Association of ABCB1 T-129C polymorphism and multiple myeloma risk in Polish population

Introduction

Plasma cell myeloma (multiple myeloma – MM) is a cancer of blood cells derived from bone marrow. It belongs to the group of malicious monoclonal gammapathy. Memory B cells are considered to be responsible for clonal proliferation. The expansion of neoplastic cells in bone marrow and the production of monoclonal immunoglobulin are responsible for disease symptoms manifestation [1]. As a result of disseminated marrow infiltrated by tumor cells, its function is disturbed – anemia, coagulation disorders, immunity disorders. Other characteristic symptoms of myeloma are osteolytic lesions, hypercalcemia, and renal failure [2]. Multiple myeloma is the second cause of neoplasms of the hematopoietic system in terms of the frequency of disease, it accounts for 1% of all cancers and approximately 10% of all hematologic malignancies. In Europe, 4.6/100 000 males and 3.2/100 000 females develop MM every year. The median age of patients at the time of diagnosis is about 65 years [3, 4]. The molecular mechanism of disease development has not yet been fully understood, attention is paid to the simultaneous participation of many environmental and genetic factors. Genetic variants like SNPs may be a protective or risk factor in the cancer development and be correlated with response to chemotherapy [5, 6].
ATP-binding cassette transporters play an important role in outflowing of xenobiotics and toxic compounds from cells [7]. ATP-binding cassette sub-family B member 1 – ABCB1 gene, also named as MDR1 gene - Multidrug Resistance Gene 1 is located on the chromosome 7q21.1 ABCB1 consists of 28 introns and 28 exons and encodes transmembrane P-gp – P-glycoprotein with molecular weight 170 kDa or 1280 amino acids [7]. Firstly this protein was described in drug-resistant cells [8]. P-gp occurs in both normal tissues and cancer cells. The ABCB1 gene is widely expressed in human organs and tissues, significantly in the apical membranes of excretory tissues: brain microvessel endothelium cells, intestinal epithelial cells, renal proximal tubular epithelial cells, placenta and testes [5, 9]. This protein is responsible for efflux endogenous metabolites and toxic xenobiotics, including carcinogens from cells, what is a protective mechanism against carcinogenesis. On the other hand, P-gp plays an important role in drug response trough the reduction the effect of the drug through modulating absorption, metabolism and promoting elimination from cells [10, 11]. P-gp also plays a role in the development of immune response by activating lymphocytes. Among the substrates for P-gp are: lipids, sterols, analgesics, antidepressants, anticancer drugs and immunomodulators such as doxorubicin, vinblastine, vincristine, epirubicin, etoposide and imatinib, are widely used in chemotherapy, including multiple myeloma therapy [11, 12, 13].
Genetic changes that affect the activity or expression of P-gp may contribute to the risk of developing cancer as well as potential response to chemotherapy [14, 15, 16, 17]. Recent studies suggest that genetic components may play an important role in the ethiopahology of MM [17]. SNPs in ABCB1 gene are highly diverse in different ethnic populations. So far, more than 50 SNPs have been identified in ABCB1 gene [11, 18]. Some of them have been studied more widely, others not enough. The aim of this study was to determine the potential significance of SNP T-129C of ABCB1 gene (rs3213619) in the development of multiple myeloma. According to the state of our knowledge, the role of this polymorphism in promoter region at ABCB1 gene in multiple myeloma has not been studied in the Polish population.

Material and methods

Study subject

91 blood samples collected from patients (50 females; 41 males, median age of the group: 63 years) with multiple myeloma diagnosed at the Cathedral of Hematology Medical University of Lodz, Poland were recruited to the study. Various treatment regimens have been used in the therapy of patients, such as: MP (melphalan; cisplatin), VAD (vincristine; doxorubicin; dexamethasone). In almost half of the study group, the clinical stage was assessed to III, according to Durie-Salomon classification.
The healthy control group consisted of 94 blood samples obtained from healthy individuals (56 females, 38 males, median age of group: 33 years) from the local blood bank were ethnic and geographically matched with the group of multiple myeloma patients. The investigation was in accordance with the Declaration of Helsinki, the Good Laboratory Practice rules and was approved by the Ethical Committee of the Medical University of Lodz No: RNN/88/16/KE. All patients provided a written informed consent before their inclusion in the study.

