Department of Dermatology and 1 Department of Occupational and Social Medicine, Georg-August-University, Goettingen, Germany, 2 Institute of Medical Biometry and Statistics, University Hospital Schleswig-HolsteinCampus Lübeck, Germany, 3 Basic Research Laboratory, Center for Cancer Research, National Cancer Institute/NIH, Bethesda, MD, USA and 4 Department of Dermatology, Ludwig-Maximilians-University, Munich, Germany
* To whom correspondence should be addressed at: Department of Dermatology, Georg-August-University Goettingen, Von-Siebold-Strasse 3, 37075 Goettingen, Germany. Tel: +49 551 396410; Fax: +49 551 392414; E-mail: semmert{at}gwdg.de
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Abstract |
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Abbreviations: CDK, cyclin D kinase; NER, nucleotide excision repair; SNP, single nucleotide polymorphism; XP, xeroderma pigmentosum; XPV, XP variant
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Introduction |
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Xeroderma pigmentosum (XP) is an autosomal recessive disease with defective NER. XP patients exhibit an extreme sensitivity to UV light, resulting in a high incidence of skin cancers (1000 times that of the general population), including basal cell carcinomas, squamous cell carcinomas and melanomas (2,3). The clinical findings are associated with cellular defects, including sensitivity to killing and mutagenic effects of UV and the inability of XP cells to repair UV-induced DNA damage (4). Seven different DNA NER genes that correct seven distinct genetic XP complementation groups (XPAG) have been identified (2). In addition, XP variant (XPV) patients have a defect in DNA polymerase eta, which is responsible for the error-free bypass of UV-induced DNA damage (5).
A normal DNA repair capacity is necessary to maintain normal cellular functions. Polymorphisms in the DNA repair genes may contribute to variations in the DNA repair capacity in the general population and may affect genetic susceptibility to cancer. Even a slight reduction in DNA repair capacity could result in a significantly increased risk of cancer. For example, a reduced DNA repair capacity has been found in patients with lung cancer, head and neck squamous cell cancer and patients with basal cell carcinomas (6) using host cell reactivation and the lymphocytes of the patients (7,8). An increase in the incidence of skin cancer has also been reported in some families of patients with XP whose members would have been expected to be XP heterozygotes (carriers) and thus, might have a slightly reduced NER capacity. The blood relatives of patients with XP in four families compared with spouse controls were reported to have a 16-fold increase in skin cancer (9). In addition, Wei et al. (10) recently demonstrated that reduced DNA repair capacity is an independent risk factor for the development of cutaneous melanoma in the general population.
Cutaneous melanoma is the most serious form of skin cancer. There was a progressive worldwide rise in the incidence of cutaneous melanoma between the years 1940 and 2000 (11). The mortality rate of melanoma has been climbing steadily from 2/100 000 in 1969 to 3/100 000 in 1999, and most of this increase was owing to an increased mortality among men aged 65 years and older (12). In the USA, 59 100 new melanoma cases and 7900 related deaths are predicted for the year 2004 (13). The risk of melanoma increases with age (14) and other risk factors such as sunlight exposure (15), particularly an intermittent exposure (14), family history of melanoma, dysplastic nevi or atypical nevi, number of nevi, skin sensitivity to sun, freckling, fair hair, eye and skin color (11).
The XP complementation group C is the most prevalent form among North Americans and Europeans (16). The XPC gene is located on chromosome 3p25 and encodes a 940 amino acid protein involved in DNA damage recognition during the early steps of the NER process (2). Shen et al. (17) showed that an intronic Poly-AT polymorphism (PAT) within intron 9 of the XPC gene was associated with the risk of squamous cell carcinoma of the head and neck. In addition, a single nucleotide polymorphism (SNP) in exon 15 of the XPC gene (A2920C) that leads to an amino acid change (Lys939Gln) was shown to be associated with bladder cancer in a Swedish population (18). We hypothesize that variations in the DNA repair owing to inherited polymorphisms in the XPC gene might be associated with a susceptibility to melanoma in the normal population.
In the present hospital-based casecontrol study, we tested the hypothesis that three XPC polymorphisms, a Poly-AT insertiondeletion polymorphism in intron 9 (PAT), an SNP in exon 15 (A2920C) and a newly identified splice acceptor site SNP in intron 11 (C6A) are associated with the risk of developing cutaneous melanoma. In a previous study, it was demonstrated that these three polymorphisms are in a linkage disequilibrium and that the splice acceptor polymorphism in intron 11 leads to an increased skipping of exon 12 and a reduced DNA repair function of XPC (19).
