|Molecular Vision 2007;
Received 4 September 2007 | Accepted 16 November 2007 | Published 26 November 2007
No association between OPA1 polymorphisms and primary open-angle glaucoma in three different populations
Janey L. Wiggs,3
Michael A. Hauser,1,2
R. Rand Allingham1,2
1Center for Human Genetics, Duke University Medical Center, Durham, NC; 2Department of Ophthalmology, Duke University Medical Center, Durham, NC; 3Department of Ophthalmology, Harvard Medical School, Boston, MA
Correspondence to: R. Rand Allingham, MD, Duke University Eye Center, DUMC Box 3802, Erwin Road, Durham, NC, 27710; Phone: (919) 684-2975; FAX: (919) 681-8267; email: firstname.lastname@example.org
Purpose: To investigate whether recently described polymorphisms in the optic atrophy 1 gene (OPA1) are associated with primary open-angle glaucoma (POAG) with elevated intraocular pressure in the Caucasian, African-American, and Ghanaian (West African) populations.
Methods: POAG was defined as the presence of glaucomatous optic nerve damage, associated visual field loss, and elevated intraocular pressure (>21 mm of mercury in both eyes). We used TaqMan allelic discrimination assays to genotype two single nucleotide polymorphisms (SNPs, rs10451941 and rs166850) in OPA1 in the Caucasian (279 cases, 227 controls), African American (193 cases, 97 controls), and Ghanaian (170 cases, 138 controls) populations. Allele, genotype, and haplotype frequencies were compared between the cases and controls from each population.
Results: There was no significant difference in OPA1 allele or genotype frequencies between POAG patients and controls at the rs10451941 and rs166850 SNPs in any population (p>0.05). Haplotype analysis also failed to demonstrate a significant association with POAG. The age-of-onset distribution in the Caucasian POAG patients was independent from genotypes at rs10451941.
Conclusions: There was no association between two previously implicated OPA1 polymorphisms and a POAG phenotype that includes elevated intraocular pressure. This represents the first association analysis of OPA1 in high tension glaucoma in the African American and Ghanaian populations and is the largest study to date on the investigation of the potential association between OPA1 and POAG with elevated intraocular pressure. OPA1 association with POAG may be limited to patients with normal tension glaucoma in these populations.
Glaucoma is the second leading cause of blindness worldwide and affects more than 60 million people worldwide including 2.2 million Americans [1,2]. Primary open-angle glaucoma (POAG), the most common form of glaucoma, is clinically characterized by progressive optic neuropathy with characteristic visual field changes. Major risk factors for POAG include elevated intraocular pressure (IOP), increased age, positive family history of POAG, and race (African American) . POAG is the leading cause of blindness in the African American population and has a prevalence that is three times greater than that in the Caucasians . Elevated IOP is present in 2/3 of Caucasians, African Americans, and individuals of West African ancestry who have POAG [4-6]. Normal tension glaucoma (NTG), where IOP remains within a statistically normal range, is an important subset of POAG and accounts for the remaining 1/3 of POAG cases [4-6].
POAG is a complex trait and genetically heterogeneous with multiple genes independently or in concert contributing to an individual's susceptibility to glaucomatous optic neuropathy. It has been found that mutations in the myocilin, optineurin, and WDR36 genes account for a relatively small proportion (<10%) of POAG [7-10]. Mutations in the optic atrophy 1 (OPA1) gene have been associated with optic atrophy type 1 (OMIM 605290), a dominantly inherited form of optic neuropathy [11,12]. OPA1 is expressed in the optic nerve and has been analyzed as a candidate gene for POAG [11,12]. Polymorphisms of OPA1 have been associated with NTG in the Caucasian and Japanese populations and were found to influence age-of-onset of high tension glaucoma (HTG) in the Japanese population [13-15]. However, no association has been found between OPA1 polymorphisms and NTG in the Korean or African Caribbean population of Barbados in the West Indies or with HTG in the Caucasian population [16-18]. Therefore, it is very important to confirm whether the OPA1 polymorphisms are associated with POAG. The most commonly reported OPA1 polymorphisms related with POAG are rs166850 and rs10451941. In the present study, we have tested the association between these two most commonly reported OPA1 polymorphisms and primary open angle glaucoma (POAG) with elevated intraocular pressure in three different case/control populations, the Caucasian, African American, and Ghanaian (West African).
