RU2473912C1 - Method for prediction of blood pressure level in pregnant women depending on genetic endothelin 1 polymorphism - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for prediction of blood pressure level in pregnant women involves DNA recovery from peripheral venous blood lymphocytes and analysis of endothelin 1 (ET-1) K198N gene polymorphism by polymerase chain reaction. Observing the 198K allele of K198N ET-1 polymorphism enables predicting a risk of blood pressure increase in pregnant women on their 37-40 weeks of pregnancy.

EFFECT: invention provides producing an effective assessment criterion of blood pressure variation in pregnant women enable a pre-pregnancy possibility of upward blood pressure variation and applying a specific approach to pregnant woman management over the whole period of gestation.

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Description

The invention relates to the field of medical diagnosis, can be used to predict the level of blood pressure in pregnant women depending on the genetic polymorphism of endothelin 1.

Physiological adaptive changes in the pregnant woman's body have a significant effect on her cardiovascular system, which functions with increased load [1].

Changes in the cardiovascular system during pregnancy are due to the following reasons [2]:

- the emergence of a new circle of blood circulation in the mother-placenta-fetus system;

- an increase in the volume of circulating blood (BCC);

- increase in total body weight;

- increase in intra-abdominal pressure.

Normal pregnancy occurs with low blood pressure (or normal), severe vasodilation, low total peripheral vascular resistance, but with an increase in plasma volume and cardiac output, high renal blood flow, and high plasma renin activity [3].

The increase in blood pressure during pregnancy is due to changes in hemodynamics and kidney function, which are opposite to those that occur during normal pregnancy, that is, the volume of plasma is reduced, and the volume of extracellular fluid is not changed, stroke output is reduced, total peripheral vascular resistance is increased, blood flow in the uterus and kidneys is reduced , plasma renin activity is reduced [4]. An important role in these changes is played by the imbalance between the production of vasoconstrictors with an aggregate effect and the synthesis of vasodilators and antiplatelet agents [3, 5].

An increase in blood pressure during pregnancy is a considerable danger not only for the mother's body, but also for the fetus. Under the influence of high blood pressure, the structure of the blood vessels of the woman’s body changes, which in turn leads to a violation of the blood supply to the tissues and organs of the fetus and the woman’s body [1, 6].

To date, a wealth of information has been accumulated indicating that hereditary factors can play a significant role in the development of changes in blood pressure levels [7]. According to experimental and clinical data, an important role in these processes is played by the so-called genes - candidates for “vascular reactions”, one way or another involved in the regulation of blood pressure [8, 9-13].

The prior art method for the diagnosis of arterial hypertension caused by pregnancy, including the use of a photoplethysmographic arterial pulsation sensor and an ischemic test (application: 2005112689/14, 04/26/2005).

The disadvantage is that the method can only be used when pregnancy occurs, so it is impossible to use it to predict arterial hypertension caused by pregnancy.

The known method of "Quantitative diagnostic analysis of hypertension" according to the patent of the Russian Federation No. 2287160, including:

1) in vitro diagnostics of a genetically determined predisposition to hypertension in a sample isolated from the patient’s body by detecting single nucleotide polymorphisms (SNPs) correlating with hypertension in exon 8 (C → T) of the hsgk1 gene and in intron 6 (T → C) of the hsgk1 gene;

2) the creation of diagnostic kits containing specific reagents for the detection of both types of SNP.

The disadvantage of this method is that it is intended only to identify a genetic predisposition to hypertension, while hypertension in pregnant women is a completely different pathological condition compared to essential hypertension. An increase in the level of blood pressure in pregnant women completely disappears after the end of pregnancy, while essential hypertension, which has formed in the patient, then persists throughout his life.

There is a method for predicting the risk of a persistent increase in blood pressure during pregnancy in women with hypertension according to RU patent No. 2410020, according to which the pulse wave propagation velocity in muscle type arteries (SPWm) and the integral module of the arterial bed elasticity (Eo) at the beginning of the gestation period are examined up to 20 weeks. The disadvantage is that forecasting is carried out directly in pregnant women already suffering from hypertension.

A known method for predicting the development of hypertension by genetic risk factors (pat sig RU No. 2287158), closest to the claimed and selected for the prototype. According to the prototype, the method consists in isolating DNA from peripheral venous blood lymphocytes, using a polymerase chain reaction for DNA synthesis of amplification of PON1 and CETP gene fragments, within which polymorphic nucleotide substitutions are localized, analysis of length polymorphism of amplicon restriction fragments and identification of PON ∗ BB and CETP genotypes ∗ II predict the risk of developing hypertension.

The disadvantage is that this method cannot be used to predict the level of blood pressure in pregnant women.

The present invention is to develop a method for predicting the level of blood pressure in pregnant women according to genetic risk factors depending on the genetic polymorphism of endothelin 1.

The technical result of the use of the invention is to obtain criteria for assessing changes in blood pressure in pregnant women, allowing in a short time and before pregnancy to determine the likelihood of changes in blood pressure upward towards the end of pregnancy or its pronounced dynamics in the event of pregnancy and, therefore, apply certain management women during the entire gestation period.

