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lergic rhinitis // Eur Arch 0torhinolaryngol.-2010.-267(3).-P.391-5
8. Kubach I.,Lutter P., Bopp T.,et al. Human CD4 CD25 regulatory T cells: proteome analysis identifies galectin -10 as a novel marker essential for their anergi and suppressive function // Blood.- 2007.-110(5).-p.1550-1558
9. Mou Z, Shi J, Tan Y. Et al. Association between TIM-1 gene polymorphisms and allergic rhinitis in a Han Chinese population \\ J Invest Allergol Clin Immunol.-2010.-20(1).-P.3-8.
English version: PREVALENCE OF HAPLOTYPES OF POLYMORPHIC GENES TLR 2, TLR 4, CLC-10 AND THEIR ASSOCIATION WITH SOME IMMUNOLOGICAL PARAMETERS IN PATIENTS WITH ALLERGIC RHINITIS*
Sakevych V. D.
State Higher School of Ukraine "Ukrainian Medical Stomatological Academy", Poltava
To determine the role of specific polymorphisms of genes that control the structural and regulatory elements nonspecific resistance of the organism in the development of allergic rhinitis prevalence determined polymorphism 2258G / A gene TLR2 (rs5743708), TLR 4 gene (rs4986790) and CLC-10 gene (rs420297) in the group surveillance and population control group, the analysis of immunological parameters and clinical manifestations in patients with polymorphic variants studied genes. To clarify a possible combination of different genotypes of all genes that define haplotype analyzes . When comparing the frequencies of hapiotypes defined by polymorphisms of genes TLR2 (rs5743708), TLR 4 (rs4986790) and CLC-10 (rs420297) in patients with AR and population control were found reiiable relationship between the presence of T allele genotype haiektynu -10 gene, allele A gene TLR 2 or TLR gene allele G 4 and the development of RA. In the study of the prevalence of possible combinations of genotypes TLR 4 gene (Asp299Giy) and CLC10 (rs420297)) found that the most common haplotype AASS as in the control group (30% ) and in patients with AR ( 24%) in patients with AR and population control were found reliable relationship between the presence of T allele genotype haiektynu -10 gene and allele G TLR 4 gene with the development of RA. In patients with AR haplotype carriers TLR4 / CLC 10 containing polymorphic alleles G and T were found significantly higher levels of expression of molecules CD4 CD25 Foxp3 MPER cells ( Mann- Whitney U (n = 4 ; n = 41) = 25,50; p = 0.024 ), which was 6,29 ± 0,50% (thousands / ml) of reducing the amount of IL -10 and IL -4 increase (55,9 ± 11,33 pg / mi). The results obtained can be considered gene TLR 4 SNPs (rs4986790) and CLC-10 (rs420297), as an additional prognostic indicator in pathogenetic studies.
Keywords: polymorphism, Toll-like receptors, halektyn-10 , allergic rhinitis.
Introduction
There is a tendency to a rapid spread of allergic diseases in the world, which become a serious issue because of the widespread, annual growth of widespread disease, frequent complications, as well as a sharp decline in performance and quality of life of patients.
AR is seen as a major problem of allergology , since the disease in most cases is the first clinical manifestation of atopy , followed by transformation of bronchial asthma (BA ) [4 ].
The study of the genetic basis of atopy is a key issue, which is necessary to establish the relationship between hereditary and environmental factors in the implementation of very complex pathological phenotype and understanding the mechanisms of interaction polygenic systems in the implementation of genetic information at the level of the whole organism . Knowing the molecular and cellular mechanisms of disease, we may identify genes, whose protein products are the most significant. According to studies , the genes of atopy and associated states are concentrated mainly in the 10 areas of the human genome .
According to studies , there are data about the association of allergic rhinitis with gene polymorphisms TIM-1 [9], SD14 [5], TLR 2-4 [6], RNA se 3 [7].
Specificity of the innate immune system is realized through family Toll- like receptors (TLRs). Important structural and molecular elements of the pattern - distinctive receptor (SPR) is a Toll- like receptor 2 (TLR 2) and Toll- like receptor 4 (TLR4). The genes encoding TLR2 and TLR4 show high variability in the population [1]. Recently the information has appeared about the identification of functional polymorphisms of genes TLR, due to the replacement of single nucleotides (from Eng. Single nucleotide polymorphism - SNP). As a result of these substitutions there is a reduction of ability to recognize appropriate ligands and efficiency of signal pulses, which leads to disruption of the activation of immune cells. A functional polymorphism of TLR 2-4 gives the regulation of innate immune response that is essential imbalance T1/T2-helpers. Similar mechanism may play a crucial role in the formation of chronic inflammation and has attracted attention as a potential risk factor for the development of atopic diseases, including AR [6].
