RU2540928C1 - Method for prediction of risk of degree 2-3 chronic placental insufficiency with foetal growth delay syndrome in pregnant women - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention aims at the prediction of a risk of degree 2-3 placental insufficiency with foetal growth delay syndrome in the pregnant women. Peripheral venous blood is sampled to recover DNA. 20210G/A FII, 1691G/A FV and 10976G/A FVII coagulation factor genes are analysed. Observing 10976G FVII allele and 10976GG FVII genotype enables predicting a high risk of degree 2-3 placental insufficiency with foetal growth delay syndrome in the pregnant women. The presence of combinations of 20210GG FII genotype and 10976A FVII alleles; 20210G FII, 10976A FVII alleles with 1691GG FV genotype or 20210G FII and 10976A FVII alleles provides predicting a low risk of degree 2-3 placental insufficiency with foetal growth delay syndrome in the pregnant women.

EFFECT: effective method for the prediction of degree 2-3 placental insufficiency with foetal growth delay syndrome in the pregnant women.

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Description

The invention relates to the field of medical diagnosis, can be used to predict the risk of developing chronic placental insufficiency with fetal growth retardation syndrome of the 2nd-3rd degree in pregnant women.

Placental insufficiency (PN) is a syndrome caused by morphological and functional changes in the placenta, with the progression of which the syndrome of fetal growth retardation (SZRP) develops [Ailamazyan, E.K. Obstetrics: a textbook for honey. universities / E.K. Aylamazyan. - 4th ed., Additional. St. Petersburg: SpetsLit, 2003. - 529 s]. SZRP is defined as the lag of the size of the fetus from the expected at this gestational age or as the mass of the fetus at birth below the tenth percentile for a given gestational age. Perinatal mortality in women undergoing placental insufficiency is 10.3% among full-term newborns, and 49% among premature infants. SZRP takes the third place in the structure of causes of perinatal morbidity, later on in children with SZRP there is a lag in physical development (60%), its disharmony (80%), a delay in the rate of psychomotor development (42%) [Serov, V.N. Critical conditions in obstetrics [Text]: a guide for doctors / V.N. Serov, S.A. Markin. - Moscow: Medizdat, 2003 .-- 704 s; Rybkina, N.L. Premature babies: fetoinfantile losses, incidence, hormonal features of the adaptation period [Text]: author. Thesis (Candidate of Medical Sciences: 14.00.33 / NL Rybkina; Kazan, State Medical University - Kazan, 2000. - 24 s].

Placental insufficiency has a multifactorial nature. According to the results of a number of studies, one of the factors leading to the development of placental insufficiency and SZRP is congenital thrombophilia. Thrombophilia is understood as the pathological condition of the body, characterized by an increased tendency to intravascular thrombosis due to hereditary or acquired disorders of the hemostatic system [Makatsaria, A.D. Thrombosis and thromboembolism in the obstetric and gynecological clinic [Text]: molecular genetic. mechanisms and strategies for the prevention of thromboembolic complications: a guide for doctors / A.D. Makatsaria, V.O. Bitsadze, S.V. Akinshina. - Moscow: Honey. inform. Agency, 2007. - 1064 s].

