FokI vitamin D receptor gene polymorphism in association with multiple sclerosis risk and disability progression in Slovaks Daniel Cˇierny1, Jozef Michalik2, Egon Kurcˇa2, Dusˇan Dobrota1, Ja´n Lehotsky´1 1

Department of Medical Biochemistry, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovak Republic, 2Department of Neurology, Jessenius Faculty of Medicine and University Hospital Martin, Comenius University in Bratislava, Martin, Slovak Republic Objective: It is still unclear as to why multiple sclerosis (MS) is so devastating and rapidly progressive in one patient and less so in another. Recent data implicate vitamin D in modulation of the risk as well as the clinical course of disease. Since vitamin D acts through the vitamin D receptor (VDR), association of single nucleotide polymorphisms (SNPs) in the VDR gene might account for variations in the MS risk within populations. The aim of our study was to determine the association between FokI gene polymorphism (rs10735810) and the risk of MS in a cohort of Slovak population and to investigate possible correlations of this SNP with the rate of disease disability progression. Methods: FokI SNP was detected in 270 clinically diagnosed MS patients and 303 healthy control subjects. Genotyping was performed by polymerase chain reaction (PCR) and restriction analysis. We used multiple sclerosis severity score (MSSS) for patient’s stratification by the rate of disease disability progression. Results: We observed a significantly higher frequency of Ff genotype of FokI SNP (53.4 vs 43.7%, P 5 0.042) in women with MS compared to women of the control group. There was no significant association between FokI SNP and the rate of disease disability progression. Discussion: Although our findings suggest a weak association between VDR SNP FokI and the MS risk in women, further studies are needed to explore the role of VDR polymorphic alterations in MS disease etiology and pathogenesis. Keywords: Multiple sclerosis, MSSS, Disability progression, Vitamin D receptor, FokI gene polymorphism

Multiple sclerosis (MS) is a common chronic autoimmune inflammatory disease of the central nervous system (CNS). Focal white blood cells infiltration leads to the process of inflammation, myelin sheath breakdown, demyelination, remyelination, neuronal and axonal degeneration, and subsequent deterioration of neurological functions.1 In triggering of an autoimmune response, the genetic predisposition of an individual undergoes a strong interaction with its environment.2,3 In the past decade, much attention has been given to vitamin D and its role in MS. Vitamin D in the human body undergoes a complex metabolism. As a precursor of a hormonally active form, cholecalciferol (vitamin D3) is produced in the skin after sunlight exposure and can also be absorbed from a diet. Subsequently, cholecalciferol is hydroxylated in

Correspondence to: Jan Lehotsky, Department of Medical Biochemistry, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovak Republic. Email: [email protected]

ß W. S. Maney & Son Ltd 2015 DOI 10.1179/1743132814Y.0000000459

the liver forming 25-hydroxycholecalciferol, calcidiol. The hormonally active form of vitamin D, 1,25dihydroxycholecalciferol, calcitriol is produced by further hydroxylation especially in the kidneys but also in other tissues. In various cells, calcitriol binds to the vitamin D receptor (VDR) providing its physiological functions by modulation of the target gene’s transcription. There is a growing evidence that vitamin D regulates not only bone metabolism but also has large-scale immunomodulatory and antiinflammatory effects.4 One of the typical traits of MS is the disruption of T-helper (TH) cell balance. Murine TH cells are directly regulated by calcitriol, which inhibits TH1 cells and increase development of TH2 cells in vitro.5 Calcitriol treatment completely prevents the induction and progression of experimental autoimmune encephalomyelitis (EAE), a murine model of MS.6 In human beings, calcidiol plasma levels measurement is usually used to reflect the vitamin D status in the body. It has been shown that high calcidiol plasma levels are associated with

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an improvement of regulatory T-cells function and a shift of TH cells balance toward TH2 cells.7 In Caucasians, calcidiol plasma levels are in negative correlation with the MS risk, particularly under the age of 20 years with no difference between sexes.8 It has also been observed that vitamin D intake from dietary supplements with a daily dose of more than 400 IU is significantly associated with a decrease in MS risk.9 Besides, there is evidence that in relapsingremitting MS, calcidiol plasma levels are lower during relapses compared to the periods of remission.10 Lower calcidiol levels are also associated with relapse rates as well as high expanded disability status scale (EDSS) scores and progressive forms of MS.11 On the other hand, there are also interventional studies, in which high doses of peroral vitamin D3 supplementation was performed. It leaded to a reduction of annual relapse rates, T-cell reactivity, and proliferation12 and also to decrease in the number of gadoliniumenhancing lesions per patient but without any effect on the disease activity and progression.13 Apart from these effects of vitamin D in MS, the molecular mechanisms of vitamin D function should be considered. As mentioned earlier, vitamin D acts through the VDR. The gene for the VDR is located on the 12q13 chromosomal region and consists of 11 exons. Non-coding exons 1A, 1B, and 1C are located in the 59 end of the VDR gene and exons 2–9 encode the structural portion of the VDR gene product.14 In the VDR gene, ApaI (rs7975232), BsmI (rs1544410), FokI (rs10735810), and TaqI (rs731236) single nucleotide polymorphisms (SNPs) have biological effects on its function and are mostly studied in MS as well as in other diseases. The 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 (an 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 3 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 the transcription factor TFIIB.15,16 Vitamin D receptor’s activity is very important in suppression of the murine model of MS. Animals that lack VDR are not protected from EAE induction by calcitriol.17 It is well known that in mild climate regions, hypovitaminosis of vitamin D is rather frequent.18 Due to the complexity of MS etiopathogenesis, not all insufficient or deficient individuals develop MS. Interestingly, several studies have found an association between the VDR gene polymorphisms and a risk of MS. Differences in allele frequency of the

