RU2565407C1 - Method for predicting risk of stage iii hypertensive disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: DNA is recovered from peripheral venous blood of Russian patients born in the Central Black Earth of the Russian Federation, and TNFα gene -308G/A polymorphisms are genotyped. A risk of stage III hypertensive disease is stated depending on the detected locus variants -308G/A TNFα and other predictors of this disease. If the derived value is more than 0.50, a high risk of the disease is predicted.

EFFECT: invention enables predicting the risk of stage III hypertensive disease effectively.

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Description

The invention relates to the field of medical diagnosis, can be used to predict the risk of developing stage III hypertension.

Hypertension is the most common cardiovascular disease among the working-age population [Mancia G., Fagard R., Narkiewicz K., 2013. ESH / ESC guidelines for the management of arterial hypertension. Journal of Hypertens, 31, (7): 1281-1357]. It is known that stage III hypertension is characterized by the development of associated (concomitant) clinical conditions and is one of the most significant risk factors for myocardial infarction, acute cerebrovascular accident, which are characterized by high and early disability, as well as premature mortality of patients, which explains the great social the significance of this pathology [Lubna A. Al-Ansary, Andrea C. Tricco, Sharon E. Straus. A Systematic Review of Recent Clinical Practice Guidelines on the Diagnosis, Assessment and Management of Hypertension. Plos one. 2013; 8 (1): 537-44].

Therefore, the ability to predict the risk of developing stage III hypertension will optimize the individual management tactics of these patients with hypertension, aimed at preventing the development of stage III disease.

Hypertension is a multifactorial disease with a pronounced genetic component. According to experimental and clinical data, a significant contribution to the development of this disease is made by the cytokines involved in the inflammatory process and apoptosis mechanisms. Among cytokines, tumor necrosis factor alpha (TNFα) deserves special attention.

TNFα is a multifunctional cytokine that is synthesized by activated macrophages, monocytes, neutrophils, dendritic cells, T and B lymphocytes, endothelial, smooth muscle cells, natural killers and mast cells [Hollegaard, M.V. Cytokine gene polymorphism in human disease [Text] / M.V. Hollegaard, J.L. Bidwell // Genes Immun. - 2006. - Vol. 7. - Suppl. 3. - P. 269-276]. He is involved in the regulation of a wide range of biological processes, such as proliferation, cell differentiation [Waters J. P., Pober J. S., Bradley J. R., 2013. Tumor necrosis factor in infectious disease. J. Pathol., 230 (2): 132-147], affects insulin resistance, endothelial function, lipid metabolism [The -308G / A of tumor necrosis factor (TNF) -α and 825C / T of guanidine nucleotide binding protein 3 (GNB3) are Associated with the Onset of Acute Myocardial Infarction and Obesity in Taiwan [Text] /W.-T. Chang, Y.-C. Wang, C.-C. Chen [et al.] // Int. J. Mol. Sci. - 2012. - Vol.13, No. 2. - P. 1846-1857], takes part in the regulation of apoptosis (programmed cell death), and also activates the processes of oxidative stress of cardiomyocytes [Eliseev, M. S. The role of tumor necrosis factor alpha (TNF-α) in the development of metabolic disorders and atherosclerosis and the effect of TNF-α inhibitors on them in patients with rheumatic diseases [Text] / M. S. Eliseev, V. G. Barskova, E. L. Nasonov // Scientific and Practical Rheumatology. - 2009. - No. 2. - S. 67-72].

The gene encoding the TNFα protein includes 4 exons and 3 introns and maps to the human genome on the short arm of the sixth chromosome (6p21.3), located next to the genes of the main histocompatibility complex [Tumor Necrosis Factor and Lymphotoxin Alfa Genetic Polymorphisms and Outcome in Pediatric Patients With Non-Hodgkin's Lymphoma: Results From Berlin-Frankfurt-Münster Trial NHL-BFM 95 [Text] / K. Seidemann [et al.] // Journal of Clinical Oncology. - 2005 .-- Vol.23. - No. 33. - P. 8414-8421]. The most common type of TNFα gene mutations are single nucleotide substitutions in the promoter region. Some allelic variants of this gene increase the binding of transcription factors to the promoter and activate transcription, which subsequently leads to an increase in the expression of TNFα [Tumonochrismophthalpha-308 and lhymphothophonhomhthonhimphonhimphonhimphonhimphon /BUT. Ibráhim, H. Abdel Ráhman, M. Khorshiеd [et al.] // Leuk. Res. - 2012. - Vol. 36, No. 6. - R. 694-698].

