To examine the association of genetic polymorphisms (-308)G/A TNFα, (+250)A/G Ltα, (+36)A/G TNFR1, (+1663) A/G TNFR2 with the development of primary open angle glaucoma (POAG) among people in Central Russi

Churnosov, M.

Ovtcharova, V.

Polonikov, A.

Reshetnikov, E.

Tikunova, E.

DSpace at Belgorod State University

1490·Basic Research·Genes of tumor necrosis factors and their receptors and the primary open angle glaucoma in the population of Central RussiaEvgeniya Tikunova1, Veronika Ovtcharova1, Evgeny Reshetnikov1, Volodymyr Dvornyk2, Alexey Polonikov3, Olga Bushueva3, Mikhail Churnosov11Department of Medical Biological Disciplines, Belgorod State University, Belgorod 308015, Russia 2School of Biological Sciences, University of Hong Kong, Hong Kong, China3Department of Biology, Medical Genetics and Ecology, Kursk State Medical University, Kursk 305041, RussiaCorrespondence to: Evgeny Reshetnikov. Department of Medical Biological Disciplines, Belgorod State University, 85 Pobeda St., Belgorod 308015, Russia. reshetnikov@bsu.edu.ruReceived: 2016-10-26        Accepted: 2017-05-17Abstract● AIM: To examine the association of genetic polymorphisms (-308)G/A TNFα, (+250)A/G Ltα, (+36)A/G TNFR1, (+1663)A/G TNFR2 with the development of primary open angle glaucoma (POAG) among people in Central Russia.● METHODS: The study sample included 443 individuals, of which 252 patients with POAG and 191 individuals in the control group. Genotyping of (-308)G/A TNFα, (+250)A/G Ltα, (+36)A/G TNFR1, (+1663)A/G TNFR2 was performed using polymerase chain reaction. The distribution of alleles and genotypes of the studied DNA markers in the groups was examined by 2×2 contingency tables and χ2 with the Yates’s correction for continuity and odds ratios (OR) with 95% confidence intervals (CI).● RESULTS: Allele (-308)G TNFα (Р=0.01, OR=1.78, 95%CI 1.12-2.85) was identified as a risk factor for POAG. Homozygotes (-308) AA TNFα are at a lowest risk for development of the disease (Р=0.01, OR=0.0005). The following combination of genetic variants of cytokines were associated with a reduced risk of POAG: (+1663)A TNFR2 and (+250)G Ltα (OR=0.34)● CONCLUSION: Genetic polymorphisms (-308)G/A TNFα, (+250)A/G Ltα, (+1663)A/G TNFR2 associated with the development of POAG in the population of Central Russia.● KEYWORDS: primary open angle glaucoma; tumor necrosis factor; tumor necrosis factor receptor; gene polymorphismDOI:10.18240/ijo.2017.10.02Citation: Tikunova E, Ovtcharova V, Reshetnikov E, Dvornyk V, Polonikov A, Bushueva O, Churnosov M. Genes of tumor necrosis factors and their receptors and the primary open angle glaucoma in the population of Central Russia. Int J Ophthalmol 2017;10(10):1490-1494INTRODUCTIONG laucoma 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 etiopathogenic 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 METHODSThe 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. Genes and the primary open angle glaucomaInt J Ophthalmol,    Vol. 10,    No. 10,  Oct.18,  2017      www.ijo.cnTel:8629-82245172     8629-82210956        Email:ijopress@163.com1491The 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/Gof 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 was described elsewhere[18]. The studied loci were genotyped using TaqMan probes and primers described previously (Table 2). 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% confidence 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].RESULTSThe physical characteristics of the participants are given in Table 3. The control group is similar to the case group by Table 1 SNP function prediction of the studied polymorphisms SNP Allele TFBS Splicing (ESE or ESS)miRNA (miRanda) RP ConservationAllele frequencyAllele Europeanrs1800629 A/G + - - 0.04 0 G 0.795rs909253 G/A + - - 0.21 0 A 0.649rs767455 C/T - + - 0.52 1 T 0.505rs1061624 A/G - - + 0.24 0.001 A 0.541Data 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 polymorphismsPolymorphisms Position in the gene 5’-3’ sequence of the primers and probes References-308G/A TNFα (rs1800629) Promotor F: 5’-GAAATGGAGGCAATAGGTTTTGAG-3’R: 5’-GGCCACTGACTGATTTGTGTGTAG-3’5’-FAM-CCGTCCTCATGCC- RTQ1-3’5’-ROX-CCGTCCCCATGCC - RTQ1-3’Landi et al[19]+250A/G Ltα  (rs909253) Intron 1 F: 5’-CAGTCTCATTGTCTCTGTCACACATT-3’R: 5’-ACAGAGAGAGACAGGAAGGGAACA-3’5’-FAM:CCATGGTTCCTCTC-RTQ1-3’ 