RU2469096C2 - Method for detection of genetic predisposition to developing myocarial infarction in individuals with no clinical implications of ischemic heart disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to a method for detection of genetic predisposition in an individual with no clinical implications of ischemic heart disease (IHD) to developing myocardial infarction (MI). The invention may be used in cardiology for detection of genetic predisposition to developing myocardial infarction (MI). A combination of two pathogenetically MI-related polymorphisms representing single nucleotide substitutes C1019T in connexin-37 (Cx37) gene and G894T in endothelial NO-CHHTa3bi.(eNOS) gene are examined. A high degree of risk of developing MI in an individual being examined is stated by a combination of homozygous genotype TT in C1019T polymorphism of Cx37 gene and homozygous genotype GG in G894T polymorphism of eNOS gene.

EFFECT: invention enables specifying a group of high risk of developing MI among almost healthy persons for the purpose of taking primary prevention actions.

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Description

Introduction

The present invention relates to the field of molecular biology and genetics and can be used in medicine to predict the risk of myocardial infarction (MI). A method is proposed for determining the hereditary predisposition to the development of myocardial infarction in individuals without clinical manifestations of coronary heart disease (CHD), which involves testing the subject's DNA for the presence of a specific combination of alleles (a certain genotype) using the polymorphic site 1019 of the connexin-37 gene (Cx37) and the polymorphic site 894 of the gene endothelial NO synthase (eNOS).

Myocardial infarction is an important medical and social problem, as it has become one of the leading causes of death and disability worldwide. Currently, it is considered proven that the cause of most MI is intravascular thrombosis, which usually occurs in place of an existing atherosclerotic plaque with a damaged surface (Zaman A.G. et al., 2000). Moreover, the plaques that cause myocardial infarction are most often non-occlusive and narrow the artery lumen by an average of 48% (Ambrose J.A. et al., 1988). Such lesions, as a rule, are hemodynamically insignificant and do not lead to the appearance of typical symptoms of angina pectoris. Accordingly, people with these plaques are considered to be practically healthy in most cases, so the occurrence of MI appears to be a sudden event. The situation is aggravated by the fact that to date, reliable methods for preclinical diagnosis of MI have not been developed.

It is known that genetically determined variations in the activity of proteins involved in the pathogenesis of atherosclerosis and its complications play an important role in the development of MI (Lane D.A., Grant P.J., 2000, Damani S.B., Topol E.J., 2007, Packard R.R., Libby P., 2008). Since for many genetic factors there are no biochemical or other correlates that can be measured in patients, the method of direct genotyping seems to be the most promising direction in preclinical diagnosis and prognosis of MI in people without clinical manifestations of coronary artery disease.

State of the art

In recent years, much attention has been paid to the study of genetic risk factors for the development of CHD and MI. A significant number of studies have been carried out in which polymorphic markers in the genes of proteins associated with endothelial dysfunction, the development of inflammatory reactions, the regulation of migration and proliferation of smooth muscle cells, coagulation, fibrinolysis and many other processes have been studied (Zhu MM et al., 2000, Xu J. et al., 2010, Siegerink B. et al., 2009, Tanis BC et al., 2004, Santoso S. et al., 1999, Eriksson P. et al., 1995, Iacoviello L. et al., 1998).

Recently, one of the most widely discussed risk factors for the development of coronary heart disease and myocardial infarction is the connexin-37 gene polymorphism (Cx37). Cx37 in the human body is expressed, in particular, in the endothelium, monocytes and smooth muscle cells located under the atherosclerotic plaque (ASB). It participates in monocyte adhesion, mediates interactions between endothelium and vascular smooth muscle cells and is considered by many researchers as an important molecular factor involved in the development of atherosclerosis (Derouette J.P. et al., 2009). In 1997, Richard et al. described a single nucleotide substitution of C for T at the 1019 position of the Cx37 gene, leading to the replacement of proline with serine at position 319 of the protein amino acid sequence (Richard G. et al., 1997). Later, in a number of studies, the relationship of the mutant homozygous TT genotype with the development of MI was demonstrated (Hirashiki A. et al., 2003, Listi F. et al., 2005, Listi F. et al., 2007).

