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Kayumov Abdurakhman Abdumavlyanovich, Ph. D.,
Karimov Khamid Yakubovich, MD, professor,
Boboev Kadirzhon Tuhtabaevich, MD, Research Institute of Hematology and Blood Transfusion of the Ministry of Health of Uzbekistan E-mail: atabek2004@mail.ru
Role of polymorphism RS1800629 gene proinflammatory cytokine TNF-a in the development and clinical course of leukemia
Abstract: To evaluate the significance of genotypic variants of the poly-morphism rs1800629 gene pro-inflammatory cytokine TNF-a in the development of leukemia and associating them with complications of viral infections Epstein-Barr. Thus, we found statistically significant differences in the prevalence of genotypes of rs1800629 polymorphic variant in a gene TNFa between subgroups of the study sample. Not been identified genotype demonstrated significantly adverse effect with respect to the risk of EBV. Studies have shown that heterozygous polymorphism was more frequent (p = 0.01) in patients with hematological malignancies.
Keywords: leukemia, polymorphism 308 G/A gene TNF-a, Epstein-Barr virus.
Relevance
It is known that functional polymorphisms in the gene for pro-inflammatory cytokine TNF-a can significantly affect their production and therefore, the severity and chronicity of infection with EBV. There are many studies about role of polymorphisms of genes INF-y, TNF-alpha, IL-1 RA. in various diseases. The main mediator of inflammation is a TNF-a, stimulating immune system in many of its links, controls the expression of many cytokines and growth factors. An increased TNF-alpha production by B-lympho-cytes in response to the action ofEBV [1; 3]. M. S. Wu et al. studies, showed the presence of significant association of allele A at position — rs1800629 gene of EBV-associated gastric carcinoma [2; 4].
Increased secretion of inflammatory factors associated with latent Epstein-Barr virus (EBV) infection and pathology of EBV-associated diseases; However, knowledge of the inflammatory response and its biological significance for EBV lytic cycle remains elusive [4; 7]. BZLF1 the Early activator of proinflammatory transcription factor inhibits tumor necrosis factor alpha (TNF-alpha) by binding to the TNF-alpha promoter, and preventing the activation of NF-kV. BZLF1A207-210 deletion mutant 4 amino acids (aa) in the binding of protein-protein domain was not capable of inhibiting proinflammatory factors TNF-a and interferon-gamma (IFN-y) and reduced viral DNA replication with full transcriptional activity during lytic gene expression of EBV. Depletion of TNF-a restored virus replication mediated BZLF1A207-210. Furthermore, the combination BETA-a- and y-IFN neutralizing antibodies recovered BZLF1A207-210-mediated replication of the virus, indicating that the antiviral response is attenuated BZLF1 aid optimal lytic replication primarily by inhibiting TNF-alpha and IFN-y secretion in during the lytic cycle. These results indicate that EBV BZLF1 attenuates pro-inflammatory responses to facilitate viral replication [5; 6; 8].
However, information about the role of cytokine gene polymorphism in the development of leukemia and associating them with complications of viral infection Epstein -Barr virtually nonexistent.
Objective
To evaluate the significance of genotypic variants of the polymorphism rs1800629 gene pro-inflammatory cytokine TNF-a in the development of leukemia and associating them with complications of viral infections Epstein-Barr.
Materials and Methods
The object and subject of the study were patients with leukemia (CML and AL), DNA samples from patients and 110 healthy volunteers, TNF-a gene (6r21.3) nucleotide substitution G > A at position -308.
The study included 114 patients with AML and CML in age from 15 to 79 years, the median age was 37.8 ± 2.34 years, observed on the basis of clinic Institute of Hematology and Blood Transfusion of the Ministry of Health of Uzbekistan. The diagnosis of leukemia is based on a set of clinic, clinical, laboratory and instrumental data according to WHO criteria.
Isolation of DNA from nuclei of lymphocytes was carried out according to standard methods with some modifications (Sambrook et al., 1989). The concentration and purity of the isolated DNA were estimated by measuring the optical density of the DNA-containing solutions at a wavelength of 260 nm. and 280 nm. on a spectro-photometer against TE NanoDrop 2000 (USA). Genotyping polymorphism 308G > A TNF-a gene was performed by polymerase chain reaction on a programmable thermal cycler CG-1-96 «Cor-bett Research» (Australia) in 2720 and "an Applied Biosystems" (USA), with NGOs "Liteh" test systems (Russia) according to the manufacturer's instructions.
