RU2557944C1 - Method of predicting level of arterial pressure in women in late pregnancy - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention deals with method of predicting level of arterial pressure in women of Russian nationality, born in Central Black Earth region of Russia. Method includes separation of DNA from lymphocytes of peripheral venous blood and analysis of genetic polymorphisms. +46G/A ADRB2 and 4a/4b eNOS by method of polymerase chain reaction Level of systolic arterial pressure in women in late pregnancy is predicted by results of multiple regression equation of the following type: Y=15,455+2,544x+9,946x+0,736x+4,716x+0,185xwhere xis genetic variant in locus - 4a/4b eNOS, namely 4b4b=1; 4a4b=2; 4a4a=3; xis presence of preeclampsia in relatives: yes=0, no=1; xis level of systolic arterial pressure before pregnancy, mm Hg; xis presence of cardiovascular system pathology: yes=0, no=1; xis woman's weight before pregnancy, kg Level of diastolic arterial pressure in women in late pregnancy is predicted, for which purpose multiple regression equation of the following type is used: Y=14,200+7,768x-2,877x+7,500x+0,414x+3,668xwhere xis genetic variant in locus - 4a4b eNOS, namely 4b4b+4a4b=1, 4a4a=0; xis genetic variant in locus +46G/A ADRB2, namely GG+GA=1, AA=0; xis presence of preeclapsia in relatives:yes=0, no=1; xis systolic arterial pressure before pregnancy, mm Hg; xis presence of cardiovascular system pathology: yes=0, no=1.EFFECT: invention makes it possible to realise early prediction of increase of arterial pressure level in women in late pregnancy, will make it possible to form of women at the stage of pregravidal preparation and at early terms of pregnancy groups of high risk of developing hypertension in late pregnancy, as well as realise required therapeutic-preventive measures aimed at prevention of development of said pregnancy complication in due time.2 dwg, 2 tbl, 1 ex

Description

The invention relates to the field of medical diagnosis, can be used to predict the level of blood pressure in women at the end of pregnancy.

Arterial hypertension (AH) is one of the most common forms of pathology. The frequency of hypertensive conditions in pregnant women ranges from 15 to 20%, and in some regions of Russia up to 29% [Vikhlyaeva, E. M. Preclinical manifestations of systemic disorders, clinical outcomes and long-term consequences of preeclampsia [Text] / E. M. Vikhlyaev // Obstetrics and gynecology. - 2009. - No. 1. - S. 3-6]. Hypertensive disorders during pregnancy are one of the most important public health problems worldwide. The clinical significance of the increase in blood pressure during pregnancy is explained by the high frequency of pathological conditions that develop in pregnant women with a high level of blood pressure [Vertkin A.L., Tkacheva ON, Murashko L.E. et al. Arterial hypertension of pregnant women: diagnosis, management tactics and treatment approaches [Text] // Attending physician. - 2006. - No. 3. - S. 22-25 .; Gmurman V.E. Probability Theory and Mathematical Statistics. - M .: Higher school, 2006. - 284 p .; Makarov O.V., Nikolaev N.N., Volkova E.V. Arterial hypertension in pregnant women. Is it only gestosis? [Text] // M .: Geotar-Media, 2006. - 176 p.]. The most dangerous are preeclampsia (5.5%), premature detachment of a normally located placenta (5-10%), intrauterine fetal death associated with placental insufficiency (24.5%). According to WHO data, maternal and perinatal mortality due to complications caused by increased blood pressure in pregnant women is from 20 to 40% [Vertkin A.L., Tkacheva O.N., Murashko L.E. et al. Arterial hypertension of pregnant women: diagnosis, management tactics and treatment approaches [Text] // Attending physician. - 2006. - No. 3. - S. 22-25 .; Gmurman V.E. Probability Theory and Mathematical Statistics. - M.: Higher School, 2006. - 284 p.].

Blood pressure is regulated by various mechanisms. Along with other factors, a violation of its vascular regulation, associated with a reduced content of nitric oxide in the blood, can lead to hypertension.

