The C Allele of a Synonymous SNP ( rs 1805414 , Ala 284 Ala ) in PARP 1 is a Risk Factor for Susceptibility to Breast Cancer in Saudi Patients

Breast cancer is the most common neoplasm and the second leading cause of cancer death in Saudi women. The recent increase in incidence has made breast cancer one of the most frequently recorded diseases among Saudi women (Ferlay et al., 2010). The age-adjusted death rate because of breast cancer in Saudi Arabia is also rising, with the most rapid increase in the world from 1985 to 2008 (Saudi National Cancer Registry, 2011). This malignancy represents a heterogeneous group of tumors with characteristic molecular features, prognosis and responses to available therapy. DNA repair pathways exist in all cells for maintaining genome integrity (Hoeijmakers, 2001), and mutations within these pathways can result in cancer (Heinen et al., 2002). Interindividual variations in DNA damage and repair have been associated with an increased risk of breast cancer (Johnson et al., 2000; Tyrer et al., 2004). Poly (ADP-ribose) polymerase 1 (PARP-1) is a DNA double strand break-sensing protein, and its activation is one of the early responses to DNA damage (Schreiber et al., 2006). PARP-1 gene localizes to chromosome


Introduction
Breast cancer is the most common neoplasm and the second leading cause of cancer death in Saudi women.The recent increase in incidence has made breast cancer one of the most frequently recorded diseases among Saudi women (Ferlay et al., 2010).The age-adjusted death rate because of breast cancer in Saudi Arabia is also rising, with the most rapid increase in the world from 1985 to 2008 (Saudi National Cancer Registry, 2011).This malignancy represents a heterogeneous group of tumors with characteristic molecular features, prognosis and responses to available therapy.
DNA repair pathways exist in all cells for maintaining genome integrity (Hoeijmakers, 2001), and mutations within these pathways can result in cancer (Heinen et al., 2002).Interindividual variations in DNA damage and repair have been associated with an increased risk of breast cancer (Johnson et al., 2000;Tyrer et al., 2004).Poly (ADP-ribose) polymerase 1 (PARP-1) is a DNA double strand break-sensing protein, and its activation is one of the early responses to DNA damage (Schreiber et al., 2006).PARP-1 gene localizes to chromosome Genome Research Chair, Department of Biochemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia *For correspondence: reddyparine@gmail.com

RESEARCH ARTICLE
The C Allele of a Synonymous SNP (rs1805414, Ala284Ala) in PARP1 is a Risk Factor for Susceptibility to Breast Cancer in Saudi Patients Mohammed Alanazi, Akbar Ali Khan Pathan, Jilani P Shaik, Abdullah Al Amri, Narasimha Reddy Parine* 1q41-42, consists of 23 exons and spans 47.3 kb (Schreiber et al., 2006).It encodes a multifunctional nuclear protein, which consists of an N-terminal DNA binding domain, a central auto-modification domain and a C-terminal catalytic domain (Cottet et al., 2000).PARP-1 catalyzes poly(ADP-ribosyl)ation, an immediate DNA-damage dependent post-translational modification of itself, histones and other nuclear proteins, which is believed to play a multifunctional role in various cellular processes, including DNA-damage detection and repair, cell death pathways and mitotic apparatus function (Kim et al., 2005).PARP-1 deficiency in mice resulted in spontaneous mammary carcinomas, and additional p53 mutations shorten the latency of mammary tumor formation suggesting a possible involvement of PARP-1 in breast carcinogenesis (Tong et al., 2007).PARP-1 has been implicated in tumorigenesis (Tong et al., 2001;Masutani et al., 2003).Few studies studies indicate that PARP-1 plays an important role in suppressing malignancy in mice.Interestingly, reduced PARP-1 activity in human peripheral blood lymphocytes has been linked with human breast, colon, lung (Pero et al., 1990) and laryngeal cancers (Rajaee-Behbahani et al., 2002).
There are many identified single nucleotide polymorphism (SNP) in the PARP-1 gene, and some of which are reported to be implicated in carcinogenesis (Hao et al., 2004;Lockett et al., 2004;Shiokawa et al., 2005;Zhang et al., 2005).Several SNPs have been found in PARP1 gene (Figueroa et al., 2007).Although many studies have searched for the association between PARP-1 polymorphisms and the risk of malignancy, the results are inconsistent in different organs and in different ethnic groups.Few studies have reported positive associations between the SNP's rs1805404 and rs1805414 at position 81 and 284, have been reported to be significantly associated with an increased risk of Alzheimer's disease (Liu et al., 2010), Glioblastoma (Keller et al., 2011), protectively associated with Colorectal cancer (Berndt et al., 2007;Ogino et al., 2010).rs1805404 reported be strongly associated with Tourette syndrome risk (Wu et al., 2013).
To the best of our knowledge, till now there are no reports about the association between the SNPs of PARP-1 rs1805404 (Asp81Asp) and rs1805414 (Ala284Ala) and breast cancer in Saudi population.Therefore, in the current population based case-control study, we investigated the genotype distributions of the rs1805404 (Asp81Asp) and rs1805414 (Ala284Ala) in patients with breast cancer.

