Method for predicting risk of hysteromyoma

FIELD: medicine.

SUBSTANCE: invention represents a method for predicting a risk of isolated hysteromyoma involving sampling and studying peripheral venous blood, differing by the fact that DNA is recovered from the peripheral venous blood; genetic polymorphisms of interleukin genes are typed, and combinations of polymorphisms of interleukin-1 (-889 TT IL-1) and interleukin-5 (-703 C IL-5) genes are analysed; a high risk of isolated hysteromyoma is predicted, if observing a combination of -889 TT IL-1 allele and -703 C IL-5 allele in Russian females, native of the Central Black Earth Region.

EFFECT: invention provides obtaining criteria of the risk of isolated hysteromyoma as shown by the combination of the molecular-genetic markers 889 TT IL-1A and -703 C IL-5.

1 ex, 1 tbl, 2 dwg

 

The invention relates to the field of medical diagnostics, namely to gynecology, and can be used to predict the risk of formation of isolated uterine fibroids.

The problem of uterine fibroids in women of all age periods is one of the important in modern gynecology. This is because uterine fibroids - the most common benign tumor of the female genital organs, not only in the perimenopausal period, when it is found in more than 50% of women, but in their reproductive years. Most often uterine fibroids diagnosed in women aged 35 years, at the age of 35-55 years, accounts for 90% of the total number of diseases, and rarely uterine fibroids common in women younger than 20 and older than 70 years. Uterine fibroids cause menstrual disorders, anemia, chronic pelvic pain. From 60 to 95% of all surgical interventions in women of reproductive age with uterine cancer accounts for radical surgery, leading to loss of reproductive and menstrual function and expressed by vascular and psycho-emotional disorders. Early detection of uterine fibroids will allow for a more efficient conservative treatment and thereby reduce the risk of surgical intervention [Sidorova I. S. uterine Fibroids (modern problems of etiology, pathogenesis, diagnosis, and treatment). - M., 2003].

A special role in the mechanisms of tumor growth is given to the processes of apoptosis. Tumor growth is the sum of the dynamic balance between proliferation and cell death. Thus, growth can be associated with excessive proliferation and low index of cell death. Recently, data were obtained that the cell is able to receive signals how to divide, and to death, triggering a cascade of complex processes leading to fragmentation of the cell with the formation of the membrane surrounded the so-called apoptotic cells. This process is called apoptosis, i.e. programmed cell death. Important in the mechanisms of apoptosis is given to interleukins. Currently identified more than two dozen interleukins (interleukin-1, interleukin-2, etc.). They regulate intercellular and inter-system interactions that determine cell survival, stimulation or inhibition of their growth, differentiation, functional activity and apoptosis, and ensure the consistency of the action of the immune, endocrine and nervous system at the cellular level in normal conditions and in response to pathological effects [Golubeva O. V. Genetic factors predisposing to the development of adenomyosis / O. V. Golubeva, T. E. Ivashchenko, V. S. Baranov, E. K. aylamazyan // Journal of obstetrics and female diseases. 2007. - T. 1. - No. 2. - P. 2-38].

In the patent of Russian Federation №2355319, publ. 20.08.2012 on application No. 2007137713/14 from 11.10.2007 (Kulagina N. In., Kozlov V. V.) proposed a method of predicting the course of uterine fibroids with the development of clinical symptoms, including the determination of the age of menarche, the availability of medical abortions before the first birth, the presence of inflammatory complications of medical abortion, the presence of hypertension during the previous pregnancies, the presence of anomalies of labor activity, body mass index, presence of hypertension and presence of chronic diseases of the gastrointestinal tract and the calculation of the indicators predict the clinical course of uterine fibroids. On the basis of its value forecast for uterine fibroids with the development of clinical symptoms. The method does not allow to predict the formation of isolated uterine fibroids.

A prototype of the selected RF patent №2340897, publ. 10.12.2008 by application No. 2007126877/15 dated 16.07.2007 "a Method of predicting rapid growth of uterine leiomyoma" (Sotnikova N. Yu. (RU), A. Sosnina E. (RU), Boyko O. M. (RU)), where a method of predicting rapid growth of leiomyoma of the uterus, including the fence and the study of peripheral venous blood, determination in peripheral blood of women with uterine leiomyoma relative amount of IL-8+ monocytes and in the case that the values of this index equal to or greater than 63%, the forecast quickly�on the growth of leiomyoma during the year.

The disadvantage of the prototype is that the proposed method allows to predict the growth of uterine leiomyoma, but makes it impossible to predict the formation of isolated uterine fibroids.

The object of the present invention is to provide a method for predicting the formation of an isolated form of uterine fibroids according to combinations of genetic polymorphisms: -889 TT IL-1 together with -703 With IL-5.

The technical result of the invention - preparation of risk assessment criteria for the formation of isolated uterine fibroids in women of Russian nationality, which urozhencami of the Central Chernozem region of Russia.

In accordance with the assigned task has been developed a method of predicting the formation of isolated uterine fibroids, including:

the fence and the study of peripheral venous blood;

- extraction of DNA from peripheral venous blood;

- typing of genetic polymorphisms of interleukin (-889 C/T IL-1 and -703 C/T IL-5);

- analysis of combinations of gene polymorphisms of interleukin-1 (-889 TT IL-1) and interleukin-5 (-703 With IL-5);

- prediction of high risk of development of isolated uterine fibroids in patients in case of detection of allele combinations -889 TT IL-1 and allele -703 With IL-5.

Novelty and inventive step lies in the fact that the prior art is unknown to the ability of the forecast time�itia isolated uterine fibroids by the presence of allele combinations -889 CT of the gene of interleukin-1 and allele -703 With interleukin-5 in women of Russian nationality, which urozhencami of the Central Chernozem region of Russia.

