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Abdurakhmanova Nargiza, Independent researcher, the department of Internal diseases № 3, Tashkent Medical Academy Akhmedov Khalmurad, doctor of medical sciences, professor, Tashkent medical academy E-mail: khalmurad11968@mail.ru Rakhimova Matluba, Independent researcher, the department of Internal diseases № 3,
Tashkent Medical Academy Razikov Abdunabi,
Independent researcher, the department of Internal diseases № 3,
Tashkent Medical Academy
ASSOCIATION ANALYSIS OF THREE MDR1 (ABCB1) GENE VARIANTS (C1236T, G2677T AND C3435T) AND THEIR GENOTYPES OF THE RHEUMATOID ARTHRITIS PATIENTS
Abstract: Genetic studies of the polymorphism genotypes of the MDR1 gene in 76 patients with rheumatoid arthritis showed certain dependencies on the stability of the basic therapy. The triple genotype CT-GT-CT (C1236T-G2677T / A-C3435T) was found to be common among patients with drug resistance. From this it follows that the genotypes of polymorphisms of the MDR1 gene may have a certain effect on the course of RA..
Keywords: rheumatoid arthritis, MDR1 (ABCB1), RA., genotype, polymorphism.
Rheumatoid arthritis (RA) is the most common inflam- According to some estimates [4, 59-60], from 50 to 90% of matory rheumatic disease, characterized by a progressive adverse pharmacological responses is determined by the ge-course with the formation of deformities and severe function- netic characteristics of individuals. The study of individual al disorders of the musculoskeletal system, damage to internal genetic differences underlying the variability of the body's organs, which leads to disability and reduced life expectancy response to a particular drug is of paramount importance for of patients [8, 93-97]. Over the past decade, major changes the optimization of pharmacotherapy. It follows from this that have occurred in the management of RA patients, which have it is necessary to search for new approaches in the treatment significantly improved the results of treatment and made it of RA, i. e. the expansion of pharmacogenetic research, in par-possible to set the main goal - achieving clinical remission ticular the study of the relationship of the genotype with the (recommendations of EULAR, 2013). Targeted progress is clinically significant effect of the drug and the perverse reac-associated not only with the use of basic disease-modifying tion to the drug, as well as the study of the relationship of anti-rheumatic drugs, but also with specially developed inno- the genotype with the drug metabolism. Therefore, in recent vative genetically engineered biological preparations (GIBP). years, most attention is paid to the metabolic characteristics However, despite the entire arsenal of modern drugs in the of metatrexate in the liver under the predominant influence treatment of this disease, in 25-30% of cases, "complete clini- of transport proteins, in particular, the P-glycoprotein (P-gp) cal remission" or "low disease activity" is not achieved due group of proteins, which is encoded by the MDR1 gene (multi to refractoriness to treatment [5, 1484-1487]. This is due to drug resistancegene 1) [4, 59-60; 7, 933-936]. The product the fact that currently RA is considered as a complex disease, of the MDR1 gene is P-gp, acting as a transmembrane pump, the development of which is influenced by both genetic fac- and affects the action of many drugs [5], in particular metor-tors and environmental factors [2, 13-15; 3, 56-58]. It is well trexate. According to [1, 427-433], the activity ofthis protein known that the response to the effects of chemical factors, in- primarily depends on the polymorphism of the gene encod-cluding the response to medication, is individual and depends ing its structure. Therefore, genetic studies aimed at studying on lifestyle, age, gender, ethnicity, health, nutrition, interac- the characteristics of the protein transport system (MDR1) tions of the drugs used, individual metabolic characteristics. involved in the metabolism of basic drugs allow us to solve
problems regarding the prediction of the clinical course of RA, as well as the issues of selecting effective doses ofbasic therapy to achieve the main goal of "clinical remission" of the disease.
Materials and methods of the research. The study involved 76 patients with RA. of uzbek nationality, of which women accounted for 73 people, and men - 3 patients, aged 48.9 ± 15.9 years, with disease duration 7.5 ± 3.1 years. RA was diagnosed according to the criteria of the American College of Rheumatology (ACR). 75.6% ofpatients had high and 24.4% moderate RA activity (DAS28).
Genotyping ofABCB1 gene. Venous blood of 3 mL was drawn from patients during their visit to the the Rheumatology Department and stored in EDTA tubes. Genomic DNA was extracted from blood samples using an "RIBO-prep" reagents (AmpliSens, Russia). Genotyping A reagent kit was used to determine the MDR1 C3435T, C1236T, T3435C polymorphism. (SINTOL, Russia).
