Method of predicting development of risk of combined proliferative reproductive system diseases in women

FIELD: medicine.

SUBSTANCE: DNA from peripheral venous blood is extracted. An analysis of a combination of genetic versions of polymorphous markers of genes of cytokines of the gene regulator of the activity of normal expression and secretion of T-cells (-403 G/A RANTES), macrophage protein -1β (+1931 A/T MIP 1β), factor of stromal cells (-801 G/A SDF1), interleukin -1 (-511 C/T IL-1B), monocyte chemoattractant protein -1 (C/G MCP-1), interleukin -4 (-590 C/T IL-4) is performed. An increased risk of development of a combination of uterine myoma with endometriosis and hyperplastic processes of the endometrium is predicted if the combination of alleles 403 A RANTES, G MCP-1,+1931 A MIP 1β, -590 C IL-4 or the combination of alleles -403 A RANTES,+1931 A MIP 1β, -801 G SDF1, -511 C IL-1B is identified.

EFFECT: application of the claimed method makes it possible to detect a group of patients with a risk of developing a combination of proliferative reproductive system diseases, which makes it possible to prescribe an adequate therapy to prevent further progressing of the diseases.

3 dwg, 2 ex

 

A method of predicting risk of developing concomitant proliferative diseases of the reproductive system in women

The invention relates to the field of medical diagnostics, can be used in gynecology to predict risk of developing concomitant proliferative diseases of the reproductive system including the combination of uterine myoma, endometriosis and endometrial hyperplasia.

Despite a long history of studying the problem of proliferative diseases and benign tumors of the uterus in the last decades there has been a global increase in the frequency of occurrence of uterine fibroids, endometriosis and endometrial hyperplastic processes, many authors have noted a high frequency (85%) of a combination of these diseases. This is evidenced by similar premorbid background, identical clinical manifestations, as well as some clinical and pathogenetic features of uterine fibroids, endometriosis and endometrial hyperplasia. When combined proliferative diseases violated many of the functions of the reproductive system, being a frequent cause of both primary and secondary infertility, severe pain syndrome, dysfunction of adjacent organs, anemia as a consequence of bleeding, complications of pregnancy, childbirth and the postpartum period that in the aggregate PR�leads to a temporary, and often long-term disability. Despite the considerable number of works devoted to the study of premorbid background, pathogenesis, diagnosis and treatment of isolated forms of proliferative diseases of the uterus, the current study is the combination of uterine myoma, endometriosis and endometrial hyperplasia, as many questions remain, the interpretation of the results ambiguous and tactics of the patients is controversial. To date there are no clear criteria that predict an increased risk of developing concomitant proliferative diseases of the reproductive system [Adamyan L. V. Endometriosis. /L. Adamyan., V. I. Kulakov., Andreeva E. H. /Manual for doctors. M.: Medicine. - 2006. - 416 p.].

Proliferative diseases of the uterus are multifactorial diseases that develop as a result of complex action of genes, hormones, growth factors, cytokines when exposed to adverse environmental factors [Axenovich, T. I. Mapping the genes that determine the spread of human disease /T. I. Axenovich //Medical genetics. - 2006. - Vol. 5, No. 2. Pp. 11-15.]. Currently, worldwide there is active molecular genetic study of proliferative diseases of the uterus using a variety of methods.

Qi�okini are mediators of intercellular interactions, regulate hematopoiesis, immune response, cell cycle, involved in many physiological and pathological processes. Cytokines are rarely, if ever, act alone. As a rule, observe the final effect is the result of complex action of cytokines, sometimes possessing antagonistic properties. In recent years much attention is paid to the role of cytokines in the pathogenesis of proliferative processes of the uterus. The cytokines include chemokines, interleukins, factors tumor necrosis.

