Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide. To identify new susceptibility loci, we performed meta-analysis on genome-wide association study (GWAS) results from eight independent studies from the United States (3,853 cases and 33,480 controls) and investigated the most significantly associated SNPs in two Australian studies (1,252 cases and 2,592 controls), three European studies (875 cases and 4,107 controls) and a Singaporean Chinese study (1,037 cases and 2,543 controls). A meta-analysis of the top SNPs identified three new associated loci: rs35934224[T] in TXNRD2 (odds ratio (OR) = 0.78, P = 4.05 × 10−11) encoding a mitochondrial protein required for redox homeostasis; rs7137828[T] in ATXN2 (OR = 1.17, P = 8.73 × 10−10); and rs2745572[A] upstream of FOXC1 (OR = 1.17, P = 1.76 × 10−10). Using RT-PCR and immunohistochemistry, we show TXNRD2 and ATXN2 expression in retinal ganglion cells and the optic nerve head. These results identify new pathways underlying POAG susceptibility and suggest new targets for preventative therapies.
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NEIGHBORHOOD data collection and analysis are supported by US National Institutes of Health/National Eye Institute (NIH/NEI) grant R01EY022305 (J.L.W.). Support for recruitment in ANZRAG (Australian and New Zealand Registry of Advanced Glaucoma) was provided by the Royal Australian and New Zealand College of Ophthalmology (RANZCO) Eye Foundation and by the National Health and Medical Research Council (NHMRC) of Australia (535074, 1031362, 1023911 and 1021105). EPIC-Norfolk infrastructure and core functions are supported by grants from the UK Medical Research Council (G1000143) and Cancer Research UK (C864/A14136). BMES (Blue Mountains Eye Study) was supported by the NHMRC, the Centre for Clinical Research Excellence in Translational Clinical Research in Eye Diseases, NHMRC Senior Research Fellowships and the Wellcome Trust, UK. Collection and genotyping for the South London Case-Control cohort (UK) were supported by a National Institute for Health Research (NIHR) Senior Research Fellowship (C.J.H.), and analysis was supported by a Fight for Sight Early Career Investigator Award (P.J.H.). The Singapore study (E.N.V., T.A. and C.C.K.) was supported by a Biomedical Research Council (BMRC) grant in Singapore, reference BMRC 10/1/35/19/675. This research was also partly supported by a grant (NMRC/TCR/008-SERI/2013) from the Singapore National Research Foundation under its Translational and Clinical Research Flagship Programme and administered by the Singapore Ministry of Health's National Medical Research Council. Additional acknowledgment and funding details are in the Supplementary Note.
The authors declare no competing financial interests.
A list of members and affiliations is provided in the Supplementary Note.
Integrated supplementary information
Supplementary Figure 1 QQ plots for individual data sets meta-analyzed in NEIGHBORHOOD and the NEIGHBORHOOD meta-analysis.
The top eight panels are QQ plots for individual data sets; the bottom panel is for the NEIGHBORHOOD meta-analysis. NEIGHBORHOOD, National Eye Institute Glaucoma Human Genetics Collaboration Heritable Overall Operational Database; NHS/HPFS, Nurses Health Study/Health Professionals Follow-up Study; MEEI, Massachusetts Eye and Ear Infirmary; NEIGHBOR, National Eye Institute Glaucoma Human Genetics Collaboration; OHTS, Ocular Hypertension Treatment Study; WGHS, Women’s Genome Health Study. Individual λ values are: NHS/HPFS Affymetrix, 1.008; NHS/HPFS Illumina, 1.024; Iowa, 1.031; Marshfield, 1.003; MEEI, 1.007; NEIGHBOR, 1.051; OHTS, 1.015; WGHS, 1.011. The overall NEIGHBORHOOD λ value is 1.064.
For this analysis, each NEIGHBORHOOD data set was excluded from a meta-analysis of the other data sets. The odds ratios from the meta-analyses were compared by calculating the Pearson’s product-moment correlation coefficient between each leave-one-out analysis and the overall meta-analysis of all eight NEIGHBORHOOD data sets (stage 1). Correlations were calculated in R using the corrplot package and ellipse option. Color intensity and ellipse sharpness increase with increasing Pearson’s correlation coefficient.
SNPs located in novel regions are colored pink, and those in previously known associated regions are colored green. The P values used to create this figure were corrected for the genomic inflation factor (λ = 1.06). Regions reaching genome-wide significance include one novel region (FOXC1) and three regions previously identified (TMCO1, CDKN2BAS and SIX6). The GAS7 region was previously associated with the quantitative risk factor for POAG, IOP. AFAP1 and ABCA1 previously associated with POAG did not reach genome-wide significance. ATXN2 and TXNRD2 surpassed the significance level of P < 1 × 10−5 at stage 1, along with other interesting regions, including 1p, 2p, 2q, 5p, 6p, 6q, 10q and 20p (3,853 cases and 33,480 controls).
Supplementary Figure 4 Forest plots for NEIGHBORHOOD data sets showing association results for the top SNPs in the three novel regions identified in this study.
Square symbols are positioned along the x axis according to OR, with lines representing the confidence interval. The size of the square is inversely proportional to the width of the 95% confidence interval. A summary for all eight NEIGHBORHOOD data sets is shown as the black diamond. OHTS, Ocular Hypertension Treatment Study; MEEI, Massachusetts Eye and Ear Infirmary; MFC, Marshfield Clinic; NHS/HPFS, Nurses Health Study/Health Professionals Follow-up Study; Affy, Affymetrix; Illu, Illumina; WGHS, Women’s Genome Health Study.
