Association of Open-Angle Glaucoma Loci With Incident Glaucoma in the Blue Mountains Eye Study




Purpose


To determine if open-angle glaucoma (OAG)-associated single nucleotide polymorphisms (SNPs) are associated with incident glaucoma and if such genetic information is useful in OAG risk prediction.


Design


Case-control from within a population-based longitudinal study.


Methods


study population : Individuals aged over 49 years of age living in the Blue Mountains region west of Sydney and enrolled in the Blue Mountains Eye Study. observation : Cases for this sub-study (n = 67) developed incident OAG between baseline and 10-year visits, in either eye, while controls (n = 1919) had no evidence for OAG at any visit. All participants had an ocular examination and DNA genotyped for reported OAG risk SNPs. main outcome measure : Incident OAG.


Results


Two loci also known to be associated with cup-to-disc ratio as well as OAG (9p21 near CDKN2B-AS1 and SIX1/SIX6 ) were both significantly associated with incident OAG in the Blue Mountains Eye Study cohort ( P = .006 and P = .004, respectively). The TMCO1 locus was nominally associated ( P = .012), while the CAV1/CAV2 and 8q22 loci were not associated. Multivariate logistic regression and neural network analysis both indicated that the genetic risk factors contributed positively to the predictive models incorporating traditional risk factors.


Conclusions


This study shows that previously reported genetic variations related to OAG and cup-to-disc ratio are associated with the onset of OAG and thus may become useful in risk prediction algorithms designed to target early treatment to those most at risk of developing glaucoma.


Open-angle glaucoma (OAG) is one of the most common causes of blindness worldwide and the number of affected individuals is expected to increase as the population ages. It is characterized by the progressive loss of retinal ganglion cells, resulting in visual field defects beginning in the periphery and progressing centrally.


Current guidelines for the Screening, Prognosis, Diagnosis, Management, and Prevention of Glaucoma state that individuals at low risk of conversion from glaucoma suspect or ocular hypertension to glaucoma should be monitored, and those at high risk should be considered for treatment. The determination of who is at risk is based on a range of clinical risk factors, such as intraocular pressure, migraine, family history, and central corneal thickness. The genetic component of glaucoma risk is well recognized. Several high-penetrance genes have been described and genetic testing is available for some of these. However, most patients do not carry mutations, and thus the contribution of genetics in risk prediction is currently limited to knowledge of family history, which is notoriously unreliable.


Several common genetic variants increasing the risk of OAG have recently been identified through genome-wide association studies (GWAS; Table 1 ). Three studies of white individuals have collectively identified 5 loci. Loci reaching genome-wide significance levels include TMCO 1 on chromosome 1q24, CAV1/CAV2 on 7q31, a regulatory region on 8q22, the 9p21 locus near CDKN2B-AS1 , and SIX1/SIX6 on 14q23. Several of these loci have also been associated with OAG-related quantitative traits, including intraocular pressure (IOP) and vertical cup-to-disc ratio (VCDR). However, reports from these cross-sectional studies did not distinguish whether the SNPs are associated with the initiation or progression of OAG. Different genetic factors may be involved with these 2 phases. Two of the loci (9p21 and TMCO1 ) have been identified in an advanced OAG cohort, suggesting they could be important in disease progression leading to the observed enrichment in advanced disease. Both regions are also associated with less severe OAG cases, indicating they may also be important to the vulnerability to OAG and its initiation.



Table 1

Published Genome-Wide Significant Single Nucleotide Polymorphism Associations With Open-Angle Glaucoma and Associated Traits




























































































Locus SNP (Allele) Gene Phenotype Country Results Reference
1q24 rs4656461(G) TMCO1 Advanced POAG Australia P = 6.1 × 1 −10
OR = 1.68
Burdon et al
rs755523(C) TMCO1 IOP Netherlands P = 1.6 × 10 −8
B = 0.28
Van Koolwijk et al
7q31 rs4236601(A) CAV1/CAV2 POAG Iceland P = 5.0 × 10 −10
OR = 1.36
Thorleifsson et al
8q22 rs1521774(G) Gene desert NTG USA P = 1.35 × 10 −9
OR = 0.62
Wiggs et al
9p21 rs4977756(A) CDKN2B-AS1 Advanced POAG Australia P = 4.7 × 10 −9
OR = 1.50
Burdon et al
rs1412829(T) CDKN2B-AS1 POAG Australia P = 2.93 × 10 −10
OR = 1.45
Burdon et al
rs1063192(G) CDKN2B POAG USA P = 2.30 × 20 −17
OR = 1.43
Wiggs et al
VCDR Netherlands P = 1.96 × 10 −14
B = −0.014
Ramdas et al
14q22 rs10483727(A) SIX1/SIX6 POAG USA P = 3.87 × 10 −11
OR = 1.32
Wiggs et al
VCDR Netherlands P = 9.30 × 10 −11
B = 0.012
Ramdas et al

B = beta; IOP = intraocular pressure; NTG = normal tension glaucoma; OR =odds ratio; P = P value; POAG = primary open-angle glaucoma; SNP = single nucleotide polymorphism; VCDR = vertical cup-to-disc ratio.


