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Vitamin D and Human DNA Methylation: Global and Genome-Wide Studies

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Presentation on theme: "Vitamin D and Human DNA Methylation: Global and Genome-Wide Studies"— Presentation transcript:

1 Vitamin D and Human DNA Methylation: Global and Genome-Wide Studies
Yanbin Dong MD PhD Regents’ Professor, Pediatrics Georgia Prevention Institute Department of Pediatrics Medical College of Georgia Augusta University

2 Is Vitamin D a nontraditional CVD risk factor
Is Vitamin D a nontraditional CVD risk factor? Observational and Interventional Approaches Suboptimal Vitamin D (minimal sun exposure, skin pigmentation, diet, and adiposity) Higher cardiometabolic risk (arterial stiffness, endothelial dysfunction, hypertension, insulin resistance, short telomeres, and inflammation) Dong et al, Pediatrics, 2010; Dong et al, JCEM, 2010; Harris et al, AJH, 2011; Zhu et al, Int J Obes, 2011; Parikh et al, Diabetes Care, 2012

3 Vitamin D and Epigenome
Almost 10% of the human genome may be at least partially regulated by vitamin D. Vitamin D receptor (VDR) is viewed as a master regulator of transcription by binding to the VDRE. The binding recruits coactivaters and enzymes with histone acetylation. Causing the structural changes in chromatin, therefore facilitating gene transcription

4 Knowledge Gap Is vitamin D involved in human DNA methylation?
Does vitamin D regulate methylation globally? The human epigenome of vitamin D deficiency is to be elucidated, namely, an epigenome-wide association study (EWAS) is lacking.

5 Study 1: Global Methylation
Interventional approach We aimed to test the hypothesis that vitamin D3 supplementation will increase global DNA methylation level in African Americans with vitamin D deficiency [25(OH)D ≤50 nmol/L or 20 ng/mL] in a dose-responsive manner. In a cross-sectional study, we previously found that vitamin D deficiency was associated with global hypomethylation. Zhu et al, PLOS ONE 2016

6 Randomized Clinical Trial (NCT01583621)
Global DNA methylation level (percentage of 5-methylcytosine, %5-mC) was quantified in leukocyte DNA using the MethylFlash Methylated DNA Quantification kit (Epigentek).

7 Baseline Characteristics
Values are presented as Mean ± SEM; BMI: body mass index; 25(OH)D: Serum 25-hydroxyvitamin; %5-mc: percentage of 5-methylcytosine; PTH: plasma parathyroid hormone

8 25(OH)D changes (Δ) Bhagatwala et al BMC Obesity 2015

9 Changes in global methylation
Zhu et al, PLOS ONE 2016

10 Summary of Study 1 Vitamin D supplementation increases global DNA methylation level in a dose-responsive manner. Epigenome-wide methylation (EWAS) is warranted.

11 Cross-sectional approach
Study 2: Epigenome-Wide Methylation: Cross-sectional approach To profile the leukocyte methylome of vitamin D deficiency using EWAS; To provide previously unrecognized findings and insight; and epigenetically define inflammation associated with vitamin D deficiency

12 Extreme Phenotypes Cases (<25 nmol/L) Controls (>75 nmol/L) p
African Americans 11 - Age, years 16.2 ± 1.2 16.1 ± 1.3 0.791 Age range, years 14.2 – 18.6 Height, cm 174.6 ± 6.1 174.3 ± 8.8 0.930 Weight, kg 63.2 ± 7.4 62.5 ± 8.5 0.841 BMI, kg/m2 20.7 ± 2.5 20.6 ± 2.4 0.897 WC, cm 71.2 ± 4.7 70.9 ±4.3 0.878 SBP, mmHg 113.0 ± 5.7 112.6 ± 4.7 0.855 DBP, mmHg 58.5 ± 6.1 58.2 ± 4.5 0.940 25(OH)D, nmol/L 19.8 ±3.8 92.0 ± 12.4 <0.0001

13 Infinium HumanMethylation27 BeadChip
>27,000 CpG sites located in the promoter region >14,000 genes On average, 2 CpG sites per gene 3-20 CpG sites for >200 cancer-related and imprinted genes Single CpG resolution β value ranges from 0 (unmethylated) to 1 (fully methylated) Low sample input (500 ng or 1 ug) High sample throughput- 12 samples per array

14 Statistics The Limma package was used to analyze each CpG site for differential methylation between cases and controls Each CpG site was assigned a raw p value based on a moderated t-statistic. False discovery rate (FDR) used for correction of multiple testing. Gene Ontology analysis was conducted with FatiGO. ( Al-Shahrour et al. Bioinformatics. 2004; 4: ).

