To explore whether additional SNPs in the regions flanking SNP rs10993994 are associated with prostate cancer risk, we genotyped 16 tagging SNPs in ~65 kb genomic region at 10q11 in the CAncer of the Prostate in Sweden (CAPS) study population. The distributions of genotypes for all the 16 SNPs were consistent with Hardy–Weinberg expectations in control subjects (P
> 0.05). We also imputed 29 SNPs (with call rate >90%) in the region based on the genotyped SNPs using the computer program IMPUTE (7
). Allele frequency differences between cases and controls in CAPS were compared for these 45 SNPs using a χ2
test (Supplementary Material, Table S1
). Multiple SNPs in this ~65 kb region were significantly associated with prostate cancer risk (Fig. A, blue diamond). Specifically, many SNPs within a 25 kb region (51 194 000–51 219 000) were highly significant, including rs10993994 (P
= 0.001); however, they were no longer significant after adjusting for rs10993994 (Fig. A, pink diamond). This group of SNPs was in strong linkage disequilibrium (LD) and located in one haplotype block, referred to here as locus 1 (Fig. B). Several SNPs that are immediately telomeric of locus 1 (51 219 000–51 234 000) were not significantly associated with prostate cancer risk. It is noted that there is a 7 kb genomic region to the telomeric side of rs10993994 where no SNP is cataloged in the HapMap database.
Figure 1. A schematic view of genetic association between SNPs at 10q11 and prostate cancer risk in two study populations. (A) Allele tests for 45 SNPs at 10q11 and prostate cancer risk in CAPS. Two separate clusters of prostate cancer-associated SNPs were found (more ...)
Interestingly, multiple SNPs that were further telomeric were again associated with prostate cancer risk (Fig. A, blue diamond). More importantly, they remained significantly associated with prostate cancer risk after adjusting for rs10993994 (Fig. A, pink diamond), suggesting they are independent from locus 1. These SNPs are in strong LD and located in a single haplotype block, and are termed locus 2. SNP rs10761581 (51 238 384) was the most significant (P
= 0.0005) at locus 2 and the most significant in the entire ~65 kb region, even stronger than rs10993994 at locus 1. We estimated the recombination rate across the region among control subjects in CAPS using SequenceLDhot software (8
) and found strong evidence for a recombination hotspot between the two loci at 51 219 000–51 238 000 (P
= 1.24 × 10−15
) (Fig. C).
The recombination hotspot is also seen in the HapMap data (51 225 000–51 234 000, Release 21, Phases I and II). This recombination hotspot separates these two prostate cancer loci at 10q11. Prostate cancer risk-associated SNPs at locus 1 are in the 5′ untranslated region of the MSMB
gene, and the SNP rs10993994 is 57 bp upstream of the TSS of MSMB
. Prostate cancer risk-associated SNPs at locus 2, on the other hand, are within another gene, nuclear receptor coactivator 4 (NCOA4
). NCOA4 is also known as androgen receptor coactivator (ARA70
), which is known to enhance androgen receptor (AR) transcriptional activity in prostate cancer cells (9
We also examined the association of these SNPs with prostate specific antigen (PSA) levels among control subjects (Fig. D). Similar to the results of Eeles et al.
), we found the prostate cancer risk-associated allele of rs10993994 at locus 1 was strongly associated with higher PSA levels (P
= 0.001). Surprisingly, although many genotyped or imputed SNPs at locus 1 are in strong LD with rs10993994, none of these SNP was significantly associated with PSA levels (P
> 0.05). Similarly, none of the SNPs at locus 2 was significantly associated with PSA levels (P
When associations of SNPs at loci 1 and 2 were tested with clinicopathologic variables of prostate cancer cases (i.e. Gleason scores, TNM stages and pre-operative PSA), we did not observe any significant association (P > 0.05). However, we cannot exclude the possibility that these SNPs are weakly associated with the clinicopathologic variables. For example, the case–case study in CAPS can only detect with ≥80% power and at 5% significance level, if allele T of rs10993994 confers odds ratio (OR) ≥1.16 for aggressive prostate cancer over non-aggressive prostate cancer.
As a confirmation effort, we examined the novel locus 2, as well as the previously reported locus 1, in three additional study populations; The Johns Hopkins Hospital (JHH), Cancer Prevention Study-II (CPS-II) and Prostate, Lung, Colon and Ovarian (PLCO). One representative SNP in each locus (rs10993994 at locus 1 and rs10761581 at locus 2) was analyzed. A significant association was found for SNP rs10993994 at locus 1 in each population (Table ). The overall P
of the allelic test in four populations was 7.7 × 10−15
, exceeding a genome-wide significance level of 2 × 10−8
that accounts for multiple tests of ~2 million SNPs in the genome. It is noted that association results of rs10993994 at locus 1 in CAPS, PLCO and CPS-II have been previously published (1
). For SNP rs10761581 at locus 2, the association was confirmed only in PLCO (P
= 0.02) but not in JHH and CPS-II. The association of the SNP in PLCO remained significant after adjusting for rs10993994 at locus 1. Overall, the statistical evidence for locus 2 was weaker than that of locus 1, with a combined P
of 0.002 for an allelic test in four populations. The difference in allelic associations at locus 2 among these four study populations was marginally significant using a Breslow-Day test for homogeneity (P
= 0.05), providing some evidence that the association at this locus is heterogeneous among studies.
Association of prostate cancer risk with SNPs rs10993994 and rs10761581 (per study and combined)
The rs10993994 SNP is located at −57 bp 5′ to the TSS of the MSMB gene (Fig. A). To test the functional significance of the rs10993994 SNP, we cloned ~0.5 kb of the promoter sequence from individuals homozygous for either T or C at the rs10993994, and used these to drive luciferase expression in LNCaP cells. We found that the prostate cancer risk-associated allele T of the rs10993994 SNP had only 13% of the promoter activity compared with the C allele (Fig. B). Treatment with increasing concentrations of the synthetic androgen R1881 resulted in a dose-dependent increase in promoter activity of the C, but not the T allele of the SNP rs10993994. Because no other sequence variant within this ~0.5 kb region was found in these two clones, the different promoter activities are likely due to the variant rs10993994. A similar promoter assay was performed for rs10761581; however, we did not observe differences in promoter activity between the two alleles (data not shown).
Figure 2. The rs10993994 SNP alters basal and androgen-stimulated MSMB promoter activity. (A) Schematic of the promixal region of the MSMB promoter showing the rs10993994 SNP (bracketed and bold letters) relative to the TSS. The locations of the forward and reverse (more ...)