XPD polymorphisms: effects on DNA repair proficiency

Ruth M. Lunn1, Kathy J. Helzlsouer2, Ram Parshad3, David M. Umbach4, Emily L. Harris2,5, Katherine K. Sanford6 and Douglas A. Bell1,7

1 Laboratory of Computational Biology and Risk Analysis, National Institute of Environmental Health Sciences, National Institutes of Health, MD C3-03, PO Box 12233, Research Triangle Park, NC 27709,
2 Department of Epidemiology, The Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD,
3 Department of Pathology, Howard University College of Medicine, Washington, DC,
4 Biostatistics Branch, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC and
6 Laboratory of Cellular and Molecular Biology, Division of Cancer Etiology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
XPD codes for a DNA helicase involved in transcription and nucleotide excision repair. Rare XPD mutations diminish nucleotide excision repair resulting in hypersensitivity to UV light and increased risk of skin cancer. Several polymorphisms in this gene have been identified but their impact on DNA repair is not known. We compared XPD genotypes at codons 312 and 751 with DNA repair proficiency in 31 women. XPD genotypes were measured by PCR–RFLP. DNA repair proficiency was assessed using a cytogenetic assay that detects X-ray induced chromatid aberrations (breaks and gaps). Chromatid aberrations were scored per 100 metaphase cells following incubation at 37°C (1.5 h after irradiation) to allow for repair of DNA damage. Individuals with the Lys/Lys codon 751 XPD genotype had a higher number of chromatid aberrations (132/100 metaphase cells) than those having a 751Gln allele (34/100 metaphase cells). Individuals having greater than 60 chromatid breaks plus gaps were categorized as having sub-optimal repair. Possessing a Lys/Lys751 genotype increased the risk of sub-optimal DNA repair (odds ratio = 7.2, 95% confidence interval = 1.01–87.7). The Asp312Asn XPD polymorphism did not appear to affect DNA repair proficiency. These results suggest that the Lys751 (common) allele may alter the XPD protein product resulting in sub-optimal repair of X-ray-induced DNA damage.

Abbreviations: 95% CI, 95% confidence interval; CS, cockayne syndrome; NER, nucleotide excision repair; OR, odds ratio; TTD, trichothiodystrophy; XP, xeroderma pigmentosum.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hereditary genetic defects in DNA repair lead to increased risk of cancer. Individuals with xeroderma pigmentosum (XP), a rare autosomal recessive disease resulting from a defect in nucleotide excision repair (NER) of UV-damaged DNA, have a >1000-fold increased risk of skin cancer (1). These individuals are extremely hypersensitive to sunlight and have pigmentation abnormalities. Cell fusion analyses have identified seven genetic complementation groups (XPA to XPG) that encode for proteins participating in different steps of the NER pathway (2,3). NER deficiencies are also responsible for two other genetic diseases, Cockayne syndrome (CS), characterized by growth and mental retardation and neurological degeneration, and trichothiodystrophy (TTD), characterized by sulfur-deficient brittle hair and impaired mental and physical development (4,5).

Although CS, TTD and XP exhibit different clinical manifestations, they all have NER deficiencies. Moreover, all of these diseases can result from deficient XPD or XPB proteins. Recent elucidation of the functions of the various repair proteins encoded by the complementation groups has provided insight into these disease processes. NER is composed of two sub-pathways, global genome repair and transcription-coupled repair. Transcription-coupled repair occurs rapidly because it repairs damage to the transcribed strand of active genes, whereas global repair is much slower and repairs damage to inactive genes (6,7). XP results from defects in both transcription-coupled repair and global repair depending on the complementation group, and TTD and CS are caused by defective transcription-coupled repair and probably alterations in transcription (8). Transcription and repair are linked via the TFIIH complex, a basal transcription factor that participates in NER and transcription initiation. Moreover, XPB and XPD are components of the transcription factor, TFIIH, thus explaining their involvement in diseases with different phenotypes (9).