5’-ROX:CTGCCATGATTCC-RTQ1-3’de Jong et al[20]+36A/G TNFR1 (rs767455) Exon 1 F: 5’- AGCCCACTCTTCCCTTTGTC-3’R: 5’-CCACCGTGCCTGACCTG-3’5’- FAM: CTGCTGCCACTGGT-RTQ1 -3’5’- ROX: CTGCTGCCGCTGGT-BHQ2 -3’Chae et al[21]+1663A/G TNFR2 (rs1061624) 3'UTR F: 5’- TGACCTGCAGGCCAAGAG-3’F: 5’- CCATGGCAGCAGAGGCTTT-3’5’-FAM: CACAACCCGCTGCC - RTQ1-3’5’-ROX: CCACAACTCGCTGCC - BHQ2-3’Ferguson et al[22]Table 3 Physical characteristics of the subjects from the case and control groupsCharacteristics Cases ControlsTotal 252 191Age, a (min-max) 70.53±8.43 (46.0-89.0)a 69.24±10.14 (40.0-87.0)BMI 28.12±4.84a 29.04±6.01Males 125 (49.6%)a 93 (48.7%)Females 127 (50.4%)a 98 (51.3%)BMI: Body mass index; aP>0.05.1492gender, 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).Genes and the primary open angle glaucomaTable 4 Comorbidities in the studied groupsComorbidity (if any)POAG patients Controlsχ2 (P)n % n %Absence of comorbidity 19 7.54 25 13.09 3.15 (0.08)Hypertonia 162 64.29 120 62.83 0.05 (0.83)Arterial hypotension 8 3.17 10 5.24 0.71 (0.40)Heart atherosclerosis 55 21.83 41 21.47 0.01 (1.00)Heart ischemia 112 44.44 78 40.84 0.44 (0.51)Other heart pathology 11 4.37 13 6.81 0.83 (0.36)DiabetesType I 2 0.79 1 0.52 0.01 (1.00)Type II 38 15.08 27 14.14 0.02 (0.89)Other endocrine disorders 13 5.16 7 3.66 0.27 (0.60)Nervous system pathology 21 8.33 24 12.57 1.69 (0.19)Kidney pathology 6 2.38 3 1.57 0.07 (0.80)Digestive system pathology 25 9.92 13 6.81 0.98 (0.32)Respiratory system pathology 9 3.57 3 1.57 0.98 (0.32)Blood pathology 3 1.19 1 0.52 0.05 (0.82)Any other pathology 32 12.70 32 16.75 1.14 (0.29)Table 5 The frequency of genotypes and alleles of the studied polymorphisms in patients with POAG and in the control groupPolymorphisms Genotypes, allelesControls (n=191) POAG patients (n=252)ОR (95% СI) χ2 (P)n % n %-308G/A TNFα (rs1800629) -308GG 139 76.80 208 84.21 1.61 (0.96-2.70) 3.28 (0.07)-308GA 36 19.89 39 15.79 0.76 (0.45-1.28) 0.95 (0.33)-308АА 6 3.31 0 0 0.0005 (-) 6.08 (0.01)-308G 314 86.74 455 92.11 1.78 (1.12-2.85) 6.01 (0.01)-308А 48 13.26 39 7.89 0.56 (0.35-0.90)χ2(HWE) (P) 3.32 (>0.05) 1.81 (>0.05) -+36A/G TNFR1 (rs767455) +36АА 44 24.58 59 23.89 0.96 (0.60-1.55) 0.01 (0.96)+36АG 83 46.37 126 51.01 1.20 (0.80-1.80) 0.72 (0.40)+36GG 52 29.05 62 25.10 0.82 (0.52-1.29) 0.64 (0.43)+36А 171 47.77 244 49.39 1.07 (0.81-1.42) 0.16 (0.69)+36G 187 52.23 250 50.61 0.94 (0.71-1.24)χ2(HWE) (P) 0.90 (>0.05) 0.10 (>0.05) -+1663A/G TNFR2 (rs1061624) +1663АА 36 20.57 38 15.32 0.70 (0.41-1.19) 1.61 (0.21)+1663АG 75 42.86 128 51.61 1.42 (0.95-2.14) 2.81 (0.09)+1663GG 64 36.57 82 33.07 0.86 (0.56-1.31) 0.41 (0.52)+1663А 147 42.00 204 41.13 0.97 (0.72-1.29) 0.03 (0.86)+1663G 203 58.00 292 58.87 1.04 (0.78-1.38)χ2(HWE) (P) 2.53 (>0.05) 1.07 (>0.05) -+250A/G Ltα (rs909253) +250АА 85 46.96 139 55.83 1.43 (0.95-2.14) 2.95 (0.09)+250АG 81 44.75 89 35.74 0.69 (0.46-1.04) 3.19 (0.07)+250GG 15 8.29 21 8.43 1.02 (0.49-2.15) 0.01 (1.00)+250А 251 69.34 367 73.69 1.24 (0.91-1.69) 1.76 (0.19)+250G 111 30.66 131 26.31 0.81 (0.59-1.10)χ2(HWE) (P) 0.50 (>0.05) 1.52 (>0.05) -HWE: Hardy-Weinberg equilibrium.Int J Ophthalmol,    Vol. 10,    No. 10,  Oct.18,  2017      www.ijo.cnTel:8629-82245172     8629-82210956        Email:ijopress@163.com1493The 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).DISCUSSIONThe 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.ACKNOWLEDGEMENTSConflicts of Interest: Tikunova E, None; Ovtcharova V, None; Reshetnikov E, None; Dvornyk V, None; Polonikov A, None; Bushueva O, None; Churnosov M, None.REFERENCES1 Quigley HA, Broman AT. The number of people with glaucoma worldwide in 2010 and 2020. Br J Ophthalmol 2006;90(3):262-267.2 Distelhorst JS, Hughes GM. Open-angle glaucoma. Am Fam Physician 2003;67(9):1937-1944. 3 Tеzеl G. TNF-α signaling in glaucomatous neurodegeneration. Prоg Brain Rеs 2008;173:409-421.4 Agarwal R, Agarwal P. Glaucomatous neurodegeneration: an eye on tumor necrosis factor-alpha. 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Genes of tumor necrosis factors and their receptors and the primary open angle glaucoma in the population of Central Russia

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Genes of tumor necrosis factors and their receptors and the primary open angle glaucoma in the population of Central Russia

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