The PCR of G2677A, polymorphism was performed in a total volume of 25 mkl, using 100 ng of genomic DNA with 20 pmol primers each, 0.2mM of each dNTP, 1 x buffer, 2 mM ofMgCl2 and 1 U Taq DNA polymerase The MDR1 C3435T, C1236T, T3435C genes polymorphism was determined using the polymerase chain reaction assay. The cycling conditions performed in 7500 Fast Real time PCR system (Applied Biosystems, USA).
Table 1.- Allele and genoty
The results were subjected to static processing using the computer program EXCEL and STATISTICA 6.0., With the calculation of the arithmetic mean and deviation errors (M ± m). The significance of differences was calculated using the Wilcoxon method.
Results. In the present study, 76 patients with RA were evaluated for the presence of C1236T, G2677T and C3435T polymorphisms of the ABCB1 gene. As shown by the results of genetic analysis in patients with RA, normal and mutant homozygotes as well as heterozygotes were detected for all selected polymorphisms (Table 1). In our results, the normal homozygous polymorphism of the three isoforms of the MDR1 gene SS genotype was found in the C3435T isoform in 31.5%, in the G2677T isoform in 27.6% of patients with RA.. It is noteworthy that in RA. patients with a carrier of normal C1236T homozygotes, the genotype did not occur at all. In turn, mutant homozygotes were found in all isoforms of this gene, but there was no significant difference between them in the frequency of occurrence (p > 0.05). Thus, TT genotype was found in C3435T isoform in 29%, in G2677T isoform in 27.6%, and in patients with C1236T isoform in 27.6%.
Especially significant was the presence of heterozygotes (CT genotype), which had a high occurrence rate of 39.5% with the C3435T isoform, with the G2677T isoform at 44.8% and the C1236T isoform at 72.4%.
frequencies of RA patients
MDR1 gene Patients (n=76) %
C1236T
CC 0 0
CT 55 72.4
TT 21 27.6
Allele
C 55 36.1
T 97 63.9
G2677T
GG 22 28.9
GT 31 40.7
TT 23 30.2
Allele
C 75 49.4
T 77 50.6
T3435C
CC 24 31.5
CT 30 39.5
TT 22 29.0
Allele
C 78 51.3
T 74 48.7
The allelic frequency showed that the C allele was found in the T3435C isoform in 51.3%, in the G2677T isoform in 50% and in the C1236T isoform in 36.1% of patients. In the
When we analyzing the triple genotyping from 27 combinations of genotypes in our study, 16 did not occur at all, and the most frequently occurring genotype was CT-GT-CT, which was found in 26.32% of cases. Besides, two more genotypes were encountered more often than others: CC-TT-TT-22.36% and TT-GG-CT-21.1% (table number 2). The remaining 8 genotypes were found in an insignificant number (CC-TT-CT, CT-TT-TT, TT-GG-TT,- 1.31% genotypes, CT-GG-CT-3.94%, CT-GT-TT -5.26%, CT-TT-CT-2.63%, TT-GT-CT-6.57%. When analyzing double genotyping, CT genotype dominance was revealed (T3435C
analysis of allelic forms, the T allele was found in the T3435C isoform in 48.7%, in the G2677T isoform in 50% and in the C1236T isoform in 63.9% of patients.
/ G2677T-30.2%; T3435C / C1236T - 35.5%; G2677T / C1236T-40.7%)
Our results suggest that the CT genotype might be a susceptibility factor for the disease phenotype. In our further studies, based on the triple genotype, we plan to formulate a phenotypic characterization for RA patients.
