CYP 2 E 1 * 5 B , CYP 2 E 1 * 6 , CYP 2 E 1 * 7 B , CYP 2 E 1 * 2 , and CYP 2 E 1 * 3 Allele Frequencies in Iranian Populations

The only member of human cytochrome P450 E subfamily is CYP2E1 (Zhuge et al., 2003), located in the 10q24.3-qter region of chromosome 10. It contains 9 exons, 8 introns, and a typical TATA box and it spans 11,413 base pairs of genomic DNA (Umeno, 1988). CYP2E1 plays a key role in the metabolic activation of many low molecular weight carcinogens (e.g., benzene, N-nitrosamines, carbon tetrachloride chloroform and vinyl chloride) and producing reactive oxygen species (e.g., superoxide anion radical, hydrogen peroxide), which can affect target tissue and ultimately lead to carcinogenesis (Hou et al., 2007; Guo et al., 2010; Feng et al., 2012). CYP2E1 has several polymorphisms. Studies reported that its functional polymorphisms are associated with increased and decreased susceptibility to many cancer types, including esophageal cancer, lung cancer, nasopharyngeal carcinoma, and colorectal cancer (Cai et al., 2005; Sangrajrang et al., 2006). CYP2E1*5B (rs2031920) and CYP2E1*6 (rs6413432)


Introduction
The only member of human Cytochrome P450 E subfamily is CYP2E1 (Zhuge et al., 2003). Human CYP2E1 gene is located in 10q24.3-qter region of chromosome 10. It contains 9 exons, 8 introns, and a typical TATA box and it spans 11,413 base pairs of genomic DNA (Umeno, 1988). CYP2E1 plays a key role in the metabolic activation of many low molecular weight carcinogens (e.g., benzene, N-nitrosamines, carbon tetrachloride chloroform and vinyl chloride) and producing reactive oxygen species (e.g., superoxide anion radical, hydrogen peroxide), which can affect target tissue and ultimately lead to carcinogenesis (Hou et al., 2007;Guo et al., 2010;Feng et al., 2012).
CYP2E1 has several polymorphisms. Studies reported that its functional polymorphisms are associated with increased and decreased susceptibility to many cancer 2 named DD, DC, and CC, respectively. CYP2E1*6 doesn't affect gene transcription, but it is likely to affect CYP2E1 catalytic activity (Uematsu et al., 1991). CYP2E1*7B (rs6413420; G-71T) was also found in the promoter region of the gene. CYP2E1*7B affects the transcription of the gene and causes 1.8-fold increase in the transcriptional activity of CYP2E1 (Fairbrother et al., 1998). Therefore, it is expected that CYP2E1*6 and CYP2E1*7B influence cancer risk. CYP2E1*2 (rs72559710) and CYP2E1*3 (rs55897648) were identified in exon 2 and exon 8 of the gene, respectively. CYP2E1*2 that causes an R76H amino acid exchange, lowers enzyme synthesis and catalytic activity. The additional polymorphism, CYP2E1*3, was detected, causing the substitution of Valine to Isoleucine at position 389, but without any effect on enzyme synthesis and catalytic activity (Hu et al., 1997).
Inter-individual differences in the expression level, which can result in tumor development, have been connected with its polymorphisms. The allele frequencies of these polymorphisms differ remarkably among different human populations (Bolt et al., 2003). These polymorphisms are well characterized in different populations, but little is known about the Iranian ethnic group. This study could provide valuable data for association studies between these polymorphisms and cancer susceptibility. Therefore, the genotype and allele frequencies of CYP2E1 single nucleotide polymorphisms, including *5B, *6, *7B, *2, and *3 were presented, estimated and analyzed in this study. The present study was aimed to provide basic information about the allele and genotype distribution of CYP2E1 polymorphisms.

