New insights into the genetics of primary open-angle glaucoma based on meta-analyses of intraocular pressure and optic disc characteristics.

Primary open-angle glaucoma (POAG), the most common optic neuropathy, is a heritable disease. Siblings of POAG cases have a ten-fold increased risk of developing the disease. Intraocular pressure (IOP) and optic nerve head characteristics are used clinically to predict POAG risk. We conducted a genome-wide association meta-analysis of IOP and optic disc parameters and validated our findings in multiple sets of POAG cases and controls. Using imputation to the 1000 genomes (1000G) reference set, we identified 9 new genomic regions associated with vertical cup-disc ratio (VCDR) and 1 new region associated with IOP. Additionally, we found 5 novel loci for optic nerve cup area and 6 for disc area. Previously it was assumed that genetic variation influenced POAG either through IOP or via changes to the optic nerve head; here we present evidence that some genomic regions affect both IOP and the disc parameters. We characterized the effect of the novel loci through pathway analysis and found that pathways involved are not entirely distinct as assumed so far. Further, we identified a novel association between CDKN1A and POAG. Using a zebrafish model we show that six6b (associated with POAG and optic nerve head variation) alters the expression of cdkn1a. In summary, we have identified several novel genes influencing the major clinical risk predictors of POAG and showed that genetic variation in CDKN1A is important in POAG risk.

WNT10A exonic variant increases the risk of keratoconus by decreasing corneal thickness.

Keratoconus is a degenerative eye condition which results from thinning of the cornea and causes vision distortion. Treatments such as ultraviolet (UV) cross-linking have proved effective for management of keratoconus when performed in early stages of the disease. The central corneal thickness (CCT) is a highly heritable endophenotype of keratoconus, and it is estimated that up to 95% of its phenotypic variance is due to genetics. Genome-wide association efforts of CCT have identified common variants (i.e. minor allele frequency (MAF) >5%). However, these studies typically ignore the large set of exonic variants whose MAF is usually low. In this study, we performed a CCT exome-wide association analysis in a sample of 1029 individuals from a population-based study in Western Australia. We identified a genome-wide significant exonic variant rs121908120 (P = 6.63 × 10(-10)) in WNT10A. This gene is 437 kb from a gene previously associated with CCT (USP37). We showed in a conditional analysis that the WNT10A variant completely accounts for the signal previously seen at USP37. We replicated our finding in independent samples from the Brisbane Adolescent Twin Study, Twin Eye Study in Tasmania and the Rotterdam Study. Further, we genotyped rs121908120 in 621 keratoconus cases and compared the frequency to a sample of 1680 unscreened controls from the Queensland Twin Registry. We found that rs121908120 increases the risk of keratoconus two times (odds ratio 2.03, P = 5.41 × 10(-5)).

ARHGEF12 influences the risk of glaucoma by increasing intraocular pressure.

Primary open-angle glaucoma (POAG) is a blinding disease. Two important risk factors for this disease are a positive family history and elevated intraocular pressure (IOP), which is also highly heritable. Genes found to date associated with IOP and POAG are ABCA1, CAV1/CAV2, GAS7 and TMCO1. However, these genes explain only a small part of the heritability of IOP and POAG. We performed a genome-wide association study of IOP in the population-based Rotterdam Study I and Rotterdam Study II using single nucleotide polymorphisms (SNPs) imputed to 1000 Genomes. In this discovery cohort (n = 8105), we identified a new locus associated with IOP. The most significantly associated SNP was rs58073046 (ß = 0.44, P-value = 1.87 × 10(-8), minor allele frequency = 0.12), within the gene ARHGEF12. Independent replication in five population-based studies (n = 7471) resulted in an effect size in the same direction that was significantly associated (ß = 0.16, P-value = 0.04). The SNP was also significantly associated with POAG in two independent case-control studies [n = 1225 cases and n = 4117 controls; odds ratio (OR) = 1.53, P-value = 1.99 × 10(-8)], especially with high-tension glaucoma (OR = 1.66, P-value = 2.81 × 10(-9); for normal-tension glaucoma OR = 1.29, P-value = 4.23 × 10(-2)). ARHGEF12 plays an important role in the RhoA/RhoA kinase pathway, which has been implicated in IOP regulation. Furthermore, it binds to ABCA1 and links the ABCA1, CAV1/CAV2 and GAS7 pathway to Mendelian POAG genes (MYOC, OPTN, WDR36). In conclusion, this study identified a novel association between IOP and ARHGEF12.

