The p53 Codon 72 Polymorphism and the Risk of Oral Cancer in a Chinese Han Population

Abstract

Background: The p53 codon 72 polymorphism has been investigated extensively for its association with various cancers around the world. It is still unclear whether the p53 codon 72 polymorphism is associated with oral cancer risk. Aim: The aim of our study was to evaluate the association between the p53 codon 72 polymorphism and the oral cancer risk in Chinese Han patients. Methods: A hospital-based case-control study with 200 patients with oral cancer and 200 matched controls was conducted. Genomic DNA was isolated from peripheral blood, and gene polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism. Results: The arginine (Arg)/Arg genotype conferred 0.57 times reduced risk to oral cancer (95% confidence interval [CI]=0.36, 0.89; p=0.01). The Arg allele frequency was significantly lower (odds ratios [OR]=0.74, 95% CI=0.56, 0.98; p=0.03) in comparison with controls in patients with oral cancer. The proline allele frequency was significantly higher (OR=1.35, 95% CI=1.02, 1.79; p=0.03) in comparison with controls in patients with oral cancer. When stratified by the clinical stage, lymph node metastasis, and histological differentiation of oral cancer, no statistically significant results were observed. Conclusion: Our results thus suggest that the p53 codon 72 polymorphism modulates susceptibility to oral cancer in Chinese Han patients.

Introduction

Oral cancer is one of the most common causes of morbidity and mortality today, with more than 10 million new cases and more than 6 million deaths each year worldwide (Petersen, 2009). It is estimated that around 43% of cancer deaths are due to tobacco use, unhealthy diets, alcohol consumption, inactive lifestyles, infections, and genetic factors (Petersen, 2009). Despite advances in treatment for oral cancer, the 5-year survival rate remains poor, with survival being dependent on the stages at diagnosis, because the majority of patients are diagnosed at stage III or IV (Gomez et al., 2009). Similar to many other malignancies, oral cancer is caused by the interactions between genetic and certain epigenic or environmental factors (Kietthubthew et al., 2003). The genetic component may influence an individual's susceptibility to cancer. This component includes polymorphic genes that modulate chemical metabolism, DNA repair, and cell cycle control (Kietthubthew et al., 2003).

The p53 tumor suppressor gene, located on chromosome 17p13, is one of the most commonly mutated genes in all types of human cancer (Hollstein et al., 1991). It contributes to the maintenance of genomic stability by controlling cell cycle and facilitating DNA repair in response to DNA damage (Hollstein et al., 1991). The human p53 gene contains a single-nucleotide polymorphism that encodes either arginine (Arg) or proline (Pro) at the amino acid codon 72 of the p53 protein (Buchman et al., 1988). Each individual thus inherits a p53 genotype that can be heterozygous (Arg/Pro) or homozygous for either arginine (Arg/Arg) or proline (Pro/Pro) at p53 codon 72 (Buchman et al., 1988).

Over the last decade, there are many studies to investigate the association between the p53 codon 72 polymorphism and the risk of oral cancer, but results have been inconsistent (Hamel et al., 2000; McWilliams et al., 2000; Summersgill et al., 2000; Tandle et al., 2001; Nagpal et al., 2002; Shen et al., 2002; Katiyar et al., 2003; Kietthubthew et al., 2003; Bau et al., 2007; Chen et al., 2008; Misra et al., 2009). The aim of our study was to evaluate the association between the p53 codon 72 polymorphism and the oral cancer risk in Chinese Han patients.

Materials and Methods

Study subjects

A hospital-based case-control study with 200 oral cancer cases and 200 matched controls was conducted between July 2010 and March 2012 at the Department of Oral & Maxillofacial Surgery, Stomatological College, the Harbin Medical University in Harbin, China. The controls were recruited from residents living in the similar geographic area. Informed consents were obtained according to the Declaration of Helsinki. Informed consent was obtained from both groups that participated in this study. Each participant was personally interviewed with a questionnaire that had been approved by the university ethics committee. The information collected and used in this study were related to history of the individual's demographic background, tobacco smoking, use of smokeless tobacco, betel chewing, alcohol drinking, and other possible risk factors as occupational exposure, nutrition, oral hygiene, as well as personal and family history of various cancers. Tobacco smoking was defined as nonsmoker (smoked <100 cigarettes in lifetime) and smoker. Alcohol consumption was defined as nondrinker and drinker (consumed more than one cup, 200 mL, per day).

Genotyping

Genomic DNA was isolated from peripheral blood by use of the QIAamp DNA blood mini kit (QIAGEN, Inc., Valencia, CA). Gene polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The two primers were 5′-TTGCCGTCCCAAGCAATGGATGA-3′ and 5′-TCTGGGAAGGGACAGAAGA TGAC-3′. Each PCR mixture contained 10 pmol of each primer and genomic DNA. The PCR products were digested with 2 U of the restriction enzyme BstUI (New England Biolabs, Beverly, MA) at 60°c for 16 h. The DNA fragments were electrophoresed through a 2% agarose gel and stained with ethidium bromide.

