Polymorphism in cytochrome P450 2E1 and interaction with other genetic risk factors and susceptibility to alcoholic liver cirrhosis

Mutation Research 664 (2009) 55–63

Contents lists available at ScienceDirect

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: www.elsevier.com/locate/mutres

Polymorphism in cytochrome P450 2E1 and interaction with other genetic risk factors and susceptibility to alcoholic liver cirrhosis Anwar Jamal Khan a , Munindra Ruwali a , Gourdas Choudhuri b , Neeraj Mathur a , Qayyum Husain c , Devendra Parmar a,∗ a b c

Developmental Toxicology Division, Indian Institute of Toxicology Research (formerly ITRC), CSIR, P.O. Box 80, M.G. Marg, Lucknow 226 001, UP, India Department of Gastroenterology, Sanjay Gandhi Post Graduate Institute of Medical Science, Raebareli Road, Lucknow 226014, UP, India Department of Biochemistry, Faculty of Life Science, Aligarh Muslim University, Aligarh 202002, UP, India

a r t i c l e

i n f o

Article history: Received 3 June 2008 Received in revised form 12 February 2009 Accepted 13 February 2009 Available online 28 February 2009 Keywords: Cytochrome P450 2E1 Polymorphism Cirrhosis Alcoholics Haplotype

a b s t r a c t The association of polymorphism in cytochrome P450 2E1 (CYP2E1), the major microsomal ethanol metabolizing enzyme and its interaction with genes, involved in detoxification of reactive oxygen species, such as glutathione-S-transferases M1 (GSTM1) and alcohol intake, gamma-aminobutyric acid receptor ␥2 (GABRG2) was studied with the risk to alcoholic cirrhosis in a case–control study. A total of 160 alcoholic cirrhotic and 125 non-alcoholic cirrhotic cases, visiting the OPD facility of Gastroenterology Department of Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGI), Lucknow, India and 250 non-alcoholic and 100 alcoholic controls having no evidence of liver disease were included in the study. PCR-based RFLP methodology was followed for genotyping studies. Our data revealed that the variant genotypes of CYP2E1*5B exhibited significant association with the alcoholic liver cirrhosis when compared to non-alcoholic controls (OR: 4.3; 95%CI: 1.5–12.4; p: 0.003) or non-alcoholic cirrhosis patients (OR: 5.4; 95%CI: 1.2–24.5; p: 0.01) or alcoholic controls (OR: 4.3; 95%CI: 0.95–19.62; p: 0.04). Haplotype approach revealed that haplotype T–A–T was found to be associated with more than 5-fold increase in risk for alcoholic cirrhosis. Likewise, combination of variant genotype of CYP2E1*5B with null genotype of GSTM1, a phase II detoxification enzyme, resulted in several fold increase in risk in alcoholic cirrhotic patients when compared with non-alcoholic controls or non-alcoholic cirrhotic patients. Further, the combination of variant genotype of CYP2E1*5B with GABRG2, significantly increased the risk upto 6.5fold in alcoholic cirrhotic patients when compared with non-alcoholic controls thereby suggesting the role of gene–gene interaction in alcoholic cirrhosis. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Cirrhosis, a chronic liver disease, is mainly caused by heavy alcohol consumption or hepatitis B and C viral infections [1,2]. While nearly all heavy alcohol drinkers reveal fatty liver, only 10–20% of alcoholics develop cirrhosis [3,4]. Chronic alcohol intake is known to be associated with increase in the levels of cytochrome P450 2E1 (CYP2E1), a major component of the microsomal ethanol oxidizing system in the liver [5]. The activity of CYP2E1 can increase up to 20-fold following continuous alcohol consumption [6]. The acetaldehyde and free radicals so generated are known to react with cell membranes and this results in formation of protein adducts and an increase in collagen synthesis which ultimately leads to alcoholic liver diseases including cirrhosis [7–9].

∗ Corresponding author. Tel.: +91 522 2627586x261; fax: +91 522 2628227/2621547. E-mail address: parmar [email protected] (D. Parmar). 0027-5107/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2009.02.009

Genetic polymorphism has been reported in CYP2E1 gene, which may account for variation in CYP2E1 activity [10,11]. CYP2E1*5B (RsaI), located at 5 regulatory region (−1053C > T) is associated with higher transcription and increased enzyme activity [12–14]. The other polymorphism, known as DraI polymorphism (CYP2E1*6), located at intron 6 (7632T > A), is not known to be associated with increased expression or enzyme activity [15]. MspI polymorphism has also been identified in CYP2E1 and is located at intron 6 (6827A > G). Polymorphism is also known for glutathione-S-transferases (GSTs), the phase II enzymes, involved in detoxification of reactive oxygen species (ROS) including free radicals generated as by-products of ethanol metabolism. Homozygous gene deletion has been reported in GSTM1, a major GST isoenzyme [16,17]. Lack of GSTM1 activity due to gene deletion, may potentially increase susceptibility to alcoholic liver disease [18,19]. Similarly, polymorphism has been reported in gamma-aminobutyric acid receptor ␥2 (GABRG2) which has been shown to be associated with alcoholism [20–22]. SNP at intron 8 (84435G > A) results in G to A substitution and variant form (AA + GA) of GABRG2 has reduced