An early predictor of these disorders is endothelial dysfunction, the most important markers of which include nitric oxide and endothelin-1 levels [Vikhlyaeva, E. M. Preclinical manifestations of systemic disorders, clinical outcomes and long-term consequences of preeclampsia [Text] / E. M. Vikhlyaev // Obstetrics and gynecology. - 2009. - No. 1. - S. 3-6]. With “normal” endothelial function, the balance is always shifted towards maintaining vasodilation, which is ensured, inter alia, due to stable, sufficient expression of endothelial NO synthase. This enzyme is involved in the synthesis of NO and, therefore, in the regulation of vascular tone and blood pressure. Changes in the expression of NO synthase are associated with a number of cardiovascular diseases, including arterial hypertension [Eilamazyan E.K. Emergency care in extreme conditions in obstetric practice. [Text] // 3rd ed. SPb. 2002, p. 426-432 .; H. Critchley, A. Poston, J. Walker, Pre-eclampsia, [Text] // RCOG Press, London (2003), pp. 189-207].

The eNOS gene is located on chromosome 7q35-36. Several polymorphic regions were found in the exons and introns of the eNOS gene, two of which were most studied, namely, the minisatellite repeat in intron 4 (eNOS 4a / 4b polymorphism) and a mutation at position 298 of the protein sequence, leading to the replacement of the glutamic acid residue with aspartic acid (Glu298Asp) . In the case of the polymorphic marker 4a / 4b eNOS, a correlation was found between the genotypes and the level of nitrates and nitrites in the blood, directly related to the rate of NO production by vascular endothelium. Carriers of genotype 4b / 4b have a level of nitrates and nitrites in the blood 25% higher than carriers of genotype 4a / 4a. Thus, we can talk about the potential genetic role of genotype 4a / 4a as a risk factor for the development of atherosclerosis and diseases that lead to impaired normal production of NO [Endothelial nitric oxide synthase Glu298Asp gene polymorphism, blood pressure and hypertension in a general population sample [Text] / B . Wolff, H. J. Grabe, C. Schlüter [et al.] // J. Hypertens - 2005. - Vol.23, No. 7. - R. 1361-1366].

Among the factors influencing the level of blood pressure, β ₂ -adrenoreceptors play an important role [Widmaier, EP Vander's Human Physiology: the mechanisms of body function [Text] / EP Widmaier, H. Raff, KT Strang. - 11th ed. - Boston, MA: McGraw-Hill Higher Education, 2008. - xxviii, 770 p.]. When catecholamines excite β ₂ -adrenoreceptors encoded by the ADRB2 gene, a "inhibitory" effect is achieved, which is manifested in the expansion of blood vessels (coronary, skeletal muscles), relaxation of smooth muscles, and respiratory tract. Some studies have linked the + 46G polymorphism allele with high blood pressure and the risk of hypertension [Beta (2) -adrenergic receptor gene variation and hypertension in subjects with type 2 diabetes [Text] / K. Bengtsson, M. Orho-Melander, O. Melander [et al.] // Hypertension. - 2001. - Vol. 37, No. 5. - R. 1303-1308], other authors have not identified such associations [Kato N. et al., 2001; Herrmann S. et al., 2002]. There is a number of data showing that + 46G / A ADRB2 polymorphism reduces receptor sensitivity to ligands and may be associated with the development of arterial hypertension [Brodde, OE Beta1- and beta2-adrenoceptor polymorphisms and cardiovascular diseases [Text] / OE Brodde / / Fundam. Clin. Pharmacol - 2008. - Vol.22, No. 2. - R. 107-125].

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

In the studied medical and accessible patent literature, the authors did not find a method for predicting the level of blood pressure in women at the end of pregnancy based on data on genetic polymorphisms 4a / 4b eNOS and + 46G / A ADRB2 in combination with other factors affecting the level of blood pressure .

