RU2746234C1 - Method for predicting the risk of thrombotic complications of patients with cardiovascular pathology - Google Patents

Abstract

FIELD: medicine; cardiology.SUBSTANCE: method for predicting the risk of thrombotic complications of patients with cardiovascular pathology is proposed. In the peripheral blood of the patient, polymorphisms of the eNOS:G894T and AGTR2:1675 genes are determined. When the TT homozygote or GT heterozygote of the eNOS rs1799983 gene is combined with the AA homozygote or GA heterozygote of the AGTR2 rs1403543 gene, a high risk of thrombosis is predicted.EFFECT: invention provides increased accuracy and objectivity of predicting the risk of thrombotic complications of patients with cardiovascular pathology.1 cl, 7 dwg, 4 tbl, 3 ex

Description

The invention relates to the field of cardiology and, in particular, to the treatment of coronary heart disease, the prevention of thrombotic complications.

Cardiovascular disease (CVD) is the leading cause of death worldwide: for no other reason as many people die each year as from CVD. (WHO, 17.05.2017), (Iskakov E.B. Epidemiology of cardiovascular diseases // Iskakov Evgeniy Borisovich (2017). Epidemiology of cardiovascular diseases. Medicine and ecology, 2017; 2: 19-28).

Polymorphism of the eNOS gene can cause endothelial dysfunction, which leads to the development of ischemic heart disease, and the AT2 receptor is part of the renin-angiotensin-aldosterone system, which is involved in the regulation of blood circulation and vascular tone and also has a wide range of functions, involving mechanisms of increasing nitric oxide production (Khokhlov A.L. Polymorphism of the eNOS and AGTR2 genes as a risk factor for the development of coronary heart disease // Problems of standardization in healthcare 2015; 9-10: 46-50).

Along with modifiable risk factors, there are those that cannot be influenced: gender, genetic characteristics. Therefore, one of the urgent problems is the search for the genetic basis for the development of cardiovascular diseases. These genes include: the eNOS gene, which produces a regulator of the vascular wall - nitric oxide; type 2 angiotensin II receptor gene (AGTR2), which is an element of the renin-angiotensin-aldosterone system involved in the regulation of blood circulation and vascular tone. The clinical guidelines emphasize the special role of genes that regulate endothelial function, since their structural changes can serve as a pathophysiological basis for the development of coronary heart disease.

It is known that the endothelial NO synthase gene encodes an enzyme (Pozdnyakov, N.O. Polymorphism of the eNOS gene: prevalence and relationship with diseases / N.O. Pozdnyakov, A.L. Khokhlov // Pharmateka. - 2015. - No. 13. - Pp. 21-24), responsible for the synthesis in the vascular wall of nitric oxide - the most important vasodilator, antiplatelet agent, antimitogen and antioxidant (Belous, A.S. Assessment of the correction of morphological changes in modeling endothelial dysfunction with ultra-low doses of antibodies to eNOS in a 28-day model L -NAME of induced nitric oxide deficiency / A.S. Belous // Scientific Bulletin of Belgorod State University. Series: Medicine. Pharmacy. - 2014. - No. 11-1 (182), volume 26. - P. 127-130 .; Parshina , S. S. Biological effects of nitric oxide in the development of cardiovascular pathology as the basis for the use of terahertz therapy / S. S. Parshina, T. N. Afanasyeva, V. D. Tupikin // BMIK. - 2012. - No. 6. - P. 446-452).

Suppression or decrease in eNOS activity, as well as the manifestation of polymorphic variants of this gene (GT, TT), leads to a decrease in the level of expression of NO synthase and a lack of nitric oxide, which is manifested by endothelial dysfunction leading to the development of atherogenesis and atherothrombosis. In this regard, functional variants of the eNOS gene affect individual susceptibility to atherosclerosis (Bebyakova, N.A. Analysis of the effect of 894g> t polymorphism of the nos 3 gene on the production of vasoactive endothelial factors / N.A. Bebyakova, A.A. Kuba, O M. Feliksova, A. V. Khromova // Modern medicine: topical issues and development prospects. - 2015. - Issue II. - P. 74-77).

