Relationship between vitamin D receptor genotypes (FOK1rs2228570) and IL18 gene expression in sample of multiple sclerosis Iraqi patients

Abstract

BACKGROUND:

Multiple Sclerosis known as MS, this chronic inflammatory demyelinating condition affects the nervous system. It is a heterogenic and multifactorial disease. The goal of the current study was to investigate the relationship between MS patients’ IL18 gene expression and the vitamin D receptor gene polymorphism (FOK1rs2228570).

OBJECTIVE:

The aim of the study to investigate the association of vitamin D receptor (FOK1rs2228570) gene polymorphism and pro inflammatory cytokine (IL18) gene expression among multiple sclerosis Iraqi patients. Detection VDR polymorphism and determine whether this SNP is involved in susceptibility to multiple sclerosis and estimation IL18 gene expression and explore its relation with multiple sclerosis susceptibility.

METHODS:

Blood samples were taken from 75 MS patients in Iraq (30 men and 45 women), as well as from 75 volunteers who seemed to be in a favorable state of health and fell within the age range of 20 to 50 years. Tetra-ARMS Polymerase Chain Reaction (Tetra-ARMS PCR) was used to find polymorphisms in the vitamin D receptor (VDR) gene, and Real-time Polymerase Chain Reaction (RT-PCR) was used to measure IL18 gene expression.

RESULTS:

The findings from the analysis of VDR gene polymorphism in patients with MS indicated that the wild-type genotype T/T was present in 8 individuals, accounting for 10.6%, the heterogeneous genotype TC was 36 (48%), and the homogeneous genotype CC was 31 (41.3%), whilst T allele frequency was 52(34.6%) and C allele was 98(65.3%) with ( $P\leqslant$ 0.01) significant difference and even as in control T/T genotype was 49(65.3%), TC genotype was 21(28%), CC genotype was 5(6.66%), T allele frequency was 119(79.3%) and C allele was 31(20.6%) with significant difference ( $P\leqslant$ 0.001). While estimation of IL18 expression showed high elevation in patients’ group (2.59 $\pm$ 0.51 fold) by significance difference ( $P\leqslant$ 0.5) when compared to control group (1.35 $\pm$ 0.14 fold). The relationship between IL18 gene expression with VDR variant in MS patients demonstrated a significant rise (2.9 $\pm$ 0.51 fold) at CC genotype patients in IL18 folding gene expression, followed by (4.6 $\pm$ 0.17 fold) in TC genotype patients and finally (1.4 $\pm$ 0.08 fold) in TT genotype patients with highly significant ( $P\leqslant$ 0.01).

CONCLUSION:

The VDR(FOK1rs2228570) genotype was significantly correlated with IL18 expression in MS patients from Iraq.

Keywords

VDR IL18 genotypes MS gene expression

1. Introduction

Multiple sclerosis (MS) occurs as a result of aggressive immune attack against the myelin sheath that surrounds nerve cells [1]. Both genetic and environmental factors increase the risk of MS significantly [2]. A class of fat-soluble secosteroid hormones including vitamin D have a physiological and regulatory impact, especially on brain and spinal cord development. Vitamin D has a positive impact on the immune system. It has been demonstrated that adequate levels of vitamin D may reduce the incidence of MS. Therefore, the possible effects of vitamin D warrant further investigation, although it plays a key role in the development and maturation of immune cells, including macrophages, dendritic cells (DCs), T cells, and B cells [3, 4].

Dendritic cell maturation is inhibited by vitamin D3 via suppression of the major histocompatibility complex (MHC) class II, CD40, CD80, and CD86 expression. The synthesis of pro-inflammatory cytokines such as IL-12 is decreased, while the synthesis of anti-inflammatory cytokines such as IL-10 is increased in DCs. Vitamin D3 inhibits the expression of IL2, IL17, and IFN in T cells. It also inhibits the cytotoxicity of CD8 $+$ T cells and the action and production of CD4 $+$ cells. The production and activation of T regulatory cells are promoted, whereas B cell synthesis, variation, and Ig production is repressed [5].

Vitamin D binds to the nuclear vitamin D receptor (VDR). VDRs belong to the steroid/thyroid hormone receptor superfamily. The VDR gene is located on chromosome 12q12–14 in humans [3]. Changes in vitamin D metabolism and function are linked to specific variations in VDR gene such as exon 2 SNP FokI (rs2228570), also known as the start codon polymorphism (SCP) [6].

