Cytochrome P4502E1 ( CYP2E1 ) Genetic Polymorphisms in a Lebanese Population: Frequency Distribution and Association with Morbid Diseases

Abstract

Background: The drug-metabolizing enzyme CYP2E1 is of great interest in environmental medicine because of its involvement in the bioactivation of multiple procarcinogens. Aim: This study is aimed at determining the frequency of genetic polymorphisms of the three restriction fragment length polymorphisms: PstI and RsaI (CYP2E1*5B) and DraI (CYP2E1*6) in 216 cancer-free Lebanese individuals, as well as assessing potential association with morbid diseases in this specific population. Results: The frequency of C-T allele of CYP2E*5B was 0.7% and that of A CYP2E1*6 was 6.3%. All those who carried the CYP2E1*5B allele also carried the CYP2E1*6 allele. There was a significant decrease in coronary artery disease incidence in patients carrying a CYP2E1*6 genetic polymorphism (39.7% of the noncarriers vs. 13.6% of the carriers had coronary artery disease; p = 0.019); similar results were found with the haplotype analysis (p = 0.03) but not with CYP2E1*5B alone. Conclusion: This is the first study on the genetic polymorphism of CYP2E1 in a Lebanese population. These data will be useful for future assessment of the role of CYP2E1 polymorphisms in the cancer population in Lebanon. It is recommended that careful population selection be performed in designing case-control studies that evaluate the association between CYP2E1 and cancer incidence. The most important factors to be controlled for are sex, body mass index, environmental exposure, lifestyle habits, and possibly, history of coronary artery disease.

Introduction

Metabolism is a major route of elimination from the body for most drugs and chemical toxicants. Drug metabolism is mainly mediated by two classes of drug-metabolizing enzymes (DMEs): phase I oxidation enzymes, the most important of which is the cytochrome P450 (CYP) system, which is usually involved in the activation of toxicants; and phase II enzymes, which mostly detoxify the products to be excreted in the urine (Boobis and Davies, 1984; Nelson et al., 1993). Significant interindividual variability in the expression and activity of DMEs results in wide differences in the rate and extent of metabolism of drugs between individuals. This variability is controlled by both genetic and disease states, as well as environmental factors (Nebert and Gonzalez, 1987; Cheng and Morgan, 2001).

The CYP2E1 DME is of great interest in environmental medicine because of its involvement in the bioactivation of multiple procarcinogens, such as aniline, benzene, and vinyl chloride (Raucy et al., 1993; Tanaka et al., 2000). The CYP2E1 gene is present on chromosome 10, and its expression varies in various tissues, the highest being in the liver; it is under the control of liver-enriched transcription factors interacting with cis-acting regulatory sequences that are mainly present in the 5′-flanking region of the gene (Tanaka et al., 2000). CYP2E1 is inducible by lifestyle factors such as smoking and ethanol consumption, and several genetic polymorphisms have been found to be associated with variability in its expression and enzymatic activity. Thus, it has been found that CYP2E1 genetic polymorphisms have an impact on the incidence of hepatic and pancreatic diseases (Yang et al., 2001;Verlaan et al., 2004) as well as cancer, mainly of the head and neck, lung, and gastrointestinal tract (Bartsch et al., 2000; Danko and Chaschin, 2005). It appears that CYP2E1-induced toxicity is mediated by the activation of a state of oxidative stress (Caro and Cederbaum, 2004).

To date, more than 10 CYP2E1 genetic polymorphisms have been described (Tanaka, 1999; Ingelman-Sundberg et al., 2008), but only 3 variants have been extensively studied and shown to be linked to cancer risk (Danko and Chaschin, 2005): PstI and RsaI (CYP2E1*5B) and DraI (CYP2E1*6) (Hayashi et al., 1991; Watanabe et al., 1994). Although there is no convincing evidence on correlation between CYP2E1 gene polymorphisms and catalytic activity of the enzyme, there are obvious ethnic differences in frequency and activity, which point to an impact of genetic factors that are ultimately associated with cancer development (Lucas et al., 1995; Bolt et al., 2003; Meisel et al., 2003; Reisfeld and Yang, 2004). Therefore, one must be quite careful in extrapolating results from a specific ethnic group of subjects to another. One interesting and understudied group is the Middle Eastern population in general and the Lebanese people in particular.

