Polymorphism analysis of Glutathione S-transferase A1 in patients with hematological diseases and its effect on GST enzyme activity  
Guangyi Yu, Qingshan Yang, Guoshun Zhang, Yin Xie, Lifeng Zhang*   
School of Pharmaceutical Sciences, Shanxi Medical University, Taiyuan 030000, China
Abstract: Glutathione S-transferases (GSTs) are important drug-metabolizing enzymes that catalyze the binding of glutathione (GSH) to electrophilic substances. GST has genetic polymorphism, and the enzyme activity of GST affects the metabolism of certain drugs in vivo. In the present day, we investigated the GST enzyme activity and GSTA1 gene polymorphism in 170 patients with hematological diseases and explored their relationship. The GSTA1 gene polymorphism of the patient was analyzed by PCR- restriction fragment length polymorphism (PCR-RFLP) technique, and the base sequences of the four mutation sites (-631, -567, -69, and -52) in the promoter region were determined by DNA-Sequencer. The patient's GST enzyme activity was calculated by measuring the rate at which it catalyzed the reaction between 1-chloro-2,4-dinitrobenzene (CDNB) and GSH. The average GST enzyme activities of males and females were 5.20±0.13 and 5.17±0.12 nmol/min/mL, respectively, and the difference was not significant (P = 0.91). The frequencies of genotypes GSTA1*A*A (wild genotype), GSTA1*A*B (heterozygous genotype), and GSTA1*B*B (homozygous mutant genotype) were 75.3%, 22.9%, and 1.8%, respectively. Alleles GSTA1*A and *B were distributed at 86.8% and 13.2%, respectively. The genotype frequency distribution between males and females was no significant difference by Pearson’s chi-square test (P = 0.743). The average GST activity of the heterozygous mutant genotype (4.83±0.76 nmol/min/mL) was lower than the wild genotype (5.34±1.26 nmol/min/mL, P = 0.018), and higher than that of the homozygous mutant genotype (3.32±0.07 nmol/min/mL, P = 0.022). These findings might help us improve the individualized treatment of patients with hematological diseases in the future and promote the development of precision medicine for blood diseases.           
Keywords: Polymorphism; Glutathione S-transferase; Hematological disease; Enzyme activity; PCR   
CLC number: R968                Document code: A                 Article ID: 10031057(2019)639309
1. Introduction
Over the past few decades, the incidence of hematological diseases has an increasing trend year by year[1,2]. Malignant hematological diseases have seriouslyjeopardized the health of patients, mainly including leukemia, lymphoma, myeloma, myelodysplastic syndrome and so on[35]. The treatments of hematologic malignancies include radiotherapy, chemotherapy, targeted therapy, and bone marrow transplantation. Hematopoietic stem cell transplantation (HSCT) is the common method for hematological malignancies[68]. Busulfan combined with cyclophosphamide is commonly used in the pretreatment of HSCT[9]. The metabolisms of busulfan and cyclophosphamide are mediated by GST enzymes[7,10,11].
GST enzymes play important metabolic roles by catalyzing the nucleophilic attack of glutathione (GSH) on non-polar compounds containing electrophilic carbon, nitrogen or sulfur atoms[12,13]. Genetic polymorphisms are present in the GST enzyme, and the gene encoding GST enzyme is distributed in at least seven stains. Currently, according to the homology of N-terminal amino acid sequence of GST subunit and considering the substrate specificity, immunoreactivity and sensitivity of various GSTs to different inhibitors, mammalian GSTs are classified into alpha (A), mu (M), pi (P), theta (T) and kappa (K)[14,15]. Despite the existence of different forms of enzymes, such as GSTM1, GSTP1 and GSTT1, GSTA1 is the most expressed form of the liver and is widely distributed in the body[16]. The GST of alpha class possesses high glutathione peroxidase activity against damage from endogenous and exogenous electrophilic substances and plays a key role in the metabolic process of certain chemotherapeutic drugs for the treatment of hematological diseases[17,18]. In the human liver, the enzyme of GSTA1 genotype accounts for about half of the total amount of GST enzyme[19]. Therefore, our study focused on GSTA1 and its genetic polymorphism.
The variants were reported in the human GSTA1 gene with four SNPs in the promoter region: -631G/T, -567T, -69C, -52G, named as hGSTA1*A; and -631G, -567G, -69T, -52A, named as hGSTA1*B[20]. In order to ensure accurate and reliable results, all four SNP sites were investigated by PCR-RFLP in this study. The activity of GST enzyme is directly or indirectly involved in the metabolism of chemotherapeutic drugs in patients with hematological diseases. Therefore, we also investigated the activity of GST enzyme in patients with hematological diseases.
