Meta-analysis of genome-wide association studies identifies novel loci that influence cupping and the glaucomatous process

Glaucoma is characterized by irreversible optic nerve degeneration and is the most frequent cause of irreversible blindness worldwide. Here, the International Glaucoma Genetics Consortium conducts a meta-analysis of genome-wide association studies of vertical cup-disc ratio (VCDR), an important disease-related optic nerve parameter. In 21,094 individuals of European ancestry and 6,784 individuals of Asian ancestry, we identify 10 new loci associated with variation in VCDR. In a separate risk-score analysis of five case-control studies, Caucasians in the highest quintile have a 2.5-fold increased risk of primary open-angle glaucoma as compared with those in the lowest quintile. This study has more than doubled the known loci associated with optic disc cupping and will allow greater understanding of mechanisms involved in this common blinding condition.

For each SNP with the lowest P value (z-statistic) in individuals with European ancestry, we looked at the SNP with the lowest P value (z-statistic) in individuals with Asian ancestry within 100,000 basepairs to determine the differences between both ancestries. SNPs that showed genome-wide significant (P < 5 × 10 −8 ) association with vertical cup-disc ratio in subjects of Asian ancestry , adjusted for disc area. Note that the number of subjects is lower in the tables as several studies did not measure disc area. SNPs that showed genome-wide significant (P < 5 × 10 −8 ) association with vertical cup-disc ratio in subjects of European and Asian ancestry combined, adjusted for disc area. Note that the number of subjects is lower in the tables as several studies did not measure disc area.  SNPs that showed genome-wide significant (P < 5 × 10 −8 ) association with vertical cup-disc ratio in subjects of European ancestry , adjusted for spherical equivalent. Note that the number of subjects is lower in the tables as several studies did not measure disc spherical equivalent.  SNPs that showed genome-wide significant (P < 5 × 10 −8 ) association with vertical cup-disc ratio in subjects of Asian ancestry , adjusted for spherical equivalent. Note that the number of subjects is lower in the tables as several studies did not measure disc spherical equivalent.  SNPs that showed genome-wide significant (P < 5 × 10 −8 ) association with vertical cup-disc ratio in subjects of European and Asian ancestry combined, adjusted for spherical equivalent. Note that the number of subjects is lower in the tables as several studies did not measure disc spherical equivalent.  Proxy SNPs were used for three missing SNPs in Southampton: * = rs1192415 (G allele), ** = rs2623320 (T allele), *** = rs4918188 (C allele). A1 = reference allele; A2 = other allele; Chr. = chromosome; OR = Odds ratio; SNP = single nucleotide polymorphism. A weighted genetic risk score per individual was calculated. Standardized regression coefficients of the eighteen top SNPs from the vertical cup-disc ratio analysis were used as weighting factor. The weighted risk scores were divided into quintiles. Odds ratios were calculated for each quintile, using the first quintile as a reference. -

deCODE
We thank all the participants whose contribution made this study possible, as well as their ophthalmologists. We also thank the personnel at deCODE recruitment center and core facilities for their hard work and enthusiasm.

Australian & New Zealand Registry of Advanced Glaucoma (ANZRAG)
The Australian & New Zealand Registry of Advanced Glaucoma (ANZRAG) recruits cases of advanced glaucoma Australia-wide through ophthalmologist referral. The cohort also included participants enrolled in the Glaucoma Inheritance Study in Tasmania (GIST) that met the criteria for ANZRAG. This cohort has been described previously 8 . Advanced Primary Open Angle Glaucoma was defined as bestcorrected visual acuity worse than 6/60 due to primary open angle glaucoma, or a reliable 24-2 Visual Field with a mean deviation of worse than -22db or at least 2 out of 4 central fixation squares affected with a Pattern Standard Deviation of < 0.5%. The less severely affected eye was also required to have signs of glaucomatous disc damage. Clinical exclusion criteria included: i) pseudoexfoliation or pigmentary glaucoma, ii) angle closure or mixed mechanism glaucoma; iii) secondary glaucoma due to aphakia, rubella, rubeosis or inflammation; iv) infantile glaucoma, v) glaucoma in the presence of a known associated syndrome. In total there were 1,155 glaucoma cases and 1,992 controls. DNA was extracted from peripheral whole blood. Genotyping was conducted using Illumina omni-1M or omni-express arrays. Cases and controls genotypes for the 569,249 SNPs common to the arrays were taken forward for analysis. Principle components were computed for all participants and reference samples of known northern European ancestry (1000G British, CEU, Finland participants). Participants with PC1 or PC2 values >6 standard deviations from the known northern European ancestry group were excluded. Identity by state was computed based on autosomal markers, with individuals with relatedness >0.2 removed. Association testing was conducted using logistic regression with sex and the first 6 principle components fitted as covariates. Imputation was conducted using IMPUTE2 9 , with 1000 Genomes Phase 1 Europeans as the reference panel. Association testing was done using SNPTEST. SNPs of interest from the VCDR analysis were selected to assess association with POAG.

