The EPHX 1 rs 1051740 Polymorphism Is Associated with Childhood Acute Lymphoblastic Leukemia in a Korean Population

Microsomal epoxide hydrolase (EPHX1) is involved in the activation and detoxification of exogenous chemicals. Genetic polymorphisms in EPHX1 have been associated with the development of leukemia. To investigate an association between single-nucleotide polymorphisms (SNPs) of EPHX1 and risk factors for childhood acute lymphoblastic leukemia (ALL) in Korean children, we genotyped two SNPs, Tyr113His (rs1051740) and His139Arg (rs2234922) in 185 childhood ALL cases and 536 healthy controls. Genotyping for these two SNPs was performed by simplex pyrosequencing assay and high-resolution melt analysis, respectively. We found that the Tyr113His genotype was associated with a decreased risk of childhood ALL (odds ratio, OR = 0.64, 95% confidence interval, CI = 0.43 0.93; p = 0.02). There was no association between His139Arg and the combined genotypes and the risk of childhood ALL. These results suggest that the EPHX1 113TyrHis genotype may protect against leukemogenesis in childhood.


Introduction
Childhood acute lymphoblastic leukemia (ALL) is the most common type of cancer in children, representing nearly one-third of all pediatric cancers [1].Childhood ALL is caused by multiple factors, including genetic disorders, exposure to radiation, and immune dysfunction [2] [3].Environmental factors, such as pesticides, household chemical exposure, and parental smoking and alcohol use, have also been associated with childhood cancers, including childhood ALL [3]- [5].These factors are detoxified by drug-metabolizing enzymes, through phase I and II metabolism.
Many studies have suggested that genetic polymorphisms in the drug-metabolizing enzymes play a role in susceptibility to childhood ALL [6]- [9].Recent studies have suggested that the interaction between these genes and environmental factors may also affect the susceptibility to leukemia [10]- [12].Microsomal epoxide hydrolase (EPHX1), along with CYP, another family of xenobiotic-metabolizing enzymes, plays an important role in both the detoxification and activation of procarcinogens [13] [14] and is considered to be a protective enzyme [15].Two polymorphisms in EPHX1, Tyr113 His in exon 3 (rs1051740) and His139Arg in exon 4 (rs2234922), have been identified to affect enzyme activity.The 113His allele in exon 3 decreases enzyme activity by 40% and is called the "slow" allele, whereas the 139Arg allele in exon 4 increases enzyme activity in vitro by 25% and is referred to as the "fast" allele [16].
Previous studies have reported inconsistent findings regarding an association between EPHX1 polymorphisms and cancer risk [17]- [20].To our knowledge, only three reported studies have analyzed these polymorphisms in childhood ALL [7] [11] [21], with the results being variable.Additionally, no research on an Asian population has been published.Thus, the aim of this study was to investigate the association of these two polymorphisms and their haplotypes in EPHX1 with the risk of childhood ALL in Korean children.

Study Population
All childhood ALL less than 18 years of age were consecutively enrolled at Chonnam National University Hwasun Hospital between October 2001 and August 2012.The 185 ALL cases consisted of 105 males and 80 females, with a mean age of 6.2 ± 4.2 years (range, 3 months to 17 years).
The control group consisted of 536 unrelated individuals (267 males, 269 females) with a mean age of 7.5 ± 4.0 years (range, 1 month to 17 years) with no previous history of malignancy.The controls were selected randomly from local residents during the same time period.
At the time of peripheral blood collection, the parents of all cases and control subjects provided informed consent to participate in this study.Childhood ALL cases for this study were provided by the Chonnam National University Hwasun Hospital National Biobank of Korea, a member of the National Biobank of Korea, supported by the Ministry of Health, Welfare, and Family Affairs.This study was approved by the Chonnam National University Hwasun Hospital Institutional Review Board, Hwasun, Korea.

Statistical Analysis
The expected frequency of control genotypes was evaluated using the Hardy-Weinberg equilibrium test.Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using logistic regression models, adjusting for age and gender, to estimate the association between each genotype and childhood ALL.Subjects with wild-type genotypes were considered to represent the baseline risk.Statistical analyses were performed using the SPSS software version 21.0 (SPSS, Inc., an IBM Company, Chicago, IL, USA).

