Method for prediction of risk of thrombocytopenia development accompanying clinical course of chronic lymphatic leukaemia
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FIELD: medicine.
SUBSTANCE: method for the prediction of a risk of the thrombocytopenia development accompanying the clinical course of chronic lymphatic leukaemia involves the DNA recovery from peripheral venous blood, the analysis of interleukin 1A - 889C/T gene and interleukin 1 receptor antagonist VNTR polymorphisms.
EFFECT: high risk of the thrombocytopenia development accompanying the clinical course of chronic lymphatic leukaemia is predicted if an allele has been detected.
1 tbl, 4 dwg
The invention relates to the field of medical diagnostics, can be used to predict the risk of thrombocytopenia in the course of chronic lymphocytic leukemia.
Chronic lymphocytic leukemia (CLL) is a benign tumor, its substrate consists predominantly of morphologically Mature lymphocytes. The disease manifests lymphocytic leukocytosis, diffuse lymphocytic proliferation in the bone marrow, lymph nodes, spleen and liver [1].
The course of chronic lymphocytic leukemia is characterized by the development of various complications [2]. The most frequent complications are cytopenic syndromes, such as anemia and thrombocytopenia. Cytopenic complications in CLL is more often caused by immune system disorders in which the immune system attacks and destroys its own red blood cells. The relationship between duration of life of patients and cytopenic complications obvious. When thrombocytopenia (13-15% of cases) occur bleeding or hemorrhage in the brain that become the cause of death of patients [3].
As evidenced by the results of several studies, a significant role in the development of cytopenic complications of chronic lymphocytic leukemia played by genetic factors. Among the candidate genes, regulatory, mainly about Essy nonspecific defense, cell proliferation, hematopoiesis and apoptosis are essential genes for interleukins, in particular interleukin 1A and receptor antagonist interleukin 1 [4].
Interleukin 1A or IL-1A cytokine with a wide range of biological and physiological effects. This cytokine polifunzionale and performs at least 50 different functions, targets which are cells of almost all tissues and organs. IL-1A consists of 271 amino acid residue with a molecular mass of 17 kDa. The gene encoding IL-1A, mapped on the long arm of chromosome 2, in the promoter whose parts are in the position -889 is a single nucleotide substitution (-889C/T) [5].
Receptor antagonist interleukin 1 or IL-1Ra was first isolated from the culture medium stimulated monocytes and had the ability to inhibit the action of IL-1 on lymphocytes and fibroblasts by blocking the binding of IL-1 with cellular receptors. The gene encoding IL-1Ra, mapped on the long arm of chromosome 2 in the region q14. In the gene for IL-1Ra known minisatellite polymorphism is variability in the number 86-membered tandem repeats, VNTR in the 2nd intron, which implies the existence of five alleles, each of which corresponds to a certain number of repetitions. The most common allele of IL-1Ra∗1, containing 4 repeat in 71% of the population, and allele IL-1Ra∗2, containing 286-membered repeat 23% of the community is. The remaining alleles occur in less than 5% of cases [6].
A known method for diagnosis and prognosis of cancer, based on the study of somatic mutations in the gene multifunctional tumor suppressor (MTS) in the case of human neoplasms. U.S. patent No. 2164419, 27.03.2001. "MTS gene, Mutations of this gene and methods of diagnosis zlokachestvennykh of tumors using gene sequence MTS" (Myriad Genetics, Inc. (US), etc). The invention relates to mutations of the gene MTS in the germ line and the use of these mutations for the diagnosis of predisposition to these cancers, like melanoma, ocular melanoma, leukemia, astrocytoma, glioblastoma, lymphoma, glioma, Hodgkin's lymphoma, multiple myeloma, sarcoma, myosarcoma, loungeorama, caluclation carcinoma, chronic limfoidnye lymphocytic leukemia, and tumors of the pancreas, ovaries, uterus, testes, kidneys, stomach, colon and rectum. The invention also relates to the treatment of those human neoplasms, in which the mutation occurred in the gene MTS, including gene therapy, replacing protein therapy and the use of mimetics. The technical result of the invention is the expansion of the means of diagnosis and therapy of tumors.
The disadvantage of the method lies in the complexity analysis, its duration and to whom egovina.
A prototype of the selected RF patent №2248574 at the request of the Russian Federation No. 2003123171/15, 22.07.2003 "a Method for predicting the development and course of chronic lymphocytic leukemia" (Bakirov A.B. (RU), Bakirov B.A. (EN), where a method for forecasting the development and course of chronic lymphocytic leukemia by typing polymorphism DNA of lymphocytes isolated from the blood of the patient, TNF-alfa and TNF-beta, including the fence peripheral venous blood from the cells which secrete DNA by polymerase chain reaction of DNA synthesis, performed genotyping of the polymorphism in the promoter regions of TNF-αandβand in the detection of genotype LT∗22, characterized by the presence of mutations of the gene TNF-βin the homozygous state, identify individuals predisposed to the development of chronic lymphocytic leukemia, and when determining the combinations of genotypes of TNF∗22/LT∗22 and TNF∗12/LT∗11 in patients with chronic lymphocytic leukemia predict aggressive disease course.
