Age- and gender-independent association of glutathione S-transferase null polymorphisms with chronic myeloid leukemia

Authors

  • Abdel Rahim Mahmoud Muddathir Department of Clinical Laboratory Sciences, Faculty of Applied Medical Sciences, Taibah University, Medina, Saudi Arabia; Department of Hematology and Blood Transfusion, Faculty of Medical Laboratory Sciences, Alzaeim Alazhari University, Khartoum, Sudan
  • Elharam Ibrahim Abdallah Department of Hematology and Blood Transfusion, Faculty of Medical Laboratory Sciences, Alzaeim Alazhari University, Khartoum, Sudan
  • Omar Falah Khabour Department of Clinical Laboratory Sciences, Faculty of Applied Medical Sciences, Taibah University, Medina, Saudi Arabia; Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, Jordan University of Science and Technology, Irbid, Jordan https://orcid.org/0000-0002-3006-3104
  • Ream Elzain Abdelgader Department of Hematology and Blood Transfusion, Faculty of Medical Laboratory Sciences, Kordofan University, El-Obeid, Sudan https://orcid.org/0000-0001-7507-4774
  • Mahmoud Mohamed Elgari Department of Clinical Laboratory Sciences, Faculty of Applied Medical Sciences, Taibah University, Medina, Saudi Arabia

DOI:

https://doi.org/10.17305/bjbms.2019.4176

Keywords:

GSTs, glutathione S-transferase, null polymorphism, GSTT1, GSTM1, CML, haplotype

Abstract

The glutathione S-transferase (GST) genes encode enzymes that mediate the detoxification of xenobiotics by catalyzing the conjugation of glutathione (GSH) to xenobiotic substrates. The aim of the current study is to investigate the association between GSTT1 and GSTM1 polymorphisms and chronic myeloid leukemia (CML) among Sudanese patients. Patients with CML (n = 115) were recruited to the study from the Radiation and Isotope Centre Khartoum (RICK)-Sudan. Healthy individuals (n = 104) were included as controls. Genotyping of GSTT1 and GSTM1 polymorphisms was performed using multiplex PCR. Null deletions in the GSTT1 and GSTM1 genes are common in the Sudanese population (control group), with frequencies of 33.9% and 38.2%, respectively. The frequencies of GSTT1 (OR: 3.25, 95% CI: 1.87–5.65, p < 0.001) and GSTM1 (OR: 2.14, 95% CI: 1.25–3.67, p < 0.005) null genotypes were significantly higher in CML patients vs. controls. The distribution of GSTT1 and GSTM1 null polymorphisms was not different between male and female (p > 0.01) and young and old CML patients (p > 0.05). Hematological parameters were not affected by null polymorphisms in the patient group (p > 0.05). In addition, the frequency of GSTM1 null polymorphism was lower in advanced-phase CML patients compared to chronic-phase patients (p < 0.05). The GSTT1 and GSTM1 null polymorphisms are associated with CML among Sudanese patients, independently of their age and gender.

Downloads

Download data is not yet available.
Age- and gender-independent association of glutathione S-transferase null polymorphisms with chronic myeloid leukemia

Downloads

Additional Files

Published

2019-04-15

How to Cite

1.
Muddathir ARM, Abdallah EI, Khabour OF, Abdelgader RE, Elgari MM. Age- and gender-independent association of glutathione S-transferase null polymorphisms with chronic myeloid leukemia. Biomol Biomed [Internet]. 2019Apr.15 [cited 2023Nov.30];19(4):350-4. Available from: https://www.bjbms.org/ojs/index.php/bjbms/article/view/4176

Issue

Vol. 19 No. 4 (2019)

Section

Molecular Biology

INTRODUCTION

Glutathione S-transferases (GSTs) are enzymes that mediate the detoxification of xenobiotics in the body. GSTs catalyze the conjugation of xenobiotic substrates to glutathione (GSH) to increase the solubility of such substrates in cells [1]. In addition, GSTs may directly bind xenobiotics to facilitate their intracellular transport [2]. Eight GST genes have been identified in the human genome [3], including GSTM1 that encodes GST mu 1 and GSTT1 which encodes GST theta 1 [4]. Both genes are of clinical interest, due to the occurrence of null genotypes that lack the whole gene [5]. For example, null GSTM1 and GSTT1 polymorphisms are associated with an increased risk of several types of malignancies, including lung, blood, breast, and head and neck cancer [6-9]. Low activity of GSTs and reduced detoxification might enhance the potency of therapeutic drugs and influence the patient response to chemotherapy [6,10,11]. GSTs may also modulate some of the drug effects, such as the generation of hydroperoxides or other reactive oxygen species in the case of adriamycin, mitomycin C, and cisplatin [12].

