RU2700565C1 - Method for determining predisposition to developing obesity in children in conditions of excessive contamination with aluminum - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to paediatrics and endocrinology, and aims at determining predisposition to developing obesity in children in conditions of excessive contamination with aluminum. Blood and urine samples are taken in a child. Urine sample is used to determine content of aluminum, and blood serum – IgG level to aluminum. DNA of buccal epithelium is extracted. On the detecting amplifier using polymerase chain reaction in real time genotyping of MTNR1B (rs 10830963) and SIRT1 (rs 7069102) gene polymorphism is carried out. At simultaneous detection of the following diagnostic criteria: presence of variant homozygous or heterozygous genotypes of gene MTNR1B (rs 10830963) and gene SIRT1 (rs 7069102) under condition of simultaneous excess of aluminum concentration in urine by more than 2 times compared to reference level of aluminum content in urine, as well as when the physiological norm of IgG level to aluminum is exceeded in comparison with the upper limit, a conclusion is made on the child's propensity to develop obesity in conditions of excessive contamination with aluminum.

EFFECT: invention provides reliable assessment of aluminum effect on predisposition of manifestations of endocrine disorders in the form of obesity.

1 cl, 2 tbl, 2 ex

Description

The invention relates to medicine, namely to pediatrics, endocrinology, and can be used for early diagnosis and prediction of the toxic effects of aluminum on the manifestation of obesity and overweight in children. The invention is intended to identify the adverse effects of aluminum from the environment on the health of a child living in areas with a high content of this toxicant in ambient air and in drinking water.

The invention can be used to make a preliminary diagnosis both in specialized clinics for the examination of patients, and in conventional healthcare institutions. The results of these examinations are necessary for the development of individual prevention programs, and in addition, they can be used in the formation of sanitary and hygienic measures to prevent and eliminate the effects of harmful chemicals, in particular aluminum, which causes the formation of obesity (metabolic syndrome) in children.

The relevance of the proposed method is determined by the fact that an increased level of aluminum in the environment can cause pathology of the endocrine system.

Due to the widespread use of chemical technologies in various production cycles and areas of human activity, the attention of scientists and physicians is attracted by the problem of identifying threshold criteria that allow us to assess at an early stage that the exposure to a harmful chemical exceeds the compensatory capabilities of the body and harms human health, especially children, in connection with the fully formed compensatory capabilities. The relevance of the study of the adverse effects of aluminum is especially determined in the territories near which non-ferrous metallurgy enterprises are located.

For the tasks of assessing the degree of environmental impact on health, early diagnosis of health disorders, and also for choosing effective prevention, it is important to highlight marker indicators of specific diseases or their precursors in humans under the influence of aluminum, which can be used as indicators of endocrine disruption and as additional diagnostic criteria for the sanitary-epidemiological situation.

(48,1% случаев в структуре эндокринной заболеваемости). У детей, проживающих в условиях повышенного содержания алюминия, установлена достоверная причинно-следственная связь вероятности развития заболеваний эндокринной системы и избыточного питания с повышенным содержанием алюминия в моче (доля дисперсии зависимой переменной R²=0,47-0,7; 261,5≤F≥672,4; достоверность р=0,001).According to the results of epidemiological studies, in the Russian Federation the prevalence of overweight in children in different regions of Russia ranges from 5.5 to 11.8%, and about 5.5% of children living in rural areas and 8.5% of children are obese. - in the city. Obesity and excess body weight (according to the International Classification of Diseases ICD-10: E66.0-E67.8) in recent years for the first time came first and amounted to

(48.1% of cases in the structure of endocrine morbidity). In children living in conditions of high aluminum content, a reliable causal relationship was established between the likelihood of developing diseases of the endocrine system and excess nutrition with a high content of aluminum in the urine (the proportion of the variance of the dependent variable R ² = 0.47-0.7; 261.5≤ F≥672.4; significance p = 0.001).

Obesity - excess fat deposition in the body, can be either an independent multifactorial disease, or a syndrome that accompanies other diseases.

According to the recommendations of nutrition experts, as a highly informative indicator of overweight and obesity, the body mass index (hereinafter - BMI) or the Ketle index, defined as the ratio of body weight (in kg) to height (in meters), squared, is used. In relation to adults, obesity is said when their body mass index is greater than or equal to 30 kg / m ² (Obesity problem in the WHO European Region and strategies for its solution / Edited by F. Branca, H. Nikogosian, T. Lobsteim. - European regional WHO Bureau, 2007 - S. 1-7). BMI for adults and for children is calculated a little differently. Additional parameters when calculating the index for children are the age and gender of the child. Normal BMI in children is less than in adults - from 15 to 18.5, depending on age. This is due to the fact that children and adults have different body proportions and, accordingly, the ratio of height and weight.

