RU2574015C1 - Method for prediction risk of chronic true eczema development in males - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention aims to predict the risk of chronic true eczema (CTE) among Russian male individuals, natives of Central Black Earth Region of the Russian Federation A genomic DNA is recovered from peripheral venous blood, and t loci are analysed by a polymerase chain reaction. If observing -308A TNFα allele or -308GA TNFα genotype, or +1663G TNFR2 allele or +1663GG TNFR2 genotype, or a combination of +250G Ltα and +36A TNFR1, or -308A TNFα and +36A TNFR1, or +250G Ltα and +1663G TNFR2, or -308G TNFα and +1663G TNFR2, or -308A TNFα and +250G Ltα, or -308G TNFα and +250G Ltα and +1663G TNFR2, or -308A TNFα and +250G Ltα and +36A TNFR1, or -308A TNFα and +250G Ltα and +1663G TNFR2, the high risk of the CTE development is predicted; -308GG TNFα genotype, or +1663AA TNFR2 genotype, or a combination of +250A Ltα allele and +1663AA TNFR2 genotype, or a combination of -308GG TNFα and +250A Ltα, or -308GG TNFα and +1663A TNFR2, or +36G TNFR1 and +1663A TNFR2 shows the low risk of the CTE development.

EFFECT: invention ensures generating new criteria of the risk of the CTE development.

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Description

The invention relates to the field of medical diagnosis, can be used to predict the risk of developing chronic true eczema in males.

True eczema is a ubiquitous, recurring skin disease that is characterized by polymorphism of morphological elements and occurs at any age [Clinical Dermatovenerology: a guide for doctors: in 2 volumes / ed. Yu.K. Skripkina, Yu.S. Butova. - Moscow: GEOTAR-Media, 2009. - T. 2. - 921 p.]. The name of this disease can be explained by the property of vesicles to quickly open, like bubbles of boiling water (in Greek eczeo - to boil). An important sign of acute eczema is the presence of grouped, numerous and rapidly opening small vesicles that form serous “wells”, similar to vesicles on the surface of boiling water [Ospanova S.A. Improving the treatment of eczema taking into account endogenous intoxication: co-author. dis. ... cand. honey. Sciences: 14.00.11 / С.A. Ospanova; Kazakh. scientific researcher leather.-venerol. Institute of Health Care Rep. Kazakhstan. - Almaty, 2008. - 21 p.].

The etiological and pathogenetic aspects of the development of eczema, highlighted in modern literature, are controversial. At different stages of the development of studies on eczema, neurogenic and allergic theories, the role of the endocrine system, and hereditary factors were given great importance in the etiology and pathogenesis of eczema. It should be noted that the etiology and pathogenesis of eczema are extremely complex and in many of their aspects remain unexplored [Kosheleva I.V. The effectiveness of the complex treatment of patients with eczema using various methods of ozone therapy / I.V. Kosheleva, A.G. Kulikov // Issues of balneology, physiotherapy and physiotherapy. - 2001. - No. 5. - S. 40-42].

A significant role in the pathogenesis of eczema is played by changes in the state of prostaglandins and cyclic nucleotides, which occupy a major place in intracellular regulatory mechanisms. They mediate neuroendocrine information, turn it into a specific cell response and realize normal and pathological reactions of the body [Arushanyan EB The therapeutic possibilities of melatonin and its effect on immunological parameters in patients with eczema / E.B. Arushanyan and J. M. Alli Absi, V.V. Chebotarev // Experimental and clinical pharmacology. - 2003. - T. 66, No. 3. - S. 59-61].

Prostaglandins E1 and F201, cyclic adenosine monophosphate and guanosine monophosphate have a regulatory effect on the development of allergic and inflammatory reactions, the functional activity of the body's immune system. There are antagonistic relationships between prostaglandins E1 (PGE1, cyclic adenosine monophosphate (cAMP) and prostaglandins F2a, cyclic guanosine monophosphate (cGMP). thereby contributing to the development of allergic and inflammatory reactions [Burova SA Immunomodulators in the practice of a dermatologist / S.A. Burova, GN Makova // Actual issue Therapy for Sexually Transmitted Infections and Chronic Dermatoses, Yekaterinburg, May 30-31, 2002: abstract of the scientific and practical conference - Yekaterinburg, 2002. - P. 39. In patients with eczema, an increase in the concentration of PGE1 was established. and PHF2a in blood plasma, however, an increase in PHPa is more significant, which leads to a change in the ratio of PGE1 / PHF2 (predominantly PHF2a) and the formation of a specific deficiency of PGE1 [Degtyar Yu.S. Peculiarities of the hormonal status in patients with eczema and other dermatoses in the North / South. FROM. Degtyar, L.K. Dobrodeeva // Bulletin of Dermatology and Venereology. - 2000. - No. 5. - S. 50-53].

