Specific combinations of DNA repair gene variants and increased risk for non-small cell lung cancer

Odilia Popanda1,5, Torsten Schattenberg1, Chi Tai Phong1, Dorota Butkiewicz2, Angela Risch1, Lutz Edler3, Klaus Kayser4, Hendrik Dienemann4, Volker Schulz4, Peter Drings4, Helmut Bartsch1 and Peter Schmezer1

1 Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany, 2 Department of Tumor Biology, Center of Oncology-M. Sklodowska-Curie Memorial Institute, Gliwice, Poland, 3 Division of Biostatistics, German Cancer Research Center (DKFZ), Heidelberg, Germany and 4 Thoraxklinik Heidelberg-Rohrbach, Heidelberg, Germany

5 To whom correspondence should be addressed Email: o.popanda{at}dkfz-heidelberg.de


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several polymorphisms in DNA repair genes have been reported to be associated with lung cancer risk including XPA (–4G/A), XPD (Lys751Gln and Asp312Asn), XRCC1 (Arg399Gln), APE1 (Asp148Glu) and XRCC3 (Thr241Met). As there is little information on the combined effects of these variants, polymorphisms were analyzed in a case-control study including 463 lung cancer cases [among them 204 adenocarcinoma and 212 squamous cell carcinoma (SCC)] and 460 tumor-free hospital controls. Odds ratios (OR) adjusted for age, gender, smoking and occupational exposure were calculated for the variants alone and combinations thereof. For homozygous individuals carrying the Glu variant of APE1, a protective effect was found (OR = 0.77, CI = 0.51–1.16). Individuals homozygous for the variants XPA (–4A) (OR = 1.53, CI = 0.94–2.5), XPD 751Gln (OR = 1.39, CI = 0.90–2.14) or XRCC3 241Met (OR = 1.29, CI = 0.85–1.98) showed a slightly higher risk for lung cancer overall. In the subgroup of adenocarcinoma cases, adjusted ORs were increased for individuals homozygous for XPA (–4A) (OR = 1.62, CI = 0.91–2.88) and XRCC3 241Met (OR = 1.65; CI = 0.99–2.75). When analyzing the combined effects of variant alleles, 54 patients and controls were identified that were homozygous for two or three of the potential risk alleles [i.e. the variants in nucleotide excision repair, XPA (–4A) and XPD 751Gln, and in homologous recombination, XRCC3-241Met]. ORs were significantly increased when all patients (OR = 2.37; CI = 1.26–4.48), patients with SCC (OR = 2.83; CI = 1.17–6.85) and with adenocarcinoma (OR = 3.05; CI = 1.49–6.23) were analyzed. Combinations of polymorphisms in genes involved in the same repair pathway (XPA + XPD or XRCC1 + APE1) affected lung cancer risk only in patients with SCC. These results indicate that lung cancer risk is only moderately increased by single DNA repair gene variants investigated but it is considerably enhanced by specific combinations of variant alleles. Analyses of additional DNA repair gene interactions in larger population-based studies are warranted for identification of high-risk subjects.

Abbreviations: AC, adenocarcinoma; BER, base excision repair; CI, confidence intervals; ERCC2, excision repair cross-complementing rodent repair deficiency, complementation group 2 (xeroderma pigmentosum D); NER, nucleotide excision repair; OR, odds ratio; PY, pack years; SCC, squamous cell carcinoma; XP, Xeroderma pigmentosum


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
DNA damage induced by exogenous carcinogens or by endogenous metabolic processes can be converted into gene mutations leading to genomic instability and malignant transformation. To prevent consequences of such detrimental mutations cells have evolved mechanisms to preserve genomic integrity. The normal function of DNA repair enzymes plays a critical role as DNA repair effectively removes damaged sites from DNA (1). Human cancers of the lung are well known examples of tumors that are caused by environmental carcinogens such as tobacco smoke and environmental pollutants (2). The impact of DNA repair capacity on tumor development was shown by several studies that found reduced DNA repair capacity to be associated with increased lung cancer risk (35). However, it still has to be elucidated which DNA repair pathways are impaired in some individuals and why they show an enhanced risk for developing cancer.

