Vitamin D receptor polymorphisms and diseases

Abstract

The vitamin D endocrine system is central to the control of bone and calcium homeostasis. Thus, alterations in the vitamin D pathway lead to disturbances in mineral metabolism. Furthermore, a role for vitamin D has been suggested in other diseases, like cancer, diabetes and cardiovascular disease. Expression and nuclear activation of the vitamin D receptor (VDR) are necessary for the effects of vitamin D. Several genetic variations have been identified in the VDR. DNA sequence variations, which occur frequently in the population, are referred to as “polymorphisms" and can have biological effects. To test whether there is a linkage between VDR polymorphisms and diseases, epidemiological studies are performed. In these studies, the presence of a variation of the gene is studied in a population of patients, and then compared to a control group. Thus, association studies are performed, and a link among gene polymorphisms and diseases can be established. Since the discovery of VDR polymorphisms a number of papers have been published studying its role in bone biology, renal diseases, diabetes, etc. The purpose of this review is to summarize the vast amount of information regarding vitamin D receptor polymorphisms and human diseases, and discuss its possible role as diagnostic tools.

Introduction

The vitamin D endocrine system is central to the control of bone and calcium homeostasis. The active form of the vitamin D is 1,25-dihydroxyvitamin D (calcitriol), the circulating level of which is tightly regulated and acts through a specific receptor to mediate its genomics actions on almost every aspect of calcium homeostasis. Furthermore, it has also been shown that vitamin D plays an important role in other metabolic pathways, such as those involved in the immune response and cancer (Fig. 1) [1]. Vitamin D, derived from the diet or by bioactivation of 7-dehydrocholesterol, is inert and must be activated to exert its biological activity. Vitamin D3 is produced in the skin by an ultraviolet light-induced photolytic conversion of 7-dehydrocholesterol to previtamin D3 [2], [3] followed by thermal isomerization to vitamin D3 [4], [5]. The first step in the metabolic activation of vitamin D is hydroxylation of carbon 25. This reaction occurs primarily in the liver, although other tissues including skin, intestine, and kidney have been reported to catalyze 25-hydroxylation of vitamin D. The second and more important step in vitamin D bioactivation, the formation of 1,25(OH)₂D₃ from 25(OH)D₃ occurs, under physiological conditions, mainly in the kidney [5]. The renal enzyme responsible for producing 1,25(OH)₂D₃, 25(OH)D-1a-hydroxylase, is located in the inner mitochondrial membrane and is a cytochrome P-450 monooxygenase requiring molecular oxygen and reduced ferredoxin [6]. In recent years, many reports have demonstrated that the kidney is not unique in its ability to convert 25(OH)D₃ to 1,25(OH)₂D₃. Numerous cells and tissues express 1a-hydroxylase in vitro; however, in humans, these extrarenal sources of 1,25(OH)₂D₃ only contribute significantly to circulating 1,25(OH)₂D₃ levels during pregnancy, in chronic renal failure, and in pathological conditions such as sarcoidosis, tuberculosis, granulomatous disorders, and rheumatoid arthritis.

Most of the biological activities of 1,25(OH)₂D₃ are mediated by a high-affinity receptor that acts as a ligand-activated transcription factor. The major steps involved in the control of gene transcription by the vitamin D receptor (VDR) include ligand binding, heterodimerization with retinoid X receptor (RXR), binding of the heterodimer to vitamin D response elements (VDREs), and recruitment of other nuclear proteins into the transcriptional preinitiation complex. Thus, genetic alterations of the VDR gene could lead to important defects on gene activation, affecting calcium metabolism, cell proliferation, immune function, etc., which could be explained by changes in the protein sequence. For instance, deleterious mutations in the VDR gene cause 1,25-dihydroxivitamin D resistant rickets, a rare monogenetic disease [7]. More subtle sequence variations (polymorphisms) in the VDR gene also occur more frequently, but the significance of it has not been systematically analyzed and the effects on the VDR protein levels and function are unknown.

A polymorphism is a genetic variant that appears in at least 1% of the population. These changes can occur in non-coding parts of the gene (introns), so they would not be seen in the protein product. Changes in these regulatory parts of the gene would then affect the degree of expression of the gene, and thus the levels of the protein. For instance, changes in the 5′-promoter of the VDR gene can affect mRNA expression patterns and levels, while 3′ untranslated region (UTR) sequence variations can affect the mRNA stability and protein translation efficiency. However, the changes can take place in exonic parts of the DNA, then leading to changes in the protein sequence. Nonetheless, changes in exonic sequences of the DNA which do not alter the protein structure are also possible, and are called synonymous polymorphisms. Often these changes create or abolish sites for restriction enzymes to cut the DNA. Digestion with the enzyme then produces DNA fragments of a different length which can be detected by electrophoresis. These polymorphisms are called Restriction Fragment Length Polymorphisms (RFLPs). Developments in DNA sequencing now make it easier to look for allelic versions of a gene by sequencing samples of the gene taken from different members of a population (or from a heterozygous individual).