The established biochemical and immune disorders made it possible to develop a new concept of the pathogenesis of eczema. In individuals with a hereditary predisposition, which is confirmed by a positive association of histocompatibility system antigens (B22 and Cw1), the synthesis of prostaglandin F2a is enhanced, which stimulates the production of cyclic guanosine monophosphate, which activates the synthesis of serotonin, histamine and other allergy mediators, which contributes to the development of inflammatory and allergens vascular permeability [Degtyar Yu.S. The state of the immune status in patients with eczema in the European North of Russia / U.S. Degtyar, L.K. Dobrodeeva // Bulletin of Dermatology and Venereology. - 2001. - No. 1. - S. 44-46].

Simultaneously with an increase in the formation of prostaglandin F2a, the synthesis of prostaglandin E1 increases, but its concentration is significantly reduced in relation to the increasing concentration of prostaglandin F201. The lack of prostaglandin E1, a violation of its ratio with the content of prostaglandin F2a leads to insufficient stimulation of the synthesis of cyclic adenosine monophosphate, which inhibits the formation of allergic and inflammatory reactions, the development of allergy mediators [Dikova O.V. The state of endotoxemia in patients with eczema / O.V. Dikova, S.V. Selivanova, E.Yu. Kochetkova // Bulletin of postgraduate medical education. - 2001. - No. 1. - S. 31].

Thus, in patients with eczema, as a result of an increase in the F2a content and a violation of the ratio of PGE1 / PHF2a and cAMP / cGMP, there is a predominance of prostaglandin F2a and cyclic guanosine monophosphate, which is one of the causes of the disease [Mazitova L. Modern aspects of the pathogenesis of curing allergic dermatoses in children / L.P. Mazitova // Russian Medical Journal. - 2001. - T. 9, No. 11. - S. 457-460].

In platelets of peripheral blood of patients with eczema, an increase in the synthesis and excretion of serotonin into the bloodstream has been established. The platelet release reaction, as a result of which serotonin enters the bloodstream, is regulated by prostaglandins. Prostaglandin E1 suppresses it, and prostaglandin F2a provokes [Skin and sexually transmitted diseases: Ref. family doctor / ed. K.N. Monakhova. - Moscow; St. Petersburg: DILA, 2005. - 159 p.]. The prevalence of prostaglandin F2a causes an increase in blood serotonin, which exacerbates allergic reactions. The thyroid hormone thyrocalcitonin stimulates the activity of adenylate cyclase, an enzyme that catalyzes the synthesis of cyclic adenosine monophosphate from ATP. An increase in the content of thyrocalcitonin in patients with eczema may be a manifestation of the protective reactions of the body.

Prostaglandins and cyclic nucleotides are factors in the regulation of the immune response. Their increased synthesis leads to changes in immunological reactivity and causes profound immunological disorders, which are manifested in the suppression of cellular immunity (the content of T-ROCK, quantitative and functional indicators of T-helpers and T-suppressors are reduced, the suppressor activity of T-lymphocytes is suppressed) and non-specific defense factors (decreases the number of complementary and spontaneous neutrophils, the content of cytoplasmic granules of neutrophilic leukocytes, the percentage of phagocytosis) [L. Strachunsky Pharmacoepidemiology: Fundamental Concepts and Practices. application / L.C. Strachunsky, S.N. Kozlov, S.A. Rachina // Clinical Pharmacology and Therapy. - 2001. - T. 10, No. 4. - S. 48-53]. Changes in immune status increase allergic reactivity and, along with cyclic nucleotides and prostaglandins, lead to the formation of eczema. The severity of imbalance in prostaglandins, cyclic nucleotides and immunological reactivity is reflected in the severity of the process and clinical manifestations.

