RESEARCH ARTICLE

CYP1A1 and CYP2E1 genotypes and risk of esophageal squamouscell carcinoma in a high-incidence region, Kashmir

Gulzar Ahmad Bhat & Idrees Ayoub Shah & Muzamil Ashraf Makhdoomi &Beenish Iqbal & Rumaisa Rafiq & Sumaiya Nabi & AkbarMasood & MohdMaqbool Lone &

Nazir Ahmad Dar

Received: 27 November 2013 /Accepted: 26 January 2014# International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract The study analyzed the relationship between genet-ic polymorphisms of phase I xenobiotic metabolizing en-zymes, cytochromes P450 (CYP) 1A1 and CYP2E1 andesophageal squamous cell carcinoma (ESCC) in Kashmir,India. The different genotypes of CYP1A1 and CYP2E1 wereanalyzed by polymerase chain reaction and restriction frag-ment length polymorphism in 526 ESCC cases and equalnumber of matched controls. Conditional logistic regressionmodels were used to assess the association of various geno-types with ESCC, gene–gene, and gene–environment interac-tions. High risk of ESCC was found in participants whocarried CYP1A1 Val/Val genotype (OR=2.87; 95 % CI=1.00–8.44) and the risk increased in such individuals whenc1/c1 of CYP2E1 genotype was also present (OR=5.68; 95 %CI=1.09–29.52). Risk due to CYP1A1 Val/Val genotype wasfurther enhanced (OR=8.55; 95 % CI=1.86–42.20) when theanalysis was limited to ever smokers. Participants who carriedCYP2E1 c1/c2 genotype showed an inverse relation (OR=0.27; 95 % CI=0.17–0.43) with ESCC. The inverse associa-tion of CYP2E1 c1/c2 genotype was retained when CYP1A1Ile/Ile was also present (OR=0.18; 95 % CI=0.09–0.32), aswell as when analysis was limited to ever smokers (OR=0.45;95 % CI=0.23–0.90). Significant interaction was found be-tween CYP1A1 (Val/Val) and CYP2E1 (c1/c1) genotypes(OR=1.30; 95 % CI=1.12–1.51; P=0.001) and betweenCYP1A1 (Val/Val) and smoking (OR=1.31; 95 % CI=1.01–1.69; P=0.043). The study suggests CYP1A1 Val/Val and

CYP2E1 c1/c1 genotypes are significantly associated withESCC risk.

Keywords Polymorphism .CYP1A1 .CYP2E1 . Kashmir .

Esophageal squamous cell carcinoma . Smoking . Interaction

Introduction

Esophageal cancer is the eighth most common malignancyand the sixth common cause of cancer deaths worldwide [1]with a very vast geographical distribution [2]. Histologicallyesophageal cancer is mainly represented by esophageal squa-mous cell carcinoma (ESCC) and adenocarcinoma [3]. ESCCis very common in Asian countries, which are part of the‘esophageal cancer belt’ [4, 5]. Although some factors [6–9]are associated with ESCC risk, the environmental and geneticrisk factors are yet to be identified to completely understand itsetiology particularly in the high-risk regions including Kash-mir. In case of individuals who have same exposures, thedifferential cancer risk can be due to polymorphisms in car-cinogen metabolizing genes. Such variations may result intodifferent gene–gene and gene–environment interactions andmodify the impact of environmental risk factors in cancerdevelopment [10–13].

Cytochrome P-450, the phase I enzymes, metabolize en-dogenous products and xenobiotics including polycyclic aro-matic hydrocarbons (PAHs) [14, 15] and nitrosamines [16]present in tobacco and some dietary items commonly con-sumed in high-risk populations [17, 18]. The biological andbiochemical evidences indicate that the different polymorphicvariants of these enzymes [19] have different levels of activity[20]. Therefore, the higher risk of ESCC of an individual canbe in part due to polymorphic forms of these genes.

In Kashmir, ESCC has been the leadingmalignancy in boththe sexes [21] and reportedly population is exposed to a range

G. A. Bhat : I. A. Shah :M. A. Makhdoomi :B. Iqbal :R. Rafiq :S. Nabi :A. Masood :N. A. Dar (*)Department of Biochemistry, University of Kashmir, Srinagar,J&K 190006, Indiae-mail: nazirramzan@gmail.com

M. M. LoneDepartment of Radiation Oncology, SK Institute of MedicalSciences, Srinagar, J&K 190011, India

Tumor Biol.DOI 10.1007/s13277-014-1694-6

of xenobiotics through diet and smoking [7, 22, 23]. But therole of polymorphisms in genes involved in such xenobioticmetabolism, the interaction among them and with the envi-ronment is not studied well. Therefore, the study was designedto assess the risk of ESCC associated with various polymor-phisms in CYP2E1 and CYP1A1 in Kashmir.