Role of polymorphism RS1800629 gene proinflammatory cytokine TNF-a in the development and clinical course of leukemia
Statistical treatment
Study Design: Case-control, ie, by comparing the frequency of genotypes distribution among the sick and the healthy, the case-the case, by comparing the data among patients subgroups.
Evaluation of deviation of the distribution of genotypes from the canonical distribution of Hardy-Weinberg equilibrium (RCE) was performed using the computer program "GenePop" ("Genetics of Population"). The frequency of allele and genotype variants (f) is calculated using the formula:
f= n/2N and f = n/N, where n — the occurrence of variant (allele or genotype), N — sample size.
To calculate the ratio, "the ratio of chance" (OR — odds ratio) with 95 % confidence intervals (CI — confidence interval), x2 and p values used package «OpenEpi 2009, Version 2.3» statistical programs.
The relative deviation of expected heterozygosity from watch-ing-my (D) was calculated according to the formula:
D (hofcs hexp)/hexp ,
where h , and h — the expected and observed heterozygosity,
obs exp l J d J'
respectively.
The predictive efficacy (AUC-qualifier) studied at the E-genetic markers was determined by the standard formula:
AUC = (Se + Sp)/2, where Se and Sp — the sensitivity and specificity of a genetic marker, respectively. If the index AUC < 0.5, the marker — the occasional qualifier; AUC = 0.5-0.6 — bad; AUC = 0.6-0.7 — medium; AUC = 0.7-0.8 — good; AUC > 0.8 — great classifier (Hosmer D. W., Lemeshow S. et al, 2000.).
Results and discussion
When comparing samples between patients with CML and AL identified Article cally no significant differences in the distribution of TNF-a genotype frequencies of rs1800629 polymorphism of the gene. Therefore, in the future it will only go for the combined group.
An analysis of the frequency distribution of genotypes of rs1800629 polymorphism of TNF-a gene and their compliance with population-balance RCE Group leukemia patients and healthy donors were carried out separately. As expected, in the groups ofpatients and control the distribution of genotypes of polymorphic loci on the corresponded RCE. In the studied groups — patients and controls, the mean value of the observed heterozygote (Hobs) was equal to 0.28 and 14.55 respectively and above average, theoretically expected heterozygote (Hexp = 0.24 and 13.5 %, respectively). Thus, in patients and control groups, the amount of actual deviation from a theoretical ratio ofheterozygote at the level of > 0, as indicated by a positive value and Wright fixation index (D = 0.2 and 0.08, respectively).
Table 1. - The coefficients of the deviation of the actual heterozygote Theoretical
The frequency of alleles The frequency distribution of genotypes
Group n G A G/G G/A A/A
N % n % n % n % n %
1 Basic group 114 196 86.0 32 14.0 82 71.9 32 28.1 - 0
2 Control group 110 204 92.7 16 7.3 94 85.4 16 14.5 - 0
Indicators SS and SP of the genetic marker among the studied groups corresponded to 0.28 and 0.85. The calculated predictive eficiency of this marker was AUC = 0.57. These data show not very high indicator Classifier polymorphism rs1800629, on what basis can make a pre-theoretical conclusion that the genetic mutation 308G > A is not a high-performance classifier for predicting the development of leukemia.
To estimate the contribution of the genetic marker 308G > A gene TNF-a in the pathogenesis of leukemia, we conducted a comparative analysis of the occurrence frequencies of alleles and genotypes in patients and leukemia group of healthy controls.
Analysis of the distribution of alleles of polymorphic variants of 308G > A gene TNF-a showed that the distribution of allele frequencies in patients was significantly different from the control group (P < 0.05). In both groups, the prevalence rate was observed wild (normal) allele "G" while reducing the incidence of a rare, functionally defective allele "A". The prevalence of alleles in a population sample was as follows: "A" — 7.3 % (16/220), "G" — 14.0 % (16/220); in patients: "A" — 14.0 % (32/228), "G" — 86.0 % (196/228). According to the calculated ratio odds ratio, carrier of a rare allele "A" polymorphism 308G > A TNF-a gene by more than 2 times significantly
increases the risk of developing leukemia (x2 = 5.3; p = 0.02; OR = 2.1; 95 % CI 1.107, 3.914).
