**7. Vitamin D and its role in risk and progression of multiple sclerosis**

In the past decades, much attention has been given to vitamin D and its role in MS and other autoimmune diseases. The following sectionsare dedicated to the metabolism and structure of vitamin D, its immunological effects, serum level and mechanisms of action of vitamin D in the prevention and treatment of MS. We also describe the genetic factors that can modulate the biological effects of vitamin D.

#### **7.1. The structure and metabolism of vitamin D**

Vitamin D in the human body undergoes a complex metabolism. Cholecalciferol (vitamin D3), as a precursor of a hormonally active form, is produced in the skin from 7-dehydrocho‐ lesterol after sunlight exposure and can also be absorbed from the diet. Subsequently, chole‐ calciferol is hydroxylated in the liver forming 25-hydroxycholecalciferol, calcidiol. The hormonally active form of vitamin D, 1,25- dihydroxycholecalciferol, calcitriol, is produced by further hydroxylation especially in the kidneys and also in other tissues. The enzyme cat‐ alysing this hydroxylation is 25-hydroxyvitamin D-1α-hydroxylase, coded by *CYP27B1* (cy‐ tochrome P450 family 27 subfamily B member 1) gene [66, 67]. In various cells, the bioactive form of vitamin D binds to the vitamin D receptor (VDR) providing its physiological func‐ tions by modulation of the target gene's transcription [68]. The circulating serum level of vi‐ tamin D depends not only on environmental factors such as exposition to sunlight and vitamin D intake but also on genetic and epigenetic factors.The genetic factors can influence the effects of vitamin D through the variability of the genes participating in its activation and degradation, transport and receptor signalling [69].

#### **7.2. The effect of vitamin D on the functions of immune cells**

There is growing evidence that vitamin D not only regulates bone metabolism but also has large-scale immunomodulatory and anti-inflammatory effects. A linkage has been found be‐ tween vitamin D deficiency and increased risk of autoimmune diseases [70]. The immuno‐ competent cells—macrophages, dendritic cells, Tcells and Bcells are able to produce calcitriol and express the VDR at the high rate. Through this, vitamin D modulates the syn‐ thesis of various cytokines and immunoglobulins and is involved in the regulation of innate and adaptive immune response. Autocrine and paracrine effects of vitamin D depend also on its serum level, and individuals with hypovitaminosis D are in a state of immune system dysfunction and are predisposed to the development of autoimmune diseases [71].

In Tcells, calcitriol inhibits the production of IL-12 and IFN-γ and subsequent differentiation of TH1 lymphocytes that are the key cells involved in the MS development. Calcitriol improves the immunosuppressive functions of TREGcells and ameliorates the TH2-cell development by the activation of the promotor region of *IL-4* gene [19, 72]. Vitamin D increases the expression of IL-4, IL-5 and IL-10 that are able to activate TH2 cells, decreases the production of IFN-γ, blocks the formation of TH1cells after antigen stimulation and has positive effects on the TH1 mediated autoimmune diseases [73, 74]. Bcells, which also participate in the demyelinating process and produce intrathecalimmunoglobulins, express VDR and vitamin D hydroxylases. In Bcells, calcitriol reduces the intracellular signal pathways of nuclear transcription factor NFkappa B (NF-kB) and CD40 signalling [75]. Calcitriol inhibits the maturation and proliferation of Bcells, induces apoptosis of Bcells, inhibits the differentiation of plasma and memory cells and decreases the production of immunoglobulinsIgG and IgM [76]. Immature Bcells are more prone to regulation by calcitriol when compared to plasma cells. Calcitriol also decreases the expression of MHCII molecules and co-stimulatory molecules in Bcells [19].

