XPD exon 10 and 23 polymorphisms and DNA repair in human skin in situ

Kari Hemminki3,, Guogang Xu, Sabrina Angelini, Erna Snellman1,, Christer T. Jansen2,, Bo Lambert and Sai-Mei Hou

Department of Biosciences at Novum, Karolinska Institute, 14157 Huddinge, Sweden,
1 Department of Dermatology, Päijät-Häme Central Hospital, 15850 Lahti, Finland and
2 Department of Dermatology, University of Turku, 20520 Turku, Finland


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Forty-four Finnish volunteers who were previously studied with regard to the repair rate of UV-specific cyclobutane pyrimidine dimers in the skin were genotyped for XPD polymorphisms at codons 312 (exon 10 G->A, Asp->Asn) and 751 (exon 23 A->C, Lys->Gln). The repair rate was measured at 24 h for two different cyclobutane dimers. The data did not show consistent XPD genotype-specific differences in DNA repair rates among all subjects. The combined exon 10 AA and exon 23 CC genotype was associated with an ~50% depression of repair rate but this was of borderline statistical significance. However, the exon 23 C allele was associated with depressed repair among subjects aged 50 years or older and the result was consistent with both dimers.

Abbreviations: CPDs, cyclobutane pyrimidine dimers; NER, nucleotide excision repair; SNPs, single nucleotide polymorphisms; SSR, solar simulating radiation; UVR, UV radiation; XPD, xeroderma pigmentosum complementation group D.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One of the dilemmas of the present molecular medicine field is that it is often easier to use genotype rather than phenotype data in order to analyze associations with disease frequency. Such association studies are technically reliable if banked human specimens are available. The drawback is that only limited information may be available on the functional effects, if any, of the polymorphisms tested. Indeed, while the data on single nucleotide polymorphisms (SNPs) in various genes, including DNA repair genes, is increasing rapidly, data on their possible functional effects is lagging behind. The ultimate functional test is to show an effect in humans in vivo.

Data on DNA repair rates in humans are derived from cultured cells, because methods have not been available for quantitative assay of DNA repair rates in humans in situ(14). Semi-quantitative immunofluorescence techniques have been used and have the advantage of being able to localize the damage (5,6). A DNA repair test was recently introduced based on quantification of specific UV radiation (UVR)-induced photoproducts in human skin biopsies by the 32P post-labeling technique (7). For the determination of repair rate a single dose of UVR was administered to the buttocks of volunteers and the removal of damage was followed in biopsies taken at various times after UV exposure. UV damage was quantified as specific radioactive fractions by HPLC. Synthetic standards were available for identification of two cyclobutane pyrimidine dimers (CPDs) and two 6–4 photoproducts. The results obtained showed that repair kinetics of CPDs and 6–4 photoproducts encompassed fast and slow components, probably relating to the genomic heterogeneity of nucleotide excision repair (NER) (1,8). CPDs were removed considerably slower than 6–4 photoproducts, with 50% removal times of ~15 and 5 h, respectively. Dimers at TT sites were repaired slower than those at TC sites (8). A 20-fold inter-individual variation in repair rates was observed, but age, basal cell carcinoma and melanoma did not appear to affect repair rates when measured up to 48 h (911). Variation due to the post-labeling method is only 30% in repeated samples; even in samples taken from the same individuals 3 weeks apart the correlation coefficients were from 0.7 to 0.85 (12).

Polymorphisms in DNA repair genes are currently being actively investigated because of possible effects on susceptibility to cancer (13). NER is a complex process in which many proteins take part (1,3). The xeroderma pigmentosum complementation group D (XPD) protein is involved in the unwinding of DNA and forms a complex with transcription factor IIH during DNA repair. Mutations in XPD cause a severe but variable depression of NER (3). Several non-synonymous SNPs have been described in the XPD gene, including those at codons 312 (exon 10 G->A, Asp->Asn) and 751 (exon 23 A->C, Lys->Gln) (13). The frequencies of these variant alleles have been estimated to be ~30% in two US studies (13,14). It is not known whether these polymorphisms have functional effects. However, the more common AA genotype at exon 23 has been suggested to be a risk factor for basal cell carcinoma (15) and susceptibility to X-ray-induced chromatid aberrations (14), while no effect has been noted on lymphocyte sister chromatid exchanges (16). However, a recent study on head and neck cancers suggested that the exon 23 C allele was a risk factor and the effect was stronger among old subjects (17).

In the present study we have used the recently developed in situ DNA repair assay to functionally test the effects of the two XPD polymorphisms on the rate of repair of two specific CPDs. The human sampling was carried out in a dermatology clinic in Lahti, Finland and the 32P post-labeling analysis of UV damage in Sweden, in the context of three earlier studies (911). Genotyping analysis was carried out specifically for the present study.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study population
The subjects came from two previous studies on DNA repair rates among Finnish melanoma and basal cell carcinoma patients and healthy controls, frequency matched on age, skin type and gender (911). Many characteristics of the study populations have been reported (911).

