P53 codon 72 polymorphism, human papillomavirus infection, and their interaction to oral carcinoma susceptibility

Tumor suppressor gene p53 plays an important role in the maintenance of the genomic integrity, and mutation in the gene may alter an individual’s susceptibility to various carcinomas. P53 Arg72Pro or codon 72 polymorphism has been indicated to increase the risk of developing certain cancers such as bladder cancer and cervical cancer. Human papillomavirus (HPV) infection has been shown as a risk factor for certain cancers such as cervical cancer and oral cancer as well, and the HPV oncoprotein E6 may induce the degradation of p53 function. However, the association between p53 Arg72Pro polymorphism and the risk of oral cancer with HPV infection remains inconclusive. Therefore, this meta-analysis involving 5,614 participants was performed to investigate the relations among the p53 Arg72Pro polymorphism, HPV infection, and the risk of developing oral cancer. A search of the literature by PubMed, Embase, Web of Science, and China National Knowledge Infrastructure databases was conducted to identify studies based on the inclusion and exclusion criteria. Odds ratios with 95 % confidence intervals were combined using a random-effect model or a fixed-effect model. The current study was conducted with 13 studies consisting of 2,413 cases and 3,201 controls. Neither overall analysis nor stratified analyses detected any obvious evidence of association between p53 Arg72Pro polymorphism and oral cancer susceptibility in all genetic models. However, a significant association between p53 Arg72Pro polymorphism and the risk of oral cancer with HPV infection was detected in the Arg/Arg vs. Arg/Pro + Pro/Pro model. In the current meta-analysis which used the quantitative data synthesis for the first time, our study demonstrated that p53 Arg72Pro polymorphism together with HPV infection might jointly alter an individual’s susceptibility to the risk of oral cancer. Our results suggested that p53 Arg72Pro polymorphism may partly contribute to the pathogenesis of oral cancer development.


Background
The incident rate for oral cancer has been increasing recently. Research studies have suggested that smoking, alcohol consumption, and betel quid chewing are risk factors that predispose individuals to oral cancer [1][2][3]. Nevertheless, only some smokers, alcohol users, and betel quid users develop oral cancer, which indicated that it can be a multifactorial process associated with various risk factors for oral cancer development. These exogenous carcinogens may induce a defective DNA damage response, which may alter the expression of tumor suppressor genes apoptosis or may result in genomic instability [4,5]. Accumulative evidence indicates that individual susceptibility to oral cancer also depends on genetic predisposition and viral infection [6,7]. Therefore, both environmental and genetic factors may play an important role in the process of oral cancer development.
Many published studies have reported that oral carcinoma susceptibility is associated with gene polymorphism.
In recent years, much attention has been focused on the p53 codon 72 Arg/Pro polymorphism. The p53 tumor suppressor gene is located at human chromosome 17 and encoding a 53-kDa nuclear phosphoprotein which plays a crucial role in cell cycle regulation, maintenance of genomic integrity, apoptosis, and challenge of environmental insults [8,9]. Mutant p53 codon 72 may allow cells with environment-associated damaged DNA to enter the cell cycle, leading to the development of tumors [10,11]. In fact, there have been extensive research studies demonstrated that p53 Arg72Pro polymorphism played an important role in developing cervical cancer in HPV-positive patients. Odds of developing cervical cancer was significantly higher with the p53 Arg allele in HPV associated cervical cancer. This association was not detected in HPV-negative patients [12]. In addition, the Potentially relevant articles were identified from the database (n=33) Articles were excluded due to the fact that they have no related precise genotype data for cases and controls (n=5) Articles retrieved for more detailed evaluation (n=28) Articles were excluded because they were not case-control studies (n=7) Articles with usable information (n=21) One study published without English (n=1), and one study was an extension of another, only the latest published one was included (n=1) Articles with eligible information (n=19) Articles were excluded because they were not in agreement with HWE (n=5). One study was excluded due to a very small sample size.
Articles with eligible information ultimately (n=13)    association between p53 Arg72Pro polymorphism and oral cancer has been investigated, however, the results were inconsistent. HPV infection have been proved as an independent risk factor for the development of oral cancer [13,14]. The viral E6 protein, which encoded by two high risk HPV types named HPV-16 and HPV-18, was testified to bind and inactivate the human p53 gene product, and marking it for destruction by the ubiquitin proteasome pathway [15][16][17]. Storey et al. suggested that the p53 Arg72Pro polymorphism plays a part in the development of HPV-associated cancer in 1998 for the first time [18]. Since then, researchers have investigated the combined influence of the Arg72Pro polymorphism and HPV infection in the risk of developing oral cancer, but the results remained inconclusive [19][20][21].
Therefore, whether or not p53 Arg72Pro polymorphism can increase the risk of oral cancer with HPV infection remains unclear. Based on the above reasons, we conducted this evidence-based quantitative meta-analysis to investigate the relationship between p53 polymorphisms and the risk of HPV-related oral cancer.