Over 130 DNA repair genes are currently known (6). They are cooperating in distinct pathways that are specialized in the repair of the different types of DNA damage (7,8). The main pathways include nucleotide excision repair (NER) for the removal of UV damage and bulky DNA adducts, base excision repair (BER) for restoring minor base alterations such as oxidative damage and methylated bases, homologous recombination and non-homologous end joining for the repair of DNA double-strand breaks. The diversity of DNA damage induced by the complex mixture of carcinogens in tobacco smoke, however, requires the combined activity of all DNA repair pathways to prevent mutations.

Single nucleotide polymorphisms were found in nearly all human DNA repair genes that have been investigated so far (9), and some of them were shown to modulate levels of DNA damage, individual DNA repair capacity and cancer risk (1012). Studies on the effects of DNA repair gene polymorphisms on lung cancer risk were not conclusive so far (10,13,14), in part because of some shortcomings in these studies, e.g. small numbers or mixed ethnicities. In addition, determination of gene variants revealed that the genetic variability of DNA repair pathways seems to be higher than the variability in other sets of candidate disease-susceptibility genes such as cardiovascular or hypertension risk genes (9,15). Thus, the repair genotype can be very complex in an individual resulting in several variant peptides within the respective repair complexes. It was suggested that DNA repair function and cancer risk are significantly modulated by additive and even multiplicative effects of various variant alleles (15). The amount of variation and the complexity of the genotypes should, thus, be carefully taken into account when analyzing associations between genotype and cancer risk.

There are only a few studies where the joint effects of more than one variant allele were investigated, mainly in bladder or breast cancer (e.g. refs 1618). These studies showed that individuals with more than one variant allele were often at a considerably higher risk. Therefore, our study aimed to analyze the effects of complex genotypes on lung cancer risk and chose allele combinations based on biologic evidence. We investigated combinations of variant alleles involved in the repair of tobacco smoke-induced DNA damage, i.e. NER, BER, repair of oxidative DNA damage and double-strand breaks. Further selection criteria were suggestions in the literature that these polymorphisms were associated with altered protein functions and that the variant alleles occurred rather frequently (>30%) so to preserve power for subgroup analyses.

According to these criteria we investigated the following polymorphisms: XPA (–4G/A), XPD Lys751Gln, XPD Asp312Asn, XRCC1 Arg399Gln, APE1 Asp148Glu and XRCC3 Thr241Met (1921). The gene products of XPA and XPD are involved in NER. The XPD protein, encoded by the ERCC2 gene, is part of the transcription factor TFIIH and functions as an ATP-dependent helicase whereas XPA encodes a DNA damage-binding protein (7,8). The X-ray repair cross-complementary gene XRCC1 and the apurinic/apyrimidinic endonuclase APE1 participate in BER: XRCC1 coordinates as a scaffold protein for other BER proteins at the site of base damage repair (22), among them APE1, which cleaves 5' of DNA abasic sugar residues generated from exogenous and endogenous agents (7,8). The X-ray repair cross-complementary gene XRCC3 functions in homologous recombination repair of DNA double-strand breaks (22).

These six polymorphisms were investigated in a case-control study with lung cancer patients recruited in the Thoraxklinik in Heidelberg. Odds ratios (OR) were determined for each of the six gene variants for all study patients and for those with the histological subtypes of adenocarcinoma (AC) and squamous cell carcinoma (SCC); these subtypes are supposed to have slightly different etiologies (23). In addition, the incidence of combinations of genotypes was determined in the study population. Combinations included variant alleles of genes involved in NER (XPA and XPD) and in BER (XRCC1 and APE1). As the variant alleles of XPA (–4A), XPD 751Gln and XRCC3 241Met showed enhanced ORs, their combined effect on lung cancer risk was also analyzed.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study population
463 primary lung cancer cases and 460 randomly selected, non-matched hospital controls, all Caucasians, were recruited from the internal medicine and surgery departments of the Thoraxklinik in Heidelberg between 1996 and 2000 (for further details, see refs 24,25). The study was approved by the ethical committee of the University of Heidelberg (reference no. 201/98). Cases were all newly diagnosed patients with histopathologically confirmed primary lung cancer who were surgically treated. The cases included 204 adenocarcinomas, 212 squamous cell carcinoma cases and 47 other tumors with a variety of different pathologies (including large cell and small cell carcinomas, carcinoids and mixed types). The two histological subgroups, SCC and AC, were large enough to allow a separate statistical analysis. Controls had no previous or present history of malignant disease. They suffered mainly from: alveolitis, bronchitis, pneumonia, fibrosis, sarcoidosis, chronic obstructive pulmonary disease and emphysema.