According to modern concepts, in the development of eczema, the main role is played by T-lymphocytes, which carry specific antigen receptors on their surface and secrete a number of pro-inflammatory cytokines: IL-1, IL-2, TNF-α [Pavlova OV Eczema: etiology, pathogenesis, clinic, diagnosis and treatment / O.V. Pavlova. - Moscow: URSS Librocom, 2010. - 61 p.].

The release of biologically active substances causes the development of tissue reactions of inflammation, which is clinically manifested by an allergic reaction in the form of edema, hyperemia, and itching. Antigenic stimulation of Th1 leads to the synthesis of IL-2, with IL-2, which produces the ability of CD4 cells, in patients is higher than in healthy people.

Proinflammatory cytokines induce the expression of adhesion molecules on endothelial cells and leukocytes, as a result of which, through transendothelial migration, the influx of leukocytes from the vascular bed into the foci of inflammation is intensified [Intеrеukin-16 stimulates theexpression and human receptors. L. Mathy, W. Schaeuer, M. Lanzendörfer [et al.] // Immunolgy. - 2000. - Vol. 100, No. 1. - P. 63-69]. Further accumulation and promotion of immunocompetent cells in the focus of inflammation is controlled by chemokines, which are produced by endothelial cells and macrophages. Cellular infiltrate in the focus of inflammation, consisting of macrophages, neutrophils and eosinophils, contributes to the development of allergic inflammation. Polymorphic skin infiltrate in eczema is the result of the action of the resulting pro-inflammatory cytokines, including TNF-α.

Thus, Th1 lymphocytes, macrophages, endothelial cells and cytokines secreted by them are involved in delayed-type allergic reactions that underlie the formation of true eczema.

According to most researchers, eczema should be attributed to multifactorial diseases with a polygenic type of inheritance with a threshold effect. With the combination of certain genetic defects and environmental factors, a threshold value is reached at which the clinical picture of the disease appears. There are a large number of phenotypically healthy people with a high risk of the formation of allergic diseases in adverse environmental conditions.

To assess the current patent situation, a search was performed on the title documents for the period from 1990 to 2013. The analysis of documents was carried out in the direction: a method for predicting the risk of developing chronic true eczema in male individuals based on molecular genetic data depending on polymorphic gene markers. No similar patents have been identified.

German scientists investigated the effects of polymorphisms -238G / A TNFα and -308G / A TNFα on the development of dermatitis of the hands. The results of the work showed that in patients with dermatitis of the hands, the maximum concentration of the genotype -308GA TNFα was established (31.20%), which is statistically significantly higher than the control indicators (24.60%, OR = 1.33 95%, CI 1.05-1, 74). This molecular genetic marker is a risk factor for the development of dermatitis of the hands. The genetic factor -238GA TNFα (5.60%) in patients compared to the control (8.5%, OR = 0.57–95%, CI 0.34–0.97) serves as a protective factor for the development of dermatitis of the hands.

The article identified in the scientific and medical literature that describes a method for predicting the risk of developing chronic true eczema (hereinafter referred to as CIE) in male individuals based on data on genetic polymorphism + 250A / G Ltα [Association of lymphotoxin polymorphism α (+250 A / G Ltα) with the formation of chronic true eczema [Electronic resource] / Ya.E. Denisova, M.I. Churnosov // Modern problems of science and education. - 2014. - No. 3. - Access mode: http://www.science-education.ru/117-13677]. The method includes venous blood sampling, isolation of genomic DNA from peripheral blood, and analysis of the +250 A / G Ltα locus by polymerase chain reaction (hereinafter PCR) of DNA synthesis. The + 250GG Ltα genotype is a risk factor for the development of this disease in men, and the + 250A Ltα allele is a protective factor.

The disadvantage of the prototype is that it does not take into account the influence on the development of CIE of other genetic polymorphisms and their combinations.

The objective of this study is to expand the arsenal of diagnostic methods, namely the creation of a method for predicting the risk of CIE in male individuals according to data on genetic polymorphisms and their combinations.