Study population
A total of 195 blood samples were obtained from King Khalid University Hospital.These encompassed 99 patients with breast cancer disease and 96 healthy controls.All controls were age-matched and recruited from physical examinations after diagnostic exclusion of cancer and cancer-related diseases.Blood samples of the experimental and control groups were obtained before treatment.Histopathology and medical records were reviewed to confirm diagnosis.Controls were frequency matched to cases on age/race and recruited from the clinic population receiving routine mammography at the breast screening and diagnostic center.Eligibility criteria for controls included normal mammography results and no prior cancer history.Written informed consent was obtained from all participants, and approval was received from the King Khalid University Hospital ethics review committee.Every study participant completed a selfadministered baseline questionnaire, which included information on demographics, reproductive history, medical conditions and family history of cancer.

DNA extraction
Approximately 3 ml of blood samples were collected in sterile tubes containing ethylenediaminetetracetic acid (EDTA) from all subjects enrolled in the study.Genomic DNA was isolated from blood samples using QIAmp kit (QIAmp DNA blood Mini Kit, Qiagen, Valencia, CA) following the manufacturer's instructions.After extraction and purification, the DNA was quantitated on a NanoDrop 8000, to determine the concentration and its purity was examined using standard A260/A280 and A260/A230 ratios (NanoDrop 8000) (Sambrook et al., 1989).

Genotyping
Two SNPs (rs1805404 and rs1805414) in PARP1 gene were genotyped using TaqMan allelic discrimination assay (Livak, 1999).For each sample, 20 ng DNA per reaction was used with 5.6 µL of 2X Universal Master Mix and 200 nM primers (Applied Biosystems, Foster City, CA, USA).All genotypes were determined by endpoint reading on an ABI 7500 (Applied Biosystems, Foster City, CA, USA).Primers and probe mix were purchased directly through the assays-on-demand service of Applied Biosystems.Five percent of the samples were randomly selected and subjected to repeat analysis as a quality control measure for verification of genotyping procedures.

Statistical analysis
Genotype and allelic frequencies were computed and were checked for deviation from Hardy-Weinberg equilibrium (http://ihg2.helmholtz-muenchen.de/cgi-bin/ hw/hwa1.pl).Case-control and other genetic comparisons were performed using the chi-square test and allelic odds ratios (OR), and 95% confidence intervals (CI) were calculated by Fisher's exact test (two-tailed).Statistic analysis was done using SPSS 16.0 for Windows.We considered p-value of <0.05 as significant.

Results
A total of 99 BR cases and 96 healthy controls were included in this study.Clinical characteristics of breast cancer cases and healthy controls are given in Table 1.Out of 96 confirmed cases of breast cancer, 47 were ER positive and 43 ER negative, 49 were PR positive and 41 PR negative, 38 were HER positive and 52 HER negative (Table 1).
All the genotypic distributions were consistent with that expected in the Hardy-Weinberg model (Table 2).Homozygous ancestral allele was used as a reference to determine the odds of acquiring breast cancers in relation to the other two genotypes.The genotype distribution of  the analyzed SNPs along with the corresponding odds ratio and significance are shown in Table 3.We observed statistically significant association with one of the two SNPs (rs1805414) with breast cancer risk.
In Saudi Arabian patients, the median age of onset of breast cancer is 47 years, substantially lower than 62 years observed in the American population (Anderson et al., 2006;Saudi National Cancer Registry, 2011).To evaluate the association of the analyzed SNPs with the young age at diagnosis of breast cancer, we stratified the patients as ≤48 (n=46) or >48 (n=53) years of age.The genotype distribution for the individual SNP along with the statistical analysis are shown in Table 4. Interestingly, only PARP1 rs1805414 which showed significant association in the overall study or in the patient group with age <48 years, indicated a significant risk on patients with ≤48 years (Table 4).It was noted that, women aged ≤48 years had slightly higher risk for developing breast cancer in Saudi population with this allelic change (OR=4.333,χ 2 =4.54, p<0.03319).
We conducted the association of breast cancer risk with the individual SNPs based on the estrogen receptor (ER) status of the tumors.The genotype distribution in the ER+ (n=53) and ER-(n=43) groups were compared with each other (Tables 5).Interestingly, homozygosity of the minor allele (T) at SNP rs1805414 posed protective influence on ER+ breast cancer (OR=0.512;χ 2 =4.42; p=0.0356).This association was not observed in the ER-ve category as well as in the overall study population.The rs1805404, that was significantly associated with increased risk of breast cancer in the overall study population doesn't exhibited any association for the ER+ as well as ER-group (Tables 5).
We performed the association of breast cancer risk with the individual SNPs based on the progesterone receptor (PR) status of the tumors.The genotype distribution in the A slightly significant protective nature was observed in women with C/T+T/T in breast cancer cases compared to healthy individuals (OR=0.44,χ 2 =3.94, p<0.047).
The association of breast cancer risk with the individual SNPs based on the Human Epidermal Growth Factor Receptor 2 (HER2) status of the tumors was also evaluated.The genotype distribution in the HER+ (n=41) and HER-(n=57) groups were compared with each other disease risk.Interestingly, homozygosity of the minor allele (T) at SNP rs1805414 posed protective influence on HER2+ breast cancer (OR=0.524;χ 2 =4.53; p=0.03337).This association was not observed in the ER-ve category as well as in the overall study population.The rs1805414, that was significantly associated with increased risk of breast cancer in the overall study population doesn't exhibited any association for the ER+ as well as ER-group (Table 7).

