RU2565405C1 - METHOD FOR PREDICTION OF BLOOD PRESSURE IN DELIVERING FEMALES WITH USING 4a/4b eNOS GENETIC POLYMORPHISM - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: peripheral venous blood DNA in recovered in Russian women native of the Central Black Earth Region of Russia. A polymerase chain reaction is conducted to analyse 4a/4b eNOS genetic polymorphism. If 4a4a eNOS genetic version is observed, an increase of systolic blood pressure is predicted in the delivering women.

EFFECT: deriving an effective criteria of estimation of the blood pressure in the delivering women that enables to determine a probability of blood pressure variation and using a certain approach to the women of a risk group before pregnancy or in the beginning thereof.

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Description

The invention relates to the field of medical diagnosis, can be used in obstetrics to predict the level of blood pressure in women at the time of delivery using genetic polymorphism 4a / 4b eNOS.

Arterial hypertension (AH) in pregnant women is one of the most common forms of pathology. AH increases the frequency of fetal death of the fetus, premature birth, detachment of the normally located placenta. Perinatal mortality (30-100%) and preterm birth (10-12%) in pregnant women with hypertension significantly exceed the corresponding rates in pregnant women without hypertension [I.M. Mellin. Combined preeclampsia in pregnant women with hypertension: it is possible and necessary to prevent [Text] // Medical aspects of women's health. - 2011. - No. 4 (44). - S. 5-13]. AH in pregnant women increases the risk of neurological and cardiovascular diseases in children [Maqnussek E.B. Pregnancy cardiovascular risk as predictors of preeclampsia: population based cohort study / Maqnussek E.B. et al. // BMJ. - 2007. - No. 335. - R. 978-986]. Complications of hypertension account for 20-30% of cases in the structure of maternal mortality. Terrible complications are cerebrovascular accidents, retinal detachment, preeclampsia (PE), eclampsia, massive coagulopathic bleeding [Pavlova T.V. Morphological features of the uteroplacental blood flow during gestosis [Text] / Pavlova T.V., Semykin A.N., Petrukhin V.A., Selivanova A.V. // Russian Bulletin of the Obstetrician and Gynecologist. - 2009. - T. 9, No. 5. - S. 15-19]. In newborns whose mothers had hypertensive disorders, there are significant differences compared with children born to normotonic mothers: low birth weight (12.1% and 7.15%, respectively), early birth (1% and 0.5%, respectively ), acidosis in the umbilical artery (0.58% and 0.19%, respectively), less than 3 points on the Apgar scale after 5 minutes (0.89% and 0.66%, respectively) [Skiaerven R. et al. Recurrence of preeclampsia across generations: exploring. fetal and maternal genetic components in a population based cohort / Skiaerven R. et al. //B.M.J. - 2005 .-- Vol.331. - 877.].

Genetic factors account for 30–40% of cases of primary arterial hypertension. The development of arterial hypertension is due to the polymorphism of many genes [P.N. Veropotvelyan. Genetic aspects of gestosis / P.N. Veropotvelyan, N.P. Veropotvelyan, N.S. Elchaninova et al. // PAG. - 2008. - No. 2 (426). - S. 62-65].

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 levels of nitric oxide and endothelin-1 [Vikhlyaeva EM Preclinical manifestations of systemic disorders, clinical outcomes and long-term consequences of preeclampsia [Text] / Е.М. 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, among other things, 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. Inhibition of eNOS leads to an increase in blood pressure, and increased expression of the eNOS gene causes the development of hypotension [Solov I.A. Endothelial dysfunction as one of the pathogenetic factors of microcirculation disorders during gestosis [Text] / Solov IA, Glukhova TN, Chesnokova NP // Russian Bulletin of the Obstetrician and Gynecologist. - 2006. - T. 6, No. 6. - S. 4-9.].

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, HJ Grabe, C. Schlüter [et al.] // J. Hypertens. - 2005. - Vol. 23, No. 7. - P. 1361-1366.].

The present invention is to develop a method for predicting the level of blood pressure in pregnant women at the time of delivery, depending on the genetic polymorphism of eNOS.

In the studied scientific medical and accessible patent literature, the authors did not find a method for predicting the level of blood pressure in women at the time of delivery based on data on the genetic polymorphism 4a / 4b eNOS.

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 application for a patent of the Russian Federation No. 2012123860 (date of publication of the application December 20, 2013), a method for assessing the risk of pregnancy pathology is proposed, which consists in the analysis of human biological fluids to assess the risk of preeclampsia, premature birth, as well as preeclampsia against arterial hypertension , namely blood serum, using reversed-phase liquid chromatography with registration of the analysis result in the ultraviolet region of the spectrum, and as biomarkers of pregnancy pathology, zuyutsya hypoxanthine, xanthine, uric acid.

The disadvantage of this method is the complexity of the analysis, its duration, and genetic polymorphisms for predicting the level of blood pressure in women at the time of delivery are not considered.

Known patent of the Russian Federation No. 2473912 according to the application of the Russian Federation 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.Yu. .; Kolesnikov Yu.V., Polonikov A.V.). The inventive method, including the extraction of DNA from lymphocytes of peripheral venous blood and the analysis of K198N polymorphism of the endothelin 1 gene (ET-1) by polymerase chain reaction, provides an effective criterion for assessing changes in blood pressure in pregnant women, which allows determining the probability of a change in blood pressure to the side before pregnancy increase it and apply certain tactics of conducting a woman during the entire gestation period.

