Download Association of the DNA repair gene XPD Asp312Asn polymorphism

Carcinogenesis vol.24 no.10 pp.1671±1676, 2003
DOI: 10.1093/carcin/bgg115
Association of the DNA repair gene XPD Asp312Asn polymorphism with
p53 gene mutations in tobacco-related non-small cell lung cancer
Wei-Min Gao1, Marjorie Romkes2,7, Richard D.Day3,
Jill M.Siegfried4,7, James D.Luketich5,7, Hussam
H.Mady6,8, Mona F.Melhem6,8 and Phouthone
Keohavong1,7,9
Introduction
Abbreviations: BER, base excision repair; BDPE, benzo[a]pyrene-diolepoxides; CI, confidence interval; DRC, DNA repair capacity; MGB, minor
groove binder; NER, nucleotide excision repair; NSCLC, non-small cell lung
cancer; OR, odds ratio; PARP, poly (ADP-ribose) polymerase; PBL,
peripheral blood lymphocyte; PY, pack-year; ROS, reactive oxygen species;
SCE, sister chromatid exchange; TFIIH, transcription factor IIH; XPD,
xeroderma pigmentosum group D; XRCC, X-ray repair cross-complementing
group 1.
Lung cancer is the leading cause of cancer-related deaths in the
US. Cigarette smoking is the major cause of lung cancer (1±3).
Cigarette smoke contains a myriad of chemical carcinogens
and reactive oxygen species (ROS). Chemical carcinogens,
including polycyclic aromatic hydrocarbons, aromatic amines
and N-nitroso compounds, can form DNA bulky adducts by
covalently binding with DNA and then irreversibly result in
DNA mutations (4,5). For instance, benzo[a]pyrene can be bioactivated in vivo into benzo[a]pyrene-diol-epoxides (BPDE),
which are well-known damaging metabolites and are related to
a specific mutational spectrum in the p53 gene (6). Tobacco
smoke and tobacco itself increase the production of ROS in
cells, resulting in the production of oxidative lesions in
DNA. The accumulation of ROS may inflict oxidative DNA
damage indirectly, by inactivation of enzymes that are
involved in DNA synthesis, or directly, by generating DNA
strand breaks and base damage that can lead to mutations in
tumor suppressor genes or oncogenes (7,8). Inactivation of the
tumor suppressor gene p53 is common in a wide variety of
human cancers, including non-small cell lung cancer (NSCLC)
(9). Among NSCLC tumors, the frequency of p53 mutations
are between 20 and 50%, of which 480% occur in exons 5±8
(10±16).
The removal or repair of DNA damage plays a key role in
protecting the integrity of the genome from the insults of
cancer-causing agents. DNA repair genetic polymorphisms
may result in altered function and/or efficiency of DNA repair,
and may contribute to inter-individual variation of DNA repair
capacity (4,17,18). Bulky adduct lesions induced by smoking
chemical carcinogens are repaired through the nucleotide excision repair (NER) pathway (19). Xeroderma pigmentosum
group D (XPD) is involved in the NER pathway by functioning
as an ATP-dependent DNA helicase with its 50 ±30 activity joint
to the basal transcription factor IIH (TFIIH) (20). Three nonsynonymous single nucleotide polymorphisms that induce
amino acid changes have been found in the XPD gene at
codon 199 (Ile ! Met), codon 312 (Asp ! Asn) and codon
751 (Lys ! Gln) (21). Epidemiological studies have indicated
that the XPD 312 and 751 polymorphisms might modify the
risk of lung cancer (22,23). In addition to NER, damaged bases
and DNA single strand breaks can be repaired through the base
excision repair (BER) pathway (24). The X-ray repair crosscomplementing group 1 (XRCC1) protein is implicated in the
BER processes by serving as a molecular scaffold interacting
with poly (ADP-ribose) polymerase (PARP), DNA polymerase-b and DNA ligase IIIa (25±30). Three polymorphisms in
the XRCC1 gene that lead to amino acid substitutions have
been described at codon 194 (Arg ! Trp), codon 280 (Arg !
