TP53 p.Arg72Pro polymorphism and Breast Cancer Risk: A meta-analysis of case-control studies

Background :The effect of the p.Arg72Pro variant of the P53 gene on the risk of developmentof breast cancer remains variable in populations. However, the use of strategiessuchas pooling age-matched controls with disease cases may provide a solid meta-analysis. Our goal was to perform a meta-analysis in order to assessthe association of p.Arg72Provariant of P53 gene with breast cancer risk. Methods : Databases such as PubMed, Genetics Medical Literature, Harvard University Library, Web of Science and Genesis Library were used to search articles. Age-matched case-control studies on breast cancer that have evaluated the genotype frequencies of the p.Arg72Pro of P53 gene were selected. The fixed and random effects (Mantel-Haenszel) were calculated using pooled odds ratio of 95% CI to determine the risk of disease. Inconsistency was calculated to determine heterogeneity among the studies. The publication bias was estimated using the funnel plot. Results : Twenty-one publications with cases age-matched controls including7841disease cases and 8876controls were evaluated in this meta-analysis. Overall, our results suggested that p.Arg72ProP53 was associated with a risk for breast cancer for the dominant model (OR= 1.09, 95% CI = 1.02-1.16; P= 0.01) and the additive model (OR= 1.09, 95% CI = 1.01-1.17; P= 0.03), but not in the recessive model (OR = 1.07, 95% CI = 0.97-1.16; P= 0.19). According to the ethnic group, allele Pro has been associated with breast cancer risk in Europeans for the dominant and additive models. Conclusions : This meta-analysis found a significant association between p.Arg72Pro in the P53 gene and the risk of breast cancer. Individuals carrying at least one Pro allele of the P53 gene are more likely to have breast cancer with dominant and additive models than individualsharboringthe Arg allele. meta-analyzes, inclusion not robust the case with age-matched control studies and the non-deviation in HWE of the distribution of p.Arg72Pro in analyzes. Several studies (cases and controls, and case with age-matched controls) between p.Arg72Pro of P53 gene and the risk of improved the power of the meta-analysis of this genetic variant.

only age-matched case-control studies, to qualitatively assess the effect of p.Arg72Pro on the risk of breast cancer.

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", "p.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 p.Arg72Pro of the P53 gene and risk of breast cancer, (2) full manuscript available, (3) case-control study with age-matched, (4) distribution of genotype respecting Hardy-Weinberg equilibrium (HWE) in controls, (5) availability of the three genotypic frequencies (Arg/Arg, Arg/Pro and Pro/Pro) in the case and control groups. (6) Three investigators independently evaluated each study to determine eligibility.

Data extraction
The data were collected by an investigator and verified by a second investigator to reach consensus on all points. First author, year of publication, country, ethnicity of study population, sample size, agematched, distribution of genotype and alleles, as well as the recalculation of HWE in controls were extracted from the eligible studies. A third reviewer made a contradictory assessment to reconcile the assumptions. The data of controls evaluated with p.Arg72Pro variant were included in this metaanalysis.

Statistical analysis
Chi 2 analysis with a significance level of P < 0.05 was used to evaluate whether p.Arg72Pro polymorphism distribution of the P53 gene in controls fits Hardy-Weinberg equilibrium (HWE). The association between the p.Arg72Pro and the risk of breast cancer was evaluated by the Odd ratio (OR)of 95% CI. We evaluated the association strength of Arg72Pro polymorphism of P53 gene was made with the genetic models: dominant (Pro/Pro + Arg/Pro vs. Arg/Arg), recessive (Pro/Pro vs.Arg/Arg+Arg/Pro)and additive (Pro vs. Arg). The hypothesis of heterogeneity among the studies was assessed by I 2 statistical test [18][19]. If I 2 > 50% (presence of heterogeneity), the random effects model was used to calculate the overall OR, otherwise (lack of heterogeneity), the fixed effects method has been used. We also have examined the funnel plot to determine publication bias [20]. All statistical analyses were performed with Review Manager Software version 5.1.

Results
In the light of our results, 81case-control studies from the literature search ( Fig. 1 Table 1. Genotype distribution of TP53 p.Arg72Pro polymorphism in breast cancer cases and agematched controls in studies included. The results of combined analyses for p.Arg72Pro TP53 gene were showed in Table 2. Tableau 2. Distribution of p.Arg72Pro TP53 gene according to the genetic models Overall, after elimination of studies deviating from Hardy-Weinberg equilibrium in controls and no evidence of heterogeneity (I 2 > 50%) was found with the dataset analyzed in the recessive and dominant models. However, a modest heterogeneity was observed between the p.Arg72Pro variant and the risk of breast cancer in the combined analyses and Asians for the additive model (Table 2). 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. 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. After the exclusion of studies [39][40][41][42], no study has shown a significant influence of the pooled OR effect in each of the different genetic models ( Table 2).
The publication bias has been evaluated using the funnel plot. After excluding studies that deviated from the Hardy-Weinberg equilibrium in controls and the studies influencing the Odd ratio values, no significant publication bias was found in dominant, recessive and additive models (Figure2).