All study participants signed an informed consent form and completed a detailed questionnaire on his or her personal history, smoking habits [never-smoker, current smoker, ex-smoker, number of pack-years (PY) smoked] and occupational exposures. Occupational exposure status was established on the basis of the information given in the questionnaire in the job title (e.g. welders, drivers, mechanics, industry workers, painters) or on exposure to known or suspected lung carcinogens (asbestos and mineral fibers, metals, all kinds of dust and fumes, cement, petrol and coal products, diesel exhaust, solvents, pesticides, etc.) (26,27). The non-exposure group consisted of individuals claiming lack of exposure and/or having had jobs with no such exposure (e.g. office workers, teachers, housewives).

The demographic data for cases and controls are presented in Table I. Briefly, the median age of all patients was slightly higher than in controls (61 and 55 years, respectively). There were significantly more males (76 versus 68%), smokers (90 versus 66%) and individuals with possible occupational exposure to lung carcinogens (71 versus 58%) among all cases than among controls. The group of patients with adenocarcinoma, however, showed no significant differences to the control group regarding gender (P = 0.17) and occupational exposure (P = 0.15).


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Table I. Characteristics of lung cancer cases and controls

 
Five milliliters of peripheral blood in EDTA were collected from all participants and buffy coats were stored at –80°C. Subsequently, genomic DNA was isolated using a QIAamp DNA Blood Kit (Qiagen, Hilden, Deutschland).

Genotyping based on PCR followed by RFLP analysis
The polymorphisms XPD Lys751Gln (A to C change in nucleotide position 35931 of the ERCC2 gene, GenBank accession no. L47234) and XRCC3 Thr241Met (C to T exchange in nucleotide position 18067, GenBank accession no. AF037222) were analyzed using the primers given in Table II. The amplification of PCR products was carried out in a volume of 20 µl using 20 ng of DNA, 1x buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 1x Q solution, 1 U Taq polymerase (Qiagen, Hilden, Germany) and 10 pmol of each primer. Cycling conditions were as follows: initial denaturation at 94°C, 3 min and 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C (XPD) or 55°C (XRCC3) for 30 s, extension at 72°C for 30 s followed by one cycle of final elongation at 72°C for 10 min. Length of PCR products was 348 bp for XPD Lys751Gln and 205 bp for XRCC3 Thr241Met. PCR products were digested with 10 U of restriction enzyme: PstI for XPD Lys751Gln, or NlaIII for XRCC3 Thr241Met (purchased from New England BioLabs, Frankfurt, Germany) and separated on a 3% agarose gel. An additional restriction site was located in each PCR product to control for complete digestion. The digestion patterns observed were as follows: XPD Lys751Gln: 239 and 109 bp fragments in homozygote GG and 62, 177 and 109 bp in homozygote AA; XRCC3 Thr241Met: 37, 105 and 63 bp for homozygote CC, and 37 and 168 bp for homozygote TT. For 100 randomly selected samples, data were confirmed by independently repeated PCR amplification and RFLP analysis and 100% concordance of results was obtained.