However, there are at least two reasons why the results of a study of a polymorphism (in this case 1019T in the Cx37 gene) associated with a particular disease (in this case, MI) cannot be considered a sufficient basis for predicting the risk of developing this diseases. Firstly, the individual contribution of each individual genetic variation to the risk of developing the disease is small, so the genotyping of patients with only one polymorphism has a low predictive power. In addition, polymorphism may have different pathogenetic significance in representatives of different populations. Obviously, increasing the reliability of the results and predictive ability is provided by studying the combination of allelic variants of several genes.

As applied to MI, this approach was used by Japanese researchers Yamada et al. (WO 2004/001037). The authors selected 10 polymorphisms associated with the development of MI in men and 5 polymorphisms associated with the development of MI in women, and proposed methods for determining the genetic risk of developing MI, based on determining the combination of two or more polymorphisms from the selected list.

The closest analogue of the present invention is a method for determining the risk of developing MI, proposed by these authors and protected by US patent 7,521,181, in which predicting the development of MI includes the following stages: a) analysis of 2 polymorphisms: at position 1019 of the connexin-37 gene and at position 242 of the p22phox gene NADH / NADPH oxidase; b) genotype determination based on the information received according to the two named polymorphisms; and c) a conclusion based on the results of determining the genotype about the genetic risk of myocardial infarction according to the tables presented in the work.

It should be noted, however, that for all the advantages of the model described in this paper, it has several disadvantages. Thus, there is reason to believe that the NADPH oxidase polymorphism is a predictor of the development of MI not in all populations. In particular, for the Russian population, its relationship with the development of IHD, but not IM, is known (Samohodskaya L.M. et al., 2010). In addition, to increase the predictive power of the model, it is preferable to select polymorphisms that are combined pathogenetically and at the same time reinforce each other, however, there are no data in the literature that indicate the presence of a link between NADPH oxidase and Cx37 in the pathogenesis of MI. The most significant drawback of the method of Yamada et al. is a combination of patients with myocardial infarction, who had and did not have a clinical history of coronary heart disease, into a general group, despite the fact that genetic tests associated with the risk of myocardial infarction are most important for people without clinical manifestations of coronary heart disease.

With this in mind, the main objective of the present invention was to create, based on a study of a combination of interconnected polymorphisms, a reliable predictive model of MI in subjects without previous clinical manifestations of coronary artery disease.

Disclosure of invention

The proposed method for predicting the development of MI in individuals without clinical manifestations of coronary heart disease was based on the determination of C1019T polymorphisms of the Cx37 and G894T gene (Glu298Asp) of the type 3 NO synthase gene (endothelial NO synthase, eNOS), followed by the establishment of the genotype at the two indicated polymorphic sites .

The eNOS gene polymorphism was selected as the second indicator for the following reasons.

First, it is now known that this enzyme, and in particular its variants encoded by the 894G and 894T alleles, show a definite connection with the development of coronary atherosclerosis and MI. Replacing guanine with thymine at the 894th position of the NO synthase gene leads to the replacement of glutamine with asparagine at the 298th position of the enzyme (Glu298Asp), which is accompanied by a decrease in its catalytic activity in the vessels. Moreover, it was experimentally established that the TT genotype (298Asp homozygosity) significantly increases the risk of developing cardiovascular diseases (Hingorani A.D. et al., 1999, Samokhodskaya L.M. et al., 2010). According to some data, the homozygous state of TT also correlates with an increase in the frequency of MI (Hingorani A., 1999); however, other studies specify that such a relationship is characteristic only of MI against the background of IHD (Samohodskaya L.M. et al., 2010). As for the GG genotype, there is reason to believe that an increased concentration of NO relative to carriers of other genotypes in individuals with the GG genotype can lead to a decrease in the proliferation of smooth muscle cells involved in the synthesis of collagen ASB capsules. In such patients, the probability of plaque destabilization, including hemodynamically insignificant, may be increased.

