RU2612630C2 - Method for determination of individual genetic risk for ischemic stroke - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method is represented by detection and identification of the most promising genes polymorphisms that determine the increased risk of ischemic stroke (IS IR).

EFFECT: invention allows to create a fundamentally new way to assess genetic risk of stroke, based on 48 SNP instead of a few, as well as SNP classification according to their pathophysiological significance, and to create an algorithm evaluation and interpretation of the individual risk of stroke.

6 cl, 4 dwg, 4 tbl, 1 ex

Description

The invention relates to molecular biology and medicine, and in particular to methods for identifying and identifying the most promising polymorphisms of genes that determine an increased risk of developing ischemic stroke (PP II).

The incidence of stroke remains one of the most significant medical and social problems not only in the Russian Federation, but also in many other economically developed countries. According to the World Health Organization (WHO), in most countries of the world, 100-300 cases of stroke are recorded annually per 100,000 population. In the Russian Federation, this indicator is estimated at 350-400 cases of stroke per year per 100,000 population. Mortality in stroke is quite high: out of 100 patients in the acute period of stroke, 35-40 people die, by the end of the year the total number of deaths reaches 50. Primary stroke is on average 75%, repeated stroke - about 25% of all cases of stroke. A stroke often leaves behind severe consequences in the form of motor, speech and other disorders. According to the stroke register of the Scientific Center of Neurology of the Russian Academy of Medical Sciences (Moscow), among surviving patients, motor impairment is observed by the end of the acute period of stroke in 85%, by the end of the first year - in 70%, speech disorders (aphasia) by the end of the acute period - in 36 %, by the end of the first year - in 18% of patients.

It is now apparent that various predisposition factors determine the risk of stroke not equally. The concept of the heterogeneous causes of stroke involves dividing it into types and subtypes. So, hemorrhagic stroke occurs in 20% of cases of stroke, including brain hematoma - in 15%, subarachnoid hemorrhage - in 5%; ischemic stroke - in 80% [Vereshchagin N.V., 2004].

The individualization of stroke prevention has undoubted advantages. Despite the huge amount of evidence of the clinical significance of stroke risk factors, their timely correction is far from complete. Informing the patient based on evidence of evidence-based medicine that his personal risk of developing a stroke exceeds the average population risk, including due to the presence of objective genetic predisposition factors, should strengthen his commitment to preventive and therapeutic measures.

At the moment, it seems certain that the patient’s genetic predisposition makes a significant contribution to the risk of stroke. If we consider the predisposition to stroke as a phenotype, it should be assumed that it develops as a result of a complex network of interactions of genetic and non-genetic factors [Kovaleva et al., 2013; Titov et al. 2012].

At the elementary level, each factor is assumed to be independent and characterized by its own risk indicator, which, as a first approximation, can be estimated by the magnitude of the odds ratio (OR). In the case of a genetic factor, this will be, respectively, the stroke OR in the group of carriers of the minor allele (risk or protective) and stroke in the group of carriers of the normal (wild) allele. At the same time, the risk of homozygotes and heterozygotes for the mutant allele can be estimated using various models: dominant, recessive, or codominant (multiplicative, additive, or more complex). It can be hoped that the more risk factors taken into account, the more reliable the risk assessment.

The prior art method for the genetic diagnosis of susceptibility to cardiovascular disease (RU 2376372, C12N 15/00, 12/20/2009), based on the determination of genotypes by polymorphic variants A1166C of the gene AGTR1, A-240T and A2350G of the ACE gene, C677T of the MTHFR gene, T174M of the AGT gene, C825T of the GNB3 gene, VNTR4a / b and G894T of the NOS3 gene, G1691A of the F5 gene, PLA1 / A2 of the ITGB3 gene, G20210A of the F2 gene and risk assessment by summing the number of points assigned to each genotype. At the same time, 0 points are assigned to the genotypes of “low” risk of cardiovascular pathology, these include genotypes 1166АА of the AGTR1 gene, -240АА, 2350АА of the ACE gene, 677СС of the MTHFR gene, 174TT of the AGT gene, 825CC of the GNB3 gene, VNTR4bb and 894GG of the NG3 gene, 16GNG3, NOS3, F5, PLA1 / A1 of the ITGB3 gene, 20210GG of the F2 gene. The “medium” risk genotypes are assigned 0.5 points, these include the 1166AC genotypes of the AGTR1 gene, -240AT and 2350AG of the ACE gene, 677ST of the MTHFR gene, 174TM of the AGT gene, 825CT of the GNB3 gene, VNTR4ab and 894GT of the NOS3 gene. High-risk genotypes are assigned 1 point, these include genotypes 1166CC of the AGTR1 gene, -240TT and 2350GG of the ACE gene, 677TT of the MTHFR gene, 174MT of the AGT gene, 825TT of the GNB3 gene, VNTR4aa and 894GT of the NOS3, 1691GA / F91A gene, 1691GA and 1691GA A2 and PLA2 / A2 of the ITGB3 gene, 20210GA and 20210AA of the F2 gene. The risk of cardiovascular disease is considered "low" with a score of 0 to 3, medium - from 3.5 to 6, high - from 6.5 to 11 points.