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Table II. Primer and hybridization probes for genotype analyses

 
Genotyping by fluorescence-based melting curve analysis
Detection of four polymorphisms was performed by rapid capillary PCR with melting curve analysis using fluorescence labeled hybridization probes in a LightCycler (Roche Diagnostics, Mannheim, Germany): the XPA (–4G/A) polymorphism (G to A exchange 4 nt before the ATG start codon in nucleotide position 66, GenBank accession no. NM_000380.2), the XPD Asp312Arg polymorphism (G to A exchange in nucleotide position 23591 of the ERCC2 gene, GenBank accession no. L47234), the APE1 Asp148Glu polymorphism (T to G exchange in nucleotide position 2197, GenBank accession no. M92444) and the XRCC1 Arg399Gln polymorphism (G to A exchange in nucleotide position 28152, GenBank accession no. L34079). PCR primers and probes for melting point analyses are given in Table II. Probes were designed with the help of Tib Molbiol, Berlin, Germany. The melting point analysis uses fluorescence resonance energy transfer for detecting a polymorphic site. The sensor probe was designed for a perfect match either to the wild-type or the variant allele sequence. Thus, in the allele with the sequence deviating from the sensor, a 1 nt mismatch between sensor and target DNA sequence was formed and caused destabilization of the hybrid yielding to a melting point (Tm) shift.

Analysis was performed in 10 µl volumes in glass capillaries (Roche Diagnostics) using Qiagen reagents: 1x PCR buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 0.1% BSA in DMSO, 0.5x Q solution, 1 U Taq DNA polymerase, 1 µM of each primer, 0.15 µM of each probe and 10 ng of DNA. Reaction conditions were as follows: initial denaturation at 95°C for 3 min, then 40 cycles of denaturation at 95°C for 15 s, annealing at 62 (XPA and XPD), 58 (APE1) or 55°C (XRCC1) for 10 s and elongation at 72°C for 12 s. Melting curve analysis was performed with an initial denaturation at 95°C for 30 s, 15 s at 40°C, slow heating to 75°C with a ramping rate 0.3°C/s and continuous fluorescence detection. Melting curves were converted to melting peaks by plotting the negative derivatives of fluorescence against temperature (–dF/dT). Melting curves were evaluated by two independent observers. In addition, 100 randomly selected samples were analyzed by conventional PCR–RFLP methods to verify the LightCycler results and 100% concordance was found.

Statistical analysis
The lung cancer group and the histological subgroups of AC and SCC, each were compared with the controls for the basic characteristics of age, gender, smoking and occupational exposure (Table I). Both the population of cases and controls were tested as to whether they were in Hardy–Weinberg equilibrium using the {chi}2 test of goodness of fit with two degrees of freedom, with respect to the distribution of alleles. Lung cancer risk was analyzed for association with polymorphisms: crude ORs and their 95% confidence intervals (CI) were calculated and the corresponding statistical significance tests (null hypothesis H0: OR = 1 versus the alternative H1: OR != 1) were performed according to described methods (28). The more frequent allele was assumed to be the reference. In addition, multiple unconditional logistic regression was performed to assess the association between the occurrence of lung cancer and the prevalence of the polymorphism adjusted for age, gender, smoking and occupational exposure. Age at diagnosis was stratified into four age groups with ≤50; 51–60; 61–70; ≥71 years. Smoking was quantified by the degree of tobacco consumption categorized as PY groups with ≤20 PY and >20 PY (1 PY = 20 cigarettes/day for 1 year). Occupational exposure (see ‘Study population’) was categorized as no exposure or possible exposure. A possible difference in the effect of one polymorphism in the group of mild (≤20 PY) or heavy (>20 PY) smokers was tested by introducing a polymorphism * smoking interaction term in the logistic regression model.

In an additional analysis (not shown), data were evaluated after introducing an ‘exposure index’ instead of treating the risk factors as multi-variables by calculating a combined score (gender: male = 1, female = 0; age: three groups: 0, 1, 2; smoking: never, ever and PY ≤20, PY >20: 0, 1, 2; occupational exposure: 0, 1). There was no significant and numerically relevant difference between the ORs adjusted for this exposure index and the ORs obtained after ‘multivariable’ adjustment.

Further, lung cancer risk was analyzed for those individuals that were homozygous for more than one variant allele by calculation of crude and adjusted ORs for specific combinations. A trend of the ORs depending on the number of variant alleles was examined using ordinal regression. In all these combinations we may have combined possibly ‘at-risk’ and ‘protective’ alleles. We checked this caveat in our analysis as far as possible, given our sample size, by breaking down combinations (i.e. the ‘any out of three at-risk alleles’; Table VII) into its single three components. The results of this analysis (data not shown) were not different compared with those reported in Table VII by combining variant alleles for a trend analysis using scores.