Incidence of glaucomatous visual field loss after two decades of follow-up: the Rotterdam Study.

To determine the incidence of glaucomatous visual field loss (GVFL) two decades after the start of the Rotterdam Study, and to compare known risk factors for open-angle glaucoma (OAG) between different clinical manifestations of OAG. Of 6806 participants aged 55 years and older from the population-based Rotterdam Study, 3939 underwent visual field testing at baseline and at least one follow-up round. The ophthalmic examinations included optic disc assessment and measurements of intraocular pressure (IOP), refractive error, diastolic blood pressure (DBP), and height and weight. The incidence rate of GVFL was calculated. Associations with the risk factors age, gender, baseline IOP, family history, myopia, DBP, and body-mass index [BMI] were assessed using Cox regression, with different clinical manifestations of OAG as outcome measure (glaucomatous optic neuropathy (GON), GVFL, GVFL and GON, GVFL without GON, and GON without GVFL). Median follow-up was 11.1 (IQR 6.8-17.2; range 5.0-20.3) years. The incidence rate of GVFL was 2.9 (95% confidence interval 2.4-3.4) per 1000 person years (140 cases with incident GVFL in one (n = 113) or both (n = 27) eyes). Baseline IOP and age were significantly associated with all OAG outcomes (all p < 0.001); BMI showed a non-significant protective effect in all outcomes (p = 0.01 to p = 0.09). Gender, myopia, and DBP were not associated with any outcome. Our study provides an estimate of the long-term incidence of GVFL in a predominantly white population. The development of GVFL was strongly associated with baseline IOP and age. Risk factor profiles were similar for the different outcomes.

Meta-analysis of Genome-Wide Association Studies Identifies Novel Loci Associated With Optic Disc Morphology.

Primary open-angle glaucoma is the most common optic neuropathy and an important cause of irreversible blindness worldwide. The optic nerve head or optic disc is divided in two parts: a central cup (without nerve fibers) surrounded by the neuroretinal rim (containing axons of the retinal ganglion cells). The International Glaucoma Genetics Consortium conducted a meta-analysis of genome-wide association studies consisting of 17,248 individuals of European ancestry and 6,841 individuals of Asian ancestry. The outcomes of the genome-wide association studies were disc area and cup area. These specific measurements describe optic nerve morphology in another way than the vertical cup-disc ratio, which is a clinically used measurement, and may shed light on new glaucoma mechanisms. We identified 10 new loci associated with disc area (CDC42BPA, F5, DIRC3, RARB, ABI3BP, DCAF4L2, ELP4, TMTC2, NR2F2, and HORMAD2) and another 10 new loci associated with cup area (DHRS3, TRIB2, EFEMP1, FLNB, FAM101, DDHD1, ASB7, KPNB1, BCAS3, and TRIOBP). The new genes participate in a number of pathways and future work is likely to identify more functions related to the pathogenesis of glaucoma.

Exome sequencing and functional analyses suggest that SIX6 is a gene involved in an altered proliferation-differentiation balance early in life and optic nerve degeneration at old age.

