br Abbreviations BC breast cancer TF transcription factor
Abbreviations: BC, breast cancer; TF, transcription factor
E-mail address: [email protected] (M. Hadavi).
key DNA repair enzyme which is essential for DNA single-strand break (SSB) repair -a sub-pathway related to LDN 193189 excision repair, but also has been demonstrated to play a crucial role in non-homologous end joining (NHEJ) (Amé et al., 2004; Burkle, 2001; Krishnakumar and Kraus, 2010; Schreiber et al., 2006). PARP proteins also have been involved in various cellular processes including cell survival and death, transcrip-tional and chromatin structure regulations, telomere integrity, and cell division (D'Amours et al., 1999; Hassa and Hottiger, 2008). PARP1 is an enzyme responsible for about 90% of the ADP-ribosyl transferase ac-tivity in human cells (D'Amours et al., 1999). Dysregulation of PARP1 expression has been reported in a variety of human cancers, including breast, melanoma, colorectal, head and neck cancers (Gonçalves et al., 2011; Nosho et al., 2006; Staibano et al., 2005). Furthermore, some defects in PARP1 are enhancing cancer risk (Ossovskaya et al., 2010).
Several studies have demonstrated the association of PARP1 gene variants with the incidence risk of breast cancer. However, the findings
of these studies are still controversial and inconclusive (Alanazi et al., 2013a; Alanazi et al., 2013b; Cao et al., 2007; Smith et al., 2008; Tang et al., 2013). Therefore, the aim of this study was to investigate the association of breast cancer occurrence with three Single Nucleotide Polymorphisms (SNP) of PARP1. The association of two SNPs (rs907187 and rs4653734) at promoter region and a missense mutation (rs1136410) at active site of PARP1 and their haplotypes with BC risk were analyzed among Iranian women. To the best of our knowledge, this study is the first investigation on rs1136410 and rs907187 in Ir-anian population and the first association study of rs4653734 with BC in the world.
2. Material and methods
2.1. Sampling and genotyping
The present study included a total of 186 patients with histo-pathological and surgical conformation of BC and 200 healthy in-dividuals. The current study was conducted in accordance with the tenets of the Declaration of Helsinki. All of the subjects were consented to participate in the study. All volunteers, who met the inclusion cri-teria for participating in this study, were Iranian females who resided in Guilan province. The study subjects' ages ranged from 35 to 75 years, with an average of 58.4 years. Individuals who had a history of breast tumor or antecedents of malignancy were excluded from the control group. The control group consisted of 100 age- and ethnicity-matched healthy women.
Genomic DNA was extracted from the peripheral blood through Triton X-100 technique. For this purpose, 2 ml of venous blood from each case and control subjects were collected in EDTA-containing tubes. The purified DNA was analyzed by electrophoresis on 1% agarose gel stained with safe stain. Final DNA concentration was determined using Nanodrop (ND-1000, ABI).
2.2. Linkage disequilibrium (LD) analysis
2.3. Haplotype analysis
In order to estimate the multi-locus association of PARP1 variants with BC dependence, haplotype analysis was performed on rs907187, rs4653734 and rs1136410. Five haplotypes were predicted with fre-quencies higher than 0.5%. Haplotype analyses among case and control groups were performed based on the maximum-likelihood method with an expectation-maximization algorithm. Permutation p-values were calculated by comparing haplotype frequencies between control and case groups on the basis of 10,000 replications.
2.4. Bioinformatics analysis
To obtain the potential functions of SNPs located in the regulatory regions and determine the effects of rs4653734 and rs907187 on transcription factor (TF) binding sites, the promoter sequences of two aforementioned SNPs were analyzed by Genomatix (https://www. genomatix.de), Regulome DB (http://www.regulomedb.org/) and TFBIND (http://tfbind.hgc.jp/). Genomatix and TFBIND servers were used to identify TFs which their binding sites can be significantly af-fected by a given SNP. Also Regulome DB tool applied to verify TF bound to intended nucleotide sequence of the gene promoter. Interestingly, as rs907187 or rs4653734 were in CpG island, the present study investigated the likelihood of these two SNPs as putative CpG-SNPs by MethPrimer tool for further investigations.
2.5. Statistical analysis
To determine whether any significant differences in SNP frequencies occurred between the case and control groups, allele and genotype frequencies were compared using the chi-square method by SNPAlyze (ver. 8.1.1), SPSS (ver. 20) and MedCalc (ver. 14.8). Odds ratio (OR) and 95% confidence intervals (CI) were calculated to determine the risk of BC associated with a given PARP1 genotype. P-value of < 0.05 was considered as the statistical significance.