A Case Control Candidate Gene Study on Identification of P53 Polymorphisms/Mutations in Breast Cancer Patients

Abstract

Breast cancer is the most predominant disease and leading cause of death in women worldwide. This disease is responsible for 30% malignancy in females and around 2.3 million new cases are reported globally. In Pakistan, the incidence rate of breast cancer is very high as every 1 in 8 women are at risk of getting this disease. Inherited mutations are responsible for 10% cause of breast cancer specially in tumour suppressor genes like P53. Therefore, the aim of the current study was to identify single nucleotide polymorphisms(SNPs)/mutations in P53 gene and find the association of these mutations with breast cancer. A questionnaire was developed to collect demographic data of cancer patients. Blood samples of cancer patients (n=200) and controls (n=200) were collected for the detection of polymorphisms in P53 gene. DNA was extracted via phenol chloroform method and amplified by designed primers for the hotspot region of exon 4, 5 and 6 of P53 gene. Single strand conformation polymorphism detection was done on the variants identified by banding pattern followed by Sanger sequencing. The Sanger sequencing results were aligned with reference sequence (NG_017013.2) retrieved from NCBI, and analysed by using BIOEDIT software and Mutation Taster. Sequencing analysis revealed 2 novel SNPs at chromosomal position chr17:7578423C>GN/A, chr17:7578539T>GN/A and 1 reported SNP at chromosomal position chr17:7578645C>TN/A. In these two novel SNPs, both were non- synonymous in exon 4 and upon in silico analysis indicated that these mutations may have interactions with several other genetic factors like CCR2, HIPK1 and AXN1. These novel mutations can change interaction of P53 protein and may have role in breast cancer development and progression. These findings will contribute not only towards research on variations in P53 gene (tumour suppressor gene) but also possible candidate for development of diagnostic biomarker. Additionally, our findings provide data for breast cancer awareness, campaigns and further research on local population to address the high prevalence that causing a national disease burden.