Materials and methods

Subject recruitment and data collection

The hospital-based case–control study was carried out inKashmir from September 2008 to December 2011. A totalof 526 histopathologically confirmed ESCC cases and equalnumber of controls were recruited for the study. The incidentcases of ESCC were recruited in the Department of RadiationOncology, Sher-i-Kashmir Institute of Medical Sciences(SKIMS), the largest and the only tertiary care hospital inthe Kashmir valley. All the cases were more than 18 yearsold and had no prior history of any malignancy. For each case,we recruited one control individually matched to the case forsex, age (±5 years), and district of residence. For most of thecases, controls were enrolled from in-patient wards of districthospitals in the respective districts from where these caseswere referred. The selection of the controls was not limited toa particular set of diseases, however the patients who wereadmitted for diseases related to tobacco smoking and alcoholdrinking, the two prime risk factors for ESCC, were notrecruited as controls. The various reasons of hospitalizationand the details of wards in which controls were recruited areprovided elsewhere [6]. The controls were recruited within6 months after their respective cases were recruited. Theparticipation rate for both cases and controls were high(96 % for cases and 98 % for controls). A limited number oftrained researchers conducted the interviews and no proxieswere used. Informed consent was obtained from all partici-pants and the study was reviewed and approved by Institu-tional Ethics Committee of SKIMS.

Questionnaire for epidemiological study

Structured questionnaires were used to collect information onage, sex, ethnicity, religion, place of residence, education,smoking, family history, socioeconomic status, fruit and veg-etable consumption, and other possible confounding factors ofinterest in face to face interviews.

Collection of blood samples

Five milliliters of venous blood was collected from eachsubject in sterilized plastic vials containing EDTA (0.5 M;pH=8.0) and stored at −80ºC before DNA extraction.

DNA extraction

Genomic DNAwas extracted from blood samples by using thephenol chloroform method [24]. The DNA extracted wasquantified and stored at 4ºC until used for polymerase chainreaction (PCR) and restriction fragment length polymorphism(RFLP).

PCR–RFLP analysis of gene polymorphism

CYP1A1: The BsrDI polymorphism in CYP1A1 exon 7 wasdetermined using PCR and RFLP, according to the methodused by Cascorbi et al. [25]. The primer sequences used were:5′-CTG TCT CCC TCT GGT TAC AGG AAG C-3′(upstream) and 5′-TTC CAC CCG TTG CAG CAG GATAGC C-3′ (downstream; Sigma-Aldrich). Amplification wasperformed by initial denaturation at 95 °C for 5 min, followedby 30 cycles of denaturation at 95 °C for 30 s, annealing at72.3 °C for 30 s, extension at 72 °C for 30 s, and a finalextension at 72 °C for 7 min. Genomic DNA (150–190 ng)was amplified in a total volume of 25 μl reaction mixturecontaining 10× PCR buffer, MgCl2 (1.5 mM), each primer(20 pmol), dNTPs (200 μM), and 1 unit of DNA Taq poly-merase (Fermantas, MBI, Vilnius, Lithuania). The PCR prod-uct was digested by restriction enzyme BsrDI (Fermantas,MBI, Vilnius, Lithuania) and then resolved by 2.5 % agarosegel. When a BsrDI restriction site was present, the fragment of204 bp was digested into two fragments of 149 and 55 bp.Individuals with homozygous wild (Ile/Ile) condition had149- and 55-bp fragment, heterozygous (Ile/Val) individualshad three bands 204, 149, and 55 bp; while as homozygousmutant (Val/Val) individuals retained the larger parent band(204 bp). For 10 % of the samples the results were validatedby sequencing (Fig. 1).