Primary open-angle glaucoma (POAG) is a hereditary neurodegenerative disease, characterized by optic nerve changes including increased excavation, notching and optic disc hemorrhages. The excavation can be described by the vertical cup-disc ratio (VCDR). Previously, genome-wide significant evidence for the association of rs10483727 in SIX1-SIX6 locus with VCDR and subsequent POAG was found. Using 1000 genomes-based imputation of four independent population-based cohorts in the Netherlands, we identified a missense variant rs33912345 (His141Asn) in SIX6 associated with VCDR (Pmeta = 7.74 × 10(-7), n = 11 473) and POAG (Pmeta = 6.09 × 10(-3), n = 292). Exome sequencing analysis revealed another missense variant rs146737847 (Glu129Lys) also in SIX6 associated with VCDR (P = 5.09 × 10(-3), n = 1208). These two findings point to SIX6 as the responsible gene for the previously reported association signal. Functional characterization of SIX6 in zebrafish revealed that knockdown of six6b led to a small eye phenotype. Histological analysis showed retinal lamination, implying an apparent normal development of the eye, but an underdeveloped lens, and reduced optic nerve diameter. Expression analysis of morphants at 3 dpf showed a 5.5-fold up-regulation of cdkn2b, a cyclin-dependent kinase inhibitor, involved in cell cycle regulation and previously associated with VCDR and POAG in genome-wide association studies (GWASs). Since both six6b and cdkn2b play a key role in cell proliferation, we assessed the proliferative activity in the eye of morphants and found an alteration in the proliferative pattern of retinal cells. Our findings in humans and zebrafish suggest a functional involvement of six6b in early eye development, and open new insights into the genetic architecture of POAG.

Associations with intraocular pressure across Europe: The European Eye Epidemiology (E3) Consortium.

Raised intraocular pressure (IOP) is the most important risk factor for developing glaucoma, the second commonest cause of blindness globally. Understanding associations with IOP and variations in IOP between countries may teach us about mechanisms underlying glaucoma. We examined cross-sectional associations with IOP in 43,500 European adults from 12 cohort studies belonging to the European Eye Epidemiology (E3) consortium. Each study conducted multivariable linear regression with IOP as the outcome variable and results were pooled using random effects meta-analysis. The association of standardized study IOP with latitude was tested using meta-regression. Higher IOP was observed in men (0.18 mmHg; 95 % CI 0.06, 0.31; P = 0.004) and with higher body mass index (0.21 mmHg per 5 kg/m2; 95 % CI 0.14, 0.28; P < 0.001), shorter height (-0.17 mmHg per 10 cm; 95 % CI -0.25, -0.08; P < 0.001), higher systolic blood pressure (0.17 mmHg per 10 mmHg; 95 % CI 0.12, 0.22; P < 0.001) and more myopic refraction (0.06 mmHg per Dioptre; 95 % CI 0.03, 0.09; P < 0.001). An inverted U-shaped trend was observed between age and IOP, with IOP increasing up to the age of 60 and decreasing in participants older than 70 years. We found no significant association between standardized IOP and study location latitude (P = 0.76). Novel findings of our study include the association of lower IOP in taller people and an inverted-U shaped association of IOP with age. We found no evidence of significant variation in IOP across Europe. Despite the limited range of latitude amongst included studies, this finding is in favour of collaborative pooling of data from studies examining environmental and genetic determinants of IOP in Europeans.

Author Correction: Cross-ancestry genome-wide association analysis of corneal thickness strengthens link between complex and Mendelian eye diseases.

Emmanuelle Souzeau, who contributed to analysis of data, was inadvertently omitted from the author list in the originally published version of this Article. This has now been corrected in both the PDF and HTML versions of the Article.

Cross-ancestry genome-wide association analysis of corneal thickness strengthens link between complex and Mendelian eye diseases.