Collectively, taking patients with blood disease as research subjects, this study investigated the GSTA1 genetic polymorphism and GST enzyme activity in patients and analyzed their relationship. These findings might help us improve the individualized treatment of patients with hematological diseases in the future and promote the development of precision medicine for blood diseases.
2. Materials and methods
2.1. Materials
The UV-visible spectrophotometer was a product of INESA Analytical Instrument Co., Ltd. (Shanghai, China). The Verity 96well PCR instrument and 3730XL Gene Sequencer were obtained from Applied BiosystemsTM of ThermoFisher Scientific. (Waltham, MA, USA). The refrigerated centrifuge was purchased from Anhui USTC Zonkia Scientific Instruments Co., Ltd. The FR-980A Gel Image Analysis System was provided by Life ScienceResearch Products and System Engineering. (Shanghai, China). The glutathione S-transferase assay kit was a product of Comin Biotechnology Co., Ltd. (Suzhou, China). The EZ-10 spin column blood genomic DNA purification kit and the SanPrep column PCR product purification kit were products of Sangon Biotech. Taq Plus DNA polymerase, dNTP, 10X PCR buffer (with Mg2+),EDTA, and DNA Ladder Mix (1003000bp) weresupplied from Sangon Biotech. (Shanghai, China). BigDyeTerminator v1.1, HiDi Formamide, and POP-7TM Polymerwere products of ThermoFisher (Applied BiosystemsTM, MA, USA). All other chemicals and reagents were of the highest grade commercially available.
2.2. Subjects and blood samples
Blood samples were collected from the malignant hematologic disease population of 170 patients without direct blood relationship in Shanxi Provincial Cancer Hospital and Shanxi Dayi Hospital, including leukemia (n = 27), multiple myeloma (n = 14), non-Hodgkin's lymphoma (n = 86), Hodgkin's lymphoma (n = 34), myelodysplastic syndromes (n = 4), anaemia (n = 3), extramedullary plasmacytoma (n = 1), and Castleman’s disease (n = 1). Blood samples were conserved at 4 ºC until subsequent testing. All subjects were of ethnicHan origin. The study was authorized by the Ethics Committee of Shanxi Medical University and written informed consent were received from all subjects.
2.3. DNA extraction and genotyping study
Genomic DNA was extracted by using the DNA extractor kit mentioned above. The quality and concentration of extracted DNA were checked by electrophoresis. In order to detect genotyping of GSTA1, four SNPs (T-631G, T-567G, C-69T, G-52A) variation in the promoter region were studied by PCR-RFLP[17,20]. The gene was co-amplified by using the forward primer (GSTA1-F) and reverse primer (GSTA1-R). The sequence of the GSTA1-F was 5’-CCCTACATGGTATAGGTGAAAT-3’; GSTA1-R 5’-GTGCTAAGGACACATATTAGCA-3’[20].
The PCR reaction was carried out a 50-μL volume containing of 40 ng/μL genomic DNA, 10 × PCR Buffer, 15 mM Mg2+, 10 mM dNTPs, 5 U/μL Taq Plus DNA Polymerase, and 10 μM each primer. The DNA chains were denatured at 95 ºC for 5 min, followed by the PCR program as follows: 94 ºC for 30 s, 60 ºC for 30 s, 72 ºC for 50 s. The process was carried out for 35 cycles, and the final extension step was conducted 72 ºC for 5 min. Obtained PCR products were purified by using the purification kit mentioned above.
The sequencing of PCR products was performed in a 20-μL volume consisting of 10 ng/μL purified PCR products, 2.5 × BigDye, 5 × BigDye Seq Buffer and 3.2 pmol/μL Sequencing primers. The reaction conditionswere as follows: denaturing step at 96 ºC for 1 min, 96 ºC for 10 s, 50 ºC for 5 s, 60 ºC for 4 min, and repeated 25 cycles. The sequencer stopped the insulation at 4 ºC, finally.
2.4. Determination of GST enzyme activity
Blood samples were collected into silanized EDTA anticoagulant blood collection tube and centrifuged at 4000 × g for 15 min at 4 ºC within 2 h. The supernatant (200 μL) was taken out into a 1-mL Eppendorf tube and stored in a refrigerator at 4 ºC until analysis. The remaining blood samples were used for DNA extraction.