Inclusion and exclusion criteria
The following inclusion criteria were used for the selection of literature for meta-analysis: (1) published in English; (2) examined case-control studies investigating the association between HPV infection, Arg72Pro polymorphism, and the risk of oral cancer; (3) definite histopathologic diagnosis; and (4) genotype distribution in controls must be in Hardy-Weinberg equilibrium (HWE). Major exclusion criteria included: (1) the unpublished reports and abstracts; (2) when duplicated studies published Fig. 3 The association between p53 Arg72Pro polymorphism and the risk of oral cancer in total population (Arg/Arg vs. Pro/Pro) by the same author, only the most recent publication study was chosen.

Data extraction
All the eligible articles were independently reviewed and extracted by two reviewers (YL and WY) according to the selection criteria listed above. Disagreement was resolved by the third independent investigator (JH). The following data were extracted from the each study: the first author, year of publication, country, ethnicity, genotyping methods, source of the controls, and genotype numbers from the cases and controls.

Statistical analysis
The STATA version 11.0 software (Stata Corporation, College Station, TX) was used to conduct the statistical analyses. The combined odds ratio (OR) with a corresponding 95 % confidence interval (CI) was estimated to evaluate the relationship among p53 Arg72Pro polymorphisms, HPV infection, and the risk of oral cancer. For control groups, the goodness-of-fit test (Chi-square test or Fisher exact test) was used to test the deviations from HWE. The following statistical models were used in the meta-analysis: the allelic model (Arg72 allele vs. Pro72 allele), the codominant model (homozygote comparison: Arg/Arg vs. Pro/Pro), the dominant model (Arg/Arg + Arg/Pro vs. Pro/Pro), and the recessive model (Arg/Arg vs. Arg/Pro + Pro/Pro). Statistics Q and I 2 statistic were evaluated to investigate the between-study heterogeneity [22,23]. Either the random-effect model or the fixed-effect model was used to calculate the pooled effect estimate either in the presence or in the absence of heterogeneity, respectively [24,25]. Additionally, the Begg's funnel plot and the Egger's test were used to estimate the publication bias (p < 0.05 was considered statistically significant) [26,27].

Meta-analysis results
The association between p53 Arg72Pro polymorphism and the risk of oral cancer in total population A total of 13 studies were included in the meta-analysis to examine the association between p53 Arg72Pro polymorphism and the risk of oral cancer. There was no evidence of a significant association in any genetic model (Arg72 allele vs.  Table 3). However, significant heterogeneity across the studies was present in four genetic models (P = 0.000, 0.002, 0.013, 0.023 for the allelic genetic model, the homozygote comparison model, the dominant model and the recessive model, respectively Table 3).

The association between p53 Arg72Pro polymorphism and the risk of oral cancer in a specific population
In order to determine the major cause for the heterogeneity, a stratified analysis of the specific populations was performed. Eight studies were conducted in Asian populations and five studies were conducted in Caucasian populations. No significant association between the risk of oral cancer and p53 codon 72 polymorphism was detected in the Asian and the Caucasian groups in any genetic model (Table 3). Significant heterogeneity was detected in both groups in all genetic models, except for Pro/Pro vs. Arg/Arg + Arg/Pro in the Caucasian group ( Table 3).