Genotyping

ABCB1 T-129C (rs3213619) polymorphism was evaluated applying the PCR-RFLP technique. DNA was isolated from peripheral blood according to “Blood Mini” protocol (A&A Biotechnology, Poland). DNA samples, until analysis, were stored at –20°C. PCR reaction for both investigated and control groups was performed according to 2xPCR Super Master Mix (Biotool.com, USA) protocol in the volume 20 µl PCR mixture. The PCR mixture composed of 5 µl of 2xPCR Super Master Mix, 0.5 mM of each primer, 50 ng of DNA template and distilled water up to 20 µl. Negative control (without DNA) was included in every experiment. PCR products were evaluated during electrophoresis in 2% agarose gel with ethidium bromide. Products of PCR reaction for SNP T-129C in ABCB1 gene had the size of 258 bp.
PCR products were digested by restriction enzyme, specific to the studied polymorphism: MspII. The digestion mixture consist of: 16 µl of PCR product, 2 µl of 10× PCR buffer, 0.2 µl of specified enzyme 10 U/µl and 1.8 µl of distilled water up to 20 µl. Digestion by restriction enzyme was performed for SNP T-129C: at 37°C for 16 h. Amplified DNA fragments after digestion by restriction enzyme were underwent electrophoresis on 2% agarose with ethidium bromide. The details of PCR-RLFP method are given in Table I.

Statistical analysis

STATISTICA 10 statistical software (StatSoft Inc. 2011) was used for data analysis. The χ2 Pearson test with the Yates correction was applied to evaluate the conformity between the observed and expected genotype frequencies in the investigated and control groups according to Hardy-Weinberg equilibrium. Differences in genotype frequencies among MM patients and control group and association of the various genotypes with clinical date were determined using the χ2 Pearson test with the Yates correction test. All p-values were two-sides and p < 0.05 was considered as statistically significant.

Results

In this study all blood samples from investigated and control groups were successfully analyzed for T-129C of ABCB1 gene. The frequency of particular genotypes in both groups was consistent with the Hardy-Wineberg equilibrium (p = 1.000).
First, genotype and allele frequencies for studied SNP between group of patients with multiple myeloma and healthy individuals were compared. In both groups the dominant genotype was homozygote TT (98% – multiple myeloma patients; 96% – healthy individuals). There was no significant statistical difference between multiple myeloma cohort and healthy individuals (p = 0.4297, p = 0.4296). All results are shown in Table II.
Secondly, the group of patients with multiple myeloma was divided, according to gender, into two subgroups (Table III), than the frequencies of geno- types for SNP T-129C were compared. Similarly, there was no statistically significant differences between the presence of a specific genotype and gender (p = 0.8870).
In the next step of the analysis the multiple myeloma cohort was divided according to age. Frequencies of particular genotypes of studied SNP were compared between the subgroup of patients with age 63 years and under this and subgroup of patients with age over 63 years. No statistically significant differences were found (p = 0.1572).
In the last part of the research, the group of patients with multiple myeloma were divided according to the type of the produced by myeloma cells immunoglobulin into four subgroups. Results are summarized in Table III. Then the analysis of genotype frequencies of SNP T-129C and these subgroups of patients were performed, no statistically significant differences were found (p = 0.6901).

Discussion

The P-gp protein encoded by the ABCB1 gene is an active exporter responsible for the transport of substances from the cytoplasm outside the cell or to specific intracellular compartments. The physiological function of P-gp is protection cells against harmful substances – metabolites or toxins of both endogenous and exogenous origin. The presence of membrane P-gp contributes to the removal of xenobiotics, ensuring the proper functioning of the intestines, kidneys, liver barrier or blood-brain barrier [12]. P-gp is involved in the regulation of the immune response and the process of proliferation and differentiation of hematopoietic stem cells [19].
Previous knowledge about P-gp protein suggests that a wild variance of T-129C ABCB1 can protect cells from carcinogen accumulation [20]. Therefore, it is likely mutant form of this SNP in ABCB1 may reduce ability to eliminate carcinogens, what could promotes carcinogenesiss. On the other hand, SNPs in the ABCB1 gene might influence the efficiency of chemotherapy by inducing multidrug resistance and could become a prognostic factor [12, 21].
So far, genetic variants in ABCB1 have been studied in various diseases such as Parkinson’s disease, mood disorders, breast cancer and colorectal cancer [22, 23]. ABCB1 SNPs (C1236T, G2677T/A, C3435T) have also been studied in hematological diseases such as chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B lymphoma (DLBCL) and multiple myeloma (MM) [5, 18, 20, 24]. From the previously published data, it can be assumed that polymorphisms in the gene regulatory region can also affect the expression and function of P-gp e.g. T-2410C, T-1910C, T-692C and T-129C and could be associated with hematological tumors [8].
The T-129C SNP in ABCB1 gene is located in the promoter region. The role of this polymorphism in cancer development and progression has not been sufficiently recognized. It was confirmed that this SNP increases the risk of DNA damage in response to carcinogens such as pesticides [24]. Also some data about neuropathic pain induced by chemotherapy in multiple myeloma have been reported [25] what confirmed that investigation of genes from the group of ABC transporters could help to solve that problem. Especially, that some data from the literature indicate that some genes [26] may also be involved in sensitivity to the use of treatment. However, in case of our study we did not get information about neuropathic pain in the group of investigated patients during chemotherapy. Therefore, we could not evaluate it and this could become the next step in the future analysis.
Distribution of individual genetic variants of the SNP T-129C varies within the populations. The results obtained in our study were consistent with the distribution of the frequency of genotypes for the healthy members of the Polish population in the study conducted by Tan et al. in which the distribution of individual genotypes was as follows: TT 93.5%, CT 6.5%, CC 0% [27]. In another research on the French population, the following distribution was obtained: TT 92%, CT 8%, CC 0% [28]. The presence of a CC homozygous has not been demonstrated for the Caucasian ethnic group. In contrast to the Japanese population, where the presence of CC homozygotes has been observed with frequencies CC 1.3% [29].
Our results indicate a lack of association between the studied T-129C polymorphism and the increased risk of multiple myeloma development (p = 0.4297). Contrary to our results, Hu et al. showed that polymorphisms of ABCB1 T-129C is connected with the risk of DLBCL [30]. In turn, results obtained in our study are consistent with the data published by Komoto et al. where there were no statistically significant differences between the group of healthy people and the group of patients with oesophageal cancer or with a group of patients with colorectal cancer. Similar results were obtained by Liu et al. in the study of the role of SNP in ABCB1 gene in susceptibility to primary open-angle glaucoma. In this study there were no significant differences in ABCB1 T-129C frequencies in investigated and control groups [31]. Studies on the role of T-129C polymorphism in the ABCB1 gene were also conducted in ovarian cancer. Similarly, there were no differences in the incidence of alleles between patients with ovarian cancer and the control group for SNP T-129C [32]. In the next step it could be important to compare of studied SNPs of ABCB1 gene with others recently investigated polymorphisms, including promising CD4 gene, tested on an animal model [33].
We are aware of the limitations of the study, however, we hope that our research on the role of genetic variants in this incurable cancer will contribute to the increasing of knowledge about multiple myeloma.