For the prototype, RF patent No. 2473912 was selected according to RF application No. 20111139871/15, September 30, 2011 “A method for predicting the level of blood pressure in pregnant women depending on the genetic polymorphism of endothelin - 1” (Churnosov MI, Reshetnikov EA, Akulova L .Y .; Kolesnikov Yu.V., Polonikov A.V.). The inventive method, including the selection of DNA from lymphocytes of peripheral venous blood and analysis of K198N polymorphism of the endothelin 1 gene (ET-1) by polymerase chain reaction. In the case of the detection of the 198K allele of the K198N ET-1 polymorphism, the risk of an increase in blood pressure in pregnant women is predicted for periods of 37-40 weeks, provides an effective criterion for assessing changes in blood pressure in pregnant women, which allows determining the probability of an increase in blood pressure up to pregnancy and applying it before pregnancy certain tactics of conducting a woman during the entire gestation period.

The disadvantage of this method is that only one genetic factor is analyzed - the genetic polymorphism of endothelin -1 associated with the level of blood pressure in pregnant women and other predictors of predicting the level of blood pressure in women at the end of pregnancy are not considered.

The objective of the invention is to expand the arsenal of methods for predicting the level of blood pressure in women at the end of pregnancy.

EFFECT: obtaining evaluation criteria for predicting the level of blood pressure in women of Russian nationality who are natives of the Central Black Earth Region of Russia at the end of pregnancy, allowing, including before pregnancy, to determine the likelihood of a change in blood pressure in women when pregnancy occurs towards increase it and, therefore, implement certain tactics for the management of women at risk throughout the gestation period.

In accordance with the task, a method was developed for predicting the level of blood pressure in women of Russian nationality who are natives of the Central Black Earth Region of Russia at the end of pregnancy, including:

- DNA isolation from peripheral venous blood;

- analysis of polymorphisms by polymerase chain reaction;

- predicting the level of blood pressure in women at the end of pregnancy, depending on the identified genetic variants at the 4A / 4b eNOS and + 46G / A ADRB2 loci and the following predictors:

for GARDEN - the weight of a woman before pregnancy, the presence of PE in relatives, the presence of pathology of the cardiovascular system, the level of GARDEN before pregnancy, a genetic variant at the 4a / 4b eNOS locus;

for DBP, the weight of the woman before pregnancy, the presence of PE in relatives, the presence of pathology of the cardiovascular system, the level of SBP before pregnancy, the genetic variant at the 4a / 4b eNOS locus and the genetic variant at the + 46G / A ADRB2 locus.

- prediction of the level of systolic blood pressure in women at the end of pregnancy according to the results of the multiple regression equation of the following form:

Y ₁ = 15.455 + 2.544x ₁ + 9.946x ₂ + 0.736x ₃ + 4.716x ₄ + 0.185x _5, where x ₁ is the genetic variant at the locus - 4a / 4b eNOS (4b4b-1; 4a4b-2; 4a4a-3 ), x ₂ - the presence of preeclampsia in relatives (yes-0; no-1), x ₃ - the level of systolic blood pressure before pregnancy, mmHg, x ₄ - the presence of pathology of the cardiovascular system (yes-0; no -1), x ₅ - woman's weight before pregnancy, kg.

- predicting the level of diastolic blood pressure according to the results of the multiple regression equation of the following form:

Y ₂ = 14,200 + 7,768x ₁ -2,877x ₂ + 7,500x ₃ + 0,414x ₄ + 3,668x _5,

where x ₁ is the genetic variant at the locus - 4a4b eNOS ((4b4b + 4a4b) -1; 4a4a-0), x ₂ is the genetic version at the locus + 46G / A ADRB2 ((GG + GA) -1; AA-0) , x ₃ - the presence of preeclampsia in relatives (yes-0; no-1), x ₄ - systolic blood pressure before pregnancy, mmHg, x ₅ - the presence of pathology of the cardiovascular system (yes-0; no-1 )

The novelty and inventive step lies in the fact that the possibility of predicting the level of blood pressure in women of Russian nationality who are natives of the Central Black Earth Region of Russia at the end of pregnancy by the presence of genetic variants 4a / 4b eNOS and + 46G / A ADRB2 in combination is not known from the prior art with other predictors.

