RU2558854C1 - Method for prediction of risk of endometriosis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: DNA is recovered from peripheral venous blood; combinations of genetic versions of polymorphous markers of interleukin 6 (-174 G/C IL-6), interleukin 1B (-511 C/T IL-1B), interleukin 10 (-592 C/A IL-10) cytokine genes, stromal cell factor (-801 G/A SDF1) chemokine genes, macrophage inflammatory protein -1β (+1931 A/T MIP 1β), T-cell normal expression and secretion activity regulator (-403 G/A RANTES), interferon-inducible T-cell chemoattractant (A/G I-TAC) are typed and analysed. A high risk of isolated endometriosis is predicted if observing combinations of -174 C IL-6, -801 A SDF1, +1931 A MIP1β, -592 C IL-10 alleles, -801 A SDF1, -403 G RANTES, +1931 A MIP1β, -592 C IL-10 alleles, -801 A SDF1, -403 G RANTES, -511 C IL-1B, -592 C IL-10 alleles, -801 A SDF1, +1931 A MIP1β, -592 C IL-10 alleles, -174 C IL-6, -801 A SDF1, -403 G RANTES, -592 C IL-10 alleles, -801 A SDF1, A I-TAC, -511 CC IL-1B alleles.

EFFECT: simple non-invasive diagnostic technique for endometriosis that increases a risk of recurrence upon the completion of any therapeutic method, both conservative, and surgical.

5 dwg, 1 tbl, 1 ex

Description

The invention relates to the field of medical diagnosis, can be used in gynecology to predict the risk of the formation of isolated endometriosis.

Worldwide, approximately 176 million women suffer from endometriosis, the number of which is constantly growing, so this disease is classified as a modern epidemic. Endometriosis affects women regardless of race, socio-economic status and age - from adolescents 10-11 years old to women aged 60-70 years. In chronic pelvic pain and infertility is observed in 40-70% and 30-50%, respectively. Endometriosis is a benign growth of tissue functionally and morphologically similar to normal endometrium, which is characterized by increased local formation of estrogens and sensitivity to them, as well as resistance to progesterone. Endometriosis is a hormone-dependent disease, the complex pathogenesis of which remains to the end unsolved. Dysfunction of the immune system, impaired adhesion molecules, cell proliferation and apoptosis have been identified. An increase in the level of cytokines and inflammatory mediators is also an important link in the pathogenesis of both disorders [Adamyan L.V., Andreeva E.N. The role of modern hormone-modulating therapy in the complex treatment of genital endometriosis. Prob. Reprod. 2011; 17: 6: 66-77].

The traditional symptoms of endometriosis are dysmenorrhea, chronic pelvic pain, dyspareunia, infertility, however, in recent years, quite high rates of allergic and autoimmune diseases have been found, a possibility of malignant transformation in certain forms of endometriosis is of some concern. Concomitant inflammatory process, immunological dysregulation, inhibition of apoptosis, activation of angiogenesis are pathogenetic factors that contribute to the survival and growth of endometrioid implants [Vigano R., Infantino M., Lattuada D. et al. Intercellular adhesion molecule-1 (ICAM-1) gene polymorphisms in endometriosis. Mol Hum Reprod 2003; 9: 47-52].

Endometriosis is a difficult to diagnose disease, especially in the early stages, more often the diagnosis is made when the patient consults a doctor about infertility. Endometriosis is called a “missed” disease, since it takes 7-8 years on average from the moment the first symptoms of the disease appear to the correct diagnosis. Unfortunately, a simple non-invasive method for diagnosing endometriosis has not yet been developed, which increases the risk of relapse of the disease after the termination of any method of therapy, both conservative and surgical.

For an analogue, RF patent No. 2360605 was selected (published on July 10, 2009) according to the application: 2008102542/14 dated January 22, 2008 “A method for the diagnosis of retrocervical endometriosis and endometriosis of the sacro-uterine ligaments” (Kolpinsky G.I., Lobanova O.G.), where A method for detecting minimal foci of endometriosis by ultrasound examination with a transvaginal probe in the first three days after ovulation is proposed. The sensor is installed in the posterior vaginal fornix. In the presence of rounded foci of medium and increased echogenicity in the posterior region of the heterogeneous structure with uneven contours or in the presence of similar formations in one of the parametric regions at the level of the isthmus, retrocervical endometriosis or endometriosis of the sacro-uterine ligaments is diagnosed. The method allows to identify the minimum foci of endometriosis due to the use of ovulatory fluid as a contrast.