Coagulation factor VII (proconvertin) is one of the key factors in the blood coagulation cascade. It belongs to glycoproteins, is synthesized in the liver with the participation of vitamin K. Molecular mass - 63000 kDa. The content in the blood plasma of 80-120% of the activity [Okorokov AN, 2003]. Factor VII is involved in the external pathway of blood coagulation activation. This pathway includes tissue factor (TF), factor VII, and Ca ions. The factor VII gene is located on the long arm of the 13th chromosome (13q34). 5 variants of factor VII gene polymorphism were found, which cause fluctuations in its level within 30%. Polymorphism is the replacement of arginine with glutamine at position 353 of the Arg353Gln protein chain (R353Q). The replacement is due to a point mutation at position 10976 of one nitrogenous base of guanine (G) to another nitrogenous base of adenine (A), therefore the same polymorphism can be designated as 10976G / A. The presence of the heterozygous variant 10976GA FVII leads to a decrease in the concentration of factor VII in the blood by about 25%, and the presence of the homozygous variant 10976AA FVII leads to a decrease in the concentration of the factor by about 50% compared with the carriers of the variant 10976GG FVII. The type of inheritance is autosomal recessive. The prevalence in the European population is 10-20%). [Makatsaria, A.D. Thrombosis and thromboembolism in the obstetric and gynecological clinic [Text]: molecular genetic. mechanisms and strategies for the prevention of thromboembolic complications: a guide for doctors / A.D. Makatsaria, V.O. Bitsadze, S.V. Akinshina. - Moscow: Honey. inform. agency, 2007. - 1064 s; Okorokov, A.N. Diagnosis of diseases of internal organs [Text]: a guide for doctors: in 10 tons / A.N. Hams. - Moscow: Honey. lit., 2001-2005. - T.5: Diagnosis of diseases of the blood system. Diagnosis of kidney disease. - Moscow, 2003. - 512 p.: Ill.].

Factor V (proaccelerin) is a precursor to the active factor of accelerin. Belongs to glycoproteins of globulin fraction of blood. It is synthesized in the liver and megakaryocytes. The molecular weight is 330,000 daltons. The content in the blood plasma is 0.007-0.01 g / l or 70-150% activity. The minimum level required for hemostasis is 10-25%. The factor V gene is located on chromosome 1q21-25, consists of 6672 base pairs, including an untranslated 3′-region of 163 base pairs and an untranslated 5′-region of 97 or 103 base pairs, depending on which of the two transcription initiation sites is used . Coagulation factor V is a part of the prothrombinase complex, which occurs when blood coagulation is activated by an external or internal pathway and consists of activated factor X (factor Xa), activated factor V (factor Va) and calcium ions associated with phospholipid (PL) membranes (as a rule, these are platelet membranes). The function of the prothrombinase complex is to cleave peptide fragments from the prothrombin molecule, converting prothrombin to thrombin (an enzyme that polymerizes fibrin from fibrinogen). Fibrin is the final product of blood coagulation. The enzyme that cleaves prothrombin in the prothrombinase complex is factor Xa, but without the participation of factor V, this reaction proceeds very slowly. Activated factor V, combining with Xa on the phospholipid surface, accelerates the reaction of thrombin formation by tens of thousands of times. The most common cause of resistance to activated protein C is the Leiden mutation, when activated factor V becomes resistant to cleavage by activated protein C. The Leiden mutation V of coagulation factor V is characterized by the replacement of guanine (G) nucleotide with adenine (A) nucleotide at position 1691 (1691 G / AFV). This leads to the replacement of the amino acid arginine with the amino acid glutamine at position 506 in the protein chain, which is a product of this. Mutation is inherited according to an autosomal dominant principle. Heterozygous carriers are an average of 4-7% of the European population. An increased tendency to thrombosis manifests itself in a state of heterozygosity. Cases of homozygous carriage of the Leiden mutation in the population are extremely rare [Makatsaria, A.D. Thrombosis and thromboembolism in the obstetric and gynecological clinic [Text]: molecular genetic. mechanisms and strategies for the prevention of thromboembolic complications: a guide for doctors / A.D. Makatsaria, V.O. Bitsadze, S.V. Akinshina. - Moscow: Honey. inform. agency, 2007. - 1064 s; Okorokov, A.N. Diagnosis of diseases of internal organs [Text]: a guide for doctors: in 10 tons / A.N. Hams. - Moscow: Honey. lit., 2001-2005. - T.5: Diagnosis of diseases of the blood system. Diagnosis of kidney disease. - Moscow, 2003. - 512 p.: Ill.].

