Fall 2012 - 08.pdf 691Vol. 9 | No. 4 | Fall 2012 |U R O LO G Y J O U R N A L Association of G/A Polymorphism, rs266882, in AREI Region of the Prostate-Specific Antigen Gene with Prostate Cancer Risk and Clinicopathological Features Mohammad Samzadeh,1 Mandana Hasanzad,2 Seyed Hamid Jamaldini,3 Ali Akbar Hagh- doost,4 Mahdi Afshari,4 Seyed Amir Mohsen Ziaee1 Purpose: - phism with clinicopathologic characteristics of the disease and prostate cancer (PCa) risk. Materials and Methods: 111 subjects with benign prostatic hyperplasia (BPH), were recruited in this study. Genotyping was performed by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism method. Results: compared to AG genotype (adjusted odds ratio = 2.4; P = .03). The percentages of G alleles of polymorphisms in patients with PCa were more than that in ones with BPH (odds ratio = 1.2; P = .7). Conclusion: The GG genotype of PSA 158A/G polymorphism is a predisposing factor for PCa. But no association was observed between alleles and grade, stage, or age of diagnosis. Simi- larly, the rs266882 polymorphism was not associated with PSA plasma levels. Keywords: polymorphism Corresponding Author: Seyed Amir Mohsen Ziaee, MD Urology and Nephrology Research Center, No. 103, 9th Boustan St, Pasdaran Ave, 1666677951, Tehran, Iran Tel: +98 21 2256 7222 Fax: +98 21 2256 7282 E-mail: samziaee@hot mail.com Received December 2011 Accepted May 2012 1Urology and Nephrology Research Center, Shahid Lab- bafinejad Medical Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2Tehran Medical Branch, Islamic Azad University, Tehran, Iran 3Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran 4Research Center for Modeling of Health, Kerman University of Medical Sciences, Kerman, Iran CELLULAR AND MOLECULAR UROLOGY 692 | INTRODUCTION P rostate malignancy, the second most common can- cer in men, accounts for 32% of all types of can- cer and is the 4th most frequent cancer in the world. Although the main etiological cause of the disease is still unclear, it is known to be a multifactorial disease with a genetic component.(1) - dase 3, is a serine protease that is part of the kallikrein su- perfamily, mainly produced by prostate cells.(2) It has wide- ly been used as a diagnostic marker of the prostate cancer (PCa) since the early 1990s. Serum level of PSA increases with benign prostatic conditions, but a higher surge of it is seen with PCa and that is why it is the most commonly used clinical biomarker for PCa diagnosis and follow-up. Early detection through serum testing for PSA and im- proved procedures for surgical intervention and radiation however, there is still no effective cure for men with ad- vanced disease. Therefore, much research has been dedi- cated to identifying prognostic markers that distinguish in- dolent versus aggressive forms of PCa.(3) Recent technological advances have allowed investigators to interrogate thousands of single nucleotide polymor- genetic markers associated with risk of developing this - liferation of the prostate gland are not well-known, but the gene for PSA has been the focus of several studies.(3) The PSA gene is mapped on chromosome 19 (19q13.4).(4) androgens. Production of PSA is mediated through bind- ing of the androgen receptor (AR) to androgen response elements (ARE) in the promoter region of the PSA gene. (4) After binding of androgen to the AR, a cascade of cel- lular events happens, which causes the movement of AR to the nucleus, where it binds to AREs in the promoters of target genes to initiate PSA gene transcription. At least region.(5) The one nearest to the transcription start site is referred to as AREI. Several polymorphisms have been reported which can in- (6) A recent study men- tioned using this SNP as a predictive marker.(7) The PSA gene contains a polymorphism, substitution of a Guanine (G) by an Adenine (A), locating 158 bases upstream of the transcription site, which have transcriptional control role related to serum levels of PSA.