The problem is solved by molecular genetic typing of the endothelin 1 gene by determining the genetic polymorphism associated with the replacement of the guanine nucleotide gene in thrombol 5 at thymine at position 90 and leading to the replacement of the amino acid lysine (K) with asparagine (N) in the 198 codon of the polypeptide encoded by this gene (K198N ET-1). In accordance with the task, a method was developed to assess the level of blood pressure in pregnant women depending on the genetic polymorphism of endothelin 1, including:

1) DNA extraction from peripheral venous blood by phenol-chloroform extraction;

2) analysis of the K198N polymorphism of the endothelin 1 gene;

3) predicting the risk of increased blood pressure in pregnant women in case of detection of carrier of the 198K allele of the endothelin 1 gene (ET-1).

The novelty and inventive step consists in the fact that the prior art does not know a method for determining the level of blood pressure in pregnant women by the presence of alleles and genotypes of the endothelin 1 gene.

The method is as follows.

DNA is isolated from samples of peripheral venous blood of pregnant women in 2 stages. At the first stage, 25 ml of lysis buffer containing 320 mM sucrose, 1% Triton X-100, 5 mM MgCl ₂ , 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 suspended. Then, 0.4 ml of 10% SDS, 35 μl of proteinase K (10 mg / ml) are added and 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.

Analysis of the K198N polymorphism of the ET-1 gene (K198N ET-1) is carried out by polymerase chain reaction of DNA synthesis using the IQ5 amplifier (Bio-Rad) using locus-specific oligonucleotide primers and specific probes, followed by analysis of the polymorphism by the allele discrimination method. The polymerase chain reaction (PCR) of the synthesis of the polymorphic locus of endothelin 1 is carried out in 25 μl of the total volume of the mixture, including 67 mm Tris-Hcl (pH 8.8), 2.5 mm MgCl ₂ , 0.1 μg of genomic DNA, 10 pM each primer, 5 pcmol of fluorescently labeled probe, 200 μM dATP, dGTP, dCTP, dTTP and 1 unit of active Taq polymerase. The temperature regimes of PCR and the sequence of primers and probes are shown in table 1.

The structure of primers and probes, the temperature protocol of the amplified fragment of the locus K198N ET-1

Table 1 Gene polymorphism The sequence of primers and probes Temperature protocol F: 5'-AGGTCGGAGACCATGAGAAAC-3 ' 1.95 ° C R: 5'-AATGTGCTCGGTTGTGGGT-3 ' 2. 40 cycles: ET-1 K198N 5'-FAM-CTGAAAGGCACACCCCTTAGAGAG-RTQ1-3 ' 1 min - 61 ° С (real-time) 5'-ROX-CTGAAAGGCAATCCCTCCAGAG-BHQ2-3 ' 15 sec - 95 ° С

The present invention is characterized by the following graphic materials:

Figure 1. The graph “Discrimination of alleles at the locus 198 KN ET-1 for ... pregnant women” (where ∙ - homozygotes for the 198K allele, □ - homozygotes for the 198N allele, ▲ - 198KN heterozygotes, ◇ - negative control).

When performing PCR in an IQ5 amplifier with fluorescence detection, genotyping is carried out using TaqMan probes according to the RFU values (relative fluorescence level) of each probe shown in Figure 1. A probe with a FAM fluorescent dye corresponds to a "wild" 198K allele, a probe with a ROX mutant dye 198N.

In figure 1, two bands, vertical and horizontal, divide the graph into four sections: one for each homozygous state, one for the heterozygous state and the section without reaction. Assigning genotypes to unknown samples is determined by plotting the relative fluorescence (RFU) level for one fluorophore (198K allele on the x axis) relative to the RFU for another fluorophore (198N allele on the y axis) in the allele discrimination diagram. If the RFU values of an unknown sample are above the horizontal strip and to the right of the vertical strip, the genotype is heterozygous (198KN). 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 198N allele (the RFU allele of 198N is 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 198K allele (the 198K RFU allele is plotted along the x axis). If the RFU values of an unknown sample are below the horizontal strip and to the left of the vertical, determination of the genotype is impossible (in this case, an undefined sample is a negative control).

To assess the correspondence of the observed distribution of genotypes to the expected, based on Hardy-Weinberg equilibrium, the χ ² criterion, observed heterozygosity, expected heterozygosity, and Wright's fixation index are used. Associations of alleles and genotypes of a DNA marker with a predisposition to the development of hypertension are evaluated using the nonparametric Mann-Whitney method.

As a specific example, an analysis of the relationship of the K198N polymorphic marker ET-1 gene with blood pressure indices in 587 pregnant women for periods of 37-40 weeks (systolic (SBP), diastolic (DBP), pulse (PD) and mean (BPd) blood pressure) is given. and their dynamics associated with the occurrence of pregnancy (for this, indicators of changes in systolic blood pressure - ΔSAD, changes in diastolic blood pressure - ΔDAD and changes in mean blood pressure - ΔAD cf) were calculated. Since the distribution of the analyzed parameters, estimated using the Shapiro-Wilk test, does not correspond to the normal distribution law (p <0.05), the median (Me) and interquartile range (Q25-Q75) were used to describe the indicators of blood pressure, and when comparing individuals with different genotypes for these indicators used a nonparametric method - the Mann-Whitney test.