Discussion. As mentioned above, drug resistance is one of the important factors affecting the effectiveness of RA therapy. Individualization of pharmacotherapy, which is engaged in pharmacogenetics, consists of identifying polymorphic markers associated with changes in the body's
Table 2.- Comparative analysis of combined genotypes of RA patients
Triple genotypes Patients n=76 % Double genotypes Patients n=76 %
T3435C-G2677T- C1236T T3435C-G2677T
CC-GG-CC 0 0 CC-GG 0 0
CC-GG-CT 0 0 CC-GT 4 5.2
CC-GG-TT 0 0 CC-TT 20 26.31
CC-GT-CC 0 0 CT-GG 4 5.2
CC-GT-CT 4 5.26 CT-GT 23 30.2
CC-GT-TT 0 0 CT-TT 3 3.94
CC-TT-CC 0 0 TT-GG 18 23.68
CC-TT-CT 1 1.31 TT-GT 4 5.26
CC-TT-TT 19 25.0 TT-TT 0 0
CT-GG-CC 0 0 T3435C-C1236T
CT-GG-CT 3 3.94 CC-CC 0 0
CT-GG-TT 0 0 CC-CT 7 9.21
CT-GT-CC 0 0 CC-TT 17 22.3
CT-GT-CT 20 26.32 CT-CC 0 0
CT-GT-TT 4 5.26 CT-CT 27 35.5
CT-TT-CC 0 0 CT-TT 3 3.9
CT-TT-CT 2 2.63 TT-CC 0 0
CT-TT-TT 1 1.31 TT-CT 21 27.6
TT-GG-CC 0 0 TT-TT 1 1.3
TT-GG-CT 17 22.3 G2677T-C1236T
TT-GG-TT 1 1.31 GG-CC 0 0
TT-GT-CC 0 0 GG-CT 21 27.63
TT-GT-CT 4 5.26 GG-TT 1 1.3
TT-GT-TT 0 0 GT-CC 0 0
TT-TT-CC 0 0 GT-CT 28 36.8
TT-TT-CT 0 0 GT-TT 3 3.9
TT-TT-TT 0 0 TT-CC 0 0
TT-CT 4 5.2
TT-TT 19 25
response to drugs, developing methods for genotyping patients, and introducing this approach into practical medicine. Recently, there has been an increase in the number of reports that some cell membrane transporters may influence the distribution of drug compounds. P-gp encoded by the gene MDR1 is a transport protein that plays a key role in the removal of a number of drugs from the cell [9, 496-503]. Some researchers suggest [5, 1484-1489] that there is an association of MDR1 gene polymorphisms with the efficacy and safety of many drugs. Currently, many polymorphic markers in the MDR1 gene have been studied, but three of them, C1236T, G2677T / A, and C3435T, are preferred to study associations. The rest, as a rule, occur with a low frequency, which does not explain the widespread disturbances in the functioning of P-gp. The C3435T and C1236T polymorphisms are believed to lead to a decrease in the expression of the MDR1 gene, and the "apparent" influence of the polymorphic marker G2677T/A is attributed to the linkage effect [10]. Therefore, in our work, we investigated the relationship between the three isoforms of the gene MDR1 (ABCB1) in RA. According to the literature [6], MDR-1 was one of the MDR genes, and its polymorphism was correlated with the activity of P-gp. Indeed, according to the literature [9, 496-503], the likelihood of clinical remission of RA after therapy with methotrexate and glucocortico-steroids in patients with the carrier of the 3435TT mutant genotype is higher than in patients with the 3435CC and 3435CT genotypes. This causes a discussion, since it follows from this that mutant genes have a double effect on the metabolism of drugs. Firstly, as a gene that has been altered against the background of environmental factors, it can have an inadequate effect on the body on the one hand [12, 546-554] and, secondly, a positive one, like suppression of disease activity and its response to therapy [7, 933-937].
Therefore, the results of our studies indicate the need for further clarification of the role of the influence of mutant homozygotes on the metabolism of metatrexate in patients with RA of Uzbek nationality, since the frequency of occurrence was high in all MDR-1 isoforms.
According to the literature, there is no doubt that during the carriage [11, 1041-1049], the genotype MDR13435 CT has a bad response (p = 0.01) to methotrexate, and with the CC-gen-otype, the positive response is opposite (p = 0.01). Therefore, recently, an attempt has been made to develop a prediction or predictive model containing genetic and clinical indicators for identifying patients with drug resistance to methotrexate.
Of particular interest is the study on the identification of frequencies of polymorphic variants of MDR1 genes, because according to the results of our studies, the triple genotype CT-GT-CT (C1236T-G2677T / A-C3435T) was common among patients with drug resistance. Literature data on the role in the combination of the above ternary genotypes on the metabolism of metatrexate in RA are absent. Therefore, further research is needed to understand the relationship between polymorphisms of the ABCB1 gene and the perception of PA treatment.
Conclusions: Genetic studies of the polymorphism genotypes of the MDR1 gene in RA patients showed certain dependencies on the stability of the basic therapy. The triple genotype CT-GT-CT (C1236T-G2677T / A-C3435T) was found to be common among patients with drug resistance. From this it follows that the genotypes of polymorphisms of the MDR1 gene may have a certain effect on the course of RA. The obtained data can be used in studies devoted to the study of the functional manifestations of polymorphic variants of MDR1, in the study of individual pharmacokinetics of various drugs, as well as in population-genetic studies. The data on the distribution frequencies of the studied alleles can serve as material for comparing the results in population studies by other authors.
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