To ascertain if single nucleotide polymorphisms (SNPs) involved in the determination of central corneal thickness, optic disc area, and vertical cup-to-disc ratio (VCDR) also are associated with open-angle glaucoma (OAG).
Retrospective case-control genetic association study.
A total of 16 SNPs associated with central corneal thickness, optic disc area, and VCDR were genotyped in 876 OAG cases and 883 normal controls. To determine if the SNPs were also correlated with OAG severity, the cohort was stratified into advanced OAG (n = 326) and nonadvanced OAG (n = 550). Both the cases and controls were of European descent and were recruited from within Australia.
Two VCDR SNPs were found to be significantly associated with OAG after correction for multiple testing. The 2 SNPs were rs10483727, found adjacent to the SIX1 gene ( P = 6.2 × 10 −06 ; odds ratio, 1.38; 95% confidence interval, 1.20 to 1.59), and rs1063192, found within the CDKN2B gene ( P = 2.2 × 10 −05 ; odds ratio, 0.74; 95% confidence interval, 0.64 to 0.85). The CDKN2B variant rs1063192 also was found to be associated more strongly with advanced OAG.
The findings from this study indicate that variants influencing VCDR are also risk alleles for OAG in our Australian cohort of European descent. The identification of SIX1 and CDKN2B as susceptibility loci will assist in understanding the pathologic mechanisms involved in the development of OAG.
Open-angle glaucoma (OAG) is a progressive optic neuropathy characterized by apoptosis of retinal ganglion cells, causing cupping of the optic disc and resulting in irreversible loss of vision. OAG, most commonly an adult-onset condition, is currently one of the leading causes of permanent blindness worldwide. The pathogenic mechanisms underlying OAG are poorly understood, although there are several well-established risk factors, including elevated intraocular pressure (IOP), age, family history, and central corneal thickness (CCT). Evidence from ethnic, familial, and twin studies also indicates that OAG has a strong genetic component, with several genes associated with the condition. These include MYOC , which is mutated in approximately 3% of OAG cases, and the recent discovery from genome-wide association (GWA) studies that variants within the CAV1 / CAV2 , CDKN2B-AS1 , and TMC01 loci increase susceptibility to OAG. Despite the identification of these and other genes, much of the genetic architecture of OAG remains to be elucidated.
Understanding the genetic component of OAG is problematic because the disease arises from a range of pathologic processes. There is a broad spectrum of phenotypic heterogeneity, including differences in the appearance of the optic nerve head, variation in the pattern of damage to visual fields, large variations in IOP, and often asymmetrical loss of vision. Age of onset and response to treatment also can differ greatly among individuals. To overcome the complexity and heterogeneity of diseases such as OAG, genetic studies can benefit by choosing to focus on the identification of genetic loci associated with highly heritable quantitative traits that form part of the overall phenotype. These traits, also referred to as endophenotypes, offer the advantages of being objectively quantifiable and providing a continuum of risk, rather than a dichotomous measure of disease status, in order to potentially simplify understanding the underlying genetic architecture of the disease of interest.
Several ocular quantitative traits, including CCT, optic disc area, and vertical cup-to-disc ratio (VCDR), represent plausible OAG endophenotypes. These traits are highly heritable, and all have been identified as risk factors for the onset of OAG. In individuals with ocular hypertension, thinner CCT measurements are associated with an increased risk of developing OAG, and familial and twin studies have indicated that CCT is a highly heritable trait, with heritability estimates in excess of 90%. Both optic disc area and VCDR are measurements that reflect the size and configuration of the optic nerve, a structure integral to the pathologic features of OAG. It has been postulated that larger optic discs are more susceptible to glaucomatous damage, whereas increases in the VCDR indicate progressive degeneration of retinal ganglion cells. Heritability estimates for the optic disc area and VCDR range from 52% to 77% and 56% to 79%, respectively.
