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 particularly 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‐
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.
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).
Table 1
TP53 Arg72Pro and PIN3 Ins16bp genotypic and allelic frequencies. Association with familial and sporadic breast cancer risk.
Arg72Pro
| | | | | | | |
|
Arg/Arg
| 39 (53.4) | 256 (59.0) | Reference | 98 (56.0) | 124 (58.5) | Reference |
|
Arg/Pro
| 25 (34.2) | 142 (32.7) | 1.19 (0.68–2.08) | 61 (34.9) | 70 (33.0) | 1.26 (0.79–2.02) |
|
Pro/Pro
| 9 (12.3) | 36 (8.3) | 1.58 (0.68–3.67) | 16 (9.1) | 18 (8.5) | 1.35 (0.63–2.88) |
Alleles | | | | | | | |
|
Arg
| 103 (70.5) | 654 (75.3) | Reference | 257 (73.4) | 318 (75.0) | Reference |
|
Pro
| 43 (29.5) | 214 (24.7) | 1.28 (0.85–1.91) | 93 (26.6) | 106 (25.0) | 1.09 (0.78–1.52) |
PIN3 Ins16bp
| | | | | | | |
|
A1A1
| 46 (65.7) | 299 (68.0) | Reference | 122 (63.9) | 147 (68.1) | Reference |
|
A1A2
| 15 (21.4) | 130 (29.5) | 0.80 (0.43–1.49) | 56 (29.3) | 65 (30.1) | 1.07 (0.67–1.70) |
|
A2A2
| 9 (12.9) | 11 (2.5) | 4.40 (1.60–12.0) | 13 (6.8) | 4 (1.9) | 3.88 (1.18–12.8) |
Alleles | | | | | | | |
|
A1
| 107 (76.4) | 728 (82.7) | Reference | 300 (78.5) | 359 (83.1) | Reference |
|
A2
| 33 (23.6) | 152 (17.3) | 1.48 (0.94–2.31) | 82 (21.5) | 73 (16.9) | 1.34 (0.93–1.94) |
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 observed in A2A2 genotype frequencies comparing sporadic breast cancer cases and respective control group (p = 0.026). Our results showed that carriers of A2A2 genotype with no FH present an increased risk of breast cancer (OR = 3.88, 95% CI 1.18–12.8).
We investigated haplotype effects of the two polymorphisms studied in breast cancer risk (Table
2). Compared the common
TP53 Arg-A1 haplotype with the other expected haplotypes; we only observed statistically significant differences regarding
TP53 Arg-A2 haplotype between the familial breast cancer cases and respective control group (p = 0.028). Carriers of
TP53 Arg-A2 haplotype and presence of FH of breast cancer presented an increased risk of develop breast cancer (OR= 2.10; 95% CI 1.08–4.06).
Table 2
Expected haplotype frequencies between Arg72Pro and PIN3 Ins16bp polymorphisms. Association with familial and sporadic breast cancer risk.
Arg-A1
| 0.607 | 0.711 | Reference | 0.695 | 0.705 | Reference |
Arg-A2
| 0.091 | 0.041 | 2.10 (1.08–4.06) | 0.048 | 0.045 | 1.06 (0.53–2.12) |
Pro-A1
| 0.150 | 0.111 | 1.49 (0.86–2.58) | 0.098 | 0.119 | 0.80 (0.49–1.32) |
Pro-A2
| 0.151 | 0.137 | 1.27 (0.72–2.24) | 0.160 | 0.131 | 1.27 (0.83–1.95) |
We examined the relationship between age at onset and genotypes and found a positive correlation in the FH group. The mean age of FH patients group with A2A2 genotype was 33.43 (± 8.08) years, whereas the mean age of patients with A1A1 and A1A2 genotypes was 42.44 (± 12.14) and 44.80 (± 10.85) years, respectively (Kruskal Wallis test p = 0.056). Therefore, the carrier's status of A2A2 genotype was associated with an earlier age at onset cancer with respect to the patients with A1 genotypes. However, this difference was in the frontier of statistically significant, possibly because of the smaller size of the group (7 patients to Pro/Pro genotype). No association was observed relating age at onset and Arg72Pro polymorphism (Kruskal Wallis test p = 0.747).
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).
Discussion
Breast cancer is an heterogeneous disease, as sustained by wide variable morphological appearance, many risks factors and distinct gene expression profile [
2,
22]. Common genetic alterations (e.g. polymorphisms), with possible effects on function and/or protein expression, within genes involved in essential cellular pathways, such as carcinogen metabolism, DNA repair, cell cycle control and cell proliferation, could predispose individuals to cancer [
15,
23‐
25], including breast cancer [
15,
26‐
29].
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‐
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‐
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.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
The SC was the principal investigator; contribute to data and samples collection, developed study design, experimental plan and implementation, statistical analysis, and drafted the manuscript. DP contributed to data and samples collection and critical revision of the manuscript. DP, HR and JCT were critical to data and samples collection. RM contributed to study design, samples collection, data analysis and critical review of the manuscript. FS contributed to conception of the study hypotheses, study design and critical review of the manuscript. In addition, all the authors read and approved the final submitted manuscript.