SPECTRUM OF TP53 TUMOR SUPPRESSOR GENE MUTATIONS AND CODON 72 POLYMORPHISM IN PAKISTANI FEMALE BREAST CANCER PATIENTS

Abstract

The tumor suppressor gene TP53 encodes a nuclear protein that prevents the cells from dividing before DNA damage is repaired. Mutations in TP53 gene have effects on its biological activities. The objectives of present study aims at determining the frequency TP53 mutations in sporadic, genetic lineage and analysis of the data i.e. questionnaire collected from breast cancer patients from Pakistan, during the study. Female breast cancer patients were recruited at Shaukat Khanum Memorial Cancer Hospital & Research Centre and Mayo Hospital, Lahore Pakistan, from January 2005-December 2008. A total of 150 sporadic breast cancer patients and three families with breast cancer cases were included in the study. From all study participants, a blood sample and a piece of tissue of normal and tumor both were collected. DNA was extracted and exons 5-8 (central region) of TP53 gene were PCR amplified. Each sample was heteroduplexed with a normal control sample (confirmed by sequencing). To screen TP53 mutations Temporal Temperature Gradient Gel Electrophoresis (TTGE) was performed. The mutations were confirmed by sequencing. Restriction Fragment Length Polymorphism (RFLP) was used for understanding the status of codon 72, exon 4 of TP53 gene polymorphism (arg/arg) in Pakistan. The data was analyzed using the R15 programme, provided by International Agency for Research on Cancer. Three deleterious mutations were detected in the sporadic breast cancer patients, viz., codon 238 where TGT is mutated to TAT (cys to tyr), codon 248 where CGG is mutated to CAG ( arg to glu), and codon 278 where CCT is mutated to TCT (pro to ser). These mutations were not detected in normal breast tissue and blood samples of these patients. R15 analysis (IARC, 2011) of TP53 gene mutations showed that the mutations detected in Pakistani breast cancer patients are reported most prevalent somatic mutations (codon 238 = 79 tumors, codon 248 = 779 tumors and codon 278 = 74 tumors) in breast cancer patients of the world. Three-dimensional structures were predicted by 3D Viewer (software given on IARC website) and found that all these three mutations are in DNA binding region of TP53 and could change the structure of protein and, therefore, affect its function. TP53 mutation has not been observed in normal persons and breast cancer families blood samples. One family was detected with Li-Fraumeni syndrome characters but TP53 mutations are not found in it. Although the polymorphism arg/arg, codon 72, exon 4 of TP53 gene is reported as a functional relevant polymorphism that contributes to breast cancer development yet in the vpresent study, genotype arg/pro and pro/pro, both polymorphisms were found more significant in Pakistani breast cancer patients as compared to arg/arg with corresponding ratio of arg/pro (53.3): pro/pro (34.6): arg/arg (12). Normal controls showed about the same difference in ratio of arg/pro: pro/pro: arg/arg, (50:40:10). Correlation of TP53 mutations with clinicopathological parameters (data collected by questionnaire) was observed. Patients were divided into two groups; group 1 (TP53 non mutated) and group 2 (TP53 mutated). As both groups have not shown any difference so no prominent correlation between TP53 mutations and clinicopathological parameters was found. It is concluded that the frequency of TP53 gene mutations in DNA coding region (5-8 exon ) is low in Pakistani breast cancer patients. However, present study is in favor of the fact that the frequency of TP53 gene mutations is different in different geographical areas. Genotype arg/arg is less prevalent in the female breast cancer patients and normal population of Pakistan. There was no significant correlation between TP53 mutation and tumor aggressiveness e.g. nodal status, size, ER/PR, histopathology etc. Epidemiologically, no carcinogen was found important as a causative factor of TP53 gene mutations in Pakistani breast cancer patients.