Polymorphisms of the DNA repair gene XPD: correlations with risk of basal cell carcinoma revisited
Ulla Vogel3,,
Mohammad Hedayati1,,
Marianne Dybdahl,
Lawrence Grossman1, and
Bjørn Andersen Nexø2,
National Institute for Occupational Health, DK-2100 Copenhagen O, Denmark,
1 Department of Biochemistry, The Johns Hopkin's School of Public Health, Baltimore, MD 21250, USA and
2 Institute of Human Genetics, University of Aarhus, DK-8000 Aarhus C, Denmark
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Abstract
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The XPD gene product has a dual function in basal transcription and in nucleotide excision repair. We have previously reported that two polymorphisms in the gene, one silent mutation in codon 156 of exon 6 and one giving rise to a Lys
Gln substitution in codon 751 of exon 23, showed signs of being associated with basal cell carcinoma in a Scandinavian study group of psoriasis patients and non-psoriatics with and without basal cell carcinoma [Dybdahl, Vogel, Frentz, Wallin and Nexø (1999) Cancer Epidemiol. Biomark. Prev., 8, 7781]. In both polymorphisms, the CC genotype appeared to be protective against basal cell carcinoma. Here, we have genotyped an American study group of basal cell carcinoma patients and controls without skin cancer for the two polymorphisms. In addition, we studied an A
G polymorphism in codon 312 of exon 10, which results in an Asp
Asn substitution in a conserved region of XPD. In the whole study group, subjects carrying the AA and AC genotype in exon 6 were at 1.9-fold higher risk of basal cell carcinoma (P = 0.062, CI 0.963.75). If only subjects without a family history of non-melanoma skin cancer were included, subjects carrying AA or AC genotype were at 3.3-fold higher risk of basal cell carcinoma (P = 0.007, CI 1.358.18). Among subjects with a family history of non-melanoma skin cancer, subjects with an AG or AA genotype in codon 312 of exon 10 were at 5.25-fold increased risk of basal cell carcinoma (P = 0.027, CI 1.1523.93). A protective effect of the CC genotype in exon 23 could not be confirmed. Cases with a family history of skin cancer had statistically significantly different allele frequencies of the polymorphisms in exon 6 and exon 10 from cases without family history of non-melanoma skin cancer. Our results indicate that the exon 6A allele is a risk factor in basal cell carcinoma.
Abbreviations: BCC, basal cell carcinoma; OR, odds ratio; CI, confidence interval.
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Introduction
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Genes involved in DNA repair play a critical role in the first line of defense against cancer. This is illustrated by the rare hereditary disease xeroderma pigmentosum (1), which results in more than a 1000-fold increased risk of sunlight induced skin cancer, caused by defects in the nucleotide excision repair pathway. Another example is the relationship between defects in mismatch repair genes and familial colon cancer (2).
Associations of risk of certain cancers with the constitution of people have been obtained in studies of DNA repair capacity (36). These studies are highly suggestive of genetic or life-style factors being of importance to cancer risk through the modulation of DNA repair. However, as the factors determining DNA repair capacity are as yet unknown, these correlations do not extrapolate easily to statements about importance of genetic factors in disease occurrence.
XPD encodes a helicase, which participates in both nucleotide excision repair and basal transcription as part of the transcription factor TFIIH (7). Mutations destroying enzymatic function of the XPD protein is manifested clinically in combinations of three severe syndromes, Cockayne syndrome, xeroderma pigmentosum and trichotiodystrophy depending on the location of the mutation (8,9). In healthy individuals, the XPD protein level may also be of importance as the mRNA level of XPD has been shown to correlate with the DNA repair capacity in primary lymphoblasts (10).
