XPA polymorphism associated with reduced lung cancer risk and a modulating effect on nucleotide excision repair capacity

Xifeng Wu1,3, Hua Zhao1, Qingyi Wei1, Christopher I. Amos1, Kerang Zhang1, Zhaozheng Guo1, Yawei Qiao1, Waun K. Hong2 and Margaret R. Spitz1

1 Department of Epidemiology and
2 Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
XPA, a DNA binding protein in the nucleotide excision repair (NER) pathway, modulates damage recognition. Recently, a common single-nucleotide polymorphism (A -> G) of unknown function was identified in the 5' non-coding region of the XPA gene. Because a deficiency in NER is associated with an increased risk of lung cancer, we evaluated the role of this polymorphism in 695 lung cancer case patients and 695 age-, sex-, ethnicity- and smoking-matched control subjects. We also studied the effect of this polymorphism on NER capacity in a subset sample for whom the host cell reactivation data were available. The presence of one or two copies of the G allele was associated with a reduced lung cancer risk for Caucasians {adjusted odds ratio (ORadj) = 0.69 [95% confidence interval (CI) = 0.53–0.90]}, Mexican-Americans [ORadj = 0.32 (95% CI = 0.12–0.83)] and African-Americans [ORadj = 0.45 (95% CI = 0.16–1.22)]. In Caucasians, ever smokers with one or more copies of the G allele were observed to have a significantly reduced risk of lung cancer. Control subjects with one or two copies of the G allele demonstrated more efficient DRC than did those with the homozygous A allele. Our data suggest that the XPA 5' non-coding region polymorphism modulates NER capacity and is associated with decreased lung cancer risk, especially in the presence of exposure to tobacco carcinogens.

Abbreviations: DRC, DNA repair capacity; NER, nucleotide excision repair


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
DNA repair systems are indispensable for maintaining genomic integrity. The ability to monitor and repair carcinogen-induced DNA damage is an important determinant of susceptibility to carcinogenesis. Considerable evidence exists that reduced DNA repair capacity (DRC) may play a role in cancer development (1). We have reported previously in a case-control analysis, that reduced DRC is associated with a >2-fold increased risk of lung cancer (2). Findings from studies of twins and from other family studies have shown that DRC is genetically determined (3,4). The phenotype of suboptimal DRC was more common among relatives of cancer patients with similar poor DRC (3). The phenotype of reduced DRC involving one pathway, e.g. nucleotide excision repair (NER), is independent of the phenotype involving another pathway, e.g. double-stranded break repair (4). In humans, many genes are involved in DNA repair, including polymerases and helicases, as well as genes associated with chromosome segregation, nucleotide synthesis, DNA damage recognition and DNA damage signal transduction. Polymorphisms in any of these genes may modulate DRC and contribute to inter-individual variation in susceptibility to lung carcinogenesis (57).

The XPA gene product is a zinc-finger DNA-binding protein. It is essential in the NER pathway that is involved in removing a wide range of lesions, including benzo[a]pyrene-induced bulky DNA adducts (8,9). The XPA protein is also involved in both global genome and transcription-coupled repair pathways (10). It maintains an intricate network of contacts with core repair factors during its role in DNA repair (11,12). Although the detailed steps involved in NER are still not completely clear, below is the approximate assessment of how the NER systems appear to act (13,14). In the global genome repair pathway, the protein complex XPC–HHR23B, which appears to be essential for the recruitment of all subsequent NER factors in the pre-incision complex, binds to damaged DNA. Then, the multicomponent transcription factor TFIIH, which is responsible for unwinding the damaged region of the DNA (15,16), is recruited. Next, XPG nuclease cleaves the DNA on the 3'-end (17). Following cleavage of the DNA, XPA/RPA proteins join the complex and recruit the ERCC1–XPF complex, which cleaves the 5'-end (18,19). Finally, most of the complexes unbind, leaving only the RPA complex and making room for DNA polymerase {delta} and its cofactors to patch the gap left in the DNA. Hence, any variations that may occur in the XPA gene might have the potential to affect protein function and subsequently DRC.

