Aim: Polymorphisms in OPA1, the gene responsible for autosomal dominant optic atrophy, were recently found to be strongly associated with normal tension glaucoma (NTG). The aim of this study was to determine whether OPA1 polymorphisms affect the phenotype of NTG patients.
Methods: A retrospective analysis was performed of 108 well characterised NTG patients who had been genotyped for OPA1 variations, and who had previously undergone automated perimetry and Heidelberg retina tomography (HRT). 25 NTG patients had the at-risk OPA1 genotype (IVS 8 +4 C/T; +32 T/C) and 83 NTG patients did not. Differences between groups were sought in a wide range of structural, psychophysical, and demographic factors. These included sex, age at diagnosis, family history of glaucoma, history of ischaemic risk factors and vasospasm, laterality of glaucoma, presenting and highest diurnal intraocular pressure (IOP), initial cup-disc (CD) ratio, baseline visual field global indices, and optic disc parameters as measured by HRT. For a subgroup of patients with at least 5 years of follow up and 10 visual field tests, pointwise linear regression analysis (progressor for Windows software) was applied to the visual field series.
Results: There was no significant difference in the two groups with respect to sex, age at diagnosis, family history of glaucoma, history of ischaemic risk factors and vasospasm, or laterality of glaucoma. The comparison of IOP, CD ratio and visual field global indices, MD and CPSD in the two groups showed no significant difference. There were no differences in the mean values for any of the HRT parameters analysed. For the subgroup of patients with at least 5 years of follow up, there was also no significant difference in the number of patients with progressing locations, the mean number of progressing locations per subject, the mean slope of the progressing locations or the mean slope for whole visual field.
Conclusions: The absence of phenotypic differences in normal tension glaucoma patients with and without the OPA1 polymorphisms IVS 8 +4 C/T; +32 T/C suggest that these OPA1 polymorphisms do not underlie any major phenotypic diversity in these patients.
- normal tension glaucoma
- OPA1 polymorphisms
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Glaucoma, the leading cause of irreversible blindness worldwide,1,2 is typified by progressive loss of optic nerve axons and visual field damage. Normal tension glaucoma (NTG) is an important subtype of glaucoma in which intraocular pressures (IOP) are within the statistically normal population range, and accounts for approximately a third of all primary open angle glaucoma cases.3–6 Although there is evidence for the role of genetic factors in NTG,7–10 the molecular mechanisms underlying NTG are unknown. The discovery of optineurin as a causative gene suggests that one mechanism producing NTG may involve apoptosis of retinal and optic nerve ganglion cells.10
Autosomal dominant optic atrophy (ADOA) is another condition with slowly progressive optic nerve damage.11–14 In ADOA patients, excavation of the optic nerve may develop, and such patients are occasionally misdiagnosed as having NTG.15 Mutations in OPA1 (MIM No 605290), the gene responsible for ADOA, were recently found to result in primary degeneration of retinal ganglion cells.16 The similarities between glaucoma and ADOA (in terms of the cell type affected and the phenotype) make OPA1 a good candidate gene for glaucoma, particularly NTG.
A recent study found an association between polymorphisms in the OPA1 gene and NTG.17 Approximately 20% of NTG subjects were found to carry two single nucleotide polymorphisms (SNPs) on intervening sequence (IVS) eight of the OPA1 gene (IVS 8 +4 C/T; +32 T/C), compared to only 3.7% of control subjects (χ2 =22.04, p= 0.00001), indicating that this genotype is strongly associated with NTG, and may represent a diagnostic marker for the disease.17 This observation raises the possibility that different subgroups of NTG exist, one of which is distinguished by genetic variations in OPA1. The aim of this study was to compare clinical features of NTG patients with and without such polymorphisms in order to determine whether OPA1 polymorphisms impart a characteristic phenotype in NTG patients.
A retrospective analysis was performed of 108 white NTG patients who had been previously genotyped for OPA1 variations. There were 25 NTG patients (group 1) with, and 83 NTG patients (group 2) without the at-risk OPA1 genotype IVS 8 +4 C/T; +32 T/C. These patients attended a tertiary referral glaucoma clinic and had undergone various investigations including automated perimetry and Heidelberg retina tomography (HRT, Heidelberg Engineering, Heidelberg, Germany). NTG cases were defined by the following strict criteria18: the presence of typical glaucomatous optic neuropathy with compatible visual field loss; mean IOP without treatment that was consistently equal to or less than 21 mm Hg on diurnal testing, open drainage angles on gonioscopy, and absence of a secondary cause for glaucomatous optic neuropathy, such as a previously raised IOP following trauma, a period of steroid administration or uveitis. Only one eye from each patient was included. For bilateral cases, the right eye was analysed. The study had the approval of the Moorfield’s Eye Hospital ethics committee and was performed in accordance with the Helsinki Declaration.
Patients’ hospital records were reviewed and the following data collected: demographic characteristics including sex and age at diagnosis; family history of glaucoma; history of ischaemic risk factors such as hypertension, diabetes mellitus, ischaemic heart disease and smoking; history of vasospasm such as migraine and cold hands and feet; the presenting and highest recorded diurnal intraocular pressure (IOP); cup-disc (CD) ratio at presentation; and interocular symmetry of glaucoma.
Visual field analysis
Subjects underwent static automated white on white threshold perimetry (program 24-2, model 640, Humphrey Instruments, Dublin, CA, USA). The first visual field test for all subjects was discarded from the analyses to allow for learning effects, and the subsequent first reliable visual field was used as the baseline. The global indices such as mean deviation (MD) and corrected pattern standard deviation (CPSD) of the baseline visual fields were analysed for all cases.
