functional fen1 polymorphisms are associated with dna damage levels and lung cancer risk

Human MutationRESEARCH ARTICLE

Functional FEN1 Polymorphisms Are Associated withDNA Damage Levels and Lung Cancer Risk

Ming Yang,1,2y Huan Guo,3y Chen Wu,1,2 Yuefeng He,3 Dianke Yu,1,2 Li Zhou,3 Fang Wang,3 Jian Xu,1 Wen Tan,1,2

Guanghai Wang,1,2 Binghui Shen,4 Jing Yuan,3 Tangchun Wu,3 and Dongxin Lin1,2�

1Department of Etiology and Carcinogenesis, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical

College, Beijing, People’s Republic of China; 2Beijing Key Laboratory for Cancer Prevention, Beijing, People’s Republic of China; 3Institute of

Occupational Medicine and Ministry of Education Key Lab for Environment and Health, School of Public Health, Tongji Medical College,

Huazhong University of Science and Technology, Wuhan, People’s Republic of China; 4Department of Radiation Biology, City of Hope National

Medical Center and Beckman Research Institute, Duarte, California

Communicated by Michael DeanReceived 11 February 2009; accepted revised manuscript 18 May 2009.

Published online 9 June 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/humu.21060

ABSTRACT: Flap endonuclease 1 (FEN1) is a key enzymein maintaining genomic stability and protecting againstcarcinogenesis. This study investigated whether functionalvariations in FEN1 gene are associated with DNA damageand lung cancer risk. Thirty DNA samples were sequencedto identify variants and function of the variants wasexamined by a set of biochemical assays. DNA damagelevels were detected by comet assays in a cohort of 303coke-oven workers and 297 controls. The association withlung cancer risk was examined in two independentcase–control panels consisted of a total 1,840 lung cancerpatients and 1,958 controls. We identified two singlenucleotide polymorphisms (SNPs) located in the FEN1promoter c.�69G4A (rs174538:G4A) and 30-untransla-tional region c.4150G4T (rs4246215:G4T) that wereassociated with reduced FEN1 expression. Among coke-oven workers, DNA damage levels were significantlyhigher in the �69GG or GA carriers compared with the�69AA carriers. The �69GG or 4150GG carriers had asignificantly increased risk for developing lung cancercompared with the �69AA or 4150TT carriers. Theseresults highlight FEN1 as an important gene in humancarcinogenesis and genetic polymorphisms in FEN1 confersusceptibility to lung cancer.Hum Mutat 30:1320–1328, 2009. & 2009 Wiley-Liss, Inc.

KEY WORDS: FEN1; DNA damage; lung cancer

Introduction

Lung cancer is one of the leading causes of cancer death allaround the world, with over one million deaths each year [Parkin

et al., 2005]. It is well known that more than 75% of lung cancer isattributed to environmental carcinogen exposure, such as tobaccosmoking and certain occupational exposures [Parkin et al., 2005].Polycyclic aromatic hydrocarbons (PAHs), a well-establishedgroup of chemical carcinogens, are formed and released duringtobacco smoking and burning process [IARC, 1983; Thun et al.,1997]. Epidemiological studies have suggested an etiological linkbetween PAH exposure and a three- to sevenfold increased risk fordeveloping lung cancer in coke-oven workers [IARC, 1983].Carcinogenic PAHs cause DNA damage and genomic instabilitythrough directly forming bulky DNA adducts [Popp et al., 1997]or producing reactive oxygen species, which subsequently result ina wide variety of nonbulky base damage and single-strand breaks[Frenkel, 1992]. However, not all exposed individuals developlung cancer, suggesting that the genetic makeup is also importantin the development of this malignancy. DNA repair capability,which is known to be polymorphic among populations, has beenrecognized as the most critical mechanism in protection againstDNA damages [Conney, 1982; Hoeijmakers, 2001]. Therefore,interindividual variation in DNA repair capacity may play asignificant role in modifying lung cancer risk.

Flap endonuclease 1 (FEN1; MIM 600393) is a structure-specific nuclease best known for its involvement in efficient 50-flapremoval during long-patch base-excision repair and the matura-tion of Okazaki fragments in DNA replication [Harrington andLieber, 1994; Lieber, 1997; Shen et al., 2005]. In addition, FEN1 isalso characterized as a 50 exonuclease [Liu et al., 2004] and a gap-dependent endonuclease [Reagan et al., 1995; Zheng et al., 2005],which can be stimulated to promote apoptotic DNA fragmenta-tion in response to apoptotic stimuli. Because of its critical role inDNA repair and other multiple DNA metabolic pathways, FEN1serves as a key enzyme in maintaining genomic stability andprotecting against carcinogenesis. Previous studies reported thatthe functional impairment of yeast RAD27, the homolog ofmammalian FEN1, results in hypersensitivity to DNA-damagingagents and a marked increase in the rate of spontaneous mutations[Parrish et al., 2003; Tishkoff et al., 1997; Zheng et al., 2005].Consistent with this phenomenon, haploin-sufficient Fen1 hasbeen shown to be involved in genome instability and cancerdevelopment in mice [Kucherlapati et al., 2002]. Recently, Zhenget al. [2007] have shown that FEN1 mutations resulting in reducednuclease activity occur in human cancer cells. Approximate 70%of mice knocked-in the mutated FEN1 developed tumors in

OFFICIAL JOURNAL

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& 2009 WILEY-LISS, INC.