To date, few genetic polymorphism frequency studies have been performed in the Middle East, mainly on Turkish, Egyptian, Saudi Arab, and Iranian people (Peyvandi et al., 2002; Hamdy et al., 2003a,b). No in vivo CYP metabolism studies have been ever performed on Lebanese people; however, in vitro studies have been published (Abdel-Razzak et al., 1993, 1994). As for the association of drug metabolism with cancer, a number of studies have been performed on the Turkish population and one on the Iranian population. The focus was on CYP2E1, CYP1A1, CYP2A6, and the glutathione S-transferase (GST) superfamily and the risk of several types of cancers, mainly breast, lung, prostate, ovarian, childhood leukemia, and esophageal cancer (Kocabas et al., 2002; Sepehr et al., 2004; Ulusoy et al., 2007a).

This study was prompted by the fact that the Lebanese population remains unexplored, and it aimed at determining the frequency of genetic polymorphisms for the DME CYP2E1 in a Lebanese population, as well as assessing potential association with morbid diseases in this specific population.

Materials and Methods

Subjects

This study included 231 Lebanese patients who were initially recruited at the American University of Beirut Medical Center (AUBMC) for a study on the pharmacogenetics of anticoagulants. All patients were on a maintenance dose of an anticoagulant drug for different indications. Baseline demographics, occupational exposure, lifestyle habits, and medical history were collected. Venous blood was drawn into ethylenediaminetetraacetic acid-containing tubes and stored at −80°C for later DNA isolation and genotyping. The study was approved by the University Institutional Review Board for human rights.

Genotyping

DNA was isolated using a DNA isolation kit from Qiagen (Germantown, MD). Genotyping for CYP2E1*5B (rs3813867 and rs2031920) and CYP2E1*6 (rs6413432) was performed by restriction fragment length polymorphism. The method and primer sequences used for the different polymorphisms were based on the publications of Wu et al. (1997, 1998).

Statistical analysis

Data were analyzed using the statistical package SPSS v.16 (SPSS, Chicago, IL). Percentage frequencies and, when applicable, mean with standard deviation were calculated for the baseline characteristics of the subjects. CYP2E1 genotypes and allele frequencies were computed and tested for Hardy-Weinberg equilibrium using an online calculator (Court, 2008). Assessment of the relationship between genotype groups and patients' baseline characteristics, including age, sex, body mass index (BMI), indication for anticoagulation, smoking, alcohol intake, dyslipidemia, diabetes mellitus, hypertension, coronary artery disease, and cerebrovascular disease, was carried out. We looked at CYP2E1*5B and CYP2E1*6 separately and then combined them as a haplotype. Carriage of at least one CYP2E1*5B or CYP2E1*6 genetic polymorphism was labeled as “any” category. t-test or analysis of variance was used for the age and BMI analysis, whereas chi-square or Fisher's exact test was used for the rest of the variables. A p-value of less than 0.05 was considered significant.

Results

Of 231 patients in this study, 15 participants were diagnosed with cancer; hence, only 216 were eligible for analysis, 100 men and 116 women with a mean age of 67 years. The frequencies of the CYP2E1 alleles were found to fit Hardy-Weinberg equilibrium and are shown in Table 1. For CYP2E1*5B, 213 individuals were c1/c1 (98.6%), 3 were c1/c2 (1.4%), and no one was c2/c2. As for CYP2E1*6, 194 were DD (89.8%), 21 were DC (9.7%), and 1 was CC (0.5%). The PstI and RsaI genotypes were in complete linkage disequilibrium, and all those who carried the CYP2E1*5B allele also carried the CYP2E1*6 allele. All 15 cancer patients were found to have “wild types” for the three polymorphisms. The list of cancers comprised four prostate, four breast, two urinary tract, two colorectal, one adrenal, one liposarcoma, and one pancreatic. Of the cancer patients, four were current smokers and four drank alcohol.

