Layout 1 Vitamin D receptor gene polymorphisms in patients with relapsing multiple sclerosis Eur J Transl Myol 35 (1) 12993, 2025 doi: 10.4081/ejtm.2024.12993 Multiple Sclerosis (MS) is the predominant non- traumatic debilitating condition that impacts young adults.1 The incidence and prevalence of MS are increasing in developed and developing countries, the main cause of which is unclear.2 According to the speed of disease progression, there are four clinical stages of MS, which are: RRMS, Secondary Progressive Type, relapsing-progressive type, and primary progressive type. RRMS is the most common clinical type of MS and is characterized by many symptoms in attacks and complete recovery between attacks.3 The most significant risk factors for MS may be a combination of genetic and environmental factors. Moreover, vitamin D deficiency is a determining risk factor as well as a frequently reported environmental factor in the etiology of MS, regardless of latitude and viral infections.4 In recent years, there has been a lot of evidence that shows that vitamin D can play a role in the pathogenesis of MS, disease courses, and duration.5 Abstract The relationship between the Vitamin D Receptor (VDR) gene and many pathogenic pathways in Relapsing-Remitting Multiple Sclerosis (RRMS) remains unclear. Given the significance of the topic, we conducted this study to explore the correlation between vitamin D receptor gene polymor- phisms and clinical and inflammatory factors in patients suffering from relapsing-remitting multiple sclerosis. The current research is a case/control study conducted based on the Helsinki Ethical Prin- ciples. RRMS disease was confirmed based on history, clinical symptoms, radiological signs and neurologist diagnosis. The research population consisted of healthy people and patients with RRMS who were referred to Hazrat Rasool Akram Hospital between 2021 and 2023. For each person par- ticipating in the study (RRMS patient and healthy), five milliliters of peripheral blood containing the anticoagulant EDTA was collected. Polymerase chain reaction was performed using two specific and appropriate oligonucleotide primers. The restriction fragment length polymorphism technique was used, one of the standard methods for identifying polymorphisms. Statistical analysis was per- formed using SPSS software version 23. The odds ratio and 95% confidence limits were calculated. The SNP Analyzer software was used to analyze the allele frequency of each polymorphism in healthy and RRMS individuals and compare the values. Prism version 5 software was used to draw diagrams. In the present study, a statistically significant difference was observed between the per- centage of FokI genotypes in RRMS patients and healthy individuals. FokI polymorphism showed a significantly increased risk with an odds ratio of 7.28 in patients with the FF genotype compared to healthy individuals. ApaI, TaqI, and BsmI were not significantly different between the two groups. Based on the findings of the present study, FokI polymorphism showed a significant risk increase in RRMS patients with FF genotype compared to healthy individuals. Key Words: relapsing multiple sclerosis, vitamin D, gene polymorphisms. Eur J Transl Myol 35 (1) 12993, 2025 doi: 10.4081/ejtm.2024.12993 Vitamin D receptor gene polymorphisms in patients with relapsing multiple sclerosis Maryam Milanifard,1 Soraya Mehrabi,2 Reza Ahadi,3 Mohsen Nabiuni,4 Samaneh Azimi Souteh,5 Mohammad Taghi Joghataei2,3,6 1Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran; 2Department of Neuroscience, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran; 3Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences, Tehran, Iran; 4Department of Neurosurgery, Iran University of Medical Sciences, Tehran, Iran; 5Department of Radiology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran; 6Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (CC BY-NC 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. - 1 - Vitamin D receptor gene polymorphisms in patients with relapsing multiple sclerosis Eur J Transl Myol 35 (1) 12993, 2025 doi: 10.4081/ejtm.2024.12993 The relationship between the Vitamin D Receptor (VDR) gene and many pathogenic pathways in RRMS remains unclear. Insufficient vitamin D and issues with VDR gene expression can decrease kisspectin 10 levels in the blood and inhibit the maturation of T lymphocytes, increasing the likelihood of an MS recurrence.6 All known biological ef- fects of vitamin D require VDR, which is present in mac- rophages, monocytes, T/B cells, and dendritic