Beijing Eye Study (BES)
The All study participants underwent an ophthalmic examination including refractometry, pneumotonometry, slit-lamp biomicroscopy, and photography of the cornea, lens, optic disk, and macula. Optic disc parameters were measured using Planimetry and the vertical cup-disc ratio was manually calculated. Blood samples were taken from 2,929 (90.1%), and DNA was extracted from blood leucocytes according to standard procedures. We performed genotyping using Illumina Human610-Quad BeadChip in 988 subjects 12 . 151 individuals with cryptic relatedness were excluded during sample QC procedure. After the removal of samples, SNPs were excluded based on (i) high rates of missingness (>5%); (ii) monomorphism; (iii) gross departure from HWE of p<10 -6 . Imputation was performed using IMPUTE v2.2.2 9 on post-QC SNPs. The HapMap Phase II panel (build36, release 22 db126 JPT+CHB HapMap panel) was used for the imputation.

Blue Mountains Eye Study (BMES)
BMES was a population-based eye assessment of a representative older Australian community sample. During the period 1992-1994, 3654 residents (57% male and 43% females) aged 49-97 were examined (BMES-1). The study was approved by the Western Sydney Area Health Service Human Ethics Committee, and written, informed consent was obtained from all participants. All participants had undertaken a detailed eye examination including stereo photographs in order to calculate the diameter of the vertical optic disc and the vertical cup. Vertical disc (VDD) and cup diameter (VCD) were obtained after pupil dilation from 30º color stereoscopic optic disc photographs taken with a 99 Zeiss FF3 fundus camera (Carl Zeiss Meditec, Dublin, CA). Vertical CDR was calculated by the vertical disc and cup measurements. Further details have been described elsewhere 13 . Samples were genotyped on the Human660W-Quad. Imputation was performed with IMPUTE2 which adopts a twostage approach using both haploid and diploid reference panels. 1000 Genome pilot data was used as reference panel. The SNPs considered in this study were those present also in HapMap2.

deCODE
The Icelandic primary open angle glaucoma (POAG) cases were identified from a list of participants in the Reykjavik Eye Study 14 and from a list compiled by Icelandic ophthalmologists in 2008 that included patients 55-86 years old at the time of diagnosis, all meeting either structural (glaucomatous optic neuropathy) or functional (glaucomatous visual field defects) criteria of glaucoma or both. For visual fields measurements, Octopus 123 perimeter (Haag-Streit AG, Koniz Switzerland) was used. Intra ocular pressure was not a part of the definition. On gonioscopy the angles were found to be open and normal in appearance. The combined list includes 598 individuals, 290 men and 308 women with POAG. The diagnosis of exfoliation syndrome (XFS) was specifically evaluated and if detected the participant was excluded from the study. The control group included 98,670 Icelandic individuals without know history of glaucoma. 533 of the POAG samples and 85,689 of the controls samples were assayed with the Illumina HumanHap300, HumanCNV370, HumanHap610, HumanHap1M, HumanHap660, Omni-1, Omni 2.5 or Omni Express bead chips at deCODE genetics and genotypes for about 34 million sequence variants were imputed into the chip typed individuals based on a training set of 2,230 whole genome sequenced Icelanders using methods previously described 15 . The case-control association analysis was done on the imputed genotypes, including in addition 65 cases and 12,981 controls that were not chip typed but for which genotype probabilities were imputed using methods of familial imputation 15 . For the risk score analysis only chip typed individuals were used. The study was approved by the Icelandic National Bioethics Committee and by the Icelandic Data Protection Authority. Informed consent was obtained from all participants. The study was conducted in accordance with revised Declaration of Helsinki.

Erasmus Rucphen Family (ERF) Study
The Erasmus Rucphen Family (ERF) Study is a family-based cohort in a genetically isolated population in the southwest of the Netherlands with over 3,000 participants aged between 18 and 86 years 16

Gutenberg Health Study (GHS I, GHS II)
The GHS is a population-based, prospective, observational cohort study in the Rhine-Main Region in midwestern Germany with a total of 15,010 participants and follow-up after five years. The study sample is recruited from subjects aged between 35 and 74 years at the time of the exam. The sample was drawn randomly from local governmental registry offices and stratified by gender, residence (urban and rural) and decade of age. Exclusion criteria were insufficient knowledge of the German language to understand explanations and instructions, and physical or psychic inability to participate in the examinations in the study center. The study was approved by the Medical Ethics Committee of the University Medical Center Mainz and by the local and federal data safety commissioners. According to the tenets of the Declaration of Helsinki, written informed consent was obtained from all participants prior to entering the study.
Within GHS, DNA was extracted from buffy-coats from EDTA blood samples as described earlier 19 . Genetic analysis was conducted in the first 5,000 study participants. For these, 3 . Genotypes were not released for SNPs that had call rates less than 85%, more than 1 HapMap replicate error, cluster separation less than 0.2, more than a 3% (autosomal) or 2.2% (X chromosome) difference in call rate between genders, more than 0.4% (X chromosome) male heterozygosity, or more than a 8% (autosomal) difference in AB frequency. Imputation was performed with IMPUTE2 using the March 2012 1000 genomes as a reference panel. Logistic regression using a model included age, gender, study site and 2 eigenvectors (EV1 and 2) was to assess the association between individual SNPs and POAG was done using PLINK v1.07.

