TP53 PIN3 polymorphism associated with breast cancer risk in Iranian women

298 Indian Journal of Cancer | July-September 2011 | Volume 48 | Issue 3298

298 CMYK298 CMYK

TP53 PIN3 polymorphism associated with breast cancer risk in Iranian women

Faghani M, Ghasemi FM, Nikhbakht M1, Salehi M2

Department of Anatomical Sciences, Guilan University of Medical Sciences, Rasht, Departments of 1Anatomical Sciences and 2Genetic, Isfahan University of Medical Sciences, Isfahan, Iran

Correspondence to: Dr. Masoumeh Faghani, E-mail: [email protected]

AbstractBACKGROUND: Breast cancer is one of the most common cancers in Iranian women. The p53 gene plays a principal role in genomic stability, and its function varies according to polymorphisms. The aim of our study was to determine the relationship between the intron3 16bp duplication polymorphism of the p53 gene and breast cancer in Iranian women. MATERIALS AND METHODS: We performed a case-control study among 145 patients with invasive ductal carcinoma of the breast and 145 controls in Isfahan, Iran. The distribution of the intron3 16bp duplication polymorphism was determined by polymerase chain reaction (PCR). The relationship between clinicopathological data and the PIN3 polymorphism was examined using chi-squared analysis. RESULTS: A significant difference was observed in the polymorphism variants in breast cancer specimens compared with controls (P < .001). Among the cancer patients, 59.9% were below the age of 50 years; and 67.5% of the patients in this group had the intron3 16bp duplication polymorphism. CONCLUSIONS: PIN3 Ins 16bp duplication polymorphism is a genetically predisposing factor for breast cancer development in Iranian women and may be causal in patients under the age of 50 years.

Key words: Breast cancer, Iranian women, PIN3 polymorphism, TP53 genotypes

Breast Cancer - Original Article

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DOI: 10.4103/0019-509X.84925

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Introduction

The p53 tumor suppressor gene [Online Mendelian Inheritance in Man (OMIM) no. 191170] codes a 53 kilodalton (KD) protein, which plays an essential role in the cell cycle.[1] DNA damage increases p53 expression, resulting in cell cycle arrest, apoptosis and DNA repair.[2] Somatic mutations in p53 gene exist in more than half of human cancers.[3] It has been shown that mutations in the splice, donor and acceptor sites, intron and promoter elements, may be important in regulating gene expression.[4] In addition to mutation, p53 gene polymorphisms have a role in breast cancer development.[5] Among the 14 polymorphisms of p53, most studies are performed

on Arg72pro (rs1042522), intron3 (rs17878362) and intron6 MspI (rs17880604). [6] There is a relationship between Arg72pro polymorphism and development of cancers.[7-12] In addition, some researchers have reported polymorphism within introns or non-coding regions such as intron 3, 6.[13-15]; 16 base pair (bp) duplication in intron 3 of the TP53 gene (PIN3 Ins16bp) polymorphism may be associated with cancers. [16] Some studies have reported a relationship between PIN3 p53 polymorphism and breast cancer.[15,17] However, this is a controversial subject.[18,19]

Breast cancer is the most common cancer in women and the second leading cause of death worldwide. [20] According to the World Health Organization, 2.1 million cases are identified yearly.[21] Breast cancer is the first cancer to have been diagnosed in Iranian women (21.4% of all cancers) and the first cause of cancer-related death.[22] The age at diagnosis of breast cancer in Iranian women is approximately one decade lower than that in women of other countries,[23] and approximately 10% of these cancers have been reported in Isfahan.[24] There have been studies on the Arg72pro polymorphism in breast cancer among Iranian

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299Indian Journal of Cancer | July-September 2011 | Volume 48 | Issue 3 299

Faghani, et al.: P53 PIN3 polymorphism and breast cancer

women,[11,25,26] but there is no report about the PIN3 polymorphism in Iran. Thus we aimed to study the relationship, if any, between the PIN3 polymorphism of the p53 gene and the risk of breast cancer in Iranian women in Isfahan.

Materials and Methods

Blood samples were obtained from 145 patients with invasive ductal breast carcinoma (average age, 23-79 years; mean age, 50 years), referred by the hospital of Isfahan University of Medical Sciences in the period 2003-2006. All cases were histologically confirmed by our Pathology Department. Control group consisted of 145 age-matched women without known malignancy or autoimmune disease, from whom blood was also taken. Prior to commencement of the study, all patients provided informed consent and the study was approved by our institutional ethics committee, Samples were divided into two groups: 86 patients were premenopausal, and 59 were postmenopausal; and among controls, the corresponding numbers were 87 and 58, respectively.

