2. Vitamin D Status and Genetic Evidence in the Populations

Regarding vitamin D deficiency, several factors have been described, mainly the lack of exposure to sunlight, latitude, the season of the year, skin pigmentation, and use of sunscreen; other factors involved in vitamin D deficiency are diet, age, pharmacotherapy administered (antiepileptic and glucocorticoids), and particularly, several studies have described that genetic differences between individuals and populations such as genetic polymorphisms could influence the vitamin D deficiencies presented in all populations around the world^[10].

GC

CYP2R1

CYP27B1

VDR genes are associated with lower calcidiol serum levels^[11]. Moreover, vitamin D serum deficiencies are present in a high frequency in a healthy population, which could be related to the SNPs’ presence in these genes that may modify the response to supplementation of vitamin D in health and disease. A study conducted in healthy Iranian adolescents described the differential effect of the

CYP2R1

p = 0.002)^[12]. This is evidence of the role of polymorphisms in genes related to vitamin D metabolism in the variation of the response to vitamin D supplementation, even in healthy conditions.

3. Polymorphisms in the Main Key Genes Related to Vitamin D Metabolism

The skin produces around 80% of vitamin D in the form of cholecalciferol when exposed to ultraviolet B (UVB) light at a wavelength of 290–320 nm, and the remaining 20% is obtained from the diet as ergocalciferol from mushrooms or as cholecalciferol from fortified dairy products, fish, and eggs, mainly ^[13].

GC

Figure 1a). The

GC (rs2282679) SNP was been associated with modulation of vitamin D serum levels in several populations^[14][15][16]. In these studies, the main vitamin D circulating metabolite in blood, calcidiol, was used to evaluate vitamin D deficiencies ^[17].

Figure 12.

a

GC

GC

b

CYP2R1

CYP2R1

c

CYP27B1

CYP27B1

d

VDR

IL-17A

FOXP3.

VDR

FokI

FokI

BsmI

VDR

VDR

ApaI

VDR

VDR

TaqI

VDR, presents a change of C > T (also called T > t) and generates a synonymous change of the isoleucine amino acid in the coding sequence, therefore it does not change the encoded protein, but it could influence the stability of the mRNA. All these SNPs are related to modulating de vitamin D serum status in health and disease. Ile: isoleucine; Thr: threonine; Met: methionine VDBP: vitamin D binding protein; VDR: vitamin D receptor; RXR: retinoid X receptor; VDRE: vitamin D response elements; UTR: untranslated region; THEM4: thioesterase superfamily member 4; Th: T helper lymphocyte; VDRA: wild type full-length VDR A isoform.

CYP2R1

Figure 1b), the (rs10741657) SNP described in

CYP2R1 had also been related to modulate calcidiol serum status in several studies^{[12][14][15][18]}. Then, after the generation of calcidiol in the liver, it binds again to VDBP and subsequently interacts with the enzyme 1-α hydroxylase, encoded by the

CYP27B1

Figure 1c)^[13]. Polymorphisms in

CYP27B1, particularly the rs10877012 SNP, was associated with lower calcidiol serum levels^[19].

VDR

After the calcitriol translocation to the nucleus, this complex forms a heterodimer with the retinoid x receptor (RXR), and thus regulates vitamin D target genes through its binding to specific DNA sequences called vitamin D response elements (VDREs)^[20][21]. Since VDR is expressed in different immune cells, such as neutrophils, macrophages, dendritic cells (CDs), T and B lymphocytes, calcitriol may regulate the immune system^[8].

Calcitriol can be produced by monocytes and macrophages and generate a shift from pro-inflammatory to tolerogenic immune status^[22]. Calcitriol promotes M1 phenotype switching to M2 via the nVDR-PPARγ pathway and via the upregulation of the expression of IL-10^[23]. Particularly, calcitriol promotes a shift from Th1 and Th17 to Th2 immune profile via suppression of expression of cytokines of the Th1 (IL-2, IFN-γ, TNF-α) and Th17 (IL-17, IL-21) profiles, and induction of the expression of cytokines of Th2 profile (IL-4, IL-5, IL-9, IL-13), in order to limit inflammatory processes and autoimmune reactions ^[8][22].

VDR

FokI

BsmI

ApaI

TaqI

Figure 1d). Therefore, SNPs′ presence in key enzymes related to vitamin D metabolism may modulate calcidiol levels and calcitriol function; they may also modify the disease activity in MS, RA, and SLE through the vitamin D deficiency, and contribute to genetic susceptibility to autoimmunity. In the following sections, we will describe how polymorphisms in these genes can modulate their expressions.