56

A.J. Khan et al. / Mutation Research 664 (2009) 55–63

binding affinity for ethanol resulting in consumption of higher amounts of ethanol to produce the same inhibitory effect as produced by wild type subunit [20–22]. Ethnic variations have been reported in the distribution of CYP2E1, GSTM1 and GABRG2 genotypes [16,23–26]. Though polymorphism in CYP2E1, GSTM1 and GABRG2 has been identified in the Caucasian and Oriental population [10,11,16,25,26] only CYP2E1*5B and CYP2E1*6 polymorphism has been reported in the Oriental population [14,27,28]. CYP2E1*5B (RsaI) has been found to be associated with alcoholic liver disease in Oriental population [14,27,28]. However, this association was not found to be consistent in Caucasians [11,29–31]. No significant association of CYP2E1*6 (DraI) polymorphism has been observed with alcoholic liver disease in majority of the studies [8,10,11,15,32]. Similarly, no association of MspI polymorphism with alcoholic liver cirrhosis has been observed in any population [10]. Likewise, GSTM1 null genotype has been found to be associated with alcoholic liver diseases in some studies [18,19,33,34] though there are several reports, which do not show association of GSTM1 null genotype with the alcoholic liver disease [35,36]. However, not much information is available on association of polymorphic variants of CYP2E1, GABRG2 and GSTM1 with susceptibility to alcoholic cirrhosis in Indian population. The present case–control study was therefore initiated to investigate the association of polymorphism in CYP2E1 gene with alcoholic liver cirrhosis in North Indian population. Attempts were also made to investigate the interaction of CYP2E1 with other genetic risk factors such as GSTM1 and GABRG2 in enhancing the susceptibility to alcoholic liver cirrhosis. Our data showed that the variant genotypes of CYP2E1*5B, GABRG2 and GSTM1 (null) genotype are associated with alcoholic liver cirrhosis. Cases carrying combination of variant genotype of CYP2E1*5B with null genotype of GSTM1 or variant genotype of GABRG2 resulted in further increased risk in alcoholic cirrhotic patients when compared with non-alcoholic controls. 2. Materials and methods A case–control study was designed to investigate the association of functionally important polymorphisms in CYP2E1 (CYP2E1*5B, CYP2E1*6 and MspI) with alcoholic liver cirrhosis. Patients of alcoholic liver cirrhosis (n = 160) and non-alcoholic liver cirrhosis (n = 125) visiting the OPD facility of Gastroenterology, Department of Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow, India were included in the study. Patients with alcoholic and non-alcoholic liver cirrhosis were diagnosed on the basis of their liver biopsy and hepatitis B surface antigen and antibodies against hepatitis C virus. Control group consisted of nonalcoholic (n = 250) and alcoholic (n = 100) healthy men having no evidence of liver disease as judged by physical examination and normal liver function test. All the cases and controls included in the study belonged to the same ethnic group of North India and the average ages of non-alcoholic controls, non-alcoholic cirrhosis, alcoholic controls and alcoholic cirrhosis patients were 42 ± 13.6, 47 ± 14.2, 49 ± 11.4 and 52 ± 12.5, respectively. The protocol for research work was approved by the Human Ethics Committee of Indian Institute of Toxicology Research (IITR), Lucknow and it conforms to the provisions of the declaration of Helsinki in 1995. Informed consent was obtained from the study subjects for inclusion in the study. Before the collection of blood samples it was ensured that the subject anonymity was preserved. The controls and patients were asked to fill up the detailed questionnaire regarding their family history, medical history, life style habits, etc. The questionnaire also included other details such as frequency of alcohol intake. Subjects who consumed less than 10 g/day of alcohol were classified as non-alcoholics. Subjects who consumed more than 80 g/day of alcohol for more than 10 years were considered as alcoholics [10,12,27].

genotyping MspI polymorphism of CYP2E1 the method of Savolainen et al. [10] was followed. In brief, the reaction mixture in 50 ␮l contained 1× buffer, 200 ␮M of each dNTPs, 200 nM of each primer, 1.5 U of Taq polymerase (MBI Fermentas, Germany), 100 ng of genomic DNA and sterile MilliQ water. Amplification was performed on Gene Amp PCR system 9700 of Applied Biosystem using the following protocol: 94 ◦ C for 5 min for initial denaturation followed by 35 cycles of 94 ◦ C for 30 s, annealing at 60–64 ◦ C for 30 s to 1 min (64 ◦ C for 30 s CYP2E1*5B; 63 ◦ C for 30 s CYP2E1*6; 60 ◦ C for 1 min MspI polymorphism), extension at 72 ◦ C for 1 min for CYP2E1*5B and CYP2E1*6 or 1.5 min for MspI polymorphism, and final elongation step of 72 ◦ C for 10 min. PCR reaction resulted in a 410 bp product for CYP2E1*5B, 375 bp for CYP2E1*6 and 480 bp for MspI polymorphism. PCR products (15 ␮l) were digested with 10 U of RsaI, DraI and MspI restriction enzyme (MBI Fermentas, Germany) to identify the presence of polymorphic sites in CYP2E1 gene. Digestion of 410 bp PCR product of CYP2E1*5B into two fragments of 290 and 120 bp indicates the presence of wild type genotype of CYP2E1*5B. The presence of fragments of three sizes (410, 290 and 120 bp) was indicative of the heterozygous genotype while the undigested 410 bp PCR fragment was indicative of homozygous mutant genotype CYP2E1*5B. Similarly, digestion of 375 bp PCR product of CYP2E1*6 into two fragments of 210 and 165 bp indicates the presence of wild type genotype of CYP2E1*6. The presence of fragments of three sizes (375, 210 and 165 bp) was indicative of the heterozygous genotype while the undigested 375 bp PCR fragment was indicative of homozygous mutant genotype of CYP2E1*6. Likewise, undigested 480 bp PCR fragment of MspI polymorphism was indicative of wild type genotype. The presence of fragments of three sizes (480, 301 and 179 bp) was indicative of the heterozygous genotype while the digestion of 480 bp PCR products into two fragments of 301 and 179 bp indicates the presence of homozygous mutant genotype of MspI polymorphism. 2.3. Detection of GSTM1 polymorphism by multiplex PCR method GSTM1 genotypes were determined by the method of Arand et al. [38]. In brief, the PCR reaction was carried out with 1× buffer, 200 ␮M of each nucleotide, 200 nM of each of the primers of GSTM1, GSTT1 and albumin taken as internal control, 1.5 U of Taq polymerase (MBI Fermantas, Germany), 100 ng of genomic DNA and sterile MilliQ water in a final volume of 25 ␮l. Amplification was performed at 94 ◦ C for 5 min for initial denaturation followed by 35 cycles of 94 ◦ C for 1.0 min, annealing at 64 ◦ C for 1.0 min and extension at 72 ◦ C for 1.0 min. Final elongation step at 72 ◦ C for 10 min was also carried out. PCR product (5 ␮l) was analyzed on 2.5% agarose gel containing ethidium bromide for the presence of positive or null genotype of GSTM1. A PCR product of 215 bp indicates the positive genotype of GSTM1 and absence of band indicates the null genotype of GSTM1. 2.4. Detection of GABRG2 polymorphism GABRG2 polymorphism was identified by the method of Sander et al. [26]. PCR reaction was performed in a final volume of 50 ␮l containing 100 ng of genomic DNA, 20 pmol/␮l of each primer, 1.25 U Taq polymerase, 2 mM dNTP, 25 mM MgCl2 and 1× PCR buffer. Amplification conditions involved an initial denaturation of DNA at 95 ◦ C for 5 min, followed by 40 cycles of denaturation at 95 ◦ C for 30 s, annealing at 60 ◦ C for 30 s, extension at 72 ◦ C for 40 s and final extension at 72 ◦ C for 10 min. The resulting 287 bp PCR product was digested with the restriction endonuclease NciI (New England BioLabs). The digestion of 287 bp PCR fragment into two fragments of 263 and 24 bp was indicative of wild type (GG) genotype while undigested 287 bp PCR fragment was of homozygous mutant (AA) genotype. A mixture of all the fragments (287, 263 and 24 bp) was indicative of the heterozygous genotype. 2.5. Statistical analysis Genotype or allele frequencies of CYP2E1 among cases and controls were determined for Hardy–Weinberg equilibrium (HWE) using standard ␹2 statistics. The haplotype analyses (haplotype frequency estimation and pair wise linkage disequilibrium between the SNPs) were carried out using Haploview (www.broad.mit.edu/mpg/haploview/). Using binary logistic regression models, we determined the relationship of CYP2E1, GSTM1 and GABRG2 polymorphisms with risk of cirrhosis after adjusting for age. All statistical analysis was performed with the SPSS software package (version 11.0 for windows; SPSS Chicago, IL). The power of the present study was found to be >80% as analyzed by power genetic association analysis software (http://dceg.cancer.gov/bb/tools/pga) at the level of significance ˛ = 0.05 with sample size of 250 in non-alcoholic controls, 125 non-alcoholic cirrhosis, 100 alcoholic controls and 160 alcoholic cirrhotic patients.