However, ultrasound does not eliminate all acoustic interference from the rectum, which makes it difficult to verify small foci of retrocervical endometriosis, allows diagnosing uterine endometriosis only in the active form, the frequency of occurrence of which is 66%, which excludes the possibility of diagnosing an inactive form of the disease, there is no data on the accuracy of using this method and does not make it possible to predict the formation of endometriotic foci.

In the patent of the Russian Federation No. 2161311 (publ. 27.12.2000) according to the application No. 99107082/14, 04/07/1999 "A method for predicting the development of endometriosis leading to impaired reproductive function in girls with algomenorrhea" (Filonova L.V. (RU), Brusnitsina B .U. (RU), Aleksandrova NN (RU)), selected for the prototype, which proposed a method for predicting the course of endometriosis with the development of clinical symptoms, including the collection of peripheral blood and the study of a number of indicators of the immune system responsible for the formation of the pathogenesis of endometriosis, in particular class M immunoglobulins, circulating immune complexes, the level of T-lymphocytes (-CD3). The value of the diagnostic coefficient F is determined by the formula F = -K10.00793 + K20.013-K30.043 + 3.09, where K1 is the IgM indicator, K2 is the CEC indicator, K3 is the T-cell indicator (-CD3). If F> 0, then the patient is predicted the risk of developing endometriosis, with F <0 - the occurrence of endometriosis is unlikely. To predict the isolated development of endometriosis, the sensitivity and specificity of this method is insufficient.

The objective of this study is to expand the arsenal of diagnostic methods, namely, to create a method for predicting the risk of forming isolated endometriosis by combinations of combinations of genetic polymorphisms: alleles -174 C IL-6, -801 A SDF1, +1931 A MIP1β, -592 C IL-10; alleles -801 A SDF1, -403 G RANTES, + 1931 A MIP1β, -592 C IL-10; alleles -801 A SDF1, -403 G RANTES, -511 C IL-1B, -592 C IL-10; alleles -801 A SDF1, +1931 A MIP1β, -592 C IL-10; alleles of -174 C IL-6, -801 A SDF1, -403 G RANTES, -592 C IL-10; alleles -801 A SDF1, AI-TAC, -511 CC IL-1B.

The technical result of using the invention is to obtain criteria for assessing the risk of predicting isolated endometriosis in women of Russian nationality who are natives of the Central Black Earth Region of Russia.

In accordance with the task, a method for predicting the formation of isolated endometriosis was developed, including:

- peripheral blood sampling;

- DNA isolation from peripheral venous blood;

- typing of genetic polymorphisms of the genes for interleukin 6 (-174 G / C IL-6), interleukin 1B (-511 C / T IL-1B), interleukin 10 (-592 C / A IL-10), genes for chemokines of the stromal cell factor ( -801 G / A SDF1), a macrophage inflammatory protein -1β (+1931 A / T MIP1β), a regulator of the activity of normal expression and secretion of T cells (-403 G / A RANTES), an interferon of an inducible chemoattractant T-cell (A / GI -TAC);

- analysis of combinations of polymorphisms of the gene for interleukin 6 (-174 C IL-6), interleukin 1B (-511 C IL-1B), interleukin 10 (-592 C IL-10), chemokine genes for stromal cell factor (-801 A SDF1) , macrophage inflammatory protein -1β (+1931 A MIP1β), a regulator of the activity of normal expression and secretion of T-cells (-403 G RANTES), interferon-inducing chemoattractant T-cells (A I-TAC) - predicting an increased risk of developing isolated endometriosis in patients in the case of combinations of combinations of the alleles -174 C IL-6, -801 A SDF1, +1931 A MIP1β, -592 C IL-10; alleles -801 A SDF1, -403 G RANTES, +1931 A MIP1β, -592 C IL-10; alleles -801 A SDF1, -403 G RANTES, -511 C IL-1B, -592 C IL-10; alleles -801 A SDF1, +1931 A MIP1β, -592 C IL-10; alleles of -174 C IL-6, -801 A SDF1, -403 G RANTES, -592 C IL-10; alleles -801 A SDF1, AI-TAC, -511 CC IL-1B.