Calcitriol formed in macrophages inhibits the immune response by suppressing proliferation of TH1- and TH17cells and promoting the functions of TH2- and TREGcells [71]. Calcitriol inhibits the secretion of IL-12 by antigen-presenting cells and monocytes [77]. Vitamin D blocks the differentiation of immature dendritic cells and the expression of co-stimulatory molecules CD40, CD80, CD86 and MHC II, thus decreasing the capacity of dendritic cells to activate autoreactive Tcells. Vitamin D also ameliorates the spontaneous apoptosis of mature dendritic cells [73]. In macrophages, calcitriolsuppresses intracellular oxidative burst and listericidal activity. It also suppresses the expression of Fc and TLR receptors induced by IFN-γ that are important for antigen recognition [78]. Vitamin D suppresses the proliferation of antigenspecific Tcells and chemotaxisof dendritic cells by decreasing the expression of differentiation antigens CD80, CD86 and HLA-DR molecules [79].

#### **7.3. The murine model of MS and vitamin D**

We have shown for the first time in a Central European Slovak population that allele C of rs6897932 is associated with the risk of MS, and allele T has a protective additive effect against MS susceptibility. Moreover,we revealed that minor allele T and genotype TT of rs6897932 in

the *IL7Ra* gene are protective against rapid disease disability progression in MS [65].

**7. Vitamin D and its role in risk and progression of multiple sclerosis**

the biological effects of vitamin D.

10 Trending Topics in Multiple Sclerosis

**7.1. The structure and metabolism of vitamin D**

and degradation, transport and receptor signalling [69].

**7.2. The effect of vitamin D on the functions of immune cells**

In the past decades, much attention has been given to vitamin D and its role in MS and other autoimmune diseases. The following sectionsare dedicated to the metabolism and structure of vitamin D, its immunological effects, serum level and mechanisms of action of vitamin D in the prevention and treatment of MS. We also describe the genetic factors that can modulate

Vitamin D in the human body undergoes a complex metabolism. Cholecalciferol (vitamin D3), as a precursor of a hormonally active form, is produced in the skin from 7-dehydrocho‐ lesterol after sunlight exposure and can also be absorbed from the diet. Subsequently, chole‐ calciferol is hydroxylated in the liver forming 25-hydroxycholecalciferol, calcidiol. The hormonally active form of vitamin D, 1,25- dihydroxycholecalciferol, calcitriol, is produced by further hydroxylation especially in the kidneys and also in other tissues. The enzyme cat‐ alysing this hydroxylation is 25-hydroxyvitamin D-1α-hydroxylase, coded by *CYP27B1* (cy‐ tochrome P450 family 27 subfamily B member 1) gene [66, 67]. In various cells, the bioactive form of vitamin D binds to the vitamin D receptor (VDR) providing its physiological func‐ tions by modulation of the target gene's transcription [68]. The circulating serum level of vi‐ tamin D depends not only on environmental factors such as exposition to sunlight and vitamin D intake but also on genetic and epigenetic factors.The genetic factors can influence the effects of vitamin D through the variability of the genes participating in its activation

There is growing evidence that vitamin D not only regulates bone metabolism but also has large-scale immunomodulatory and anti-inflammatory effects. A linkage has been found be‐ tween vitamin D deficiency and increased risk of autoimmune diseases [70]. The immuno‐ competent cells—macrophages, dendritic cells, Tcells and Bcells are able to produce calcitriol and express the VDR at the high rate. Through this, vitamin D modulates the syn‐ thesis of various cytokines and immunoglobulins and is involved in the regulation of innate and adaptive immune response. Autocrine and paracrine effects of vitamin D depend also on its serum level, and individuals with hypovitaminosis D are in a state of immune system

In Tcells, calcitriol inhibits the production of IL-12 and IFN-γ and subsequent differentiation of TH1 lymphocytes that are the key cells involved in the MS development. Calcitriol improves

dysfunction and are predisposed to the development of autoimmune diseases [71].

In mice that lack the *VDR* gene or the gene of the enzyme catalysing vitamin D activation, an abnormal development and function of TH1-lymphocytes and deficiency of peripheral Tlymphocytes have been observed [80, 81]. Calcitriol treatment can prevent the induction and progression of autoimmune diseases including experimental autoimmune encephalomyelitis (EAE), a murine model of MS [82, 83]. Calcitriol can also decrease the severity of EAE symp‐ toms, and its deficiency causes an increased susceptibility of animals to EAE induction [77, 82]. In mice with chronic EAE, vitamin D administration suppresses the proliferation of specific TH1 cells, inhibits IL-12 dependent production of IFN-γ, prevents relapses and reduces perivascular infiltration, demyelination plaque formation and axonal degeneration in the brain and spinal cord [84].