Chronic myeloid leukemia (CML) is a malignant disease of the blood-forming cells of the bone marrow [13]. The disease is characterized by the presence of the fusion gene BCR-ABL1 in immature myeloid cells [14]. About 10% of all leukemias belong to this CML type [15]. Exposure to xenobiotics has been shown to increase the risk of cancer, including CML [16]. Since GSTs play a role in the detoxification of xenobiotics, polymorphisms in GSTs might modulate susceptibility to cancer and other diseases [17,18] and affect their onset [19]. In this study, we investigated the association between GSTM1 and GSTT1 null polymorphisms and CML among Sudanese patients. In addition, we compared the frequencies of these polymorphisms in the Sudanese population with those reported in other populations.

MATERIALS AND METHODS

Study design and patients

This case-control study was conducted on 115 patients with CML (50 females and 65 males, age range: 18–81 years) who attended the Radiation and Isotope Centre Khartoum (RICK) in Sudan for prognosis. Healthy individuals (n = 104, 49 females and 55 males, age range: 18–84 years) were included as a control group. The participants were recruited from May 2015 to June 2017.

A hematopathologist made the diagnosis of CML based on complete blood count (CBC), presence of a full spectrum of myeloid cells in peripheral blood film, reduced leukocyte alkaline phosphatase (LAP), and detection of t(9;22)(q34;q11) by routine cytogenetic analysis [20]. CML patients were classified into three groups (CML phases) according to the World Health Organization (WHO) criteria, including chronic (n = 93), accelerated (n = 20), and blast crisis phase (n = 2). The study was approved by the Institutional Review Boards of Alzaeim Alazhari University and Ministry of Health which utilizes the ethical principles of the Declaration of Helsinki to evaluate research proposals (approval ID: EC-AAU 25/2014). All participants signed written informed consent before inclusion in the study.

Collection of blood samples

A total of 2.5 ml venous blood samples were collected from each participant in vacutainer tubes containing the anticoagulant ethylenediaminetetraacetic acid (EDTA). Blood samples were stored at -20°C until used.

DNA extraction

DNA was extracted from EDTA-blood samples by a modified Chelex (100) protocol as described previously [21]. Briefly, a total of 500 µl of whole blood was added to an Eppendorf tube containing 1000 µl of red cell lysis buffer. The tube was mixed well and centrifuged at 12000 × g for two minutes. Then, the supernatant was discarded and the pellet was washed four times with 1000 µl of red cell lysis buffer. Next, the cleared pellet was washed with 1000 µl of phosphate-buffered saline, centrifuged at 12000 × g for two minutes, and the supernatant was removed. The pellet was then mixed with 200 µl of Chelex by vortexing and heated at 100°C for 20 minutes with vortexing every five minutes. After that, the samples were centrifuged at 14000 × g for two minutes and the supernatant was transferred into a clean Eppendorf tube. DNA samples were checked for purity using a spectrophotometer (Amersham Biosciences, Biochrom Ltd., Cambridge, UK) and stored at -20°C until used.

Genotyping of GSTT1 and GSTM1 polymorphisms

Multiplex polymerase chain reaction (multiplex PCR) was used for genotyping GSTT1 and GSTM1 null polymorphisms. The following primers were used: GSTT1, forward 5¢-TTCCTTACTGGTCCTCACATCTC-3¢ and reverse 5¢-TCACCGGATCATGGCCAGCA-3¢; GSTM1, forward 5¢-GAACTCCCTGAAAAGCTAAAGC-3¢ and reverse 5¢-GTTGGGCTCAAATATACGGTGG-3¢ [22,23]. PCR was carried out in a 25-µl reaction volume containing 100 ng of template DNA, 100 pmol of each primer, and ready-to-use master mix (12.5 µl Maxime PCR PreMix series, Intron Biotechnology, Korea). Amplification was performed in a BIO-RAD thermocycler (Mexico) using the following thermal cycle conditions: 94°C for five minutes, followed by 35 cycles of 94°C for 50 seconds, 61°C for 60 seconds, and 72°C for 60 seconds. The reaction was terminated by an extension time of seven minutes at 72°C. In a separate PCR reaction, a 436-bp long fragment of the GSTP1 gene was amplified as a positive control. PCR products were stained with EtBr and visualized on a 2.5% agarose gel. The absence of the 480-bp PCR product for GSTT1 and 219-bp PCR product for GSTM1 indicated the presence of homozygous null genotypes in those samples (Figure 1). The absence of the GSTP1 436-bp PCR product on a gel indicated a failure of PCR reaction. We should mention here that this genotyping method does not discriminate between the homozygotes (+/+) for the wild type GST alleles and heterozygotes (+/–).