Study population
Samples from 200 genetically unrelated, healthy individuals blood donors were taken which consisted of 100 males between the ages of 1 and 80 (mean age 41.9±22.3) and 100 females between the ages of 3 and 84 (mean age 40.87±19.97) were examined in this study. Blood samples were collected at the University hospital of Jundishapur and an accredited medical diagnostic laboratory in Ahvaz city (southwest of Iran). The population study was composed of two ethnic subgroups, including non-Arabs (66%) and Arabs (34%). Since these two groups of individuals were seen homogenous in relation to the allelic and genotypic frequencies and both were in Hardy-Weinberg equilibrium, they were considered as one group.
This investigation was approved by the Ethics Committee of Jundishapur University of Medical Sciences.

Genotype analysis
Genomic DNA was extracted from 100 μl of whole blood using the Diatom DNA Kit (IsoGene, Russia) according to the manufacturer's instructions. The extracted DNA was visualized on 1% agarose gel and stored at −20°C until genotyping was performed. Two different methods were used to detect five single nucleotide polymorphisms (SNPs) of CYP2E1, CYP2E1*5B, CYP2E1*6, and CYP2E1*7B. Polymorphisms were determined by polymerase chain reaction (PCR) based on the restriction fragment length polymorphism (RFLP) method with designed primer pairs. The PCR products of CYP2E1*5B, CYP2E1*6, and CYP2E1*7B were digested with the restriction enzymes PstI (Vivantis, Malaysia), DraI (Roche, Germany), and DdeI (Fermentas, UK), respectively. PCR products and restriction fragments were visualized by electrophoresis in 1.5% and 2.5% agarose gels, respectively. The information about sequence of primer pairs, amplification conditions, size of PCR products and digested products with restriction enzymes is listed in Table 1.
Tetra-ARMS PCR (amplification refractory mutation system) method was applied for genotyping of CYP2E1*2 and CYP2E1*3 because they don't have a restriction site by which alleles of CYP2E1 can be distinguished from one another. The fragment of the CYP2E1 gene that contains either *2 or *3 polymorphism was amplified by the two outer primers and the inner primers which amplified the two allelic states (Ye et al., 2001). Tetra-ARMS PCR products were visualized by electrophoresis in 2.5% agarose gel for CYP2E1*2 and CYP2E1*3 regions. The details about PCR condition and primer sequences are listed in Table 2. Several samples were randomly selected for direct sequencing of amplified products to validate the results of genotyping by PCR/RFLP and Tetra-ARMS  (Figures 1 and 2).

Statistical analysis
The statistical analysis by χ 2 -test was done to determine if the genotype frequencies of every polymorphism fit the Hardy-Weinberg equilibrium. Also, the estimated genotype frequencies were compared between the two genders and between Arabs and non-Arabs populations. Finally, the results of this study were compared with other populations by the same test. Differences with P-values<0.05 were considered statistically significant.