Additional Supporting Information may be found in the online version of this article.yMing Yang and Huan Guo contributed equally to this work.�Correspondence to: Tangchun Wu, School of Public Health, Tongji Medical College,

Huazhong University of Science and Technology, Wuhan 430030, P.R. China. E-mail:

[email protected]; or Dongxin Lin, Department of Etiology and Carcinogenesis,

Cancer Institute, Chinese Academy of Medical Sciences, Beijing 100021, P.R. China.

E-mail: [email protected]; [email protected]

multiple organs, predominantly in the lung [Zheng et al., 2007].Therefore, it is conceivable that abnormal expression and/orenzymatic function of FEN1 resulting from naturally occurringgenetic changes, such as single nucleotide polymorphisms (SNPs),may contribute to cancer susceptibility.

To date, little or nothing has been known about the SNPs andtheir functional significance in the FEN1 locus. On the basis ofprevious findings mentioned above, we hypothesized that thefunctional genetic variants in the FEN1 gene may affect FEN1expression and/or its protein function, which in turn, mayinfluence FEN1-mediated DNA repair capacity and consequentialrisk of developing lung cancer. To test this hypothesis, we screenedall SNPs in the FEN1 gene by sequencing and conducted two largeindependent case–control studies to investigate the associationbetween FEN1 genotypes and risk for developing lung cancer. Wealso examined the association between FEN1 genotype and DNAdamage/repair phenotype caused by occupational exposure toPAHs in coke-oven workers.

Materials and Methods

SNP Screening

Thirty DNA samples derived from blood lymphocytes ofrandomly selected healthy subjects (all were Han Chinese) wereused to search for SNPs within the 50-flanking region, 50-UTR,coding regions, and 30-UTR of FEN1 (GenBank accessionNM_004111.4). These samples included 60 chromosomes, providingat least a 95% confidence level to detect alleles with frequency of45%. Seven sets of PCR primers were designed for SNP screeningin reference to the FEN1 gene sequences (NC_000011) (Supp. TableS1). The seven fragments of FEN1 from each subject were amplified,and SNPs were identified by directly sequencing the PCR productsin an ABI 3730 sequencer (Applied Biosystems, Foster City, CA).Finally, we used the Mutation Explorer program (Todaysoft Inc.,Beijing) to identify SNP candidates that were further confirmed byresequencing the SNP sites from the opposite strand.

Study Subjects

This study recruited three sets of subjects who were all HanChinese. To analyze the association between FEN1 genotypes andDNA damage levels in peripheral blood lymphocytes, 303 coke-oven workers and 297 noncoke-oven workers, who worked in thesame steel company in Wuhan, Hubei Province, China, wereenrolled. Coke-oven workers had at least 6-month exposure to thecoke-oven emission by working on different workplaces includingthe top, side, and bottom of the coke ovens. Control subjects werestaff members of the offices and hospitals of the same steel companywithout such emission exposure. The workers exposed to knownDNA-damaging agents, such as radiotherapy and chemotherapy inthe last 3 months, were excluded. After providing written informedconsents, each subject was interviewed using a questionnaire tocollect information on demographic characteristics, smoking,drinking, diet, and occupational exposure status. The number ofpack-years smoked was determined as an indication of thecumulative cigarette dose level [pack-years 5 (cigarettes per day/20)� (years smoked)]. Each subject donated 5.0 ml venous blood atthe end of the work shift, which was used for comet assay and FEN1genotyping. This study was approved by the Institutional ReviewBoard of Tongji Medical College.

Case–control analysis consisted of two independent sets ofpatients with lung cancer and cancer-free controls. The first set

included 1,013 lung cancer patients and 1,131 controls, which hasbeen described in a previous study [Sun et al., 2007]. Briefly,patients were recruited at Cancer Hospital, Chinese Academy ofMedical Sciences (Beijing), a tertiary referral cancer center inNorthern China. They were from Beijing city and surroundingprovinces and were believed to be a good representation of allcases in this region of Northern China. All eligible patients wererecruited between January 1997 and July 2003, with a responserate of 87%. Controls were cancer-free individuals selected from acommunity nutritional survey of 6,450 individuals that wasconducted in the same region during the same period asrecruitment of case. The participation response rate for controlswas 83%. Control subjects were selected based on physicalexaminations and frequency-matched for age (75 years) andsex to the patients. The second set consisted of 827 patients withlung cancer and 827 controls. Patients were recruited from fourtertiary referral Hospitals at Wuhan city, Hubei Province inSouthern China, between November 2004 and July 2008. Controlswere randomly selected from 4,073 individuals participated in acommunity screening program for early detection of cancer andchronic noninfectious disease conducted in the same regionsduring the same period as the cases were recruited. Controls hadno history of cancer and were frequency-matched to case patientson age and sex. The response rates for cases and controls were 90and 84%, respectively. This Southern case–control cohort has beendescribed elsewhere [Bai et al., 2007]. At recruitment, informedconsent was obtained from each subject and this study wasapproved by the institutional review boards of Chinese Academyof Medical Sciences Cancer Institute and Tongji Medical College.

Comet Assay

Lymphocytes were isolated from 5.0 ml of heparinized bloodand suspended in 1.0 ml ice-cold phosphate-buffered saline (pH7.4). Comet assays were done according to the published protocol[Chen et al., 2006]. DNA damage was measured using an imageanalysis system (version 1.0, IMI Comet Analysis Software,China). Fifty cells were analyzed per slide, and the Olive tailmoment (Olive TM) value was used as a measurement of DNAdamage level [Singh et al., 1988].

