RU2445625C1 - Method for prediction of risk of multinodular lesions accompanying uterine leiomyoma - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves DNA recovery from peripheral venous blood, analysis of lymphotoxin α gene polymorphism, and a homozygous genotype shown by high-producing allele (+250GG) Ltα enables to predict a risk of multinodular lesions accompanying uterine leiomyoma.

EFFECT: higher prediction accuracy.

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Description

The invention relates to the field of medical diagnosis, can be used to predict the risk of developing multinodular uterine lesions in women and the clinical features of the disease.

The main task of molecular genetic typing of loci of cytokine genes is the determination of single nucleotide substitutions in genes that cause changes in the level of cytokine production, which can affect the development of the inflammatory process [1].

Uterine leiomyoma is a benign neoplasm that develops from the muscle and connective tissues of the uterus [2]. Uterine leiomyoma, as most studies talk about, develops from immature smooth muscle cells of the uterus (myometrium). The pathogenesis of uterine leiomyoma, despite a large number of studies [3, 4, 5, 6, 7], remains controversial so far. Uterine leiomyoma is a hormone-dependent tumor, that is, its development is associated with hormonal dysfunctions of a woman’s body - metabolism, synthesis and the ratio of sex hormones are disturbed. Leiomyoma can consist of several, different in size, nodes located in the myometrium layer. In medical practice, it is customary to evaluate the size of the uterine leiomyoma by analogy with the size of the developing fetus for weeks. Uterine leiomyoma has recently been considered the most common gynecological disease. According to various sources, among the examined patients, uterine leiomyoma is detected in 10-30%, and in some cases up to 50%. Recent observations show that there is a decrease in the average age of patients with uterine leiomyoma - cases of the disease at the age of 20-30 are not uncommon [8]. Much more often, uterine leiomyoma is affected by urban residents, unlike the rural population [9]. The growth of leiomyoma is significantly accelerated during pregnancy, which can lead to miscarriages or premature births. The frequency of detection of uterine leiomyoma increases significantly with age, in the period preceding menopause, that is, at 45-55 years. Frequent facts of the detection of uterine leiomyoma during preventive examinations by a gynecologist suggest that at the initial stage, uterine leiomyoma can develop asymptomatically. As a result of the asymptomatic or low-symptomatic course of uterine leiomyoma, the period of conservative (without surgical intervention) treatment of this insidious disease may be missed.

The development of uterine leiomyoma is based on complex multi-stage immunopathological mechanisms. One of the key links in the implementation of the cascade of these mechanisms is the interaction of cytokines. In this case, the central place in these interactions is occupied by tumor necrosis factors and, in particular, lymphotoxin α. According to the literature, lymphotoxin α (Lt-α) is synthesized in cells as a precursor constructed from 247 amino acid residues. The mature form consists of 171 amino acids and has a molecular weight of 18.66 kD. The Lt-α gene is localized on the 6th chromosome (6p21.3) and is part of the genes of the main histocompatibility complex [4]. Lt-α is produced by activated T-lymphocytes [10]. Lt-α acts as an endogenous tumor promoter in various tumor processes, in particular in cancer of the mammary gland, ovaries, etc., with an increase in the expression of genes that control the cell cycle. It was shown that Lt-α induces the expression of angiogenic factors promoting invasion and metastasis [12, 13].

The objective of this study is to develop a method for predicting the risk of multinodular lesions in uterine leiomyoma based on data on the polymorphism of the lymphotoxin gene α + 250 A / G Ltα.

The technical result of the use of the invention is to obtain criteria for assessing the risk of developing multinodular forms of uterine leiomyoma, allowing to determine the clinical prognosis and treatment strategy for this disease in the shortest possible time.

In accordance with the task, a method was developed to assess the risk of developing uterine leiomyoma, including:

- DNA isolation from peripheral venous blood;

- analysis of polymorphism of the lymphotoxin α gene;

- prediction of the risk of multinodular lesions with uterine leiomyoma in case of detection of a homozygous genotype for the highly producing allele (+ 250GG) Ltα.

The novelty and inventive step consists in the fact that the possibility of determining the nature of the lesion of the uterine myomatous nodes by the presence of alleles and genotypes of the lymphotoxin gene α + 250 A / G Ltα is not known from the prior art.

The method is as follows.

DNA is isolated from samples of peripheral venous blood of patients with uterine leiomyoma 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, resuspended. Then 0.4 ml of 10% SDS, 35 μl of proteinase K (10 mg / ml) are added. 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 (table 1).

The polymerase chain reaction (PCR) of the synthesis of a polymorphic locus of tumor necrosis factor +250 A / G Ltα is performed 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 (4 min at 94 ° С), 32 amplification cycles are performed according to the scheme: denaturation - 30 sec at 94 ° С; primer annealing - 30 sec at 68 ° С; elongation - 42 sec at 74 ° C. Then the samples are kept for 10 min at 72 ° C and cooled.

