br Corresponding author br One crucial
One crucial gene for maintaining genome integrity is TP53 (Vazharova and Kremensky, 2016). It is one of the most mutated tumor suppressor gene (TSG) (in 50% of all cancers) (Leroy et al., 2017). It is located on chromosome 17p13.1 and encodes a tetrameric protein (Kamada et al., 2016; Pfister and Prives, 2016). It comprises 11 exons with first being non-coding (Joruiz and Bourdon, 2016). Due to hy-poxia, oxidative stress, hyperproliferation signals expressed as a tran-scription factor, binds to various gene promoters induce Thymoquinone ar-rest, apoptosis and DNA repair (Parrales and Iwakuma, 2015). Especially, the aﬃnity of the protein determined by structural features. Any variation intervenes tetramer formation results in reduced activity and altered tumor suppressive function in response to apoptotic stimuli (Brázda and Coufal, 2017).
N. Pouladi et al.
1.3. WRAP53; overlapping structure
TP53 is overlapped by an upstream antisense gene, WRAP53, which lies on opposite strand in a head-to-head manner (Mahmoudi et al., 2009; Farnebo, 2009). This genomic structure provides a regulatory mechanism by means of antisense transcription (Pouladi et al., 2013). WD repeat-containing antisense to p53, WRAP53 (also denoted as TCAB1/WDR79), encodes a gene with 13 exons and three alternative exons, 1α, 1β, and 1ϒ which give rise to WRAP53α, WRAP53β and WRAP53ϒ respectively (Rassoolzadeh et al., 2016). WRAP53α encodes an antisense transcript, overlapped by 227 bp with the first exon of TP53, which binds to 5′ untranslated region of p53 mRNA and pro-tecting it from degradation. The exact mechanism by which this reg-ulation improve p53 stability has not been revealed so far but some proposed that it might be the results of enhanced p53 mRNA folding or inability of destabilizing sequences in p53 mRNA to act properly (Pouladi et al., 2013). It should be noticed that WRAP53β encodes a scaﬀold protein which plays a role in DNA double-strand break repair independent of p53 (Rassoolzadeh et al., 2016).
Altogether, any alteration in the genomic structure at TP53 and WRAP53 genes could result in aberrant p53 response to DNA damage and leads to tumorigenesis and poor prognosis as well as vital flaws in cell lifetime (Parrales and Iwakuma, 2015). Although many Single Nucleotide Polymorphisms (SNPs) have been proven to have a role in disease probability, more studies have to be performed for the eva-luation of these genetic variations on cancer susceptibility (Cao et al., 2016; Feng et al., 2011; Hossein Pour Feizi et al., 2011; Lv et al., 2017). With this regards, SNPs rs1042522 in TP53 and rs2287499 in WRAP53 have been evaluated in relation to breast cancer risk and their prog-nostic value in Iranian-Azeri population. Codon 72 of TP53 is located in exon 4, is a missense substitution which changes arginine to proline by shifting Guanine to Cytosine (R72p) (Tian et al., 2017). This variation is one of the well-studied SNPs and has shown to be related to many cancers such as hepatocellular carcinoma (Qiu et al., 2016) and oral carcinoma (Hou et al., 2015), cervical (Bansal et al., 2016; Li et al., 2015), bladder (Hosen et al., 2015), prostate (Michopoulou et al., 2014), nasopharyngeal (Sahu et al., 2016) and breast cancer (Osorio et al., 2008). The other SNP is also a missense substitution in codon 68 of exon 1 of WRAP53 which is manifested by a changed arginine to glycine due to Cytosine to Guanine replacement (R68G) (Cao et al., 2016). Unlike TP53 SNP, WRAP53 SNP has not been studied widely in relation to cancer contingency but there is strong evidence of the probable role of this SNP in some diseases such as ovarian (Medrek et al., 2013) and breast cancer (Bonab et al., 2014). Association of a haplotype including (rs2287499, rs1042522) with lung cancer (Jung et al., 2008) and melanoma predisposition (Alonso et al., 2010) have been conducted but no roles have been found. Here, we are to rule out the involvement of these variations and their relying haplotype in susceptibility to breast cancer and check for their prognostic value.
2. Material and methods
2.1. Sample collection
Peripheral blood and tumor tissue sample collections from 206 pa-tients newly diagnosed with the mean age of 46.90 were obtained at Nour-Nejat hospital of Tabriz-Iran and peripheral blood from 180 Azeri controls without any history of cancer with the mean age of 46.5 were obtained at the genetic laboratory of natural science department of Tabriz University. DNA extraction was carried out using SDS/protei-nase-K method. This research was approved by the Ethics Committee of Tabriz University of Medical Sciences research center (www.tbzmed.ac. ir/Research). Meta Gene 19 (2019) 117–122
Single-stranded amplicons were loaded onto 22% polyacrylamide gel consisted of 5 ml acrylamide–bisacrylamide solution (40%), 13.5 ml deionized-distilled H2O, 3.5 ml Tris-Borate–EDTA buﬀer (TBE.5×), 300 μl ammonium persulphate (10%) and 30 μl tetramethylenediamine. After that, vertical electrophoresis was conducted for 7 h at 100 V/cm in TBE buﬀer (0.6×) at 4 °C. After separation, gel subjected to silver nitrate staining to demonstrate the SSCP bands.