The HLA rs9267649 and CYP24A1 rs2248359 Variants are Associated with Multiple Sclerosis: A Study on Iranian Population

Document Type : Research Paper


1 Department of Medical Genetics, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran

2 SGS Canada, 6490 Vipond Dr. Mississauga, ON, LT5 1W8, Canada

3 Neurologist, Member of Scientific Committee of Iranian MS Society, Tehran, Iran


Background: Studies have shown that MS results from synergism between genetic and environmental factors. As a genetic factor, the rs9267649 variant through the regulatory effect on the HLA-DRB1 expression is involved in the MS development. In addition, vitamin D deficiency through involvement of rs2248359 variant of CYP24A1 has shown to play important role in the risk of MS.
Objectives: The aim of this study was to investigate both the HLA rs9267649 and CYP24A1 rs2248359 variants with risk of multiple sclerosis (MS) in Iranian population.
Materials and Methods: The rs9267649 and rs2248359 variants were genotyped in 82 Iranian Relapsing-Remitting Multiple Sclerosis (RRMS) patients and 100 matched healthy controls, using the PCR-RFLP method. The genotype and allele frequencies were calculated and statistically analyzed.
Results: A significant difference was found in the allele distribution for the both rs9267649 and rs2248359 variants, such that the A allele of rs9267649 and the C allele of rs2248359 were found to be more frequent in MS patients than in the healthy controls (p-value: 0.009, OR: 2.264, 95% CI: 1.211-4.231 and p-value: 0.028 OR: 1.594, 95% CI: 1.052-2.415), respectively.
Conclusions: The present research results provide further evidence on the association of the two variants: rs9267649 of the HLA and rs2248359 of the CYP24A1 gene with MS etiology and an increased risk of MS in Iranian RRMS patients. However, further large-scale investigations in various ethnicities and in the functional genomics level are demanded to confirm our findings.


Main Subjects

1. Background

It is estimated that more than 2.5 million people are affected by MS worldwide. Similar to many other type of diseases, the prevalence of MS is influenced by the differences in the ethnic origin, age and size of the population, as well as environmental and geographical variations. Regarding Iran, recent reports have proposed a trend toward high-prevalence for the MS ( 1 , 2 ).

With an unknown detailed molecular etiology, MS is suggested as a multifactorial disorder; a chronic autoimmune disease, and the leading cause of neurological disability in young adults characterized by inflammation, neurodegeneration, and demyelination of the central nervous system (CNS) ( 3 , 4 ). Among the genetic risk factors, the human leukocyte antigen (HLA) super locus on chromosome 6p21 composed of Class I, II, and III has widely been recognized as the most influential factor in the MS etiology. Belonging to the class II genes, the HLA-DRB1*15:01 allele is widely recognized as the most significant factor with a key role in the immune system function through encoding and involvement in the antigenic peptide presentation to the T cells. It has been proposed that HLA-DRB1*15:01 allele alters the beta-chain amino acid repertoire of the HLA class II, as a result, structural alteration of the antigenic peptide-binding groove results in a change in the T cell response and consequently the autoimmune responses in the central nervous system (CNS) ( 5 , 6 )Other factors could play a profound role in the MS etiology among which the degree of gene methylation in case of HLA-DRB1. In an unmethylated state, HLA-DRB1 shows a higher level of expression in monocytes in DRB1*15:01 carriers. In agreement with this notion recent studies have shown that rs9267649 variant is associated with an increased DNA methylation of the HLA-DRB1; as a result a reduced expression of HLA-DRB1 which modulates the DRB1*15:01 effects ( 7 ).

Among the environmental risk factors, low blood vitamin D levels in the majority of MS patients have shown to be associated with the pathogenesis of MS, presumably through the influence of vitamin D on immune cells by shifting cytokines ratio towards an anti-inflammatory state ( 8 , 9 ). The most active form of vitamin D, the 1,25-Dihydroxyvitamin D3 (Calcitriol), reduces myelin loss and promotes remyelination in the CNS. Calcitriol level is regulated by 24-hydroxylase, an enzyme coded by cytochrome P450 family 24 subfamily A member1 (CYP24A1) gene. This enzyme is responsible for hydroxylation of C24, thus, conversion of calcitriol to an inactivated form (i.e. calcitroic acid) ( 10 , 11 ).