His) and codon 399 (Arg ! Gln) (21). The XRCC1 Gln399
polymorphism resulting in single base substitution may
affect binding with PARP, leading to a deficiency of DNA
repair (31).
Carcinogenesis vol.24 no.10 # Oxford University Press; all rights reserved.
1671
1
Department of Environmental and Occupational Health, 2Department of
Medicine, 3Department of Biostatistics, 4Department of Pharmacology,
5
Department of Surgery, 6Department of Pathology and 7The University of
Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA 15260,
USA and 8The Veterans Administration Health Care System, Pittsburgh, PA
15240, USA
9
To whom correspondence should be addressed
Email: [email protected]
Lung cancer, a disease related mostly to tobacco smoke
exposure and a leading cause of cancer-related death in
industrialized countries, is frequently associated with
mutations in the p53 tumor suppressor gene. Genetic differences resulting in inter-individual variation in DNA
repair capacity may in part account for susceptibility of a
cell to genotoxic agents leading to somatic mutations,
including p53 mutations, and eventual transformation of
a normal cell into a malignant phenotype. The objective of
this study is to investigate the relationship between the
polymorphisms of two DNA repair genes, the nucleotide
excision repair xeroderma pigmentosum group D (XPD)
gene (codons 312 and 751) and the base excision repair
X-ray repair cross-complementing group 1 (XRCC1) gene
(codon 399), and p53 mutations among lung cancer
patients. Lung tumors from 204 smokers with non-small
cell lung cancer (NSCLC) were analyzed for mutations in
exons 5±8 of the p53 gene and genotypes of XPD and
XRCC1. p53 mutations were found in 20% (40/204) of the
patients. Patients with the XPD codon 312 Asn allele were
less likely to have p53 mutations (13.8%) than XPD 312
Asp/Asp (27.3%) [odds ratio (OR) 0.43, 95% confidence
interval (CI) 0.20±0.89, P ˆ 0.023]. No association was
found between p53 mutations and either XPD Lys751Gln
or XRCC1 Arg399Gln. However, the p53 mutation frequency increased with the increased number of the combined genotypes among XPD 312WT (Asp/Asp), XPD
751VT (Lys/Gln or Gln/Gln) or XRCC1 399VT (Arg/Gln
or Gln/Gln) (P ˆ 0.01, trend test). These results suggest
that individuals who smoke and have the XPD codon 312
Asp/Asp genotype may be at a greater risk of p53 mutations, especially if combined with other polymorphisms
that may result in deficient DNA repair.
W.-M.Gao et al.
There have been extensive studies investigating the prevalence of p53 mutations and their role in lung cancer risk
(9±16). A few studies reported the relationship between polymorphisms of DNA repair genes, specifically XPD and
XRCC1, and lung cancer risk (22,23,32,33). These studies
have suggested that there is an association between XPD and
XRCC1 polymorphisms and risk for lung cancer. Functional
changes in repair capacity due to inheritance of certain polymorphisms could increase the chance that adducts produced
from tobacco carcinogens result in p53 mutations. In this
study, we investigated the relationship between polymorphisms in XPD and XRCC1 genes and presence of p53 mutations in specimens obtained from 204 smoking patients with
NSCLC.
Materials and methods
Patients and tissue specimens
Patient recruitment and sample collection have been described elsewhere (10).
Briefly, histologically confirmed incident adult lung cancer patients assessed
at the University of Pittsburgh Medical Center between 1990 and 2002 were
recruited. Lung tumor tissue samples were obtained from 204 Caucasian
smokers with NSCLC (193 current smokers and 11 former smokers), including
paraffin-embedded lung tissues from 37 patients and fresh-frozen lung tumor
tissues from 167 patients. These patients consisted of 130 males and 74
females. Complete data for calculating total smoking exposure were available
for 151 smokers. The patients' mean pack-year (PY) ranged from 5 to 165
(median ˆ 50), where one PY is defined as one pack of cigarettes per day for
1 year. Patient age at diagnosis ranged from 38 to 92 years (median ˆ 66).