cells. Structurally, this receptor is placed in the family of steroid receptors, which exerts the effect of vitamin D by intracel- lularly regulating several genes.7 There are 9 exons in the VDR gene, and each one has several polymorphisms. FOKI RFLP is in exon 2, BSMI and ApaI are in intron 8, and TaqI RFLP is in exon 9. So, the 4 known polymorphisms of vi- tamin D receptor (VDR) are: BSMI (A/G), TaqI(T/C), APaI (A/C) in the 3’ region of the VDR gene, and FOKI(A) poly- morphism (C) in exon 2.8 The vitamin D receptor gene is located on the long arm of chromosome 12. Vitamin D’s main function is to regulate calcium homeostasis, bone for- mation, and resorption, but it also has other effects on the immune system. This vitamin suppresses lymphocyte pro- liferation and immunoglobulin production.9 This vitamin also inhibits the activity of the pro-inflammatory factor NF- KaPPaB as well as the production of various cytokines, such as interleukin 2 and 12 and interferon gamma.10 The presence of nuclear and membrane VDRs in fat cells sug- gests that extra fat may change the VDR, making it harder for the body to absorb vitamin D and raising the risk of MS. This is still being studied. On the other hand, vitamin D has an anti-inflammatory effect on fat cells.11,12 Obesity in early life is associated with an increased risk of developing Mul- tiple Sclerosis (MS).13 Given the significance of the topic, we conducted this study to explore the correlation between vitamin D receptor gene polymorphisms and clinical and inflammatory factors in patients suffering from relapsing- remitting multiple sclerosis. Materials and Methods The current research is a case/control study that was con- ducted based on the ethical principles of Helsinki14 and was approved by the Medical Ethics Department of Iran Uni- versity of Medical Sciences with no. IR.IUMS.FMD. REC.1401.429. RRMS disease was confirmed based on history, clinical signs, radiological signs, and neurologist’s diagnosis. The research population consisted of healthy people and patients with RRMS referring to Hazrat Rasool Akram Hospital between 2021 and 2023 who met the crit- eria for entering the research. Inclusion criteria The study included patients with relapsing-remitting mul- tiple sclerosis (RRMS) who have been diagnosed with McDonald’s 2017 criteria,15 both female and male gender, age group from 18 to 65 years. We also included newly ad- mitted patients using no drugs in the relapse phase and pa- tients only treated with intron beta and with a gap of at least 3 months from the relapse phase were evaluated. Patients were examined by a specialist in terms of plaques in MRI, disability progress status, and blood markers at least in two four-month intervals. The participants showed an EDSS be- tween 1 and 3. The patients did not take vitamin D in the preceding 6 months. Exclusion criteria We included the following categories: i) patients in other phases of RRMS except relapse; ii) patients with diabetes, liver disorders (cirrhosis and types of hepatitis), kidney dis- orders (kidney failure), congestive heart failure, high blood pressure, and cancer; iii) patients taking other drugs. Sample size determination The required sample size for the present study was cal- culated through the ratio comparison formula of two inde- pendent groups. Considering the confidence level of 95% (significance level of 5%), the power of the study was esti- mated to be 80%, according to the almost similar study of 21 people for each study group. In order to increase the power of the study and also to consider the possibility of dropping out, the minimum sample size required for the study group was 25 RRMS patients and 12 Healthy controls. α=0.05; β=0.2; Zα = 1.95; Zβ = 0.84; Mean in group 1(µ1) =2.77; Standard deviation in group 1(σ1) =1.45; Mean in group 2(µ2) =4.11; Standard deviation in group 2(σ2) =1.41; r=1. In this case/control study, 25 patients with RRMS (test group) and 12 healthy people (control group) were evaluated between 2021 and 2023. A written consent was obtained from all the people participating in the study, stating that this study did not impose any additional costs, threats, or prob- lems for them, and whenever they were unable to cooperate and continue to participate, they were excluded from the study, and there was no negligence. It does not take place when handling them. We fully explained the situation to the patient’s legal representative, child, or first-degree relative if he did not understand or was not sufficiently literate, and obtained consent in their presence and with