We have previously presented data to suggest that two known polymorphisms in the nucleotide excision repair gene XPD were associated with increased risk of basal cell carcinoma (BCC) amongst Scandinavian psoriasis patients, who through their treatment for psoriasis were exposed to very genotoxic agents such as coal tar, psoralen and UV light, X-rays and methotrexate. For the polymorphism in exon 23, an association to BCC was suggested among psoriatics as well as non-psoriatics (OR = 4.3, P = 0.075), whereas for the exon 6 polymorphism an association was only suggested in psoriatics (OR = 5.3, P = 0.078) (11). Subjects carrying AA genotype were in both cases at higher risk of BCC. In addition, we have included a G
A polymorphism in codon 312 of exon 10 which results in an Asp
Asn substitution in an evolutionally conserved region (1214).
In the present study, we have analyzed three XPD polymorphisms in a well-described study group of American BCC patients and controls recruited at dermatological clinics (4). We confirm that the XPD exon 6CC genotype is protective against basal cell carcinoma. This protective influence mainly occurs among persons without a family history of non-melanoma skin cancer. Among subjects with a family history of non-melanoma skin cancer, the A-allele of the exon 10 polymorphism was associated with increased risk of BCC.
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Materials and methods
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Study subjects
Details of the study population and subject selection have been described previously (4). Briefly, the study was a clinic based casecontrol study at the Johns Hopkin's Hospital, which serves multiple participating dermatologists in Maryland. Cases were histopathologically confirmed primary BCCs and were diagnosed between 19871990. The controls were patients from the same physician practices and had a diagnosis of mild skin disorders. All participants were Caucasians living near Baltimore and were between 20 and 60 years of age. The controls were frequency matched to the cases by age and sex. Cases and controls with any other form of cancer were excluded. In the questionnaire, the study subjects were asked if they had any blood relatives with skin cancer, and were asked to specify the type of cancer. Study subjects with relatives with basal cell carcinoma and squamous cell carcinoma and `skin cancer' were included in the group of subjects with a family of skin cancer. Subjects with relatives with melanoma were not included. At the clinic visit the subjects gave informed consent, were examined by dermatologists, completed a structured questionnaire and provided blood. Available frozen lymphocytes were genotyped. This resulted in 70 cases and 117 controls for exon 23, 68 cases and 105 controls for exon 10 and 66 cases and 111 controls for exon 6. Since previous studies (11) had indicated that the exon 23 polymorphism had the strongest association to cancer we determined this genotype first.
DNA extraction, PCR and restriction enzyme analysis
DNA was extracted from ~106 lymphocytes using Puregene DNA Isolation kit (Gentra Systems, Minneapolis, MN). PCR for genotyping exons 6 and 23 reactions were performed as described previously (11). In approximately one third of the samples, only a few lymphocytes were available for DNA purification, resulting in very little DNA. Quality control of the DNA and quantification of the amount of DNA was done by electrophoresis. When only a small amount of DNA was available, nested PCR was used.
Exon 23
In the first PCR reaction, primers 5'-GTC CTG CCC TCA GCA AAG AGA A-3' and 5'-TTC TCC TGC GAT TAA AGG CTG T-3' were used. PCR reactions were performed in a 25 µl volume containing 10 mM TrisHCl pH 9.2, 50 mM KCl, 2 mM MgCl2, 0.2 mM each deoxynucleoside triphosphate, 0.1 µM each primer, 0.5 U Taq polymerase (Life Technologies, Denmark) and 5 µl genomic DNA. The cycling conditions were initial denaturation at 96°C for 60 s, followed by 30 cycles of 30 s at 94°C, 30 s at 60°C and 72°C for 60 s. The second PCR reaction was performed as described previously using 0.251 µl PCR reaction 1.
Exon 6
In the first PCR reaction, the primers 5'-CAC ACC TGG CTC ATT TTT GTA T-3' and 5'-TCA TCC AGG TTG TAG ATG CCA-3' were used. PCR reactions were performed in a 25 µl volume containing 10 mM TrisHCl pH 8.9, 50 mM KCl, 1 mM MgCl2 and 5 µl genomic DNA. The cycling conditions were initial denaturation at 96°C for 60 s, followed by 30 cycles of 30 s at 94°C, 30 s at 58°C and 72°C for 60 s. The second PCR reaction was performed as described previously using 0.251 µl PCR reaction 1.