Patent RU 2422523 (06/27/2011, IPC C12N 15/00) describes the use of the SNP41 allele of the PDE4D gene for predicting an individual predisposition to stroke in the Russian population, the use of the molecular genetic marker of an individual predisposition to stroke, and a method for predicting an individual predisposition to stroke.

Patent RU 2453606 (06/20/2012; IPC C12Q 1/68, G01N 33/48) discloses a method for extended screening of a predisposition to cardiovascular diseases based on the detection of point nucleotide substitutions in the human genes responsible for the predisposition and development of cardiovascular diseases. The method includes the step of isolating genomic DNA from a clinical sample, amplification of regions of the ACE, APOA4, APOA5, APO, EDN1, MEF2A, FGA, ADRB1, TNBS4, APOE, FBN1, AGXT, MTHFR, CCR2, F5, F2, F7, ABCA1, ITGB3 genes , AGTR2, B2R, DES, TLR4, NOS3, KDR, MMP9, THBS2, LPL, MPO containing sequences of polymorphic loci and obtaining a single chain fluorescently labeled product by nick translation and restriction. Interpret the results of hybridization by comparing the intensity of fluorescent signals obtained with perfect and imperfect hybridization.

Existing methods for determining the genetic risk of developing cardiovascular diseases and, in particular, the above methods involve the study of only a few genetic markers (SNPs). The present invention for the first time proposes a method for assessing the genetic risk of developing a stroke based on 48 SNPs distributed in six groups according to their pathophysiological relationship with stroke or a statistically significant association with a specific subtype of stroke identified in a meta-analysis or GWAS study.

The objective of the present invention is the creation of a fundamentally new more informative way of assessing the genetic risk of stroke, as well as the classification of SNP in accordance with their pathophysiological value and the creation of an algorithm for assessing and interpreting the individual risk of stroke.

The technical result of the invention is to increase the reliability and information content of the results of the assessment of the genetic risk of stroke.

The problem is solved, and the technical result is achieved by the fact that the method for determining the genetic risk of stroke is based on the determination of single nucleotide polymorphisms and includes:

(a) isolation of the full genome DNA of the patient being examined;

(b) determination of alleles of single nucleotide polymorphisms (SNPs) in a DNA sample related to the following gene groups:

“Hemostasis” (G-II), is a group of genes related to hemostasis and thrombus formation, including genes: F2 (rs1799963), F5 (rs6025), F7 (rs6046), F13A1 (rs5958), SERPINE1 (rs1799768), FGB ( rs1800790), GP1BA (rs6065) and ITGB3 (rs5918),

“Lipid metabolism” (LO-II) is a group of genes related to metabolism, including genes and loci: APOE (rs429358), APOE (rs7412), APOA5 (rs662799), LPA (rs10455872), LPL (rs328), MTHFR ( rs1801133), PON1 (rs662) and 9p21.3 (rs2383207),

“Cellular interactions” (B-CL-II), is a group of genes involved in intercellular interactions, including the genes: CELSR1 (rs6007897), ICAM1 (rs1799969), IL1B (rs1694), LTA (rs909253), LTC4S (rs730012), LTC4S (rs3776944) and SELE (rs5355),

"Stroke-1" (II-1), is an SNP group that is an indicator of the ratio of the odds of getting a stroke, obtained by comparing patients with a cardiogenic embolic stroke and individuals, without a history of stroke, including genes and loci: CELSR1 (rs4044210 ), NOS3 (rs1799983), PDE4D (rs702553), 16q22.3 (rs7193343), 16q22.3 (rs12932445), 22ql2.3 (rs5998322), 6p21.1 (rs556512) and 4q25 (rs2200733),