Various prognostic approaches to establishing possible obesity and overweight are known in the art. Some of them are based on the use of blood indicators as diagnostic markers and, more rarely, genetic criteria.

For example, there is a method for predicting the progression of abdominal obesity in patients with metabolic syndrome, in which the level of C-peptide is determined in the blood (RF Patent No. 2297002). With its value of more than 3.5 ng / ml, the progression of abdominal obesity is predicted. However, this known method takes into account the risk of progression of obesity only in cases of metabolic syndrome and does not consider such a probability long before metabolic disorders, which reduces its prognostic value and the possibility of use in the preclinical period.

Also known is a method for differential diagnosis of exogenous constitutional and hypothalamic forms of primary obesity in women (RF Patent No. 1442189), according to which the dynamics of prolactin secretion for the administration of the neuropeptide luliberin is studied by determining the initial level of the hormone in the venous blood sample and subsequent intravenous administration of luliberin in a dose 100 mcg once. Then, blood is examined for prolactin at 30, 60, 90 and 120 minutes after administration of the drug. And with an increase in the level of secretion compared to the initial one, a conclusion is made about the exogenously constitutional form of obesity, and at a stable level in comparison with the initial one, the hypothalamic form of primary obesity is determined. The method is aimed at making the correct diagnosis in controversial cases.

The disadvantages of this method is that this technique only clarifies the clinical diagnosis, without determining the likelihood of developing disorders of carbohydrate and fat metabolism associated with exposure to negative environmental factors.

The prior art describes a method for evaluating indications for drug therapy of obesity in combination with insulin resistance in children (RF Patent No. 2279080). The essence of the method: in the erythrocytes of capillary blood in children with obesity and confirmed insulin resistance, the ratio of water fractions is determined - the hydration coefficient, and when its value is equal to or higher than 5.36, the state is regarded as requiring metformin drug therapy. The application of the method, with ease of implementation, allows for an individual approach to the administration of metformin in children with obesity and formed insulin resistance.

Its disadvantages are that this method is applicable only for treatment correction, and not for determining individual sensitivity and the whole range of medical and preventive measures (formation of risk contingents, selection of an individual diet and physical activity, drug correction, etc.).

From RF Patent No. 2452966, a method for predicting the risk of obesity in practically healthy people is known, according to which the concentration of α-fetoprotein (AFP) and the content of T-lymphocytes with transferrin receptor (CD71 +) are determined in the blood of people. Based on the data obtained, a prognostic index (PI) is calculated as the result of the ratio of the AFP concentration in IU / L to the absolute content of CD71 + lymphocytes in cells / L (PI = AFPL1U71). With a PI value of 8.1 or more, the risk of obesity is predicted. The claimed method can be used to identify the risk of obesity in people leading a sedentary lifestyle, as well as in the appointment of glucocorticoids and immunosuppressive therapy, with inherited pathology with a metabolic defect. Its disadvantage is that this method does not take into account the likelihood of obesity in children under the influence of negative environmental factors.

или вероятность снижения

его относительно физиологической нормы; устанавливают для прогностического показателя общую вероятность

превышения над нормой, либо общую вероятность

снижения относительно нормы в возрасте человека t; производят расчет общей вероятности развития у человека ожирения и по величине индивидуального риска прогнозируют риск развития ожирения у человека.From the patent of the Russian Federation No. 2677274, a method is known for predicting the individual risk of developing obesity for different periods of life in a person living in a polluted environment with aromatic hydrocarbons and metals. The method consists in the fact that the content of chemical elements that are dangerous for the development of obesity is determined in human biological media; conduct a survey; calculate the initial probability value p (t _o ) of the occurrence of obesity in a person at the current person age t _o at the time of the examination; for each quantitative diagnostic indicator, except for HDL and a qualitative indicator, find the function Ф _{i of} its deviation from the physiological norm; each specified diagnostic indicator is given the following weight coefficient φ _I ; calculate the initial probability value p (t ₀ ) of the occurrence of obesity for the current age of the person; using an iterative procedure, the individual risk of developing obesity in a person is predicted with a time step of 1 day; in the examined person, prognostic indicators in the blood and urine are determined for the current age; compare their level with the physiological norm; for the prognostic indicator, the probability of exceeding

or probability of decline

its relative physiological norm; set the prognostic indicator total probability

excess over the norm, or overall probability

decrease relative to normal in a person's age t; they calculate the overall likelihood of a person developing obesity and predict the risk of developing obesity in a person by the magnitude of the individual risk.

However, this method is very difficult to implement. And besides, it is not applicable to children.

