Genetic polymorphisms in DNA repair genes and risk of lung cancer

Dorota Butkiewicz, Marek Rusin, Lindsey Enewold1, Peter G. Shields1,2, Mieczyslaw Chorazy and Curtis C. Harris2,3

Department of Tumor Biology, Centre of Oncology, M.Sklodowska-Curie Memorial Institute, 44–101 Gliwice, Poland,
1 Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC 20007 and
2 Laboratory of Human Carcinogenesis, Division of Basic Science, National Cancer Institute, NIH, 37 Convent Drive, Building 37, Room 2C05, Bethesda, MD 20892-4255, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Polymorphisms in DNA repair genes may be associated with differences in the repair efficiency of DNA damage and may influence an individual's risk of lung cancer. The frequencies of several amino acid substitutions in XRCC1 (Arg194Trp, Arg280His and Arg399Gln), XRCC3 (Thr241Met), XPD (Ile199Met, His201Tyr, Asp312Asn and Lys751Gln) and XPF (Pro379Ser) genes were studied in 96 non-small-cell lung cancer (NSCLC) cases and in 96 healthy controls matched for age, gender and cigarette smoking. The XPD codon 312 Asp/Asp genotype was found to have almost twice the risk of lung cancer when the Asp/Asn + Asn/Asn combined genotype served as reference [odds ratio (OR) 1.86, 95% confidence interval (CI), 1.02–3.40]. In light cigarette smokers (less than the median of 34.5 pack-years), the XPD codon 312 Asp/Asp genotype was more frequent among cases than in controls and was associated with an increased risk of NSCLC. Compared with the Asn/Asn carriers, the OR in light smokers with the Asp/Asn genotype was 1.70 (CI0.35 0.43–6.74) and the OR in those with the Asp/Asp genotype was 5.32 (CI0.35–21.02) (P trend = 0.01). The 312 Asp/Asp genotype was not associated with lung cancer risk in never-smokers or heavy smokers (>34.5 pack-years). The XPD-312Asp and -751Lys polymorphisms were in linkage disequilibrium in the group studied; this finding was further supported by pedigree analysis of four families from Utah. The XPD 312Asp amino acid is evolutionarily conserved and is located in the seven-motif helicase domain of the RecQ family of DNA helicases. Our results indicate that these polymorphisms in the XPD gene should be investigated further for the possible attenuation of DNA repair and apoptotic functions and that additional molecular epidemiological studies are warranted to extend these findings.

Abbreviations: CI, confidence interval; HNPCC, hereditary nonpolyposis colon cancer; NER, nucleotide excision repair; NSCLC, non-small-cell lung cancer; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
DNA repair maintains the integrity of the human genome by reducing the mutation frequency of cancer-related genes (1). A deficiency in DNA damage repair is associated with an increased cancer risk; for example, germline mutations in mismatch repair genes lead to hereditary nonpolyposis colon cancer (HNPCC), while an inherited defect in nucleotide excision repair (NER) genes leads to xeroderma pigmentosum (24). Variations between individuals in DNA repair capacity occur in humans and may be a risk factor for cancer (59). Although tobacco smoking is the primary cause of lung cancer, a genetic susceptibility factor also can modulate the risk of smoking-related lung cancer (1013). For example, the DNA repair capacity was found to be significantly lower in lung cancer cases than in healthy controls and it was especially pronounced among younger patients and smokers (8). Individuals with a reduced DNA repair capacity also have a high level of carcinogen–DNA adducts in their tissues (14).

Recently, nine amino acid substitution variants in several DNA repair genes (e.g., in XPD and XPF genes belonging to the NER pathway, and in XRCC1 and XRCC3 genes associated with double-strand/recombination repair) were identified and estimated as occurring at the polymorphic frequency in the population studied (15). Five additional single-nucleotide polymorphisms in the coding regions of the XPF gene have been described by Fan et al. (16). The biological function of these amino acid substitutions has not yet been elucidated, but some of these variants may be associated with a reduced repair capacity and increased cancer susceptibility.