2.1. DNA isolation and genotype analysis 1 ml of blood was collected into citrate containing tubes from all the subjects. DNA was isolated from whole blood with the Flexi Gene DNA kit (Qiagen, CA) following the manufacturers protocol. Isolated DNA was subsequently used for genotyping studies. 2.2. Detection of CYP2E1*5B (RsaI), CYP2E1*6 (DraI) and MspI polymorphism The method of Liu et al. [37] and Vidal et al. [11] was followed for determining the CYP2E1 RsaI (CYP2E1*5B) and DraI (CYP2E1*6) polymorphism, respectively. For

3. Results Both the genotype and allele frequency of CYP2E1 in alcoholic and non-alcoholic controls were found to be in Hardy–Weinberg equilibrium. As the frequency of the homozygous mutant genotype of both CYP2E1*5B and CYP2E1*6 were very rare, the heterozygous and homozygous genotypes were clubbed together and are referred to as variant genotypes. The frequency of variant genotypes

Table 1 Distribution of genotype and allele frequency of CYP2E1 (CYP2E1*5B, CYP2E1*6 and MspI) among controls and cirrhotic patients. Sample (n)

Alcoholic cirrhosis (160)

CYP2E1*6 (DraI) Non-alcoholic control (250) Non-alcoholic cirrhosis (125) Alcoholic control (100)

Alcoholic cirrhosis (160)

MspI Non-alcoholic control (250) Non-alcoholic cirrhosis (125) Alcoholic control (100) Alcoholic cirrhosis (160)

Crude OR, (95%CI), p value

Adjusted ORa , (95%CI), p value)

245 (98%) 123 (98%) 98 (98%)

05 (2%) 02 (2%) 02 (2%)

1 (Ref.) 0.80 (0.15–4.2), 0.78 1.0, (0.19–5.24), 1.0

1 (Ref.) 0.85, (0.16–4.56), 0.85 1.1, (0.21–6.0), 0.88

147 (92%)

13 (8%)

4.3, (1.5–12.4), 0.003*

4.7, (1.6–13.8), 0.002*

182 (73%) 90 (72%) 67 (67%)

68 (27%) 35 (28%) 33 (33%)

1 (Ref.) 1.0, (0.64–1.68), 0.87 1.3, (0.78–2.17), 0.28

1 (Ref.) 0.99, (0.60–1.60), 0.96 1.3, (0.76–2.1), 0.36

118 (74%)

42 (26%)

0.95, (0.60–1.5), 0.83

0.91, (0.57–1.44), 0.70

219 (88%) 111 (89%) 88 (88%)

31 (12%) 14 (11%) 12 (12%)

1 (Ref.) 0.90, (0.45–1.74), 0.73 0.96, (0.47–1.96), 0.92

1 (Ref.) 0.86, (0.43–1.70), 0.65 0.97, (0.47–1.97), 0.93

139 (87%)

21 (13%)

1.0, (0.59–1.9), 0.83

1.1, (0.60–2.01), 0.75

Crude OR, (95%CI), p value

Adjusted ORa , (95%CI), p value

1 (Ref.) 1.2, (0.17–9.0), 0.82 5.4, (1.2–24.5), 0.01*

1 (Ref.) 1.2, (0.17–9.0), 0.82 5.5, (1.2–25.26), 0.01*

1 (Ref.) 1.3, (0.71–2.24), 0.41

1 (Ref.) 1.3, (0.72–2.30), 0.39 0.91, (0.53–1.54), 0.72

0.92, (0.54–1.54), 0.74

1 (Ref.) 1.1, (0.47–2.45), 0.85 1.2, (0.58–2.46), 0.62

1 (Ref.) 1.1, (0.48–2.50), 0.83 1.2, (0.60–2.57), 0.56

Crude OR, (95%CI), p value

Adjusted ORa , (95%CI), p value

1 (Ref.)

1 (Ref.)

4.3, (0.95–19.62), 0.04*

4.9, (1.0–22.62), 0.034*

1 (Ref.)

1 (Ref.)

0.72, (0.42–1.24), 0.24

0.71, (0.41–1.23), 0.22

1 (Ref.)

1 (Ref.)

1.1, (0.52–2.36), 0.79

1.0, (0.49–2.26), 0.89

Allele frequency Major

Minor

0.99 0.99 0.99

0.01 0.01 0.01

0.96

0.04

0.86 0.85 0.82

0.14 0.15 0.18

0.87

0.13

0.94 0.94 0.94

0.06 0.06 0.06

0.93

0.07

A.J. Khan et al. / Mutation Research 664 (2009) 55–63

CYP2E1*5B (RsaI) Non-alcoholic control (250) Non-alcoholic cirrhosis (125) Alcoholic control (100)

Variant, n (%)

Wild, n (%)

Ref: reference category; OR: odds ratio; 95%CI: 95% confidence interval. a OR adjusted with age in binary logistic regression models. * p < 0.05 is considered statistically significant.

57

1.0 (Ref.) 0.94, (0.61–1.44), 0.76 1.0, (0.55–1.84), 0.96 5.3, (1.46–19.60), 0.005* 3.2, (0.29–35.52), 0.31 1.6, (0.09–25.73), 0.73 1.6, (0.09–25.73), 0.73 249 (77.8%) 38 (12%) 19 (6%) 10 (3%) 2 (0.6%) 1 (0.3%) 1 (0.3%) 1.0 (Ref.) 1.37, (0.87–2.16), 0.17 1.05, (0.52–2.10), 0.89 0.87, (0.09–8.47), 0.91 2.6, (0.16–42.23), 0.47 – – 152 (76%) 34 (17%) 12 (6%) 1 (0.5%) 1 (0.5%) – – Ref: reference category; OR; odds ratio; 95%CI; 95% confidence interval. * p < 0.05 is considered statistically significant.