Sampling of skin biopsies
A broadband Philips (HP 411/A) solar simulating lamp was used as the solar simulating radiation (SSR) source. The spectral distribution of the SSR resembled the spectrum of noon summer sunlight in Helsinki (911). All participants were irradiated with an erythemally weighted UV dose of 40 mJ/cm2 CIE (Commission Internationale de l'Éclairage) (18) of SSR on a buttock skin site. One skin biopsy was taken immediately after irradiation (within 5 min) from each subject and put on dry ice at once, then kept in a –20°C freezer until DNA extraction. At 24 and 48 h another two skin biopsies were taken at the exposure sites.

DNA extraction and CPD measurement
DNA extraction from epidermis was performed as described previously (7,8). The levels of SSR-induced CPDs were quantified using the 32P post-labeling method, which has been described elsewhere (10). CPDs (T=C and T=T) were assayed as trinucleotides with an unmodified nucleotide (thymine) on the 5'-side, i.e. TT=C and TT=T. For each 32P post-labeling assay 3 µg DNA was digested and labeled. Each sample was measured twice. After labeling, 10 µl of water was added to each sample and then injected into a HPLC system for analysis. The products were detected with an online Beckman radioisotope detector. For calculation of CPD levels the HPLC peaks of interest were integrated with Beckman System Gold software and the external synthetic standards of TT=C and TT=T were used for quantification.

The repair of UV photoproducts is complex in human skin, apparently composed of many components (8). In the present study we determined the rate of repair at 24 h because at this time somewhat >50% of TT=C and somewhat <50% of TT=T is repaired (811). The time required for 50% repair of these dimers is also independent of the initial level of damage (8). Thus the variation in individual rates is most optimally gauged at 24 h.

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

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

Ten microliters of the PCR product was then digested with 10 U TaqI (Fermentas, Vilnius, Lithuania) for 4 h at 65°C in a total volume of 20 µl. The digested products were separated by electrophoresis in a 6% polyacrylamide gel. The G allele was represented by a 166 bp fragment and the A allele by a 188 bp band.

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

Five microliters of the PCR product was then digested with 10 U PstI (Promega, Madison, WI) for 4 h at 37°C in a total volume of 15 µl, followed by electrophoresis in a 5% polyacrylamide gel. The A allele was cut into two fragments (104 and 218 bp) while the C allele was cut into three fragments (104, 155 and 63 bp).

Statistical methods
The disease status had no effect on the rate of DNA repair measured at 24 and 48 h (911). Thus there was no need to stratify the subjects by disease status in the present study. The repair rates were calculated as percent CPDs repaired at 24 h compared with the CPD level at 0 h, immediately after UV exposure. The relationship between repair of the two CPD types was quantified using Pearson's correlation coefficient. Differences in allelic distribution were tested by {chi}2 analysis. Differences in repair rate between the two genotype groups were assessed using the Wilcoxon rank sum test (two-sided). The non-parametric trend test (two-sided) was used to evaluate any possible trend in repair rate across groups with increasing number of variant alleles.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 44 subjects with previously known repair rates for TT=C or TT=T were genotyped. There was no difference in the allelic distribution between patients and controls. However, because the melanoma group included 17 subjects and the basal cell carcinoma 10 subjects, we cannot make definite statements about allele distributions in these groups. The frequency of the exon 10 A allele was 33% (29/88), which did not differ (P = 0.5) from that (24/84) previously reported for healthy Americans (13,14). For the exon 23 polymorphism the variant C allele had a frequency of 48% (42/88), which was significantly (P = 0.01) more common than the overall frequency (53/166) reported for other Caucasian populations (1315).

For one individual with an exon 10 GG and exon 23 AC genotype only TT=C repair was measured (Table IGo), whereas for another individual with an exon 10 GA and exon 23 AC genotype only TT=T repair was measured (Table IIGo). Thus the effects of the exon 10 and exon 23 polymorphisms on repair rate were analyzed in all but one individual for either of the CPD types.