A known method for assessing the risk of developing metabolic syndrome (MS) in children based on genetic and biochemical markers (RF Patent No. 2492485). According to this method, after studying the biochemical parameters of HOMA-IR, an atherogenic coefficient (KA) is studied. By the coefficient of 3xKA + HOMA-IR, a conclusion is drawn about the development of metabolic syndrome (MS) and the identification of a risk group for the development of the disease among children. The range of coefficient values up to 10 corresponds to healthy children; from 10 to 14.5 - children with individual metabolic disorders and obesity; above 14.5 - diagnosed with MS. In the group of children with values calculated by the formula greater than 10 but less than 14.5, the risk of MS is additionally determined using the PCR method of the methylenetetrahydrofolate reductase (MTHFR) gene sections with further treatment with restriction endonucleases and the identification of the C / T genotype of the 677C / T polymorphic variant of the gene MTHFR of these children are at risk for developing MS. The invention allows to begin therapeutic and preventive measures at the earliest stages of the development of this disease.

The disadvantage of this method is that the diagnostic unit of this methodological approach to identifying the metabolic syndrome is based on the determination of the HOMA-IR index and atherogenicity coefficient (KA), which already characterize the presence (fact) of biochemical changes in carbohydrate and fat metabolism, that is, attribute these disorders to the early is impossible - the authors do not take into account energy metabolism indicators, and the determination of the polymorphism of the MTHFR gene will characterize only the predisposition to hyperhomocysteinemia and Lee anemia, i.e. to conditions not directly related to obesity (obesity and metabolic syndrome are not included in the list of associations with diseases of polymorphic variants 677C / T of the MTHFR gene).

A known method for assessing the risk of metabolic syndrome, based on the identification of the 4G / 4G genotype (option - 675 4G / 5G) by the PA1 gene. It is aimed at forming a risk group, but does not allow differential diagnosis (Berberoglu) M. et al. Plasminogen activator inhibitor (PAI-1) gene polymorphism (-675 4G / 5G) associated with obesity and vascular risk in children. J / Pediatr. Endocrinol Metab. 2006 May; 19 (5): 741-8).

There is a method of assessing the individual risk of overweight and obesity in children who consume drinking water with a high content of chloroform and carbon tetrachloride (RF Patent No. 2619872). Urine samples are taken, the concentration of chloroform and carbon tetrachloride is determined in it, and the levels of total cholesterol and low density lipoprotein (LDL) are determined. An anamnestic examination of the child is carried out on a three-point scale and genotyping. The risk of overweight in a child is predicted if total cholesterol rises above 3.9 mmol / L and LDL levels exceed 1.8 mmol / L, if the child has 6 or more points during an anamnestic examination, and the presence of a heterozygous HTR2A gene genotype (rs7997012 ), increasing the concentration of carbon tetrachloride in the urine above the reference and the concentration of chloroform in the range of 5.17-5.59 μg / L. The risk of obesity is predicted at a concentration of chloroform equal to or more than 5.79 μg / L. The invention provides an effective way of assessing the individual risk of overweight and obesity in children, but it is not intended to examine endocrine disorders in children living in conditions of high aluminum content in the environment.

There is also a method for detecting a genetic predisposition to obesity by establishing the presence of the FTO gene (fat mass and obesity associated), which encodes an FTO protein involved in energy metabolism and affecting metabolism in general (information from the Internet site: https://helix.ru/ kb / item / 18-051). Gene expression occurs mainly in the hypothalamic cells and is regulated by processes responsible for feelings of fullness and hunger. In the European population, two allelic variants of the FTO gene are found by the genetic marker G (45 + 52261) A (rs1861868), which is associated with increased body weight and obesity. In the regulatory region of the gene there is a polymorphic region in which guanine (G) is replaced by adenine (A). If guanine (G) is in this position, this variant of the gene is designated as the G-allele, and if adenine (A) - A-allele. Studies show that carriers of allele A show an increase in body weight and waist circumference compared with carriers of G alleles. The risk of obesity in people with the A / A genotype is significantly higher than in people with the G / G and G / A genotype.

Moreover, the known methods for determining a predisposition to the development of obesity in children precisely under conditions of excessive aluminum contamination have not been identified from the prior art, therefore, it is not possible to choose the closest analogue to the claimed object.

The technical result achieved by the present invention is to provide a reliable assessment of the effect of aluminum on the predisposition of manifestations of endocrine disorders in the form of obesity, with the possibility of subsequently judging the development of such conditions in children already in the early stages of their formation.

