RU2770869C1 - Method for predicting the risk of developing fetal growth retardation syndrome in women with a burdened family history - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to the field of biotechnology. A method is proposed for predicting the risk of developing fetal growth retardation syndrome in pregnant women who are natives of the Central Chernozem region of Russian nationality and have an aggravated family history of fetal growth retardation. This method includes isolation of DNA from peripheral venous blood and analysis of polymorphism of the epidermal growth factor (EGF) gene.EFFECT: improvement of method for predicting the risk of developing fetal growth retardation syndrome in pregnant women.1 cl, 1 dwg, 1 tbl, 1 ex

Description

The invention relates to the field of medical diagnostics, can be used to predict the risk of developing fetal growth retardation syndrome in women with a burdened family history.

In the structure of pregnancy complications, a significant place is occupied by fetal growth restriction (hereinafter referred to as FGR) [Devaskar S.U., Chu A. Intrauterine growth restriction: hungry for an answer. Physiology (Bethesda). 2016;31(2):131–46]. If the fetus does not achieve enough weight and height indicators in accordance with the gestational age, taking into account gender and ethnicity, we can talk about fetal growth retardation [Intrauterine Growth Restriction: Antenatal and Postnatal Aspects [Text]. Sharma D, Shastri S, Sharma P. Clinical Medicine Insights: Pediatrics. 2016;10:67-83]. Among all pregnancies, the incidence of IUGR can reach 8-10%. IGR has adverse consequences both for the perinatal period (increases the risk of asphyxia, meconium aspiration, respiratory distress syndrome, and other diseases, as well as mortality during this period), and for adulthood (increases the risk of cardiovascular disease, metabolic disorders and [Malhotra A., Allison B.J., Castillo-Melendez M. et al. Neonatal morbidities of fetal growth restriction: pathophysiology and impact. Front Endocrinol (Lausanne) 2019;10:55].

IGR has a heterogeneous nature and can be the result of a number of different placental factors (morphofunctional and epigenetic disorders in the placenta, etc.), fetal (genetically determined defects) and maternal (heart and vascular diseases, nutritional deficiency and imbalance, hypoxic effects, the impact of toxic substances, etc.) origin. At present, there is no doubt about the significant role of hereditary factors among the causes of IGR, including the genetic factors of the maternal organism [Efremova O.A. Study of the association of polymorphic loci of folate cycle genes with the development of fetal growth retardation syndrome of 2-3 degrees. Scientific results of biomedical research. 2020;6(1):37-50]. According to modern data, the weight of a newborn is 40% associated with hereditary factors [Golovchenko O, Abramova M, Ponomarenko I, et al. Functionally significant polymorphisms of ESR1and PGR and risk of intrauterine growth restriction in the population of Central Russia. Eur J Obstet Gynecol Reprod Biol. 2020;253:52-57]. However, it should be noted that the available molecular genetic studies do not provide an unambiguous answer about the role of specific polymorphic loci of various gene groups in the formation of IGR. Therefore, the search for genetic markers for the early diagnosis of this pregnancy complication remains relevant.

The EGF gene (chromosomal localization 4q25, HGNC ID: 3229) belongs to the superfamily of epidermal growth factor genes, and from the encoded preproprotein, due to proteolytic transformation, a peptide is formed - epidermal growth factor (consists of 53-amino acids and has a molecular weight of 6.0 kD , OMIM 131530). This protein acts as a powerful mitogenic factor that plays an important role in the growth, proliferation and differentiation of numerous cell types (ectodermal and mesodermal origin). The biological effects of epidermal growth factor are realized through its binding to its specific receptor (epidermal growth factor receptor, EGFR)located on the cell membrane [Zeng F, Harris RC. Epidermal growth factor, from gene organization to bedside. Semin Cell Dev Biol. 2014;28:2-11]. As a mitogenic growth factor, EGF plays an important role in the development of the embryo already from the moment of preimplantation [Wei Z, Park KW, Day BN, Prather RS. Effect of epidermal growth factor on preimplantation development and its receptor expression in porcine embryos. Mol Reprod Dev. 2001]. EGF has been shown to promote preimplantation embryonic growth as well as trophoblast invasion and postimplantation embryonic growth [Dadi TD, Li MW, Lloyd KC. EGF and TGF-alpha supplementation enhances the development of cloned mouse embryos. Cloning Stem Cells. 2007;9:315–326]. Studies aimed at studying the involvement of the rs4444903 locus of the EGF gene in the formation of pregnancy complications have not been found in the available literature.

In the Russian Federation, studies of the involvement of the epidermal growth factor (EGF) gene in the formation of a predisposition to the development of fetal growth retardation syndrome are scarce, and there are no data on the role of the EGF gene rs4444903 polymorphic locus in the formation of fetal growth retardation syndrome in pregnant women with a burdened family history.

In the studied scientific medical and available patent literature, the authors did not find a way to predict the risk of developing fetal growth retardation syndrome in pregnant women with a burdened family history of fetal growth retardation, based on data on the rs4444903 polymorphism of the EGF gene.

