RU2738685C1 - Method for prediction of newborn's weight in pregnant women with pre-eclampsia and a family history of pre-eclampsia - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and is intended to predict of newborn's weight in pregnant Russian nationals, who are natives of Central Black Earth Region of Russia, with preeclampsia and a burdened pre-eclampsia family history. DNA is recovered from peripheral venous blood and analyzed polymorphism of vascular endothelium growth factor A - rs833061 VEGFA. If observing an allele of T polymorphism rs833061 of the VEGFA gene, the normal weight of the newborn is predicted. If observing allele C polymorphism rs833061 of VEGFA gene low birth weight is predicted.

EFFECT: invention provides the criteria for estimating the prediction of the newborn's birth weight in pregnant Russian nationals, who are natives of the Central Black Earth Region, having preeclampsia and a burdened family history of preeclampsia on the basis of data on polymorphism rs833061 of the VEGFA gene.

1 cl, 1 dwg, 4 ex

Description

The invention relates to the field of medical diagnostics, can be used to predict the weight of a newborn in pregnant women of Russian nationality who are natives of the Central Black Earth Region of Russia, with preeclampsia (hereinafter PE) and a family history of preeclampsia.

Anthropometric parameters (weight, height) are one of the key indicators of the state of the newborn, which, firstly, characterize the course of the past pregnancy - embryonic growth, as you know, is an important indicator of the outcome of pregnancy and reflects the interaction of physiological and pathological factors affecting the fetus during intrauterine development [Placental insufficiency: Pathogenesis. Forecasting. Diagnostics. Prevention. Obstetric tactics [Text] / Strizhakov AM, Lipatov IS, Tezikov Yu.V. // Samara: LLC "OFORT". 2014. - 239 p.]. According to WHO, the average weight of a newborn is 3200 g for girls and 3300 g for boys, children weighing less than 2500 g are considered low birth weight [https://www.who.int/ru/]. Often such complications of pregnancy as placental insufficiency and preeclampsia lead to the birth of low birth weight children and in some cases to the development of 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.]. Secondly, low birth weight in newborns is a known risk factor for perinatal morbidity (development of distress syndrome, meconial aspiration, necrotizing enterocolitis, etc.) and mortality [Neonatal Morbidities of Fetal Growth Restriction: Pathophysiology and Impact [Text] / Malhotra A, Allison BJ, Castillo-Melendez M, Jenkin G, Polglase GR, Miller SL. // Front Endocrinol (Lausanne). 2019; 10: 55.]. Thirdly, low birth weight children have an increased risk of developing various diseases (cardiovascular, dyslipidemia, metabolic syndrome, etc.) in adulthood [Intrauterine Growth Restriction: Hungry for an Answer [Text] / Devaskar SU, Chu A. // Physiology (Bethesda). 2016; 31 (2): 131-146 .; Molecular genetic and epigenetic aspects of impaired endometrial receptivity in women with low birth weight [Text] / Melkozerova O.A., Bashmakova N.V., Tretyakova T.B., Shchedrina I.D.// Questions of gynecology, obstetrics and perinatology. 2019; 18 (4): 35-43].

According to the literature, maternal factors (vascular and metabolic diseases, thrombophilic conditions, nutritional disorders, drugs, etc.) are important in the formation of the anthropometric characteristics of the newborn (weight, height) [Fetal growth restriction: current knowledge.Nardozza [Text] / LMM , Caetano, ACR, Zamarian, ACP, Mazzola, JB, Silva, CP, Marçal, VMG, et al. // Arch. Gynecol. Obstet. 2017. 295, 1061–1077], including the genetic determinants of the maternal organism [Genetic markers for inherited thrombophilia are associated with fetal growth retardation in the population of Central Russia [Text] / Reshetnikov E, Zarudskaya O, Polonikov A, Bushueva O, Orlova V, Krikun E, Dvornyk V, Churnosov M. // J. Obstet. Gynaecol. Res.// 2017; 43 (7): 1139-1144]. In recent studies (including those carried out at the genome-wide level), the relationship between polymorphism of a number of candidate genes of the “maternal” genome with the height and weight of the newborn has been shown [Genome-wide associations for birth weight and correlations with adult disease [Text] / Horikoshi M. et al.// Nature. 2016.538 (7624): 248-252].