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Materials and methods |
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XPC intron 9 PAT genotyping
The insertion of 83 bp consisting of As and Ts [poly(AT); PAT] and a 5 bp deletion GTAAC at position 14571461 in the intron 9 of XPC was analyzed as described (21). Briefly, a 266 bp fragment indicated the absence of the insertiondeletion polymorphism (PAT) and a 344 bp fragment indicated the presence of the insertiondeletion polymorphism (PAT+). A 621 bp long ß-actin fragment was amplified as an external standard. PCR was performed with the Clontech Advantage 2 Kit (Clontech Laboratories, Heidelberg, Germany). The total reaction volume of 20 µl contained 1x Clontech Advantage 2 PCR buffer, 56 pmol of each Primer N1m and N2n, 6 pmol of each ß-actin sense and antisense primer, 0.2 mM dNTP Mix and 1x Clontech Advantage 2 Polymerase Mix. Finally, 1 µl (100 ng) of DNA was added. The PCR steps were as follows: 95°C for 15 min, 35 cycles of amplification (95°C for 30 s, 66°C for 3 min) and finally 66°C for 3 min.
XPC intron 11 C-6A genotyping
The XPC intron 11 splice acceptor site region contains an SNP at position 6 (C6A). PCR was conducted as described (19) in a 20 µl reaction volume including 1x PCR Rxn buffer (Invitrogen, Karlsruhe, Germany), 1.5 mM MgCl2, 8 pmol of each primer E1 and H2, 0.2 mM dNTP-Mix, and 2.5 U of Taq polymerase (Invitrogen, Karlsruhe, Germany). Finally, 1 µl (100 ng) of DNA was added. PCR steps were as follows: 3 min at 94°C, 35 cycles (94°C for 15 s; 58°C for 30 s; 72°C for 30 s), ending with 5 min at 72°C. Five microliteres of the PCR reaction were digested with 0.2 U FauI (MBI Fermentas, St Leon-Rot, Germany) at 37°C for 1 h. The C at position 6 creates a FauI restriction site. FauI digestion converts the 203 bp PCR product into two fragments of 43 and 160 bp.
XPC exon 15 A2920C genotyping
The A2920C polymorphism region in exon 15 of the XPC gene was amplified as described (21). PCR was performed in a reaction volume of 20 µl, containing 1x MBI PCR buffer with (NH4)2SO4 (MBI Fermentas), 2.5 mM MgCl2, 0.2 mM dNTP mix, 2 U of MBI recombinant Taq polymerase, 5% dimethyl sulfoxide (DMSO), 6 pmol of primer Exon15F and 7 pmol of primer 3'ntcDNAR, and 1.5 µl of DNA (150 ng). The PCR steps were conducted as follows: 95°C for 3 min, 35 cycles (45 s at 95°C, 45 s at 56°C, 1 min at 72°C), concluding with 72°C for 5 min. The SNP A2920C within XPC exon 15 creates a new restriction site for PvuII (MBI Fermentas) in the presence of C. PvuII digestion converts the 765 bp PCR product into two fragments of 585 and 180 bp.
All gels were evaluated independently by two observers (S.B. and P.L.). PCR products were initially sequenced. We ensured that in every RFLP (restriction fragment length polymorphism) experiment at least one sample showed a restriction enzyme digestion, serving as a positive control.
Statistics
To evaluate deviation from the HardyWeinberg equilibrium, observed and expected genotype frequencies were compared using a Monte Carlo goodness-of-fit test in patients and controls. ORs and exact 95% CIs were calculated to compare genotype frequencies. For a univariate description, genotype frequencies (two variant alleles versus no or one variant allele) were compared using the Fisher's exact test rendering descriptive P-values.
