Vitamin D Pathway Gene Variants and Prostate Cancer Risk

Introduction

Vitamin D has been shown to reduce cellular proliferation, increase apoptosis, and inhibit angiogenesis (1, 2). The link to prostate cancer is supported by ecological studies demonstrating an inverse relationship between prostate cancer incidence and UV exposure, which is the primary source of vitamin D (3). Serum studies, which have provided inconsistent results for a relationship between vitamin D status and prostate cancer risk, may not capture the relevant exposure period because not only does prostate carcinogenesis most likely begin decades prior to measurement, but 1-α-hydroxylase, the enzyme that activates vitamin D, is down-regulated early in the neoplastic process of prostate cancer cells (4-6).

This study did a comprehensive analysis of three genes in the vitamin D metabolism pathway: CYP27B1, encoding for 1-α-hydroxylase, which converts into the active form of the hormone 1,25-dihydroxy-vitamin D [1,25(OH)₂D]; VDR, encoding for the nuclear vitamin D receptor, which mediates all functions of 1,25(OH)₂D; and, CYP24A1, encoding for 24-hydroxylase, which catabolizes 1,25(OH)₂D into its excretion product. This population-based case-control study was completed to follow-up on our prior study which reported for two VDR loci, rs2107301 and rs2238135, carriers of the less common allele had higher risks of prostate cancer [odds ratios (OR), 2.47; 95% confidence interval (CI), 1.52-4.00 and OR, 1.95; 95% CI, 1.17-3.26, respectively] and reported no association with prostate cancer risk for the most frequently studied VDR polymorphisms FokI (rs10735810), BsmI (rs1544410), ApaI (rs7975232), and TaqI (rs731236; ref. 7).

Materials and Methods

Study Population

Study subjects were enrolled in a population-based prostate cancer case-control study that has been described previously (8). Eligible individuals were Caucasian or African American men. Cases were diagnosed with histologically confirmed prostate cancer between ages 35 to 74 years from January 1, 2002 to December 31, 2005. Prostate cancer cases were identified from the metropolitan Seattle-Puget Sound population-based tumor registry that is operated as part of the National Cancer Institute's Surveillance, Epidemiology, and End Results program. Of the 1,001 eligible interviewed cases, 827 (82.6%) had peripheral blood leukocyte samples collected. Eligible controls were recruited evenly throughout the ascertainment period for cases using random-digit telephone dialing and frequency-matched to cases by 5-year age groups. Of the 942 eligible interviewed controls, 787 (83.5%) had peripheral blood leukocyte samples collected. This study was approved by the Fred Hutchinson Cancer Research Center's Institutional Review Board and genotyping was approved by the Internal Review Board of the National Human Genome Research Institute.

Single Nucleotide Polymorphism Selection and Genotyping

Single nucleotide polymorphisms (SNP) capturing genetic variability in the VDR (9) and CYP27B1 (10) genes were selected using resequencing data, whereas SNP selection for CYP24A1 (11) used publicly available data from the HapMap consortium.⁴ SNP selection and genotyping duplicated methods described in the previous case-control study (7). The percentage of agreement of blind duplicates was ≥98% for all tagSNPs.

Statistical Analysis

Statistical methods were identical to our previous study (7). All SNPs, except for rs912505 in CYP24A1, were consistent (P > 0.05) with Hardy-Weinberg equilibrium (HWE) among Caucasian controls. Data were analyzed using unconditional logistic regression to calculate ORs as estimates of relative risk of prostate cancer associated with SNP genotypes. We included age and stratified by race in all regression models. We assessed possible confounding effects of variables listed in Table 1 and found thst none appreciably altered risk estimates, and thus, did not include them as covariates. Global tests of association, which were estimated by comparing an adjusted model that included all SNPs for a given gene to the null model that only included adjustment covariates, automatically adjusted for multiple testing based on degrees of freedom of the corresponding χ² test (12). Multiple comparisons were also accounted for by using permutations to calculate exact P values for each significant individual SNP (α = 0.05). All analyses were done using STATA statistical package (version 9.2, STATA Corp.).

Results

Cases and controls were similar in age (mean in cases, 61.7 years; in controls, 61.1 years). Cases had a higher percentage of African Americans, subjects reporting a family history of prostate cancer, and subjects with a history of Prostate-Specific Antigen (PSA) testing (Table 1). The majority of prostate cancers were local stage tumors with low/moderate Gleason scores.

There was no strong evidence of altered risk of developing prostate cancer for any tagSNPs (Table 2 ) or haplotypes evaluated (data not shown). In Caucasians, two loci (VDR, rs4760674; and CYP27B1, rs4809960) showed slightly lower relative risks of prostate cancer for homozygous variant versus homozygous wild-type carriers (OR, 0.68; 95% CI, 0.48-0.95; and OR, 0.77; 95% CI, 0.62-0.96, respectively), but after adjusting for multiple comparisons, these associations were no longer significant. Stratification by measures of tumor aggressiveness, such as Gleason score or stage, did not reveal significant associations of risk with genotypes. Lastly, there was no evidence of effect modification by total vitamin D or calcium intake.

Discussion

One goal of this study was to evaluate earlier findings by our group which showed VDR tagSNPs rs2107301 and rs2238135 to be significantly associated with prostate cancer (7). We did not replicate our earlier findings or identify any additional genotypes associated with prostate cancer risk in this comprehensive group of tagSNPs for VDR, CYP27B1, and CYP24A1 genes. Our findings corroborate a lack of an association between prostate cancer risk and frequently studied VDR polymorphisms BsmI (rs1544410), TaqI (rs731236), ApaI (rs7975232), and FokI (rs10735810; refs. 7, 13, 14).

This study attempted to capture genetic variation within a pathway of genes. Assuming 80% power, the minimal detectable OR was 0.46 or 1.76 for evaluating tagSNPs with a minor allele frequency of 5% or greater. We were underpowered to examine risk within African Americans, moreover, because polymorphic alleles in VDR, CYP27A1, and CYP24B1 differ by ethnicity, and tagSNP selection was based on a Caucasian population, gene coverage for an African American population is not complete. We did not find any evidence that vitamin D intake was an effect modifier; however, we were unable to account for plasma vitamin D levels or UV light exposure.

Our findings suggest that common genotypic variation found in VDR, CYP27A1, and CYP24B1 has little or no effect on overall prostate cancer risk. Future studies may reveal that these genotypes affect disease progression rather than the risk of developing disease.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.