The impaired production of cytokines, as they are Hypo - and Hyper-production, and the disruption of the process of their reception can lead to various diseases. Developing in diseases cytokine imbalance, in turn, is able to act as a factor aggravating their course [Can cavity matrix metalloproteinases and cytokines in patients with leiomyoma, adenomyosis or endometrial polyp /Inagaki N., Ung L., Otani, T., Ikeo T. //Eur. J. Obstet. Gynecol. Reprod. Biol. 2003. Vol.111, No. 2. P. 197-203.].

A known method for the diagnosis and prognosis of cancer, based on the study of somatic mutations in the gene for the multifunctional tumor suppressor (MTS) in the case of human neoplasias. U.S. patent No. 2164419, 27.03.2001. "MTS gene, Mutations of this gene and methods of diagnosis of malignant tumors using gene sequences MTS" (Myriad Genetics, Inc. (US), etc.). The invention relative�tsya to the MTS gene mutations in germ line and use of these mutations for the diagnosis of predisposition to such cancers, as melanoma, ocular melanoma, leukemia, astrocytoma, glioblastoma, lymphoma, glioma, Hodgkin's lymphoma, multiple myeloma, sarcoma, myosarcoma, loungeorama, cheshuichatye carcinoma, chronic lymphoid leukemia, and tumors of the pancreas, ovaries, uterus, testes, kidneys, stomach, colon and rectum. The invention also relates to therapy of human neoplasias, in which the mutation occurred in the gene for the MTS, including gene therapy, protein substitute therapy and the use of mimetics. The technical result of the invention is to expand the Arsenal of tools for diagnosis and therapy of tumors.

The disadvantage is the complexity of the analysis, its duration and cost.

A prototype of the selected RF patent №2466390 on application No. 2011105301/15, 20.08.2012, "a method for predicting the development of cancer in pathological processes of the endometrium in women of reproductive age" (Sidorova I. S. Unanyan A. L. (RU), Vlasov R. S. (RU), etc.), proposed a method for predicting the development of endometrial cancer, including the identification of clinical signs and conduct of methyl-sensitive PCR (MCH-PCR) genes MLH1, RASSF1, GSTP1, P16, RAR-b, CDX1, and calculate the probability of development of cancer of the uterine body. When the value of R greater than 0.6 predict a high likelihood of developing cancer of the body mA�Ki, when p equal to 0.3 and 0.59, - moderate probability of cancer, at p below 0,29 - low probability of cancer.

The disadvantage of the prototype is that it improves the accuracy of forecasting of development of cancer of the uterine body while existing pathological processes of the endometrium in women of reproductive age and gives the opportunity to predict the propensity of patients to the combination of proliferative diseases of the reproductive system.

The objective of this study is to expand the Arsenal of methods of diagnosis, namely the creation of a method for the prediction of proliferative diseases of the reproductive system by combinations of genetic polymorphisms: -403 And RANTES, G, MCP-1,+1931 A MIP 1β, -590 C IL-4 and -403 And RANTES,+1931 A MIP 1β, -801, G SDF1, -511 C IL-1B.

The technical result of the invention - to provide criteria for the evaluation of risk of development of a combination of proliferative diseases of the reproductive system in women of Russian nationality who are nationals of the Central Chernozem region of Russia.

In accordance with the assigned task has been developed a method of predicting the combination of proliferative diseases of the reproductive system, including:

- extraction of DNA from peripheral venous blood;

- typing of genetic polymorphisms of the gene regulator activity of normal expression and secretion of T-cells (-03 G/And RANTES), macrophage inflammatory protein-1β (+1931 A/T MIP-1β), factor stromal cells (-801 G/A SDF1), interleukin-1 (-511 C/T IL-1B), monocyte chemoattractant protein -1 (C/G MCP-1), interleukin-4 (-590 C/T IL-4);

- analysis of combinations of polymorphisms of the gene regulator activity of normal expression and secretion of T-cells (-403 And RANTES), macrophage inflammatory protein-1β (+1931 A MIP 1β), factor stromal cells (-801 G SDF1), interleukin-1 (-511 C IL-1B), monocyte chemoattractant protein-1 (G MCP-1), interleukin-4 (-590 IL-4);

- prediction of increased risk of development of a combination of proliferative diseases of the reproductive system such as uterine fibroids with endometriosis and endometrial hyperplasia in the case of combinations -403 And RANTES, G, MCP-1,+1931 A MIP 1β, -590 C IL-4, or combinations -403 And RANTES,+1931 A MIP 1β, -801, G SDF1, -511 C IL-1B.