XPD protein possesses both single-strand DNA-dependent ATPase and 5'–3' DNA helicase activities and is thought to participate in DNA unwinding during NER and transcription (9,10). NER repairs DNA damage induced by UV radiation and bulky DNA adducts. However, because XPD is involved in both transcription and NER, it may contribute to repair of other types of damage, such as ionizing radiation. Studies using lymphocytes containing mutant XP genes have an elevated chromatid aberration frequency after exposure to ionizing radiation, suggesting a role for NER proteins in the repair of ionizing radiation-induced damage (11). Ionizing radiation induces oxidative damage and several studies suggest XP proteins may participate in the repair of this type of damage (12,13).

Because XPD is important in multiple cellular tasks and rare XPD mutations result in genetic diseases, XPD polymorphisms may operate as genetic susceptibility factors. As a preliminary test of functionality, we studied the association of three XPD polymorphisms located at codons 199 (Ile->Met), 312 (Asp->Asn) and 751 (Lys->Gln) with proficiency for repair of X-ray-induced chromatid breaks and gaps. Reduced DNA repair as measured in this assay has been associated with a high degree of cancer incidence in family members (1416). We found that the Lys/Lys751 was associated with reduced repair of X-ray-induced DNA damage.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
We identified XPD genotypes and examined their association with DNA repair proficiency as measured previously in 31 Caucasian women from the Breast Surveillance Service at The Johns Hopkins Medical Institutions and female employees at the same institution (14). One woman had breast cancer, whereas the other women had no previous diagnosis of cancer and were categorized as either high risk (n = 15), defined as having at least one first-degree relative or two second-degree relatives on the same side of the family with breast cancer, or low risk (n = 15) for breast cancer.

DNA repair proficiency
DNA repair proficiency was assessed previously in a masked fashion using the assay developed by Sanford and co-workers (17,18). This assay measures unrepaired DNA (breaks and gaps) in cytogenetic preparations of metaphase lymphocytes isolated from freshly drawn blood of the subjects. The lymphocytes were exposed to X-rays and incubated to allow for repair. Colcemid was added after 0.5 h, the cells were then incubated for another 1 h and then lysed. Since the distribution of breaks and gaps is bimodal, individuals can be categorized as having normal (less than 60) and sub-optimal (greater than 60) repair proficiency based on the sum of breaks and gaps per 100 metaphase cells (14). Helzlsouer et al. (14) reported that sub-optimal DNA repair was more prevalent among women with a family history of breast cancer in this study population.

Genotyping
XPD genotypes were determined using a PCR–RFLP technique. Polymorphisms located at amino acids 199, 312 and 751, were amplified from 50 ng DNA using 200 µM of each dNTP, 0.5 U Taq (Promega, Madison, WI) + TaqStart Antibody (Sigma, St Louis, MO), 0.8 µM primer (Table IGo) and either 1.5 mM (codons 199 and 312) or 2.0 mM (codon 751) MgCl2 in 1x PCR buffer (Promega). Codons 199 and 312 were amplified together using 5% DMSO as an additive, whereas codon 751 was amplified in a separate PCR reaction. Both reactions used the same PCR program which consisted of a 4 min denaturation step at 94°C followed by 30 cycles of 30 s at 94°C, 30 s at 60°C and 60 s at 72°C. The PCR amplicons were digested for 2 h at 37°C with a discriminating restriction enzyme, and the digestion products were separated by agarose gels. Table IGo delineates these conditions.


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Table I. PCR–RFLP: primers restriction enzymes and fragment sizes
 
Statistical analysis
We compared the number of chromatid aberrations (per 100 metaphase cells) for each XPD genotype using the Mann–Whitney Rank Sum test. We also categorized individuals as having either normal or sub-optimal DNA repair proficiency as defined earlier and examined whether the risk of sub-optimal repair proficiency was associated with XPD genotype. We stratified by age (dichotomized at the mean age of 42 years for our subjects). Exact odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated via conditional maximum likelihood methods for 2x2x2 tables using LogXact software (Cytel Software Corporation, Cambridge, MA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We found no variants at codon 199 in our study population, precluding further analysis of that locus. The XPD 312 variant (Asn) allele occurred with a frequency of 0.42, similar to that reported by Shen et al. (19). The 751 variant (Gln) allele occurred with a frequency of 0.26 which is also comparable with previous reports (19,20) and to an independent North Carolina genotyping study conducted in our laboratory (unpublished results).

