The XPD variant alleles are associated with increased aromatic DNA adduct level and lung cancer risk

Sai-Mei Hou1,3, Susann Fält1, Sabrina Angelini1, Ke Yang1, Fredrik Nyberg2, Bo Lambert1 and Kari Hemminki1

1 Department of Biosciences at Novum, Karolinska Institute, S-141 57 Huddinge, Sweden and
2 Division of Environmental Epidemiology, Institute of Environmental Medicine, Karolinska Institute, Box 210, S-171 77 Stockholm, Sweden

Abstract

The DNA repair protein xeroderma pigmentosum complementation group D (XPD) is involved in the nucleotide excision repair of DNA lesions induced by many tobacco and environmental carcinogens. In order to study the functional impact of the common polymorphisms in XPD exon 10 (G > A, Asp312Asn) and exon 23 (A > C, Lys751Gln), we have genotyped 185 Swedish lung cancer cases (97 smokers and 88 never-smokers) and 162 matched population controls (83 smokers and 79 never-smokers). Presence of one or two variant alleles was associated with increased risk for lung cancer among never-smokers only, in particular younger (<70 years) never-smokers [odds ratio (OR) = 2.6, 95% confidence interval (CI) = 1.1–6.5 for exon 10; OR = 3.2, 95% CI = 1.3–8.0 for exon 23, adjusted for age, gender and environmental tobacco smoke]. Aromatic DNA adduct level (AL) in peripheral lymphocytes was found to be similar between cases and controls, but significantly increased by current or recent smoking. Overall, there was a significant trend for increasing AL with increasing number of variant alleles in exon 10 (P = 0.02) or in exon 23 (P = 0.001). In addition, subjects with the combined exon 10 AA and exon 23 CC genotype showed a significantly higher AL compared with all those with any of the other genotypes (P = 0.02). We conclude that the XPD variant alleles may be associated with reduced repair of aromatic DNA adducts in general and increased lung cancer risk among never-smokers.

Abbreviations: AL, adduct level; CI, confidence interval; ETS, environmental tobacco smoke; NER, nucleotide excision repair; OR, odds ratio; PAH, polycyclic aromatic hydrocarbons; XPD, xeroderma pigmentosum complementation group D.

Introduction

Genetic variations in the ability to repair DNA lesions induced by tobacco or environmental carcinogens may contribute to the inter-individual variation in lung cancer risk. Common polymorphisms have recently been identified in several DNA repair genes (1) including genes involved in nucleotide excision repair (NER). NER repairs bulky DNA adducts as well as UV-induced DNA damage, and is composed of two subpathways, global genome repair and transcription-coupled repair. The latter rapidly repairs damage on the transcribed strand of active genes (2). The rare autosomal recessive disease xeroderma pigmentosum (XP) results from defects in both repair pathways of UV-damaged DNA (depending on the complementation group), and individuals with XP have a >1000-fold increased risk of skin cancer (3). Given this strong association between DNA repair capacity and cancer, functional and common sequence variations of DNA repair genes may be potential cancer susceptibility factors in the general population exposed to environmental carcinogens such as polycyclic aromatic hydrocarbons (PAH).

The XPD (XP complementation group D) protein takes part in the unwinding of DNA and forms a complex with the basal transcription factor TFIIH during transcription-coupled repair. Mutations in XPD cause a severe but variable depression of NER (4). Several non-synonymous single nucleotide polymorphisms have been described in the XPD gene, including those at codons 312 (Asp > Asn, exon 10, position 23591, G > A) and 751 (Lys > Gln, exon 23, position 35931, A > C) (1). The frequencies of these variant alleles have been estimated to be ~30% in two USA studies (1,5). It is not known whether these polymorphisms have functional effects. However, the more common exon 23 AA genotype has been suggested to be a risk factor for basal cell carcinoma (6) and susceptibility to X-ray-induced chromatid aberrations (5). Recently, however, the variant exon 23 CC genotype has been associated with an increased risk (of borderline-significance) of head and neck cancer (7).

