Association of TP53 PIN3 polymorphism with breast cancer in Moroccan population

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RESEARCH ARTICLE

Association of TP53 PIN3 polymorphism with breast cancer in Moroccan population

Chaymaa Marouf&Amal Tazzite&Brehima Diakité&

Hassan Jouhadi_&Abdellatif Benider_&Sellama Nadifi

Received: 20 May 2014 / Accepted: 26 August 2014

#International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract

TP53 is a tumor suppressor gene involved in cell cycle progression control, DNA damage repair, genomic sta- bility, and apoptosis. Some polymorphisms in this gene have been associated with the development of a number of cancers including breast carcinoma. PIN3 Ins16bp polymorphism has been widely studied in different populations for an association with breast cancer risk. In most case-control studies, a dupli- cated allele has been more frequent in cases rather than con- trols but there are also inconsistent results. The present study aimed to assess the association of PIN3 Ins16bp polymor- phism of p53 with breast cancer risk in Moroccan population.

This case-control study was performed on 105 female patients with confirmed breast cancer and 114 healthy controls. The genotype frequency was 69.5 % (A1A1), 26.7 % (A1A2), and 3.8 % (A2A2) in patients and 68.4 % (A1A1), 24.6 % (A1A2), and 7 % (A2A2) in controls. No statistically signif- icant association was observed between PIN3 Ins16bp poly- morphism and breast cancer risk with odds ratio of 1.07 (confidence interval (CI)=0.58–1.97,

p=0.83) for the hetero-

zygous A1A2 and 0.53 (CI=0.15–1.85,

p

=0.32) for mutated homozygous A2A2.According to our preliminary genetic analysis, PIN3 Ins16pb polymorphism could not be assessed as a marker of risk factor for predisposition to breast cancer in Moroccan population. However, a high frequency of A2 allele

(19.3 %) in our population suggested that PIN3 Ins16pb polymorphism may be a valuable marker for study in other cancers with larger groups.

Keywords

TP53 . PIN3 Ins16bp polymorphism . Breast carcinoma . Morocco

Introduction

Breast cancer (BC) is the most prevalent malignancy and primary cause of death in women worldwide, both in the developed and developing countries. Genetic linkage studies and positional cloning have identified two major genes associated to hereditary breast cancer susceptibility.

BRCA1

and

BRCA2

are two major tumor suppressor genes associated with hereditary breast and ovarian carcinoma.

Mutations in these genes explain only a part of the famil- ial aggregation of breast cancer. Indeed, other susceptibil- ity genes have been involved in hereditary breast cancer not associated to

BRCA1

and

BRCA2

mutations [1].

Among these genes,

TP53

is a tumor suppressor gene located on chromosome 17p13.1 encoding a nuclear phos- phoprotein (p53) which acts as transcription factor in- volved in cell cycle progression control, DNA damage repair, genomic stability, senescence, and apoptosis [2].

In addition, several posttranslational modifications modu- late

p53

activity especially phosphorylation and acetyla- tion. The

p53

function is lost in various human cancers either through mutation of the gene encoding the protein or through loss of cell signaling upstream or downstream of

p53

[3–5].

TP53

has been described to be a high penetrance breast cancer susceptibility gene [6] and the most frequently mutated gene in different human cancers [7].

TP53

is important as a clinical marker, because its status in a tumor could

Chaymaa Marouf and Amal Tazzite contributed equally to this work.

C. Marouf

:

A. Tazzite

:

B. Diakité

:

S. Nadifi

Genetics and Molecular Pathology Laboratory, Medical School of Casablanca, University Hassan II, Casablanca, Morocco H. Jouhadi

:

A. Benider

Mohammed VI Center for Cancer Treatment, Ibn Rochd University Hospital, Casablanca, Morocco

C. Marouf (*)

Klausenpfad 18, 69121 Heidelberg, Germany e-mail: maroufchaymaa@gmail.com DOI 10.1007/s13277-014-2556-y

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significantly affect the prognosis of patients and response to treatment [8,

