INTRODUCTION
Glaucoma is a heterogeneous group of diseases characterized by progressive optic neuropathy and the typical reduction in visual function, being the second leading cause of irreversible blindness[1]. Primary open angle glaucoma (POAG)is the most prevalent clinical form of the disease accounting for 90% of all forms of glaucoma[2].
Currently, the etio pathogenic significance in POAG is thought to be due to violations in apoptosis. Tumor necrosis factors and their receptors are among key elements in this process[3-4].
Therefore, several genetic polymorphisms of tumor necrosis factors and their receptors have been analyzed for their possible association with POAG[5-11]. Among those, (-308)G/A TNFα (guanine to adenine substitution at position 308 of the TNFα promoter region) was the most commonly studied.The data on its association with the development of POAG were contradictory. For example, while in some populations no association of this polymorphism with the emergence of POAG was found[12-15], such an association was reported in populations from China, Iran, Egypt and Saudi Arabia[5,7,16-17].
In this study, we analyzed four polymorphisms of cytokines,(-308)G/A TNFα, (+250)A/G Ltα, (+36)A/G TNFR1, and(+1663)A/G TNFR2, for their possible association with development of POAG in the population of Central Russia.
SUBJECTS AND METHODS
The total study sample consisted of 443 participants, including 252 patients with POAG and 191 individuals in the control group. The patients with POAG were enrolled in the study according to the following criteria: open anterior chamber angle, increased intraocular pressure (≥21 mm Hg), characteristic changes in the optic disc (notching, neuroretinal rim thinning,increased the ratio of excavation/optic disc), visual field defects characteristic of glaucoma (arcuate scotoma, narrowing of the field of view with the nose, paracentral scotoma) and the absence of conditions leading to secondary glaucoma.
Table 1 SNP function prediction of the studied polymorphisms
Data is obtained using the online program from the site the National Institute of Environmental Health Sciences (https://snpinfo.niehs.nih.gov/snpinfo/snpfunc.html). TFBS: Transcription factor binding site; ESE: Exonic splicing enhancer; ESS: Exonic splicing silencer;RP: Regulatory potential.
Table 2 Primers and probes used to genotype the studied polymorphisms
The control group included individuals without POAG, acute diseases of the eye at the time of the survey, or any somatic pathology resulting in secondary injury to the eyes. All study participants signed a written informed consent in accordance with the principles of the Helsinki Declaration. The participants were examined at the Department of Eye Microsurgery of St.Iasaf Belgorod Regional Clinical Hospital.
All participants were genotyped for the following polymorphisms: (-308)G/A of TNFα (rs1800629), (+250)A/G of Ltα (rs909253), (+36)А/G of ТNFR1 (rs767455) and(+1663)A/G of ТNFR2 (rs1061624). There are data about association of TNFα (rs1800629) and Ltα (rs909253) with POAG in populations from China, Iran, Egypt and Saudi Arabia)[5,7,16-17]. TNFR1 and TNFR2 encode the respective receptors. Furthermore, the selected polymorphisms may be of functional significance. Specifically, according to the National Institute of Environmental Health Sciences (NIEHS) (https://snpinfo.niehs.nih.gov/snpinfo/snpfunc.html), rs767455 is located at the splicing site and has high regulatory potential(0.52), rs1061624 is located in the miRNA-binding site,both rs909253 and rs1800629 are located at the transcription factor binding sites (Table 1). Yet, rs1800629, rs909253, and rs1061624 are tag single nucleotide polymorphism (SNPs).
Genomic DNA was isolated from blood drawn from cubital vein of a proband. The protocol of DNA isolation wasdescribed elsewhere[18]. The studied loci were genotyped using TaqMan probes and primers described previously (Table 2).
Table 3 Physical characteristics of the subjects from the case and control groups
BMI: Body mass index; aP>0.05.