Factor II (prothrombin) is a glycoprotein belonging to α2-globulins with a molecular weight of 68000-72000 daltons. It is synthesized in the liver with the participation of vitamin K. The content in the blood plasma is about 0.1 g / l or 80-120% of activity. The minimum level necessary for hemostasis is 20-40%. The prothrombin gene is located on the eleventh chromosome and contains 14 exons and 13 introns. Under the action of factor Xa, prothrombin is cleaved in the prothrombinase complex to form thrombin. One of the most common causes of congenital thrombophilia is the prothrombin 20,210 G / A gene mutation. It is characterized by the replacement of the guanine nucleotide (G) with the adenine nucleotide (A) at position 20210. A feature of this mutation is that the nucleotide is replaced in the 3′-untranslated region located at the end of the DNA sequence of the gene that is not translated. This means that the nucleotide sequence of the altered region is not involved in the coding of the amino acid sequence of the prothrombin gene. Therefore, no chemical changes in prothrombin itself in the presence of this mutation does not occur. However, in the presence of this mutation, increased amounts of chemically normal prothrombin are detected. The level of prothrombin can be one and a half to two times higher than normal. The mutation of the prothrombin 20210 G / A gene is inherited in an autosomal dominant manner. Heterozygous carriers are 2-3% of the representatives of the European race. Among hereditary thrombophilia, this mutation is 10-15%. Thrombophilia occurs even in a heterozygous carrier of an altered gene. A homozygous variant of a mutation is a very rare find. This mutation is a risk factor for all complications associated with the Leyden mutation, such as miscarriage, placental insufficiency, fetal death, gestosis, placental abruption. The risk of thrombosis increases dozens of times during pregnancy [Makatsaria, A.D. Thrombosis and thromboembolism in the obstetric and gynecological clinic [Text: molecular genetic. mechanisms and strategies for the prevention of thromboembolic complications: a guide for doctors / A.D. Makatsaria, V.O. Bitsadze, S.V. Akinshina. - Moscow: Honey. inform. agency, 2007. - 1064 s; Okorokov, A.N. Diagnosis of diseases of internal organs [Text]: a guide for doctors: in 10 tons / A.N. Hams. - Moscow: Honey. lit., 2001-2005. - T.5: Diagnosis of diseases of the blood system. Diagnosis of kidney disease. - Moscow, 2003. - 512 p.: Ill .; Serov, V.N. Critical conditions in obstetrics [Text]: a guide for doctors / V.N. Serov, S.A. Markin. - Moscow: Medizdat, 2003. - 704].

In the studied scientific and medical and accessible patent literature, the authors did not find a method for predicting the risk of developing chronic placental insufficiency with 2-3-degree fetal growth retardation syndrome in pregnant women based on data on genetic polymorphisms 10976 G / AF VII, 20210G / A FII, 1691 G / A FV and their combination.

To assess the current patent situation, a search was performed on the title documents for the period from 1990 to 2012. The analysis of the documents was carried out in the direction: a method for predicting the risk of developing placental insufficiency with SZRP based on molecular genetic data depending on polymorphic markers of genes for coagulation factor VII and in combination with V and II coagulation factors.

Known patent of the Russian Federation No. 2148256 according to the application of the Russian Federation No. 99100806/14, 12.01.1999, publ. 04/27/2000 g, for the invention "A method for preclinical prediction of placental insufficiency", including the collection of peripheral venous blood and the determination of the content of medium-weight molecules using a spectrophotometer. With the obtained indicators more than 0.220; 0.230 and 0.240 conventional units respectively, in the first, second and third trimesters of pregnancy, the development of placental insufficiency is predicted.

The disadvantage of the prototype is that it provides for the prediction of placental insufficiency in the presence of pregnancy and is not applicable at the pregravid stage. Also, this prototype does not take into account the possibility of predicting extreme degrees of placental insufficiency with SZRP 2-3 degrees, which cause an adverse perinatal outcome.

