RU2794991C1 - Method for predicting the development of childhood obesity in the republic of bashkortostan - Google Patents

Abstract

FIELD: medicine; pediatrics.

SUBSTANCE: invention can be used to predict the development of obesity in children living in the Republic of Bashkortostan. DNA is isolated from peripheral venous blood lymphocytes. Amplification of the rs3803300 locus of the AKT1 protein kinase 1 gene and the rs279845 locus of the GABRA2 gamma-aminobutyric acid receptor gene is carried out. When AG or AA genotypes of the rs3803300 polymorphic locus of the AKT1 gene and AT or AA genotypes of the rs279845 polymorphic locus of the GABRA2 gene are detected, the risk of developing obesity in a child is predicted.

EFFECT: method provides an increase in the accuracy of the prediction by identifying factors predisposing to the development of the disease.

1 cl, 2 tbl, 5 ex

Description

The present invention relates to medicine, namely to pediatrics, and can be used to predict the development of obesity in childhood in the Republic of Bashkortostan.

Currently, there is an increase in childhood obesity throughout the world and in Russia [Bocharova O.V., Teplyakova E.D. Obesity in children and adolescents is a public health problem of the 21st century. Kazan honey. and. 2020. Volume 101, No. 3. S. 381-388]. The causes of childhood obesity include eating disorders, diseases, medication, physical inactivity, hereditary predisposition. The presence of childhood obesity causes the risk of metabolic syndrome, diabetes mellitus 2, insulin resistance [Peterkova V.A., Vasyukova O.V. On the issue of a new classification of obesity in children and adolescents // Problems of Endocrinology. 2015. Volume 61, No. 2. S. 39 44]. According to WHO, 50% of overweight children in childhood are obese in adulthood, and in adolescence this probability increases to 80% [Saidova L.B., Badritdinova M.N., Raupov A.A. Epidemiology and the state of detectability of a number of extragenital diseases, metabolic syndrome among women of childbearing age (literature review) // Actual problems of the humanities and natural sciences. 2018. No. 6. S. 131-141].

A known method for assessing the individual risk of overweight and obesity in children consuming drinking water with a high content of chloroform and carbon tetrachloride, characterized by the fact that they take blood samples, determine the concentration of chloroform and carbon tetrachloride in it, determine the levels of total cholesterol and low density lipoprotein ( LDL). Conduct anamnestic examination of the child on a three-point scale and genotyping. The risk of overweight in a child is predicted in case of an increase in total cholesterol above 3.9 mmol / l and LDL levels above 1.8 mmol / l, the child has 6 or more points in anamnestic examination, the presence of a heterozygous genotype of the HTR2A gene rs7997012, increase in the concentration of carbon tetrachloride in the blood above the reference and the concentration of chloroform in the range of 5.17-5.59 µg/l. The risk of obesity formation is predicted at a chloroform concentration equal to or more than 5.79 µg/l [Patent RU 2619872, 2017]. The disadvantage of this method is that it can only be used in a group of children who drink water with a high content of chloroform and carbon tetrachloride.

The closest analogue of the invention is a method for predicting the risk of developing obesity in childhood, which consists in determining the risk factors and calculating by the formula. From the anamnesis, such risk factors as aggravated heredity for arterial hypertension, aggravated heredity for obesity, aggravated heredity for metabolic disorders - obesity and diabetes mellitus in persons of I, II degree of kinship, pathological course of pregnancy - preeclampsia, single-parent family, low physical activity, carriage E4 isoforms of the APOE gene, carriage of the G-allele of the PPARG gene. Find out the duration of exclusive breastfeeding. The risk of developing obesity is determined by the formula [Patent RU No. 2696446, 2019]. The disadvantages of the method are laboriousness due to the collection of multiple information about the person being examined, as well as low accuracy, since it is not always possible to assess hereditary burden for all of the listed characteristics.

The objective of the invention was to develop an objective, highly informative method for predicting the development of obesity in childhood, allowing to quantify the risk of this disease in children living in the Republic of Bashkortostan.