Novelty and inventive step lies in the fact that the prior art not known to predict the possibility of development of proliferative diseases according to the presence of combinations of the -403 a allele And gene regulator activity of normal expression and secretion of T-cells (polymorphism -403 And RANTES), allele+A 1931 gene macrophage inflammatory protein-1β (polymorphism+1931 A MIP 1β), allele -801 G gene factor stromal cells (polymorphism -801 G SDF1), -511 allele C of the gene interleukin-1 (-511 polymorphism C IL-1B), G allele monocytic heh�of attractant protein-1 (G polymorphism MCP-1), allele 590 C gene interleukin-4 (polymorphism With IL-4).

The invention is characterized by the following graphic materials:

Fig. 1. Discrimination of alleles for the locus -590 C/T, IL-4, wherehomozygotes for an allele-S,homozygotes for an allele -590 T,heterozygotes -590 ART,- negative control.

Fig.2. Discrimination of alleles for the locus-801G/A SDF-1, wherehomozygotes for an allele -801 (A,homozygotes for an allele -801 AGheterozygotes -801, G,- negative control.

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

The method is as follows

DNA extracted from samples of peripheral venous blood of the patient by the method of phenol-chloroform extraction in 2 stages. In the first stage to 4 ml of blood add 25 ml of lyse buffer containing 320 mm sucrose, 1% Triton X-100, 5 mm MgCl210 mm Tris-HCl (pH=7,6). The resulting mixture was stirred and centrifuged at 4 º C, 4000 rpm for 20 minutes. After centrifugation the supernatant decanted, to the residue add 4 ml of a solution containing 25 mm EDTA (pH=8.0) and 75 mm NaCl, resuspension�. Then add 0.4 ml 10% SDS, 35 ál proteinase K (10 mg/ml) and incubated the sample at 37º for 16 hours.

In the second phase from the resulting lysate sequentially perform the extraction of DNA equal volumes of phenol, phenol-chloroform (1:1) and chloroform by centrifugation at 4000 rpm for 10 minutes. After each centrifugation produce a selection of the aqueous phase. DNA is precipitated from the solution with two volumes of chilled 96% ethanol. Formed DNA is dissolved in twice-distilled, deionized water and stored at-200C.

The selected DNA is then subjected to polymerase chain reaction using standard oligonucleotide primers.

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

On the amplifier IQ5 (Bio-Rad) conduct analysis of polymorphisms +1931 A/T MIP-1, C/G MCP-1, -801, G/A, SDF-1, -403 G/A RANTES, -590 C/T IL-4 by polymerase chain reaction of DNA synthesis using standard oligonucleotide primers and specific probes with subsequent analysis of the polymorphism method for the discrimination of alleles. The reaction mixture volume of 25 μl includes: 6.7 mm Tris-Hcl (pH 8,8), 2.5 mm MgCl2, 0.1 μg of genomic DNA, 10 PM of each primer, 5 pcmall each probe, 200 μm dATP, dGTP, dCTP, dTTP, and 1 unit of active Taq polymerase. After Denat�the radio perform 40 cycles of amplification under the scheme: a primer annealing and denaturation.

