P 53 Arg 72 Pro Polymorphism and Bladder Cancer Risk-Meta-analysis Evidence for a Link in Asians but not Caucasians

OBJECTIVE
Individual studies of the associations between P53 codon 72 polymorphism (rs1042522) and bladder cancer susceptibility have shown inconclusive results. To derive a more precise estimation of the relationship, we performed this systemic review and meta-analysis based on 15 publications.


METHODS
We used odds ratios (ORs) with 95% confidence intervals (CIs) to assess the strength of the association.


RESULTS
We found that there was no association between P53 codon 72 polymorphism and bladder cancer risk in the comparisons of Pro/ Pro vs Arg/Arg; Pro/Arg vs. Arg/Arg; Pro/Pro plus Pro/Arg vs. Arg/Arg; Arg/Arg vs. Pro/Arg plus Arg/Arg (OR=1.06 95%CI 0.81-1.39; OR=1.06 95%CI 0.83-1.36; OR=0.98 95%CI 0.78-1.23; OR=1.06 95%CI 0.84-1.32). However, a significantly increased risk of bladder cancer was found among Asians in the homozygote comparison (Pro/Pro vs. Arg/Arg, OR=1.36 95%CI 1.05-1.75, P=0.790 for heterogeneity) and the dominant model (Arg/Pro plus Pro/Pro vs. Arg/Arg, OR=1.26 95%CI 1.05-1.52, P=0.564 for heterogeneity). In contrast, no evidence of an association between bladder cancer risk and P53 genotype was observed among Caucasian population in any genetic model. When stratifying for the stage of bladder, no statistical association were found (Pro/Pro vs. Arg/Arg, OR=0.45 95%CI 0.17-1.21; Pro/Arg vs. Arg/Arg, OR=0.60 95%CI 0.28-1.27; Dominant model, OR=0.56 95%CI 0.26-1.20; Recessive model, OR=0.62 95%CI 0.35-1.08) between P53 codon 72 polymorphism and bladder cancer in all comparisons.


CONCLUSIONS
Despite the limitations, the results of the present meta-analysis suggest that, in the P53 codon 72, Pro/Pro type and dominant mode might increase the susceptibility to bladder cancer in Asians; and there are no association between genotype distribution and the stage of bladder cancer.


Introduction
An estimated 386,300 new cases of urinary bladder cancer were diagnosed and 1,520,200 deaths in 2008 worldwide (Jemal et al., 2010).Urinary bladder cancer ranks ninth in worldwide cancer incidence.Risk factors for the development of bladder cancer could be classified into three types: chemical or environment exposure, genetic and molecular abnormalities, and chronic irritation (Williams et al., 2004;Bryan et al., 2005;Kaufman et al., 2009).However, the genetic factors determine the progression to bladder cancer remain to be investigated.The most common type of bladder cancer is transitional cell carcinoma (TCC), which accounts for 90%.Approximately 70% of newly detected cases are exophytic papillary tumors confined largely to the mucosa (Ta) (70%) or, less often, to the submucosa (T1) (30%) (Herr et al., 2001).10%-20% of those superficial tumors will progress to muscularies propria invasive bladder cancer.Bladder carcinogenesis and progression from non-muscle-invasive type to muscle-invasive type are complex, multistep and multifactor processes, in which genetic factors affect RESEARCH COMMUNICATION P53 Arg72Pro Polymorphism and Bladder Cancer Risk -Metaanalysis Evidence for a Link in Asians but not Caucasians Ting Xu 1& , Zi-Cheng Xu 1& , Qin Zou 1 , Bin Yu 1 , Xin-En Huang 2 * mostly.Tumor suppressor gene P53 is involved in the development and progression of bladder cancer.
The P53 gene located at 17p13, is a prototypical tumor suppressor gene encoding a 53 kDa protein (P53) with important functions in cell cycle control, apoptosis, and maintenance of DNA integrity (Sager et al., 1989;Levine et al., 1997;Xu et al., 2001).The function of P53 is to reduce the incidence of cancers by mediating apoptosis in cells.That has activated oncogenic pathways.DNA damage or genotoxic stress may case the induction of P53, leading to growth arrest or apoptosis (Meek et al., 2004).Although P53 contains several polymorphic sites, the codon 72 polymorphism located on exon 4 is the most common candidate gene.The polymorphism consists of a single base pair change of either arginine (Arg, CGC) or proline (Pro, CCC), which creates three distinct genotypes, including homozygous for arginine (Arg/Arg), homozygous for proline (Pro/Pro) and heterozygote (Pro/ Arg) (Klug et al., 2009).
Bulks of epidemiologic studies have addressed the influence of this polymorphism on cancer risk for most common cancer types, including bladder cancer.However, small sample sizes and deficiencies in study design might contribute to the conflicting results.To conduct a comprehensive evaluation of the potential association of the P53 codon 72 polymorphism (rs1042522) and bladder cancer susceptivity, we perform this systemic review and meta-analysis of candidate genetic association studies.

