Polymorphic locus rs1061624 of the ТNFR2 gene is associated with the development of arterial hypertension in males

Aim To study the involvement of cytokine polymorphous loci in development of arterial hypertension (AH) in men from the Central Black Earth region of Russia. Materials and methods 821 men were evaluated, including 564 patients with AH and 257 individuals of the control group. Analysis of 8 cytokine mononucleotide polymorphisms (MNP) was performed using the real-time polymerase chain reaction with TagMan probes. Statistical analysis was performed with the STATISTICA (v.10.0) and PLINK (v.1.06) software. The regulatory potential of MNP was analyzed with the HaploReg (v.4.1) service (http://archive.broadinstitute.org). transcriptional factors and histones that mark enhancers and promoters in different organs and tissues. Conclusion The rs1061624 ТNFR2 gene polymorphism is involved in the development of AH in men of the Central Black Earth region of Russia.


Introduction
Hypertension is the most common cardiovascular disease. Each year it causes fatal complications in more than 9.4 million people worldwide, Elevated blood pressure (BP) levels are registered in 1.13 billion people in the general population, of whom 52.8 % are male [1].
The molecular mechanisms of hypertension are not fully understood. However, the latest data shows an essential pathogenetic role of non-specific inflammation [2,3]. The endothelium is known to be involved in the initiation and development of vascular wall inflammation. The inflammatory cascade adversely affects the endotheliumdependent processes and the mechanical properties of arteries [4]. The inflammation process is inevitably accompanied by the active production of inflammation mediators, such as cytokines [5]. Cytokines are a large group of low-molecular-weight proteins that regulate inflammation, angiogenesis, non-specific protective reactions of the body, and induce cell growth and differentiation and tissue regeneration [6].
Previous studies have shown that cytokine gene polymorphic loci are involved in developing hypertension and its complications [7][8][9][10]. However, this issue requires further research.

Objective
To study the involvement of cytokine polymorphic loci in the development of hypertension in male patients in the Central Black Earth Region of Russia.

Material and Methods
The study included 821 male patients: 564 patients with hypertension and 257 control individuals. Male patients were included in the study after diagnosis of hypertension ORIGINAL ARTICLES § was confirmed by laboratory, instrumental, and clinical examination methods under current guidelines [11]. The inclusion criteria in the hypertension group were systolic blood pressure (SBP) ≥140 mm Hg and / or diastolic blood pressure (DBP) ≥90 mm Hg; the absence of symptomatic hypertension, hepatic and renal failure. Inclusion criteria in the control group were SBP <140 mm Hg and DBP <90 mm Hg, the absence of metabolic syndrome, autoimmune diseases, and cancer. The study included patients of Russian ethnic origin, native to the Central Black Earth Region of Russia, who were not related. Hypertension and control groups were formed between 2013 and 2016 in the Cardiology Department of the St. Joasaph Belgorod Regional Clinical Hospital. The mean age of patients with hypertension was 57.60 ± 8.36 years, healthy individuals 57.54 ± 9.73 years, and comparable (Mann-Whitney U-test) (r=0.86). The clinical characteristics of the study groups have been described earlier [12]. It should be noted that of the patients with hypertension included in the study, 145 (25.71 %) had a history of ischemic stroke, 24 (4.25 %) had a myocardial infarction, and 69 (12.23 %) had coronary artery disease. The study was carried out following the Good Clinical Practice and the Declaration of Helsinki. The Ethics Committee of the Medical Institute under the Belgorod State National Research University approved the study. All subjects signed an informed consent.
Genomic DNA was isolated from the peripheral blood leukocytes in a standard phenol-chloroform extraction [13]. The polymorphic cytokine markers were analyzed by means of a polymerase chain reaction, DNA synthesis in a CF-96 Real-Time System (Bio-Rad, USA) using oligonucleotide primers and probes (OOO «Synthol», Russia). 100 % reproducibility was registered in a repeat genotyping of 5 % of samples randomly selected in the hypertension and control groups. Correspondence of the distribution of genotype and allele frequencies with the Hardy-Weinberg equilibrium was evaluated using the χ 2 test. The frequencies of genotypes and alleles in the study groups were analyzed in the 2×2 contingency tables and the Yates' χ 2 test.
The results obtained were processed using STATISTICA for Windows 10.0. The Bonferroni amendment equal to 8 was made to correct the number of SNPs analyzed, after which rbonf≤0.006 was considered statistically significant. The nature of the associations of polymorphisms with hypertension was estimated using the odds ratio (OR), and its 95 % confidence interval (95 % CI). SNP associations with hypertension were analyzed using logistic regression analysis in three genetic models (dominant, recessive, additive) using the Plink 1.06 software (http://pngu.mgh.harvard.edu / Èpurcell / plink). An adapti ve permutation test was performed to minimize false positi ves with r perm ≤0.05 being statistically significant. The power of associations in the genetic models was analyzed using Quanto v.1.2.4 (http://biostats.usc.edu / Quanto. html) and a two-tailed test taking into account the prevalence of hypertension in the adult Russian population (40 %), as well as the probability of a false positive equal to 5 % (α=0.05). The regulatory potential of the cytokine polymorphic loci was analyzed in Haploreg v4.1 (http://archive.brodinstitute.org / haploreg.php).

