Single Nucleotide Polymorphisms Associated With Prostate Cancer Development in Saudi Males

Background: Genome-wide association studies have demonstrated that single nucleotide polymorphisms (SNPs) are important risk factors for the development of prostate cancer (PC). In this study, we investigated a group of SNPs in Saudi individuals and compared their frequency in patients and healthy normal controls. Methods: A total of 320 individuals were included in the study: 85 with PC, 120 with benign nodular hyperplasia, and 115 healthy normal controls. DNA was extracted from paran-embedded formalin-xed tissue from PC and nodular hyperplasia patients and from whole blood of healthy controls. A total of thirteen SNPs were genotyped using the TaqMan® MGB PCR assay. Results: The rs16901979A, s629242T and rs1447295A alleles were found at signicantly higher frequencies in PC patients than in controls (p < 0.05). The C/A genotype of the rs16901979 SNP was observed signicantly more frequently in PC patients than in controls (43% vs 14%, OR = 0.2, p value = 0.0001) and more frequently in PC patients than in the benign hyperplasia group (43% vs 25%, OR = 2.3, p value = 0.03). Conclusion: Our study highlighted several SNP genotypes associated with PC development in Saudi males. These ndings have important implications for diagnosing PC and screening unaffected family members of Saudi patients.

Genome-wide association studies (GWAS) have been used over the last several years to measure and analyze the DNA sequence variations across the PC genome and identify possible genetic risk factors for disease associations. No single gene is responsible for PC, but some studies have demonstrated that BRCA1 and BRCA2 mutations, which are associated with ovarian and breast cancers in women, are also risk factors for PC. Men with positive BRCA2 mutations are vefold more likely to have PC than negative individuals (10,11). A group of studies have demonstrated that single nucleotide polymorphisms (SNPs) are risk factors for PC (12)(13)(14)(15). Carriers of the homozygous T allele of the rs10993994 SNP were found to have a higher risk for PC than those having the homozygous C allele (16). In this study, we investigated a group of SNPs in Saudi individuals with the aim of determining their possible genetic variations and comparing the outcomes between patients and healthy normal controls.

Patients and control
A total of 211 patients were initially recruited for this study, 16 of whom were later excluded because they were duplicates or were not Saudi nationals. The remaining patients were categorized as patients with PC (group 1) and patients with nodular hyperplasia (group 2). Para n imbedded tissue sections were obtained for each patient group. Peripheral blood in EDTA, obtained from blood bank donors, was collected from healthy normal individuals (with no history of PC) as unrelated Saudi controls (group 3). The study was approved by the institution's IRB.
DNA extraction from whole blood and para n embedded tissue Extracted DNA from archived para n embedded tissue has been used in the study of normal/diseased tissues utilizing various genetic techniques, including genome-wide pro ling technologies, such as microarrays (17)(18)(19). A MagNa pure compact instrument (Roche Diagnostics GmbH, Roche Applied Science, 68298 Mannheim, Germany) was used for DNA extraction from whole blood, and the ReliaPrep™ FFPE gDNA Miniprep System kit (https://worldwide.promega.com) was utilized for DNA extraction from para n-embedded blocks. Both methods were performed according to the manufacturer's instructions. A Nanodrop® 2000c spectrophotometer (Thermo Fisher Scienti c, Wilmington, DE, USA) was used to determine DNA quality and quantity, and a concentration of 20 ng/µl of DNA with a ratio of 260/280 between 1.6 and 2.0 was accepted for the assay genotyping.

SNP Genotyping
Thirteen SNPs, including rs4430796, rs1859962, rs16901979, rs6983267, rs1447295, rs1571801, rs4054823, rs1545985, rs7652331, rs629242, rs13149290, rs251177 andrs10492519, were genotyped. Applied Biosystems kits (Foster City, CA, 94404) were utilized, and the SNP assay contains sequencespeci c forward and reverse primers with two TaqMan® MGB probes and their dyes (VIC™ and FAM™) used to detect alleles 1 and 2 for each SNP. The procedure was carried out according to the manufacturer's instructions, and a Light Cycler®480 instrument (Roche Diagnostics Ltd, Ferrenstrasse, CH-6343 Rotkreus, Switzerland) was used for data collection. Multiple DNA samples were replicated to ensure the quality of the tested samples, and positive and negative samples were also included in each batch as controls.

Statistical analysis
Conformation for Hardy-Weinberg equilibrium (HWE) was analyzed in control data implementing the x 2 distribution (degree of freedom = 1) to detect the differences between the expected and observed values in the tested SNPs, as described by Rodrigquez et al. (20). The frequencies of alleles, genotypes and the genotype models for each SNP (dominant and recessive traits) were derived by an algorithm based on the direct counting method using SNPStats software (https://www.snpstats.net/start.htm), and the results were expressed as percentages at 95% con dence intervals (CIs). Fisher's exact test was used to compare patients and control data. The log of the odds (logit) for the presence of each SNP was calculated to determine signi cant differences at p values < 0.05.