There have been no previous reports seeking to examine genetic risk associated with the onset of OAG. To fill in this gap of knowledge, we have undertaken an analysis in an older Australian cohort from the Blue Mountains Eye Study (BMES), to determine whether genetic analysis could inform on the likelihood of an individual’s being diagnosed with glaucoma in the future. The BMES is a well-known longitudinal population-based study of ophthalmic health and disease that includes baseline and 5-year and 10-year follow-up data. This population is considered to be stable, homogeneous, and representative of the Australian population, but older compared to the average for the state of New South Wales, where it was recruited.


Methods


The Blue Mountains Eye Study was approved by the Human Research Ethics Committee of the University of Sydney for investigation of the epidemiology and genetics of ocular disease. The BMES has been described previously. Briefly, the BMES is a population-based study of individuals living in the Blue Mountains region west of Sydney, Australia. Any permanent, noninstitutionalized resident of the defined geographic region born before January 1, 1943 (aged over 49 years at time of recruitment) and able to give written informed consent was eligible for enrollment in BMES and was contacted by door-to-door canvassing. Participants underwent a baseline visit, with follow-up at 5 years and at 10 years. At baseline, all participants received a detailed eye examination, including applanation tonometry, suprathreshold automated perimetry (Humphrey 76-point test, followed by 30-2 fields [Humphrey Visual Field Analyser 630 with StatPac 2, Humphrey Instruments, Inc, San Leandro, California, USA]), and stereoscopic optic disc photography (Carl Zeiss Australia, Sydney, New South Wales, Australia). The current sub-study consisted of a case-control design from within the BMES cohort study. Participants with normal threshold or suprathreshold field tests and no sign of glaucoma at the baseline visit were included in the current study. Participants with OAG at baseline (prevalent OAG) were excluded. As previously reported, incident OAG cases were defined as participants free of OAG at baseline who showed glaucomatous field loss on full-threshold perimetry (Humphrey 24-2 or 30-2), which matched the optic disc appearance, at either the 5-year or 10-year follow-up visit, without reference to intraocular pressure. Patients with pseudoexfoliation syndrome were not excluded (n = 7). DNA was extracted from peripheral whole blood using standard techniques. Genotyping was performed on the HumanHap670 array (Illumina, San Diego, California, USA) as part of the Wellcome Trust Case Control Cohort 2 Genome-Wide Association Study. Data were cleaned and genotypes called as previously described. No significant population stratification was detected in this population.


Single nucleotide polymorphisms (SNPs) were selected for analysis if they had been previously reported to be associated with OAG (including normal tension glaucoma) at genome-wide significance in white populations. The reported SNPs with the smallest P values at each locus were chosen for this analysis. In the case of the 9p21 locus reported independently in 2 papers, the top SNP from each paper was chosen, as well as a third SNP at genome-wide significance in the replication cohorts of Burdon and associates (rs1412829). We hypothesize that if this SNP had been typed in the discovery cohort for this study, it would likely have been the top-ranked SNP at this locus. Seven SNPs at 5 loci were chosen for analysis in total.


Power calculations were conducted with the Genetic Power Calculator. This study had 77% power to detect an association at a SNP with an allele frequency of 30% and an odds ratio of 1.6 under an additive model at a P value of .007, assuming a population disease prevalence of 5.67%. These parameters are similar to those reported for most of these loci in cross-sectional studies of OAG genetics.


Differences in the demographics of the available cohort were assessed using IBM SPSS Statistics V20. Association analysis was conducted under a univariate allelic model and also using logistic regression under an additive model adjusted for baseline measurements of age, sex, mean IOP of both eyes, mean cup-to-disc ratio of both eyes, mean disc diameter of both eyes, and systolic and diastolic blood pressure using Plink. Statistical significance was set to P < .007 under a Bonferroni correction, to account for the 7 SNPs tested. One associated SNP from each significant or nominally significant locus and the clinical variables were included in a logistic regression model using IBM SPSS Statistics V20. SNPs were coded to the number of OAG risk alleles carried by each participant at each SNP (0, 1, or 2). Collinearity between variables in the model were assessed by calculating the tolerance and the variance inflation factor (VIF). No collinearity was detected (no VIF >2).


The rank importance of each model component was also assessed using a large population of neural networks (produced using Matlab; The MathWorks, Inc, Natick, Massachusetts, USA). A neural network can be thought of as a small machine capable of learning. It is trained by exposure to a dataset comprising inputs (for example, the characteristics of horses in a race) and outputs (the winning horse). After each round of training, the link strengths within the network are changed, and further training is undertaken until its predictive performance on a previously unseen “validation” dataset no longer improves. The resulting network’s performance is then measured using a final, also unseen “test” dataset. In this study, each neural network drew its inputs from unique subset of 7 SNPs and 7 clinical variables (age, sex, diastolic and systolic blood pressure, cup-to-disc ratio, IOP, and disc diameter). To cover all possible permutations of these 14 inputs, 16 383 neural networks were required.


