RU2568891C1 - Method for prediction of risk of preeclampsia by combination of cytokine genes - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: prediction of a risk of preeclampsia in Russian females, natives of the Central Black Earth Region is ensured by recovering DNA from peripheral venous blood to analyse cytokine gene polymorphism. The minor risk of preeclampsia is predicted if observing a combination of three genetic variations of four gene polymorphisms: G I-TAC (rs4512021) and +36 GG TNFR1; +250 A Ltα, G I-TAC (rs4512021) and +36 GG TNFR1; +250 G Ltα (rs909253), +36 A TNFR1 (rs767455), -403 G/A RANTES (rs2107538).

EFFECT: using the invention is expected to provide the more effective identification of the risk of preeclampsia.

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Description

The invention relates to the field of medical diagnostics, can be used in obstetrics and gynecology to predict the risk of preeclampsia based on cytokine genes.

Preeclampsia (PE) or gestosis is a pregnancy complication that occurs in its second half and is characterized by the appearance of edema, proteinuria of arterial hypertension. At present, polyetiology of PE is considered generally accepted. This pregnancy-specific complication and its development is associated with the features of the gestational process itself. Despite the high interest in the problem, the genesis of this pathology remains poorly understood [Sidorova, I.S. Endothelial dysfunction in the development of gestosis [Text] / I.S. Sidorova, I.L. Galinova // Questions of gynecology, obstetrics and perinatology. - 2006. - T. 5, No. 1. - S. 75-81; Characterization of five marker levels of the hemostatic system and endothelial status in normotensive pregnancy and preeclampsia [Text] / M. Hayashi, T. Inoue, K. Hoshimoto [et al.] // Eur. J. Haematol. - 2007. - Vol. 69, No. 5-6. - P. 297-302].

PE is characterized by a triad of typical clinical symptoms: arterial hypertension, proteinuria, edema, as well as deep disorders of the vascular system, hemostasis, immunity, hemodynamics and microcirculation, fetoplacental insufficiency, impaired renal function, liver, lungs, in fact, severe multiorgan develops with severe forms of preeclampsia insufficiency and endogenous intoxication.

According to statistical studies, the frequency of PE has not decreased over the past twenty years and according to different authors is 7-20% [Novel approaches for mechanistic understanding and predicting preeclampsia [Text] / S. Kalkunte, Z. Lai, W. Norris [ et al.] // J. Reprod. Immunol. - 2009. - Vol. 83, No. 1-2. - P. 134-138; Kachalina, T.S. Antioxidant therapy of placental insufficiency in gestosis [Text] / TS Kachalina, N.V. Lebedeva, L.N. Ilyina // Practical medicine. - 2010. - No. 43. - S. 98-99; Radzinsky, V.E. Immunochemical assessment of perinatal risk [Text] / V.E. Radzinsky, S.G. Morozov, L.A. Chugunova // Bulletin of the Peoples' Friendship University of Russia. Ser. The medicine. - 2010. - No. 5. - S. 7-12].

According to literature data, the risk factors for the development of this pathology of pregnancy include: PE during a previous pregnancy, age, first pregnancy, multiple pregnancy, genetic factors, social aspects, occupational hazards, bad habits, poor environmental conditions, insufficient and unbalanced nutrition, complicated obstetric and gynecological history inflammatory diseases of the genitals, which, as a rule, are combined with lesions of the urinary tract, pathology of the vascular system [C dorova, IS Endothelial dysfunction in the development of gestosis [Text] / I.S. Sidorova, I.L. Galinova // Questions of gynecology, obstetrics and perinatology. - 2006. - T. 5, No. 1. - S. 75-81; Aylamazyan E.K., Mozgovaya E.V. [Text] Gestosis: theory and practice. M .: MEDpress-inform, 2008.272 p .; Risk factors for a prolonged length of stay in women hospitalized for preeclampsia in Texas / Z.D. Mulla, B.S. Nuwayhid, K.M. Garcia [et al.] [Text] // Hypertens Pregnancy. 2010. Vol. 29, No. 1. P. 54-68].