The objective of this study is to expand the arsenal of diagnostic methods, namely, to create a method for predicting the development of placental insufficiency with a second-to-second degree of SROP based on data on genetic polymorphisms 10976 G / AF VII, 20210 G / AF II, 1691 G / A FV and their combination.

The technical result of using the invention is to obtain criteria for assessing the risk of developing chronic placental insufficiency with fetal growth retardation syndrome of the 2nd to 3rd degree in pregnant women and in non-pregnant patients at the pregravid stage.

In accordance with the task, a method was developed for predicting the risk of developing chronic placental insufficiency with fetal growth retardation syndrome of the 2nd to 3rd degree in pregnant women, including:

- collection of peripheral venous blood;

- DNA isolation from peripheral venous blood;

- analysis of coagulation factor gene polymorphisms 10976 G / A FVII, 1691 G / A FV, 20210 G / A FII;

- predicting an increased risk of developing chronic placental insufficiency with a fetal growth retardation syndrome of the 2nd or 3rd degree in pregnant women in case of detection of the 10976G FVII allele, genotype 10976GG FVII and a low risk of this pregnancy complication when the following combinations are detected: genotype 20210GG FII and 10976A FVII allele ; alleles 20210G FII, 10976A FVII with genotype 1691GGFV; alleles 20210G FII and 10976A FVII.

The novelty and inventive step is that the prior art does not know the possibility of predicting the risk of developing chronic placental insufficiency with fetal growth retardation syndrome of the 2nd or 3rd degree in pregnant women by the presence of genetic variants of polymorphic factors 10976 G / AF VII, 20210 G / A FII, 1691 G / A FV and their combination.

The method is as follows:

DNA is isolated from samples of peripheral venous blood of patients 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 decanted, 4 ml of a solution containing 25 mM EDTA (pH 8.0) and 75 mM NaCl are added to the precipitate 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 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 polymerase chain reaction using standard oligonucleotide primers.

Analysis of 10976 G / A polymorphisms of coagulation factor VII, 1691 G / A of coagulation factor V, 20 210 G / A of coagulation factor II was carried out by polymerase chain reaction of DNA synthesis using an IQ5 amplifier (Bio-Rad) using a ready-made kit of reagents manufactured by Synthol LLC in accordance with the manufacturer's instructions (see table).

Genotyping is carried out using the Tag Man method of probes according to the RFU values (relative fluorescence level) of each probe. A probe with HEX fluorescent dye corresponds to allele A, a probe with FAM dye corresponds to allele G.

Kits used for genotyping of DNA markers by PCR Gene Polymorphism Set Name Series Coagulation Factor V Gene (FV) A set of reagents for determining the G1691A polymorphism of the F5 gene (Factor Leiden) manufactured by Syntol LLC 1691 G / A (rs 6025) 200312 Prothrombin gene (FII) A set of reagents for determining the G20210A polymorphism of the F2 gene (Protrombin) manufactured by Syntol LLC 200312 20210 G / A (rs 1799963) Proconvertin Gene (FVII) A set of reagents for the determination of Arg353Gln polymorphism of the F7 gene manufactured by Syntol LLC 090412 10976 G / A (rs 6046)

The invention is described in the following drawings:

- figure 1, which shows the discrimination of alleles at the locus 10976 G / A of coagulation factor VII, where ○ - homozygotes 10976 GG FVII, □ - homozygotes 10976AA FVII, Δ - heterozygotes 10976 GA FVII, ◇ - negative control;

- figure 2, which shows the distribution of the allele 10976G FVII among pregnant women with SZRP of varying severity and pregnant control group;

- figure 3, which shows the distribution of genotype 10976GG FVII among pregnant women with SZRP of varying severity and pregnant control group.

In Fig. 1, two bands, vertical and horizontal, divide the graph into four sections: one for each homozygous state, one for the heterozygous state and the section without reaction. Assigning genotypes to unknown samples is determined by plotting the RFU for one fluorophore (on the x axis) relative to the RFU for another fluorophore (on the y axis) in the allele discrimination diagram.

