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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 A->G 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 1AGo).



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Fig. 1. PCR-based SSCP genotyping for CHK2/hCds1 codon 84 A->G polymorphism (A) and sequencing chromatograms (B) showing the AA, AG and GG genotypes.

 
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 1Go shows representative SSCP-band shifts. Based on these band-shift patterns and direct sequencing (Figure 1BGo), 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 {chi}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).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 IGo, 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.


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Table I. Distribution of selected variables in SCCHN patients and controls
 
As shown in Table IIGo, 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
 
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 IIIGo). The only GG homozygote was a native Chinese subject (Figure 1BGo). 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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Table III. Distribution of hCHK2/hCds1 allele and genotype frequency by four ethnic groups
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 G1 (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.


    Notes
 
4 To whom correspondence should be addressed Email: qwei{at}mdanderson.org Back


    Acknowledgments
 
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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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Received May 30, 2001; revised August 7, 2001; accepted August 24, 2001.





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