Each neural network was trained and tested with a cohort comprising glaucoma patients (n = 67) and an equal number of randomly selected controls: 70% of the cohort was used to train the network, 15% to validate its performance during training, and the remaining 15% were unseen during training and were used to test the final performance of each network. Each neural network was trained and tested 20 times. In separate analyses, controls were either age matched to within 2 years of incident cases or not age matched. An error score was calculated to describe the performance of each neural network: the error score was the proportion of cases incorrectly classified (as glaucoma or as not glaucoma) by the network. Ranking of the importance of input variables (clinical parameters and SNPs) was achieved by ranking their influence on neural network error score. If the presence of a particular SNP or clinical variable (among the neural network’s input variables) reduced the error score, that SNP or variable can be considered to make a positive contribution to the performance of the network (ie, it is of useful predictive value).




Results


The BMES cohort consisted of 1986 individuals with follow-up phenotype data at either the 5-year, 10-year, or both visits with genotypes available ( Table 2 ). Of the 1986 participants, there were 67 incident OAG cases over the full 10-year follow-up period. At baseline, the incident OAG cases were significantly older than controls ( P < .001) and had a higher proportion of female subjects ( P = .009). IOP and VCDR at the baseline visit were also significantly different between those who later developed OAG and those who did not ( Table 2 ), as was systolic blood pressure. These features of this cohort have been previously reported.



Table 2

Baseline Characteristics of the Eligible Blue Mountains Eye Study Participants by Glaucoma Status

















































Incident OAG (Cases) No OAG (Controls) P Value
N 67 1919
Sex (% female) 73% 57% .009
Age 68.9 ± 7.9 63.8 ± 8.3 <.001
Mean IOP (mm Hg) 17.6 ± 2.8 15.9 ± 2.6 <.001
Mean VCDR 0.53 ± 0.11 0.42 ± 0.12 <.001
Mean DD (mm) 1.52 ± 0.18 1.51 ± 0.17 .570
Systolic BP (mm Hg) 151.6 ± 21.2 144.0 ± 20.4 .005
Diastolic BP (mm Hg) 85.7 ± 9.5 83.4 ± 9.6 .053

BP = blood pressure; DD = disc diameter; IOP = intraocular pressure; OAG = open-angle glaucoma; VCDR = vertical cup-to-disc ratio.

Data presented as mean ± standard deviation.


Association analysis indicates that incident OAG was associated with SNPs at 3 of the 5 loci tested ( Table 3 ). Significant association under an allelic test was seen at rs1412892 ( P = .006) at the 9p21 locus as well as rs10483727 ( P = .004) at the SIX1/SIX6 locus. Additional SNPs at 9p21 and also at TMCO1 were nominally significant but did not survive after correction for multiple comparisons. The SNPs at the 8q22 and CAV1/CAV2 loci did not show association with incident glaucoma. Adjustment for covariates under an additive genetic model showed association at the same SNPs, although only SIX1/SIX6 remained significant after correction for testing 7 SNPs ( P ≤ .007) ( Table 3 ).



Table 3

Association of Genome-Wide Significant Open-Angle Glaucoma Loci With 10-Year Incident Glaucoma in the Blue Mountains Eye Study








































































































Locus CHR SNP Position (bp) a A1/A2 Frequency A1 Univariate Analysis Multivariate Analysis b
Cases Controls P Value OR (95% CI) P Value OR (95% CI)
TMCO1 1 rs4656461 163953829 G/A 0.187 0.116 .013 1.74 (1.12–2.72) .022 1.79 (1.09–2.95)
CAV1/CAV2 7 rs4236601 115949965 A/G 0.313 0.268 .239 1.25 (0.86–1.81) .329 1.21 (0.82–1.78)
8q22 8 rs1521774 106048166 G/A 0.366 0.324 .314 1.20 (0.84–1.72) .443 1.16 (0.80–1.67)
9p21 9 rs1063192 21993367 A/G 0.642 0.549 .033 1.47 (1.03–2.11) .103 1.37 (0.94–2.01)
9p21 9 rs1412829 22033926 A/G 0.687 0.568 .006 1.67 (1.15–2.42) .025 1.57 (1.06–2.32)
9p21 9 rs4977756 22058652 A/G 0.687 0.591 .027 1.52 (1.05–2.20) .109 1.37 (0.93–2.02)
SIX1/SIX6 14 rs10483727 60142628 A/G 0.508 0.384 .004 1.66 (1.17–2.34) .007 1.70 (1.16–2.50)

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Jan 7, 2017 | Posted by in OPHTHALMOLOGY | Comments Off on Association of Open-Angle Glaucoma Loci With Incident Glaucoma in the Blue Mountains Eye Study

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