FIGURE 1: Genotyping of GSTM1 and GSTT1 null polymorphisms. PCR amplification of GSTM1 and GSTT1 gene fragments and their visualization using 2% agarose gel electrophoresis. PCR products of 219 bp and 480 bp indicate the presence of GSTM1(+) and GSTT1(+) genotypes, respectively. Lane 8: DNA ladder of 100–1500 bp fragments. Lanes 1 and 5: GSTM1+/GSTT1+. Lane 2: GSTM1 null/GSTT1 null. Lanes 3 and 4: GSTM1 null/GSTT1+. Lane 6 and 7: GSTM1+/GSTT1 null. GST: Glutathione S-transferase; GSTT1: GST theta 1; GSTM1: GST mu 1; PCR: Polymerase chain reaction.

Statistical analysis

IBM SPSS Statistics for Windows, Version 21.0. (IBM Corp., Armonk, NY) was used to analyze the data. The frequencies of GSTT1 and GSTM1 genotypes were compared between patients and controls by Chi-square test. A p value of less than 0.05 was used as a threshold for significance.

RESULTS

Table 1 shows demographic characteristics of participants. The CML and control groups were not statistically different with respect to age, gender, and ethnicity (p > 0.05). The frequencies of GSTT1 and GSTM1 null genotypes in CML group were 66.1% and 62%, respectively, while in control group they were 33.9% and 38%, respectively (Table 2). The frequencies of GSTT1 (OR: 3.25, 95% CI: 1.87–5.65, p < 0.001) and GSTM1 (OR: 2.14, 95% CI: 1.25–3.67, p < 0.005) null genotypes were significantly higher in CML patients vs. controls. Similarly, when the combination of GSTT1 and GSTM1 null genotypes was considered, a significant difference was observed between CML patients and controls (OR: 2.57, 95% CI: 1.44–4.56, p < 0.01; Table 2).

TABLE 1: Demographic characteristics of participants
TABLE 2: Association of GST null polymorphisms with CML

We further analyzed the frequencies of GSTT1 and GSTM1 null genotypes among CML patients in relation to their gender, age, and CML phases (Table 3). The distribution of GSTT1 and GSTM1 null genotypes was not different between male and female (p > 0.01) and young and old CML patients (p > 0.05). However, the frequency of GSTM1 null genotype was lower in advanced-phase CML patients compared to chronic-phase patients (p < 0.05).

TABLE 3: Distribution of GSTT1 and GSTM1 null polymorphisms among CML patients according to gender, mean age, and CML phase

Table 4 shows hematological parameters of CML patients. Hematological parameters, including hemoglobin (Hb), platelet count, and white blood cell (WBC) count were not affected by GST null polymorphisms in CML patients (p > 0.05).

TABLE 4: Effect of GSTM1 and GSTM1 null polymorphisms on hematological parameters of CML patients

DISCUSSION

The current study showed a strong association between GSTT1 and GSTM1 null polymorphisms and CML in Sudanese patients.

Exposure to xenobiotics can lead to genetic alterations that increase the risk of cancer development [24,25]. GSTs are a group of enzymes which play an important role in the detoxification and elimination of xenobiotics from the body [19]. Therefore, genetic variations that affect the expression of GST genes are expected to modulate the risk of cancer, including CML [26]. Examples of such variations are GSTT1 and GSTM1 null polymorphisms that result in the complete deletion of these genes [27]. The results of our study showed a strong association between GSTT1 and GSTM1 null polymorphisms and the risk of CML among Sudanese patients. This finding is consistent with the majority of previous studies involving other populations (Table 5). For example, a strong association between GSTT1 and GSTM1 null polymorphisms and CML was reported in Egyptian [23], Syrian [28], German [29], and some other populations [30]. A positive association between GSTT1 null polymorphism and CML was reported in Turkish [31], Indian [32], and Kashmiri [22] population. On the other hand, several other studies reported a lack of association between CML and GSTT1 and GSTM1 null polymorphisms [33], which may be due to the differences in ethnicity and/or sample size.

TABLE 5: Frequency of GSTM1 and GSTT1 null polymorphisms in other populations

The present study showed that the association between CML and GSTT1 and GSTM1 null polymorphisms was not affected by the gender and age of patients, which is consistent with previous reports [34,35]. However, our results indicated a significantly lower frequency of the GSTM1 null genotype in advanced-phase CML patients compared to chronic-phase patients. Still, this finding needs to be confirmed in studies with larger sample sizes, as the number of CML patients in advanced phase was very small in our study. We showed no significant differences in Hb, platelet count, and WBC count between CML patients with and without the GST null polymorphisms and this is consistent with previous studies involving Egyptian and Indian populations [34,35].

