RU2795660C1 - Method for predicting the risk of developing preeclampsia in women based on genetic markers - Google Patents

Abstract

FIELD: medicine; medical diagnostics.

SUBSTANCE: invention can be used to predict the risk of developing preeclampsia in women of Russian nationality. DNA from the peripheral venous blood is isolated. The analysis of polymorphic variants rs1799945 of the HFE gene and rs805303 of the BAG6 gene is carried out. When a combination of genotypes GG rs1799945 of the HFE gene and GG rs805303 of the BAG6 gene is detected, a high risk of developing preeclampsia is predicted.

EFFECT: method provides for obtaining new criteria for assessing the risk of developing preeclampsia in women of the Russian nationality based on data on interlocus interaction of polymorphic variants rs1799945 of the HFE gene and rs805303 of the BAG6 gene.

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Description

The invention relates to the field of medical diagnostics and can be used to predict the risk of developing preeclampsia based on genetic testing in women of Russian nationality.

At present, the issues of reproductive health of the population and the factors that determine it are considered as one of the most significant for the modern population [Abramova M.Yu., Churnosov M.I. Modern ideas about the etiology, pathogenesis and risk factors of preeclampsia. Journal of Obstetrics and Women's Diseases. 2021;70(5):105 -116]. Particular attention is paid to indicators of maternal and perinatal mortality, as key parameters that reflect the economic and social well-being of the population of individual countries. Hypertensive disorders of pregnancy complicate the course of gestation in 5-15% of all cases and annually occupy a leading position among the causes of maternal morbidity and mortality [Bovee E.M., Gulati M., Maas A.N. Novel Cardiovascular Biomarkers Associated with Increased Cardiovascular Risk in Women With Prior Preeclampsia/HELLP Syndrome: A Narrative Review. Eur Cardiol. 2021;16:e36]. Preeclampsia (PE) occupies a special place in this group of diseases as the most dangerous and unpredictable complication of pregnancy, since the pathomorphological processes underlying the pathogenesis of PE occur long before the manifestation of pronounced clinical signs of this gestational complication [Ives C.W., Sinkey R., Rajapreyar I ., et al. Preeclampsia-Pathophysiology and Clinical Presentations: JACC State-of-the-Art Review. J Am Call Cardiol. 2020; 76 (14): 1690-1702].

Women with a burdened history of PE belong to the group of patients with a higher risk of developing thrombosis and stroke, type II diabetes mellitus, arterial hypertension (AH), renal failure, cardiomyopathies, etc. in the distant future [Chourdakis E., Oikonomou N. , Fouzas S. et al. Preeclampsia Emerging as a Risk Factor of Cardiovascular Disease in Women // High Blood Press Cardiovasc. Prev. 2021. V. 28. No. 2. P. 103-114. doi:10.1007/s40292-020-00425-7]. Also, maternal PE significantly increases the risk of early neonatal death, the birth of children with low body weight, the development of respiratory distress syndrome and peri- and intraventricular hemorrhages in newborns [Khader Y.S., Batieha A., Al-Njadat R.A., Hijazi S.S. Preeclampsia in Jordan: incidence, risk factors, and its associated maternal and neonatal outcomes // J. Matern. Fetal Neonatal Med. 2018. V. 31. No. 6. P. 770-776. doi:10.1080/14767058.2017.1297411].

Severe PE is associated with a higher risk of adverse effects. So, in 42% of women with severe PE, persistent arterial hypertension (AH) persists for a year after delivery, and in the long term, the risk of developing cardiovascular diseases is more than 10 times higher compared to the late formation of moderate PE [Hauspurg A. , Countouris M.E., Catov J.M. Hypertensive Disorders of Pregnancy and Future Maternal Health: How Can the Evidence Guide Postpartum Management? Curr Hypertens Rep. 2019; 21 (12): 96].

