Influence of Vitamin D Status and Vitamin D₃ Supplementation on Genome Wide Expression of White Blood Cells: A Randomized Double-Blind Clinical Trial

<div><p>Background</p><p>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).</p> <p>Methods and Findings</p><p>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.</p> <p>Conclusion/Significance</p><p>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.</p> <p>Trial Registration</p><p>ClinicalTrials.gov <a href="http://clinicaltrials.gov/ct2/show/NCT01696409" target="_blank">NCT01696409</a></p> </div

Principal Component Analysis across 16 microarray samples.

<p>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.)</p

The motif sequences of 17 genes that were most response to vitamin D₃ supplementation that is identical or similar to other known VDRE sequences.

<p>The positions and sequences of nucleotide motifs from 5′ upstream to the transcriptional start site for 17 genes identified as being most affected by vitamin D₃ supplementation. The similarity of the candidate VDREs with known VDREs is shown.</p

Subject demographics and total 25(OH)D levels before and after 400 IU/d or 2000 IU/d of vitamin D₃ supplementation for 8 weeks.

<p>Demographic information including sex, average age and 25(OH)D levels are included (mean ± standard deviation).</p

Verification of microarray gene expression by Real-time PCR.

<p>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.</p

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

<p>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.</p

List of biological functions of the 291 genes whose expression was influenced by vitamin D₃ supplementation.

<p>List of biological functions of the 291 genes whose expression was influenced by vitamin D₃ supplementation.</p

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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058725#pone.0058725.s002" target="_blank">Table S1</a>.</p

Similarities and differences between our dataset and other bronchial airway datasets

GSEA was used to determine if there was a gene expression relationship between other airway datasets (see Table 3 for a description of conditions 1 and 2) and our dataset based on the genes we identified to be regulated by smoking. The normalized enrichment score is plotted for datasets that had a FDR < 0.25. Gene lists derived from functional categories and chromosomal locations found to be over-represented by EASE analysis in our dataset were tested for enrichment in our dataset and the other ten datasets using GSEA. A false-color heatmap of the positive (red) and negative (blue) normalized enrichment scores (with a FDR < 0.25) is shown for each category. An asterisk indicates the results passed a stricter FDR < 0.05. The nine datasets and conditions that yielded significant results in either (a) or (b) are indicated in Table 3 by the presence of a single asterisk.<p><b>Copyright information:</b></p><p>Taken from "Reversible and permanent effects of tobacco smoke exposure on airway epithelial gene expression"</p><p>http://genomebiology.com/2007/8/9/R201</p><p>Genome Biology 2007;8(9):R201-R201.</p><p>Published online 25 Sep 2007</p><p>PMCID:PMC2375039.</p><p></p

Quantitative real time PCR results for select genes across never, former, and current smokers

For each graph sample identifiers for never (orange), former (purple), and current (green) smokers are listed along the x-axis. The sample identifications P1, P2, and P3 refer to three samples collected prospectively from never smokers that do not have corresponding microarrays. The months since smoking cessation are listed below each former smoker. The relative expression level on the y-axis is the ratio of the expression level of a particular sample versus that of a dummy reference sample. Plots of two rapidly reversible genes, and . Plots of two irreversible genes, and .<p><b>Copyright information:</b></p><p>Taken from "Reversible and permanent effects of tobacco smoke exposure on airway epithelial gene expression"</p><p>http://genomebiology.com/2007/8/9/R201</p><p>Genome Biology 2007;8(9):R201-R201.</p><p>Published online 25 Sep 2007</p><p>PMCID:PMC2375039.</p><p></p