The disadvantage of the prototype is that it does not enable to predict the propensity of a patient to thrombocytopenia.
The goal of this research is to expand the Arsenal of diagnostic methods, namely a method of predicting the risk of thrombocytopenia in the course of chronic lymphocytic leukemia according to genetic polymorphisms-889C/T gene interleukin 1A and VNTR antagonist of the receptor for interleukin 1.
The technical result of the use of the invention is the obtaining of the criteria for the assessment of the risk of thrombocytopenia in the course of chronic lymphocytic leukemia.
In accordance with the assigned task has been developed a method for predicting the risk of thrombocytopenia in the course of chronic lymphocytic leukemia, including:
- isolation of DNA from peripheral venous blood;
analysis of polymorphisms-889C/T gene interleukin 1A and VNTR receptor antagonist interleukin 1;
- prediction of the development of thrombocytopenia in patients with chronic lymphocytic leukemia in case of detection of allele-T IL-1A and/or allele of IL-1Ra∗1.
Novelty and inventive step lies in the fact that the prior art is not known to be able to predict the development of thrombocytopenia in the course of chronic lymphocytic leukemia by the presence of allele-889T gene interleukin 1A (polymorphism-889C/T IL-1A) and/or allele of IL-1Ra∗1 gene receptor antagonist interleukin 1 (VNTR polymorphism of IL-1Ra).
The method is as follows:
DNA extracted from peripheral venous blood samples of patients with chronic lymphocytic leukemia in 2 stages. At the first stage to 4 ml of blood add 25 ml of lyse buffer containing 320 mm sucrose, 1% Triton X-100, 5 mm MgCl2, 10 mm Tris-HCl (pH=7,6). The resulting mixture was stirred and centrifuged at 4°C, 4000 rpm for 20 minutes. After centrifuge is of the supernatant liquid is poured, to the precipitate add 4 ml of a solution containing 25 mm EDTA (pH=8,0) and 75 mm NaCl, resuspension. Then add 0.4 ml of 10% SDS, 35 ál of proteinase K (10 mg/ml) and incubate the sample at 37°C for 16 hours.
In the second stage of the lysate obtained consistently carried out the DNA extraction with equal volumes of phenol, phenol-chloroform (1:1) and chloroform by centrifugation at 4000 rpm for 10 minutes. After each centrifugation produce the 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 -20°C.
The selected DNA is then subjected to polymerase chain reaction using standard oligonucleotide primers (table 1).
| Table 1 | |||
| The structure of the primers and probes used for genotyping of the investigated DNA markers | |||
| Gene | Polymorphism and its localization in the gene | The structure of the primers and probes | Enzyme restriction | Literature |
| IL-1A | -S/T | 5'-aagcttgtctaccacctgaactaggc-3' | Bsp 19I | [8] |
| (promotor) (rs 1800587) | 5'-ttacatatgagccttccatg-3' | ||
| IL-1Ra | VNTR | 5'-ctcagcaacactcctat-3' | - | [9] |
| (2 intron) | 5'-tcctggtctgcaggtaa-3' | ||
The invention is characterized in the following figures:
Figure 1. Electrophoretic separation of the products of the restriction gene -889 C/T IL-1A, where the numbers 2, 3, 4 homozygotes - T; figures 5, 6, 10 - heterozygote-SS, figures 1, 7-9, 11-14 heterozygotes-ST.
Figure 2. Electrophoretic separation of the amplification products VNTR polymorphism of IL-1Ra, where the numbers 1, 2, 10-13 heterozygotes 2R/4R; figures 3-7, 9, 14 homozygotes 4R/4R, figure 8 - heterozygote 2R/5R.
Fig. Allele frequency-T IL-1A among CLL patients depending on the presence of thrombocytopenia at the time of the survey and in the control group, %.
Figure 4. Allele frequency of IL-1Ra∗1 among CLL patients depending on the presence of thrombocytopenia at the time of the survey and in the control group, %.
The study of the polymorphic locus of interleukin 1A (-C/T IL-1A) was carried out as follows. The reaction was performed in a 12.5 µl total volume with the art, 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 (3 min at 95°C) was performed with 35 cycles of amplification under the scheme: denaturation 1 min at 95°C; annealing of primers for 1 min at 55°C; elongation -1 min at 72°C. Then the sample was kept 5 min at 72°C and cooled. After RFLP analysis of the products of the enzyme were analyzed by 3%agarose gel, bromide stained by ethidium, within 1 hour 30 minutes at 80V. As an electrophoretic buffer used 1×TAE (Tris-acetate buffer). Then the samples identified in the transmitted UV light. Fragments of length 99 base pairs (BP) corresponded to the allele-S gene T/IL-1A, 83 and 16 BP - allele-T, fragments of length 99, 83 and 16 BP was observed in heterozygotes-ST (figure 1).