Restriction enzyme analysis
Restriction enzyme analysis was performed as described previously (11). In exon 6, the A- but not the C-allele has a TfiI restriction site within the 652 bp amplified PCR product. In addition, there is a second TfiI restriction site within the amplified fragment that serves as an internal control for digestion. The three possible genotypes are defined by three distinct banding patterns: CC (56 and 596 bp fragments), CA (56, 114, 482 and 596 bp fragments) and AA (56, 114 and 482 bp fragments).
In exon 23, the C- but not the A-allele has a PstI restriction site within the 324 bp PCR product. In addition, there is a second PstI restriction site within the amplified fragment that serves as an internal control for digestion. The three possible genotypes are defined by three distinct banding patterns: CC (66, 100 and 158 bp fragments), CA (66, 100, 158 and 224 bp fragments) and AA (100 and 224 bp fragments).
Exon 10
The exon 10 polymorphism (A23591G) was typed using a LightcyclerTM (Roche, Switzerland) to amplify the genomic DNA in a real-time PCR reaction and then determine the temperature profile of the fluorescent energy-transfer between an anchor and a sensor probe. The primers and probes were as follows: forward primer, 5'-GATCAAAGAGACAGACGAGC-3'; reverse primer, 5'-GAAGCCCAGGAAATGC-3'; anchor probe, 5'-CGCAGTACCAGCATGACACCAGCCT-Fluorescein-3'; sensor probe, 5'-LCRed640-CC- CCACTGCC- GATTCTATGAGG-p-3'. The reaction mix contained 500 nM each primer, 200 nM anchor and sensor probes, 10% DNA Master Hybridization Probes mix (Roche) which contains DNA polymerase, dNTP and buffer (3.5 mM MgCl2 and 5% DMSO). After an initial denaturation of 2 min at 95°C, the regimen consisted of 45 cycles of 10 s at 95°C, 15 s at 53°C and 30 s at 72°C. The last cycle was extended by 2 min at 72°C for polishing. To read the temperature profile of the energy transfer the PCR products were denatured for 10 s at 95°C, cooled to 50°C for 30 s and subjected to a temperature ramp from 50 to 95°C with a slope of 0.1°C/s. The difference in melting temperature between the allele having a perfect match with the sensor (the A-allele) and the allele with 1 base mismatch (the G-allele) was 8°C.
Statistical methods
Data analysis and
2 tests were performed using Excel (Microsoft). Odds ratios and confidence intervals were calculated manually.
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Results
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We have performed a study of BCC in relation to XPD gene polymorphisms in patients with BCC and in controls without BCC recruited in America. The study group is described in detail elsewhere (4). Table I
summarizes the distribution of age and gender for this population. We have analyzed three previously described polymorphisms (68,14), two giving rise to non-conservative amino acid substitutions in codons 312 and 751, and one silent nucleotide substitution in codon 156. The distribution of the genotypes in the study groups is shown in Table II
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Exon 6, codon 156
The C
A polymorphism in exon 6 at nucleotide 22541 does not result in an amino acid change at codon 156. The average frequency of the A-allele in the whole cohort was 0.44, which is in agreement with previous reports (11,1416). The distribution of the genotypes in the control group was in HardyWeinberg equilibrium. When the whole study group was considered, the CC genotype was mildly protective against basal cell carcinoma. Subjects carrying an AC genotype were at an ~2-fold increased risk of BCC (P = 0.056, CI 0.984.13) (Table II
). However, when only subjects with no family history of basal cell carcinoma were included (Table III
), the C-allele was protective against BCC. Thus, subjects carrying AC or AA genotypes were at 3.3-fold increased risk of BCC (P = 0.007, CI = 1.358.18).