In the studied medical and patent literature, the authors did not find a method for predicting the risk of stage III development in patients with hypertension based on data on the genetic polymorphism of -308G / A TNFα and other predictors of the development of the disease.

To assess the current patent situation, a search was performed on the title documents for the period from 1990 to 2013. The analysis of documents was carried out in the direction: a method for predicting the risk of developing stage III hypertension based on molecular genetic data depending on the polymorphic markers of the TNFα gene.

RF patent No. 23239503 (publication date July 20, 2008) proposes a method for assessing the risk of progression of arterial hypertension in women, which consists in blood sampling and analysis of biochemical parameters of energy metabolism of red blood cells, according to which, depending on the age of women, the prognosis tables determine the percentage of the risk of weight loss hypertension in the next 5 years.

The disadvantage of this method is the complexity of the analysis and the low availability of the equipment used, in addition, this method is applicable only to women and does not consider genetic polymorphisms for predicting the risk of developing stage III hypertension.

For the prototype, RF patent No. 2456608 was selected (publication date 07/20/2012) "A method for predicting the risk of hypertension in men." The inventive method involves the isolation of genomic DNA from the peripheral venous blood of patients, the identification of polymorphic variants of genes and allows to predict the development of hypertension based on the genotyping of polymorphic loci of genes of flavin monooxygenase type 3, cytochrome P-450 1 B1, N-acetyltransferase type 2 and analysis of environmental risk factors such as the pattern of alcohol consumption, the fact of smoking, and exposure to harmful chemicals using logistic regression analysis.

The disadvantage of this method is that it considers the risk of developing hypertension only in men, and according to published data, hypertension develops more often in women [Epidemiology of arterial hypertension in Russia. The results of federal monitoring in 2003-2010. / R.G. Oganov [et al.] // Cardiovascular therapy and prevention. - 2011. - No. 1. - S. 9-13]. In addition, candidate genes associated with the inflammatory mechanism of the development of hypertension and its course are not considered, and the risks of the development of associated clinical conditions, i.e. III stage.

The objective of this study is to expand the arsenal of methods for diagnosing stage III hypertension, namely, creating a method for predicting the risk of developing stage III hypertension based on data on the genetic polymorphism -308G / A TNFα and other predictors of the development of the disease.

The technical result of using the invention is to obtain criteria for assessing the prognosis of the development of stage III hypertension in individuals of Russian nationality, natives of the Central Chernozem region.

In accordance with the task, a method for predicting the risk of developing stage III hypertension was developed, including:

- DNA isolation from peripheral venous blood;

- determination of polymorphic variants of the -308G / A TNFα gene;

- predicting the risk of developing stage III hypertension depending on the identified variants at the locus -308G / A TNFα and other predictors of the development of this disease according to the logistic regression equation of the following form:

y = 11-0.399 x ₁ -0.255 x ₂ +1.290 x ₃ -0.419 x ₄ +0.912 x ₅ -1.156 x _6,

where x ₁ - the duration of the disease, years; x ₂ - body mass index, kg / m ² ; x ₃ - the presence of metabolic syndrome (no - x ₃ = 1, yes - x ₃ = 2); x ₄ - cholesterol, mmol / l; x ₅ - low density lipoproteins, mmol / l, x ₆ - genetic variant locus -308G / A TNFα (AA - x ₆ = 1, AG - x 2 = _6, GG - x ₆ = 3).

- predicting a high risk of developing stage III hypertension if the value of y is above 0.50.

The novelty and inventive step lies in the fact that the possibility of predicting the risk of developing stage III hypertension by the presence of genetic variants at the -308G / A TNFα locus in combination with the above predictors is not known from the prior art.

The method is as follows:

DNA is isolated from samples of peripheral venous blood of individuals in 2 stages. At the first stage, 25 ml of lysis buffer containing 320 mM sucrose, 1% Triton X-100, 5 mM MgCl2, 10 mM Tris-HCl (pH = 7.6) is added to 4 ml of blood. The resulting mixture was stirred and centrifuged at 4 ° C, 4000 rpm for 20 minutes. After centrifugation, the supernatant is drained, 4 ml of a solution containing 25 mM EDTA (pH = 8.0) and 75 mM NaCl are added to the precipitate, and they are resuspended. Then, 0.4 ml of 10% SDS, 35 μl of proteinase K (10 mg / ml) was added and the sample was incubated at 37 ° C for 16 hours.

At the second stage, DNA is sequentially extracted from the obtained lysate in equal volumes of phenol, phenol-chloroform (1: 1) and chloroform with centrifugation at 4000 rpm for 10 minutes. After each centrifugation, the aqueous phase is selected. DNA is precipitated from solution in two volumes of chilled 96% ethanol. The formed DNA is dissolved in bidistilled, deionized water and stored at -20 ° C. Isolated DNA was used to carry out the polymerase chain reaction of DNA synthesis.