On the amplifier TP-PCR-01-TERZIC" analyze gene polymorphism and -511 C/T IL-1B by polymerase chain reaction of DNA synthesis using standard oligonucleotide primers. The reaction is carried out in a 12.5 µl total volume of the mixture containing 33.5 mm Tris-Hcl (pH 8,8), 1.25 mm MgCl2, 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 perform a 32 cycle amplification scheme: denaturation, primer annealing and elongation. Then, the samples incubated for 5 min at 72°C and cooled. The amplification products analyzed in 2% agarose gel, stained with ethidium bromide for 30 minutes at 160V. As the electrophoretic buffer used 1×TAE (Tris-acetate buffer). Then, the samples identified in transmitted UV light.

Genotyping is carried out by the method of discrimination of alleles using the Tag Man probes. When conducting PCR amplifier with fluorescence detection (amplifier IQ5) genotyping is performed by method Man Tag probes according to the values of OEF (relative fluorescence unit) of each probe.

Two lanes, vertical and horizontal, divide the graph into four sections: one for each homozygous state, one for the heterozygous state and the section without reaction. The assignment of genotypes to unknown samples opredelaetsa� tracing of OEF for a single fluorophore on the x-axis relative to OEF for another fluorophore on the y-axis on the chart discrimination of alleles.

• If the values of OEF of the unknown sample are above the horizontal stripes and the right vertical stripes, genotype heterozygote (GA).

• If the values of OEF of the unknown sample are above the horizontal stripes and vertical bars to the left, genotype homozygote for an allele 2 (RFU allele 2 pending on the y-axis).

• If the values of OEF an unknown sample are below the horizontal bars and to the right of vertical, genotype homozygote for an allele of 1 (RFU allele 1 pending on the x-axis).

• If the values of OEF an unknown sample are below the horizontal bars and to the left of vertical, the determination of the genotype of the impossible (in this case, the uncertain sample - negative control)

Genotyping of DNA markers for the locus -511 C/T IL-1B is produced by analyzing the polymorphism of the lengths of restriction fragments (RFLP) of PCR products amplification of specific genomic regions (Fig.3) using appropriate restriction enzymes production company "Simanim" (Novosibirsk).

After incubation, the restriction mixture for 16 hours at 37°C is carried out the separation of DNA fragments by horizontal electrophoresis in an electrophoretic cell production company "Helikon" (Russia). Depending on the size of the partial DNA fragments using agarose gel 2-3% concentration�, prepared on the basis of TBE buffer, stained with ethidium bromide solution (0,01%). Visualization of trigram performed in a dark box with a transilluminator company UVP (Sweden).

Carrying out quality control of genotyping using positive control DNA samples with different genotypes and negative control sample, in which instead DNA to the reaction mixture add water.

Creation of database and statistical calculations carried out using the program "STATISTICA 6.0" [Endometrial hyperplasia: a review /J. L. Brun, E. Descat, Boubli V., Dallay D. //J. Gynecol. Obstet. Biol. Reprod. (Paris). TRANS. from French., 2006. Vol.35, No. 6. P. 542-550].

Study of the role of combinations of genetic variants of polymorphic markers of genes of cytokines in the formation of proliferative processes of the uterus is performed using software APSampler [http:sources.redhat.com/cygwin/] using Monte-Carlo Markov chains and Biesbosch nonparametric statistics [A. V. Favorov 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.] //ioinformatics. - 2005. - Vol.21, No. 10. - R. 2240-2245].

Assessment of the level of statistical significance of the obtained results is carried out using the Bonferroni (pcor), i.e., amendments that minimizes the probability of false positive results [Rebrova O. Yu. Statistical analysis IU�izinski data. https://sites.google.com/site/oyurebrova/book" t "_blank /O. Y. Rebrova//M: media sphere. - 2006 - 312 p.].

The possibility of using the proposed method to assess the risk of development of proliferative diseases of the uterus confirms the results of the observations 713 patients with different proliferative processes of the reproductive system, including uterine fibroids, endometriosis, endometrial hyperplastic processes. Among surveyed 713 78 women there is a combination of uterine fibroids, endometriosis, endometrial hyperplastic processes. The population control group consisted of 500 people. Patients were included in the relevant group of patients only after diagnosis of the disease, confirmed by clinical, laboratory and instrumental methods of examination and confirmed by histology data.