The genetic architecture of CCT, optic disc area, and VCDR have become the focus of several recent GWA studies, with numerous loci found to be associated significantly with each trait. The genes COL5A1 , FOX01 , and ZNF469 have been identified as determinants of normal CCT variation in white cohorts from Australia, Britain, and Croatia. Furthermore, 2 studies, one based in the Netherlands and the other in Australia, concurrently investigated the genetic determinants of both optic disc area and VCDR. Genes or loci found to be associated with optic disc area included ATOH7 , CDC7 / TGFBR3 , and SALL1 , whereas ATOH7 , CDKN2B , CHEK2 , DCLK1 , SCYL1 , and SIX1 all were associated with VCDR. Given the identification of genetic factors that determine CCT, optic disc area, and VCDR, it is now possible to ascertain the endophenotypic status of these traits in relation to OAG, because a genetic correlation must be evident between the trait and disease for it to be classified as an endophenotype. Studies recently published by Ramdas and associates and Fan and associates have investigated optic disc area and VCDR genes in European and American OAG cohorts, respectively, although CCT susceptibility loci were not assessed. The purpose of this study was to evaluate whether associations between OAG and CCT, optic disc area, and VCDR genes were evident in an white Australian case-control cohort.
The study sample consisted of adults of European descent older than 17 years and living in Australia between 1991 and 2010. Each participant belonged to one of the following cohorts: the Australia and New Zealand Registry of Advanced Glaucoma (ANZRAG), the Blue Mountains Eye Study (BMES), the Glaucoma Flinders Medical Centre (GFMC) study, the Glaucoma Inheritance Study Tasmania (GIST), the Launceston Nursing Home (LNH) study, and the Normal South Australia (NSA) study.
The control group consisted of 883 participants from the BMES, LNH, and NSA studies. The BMES (n = 502 included in the current study design) is a population-based survey of vision and common eye diseases in the Blue Mountains region, west of Sydney, Australia. Details of the population and full recruitment methodology have been described in detail previously. The criteria for inclusion as a control sample from the BMES included a normal IOP, optic disc, and visual fields, along with no known family history of OAG. Some BMES samples used in this study were included previously in a GWA study of CCT. The LNH study (n = 99) consisted of normal elderly controls recruited from nursing homes within the Tasmanian city of Launceston, Australia. To qualify for inclusion in the cohort, participants needed to be free of ocular hypertension and to have a normal optic disc and visual fields. The NSA cohort (n = 282) consisted of healthy elderly controls ascertained from the Flinders Eye Centre and residential retirement villages and nursing homes within Adelaide, Australia. Because the NSA study was initiated to recruit controls for an OAG study, all participants were required to have no known family history of glaucoma, as well as a normal IOP, optic disc, and visual field. Further details on the BMES, LNH, and NSA participants assessed in this study can be found in Table 1 .
|Phenotype||Cohort||No.||Female (%)||Age at Recruitment (y)|
|Range||Mean ± SD|
|Control||BMES||502||47.2||77 to 98||81.9 ± 4.1|
|LNH||99||68.7||67 to 107||86.3 ± 6.8|
|NSA||282||55.7||42 to 96||75.9 ± 8.3|
|Total||883||52.3||42 to 107||80.5 ± 5.7|
|OAG||ANZRAG||141||52.6||33 to 99||75.8 ± 11.9|
|BMES||93||9.7||52 to 94||76.5 ± 9.4|
|GFMC||153||64.9||25 to 105||72.1 ± 12.6|
|GIST||489||58.5||17 to 103||72.8 ± 11.9|
|Total||876||53.5||17 to 105||73.5 ± 11.8|
Open-angle Glaucoma Group
The OAG group consisted of 876 participants from the ANZRAG, GFMC, GIST, and BMES cohorts. The ANZRAG (n = 141 included in the current study design) is based at the Flinders Medical Centre in Adelaide, Australia, and aims to recruit cases of advanced glaucoma Australia-wide through ophthalmologist referral. Further information on the ANZRAG can be found at www.anzrag.com . Enrollment in the ANZRAG was defined by severe visual loss resulting from OAG. This included best-corrected visual acuity worse than 6/60 resulting from OAG or reliable Humphrey 24-2 visual field results (Carl Zeiss Meditec, Inc, Dublin, California, USA), with a mean deviation worse than −22 dB or at least 2 of 4 central fixation squares affected with a pattern standard deviation of less than 0.5%. The field loss must have been the result of OAG, and the less severely affected eye also was required to have signs of glaucomatous disc damage. The GFMC cohort (n = 153) was established through the recruitment of nonadvanced OAG cases from the ophthalmology clinic at the Flinders Medical Centre. The definition of OAG for the GFMC was concordant findings of typical glaucomatous visual field defects on the Humphrey 24-2 test, along with optic disc rim thinning with an enlarged cup-to-disc ratio (≥ 0.7) or cup-to-disc ratio asymmetry (≥0.2) between the 2 eyes. The GIST (n = 489 included in the current study design) aimed to recruit all cases of OAG in Tasmania, an island state of Australia. The OAG definition criteria used for the GIST was the same as that for the GFMC study, and a full description of the recruitment methodology has been published in detail previously. Because the BMES was a population-based survey, participants with OAG (n = 93) also were recruited and were included in this study. Visual field defects in the BMES were assessed using the Humphrey 30-2 test, with other OAG definition criteria the same as that used in the GIST. Clinical exclusion criteria for each cohort were: (1) pseudoexfoliation glaucoma; (2) pigmentary glaucoma; (3) angle closure or mixed mechanism glaucoma; (4) secondary glaucoma resulting from aphakia, rubella, rubeosis, or inflammation; (5) congenital or infantile glaucoma; (6) glaucoma in the presence of a known syndrome associated with glaucoma; or (7) mutation in the MYOC gene (all samples were screened by direct sequencing of exon 3). To ascertain if particular variants were associated more strongly with advanced or nonadvanced OAG, a subanalysis was performed. Advanced OAG cases (n = 326) were selected from the entire OAG cohort based on the ANZRAG criteria for advanced OAG stated above and comprised participants from the ANZRAG (n = 141) and GIST (n = 185). The nonadvanced OAG cohort consisted of the remaining participants who did not meet the criteria for advanced OAG. Further details on the ANZRAG, GIST, GFMC, and BMES participants assessed in this study can be found in Table 1 .
Genomic DNA was extracted from peripheral blood of all participants according to standard methods and was masked as to phenotype. All samples were genotyped using the Sequenom MassARRAY platform (Sequenom, San Diego, California, USA). The MassARRAY platform uses the iPLEX GOLD chemistry on a Sequenom Autoflex mass spectrometer. This methodology was performed at the Australian Genome Research Facility, Brisbane, Australia. A total of 16 single nucleotide polymorphisms (SNPs) were genotyped, representing 11 different genes or loci associated with the traits CCT, optic disc area, and VCDR ( Table 2 ). The SNPs included in this study were selected based on having a genome-wide significant association with their respective trait at the time of study design.
|SNP||Chromosome Location||Nearest Gene (Symbol; Name)||Trait||Study|
|rs1536482||9q34.3||COL5A1 ; collagen, type V, α 1||CCT||Vitart and associates|
|rs7044529||9q34.3||COL5A1 ; collagen, type V, α 1||CCT||Vitart and associates Vithana and associates|
|rs2721051||13q14.11||FOXO1 ; forkhead box O1||CCT||Lu and associates|
|rs2755237||13q14.11||FOXO1 ; forkhead box O1||CCT||Lu and associates, Vitart and associates|
|rs9938149||16q24.2||ZNF469 ; zinc finger protein 469||CCT||Lu and associates, Vithana and associates|
|rs12447690||16q24.2||ZNF469 ; zinc finger protein 469||CCT||Lu and associates, Vitart and associates, Vithana and associates|
|rs1192415||1p22||CDC7/TGFBR3; cell division cycle 7, homolog/transforming growth factor, β receptor III||Optic disc area||Ramdas and associates|
|rs1900004||10q21.3||ATOH7; atonal homolog 7||Optic disc area/VCDR||Macgregor and associates, Ramdas and associates|
|rs3858145||10q21.3||ATOH7; atonal homolog 7||Optic disc area||Macgregor and associates|
|rs12571093||10q21.3||ATOH7 / PBLD ; atonal homolog 7/Phenazine biosynthesis-like protein domain containing||Optic disc area||Macgregor and associates|
|rs1362756||16q12.1||SALL1 ; sal-like 1||Optic disc area||Ramdas and associates|
|rs1063192||9p21||CDKN2B ; cyclin-dependent kinase inhibitor 2B||VCDR||Ramdas and associates|
|rs17146964||11q13||SCYL1 ; SCY1-like 1||VCDR||Ramdas and associates|
|rs1926320||13q13||DCLK1 ; doublecortin-like kinase 1||VCDR||Ramdas and associates|
|rs10483727||14q23.1||SIX1 ; SIX homeobox 1||VCDR||Ramdas and associates|
|rs1547014||22q12.1||CHEK2 ; CHK2 checkpoint homolog||VCDR||Ramdas and associates 34|
Differences in the sex ratio of the control and OAG cohorts were assessed using a chi-square test, whereas a Mann–Whitney U test was used to assess for differences in mean age. Both of these tests were performed in IBM SPSS Statistics software version 18.0 (Chicago, Illinois, USA). Statistical significance was accepted as P < .05. All genetic association and Hardy-Weinberg equilibrium tests were undertaken using the genetic analysis program PLINK version 1.06. The minor allele counts of each SNP in the control and OAG participants were compared using a Fisher exact test. Using logistic regression, the association of each SNP with OAG was examined after adjustment for age and sex, with the additive inheritance model investigated. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using logistic regression. To adjust for multiple testing, the Bonferroni correction method was applied for the 16 genotyped SNPs (corrected P value of .003; 0.05/16 SNPs).