Table 1.

Minor Allele Frequencies of the CYP2E1*5B and CYP2E1*6 Genetic Polymorphisms in Cancer-Free Populations

		Minor allele frequency (%)
Population	N	CYP2E1*5B	CYP2E1*6	Reference
Persian Zoroastrian	106	6	—	Sepehr et al. (2004)
Italian	114	4.5	9.3	Ingelman-Sundberg et al. (1993)
German	373	2.9	6.5	Brockmoller et al. (1996)
French	172	2.3	6.4	Bouchardy et al. (2000)
Spanish	920	2.2	—	Agudo et al. (2006)
Turkish	206	1.9	8.3	Ulusoy et al. (2007b)
British	155	1.6	8.7	Yang et al. (2001)
Egyptian	247	0.9	—	Hamdy et al. (2002)
Lebanese	216	0.7	5.3	This study

Table 2 shows the baseline characteristics of all the study population as well as when stratified by CYP2E1 haplotype. There was a significant decrease in coronary artery disease incidence in patients carrying a CYP2E1*6 genetic polymorphism (p = 0.019); similar results were found with the haplotype analysis (p = 0.03) but not with CYP2E1*5B alone (p = 0.893).

Table 2.

Baseline Characteristics of the Lebanese Subjects by CYP2E1 Haplotype

		CYP2E1 polymorphisms ^a
Variable	All (N = 216)	No polymorphism (N = 194)	Any polymorphism^b (N = 22)	p-Value^c
Age (mean ± SD)	66.89 ± 13.516	67.58 ± 13.28	60.91 ± 14.369	0.947
BMI (mean ± SD)	30.18 ± 12.851	30.33 ± 13.285	8.48 ± 6.202	0.827
Sex
Female	46.3	44.8	59.1	0.260
Male	53.7	55.2	40.9
Smoking (Yes)	21.8	20.1	36.4	0.101
Alcohol intake (Yes)	24.5	25.3	18.2	0.605
Dyslipidemia (Yes)	43.5	43.8	40.9	0.825
Diabetes mellitus (Yes)	20.8	21.1	18.2	1
Hypertension (Yes)	58.8	59.8	50	0.494
Coronary artery disease (Yes)	37	39.7	13.6	0.019
Cerebrovascular disease (Yes)	20.4	21.6	9.1	0.262

CYP2E1*5B = −1293 G>C (rs3813867) associated with PstI restriction enzyme site and −1053 C>T (rs2031920) associated with RsaI restriction enzyme site; CYP2E1*6 = 7632 T>A (rs6413432) associated with DraI restriction enzyme site.

Presence of at least one CYP2E1*5B or CYP2E1*6 polymorphism. Values are percentages except for age and BMI.

p-Value was generated using t-test, exact sig. test, or chi-square test where applicable.

SD, standard deviation; BMI, body mass index.

Discussion

This is the first study describing the frequency of CYP2E1 genetic polymorphisms in a Lebanese population and assessing for potential association with comorbid diseases.