Results
A total of 320 individuals were recruited for this study; 85 were patients with PC, with ages ranging from 60 to 70 years (mean = 69.5, median = 71); 120 were patients with benign nodular hyperplasia, with ages ranging from 62 to 76 years (mean = 67.5, median = 70); and 115 were healthy normal controls, with ages ranging from 45 to 67 years (mean = 52.6, median = 60).
Tested HWE for allele frequencies on healthy control data showed a conformation to expected distribution in 11 SNPs out of 13; rs1447295 and rs1545985 SNPs were not found within the expected distribution for HWE (p values < 0.01). The comparison of the allele frequencies in the three different groups is shown in Table 1. The rs16901979A allele was found at a signi cantly higher frequency in PC patients than in healthy normal controls (23% vs 8%, OR = 0.3, 95% CI = 0.2,0.6; p value = 0.0002); this result indicates that individuals carrying this allele are vefold more susceptible to developing PC than negative individuals. The rs16901979A allele was also found at a signi cantly greater frequency in PC patients than in benign hyperplasia patients (23% vs 14%; OR = 0.4; 95% CI = 0.3, 1.0; p value = 0.049) with 2.5-fold higher disease susceptibility. Regarding the s629242 SNP, the T allele was found at a signi cantly higher level in PC patients than in healthy controls (31% vs 22%; OR = 0.6, 95% CI = 0.4, 1.0; p value = 0.03). The rs1447295A allele was detected at a signi cantly higher level in PC patients than in benign hyperplasia patients (12% vs 5%; OR = 0.4; p value = 0.0027) and in benign hyperplasia patients than in normal healthy controls (11% vs 5%; OR = 2.1; p value = 0.045). No signi cant differences were demonstrated in other allele frequencies among these three different groups. The comparison of genotype analysis is shown in Table 2. In this regard, we found the C/A genotype of the rs16901979 SNP at a signi cantly higher frequency in PC patients than in normal healthy controls (43% vs 14%, OR = 0.2, p value = 0.0001) and in PC patients compared to the benign hyperplasia group (43% vs 25%, OR = 2.3, p value = 0.03).

Discussion
SNPs may have a functional role in causing amino acid changes, mRNA transcript instability and transcription factor binding a nity variations (21)(22)(23). In this study, we characterized 13 different SNPs utilizing TaqMan Real Time PCR assays. The rs16901979 SNP showed signi cant differences in allele and genotype frequencies. The rs16901979A allele was found at a higher frequency in PC patients than in normal healthy controls, and individuals with this allele were 3.3 times more susceptible to PC. However, no signi cant difference was observed between the nodular hyperplasia patients and normal healthy controls. Accordingly, this SNP may be considered a risk genetic factor for PC disease but not for nodular hyperplasia development. A comparison of the genotype model for rs16901979 (C/C vs A/C) has shown that individuals with the A/C genotype are at 5 times higher risk for PC development than those with the C/C genotype but not for nodular hyperplasia. Additionally, we observed a minor presence of the A/A genotype among the three groups. The presence of this genetic component may indicate its lethal effect for carrier individuals among the Saudi population. The rs16901979 SNP was found to be associated with PC cancer in a case control study by Robbins et al. that included 490 prostate cancer patients and 567 healthy controls of African American individuals (24). A similar association was found in a Taiwanese study by Chen et al. (25). In a multicenter study of the Swedish population that included 2893 patients with PC and 1781 control subjects, rs16901979 was also found to be associated with PC (12). The Rs16901979 SNP is present within the non-protein-coding region of the CASC8 gene, located at 8q24 of the human genome map. GWAS and several case-control studies have demonstrated the association of speci c variants in the CASC8 gene with prostate cancer (26), and it has been suggested that 8q24 regions can independently in uence the risk for prostate cancer development and advanced disease status in particular (27).
Additionally, the rs629242T allele was found at a signi cantly higher frequency among PC patients than among healthy normal controls. In general, rs629242T is observed less frequently than rs629242C, which is classi ed as the mutated allele for the rs629242 SNP (23) and is considered a risk factor for PC occurrence with a 1.6 odds ratio. A study investigating the role of rs629242 in PC found that the rs629242T allele infers a 29% increased risk for disease progression in the African American population (28). The rs629242 SNP is present within the KIAA1211 gene located at the 4q12 region. This gene encodes an actin cytoskeletal regulator that plays a role in maintaining the integrity of epithelial cells and suppressing tumorigenesis (29).
The rs1447295A allele was found at a signi cantly lower rate in benign nodular hyperplasia patients than in normal healthy controls or PC groups but with no difference between the healthy controls and PC. This result is in keeping with ndings obtained by Robbins et al., who concluded that there is no association between the rs1447295 SNP and PC and among the African American population (24,30). However, the rs1447295 SNP was demonstrated to be associated with PC and related clinical covariables in northern Chinese men (31) and in a meta-analysis study performed by Cheg et al. (27). These contradictions may be observed because the interaction between genetic susceptibility and environmental factors plays an important role in disease development (24,30). Based on our ndings, we suggest that this rs1447295 SNP may be used as a genetic marker to differentiate between benign and malignant tumors in Saudi males with prostatic disease.

Conclusions
Our study has highlighted the association of several SNP genotypes with PC development in Saudi males. In addition to the potential utility of these results in PC diagnosis, these ndings may be useful in screening unaffected family members of patients. Availability of data and materials The datasets used in the current study are available from the corresponding author on reasonable request.

Ethics declarations
This study was approved by Institutional Review Board at King Fahad Medical City, Riyadh (IRB Log No. . Patients and Controls consent were exempt by IRB committee because we used leftover samples after clinical tests were performed.

Consent for publication
Not applicable