The mechanisms of PE formation are widely studied all over the world, there are more than 30 hypotheses for the development of this pregnancy complication, in most of which placentation disorders play a key role [Wilkerson R.G., Ogunbodede A.C. Hypertensive Disorders of Pregnancy. Emerg Med Clin North Am. 2019; 37(2):301-316]. Violation of extravillous cytotrophoblast invasion and defective remodeling of the spiral uterine arteries leads to pathomorphological changes in uteroplacental blood flow and early manifestation of PE, mainly of severe course [Syundyukova E.G., Chulkov V.S., Ryabikina M.G. Preeclampsia: the current state of the problem. Doctor.Ru. 2021; 20(1):11-16]. The development of late PE is mainly due to the presence of features of the functioning of the cardiovascular system and maternal hemodynamics and is accompanied by a moderate course [McLaughlin K., Snelgrove J.W., Sienas L.E., et al. Phenotype-Directed Management of Hypertension in Pregnancy. J Am Heart Assoc. 2022;11(7):e023694]. The role of hereditary predisposition as a fundamental risk factor for the development of PE is undoubted. According to the literature data, the contribution of genetic determinants to the formation of this gestational complication is more than 50%, among which the dominant role belongs to the maternal genome - more than 35% [Burton GJ, Redman CW, Roberts JM, et al. Pre-eclampsia: pathophysiology and clinical implications. BMJ. 2019; 366:l2381]. Many research teams are studying the contribution of various groups of candidate genes whose biological products are involved in the pathogenesis of this complication of gestation. Replicative studies of single-nucleotide polymorphic variants of various genes are widely carried out, showing significant associations with diseases that have common biological mechanisms of development with PE [Serebrova V.N., Trifonova E.A., Stepanov V.A. Evolutionary genetic analysis of the role of regulatory regions of the CORO2A gene in the formation of hereditary predisposition to preeclampsia in Russians and Yakuts. Scientific results of biomedical research. 2018;4(3):38-48]. A special niche among these works is occupied by replicative studies of GWAS-significant polymorphic loci associated with the formation of hypertension, as one of the “key” clinical symptoms of PE [Hou B., Jia X., Deng Z., et al. Exploration of CYP21A2 and CYP17A1 polymorphisms and preeclampsia risk among Chinese Han population: a large-scale case-control study based on 5021 subjects. Hum Genomics. 2020;14(1):33]. However, it should be noted that the available molecular genetic studies do not provide an unambiguous answer about the role of specific GWAS-significant for the development of AH polymorphic loci in the formation of PE, which makes it urgent to search for new genetic markers for the early diagnosis of PE.

The rs1799945 polymorphic variant of the HFE gene showed statistically significant associations (p ≤ 5 × 10 ^-8 ) with the development of arterial hypertension and various parameters of blood pressure and hypertension according to eight GWAS studies (at the time of accessing the GWAS catalog - June 2022). It was found that the G allele of the studied polymorphic variant is “risk” and is associated with an increase in blood pressure (SBP, DBP, MAP), while the C allele has a projective value in relation to the development of hypertension and is associated with lower digital values of blood pressure parameters.

According to the GWAS catalog, the rs805303 polymorphic variant of the BAG6 gene showed associations with various parameters of blood pressure (BP), which reached a genome-wide level of significance (p ≤ 5×10 ^-8 ), according to two GWAS studies (at the time of accessing the GWAS- catalog - March 2022) [14, 24]. It was found that the G allele of the polymorphic locus variant rs805303 of the BAG6 gene is associated with higher levels of SBP (p = 1.5×10 ⁻¹¹ ), DBP (p = 3.0×10 ⁻¹¹ ) and the development of AH (p = 1.1×10 ⁻¹⁰ ) in the European population, and the “protective” allele A rs805303 of the BAG6 gene is associated with a decrease in SBP.

In the studied scientific medical and available patent literature, the authors did not find a way to predict the risk of developing preeclampsia in women of Russian nationality based on data on the interlocus interaction of polymorphic variants rs1799945 of the HFE gene and rs805303 of the BAG6 gene.

