RU2433403C2 - Method of predicting development of arterial hypertension in pregnant women - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method of predicting development of arterial hypertension in pregnant women consists of realisation of DNA separation from peripheral venous blood, carrying out polymerase chain reaction, finding out data about presence of polymorphism of α-adducin 1 gene. If carrying genotype 460WW of gene of α-adducin 1 ADD1 G460W is detected, conclusion about risk of hypertension development in pregnant women is made.

EFFECT: increased efficiency of predicting arterial hypertension development in pregnant women.

1 ex, 1 tbl, 2 dwg

Description

The invention relates to the field of medical diagnosis, can be used to predict the development of arterial hypertension in pregnant women.

Arterial hypertension (AH) is a disease that is characterized by a persistent increase in blood pressure. Changes in the body during pregnancy predispose to the development of hypertension and therefore, pregnant women have a higher risk of developing AH than among the general population [1]. Arterial hypertension during pregnancy occurs in 5-15% of women and significantly complicates its prognosis; it is the main cause of maternal mortality, premature birth, perinatal loss and cardiovascular disease [2]. AH 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 tissues and organs [3]. Preeclampsia / eclampsia syndrome, the main element of which is arterial hypertension of pregnancy, is just characterized by impaired blood circulation and kidney damage. It is worth noting that eclampsia refers to extremely dangerous pregnancy conditions [1]. To date, a wealth of information has been accumulated indicating that hereditary factors can play a significant role in the development of hypertension [4]. Moreover, this pathology is polygenic in nature. According to experimental and clinical data, an important role in the development of pathology is played by so-called genes - candidates for “vascular reactions” involved in the regulation of blood pressure [5, 6, 7, 8, 9].

The authors in the available scientific and medical and patent literature did not find a way to assess the prognosis of the development of arterial hypertension in pregnant women based on data on the genetic polymorphism of the α-aducin 1 gene.

The objective of this study is to develop a method for assessing the prediction of the development of arterial hypertension in pregnant women based on data on the polymorphism of the α-aducin 1 gene.

The main task of molecular genetic typing of the α-aducin 1 gene locus is the determination of a single nucleotide substitution in the gene, which causes the replacement of the glycine amino acid with trypophan in the 460th codon of the polypeptide molecule, which can affect the development of hypertensive reactions during pregnancy.

The technical result of the use of the invention is to obtain criteria for assessing the development of hypertension in pregnant women, which make it possible to quickly determine the clinical prognosis and treatment strategy for this disease.

In accordance with the task, a method was developed for assessing the state of the cardiovascular system in pregnant women based on the analysis of polymorphism of the α-aducin 1 gene, which consists in isolating DNA from peripheral venous blood and conducting a polymerase chain reaction. The conclusion about the risk of developing hypertension in pregnant women is made in case of identification of the carrier state of the 460WW genotype of the α-adducin 1 ADD1 G460W gene.

The novelty and inventive step lies in the fact that the prior art does not know a method for determining the risk of developing hypertension in pregnant women by the presence of alleles and genotypes of the α-aducin 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 they are resuspended. Then add 0.4 ml of 10% SDS, 35 μl of proteinase K (10 mg / ml) and incubate 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 real-time polymerase chain reaction (PCR) using standard oligonucleotide primers and TaqMan type oligonucleotide probes.

The polymerase chain reaction (PCR) of the synthesis of the polymorphic locus of α-aducin 1 is carried out in 25 μl of the total volume of the mixture containing 65 mm Tris-HCl (pH = 8.8), 2.5 mm MgCl ₂ , 1 μg of genomic DNA, 10 pM each primer and probe, 100 μM dATP, dGTP, dCTP, dTTP and 2 units of active Taq polymerase. The temperature regimes of PCR and the sequence of primers and probes are shown in the table.

The sequence of primers and probes, temperature protocol, the length of the amplified fragment of the locus G460W ADD1 The sequence of primers and probes Amplified fragment length Temperature protocol F: 5'-GTCTTCGACTTGGGACTGCTT-3 ' 83 bp 5 min - 95 ° С 35 cycles: 1 min - 50 ° С (real-time) 15 sec -95 ° С R: 5'-GAGAAGACAAGATGGCTGAACTCT-3 5'-ROX-CATTCTGCCCTTCCTC-RTQ-1-3 ' 5'-FAM- ATTCTGCCATTCCTC-RTQ-1-3 '

Genotyping is carried out using the BioRad IQ5 software on the basis of data on the relative fluorescence level for each probe. The mutant allele 460W of α-aducin 1 causes a change in the primary structure of the protein, which can lead to the development of hypertensive reactions in pregnant women with gestosis.