This study was supported by statutory funds of the Department of Pharmaceutical Biochemistry and Molecular Diagnostics, Medical University of Lodz no. 503/3-015-02/503-31-001.
The authors declare no conflict of interests.

References

1. Matsui W, Wang Q, Barber JP, et al. Clonogenic multiple myeloma progenitors, stem cell properties, and drug resistance. Cancer Res 2008; 68: 190-197.
2. Eslick R, Talaulikar D. Multiple myeloma: from diagnosis to treatment. Aust Fam Physician 2013; 42: 684-688.
3. Martino A, Campa D, Buda G, et al. Polymorphisms in xenobiotic transporters ABCB1, ABCG2, ABCC2, ABCC1, ABCC3 and multiple myeloma risk: a case-control study in the context of the International Multiple Myeloma rESEarch (IMMEnSE) consortium. Leukemia 2013; 27: 1615-1616.
4. Vincent Rajkumar S. Multiple myeloma: 2014 Update on diagnosis, risk-stratification, and management. Am J Hematol 2014; 89: 999-1009.
5. Ghafouri H, Ghaderi B, Amini S, et al. Association of ABCB1 and ABCG2 single nucleotide polymorphisms with clinical findings and response to chemotherapy treatments in Kurdish patients with breast cancer. Tumour Biol 2016; 37: 7901-7906.
6. Landgren O, Weiss BM. Patterns of monoclonal gammopathy of undetermined significance and multiple myeloma in various ethnic/racial groups: support for genetic factors in pathogenesis. Leukemia 2009; 23: 1691-1697.
7. Zhai X, Wang H, Zhu X et al. Gene polymorphisms of ABC transporters are associated with clinical outcomes in children with acute lymphoblastic leukemia. Arch Med Sci 2012; 8: 659-671.
8. Balcerczak E, Panczyk M, Piaskowski S, et al. ABCB1/MDR1 gene polymorphisms as a prognostic factor in colorectal cancer. Int J Colorectal Dis. 2010; 25: 1167-1176.
9. Li Y, Pang S, Huang W, et al. Novel and functional ABCB1 gene variant in sporadic Parkinson’s disease. Neurosci Lett 2014; 566: 61-66.
10. Ryu HC, Kwon HY, Choi IK, et al. Analyses of single nucleotide polymorphisms and haplotype linkage of the human ABCB1 (MDR1) gene in Korean. Arch Pharm Res 2006; 29: 1132-1139.
11. Llaudo I, Colom H, Gimenez-Bonafe P, et al. Do drug transporter (ABCB1) SNPs and P-glycoprotein function influence cyclosporine and macrolides exposure in renal transplant patients? Results of the pharmacogenomic substudy within the symphony study. Transpl Int 2013; 26: 177-186.
12. Hodges LM, Markova SM, Chinn LW, et al. Very important pharmacogene summary: ABCB1 (MDR1, P-glycoprotein). Pharmacogenet Genomics 2011; 21: 152-161.
13. Li H, Krstin S, Wang S, et al. Capsaicin and piperine can overcome multidrug resistance in cancer cells to doxorubicin. Molecules 2018; 23: E557.
14. Zhao L, Li K, Li W, et al. Association between the C3435T polymorphism of ABCB1/MDR1 gene (rs1045642) and colorectal cancer susceptibility : a meta-analysis based on 11,339 subjects. Tumour Biol 2013; 34: 1949-1957.
15. Wang J, Guo X, Yu S, et al. MDR1 C3435T polymorphism and inflammatory bowel disease risk: a meta-analysis. Mol Biol Rep 2014; 41: 2679-2685.
16. Zhang BB, Xuan C, Deng KF, et al. Association between the MDR1 gene variant C3435T and risk of leukaemia: a meta-analysis. Eur J Cancer Care (Engl) 2013; 22: 617-625.