Currently, over 30 candidate genes for preeclampsia are known. Local gene networks of preeclampsia include genes for metabolism, genes for endothelial dysfunction, genes for the vascular system, genes for growth factors and cytokines, genes for the endocrine system, genes for the main histocompatibility complex [Genetic passport - the basis of individual and predictive medicine [Text]: [collective. monograph / V.S. Baranov, A.S. Glotov, T.E. Ivashchenko et al.]; under the editorship of V.S. Baranova. - St. Petersburg: Publishing House NL, 2009. - 527 pp., Ill.]. To date, associations of various genetic polymorphisms with PE have been identified [A low COMT activity haplotype is associated with recurrent preeclampsia in a Norwegian population cohort (HUNT2) [Text] / L.T. Roten, M.H. Fenstad, S. Forsmo [et al.] // Mol. Hum. Reprod. - 2011 .-- Vol. 17, No. 7. - P. 439-446; Young, B.C. Pathogenesis of preeclampsia [Text] / B.C. Young, R.J. Levine, S.A. Karumanchi // Annu Rev. Pathol. - 2010 .-- Vol. 5. - P. 173-192].

According to the literature, Ltα is a glycoprotein that is produced by mitogen-stimulated T-lymphocytes and leukocytes, and is also secreted by fibroblasts, astrocytes, myeloma cells, epithelio- and endotheliocytes [Ketlinsky SA, Kalinina NM, 1995; Titov V.N., 2003]. The Ltα gene is located on the sixth chromosome (6p21.3), contains 4 exons. Ltα has the ability to cause hemorrhagic necrosis of tumors [Hollegaard M.V. et al., 2006; Brian E.C., 2009], exhibits partial agonistic activity. Ltα is a chemoattractant for neutrophils [Suzuki J., 1998; Mentula P., 2005; Ketlinsky S.S. et al. 2008].

The human TNFR1 gene is located on chromosome 12p13 [Idriss N.T., Naismith J.H., 2000; Brian, E.S., 2009]. The tumor necrosis factor receptor of the first type is responsible for the acute inflammatory response and is expressed in most cell types. Soluble TNFR1 stabilizes circulating TNF and increases the half-life of this cytokine [Aggarwal Century B.V. et al., 2000; Chan F.M. et al., 2000; Paludan S.R. et al., 2001; Eissner G. et al. 2004; Heinitz O.A. et al., 2004; Brian E.C., 2009].

CCL5 / RANTES (Regulated on Activation Normal T-cell Expressed and Secreted / Regulator of the activity of normal expression and secretion of T cells).

A low molecular weight protein (the molecular weight in humans varies between 7.8 and 8.7 kDa) is located on chromosome 17q12. Belongs to the b-family of SS chemokines. Regulates the activity and secretion of T-lymphocytes.

The chemoattractant is selective for subpopulations of CD4 ⁺ / CD45RO ⁺ -T-lymphocytes, as well as monocytes and eosinophilic granulocytes. The chemokine RANTES affects the biological activity of leukocytes [Kaburagi Y et al., 2001].

I-TAC (CXCL11) (Interferon-induced T cell alpha chemoattractant) is a member of the chemokine family of small secretory proteins involved in immune and inflammatory reactions. The I-TAC gene is located on chromosome 4q11.12. This chemokine plays a key role in inflammatory processes by activating various subpopulations of leukocytes [Mackay 2001], induces anti-inflammatory cytokines in neutrophils, monocytes, macrophages, T cells, astrocytes, fibroblasts and endothelial cells [Fernandez E.J. et al. 2002].

From the field of technology known "Method for predicting gestosis" according to the patent of the Russian Federation No. 2191384 according to the application No. 2000115821/14, 06.16.2000. The claimed method allows to predict the development of gestosis 2-4 weeks before its clinical manifestations (arterial hypertension, proteinuria, edema). The time of erythrocyte resistance to peroxide hemolysis, determined in pregnant women, characterizes the total indicator of the state of cell membranes.

The disadvantage of this method is that it does not consider genetic polymorphisms and their combination with the risk of developing PE.

For the prototype, the “Method for assessing the severity of preeclampsia” was selected according to the patent of the Russian Federation No. 2478966, published on 04/10/2013, including an analysis of the plasma of peripheral venous blood of pregnant women bearing a male fetus, in which after 20 weeks of pregnancy the concentration of fetal DNA in the plasma of venous blood is determined when its content is less than 0.9 ng / ml, an uncomplicated pregnancy is predicted, when the concentration of fetal genetic material is from 1.8 to 2.6 ng / ml, moderate PEs are predicted, and at a concentration of 2.6 ng / ml and above, severe PE degrees.

The disadvantage of this method is that it does not consider genetic polymorphisms and their combination with the risk of developing PE.

The objective of this study is to expand the arsenal of diagnostic methods, namely the creation of a method for predicting the risk of developing PE based on combinations of cytokine genes.

The technical result of using the invention is to obtain criteria for assessing the risk of preeclampsia based on cytokine genes.