The visual fields of a subgroup of 88 subjects (18 from group 1 and 70 from group 2) who had at least 5 years of follow up with at least 10 visual field tests performed during this time were also analysed for progression. Pointwise linear regression analysis was applied to the field series of each of these subjects using progressor for Windows software.19 Progression was defined as the presence of a significant regression slope (p<0.01) showing 1 dB per year or more of sensitivity loss at the same test location with the addition of two out of three successive field tests to the series starting with the first three. The mean number of progressing locations per subject, the mean slope for the progressing locations as well as the mean slope of the whole visual field per year was evaluated.
Optic disc analysis
The Heidelberg retina tomograph (HRT, Heidelberg Engineering, Heidelberg, Germany) was used to image the optic disc and the baseline optic disc parameters were analysed. The mean topography of three images was generated in the 10 × 10 degree frame and the disc edge delineated on the mean image by a single trained observer (DP), using a drawn contour line. Images with significant movement artefact were rejected. Global and segmental disc and cup areas were analysed directly by means of HRT software (version 2.01b) using the standard reference plane. Rim area was calculated by subtracting the cup area from the disc area. Six predefined segments were used (0 degrees always temporal, 90 degrees always superior): temporal quadrant (−45 to +45 degrees), temporal superior octant (+45 to +90 degrees), temporal inferior octant (−90 to −45 degrees), nasal quadrant (+135 to +225 degrees), nasal superior octant (+45 to +90 degrees), and nasal inferior octant (−135 to −90 degrees).
Statistical analysis was carried out using Statistical Package for Social Sciences version 9.0 (SPSS Inc, Chicago, IL, USA). Parametric and non-parametric tests of significance were carried out where appropriate. Comparisons between groups were done with Mann-Whitney U tests for continuous variables that were not normally distributed. χ2 analysis was used for comparison of proportions. Statistical significance was assumed at the p <5% level.
There was no significant difference in the two groups with respect to demographic factors such as sex and age at diagnosis. There was also no significant difference with respect to family history of glaucoma, history of ischaemic risk factors, history of vasospasm, or laterality of glaucoma (Table 1).
The comparison of IOP, CD ratio and visual field global indices, MD, and CPSD in the two groups is summarised in Table 2. There was no significant difference found although the difference in mean highest diurnal IOP between the two groups approached significance (p = 0.06).
The mean HRT parameters are summarised in Table 3. There were no differences in the mean values for any parameter analysed.
Comparing the visual fields of the subgroup of 88 subjects (18 from group 1 and 70 from group 2) who had at least 5 years of follow up (with at least 10 visual field tests performed during this time) revealed no difference in the number of patients with progressing locations, the mean number of progressing locations per subject, the mean slope of the progressing locations, or the mean slope for whole visual field (Table 4).
This study did not detect a significant difference in a range of phenotypic features in normal tension glaucoma patients with and without the OPA1 polymorphisms IVS 8 +4 C/T; +32 T/C. This suggests that these specific genetic variations in OPA1 do not underlie any major phenotypic diversity in NTG, although the possibility of more subtle phenotypic differences, such as variable rates of response to treatment cannot be excluded. The mean presenting IOP and the highest diurnal IOP appeared to be lower in NTG patients with the OPA1 polymorphisms (almost reaching significance for the latter), which may indicate that the glaucoma in such patients is less IOP dependent. If this were to be the case, it would suggest that NTG patients have different IOP thresholds for glaucomatous damage to occur, and that those with OPA1 polymorphisms may be at risk of glaucoma at lower IOP levels.
Polymorphisms are associated with a variety of other diseases including Alzheimer’s dementia,20 age related macular degeneration,21 diabetes mellitus,22 and schizophrenia.23 Although intronic polymorphisms, of which OPA1 (IVS 8 +4 C/T and +32 T/C) is an example, are associated with conditions like intracerebral haemorrhages and cerebral aneurysms,24 little is known about how intronic polymorphisms influence disease phenotype. Possible mechanisms include regulation of transcription,25,26 effects on protein function, or indirectly by conferring susceptibility in patients to other factor(s) that mediate disease.17
The biochemical mechanisms by which OPA1 may influence NTG remain obscure. The pathogenic characteristics of OPA1 resemble those of Leber hereditary optic neuropathy, which results from a defect of the mitochondrion. Alexander et al hypothesised that mutations in the OPA1 gene affect mitochondrial integrity, resulting in an impairment of energy supply.16 Occurring in the highly energy demanding neurons of the optic nerve, notably the papillomacular bundle, this would presumably lead to damage of retinal ganglion cells and visual loss.
A variety of factors may contribute to the development of optic neuropathy in glaucoma. In the apparent absence of elevated IOP, which is the main risk factor identified for glaucoma, non-IOP related factors are advocated to predominate in eyes with NTG including abnormal blood flow,27–29 systemic hypotension,30–32 and an abnormal coagulability profile.33–36 A strong genetic component is likely to be significant in NTG.7–10 It is hypothesised that several interacting genes contribute to the development of disease, with the putative role of each polymorphic sequence variation influenced by an individual’s genetic and environmental background. However the number and identity of genes contributing to NTG has yet to be fully determined. Much remains to be learned about the phenotypic effects of specific genes and alleles in this condition. It is hoped that further research efforts will be directed towards investigating the association of OPA1 polymorphisms found in a substantial proportion of NTG patients, as it may lead to greater understanding of the factors underlying this important cause of blindness.
Grant support: The work was supported in part by the International Glaucoma Association, Moorfields Special Trustees, and the Glaucoma Research Foundation. Dr Okada is supported by Hiroshima University Faculty of Medicine, and Dr Aung by the National Medical Research Council of Singapore and the Singapore National Eye Centre.
T Aung and K Okada contributed equally to the paper and are joint first authors.