Using high pure polymerase chain reaction (PCR) template preparation DNA isolation kit (Roche), genomic DNA was extracted from blood samples, according to manufacturer’s instructions. As much as 100 to 300 ng of DNA was used as template in a 25- mL PCR reaction mixture containing 1.5 mmol MgCl2, 1 U Taq polymerase (Cinnagen, Iran) and 2 mmol either of the primer pairs. For negative control, no DNA was added or included in each run. The forward and reverse primers sequences were used to amplify the TP53 PIN3 Ins16bp polymorphism in intron3 as follows[27]:F: 5'-CTGAAAACAACGTTCTGGTA-3'R: 5'-AAGGGGGACTGTAGATGGGTG-3'

PCR cycling conditions were implemented with an initial denaturation step for 5 minutes at 95°C, followed by 35 cycles: Denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds with extension at 72°C for 60 seconds. A final extension step was performed at 72°C for 5 minutes. The PCR product was electrophoresed on 15% polyacrylamide gels and stained with ethidium bromide. For detection of intron3 polymorphism, we considered that the amplification yielded 119 or A1 allele (no duplication) and the variant 135-bp fragments or A2 allele (with 16-bp duplication) for the wild-type, depending on the absence or the presence of the 16-bp duplication in the genomic DNA. We observed both bands in the heterozygote [Figure 1]. Data such as tumor grade and size, family history, date of diagnosis, lymph node

metastasis, and progesterone and estrogen receptor were also recorded.

For statistical analysis, we used both SPSS (version 16) program and Hardy-Weinberg Equilibrium calculator. The Pearson chi-squared test was used to assess statistical significance. The odds ratio with 95% confidence interval (95% CI) was used to confirm the association between TP53 polymorphic genotypes and breast cancer risk. We used Kruskal-Wallis ANOVA test to determine the correlation between clinicopathological data and PIN3 polymorphisms. The distribution of two alleles in our groups was tested by Hardy-Weinberg Equilibrium calculator (http://ihg.gsf.de/cgi-bin/hw/hwa1.p1.).

Results

The distribution of PIN3 genotype frequencies among the two groups is not in agreement with that expected under Hardy-Weinberg Equilibrium [Table 1]. Histopathologic tumor grade, tumor size, and other clinicopathological data are presented in Table 2. There was a statistically significant difference in the distribution of the A1 and A2 alleles between the case and control groups. The genotype distribution for insertion of the 16-bp duplication polymorphism was 14.5% for homozygotes (NoIns), 85.5% for heterozygotes and 0% for homozygotes (Ins) in breast cancer patients [Table 1]. In the control group, the genotype distribution was 42.8%, 57.2% and 0% for the homozygotes (NoIns), heterozygotes and homozygotes (Ins), respectively. We found that the A1/

Figure 1: Detection of the P53 PIN3 polymorphism by PCR amplification. The PCR products were electrophoresed on 15% polyacrylamide gel and were stained with ethidium bromide. Lanes 1, 2, 3: Positive for A1 allele (no duplication) Lanes 4, 5: Positive for A2 allele (16-bp duplication) and A1 allele Lane N: Negative control. L: Gene ruler TM DNA ladder. The sizes of the bands are indicated beside the gel photo



Discussion

Breast cancer is a heterogeneous disease, reflected in the varying responses to adjuvant therapy.[28] Molecular changes, including the P53 gene mutation and polymorphism, are related to growth and development of different cancers.[29] Researchers have shown that the Arg72pro P53 gene polymorphism is a risk factor in lung, colorectal, bladder and breast cancers.[7,9,19,30,31] Polymorphisms can affect protein function or expression; polymorphisms in genes involved in DNA repair, cell-cycle control, estrogen production and carcinogen metabolism could predispose individuals to developing breast malignancy.[27] Past studies have reported that genetic variants in the promoter or intron regions could alter gene expression; this variant may not have direct functional implications but can be linked to other polymorphisms and can affect the function related to wild-type sequence.[27]

Our results are in agreement with those of the study by Sayhan and Wn, who described the association between PIN3 Ins 16-bp duplication polymorphisms and risk of ovarian and lung cancer.[32,14] However, contrary to the finding of our study, there was no correlation reported about this polymorphism in Caucasian Americans with ovarian or bladder cancer or in Turkish women with breast cancer.[33,15]

