Carcinogenesis, Vol. 22, No. 12, 2005-2008,
December 2001
© 2001 Oxford University Press
MOLECULAR EPIDEMIOLOGY AND CANCER PREVENTION |
Polymorphic hCHK2/hCds1 codon 84 allele and risk of squamous cell carcinoma of the head and neck—a case-control analysis
Yuxin Zheng1,
Lei Li2,
Hongbing Shen1,
Erich M. Sturgis1,3,
Susan A. Eicher3,
Sara S. Strom1,
Margaret R. Spitz1 and
Qingyi Wei1,4
1 Department of Epidemiology,
2 Department of Experimental Radiation Oncology and
3 Department of Head and Neck Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA
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Abstract
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Checkpoint kinase 2 (
hCHK2/hCds1) is a tumor suppressor gene
involved in cell-cycle control. A
hCHK2/hCds1 polymorphism in
codon 84 (A
G at nucleotide 252) was recently identified in Li-Fraumeni
syndrome patients. Because cell cycle regulates DNA repair that
is associated with cancer risk, we hypothesized that this new
polymorphism exists in the general population and is associated
with cancer risk. To test this hypothesis, we evaluated the
role of this polymorphism in a case-control study of 215 non-Hispanic
white patients with newly diagnosed squamous cell carcinoma
of the head and neck (SCCHN) and 229 frequency-matched cancer-free
controls. We found that the
hCHK2/hCds1 codon 84 variant was
rare and less frequent in non-Hispanic white cases (0.0186)
than in controls (0.0437;
P = 0.033). Although no variant homozygotes
were detected in these cases and controls, heterozygosity protected
against SCCHN, representing a 60% reduction of risk (adjusted
odds ratio = 0.40; 95% confidence intervals, 0.17–0.93)
compared with wild-type homozygotes. The variant allele was
also rare in other ethnic groups (0.0487, 0.0095 and 0.0541
in 115 African Americans, 105 Hispanic Americans and 111 native
Chinese, respectively), and only one variant homozygous individual
(a Chinese subject) was identified. These results suggest that
this
hCHK2/hCds1 codon 84 polymorphism is rare and may have
a protective role in the aetiology of SCCHN in non-Hispanic
whites. Larger studies are warranted to confirm this finding
and further mechanistic studies are needed to understand biological
relevance of this polymorphism.
Abbreviations: ATM, ataxia telangiectasia mutant; CI, confidence interval; hCHK2/hCds1, checkpoint kinase 2; OR, odds ratio; PCR, polymerase chain reaction; SCCHN, squamous cell carcinoma of the head and neck; SSCP, single-strand conformation polymorphism
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Introduction
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Although smoking and alcohol use are major risk factors for
squamous cell carcinoma of the head and neck (SCCHN) (
1), only
a fraction of smokers and drinkers develop this disease, suggesting
that there is genetic susceptibility to SCCHN. Recent molecular
epidemiological studies have provided some clues to the molecular
mechanisms underlying such genetic susceptibility. In response
to DNA damage induced by carcinogens, cells undergo a series
of events, including cell-cycle arrest, scheduled DNA repair
and apoptosis (
2,3). Constitutional variants of genes involved
in these processes such as DNA repair genes
XRCC1 and
XPD/ERCC2 (
4,5) probably contribute to such inter-individual variation
in susceptibility to SCCHN.
Cell-cycle control is an important process in cellular response to DNA damage, requiring the participation of many different genes, including p53, BRCA1 and ataxia-telangiectasia mutated (ATM) gene. Mutations in these genes have been implicated in human carcinogenesis (2,6). Recently, a new tumor suppressor gene, human checkpoint kinase 2 (hCHK2/hCds1) was identified (7), and mutations in this gene were subsequently described in individuals with Li-Fraumeni syndrome (8). hCHK2/hCds1 codes for a protein that functions as a DNA damage-activated protein kinase in the DNA damage-related replication checkpoints (9–12). A polymorphism of hCHK2/hCds1 in codon 84 resulting in an AG transition was also identified, but its functional relevance remains to be determined (8). Because hCHK2/hCds1 is involved in the DNA damage–response pathway in which it interacts with p53, BRCA1 and ATM, we hypothesized that this new polymorphism might be associated with cancer susceptibility. To test this hypothesis, we evaluated the association between the hCHK2/hCds1 codon 84 polymorphism and risk of SCCHN in a hospital-based case-control study of 215 patients with newly diagnosed SCCHN and 229 frequency-matched cancer-free controls.