Genotyping

Genomic DNA samples were extracted from peripheral bloodlymphocytes of all subjects. FEN1 c.�69G4A (rs174538: G4A;NM_004111.4) and c.4150G4T (rs4246215: G4T;NM_004111.4) genotypes were analyzed using polymerase chainreaction (PCR)-based restriction fragment length polymorphism(RFLP) or TaqMan allelic discrimination assay, respectively.Nucleotide numbering reflects cDNA numbering with11corresponding to the A of the ATG translation initiation codonin the reference sequence, according to journal guidelines(www.hgvs.org/mutnomen). The initiation codon is codon 1. InPCR-RFLP genotyping, the primer pairs used for amplifying DNAcontaining the FEN1 c.�69G4A or c.4150G4T sites were50-ggaggttccaggagcgtcta-30/50-ttctccaccgcttgtccc-30 or 50-tatgtcagg-ctcaaaccac-30/50-cagccagtaatcagtcacaa-30, respectively. Restrictionenzymes SalI (New England Biolabs, Beverly, MA) or Alw26I(Fermentas, Vilnius, Lithuania) was used to distinguish the FEN1c.�69G4A or c.4150G4T genotypes, respectively (Supp. Fig.S1). TaqMan assays were performed in 384-well format on an ABI7900HT system (Applied Biosystems) with primer pair 50-caagtccctcaatgccacttg-30/50-tcgcatctccgtctggaact-30 and probes

HUMAN MUTATION, Vol. 30, No. 9, 1320–1328, 2009 1321

FAM-ctgctcctgccgacgtgttcttc-TAMRA (for �69G) and HEX-cgcctgctcctgtcgacgtgt-TAMRA (for �69A) for the c.�69G4ASNP, and primer pair 50-gccagccaggaaatcatttct-30/50-atatgtcaggct-caaaccacttctc-30 and probes FAM-catcttttgtctccccct-MGB (for4150G) and HEX-catcttttgtatccccctt-MGB (for 4150 T) for thec.4150G4T SNP (GeneCore BioTechnology, Shanghai) (Supp.Fig. S2). Genotyping was performed independently in twolaboratories (Chinese Academy of Medical Sciences and TongjiMedical College) without knowledge of the case–control status. A15% random sample was reciprocally tested by different personsin the two laboratories, and the reproducibility was 99.1%.

Plasmid Construction

The FEN1 �69G and �69A allelic reporter constructs wereprepared by amplifying the 479 bp FEN1 core promoter region (from�128 to 351 bp relative to the transcriptional start site) in DNA fromsubjects homozygous for the �69GG or �69AA genotype, usingprimers of 50-cggggtacctctcaccattttgccccgcga-30 and 50-ccgctcgagctggcctttgggacacgcgg-30, which includes the KpnI and XhoIrestriction sites (underlined sequences). The PCR products weredigested with KpnI and XhoI (TaKaRa, Dalian, China) and ligated,respectively, into an appropriately digested pGL3-Basic vector(Promega, Madison, WI). The constructs were designated aspGL3b-69G and pGL3b-69A, respectively. The FEN1 30-UTR region(from 3827 to 4561 bp relative to the transcriptional start site)containing the c.4150G4T polymorphism was generated by PCRusing DNA sample from subject homozygous for the 4150GG or TTgenotype, with primers of 50-ctagtctagaatgtgtttccccattatacctccttc-30 and50-ctagtctagattacttttagaattttattgac-30, both of which contain XbaI site(underlined sequences). After digestion with XbaI (TaKaRa), thePCR products were cloned into XbaI digested vector, and theresultant constructs were designated as pGL3c-4150G or pGL3c-4150T. Restriction analysis and complete DNA sequencingconfirmed the orientation and integrity of each constructs’ inserts.

Transient Transfection and Luciferase Assays

HeLa and A549 cells (6� 104) were placed in 24-well plates andtransfected with 50 ng of reporter plasmid using LipofectamineTM

2000 (Invitrogen, Carlsbad, CA) when grown to 70% confluence.pRL-SV40 (1 ng) (Luciferase Assay System; Promega) containingrenilla reniformis luciferase was cotransfected to standardizetransfection efficiency. Luciferase activity was determined at 24-hr(pGL3b–69G and pGL3b–69A) or 48 hr (pGL3c–4150G andpGL3c–4150 T) after transfection using a luciferase assay system(Promega) as previously described [Yang et al., 2007]. For eachplasmid construct, three independent transfection experimentswere performed, and each was done in triplicate. Fold increase wascalculated by defining the activity of empty pGL3-Basic or pGL3-Control vector as 1.

Electrophoretic Mobility-Shift Assays

Synthetic double-stranded and 30 biotin-labeled oligonucleo-tides corresponding to the FEN1 �69G or �69A sequences andHeLa or A549 cell nuclear extracts were incubated at 251C for20 min using the Light Shift Chemiluminescent EMSA Kit (Pierce,Rockford, IL). The reaction mixture was separated on 6% PAGE,and the products were detected by Stabilized Streptavidin-Horseradish Peroxidase Conjugate (Pierce). For competitionassays, unlabeled probes at 200-fold molar excess were added tothe reaction mixture before the addition of biotin-labeled probes.

Quantification of the band density of DNA–protein complexes inEMSA was performed by using a UVP GDS-8000 image analysissystem (UVP, Inc., Upland, CA).