their approval. The project manager fully communicated all the study re- sults, including genetic test results and clinical and inflam- matory factor examination results, to the patients through phone calls or face-to-face interactions, significantly aiding in their disease treatment process. The following clinical variables were considered for anal- ysis at sample collection: age of onset (years), duration of MS disease (months), Extended Disability Status Scale Score (EDSS/1-3), Multiple Sclerosis Severity Score (MSSS), annual relapse The rate since the onset of the dis- ease, the number of relapses two years ago and the doctors at the time of the examination, gender, disease and smoking history, vitamin D consumption in the last 6 months, be- havioral tests and cognitive disorders were evaluated. The EDSS provides a total score on a scale of 0 to 10. EDSS levels 1.0 to 4.5 refer to fully ambulatory patients and the - 2 - Vitamin D receptor gene polymorphisms in patients with relapsing multiple sclerosis Eur J Transl Myol 35 (1) 12993, 2025 doi: 10.4081/ejtm.2024.12993 exact step count is defined by the functional system score(s). EDSS stages 5.0 to 9.5 are defined by movement disorders and common equivalents are provided in the func- tional systems scores. MSSS is a useful measure of MS severity that includes EDSS and disease duration. Achieving an MSSS score ≥ 4.8 indicates a severe phase of the disease and an MSSS < 4.8 indicates a mild phase of the disease. For each person participating in the study (RRMS patient and healthy), five milliliters of peripheral blood with EDTA anticoagulant were taken. All samples were collected from 8 am to 11 am. Two tubes of cell preparation were collected with sodium citrate for cell and plasma separation. Periph- eral blood mononuclear cells were isolated using gradient centrifugation (920 g, 30 min). They were frozen in serum with DMSO (10%) and stored in liquid nitrogen (−196 ◦C). Serum was separated by centrifugation (920 g, 15 min, room temperature) in serum separator tubes. After centrif- ugation, plasma and serum samples were divided into each part and stored at −80◦ temperature. DNA of blood samples was extracted using a genomic DNA column extraction kit manufactured by MBST company in Iran. The DNA puri- fication method in this kit is based on the specific binding of nucleic acids to the silica column in the presence of che- motropic salts. The non-nucleic acid materials that could bind to the column membrane were washed from the col- umn during different washing steps and by centrifugation. Then, the DNA attached to the column was dissolved and separated by adding an elution buffer, and after checking the purity and quality, it was frozen until used for the next steps. In the next step, a Polymerase Chain Reaction (PCR) was performed using two specific and appropriate oligonu- cleotide primers. We designed the back-and-forth primers using Beacon soft- ware, confirmed their correct connection to their own sequences in the NCBI nucleotide database, and ordered the synthesis from Tekapo Bio Company. The primers were lyophilized from the delivery company and according to the manufacturer’s protocol, they were diluted with DNase/RNase-free distilled water to a concentration of 100 pmol/μL. Amplification of Taq DNA polymerase and dNTPs was done by ABI thermocycler (Veriti 96 Well Thwrmal Cycle device) (Applied Biosystem, ABI, com- pany, USA) in three successive temperature steps. This op- eration was done for each of the enzymes separately, and the used items were optimized, and then four PCR variants were placed for each sample. To check the size of the am- plified fragments and the specificity of the products, their electrophoresis was performed on agarose gel, and after the completion of the PCR reaction, the products were electro- phoresed on 2% agarose gel. Then, the PCR-RFLP (Restric- tion Fragment Length Polymorphism) technique, which is one of the standard methods for identifying polymorphisms, was used. To achieve this, we mixed the PCR product with restriction enzymes as per the manufacturer’s instructions, and selected the optimal cutting temperature based on the enzyme’s cutting time specified in the recipe. After the incubation time of the product, electrophoresis was performed on a 2% agarose gel, and by using the pat- tern obtained from restriction enzyme cleavage, the differ- ent polymorphisms of the vitamin D receptor gene studied in each participant were separated and the gels electropho- resed with the Gel machine. Figures 1 to 4 display the ge- notype