CYP2E1: The RsaI polymorphism in 5′ flanking region ofthe CYP2E1 was determined using PCR and RFLP, accordingto the method used by Hayashi et al. [26]. The primer

Fig. 1 Chromatogram of CYP1A1 genotypes

Tumor Biol.

sequences used were: 5′-CCA GTC GAG TCT ACA TTGTCA-3′ (upstream) and 5′-AGA CCT CCA CAT TGA CTAGC-3′ (downstream; Sigma-Aldrich). Amplification was per-formed by initial denaturation at 95 °C for 5 min, followed by30 cycles of denaturation at 95 °C for 30 s, annealing at 58 °Cfor 30 s, extension at 72 °C for 30 s, and a final extension at72 °C for 10 min. Genomic DNA (150–190 ng) was amplifiedin a total volume of 25 μl reaction mixture containing 10×PCR buffer, MgCl2 (1.5 mM), each primer (20 pmol), dNTPs(200 μM), and 1 unit of DNA Taq polymerase (Fermantas,MBI, Vilnius, Lithuania). The final product was digested byrestriction enzyme RsaI (Fermantas, MBI, Vilnius, Lithuania)and resolved by 2.0 % agarose gel electrophoresis. When aRsaI restriction site was present, the fragment of 552 bp wasdigested into two fragments of 352 and 200 bp. Individualswith homozygous wild (c1/c1 or Val/Val) genotype had the352- and 200-bp fragment, heterozygous (c1/c2 or Val/Ile)individuals had three bands; and individuals with homozy-gous variant (c2/c2 or Ile/Ile) genotype retained the largerparent band (552 bp). The results obtained by PCR-RFLPwere confirmed by sequencing in randomly picked 10 % ofsamples.

Statistical analysis

Categorical variables were set for presenting and calculatingnumbers and percentages for different variants of CYP1A1and CYP2E1. Subjects were stratified into various groupsbased on smoking habit and various possible genotype com-binations. Conditional logistic regression models were used tocalculate odds ratios (ORs) and corresponding 95 % confi-dence intervals (CIs) to assess the association of the genotypeswith ESCC risk and to assess the possible gene–gene andgene–environment interactions. Adjustment was made withknown risk factors like age, sex, residence, education level,socioeconomic status, fruit and vegetable consumption, andsmoking. All statistical analysis was done using Stata soft-ware, version 12 (STATA Corp., College Station, TX, USA).Two sided P values<0.05 were considered as statisticallysignificant.

Results

Table 1 summarizes the selected demographic characteristicsof cases and controls. The mean ages (standard deviation) ofcases and controls were 60.88 (±11. 25) and 61.26 (±11.17),respectively. Distribution of education, socioeconomic status,fruit and vegetable intake, hot beverages, and smoking invarious forms was significantly different in cases from con-trols. The minor allele frequencies observed among the casesand controls were 29.56 and 22.26 % for CYP1A1 and 16.35and 31.37 % for CYP2E1, respectively and the difference in

allele distribution between cases and controls was statisticallysignificant.

The representative gel picture showing RFLP results ofCYP1A1 and CYP2E1 genotypes are shown in Figs. 2 and 3.The wild homozygous, variant homozygous, and heterozy-gous genotypic frequencies ofCYP1A1 andCYP2E1 in ESCCcases and controls are summarized in Table 2. High risk ofESCC was found in participants who carried CYP1A1Val/Val(mutant) genotype (OR=2.87; 95 % CI=1.00–8.44) and therisk increased in such individuals when c1/c1 of CYP2E1(wild) genotype was also present (OR=5.68; 95 % CI=1.09–29.52; Table 2). The risk of ESCC was further enhanced(OR=8.55; 95 % CI=1.86–42.20) on limiting the analysis toever smokers among the CYP1A1 Val/Val carrying genotypecases (Table 3).

Table 1 Demographic characters of esophageal cancer cases and controls

Characteristics Cases n (%) Controls n (%) P valuea

Total 526 (100) 526 (100) 0.001

Age (years mean ± SD) 60.88±11.25 61.26±11.17

Fruit and vegetables 1.84 (1.16) 3.23 (1.16)

Hot beverages

Warm 192 (37.07) 243 (47.18) 0.001

Hot 326 (62.93) 272 (52.82)

Place of residence

Urban 23 (4.37) 47 (8.94) <0.001

Rural 503 (95.63) 479 (91.06)

Education

No formal school 467 (89.0) 333 (64.6) <0.001

Primary 23 (4.37) 64 (12.17)

Middle 18 (3.42) 40 (7.60)

High school 15 (2.85) 50 (9.51)

College or above 3 (0.57) 39 (7.41)

Smoking

Ever 326 (61.98) 270 (51.33) <0.001

Never 200 (38.02) 256 (48.67)