(3,6,8-10) With regard to the role of PSA G158A SNP, a number of surveys have been focused on the association between this SNP, serum level of PSA, and PCa susceptibility in differ- ent populations. Since the PSA test in screening patients who had to undergo prostate biopsy encounters many chal- lenges, using the PSA 158 A/G polymorphism genotypes combined with the result of serum PSA is a method for de- creasing unnecessary biopsies. This study investigated any relation between PSA 158 G/A polymorphism and risk of PCa, tumor stage and grade, and patients with PCa. MATERIALS AND METHODS This case-control study consisted of 206 subjects, including 95 patients with PCa and 111 controls with benign prostatic hyperplasia (BPH), who were recruited from department of Iran, between February 2010 and April 2011. The present study was approved by the Urology and Neph- rology Research Center Review Board (www.unrc.ir) af- Tehran, Iran. After a written informed consent was obtained from each participant, a structured questionnaire was com- pleted to gather information on potential risk factors, in- st-degree relatives, blood group, and total and free PSA level. Blood samples were taken using sample tubes containing - tion was performed according to the standard protocol.(11) C. Open laparoscopic or radical prostatectomy was used to determine the tumor grade and stage and perineural and vascular invasion. Tumor stage and grade are determined according to 1997 TNM guideline(12) and Gleason grading Cellular and Molecular Urology 693Vol. 9 | No. 4 | Fall 2012 |U R O LO G Y J O U R N A L rs266882 Polymorphism on Prostate Cancer Risk | Samzadeh et al system, from 1 (most differentiated) to 5 (least differenti- ated), respectively. Control groups with BPH had the following criteria in order to rule out the false diagnosis of PCa: 1) Either serum PSA < 4.0 ng/mL or negative pathological report of the pros- tate biopsy with serum PSA > 4.0 ng/mL; 2) Normal digital malignancy in resected prostatic tissues from open surgi- of PCa in control group, consuming any PSA decreasing medication, hormone therapy, orchiectomy, and non-adeno- carcinoma of the prostate. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Analysis Polymerase chain reaction (PCR) was performed using 20 pmol of forward primer (5'-TTG TAT GAA GAA TCG GGG ATC GT-3') and reverse primer (5'- TCC CCC AGG AGC CCT ATA AAA-3') in a 50-μL reaction volume con- taining 2 mM MgCl 2 . The PCR program was 94 °C for 10 min followed by 35 cycles at 94 °C for 1 min, 59 °C for 1 for 10 min. After PCR, a restriction fragment length poly- morphism (RFLP) method was used with NheI restriction enzyme (Roche), then, separated on a 2% agarose gel. The three genotypes were determined according to their size: AA (300 bp), AG (150, 300 bp), and GG (150 bp) (Figure 1). by direct sequencing of several PCR samples with each genotype (Figure 2). Statistical Analyses Data analysis was performed by STATA (V.11) software. Chi-Square test and Student’s t test were used for evaluat- ing the association between categorical variables and mean values of variables, respectively. Serum levels of PSA were estimate the effect of polymorphisms on serum total PSA. Multivariable logistic regression models were used to de- termine odds ratio (OR) for categorical dependent variables adjusting for potential confounders, such as age, family his- tory of cancer, and grade and stage of tumor. Results were P value was less than .05. Using the PS Power and Sample Size Calculations software, Version 3.0, January 2009 (http://biostat.mc.vanderbilt. edu/PowerSampleSize) (William D. Dupont and Walton D. Plummer, 2009), our sample size in different subgroups .05) to detect an OR of 2.4. RESULTS Subjects’ Characteristics The average ± standard deviation age of the patients was 67 ± 8.7 years (range, 47 to 89 years). Total PSA levels were serum total PSA was 467 ng/dL more than that in patients with BPH (P < .001). In this group, the percentage of posi- that in patients with BPH (17.4% versus 1%; P < .001). In overall, the education level of patients with PCa was sig- P < .001; Table 1) Among patients, 31% had poorly differentiated tumors diagnosis (TNM stage III and IV) (Table 2). PSA and Risk of PCa Having considered the AG genotype as the reference group between PSA polymorphisms, patients with GG genotype had 1.2-fold greater risk of developing high-grade (Gleason - P = .7). The OR between GG and AA genotypes and stages of PCa was 0.75 and 0.78, respectively, indicating that these polymorphisms had a protective effect on the high level of disease stages compared to AG genotype as the refer- ence group. But the adjusted OR was slightly more than null value (Table 3). Results showed that there was not a statistical association between polymorphisms and vascular or perineural invasion. The mean age in those who had different polymorphisms indicated that patients with GG and AA genotypes had higher mean ages compared to the reference group (AG genotype). It means that patients with these polymorphisms could get the PCa at higher ages compared to those without this polymorphism. In other words, it can be said that poly- 694 | reducing the onset and also the time of diagnosis