From the field of technology known "Method for assessing the functional state of the fetus using cardiotocography" according to RF patent No. 2628240 from 15.07.2016. The method includes performing cardiotachography and analyzing the obtained cardiotachograms with determining the characteristics of the heart rate (HR) and isolating one of three types of cardiotachograms corresponding to the fetal states: normal, suspicious and pathological. The assessment is carried out in the II trimester of pregnancy according to the following indicators: short-term heart rate variability, long-term heart rate variability, episodes of high variability, episodes of low variability, deceleration. The criterion for the normal type of fetal cardiotachogram is the simultaneous achievement of the following normal values of indicators: short-term heart rate variability - not less than 3.5 ms, long-term heart rate variability - not less than 37 ms, episodes of high variability - more than 5 minutes, episodes of low variability - not more than 4 min, deceleration - no more than 3. The criterion for a suspicious type of fetal cardiotachogram is a deviation from the normal value of at least one of the indicators: episodes of high variability, episodes of low variability, deceleration. The criterion of the pathological type of cardiotachogram is the identification of at least one of the conditions: a deviation from the norm of short-term heart rate variability; deviation from the norm of long-term heart rate variability; simultaneously episodes of high variability are less than 5 minutes, episodes of low variability are more than 5 minutes, deceleration is more than 3; at the same time, episodes of high variability are 0, episodes of low variability are more than 5 minutes, deceleration is more than 3. The method allows predicting and detecting fetal pathologies associated with placental insufficiency in the II trimester of pregnancy, incl. intrauterine growth retardation. The disadvantages of this method are: 1) the possibility of using only in the II trimester of pregnancy; 2) the method does not include genetic markers, which excludes early diagnosis and preventive measures to prevent the birth of low birth weight children.

For the prototype, the patent of the Russian Federation No. 2557952 was chosen according to the application No. 2014124787/15 dated 06/18/2014 "Method for predicting the weight of a newborn taking into account polymorphic variants of the locus 10976 G / A FVII". SUBSTANCE: method includes isolation of DNA from peripheral venous blood and analysis of polymorphism of genes of coagulation factor VII 10976 G / A FVII. The body weight of a newborn at birth is predicted at 37 weeks or more of pregnancy in women of Russian nationality, who are natives of the Central Black Earth Region of Russia. In women who give birth not for the first time, the body weight of the newborn is determined by the equation: y = 6123.431-25.579x1 + 0.267x2 + 205.739x3, where y is the predicted weight of the newborn, x1 is the woman's height in centimeters; x2 is the weight of the child in previous births in grams, x3 is the genetic variant of the locus 10976G / A FVII, while x3 = 1 for the genotype 10976 GG FVII, x3 = 2 for the genotypes 10976 GA and 10976 AA FVII. In primiparous women, the body weight of a newborn is determined by the equation: y = 6278.037-21.739x1 + 232.170x2, where x1 is the woman's height in centimeters; x2 is a genetic variant of the locus 10976 G / A FVII, while x2 = 1 for genotype 10976 GG FVII, x2 = 2 for genotypes 10976 GA and 10976 AA FVII. The disadvantage of this method is the impossibility of predicting the weight of the newborn in early pregnancy in women with PE, who are at high risk for the birth of children with low body weight.

The objective of the present study is to expand the arsenal of methods for predicting the weight of a newborn, namely, to create a method for predicting the weight of a newborn in pregnant women with PE and with a family history of PE.

The technical result of using the invention is to obtain criteria for assessing the prognosis of the weight of a newborn in pregnant women of Russian nationality who are natives of the Central Chernozem region, with preeclampsia and a burdened family history of PE based on data on the rs833061 polymorphism of the VEGFA gene.