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BsmI polymorphism in the VDR gene were found in the Japanese population in 1999.19 This study pointed out for the first time the involvement of the VDR gene polymorphisms in the pathogenesis of MS. Subsequently, the positive association of the VDR gene polymorphisms have been confirmed in cohorts of MS patients from Japan,20 United Kingdom (UK),21,22 Australia,22,23 and the United States.24 On the other hand, no association of the VDR gene polymorphisms with the risk of MS was found by other studies in MS patients from Canada,25 Netherlands,26 Greece,27 Spain,28or Tasmania.29 It is obvious that the role of VDR polymorphisms in the MS risk and development is still not completely clear and results of published studies suggest for a possible ethnicity variation. On that account, the purpose of our study was to investigate the association of allele variants of the VDR gene polymorphism FokI with MS susceptibility in the cohort of Central European Slovak population. In addition to the described potential association of VDR gene polymorphisms in MS risk, FokI genotype was also found to be a predictor of calcidiol plasma levels. For example, in 150 Canadian subjects30 and in Dutch,31 a lower serum concentration of calcidiol was determined in individuals with the FF genotype. Calcidiol plasma levels correlate with the occurrence of relapses in relapsing-remitting MS,10 with high EDSS scores and different forms of MS.11 Apart from these facts, only several studies have shown the direct association between the variants of the VDR gene polymorphisms and the MS disease course. An association of lower frequency of ff genotype of FokI gene polymorphism with high EDSS scores was found in patients with MS residing in UK.36 In contrast, no association of FokI polymorphism with different MS phenotypes was found in the Spaniards.28 Thus, we focused on the identification of a potential involvement of VDR gene polymorphism FokI in the modulation of the rate of disease disability progression in the cohort of Central European Slovak population. We tried to identify whether there are different FokI allele and gene variants in a group of patients with rapidly progressing MS compared to a group of patients with slow progressing MS.

Materials and Methods Patients and controls Participants of our study consisted of 270 patients clinically diagnosed with MS and 303 healthy control subjects. The study was approved by the ethical committee of the Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin. An informed consent was given by all participating individuals. The healthy, age-matched control group

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consisted of volunteers free from any disease of the central nervous system (CNS). In MS patients, clinically definitive MS diagnosis was established according to the McDonald’s criteria in The Centre for Demyelinating Diseases at the Department of Neurology, University Hospital Martin and Jessenius Faculty of Medicine in Martin, Slovakia. Clinical data and blood samples were taken over a period from November 2012 to May 2014 only at regular medical checkups to minimize trauma to patients. General physical characteristics (sex and age) of the study group are shown in Table 1. All individuals involved in the study were Slovaks living in the central northern part of Slovakia.

Stratification of MS patients In our study, we stratified the MS patients to three groups according to the rate of disease disability progression. After obtaining an EDSS score and disease duration data from medical documentation, we ascertained an multiple sclerosis severity score (MSSS) of each patient from the global MSSS table.32 Patients with defined slow progressing pattern of MS progression were located in the first three deciles of the table with MSSS , 3 (n 5 82). Patients with a rapidly progressing pattern of MS progression were located in the last four deciles of the table with MSSS . 6 (n 5 58). MS patients who did not fit with our strict criteria defined by MSSS were characterized as mid-rate progressing MS patients with MSSS 3–6 (n 5 130). To ensure potential differences between the subgroups of slow progressing and rapidly progressing MS patients, the midrate progressing MS patients were excluded from the comparison and statistical analysis. Clinical characteristics of the MS patient groups are summarized in Table 2. The recorded parameters

FokI vitamin D receptor gene polymorphism and MS

include mean age at which the first symptoms were observed, mean disease duration, mean EDSS score, mean MSSS, progression index (EDSS divided by disease course in years), number of patients with certain MS phenotype, and rate of disease disability progression.