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Table VII. Lung cancer risk in individuals homozygous for more than one variant "risk" allele: Accumulation of variant alleles which indicated a trend for lung cancer risk when tested separately: XPA(–4)AA + XPD-751Gln + XRCC3-241Met

 
Calculation and statistical tests for crude ORs were performed using the statistical analysis system ADAM of the Biostatistics Unit of the German Cancer Research Center. Multiple logistic regression analysis and calculation of CI was done with SAS statistical evaluation system (SAS Institute, Cary, NC).

The power of the study was estimated comparing two test populations: (i) the group of heterozygous individuals and (ii) that of individuals homozygous for the variant allele. Both groups were compared with the reference group (individuals homozygous for the more frequent wild-type). (i) With a total of 463 cases and 460 controls, the study had a power ranging between 77 and 81% to detect an OR of 1.5 and higher at the significance level of 5% when the prevalence of the reference group of the polymorphism ranged between 35 and 49% compared with the prevalence of the heterozygous test group ranging between 65 and 51%. The power to detect an OR of 2.0 was in all cases higher than 99% (NCSS trial and Power Analysis and Sample Size calculation program. PASS 2002, Kaysville, UT). (ii) The power ranged between 39 and 62% when the prevalence of the reference group of the polymorphism ranged between 57 and 80% compared with that of the homozygous test group ranging between 43 and 20%. In this case, the power to detect an OR of 2.0 ranged between 81 and 97%. With a total of 204 AC cases and 460 controls, the study had a power ranging between 53 and 89% to detect at the significance level of 5% an OR of 2.0 and higher when the prevalence of the reference group of the polymorphism ranged between 56 and 79% compared with the prevalence of the homozygous test group ranging between 44 and 21%.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Six genotypes were determined for all lung cancer cases and controls (Table III) and for the histological tumor subtypes AC (Table IV) and SCC (data not shown). For all polymorphisms, the more common allele was considered to be the reference genotype whereas the less common allele was examined as the variant. In controls, frequencies of the variant alleles were 0.33 for A in nucleotide position (–4) of XPA, 0.37 for Asn of XPD in codon 312, 0.37 for Gln of XPD in codon 751, 0.49 for Glu of APE1 in codon 148 and 0.39 for Gln of XRCC1 in codon 399 as well as for Met of XRCC3 in codon 241. All allele distributions were consistent with Hardy–Weinberg equilibrium.


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Table III. Distribution of genotypes and odds ratios (OR) determined for all lung cancer cases and controls

 

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Table IV. Distribution of genotypes and odds ratios (OR) for adenocarcinoma of the lung and controls

 
The distribution of gene variants was similar between all cases and controls (Table III), a maximal difference of 5% was found for the XRCC3 polymorphism with the heterozygous genotype being less frequent in cases (43%) than in controls (48%). These differences were, however, not significant (XRCC3 P > 0.05). No change in OR was found for XPD Asp312Asn and XRCC1 Arg399Gln. The variant allele of APE1 148Glu showed a protective effect with an OR of 0.73 (Glu/Glu). Slightly increased ORs were calculated for individuals homozygous for the variant alleles of XPA (–4A), XPD 751Gln and XRCC3 241Met (OR = 1.22, 1.37 and 1.20, respectively) indicating that these alleles might enhance lung cancer risk. However, crude OR analysis did not show a statistically significant association between the genotype and cancer risk for any of the polymorphisms. As the study population differed in age, gender, smoking and occupational exposure status, ORs were adjusted for these factors. Adjustment did not alter the ORs of the polymorphisms XPD 312 and 751, APE1 148, XRCC1 399 and XRCC3 241, but the OR of XPA (–4A) was enhanced and reached borderline statistical significance (OR = 1.53, 95% CI 0.94–2.50; P = 0.09).

The analysis of the histological subtype AC (Table IV) revealed slightly higher differences between cases and controls than the analysis that included all cancer cases. ORs for the homozygous variants of XPA (–4A), XPD 751Gln and XRCC3 241Met, were higher (1.39, 1.61 and 1.51, respectively). The OR was found to be reduced for the Glu variant in codon 148 of APE1, but the reduction was not statistically significant. After adjustment for confounding factors, an OR close to significance (P = 0.054) was found for XRCC3 (OR = 1.65, 95% CI 0.99–2.75).