17. Pongstaporn W, Pakakasama S, Chaksangchaichote P, et al. MDR1 C3435T and C1236T polymorphisms: association with high-risk childhood acute lymphoblastic leukemia. Asian Pac J Cancer Prev 2015; 16: 2839-2843.
18. Au A, Aziz Baba A, Goh AS, et al. Association of genotypes and haplotypes of multi-drug transporter genes ABCB1 and ABCG2 with clinical response to imatinib mesylate in chronic myeloid leukemia patients. Biomed Pharmacother 2014; 68: 343-349.
19. Ross DD. Modulation of drug resistance transporters as a strategy for treating myelodysplastic syndrome. Best Pract Res Clin Haematol 2004; 17: 641-651.
20. Yin G, Xiao Z, Ni Y, et al. Association of MDR1 single-nucleotide polymorphisms and haplotype variants with multiple myeloma in Chinese Jiangsu Han population. Tumour Biol 2016; 37: 9549-9554.
21. Wu H, Liu Y, Kang H, et al. Genetic variations in ABCG2 gene predict breast carcinoma susceptibility and clinical outcomes after treatment with anthracycline-based chemotherapy. Biomed Res Int 2015; 2015: 279109.
22. Jin SS, Song WJ. Association between MDR1 C3435T polymorphism and colorectal cancer risk: A meta-analysis. Medicine (Baltimore) 2017; 96: e9428.
23. Fung KL, Gottesman MM. A synonymous polymorphism in a common MDR1 (ABCB1) haplotype shapes protein function. Biochim Biophys Acta 2009; 1794: 860-871.
24. Chen CC, Huang CH, Wu MT, et al. Multidrug resistance 1 gene variants, pesticide exposure, and increased risk of DNA damage. Biomed Res Int 2014; 2014: 965729.
25. Wang Q, Wang J, Gao D, Li J. Inhibition of PAR2 and TRPA1 signals allewiates neuropathic pain evoked by chemotherapeutic bortezomib. J Biol Regul Homeost Agent 2017; 31: 977-983.
26. Zhang Y, Wang Z, Zhang L, et al. Impact of connexin 43 coupling on survival and migration of multiple myeloma cells. Arch Med Sci 2017; 13: 1335-1346.
27. Tan EK, Drozdzik M, Bialecka M, et al. Analysis of MDR1 haplotypes in Parkinson’s disease in a white population. Neurosci Lett 2004; 372: 240-244.
28. Jeannesson E, Albertini L, Siest G, et al. Determination of ABCB1 polymorphisms and haplotypes frequencies in a French population. Fundam Clin Pharmacol 2007; 21: 411-418.
29. Komoto C, Nakamura T, Sakaeda T, et al. MDR1 haplotype frequencies in Japanese and Caucasian, and in Japanese patients with colorectal cancer and esophageal cancer. Drug Metab Pharmacokinet 2006; 21: 126-132.
30. Hu LL, Yu B, Yang J. MDR1 polymorphisms associated with risk and survival in diffuse large B-cell lymphoma. Leuk Lymphoma 2013; 54: 1188-1193.
31. Liu H, Yang ZK, Li Y et al. ABCB1 variants confer susceptibility to primary open-angle glaucoma and predict individual differences to latanoprost treatment. Biomed Pharmacother 2016; 80: 115-120.
32. Kufelnicka-Babout M, Beata S, Kulig A, et al. The significance of T129C and G2677T polymorphism of the MDR1 gene in ovarian cancer patients. Prz Menopauz 2008; 6: 295-300.
33. Xu J, Zhang H, Gu Y, et al. Correlation anlysis of CD4 gene polymorphism and blood routine indexes in pigs (Sus scorfa). J Biol Regul Homeost Agents 2017; 32: 327-333.

Address for correspondence

Marta Żebrowska
Laboratory of Molecular Diagnostic and Pharmacogenomics
Medical University of Lodz
Muszynskiego 1
90-151 Lodz, Poland