1.0 (Ref.) 1.1, (0.72–1.74), 0.61 1.0, (0.53–1.92), 0.96 0.67, (0.69–6.53), 0.73 2.0, (0.12–32.55), 0.61 – – 399 (79.8%) 65 (13%) 30 (6%) 3 (0.6%) 1 (0.2%) 1 (0.2%) 1 (0.2%) C–A–T C–A–A C–G–T T–A–T T–A–A C–G–A T–G–A

197 (78.8%) 36 (14.4%) 15 (6%) 1 (0.4%) 1 (0.4%) – –

Haplotypes alcoholic cirrhosis 320 OR, (95%CI), p value Haplotypes alcoholic control 200 OR, (95%CI), p value Haplotypes non-alcoholic cirrhosis 250 Haplotypes non-alcoholic control 500 CYP2E1 haplotypes (RsaI C-1053T, MspI A6827G, DraI T7632A)

of CYP2E1*5B was higher (8%) in alcoholic cirrhotic patients (ACPs) when compared with non-alcoholic controls (2%), or alcoholic controls (2%) or non-alcoholic cirrhotic patients (NACPs, 2%). The crude odds ratio (OR) for variant genotypes was found to be significantly increased when the frequency of variant genotype in ACP was compared with non-alcoholic controls (Table 1). Similarly, when the frequency of the variant genotype of CYP2E1*5B in ACP was compared with NACP, an increased risk was observed in ACP (OR: 5.4; 95%CI: 1.2–24.5; p: 0.01) which was statistically significant. Likewise, when the frequency of variant genotype of CYP2E1*5B in ACP was compared with alcoholic controls, a statistically significant increase in risk (OR: 4.3; 95%CI: 0.95–19.62; p: 0.04) was observed in the patients (Table 1). However, no risk was observed when the frequency of variant genotype of CYP2E1*5B in either alcoholic controls or NACP was compared with non-alcoholic controls. Likewise, when the frequency of variant genotypes of CYP2E1*5B in alcoholic controls was compared with NACP, no significant increase in risk was observed (Table 1). As evident from Table 1, no significant change was also observed in the OR associated with the variant genotype of CYP2E1*6 in ACP (Table 1). Likewise, when comparing the frequency of variant genotype of CYP2E1*6 in NACP with non-alcoholic controls, no significant change in OR was observed (Table 1). Similar to the DraI polymorphism of CYP2E1, the frequency of variant genotype of MspI polymorphism in ACP (13%) was similar to that observed in non-alcoholic controls (12%), NACP (11%) and alcoholic controls (12%) and no significant risk was observed in ACP. Likewise, on comparing the frequency of variant genotype of MspI polymorphism in alcoholic controls with either non-alcoholic controls or NACP, no risk was observed (Table 1). Haplotype approach was also followed to study the association of combined effect of three SNPs of CYP2E1 with cirrhosis. Out of 8 possible haplotypes, 7 were observed in the non-alcoholic controls and alcoholic cirrhotic patients and only 5 haplotypes were observed in non-alcoholic cirrhotic patients and alcoholic controls (Table 2). The haplotype, C–A–T was considered to be the reference carrying wild type alleles. The higher frequency of T–A–T haplotype, carrying variant allele of CYP2E1*5B and wild type allele of both MspI and DraI polymorphism, in ACP resulted in significant increase in risk (OR: 5.3; 95%CI: 1.46–19.60; p: 0.005) when compared with non-alcoholic controls (Table 2). Though the frequency of other haplotypes (T–A–A, C–G–A, T–G–A) were also higher in ACP when compared with non-alcoholic controls or NACP or alcoholic controls, the increase in OR associated with these haplotypes was not found to be statistically significant. Likewise, the three haplotypes (C–A–A, C–G–T and T–A–A) in NACP and alcoholic controls exhibited slightly increased risk, though not statistically significant when compared with non-alcoholic controls (Table 2). The frequency of the null genotype of GSTM1 was found to be higher in ACP (43%) when compared with non-alcoholic controls (32%) or NACP (35%) or alcoholic controls (30%) which resulted in a significant increase in relative risk in ACP when compared with nonalcoholic controls (Table 3). A slightly increased OR (1.4), though not statistically significant, was also observed in ACP when the frequency of null genotype of GSTM1 in ACP was compared with NACP. Similarly, on comparing the frequency of null genotype of GSTM1 in ACP with alcoholic controls a statistically significant increase in risk (OR: 1.7) was observed. No risk was however, observed on comparing the frequency of the null genotype of GSTM1 in alcoholic controls with either non-alcoholic controls or NACP. Similarly, no risk was found in NACP when frequency of the null genotype of GSTM1 was compared with non-alcoholic controls (Table 3). Table 4 summarizes the genotype and allele frequencies of GABRG2 gene. The frequency of the variant genotype of GABRG2 was found to be higher in ACP (75%) when compared with nonalcoholic controls (64%) or NACP (67%) and was almost similar

OR, (95%CI), p value

A.J. Khan et al. / Mutation Research 664 (2009) 55–63

Table 2 Distribution of CYP2E1 haplotypes among controls and cirrhotic patients.

58

Table 3 Distribution of genotypes and frequency of GSTM1 among controls and cirrhotic patients. Sample (n)

Non-alcoholic control (250) Non-alcoholic cirrhosis (125) Alcoholic control (100) Alcoholic cirrhosis (160)

Crude OR, (95%CI), p value

Adjusted ORa , (95%CI), p value

79 (32%)

1 (Ref.)

1 (Ref.)

81 (65%)

44 (35%)

1.1, (0.71–1.80), 0.60

1 (Ref.)

1 (Ref.)

70 (70%)

30 (30%)

0.93, (0.56–1.54), 0.77

0.79, (0.44–1.38), 0.41

0.78, (0.44–1.37), 0.39

1 (Ref.)

92 (57%)

68 (43%)

1.2, (0.74–1.85), 0.49 0.93, (0.56–1.53), 0.77 1.6, (1.0–2.41), 0.025*

1.6, (1.05–2.42), 0.028*

1.4, (0.84–2.20), 0.21

1.4, (0.84–2.27), 0.20

1.7, (1.0–2.93), 0.044*

Positive n (%)

Null n (%)

171 (68%)

Crude OR, (95%CI), p value

Adjusted ORa , (95%CI), p value

Crude OR, (95%CI), p value

Adjusted ORa , (95%CI), p value

Frequency Positive

Null

0.68

0.32

0.65

0.35

1 (Ref.)

0.70

0.30

1.7, (1.0–2.93), 0.048*

0.57

0.43

A.J. Khan et al. / Mutation Research 664 (2009) 55–63

Ref: reference category; OR: odds ratio; 95%CI: 95% confidence interval. a OR adjusted with age in binary logistic regression models. * p < 0.05 is considered statistically significant.

Table 4 Distribution of genotypes & allele frequency of GABRG2 among controls and cirrhotic patients. Sample (n)

Wild (GG), n (%)

Variant (AA + GA), n (%)

Crude OR, (95%CI), p value

Adjusted ORa , (95%CI), p value

Non-alcoholic control (250) Non-alcoholic cirrhosis (125) Alcoholic control (100) Alcoholic cirrhosis (160)

90 (36%)

160 (64%)

1 (Ref.)