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Materials and methods
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Study subjects
The recruiting of the cases and controls has been described
elsewhere (
4,5). Briefly, patients with newly diagnosed and
histologically confirmed SCCHN (primaries of the oral cavity,
oropharynx, hypopharynx and larynx) were recruited from the
Department of Head and Neck Surgery of M. D. Anderson Cancer
Center. Cancer-free control subjects were recruited from a local
managed-care organization with multiple clinics throughout the
Houston metropolitan area and were frequency-matched to the
cases by age (±5 years), sex, ethnicity and smoking status.
Because only a few patients of other ethnic groups were recruited,
this study was limited to non-Hispanic whites. The frequency
matching was designed to detect a main effect of the polymorphism.
Each eligible subject was interviewed to obtain data on age,
sex, ethnicity, smoking status and alcohol consumption (before
the onset of disease for the cases and at the time of interview
for the controls). After informed consent was obtained, each
subject donated 30 ml of blood that was collected in heparinized
tubes. From previous studies (
4,5,13), additional DNA samples
were available from randomly selected healthy subjects of other
ethnic groups including African Americans, Hispanic Americans
and native Chinese subjects and used to estimate
hCHK2/hCds1 allele and genotype frequencies. The research protocol was approved
by our Institutional Review Board.
Genotyping
From each blood sample, a leukocyte cell pellet obtained from the buffy coat by centrifugation of 1 ml of whole blood was used for DNA extraction with the QIAGEN DNA Blood Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. We then developed a polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) method to type the samples for the hCHK2/hCds1 codon 84 polymorphism, which was confirmed by direct sequencing. Briefly, we designed a pair of primers (forward, 5'-CACGATGCCAAACTCCAGCCA-3' and reverse, 5'-AAATCCATCCTGAAGGGCCCA-3') from the hCHK2/hCds1 sequence in GenBank (accession no. AF08904) to amplify a fragment of 178 bp that contains the polymorphic site (A252G). The targeted fragment was amplified in a 25 ml reaction mixture containing ~50 ng of genomic DNA template, 20 pmol of each primer, 0.1 mM each dNTP with 1 mCi of [32P]dCTP, 1x PCR buffer (50 mM KCl, 10 mM Tris–HCl, and 0.1% Triton X-100), 1.5 mM MgCl2 and 1.0 U of Taq polymerase (Sigma Chemical Co., St Louis, MO, USA). The PCR profile consisted of an initial melting step of 95°C for 5 min; 30 cycles of 94°C for 30 s, 62°C for 30 s and 72°C for 30 s; and a final extension step of 72°C for 10 min.
For SSCP analysis, 4 µl of PCR product was mixed with 4 µl of loading buffer (95% formamide, 20 mM EDTA, 0.05% xylene cyanol and 0.05% bromophenol blue). The mixture was denatured at 95°C for 5 min and then immediately put on ice. Four microliters of the mixture were loaded on a mutation detection enhancement gel (BioWittaker Molecular Applications, Rockland, ME, USA) for electrophoresis at 35 W for 5 h. After electrophoresis, the gel was dried and imaged by exposure to X-ray. The band-shift patterns were visualized by autoradiography (Figure 1A
).
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Fig. 1. PCR-based SSCP genotyping for CHK2/hCds1 codon 84 AG polymorphism (A) and sequencing chromatograms (B) showing the AA, AG and GG genotypes.