Family history

Yes 180 (34.22) 43 (8.17) <0.001

No 346 (65.78) 483 (91.83)

Allele type

CYP1A1

A 741 (70.44) 814 (77.37) 0.0002

G 311 (29.56) 238 (22.26)

CYP2E1

C1 880 (83.65) 722 (68.63) <0.0001

C2 172 (16.35) 330 (31.37)

n number of individualsa Chi-square test (χ2 ) was used to calculate P values for categoricalvariables

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An inverse relationship turned out in cases who carriedCYP2E1 c1/c2 genotypes alone (OR=0.27; 95 % CI=0.17–0.43) or in combination with CYP1A1 Ile/Ile (OR=0.18; 95 %CI=0.09–0.32). When the analysis was restricted to eversmokers, the inverse effect of CYP2E1 c1/c2 genotype wasretained and an association with ESCC turned out in case ofthe CYP2E1 c1/c1 genotype (Table 3).

On assessing the possible gene–gene and gene–environ-ment interactions, we found significant interactions betweenCYP1A1 and CYP2E1 genotypes (OR=1.30; 95 % CI=1.12–1.51; P=0.001) as well as between CYP1A1 genotype andsmoking (OR=1.31; 95 % CI=1.01–1.69; P=0.043). How-ever, no statistically significant interaction was observed be-tween CYP2E1 genotypes and smoking (Table 4).

Discussion

The present study revealed that CYP1A1 and CYP2E1 geno-types alone or in combination either increase or decrease therisk of ESCC in Kashmiri population. The risk was increasedwhen mutant genotype (Val/Val) of CYP1A1 is taken alone orin combination with wild type (c1/c1) of CYP2E1. Similarly,unlike other combinations the heterozygous (CYP2E1 c1/c2)genotype’s inverse relationship was not affected when thecases also carried wild type (Ile/Ile) of CYP1A1. There is asynergistic interaction among the studied polymorphisms in

CYP1A1 and CYP2E1 genes and smoking. Moreover, the riskassociated with CYP1A1 Val/Val genotype and the inverseassociation due to CYP2E1 c1/c2 genotype disappeared whenthe individuals carried both the genotypes that suggest adilution of each other’s effect.

Polymorphisms in genes including CYP1A1 and CYP2E1that participate in the metabolic activation of carcinogens likenitrosamines and PAHs play an important role in cancerdevelopment. The role of such variations is explained by theireffect on the activity or expression of the enzymes [27, 28].TheCYP1A1 Ile-Val substitution in exon 7 results in a twofoldincrease in microsomal enzyme activity [29], thus can explainthe risk associated with the Val/Val genotypes in CYP1A1 inthe present study. The enhanced activity of the enzyme canresult into accumulation of DNA adducts which ultimatelylead to formation of carcinogens and hence favors carcino-genesis [30, 31]. When CYP1A1 Val/Val genotype is present,the expression of the enzymes in ESCC tumorous tissue ascompared to non-tumorous tissue is higher which also canexplain its increased activity [28, 32]. The effect of the en-zyme CYP1A1 Val/Val genotype in increasing the cancer riskcan be indirectly through the induction of aryl hydrocarbonhydroxylase enzyme which is involved in metabolism of PAH[33].

Individuals with CYP2E1 c1/c2 have a lower basalCYP2E1 activity or expression [34] which may decrease theability to activate mutagens and carcinogens [35, 36] and is

Fig. 2 PCR-RFLP analysis ofCYP1A1 A4889G polymorphism.The 149 and 55 bp bandsindicates CYP1A1 Ile/Ile(homozygous wild) genotype;presence of undigested parentband (204 bp) indicate CYP1A1Val/Val (homozygous mutant)genotype while asCYP1A1 Ile/val(heterozygous) genotype containall the three bands, i.e., 204, 149,and 55 bp

Fig. 3 PCR-RFLP analysis ofCYP2E1 C1053T polymorphism.The 352 and 200 bp bandsindicates CYP2E1 c1/c1(homozygous wild) genotype;presence of undigested parentband (552 bp) indicate c2/c2(homozygous mutant ) genotypewhile as CYP2E1 c1/c2 (heterozygous) genotype containall the three bands, i.e., 552, 352,and 200 bp

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therefore a plausible explanation for the inverse relation ofheterozygous (CYP2E1 c1/c2) genotype in our study. Weobserved a higher risk for developing ESCC in the subjectswho carried CYP1A1 Val/Val (mutant) genotype and CYP2E1c1/c1 (wild) genotype. This gene–gene interaction in the studypopulation needs further studies to elucidate precisely the roleof these polymorphisms in development of ESCC.