Genotyping Peripheral venous blood samples were taken and dispensed into 3 ml tubes containing 5.4 mg of EDTA. DNA was isolated from white blood cells using The WizardH Genomic DNA Purification Kit (Promega) and stored at 220uC. Restriction fragment length polymorphism (RFLP) FokI of the VDR gene was genotyped by restriction analysis. Polymorphic region of DNA was amplified by polymerase chain reaction (PCR), which was performed in a total volume of 15 ml. The reaction volume consisted of 7 ml of REDTaq ReadyMixTM PCR reaction mix with MgCl2 (Sigma-Aldrich, St. Louis, USA), 50– 150 ng of genomic DNA sample and 0,25 ml of 25 mM primers (Microsynth) filled in to total volume of 15 ml by redistilled water. The oligonucleotide primers were: forward – 59 GATGCCAGCTGGCCC TGGCACTG 39and reverse – 59 ATGGAAACACCT TGCTTCTTCTCCCTC 39.23 The FokI polymorphism PCR cycles consisted of 4 minutes of initial denaturation at 94uC followed by 30 cycles of 94uC for 1 minute, 69uC for 1 minute, 72uC for 1 minute, and 72uC for 7 minutes. Polymerase chain reaction products were digested in a total volume of 20 ml using FastDigest endonuclease (ThermoScientific) FokI (1 ml in 37uC for 10 minutes). Digested DNA fragments were separated by electrophoresis on a 2% agarosis gel and visualized by ethidium bromide under UV light. FokI genotypes were determined as FF (272 bp), Ff (272,

Table 1 Study group characteristics Factor Sex Mean age (years)

Patients (n 5 270) 66 Men (24.4%) 41.4 ¡ 11.2 41.3 ¡ 10.8

Controls (n 5 303) 204 Women (75.6%) 41.3 ¡ 10.7

74 Men (24.4%) 35.0 ¡ 9.3 38.7 ¡ 13.6

229 Women (75.6%) 39.9 ¡ 14.5

Table 2 Clinical characteristics of MS group Factor

Patients (n 5 270)

Age of first symptom (years) 29.2 ¡ 10.0 Disease course (years) 12.1 ¡ 7.1 EDSS (points) 3.5 ¡ 1.6 MSSS (points) 4.25 ¡ 2.08 Progression index 0.386 ¡ 0.292 MS phenotype Relapsing-remitting 230 (85.2%) Secondary progressive 40 (14.8%) Rate of disease disability progression Slow progressing MS 82 (30.4%) Mid-rate progressing MS 130 (48.1%) Rapidly progressing MS 58 (21.5%)

Patients – men (n 5 66)

Patients – women (n 5 204)

30.3 ¡ 10.7 11.1 ¡ 7.2 3.8 ¡ 1.7 4.91 ¡ 2.34 0.471 ¡ 0.324

28.8 ¡ 9.8 12.5 ¡ 7.0 3.4 ¡ 1.6 4.04 ¡ 1.95 0.358 ¡ 0.276

51 (77.3%) 15 (22.7%)

179 (87.7%) 25 (12.3%)

15 (22.8%) 29 (43.9%) 22 (33.3%)

67 (32.8%) 101 (49.5%) 36 (17.7%)

MS: multiple sclerosis; EDSS: expanded disability status scale; MSSS: multiple sclerosis severity score.

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198, and 74 bp), and ff (198 and 74 bp).23 To control the quality of genotype determination, 20 samples were randomly selected and repetitively genotyped.

Statistical analysis Continuous data were presented as mean ¡ standard deviation and discrete data as percentages. From analyzed genotyped counts, we calculated genotype and allele frequencies. To assess the association of individual VDR SNP genotype and allele variants with MS risk and rate of disease disability progression, we used conventional two-by-two contingency table analysis with an incorporated standard chisquared or Fisher’s exact test when appropriate. Statistic significance was considered to be P # 0.05. For genetic risk quantification, odds ratios (OR) and 95% confidence intervals (95% CI) were calculated.

Results First, we determined commonly studied FokI polymorphism in VDR gene – in 270 MS patients and 303 healthy control subjects. The genotype and allele frequencies of FokI VDR gene polymorphism in MS patients and the control group are shown in Table 3. We found no statistically significant differences in the proportions of FokI genotypes or allele frequencies between whole MS patient and the control group.

Since it is well known that MS prevalence is also affected by gender and it is more frequent in women, we also compared genotypes and allele frequencies of FokI SNP in male MS patients and their male control group and between women MS patients and their women control group. The genotype and allele frequencies of FokI VDR gene polymorphism in men with MS and their male control group are shown in Table 4 and in women with MS and their women control group in Table 5. No statistically significant differences in the proportions of FokI genotypes or allele frequencies were found between male MS patients and the male control group. Interestingly, we have observed significant differences in the FokI genotype distribution between women with MS and the women control group (P 5 0.042). Our results have showed a significantly higher frequency of heterozygous Ff genotypes in FokI polymorphism to be 53.4% in women MS group as compared to 43.7% in the women control group (OR 5 1.48, 95% CI 5 1.01–2.16). In order to explore the proposed differences in genotype and allele frequencies of FokI VDR gene polymorphism, we compare these parameters in subgroups of MS patients with different rate of disease disability progression. Distributions of the

