XPD/ERCC2 polymorphisms and risk of head and neck cancer: a case-control analysis

Erich M. Sturgis1,2, Rong Zheng2, Lei Li3, Edward J. Castillo2, Susan A. Eicher1, Minhui Chen2, Sara S. Strom2, Margaret R. Spitz2 and Qingyi Wei2,4

Departments of
1 Head and Neck Surgery,
2 Epidemiology and
3 Experimental Radiation Oncology, The University of Texas M.D.Anderson Cancer Center, Houston, TX 77030, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
DNA repair capacity is central in maintaining normal cellular functions. Variants of several DNA repair genes,including the nucleotide excision repair gene XPD, have been described recently. Because we previously reported that patients with squamous cell carcinoma of the head and neck (SCCHN) had lower DNA repair capacity than healthy controls, we hypothesized that inherited polymorphisms of XPD may contribute to genetic susceptibility to SCCHN, a tobacco-related cancer. To test this hypothesis, we conducted a hospital-based case-control study of 189 SCCHN patients and 496 cancer-free controls who were frequency-matched on age, gender and smoking status. All subjects were non-Hispanic whites. Two XPD polymorphisms (C22541A and A35931C) were typed using the restriction enzymes TfiI and PstI, respectively. Multivariate logistic regression analysis was performed to calculate adjusted odds ratios (ORs) and 95% confidence intervals (CIs). In the controls, the frequencies of the variant 22541A and 35931C alleles were 44.7% and 33.8%, respectively. The frequency of the 22541A homozygous genotype (22541AA) was lower in cases (15.9%) than in controls (20.4%) but was not associated with risk (adjusted OR = 0.90; 95% CI = 0.52–1.56) for SCCHN. The frequency of the 35931C homozygous genotype (35931CC) was higher in cases (16.4%) than in controls (11.5%) and associated with a borderline increased risk (adjusted OR = 1.55; 95% CI = 0.96–2.52) for SCCHN. The risk was higher in older subjects (OR = 2.22; 95% CI = 1.03–4.80), current smokers (OR = 1.83; 95% CI = 0.79–4.27) and current drinkers (OR = 2.59; 95% CI = 1.25–5.34) in the stratification analysis. These results suggest a gene–environment interaction, but this did not reach statistical significance. The findings are limited due to the relatively small numbers in the subgroups and need to be verified by further investigations.

Abbreviations: BPDE, benzo[a]pyrenediol epoxide; CI, confidence interval; OR, odds ratio; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; SCCHN, squamous cell carcinoma of the head and neck; TFIIH, transcription factor IIH.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Squamous cell carcinoma of the head and neck (SCCHN) is one of the five most common cancers worldwide, accounting for >250 000 deaths annually (1); there are 40 300 new cases annually in the USA (2). Studying head and neck carcinogenesis is an excellent model for studying tobacco-induced carcinogenesis in general, because the two processes have many similar characteristics in terms of demography, exposure and molecular biology. As the survival rates for these cancers have not changed over the last 30 years (2), more emphasis on prevention seems warranted. Relatively few smokers develop cancer, so identifying those at greatest risk of tobacco-induced carcinogenesis would greatly improve the efficiency of prevention and eventually chemoprevention programs.

Genetic differences in the ability to metabolize carcinogens, repair DNA damage and induce apoptosis may contribute to the variation in cancer risk in the population at large. We have previously reported an increased risk of SCCHN associated with variants of carcinogen detoxifying genes (GSTM1 and GSTT1) (3) and of the DNA repair gene XRCC1 (4). Recently, the entire coding region of the DNA repair gene XPD/ERCC2 was resequenced in 12 normal individuals; six polymorphic variants were described (5). The XPD protein is an evolutionarily conserved helicase, a subunit of transcription factor IIH (TFIIH) that is essential for transcription and nucleotide excision repair (6). Mutations in XPD prevent its protein product from interacting with p44, another subunit of TFIIH (7) and reduce its helicase activity, resulting in a defect in nucleotide excision repair. Mutations at different sites result in three distinct clinical phenotypes: xeroderma pigmentosum, trichothiodystrophy and xeroderma pigmentosum combined with Cockayne sydrome (8). Studies have shown that XPD is involved in repairing genetic damage induced by tobacco carcinogens and other carcinogens (9).

