Molecular Vision 2003; 9:460-464 <>
Received 22 May 2003 | Accepted 20 September 2003 | Published 22 September 2003

Polymorphisms in OPA1 are associated with normal tension glaucoma

Brenda L. Powell,1 Carmel Toomes,2 Sheila Scott,2 Anna Yeung,1 Nicola J. Marchbank,2 Paul G. D. Spry,1 Rosemary Lumb,1 Christopher F. Inglehearn,2 Amanda J. Churchill1

1Division of Ophthalmology, University of Bristol, Bristol, United Kingdom; 2Molecular Medicine Unit, St James's University Hospital, Leeds, United Kingdom

Correspondence to: Amanda J. Churchill, Bristol Eye Hospital, Lower Maudlin Street, Bristol, BS1 2LX, UK; Phone: +44-117-928-4949; FAX: +44-117-925-1421; email:


Purpose: To confirm whether specific polymorphisms in intron 8 (IVS8) of the OPA1 gene are found more commonly in patients with normal tension glaucoma (NTG) compared to normal controls.

Methods: This is a cohort study of 61 patients with NTG, 49 known healthy controls and 119 individuals from the general population. The DNA sequence was determined at the +4 and +32 positions of IVS8 of the OPA1 gene. Hardy-Weinberg equilibrium was confirmed in our population by comparing the allele frequencies in two additional genes, TP53 and TYRP1. Genotypes for the NTG and control groups were compared for statistically significant differences.

Results: There were no differences in the OPA1 genotypes of the NTG and control groups at the +4 location, as had been suggested in a previous study, but a significant difference was observed at the +32 location of IVS8. The CC genotype was found in 28% of NTG patients compared to 13% of controls (p=0.006). The TC genotype was more prevalent in the control population (p=0.02) but this difference did not reach statistical significance when the Bonferroni adjustment was made for multiple analyses.

Conclusions: We have refined the previously reported association between OPA1 sequence changes and NTG by identifying a specific CC genotype at position +32 in IVS8 of the OPA1 gene that acts as a marker for NTG. At the current time, NTG is frequently diagnosed late when loss of neurons has already caused significant and irreversible peripheral field loss. If a test could be designed to identify those people at risk of developing NTG, then careful screening might detect earlier signs of disease allowing commencement of treatment before significant field loss has occurred.


Glaucoma is a major contributor to global blindness. Approximately half of all cases are of the angle closure type, prevalent among Asian populations [1]. The remaining half consists of open angle glaucoma (OAG), which is characterized by optic disc excavation and the loss of retinal ganglion cells, resulting in a gradual reduction of peripheral vision [2]. Normal tension glaucoma (NTG) is a subset of OAG and accounts for approximately one third of OAG cases [3]. While many OAG patients have high intra-ocular pressures (IOP), patients with NTG have statistically normal IOPs. Because of this, NTG can be difficult to diagnose and usually presents late in life when visual field loss has already occurred. To assist in the early diagnosis of NTG, a genetic approach to identify those at risk of developing the disease is very attractive.

Primary glaucoma is genetically heterogeneous, with at least eight loci found to be associated with the disease. So far only three genes have been identified: CYP1B1 (primary congenital glaucoma), MYOC (Juvenile Primary OAG), and recently, OPTN (Primary OAG/NTG) [4-6]. Aung et al. reported that a combination of two polymorphic markers in the gene known to cause dominant optic atrophy (OPA1) are associated with NTG [3]. Dominant optic atrophy and NTG share some common clinical and phenotypic features including optic neuropathy, normal IOPs and premature degeneration of ganglion cells. Based on these similarities, Aung and colleagues considered the OPA1 gene to be a suitable candidate for genetic screening in NTG patients. A screen of the coding region revealed no disease-causing mutations. The presence, however, of two common polymorphisms in intervening sequence (IVS) 8 of the OPA1 gene, a C/T change at +4 and a T/C change at +32, was found to be associated with NTG. This association was strongest when the polymorphisms occurred together. Twenty per cent of NTG patients were compound heterozygotes for both associated alleles, in comparison with only 4% of controls [3].

If confirmed, such a finding would be beneficial in screening patients thought to be at risk of developing NTG. As this was the first study to report an association with NTG, we undertook a validation study on a series of NTG patients and a random population of normal controls. To ensure that there was no selection bias in the populations studied, polymorphisms in two unrelated genes (TP53 and TYRP1) were also analysed.


Patient selection

Cases (n=61) were recruited from the glaucoma clinic at the Bristol Eye Hospital. All cases had intraocular pressures less than 21 mmHg (measured by Goldmann tonometry) in the presence of visual field loss and/or glaucomatous optic disc changes. Patient ages ranged from 49-94 years. Sex distribution was 43% male and 57% female. Two normal populations were analysed. The first group were unaffected spouses and healthy volunteers in Bristol and Leeds (n=49) consisting of 65% males and 35% females. The second group (parents of children affected by cystic fibrosis) were obtained from St James's University Hospital, Leeds (n=119) and had an equal distribution of male and females. All participants were Caucasians of Northern European origin. Ethics approval for the study was obtained from the United Bristol Healthcare Trust. A venous blood sample was taken from each participant after informed consent was obtained.