"Stroke-2" (II-2) is the SNP group, which is an indicator of the ratio of the odds of getting a stroke, obtained by comparing patients with an atherothrombotic stroke and people without a history of stroke, including genes and loci: LPL (rs328), AGTR1 (rs5186), HDAC9 (rs11984041), LPA (rs10455872), 9p21.3 (rs1537378), 6p21.1 (rs556621), 22ql2.3 (rs4479522) and 4q25 (rs1906591),

“Arterial hypertension” (AH) is a group of SNPs significantly associated with the development of hypertension as one of the clinical risk factors for stroke, including genes and loci 12p12 (rs7961152), 12q23 (rs11110912), 13q21 (rs1937506), 15q26 (rs2398162) , lq43 (rs2820037), 8q24 (rs6997709), FGF5 (rs16998073), MTHFR (rs17367504), NOS3 (rs3918226), CSK (rs1378942) and cl0orf107 (rs1530440);

(c) determination of the individual genetic risk of stroke based on the analysis of identified alleles in the tested SNP, in which:

(i) calculate the average population risk (APR) for each SNP according to the formula

APR = F ₁ × OR ₁ + F ₂ × OR ₂ + F ₃ × OR ₃ ,

where F ₁ - the frequency of homozygotes for the "wild" allele,

F ₂ - the frequency of heterozygotes,

F ₃ - frequencies of homozygotes for the mutant allele,

OR ₁ - an indicator of the odds ratio for the homozygous wild-type allele genotype, equal to 1,

OR ₂ - an indicator of the odds ratio for the heterozygous genotype,

OR ₃ is an indicator of the odds ratio for the homozygous genotype for the mutant allele;

(ii) calculate the relative risks (RR) for each SNP based on the genotype of the study patient according to the following formulas:

for the homozygous wild-type allele genotype: RR = 1 / APR,

for heterozygous genotype: RR = OR ₂ / APR,

for the homozygous genotype for the mutant allele RR = OR ₃ / APR;

(iii) calculate the generalized relative risks (GRR) due to the combination of alleles in the genes combined in the said groups according to the formula:

GRR _combinations = RR ¹ × RR ² × RR ³ ... × RR ⁱ ,

where RR ¹ ... RR ⁱ are the relative risks for each SNP included in the corresponding group,

(d) an assessment of the individual genetic risk of stroke for each of the mentioned gene groups based on the values of the generalized relative risks of the GRR _combination : if the GRR value of the _{combination is} <1, the risk is reduced relative to the average generalized relative risk of the population and if the GRR value of the _{combination is} > 1, the risk is increased in relation to the indicated average value.

In a preferred embodiment of the invention, based on the obtained values of the generalized relative risks of GRR _combinations , the degree of genetic risk for each of the mentioned groups of genes is assigned to one of the categories: “low risk”, “low risk”, “medium risk”, “increased risk” “High risk” and “very high risk” as follows:

for the G-II group: with a GRR value of 0.43-0.76 _combinations , the category of “low risk” is established, with 0.76-0.89 - “reduced risk”, with 0.89-1.02 - “medium risk” ", At 1.02-1.23 -" increased risk ", at 1.23-1.55 -" high risk ", at> 1.55 -" very high risk ";

for the LO-II group: when the GRR value is 0.64-0.79, the _combinations establish the category “low risk”, with 0.79-0.85 - “low risk”, with 0.85-0.91 - “medium risk ", At 0.91-1.15 -" increased risk ", at 1.15-1.75 -" high risk ", at> 1.75 -" very high risk ";

for the B-KL-II group: when the SLE combination value is 0.30-0.53, the category “low risk” is established, at 0.53-0.66 - “reduced risk”, with 0.66-0.83 - “Medium risk”, at 0.83-1.16 - “increased risk”, at 1.16-2.30 - “high risk”, at> 2.30 - “very high risk”;

for group II-1: with a GRR value of a _combination in the range of 0.58-0.70, the category of "low risk" is set, with 0.70-0.78 - "low risk", with 0.78-0.93 - " medium risk ", at 0.93-1.22 -" increased risk ", at 1.22-1.83 -" high risk ", at> 1.83 -" very high risk ";

group II-2: the value of 0,21-0,53 GRR _combination set category of "low risk" when 0,53-0,77 - "low risk" when 0,77-0,96 - "medium risk ", At 0.96-1.34 -" increased risk ", at 1.34-2.16 -" high risk ", at> 2.16 -" very high risk ";

for the AH group: with a GRR value of 0.18-0.65 _combinations , the category “low risk” is established, with 0.65-0.81 - “low risk”, with 0.81-0.99 - “medium risk”, at 0.99-1.27 - "increased risk", at 1.27-1.84 - "high risk", at> 1.84 - "very high risk".