The specified technical result is achieved by the proposed method for determining a predisposition to the development of obesity in children under conditions of excessive aluminum contamination, characterized in that they take blood and urine samples from the child, determine the aluminum content in the urine sample, and the level of immunoglobulin G (IgG) in the blood serum to aluminum, a sample of buccal epithelium is also taken from the indicated child, deoxyribonucleic acid (DNA) is extracted from the indicated sample, then on a detecting amplifier with Real-time polymerase chain reaction is used to genotypic the polymorphism of the MTNR1B and SIRT1 genes, using a DNA segment as a primer by examining the genotypes of the MTNR1B gene (rs 10830963) and the SIRT1 gene (rs 7069102), setting for each of these genes: heterozygous C , normal homozygous SS, variant homozygous GG for the MTNR1B gene (rs 10830963); heterozygous CG, normal homozygous SS, variant homozygous GG for the SIRT1 gene (rs 7069102), and at the same time the following diagnostic criteria are detected in a child: the presence of a variant homozygous or heterozygous MTNR1B gene genotypes (rs 10830963) and SIRT1 gene (rs 108699), (at 7069) simultaneous excess aluminum concentration in the urine of more than two times compared with the reference level of aluminum content in the urine of 0.01 mg / dm ^3, as well in excess compared with the upper limit of the physiological level of IgG standards to aluminum, do concluded that there was a child's predisposition to the development of obesity in terms of excess contamination with aluminum.

The stated technical result is achieved due to the following: identification of genetic disorders in the form of polymorphic variants of candidate genes (sirtuin gene - SIRT1 rs7069102; melatonin receptor B1 gene - MTNR1B rs10830963), which form an energy imbalance and circadian arrhythmia (the need to eat during inopportune - evening and evening time) leading to abdominal obesity, with simultaneous identification of the triggering (implementing) constitutional (genetic) changes in the factor - aluminum, the action of which confirms I the presence of specific antibodies (IgG to aluminum).

The role of excess aluminum in the body is manifested in the effect on the metabolism, especially the mineral one (in particular, it causes disturbances in the phosphorus-calcium metabolism and reduces the absorption of iron); on the functions of the nervous system (decreased or lost memory, cramps, etc.); in the ability to act directly on cells - their reproduction and growth; prolonged inhalation of aluminum compounds leads to fibrosis of the lung tissue. The high ability of aluminum to form complex compounds determines its role in reducing the activity of many enzymes and their systems. It was found that aluminum accumulates in plasma and tissues and has a toxic effect on patients with chronic renal failure. There is evidence of mutagenic activity of aluminum.

An important role in the pathogenesis of aluminum intoxication is played by its competitive relations with phosphorus, calcium and iron. Technogenic and abnormal geochemical loci of the excessive presence of aluminum in soil and drinking water are often found at low concentrations of calcium and magnesium in these environments. The mobility of aluminum ions increases sharply with increasing acidity (pH) of natural environments, as a result of, for example, exposure to acid rain. The main sources of aluminum intake into the environment and, then, into the human body are emissions in the areas where aluminum plants are located and wastewater from the mining industry, paints and varnishes, paper, textile and some other industries.

As a diagnostic criterion for the occurrence of endocrine dysfunctions (obesity) in children under conditions of excessive aluminum contamination, it is recommended to use the modified genotype of the sirtuin gene (SIRT1 rs7069102), which is responsible for the expression of evolutionarily conserved NAD-dependent proteins with deacetylase or ADP-ribosyltransferase activity, and regulation of important cellular processes and metabolic pathways, when, in the case of a mutation, conditions are created for the formation of energy disorders, contributing to x accumulation of adipose tissue (adipocytes). The exposure conditions to aerosols containing aluminum contribute to the replacement of phosphorus in the molecules of cyclic adenosine monophosphate (hereinafter - cAMP) and the nicotinamide adenine dinucleotide phosphate (NAD-F) and adenosine triphosphate (ATP) systems present in the cells of a living organism.

It is also proposed to use simultaneously as a diagnostic criterion the changed genotype of the melatonin receptor B1 gene (MTNR1B rs10830963), which is responsible for circadian biorhythms of the daily regimen and food intake, the combined manifestations of polymorphism of which contribute to the formation of energy balance disorders and the phenomenon of abdominal obesity.

Also, an indicator characterizing the phenomenon of impaired energy metabolism itself is cAMP, which acts as a secondary intracellular mediator in the action of primary mediators (substances with a short biodegradation period) - for example, a number of hormones, neurotransmitters (melatonin), sirtuin. cAMP mediates the biological function of biologically active mediators by activation (inactivation) of cellular protein kinases (phosphatases). Protein kinases, in turn, phosphorylate effector proteins and alter (increase or decrease) their activity.

Modulating these processes, the aluminum ions in this invention will characterize two criteria - the content of aluminum in the urine and the response to its contamination - the level of serum IgG in aluminum.

A confirming factor in impaired energy metabolism and the formation of obesity will be the inhibition of cAMP and an increase in the level of specific response to aluminum by the criterion of IgG content to aluminum.

The exposure of aluminum exceeding the reference concentration is used to implement or start the process of phosphorus metabolism disturbance (an indicator of 0.01 mg / dm ³ (the upper limit of the reference limits of 6.5 ± 3.5 μg / l was taken as the reference value of the aluminum content in the urine according to N. Tits, 2003, p. 21).