Central corneal thickness (CCT) is a highly heritable trait associated with complex eye diseases such as keratoconus and glaucoma. We perform a genome-wide association meta-analysis of CCT and identify 19 novel regions. In addition to adding support for known connective tissue-related pathways, pathway analyses uncover previously unreported gene sets. Remarkably, >20% of the CCT-loci are near or within Mendelian disorder genes. These included FBN1, ADAMTS2 and TGFB2 which associate with connective tissue disorders (Marfan, Ehlers-Danlos and Loeys-Dietz syndromes), and the LUM-DCN-KERA gene complex involved in myopia, corneal dystrophies and cornea plana. Using index CCT-increasing variants, we find a significant inverse correlation in effect sizes between CCT and keratoconus (r = -0.62, P = 5.30 × 10-5) but not between CCT and primary open-angle glaucoma (r = -0.17, P = 0.2). Our findings provide evidence for shared genetic influences between CCT and keratoconus, and implicate candidate genes acting in collagen and extracellular matrix regulation.

A Genome-Wide Scan for MicroRNA-Related Genetic Variants Associated With Primary Open-Angle Glaucoma.

Purpose: To identify microRNAs (miRNAs) involved in primary open-angle glaucoma (POAG), using genetic data. MiRNAs are small noncoding RNAs that posttranscriptionally regulate gene expression. Genetic variants in miRNAs or miRNA-binding sites within gene 3'-untranslated regions (3'UTRs) are expected to affect miRNA function and contribute to disease risk. Methods: Data from the recent genome-wide association studies on intraocular pressure, vertical cup-to-disc ratio (VCDR), cupa area and disc area were used to investigate the association of miRNAs with POAG endophenotypes. Putative targets of the associated miRNAs were studied according to their association with POAG and tested in cell line by transfection experiments for regulation by the miRNAs. Results: Of 411 miRNA variants, rs12803915:A/G in the terminal loop of pre-miR-612 and rs2273626:A/C in the seed sequence of miR-4707 were significantly associated with VCDR and cup area (P values < 1.2 × 10-4). The first variant is demonstrated to increase the miR-612 expression. We showed that the second variant does not affect the miR-4707 biogenesis, but reduces the binding of miR-4707-3p to CARD10, a gene known to be involved in glaucoma. Moreover, of 72,052 miRNA-binding-site variants, 47 were significantly associated with four POAG endophenotypes (P value < 6.9 × 10-6). Of these, we highlighted 10 variants that are more likely to affect miRNA-mediated gene regulation in POAG. These include rs3217992 and rs1063192, which have been shown experimentally to affect miR-138-3p- and miR-323b-5p-mediated regulation of CDKN2B. Conclusions: We identified a number of miRNAs that are associated with POAG endophenotypes. The identified miRNAs and their target genes are candidates for future studies on miRNA-related therapies for POAG.

Automated Segmentability Index for Layer Segmentation of Macular SD-OCT Images.

PURPOSE: To automatically identify which spectral-domain optical coherence tomography (SD-OCT) scans will provide reliable automated layer segmentations for more accurate layer thickness analyses in population studies. METHODS: Six hundred ninety macular SD-OCT image volumes (6.0 × 6.0 × 2.3 mm3) were obtained from one eyes of 690 subjects (74.6 ± 9.7 [mean ± SD] years, 37.8% of males) randomly selected from the population-based Rotterdam Study. The dataset consisted of 420 OCT volumes with successful automated retinal nerve fiber layer (RNFL) segmentations obtained from our previously reported graph-based segmentation method and 270 volumes with failed segmentations. To evaluate the reliability of the layer segmentations, we have developed a new metric, segmentability index SI, which is obtained from a random forest regressor based on 12 features using OCT voxel intensities, edge-based costs, and on-surface costs. The SI was compared with well-known quality indices, quality index (QI), and maximum tissue contrast index (mTCI), using receiver operating characteristic (ROC) analysis. RESULTS: The 95% confidence interval (CI) and the area under the curve (AUC) for the QI are 0.621 to 0.805 with AUC 0.713, for the mTCI 0.673 to 0.838 with AUC 0.756, and for the SI 0.784 to 0.920 with AUC 0.852. The SI AUC is significantly larger than either the QI or mTCI AUC (P < 0.01). CONCLUSIONS: The segmentability index SI is well suited to identify SD-OCT scans for which successful automated intraretinal layer segmentations can be expected. TRANSLATIONAL RELEVANCE: Interpreting the quantification of SD-OCT images requires the underlying segmentation to be reliable, but standard SD-OCT quality metrics do not predict which segmentations are reliable and which are not. The segmentability index SI presented in this study does allow reliable segmentations to be identified, which is important for more accurate layer thickness analyses in research and population studies.

