Influence of Vitamin D Status and Vitamin D3 Supplementation on Genome Wide Expression of White Blood Cells: A Randomized Double-Blind Clinical Trial
PLoS ONE 8(3): e58725. doi:10.1371/journal.pone.0058725
Arash Hossein-nezhad, Avrum Spira, Michael F. Holick
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
- YRNA and
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.
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 16.264.2 ng/ml (dark purple) and the other four subjects were insufficient or sufficient with a 25(OH)D of 27.568.4 ng/ml (light purple). After supplementation (dark green) serum levels of 25(OH)D in vitamin D insufficient/sufficient subjects increased to 35.268.2 ng/ml (light purple) and in the vitamin deficient subjects increased to 25.1 6 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. doi:10.1371/journal.pone.0058725.g003 that was stimulated 1.6 fold by vitamin D3 supplementation has five VDREs (Table 2) that one of them is shown in Figure 6D located at position -1027, the TATA box located at -276 and location of other transcription factor sites near this VDRE was determined (6D).
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 16.264.2 ng/ml (dark purple) and the other four subjects were insufficient or sufficient with a 25(OH)D of 27.568.4 ng/ml (light purple). After supplementation (dark green) serum levels of 25(OH)D in vitamin D insufficient/sufficient subjects increased to 35.268.2 ng/ml (light purple) and in the vitamin deficient subjects increased to 25(OH)D of 25.164.7 ng/ml (light purple). Two groups of gene-expression changes are seen based on stimulation (brown) or iinhibition (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 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 stres s 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.
Table 3. List of biological functions of the 291 genes whose expression was influenced by vitamin D3 supplementation.
|Biological functions||Gene symbol|
|Apoptosis and immune response||OSM, AXUD1, CD83, PHLPP, TNFAIP3,NFKBIA,ZNF287, PTRH2, XIAP, TNFAIP8L2, ZDHHC16, TIA1,NUDCD1, KLF11,RASA1,EGR1,|
|Mineralization and bone development||JUND, SBDS, ZNRD1,MINPP1|
|Transcriptional regulation||ORC2L,KLF10,TRIM27,EGR1,NFIL3,JUN,NR4A2,ZNF225,ZNF607,ZNF780B,ZNF616RASA1,ZNF397, ZNF284,ZFP62,HOMEZ,ZNF701, GTF2E1,ZNF232, ZNF473,TAF1A, ZNF587,MIZF,ZNF223, ZNF175,MED7, ZNF320,ZNF17,ZNF45,ZFP3,ZNF283, EGR1,MED17,ZNF235,NF780A,ZNF322A, KLF11, SUV420H1,ZNF852, HCFC2,NAPC3,TRIP11,JRKL,ZNF234,ZNF260,JUNB, KLF10, TRIM27|
|Metabolic processes||TGDS, NAPEPLD, KIAA0859, ARSK, TMEM68INSIG2, GALK2, FPGT, HMGCL, HSD17B7P2, HSD17B7|
|Response to stress and DNA repair||FANCF, MSH5, PXDNL,ATF4,STIP1,HSPA4,HSPH1,POLA2, SOS1|
|RNA processing||CCDC76, MTO1, C1orf25, PUS3, RBM5, CSTF3, ZFP36, RNASEL, NUP107, ZCCHC8,POP1,INTS7, GEMIN6|
|Ion and protein transporter||SLC39A7, SLC30A6, SLC30A5, COPB2, NUP43, GOPC, SLC35B3, BET1, USO1, PIGM, TRAPPC6B|
|Biological process||TGDS, NAPEPLD, KIAA0859, ARSK, TMEM68, INSIG2, GALK2, FPGT, HMGCL, HSD17B7P2, HSD17B7|
|Epigenetic modification||HIST1H1E, H1FX, ALKBH1,UTP3, N6AMT1, METTL4|
|Cell cycle and DNA replication||FGF5, IRS2, MIS12, C10orf2, ORC2L, HELB, CUZD1, KIAA1009, POLA2, CETN3,CEP110, POLA2, PTP4A1|
|Signal transduction||GRASP, GNRH1, TAS2R4, TAS2R3, CXCR7, RIC8B, SOS1, BBS10|
|Protein modification||PIM3, PTP4A1, RNF139, PDP2, GGCX, PPID, TTC9C, SIK1, STK38|
|Development and cell differentiation||PDE4DIP, TUBD1, KEAP1, BBS7, UTP3, C11orf73, MKKS, BPNT1,NOC3L|
PDF is attached at the bottom of this page
Note: The 400 IU group appears to be the control group. That is, 400 IU of vitamin D was not enough to alter genes
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- GC, CYP2R1 and DHCR7 genes associated with low vitamin D levels in China – 2012, 2013
- Transgeneration vitamin D deficiency related to MS was found in mice – Aug 2012
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- Off topic: Ribosomes also contain genetic information another source
- Genetic differences explain some of vitamin D variation in African-Americans Dec 2011
- 2,776 vitamin D receptor binding sites - April 2014
- Potential Immune Benefits of Strong Vitamin D Status in Healthy Individuals Report on this study. Science Daily March 2013
- Holick on this study of Gene Expression by Vitamin D April 2013 webinar
- Google Scholar has 116 references to this study as of Jan 2017
Short url = http://is.gd/291genes291 genes improved expression by 2000 IU of vitamin D – RCT March 2013
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