Background

Vitamin D may protect against poor health outcomes, including heart disease, diabetes, certain cancers, and overall mortality.^[1–5] Its biological properties include regulation of cell proliferation and immune function, as well as increased cell differentiation and apoptosis.^[6–10] These mechanisms are controlled by the active metabolite 1,25-dihydroxyvitamin D (1,25(OH)₂D) and the vitamin D receptor (VDR), often in conjunction with retinoid X receptor alpha (RXRA).^[11] This 1,25(OH)₂D-VDR-RXRA complex binds to vitamin D response elements that can activate or repress gene transcription.^[12]

Circulating vitamin D levels could affect DNA methylation via transcriptional regulation or other mechanisms.^[13] In mammals, DNA methylation is an epigenetic process by which a methyl group is transferred onto the C5 position of a cytosine, forming 5-methylcytosine. Increased methylation at CpG sites in promoter regions is associated with gene inactivation and transcriptional repression, while increased methylation at CpGs in gene bodies is associated with actively transcribed genes.^[14,15] Examples of other environmental exposures associated with methylation changes include smoking (for both smokers^[16] and their offspring^[17–19]), as well as body mass index (BMI),^[20,21] alcohol consumption,^[22] and nutrients such as folate, vitamin B12, and retinoic acid.^[23–27]

Some empirical evidence supports a link between vitamin D exposure and DNA methylation. Candidate gene approaches have observed that vitamin D is associated with methylation of CYP24A1,^[28,29]BMP2,^[30]PTEN,^[31] and DKK1.^[32] Additionally, one epigenome-wide association study (EWAS) conducted among adolescent African-American males identified two sites (cg16317961 (MAPRE2) and cg04623955 (DIO3)) that were significantly associated with serum levels of the stable precursor to 1,25(OH)₂D, 25-hydroxyvitamin D (25(OH)D).^[33] However, those findings did not replicate in a subsequent EWAS conducted among Caucasian men, nor did that subsequent EWAS identify any novel associations.^[34] Another EWAS observed no noteworthy associations between maternal 25(OH)D levels and methylation in cord blood,^[35] and an epigenome-wide in-vitro study identified no detectable methylation changes in blood mononuclear cells treated with vitamin D.^[36] Several studies of the association between vitamin D and LINE-1 global methylation levels have also been negative.^[37–39]

Methods: We studied the relationships between serum vitamin D, DNA methylation, and breast cancer using a case-cohort sample (1070 cases, 1277 in subcohort) of non-Hispanic white women. For our primary analysis, we used robust linear regression to examine the association between serum 25-hydroxyvitamin D (25(OH)D) and methylation within a random sample of the cohort (“subcohort”). We focused on 198 CpGs in or near seven vitamin D-related genes. For these 198 candidate CpG loci, we also examined how multiplicative interactions between methylation and 25(OH)D were associated with breast cancer risk. This was done using Cox proportional hazards models and the full case-cohort sample. We additionally conducted an exploratory epigenome-wide association study (EWAS) of the association between 25(OH)D and DNA methylation in the subcohort.

Results: Of the CpGs in vitamin D-related genes, cg21201924 (RXRA) had the lowest p value for association with 25(OH)D (p = 0.0004). Twenty-two other candidate CpGs were associated with 25(OH)D (p < 0.05; RXRA, NADSYN1/DHCR7, GC, or CYP27B1). We observed an interaction between 25(OH)D and methylation at cg21201924 in relation to breast cancer risk (ratio of hazard ratios = 1.22, 95% confidence interval 1.10–1.34; p = 7 × 10⁻⁵), indicating a larger methylation-breast cancer hazard ratio in those with high serum 25(OH)D concentrations. We also observed statistically significant (p < 0.05) interactions for six other RXRA CpGs and CpGs in CYP24A1, CYP27B1, NADSYN1/DHCR7, and VDR. In the EWAS of the subcohort, 25(OH)D was associated (q < 0.05) with methylation at cg24350360 (EPHX1; p = 3.4 × 10⁻⁸), cg06177555 (SPN; p = 9.8 × 10⁻⁸), and cg13243168 (SMARCD2; p = 2.9 × 10⁻⁷).

Conclusions: 25(OH)D concentrations were associated with DNA methylation of CpGs in several vitamin D-related genes, with potential links to immune function-related genes. Methylation of CpGs in vitamin D-related genes may interact with 25(OH)D to affect the risk of breast cancer.