Table 3 Distribution of genotype and allele frequencies of FokI VDR gene polymorphism in MS patients and the control group

Genotypes FF Ff ff Alleles F f

Controls (n 5 303)

Patients (n 5 270)

OR (95% CI)

P value

118 (38.9%) 143 (47.2%) 42 (13.9%)

96 (35.5%) 143 (53.0%) 31 (11.5%)

0.87 (0.62–1.21) 1.26 (0.91–1.75) 0.81 (0.49–1.32)

0.403 0.168 0.393

379 (62.5%) 227 (37.5%)

335 (62.0%) 205 (38.0%)

0.98 (0.77–1.24) 1.02 (0.80–1.30)

0.862 0.862

Table 4 Distribution of genotype and allele frequencies of FokI VDR gene polymorphism in male MS patients and controls

Genotypes FF Ff ff Alleles F f

Controls – men (n 5 74)

Patients – men (n 5 66)

OR (95% CI)

P value

28 (37.8%) 43 (58.1%) 3 (4.1%)

25 (37.9%) 34 (51.5%) 7 (10.6%)

1.00 (0.51–1.99) 0.77 (0.39–1.49) 2.81 (0.70–11.34)

1 0.435 0.191

99 (66.9%) 49 (33.1%)

84 (63.6%) 48 (36.4%)

0.87 (0.53–1.42) 1.15 (0.71–1.89)

0.566 0.566

Table 5 Distribution of genotype and allele frequencies of FokI VDR gene polymorphism in women with MS and controls

Genotypes FF Ff ff Alleles F f

304

Controls – women (n 5 229)

Patients – women (n 5 204)

OR (95% CI)

P value

90 (39.3%) 100 (43.7%) 39 (17.0%)

71 (34.8%) 109 (53.4%) 24 (11.8%)

0.82 (0.56–1.22) 1.48 (1.01–2.16) 0.65 (0.38–1.12)

0.335 0.042 0.121

280 (61.1%) 178 (38.9%)

251 (61.5%) 157 (38.5%)

1.02 (0.77–1.34) 0.98 (0.75–1.29)

0.920 0.920

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genotypes and alleles of the FokI gene polymorphism in the slow progressing, rapidly progressing, and midrate progressing MS group are summarized in Table 6. As mentioned before, we have found a higher frequency of Ff genotype in women with MS in comparison to women healthy control subjects. In spite of this fact, when we compared the subgroup of rapidly progressing MS patients to the subgroup of slow progressing MS patients, observed allele and genotype counts were without any significant differences (Table 6, 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%). Interestingly, we observed a trend of higher frequency of homozygotes FF to be 41.4% in MS patients with rapid progression of disease as compared to 26.8% in slow progressing MS patients (OR 5 1.93, 95% CI 5 0.94–3.94) with a marginal significance (P 5 0.071).

Discussion The etiology of MS is not yet completely known. Current results support the pivotal role of vitamin D insufficiency in the MS pathogenesis and progression.8–12 Pathological and clinical data collected in the past decade reveal that MS pathological mechanisms may vary according to the stage of the disease. Recently, the concept of MS as a biphasic disease has been divided into the inflammatory relapsing-remitting phase and a degenerative secondary progressive phase.33 Binding of vitamin D to its receptor is required for the proper physiological function and immune response at the multiple levels. However, the results from previous experimental studies are inconclusive, and it is not clear how the VDR function contributes to the occurrence of relapses in relapsingremitting MS patients or eventually to further degeneration in secondary progressive form of MS. In order to better understand these mechanisms, we performed a SNP analysis in the VDR gene to identify the genetic contributions to the risk of MS and to the alteration of the rate of disease disability progression. In the present exploratory study of the cohort of Slovak population, we determined gene

FokI vitamin D receptor gene polymorphism and MS

polymorphism FokI in a group of 270 MS patients and 303 healthy control subjects. First, we focused on the possible significance of the FokI gene polymorphism to the risk of MS. We did not find any significant differences by comparing the distribution of the FokI allele and gene variants in the whole MS group to the control group. Furthermore, a recent study by Cox et al.22 involving 1153 trio families from UK and in 726 MS cases from UK and Australia did not prove a direct association of the FokI gene polymorphism with the risk of MS. Similarly, Dickinson et al.29 did not detect any significant association between FokI VDR genotype and MS in 136 Tasmanian MS cases and 235 controls. Also, Garcı´a-Martı´n et al.28 investigated FokI genotype and allelic variants in 303 Spanish MS patients (94 men and 209 woman) and in control group consisting of 310 individuals. They made multiple comparisons but they found no association between FokI genotype and the MS risk neither in the whole group of MS patients and controls nor in men and women. Our results are in agreement with meta-analysis of Huang and Xie.34 They analyzed six Caucasian studies that enrolled together 1775 cases and 1830 controls, but no significant association between FokI polymorphism of VDR and the risk for MS was found. Another meta-analysis of Garcı´aMartı´n et al.28 that included the same six Caucasian studies analyzed previously by Huang and Xie34 plus their own study (together 2096 MS patients and 2193 controls) suggests that FokI genotype and allelic variants are not related with the risk for MS. On the other hand, our results do not correspond with the study of Partridge et al.,21 who reported an association of the ff genotype with a diminished risk of MS in a group of 419 U.K. MS cases. In addition to these findings, the study by Simon et al.24 in 214 American nurses reported a protective effect of exogenous intake of vitamin D that was obvious only in individuals with ff genotype. As the study demonstrated, a dose of more than 400 I.U. per day causes as much as 80% decrease of the MS risk in individuals homozygous for the minor allele.