The technical result when using the invention is to increase the accuracy of the forecast.

The most effective way to prevent the explosive growth of childhood obesity is preventive measures. Primary prevention refers to the identification of high-risk groups for obesity among children using data on molecular genetic biomarkers of susceptibility. The application in practice of the developed method will ensure the identification of children with a high risk of developing obesity.

The proposed method for predicting the risk of developing childhood obesity in the Republic of Bashkortostan is carried out as follows.

DNA is isolated from peripheral blood lymphocytes. As a preservative, a solution of the following composition is used: 0.48% citric acid, 1.32% sodium citrate, 1.47%) glucose. When taking blood, 6 ml of blood is added to 1 ml of preservative and mixed well.

To obtain DNA of the required degree of purity and sufficient molecular weight, the method of isolating DNA from blood by phenol-chloroform extraction, described by Matthew (Mathew C.C. The isolation of high molecular weight eukaryotic DNA // Methods in Molecular Biology / Ed. Walker J.M., N.Y. , L.: Human Press. - 1984. - V. 2. - P. 31-34).

1. The blood in a test tube with a preservative is thoroughly mixed and poured into a 100 ml centrifuge beaker, 50 ml of chilled lysis buffer containing 320 mM sucrose, 1% Triton X-100 solution, 5 mM MgCl2, 10 mM trisHCl (pH 7, 6).

2. The mixture is centrifuged for 20 minutes. at 4000 rpm

3. The supernatant liquid is drained, 8 ml of 25 mM EDTA, pH 8.0, is added to the resulting precipitate, and suspended.

4. Add 0.8 ml of 10% SDS and proteinase K (concentration - 10 mg/ml) to the suspension. The mixture for lysis is left overnight in a thermostat at a temperature of 38°C.

DNA extraction is carried out in the following order.

1. For deproteinization, 0.5 ml of 5M sodium perchlorate and 8 ml of phenol saturated with 1M TrisHCl to pH 7.8 are added to the lysate.

2. The mixture is centrifuged at 3000 rpm. within 10 min.

3. Select the aqueous phase containing DNA, RNA and undenatured proteins.

4. The selected phase is treated with a mixture of phenol-chloroform (1:1), and then with chloroform.

5. The preparations are precipitated with two volumes of 96% ethanol.

6. The resulting DNA precipitate is dissolved in 1.5 ml of deionized H20; the solution is stored at -20°C.

Subsequently, the resulting DNA is used as a template for polymerase chain reaction (PCR) for amplification of the rs3803300 locus of the AKT1 protein kinase 1 gene and the rs279845 locus of the GABRA2 gamma-aminobutyric acid receptor gene. Specific sequences of oligonucleotide primers and conditions for PCR analysis of polymorphic loci were selected using the DNA Star 5.05 software package and http//www.ncbi.nlm.nih.gov/snp databases.

The composition of the reaction mixture for PCR was as follows: 0.1–1 μg of genomic DNA, 0.5 μM of each oligoprimer, 250 μM of each deoxynucleoside triphosphate (Promega, USA) were placed in 10 μL of a single PCR buffer of the following composition: 60 mM Tris-HCl, pH 8.5 at 25°С, 1.5 mM MgCl2, 25 mM KCl; 10 mM 2-mercaptoethanol; 0.1% Triton X-100. Next add 5 units of Taq-DNA polymerase, 20-30 µl of mineral oil. Amplification mode: 30 cycles with the following parameters: 94°C - 5 min, 95°C - 30 seconds, 60°C -30 seconds, 72°C - 30 seconds. After the 30th cycle, incubation was carried out at 72°C for 5 minutes.