The method is as follows:

DNA is isolated from samples of the patient's peripheral venous blood in 2 stages. At the first stage, 25 ml of lysis buffer containing 320 mM sucrose, 1% Triton X-100, 5 mM MgCl2, 10 mM Tris-HCl (pH = 7.6) is added to 4 ml of blood. The resulting mixture was stirred and centrifuged at 4 ° C, 4000 rpm for 20 minutes. After centrifugation, the supernatant is decanted, 4 ml of a solution containing 25 mM EDTA (pH = 8.0) and 75 mM NaCl are added to the precipitate, and they are resuspended. Then add 0.4 ml of 10% SDS, 35 μl of proteinase K (10 mg / ml) and incubate the sample 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 ⁰ C.

The isolated DNA is then subjected to polymerase chain reaction using standard oligonucleotide primers (table 1).

The structure of primers and probes used for genotyping of the studied DNA markers

Table 1

Title 5'-3 'primer sequence
and probes Literary source VNTR 4a / 4b eNOS (rs699) F: 5-agg ccc tat ggt agt gcc ttt-3
R: 5-tct ctt agt gct gtg gtc ac-3 Spiridonova M.G. et al., 2002, 2006 + 46G / A ADRB2
(rs1042713) F: 5'-cgg cag cgc ctt ctt g-3 '
R: 5'-tgc gtg acg tcg tgg tc-3 '
5'- ROX -cac cca atg gaa gcc- BHQ2-3 '
5'- FAM -cac cca ata gaa gcc- RTQ1-3 ' Lanfear D.E.et al., 2005

The invention is characterized by:

- гомозиготы по аллелю+46А,

- гомозиготы по аллелю+46G,

- гетерозиготы+46GA,

- отрицательный контрольFigure 1. Alleles discrimination at the + 46G / A ADRB2 locus by TaqMan detection of probes according to the UOF values (relative fluorescence level) of each probe on an IQ5 amplifier with a real-time detection system, where

- homozygotes for the allele + 46A,

- homozygotes for the + 46G allele,

- heterozygotes + 46GA,

- negative control

Figure 2. Electrophoretic separation of amplification products of VNTR polymorphism 4a / 4b eNOS., Where 3, 5, 6 - homozygotes 4b4b; 2 - homozygotes 4a4a; 1, 4 - heterozygotes 4a4b.

The possibility of using the proposed method for assessing the level of blood pressure in women at the end of pregnancy is confirmed by an analysis of the results of the examination of 452 women: 249 pregnant women who had an increase in pressure by the end of pregnancy, and 203 women in the control group with a normal pregnancy. The studied samples included individuals of Russian nationality, who are natives of the Central Black Earth region of Russia and have no kinship with each other. The average age of the main group was 27.11 ± 6.42 years (varied from 18 to 44 years), in the control group - 26.5 ± 6.36 years (varied from 18 to 42 years) (p> 0.05). Thus, the population control group did not differ from the main group by age, gender, nationality and place of birth. All clinical and clinical laboratory studies were carried out on the basis of the Perinatal Center of the Belgorod Regional Clinical Hospital of St. Joasaph, with the informed consent of patients on the use of materials of medical diagnostic measures carried out during the hospitalization period and after for research purposes and were recorded according to the standards of the ethical committee of the Russian Federation .

Using the methods of mathematical modeling, multiple regression established a significant effect of genetic, biomedical and other factors on the level of blood pressure in pregnant women (Table 2).

Multiple regression coefficients and significance level of indicators used to predict the level of blood pressure in women at the end of pregnancy.

table 2

Independent (explanatory) signs (X _i ) and their gradations Regression coefficients for SBP, and _i, (p) Regression coefficients for DBP, and _i, (p) Constant 15,455 (0,05) 14,200 (0.05) Genetic variant locus 4a / 4b eNOS
(4b4b-1; 4a4b-2; 4a4a-3) 2.544 (0.04) - Genetic variant locus 4a / 4b eNOS
((4b4b + 4a4b) -1; 4a4a-0), - 7.768 (0.001) Locus variant + 46G / A ADRB2
((GG + GA) -1; AA-0), - -2.877 (0.03) The presence of preeclampsia in relatives (yes-0; no-1) 9.946 (0.00000) 7,500 (0,00000) Systolic blood pressure before pregnancy, mmHg Art. 0.736 (0.0000) 0.414 (0.0000) The presence of pathology of the cardiovascular system
(yes-0; no-1) 4.716 (0.01) 3.668 (0.003) Weight of a woman before pregnancy, kg 0.185 (0.002) -