- If the RFU of an unknown sample is above the horizontal strip and to the right of the vertical strip, the genotype is heterozygous (GA).

- If the RFU of the unknown sample is above the horizontal strip and to the left of the vertical strip, the genotype is homozygous for the A allele (the RFUs of the A allele are plotted along the y axis).

- If the RFUs of an unknown sample are below the horizontal strip and to the right of the vertical, the genotype is homozygous for the G allele (the RFUs of the G allele are plotted along the x axis).

- If the RFU values of an unknown sample are below the horizontal strip and to the left of the vertical, determination of the genotype is impossible (in this case, an undefined sample is a negative control).

The possibility of using the proposed method for assessing the risk of developing chronic placental insufficiency with a second-to-second degree of SROP in pregnant women is confirmed by an analysis of the results of observations of 250 patients with placental insufficiency and a first-degree of heart failure and 247 pregnant women in the control group. The study group included individuals of Russian nationality, who are natives of the Central Black Earth Region of Russia and not related to each other. Clinical and laboratory examination of pregnant women was carried out on the basis of the perinatal center of the Belgorod Regional Clinical Hospital of St. Joasaph. Diagnosis of intrauterine growth retardation syndrome was carried out on the basis of clinical data - the correspondence of the height of the uterine fundus (VDM) to the gestational age. The lag in the size of the VDM by 2 cm or more compared with the norm or the absence of growth of this indicator for 2-3 weeks during dynamic observation made it possible to suspect FPS. The data of the clinical examination were confirmed by the results of ultrasound fetometry with the determination of the biparietal size of the fetal head, circumferences of the chest and abdomen, the length of the humerus and femur. The fetometric parameters obtained by ultrasound were compared with F. Hadlock nomograms and gestational age, which made it possible to confirm the presence of SZRP, its severity and form of SZRP.

Out of 250 pregnant women with placental insufficiency, 133 (53.20%) patients had mild and severe seizures, and 107 (46.80%) individuals had second- and third-degree seizures. The typing of molecular genetic markers was carried out in the laboratory of "Molecular Human Genetics" of the medical faculty of the Belgorod State National Research University.

The formation of the database and statistical calculations were carried out using the program "STATISTICA 6.0". Associations of alleles and genotypes of the studied DNA markers with the development of placental insufficiency with a second-to-third degree of SZRP were evaluated using the analysis of 2 × 2 contingency tables with calculation of the χ ² criterion with Yates correction for continuity and odds ratios (OR) with 95% confidence intervals (CI). In order to minimize errors of the first kind associated with obtaining false-positive results during multiple comparisons, the Bonferroni correction was introduced - the significance level p was recalculated for multiple pairwise comparisons according to the formula: p _cor = p × n, where p is the obtained level of statistical significance, n is the number of paired comparisons. For a statistically significant level, p _cor ≤0.05. A study of the role of combinations of genetic variants of coagulation factors 1691 G / A FV, 20210 G / A FII, 10976 G / A FVII in the formation of placental insufficiency with a second-to-second degree of SARP was carried out using the APSampler software [http: //sources.redhat .com / cygwin /], using the Monte Carlo method of Markov chains and Bayesian nonparametric statistics [Favorov A.V. et al., 2005].

Figure 2 shows that patients with placental insufficiency and fetal growth retardation syndrome of the 2nd to 3rd degree have the highest allele frequency 10976G FVII (92.99%) compared with the control group - 77.07% (χ2 = 18.90 ; OR = 3.45; 95% CI 1.90-6.39; p = 0.001) and a group of pregnant women with mild severe severity of respiratory infections - 85.16% (χ2 = 6.39; p = 0.01).

Figure 3 shows that in the group of pregnant women with SZRP of the 2nd and 3rd degrees of severity, there is a high prevalence of the 10976GG FVII genotype (85.98%) in comparison with women in the control group - 56.10% (χ2 = 26.80 ; OR = 4.80; 95% CI 2.51-9.29; p = 0.001; pcor = 0.003) and pregnant women with mildly severe seizure disability - 71.09% (χ2 = 6.66; p = 0.01 ; pcor = 0.03).