3.1. Vitamin D Binding Protein (VDBP) (SNP rs2282679

GC)

Vitamin D binding protein (VDBP) is the main carrier protein for vitamin D, which binds 85 to 90% of the total circulating calcidiol ^[24]. This protein is encoded by the

GC gene located in chromosome 4, position 13.3 (4q13.3), and has 55,136 bp size ^[25]. The

GC gene consists of 13 exons and the rs2282679 SNP was described located at the position 71,742, presenting an A ˃ C change (ancestral allele: A) in the intron 12, near to the actin III subdomain^[26] and the endonuclease enzyme commonly used to identify this

GC

FokI

Flavobacterium okeanokoites) ^[27]. According to rs2282679 SNP intron location, its hypothetical functional effect may alter genetic mRNA expression due to modification of the splicing process (44). A complete GWAS showed that

GC (rs2282679) C allele was associated with lower serum calcidiol and VDBP levels in a study carried out in approximately 30,000 subjects with European ancestry included in 15 cohorts^[28]. Additionally, various studies have shown the association of the

GC (rs2282679) SNP with genetic susceptibility and disease modulation in MS, RA, and SLE^{[29][30][31][32]} (

Figure 1a).

3.2. Vitamin D 25-Hydroxylase (SNP rs10741657

CYP2R1)

The enzyme vitamin D 25-hydroxylase is a protein of around 500 amino acids with a molecular weight of 50–55 kilo-daltons (kDa). The liver is the main site where it is synthesized and performs its function of 25-hydroxylation enzyme activity, besides this enzyme activity was also described in the kidney and intestines^[33], but cholecalciferol and ergocalciferol are metabolized mainly by the vitamin D 25-hydroxylase to calcidiol in the liver^[34].

The enzyme vitamin D 25-hydroxylase is encoded by the

CYP2R1

CYP2R1 is positioned at 14,893,332 pb in the gene, it displays an A ˃ G change in the 5′ UTR region (ancestral allele: A)^[18].

In order to recognize the presence of this SNP, the endonuclease restriction enzyme

MnlI

Moraxella nonliquefaciens) is commonly used^[35]. According to the SNP location in 5′ UTR, the rs10741657 SNP may regulate gene expression by modifying translation initialization and transcript stabilization of mRNA, and therefore, modulate 25-hydroxylase expression and enzymatic activity rate^[36].

A meta-analysis that included 16 articles with 52,417 participants showed that the

CYP2R1 (rs10741657) GG genotype shows trends of low calcidiol serum levels compared to the AA genotype in the Caucasian and Asian population^[18] and when healthy persons are supplemented with cholecalciferol, carriers of the AA genotype showed a 2.5-fold increase in calcidiol levels compared to GG genotype carriers ^[12], which highlights the role of

CYP2R1

Figure 1b).

3.3. Vitamin D 1-α Hydroxylase (SNP rs10877012

CYP27B1)

The enzyme vitamin D 1-α hydroxylase corresponds to a P450 protein of 507 amino acids of around 55 kDa^[37]. This enzyme metabolizes calcidiol to calcitriol, the active form of vitamin D^[34], and it is encoded by the

CYP27B1

CYP27B1 is located at the non-coding region -1260 and is characterized by a change of G ˃ T (ancestral allele: G)^[38][39]. The restriction endonuclease enzyme

HinfI

Haemophilus influenza) is commonly used for its detection ^[27][35]. Due to its location in the 3′ UTR region, this SNP may alter transcript stabilization regulation and its localization in the cytoplasm ^[36].

A study carried out in 253 German patients with differentiated thyroid carcinoma showed that patients with the presence of the GG genotype (Referred as CC in this study by its position in the negative DNA strand) was associated with lower calcitriol serum levels than patients carrying the TT genotype (Referred as AA in this study by its position in negative DNA strand) (60 pmol/mL vs. 72 pmol/mL, respectively)^[38].

In another study carried out in Caucasian German patients with gestational diabetes, the GG (CC) genotype was also associated with lower calcidiol levels^[19]. Likewise, another study carried out in a healthy Caucasian British population demonstrated an association between the

CYP27B1 (rs10877012) G (C) allele and lower serum calcidiol levels^[40].

Besides, in type 1 diabetes in Caucasian patients from Germany, those carrying the GG (CC) genotype had a reduced amount of mRNA from

CYP27B1

p = 0.0220)^[19]. Therefore, the SNP rs10877012

CYP27B1

Figure 1c).