9]. In fact, mutations in this gene have been

associated with an increased risk for developing diverse ma- lignancies including 40 % of breast cancer cases [10]. Several polymorphisms have been identified in coding and noncoding regions of

p53

that some of them have been associated with increased risk of cancer [11]. Three p53 well studied function- al polymorphisms include Arg/Pro polymorphism in exon 4, 16bp duplication in intron 3 (PIN3), and intron 6 G13964C alteration [12], although two studies in India showed no link between polymorphisms and breast cancer [13,

14]. Further-

more, some intronic polymorphisms such as the 16 base pair (bp) duplication in intron 3 have been described to have a functional significance [15,

16]. The 16bp duplication in

intron 3 of the

TP53

gene (PIN3 Ins16bp) has been reported to affect mRNA splicing and decreased apoptotic indices and DNA repair capacity in lymphoblastoid cell lines [16,

17]. It is

therefore involved in regulation of gene expression and DNA protein interactions resulting in a defective protein. Thereby, PIN3 Ins16bp has been correlated with an increased risk of various cancers, including ovarian [18], lung [17], colon [16], breast [19], esophageal, and gastric cancer [20]. Recently, several meta-analyses have highlighted the association be- tween PIN3 and increased risk of cancer predisposition [21

–24]. He et al. (2011) indicated that PIN3 is likely an

important genetic marker, contributing to susceptibility of breast cancer. However, Sagne et al. (2013) and He et al.

(2011) showed that PIN3-related cancer risk is dependent on ethnicity and geographical origin [22,

23].

In Morocco, breast cancer seems to be a major public health problem. It has become the most common cancer, accounting for 36.5 % of all female cancers and with a mortality rate of 19.7 % [25]. The highest cancer incidence rate recorded among women at The Cancer Registry of the Grand Casablanca and Rabat is breast cancer (ASR per 100,000 women for BC in the years 2005–08) were 36.4 and 39.9 %, respectively [26,

27]. Although this incidence

appears higher compared to the other Maghreb countries [28–30], it remains low compared to developed countries [31–33]. In the greater area of Casablanca, 57 % of the cases were under 50 years of age [26].

Moreover, Morocco is a country of northwestern Africa composed predominantly of Berber and Arab ethnic groups.

The contribution of these ethnic groups to the genetic diversity of Moroccan population is evident. Therefore, it is crucial that we improve our understanding, both on the subject of genetic susceptibilities and environmental risk factors. To our knowl- edge, the association between breast cancer risk and the PIN3 Ins16bp polymorphism of

TP53

gene is not yet clear in Morocco. Thereby, we conducted this case-control study in a sample of Moroccan women in order to better understand the implication of this polymorphism in breast cancer develop- ment in our population.

Materials and methods

Subjects

We performed a case-control study involving 105 unrelated subjects, with clinically and histopathologically confirmed primary breast cancer, recruited from Mohammed VI center for Cancer treatment of Ibn Rochd University Hospital of Casablanca during 2009–2010. The control group included a total of 114 healthy women with no personal history of cancer diseases selected from DNA bank volunteers of the Genetics and Molecular Pathology Laboratory. Clinico-pathological parameters including age at diagnosis, menopause status, histology type, tumor size, Scarff-Bloom-Richardson (SBR) grade, lymph nodes status, and hormone receptors status were obtained from patients

’

medical records.

The study was approved by our institutional ethics com- mittee and written informed consent was obtained from both cases and controls.

DNA isolation and PCR amplification

Genomic DNA was extracted from peripheral blood leuko- cytes using the salting out procedure [34]. Genomic DNA was dissolved in TE (10 mM Tris-HCL and 0.1 mM EDTA, pH 8.0). Spectrophotometry was used to quantify DNA using the Nanovue TM Plus spectrophotometer.

Polymerase chain reaction (PCR) was carried out to ampli- fy

TP53

gene in a final volume of 25

μl containing 10×

reaction buffer, 25 mM MgCl2, 5 mM dNTPs, 5

μM primers,

500 U Taq DNA polymerase, and 100 ng genomic DNA. The PCR amplification parameters were 35 cycles of 30 s of denaturing at 94 °C, 30 s of annealing at 59 °C, and 1 min of extension at 72 °C.