In the subgroup of SCC, the distribution of genotypes was very similar among cases and controls for all polymorphisms. Crude ORs close to 1 were calculated for the variant homozygotes and these values were not affected significantly by adjustment for confounding factors (data not shown).

By analyzing the interaction of the polymorphisms with smoking in a logistic regression model, we tested for a possible difference in the effect of each polymorphism in mild or heavy smokers. The resulting interaction term was not significant for any of the polymorphisms and, therefore, a further subgroup analysis for the confounding factor smoking was not performed in this paper.

When comparing the incidence of the different polymorphisms in the study population, it became evident that more than one gene variant occurred in a considerable number of individuals. The frequency analysis of the simultaneous incidence of two gene variants in the study population revealed that these two polymorphisms in the XPD gene were highly correlated (P < 0.0001 for controls, all cancer cases, adenocarcinoma and SCC) suggesting linkage disequilibrium (as already reported in refs 29,30). In addition, the presence of the XPA (–4G/A) and the XPD Asp312Asn alleles were closely correlated (P < 0.0001) in squamous cell carcinoma. A lower correlation was found for XPA (–4G/A) and XRCC3 Thr241Met in adenocarcinoma (P = 0.0028), and for XPA (–4G/A) and the XPD Asp312Asn in all lung cancer cases (P = 0.0096) but not in controls. Combinations of other polymorphisms did not show a correlation (P > 0.05).

Further analysis of combinations of gene variants was concentrated on individuals that were homozygous for the variant alleles because these individuals exhibited stronger effects of these alleles compared with heterozygous individuals for whom ORs were close to 1 (Tables III and IV). The reference population consisted of all individuals who were not homozygous for at least one of the variant alleles analyzed in combination. For the following combinations, crude ORs and ORs adjusted for age, gender and smoking habits were calculated.

  1. When the three variant alleles of the NER genes, XPA (–4A/A), XPD 312Asn/Asn and XPD 751Gln/Gln, were present in individuals, enhanced ORs were found (OR = 1.97; CI 0.67–5.78; Table V). This effect was significant in SCC patients (OR = 5.35; CI 1.49–19.17; P = 0.01). An increased OR of 3.77 (CI = 1.12–12.75; P = 0.035) was also calculated for SCC patients with only the two variant alleles XPA (–4A/A) and XPD 751Gln/Gln only (detailed data not shown). For AC cases, ORs were not found to be significantly altered.
  2. When the two variant alleles in BER genes, APE1 148Glu/Glu and XRCC1 399Gln/Gln, were combined, ORs were reduced (OR = 0.69; CI 0.28–1.73), especially in the SCC group (OR = 0.39; CI. 0.10–1.53; Table VI). However, the effect was not statistically significant because of the small number of individuals matching the criteria for this combination. In AC patients, no effect of the BER variants was found (OR = 1.06; CI 0.38–2.92).
  3. The analysis of the simultaneous incidence of the three potential ‘risk alleles’, which exhibited enhanced ORs for the single polymorphisms, XPA (–4A/A), XPD 751Gln/Gln and XRCC3 241Met/Met, revealed that up to 9% of individuals were homozygous for at least two of these polymorphisms (Table VII). Due to the rarity of individuals with all three risk alleles, individuals with two or three risk alleles were evaluated as one group. The ORs calculated for this combination were significantly enhanced for all cases (OR = 2.37; CI 1.26–4.48; P = 0.008), SCC patients (OR = 2.83; 1.17–6.85; P = 0.02) and AC patients (OR = 3.05; CI 1.49–6.23; P = 0.002). For this combination of risk alleles, the difference in OR between the different histological subtypes of lung cancer was smaller than that observed for the NER variants.