Association results for NTG in NEIGHBORHOOD (stage 1). Regions reaching genome-wide significance include one novel region (C12orf23) and two regions previously identified (8q22 and CDKN2BAS) (725 NTG cases and 11,145 controls).
Regions reaching genome-wide significance include one novel region (FOXC1) and two regions previously associated with POAG (TMCO1 and SIX6) (1,868 cases and 31,497 controls).
Seven SNPs in this region reach genome-wide significance (P < 5 × 10−8) in the POAG meta-analysis (stage 2), indicated in black type at the top of the figure. Cell types: GM12878 (B lymphocytes, lymphoblastoid), H1-hESC (embryonic stem cells), K562 (leukemia), HepG2 (hepatocellular carcinoma), HUVEC (umbilical vein endothelial cells), HMEC (mammary epithelial cells), HSMM (skeletal muscle myoblasts), NHEK (epidermal keratinocytes), NHLF (lung fibroblasts). ENCODE annotation: bright red, active promoter; light red, weak promoter; purple, inactive/poised promoter; orange, strong enhancer; yellow, weak/poised enhancer; blue, insulator; dark green, transcriptional transition and transcriptional elongation; light green, weakly transcribed; gray, Polycomb repressed; light gray, heterochromatin, low signal.
Six of the 22 SNPs significantly associated with POAG in the TXNRD2 region (rs6518585, rs9754418, rs7285948, rs7288170, rs8141610 and rs12158214) on chromosome 22 are eQTLs in lymphoblasts and skin in MuTHER Twins (Grundberg et al., 2012) (GENEVAR http://www.sanger.ac.uk/science/tools/genevar-gene-expression-variation-archive). These data were obtained using microarrays for expression and HapMap 2 imputation. Shown here are the results for rs7288170, which is representative of similar results for all six SNPs. The –log(P value) for the association of the tested SNP (in this example, rs7288170) with RNA level is plotted on the y axis, and the location of the RNA is plotted on the x axis. The TXNRD2 region under the expression peak (chr22:18,229,251–18,251,941; NCBI36/hg18) includes all six eQTL SNPs (expanded region). This region includes a number of regulatory elements annotated by ENCODE. Cell types: GM12878 (B lymphocytes, lymphoblastoid), H1-hESC (embryonic stem cells), K562 (leukemia), HepG2 (hepatocellular carcinoma), HUVEC (umbilical vein endothelial cells), HMEC (mammary epithelial cells), HSMM (skeletal muscle myoblasts), NHEK (epidermal keratinocytes), NHLF (lung fibroblasts). ENCODE annotation: bright red, active promoter; light red, weak promoter; purple, inactive/poised promoter; orange, strong enhancer; yellow, weak/poised enhancer; blue, insulator; dark green, transcriptional transition and transcriptional elongation; light green, weak transcribed; gray, Polycomb repressed; light gray, heterochromatin, low signal.
All 22 SNPs located in the TXNRD2 region significantly associated with POAG after stage 2 are significant cis eQTLs for TXNRD2 in thyroid tissue (shown in the figure), and 19 are also significant cis eQTLs in tibial nerve (P values for expression correlation < 1 × 10−4) in the GTEx database (GTEx Consortium, 2015). The –log10 (P value) is shown on the y axis. rs6518585, rs12158214 and rs3424993 are only significant in thyroid tissue. The location of the significant SNPs is shown in this figure as red dots. Gray dots in the figure are cis-eQTL SNPs in thyroid tissue that did not reach genome-wide significance for association with POAG. The most significant cis-eQTL SNPs are also those that are significantly associated with POAG. The location of the TXNRD2 exons and introns is shown in blue as depicted in the UCSC Genome Browser (http://genome.ucsc.edu/).
Locus zoom plots for the FOXC1 associated region before (top panel) and after (middle panel) analysis conditional on the top GMDS SNP (rs11969985) (Gharahkhani et al., 2014). The bottom panel shows the effect of conditioning on the top FOXC1 SNP from this study (rs2745572). The OR and P value for the top SNP rs2745572 is OR = 1.254, 95% CI = (1.164, 1.351), P = 2.36 x 10−9 before conditioning and OR = 1.252, 95% CI = (1.159, 1.352), P = 1.03 x 10−8 after conditioning on rs11969985.
Lanes (from left to right): 1, Low mass DNA ladder (100 bp, 200 bp, 400 bp, 800 bp, 1200 bp and 2000 bp); 2, cornea #1; 3, cornea #2; 4, cornea #3; 5, cornea #4; 6, trabecular meshwork #1; 7, trabecular meshwork #2; 8, trabecular meshwork #3; 9, ciliary body #1; 10, ciliary body #2; 11, retina #1; 12, retina #2; 13, optic nerve head #1; 14, optic nerve head #2; 15, optic nerve head #3; 16, low mass DNA ladder. Multiple bands indicate the presence of alternative transcripts for the amplified cDNA. The expected PCR products were 400-500 bp in size. Abbreviations: L, is for low mass DNA ladder, C is for cornea, TM is for trabecular meshwork, CB is for ciliary body, R is for retina, and ON is for optic nerve head.
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Bailey, J., Loomis, S., Kang, J. et al. Genome-wide association analysis identifies TXNRD2, ATXN2 and FOXC1 as susceptibility loci for primary open-angle glaucoma. Nat Genet 48, 189–194 (2016). https://doi.org/10.1038/ng.3482
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