Also in preferred embodiments, genetic risk assessment is made based on the expected distribution GRR values for each _combination of groups of genes in accordance with the calculated values of the generalized indicators genetically determined risk by dividing the range of possible _combinations of values of GRR to 5 bands, 20% percentile: 1- th range of "low risk" - 20%; 2nd range of "low risk" - 20%; 3rd range of “medium risk” - 20%; The 4th range of "increased risk" is 20% and the 5th range, including the areas of "high risk" - 15% and "very high risk" - 5%.

Also in preferred embodiments of the invention:

- the results of the genetic risk assessment are presented in the form of a petal diagram in which on the six axes corresponding to the mentioned six groups of genes and formed by rays drawn from the center of the regular hexagon to its vertices, the GRR _{combinations are} laid off; drawing lines connecting the points on the scales corresponding to the boundary GRR values of the _combination for each of the mentioned risk categories in the corresponding group, and forming an individual profile of the patient's genetic predisposition by plotting points on the scales corresponding to the calculated GRR values of the _combination for this patient and connecting these points with lines;

- genomic DNA is isolated from blood cells or buccal epithelium;

detection of the presence of allelic single nucleotide polymorphisms is carried out by real-time PCR or by pyrosequencing.

Unlike analogues in the proposed method, not several (5-10), but 48 SNPs are used as indicators of genetic risk. Moreover, these SNPs are divided into groups of genes that are responsible for certain factors, which allows us to assess the risk of stroke separately for each gene group. These differences make it possible to increase the reliability and information content of the results of risk assessment.

In the proposed method, combinations of 48 genetic risk factors are considered. In this connection, at the next level, risk factors are grouped on the basis of the similarity of their pathophysiological relationship with stroke (for example, a group of factors contributing to the development of thrombophilic conditions), or a statistically significant relationship with a particular subtype of stroke. For each group, a generalized risk indicator can be calculated. Moreover, the absolute value of such an indicator is not fundamental. It is important to classify a specific individual as having a higher or lower risk compared with the distribution of the risk indicator in the population. At the third level, six generalized risk indicators form a “status of a predisposition to stroke”: “low risk”, “low risk”, “medium risk”, “increased risk”, “high risk” and “very high risk”.

The results of the genetic examination can clarify and additionally justify the therapeutic or prophylactic tactics selected taking into account the whole range of applied instrumental and laboratory techniques.

“Increased risk” for one group of risk factors is a worrying circumstance and requires consideration of the need for preventive measures to prevent primary stroke and the intensification of preventive measures if necessary to prevent re-stroke. First of all, attention is drawn to the “increased risk” for one of the three most informative groups of factors: “Stroke-1”, “Stroke-2” and “Cellular interactions”. When identifying an “increased risk” by the groups of factors “Lipid metabolism”, “Hemostasis” and “Arterial hypertension”, preventive measures can be, first of all, aimed at eliminating the leading cause of risk.

“High” and “very high” risk for one of the groups of factors or “increased risk” for two or more groups of factors are prognostically unfavorable indicators.

"High" and "very high" risks for two or more groups of factors statistically reliably indicate a risk of stroke and, undoubtedly, require preventive measures to prevent primary stroke and intensify preventive measures if it is necessary to prevent re-stroke.

The method for determining the genetic risk of stroke is based on the determination of SNP alleles shown in the table (see table. 1). For the selection of SNP used data from GWAS studies [Bellenguez S., 2012; Gudbjartsson D.F., 2009; Holliday E.G., 2012; Newton-Cheh S., 2009; Salvi E., 2012; WTCCC, 2007]. In most cases, the selection of SNPs and the assessment of the associated OS for the development of the proposed integrated methodology was based on the results of a meta-analysis [R. Bentley, 2010; Casas JP, 2004; Holmes M.V., 2011; Liu H., 2013; Pi Y., 2012; Tao N.M., 2009; Wang S., 2011; Wang X., 2009; Xin X.Y., 2009; Ye F., 2012; Yoon D., 2011].

Table 1. SNPs used in the method for determining the genetic risk of stroke

Individual genetic risk is calculated by SNP group. For each genotype, the frequencies of genotypes and OR are shown in the table. 2.

Group designations:

G-II - a group of genes related to hemostasis and thrombosis.

LO-II - a group of genes related to metabolism, primarily lipid metabolism.

B-KL-II - a group of genes involved in intercellular interactions, primarily in intercellular interactions in the inflammatory process.