Second, there has recently been evidence of in vitro experiments (Pfenniger A. et al., 2010), indicating the possibility of direct interaction of Cx37 with endothelial NO synthase (eNOS) and, accordingly, indicating a high probability of the participation of these proteins in some the same processes, in particular in the process of regulating the proliferation of smooth muscle cells in an atherosclerotic plaque.

When developing the proposed method for determining the genetic predisposition of individuals without clinical manifestations of coronary heart disease to the development of MI, we performed DNA analysis in 155 patients who underwent MI. To assess the prevalence of the above polymorphisms in the Russian population, a control group of 226 practically healthy donors was formed (population control). The results of the studies showed the following.

a) TT genotype of the Cx37 gene correlates with the risk of MI in individuals without clinical manifestations of coronary artery disease. It occurs in 42.5% of patients who had myocardial infarction without clinical signs of coronary artery disease in history, and only in 10.7% of patients who had myocardial infarction with clinical manifestations of coronary artery disease (p = 0.003) (see example 2, table 2).

b) The GG genotype of the eNOS gene also correlates with the risk of MI in individuals without clinical manifestations of coronary artery disease. It occurs in 54.3% of patients who had myocardial infarction without clinical signs of coronary artery disease in history, and only 28.6% of patients who had myocardial infarction with clinical manifestations of coronary artery disease (p = 0.02) (see example 3, table 3 )

c) The combination of the two above genotypes (TT gene Cx37 and GG gene eNOS) corresponds to the maximum risk of MI in people without clinical manifestations of coronary artery disease. The indicated combination is three times more common in the group of patients who underwent MI, in comparison with population control (see example 4, table 4). In this case, the most significant fact is that among all patients with MI, such a combination of genotypes was detected exclusively in individuals who underwent MI without previous manifestations of coronary artery disease (see Table 5-7).

Thus, the task of developing a predictive model of MI in people without clinical manifestations of coronary heart disease was solved by using a new combination of polymorphisms determined during DNA testing in such a model. The proposed method includes a) obtaining a sample of the genomic DNA of the subject; b) analyzing this sample for C1019T polymorphisms in the Cx37 gene and G894T in the eNOS gene; c) determining the genotype from the two named polymorphic sites; and d) drawing a conclusion about the high probability of developing MI without previous coronary artery disease in the case of the simultaneous presence of the TT genotype for the connexin-37 gene and the GG genotype for the eNOS gene.

The proposed method is quite simple to implement, characterized by high sensitivity and has a high predictive power .

Brief Description of the Figures

FIG. 1. Agarose gel electrophoresis after treatment of the corresponding PCR product with restriction enzyme MboI. GG genotype - one fragment 152 bp, GT genotype - three fragments: 152, 92 and 60 bp, TT genotype - two fragments: 92 and 60 bp

FIG. 2. Agarose gel electrophoresis after treatment of the corresponding PCR product with restriction enzyme DrdI TT genotype - two fragments: (not shown), CT genotype - three fragments: 275, 240 and 35 bp, SS genotype - one fragment of 275 bp

The implementation of the invention

Materials and methods

1. Criteria for selecting patients for analysis

The study included male patients with a clinically and instrumentally confirmed diagnosis of MI; the age of MI development is less than 55 years. The diagnosis of myocardial infarction was confirmed by a deviation from the norm of biochemical markers (an increase in MV-CPK more than two times higher than normal, an increase in cardiac troponins T and I above the 99th percentile for the control group), typical ECG changes (ST segment shift by 0.1 mV or inversion of the T wave in two or more adjacent leads, ST segment elevation, pathological Q wave or QS complex). Exclusion criteria: impaired glucose tolerance, diabetes mellitus.