Table 6 Distribution of the genotype and allele frequencies of the FokI VDR gene polymorphism in MS patients with different rate of disease disability progression Slow vs rapidly progressing MS Mid-rate progressing MS (n 5 130) Genotypes FF 50 (38.4%) Ff 66 (50.8%) ff 14 (10.8%) Alleles F 166 (63.8%) f 94 (36.2%)

Slow progressing MS (n 5 82)

Rapidly progressing MS (n 5 58)

OR (95% CI)

P value

22 (26.8%) 49 (59.8%) 11 (13.4%)

24 (41.4%) 28 (48.3%) 6 (10.3%)

1.93 (0.94–3.94) 0.63 (0.32–1.24) 0.74 (0.26–2.14)

0.071 0.179 0.584

93 (56.7%) 71 (43.3%)

76 (65.5%) 40 (34.5%)

1.45 (0.89–2.37) 0.69 (0.42–1.13)

0.138 0.138

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Subsequently, the authors reported an intermediate and dose-dependent effect also in individuals with the Ff genotype. Our analysis uncovered a significantly higher frequency of genotype Ff in women with MS when compared to women controls to be 53.4 and 43.7%, respectively. These results suggest that there is an association of Ff genotype with an increased risk of MS in Slovak women (OR 5 1.48, 95% CI 5 1.01–2.16, P 5 0.042). When we consider a dose-dependent effect of vitamin D to the MS risk as reported by Simon et al.24 in American nurses with this genotype, we may speculate that the Ff genotype can have an effect on the MS risk in Slovak women through the interaction with vitamin D plasma levels. The FokI genotype has also been confirmed to be a possible predictor of calcidiol plasma levels. An association of lower serum concentrations of calcidiol in individuals with the FF genotype was found in 150 Canadian subjects30 and in 212 Dutch MS patients.31 The complex genetic regulation of vitamin D levels in the human body was described in the study of Lin et al.35 In a cohort of 198 MS patients in southern Australia, they pointed out the relevant association of level of 25-hydroxyvitamin D as well as hazard of MS relapse with polymorphisms in genes involved in cell adhesion (rs11581062), protein kinase C signaling in platelets (rs7595037), and nutrient and growth factor signaling (rs180515). Moreover, SNP rs2248359 in CYP24A1 gene that demonstrates VDR binding modified the relationship between 25-hydroxyvitamin D and the hazard of relapse. Despite of this, magnitude of the effect of some SNPs on vitamin D levels was too small to mediate the effect on relapse. It is obvious that a proper vitamin D function can be affected also by other genetic markers, which are not directly connected to the VDR or vitamin D metabolism by the cumulative genotype effects. Based on the known role of vitamin D in etiopathogenesis of MS, in the next part of our study, we hypothesized whether allele variants of the VDR gene polymorphism FokI could have any influence on the rate of disease disability progression. As previously described in a large cohort of 512 North European Caucasian MS patients,36 there is an association of lower frequency of the ff genotype of FokI gene polymorphism with EDSS § 6 when compared to the patients with EDSS , 6. In our study, we evaluated EDSS scores and disease duration of all our MS patients to obtain an MSSS score for each patient. Consecutively, we used MSSS scores for patient’s stratification by the rate of disease disability progression. From all our 270 MS patients, those characterized as mid-rate progressing MS (n 5 130) were excluded from the comparison. We selected only patients with the rapid disease disability progression (n 5 58) and slow disease disability