In order to verify the functional role of the common variant alleles in the XPD gene, we have genotyped 185 Swedish non-smoking and smoking lung cancer cases and 162 matched population controls with regard to the XPD exon 10 and exon 23 polymorphisms. The vast majority of subjects have been studied previously with regard to the aromatic DNA adduct level (AL) in peripheral lymphocytes (8). We found no difference between cases and controls, no effect of environmental tobacco smoke (ETS) among never-smokers, but a significant effect of current or recent smoking on ALs. In the present study, we have studied the influence of XPD genotypes on both lung cancer risk and ALs in smokers and non-smokers.

Materials and methods

Study subjects
The current study population originated from a larger epidemiological study designed to investigate the effect of passive smoking on lung cancer risk (9). Cases were recruited during 1992–1995 at the three major county hospitals in Stockholm County responsible for the diagnosis and treatment of lung cancer. Lung cancer patients were asked to participate in the study when they were newly diagnosed. Each never-smoking case was used as an index case for next diagnosed ever-smoking case of the same gender and age (30–49, 50–69 and >=70 years) in the same hospital.

Healthy population controls were extracted from the Stockholm residence files every 6 months and frequency matched to cases with regard to hospital catchment area, gender, age group as well as broad smoking categories: `smoker' (current or recent—quit within 2 years), former smoker (quit >2 years ago) and never-smoker. Never-smokers were defined as those who never smoked regularly (<1 cigarette/day during a year).

Detailed exposure data on smoking, ETS (from spouse, work or other places), dietary habits, as well as residential and working histories were mainly collected by personal interview according to a standard questionnaire. Blood samples were taken before radiotherapy or chemotherapy in all but 10 cases (seven of them with ALs measured). Lymphocytes and granulocytes were isolated after density separation in Polymorphprep (Pharmacia, Sweden). The former were frozen (–135°C) in aliquots for subsequent adduct measurement, and the latter were freshly used for DNA isolation (using saturated NaCl) and genotyping.

Adduct measurement
The [32P]-TLC (thin-layer chromatography) assay of aromatic DNA adducts was carried out as described previously (10). In brief, DNA was extracted from the crude nuclei using organic solvents after degrading RNAs and proteins, and digested by micrococcal nuclease and spleen phosphodiesterase to 3' nucleotides. Adducts were then enriched by nuclease P1 treatment. A post-labelling reaction was carried out and applied on a TLC plate for adduct separation in three dimensions. Following autoradiography, the adduct spots were excised from the TLC plate for counting of radioactivity. Two to five assays were carried out for each sample.

The detection limit of the assay was ~1 adduct/109 nt. Based on the TLC systems used, the method detected aromatic types of DNA adducts. Benzo[a]pyrene diol epoxide modified DNA was used as an external standard.

XPD exon 10 genotyping
Analysis of the XPD Asp312Asn (G > A) polymorphism in the end of exon 10 was performed by restriction analysis of a 188 bp genomic PCR product. A forward primer with a C > T mismatch (D10f: 5'-ACC TGG CCA ACC CCG TGC TGC TC-3') was designed in order to create a restriction site for TaqI (TVCGA) in the PCR product of the G-allele. The reverse primer was designed according to the wild-type sequence within exon 11: 5'-ACT TCA CGT ACT CCA GCA G-3' (D11r).

Approximately 50 ng DNA was used in a 20 µl PCR reaction containing 20 mM Tris–HCl (pH 9.0), 100 mM KCl, 0.2% Triton X-100, 10% DMSO, 1.5 mM MgCl2, 0.25 mM dNTP and 0.5 µM of each primer. After a 5 min hot start at 95°C, 1.5 U Taq polymerase (Promega, Madison, WI, USA) was added (at 85°C) to each tube. The DNA was then amplified by 35 cycles of denaturation (94°C, 30 s), annealing (56°C, 30 s) and elongation (72°C, 30 s, the last cycle 7 min extra) in a thermocycler (DNA-Engine, MJ Research, Waltham, MA).