Table 1
Primer structure and restriction enzyme used for genotyping of the studied DNA marker Gene Polymorphism and its localization in the gene Primer structure Restriction enzyme Literature Ltα + 250A / G F: 5'-CCGTGCTTCGTGCTTTGGACTA-3 ' Bsp 19I (Sugiura et al., 2002) (1 intron) R: 5'-AGAGGGGTGGATGCTTGGGTTC-3 '

The amplification obtained during the PCR of the tumor necrosis factor +250 A / G Ltα gene is subjected to restriction (RFLP analysis) by adding 5 units to 12.5 μl of the amplification. restriction enzyme activity and incubated in a thermostat at a temperature of 37 ° C for 16 hours. For the analysis of the polymorphism of the tumor necrosis factor +250 A / G Ltα gene, the restriction endonuclease Bsp 191 SibEnzyme is used.

After RFLP analysis, the restriction products were analyzed on a 2% ethidium bromide stained agarose gel for 30 minutes at 150 V. 1xTAE (Tris Acetate Buffer) was used as electrophoresis buffer. Samples were then identified in transmitted UV light. Fragments 782 bp long corresponded to the allele (+ 250A) of Ltα, 586 and 196 bp - allele (+ 250G) Ltα, fragments 782, 586 and 196 bp in length were observed in heterozygotes (+ 250AG) Ltα.

The nucleotide sequence of the lymphotoxin α gene is as follows:

1 ccgtgcttcg tgctttggac taccgccccg cagtgtcctg ccctctgcct gggcctcggt

61 ccctcctgca cctgctgcct ggatccccgg cctgcctggg cctgggcctt ggtgggtttg

121 gttttggttt ccttctctgt ctctgactct ccatctgtca gtctcattgt ctctgtcaca

181 cattctctgt ttctgccatg attcctctct gttcccttcc tgtctctctc tgtctccctc

241 tgctcacctt ggggtttctc tgactgcatc ttgtcccctt ctctgtcgat ctctctctcg

301 ggggtcgggg ggtgctgtct cccagggcgg gaggtctgtc ttccgccgcg tgccccgccc

361 cgctcactgt ctctctctct ctctctctct ttctctgcag gttctcccca tgacaccacc

421 tgaacgtctc ttcctcccaa gggtgcgtgg caccacccta cacctcctcc ttctggggct

481 gctgctggtt ctgctgcctg gggcccaggt gaggcagcag gagaatgggg gctgctgggg

541 tggctcagcc aaaccttgag ccctagagcc cccctcaact ctgttctccc ctaggggctc

601 cctggtgttg gcctcacacc ttcagctgcc cagactgccc gtcagcaccc caagatgcat

661 cttgcccaca gcaccctcaa acctgctgct cacctcattg gtaaacatcc acctgacctc

721 ccagacatgt ccccaccagc tctcctccta cccctgcctc agg aacccaa gcatccaccc

781 ctct

The ccatgg restriction site for Bsp19I restriction enzyme is bold. The primers are underlined by direct (F: 5'-CCGTGCTTCGTGCTTTGGACTA-3 ') and reverse (R: 5'-AGAGGGGTGGATGCTTOGGTTC-3').

The formation of the database and statistical calculations were carried out using the program "STATISTICA 6.0". The degree of damage to the uterine myomatous nodes in women with different genotypes by the analyzed gene was compared.

As a specific example, an analysis of the results of observations of 221 women with uterine leiomyoma was carried out. Patients were included in the examination group only after a diagnosis of the disease was confirmed, using clinical and laboratory-instrumental examination methods.

All patients, depending on the presence or absence in the genotype of the high secretory allele (+ 250G) Ltα, which causes higher production of tumor necrosis factor, were divided into 3 groups. The 1st group includes individuals with the genotype (+ 250GG) Ltα (n = 28), the 2nd group is made up of patients with one high-secretory allele genotype (+ 250AG) Ltα (n = 73), the third group is made up of patients homozygous according to the low secret allele (+ 250АА) Ltα (n = 120).

As a result of studying the nature of the lesion by uterine myomatous nodes, it was found (Table 2) that in women whose genotype contains two highly producing alleles (+ 250GG) of Ltα, the frequency of occurrence of multinodular forms of fibroids (4 nodes or more) is 25%, which is 1.5 times higher than the occurrence of this trait in women with one high secretory allele (+ 250AG) Ltα (15%) in the genotype and 2.5 times higher than the occurrence of multi-site forms of leiomyoma in women without a high secretory allele (+ 250AA Ltα genotype).

table 2
The dependence of the occurrence of multinodular lesions in uterine leiomyoma depending on the genotype of a woman according to the lymphotoxin gene α Genotype Number of women % occurrence of multinodal lesions Gg 28 25% GA 73 fifteen% AA 120 10%

The use of the χ ² criterion made it possible to identify significant differences (χ ² = 6.79; p = 0.01, OR = 3.0; CI = 1.27-7.18) in terms of the incidence of multinodular forms of uterine leiomyoma (4 nodes or more ) between women with two high secretory lymphotoxin α alleles and women with two low secretory lymphotoxin α alleles in the genotype. A significantly higher frequency of occurrence of multinodular forms of uterine leiomyoma (4 nodes or more) with carriage of highly productive alleles of the lymphotoxin α gene in the homozygous state - 2.5 times more often than in their absence, indicates the possible prognostic significance of this genetic marker.

Literature.

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Claims (1)

A method for predicting the development of multinodular lesions in uterine leiomyoma, including the extraction of DNA from peripheral venous blood, characterized in that the polymorphism of the lymphotoxin α gene is analyzed and the risk of multinodular lesions in uterine leiomyoma is predicted if a homozygous genotype is detected by a highly producing allele (+ 250GG).