RU2751594C1 - Method for detecting predisposition to comorbidity of pancreatic dysfunction in adults with arterial hypertension in conditions of blood contamination with benzene - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology. Blood samples are taken and the benzene content and the level of immunoglobulin G (IgG) to benzene are determined. A sample of the buccal epithelium is taken from the specified patient and deoxyribonucleic acid (DNA) is isolated from the specified sample. Genotyping of the eNOS gene polymorphism (rs1799983) and genotyping of the HLA-DRA gene polymorphism (rs3135388) are performed by PCR. With the simultaneous detection of diagnostic markers: the presence of a heterozygous GT genotype of the eM%* gene (rsl799983), the presence of a homozygous CC genotype of the gene (rs3135388), provided the presence of benzene in the blood is not lower than 0.00064 mg/dm3, as well as in excess of at least 2.0 times the level of IgG to benzene in comparison with the upper limit of the physiological norm, it is concluded that the patient is highly susceptible to comorbidity of pancreatic dysfunction associated with arterial hypertension in the patient under conditions of excessive blood contamination with benzene.EFFECT: invention ensures possibility of predicting the predisposition to the manifestation of comorbidity of pancreatic dysfunction in adults with arterial hypertension under conditions of benzene contamination through the use of genetic markers and the expansion of the number of information indicators.1 cl, 1 tbl, 2 ex

Description

The invention relates to the field of medical diagnostics and therapy, and can be used to predict a high degree of genetic predisposition to the manifestation of comorbidity of pancreatic dysfunction associated with the development of arterial hypertension in adults living in an area characterized by an increased content of benzene in the ambient air.

This method can be used for early detection of the likelihood of manifestations of comorbidity of the pancreatic pathology in patients with arterial hypertension living in unfavorable conditions of aerogenic exposure to benzene, as well as to increase the effectiveness of a set of preventive measures aimed at reducing the harmful effects of benzene on immune events manifested, for example , from the digestive system in the form of aggressive inflammatory reactions (chronic pancreatitis).

Benzene is a representative of the class of aromatic hydrocarbons and belongs to highly hazardous chemical substances (2nd hazard class according to GOST 12.1.005). A characteristic feature of this compound is a significant prevalence in the atmospheric air of populated areas and a high damaging ability when it enters the body, due to the formation of aggressive metabolites in the process of biotransformation.

The main route of benzene entering the body is inhalation. It should be borne in mind that benzene is a true xenobiotic and is identified in the biological media of the body only upon exogenous intake.

In the prior art, two approaches are known to diagnose predisposition to diseases of both the pancreas and arterial hypertension. One of them is to establish laboratory parameters, which are used to judge the possible risk of the disease, and the second way is genetic diagnosis through establishing the presence of genetic polymorphisms.

Considering that the present invention relates to the second type, below we present technical solutions known from the prior art, aimed at identifying a genetic predisposition to these diseases. It should be noted that the diagnosis of these comorbid diseases: disorders of the pancreas and arterial hypertension, medical genetics provides separately, i.e. not combined.

From the prior art (RF Patent No. 2732974), a method for differential diagnosis of diseases of the pancreas is known. At the stage of examination, the content of dehydroepiandrosterone sulfate - DHEA-S and 17-hydroxyprogesterone - 17-OHP is determined in the blood of a patient with pancreatic disease by the method of immunophenotyping. At the level of DHEA-S = 2.4 ± 0.2 and 17-OHP = 0.42 ± 0.40, the patient is diagnosed with a neuroendocrine tumor of the pancreas. When the level of DHEA-S = 3.7 ± 0.37 and 17-OHP = 1.9 ± 0.19, adenocarcinoma of the pancreas is diagnosed. At the level of DHEA-S = 6.42 ± 0.69 and 17-OHP = 2.8 ± 0.32, chronic pancreatitis is diagnosed. The method provides the possibility of differential diagnosis of diseases of the pancreas at the stages of primary examination.

Also from another RF Patent No. 2731693 a method for predicting the development of acute pancreatitis in residents of the Republic of Bashkortostan is known. When implementing the method, DNA is isolated from lymphocytes of peripheral venous blood, genotyping by allele-specific polymerase chain reaction of DNA synthesis. A polymorphic variant of the Arg47His gene of alcohol dehydrogenase ADH1B, a polymorphic variant of Glu487Lys of the gene of aldehyde dehydrogenase ALDH2 are investigated. If the Arg allele of the ADH1B gene is found in males and females of the Russian nationality, in females of the Russian, Bashkir and Tatar nationalities the Lys allele of the ALDH2 gene is predicted to have a high risk of developing acute pancreatitis.