of PCa. The mean total PSA in those who had GG and AA geno- types was around 1.2 and 0.2 ng/dL more than that in those with AG genotype (P = .9 and P = .2, respectively); adjust- ing for other factors did not change this difference consider- ably (Table 3). PSA Genotypes and PCa and control groups for any of the PSA ARE-I genotypes. However, some kinds of polymorphisms, like GG geno- type, had more frequencies in patients with PCa (32%) than in BPH group (22.6%). The OR between GG polymorphism and AG polymorphism (reference) was 1.76. Having ad- justed for potential confounders, such as age and stage and grade of tumor, this gap became more prominent and sta- P = .03); it means 2.4-fold greater risk of developing PCa in patients with GG geno- Cellular and Molecular Urology Figure 1. Representative screening for the prostate-specific antigen genotypes. Left to right: line 1: GG, line 2: GG, line 3: AG, line 4: AG, line 5: AA, and line 6: AA Figure 2. A sample of sequencing result for confirming prostate-specific antigen genotyping. Arrow is the position of rs266882. 695Vol. 9 | No. 4 | Fall 2012 |U R O LO G Y J O U R N A L rs266882 Polymorphism on Prostate Cancer Risk | Samzadeh et al was observed between AA genotype and PCa (Table 4). The Allele Frequency of PSA Gene Patients with PCa had a greater percentage of G alleles than the control BPH ones; however, it was not statistically sig- - ment of PCa was 1.22, which means 1.2-fold greater risk of developing PCa compared to A allele, but that was not Table 2. Frequency of different stages and grades of prostate cancer in the study population. Grade and Stage Number Percent Grade Gleason < 7 Gleason > 7 58 70.65 26 29.35 TNM Stage I and II III and IV 63 78.16 17 21.84 Table 1. Comparison of main variables in patients with prostate cancer and benign prostatic hyperplasia.* Variables Cancer Benign prostatic hyperplasia P number percent number percent Smoking Never Stop Currently 63 67.7 88 83 .02911 11.8 9 8.5 19 20.4 9 8.5 Drug history (finasteride) 14 14.7 55 49.5 < .001 Marital status 91 97.8 104 98 .89 Family history of prostate cancer 16 17.4 1 1 < .001 > 40 year’s son (> 2 sons) 0 0 6 15.4 < .001 Education Illiterate Primary Diploma Academic 15 16.3 32 31.7 < .001 36 39 4 53.5 23 25 11 10.9 18 19.6 4 4 Blood group O A B AB 32 36.8 31 36 .78 39 45 34 39.5 15 17.2 20 23.3 1 1.1 1 1.2 Other disease None CVD HLP Breast cancer DM 75 78.9 102 92 .76 14 14.7 7 6.3 2 2.1 0 0 1 1.1 0 0 3 3.2 2 1.8 Abnormal PSA Ratio 45 50.6 14 17.9 < .001 Mean (SD) Mean (SD) Total PSA 474.4 (4299) 7.3 (7.11) < .001 Age 63.2 (7.34) 70.3 (8.53) < .001 Body mass index* 25 (3.11) 25.2 (3.50) .67 *CVD indicates cardiovascular disease; HLP, hyperlipidemia; DM, diabetes mellitus; PSA, prostate-specific antigen; and SD, standard devia- tion. 696 | DISCUSSION In previous studies, it was demonstrated that PSA gene ho- mozygous for the G allele is associated with higher serum PSA concentration and some other tumors(3,6,13-15) whereas the homozygous for the A allele is associated with a higher level of total PSA in men with or without PCa.(9,10) In a case-control analysis of 500 Caucasian cases and 676 con- and total and free PSA plasma levels.(16) In the present study, the total PSA levels in those who had GG genotypes Cellular and Molecular Urology Table 3. Relationship between AREI (PSA-158 G/A) genotype and prostate cancer stage and grade, total PSA level, perineural and vascular invasion of cancer, and age at diagnosis.* GG GenotypeAG GenotypeAA GenotypeOverall PProstate Cancer 0.9 (Based on Chi-Square)Stage 21 (33)22 (35)20 (32) Stages 1and 2, n (%) 5 (29)7 (41)5 (29) Stages 3 and 4, n (%) 0.75 ( .7) 1.04 ( .9) 1 1 0.78 ( .7) 1.04 ( .9) OR (P) Crude Adjusted 0.9 (Based on Fisher’s Exact test)Grade 18 (31)22 (37.9)18 (31) Gleason < 7, n (%) 9 (34.6)9 (34.6)8 (30.7) Gleason ≥ 7, n (%) 1.2 ( .7) 1.80 ( .3) 1 1 1.08 ( .9) 1.51 ( .5) OR (P) Crude Adjusted 0.4 (Based on Fisher’s Exact test)Vascular invasion 20 (31)24 (37)20 (31) Negative, n (%) 0 (0)3 (75)1 (25) Positive, n (%) - - 1 1 0.4 ( .4) 0.6 ( .7) OR (P) Crude Adjusted 0.5 (Based on Fisher’s Exact test)Perineural invasion 3 (37)4 (50)1(12) Negative, n (%) 19 (27)26 (37.7)24 (34.8) Positive, n (%) 0.97 ( .9) 1.2 ( .8) 1 1 3.7 ( .2) 5.8 ( .15) OR (P) Crude Adjusted 0.7 (Based on ANOVA test)Age of diagnosis 497758 Number 66.9 (7.9)66.8 (9)67.9 (8.9) Mean age (SD) 3.3 ( .09) 3.9 ( .05) 0 0 0.9 ( .6) 1.9 ( .3) Mean difference (P) Crude Adjusted 0.6Total PSA level 456346 Number 15.7 (24)13 (17)12 (15.6) Mean PSA (SD) 1.2 ( .9) 1.06 ( .9) 0 0 0.2 ( .2) 0.2 ( .2) Mean difference Crude Adjusted *OR indicates odds ratio; SD, standard deviation; and PSA, prostate-specific antigen. 