Real-time Analysis of FEN1 RNA

Thirty-eight normal lung tissues adjacent to the tumors wereobtained from surgically removed specimens of patients (28 malesand 10 females). The normal tissues sampled at least 5 cm awayfrom the margin of the tumor were immediately stored in liquidnitrogen. Total RNA was isolated and converted to cDNA using anoligo(dT)15 primer and Superscript II (Invitrogen). Relative geneexpression quantitation for FEN1 and b-actin as an internalreference gene was carried out using the ABI 7900HT real-timePCR system in triplicate, based on the SYBR-Green method. Theprimers used for FEN1 were 50-ctgtggacctcatccagaagca-30 and 50-ccagcacctcaggttccaaga-30; and for b-actin were 50-ggcggcaccaccatg-taccct-30 and 50-aggggccggactcgtcatact-30. PCR specificity wasconfirmed by dissociation curve analysis and gel electrophoresis.All analysis was performed in a blinded fashion with thelaboratory persons unaware of genotyping data. The expressionof individual FEN1 measurements was calculated relative toexpression of b-actin using a modification of the method asdescribed previously [Lehmann and Kreipe, 2001].

Statistical Analysis and Haplotype Construction

Olive TM values were normalized by natural logarithm (ln)transformation. The associations between the FEN1 genotypes andthe ln-transformed Olive TM values were tested by analysis ofcovariance, followed by a Bonferroni correction for multiplecomparisons with adjustment for age, sex, work site, drinking status,and pack-years of cigarette smoking. The associations between FEN1genotypes and risk of lung cancer were estimated by odds ratios(ORs) and their 95% confidence intervals (95% CIs) computed bylogistic regression models. All ORs were adjusted for age, sex, andsmoking, where it was appropriate. A more than multiplicativegene–environment interaction was evaluated by logistic regressionanalysis including main effect variables and their product terms.When the test for multiplicative interaction was not rejected, furthertest for an additive interaction was performed by a bootstrapping testof goodness of fit of the null hypothesis for no departure from anadditive model versus an alternative hypothesis for a departure froman additive model [Brennan, 2002]. A Student’s t test was used toexamine the differences in luciferase reporter gene expression, andMann-Whitney U-test was used to compare pack-years smoked orenvironmental particulate-B[a]P concentration between coke-ovenworkers and noncoke-oven workers and assess differences in FEN1transcript abundance with different genotypes. These statisticalanalyses were implemented in Statistic Analysis System software(version 8.0, SAS Institute, Cary, NC) and Statistical Package forSocial Sciences (version 12.0, SPSS Inc., Chicago, IL). Haploview 3.2software was used to construct the haplotypes and Haplo.statssoftware package developed using the R language was used toestimate adjusted ORs and 95% CIs for each haplotype [Schaid et al.,2002]. Simulations were run for 1,000 times for empirical p values.

Results

Identification of FEN1 SNPs

By sequencing the 50-flanking region (�2 kb), 50-untranslatedregions (UTR), coding regions, and 30-UTR of FEN1 from 30

1322 HUMAN MUTATION, Vol. 30, No. 9, 1320–1328, 2009

healthy individuals, we identified two SNPs, c.�69G4A(rs174538: G4A) and c.4150G4T (rs4246215: G4T), whichare located in the promoter region and 30-UTR, respectively(Supp. Fig. S1). The allelic frequencies for the �69A and 4150 Twere 0.368 and 0.372 among 1,131 controls in northern Chinesepopulation, and 0.457 and 0.456 among 827 controls in southernChinese population. The distribution of allelic frequencies of bothSNPs differed significantly between northern and southernChinese populations (po0.0001). Linkage disequilibrium analysisshowed that these two SNPs are in strong linkage, with D05 0.94and r2 5 0.83 in northern Chinese population and D05 0.98 andr2 5 0.96 in southern Chinese population.

Effects of FEN1 SNPs on FEN1 Expression

Because c.�69G4A polymorphism is located in the corepromoter region of FEN1, we evaluated whether this variant hasallele-specific effect on FEN1 transcriptional activity. As shown inFigure 1A, relative luciferase expression driven by the �69G-containing promoter were 37–59% of those driven by the �69A-containing promoter in the two types of cell lines (all po0.001).Similar results were obtained when reporter gene expressiondriven by different 30-UTR constructs, pGL3c–4150 T orpGL3c–4150G, was compared, with the expression levels being16 to 21% greater for pGL3c–4150 T than pGL3c–4150G (allpo0.001; Fig. 1B). These results suggest that the FEN1 �69A or4150 T variant may have heightened expression in vivo. Wetherefore examined the association between FEN1 genotypes atthese SNP sites and FEN1 RNA expression levels in lung tissuesdetermined by real-time RT-PCR. We found that subjects with the�69AA genotype had significantly higher FEN1 RNA levels(mean7SE) than those with the �69GG genotype [0.19770.035

(n 5 9) vs. 0.05770.008 (n 5 17); p 5 0.004]. However, the�69GA genotype carriers had a level [0.13870.020 (n 5 12)]similar to that of the AA genotype carriers (p 5 0.142). When theGA and AA genotype were pooled together in analysis, thedifference between the GA1AA genotype and GG genotype wasalso significant (0.16370.020 vs. 0.05770.008; po0.001). Similarresults were observed when the FEN1 RNA levels were comparedas a function of 4150G4T genotypes (data not shown).

