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Karimov Khamid Yakubovich, Abdurakhmanova Nigora Nazimovna, Boboev Kadir Tuxtabayevich Scientific research institute of hematology and blood transfusion, Republic Uzbekistan E-mail: dok.mzt@mail.ru
Analysis of association of polymorphism rs1045642 of мdr1 gene with development of myeloproliferative diseases
Abstract: The authors to evaluate of the role of C3435T polymorphism of MDR1 gene in the pathogenesis of chronic myeloid leukemia (CML) and erythremia observed the 138 patients with CML and erythremia. The investigation has showed that rs1045642 polymorphism of MDR1 gene is associated with a risk of MPD developing. The prognostic value of genotyp-ing of rs1045642 polymorphism of -MDR1 gene demonstrates a high level of efficiency by classifier as an independent gene-determinant in developing MPD, at significantly higher values.
Keywords polymorphism, МDR1 gene, myeloid leukemia, erythremia.
Introduction. Myeloproliferative diseases (MPD) are clonal diseases arising at the hematopoietic stem cell level [2]. Etiological factors and mechanisms, which provoke MPD development, are not still fully studied. The leading hypothesis is that, there are multiple stages in the development of the disease, where the predisposition to the disease emerges under the effect of external carcinogens, damaging by gene of normal cells, and causes to its malignant transformation [3]. Nowadays the facilities of basic study of MPD are not limited to the research diagnostic genetic mutations, such as BCR/ABL, JAK2, MPL and others. At the same time, over recent years, there is paid more attention to polymorphic variants of genes of xenobiotics biotransformation system. Determination of correlation between detection of the specific genotype of these genes and definite forms of MPD may lead us to better understand and comprehend the mechanisms of formation of various forms of diseases [4].
MDR1 gene with length of209660 p. n. is localized at the short seventh chromosome's arm (7q21.1). It contains 29 exons and is expressed by formation of the transcript w ith length of4916n [5]. The MDR1 gene encodes synthesis of P-glycoprotein transfer protein, which is involved in biotransformation of xenobiotics, protecting by this way cells from the toxic effects of various compounds [6]. At the present time, there have been several polymorphous types of MDR1 gene investigated, but the preference is given to C3435T polymorphism (rs1045642) in investigation of associations with cancer pathology [7].
In world literature there is evidence of the involvement of MDR1 gene in the development of malignant diseases, such as various types of leukosis, rectal cancer, endometrial cancer, Hodgkin's diseases (lymphoma) and so on [8-10; 11; 13].
In spite of this, it is not yet completely clear functional and clinical significance of the different polymorphic variants of the gene under the distribution of toxic substances and, as a consequence, the occurrence of susceptibility to development of malignant neoplasms. In addition, to date, yet there is no consensus about the role of rs1045642 polymorphic marker of MDR1 gene with manifestation of the different options of MPD.
The aim of the study is evaluation of the role of C3435T polymorphism of MDR1 gene in the pathogenesis of chronic myeloid leukemia (CML) and erythremia.
Materials and methods. The study is performed on DNA samples purified from the peripheral blood of patients, who were observed at the clinic of Scientific research institute of hematology
and blood transfusion of Uzbekistan. There have been 138 patients (109 patients with CML, 29 ones with erythremia) observed and studied. The control group was 86 persons of Uzbek nationality, without any cancer.
DNA purification from whole blood was performed using a standard set of Ribosorb production (AmpliSens®, Russia). The concentration and purity of the purified DNA was determined by the apparatus of NanoDrop 2000 (USA). Genotyping (genetic typing) ofrs1045642 polymorphic variant ofMDR1 gene was performed by standard PCR using primers structures that were described on the website http://www.ncbi.nlm.nih.gov/SNP (Table 1).
Table 1. - The structure of oligonucleotide primers
No. Localiza- Polymor- Structure of oligonucleotide
tion of the phism primers
gene
1 MDR1 rs1045642 F: 5-AGAGAGACTTA-CATTAGGCAG-3' R:5'-R 5AGTGGCTCCGAGCA-CACC-3'.
Testing was conducted on a programmable thermal cycler of "Applied Biosystems" company (USA), using the test-kit of "Liteh" company (Russia), according to manufacturer's instructions (Fig. 1).
Statistical analysis of the results was carried out using statistical software package OpenEpi 2009, 2.3 Version.
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. W M • - U —> ■ - ■» _A J.ir
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Figure 1. Electrophoregram for detection of rs1045642 polymorphism of MDR1 gene
Results and discussion. According to NCBI data MDR1 gene has more than 50 polymorphisms (SNPs). Among them, the great-
Analysis of association of polymorphism rs1045642 of Mdrl gene with development of myeloproliferative diseases
est interest was shown to the polymorphic marker C3435T. According to the authors' data, the frequency of heterozygous genotypes of this polymorphism in the European population was 48.3% and homozygotes in the same group — 23.9% [1, 12].