In the NOS3 gene in the 4th intron, the polymorphism of the promoter region of the gene is 786T> C and the structural replacement in the 7th exon 894G> T. This variant of the eNOS 894G> T gene polymorphism is important because the T allele leads to a decrease in eNOS activity (Hinz, J. The eNOS 894G / T gene polymorphism and its influence on early and long-term mortality after on-pump cardiac surgery / J . Hinz // Journal of Cardiothoracic Surgery. - 2013. - No. 8. - P. 199.) and a decrease in the level of NO (Mackawy, AMN. Association of the endothelial nitric oxide synthase gene G894T polymorphism with the risk of diabetic nephropathy in Qassim region , Saudi Arabia-A pilot study / AM.H. Mackawy, AA Khan, ME-S Badawy // Meta Gene. - 2014. - No. 2. - P. 392-402; Gu, Z. Promoter polymorphism T-786C; , 894GAT at exon 7 of endothelial nitric oxide synthase gene are associated with risk of osteoporosis in Sichuan region male residents / Z. Gu, Y. Zhang, G. Qiu // Int J Clin Exp Pathol. - 2015. - No. 8 (11 - P. 15270-4; Liu, D. Association between the - 786T > C lpolymorphism in the promoter region of endothelial nitric oxide synthase (eNOS) and risk of coronary artery disease: as ystematic review and meta-analysis / D. Liu, Z. Jiang, L. Dai [et al.] // Gene. - 2014. - No. 545 (1). - P. 175-83).

It is known that in the development of essential AT, the leading role is assigned to the polymorphism of the following genes: REN (renin gene), ACE (angiotensin-converting enzyme gene), AGT (angiotensinogen gene), AGTR1 (type 1 receptor gene for angiotensin II), AGTR2 (receptor gene Type 2 to angiotensin II), BKR2 (type 2 bradykinin receptor gene), ADRB1 (β1 adrenergic receptor gene), ADRB2 (β2 adrenergic receptor gene), MTHFR (5,10 methylenetetrahydrofolate reductase gene), NOS3 (NO synthase gene) 3 types). The products of these genes provide different steps in the same metabolic chain.

In recent years, the interest of many researchers has focused on the function of type 2 receptors for ATP, which are present in almost all tissues, especially in the vascular endothelium. The gene for the type 2 receptor for angiotensin II (AGTR2) is located on the long arm of the X chromosome at the Xq22-q23 locus and contains 3 exons. The AGTR2 gene is expressed primarily under estrogen control. Like AGTR1, AGTR2 is also involved in angiotensin II-mediated reactions, but, being its antagonist, it predominantly controls vasodilatory functions. 5 polymorphic variants of the AGTR2 gene have been described. The most studied is the 3123 OA polymorphism linked to the + 1675G> A variant in intron 1, which affects the start of transcription. An association of the 3123A variant with hypertension in adult women and in boys with hypertension has been shown, that is, variants of homo- (AA) and heterozygotes (GA) are associated with the development of hypertension (OV Shevchenko Genetic bases of the pathogenesis of essential arterial hypertension (review) / O V. Shevchenko, A. A. Svistunov, V. B. Borodulin, A. V. Ruta, E. N. Bychkov // Saratov Journal of Medical Scientific Research. - 2011. - Volume 7 - No. 1. - P. 83 -87).

The purpose of the proposed method for predicting the risk of thrombotic complications in patients with cardiovascular disease is to improve accuracy and objectivity.

This goal is achieved by the fact that patients with cardiovascular pathology in the peripheral blood determine the polymorphism of the eNOS rs 1799983 and AGTR2 rs 1403543 genes. And if a combination of eNOS rs 1799983 and AGTR2 rs 1403543 gene polymorphisms is detected in the form of homo- and / or heterozygotes, a high risk of thrombotic complications.

The novelty of the proposed method lies in the fact that the prediction of thrombotic complications is carried out taking into account the frequency of occurrence of polymorphisms of the eNOS rs 1799983 and AGTR2 rs 1403543 genes responsible for the development of this pathology.

Technical solutions that have features that coincide with the distinctive features of the proposed method have not been identified, which allows us to conclude that the proposed method meets the criterion of "inventive step".

The proposed method is carried out as follows.