Ten microlitres of the PCR product was then digested by 10 U TaqI (Fermentas, Vilnius, Lithuania) for 4 h at 65°C in a total volume of 20 µl. The digested products were separated by electrophoresis in a 6% polyacrylamide gel. The G-allele was represented by a 166 bp band and the A-allele by a 188 bp band. To ensure proper digestion of the PCR product, a subset of the samples were re-genotyped and identical results were obtained.

XPD exon 23 genotyping
The method for genotyping of the exon 23 polymorphism (Lys751Gln, A > C) was modified from Dybdahl et al. (6). A 322 bp fragment including the 84 bp long exon 23 was amplified using two intronic primers flanking exon 23: 5'-ATC CTG TCC CTA CTG GCC ATT C-3' (D23f) and 5'-TGG ACG TGA CAG TGA GAA AT-3' (D23r). Approximately 50 ng DNA was used in a 25 µl PCR reaction containing 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl2, 0.25 mM dNTP and 1 µM of each primer. The DNA was amplified by adding 1 U Taq polymerase after a 5 min hot start, followed by 35 cycles of denaturation (94°C, 15 s), annealing (60°C, 15 s) and elongation (72°C, 15 s, the last cycle 7 min extra).

Five microlitres of the PCR product were then digested by 10 U PstI (Promega) for 4 h at 37°C in a total volume of 15 µl, followed by electrophoresis in a 5% polyacrylamide gel. The A-allele was cut into two fragments (104 and 218 bp) while the C-allele was cut into three fragments (104, 155 and 63 bp). Presence of the 104 bp fragment was considered as an internal positive control for a successful digestion of the sample.

Statistical methods
The chi-squared ({chi}2) analysis was used to test the difference between groups in allelic or genotypic distribution. Multiple logistic regression was carried out to calculate genotype-associated lung cancer risk [odds ratio (OR), with 95% confidence interval (CI)], with adjustment for potential confounding factors, such as age, gender, ETS exposure (non-smokers) or packyears (smokers). Age and packyears were strongly correlated to each other and thus not used in the same model.

The difference in ALs between two groups was assessed using the Wilcoxon rank-sum (Mann–Whitney) test. The non-parametric trend test (two-sided) was used to evaluate any possible trend in AL across groups with an increasing number of variant alleles. The distribution of AL was then normalized by ln-transformations, and the influence of variant allele on lnAL (presented by the coefficient ß) was studied by using multiple linear regression adjusting for age, gender, case status and smoking category (current, recent, former and never). All trend effects were verified by converting the number of variant alleles from one categorical variable to several dichotomous indicator variables.

Results

Table IGo shows the frequency distribution of various constitutional or measured parameters in XPD-genotyped lung cancer cases and population controls. A total of 185 cases and 162 controls were genotyped with regard to XPD exon 23 polymorphism. The age distribution among XPD-genotyped subjects was very similar in the groups of patients (median 69, range 30–92) and controls (median 68, range 30–89) as a result of matching. The ever-smoking patients had, however, a significantly higher number of packyears of smoking (PY, 1 pack of cigarettes/day for 1 year) than the corresponding controls. Over 70% of the subjects were women. The majority of cases had adenocarcinoma (50.3%) or squamous cell carcinoma (23.2%). Ever-smokers had a significantly higher proportion of squamous cell carcinoma than never-smokers (38.1 versus 8.8%, P = 0.0001), whereas adenocarcinomas were significantly more frequent among never-smokers than among ever-smokers (62.5 versus 39.2%, P = 0.002).