The GST enzyme catalyzes the binding of glutathione (GSH) to 1-chloro-2,4-dinitrobenzene (CDNB), andthe binding product has a specific light absorption at a wavelength of 340 nm. The enzyme activity of GST was calculated by measuring the rate of increase in absorbance at 340 nm[20]. Concisely, the reaction system was carried out in a 220-μL volume consisting of 20 μL blood supernatant, 180 μL of 1 mM CDNB, and 20 μL of 10 mM GSH. The GST enzyme activity of each sample was computed by testing the rate of change of absorbance for 5 min and repeated three times to calculate the average.
The precision and accuracy of the UV spectrophotometerand method were assessed by determining three samples representing low, middle, and high activities levels for three batches on three divided days. The intra-day relativestandard deviation (R.S.D) of the three activity levels were 4.12%, 6.32% and 8.19%. The inter-day relative standard deviation (R.S.D) of the three activity levels were 3.89%, 7.68% and 7.97%. All R.S.D values were less than 10%, and the method satisfied the analysis requirements.
2.5. Data analysis
DNA sequencing peak maps of four SNPs loci were analyzed by using Chromas (Version 2.6.6), and sequence alignment was performed by Seqman of DNAStar (Version 8.0). Statistical analysis of all data was using IBM SPSS Statistics (Version 22). The relationshipbetween GST genotype and enzyme activity was studiedby ANOVA analysis. The χ2-test was used to analyze the gene frequency distribution of different genders. The multiple comparisons of several samples were analyzed by the least significant difference test (LSD-test). All hypothesis tests were performed at a level of P<0.05.
3. Results
3.1. Genotyping analysis of GSTA1
Gene sequences of four SNPs (-631, -567, -69, -52) were tested, and Figure 1 shows the base sequences of different genotypes. In part II of Figure 1, two different color peaks were detected at -69 site: the wild type (C/C) produced only a blue peak, the homozygous mutant genotype (T/T) appeared only a red peak, and heterozygous genotype (C/T) was identified by red and blue peaks. Combining the base sequences of the other three loci, the genotypes of subjects were determined as shown in Table 1. The frequencies of genotypes GSTA1*A*A (wild genotype), GSTA1*A*B (heterozygous genotype),and GSTA1*B*B (homozygous mutant genotype) were75.3%, 22.9%, and 1.8%, respectively. Alleles GSTA1*Aand *B were distributed at 86.8% and 13.2%. The results showed no difference in genotype frequency distribution between males and females by Pearson χ2-test (P = 0.743),which was consistent with the Hardy-Weinberg equilibrium.
Figure 1. The sequences in the promoter region of four SNP sites. Parts I, II, III, and IV represent sites -52, -69, -567, and -631, respectively. Peaks of different colors represent different base types. Red, blue, green, and black peaks indicate T, C, A, and G bases, respectively.
Table 1. Genotypic frequency distribution of GSTA1 polymorphism in patients with hematological diseases.  
3.2. GST enzyme activity analysis
GST enzyme activity presented a positively skewed distribution as shown in Figure 2 (P<0.05). The GST enzyme activities were investigated in different genders and age groups. As illustrated in Table 3, the average GST enzyme activities of male and female were 5.20±0.13 and 5.17±0.12 nmol/min/mL, respectively, and the difference was not significant (P = 0.91). The multiple comparisons demonstrated that there was no difference in GST activity among different age groups with all P-values greater than 0.05.
Figure 2. The frequency distribution of GST enzyme activity in patients with hematological diseases. 
3.3. The relationship between GSTA1 polymorphism and GST activity
Results indicated that the average GST activity of the heterozygous mutant genotype (4.83±0.76 nmol/min/mL) was lower than of the wild genotype (5.34±1.26 nmol/min/mL,P = 0.018), but higher than that of the homozygous mutant genotype (3.32±0.07nmol/min/mL, P = 0.022) as shown in Table 2. 
Table 2. Comparison of GST activities of different GSTA1 genotypes (nmol/min/mL) in patients with hematological diseases.
Table 3. GST enzyme activity in different genders and age groups (nmol/min/mL) in patients with hematological diseases.