The anti-inflammatory and anti-proliferative properties of vitamin D are well established. IL-18, an immunostimulatory cytokine, is synthesized primarily by the initiated macrophages, although it may also be expressed by other cells, such as Kupffer cells, T and B cells. These activities are, at least in part, mediated via regulation and interaction with pro-inflammatory cytokines like IL-18. Due to its expression on natural NK cells, monocytes, DCs, T and B cells, both inborn and acquired immune responses can be controlled by IL-18. Its biological effects are attributed to its ability to promote innate immunity via combination with Th1 and Th2 cells. When combined with other inflammatory cytokines, it induces the synthesis of interferon (IFN) by NK, T cells, and perhaps other cells. It stimulates the expression and production of multiple chemokines, including Fas Ligand, TNF, and IL-1 [7, 8], the Th-1 immune response and IFN- $\gamma$ synthesis [9]. IL-18 has been detected in MS lesions. The prevention of experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, has been demonstrated by antibodies against IL-18 [10]. Therefore, it can be reasonably assumed that IL-18 plays a key role in the immunopathogenesis of the disease. IL-18 has been shown to play a crucial role in MS. It acts as a pleiotropic cytokine that mediates a variety of physiological and pathological activities in the immune and central nervous systems (CNS).

Elevated IL-18 levels are typically observed in the case of MS disease [11, 12]. With MS being a critical health issue of great significance. The purpose of the present study was to look into the link among IL18 gene expression and the vitamin D receptor (FOK1rs2228570) gene polymorphism in MS patients from Iraq.

2. Material and methods

The study was approved by the Ethics Committee of our institution. Venous blood (3 mL) was drawn from each of the 75 patients diagnosed with MS (30 men and 45 women). The patients were aged between 20 years and 50 years. They were age- and gender-matched with 75 apparently healthy volunteers, who served as a control group.

Table 1
The VDR (rs2228570) primer sequences for Tetra-ARMS PCR

Primer name	Sequence 5’–3’	Reference
Forward inner (allele C)	CCGTGGCCTGCTTGCTGTTCTTACAGGTAC	[32]
Reverse inner (allele T)	GGAAGTGCTGGCCGCCATTGCCTACA
Forward outer	CGAATCATGTATGAGGGCTCCGAAGGCA
Reverse outer	AGAGCCTGGGAGGAGGGCTCACCTGAAG

Table 2

Amplification fragments PCR program of VDR (rs2228570)

Step	^∘C	Min/sec	Cycle
Initial denaturation	95	05:00	1
De-naturation	95	00:30	35
Annealing	61	00:30
Extension	72	00:30
Final extension	72	05:00	1

Each sample was divided into two portions. The first 2.75 mL of blood was transferred into EDTA anti-coagulant tubes, mixed gently, and stored at $-$ 20^∘C until the DNA was purified as instructed by the kit manufacturer (Promega/USA) [13]. The VDR (rs2228570) polymorphism was detected via Tetra-ARMS PCR, which is a modest and inexpensive method used to identify single nucleotide polymorphisms (SNPs). Four primers in a single PCR were based on gel electrophoresis. The primers were used to amplify the denatured DNA, as shown in Table 1. The PCR amplification was achieved using the program presented in Table 2. Electrophoresis was used to separate the PCR fragments (363 bp, 222 bp, and 197 bp) on a 2.5% agarose gel.

Table 3

PCR program for cDNA conversion

Step	^∘C
Priming	25 for 10 minutes
Transcription in reverse	50 for 60 minutes
Inactivation of RT	80 for 5 minutes
hold	13 $\infty$

Table 4

Primers of IL18 and GADPH genes with their sequences

Primer name	Sequence 5’–3’	Reference
IL-18-F	CCCTTTGCTCCCCTGGCGAG	[33]
IL-18-R	AGACTGCAGCAGGTGGCAGC
GADPH-F	TGCACCACCAACTGCTTAGC	[34]
GADPH-R	GGCATGGACTGTGGTCATGAG

The remaining 250 $\mu$ L of blood was obtained from each patient and transferred into 750 mL of TRIzol for RNA extraction using the appropriate RNA purification kit (Promega/USA). Estimates of DNA and RNA purity and concentration were obtained as reported previously [13] using Nanodrop (Bioneer/Korea). The IL18 gene expression was detected using a two-step RT-qPCR. In the first step, RNA was converted to cDNA using the AddScript Reverse Transcriptase Kit (ADD BIO Inc, Korea), according to the programme shown in Table 3. Subsequently, in the second step, the RT-qPCR was carried out following the method outlined by Mohammed [14]. The laboratory method for the purification of biological molecules was based on PCR. It was used to monitor the magnification of the target DNA during conventional PCR. The use of real-time PCR facilitates quantitative and semi-quantitative applications for DNA amplification employing specific primers provided by Macrogen (Korea), as shown in Table 4, based on information supplied by NCBI. Utilising RT-qPCR (Molecular System, Australia), the PCR amplification was carried out in accordance with the programme, which is presented in Table 5.