of VDR gene polymorphisms based on PCR-RFLP. Table 1 summarizes the visualization of RFLP bands using 3% agarose gel electrophoresis. Table 1 summarizes the SNP primer sequences and amplicon sizes. Statistical analysis was performed using SPSS software ver- sion 23. Differences with a p value <0.05 were considered significant. To check the normality of the data, the Kolmo- gorov-Smirnov test was used. Two study groups were ex- amined for demographic variables. The Mann-Whitney U test was used to evaluate the differences between case and control groups. Spearman’s rank correlation coefficient was used to determine continuous correlation. To measure the relationship between the factors and MS risk, odds ratio and 95% confidence limits were calculated. The relationship be- tween MS patients and clinical factors was evaluated by the chi-square test and Fisher’s exact test. An odds ratio and 95% confidence limits are calculated. The SNP Analyzer software was used to analyze the allele frequency of each - 3 - Figure 1. FokI: lane 1: FF, lane 2: FF, and lane 3: ff. Figure 2. BsmI: line 1: BB, lines 2, 4: bb, and line 3: Bb. Vitamin D receptor gene polymorphisms in patients with relapsing multiple sclerosis Eur J Transl Myol 35 (1) 12993, 2025 doi: 10.4081/ejtm.2024.12993 polymorphism in healthy and RRMS individuals and com- pare the values. Prism version 5 software was used to draw diagrams. Findings The mean and standard deviation of the age of the partic- ipants in this study in the test and control groups were 32.16±5.87 years and 31.58±8.14 years, respectively. The independent t-test did not demonstrate a statistically sig- nificant difference in the mean age of the two groups, and both groups were similar in age (p=0.829). 72% of the test group and 41.6% of the control group were women (p=0.364). The mean and standard deviation of the dura- tion of disease in the patients participating in this study in the test group was 45.44±41.74 months. The mean and standard deviation of the age of disease onset in the pa- tients participating in this study in the test group was 28.52±5.07 years. 28% of newly admitted patients were in the relapse phase while taking no medication, and 72% of the patients were only treated with intron beta and with a gap of at least 3 months from the relapse phase. The average and deviation of the time since the last attack in the patients participating in this study in the test group was 3.60±2.44 months. In the test group, 88% of the participants had a deficiency of 25-hydroxyvitamin D level, and in the control group, 66.7% of the participants had insufficient vitamin D. Ac- cording to the results of the Spearman test, the two groups had a statistically significant difference in terms of the level of 25 hydroxy vitamin D (p<0.001). According to Table 2, - 4 - Figure 3. ApaI: lane 1, 2: aa, lane 3, 4: Aa, and lane 5, 6: AA. Figure 4. TaqI: lane 1: tt, lane 2: TT, and lane 3: TT. Table 1. RFLP bands with 3% agarose gel electro- phoresis. SNP Size (bp) The number Geno- type of bands FokI 265 1 FF 265، 196، 69 3 Ff 196، 69 2 ff BsmI 358 1 BB 358، 192،166 3 Bb 192، 166 2 bb ApaI 740 1 AA 740،530، 210 3 Aa 530، 210 2 aa TaqI 495، 245 2 TT 495، 290، 245، 205 4 Tt 290، 245، 205 3 tt Table 2. Polymorphisms and haplotypes of the VDR gene. SNP Primers sequences Amplicon (bp) FokI F 5′-AGC TGG CCC TGG CAC TGA CTC TGC TCT-3′ 265 R 5′-ATG GAA ACA CCT TGC TTC TTC TCC CTC-3′ BsmI F 5′-GGG AGA CGT AGC AAA AGG-3′ 358 R 5′-AGA GGT CAA GGG TCA CTG-3′ ApaI F 5′-CAG AGC ATG GAC AGG GAG CAA-3′ 740 R 5′-GCA ACT CCT CAT GGC TGA GGT CTC-3′ TaqI F 5′-CAG AGC ATG GAC AGG GAG CAA-3′ 495, 245 R 5′-GCA ACT CCT CAT GGC TGA GGT CTC-3′ Vitamin D receptor gene polymorphisms in patients with relapsing multiple sclerosis Eur J Transl Myol 35 (1) 12993, 2025 doi: 10.4081/ejtm.2024.12993 the mean and standard deviation of 25 hydroxy vitamin D levels of the patients participating in this study in the control and control groups were 16.20±4.01 ng/mL and 25.50±4.87 ng/ml, respectively. According to the Mann-Whitney sta- tistical test, there was a statistically significant difference in terms of the level of 25 hydroxy vitamin D between the test and control groups (p<0.001), so in the test group, vi- tamin D deficiency was observed, and in the control group, insufficient vitamin D in the company. There was the mean serum level of 25-hydroxyvitamin D in patients with the disease was significantly lower than that of healthy subjects (p<0.001) (Table 3). According to Table 4, in the present study, the value of Spearman’s correlation coefficient