697Vol. 9 | No. 4 | Fall 2012 |U R O LO G Y J O U R N A L rs266882 Polymorphism on Prostate Cancer Risk | Samzadeh et al was 1.2 ng/dL more than that in those who had A/G, but like Turkish and Japanese population, it was not statistically (17) There are two different reports on the association of this SNP with serum PSA level in Japanese population. One showed association between higher PSA level and GG gen- otypes and the other showed no association between -158 G/A polymorphism and the serum PSA level.(18,19) A functional study has indicated that the PSA -158 G/A polymorphism has no functional effect on the activity of the PSA promoter in vitro and in vivo,(20) which supports It has been proposed that rs266882 polymorphism is asso- ciated with disease stage or grade.(6,9,21-26) The GG geno- type in Taiwanese is associated with larger tumor volume and higher pathological stage.(14) A large case-control study of Caucasian-Australians (821 patients and 734 controls) stage III to IV tumors,(15) whereas a larger case-control study on a white American population found that the GG genotype is associated with the lower stage of the disease. (22) - tween rs266882 and high grade or advanced stage of the disease. The GG genotype was associated with a 1.2-fold PCa (OR = 1.2; P = .7). Another study which investigated the association of rs266882 SNP with PCa risk found that this SNP was not associated with cancer grade.(27) Several investigators have studied the relation of the rs266882 polymorphism with PCa susceptibility. An ini- tial case-control study of non-Hispanic white men (57 patients and 156 controls) reported a positive association of homozygous variant GG genotype of rs266882 with a three-fold risk of advanced cancer suggesting that the PSA promoter activity under the control of allelic variation is an androgen-dependent event.(28) Subsequent studies on this polymorphism also reported increased risk of PCa with the G allele in Taiwan (122 patients and 84 controls),(14) Scotland (97 patients and 144 controls),(24) and Turkey (49 patients and 47 controls). In a larger sibling-based case- control study on a predominantly white American popula- tion, the association between PCa susceptibility and the G (22) A case-control study on patients with Turkish origin re- (29) Another study in 2008 found that the GG genotype carriers have a higher risk of developing PCa than those with the AG and AA genotypes. (30) - ciation between the A allele and increased risk of Pca.(10,23) Similarly, a larger study of Australian Caucasian men (209 patients and 223 controls) found a three-fold risk of PCa with the AA allele.(25) - Table 4. Genotype and allele frequency and odds ratio between different polymorphisms in patients with prostate cancer and be- nign prostatic hyperplasia.£ Polymorphism Cancer, no (%) Benign prostatic hyperplasia, no (%) Overall P* 95%CI Odds Ratio (P) Crude Adjusted PSA Polymorphism AG GG AA Polymorphism Allele G A .27 33 (38) 50 (47) 1 1 28 (32.2) 24 (22.6) 1.76 ( .1) 2.4 ( .03) 26 (29.9) 32 (30.2) 1.23 ( .5) 1.54 ( .3) .389 (51) 98 (46.2) 85 (49) 114 (53.8) *Based on Chi-Square test. £CI indicates confidence interval; and PSA, prostate-specific antigen. 698 | cantly increased the risk of cancer more than 2-fold com- pared to AG genotype (Adjusted OR = 2.4; P = .03), which is in line with several studies.(18,31,32) The percentages of G alleles of polymorphisms in patients with PCa were more than that in those with BPH. However, we found a 70% increased risk of PCa with GG polymorphism. In patients with PCa, allele frequency of the PSA polymor- phism at position -158 (A 0.49, G 0.51) was similar to Af- rican American men (A 0.52, G 0.48), non-Hispanic white men (A 0.48, G 0.52), and Hispanic white men (A 0.37, G 0.63),(9) and different from Turkish (A 0.63, G 0.36) and Japanese men (A 0.22 G 0.78). polymorphisms provided such inconsistent results cannot be fully understood, but some factors, such as ethnicity, life-style, and/or gene–gene, and gene–environment inter- by studies with larger number of advanced cases. CONCLUSION genotype of PSA polymorphism and PCa risk compared to AG genotype. But no association was observed between al- leles and grade, stage, or age of diagnosis. Similarly, the rs266882 polymorphism was not associated with PSA plas- ma levels and cancer risk. The difference in results for PSA AREI polymorphisms between studies may be minimized using larger study groups. 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