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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.
References:
1. GeneCard for gene TNF//[Electronic resource]. - Available from: http://bioinfo.weizmann.ac.il/cards-bin/carddisp?TNF 18.
2. Bidwell J., Keen L., Gallagher G. et al. Cytokine gene polymorphism in human disease: on-line databases//Genes and Imuunity. -1999. - № 1. - P. 3-19.
3. Bidwell J., Keen L., Gallageher G. et al. Cytokine gene polymorphism in human disease: on-line databases. Supplement 1//Genes Immun. - 2001. - Vol. 2, № 2. - P. 61-70.
4. Mori A., Takao S., Pradutkanchana J., Kietthubthew S., Mitarnun W., Ishida T. High tumor necrosis factor-alpha levels in the patients with Epstein-Barr virus-associated peripheral T-cell proliferative disease/lymphoma//Leuk Res. - 2003, Jun. - 27(6): 493-498.
5. Li Y., Long X., Huang L. et al. Epstein-Barr Virus BZLF1-Mediated Downregulation of Proinflammatory Factors Is Essential for Optimal Lytic Viral Replication//J Virol. - 2015, Nov. - 90(2): 887-903. doi: 10.1128/JVI.01921-15.
6. Hui-Hui Li., Hui Zhu et al. Tumour Necrosis Factor-a Gene Polymorphism Is Associated with Metastasis in Patients with Triple Negative Breast Cancer. - Scientific Reports 5. - Article number: 10244 (2015).
7. Au W. Y., Fung A., Wong K. F., Chan C. H., Liang R. Tumor necrosis factor alpha promoter polymorphism and the risk of chronic lymphocytic leukemia and myeloma in the Chinese population//Leuk Lymphoma. - 2006, Oct. - 47(10): 2189-2193.
8. Seidemann Kathrin, Zimmermann Martin et al. Tumor Necrosis Factor and Lymphotoxin Alfa Genetic Polymorphisms and Outcome in Pediatric Patients With Non-Hodgkin's Lymphoma: Results From Berlin-Frankfurt-Munster Trial NHL-BFM 95//Journal of clinical oncology. - 2005. - 01.2179.
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: [email protected]
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,
Studying frequency of CTLA4 gene polymorphism in patients with hematological malignancies
which is accompanied by involvement in systemic inflammation cytokines, imbalances manifest production and regulation of these biologically active substances [1; 4]. Cellular immune required costimulatory activity to initiate or inhibit antigen-specific T- cell response. CTLA-4 is expressed in the inhibitory receptor and activated regulatory T-cells. Single nucleotide polymorphism (SNP), + 49 A/G gene CTLA-4 alters the intracellular distribution of CTLA-4, interleukin-2, and, as a consequence, the proliferation of T-cells [9]. CTLA4 is an inhibitory receptor expressed on a subset of T-lymphocytes. Single nucleotide polymorphisms CTLA4 gene involved in autoimmune diseases, inverted, CTLA4 variations associated with chronic infections such as herpes virus infection [10].
Objective
To study the frequency of the gene polymorphism of CTLA 4 49G > A in patients with hematological malignancies.
Materials and methods
The object and subject of the study were patients with leukemia (CML and AL), DNA samples from patients and 110 healthy blood donors, gene CTLA4 49G > A (6r21.3) nucleotide substitution G > A at position - 308. The study were 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.
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 spectrophotometer against TE NanoDrop 2000 (USA). Genotyping gene polymorphism CTLA4 49 G > A 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.
Statistical processing
Study Design: Case — control, i.e, 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").
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 predictive efficacy (AUC-qualifier) we studied genetic markers was determined by the standard formula:
AUC = (Se + Sp)/2, where Se and Sp — the sensitivity and specificity of the 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 (Hos-mer D. W., Lemeshow S. et al., 2000).
Results
Patients included in the study were diagnosed on clinical signs, as the morphology of bone marrow cells as well as molecular, immu-nophenotypic, and cytogenetic data. When studying the expected and observed genotypes frequency distribution of patients no significant differences were detected (P = 0.24).
Evaluation of gene CTLA 4 polymorphism rs231775 in both examined groups the observed distribution of genotypes of polymorphism rs231775 CTLA4 gene, consistent with the expected law on the Hardy-Weinberg equilibrium (P > 0.05). The proportion of heterozygous polymorphism rs231775 gene CTLA4 in the population, for each locus were calculated observed heterozygosity (Hobs), expected heterozygosity (Hexp) and the coefficient of variation of Hexp Hobs (F).
Table 1. - Estimated and observed frequency distribution genotype RH in the group of patients
Genotypes genotype frequency x2 Р
expected observed
A/A 40.45 42.98 0.181 0.24
А/G 46.30 41.23 0.634
G/G 13.25 15.79 0.554
Total 100.00 100.00 1.369
For the polymorphism rs231775 gene CTLA4 in the study group demonstrated a positive value relative deviation F = 0.1 with the observed frequency of the minor allele is lower than expected — 0.46 and 0.41 respectively. Polymorphism of CTLA +49 A/G is an important genetic factor associated with risk or protection against the development of various diseases and has an impact in the pathogenesis of autoimmune diseases. However, other closely linked candidate genes in equilibrium with the clutch CTLA4, such as CD28 and ICOS, may be associated with the development of autoimmune and infectious diseases [2]. Literary analysis results suggest that at position +49 CTLA4 gene can significantly increase the risk of chronic viral infection, while G in position +49 can positively affect viral clearance [3].