Discussion
Iran is a country which has a population with different ethnic identities and different languages. CYP2E1 polymorphisms shows inter-ethnic and interracial differences, significant contribution to individual differences in susceptibility to cancer development. Estimation probabilities of cancer development both for individuals and ethnic groups can be performed by using SNPs as genetic markers (Danko and Chaschin, 2005). Therefore, the identification of polymorphisms in different populations such as Iranians could be useful for assessing genetic cancer susceptibility. The present results are the first to evaluate genotype and allele frequencies of CYP2E1 polymorphisms. The next step after the initial data is to study the association of these polymorphisms with cancer. If such a conclusion is true, particularly among industrial workers, this is recommended because it is possible that the pattern of polymorphism is different. Therefore, whether a polymorphism is a crucial or has no significant differences, a specific test will be performed specifically among the high risk groups.
CYP2E1 polymorphisms could also affect susceptibility to adverse drug reactions (ADRs) (Costa et al., 2012). Accordingly, this study provides valuable information not only for further investigation of association between CYP2E1 polymorphisms and susceptibility to several types of cancer but also for the study of adverse drug reactions.
Since SNPs of autosomal chromosomes can be changed by effects of many factors in the evolutionary history, the investigation of single nucleotide polymorphisms provide valuable data regarding relations between populations (Kayaalti and Soylemezoglu, 2010). Ahvaz city lies in the southwest of Iran between Iraqi border and the Zagros Mountains. Centuries ago, Arabs migrated from neighboring countries to Iran. In the current study, the genotype distributions were compared between Arabs and non-Arabs. CYP2E1 polymorphisms had similar allele frequencies for two ethnic subgroups of the study population. Therefore, two subgroups might have the same cancer susceptibility.
CYP2E1 genotype distribution was also compared between Iranian and other populations. The genotype distribution of CYP2E1*5B was similar to Europeans such as German (P-value=0.11) , British (P-value=0.88) , French (P-value=0.29) , and Poland Caucasians (P-value=0.08) , but it was different from Italian (P-value=0.03) . There were no significant differences between this study and studies on American (P-value=0.6)  and Turkish (P-value=0.54)  populations. But, the results of this population differed significantly from those of Chilean ), Mexican Mestizos, Mexican Huichols (Gordillo-Bastidas et al. 2010, and Brazilian (Marques et al., 2006( (P-value<0.0001). The genotype frequencies of CYP2E1*5B showed significant differences between Iranian and other Asians, including Japanese , Thai (Sangrajrang et al., 2006), and Northeastern Chinese (Guo et al., 2012) (P-value<0.0001). On the other hand, the results were similar to Indian (P-value=0.31)   (Table 4).
There was a significant correlation between c2/c2 genotype and head and neck cancer in Asians, but not in Caucasians (Tang et al., 2010). The other study found that c2/c2 homozygote genotype was associated with the increased risk of colorectal cancer in Caucasians (Zhou et al., 2010). Therefore, we expect the same association study results in Iranian population because CYP2E1*5B genotype distribution was similar to Caucasians.
A meta-analysis showed a protective effect of DraI C/D and C/C genotypes for lung cancer in the Asian population (Wang et al., 2010). On the other hand, homozygote genotype of DraI polymorphism was associated with head and neck cancer in Asians (Tang et al., 2010). Thus, this polymorphism may have an impact on cancer susceptibility. Accordingly, the risk of cancer development in Iranian population might be similar to other Asian countries.
The genotype frequencies of CYP2E1*7B were compared between this study and studies done on other populations. The results of Chi-square test showed that genotype distribution of CYP2E1*7B was significantly different from European populations, including German , British , and Swedish (Ernstgard et al., 2004) (P-value<0.0001). Also, the results were significantly different from those of Turkish population (P-value<0.0001) Kayaalti and Soylemezoglu, 2010) (Table 6).
In the case of CYP2E1*7B, the data was limited. Accordingly, the results were only compared with Europeans and the comparison showed significant differences.
For CYP2E1*2 polymorphism, all subjects carried the common homozygote genotype (GG) and the GA or AA genotypes were not detected. CYP2E1*2 and CYP2E1*3 polymorphisms occur at very low frequencies in both Caucasians and Orientals (Ingelman-Sundberg, 2001). Thus, Iranian population is expected to have the same susceptibility to cancer in relation to xenobiotic effects. Similarly, Iranians exhibit low frequency of the CYP2E1*2 (exon 2) and *3 (exon 8) alleles. This results emphasizes that the coding sequence of CYP2E1 gene is highly conserved and CYP2E1 isoenzyme is an important physiologically (Hu et al., 1997).
In conclusion, ethnic and geographic differences may explain discrepancies in the prevalence of CYP2E1 polymorphisms. Genotype distribution studies could provide valuable information to help further investigations of association between polymorphisms and several types of cancer and other diseases. Studies in larger groups are recommended to confirm our results. A larger data base may allow for a more precise estimate of the population frequency for these polymorphisms among normal samples.