In studies of foreign scientists the association of allergic rhinitis with gene polymorphisms halektyn -10 (CLC-
* To cite this English version: Sakevych V. D. Prevalence of haplotypes of polymorphic genes TLR 2, TLR 4, CLC-10 and their association with some immunological parameters in patients with allergic rhinitis - // Problemy ekologii ta medytsyny. - 2013. - Vol 17, № 5-6. - P. 20 -23.
npoSAeMH eKOAorii Ta MejHUHHH
10) is studied [3]. Halektyn -10 is representative of a family of endogenous lectins (known as lizofosfolipaza , protein Charcot - Leyden) , found in eosinophils and basophils. Exact studies have shown a significant intracellular expression of halektynu -10 to SD25+ Treg cells. In this connection, it does not participate directly in the suppression SD25+ Treg cells, but the specific blockade of halektyn -10 restores proliferative capacity SD25+ Treg cells and enhances their suppressive function. So halektyn -10 is necessary for the regulatory activity of Treg cells [8].
To determine the role of specific polymorphisms of genes that controls the structural and regulatory elements nonspecific resistance of the organism in the development of allergic rhinitis prevalence determined polymorphism 2258G / A gene TLR2 (rs5743708), TLR 4 gene (rs4986790) and CLC-10 gene (rs420297) in the group surveillance and population control group, the analysis of immunological parameters and clinical manifestations in patients with polymorphic variants studied genes.
Materials and methods.
To address the challenges put forward were examined 45 patients with AR aged 19 to 65 years (35,6 ± 1,57) (men accounted for 51 % (23 patients) , and women - 49% (22 patients )). At the time of the survey , patients were in clinical remission stage and stopped receiving allergy medications 72 hours , the patients had severe comorbidity.
Diagnosis is established based on the AR diagnostic criteria ARIA (2008) diagnostic algorithm adopted in Ukraine and approved by the Ministry of Health of Ukraine. Quality of life of patients was determined using generally recognized questionnaires (Adult Rhinocon-junctivitis Quality of Life Questionnaire).
Sensitization to allergens diagnosed on the basis of complex allergy diagnostic testing: collection allergic history, a positive skin scarificating test to allergens using standard sets (of "Immunologist ', Vinnitsa, Ukraine).
According to the standard procedure was conducted to determine the number of white blood cells in the blood and counting of blood cells in smears. Lymphocyte phe-notype was analyzed in venous blood using monoclonal antibodies to CD4, SD25 (production of "sorbent", Russia) and intracellular protein Foxp3 («Bioscience», USA) by flow tsytoflyuorymetriyi by flow tsytoflyuorymetra EPIX LX-MCL (Beckman Coulter, USA) using a program called System II TM software.
The levels of total IgE, interleukin-4 (IL-4) and inter-leukin-10 (IL-10) were determined using ELISA test kits (of "Ukrmed Don", Ukraine) using ELISA analyzer "Stat -Fax 2100" (USA).
To determine gene polymorphism rs420297 CLC-10 conducted the selection of genomic DNA from peripheral blood of examined using a set of "DNA Express-blood" (OOO NPF «Lyteh", Russia). Determination of gene polymorphism TLR2 (rs5743708) and TLR 4 (rs4986790) conducted by polymerase chain reaction [1].
Type allele (C \ T) CLC-10 gene was amplified using allele-specific polymerase chain reaction in 35 ml reaction mixture contained: 2.5 ml 10 x Buf for amplification, and 2 mM magnesium chloride, 0.2 mM of each dNTP , 2.5 units. Tag DNA polymerase with the addition of 5 pkmol specific primers: CLC_up 5'-CCC AGC AAC CAT
GCT TCT TGT TAC-3 '; CLC_low 5'-TGA GCA AAC CCA CCT-3' and 5 pkmol specific probes labeled with fluorescent dyes FAM and R6G from the 5 'end and BHQ-1, BHQ-2 from the 3' end, respectively:
CLC_wt (FAM) CG-CTG-GAG-GAA-CAG-GAA-AA (BHQ-1);
CLC_m (R6G) CG-CTG-GAG-GAA-CAA-GAA-AAA (BHQ-2)
Genomic DNA of examined of 20-50 ng was added to the mixture. Gene amplification halektynu-10 was performed on Thermocyclers detecting DT-322 (OOO "NPO DNA-technology", Russia) in real time, as follows:
- First cycle - 95°C / 3 minutes;
- 40 cycles - 95°C/15 seconds;
63°C/40 seconds
Products gene amplification CLC-10 were identified using fluorescent registration accumulation of DNA fluorescence channels: for the "wild" allele (wt): 1 channel -dye FAM, for the mutant allele (m): 2 - channel dye R6G, directly in the reaction.