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

The closest method is described in the source (Reshetnikov EA Polymorphism of the genes of vasoactive hormones and changes in the functioning of the cardiovascular system in women during pregnancy, abstract of the dissertation for the candidate of biological sciences), which states that women of Russian nationality, who are natives of the Central Black Earth Region of the Russian Federation having the AA genotype of the 4a / 4b eNOS locus are characterized by higher values of diastolic pressure and, accordingly, higher values of mean arterial pressure Iya at the end of pregnancy.

The objective of the invention is the expansion of the arsenal of technical tools for assessing the prognosis of blood pressure in women at the time of delivery, depending on the genetic polymorphism of eNOS.

The technical result of the use of the invention is to obtain criteria for assessing the prognosis of high blood pressure in women of Russian nationality who are natives of the Central Black Earth Region of Russia at the time of delivery, which allows for the implementation of certain tactics for managing women at risk throughout pregnancy during 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 time of delivery, including:

- DNA isolation from peripheral venous blood;

- analysis of polymorphisms by polymerase chain reaction;

- predicting an increase in blood pressure in women at the time of delivery in the case of a detected genetic variant at the 4a / 4b eNOS locus.

The novelty and inventive step consists in the fact that the possibility of predicting an increase in blood pressure in women at the time of delivery by the presence of the genetic variant 4a / 4b eNOS is not known from the prior art.

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 drained, 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 ° C.

The isolated DNA is then subjected to polymerase chain reaction using standard oligonucleotide primers shown in 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

The invention is characterized by:

Figure 1 - Electrophoretic separation of amplification products of the locus 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 time of delivery is confirmed by the analysis of the results of the examination of 349 women: 249 of them are pregnant, who had an increase in pressure at the time of delivery, and 100 women from the control group with 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. All clinical and clinical and laboratory studies were carried out at the Perinatal Center of the Belgorod Regional Clinical Hospital of St. Joasaph, with the informed consent of patients to use the materials of treatment and diagnostic measures during the hospitalization period and after, for scientific research purposes and were recorded according to the standards of the Russian Ethics Committee Federation.

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 polymorphic marker 4a / 4b eNOS with indicators of blood pressure in women at the time of delivery is given.

It was found that women with different genotypes at the 4a / 4b eNOS locus have significant differences in blood pressure at the end of pregnancy.

In order to solve the problem of multiple comparisons associated with obtaining false positive results (error of the first kind), the Bonferroni correction was introduced, i.e. recalculated the significance level p for multiple paired comparisons according to the formula [Rebrova O.Yu., 2006]: pcor = p · n, where p is the obtained level of statistical significance, n is the number of paired comparisons. For a statistically significant level, pcor≤0.05 was taken.

Table 2 shows the association of 4a / 4b eNOS genetic polymorphism with blood pressure: pregnant women with the 4A4a eNOS genotype have higher median systolic, diastolic and mean blood pressure at the end of pregnancy (150.0 mmHg 100.0 mmHg and 113.3 mmHg, respectively) compared with women with the 4b4b genotype (SBP - 130.0 mmHg, p = 0.007, taking into account the Bonferroni correction p _cor = 0.021; DBP - 80.0 mm Hg; p = 0.004; p _cor = 0.012; BP mean - 100.0 mm Hg; p = 0.004; p _cor = 0.012) and genotype 4a4b (SBP - 130.0 mmHg; p = 0.01, including pop Bonferroni equalities p _cor = 0.03; DBP - 90.0 mm Hg; p = 0.006; p _cor = 0.018; BP mean - 103.3 mm Hg; p = 0.006; p _cor = 0.018 )

Associations of genetic polymorphism 4a / 4b eNOS with the level of blood pressure in pregnant women (Me, Q25-Q75)

Specific example

Venous blood was taken from pregnant A., a Russian nationality, a native of the Central Black Earth Region, and genotype 4a4a was detected at the 4a / 4b eNOS locus when genotyping DNA markers. By the end of gestation in pregnant A., the level of blood pressure increased and amounted to:

GARDEN - 170.0 mm Hg, DBP - 110.0 mm Hg and HELL cf. - 130 mmHg

The given example confirms that the task - the development of a method for predicting an increase in blood pressure in women of Russian nationality who are natives of the Central Black Earth Region of Russia at the time of delivery using genetic polymorphism 4a / 4b eNOS - has been solved. Based on the identification of a woman’s genetic variant 4a4a eNOS polymorphism 4a / 4b eNOS, they can predict higher blood pressure by the end of gestation for periods of 37-40 weeks.

Claims (1)

A method for predicting the level of blood pressure at the time of delivery in women of Russian nationality who are natives of the Central Black Earth region of Russia, including the extraction of DNA from peripheral venous blood and the analysis of polymorphisms, is carried out by polymerase chain reaction analysis of genetic polymorphism 4a / 4b eNOS, characterized in that predict increased indicators of systolic blood pressure in women at the time of delivery in the detection of the genetic variant 4a4a eNOS.

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