2. Objectives

In the present study, we have probed the association between these two variants: rs9267649 and rs2248359 with MS in Iranian patients. The first variant is located on the HLA region of the HLA class III loci on the short arm of human chromosome 6 and the second in the promoter region of CYP24A1 gene on the long arm of human chromosome 20. Applying PCR-RFLP, herein we report the association of these two variants and the related alleles with the etiology of RRMS.

3. Materials and Methods

3.1. Type of MS and Sample Size

In the present investigation, a total number of 182 subjects were included in the study consisting of 82 RRMS; the most common type of MS. The disease was diagnosed by the neurologist according to the McDonald criteria. The control group was consisted of 100 healthy subjects without previous history of autoimmune disease. The two groups were also matched for age, gender, as well as ethnicity.

All participants were informed about the purpose of the study and the written consent was obtained. The ethics of the study was approved by the Ethical Committee at the National Institute of Genetic Engineering and Biotechnology, Iran.

3.2. Sample Collection and Genomic DNA Extraction

A total volume of 3 mL of whole blood was collected by venipuncture in a sterile tube containing EDTA (1.5 mg.mL-1). Genomic DNA was isolated from 200 µL of the whole blood using Gene All DNA Blood Mini Kit (Exgene Clinic SV, Korea), and concentration of the extracted DNA was determined applying spectrophotometer (NanoDrop 2000 Thermo-Fisher Scientific, USA). The quality of the extracted DNA samples was assessed through running on a 1% agarose gel and stored at –20 °C until use.

3.3. Genotyping

Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) was used for single nucleotide polymorphism identification in the HLA region and CYP24A1 gene, respectively. The primers used for genotyping were either designed applying OLIGO7 (i.e. rs9267649 primer set) and tested with the UCSC In-Silico PCR program available online ( or obtained through referring to the publications (for rs2248359; Skalli, et al) ( 12 ).

Polymerase Chain Reaction PCR was performed in a total volume of 25µL under standard reaction condition using thermal cycler (TechneFlexigene, UK). The primer sequences and details of PCR conditions are presented in Table1.

SNP Primer sequence (5’-3’) Product length (bp) PCR condition Restriction enzyme (Restriction site) Length of fragments following RFLP
rs9267649 F: ACCTGACCCTTCCCTGTGTAT 400 35 cycles of 95 °C for 30 sec, 63 °C for 20 sec and 72 °C for 30 sec RsaI (G/A) 318 bp and 82 bp
rs2248359 F: AGTTAGGAAATGCGCCTTGAG 326 35 cycles of 95 °C for 30 sec, 60 °C for 30 sec and 72 °C for 30 sec SacII (C/T) 228 bp and 98 bp
Table 1.Primer’s sequences, PCR condition and the expected product length before and after digestion

The PCR-generated amplicons were analyzed by running on a 1.5% agarose gel electrophoresis. Subsequently, PCR products of the both rs9267649 and rs2248359 were digested overnight at 37 °C applying RsaI and SacII restriction enzymes (Fermentas) for analysis of the both variants, respectively.

Applying RsaI restriction enzyme on the PCR product of the HLA results in the generation of two blunt-ended products. This enzyme recognizes the G allele and digests double-stranded DNA at the 5´-GTAC-3’ restriction site. The products with the approximate sizes of 82 bp and 318 bp are identifiable in the subsequent an agarose electrophoresis which represents the GG (ancestral) genotype. A single band of 400 bp represents the AA genotype (variant), while, production of all the three bands represents the GA (heterozygous) genotype, respectively.

The SacII restriction enzyme recognizes 5´-CCGCGG-3’ restriction site of the CYP24A1 gene. Following enzymatic digestion, those CYP24A1 variants which are carrier of the first C nucleotide give rise to two sticky end restriction products (98 bp and 228 bp). On the other hand, in T allele carriers, this recognition site is lost and a single band will be generated when exposed to the restriction enzyme with an approximate size of 326 bp in the subsequent electrophoresis. All of the digested products were separated on a 2.5% agarose gel. The results obtained through restriction fragment length polymorphism analysis are summarized in Table 1. Further verification on the obtained genotypes was obtained through application of direct Sanger sequencing (ABI 370Xl Analyzer, Applied Biosystems) on the PCR products. Subsequently, chromatograms were checked using Chromas2.6.6 (Technelysium Pty Ltd, Australia) ( and aligned against multiple sequences through application of ClustalW algorithm of the MEGA7.0.26 software ( ( 13 ).