15 Results Mean methylation differences between cases and controls
Zhu et al Journal of Pediatrics 2013

16 Implicated in immune cell activation, regulation and
Implicated in immune cell activation, regulation and inflammatory response.

17 Gene Ontology Analysis

18 Summary of Study 2 We identified modest but significant methylation differences in leukocyte DNA between vitamin D deficient cases and controls. More CpG sites are associated with reduced DNA methylation levels. Our study suggests that DNA methylation may play an important role in vitamin D-mediated inflammation and immune regulation.

19 Randomized Clinical Trial (NCT01583621)
EWAS studies are recently funded by American Heart Association Ongoing project Please stay tuned!

20 Study 3: Genome-Wide Methylation Maternal vitamin D supplementation
Whether maternal vitamin D supplementation has any effect on neonatal epigenome remains unclear. We aimed to examine the association of maternal vitamin D supplementation and cord blood DNA methylation in a genome-wide fashion. In collaboration with Dr. Carol Wagner at Medical University South Carolina

21 Study Design This is an ongoing placebo-controlled randomized clinical trial (RCT) at the Medical University of South Carolina designed to examine maternal and neonatal health status as a function of vitamin D supplementation 300 pregnant women enrolled between weeks of gestation and followed until delivery Each mother is randomized to receive either 4000 IU/day vitamin D3 or placebo; in addition to a daily standard prenatal vitamin containing 400 IU vitamin D3

22 Clinical Characteristics of Cord Blood Samples
Means ± SD

23 Approaches Maternal blood samples were collected at each trimester.
Cord blood was collected at delivery. Both maternal and cord blood total 25(OH)D concentrations were measured (Diasorin RIA, Stillwater, MN).

24 Infinium HumanMethylation450 BeadChip
>485,000 CpG sites at single-nucleotide resolution Located in the promoter region > Covers 99% of RefSeq genes On average, 17 CpG sites per gene region across promoter, 5’UTR, first exon, gene body, and 3’UTR Covers 96% of CpG islands, and island shores and the regions flanking them β value ranges from 0 (unmethylated) to 1 (fully methylated) Low sample input (500 ng or 1 ug) High sample throughput- 12 samples per array

25 Volcano Plots of Differential Methylation
Cord blood 25(OH)D Maternal 25(OH)D

26 Results A total of 2,027 CpG sites had raw p value <0.01 for cord blood DNA methylation and 2,427 CpG sites had raw p value<0.01 for mothers’ DNA methylation. 1,375 CpG sites had both raw p value <0.01. The top 10 genes include GRIK2, GRIN2D, B3GNTL1, SLC2A1, PF4V1, TSPAN13, KLK5, PRICKLE1, PCNX, AGBL5. The pathway analysis for the top 200 genes showed that these genes were enriched in neurogenesis, the regulation of metabolic process, antigen processing and presentation, inflammation, regulation of cell death, cell proliferation, transmission of nerve impulse, neurogenesis, neuron differentiation and transcription factor activity.

27 Top 10 differentially methylated genes

28

29 Summary and Future Work
Maternal vitamin D supplementation and resultant cord blood vitamin D levels are associated with DNA methylation changes. Further analyses in the entire cohort post to the completion of this ongoing RCT are warranted to validate these findings. Future studies are to examine whether the DNA methylation of identified CpG sites are associated with phenotypes in babies at two year follow-up (i.e. neurodevelopment measures, etc).

30 Conclusions/Discussions of All Studies
Increased DNA methylation in peripheral leukocytes, and subsequently increased genomic and chromosomal stability, may be one underlying mechanism by which vitamin D supplementation exerts its anti-inflammatory and other beneficial effects. Vitamin D is simple, safe and inexpensive, which can be used to activate or silence gene expressions in peripheral leukocytes by “resetting” the chromatin to its normal status, to attenuate or counterbalance inflammation.

31 Acknowledgement Collaborators Haidong Zhu Huidong Shi Xiaoling Wang
Shaoyong Su Bernard Gutin Gregory Harshfield Harold Snieder Carol Wagner Bruce Hollis Lab Personnel Samip Parikh Jigar Bhagatwala De-huang Guo Ishita Kotak Ying Huang Jeffrey Thomas Robyn Haven Anas Raed Funding: 14GRNT AHA-Grant-in-Aid (Dong), 16GRANT AHA Grant-in-Aid (Zhu) R01 HL (Dong/Zhu, Co-PI)

32 Team


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