We have investigated the frequency of single nucleotide substitutions in the coding regions of the XRCC1, XRCC3, XPD and XPF genes in 192 inhabitants of a highly industrialized and polluted region of Upper Silesia, Poland [96 non-small-cell lung cancer (NSCLC) patients and 96 healthy controls]. The following amino acid substitutions were analyzed: those encoded by exons 6, 9 and 10 of the XRCC1 gene (Arg194Trp, Arg280His and Arg399Gln, respectively); exon 7 of the XRCC3 gene (Thr241Met); exons 8, 10 and 23 of the XPD gene (Ile199Met, His201Tyr, Asp312Asn and Lys751Gln, respectively); and exon 7 of the XPF gene (Pro379Ser). Pedigree analysis, including 52 members of four, three-generation families from Utah (who were CEPH reference families), was also performed to check for the presence of possible linkage between the polymorphisms and to identify haplotypes. We investigated the hypothesis that the studied polymorphisms were indicators of lung cancer risk.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study groups
The 96 cases were males with primary NSCLC who had undergone surgery at the Silesian Medical Academy Hospital in Zabrze, Poland, between 1991 and 1995 (17,18). There were 65 (68%) cases of squamous cell carcinoma, 25 (26%) adenocarcinomas and six (6%) large cell carcinomas, all diagnosed according to the WHO classification (19). The mean age of cases was 56.8 ± 8.7 years (mean ± SD, range 39–76 years). The group included 17 (18%) never-smokers, 21 (22%) ex-smokers and 58 (60%) current smokers (57.0 ± 33.0 pack-years, mean ± SD, data for smokers). The control group included 96 unrelated healthy males (56.3 ± 8.8 years, mean ± SD, range 39–79 years), matched to the cases by age, smoking habit and occupational exposure. These individuals were selected partly from the Upper Silesian population recruited previously for occupational studies (20,21). There were 17 (18%) never-smokers, 26 (27%) ex-smokers and 53 (55%) current smokers (55.7 ± 34.9 pack-years, mean ± SD, data for smokers). Sixty-one (64%) of the cases and 60 (62%) of the controls had been occupationally exposed to fossil fuel-derived substances; they included coal miners, coke oven workers, car drivers and mechanics exposed to diesel exhaust, coal and oil derivatives, and coal dust. A trained interviewer collected detailed information on age, occupational history, cigarette smoking, lifestyle, family cancer history, exposure and dietary habits from all cases and controls with a standardized questionnaire. All individuals enrolled in the study were inhabitants of a highly industrialized and polluted region of Poland (Upper Silesia) (22).

Fifty-two members of four families (numbers 1345, 1333, 1331 and 1340) from Utah, all of which were CEPH reference families, were also included in the study (National Institute of General Medical Sciences, Human Genetics Mutant Cell Respiratory, Coriell Institute, Camden, NJ).