Considering its affinity for damaged DNA and its ability to interact with many (core) repair factors, XPA is likely to play a central role in positioning the repair machinery correctly around these lesions. Recently, a common single-nucleotide polymorphism (A -> G) in the 5' non-coding region (4 nucleotides upstream from the start codon) of the gene was identified (20) but its functional relevance remains to be determined. Because a deficiency in NER is associated with increased risk of lung cancer (2) and because some polymorphisms in NER genes may predict repair phenotype (6), we evaluated the role of the common polymorphism in the 5' non-coding region in lung cancer susceptibility and the effect of this polymorphism on NER capacity.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study population and data collection
In this study, 1390 subjects were included: 695 lung cancer case patients and 695 healthy control subjects. The details of the recruitment of subjects were the same as those described previously (21). The case patients had newly diagnosed, previously untreated and histologically confirmed lung cancer and were enrolled at The University of Texas M. D. Anderson Cancer Center (Houston, TX) between 1995 and 2000. The control subjects had no history of cancer and were selected from a contact database of a large multi-speciality physician group in the Houston, Texas metropolitan area. Control subjects were frequency matched to the case patients by age (± 5 years), sex, ethnicity and smoking status. There were no exclusions on the basis of age, sex or ethnicity. All subjects signed a consent form and were interviewed using a structured questionnaire that elicited epidemiological data, including demographic data and smoking history. At the end of the interview, 30 ml of peripheral blood was drawn into heparinized tubes. The tubes were then coded with a unique study identification number and immediately hand delivered to the lab. Six milliliters of blood was used to isolate DNA for genetic analysis and 15 ml of blood was used to isolate lymphocytes for the DNA repair assay. The extra blood was retained for other genetic susceptibility marker analyses. The study was approved by the institutional review boards of The University of Texas M. D. Anderson Cancer Center and the Kelsey-Seybold Foundation.

XPA genotyping
Genomic DNA was first isolated from peripheral blood lymphocytes by proteinase K digestion followed by isopropanol extraction and ethanol precipitation, then subjected to restriction fragment length polymorphism–polymerase chain reaction (RFLP–PCR) analysis. Two primers 5'-CTAGGTCCTCGGAGTGGTCC-3' and 5'-GCCCAAACCTCCAGTAGCC-3' (GenoSys, The Woodlands, TX) were used to amplify the 204 bp fragment containing the A/G polymorphic site. The A -> G single nucleotide substitution in the 5' flanking region creates a BspEI restriction site. Briefly, the PCR reaction was performed in a 25 µl reaction mixture containing 10x PCR buffer (500 mM KCl, 100 mM Tris–HCl, 1.0% Triton X-100; Promega, Madison, WI), 2.5 mM MgCl2, 0.25 mM dNTPs, 0.5 µM each primer, 50 µg of template DNA, 2% DMSO and 2 U of Taq polymerase in storage buffer B (20 mM Tris–HCl, 100 mM KCl, 0.1 mM EDTA, 1 mM 1,4-dithiothreitol, 50% glycerol, 0.5% Nonidet-P40 and 0.5% Tween 20; Promega). The PCR cycling conditions for the assay were 94°C for 5 min, followed by 30 cycles at 94°C for 30 s, 57°C for 30 s and 72°C for 60 s, with a final extension step at 72°C for 7 min. Next, 15 µl of the PCR product was digested with 12 U of BspEI (New England Biolabs, Beverly, MA) overnight at 37°C and resolved for 30 min at 220 V on 4% agarose gel stained with ethidium bromide. The AA genotype yielded a 204 bp fragment, the AG genotype yielded 204, 185 and 19 bp fragments and the GG genotype yielded 185 and 19 bp fragments.