From the field of technology, patent No. 2646505 "Method for detecting a hereditary predisposition to the development of fetal growth retardation in smoking women" is known according to application No. 2017115003 dated 04/27/2017. The method is a study of peripheral venous blood, including DNA isolation, polymerase chain reaction and analysis of polymorphism of women's genes, characterized in that they analyze the polymorphism of the IL-10 G-1082A gene and identify the genotypes IL-10-1082A/A or IL- 70-1082G/A conclude that there is a hereditary predisposition to fetal growth retardation in female smokers. The disadvantage of this method is the ability to predict the risk of fetal growth retardation only in a limited group of pregnant women, namely, only in women who smoke.

For the prototype, patent No. 2540928 was taken according to application No. 2013148878/15 dated 10/31/2013 "A method for predicting the risk of developing placental insufficiency with fetal growth retardation syndrome of the 2nd-3rd degree in pregnant women", including the collection of peripheral venous blood, and differing in that that after DNA isolation, analysis of polymorphisms of genes of coagulation factors 20210G/A FII, 1691G/A FV, 10976G/A FVII is carried out and an increased risk of developing placental insufficiency with fetal growth retardation syndrome of 2-3 degree in pregnant women is predicted in case of detection of the 10976G FVII allele and genotype 10976GG FVII, and low risk is predicted in the presence of the following combinations: genotype 20210GG FII and allele 10976A FVII; alleles 20210G FII, 10976А FVII with genotype 1691GG FV; alleles 20210G FII and 10976A FVII. Methodical complexity: DNA isolation and analysis of several coagulation factor genes. To study them, a modern genetic laboratory is needed. The disadvantages of this method are the methodological complexity and complexity of the study. In addition, similar analytical studies have not been conducted in women with placental insufficiency, but without the formation of the IGR syndrome.

The objective of this study is to expand the arsenal of diagnostic methods, namely, to create a method for predicting the risk of developing fetal growth retardation syndrome in Russian pregnant women who are natives of the Central Chernozem Region and have a burdened family history of fetal growth retardation, based on data on the rs4444903 polymorphic locus of the epidermal factor gene. EGF growth.

The technical result of using the invention is to obtain criteria for assessing the risk of developing fetal growth retardation syndrome in pregnant women of Russian nationality, who are natives of the Central Chernozem Region and have a burdened family history of IGR, based on data on the rs4444903 polymorphic locus of the epidermal growth factor EGF gene, including:

- isolation of DNA from peripheral venous blood;

- analysis of polymorphism of the gene of epidermal growth factor rs4444903 EGF;

- predicting a high risk of developing fetal growth retardation syndrome in pregnant women with a family history of FGR when a polymorphic growth locus rs4444903 of the EGF gene is detected in the G allele genotype.

The novelty and inventive step lies in the fact that the prior art does not know the possibility of predicting the development of fetal growth retardation syndrome in pregnant women of Russian nationality, who are natives of the Central Chernozem Region and have a burdened family history of IGR based on data on the rs4444903 polymorphic locus of the epidermal growth factor gene ( EGF);

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 MgCl ₂ , 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 obtained lysate with equal volumes of phenol, phenol-chloroform (1:1) and chloroform with centrifugation at 4000 rpm for 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.

Analysis of the rs4444903 polymorphism of the EGF gene is carried out by PCR DNA synthesis on a CFX96 amplifier (Bio-Rad) using standard oligonucleotide primers and probes, followed by analysis of polymorphism by the allele discrimination method. 10 µl reaction mixture includes: 67 mM Tris-HCl (pH=8.8), 2.5 mM MgCl ₂ , 0.1 µg of genomic DNA, 10 pM of each primer, 5 pmol of each probe, 200 µM of dATP, dGTP, dCTP, dTTP and 1 unit of active Taq polymerase. After denaturation (2 min at 95°C), 40 cycles of amplification are performed according to the scheme: primer annealing - 30 sec at t=60°C; denaturation - 10 sec at t=94°C. When conducting PCR in an amplifier (CFX96) with fluorescent detection, genotyping is carried out by the Tag Man probe method according to RFU values (relative fluorescence units). For rs4444903 EGF, the probe with the fluorescent dye VIC corresponds to the A allele, the probe with the FAM dye corresponds to the G allele (Fig. 1).

The invention is characterized by figures.

- AA,

- GG,

- AG, ♦ - 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 ITGB3 polymorphism (rs5918):

-AA,

- GG,

- AG, ♦ - negative control.

To analyze the association of the studied polymorphic locus with the risk of developing fetal growth retardation in pregnant women with a family history of IGR, the odds ratios (OR) and their 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 (allelic, additive, recessive, dominant) with the introduction of corrections for covariates (age and the woman's body mass index before pregnancy) and multiple comparisons (an adaptive permutation test was used). pperm<0.05 was used as a statistically significant level.