The effect of multiple factors on study parameters was determined using the Statistical Analysis System (SAS) programme [15]. The Chi-square test was used to compare the distribution of patients and control subjects according to gender and autoimmune disease as well as genotype frequencies and alleles between sick and control groups. $T$ -test was used to compare the mean gene expression of patients and control for significant differences between percentages (at probabilities of 0.05 and 0.01). The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to estimate the risk of MS with variable genotypes.

3. Results

Table 5
PCR program for IL18 gene amplification fragments

Steps	^∘C	Min/sec	Cycle
Initial denaturation	95^∘C	5 min	1
De_naturation	94^∘C	30 sec	45
Annealing IL18	56^∘C	30 sec
Extention	72	20 sec

Table 6

Comparison of patients according on gender

Gender	Patients No. (%)
Female	45 (60%)
Male	30 (40%)
Total	75
Chi-square	3.95
$P$ -value	0.04^*

^*( $P\leqslant$ 0.05).

The spreading of MS patients by gender showed that women were more impacted by the disease 45 (60%) than men 30 (40%) significantly changed ( $P\leqslant$ 0.05) shown in Table 6.

Table 7

Distribution of MS patients based on history of autoimmune disease

Auto immune disease	No	Percentage (%)
Yes	15	20%
No	60	80%
Total	75	100%
Chi-square	27.0
$P$ -value	0.0001^**

^**( $P\leqslant$ 0.01).

The results of patient distribution based on historiological MS family revealed that 15 (20%) of patients had the illness, whereas 60 (80%) did not, with the variance being highly substantial ( $P\leqslant$ 0.01) as clarified in Table 7.

Figure 1.

Gel electrophoresis showing FOKI (rs2228570) polymorphism analyzed on 2.5% agarose gel. Lane 1: DNA Ladder marker (1000bp-100bp), Lane 2: TT genotype (363, 197) bp, Lane 3: CT genotype (363, 222,197) bp, Lane 5: CC genotype (363, 222) bp.

The result of T-ARMS PCR for e VDR gene SNP (FOK1: rs2228570) showed that the T-ARMS-PCR product was divided into three bands: 363 bp represented outer PCR which served as a DNA template for the inner allele T (197 bp) and the allele C (222 bp). The wild-type genotype (TT) had only two bands 363and 197 bp. The heterozygous -mutant genotype (TC) would have three bands of 363, 197and 222 bp, and homogenotybe “mutant” (CC) had two bands 363 and 122 bp as shown in (Fig. 1).

Table 8

Genotype distribution and allele frequency in the patient and control groups for the VDR SNP (FOKI rs2228570)

Genotype T/C	Patients no. (%)	Control no. (%)	Chi-square ( $\chi^{2})$	O.R. (C.I.)	$P$ -value
Wild: TT	8 (10.6%)	49 (65.3%)	Ref	0.85 (95%)	0.0062^**
Mutant heterogeneous: TC	36 (48%)	21 (28%)	3.95	1.26 (0.49–2.03)	0.03^*
Mutant homogeneous: CC	31 (41.3%)	5 (6.66%)	6.84	2.19 (0.58–2.74)	0.005^**
Total	75 (100%)	75 (100%)
Chi-square	11.74	9.45
$P$ -value	0.003	0.009
Allele	Frequency		Chi-square	O.R. (C.I.)	$P$ -value
T	52 (34.6%)	119 (79.3%)	Ref	0.84 (0.52–1.34)	0.002^**
C	98 (65.3%)	31 (20.6%)	0.52	1.66 (0.72–1.86)	0.007^**
Chi-square	12.34	0.60
$P$ -value	0.0004^**	0.001^**

^**( $P\leqslant$ 0.01); ^*( $P\leqslant$ 0.05).