Detection of the polymorphisms
DNA was isolated from frozen non-tumorous lung tissue (cases) and blood (controls) samples using standard procedures with sodium dodecyl sulfate (SDS)–proteinase K–RNase digestion and phenol–chloroform extraction. For the family members, DNA samples isolated from immortalized lymphoblasts were purchased from Coriell Cell Repositories. The polymorphisms were analyzed by polymerase chain reaction (PCR) assays combined with restriction fragment length polymorphism (RFLP) assays or DNA sequencing. PCR primer sequences used for amplification were identical to those published by Shen et al. (15), except for the XRCC1 codon 280 (5'-CCAGTGGTACTAACCTAATC-3', 5'-CACTCAGCACCAGTACCACA-3') and XRCC1 codon 399 polymorphisms (5'-TAAGGAGTGGGTGCCGGACTGTC-3', 5'-AGTAGTCTGCTGGCTCTGG-3'). To detect polymorphisms in XRCC1 codons 194, 280 and 399 XRCC3 codon 241, XPF codon 379 and XPD codons 312 and 751, 50 ng of DNA was used as a template in the PCR reaction with 1x PCR standard buffer II (Perkin Elmer), 1.5 mM MgCl2, 0.2 mM of each dNTP (Pharmacia), primers (12.5 pmol each) and 2 U AmpliTaq polymerase (Perkin Elmer) in a total volume of 25 µl. Amplification of the XPD exon 10 fragment was carried out with 5% dimethylsulfoxide (DMSO). PCR for XPD exon 8 was performed in a total volume of 30 µl containing 100 ng of DNA and 1.5 U AmpliTaq Gold (Perkin Elmer). We used the following thermal cycling conditions: denaturation, 94°C for 4 min followed by 35–40 cycles of denaturation at 94°C for 30 s; primer annealing, 30 s at 55°C for XRCC1-280, XPF-379 and XPD-751; at 57°C for XPD-199 and -201; at 60°C for XRCC1-194, XRCC1-399 and XRCC3-241; or at 64°C for XPD-312; primer extension, 72°C for 30 s. The cycles were followed by a final extension step at 72°C for 4 min. The PCR products (15 µl) were then digested according to the manufacturer's instructions (New England Biolabs) with the following restriction enzymes: 10 U of PstI (the restriction site is present in the XPD-751Gln allele), 5 U of NlaIII and 2 U of Sau3AI (the NlaIII restriction site is lost in the XPD-199Met allele, and the Sau3AI restriction site is lost in the XPD-201Tyr allele), 10 U of MspI (this restriction site is lost in the XRCC1-194Trp and XRCC1-399Gln alleles), 5 U of RsaI (this restriction site is lost in the XRCC1-280His allele) and 5 U NlaIII (this restriction site is lost in the XRCC3-241Thr allele). The digestion products were separated using 3–4% agarose gels (FMC BioProducts). Polymorphisms in XPD-312 and XPF-379 were detected by direct DNA sequencing of PCR products that were previously enzymatically purified with exonuclease and shrimp alkaline phosphatase (Amersham Life Science). The XPF exon 7 PCR fragment was sequenced using a Thermo Sequence Dye Terminator Cycle Sequencing Pre-mix kit version 2.0 (Amersham Life Science) and the same sense primer that was used for the PCR reaction. A BigDye Terminator Cycle sequencing kit (Perkin Elmer) and an additional sense primer (5'-AGAAGACGGTGCTCAGGTG-3') were used for sequencing the XPD exon 10 PCR fragment. The sequencing products were analyzed with the ABI Prism 377 DNA automated sequencer (Perkin Elmer).

Statistical analysis
Unconditional logistic regression was performed to assess the association of the genotype and lung cancer risk using the SAS statistical package (Proc Logistic, Cary, NC). The dependent variable was lung cancer case status and the independent variables were genotype (separately and grouped together for Asp/Asn and Asn/Asn, as shown). The models included the independent variables of smoking categorization by pack-years (three categories: never-smokers and smokers who had smoked less than or more than the median in controls, i.e. 34.5 pack-years) and age. The association between XPD-312 and XPD-751 was assessed using Fisher's exact test (SAS software).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The frequencies of the polymorphisms in cancer cases and controls are shown in Table IGo. The distributions of the XPD-312, XPD-751, XRCC1-399 and XRCC3-241 genotypes were in Hardy–Weinberg equilibrium. There was a statistically significant association between the XPD-312 genotypes and lung cancer risk (Table IIGo). To assess the relationship, adjusted odds ratios (OR) were calculated using both the XPD-312Asn/Asn homozygous genotype alone or combined with the Asp/Asn heterozygous genotype as the reference groups. While the ORs for the Asp/Asp and Asp/Asn genotypes were not significantly different from that for the Asn/Asn reference group, there was a difference for the Asp/Asp genotype when compared with the Asp/Asn + Asn/Asn genotypes [OR = 1.86, 95% confidence interval (CI) = 1.02–3.40]. When the distribution of genotypes (wild-type homozygotes, heterozygotes and polymorphic homozygotes) in cancer patients and controls was examined in more detail, it was found that there was a higher frequency of the XPD-312Asp/Asp genotype in cases with Kreyberg type I carcinoma (49%, 35/71) (squamous and large cell carcinomas combined) than in controls (31%, 29/94) (P = 0.04; data not shown). When smoking habits were considered, light smokers (those smoking for <34.5 pack-years) had a statistically significant increased risk by genotype that was consistent with a gene-dose effect, but similar findings were not seen for never-smokers or persons who had smoked for >34.5 pack-years (Table IIIGo). In light smokers, the risk of NSCLC was higher in those with the Asp/Asp genotype than in those with the Asn/Asn genotype (OR = 5.32, 95% CI = 1.35–21.02) or with the Asp/Asn + Asn/Asn combined genotype (OR = 3.89, 95% CI = 1.32–11.5).