The problem is solved by the proposed method, which includes:

- isolation of DNA from peripheral venous blood;

- analysis of the gene polymorphism of vascular endothelial growth factor A rs833061 VEGFA;

- prediction of the newborn's weight: when the T allele of the rs833061 polymorphism of the VEGFA gene is detected, the birth of a fetus with a normal body weight is predicted. When the C allele rs833061 VEGFA is detected, a low birth weight is predicted.

The novelty and inventive step lies in the fact that the prior art does not know the possibility of predicting the weight of a newborn in pregnant women with preeclampsia and a family history of PE, taking into account the rs833061 polymorphism of the VEGFA gene.

The method is carried out as follows:

Isolation of genomic DNA from peripheral blood is carried out 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 MgCl2, 10 mM Tris-HCl with pH = 7.6 are added to 4 ml of blood with EDTA. The resulting mixture is stirred and centrifuged at 4 ° C, 4000 rpm. within 20 minutes. After centrifugation, the supernatant is decanted, 4 ml of a solution containing 25 mM EDTA with pH = 8.0 and 75 mM NaCl is added to the sediment, and the solution is resuspended. Then add 0.4 ml of 10% SDS, 35 μl of proteinase K, 10 mg / ml, and the sample is incubated at 37 ° C for 16 hours.

At the second stage, DNA extraction is carried out sequentially from the obtained lysate with equal volumes of phenol, phenol-chloroform (1: 1) and chloroform with centrifugation at 4000 rpm. within 10 minutes. After each centrifugation, the aqueous phase is sampled. 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 minus 200 ° C. The isolated DNA is used to carry out the polymerase chain reaction of DNA synthesis.

Analysis of the polymorphism of the vascular endothelial growth factor A rs833061 VEGFA is carried out by PCR synthesis of DNA on a CFX96 amplifier (Bio-Rad) using standard oligonucleotide primers and probes, followed by analysis of polymorphism by allele discrimination. The reaction mixture with a volume of 25 μL includes: 67 mM Tris-HCl with pH = 8.8, 2.5 mM MgCl2, 0.1 μg of genomic DNA, 10 pM of each primer, 5 pmol of each probe, 200 μM dATP, dGTP, dCTP, dTTP and 1 unit of active Taq polymerase. After denaturation for 5 min at 95 ° C, 40 amplification cycles are performed according to the scheme: primer annealing - 1 min. at t = 54 ° C; denaturation - 15 sec at t = 95 ° С. When carrying out PCR in a CFX96 amplifier with fluorescence detection, genotyping is carried out by the Tag Man probe method according to the data of RFU values (relative units of fluorescence). For rs833061 VEGFA, the ROX fluorescent dye probe corresponds to the C allele, the FAM dye probe corresponds to the T allele (Fig. 1).

Genotyping of the rs833061 polymorphism of the VEGFA gene is carried out by the method of detecting TagMan probes according to the data of the RFU values (relative fluorescence units) of each probe on a CFX96 amplifier with a detection system in real time.

The invention is characterized by figures.

FIG. 1. Discrimination of alleles by the TaqMan probe detection method according to the RFU values (relative fluorescence units) of each probe on the CFX96 amplifier with a real-time VEGFA polymorphism detection system (rs833061): - CC, - TT, - CT, • - negative control.

To study the association of the polymorphic locus rs833061 VEGFA with the newborn's weight, we used log-linear regression analysis (allelic, additive, recessive, and dominant genetic models were considered). The calculations were performed using the PLINK v. 2.050 (http://zzz.bwh.harvard.edu/plink/). The regression coefficients (β) and their errors (SE) were calculated, characterizing the direction of change in the studied quantitative indicator (newborn weight) to one polymorphic genetic variant (minor allele).