1 (Ref)

41 (33%)

84 (67%)

1.2, (0.73–1.8), 0.54

1.1, (0.69–1.73), 0.70

26 (26%)

74 (74%)

1.6, (0.95–2.68), 0.07

1.6, (0.94–2.68), 0.08

40 (25%)

120 (75%)

*

1.7, (1.0–2.62), 0.02

*

1.6, (0.99–2.46), 0.048

Crude OR, (95%CI), p value

Adjusted ORa , (95%CI), p value

Crude OR, (95%CI), p value

Adjusted ORa , (95%CI), p value

Allele frequency G

A

0.57

0.43

0.55

0.45

1 (Ref.)

1 (Ref.)

1.4, (0.77–2.48), 0.27

1.4, (0.79–2.54), 0.24

1 (Ref.)

1 (Ref.)

0.48

0.52

1.5, (0.87–2.45), 0.14

1.5, (0.88–2.52), 0.13

1.1, (0.59–1.9), 0.85

1.0, (0.56–1.81), 0.96

0.47

0.53

Ref: reference category; OR: odds ratio; 95%CI: 95% confidence interval. a OR adjusted with age in binary logistic regression models. * p < 0.05 is considered statistically significant.

59

– 00 (–) Ref: reference category; OR: odds ratio; 95%CI: 95% confidence interval. * p < 0.05 is considered statistically significant.

02 (2%) 02 (1%) 08 (5%)

7.7, (1.60–37.16), 0.003*

3.2, (0.66–15.64), 0.12

2.2, (0.42–12.03), 0.32 02 (1.5%) – 00 (–) 03 (1%) 05 (3%)

3.2, (0.75–1.78), 0.09

1.2, (0.75–2.02), 0.39 44 (35%) 1.7, (0.99–2.98), 0.052 28 (28%) 77 (31%) 60 (37.5%)

1.5, (0.98–2.30), 0.06

79 (63.5%) 1.0 (Ref.) 70 (70%) 1.0 (Ref.) 168 (67%) 87 (54.5%)

CYP2E1*5B (Wild) & GSTM1 (+/+) CYP2E1*5B (Wild) & GSTM1 (−/−) CYP2E1*5B (Variant) & GSTM1 (+/+) CYP2E1*5B (Variant) & GSTM1 (−/−)

Non-alcoholic control (250), n (%) Alcoholic cirrhosis (160), n (%)

Consistent with the earlier studies [12,29,39,40], our data have shown that polymorphisms exist in the CYP2E1 gene in North Indian population. The frequency of minor allele (1%) of CYP2E1*5B (RsaI) was found to be similar to that reported earlier in North Indian population [39,40]. Similar frequency of the minor allele of CYP2E1*5B is also reported in the Caucasians (1–4%) population [12,29,31] while the Oriental (Chinese and Japanese) population carry much higher frequency (20–30%) of the minor allele [41,42]. Similarly, the minor allele frequency (16%) of CYP2E1*6 (DraI) was quite similar to that reported earlier in the Indian population [39,40]. The Caucasians are reported to carry much lower frequency (7–12%) of the minor allele [10,31,43], while the same for CYP2E1*6 is relatively higher in Oriental (20–30%) population [41,43]. Our data further revealed that MspI polymorphism is present in North Indian population, though its minor allele frequency is higher (6%) than reported in the Caucasians (2%) [10]. The significant increase in the frequency of variant genotype of CYP2E1*5B in alcoholic cirrhotic patients when compared with non-alcoholic controls suggests that polymorphism in CYP2E1 may modify the susceptibility of an individual to alcoholic liver cirrhosis. Since the variant genotype of CYP2E1*5B is known to increase the CYP2E1 enzyme activity, the increase in frequency of CYP2E1*5B may lead to increased metabolism of alcohol in alcoholic liver cirrhotic cases when compared with non-alcoholic controls resulting in increased formation of ROS [13,14]. Significant association of CYP2E1*5B with alcoholic cirrhosis and other alcoholic liver diseases has been shown in the Oriental population [14,27,28]. However, this association was not found to be consistent in the Caucasians [11,15,29–31] and has been partly attributed to the smaller sample size, lower frequency of variant genotype, and ethnic variations among them [11,15,31]. Recent meta-analysis also showed no significant association between the CYP2E1 polymorphism and the risk of developing alcoholic liver disease [44].

Table 5 Genotype combinations of CYP2E1*5B (RsaI) and GSTM1 in controls and cirrhotic patients.

4. Discussion

OR, (95%CI), p value

Alcoholic control (100), n (%)

OR, (95%CI), p value

Non-alcoholic cirrhosis (125), n (%)

OR, (95%CI), p value

to that observed in alcoholic controls (74%). When the frequency of variant genotype of GABRG2 in ACP was compared with nonalcoholic controls the crude odds ratio was found to be 1.7 which was statistically significant. A slight increase in risk was also observed in ACP when the frequency of variant genotype of GABRG2 was compared with NACP, though the increase in risk was statistically not significant. Similarly, when the frequency of variant genotype of GABRG2 in alcoholic controls was compared with either non-alcoholic controls or NACP, no significant increase in risk was observed (Table 4). The distribution of genotype combinations of CYP2E1*5B and GSTM1 are summarized in Table 5. Even though the frequency of variant genotype combination of CYP2E1*5B and GSTM1 (null) was very low in ACP (5%) and non-alcoholic controls (1%), the carriers of both the putative high risk genes in ACP had an OR of 7.7 when compared with non-alcoholic controls (Table 5). However, no significant increase in OR was observed in alcoholic controls or NACP carrying both the putative high risk genes when compared with non-alcoholic controls (data not shown). Table 6 summarizes the genotype combinations of CYP2E1*5B and GABRG2 genes. The carriers of both the putative high risk genes in ACP had an OR of 6.4 when compared with non-alcoholic controls. The OR was further found to be increased (OR: 11.5) in ACP carrying both the putative high risk genes in ACP when compared with NACP. An increase in the risk (OR: 1.6) was also found in ACP carrying wild type genotype of CYP2E1*5B and variant genotype of GABRG2 when compared with non-alcoholic controls (Table 6). However, no significant increase in OR was observed in alcoholic controls or NACP carrying both the putative high risk genes when compared with non-alcoholic controls (data not shown).

1.0 (Ref.)

A.J. Khan et al. / Mutation Research 664 (2009) 55–63

Genotype

60

11.5, (1.42–94.06), 0.005* 01 (0.75%) 3.7, (0.77–18.40), 0.08 02 (2%)

2.1, (0.18–24.18), 0.54 01 (0.75%) –

Ref: reference category; OR: odds ratio; 95%CI: 95% confidence interval. * p < 0.05 is considered statistically significant.

04 (1.5%) 11 (7%)

6.4, (1.92–21.50), 0.000

01 (0.5%) 02 (1.5%)

4.7, (0.41–53.22), 0.17

156 (68%) 109 (68%)

89 (35.5%) 38 (23.5%)

CYP2E1*5B (Wild) & GABRG2 (GG) CYP2E1*5B (Wild) & GABRG2 (AA + GA) CYP2E1*5B (Variant) & GABRG2 (GG) CYP2E1*5B (Variant) & GABRG2 (AA + GA)

OR, (95%CI), p value Non-alcoholic control (250), n (%) Alcoholic cirrhosis (160), n (%) Genotype

Table 6 Genotype combinations of CYP2E1*5B (RsaI) and GABRG2 in controls and cirrhotic patients.

