A Genome-Wide Methylation Study of Severe Vitamin D Deficiency in African American Adolescents

Objectives To test the hypothesis that changes in DNA methylation are involved in vitamin D deficiency-related immune cell regulation using an unbiased genome-wide approach combined with a genomic and epigenomic integrative approach. Study design We performed a genome-wide methylation scan using the Illumina HumanMethylation 27 Bead-Chip on leukocyte DNA of 11 cases of vitamin D deficiency (serum 25-hydroxyvitamin D [25(OH) D] <= 25 nmol/L) and 11 age-matched controls ([25(OH) D] > 75 nmol/L); the subjects were African American normal-weight (body mass index <85th percentile) males aged 14-19 years. The Limma package was used to analyze each CpG site for differential methylation between cases and controls. To correct for multiple testing, the set of raw P values were converted to false discovery rates (FDRs). We also compared our findings with the recent data from Genome-Wide Association Studies of circulating 25(OH) D levels and then performed a permutation test to examine whether the double hit genes were randomly enriched. Results A total of 79 CpG sites achieved raw P<.001. Of the 79 CpG sites, 2 CpG sites survived multiple testing: cg16317961 (raw P = 3.5 x 10(-6), FDR = 0.078, in MAPRE2) and cg04623955 (raw P = 5.9 x 10(-6), FDR = 0.078, in DIO3). Furthermore, 3 out of the 4 genes previously identified in the 2 Genome-Wide Association Studies were also significant at the methylation level (DHCR7: cg07487535, P=.015 and cg10763288, P=.017; CYP2R1: cg25454890, P=.040; CYP24A1: cg18956481, P=.022), reflecting significant enrichment (P=.0098). Conclusion Severe vitamin D deficiency is associated with methylation changes in leukocyte DNA. The genomic and epigenomic approach reinforce the crucial roles played by the DHCR7, CYP2R1, and CYP24A1 genes in vitamin D metabolism. (J Pediatr 2013;162:1004-9).