The Genotype TT which represented as wild type of the patients group found in only 8 samples which represented at percentage 10.6%, when compared to the control group 49 samples which represented at percentage 65.3%, with highly significant ( $P\leqslant$ 0.01).The genotype TC, the heterozygote frequented 36 times in patient group represented at percentage 48% and 21 sample in the control group represented at percentage 28%, with highly significant ( $P\leqslant$ 0.01), also the genotype CC, was repeated 31 times in the patient group represented at percentage (41.3%), and was 5 for the control group represented at percentage 6.66%, with significant ( $P\leqslant$ 0.01). The T allele frequency was 52 for patients group represented at percentage 34.6% and 119 for the control group represented at percentage 79.3%, the C allele frequented 98 in the patients group represented at percentage 65.3%, as well as for the control group 31 represented at percentage 20.6% with very significant ( $P\leqslant$ 0.01) as illustrated in Table 8.

Table 9

Comparison of the expression of the genes GADPH and IL18 in the MS patient and control

Group	Mean $\pm$ SE
	CT (GADPH)	CT (IL18)	Delta CT	Delta CT	IL18 Folding
Patients	27.62	23.90	$-$ 3.71	0.88	2.59 $\pm$ 0.51
Control	26.1	22.47	$-$ 3.94	0.65	1.35 $\pm$ 0.14
$P$ -value	–	–	–	–	0.024^*
$T$ -test	–	–	–	–	2.30

^*( $P\leqslant$ 0.05).

The results of comparing the expression of the IL18 gene in the patient and control groups showed that the patient group had a considerable increase in IL18 average folding (2.59 $+$ 0.51 fold) compared to the control group (1.35 $+$ 0.14-fold), with differences that were statistically significant ( $P\leqslant$ 0.05) as elucidated in Table 9 and (Fig. 2).

Figure 2.

Gene expression of GADPH and IL18 in patients and control groups.

Table 10

Relationship between FOK1 genotype and IL18 gene expression

Genotype	Mean $\pm$ SE
	IL18 gene expression (folding)
TT	1.4 $\pm$ 0.08
TC	4.6 $\pm$ 0.17
CC	2.9 $\pm$ 0.51
LSD value	0.522^**
$P$ -value	0.0039

^**( $P\leqslant$ 0.1).

The investigation into the association between IL18 gene expression and VDR genotype among multiple sclerosis (MS) patients elucidated a considerable upregulation, measured at 2.9 $\pm$ 0.51 fold in IL18 gene expression among individuals with the CC genotype. Subsequently, a more pronounced elevation at 4.6 $\pm$ 0.17 fold was observed in patients with the TC genotype, followed by a comparatively lower increase at 1.4 $\pm$ 0.08 fold in those with the TT genotype. This observed variation demonstrated statistical significance ( $P\leqslant$ 0.01), as depicted in Table 10.

4. Discussion

The results of the current study reveal that the susceptibility to MS among females was higher than in males. Numerous physiological and biological factors, including menstruation, pregnancy, and breast-feeding affected the performance of the immune system in females. These factors also altered the hormone status, which in turn affected the immune system. Some studies suggest that the two X chromosomes in females are the key to myelin proteo-lipid protein (PLP). The X chromosome may affect the gene expression and inactivation of specific genes that protect against autoimmunity or the overexpression of a susceptibility gene, thereby increasing the risk of autoimmune disease. Myelin, the most abundant protein in the central nervous system, is encoded by genes located on the X chromosome and is likely to be the target of immune attack in MS [16, 17, 18].

The present findings are consistent with the study conducted by Al-Hamadai [19]who reported a higher incidence of MS onset during the teenage years in females compared with males. The study findings are also in agreement with those reported by Hammood et al. [20], who showed significant gender-based differences in the incidence of MS (40% male and 60% female), as well as variations in disease severity.

The result of the absence of a family history of the disease may be due to the epigenetic, which have recently proven their relationship to the occurrence of many diseases, especially autoimmune diseases, so it was found that the lifestyle of nutrition, sports, smoking, drinking alcohol, drugs, instability of the sympathetic state and poor mental health may have the greatest impact on susceptibility to disease [21, 22].

Results of genotypes, VDR allele frequency, and distribution of SNP (FOKI rs2228570) in patients and control groups, showed that patients carrying the (C) allele at least once had a greater probability of developing MS. Among the various genetic variations associated with disease susceptibility, SNPs are the most extensively investigated. Fok-I polymorphism is of particular functional significance. It is defined by an exon 2 and translation start site of the VDR gene T/C allele variant. Additionally, a fascinating interplay between the effect of the VDR Fok-I polymorphism on MS risk and the dietary intake of vitamin D was observed. Vitamin D carrying the T allele in females had a substantially robust protective effect against MS [23, 24, 25].