Although we do not know the functional significance of the newly identified XPD variants, we hypothesized that genetic variants in XPD may be markers for SCCHN susceptibility and therefore would exist at different frequencies in individuals with and without cancer. To test this hypothesis, we performed a hospital based case control study using a polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) assay to genotype two variants of the XPD gene in 189 patients with SCCHN and 496 cancer-free controls. The two XPD variants, C22541A at codon 156 of exon 6 and A35931C at codon 751 of exon 23, were chosen because their allele frequencies are higher (>=25%) than those of other reported variants (5) and because it has been suggested that the C22541A and A35931C polymorphisms are associated with risk for non-melanoma skin cancer in psoriasis patients (10).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study subjects
From May 1995 to December 1999, patients with histopathologically confirmed SCCHN (having primary tumors of the oral cavity, oropharynx, hypopharynx and larynx) were recruited from patients seen at the Department of Head and Neck Surgery of The University of Texas M.D.Anderson Cancer Center. Patients with primary tumors of the nasopharynx or sinonasal tract, primary tumors outside the upper aerodigestive tract, cervical metastases of unknown primary origin and a histopathological diagnosis of cancer other than squamous cell carcinoma were excluded. Effort was made to obtain blood samples from each eligible (incident) case before treatment. Patients who had already received treatment before blood samples were taken were excluded from the analysis. Healthy control subjects were recruited from a local managed care organization with multiple clinics throughout the Houston metropolitan area during a similar time period (11). These control subjects were first surveyed by a short questionnaire for willingness to participate in research studies and to provide information on smoking behaviors and demographics. A computer database of >50 000 potential control subjects was constructed and used to identify eligible control subjects who were frequency-matched to the patients by age (within 5 years), gender, ethnicity and smoking status. Once identified and contacted, the participation rate was 73.3%. Each eligible subject was then scheduled for an interview. Data on age, gender, ethnicity, smoking status, and alcohol consumption before the onset of disease were derived from questionnaires administered by interviewers. Because genotype frequencies can vary between ethnic groups and few minority patients were recruited, only non-Hispanic whites were included in this study. After informed consent was obtained, each subject donated 30 ml of blood in heparinized tubes. The research protocol was approved by the M.D.Anderson Institutional Review Board.

Genotyping
From each blood sample, a leukocyte cell pellet was obtained from the buffy coat by centrifugation of 1 ml of whole blood and used for DNA extraction. PCR assays were used to amplify the exons of XPD/ERCC2 containing the polymorphisms of interest. We focused on two polymorphisms that have relatively high frequencies of variant alleles with a restriction site. The primers for exon 6 were 5'-TGGAGTGCTATGGCACGATCTCT-3' and 5'-CCATGGGCATCAAATTCCTGGGA-3', which generate a 644 bp fragment. The primers for exon 23 were 5'-TCAAACATCCTGTCCCTACT-3' and 5'-CTGCGATTAAAGGCTGTGGA-3', which generate a 344 bp fragment. These fragments were amplified separately but under the same conditions in a 50 µl reaction mixture containing ~50 ng of genomic DNA, 12.5 pmol of each primer, 0.1 mM each dNTP, 1x PCR buffer [50 mM KCl, 10 mM Tris–HCl (pH 9.0 at 25°C), and 0.1 % Triton X-100], 1.5 mM MgCl2 and 2.25 units of Taq polymerase (Promega Corporation, Madison, WI). The PCR regime consisted of: (i) an initial melting step of 95°C for 5 min; (ii) 35 cycles of 95°C for 30 s, 61°C for 35 s and 72°C for 45 s; and (iii) a final elongation step of 72°C for 10 min. The PCR products were checked on a 1% agarose gel, photographed with Polaroid film and then subjected to RFLP analysis.

The restriction enzyme TfiI (New England BioLabs, Inc., Beverly, MA) was used to distinguish the C22541A polymorphism of exon 6 in which the gain of a TfiI restriction site occurs in the polymorphic allele (10). The wild-type allele has a single TfiI restriction site resulting in two bands (587 and 57 bp), whereas the variant allele produces three fragments (474, 113, and 57 bp). The restriction enzyme PstI (New England BioLabs) was used to type the A35931C polymorphism of exon 23, in which the gain of a PstI restriction site occurs in the variant allele. The wild-type allele has a single PstI restriction site resulting in two bands (234 and 110 bp), whereas the variant allele results in three fragments (171, 110, and 63 bp). The entire 50 µl PCR reaction volume was digested with 2 units of TfiI or 10 units of PstI (for each variant) and the 1x buffer supplied with each restriction enzyme at 37°C overnight with the exception of TfiI (65°C for 1 h). The digestion products were separated on a 3% NuSieve 3:1 agarose (FMC BioProducts, Rockland, ME) gel and photographed with Polaroid film.