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progression (n 5 82) verified by MSSS (see Stratification of MS patients). In spite of the strict discrimination between both groups, no significant differences were found in allele frequencies of FokI VDR variants between rapid progressing and the slow progressing MS groups. We found the frequency of allele F to be 65.5% in rapidly progressing MS patients and 56.7% in slow progressing MS patients (OR 5 1.45, 95% CI 5 0.89–2.37, P 5 0.138). Frequency of allele f was 34.5% in rapidly progressing MS patients and 43.3% in slow progressing MS patients (OR 5 0.69, 95% CI 5 0.42–1.13, P 5 0.138). In the distribution of genotype ff and Ff in rapidly progressing MS patients compared to slow progressing MS patients, we did not identify any significant differences (ff: 10.3 vs 13.4%, OR 5 0.74, 95% CI 5 0.26–2.14, P 5 0.584 and Ff: 48.3 vs 59.8%, OR 5 0.63, 95% CI 5 0.32–1.24, P 5 0.179). However, the analysis uncovered a trend of higher frequency of homozygotes FF in MS patients with rapid disease disability progression to be 41.4% when compared to the group of MS patients with slow disease disability progression to be 26.8% (OR 5 1.93, 95% CI 5 0.94–3.94, P 5 0.071). The non-significant trend of higher frequency of FF genotype in MS patients with rapid disease disability progression is consistent with the findings of Mamutse et al.36 who found a higher frequency of the ff genotype in patients with reduced disability measured by either EDSS or MSSS. We suggest that FokI VDR gene polymorphism in our cohort of Slovak MS population may not be taken as a single genetic marker associated with the rate of disease disability progression. We should remark that our group of MS patients consisted only of patients with a relapsing-remitting form of MS (n 5 230) and the secondary progressive form of MS (n 5 40). The recent study of Garcı´a-Martı´n et al.28 in 303 Spanish MS patients showed no differences of genotype and allelic frequencies of FokI gene polymorphisms among patients with relapsing-remitting, primary progressive, and secondary progressive form of MS. More importantly, as can be seen from clinical practice, patients with the relapsing-remitting form commonly sooner or later progress to the next stage of the disease known as a secondary progressive form of MS. The probability of entering the progressive phase is affected by many factors.33 Regarding this fact, we did not make any comparisons between groups of patients with these two forms of MS and we mainly focused on the identification of contributions of the VDR gene polymorphisms to the rate of disease disability progression objectively defined by MSSS. In Australian cohort of MS patients, they found no consistent evidence for an association of 60 MS risk-associated SNPs with

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progression in disability (measured by MSSS, change in EDSS and Scripps neurologic rating scale). Despite of this, they observed the relationship between SNPs and hazard of relapse.35 In our study, we presumed that VDR gene polymorphisms could be associated with MS disability progression through VDR and vitamin D immunomodulatory effects. Anyway, our findings considering the association between FokI VDR gene polymorphism and the MS disability progression are consistent with the results of Lin et al.35 They hypothesized that different genetic susceptibilities in MS relapse are driven by acute inflammation while those in disability progression by the process of neurodegeneration. Further studies are needed to explore the role of VDR polymorphic alterations in MS disease etiology and pathogenesis. In conclusion, the findings of our present study in the MS patients from the Central–North region of Slovakia have confirmed the association of FokI heterozygous genotype Ff with the risk of MS in women. Our study has not shown any significant association between FokI VDR gene polymorphism and the rate of disease disability progression in our cohort of Slovak MS patients. It seems that contributions from genetic and allelic variants of FokI VDR gene polymorphism have only a small impact in such a complex disease as MS. Thus, the role of the FokI VDR gene polymorphism in etiopathogenesis of MS remains still controversial and can not be thought as an apparent single genetic marker. The discrepancy in our results and in other performed studies might be caused by factors as not uniform criteria for diagnosis of MS, missing gender consideration, inconsistent methods of genotyping, ethnic, and geographical factors, and finally, the small sample size.34 We may only speculate whether the observed gene polymorphism in the VDR gene via a complex interaction with other environmental and genetic factors play a role in MS. If any VDR genotype could increase the MS risk or the risk of more severe disability progression, there are still many other environmental factors, such as sun exposure and skin type, vitamin D intake from food and supplements, physical activity, infections, and smoking that can interact together and modify the risk through many possible mechanisms. For example, in Tasmania, Dickinson et al.29 identified melanin density that can affect the interaction of Cdx-2 polymorphism of VDR with higher risk of MS in individuals with low winter sun exposure during childhood. From genetic factors, VDR polymorphisms can interact with several non-HLA genes35 as well as with HLA-genes. As an example, Cox et al.22 reported gene–gene interaction between FokI SNP and rs3135388 in HLA-gene with MS risk (P 5 0.011). They found that risk of MS appears to be

FokI vitamin D receptor gene polymorphism and MS

increased with the increased number of FokI C alleles in patients homozygous for rs3135388 A allele (i.e., DRB1*1501 positive). This observation supports the theory that the VDRE on HLADRB1*1501 gene can be affected by the more active form of the FokI allele. It is obvious that it is difficult to evaluate the overall contribution of those many factors to MS pathogenesis in an individual. In summary, because of complexity of the MS etiopathology, it is still difficult to come to a firm conclusion about the association of VDR gene polymorphisms with the MS. Further genetic, biochemical, and immunological studies are needed to clarify mechanisms of the previously clinically proved vitamin D efficacy to halt or to slow the progression of MS disease.