The technical result of using the invention is to obtain criteria for assessing the risk of developing chronic true eczema in male individuals of Russian nationality, natives of the Central Black Soil of the Russian Federation, according to genetic polymorphisms -308G / A TNFα, + 250A / G Ltα, + 36A / G TNFR1 and + 1663A / G TNFR2 and combinations thereof.

In accordance with the task, a method for predicting HIE was developed, including:

- venous blood sampling,

- isolation of genomic DNA from peripheral blood,

- analysis of the locus + 250A / G Ltα by polymerase chain reaction of DNA synthesis,

which has the following new features:

- analysis of gene polymorphisms of tumor necrosis factor α genes (-308G / A TNFα), type 1 tumor necrosis factor receptor (+ 36A / G TNFR1) and type 2 tumor necrosis factor receptor (+ 1663A / G TNFR2);

- predict an increased risk of developing CIE in male individuals of Russian nationality, natives of the Central Black Earth Region of the Russian Federation, in the presence of the -308A TNFα allele or the -308GA TNFα allele, or the + 1663G TNFR2 allele or the + 1663GG TNFR2 genotype, or a combination of + 250G Ltα alleles 36A TNFR1, or a combination of the -308A TNFα c alleles + 36A TNFR1, or a combination of the + 250G Ltα c + 1663G TNFR2 alleles, or a combination of the -308G TNFα c + 1663G TNFR2 alleles, or a combination of the -308A TNFα alleles, or a combination of +30G Ltα alleles, or -308G TNFα s + 250G Ltα s + 1663 G TNFR2, or a combination of alleles -308A TNFα with + 250G Ltα s + 36A TNFR1, or a combination of alleles -308A TNF with a + 250G Ltα + 1663G TNFR2;

- predict a reduced risk of developing CIE in male individuals of Russian nationality, natives of the Central Black Earth Region of the Russian Federation, in the presence of the genotype -308GG TNFα, or the genotype + 1663AA TNFR2, or the combination of the + 250A Ltα allele + + genotype 1663AA TNFR2, or a combination of the TNFR230GG genotype -308G with the + 250A Ltα allele, or a combination of the -308GG TNFα genotype with the + 1663A TNFR2 allele, or a combination of + 36G TNFR1 alleles with + 1663A TNFR2.

The novelty and inventive step consists in the fact that for the first time the involvement of genetic polymorphisms of tumor necrosis factor α (-308 G / A TNFα), tumor necrosis factor receptor type 1 (+36 A / G TNFR1) and tumor necrosis factor receptor 2- type (+ 1663А / G ТNFR2) and their combinations, including with lymphotoxin α (+ 250A / G Ltα), in the formation of chronic true eczema in male individuals of Russian nationality, natives of the Central Black Earth Region of the Russian Federation, which will increase the reliability of the forecast .

The invention is characterized by:

- 308АА TNFα,

- 308GG TNFα,

- 308GA TNFα, ♦ - контрольный образец.Figure 1. Allele discrimination by the method of detecting TaqMan probes according to the values of the relative fluorescence level (hereinafter UOF) of each probe on an IQ5 amplifier with a real-time detection system of the locus -308 G / A TNFα, where

- 308AA TNFα,

- 308GG TNFα,

- 308GA TNFα, ♦ - control sample.

- +250GG Ltα,

- +250AA Ltα,

- +250AG Ltα, ♦ - контрольный образец.Figure 2. Allele discrimination by the detection method of TaqMan probes according to the UOF values of each probe on an IQ5 amplifier with a real-time detection system of the locus +250 A / G Ltα, where

- + 250GG Ltα,

- + 250AA Ltα,

- + 250AG Ltα, ♦ - control sample.

- +36AA TNFR1,

- +36GG TNFR1,

- +36AG TNFR1, ♦ - контрольный образец.Figure 3. Allele discrimination by the detection method of TaqMan probes according to the UOF values of each probe on an IQ5 amplifier with a real-time detection system of the locus +36 A / G TNFR1, where

- + 36AA TNFR1,

- + 36GG TNFR1,

- + 36AG TNFR1, ♦ - control sample.