In the current study included individuals of Russian nationality who are nationals of the Central Chernozem region of Russia and have no relationship among themselves.

Found that the combination of molecular genetic markers -403 And RANTES, G, MCP-1,+1931 A MIP 1β, -590 C IL-4 occurs in 28,07% of patients, which is 3.5 times more often than in the control group (8,08%, p=0,000007, pcor=0,05, OR=4,43 95%CI 2,21 - 8,92). Also in patients with concomitant proliferative diseases of the reproductive system (uterine fibroids, endometriosis, gipe�plastic processes in the endometrium) is found the combination of genetic variants -403 And RANTES,+1931 A MIP 1β, -801 G SDF1, -511 C IL-1B (50,72%) in 2.1 times more often in comparison with the control (24,45%, p=0,000007, pcor=0,05, OR=3,18, 95%CI 1,78 - 5,67).

Specific examples.

Example 1.

Patient M. of Russian nationality, born of the Central Chernozem region of Russia, was held the fence peripheral venous blood, extraction of DNA from peripheral venous blood; the typing of genes+1931 A/T MIP-1, C/G MCP-1, -801, G/A, SDF-1, -403 G/A RANTES, -590 C/T IL-4 by polymerase chain reaction.

It is revealed that due to the presence of combinations of alleles-403 And RANTES, G, MCP-1,+1931 A MIP 1β, -590 C IL-4, the patient can be attributed to the risk group (OR=4,43).

Example 2.

Mrs. K. has Russian nationality, a native of the Central Chernozem region of Russia, was held the fence peripheral venous blood, extraction of DNA from peripheral venous blood; the typing of genes -403 G/A, RANTES,+1931 A/T MIP-1β, -801, G/A SDF1, -511 C/T IL-1B. It is revealed that due to the presence of combinations of alleles-403 And RANTES,+1931 A MIP 1β, -801, G SDF1, -511 C IL-1B, the patient can be attributed to the risk group (OR=3,18).

Quality control of genotyping using positive control DNA samples with different genotypes and negative control sample, in which instead DNA to the reaction mixture water was added, showed the almost complete coincidence of the results of the study (98.5%), and no contamination of the negative control sample.

Thus, obtained is given�s confirm what combination of alleles -403 And RANTES, G, MCP-1,+1931 A MIP 1β, -590 C IL-4, or combinations of alleles -403 And RANTES,+1931 A MIP 1β, -801, G SDF1, -511 C IL-1B are risk factors for the development of uterine fibroids in combination with endometriosis and endometrial hyperplasia (OR=4,43 and OR=3,18, respectively).

Application of this method allows to form a group of patients with an increased risk of developing concomitant proliferative diseases of the reproductive system, this knowledge will help to understand the doctor in a specific clinical situation, just assign adequate therapy that will serve as a prevention of further progression/recurrence of the disease and improve the patient's quality of life in General.

A method of predicting risk of developing concomitant proliferative diseases of the reproductive system in women of Russian nationality, which urozhencami of the Central Chernozem region of Russia, including extraction of DNA from peripheral venous blood, characterized in that carry out the analysis of combinations of genetic variants of polymorphic markers of cytokine gene regulator activity of normal expression and secretion of T-cells (-403 G/a And RANTES), macrophage inflammatory protein-1β (+1931 A/T MIP-1β), factor stromal cells (-801 G/A SDF1), interleukin-1 (-511 C/T IL-1B), monocyte chemoattractant protein -1 (C/G MCP-1), interleukin-4 (-590 C/T IL-4) and about�will nezirut an increased risk of developing combination of uterine myoma, endometriosis and endometrial hyperplasia in identifying combinations of alleles -403 And RANTES, G MCP-1,+1931 A MIP 1β, -590 C IL-4, or combinations of alleles -403 And RANTES,+1931 A MIP 1β, -801, G SDF1, -511 C IL-1B.