The effectiveness of the gene polymorphism rs231775 CTLA4 as an independent marker is low. SE is median corresponds to 0.57, the SP is equal to 0.33, and the assessment of the likelihood that this marker can be distinguished from a healthy patient the AUC, is very low and is equal to AUC = 0.45 (random token). 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 [7].
Studies on the analysis of the distribution of frequencies of alleles and genotypes of polymorphic variants show that homozygous AA genotype in patients meets 43.0 % (49/114), while the control group, 32.7 % (36/114), respectively. This chance ratio (x2 = 2.5; P = 0.1; OR = 1.5; 95 % CI 0.8993-2.67). Heterozygous AG genotype in the control group was 50.0 %, 47 cases, patients with less than 9 % (41.0 %) of 55 cases, respectively (x2 = 1.7; P = 0.2; OR = 0.7; 95 % CI 0.4138-1.189), results were statistically unreliable. Homozygous GG genotype in the control and in the group of patients met about the same amount, and statistical differences were found, was 17.3 % and 15.8 %, respectively. At the same time OR odds ratio was 0.6 (x2 = 2.5; P = 0.1; OR = 0.6; 95 % CI 0.3745-1.112), respectively.
Conclusion
The first studies of the effect of blocking CTLA-4 to enhance the immune response were carried out in 1995 by a group M. K. Jenkins [5; 6]. Based on the data obtained, the authors suggested that the use of blocking antibodies against CTLA-4 can reactivate the antigen-specific T-cell response in the tumor, contributing to its
destruction [7; 8]. The modern anti-cancer therapy is based on the targeted therapy or the new generation Immuno drugs that may enhance the anti-tumor immune response. The results show that the polymorphism rs231775 CTLA4 gene not found significant differences in the distribution of genotypes and allele frequencies between the main group and the control group. However, there is
a slight tendency to increase the number of heterozygous A/G and A allele of rs231775 * in the control group compared to patients (50.0 % and 41.2 %, respectively) that requires confirmation on larger sample of patients. Also, the study of interactive regulation of systemic inflammation cytokines to create new approaches treat hematological diseases.
2.
3.
4.
5.
6.
7.
10.
A/A A/G G/G
Fig. 1. The frequency of alleles and genotypes distribution of polymorphism A49G gene CTLA 4 in the groups of patients
References:
Боголюбова А. В., Ефимов Г. А., Друцкая М. С., Недоспасов С. А. Иммунотерапия опухолей, основанная на блокировке иммунологических контрольных точек//Медицинская иммунология. - 2015. - Т. 17, № 5. - С. 395-406.
Howard T. A., Rochelle J. M., Seldin M. F. CD28 and CTLA-4, two related members of the Ig supergene family, are tightly linked on proximal mouse chromosome//Immunogenetics. - 1991. - Vol. 33, no. 1. - Р. 74-76.
Ishida M., Iwai Y., Tanaka Y., Okazaki T., Freeman G.J., Minato N., Honjo T.//DiMed. - 2010. - Vol. 363, no. 8. - Р. 711-723. Клясова Г. А. Инфекция при гемобластозах и депрессиях кроветворения: клиника, диагностика и лечение. Автореф. - М., 2009. Weber G., Caruana I., Rouce R. H., Barrett A.J., Gerdemann U., Leen A. M. et al. Generation of tumor antigen-specific T cell lines from pediatric patients with acute lymphoblastic leukemia: implications for immunotherapy//Clin Cancer Res. - 2013. - 19: 5079e91. Bollard C. M., Aguilar L., Straathof K. C., Gahn B., Huls M. H., Rousseau A. et al. Cytotoxic T lymphocyte therapy for Epstein-Barr virusю Hodgkin's disease//J Exp Med. - 2004. - 200: 1623e33.
Bollard C. M., Gottschalk S., Torrano V., DioufO., Ku S., Hazrat Y. et al. Sustained complete responses in lymphoma patients receiving autologous cytotoxic T lymphocytes targeting Epstein-Barr virus latent membrane protein (published online ahead of print December 16, 2013)//J Clin Oncol.
Grupp S. A., Kalos M., Barrett D., Aplenc R., Porter D. L., Rheingold S. R. et al. Himeric antigen receptor-modified T cells for acute lymphoid leukemia//N Engl J Med. - 2013. - 368: 1509e18.
Fernández-Mestre M., Sánchez K., Balbás O. et al. Influence of CTLA-4 gene polymorphism in autoimmune and infectious diseases// Hum Immunol. - 2009, Jul. - 70(7): 532-535.
Schott E., Witt H., Pascu M., van Boemmel F., Weich V. Association of CTLA4 single nucleotide polymorphisms with viral but not autoimmune liver disease//Eur J Gastroenterol Hepatol. - 2007, Nov. - 19(11): 947-951.
Kamilova Umida,
Republican Specialized Scientific-Practical Medical Center of Therapy and Medical Rehabilitation JSC, Prof., Uzbekistan E-mail: [email protected] Usupov Donyor,
Fergana Branch of the Republican Emergency Care Research Center
Evaluation of endpoints in patients with myocardial infarction
Abstract: the aim of the study of evaluation of endpoints in patients with myocardial infarction. Determination of early predictors ofpoor prognosis in patients with myocardial infarction identifies patients at high cardiovascular risk and poor prognosis. Keywords: myocardial infarction, prognosis, endpoints.