However, these methods do not allow preliminary diagnosis in the early stages of comorbidity of pancreatic disease (hereinafter referred to as the pancreas) in a patient with arterial hypertension (hereinafter referred to as AH).

A method for predicting the development of a comorbid form of arterial hypertension (AH) and chronic obstructive pulmonary disease (COPD) is known from RF patent No. 2620566. In a patient with AH or COPD, the genotypes of the polymorphic locus M235T of the angiotensinogen gene (AGT) and the polymorphic locus A1166C of the type I receptor gene of agiotensin II (AGTR1) are determined. If a patient has the T / T genotype of the M235T polymorphic locus of the AGT gene and the A / C genotype of the A1166C polymorphic locus of the AGTR1 gene, a high probability of comorbid hypertension and COPD is predicted within 10 years after the onset of the first disease.

A method for determining the risk of developing arterial hypertension is also known from the prior art (RF Patent No. 2600442). This method involves sampling venous blood, isolating genetic material, carrying out polymerase chain reaction (PCR) in real time and determining the insertion / deletion (I and D alleles) of the Alu fragment of the angiotensin converting enzyme gene. The risk of developing arterial hypertension in those working in conditions of exceeding the noise level above the maximum permissible for genotype I / I of the angiotensin-converting enzyme gene is estimated below 50%, with genotype I / D - 50-70%, and with genotype D / D - above 70%. The invention provides an increase in the rate of determination of the risk of arterial hypertension in persons exposed to noise, and simplification of the method while increasing the degree of its reliability.

From the patent of the Russian Federation No. 2627443 a method is known for predicting the risk of arterial hypertension in the Shors - the indigenous inhabitants of mountain Shoria, who have renal dysfunction (PD). The method includes taking into account the age, body weight, waist circumference (OT), the ratio of the waist circumference to the thigh circumference (ITB), the presence of hyperuricemia, salt abuse, the level of total cholesterol (TC), the level of triglycerides (TG), the level of low-density lipoprotein cholesterol (LDL) -LPNP). Additionally, markers are determined: growth, albuminuria (AU), high-density cholesterol (HDL-C), genetic markers: genes of endothelial nitric oxide synthase (eNOS) systems, angiotensin-converting enzyme (ACE), methylenetetrahydrofolate reductase (MTHFR). Then, for each factor, the prognostic coefficient (PC) is set in points. The resulting PCs are summed up. If the PC sum is +6 points and above, a predisposition to the development of hypertension is predicted in the Shors - the indigenous inhabitants of Gornaya Shoria with renal dysfunction.

All of these known methods do not provide the diagnosis of a predisposition to comorbid forms of pancreatic dysfunction and arterial hypertension, which does not allow their use in these pathologies with their comorbidity.

At the same time, the prior art did not reveal the known methods for assessing the predisposition to the manifestation of comorbidity of pancreatic dysfunction associated with arterial hypertension in adults under conditions of benzene contamination, therefore, it is not possible to choose the closest analogue to the claimed object.

The technical result achieved by the present invention consists in providing the possibility of predicting a predisposition to the manifestation of comorbidity of pancreatic dysfunction in adults with arterial hypertension under conditions of benzene contamination, through the use of genetic markers, expanding the number of information indicators, as well as those associated with the complex use of the content in the blood toxicant - benzene.

The specified technical result is achieved by the proposed method for revealing a predisposition to the manifestation of comorbidity of pancreatic dysfunction in adults with arterial hypertension under conditions of blood contamination with benzene, according to which blood samples are taken from the patient, the benzene content and the level of immunoglobulin G (IgG) to benzene are determined in the sample, also, a sample of the buccal epithelium is taken from the specified patient, deoxyribonucleic acid (DNA) is isolated from the specified sample, then, by means of the polymerase chain reaction, genotyping of the eNOS gene polymorphism (rs1799983) is carried out, thus establishing the homozygous GG or heterozygous GT state of the genotype for the specified gene, and genotyping of the HL4-D7L4 gene polymorphism (rs3135388) is carried out, while establishing the homozygous CC or heterozygous CT state of the genotype for the specified gene, and with the simultaneous detection of the following diagnostic markers: the presence of heterozygous G T genotype of the eNOS gene (rs1799983), the presence of a homozygous CC genotype of the HLA-DRA gene (rs3135388), provided that the presence of benzene in the blood is not lower than 0.00064 mg / dm ³ , as well as if the level is not less than 2.0 times higher IgG to benzene compared with the upper limit of the physiological norm, it is concluded that the patient is highly susceptible to comorbidity of pancreatic dysfunction associated with arterial hypertension in adults under conditions of excessive blood contamination with benzene.