The possibility of using the proposed method for predicting the weight of a newborn in pregnant women with preeclampsia and a burdened family history of PE is confirmed by an analysis of the observation results of 190 patients with preeclampsia (mean age 26.88 ± 5.37 years). The study group included women of Russian nationality, who are natives of the Central Black Earth Region of Russia and have no relationship with each other. The diagnosis of preeclampsia was based on the presence of: edema, arterial hypertension and proteinuria. The exclusion criteria included: diseases of the uterus (fibroids of the uterus, anomalies in the development of internal genital organs), other pathologies of pregnancy (anomalies of attachment and location of the placenta, fetal growth retardation, Rh-conflict), fetal pathology (CDF), multiple pregnancy. Clinical and clinical laboratory examination of pregnant women was carried out at the time of delivery at the perinatal center of the Belgorod Regional Clinical Hospital of St. Joasaph. Newborn somatometry was performed using standard methods. The typing of molecular genetic markers was carried out in the laboratory of "Human Molecular Genetics" of the Medical Faculty of the Belgorod State National Research University. This study was carried out under the supervision of the Ethics Committee of the Faculty of Medicine of NRU BelSU (informed consent was obtained from each woman included in the study).

When conducting a log-linear regression analysis, an association of the rs833061 polymorphism of the VEGFA gene with the newborn weight in women with preeclampsia and a burdened family history was established: the minor T allele of the rs833061 polymorphism of the VEGFA gene was significantly associated with the birth of a fetus with normal body weight (for the allelic model, β = 0.17 = 0.003, pperm = 0.005, additive model β = 0.176, p = 0.002, pperm = 0.002, dominant model β = 0.212, p = 0.02, pperm = 0.02, recessive model β = 0.248, p = 0.008, pperm = 0.009); the reference allele C rs833061 VEGFA is associated with low birth weight. If a woman has the TT genotype of the rs833061 polymorphism, the weight of the newborn is maximum, and when determining the CC genotype, the weight of the newborn is minimal.

Examples of using the proposed method.

Example 1. A pregnant woman A. was diagnosed with moderate preeclampsia. The history of the disease determined the presence of a burdened family history. According to the data of molecular genetic examination, the TT genotype of the rs833061 VEGFA polymorphism was revealed, which made it possible to assign it to the group of pregnant women with a low risk of giving birth to a low birth weight fetus. Further observation of this patient showed that the weight of a newborn born at a period of 40-41 weeks was 3350 grams.

Example 2. A pregnant woman V. was diagnosed with moderate preeclampsia. The history of the disease determined the presence of a burdened family history. Based on the data of molecular genetic examination, the genotype of CT of the rs833061 polymorphism of the VEGFA gene was determined, which made it possible to assign it to the group with an increased risk of giving birth to a low birth weight fetus. Further observation of this patient showed that the weight of a newborn, born at 38-39 weeks, was 2600 grams.

Example 3. A pregnant woman Z. was diagnosed with severe preeclampsia. The history of the disease determined the presence of a burdened family history. Based on the data of molecular genetic examination, the CC genotype of the rs833061 VEGFA polymorphism was established, which made it possible to assign it to the group of high risk of having a fetus with low body weight. Further observation of this patient showed that the weight of a newborn, born at 37-38 weeks, was 2150 grams.

Example 4. A pregnant woman L. was diagnosed with moderate preeclampsia. The history of the disease determined the presence of a burdened family history. According to the data of molecular genetic examination, the CT genotype of the rs833061 VEGFA polymorphism was revealed, which made it possible to assign it to the group of pregnant women with an increased risk of having a fetus with low birth weight. Further observation of this patient showed that the weight of a newborn born at 39-40 weeks was 2500 grams.

Thus, the proposed method makes it possible to predict the weight of a newborn in women with preeclampsia of Russian nationality, who are natives of the Central Black Earth Region of Russia and have a burdened family history of PE, taking into account the rs833061 polymorphism of the VEGFA gene.

Predicting the birth of children with low birth weight will help improve the range of measures aimed at preventing and treating this complication of pregnancy, which will improve perinatal outcomes.

Claims (1)

A method for predicting the weight of a newborn in pregnant women of Russian nationality who are natives of the Central Black Earth Region of Russia, with preeclampsia and a burdened family history of preeclampsia, including DNA isolation from peripheral venous blood, analysis of vascular endothelial growth factor polymorphism A - rs833061 VEGFA, prediction of normal birth weight with normal weight T of the rs833061 polymorphism of the VEGFA gene, prediction of low birth weight in the detection of the C allele of the rs833061 polymorphism of the VEGFA gene.

Images