1. Introduction

Congenital glaucoma (CH) continues to be one of the main causes of irreversible blindness and visual disability, and therefore the treatment of this disease remains a very urgent problem in pediatric ophthalmology [4,8]. Surgery is the main treatment for congenital glaucoma. The importance and value of surgical treatment is confirmed by the results of studies conducted by the European Glaucoma Society (2011), which indicate that surgical methods are more effective in reducing and normalizing intraocular pressure (IOP) [1].

In recent years, in ophthalmic pathology, much attention has been paid to the study of the role of cytokine polymorphisms in predicting healing and early scarring after surgery. The primary postoperative immune response is mediated by the production of proinflammatory cytokines, in particular, tumor necrosis factor alpha (TNF-α) [2,7].

Tumor necrosis factor alpha (TNF-α) is a protein that is a product of monocytes, macrophages, endothelial, mast and myeloid cells, neuroglia cells and has a wide spectrum of biological effects. This cytokine plays a role in the development of the inflammatory response, and also stimulates the proliferation of T and B lymphocytes [3,5,6].

The TNF gene encoding TNF-α is located on the short arm of chromosome 6 (6p21.1 6p21.3) within the cluster of class III genes of the major histocompatibility complex. One of the most studied polymorphic variants in this gene is rs1800629 (G-308A), located in the gene promoter, associated with the level of gene expression and production of TNF-α - allelic variant A-308 is associated with a high concentration [9,10].

In connection with the above, it seems relevant to study the genetic polymorphisms of cytokine genes in children with congenital glaucoma to expand the understanding of the pathogenetic mechanisms of the formation of the immune response in glaucomatous processes and the postoperative outcome of the disease in carriers of polymorphic mutations.

Purpose of the study – to identify the role of genetic polymorphism of the TNF-α gene (G-308A) in the healing of drainage.

2. Materials and Methods

Immunological and genetic studies were carried out in the laboratory of immunoregulation of the Institute of Immunology and Human Genomics of the Academy of Sciences of the Republic of Uzbekistan. The study included 115 patients aged 2-14 years with refractory glaucoma and 45 apparently healthy children of the same age, who made up the comparison group.

Measurement of the concentration of TNFα was carried out by the method of enzyme-linked immunosorbent assay (ELISA) using commercial reagent kits LLC "Vector-Best" (Russia, Novosibirsk).

The data were statistically processed using the Statistica 6.0 computer program. The significance of differences in the mean values of the compared indicators was assessed by the Student's test (t).

The SNP of the TNFα gene promoter -308G / A (rs1800629) was determined by the polymerase chain reaction (PCR) method using kits for the determination of polymorphisms in the human genome, OOO NPF Litekh (Moscow), by the method of allele-specific polymerase chain reaction with electrophoretic detection.

The correspondence of the observed distributions of genotype frequencies to theoretically expected by the Hardy-Weinberg equation was assessed using the χ2 test. To assess associations, the relative risk (OR) was calculated with a confidence interval (CI) at a 95% significance level.

3. Results and Its Discussion

As a result of the comparative analysis, an increased content of TNF-α in sick children with congenital glaucoma was found in relation to the indicators of the control group. The serum concentration of the studied cytokine in children of the main group before surgery averaged 124.06 ± 2.42 pg / ml, which was significantly higher than this indicator in the control group 20.37 ± 2.14 pg / ml (P <0, 05) and this indicated a pronounced hypercytokinemia in the development of the glaucomatous process.

Comparative analysis of TNFα-308G / A genotypes for the GG genotype revealed significant differences between the general group of patients with GV and the control group (OR = 0.41; 95% CI: 0.213> 0.41> 0.789; χ2 = 7.352). Analysis of the heterozygous GA genotype revealed differences between the frequency of occurrence in patients with GV and the control group (33.06% and 16.84%, respectively; OR = 2.439; 95% CI: 1.267> 2.439> 4.694; χ2 = 7.352).

The data obtained indicate that the -308 (G / A) TNF polymorphism makes a significant contribution to the susceptibility to disease.

The proinflammatory cytokine TNF-α influences the development of such postoperative complications as early scarring, and its level affects the duration of these complications. It was found that the higher the serum TNF-α level, the higher the frequency, severity and duration of reactive complications after drainage.

Analysis of the results after surgery allowed us to identify situations, the most favorable and unfavorable course of the postoperative period, on the basis of which two groups were formed. The main criteria for selecting patients by groups were factors of unsatisfactory (poor) and satisfactory (good) healing, based on determining the concentration of the studied mediator-immune response in blood serum.

To identify a more accurate state of drainage healing, we carried out a comparative analysis of the TNF-α content in the blood serum over time, on the 3rd, 7th and 14th days after surgery (Fig. 1).

Figure 1. TNF-α content in groups of children before and after surgical intervention

The postoperative analysis of the immune status was studied taking into account the clinical course. It should be noted that the significant expression of regulatory cytokines in the groups manifested itself in the postoperative period.