- +1663GG TNFR2,

- +1663AA TNFR2,

- +1663AG TNFR2, ♦ - контрольный образец.Figure 4. Allele discrimination by the detection method of TaqMan probes according to the UOF values of each probe on an IQ5 amplifier with a real-time detection system of the locus + 1663A / G TNFR2, where

- + 1663GG TNFR2,

- + 1663AA TNFR2,

- + 1663AG TNFR2, ♦ - control sample.

FIG. 5, in which the table shows the prevalence of combinations of certain alleles / genotypes of cytokine genes in patients with CIE and in males of the control group.

In figures 1-4, two bands, vertical and horizontal, divide the graph into four sections: one for each homozygous state, one for the heterozygous state and the section without reaction. Assigning genotypes to unknown samples is determined by plotting the relative fluorescence level for one fluorophore on the x axis, relative to the UVR for another fluorophore on the y axis on the allele discrimination diagram. A probe with a fluorescent dye ROX corresponds to allele A, a probe with a FAM dye corresponds to allele G.

• If the UVR values of an unknown sample are above the horizontal strip and to the right of the vertical strip, the genotype is heterozygous (GA).

• If the PFV of an unknown sample is above the horizontal strip and to the left of the vertical strip, the genotype is homozygous for the A allele (the PF of the A allele is plotted along the y axis).

• If the PFV of an unknown sample is below the horizontal strip and to the right of the vertical one, the genotype is homozygous for the G allele (PF of the G allele is plotted along the x axis).

• If the UVR values of an unknown sample are below the horizontal strip and to the left of the vertical, genotype determination is not possible: in this case, an undefined sample is a negative control.

The method is as follows:

Genomic DNA is isolated using the standard phenol-chloroform extraction method. Stage 1: 25 ml of lysis buffer, which contains 320 mM sucrose, 1% Triton X-100, 5 mM MgCl ₂ and 10 mM Tris-HCl (pH = 7.6), is added to 4 ml of blood [Mathew C. C ., 1984]. The resulting mixture at 4 ° C, 4000 rpm for 20 minutes is stirred and centrifuged. After centrifugation, the supernatant is drained, 4 ml of the solution are added to the resulting precipitate, with 25 mM EDTA (pH = 7.8) and 75 mM NaCl, then resuspended. After that, 0.4 ml of 10% SDS, 35 μl of proteinase K (10 mg / ml) are added and the sample is incubated for 37 hours at 37 ° C.

At the 2nd stage, for 10 minutes at 4000 rpm, DNA is sequentially extracted from the obtained lysate in equal parts of phenol, phenol-chloroform (1: 1) and chloroform with centrifugation. The selection of the aqueous phase after each centrifugation. DNA was precipitated from the solution in 2 volumes of chilled 96% ethanol. The resulting DNA is dissolved in deionized, double-distilled water and stored at t = -20 ^° C. The isolated DNA is used for the polymerase chain reaction (PCR) of DNA synthesis.

Analysis of -308G / A TNFα, + 250G / A Ltα, + 36A / G TNFR1, + 1663A / G TNFR2 is carried out by PCR synthesis of DNA on CFX 96 with real-time PCR system. Thermus aquaticus DNA polymerase (manufacturer Sileks-M) and oligonucleotide primers and probes (synthesized by Syntol) are used.

The reaction mixture (25 μl volume) includes: 67 mM Tris-HCl (pH = 8.8), 2.5 mM MgCl ₂ , 0.1 μg of genomic DNK, 10 pM of each primer, 5 pm of each probe, 200 mkM dATP, dGTP, dSTP, dTTP and 1 unit of active Taq polymerase. For each pair of primers, the optimal temperature and time conditions of annealing are calculated and the corresponding concentration of MgCl _{2 is} selected.

Genotyping is carried out using the Tag Man method of probes based on the data of the relative fluorescence level (hereinafter referred to as UOF) for each probe during PCR in the IQ5 amplifier.

The possibility of using the proposed method for assessing the risk of developing chronic true eczema is confirmed by an analysis of the results of the examination of 205 men: 84 patients with CIE and 121 individuals in the control group.

The studied groups included persons of Russian nationality, who were natives of the Central Black Soil of the Russian Federation and had no kinship with each other. Clinical, clinical and laboratory examination of patients was carried out on the basis of the outpatient department of OGBUZ “Skin and venereologic dispensary”.