The PIN3 Ins16bp polymorphism (rs17878362) was de- tected 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 product was separated by electrophoresis in a 4.5 % agarose gel and visualized by ethidium bromide staining. A1 wild-type alleles (NoIns) resulted in 119bp fragment, and A2 mutated alleles (Ins) resulted in 135bp fragment. Both frag- ments were observed for heterozygous genotype (NoIns/Ins).

Statistical analysis

Statistical analysis was performed using SPSS 19.0 software.

To measure the distribution of PIN3 Ins16bp polymorphism,

Hardy Weinberg equilibrium was tested by comparing ob-

served and expected genotype frequencies in both cases and

controls using

χ2 test. Odds ratio with a confidence interval

(CI) of 95 % were used to assess the strength of the association

between the studied polymorphism and breast cancer risk. The

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χ2 test or Fisher test was used to evaluate the correlation

between the studied polymorphism and the clinico- pathological parameters. The

P

value (<0.05) was considered statistically significant.

Results

We analyzed 105 breast cancer patients and 114 healthy Moroccan subjects. The mean age was 41 ±2 and 36.5 ± 2.81 years for patients and healthy controls, respectively. No correlation was observed between PIN3 Ins16bp polymor- phism and clinico-pathological parameters of breast cancer cases either in the age at diagnosis, menopause status, histol- ogy type, tumor size, SBR grade, lymph nodes status, and hormone receptors status (Table

1).

The distribution of PIN3 genotype frequencies is in agree- ment with those expected under Hardy Weinberg equilibrium

among cases and controls (χ2=0.40,

p=0.82; χ2=5.26,p

= 0.07). Frequencies of

TP53

PIN3 Ins16bp polymorphism genotypes were 69.5 % for wild-type homozygous (A1A1), 26.7 % for heterozygous (A1A2), and 3.8 % for mutated homozygous (A2A2) in breast cancer cases. In healthy con- trols, we observed frequencies of 68.4, 24.6, and 7 % for wild- type homozygous (A1A1), heterozygous (A1A2), and mutat- ed homozygous (A2A2), respectively (Table

2).

No statistically significant association was observed be- tween PIN3 Ins16bp polymorphism and breast cancer risk with odds ratio of 1.07 (CI=0.58–1.97,

p

=0.83) for the het- erozygous A1A2 and 0.53 (CI=0.15–1.85,

p

=0.32) for the mutated homozygous A2A2 (Table

2).

Table

2

also shows that the allelic frequencies of PIN3 Ins16pb polymorphism did not present significant difference between breast cancer cases and controls (odds ratio (OR)=

0.87; 95 % CI=0.53–1.41;

p=0.56). A1 wild-type and A2

mutated allele frequencies were respectively 82.9 and 17.1 % in cases, 80.7 and 19.3 % in controls.

Table 1 Frequencies of PIN3 Ins16bp polymorphism in patients with breast cancer according to clinico-pathological features

A1A1wild-type genotype,A1A2 heterozygous,A2A2mutated homozygous,Ntotal number,–ze- ro,IDCinvasive ductal carcinoma,ILCinvasive lobular carcinoma,ERestrogen receptors,PR progesterone receptors

Variable N PIN3 Ins16bp polymorphism Pvalue

A1A1 A1A2 A2A2

Age group 105 0.70

<40 years 59 43 (72.9 %) 14 (23.7 %) 2 (3.4 %)

≥40 years 46 30 (65.2 %) 14 (30.4 %) 2 (4.3 %)

Menopausal status 105 0.40

Premenopausal 76 51 (67.1 %) 21 (27.6 %) 4 (5.3 %)

Postmenopausal 29 22 (75.9 %) 7 (24.1 %) –

Histology 105 0.82

IDC 98 67 (68.4 %) 27 (27.6 %) 4 (4.1 %)

