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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.

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Figure 1: Association results for the regions reaching genome-wide significance.
Figure 2: Distribution of the ABCA1 protein in human ocular tissues.

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Acknowledgements

Australian and New Zealand Registry of Advanced Glaucoma (ANZRAG): support for recruitment of ANZRAG was provided by the Royal Australian and New Zealand College of Ophthalmology (RANZCO) Eye Foundation. Genotyping was funded by the National Health and Medical Research Council (NHMRC) of Australia (#535074 and #1023911). This work was also supported by funding from NHMRC #1031362 awarded to J.E.C., NHMRC #1037838 awarded to A.W.H., NHMRC #1048037 awarded to S.L.G., NHMRC #1009844 awarded to R.J.C. and I.G., NHMRC #1031920 and an Alcon Research Institute grant awarded to D.A.M., an Allergan Unrestricted grant awarded to A.J.W., the BrightFocus Foundation and a Ramaciotti Establishment Grant. The authors acknowledge the support of B. Usher-Ridge in patient recruitment and data collection and P. Danoy and J. Hadler for genotyping.

Controls for the ANZRAG discovery cohort were drawn from the Australian Cancer Study, the Study of Digestive Health and a study of inflammatory bowel diseases. The Australian Cancer Study was supported by the Queensland Cancer Fund and the NHMRC of Australia (program number 199600, awarded to D.C.W., A.C. Green, N.K. Hayward, P.G. Parsons, D.M. Purdie and P.M. Webb, and program number 552429, awarded to D.C.W.). The Study of Digestive Health was supported by grant number 5 R01 CA 001833 from the US National Cancer Institute (awarded to D.C.W.).

The Barrett's and Esophageal Adenocarcinoma Genetic Susceptibility Study (BEAGESS) sponsored the genotyping of cases with esophageal cancer and Barrett's esophagus, which were used as unscreened controls in the ANZRAG discovery cohort. BEAGESS was funded by grant R01 CA136725 from the US National Cancer Institute.

Genotyping for part of the Australian twin control sample included in the ANZRAG replication cohort was funded by an NHMRC Medical Genomics Grant. Genotyping for the remainder of twin controls was performed by the US National Institutes of Health (NIH) Center for Inherited Research (CIDR) as part of NIH/National Eye Institute (NEI) grant 1RO1EY018246, and we are grateful to C. Day and staff. We acknowledge with appreciation all women who participated in the QIMR endometriosis study. We thank Endometriosis Associations for supporting study recruitment. We thank S. Nicolaides and the Queensland Medical Laboratory for pro bono collection and delivery of blood samples and other pathology services for assistance with blood collection. The QIMR twin and endometriosis studies were supported by grants from the NHMRC of Australia (241944, 339462, 389927, 389875, 389891, 389892, 389938, 443036, 442915, 442981, 496610, 496739, 552485 and 552498), the Cooperative Research Centre for Discovery of Genes for Common Human Diseases (CRC), Cerylid Biosciences (Melbourne) and donations from N. and S. Hawkins. We thank M.J. Wright, M.J. Campbell, A. Caracella, S. Gordon, D.R. Nyholt, A.K. Henders, B. Haddon, D. Smyth, H. Beeby, O. Zheng and B. Chapman for their input into project management, databases, sample processing and genotyping. We are grateful to the many research assistants and interviewers for assistance with the studies contributing to the QIMR twin collection.

Blue Mountains Eye Study (BMES): BMES was supported by the NHMRC, Canberra Australia (974159, 211069, 457349, 512423, 475604 and 529912), the Centre for Clinical Research Excellence in Translational Clinical Research in Eye Diseases, NHMRC Senior Research Fellowships (358702 and 632909 to J.J.W.) and the Wellcome Trust, UK, as part of Wellcome Trust Case Control Consortium 2 (A.C.V., P. McGuffin, P. Mitchell, F.T. and P.J.F.) for genotyping costs of the entire BMES population (085475B08Z, 08547508Z, 076113). P.J.F. is also supported by Medical Research Council (MRC) G0401527, Research Into Ageing (Ref 262) and NIHR (UK) Biomedical Research Centre at Moorfields Eye Hospital and University College London Institute of Ophthalmology (BRC2_009) funds.

The BMES acknowledges E. Rochtchina from the Centre for Vision Research, Department of Ophthalmology and Westmead Millennium Institute University of Sydney, J. Attia, R. Scott and E.G. Holliday from the University of Newcastle, J. Xie and P.N. Baird from the Centre for Eye Research Australia, University of Melbourne, M.T. Inouye, Medical Systems Biology, Department of Pathology and Department of Microbiology and Immunology, University of Melbourne and X. Sim, National University of Singapore.