Thus, to establish early manifestations and the formation of a specific disturbance in energy metabolism in the form of obesity associated with an increased aluminum content in the urine, it is recommended to use a methodological approach based on the determination of candidate genotypes in the case of heterozygotes and / or variant homozygotes of MTNR1B gene regions (rs10830963 ) and SIRT1 (rs7069102), deviation from the physiological norm of the content of IgG specific for aluminum in the form of its overproduction, excess aluminum in the urine, exceed reference level it is not less than 2 times, record the presence formed disorders of energy metabolism, which is expressed obesity associated with exposure of the aluminum. This is proved by a decrease in cAMP expression, as cyclic adenosine monophosphate (cAMP), a derivative of the main energy source in the body - ATP, is a mediator that transmits the effect of stimulating extracellular receptors to the internal structures of the cell, depends on sirtuin polymorphism, which reduces the activity of glucose-sensitive cAMP, as well as on the level of Al contamination when its competitive relations with phosphorus, which contributes to the replacement of phosphorus in c-AMP molecules and the inactivation of cellular protein kinases.

Thanks to the use of blood and urine samples as the test material, as well as standard methods for studying genetic parameters, the simplicity, reliability and accessibility of studies, as well as obtaining the results of the necessary information content, are ensured.

The determination of the content of a chemical contaminant - aluminum, in the urine is due to the fact that urine is a medium with referenced (background) levels in relation to technogenic chemicals, as well as a standard medium used for intoxication protocols caused by metals.

Thanks to the use of buccal epithelium as a test material (a sample of biological material from the cheek mucosa), the simplicity and reliability of studies are ensured, as well as the necessary information content in terms of the isolation of deoxyribonucleic acid (DNA) from the indicated sample and through polymerase chain reaction (PNR) polymorphism genotyping the indicated genes MTNR1B and SIRT1. At the same time, it is proposed to use as a primer a portion of the DNA of the MTNR1B gene (rs10830963) and the SIRT1 gene (ts7069102), setting for each gene one of the following states: heterozygous, or normal homozygous, or variant homozygous.

In the MTNR1B gene (rs10830963) and in the SIRT1 gene (rs7069102): CC is the normal homozygous genotype; CG - heterozygous genotype; GG is the variant homozygous genotype.

Due to the fact that in the inventive method, it is proposed to use precisely the polymorphism of the MTNR1B gene (rs10830963) and the SIRT1 gene (rs7069102) as diagnostic criteria at the same time. the sirtuin SIRT1 gene belongs to the family of energy-dependent and energy-mediating proteins (NAD and ADP), expression disorders of which lead to the formation of a ligament - impaired glycolysis - diabetes mellitus-obesity. The melatonin MTNR1B gene is a hypothalamic receptor that performs central regulation of circadian biorhythms (polymorphic gene changes and impaired receptor expression leads to an irresistible desire to eat at inopportune times (evening, night), which, together with altered sirtuin expression, reduce the expression processes of cAMP, ATP, ADP etc., and lead to the accumulation and redundancy of adipocytic elements, which is also facilitated by the activity of aluminum ions, which are observed in excess in urine. Therefore, according to these indicators, one can judge the gene cally deterministic nature of endocrine disorders as obesity and develop individual programs determine the predisposition to obesity and correction.

Moreover, from the prior art, unknown methods for identifying a predisposition to the development of obesity in children under conditions of excessive contamination with aluminum using both polymorphisms of these genes at the same time.

The level of IgG to aluminum characterizes the very phenomenon of specific sensitization.

Thus, when assessing the effect of aluminum on the predisposition to obesity in a child in the proposed method, it is recommended to use the following criteria:

- the simultaneous presence of a variant homozygous or heterozygous genotypes of the MTNR1B gene (rs10830963) and the presence of a variant homozygous or heterozygous genotypes of the SIRT1 gene (rs7069102);

- excess of aluminum concentration in urine above the reference level by more than 2 times,

- and the excess compared with the upper limit of the physiological norm of the level of IgG to aluminum,

It is thanks to the expansion of information indicators associated with polymorphic variants of these genes associated with a genetic defect in the endocrine system and an innate sensitivity to external factors (for example, aluminum), simultaneously with the amount of the chemical toxicant - aluminum in the urine, and an excess of the level of IgG to aluminum compared with physiological norm, the accuracy of evaluating the modifying effect of aluminum on the potential development of obesity in a child will be ensured.

Based on the foregoing, we can conclude that the technical result is achieved due to the totality of the operations of the proposed method, their sequence and modes of its implementation.

The proposed method is implemented as follows.