The possibility of using the proposed method to assess the risk of developing fetal growth retardation syndrome in pregnant women with fetal growth retardation confirms the analysis of the results of observations of 196 pregnant women with IGR and 324 pregnant women of the control group. The average age of women with FGR (26.63±4.41 years) and women in the control group with the physiological course of gestation (26.17±4.98 years) were comparable (p > 0.05). The basis for the diagnosis of IGR was the presence of a discrepancy between the estimated fetal weight (with a deviation of more than 10th percentile) in comparison with the norm for a given gestational age. Assessment of the anthropometric characteristics of the newborn was used to confirm the presence of IUGR. Samples for the study were formed in the specialized departments of the perinatal center of the Belgorod Regional Clinical Hospital of St. Joasaph in the period 2008–2015. The basis for inclusion in the main group was the presence of an isolated IGR in a pregnant woman, the control group was formed from pregnant women with physiological gestation. Exclusion criteria: gestational age <37 weeks. and >40 weeks, multiple pregnancy, the presence of other pathology of pregnancy (preeclampsia, anomalies of attachment and location of the placenta, the presence of Rh-conflict), congenital malformations in the fetus, benign proliferative diseases of the uterus (uterine fibroids) and its developmental anomalies, severe somatic pathology, refusal women from participating in the present study.

This study was performed according to the standards of good clinical practice and in accordance with the principles of the Declaration of Helsinki. This work was approved by the ethics committee of the Medical Institute of the National Research University BelSU (protocol No. 2 of February 13, 2008) and was carried out with the written voluntary informed consent of all the examined women.

Examination of pregnant women was carried out at the time of delivery and included: general clinical analysis of blood and urine, biochemical blood test, coagulogram, determination of the protein level in daily urine. Performed CTG, fetal ultrasound, Doppler with the assessment of blood flow in the vessels of the umbilical cord and uterus. The biochemical analyzer Architect c4000 (Abbotte, USA) was used for the study. Ultrasonic fetometry was performed on an expert-class ultrasonic device TOSHIBA XARIO SSA-660A (Toshiba, Japan). When diagnosing IGR, the differences between the obtained fetometric indicators and F. Hadlock nomograms were assessed. Somatometry of the newborn was carried out by the standard method.

It was found that the G allele of the polymorphic locus rs4444903 of the EGF gene is associated with a high risk of developing fetal growth retardation within the following genetic models of allele interaction: additive (OR=1.33, 95% CI 1.02-1.75, p=0.038, pperm =0.039, 512 permutation procedures were performed) and dominant (OR=1.62, 95% CI 1.06-2.47, p=0.026, pperm=0.031, 655 permutation procedures were performed). The results are shown in table 1.

Table 1

Thus, the data obtained indicate that the G allelic variant of the rs4444903 polymorphic locus of the EGF gene is a risk factor for the development of fetal growth retardation syndrome in pregnant women with a family history of IGR.

Specific Implementation Examples

In a pregnant woman K., a native of the Central Chernozem region of Russian nationality, who has a burdened family history, venous blood was taken in the early stages of pregnancy, the genotyping of DNA markers revealed the G allele in the AG genotype of the rs4444903 polymorphic locus of the EGF gene, which allowed her to be assigned to the group pregnant women at high risk of developing sdfd. This was confirmed by further observation: at a period of 30 weeks, a diagnosis of fetal growth retardation of the 1st degree was established.

In a pregnant woman Zh., a native of the Central Chernozem region of Russian nationality, who has a burdened family history, venous blood was taken in the early stages of pregnancy; genotyping of DNA markers revealed the absence of the G allele, genotype AA, polymorphic locus rs4444903 of the EGF gene, which made it possible to attribute her in a group of pregnant women with a low risk of developing sdfd. This was confirmed by further observation: during pregnancy and after childbirth, no signs of FGR were detected.

In a pregnant woman M., a native of the Central Chernozem region of Russian nationality, who has a burdened family history, venous blood was taken at the stage of pregravid preparation; during genotyping of DNA markers, the G allele was detected in the GG genotype of the rs4444903 polymorphic locus of the EGF gene, which allowed her to be assigned to the group pregnant women at high risk of developing sdfd. This was confirmed by further observation: at the onset of pregnancy at a period of 30 weeks, a diagnosis of fetal growth retardation of the II degree was established.

Pregnant woman D., a native of the Central Chernozem region of Russian nationality, who has a burdened family history, had venous blood taken at the stage of pregravid preparation; genotyping of DNA markers revealed the absence of the G allele, genotype AA, polymorphic locus rs4444903 of the EGF gene, which made it possible to attribute her in a group of pregnant women with a low risk of developing sdfd. This was confirmed by further observation after the onset of pregnancy: during pregnancy and after childbirth, no signs of FGR were detected.

Thus, the proposed method makes it possible to predict the risk of developing fetal growth retardation syndrome in Russian pregnant women who are natives of the Central Black Earth Region of Russia and have a burdened family history of IGR, taking into account the rs4444903 polymorphism of the epidermal growth factor (EGF) gene.

Claims (1)

A method for predicting the risk of developing fetal growth retardation syndrome in pregnant women who are natives of the Central Chernozem region of Russian nationality and have a burdened family history of fetal growth retardation, including DNA extraction from peripheral venous blood and analysis of polymorphism of the epidermal growth factor EGF gene, a high risk of developing fetal growth retardation syndrome in pregnant women with a burdened family history, it is determined when the G allele of the polymorphic locus rs4444903 of the EGF gene is detected.

Images