The average age of patients with chronic true eczema was 45.91 ± 2.68 (varied from 18 to 86 years), and the control sample was 43.62 ± 3.46 (varied from 20 to 76 years) (p> 0.05). Therefore, the group of patients did not differ from the control group by age, place of birth and nationality.

In order to solve the problem of multiple comparisons associated with obtaining false positive results (error of the first kind), the Bonferroni correction is introduced, i.e. recalculate the significance level p for multiple paired comparisons according to the formula: p _cor = p · n, where p is the obtained level of statistical significance, n is the number of paired comparisons. For a statistically significant level, p _cor ≤0.05. The analysis of associations of combinations of genetic variants with CIE is carried out using the APSampler software (http://sources.redhat.com/cygwin/), using the Monte Carlo method of Markov chains and Bayesian nonparametric statistics. The p level obtained after the permutation test p _rrm ≤0.05 (100 permutations performed) is taken as statistically significant.

As a result of the study, in the group of men the concentration of the -308GG TNFα genotype (62.03%) is lower than in the control group, where this indicator was 79.83% (χ ² = 6.72, p = 0.01, p _cor = 0.03, OR = 0.41 95% CI 0.21-0.82).

It was also found that among patients with CIE, the genotype frequency + 1663AA TNFR2 (19.18%) is lower than in the control group, where this indicator was 39.17% (χ ² = 7.49, p = 0.007, p _cor = 0.021, OR = 0.37 95% CI 0.17-0.77).

Thus, the obtained data indicate that the genotypes -308GG TNFα (OR = 0.41) or + 1663АА TNFR2 (OR = 0.37) have a protective value.

As a result of a comprehensive analysis of the carriage of combinations of alleles and genotypes of the studied loci of tumor necrosis factors and their receptors, a number of significant differences were revealed between 84 men with CIE and 121 men in the control group (table in figure 5). All four examined genetic polymorphisms -308G / A TNFα, + 250A / G Ltα, + 36A / G TNFR1, + 1663A / G TNFR2 are involved in the formation of 12 significant combinations of genetic variants that distinguish men with CIE from men in the control group.

It should be noted that 4 associations of combinations (OR = 0.32-0.45) have a protective orientation with the formation of chronic true eczema.

- the combination of the + 250A Ltα allele with the + 1663AA TNFR2 genotype (combination A in the table in Fig. 5) (OR = 0.32 95% CI 0.16-0.66). In 14.29% of patients with CIE, this combination of genetic markers is observed, while in the control group it was detected in 38.02%.

- a combination of the -308GG TNFα genotype with the + 250A Ltα allele (combination B), which is observed in 52.38% of patients and 77.69% of the population control. The level of statistical significance of differences in the frequencies of combinations of these genetic markers between patients and control is also quite high (p _perm = 0.002). The odds ratio for combination B is 0.34 (95% CI 0.18-0.65).

- a combination of the -308GG TNFα genotype with the + 1663A TNFR2 allele (combination C). The odds ratio for this combination of polymorphic variants is 0.35 with a 95% confidence interval of 0.19-0.65 (p _perm = 0.002). Combination C occurs in 64.46% of men in the control group and only in 34.52% of men with CIE.

- combination of the + 36G TNFR1 allele with the + 1663A TNFR2 allele (combination D). The odds ratio for this combination of polymorphic variants is 0.45 with a 95% confidence interval of 0.25-0.81 (p _perm = 0.002). Combination D occurs in 63.64% of men in the control group and 38.09% of men with CIE.

As a result of the study, a group of men revealed differences in the distribution of molecular genetic markers at the -308G / A TNFα loci and + 1663A / G TNFR2 between the control and patients with chronic true eczema. So, among patients with CIE, the frequencies of the -308A TNFα allele (19.62%) and the -308GA TNFα genotype (36.71%) were higher than in the control group, where these indicators were 10.50% (χ ² = 5.77 , p = 0.02, OR = 2.08 95% CI 1.13-3.83) and 19.33% (χ ² = 6.54, p = 0.01, p _cor = 0.03, OR = 2.42 95% CI 1.21-4.86), respectively. It was also found that among patients with CIE, the concentrations of genetic variants + 1663G TNFR2 (59.59%) and + 1663GG TNFR2 (38.35%) were higher than in the control group, where these indicators were 40.83% (χ ² = 12.06, p = 0.001, OR = 2.14 95% CI 1.38-3.32) and 20.83% (χ ² = 6.14, p = 0.01, p _cor = 0.03, OR = 2.36 95% CI 0.18-4.75), respectively.