GSTT1 and GSTM1 null polymorphisms have been shown to increase the risk of different cancers such as oral [9], head and neck [8], prostate [7], and cervical cancer [36]. Moreover, these polymorphisms were associated with treatment outcomes in breast [10], non-small cell lung [6], and bladder cancer [11]. One of the possible mechanisms explaining the effect of GST null polymorphisms on treatment outcomes in cancer is the involvement of GST enzymes in the detoxification of environmental and food xenobiotics and the subsequent elimination of their mutagenic potential in cells [10].

Overall, our study suggests that GSTT1 and GSTM1 null polymorphisms are associated with CML among Sudanese patients independently of their age and gender.

Acknowledgements

ACKNOWLEDGMENTS

The authors would like to thank the Faculty of Applied Medical Sciences at Taibah University for its support. Authors also would like to thank all staff of National Centre for Research, Khartoum, Sudan for their help in recruitment of patients.

DECLARATION OF INTERESTS

The authors declare no conflict of interests.

REFERENCES

  1. , (). Structure, function and disease relevance of Omega-class glutathione transferases. Arch Toxicol. https://doi.org/10.1007/s00204-016-1691-1
  2. , , , (). Intracellular binding and transport of hormones and xenobiotics by glutathione-S-transferases. Drug Metab Rev. https://doi.org/10.3109/03602538808994138
  3. , , (). The association between gene-environment interactions and diseases involving the human GST superfamily with SNP variants. Int J Environ Res Public Health. https://doi.org/10.3390/ijerph13040379
  4. , (). Genetic polymorphism and function of glutathione S-transferases in tumor drug resistance. Curr Opin Pharmacol. https://doi.org/10.1016/j.coph.2007.06.009
  5. (). Utilization of glutathione S-transferase Mu 1- and Theta 1-null mice as animal models for absorption, distribution, metabolism, excretion and toxicity studies. Expert Opin Drug Metab Toxicol. https://doi.org/10.1517/17425255.2013.780027
  6. , , , (). Glutathione S-transferase pi polymorphism contributes to the treatment outcomes of advanced non-small cell lung cancer patients in a Chinese population. Genet Mol Res. https://doi.org/10.4238/gmr.15037498
  7. , , , , (). GSTT1 and GSTM1 polymorphisms and prostate cancer risk in Asians:a systematic review and meta-analysis. Tumour Biol. https://doi.org/10.1007/s13277-013-0778-z
  8. , , (). Genetic variations and head and neck cancer risks. Mol Biol Rep. https://doi.org/10.1007/s11033-014-3125-6
  9. , , , , , (). Single nucleotide polymorphisms as markers of genetic susceptibility for oral potentially malignant disorders risk:Review of evidence to date. Oral Oncol. https://doi.org/10.1016/j.oraloncology.2016.08.005
  10. , , , , , (). GSTT1 and GSTM1 polymorphisms predict treatment outcome for breast cancer:a systematic review and meta-analysis. Tumour Biol. https://doi.org/10.1007/s13277-015-4401-3
  11. , , , , , (). Glutathione S-transferase M1 and T1 polymorphisms:susceptibility and outcomes in muscle invasive bladder cancer patients. Eur J Cancer. https://doi.org/10.1016/j.ejca.2013.05.019
  12. , (). Relevance of the deletion polymorphisms of the glutathione S-transferases GSTT1 and GSTM1 in pharmacology and toxicology. Curr Drug Metab. https://doi.org/10.2174/138920006778017786
  13. , , (). Laboratory monitoring of chronic myeloid leukemia in patients on tyrosine kinase inhibitors. Indian J Hematol Blood Transfus. https://doi.org/10.1007/s12288-018-0933-1
  14. , , (). Considerations for successful treatment-free remission in chronic myeloid leukemia. Clin Lymphoma Myeloma Leuk. https://doi.org/10.1016/j.clml.2017.11.006
  15. , , (). Enhancer dysfunction in leukemia. Blood. https://doi.org/10.1182/blood-2017-11-737379
  16. , , , , , (). Lifestyle and dietary factors in relation to risk of chronic myeloid leukemia in the NIH-AARP Diet and Health Study. Cancer Epidemiol Biomarkers Prev. https://doi.org/10.1158/1055-9965.EPI-13-0093