The control group was consisted of 95 healthy individuals from a database of genetic samples SRI Genetic and immunological bases of pathology and pharmacogenetics VDNZU "UMSA." In the control group for the study of gene polymorphism SLC-10 were selected 45 DNA samples of persons not suffering from allergic diseases. The study was conducted in accordance with provided written consent to the inspection and conclusion of the Commission on ethical issues and bioethics Ukrainian Medical Dental Academy.
Mathematical analysis of the data was carried out using the program «STATISTICA 6.0» (StatSoft Inc). Comparison of genotype frequencies between the study groups was performed by analysis of contingency tables using Fisher's exact test. To compare allele frequencies used criterion x2. To assess the reliability of differences between groups using Fisher's exact two-sided test (for small groups). For all types of analysis considered differences statistically significant at p <0.05.
Results and discussion.
In the study of family allergic history in patients with AR were found various manifestations of allergy in the family in 76%. The presence of allergic diseases in relatives and II degree relatives of the mother was found in 35% of the father - in 30% of both parents - 11% of all patients examined in AR. There were no data on the burdened history of allergy in 24% of patients with RA. The results are consistent with data indicating preferential relationship with atopic diseases of the mother [2].
As noted above , as a result of observation of the dynamics of the disease in patients with AR were installed AR severity: mild course - in 11 (25%) , medium -heavy -in 32 (71%), heavy - in 2 (4%). Also was revealed the presence of genetic predisposition to allergic relatives and II degree relatives of the mother was found in 35% of the father - in 30% of both parents - 11% of all patients examined in AR. In 44% of the course AR has been associated with various nosological forms of allergic disease. In 20% of the patients on concomitant AR was established diagnosis of asthma, 15% present symptoms of AD, the full triad of atopy was found in 11% of patients surveyed by us in AR. With Allergic examination of patients with AR in 89% of patients were found positive skin
tests to pollen, fungal, household, epidermal and food allergens. Moreover, 7% occurred sensitization to one allergen group, 29% - to two groups, 36% - up to three groups, 13% - to the four groups, and 4% - of all five groups of allergens. In 11 % of patients had negative skin tests to all allergens used .
The distribution of haplotype frequencies polymorphism TLR 2,
To clarify a possible combination of different genotypes of all genes that define haplotypes were analyzed. We found that the most common haplotype GGAASS as in the control group (30%) and in patients with AR (23%) (Table 1).
Table 1.
TLR 4 and halektynu-10 gene among Poltava population and patients
with allergic rhinitis,% (n)
CLC10 TLR 4 TLR 2 GG AA CC GA AA CC C C < < A A GG AA CT GA AA CT AA AA CT GG AA TT GA AA TT AA AA TT GG AG CC GA AG CC AA AG CC GG AG CT GA AG CT AA AG CT GG AG TT GA AG TT AA AG TT GG GG CC GA GG CC AA GG CC GG GG CT GA GG CT AA GG CT GG GG TT GA GG TT AA GG TT
Group control (n=45) 66,7 (30) 4,4 (2) 0 17,8 (8) 2,2 (1) 0 2,2 (1) 0 0 4,4 (2) 0 0 2,2 (1) 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Patients in AR (n=45) 51,1 (23) 2,2 (1) 0 20,0 (9) 0 0 11,1 (5) 4,4 (2) 0 4,4 (2) 0 0 4,4 (2) 0 0 2,2 (1) 0 0 0 0 0 0 0 0 0 0 0
CLC10 CT/TT allele carriers T x2 2,45; BW = 2,26 (0,92-5,56); p = 0,118
TLR 4 896A/G AG/GG allele carriers G x2 0,49; BW = 2,15 (0,50-9,21); p = 0,482
TLR 2 2258G/A GA/AA allele carriers A x2 0,18; BW = 1,00 (0,19-5,24); p = 0,673
In comparison of the frequencies of haplotypes defined by polymorphisms of genes TLR2 (rs5743708), TLR 4 (rs4986790) and CLC-10 (rs420297) in patients with AR and population control were found reliable relationship between the presence of T allele genotype halek-tynu -10 gene, allele A gene TLR 2 or TLR gene allele G 4 and the development of RA.
