin indicates the intensity of elastin staining from red (increased expression) to purple (decreased expression). c) Immunofluorescent staining of HA-tagged LOXL1 variants overexpressed in HLEC cells labelled with anti-HA for detection of overexpressed forms of LOXL1 (red) and collagen IV (green). Cell nuclei are stained in blue.. The heat map for collagen IV indicates the intensity of collagen IV staining from red (increased expression) to purple (decreased expression). d) Immunofluorescent staining of HA-tagged LOXL1 variants overexpressed in HLEC cells labelled with anti-HA for detection of overexpressed forms of LOXL1 (red) and fibrillin 1 (green). Cell nuclei are stained in blue. The heat map for fibrillin 1 indicates the intensity of fibrillin 1 staining from red (increased expression) to purple (decreased expression). e) Cumulative average of impedance values (as a surrogate for cellular adhesion strength) measured over 35h post nucleofection of HLECs overexpressing the four tested LOXL1 haplotypes. Data represent mean ± s.e.m. of four independent experiments. ^** represents P<0.01 when compared against the rare, protective LOXL1 p.407F-carrying G-A-T haplotype. The four haplotypes tested were LOXL1-(G-A-T), - (G-A-A), -(T-G-A) and -(G-G-A). This experiment was further validated in Supplementary Figure 7.

Figure 2

Manhattan plot of the results from the GWAS discovery plus replication meta-analysis comprising 13,620 XFS cases and 109,837 controls. Genetic markers are plotted according to chromosomal location on the horizontal axis and statistical significance on the vertical axis. SNP markers at seven independent loci surpass genome-wide significance (defined as P<5×10⁻⁸). They are LOXL1, CACNA1A, POMP, TMEM136, AGPAT1, SEMA6A and RBMS3.

Figure 3

Expression of POMP protein in ocular tissues of normal human donor eyes and donor eyes with XFS, as determined by Western blotting and immunohistochemistry. Immunofluorescence labelling of normal eye tissues shows punctate POMP immune-positivity (green fluorescence) in the cytoplasm of the corneal epithelium (A), the corneal endothelium (B), limbal epithelium and stromal cells (C), trabecular meshwork endothelium (D), ciliary epithelium (E), and retinal cell layers (F). Reduced POMP protein expression levels in iris and ciliary body tissues of XFS eyes compared to age matched controls are shown by Western blot analysis (G), and by immunofluorescence labelling of iridal (H,L) and ciliary epithelia (J,M) as well as vascular endothelia in the iris (K,N). Reduced staining intensity in XFS tissues is associated with LOXLI-positive exfoliation material accumulations (red immunofluorescence) on the surface of the iris pigment epithelium (L), ciliary epithelium (M) and iris blood vessel walls (N). Western blot (cropped images) and densitometry analysis shows reduced POMP protein expression in iris and ciliary body tissue lysates of XFS eyes compared to control eyes (G). Data are shown as the POMP/B-actin ratio (n=6 for each group; mean ± standard deviation; *P<0.01; **P<0.005); uncropped versions of all Western blots are shown in Supplementary Figure 16. (BV blood vessel, CE ciliary epithelium, CoE corneal epithelium, DM Descemet membrane, GCL retinal ganglion cell layer, INL inner nuclear layer, IPE iris pigment epithelium, LE limbal epithelium, ONL outer nuclear layer, SC Schlemm’s canal, ST stroma, TM trabecular meshwork; DAPI nuclear counterstain in blue; scale bars = 100 μm in C,D,F and 20 μm in A,B,E,H-N).

Figure 4

Expression of TMEM136 protein in ocular tissues of normal human donor eyes and donor eyes with XFS, as determined by Western blotting and immunohistochemistry. Immunofluorescence labelling of normal eye tissues shows cytoplasmic TMEM136 immunopositivity (green fluorescence) in limbal blood vessels (A), trabecular meshwork and Schlemm’s canal endothelium (B), walls of aqueous veins (arrows) (C), blood vessels of the iris (arrows) (D), blood vessels and epithelia of the ciliary body (E), and retinal blood vessels and cell layers (F). Reduced TMEM136 protein expression levels in iris and ciliary body tissues of XFS eyes compared to age matched controls are shown by Western blot analysis (G), and by immunofluorescence labelling of iridal (H,L) and ciliary epithelia (J,M) as well as vascular endothelia in the iris (K,N). Reduced staining intensity in XFS tissues is associated with LOXL1- positive exfoliation material accumulations (red immunofluorescence) on the surface of the iris pigment epithelium (L), ciliary epithelium (M) and iris blood vessel walls (N). Western blot (cropped images) and densitometry analysis shows reduced TMEM136 protein (isoform 1 at 28 KD and isoform 3 at 31 KD) expression in iris and ciliary body tissue lysates of XFS eyes compared to control eyes (G). Data are shown as the TMEM136/B-actin ratio (mean ± standard deviation; n=6 for each group; *P<0.01; **P<0.005); uncropped versions of all Western blots are shown in Supplementary Figure 17. (AV aqueous vein, BV blood vessel, CE ciliary epithelium, DIL dilator muscle, GCL retinal ganglion cell layer, INL inner nuclear layer, IPE iris pigment epithelium, LE limbal epithelium, ONL outer nuclear layer, SC Schlemm’s canal, ST stroma, TM trabecular meshwork; DAPI nuclear counterstain in blue; scale bars = 200 μm in A, 100 μm in B-F and 20 μm in H-N).