Nucleotide substitutions at a position in the promoter region of the AKT1 gene and the intron polymorphic marker of the GABRA2 gene are detected using PCR-RFLP analysis. To do this, 7 μl of the amplificate is mixed with 5 units. corresponding restriction endonucleases, the mixture is kept at 37°C for 10-12 hours in a thermostat for restrictases Alw26 I and Hinf I. Then electrophoresis is carried out in a vertical 7% polyacrylamide gel. As an electrolyte for electrophoresis, 1X borate buffer (0.089 M Tris HCl pH=7.8; 0.089 M boric acid, 0.002 M EDTA pH=8.0) is used. Data on the sizes of the resulting fragments are presented in Table 1.

Statistical processing of the study results was carried out using MS Office Excel software. In pairwise comparison of allele and genotype frequencies in the groups of patients and controls, the ^χ2 test was used for contingency tables 2×2 with Yates correction for continuity [http://www.biometrica.tomsk.ru/]. If statistically significant differences (p<0.05) were found between the studied samples, the odds ratio (odds ratio, OR) was evaluated, as well as the boundaries of its 95% confidence interval (CI 95%). The observed distribution of allele and genotype frequencies for all studied loci corresponds to those expected from the Hardy-Weinberg equation.

When AG or AA genotypes of the rs3803300 polymorphic locus of the AKT1 gene and AT or AA genotypes of the rs279845 polymorphic locus of the GABRA2 gene are detected, the risk of developing obesity in a child is predicted.

We examined DNA samples from 263 obese children and 409 non-obese children. The average age of obese subjects was 6.5 years, children in the control group 7.2 years.

The study of polymorphic variants rs3803300 of the AKT1 gene and rs279845 of the GABRA2 gene among obese children revealed the following results (Table 2). In the group of children with obesity, the frequency of AG-AA rs3803300 genotypes of the AKT1 gene and the frequency of AT-AA genotypes of the rs279845 polymorphic locus of the GABRA2 gene were statistically significantly increased. An increased risk of childhood obesity is associated with the AG-AA locus rs3803300 of the AKT1 gene and the AT-AA locus rs279845 of the GABRA2 gene.

To quantify the relative risk of developing the disease for each of the above genetic markers, we calculated an odds ratio (oddsratio - OR). To do this, we used the formula proposed by Bland [Bland, J.M. The odds ratio / J.M. Bland, D.G. Altaian // Br. Med. J. - 2000. - Vol.320. - P. 1468]: OR=(a×d)/(b×c), where a is the number of persons with the presence, b - with no marker (allele or genotype) among patients; c and d - the number of individuals, respectively, with the presence and absence of the marker among healthy people. An increased risk of developing childhood obesity is diagnosed with an OR greater than 1.0.

We give an example of a specific calculation. In the group of children with obesity, 53 children with AG-AA risk genotypes were identified for the rs3803300 polymorphic locus of the AKT1 gene, i.e. a=53. In 210 obese children no risk genotypes were identified, so b=210. In the control group, 55 subjects with AG-AA genotypes were identified, therefore, c=55. In the control group, risk variants are absent in 354 children, which means that d=354. Substituting these values into the above formula, we get:

OR=(53×354)/(210×55)=18762/11550=1.62. Consequently, in individuals with variants of homozygotes for a rare allele and heterozygotes for AG-AA, the risk of developing childhood obesity is increased by 1.62 times compared with the control. The OR values calculated based on the results of the study are further used in the course of developing the patient's genetic passport. The OR indices obtained in our study in the presence of risk genotypes for the AKT1 rs3803300 and GABRA2 rs279845 loci are presented in Table 2.

The invention is illustrated by the following clinical examples.