In the analysis of VNTR polymorphism of IL-1Ra reaction was performed 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 (4 min at 95°C) was performed with 32 cycles of amplification under the scheme: denaturation - 40 sec at 94°C; annealing of primers to 40 sec at 60°C; elongation - 1 min at 72°C. Then the sample was kept 5 min at 72°C and cooled. Amplification products were analyzed in 2%agarose gel, stained bromide is m ethidium, for 30 minutes at 160V. As an electrophoretic buffer used 1×TAE (Tris-acetate buffer). Then the samples identified in the transmitted UV light. As a result of amplification of the identified DNA fragments by length 240-595 P.N., 2, 3, 4, 5 or 6 copies of tandem repeats. These alleles were designated as 2R, 3R, 4R, 5R and 6R and met alleles of IL-1Ra 2, 3, 1, 4, and 5, respectively (figure 2).
Formation of database and statistical calculations were performed using the STATISTICA 6.0". Association of alleles and genotypes of the studied DNA markers with the development of thrombocytopenia in the course of chronic lymphocytic leukemia was evaluated using analysis of contingency tables 2×2 with the calculation of χ2 with the amendment of the Yates continuity and odds ratios (OR) with 95% confidence intervals (CI) [7].
The possibility of using the proposed method for assessing the risk of thrombocytopenia in the course of chronic lymphocytic leukemia confirms the analysis of observations 206 patients with chronic lymphocytic leukemia and 307 human population control. Patients were included in the appropriate group of patients only after diagnosis of the disease, confirmed using clinical and laboratory and instrumental methods of examination.
The studied group included individuals of Russian nationality whom the Xia natives of Central Chernozem region of Russia and do not have a relationship with each other.
It is established that patients with significant thrombocytopenia at the time of the survey have a maximum concentration of molecular genetic marker T IL-1A (36,54%) (figure 3), which is statistically significantly higher than the rate in the control group (22,77%, χ2=4.26 deaths, p=0.03, OR=1,95, 95% CI of 1.03-3,67).
Similar orientation data were obtained during the study of the distribution of allele IL-1Ra∗1 patients developed thrombocytopenia at the time of the survey (84,62%) compared with control (70,31%, χ2=4,11, p=0.04, OR=2,32, 95% CI of 1.02-5,46) (figure 4).
Thus, the obtained data suggest that allele-T gene IL-1A, allele IL-1Ra∗1 gene receptor antagonist interleukin 1 are risk factors for the development of thrombocytopenia in the course of chronic lymphocytic leukemia (OR=1,95 and OR=2,32, respectively).
Using this technique will allow us to take preventive measures to prevent the occurrence of thrombocytopenia in the course of chronic lymphocytic leukemia, as well as select individual tactics patient with chronic lymphocytic leukemia.
Literature
1. Vorob'ev A.I. Tumors of the lymphatic system / A.I. Vorobiev, A. M. Kremenets, D.V., Kharazishvili // the Hematology and Transfusiology. - 2000. - Vol.45, No. 3. - P.3-14.
2. Doronin, VA Modern aspects of pathogenesis and diagnosis of chronic lymphocytic leukemia: a review of the literature. / VA Doronin // Clinicas the th laboratory diagnostics. - 2003. No. 4. - P.24-25.
3. Nikitin, E.A. Chronic lymphocytic leukemia / E. Nikitin // Clinical Oncohematology. Basic research and clinical practice. - 2009. - Vol.2, No. 1. - P.87-91.
4. Axenovich TI Mapping of genes determining common human disease / TI Axenovich // Medical genetics. - 2006. - V.5, №2. - C.11-15.
5. Genetic susceptibility variants for chronic lymphocytic leukemia / Slager, S.L., L.R. Goldin, Strom SS [et al.] // Cancer Epidemiol. Biomarkers Are Prev. - 2010. - Vol.19, No. 4. - P.1098-1102.
6. Association of polymorphic genes interleukins with immunopathology of Teleuts / A.V. Shabalin, A.V. Stupava, A.N. Glushkov [and other] // Immunology. - 2007. - V.28, No. 1. - P.3-7.
7. Borovikov Century Statistica: the art of data analysis on the computer / Century Borovikov. - 2nd ed. - SPb.: Peter, 2003. - 688 S.: ill. - (For professionals).
8. Polymorphisms in the IL-1a and IL-1b Genes Are Not Associated with Susceptibility to Chronic Periodontitis in a Brazilian Population / P.C. Trevilatto, R. Scarel-Caminaga, R.B. Brito [et al.] // Braz. J. Oral. Sci. - 2003. - Vol.2, No. 7. - P.348-352.
9. Interleukin-1 receptor antagonist gene polymorphism is associated with increased risk of epithelial ovarian cancer / J. Sehouli, A. Mustea, Koensgen d [et al.] // Ann. Oncol. - 2003. - Vol.14, No. 10. - P.1501-1504.
A method for predicting the risk of thrombocytopenia in the course of chronic lymphocytic leukemia, including DNA isolation from peripheral venous blood and the analysis of polymorphisms - S/T gene interleukin 1A and VNTR receptor antagonist interleukin 1, which predict an increased risk of developing thrombocytopenia is in the course of chronic lymphocytic leukemia in case of detection of allele - T IL-1A and/or allele of IL-1 Ra∗1.