Susceptibility Loci Associations with Prostate Cancer Risk in Northern Chinese Men

Prostate cancer (PCa) is the most commonly diagnosed noncutaneous cancer in the United States men with over 200,000 new cases and 30,000 deaths estimated in 2010 (Jemal et al., 2010). Amundadottir et al. (2006) evidenced a greater estimated Population attributable risk (PAR) (16%) that may contribute to higher incidence of PCa in African American men than in men of European ancestry (Amundadottir et al., 2006). The prevalence of PCa in Chinese population in mainland of China was low before, but with population ageing, the prevalence is increasing by years recently (Sun et al., 2004). PCa incidence was in the third place of malignant tumors in Chinese population and it has become a important risk to expectancy life of Chinese men over the age of 50 years old (Sun et al., 2004).

To determine whether those variants were also associated with prostate cancer risk in the northern Chinese population, we evaluated 6 prostate cancer susceptibility loci in a population based case-control study. All subjects were from Beijing and Tianjin, including 266 prostate cancer cases and 288 population controls. We also explored the relationship between PCa clinical covariates and the genetic risk variants to infer their impact on occurrence and aggression of prostate cancer in northern Chinese men.

Study Population
Permanent residents of Beijing and Tianjin involved in this study.The cases were diagnosed with histologically confirmed PCa at the Department of Urology, Beijing Hospital, Ministry of Health or Tianjin Urology Institute, Second People's Hospital of Tianjin Medical University between January 1, 2000 and December 1, 2010. PCa related clinical data, including age at diagnosis, serum PSA,Gleason score and tumor stage , were acquired from a medical record review. PSA more than 20 ng/mL, Gleason score 8 or higher, and/or pathological stage III or higher with PCa patients were defined as aggressive. Local residents participating in routine physical examination were defined as controls with age matched for this research in the Beijing and Tianjin. Those residents with PSA less than 4.0 ng/mL, negative digital rectal examination and no family history of PCa were also included in the control group. The study were a total of 266 PCa cases and 288 controls . All participants were men of unrelated Northern Han Chinese ancestry. We conducted a Consolidated Standards of Reporting Trials diagram of the flow of participants through each stage of the study. The ethics committee at the 2 participating hospitals approved this study and all subjects signed informed consent .None of the 288 controls had the family history with tumor.

SNP Selection for Genotyping
6 SNPs with PCa risk in previous GWASs of European and American populations were selected randomly. These loci included rs13385191, rs12653946, rs1983891, rs339331, rs9600079 and rs2735839. Whole blood genomic DNA extraction kit (Biochain (Beijing) Science-Technology Beijing, People's Republic of China) was used to extract blood genomic DNA. PCR-high resolution melting curves of small amplicons were done according to a previous study. PCR was performed using a PTC-225 Tetrad ® DNA Thermal Cycler under certain conditions, including initial denaturation at 95 ℃ for 3 minutes, followed by 45 cycles at 95℃ for 30 seconds, annealing for 30 seconds, extension at 72℃ for 4 seconds and completion at 72℃ for 2 minutes. After 2 cycles at 94℃ for 30 seconds and at 24℃ for 2 minutes. PCR products were genotyped automatically and verified manually using a LightScanner ® TMHR-I 96. Six samples were randomly selected from the 3 verified genotypes of each risk variant, amplified by PCR and sequenced elsewhere to confirm genotyping results. The PCR procedure involved initial denaturation at 95℃ for 3 minutes, followed by 35 cycles at 95℃ for 30 seconds, annealing for 30 seconds, extension at 72℃ for 30 seconds and completion at 72℃for 2 minutes.

Statistical Analysis
Pearson's chi-square test was used to test the Hardy-Weinberg equilibrium for each SNP separately among controls using a cut off of p>0.05. The OR and 95% CI were estimated for each risk allele vs each nonrisk allele. OR and 95% CI in models were calculated to compare genotype frequencies between PCa case and control using Pearson's chi-square or Fisher's exact test. Statistical analysis was done using SPSS ® , version 16.0 with <0.05 considered significant.