The specimens' histologies included in this investigation were as follows:
153 adenocarcinomas, 40 squamous cell carcinomas, seven adenosquamous
carcinomas and four large cell carcinomas.
Analysis of mutations on the p53 gene
DNA was extracted from 167 fresh-frozen lung tissues as described previously
(10). In addition, 37 paraffin-embedded tissue sections (25 mm) were individually deparaffinized in xylene, then ethanol and rinsed with water. The
DNA was then extracted using the same DNA extraction method as was used
for fresh-frozen tissues. p53 mutations occurring in exons 5±8 of the p53 gene
were analyzed by PCR±SSCP as described previously (10).
Genotyping for DNA repair genes
All polymorphisms were analyzed in genomic DNA isolated from tumors
using the ABI Prism 7700 sequence detector (TaqMan; Applied Biosystems,
Foster City, CA). PCR primers and minor groove binder (MGB) probes were
designed using the Primer Express 1.5 software (Applied Biosystems). Briefly,
the 25 ml genotyping reaction mix contained 10±20 ng of genomic DNA, 1
TaqMan Universal PCR Master Mix (Applied Biosystems), 300 nM of each
primer, 100±500 nM of one pair of specific oligonucleotide probes labeled
with the reporter dye 6-carboxy-fluorescein (FAM), corresponding to wildtype homozygotes (XPD-312Asp/Asp, XPD-715Lys/Lys or XRCC1-399Arg/
Arg) and with the reporter VICTM corresponding to the variant homozygotes
(XPD-312Asn/Asn, XPD-751Gln/Gln or XRCC1-399Gln/Gln), at the 50 end
and with a non-fluorescent quencher and MGB at the 30 end. The PCR reaction
was carried out as follows: 50 C, 2 min, for 1 cycle; 95 C, 10 min, for 1 cycle;
95 C, 15 s, 57±59 C, 1 min, for 45 cycles. Amplified DNA exhibiting each
genotype was electrophoresed on an acrylamide gel to confirm amplimer size
and sequenced to confirm each genotype. For quality control, genotype determinations were run as duplicates and we observed a 100% concordance rate. In
every assay, oligonucleotide controls for the wild-type homozygotes, heterozygotes and variant homozygotes were included. After PCR, the genotype was
analyzed with Sequence Detection System according to the fluorescence yield
for the two different dyes. The genotyping of the DNA repair genes was
performed with DNA from tumor tissue for all 204 patients. Furthermore, for
22 of these patients, we had both tumor tissue and matched peripheral blood
lymphocyte (PBL) samples available that were also analyzed for the panel of
genetic polymorphisms. Our results showed that the genotype identified in the
tumor tissue was identical to the genotype found in the matched PBL for these
22 patients (data not shown). Therefore, although we cannot exclude the
possibility that chromosomal loss at the repair gene locus might occur in
some of the tumors, our results indicate such a fortuitous loss, if it occurs,
may involve only a small fraction of the tumors and is highly unlikely to affect
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our overall results. For each polymorphism screened, individuals were grouped
as WT (homozygous wild-type) and VT (homozygous and heterozygous variant). WT was defined as XPD 312Asp/Asp, XPD 751Lys/Lys and XRCC1
399Arg/Arg. VT includes XPD 312Asp/Asn or XPD 312Asn/Asn, XPD
751Lys/Gln or XPD 751Gln/Gln and XRCC1 399Arg/Gln or XRCC1
399Gln/Gln.
Statistical analysis
Fisher's exact and c2 (Pearson's correlation) tests were employed to test the
association between genotypes and p53 mutation frequency dichotomized as
mutation negative and mutation positive. The possible associations were
evaluated by stepwise unconditional multivariate logistic regression models
controlling for age and sex [odds ratio (OR) and 95% confidence interval (CI)]
where appropriate. A trend test (a non-parametric test for trend across ordered
groups) for the ordered groups generating from the genotype combination was
also analyzed. All statistical tests were two-sided. A P of 0.05 was considered statistically significant. Analysis was performed using the STATA 6.0
software for Windows.