2. DNA isolation

Genomic DNA was isolated from a blood sample using a modified method with proteinase K and phenol-chloroform extraction. To do this, 700 μl of the analyzed blood sample and 700 μl of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) were added to a 1.5 ml tube (Eppendorf) for erythrocyte hemolysis. The procedure was repeated 2 times, and then 400 μl of Proteinase K buffer containing 200 μg / ml Proteinase K was added to the cell pellet, and then incubated for 4 hours at 60 ° C with a constant temperature shaker (850 rpm). Then phenol-chloroform DNA extraction was carried out, followed by precipitation with 96% ethanol and centrifugation for 10 minutes at 10,000 rpm. After a series of washes, DNA was eluted with TE buffer (200 μl). The concentration of extracted DNA ranged from 30 to 50 ng / μl.

3. PCR amplification and analysis of PCR products

To determine the polymorphisms in the studied genes, the RFLP method (restriction fragment length polymorphism) was used. PCR was performed in a Master Cycler thermal cycler (Eppendorf). A sample of the following composition was used: 100 mM Tris-HCl, pH 8.3 (25 ° C), 50 mM KCl, MgCl ₂ (the concentration was selected individually for each pair of primers), four deoxynucleoside triphosphates (200 μM each), Taq- polymerase (0.5 u / reaction) and 1-2 μg of genomic DNA and primers (4-8 pmol / reaction). The final volume of the reaction mixture was 25 μl. When setting up PCR, a hot start technique was used (Hebert B, Bergeron J, Potworowski EF, Tijssen P., 1993).

The reaction conditions were slightly different depending on the determined allele (see examples 2-3).

Restriction analysis of PCR products was carried out using an appropriate restriction enzyme for 6 hours.

The restriction products were analyzed by agarose gel electrophoresis, the concentration of which was selected depending on the length of the restriction fragments.

4. Statistical processing of results

Data were presented as arithmetic mean with standard deviation. The statistical significance of differences for quantitative traits in the case of a normal distribution of the latter was evaluated using the t-test. The statistical significance of differences in qualitative characteristics in the compared groups was evaluated using the χ ² criterion with Yates correction and the Fisher exact criterion. A significance level was adopted p <0.05. For statistical processing, the Statistica 6.0 software package was used.

Examples

Example 1 Characterization of a group of patients selected for DNA testing.

In accordance with the above criteria, 155 patients were selected, of which 127 had MI without previous clinical manifestations of IHD, and 28 had IHD before MI. In the frequency of occurrence of the main risk factors for cardiovascular diseases, the groups did not significantly differ.

Table 1. Comparative characteristics of the examined patients.

Example 2. Determination of C1019T polymorphism of the Cx37 gene.

Genomic DNA isolated from each blood sample was subjected to PCR amplification as described in the Materials and Methods section.

After a hot start and first denaturation (94 ° C, 5 min), amplification of 30 cycles was carried out, each of which included denaturation at 94 ° C for 10 sec, annealing of the primers at 60 ° C for 60 sec and elongation at 72 ° C within 60 sec. The primers used were 5'-CTG GAC CCA CCC CCT CAG AAT GGC CAA AGA-3 'and 5'-AGG AAG CCG TAG TGC CTG GTG G-3'. At the end of the reaction, 8.5 μl of the PCR product (275 bp) was incubated at 37 ° C with 0.5 μl (5 U) of DrdI restrictase and 1 μl of 10-fold restriction buffer. Restriction products were visualized by electrophoresis on a 2% agarose gel containing 1 μg / ml ethidium bromide. In the presence of the C allele, fragments of sizes 240 and 35 bp were formed. In the presence of the T allele, the restriction site is absent; therefore, a fragment of 275 bp is preserved. (Fig. 1). After analysis of all samples, the frequency of occurrence of genotypes in the analyzed groups was calculated. The results are presented in table. 2.

Table 2. The frequency of occurrence of C1019T polymorphism genotypes of the Cx37 gene in patients with MI and in the population

* differences in the frequency of occurrence of a given genotype in comparison with the total frequency of two other genotypes. p _1-2 is the statistical level of significance for the differences between the group of patients with myocardial infarction without a clinical history of coronary artery disease and a group of patients with myocardial infarction with a history of coronary artery disease, p _1-3 is the statistical level of significance for the differences between the group of patients with myocardial infarction without a clinical history of coronary artery disease and the population control group, p _2-3 - statistical significance level for the differences between the group of patients with myocardial infarction with a history of IHD and the group of population control.