#### **7.4. Serum level of vitamin D and dose management**

To reflect vitamin D status in the human body, calcidiol plasma level measurement is usually used. Calcidiol is the main circulating form of vitamin D in plasma, and its biological half-time is 19 days [85]. The recommended daily dose of vitamin D is approximately 10times higher than in the past. The optimal serum level of vitamin D is 75–250 nmol/l (30–100 ng/ml). In countries with less sunny climate, the necessary daily dose of vitamin D is 1000–4000 IU/day (1 μg = 40 IU) [86].

The risk of vitamin D overdosing is hypercalcaemia and subsequent organ and tissue damage. Whole body exposure to sunlight results in the production of around 10,000 IU of vitamin D, so it is not simple to cause vitamin D intoxication by its short-term peroral supplementation. The results of several studies suggest that even high-dose vitamin D3 supplementation in MS patients is safe and clinically useful. Burton et al. [87] administered high peroral doses of vitamin D to healthy individuals and MS patients. The initial dose was 40,000 IU/dayduring 28 weeks, followed by 10,000 IU/day during 12 weeks; later it was gradually decreased to 0 IU/day, combined with 1.2 grams of calcium per day. During the period of 40,000 IU of vitamin D per day, the serum levels reached 413 nmol/l, which was higher than the conventional limit established for vitamin D toxicity (250 nmol/l). Calcidiol serum levels remained around this limit for 18 weeks without any observed negative effects. The serum level of calcium was in the physiological reference range during the whole study duration. Moreover, no cardiac rhythm abnormalities or impairment of hepatic or renal functions was observed. Kimball et al. [88] administered 4000–40,000 IU/day to patients in the active phase of MS together with 1.2 grams of calcium. Medium serum level of calcidiol was 78 ± 35 nmol/l and rose to 386 ± 157 nmol/l. Serum calcium level and urinary calcium to creatinine ratio did not exceed the physiological reference values. Vitamin D supplementation in this study did not cause any change in the serum level of hepatic enzymes, creatinine, electrolytes, proteins and parathor‐ mone. Although the serum level of calcidiol doubled the physiological upper range value, hypercalcaemia or hypercalciuria was not observed.

Although the significant toxicity of vitamin D3 was not observed even in doses of 40,000 IU/ day, its daily dose in healthy individuals should not exceed 2000 IU. The optimal daily dose of vitamin D3 that should be routinely recommended to women during pregnancy and lactation is 1000 IU. Children born in families with MS history should be administered daily 200–400 IU of vitamin D3 [66].

#### **7.5. Vitamin D and the course, prevention and treatment of MS**

The role of vitamin D in the prevention of MS development has been confirmed by many experimental, epidemiological, genetic and immunological studies. Vitamin D insufficiency during the whitematter development can alter the pathways of axonal differentiation and adhesion and increase the apoptosis of oligodendrocytes that express VDR. This results in local microenvironmental changes and altered regeneratory and remyelinating capacity [66]. In individuals with an increased genetic risk of MS, it is possible to prevent the demyelina‐ tion process by preventive vitamin D administration. This preventive strategy would be bet‐ ter than reparation of already developed myelin and axonal damage [12, 66].

High-dose peroral vitamin D intake has been found to be inversely associated with the risk of MS in a cohort of more than 90,000 women. Peroral vitamin D supplementation in a dose higher than 400 IU/day leads to the reduction of MS risk when compared to the individuals with no vitamin D intake (RR = 0.59, 95% CI = 0.38–0.91,*p* = 0.006) [89]. Also,calcidiol plasma levels are inversely correlated with MS risk. This association is particularly obvious in whites, while among blacks and Hispanics with lower 25-hydroxyvitamin D levels than whites, there was no significant association between vitamin D and MS risk [90]. Vitamin D also has reparative effects for the nervous tissue, especially in patients in the early phases of the disease. In countries with low sun exposure, food supplementation of vitamin D could be a simple and cheap method of MS prevention [86]. The incidence of MS could be reduced by the adminis‐ tration of vitamin D to pregnant women, and all children living in mild climates should be more exposed to sunlight and should be on a vitamin D–rich diet [66].

