Importance of TP53 codon 72 and intron 3 duplication 16bp polymorphisms in prediction of susceptibility on breast cancer

Background TP53 is one of major tumour suppressor genes being essential in preservation of genome integrity. Two very common polymorphisms have been demonstrated to contribute to cancer susceptibility and tumour behaviour. The purpose of this study was to evaluate the role of Arg72Pro and PIN3 Ins16bp polymorphisms in TP53 gene as genetic susceptibility and predictive markers to breast cancer. Methods We analysed DNA samples from 264 breast cancer patients and 440 controls, for TP53 Arg72Pro and PIN3 Ins16bp polymorphisms using PCR-RFLP. Results We observed that women with A2A2 genotype have increased risk for developing breast cancer, either in women with or without familial history (FH) of the disease (OR = 4.40, 95% CI 1.60–12.0; p = 0.004; OR = 3.88, 95% CI 1.18–12.8; p = 0.026, respectively). In haplotype analysis, statistically significant differences were found between TP53 Arg-A2 haplotype frequencies and familial breast cancer cases and the respective control group (OR = 2.10, 95% CI 1.08–4.06; p = 0.028). Furthermore, both TP53 polymorphisms are associated with higher incidence of lymph node metastases. Conclusion Our findings suggest TP53 PIN3 Ins16bp polymorphism as a real risk modifier in breast cancer disease, either in sporadic and familial breast cancer. Furthermore, both TP53 polymorphisms are associated with higher incidence of lymph node metastases.


Background
Breast cancer have been associated with well-established risk factors, such as high estrogen exposure, environmental factors (e. g. diet and ionizing radiation) and family history [1,2]. Family history of breast cancer is a particu-larly important high risk factor for this disease. Two genes were identified as the major susceptibility genes in high risk families, namely BRCA1 and BRCA2. However, these genes account for only a minority of the overall family risk of breast cancer [3]. Furthermore, approximately only 10% of all breast cancer cases exhibit a familial pattern of incidence [4,5]. In this way, the remaining familial and sporadic risk may be due to common low to moderate penetrance genetic variants, which are also referred as genetic polymorphisms. One strong candidate for genetic susceptibility factor to familial and/or sporadic breast cancer is the TP53 gene. This gene is frequently somatically mutated in breast cancer [6,7] and TP53 germline mutations are associated with increased risk for developing diverse malignancies, including 25-30% of hereditary breast cancer cases associated with Li-Fraumeni syndrome [8]. Furthermore, based on its pivotal role in DNA damage repair and its physical and functional interactions with BRCA1 and BRCA2 proteins [9,10], TP53 seems to be a strong candidate breast cancer predisposition.
The TP53 tumour suppressor gene, also designated the guardian of the genome, is essential in preservation of genome integrity. From the numerous biological functions of p53 protein, inhibition of cell cycle progression, DNA repair and apoptosis are the major cellular pathways where it is involved [6].
TP53 gene mutations are widely detected in breast cancer, being correlated with specific clinical phenotypes [11,12].
Predisposition to several human cancers has been associated with genetic polymorphisms, which may represent an important contribution to cancer susceptibility and tumour behaviour [13][14][15][16]. Several polymorphisms have been identified within TP53 gene, both in non-coding and coding regions [17]. One of the most well studied TP53 gene polymorphism is Arg72Pro, located in codon 72 on exon 4, leading to arginine-proline substitution, which in its turn results in a structural alteration of the protein [18]. Another common polymorphism is 16 base pair (bp) duplication in intron 3 of the TP53 gene (PIN3 Ins16bp).
In this case-control study, we hypothesize that the two common polymorphisms of TP53 gene play a role either apoptosis, cell cycle control efficiency, as well as DNA repair capacity, which ultimately may contribute to an increase of breast cancer susceptibility within familial and/or sporadic cases, as well as represent an additional tool for prognosis prediction.