DNA repair capacity
The host cell reactivation assay measures the activity of the CAT gene, a bacterial drug resistance gene, in host cells. DRC is expressed as the ratio of CAT activity of the cells transfected with benzo[a]pyrene diolepoxide (BPDE)-treated plasmid to that of the cells transfected with untreated plasmids. Because a single unrepaired DNA adduct can effectively block CAT transcription, the change in CAT activity will reflect the ability of the transfected cells to remove BPDE-induced adducts from the plasmids (2,22).

Lymphocyte processing.
The isolated lymphocytes were frozen in a freezing medium containing 50% fetal bovine serum, 40% RPMI 1640 and 10% DMSO (Fisher Scientific, Pittsburgh, PA) and later thawed in a thawing medium containing 50% fetal bovine serum, 40% RPMI 1640 and 10% dextrose (Sigma Chemical, St Louis, MO), ensuring ~90% viability after thawing.

Plasmid treatment.
The pCMVcat (chloramphenicol acetyltransferase) plasmid (500 mg/ml) was treated with either 0 (control) or 60 mM of BPDE for 3 h in a dark room. The BPDE forms adducts with the plasmid, preventing CAT gene transcription, and these adducts are removed by the NER pathway. The plasmids were then precipitated, washed three times in 70% ethanol, dissolved in Tris–EDTA buffer to yield a final plasmid concentration of 50 µg/ml, and checked for conformational changes on a 0.8% agarose gel. Aliquots of treated plasmid were stored at -80°C for later use, and all plasmids used in this study were from a single treatment.

Transfection.
The frozen cells were briefly thawed, mixed with 9 ml of thawing medium, and then incubated in RPMI 1640 supplemented with 20% fetal bovine serum and 112.5 µg/ml phytohemagglutinin (Murex Diagnostics, Norcross, GA) at 37°C for 72 h. For transfection, 2x106 cells were washed in Tris–EDTA buffered saline solution with calcium (0.7 µM) and magnesium (0.5 µM) and then transfected with 0.25 µg of either treated or untreated plasmids using diethylaminoethyl-dextran (Pharmacia Biotech, Piscataway, NJ). Duplicate transfections were performed with both treated and untreated plasmids. CAT gene expression (and thus NER capacity) was measured 40 h after transfection. Briefly, the transfected cells were collected and washed twice with Tris-buffered saline and resuspended in 31.5 µl with 0.25 M Tris-buffered saline in a 1.5 ml tube. The cells were lysed by three 10 min cycles of freezing and thawing in a dry ice–ethanol bath and a 37°C water bath. Cell extracts were then assayed for CAT expression or activity. The activity of the repaired CAT gene was measured by a scintillation counter for the formation of [3H]monoacetylated and [3H]diacetylated chloramphenicols through the reaction between chloramphenicol and [3H]acetyl coenzyme A catalyzed by CAT protein in the cell extract. DRC was measured as the ratio of reporter gene activity in cells transfected with damaged plamids to that in cells transfected with undamaged plamids. The method for calculation of DRC has been described previously (2).

Statistical analysis
STATA software was used to perform statistical analyses. In the first step of analysis, Pearson’s {chi}2 test was used to compare the distribution of select demographic variables such as age, sex, ethnicity, smoking status and the XPA genotypes between lung cancer case patients and control subjects. Student’s t-test was used to compare mean age and mean pack-years between case patients and control subjects. Patients were stratified into three categories of smoking: never-, former- and current smoker. A never smoker was defined as one who had never smoked or had smoked fewer than 100 cigarettes in his/her lifetime. A former smoker was defined as one who had a history of smoking but had stopped at least 1 year before being diagnosed (or at least 1 year before enrollment into the study, for control subjects). Pack-years were calculated using the following formula: pack-years = (number of cigarettes smoked per day/20 cigarettes)xnumber of years smoked. Hardy-Weinberg equilibrium was tested using the goodness-of-fit {chi}2 test to compare the observed allele frequencies with the expected frequencies in control subjects. In the second step of the analysis, odds ratios (OR) and 95% confidence intervals (95% CI) were used to estimate risk associated with the XPA genotypes using both univariate and unconditional multivariate logistic regression models, and in the latter model, adjusting for confounding factors such as age, sex and smoking status or pack-years. P-values <0.05 were considered significant. DRC was analyzed as a continuous variable. A Student’s t-test was used to compare the differences in repair capacity between different genotypes and by case/control status.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Characteristics of study population
A total of 695 confirmed lung cancer case patients and 695 control subjects were included in this study. Select characteristics of the study population are shown in Table IGo. The case patients and control subjects were generally well matched on the basis of sex, age, ethnicity and smoking status. The study is still ongoing and particularly for the relatively small sample sizes of both Mexican-American and African-American cases and controls, close matching has not yet been achieved especially on age.


