Acute lymphoblastic leukemia (ALL) is the most common type of childhood cancer . Genetic susceptibility has been studied as a risk factor since environmental exposure to xenobiotics induces the accumulation of somatic mutations . Drug detoxification enzymes are divided into phase I and phase II enzymes . Glutathione S-transferases (GST) are categorized as phase II drug detoxification enzymes that facilitate the conjugation of glutathione (GSH) using several endogenous and exogenous substances. They are indispensable for cellular protection against oxidative stresses . GST gene polymorphisms cause alterations in the enzyme activity that may modify the individual susceptibility to cancer .
The present case-control study aimed to investigate the potential association of GSTO1*A140D (rs4925) and GSTO2*N142D (rs156697) polymorphisms with the susceptibility to childhood ALL and their impact on ALL prognosis in the Egyptian population. Genotyping was performed by PCR amplification of the target gene followed by the RFLP technique. The study included 96 pediatric ALL patients and 99 age- and sex-matched control subjects.
The ALL population was a selection of all patients attending the pediatric hematology clinic, New Children Hospital, Cairo University (NCHCU) during the period from April 2015 to October 2017. Selected patients were Egyptian ALL patients of the pediatric age group. The patients’ ages ranged from 1.5 and 14 years with a mean of 6.95 ± 3.01 years and a median of 7 years. Among the 96 patients, 69 (72%) were males and 27 (28%) were females with a male to female ratio = 2.5:1. The diagnosis of ALL was based on WHO criteria. Sixty-one (63.5%) patients were classified as B-ALL while 35 (36.4%) patients as T-ALL. t(1;19) was found in 3 (3.1%) patients while all cases were negative for t(4;11), t(12;21), t(9;22) p190kD, and t(9;22) p210kD. The number of patients with the most common ALL translocations is very low. That might be attributed to the low sensitivity of the used technique. Out of the studied 96 ALL patients, 46 (48%) patients were de novo ALL cases.
In the present study, GSTO1*A140D (rs4925) genotyping revealed a statistically non-significant distribution among ALL patients and controls [CC (44.8% vs. 56.6%), CA (46.9% vs. 40.4%), and AA (8.3% vs. 3.0%), respectively] (0.106). GSTO1*A140D variant genotypes' (CA + AA) frequency did not differ significantly between ALL cases and controls (55.8% vs. 43.4%) [OR (95%CI): 1.643 (0.932–2898)] (0.085). However, comparing the de novo ALL cases to controls revealed a statistically significant different GSTO1*A140D genotype distribution [CC (37% vs. 56.6%), CA (47.8% vs. 40.4%), and AA: (15.2% vs. 3.0%), respectively] (0.008). GSTO1*A140D variant genotypes' (CA + AA) frequency was significantly higher in the de novo group than in controls (63% vs. 43.4%) [OR (95%CI): 2.222 (1.038–4.558)] (0.028).
The MAF of GSTO1*A140D-A was higher in ALL patients than in controls; however, the difference did not reach a statistically significant level (0.31 vs. 0.23) [OR (95%CI): 1.563 (0.997–2.488)] (0.051). However, in the de novo group, it was significantly higher than controls (0.39 vs. 0.23) [OR (95%CI): 2.124 (1.24–3.620)] (0.005) (Table 3).
GSTO2*N142D (rs156697) genotyping showed a statistically significant different distribution between ALL patients and controls [AA (26% vs. 36.3%), AG (62.5% vs. 61.6%), and GG: (11.4% vs. 2.0%), respectively] (0.017) and between de novo ALL patient group and controls [AA (37% vs. 36.3%), AG (45.7% vs. 61.6%), and GG: (17.3% vs. 2.0%), respectively] (0.002). The MAF of GSTO2*N142D-G was significantly higher in ALL patients than in controls (0.42 vs. 0.32) [OR (95%CI): 1.521 (1.006–2.298)] (0.046) (Table 3).
Based on these findings, we concluded that GSTO1*A140D (rs4925) and GSTO2*N142D (rs156697) polymorphisms are associated with an increased risk of de novo childhood ALL in the Egyptian population. Our results are in accordance with those of Pongstaporn et al. , who investigated the association of GSTO polymorphisms with the susceptibility to ALL in the pediatric Thai population. They reported that GSTO1*A140D (rs4925) was associated with the susceptibility to ALL [OR (95%CI): 2.24 (1.16–4.35); 0.009] while GSTO2*N142D (rs156697) was associated with the high-risk group of ALL patients [OR (95%CI): 5.52 (1.72–17.71); 0.004], thus, suggesting that GSTO polymorphisms may play a role in the occurrence of ALL.
However, our results were discordant with the findings of Rezazadeh et al.  who studied the association of GSTO polymorphisms with the susceptibility to pre-B-ALL in the pediatric Iranian population. They found that GSTO genotype distribution was similar among pre-B-ALL patients and controls. The difference between their results and ours may be attributed to the ethnic variation of the GSTO polymorphisms as variant genotypes are variably expressed among different ethnic populations as shown in previous studies [25,26,27].
The MAF of GSTO1*A140D-A in the Europeans (Italian and Spaniard) and European ancestors (Australian) ranged from 0.302 to 0.399 while in Asians (Chinese, Japanese and Mongolian), it ranged from 0.108 to 0.165. In the Africans (Bantu and Ovambo), it was much lower than those of the Europeans and Asians (ranging from 0.040 to 0.081). In contrast, the Turkish population followed a pattern similar to that of Africans. On the other hand, the MAF of GSTO2*N142D-G in Asians (ranging from 0.173 to 0.285) was lower than that of the Europeans (ranging from 0.310 to 0.341). In the Turkish population, it was similar to that of the Asians while in the Africans, it was higher than that of Asians, ranging from 0.583 to 0.855 [15, 26, 27, 31]. In our Egyptian control group, the MAF of GSTO1*A140D-A was 0.23 while that of GSTO2*N142D-G was 0.32. The latter is similar to that of the Europeans. In conclusion, for GSTO1*A140D (rs4925) and GSTO2*N142D (rs156697), the MAFs were different among different ethnic populations suggesting that these polymorphisms could be useful genetic markers but the association of cancer with a particular mutation in one population might be of limited value as a cancer biomarker into another population. Further detailed studies on different ethnic populations are needed to clarify the geographical distribution of these genetic markers and the possible association between GSTO polymorphism, ethnicity, and the occurrence of ALL. In addition, studying the physiological role of GSTO1*A140D (rs4925) and GSTO2*N142D (rs156697) will lead to a greater insight into the significance of GSTO polymorphism.
ALL patients were categorized into high-risk and standard-risk groups based on the age, WBCs, and immune-phenotypic analysis. The high-risk group is more than 10 years old or had WBCs > 50 × 109/L or T cell immune-phenotype while the standard-risk group is less than 10 years old or had WBCs < 50 × 109/L or had no T cell immune-phenotype . The present study revealed that the frequency of GSTO1*A140D (rs4925) and GSTO2*N142D (rs156697) variant genotypes in the high-risk ALL group was higher than the corresponding wild genotypes; moreover, the frequency of the combined GSTO1*A140D and GSTO2*N142D polymorphisms in the high-risk ALL group was higher than single polymorphisms [GSTO1*A140D or GSTO2*N142D] and wild genotypes, but these differences did not reach a statistical significance (Table 4). However, Pongstaporn et al.  found that GSTO2*N142D (rs4925) polymorphism was associated with the high-risk group of childhood ALL (0.004), indicating that GSTO2*N142D is associated with the susceptibility to high-risk ALL and can be used as an indicator of the disease severity.