This study adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained from all participating individuals. The research was reviewed and approved by the Institutional Review Board from all participating institutions including Duke University Medical Center (Durham, NC), the Tufts New England Medical Center (Boston, MA), and the Massachusetts Eye and Ear Infirmary (Boston, MA). All study subjects including the controls were examined or their medical records reviewed by one of the investigators (J.L.W., R.R.A.). Ghanaian patients seen at the Emmanuel Eye Clinic in Accra, Ghana were recruited for this study . Recruitment efforts have been ongoing since 1999. All POAG patients met the following three inclusion criteria: (1) intraocular pressure greater than 21 mmHg in both eyes without medications or greater than 19 mmHg on two or more medications; (2) glaucomatous optic neuropathy in both eyes; and (3) visual field loss consistent with optic nerve damage in at least one eye . Glaucomatous optic nerve damage was defined as cup-to-disc ratio higher than 0.7 or focal loss of the nerve fiber layer (notch) associated with a specific visual field defect. Visual fields were performed using automated perimetry and were scored with a modified six-stage system adapted from that published by Quigley . The controls consisted of unaffected spouses or were enrolled through local advertisements. The following criteria were required for controls: (1) no first degree relative with glaucoma; (2) no first degree relative within control data set; (3) intraocular pressure without treatment less than 21 mmHg tested by applanation tonometry on two occasions; (4) no evidence of glaucomatous optic nerve disease; and (5) normal visual field testing (e.g. Humphrey SITA Fast or similar screening program).
Genomic DNA genotyping
Genomic DNA from peripheral blood was prepared from all individuals by using standard techniques (Gentra, Minneapolis, MN). TaqMan allelic discrimination assays were employed for genotyping single nucleotide polymorphisms (SNPs), rs10451941 and rs166850, by the use of Assays-On-Demand products according to the standard protocols from the manufacturer (ABI, Foster City, CA). For quality-control (QC) purposes, two CEPH (Centre d'Etude du Polymorphisme Humain) standards were included in each 96-well plate, and samples from six individuals were duplicated across all plates with the laboratory technicians blinded to their identities. Genotype submission to the analysis database required matching QC genotypes within and across plates and with at least 95% genotyping efficiency.
In each of the three populations, genotype frequencies of POAG cases and controls were compared by logistic regression with adjustment for age and sex using SAS software (SAS Institute Inc., Cary, NC). Analysis of Hardy-Weinberg equilibrium (HWE) was performed separately for patients and controls using GDA software according to previously described methods . SNP genotypes were coded according to a log-additive model in which the risk of carrying a single variant (minor) allele was assumed to be intermediate between the risk of carrying zero (reference genotype) or two variant alleles on the logarithmic scale. Haplotype analysis was performed with the haplo.stats package [22,23]. Power calculations were performed with QUANTO software using previously described methods and assuming a population prevalence of 10% and a log-additive risk model . Two-sample t-tests and the analogous nonparametric tests (Wilcoxon rank sum) were used to test whether the mean age of onset differed by genotype at rs10451941 (carrying at least one C allele versus none) as previously reported [13,25].
This study included 279 Caucasian cases and 227 controls, 193 African American cases and 97 controls, and 170 Ghanaian African cases and 138 controls. A detailed summary of the phenotypes with these three data sets was listed in Table 1. The mean age of onset or diagnosis was 58.7±12.8 years for the Caucasians, 55.3±13.3 years for the African Americans, and 55.4±13.8 years for the Ghanaians. Both SNPs were in HWE in cases (p>0.05) and controls (p>0.05) from all three populations.
There were no significant genotype or allele frequency differences at rs10451941 or rs166850 between POAG cases and controls in any of the three populations (Table 2). Neither SNP was found to have a statistically significant association with POAG. There was no significant difference in the age of onset between POAG patients with or without the C allele at rs10451941 in any population (p>0.25). It was noted that SNP rs166850 was monomorphic in the Ghanaian population (only the C allele observed), which was consistent with HapMap results from the Nigerian (Yoruba) population . Since the compound genotype at rs166850 and rs10451941 was previously reported to be strongly associated with NTG in other populations, the haplotypes formed by these two SNPs were examined further in the Caucasian population. No significant haplotype frequency differences were observed (p=0.32).