PCR amplification

DNA was extracted using either standard phenol/chloroform extraction, or by the Genomic DNA Purification System (Whatman BioScience, Maidstone, UK). Conditions and primers for PCR amplification of IVS8 of the OPA1 gene, IVS3 of the TP53 gene, and exon 2 of the TYRP1 gene are detailed in Table 1.

PCR for OPA1 IVS8 was carried out in a reaction volume of 50μl using 1X PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl), 2.5 mM MgCl2, 100μM dNTPs (Applied Biosystems, Warrington, UK), 0.7 U Taq polymerase (Amplitaq Gold, Applied Biosystems), 1 μM M13 modified primers (MWG Biotech AG, Ebersberg, Germany) and 50-100 ng of genomic DNA. PCR for TP53 IVS3 and TYRP1 exon 2 were carried out in a reaction volume of 15μl using 1X PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl), 2.0/1.5 mM MgCl2 (respectively), 200μM dNTPs (Applied Biosystems), 0.25 U Taq polymerase (Amplitaq Gold, Applied Biosystems), 1μM primers (Cruachem Ltd., Glasgow, Scotland) and 50-100 ng of genomic DNA. Cycling parameters were 5 min at 94 °C, followed by 30/35 cycles of denaturation at 94 °C for 1 min, annealing at 55/60 °C for 1 min and extension at 72 °C. Final extension was at 72 °C for 5 min.


PCR products were purified using QIAquick columns (Qiagen, Hilden, Germany) before being sequenced bidirectionally on a LICOR 4200 DNA sequencer. Sequencing reactions were carried out using the Thermo Sequenase fluorescent labelled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech, Little Chalfont, UK). M13 sequencing primers were synthesised by MWG-Biotech AG (Ebersberg, Germany). Sequencing reactions were loaded onto a polyacrylamide Sequagel XR gel (National Diagnostics-Flowgen, Ashby de la Zouch, UK) and run at 2000 V, 50 mA and 50 °C.

dHPLC Analysis

Denaturing high performance liquid chromatography (dHPLC) on the Transgenomic Wave® nucleic acid fragment analysis system (Transgenomic, Crewe, UK) was used to screen samples for polymorphisms. A common SNP in exon 2 of TYRP1 and a 16 base pair (bp) polymorphic repeat in IVS3 of the TP53 gene were analysed [7,8]. Following PCR, the TYRP1 samples were heated to 95 °C for 5 min, then cooled to 34 °C over a 40 min period to induce heteroduplex formation. TYRP1 analysis was performed using an 8 μl injection volume, the standard protocol for the Transgenomic Wave® heteroduplex detection method at a melting temperature of 63 °C and a slope of 2.0% increase in buffer B (0.1 M TEAA, 25% acetonitrile) per minute. The resulting chromatograms were studied for deviation from the wild-type homozygous pattern.

TP53 PCR samples were analysed using an 8 μl injection volume, the Transgenomic Wave® double stranded, single fragment sizing technique (135 bp) at 50 °C and a slope of 2.0% increase in buffer B (0.1 M TEAA, 25% acetonitrile) per minute. The wild-type homozygote fragment of 119 bp was easily discernible from the 119/135 heterozygote or the 135/135 bp homozygote on the resulting chromatograms.

Statistical analyses

Genotype frequencies for individual polymorphisms were calculated by χ2 analysis, with statistical significance assumed when p<0.05. Statistical analyses of the compound OPA1 IVS8 polymorphisms were conducted using standard χ2 analyses. Due to the multiple analyses carried out on this data, the Bonferroni adjustment was applied (number of analyses was 6), giving a p value for significance of 0.008.


The study population was in Hardy-Weinberg equilibrium for all alleles investigated. There was no statistically significant difference in genotype frequencies between the two groups of normal controls (results not shown), allowing them to be combined for the final analyses. Distribution of the TP53 IVS3 polymorphism and the TYRP1 exon 2 polymorphisms were similar between the normal and NTG populations, suggesting no selection bias was present (Table 2).

Genotype analysis of OPA1 IVS8 showed no difference in distribution between the normal population and the NTG patients at the +4 location, however a significant difference was observed between the two populations at the +32 location (Table 3). The CC genotype was observed more frequently in the NTG patients (odds ratio [OR]=2.70; 95% confidence intervals [CI] [1.31,5.57]; p=0.006), while the TC genotype was more prevalent in the control population (OR=0.49; 95% CI [0.27,0.88]; p=0.02).

Analysis of the combined genotypes revealed no difference between the normal and diseased population with respect to the CT/TC genotype (OR=0.84; 95% CI [0.39,1.77]; p=0.64), as previously reported [3]. However differences in distribution of the CC/CC and CC/TC genotypes were observed (Table 4). The CC/CC genotype occurred more frequently in NTG patients (OR=2.70; 95% CI [1.31,5.57]; p=0.006), while the CC/TC genotype was more common in the control group (OR=0.51; 95% CI [0.27,0.98]; p=0.04).