Effect of FEN1 Promoter SNP on Nuclear Protein BindingActivity

We then examined whether the FEN1 c.�69G4A SNP changesthe binding pattern of transcriptional factors. Electrophoreticmobility-shift assays showed that nuclear proteins prepared fromA549 cells were able to bind to both �69G and �69A probes(Fig. 2, bands I and II). However, under the same experimentalconditions, the levels of the nuclear proteins bound to the �69Gprobe were 2.58-fold (band I ) and 2.66-fold (band II) higher thanthose bound to the �69A probe (Fig. 2, lane 2 vs. lane 6) asquantified by UVP GDS-8000 image analysis system. Competitionassays showed that the addition of unlabeled �69G or �69A probeto the reaction mixture completely eliminated these DNA–proteincomplexes, indicating that the binding are sequence-specific(Fig. 2, lanes 3, 4 and 7, 8). Similar results were seen in assayswith nuclear proteins derived from HeLa cells (data not shown).

Association of FEN1 SNPs with DNA Damage amongCoke-Oven Workers

DNA damage levels among 303 coke-oven workers and 297controls were determined by comet assays. The general character-

Figure 1. Transient luciferase reporter gene expression assays with constructs containing different alleles of FEN1 promoter (A) or 30-UTR(B) in HeLa and A549 cells. Upper: schematic representation of different reporter gene constructs; Lower: luciferase expression of theseconstructs. The cells were cotransfected with pRL-SV40 to standardize transfection efficiency. Fold increase was measured by defining theactivity of the empty pGL3-Basic or pGL3-Control vector as 1. All experiments were performed in triplicates at least in three independenttransfection experiments and each value represents mean7SD. �po0.001 compared with each of the construct.


istics and Olive TM of the subjects are shown in Table 1. Themedian Olive TM value (range) in coke-oven workers wassignificantly higher than that in noncoke-oven workers [�0.85(range, �2.58–1.45) vs. �1.12 (range, �3.51–1.15); Po0.001].The median values of air benzene soluble mater (BSM,0.85 mg/m3) and particulate-B[a]P (630 ng/m3) in coke-ovenworking environment were significantly higher than those innoncoke-oven working environment (BSM, under detection limit;B[a]P, 40 ng/m3; both Po0.001), with the highest being at the topof the oven (BSM, 4.47 mg/m3; B[a]P, 3960 ng/m3) [Tan et al.,2006]. Figure 3 shows the association between Olive TM valuesand FEN1 genotypes. Among coke-oven workers, the medianOlive TM values were significantly higher in the �69GG [�0.59(range, �2.28–1.45), n 5 126; po0.001] and �69GA [�0.90(range, �2.02–1.35), n 5 123; p 5 0.023] genotypes than in the�69AA genotype [�1.11 (range, �2.58–0.93), n 5 54] adjustedfor age, sex, work site, drinking, and smoking. However, no suchdifferences were found among noncoke-oven workers (Fig. 3A).Similar results were also observed when subjects were categorizedby the c.4150G4T genotype (Fig. 3B). These results clearlyindicated that DNA damage levels in exposed subjects wereassociated with the FEN1 �69G or 4150G variant in an alleledose-dependent manner.

Association between FEN1 SNP and Lung Cancer Risk

To assess the association between FEN1 SNPs and risk ofdeveloping lung cancer, two case–control analyses were conductedindependently by two laboratories at Chinese Academy of MedicalSciences in northern (Beijing) and Tongji Medical College insouthern (Wuhan, Hubei Province) Chinese populations. Thesubject characteristics are shown in Table 2. All observed genotypefrequencies in both controls and patients conform to Hardy-Weinberg equilibrium. In the northern Chinese population,frequencies of the �9AA, GA and GG genotypes in cases weresignificantly different from those in controls (w2 5 18.92;po0.0001; df 5 2; Table 3). Similarly, frequencies of the 4150TT,GT, and GG genotypes were also significantly different betweencases and controls (w2 5 7.86; p 5 0.02; df 5 2). Logistic regressionanalysis showed that the �69GG genotype had a 1.66-foldincreased risk for lung cancer compared with the �69AA genotype(95% CI, 1.24–2.22), and the 4150GG genotype had a 1.44-foldincreased risk compared with the 4150TT genotype (95% CI,1.09–1.91). However, the heterozygous genotypes of both poly-morphisms were not associated with increased risk. The associa-tion was confirmed in southern Chinese population where the ORfor the �69GG or 4150GG genotype was 1.37 (95% CI, 1.03–1.83)or 1.40 (95% CI, 1.05–1.86) compared with the �69AA or 4150TTgenotype, respectively. Interestingly, in this population, increasedlung cancer risk was also associated with the heterozygous �69GA(OR, 1.32; 95% CI, 1.07–1.63) and 4150GT (OR, 1.35; 95% CI,1.03–1.77) genotypes. Adjustment for age, sex, and smoking didnot significantly change the respective ORs.

Because c.�69G4A and c.4150G4T polymorphisms are inlinkage disequilibrium, we constructed two-marker haplotypes(Table 4). Compared with the A-69T4150 haplotype, all the otherthree haplotypes were associated with increased risk of developinglung cancer in Beijing cohort, with the adjusted ORs being 1.30(95% CI, 1.14–1.48; P 5 0.031), 2.12 (95% CI, 1.30–3.48;P 5 0.021) and 2.67 (95% CI, 1.87–3.80; po0.001) for theG-69G4150, A-69G4150, and G-69T4150 haplotypes, respectively.However, we found that in Wuhan cohort only the G-69G4150

haplotype was associated with significantly increased risk ofdeveloping lung cancer (OR, 1.18, 95% CI, 1.03–1.35) comparedwith the A-69T4150 haplotype.