Results and Discussion
For all cytokine polymorphic loci analyzed (p>0.05), the observed distribution of genotypes corresponded to the anticipated distribution in the Hardy-Weinberg equilibrium. The allele frequencies of the cytokine genes SNPs in patients with hypertension and the control group are presented in Table 1. The rs1061624 TNFR2 and rs909253 TNFβ differences were significant.
It was identified that allele A (OR=0.73) and genotypes AA (OR=0.65), GG (OR=1.51) of polymorphic locus rs1061624, and allele G (OR=0.79) and genotypes AG (OR=0.71), AA (OR=1.43) of locus rs909253 (r<0.05) were associated with hypertension in male patients. However, the differences remained significant after correction for multiple comparisons (rbonf≤0.006) only for allele A of rs1061624 of TNFR2 acting as a protective factor in the development of the disease (OR<1). The analysis showed no associations of rs1800629 TNFα, rs767455 TNFR1, rs833061 VEGFA, rs2981582 FGFR-2, rs6214 IGF-1, and rs1800469 TG Fβ-1 with hypertension in male patients.
Results of the logistic regression analysis of the associations of cytokine genotypes with hypertension are provided in Table 2.
The rs1061624 TNFR2 polymorphism was shown to be associated with hypertension in male patients in the recessive (p perm =0.0004, power 57.21 %) and additive (p perm =0.0006, power 83.08 %) genetic models and has a protective effect in the development of the disease (OR=0.33-0.50).
According to the HaplReg (v4.1) database, the rs1061624 TNFR2 polymorphism is located in the DNA region hypersensitive to DNase-1 in stem cells and peripheral blood monocytes. This SNP is located in DNA fragments which bind to modified histones (H3K4me1 and H3K4me3) that label enhancers and promoters in 12 different organs and tissues. These include the peripheral ORIGINAL ARTICLES § blood cells, the heart, the brain, the digestive system, etc. rs1061624 was shown in the field of regulatory DNA motifs. Its allele A reduces the affinity to transcriptional factors BCL-disc9, Myc-disc10, NRSF-disc9, and VDR-2 (http://archive.brodinstitute.org / haploreg.php). The associa tions of rs1061624 TNFR2 with hypertension may be based on the established regulatory effects of this SNP and the general biological functions of the tumor necrosis factor receptor type II. According to the GeneCards database, TNFR2 is synthesized in circulating T lymphocytes, endotheliocytes, macrophages and induces cell proliferation and migration. Tumor necrosis factor receptor type II and TNFR1 form a heterocomplex with ubiquitin ligase activity protecting cells from apoptosis by stimulating antioxidant pathways (http://www.genecards.org / ).
The impaired production of TNFR2 and other cytokines was shown to aggravate endothelium-dependant vasodilation and induce the vasoconstrictor synthesis. This leads to the rigidity of the vascular wall and the retention of BP at high levels [14]. Shai et al. showed that an increase in the TNFR2 serum levels correlates with a high risk of myocardial infarction (OR=2.48, r=0.034) and coronary artery disease (OR=2.02, r=0.003) in the North American population [15].

ORIGINAL ARTICLES §
There are few studies of the contribution of tumor necrosis factor receptor genes in the development of cardiovascular diseases. For example, Allen et al. studied the association of rs1061624 TNFR2 and rs4149570 TNFR1 with the development of coronary artery disease in the British population (n=430) although they did not find any significant associations (p>0.05) [10]. However, data has been published showing the association of TNFα polymorphisms with hypertension, with TNFα encoding tumor necrosis factor and acting through TNFR2. In the Asian population, Liaquat et al. established the associations of polymorphic marker -238G / A TNFα with cardiomyopathy in hypertension (p=0.01) [7], and Tong et al. showed that locus -308GA TNFα was involved in the development of ischemic stroke (p=0.03) [9]. Interestingly, Conen et al. found no associations of rs909253 TNFβ with hypertension in the American population (p=0.53) [16], which is consistent with our findings.

Conclusion
We analyzed the involvement of cytokine gene polymorphisms in the development of hypertension in male patients. Significant associations of rs1061624 TNFR2 with hyper tension were established in the recessive (OR=0.33) and additive (OR=0.50) genetic models. Single nucleotide polymorphism TNFR2 is characterized by high regulatory potential. It is located in DNA fragments hypersensitive to Dnase-1 and the fragments to which transcription factors and histones, labelling promoters and enhancers in various organs, bind.

Funding
This study was supported by a grant from the President of the Russian Federation for leading scientific schools of the Russian Federation (project NSh-2609.2020. 7).

No conflict of interest is reported.
The article was received on 12/01/2020 Produced by Plink software; OR, odds ratio; CI, confidence interval; p, significance level, significant differences are marked with an asterisk.