To evaluate the association of the assessed risk factors with melanoma, logistic regression analyses were performed predicting melanoma from age, number of nevi and skin type as continuous covariates, as well as gender, hair color and eye color as categorical covariates. All possible two-way interactions were additionally analyzed. A manual backward selection of covariates with eliminating covariates with P > 0.05 for Wald's 2 was conducted. To determine whether an association with XPC polymorphisms might depend on this background of risk factors, we investigated the association of XPC polymorphisms with melanoma while controlling for these risk factors. In logistic regression analyses, the XPC polymorphisms were added singly to the models derived in the previous step. Although we tested three polymorphisms for statistical significance, they are highly interrelated owing to their high linkage disequilibrium. As a consequence, adjustments for multiple testing would be overly conservative, and we refrained from adjusting the P-values accordingly. However, any results have to be validated in independent samples in order to guard against false positive findings.
For further exploratory analyses, we divided the cases into subgroups according to the presence of multiple primary melanomas and Breslow thickness of melanomas at primary diagnosis. The association of risk factors and XPC polymorphisms with the presence of multiple primary melanomas (0 = control group, 1 = no multiple melanoma, 2 = multiple melanoma) as well as with Breslow tumor thickness (0 = control group, 1 = thickness 1 mm, 2 = thickness >1 mm) was investigated in exploratory ordinal regressions analogously to the above mentioned procedure.
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Results |
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On applying the logistic regression analysis, there were no independent associations of gender, hair color and eye color with the risk for melanoma development in our study population. A positive family history of melanoma was too rare to analyze. However, the frequencies of the phenotypic melanoma risk factors, number of nevi and skin type as well as age were different in the melanoma patient group compared with the control group (Table I). For example, control probands had a lower number of nevi than patients with melanoma [mean (SD): 11.0 (17.3) versus 19.6 (20.9)] and were younger [mean age (SD): 37.1 (13.7) years versus 53.1 (15.4) years]. In addition, only 6.5% of the control subjects were of skin type 1, whereas 20.1% of the patients with melanoma had this skin type.
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XPC polymorphisms and melanoma risk
The three XPC polymorphisms were not found to be associated with the risk of melanoma using Fisher's exact test (Table III). However, using multivariate logistic regression analyses while controlling for a linear increase in age, skin type and number of nevi, the XPC intron 9 PAT+, intron 11 6A and exon 15 2920C polymorphisms were associated with increased risks of melanoma: OR 1.87 (95% CI: 1.103.19; descriptive P = 0.022), OR 1.83 (95% CI: 1.073.11; descriptive P = 0.026) and OR 1.82 (95% CI: 1.073.08; descriptive P = 0.026), respectively (Table III).
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One of the most important prognostic factors in primary melanoma is tumor thickness. A linear increase of the risk of death is observed with increasing tumor thickness (23). We utilized the commonly used cutoff of 1 mm Breslow tumor thickness. Exploratory multivariate ordinal regression analyses assessing the impact of the XPC polymorphisms while controlling for age, skin type and number of nevi revealed no independent association of the XPC polymorphisms with tumor thickness (data not shown).
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Discussion |
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DNA repair polymorphisms and cancer risk
NER proteins not only repair UV light induced DNA photoproducts but also remove DNA adducts induced by other carcinogens such as benzo[a]pyrene diol epoxide found in tobacco smoke (29). Therefore, polymorphisms in NER genes may affect the DNA repair phenotype, which contributes to the risk of developing cancers of the skin (30), the lung (6,31) and the head and neck (7). For example, Wu et al. (32) showed that an XPA 5' non-coding region polymorphism modulates NER capacity and is associated with a decreased lung cancer risk, particularly in the presence of exposure to tobacco carcinogens. Among 40 basal cell carcinoma patients and 40 controls, donors carrying two A alleles in XPD exon 23 (Lys751) (33,34) had a 4.3-fold higher (but not statistically significant) risk of basal cell carcinoma of the skin than donors with two C alleles (Gln751) (P = 0.075; 95% CI 0.7923.57) (35). In agreement with this finding, Tomescu et al. (36) found a significant association of three polymorphisms in the XPD gene in exons 6 (A22541C), 22 (C353326T) and 23 (A35931C) with melanoma.