was -0.435. Because the p-value in this study was calculated as 0.030 and this value is smaller than 0.05, then this correlation coefficient is sig- nificant at the 0.05 level. So, it can be concluded that there is a statistically significant relationship between the EDSS scale scores and the level of 25 hydroxy vitamin D in MS patients participating in the study. The negative sign of the correlation coefficient indicates that the relationship is not aligned. This means that MS patients with lower levels of 25-hydroxyvitamin D had higher scores and patients with higher levels of 25-hydroxyvitamin D had lower scores. As seen in Table 4, there was. a significant negative trend be- tween serum levels of 25 hydroxy vitamin D and EDSS score (p<0.001). According to Table 5, in univariate conditional logistic re- gression analysis, the odds ratio (OR) was 1.55 (95% CI, 1.187–2.040; p<0.001), indicating that low levels of 25- hydroxyvitamin D can 1.55 to increase the odds of MS disease, this relationship was statistically significant, and a strong relationship was observed (p<0.001). According to Table 6, the mean and standard deviation of 25 hydroxy vitamin D levels in patients participating in this study in the mild and severe groups were 18.42±3.64 ng/mL and 15.33±3.89 ng/mL, respectively. According to the Mann-Whitney statistical test, a statistically significant dif- ference was observed in terms of the level of 25-hydroxy- vitamin D between the two mild and severe groups - 5 - Table 3. Mean values of 25 hydroxy vitamin D levels in patients with relapsing-remitting MS in both experimental and control groups. Groups Test (n=25) Witness (n=12) P-value Number (percent) Number (percent) 25 hydroxy vitamin D level (ng/mL) 1 (3/8) Spearman correlation >0.001 22 (88) 8 (7/66) Shortage 3 (12) 3 (25%) Insufficient vitamin D 0 87/4 ± 50/25 Mann-Whitney test >0.001 Table 4. Correlation between serum levels of 25 hydroxy vitamin D and Extended Disability Status Scale (EDSS) score. Vitamin D serum level EDSS Spearman’s statistical test Vitamin D serum level Correlation 1/100 -0/435 p-value 0/030 Number 25 25 Spearman’s statistical test EDSS Correlation -0/435 1/000 p-value 0/030 Number 25 25 Table 5. Odds ratio of MS disease with 25 hydroxy vitamin D levels. B S.E. Wald df Sig. Exp (B) 95% C.I. for EXP(B) Lower Upper Vitamin D 442.0 0.138 10.23 1 0.001 1.55 1.187 2.040 Constant -9.76 2.93 11.07 1 0.001 0 Vitamin D receptor gene polymorphisms in patients with relapsing multiple sclerosis Eur J Transl Myol 35 (1) 12993, 2025 doi: 10.4081/ejtm.2024.12993 (p<0.001), so the level of vitamin D in the severe group was significantly lower than the mild group. According to Table 7, the FokI genotype distribution in RRMS patients was as follows: FF (wild) =72%, Ff (het- erozygous) =28% and ff (mutant) =0%; in the control group: FF=25%, Ff=41.7%, and Ff=33.3%. The statistically significant difference was observed be- tween the percentage of genotypes in both test and control groups (p=0.003). Statistical analysis of FokI polymorphism showed a signif- icant increase in risk in patients with FF genotype compared to the control group (OR=7.28: 95% CI; 1.86, 28.41). The odds ratio was 7.28. The distribution of ApaI genotype in RRMS patients was as follows: AA (wild) =40%, Aa (heterozygous) =56% and aa (mutant) =4%; in the control group: AA=50%, Aa=25%, and aa=25%. No statistically significant difference was observed between the percentage of genotypes in the two test and control groups (p=0.075). Statistical analysis of ApaI polymorphism showed no sig- nificant risk increase in patients with AA genotype com- pared to the control group (OR=1.28: 95% CI; 0.45, 3.62). The odds ratio was 1.28. The distribution of TaqI genotype in RRMS patients was as follows: TT (wild) =48%, Tt (heterozygous) =40% and tt (mutant) =12%; in the control group: TT=33.3%, Tt=33.3% and tt=33.3%. No statistically significant difference was observed between the percentage of genotypes in the two test and control groups (p=0.294). Statistical analysis of TaqI polymorphism showed no sig- nificant increased risk in patients with TT genotype com- pared to controls (OR=1.89: 95% CI; 0.74, 4.84). The odds ratio was 1.89. The distribution of BsmI genotype in RRMS patients was as follows: BB (wild)=40%, Bb (heterozygous)=24% and bb (mutant)=36%; in the control group: BB=41.7%, Bb=25%, and bb=33.3%. No statistically significant difference was observed between the percentage of genotypes in the two test and control groups (p=0.987). Statistical analysis of BsmI polymorphism showed no