Statistical analysis

STATA program version 11.0 was used to perform all the statistical analyses (StataCorp LP, College Station, TX). The Chi-squared test was used for the comparison of proportion of categorical variables, and the t-test was used to compare age. A p-value<0.05 was considered statistically significant.

Results

Demography and risk factors in patients with oral cancer and controls are presented in Table 1. They were well matched based on the age (p=0.21) and sex (p=0.46). Tobacco smoking (p<0.001), alcohol consumption (p<0.001), and betel nut chewing (p<0.001) were risk factors of oral cancer (Table 1). The distributions of genotype in both groups were in the Hardy-Weinberg equilibrium.

Table 1.

Demography and Risk Factors in Patients with Oral Cancer and Controls

Variable	Cases (200) n (%)	Controls (200) n (%)	p
Age	56.3±11.4	54.9±10.7	0.21
Sex			0.46
Male	162 (81.0)	156 (78.0)
Female	38 (19.0)	44 (22.0)
Tobacco smoking			<0.001
Ever	158 (79.0)	60 (30.0)
Never	42 (21.0)	140 (70.0)
Alcohol consumption			<0.001
Ever	135 (67.5)	79 (39.5)
Never	65 (32.5)	121 (60.5)
Betel nut chewing			<0.001
Ever	117 (58.5)	17 (8.5)
Never	83 (41.5)	183 (91.5)
Human papillomavirus			0.32
Positive	61 (30.5)	52 (26.0)
Negative	139 (69.5)	148 (74.0)
Clinical stage
Stage I/II	99 (49.5)	/	/
Stage III/IV	101 (50.5)	/	/
Pathologic T stage
T1/T2	124 (62.0)	/	/
T3/T4	76 (38.0)	/	/
Lymph node metastasis
Negative	143 (71.5)	/	/
Positive	57 (28.5)	/	/
Histological differentiation
Well	65 (32.5)	/	/
Moderate	125 (62.5)	/	/
Poor	10 (5.0)	/	/

/, not applicable.

The Arg/Arg genotype conferred 0.57 times reduced risk to oral cancer (95% confidence interval [CI]=0.36, 0.89; p=0.01) (Table 2). The Arg allele frequency was significantly lower (odds ratios [OR]=0.74, 95% CI=0.56, 0.98; p=0.03) in comparison with controls in patients with oral cancer (Table 2). The Pro allele frequency was significantly higher (OR=1.35, 95% CI=1.02, 1.79; p=0.03) in comparison with controls in patients with oral cancer (Table 2). When stratified by the clinical stage, lymph node metastasis, and histological differentiation of oral cancer, no statistically significant results were observed (Table 3).

Table 2.

Genotype Frequencies of p 53 Codon 72 Polymorphisms Among Oral Cancer Patients and Controls

Genotype	Cases n (%)	Controls n (%)	OR (95% CI)	p
Arg/Arg	43 (21.5)	65 (32.5)	0.57 (0.36, 0.89)	0.01
Arg/Pro	81 (40.5)	67 (33.5)	1.35 (0.90, 2.03)	0.15
Pro/Pro	76 (38.0)	68 (34.0)	1.19 (0.79, 1.79)	0.41
Arg allele frequency	167 (41.8)	197 (49.3)	0.74 (0.56, 0.98)	0.03
Pro allele frequency	233 (58.2)	203 (50.7)	1.35 (1.02, 1.79)	0.03

OR, odds ratios; CI, confidence interval; Arg, arginine; Pro, proline.

Table 3.

Stratification Analysis of the p 53 Codon 72 Genotype Frequency in Oral Cancer

		Arg/Arg			Arg/Pro			Pro/Pro
Variable	Cases	n (%)	OR (95% CI)	p	n (%)	OR (95% CI)	p	n (%)	OR (95% CI)	p
Clinical stage	200	43 (21.5)	1 (Reference)		81 (40.5)	1 (Reference)		76 (38.0)	1 (Reference)
Stage I/II	99	22 (22.2)	1.03 (0.59, 1.82)	0.91	38 (38.4)	0.95 (0.60, 1.49)	0.82	39 (39.4)	1.04 (0.66, 1.63)	0.88
Stage III/IV	101	21 (20.8)	0.97 (0.55, 1.72)	0.91	43 (42.6)	1.05 (0.68, 1.63)	0.82	37 (36.6)	0.96 (0.61, 1.53)	0.88
Pathologic T stage	200	43 (21.5)	1 (Reference)		81 (40.5)	1 (Reference)		76 (38.0)	1 (Reference)
T1/T2	124	28 (22.6)	1.05 (0.62, 1.78)	0.86	52 (41.9)	1.04 (0.68, 1.57)	0.87	44 (35.5)	0.93 (0.61, 1.44)	0.76
T3/T4	76	15 (19.7)	0.92 (0.48, 1.75)	0.80	29 (38.2)	0.94 (0.57, 1.55)	0.82	32 (42.1)	1.11 (0.68, 1.81)	0.68
Lymph node metastasis	200	43 (21.5)	1 (Reference)		81 (40.5)	1 (Reference)		76 (38.0)	1 (Reference)
Negative	143	33 (23.1)	1.07 (0.65, 1.77)	0.78	55 (38.5)	0.95 (0.63, 1.42)	0.80	55 (38.5)	1.01 (0.67, 1.52)	0.95
Positive	57	10 (17.5)	0.82 (0.39, 1.73)	0.59	26 (45.6)	1.13 (0.66, 1.92)	0.66	21 (36.8)	0.97 (0.55, 1.71)	0.92
Histological differentiation	200	43 (21.5)	1 (Reference)		81 (40.5)	1 (Reference)		76 (38.0)	1 (Reference)
Well	65	16 (24.6)	1.15 (0.61, 2.17)	0.68	26 (40.0)	0.99 (0.59, 1.67)	0.96	23 (35.4)	0.93 (0.54, 1.60)	0.80
Moderate	125	25 (20.0)	0.93 (0.54, 1.60)	0.79	51 (40.8)	1.01 (0.67, 1.53)	0.97	49 (39.2)	1.03 (0.68, 1.58)	0.89
Poor	10	2 (20.0)	0.93 (0.20, 4.40)	0.93	4 (40.0)	0.99 (0.30, 3.24)	0.98	4 (40.0)	1.05 (0.32, 3.46)	0.93