3.4. Statistical Analysis

The association of the two variants; rs9267649 and rs2248359 with the risk of MS was evaluated by comparing genotypic and allelic frequencies between the cases and controls. The difference in the genotypic and allelic distribution was determined by using Chi-Square statistical method of analysis. Furthermore, the risk estimates were calculated using logistic regression and odds ratios (ORs) with 95% confidence intervals (CIs) for the dominant, co-dominant, recessive, and allelic models. Statistical analysis was conducted using SPSS software (version 24, SPSS Inc., Chicago, IL, USA). Data with p-values ≤ 0.05 were considered statistically significant.

4. Results

4.1. Demographic Characteristics

Table 2 represents a summary of the demographic and clinical features of the participants in the present study. Running Shapiro-Wilk normality test indicated an abnormal distribution of the age data between the case and control groups (p-value of 0.000). Checking for the significance of the age difference between the two groups, Mann-Whitney test was used; result of which showed that there isn’t a significant difference in the mean age between the two groups (p-value of 0.117). As well, we could not find a significant difference between the sexes in the two groups using Chi-Square test (p-value of 0.795).

Characteristics Healthy Controls (n=100) MS Patients (n=82)
Female 82 66
Male 18 16
(Female/Male ratio) 4.55 4.12
Age (years)(mean±SD) 31.4±6/03 33.01±7.03
Disease diagnosis (years)(mean±SD) n/a 3.7±2.1
EDSS*(mean± SD) n/a 2.54±1.43
MS-type (n) n/a RRMS(82)
*Expanded disability status scale.
Table 2.Demographic and clinical characteristics of the study participants in the present study.

4.2. Target Polymorphisms and Association with MS

The frequencies of the rs9267649 and rs2248359 variants were examined for Hardy-Weinberg equilibrium (HWE) through application of the Chi-Square respectively; all of which were found to be consistent with HWE (p-value>0.05).

The number and sizes of the PCR-RFLP fragments for the two polymorphisms: the rs9267649 of the HLA system and rs2248359 of the CYP24A1 were determined by digestion of the PCR products with the respective restriction enzymes; RsaI (for rs9267649) and SacII (in case of rs2248359) and subsequent running on a 2.5% agarose gel.

As Figure 1 represents the presence of all the three respective fragments (i.e., 400, 318, and 82 bp for rs9267649 variant and 326, 228, and 98 bp regarding rs2248359 variant) could be observed on the studied subjects in the electrophoresis.

Additionally, applying direct Sanger sequencing confirmed PCR-RFLP pattern and the obtained electropherograms have indicated the respective genotypes for the rs9267649 and rs2248359 variants, as well (Fig. 2).

The allelic distribution and genotypic frequencies of the rs9267649 and rs2248359 variants are summarized in Table 3. The estimated frequencies for all genotypes of the rs9267649 variant (i.e., GG, GA, and AA) in cases were statistically different from those of controls (68.3% vs 83.0% (GG), 26.8% vs 16.0% (GA) and 4.9% vs 1.0% (AA), with a p-value of 0.043). Accordingly, a significant difference was found between the genotypes of rs2248359 variant (CC, CT, TT) in the subjects when compared to those of controls (28.0% vs 24.0% (CC), 56.1% vs 41.0% (CT), 15.9% vs 35.0% (TT), and p-value of 0.013) (Fig. 3).

