Abstract
Background: Glaucoma is a heterogenous group of optic neuropathies leading to progressive degeneration of the optic nerve and vision loss. Over the past decades, disease-causing genes have been identified and multigenic inheritance theory has-beens investigated for many cases of glaucoma. The purpose of this study was to investigate the distribution of mutations in glutathione S-transferase M1 (GSTM1) and optic atrophy 1 (OPA1) genes in a series of patients of Greek origin. Patients and Methods: This was a case–control study of 106 patients with primary open-angle glaucoma (POAG) and 120 healthy controls of Greek origin, surveyed for polymorphisms with potential correlation to POAG. A DNA sample from each individual was genotyped for GSTM1 and OPA1 (rs166850, rs10451941) polymorphisms. Results: GSTM1 null genotype carriers seem to have an increased risk of developing POAG (odds ratio=1.86, 95% confidence interval=1.07-3.21; p=0.03). The results indicate that the OPA1 genotype (rs166850 and rs10451941 polymorphisms) is not significantly associated with POAG. Conclusion: The GSTM1 null genotype might be associated with increased risk of development of POAG in the Greek population. No significant correlation was found between OPA1 polymorphisms and POAG.
The term glaucoma corresponds to a heterogeneous group of diseases characterized by degeneration of the optic nerve and retinal ganglion cell death. Glaucoma is categorized according to whether it is congenital or acquired, whether the configuration of the iridocorneal angle is open or closed, and whether a direct cause is recognized (secondary) or not (primary). Primary open-angle glaucoma (POAG) is the most common form of glaucoma in the Western world. By 2020, there will be 79.6 million people with glaucoma and 74% of those will develop open-angle glaucomas (1).
POAG is mainly characterized by progressive optic neuropathy with characteristic visual field changes. The main risk factors are elevated intraocular pressure (IOP), smoking, myopia, aging, vascular factors (hypertension, diabetes, ischemia) and family history (2). POAG is classified into high-tension glaucoma (HTG) and normal-tension glaucoma (NTG). The main feature of HTG is a raised IOP. In NTG, IOP is within the statistically normal range and NTG cases account for approximately 20% of all POAG (3).
Even if POAG is considered to be a multifactorial disease, a significant heritable component has been suggested since the beginning of the 20th century (4-7). Genetic studies have defined three main causative genes in POAG, the optineurin (OPTN) (10p14-15) (8), myocilin (MYOC) (1q24-25) (9), and WD repeat domain 36 (WDR36) (5q21-22) (10). These genes are responsible only for a small proportion (approximately 10%) of POAG cases worldwide (11). However, recent genome-wide association studies have identified several new genetic variations that might contribute to POAG pathogenesis, including neurotrophin-4 (NTF4) (12, 13), caveolin 1 (CAV1) and caveolin 2 (CAV2) (14), S1 RNA binding domain 1 (SRBD1) (15, 16), transmembrane and coiled-coil domains 1 (TMCO1) (17), cyclin-dependent kinase inhibitor 2B–antisense RNA 1 (CDKN2B–AS1) (18) and more recently transforming growth factor, beta receptor 3 (TGFBR3) (19), variants near ATP-binding cassette, sub-family A (ABCA1) and in phosphomannomutase 2 (PMM2) (20). Among them polymorphisms in glutathione S-transferase M1 (GSTM1) and optic atrophy 1 (OPA1) genes are also believed to contribute in the pathogenesis of POAG (21-23).
GSTM1 polymorphism is considered to be risk factor for POAG and other types of glaucoma. GST is a multigenetic group of enzymes playing important roles in antioxidative processes and elimination of carcinogens, toxins and oxidants (24). Studies have been conducted in various populations, however, results remain controversial. Several studies revealed possible correlation between GSTM1 polymorphisms and POAG pathogenesis (25-30), although in some, results were insignificant probably due to small subject numbers (25); yet others studies showed no association between POAG and GSTM1 polymorphisms (31, 32). However, it is important to notice that Yang et al. reported that a GST antigen was present at significantly higher levels in patients with glaucoma compared to controls. Their findings suggested that people expressing GSTM1 are at an increased risk of developing auto-antibodies against GST and may have an increased risk of glaucoma (33).
The OPA1 gene on chromosome 3q28 is responsible for autosomal dominant optic atrophy (ADOA) (34). Its product is a dynamin-related guanosine triphosphatase and has an important role in mitochondrial biogenesis and membrane integrity. As it is expressed in retinal ganglion cells, mutations of OPA1 are suspected to lead to their apoptosis and optic neuropathy (35,36). Concerning POAG, although OPA1 is a candidate genetic risk factor, results so far remain controversial. Mabuchi et al. reported that OPA1 IVS 8 +32 T/C polymorphism may be genetic risk factor for both NTG and HTG (37). Several studies presented association of OPA1 polymorphisms with NTG (38-40) but not with HTG (41-43) as well; therefore it is still debated whether OPA1 polymorphisms are involved in POAG pathogenesis.
Given these considerations, and since it is known that there are variations between ethnic populations, the purpose of our study was to investigate the potential association between OPA1 (rs166850, rs10451941) and GSTM1 gene poly-morphisms and POAG susceptibility in a well-defined Greek cohort. To our knowledge, this is the first study conducting such genotyping in population of Greek patients with POAG.
Materials and Methods
Patients. Familial (n=44) and sporadic (n=62) cases of POAG, primarily diagnosed at least 10 years before recruitment into this study, as well as 120 unaffected healthy controls, with a history free of other ophthalmological or systematic diseases were recruited from the Glaucoma Unit of the First Department of Ophthalmology, University of Athens. All participants gave their written consent and had a blood sample taken for genetic analysis.