sig- nificant increased risk in patients with TT genotype com- pared to the control group (OR=0.94: 95% CI; 0.42, 2.08). The odds ratio was 0.94. - 6 - Table 6. Determining the relationship between 25-hydroxyvitamin D levels and MS disease progression. Groups Mild (n=7) Intense (n=18) 25 hydroxy vitamin D level (ng/mL) Standard deviation ± mean 18.42 ± 3,64 15.33 ± 3.89 Z -2.56 P-value 0.010 Table 7. Relationship between polymorphisms and haplotypes of the VDR gene and the odds ratio of polymor- phisms. SNPs Genotype Genotypic frequency (%) Significance Odds ratio (95% CI) RRMS (n=25) Control (n=12) FokI rs2228570 FF 18 (72) 3 (25) χ2= 11.96 7.28 (1.86, 28.41) Ff 7 (28) 5 (41.7) P= 0.003 ff 0 4(33.3) ApaI rs7975232 AA 10 (40) 6 (50) χ2=5.19 1.28 (0.45, 3.62) Aa 14 (56) 3 (25) P=0.075 aa 1 (4) 3 (25) TaqI rs731236 TT 12 (48) 4 (33.3) χ2=2.44 1.89 (0.74, 4.84) Tt 10 (40) 4 (33.3) P=0.294 tt 3 (12) 4 (33.3) BsmI rs1544410 BB 10 (40) 5 (41.7) χ2=0.025 0.94 (0.42, 2.08) Bb 6 (24) 3 (25) P=0.987 bb 9 (36) 4 (33.3) χ2 chi-square test; p, p-value; Significant at P ≤0.05. Vitamin D receptor gene polymorphisms in patients with relapsing multiple sclerosis Eur J Transl Myol 35 (1) 12993, 2025 doi: 10.4081/ejtm.2024.12993 Discussion In the present study, a statistically significant difference was observed between the percentage of FokI genotypes in RRMS patients and healthy individuals. FokI polymor- phism showed a significantly increased risk with an odds ratio of 7.28 in patients with the FF genotype compared to healthy individuals. ApaI, TaqI, and BsmI were not signif- icantly different between the two groups. In previous studies, the association between VDR gene polymorphisms and MS was investigated in several populations.15-17 The re- sults of the present study are consistent with some studies conducted on different populations. A study in Iran found a big difference in the types of genes found in MS patients and healthy people when it comes to polymorphism and al- lelic frequency for ApaI, BsmI, and TaqI.18 A Japanese group found an increased frequency of the ApaI AA geno- type and A allele in MS.18 Sioka et al. showed no association between ApaI, BsmI, and TaqI polymorphisms of the VDR gene.19 The results of our study are consistent with those found by Sioka et al.19 Previous epidemiological and biological evidence has sup- ported the protective effect of vitamin D on the underlying disease course of MS.20,21 The present study’s findings re- vealed low serum levels of 25-hydroxyvitamin D in MS pa- tients. Patients with MS had a significantly lower mean serum level of 25-hydroxyvitamin D than healthy individ- uals, which was considered statistically significant. The present study observed a statistically significant relationship between low serum levels of 25-hydroxyvitamin D and the progression of MS. Fitzgerald et al. also linked low levels of 25-hydroxyvita- min D in the blood to a higher risk of MS, higher disease activity, and a faster rate of progression in early MS and clinically isolated syndromes, in line with the findings of this study.22 Similarly, the present study observed a signif- icant association between low levels of 25-hydroxyvitamin D and increased disease activity, as measured by the EDSS score. Although Mowry et al. reported that vitamin D levels were inversely associated with MS activity on brain MRI.23 Another study reported that there is no relationship between 25-hydroxyvitamin D levels and disability scores in MS pa- tients in Iran.24 However, the study by Zhang et al. in China reported the same findings as the present study.25 Sutherland et al. illustrated that vitamin D deficiency is common in MS patients, affecting approximately 50% of MS patients in Europe and 77% in African Americans.26 Dong et al. reported that 76% of Kuwaiti MS patients were vitamin D deficient.27 Similarly, Braidy et al. in their study showed that, more than half of the MS patients had vitamin D deficiency.28 In addition, the serum level of 25(OH) D was lower in MS patients compared to the control group. According to Beecham et al. study serum vitamin D levels were low in Japanese MS patients.29 Al-Temaimi et al. found that there was no significant dif- ference in vitamin D levels between MS and healthy sub- jects. In this study, the small sample (N=50) and the high rate of vitamin D deficiency (82%) in the control group should be considered.30 Niino et al. showed that the serum level of 25(OH) D was higher in MS than in the control group (healthy subjects).31 Vickaryous et al. illustrated that population-based prospec- tive