The invention is characterized by the following graphical image.

Fig. 1. Electrophoretic separation of the products of restriction gene -703 C/T IL-5., where 1, 3-8, 12, 15 homozygotes -703 MOP; 10, 11, 13, 14 heterozygotes -703 ST; 2, 9 homozygotes -703 TT.

Fig. 2. Electrophoretic separation of the products of restriction gene -889 C/T IL 1A., where 2, 3, 4 homozygotes -889 CT; 5, 6, 10 - homozygote -889 SS, 1, 7-9, 11-14 heterozygotes -889 CT.

The method is as follows.

DNA extracted from samples of peripheral venous blood of patients with proliferative diseases of the uterus (uterine fibroids with endometriosis, endometrial hyperplasia) 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°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 the sample was incubated at 37°C 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) � of 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 -200°C.

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

Molecular genetic analysis is carried out by polymerase chain reaction of DNA synthesis. PCR is performed on the amplifier TP-PCR-01-TERZIC". Analysis of polymorphism of genes -889 C/T IL-1A, -703 C/T IL-5 is carried out by polymerase chain reaction of DNA synthesis using standard oligonucleotide primers by the method, described in reference [Hulkkonen, J. Inflammotory cytokines and cytokine gene polymorphisms in chronic lymphocytic leukaemia, in primary Sjögren''s Syndrome and Haelthy Subjects: [acad. diss.] / J. Hulkkonen; University of Tampere, Medical School Tampere University Hospital. - Tampere, 2002. - 81 p.].

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, okrasa�nom with ethidium bromide, for 30 minutes at 160 V. as electrophoretic buffer used 1×TAE (Tris-acetate buffer). Then, the samples identified in transmitted UV light.

Genotyping of DNA markers (loci -889 C/T IL-1A, -703 C/T IL-5) produced by the method of analysis of the polymorphism of the lengths of restriction fragments (RFLP) of PCR products amplification of specific genomic regions using appropriate enzymes produced by "Simanim" (Novosibirsk).

After incubation, the restriction mixture for 16 hours at 37°C carry 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%). Results visualization of trigram carried out in a dark box with a transilluminator company UVP (Sweden), shown in Fig. 1 and Fig. 2.

Creation of database and statistical calculations were performed using the program STATISTICA 6.0 [Borovikov, V. Statistica: the art of computer data analysis / V. Borovikov. - 2nd ed. - SPb.: Peter, 2003. - 688 p.: ill. - (For professionals)].

Study of the role of combinations of genetic variants of polymorphic markers �enow interleukins in the formation of isolated uterine fibroids was performed using the software APSampler [http:sources.redhat.com/cygwin/], using Monte-Carlo Markov chains and Biesbosch nonparametric statistics.

Assessment of the level of statistical significance of the obtained results was performed using the Bonferroni (pcor), i.e. amendments that minimizes the probability of false positive results [Rebrova O. Yu., Statistical analysis of medical data. The use of the Statistica / O. Y. Rebrova // M: media sphere. - 2006 - 312 p.].

The possibility of using the proposed method for assessing increased risk of isolated uterine fibroids confirms the results of the observations 713 patients with proliferative processes of the reproductive system, as well as 500 people population control. Among surveyed 713 women isolated uterine cancer was diagnosed in 117 patients. 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.

It is established that the combination of two genetic variants -889 TT IL-1A and -703 With IL-5 in the patient group�to with isolated uterine cancer occurs (14,47%) significantly more often (in 6.8 times), than in the control group (2,12%, p=0.0001, pcor=0,02). These data suggest a significant contribution of combinations of polymorphic variants of genes -889 C/T IL-1A and -703 C/T IL-5 in the formation of an isolated form of uterine fibroids (OR=7,78, 95% CI 2,61-23,21).

A specific example of the proposed method

Patient X Russian nationality, a native of the Central Chernozem region of Russia, were held the fence and the study of peripheral venous blood, extraction of DNA from peripheral venous blood; the typing of genetic polymorphisms of interleukins -889 C/T IL-1 and -703 C/T IL-5 and analysis of combinations of polymorphisms -889 TT IL-1 and -703 With IL-5. The results are presented in Fig. 1 and Fig. 2.

It is revealed that due to the presence of a combination of molecular genetic markers -889 allele TT IL-1A and allele -703 With IL-5 woman can be attributed to the risk group of the formation of isolated uterine fibroids and pick up individual tactics to conservative therapy and reduce the risk of surgical intervention.

Thus, the proposed method solves the problem and ensures the achievement of the technical result - obtaining criteria for the assessment of risk of formation of isolated uterine fibroids on a combination of molecular genetic markers -889 TT IL-1A and -703 With IL-5 (OR=7,78).

The use of this method will allow you to identify the risk group R�svitil isolated uterine fibroids in patients of Russian nationality, of female of the Central Chernozem region of Russia, and to pick up individual tactics to take remedial measures to conduct conservative treatment and reduce the risk of surgical intervention.

A method of predicting risk of development of isolated uterine fibroids, including the fence and the study of peripheral venous blood, characterized in that from the peripheral venous blood DNA isolated, carried out the typing of genetic polymorphisms of interleukins and analysis of combinations of gene polymorphisms of interleukin-1 (-889 TT IL-1) and interleukin-5 (-703 With IL-5), predict a high risk of isolated uterine fibroids in case of detection of allele combinations -889 TT IL-1 and allele -703 With IL-5 in women of Russian nationality, of female the Central Chernozem region of Russia.



 

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