The clinical examination protocol included a full medical and ophthalmologic disease history. The anterior segment of the eye was examined by slit lamp, including gonioscopy; fundus examination including evaluation of the appearance of the optic disc and cup/disc ratio measurement were assessed with a stereoscopic fundus lens (Volk Optical Inc., Mentor, OH, USA). IOP was measured with Goldmann applanation tonometer (Haag Streit AG, Bern, Switzerland); ocular hypertension was defined as ≥21 mmHg. Automated perimetry was performed with a Humphrey Automated Field Analyzer (Humphrey Inc., San Leandro, CA, USA) using 30-2 SITA protocol in order to determine visual field defects in the POAG patient group (44, 45). Both patients and controls underwent automated perimetry. No glaucomatic defects were detected in controls.
Genotyping. Genomic DNA was prepared from blood samples using the PureLink™ Genomic DNA kit (Invitrogen Inc., Rockville, MD, USA). The GSTM1 polymorphism was evaluated by polymerase chain reaction (PCR) as previously described (46). Primer sequences for GSTM1 were 5’-GAACTCCCTGAAA AGCTAAAGC-3’ (forward primer) and 5’-GTTGGGCTCAAATAT ACGGTGG-3’ (reverse primer), which produced a 219 bp band. GSTM1 null genotype (−) is indicated by the absence of a 219 bp band. Genotyping for OPA1 rs166850 and rs10451941 was performed by restriction fragment-length polymorphism analysis as previously described with specific primers for both genotypes (forward primer, 5’-CCC TTT TAG TTT TTA CGA TGA AGA-3’; reverse primer, 5’-TTG CTT AAG ACA TTA CTT GGA ACA-3’). The restriction enzymes RsaI and FspBI (Fermentas, York, UK) discriminated the respective genotypes (40). In all cases, as an internal positive control for successful PCR, β-globin (268 bp) was amplified with the primers 5’-CAACTTCATCCACGTTCACC-3’ (forward primer) and 5’-GAAGAGCCAAGGACAGTTAC-3’ (reverse primer).
Statistical analysis. Frequency and susceptibilities of mutations were compared using the χ2 test. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated with the corresponding χ2 distribution test. The p-values obtained were two-tailed and determined to be significant when p<0.05. Hardy–Weinberg equilibrium was verified by calculating the expected frequencies and numbers and was tested separately in patients and controls using the goodness-of-fit χ2 test. All the comparisons were performed using GraphPad version 3.00 (GraphPad Software Inc., San Diego, CA, USA).
Results
The demographic characteristics of patients and controls are presented in Table I. The genotyping results are presented in Table II. The patients with POAG had a significantly higher IOP than the healthy controls (p<0.0001). When analyzing the Hardy–Weinberg equilibrium, we observed no significant deviation from expected numbers of both cases and controls. The GSTM1 null genotype frequency was significantly higher in patients with POAG (44.34%) than in controls (30%). The carriers of the GSTM1 null genotype seemed to have an increased risk of developing POAG (p=0.03). As illustrated in Table II, there was no significant difference in OPA1 genotypic frequencies between patients and controls for the rs10451941 and rs166850 polymorphisms. No significant association was found between IOP and the genotypes of the GSTM1 and OPA1 genes tested.
Discussion
Even if the pathophysiology of POAG is not well understood, it has been supported that genetic factors might play a crucial role in POAG development (8-11,47-48). This study aimed to determine whether previous findings of OPA1 (rs166850, rs10451941) and GSTM1 gene polymorphisms could be replicated in a Greek cohort of patients with POAG.
Demographic characteristics of patients and controls.
Genotype distribution of optic atrophy 1 (OPA1) (rs166850, rs10451941) and glutathione S-transferase M1 (GSTM1) polymorphisms in primary open-angle glaucoma (POAG) patients and controls.
Concerning the GSTM1 gene, several studies supported that there is a significant association of GSTM1 null genotype with POAG in several ethnic populations (25-30), indicating that oxidative damage, which is significantly increased in the trabecular meshwork of patients with glaucomas, as stated by Izzotti et al. (49-50), may be implicated in POAG development (27). Conversely, Jansson et al. found no evidence of association between GSTM1 genotype and POAG development in a Swedish population (31), and Barbosa et al. (25) stated that although patients with POAG had a higher frequency of GSTM1 null genotype, the difference was not significant (p=0.0874).
Our results confirm the association of GSTM1 null genotype with POAG in this Greek population. Rocha et al. demonstrated correlation between GSTM1 null genotype and POAG in a Brazilian population (27); their study also suggested that GSTM1 null genotype was associated with higher IOP. However, even if Greek patients with POAG had increased IOPs compared to healthy controls, no association of IOP with a specific genotype was found. Regarding the association of the rs166850, rs10451941 polymorphisms of the OPA1 gene with POAG risk, in agreement with previous studies (40, 42-43), our data could not support an association of these polymorphisms with the development of POAG in this Greek population. Furthermore, there was no association between those polymorphisms and elevated IOP.
In conclusion, our study did not find any significant association between OPA1 polymorphisms and POAG, however, our results suggest that GSTM1 null genotype might be a risk factor for POAG in the Greek population. It is hypothesized that several genes and lifestyle/ environmental factors may contribute to the development of POAG. However, the identity and number of these genes has yet to be defined. We hope that further studies will be directed towards investigation of GSTM1 and OPA1 polymorphisms as they might lead to better understanding of the pathophysiology of this important cause of irreversible blindness.
Footnotes
Conflicts of Interest
The Authors declare that they have no competing interests in regard to this study.
- Received January 12, 2016.
- Revision received March 28, 2016.
- Accepted March 31, 2016.
- Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