Discussion

Many polymorphisms of difference genes have been related to the tumorigenesis of oral cancer. A meta-analysis, including 12 published case-control studies of 1259 patients with oral cancer and 2262 controls, demonstrates that the CYP2E1 Rsa I/Pst I c2 allele may be a biomarker for oral cancer, especially among Asian populations (Niu et al., 2012). A HuGE review and meta-analysis of 28 case-control studies suggested that the glutathione S-transferase M1 (GSTM1) null genotype may be associated with a higher risk of oral cancer in Asians, but not in Caucasians, and this effect may be modified by the smoking status (Zhuo et al., 2009; Zhang et al., 2011b). A meta-analysis, including 1326 cases and 3130 controls, suggested that the Arg194Trp polymorphism in the XRCC1 gene might be a biomarker of oral cancer susceptibility among the Asian population (Zhou et al., 2009). However, the case-control study conducted by Balaji et al. (2011) showed that the EPHX1 gene polymorphisms and haplotypes were not involved in the susceptibility to oral cancer in South Indian subjects.

The association of the P53 codon 72 polymorphism with some other cancers has also been studied. A hospital-based case-control study suggested that the p53 codon 72 Arg/Arg genotype and the Arg allele were associated with a lower risk of bladder cancer in the Chinese population (Zhang et al., 2011a). A meta-analysis of 10 case-control studies involving 3549 subjects suggested that the p53 Arg72 allele was a protective factor, and that the Pro/Pro genotype might increase the susceptibility to bladder cancer in Asians (Jiang et al., 2010). A hospital-based case-control study suggested that the p53 codon 72 polymorphism may be associated with gastric cancer in Chinese Han patients, and that difference in genotype distribution may be associated with the location and the stage of gastric cancer (Zhou et al., 2010). A meta-analysis, including 12 case-control studies, suggested that the p53 codon 72 polymorphism may be associated with gastric cancer among Asians, and that difference in genotype distribution may be associated with the location, stage, and histological differentiation of gastric cancer (Zhou et al., 2007). A meta-analysis based on 39 case-control studies suggested that p53 codon 72 polymorphism may contribute to susceptibility to breast cancer, especially in Europeans (Zhang et al., 2010). A meta-analysis of 37 studies suggested that the p53 codon 72 polymorphism was associated with an increased risk for invasive cervical cancer (Jee et al., 2004).

Although there is accumulating evidence that p53 genetic alteration is closely associated with oral carcinogenesis, the clear mechanism of this association is unclear. The p53 Arg72 protein induces apoptosis faster and suppresses transformation more efficiently than the p53 Pro72 protein (Thomas et al., 1999). In Pim's study, p53 polymorphic variants at codon 72 exerted different effects on cell cycle progression (Pim and Banks, 2004). The codon 72 polymorphic variants of p53 have markedly different apoptotic potential (Dumont et al., 2003). Mutations of the p53 gene were found in 35% of oral cancer cases, suggesting that mutation of p53 is involved in the carcinogenesis of the oral cavity (Yook and Kim, 1998).

Some shortcomings of this study should be mentioned. First, this is a hospital-based case-control study, so the selection bias may not be avoidable, and the subjects may not be representative of the general population. Second, the relatively small patient population is one of the potential weaknesses. Finally, the population was only from China; further study is required to validate this association in different populations.

In conclusion, our study suggests that the p53 codon 72 polymorphism modulates susceptibility to oral cancer in Chinese Han patients. Additional well-designed large studies are required to validate this association in different populations.

Footnotes

Acknowledgment

Financial Support: The Scientific Research Foundation of Heilongjiang Provincial Health Department (2009-094).

Author Disclosure Statement

No competing financial interests exist.

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