For these groups, the used odds ratios were obtained by comparing patients with ischemic stroke and those without a history of stroke.

II-1 - SNP group, the pathophysiological significance of which may be unclear. For these groups, whenever possible, the relationship of the chances of developing a stroke is used, obtained by comparing patients with a cardiogenic embolic stroke and people without a history of stroke.

II-2 is a group of SNPs whose pathophysiological significance may be unclear. For these groups, whenever possible, the relationship of the chances of developing a stroke is used, obtained by comparing patients with atherothrombotic stroke and people without a history of stroke.

AH is a group of SNPs significantly associated with the development of arterial hypertension, that is, one of the clinical risk factors for stroke.

Further, these groups will be conditionally designated as “Hemostasis”, “Lipid metabolism”, “Cellular interactions”, “Stroke-1”, “Stroke-2” and “Arterial hypertension”, respectively.

Depending on the identified spectrum of genotypes in the patient, the individual genetic risk of stroke is calculated for the corresponding SNP groups using the following procedure.

1) Calculation of the average population risk (APR) for each SNP

For each SNP used in this methodology, the heterozygotes (OR ₂ ) and homozygotes (OR ₃ ) are assumed to be known by the mutant allele relative to the homozygous by the “wild” allele and the frequency of occurrence of genotypes in the population (F ₁ , F ₂ , and F ₃ , i.e. frequencies of homozygotes for the “wild” allele, heterozygotes and homozygotes (OR ₃ ) for the mutant allele, respectively). The values of OR ₂ and OR ₃ are taken directly from the publications given in table. 1 in the column "main source"; the values of F ₁ , F ₂ , and F ₃ for the Caucasian population (Caucasian) are taken from the NCBI database [http://www.ncbi.nlm.nih.gov/snp/].

Average population risk (APR) for an individual SNP is calculated by the formula

By definition, the average population risk is greater than 1 if the mutant allele is a risk allele, and less than 1 if the mutant allele is protective (reduces stroke PP).

2) Calculation of relative risk (RR) for each SNP

The relative risk of each of the three genotypes for a separate SNP is calculated by the formulas:

for the homozygous genotype for the "wild" allele: RR = 1 / APR,

for heterozygous genotype: RR = OR ₂ / APR,

for the homozygous genotype for the mutant allele RR = OR ₃ / APR.

With this definition, the relative risk of the genotype is normalized to the average population risk for the considered SNP, in other words, the average value of the relative risk for each SNP in the population is 1.

3) Calculation of the relative risks of different genotypes for the SNP group The relative risk due to the combination of alleles from seven to eleven SNPs combined into a group cannot be estimated by case-control studies, since in a group of 7 SNPs there are 3 ⁷ , that is, 2187, combinations alleles, and in a group of 11 SNPs, 3 ¹¹ (177147) variants of allele combinations are possible. Thus, the study would have to include millions of people. Even the most common variant of combinations (“wild”, that is, the most common, allele for all 7-11 SNPs and, as a result, the most common combination) would occur in only about 1-5% of the subjects. Therefore, in the proposed methodology, the generalized relative risk (GRR), due to the combination of alleles combined into a group, is determined in accordance with the multiplicative model according to the formula:

where RR ¹ ... RR ⁱ - relative risks for each of the SNP included in the corresponding group.

In a similar way, the total relative risk (SRR) due to the combination of the alleles of all 48 SNPs can be calculated:

where RR ¹ ... RR ⁴⁸ are the relative risks for each of the 48 SNPs.

Distribution of possible values of generalized relative risk in a population

The generalized relative risk is also a normalized indicator, that is, its value more than 1 corresponds to values above the average generalized relative risk in the population, and the value is less than 1, respectively, to values below the average. The distribution of the values of the generalized relative risk in the population is determined by the frequency of occurrence of the corresponding genotypes in the population. The analysis shows that these distributions are non-Gaussian (“not normal”) in nature and differ in their slanting to the region of large and very large values. The distributions for the six selected groups of risk factors are shown in FIG. one.