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Table I. Repair rate of TT=C at 24 h related to XPD polymorphisms [mean ± SD per 106 nt (n)]
 

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Table II. Repair rate of TT=T at 24 h related to XPD polymorphisms [mean ± SD per 106 nt (n)]
 
Table IGo shows the extent of TT=C repair in the skin of 43 subjects stratified by exon 10 and 23 genotypes. For exon 10 the six individuals with the homozygous AA genotype showed a significantly reduced rate of DNA repair compared with those with the GG or GA genotypes (P = 0.01, Wilcoxon test). All six individuals actually had repair rates below the overall median of 58% (sub-normal). For exon 23 the rate of repair was uniform over all genotypes. Also the four double homozygotes, AA for exon 10 and CC for exon 23, differed significantly from the others in rate of DNA repair (P = 0.04). These four individuals had sub-normal repair and the mean rate (27.3%) was ~50% of the rates for other genotypes (overall mean 55.4%). Two of the double homozygotes were below age 50 years and two were older, with mean repair rates of 46.1 and 8.5%, respectively. Thus the depressed repair of TT=C seems to be explained by both the exon 10 polymorphism and a greater age.

A similar tabulation is shown for the rate of TT=T repair (Table IIGo). Again, only the four double homozygotes (exon 10 AA and exon 23 CC) differed from the others (P = 0.09). These four had repair rates below the overall median of 34%. The mean rate for these individuals (15.8%) was <50% of the overall mean for subjects with other genotypes (36.2%). Two of the double homozygotes were below age 50 years and two were older, with mean repair rates of 27.8 and 3.8%, respectively. The uniform findings on the age effect in Tables I and IIGoGo lend support to each other, as the repair of TT=T was significantly correlated with the repair of TT=C (r = 0.65, P < 0.0001). There was, however, no apparent effect of the exon 10 polymorphism on TT=T.

The data on repair rates was then stratified by age (Table IIIGo). Among the 43 subjects analyzed in each repair assay 18 were younger than 50 years at the time of sampling. Age did not modify the effect of the exon 10 polymorphism on repair rate. The repair rate, however, decreased systematically with presence of the exon 23 C allele among those who were at least 50 years old (P = 0.05 for TT=C and P = 0.02 for TT=T, trend test). Within the group of exon 23 CC homozygotes the lowest repair rates for both TT=C and TT=T were shown by three of the four older subjects. Two of them were the same subjects as the double homozygotes with depressed repair in Tables I and IIGoGo.


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Table III. XPD polymorphisms and age related to repair rate of CPD at 24 h [mean ± SD per 106 nt (n)]
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There has been much recent interest in DNA repair polymorphisms as a risk factor for human cancer but the results have remained contradictory (1317). However, none of the published studies have used a direct DNA repair test as an end-point and this is the novelty of the present study. One clear result from the published studies using the new DNA repair test has been the marked and largely unexplained inter-individual differences in repair rates between healthy human subjects (911). The biological mechanisms underlying such differences are challenging and the possible modifying effects of repair polymorphisms could readily be invoked. However, the downside of such large differences is that small effects of single susceptibility factors are likely to go unnoticed unless large populations can be studied. This may partly explain why we have observed no clear decrease in DNA repair rates with age (9) or disease status (10,11) among patients with basal cell carcinoma or melanoma and matched controls who were also included in the present study.

The present results showed no association between DNA repair rate and individual genotype among all subjects. The combined exon 10 AA and exon 23 CC genotype was associated with depressed repair, ~50% of that for the other genotypes. However, this decrease was only observed among those who were 50 years or older, suggesting that age was a modifying factor. The decrease in repair was observed for both TT=C and TT=T, showing technical consistency. Older subjects turned out to have depressed repair of both TT=C and TT=T if they had the exon 23 CC genotype. This genotype-mediated age effect has no obvious biological explanation and remains tentative. On the other hand, it may suggest that the exon 23 C allele is associated with depressed repair when combined with greater age. This is supported by the significant trend of decreasing repair rate for both TT=C and TT=T with increasing numbers of the variant allele in exon 23 among older subjects. The present results are not in accord with previous studies on basal cell carcinoma and chromatid aberration in vitro, which associated the common exon 23 A allele with risk, however, the end-points of all these studies were different (14,15). However, the results are in line with a recent study associating the exon 23 C allele with risk of head and neck cancer (17). Interestingly, greater age was associated with the increased risk, exactly as in our study.

In summary, our data do not show consistent XPD genotype-specific differences in DNA repair rates among all subjects, but the exon 23 C allele may be associated with depressed repair among older subjects. This finding, however, needs to be confirmed in larger studies.