With the help of bioinformation studies, a number of protective combinations of the studied genetic markers of coagulation factors have been identified in relation to the development of placental insufficiency with a second-to-second degree of severity of severity. Thus, the combination of the 20210GG FII genotype and the 10976A FVII allele among pregnant women with moderate to severe severe respiratory infections occurs in 13.08%, compared with 46.82% of the control group (p = 0.0000001, pcor = 0.0000006, OR = 0 , 17, 95% CI 0.09-0.32). The prevalence of the combination of the 20210G FII, 10976A FVII alleles with the 1691GG FV genotype in the group of pregnant women with moderate to severe severe respiratory infections is 13.21% compared with 46.99% in pregnant women without a high-risk shock syndrome (p = 0.0000002, pcor = 0.0000024, OR = 0.17, 95% CI 0.09-0.33). The combination of 20210G FII and 10976A FVII alleles among patients with moderate and severe severe respiratory infections was 14.02%, while in the control group this indicator was 47.40% (p = 0.0000003, pcor = 0.000012, OR = 0, 18, 95% CI 0.10-0.33).

Thus, the obtained data indicate the involvement of the genetic polymorphism of 10976 G / A FVII coagulation factor in the formation of placental insufficiency with a second-to-second degree of SROP. A marker of the development of placental insufficiency with SZRP of the 2nd and 3rd degree is the 10976G FVII allele (OR = 3.45) and the 10976GG FVII genotype (OR = 4.80).

A combination of the 10976A FVII allele with the 20210GG FII genotype (OR = 0.17) plays a protective role in relation to the development of placental insufficiency with a 2-3-degree SZRP; alleles 10976A FVII and 20210G FII with genotype 1691GG FV (OR = 0.17); alleles 10976A FVII and 20210G FII (OR = 0.18).

Using this method allows us to predict the risk of placental insufficiency with a second-degree SZRP in pregnant women and, on the basis of this, determine the complex of measures of pregravid preparation and management tactics of pregnant patients. When pregnant women are diagnosed with a genetic risk factor for placental insufficiency with a second-degree SZRP (10976G FVII allele and 10976GG FVII genotype), dynamic monitoring of fetal growth should be recommended (regular visits to an obstetrician-gynecologist with measurement of the height of the uterine fundus and ultrasound determination of fetometric indicators, determination of the intrauterine condition of the fetus according to Doppler and cardiotocography) for the purpose of early diagnosis and timely pathogenetic treatment of placental insufficiency and SZRP (low molecular weight heparins), as well as choosing the optimal time and method of delivery. When carrying out pregravid preparation in women at risk for the development of placental insufficiency with FGR, taking into account the identified genetic markers, the appointment of pathogenetically substantiated anticoagulant therapy.

In the presence of a combination of the 10976A FVII allele with the 20210GG FII genotype; alleles 10976A FVII and 20210G FII with genotype 1691GG FV; the 10976A FVII allele with the 20210G FII allele, the risk of developing placental insufficiency with a second to third degree of SROP should be considered low.

Claims (1)

A method for predicting the risk of developing placental insufficiency with a fetal growth retardation syndrome of the 2nd-3rd degree in pregnant women, including the collection of peripheral venous blood, characterized in that after DNA extraction, coagulation factor gene polymorphisms are analyzed20210G / A FII,1691G / A FV,10976G / A FVIIand predict an increased risk of placental insufficiency with fetal growth retardation syndrome of the 2nd-3rd degree in pregnant women in case of detection of an allele10976G FVIIand genotype10976GG FVII, and low risk is predicted in the presence of the following combinations: genotype20210GG FIIand allele10976A FVII; alleles20210G FII,10976A FVIIwith genotype1691GG FV; alleles20210G FIIand10976A FVII.

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