However, the study of family allergic history in this group of patients with Ar found various manifestations of allergy in the family in 100%. As a result of observation of the dynamics of the disease in patients was found severity of AR (medium-difficult - 89%, mild course - 11%) according to the clinical form of AR (year-round (or persistent) - 100%). When Allergic to 100% of patients were found positive skin tests to pollen, fungal, household, epidermal and food allergens.
Analysis of the clinical course of disease in these patients showed the presence of comorbidity: frequent SARS that were more prolonged course and complications of the bronchopulmonary system (bronchitis, pneumonia) - 83%; polypous rhinosinusitis - 67% gastroduo-denitises - 50%.
In the analysis of immunological parameters including carrier haplotypes revealed a difference in the level of statistical trend (p < 0,06) in terms of CD4 / 25 / Foxp3 in carriers of the mutant allele haplotypes.
In order to better study and understanding of genetic predisposition to the emergence of AR reasonable is to study the prevalence of possible combinations of genotypes 4 and TLR genes CLC10 (Table 2).
Table 2.
Frequency distribution of haplotypes 4 and TLR polymorphisms halektynu-10 gene among^ Poltava
population and patients with allergic rhinitis,% (n)
CLC 10 TLR4 AA CC AA CT AA TT AG CC AG CT AG TT GG CC GG CT GG TT
Group control (n=45) 66,7 (30) 17,8 (8) 2,2 (1) 4,4 (2) 2,2 (1) 0 0 0 0
Patients in AR (n=45) 53,3 (24) 20 (9) 13,3 (6) 4,4 (2) 4,4 (2) 4,4 (2) 0 0 0
(CLC10) CT/TT, ( TLR 4) AG/GG X2 = 0,26; BW = 3,14 (0,31-31,42); p = 0,609
We found that the most common haplotype AASS as in the control group (30%) and in patients with AR (24%). When comparing the frequencies of haplotypes defined by polymorphisms of genes TLR 4 (rs4986790) and CLC-10 (rs420297) in patients with AR and population control were found reliable relationship between the presence of T allele genotype halektynu -10 gene and allele G TLR 4 gene with the development of RA.
For a more detailed assessment of cellular and humoral immunity in patients with AR depending on TLR4 polymorphisms and haplotypes CLC 10 immunological parameters were evaluated in patients with AR haplotype TLR4 CLC 10 (Table 3) in comparison with those of the patients in the AR that are not carriers of these haplotypes.
As noted earlier, the imbalance T1\T2 -helper cells with a predominance of T-2 immune response plays a major role in the development of RA. In response to exposure to allergens in patients with AR is the release of T2- induced cytokines and activation of eosinophils, mast cells and increased synthesis of IgE. There is information of mentioned reactions that occur indirectly via TLR4. Halektyn -10 does not directly take part in the suppression SD25+ Treg cells, but is necessary for the regulatory activity of Treg cells [ 8]. Immunological parameters were evaluated in patients with AR haplotype TLR4/CLC 10 (Table 3) in comparison with those of the patients in the AR which is not specified haplotype carriers .
npoSAeMH eKOAorii Ta MejHUHHH
Table 3.
