Association between the Prostate-Specific Antigen Gene and the Risk of Prostate Cancer in a Taiwanese Population
Marcelo Chen1,2, Yu-Chuan Huang3, Stone Yang4, Yi-Ming Arthur Chen5
1Department of Urology, Mackay Memorial Hospital, Hsinchu, Taiwan
2Division of Preventive Medicine, Institute of Public Health, National Yang-Ming University, Taipei, Taiwan
3Genetics Center, Department of Medical Research, China Medical University Hospital, Taichung, Taiwan
4Department of Urology, Mackay Memorial Hospital, Taipei, Taiwan
5Institute of Microbiology and Immunology, School of Medicine, National Yang-Ming University, Taipei, Taiwan
Running Title: PSA Gene and Risk of Prostate Cancer in Taiwan Address reprint requests and correspondence to:
Yi-Ming Arthur Chen
School of Medicine
National Yang-Ming University 155, Sec. 2, Li-Noun Street Shih-Pai, Taipei 112 Taiwan
Telephone number: 886-2-28267193 Fax number: 886-2-28270576
Abstract
Objective: To determine the association between a G/A polymorphism at position -158 of the prostate-specific antigen (PSA) gene and the risk of prostate cancer in Taiwanese men.
Materials and Methods: We genotyped 149 prostate cancer patients and 176 healthy controls. Logistic regression was used to estimate odd ratios (OR) and confidence intervals (CI).
Results: The G allele was more frequent than the A allele in both cases and controls. The A allele was associated with a statistically insignificant higher risk of prostate cancer (OR=1.17, CI=0.78-1.76). The GA (OR=1.18) and AA (OR=1.19) genotypes were associated with a statistically insignificant higher risk of prostate cancer. In an analysis by disease aggressiveness, aggressive disease had a higher OR than that for nonaggressive disease (1.38 vs. 0.77); however, these associations were not statistically significant.
Conclusion: No association was found between the polymorphism and the risk of prostate cancer. The A allele may be associated with aggressive prostate cancer. Key words: prostate cancer, genetic polymorphism, prostate-specific antigen gene
Introduction
Prostate cancer is the sixth most common type of cancer and it is the seventh leading cause of cancer death in Taiwanese men [1]. Its incidence in 2004 was 16.35 per 100,000 and its mortality rate was 7.12 per 100,000.
Age, race, genetics, diet, environment, infectious agents, physical activity, sexual activity, smoking and vasectomy have been reported to be potential risk factors of prostate cancer.
The association between genetic polymorphisms and prostate cancer has been extensively studied. Polymorphisms at the -158 position of the prostate-specific antigen (PSA) gene have been proposed to be associated with an increased risk of prostate cancer [2]. We therefore conducted a case control study to verify this occurrence in a Taiwanese population.
Materials and Methods
Peripheral blood lymphocytes were collected from 149 prostate cancer patients and 176 healthy controls from Mackay Memorial Hospital in Taiwan. Demographic and clinical data were obtained from medical records. Men of ethnicity other than Chinese Han were excluded from this study. This subject was approved by the Institutional Review Board of Mackay Memorial Hospital and written informed consent was obtained from all study subjects.
A 5-ml sample of peripheral blood was obtained from each prostate cancer patient and control subject. Buffy coat samples were prepared immediately and stored at -80℃ until extraction of the genomic DNA. Genomic DNA was obtained by conventional phenol/chloroform extraction, followed by ethanol precipitation, and stored at -20℃ until used for genotyping by polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP).
The NheI restriction enzyme was used to distinguish the allele of the G/A polymorphism at position -158 in the promoter region of the PSA gene [2]. Amplification of the polymorphic site was performed by use of a forward
primer (5’-TTG TAT GAA GAA TCG GGG ATC GT-3’) and a reverse primer (5’-TCC CCC AGG AGC CCT ATA AAA-3’). Polymerase chain reaction was carried out with 20 ng of genomic DNA and 10 pmol of each primer in a 20-μl reaction volume containing 2 mM MgCl2 under the following conditions: 93℃ for 10 minutes, 35 cycles at 94℃ for 1 minute, 59℃ for 1 minute, 72℃ for 40 seconds, and a final cycle at 72℃ for 10 minutes. Digestion of a 7-μl aliquot was conducted with 1.5 units of NheI restriction enzyme (New England Biolabs, Beverly, MA, USA) in 1.5μl of 10× BSA and 6μl of water at 37℃ for over 3 hours and the fragments were then separated on a 2.5% agarose gel. The three distinct banding patterns seen represented the three possible genotypes: AA (300 base pairs), AG (150 and 300 base pairs), and GG (150 base pairs).