Thus, the data obtained indicate the involvement of the genetic markers -308G / A TNFα and + 1663A / G TNFR2 in the formation of susceptibility to chronic true eczema in men. Factors for an increased risk of developing CIE in men are -308A TNFα (OR = 2.08), or -308GA TNFα (OR = 2.42), or + 1663G TNFR2 (OR = 2.14), or + 1663GG TNFR2 (OR = 2.36).

It should be noted that 8 combinations are risk factors for the development of CIE in men (OR = 2.18-3.89).

Significant differences were found in the concentrations of combinations of + 250G Ltα genotypes with + 36A TNFR1 (combination E) between men with CIE (46.43%) and control men (31.40%). This combination is a risk factor for the development of CIE (p _perm = 0.02, OR = 2.18, 95% CI 1.21-3.93). There were also differences in the distribution of combinations of two genetic markers -308A TNFα with + 36A TNFR1 (combination F) between men with CIE (30.95%) and men in the control group (15.70%). This combination of polymorphic gene variants is also a risk factor for the development of chronic true eczema (p _perm = 0.01, OR = 2.68, 95% CI 1.36-5.30).

Differences were found in the frequency of combinations of + 250G Ltα alleles with + 1663G TNFR2 (combination G) between men with CIE and control men. The concentration of this combination of genetic variants in the control group is 23.97%, while among sick men this indicator is 40.48% (p _perm = 0.002, OR = 2.81 95% CI 1.51-5.24).

The combination of genetic markers, which is observed in 69.05% of men with CIE and 59.50% of men in the control group, includes a combination of two genetic markers: -308G TNFα with + 1663G TNFR2 (combination H) (p _perm = 0.01). This combination is a risk factor for the development of CIE (OR = 2.85, 95% CI 1.41-5.77).

Combination I, which is observed in 33.33% of sick men and 15.70% of men in the control group, includes a combination of two genetic factors: -308A TNFα with + 250G Ltα. The level of statistical significance of differences in the frequencies of combinations of these genetic markers between sick men and the control is also quite high (p _perm = 0.01). The odds ratio for combination I is 2.95 (95% CI 1.50-5.78).

The risk factors for the development of CIE are combinations of genetic factors J, K and L. The combination of J, represented by the combination of the -308G TNFα alleles with + 250G Ltα with + 1663G TNFR2, is found in men of control (23.14%) 1.75 times less than among patients with CIE (40.48%, p _perm = 0.001), the odds ratio is 3.04 (at 95% CI 1.61-5.71).

Also, in men of the control group, combinations of K and L are significantly less frequent (1.36 and 1.25 times, respectively) compared with patients with CIE. The concentrations of these combinations of genetic variants in control men are 12.40% and 9.09%, respectively, whereas among sick men, these indicators are 29.76% (p _perm = 0.002) and 23.81% (p _perm = 0.002), respectively . The odds ratios for these combinations are 3.40 and 3.89.

Thus, summarizing the results obtained, it can be concluded that male individuals of Russian nationality, natives of the Central Black Earth Region of the Russian Federation have combinations of all four analyzed genetic polymorphisms of cytokines -308G / A TNFα, + 250A / G Ltα, + 36A / G TNFR1 and + 1663A / G TNFR2 determines the predisposition to the occurrence of CIE. They form 12 significant combinations of genetic variants of tumor necrosis factors and their receptors associated with the development of CIE. Moreover, 4 combinations have a protective focus (OR = 0.32-0.45), and 8 combinations are risk factors for the development of CIE (OR = 2.18-3.89).

Examples confirming the feasibility of the proposed invention.

1. In the male patient A., the -308A TNFα allele was detected. Upon further observation, chronic true eczema occurred in this patient.

2. In male patient B., the genotype -308GA TNFα was detected. Upon further observation, chronic true eczema occurred in this patient.

3. In the male patient U., the + 1663G TNFR2 allele was detected. Upon further observation, chronic true eczema occurred in this patient.