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Table I. Frequency distribution of various subject characteristics in lung cancer cases and population controls genotyped with regard to XPD polymorphisms
 
The vast majority of XPD-genotyped subjects had enough numbers of lymphocytes isolated to get the level of aromatic DNA adducts measured (171 cases and 146 controls, Table IGo). There was no significant difference in the AL between cases and controls although packyears of smoking were significantly higher in cases than in controls. This might be explained by a persistence of smoking-induced elevation in DNA repair activity and a more complete clearance of smoking-induced DNA adducts in cases who smoked longer but stopped smoking after developing symptoms or receiving diagnosis (8). Current or recent smokers showed a significantly higher AL than former/never-smokers overall, without (Wilcoxon's P = 0.0006 for AL) or with (ß = 0.21, P = 0.0001 for lnAL) adjustment for age, gender and case status. This is consistent with the short turnover of DNA adducts.

The XPD exon 10 genotyping was successful in all but one ever-smoking case (Table IGo). The distribution of genotypes among controls was in Hardy–Weinberg equilibrium for both polymorphisms ({chi}2 test, P = 0.9 for observed versus expected exon 10 genotype frequencies and P = 0.4 for exon 23). Patients did not differ from controls in the distribution of XPD genotypes or alleles in exon 10 or exon 23. Overall, the polymorphism in exon 10 is in linkage disequilibrium with the polymorphism in exon 23 ({chi}2 test, P < 0.0001). The vast majority (72%) of the exon 10 AA homozygotes (33/46) had the exon 23 CC genotype, and most (80%) of the exon 10 GG homozygotes (107/134) had the exon 23 AA genotype. A large proportion of individuals (112/346 = 32%) were heterozygous for the nucleotide variations in both exon 10 and exon 23.

Table IIGo shows the lung cancer risk related to XPD genotypes in ever-smokers and never-smokers stratified by age (with cut point at 70 years, close to the overall median of 69). The wild-type homozygotes were used as a reference category and the risk associated with the presence of one or two variant alleles was adjusted for packyears and gender (for smokers), or for age, gender and ETS exposure (for never-smokers). Presence of at least one variant allele in exon 10 was associated with increased cancer risk among younger never-smokers only (OR = 2.6, 95% CI = 1.1–6.5). A similar risk elevation was obtained for the exon 23 variant allele in this subgroup (OR = 3.2, 95% CI = 1.3–8.0). The OR in all other age and smoking subgroups were close to or below unity. The risk for squamous cell carcinoma was similar (non-significant) to that for adenocarcinoma, both among ever-smokers or never-smokers and overall (data not shown).


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Table II. Lung cancer risk related to XPD polymorphisms in ever-smokers and never-smokers stratified by age
 
Table IIIGo shows the pair wise distribution of AL in relation to the two polymorphisms, for cases and controls separately and combined. The linkage between the two polymorphisms can also be seen here. There was a significant trend for increasing AL with increasing number of variant alleles in exon 23 overall (P = 0.001), which was mainly due to the trend among controls (P = 0.01). The corresponding trend for exon 10 was also significant (P = 0.02), again, due to that in the controls (P = 0.02). In addition, subjects with the combined exon 10 AA and exon 23 CC genotype showed a significantly higher AL compared with all those with any of the other genotypes (P = 0.02), in particular among controls (P = 0.001). Even after adjustment for age, gender, case status and smoking category in the total sample, lnAL was significantly increased by the number of variant alleles in exon 10 (ß = 0.11, P = 0.01) or exon 23 (ß = 0.11, P = 0.005) or by the AA/CC double homozygosity (ß = 0.23, P = 0.02).


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Table III. Aromatic DNA AL (per 108 nt) in relation to XPD polymorphisms in exon 10 and exon 23
 
Discussion

The variant A allele in XPD exon 10 of our controls was more frequent (37.0%) than that reported previously (164/598 = 27.4%) in three American studies (1,5,11). This difference is, however, not statistically significant. The exon 10 allele A frequency in our study was very similar to that (33%) reported in a forth American study (12). For the exon 23 polymorphism, the frequency of the variant C allele in our controls (37.3%) was similar to the overall frequency (655/1944 = 33.7 %) reported for healthy Caucasians (1,5–7,11,13).