The achieved technical result is ensured by the following.

At present, much attention is paid to the problem of studying the influence of harmful chemical factors on the formation of public health. For example, the occupational morbidity of oil industry workers is caused by a complex of unfavorable factors of the working environment, including air pollution of the working area with harmful chemical substances of the 1st and 2nd hazard classes. The presence of a combined action of production factors (hard physical labor, exposure to chemicals, increased neuro-emotional stress) is the main cause of comorbid conditions, including hypertension and pancreatic pathology. Evaluation of the genotoxic effect of an aromatic hydrocarbon such as benzene has shown that occupational exposure to these products is a source of oxidative stress and DNA damage and plays a role in the benzene-mediated carcinogenic effect (pancreatic cancer). The same effects are applicable, in addition to oil workers, and for other people living in an environmentally unfavorable territory.

The task of studying the influence of anthropogenic chemical factors on the formation of morbidity in the urbanized population under the conditions of its deterministic genetics is extremely urgent today. Such diseases can be monogenic or polygenic. For their implementation, it is not enough just the corresponding genetic constitution of the individual - you also need a factor or a complex of environmental factors that trigger the formation of a phenotype mediated by a specific genotype. Thus, with the participation of the environmental factor, a hereditary predisposition is realized.

Hypertension is considered one of the reasons for the development of the pathology of the digestive system. Often, an increase in blood pressure provokes pancreatitis, an inflammatory disease that occurs in the pancreas. Based on this, it should be understood that the process of normalizing the digestive system should begin with the elimination of the root cause, an increase in blood pressure that entailed hypertension, in this case - with the treatment of the specified disease - hypertension.

Hypertension develops against the background of an improper diet, when people withstand long breaks between meals, abuse salty, spicy food, consume a lot of strong tea and coffee. This leads to fluid retention in the body and vasospasm, as well as to severe pain in the pancreas, affecting pressure. Sometimes hypertension is diagnosed against the background of pancreatitis, if severe disorders have occurred in the pancreas, resulting in a failure in the production of substances necessary to maintain normal blood sugar levels. And vice versa, the occurrence of pressure surges occurs due to intoxication with decay products.

In turn, the chronic inflammatory process in the pancreas - pancreatitis, develops for a long time, due to dystonia of the vessels feeding the pancreas and, above all, those mapped in the area of the sphincter apparatus of the parenchymal organ. Chronic inflammation in the gland can also occur as a result of long-standing vascular diseases - atherosclerosis and hypertension.

Carrying out studies of the polymorphism of candidate genes in comorbid conditions makes it possible to establish not only the etiological genetic factor, but also to analyze the possible mechanism of disease formation in an anthropogenically altered environment, which is important for both early diagnosis and correction of such conditions. Many diseases with a genetic predisposition are manifested in the form of syndromes or a complex of pathological signs, seemingly unrelated to each other, which is determined by the multifunctionality of the genes encoding these signs.

These polyfunctional genes include the genes for endothelial NO synthase (eNOS), as well as the major histocompatibility complex (HLA-DRA) gene, which are associated with diseases of the vascular system, as well as the pancreas.

Nitric oxide (NO) is the main endothelial relaxation factor involved in maintaining the tone of the vascular wall, thrombogenesis. The wild allelic variant of this gene leads to a decrease in the level of expression of NO synthase and, as a consequence, to a decrease in the body's resistance to hypertensive influences from the external and internal environment. The enzyme nitric oxide synthase (NOS) synthesizes a metastable free radical nitric oxide, which causes vasodilation, disrupts oxygen transport and leads to a deterioration in the oxygen status of the body, NO also stimulates platelet sequestration, increases the permeability of various membranes, causes metabolic and structural damage to endothelial cells, is cytotoxic effect.

