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Serum 25(OH)D is an objective measure of assessing vitamin D deficiency or toxicity. Some disagreement exists regarding the exact serum 25(OH)D levels in defining vitamin D deficiency, insufficiency, and sufficiency. It is widely proposed that the optimal level of serum 25(OH)D for bone health is 75 nmol/L. Data from cohort studies suggest that optimal levels may be higher for prevention of disorders other than osteoporosis. Serum 25(OH)D level >500 nmol/L is toxicity possible.
Vitamin D deficiency or insufficiency [25(OH)D<75 nmol/L] is a common health problem affecting people of all ages and both genders. About 1 billion people worldwide have vitamin D deficiency or insufficiency. More than 60% of North Americans have blood 25(OH)D levels lower than the acceptable level (75 nmol/L). The incidence of deficiency and insufficiency continues to grow because of the scarcity of vitamin D in food, fervent sunscreen usage, and increasing indoor working habits. Given the common vitamin D deficiency and the powerful effect of vitamin D in human beings, "it is reasonable for everyone to have his or her blood 25(OH)D concentration measured once a year" in order to achieve the optimal vitamin D level (Holick, Am J Clin Nutr, 2002).
It is well established that genetic factors are involved in regulation of serum 25(OH)D concentration. Numerous studies have shown that serum 25(OH)D is highly genetically determined. Due to inter-individual genetic variation, people who are exposed to a high amount of sunlight or taking high dose of vitamin D supplementation may also suffer from low serum 25(OH)D level (<75nmol/L). Therefore, it is important to identify genetic factors that account for serum 25(OH)D variation. Currently, such specific genes are largely unknown. We have initiated projects to understand genetic and epigenetic factors that contribute to serum 25(OH)D variation. Understanding these factors will lead to "personalized" vitamin D deficiency treatment, and cost-effective prevention of osteoporosis.
Figure 1. Vitamin D metabolism, transportation, and signaling pathways. Selected candidate genes are labeled in the blue rectangles.
Circulating monocytes play important roles for vitamin D metabolism. Enzymes essential for vitamin D metabolism, such as CYP
The objective of study is to identify cellular biomarkers for vitamin D dose-response variation. Here vitamin D dose-response variation is calculated as the difference between measurements of serum 25(OH)D at baseline and at the end of 12-month vitamin D3 intervention, divided by the total vitamin D intake.
We hypothesize that the differentially expressed genes, proteins, and DNA methylation levels in circulating monocytes provide biomarkers to predict vitamin D dose-response variation.
To reach our objective, we took advantage of a completed NIH human project (5R01AG014683) (Calcium and Vitamin D Malnutrition in Elderly Women Study, called CaMEWS hereafter). The CaMEWS was a population-based, randomized, placebo-controlled clinical trial. In total, 446 postmenopausal women with age ≥55 years were treated with calcium (1500 mg/day) and vitamin D3 (1100 IU/day) for at least 12-months. Vitamin D3 treatment compliance rate is 85.7%. For each subject, the serum 25(OH)D level was measured at the baseline and again after 12-month vitamin D3 treatment.
Using the 12-month increase of serum 25(OH)D values, we have identified women who responded to vitamin D supplementation (responders) and women who did not respond to vitamin D supplementation (non-responders) from the CaMEWS.The responders and non-responders were subjects at the extreme tails of the distribution of the 12-month increase of serum 25(OH)D level (Figure 2). The responders are subjects who have the highest vitamin D dose-response values. The non-responders are subjects who have the lowest vitamin D dose-response levels.
We have recruited 30 responders and 30 non-respondersfrom the 446 CaMEWS participants. Blood samples were collected. Monocytes were isolated and stored in liquid nitrogen. Using the unique sample, we will conduct epigenetics, gene expression, protein expression, genetic/genomic data analyses to identify cellular biomarkers for vitamin D dose-response variation.
Figure 2. Frequency distribution of the participants of the CaMEWS, n=446.
DNA methylation is one of the most common epigenetic mechanisms in humans. Studiesreport that vitamin D affects DNA methylation levels in vitro in vitamin D genes, such as the CYP27B1 gene. The relationship between vitamin D supplementation and the DNA methylation status are unknown in humans. Identifying this relationshipis important for helping physicians tailor vitamin D treatment to individual patient's needs.
The sample for the project comes from the completed vitamin D3 intervention study, CaMEWS. For each participant of the CaMEWS, we archived 2 ml serum sample in -80oC freezers in year 2000-2005. From the 446 subjects who received calcium and vitamin D3 treatment, we selected 18 responders and 18 non-responders from the 446 subjects, at the two extreme tails of the distribution of the 12-month increase of serum 25(OH)D (Figure 2). For each subject, genomic DNA before and after 12-month vitamin D3 intervention was extracted from frozen serum. Genes (CYP27A1, CYP2R1, CYP27B1, CYP24A1) important for vitamin D metabolism are selected for the research. By measuring DNA methylation level in the selected candidate genes, we will test: 1) whether a vitamin D supplementation affects DNA methylation level; 2) whether the DNA methylation status of these genes is associated with the dose-response of serum 25(OH)D to vitamin D supplementation.
Our preliminary data shows that the average methylation ratio of the CYP2R1 gene in the non-responder group (30%) was significantly higher than that in the responder group (8%) (p=0.004) (Figure 3). In both groups, the DNA methylation ratio was unaffected by vitamin D3 intervention (Figure 3). The hypermetylation in the non-responder group may contribute to gene silencing of CYP2R1, leading to lower increase of serum 25(OH)D levels. The data suggest that the methylation status of the CYP2R1 may be useful in predicting vitamin D dose-response variation in serum 25(OH)D.
We are testing the relationship between vitamin D supplementation and DNA methylation status of the four candidate genes in expanded samples.
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