Statistical analysis
Univariate analysis was first performed to calculate the frequency of each genotype. The concordance between the genotype frequencies of two polymorphisms was also examined by tabulation. The observed genotype frequencies were compared with those calculated from Hardy Weinberg disequilibrium theory (p2 + 2pq + q2, where p is the frequency of the variant allele and g = 1 – P). The crude odds ratios (ORs) and their 95% confidence intervals (CIs) for the C22541A and A35931C genotypes were calculated by logistic regression analysis with adjustment for age, gender, smoking status and alcohol use. Subjects who had smoked >100 cigarettes in their lifetimes (before diagnosis for the cases) were defined as the `ever smokers'; of this group, those who had given up smoking >1 year previously were defined as `former smokers' and the others as `current smokers'. Subjects who had drank alcoholic beverages at least once a week >1 year previously (before diagnosis for the cases) were defined as `ever drinkers'; of this group, those who had given up drinking for >1 year were defined as `former drinkers' and the others as `current drinkers'. For logistic regression analysis, the XPD genotype was recoded as a dummy variable. To evaluate gene–environment interaction, the products of XPD genotypes with variables for age group, smoking status or alcohol use were included in the multivariate logistic regression models in the presence of age, gender, smoking status, alcohol use and XPD genotypes. All of the statistical analyses were performed with Statistical Analysis System software (Version 6; SAS Institute Inc., Cary, NC).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As shown in Table IGo, the analysis included 189 cases (site of cancer: oral cavity, 70; pharynx, 75; larynx, 44) and 496 controls. Although effort was made to frequency-match cases and controls by age, gender and smoking status, the cases were younger than controls. However, there was no significant difference in the mean age between the cases (mean ± SD, 58.3 ± 13.0, range 20–89 years) and the controls (59.7 ± 11.3, range 26–87 years) (P = 0.160). There were more males in the cases (70.4%) than in the controls (59.9%) and more current smokers (33.9%) and current drinkers (48.2%) in the cases than in the controls (26.2% and 38.9%, respectively). These differences were controlled for in the multivariate logistic regression models.


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Table I. Select demographic and risk factors in HNSCC patients and healthy controls
 
The concordance of the two XPD/ERCC2 variants was first examined as shown in Table IIGo. While haplotyping was not possible with the PCR–RFLP method, no control subject was homozygous for both the 22541A (codon 156 of exon 6) and 35931C (condon 751 of exon 23) alleles (Table IIGo). Control subjects homozygous for the 22541A alleles (i.e. genotype 22541AA) were usually (89.1% of the time) also homozygous for the 35931A allele, and control subjects with the 35931CC genotype were often (75.4% of the time) homozygous for the 22541C allele (Table IIGo). The cases had a similar pattern of genotype distribution. These findings are also consistent with an earlier report suggesting linkage disequilibrium of these two polymorphisms (10).


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Table II. Concordance between two XPD/ERCC2 polymorphisms in SCCHN patients and healthy controls; the number (%) in each group is shown
 
The frequency of the 22541A allele was lower, but not significantly, in the cases (41.5%) than in the controls (44.7%). This allele frequency of the controls (44.7%) was similar to those reported (43–50%) from much smaller control populations (N = 40 or 70) (10,12). The frequency of the XPD 35931C allele was non-significantly higher in the cases (38.4%) than in the controls (33.8%). Again, this allele frequency in controls (33.8%) was similar to those (29–38%) reported for smaller control groups (N = 12–40) (5,10,12). The observed genotypes of the two variants in the controls were not statistically different from those expected from the Hardy Weinberg disequilibrium theory.

To evaluate the association between the two XPD variants and risk of SCCHN, crude and adjusted ORs and their 95% CIs were calculated using both the homozygous genotypes of the XPD 22541CC and 35931AA or combined with their heterozygous genotypes as the reference groups, respectively. As shown in Table IIIGo, the 22541AA genotype was less frequent in the cases (15.9%) than in the controls (20.4%), whereas the 22541C/A and C/C genotypes were more frequent in the cases (51.3% and 32.8%, respectively) than in the controls (48.6% and 31.1%, respectively). Compared with both the 22541C/C genotype and the (22541C/C + C/A) genotypes, the 22541AA genotype was associated with a slightly protective effect (adjusted OR = 0.90, 95% CI = 0.52–1.56 for former comparison; OR = 0.92, 95% CI = 0.65–1.32 for latter comparison) against SCCHN. These findings are consistent with a non-significantly elevated risk associated with the XPD 22541CC genotype reported for non-melanoma skin cancer (10).