Mean and/or median chromatid breaks, gaps and totals (breaks + gaps) were calculated for each XPD genotype (Table IIGo). Because variant homozygotes were rare (n = 2 and n = 1 for XPD 312 and 751, respectively), we combined them with heterozygotes and compared that pooled genotype with the more common homozygote genotype. Individuals homozygous for the Lys751 (common) allele had significantly (P = 0.01) higher chromatid aberrations (median = 132) than those having a Gln751 allele (median = 34). The median number of chromatid aberrations was also higher in individuals homozygous for the more common Asp312 allele than those having at least one variant Asn312 allele (132 and 50 breaks and gaps, respectively). However, this difference was not statistically significant (P = 0.22).


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Table II. XPD genotypes and chromatid aberrations
 
Figure 1Go depicts chromatid aberrations plotted for each Lys751Gln genotype group and stratified by familial breast cancer risk status. Women having the Lys/Lys751 genotype had higher median chromatid aberrations than those with genotypes containing the variant Gln allele whether they came from the low-risk group (89 versus 34, respectively; P = 0.34) or from the high-risk group (136 versus 38, respectively; P = 0.05). While the difference is statistically significant only in the high-risk group, the number of individuals in each group is small. Interestingly, all the women (n = 7) having both the Lys/Lys751 genotype and categorized as having high risk for breast cancer had sub-optimal repair.



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Fig. 1. XPD Lys751Gln genotypes in low and high breast cancer risk groups. Total chromatid aberrations (breaks and gaps) are plotted for XPD Lys751Gln genotypes (Lys/Lys and Lys/Gln + Gln/Gln) stratified according to familial breast cancer risk status. Open circles, data points for each individual; closed circles, median values for each group. The difference in chromatid aberrations between XPD genotypes, Lys/Lys versus Lys/Gln + Gln/Gln, stratified according to familial breast cancer risk was tested using the rank sum test (P = 0.34 and P = 0.05 for the low- and high-risk groups, respectively). The dotted line represents the bimodal division between good repair and sub-optimal repair (60 chromatid aberrations).

 
To assess the risk of sub-optimal repair due to different XPD alleles, we categorized individuals into two repair proficiency groups (sub-optimal and normal repair) based on the number of chromatid aberrations (Table IIIGo). In our study population, age was related to the Lys751Gln polymorphism (P = 0.01) but not to risk status (P = 0.6) or to the Asn312Asp polymorphism (P = 0.53). We observed a significant increased risk of sub-optimal DNA repair for women having the Lys/Lys751 genotype (OR = 7.2, 95% CI = 1.01–87.7; P = 0.035), but not for the Asp/Asp312 genotype (OR = 1.8, 95% CI = 0.3–11.0, P = 0.47).


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Table III. XPD genotypes and risk of sub-optimal DNA repair
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, the Lys/Lys751 genotype was associated with sub-optimal repair of DNA damage induced by X-irradiation. DNA repair was assessed using a cytogenetic assay that measures chromatid aberrations 0.5–1.5 h after treatment with X-rays. Chromatid aberrations result from unrepaired DNA strand breaks caused directly by X-irradiation, or indirectly as a result of repair of other X-ray-induced damage such as base damage (16). Persistence of these breaks suggests a deficiency in DNA repair. DNA repair proficiency, as measured by this cytogenetic assay, has been associated with predisposition to cancer (1416). Parshad et al. (21) reported the sensitivity of the chromatid aberration assay was sufficient to detect XP carriers (heterozygotes) who were clinically normal. While the cytogenetic assay is well documented to detect deficient DNA repair, we need to address three points concerning our findings: (i) the relationship between X-ray irradiation and XP; (ii) possible discordance between the present work and results from previous studies of rare mutant XPD genotypes; and (iii) the high frequency of the 751 Lys allele (Lys/Lys751 is the most common genotype).

Clinically, XP is due to deficient NER repair of UV-induced damage. Nevertheless, XP mutant cell lines are deficient in the repair of ionizing radiation as measured by chromatid aberrations (11). XP proteins probably participate in the repair of oxidative damage induced by ionizing radiation. Satoh et al. (12) reported that extracts from XP cells were unable to repair a specific class of oxygen free radical induced base lesions. Oxidative damage induced by ionizing radiation is preferentially repaired on the transcribed strand, suggesting a role for enzymes such as XPD which participate in transcription-coupled repair (13). Thus, there is a plausible role for the XPD protein in the repair of some types of radiation-induced damage.