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After the size of each PCR product was confirmed by gel electrophoresis,
the band containing the PCR product was cut off and collected
for purification with a QIAEX II Gel Extraction Kit (Qiagen,
Chatsworth, CA, USA) according to the manufacturer's instruction.
The sequencing analysis was performed with an Automated Model
373A Sequencer (Applied Biosystem, Foster City, CA, USA). Figure
1
shows representative SSCP-band shifts. Based on these band-shift
patterns and direct sequencing (Figure 1B
), subjects were typed
as AA, AG or GG. About 10% of the samples were randomly selected
for repeated assays, and the results were 100% concordant.
Statistical analysis
Differences between the cases and controls in the distributions of selected demographic variables, smoking, alcohol consumption and hCHK2/hCds1 genotype frequencies were evaluated by the 
2-test. The association between the hCHK2/hCds1 codon 84 allele and SCCHN was estimated by computing odds ratios (ORs) and 95% confidence intervals (CIs) from both univariate and multivariate logistic regression analyses. Those subjects who had smoked >100 cigarettes in their lifetimes but had quit smoking >1 year previously were defined as former smokers and the remainder of the smokers were defined as current smokers. Those who drank alcoholic beverages at least once a week for >1 year in previous years but had quit drinking for >1 year prior to recruitment were defined as former drinkers and the remainder of the drinkers were defined as current drinkers. For logistic regression analysis, the hCHK2/hCds1 codon 84 genotype was recoded as a dummy (0, 1) variable. All of the statistical analyses were performed with Statistical Analysis System software (Version 6.12; SAS Institute, Cary, NC, USA).
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Results
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To investigate the association between the
hCHK2/hCds1 codon
84 polymorphism and risk of SCCHN, we evaluated the frequency
of the polymorphism in 215 non-Hispanic white SCCHN cases and
229 non-Hispanic white controls. As shown in Table I
, there
were no statistically significant differences in the distributions
of the matching variables (age, sex, smoking status and alcohol
consumption) between cases and controls, suggesting that the
frequency matching on these variables was adequate.
As shown in Table II
, the frequencies of the variant G allele
were 0.0186 and 0.0437 for cases and controls, respectively,
and the difference was statistically significant (
P = 0.033).
Although there were no GG homozygotes among these non-Hispanic
white subjects, the heterozygous AG genotype was less frequent
in the cases (3.7%) than in the controls (8.7%) and was associated
with a crude OR of 0.40 (95% CI, 0.17–0.94), which was
nearly unchanged after adjustment for age, sex, smoking status,
and alcohol use (OR = 0.40; 95% CI, 0.17–0.93).
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Table II. Distribution of hCHK2/hCds1 genotype and allele frequency and associated risk in SCCHN patients and controls
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To further investigate whether there were ethnic differences
in the allele and genotype frequencies of this
hCHK2/hCds1 codon
84 polymorphism, we performed the genotyping assays for selected
healthy subjects from three other ethnic groups (115 African
Americans, 105 Hispanic Americans and 111 native Chinese subjects)
with similar age and sex distributions to that of the healthy
non-Hispanic whites (
n = 229) used in the case-control analysis.
The distributions of age and sex among these four ethnic groups
were similar (data not shown;
P > 0.05). The mean age (±SD)
for non-Hispanic whites, African Americans, Hispanic Americans
and native Chinese subjects were 56.3 ± 12.1, 53.5 ±
11.1, 55.5 ± 10.9 and 58.2 ± 9.5 years, respectively,
and the variant allele frequency was also similar for three
ethnic groups but was lower in Hispanic Americans (Table III
).
The only GG homozygote was a native Chinese subject (Figure
1B
). The significance of this ethnic difference needs to be
further investigated. Although all allele frequencies of the
four ethnic groups were in agreement of Hardy–Weinberg
equilibrium (data not shown), further comparisons between the
expected and observed genotype distributions among these four
ethnic groups were not statistically possible because only one
variant homozygote was identified.