NEI Glaucoma Human Genetics Collaboration (NEIGHBOR): genotyping services for the NEIGHBOR study were provided by the CIDR and were supported by the NEI through grant HG005259-01 (J.L.W.). Additionally, CIDR is funded through a federal contract from the NIH to The Johns Hopkins University, contract number HHSN268200782096C. Collecting and processing samples for the NEIGHBOR data set was supported by the NEI through American Recovery and Reinvestment Act (ARRA) grants 3R01EY015872-05S1 (J.L.W.) and 3R01EY019126-02S1 (M.A.H.). Genotype imputation and meta-analysis were supported by EY022305 (J.L.W.). Funding for the collection of cases and controls was provided by the following NIH grants: EY015543 (R.R. Allingham); EY006827 (D. Gaasterland); HL73042, HL073389, EY13315, EY023646 (M.A.H.); CA87969, CA49449, CA55075 (J.H. Kang); EY009149 (P.R. Lichter); HG004608 (C. McCarty); EY008208 (F.A. Medeiros); EY015473 (L.R.P.); EY012118 (M. Pericak-Vance); EY015682 (A. Realini); EY011671, EY09580 (J.E. Richards); EY013178 (J.S. Schuman); RR015574, EY015872, EY010886, EY009847, EY014104 (J.L.W.); EY011008, EY144428, EY144448 and EY18660 (K. Zhang). J.L.W. and L.R.P. are also supported by the Harvard Glaucoma Center for Excellence and the Margolis Fund. Y. Liu is supported by the Glaucoma Research Foundation, the American Health Assistance Foundation and the Glaucoma Foundation. J.L.W., L.R.P., D.C. Musch and J.E. Richards are supported by Research to Prevent Blindness. J.N.C.B. is supported by NIH T32 EY007157 (CWRU) and T32 EY21453-2 (VUMC).

MEEI case-control sample: genotyping for the Massachusetts Eye and Ear Infirmary (MEEI) case-control sample was performed at the Broad Institute of MIT and Harvard with funding support from the NIH GEI (Gene Environment Initiative) (U01HG04424 and U01HG004728). The GENEVA Coordinating Center (U01HG004446) assisted with genotype cleaning. Imputation was supported by NIH EY022305. Collection of cases and controls was supported by NIH EY015872.

Support for molecular analysis of the associated genes was provided by the Ophthalmic Research Institute of Australia. The authors acknowledge the support of M. Philpott in collection of cadaveric human eye tissues and N. Mabarrack for initial optimization of the antibody to AFAP.

S. MacGregor is supported by Australian Research Council (ARC) and NHMRC Fellowships. G.W.M., M.A.B., K.P.B., D.C.W. and J.E.C. are supported by Australian NHMRC Fellowships. D.C.W. was funded by the ARC and G.R.-S. was funded by NHMRC during the period of this study. The authors acknowledge C. Abbot (Alcon Research Ltd.) for providing normal and glaucomatous trabecular meshwork cell lines, NTM-5 and GTM-3, respectively, as a kind gift.

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K.P.B., S. MacGregor and J.E.C. were involved in designing the study. A.W.H., K.C., L.R.P., M.A.H., A.C.V., P. McGuffin, F.T., P.J.F., J.J.W., G.W.M., N.G.M., G.R.-S., D.C.W., M.A.B., J.L.W., D.A.M., P. Mitchell and J.E.C. were involved in participant recruitment, sample collection or genotyping. Analysis was performed by P.G., R.F., K.P.B., S.S., M.H.L., J.N.C.B., S.J.L., L.R.P., J.L.H., J.L.W. and S. MacGregor. Designing and conducting the laboratory experiments were performed by K.P.B., S.S., S. Martin and R.F. Clinician assessments were performed by S.L.G., R.J.C., M.C., A.J.W., T.Z., E.S., J.L., J.T.F., S.K., J.B.R., I.G., P.R.H., R.A.M., D.A.M. and J.E.C. The initial draft was written by P.G., K.P.B., S.S. and S. MacGregor.

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Correspondence to Puya Gharahkhani or Jamie E Craig.

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Supplementary Figure 1 Quantile-quantile plot in the discovery cohort.

This figure shows the Q-Q plot created in R for two-sided P-values obtained from the association analysis of the genome-wide SNPs (using logistic regression with sex and the first 6 principal components fitted as covariates) with the development of primary open-angle glaucoma (POAG). The x-axis shows the expected distribution of -log10(P-values) under the null hypothesis of no association. The y-axis show the observed -log10(P-values) in the association analysis in the discovery cohort in this study (1,155 advanced POAG cases and 1,992 controls). The P-values were corrected for the genomic inflation factor lambda (λ = 1.06 for imputed SNPs). The curves are made of dots each being an observed -log10(P-values) calculated for one or more SNPs. The red indicator lines show where x = y.

Supplementary Figure 2 Manhattan plot for association of genome-wide SNPs with primary open-angle glaucoma.