cohort study of 145 people with RRMS living in south- ern Tasmania and Australia from 2002 to 2005 found that higher 25(OH) D levels were associated with a reduced risk of relapse.32 Wasnik et al. showed that Every 10 nmol/L increase in 25(OH) D results in a 12% reduction in the risk of recur- rence. The radiological signs of MS disease activity also correlate with these clinical findings.4 According to Simpson Jr. et al. Results from a prospective cohort study of 1,482 MS patients in the BEYOND study who were administered interferon beta 1b found that a 50.0 nmol/L increase in blood 25(OH)D levels was associated with a 31% was associated with a lower rate of new lesions on MRI. Patients with 25(OH) D levels above 100.0 nmol/L had the lowest rate of new lesions on MRI. However, we did not observe any significant association with the recur- rence rate.33 The current study had limitations such as the small sample size and the need to select more studies with a larger sample size; It is also very important to select the sample size from different regions. Conducting a study to investigate poly- morphic translocation of the VDR gene is necessary to un- derstand the function of the VDR and increase the likelihood of detecting alleles that contribute to the risk of common diseases, as well as further research involving more cases to obtain results This can be distorted by small samples or low frequencies. Minor alleles are required. Conclusions Based on the findings of the present study, FokI polymor- phism showed a significant risk increase with an odds ratio of 7.28 in RRMS patients with FF genotype compared to healthy individuals (OR=7.28: 95% CI; 1.86, 28.41). List of abbreviations VDR, Vitamin D Receptor RRMS, Relapsing-Remitting Multiple Sclerosis MS, Multiple Sclerosis PCR, Polymerase Chain Reaction MSSS, Multiple Sclerosis Severity Score RFLP, Restriction Fragment Length Polymorphism Conflict of interest The authors declare no potential conflict of interest. Ethics approval This research was approved by the Medical Ethics Depart- ment of Iran University of Medical Sciences with no. IR.IUMS.FMD.REC.1401.429 Informed consent and patient consent for publication All patients participating in this study signed a written in- formed consent form for participating in this study. - 7 - Vitamin D receptor gene polymorphisms in patients with relapsing multiple sclerosis Eur J Transl Myol 35 (1) 12993, 2025 doi: 10.4081/ejtm.2024.12993 Availability of data and materials All data generated or analyzed during this study are in- cluded in this published article. Corresponding author Mohammad Taghi Joghataei, Department of Neuroscience, Faculty of Advanced Technologies in Medicine, Iran Uni- versity of Medical Sciences, Tehran, Iran. ORCID ID: 0000-0003-0492-8239 E-mail: m_joghataei@yahoo.com Co authors Maryam Milanifard ORCID ID: 0000-0002-0888-8847 E-mail: marayammilani837@yahoo.com Soraya Mehrabi ORCID ID: 0000-0001-8555-726X E-mail: Soraya.mehrabi@gmail.com Reza Ahadi ORCID ID: 0000-0002-3484-9606 E-mail: rezaahadi1353@gmail.com Mohsen Nabiuni ORCID ID: 0000-0002-0841-6612 E-mail: nabiuni_m@yahoo.com Samaneh Azimi Souteh ORCID ID: 0000-0001-7703-1671 E-mail: s.azimi.s1991@gmail.com References 1. Dobson R, Giovannoni G. Multiple sclerosis – a review. Eur J Neurol 2019;26:27-40. 2. Elssayed A, AlRgaiba RI, AlZalbani MK, et al. Review on diagnosis and management approach of multiple sclerosis. Int J Pharm Res Allied Sci 2023;12:100-5. 3. Filippi M, Preziosa P, Barkhof F, et al. Diagnosis of pro- gressive multiple sclerosis from the imaging perspec- tive: a review. JAMA Neurol 2021;78:351-64. 4. Wasnik S, Sharma I, Baylink DJ, Tang X. Vitamin D as a potential therapy for multiple sclerosis: Where are we? Int J Mol Sci 2020;21:3102. 5. Hartl C, Obermeier V, Gerdes LA, et al. Seasonal vari- ations of 25-OH vitamin D serum levels are associated with clinical disease activity in multiple sclerosis pa- tients. J Neurol Sci 2017;375:160-4. 6. Díez BC, Pérez-Ramírez C, del Mar Maldonado- Montoro M, et al. Association between polymor- phisms in the vitamin D receptor and susceptibility to multiple sclerosis. Pharmacokinetics Genomics 2021;31:40-7. 7. Usategui-Martín R, De Luis-Román DA, Fernández- Gómez JM, et al. Vitamin D receptor (VDR) gene poly- morphisms modify the response to vitamin D supplementation: a systematic review and meta-analy- sis. Nutrients 2022;14:360. 8. Aga SS, Banday MZ, Nissar S, et al. Vitamin D Re- ceptor Polymorphisms and Diseases. In: Aga SS, Ban- day MZ, Nissar S (eds.). Genetic Polymorphism and Disease: CRC Press; 2022. p. 185-98. 