Disclaimer Statements Contributors I would like to declare that all authors, namely, Daniel Cierny, Jozef Michalik, Egon Kurca, Dusan Dobrota, and Jan Lehotsky contributed and have taken full author role in this original research paper. Funding Ministry of Education of the Slovak Republic, Ministry of Health of the Slovak Republic, EU sources and European Regional Development Fund. Conflicts of interest The authors have no conflict of interest to declare. Ethics approval The study was approved by the ethical committee of the Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin.

Acknowledgements The authors would like to thank T. Jaunky, Dr N. A. Yeboah, and Dr S. Mahmood for editing English grammar in the manuscript. This study was supported by the grants VEGA 213/12 from the Ministry of Education of the Slovak Republic, 2012/30UKMA-7: Biological and molecular markers of multiple sclerosis from Ministry of Health of the Slovak Republic and by the project ‘‘Identification of novel markers in diagnostic panel of neurological diseases’’ code: 26220220114 co-financed from EU sources and European Regional Development Fund.

References 1 Compston A, Coles A. Multiple sclerosis. Lancet. 2008; 372(9648):1502–17. 2 Nischwitz S, Mu¨ller-Myhsok B, Weber F. Risk conferring genes in multiple sclerosis. FEBS Lett. 2011;585(23):3789–97. 3 Kakalacheva K, Lunemann JD. Environmental triggers of multiple sclerosis. FEBS Lett. 2011;585(23):3724–9. 4 Adorini L, Penna G. Control of autoimmune diseases by the vitamin D endocrine system. Nat Clin Pract Rheumatol. 2008;4(8):404–12. 5 Boonstra A, Barrat FJ, Crain C, Heath VL, Savelkoul HF, O’Garra A. 1a,25-Dihydroxyvitamin D3 has a direct effect on

Neurological Research

2015

VOL .

37

NO .

4

307

Cˇ ierny et al.

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

308

FokI vitamin D receptor gene polymorphism and MS

naive CD4z T cells to enhance the development of Th2 cells. J Immunol. 2001;167(9):4974–80. Cantorna MT, Hayes CE, DeLuca HF. 1,25-Dihydroxyvitamin D3 reversibly blocks the progression of relapsing encephalomyelitis, a model of multiple sclerosis. Proc Natl Acad Sci U S A. 1996;93(15):7861–4. Smolders J, Thewissen M, Peelen E, Menheere P, Cohen Tervaert JW, Damoiseaux J, et al. Vitamin D status is positively correlated with regulatory T cell function in patients with multiple sclerosis. PLoS One. 2009;4(8):e6635. Munger KL, Levin LI, Hollis BW, Howard NS, Ascherio A. Serum 25-hydroxyvitamin D levels and risk of multiple sclerosis. JAMA. 2006;296(23):32–8. Munger KL, Zhang SM, O’Reilly E, Herna´n MA, Olek MJ, Willett WC, et al. Vitamin D intake and incidence of multiple sclerosis. Neurology. 2004;62(1):60–5. Soilu-Ha¨nninen M, Airas L, Mononen I, Heikkila¨ A, Viljanen M, Ha¨nninen A. 25-Hydroxyvitamin D levels in serum at the onset of multiple sclerosis. Mult Scler. 2005;11(3):266–71. Smolders J, Menheere P, Kessels A, Damoiseaux J, Hupperts R. Association of vitamin D metabolite levels with relapse rate and disability in multiple sclerosis. Mult Scler. 2008;14(9):1220–4. Burton JM, Kimball S, Vieth R, Bar-Or A, Dosch HM, Cheung R, et al. A phase I/II dose-escalation trial of vitamin D3 and calcium in multiple sclerosis. Neurology. 2010;74(23):1852–9. Kimball SM, Ursell MR, O’Connor P, Vieth R. Safety of vitamin D3 in adults with multiple sclerosis. Am J Clin Nutr. 2007;86(3):645–51. Miyamoto K, Kesterson RA, Yamamoto H, Taketani Y, Nishiwaki E, Tatsumi S, et al. Structural organization of the human vitamin D receptor chromosomal gene and its promoter. Mol Endocrinol. 1997;11(8):1165–79. Uitterlinden AG, Fang Y, Van Meurs JB, Pols HA, Van Leeuwen JP. Genetics and biology of vitamin D receptor polymorphisms. Gene. 2004;338(2):143–56. Jurutka PW, Remus LS, Whitfield GK, Thompson PD, Hsieh JC, Zitzer H, et al. The polymorphic N terminus in human vitamin D receptor isoforms influences transcriptional activity by modulating interaction with transcription factor IIB. Mol Endocrinol. 2000;14(3):401–20. Meehan TF, DeLuca HF. The vitamin D receptor is necessary for 1alpha,25-dihydroxyvitamin D(3) to suppress experimental autoimmune encephalomyelitis in mice. Arch Biochem Biophys. 2002;408(2):200–4. Holick MF, Chen TC. Vitamin D deficiency: a worldwide problem with health consequences. Am J Clin Nutr. 2008; 87(4):1080S–6S. Fukazawa T, Yabe I, Kikuchi S, Sasaki H, Hamada T, Miyasaka K, et al. Association of vitamin D receptor gene polymorphism with multiple sclerosis in Japanese. J Neurol Sci. 1999;166(1):47–52. Niino M, Fukazawa T, Yabe I, Kikuchi S, Sasaki H, Tashiro K. Vitamin D receptor gene polymorphism in multiple sclerosis and the association with HLA class II alleles. J Neurol Sci. 2000;177(1):65–71. Partridge JM, Weatherby SJ, Woolmore JA, Highland DJ, Fryer AA, Mann CL, et al. Susceptibility and outcome in MS: associations with polymorphisms in pigmentation-related genes. Neurology. 2004;62(12):2323–5.