Analysis of the -308G / A TNFα polymorphism is carried out by polymerase chain reaction (PCR) DNA synthesis. PCR was performed on an IQ5 apparatus (Bio-Rad) in real time using Thermus aquaticus DNA polymerase manufactured by Sileks-M and standard oligonucleotide primers and probes synthesized by Syntol followed by analysis of polymorphisms by allele discrimination (Fig. one). To discriminate alleles, the Bio-Rad program “IQ5-Standart Edition” is used.

Genotyping is carried out using the Tag Man method of probes according to the RFU values (relative fluorescence level) of each probe. A probe with a fluorescent dye ROX corresponds to allele G, a probe with a dye FAM corresponds to allele A.

Two bands, vertical and horizontal, divide the graph into four sections: one for each homozygous state, one for the heterozygous state and a section without reaction. Assigning genotypes to unknown samples is determined by plotting the RFU for one fluorophore (on the x axis) relative to the RFU for another fluorophore (on the y axis) in the allele discrimination diagram.

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

• If the RFU values of an unknown sample are above the horizontal strip and to the left of the vertical strip, the genotype is homozygous for the G allele (the RFUs of the G allele are plotted along the y axis).

• If the RFU values of an unknown sample are below the horizontal strip and to the right of the vertical, the genotype is homozygous for the A allele (the RFUs of the A allele are plotted along the x axis).

• If the RFU of an unknown sample is below the horizontal bar and to the left of the vertical, genotype determination is not possible (in this case, an undefined sample is a negative control).

 The invention is characterized in the following figures:

FIG. 1, where discrimination of alleles at the locus -308G / A TNFα is presented, where ○ - homozygotes -308AA, □ - homozygotes -308GG, Δ - heterozygotes -308GA, ◊ - negative control.

FIG. 2, where the table shows the coefficients of logistic regression and the significance level of indicators used to predict stage III hypertension.

The possibility of using the proposed method for predicting the risk of developing stage III hypertension is confirmed by an analysis of the observation results of 452 individuals: 143 patients with hypertension with stage III disease and 309 patients with stages I and II of hypertension.

Criteria for inclusion in the studied samples:

1. Individuals of Russian nationality, who are natives of the Central Black Soil of the Russian Federation and do not have a relationship between themselves.

2. Voluntary patient consent for the study.

3. Individuals after the diagnosis of hypertension were included in the group of patients, which was confirmed by clinical and laboratory-instrumental methods of examination, in accordance with the mandatory diagnostic standards recommended by GFCF [GFCF Recommendations, 2010].

Exclusion criteria from the studied samples:

1. Patients with symptomatic arterial hypertension.

2. The presence of severe somatic pathology (liver, kidney failure, autoimmune and oncological diseases).

3. Patients of non-Russian nationality born outside the Central Black Earth Region of Russia.

4. Individuals who abandoned the study.

Clinical and laboratory examination of individuals was carried out on the basis of the cardiology department of the Belgorod Regional Clinical Hospital of St. Joasaph. Molecular genetic markers were typed in the laboratory of Human Molecular Genetics at the Medical Institute of the Belgorod State National Research University. The formation of the database and statistical calculations were carried out using the program "STATISTICA 6.0".

A mathematical model for predicting the risk of developing stage III hypertension using the method of binary logistic regression [Rebrova, O.Yu. Statistical analysis of medical data. Application of the STATISTICA application package [Text] /O.Yu. Rebrov. - [3rd ed.]. - Moscow: Media Sphere, 2006. - 305 p.: Ill .; Borovikov, V.P. Statistica: the art of computer data analysis [Text] / V.P. Borovikov. - 2nd ed. - St. Petersburg: Peter, 2003. - 688 p.].

During the logistic regression, the following statistical model was developed for predicting the risk of developing stage III hypertension, including the following factors: genetic variant at the locus -308G / A TNFα, duration of the disease, body mass index, presence of metabolic syndrome, cholesterol and low density lipoproteins (Fig. 2). The significance levels of the regression coefficients for all predictors were less than 0.05, i.e. each of them had a statistically significant effect on the risk of developing stage III hypertension. The value of the odds ratio for the presented regression model is 32.45, demonstrating that with an increase in the value of the i-th sign by a unit, the chance of the occurrence of stage III hypertension increases by more than 32 times.

Using the obtained logistic regression coefficients, based on data on the genetic variants of the -308G / A TNFα locus, disease duration, body mass index, presence of metabolic syndrome, cholesterol and low density lipoproteins, the risk of developing stage III hypertension can be predicted.

