Analysis of the Vitamin D Receptor Gene Sequence Variants in Type 1 Diabetes

The molecular mechanisms underlying type 1 diabetes are only partly understood. It develops as a result of a complex interaction of many genetic and environmental factors leading to the immune destruction of the insulin-producing β-cells (1). Three disease loci have been identified so far that contribute to the etiology of type 1 diabetes, the HLA complex, the variable number of tandem repeats locus located in the promoter region of the insulin (INS) gene, and the cytotoxic T-cell–associated antigen-4 gene (CTLA4) (1,2).

1α,25-dihydroxyvitamin D₃, the hormonally active form of vitamin D, has been successfully used to prevent autoimmune insulitis and reduce diabetes incidence in the mouse model of type 1 diabetes, as well as in animal models of other autoimmune diseases (3–6). In humans, population studies suggest that vitamin D supplementation in early childhood decreases type 1 diabetes incidence (7,8), raising hopes that it may be used as a type 1 diabetes preventive treatment. Vitamin D has been long known to play a central role in bone and mineral metabolism. Now it is widely recognized to regulate growth and differentiation in many target tissues and act as a modulator in the immune system (9). Its effects are mediated by the vitamin D receptor (VDR), a member of the nuclear receptor superfamily of transcriptional regulators. VDR is found in >30 different tissues, including islet cells of the pancreas, circulating monocytes, dendritic cells, and activated T-cells (9). Upon binding 1α,25-dihydroxyvitamin D₃, VDR regulates gene expression by direct interaction with specific sequence elements in the promoter region of hormone-responsive target genes. In the immune system, 1α,25-dihydroxyvitamin D₃ was shown to suppress production of the interleukin (IL)-12, IL-2, tumor necrosis factor-α, and interferon-γ cytokines and activate expression of transforming growth factor-β1 and IL-4 cytokines, thereby inhibiting Th1-type responses and to induce regulatory T-cells (9). It can also regulate differentiation and maturation of dendritic cells critical in the induction of T-cell–mediated immune responses (10). These pathways may explain the beneficial effects of vitamin D in autoimmune diseases (6).

The VDR gene is located on chromosome 12q12-q14 and includes eight protein-coding exons (exons 2–9) and six untranslated exons (exons 1a–1f), which are alternatively spliced, and two promoter regions (11). Four common single nucleotide polymorphisms (SNPs) in the VDR gene have been studied intensively (12): FokI T>C (rs10735810), BsmI A>G (rs1544410), ApaI G>T (rs7975232), and TaqI C>T (rs731236). Allele T of the FokI SNP creates an alternative ATG codon leading to a three-amino-acid–longer VDR protein (13). SNPs BsmI and ApaI are located in an intron, and TaqI is a silent SNP in exon 9. These four SNPs have been tested for association with various human traits and diseases and have been reported (12) to affect risk of cancers and bone density–related and immune-mediated disorders. Several studies reported association of type 1 diabetes with one of these four SNPs. However, the reported associations are inconsistent between studies (14–19). Since these SNPs, with potential exception of the FokI variant, have no known functional role, these results may indicate that they are merely markers in linkage disequilibrium with a true causal variant(s), which remains unknown. Recently, we performed a comprehensive identification (20) of the sequence polymorphisms in the VDR gene region and developed its dense SNP map. Here we report a study of association between 98 SNPs in the VDR gene region and type 1 diabetes.

We genotyped a set of 458 U.K. families with two type 1 diabetic offspring in each for 98 VDR SNPs, including the four commonly studied SNPs (FokI, BsmI, ApaI, and TaqI). Additionally, we genotyped a set of 307 U.S. families with two type 1 diabetic offspring in each family for 40 out of these 98 SNPs. We found that four SNPs, rs4303288, rs11168275, rs12721366, and rs2544043, which were tested here for the first time, showed evidence of association with type 1 diabetes (P = 0.01–0.03) (Figs. 1 and supplementary table [available in an online appendix at http://diabetes.diabetesjournals.org). We genotyped these four SNPs, as well as SNPs FokI, BsmI, ApaI, and TaqI, in an additional set of type 1 diabetic families with at least one affected offspring from the U.K., Finland, Norway, Romania, and U.S. In the analysis of the combined family set, none of the four commonly studied SNPs were associated (P > 0.05) (Table 1). Among SNPs first tested by us, rs12721366, rs2544043, and rs4303288 showed some evidence of association with type 1 diabetes in the combined family set (P < 0.05) (Table 1). These associations cannot be viewed as statistically significant on a genome-wide scale (21,22). Therefore, we analyzed these three SNPs in an additional independent set of up to 1,587 type 1 diabetic patients and 1,827 control subjects from the U.K. and did not find any evidence of association (Table 2). These results indicate that a major effect, such as odds ratio (OR) > 1.5 for a common allele, is unlikely to exist for the VDR polymorphisms in type 1 diabetes. Population substructure within case-control samples could affect association studies (23,24). However, given that we have found no convincing evidence of association between the VDR SNPs and type 1 diabetes in the large family collection, potential population substructure in the British case-control sample should not greatly alter our conclusion.

SNPs BsmI, ApaI, and TaqI are in linkage disequilibrium, and their haplotypes were previously shown (15) to be associated with type 1 diabetes in the German population. Therefore, in 1,811 type 1 diabetic families who were genotyped for all three of these SNPs we tested association of the BsmI-ApaI-TaqI, BsmI-TaqI, and ApaI-TaqI haplotypes (15) and found no association (7 degrees of freedom [df], P = 0.11; 3 df, P = 0.18; and 3 df, P = 0.16, respectively).

Our study does not support previous reports (14–17) in much smaller samples, which showed some evidence of type 1 diabetes association for the FokI, BsmI, ApaI, or TaqI SNPs or their haplotypes. Spurious association and publication bias is a possible explanation of the previous positive results, particularly given that there is no consistency regarding the findings of the associated SNPs and their alleles between different studies.

Environmental factor(s), specific to some groups of type 1 diabetic patients, may alter the risk associated with particular SNPs in the VDR gene. For instance, VDR functions together with 1α,25-dihydroxyvitamin D₃, the level of which is dependent on various environmental factors (25). These factors include vitamin D intake in the diet or as a supplement and its synthesis from precursors in skin under ultraviolet light exposure. Such environmental factors may modulate risk associated with the sequence variation in the VDR gene, e.g., certain variants may only be functionally important among a subpopulation of subjects with vitamin D insufficiency. While it is difficult to design a study to evaluate such potential interaction directly, it may manifest itself as regional or temporal heterogeneity in association between patients, who would have developed type 1 diabetes in different environments. We therefore analyzed the effect of the country of origin of a patient and a combined effect of the patient’s year of birth and a country of origin on association between type 1 diabetes and eight VDR SNPs that we studied in five different populations. We did not find any evidence for significant heterogeneity of the type 1 diabetes association (Table 1). However, environmental factors that influence levels of active vitamin D forms in humans are complex, and their effect may not be excluded by our analysis.