In addition to its central role as a vasodilator, nitric oxide performs the function of a neurotransmitter and plays an important role in the long-term potentiation of neuronal memory, inhibits the adhesion of blood cells to the endothelium.

The gene of the main histocompatibility complex HLA-DRA - a large region of the genome responsible for the preservation of biological individuality, plays a vital role in the formation of exocrine, endocrine and autoimmune pathology, in particular, the pancreas, which in the case of overexpression of the gene leads to the formation of not only the pathology of the tail region pancreas and diabetes, but also aggressiveness towards its exocrine function, determining the course of pancreatitis. Genetic vascular and autoimmune compartments of the pathogenesis of the inflammatory process, including in the pancreas, are triggered by the permissive action of external anthropogenic chemical factors, including aromatic hydrocarbons such as benzene.

The HLA-DRA is one of the HLA Class II Alpha chain paralogs. This class II molecule is a heterodimer composed of alpha and beta chains attached to a membrane. It plays a central role in the immune system, presenting peptides derived from extracellular proteins.

It is recommended to use the heterozygous GT state of the genotypes of the endothelial NO synthase gene eNOS (rs1799983), which is responsible for the formation of endothelial synthase type 3, creating conditions for conversion of nitric oxide into stable NO-metabolites, in particular the formation of endogenous nitrite; however, the conditions of exogenous exposure to benzene block the conversion of nitrates into a stable form. The formation of redundancy of aggressive unstable oxides determines the start and maintains of inflammatory processes in the pancreas, translating them into a chronic course. The developing insufficiency of the enzymatic functional of the pancreas affects digestion with the formation of an excess of easily digestible carbohydrates and causes the risk of an atherosclerotic process and hypertonicity phenomena, accelerated by additional haptenic exposure to aromatic hydrocarbons.

At the same time, homozygous CC variants of the genotype of the gene of the main histocompatibility complex HL4-DRA (rs3135388), which is responsible for the functioning of cellular formations of innate immunity, are associated with hyperergic immune events manifested from the digestive system in the form of aggressive inflammatory reactions (chronic pancreatitis). An increase in the indicator characterizing the very phenomenon of specific sensitization according to the criterion of IgG content to benzene, as well as an increase in the level of nitric oxide, as a realization of a genetic defect of nitric oxide synthase, serves as a confirming factor of immune hyperergy.

Thus, the combination of the polymorphism of the genes of the major histocompatibility complex HLA-DRA (CC genotype) and endothelial NO synthase eNOS (GT heterozygous genotype) are pathogenetically significant for the realization of the predisposition to the development of comorbid conditions - pancreatic diseases associated with arterial hypertension in conditions of excessive congestion. the patient's biological environment with benzene.

Based on the foregoing, it can be concluded that the delivered technical result is ensured by the combination of operations of the proposed method, their sequence and modes of its implementation.

The proposed method is implemented as follows.

1. On the territory with a high content of benzene in the atmospheric air (0.3 mg / m3 is the standard in the atmospheric air, it is 1 MPC _s.s. ) in environmental objects, patients were selected, of the same ethnic population, permanently residing in this territory ...

2. A whole blood sample was taken from them into special tubes with an anticoagulant (trizol was used to stabilize RNA, and EDTA (Ethylenediaminetetraacetic acid) (0.05M EDTA solution in a ratio of 500 μl of blood to 50 μl of anticoagulant) was used to isolate blood cells and studying markers of cell differentiation.

3. In the whole blood of patients, the content of benzene was determined by the traditional method of gas chromatographic analysis “Method of gas chromatography with flame ionization detection. Equipment: chromatograph "Chromatek-Crystal 5000.2"

4. Also, a sample of the buccal epithelium (in the form of a smear from the buccal mucosa) is taken from the specified patient. After taking the material, the swab (the working part of the probe with a cotton swab) is placed in a sterile Eppendorf tube with 500 μl of transport medium (sterile 0.9% NaCl solution). Close the tube with the solution and the working part of the probe.

