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"Child`s Health" 6 (49) 2013

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Genetic polymorphism asp299gly gene tol-like receptor 4 in children with helicobacter pylori infection

Authors: Abaturov A.Ye., Gerasymenko O.N. - State institution «Dnepropetrovsk Medical Academy of the Ministry of Health of Ukraine», Dnipropetrovsk, Ukraine; Shlykova O.A., Kaidashev I.P. - Higher State Educational Establishment of Ukraine «Ukrainian Medical Stomatological Academy», Research Institute for Genetics and Immunological Grounds of Pathology and Pharmacogenetics, Poltava, Ukraine

Categories: Family medicine/Therapy, Pediatrics/Neonatology

Sections: Clinical researches

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Keywords

helicobacter pylori infection, polymorphism asp299gly gene tlr 4, children.

Introduction: Nature of the inflammatory response and specific immunological reactions depends on differences in the genes that control organism protective reactions, especially gene regulatory molecules provide the first stages of the inflammatory response: pathogen recognition, activation of intracellular path of excitation and synthesis of inflammatory mediators.

Analysis of the human TLR4 gene identified 29 different SNP, most of which are located in the extracellular domain, providing the recognition of LPS, but only two of them are associated with changes in response to LPS function, primarily, SNP Asp299Gly. AG mutation at position 896 of a gene that encodes TLR4, leads to the substitution of asparagine by glycine at amino acid position 299 (Asp299Gly), changing the structure of the extracellular domain of TLR4).

The purpose of the study was to determine the characteristics of H. pylori infection in children with polymorphisms ASP299GLY of Toll-like receptor (TLR4) gene.

Methods. 31 children aged 9 to 17 years with chronic gastroduodenal pathology were observed. The control group included 95 healthy individuals.

Methods: endoscopy of the esophagus, stomach and duodenum was conducted for all children according to the standard technique of biopsy mucosa of antrum and immunological investigations that reflect the level of interest of H. pylori infection in the pathogenesis of the disease. Determination of expression level of TLR4 and of Asp299Gly TLR4 gene polymorphism was performed by polymerase chain reaction (PCR) in the Institute of Genetic and immune-based diseases and the development of pharmacogenetics (Poltava). The material for the study was peripheral venous blood. Genomic DNA was isolated using the kit for the DNA / RNA from serum or plasma (LitTeh, Russia). Gene polymorphism Asp299Gly TLR 4 was amplified by PCR in 25 l of reaction mixture containing 2.5 l of 10 x Buf amplification, 2 mM magnesium chloride, 0.2 mM of each dNTP; from 66 ng primer Asp299Gly:

F: 5'-GATTAGCATACTTAGACTACTACCTCCATG-3 '

R: 5'-GATCAACTTCTGAAAAAGCATTCCCAC-3 '

2.5 units. DNA Polymerase Tag; 20-50 ng of genomic DNA.

Method of enzyme-linked immunosorbent assay (ELISA) was used to estimate the concentration of serum sCD14, (ELISA test kit, Diaclone, France).

Results. Among children with chronic gastroduodenal pathology, 16.13% possessed heterozygous genotype Asp/Gly, which is higher than in the control group (P = 0.01). "Wild" genotype Asp / Asp was seen in 83.87% (26), which was not significantly different from the control group (OR = 0,11, P = 0,01). Patients with homozygous genotype for Gly alleles were not detected. Perhaps this is due to the small group studied, or can be the evidence of specific protection, the value of having such a protective genotype in the human genome and can be considered as a factor of stability in the development of chronic inflammation of the stomach.

It was shown that in H. pylori positive children the frequency of the heterozygous genotype Asp/Gly was significantly higher than in H. pylori negative patients (17.4% and 12.5%, respectively, OR = 1,48; P = 0,75). Among H. pylori-positive children heterozygous genotype Asp/Gly was detected more frequently in children infected with СagA-positive strains of H. pylori, compared with patients with СagA - negative status (p> 0.5). It is proved that in children with genotype Asp/Gly polymorphism of TLR 4 a decrease in the expression of TLR 4 in biopsy of the gastric mucosa and water-soluble sCD14 levels was observed, together with prominent inflammatory mucosal changes (Pu<0,05). The presence of children with heterozygous variant SNP Asp299Gly of TLR 4 gene (genotype Asp / Gly) probably causes a violation of TLR4 expression in the biopsy of the gastric mucosa, production of sCD14 and promotes invasion of H. pylori. It can also be assumed that the onset of the inflammatory process through the activation of other intracellular molecular pathways, perhaps, NOD-associated, occurs.

Conclusion. Among children with chronic inflammatory gastroduodenal diseases, genotype Asp / Gly SNP Asp299Gly of the gene TLR4 is observed significantly higher than in healthy population. Presence of polymorphism Asp299Gly TLR 4 gene of genotype Asp / Gly in children predisposes to infection with H. pylori.



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