Discussion
DNA repair mechanisms play a major role in protecting against carcinogenesis and genetic defect in DNA repair can cause human cancer (Heinen et al., 2002).Through repairing DNA damage and maintaining genetic stability, PARP-1 has played an important role in prevention of carcinogenesis.Our results suggest that, despite the strong biological plausibility, PARP1 is a susceptibility locus for breast cancer in Saudi population.The human PARP1 gene consists of 23 exons spanning 43 kb and has been localized to chromosome 1q41-q42.The PARP-1 rs1805404 (Asp81Asp) and rs1805414 (Ala284Ala) polymorphism has been implicated in cancer susceptibility.In this study, we found for the first time that the PARP-1 rs1805414 (Ala284Ala) genotype significantly contributes to breast cancer susceptibility in Saudi population, which further extend the important role of PARP-1 in carcinogenesis.
Our findings suggests that, homozygotes at PARP1 locus (Ala284Ala) may be associated with an increased risk of Breast cancer (Table 3).Neither variant is likely to influence the activity of the protein itself: the T allele at Ala284Ala does not affect the amino acid sequence of the protein.Thus, our results supports Berndt et al. (2007) findings in which they suspected that the association that we observed with colorectal adenoma may be due to linkage disequilibrium with another variant in the region, possibly in the PARP1 promoter.Because PARP1 activity is highly regulated by its promoter, variants within transcription binding sites in the promoter may influence its expression (Berndt et al., 2007).In a similar way Milani et al. (2007) reported that PARP1 contains contain functional regulatory SNPs in their promoter regions and SNP rs1805414 expression level is effected by allelic imbalance in cancer cells.We have observed that rs1805414 is associated with the Age and breast cancer susceptibility (Table 4 and 5).Cao et al., (2007) reported that the Ala284Ala (TqC) PARP-1 variant was likely associated with loss of estrogen-and progesteronereceptor expression.This implies that genetic variants of PARP-1 may contribute to breast cancerogenesis and that the PARP-1 Ala284Ala variant protein may influence hormonal therapy of breast cancer (Cao et al., 2007).But interestingly rs1805414 showed protective nature in ER positive samples in Saudi population when compared to ER negative samples.
Our study has some strengths: patients and controls came from the same geographical area; genotyping errors were avoided using duplicate samples; markers were tested to assure a true association.For the association testing, we also considered multiple genetic models, adjusted our analyses for possible confounders (age at study), and stratified our sample for multiple variables (age at study, ER, PR, HER status) to explore possible effect modifications.
In summary, our study firstly shows a significant association between the PARP-1 Ala284Ala (rs1805414) genotypes and increased risk of breast carcinoma in Saudi patients.These findings suggest that the SNP rs1805414 may modulate the occurrence of PARP1 mutations and contribute to breast carcinogenesis.Our findings suggest that PARP-1 dysfunction may play an important role in the development of breast carcinoma.Despite our data supports for a clear association between PARP1 and breast cancer in Saudi population and PARP1 gene plays a major role in the susceptibility to the disease.As the sample size of this study is not sufficiently large and is restricted to Saudi population, the present data should be validated in larger samples and in other ethnic groups.Additional functional as well as association studies investigating gene-gene interactions are required to elucidate this issue, and it remains possible that PARP1 variability may affect disease progression or the susceptibility to develop breast cancer.Additional studies are needed to confirm these findings and to further characterize the specific genetic variants that alter adenoma risk.