Next, DNA is extracted from the sample. For this, samples in an amount of 100 μl are lysed with 300 μl of lysis solution, which is a 0.5% solution of sarcosyl and proteinase K (20 mg / ml) in acetate buffer (pH 7.5). Then a sorbent (kaolin) is added and by successive washing procedures the samples are washed with phosphate-saline buffer (pH 7.2) from proteins and with a mixture of isopropyl alcohol: acetone to remove lipids. In this case, the nucleic acids remain on the sorbent. Next, the DNA adsorbed on the sorbent from the sample are extracted with a TE buffer, which is a mixture of 10 mM Tris-HCl and 1 mM EDTA (pH 8.0). The extract is centrifuged. After centrifugation of the tube, the supernatant contains purified DNA.

The resulting material is ready for polymerase chain reaction (PCR). PNR is carried out on a detecting amplifier with hybridization-fluorescence detection in "real time" using ready-made sets of primers and probes produced by CJSC Syntol, Russia, in which DNA regions of the eNOS (rs1799983) and HL4-DRA8 (rs313538 ).

An amplification reaction is carried out, this is achieved by preparing a reaction mixture for the study of the allelic state of each gene in an individual patient. 0.1 μl of the prepared mixture of primers (a genetically accepted term denoting the final nucleotides with a label, limiting (cutting off) the amplified nucleotide chain of a gene) and probes for the selected eNOS genes (rs1799983) and (rs3135388) (reagent kits used for determination of the polymorphism of these genes (CJSC "Syntol", Russia). The remaining components necessary for PCR are added to each tube: nucleotides (deoxynucleoside triphosphates: 10 mM dATP, dTTP, dGTP, dCTP each), buffer (100 mM Tris-HCl buffer, 500 mM KCl, 40 mM MgCl ₂ ) and Tag F- polymerase. The sample is added in the amount of 10 μl. Thus, the total volume of the reaction mixture is 25 μl. Each tube is tightly closed with a stopper and placed in the thermocycler.

When carrying out PCR, amplification and detection are carried out on a detecting amplifier CFX96 from Bio-Rad.

A universal amplification program is used, selected by the manufacturer of the reagents. It includes several stages: Stage 1 - activation of TaqF-polymerase (“hot start” mode) lasts 15 min at 95 ° C; Stage 2 - setting cycles of amplification without measuring fluorescence (5 cycles); Stage 3 - working cycles of amplification with measurement of fluorescence (40 cycles).

Each amplification cycle includes DNA denaturation (5 s at 95 ° C), primer annealing (20 s at 60 ° C), and the DNA polymerization reaction itself (15 s at 72 ° C).

Registration of the fluorescence signal arising from the accumulation of amplification products of DNA regions is carried out in "real time" after the stage of annealing of primers for selected genes via the VIC channel to detect one of the allelic gene variants, and via the FAM channel for an alternative variant.

The results are interpreted based on the presence (or absence) of the intersection of the fluorescence curve with the set threshold line, which corresponds to the presence (or absence) of the threshold cycle value (N) in the corresponding column in the results table displayed in the software for the CFX96 amplifier.

According to the ratio of the threshold cycles obtained by two channels of detection, the state of the gene in the investigated DNA region is determined (the method of allelic discrimination). There were two possible variants of the gene state: homozygous - in the case when one of the values of the threshold cycle is not determined (below the threshold line) and heterozygous - in the case when two values of the threshold cycles were obtained and parabolic fluorescence curves were obtained through these channels. Depending on the accumulation of which amplification product occurs in the reaction, a heterozygous, or normal homozygous, or variant homozygous state of the gene is established.

5. And if the following conditions are met: the presence of a heterozygous GT genotype of the eNOS gene (rs1799983), the presence of a homozygous CC genotype of the HLA-DRA gene (rs3135388), the presence of benzene in the blood of at least 0.00064 mg / dm ³ , as well as in excess of at least than 2.0 times the level of IgG to benzene compared to the upper limit of the physiological norm, it is concluded that the patient is highly susceptible to comorbidity of pancreatic dysfunction associated with arterial hypertension under conditions of excessive benzene contamination.

When conducting tests for the implementation of the proposed method, a survey of the population was carried out under various conditions for the content of benzene in bisreds (blood).

Two groups were formed: an observation group, adults with the presence of benzene in the blood, and a comparison group with a low or no benzene content in the blood. The observation group included 56 men (mean age 43.1 ± 0.7 years). The comparison group included 61 men (mean age 45.4 ± 0.8 years). The study groups were comparable in terms of age, gender, ethnic composition, comorbidity, socioeconomic level, quality and nutritional composition.