    Notes
 
3 To whom correspondence should be addressed Email: kari.hemminki{at}cnt.ki.se Back


    Acknowledgments
 
This study was supported by the Swedish Radiation Protection Institute and the Swedish Cancer Society.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Balajee,A.S. and Bohr,V.A. (2000) Genomic heterogeneity of nucleotide excision repair. Gene, 250, 15–30.[ISI][Medline]
  2. Berwick,M. and Vineis,P. (2000) Markers of DNA repair and susceptibility to cancer in humans: an epidemiologic review. J. Natl Cancer Inst., 92, 874–897.[Abstract/Free Full Text]
  3. de Boer,J. and Hoeijmakers,J.H. (2000) Nucleotide excision repair and human syndromes. Carcinogenesis, 21, 453–460.[Abstract/Free Full Text]
  4. Hemminki,K., Xu,G. and Le Curieux,F. (2000) Re: markers of DNA repair and susceptibility to cancer in humans: an epidemiologic review. J. Natl Cancer Inst., 92, 1536–1537.[Free Full Text]
  5. Roza,L., de Gruijl,F.R., Henegouwen,J.B., Guikers,K., van Weelden,H., van der Schans,G.P. and Baan,R.A. (1991) Detection of photorepair of UV-induced thymidine dimers in human epidermis by immunofluorescence microscopy. J. Invest. Dermatol., 96, 903–907.[Abstract]
  6. Young,A.R., Chadwick,C.A., Harrison,G.I., Hawk,J.L., Nikaido,O. and Potten,C.S. (1996) The in situ repair kinetics of epidermal thymidine dimers and 6–4 photoproducts in human skin types I and II. J. Invest. Dermatol., 106, 1307–1313.[Abstract]
  7. Bykov,V.J., Jansen,C.T. and Hemminki,K. (1998) High levels of dipyrimidine dimers are induced in human skin by solar-simulating UV radiation. Cancer Epidemiol. Biomarkers Prev., 7, 199–202.[Abstract]
  8. Bykov,V.J., Sheehan,J.M., Hemminki,K. and Young,A.R. (1999) In situ repair of cyclobutane pyrimidine dimers and 6–4 photoproducts in human skin exposed to solar simulating radiation. J. Invest. Dermatol., 112, 326–331.[Abstract/Free Full Text]
  9. Xu,G., Snellman,E., Bykov,V.J., Jansen,C.T. and Hemminki,K. (2000) Effect of age on the formation and repair of UV photoproducts in human skin in situ. Mutat. Res., 459, 195–202.[ISI][Medline]
  10. Xu,G., Snellman,E., Bykov,V.J., Jansen,C.T. and Hemminki,K. (2000) Cutaneous melanoma patients have normal repair kinetics of ultraviolet-induced DNA repair in skin in situ. J. Invest. Dermatol., 114, 628–631.[Abstract/Free Full Text]
  11. Xu,G., Snellman,E., Jansen,C.T. and Hemminki,K. (2000) Levels and repair of cyclobutane pyrimidine dimers and 6–4 photoproducts in skin of sporadic basal cell carcinoma patients. J. Invest. Dermatol., 115, 95–99.[Abstract/Free Full Text]
  12. Bykov,V.J., Marcusson,J.A. and Hemminki,K. (2001) Protective effects of tanning on cutaneous DNA damage in situ. Dermatology, 202, 22–26.[ISI][Medline]
  13. Shen,M.R., Jones,I.M. and Mohrenweiser,H. (1998) Nonconservative amino acid substitution variants exist at polymorphic frequency in DNA repair genes in healthy humans. Cancer Res., 58, 604–608.[Abstract]
  14. Lunn,R.M., Helzlsouer,K.J., Parshad,R., Umbach,D.M., Harris,E.L., Sanford,K.K. and Bell,D.A. (2000) XPD polymorphisms: effects on DNA repair proficiency. Carcinogenesis, 21, 551–555.[Abstract/Free Full Text]
  15. Dybdahl,M., Vogel,U., Frentz, G, Wallin,H. and Nexo,B.A. (1999) Polymorphisms in the DNA repair gene XPD: correlations with risk and age at onset of basal cell carcinoma. Cancer Epidemiol. Biomarkers Prev., 8, 77–81.[Abstract/Free Full Text]
  16. Duell,E.J., Wiencke,J.K., Cheng,T.-J., Varkonyi,A., Zuo,Z.F., Ashok,T.D.S., Mark,E.J., Wain,J.C., Christiani,D.C. and Kelsey,K.T. (2000) Polymorphisms in the DNA repair genes XRCC1 and ERCC2 and biomarkers of DNA damage in human blood mononuclear cells. Carcinogenesis, 21, 965–971.[Abstract/Free Full Text]
  17. Sturgis,E.M., Zheng,R., Li,L., Castillo,E.J., Eicher,S.A., Chen,M., Strom,S.S., Spitz,M.R. and Wei,Q. (2000) XPD/ERCC2 polymorphisms and risk of head and neck cancer: a case–control analysis. Carcinogenesis, 21, 2219–2223.[Abstract/Free Full Text]
  18. McKinlay,A.F. and Diffey,B.L. (1987) A reference action spectrum for ultraviolet induced erythema in human skin. Comm. Int. Éclairage J. Res. Note, 6, 17–22.
Received January 4, 2001; revised February 28, 2001; accepted April 11, 2001.