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Discussion
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hCHK2/hCds1 plays an important role in the DNA damage–response
pathway, in which it is first activated in response to DNA damage
and then directly phosphorylates serine 20 of p53 (
10) and serine
988 of BRCA1, leading to cell-cycle arrest in G
1 (
9,10). hCHK/hCds1
activation can be mediated by both ATM-dependent and -independent
pathways (
14). In response to ionizing radiation (
7) but not
UV radiation (
15), the Ser-Gln/Thr-Gln cluster domain of hCHK2/hCds1
is directly phosphorylated by ATM. Identification of mutations
in
hCHK/hCds1 in Li-Fraumeni syndrome patients established the
gene's role as a tumor suppressor gene (
8). Because hCHK2/hCds1
is pivotal in regulating the cell cycle, particularly in response
to various kinds of DNA damage, loss of its function is detrimental
to the maintenance of genetic integrity. Searching for genomic
mutations in
hCHK2/hCds1 has been difficult because the 3' portion
of the gene is duplicated (
16). Kimura
et al. (
17) tested for
germline mutations of
hCHK2/hCds1 in 25 patients with familial
gastric cancer but did not find any mutations. Harruki
et al.
(
18) found that somatic
hCHK2/hCds1 mutation is infrequent in
small cell lung cancer. Bell
et al. (
8) identified four germline
mutations in 22 patients with Li-Fraumeni syndrome and 49 sporadic
cancer cell lines: two frameshifts in the C-terminal domain
resulting in loss of kinase activity and another two missense
mutations within the forkhead homology-associated domain, one
that reduced activity and one that affected protein–protein
interaction (
19). In addition, the A
G polymorphism at its codon
84 was also identified in four of 28 normal lymphoblastoid cell
lines (
8). It was considered to be a silent polymorphism because
no amino acid substitutions in the
hCHK2/hCds1 coding sequence
were observed in control specimens (
8). The low frequencies
of mutations in
hCHK2/hCds1 in cancer cell lines, compared with
that of other tumor suppressor genes such as
p53 (
2), further
emphasizes the role of normal
hCHK2/hCds1 in maintaining cell
functions. These findings lead to the notion that polymorphisms
in
hCHK2/hCds1 rather than a specific disease-related mutation
may play a role in cancer susceptibility. Therefore, we conducted
this case-control analysis to evaluate the relationship between
this newly identified polymorphism and risk of SCCHN.
In the study presented here, we developed a new PCR-based SSCP method to detect the newly reported hCHK/hCds1 codon 84 variant in genomic DNA. We confirmed that this hCHK/hCds1 variant existed in the general population. Although the variant was rare, particularly for the homozygous variant, it appeared to be protective against SCCHN in the non-Hispanic white population. We also provided estimates for the variant allele frequency in four different ethnic groups, and we found that Hispanic Americans were least likely to have the variant allele. Because the polymorphism is rare and the results of our study may be due to chance, larger studies are needed to confirm our findings. Although the biological relevance of the variant is unclear and this polymorphism reportedly does not affect the protein's functions (8), the variant is probably in linkage disequilibrium with functional polymorphisms at other sites of the genome. Therefore, further studies are needed to determine the role of this variant in susceptibility to other types of cancer.
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Notes
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4 To whom correspondence should be addressed Email:
qwei{at}mdanderson.org 
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Acknowledgments
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We thank Dr Maureen Goode for her scientific editing, Ms Li-E
Wang and Ms Min Fu for their technical support, Ms Margaret
Lung for her recruitment of the subjects, and Ms Joanne Sider
and Ms Joyce Brown for manuscript preparation. This study was
supported in part by National Institute of Health research grants
CA 70334 and ES 11740 (to Q.W.), CA55769 and CA 60374 (to M.R.S.)
and CA 16672 (to M. D. Anderson Cancer Center); by National
Institute of Environmental Health Sciences grant ES 07784; and
by funds collected pursuant to the Comprehensive Tobacco Settlement
of 1998 and appropriated by the 76th Legislature to The University
of Texas M. D. Anderson Cancer Center.
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Received May 30, 2001;
revised August 7, 2001;
accepted August 24, 2001.
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