Statistically significant differences were found when comparing the frequencies of detection of genotypes of rs1800629 polymorphism in the gene TNF- a (P < 0.05). Among the surveyed groups of patients had an increase in the proportion of homozygous genotype G/G (71.9 %), by reducing the frequency straight and homozygous genotypes (28.1 % and 0.0 %, respectively). The frequency distribution of "G/G" and "A/G" genotypes was 85.4 % in the control group (32/114) and 14.5 % (16/110), respectively. According to the calculated ratio chance genotype of the ratio "A/G" significantly increases the risk of leukemia in more than 2 times (x2 = 6.1; p = 0.01; OR = 2.3; 95 % CI 1.174, 4.477).
It should be emphasized that the polymorphism 308G > A gene in TNF-a A/A homozygote or in patients nor in the control group was not detected.
Thus, the polymorphism rs1800629 gene TNF-a is of some importance in the development ofleukemia, which suggests the need to continue a comprehensive study of the role of gene families of cytokines in the pathogenesis ofleukemia. These data can complement the overall picture of the genetic basis of susceptibility to these diseases.
Table 2. - The frequency distribution of allele and genotype polymorphism rs1800629 TNF-a gene in a group of patients and controls
The frequency of alleles The frequency distribution of genotypes
Groups n G A G/G G/A A/A
N % n % n % n % n %
1 Basic group 114 196 86.0 32 14.0 82 71.9 32 28.1 - 0
2 Control group 110 204 92.7 16 7.3 94 85.4 16 14.5 - 0
The next stage of our study was to analyze the association of rs1800629 polymorphism genotypic variants of the gene TNF-a complications of viral infection with Epstein-Barr virus. Frequencies of genotype studied genes in subgroups of patients with and without EBV are presented in Table 3.
Table 3. - Frequency distribution of genotypes in patients with and without EBV
Genotypes Epstein-Barr virus
Positive n = 12 Negative n = 102 OR x2 P
G/G 10 82
G/A 2 20 0.8 0.02 > 0.05
A/A 0 0
ALL 12 102
Among the 114 patients studied, 12 patients were carriers of EBV, which amounted to 10.5 %. Analysis of the distribution of genotypes of the studied polymorphisms showed that among these carriers significantly more frequently detected wild genotype G/G, than among patients without EBV (83.3 % vs. 80.4 %,
respectively). Patients with heterozygous G/A genotype was 16.7 % (2/12). It is interesting to note that among the patients without EBV this genotype was detected more frequently than in patients with EBV (20/102; 19.6 % vs. 16.7 %, respectively). Accordingly, the calculation of odds ratios also showed a negative association unfavorable genotype G/A carriage with VEB (x2 = 6.1; p = 0.01; OR = 2.3; 95 % CI 1.174-4.477).
Thus, we found statistically significant differences in the prevalence of genotypes of rs1800629 polymorphic variant in a gene TNFa between subgroups of the study sample. Not been identified genotype demonstrated significantly adverse effect with respect to the risk of EBV. Studies have shown that heterozygous polymorphism was more frequent (p = 0.01) in patients with hematological malignancies. This fact can be concluded that the gene polymorphism of TNF alpha by disturbances in the immune regulation could play a role in the formation of leukemia. Despite the differences found among the patients showed no significant differences, indicating that changes in one-pointedness of humoral immunity and points to a comprehensive study of all the links of pro- and anti-inflammatory cytokines.
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Kayumov Abdurakhman Abdumavlyanovich, Ph. D., Karimov Khamid Yakubovich, MD, professor, Boboev Kadirzhon Tuhtabaevich, MD, Research Institute of Hematology and Blood Transfusion of the Ministry of Health of Uzbekistan E-mail: atabek2004@mail.ru
Studying frequency of CTLA4 gene polymorphism in patients with hematological malignancies
Abstract: To study the frequency of the gene polymorphism of CTLA 4 49G > A in patients with hematological malignancies. These indicators show an extremely low degree of predictions of the studied polymorphism rs231775 CTLA4 gene. Studying cytokines, one might think that an isolated form, they are not involved in immune responses may interact with each other, can not only create the optimal immune responses, but also in using this approach, you can answer the fundamental questions of modern tumor immunology and the interaction of immune and neoplastic cells Keywords: hematological malignancies, cytokine polymorphisms, of CTLA4.
Despite advances in diagnosis and treatment, which allowed complications from systemic inflammation position. According to considered hemoblastoses potentially curable disease still remain some authors, in the development of malignancy is a violation of many questions about diagnosis, pathogenesis of leukemia and the interaction of the tumor cells and cells of the immune system,