Vitamin D is not only a factor modifying MS risk, but it can also have a role in the modulation of disease course. It has been observed that in relapsing remitting MS, calcidiol plasma levels are lower during relapses compared to the periods of remission [91]. In addition, there is evidence that lower calcidiol levels are associated with higher relapse rates and higher risk of exacerbation, as well as higher expanded disability status scale (EDSS) scores and progressive forms of MS [92–94]. Vitamin D can improve memory and cognitive impairments in patients with MS, Alzheimer disease and in patients after chemotherapeutical treatment [95]. Highdose peroral vitamin D supplementation has immunomodulatory effects and leads to reduc‐ tion in the number of relapses and suppression of the inflammatory activity and proliferation of Tcells [87], as well as the decrease in the number of gadolinium-enhancing lesions in brain [88].

In our study, we examined the serum levels of calcidiol in a group of MS patients from the Central-Northern part of Slovakia. We found that hypovitaminosis D is more frequent in MS patients, when compared to healthy individuals. Serum levels of calcidiol were significantly lower in MS patients when compared to controls (15.0 ± 6.1 ng/ml vs. 18.2 ± 8.3 ng/ml, *p*(*K*−*W*) = 0.001). Moreover, we found that there is an association of the serum level of vitamin D with the rate of MS disability progression (*p*(*K*−*W*) = 0.000). We detected similar serum levels of calcidiol in slow progressing and mid-rate progressing MS patients (15.7 ± 5.0 ng/ml vs. 15.8 ± 6.6 ng/ml), but interestingly we noticed a marked decrease of calcidiol serum levels in rapidly progressing MS patients (12.8 ± 5.9 ng/ml). In addition, calcidiol levelwas significantly lower in all subgroups of MS patients when compared to controls (18.2 ± 8.3 ng/ml). Thus we can conclude that decreased serum level of calcidiol in MS patients can be one of the factors related to increased risk of MS development, as well as increased risk of rapid disease disability progression.

#### **7.6. Genetic factors related to vitamin D effects in MS**

countries with less sunny climate, the necessary daily dose of vitamin D is 1000–4000 IU/day

The risk of vitamin D overdosing is hypercalcaemia and subsequent organ and tissue damage. Whole body exposure to sunlight results in the production of around 10,000 IU of vitamin D, so it is not simple to cause vitamin D intoxication by its short-term peroral supplementation. The results of several studies suggest that even high-dose vitamin D3 supplementation in MS patients is safe and clinically useful. Burton et al. [87] administered high peroral doses of vitamin D to healthy individuals and MS patients. The initial dose was 40,000 IU/dayduring 28 weeks, followed by 10,000 IU/day during 12 weeks; later it was gradually decreased to 0 IU/day, combined with 1.2 grams of calcium per day. During the period of 40,000 IU of vitamin D per day, the serum levels reached 413 nmol/l, which was higher than the conventional limit established for vitamin D toxicity (250 nmol/l). Calcidiol serum levels remained around this limit for 18 weeks without any observed negative effects. The serum level of calcium was in the physiological reference range during the whole study duration. Moreover, no cardiac rhythm abnormalities or impairment of hepatic or renal functions was observed. Kimball et al. [88] administered 4000–40,000 IU/day to patients in the active phase of MS together with 1.2 grams of calcium. Medium serum level of calcidiol was 78 ± 35 nmol/l and rose to 386 ± 157 nmol/l. Serum calcium level and urinary calcium to creatinine ratio did not exceed the physiological reference values. Vitamin D supplementation in this study did not cause any change in the serum level of hepatic enzymes, creatinine, electrolytes, proteins and parathor‐ mone. Although the serum level of calcidiol doubled the physiological upper range value,