Power calculations were conducted for the case-control analysis using the Genetic Power Calculator ( pngu.mgh.harvard.edu/∼purcell/gpc/ ). Calculations were performed with risk allele frequencies of 0.1, 0.2, 0.3, and 0.4. Genotype relative risks for the heterozygous and homozygous genotypes were set at 1.5 and 2.25, respectively. Linkage disequilibrium between the marker and risk allele was set at D′ = 1.0, and OAG prevalence was set at 2%. Under these criteria, the cohort had a power of 97.82% at a risk allele frequency of 0.1, 99.94% at a risk allele frequency of 0.2, 99.99% at a risk allele frequency of 0.3, and 100% at a risk allele frequency of 0.4 when calculated for an allelic model.
There was no difference in the sex ratios of the control and full OAG cohort ( P = .641). However, the control cohort was older than the full OAG cohort, with a mean age of 80.5 ± 5.7 years, compared with the mean age of 73.5 ± 11.8 years for the full OAG cohort ( P < .001). Minor allele frequencies from the control and OAG cohorts and results of the association tests for each SNP can be found in Table 3 , Table 4 , and Table 5 . Only 1 SNP, rs2755237 from the FOX01 gene, was found not to be in Hardy-Weinberg equilibrium ( P < .003).
|SNP: Minor Allele||Gene||Trait||MAF||Allelic Association||Adjusted for Age and Sex|
|OAG||Control||P Value||OR (95% CI)||P Value||OR (95% CI)|
|rs10483727: T||SIX1||VCDR||0.45||0.37||6.2 × 10 –06||1.38 (1.20 to 1.59)||4.2 × 10 –05||1.36 (1.17 to 1.57)|
|rs1063192: G||CDKN2B||VCDR||0.37||0.44||2.2 × 10 –05||0.74 (0.64 to 0.85)||7.1 × 10 –04||0.78 (0.67 to 0.90)|
|rs1192415: G||CDC7/TGFBR3||ODA||0.20||0.16||.01||1.27 (1.06 to 1.51)||.03||1.22 (1.02 to 1.47)|
|rs2721051: A||FOXO1||CCT||0.11||0.09||.04||1.26 (1.01 to 1.58)||.11||1.21 (0.96 to 1.54)|
|rs3858145: G||ATOH7||ODA||0.30||0.27||.08||1.15 (0.98 to 1.33)||.12||1.13 (0.97 to 1.33)|
|rs2755237: C||FOXO1||CCT||0.17||0.15||.11||1.17 (0.97 to 1.41)||.24||1.12 (0.93 to 1.36)|
|rs12571093: A||ATOH7||ODA||0.17||0.15||.12||1.16 (0.96 to 1.40)||.17||1.15 (0.94 to 1.41)|
|rs1926320: C||DCLK1||VCDR||0.20||0.22||.13||0.88 (0.74 to 1.04)||.24||0.90 (0.76 to 1.07)|
|rs1900004: A||ATOH7/PBLD||ODA||0.27||0.25||.17||1.12 (0.95 to 1.31)||.18||1.12 (0.95 to 1.33)|