Results
Genotype and allele frequencies for XPD and XRCC1
polymorphisms
The genotypes and frequency distribution of alleles among the
204 cases are presented in Table I. The XPD-312Asn allele,
XPD-751Gln allele and XRCC1-399Gln allele represented 34,
37 and 36% of the 204 patients, respectively.
Characterization of the roles of XPD and XRCC1 polymorphisms in p53 mutations
In order to investigate whether a variation in DNA repair
capacity associated with XPD and XRCC1 polymorphisms
affect the occurrence of mutations in the p53 gene, we compared the XPD and XRCC1 genotypes with p53 mutation
frequencies in lung tumors from NSCLC patients (Table II
and Figure 1A). p53 mutations were detected in 40 of 204
(20%) tumors. Figure 1A shows the frequency of p53 mutations in relation to XPD 312 genotype. Individuals containing
the XPD 312 WT had a 27.3% p53 mutation frequency. For
comparison, individuals who were homozygous (Asn/Asn) or
heterozygous (Asp/Asn) for the XPD 312 Asn allele had a
lower p53 mutation frequency of 13.8 and 13.6%, respectively.
Individuals with the XPD 312VT, which combined patients
who were homozygous or heterozygous for the XPD 312 Asn
allele, had a mutation frequency of 13.8%. Therefore, patients
with XPD 312VT were less likely to have p53 mutations than
those with an XPD 312WT (OR, 0.43; 95% CI, 0.20±0.89;
Table I. XPD and XRCC1 genotypes and allele frequency
Genotypes
No. of patients
Frequency (%)
XPD Asp312Asn
Asp/Asp
Asp/Asn
Asn/Asn
Asn allele frequency
88
94
22
43
46
11
34
XPD Lys751Gln
Lys/Lys
Lys/Gln
Gln/Gln
Gln allele frequency
82
94
28
40
46
14
37
XRCC1 Arg399Gln
Arg/Arg
Arg/Gln
Gln/Gln
Gln allele frequency
85
90
29
42
44
14
36
p53 mutations and DNA repair genes in lung cancer
Table II. The association between XPD and XRCC1 polymorphisms and p53 mutation
p53 mutation
XPD Asp312Asn
Asp/Asp
Asp/Asn
Asn/Asn
Asp/Asn‡Asn/Asnc
XPD Lys751Gln
Lys/Lys
Lys/Gln
Gln/Gln
Lys/Gln‡Gln/Glnc
XRCC1 Arg399Gln
Arg/Arg
Arg/Gln
Gln/Gln
Arg/Gln‡Gln/Glnc
Crude OR
95% CI: P
Adjusted ORa
95% CI: P
Negative
Positive
64
81
19
100
24
13
3
16
1.00b
0.43
0.42
0.43
0.20±0.91; 0.027
0.11±1.55; 0.194
0.21±0.86; 0.018
1.00b
0.43
0.39
0.43
0.20±0.96; 0.038
0.10±1.48; 0.168
0.20±0.89; 0.023
67
76
21
97
15
18
7
25
1.00b
1.06
1.49
1.15
0.49±2.26; 0.885
0.54±4.14; 0.445
0.56±2.35; 0.698
1.00b
0.99
1.38
1.08
0.45±2.18; 0.978
0.49±3.88; 0.538
0.52±2.26; 0.828
69
68
27
95
16
22
2
24
1.00b
1.40
0.32
1.09
0.68±2.88; 0.368
0.07±1.48; 0.145
0.54±2.20; 0.812
1.00b
1.34
0.33
1.06
0.63±2.85; 0.448
0.07±1.56; 0.162
0.51±2.20; 0.883
a
OR adjusted for age, sex.
Reference group.
c
The separate logistic regression was run to combine the heterozygous and variant homozygous.
b
P ˆ 0.023; Table II). No evidence of association was found
between the presence of p53 mutations and the XPD 751 or
XRCC1 399 genotypes, even when heterozygous were
combined with homozygous variant genotypes (P 4 0.05)
(Table II).