Although the significant toxicity of vitamin D3 was not observed even in doses of 40,000 IU/ day, its daily dose in healthy individuals should not exceed 2000 IU. The optimal daily dose of vitamin D3 that should be routinely recommended to women during pregnancy and lactation is 1000 IU. Children born in families with MS history should be administered daily

The role of vitamin D in the prevention of MS development has been confirmed by many experimental, epidemiological, genetic and immunological studies. Vitamin D insufficiency during the whitematter development can alter the pathways of axonal differentiation and adhesion and increase the apoptosis of oligodendrocytes that express VDR. This results in local microenvironmental changes and altered regeneratory and remyelinating capacity [66]. In individuals with an increased genetic risk of MS, it is possible to prevent the demyelina‐ tion process by preventive vitamin D administration. This preventive strategy would be bet‐

High-dose peroral vitamin D intake has been found to be inversely associated with the risk of MS in a cohort of more than 90,000 women. Peroral vitamin D supplementation in a dose higher than 400 IU/day leads to the reduction of MS risk when compared to the individuals with no vitamin D intake (RR = 0.59, 95% CI = 0.38–0.91,*p* = 0.006) [89]. Also,calcidiol plasma levels are

(1 μg = 40 IU) [86].

12 Trending Topics in Multiple Sclerosis

hypercalcaemia or hypercalciuria was not observed.

**7.5. Vitamin D and the course, prevention and treatment of MS**

ter than reparation of already developed myelin and axonal damage [12, 66].

200–400 IU of vitamin D3 [66].

Nucleotide exchange in DNA sequence can cause the production of protein products with different activities. Polymorphisms of the genes involved in the activation, transport, signal‐ ling and degradation of vitamin D can, together with other factors, modify the individual immune response and thus can be related to MS. Because of the beneficial effects of vitamin D, in individuals with genes predisposing to its higher serum levels, the risk of MS should be reduced [96]. The serum level of vitamin D can be modified by *VDR* gene polymorphisms [97– 99]. The fact that serum levels of vitamin D are similar in twins, and especially when they are monozygotic twins, speaks in favour of a genetic regulation. Gene polymorphisms FokI in *VDR* gene, rs4646536 and rs703842 in the *CYP27B1* gene and rs10741657 in the *CYP2R1* gene are the significant predictors of caldiciol serum level [99]. Hypovitaminosis D is common in higher latitudes because of the lack of sun exposure [100]. The fact that not all vitamin D– deficient individuals develop MS is probably the result of the complexity of the etiopathoge‐ neis of MS and the interaction of many factors. The positive effects of vitamin D in MS can be dampened for example by the allele HLA-DRB1\*15 [96]. In MS patients, it is necessary to find out the link between the genotype and the vitamin D serum level and also the genetic inter‐ actions among the genes *CYP27B1, VDR* and *HLA* [19]. The gene polymorphisms associated with vitamin D metabolism are summarized in **Table 2**.