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Table I. Distribution of select characteristics among study subjects
 
Genotype distribution of XPA
There were significant differences in XPA genotype distribution in the control subjects among three ethnic subgroups (P = 0.033). The genotype frequencies among the control subjects were in Hardy-Weinberg equilibrium for Mexican-Americans (P = 0.294) and African-Americans (P = 0.548), but not for Caucasians (P = 0.035). There was a significant difference in genotype distribution between the cases and the controls among Caucasians (P = 0.015), a borderline significant difference for Mexican-Americans (P = 0.070), and no difference among African-Americans (P = 0.378). For all three ethnic groups, the GG and GA genotypes were more common in the control subjects than in the case patients. In addition, the G allele frequency was higher in the control subjects than in the cases for all three ethnic groups (Caucasians: 0.55 versus 0.52; Mexican-Americans: 0.61 versus 0.47; African-Americans: 0.70 versus 0.63 controls versus cases). Among the control subjects, African-Americans exhibited the highest G allele frequency, and Caucasians the lowest.

Risk estimates among ethnic groups
The risk estimates for individuals with one or two copies of the G allele are shown in Table IIGo. Compared with individuals with AA genotypes, Caucasians with one or two copies of the G allele were at a significantly reduced risk for lung cancer, with adjusted ORs of 0.65 (95% CI = 0.48–0.87) and 0.74 (95% CI = 0.55–1.01), respectively. A similar but even stronger association was observed for Mexican-Americans with ORs of 0.27 and 0.37, respectively. For AfricanAmericans, there was a 50% reduction in risk, although this was not statistically significant. We also present risks for combined AG and GG genotypes in Table IIGo.


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Table II. Risk estimates for XPA polymorphisms in cases and controls among ethnic groups
 
Risk estimates in Caucasians by smoking status
Caucasians were further stratified by smoking status (Table IIIGo). This stratification was not feasible for Mexican-Americans and African-Americans, owing to the small number of subjects in these groups. Ever smokers (but not never smokers) with at least one copy of the G allele showed a reduced risk (AdjOR = 0.64; 95% CI = 0.47–0.87 for one copy of the G allele, AdjOR = 0.75; 95% CI = 0.54–1.04 for two copies of the G allele and AdjOR = 0.68; 95% CI = 0.51–0.91 for combining one and two copies of the G allele). Whenever smokers were further stratified into former and current smokers, a similarly reduced risk was observed for both groups (data not shown).


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Table III. Analysis of XPA by smoking status in Caucasians
 
DNA repair capacity in Caucasians
The DRC among Caucasian subjects is shown in Table IVGo. Because of the small number of subjects in other ethnic groups, we restricted analyses to Caucasian subjects. In the control group, individuals with one or more copies of the G allele showed a significantly higher mean DRC (9.53 ± 4.81) than did those without the G allele (8.29 ± 2.81) (P = 0.03). No difference in repair capacity was observed in case patients. Also, similar trends were found when we compared the natural log-transformed DRC among different XPA genotypes (data not shown).


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Table IV. DNA repair capacity (DRCa) in Caucasians by XPA genotypes
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There is an expanding body of evidence suggesting that polymorphisms in DNA repair genes are associated with cancer risk. For example, of the five polymorphisms in the XPD gene, the Asn312Asp polymorphism is shown to be associated with a lower risk of lung cancer (7) and basal cell carcinoma (23), and the Lys751Gln polymorphism is associated with a decreased risk of basal cell carcinoma (24), but an increased risk of head and neck cancer (5).

