Genome-wide association studies have identified more than 100 common single nucleotide polymorphisms (SNPs) that are associated with prostate cancer risk. However, the vast majority of these SNPs lie in noncoding regions of the genome. To test whether these risk SNPs regulate their target genes through long-range chromatin interactions, we applied capture-based 3C sequencing technology to investigate possible cis-interactions at ten prostate cancer risk loci in six cell lines. We identified significant physical interactions between risk regions and their potential target genes including CAPG at 2p11.2, C2orf43 at 2p24.1, RFX6 at 6q22.1, NFASC at 1q32.1, MYC at 8q24.1 and AGAP7P at 10q11.23. Most of the interaction peaks were co-localized to regions of active histone modification and transcription factor binding sites. Expression quantitative trait locus (eQTL) analysis showed suggestive eQTL signals at rs1446669, rs699664 and rs1078004 for CAPG (p < 0.004), rs13394027 for C2orf43 (p = 2.25E-27), rs10993994 and rs4631830 for AGAP7P (p < 8.02E-5). Further analysis revealed an enhancer activity at genomic region surrounding rs4631830 which was expected to disrupt HOXB-like DNA binding affinity. This study identifies a set of candidate genes and their potential regulatory variants, and provides additional evidence showing the role of long-range chromatin interactions in prostate cancer etiology.

In the past twenty-five years, over 700 case-control association studies on the risk of prostate cancer have been published worldwide, but their results were largely inconsistent. To facilitate following and explaining these findings, we performed a systematic meta-analysis using allelic contrasts for gene-specific SNVs from at least three independent population-based case-control studies, which were published in the field of prostate cancer between August 1, 1990 and August 1, 2015. Across 66 meta-analyses, a total of 20 genetic variants involving 584,100 subjects in 19 different genes (KLK3, IGFBP3, ESR1, SOD2, CAT, CYP1B1, VDR, RFX6, HNF1B, SRD5A2, FGFR4, LEP, HOXB13, FAS, FOXP4, SLC22A3, LMTK2, EHBP1 and MSMB) exhibited significant association with prostate cancer. The average summary OR was 1.33 (ranging from: 1.016-3.788) for risk alleles and 0.838 (ranging from: 0.757-0.896) for protective alleles. Of these positive variants, FOXP4 rs1983891, LMTK2 rs6465657 and RFX6 rs339331 had not been previously meta-analyzed. Further analyses with sufficient power design and investigations of the potential biological roles of these genetic variants in prostate cancer should be conducted.

PURPOSE: Prostate cancer (PCa) is one of the most common malignancies in males, and multiple genetic studies have confirmed association with susceptibility to PCa. However, the risk conferred in men living in China is unkown. We selected 6 previously identified variants as candidates to define their association with PCa in Chinese men.
METHODS: We genotyped 6 single nucleotide polymorphisms (SNPs) (rs1465618, rs1983891, rs339331, rs16901966, rs1447295 and rs10090154) using high resolution melting (HRM) analysis and assessed their association with PCa risk in a case-control study of 481 patients and 480 controls in a Chinese population. In addition, the individual and cumulative contribution for the risk of PCa and clinical covariates were analysed.
RESULTS: We found that 5 of the 6 genetic variants were associated with PCa risk. The T allele of rs339331 and the G allele of rs16901966 showed a significant association with PCa susceptibility: OR (95%CI)= 0.78 (0.64-0.94), p<0.009 and OR (95%CI)= 0.66 (0.54-0.81), p<0.0001, as well as A allele of rs1447295 (OR [95%CI]=1.46 (1.17-1.84), p<0.001) and T allele of rs10090154 (OR [95%CI]= 0.58 (0.46-0.74), p<0.0001). rs339331(T) was associated with a 0.71-fold and 1.42-fold increase of PCa risk by dominant model (p=0.007) and recessive model (p=0.007). rs16901966 (G) was associated with a 0.51-fold and 1.98-fold increase of PCa risk by dominant model (p=0.006) and recessive model (p=0.0058). rs10090154 (T) was associated with a 1.89-fold and 0.53-fold increase of PCa risk by dominant model (p=0.000006) and recessive model (p=0.000006). And, rs1983891(C) was associated with a 0.77-fold increase of PCa risk by recessive model (p=0.045). rs1447295 was associated with a 1.57-fold increase of PCa risk by dominant model (p=0.008). rs1465618 showed no significant association with PCa. The cumulative effects test of risk alleles (rs rs1983891, rs339331, rs16901966, rs1447295 and rs10090154) showed an increasing risk to PCa in a frequency-dependent manner (ptrend=0.001), and men with more than 3 risk alleles had the most significant susceptibility to PCa (OR=1.99, p=0.001), compared with those who had one risk allele (OR=1.17, p=0.486).
CONCLUSION: Our results provide further support for association of the THADA, FOXP4, GPRC6A/RFX6 and 8q24 genes with Pca in Asian populations. Further work is still required to determine the functional variations and finally clarify the underlying biological mechanisms.