In accordance with the task, a method was developed for predicting the risk of preeclampsia in women of Russian nationality, a native of the Central Black Earth Region, based on combinations of cytokine genes, including

- DNA isolation from peripheral venous blood;

- analysis of cytokine gene polymorphisms +250 A / G Ltα (rs909253), +36 A / G TNFR1 (rs767455), -403 G / A RANTES (rs2107538), A / G I-TAC (rs4512021);

- predicting the minimum risk of developing preeclampsia based on cytokine genes for three combinations of genetic variants of four genetic polymorphisms: G I-TAC (rs 4512021) and +36 GG TNFR1; +250 A Ltα, G I-TAC (rs4512021) and +36 GG TNFR1; +250 G Ltα (rs909253), +36 A TNFR1 (rs767455), -403 G / A RANTES (rs2107538).

The novelty and inventive step is that the prior art does not know the possibility of predicting the development of preeclampsia by the presence of various combinations of genetic variants of polymorphic loci based on combinations of cytokine genes + 250A / G Ltα (rs909253), +36 A / G TNFR1 (rs767455), -403 G / A RANTES (rs2107538), A / G I-TAC (rs4512021).

The method is as follows.

Genomic DNA was isolated from peripheral blood by phenol-chloroform extraction (Mathew, 1984) in two 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) was 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 was decanted, 4 ml of a solution containing 25 mM EDTA (pH = 8.0) and 75 mM NaCl were added to the precipitate, and resuspended. Then, 0.4 ml of 10% SDS, 35 μl of proteinase K (10 mg / ml) was added and the sample was incubated at 37 ° C for 16 hours.

At the second stage, DNA was 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 was selected. DNA was precipitated from solution in two volumes of chilled 96% ethanol. The formed DNA was dissolved in bidistilled, deionized water and stored at -20 ° C. Isolated DNA was used to carry out the polymerase chain reaction of DNA synthesis.

Genotyping of DNA marker loci +250 A / G Ltα (rs909253), +36 A / G TNFR1 (rs767455), -403 G / A RANTES (rs2107538), A / G I-TAC (rs4512021) was performed by restriction length polymorphism analysis fragments (RFLP) of PCR amplification products of specific genome regions using the corresponding restriction enzymes manufactured by Sibenzym (Novosibirsk) (Table 1).

The invention is characterized by figures.

FIG. 1. Locus discrimination of alleles - 403 G / A RANTES (rs2107538) (where: ● - homozygotes - 403GG, ■ - homozygotes - 403AA, ▲ - heterozygotes - 403GA, ♦ - negative control).

FIG. 2. Discrimination of alleles at the G / A I-TAC locus (rs4512021) (where: ● - GG homozygotes, ■ - AA homozygotes, ▲ - AG heterozygotes, ♦ - negative control).

FIG. 3. Discrimination of alleles at the locus +250 A / G Ltα (where: ● - homozygotes + 250GG, ■ - homozygotes + 250AA, ▲ - heterozygotes + 250AG, ♦ - negative control).

FIG. 4. Allele discrimination at the locus +36 A / G TNFR1 (rs767455) (where: ● - homozygotes + 36AA TNFR1, ■ - homozygotes + 36GG TNFR1, ▲ - heterozygotes + 36AG TNFR1, ♦ - undefined sample).

Loci genotyping of +250 A / G Ltα (rs909253), +36 A / G TNFR1 (rs167455), -403 G / A RANTES (rs2107538), A / G I-TAC (rs4512021) was carried out by the detection method of TagMan probes according to RFU values (relative fluorescence level) of each probe on an IQ5 amplifier with a real-time detection system. To discriminate the alleles, the Bio-Rad IQ5-Standart Edition program was used.

An analysis of the associations of combinations of genetic variants with preeclampsia was performed using the APSampler software (http://sources.redhat.com/cygwin/), using the Monte Carlo method of Markov chains and Bayesian nonparametric statistics.

Statistical data processing was carried out on a personal computer using the software packages "STATISTICA for Windows 6.0" and "Microsoft Excel 2002".

The possibility of using the proposed method for assessing the risk of the occurrence and development of PE is confirmed by the analysis of the observation results of 247 patients with PE and 245 women in the control group. The total sample size was 492 people. The average age of women with PE was 31.2 ± 7.5 years (varied from 18 to 42 years), and in the control group 31.7 ± 7.3 years (varied from 18 to 43 years) (p> 0.05) . The studied samples included individuals of Russian nationality who are natives of the Central Black Earth Region of Russia and have no kinship between themselves. Thus, the control group did not differ from the group of pregnant women with PE by sex, age (p> 0.05), place of birth and nationality.