Validity of Automated Choroidal Segmentation in SS-OCT and SD-OCT.

PURPOSE: To evaluate the validity of a novel fully automated three-dimensional (3D) method capable of segmenting the choroid from two different optical coherence tomography scanners: swept-source OCT (SS-OCT) and spectral-domain OCT (SD-OCT). METHODS: One hundred eight subjects were imaged using SS-OCT and SD-OCT. A 3D method was used to segment the choroid and quantify the choroidal thickness along each A-scan. The segmented choroidal posterior boundary was evaluated by comparing to manual segmentation. Differences were assessed to test the agreement between segmentation results of the same subject. Choroidal thickness was defined as the Euclidian distance between Bruch's membrane and the choroidal posterior boundary, and reproducibility was analyzed using automatically and manually determined choroidal thicknesses. RESULTS: For SS-OCT, the average choroidal thickness of the entire 6- by 6-mm2 macular region was 219.5 µm (95% confidence interval [CI], 204.9-234.2 µm), and for SD-OCT it was 209.5 µm (95% CI, 197.9-221.0 µm). The agreement between automated and manual segmentations was high: Average relative difference was less than 5 µm, and average absolute difference was less than 15 µm. Reproducibility of choroidal thickness between repeated SS-OCT scans was high (coefficient of variation [CV] of 3.3%, intraclass correlation coefficient [ICC] of 0.98), and differences between SS-OCT and SD-OCT results were small (CV of 11.0%, ICC of 0.73). CONCLUSIONS: We have developed a fully automated 3D method for segmenting the choroid and quantifying choroidal thickness along each A-scan. The method yielded high validity. Our method can be used reliably to study local choroidal changes and may improve the diagnosis and management of patients with ocular diseases in which the choroid is affected.

Population-based evaluation of retinal nerve fiber layer, retinal ganglion cell layer, and inner plexiform layer as a diagnostic tool for glaucoma.

PURPOSE: We determined the glaucoma screening performance of regional optical coherence tomography (OCT) layer thickness measurements in the peripapillary and macular region, in a population-based setting. METHODS: Subjects (n = 1224) in the Rotterdam Study underwent visual field testing (Humphrey Field Analyzer) and OCT of the macula and optic nerve head (Topcon 3-D OCT-1000). We determined the mean thicknesses of the retinal nerve fiber layer (RNFL), retinal ganglion cell layer (RGCL), and inner plexiform layer for regions-of-interest; thus, defining a series of OCT parameters, using the Iowa Reference Algorithms. Reference standard was the presence of glaucomatous visual field loss (GVFL); controls were subjects without GVFL, an intraocular pressure (IOP) of 21 mm Hg or less, and no positive family history for glaucoma. We calculated the area under the receiver operating characteristics curve (AUCs) and the sensitivity at 97.5% specificity for each parameter. RESULTS: After excluding 23 subjects with an IOP > 21 mm Hg and 73 subjects with a positive family history for glaucoma, there were 1087 controls and 41 glaucoma cases. Mean RGCL thickness in the inferior half of the macular region showed the highest AUC (0.85; 95% confidence interval [CI] 0.77-0.92) and sensitivity (53.7%; 95% CI, 38.7-68.0%). The mean thickness of the peripapillary RNFL had an AUC of 0.77 (95% CI, 0.69-0.85) and a sensitivity of 24.4% (95% CI, 13.7-39.5%). CONCLUSIONS: Macular RGCL loss is at least as common as peripapillary RNFL abnormalities in population-based glaucoma cases. Screening for glaucoma using OCT-derived regional thickness identifies approximately half of those cases of glaucoma as diagnosed by perimetry.

