Association of Six Susceptibility Loci with Prostate Cancer in Northern Chinese Men

Prostate cancer (PCa) is one of the most common cancers affecting men worldwide. It has different morbidity rates in different countries. Although the American population has a higher morbidity than the Chinese population, the prevalence of PCa in China requires attention (Jamal et al., 2007; Zhang et al., 2011). Risk factors for PCa increase with age, ethnic background, and familial history of PCa (Chung et al., 2011). A genome-wide association study (GWAS) found


Introduction
Prostate cancer (PCa) is one of the most common cancers affecting men worldwide.It has different morbidity rates in different countries.Although the American population has a higher morbidity than the Chinese population, the prevalence of PCa in China requires attention (Jamal et al., 2007;Zhang et al., 2011).
Risk factors for PCa increase with age, ethnic background, and familial history of PCa (Chung et al., 2011).A genome-wide association study (GWAS) found

Study Population
To 2010 August, data were collected on 289 PCa patients (214 with nonaggressive PCa, Gleason score < 8, and disease stage < III; 75 with aggressive PCa, Gleason score ≥ 8, and/or disease stage ≥ III) and 288 controls among men of Chinese ancestry.The investigators conducted a case-control study.The mean age of the patients with PCa was 72.3 ± 7.48 years, and the mean age of the total control population was 70.5 ± 7.9 years.Patients were recruited at Beijing Hospital and Tianjin Urology Research Institute during the same period.
All PCa patients were diagnosed by histopathology.Blood samples and clinical information (family history of PCa, diagnostic age, body mass index [BMI], Gleason score, prostate-specific antigen [PSA] level, and neoplasm staging) were collected.Standards of controls were male, no family history of PCa, negative digital rectal examination, and PSA level < 4 ng/mL.Each tumor was graded using the Gleason score system and staged using the tumor-node-metastasis system.This study was approved by the Ethics Committee of Beijing Hospital and Tianjin Urology Research Institute, and informed consent was obtained from all study participants.

Reagents
Kits for extracting DNA were purchased from Bio Chain Company in Beijing.Taq DNA polymerase and deoxyribonucleotide triphosphates were purchased from Beijing Dingguo biotechnology Co. Ltd.LC-green PLUS saturated fluorescent dye was obtained from American Idaho Company.Synthetic primers were purchased from Shanghai Shenggong Biotechnology Co. Ltd.

SNP Selection for Genotyping
Six SNPs were selected from European decent risk loci (MSMB, rs10993994, T; 11p15, rs7127900, A; 11q13, rs7931342, T; HNF1B, rs4430796, A; 17q24, rs11859962, G; and KLK2, rs2735839, A).Blood genomic DNA was extracted using a whole blood genomic DNA extraction kit (Biochain Science-Technology, Beijing, China).Polymerase chain reaction (PCR) amplification and highresolution melting curve analysis of small amplicons were performed according to a study by Liew et al. (Liew et al., 2004).PCR was performed in a PTC-225 Tetrad® DNA Thermal Cycler under the following conditions: initial denaturation at 95°C for 5 min; 35 cycles at 95°C for 30 s, annealing for 30 s, and extension at 72°C for 6 s; and completion at 72°C for 7 min, followed by 2 cycles at 94°C for 30 s and at 24°C for 2 min.The automatically and manually verified PCR products were genotyped using the Light Scanner® TMHR-I 96.The PCR procedure included denaturation at 95°C for 5 minutes; 35 cycles at 95℃ for 30 seconds, annealing for 30 seconds, and extension at 72°C for 45 seconds; and completion at 72°C for 7 minutes (Liu et al., 2012).

Statistical Analysis
Fisher's exact test was used to evaluate Hardy-Weinberg equilibrium (HWE) for each SNP among cases and controls.Hardy-Weinberg balance testing reveals the representative of the sample group.Allele frequency and genotype differences between cases and controls were tested for each SNP using the chi-square test with 1 degree of freedom (Jielin et al., 2008).Odds ratios (ORs) and 95% confidence intervals (CIs) of the effect of each variant on PCa risk were computed using SHEsis and SPSS version 11.5 (P < 0.05).

Results
No significant difference was noted in mean age between cases and controls (72.3 ± 7.48 years versus 70.5 ± 7.9 years).There were 49 patients with a BMI value of 25 or greater and 85 patients with a BMI value of less than 25.Seventy-four patients had a PSA value of 20 or greater and 102 patients had a value of less than 10; a PSA value between 10 and 20 was reported in 39 patients.Fortytwo patients had a Gleason score of 8 or greater and 101 patients had a score of less than 8. Tumor stage ≥ III was reported in 57 patients and stage < III was reported in 78 patients (Table 1).
HNF1B is located on chromosome 17q12.It is a transcription factor that encodes 3 isoforms: transcriptional activators A and B and transcriptional repressor C. Two risk alleles for PCa have been detected in the HNF1B non-coding sequence (Gudmundsson et al., 2007;Waters et al., 2009;Setiawan et al., 2012). Jielin et al. (Jielin et al., 2008) first found PCa risk-associated SNPs at 17q12 (HNF1B) and 17q24.3.Furthermore, they showed that 2 17q SNPs had a risk genotype AA that plays a part in PCa.However, they did not find the association with clinical characteristics in their research.They believed that the SNPs at 17q12 and 17q24.3 were related to risk for more and less aggressive tumors; these SNPs likely influenced aspects of PCa initiation rather than progression (Sun et al., 2008).In our study, patients carrying TG on 17q24 (rs1859962, G) were negatively associated with an increased BMI (P = 0.03, OR = 0.44, 95% CI = 0.21-0.92).AG on HNF1B (rs4430796, A) was related to PSA increase (P = 0.002).In another study, researchers concluded from GWAS that HNF1B (rs4430796, A) was related to endometrial cancer risk in women of European background (Spurdle et al., 2011).HNF1B had also been reported to be associated with maturity-onset diabetes of the young subtype 5 (MODY5), renal cysts, pancreatic atrophy, and uterine abnormalities caused by incomplete Mullerian duct fusion and Mullerian duct aplasia.Interestingly, diabetes and PCa had a common biotic link; the former was associated with a decreased