LYON, FRANCE—Researchers are uncovering an increasing amount of evidence that vitamin D plays a role in multiple sclerosis (MS), according to a review presented at the 28th Congress of the European Committee for Treatment and Research in MS. Recent findings indicate that vitamin D may be influential during interactions between genes and the environment and may have long-term epigenetic effects.

Sreeram Ramagopalan, postdoctoral fellow at Oxford University in the United Kingdom, described the results of various investigations that shed further light on how vitamin D levels may affect gene expression and, ultimately, the risk of developing MS.

A Threshold Effect on Activation of the Vitamin D Receptor
To investigate the relationship between vitamin D receptor binding and vitamin D levels in serum, Dr. Ramagopalan and his colleagues examined nine healthy individuals. Using flow sorting and chromatin immunoprecipitation sequencing, the researchers observed a strong correlation between serum hydroxyvitamin D levels and vitamin D receptor binding in the subjects' CD4+ T-cells. "There seems to be a threshold effect on activation of the vitamin D receptor," said Dr. Ramagopalan.

Healthy individuals with serum 25-hydroxyvitamin D levels greater than 75 nmol/L had a mean of approximately 4,500 vitamin D receptor binding sites. Individuals with serum vitamin D levels lower than 75 nmol/L had a mean of approximately 500 binding sites.

A hierarchical clustering analysis revealed that individuals with serum hydroxyvitamin D levels greater than 75 nmol/L tended to have intronic and intragenic binding sites. Promoter-based binding sites were more common in subjects with lower levels of hydroxyvitamin D. The Vitamin D Council recommends a minimum serum vitamin D level of 75 nmol/L for health, noted Dr. Ramagopalan.

Further testing indicated that vitamin D receptor binding sites were highly enriched for genes associated with MS in individuals with serum hydroxyvitamin D levels greater than 75 nmol/L. The investigators found no enrichment for vitamin D receptor binding sites in individuals who had serum hydroxyvitamin D levels lower than 75 nmol/L. In addition, the researchers detected "a big difference in gene expression, and this also related to MS-associated genes," between individuals with serum hydroxyvitamin D levels above and below 75 nmol/L, said Dr. Ramagopalan.

Methylation May Affect Vitamin D's Influence on MS
Dr. Ramagopalan and his colleagues are examining sets of twins as part of ongoing studies of the epigenetics of MS. Each set includes one twin with MS and one without. Recently, the investigators studied DNA methylation in the twins' CD4+ T-cells using an Illumina 27k array, which covers approximately 0.1% of DNA methylone. They found "about 120 differentially methylated regions or methylation-variable positions," including hypomethylation and hypermethylation in the affected twins, said Dr. Ramagopalan.

After examining three of these twin pairs to determine whether vitamin D receptor binding was affected by DNA methylation, the group found that methylation at promoter sites correlated with the absence of vitamin D receptor binding. Conversely, lack of methylation correlated with high levels of vitamin D receptor binding. Methylation associated with MS also appeared to affect vitamin D receptor binding. In affected twins, hypermethylation correlated with little or no vitamin D receptor binding. "Methylation associated with MS may affect the influence of vitamin D in patients with MS," said Dr. Ramagopalan.

Vitamin D May Influence the Expression of Thousands of Genes
These results extend and complement the findings of Dr. Ramagopalan's previous studies. In 2010, he and his colleagues investigated whether the vitamin D receptor was more or less likely to bind to genes associated with 47 diseases. The researchers found that genes associated with autoimmune diseases such as type 1 diabetes, Crohn's disease, MS, and colorectal cancer were unusually enriched for vitamin D receptor binding sites.

At the time, approximately 10 genes were associated with MS, and more have been associated with the disease since then. In the group's updated analysis, performed in 2012, 80% of the more than 60 MS-associated genes had vitamin D receptor binding near them.

The vitamin D receptor tends to bind to intragenic or intronic binding sites, and Dr. Ramagopalan's group wanted to understand how this tendency affects gene expression. The investigators found that vitamin D receptors bound to strong enhancer sites and active promoter sites at more than 60 times the rate that would result from chance. "This suggested that the binding of vitamin D receptors to these regions should influence gene expression," said Dr. Ramagopalan. The investigators also found that when the Epstein–Barr virus protein EBNA-3 was knocked out, vitamin D was associated with the differential expression of approximately 4,000 genes.

In subsequent studies, the researchers exposed in utero mice to vitamin D deficiency. The number and type of genes expressed were different in these mice, compared with control mice. Genes that were upregulated in mice exposed to vitamin D deficiency were also upregulated in their offspring, and the same correspondence was true for genes that were downregulated in the parent mice. "This suggests that vitamin D impacts gene expression until adulthood, and this impact also lasts until the next generation," said Dr. Ramagopalan. "We're actively trying to study the mechanism and how this potentially may relate to complex disease."

—Erik Greb
Senior Associate Editor

Research Helps Clarify the Role of Vitamin D in Multiple Sclerosis