Comparative analysis of the systemic cytokine status after surgery revealed insignificant changes in the synthesis of proinflammatory cytokines in the group with poor healing ability.

In our work, the content of TNF-α slightly decreased after 3 days by 6%, after 7 days by 11.5%, and after 14 days by 21.7% compared with preoperative indicators (3 - 116.82 ± 3 , 1 pg / ml (P <0.01), 7 - 109.9 ± 5.8 pg / ml (P <0.01), 14 - 97.24 ± 1.7 pg / ml (P <0, 01) versus 124.06 ± 2.42 pg / ml.

Normally, TNF-α production significantly increases in the first few hours to several days after surgery, then decreases to the initial level, but in our case, increased expression before surgery, relatively increased synthesis, and a pronounced slight decrease indicate an initial pathological state, which is probably genetically conditioned.

Further, the analysis of allele frequencies and genotypes of the TNF-α -308 G / A gene was carried out in patients in the group with poor healing ability. Comparative analysis of TNFα-308G / A genotypes for the GG genotype revealed significant differences between patients with GV and the control group. So the frequency of occurrence of the dominant genotype GG was more often in the healthy control group -83.16% (OR = 0.41; 95% CI: 0.213> 0.41> 0.789; χ2 = 7.352) than in the cohort of GV patients with poor healing - 58.49% (OR = 0.285; 95% CI: 0.133> 0.285> 0.614; χ2 = 10.847).

Analysis of the heterozygous GA genotype revealed differences between the frequency of occurrence in patients with GV and the control group (41.51% and 16.84%, respectively; OR = 3.504; 95% CI: 1.628> 3.504> 7.54; χ2 = 10.847).

At the same time, associations of the polymorphism of the studied genetic marker with the content of TNF-α in the blood serum in patients with poor healing ability were revealed and indicate the presence of a relationship between immune disorders and their genetic determination. The results obtained once again prove that the -308 polymorphism increases the transcriptional activity of the TNF-α gene and, accordingly, the production of a cytokine, which directly affects early scarring.

In genotypic analysis, no homozygous AA genotype was recorded.

The above listed endogenous polypeptide mediators of intercellular interaction are actively involved in the development and maintenance of already impaired immune homeostasis in congenital glaucoma. We tend to consider a slow decline as well as a possible increase in their concentrations as a negative factor, the implementation of which can initiate enhanced synthesis of extracellular matrix proteins, remodeling of the damaged trabecular network, which will lead to an increase in resistance to the outflow of IHF and back to a renewed increase in intraocular pressure.

The study of the concentration of TNF-α showed that the concentration of tumor necrosis factor-alpha in the blood serum of children of this group averaged 102.71 ± 4.2 pg / ml versus 124.06 ± 2.42 pg / ml (P <0, 05). This indicates a decrease in the synthesis of this mediator by 17.5%. After 7 days, the level of TNF-α averaged 82.53 ± 3.7 pg / ml, which is 33.5% less than the preoperative data (P <0.01). 14 days after surgery, the analysis of the study of the pro-inflammatory cytokine showed that the level of TNF-α synthesis decreased by 57.8% and averaged 52.45 ± 2.9 pg / ml (P <0.01).

Analysis of allele frequencies and genotypes of the TNF-α -308G / A gene in patients in the group with good healing ability did not reveal any significant differences. Thus, the frequency of occurrence of the dominant GG genotype was 73.24% (OR = 0.554; 95% CI: 0.261> 0.554> 1.175; χ2 = 2.402) versus the values of the control group and the group of children with poor healing GV, respectively. Analysis of the carriage of the heterozygous GA genotype also did not reveal significant differences. The frequency of occurrence of the heterozygous GA genotype in the group of children with CH with good healing was 26.76% (OR = 1.804; 95% CI: 0.851> 1.804> 3.825; χ2 = 2.402), compared with the group of CH children with poor healing and control group.

The homozygous AA genotype was also not found in the group of children with good healing ability.

Thus, the assessment of TNF-α production in children with CH after the use of the Glautex drainage makes it possible to clarify the pathogenetic mechanisms that determine postoperative healing and to establish one of the causes of early scarring. Thus, upon admission, children with CH were found to have a 6-fold increase in the level of TNF-α in the blood serum compared with similar indicators in healthy children. A postoperative study in dynamics revealed a slight decrease in the synthesis of this cytokine, which is associated with genetic determination.

4. Conclusions

1. It was revealed that in children with GV before surgery, the concentration of TNF-α in the blood serum is 6 times higher than in the control group.

2. It was found that in the group of children with poor healing ability, surgical treatment did not reduce the expression of the studied mediator, and the analysis of TNF-α in dynamics revealed a slight decrease in the synthesis of this cytokine, which is associated with genetic determination.

3. Certainly, the obtained typing data of the TNF-α gene indicate that the -308 (G / A) TNF polymorphism, in particular, heterozygous carriage, makes a significant contribution to the predisposition of early scarring, as it promotes adhesion of the wound canal and vice versa leads to the accumulation of intraocular fluid.