*

00 (–)

1.4, (0.81–2.34), 0.23 83 (66.5%) 72 (72%) 1.6, (1.04–2.57), 0.03*

1.0, (0.57–1.80), 0.90

1.0 (Ref.) 40 (32%) 1.0 (Ref.) 26 (26%) 1.0 (Ref.)

Alcoholic control (100), n (%)

OR, (95%CI), p value

Non-alcoholic cirrhosis (125), n (%)

OR, (95%CI), p value

A.J. Khan et al. / Mutation Research 664 (2009) 55–63

61

That the variant genotype (Rsa1) of CYP2E1 leads to an increased oxidative stress in alcoholic cirrhotic cases was further shown with statistically significant increase in risk (OR: 5.4; 95%CI: 1.2–24.5; p: 0.01) in cases when compared with non-alcoholic cirrhotic cases carrying the variant genotype of the same. Non-alcoholic cirrhosis has been primarily attributed to hepatitis B and C virus infections which increase the expression of CYP2E1 in liver [2] but not to the extent as observed in alcoholics due to excess alcohol intake [5]. Further evidence that the increased frequency of the variant genotypes (Rsa1) of CYP2E1 may lead to increased oxidative stress in alcoholic liver cirrhosis was demonstrated by significant increase in risk (OR: 4.3; 95%CI: 0.95–19.62; p: 0.04) in cases when compared with the alcoholic controls. Further, the frequency of variant genotype (Rsa1) of CYP2E1 in alcoholic and non-alcoholic controls was almost similar suggesting that the variant genotype (Rsa1) of CYP2E1 is involved in alcoholic liver diseases [11,32]. Our data further supports the earlier studies [10,11,31,32], that CYP2E1*6 and MspI polymorphisms are not associated with alcoholic cirrhosis. As observed with CYP2E1*5B, a statistically significant risk for null genotype of GSTM1 was found in alcoholic cirrhotic patients when compared to non-alcoholic and alcoholic controls. Null genotype of GSTM1 has been found to be associated with alcoholic liver diseases [18,19,33], though there are few reports which do not show significant association with the disease [35,36]. Likewise, gamma-aminobutyric acid ␥2 receptor (GABRG2) polymorphism was also found to significantly increase the risk in alcoholic cirrhotic patients when compared with non-alcoholic controls. Variant form of GABRG2 has been shown to have less binding affinity for ethanol than wild type (␥2L) receptor resulting in consumption of higher amount of ethanol to produce the same inhibitory effect as produced by wild type subunit [21,22]. This increased intake of ethanol due to variant genotype of GABRG2 receptor may lead to increased generation of reactive oxygen species and oxidative stress in the patients due to metabolism of ethanol by CYP2E1 which may result in greater liver damage. Haplotype analysis has revealed that RsaI and DraI polymorphism of CYP2E1 do not exhibit linkage disequilibrium (LD) in either controls or patients. Similar results were observed in other populations [45,46], though there are reports indicating LD between these two polymorphisms in Taiwanese, European and Mexican American population [47,48]. Haplotype data also demonstrated that the frequency of haplotype T–A–T and T–A–A, C–G–A and T–G–A are increased in the alcoholic cirrhotic patients. The haplotype T–A–T, which contains variant genotype of CYP2E1*5B, was found to be associated with several fold increased risk (OR: 5.3; 95%CI: 1.46–19.60; p: 0.005) for alcoholic cirrhosis. No such association of CYP2E1 haplotypes has been reported earlier with alcoholic cirrhosis in other populations. Our study further provided evidence that combination of SNPs could be involved in increasing the risk to alcoholic liver cirrhosis indicating relatively much higher increase in the risk (OR: 7.7) in the alcoholic cirrhotic patients carrying combination of variant genotype of CYP2E1*5B and null genotype of GSTM1 when compared with the non-alcoholic controls. This may be due to the increase in ROS generation which is not effectively detoxified in these alcoholic cirrhotic patients simultaneously carrying the null genotype of GSTM1. Likewise, increase in the OR (6.4) in patients with alcoholic cirrhosis carrying combination of variant genotypes of CYP2E1*5B and GABRG2 when compared with non-alcoholic controls or non-alcoholic cirrhotic patients have further suggested that increased intake of ethanol due to variant genotype of GABRG2 receptor may lead to increased generation of ROS and oxidative stress in these patients as they also carried variant genotypes of CYP2E1*5B. However, this increase in risk in alcoholic cirrhotic cases must be interpreted with caution due to only a few cases carrying the combination of SNPs. Due to the rare frequency of