The association between combined effect of p53 Arg72Pro polymorphism with HPV infection and the risk of oral cancer in total population
A total of five studies, including 396 cases and 213 controls, were included to evaluate the relations among HPV, p53 Arg72Pro polymorphism, and oral cancer susceptibility. The result showed that the association of HPV with p53 Arg72Pro variant genotypes displayed a statistical significance on oral cancer risk in the Arg/Arg  (Fig. 2, Table 3). There was no significant heterogeneity among these studies (Q = 0.93, I 2 = 0.0 %, P = 0.92; Table 3).

Publication bias
Begg's funnel plots seemed to be approximately symmetrical in all meta-analyses (data not shown). Additionally, Egger's tests did not reveal any obvious evidence of publication bias either (Table 3).

Discussion
Since the identification of the p53 codon 72 polymorphism, many studies have been devoted to explore the genetic effect of p53 Arg72Pro polymorphism on susceptibility of oral cancer. However, the evidence regarding the role of single nucleotide polymorphism of p53 Arg72Pro gene as a genetic marker for the risk of oral cancer is inconsistent. This prompted us to undertake the present meta-analysis to explore a more robust estimate of the relationship between p53 Arg72Pro genetic variant and the oral cancer susceptibility. In this study, we found that individuals who have genetic variants (Arg/Pro genotype or Pro/Pro genotype) may not have induced modification of oral cancer risk compared with those who carry wild-type genotype (Arg/Arg genotype). Same SNP may play different roles in the development of cancer in different ethnic populations. Therefore, the relation of p53 Arg72Pro polymorphism with oral cancer susceptibility might be affected by the different ethnic groups. Nevertheless, neither Arg/Arg genotype individuals nor Pro carriers have a significant association with oral carcinoma in the Asian group or the Caucasian group.
HPV belongs to a large virus family, the PAPOVA virus family. There are nearly a hundred types of HPV discovered in human [38]. In this family, some of the members are known to be high-risk oncogenic HPV type, such as HPV-16, HPV-18, HPV-33, and HPV-58. Through encoding oncogenic protein E6, high-risk HPV types inhibit p53 cell cycle tumor suppressor. The viral E6 protein has a powerful binding affinity for p53 protein resulting in its ubiquitination and destruction, thereby inducing degradation of p53 function and cell cycle out of control [15]. Therefore, p53 gene may have some interaction with HPV infection in susceptibility to HPV-associated oral cancer. Some investigators have found that joint action of the p53 codon 72 polymorphism with HPV is associated with the risk of oral cancer  [19,20,30], but different conclusions were obtained by other investigators [21,36]. Considering the above mentioned conflicting conclusions, a subgroup analysis of interaction of p53 gene polymorphism with HPV infection on oral cancer susceptibility was performed. Our study demonstrated a significant interaction between HPV infection and p53 Arg72Pro polymorphism on the risk of developing oral cancer in p53 Arg/Arg genotype carriers compared with p53 72Pro carriers. The small sample size is a major limitation in this study. There were only five research articles investigating the interaction between the infection with HPV and p53 codon 72 polymorphism on the risk of oral carcinoma. Thus, additional studies with larger sample size are needed to further evaluate the impact of HPV infection and p53 Arg72Pro polymorphism on HPV-associated oral cancer susceptibility.

Conclusion
For the first time, the current study provided the quantitatively synthesized estimates for the effect of interaction between HPV infection and p53 Arg72Pro polymorphism on the risk of developing oral cancer. This combined effect might together alter an individual's susceptibility to oral cancer. Our results suggested that p53 Arg72Pro polymorphism may partly contribute to the pathogenesis of oral cancer development. Further well-designed studies with reference to the interactions of gene-gene and gene-environment on p53 codon 72 polymorphism to oral carcinoma susceptibility are required.