Discussion
Breast cancer is a multifactorial disease and its occurrence depends on the synergistic action of clinical, biological and environment factors and mechanisms [43][44]. In addition to these risk factors, the role of specific genes in the pathology of breast cancer is increasingly evident.
The protein and the TP53 gene have been widely studied for their associations with cancers, especially breast cancer, because they play a key role in DNA repair, cell cycle control and apoptosis [45]. 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 [46][47]. Several functional genetic variants p.Arg72Pro, p.Pro47Ser, 16-bp-Insertion allele) have been described and speculated to be associated with the risk of breast cancer incidence [13,[46][47][48][49]. Although numerous epidemiological studies have been conducted to assess the role of SNP TP53 in the risk of breast cancer in different populations. However, these results are controversial. The meta-analysis can be an adequate tool to detect the effect of a gene in diseases with a great power of confidence. Our meta-analysis evaluated the association between the variant p.Arg72Pro of the tumor suppressor P53 gene and breast cancer with eligibility criteria of case-control studies that had age-matched controls in HWE. In this metaanalysis, we found that the Pro allele of p53 gene was associated with risk of breast cancer risk for Europeans and Africans when compared to Arg allele. This finding is consistent in part with a previous meta-analysis which investigated breast cancer risk in 41 cases unmatched control studies [50], but discordant with the finding reported by Hou et al 2013 [51], Zhuo et al [52] and Hao et al [53]. The difference between these results can be explained by the presence of heterogeneity between studies, and the mixture studies with age-matched and/or unmatched controls in their analysis. This heterogeneity may be due to the difference in ethnicity. The effects of ethnicity can be explained by several factors including allelic heterogeneity between populations, ie the same locus, but different causal variants can influence the risk of cancer from one population to another or gene-gene and gene-environment interaction and the variation of linkage disequilibrium among population [54,55,56]. However, in the previous meta-analyzes, the inclusion criteria of studies were not robust enough, the case with age-matched control studies and the non-deviation in HWE of the distribution of p.Arg72Pro in controls were not taken into account in the analyzes. Several studies (cases and controls, and case with age-matched controls) between p.Arg72Pro of P53 gene and the risk of breast cancer have been reported, which improved the power of the meta-analysis of this genetic variant.
The analysis of subtypes studies based on the case and age matched controls and ethnicity being possible now. So, to have a more precise estimate of genetic associations, we have updated this meta-analysis. In addition, the meta-analyzes of Goncalves et al. 2013 [50], He et al. 2011 [57] and Ma et al. 2011 [58] showed that the Arg allele of the P53 gene was not associated with the risk of breast cancer, which is consistent with our findings. The literature is composed of contradictory conclusions regarding the association of Arg72Pro P53 gene with breast cancer risk, but most of the previous meta-analyses focused on the presence or absence of the wild-type (Arg) allele in these genetic models: dominant (Arg/Arg+Pro/Arg vs. Pro/Pro), recessive (Arg/Arg vs. Arg/Pro+Pro/Pro) and additive (Arg vs. Pro) [57][58]. However, we have found some bias in certain studies regarding the criteria for inclusion of scientific articles, which may have influenced the interpretation of these metaanalyzes. This bias existed in mostly studies whose distribution of Arg/Arg, Arg/Pro and Pro/Pro genotypes in controls was not in HWE [59][60][61][62][63][64][65][66][67][68][69][70][71][72].We also performed an in-depth sensitivity analysis by removing each single study from the pooled data and the results showed that there was no influence of the individual data on the overall results. In addition, we also calculated the overall combined OR on the additive model (Pro vs Arg) with and without the four studies which influenced the values of the Odds ratio [39][40][41][42], and in both cases, we found that the Pro allele was associated with a high risk of developing breast cancer. The major advantage of this meta-analysis was the inclusion of a large number of samples including very selective criteria. in order to identify a statistically significant association in one of the genetic models. However, several limitations need to be highlighted. Case age-matched control studies were rare in some ethnic groups, only one African study met the inclusion criteria and two studies in Americans.

Conclusions
In the light of this meta-analysis, individuals carrying at least one Pro allele of the P53 gene are more likely to have breast cancer with dominant and additive models than individuals carrying the Arg wild-

Consent for publication
Not applicable.

Availability of data and materials
The dataset analyzed for this study is available from the table 1.

Competing interests
The authors declare that they have no competing interests.

Funding
In prelude to the Genetic and Epigenetic aspects of Breast cancer in Mali project, financially supported by HBNU Global Health Fellowship programs we conducted this work to justify gene selection.   Figure 1 Flow diagram of the studies evaluated for meta-analysis