Effects of Gene–Smoking Interaction

Because DNA damage caused by tobacco carcinogens includingPAHs is believed to be an important mechanism underlying lungcarcinogenesis [Hecht, 1999], we thus investigated whether aninteraction exists between the FEN1 polymorphisms and smoking

Figure 2. Electrophoretic mobility shift assays with biotin-labeledoligonucleotides containing the �69G or �69A allele and nuclearextracts from A549 cells. Lanes 1 and 5, probes only; lanes 2 and 6,probes and nuclear extracts; lanes 3 and 7, or 4 and 8, probes andnuclear extracts plus 200-fold �69A or �69G unlabeled competitors,respectively. The arrow localizes the two major oligonucleotide/nuclear protein complexes (Band I and Band II).

Table 1. Characteristics of Study Subjects for DNA Damage Analysis by Exposure Status

Variable Coke-oven worker (n 5 303) Noncoke-oven worker (n 5 297) p

Sex (male/female) 274/29 253/44 0.050a

Age (year, mean7SD) 38.873.8 39.378.6 0.537b

Smoking status (yes/no) 206/97 179/118 0.049a

Median pack-years smoked (range) 8.0 (0.0–62.0) 5.6 (0.0–80.0) 0.012c

Drinking (yes/no) 153/150 144/153 0.622a

Median Olive TM (range) �0.85 (�2.58–1.45) �1.12 (�3.51–1.15) o0.001b

Benzene soluble mater [BSM, mg/m3, median (range)] 0.85 (0.68–4.47) UDL NC

Particulate-B[a]P [ng/m3, median (range)] 630 (200–3960) 40 (35–42) o0.001c

UDL, under detection limit; NC, not calculated.aTwo-sided w2 test for comparison between two groups.bStudent’s t tests for comparison between two groups.cMann-Whitney U-test for comparison between two groups.


in lung cancer risk in both Beijing and Wuhan cohorts (Table 5).It is interesting to note that among nonsmokers in the twocohorts, both �69GA and �69GG variant genotypes were notassociated with increased risk of lung cancer. However, smokershaving �69GG genotype had much higher risk, with the ORsbeing 3.25 (95% CI, 2.20–4.82) in Beijing cohort and 2.43 (95%CI, 1.62–3.66) in Wuhan cohort, compared with smokers havingthe �69AA genotype (OR, 1.49; 95% CI, 0.92–2.44 and OR, 1.47;95% CI, 0.94–2.30; homogeneity test P 5 0.0001 and P 5 0.0049,respectively). Similar results were also seen for the 4150GGgenotype; smokers having the 4150GG genotype had ORs of 2.90(95% CI, 1.97–4.26) in Beijing cohort and 2.59 (95% CI,1.72–3.90) in the Wuhan cohort, which were respectively higher

than that for smokers having the 4150TT genotype (OR, 1.67; 95%CI, 1.05–2.67 in the Beijing cohort and OR, 1.58; 95% CI,1.01–2.47 in the Wuhan cohort; homogeneity test p 5 0.0038 andP 5 0.0059, respectively). In the Beijing cohort, a test for additiveinteraction between smoking (nonsmoker vs. smoker) and thec.�69G4A SNP was significant (p 5 0.020). A similar trend wasobserved for the interaction between smoking and the c.4150G4TSNP, which is in strong linkage to the c.�69G4A SNP (p 5 0.021;Table 5). However, we did not observe such an interaction betweensmoking and genotypes in Wuhan cohort (Table 5). On the otherhand, stratification analysis did not show any significant effect ofage, sex, or pack-years on risk of lung cancer related to these twogenetic polymorphisms (data not shown).

Figure 3. Box plot of ln-transformed Olive tail moment (Olive TM) values by FEN1 genotypes. A: the c.�69G4A variant. B: The c.4150T4Gvariant. In coke-oven workers, subjects with �69GG (n 5 126) and GA (n 5 123) genotypes, or 4150GG (n 5 125) and GT (n 5 124) genotypes hadsignificantly higher median values than subjects with the �69AA (n 5 54) or 4150TT (n 5 54) genotype (all po0.05), whereas no such effect wasseen in noncoke-oven workers. The line inside each box represents the median; the upper and lower limits of the box are the 75th and 25thpercentiles, respectively, and the vertical bars above and below the box indicate the maximum and minimum values, respectively. The solidcircles are outlier values.

Table 2. Distribution of Selected Characteristics among Cases and Controls in Chinese Populations

Beijing cohort Wuhan cohort

Cases (n 5 1013) Controls (n 5 1131) Cases (n 5 827) Controls (n 5 827)

Variable n (%) n (%) pa n (%) n (%) pa

Age (year) 0.162 0.840

o50 218 (21.5) 251 (22.2) 182 (22.0) 190 (23.0)

51–60 336 (33.2) 342 (30.2) 244 (29.5) 236 (28.5)

61–70 334 (33.0) 417 (36.9) 256 (31.0) 246 (29.7)

470 125 (12.3) 121 (10.7) 145 (17.5) 155 (18.7)

Sex 0.556 0.390

Male 683 (67.4) 776 (68.6) 668 (80.8) 654 (79.1)

Female 330 (32.6) 355 (31.4) 159 (19.2) 173 (20.9)

Smoking status o0.001 o0.001

No 414 (40.9) 639 (56.5) 238 (28.8) 402 (48.6)

Yes 599 (59.1) 492 (43.5) 589 (71.2) 425 (51.4)

Pack-years smoked o0.001 o0.001

r20 (or r24)b 174 (29.0) 294 (59.8) 158 (26.8) 212 (49.9)

420 (or 424)b 425 (71.0) 198 (40.2) 431 (73.2) 213 (50.1)

Histological type

Squamous cell carcinoma 406 (40.1) 295 (35.7)

Adenocarcinoma 358 (35.3) 314 (37.9)

Otherc 249 (24.6) 218 (26.4)

aTwo-sided w2 test.bLight and heavy smokers were categorized by the 50th percentile pack-years value among controls, o20 or 420 pack-years in Northern population and o24 or 424 pack-years in Southern population.cOthers includes small cell lung cancer, undifferentiated cancer, bronchioalveolar carcinoma, and mixed cell carcinoma.