Statistically significant (p <0.05) factors affecting the level of blood pressure in women at the end of pregnancy include: for SBP - the weight of the woman before pregnancy, the presence of PE in relatives, the presence of pathology of the cardiovascular system, the level of SBP before pregnancy, genetic locus variant 4a / 4b eNOS; for DBP, the weight of the woman before pregnancy, the presence of PE in relatives, the presence of pathology of the cardiovascular system, the level of SBP before pregnancy, the genetic variant at the 4a / 4b eNOS locus and the genetic variant at the + 46G / A ADRB2 locus. The result of multiple regression analysis is the calculation of estimates of the regression coefficients a ₁ , a ₂ , ... and _i equations [Rebrova O.Yu., 2006]:

Y = C + a ₁ x ₁ + a ₂ x ₂ + a ₃ x ₃ + ... + a _n x _n , where x _i are informative features, values a _i are coefficients for these features, C is a constant.

The regression model for predicting the level of systolic blood pressure includes the following predictors: the weight of the woman before pregnancy (t = 3.03; p = 0.002), the presence of PE in relatives (t = 6.72; p <0.0001), the presence of cardiac the vascular system (t = 2.50; p = 0.012), the level of SBP before pregnancy (t = 10.16; p <0.0001), the genetic variant at the 4A / 4b eNOS locus (t = 2.02; p = 0 , 04). These factors determine = 40.30% of the variability in the level of systolic blood pressure in women at the end of pregnancy F (5,340) = 45.90; p <0.0001. It should be noted that, in monofactor models, the contribution of these predictors to systolic blood pressure variability in pregnant women at 37–40 weeks, estimated using simple regression, is significantly lower and amounts to h ² = 10.27% for a woman’s weight before pregnancy (t = 6, 29; p <0.0001), h ² = 14.02% for the presence of PE in relatives (t = 7.52; p <0.0001), h ² = 26.61% for the level of systolic blood pressure before pregnancy ( t = 11.22; p <0.0001).

The multiple regression equation for predicting the level of systolic blood pressure in women at the end of pregnancy has the following form:

Y ₁ = 15.455 + 2.544x ₁ + 9.946x ₂ + 0.736x ₃ + 4.716x ₄ + 0.185x _5,

where x ₁ is the genetic variant at the locus - 4a / 4b eNOS (4b4b-1; 4a4b-2; 4a4a-3), x ₂ is the presence of preeclampsia in relatives (yes-0; no-1), x ₃ is the level of systolic arterial pressure before pregnancy, mmHg, x ₄ - the presence of pathology of the cardiovascular system (yes-0; no-1), x ₅ - woman's weight before pregnancy, kg.

The obtained regression model for predicting the level of diastolic blood pressure includes five predictors: the level of systolic blood pressure before pregnancy (t = 8.63; (p <0.00001), the presence of PE in relatives (t = 7.47; p <0.00001) , the presence of pathology of the cardiovascular system (t = 2.94; p = 0.003), the genetic variant at the 4a / 4b eNOS locus (t = 3.31; p = 0.001), the genetic variant at the locus + 46G / A ADRB2 (t = 2.06; p = 0.03), which determine 34.68% of variability in the level of diastolic blood pressure in women at the end of pregnancy F (5.335) = 35.57; p <0.0001. ence these predictors comprise h ² = 14.02% availability for PE relatives (t = 7,94; p = 0,0000 ), h ² = 19.79% for systolic blood pressure prior to pregnancy (t = 9,25 ; p <0.0001).