The novelty and inventive step lies in the fact that the possibility of predicting the development of isolated endometriosis in women of Russian nationality who are natives of the Central Black Earth Region of Russia by the presence of combinations of the -174 C alleles of the interleukin 6 gene (polymorphism -174 C IL-6) is not known from the prior art. , allele -511 C interleukin 1 B (polymorphism -511 C IL-1B), alleles -592 C interleukin 10 (polymorphism -592 C IL-10), alleles -801 A stromal cell factor (polymorphism -801 A SDF1), alleles 1931 A macrophage inflammatory protein -1β (gender imorphism + 1931 A MIP1β), -403 G alleles of the regulator of normal expression and T-cell secretion activity (-403 G RANTES polymorphism), A interferon alleles of the inducible chemoattractant T-cell (A I-TAC polymorphism).

The invention is characterized by the following graphic images:

FIG. 1. Allele discrimination at the locus + 1931 A / T MIP-lp, where ● - homozygotes + 1931AA, ■ - homozygotes + 1931TT, ▲ - heterozygotes +1931 AT, ♦ - negative control.

FIG. 2. Discrimination of alleles at the locus -801 G / A SDF1, where ● - homozygotes -801GG, ■ - homozygotes -801AA, ▲ - heterozygotes -801GA, ♦ - negative control.

FIG. 3. Allele discrimination at the locus -403 G / A RANTES, where ● - homozygotes -403GG, ■ - homozygotes -403AA, ▲ - heterozygotes -403GA, ♦ - negative control.

FIG. 4. Electrophoretic separation of restriction products of the gene IL-1B -511, where 1, 9 - homozygotes -511TT; 2, 3, 5, 7, 8 - heterozygotes -511ST, 4, 6, - homozygotes -511SS.

FIG. 5. Electrophoretic separation of the restriction products of the -592C / A gene of IL-10, where 1, 8-10 - homozygotes - 592СС, 4-7, 11-12 - heterozygotes - 592CA, 2 - homozygotes - 592AA.

The method is as follows:

DNA is isolated from samples of peripheral venous blood of patients with isolated endometrial hyperplastic processes by phenol-chloroform extraction 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, 0.4 ml of 10% SDS, 35 μl of proteinase K (10 mg / ml) are added and the sample is incubated 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 min. 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 -200 ° C.

The isolated DNA is then subjected to polymerase chain reaction using standard oligonucleotide primers (Table 1).

The structure of primers and probes used for genotyping of the studied DNA markers

Molecular genetic analysis is carried out by polymerase chain reaction of DNA synthesis. PCR is carried out on amplifiers IQ5 (BioRad) and TP4-PCR-01- "TERTSIK".

IQ5 (Bio-Rad) amplifiers analyze gene polymorphisms +1931 A / T MIP1β, -801 G / A SDF-1, -403 G / A RANTES, A / G I-TAC, -174 G / C IL-6 polymerase chain reaction of DNA synthesis using standard oligonucleotide primers and specific probes, followed by analysis of polymorphism by allele discrimination. The reaction mixture with a volume of 25 μl includes: 6.7 mm Tris-HCl (pH 8.8), 2.5 mm MgCl ₂ , 0.1 μg of genomic DNA, 10 pM of each primer, 5 pcmol of each probe, 200 μM dATP, dGTP, dCTP, dTTP and 1 unit of active Taq polymerase. After denaturation, 40 amplification cycles are performed according to the scheme: primer annealing and denaturation.

The TP4-PCR-01- “TERTSIK” amplifier analyzes the polymorphism of the -511 C / T IL-1B, -592 C / A IL-10 gene polymerase by the polymerase chain reaction of DNA synthesis using standard oligonucleotide primers according to the method described in (10 ) The reaction is carried out in 12.5 μl of the total volume of the mixture containing 33.5 mm Tris-HCl (pH 8.8), 1.25 mm MgCl ₂ , 0.5 μg of genomic DNA, 5 pM of each primer, 100 μM dATP , dGTP, dCTP, dTTP and 1 unit of active Taq polymerase. After denaturation, 32 amplification cycles are performed according to the scheme: denaturation, annealing of primers and elongation. Then the samples are kept for 5 min at 72 ° C and cooled. The amplification products are analyzed on a 2% agarose gel stained with ethidium bromide for 30 min at 160V. As electrophoresis buffer use 1xTAE (Tris-acetate buffer). Samples are then identified in transmitted UV light.