4. Discussion
GSTs exert detoxification metabolism by catalyzing the binding of electrophilic compounds to GSH, includingcarcinogens and certain chemotherapeutic drugs[13]. Busulfan combined with cyclophosphamide is commonly used in the pretreatment of hematopoietic stem cell transplantation[7,9,22]. The main pathway of busulfan biotransformation is to form a complex with glutathione (GSH) under the catalysis of GST enzyme in the liver, which is further decomposed into tetrahydrothiophene (THT) and EdAG, and EdAG reacts with GSH to form GSG[11]. Cyclophosphamide is first converted to the active metabolite 4-hydroxy-cyclophosphamide under the catalysis of the CYP2B6 enzyme, and the subsequent metabolism of phosphoramide mustard is mediated by the GST enzyme[23]. The activity of GST enzyme in patients with hematological diseases directly affects the therapeutic effect of chemotherapy drugs. There are many methods for measuring GST enzyme activity, but this study was more intuitive and effective by testing the activity of GST enzyme in patients’ serum for patients with hematological diseases[24]. The results showed that there was no significant difference in GST enzyme activity between different genders (P = 0.91) and age groups (all P>0.05). This might, to a certain extent, mean that the GST enzyme activity of different individuals is congenitally determined by the gene encoding the GST enzyme. Genetic polymorphism may be one of the causes of differences in individual GST enzyme activity. It has also been reported that the genetic polymorphism of GSTA1 affects the metabolism of drugs such as busulfan[17,25].
Previously, the GSTA1 gene polymorphism has been reported in healthy populations of different countries and races[2628]. However, this research focused on patients with hematological diseases and was closely linked to clinical practice. The base type of the four SNP sites (-631, -567, -69, and -52) in the promoter region was detected in our study to determine the genotype of GSTA1. Compared with the traditional methods for determining GST genotype based on the strip on the dextran gel, this research used a DNA Sequencer to directly test the base sequence, and the results were more intuitive and accurate. As revealed in Table 1, the frequency distribution of GSTA1 genotypewas not significantly different between male and female (P=0.743). This finding signified that the probability of GSTA1 mutation occurring in different genders was similar. In combination with the GST enzyme activity, the average GST activity of the heterozygous mutant genotype was lower than the wild genotype and higher than the homozygous mutant genotype, as shown in Table 2. Individuals with different GSTA1 genotypes had different GST enzyme activities, which could provide a basis for clinical precision treatment.
In summary, we studied the GSTA1 gene polymorphism and GST enzyme activity in patients with hematological diseases. The results demonstrated that the GSTA1 gene polymorphisms affect GST enzyme activity in patients. These findings might help us improve the individualized treatment of patients with hematological diseases in the future and promote the development of precision medicine for blood diseases.
Each author mentioned in the paper contributes to this research. This experiment was designed by Lifeng Zhang, and the specific experiment was carried out by Guangyi Yu. We would like to acknowledge Qingshan Yang forcollecting blood samples, Guoshun Zhang for providing genetic information, Yin Xie for providing instrument supports during this study. 
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喻光燚, 杨青山, 张国顺, 谢茵, 张丽锋*
山西医科大学药学院临床药学教研室, 山西 太原030000    
摘要: 谷胱甘肽硫转移酶(GSTs)是体内重要的药物代谢酶, 且存在基因多态性的现象。本研究调查了170例血液病患者的GST酶活性和GSTA1基因多态性, 并探讨了它们之间的关系。通过PCR-RFLP技术分析患者的GSTA1基因多态性, DNA测序仪确定启动子区域中的四个突变位点(-631, -567, -69-52)的碱基序列; 通过测量CDNBGSH之间催化反应的速率来计算患者的GST酶活性。结果显示, 男性和女性的平均GST活性分别为5.20±0.135.17±0.12 nmol/min/mL, 差异不显(P=0.91)。基因型GSTA1*A*AGSTA1*A*BGSTA1*B*B的频率分别为75.3%, 22.9%1.8%; 等位基因GSTA1*A*B频率为86.8%13.2%。通过卡方检验, 男性和女性之间的基因型频率分布没有显着差异(P = 0.743)。杂合突变基因型的平均GST活性(4.83±0.76 nmol/min/mL)低于野生基因型(5.34±1.26 nmol/min/mL, P = 0.018), 高于纯合突变基因(3.32±0.07 nmol/min/mL, P = 0.022)。这些发现可能有助于改善血液病患者的个体化治疗, 并促进血液病精准医学的发展。 
关键词: 基因多态性; 谷胱甘肽硫转移酶; 血液病; 酶活; PCR  
Received: 2019-03-18; Revised: 2019-04-27; Accepted: 2019-05-04.
*Corresponding author. Tel.: +86-13513641356, E-mail: zhlf7@163.com  

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