1. Choose the territory of environmental risk, characterized by the presence of chemical toxicants due to the ecological environment. Studies were conducted on the territory of one of the industrial cities of the Russian Federation, characterized by the presence in the atmospheric air of increased aluminum concentrations (up to 2 maximum permissible concentration)

2. In this territory, a group of children of one ethnic population is selected. Then a urine sample is taken from the examined child to determine the aluminum content. The aluminum content in the urine is determined according to the Method “Control Methods. Chemical factors. Determination of the content of chemical elements in diagnosed biosubstrates, preparations and biologically active additives by the method of mass spectrometry with inductively coupled argon plasma ”, described in MUK 4.1.1483-03, using a mass spectrometer with inductively coupled plasma Agilent 7500xx with an octopole reaction cell (USA ) It is established whether there is an excess of the concentration of this contaminant in the urine sample over its reference level (in the example of a specific implementation in the territory, the reference value for aluminum was an indicator equal to 0.01 mg / dm ³ ).

3. Then determine the content of IgG to aluminum in the blood sample according to the method: Zaitseva N.V., Belyaev E.N., Dolgikh O.V. Methods for the diagnosis of sensitization to low molecular weight chemical compounds / Methodical recommendations. - M., 2002. - P. 29. They also determine the content of cAMP in the blood sample by enzyme-linked immunosorbent assay using a commercial Assay Designs Correlate-EIA cyclic AMP kit (Biochemmak, Moscow).

4. Also, a sample of buccal epithelium (in the form of a smear from the mucous membrane of the cheek) is taken from the indicated child. After taking the material, the tampon (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). The tube with the solution and the working part of the probe is closed.

Next, DNA is extracted from the sample. For this, samples in an amount of 100 μl are lysed with 300 μl of a lysing 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 the washing samples are washed with phosphate-buffered saline (pH 7.2) from proteins and a mixture of isopropyl alcohol: acetone from lipids. Nucleic acids remain on the sorbent. Next, the DNA adsorbed on the sorbent from the sample is extracted with 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 the production of polymerase chain reaction (PCR). PCR is carried out on a detecting amplifier with hybridization-fluorescence detection in real time using ready-made sets of primers and probes manufactured by Syntol CJSC, Russia, in which MTNR1B (rs10830963) and SIRT1 (rs1026969) gene DNA segments were used as primers .

An amplification reaction is carried out, this is achieved by the fact that in order to study the allelic state of each gene in an individual child, their reaction mixture is prepared. 0.1 μl of the prepared primer mixture (the term used in genetics for the labeled final nucleotides that limit (cut off) the amplified gene nucleotide strand) and probes for the selected MTNR1B (rs10830963) and SIRT1 (rs7069102) genes is used (reagent kits are used) to determine the polymorphism MTNRJB (rs10830963) and SIRT1 (rs7069102) Synthol CJSC, Russia). The remaining components necessary for PCR are added to each tube: nucleotides (deoxynucleoside triphosphates: 10 mM dATP, dTTP, dGTP, dCTP), buffers (100 mM Tris-HCl buffer, 500 mM KCl, 40 mM MgCl ₂ ) and Tag F- polymerase. Make a sample in an amount of 10 μl. Thus, the total volume of the reaction mixture is 25 μl. Each tube is tightly closed by a stopper and installed in the amplifier.

During PCR, amplification and detection are carried out on a Bio-Rad detection amplifier CFX96.

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

The fluorescence signal arising from the accumulation of amplification products of DNA regions is recorded in the “real time” mode after the annealing of the primers for the selected genes via the VIC channel to detect one of the allelic variants of the genes, and the FAM channel for an alternative.

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

The ratio of the threshold cycles obtained through the two detection channels determines the state of the MTNR1B gene in the studied DNA region rs10830963, as well as the SIRT1 gene in the studied DNA region rs7069102 (allelic discrimination method). There were two possible variants of the state of the gene: 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 from 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 MTNR1B gene (rs10830963) and the SIRT1 gene (rs7069102) is established.

5. And under the following conditions: the presence of a variant homozygous or heterozygous genotypes of the MTNR1B gene (rs10830963) and the SIRT1 gene (rs7069102), the excess of aluminum concentration in the urine is more than 2 times higher than the reference level, and also an excess compared with the upper physiological limit norms of the level of IgG in the blood serum to aluminum, conclude that the child is predisposed to develop obesity in conditions of excessive aluminum contamination.

When conducting tests to implement the proposed method, a survey was carried out of the child population living in different environmental conditions of aluminum.

Two groups were formed: the observation group, whose children lived in a territory unsuccessful with regard to aluminum, and the comparison group, living in conditions of sanitary and hygienic well-being. The observation group included 33 children (average age 6.2 ± 0.6 years; 51% of boys and 49% of girls). The comparison group included 31 children (average age 6.4 ± 0.3 years; 48% of boys and 52% of girls). The study groups were comparable in age, gender, ethnic composition, concomitant pathology, socio-economic level of the family, quality and composition of nutrition.

In the territories where the children of the observation group were living, an excess of aluminum content of up to 2.1 MPC m.r.