Example 1

Patient R., aged 16, has been obese since the age of 10. The presence of risk factors was revealed in the family: the mother suffers from grade III obesity. The biological material of 4 ml of blood was taken from the child. The results of the molecular genetic analysis followed by DNA isolation by phenol-chloroform extraction and amplification of AKT1 rs3803300, GABRA2 rs279845 gene fragments in a reaction mixture containing 0.1-1 μg of genomic DNA, 1 u. Taq polymerase, 0.25 µM of each oligoprimer, 250 µM of each deoxynucleoside triphosphate in 25 µl of 1 x PCR buffer. After amplification, restriction was performed, followed by electrophoresis of the fragments at a constant voltage of 250–300 volts after 15 minutes of preelectrophoresis. After the end of electrophoresis, the gel was stained with a solution of ethidium bromide for 10 minutes and analyzed under ultraviolet light on a transilluminator. The study of the polymorphic locus AKT1 rs3803300 revealed the AA genotype, in which the odds ratio for the development of obesity is 3.37 (Table 2). At the GABRA2 rs279845 locus, the AA risk genotype was also identified; in this case, the probability of developing obesity increases by 0.81 (Table 2).

The patient's parents were offered measures to correct the child's weight, medications, and an increase in physical activity. These recommendations were not fully implemented, and during the examination two years later, the child was diagnosed with type 2 diabetes mellitus, and the weight continued to increase.

Example 2

The mother of patient U. has been ill with metabolic syndrome and obesity for the past two years. I applied for genetic counseling to my 6-year-old daughter, who was also obese from birth. For genetic testing, 4 ml of blood was taken from mother and daughter. DNA extraction was carried out using the phenol-chloroform extraction method, followed by PCR-RFLP analysis of the AKT1 rs3803300, GABRA2 rs279845 loci

4 ml of venous blood was taken from the daughter, followed by DNA isolation by phenol-chloroform extraction and amplification of AKT1 rs3803300, GABRA2 rs279845 gene fragments in a reaction mixture containing 0.1-1 μg of genomic DNA, 1 u. Taq polymerase, 0.25 µM of each oligoprimer, 250 µM of each deoxynucleoside triphosphate in 25 µl of single PCR buffer for each locus. The resulting amplifications of the rs3803300 markers of the AKT1 gene were uncoupled with the addition of the Alw26 I enzyme and rs279845 of the GABRA2 gene.

After amplification, electrophoresis of gene fragments AKT1 rs3803300, GABRA2 rs279845 was performed at a constant voltage of 250-300 volts after 15-minute preelectrophoresis. After the end of electrophoresis, the gel was stained with a solution of ethidium bromide for 10 minutes and analyzed under ultraviolet light on a transilluminator.

In the study of the AKT1 rs3803300 polymorphic locus, the AA genotype was identified, in which the odds ratio for developing obesity is 3.37 and the AT rs279845 genotype of the GABRA2 gene, in this case, the risk is 0.66 (Table 2).

Given the high genetic risk of obesity in a child, parents were offered preventive measures to correct the child's weight, lifestyle changes, diet therapy, and increased physical activity. These recommendations were not fully implemented, the patient's daughter continued to gain weight.

Example 3

Patient N., aged 14, has been obese since the age of 8. The presence of risk factors was revealed in the family: the mother suffers from grade III obesity. The father of the child has II degree obesity and arterial hypertension. At a consultation with an endocrinologist, the child's parents were offered a molecular genetic study to determine the risk of developing childhood obesity. The patient was taken biological material 4 ml of blood. The results of the molecular genetic analysis followed by DNA isolation by phenol-chloroform extraction and amplification of AKT1 rs3803300, GABRA2 rs279845 gene fragments in a reaction mixture containing 0.1-1 μg of genomic DNA, 1 u. Taq polymerase, 0.25 µM of each oligoprimer, 250 µM of each deoxynucleoside triphosphate in 25 µl of 1 x PCR buffer. After amplification, restriction was performed, followed by electrophoresis of the fragments at a constant voltage of 250–300 volts after 15 minutes of preelectrophoresis. After the end of electrophoresis, the gel was stained with a solution of ethidium bromide for 10 minutes and analyzed under ultraviolet light on a transilluminator. In the study of the AKT1 rs3803300 polymorphic locus, the AG genotype was identified, in which the odds ratio for developing obesity is 1.56 and the AA rs279845 genotype of the GABRA2 gene, in this case, the risk is 0.81 (Table 2).