As compared to never smokers, ever smokers have anadditional burden of the carcinogenic exposure from tobaccosmoking. The increased risk of smokers can be due to the highexposure and the high activity of CYP1A1 (Val/Val) andCYP2E1 (c1/c1). This shows that the risk due to smoking ismodified by a genotype, which may be because of the gene–environment interaction. Thus two individuals having sameexposure cannot have the same chances of developing cancerwhen harboring different genotypes.

Our results are in agreement with previous studies thatsupport the positive association of CYP1A1 and CYP2E1genotypes with ESCC [37, 38]. However, insignificant

association of these genes with ESCC in some studies[39, 40] could be explained due to the ethnicity differ-ences and exposures to different carcinogens [41–43].Also, unlike our study the earlier studies had smallsample size and some were not conducted in high-riskregions. To the best of our knowledge no genome wideassociation study has reported any association of thegenotypes analyzed in this study [44–46].

Little is known about the effect of CYP1A1 and CYP2E1genetic polymorphisms on the susceptibility to ESCC inKashmiri population. Our results are not in agreement with aprevious report from Kashmir which showed CYP2E1 c1/c2variant enhanced the risk of ESCC but did not associateCYP1A1 polymorphism with ESCC risk [47]. Unlike ourstudy, the report was based on a small sample size and alsothe results were not adjusted with confounding factors ofESCC. This is the first large case control study that attemptedto study the impact of genetic polymorphism of CYP1A1 andCYP2E1 on the risk of ESCC in Kashmir valley.

Table 2 Independent and combined distribution of CYP1A1 and CYP2E1 genotypes in ESCC cases and matched controls

Genotypes Cases n (%) Controls n (%) Unadjusted ORa (95 % CI) Adjusted ORi (95 % CIb)

Total 526 (100) 526 (100) – –

CYP1A1 Ile/Ilec 253 (48.10) 300 (57.03) Referent Referent

CYP1A1 Ile/Vald 235 (44.68) 214 (40.68) 1.30 (1.01–1.67) 1.30 (0.84–1.99)

CYP1A1 Val/Vale 38 (7.22) 12 (2.28) 3.57 (1.84–6.91) 2.87 (1.00–8.44)

CYP2E1 c1/c1f 366 (69.60) 207 (39.35) Referent Referent

CYP2E1 c1/c2g 148 (28.14) 308 (58.56) 0.27 (0.20–0.36) 0.27 (0.17–0.43)

CYP2E1 c2/c2h 12 (2.28) 11 (2.10) 0.62 (0.26–1.49) 0.25 (0.04–1.62)

Combined genotypesj

Ile/Ile+c1/c1 197 (37.45) 111 (21.10) Referent Referent

Ile/Ile+c1/c2 52 (9.89) 184 (34.98) 0.14 (0.09–0.22) 0.18 (0.09–0.32)

Ile/Ile+c2/c2 04 (0.76) 05 (0.95) 0.63 (0.16–2.48) 0.28 (0.05–1.52)

Ile/Val+c1/c1 151 (28.71) 94 (17.87) 0.84 (0.57–1.22) 1.09 (0.67–1.78)

Ile/Val+c1/c2 77 (14.64) 118 (22.43) 0.38 (0.25–0.56) 0.29 (0.18–0.50)

Ile/Val+c2/c2 07 (1.33) 02 (0.36) 1.07 (0.25–5.60) 1.29 (0.19–8.87)

Val/Val+c1/c1 18 (3.42) 02 (0.38) 5.59 (1.26–24.81) 5.68 (1.09–29.52)

Val/Val+c1/c2 19 (3.61) 6 (1.14) 1.45 (0.54–3.89) 2.02 (0.55–7.36)

Val/Val+c2/c2 1 (0.19) 4 (0.76) 0.18 (0.02–1.67) –

a OR=odds ratio. ORs (95 % CIs) were obtained from conditional logistic regression modelsb CI=confidence intervalc Ile/Ile=CYP1A1 wild genotyped Ile/Val=CYP1A1 heterozygous genotypee Val/Val=CYP1A1 mutant genotypef c1/c1=CYP2E1 wild genotypeg c1/c2=CYP2E1 heterozygous genotypeh c2/c2=CYP2E1 mutant genotypei Adjusted ORs (95 % CIs) was obtained in conditional logistic regression models with adjusted for age, family history, education level, daily fruit andvegetable consumption, place of residence, socioeconomic status, and smokingj Genotype effects of both genes