From the data presented, it is clear that the risk of developing MI in patients without a clinical manifestation of coronary artery disease in the history correlates with the presence of the TT genotype. In patients who underwent myocardial infarction against the background of clinical manifestations of coronary heart disease, on the contrary, this genotype is found 3-5 times less often than the CC and CT genotypes in the same group of patients and about 4 times less than the TT genotype in the group of patients with myocardial infarction without coronary history . The frequencies of different genotypes in the population control group did not practically differ from the frequencies in the group of patients with myocardial infarction with a history of IHD.

Example 3 Determination of the G894T polymorphism of the NO synthase gene.

Genomic DNA isolated from each blood sample was subjected to PCR amplification as described in the Materials and Methods section.

After a hot start and first denaturation (94 ° C, 5 min), amplification of 35 cycles was carried out, each of which included denaturation (95 ° C, 10 sec), primer annealing (61 ° C, 30 sec), elongation (72 ° C , 45 sec.). The primers used were 5'-GGC TGG ACC CCA GGA AAC-3 'and 5'-CCA CCC AGT CAA TCC CTT TG-3' primers. After amplification was completed, 8.5 μl of the PCR product (198 bp) was incubated at 37 ° C with 0.5 μl (10 U) of MboI restrictase and 1 μl of 10-fold restriction buffer. Restriction products were visualized by electrophoresis on a 2% agarose gel containing 1 μg / ml ethidium bromide. The presence of the G allele was detected by the presence of one fragment of 152 bp in size, and the T allele by the presence of two fragments of 92 and 60 bp. (Fig. 2) . After analysis of all samples, the frequency of occurrence of genotypes in the analyzed groups was calculated. The results are presented in table. 3.

Table 3. Frequency of occurrence of genotypes of G894T polymorphism of the NO synthase gene in patients with MI and in the population

* differences in the frequency of occurrence of a given genotype in comparison with the total frequency of two other genotypes. p _1-2 is the statistical level of significance for the differences between the group of patients with myocardial infarction without a clinical history of coronary artery disease and a group of patients with myocardial infarction with a history of coronary artery disease, p _1-3 is the statistical level of significance for the differences between the group of patients with myocardial infarction without a clinical history of coronary artery disease and the population control group, p _2-3 - statistical significance level for the differences between the group of patients with myocardial infarction with a history of IHD and the group of population control.

From the obtained data, a relationship follows between the development of MI in subjects without a clinical history of IHD and the presence of the 894G allele of the eNOS gene in a homozygous state. In patients who underwent myocardial infarction against the background of previous coronary heart disease, this genotype (GG) is approximately two times less common than in patients without a coronary history. It should be noted that the differences between the groups of patients with MI and the group of population control were not statistically significant, i.e. By itself, the determination of the G894T polymorphism of the eNOS gene is not a reliable enough test to determine a predisposition to the development of MI.

Example 4 Analysis of the frequency of occurrence of combinations of genotypes and predicting the possibility of developing MI in individuals without clinical manifestations of IHD.

In order to determine the combinations that provide the most accurate prediction of the likelihood of developing MI in people without manifestations of IHD, all possible combinations of genotypes were analyzed for the two polymorphisms studied. The results are presented in table. four.

Table 4. Frequency of occurrence of various combinations of genotypes according to Cx37 and eNOS polymorphisms in patients with MI, depending on the presence of clinical manifestations of IHD in history.