Literature search strategy
PUBMED, Embase databases were searched to retrieve papers linking P53 codon 72 polymorphism and bladder cancer risk available up to January 2012, using the following keywords: 'P53', 'Polymorphism', 'single nucleotide polymorphism', 'bladder cancer', 'bladder neoplasm' and 'bladder tumor' separately and combined.The research was restricted on human subjects only.We also hand-searched the reference lists for all retrieved studies and relevant review articles for additional data.We only considered studies published in English.

Inclusion and exclusion criteria
Studies using an analytic design (case-control, nested case-control, or cohort) and employing validated genotyping methods to examine the frequency of rs1042522 among bladder cancer patients and controls were eligible for inclusion.Studies should include sufficient genotype data for analysis.Case reports, letters, reviews and editorial articles were excluded.When there was overlapping study population, only the mostly published study was included.Family-based studies were not considered eligible owing to different design considerations.

Data Extraction
Using a standardized form, data from published studies were extracted independently by two reviewers (Xu T and Xu ZC) to populate files with the necessary information.The following information was extracted from each of the included articles: first author's surname, year of publication, country of origin, ethnicity, source of control and case groups, genotyping methods, total number of cases and controls, and detailed numbers of Arg/Arg, Arg/ Pro and Pro/Pro genotype respectively.Disagreement was resolved by consensus with a third reviewer (Yu B).

Statistic analysis and Publication bias
For our main analysis, the following contrasts for P53 codon 72 polymorphism were evaluated: codominant model (homozygote comparison Arg/Arg vs. Pro/Pro, heterozygote comparison Arg/Pro vs. Pro/Pro), dominant model (Arg/Pro plus Pro/Pro vs. Arg/Arg) and recessive model (Arg/Arg plus Arg/Pro vs. Pro/Pro).All associations were presented as odds ratios (OR) with their corresponding 95% confidence intervals (95%CI).Subgroup analyses were conducted on the basis of ethnic group, source of control, genotyping methods, and tumor stage.Between-study heterogeneity was tested using the Χ2-based Q-test.We separately used random-effects and fixed-effects models to analyze the data for accessing the stability of the results (Mantel et al., 1959;DerSimonian et al., 1986).We also tested whether genotype frequencies of controls were in Hardy-Weinberg equilibrium (HWE) using the chi-square test.
Publication bias was investigated by Begg's funnel plot, and funnel polt asymmetry was assessed by the Egger linear regression test (Egger et al., 1997); statistical significance was considered when the P value of the Egger test was < 0.05.

Study characteristics
According to the inclusion and exclusion criteria, 15 studies were evaluable in the meta-analysis (Wu et al., 1995;Biro et al., 2000;Chen et al., 2000;Toruner et al., 2001;Soulitzis et al., 2002;Kuroda et al., 2003;Mabrouk et al., 2003;Chung et al., 2008;Horikawa et al., 2008;Ye et al., 2008;Pandith et al., 2010;Srivastava et al., 2010;Lin et al., 2011;Santos et al., 2011;Zhang et al., 2011).The first one was published in 1995 (Wu et al., 1995) and the last in 2011 (Lin et al., 2011).Detailed study characteristics are presented in Table 1.A total of 15 studies investigated 2,250 bladder cancer cases and 3,057 controls were included in the analysis.All studies were case-control studies that evaluated the association between P53 codon 72 polymorphism and bladder cancer risk.Seven studies were conducted on Asian population, 1 was in African, and the remaining seven studies were conducted in Caucasians population.Restriction Fragment Length Polymorphism was performed in 9 publications, polymerase chain reaction (PCR) was performed in the remaining 6 studies.The distribution of genotypes in the controls of all studies was consistent with HWE except for three studies (Soulitzis et al., 2002;Ye et al., 2008;Pandith et al., 2010)
We assessed the source of heterogeneity and we found there were three studies (Chen et al., 2000;Soulitzis et al., 2002;Ye et al., 2008) whose controls deviated from Hardy-Weinberg equilibrium were the main origin of heterogeneity.After excluding the three investigations, the heterogeneity was effectively decreased and the crude ORs were still stable, all the comparisons were performed under both random-effects model and fixed-effects model; as shown in Table 2.