|rs17146964: G||SCYL1||VCDR||0.20||0.18||.20||1.12 (0.94 to 1.33)||.18||1.14 (0.94 to 1.36)|
|rs1536482: A||COL5A1/RXRA||CCT||0.30||0.32||.31||0.92 (0.79 to 1.08)||.46||0.94 (0.81 to 1.10)|
|rs12447690: C||ZNF469||CCT||0.35||0.33||.33||1.08 (0.93 to 1.25)||.91||1.01 (0.87 to 1.18)|
|rs1547014: T||CHEK2||VCDR||0.30||0.31||.41||0.94 (0.81 to 1.09)||.77||0.98 (0.84 to 1.14)|
|rs7044529: T||COL5A1||CCT||0.14||0.13||.68||1.05 (0.86 to 1.28)||.98||1.00 (0.81 to 1.24)|
|rs9938149: C||ZNF469||CCT||0.35||0.35||.85||0.98 (0.85 to 1.14)||.46||0.94 (0.81 to 1.10)|
|rs1362756: G||SALL1||ODA||0.30||0.30||.85||1.02 (0.88 to 1.18)||.96||1.00 (0.85 to 1.17)|
|SNP: Minor Allele||Gene||Trait||MAF||Allelic Association||Adjusted for Age and Sex|
|OAG||Control||P Value||OR (95% CI)||P Value||OR (95% CI)|
|rs1063192: G||CDKN2B||VCDR||0.33||0.44||3 × 10 –06||0.63 (0.52 to 0.77)||1.1 × 10 –04||0.68 (0.56 to 0.83)|
|rs10483727: T||SIX1||VCDR||0.44||0.37||.002||1.35 (1.12 to 1.63)||.002||1.35 (1.11 to 1.64)|
|rs1192415: G||CDC7/TGFBR3||ODA||0.20||0.16||.04||1.28 (1.01 to 1.62)||.05||1.27 (0.99 to 1.61)|
|rs1926320: C||DCLK1||VCDR||0.19||0.22||.07||0.80 (0.64 to 1.01)||.24||0.87 (0.69 to 1.10)|
|rs1547014: T||CHEK2||VCDR||0.27||0.31||.11||0.84 (0.69 to 1.04)||.26||0.89 (0.72 to 1.09)|
|rs12571093: A||ATOH7||ODA||0.18||0.15||.12||1.22 (0.95 to 1.56)||.14||1.21 (0.94 to 1.57)|
|rs17146964: G||SCYL1||VCDR||0.21||0.18||.23||1.15 (0.92 to 1.45)||.18||1.18 (0.93 to 1.49)|
|rs1536482: A||COL5A1/RXRA||CCT||0.29||0.32||.25||0.88 (0.72 to 1.08)||.28||0.89 (0.73 to 1.10)|
|rs2755237: C||FOXO1||CCT||0.17||0.15||.27||1.16 (0.90 to 1.49)||.32||1.13 (0.89 to 1.45)|
|rs1900004: A||ATOH7/PBLD||ODA||0.27||0.25||.28||1.12 (0.91 to 1.39)||.33||1.11 (0.90 to 1.38)|
|rs7044529: T||COL5A1||CCT||0.15||0.13||.34||1.14 (0.88 to 1.48)||.49||1.10 (0.84 to 1.43)|
|rs3858145: G||ATOH7||ODA||0.29||0.27||.43||1.09 (0.89 to 1.33)||.46||1.08 (0.88 to 1.33)|
|rs2721051: A||FOXO1||CCT||0.10||0.09||.47||1.12 (0.82 to 1.53)||.51||1.11 (0.81 to 1.53)|
|rs9938149: C||ZNF469||CCT||0.34||0.35||.69||0.96 (0.79 to 1.16)||.39||0.92 (0.75 to 1.12)|
|rs12447690: C||ZNF469||CCT||0.34||0.33||.76||1.03 (0.85 to 1.26)||.76||0.97 (0.79 to 1.19)|
|rs1362756: G||SALL1||ODA||0.30||0.30||1.00||1.00 (0.81 to 1.22)||.80||0.97 (0.79 to 1.20)|