The multiple regression equation for predicting the level of diastolic blood pressure in women at the end of pregnancy has the following form:

Y ₂ = 14,200 + 7,768x ₁ -2,877x ₂ + 7,500x ₃ + 0,414x ₄ + 3,668x _5,

where x ₁ is the genetic variant at the locus - 4a4b eNOS ((4b4b + 4a4b) -1; 4a4a-0), x ₂ is the genetic version at the locus + 46G / A ADRB2 ((GG + GA) -1; AA-0) , x ₃ - the presence of preeclampsia in relatives (yes-0; no-1), x ₄ - systolic blood pressure before pregnancy, mmHg, x ₅ - the presence of pathology of the cardiovascular system (yes-0; no-1 )

In order to assess the performance of specific regression models, we examined the patient with respect to these factors affecting the level of blood pressure.

So, in pregnant E. of Russian nationality, who is a native of the Central Black Earth region of Russia, the following indicators were determined: the presence of pathology of the cardiovascular system - (1), the presence of PE in relatives - (1), GARDEN before pregnancy - 90 mm Hg , the weight of a woman before pregnancy is 71 kg., the genetic version at the locus is 4a / 4b eNOS (4b4b-1), the genetic version at the locus is + 46G / A ADRB2 (AA-1). We substitute these attribute values into the two above equations and find the value of y in each equation:

For GARDEN:

Y ₁ = 15.455 + 2.544 * 1 + 9.946 * 1 + 0.736 * 90.0 + 4.716 * 1 + 0.185 * 71 = 112.036.

For DBP:

Y ₂ = 14,200 + 7,768 * 1 - 2,877 * 0 + 7,500 * 1 + 0,414 * 90 + 3,668 * 1 = 70,396.

Further monitoring of this patient showed that the level of SBP and DBP at the end of pregnancy was 110.00 mm Hg. and 70.00 mmHg respectively.

Therefore, the use of the proposed method allows to predict an increase in blood pressure in women at the end of pregnancy.

Thus, the models we developed make it possible to predict elevated blood pressure in women of Russian nationality who are natives of the Central Black Earth Region of Russia at the end of pregnancy, depending on the genetic variants of the 4A / 4B eNOS and + 46G / A ADRB2 loci in combination with other predictors. Early prediction of an increase in blood pressure in women at the end of pregnancy will make it possible to form among women with pregravid preparation and in early pregnancy high-risk groups for the development of hypertension at the end of pregnancy and timely implement the necessary preventive and preventive measures in these groups to prevent the development of this pregnancy complication.

Claims (1)

A method for predicting the level of blood pressure in women of Russian nationality who are natives of the Central Black Earth Region of Russia at the end of pregnancy, including the extraction of DNA from peripheral venous blood, analysis of polymorphisms by polymerase chain reaction, characterized in that they analyze genetic polymorphisms + 46G / A ADRB2 and 4a / 4b eNOS in combination with other predictors and predict the level of systolic and dystolic blood pressure in women at the end of pregnancy, while to predict ur vnya systolic blood pressure in women in late pregnancy using multiple regression equation of the following form:
Y ₁ = 15.455 + 2.544x ₁ + 9.946x ₂ + 0.736x ₃ + 4.716x ₄ + 0.185x _5,
where x ₁ is the genetic variant at the locus - 4a / 4b eNOS, namely 4b4b = 1; 4a4b = 2; 4a4a = 3; x ₂ - the presence of preeclampsia in relatives: yes = 0, no = 1; x ₃ - systolic blood pressure before pregnancy, mmHg; x ₄ - the presence of pathology of the cardiovascular system: yes = 0, no = 1; x ₅ - woman's weight before pregnancy, kg;
- to predict the level of diastolic blood pressure in women at the end of pregnancy, use the multiple regression equation of the following form:
Y ₂ = 14,200 + 7,768x ₁ -2,877x ₂ + 7,500x ₃ + 0,414x ₄ + 3,668x _5,
where x ₁ is the genetic variant at the locus - 4a4b eNOS, namely 4b4b + 4a4b = 1, 4a4a = 0; x ₂ is the genetic variant at the locus + 46G / A ADRB2, namely GG + GA = 1, AA = 0; x ₃ - the presence of preeclampsia in relatives: yes = 0, no = 1; x ₄ - systolic blood pressure before pregnancy, mm Hg; x ₅ - the presence of pathology of the cardiovascular system: yes = 0, no = 1.

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