Publication bias
The publication bias of the meta-analysis of the association between P53 codon 72 polymorphism and bladder cancer risk was detected by Begg's test and Egger's test.All graphical funnel plots of the included studies appeared to be symmetrical.The Egger test also showed that there was non-significance in all evaluation of publication bias (P > 0.05).Information concerning the Egger publication bias test is given in figure.

Discussion
Many studies have investigated the association between P53 codon 72 polymorphism and bladder cancer risk in the last two decades, but the results were inconclusive.Soulitzis et al. (2002) found individuals harboring the Arg/Arg genotype have an increased risk (P < 0.00002; OR=4.69 95%CI 2.13-10.41) of developing bladder cancer in whites.But Zhang et al.' (2011) research data suggest that P53 codon 72 Arg/Arg genotype and Arg allele are associated with a lower risk (P=0.02,OR=0.53 95%CI 0.31-0.89) of bladder cancer in Asian population.
The polymorphism of TP53 codon 72 occurs in a proline-rich region that has been reported to play a vital role in the growth repression and apoptotic functions of P53 protein (Dumont et al., 2003).The polymorphic variants from Arg to Pro, differ in their capability of binding the transcriptional protein, activating transcription, and suppressing the transformation (Chang et al., 2002).Arg variant was reported to induce cell apoptosis and suppress transformation more efficiently than Pro variant do, which may be due to the ability of the Arg variant to localize in mitochondria that regulates the release of cytochrome C, which plays an important role in apoptosis (Dumont et al., 2003).However, in our meta-analysis based on 15 studies, there was no association between P53 codon 72  The discrepancy between the results of Asians and Caucasians might be due to differences of genetic backgrounds and the environment existed among different races, we further conducted subgroup analysis by ethnicity.We found that patients with bladder cancer had a significantly higher frequency of Pro/Pro, Pro/Pro plus Pro/Arg than none cancer patients among Asians.The hospital-based studies showed an increased risk in all genetic model, which may be caused by selection bias.Considering possible impact of P53 codon 72 polymorphism on the development and progression of bladder cancer, we extracted data concerning stage of bladder cancer which divided into two groups: superficial and invasive, and further performed meta-analysis.The results failed showing a significant association of P53 codon 72 polymorphisms with stage of bladder cancer, suggesting that P53 codon 72 polymorphism might not contribute to progression of developing bladder cancer.
However, there are several limitations in this meta-analysis.First, the controls were not uniformly defined; some controls were hospital-based.Hence, nondifferential misclassification bias is possible.Secondly, all published studies written in English.It is possible that some related published or unpublished studies that might meet the inclusion criteria were missed.Hence, some inevitable publication bias might exist in the results, though the funnel plots as well as Egger's linear regression tests indicated on remarkable publication biases in the meta-analyses.Third, in the subgroup analysis by ethnicity, the population of Caucasians comes from three continents involving North American, South American and Europe, which may cause the selection bias.It may be underpowered to explore the real association.Only one study reported on African (Horikawa et al., 2008), reflecting the current lack of epidemiologic studies in these populations.Furthermore, we were also unable to examine the interactions among gene-environment, lacking of the original data of the included studies limited our further evaluation of potential interactions, which may be an important component of the association between P53 codon 72 polymorphism and environment and bladder cancer risk.
In summary, despite the limitations, the results of the present meta-analysis suggest that, in the P53 codon 72, Pro/Pro type and dominant mode might increase the susceptibility to bladder cancer in Asians; and there are no association between genotype distribution and the stage of bladder cancer.

Figure 1 .
Figure 1.Forest Plots of Cancer Risk Associated with the P53 Codon 72 Polymorphisms in the Stratified Analyses by Ethnicities and Overall Cancer Risk.The plots of Pro/Pro vs. Arg/Arg were given (Pandith et al., P=0.0295; Ye et al.,  P=0.0001; Souitzis et al., P=0.0014).

Figure 3 .
Figure 3. Forest Plots of Cancer Risk Associated with the P53 Codon 72 Polymorphisms in the Stratified Analyses by Ethnicities and Overall Cancer Risk.The plots of dominant model were given

Table 2 . The Main Results of the Meta-analysis of p53 Arg72Pro Polymorphism and Bladder Carcinoma Risk
Dominant model, Pro/Pro+ Pro/Arg vs. Arg/ Arg; Recessive model, Arg/ Arg vs. Pro/Arg + Arg/ Arg; P*,value of Q-test for heterogeneity