The calculation of generalized relative risks was carried out according to the multiplicative model, while the real patterns of risk accumulation in the presence of two or more formally independent risk factors (genetically unlinked SNPs) are unknown. Therefore, one should not assume that an individual with a GRR of 5 is five times more likely to develop a stroke than individuals with GRR = 1. Using statistical terminology, we can say that the GRR variable is not described by an interval scale, but it is ordinal. In other words, the value of GRR = 5 is greater than the value of GRR = 2, and GRR = 2, in turn, is greater than the value of GRR = 0.8, but the question “how many times more?” Does not make sense. Therefore, for each of the six groups of factors, the whole area of possible GRR values is divided into 5 ranges (20% percentiles). Thus, 20% of the lowest GRR values fall into the "low risk" range, 20% of the next largest values fall into the "low risk" range, and so on. Within the highest 20% percentile, the ranges of "high risk" (15%) and "very high risk" are highlighted - 5% of the highest values (the ranges of risk values are clearly shown in Fig. 2). The boundaries of the ranges for six groups of genetic polymorphisms - risk factors are shown in table 3.

Assessment and interpretation of individual risk

The results of the analysis of calculations of stroke PP indicators for each of the 48 SNPs and generalized risk indicators for 6 groups of risk factors. The groups are conventionally designated as “Hemostasis”, “Lipid metabolism”, “Cellular interactions”, “Stroke-1”, “Stroke-2” and “Arterial hypertension” (see Fig. 1).

The interpretation of quantitative values of generalized risk indicators is based on the estimated distribution of these values in the population and is presented graphically in the form of a petal chart (Fig. 3). For each of the 6 generalized indicators of genetically determined risk, an individual formally belongs to one of six categories: “low risk”, “low risk”, “medium risk”, “high risk”, “high risk” and “very high risk” of stroke , for each of which the boundaries of the ranges are indicated. The maximum possible quantitative risk indicators range from 5 for the group of factors “Hemostasis” to 85 for the group of factors “Cellular interactions”.

Gray lines show certain values of generalized risk indicators (0.5; 1; 1.5 and 2); these lines are intended for additional orientation when applying individual risk values to the petal chart.

The following is an example embodiment of the invention with reference to the accompanying figures.

An example embodiment of the invention

Clinical example 1

A study of the individual genetic risk of a patient aged 73 years was conducted. DNA was isolated and, using real-time PCR technology, alleles for each of the 48 SNPs were determined. The results of the determination of genotypes are presented in table 4.

The sequence of calculation of individual risk.

1. The calculation of the average population risk for each SNP (APR) was performed using the values from table 2 as follows.

For rs3918226 APR, the homozygous frequency (CC) is 0.833 × 1 + the heterozygous (CT) frequency is 0.133 × 1.34 + the homozygous frequency (TT) is 0.034 × 2.68 = 0.833 + 0.17822 + 0.9112 = 1.10234.

For rs11110912 APR, the homozygous frequency (CC) is 0.611 × 1 + the heterozygous frequency (CG) is 0.354 × 1.33 + the homozygous frequency (GG) is 0.035 × 1.34 = 0.611 + 0.47082 + 0.0469 = 1.12872.

Similarly, calculations were performed for all other SNPs.

2. After calculating the average population risk (APR) for all SNPs, the relative risk (RR) for each SNP was calculated using the results of patient genotyping (Table 4). Relative risk was defined as the ratio of the individual risk of the identified SNP genotype (OR) to its average population risk (APR). So, for the genotype CC locus rs3918226 RR = 1 / 1.10234 = 0.90716; for the GG genotype of the locus rs11110912 RR = 1.33 / 1.12872 = 1.178326.

The relative risk for all SNPs was calculated similarly.

3. Next, we calculated the generalized relative risk (GRR) for each SNP group (in accordance with Table 2) by multiplying all the relative risk (RR) values of the loci of one group. Thus, for the AG group, GRR = 0.90716 × 1.178326 × 0.845208 × 1.10166 × 0.76979 × 0.86505 × 0.9006 × 1.0382 × 0.96238 × 1.01636 × 1.03292 = 0.6261.

For the groups “II-1”, “II-2”, “LO-II”, “G-II” and “V-KL-II”, the similarly calculated generalized risk (GRR) amounted to 0.6264; 4,9856; 0.8318; 0.9342; 1.4726, respectively.

4. The total relative risk (SRR) was calculated as the product of all group values of the generalized relative risk (GRR), and in the presented case it was 0.6264 × 4.9856 × 0.8318 × 0.9342 × 1.4726 × 0.6261 = 2.24 (pronounced case of predisposition).

“Very high” risk for the “Stroke-2” group of factors, “high” risk for the “Cellular interactions” group of factors, and for other groups of factors, risk indicators are in the range of average population values or lower. The risk is due to the combined contribution of polymorphisms in the genes AGTR1, HDAC9, 6p21.1, 22q12.3 / APOL2.

A genetic predisposition profile for six groups of risk factors for stroke is shown in FIG. four.