Frequency distribution

In the population under study, and as expected, CYP2E1*5B polymorphism was found to be less common than CYP2E1*6. It is interesting that the allele frequencies of both polymorphisms were the lowest when compared with representative countries of the relevant known migration routes to Lebanon, and CYP2E1*5B minor allele frequency was close to frequencies from Egyptians only. Lebanon is a small country located on the eastern coast of the Mediterranean Sea and has a population of 4.1 million (United Nations Development Program, 2008). Its central location within the ancient world of the Phoenicians, Egyptians, Persians, Greeks, Arabs, Crusaders, and Ottomans contributed to its diverse demography. Currently, its population is composed of different ethnic groups based on religious affiliations, mainly Christians, Sunnite and Shiite Muslims, and Druses. It has been reported that Muslim expansion from the Arabian Peninsula during the 7th century lead to the current Lebanese Muslims. The Crusaders in the 11th-13th centuries introduced European lineages to already existing Lebanese Christians who have inhabited the eastern coast of the Mediterranean area and the nearby Mount Lebanon since the early onset of Christianity (Zalloua et al., 2008; El-Sibai et al., 2009). Most of our research participants were Muslims who constitute the majority of the AUBMC neighborhood; therefore, our results may reflect the effect of the immigration of Muslims from the Arabian Peninsula to both Lebanon and Egypt.

Association with diseases

Although there is an abundant literature on the association of CYP2E1 polymorphisms with the incidence of cancer, few studies examined the relationship with other diseases such as diabetes and atherosclerosis. In most of the studies, the controls were described to be cancer free, but information about concomitant diseases has not been detailed (Wu et al., 1997, 1998, 2006; Eom et al., 2009), even when controls were recruited from a pool of hospital patients (Nishimoto et al., 2000; Boccia et al., 2008). The data in this study show that there is no relationship between most morbid diseases such as diabetes mellitus, hypertension, and hyperlipidemia and CYP2E1 polymorphisms. There was equally no relationship with BMI and sex, yet it is still recommended that case-control studies be matched for sex and BMI; for instance, Lucas et al. (1995) had suggested that there is a sex difference in the activity of CYP2E1 in the DraI polymorphism; further, obesity was shown to induce the activity of CYP2E1 (O'Shea et al., 1994). Kim and O'Shea (1995) genotyped 70 Caucasians for RsaI and DraI and showed that there was a difference in the clearance of chlorzoxazone according to sex, but this difference disappeared after adjusting for body weight. As for atherosclerosis, Salama et al. (2002) have shown that CYP2E1*5B genetic polymorphism may be associated with an increased risk of atherosclerosis among cigarette smokers, and Marusin et al. (2007) also showed similar results with the PstI c2 allele. Note that we had a relatively low CYP2E1*5B allele frequency, and hence, our study may have been underpowered to get significant results with this polymorphism. Nevertheless, the three patients who carried the CYP2E1*5B allele also carried the CYP2E1*6 allele. Interestingly, it has been suggested that DraI and other genes potentially affect serum high-density lipoprotein, which is a known protective factor against atherosclerosis (Grundy et al., 2004). We unfortunately do not have data on high-density lipoprotein levels in our study population. Finally, Yamada et al. (2002) have reported a positive association between CYP2E1*5B polymorphism with hypertension in patients who excessively consume alcohol. In this study, only a quarter of the subjects consume alcohol, and most were categorized as “occasional” drinkers. There was no significant correlation between the incidence of coronary artery disease and smoking (p = not statistically significant). It is important to note that our finding may be secondary to selection bias and that an appropriate case-control study is needed.

Association with cancer

The association between CYP2E1 genetic polymorphisms and the risk of cancer has been extensively studied in case-control studies. In this study, all patients who had cancer did not carry any of the studied polymorphisms and many of them were nonsmokers and nonalcohol drinkers.

Conclusion

Although the findings are based on small figures and the sample is not representative of the Lebanese population, this study is a starting point for determining the frequency of CYP2E1 genetic polymorphisms and exploring a potential associated risk with cancer development and comorbid diseases in the Lebanese population. It is recommended that careful population selection be performed in designing case-control studies that evaluate the association between CYP2E1 and cancer incidence. The most important factors to be controlled for are age, sex, BMI, environmental exposure, lifestyle habits, and possibly, history of coronary artery disease.

Footnotes

Acknowledgments

The authors are grateful to Dr. Joseph Simaan for reviewing this manuscript. This study was funded by the American University of Beirut Medical Practice Plan and University Research Board and by the Lebanese National Council for Scientific Research.

Disclosure Statement

No competing financial interests exist.

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