Further statistical analysis was performed among subgroups
of patients with the combinations of XPD and XRCC1 polymorphisms. As it has been shown previously that XPD
312WT, XPD 751VT and XRCC1 399VT genotypes resulted
in deficient DNA repair (22,23,32,33), they were defined as
defective genotype subgroups in this analysis. The proportion
of p53 mutations among the four groups, carrying 0, 1, 2 and 3
defective genotypes (XPD 312WT, XPD 751VT and XRCC1
399VT), were 0 (0/6), 14.7 (11/75), 21.7 (25/115) and 50%
(4/8), respectively (Figure 1B). Individuals carrying defective
genotypes had a higher risk of having p53 mutations than those
without defective genotypes with a p53 mutation frequency
increasing with an increased number of defective genotypes in
these patients (P ˆ 0.01, trend test).
Discussion
We investigated the relationship between polymorphisms of
DNA repair genes, XPD and XRCC1, and p53 mutations in
lung tumors of patients with active smoking history with
NSCLC. We hypothesized that if there were functional relevance for the polymorphic DNA repair enzymes in the removal
of DNA damage caused by tobacco smoke carcinogens, we
would detect differences in p53 mutation frequencies in lung
tumors of smoking lung cancer patients.
Our results showed that the XPD-312Asn and XPD-751Gln
variant alleles occurred at a frequency of 0.34 and 0.37,
respectively. These frequencies were similar to those reported
previously for NSCLC by Butkiewicz et al. (0.37 and 0.40,
respectively) and lung cancer patients by Spitz et al. (0.29 and
0.36, respectively) (22,23). The XRCC1-399Gln allele frequency observed in our study (0.36) was also consistent with
those of previous studies (22,32,34). In the present study, the
p53 mutation frequency in NSCLC of 20% (40/204) was lower
than that reported for these cancers in other studies (11±16).
The difference in frequency may be due to several factors,
including the level of sensitivity of the mutation detection methods used by the different groups, and/or the geographical differences of patients investigated in these
studies (10).
We first evaluated the association between XPD 312 polymorphism and the presence of p53 mutations in lung tumors.
Individuals with either an XPD 312 Asp/Asn or Asn/Asn
genotype had a lower risk of having p53 mutations than
those with XPD 312 Asp/Asp genotype (OR ˆ 0.43; P ˆ
0.023). However, we did not find any significant association
between the XPD 751 polymorphism or XRCC1 399 polymorphism and the presence of p53 mutations in the patients'
lung tumors. These results showed a significant association
between DNA repair XPD gene codon 312 and p53 mutation
frequency, suggesting this polymorphism may modulate the
relationship between cigarette smoking and incidence of p53
mutations.
There have been a few studies evaluating the relationship
between polymorphisms of DNA repair genes and lung cancer
risk (22,23,32,33). The results of our study support some of the
studies showing that XPD 312 polymorphism may affect the
carcinogenesis process. For instance, XPD 312 Asn/Asn has a
protective effect against lung cancer (22), and glioma (35).
The molecular mechanism by which this polymorphism affects
lung cancer risk is not understood. It has been shown that
individuals with the 312Asn alleles had a lower level of
DNA damage (chromatid aberrations) to UV (36), and a higher
apoptotic response than the 312 Asp/Asp genotype (37). In
addition, the latter genotype was associated with an increased
BPDE±DNA adduct level (34). We have shown in this study
that patients with XPD 312 Asp/Asn or Asn/Asn had a lower
p53 mutation frequency than that of patients with XPD 312
Asp/Asp. This may be explained by the fact that a higher
apoptotic response can eliminate mutation-prone cells and
why individuals with an Asn allele are less susceptible to
cancer (37). However, our results are in disagreement with
those of Spitz et al. (23) who reported that this XPD 312
Asn/Asn genotype was associated with less optimal DNA
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W.-M.Gao et al.