In 2000, a common biallelic polymorphism (PAT) in the XPC DNA repair gene consisting of an insertion of 83 bases of A and T [poly(AT)] and a five base deletion within intron 9 was discovered at a frequency of 40% for the presence of the A/T insertions (PAT+) (21). Additionally, it was found that an SNP near the end of the XPC cDNA existed (A2920C) at a comparable frequency for 2920C resulting in an amino acid change from Lys to Gln (37). The function of this XPC exon 15 SNP was assessed in an allele-specific post-UV host cell reactivation assay. The XPC 2920C allele was equally efficient as the 2920A allele when the ability of these XPC cDNAs to complement the DNA repair defect in XPC cells was assessed (21). Interestingly, the intronic PAT polymorphism was found to be associated with the risk of developing squamous cell carcinoma of the head and neck in a casecontrol study comprising 287 cases and 311 controls (17). In addition, the XPC exon 15 2920C allele was reported to be associated with the development of bladder cancer in 305 Caucasian patients and 246 healthy controls (18). Vodicka et al. (38) found that the XPC 2920C/C genotype was associated with a reduced repair rate of single strand breaks.
In 2002, a new common C-6A SNP in the XPC intron 11 splice acceptor site was discovered (19). The intron 11 6A polymorphism decreased the information content of the splice acceptor site from 7.5 to 5.1 bits (39). Fibroblasts homozygous for A/A had significantly higher levels (2.6-fold) of an XPC mRNA isoform that skips exon 12 than those homozygous for C/C. This abnormally spliced XPC mRNA isoform has a diminished DNA repair function. Normal fibroblasts harboring the homozygous A/A genotype roughly showed a 50% reduction in their nucleotide excision DNA repair capability (19). This reduction in DNA repair function may explain the association of the XPC PAT+/intron 11 6A/exon 15 2920C haplotype with the development of different cancers (19). In another study, Qiao et al. (40) reported that in 102 healthy individuals all subjects with the homozygous PAT+/+ polymorphism had a relative reduction in NER capacity of 23.4% compared with the PAT/ individuals (P = 0.02). Other studies identified a 10% reduction of DNA repair capacities in patients with basal cell carcinoma (30), a 25% reduction in patients with lung cancer (32) and a 30% reduction in patients with head and neck squamous cell cancer (7) compared with healthy controls.
The distribution of the XPC intron 11 splice acceptor polymorphism was compared with that of the other polymorphisms in the XPC gene: PAT and the SNP in exon 15 A2920C. These markers are 9 kb apart in the XPC gene. Among 97 normal donors, these three markers were in a linkage disequilibrium and are consistent with a haplotype of intron 9 PAT+/intron 11 6A/exon 15 2920C in 40% of the donors. The present study confirms and extends these findings. We confirmed in a European population of 669 subjects that the three polymorphisms in the XPC gene indeed form a haplotype at a frequency of
40% for XPC intron 9 PAT+/intron 11 6A/exon 15 2920C. These genotype frequencies are consistent with those reported by Khan et al. (19,21) and others (17,18). In our population of 669 study subjects, only 11 genotypes (1.6%) did not fit the prediction of the polymorphisms XPC intron 9 PAT+/intron 11 6A/exon 15 2920C forming a haplotype (Table II). In the future, genetic profiles of cancer risks will have to be constructed to develop risk models incorporating the combinations of many polymorphisms in many genes at once. Our data indicate that only one of the three XPC polymorphisms would be sufficient to be included into risk assessment studies and statistical modeling. The XPC PAT polymorphism presents itself for this purpose, because the additional step of restriction enzyme digestion is not needed for its detection.
XPC polymorphisms and melanoma risk
We found an association of XPC intron 9 PAT+, intron 11 6A and exon 15 2920C polymorphisms with the risk of melanoma, using multivariate logistic regression analysis while controlling for age, skin type and number of nevi (Table III). This finding is similar to that reported by Shen et al. (12), who did find an overall association between p53 gene codon 72 Arg homozygotes and increased risk of cutaneous melanoma.
In exploratory multivariate analyses, we found an association of the intron 9 PAT+, intron 11 6A and exon 15 2920C polymorphisms with the risk of melanoma in patients with sporadic multiple primary melanomas and absence of other risk factors. It is a well accepted fact that patients with melanoma are prone to develop further melanomas (22). In a cohort of 535 patients with melanoma from Germany, Nashan et al. (41) found that melanoma patients have at least a 30-fold increased risk for the development of a second melanoma compared with individuals with no melanoma in their personal medical history. Seventy percent of patients with multiple primary melanomas revealed none of the known predispositions for melanoma such as multiple or atypical moles or a history of familial melanomas. Approximately 812% of all melanoma cases occur in individuals with a history of familial cutaneous melanoma (42,43). In only about half of those familial melanoma cases, there is a genetic defect in the CDKN2 gene encoding a protein (p16) which is a cell cycle control protein and as an inhibitor of cyclin D kinase (CDK) acts as a brake on the first part of the cell cycle, phase G1 (44). In our study, most of the patients with multiple primary melanoma developed sporadic multiple primary melanomas without a family history of melanomas, multiple nevi or dysplastic nevi. Clearly, the group of patients with multiple primary melanoma is a heterogeneous group in terms of pathogenesis of melanomas. A revision of pathogenesis is needed as the clustering of melanomas in these patients suggests unrecognized inherited or acquired risk factors, which are conducive to the development of several melanomas (41). Reduced DNA repair may constitute such a risk factor. Further research is needed to assess how variations in DNA repair genes affect molecular events involved in the development of melanoma.