Patent RU No. 2648872 (publ. 03/28/2018) "Method for early prediction of the development of preeclampsia" is known from the field of technology. This method includes the collection of anamnestic data: family, social, obstetric-gynecological and somatic history; determination of indicators of red blood, platelets, leukoformula; biochemical and immunological analysis; assessment of peripheral hemodynamics by Doppler metry in the uterine arteries, characterized in that the prediction is carried out at a period of 11-14 weeks of pregnancy, the serum level of the hormone erythropoietin is additionally determined with the calculation of the coefficient of adequacy of its production and the assessment of the degree of adequacy of erythropoietin production, the blood flow velocity in the right and left uterine arteries with the determination of the systolic-diastolic ratio, on the basis of the data obtained, the coefficient of predicting the development of preeclampsia G1 and G1 is determined, and if at least one value of the coefficient G is greater than or equal to 0, a high risk of developing preeclampsia is predicted. Significant disadvantages of this method for early prediction of the development of preeclampsia are: 1) a large number of included parameters, which determines the low reproducibility of the proposed method, as well as the difficulty in carrying out a large number of required manipulations and their high cost; 2) The impossibility of predicting preeclampsia at the pregravid stage (before pregnancy); 3) This method does not provide for the inclusion in the calculation of the risk assessment of the development of preeclampsia of genetic markers, whose contribution to the formation of PE is more than 50%.

Patent RU No. 2723627 (published on June 17, 2020) describes "A method for predicting severe preeclampsia in pregnant women with a prothrombin gene mutation, genotype F2G20210A". The method includes a study of the blood of pregnant women in the period of 7-8 weeks, characterized in that the activity of prothrombin is determined by using plasma deficient in the substrate, and when prothrombin activity is detected > 180%, provided that 100% corresponds to a prothrombin concentration in plasma of 1 IU / ml, draw a conclusion about the risk of developing severe preeclampsia. The disadvantages of the method are: 1) The ability to predict only the severe course of preeclampsia; 2) This method is informative only in a cohort of women carriers of the F2G20210A prothrombin gene mutation; 3) The impossibility of predicting the risk of developing preeclampsia at the pregravid stage.

Patent RU No. 2754956 (publ. 09/08/2021) "Method for predicting the risk of developing preeclampsia in pregnant women with fetal growth retardation syndrome" was chosen as the prototype. This method includes isolation of DNA from peripheral venous blood and analysis of the polymorphism of the platelet glycoprotein IIIa ITGB3 gene; a high risk of preeclampsia is predicted when the C allele of the rs5918 polymorphic locus of the ITGB3 gene is detected. The disadvantages of this method are: 1) The possibility of its use only in a limited cohort of patients, namely, in pregnant women diagnosed with fetal growth retardation syndrome; 2) When assessing the risk of developing preeclampsia, only one polymorphic locus is included, while interlocus interactions have a significant impact on the risk of any pathology.

The objective of this study is to expand the arsenal of diagnostic methods, namely, to create a method for predicting the risk of developing preeclampsia in women of Russian nationality based on data on the interlocus interaction of polymorphic variants rs1799945 of the HFE gene and rs805303 of the BAG6 gene.

The technical result of using the invention is to obtain criteria for assessing the risk of developing preeclampsia in women of Russian nationality based on data on interlocus interaction of polymorphic variants rs1799945 of the HFE gene and rs805303 of the BAG6 gene, including:

- isolation of DNA from peripheral venous blood;

- analysis of polymorphic loci rs1799945 of the HFE gene and rs805303 of the BAG6 gene;

- predicting a high risk of developing preeclampsia in women of Russian nationality when a combination of genotypes rs1799945 GG HFE × rs805303 GG BAG6 is detected.

The novelty and inventive step lies in the fact that the prior art does not know the possibility of predicting the development of preeclampsia in women of Russian nationality based on data on interlocus interaction of polymorphic variants rs1799945 of the HFE gene and rs805303 of the BAG6 gene.