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Table3

ORand95

%CIof

CYP

1A1andCYP

2E1genotypesin

ESC

Ccasesandcontrolsstratifiedby

smoking

Variables

CYP1A

1CYP2E

1

Cases

(%)

Controls(%

)UnadjustedOR(95%

CI)

AdjustedOR(95%

CI)

Cases

(%)

Controls(%

)UnadjustedORa(95%

CIb)

AdjustedORi(95%

CI)

Wild

+neversmokerc,d

101(19.20)

134(25.48)

Referent

Referent

140(26.62)

97(18.44)

Referent

Referent

Heterozygous+

neversmokere,f

83(13.78)

114(21.67)

0.98

(0.67–1.43)

0.86

(0.44–1.66)

58(11.03)

154(29.28)

0.28

(0.18–0.43)

0.33

(0.16–0.65)

Mutant+neversmokerg,h

16(3.04)

08(1.52)

2.4(0.99–5.84)

0.77

(0.15–3.82)

02(0.38)

05(0.95)

0.42

(0.08–2.30)

0.02

(0.00–0.47)

Wild

+eversm

oker

152(28.90)

166(31.56)

1.3(0.90–1.90)

1.10

(0.57–2.16)

226(42.97)

110(20.91)

1.65

(1.12–2.42)

1.89

(1.01–3.55)

Heterozygous+

eversm

oker

152(28.90)

100(19.01)

2.11

(1.44–3.10)

2.01

(1.06–3.81)

90(17.11)

154(29.28)

0.44

(0.29–0.66)

0.45

(0.23–0.90)

Mutant+eversm

oker

22(4.18)

04(0.76)

7.28

(2.42–21.92)

8.55

(1.86–42.20)

10(1.90)

06(1.14)

1.09

(0.36–3.30)

1.53

(0.14–16.73)

aOR=odds

ratio

.ORs(95%

CIs)wereobtained

from

conditionallogisticregression

models

bCI=

confidence

interval

cWild

=CYP1A

1Ile/Ile

dCYP2E

1c1/c1genotype

eHeterozygous=

CYP

1A1Ile/Val

fCYP

2E1c1/c2genotype

gMutant=CYP1A

1Val/Val

hCYP2E

1c2/c2genotype

iAdjustedORs(95%

CIs)was

obtained

inconditionallogisticregression

modelswith

adjusted

forage,family

history,educationlevel,daily

fruitandvegetableconsum

ption,

placeof

residence,and

socioeconomicstatus

Tumor Biol.

The major strengths of this study include relatively a largesample size, histological verification of ESCC, and adjust-ments of the results for multiple potential confounding factors.Similar to other hospital-based case–controls studies withretrospective exposure assessments, recall and selection biascan be weak points of this study.

In conclusion our study suggests that CYP2E1 andCYP1A1 genotypes are associated with the risk of ESCC inKashmir. Further there is a synergistic effect of the smokingwith Val/Val genotype of CYP1A1 in the development ofESCC.

Acknowledgement We are highly obliged to all the subjects whoparticipated in this study. We are also thankful to all the consultants andall the paramedical staff at SKIMS for their valuable help during subjectrecruitment and sample collection. We are extremely grateful of AmirAlmasi-Hashiani, Department of Epidemiology and Biostatistics, TehranUniversity of Medical Sciences, Tehran for his statistical assistance.

Conflict of interest None.

Funding The study was financially supported by research grant fromIndian Council of Medical Research (ICMR), New Delhi under IRIS ID5/13/37/2007/-NCD-111.

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Table 4 Results for gene–gene and gene–environment interaction

Risk factor Standard error P value OR 95 % CI

Smoking 0.29 0.99 0.99 0.56–1.77

CYP1A1 smokinga 0.17 0.043 1.31 1.01–1.69

CYP2E1 smokingb 0.14 0.827 0.97 0.74–1.27

CYP1A1 2E1c 0.10 0.001 1.30 1.12–1.51

a Interaction between smoking and CYP1A1 Val/Val genotypeb Interaction between smoking and CYP2E1 c1/c1 genotypec Interaction between CYP1A1 and CYP2E1 genotypes

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