Study Population
We analysed a total of 264 DNA breast cancer cases: 73 unrelated familial breast cancer cases were selected from the Oncology and Surgical Departments from S. João Hospital at Porto and Vigo Hospital, and 191 unrelated sporadic breast cancer cases were recruited from IPO-Porto (Oncology Portuguese Institute), during 1998-2003, from patients that were receiving treatment. All cases were histological confirmed at the Department of Pathology. Clinico-pathological parameters were obtained when possible from hospital clinical records. Familial case group presented a mean age of 42.07 years, with an age range of 24-77 years. The high-risk familial breast cancer group, also designated by us as family history (FH) breast cancer cases, included women with the follow features, based on the Breast Cancer Linkage Consortium criteria [19]: early onset (≤40 years) and/or bilaterality; or more than three cases of breast cancer in the family; or more than one case of ovarian cancer in the family; or more than two firstdegree relatives involved; or male breast cancer. These high-risk breast cancer cases are BRCA1/BRCA2 mutations non-carriers [20]. Sporadic cases group (with no presence of FH) presented a mean age of 53.41 years, with an age range of 41-88 years. Control women were randomly selected from blood banks in the same region during the same time period as the cases were collected. The selection criteria include no prior history of cancer, and controls were frequency matched to the cases by age (± 5 years). A total of 440 healthy women presenting a median age of 42.29 years and an age range of 21-85 years, were used as control group of familial breast cancer cases. From the above control group 216 healthy women were selected according to the age of diagnosis of the sporadic breast cancer patients group (higher than 40 years), with a median age of 53.05 and an age range of 41-85, being used as control group of sporadic breast cancer cases. All participants provided informed consent. Ethical approval was obtained by the specific institutions ethical committees.

Laboratory Methods
Genomic DNA was isolated from lymphocytes of peripherical blood using Puregene ® DNA Purification Kit (Gentra Systems, Minneapolis, USA). All the polymorphisms were assessed by PCR-RFLP technique. TP53 Arg72Pro polymorphism (rs1042522) was detected by amplifying genomic DNA with the forward primer 5'-GAA GAC CCA GGT CCA GAT GA-3' and the reverse primer 5'-CTG CCC TGG TAG GTT TTC TG-3'. The PCR amplification parameters were 32 cycles each of 30 sec at 94°C, 30 sec at 54°C, and 30 sec at 72°C. The 152 bp PCR product was digested with Bsh1236I (Fermentas, Ontario, Canada) at 37°C overnight. Digested products were separated by electrophoresis in a 3% agarose gel (Seakem ® LE Agarose, Rockland, USA) and visualized by ethidium bromide staining. Wild type alleles resulted in 50 and 102 bp fragments and the variant alleles resulted in 152 bp fragment following restriction enzyme digestion. TP53 PIN3 Ins16bp polymorphism (rs17878362) was detected by amplifying genomic DNA with the forward primer 5'-CTG AAA ACA ACG TTC TGG TA-3' and the reverse primer 5'-AAG GGG GAC TGT AGA TGG GTG-3'. The PCR amplification parameters were 32 cycles each of 30 sec at 94°C, 30 sec at 60°C, and 30 sec at 72°C. The PCR product was separated by electrophoresis in a 4% agarose gel (Seakem ® LE Agarose, Rockland, USA) and visualized by ethidium bromide staining. Wild type alleles, designated A1 allele (no duplication) resulted in 119 bp fragment and the variant alleles, designated A2 allele (with 16 bp duplication) resulted in 135 bp fragment.
To ensure quality control of all genotyping results, 10% of the samples was randomly selected and sequenced using an ABI automated sequencer. We obtained a 98% of concordance between the genotyping results acquire by the two methods.