Tables 2 and 3 demonstrate the findings of comparative studies on the genetic structure of rs1045642 polymorphism of MDR1 gene in samples of patients with MPD and control. The frequency distribution of genotypes of rs1045642 polymorphic
locus of MDR1 gene among patients group and control group complied with the estimated results when Hardy-Weinberg equilibrium (P> 0.05). There was found high rate of estimated heterozygosity (0.47%) in a population sampling. At diallel polymorphism this index is an indicator of significant diversity of the population, equal to the maximum (0.5%). This means that about 50% of individuals in our population carry T -allele in hetero or homozygous state.
Table 2. - Estimated and observed distribution frequency of genotypes for Hardy-Weinberg principle of rs1045642 polymorphism -MDR1 gene in the main group of patients:
Genotypes Genotypes frequency x2 P
observed estimated
C/C 26.81 25.00 0.181 0.3946
C/T 46.38 50.00 0.362
T/T 26.81 25.00 0.181
Total 100.00 100.00 0.725
Table 3. - Estimated and observed distribution frequency of genotypes for Hardy-Weinberg principle of rs1045642 polymorphism -MDR1 gene in population group
Genotypes Genotypes frequency x2 P
observed estimated
C/C 39.53 39.43 0.000 0.9658
C/T 46.51 46.73 0.001
T/T 13.95 13.85 0.001
Total 100.00 100.00 0.002
As regards the effectiveness of this locus as an independent marker, it must be noted the relatively high level of specificity with the index SP = 0.73 with SE = 0.39 (95% CI 1.006-3.166). The calculated ratio AUC (0.73) demonstrates the high level of efficiency on classifier of this polymorphism as an independent gene-
candidate, at significantly high values (OR = 1.8; P <0.05). The Comparison of frequencies of rs1045642 alleles and genotypes of MDR1 gene among patients with MPD and comparison groups was conducted by the randomized controlled method.
Table 4. - Distribution frequency of alleles and genotypes of rs1045642 polymorphism of MDR1 gene among studied patients group and population sampling.
No. Group N Alleles Distribution frequency of g enotypes
C T C/C C/T T/T
n % n % n % n % n %
1 Main Group 138 138 50,0 138 50.0 37 26.8 64 46.4 37 26.8
1.1 CML 109 106 48.6 112 51.4 28 25.7 50 45.9 31 28.4
1.2 Erythremia 29 32 55.2 26 44.8 9 28.1 14 43.7 6 18.7
2 Control group 86 108 62.8 64 37.2 34 39.5 40 46.5 12 13.9
The analysis revealed significant differences in the distribution frequencies of occurrence of genotypes and alleles of rs1045642 polymorphism of MDR1 gene in patients with MPD and in the control group (Table 4). The combined sample of patients displayed significantly more frequent occurrence of T allele compared to the control group (50.0% and 51.4%, respectively; x2= = 6.5; p = 0.01; OR = 1.9; 95% CI 1.144, 2.49). The frequency of this allele is 1.8 times substantially higher in CML patients subgroup than in the in healthy donors (x2 = 7.8; p = 0.005; OR = 1.8; 95% CI 1.186, 2.68). At the same time the difference in frequency of this allele between erythremia patients and control subgroups was unessential (44.8% and 37.2%, respectively; p> 0.05).
In the combined sample and in CML patients subgroups the frequency of unfavorable genotype T/T also significantly dominated over its level in the control group (26.8% and 28.4% vs. 13.9% respectively). According to the calculated odds ratio the presence of the present genotype 2.3 times increased the risk of MPD development (x2 = 5.1; p = 0.02; OR = 2.3; 95% CI 1.103, 4.626). It was noted a slight increase in the frequency of occurrence of the present genotype in the subgroup of patients with erythremia as
well, in comparison with the control group, that indicates the availability of a trend towards association of unfavorable genotype with MPD formation (18.7% vs. 13.9%, respectively; p> 0.05). Homozygous genotype frequency for allele-C, in contrast was significantly lower in the group of patients (26.8%) than in the control group (39.5%), that is an evidence of a favorable protective effect of present genotype on the development of disease (x2 = 3,9; P = 0.046; OR = 0.6; 95% CI 0.3158. 0.994).
There was the tendency to reduce the frequency of this genotype in erythremia patients subgroup revealed compared to the control group (28.1% vs. 39.5%, respectively). At the same time, the differences did not reach the threshold level of significance (x2= = 0.6; P = 0.4; OR = 1.4; 95% CI 0.5922, 3.565).
Also it is worth noting that when comparing patients subgroups and control groups there was a significant increase in the frequency of genotype C/C in a population sampling revealed in comparison with the subgroup of patients with CML (39.5% vs. 25.7%, respectively; x2 = 4.2; P = 0.04; OR = 0.5; 95% CI 0.2874. 0.9725).
Moreover, the frequency of heterozygous genotype C/T rs1045642-of MDR1 gene in all groups studied (patients and con-
trolled) was similar and did not reach statistically significant differences versus control group (P> 0.05).
Thus, we can conclude that the homozygous genotype T/T of rs1045642 polymorphism -MDR1 gene is an important determinant of increased risk for MPD development in Uzbekistan (P <0.05). These data further reinforce the position of rs1045642 polymorphism of -MDR1 gene as a marker, causing disorder of the regulatory function of P-glycoprotein and processes of biotransformation of xenobiotics, and perhaps associated with the formation of cancer processes.