Immunological parameters in patients with AR depending on TLR4 polymorphisms and haplotypes CLC 10
Index Indicators of healthy individuals Patients with AR haplotypes AG/(CT+TT), (n=4) Patients with RA, with haplotype AA+AG/CC+TT+CT (n=41)
L, 10a/n 4,0 - 8,8 4,72 ± 0,60 5,69 ± 0,33
Lymphocytes, % 18 - 40 28,75 ± 3,75 28,73 ± 1,19
Eosinophils, % 0 - 5 6,25 ± 1,03 4,10 ± 0,45
Total IgE, IU / ml 0 - 130 206,78 ± 5,08 197,41 ± 12,53
CD4+, % 39 ± 5 41,68 ± 3,14 40,43 ± 1,30
CD4+/CD25+,% 9,4 ± 2,05 13,83 ± 2,87 17,21 ± 2,10
CD4+CD25+Foxp3+,% 5-10% Big CD4+ 6,29 ± 0,50 4,5 ± 0,40
IL-10, pg / ml 0- 50 nr/Mn 0,34 ± 0,02 0,36 ± 0,02
IL-4, pg / ml 0-20nr/Mn 55,9 ± 11,33 49,84 ± 3,80
In the study of peripheral blood of patients with AR carriers listed haplotypes was revealed that the level of white blood cells averaged 4,72 ± 0,60 * 109 / L , that did not go beyond the parameters of healthy individuals (4.0 - 8.8 * 109 / l). The relative number of lymphocytes in the average of 28,75 ± 3,75%, which is also not beyond the parameters of healthy individuals (18 - 40%) revealed mild eosinophilia with an increase in the relative number of eosinophils that averaged 6,25 ± 1, 03 %. According to a survey of patients with AR haplotype carriers TLR4 CLC 10 immunological parameters in the expression of CD4 molecules in patients with AR had a tendency to increase, and the average was 41,68 ± 3,14% in terms of healthy subjects 39 ± 5% level expression of CD4 / CD25 was 13,83 ± 2,87%, higher than that of healthy people
(9,4 ± 2,05%). Indicator of the level of expression of CD4 CD25 Foxp3 molecules tended to increase ( on average was 6,29 ± 0,50%). In determining the level of total IgE in the average concentration in patients with AR haplotype carriers TLR4 CLC 10 was 206,78 ± 5,08 IU / ml at performance in healthy persons 0 - 130 IU / ml.
The content of serum IL-10 in patients with AR is averaged 0,34 ± 0,02 pg / ml, which is not higher than in healthy people 0 - 50 pg / ml; marked increase in the content of iL-4 and serum was 55, 9 ± 11,33 pg / ml at performance in healthy persons 0 - 20 pg / ml.
In order to detect differences in immunological parameters in patients with AR haplotype carriers TLR4 CLC 10 compared the respective groups using the MannWhitney (tab.4).
Tab.4
Differences in immunological parameters in patients with AR haplotype carriers TLR4 CLC 10
Index Patients with AR haplotypes TLR4 CLC 10 , (n=4) Patients with RA, (n=41)
CD4+CD25+Foxp3+,% 6,29 ± 0,50 4,5 ± 0,40
U, P U(n=4;n= 41) = 25,50; p=0,024
U, p - differences between groups by Mann-Whitney.
In clarifying the dependence of the level of expression of CD4 CD25 Foxp3 molecules in patients with AR Media TLR4 CLC 10 haplotypes observed in this group rate was statistically significantly higher (Mann-Whitney U (n = 4; n = 41) = 25.50; p = 0,024) and was 6,29 ± 0,50% (thousands / ml).
The results can considered gene TLR 4 SNPs (rs4986790) and CLC-10 (rs420297), as an additional prognostic indicator in pathogenetic studies.
Conclusions
1. When comparing the frequencies of haplotypes defined by polymorphisms of genes TLR2 (rs5743708), TLR 4 (rs4986790) and CLC-10 (rs420297) in patients with AR and population control were found reliable relationship between the presence of T allele genotype halek-tynu -10 gene, allele A gene TLR 2 or TLR gene allele G 4 and the development of RA.
2. In the study of the prevalence of possible combinations of genotypes TLR 4 gene (rs4986790) and CLC10 (rs420297)) was found that the most common haplotype AASS as in the control group (30%) and in patients with AR (24%) in patients with AR and population control were found reliable relationship between the presence of T allele genotype halektynu -10 gene and allele G TLR 4 gene with the development of RA.
3. In patients with AR haplotype carriers TLR4 / CLC-10 containing polymorphic alleles G and T were found significantly higher levels of expression of molecules CD4 CD25 Foxp3 MPER cells (Mann-Whitney U (n = 4; n =
41) = 25,50; p = 0.024), which was 6,29 ± 0,50% (thousands / ml) of reducing the amount of IL-10 and IL-4 increase (55,9 ± 11,33 pg / ml).
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9. Mou Z, Shi J, Tan Y. Et al. Association between TIM-1 gene polymorphisms and allergic rhinitis in a Han Chinese population \\ J Invest Allergol Clin Immunol.-2010.-20(1).-P.3-8.
Marepian HaaminoB go peaamii 3.12.2013