ILC 4 3 (75.0 %) 1 (25.0 %) –

Others 3 3 (100.0 %) – –

Tumor size 105 0.31

<2 cm 14 9 (64.3 %) 4 (28.6 %) 1 (7.1 %)

>2 cm 51 40 (78.4 %) 9 (17.6 %) 2 (3.9 %)

>5 cm 20 14 (70.0 %) 6 (30.0 %) –

Tumor of any size with extension 20 10 (50.0 %) 9 (45.0 %) 1 (5.0 %)

SBR 105 0.37

I 6 4 (66.7 %) 1 (16.7 %) 1 (16.7 %)

II 71 50 (70.4 %) 18 (25.4 %) 3 (4.2 %)

III 28 19 (67.9 %) 9 (32.1 %) –

Node involvement 105 0.46

N− 43 27 (62.8 %) 4 (22.6 %) 2 (3.2 %)

N+ 62 46 (74.2 %) 14 (32.6 %) 2 (4.7 %)

PR 105 0.16

PR− 48 29 (60.4 %) 17 (35.4 %) 2 (4.2 %)

PR+ 57 44 (77.2 %) 11 (19.3 %) 2 (3.5 %)

ER 105 0.78

ER− 41 27 (65.9 %) 12 (29.3 %) 2 (4.9 %)

ER+ 64 46 (71.9 %) 16 (25.0 %) 2 (3.1 %)

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Discussion

Mutations and polymorphisms in

TP53

gene have been wide- ly associated with the development of a number of cancers [7,

35]. Indeed, the DNA damage causes accumulation of the p53

protein and cell cycle arrest in G1 phase, awaiting repair or induction of apoptosis, thereby protecting the organism from potentially carcinogenic cells proliferation [36,

37].

Thus, variants located in the promoter regions or intronic regions of this gene could alter gene expression. The majority of reported mutations at the tumor suppressor gene

p53

are missense mutations [38–41]. However, other common poly- morphisms in the noncoding region of the

TP53

gene that may also play an important role in regulating gene expression have been described.

The PIN3 Ins16bp polymorphism has been frequently studied for an association with breast cancer risk in different populations. However, studies have yielded inconsistent re- sults. While a number of research have revealed a positive association between increased breast cancer risk and PIN3 Ins16bp polymorphism [42–45,

24], others have found no

significant differences [46–53, 19]. These conflicting results about the relationship between PIN3 Ins16bp polymorphism and breast cancer risk could be related to some factors such as sample size, ethnicity, genetic background, and environmental factors.

In the present study, we investigated the frequency of intron3 16bp duplication polymorphism of the

TP53

gene in 105 female patients with confirmed breast cancer and 114 healthy controls. Our results did not reveal significant associ- ation between this polymorphism and breast cancer risk in the present sample. This result is in agreement with some studies which showed that PIN3 Ins16bp was not associated with increased breast cancer risk [46–55].

Nevertheless, there are other reports showing an important role of this polymorphism in breast cancer. In fact, some authors found significantly different genotypic frequencies of this polymorphism in breast cancer cases compared to controls [42]. On the other side, homozygous genotype of minor allele of the PIN3 Ins16bp polymorphism was associ- ated with sporadic breast cancer (OR=2.15, 95 %; CI=1.80

–

2.56;

p=0.006) in the Croatian population [43]. Costa et al.

[19] showed PIN3 Ins16bp polymorphism as a real risk mod- ifier in breast cancer disease either in sporadic and familial breast cancer. Similarly, PIN3 Ins16bp polymorphism has been found to be a genetically predisposing factor for breast cancer development in Iranian women especially in patients under the age of 50 years [44]. In addition, a recent meta- analysis [24] has suggested that the 16bp duplication poly- morphism of

p53

may contribute to susceptibility to breast cancer. However, they indicated that additional well-designed large studies are required to validate this result.

Furthermore, in the present study, we observed that the wild genotype A1A1 is more frequent in cases of breast cancer in comparison to the control group (69.5 vs. 68.4 %, respec- tively). This result was also observed in other investigations.