 

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1 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: the present innovation deals with studying and treating diseases of inflammatory, autoimmune and degenerative genesis. One should perform sampling of heparinized blood followed by its sedimentation to obtain blood plasma with leukocytes and centrifuging to isolate the latter which are washed against erythrocytic and serumal admixtures, and, also, it deals with calculating the number of cells in samples out of leukocytic suspension after incubation (B) for 1.5 h at 37 C in holes of plastic microplotting board, out of leukocytic suspension one should additionally prepare two samples, one should be applied to calculate total number of leukocytes before incubation (A), the second sample undergoes incubation at the same mode at addition of autoserum to calculate the number of cells remained after incubation (C). One should state upon adhesive properties of leukocytes by the index of spontaneous adhesion (D), where D=(A-B)/B.100%, and effect for enhanced cellular adhesion under the impact of autoserum should be detected by the value of K=(B-C)/C.100% at K ≥ 30%, where B - C - the number of cells undergone additional adhesion after addition of autoserum. The present innovation widens functional possibilities of the suggested method due to obtaining additional values depicting adhesive properties of blood leukocytes.

EFFECT: higher accuracy of detection.

FIELD: medicine, immunology.

SUBSTANCE: one should carry out reaction of blast-transformation, detect proliferation of T-lymphocytes activated with antibodies to CD3 in the presence of interleukin-7 (ACT IL-7) and in the presence of interleukin-7 and dexametazone (ACT IL-7 D), calculate the index for dexametazone action as the ratio of ACT IL-7 to ACT IL-7 D, moreover, the value of dexametazone action index being above 1.2 indicates increased production of cytokins that suppress T-lymphocytes in neonatals. The method enables to detect functional defect of immune system that characterizes neonatal period.

EFFECT: higher efficiency of detection.

2 ex

FIELD: medicine.

SUBSTANCE: method involves measuring forced exhalation volume per 1 s (FEV1) in l, full right ventricle evacuation time (RVE) in ms and angiotensin II value (AII) in ng/l. Discriminant relationship is built as D=0.504·RVE+3.038·FEV1 - 2.0·AII. D being less than 83.88, pulmonary hypertension occurrence is predicted within 1 year. D being equal to or greater than 83.88, no pulmonary hypertension is predicted to occur.

EFFECT: enhanced accuracy of prediction.

FIELD: medicine, medicinal immunology.

SUBSTANCE: method involves determination of heterophilic antibodies in human serum blood by the Paul-Bunnel's method relatively the level of circulating immune complexes, complement-activating properties of heterophilic antibodies by incubation of standardized ram erythrocytes with 0.8% serum for 30 ± 5 min and the following measurement of the erythrocytes lysis degree. The measurement of the effector function coefficient of heterophilic antibodies is carried out by the complement system Keff.f.h.a.-c.s. by the formula: Keff.f.h.a.-c.s. = Y/Tg.a. wherein Y means a lysis degree, %; Tg.a. means a reverse titer of heterophilic antibodies to ram erythrocytes. The damage assay is carried out by comparison of the immune status with the relative level of circulating immune complexes in serum. Method provides detection of preclinic from of immunodeficiency and autoimmune diseases that opens the possibility for their prophylaxis at most early stages of development. Invention can be used for assay of damage in the immune status in human serum blood.

EFFECT: improved method for assay.

5 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: method involves concurrently examining anti-inflammatory IL-4 level in blood serum and lacrimal fluid. The value being within the limits of 60-70 pg/l in blood serum and 5-15 pg/l in lacrimal fluid, disease prognosis is considered to be unfavorable. The IL-4 concentration being within the limits of 90-100 pg/l in blood serum and 20-30 pg/l in lacrimal fluid, disease prognosis is considered to be favorable.

EFFECT: high accuracy of diagnosis.

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