Raine Study
The Western Australian Pregnancy Cohort (Raine) Study is an ongoing prospective cohort study of pregnancy, childhood, adolescence and young adulthood in Perth, Western Australia 22 . At the initiation of the study, 2,900 pregnant women were recruited at 16-18 weeks' gestation from the state's largest public women's hospital and surrounding private practices for a randomized clinical trial investigating effects of intensive ultrasound and Doppler studies in pregnancy outcomes. Following this study, the offspring of the recruited individuals have been evaluated in detail during childhood and adolescence. At the 20-year review of the cohort, Raine participants underwent a comprehensive ocular examination for the first time 23 . As part of this examination, a baseline glaucoma analysis was done on each participant using the Heidelberg Retina Tomography 3 (Heidelberg Engineering, Heidelberg, Germany). Participant was appropriately positioned in front of the camera and instructed to stare at the flashing light. The position of the camera was adjusted to illuminate and sharpen the image of the optic disc. Poor images were repeated. Each scan was reviewed at the end and the mean standard deviation of less than 20 μm was maintained for quality check. DNA samples and consents for GWAS studies were available from the previous assessments. Genotype data was generated using the genome-wide Illumina 660 Quad Array at the Centre for Applied Genomics (Toronto, Ontario, Canada). As part of quality control (QC), we investigated for any individuals who were related with a π > 0.1875 (second or third degree relatives) and excluded the individuals with the higher proportion of missing data. We also excluded people who had a high degree of missing genotyping data (> 3%). The data was filtered for a Hardy-Weinberg equilibrium pvalue > 5.7x10-7, SNP call rate >95%, and a minor allele frequency >0.01. We performed the GWAS imputation in the MACH v1.0.16 (http://www.sph.umich.edu/csg/yli/mach/index.html) software using the CEU samples from HapMap phase2 build 36 release 22 (http://hapmap.ncbi.nlm.nih.gov/index.html.en). This study was approved by the Human Research Ethics Committee of the University of Western Australia. The study was conducted in accordance with the Declaration of Helsinki and informed consent was obtained from all participants.

Rotterdam Study (RS-I, RS-II, RS-III)
The Rotterdam Study (RS) is a prospective population-based cohort study in the elderly living in Ommoord, a suburb of Rotterdam, the Netherlands 24 . In brief, the Rotterdam Study consists of 3 independent cohorts: RS-I, RS-II, and RS-III. Participants underwent multiple physical examinations with regular intervals from 1991 to present. The optic nerve head was assessed with ImageNet (RS-I and RS-II) or Heidelberg Retina Tomograph 2 (RS-III). Details of this assessment have been described elsewhere 18 . All measurements in RS-I, RS-II and RS-III were conducted after the Medical Ethics Committee of the Erasmus University had approved the study protocols and all participants had given a written informed consent in accordance with the Declaration of Helsinki. DNA was extracted from blood leucocytes according to standard procedures. Genotyping of SNPs was performed using the Illumina Infinium II HumanHap550 chip v3.0 array (RS-I); the HumanHap550 Duo Arrays and the Illumina Human610-Quad Arrays (RS-II), and the Human 610 Quad Arrays Illumina (RS-III). Samples with low call rate (<97.5%), with excess autosomal heterozygosity (>0.336), or with sex-mismatch were excluded, as were outliers identified by the identity-by-state clustering analysis (outliers were defined as being >3 s.d. from population mean or having identity-by-state probabilities >97%). We used genomic control to obtain optimal and unbiased results and applied the inverse variance method of each effect size estimated for both autosomal SNPs that were genotyped and imputed in both cohorts. A set of genotyped input SNPs with call rate >98%, with minor allele frequency >0.01, and with Hardy-Weinberg P value >10 −6 was used for imputation. We used the Markov Chain Haplotyping (MACH) package version 1.0.15 software (Rotterdam, The Netherlands; imputed to plus strand of NCBI build 36, HapMap release #22) for the analyses. For each imputed SNP, a reliability of imputation was estimated as the ratio of the empirically observed dosage variance to the expected binomial dosage variance (O/E ratio). GWAS analyses were performed using GRIMP 25 .

Singapore (SCES, SIMES, SINDI)
Singapore Malay Eye Study (SIMES) is a population-based prevalence survey of Malay adults aged 40 to 79 years living in Singapore that was conducted between August of 2004 and June of 2006 26 . From a Ministry of Home Affairs random sample of 16,069 Malay adults in the Southwestern area, an agestratified random sampling strategy was used in selecting 1400 from each decade from age 40 years onward (40-49, 50-59, 60-69, and 70-79 years).The 4,168 eligible participants from the sampling frame, while 3280 (78.7%) participated. Genome-wide genotyping was performed in 3,072 individuals 12,27 . Total of 3,072 DNA samples were genotyped using the Illumina Human 610 Quad Beadchips 27,28 . Using the same quality control criteria, we omitted a total of 530 individuals including those of subpopulation structure (n=170), cryptic relatedness (n=279), excessive heterozygosity or