Genotyping is carried out by allele discrimination using Tag Man probes. When performing PCR in a thermocycler with fluorescence detection (IQ5 amplifier), genotyping is carried out using the Tag Man method of probes according to the RFU values of each probe, where RFU is the level of relative fluorescence.

Two bands, vertical and horizontal, divide the graph into four sections: one for each homozygous state, one for the heterozygous state and a 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 values of an unknown sample are above the horizontal strip and to the left of the vertical strip, the genotype is homozygous for allele 2 (RFU for allele 2 is plotted along the y axis).

If the RFU values of an unknown sample are below the horizontal strip and to the right of the vertical, the genotype is homozygous for allele 1 (the RFU of allele 1 is 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).

Genotyping of DNA markers (locus -511 C / T IL-1B, -592 C / A IL-10) is carried out by analysis of restriction fragment length polymorphism (RFLP) of PCR amplification products of specific genome sections using appropriate restriction enzymes manufactured by Sibenzim "(Novosibirsk).

After incubation of the restriction mixture for 16 hours at a temperature of 37 ° C, DNA fragments are separated by horizontal electrophoresis on electrophoretic cells manufactured by Helikon (Russia). Depending on the size of the separated DNA fragments, an agarose gel of 2-3% concentration is used, prepared on the basis of a TBE buffer, stained with ethidium bromide solution (0.01%). Visualization of phoreograms is carried out in a dark box with a transilluminator from UVP (Sweden).

Quality control of genotyping is carried out using positive control DNA samples with different genotypes and a negative control sample, where instead of DNA, water is added to the reaction mixture.

The formation of the database and statistical calculations are carried out using the program "STATISTICA 6.0" [Rebrova O.Yu. Statistical analysis of medical data. Application of the Ststistica software package / O.Yu. Rebrova // M .: MediaSfera. - 2006. - 312 p.].

The role of combinations of genetic variants of polymorphic markers of interleukin genes in the formation of isolated uterine fibroids is studied using the APSampler software [http: sources.redhat.com/cygwin/], using the Monte Carlo method of Markov chains and Bayesian nonparametric statistics [Favorov A.V. A Gibbs sampler for identification of symmetrically structured, spaced DNA motifs with improved estimation of the signal length / A.V. Favorov, M.S. Gelfand, A.V. Gerasimova [et al.] // Bioinformatics. - 2005. - Vol. 21, No. 10. - P. 2240-2245].

The level of statistical significance of the obtained results is estimated using the Bonferroni correction (p _cor ), i.e. corrections that minimize the probability of obtaining false positive results [Borovikov, V. Statistica: the art of computer data analysis / V. Borovikov. - 2nd ed. - SPb .: Peter, 2003 .-- 688 p.: Ill. - (For professionals)].

The possibility of using the proposed method for assessing the risk of developing isolated endometriosis is confirmed by an analysis of the observation results of 713 patients with various proliferative processes of the reproductive system (uterine fibroids, endometriosis, endometrial hyperplastic processes) and a population control group of 500 people. Among 713 examined patients, 104 women had endometriosis. Patients were included in the appropriate group of patients only after a diagnosis of the disease was confirmed, confirmed by clinical, laboratory and instrumental methods of examination and the confirmed result of histological examination. The studied groups included individuals of Russian nationality, who are natives of the Central Black Earth Region of Russia and have no kinship with each other.

It was found that in patients with an isolated form of endometriosis, combinations of genetic variants of alleles:

-174 CIL-6, -801 A SDF1, + 1931 A MIP1β, -592 CIL-10 occurs in 40.00%, which is 3.4 times more likely than in the control group (11.72%, p = 0, 0000008, p _cor = 0.006, OR = 5.02 95% CI 2.52-10.01),

-801 A SDF1, -403 G RANTES, +1931 A MIP1β, -592 C IL-10 occurs in 49.18%, which is 2.5 times more likely than in the control group (19.44%, p = 0, 000001, p _cor = 0.009, OR = 4.00),

-801 A SDF1, -403 G RANTES, -511 С IL-1B, -592 С IL-10, occurs in 41.77%, which is 2.4 times more likely than in the control group (16.77%, p = 0.000004, p _cor = 0.03, OR = 3.55),

-174 C IL-6, -801 A SDF1, -403 G RANTES, -592 CIL-10 occurs in 37.31%, which is 2.7 times more often compared to the control group (; 13.84%, p = 0.000006, p _cor = 0.05, OR = 3.70).