Genetic polymorphisms have been reported to be associated with a variety of diseases, including age-related macular degeneration, diabetes, and cancer [9]. Identification of susceptibility alleles for disease may aid in screening at-risk populations, leading to earlier diagnosis and more effective treatments. A double polymorphism in the OPA1 gene has recently been reported to be associated with NTG [3]. In an attempt to confirm this finding, we screened a series of NTG patients and a random population of normal controls.

In contrast to the results reported by Aung and colleagues [3], we found no association of NTG with the compound heterozygote at +4/+32 position in IVS8 of OPA1. We report six possible combined genotypes at the +4 and +32 positions. Aung et al. observed only four genotypes since they saw no CC/CC or TT/TT homozygotes. Neither study observed CT/CC or TT/CC genotypes (Table 4). It is interesting to note that there was no difference in the frequency of the CT/TC genotype in the NTG cohorts of the present study and that reported by Aung. There was, however, a significant difference in the CT/TC frequency observed in the normal populations from the two studies (χ2=24.6, p<0.0001). It is not clear why such a difference should exist, as recruitment of the normal controls in both studies was similar. We used 2 control populations: one group (Bristol) were unaffected friends/spouse of study individuals examined for glaucomatous optic disc changes (as per the Aung et al Study). The other control group were not examined (Leeds), but since the prevalence of NTG approximates to 1% in the general population this group could conceivably contain a maximum of 2 individuals who may have NTG. The results of the 2 control groups were statistically similar and illustrate that when a disease prevalence is very low, it is acceptable to use a control group randomly chosen from the general population. All subjects and controls were white Caucasians of British descent.

When we analysed the genotypes at each locus individually, we found no association with NTG for any genotype at the +4 locus (Table 3). This contradicts the findings by Aung et al and is accounted for not by a fall in the frequency of the putative NTG susceptibility allele T but by an increase in the prevalence of the T allele in control samples. We did, however, find a significant association with NTG at the +32 locus (p=0.006 for the CC genotype). Incidence of the CC genotype in this position in the NTG group (27.9%) was more than twice that in the normal population (12.5%).

Analysis of polymorphisms in the control genes, TP53 and TYRP1, showed that no selection bias existed between the NTG and normal control groups in our study (Table 2). We observed two distinct and reproducible chromatogram patterns that deviated from the wild-type in our analysis of the TYRP1 gene. Thus we demonstrated two polymorphisms in exon 2 of the TYRP1 gene. This was an unexpected finding since there is only one polymorphism in exon 2 described in the literature [7]. Interestingly, the novel polymorphism was only found in the normal populations studied and at only a very low frequency (5.4%).

There have recently been four further studies involving the CT/TC polymorphism in IVS8 of the OPA1 gene [10-13]. In the first study [10], comparison of the frequency of the double polymorphism between NTG patients, POAG patients, and normal controls revealed no association between the double polymorphism and POAG. The authors suggest therefore that genetic differences clearly exist between NTG and POAG. Since we have found no association between the CT/TC genotype and NTG, it would now be interesting to look at the frequency of the +32 CC genotype in POAG. The second study, by Sato et al. [11], corroborates our findings that there is no statistical difference in the distribution frequencies of the CT/TC in 134 Japanese NTG patients and 90 age-matched controls. Kim et al. [12], studied the IVS8 +4 CT polymorphism in 42 Korean NTG patients and 42 healthy controls. They found no association between the +4 CT genotype and NTG. Finally, in the fourth study [13], the NTG population (which had previously been genotyped for the CT/TC OPA1 polymorphism) was analysed with respect to phenotype. There was no association between the compound heterozygote polymorphism and the phenotypic features analysed. This could be interpreted as support for the hypothesis that there is no association between the CT/TC genotype and NTG, but might also reflect a common phenotypic endpoint resulting from multiple genetic lesions, as has been observed for retinitis pigmentosa.

This study supports the hypothesis that the OPA1 IVS8 +32 CC genotype is associated with NTG. It is possible that this genotype may predispose ganglion cells to apoptosis, resulting in the premature loss of optic nerve axons. We would emphasize, however, that the results of single nucleotide polymorphism analyses should always be interpreted with caution and verification studies are essential to confirm initial findings. It is not yet clear how this polymorphism might act and further work will be necessary, both to clarify the function of the OPA1 protein, and to understand the effect (if any) of the OPA1 IVS8 +32 CC genotype on protein expression and/or function.


The authors would also like to thank Dr. Linda Tyfield and Maggie Williams from the Lewis Laboratories in Southmead, Bristol for use of the LICOR 4200 DNA sequencer. This work was supported by The Charitable Trusts for the United Bristol Hospitals and the National Eye Research Centre and was presented, in part, as a poster at the Association for Research in Vision and Ophthalmology (ARVO) meeting May 2002, Fort Lauderdale. No author has a commercial interest.


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Powell, Mol Vis 2003; 9:460-464 <>
©2003 Molecular Vision <>
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