Arg72Pro polymorphism in P53 gene and Breast Cancer Risk Population: a meta-analysis of case-control studies matched

Background: The effect of the Arg72Pro variant of the P53 gene on the risk of developing breast cancer remains variable in populations. However, using strategies like grouping age-matched controls with disease cases may provide a strong meta-analysis. Our goal was to perform a meta-analysis in order to study the association of Arg72Pro variant of P53 gene and breast cancer. Methods: Databases such as PubMed, Genetics Medical Literature, Harvard University Library, Web of Science and Genesis Library were used to search articles. Age-matched control studies on breast cancer that evaluated the genotype frequencies of the Arg72Pro P53 gene were selected. Fixed and random effects (Mantel-Haenszel) were calculated using pooled odds ratio of 95% CI to determine the disease risk. Hardy-Weinberg equilibrium test was used to measure deviation in the distribution of genotypes in controls. Inconsistency was calculated to determine heterogeneity among the studies. Estimated publication bias was performed through the funnel plot and Egger’s test. Results: Nine publications with controls age-matched cases including 4684 disease cases and 4636 controls were evaluated in this meta-analysis, all were in the Caucasian population. Our results suggested that Arg72Pro P53 was associated with a risk for breast cancer in the dominant model (Pro/Pro+Arg/Pro vs. Arg/Arg: OR= 1.16, 95% CI = 1.04-1.31) and the additive model (Pro vs. Arg: OR= 1.13, 95% CI = 1.03-1.24, P= 0.007), but not in the recessive model (Pro/Pro vs. Arg/Arg + Arg/Pro, OR = 1.18, 95% CI = 0.96-1.44; P= 0.12). Conclusions: This meta-analysis found significant association between the Arg72Pro polymorphism in the P53 gene and the breast cancer risk. Individuals carrying at least one Pro allele of the P53 gene are more likely to have breast cancer with dominant and additive

substitution of Arginine by Proline at position 72. Arg72Pro variant is located in exon 4 and has been shown to be associated with many pathologies including cancer [10][11]. Although many association studies on candidate genes have investigated the relationship between the Arg72Pro variant of the P53 gene and the risk of breast cancer, the reports from these studies remain contradictory. Some studies have shown that Arg72Pro gene is associated with the risk of breast cancer, while others found no associations. The studies carried out by Menzel et al. 2004 [12] and Akkiprik et al. 2009 [13] have shown a link between Arg72Pro and breast cancer risk in the Caucasian populations. However, another age-unmatched casecontrol study in the similar population concluded that Arg72Pro P53 was not associated with the risk of breast cancer [14]. This inconsistency in the relationship between Arg72Pro P53 gene and breast cancer risk may be explained by a very high heterogeneity in the frequency of mutations. This heterogeneity is likely related to the geographical origin of patients, the ethnicity [15][16][17] and the age-unmatched controls with patients' group of the same population. In view of all these observations, the present meta-analysis will include only studies with age-matched controls with patients, to qualitatively assess the effect of Arg72Pro on the risk of breast cancer in the Caucasian population.

Results
In the light of our results, 87 case-control studies from the literature search ( Fig. 1) that investigated the association of Arg72Pro of P53 gene in the context of breast cancer were included, of which only 26 studies had a genotype distribution of control population that met Hardy-Weinberg equilibrium, and 9 out of the 26 studies have age-matched controls, all of which were from Caucasian populations (Table 1). This analysis was very selective with a total of seventy-eight studies that did not meet inclusion criteria of this meta-analysis. Genotype distribution of the control population that met Hardy-Weinberg equilibrium was a minimum requirement for studies to be included.

Test for Heterogeneity
After elimination of studies deviating from Hardy-Weinberg equilibrium in controls, no evidence of heterogeneity (I 2 > 50%) was found with the dataset analyzed in the different genetic models. According to the genetic models, the fixed effects models (p < 0.05 for Q test) were used for the interpretation of the pooled OR in this metaanalysis ( Table 2) (Supplementary material). In addition, we compared the pooled OR of the fixed and random effects, and found no statistically significant difference between the two effects, which supports strongly the consistency of the present study's data.

Analysis's Influence
To maintain the stability of the meta-analysis after the non-inclusion of deviant studies of HWE and sensitivity analysis, we evaluated the influence of each study on pooled OR. No study has shown a significant influence of the pooled OR effect in each of the different genetic models (Table 2).

Publication Bias
The publication bias has been evaluated using the funnel plot. After excluding studies that disagree with the Hardy-Weinberg equilibrium in controls and the sensitivity analysis, no significant publication bias was found in dominant, recessive and additive models (Fig. 2).

Discussions
Breast cancer is a multifactorial disease and its occurrence depends on the synergistic action of clinical, biological and environment factors and mechanisms [24][25]. In addition to these risk factors, the role of specific genes in the pathology of breast cancer is increasingly evident. The P53 gene encodes a transcription factor that binds to DNA and promotes the expression of genes that would repair cellular damage. Therefore, P53 is a tumor suppressor that sounds the alarm when DNA damage prevents the cell from turning into a cancer cell, or even inducing cell death. In the presence of a mutation, P53 gene can no longer repair the damaged DNA, which will lead to appearance of the malignant cells responsible for tumorigenesis [26][27]. In the recent decade, many studies have been conducted to assess the correlation between the polymorphism of the Arg72Pro P53 gene and the  [34] showed that the Arg allele of the P53 gene was not associated with the risk of breast cancer, which is consistent with our findings.

Literature search
The Pubmed Genetics Medical Literature Database, the Harvard University Library, and the Web of Science and Genesis Library were used to identify available articles published in English. The keywords "P53", "Arg72Pro" and "polymorphism" or ''mutation'' or ''gene'' and "breast cancer" cited in the genetic association studies were used to detect and select scientific manuscripts in these databases. We also reviewed references cited in these studies to identify additional articles that were not identified by our research in the databases.

Inclusion criteria
The inclusion criteria were: (1) published case-control studies as an original article to evaluate the association between Arg72Pro polymorphism of the P53 gene and risk of breast cancer, (2) full manuscript available, (3) case-control study with age-  Figure 1 Flow diagram of the studies evaluated for meta-analysis  Additional file 1.docx