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Table I. Allelic frequency of the XPD, XRCC1 and XRCC3 polymorphisms in Polish, Danish and American populations
 

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Table II. The XPD-312 genotypes and lung cancer risk
 

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Table III. The XPD-312 genotypes and lung cancer risk by smoking status
 
The polymorphisms in codons 312 and 751 of the XPD gene appeared to be in linkage disequilibrium in our study (p = 2.02 x 10–17 in controls; p = 6.95 x 10–18 in cases). Carriers of the XPD-751Lys/Lys genotype tended to have the XPD-312Asp/Asp genotype in cases as well as in controls. The recombinant haplotypes were found in both studied groups; there was no difference in inferred haplotype distribution between cases and controls. Pedigree analysis of the XPD genotypes in the members of four, three-generation families also showed the linkage and confirmed the presence of four haplotypes in this group. The approximate frequencies (based on genotypes of the grandparents only; n = 14) were: 312Asp/751Lys, 57%; 312Asn/751Gln, 32%; 312Asn/751Lys, 4%; and 312Asp/751Gln, 7%.

The distribution of genotypes associated with polymorphisms of XPD-751, XRCC3 and XRCC1 did not differ between cases and controls.

We compared the frequency of the common genetic polymorphisms observed in the Polish population with the frequencies found in other reported populations (Table IGo). The XPD-199 and -201 polymorphisms and the XPF-379 polymorphism were not found in this sample of the Polish population. The XRCC1-194 and -280 polymorphisms were infrequent in our group (the frequencies of the 194Trp and 280His alleles were 0.04–0.05 and 0.02–0.05 respectively), whereas the XRCC3-241 polymorphism had a very similar frequency to that observed by Shen et al. (15) (Met allele frequency 0.32).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We examined the frequency of recently reported polymorphisms in DNA repair genes (XRCC1, XRCC3, XPD and XPF) in NSCLC cases and healthy controls from Poland and investigated the potential relationship between the polymorphisms and lung cancer risk. Generally, similar frequencies of the studied polymorphisms were observed in cases and in controls, the exception being the Asp312Asn polymorphism in exon 10 of XPD. The XPD gene product and the related XPB protein are DNA helicases, components of the basal transcription factor TFIIH complex; they are involved in transcription and NER (23,24) and in the p53-dependent apoptotic pathway (25,26). Hereditary defects in the XPD gene can result in three distinct human disorders: xeroderma pigmentosum complementation group D, trichothiodystrophy and Cockayne syndrome (27). The XPD-312 polymorphism is characterized by a 23591G->A substitution, causing an Asp->Asn amino acid exchange at codon 312 of XPD; this residue has been highly conserved through evolution, so this substitution may be functionally significant. We searched several databases (SwissProt, trEMBL and trEMBENEW) with the BLAST2.0 program (28) for homologs of amino acids 301–332 (containing the polymorphic 312 site) of the human XPD protein; the analysis revealed eight similar proteins (Table IVGo). Four of them are XPD/ERCC2 proteins from mouse, hamster, fish and Drosophila. The others include the following: REPD protein from Dictyostelium (slime mold), F19K9.20 protein from Arabidopsis, RAD15 from Schizosaccharomyces pombe and RAD3 from Saccharmoyces cerevisiae. All of these proteins also show other regions of extensive homology including the perfectly conserved DEAH helicase motif (not shown). The polymorphic residue 312 is conserved in vertebrates. In Drosophila and Dictyostelium the homologous residue is occupied by an amino acid that is coded by the human polymorphic allele (312Asn). Interestingly, all compared proteins that do not have aspartic acid at residue 312 (invertebrates and non-animals) have aspartic acid at residue 313 (Table IVGo).