SNP Genotype/Allele Case (n=82) (%) Control (n=100) (%) X2 P value OR (95%CI)
rs9267649 GG 56 (68.3%) 83 (83%) 6.273 0.043 df=2
GA 22 (26.8%) 16 (16.0%)
AA 4 (4.9%) 1 (1.0%)
Recessive genetic model AA 4 (4.9%) 1 (1.0%) 2.536 0.111 df=1 5.077 (0.556-46.340)
GA+GG 78 (95.1%) 99 (99.0%)
Dominant genetic model AA+GA 26 (31.7%) 17 (17.0%) 5.401 0.020 df=1 2.267 (1.127-4.560)
GG 56 (68.3%) 83 (83.0%)
Codominant genetic model GA 60 (41.7%) 84 (58.3%) 3.198 0.074 df=1 0.519 (0.252-1.072)
GG+AA 22 (57.9%) 16 (42.1%)
Allelic model A 30 (18.3%) 18 (9.0%) 6.797 0.009 df =1 2.264 (1.211-4.231)
G 134 (81.7%) 182 (91.0%)
rs2248359 CC 23 (28.0%) 24 (24.0%) 8.697 0.013 df =2
CT 46 (56.1%) 41 (41.0%)
TT 13 (15.9%) 35 (35.0%)
Recessive genetic model CC 23 (28.0%) 24 (24.0%) 0.386 0.535 df =1 1.234 (0.635-2.401)
CT+TT 59 (72.0%) 76 (76.0%)
Dominant genetic model CC+CT 69 (84.1) 65 (65%) 8.506 0.004 df =1 2.858 (1.390-5.878)
TT 13 (15.9%) 35 (35%)
Codominant genetic model CT 46 (56.1%) 41 (41.0%) 4.116 0.042 df =1 1.839 (1.018-3.320)
CC+TT 36 (43.9%) 59 (59.0%)
Allelic model C 92 (56.1%) 89 (44.5%) 4.848 0.028 df =1 1.594 (1.052-2.415)
T 72 (43.9%) 111 (55.5%)
Table 3.Distribution of the allele and genotype frequencies of the rs9267649 and rs2248359 studied in the RRMS cases and controls.

Furthermore, a significant difference in the genotype frequency was detected in the dominant model of the rs9267649 variant, dominant, and co-dominant models of the rs2248359 variant, respectively (Table 3). We also were able to find a significant difference in the allelic frequencies for the rs9267649 and rs2248359 variants between cases and controls. The A allele of the rs9267649 variant and the C allele of the rs2248359 variant were associated with the risk of MS (p-value of 0.009, OR: 2.264, 95% CI: 1.211-4.231, and p-value of 0.028, OR: 1.594, 95% CI: 1.052-2.415, respectively).

Figure 1. The electrophoretic pattern of PCR-RFLP; A) rs9267649 variant; 1: heterozygous product (3 bands; 82 bp, 318 bp and 400 bp), 2: undigested PCR product (1 band; 400 bp), 3,4: homozygous products (2 bands; 82 bp and 318 bp), 5: DNA ladder 50 bp (17 bands; 50-1500 bp) from left to right, respectively. B) rs2248359 variant; 1,2: heterozygous products (3 bands; 98 bp, 228 bp and 326 bp), 3: undigested PCR product (1 band; 326 bp), 4: homozygous product (2 bands; 98 bp and 228 bp), 5: DNA ladder 50 bp (17 bands; 50-1500 bp) from left to right, respectively.

Figure 2. Direct Sanger sequencing; PCR was carried out and product was subjected to the Direct Sanger sequencing for both variants: rs9267649 and rs2248359. Panels: A) and B) indicate the genotypes of GG and AA homozygotes of the rs9267649 variant. Panel C), and D) indicate the genotypes of CC homozygote and the CT heterozygote of the rs2248359, respectively.In all panels; Black graph: G nucleotide, Green graph: A nucleotide, Blue graph: C nucleotide, Red graph: T nucleotide

Figure 3. Genotypes’ frequency distribution in RRMS cases and healthy controls counterparts. A) The estimated frequencies for all genotypes of the rs9267649 variant (GG, GA, AA) were statistically different from those in the controls (estimated p value: 0.043). And B) The estimated frequencies for all genotypes of the rs2248359 variant (CC, CT, TT) were statistically different from those in the controls (estimated p value: 0.013).

5. Discussion

Studies conducted through application of Genome-Wide Association Studies (GWAS) have led us to the identification of a number of variants such as SNPs involved in susceptibility to the MS. Thus far, around 200 loci have been found to play a role in the susceptibility to MS development. While HLA region was proposed as the main susceptibility locus for MS; as shown by GWAS, nevertheless several other genes within non-HLA region have also been proposed to contribute in the disease development ( 14 , 15 ). Among the two variants analyzed in the present study, a strong association was reported formerly by Kular et al between rs9267649 variant and MS. According to their study, rs9267649 is associated with an increased HLA-DRB1 DMR methylation, resulting in a reduced expression of the gene, and a modulated risk effect of DRB1*15:01 ( 7 ).

Formerly, rs9267649 variant of the HLA system has been subject of study in the other parts of the world. For instance, Kular et al., have carried out a meta-analysis to investigate the association between rs9267649 and MS through three cohort studies carried out in Sweden, Germany, and Iceland on a total number of 14,259 cases and a larger population of the control (i.e. 171,347) on European ethnicities ( 7 ). Nevertheless, the association of this variant with MS has not been well studied in the other parts of the world such as Asian ethnicities and Persians (Iranian) in particular. Our study shows a positive association for the A allele of rs9267649 with the risk of MS (18.3% vs. 9.0%, with a p-value of 0.009).