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Table III. XPD polymorphisms in codon 156 (exon 6) and risk of basal cell carcinoma in relation to family history of non-melanoma skin cancer
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The DNA repair capacity has previously been measured in this study group (4). In the whole study group, subjects with a CC genotype had on average a slightly higher DNA repair capacity (7.83% cat activity at 700 J/m2) than subjects carrying an AC (7.76%) or AA (7.46%) genotype, although the difference was not statistically significant. This tendency was enhanced among subjects without a family history of skin cancer, as subjects with a CC genotype had an average DNA repair capacity of 7.99% cat activity at 700 J/m2, whereas subjects carrying an AC had on the average 7.65% and subjects carrying AA genotype had an average activity of 7.39% at 700 J/m2. None of the differences were statistically significant. Among subjects with a family history of BCC, there was no association between exon 6 polymorphism and cancer status (Table III
). In a previous Scandinavian study (11), it was found that the C-allele was protective against BCC among psoriasis patients (P = 0.078), but not among non-psoriatic patients. Exclusion of subjects with a family history of non-melanoma skin cancer from the Scandinavian study group (including both psoriatics and non-psoriatics) increased the protective effect of the C-allele in the Scandinavian study group, although the protection was not statistically significant (results not shown).
Exon 10, codon 312
The G
A polymorphism in codon 312 of exon 10 results in an Asp
Asn substitution in an evolutionally conserved region (12,13). The average frequency of the C-allele was 0.38, which is in agreement with previous findings (13,14). The distribution of the genotypes in the control group was in HardyWeinberg equilibrium. There was no difference in DNA repair capacity between the different genotypes (data not shown). When the whole study group was considered, there was no difference in allele frequencies among BCC cases and controls (Table II
). However, among subjects with a family history of BCC, subjects carrying AG or AA genotypes were at 5.25-fold increased risk of BCC (P = 0.027, CI = 1.1523.93) (Table IV
). Among subjects without family history of non-melanoma skin cancer, there was no difference in genotype distribution between BCC cases and controls.
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Table IV. XPD polymorphism in codon 312 (exon 10) and risk of basal cell carcinoma in relation to family history of non-melanoma skin cancer
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Exon 23, codon 751
The A
C polymorphism in exon 23 at nucleotide position 35931 gives rise to the amino acid substitution Lys
Gln in codon 751. The average frequency of the C-allele was 0.39, which is at the high end of the previously reported allele frequencies (0.290.40) (11,1315). The distribution of the genotypes in the control group was in HardyWeinberg equilibrium. When the whole group was considered, there was no correlation between genotype and risk of basal cell cancer, as the allele frequency was almost identical in the two groups (Table II
). However, when only subjects with a family history of BCC were considered, the A-allele was mildly protective (Table V
). Thus, subjects carrying an AC or CC genotype had a 3.6-fold increased risk of BCC (P = 0.092, CI 0.7817.00). This is opposite of the tendency in the Scandinavian study group of psoriasis patients, where it was found that the C-allele was mildly protective (11). There was no statistically significant difference in DNA repair capacity between the different genotypes.
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Table V. XPD polymorphism in codon 751 (exon 23) and risk of basal cell carcinoma in relation to family history of non-melanoma skin cancer
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The association of a family history of non-melanoma skin cancer with the allele frequency is listed in Table VI
. The A-allele frequency in exon 6 among BCC cases with a family history of non-melanoma skin cancer is lower than the A-allele frequency among BCC cases without a family history of non-melanoma skin cancer (P = 0.013). Likewise, the allele frequency of the A-allele in exon 10 is higher among BCC cases with a family history of non-melanoma skin cancer than among subjects without family history of non-melanoma skin cancer (P = 0.042).
We have previously found that the polymorphisms in exons 6 and 23 of the XPD gene are closely linked in a Scandinavian study group (11). The two polymorphisms are also closely linked in this study group with a P value of 2x1012. The present study group is more heterogeneous than the Scandinavian study group as the haplotype exon 6A exon 23C was not found in the Scandinavian study group, but was frequently found in this study group, almost exclusively in persons of central and eastern European and Russian descent. Likewise, the polymorphism in exon 10 is in linkage disequilibria with both the polymorphism in exon 6 and the polymorphism in exon 23.