Genotypes Patients with myocardial infarction without clinical manifestations of coronary artery disease in history
100% (n = 127) Patients with a history of IHD clinic,
100% (n = 28) Population control, 100% (n = 228) Cx37 eNOS SS Gg 12.6 (n = 16) 7.15 (n = 2) 15.49 (n = 35) SS GT 9.45 (n = 12) 21.43 (n = 6) 23.45 (n = 53) SS TT 0.79 (n = 1) 7.15 (n = 2) 2.65 (n = 6) ST Gg 18.11 (n = 23) 21.43 (n = 6) 22.57 (n = 51) ST GT 14.95 (n = 19) 28.57 (n = 8) 16.37 (n = 37) ST TT 1.58 (n = 2) 3.57 (n = 1) 5.75 (n = 13) TT Gg 23.62 (n = 30) 0 (n = 0) 7.02 (n = 16) TT GT 18.11 (n = 23) 10.7 (n = 3) 6.19 (n = 14) TT TT 0.79 (n = 1) 0 (n = 0) 0.44 (n = 1)

From the data obtained, it clearly follows that in the group of patients with myocardial infarction without a coronary artery disease clinic, a combination of TT / GG genotypes is the most common in history. The proportion of this genotype in patients of this group is 23.62%, while the representation of this combination in the control group is three times lower (7.02%). At the same time, an unexpected feature was revealed in the distribution of the TT / GG combination between two groups of patients: this combination occurs exclusively in patients with myocardial infarction without clinical history of IHD (see row 7 in table 4), i.e. quite specifically determines the genetic predisposition to the development of MI without previous symptoms of coronary heart disease (data on the combination of TT / TT genotypes are not taken into account due to their inaccuracy).

Table 5 presents data on the comparison of patient groups by the frequency of occurrence of the combination of TT / GG and other combinations of genotypes using Fisher's exact test

Table 5. The frequency of occurrence of a combination of TT genotypes of the Cx37 gene and GG of the eNOS gene in comparison with other combinations in patients with MI, depending on the history of clinical manifestations of coronary artery disease.

Tables 6 and 7 present comparison data for each of the two groups of patients with MI (with clinical manifestations of IHD in the history and without them) with population control.

Table 6. Frequency of occurrence of a combination of TT genotypes of the Cx37 gene and GG of the eNOS gene in comparison with other combinations in patients with MI without clinical manifestations of coronary artery disease in history and in the population control group.

Table 7. The frequency of occurrence of a combination of TT genotypes of the Cx37 gene and GG of the eNOS gene in comparison with other combinations in patients with MI, depending on the presence of clinical manifestations of IHD.

The combination of two genotypes (TT on the polymorphic site 1019 of the Cx37 gene and GG on the polymorphic site 894 of the eNOS gene), the unfavorable role of each of them in the development of MI was shown earlier, corresponds to the maximum risk of MI in people without clinical manifestations of coronary artery disease. The indicated combination is statistically significantly more common in the group of patients who underwent MI and did not have a history of coronary artery disease, compared with both the population control and the group of patients without manifestations of coronary artery disease. The distribution of various combinations in the group of patients with myocardial infarction who had a history of IHD clinic does not have statistically significant differences from their distribution in the control group.

Thus, the proposed method for determining the risk of MI development, based on the analysis of a combination of polymorphisms of the Cx37 and eNOS genes, allows us to distinguish among practically healthy individuals a high risk group for the development of MI based on the status of the carrier of the TT genotype (position 1019 of the Cx37 gene) / GG (position 894 of the gene eNOS). Since according to our data, MI develops in individuals with the indicated genotype without any clinical precursors, such individuals should initially be recommended regular cardiac examinations and primary prevention measures.

Claims (1)

A method for determining the hereditary predisposition of a subject without clinical manifestations of coronary heart disease (CHD) to the development of myocardial infarction (MI), which involves analyzing a DNA sample for the presence of two polymorphisms that are significant for the development of MI, one of which is C1019T polymorphism of the connexin-37 gene (Cx37), determination genotype for each of the studied polymorphic sites and drawing up conclusions about the likelihood of developing MI based on the established combination of genotypes, characterized in that the second analyzed polymorph IOM is G894T eNOS gene polymorphism, wherein the detection of homozygous GG genotype of this polymorphism in combination with the homozygous genotype TT polymorphism S1019T Sh37 gene forms the basis for the conclusion of a high degree of risk of myocardial infarction without previous clinical coronary artery disease symptoms.

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