Subjective clinical data
The mean ages in case with PCa and control individuals were 72.17±7.72 years vs 70.47±7.60 years separatively. Total serum PSA mean levels in case with PCa and these in control individuals were 46.42±143.11 and 1.20±1.01 ng/ml respectively, and no significant difference between both of them (p = 0.129).
The Table1 lists the clinical baseline characteristics of the 266 cases,including Gleason score, tumor stage and aggressive status of patients etc.

PCa-associated allele
The genotype frequency of all SNPs among controls did not deviate from the Hardy-Weinberg equilibrium (p>0.05). Comprizing the allelic frequency between cases with PCa and controls showed that only RFX6 gene(rs339331,C) was associated with PCa (OR= 0.73, 95% CI =0.57-0.94, P = 0.013) by genetic statistical analysis of the 6 loci.    PCa-associated genotypes We found subjects carrying (CC+CT) genotypes had a decreased risk for prostate cancer compared to those carrying the TT homozygote (OR =0.64, 95% CI = 0.45-0.90, P = 0.008) in RFX6 gene (rs339331, C) ( Table 2) .

Analysis of PCa-associated risk factors
We analyzed the association of the 6 SNPs with the clinical covariants (phenotypes) prostate cancer through case-control comprision, including the PSA levels, ages at diagnosis, Gleason scores, Tumor stages and aggressive in patients with PCa by stratification analysis. The result showed that only the genotypes TT of in 13q22 (rs9600079, T) was associated with tumor stage (P=0.044,OR=2.34, 95% CI=0.94-5.87). For the other SNPs we not observed significantly associated with any clinical covariants in cases with prostate cancer (P > 0.05) ( Table 3).

Discussion
The products of KLK3 gene are as the important biomarkers i.e. human prostate-specific antigen (PSA) using in the clinical diagnosis of prostate cancer (PCa). The G protein-coupled receptor RFX6, C2orf43 and FOXP4 signaling play the important role in the development of PCa. We studied six loci involving rs2735839(G) located in KLK3, rs13385191(G) of C2orf43, rs1983891(T) in FOXP4, rs339331(C) in RFX6 gene, rs12653946(C) in 5p15 and rs9600079(T) in 13q22 to investigate the association of these genetic variations with PCa patients in Northern Chinese, to infer their impact on occurrence and aggression of prostate cancer in northern Chinese men.
Kallikrein-related peptidase 3 (Previous names: kallikrein 3, (prostate specific antigen) ) is one of the fifteen kallikrein subfamily members located in a cluster on chromosome 19. Its protein product is a protease present in seminal plasma. Serum level of this protein, called PSA in the clinical setting, is useful in the diagnosis and monitoring of prostatic carcinoma. Waters et al. (2009) produced the Multiethnic Cohort (African Americans, European Americans, Latinos, Japanese, Americans, and Native Hawaiians). They found KLK2/3, rs2735839 (OR, 1.06; 95% CI, 0.97-1.16; P < 0.05) variant was positively associated with PCa risk, with statistically significant associations from ethnic-pooled analyses. While our populational study haven't found statistically significant associations with PCa risk in KLK3 gene.
The function of Forkhead box P4 (FOXP4) is transcriptional repressor that represses lung-specific expression. Regulatory factor X, 6 (RFX6), transcription factor required to direct islet cell differentiation during endocrine pancreas development. Specifically required for the differentiation of 4 of the 5 islet cell types and for the production of insulin. Not required for pancreatic PP (polypeptide-producing) cells differentiation. Acts downstream of NEUROG3 and regulates the transcription factors involved in beta-cell maturation and function, thereby restricting the expression of the beta-cell differentiation and specification genes, and thus the beta-cell fate choice. Activates transcription by forming a heterodimer with RFX3 and binding to the X-box in the promoter of target genes. Lindström et al. (2012) found SNPs (rs13385191, rs12653946, rs1983891, and rs339331) were significantly associated with prostate cancer risk (P values ranging from 0.01 to 1.1×10 -5 ). Allele frequencies and OR were overall lower in population of European descent than these in the discovery Asian population.
In conclusion, our results shows that the RFX6 gene (rs339331, C) variant is associated with PCa risk, the 13q22 (rs9600079, T) variant in is related to advanced tumor stage in northern Chinese men. However, the other loci in this study were failed to be replicated, probably are not risk alleles associated with PCa in northern Chinese men,which maybe harbor genetic differences from the other populations.