Breslow's vertical tumor thickness is one of the strongest prognostic factors in melanoma and a marker for tumor progression (23,45). A tumor thickness of 1 mm indicates that the primary melanoma tumor has invaded the dermis. The multistage theory of tumor development and progression has been proposed as a general model for environmental oncogenesis (46). Tumor initiation is followed by tumor promotion and then progression leading to a gradual increase in cellular genetic instability over time. Numerous links have been identified between oncogenesis and acquired or inherited faulty genome guardians that cause a mutator phenotype, highlighting the key role of DNA protection systems, DNA repair being one of the most relevant systems, in tumor prevention (47). Using exploratory multivariate ordinal regression analyses, we found no independent association of the XPC polymorphisms with melanoma tumor thickness and thus, for melanoma progression.
A linear increase in age was an independent risk factor for the development of melanoma in our study population. Two investigations on basal cell carcinoma (35) and head and neck cancer (48) suggested that carriers of an XPD polymorphism that is associated with reduced DNA repair (49) may be at risk for cancer at an older age. Shen et al. (12) found an association of a polymorphism in the p53 gene with an increased risk of cutaneous melanoma in patients >50 years of age. However, we found no interactions between age and the presence of the XPC polymorphisms with respect to the development of melanoma in our study (data not shown). We cannot exclude the possibility that the older cohort of patients with melanoma is enriched for the XPC haplotype relative to controls, because control individuals with this XPC haplotype may already have died from other types of cancers.
Age plays a major role in the vulnerability to photocarcinogens (14). The risk of melanoma increases steeply with age (50). During aging, DNA accumulates changes that activate proto-oncogenes and inactivate tumor suppressor genes (47). The genetic instability driving oncogenesis is fuelled by DNA damage and errors made by the DNA machinery (47,51). Other critical steps in the development of cancer may be age-related, pro-oncogenic changes in the epithelial stroma milieu (52). As age increases, defence mechanisms against sunlight exposure, such as skin pigmentation (51), epidermal thickness (51) and importantly, DNA repair efficiency (53,54) may decrease. Moriwaki et al. (53) found an age-related decline in post-UV DNA repair capacity [measured by the ability to repair a UV-treated plasmid (pCMVcat)] of 0.6% per year (P = 0.0001) in cultured primary skin fibroblasts from normal donors from the first to the tenth decade of life. There was a corresponding age-related increase in post-UV mutability [measured as mutations introduced into a transfected, UV-treated plasmid (pSP189)] of +0.6% per year (P = 0.001) in lymphoblastoid cell lines from normal donors of the same age range. Goukassian et al. (55) found a similar significant decrease with aging in the repair rates of DNA photoproducts, thymine dimers as well as 64 photoproducts, in normal human dermal fibroblasts. The risk of virtually all cancers increases with the age of the patient. In older subjects, the equilibrium between DNA damage and repair may be altered and further destabilized in the presence of variant alleles in DNA repair genes, which may be otherwise negligible.
In conclusion, our casecontrol study supports the hypothesis that the XPC intron 9 PAT+, intron 11 6A and exon 15 2920C haplotype may contribute to the risk of developing cutaneous melanoma by increasing the rate of an alternatively spliced XPC mRNA isoform that skipped exon 12 and leads to a reduced DNA repair function. Further studies are necessary to elucidate the interactions between altered DNA repair and the risk of developing cutaneous melanoma at least in certain subgroups of patients with melanoma patients. Our results have do be validated in independent samples in order to guard against false positive findings.
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Acknowledgments |
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Conflict of Interest Statement: None declared.
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References |
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