On the territory of residence of the observation group, an excess of the benzene content in the air of more than 1 MPC d.w. was noted (0.3 mg / m3 standard in the atmospheric air - 1 MPC d.w.).

A blood sample was taken from all examined adults, the study of which was carried out according to the above scheme. A sample of the buccal epithelium was also taken to establish the polymorphism of the genes under study by the PCR reaction.

To prove the reliability of the declared criteria for diagnosing a patient's predisposition to comorbidity of pancreatic dysfunction associated with chronic arterial hypertension under conditions of excessive benzene contamination, the content of nitric oxide was established in a blood serum sample (according to the method of "Quantitative determination of nitric oxide in the culture medium, serum, plasma and urine "by spectrophotometric analysis, Cat.N KGE001" BioChemMac ", Moscow) and IgG to benzene. These indicators are a confirming factor of excessive loading (contamination) with benzene associated with a feature of the genotypes of the eNOS and HLA-DRA genes, the polymorphism of which is involved in the formation of combined pathology of the pancreas and hypertension.

The data obtained as a result of the research are shown in table 1.

The data given in the specified table 1 show that with the simultaneous combination of the heterozygous state of the GT genotype of the eNOS gene (rs1799983), the homozygous state of the CC genotype of the allele of the HLA-DRA gene (rs3135388), the concentration of the “guilty” factor (benzene) in the blood is not lower than 0 , 00064 mg / dm ^3, and in excess of not less than 2.0 times the level of IgG to benzene as compared with the upper limit of the physiological norm (0-0.1 units), patients there is an increase, and more than 10% compared to the upper limit of the physiological norm, the content of nitric oxide (patients 1-3). And this means that at the same time, the implementation of a negative genetic program in a person by benzene is recorded, which is expressed in the risk of comorbidity in the form of pancreatic pathology associated with arterial hypertension in a patient. All these patients had persistent hypertension, and after 6-12 months of medical observation, signs of dysfunction of the pancreas appeared, manifested by painful sensations in the left hypochondrium, supplemented by palpitations, excessive sweating, general weakness, and dyspeptic disorders.

Whereas in the absence of benzene in the blood, even under the conditions of inheritance of the genotypes of the indicated genes GT eNOS (rs1799983) and CC HLA-DRA (rs3135388), the IgG level and the content of nitric oxide are within the physiological norm (patient 4), which proves the inducing effect precisely benzene to the indicated genes.

In the case of the presence of benzene in the blood less than 0.00064 mg / dm ³ , but in the presence of a homozygous GG variant of the eNOS gene (rs1799983) and at the same time a heterozygous CT variant of the HLA-DRA gene (rs3135388) (patient 5), an excess of IgG level and oxide content nitrogen in the blood above the upper limit of the physiological norm is less than 10%, which is evidence that with these sets of genes the comorbidity of these states is not realized.

In patients with homozygous genotypes of both of these genes GG eNOS (rs1799983) and CC HLA-DRA (rs3135388) and an absent level of benzene in the blood, the level of nitric oxide is within the physiological norm (patients 10, 12-13).

In patients 7 and 8, who are characterized by the content of benzene in the blood and at the same time have inheritance of the heterozygous genotype of both of these genes, the conclusion about a predisposition to dysfunction of the pancreas in the presence of hypertension was not diagnosed, while the level of IgG and the content of nitric oxide are within the normal range , which indicates the absence of the necessary genetic conditions (predisposition) for the implementation of the existing impact in the form of a comorbid disorder. The same picture is observed in the absence of benzene in the blood (patients 6 and 9).

Moreover, it should be noted that all patients from both observation and comparison groups were under medical supervision. As a result, it was found that in patients, even with the presence of hypertension, but with the absence of the simultaneity of the declared diagnostic criteria, no dysfunction of the pancreas was detected during the observation period.

To illustrate the implementation of the proposed method, two examples are given for specific patients of the same age and ethnicity from the group with an increased content of benzene in the blood and with the absence of benzene in the blood.