The discovery of genetic variants linked with susceptibility of diseases can be the key to advances in preventive medicine. In general, we use association studies to test whether a polymorphism occurs more frequently in the cases studied than in the controls. If a relationship with the disease emerges from association studies, this finding would strongly support the idea that the candidate gene is in some way involved in the disease. The interpretation of the association studies however is sometimes hindered by the fact that most of the polymorphisms used have an unknown functional effect, so it is expected a linkage to truly functional polymorphisms elsewhere in the same or a nearby gene. Thus, in addition to knowing which polymorphisms are present in a candidate gene area, it is important to understand how they relate to each other, both in a genetic and in a functional level.

The existence of several RFLPs in the VDR gene has been described using different restriction enzymes. Examples of these include the Tru9I [8], TaqI [9], BsmI [10], EcoRV [10] and ApaI [11]. All these RFLPs are located between the 8 and 9 exons and lay in an area with unknown function. A different case of RFLP is the so-called FokI. This polymorphism was described in the early nineties [12], [13] in the exon 2, and consisted of a T to C change. The change is inside a start codon (ATG), so when the C variant is present, an alternative start site is used leading to a protein with different size. Most of the experiments conducted so far point to the fact that the shorter form of the protein (424 aa) is more active than the long form (427 aa) in terms of its transactivation activity as a transcription factor. However, it seems to be a gene-specific and cell type-specific effect. Thus, some genes and some cell types will be more sensitive to the effect of the polymorphism than others [14].

Using the sequencing approach, a number of new polymorphisms have been found. For instance, Brown et al. [15] found a C to T change near the exon 2 and a insertion/deletion of a G after exon 7. Arai et al. in 2001 [16] detected a new polymorphism (Cdx2) using the same technique in Japanese women. This polymorphism consisted in a G to A change in the promoter region of the VDR gene. That change is within the binding site for an intestinal-specific transcription factor called CDX2. Recently the polymorphism has also been described among different racial groups [17].

The 3′ UTR of the VDR gene is also a source of several different polymorphisms. However, conflicting reports over the number and position of the polymorphisms exist in the literature. Morrison et al. [9] and Durrin et al. [18] reported 13 and 7 different polymorphisms, respectively. Surprisingly, only two sequences were common in both papers.

As we stated before, the association of a certain polymorphism with a phenotype does not necessarily mean that the polymorphism is causing it. The association of alleles of different polymorphisms with each other within a population is called linkage disequilibrium (LD) [19]. The low level of recombination over time in a certain area of a gene, leads to the presence of certain polymorphisms with a high level of association. In practice this means that we can predict the presence of a certain allele by the presence of an adjacent linked one. In cases of high levels of LD, this leads to blocks of alleles that are present together forming what we call a haplotype. Those blocks vary in size, having an average size of 10–20 kb, and they could be very useful to determine the causes of certain genetic diseases. One of the advantages is that we only need to determine the presence of a few polymorphisms to know the presence of all the alleles associated with the haplotype. Then, once we know which haplotype carries the risk allele, we can use different techniques to determine which polymorphism is truly responsible for the phenotype observed. Several studies have been performed to determine the degree of LD among the known polymorphisms of the VDR gene. So far, the information available is very limited but a strong degree of LD has been found among the TaqI, BsmI, EcoRV and ApaI RFLPs [9] with five different haplotypes [20]. Furthermore, Uitterlinden et al. [21] have shown a strong LD between the BsmI RFLP and a polymorphism present in the 3′ UTR (VNTR) [22]. Thus, the study of the LD among the different VDR polymorphisms could give new insights in the etiology of certain diseases.

In a recent paper, Nejentsev et al. [23] sequenced 94 kb in a 164 kb region of chromosome 12q12-q14 around the VDR gene. They found 245 polymorphisms that seem to be in three LD blocks. Polymorphism within each block showed little, if any, LD with polymorphisms in a different block. They compared the LD blocks among four different populations, finding remarkable similarity on the LD patterns in all the European populations but not with the African population. Therefore, they concluded that European populations show relative advantage to detect initial disease association, because fewer tag polymorphisms are required to characterize a common variation. They also showed that African populations have higher haplotype diversity. Analysis of additional haplotypes may further help the fine mapping of a casual variant. Therefore, genetic analysis in populations of different origin would facilitate the study of complex diseases.

Very recent studies have started to shed some light on the functional effects of polymorphisms in the non-coding regions of the VDR gene. Fang et al. [24] analyzed 15 haplotypes in the 5′ 1a/1e, 1b promoter region and in the 3′ UTR and they found very strong association with risk of bone fracture. Furthermore, they performed functional analysis showing that variants carrying the 1e/1a promoter with increased risk had lower mRNA levels. They also showed that, in an osteoblast cell line, the presence of the 3′ UTR risk haplotype caused a 30% increase in mRNA decay. D'Alesio et al. [25] analyzed two polymorphisms in the promoter region of the VDR gene (G-1521C and A-1012G). They found that one base change in any of the variant sites led to a dramatic change in protein-DNA complex formation and a smaller height from 11 years of age up to adult height.