First, peripheral blood is drawn from the patient. Then, the patient's DNA is isolated from the blood by the method of polymerase chain reaction (PCR). To obtain DNA from the analyzed material, a set of reagents for isolation (from Syntol) is used. To assess the amount of isolated DNA, a set of reagents for PCR amplification of human genomic DNA in real time (KVM) manufactured by Syntol is used.

Further, using a set of reagents intended for use with detecting amplifiers (Syntol company), polymorphism of the eNOS rs 1799983 and AGTR2 rs 1403543 genes is determined.

After that, interpretation of the results obtained is performed. With a combination of polymorphisms of the eNOS rs1799983 and AGTR2 rs 1403543 genes in the form of homo- and / or heterozygotes, a high risk of thrombosis is predicted.

Clinical studies of the method were carried out on 200 patients with various forms of cardiovascular pathology: myocardial infarction, unstable and stable forms of angina pectoris with concomitant conditions: stroke, atherosclerosis of brachiocephalic vessels, leg vessels, venous thrombosis.

To determine the possible associations of the eNOS and AGTR2 genes with the risk group, all patient groups were divided into 3 subgroups in accordance with the allelic variants GG, GA, AA for the analysis of the AGTR2 gene; and in accordance with allelic variants GG, GT, TT for eNOS gene analysis. The frequency of occurrence of alleles of the eNOS gene in the first subgroup of patients of very high risk is: GG - 8 (22.2%), GT - 23 (63.9%), TT - 5 (13.9%), in the second, high risk - GG - 34 (39.5%), GT - 50 (58.2%), TT - 2 (2.3%), in the third - low and medium risk - GG - 52 (66.7%), GT - 24 (30.8%), TT - 2 (2.5%) (Table 1.1).

The calculation was performed by the chi-square method using correlation analysis (Table 1.2).

The minimum expected number is 1.62.

The frequency of occurrence of alleles of the AGTR2 gene in the first subgroup of patients is: GG - 12 (33.3%), GA - 6 (16.7%), AA - 18 (50%), in the second - GG - 26 (30.2% ), GA - 21 (24.4%), AA - 39 (45.3%), in the third - GG - 46 (59%), GA - 17 (21.8%), AA - 15 (19.2 %) (Tables 2.1, 2.2)

The calculation was performed by the chi-square method using correlation analysis (Table 2.2).

The minimum estimated number is 7.92.

A direct correlation was found between risk groups with genes eNOS (0.639, p <0.0001) and AGTR2 (0.270, p <0.0001), both homo- and heterozygote, in which the percentage of the polymorphic homozygous gene was noted for risk groups ( allelic variant of TT gene eNOS rs 1799983 and AA gene AGTR2 rs1403543), (see Fig. No. 1), which shows the percentage of polymorphic heterozygous gene by risk groups (allelic variant of GT gene eNOS rs 1799983 and GA gene AGTR2 rs1403543) (see Fig. No. 2). Statistical processing was performed by the chi-square method (p <0.005).

Thus, in patients with cardiovascular pathology, the occurrence of thrombotic complications correlates with the polymorphism of the eNOS rs 1799983 and AGTR2 rs1403543 genes.

When conducting conjugate analysis through chi-square, it was proved that the combination of polymorphism of the eNOS rs 1799983 and AGTR2 rs 1403543 genes in the form of homo- and / or heterozygotes is a predictor of thrombotic complications (Fig. 1-7).

FIG. 1 shows the ratio of a polymorphic homozygous gene with risk groups (TT of the eNOS rs1799983 gene and AA of the AGTR2 rs1403543 gene).

FIG. 2 shows the ratio of the polymorphic heterozygous gene with risk groups (GT of the eNOS gene rs 1799983 and GA of the AGTR2 gene rs1403543).

FIG. 3 shows the ratio of the polymorphic homozygous gene (TT gene eNOS rs 1799983) and heterozygous (GA gene AGTR2 rs 1403543) with risk groups.

FIG. 4 shows the ratio of the polymorphic heterozygous gene (GT gene eNOS rs1799983) and homozygous (AA gene AGTR2 rs1403543) with risk groups.

FIG. 5 shows the ratio of normal genes (GG gene eNOS rs1799983) and (GG gene AGTR2 rs1403543) with risk groups.

FIG. 6 shows the ratio of the normal eNOS gene (GG of the eNOS gene rs1799983) with the homo and heterozygotes AGTR2 (GA, AA of the AGTR2 gene rs1403543).