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Table V. Lung cancer risk in individuals homozygous for more than one variant allele: Combination of variant alleles from genes participating in nucleotide excision repair: XPA(–4)AA + XPD-312Asn + XPD-751Gln

 

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Table VI. Lung cancer risk in individuals homozygous for more than one variant allele: Combination of variant alleles from genes which participate in the base excision repair: APE1-148Glu + XRCC1-399Gln

 
For the combination of risk alleles, the increase in ORs depending on the number of variant alleles was found to be statistically significant for all cases (P = 0.03) and for the AC patients (P = 0.003). For all other gene combinations evaluated, the trend for the change of the ORs was not significant.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Based on our results the homozygous variant alleles, the (–4A) allele of the XPA gene, the Gln variant in codon 751 of XPD and the Met variant in codon 241 of XRCC3, might contribute to lung cancer risk, whereas the variant allele Glu of APE1 in codon 148 exerted a protective effect. As the power of our study was higher than 2 to detect a risk increase, we conclude that the contribution to relative risk of the four genetic polymorphisms analyzed is moderate. Our results are, however, in agreement with other studies (10,14,31,32), which reported associations between cancer risk in white ethnicities and individual repair gene variants with ORs between 1 and 2. Because in our study matching of cases and controls was not perfect, ORs were adjusted for age, gender, smoking habits and occupational exposure. Following multiple logistic regression analysis differences between crude and adjusted ORs, however, were small. It is still conceivable that the genetic contribution would be more pronounced if cases and controls could be better matched. In addition, the ORs we found may be an underestimate of lung cancer risk as we have used hospital controls with non-malignant lung diseases. Some of them, especially those associated with chronic inflammatory processes are suspected to be predisposing factors for lung cancer (33,34). However, from a review by Goode et al. (10), it is apparent, that ORs determined in repair-polymorphism studies with population-based controls were in general not higher than ORs obtained with hospital-based controls. Similarly, no differences between hospital- and population-based controls (n ~15 000) were found in case-control studies on polymorphic metabolism genes (35).

For XPD Asp312Asn and XRCC1 Arg399Gln variants, we found no effect on lung cancer risk. There is no obvious explanation for the different impact on cancer risk of the polymorphisms analyzed because there is experimental evidence that all polymorphisms can impair DNA repair: subjects homozygous for the variant genotypes of XPD and XPA (–4A) showed sub-optimal DNA repair capacity (11,30,32) and the variant allele of the APE1 polymorphism in codon 148 was correlated with prolonged cell-cycle delay after irradiation (16). A study with breast cancer patients revealed that smokers homozygous for Met in position 241 of the XRCC3 protein exhibited a significantly higher level of DNA adducts than individuals with only the Thr allele (36). For XRCC1 Arg399Gln, genotype/phenotype studies reported associations of the variant allele with increase in sister chromatide exchanges, aflatoxin B1-DNA adducts and mutagen sensitivity (3639). However, the potential of a polymorphism to impair DNA repair activity may not always parallel its impact on cancer risk as other response modifiers can interfere.

The markedly increased ORs reported for the polymorphisms in XRCC1 codon 399 in white AC (40) and in SCC from Korea (41), and for the XPD codon 312 in Chinese lung cancer patients (29) were not confirmed in our study, probably because of ethnic differences in the populations studied. In Asians, polymorphisms occur at different allelic frequencies and might be linked with other risk markers than in Caucasians. Additional sequence variants were found in both genes, e.g. a variant in codon 751 of the XPD gene and variants in codons 194 and 280 of the XRCC1 gene (18). The latter two polymorphisms were not included in our study because allele frequencies were only ~10%. Nevertheless, the impact of these two variants, and other not yet identified risk alleles, might conceal the effect of the XRCC1 polymorphism in codon 399.

Increased ORs for single genotypes were found in our study for the lung cancer subtype AC, but not for SCC. Again, in this subtype analysis, our study had the power to detect ORs higher than 2. AC is suggested to have a somewhat different etiopathology than other histological subtypes: smoking increases the risk for all histological types of lung cancer, although this effect is thought to be less strong for AC than for SCC and small cell carcinoma (23). Tissue and organ-specific differences in mRNA levels were observed for various NER genes, including XPA and XPD, which may influence the repair rate in a given tissue (42,43). It is, however, not clear whether such tissue-specific differences in DNA repair capacity and/or expression of DNA repair proteins are associated with genetic predisposition.