An assessment of the risk to children's health showed that, with chronic exposure to contaminants, hazard factors for aluminum were up to 1.12. It was established that the chronic exposure to aluminum forms a danger of developing disorders of the endocrine system (hazard index HI - up to 1.97).

The risk assessment of the development of diseases is carried out according to a standardized method in accordance with the “Guidelines for assessing the risk to public health when exposed to chemicals that pollute the environment” (R 2.1.10.1920-04).

A urine sample and a venous blood sample were taken from all examined children, the study of which was carried out according to the above scheme. A sample of buccal epithelium was also taken to establish the polymorphism of the studied genes by the PCR reaction.

To prove the reliability of the claimed criteria for the diagnosis of a predisposition to obesity with increased aluminum contamination, the content of cAMP was established in the blood serum sample. This cAMP indicator is a confirming factor for excessive load (contamination) of aluminum on the one hand (phosphorus replacement in c-AMP molecules and inactivation of cellular protein kinases) and impaired expression of sirtuin, on the other (sirtuin mutations reduce the activity of glucose-sensitive cAMP), since the observed deficiency of the adenosine triphosphate derivative - cAMP characterizes a decrease in the intensity of energy processes that ensure the completeness of glucose and cholesterol-dependent reactions, a violation of which leads to uw overweight.

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

The results are shown in table 1 in the form of five blocks, each of which shows a different combination of polymorphisms of these genes, the content of aluminum in the urine, the level of IgG in serum to aluminum and the content of camp in the blood.

The data shown in Table 1 show that, with the simultaneous combination of the variant (heterozygous or variant homozygous state of the genotype) allele of the MTNR1B gene (rs10830963) and the SIRT1 gene (rs7069102), the concentration of the “guilty” factor (aluminum) in the urine is higher than the reference concentration ( 0.01 mg / dm ³ ) more than 2 times, and exceeding the physiological norm of the level of IgG to aluminum (patients 1-4), there is a decrease compared with the physiological norm of cAMP content. And this means that at the same time, the implementation of aluminum's negative genetic program in the child, expressed in violation of the endocrine system through the manifestation of obesity, is recorded.

Whereas at an acceptable (at the reference concentration level) aluminum content in the urine, even under conditions of inheritance of the heterozygous or variant genotype of these genes (patients 8-11), the IgG level and cAMP content are within the physiological norm.

And, conversely, if the aluminum content in the urine is more than 2 times higher than the reference level, but in the absence of polymorphic changes in these genes (patients 12-13), the IgG level and cAMP content are within the physiological norm.

In patients 5-7, who are characterized by an excess of aluminum in the urine more than 2 times compared with the reference level, an excess of the level of IgG to aluminum compared to the norm, but at the same time they have the inheritance of the heterozygous genotype of only one of these genes, and not simultaneously both, the conclusion about a predisposition to obesity is not diagnosed, while the cAMP content is within normal limits, which indicates the absence of the necessary genetic conditions (predisposition) for the implementation of the existing exposure in the form of an endocrine disorder.

Thus, this proves that only a simultaneous excess of aluminum content in urine by more than 2 times, relative to the reference level, an excess of IgG to aluminum is higher than normal and only the simultaneous presence of polymorphic genotypes of the MTNR1B gene (rs10830963) and the SIRT1 gene (rs7069102 ), is characterized by the appearance of negative effects in the form of a deviation from the norm of cAMP content, which characterizes the violation of energy metabolism, a decrease in which leads to the formation of obesity, and thus the accuracy and reliability of the proposed way.

To illustrate the implementation of the proposed method, two examples are given for specific patients of the same age and ethnicity from a group of children with a high content of aluminum in the urine and children with an aluminum content in the urine within the reference concentration.

Example 1. Patient, 6 years old, Russian, metabolic syndrome. The level of aluminum in urine was established more than 2 times higher than the reference level - 0.04 mg / dm ³ . The presence of the heterozygous genotype of the MTNR1B gene (rs10830963) - CG and the variant homozygous genotype of the SIRT1 gene (rs7069102) - GG was established. The content of IgG to aluminum: 0.205 cu, i.e. 2.0 times higher than the upper limit of the norm. The content of cAMP in the blood is a marker of the intensity of energy metabolism, 5.5 pmol / cm ³ , i.e. below the normal range (5.9-10.9 IU / ml). Thus, high levels of IgG to aluminum and a deficiency of cAMP against a background of an increase in urine aluminum content more than 2 times higher than the reference level, as well as the presence of heterozygous polymorphism of the MTNR1B gene (rs10830963) - CG and a variant homozygous SIRT1 gene genotype (rs7069102) - GG encoding proteins responsible for circadian biorhythms of the daily regimen and eating and expression of evolutionarily conserved proteins with deacetylase or ADP-ribosyltransferase activity, which indicates the formation of energy disorders, when in the case of a mutation, conditions are created that facilitate the accumulation of adipose tissue initiated by the action of aluminum.