During the analysis in this study, the group of heterozygotes is combined with the group of homozygotes that have no differences in the values of medians and interquartile intervals from heterozygous carriers, or they were minimal, which, by increasing the volume of one of the compared groups, reduces the likelihood of an error of the second kind, i.e. . errors associated with obtaining false negative results (with a small sample size). Secondly, initially leveling the problem of multiple comparisons, the probability of an error of the first kind decreases, i.e. errors associated with obtaining false positive results.

When studying the associations of the K198N polymorphism of the endothelium 1 gene with blood pressure indicators in women at the end of pregnancy, it was found that women with the 198K allele in the genotype (198KK and 198KN genotypes) are characterized by large values of systolic and diastolic blood pressure (median 135.0 mmHg) Art. and 85.0 mm Hg, respectively) compared with individuals with the genotype 198NN (115.0 mm Hg, p = 0.001 and 70.0 mm Hg, p = 0.001, respectively ) (Table 2).

Association of K198N polymorphism of endothelin 1 gene with blood pressure in women at the end of pregnancy. Me (Q25-Q75)

table 2 Indicators Genotypes R 198KK + 198KN (n = 572) 198NN (n = 15) GARDEN, mmHg 135.0 (120.0-145.0) 115.0 (110.0-130.0) 0.001 DBP, mmHg 85.0 (75.0-90.0) 70.0 (70.0-80.0) 0.001 PD, mmHg 50.0 (40.0-55.0) 40.0 (40.0-50.0) 0,03 HELL cf., mmHg 103.3 (88.3-110.0) 86.7 (83.3-96.7) 0,0007 ΔAD cf., mmHg 13.3 (5.0-23.3) 5.0 (0.0-16.7) 0.006 ΔCAD, mmHg 20.0 (10.0-30.0) 5.0 (0.0-20.0) 0.004 ΔDAD, mmHg 10.0 (0.0-20.0) 5.0 (0.0-15.0) 0.02

In women with the 198KK and 198KN genotypes, the medians of the pulse pressure and mean arterial pressure are 50.0 and 103.3 mmHg, which is more than similar values among pregnant women with the 198NN genotype (40.0 mmHg, p = 0.03 and 86.7 mmHg, p = 0.0007, respectively). Also, individuals with the 198KK and 198KN genotypes are characterized by a higher dynamics of blood pressure indicators by the end of pregnancy: the median changes in systolic, diastolic and mean blood pressure are 20.0 mm Hg, 10.0 mm Hg. and 13.3 mmHg, respectively, in women with the 198NN genotype, 5.0 mmHg each (p = 0.004-0.01).

In the studied groups of women before pregnancy, no significant associations of the polymorphic marker 198KN ET-1 with blood pressure indicators were revealed (Table 3).

The association of K198N endothelin 1 gene polymorphism with blood pressure in women outside pregnancy. Me (Q25-Q75)

Table 3 Indicators Genotypes R 198KK + 198KN (n = 572) 198NN (n = 15) GARDEN, mmHg 110.0 (110.0-120.0) 110.0 (100.0-120.0) 0.26 DBP, mmHg 70.0 (70.0-80.0) 70.0 (60.0-70.0) 0.18 PD, mmHg 40.0 (40.0-45.0) 40.0 (40.0-40.0) 0.63 HELL cf., mmHg 83.3 (83.3-90.0) 81.7 (76.7-86.7) 0.09

Thus, it was confirmed that pregnant women with the 198K allele of the endothelin 1 gene in the genotype are characterized by high blood pressure at 37–40 weeks of gestation and pronounced dynamics of blood pressure by the end of pregnancy compared with women with the 198NN genotype who have lower rates Blood pressure at 38-40 weeks of gestation and less pronounced dynamics of blood pressure at the end of pregnancy.

The above examples confirm that the task is to develop a method for predicting the level of blood pressure in pregnant women depending on the genetic polymorphism of endothelin 1 solved. Based on the identification of a 198K allele in a woman with the K198N ET-1 polymorphism, they can predict pronounced dynamics of blood pressure during pregnancy and high blood pressure by the end of gestation for periods of 37-40 pedals.

Literature

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Claims (1)

A method for predicting the level of blood pressure in pregnant women depending on the genetic polymorphism of endothelin 1, including the isolation of DNA from peripheral venous blood lymphocytes, the analysis of polymorphism by the polymerase chain reaction of DNA synthesis, characterized in that they analyze the K198N polymorphism of the endothelin 1 gene in the case of detection of the 198K allele K198N ET-1 polymorphism predicts the risk of increased blood pressure in pregnant women for periods of 37-40 weeks.

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