Many of the genes involved in DNA repair exist as multiple genetic variants, which may have additive effects on DNA repair activity and lung cancer risk (15). Therefore, genotypes and phenotypes in different repair pathways must be evaluated simultaneously to fully assess their impact on cancer susceptibility. Data obtained from this study suggest a potential gene–gene interaction among the variant alleles of XPA (–4A/A), XPD 751Gln/Gln and XRCC3 241Met/Met increasing significantly lung cancer risk overall and in AC and SCC up to 3-fold. This increase in risk is supported by biological evidence. Smoking, the main cause of lung cancer, induces a great diversity of DNA damage, which must be repaired by more than one repair pathway such as NER (represented by XPA and XPD) and homologous recombination (represented by XRCC3). The combined occurrence of genetic variants in these two repair pathways may, thus, contribute to a greater lung cancer risk.

In addition, we found a >5-fold increased risk for SCC in individuals with three variant NER alleles [XPA (–4A/A) and the XPD 312Asn/Asn and 751Gln/Gln] and a reduction of risk in subjects with two variant BER alleles (APE1 148Glu/Glu and XRCC1 399Gln/Gln) indicating that combinations of variant alleles within a specific pathway can affect the risk for SCC much stronger than the single variants. In addition, our results underline the important role both NER and BER play in the removal of DNA lesions induced by tobacco-specific nitrosamines, polycyclic aromatic hydrocarbons and oxidative processes (44). Interestingly, the allele combinations within a repair pathway had no effects on AC risk, which is in contrast to the increased ORs found in the analysis of single variants in AC. As we found different distributions of allele combinations in AC and SCC cases, our data suggest that polymorphisms in repair genes contribute to the differences that exist between the susceptibility for SCC and AC based on the response to carcinogenic exposures.

The impact of allele combinations on lung cancer risk cannot be explained by haplotypes as four of the five genes are located on different chromosomes (XPA, 9q22.3; XPD, 19q13.2; XRCC3, 14q33.3; XRCC1, 19q13.2; and APE1, 14q12; ref. 45). This might suggest that specific gene–gene interactions either within one repair pathway or between different pathways are affecting lung cancer risk. As the functional impact of a single variant is low, the interaction of several variant proteins with slightly reduced functional activity may be necessary to significantly decrease DNA repair activity and to increase cancer risk. The analysis of allele combinations presented here extends a few studies already reported (see ‘Introduction’), which analyzed such combined effects with regard to cancer risk or repair function. However, given the great variety of genotype combinations, only a limited number of individuals with a specific genotype combination could be studied in our cohort. Our results require validation in larger population-based studies including a wider range of genetic variants.

The relatively low impact of single variant alleles on cancer risk that we and others have found contrasts the marked increased risk associated with a reduced phenotypic DNA repair capacity (reviewed in refs 5,15,46). Here, one must consider that complex environmental carcinogen exposure generates various types of DNA damage, which is repaired by different pathways and whereby repair proteins can be active in more than one pathway. Therefore, the DNA repair capacity determined for example in carcinogen-treated human lymphocytes is an integrative measure of the functional impact of all repair variants. In our current studies we are measuring DNA repair capacity to see how it reflects the complete ‘DNA repair genotype’, which cannot be easily characterized by today's genotyping methods.


    Acknowledgments
 
The authors wish to thank Peter Waas and Birgit Jäger (Division of Toxicology and Cancer risk factors, DKFZ) for their excellent technical assistance and Renate Rausch (Division of Biostatistics, DKFZ) for her valuable help with the statistical analysis. We thank O.Landt (Tib Molbiol, Berlin, Germany) for helping with the LightCycler probe design. We are also grateful to all patients and staff at the Thoraxklinik in Heidelberg involved in sample and data collection. This work was partly supported by a scholarship granted by the ‘Deutsches Krebsforschungszentrum’ (D.B.), and funding from the ‘Verein zur Förderung der Krebsforschung in Deutschland e.V.’ (A.R.) and the ‘Deutsche Krebshilfe’ (sample collection).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 10, 2004; revised July 30, 2004; accepted August 3, 2004.