Discussion

In the present study, we employed a gene-based approach toinvestigate the associations between SNPs in the FEN1 locus andDNA damage in coke-oven workers and risk of developing lungcancer in a case–control design. We identified two SNPs, c.�69G4Aand c.4150G4T, located in the promoter region and 30-UTR ofFEN1, which are in strong linkage disequilibrium. We found thatthese SNPs were significantly associated with reduced FEN1expression, elevated DNA damage in PAH-exposed coke-ovenworkers and elevated risk of developing lung cancer in two generalChinese populations. Moreover, an additive interaction between theseSNPs and smoking in intensifying risk of lung cancer was observed.The SNPs displayed their effects only in smokers but not nonsmokers.

These results are in agreement with our previous studies indicatedthat functional SNPs in the base-excision repair genes conferpredisposition to lung cancer [Hao et al., 2006; Zhang et al., 2005].These results also support the notion that genetic polymorphismsinfluencing DNA repair capacity play a role in human carcinogenesis.

As a multifunctional nuclease, FEN1 has been found acrossdifferent animal kingdoms from archaebacteria to humans [Shenet al., 2005]. This structurally and functionally conserved proteinplays a very important role in genome maintenance. InSaccharomyces cerevisiae, FEN1-null mutants (DRad27) exhibit acomplex phenotype. If deletions of other important DNAmetabolic genes (i.e., RAD9, RAD17, or RAD24) happen at thesame time, DRad27 is synthetic lethal [Paulovich et al., 1998]. Inmice, Fen1 homozygous knockout is embryonic lethal; however,Fen1 heterozygous knockout, combined with a mutation of theadenomatous polyposis coli (APC) gene, causes gastrointestinaltract adenocarcinomas with decreased animal survival [Kucherla-pati et al., 2002]. Recently, a transgenic mouse model harboringthe E160D mutation in Fen1, which represents some frequentlyoccurring mutations in certain types of human cancer, wasestablished [Zheng et al., 2007]. The strong mutator phenotypeand accumulation of incompletely digested apoptotic DNAfragments resulting from Fen1 deficiency, which leads to genomeinstability, chronic inflammation, and initiation of cancer, wereobserved in the animal model [Zheng et al., 2007]. All thesefindings indicate that FEN1 is a cancer susceptibility gene and,thus, it would be expected that SNPs affecting FEN1 expressionand/or function may modify cancer risk.

Because FEN1 is an important tumor suppressor [Henneke et al.,2003a], regulation of its function at the posttranslational level, such asacetylation [Hasan et al., 2001], phosphorylation [Henneke et al.,2003b], and protein–protein interactions [Chapados et al., 2004; Guoet al., 2008], has been extensively studied. However, regulation atFEN1 RNA transcriptional level has not been illustrated yet.Accumulating evidence demonstrates that genetic polymorphisms inthe gene promoter and 30-UTR regions may affect transcriptional andposttranscriptional expression. The region between �128 base pairand 351 base pair has been identified and shown to be essential forbasal activity of the FEN1 promoter [Singh et al., 2008]. Interestingly,our results showed that the c.�69G4A SNP located in this regioncauses increased promoter activity, which is most likely to be due to ahigher binding activity of the G allele toward some unknowntranscriptional inhibitors. In addition, the c.4150G4T SNP is alsoassociated with differential levels of FEN1 RNA expression. Werationalized that the elevated FEN1 RNA expression in 4150 T allelemight be attributed to the destruction of some repressive microRNAbinding site within the 30-UTR [Filipowicz et al., 2008; Hagan andCroce, 2007; Mayr et al., 2007], although further studies are needed toexamine this postulation. Moreover, consistent with previous report[Ma et al., 2000], we did not find any SNPs in the coding region ofthis highly conserved DNA repair gene. Taken together, these findingsfurther indicated that naturally occurring genetic polymorphisms inthe regulation regions of cancer-related genes may underliephenotypic variations in susceptibility to cancer.

Exposure to PAHs has been associated etiologically with lung cancerin several occupational circumstances, such as coke production [IARC,1983] or tobacco smoking [Parkin et al., 2005]. In this study, we foundthat DNA damage levels among coke-oven workers were significantlyhigher than those among noncoke-oven workers, which is in line withprevious reports [Chen et al., 2006; Leng et al., 2004; Yang et al., 2008],and indicated that DNA damage in coke-oven workers was due toexposure to coke-oven emission. However, although exposed subjectshad higher DNA damage levels than unexposed subjects, higher Olive

Table 3. Genotype Frequencies of FEN1 among Cases andControls and Their Association with the Risk of Lung Cancer inBeijing and Wuhan Case–Control Cohorts

Cohort Genotype Cases, n (%)

Controls,

n (%) ORa (95% CI) p

c.–69G4A

Beijing n 5 1013 n 5 1131

AA 106 (10.4) 168 (14.8) 1.00 (Reference)