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Table III. Logistic regression analysis of XPD/ERCC2 polymorphisms in SCCHN
 
The XPD 35931CC variant genotype was more frequent in the cases (16.4%) than in the controls (11.5%), whereas the XPD 35931AA or AC genotypes were almost identical between the cases and the controls (Table IIIGo). Compared with the 35931AA genotype, the variant 35931CC genotype was associated with a borderline increased risk (adjusted OR = 1.65; 95% CI = 0.98–2.77) of SCCHN, whereas no increased risk (adjusted OR = 1.12; 95% CI = 0.77–1.62) was associated with the 35931AC genotype. Compared with the combined group of 35931AA + 35931AC genotypes, the 35931CC genotype remained associated with a borderline increased risk (OR = 1.55; 95% CI = 0.92–2.52) for SCCHN.

Finally, using the combined reference groups [i.e. (22541CC + 22541CA) or (35931AA + 35931AC)], we analyzed the associations between the two XPD variant genotypes of 35931CC and 22541AA and subgroups for age, gender and smoking/drinking status (Table IVGo). The 22541AA genotype was more frequent among most of the subgroups in the cases than in the controls, except for the `never smokers' and `never drinkers'. Although a significant protective effect was observed for the subgroup of former drinkers (OR = 0.44; 95% CI = 0.23–0.86), no significant protective effect was observed for other subgroups. The 35931CC genotype was more frequent among smokers or drinkers in the cases than in the controls, but was less frequent among the `never drinkers' in the cases than in the controls. Compared with the combined 35931AA+35931AC genotypes, the 35931CC genotype was associated with a borderline increased risk in current smokers (adjusted OR = 1.83; 95% CI = 0.79–4.27), but a significantly increased risk in current drinkers (adjusted OR = 2.59; 95% CI = 1.25–5.34), as well as in subjects aged >65 years (adjusted OR = 2.22; 95% CI = 1.03–4.80). These findings are consistent with the notion that a gene environment interaction may exist for 35931CC genotype, because the older age is also an indication of higher lifetime cumulative exposure. However, this suggestive evidence for interaction between exposure and the 35931CC genotype did not reach a level of statistical significance as assessed in the multivariate logistic regression analysis (P = 0.295, 0.680 and 0.059 for the 35931CC genotype with age, smoking status and alcohol use, respectively).


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Table IV. Stratification analysis of XPD/ERCC2 genotype frequencies, adjusted OR and 95% CI in SCCHN patients and controls
 
There was no difference in risk associated with the 22541AA or 35931CC genotype among subgroups of cases (i.e. those with cancer of the oral cavity, pharynx or larynx) (data not shown), suggesting that these polymorphisms are constitutional markers for susceptibility to cancer, rather than markers of the tumors.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study is the first analysis of two reported XPD/ERCC2 polymorphisms in a case–control study of SCCHN and it provides estimates of these variant allele frequencies from nearly 500 cancer-free controls. Overall, no statistically significant differences were found between cases and controls for the two variant genotypes, although a borderline increased risk was associated with the XPD 35931CC (Gln751) compared with the XPD 35931AA (Lys751) of exon 23. In particular, this risk was further increased for the subgroup of current smokers and was statistically significant for the subgroups of older subjects and current drinkers, indicating a possible gene–environment interaction. Taken together, these findings suggest that the XPD 35931CC genotype may have some biological relevance, whereas the XPD C22541A variant at codon 156 of exon 6 has no biological relevance in the etiology of SCCHN.

XPD codes for a DNA helicase, a component of transcription factor TFIIH involved in nucleotide excision repair (5). Mutations, such as a nucleotide substitution in codon 541 causing Ser to be changed to Arg in the helicase domain IV of the XPD protein, can result in defective repair phenotypes (13,14). A recent study of 34 psoriasis patients suggested that lymphocytes from psoriasis patients with basal cell carcinomas and the 35931AA+35931AC genotypes or the 22541AA+ 22541AC genotypes had increased levels of DNA strand-breaks, an indicator of incision levels resulting from incomplete DNA repair measured by the comet assay, after ultraviolet C irradiation (15). This suggests that people with the XPD 35931CC and 22541CC genotypes had a higher DNA repair level. The nucleotide variation at A35931C is located ~50 bases upstream from the poly(A) signal, which may alter the function of the XPD protein. The observed effect was also suggested to be possibly attributed to other repair proteins encoded by genes such as XRCC1, ERCC1 and LIG1, which are located close to XPD as a result of cosegregation with a polymorphism in one of these genes (15). Another study of 31 women suggested that individuals with the XPD 35931AA genotype (encoding Lys/Lys at codon 751) genotype also had a higher level of chromatid aberrations induced by X-ray than those having the XPD 35931CC genotype (encoding Lys/Gln) (16). This finding appears to be consistent with the study of psoriasis patients (15). To the contrary, another study of 76 healthy subjects suggested that the 35931CC genotype was unrelated to either the frequency of smoking-induced sister chromatid exchange or polyphenol DNA adducts (n = 61) (17). As we demonstrated before, xeroderma pigmentosum cells deficient in nucleotide excision repair are sensitive to UV light, which causes DNA modification, but resistant to gamma radiation, which causes DNA strand breaks (18). Therefore, the discrepancy in the effect of the XPD A35931C polymorphism at codon 751 of exon 23 in these three studies may be due either to the difference in DNA damage induced by different carcinogens or to the relative small samples used for the analysis.