As well, this finding indicates a protective effect of the  G allele of rs9267649 on the development of MS, if not at all, but at least among Iranians.

Located on the promoter region of the CYP24A1, the rs2248359 variant which is highlighted by the involvement in the vitamin D metabolism ( 16 ) is among the non-HLA loci predicted by the GWAS to have a strong linkage potential with the MS ( 15 ).

Vitamin D modulates immune response; a function supported by many studies that serum vitamin D deficiency and polymorphisms in the genes involved in vitamin D metabolism could lead to  autoimmune diseases ( 17 ). While GWAS has been conducted in a large population of the European ancestry, however, due to the diversity of the polymorphisms worldwide, in addition to the socio-behavioral factors such as vitamin D deficiency, insufficient exposure to sunlight, melanin composition of the skin, etc., additional studies on the other populations and ethnicities, and among which, Iranians is mandatory. Our investigation shows an increased C allele frequency in the rs2248359 variant in MS patients. The C allele carriers (hetero or homozygous carriers) are more likely to develop MS than non-carriers (56.1% vs 44.5%, p-value 0.028) through changes in the CYP24A1 promoter efficiency; resulting in the transcriptional output and mRNA levels.

Studies by other investigators suggest that the C allele of rs2248359 variant leads to an enhanced CYP24A1 expression in the human brain. As a result, an increased 24-hydroxylase expression and an exacerbated degradation of the 1,25(OH)2D3 (Calcitriol) through hydroxylation confers susceptibility to MS ( 10 , 16 ). While we carried out our study on a small population size, however, our results are in conformity with the GWAS study conducted by IMSGC/WTCCC2 on a large number of European subjects (9,772 MS cases and 17,376 controls) ( 15 ) as well as the study of Beechman et al., (14,498 MS cases vs. 24,091 controls) ( 14 ), in addition to the study carried out on African Americans subjects (803 MS cases vs. 1516 controls) ( 18 ). In contrast, other studies carried out on Germans ( 19 ), Chinese ( 20 ) Danish ( 21 ), Moroccan ( 12 ) and Atlai ethnicities ( 22 ) indicate a lack of a significant association between rs2248359 and MS risk. Also, a recent study among European population did not show a significant association between rs2248359 and MS AAO (age at onset) ( 23 ). Such disparity could be attributed to the ethnic, socioeconomics differences, as well as geographical and environmental influences, respectively.

According to the previous study among the Iranian population to analyze CYP24A1 gene expression status in 50 RRMS patients, the expression level of CYP24A1 compared with a normal individual has shown a decrease, however, no reached statistical significance. Besides, that study did not observe a gene expression-phenotype correlation for CYP24A1 which may be due to limited statistical power as a result of the small sample size. So, further studies are required to identify the contribution of genes involved in MS, particularly CYP24A1 ( 24 ).

The involvement of the rs2248359 variant is not only limited to the MS but also studies on several other inflammatory diseases have documented a significant correlation between this variant and those other diseases, among which, Asthma ( 25 ), Atopic dermatitis ( 26 ), Type 2 diabetes and Hepatitis C ( 28 ).

The relationship of the gene-environment with respect to Vitamin D was shown in other studies, among which, the interaction between vitamin D and the HLA DRB1*1501 allele, as vitamin D response element (VDRE), has been identified in the promoter region of the HLA DRB1*1501 gene ( 29 ).

Therefore, it could be concluded that SNPs related to vitamin D metabolism can affect MS susceptibility through gene-environment and gene-gene interactions.

6. Conclusion

In the present study, we have attempted to investigate both the HLA and non-HLA region variations in parallel. In conclusion, our data suggest that the HLA rs9267649 A/G genotype and the non-HLA rs2248359 C/T genotype are associated with the increased susceptibility to MS. However, large-scale investigations with more sample sizes among Iranian and various ethnicities are required to confirm our results. Also, further studies at the functional genomics level will help us to clarify the underlying correlation between DNA polymorphisms and susceptibility to MS.


We thank all the participants, especially MS patients and staffs of MS Clinic of Imam Hossein Hospital, Tehran, Iran, for their kind helps which made the progress of the present investigation possible.


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