Brief Description of the Drawings

In FIG. Figure 1 shows histograms of the distribution of relative stroke risk values in a population for six SNP groups:

a) the distribution of the values of the relative risk of stroke in the population for the II-1 group, calculated according to the group of risk factors associated mainly with cardiogenic embolic stroke;

b) the distribution of the values of the relative risk of stroke in the population for the II-2 group, calculated according to the group of risk factors associated mainly with atherosclerotic stroke;

c) distribution of values of the relative risk of stroke in the population for the B-CL-II group, calculated according to the group of risk factors “Cellular interactions”;

d) distribution of values of the relative risk of stroke in the population for the LO-II group, calculated according to the group of risk factors “Lipid metabolism”;

d) the distribution of the values of the relative risk of stroke in the population for the G-II group, calculated according to the group of risk factors "Hemostasis";

f) distribution of the relative risk of stroke in the population for the hypertension group, calculated according to the group of risk factors “Arterial hypertension”.

In FIG. Figure 2 presents a histogram of the distribution of the values of the relative risk of stroke in the population for the II-2 group, with a clear indication of the ranges of risk values.

In FIG. Figure 3 shows the genetic predisposition profile for six groups of risk factors for stroke.

In FIG. Figure 4 presents an example of an individual profile of a genetic predisposition for six groups of risk factors for stroke.

Claims (37)