Our Caucasian sample had >99% power at 5% significance level to detect the previously reported effect size (odds ratio estimates ranging from 2.3 to 3.1) for the allele frequencies observed in the controls . It had 79% power at 5% significance level to detect an odds ration (OR) of 1.6 or greater. For the African American sample, power ranged from 50% for an OR of 2.3 to 78% for an OR of 3.1 for rs166850 due to the lower allele frequency. For both African American and Ghanaian samples, the power was >99% for rs10451941.
Mutations in OPA1 are believed to cause primary degeneration of the retinal ganglion cells, which play a key role in the vision loss associated with POAG [11,12]. Several previous studies reported an association between OPA1 polymorphisms and normal tension glaucoma (NTG) in the Japanese and Caucasian populations [13-15] (Table 3). In this study, we examined the possible associations between two particular polymorphisms of OPA1 and POAG patients of Caucasian, African American, and Ghanaian ethnicity. We did not find an association between these variants of OPA1 with high tension glaucoma in any population. Failure to replicate the previous reportedly OPA1 association with glaucoma in the Caucasian population may be due to the fact that our cases are high tension glaucoma instead of normal tension glaucoma. Mabuchi et al.  recently suggested that rs10451941 may influence the age at diagnosis of POAG in the Japanese population, suggesting a potential genetic risk factor for HTG  while Yao et al.  and Aung et al.  did not find any significant association between OPA1 polymorphisms and POAG with elevated intraocular pressure in the Barbados or Caucasian populations [17,18] (Table 3). However, the studies by Yao and Aung were based on small numbers of POAG patients (48 and 90 individuals, respectively) and may have lacked statistical power to detect an association [17,18]. The data sets examined here were larger (Table 2 and Table 3), and this is the largest study to date on HTG with OPA1. Our power calculations demonstrate that it is unlikely for the two variants we genotyped to have a major effect (OR is greater than or equal to 1.6 in Caucasians) on HTG. We cannot exclude the possibility that these variants may have substantially smaller effects. Given the sample sizes and the availability of samples from three different populations, our study is one of the most comprehensive association studies of OPA1 to date. Our study and the previous reports have demonstrated large allele frequency differences of OPA1 polymorphisms with a reported minor allele frequency at rs10451941 of 15% in Japanese individuals  compared to 41% in Caucasians (Table 2) and a minor allele frequency at rs166850 of 15% in Caucasians compared to <4% in those of African ancestry (Table 2). The linkage disequilibrium (LD) plot from the HapMap database showed strong LD within OPA1 in the population of both Caucasian and African (Yoruba, Nigeria; Figure 1). These plots were generated using HaploView3.32 based on HapMap genotyping data. The high LD of OPA1 with both Caucasian and African population suggests the two polymorphisms we genotyped might capture most of the variation in OPA1. Therefore, it seems that the other polymorphisms in OPA1 might not contribute to POAG in these populations.
In conclusion, our study did not find any significant association between OPA1 polymorphisms and POAG with increased intraocular pressure in the Caucasian, African American, and Ghanaian populations. This represents the first association analysis of OPA1 in high tension POAG in African Americans and West Africans (Ghanaian). In these populations, the frequency of the minor allele at rs166850 is very low while the frequency of the minor allele at rs10451941 is higher than in Caucasian populations. Our study suggests that an OPA1 association with POAG may be limited to patients with normal tension glaucoma.
We thank the study participants for their participation. We thank Dr. Stephen Akafo at the University of Ghana; Dr. Leon Herndon and Dr. Pratap Challa at Duke University; and the study staff at the Duke Center for Human Genetics and Department of Ophthalmology for their integral role in this project. This research was supported by NIH grants R01EY013315 (M.A.H.), R01EY015872 (J.L.W.), R03EY014939 (R.R.A.), R01EY015543 (R.R.A.), and Research to Prevent Blindness (R.R.A.).
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