The risk factors for BCC have previously been described in detail for this study group (5). A number of risk factors were identified for the development of BCC including age, previous family history of skin cancer, skin type and the lifetime number of sunburns. Other studies have also identified the number of lifetime sunburns as a risk factor for BCC (17). In order to determine a possible geneenvironment interaction, the lifetime number of sunburns was correlated to the genotype and BCC. No interaction between exposure and genotypes was found for any of the three polymorphisms.
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Discussion
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This investigation reiterates that the silent polymorphism in codon 156 of exon 6 influences the risk of developing BCC. The C-allele seems to be protective. We also found suggestions of an association between the codon 312 polymorphism in exon 10 and risk of BCC among subjects with a family history of non-melanoma skin cancer. No statistically significant effect of the exon 23 polymorphism was found, indicating that the polymorphism may not be associated with risk of BCC. Recently, diverging results have been published as to which allele is protective in the exon 23 polymorphism. The exon 23C allele was found to be marginally associated with increased risk of cancer of the neck and head (15). On the other hand, the exon 23A allele has been shown to be associated with suboptimal DNA repair activity (X-ray induced chromatid aberrations) (12), and to risk of BCC and the age of onset of BCC (11).
The exon 6 polymorphism does not result in an amino acid change. It is therefore unlikely that the enzymatic function of XPD is affected by the mutation. The mutation may influence the rate of translation by altering codon usage or reduce XPD protein levels through an effect on mRNA stability (11). Alternatively, the exon 6 polymorphism may be linked to another mutation. If the biologically active mutation is outside of the XPD gene, it should be located upstream of XPD, since the association of BCC occurrence to exon 6 is stronger than the association of BCC occurrence to exon 23. The 13.3 region of chromosome 19 encompassing the XPD gene encodes several other genes involved in DNA repair, for example, ERCC1, XRCC1 and LIG1.
The polymorphism in exon 10 gives rise to an amino acid substitution and may therefore affect the enzymatic activity of XPD. However, in a study comparing genotypes and DNA repair capacity, DNA repair capacity was found to correlate with the polymorphism in codon 751, but not with the polymorphism in codon 312 (12). In the present work the polymorphism is only associated with BCC among subjects with a family history of non-melanoma skin cancer, and not among those without a family history, and it is therefore unlikely that the effective mutation is the polymorphism itself. Rather, the polymorphism is probably linked to the biologically important mutation. A putative tumor suppressor gene has recently been mapped to this area (18).
UV light exposure is undoubtedly the origin of most BCCs in this study group (4,5). We found no interaction between genotypes and the number of lifetime sunburns in this study group, which might reflect that sun-exposure is difficult to assert by questionnaires.
Interestingly, it appeared that a family history of non-melanoma skin cancer influenced the genotype distributions. The allele frequencies in both the exon 6 and 10 polymorphisms seemed to be different in BCC cases with and without family history of non-melanoma skin cancer (Table VI
). The protective influence of exon 6C was exclusively present in the persons without a family history of skin cancer. Conversely, the protective influence of exon 10G was exclusively present among the subjects with a family history of skin cancer. The fact that the two factors are not additive could suggest that family history overrides the importance of the exon 6 polymorphism. However, the two polymorphisms are genetically tightly linked, and the protective exon 6C allele co-segregates with the risk allele exon 10A (Table VII
). Thus, the two linked alleles have opposite effects, so we would only see the effect of one of the protective alleles at a time. This may explain why the allele frequency of the protective exon 6C allele is higher among subjects with a family history of non-melanoma skin cancer than among subjects without a family history of non-melanoma skin cancer, even though family history of skin cancer is a risk factor for BCC (Table VI
).
In summary, our results indicate that exon 6A is a risk factor for BCC.
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Notes
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3 To whom correspondence should be addressed Email: ubv{at}ami.dk 
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Acknowledgments
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The authors wish to thank Anne-Karin Jensen, Birgitte Korsholm and Thrine Schneidermann for excellent technical assistance. The work is supported by the Danish Medical Research Council Grant 9600259 and the Danish SUE program (J. no. 9800647-67).
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Received August 8, 2000;
revised February 15, 2001;
accepted February 22, 2001.