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

In total 1,155 glaucoma cases and 1,992 controls, genotyped on Illumina Omni1M or OmniExpress arrays and imputed to the 1000 Genomes Phase 1 Europeans panel, were used as discovery cohort in this study to perform a genome-wide association study for Primary Open Angle Glaucoma (POAG) (stage 1). The association results for the top SNPs from the discovery cohort were replicated in stage 2 and then stage 3 replication cohorts. The cohort details, genotyping platforms for each cohort and diagnostic criteria are listed in the Supplementary Notes . In addition, we performed a meta-analysis for the top SNPs in the discovery and replication cohorts. In this method section we have described the methods used for imputation and statistical analysis for the discovery cohort. Methods used for each replication cohort are present in the Supplementary Notes . The QC for the discovery cohort was performed in PLINK 34 by removing individuals with more than 3% missing genotypes, SNPs with call rate <97%, minor allele frequency (MAF) <0.01, and Hardy-Weinberg equilibrium P <0.0001 in controls and P <5e-10 in cases. We used the same QC protocol before merging the cases and controls in our discovery cohort to avoid mismatches between the merged datasets. Following merging, the genotypes for 569,249 SNPs common to the arrays were taken forward for analysis. Identity by descent was computed in PLINK based on autosomal markers, with one of each pair of individuals with relatedness of > 0.2 removed. Principal components were computed for all participants and reference samples of known northern European ancestry (1000G British, CEU, Finland participants) using smartpca package from EIGENSOFT software 35 , 36 . Participants with PC1 or PC2 values > 6 standard deviations from the known northern European ancestry group were excluded. Imputation was conducted using IMPUTE2 37 in 1Mb sections, with the 1000 Genomes Phase 1 38 Europeans (March 2012 release) as the reference panel. Genotyped SNPs who were strand ambiguous (e.g. A/T, C/G) were dropped from the input genotype panel prior to imputation; given these are deliberately under-represented on Illumina arrays this has limited effects on ability to impute data but gives greater confidence in the imputation's quality. Imputation was performed with the recommended settings for IMPUTE2 including a 250kb buffer flanking imputation sections; and the effective size of the sampled population as 20,000 37 . Reference panel SNPs with a minor allele 0.8 and MAF> 0.01 were carried forward for analysis. Association testing on the imputed data was performed in SNPTEST 39 , 40 using additive model (-frequentist 1) and full dosage scores (-method expected) with sex and the first 6 principal components fitted as covariates. Genomic inflation factor lambda was calculated to investigate the presence of population stratification and inflation. The P -values were corrected for genomic inflation factor lambda. Q-Q and Manhattan plots were created in R 41 . Regional association plots for the regions reaching genome-wide significance were created using LocusZoom 42 . In order to investigate whether any hits identified in the discovery cohort were driven by a subset of controls affected by the other diseases (oesophageal cancer, Barrett's oesophagus, and inflammatory bowel diseases) we also performed a genome-wide association analysis after removing the controls who were affected by the other diseases (refer to the Supplementary Notes for the structure of controls in the discovery cohort). This analysis included 1,155 glaucoma cases and 1,147 controls. Associations of top autosomal SNPs in the discovery cohort ( P < 1×10 -4 ) (stage 1) were investigated in the replication cohorts (stage 2 and 3) (refer to the Supplementary Notes for the structure of replication cohorts, QC and statistical analysis for each cohort). Stage 2 included two Australian replication data sets (total 932 cases, 6,862 controls) and stage 3 included two US cohorts (total 2,616 cases, 2,634 controls). For replication in stage 2, twenty four SNPs with the best P -values in the discovery cohort were used as the lead SNPs for the autosomal regions with P < 1×10 -4 ( Supplementary Table 4 ). The SNPs that were nominally replicated in stage 2 ( P <0.05) were taken forward for replication in stage 3. To make maximum valid use of our cohorts, for replication we focused on SNPs directly genotyped on the Illumina Human610/670 arrays. Since a portion of the stage 2 cases were genotyped on a non-genomewide