The method is carried out as follows:

Isolation of genomic DNA from peripheral blood is carried out by the method of 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 MgCl2, 10 mM Tris-HCl (pH=7.6) is added to 4 ml of blood with EDTA. The resulting mixture is 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 is added to the sediment, and 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 resulting lysate with equal volumes of phenol, phenol-chloroform (1:1) and chloroform with centrifugation at 4000 rpm. within 10 minutes. After each centrifugation produce the selection of the aqueous phase. DNA is precipitated from solution with two volumes of chilled 96% ethanol. After lyophilization, the resulting DNA is dissolved in bidistilled, deionized water and stored at -20°C. The isolated DNA is used to carry out the polymerase chain reaction of DNA synthesis.

The analysis of polymorphic markers rs1799945 of the HFE gene and rs805303 of the BAG6 gene was carried out by polymerase chain reaction (PCR) on a CFX-96 Real-Time System thermal cycler (Bio-Rad, USA) using standard oligonucleotide primers and probes (synthesized at Test - Gen" (Ulyanovsk). Amplification of genomic DNA was carried out in a reaction mixture with a total volume of 10 μl, including a mixture for PCR - 4 μl, Taq polymerase - 2 μl, the test sample (~30 ng DNA / μl) - 1 μl, deionized water - 3 µl Genotyping of the studied samples was carried out using the software "CFX-Manager ™" by the method of discrimination of alleles by the values of relative fluorescence units (RFU) For the rs1799945 polymorphism of the HFE gene, the probe with the fluorescent dye VIC corresponds to the allele C, the probe with the FAM dye corresponds to the allele G (Fig. 1), for the rs805303 BAG6 gene, the probe with the fluorescent dye VIC corresponds to the G allele, the probe with the FAM dye corresponds to the A allele (Fig. 2).

The invention is characterized by figures.

-СС,

-GG,

-CG, ♦-отрицательный контроль.Fig. 1. Allele discrimination by detection of TaqMan probes according to the RFU values (relative fluorescence units) of each probe on a CFX96 amplifier with a real-time detection system for HFE polymorphism (rs1799945):

-SS,

-GG,

-CG, ♦-negative control.

-GG,

-AA,

-AG, ♦-отрицательный контроль.Fig. Fig. 2. Allele discrimination by detection of TaqMan probes according to the RFU values (relative fluorescence units) of each probe on a CFX96 amplifier with a real-time BAG6 polymorphism detection system (rs805303):

-GG,

-AA,

-AG, ♦-negative control.

To analyze the association of the studied polymorphic loci with the risk of developing preeclampsia in Russian women, the odds ratio (OR) and its 95% confidence interval (95% CI) were calculated. Calculations were performed by the method of logistic regression in the statistical package of the PLINK program (available on the electronic resource http://zzz.bwh.harvard.edu/plink/) according to four genetic models with the introduction of corrections for covariates (age and body mass index of a woman before pregnancy) and multiple comparisons (an adaptive permutation test was used). To study interlocus interactions associated with the development of preeclampsia, a modification of the Multifactor Dimensionality Reduction (MDR) method, MB-MDR, was used. The execution of MB-MDR was carried out in the program of the same name (version 2.6) in the R environment.

The possibility of using the proposed method to assess the risk of developing preeclampsia in women of Russian nationality is confirmed by the analysis of the results of observations of 950 pregnant women: 452 women with PE and 498 individuals with physiological gestation. Verification of the diagnosis of PE was carried out on the basis of the presence of arterial hypertension and proteinuria. The samples included women of the Russian ethnic group, born and living in the Central Black Earth Region of the Russian Federation, who do not have family ties. The study excluded women with multiple pregnancies, having other pathology of pregnancy (abnormalities of attachment and location of the placenta, placental insufficiency, Rh conflict, congenital malformations of the fetus) and severe somatic pathology (AH, DM, etc.), who refused to participate in the study. When included in the study, the woman gave voluntary informed consent. Permission was obtained from the ethical committee of the Medical Institute of the National Research University of BelSU to conduct the study.