**Table 2.** The gene polymorphisms associated with vitamin D metabolism [19, 98, 99, 101].

#### *7.6.1. Genetic variants in vitamin D receptor gene in MS*

According to the effects of vitamin D in MS, the molecular mechanisms of vitamin D function should be considered. As mentioned earlier, vitamin D executes its physiological effect via binding and activation of VDR. Interestingly, the activation of VDR by calcitriol can suppress the induction of EAE, while animals that lack VDR are not protected against EAE [102]. The gene for VDR is located on the 12q13 chromosomal region and consists of 11 exons. Non-coding exons 1A, 1B and 1C are located in the 5′ end of the VDR gene, and exons 2–9 encode the structural portion of the VDR protein [103]. VDR sequence is similar to that of the receptors for steroid hormones and hormones of the thyroid gland. VDR is a regulatory transcription factor and consists of highly conservative DNA-binding and ligand-binding domains. The signal pathways associated with the VDR regulate the transcription of genes involved in the regulation of bone metabolism, immune response and cancer [83]. The polymorphisms in the initiation codon of the *VDR* gene can cause the formation of transcription variants coding different proteins [104]. In the *VDR* gene, SNPs ApaI (rs7975232), BsmI (rs1544410), FokI (rs10735810) and TaqI (rs731236) have functional biological effects and are mostly studied in MS as well as in other diseases. These gene polymorphisms can alter mRNA level, its stability and alternative splicingand also the stability of the final gene product, amount of protein isoforms and their interactions [105]. FokI gene polymorphism is located in exon 2 of the *VDR* gene, and its variants result in a change of protein structure. There are two possible allele variants, f (presence of a restriction site for FokI endonuclease) and F (absence of a restriction site for FokI endonuclease). It has been confirmed that the f (T) allele leads to the expression of a VDR protein, which is three amino acids longer (427 amino acids) than the F (C) allele (424 amino acids). The shorter isoform of the receptor is more transcriptionally potent through a more efficient interaction with transcription factor TFIIB [105, 106]. Near the 3′ end of the *VDR* gene, we can find the ApaI and BsmI polymorphism in the intron between exon 8 and 9 and TaqI gene polymorphism in exon 9 [107]. The allele variants of these gene polymorphisms and their combinations regulate the functions of VDR through the modulation of mRNA stability. In Caucasians, TaqI, ApaI and BsmI polymorphisms are in strong linkage disequilibrium and are present in five haplotype blocks. Haplotype2 (t-A-B) probably results in a lower number of 'A' in polyA variable number of tandem repeats (VNTR), while haplotype 1 (T-A-b) is connected to a large number of 'A', thus modulating mRNA stability [106]. Morrison et al. [108] found that allele b (G) of the BsmI polymorphism causes a decreased expression of VDR mRNA.

*VDR* gene, rs4646536 and rs703842 in the *CYP27B1* gene and rs10741657 in the *CYP2R1* gene are the significant predictors of caldiciol serum level [99]. Hypovitaminosis D is common in higher latitudes because of the lack of sun exposure [100]. The fact that not all vitamin D– deficient individuals develop MS is probably the result of the complexity of the etiopathoge‐ neis of MS and the interaction of many factors. The positive effects of vitamin D in MS can be dampened for example by the allele HLA-DRB1\*15 [96]. In MS patients, it is necessary to find out the link between the genotype and the vitamin D serum level and also the genetic inter‐ actions among the genes *CYP27B1, VDR* and *HLA* [19]. The gene polymorphisms associated

*Gene function Localization SNP Allele*

*DBP (vitamin D binding protein)* Transport in plasma 4q12 rs7041 G/T

**Table 2.** The gene polymorphisms associated with vitamin D metabolism [19, 98, 99, 101].

*7.6.1. Genetic variants in vitamin D receptor gene in MS*

*VDR (vitamin D receptor)* Receptor 12q13 rs1544410 (BsmI) A/G (B/b)

According to the effects of vitamin D in MS, the molecular mechanisms of vitamin D function should be considered. As mentioned earlier, vitamin D executes its physiological effect via

Hydroxylation 12q13 rs703842 C/T

Hydroxylation 11p15 rs10741657 A/G

Deactivation 20q13 rs2296241 A/G

rs10877012 G/C rs4646536 C/T rs10877015 A/G rs118204009 A/G rs118204012 A/G rs118204011 C/T

rs10500804 G/T rs12794714 A/G

rs4588 A/C

rs7975232 (ApaI) T/C (A/a) rs731236 (TaqI) T/C (T/t) rs10735810 (FokI) C/T (F/f) rs11568820 (Cdx2) G/A rs2254210 A/G rs98784 C/T

with vitamin D metabolism are summarized in **Table 2**.