platform (Sequenom) we could not accurately evaluate the imputed SNPs from stage 1. Hence the most-associated SNP upstream of the ABCA1 (rs2472493), and SNPs in high LD with the most-associated SNP near ARHGEF12 (rs11217878 and rs2276035, r 2 =1 and r 2 =0.94, respectively with rs11827818) were used in the replication studies. Similarly, SNPs with high LD with the most-associated SNP in GMDS (rs2761233 and rs11969985, r 2 =0.93 and r 2 =0.87, respectively) with rs114096562, the most associated SNP in GMDS , were used for replication studies. Fixed-effects meta-analysis for the top SNPs was performed between the discovery and replication cohorts in METAL 43 using the effect sizes and their standard errors for the risk alleles. Presence of heterogeneity between the cohorts for effect sizes of risk alleles was investigated using the I2 statistic, as implemented in METAL. Candidate genes in the regions of association were selected based on the location and function of the genes, the pathways that the genes are involved in, tissue location of the expression of the gene, and whether similar phenotypes were reported to be caused by mutations in these genes. This information was found in Ensembl 44 , NCBI , UCSC genome Bioinformatics 45 , Genecards 46 , and UniprotKB 47 , as well as available published data. To predict functional effects of the top POAG associated SNPs identified in this study, we used the ENCODE project data 48 and the associated databases, RegulomeDB 49 and HaploReg v2 50 . We used Genevar database 51 to investigate expression quantitative trait loci within genetic regions of interest. Ocular tissues from post-mortem human eyes were obtained through the Eye Bank of South Australia, according to guidelines of the Southern Adelaide Clinical Human Research Ethics Committee. Normal and glaucomatous trabecular meshwork cell lines, NTM-5 and GTM-3, were a kind gift from Dr Clark Abbot, Alcon Research Ltd., USA. Both the cell lines tested negative for mycoplasma contamination. Total RNA was extracted using the RNeasy Mini Kit (Qiagen Pty Ltd., Doncaster, Australia). First strand cDNA was synthesised using the Superscript III reverse transcriptase (Invitrogen, Life Technologies Australia Pty Ltd., Mulgrave, Australia) and random hexamers. PCR was performed using the Hot Star Taq Plus polymerase (Qiagen) and gene-specific primers ( Supplementary Table 9 ). PCR was performed at the conditions specified in Supplementary Table 9 . The enzyme was activated at 95°C for 5 min, denaturation was at 95°C for 30 sec, and elongation at 72°C. Additional elongation at 72°C for 5 min was allowed after completion of the amplification cycles. Specificity of each amplified product was confirmed by sequencing. Eye tissue was fixed in neutral buffered formalin and embedded in paraffin. For immunolabelling, 4 μm sections were blocked with 5% normal goat serum and incubated with the mouse anti-ABCA1 (1:2000, cat# Ab66217, Sapphire Biosciences, NSW, Australia) or anti-AFAP (1:1000, BD Transduction Laboratories, CA, USA, cat# 610200) primary antibody at 4°C overnight. Primary antibody binding was detected with the Novolink Polymer detection kit (Leica Microsystems, Bannockburn, IL, USA) and Chromogen substrate coloration (Dako, Glostrup, Denmark). Sections were counterstained with haematoxylin and mounted in dePeX (Merck KGaA, Darmstadt, Germany). Light microscopy was performed on Olympus BX50 brightfield upright microscope attached with a Q-Imaging colour CCD camera; images were taken using the QCapture software (Q-Imaging Corporate, Surry, BC, Canada). For Western blotting, proteins from NTM-5 and GTM-3 human trabecular meshwork cells, respectively, established from a normal and an individual with glaucoma, were extracted in RIPA buffer, analysed by SDS-PAGE using the mini-PROTEAN TGX gel and transferred onto PVDF membrane (Bio-Rad Laboratories Pty. Ltd., NSW, Australia). Western blotting was performed using the mouse anti-ABCA1 (1:500, cat# Ab66217, Sapphire Biosciences, NSW, Australia) or anti-AFAP (1:250, BD Transduction Laboratories, CA, USA, cat# 610200) primary antibody followed by hybridisation with the hydrogen peroxide conjugated goat anti-mouse IgG secondary antibody (1:1000, Jackson ImmunoResearch Laboratories Inc., Brisbane, Australia, cat# 115-035-003). ABCA1 antibody binding was detected using the Pierce SuperSignal West Pico (Jackson ImmunoResearch Laboratories Inc., Brisbane, Australia) and AFAP1 antibody binding using ECL Prime (GE Healthcare Australia and New Zealand, Sydney, Australia), chemiluminescence reagents.