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Table IV. Conservation of the amino acid residues at and around the site of the human XPD polymorphism Asp312->Asn
 
In this study, the frequency of the XPD-312Asp allele among lung cancer patients was higher than in healthy controls (0.63 and 0.56, respectively). The XPD-312Asp/Asp genotype was associated with almost double the risk of lung cancer compared with the Asn/Asn + Asp/Asn genotypes combined. Thus, the minor allele of XPD (312Asn) may be considered as an allele that reduces lung cancer risk. The association between the XPD genetic polymorphism and lung cancer risk is biologically plausible, because the XPD protein functions as a 5'–3' helicase in the NER mechanism that is responsible for the repair of many DNA lesions induced by genotoxic agents present in the environment (e.g. bulky adducts) (2931) and plays a role in activating apoptosis through the interaction between p53 and TFIIH to remove damaged cells (25,26). In our study, the Asp/Asp genotype was associated with a 5-fold higher risk of lung cancer in light smokers (Table IIIGo). Our observation that the XPD polymorphism had the clearest effect at a low-dose level of cigarette smoking might imply a gene–environment interaction. The basis for this low-level effect is not clear.

The XPD-312 polymorphism was in linkage disequilibrium with the XPD-751 polymorphism in our study. A statistically significant proportion of individuals carrying the XPD-312Asn allele also had the XPD-751Gln allele, while most XPD-312Asp allele carriers had the XPD-751Lys allele.

The XPD-751 polymorphism was associated recently with an increased risk of basal cell carcinoma in a Danish study (32) and with squamous cell carcinoma of the head and neck in an American study (33). Lunn et al. (34) found that in individuals carrying the XPD-751Lys/Lys genotype there were significantly more ionizing radiation-induced chromatid aberrations than in individuals homozygous for the XPD-751Gln allele. This is in contrast with our study, which did not show any significant association between the XPD-751 polymorphism and lung cancer risk. In the report by Lunn et al. (34), individuals with the XPD-312Asp/Asp genotype also showed more chromatid aberrations than XPD-312Asn carriers. Although the difference was not statistically significant, it is consistent with our results showing that carriers of the 312Asp/Asp genotype are at twice the risk of developing smoking-associated lung cancer. Because residue 751 in the carboxyl terminus of the XPD protein is not evolutionarily conserved, the association of the XPD-751 polymorphism with variations in DNA repair capacity and cancer risk is somewhat puzzling. Perhaps the XPD-312 polymorphism modulates the apoptotic function of the protein whereas the XPD-751 polymorphism modulates only its DNA repair efficiency. Although the functional significance of the XPD polymorphisms has not yet been elucidated, it is possible that they may be responsible, in part, for the interindividual DNA damage repair variations in the general population and for a low DNA repair capacity phenotype characteristic of cancer patients (7,8,14).

Polymorphisms in XRCC1 codons 194 and 280, XPF codon 379 and XPD codons 199 and 201 were rare (or absent) in our study population. The allelic distributions found in our study were similar to those observed in Caucasians from North Carolina (35) and a healthy population from Denmark (32) (see Table IGo). There were also differences between allele frequencies of the polymorphisms examined in our study and those reported previously. The XRCC1-194 polymorphism was rare in our group (4–5%) when compared with a group of 12 unidentified individuals (25%) studied by Shen et al. (15), whereas the XRCC3-241Met polymorphism had a very similar frequency in the Polish group and in the report by Shen et al. (15). The differences in allele frequency are consistent with the hypothesis that polymorphisms in DNA repair genes exhibit ethnic variations similarly to polymorphisms of carcinogen-metabolizing enzymes.

Although based on relatively small numbers, the above results show that the polymorphic XPD variants should be investigated for the modulation of NER and apoptotic functions. Additional molecular epidemiological studies of cancer risk are also warranted.


    Notes
 
3 To whom correspondence should be addressed Email: curtis_harris{at}nih.gov Back


    Acknowledgments
 
We thank Prof. K.Czyzewski and Dr. J.Harasim from the Silesian Medical Academy Hospital in Zabrze and Dr. E. Grzybowska, Dr. M.Klementowicz, G.Dziedzic and M.Patera for providing the samples and questionnaire data. The excellent technical assistance of A.Kostowska, I.Matuszczyk, H.Paterak and J.Wiatrak in collecting material and isolating DNA is appreciated. Dr. X.Wang provided helpful criticism of the manuscript. The editorial assistance of D.Dudek and technical support by E.Bowman are appreciated also. This work was supported, in part, by the Polish-American MSC Fund II grant no. MZ/NIH-97-313 and the Polish State Committee for Scientific Research KBN grant no. 4 P05A 06217.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 14, 2000; revised November 23, 2000; accepted November 24, 2000.