*CYP27B1 (cytochrome P450 family 27 subfamily*

*CYP2R1 (cytochrome P450 family 2 subfamily R member 1, vitamin D325-hydroxylase)*

*CYP24A1 (cytochrome P450 family 24 subfamily A*

*polypeptide, vitamin D 24-hydroxylase)*

*B member 1, 25-hydroxyvitamin D3*

14 Trending Topics in Multiple Sclerosis

*1-alpha-hydroxylase)*

Interestingly, several studies have found an association between *VDR* gene polymorphisms and the risk of MS. Differences in allele frequency of the BsmI polymorphism in the *VDR* gene were found in Japan by Fukazawa et al. [109], who for the first time pointed out the involve‐ ment of *VDR* gene polymorphisms in the pathogenesis of MS. The association of *VDR* gene polymorphisms with MS has been confirmed in cohorts of MS patients from Japan [110], theUK [111, 112], Australia [107] and the USA [98]. On the contrary, no association of VDR gene polymorphisms with the risk of MS was found by studies in MS patients from Canada [113], Netherlands [114], Greece [115], Spain [116, 117], Tasmania [118] and Iran [119]. The presence of specific haplotypes of the *VDR* gene can increase the risk of MS development, especially its progressive forms. Tajouri et al. [107] in Australia found haplotype A-t (T-C) of ApaI and TaqI polymorphism to increase the risk of MS development, especially its progressive forms. The carriership of allele t (C) in their study increased MS risk twice. Fukazawa et al. [109] found allele b (G) and genotype bb (GG) of BsmI polymorphism to increase MS risk, but without any association with the form and severity of MS (EDSS, magnetic resonance imaging (MRI)). Allele b (G) of BsmI polymorphism of VDR has been found to be associated with MS risk in combi‐ nation with allele A (T) of ApaI polymorphism by Niino et al. [110]. However, in their study, they did not find any association of ApaI gene polymorphism with clinical form and severity of MS evaluated by the EDSS score, disease duration and MRI findings. Agliardi et al. [120] in Italy found that allele T (T) and genotype TT (TT) are protective against MS development, supported by the finding that the expression of VDR mRNA is increased four times by genotype Tt (TC) and eight times by genotype TT (TT) when compared to genotype tt (CC). The observed effect is present especially when the protective allele Tis present in the combi‐ nation with HLA-DRB1\*15 allele.

The role of VDR gene polymorphisms is still not completely understood, and it seems to vary among different populations. For proper cell signalling to decrease the risk of MS, it is probably necessary to reach a certain level of the transcriptional activity of VDR that is also modified genetically. For proper immunoregulation, the individuals that have the genotype causing the decreased VDR protein activity can need a higher peroral vitamin D intake or higher level of sun exposure. Contrarily, in individuals with higher transcriptional activity of VDR, a lower sun exposure or vitamin D intake can be sufficient for proper immune system regulation.

The findings of our previous study in MS patients from the Central-Northern region of Slovakia have confirmed the association of FokI heterozygous genotype Ff with an increased risk of MS in women [10]. Although we found no statistically significant differences in the proportions of FokI genotypes or allele frequencies between total MS patient and the control group, we have observed significant differences in the FokI genotype distribution between women with MS and the female control group (*p* = 0.042). Our results have shown a signifi‐ cantly higher frequency of heterozygous Ff genotypes in FokI polymorphism in the female MS group (53.4%) as compared to 43.7% in the female control group (OR = 1.48, 95% CI = 1.01– 2.16). In spite of this fact, when we compared the subgroup of rapidly progressing MS patients with the subgroup of slow progressing MS patients, allele and genotype counts were not significantly different between them (allele f: 34.5 vs. 43.3%, allele F: 65.5 vs. 56.7%, genotype ff: 10.3 vs. 13.4%, genotype Ff: 48.3 vs. 59.8%). Since we have not shown any significant association between FokI VDR gene polymorphism and the rate of disease disability progres‐ sion in our cohort of Slovak MS patients, we observed a trend of higher frequency of homo‐ zygotes FF to be 41.4% in MS patients with rapid progression of disease as compared to 26.8% in slow progressing MS patients (OR = 1.93, 95% CI=0.94–3.94) with a marginal level of significance (*p* = 0.071). From the results of our study, it seems that contributions from genetic and allelic variants of FokI VDR gene polymorphism have only a small impact in a disease as complex as MS, andits role in the etiopathogenesis of MS still remains controversial.