Hrstka and Trifa could not find a relationship between the PIN3 polymorphism and breast cancer, and attributed differences to race or geographical distribution.[18,19]

In our study, we investigated the frequency of 16 bp duplication polymorphism intron3 of p53 in 145 female patients with invasive ductal breast carcinoma and 145 healthy controls from the central part (Isfahan province) of Iran. We observed an association between the A1/A2 genotype and higher risk of breast cancer among patients, showing that TP53 PIN3 Ins 16-bp duplication polymorphism increased susceptibility for breast cancer in Iranian women. The frequency of the inherited 16bp insertion statistically differed between patients and controls. Our results confirmed those of studies performed on ovarian, lung and breast cancers,[14,32,34] but were contrary to those of other studies.[15,33] The age range of patients was 23-79 years, and most of the younger women (<50 years) had the TP53 PIN3 Ins 16-bp duplication polymorphism. Data in Iranian patients shows that age of patients at diagnosis of breast cancer in Iran is lower than that of patients in other countries and that tumors are more aggressive compared to those in patients in other countries.[23,35] Our data,

Table 1: Demographics of breast cancer patients (n = 145) and controls (n = 145)Polymorphism genotype1-1 1-2 2-2 P

Allele frequency P HW Total 1 2

Intron 3Control 62 83 0 Cancer 21 124 0 0.0002

207 83 0.00001a 145166 124 0.000 0.000002b 145

a: Exact HW = 1.377e-06; b: Exact HW = 2.364e-19

Table 2: Association of clinicopathological variables with p53 PIN3 genotypesVariable Total PIN3 Ins 16-bp

P-valueHistologic gradea

G1 45 0.206G2 66G3 34

Nodal statusa

Negative 68 0.001Positive 77

Tumor sizeb 145 0.625ER statusa


PgR statusa


Site of tumora

Left 52 0.236Right 79Bilateral 14

a: Pearson c2 test; b: Kruskal-Wallis ANOVA test

A2 genotype carriers had a 4.41-fold (95% CI, 2.5-7.78; P < .001) increased risk of breast cancer. The allelic frequency was strongly associated with the A1/A2 genotype (P = .001; odds ratio= 1.85, 1.37-2.5). In addition, out of 145 patients, we used the data of 77 (53.1%) patients with lymph node metastasis; 70.5% of these had the A1/A2 genotype.

Our data showed that neither estrogen nor progesterone receptor expression was associated with the presence of a specific genotype [Table 2]. On the other hand, we considered the relationship between the PIN3 polymorphism and menopause, tumor grade and tumor size. No relationship was found between histologic type and tumor size, genotype and menopause (P > .05). These data showed that 64.1% of cancer patients were in the “before menopause” age category and 67.5% of this group had the A1/A2 genotype. The Chi-squared test revealed no significant differences between the two menopausal groups and the genotypes of the p53 gene (OR = 1.02; 95% CI, 0.64-1.64; P = .5).

301Indian Journal of Cancer | July-September 2011 | Volume 48 | Issue 3 301


which showed a high level of lymph node metastasis (53.1%), confirmed this claim.

It has been suggested that PIN3 Ins 16-bp duplication polymorphism and other polymorphisms of p53 (72Arg) affect the DNA repair gene, resulting in alteration of p53 function. Some investigators have reported that the Arg-A2 haplotype has a positive association with cancer[14,36] and that the A2 allele is associated with decreased apoptotic and DNA repair functions.[34] Due to the high frequency of the A2 (42.8%) and Arg (75.4%) alleles (data not shown) in our patients, it appears that linkage between the two aforementioned factors possibly has greater effect in the development of invasive cancers such as ductal carcinoma in Iranian (Isfahanian) women. In addition, in our previous work we showed that the Arg/Arg polymorphism of p53 codon 72 is related to breast cancer development (data not shown).[11]

In conclusion, our findings indicate that Iranian women with TP53 PIN3 Ins16bp polymorphism and Arg/Arg polymorphism may have a higher risk for developing breast cancer; these findings require confirmation with a larger sample size. Because polymorphisms can be considered potent factors for breast cancer development, this data can be considered as part of a genetic profile in high-risk women and would create an effective approach to aid in early diagnosis and treatment.

Acknowledgments

We thank the Deputy Research Isfahan University of Medical Sciences for financial support under grant number 185191. We also thank the faculty of the Biotechnology Department of Pharmacy, Isfahan University of Medical Sciences; and Ms. Fatemeh Moazzen for their technical support.

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TP53 PIN3 polymorphism associated with breast cancer risk in Iranian women

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