Fig. 1. (A) Frequency of p53 mutation in relation to XPD genotypes at codons
312. Asp/Asn ‡ Asn/Asn was the combination of Asp/Asn and Asn/Asn.
Asterisk markers showed that the differences were statistically significant
compared with the Asp/Asp group (P 5 0.05). (B) Frequency of p53 mutation
in relation to the gene±gene interaction. The numbers in parentheses are the
total numbers of subjects. The groups of the population were defined as
follows: 0, individuals carrying the combination of XPD 312 VT, XPD 751 WT
and XRCC1 399 WT; 1, individuals carrying either one of the XPD 312 WT,
XPD 751 VT or XRCC1 399 VT; 2, individuals carrying either two of the XPD
312 WT, XPD 751 VT or XRCC1 399 VT; 3, individuals carrying the
combination of XPD 312 WT, XPD 751 VT and XRCC1 399 VT. Trend test for
the ordered groups showed the statistically significant (P ˆ 0.01).
repair capacity (DRC) in cultured cells. The occurrence of lung
cancer in XPD 312VT subjects with a low frequency of p53
mutation also suggests that a different cancer pathway is activated in these individuals.
Our results showed there was no association between the
XPD Lys751Gln polymorphism and p53 mutation status of the
tumor. Indeed, a few studies reported an absence of correlation
of XPD Lys751Gln polymorphism with lung cancer risk
(22,38). There is also a lack of association between this
polymorphism with the levels of polyphenol DNA adducts
(39), or the risk of bladder cancer (40). However, a few studies
suggested that XPD 751 polymorphism may be related to
cancer risk. For instance, XPD 751 Gln allele might be
associated with a high risk of lung cancer (23), and squamous
cell carcinoma of the head and neck (41). On the other hand,
other studies showed that individuals with the XPD 751 Lys/
Lys genotype were at a higher risk of basal cell carcinoma (42),
or associated with a less suboptimal DRC (36).
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The observed differential effects of XPD 312 and 751 polymorphisms on p53 mutation frequencies are not completely
understood because of the currently limited information available on the functional significance of the XPD polymorphisms.
Nevertheless, it has been shown that the 312 Asp amino acid of
XPD is highly conserved through evolution (19,22), and may
thus be functionally important for the XPD enzyme. Conversely, the 751 Lys amino acid in the C-terminus of the XPD
protein is not evolutionarily conserved. Also, as a member of
the multiprotein complex TFIIH, XPD has multiple cellular
functions and may in this way interact with various proteins
(43). Amino acid variation between codons 312 and 751 of
XPD may also lead to different protein interactions, resulting
in the expression of different functional phenotypes (36).
Finally, it may be possible that the sequence variation at
codon 312 could affect RNA stability or influence protein
synthesis, leading to differences in apoptosis (23).
We also observed no association between XRCC1
Arg399Gln and p53 mutation frequencies. This result is in
agreement with those of Butkiewicz et al. (22), who found
no association between lung cancer risk and XRCC1 399
polymorphism in the Polish population. In disagreement with
our results, other studies showed that XRCC1 399 polymorphism may play a role in cigarette smoking-induced lung cancer.
Divine et al. (32) found a positive association of XRCC1
399Gln allele with an increased risk of lung adenocarcinoma.
Furthermore, Park et al. (44) reported that XRCC1 399Gln was
associated with an increased risk for squamous cell carcinoma
of lung with lower degrees of cigarette use in the Korean
population. XRCC1 399 Gln/Gln genotype was associated
with higher levels of placental aflatoxin-B1 adducts and glycophorin A mutations in erythrocytes (45), higher frequencies
of DNA damage (sister chromatid exchange, SCE) in smokers
(38), or a lower repair capacity of NNK-induced genetic
damage (SCE) in cultured human lymphocytes from nonsmoking volunteers, compared with those carrying the
XRCC1 399 Arg/Arg genotype (46). The absence of an association of XRCC1 399 polymorphism with the p53 mutation
status in our study may be due to several factors, including the
small number of patients, or the histological subtypes of lung
cancer analyzed in this study.
In addition to individual polymorphisms, we investigated the
combination of the XPD and XRCC1 polymorphisms. We
observed no p53 mutations among the six patients who had
the combined genotypes with XPD 312VT, XPD 751WT and
XRCC1 399WT. There are eight patients who carried all three
defective genotypes with XPD 312WT, XPD 751VT and
XRCC1 399VT, and half (50%) contained p53 mutations.