With regard to the second point, we observed an increased chromatid aberration frequency in cells from individuals having the Lys/Lys751 XPD polymorphism; however, Sanford et al. (22), using the same cytogenetic assay, reported that the chromatid aberration frequency was not elevated in cells containing a rare XPD mutation (obtained from an XP patient). This discrepancy may be due to the different location of the polymorphism and the mutation; the polymorphism occurs at codon 751 whereas most XP individuals in complementation group D have a mutation at codon 683, the putative nuclear location signal which is believed to be responsible for XP symptoms (8,2325). XPD is part of the TFIIH transcription factor, which is a multi-protein complex involved in many different functions, including transcription, NER, transcription-coupled repair, apoptosis and cell cycle regulation (13). Thus, XPD interacts with many different proteins as part of this complex. Amino acid variants in different domains, such as 683 and 751, of XPD may affect different protein interactions, and result in the expression of different phenotypes. While we have no additional data to support this hypothesis, it is possible that the 751 Lys allele could have different effects in different DNA repair pathways (as assessed using other DNA repair assays).

Thirdly, we unexpectedly found the more common allele (Lys751) was associated with higher levels of chromatid aberrations than the variant allele (Gln751). Dybdahl et al. (20) also reported that individuals with the common allele (Lys751) had an elevated, but not significantly so, risk of basal cell carcinoma (OR = 4.2, 95% CI = 0.8–24). While one might expect the common allele to confer protection rather than risk, putative `at risk' genotypes of metabolism genes such as glutathione S-transferase M1 have frequencies in excess of 60% in some populations (26). In addition, the effect of a given allele on repair may depend on the exposure and interaction with other genes participating in DNA damage recognition, repair and cell cycle regulation.

We found that all of the individuals with both the Lys/Lys751 genotype and a family history of breast cancer had sub-optimal repair (data grouped in the upper right corner of Figure 1Go), as defined by an increase in chromosomal aberrations induced by G2 X-irradiation. This chromosomal radiosensitivity has been shown to be associated with individuals reporting cancer, including breast cancer, in their family. Roberts et al. (27) studied cellular radiosensitivity, using a cytogenetic assay similar to ours, in family members of radiosensitive (breast cancer patients) and non-sensitive individuals. Segregation analysis suggested that the radiosensitivity was heritable with a single major gene accounting for 82% of the variance among family members. The addition of a second, rarer gene to the model resulted in a better fit of the data. The authors postulated that these cancer-predisposing genes were common, low penetrance alleles found in normal populations (27), such as polymorphisms in DNA repair genes. The present finding is consistent with the hypothesis that multiple low-penetrance alleles could be involved in heritable radiosensitivity. The XPD Lys/Lys751 polymorphisms, specifically, and underlying genetic determinants of familial risk, in general, would be possibilities. Because of the small study population (n = 31), however, further statistical analysis of possible interrelationships among XPD genotype and familial risk status using the data available to us would not be informative. Also, the confidence intervals around the risk estimates are wide, indicating a need to confirm both the qualitative and quantitative results of this work by extending it to a much larger study population. Nevertheless, the finding that the Lys/Lys751 genotype is associated with sub-optimal repair provides sufficient evidence to justify further phenotype/ genotype studies as well as determining the impact of the Lys751Gln polymorphism on cancer risk.


    Acknowledgments
 
We would like to express our appreciation to Drs Harvey Mohrenweiser and Richard Shen (Lawrence Livermore National Laboratory) for sharing information about XPD polymorphisms. We also thank Drs Mariana Stern (NIEHS) and William Kaufmann (University of North Carolina, Chapel Hill) for critical review of the manuscript. This research was supported in part by Public Health Service (PHS) grant CA36390 from the National Cancer Institute, National Institutes of Health and Department of Defense grant DAMD 17-97-1-7072.


    Notes
 
5 Present address: Kaiser Permanente Center for Health Research, Portland, OR, USA Back

7 To whom correspondence should be addressed Email: bell1{at}niehs.nih.gov Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 14, 1999; revised February 7, 2000; accepted February 10, 2000.