Conclusion:
1. rs1045642 polymorphism ofMDR1 gene is associated with a risk ofMPD developing. Functionally unfavorable genotype T/T is a predisposing marker to damage the expression level of P-glycoprotein and elimination ofvarious toxins and carcinogens from the body. On the contrary, the carriage of wild genotype C/C was significantly associated with a protective effect against MPD development (P <0.05).
2. The prognostic value of genotyping of rs1045642 polymorphism of -MDR1 gene demonstrates a high level of efficiency by classifier as an independent gene-determinant in developing MPD, at significantly higher values (P <0.05).
References:
1. Sychev D. A., Ramenskaya G. V., Ignatiev I. V., Kukes V. G., Clinical pharmacogenetics: Teaching medium/under Ed. of V. G Kukes, N.P Bochkova. - M.: GEOTAR Media, 2007. - P. 9.
2. Deininger M. W., Goldman J. M., Melo J. V. The molecular biology of chronic myeloid leukemia. Blood.2000; 96: P 3343-3356.
3. Rumjanek V.M, Vidal R.S, Maia R. C. Multidrug resistance in chronic myeloid leukaemia Biosci Rep.2013;33 P.
4. Mabel Lardo, Marcelo Castro, Beatriz Moiraghi, Francisca Rojas. MDR1/ABCB1 gene polymorphisms in patients with chronic myeloid leukemia 2015 sep; 50 (3): P. 154-159.
5. Callen D. F., Baker E., Simmers R. N., Seshadri R., Roninson I. B. 1987. Localization of the human multiple drug resistance gene, MDR1, to 7 q 21.1. Hum. Genet. 77, 142-144 p.
6. Chin J. E., Soffir R., Noonan K. E., Kyunghee C., Roninson I. B. Structure and expression of the human MDR (P-glycoprotein) gene family.//Molecular and Cellular Biology. 1989. 9, P. 3808-3820.
7. Chen C., Clark D., Ueda K., Pastan I., Gottesman M. M., Roninson I. B. 1990. Genomic organization of the human multidrug resistance (MDRl) gene and origin of P-glycoproteins. J. Biol. Chem. 265, P. 506-514.
8. Rao D. N., Anuradha C., Vishnupriya S., Sailaja K., Surekha D., Raghunadharao D., Rajappa S. Association of an MDR1 gene (C3435T) polymorphism with acute leukemia in India. Asian Pac J Cancer Prev. 2010; 11 (4): P. 1063-1066.
9. Qian X., Cao S., Yang G., Dong J., Jin G., Shen Y., Hu Z. Variant genotypes of MDR1 C3435T increase the risk of leukemia: evidence from 10 case-control studies. Leuk Lymphoma. 2012 Jun; 53 (6): P. 1183-7.
10. Andersen V., Ostergaard M., Christensen J., Overvad K., Tjonneland A., Vogel U. Polymorphisms in the xenobiotic transporter Multidrug Resistance MDR1) and interaction with meat intake in relation to risk of colorectal cancer a Danish prospective case-cohort study. BMC Cancer. 2009 Nov 21;9: P. 407.
11. Mrozikiewicz P. M., Seremak-Mrozikiewicz A., Semczuk A., Landt O., Breborowicz G. H., Drews K. The significance of C3435T point mutation of the MDR1 gene in endometrial cancer. Int J Gynecol Cancer. 2007 May-Jun;.17. (3):.P 728-31.
12. Nizar M., Mhaidat, Osama Y Alshogran, Omar F Khabour, Karem H Alzoubi, Ismail I Matalka, William J Haddadin, Ibraheem O Ma-hasneh, and Ahmad N Aldaher. Multi-drug resistance 1 genetic polymorphism and prediction of chemotherapy response in Hodgkin's Lymphoma. J Exp Clin Cancer Res. 2011; 30 (1): P. 68.
13. Hoffmeyer S., Burk O., von Richter O., Arnold H. P., Brockmoller J., Johne A., Cascorbi I., GerloffT., Roots I., Eichelbaum M., Brinkmann U. 2000. Functional polymorphisms of the human multidrug-resistance gene: Multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo. Proc. Natl. Acad. Sci. USA. 97, P. 3473-3478.
Kahhorov, Jamoliddin Professor, MD. PhD., Atakhanova Nigora, Professor, MD. PhD., Shayusupov Nariman, Professor, MD. PhD., Kakhkharov Alisher Dr., Tashkent Medical Academy, Oncology and radiology department Tashkent City Oncology Center, Breast cancer unit E-mail: alisher1510@mail.ru
Tumor-to-breast ratio in forecast of breast cancer
Abstract: The role of tumor-to-breast ratio is determined in breast cancer forecast. Case histories of 49 patients with breast cancer were analyzed. The undergone analysis showed that tumor-to-breast ratio has a statistically significant impact on the outcome of the treatment of breast cancer.
Keywords: forecast, tumor-to-breast ratio, breast MRI, TNM system.