The patient's parents were offered measures to correct the child's weight: diet therapy, medications, increased physical activity. These recommendations were not fully implemented; during the examination two years later, the child was diagnosed with arterial hypertension, obesity persisted.

Example 4

Patient B., 8 years old, was treated in a hospital for obstructive bronchitis. Additionally, endogenous-constitutional obesity of the 1st degree was diagnosed. The patient's mother has been diagnosed with metabolic syndrome and obesity over the past ten years. The family was advised to consult an endocrinologist. The patient was offered a molecular genetic study to determine the risk of developing childhood obesity. For molecular genetic analysis, 4 ml of venous blood was taken from patient K., followed by DNA isolation by phenol-chloroform extraction and amplification of gene fragments AKT1 rs3803300, GABRA2 rs279845 in a reaction mixture containing 0.1-1 μg of genomic DNA, 1 unit . Taq polymerase, 0.25 µM of each oligoprimer, 250 µM of each deoxynucleoside triphosphate in 25 µl of 1 x PCR buffer. After amplification, electrophoresis of the fragments was carried out at a constant voltage of 250-300 volts after a 15-minute pre-electrophoresis. After the end of electrophoresis, the gel was stained with a solution of ethidium bromide for 10 minutes and analyzed under ultraviolet light on a transilluminator. In the study of the AKT1 rs3803300 polymorphic locus, the AG genotype was identified, in which the odds ratio for developing obesity is 1.56 and the AT rs279845 genotype of the GABRA2 gene, in this case, the risk is 0.66 (Table 2).

The child's parents were offered preventive measures to correct the child's weight through diet therapy and increased physical activity. These recommendations were not fully implemented, the patient's son continued to gain weight, which was recorded at a second consultation with an endocrinologist a year later.

Example 5

Patient K., 4 years old, consulted a gastroenterologist about abdominal pain. Was diagnosed with biliary dyskinesia and obesity I degree. The family was advised to consult an endocrinologist. The patient was offered a molecular genetic study to determine the risk of developing childhood obesity. For molecular genetic analysis, 4 ml of venous blood was taken from patient K., followed by DNA isolation by phenol-chloroform extraction and amplification of gene fragments AKT1 rs3803300, GABRA2 rs279845 in a reaction mixture containing 0.1-1 μg of genomic DNA, 1 unit . Taq polymerase, 0.25 µM of each oligoprimer, 250 µM of each deoxynucleoside triphosphate in 25 µl of 1 x PCR buffer. After amplification, electrophoresis of the fragments was carried out at a constant voltage of 250-300 volts after a 15-minute pre-electrophoresis. After the end of electrophoresis, the gel was stained with a solution of ethidium bromide for 10 minutes and analyzed under ultraviolet light on a transilluminator. The study of the polymorphic AKT1 rs3803300 locus revealed the reduced risk GG genotype (the odds ratio for developing obesity in this case is 0.61), and the study of the GABRA2 polymorphic locus rs279845 revealed the protective TT genotype (OR=1.60) (Table 2).

For these options, the prognosis for the development of obesity in the future is favorable. Subsequently, the parents did not turn to an endocrinologist.

These examples demonstrate the prognostic and diagnostic significance of the proposed method. The method allows predicting the risk of developing childhood obesity before its clinical manifestations and the development of comorbid conditions of the disease.

Claims (1)

A method for predicting the development of obesity in children living in the Republic of Bashkortostan, including DNA isolation from peripheral venous blood lymphocytes, genotyping by polymerase chain reaction DNA synthesis, characterized in that amplification of the rs3803300 locus of the AKT1 protein kinase 1 gene and the rs279845 locus of the gamma-aminobutyric acid receptor gene GABRA2 and detection of AG or AA genotypes of the rs3803300 polymorphic locus of the AKT1 gene and AT or AA genotypes of the rs279845 polymorphic locus of the GABRA2 gene predict the risk of developing obesity in a child.