9. Taymans SE, Pack S, Pak E, et al. The human vitamin D receptor gene (VDR) is localized to region 12cen‐q12 by fluorescent in situ hybridization and radiation hybrid mapping: genetic and physical VDR map. J Bone Min- eral Res 1999;14:1163-6. 10. Wöbke TK, Steinhilber D. Vitamin D in inflammatory diseases. Frontiers Physiol 2014;5:97223. 11. Vanlint S. Vitamin D and obesity. Nutrients 2013;5: 949-56. 12. Walsh JS, Bowles S, Evans AL. Vitamin D in obesity. Curr Opinion Endocrinol Diabetes Obesity 2017;24: 389-94. 13. Hwang J, Yoo JA, Yoon H, et al. The role of leptin in the association between obesity and psoriasis. Biomol Ther (Seoul) 2021;29:11-21. 14. Goodyear MD, Krleza-Jeric K, Lemmens T. The dec- laration of Helsinki. Br Med J 2007;335:624-5. 15. Chen XL, Zhang ML, Zhu L, et al. Vitamin D receptor gene polymorphisms and the risk of multiple sclerosis: An updated meta-analysis. Microbial Pathogenesis 2017;110:594-602. 16. Ben-Selma W, Ben-Fredj N, Chebel S, et al. Age- and gender-specific effects on VDR gene polymorphisms and risk of the development of multiple sclerosis in Tu- nisians: a preliminary study. Int J Immunogenet 2015; 42:174-81. 17. García-Martín E, Agúndez JA, Martínez C, et al. Vita- min 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:e65487. 18. Abdollahzadeh R, Fard MS, Rahmani F, et al. Predis- posing role of vitamin D receptor (VDR) polymor- phisms in the development of multiple sclerosis: A case-control study. J Neurol Sci 2016;367:148-51. 19. Sioka C, Papakonstantinou S, Markoula S, et al. Vita- min D receptor gene polymorphisms in multiple scle- rosis patients in northwest Greece. J Negat Results Biomed 2011;10:3. 20. Ascherio A, Munger KL, Lünemann JD. The initiation and prevention of multiple sclerosis. Nature Rev Neurol 2012;8:602-12. 21. Rodríguez Murúa S, Farez MF, Quintana FJ. The im- mune response in multiple sclerosis. Annual Review of Pathology: Mechanisms Dis. 2022;17:121-39. 22. Fitzgerald KC, Munger KL, Köchert K, et al. Associa- tion of vitamin D levels with multiple sclerosis activity and progression in patients receiving interferon beta-1b. JAMA Neurol 2015;72:1458-65. 23. Mowry EM, Waubant E, McCulloch CE, et al. Vitamin D status predicts new brain magnetic resonance imaging ac- tivity in multiple sclerosis. Ann Neurol 2012;72:234-40. 24. Karampoor S, Zahednasab H, Ramagopalan S, et al. 25- hydroxyvitamin D levels are associated with multiple - 8 - Vitamin D receptor gene polymorphisms in patients with relapsing multiple sclerosis Eur J Transl Myol 35 (1) 12993, 2025 doi: 10.4081/ejtm.2024.12993 sclerosis in Iran: A cross-sectional study. J Neuroim- munol 2016;290:47-8. 25. Zhang Y, Liu G, Han X, et al. The association of serum 25-hydroxyvitamin D levels with multiple sclerosis sev- erity and progression in a case-control study from China. J Neuroimmunol 2016;297:127-31. 26. Sutherland JP, Zhou A, Leach MJ, Hyppönen E. Differ- ences and determinants of vitamin D deficiency among UK biobank participants: A cross-ethnic and socioeco- nomic study. Clin Nutr 2021;40:3436-47. 27. Dong H, Asmolovaite V, Marseal N. Vitamin D status and dietary intake in young university students in the UK. Nutrition Food Sci 2022;52:616-26. 28. Braidy S, Matar AM, Borjac J. Association of CYP27B1 Polymorphism and Vitamin D Levels with Multiple Sclerosis Development in Lebanese Pop- ulation of Bekaa Region: A Preliminary Study. Genetics Applic 2021;5:18-25. 29. Beecham AH, Amezcua L, Chinea A, et al. The genetic diversity of multiple sclerosis risk among Hispanic and African American populations living in the United States. Mult Scler 2020;26:1329-39. 30. Al-Temaimi RA, Al-Enezi A, Al-Serri A, et al. The as- sociation of vitamin D receptor polymorphisms with multiple sclerosis in a case-control study from Kuwait. PloS One 2015;10:e0142265. 31. Niino M, Sato S, Fukazawa T, et al. Decreased serum vitamin D levels in Japanese patients with multiple scle- rosis. J Neuroimmunol 2015;279:40-5. 32. Vickaryous N, et al. Remote testing of vitamin D levels across the UK MS population—A case control study. Plos One 2020;15:e0241459. 33. Simpson S Jr, Taylor B, Blizzard L, et al. Higher 25‐hy- droxyvitamin D is associated with lower relapse risk in multiple sclerosis. Ann Neurol 2010;68:193-203. Disclaimer All claims expressed in this article are solely those of the authors and do not necessarily represent those of their af- filiated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. Submitted: 30 August 2024. Accepted: 16 September 2024. Early access: 21 November 2024. - 9 -