Results
The distributions of polymorphisms in EPHX1 among the childhood ALL cases and the controls are shown in Table 1.Two major polymorphisms, Tyr113His in exon 3 and His139Arg in exon 4, were analyzed.The control genotype frequencies were within Hardy-Weinberg equilibrium and did not differ significantly between males and females (data not shown).EPHX1 113Tyr/His and the combined Tyr/His and His/His genotypes were asso-ciated with a decreased risk for childhood ALL (OR TC = 0.64, p = 0.02; OR TC+CC = 0.68, p = 0.03).No association was found between the His139Arg polymorphism and the risk of childhood ALL.The distribution of the combined genotypes of EPHX1 Tyr113His and His139Arg polymorphisms with the imputed phenotypes [16] between the cases and controls are shown in Table 2. Eight genotype combinations, the exceptions being 113 His/His and 139Arg/Arg, are listed.No association was found between the combined genotypes with imputed phenotypes and the risk of childhood ALL compared with normal EPHX1 enzyme activity.

Discussion
In the present study, we investigated whether two polymorphisms in EPHX1 genes were associated with a risk of developing childhood ALL in a Korean population.The Tyr113His genotype was associated with a decreased risk of childhood ALL.There was no association between the His139Arg polymorphism and the combined genotypes and the risk of childhood ALL.
EPHX1 is an important metabolizing enzyme that is involved in detoxification by catalyzing the hydrolysis of arene and aliphatic epoxides from PAHs and activation by generating carcinogenic compounds from epoxides [13] [14].Two major polymorphisms, the 113 His allele in exon 3 (rs1051740) and the 139 Arg allele in exon 4  (rs2234922), are known to reduce and increase enzyme activity, respectively [16].To date, these two polymorphisms were considered to be associated with cancer risk.However, the many studies that have investigated this have produced inconsistent findings.The Tyr113His polymorphism has been associated with, in most studies, an increased risk for various cancers, such as lung [20] [24] [25], ovarian [26], esophageal [27], breast [28], and bladder [29] cancer and, in a few studies, a decreased risk of childhood ALL [7] and lung cancer [30].In contrast, the His139Arg polymorphism has no association with many [11] [27] [29] [31].These results suggest that the decreased enzyme activity of the His allele in the Tyr113His polymorphism affects the risk of cancer to a greater degree than the increased enzyme activity due to the Tyr allele in the Tyr113His polymorphism.
In the present study, the 113Tyr/His and the combined Tyr/His and His/His genotypes were associated with a decreased risk of childhood ALL, while the His139Arg polymorphism had no effect.Three studies of childhood ALL risk have also reported no association with His139Arg, while Tyr113His showed a decreased risk [7] in Brazil, an increase [21] in Turkey, and no association [11] in France.Our results are similar to those of Silveira Vda et al. [7].
The conflicting results for the associations between the polymorphisms and the risk for ALL may be due to differing genotype frequencies, ethnicities, and environmental factors.In the four studies, including ours, the frequencies of the 113 His/His genotype in the control groups ranged from 2% to 18.4%.However, the frequencies of the 139 Arg/Arg genotype in the control groups were less diverse, ranging from 2.1% to 4.4%.
Correlations between EPHX1 enzymatic activity and different combinations of the two variants were reported by Hasset et al. [16], and activity was suggested to be a cancer risk factor.Tumer et al. reported that only the very low activity of the 113 His/His and 139 His/His combinations significantly increased the risk of childhood ALL [21].However, in our study, no associations were found between these combinations and childhood ALL.
Our study had several limitations.First, we did not evaluate gene-environment interactions.Zhou et al. reported that lung cancer risk decreased as cumulative smoking increased and suggested that smoking plays an important role in the association of the His/His genotype with lung cancer [32].Several studies have suggested that the interaction between drug-metabolizing genes and parental smoking and alcohol might affect childhood leukemia [11] [12] [33].Thus, additional studies with stratification according to chemical exposure and with larger sample sizes in local areas should be conducted to further explore the associations between EPHX1 polymorphisms and childhood leukemogenesis.Second, it is possible that the null association between His139Arg and childhood ALL may have been due to the small sample size.

Conclusion
In conclusion, the EPHX1 113 Arg/His genotype was associated with a decreased risk of childhood ALL in Korean children.These results suggest that Arg113His polymorphism may have some protective function against leukemogenesis in childhood.

Table 1 .
Association between polymorphisms in EPHX1 and risk of childhood ALL.

Table 2 .
Association between the combined EPHX1 Tyr113His and His139Arg polymorphism genotypes and risk of childhood ALL.