The allelic and genotypic frequencies of the PSA gene were then determined. Statistical methods were used to determine the significance of the differences of the allele and genotype frequencies between the case and control groups. Logistic regression models were used to estimate odd ratios (OR) and their corresponding 95% confidence intervals (CI) between the case and control groups. The Statistical Package for the Social Sciences, version 11 (SPSS, Inc., Chicago, Illinois, USA) was used for statistical
Results
In this study, 149 prostate cancer patients and 176 controls were recruited. The mean age of the prostate cancer patients was 75.48 ± 5.31 years, and the mean age of the control group was 72.79 ± 7.01 years (p<0.01). The mean Gleason score of the prostate cancer patients was 6.84 ± 1.56.
The allelic frequencies of the G and A alleles at position -158 of the PSA gene are shown in Table 1. The frequency of the G allele was higher than that of the A allele in both the prostate cancer (81.9% vs. 18.1%) and control groups (84.1% vs. 15.9%). The control group had a slightly higher frequency of the G allele than the prostate cancer group (84.1% vs. 81.9%). The A allele appeared to be associated with a higher risk of prostate cancer (OR=1.17), but this association was not found to be statistically significant (CI=0.78-1.76).
The genotypic frequencies of the GG, GA, and AA single nucleotide polymorphisms are also shown in Table 1. The control group had a slightly higher frequency of the GG genotype (71.6% vs. 67.1%). When compared to the GG genotype, the GA genotype (OR=1.18) and AA genotype (OR=1.19)
were associated with a higher risk of prostate cancer. However, these associations were not found to be statistically significant (CI=0.71-1.96 and 0.35-4.08, respectively). Because the number of subjects with the AA genotype was relatively small, subjects with the GA and AA genotypes were pooled together and compared with the GG genotype. However, the increased risk of prostate cancer (OR=1.18) was also not found to be statistically significant (CI=0.72-1.92). PSA level appeared to be slightly higher in the GG genotype group (382.2±1581.5 ng/ml) when compared with the GA and AA genotype group (301.2±697.0 ng/ml); however, this difference was not statistically significant (p=0.74).
With regard to disease aggressiveness, aggressive disease appeared to have a higher OR than nonaggressive disease (1.38 vs. 0.77) (Table 2). Even though these associations were not statistically significant, the risk allele appeared to be associated with more aggressive prostate cancer.
Discussion
The PSA gene is a kallikrein-like protease synthesized by the luminal epithelial cells of the prostate [3]. PSA expression is regulated by androgens via the androgen receptor. The androgen response element I (ARE-I) is one of three AREs identified on the PSA gene promoter. A single nucleotide polymorphism, an adenine to guanine substitution at position –158 in the ARE-I has been described [4], and Xue et al have proposed that the androgen receptor binds the A and G alleles with differing affinity thereby influencing prostate cancer risk [2].
Several studies have investigated the relationship between this polymorphism and the risk of prostate cancer with contradictory results. While several studies have shown an association between the GG genotype and advanced prostate cancer at the time of diagnosis [2, 5-7], our results showed that the GG genotype was not associated with advanced disease.
Some studies have shown that the GG genotype conferred an increased risk of prostate cancer [2, 7], others have reported no significant association [8-10], while a study suggested that the G allele had a protective effect against prostate cancer [5].
In a study of a Taiwanese population, Chiang et al reported significantly greater frequencies of the G allele (87.3% vs. 61.9% in controls) and GG genotype (78.7% vs 61.9% in controls) in prostate cancer patients [11]. However,
our study contradicted these results as the G allele and GG genotype were found in greater frequencies in controls. Our findings, however, were not statistically significant. We cannot provide an adequate explanation for this contradiction, but we propose that the association between this polymorphism and the risk of prostate cancer in a Han population may be weaker than previously thought.
Allelic frequencies may also vary according to ethnicity. The A and G alleles have been reported to occur in equal frequencies in Caucasian and African-American populations. [4] A Japanese study by Wang et al reported that the A allele was less common (22% vs 78% for the G allele) [8], and the Taiwanese study by Chiang et al showed that the A allele was present in only 13% of prostate cancer patients [11]. Our study results corroborated this finding as the A allele frequency in prostate cancer patients was calculated to be 18%.
The main limitations of this study were that PSA level was not obtained in control subjects and that age-matching was not performed between controls and cases. Even though elevated PSA could also be caused by infection, benign prostatic enlargement or instrumentation, markedly elevated PSA may have warranted pathological examination to rule out the presence of prostate cancer. It is therefore possible that some of the subjects grouped as controls may harbor prostate cancer. The control subjects were on average about 2 years younger than
the prostate cancer cases. The incidence of prostate cancer increases with age, so it is possible that some of the subjects originally grouped as controls could develop prostate cancer as they grow older.