1. A method for determining the genetic risk of developing ischemic stroke, based on the determination of single nucleotide polymorphisms, including: (a) isolation of the full genome DNA of the patient being examined; (b) determination of alleles of single nucleotide polymorphisms (SNPs) in a DNA sample related to the following gene groups: “Hemostasis” (G-II), is a group of genes related to hemostasis and thrombus formation, including genes: F2 (rs1799963), F5 (rs6025), F7 (rs6046), F13A1 (rs5958), SERPINE1 (rs1799768), FGB ( rs1800790), GP1BA (rs6065) and ITGB3 (rs5918), “Lipid metabolism” (LO-II) is a group of genes related to metabolism, including genes and loci: APOE (rs429358), APOE (rs7412), APOA5 (rs662799), LPA (rs10455872), LPL (rs328), MTHFR ( rs1801133), PON1 (rs662) and 9p21.3 (rs2383207), "Cellular interactions" (B-CL-II), is a group of genes involved in intercellular interaction, including the genes: CELSR1 (rs6007897), ICAM1 (rs1799969), IL1B (rs1694), LTA (rs909253), LTC4S (rs730012), LTC4S (rs3776944) and SELE (rs5355), "Stroke-1" (II-1), is an SNP group that is an indicator of the ratio of the odds of getting a stroke, obtained by comparing patients with a cardiogenic embolic stroke and individuals, without a history of stroke, including genes and loci: CELSR1 (rs4044210 ), NOS3 (rs1799983), PDE4D (rs702553), 16q22.3 (rs7193343), 16q22.3 (rs12932445), 22q12.3 (rs5998322), 6p21.1 (rs556512) and 4q25 (rs2200733), "Stroke-2" (II-2) is the SNP group, which is an indicator of the ratio of the odds of getting a stroke, obtained by comparing patients with an atherothrombotic stroke and people without a history of stroke, including genes and loci: LPL (rs328), AGTR1 (rs5186), HDAC9 (rs11984041), LPA (rs10455872), 9p21.3 (rs1537378), 6p21.1 (rs556621), 22q12.3 (rs4479522) and 4q25 (rs1906591), “Arterial hypertension” (AH) is a group of SNPs significantly associated with the development of hypertension as one of the clinical risk factors for stroke, including genes and loci 12p12 (rs7961152), 12q23 (rs11110912), 13q21 (rs1937506), 15q26 (rs2398162) , 1q43 (rs2820037), 8q24 (rs6997709), FGF5 (rs16998073), MTHFR (rs17367504), NOS3 (rs3918226), CSK (rs1378942) and c10orfl07 (rs1530440); (c) determination of the individual genetic risk of stroke based on the analysis of identified alleles in the tested SNP, in which: (i) calculate the average population risk (APR) for each SNP according to the formula APR = F ₁ × OR ₁ + F ₂ × OR ₂ + F ₃ × OR ₃ , where F ₁ - the frequency of homozygotes for the "wild" allele, F ₂ - the frequency of heterozygotes, F ₃ - frequencies of homozygotes for the mutant allele, OR ₁ - an indicator of the odds ratio for the homozygous wild-type allele genotype, equal to 1, OR ₂ - an indicator of the odds ratio for the heterozygous genotype, OR ₃ is an indicator of the odds ratio for the homozygous genotype for the mutant allele; (ii) calculate the relative risks (RR) for each SNP based on the genotype of the study patient according to the following formulas: for the homozygous wild-type allele genotype: RR = 1 / APR, for heterozygous genotype: RR = OR ₂ / APR, for the homozygous genotype for the mutant allele RR = OR ₃ / APR; (iii) calculate the generalized relative risks (GRR) due to the combination of alleles in the genes combined in the said groups according to the formula GRR _combinations = RR ¹ × RR ² × RR ³ ... × RR ⁱ , where RR ¹ ... RR ⁱ are the relative risks for each SNP included in the corresponding group; (d) an assessment of the individual genetic risk of stroke for each of the mentioned gene groups based on the values of the generalized relative risks of the GRR _combination : if the GRR value of the _{combination is} <1, the risk is reduced relative to the average generalized relative risk of the population and if the value of the GRR _{combination is} > 1 increased in relation to the indicated average value. 2. The method according to p. 1, in which, based on the obtained values of the generalized relative risks of GRR _combinations , the degree of genetic risk for each of the mentioned groups of genes is assigned to one of the categories: "low risk", "low risk", "medium risk" , “Increased risk”, “high risk” and “very high risk” as follows: for the G-II group: with a GRR value of 0.43-0.76 _combinations , the category of “low risk” is established, with 0.76-0.89 - “reduced risk”, with 0.89-1.02 - “medium risk” ", At 1.02-1.23 -" increased risk ", at 1.23-1.55 -" high risk ", at> 1.55 -" very high risk "; for the LO-II group: with a GRR value of 0.64-0.79 _combinations , the category of “low risk” is established, with 0.79-0.85 - “low risk”, with 0.85-0.91 “medium risk” , at 0.91-1.15 - "increased risk", at 1.15-1.75 - "high risk", at> 1.75 - "very high risk"; for group B-CL-AI: at a value of 0,30-0,53 GRR _combination set category of "low risk" when 0.53-0.66 - "low risk" when 0,66-0,83 - " medium risk ", at 0.83-1.16 -" increased risk ", at 1.16-2.30 -" high risk ", at> 2.30 -" very high risk "; for group II-1: with a GRR value of a _combination in the range of 0.58-0.70, the category of "low risk" is set, with 0.70-0.78 - "low risk", with 0.78-0.93 - " medium risk ", at 0.93-1.22 -" increased risk ", at 1.22-1.83 -" high risk ", at> 1.83 -" very high risk "; group II-2: the value of 0,21-0,53 GRR _combination set category of "low risk" when 0,53-0,77 - "low risk" when 0,77-0,96 - "medium risk ", At 0.96-1.34 -" increased risk ", at 1.34-2.16 -" high risk ", at> 2.16 -" very high risk "; for the AH group: with a GRR value of 0.18-0.65 _combinations , the category “low risk” is established, with 0.65-0.81 - “low risk”, with 0.81-0.99 - “medium risk”, at 0.99-1.27 - "increased risk", at 1.27-1.84 - "high risk", at> 1.84 - "very high risk". 3. The method of claim. 2, wherein the genetic risk assessment is made based on the expected distribution GRR values for each _combination of groups of genes in accordance with the calculated values of the generalized indicators genetically determined risk by dividing the range of possible _combinations of values GRR 5 ranges of 20% percentile: 1st range of "low risk" - 20%; 2nd range of "low risk" - 20%; 3rd range of “medium risk” - 20%; The 4th range of "increased risk" is 20% and the 5th range, including the areas of "high risk" - 15% and "very high risk" - 5%. 4. The method according to claim 2 or 3, in which the results of the genetic risk assessment are presented in the form of a petal diagram in which GRR values are laid off on six axes corresponding to the six gene groups and formed by rays drawn from the center of the regular hexagon to its vertices _combinations , draw lines connecting the points on the scales corresponding to the boundary GRR values of the _combination for each of the mentioned risk categories in the corresponding group, and form an individual profile of the genetic predisposition the patient’s personality by plotting points on the scales that correspond to the calculated GRR values of the _combination for the patient and connecting these points with lines. 5. The method according to any one of paragraphs. 1-3, in which full genomic DNA is isolated from blood cells or buccal epithelium. 6. The method according to any one of paragraphs. 1-3, in which the detection of the presence of allelic single nucleotide polymorphisms is carried out by real-time PCR or by pyrosequencing.

Images