The logistic regression equation has the following form [Rebrova, O.Yu., 2006]:

y = b ₀ + b ₁ x ₁ + b ₂ x ₂ + b ₃ x ₃ + ... + b _n x _n ,

where x ₁ -x _n informative signs (risk factors), b ₀ -b _n - regression coefficients for these signs.

P = e ^y / (1 + e ^y ),

where e is a mathematical constant approximately equal to 2.72.

In our case, the logistic regression equation will be as follows:

y = 11-0.399 x ₁ -0.255 x ₂ +1.290 x ₃ -0.419 x ₄ +0.912 x ₅ -1.156 x _6,

where x ₁ - the duration of the disease, years; x ₂ - body mass index, kg / m ² ; x ₃ - the presence of metabolic syndrome (no - x ₃ = 1, yes - x ₃ = 2); x ₄ - cholesterol, mmol / l; x ₅ - low density lipoproteins, mmol / l, x ₆ - genetic variant at the locus -308G / A TNFα (AA - x ₆ = 1, AG - x ₆ = 2, GG - x ₆ = 3).

In order to verify the model’s performance, two additional patients with Russian hypertension, who are residents of the Central Chernozem Region, were additionally included in the analysis. Patient A. identified the following indicators: disease duration - 7 years (x ₁ ), body mass index - 32.0 kg / m ² (x ₂ ), the presence of metabolic syndrome - yes (x ₃ = 2), cholesterol - 6, 0 mmol / L (x ₄ ), low density lipoproteins - 4.5 mmol / L (x ₅ ), the genetic version of the locus is -308G / A TNFα - AA (x ₆ = 1).

For patient B., the following indicators were determined: the duration of hypertension - 10 years (x ₁ ), body mass index - 31.2 kg / m ² (x ₂ ), the presence of metabolic syndrome - no (x ₃ = 1), cholesterol - 5.4 mmol / l (x ₄ ), low density lipoproteins - 2.6 mmol / l (x ₅ ), the genetic version of the locus is -308G / A TNFα - GG (x ₆ = 3).

Substituting the values of the corresponding indicators into the regression equation, we obtain for patient A.

y = 11-0.399 * 7-0.255 * 32.0 + 1.290 * 2-0.419 * 6.0 + 0.912 * 4.5-1.156 * 1 = 3.061

accordingly, P = 2.72 ^3.061 / (1 + 2.72 ^3.061 ) = 0.9553, i.e. the risk of developing stage III hypertension in patient A. is high, because a value of y is above 0.50.

For patient B .:

y = 11-0.399 * 10-0.255 * 31.2 + 1.290 * 1-0.419 * 5.4 + 0.912 * 2.6-1.156 * 3 = -3.0774,

respectively P = 2.72 ^-3.0774 / (1 + 2.72 ^-3.0774 ) or (1 / 2.72 ^3.0774 ) / 1 + (1 / 2.72 ^3.0774 ) = 0.0460 , i.e. the risk of developing stage III hypertension in patient B. is extremely low, because a value of y is below 0.50.

Thus, we can conclude that this model of logistic regression is workable and allows you to effectively predict the risk of developing stage III hypertension, i.e. associated clinical conditions, depending on the genetic variants of tumor necrosis factor α, duration of the disease, body mass index, presence of metabolic syndrome, cholesterol and low density lipoproteins in order to determine the management tactics of a patient with hypertension to prevent the development of associated clinical conditions.

Claims (1)

A method for predicting the risk of developing stage III hypertension, including the isolation of genomic DNA from the peripheral venous blood of patients, characterized in that patients of Russian nationality who are natives of the Central Black Earth Region of the Russian Federation determine the TNFα gene polymorphism genotypes -308G / A; they predict the risk of developing stage III hypertension depending on the identified variants at the locus -308G / A TNFα and other predictors of the development of this disease according to the logistic regression equation of the following form:
y = 11-0.399x ₁ -0.255x ₂ + 1.290x ₃ -0.419x ₄ + 0.912x ₅ -1.156x _6,
where x ₁ - the duration of the disease, years; x ₂ - body mass index, kg / m ² ; x ₃ - the presence of metabolic syndrome (no - x ₃ = 1, yes - x ₃ = 2); x ₄ - cholesterol, mmol / l; x ₅ - low density lipoproteins, mmol / l; x ₆ is the genetic variant at the locus -308G / A TNFα (AA - x ₆ = 1, AG - x ₆ = 2, GG - x ₆ = 3), and a high risk of developing stage III hypertension is predicted if above 0.50.

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