Influence of vitamin D status and vitamin D3 supplementation on genome wide expression of white blood cells: a randomized double-blind clinical trial

Abstract

Background: Although there have been numerous observations of vitamin D deficiency and its links to chronic diseases, no studies have reported on how vitamin D status and vitamin D3 supplementation affects broad gene expression in humans. The objective of this study was to determine the effect of vitamin D status and subsequent vitamin D supplementation on broad gene expression in healthy adults. (Trial registration: ClinicalTrials.gov NCT01696409).

Methods and findings: A randomized, double-blind, single center pilot trial was conducted for comparing vitamin D supplementation with either 400 IUs (n = 3) or 2000 IUs (n = 5) vitamin D3 daily for 2 months on broad gene expression in the white blood cells collected from 8 healthy adults in the winter. Microarrays of the 16 buffy coats from eight subjects passed the quality control filters and normalized with the RMA method. Vitamin D3 supplementation that improved serum 25-hydroxyvitamin D concentrations was associated with at least a 1.5 fold alteration in the expression of 291 genes. There was a significant difference in the expression of 66 genes between subjects at baseline with vitamin D deficiency (25(OH)D<20 ng/ml) and subjects with a 25(OH)D>20 ng/ml. After vitamin D3 supplementation gene expression of these 66 genes was similar for both groups. Seventeen vitamin D-regulated genes with new candidate vitamin D response elements including TRIM27, CD83, COPB2, YRNA and CETN3 which have been shown to be important for transcriptional regulation, immune function, response to stress and DNA repair were identified.

Conclusion/significance: Our data suggest that any improvement in vitamin D status will significantly affect expression of genes that have a wide variety of biologic functions of more than 160 pathways linked to cancer, autoimmune disorders and cardiovascular disease with have been associated with vitamin D deficiency. This study reveals for the first time molecular finger prints that help explain the nonskeletal health benefits of vitamin D.

Trial registration: ClinicalTrials.gov NCT01696409.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Flow Diagram of Study Subjects.

Figure 2. Principal Component Analysis across 16 microarray samples.

There is no grouping of samples along the first or second principal components (representing 18.6% and 17.9% of the variance in gene expression, respectively) based on the expression of these genes. Sample types of each group before or after vitamin D3 supplementation are color-coded for the dose of vitamin D3 supplementation. Red =  2000 IUs and blue =  400 IUs (PoV  =  Possibility of Variance.)

Figure 3. Heatmaps of vitamin D responsive genes whose expression levels change after 2 months vitamin D3 supplementation.

Before supplementation (light green) four subjects were vitamin D deficient with 25(OH)D of 16.2±4.2 ng/ml (dark purple) and the other four subjects were insufficient or sufficient with a 25(OH)D of 27.5±8.4 ng/ml(light purple). After supplementation (dark green) serum levels of 25(OH)D in vitamin D insufficient/sufficient subjects increased to 35.2±8.2 ng/ml (light purple) and in the vitamin deficient subjects increased to 25.1± 4.7 ng/ml(dark purple). Two groups of gene-expression changes are seen based on stimulation (brown) or inhibition (blue) of gene expression post vitamin D3 supplementation. (Colors ranged from blue to brown; High expression  =  brown, average expression  =  white, low expression  =  blue). Clustering of the 291 genes affected by vitamin D3 supplementation was based on stimulation (brown) or inhibition (blue) of gene expression. The list of the 291 genes is shown in Table S1.

Figure 4. Verification of microarray gene expression by Real-time PCR.

For verification of gene expression real-time PCR was performed for four genes including CD83, TNFAIP3, KLF10 and SBDS. Relationship between two sets of data from microarray and real-time PCR is shown by linear regression with 95% mean prediction interval. The results showed the relative expression of these genes was consistent with the expression observed from the broad gene expression by microarray.

Figure 5. Heatmaps of vitamin D responsive genes affected by vitamin D status.

Before supplementation (light green) four subjects were vitamin D deficient with 25(OH)D of 16.2±4.2 ng/ml (dark purple) and the other four subjects were insufficient or sufficient with a 25(OH)D of 27.5±8.4 ng/ml(light purple). After supplementation (dark green) serum levels of 25(OH)D in vitamin D insufficient/sufficient subjects increased to 35.2±8.2 ng/ml (light purple) and in the vitamin deficient subjects increased to 25(OH)D of 25.1±4.7 ng/ml(dark purple). Two groups of gene-expression changes are seen based on stimulation (brown) or inhibition (blue) of gene expression post vitamin D3 supplementation. (Colors ranged from blue to brown; High expression  =  brown, average expression  =  white, low expression  =  blue).Expression of 66 genes before supplementation was significantly different in the vitamin D deficient group (dark purple) compared to the vitamin D insufficient/sufficient group (light purple). Clustering of the 66 genes affected by vitamin D status and vitamin D3 supplementation was based on stimulation (brown) or inhibition (blue) of gene expression.

Figure 6. Sequence of candidate VDREs compared with known VDREs.

(A) The candidate sequences of VDREs (B), motifs created based on known VDRE sequences previously reported and (C) motifs based on the sum of these sequences and (D) the location of candidate VDREs of pseudouridylate synthase 3 (PUS3) and the location of other transcription regulation sites in this gene including TATA box, SF1and CCAAT. The major structure of candidate VDREs are based on the consensus sequence RGKTSA (R  =  A or G, K  =  G or T, and S  =  C or G).

Figure 7. Biological functions for genes whose expression levels were altered after 2 months of vitamin D3 supplementation.

After receiving vitamin D3 supplementation we identified 291 genes whose expression was significantly decreased or increased. Some of these genes influence several pathways that are involved in response to stress and DNA repair, DNA replication, immune regulation, epigenetic modification, transcriptional regulation and other biological functions. In addition vitamin D3 supplementation influenced the expression of Y RNA and CETN3 that are involved in DNA repair in response to UVR exposure.