The allele frequencies were analyzed for their correspondence to the Hardy-Weinberg equilibrium (HWE) using the χ2 criterion. The distribution of alleles and genotypes in groups was assessed using 2×2 contingency tables, odds ratios (OR)with 95% con fidence intervals (CI), and the Yates’s χ2 test. A contribution of the genetic variants and their combinations POAG was analyzed, using the MCMC and Bayesian nonparametric statistics methods suggested by Favorov et al[23] and implemented in APSampler. The error of the first kind from multiple comparisons was corrected by a permutation test (pperm)[24].
RESULTS
The physical characteristics of the participants are given in Table 3. The control group is similar to the case group bygender, age and prevalence of various comorbidities (Table 4).Allele (-308)G TNFα was associated with an increased risk for POAG (Р=0.01, OR=1.78, 95% CI 1.12-2.85) (Power 0.72), whereas homozygotes (-308)АА TNFα had a lower risk for development of POAG (Р=0.01, OR=0.0005, 95% CI -)(Power 0.79) (Table 5).
Table 4 Comorbidities in the studied groups
Table 5 The frequency of genotypes and alleles of the studied polymorphisms in patients with POAG and in the control group
HWE: Hardy-Weinberg equilibrium.
The combination of the following genetic variants also showed association with a lower risk of POAG: (+1663)А TNFR2 and (+250)G Ltα (Case 5.65%, Control 15.12%, Р=0.001,рperm=0.0003, OR=0.34, 95% CI 0.17-0.66).
DISCUSSION
The study identified the risk and protective genetic variants for POAG. Along with the statistically significant association of individual SNPs, i.e. (-308)G TNFα is a risk factor for POAG and (-308)AA TNFα as a protective factor, a significant contribution to the combination of genetic variants (+1663)А TNFR2 and (+250)G Ltα in lowering a risk for POAG was found.
The role of tumor necrosis factors in POAG is of a great interest, because they contribute to development of optic neuropathy. TNFα, binding to TNFR1, either directly induces apoptosis of retinal ganglion cells or indirectly participates in the progression of glaucomatous neurodegeneration. Elevated intraocular pressure and vascular factors leads to the activation of astrocytes that produce TNFα, which in turn initiates the process of cell death. Moreover, activated astrocytes produce nitric oxide synthase (NOS-2) and thereby affect the formation of NO. Hyperproduction of NO and its transformation into peroxynitrite also induce apoptosis of retinal ganglion cells[3-4]. Thus, tumor necrosis factors may be involved in the pathogenesis of POAG as the main mediators of apoptosis, but the exact mechanism of the involvement of these cytokines in developing POAG remains unclear.
The available data about a probable role of the studied polymorphisms in development of POAG are inconsistent. For example, Chinese researchers determined higher frequency of allele G of the (-308)G/A TNFα polymorphism in POAG patients as compared to the controls (OR=1.89, 95% CI 1.14-3.13)[6], which is in agreement with our results. On the other hand, several studies reported that another allele, (-308)A TNFα, is a risk factor for POAG[5,7,11]. Finally, some studies did not found any association between the (-308)G/A TNFα polymorphism and POAG[9-10].
The data about possible contribution of (+250)А/G Ltα, (+36)A/G TNFR1 and (+1663)A/G TNFR2 to POAG are extremely scarce. Al-Dabbagh et al[7] reported that frequency of genotype+250GG Ltα was higher in the sample of 200 POAG patients from Saudi Arabia (P=0.001, OR=3.28), whereas genotype+250AG Ltα was more common in the controls (P=0.001,OR=0.33). These results differ from ours, which may be due to the different population genetic structure and/or study design.One study determined no association of the +36A/G TNFR1 locus with development of POAG[5]. No studies on association of the (+1663)A/G TNFR2 polymorphism with POAG was found in the available literature.
In summary, the results of the present study suggest that allele(-308)G TNFα is a risk factor for POAG in a population of Central Russia, whereas carriers of the (-308)АА TNFα genotype and haplotype (+1663)А TNFR2/(+250)G Ltα(OR=0.34) have a lower risk to develop POAG.
ACKNOWLEDGEMENTS
Conflicts of Interest: Tikunova E, None; Ovtcharova V,None; Reshetnikov E, None; Dvornyk V, None; Polonikov A, None; Bushueva O, None; Churnosov M, None.
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