In agreement with the previous report (12), the polymorphism in exon 10 is in linkage disequilibrium with the polymorphism in exon 23 in our study. The exon 10 or exon 23 polymorphism has also been found to be closely linked to a silent polymorphism in exon 6 that has been associated with increased risk of basal cell carcinoma (6,12). In contrast to the A > C polymorphism in codon 751 (Lys > Gln) of exon 23, the G > A polymorphism in codon 312 of exon 10 results in a non-conservative amino acid replacement (Asp > Asn) in an evolutionary highly conserved region (1). This is indicative of a stronger functional role of the exon 10 polymorphism compared to the exon 23 polymorphism.

We found that presence of one or two variant alleles was associated with an increased risk for lung cancer among never-smokers only, in particular those who were younger than 70 years (Table IIGo). In addition, there were significant trends of increasing ALs with increasing number of variant alleles in both exons of XPD, in particular among controls (Table IIIGo) and among former or never-smokers (data not shown). Thus, the XPD variant alleles may be associated with a reduced repair of aromatic DNA adducts in general. Because of the small sample size, especially after multiple subdivisions, the results obtained may be due to chance. Nevertheless, the exon 23 CC homozygosity has recently been associated with high DNA AL among healthy never-smokers (14). These results support our previous notion that common genotype alterations for enzymes involved in detoxification or repair may confer increased susceptibility to tobacco carcinogens when age or exposure level is low (15).

In contrast to our findings, individuals carrying two exon 23 A alleles (wild-type homozygotes) have been shown previously to have a reduced repair proficiency as measured by a higher frequency of chromatid aberrations induced by X-ray in vitro (5). The AA genotype or the A allele in exon 23 has also been associated with an increased risk for basal cell carcinoma (6) and melanoma (13), respectively. These studies were, however, very small and no effect of XPD polymorphism was seen in a much bigger study on melanoma (16). In addition, a protective effect of the CC genotype in exon 23 could not be confirmed in a recent American study of basal cell carcinoma (12).

The presence of one or two variant A alleles in exon 10 was, however, significantly associated with an increased risk of basal cell carcinoma among subjects with a family history of non-melanoma skin cancer (12). The exon 10 polymorphism did not appear to affect DNA repair proficiency (5). Our recent data (17) showed, however, that the combined exon 10 AA and exon 23 CC genotype (variant homozygotes) was associated with depressed repair rates of UV-induced DNA cyclobutane dimers in the skin of volunteers after solar irradiation. The decrease was only observed among those who were 50 years or older and there was a significant trend of decreasing repair rate with increasing number of variant alleles in exon 23 among older subjects. This may suggest that the exon 23 C allele is associated with a depressed repair of UV-induced DNA damages when combined with high age. Difference in age distribution may thus explain some discrepancies between different reports.

The depressed repair of UV-induced DNA damage in variant homozygotes was consistent with our present findings among lung cancer patients and population controls, who had an overall high age (mean 67 and 65 years, respectively). In agreement with our results, a recent study on head and neck cancer revealed a higher frequency of the XPD exon 23 CC genotype in cases than in controls, in particular among older subjects (7). More recently, subjects with at least two variant alleles in exon 10 and exon 23 of XPD were shown to have a significantly increased risk for lung cancer (11). In addition, lung cancer cases homozygous for the variant allele in either exon had a significantly reduced repair capacity (as measured by the host cell reactivation assay) against DNA damage induced by benzo[a]pyrene, a major constituent of cigarette smoke. Taken together, it seems that the variant alleles of XPD exon 10 and exon 23 are associated with decreased repair of aromatic DNA adducts and increased risk for smoke-related cancer.

Notes

3 To whom correspondence should be addressed
Email: saimei.hou{at}cnt.ki.se Back

Acknowledgments

This study was supported by the Swedish Cancer Society and the Swedish Match Medical Research Fund.

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Received September 26, 2001; revised January 2, 2002; accepted January 4, 2002.