Indeed, Suspitsin et al. [47] had reported a frequency of 77 % of wild-type genotype A1A1 in breast cancer cases and 75 % in healthy women. On the other hand, higher frequency of PIN3 Ins16bp A2A2 genotype has been observed in healthy controls (7 %) as compared to breast cancer patients (3.8 %).

Our result is in agreement with Alawadi et al. [56] which reported an A2/A2 frequency of 10.5 % in controls and 4.8 % in cases. Besides, another Tunisian investigation has revealed an A2A2 genotype frequency of 0 % in familial breast cancer vs. 3.8 % in controls [52].

Regarding allele frequencies, A1 wild-type allele frequen- cy was 82.9 % in our breast cancer group. Nearly, the same frequency was observed in other populations such as Jordani- an and Pakistani breast cancer cases [57,

58]. Based on our

results, A2 mutated allele is not associated to breast cancer risk with an odds ratio of 0.87 (CI= 0.53–1.41); without significant statistical difference. This result is not in accor- dance with other German studies that found Ins16bp allele increases breast cancer risk appreciably in women by the age of 50 years with a first-degree family history of breast cancer [42,

59]. Similarly, the intron 3 Ins16bp rare alleles have also

been associated with cancer risk in American-Caucasian breast cancer patients [46]. In addition, Gemignani et al. [16]

found that this 16bp insertion was associated with lower levels of

TP53

transcripts, suggesting that this polymorphism causes an alteration in mRNA processing, which provides a possible

Table 2 Genotype and allele frequencies of PIN3 Ins16bp polymorphism in breast cancer patients and healthy controls

Genotype Cases (N=105) Controls (N=114) OR (95 % CI) Pvalue

A1A1 73 (69.5 %) 78 (68.4 %) Reference

A1A2 28 (26.7 %) 28 (24.6 %) 1.07 (0.58–1.97) 0.83

A2A2 4 (3.8 %) 8 (7 %) 0.53 (0.15–1.85) 0.32

Allele A1 174 (82.9 %) 184 (80.7 %) Reference

Allele A2 36 (17.1 %) 44 (19.3 %) 0.87 (0.53–1.41) 0.56

A1A1wild-type genotype,A1A2heterozygous,A2A2mutated homozygous,A1wild-type allele,A2mutated allele,ORodds ratio,CIconfidence interval

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molecular basis for increase cancer risk. Yet, the molecular mechanism and the biological effect of this polymorphism have not been completely elucidated in breast cancer.

Our results did not reach statistical significance. This is may be due to the limited statistical power to detect a small effect because of our small sample size. Indeed, Wu et al. [24]

have observed an evidence for the association between the 16bp Ins allele and breast cancer risk among large sample studies including more than 500 subjects but not among small sample studies (≤500 subjects). For these reasons, results of the present study must be interpreted with caution and cannot be generalized.

Conclusion

Our preliminary genetic analysis indicates that there is no correlation between intron 3 genotype and the risk of breast cancer. However, breast cancer is a multifactorial disease with critical interaction between genetic and environmental factors.

Sagne et al showed that the ethnicity and geographical origin are important parameters in PIN3-related cancer risk. In most cases, genetic factors create the potential for cancer, while environmental triggers are required to initiate a cascade of cellular activities responsible for cancer initiation, promotion, and progression [60]. The environmental factors like diet and air pollution are rapidly changing in most developing coun- tries by westernization as the lifestyle (such as the first birth

>30 years of age, less breastfeeding, early menarche, late menopause, etc.) has been changed during the last decades in Moroccan population. Additional research is needed to investigate the potential function of these polymorphisms in response to different types of drug regimen being used in breast cancer apart from their role in tumor behavior.

Acknowledgments We would like to thank all the staff of the Oncol- ogy Department of Ibn Rochd University Hospital and the Genetic and Molecular Pathology Laboratory for their collaboration.

Conflicts of interest The authors have no conflicts of interest to declare.

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