Statistical Analysis
Analysis of data was performed using the computer software SPSS version 14.0 (SPSS Inc., Chicago, USA). Chisquare (χ2 test) analysis was used to compare categorical variables. Whenever necessary, the Fisher test was used when number of samples was equal or inferior to 5. A 5% level of significance was used in the analysis. The Odds Ratio (OR) and its 95% confidence interval (CI) were calculated to measure the association between TP53 polymorphic genotypes and breast cancer risk. Logistic regression analysis was used to calculate the adjusted OR and 95% CI for the influence of TP53 genotypes in the risk of breast cancer, adjusted for age and/or FH. Whenever appropriate, the observed number of each genotype in control groups were compared with that expected for a population in the Hardy-Weinberg Equilibrium by using a goodness of fit χ2 test. The frequencies of expected haplotypes were estimated by using the statistical methodologies implemented by HPlus software [21], which infers haplotypes based on expectation-maximization with a modified progressive ligation computational algorithm. The nonparametric test, Kruskal-Wallis test, was used to compare mean age of diagnosis between the different genotype polymorphisms.

Results
The distribution of the genotype frequencies in PIN3 Ins16bp polymorphisms among control group (p = 0.478) and in Arg72Pro and PIN3 Ins16bp among control subgroup (p = 0.082 and p = 0.294) is in agreement with those expected under Hardy-Weinberg equilibrium, excepted for Arg72Pro in the overall control group (p = 0.013).
Concerning TP53 Arg72Pro polymorphism in the familial breast cancer cases, frequencies of Arg72Arg, Arg72Pro and Pro72Pro were 53.4%, 34.2% and 12.3%, respectively. In sporadic breast cancer, 56.0%, 34.9% and 9.1% were homozygous for 72Arg allele, heterozygous and homozygous for 72Pro allele, respectively. No statistically significant associations were found between the TP53 Arg72Pro polymorphism and risk of familial and sporadic breast cancer risk ( Table 1).
Frequencies of TP53 PIN3 Ins16bp polymorphism genotypes were 65.7% to A1A1, 21.4% to A1A2 and 12.9% to A2A2, in familial breast cancer cases. Regarding sporadic breast cancer group, we observed 63.9%, 29.3% and 6.8% frequencies for homozygous for A1 allele, heterozygous and homozygous for A2 allele, respectively. We observed that A2A2 genotype carriers with positive FH were at a 4.40-fold (95% CI = 1.60-12.0; p = 0.004) increased risk of breast cancer compared with the respective control group. Moreover, statistically significant differences were We investigated haplotype effects of the two polymorphisms studied in breast cancer risk ( The analysis of the TP53 polymorphisms with respect to some clinical pathological showed a significant association of Pro or A2 genotypes with the presence of lymph node metastases (p = 0.009 and p < 0.001, respectively, adjusted for age and breast cancer family history, using logistic regression analysis).
The TP53 is one of the major tumour suppressor genes which carry out essential functions in preservation of genome integrity. Thus, when the cell is under stress, particularly stress which will involve DNA damage, p53 promotes growth arrest, allowing the cell to repair the DNA lesions. If the damage is excessively hazardous, then p53 will lead to cell apoptosis. Several genetic polymorphisms have been described in TP53 gene [18] and some of these variants seem to confer different functions among the p53 [30][31][32].
In the present study, we evaluated two separate TP53 polymorphisms, Arg72Pro and PIN3 Ins16bp, in two groups of breast cancer, familial and sporadic cases, as well as in matching control groups. The allelic frequencies of our control group for the different polymorphisms are in accordance with earlier reports from European populations [16,33]. We found a deviation from Hardy-Weinberg equilibrium in overall group of controls for Arg72Pro P53 polymorphism genotypes. Hardy-Weinberg equilibrium depends on a series of features about the tested population, including, for example the sample population size, random mating, no migration, no genetic drift and no selection taking place [34]. Thus, this deviation could be due to chance or violation of these assumptions, being the possibility of genotyping errors lower, since 10% our control sample genotyping were confirmed by direct sequence and its were similar with other European populations.