Finally, we observed that the p53 mutation frequency
increased with the increased number of defective genotypes
the patients carried (Figure 1B, P ˆ 0.01, trend test). These
results suggested that the presence of certain gene±gene combinations might influence the levels of p53 mutations. It may
be that a combined inefficient NER and BER further enhance
genetic damage, resulting in the increased p53 mutation frequencies that we observed.
Previously, Hou et al. (47) reported a marginally significant increase in transversions of p53 mutations among
patients with at least one XPD variant allele, compared with
the wild-type homozygotes in the group of combined neversmokers and ever-smokers (P ˆ 0.095 for XPD 312 and
0.085 for XPD 751), but not in any of the separate groups
of ever-smokers or never-smokers. In our study, the 40 (20%)
p53 mutations and DNA repair genes in lung cancer
Table III. Types of p53 mutations in 40 NSCLC patients with XPD and
XRCC1 genotypes
p53 mutation
XPD 312
a
Transversions (n ˆ 20)
Transitions (n ˆ 17)
Deletions (n ˆ 3)
Total
XPD 751
b
a
XRCC1 399
b
WT
VT
WT
VT
WTa
VTb
13
9
2
24
7
8
1
16
9
5
1
15
11
12
2
25
6
9
1
16
14
8
2
24
a
WT: XPD 312Asp/Asp, XPD 751Lys/Lys and XRCC1 399Arg/Arg.
VT: XPD 312Asp/Asn or XPD 312Asn/Asn, XPD 751Lys/Gln or XPD
751Gln/Gln and XRCC1 399Arg/Gln or XRCC1 399Gln/Gln.
b
p53 mutations identified included 20 (50%) transversions, 17
(43%) transitions and three (7%) deletions (Table III), compared with the 11 p53 mutations (27%), including eight
(73%) transversions and three (27%) transitions, identified
by Hou et al. (47). Therefore, both the frequencies and types
of p53 mutations among ever-smokers are not significantly
different between the two studies. As shown in Table III, 13
of the 20 (75%) transversions and nine of the 17 (53%)
transitions had the XPD 312 Asp/Asp genotype. Eleven of
the 20 (55%) transversions and 12 of the 17 (71%) p53
transitions had each at least one allele Gln for the XPD 751
polymorphism. Fourteen of the 20 (70%) transversions and
eight of the 17 (47%) p53 transitions each had at least one
allele Gln in XRCC1 399. There is no association between
the XPD 312, XPD 751 or XRCC1 399 genotypes and any of
the type of p53 mutations (P 4 0.05, two-sided Fisher's
exact test). This observation is not in disagreement with the
result reported by Hou et al. (47) among ever-smokers.
Furthermore, we observe no association between the hotspot
mutations (codons 245, 248 and 249) identified in this study
and the individually XPD 312, XPD 751 and XRCC1 399
genotypes, or the combination of XPD and XRCC1
polymorphisms (data not shown). Additional studies involving a larger number of patients are needed to confirm
the role of these polymorphisms in relation to the type of p53
mutations in both ever-smokers and non-smokers.
In summary, the results of this study showed a significant
association in smoking patients with NSCLC between presence of a defective XPD 312 Asp/Asp, or the combination
of defective genotypes of XPD 312WT, XPD 751VT and
XRCC1 399VT, and an increased risk for p53 mutation frequency in lung tumors. Individuals who smoke and have XPD
312 Asp/Asp genotype may be at a greater risk for production
of p53 mutations, which could increase the chance for lung
tumor formation. Future studies are needed to characterize the
biological consequences of these polymorphisms as well as to
consider additional polymorphisms in metabolic genes and
other DNA repair genes that could influence formation of
DNA adducts and cytogenetic damage.
Acknowledgement
This work was supported by a grant from the American Cancer Society (RPG99-161-01-CNE) and the SPORE grant (P50 CA090440).
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Received March 14, 2003; revised June 6, 2003; accepted June 28, 2003