This plot was created using R software for association of genome-wide SNPs (using logistic regression with sex and the first 6 principal components fitted as covariates) with the development of primary open-angle glaucoma in stage 1 of this study using the discovery cohort (1,155 advanced POAG cases and 1,992 controls). The SNPs have been plotted against their chromosomal positions (the x-axis) and -log10(P-values) calculated through the genome-wide association analysis (the y-axis). All the SNPs on each chromosome have been shown in the same color but a distinct color from that of the adjacent chromosome in the plot. Chromosome 23 is the X chromosome. The texts in the figure indicate the chromosome, position (base pair) and the name of the adjacent genes for the top hits. The horizontal line in the figure indicates the genome-wide significance level (-log10(P-value) = 7.30). The two-sided P-values used to create this figure were corrected for the genomic inflation factor lambda (λ = 1.06 for imputed SNPs). Two regions on chromosome 9, and one region on chromosome 11 reached genome-wide significance (P < 5 × 10-8; -log10P > 7.30). In addition, one region on chromosome 6 also came close to genome-wide significance (P = 7.0 × 10-8; -log10P = 7.15). The previously identified region at TMCO1 did not reach genome-wide significance in the stage 1 analysis (control sample was changed relative to previous work to allow a larger set of SNPs to be used as the basis of imputation in stage 1) but TMCO1 SNPs exceeded P < 1 × 10-5, with P < 5 × 10-8 obtained at TMCO1 when stage 1 and 2 samples were combined.

Supplementary Figure 3 Expression analysis of the ABCA1, AFAP1 and GMDS genes in human ocular tissues.

(a-d) Expression of the ABCA1 (a), AFAP1 (b,c) and GMDS (d) mRNA was analyzed by RT-PCR using gene specific primers. (a) The 913 bp PCR product amplified with ABCA1-specific primers in iris, ciliary body (CB), retina, optic nerve head (ONH), optic nerve (ON) and normal (NTM) and glaucomatous (GTM) trabecular meshwork cell lines (respectively, NTM-5 and GTM-3) corresponds to the expected size of the predominant transcript encoded by the gene. The 770 bp amplicon in the ciliary body, retina, optic nerve head and normal and glaucomatous trabecular meshwork cells represents the alternate transcript lacking exon 4. The origin of the >1000 bp amplified product in the ciliary body and retina is unclear. (b) The 408 bp PCR product amplified with AFAP1-specific primers in the indicated tissues corresponds to the expected size of the ubiquitously expressed B isoform. (c) Upon increasing the amplification cycles, in addition to the 408 bp amplicon, the 660 bp product in the retina corresponds to the neuronal cell specific, A isoform of AFAP1. The product marked with an asterisk was found to be non-specific by sequencing. Amplification from three independent cDNA preparations is shown. (d) The 300 bp PCR product amplified with primers specific to the GMDS variant 1, in all the analyzed tissues corresponds to the expected size of this variant. Representative data from two independent experiments are shown. M, molecular size markers; RT-, without reverse transcriptase control; bp, base pair.

Supplementary Figure 4 Western blot showing specificity of the ABCA1 antibody.

Protein lysates of NTM-5 (NTM) and GTM-3 (GTM) human trabecular meshwork cells, respectively, from a normal control and an individual with glaucoma were analyzed by SDS-PAGE and western blotting performed using the mouse anti-ABCA1 antibody. The >250 kDa protein band detected in both the cell types is greater than the expected size of ABCA1 (254 kDa) and corresponds to the post-translationally modified form of the protein. The 70 kDa band detected in both the samples has been reported previously but its identity remains unknown. The blot was overexposed to improve visibility of the expected protein species. Red indicates oversaturation of signal. The minor protein bands seen in both the cell types are likely degradation products of the full-length protein and were not visible in a shorter exposure. kDa = kilodaltons.

Supplementary Figure 5 Distribution of the AFAP1 protein in human ocular tissues.

(a-f) Sections of a normal human eye were immunolabelled with the anti-AFAP1 antibody (brown) (see Supplementary Figure 6 for antibody specificity) and counterstained with hemotoxylin to visualize nuclei (blue). Positive immunolabelling was detected in the trabecular meshwork (a,b), retina (c,d) and optic nerve (e,f). In the retina (c,d), AFAP1 immunolabelling was prominent in the photoreceptor inner segment (IS), in some cells in the inner nuclear layer (INL), in retinal ganglion cells in the ganglion cell layer (GCL) and retinal blood vessel wall (not shown). In the optic nerve (e,f), AFAP1 protein was primarily present in the astrocytes. (g,h) In sections of a glaucomatous eye, similar AFAP1 distribution was observed (data not shown). Labelling in the layers of the retina (g,h) was also similar to that in a normal eye. Positive labelling in the blood vessel wall is marked with an arrow. The experiment was repeated for reproducibility. sc, Schlemm’s canal; IPL, inner plexiform layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium. Scale bar = 100 μm (a-d, f-g); 500 μm (e).

Supplementary Figure 6 Western blot showing specificity of the AFAP1 antibody.

Protein lysate of NTM-5 (NTM) human trabecular meshwork cells from a normal individual were analyzed by SDS-PAGE and western blotting performed using the mouse anti-AFAP1 antibody. The detected 110 kDa protein band corresponds to the expected size of the AFAP1 B isoform.

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Gharahkhani, P., Burdon, K., Fogarty, R. et al. Common variants near ABCA1, AFAP1 and GMDS confer risk of primary open-angle glaucoma. Nat Genet 46, 1120–1125 (2014). https://doi.org/10.1038/ng.3079

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