62

A.J. Khan et al. / Mutation Research 664 (2009) 55–63

CYP2E1*5B in the controls (5 out of 250), the number of individuals who carried combination of variant genotypes of CYP2E1*5B and GABRG2 (4 out of 250) or null genotype of GSTM1 and CYP2E1*5B (2 out of 250) was further reduced because of which the risk appeared to be relatively much higher when compared with the patients. Caution may further be taken because of admixture observed in the different populations even though the ethnicity of control and patients was similar. The Indian population in general is considered to be homogenous with population being stratified on the basis of geographical zone and linguistic basis [49]. A recent study has shown mixing among the Indian population though minimum mixing was observed in Indo-European population which was included in the present study [50]. However, even then the founder effects cannot be ignored. These issues of selecting appropriate individuals can be circumvented only through the use of family studies, though including the relatives for such studies itself introduces problems in genetic study [51]. In conclusion, our data suggests that polymorphism in CYP2E1 particularly CYP2E1*5B is associated with an increased susceptibility to alcoholic cirrhosis. Haplotype analysis revealed that haplotype T–A–T, carrying variant allele of CYP2E1*5B, was associated with increased risk for alcoholic cirrhosis. Likewise, several fold increased risk in the alcoholic cirrhotic cases carrying variant genotype combinations have revealed that interaction of CYP2E1*5B with genes which leads to increased alcohol intake (GABRG2) or increased oxidative stress (GSTM1 null genotype) play an important role in enhancing the susceptibility to alcoholic cirrhosis. Conflict of interest None. Acknowledgements The authors are grateful to the Director, Indian Institute of Toxicology Research (formerly ITRC), Lucknow for his keen interest and support in carrying out the study. Mr. Anwar J. Khan is grateful to ICMR, New Delhi for providing a Senior Research Fellowship. Mr. Munindra Ruwali is thankful to UGC, New Delhi for providing Senior Research Fellowship. The financial support of CSIR Network project CMM-0016 (Predictive Medicine using repeat and single nucleotide polymorphisms) in carrying out the above studies is gratefully acknowledged. The technical assistance of Mr. B. S. Pandey and Mr. Rajesh Misra and computer help of Mr. Mohd Aslam is also gratefully acknowledged. ITRC Communication Number: 2661. References [1] K. Walsh, G. Alexander, Alcoholic liver disease, Postgrad. Med. J. 76 (2000) 280–286. [2] J. Choi, J-H.J. Ou, Mechanisms of liver injury. III. Oxidative stress in the pathogenesis of hepatitis C virus, Am. J. Physiol. Gastrointest. Liver Physiol. 290 (2006) G847–G851. [3] R.E. Mann, R.G. Smart, R. Govoni, The epidemiology of alcoholic liver disease, Alcohol Res. Health 27 (2003) 209–219. [4] V.T. Savolainen, M. Perola, K. Lalu, A. Penttila, I. Virtanen, P.J. Karhunen, Early centrilobular fibrogenesis precirrhotic lesions among moderate alcohol consumers and chronic alcoholics, J. Hepatol. 23 (1995) 524–531. [5] T. Takahashi, J.M. Lasker, A.S. Rosman, C.S. Lieber, Induction of cytochrome P4502E1 in the human liver by ethanol is caused by a corresponding increase in encoding messenger RNA, Hepatology 17 (1993) 236–245. [6] F. Stickel, C.H. Osterreicher, The role of genetic polymorphism in alcoholic liver disease, Alcohol Alcohol. 41 (2006) 209–224. [7] J. Agundez, M. Ladero, D. Rubio, J. Benitez, Rsa I polymorphism at the cytochrome P4502E1 locus is not related to the risk of alcohol-related severe liver disease, Liver 16 (1996) 380–383. [8] D. Lucas, C. Menez, F. Floch, Y. Gourlaouen, O. Sparfel, I. Joannet, et al., Cytochromes P4502E1 and P4501A1 genotypes and susceptibility to cirrhosis or upper aerodigestive tract cancer in alcoholic Caucasians, Alcohol Clin. Exp. Res. 20 (1996) 1033–1037.

[9] T. Castillo, D.R. Koop, S. Kaminura, G. Triadafilopoulos, H. Tsukamoto, Role of cytochrome P-450 2E1 in ethanol–carbon tetrachloride- and iron-dependent microsomal lipid peroxidation, Hepatology 16 (1992) 992–996. [10] V.T. Savolainen, J. Pajarinen, M. Perola, A. Penttila, P.J. Karhunen, Polymorphism in the cytochrome p4502E1 gene and risk of alcoholic liver disease, J. Hepatol. 26 (1997) 55–61. [11] F. Vidal, A. Lorenzo, T. Auguet, M. Olona, M. Broch, C. Gutierrez, et al., Genetic polymorphisms of ADH2, ADH3, CYP4502E1 Dra-I and Pst-I, and ALDH2 in Spanish men: lack of association with alcoholism and alcoholic liver disease, J. Hepatol. 41 (2004) 744–750. [12] J. Grove, A.S. Brown, A.K. Daly, M.F. Bassendine, O.F. James, C.P. Day, The RsaI polymorphism of CYP2E1 and susceptibility to alcoholic liver disease in Caucasians: effect on age of presentation and dependence on alcohol dehydrogenase genotype, Pharmacogenetics 8 (1998) 335–342. [13] S. Hayashi, J. Watanabe, K. Kawajiri, Genetic polymorphisms in the 5 -flanking region change transcriptional regulation of the human cytochrome P450IIE1 gene, J. Biochem. 110 (1991) 559–565. [14] M. Tsutsumi, A. Takada, J.S. Wang, Genetic polymorphisms of cytochrome P4502E1 related to the development of alcoholic liver disease, Gastroenterology 107 (1994) 1430–1435. [15] A. Parsian, C.R. Clonigner, Z.H. Zhang, Association of polymorphisms of CYP2E1 gene in alcoholics with cirrhosis, antisocial personality, and normal controls, Alcohol: Clin. Exp. Res. 22 (1998) 888–891. [16] S.E. Pemble, A.F. Wardle, J.B. Taylor, Glutathione S-transferase class kappa: characterization by the cloning of rat mitochondrial GST and identification of a human homologue, Biochem. J. 319 (1996) 749–754. [17] F.F. Parl, Glutathione S-transferase genotypes and cancer risk, Cancer Lett. 221 (2) (2005) 123–129. [18] V.T. Savolainen, J. Pajarinen, M. Perola, A. Penttila, P.J. Karhunen, Glutathione-Stransferase GSTM ‘null’ genotype and the risk of alcoholic liver disease, Alcohol: Clin. Exp. Res. 20 (1996) 1340–1345. [19] J.M. Ladero, C. Martinez, E. Garcia-Martin, M. Fernández-Arquero, G. LópezAlonso, E.G. de la Concha, Polymorphisms of the glutathione S-transferases mu1 (GSTM1) and theta-1 (GSTT1) and the risk of advanced alcoholic liver disease, Scand. J. Gastroenterol. 40 (2005) 348–353. [20] P. Whiting, R.M. McKernan, L.L. Iversen, Another mechanism for creating diversity in gamma-aminobutyrate type A receptors: RNA splicing directs expression of two forms of ␥2 phosphorylation site, Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 9966–9970. [21] K.A. Wafford, D.M. Burnett, N.J. Leidenheimer, D.R. Burt, J.B. Wang, P. Kofuji, et al., Ethanol sensitivity of the GABAA receptor expressed in Xenopus oocytes requires 8 amino acids contained in the gamma2 subunit, Neuron 7 (1991) 27–33. [22] R.A. Harris, W.R. Proctor, S.J. McQuilkin, R.L. Klein, M.P. Mascia, V. Whatley, P.J. Whiting, T.V. Dunwiddie, Ethanol increases GABAA responses in cells stably transfected with receptor subunits, Alcohol Clin. Exp. Res. 19 (1995) 226–232. [23] C.S. Lieber, Alcohol and the liver update, Hepatology 106 (1994) 1085–1105. [24] S. Kato, P.G. Shields, N.F. Caporaso, R.N. Hoover, B.F. Trump, H. Sugimura, A. Weston, et al., Cytochrome P450IIE1 polymorphisms, racial variation and lung cancer risk, Cancer Res. 52 (1993) 6712–6715. [25] J. Seidegaard, R.W. Pero, B. Stille, Identification of the trans-stilbene oxide-active glutathione transferase in human mononuclear leukocytes and in liver as GST1, Biochem. Genet. 27 (3-4) (1989) 253–261. [26] T. Sander, D. Ball, R. Murray, J. Patel, J. Samochowiec, G. Winterer, et al., Association analysis of sequence variants of the GABAA ␣6, ␤2, and ␥2 gene cluster and alcohol dependence, Alcohol.: Clin. Exp. Res. 23 (1999) 427–431. [27] F. Tanaka, Y. Shiratori, O. Yokosuka, F. Imazeki, Y. Tsukada, M. Omata, Polymorphism of alcohol-metabolizing genes affects drinking behavior and alcoholic liver disease in Japanese men, Alcohol.: Clin. Exp. Res. 21 (1997) 596–601. [28] Y.F. Piao, J.T. Li, Y. Shi, Relationship between genetic polymorphism of cytochrome P450IIE1 and fatty liver, World J. Gastroenterol. 9 (2003) 2612–2615. [29] M. Pirmohamed, N.R. Kitteringham, L.J. Quest, R.L. Allott, V.J. Green, I.T. Gilmore, Genetic polymorphism of cytochrome P4502E1 and risk of alcoholic liver disease in Caucasians, Pharmacogenetics 5 (1995) 351–357. [30] H. Cichoz-Lach, J. Partycka, I. Nesina, K. Celinski, M. Slomka, The influence of genetic polymorphism of CYP2E1 on the development of alcohol liver cirrhosis, Wiad. Lek. 59 (2006) 757–761. [31] N.A.C.S. Wong, F. Rae, K.J. Simpson, G.D. Murray, D.J. Harrison, Genetic polymorphism of cytochrome P4502E1 and susceptibility to alcoholic liver disease and hepatocellular carcinoma in a white population: a study and literature review, including meta-analysis, J. Clin. Pathol./Mol. Pathol. 53 (2000) 88–93. [32] A.J.M. Loza, M.T.R. Iglesias, I.P. Diaz, S.C. Castellanos, C.G. Andrade, M.E.M. Mora, et al., Association of alcohol-metabolizing genes with alcoholism in a Mexican Indian (Otomi) population, Alcohol 39 (2006) 73–79. [33] V.S. Baranov, T. Ivaschenko, B. Bakay, M. Aseev, R. Belotserkovskaya, H. Baranova, et al., Proportion of the GSTM1 genotype in some Slavic populations and its correlation with cystic fibrosis and some multifactorial diseases, Hum. Genet. 97 (1996) 516–520. [34] S. Harada, S. Takase, N. Horiike, K. Ishii, N. Ishii, A. Takada, Genetic and epidemiologic studies on alcoholic liver diseases, Arukoru Kenkyuto Yakubutsu Ison 28 (1993) 400–413. [35] L. Rodrigo, V. Alvarez, M. Rodriguez, R. Perez, R. Alvarez, E. Coto, Nacetyltransferase-2, glutathione-S-transferase M1, alcohol dehydrogenase, and

