Common variants near ABCA1, AFAP1 and GMDS confer risk of primary open-angle glaucoma

Abstract

Primary open-angle glaucoma (POAG) is a major cause of irreversible blindness worldwide. We performed a genome-wide association study in an Australian discovery cohort comprising 1,155 cases with advanced POAG and 1,992 controls. We investigated the association of the top SNPs from the discovery stage in two Australian replication cohorts (932 cases and 6,862 controls total) and two US replication cohorts (2,616 cases and 2,634 controls total). Meta-analysis of all cohorts identified three loci newly associated with development of POAG. These loci are located upstream of ABCA1 (rs2472493[G], odds ratio (OR) = 1.31, P = 2.1 × 10(-19)), within AFAP1 (rs4619890[G], OR = 1.20, P = 7.0 × 10(-10)) and within GMDS (rs11969985[G], OR = 1.31, P = 7.7 × 10(-10)). Using RT-PCR and immunolabeling, we show that these genes are expressed within human retina, optic nerve and trabecular meshwork and that ABCA1 and AFAP1 are also expressed in retinal ganglion cells.

Figure 1. Association results for the regions reaching genome-wide significance

These plots show the regional association (using logistic regression with sex and the first 6 principal components fitted as covariates) and recombination rates for the top SNPs in the discovery dataset (1,155 advanced POAG cases and 1,992 controls). In each plot, the solid diamond indicates the top-ranked SNP in the region based on two-sided P-value. The colour box at the right or left corner of each plot indicates the pairwise correlation (R²) between the top SNP and the other SNPs in the region. The blue spikes show the estimated recombination rates. The box underneath each plot shows the gene annotations in the region. Each plot was created using LocusZoom (http://csg.sph.umich.edu/locuszoom/) for the top-ranked SNP in each region with 400-kb region surrounding it. (a) The top-ranked SNP for this plot is rs2472493 on chromosome 9 upstream of ABCA1 gene with P=2.0×10^-10. (b) The top-ranked SNP for this plot is rs11827818 on chromosome 11 near ARHGEF12 with P = 9.2 × 10⁻⁹. (c) The top-ranked SNP for this plot is rs114096562 on chromosome 6 in GMDS gene with P=7.0×10^-8. (d) This plot is centred on rs4619890 SNP on chromosome 4 in AFAP1 gene with P=9.7×10^-6. This SNP clearly reached genome-wide significance (P=7.0×10^-10) in the meta-analysis of the results between the discovery and replication cohorts.

Figure 1. Association results for the regions reaching genome-wide significance

These plots show the regional association (using logistic regression with sex and the first 6 principal components fitted as covariates) and recombination rates for the top SNPs in the discovery dataset (1,155 advanced POAG cases and 1,992 controls). In each plot, the solid diamond indicates the top-ranked SNP in the region based on two-sided P-value. The colour box at the right or left corner of each plot indicates the pairwise correlation (R²) between the top SNP and the other SNPs in the region. The blue spikes show the estimated recombination rates. The box underneath each plot shows the gene annotations in the region. Each plot was created using LocusZoom (http://csg.sph.umich.edu/locuszoom/) for the top-ranked SNP in each region with 400-kb region surrounding it. (a) The top-ranked SNP for this plot is rs2472493 on chromosome 9 upstream of ABCA1 gene with P=2.0×10^-10. (b) The top-ranked SNP for this plot is rs11827818 on chromosome 11 near ARHGEF12 with P = 9.2 × 10⁻⁹. (c) The top-ranked SNP for this plot is rs114096562 on chromosome 6 in GMDS gene with P=7.0×10^-8. (d) This plot is centred on rs4619890 SNP on chromosome 4 in AFAP1 gene with P=9.7×10^-6. This SNP clearly reached genome-wide significance (P=7.0×10^-10) in the meta-analysis of the results between the discovery and replication cohorts.

Figure 2. Distribution of the ABCA1 protein in human ocular tissues

Sections of a normal human eye were immunolabelled with the anti-ABCA1 antibody (brown) and counterstained with haemotoxylin to visualise nuclei (blue). Positive immunolabelling was detected in the trabecular meshwork (a and b), throughout the retina (c and d) and in the optic nerve (e and f). In the retina (c), comparatively pronounced ABCA1 immunolabelling was observed at the tips of photoreceptors, in the outer limiting membrane (OLM), outer plexiform layer (OPL) and nerve fibre layer (NFL). (d) Labelling was also pronounced in some cells in the inner nuclear layer (INL; arrow), in retinal ganglion cells in the ganglion cell layer (GCL; arrowhead) and retinal blood vessel wall (not shown). In the optic nerve (e and f), the protein was distributed in the nerve fibre bundles (e, asterisk) and at the cell boundary of astrocytes in the glial columns (f, arrow). In sections of a glaucomatous eye, (data not shown), including in the retina (g), similar distribution of the protein to that in the normal eye, was observed. The experiment was repeated for reproducibility. (h) Section hybridised with the secondary detection reagent alone as negative control. sc, Schlemm's canal; RPE, retinal pigment epithelium; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; IPL, inner plexiform layer. Scale bar=100μm.