Neurological Research

2015

VOL .

37

NO .

4

22 Cox MB, Ban M, Bowden NA, Baker A, Scott RJ, Lechner-Scott J. Potential association of vitamin D receptor polymorphism Taq1 with multiple sclerosis. Mult Scler. 2012;18(1):16–22. 23 Tajouri L, Ovcaric M, Curtain R, Johnson MP, Griffiths LR, Csurhes P, et al. Variation in the vitamin D receptor gene is associated with multiple sclerosis in an Australian population. J Neurogenet. 2005;19(1):25–38. 24 Simon KC, Munger KL, Yang X, Ascherio A. Polymorphisms in vitamin D metabolism related genes and risk of multiple sclerosis. Mult Scler. 2010;16(2):133. 25 Steckley JL, Dyment DA, Sadovnick AD, Risch N, Hayes C, Ebers GC. Genetic analysis of vitamin D related genes in Canadian multiple sclerosis patients. Canadian Collaborative Study Group. Neurology. 2000;54(3):729–32. 26 Smolders J, Damoiseaux J, Menheere P, Tervaert JW, Hupperts R. Association study on two vitamin D receptor gene polymorphisms and vitamin D metabolites in multiple sclerosis. Ann N Y Acad Sci. 2009;1173:515–20. 27 Sioka C, Papakonstantinou S, Markoula S, Gkartziou F, Georgiou A, Georgiou I, et al. Vitamin D receptor gene polymorphisms in multiple sclerosis patients in northwest Greece. J Negat Results Biomed. 2011;10:3. 28 Garcı´a-Martı´n E, Agu´ndez JAG, Martı´nez C, Benito-Leo´n J, Milla´n-Pascual J, Calleja P, et al. Vitamin D3 receptor (VDR) gene rs2228570 (Fok1) and rs731236 (Taq1) variants are not associated with the risk for multiple sclerosis: results of a new study and a meta-analysis. PLoS One. 2013;8(6): e65487. 29 Dickinson JL, Perera DI, van der Mei AF, Ponsonby AL, Polanowski AM, Thomson RJ, et al. Past environmental sun exposure and risk of multiple sclerosis: a role for the Cdx-2 vitamin D receptor variant in this interaction. Mult Scler. 2009;15:563–70. 30 Orton SM, Morris AP, Herrera BM, Ramagopalan SV, Lincoln MR, Chao MJ, et al. Evidence for genetic regulation of vitamin D status in twins with multiple sclerosis. Am J Clin Nutr. 2008;88(2):441–7. 31 Smolders J, Damoiseaux J, Menheere P, Tervaert JW, Hupperts R. Fok-I vitamin D receptor gene polymorphism (rs10735810) and vitamin D metabolism in multiple sclerosis. J Neuroimmunol. 2009;207(1–2):117–21. 32 Roxburgh RH, Seaman SR, Masterman T, Hensiek AE, Sawcer SJ, Vukusic S, et al. Multiple sclerosis severity score. Using disability and disease duration to rate disease severity. Neurology. 2005;64(7):1144–51. 33 Scalfari A, Neuhaus A, Daumer M, Muraro PA, Ebers GC. Onset of secondary progressive phase and long-term evolution of multiple sclerosis. J Neurol Neurosurg Psychiatry. 2014; 85(1):67–75. 34 Huang J, Xie ZF. Polymorphisms in the vitamin D receptor gene and multiple sclerosis risk: a meta-analysis of case-control studies. J Neurol Sci. 2012;313(1–2):79–85. 35 Lin R, Taylor BV, Simpson S Jr., Charlesworth J, Ponsonby AL, Pittas F, et al. Association between multiple sclerosis riskassociated SNPs and relapse and disability – a prospective cohort study. Mult Scler. 2014;20(3):313–21. 36 Mamutse G, Woolmore J, Pye E, Partridge J, Boggild M, Young C, et al. Vitamin D receptor gene polymorphism is associated with reduced disability in multiple sclerosis. Mult Scler. 2008;14(9):1280–3.