All clinical and clinical and laboratory studies were carried out on the basis of the Perinatal Center of the Belgorod Regional Clinical Hospital, with the informed consent of patients on the use of materials from diagnostic and treatment measures carried out during the hospitalization and after associated with PE for research purposes and were recorded according to ethical standards Committee of the Russian Federation. An analysis of the associations of combinations of genetic variants with PE was carried out using the APSampler software (http://sources.redhat.com/cygwin/) using the Monte Carlo Markov chain method and Bayesian nonparametric statistics [A Gibbs sampler for identification of symmetrically structured, spaced DNA motifs with improved estimation of the signal length [Text] / AV Favorov, M.S. Gelfand, A.V. Gerasimova [et al.] // Bioinformatics. - 2005. - Vol. 21, No. 10. - P. 2240-2245].

According to the results of this study, the features of the “genetic constitution” of pregnant women with PE based on combinations of cytokine genes were revealed (Table 2).

It was found that the combination of +250 A Ltα and G I-TAC alleles (rs4512021) with the +36 GG TNFR1 genotype is observed in 13.38% of pregnant women with PE, whereas in the control group it was detected in 20.00% (p = 0, 03). This combination has a protective value for the development of PE (OR = 0.62, 95% CI 0.38-1.00). Along with this, the combination of the G I-TAC allele (rs4512021) with the genotype +36 GG TNFR1 is observed among pregnant women with PE 1.5 times less than in the control group (13.75% and 20.00%, respectively, p = 0 , 04). With this combination of polymorphic markers of cytokine genes, the risk of developing PE is significantly reduced (OR = 0.64, 95% CI 0.38-1.06). A protective trend was also registered in the following combination of the studied polymorphisms of cytokine genes. In the control group, the combination of +250 G Ltα alleles (rs909253), +36 A TNFR1 (rs767455), -403A RANTES (rs2107538) (11.2%) is 1.7 times more likely than pregnant women with PE (6.35 %, p = 0.05, OR = 0.55, 95% CI 0.27-1.11).

Examples of specific application.

1. Venous blood was taken from a pregnant A., a Russian nationality, a native of the Central Black Earth Region, and genotyping of DNA markers revealed a combination of two genetic variants G I-TAC (rs4512021) and +36 GG TNFR1, which allowed her to be assigned to the group of pregnant women with a reduced risk of developing PE. This confirmed further observation. During pregnancy, she showed no signs of PE.

2. Woman B., during pregravid preparation, venous blood was taken, genotyping of DNA markers revealed a combination of three genetic variants +250 A Ltα, G I-TAC (rs4512021) and +36 GG TNFR1, which allowed her to be assigned to the group pregnant women with a reduced risk of developing PE. This confirmed further observation. When pregnancy occurred, she showed no signs of PE.

3. Venous blood was taken from a pregnant E., a Russian nationality, a native of the Central Chernozem Region, a combination of the +250 G Ltα (rs909253), +36 A TNFR1 (rs767455), -403А RANTES (rs2107538) alleles was detected during genotyping of DNA markers , which allowed her to be included in the group of pregnant women with a reduced risk of developing PE. This confirmed further observation. She showed no signs of PE.

4. Venous blood was taken from a pregnant G., a Russian nationality, a native of the Central Chernozem Region, a combination of the +250 G Ltα (rs909253), +36 A TNFR1 (rs767455), -403А RANTES (rs2107538) alleles was detected during genotyping of DNA markers , which allowed her to be included in the group of pregnant women with a reduced risk of developing PE. This confirmed further observation. She showed no signs of PE.

The use of this method will allow to form at-risk groups among women with pregravid preparation and in the early stages of pregnancy and to timely implement the necessary therapeutic and preventive measures in these groups to prevent the development of PE.

Claims (1)

A method for predicting the risk of developing preeclampsia in women of Russian nationality, a native of the Central Chernozem region, including DNA extraction from peripheral venous blood, analysis of cytokine gene polymorphisms and predicting the minimum risk of preeclampsia based on cytokine genes using three combinations of genetic variants of four genetic polymorphisms: G I-TAC ( rs4512021) and +36 GG TNFR1; +250 A Ltα, G I-TAC (rs4512021) and +36 GG TNFR1; +250 G Ltα (rs909253), +36 A TNFR1 (rs767455), -403 G / A RANTES (rs2107538).

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