A.J. Khan et al. / Mutation Research 664 (2009) 55–63

[36]

[37]

[38]

[39]

[40]

[41]

[42]

cytochrome P450IIE1 genotypes in alcoholic liver cirrhosis: a case–control study, Scand. J. Gastroenterol. 34 (1999) 303–307. R.V. Burim, R. Canalle, L. Martinelli Ade, et al., Polymorphisms in glutathione Stransferases GSTM1 GSTT1 and GSTP1 and cytochromes P4502E1 and CYP1A1 and susceptibility to cirrhosis or pancreatitis in alcoholics, Mutagenesis 19 (2004) 291–298. S. Liu, J.Y. Park, S.P. Schantz, J.C. Stern, P. Lazarus, Elucidation of CYP2E1 5 regulatory RsaI/PstI allelic variants and their role in risk for oral cancer, Oral oncol. 37 (2000) 437–445. M. Arand, R. Muhlbauer, J. Hengstler, E. Jager, J. Fuchs, L. Winkler, F. Oesch, A multiplex polymerase chain reaction for the simultaneous analysis of the glutathione S-transferase GSTM1 and GSTT1 polymorphisms, Anal. Biochem. 236 (1996) 184–186. R.D. Mittal, D.S. Srivastava, A. Mandhani, B. Mittal, Genetic polymorphism of drug metabolizing enzymes (CYP2E1, GSTP1) and susceptibility to bladder cancer in North India, Asian Pac. J. Cancer Prev. 6 (2005) 6–9. S.S. Soya, N. Padmaja, Adithan, Genetic polymorphism of CYP2E1 and GSTP1 in a South Indian population—comparisons with North India, Caucasians and Chinese, Asian Pac. J. Cancer Prev. 6 (2005) 315–319. A. Hildesheim, L.M. Anderson, C.J. Chen, Y.J. Cheng, L.A. Brinton, A.K. Daly, et al., CYP2E1 genetic polymorphisms and risk of nasopharyngeal carcinoma in Taiwan, J. Natl. Cancer Inst. 89 (1997) 1207–1212. Y.C. Chao, T.H. Young, H.S. Tang, C.T. Hsu, Alcoholism and alcoholic organ damage and genetic polymorphisms of alcohol metabolizing enzymes in Chinese patients, Hepatology 25 (1997) 112–117.

63

[43] S. Garte, L. Saspari, A.K. Alexandrie, C. Ambrosone, H. Autrup, J.L. Autrup, et al., Metabolic gene polymorphism frequencies in control populations, Cancer Epidemiol., Biomarker Prev. 10 (2001) 1239–1248. [44] E. Zintzaras, I. Stefanidis, M. Santos, F. Vidal, Do alcohol-metabolizing enzyme gene polymorphisms increase the risk of alcoholism and alcoholic liver disease? Hepatology 43 (2006) 352–361. [45] S. Kato, P.G. Shields, N.E. Caporaso, H. Sugimura, G.E. Trivers, M.A. Tucker, et al., Analysis of cytochrome P450 2E1 genetic polymorphisms in relation to human lung cancer, Cancer Epidemiol. Biomarkers Prev. 3 (1994) 515–518. [46] X. Wu, C.I. Amos, B.L. Kemp, H. Shi, H. Jiang, Y. Wan, et al., Cytochrome P450 2E1 DraI polymorphisms in lung cancer in minority populations, Cancer Epidemiol. Biomarkers Prev. 7 (1998) 13–18. [47] M.W. Yu, A. Gladek-Yarborough, S. Chiamprasert, R.M. Santella, Y.F. Liaw, C.J. Chen, Cytochrome P450 2E1 and glutathione S-transferase M1 polymorphisms and susceptibility to hepatocellular carcinoma, Gastroenterology 109 (1995) 1266–1273. [48] M. Yang, J. Tsuang, Y.J.Y. Wan, A haplotype analysis of CYP2E1 polymorphisms in relation to alcoholic phenotypes in Mexican Americans, Alcohol.: Clin. Exp. Res. 31 (2007) 1991–2000. [49] The Indian Genome Variation Consortium, The Indian Genome Variation database (IGVdb): a project overview”, Hum. Genet. 118 (2005) 1–11. [50] The Indian Genome Variation Consortium, Genetic landscape of the people of India: a canvas for disease gene exploration, J. Genet. 87 (2008) 3–20. [51] M.F. Bassendine, C.P. Day, The inheritance of alcoholic liver disease, Ballieres Clin. Gastroenterol. 12 (1998) 317–335.