4. In the male patient K., the genotype + 1663GG TNFR2 was detected. Upon further observation, chronic true eczema occurred in this patient.

5. In patient Male N., a combination of + 250G Ltα with + 36A TNFR1 was detected. Upon further observation, chronic true eczema occurred in this patient.

6. In male patient D., a combination of -308A TNFα with + 36A TNFR1 was detected. Upon further observation, chronic true eczema occurred in this patient.

7. A combination of + 250G Ltα with + 1663G TNFR2 was found in male patient Sh. Upon further observation, chronic true eczema occurred in this patient.

8. A combination of -308G TNFα with + 1663G TNFR2 was detected in male O. Upon further observation, chronic true eczema occurred in this patient.

9. A combination of -308 A TNFα with + 250G Ltα was detected in male patient B. Upon further observation, chronic true eczema occurred in this patient.

10. A combination of -308G TNFα, + 250G Ltα with + 1663G TNFR2 was detected in male patient E. Upon further observation, chronic true eczema occurred in this patient.

11. A combination of -308A TNF, + 250G Ltα with + 36A TNFR1 was detected in male male L. Upon further observation, chronic true eczema occurred in this patient.

12. The male patient P. showed a combination of -308A TNFα, + 250G Ltα with + 1663G TNFR2. Upon further observation, chronic true eczema occurred in this patient.

13. A male genotype -308GG TNFα was found in male patient I. With further observation of this patient, the occurrence of chronic true eczema was not detected.

14. In male patient F., genotype + 1663AA TNFR2 was detected. With further observation of this patient, the occurrence of chronic true eczema was not detected.

15. A combination of + 250A Ltα with + 1663АА TNFR2 was found in male patient J. With further observation of this patient, the occurrence of chronic true eczema was not detected.

16. A combination of -308GG TNFα with + 250A Ltα was found in male male N. With further observation of this patient, the occurrence of chronic true eczema was not detected.

17. A combination of -308GG TNFα with + 1663A TNFR2 was found in male C. male patient. With further observation of this patient, the occurrence of chronic true eczema was not detected.

18. A combination of + 36G TNFR1 with + 1663A TNFR2 was found in male patient Ch. With further observation of this patient, the occurrence of chronic true eczema was not detected.

The application of this method will allow for timely implementation among male individuals of Russian nationality, natives of the Central Black Earth Region of the Russian Federation who are at risk, the necessary preventive measures to prevent the development of chronic true eczema: the exception of too frequent hand washing, contact with irritating detergents, cleaning products, the use of delicate soap and moisturizing the skin.

Claims (1)

A method for predicting the risk of chronic true eczema in male individuals of Russian nationality, natives of the Central Black Earth Region of the Russian Federation, including the isolation of genomic DNA from peripheral blood and analysis of the + 250A / G Ltα locus by the method of polymerase chain reaction of DNA synthesis, characterized in that they additionally analyze genetic -308G / A TNFα polymorphisms, + 36A / G TNFR1, + 1663A / G TNFR2 and their combinations predict an increased risk of CIE in the presence of the -308A TNFα allele or the -308GA TNFα genotype or the + 1663G TNF allele R2 or genotype + 1663GG TNFR2, or a combination of + 250G Ltα c + 36A TNFR1 alleles, or a combination of -308A TNFα c + 36A TNFR1 alleles, or a combination of + 250G Ltα c + 1663G TNFR2 alleles, or a combination of -308G TNFF cR 1663 alleles , or a combination of -308A TNFα alleles with + 250G Ltα, or a combination of -308G TNFα c + 250G Ltα alleles with + 1663G TNFR2, or a combination of -308A TNFα c + 250G Ltα alleles with + 36A TNFR1, or a combination of -308A TNFα c + 250G Ltα s + 1663G TNFR2; predict a reduced risk of CIE in the presence of the -308GG TNFα genotype, or + 1663AA TNFR2 genotype, or a combination of the + 250A Ltα allele with the + 1663AA TNFR2 genotype, or a combination of the -308GG TNFα genotype with the + 250A Ltα allele, or a combination of the TNF α -830GG and 308GF + 1663A TNFR2, or a combination of the + 36G TNFR1 allele with the + 1663A TNFR2 allele.

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