The vast majority of disease-associated single-nucleotide polymorphisms (SNPs) mapped by genome-wide association studies (GWASs) are located in the non-protein-coding genome, but establishing the functional and mechanistic roles of these sequence variants has proven challenging. Here we describe a general pipeline in which candidate functional SNPs are first evaluated by fine mapping, epigenomic profiling, and epigenome editing, and then interrogated for causal function by using genome editing to create isogenic cell lines followed by phenotypic characterization. To validate this approach, we analyzed the 6q22.1 prostate cancer risk locus and identified rs339331 as the top-scoring SNP. Epigenome editing confirmed that the rs339331 region possessed regulatory potential. By using transcription activator-like effector nuclease (TALEN)-mediated genome editing, we created a panel of isogenic 22Rv1 prostate cancer cell lines representing all three genotypes (TT, TC, CC) at rs339331. Introduction of the 'T' risk allele increased transcription of the regulatory factor 6 (RFX6) gene, increased homeobox B13 (HOXB13) binding at the rs339331 region, and increased deposition of the enhancer-associated H3K4me2 histone mark at the rs339331 region compared to lines homozygous for the 'C' protective allele. The cell lines also differed in cellular morphology and adhesion, and pathway analysis of differentially expressed genes suggested an influence of androgens. In summary, we have developed and validated a widely accessible approach that can be used to establish functional causality for noncoding sequence variants identified by GWASs.

A new study shows that HOXB13 is preferentially recruited to the risk allele of a prostate cancer-associated SNP, enhancing the expression of RFX6, a driver of prostate cancer cell migration and predictor of disease progression. The work illustrates how a single risk locus contributes both to prostate cancer incidence and, through functional follow-up, to disease progression.

Genome-wide association studies have identified thousands of SNPs associated with predisposition to various diseases, including prostate cancer. However, the mechanistic roles of these SNPs remain poorly defined, particularly for noncoding polymorphisms. Here we find that the prostate cancer risk-associated SNP rs339331 at 6q22 lies within a functional HOXB13-binding site. The risk-associated T allele at rs339331 increases binding of HOXB13 to a transcriptional enhancer, conferring allele-specific upregulation of the rs339331-associated gene RFX6. Suppression of RFX6 diminishes prostate cancer cell proliferation, migration and invasion. Clinical data indicate that RFX6 upregulation in human prostate cancers correlates with tumor progression, metastasis and risk of biochemical relapse. Finally, we observe a significant association between the risk-associated T allele at rs339331 and increased RFX6 mRNA levels in human prostate tumors. Together, our results suggest that rs339331 affects prostate cancer risk by altering RFX6 expression through a functional interaction with the prostate cancer susceptibility gene HOXB13.

BACKGROUND: KLK3 gene products, like human prostate-specific antigen (PSA), are important biomarkers in the clinical diagnosis of prostate cancer (PCa). G protein-coupled receptor RFX6, C2orf43 and FOXP4 signaling plays important roles in the development of PCa. However, associations of these genes with PCa in northern Chinese men remain to be detailed. This study aimed to investigate their impact on occurrence and level of malignancy.
METHODS: All subjects were from Beijing and Tianjin, including 266 cases with prostate cancer and 288 normal individuals as controls. We evaluated associations between clinical covariates (age at diagnosis, prostate specific antigen, Gleason score, tumor stage and aggressive) and 6 candidate PCa risk loci, genotyped by PCR- high resolution melting curve and sequencing methods.
RESULTS: Case-control analysis of allelic frequency of PCa associated with PCa showed that one of the 6 candidate risk loci, rs339331 in the RFX6 gene, was associated with reduced risk of prostate cancer (odds ratio (OR) = 0.73, 95% confidence interval (CI) =0.57-0.94, P = 0.013) in northern Chinese men. In addition, subjects with CX (CC+TC) genotypes had a decreased risk for prostrate cancer compared to those carrying the TT homozygote (OR =0.64, 95% CI = 0.45- 0.90, P = 0.008). The TT genotype of 13q22 (rs9600079, T) was associated with tumor stage (P=0.044, OR=2.34, 95% CI=0.94-5.87). Other SNPs were not significantly associated with clinical covariates in prostate cancer (P > 0.05). CONCLUSIONS. rs339331 in the RFX6 gene may be associated with prostate cancer as a susceptibility locus in northern Chinese men.