FIG. 7 shows the ratio of the normal AGTR2 gene (GG of the AGTR2 gene rs1403543) with the homo- and heterozygote eNOS (TT, GT of the eNOS gene rs1799983).

Note: * p <0.005. Statistical processing was carried out by the chi-square method.

We give clinical examples of the application of the proposed method. The first and third examples - with an extreme degree of deviations (correspond to Fig. 1 and Fig. 5, respectively), the second example corresponds to intermediate variants of gene polymorphism (Fig. 2, 3, 4, 6, 7).

Example # 1. Patient M., 64 years old, was admitted to the cardiology department of the primary vascular center with a diagnosis of acute coronary syndrome, then Q-forming myocardial infarction developed. Diagnosis: Ischemic heart disease: acute Q-forming myocardial infarction. Atherosclerosis of the aorta, valves, brachiocephalic arteries with stenosis up to 60%. Essential hypertension, stage 3, stratification risk 4. NK II according to Killip. Paroxysmal atrial fibrillation. CHF 2a FC 3. Condition after suffering from stroke. Atherosclerosis of the vessels of the legs.

Genetic testing revealed the carriage of eNOS rs1799983 gene polymorphism in the form of TT homozygote and AGTR2 rs 1403543 in the form of AA homozygote, which indicates a high risk of thrombotic complications.

This was confirmed by the fact that a year later the patient developed a recurrent Q-forming myocardial infarction and during coronary angiography revealed multiple atherosclerotic lesions (plaques).

Example # 2. Patient G., 55 years old, was admitted to the cardiology department of acute coronary syndrome, which turned into unstable angina pectoris with an outcome in angina pectoris of a high functional class - 3 FC. She has a history of PE, post-thrombotic leg vein disease (PTD), BCA atherosclerosis. The diagnosis of ischemic heart disease: unstable angina with an outcome in angina pectoris of 3 FC. Atherosclerosis of the aorta, valves, brachiocephalic arteries with stenosis up to 30%. Essential hypertension, stage 3, stratification risk 4. NK I according to Killip. CHF 2 FC 2. Condition after postponed pulmonary embolism. PTB legs.

Genetic testing revealed the carriage of eNOS rs1799983 gene polymorphism in the form of GT heterozygote and AGTR2 rs 1403543 in the form of GA heterozygote, which indicates a high risk of thrombotic complications.

This was confirmed by the progression of atherosclerosis against the background of adequate therapy: the development of acute myocardial infarction after 1 year and 8 months, an increase in atherosclerotic plaques up to 40%.

Example No. 3. Patient F., 74 years old, was admitted to the cardiology department with acute coronary syndrome, which turned into angina pectoris of a low functional class - 2 FC. Permanent atrial fibrillation also occurs. History of atherosclerosis of the brachiocephalic arteries. Diagnosis: ischemic heart disease: unstable angina with outcome in angina pectoris 2 FC. Atherosclerosis of the aorta, valves, brachiocephalic arteries. Atrial fibrillation is a permanent form. Widespread osteochondrosis of the spine, thoracalgia.

Genetic testing revealed the carriage of polymorphism of the eNOS gene - homozygote GG, gene AGTR2 in the form of homozygote GG, which indicates a low risk of thrombotic complications in cardiovascular pathology. This was confirmed by a long follow-up period: during 5 years of follow-up, the patient had no significant progression of atherosclerosis according to the USAS BCA data, there was no progression of the functional class of angina pectoris and the development of other cardiovascular complications.

The proposed method for predicting the risk of thrombotic complications in patients with cardiovascular pathology was used in OGBUZ "District hospital of the Kostroma district No. 1", NUZ "Road clinical hospital at st. Yaroslavl JSC "Russian Railways".

Claims (1)

A method for predicting the risk of thrombotic complications in patients with cardiovascular pathology, including the determination of gene polymorphisms in the patient's peripheral blood, characterized in that polymorphisms of the eNOS genes are determined in the patient's peripheral blood: G894T and AGTR2: 1675 and when a TT homozygote or a GT gene heterozygote is combined eNOS rs1799983 and homozygotes AA or heterozygotes GA of the AGTR2 gene rs1403543 predict a high risk of thrombosis.

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