  17. , , , , , (). GSTT1 null genotype contributes to hepatocellular carcinoma risk:a meta-analysis. Tumour Biol. https://doi.org/10.1007/s13277-013-1026-2
  18. , , , (). Genetic polymorphisms of glutathione S-transferase genes GSTM1, GSTT1 and risk of hepatocellular carcinoma. PLoS One. https://doi.org/10.1371/journal.pone.0048924
  19. , , , (). Glutathione transferases:substrates, inihibitors and pro-drugs in cancer and neurodegenerative diseases. Oncogenesis. https://doi.org/10.1038/s41389-017-0025-3
  20. , (). Chronic myeloid leukemia:2014 update on diagnosis, monitoring, and management. Am J Hematol. https://doi.org/10.1002/ajh.23691
  21. , , (). An evaluation of the performance of five extraction methods:Chelex(R) 100, QIAamp(R) DNA Blood Mini Kit, QIAamp(R) DNA Investigator Kit, QIAsymphony(R) DNA Investigator(R) Kit and DNA IQ. Sci Justice. https://doi.org/10.1016/j.scijus.2015.01.005
  22. , , , , , (). Polymorphic variation in glutathione-S-transferase genes and risk of chronic myeloid leukaemia in the Kashmiri population. Asian Pac J Cancer Prev. https://doi.org/10.7314/APJCP.2012.13.1.069
  23. , , , (). Glutathione S transferase (GSTP 1, GSTM 1, and GSTT 1) gene polymorphisms in Egyptian patients with acute myeloid leukemia. Indian J Cancer. https://doi.org/10.4103/0019-509X.178408
  24. , , , , , (). Environmental carcinogen exposure and lifestyle factors affecting cancer risk in Qatar:findings from a qualitative review. East Mediterr Health J. https://doi.org/10.26719/2016.22.3.219
  25. , , , , , (). Nicotine, carcinogen, and toxin exposure in long-term e-cigarette and nicotine replacement therapy users:a cross-sectional study. Ann Intern Med. https://doi.org/10.7326/M16-1107
  26. , , , , , (). Genetic polymorphism in glutathione transferases (GST):population distribution of GSTM1, T1, and P1 conjugating activity. J Toxicol Environ Health B Crit Rev. https://doi.org/10.1080/10937400903158375
  27. , , , , , (). GSTT1 and GSTM1 polymorphisms predict treatment outcome for acute myeloid leukemia:a systematic review and meta-analysis. Ann Hematol. 90https://doi.org/10.1007/s00277-014-2050-z
  28. , , , (). Influence of CYP1A1, GST polymorphisms and susceptibility risk of chronic myeloid leukemia in Syrian population. Med Oncol. https://doi.org/10.1007/s12032-014-0889-4
  29. , , , , , (). Increased risk for therapy-associated hematologic malignancies in patients with carcinoma of the breast and combined homozygous gene deletions of glutathione transferases M1 and T1. Leuk Res. https://doi.org/10.1016/S0145-2126(01)00124-2
  30. (). Glutathione S-transferase M1 and T1 genes and susceptibility to chronic myeloid leukemia:a meta-analysis. Genet Test Mol Biomarkers. https://doi.org/10.1089/gtmb.2009.0079
  31. , , (). Variations in glutathione-S-transferase genes influence risk of chronic myeloid leukemia. Hematol Oncol. https://doi.org/10.1002/hon.1018
  32. , , (). Increased frequencies of glutathione-S-transferase (GSTM1 and GSTT1) null genotypes in Indian patients with chronic myeloid leukemia. Leuk Res. https://doi.org/10.1016/j.leukres.2007.02.003
  33. , , , , , (). CAT, GPX1, MnSOD, GSTM1, GSTT1, and GSTP1 genetic polymorphisms in chronic myeloid leukemia:a case-control study. Oxid Med Cell Longev. https://doi.org/10.1155/2014/875861
  34. , , (). Glutathione-S-transferase P1 as a risk factor for Egyptian patients with chronic myeloid leukemia. Egypt J Haematol. https://doi.org/10.4103/1110-1067.186408
  35. , , , , , (). Glutathione S-transferase M1 and T1 null genotype frequency in chronic myeloid leukaemia. Eur J Cancer Prev. https://doi.org/10.1097/00008469-200506000-00014
  36. , (). Meta-analysis of association between GSTM1 gene polymorphism and cervical cancer. Asian Pac J Trop Med. https://doi.org/10.1016/S1995-7645(12)60083-2
  37. , , , , , (). Association between methylene tetrahydrofolate reductase and glutathione S-transferase M1 gene polymorphisms and chronic myeloid leukemia in a Brazilian population. Genet Mol Res. https://doi.org/10.4238/2012.April.19.6