Example 1. Patient, 46 years old, Russian, works as an operator of a well installation, suffers from a constant high level of blood pressure (AH) - systolic - 140-160 mm Hg. The level of benzene in the blood was set at 0.00085 mg / dm ³ . The specified patient was found to have a heterozygous GT genotype of the eNOS gene (rs1799983) and a homozygous CC genotype of the HLA-DRA gene (rs 3135388). The value of the IgG content in the blood to benzene is 0.201 c.u., i.e. 2 times higher than the upper limit of the norm. The content of nitric oxide in the blood - a marker of disorders of the endothelium of the vascular bed and the sphincter apparatus of the pancreas, 208.5 μmol / l, i.e. above the normal range (70.4-150 μmol / l). Taking into account the presence of a combination of these genetic markers together with the presence of benzene in the blood and a high level of IgG to benzene, it was suggested that there is a possibility of comorbidity of pancreatic dysfunction against the background of the course of hypertension. After 6 months, during the physical examination, the patient was found to have periodic pain in the form of girdle pain in the left hypochondrium after eating. The performed ultrasound examination (US) showed a slight change in the echostructure of the pancreas, which made it possible to establish a dysfunction of the pancreas. To clarify the diagnosis, a medical observation of this patient was carried out first on an outpatient basis, then in an inpatient department of gastroenterology. It was found that during the observation the specified patient had 6 episodes of exacerbation of chronic pancreatitis, which required inpatient treatment. Thus, the accuracy of the set of diagnostic criteria of the proposed method in predicting the predisposition of such a patient to comorbidity of pancreatic dysfunction (pancreatitis) in combination with the presence of arterial hypertension was proved.

This proves that in this patient the condition from exposure to benzene is assessed as the formation of manifestations of pancreatic pathology associated with hypertension, with a genetic predisposition.

Example 2. Patient, 45 years old, Russian, no pathology of the pancreas was detected, the presence of rare increases in systolic blood pressure up to 135 mm Hg.

The content of benzene in the blood is 0.00035 mg / dm ³ . The presence of heterozygous GT genotypes of the eNOS gene (rs1799983) and the CT genotype of the HLA-DRA gene (rs 3135388) was established. The value of the IgG content to benzene is 0.1 c.u., i.e. the upper limit of the physiological norm and there is no excess. The content of nitric oxide in the blood is -144.4 μmol / l, i.e. in the area of the upper limit of the normal range (70.4-150 μmol / l).

Thus, the absence of the simultaneous presence of the heterozygous GT genotype of the eNOS gene (rs1799983) and the homozygous CC gene HLA-DRA (rs 3135388), along with the absence of an increased IgG level even against the background of the presence of benzene in the blood, does not lead to the development of negative effects in the form of disorders with side of the pancreas, although there are some deviations from the functioning of the cardiovascular system in the form of an episodic increase in blood pressure above 140 mm Hg.

Thus, the given data show that when the proposed method is implemented using the proposed criteria, its purpose is ensured.

The inventive method allows to establish with sufficient reliability the precursors of pancreatic pathology occurring against the background of arterial hypertension (AH), associated with the influence of benzene, according to the claimed genetic criteria, the content of benzene in the blood and the level of IgG to benzene, and to start preventive measures in advance.

Claims (1)

A method for detecting a predisposition to the manifestation of comorbidity of pancreatic dysfunction in adults with arterial hypertension under conditions of blood contamination with benzene, characterized by the fact that blood samples are taken from the patient, the benzene content and the level of immunoglobulin G (IgG) to benzene are determined in the sample, also in the specified a sample of the buccal epithelium is taken from the patient, deoxyribonucleic acid (DNA) is isolated from the specified sample, then the eNOS gene polymorphism (rs1799983) is genotyped by the polymerase chain reaction, while the homozygous GG or heterozygous GT state of the genotype is established for the specified gene, and the polymorphism is genotyped gene (rs3135388), while establishing a homozygous CC or heterozygous CT state of the genotype for the specified gene, and simultaneously detecting the following diagnostic markers: the presence of a heterozygous GT genotype of the eNOS gene (rs1799983), the presence of a homozygous CC genotype of the HLA-DRA gene (rs3135388), subject to the presence of benzene in the blood at least 0.00064 mg / dm ³ , as well as if the level of IgG to benzene is at least 2.0 times higher than the upper limit of the physiological norm, the conclusion is drawn about a high degree of the patient's predisposition to the manifestation of comorbidity of pancreatic dysfunction associated with arterial hypertension in adults, in conditions of excessive blood contamination with benzene.