This suggests that, according to the proposed method, in this child, the condition from exposure to aluminum is assessed as the formation of early manifestations of the metabolic syndrome (obesity), which has a genetic predisposition, realized in conditions of excessive aluminum contamination.

To prove this conclusion, the following metabolic indicators were established for this child, which had the following values: glucose level - 6.1 mmol / dm ³ (physiological norm 3.3-5.5 mmol / dm ³ ); the LDL level is 4.2 mmol / dm ³ (the norm is 1.5-3.9 mmol / dm ³ ).

Analyzing the presented data, we can conclude that the patient has changes in carbohydrate and fat metabolism, in particular, the presence of excessive glucose and low density lipoproteins (LDL), which characterizes the presence of violations of their utilization, that is, reduced energy metabolism, which triggers the processes metabolic disorders leading to the formation of obesity associated with aluminum contamination.

Example 2. Patient, 7 years old, Russian. The level of aluminum in the urine is determined more than 2 times higher than the reference level - 0.05 mg / dm ³ . There are no polymorphic (variant) genotypes of the MTNR1B gene (rs10830963) - CC and the SIRT1 gene (rs7069102) - CC. The content of IgG to aluminum: 0,049 cu - in the normal range. The content of cAMP in the blood is a marker of the intensity of energy metabolism - 5.9 pmol / cm, in the normal range (normal 5.9-10.9 IU / ml). Thus, the absence of polymorphism of the genes - the MTNR1B gene (rs10830963) - SS and the SIRT1 gene (rs7069102) - SS against the background of an increase in the urine aluminum content in relation to the reference level, more than 2 times, does not lead to the development of negative effects in the form deviations from the normal level of IgG to aluminum and cAMP, that is, in the absence of a negative genetic program, an excess of the reference concentration of aluminum in the urine does not form a deficiency in energy-dependent processes, and does not lead to the development of obesity in this patient.

To prove the accuracy of this conclusion - the absence of disturbances in carbohydrate and fat metabolism, the following metabolic indicators were established for this child, which had the following values: glucose level - 5.1 mmol / dm ³ (physiological norm 3.3-5.5 mmol / dm ³ ); LDL - 3.2 mmol / dm ³ (normal 1,5-3,9 mmol / dm ^3).

Analyzing the presented data, we can conclude that the examined patient has no disturbances in energy and related carbohydrate and fat metabolism. Thus, in the analysis of metabolic markers, changes in energy metabolism due to the action of aluminum were not detected.

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

To prove the reliability of the proposed method, a genetic examination of two groups of children living in the territory with a high content of aluminum in the air and drinking water was carried out. Moreover, the first group (22 children) was obese, the second group (19 children) was of normal weight. In these children, a comparative analysis of polymorphic changes in the genotypes of the melatonin receptor and sirtuin was carried out. The data obtained during this survey are shown in table 2.

The results of a genetic examination of children showed that in the group of children suffering from obesity, the carriers of mutations in the heterozygous genes MTNR1B (rs10830963) and SIRT1 (rs7069102) are the majority, while this pattern was not found in the group of children with normal weight.

The inventive method allows with sufficient reliability to establish the precursors of obesity associated with the influence of aluminum, according to the claimed genetic criteria, the aluminum content in the urine and the level of IgG to aluminum, and start preventive measures.

Claims (1)

A method for determining a predisposition to the development of obesity in children under conditions of excessive aluminum contamination, characterized in that they take blood and urine samples from the child, determine the aluminum content in the urine sample, and the level of immunoglobulin G (IgG) to aluminum in the blood serum, also of the specified child, a sample of buccal epithelium is taken, deoxyribonucleic acid (DNA) is extracted from the specified sample, then on a detecting amplifier using a real-time polymerase chain reaction The belts carry out genotyping of the polymorphism of the MTNR1B and SIRT1 genes, using a DNA fragment as a primer by examining the genotypes of the MTNR1B gene (rs 10830963) and the SIRT1 gene (rs 7069102), establishing conditions for each of these genes: heterozygous CG, normal homozygous SS, homozygous GG for the MTNR1B gene (rs 10830963); heterozygous CG, normal homozygous SS, variant homozygous GG for the SIRT1 gene (rs 7069102), and at the same time the following diagnostic criteria are found in a child: the presence of a variant homozygous or heterozygous MTNR1B gene genotypes (rs 10830963) and the SIRT1 gene (rs10869) exceeding the concentration of aluminum in the urine by more than 2 times compared with the reference level of aluminum in the urine, equal to 0.01 mg / DM ³ , as well as when exceeding the upper limit of the physiological norm of the level of IgG to aluminum, do the conclusion that the child has a predisposition to the development of obesity in conditions of excessive aluminum contamination.