br The region containing TP polymorphism was PCR amplified
The region containing TP53 polymorphism was PCR-amplified by ARMS-PCR method using forward and reverse primers 5′- TCCCCCTT GCCGTCCCAA-3′ and 5′-CTGGTGCAGGGGCCACGC-3 respectively for Arginine, forward and reverse primers 5′-GCCAGAGGCTGCTCCCCC-3′ and 5′-CGTGCAAGTCACAGACTT-3′ respectively for Proline geno-typing. PCR reaction was performed in a total volume of 25 μl con-taining 1 to 2 μl DNA (200 ng), 2.5 ml of 10× PCR buﬀer, 1 μl MgCl2, 0.5 μl dNTP, 0.75 μl of each forward and reverse primers (10 pmol) and
0.15 μl of Taq sybr safe enzyme. Argenine specific band with 144 bp and proline specific band with 177 bp were identified on 2% agarose gel.
2.4. Statistical and in silico analysis
To determine the association of these genotyped SNPs with breast cancer susceptibility, we assessed the data with javastat online statistics package (http://statpages.info/ctab2x2.html) for statistical tests. Although, we analyzed these polymorphism data including genotype and allelic frequencies in relation to clinicopathological features such as age, aﬀected the side of the breast, histological features, tumor size and stage of cancer with SPSS software V.16 through Pearson's chi-square test or Fischer's exact test with p-value being < 0.05 as statistically significant. The frequency of these two polymorphisms as pairwise haplotypes was evaluated by calculating the linkage disequilibrium by Haploview software v4.2 based on p-values smaller than 0.05 as sig-nificant. in silico analysis conducted for investigating deleterious and harmful, disease and cancer-associated SNPs and SNP eﬀects on RNA secondary structure by SIFT, Polyphen2, Fanthmm, RNAsnp, and SNP& GO online servers.
Single nucleotide polymorphisms (SNPs) in crucial genes can in-fluence the cellular behavior and results in disease and cancer occur-rence (Devi et al., 2015). Significantly, many polymorphisms in TP53 and its neighbor overlapping gene, WRAP53, might have a significant impact on various molecular pathways and might confer an abnormal behavior which results in diﬀerent cancers (Qiu et al., 2016; Bansal
N. Pouladi et al.
Genotypes and allelic frequencies of the two polymorphisms.
Cases Controls p-Value
The relationship between the two polymorphisms and clinicopathological parameters.
CC CG GG p-Value CC CG GG p-Value
Genotype combinations of the polymorphisms in cases and controls.
Codon 68 Codon 72 Cases Controls p-Value
Pairwise haplotype frequency distribution in cases and controls.
Pairwise haplotype Cases% Controls% p-Value
Fig. 1. LD visualization for putative block consists of SNP1 (rs2287499) and SNP2 (rs1042522). The number in the square is D′ value which indicates the degree of linkage disequilibrium.
SNPs in silico prediction.
Neutral Neutral Polyphen2 100 100
Significant Not significant SNP&GO RI = 9 RI = 9
A total of 206 breast cancer patients and 180 controls from Azeri population were genotyped for both codon 68 of WRAP53 and codon 72 of TP53 genes. No deviation from Hardy-Weinberg equilibrium was seen for both polymorphisms (p-value = 0.235, p-value = 0.199 for rs2287499 and rs1042522 respectively). The genotype and allelic fre-quencies of these two polymorphisms then evaluated for their re-spective association with breast cancer susceptibility. The genotype and allelic distribution data have been shown in Table 1. As data displays, there were no significant diﬀerences in genotypes of these single nu-cleotide variations.
In this assessed population, the frequency of age distribution showed that 63 (30.6%) of cases were had equal or < 40 years old and 143 (69.4%) of them were had higher than 40 years old with a range between 25 and 83 years of age. In addition, histopathology
Fig. 2. RNAsnp prediction for two SNPs. local regions of a) R72P substitution b) R68G substitution. Black line indicates a not significant changes (p-value > 0.02) but other colors illustrate diﬀerent significant values (c). Secondary structure of d1) wild type p53 RNA d2) mutant type p53 RNA e1) wild type WRAP53 RNA e2) mutant type WRAP53 RNA.
manifestations showed 187 cases of in-situ ductal carcinoma (IDC), 10 cases of in-situ lobular carcinoma (ILC) and 9 cases of (DCIS). Furthermore, no significant relation between polymorphisms with the side of involved breast, histopathology, and stage of cancer was ob-served (Table 2). Even though there were no significant diﬀerences between codon 68 of WRAP53 with the size of the tumor but there was a significant correlation between codon 72 of TP53 and the size of the tumor with the p-value of 0.038.