BACKGROUND: Prostate cancer represents the leading cause of male death across the world. A recent genome-wide association study (GWAS) identified five novel susceptibility loci for prostate cancer in the Japanese population. This study is to replicate and fine map the potential association of these five loci with prostate cancer in the Chinese Han population.
METHODS: In Phase I of the study, we tested the five single nucleotide polymorphisms (SNPs) which showed the strongest association evidence in the original GWAS in Japanese. The study sample consists of 1,169 Chinese Hans, comprising 483 patients and 686 healthy controls. Then in phase II, flanking SNPs of the successfully replicated SNPs in Phase I were genotyped and tested for association with prostate cancer to fine map those significant association signals.
RESULTS: We successfully replicated the association of rs13385191 (located in the C2orf43 gene, P = 8.60×10(-5)), rs12653946 (P = 1.33×10(-6)), rs1983891 (FOXP4, P = 6.22×10(-5)), and rs339331 (GPRC6A/RFX6, P = 1.42×10(-5)) with prostate cancer. The most significant odds ratio (OR) was recorded as 1.41 (95% confidence interval 1.18-1.68) for rs12653946. Rs9600079 did not show significant association (P = 8.07×10(-2)) with prostate cancer in this study. The Phase II study refined these association signals, and identified several SNPs showing more significant association with prostate cancer than the very SNPs tested in Phase I.
CONCLUSIONS: Our results provide further support for association of the C2orf43, FOXP4, GPRC6A and RFX6 genes with prostate cancer in Eastern Asian populations. This study also characterized the novel loci reported in the original GWAS with more details. Further work is still required to determine the functional variations and finally clarify the underlying biological mechanisms.

A recent genome-wide association study has identified five new genetic variants for prostate cancer susceptibility in a Japanese population, but it is unknown whether these newly identified variants are associated with prostate cancer risk in other populations, including Chinese men. We genotyped these five variants in a case-control study of 1524 patients diagnosed with prostate cancer and 2169 control subjects from the Chinese Consortium for Prostate Cancer Genetics (ChinaPCa). We found that three of the five genetic variants were associated with prostate cancer risk (P = 4.33 × 10(-8) for rs12653946 at 5p15, 4.43 × 10(-5) for rs339331 at 6q22 and 8.42 × 10(-4) for rs9600079 at 13q22, respectively). A cumulative effect was observed in a dose-dependent manner with increasing numbers of risk variant alleles (P(trend) = 2.58 × 10(-13)), and men with 5-6 risk alleles had a 2-fold higher risk of prostate cancer than men with 0-2 risk alleles (odds ratio = 2.26, 95% confidence interval = 1.78-2.87). Furthermore, rs339331 T allele was significantly associated with RFX6 and GPRC6A higher messenger RNA expression, compared with the C allele. However, none of the variants was associated with clinical stage, Gleason score or family history. These results provide further evidence that the risk loci identified in Japanese men also contribute to prostate cancer susceptibility in Chinese men.

Prostate cancer is one of the most common malignancies in males throughout the world, and its incidence is increasing in Asian countries. We carried out a genome-wide association study and replication study using 4,584 Japanese men with prostate cancer and 8,801 control subjects. From the thirty-one associated SNPs reported in previous genome-wide association studies in European populations, we confirmed the association of nine SNPs at P < 1.0 x 10(-7) and ten SNPs at P < 0.05 in the Japanese population. The remaining 12 SNPs showed no association (P > 0.05). In addition, we report here five new loci for prostate cancer susceptibility, at 5p15 (lambda-corrected probability P(GC) = 3.9 x 10(-18)), GPRC6A/RFX6 (P(GC) = 1.6 x 10(-12)), 13q22 (P(GC) = 2.8 x 10(-9)), C2orf43 (P(GC) = 7.5 x 10(-8)) and FOXP4 (P(GC) = 7.6 x 10(-8)). These findings advance our understanding of the genetic basis of prostate carcinogenesis and also highlight the genetic heterogeneity of prostate cancer susceptibility among different ethnic populations.