To analyze the association of polymorphic markers rs1799945 of the HFE gene and rs805303 of the BAG6 gene with the risk of developing preeclampsia in Russian women, the odds ratio (OR) and its 95% confidence interval (95% CI) were calculated. Calculations were performed by the method of logistic regression in the statistical package of the PLINK program according to allelic, additive, recessive, dominant genetic models with the introduction of corrections for covariates and multiple comparisons (at least 1000 permutation procedures were performed). The value of p _perm <0.05 was used as a statistically significant level. To study interlocus interactions associated with the development of preeclampsia, a modification of the Multifactor Dimensionality Reduction (MDR) method, MB-MDR, was used. The execution of MB-MDR was carried out in the program of the same name (version 2.6) in the R environment.

The study found that the combination of genotypes rs1799945 GG HFE × rs805303 GG BAG6 within the epistatic model of two-locus interaction (β=1.43; p=0.027) is associated with a high risk of developing preeclampsia in Russian women.

As examples of the specific application of the developed method, a survey of women of Russian nationality is given, a molecular genetic examination was performed at the rs1799945 loci of the HFE gene and rs805303 of the BAG6 gene.

After taking venous blood from the cubital vein and subsequent genotyping of the isolated DNA, the combination of genotypes rs1799945 CC HFE × rs805303 GG BAG6 was detected in a woman S., who applied to a doctor for pregnancy planning. According to the genotyping data, patient S. is not included in the group of individuals with a high risk of developing preeclampsia. Upon further observation of this woman during pregnancy, there were no clinical signs of preeclampsia during the entire gestation period.

In woman T., at the stage of pregravid preparation after taking venous blood from the cubital vein and subsequent genotyping of the isolated DNA, a combination of genotypes rs1799945 GG HFE × rs805303 GG BAG6 was established. According to the results of genetic analysis, patient T. is included in the group of individuals with a high risk of developing preeclampsia. Upon further observation, this woman was diagnosed with moderate preeclampsia at 31 weeks.

In woman Z., at the stage of pregravid preparation after taking venous blood from the cubital vein and subsequent DNA genotyping, a combination of genotypes rs1799945 GG HFE × rs805303 GG BAG6 was detected. According to the results of molecular genetic analysis, patient Z. is included in the group of individuals with a high risk of developing preeclampsia. Upon further observation, this woman was diagnosed with moderate preeclampsia at 33 weeks.

After taking venous blood from the cubital vein and subsequent genomic DNA genotyping, a combination of genotypes rs1799945 CG HFE × rs805303 AG BAG6 was detected in a woman M., who applied to a doctor for pregnancy planning. According to the genotyping data, patient M. is not included in the group of individuals with a high risk of developing preeclampsia. Upon further observation of this woman during pregnancy, there were no clinical signs of preeclampsia during the entire gestation period.

In woman L., at the stage of pregravid preparation after taking venous blood from the cubital vein and subsequent genotyping of the isolated DNA, a combination of genotypes rs1799945 GG HFE × rs805303 AA BAG6 was detected. According to the genotyping data, patient L. is not included in the group of individuals with a high risk of developing preeclampsia. Upon further observation of this woman during pregnancy, there were no clinical signs of preeclampsia during the entire gestation period.

After taking venous blood from the cubital vein and subsequent genotyping of the isolated DNA, the combination of genotypes rs1799945 GG HFE × rs805303 GG BAG6 was found in a woman D., who turned to a doctor for pregnancy planning. According to the genotyping data, patient S. is included in the group of individuals with a high risk of developing preeclampsia. Upon further observation of this woman at the onset of pregnancy for a period of 27 weeks, the diagnosis was verified: severe preeclampsia.

The use of this method will allow at the preconception stage to form risk groups among women of Russian nationality and timely implement the necessary therapeutic and preventive measures in these cohorts to prevent the development of preeclampsia.

Claims (1)

A method for predicting the risk of developing preeclampsia, taking into account genetic markers in women of Russian nationality, including isolation of DNA from peripheral venous blood, analysis of polymorphisms, characterized in that in the study of polymorphic variants rs1799945 of the HFE gene and rs805303 of the BAG6 gene, a high risk of developing preeclampsia is predicted when a combination of GG genotypes is detected rs1799945 of the HFE gene and GG rs805303 of the BAG6 gene.

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