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.

Genome-wide analysis of multi-ancestry cohorts identifies new loci influencing intraocular pressure and susceptibility to glaucoma.

Elevated intraocular pressure (IOP) is an important risk factor in developing glaucoma, and variability in IOP might herald glaucomatous development or progression. We report the results of a genome-wide association study meta-analysis of 18 population cohorts from the International Glaucoma Genetics Consortium (IGGC), comprising 35,296 multi-ancestry participants for IOP. We confirm genetic association of known loci for IOP and primary open-angle glaucoma (POAG) and identify four new IOP-associated loci located on chromosome 3q25.31 within the FNDC3B gene (P = 4.19 × 10(-8) for rs6445055), two on chromosome 9 (P = 2.80 × 10(-11) for rs2472493 near ABCA1 and P = 6.39 × 10(-11) for rs8176693 within ABO) and one on chromosome 11p11.2 (best P = 1.04 × 10(-11) for rs747782). Separate meta-analyses of 4 independent POAG cohorts, totaling 4,284 cases and 95,560 controls, showed that 3 of these loci for IOP were also associated with POAG.

Genome-wide association analyses identify multiple loci associated with central corneal thickness and keratoconus.

Central corneal thickness (CCT) is associated with eye conditions including keratoconus and glaucoma. We performed a meta-analysis on >20,000 individuals in European and Asian populations that identified 16 new loci associated with CCT at genome-wide significance (P < 5 × 10(-8)). We further showed that 2 CCT-associated loci, FOXO1 and FNDC3B, conferred relatively large risks for keratoconus in 2 cohorts with 874 cases and 6,085 controls (rs2721051 near FOXO1 had odds ratio (OR) = 1.62, 95% confidence interval (CI) = 1.4-1.88, P = 2.7 × 10(-10), and rs4894535 in FNDC3B had OR = 1.47, 95% CI = 1.29-1.68, P = 4.9 × 10(-9)). FNDC3B was also associated with primary open-angle glaucoma (P = 5.6 × 10(-4); tested in 3 cohorts with 2,979 cases and 7,399 controls). Further analyses implicate the collagen and extracellular matrix pathways in the regulation of CCT.

Optimizing the information yield of 3-D OCT in glaucoma.

PURPOSE: To determine, first, which regions of 3-D optical coherence tomography (OCT) volumes can be segmented completely in the majority of subjects and, second, the relationship between analyzed area and thickness measurement test-retest variability. METHODS: Three-dimensional OCT volumes (6 × 6 mm) centered around the fovea and optic nerve head (ONH) of 925 Rotterdam Study participants were analyzed; 44 participants were scanned twice. Volumes were segmented into 10 layers, and we determined the area where all layers could be identified in at least 95% (macula) or 90% (ONH) of subjects. Macular volumes were divided in 2 × 2, 4 × 4, 6 × 6, 8 × 8, or 68 blocks. We placed two circles around the ONH; the ONH had to fit into the smaller circle, and the larger circle had to fit into the segmentable part of the volume. The area between the circles was divided in 3 to 12 segments. We determined the test-retest variability (coefficient of repeatability) of the retinal nerve fiber layer (RNFL) and ganglion cell layer (RGCL) thickness measurements as a function of size of blocks/segments. RESULTS: Eighty-two percent of the macular volume could be segmented in at least 95% of subjects; for the ONH, this was 65% in at least 90%. The radii of the circles were 1.03 and 1.84 mm. Depending on the analyzed area, median test-retest variability ranged from 8% to 15% for macular RNFL, 11% to 22% for macular RGCL, 5% to 11% for the two together, and 18% to 22% for ONH RNFL. CONCLUSIONS: Test-retest variability hampers a detailed analysis of 3-D OCT data. Combined macular RNFL and RGCL thickness averaged over larger areas had the best test-retest variability.