To assess the correspondence of the observed distribution of genotypes to the expected, based on Hardy-Weinberg equilibrium, the χ ² criterion, the observed heterozygosity, the expected heterozygosity, and the Wright fixation index were used. Associations of alleles and genotypes of a DNA marker with a predisposition to the development of hypertension are evaluated by analyzing a 2 × 2 contingency table with the calculation of the χ ² criterion with Yates correction for continuity and odds ratios (OR) with 95% confidence intervals (CI).

The invention is confirmed by the images presented in the drawings:

Figure 1 - chart "Distribution of pregnant women having an allele 460G locus

G460W ADD1, in terms of blood pressure ";

Figure 2 - chart "Distribution of pregnant women having the genotype 460WW of the locus G460W ADD1, according to the level of blood pressure."

As a specific example, the analysis of the results of observations of 241 pregnant women was carried out. A sample of pregnant women was formed on the basis of the perinatal center of the regional clinical hospital in Belgorod. All women were divided into 3 groups, depending on the level of blood pressure: 1)> 140/90 mm Hg. - hypertension (n = 97); 2) <90/60 mm Hg - hypotension (n = 7); 3) 90-140 / 60-90 mmHg - normotonic (n = 137).

As can be seen from the diagrams presented in figure 1 and figure 2, the presence of the 460G allele (genotypes 460GG and 460GW) is associated with normotonia - in this group the largest percentage of pregnant women with normal blood pressure (58.1% versus 14.3% in carriers of the 460WW genotype, OR = 8.3 (Cl 1.02-189.43), p = 0.05). On the contrary, the presence of the 460W allele in a homozygous state causes the development of hypertension - the vast majority of pregnant women with a mutant genotype have high blood pressure (85.7% versus 38.9% in carriers of the 460G allele, OR = 0.1 (Cl 0.005-0.91) , p = 0.036).

Thus, the presence of the 460WW genotype of the G460W ADD1 locus can be considered an unfavorable factor in the development of arterial hypertension in pregnant women and indicates the possible prognostic significance of this genetic marker.

Literature

1. Shekhtman M.M., Burduli G.M. Respiratory and circulatory diseases in pregnant women. - M: "Triad-X", 2002. - 232 p.

2. Tkacheva O.N., Barabashkina A.V. Actual issues of the pathogenesis, diagnosis and pharmacotherapy of arterial hypertension in pregnant women. - M: "PAGRI", 2006 - 140 p.

3. Savelyev G. M. Obstetrics. - M .: Medicine, 2000 .-- 160 p.

4. Torshin I.Yu., Gromova O.A. Vascular diseases of the heart, brain and molecular genes. Part 3: the role of molecular genes in vasoconstriction, vasodilation, electrolyte metabolism and vascular remodeling // Difficult patient.- 2008. - v.6, No. 5-6. - S.8-11.

5. Vshse M. Psaty, Nicholas L. Smith, Susan R. Heckbert. et al. Diuretic Therapy, the alpha-Adducin Gene Variant, and the Risk of Myocardial Infarction or Stroke in Persons With Treated Hypertension // JAMA. - 2002, April 3. - v.287, No. 13. - P.1680-1689.

6. Iwai C., Akita H., Kanazawa K. et al. Arg389 Gly polymorphism of the human betal-adrenergic receptor in patients with nonfatal acute myocardial infarction // Am Heart J. - 2003. - v. 1, No. 146. - R. 106-109.

7. Min-Ho Shin, Eun-Kyung Chung, Hee-Nam Kim et al. Alpha-adducin Gly460 Trp and Essential Hypertension in Korea // J Korean Med Sci. - 2004. - No. 19. - P. 812-814.

8. Rubattu S., Stanzione R., Di Angelantonio E., Zanda B. et al. Atrial natriuretic peptide gene polymorphisms and risk of ischemic stroke in humans // Stroke. - 2004. - v.4, No. 35. - P.814-818.

Claims (1)

A method for predicting the development of arterial hypertension in pregnant women, including the extraction of DNA from peripheral venous blood, carrying out a polymerase chain reaction, the detection of data on the presence of gene polymorphism, characterized in that if the carrier of the 460WW genotype of the ADD1 1 AD4 gene G460W is identified, they conclude that there is a risk of development hypertension in pregnant women.

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