Our finding that the XPD 35931CC is a potential risk genotype is consistent with the reported polymorphic site that causes amino acid substitution (5) and may affect the protein function (12). Although the impact of this polymorphism on DNA repair phenotype is still unclear, our results support its possible biological significance in the etiology of SCCHN. We previously reported that SCCHN patients had a reduced repair capacity for tobacco carcinogen BPDE-induced DNA damage in a reporter gene (19), suggesting that an inefficient nucleotide excision repair may be involved in the etiology of SCCHN. A normal nucleotide excision repair function requires interaction between many DNA repair gene products including transcription factor TFIIH (6,7), which contains several subunits that include XPD. According to our results, it is possible that the A35931C polymorphism may be associated with a change in the function of the TFIIH. Clearly, functional (phenotypic) studies of DNA repair function in individuals with various genotypes of this polymorphism are needed. However, it will be difficult to detect subtle differences in DNA repair capacity due to a single polymorphism of a single gene in a very complex pathway, and protein structure. Therefore, the role of the XPD A35931C polymorphism in the etiology of SCCHN needs to be evaluated within the context of other polymorphisms of the genes involved in TFIIH. The XPD C22541A polymorphism is silent, resulting in no amino acid substitution (5). It is possible that such a sequence variation could affect RNA stability or otherwise disturb protein synthesis (10). However, our results do not support the possible biological role of the XPD C22541A polymorphism in the etiology of SCCHN.

Perhaps the most intriguing finding was the elevated risk in older subjects, current smokers and current drinkers. SCCHN is primarily a disease of smokers and drinkers (20,21) and rarely occurs in non-smokers and non-drinkers. Theoretically, SCCHN patients are genetically more susceptible to carcinogenesis in the mucosa of the upper aerodigestive tract. According to this hypothesis, cancer occurs in these individuals after relatively minor environmental exposures to carcinogens because of an inherently less efficient carcinogen detoxification process, DNA repair system or apoptotic mechanism. Therefore, early onset of disease is a feature of genetic susceptibility. However, our data suggest that current and cumulative exposure to tobacco smoke and alcohol may be more relevant in those who have the XPD/ERCC2 35931CC genotype. It is possible that this genotype results in suboptimal DNA repair capacity (15), which may only be overwhelmed by excessive damage induced by tobacco smoke and alcohol exposure. However, this hypothesis needs to be verified in future studies.

It is possible that our findings are due to chance, because of the relatively small numbers in the subgroups. Further studies with more patients and quantitative measures of tobacco and alcohol exposure will be needed to confirm these findings. As new technologies are developed for easier genotyping of several (perhaps hundreds) of genes at once, it will become possible to construct genetic profiles of risk or risk models composed of combinations of several polymorphisms in several genes, each of which contributes only slightly to the overall risk. The type of work presented here will be critical to deciding which genetic polymorphisms to include in such risk models. Ultimately, genetic profiling will improve primary and secondary prevention strategies as well as chemoprevention.


    Notes
 
4 To whom correspondence should be addressed Email: qwei{at}mdanderson.org Back


    Acknowledgments
 
We thank Dr Harvey Mohrenweiser for his technical advice, Dr Maureen Goode for her scientific editing, Ms Linda Young and Ms Margaret Lung for their assistance in recruiting patients, Ms Yongli Guan, Ms Kristina Dohlstrom and Ms Lilian Mugartegui for their technical assistance and Ms Joanne Sider and Ms Joyce Brown for manuscript preparation. This study was supported by grants from the National Institute of Health CA 55769 (to M.R.S.) and CA 70334, CA 74851 and CA 77242 (to Q.W.), by a National Cancer Institute grant (CA16672) to M.D.Anderson Cancer Center and by a grant ES07784 from the National Institute of Environmental Health Sciences. E.M.S. is a clinical research fellow supported by NIH Training grant CA 60374 (to Dr Gary Clayman).


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 10, 1999; revised June 6, 2000; accepted August 29, 2000.