GA 402 (39.7) 496 (43.9) 1.22 (0.91–1.62) 0.179

GG 505 (49.9) 467 (41.3) 1.66 (1.24–2.22) 5.9� 10�4

ptrendb 7.8� 10�5

Wuhan n 5 827 n 5 827

AA 147 (17.8) 186 (22.5) 1.00 (Reference)

GA 394 (47.6) 384 (46.4) 1.32 (1.01–1.74) 0.045

GG 286 (34.6) 257 (31.1) 1.37 (1.03–1.83) 0.029

ptrendb 0.020

c.4150G4T

Beijing n 5 1013 n 5 1131

TT 124 (12.2) 171 (15.1) 1.00 (Reference)

GT 421 (41.6) 500 (44.2) 1.13 (0.86–1.49) 0.389

GG 468 (46.2) 460 (40.7) 1.44 (1.09–1.91) 0.011

ptrendb 0.020

Wuhan n 5 827 n 5 827

TT 147 (17.8) 187 (22.6) 1.00 (Reference)

GT 394 (47.6) 383 (46.3) 1.35 (1.03–1.77) 0.032

GG 286 (34.6) 257 (31.1) 1.40 (1.05–1.86) 0.022

ptrendb 0.018

OR, odds ratio; CI, confidence interval.aData were calculated by logistic regression with adjustment for age, sex, andsmoking.bTest for trend of odds was two-sided and based on likelihood ratio test assuming amultiplicative model.

Table 4. Distribution of FEN1 Haplotypes among Patients andControls and Their Association with Lung Cancer in Beijing andWuhan Cohorts

No. of chromosome (%)

Haplotype Patients Controls ORa (95% CI) pb

Beijing cohort

A-69T4150 575 (28.4) 812 (35.9) 1.00 (Reference)

G-69G4150 1,939 (65.1) 1,400 (61.9) 1.30 (1.14–1.48) 0.031

A-69G4150 38 (1.9) 20 (0.9) 2.12 (1.30–3.48) 0.021

G-69T4150 93 (4.6) 29 (1.3) 2.67 (1.87–3.80) o0.001

Wuhan cohort

A-69T4150 676 (40.9) 746 (45.1) 1.00 (Reference)

G-69T4150 12 (0.7) 12 (0.7) 1.09 (0.50–2.36) 1.000

A-69G4150 12 (0.7) 10 (0.6) 1.17 (0.53–2.59) 0.840

G-69G4150 954 (57.7) 887 (53.6) 1.18 (1.03–1.35) 0.020

OR, odds ratio; CI confidence interval.aAdjusted for sex, age, and smoking status.bAfter 1,000 permutation tests.


TM values were seen among subjects having the FEN1 �69G or4150G allele compared with subjects having the FEN1�69A or 4150 Tallele. These results implicated that the FEN1 SNPs were positivelyassociated with differential individual DNA repair capability towardscoke-oven emission exposure. Although a high level of PAH exposureis confined to certain occupations such as coke-oven workers, tobaccosmoking is an important source of PAH exposure in generalpopulations. We found that the FEN1 SNPs confer susceptibility tolung cancer in both northern and southern Chinese populations in amanner of interaction with smoking. Because tobacco smoke containshigh levels of PAHs and numerous other DNA-damaging agents, sucha gene–environment interaction would be expected and biologicalreasonable. However, we did not detect any association between FEN1genotypes and DNA damage levels in noncoke-oven workers. Becausenoncoke-oven workers did not have high exposure to PAH and hadsignificantly lower pack-years smoked compared with coke-ovenworkers (5.6 vs. 8.0), such a result is reasonable, and may suggest thatthe difference in the DNA damage levels related to FEN1 genotypesonly occurs when exposure reaches to a certain threshold.

We observed a significant difference in the allelic and genotypefrequencies of both c.�69G4A and c.4150G4T SNPs in twoChinese populations included in the present study. The poly-morphisms displayed different action models for risk of thecancer, recessive effect in northern population and dominanteffect in southern population. These results suggest that theseSNPs in the FEN1 gene may be relatively novel, probably resultingfrom certain different environmental pressures in differentgeographical areas. Therefore, it would be interesting to conductfurther studies in different ethnic populations for comparison.However, despite of the significant difference in genotypefrequency in northern and southern Chinese populations, theseSNPs showed a consistent association with risk of lung cancer intwo independent case–control cohorts. Having relatively large

sample sizes, significantly increased odd ratios with small p values,our results are unlikely to be attributable to selection bias orunknown confounding factors. More importantly, our data onSNP function and the genotype–phenotype relationship betweenthe SNPs and DNA damage/repair all support our conclusion.

In summary, we identified two SNPs in the FEN1 gene that areassociated with increased DNA damage in subjects exposed todefined carcinogens and increased risk of developing lung cancer ina manner of interaction with tobacco smoking. These results providefurther evidence highlighting FEN1 as an important gene in humancarcinogenesis. However, because this is the first report concerningthe FEN1 polymorphisms and lung cancer risk in Chinesepopulation, independent studies are needed to validate our findings.

Acknowledgments

We thank Yuying Liu, Tong Sun, and Junniao Liu for their assistance in

recruiting patients and technical support. Contract grant sponsor:

National Natural Science Foundation (grant 30530710 to D.L.) and State

Key Basic Research Program (grant 2004CB518701 to D.L. and grant

2002CB512905 to T.W.). Conflict Interests: no conflict financial interests

exist.

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functional fen1 polymorphisms are associated with dna damage levels and lung cancer risk

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