-801 A SDF1, +1931 A MIP1β, -592 C IL-10 occurs in 48.38%, which is 2.2 times more often than in the control group (22.00%, p = 0.000005, p _cor = 0.04, OR = 3.32).

and -801 A SDF1, A I-TAC, -511 CC IL-1B is found in 24.73%, which is 3 times more often compared to the control group (8.03%, p = 0.000008, p _cor = 0 05, OR = 3.76).

A specific example of the use of the proposed method.

A patient X., of Russian nationality, who is a native of the Central Black Earth Region of Russia, underwent peripheral venous blood sampling, DNA extraction from peripheral venous blood; typing of genetic polymorphisms of interleukin genes (-174 G / C IL-6), interleukin 1B (-511 C / T IL-1B), interleukin 10 (-592 C / A IL-10), chemokine genes of stromal cell factor (-801 G / A SDF1), a macrophage inflammatory protein -1β (+1931 A / T MIP1β), a regulator of the activity of normal expression and secretion of T cells (-403 G / A RANTES), interferon of an inducible chemoattractant T-cell (A / G I- TAC) and analysis of combinations of interleukin 6 gene polymorphisms (-174 C IL-6), interleukin 1 B (-511 C IL-1B), interleukin 10 (-592 C IL-10), stromal cell factor chemokine genes (-801 A SDF1), macrophage ceiling elements inflammatory protein -1β (+1931 A MIP1β), regulator activity normal expression and secretion of T cells (-403 G RANTES), interferon indutsebelnogo chemoattractant T cell (A I-TAC).

The results are presented in FIG. 1-5.

The data obtained indicate the presence of combinations of alleles:

-174 C IL-6, -801 A SDF1, + 1931 A MIP1β, -592 C IL-10 (OR = 5.02),

-801 A SDF1, -403 G RANTES, +1931 A MIP1β, -592 С IL-10 (OR = 4.00),

-801 A SDF1, -403 G RANTES, -511 CIL-1B, -592 С IL-10 (OR = 3.55),

-801 A SDF1, +1931 A MIP1β, -592 C IL-10 (OR = 3.32),

-174 CIL-6, -801 A SDF1, -403 G RANTES, -592 С IL-10 (OR = 3.70),

-801 A SDF1, A I-TAC, -511 CC IL-1B (OR = 3.76), which confirms the need to classify the patient at risk of developing isolated endometriosis.

The use of this method will allow to form a group of women of Russian nationality who are natives of the Central Black Earth Region of Russia with an increased risk of developing isolated endometriosis. In this group of women, it is recommended to conduct a study of the concentration of tumor markers CA125, CA 19-9, CEA by enzyme-linked immunosorbent assay, dynamic examination of ultrasound of the pelvic organs, simple and advanced colposcopy, bacterioscopic examination of smears, MRI examination, priority of reproductive goals in a young reproductive age.

Claims (1)

A method for predicting the risk of developing isolated endometriosis, including the collection of peripheral venous blood, characterized in that DNA is isolated from the peripheral venous blood, after which typing and analysis of combinations of genetic polymorphisms of interleukin 6 (-174 G / C IL-6) and interleukin 1B (-511 C / T IL-1B), interleukin 10 (-592 C / A IL-10), stromal cell factor chemokine genes (-801 G / A SDF1), macrophage inflammatory protein -1β (+1931 A / T MIP 1β), a regulator of the activity of normal expression and secretion of T cells (-403 G / A RA NTES), an interferon-inducible chemoattractant of T cells (A / G I-TAC), predicts an increased risk of developing isolated endometriosis in patients of Russian nationality who are natives of the Central Black Earth Region of Russia when combinations of the -174 C-IL-6, -801 A SDF1 combination are detected , +1931 A MIP1β, -592 С IL-10, alleles -801 A SDF1, -403 G RANTES, + 1931 A MIPip, -592 С IL-10, alleles -801 A SDF1, -403 G RANTES, -511 С IL-1B, -592 C, IL-10, alleles -801 A SDF1, +1931 A MIP1β, -592 С, IL-10, alleles -174 С IL-6, -801 A SDF1, -403 G RANTES, -592 С IL-10, alleles -801 A SDF1, A I-TAC, -511 CC IL-1B.

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