Conclusion
In this study, the PSA -158 A/G polymorphism was not found to be associated with the risk of prostate cancer in a Taiwanese population. The frequency of the A allele in prostate cancer patients is lower in Asian than in Caucasian or African-American populations. The A allele may be associated with aggressive prostate cancer.
Acknowledgement
This study was supported by the Mackay Memorial Hospital and National Yang-Ming University (MMHYM) Joint Research Program (grant number MMHYM93-N010-016) from Mackay Memorial Hospital.
References
1. Department of Health, Executive Yuan, ROC (Taiwan). Summary of statistics on causes of death in Taiwan, 2004.
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3. Watt KW, Lee PJ, M’Timkulu T, Chan WP and Loor R. Human prostate-specific antigen: structural and functional similarity with serine proteases.
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4. Rao A and Cramer SD. Identification of a polymorphism in the ARE 1 region of the PSA promotor. Proc. Am Assoc Cancer Res 1999;40:65.
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6. Medeiros R, Morais A, Vasconcelos A, et al. Linkage between polymorphisms in the prostate specific antigen ARE 1 gene region, prostate cancer risk and circulating tumor cells. Prostate 2002;53:88–94.
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Table 1. Allelic and genotypic frequencies of PSA gene polymorphism in prostate cancer patients and controls.
PSA gene -158 G/A Cases Controls OR (95%CI) polymorphism n=149 (%) n=176 (%) G-allele 244 (81.9) 296 (84.1) 1.00 (referent) A-allele 54 (18.1) 56 (15.9) 1.17 (0.78-1.76) GG 100 (67.1) 126 (71.6) 1.00 (referent) GA 44 (29.5) 44 (25.0) 1.18 (0.71-1.96)* AA 5 (3.4) 6 (3.4) 1.19 (0.35-4.08)* GA+AA 49(32.9) 50 (28.4) 1.18 (0.72-1.92)* *Age-adjusted OR
Table 2.Genotypic frequencies in prostate cancer patients by Gleason score Group GG GA AA OR (95% CI) p-value n (%) n (%) n (%) Control 126 (71.6) 44 (25.0) 6 (3.4) 1.00 (Ref) Aggressiveness* Nonaggressiveness PCa 23 (76.7) 7 (23.3) 0 (0) 0.77 (0.31-1.90) 0.77 Aggressiveness PCa 77 (64.7) 37 (31.1) 5 (4.2) 1.38 (0.84-2.26) 0.21
中文抄錄 台灣人攝護腺特異性抗原基因之基因多型性與攝護腺癌之相關性研究 陳建志1,2, 黃毓銓3, 楊志東4, 陳宜民5 1新竹馬偕醫院泌尿科 2國立陽明大學公共衛生研究所預防醫學組 3中國醫藥大學附設醫院醫學研究部遺傳中心 4台北馬偕醫院泌尿科 5國立陽明大學微生物及免疫學研究所 研究目的：瞭解台灣人攝護腺特異性抗原基因在驅動子-158 位置之 G/A 基 因多型性與攝護腺癌之易罹患性。 材料與方法：我們以病例對照研究法收集馬偕醫院149 位攝護腺癌患者及 176 健康健檢者，以 PCR-RFLP 的方法定義攝護腺特異性抗原基因-158 G/A 的基因型，並利用邏輯式回歸方法分析不同基因型罹患攝護腺癌的勝算比 (odds ratio; OR)及 95%信賴區間。
結果：在攝護腺癌患者帶有G 對偶基因及 A 對偶基因的比率分別 81.9%及
18.1%；在健康健檢者則為 84.1%及 15.9%。A 對偶基因有較高的勝算比罹患 攝護腺癌，但不具統計上的顯著差異(OR, 1.17; 95% CI = 0.78-1.76)；相較於 GG 基因型，GA 及 AA 基因型有較高的勝算比罹患攝護腺癌，不過亦不具
0.35-4.08)；此外，在細胞惡性程度較高的病人(Gleason score > 7)，有 27.8% 的患者基因型為GG 型；34.1%的患者基因型為 GA 或 AA 型。 討論：由以上結果推測攝護腺特異性抗原基因在-158 G/A 的基因多型性和台 灣人罹患攝護腺癌並不相關；且相較於其他人種，亞洲人有較低的比率帶有 A 對偶基因；此外，A 對偶基因可能和細胞惡性程度較高的攝護腺癌有關。 鍵語: 攝護腺癌, 基因多型性, 攝護腺特異性抗原基因