Recently, an A -> G single-nucleotide substitution was identified in the 5' non-coding region of the XPA gene, located 4 nucleotides upstream of the ATG start codon (20). The functional significance of this polymorphism is unknown. It has been demonstrated that the 5' non-coding region may regulate gene expression through transcriptional and post-transcriptional control mechanisms (25,26) and we speculated that this polymorphism might affect the mRNA tertiary structure and stability or affect the binding between translational factors and mRNA. In addition, several site-directed mutagenesis studies have shown that mutations in the functional domains of the XPA gene could partially or completely inactivate its function (11,2732). Although there are no in vitro data to suggest any functional effect of this polymorphism, the results from this population-based study indicate that this polymorphism may have functional significance.

In this study, we showed that individuals with the A -> G substitution in the 5'-end of the non-coding region, had a reduced risk of lung cancer. This pattern was statistically significant for Caucasians (ORadj = 0.69; 95% CI = 0.53–0.90) and Mexican-Americans (ORadj = 0.32; 95% CI = 0.12–0.83), but not for African-Americans (ORadj = 0.45; 95% CI = 0.16–1.22). We also observed that the polymorphism conferred a greater protective effect among ever smokers than never smokers, suggesting that the protective effect is evident largely in the presence of carcinogenic exposure. There were also ethnic differences in distribution of the variant allele. The frequency of the G allele was higher among African-American control subjects (0.70) than among Caucasian (0.55) or Mexican-American control subjects (0.61). In 35 Caucasian individuals in Poland (20), the frequency of the G allele was 0.57, which accords well with our data in US Caucasians.

Interestingly, Park et al. recently reported a similar association between XPA A/G polymorphism and lung cancer risk in a Korean study (33). The frequency of the G allele was 0.51, which was similar to our study. They found that the GG genotype was a protective factor for lung cancer risk with an odds ratio of 0.56 (95% CI: 0.35–0.90). The protective effect was more evident in younger individuals (age <=62 years old), male and current smokers. Although the protective effect of the G variant allele did not differ by age or gender in our study (data not shown), we also found that the effect was more striking in ever smokers. Ethnicity, smoking behavior, and sample size between these two studies may contribute to the different results by age and gender.

Using a subset of the study population, our group reported previously that lung cancer cases exhibited a significantly lower DRC than controls (2). Case patients who were younger at diagnosis (<60 years old), female, lighter smokers or who reported a family history of cancer exhibited the lowest DRC and the highest lung cancer risk among their subgroups, suggesting that these subgroups may be especially susceptible to lung cancer. NER is a major player in the repair of DNA damage caused by exposure to tobacco carcinogens. As more than 30 genes are involved in NER, many factors might affect the capacity of NER. Their effects may range from a large effect to little or no effect. We have reported previously that two polymorphisms in the XPD gene were associated with reduced NER capacity and with increased risk of lung cancer (6). In this analysis, we observed that the XPA polymorphism was associated with more efficient DRC in control subjects with the variant alleles, but not in the cases. In lung cancer cases, there are likely a variety of multigenic defects that could potentially mask the effects of a single variant allele. It may be possible to determine how variant alleles contribute to DRC in lung cancer cases by studying multiple polymorphisms involved in the same DNA repair pathway.

In summary, we have demonstrated that an XPA polymorphism modulates NER capacity and is associated with reduced lung cancer risk, especially among ever smokers.


    Notes
 
3 To whom correspondence should be addressed Email: xwu{at}mail.mdanderson.org Back


    Acknowledgments
 
Dr Waun Ki Hong is an American Cancer Society Clinical Research Professor. This work was supported by NCI grants P01 CA52051, U19 CA68437 and R01 55769.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received October 3, 2002; revised December 10, 2002; accepted December 16, 2002.