Concerning the codon 72 TP53 polymorphism (Arg72Pro), we did not find any association between this polymorphism and breast cancer. Our results are in agreement with other studies [33,35,36], however, the literature remains highly controversial regarding the role of this polymorphism in breast cancer risk [37][38][39][40][41][42]. One study showed that TP53 72Pro variant induces transcription activation more efficiently than TP53 72Arg variant [40]. On the other hand, other authors revealed that TP53 72Pro variant induce cell cycle arrest better than 72Arg [31]. Other studies have showed that TP53 72Arg variant is more efficient in inducing apoptosis [32,42]. Beside apoptosis and cell cycle control, p53 protein seems to be crucial in the regulation of the different DNA repair pathways [43]. A recent study demonstrated the influence of TP53 Arg72Pro in DNA repair capacity, showing that TP53 72Pro variant activates several TP53 dependent target genes involved in DNA repair and DNA damage repair much more efficiently than the 72Arg variant expressing cells [30]. These contradictory results could be explained by the differential effects of this alteration in p53 function. Several in vitro evidences have demonstrated that both TP53 Arg72Pro variants may selectively regulate specific cellular functions.
In TP53 PIN3 polymorphism, our findings suggest an association of A2A2 genotype and increased breast cancer risk among women with FH and sporadic breast cancer, suggesting that this polymorphism contributed to enhance susceptibility for breast cancer among Portuguese population, regardless of the presence of FH. Our results are supported by previously reported studies suggesting an association of PIN3 A2 genotypes with breast cancer risk [44]. Although, the biological effect of the TP53 PIN3 Ins16bp polymorphism is currently unclear, theoretically, this polymorphism could affect mRNA splicing, altering the coding regions and therefore being implicated in regulation of gene expression and DNA-protein interactions, resulting in a defective protein [45,46]. Until now, just a single study had show PIN3 A2 allele presents reduced mRNA stability [47].
The linkage disequilibrium between TP53 polymorphisms region could be an important factor affecting the incidence of cancer in general [48,49], and breast cancer, in particular [42,44,50]. Thus, haplotype analysis would be important to confirm the significance of this variant on breast cancer susceptibility. A statistical significant association was found between Arg-A2 haplotype and breast cancer susceptibility among women with FH of breast cancer. On the other hand, a recent study has found that Pro-A1 haplotype individuals present increased breast cancer risk, however, in BRCA2 mutation carriers [42]. Nevertheless, other reports have also demonstrated a positive association of Arg-A2 haplotype with cancer [44,48]. Moreover, functional studies have shown that, in a specific haplotype combination, A2 allele is associated with decreased apoptotic and DNA repair capacity [33,48].
Our findings suggest the Pro/Pro and A2A2 TP53 genotypes as predictor factors for the presence of lymph node metastases, being in agreement with previously functional studies in the biological consequences of these variations in P53 protein functions [40,47].
The natural history of breast cancer can be influenced by several factors. We hypothesize that under the influence of TP53 genetic polymorphisms, chronic exposure to higher levels of several endogenous (e.g. estrogens) and exogenous breast carcinogens resulting in consequent higher accumulation of DNA damage during an individual's lifetime, may alter the age at onset of disease. Moreover, it has been suggested that TP53 polymorphisms are associated to familial breast cancer by the age of 50 years [33]. Our results are consistent with this hypothesis, since TP53 PIN3 Ins16bp polymorphism seems to influence directly the age to onset of familial breast cancer.

Conclusion
In conclusion, our findings suggest TP53 PIN3 Ins16bp polymorphism as a real risk modifier in breast cancer disease, either in sporadic and familial breast cancer. Subsequently, these results will be crucial in the characterization of the genetic breast cancer susceptibility profile, within familial breast cancer cases non-carriers of BRCA1/BRCA2 mutations. Furthermore, our findings suggest TP53 Arg72Pro and PIN3 Ins16bp polymorphisms as predictive factors of presence of lymph node metastases.