Our findings echoed earlier studies, which revealed an important correlation between Fok-I T/C polymorphism and MS in both the allele model (TC) and the dominant model (CC) among the Turkish population. This correlation was particularly significant when compared with Caucasian populations. Further, the C allele in the Fok-I polymorphism was linked to reduced levels of 25(OH)D. Notable associations were also established between VDR gene polymorphisms and MS severity and progression [26, 27].

The results of comparing the expression of the IL18 gene in the patient and control groups showed that the average folding was much higher in the patient group in opposition to control group. The expression of cytokines is exhibited by immune cells under the influence of various factors, including hormonal conditions, inflammation, infections, and genetic variations in cytokines [28].

The advancement of multiple autoimmune and inflammatory illnesses has been linked to the increased expression of IL18 and/or gene polymorphisms related to IL18. As a result, a pivotal role is played by IL18 in connecting innate immune responses to adaptive immune responses, thereby significantly conducting to the initiation of autoimmune diseases. Due to these connections, a direct impact on the progression of MS is made by IL18 [29].

This outcome is consistent with previous studies that demonstrated markedly higher levels of IL18 in patients with MS during the acute stage of the disease compared with those in the stable phase. Further, the studies revealed elevated levels of IL18 in patients with relapsing-remitting MS (RRMS). Thus, IL18 is particularly elevated in patients diagnosed with MS showing active lesions on MRI when compared with individuals without such lesions [30, 31].

The findings relating to the association between IL18 gene expression and VDR genotype in MS patients revealed a higher prevalence of MS in individuals harboring at least one copy of the (C) allele. Additionally, the presence of the mutant (C) allele in heterozygous (TC) and homozygous (CC) individuals with the IL18 status was noted in MS patients. The correlation between the severity of MS and vitamin D levels can be explained by the potent immune-modulating properties of vitamin D. This vitamin suppresses the activity of proinflammatory cells while concurrently promoting the activity of anti-inflammatory cells and cytokines. Based on our result we emphasize the need for future studies to include samples from multiple cities or regions to enhance the generalizability of the findings.

5. Conclusion

VDR(FOK1rs2228570) genotype has a significant impact on immunological responses and, hence, MS susceptibility. The presence of the mutant (C) allele, whether in heterozygous (TC) or homozygous (TT) genotypes, was associated with IL18 gene expression in Iraqi MS patients, and there was a greater risk of MS shown in individuals who have at least one copy of the (C) allele.

Author contributions

Each of the below authors made a significant contribution to the research and writing of this publication. Conceptualization Z. K.L.; interpretation or analysis of data Z. K.L, B. J.M., Z. K.L; soft-ware, Z. K.L; validation, Z. K.L, B. J.M.; formal analysis, Z. K.L, investigation, Z. K.L; resources, Z. K.L; data curation, Z. K.L; preparation of the manuscript, Z. K.L; writing-review and editing Z. K.L, B. J.M.; Revision for important intellectual content, Z. K.L; supervision, B. J.M. All authors have read and agreed to the published version of the manuscript.

Availability of data and materials

It is possible to obtain the data that supported the findings of this study from the Institute of Genetic Engineering and Biotechnology for Post Graduate Studies, University of Baghdad in Iraq Requests regarding the data may be made to the corresponding author.

Ethical considerations

Institute of Genetic Engineering and Biotechnology for Post Graduate Studies, University of Baghdad approved the study and of informed consent (Reference number: IGEBGS.H.2.1303, at 15-5-2022).

Footnotes

Acknowledgments

The Institute of Genetic Engineering and Biotechnology for Post Graduate Studies team is acknowledged by the authors for their assistance. We also like to express our gratitude to the referees for their careful review of the article and their illuminating comments.

Conflict of interest

The authors affirm that they have no competing interests.

References

Leray

Moreau

Fromont

and Edan

, Epidemiology of multiple sclerosis, Revue Neurologique 172 (2016), 3–13.

McKay

K.A.

and Tremlett

, Epidemiology of Multiple Sclerosis and Environmental Risk Factors, Neuroimmunology: Multiple Sclerosis, Autoimmune Neurology and Related Diseases (2021), 137–153.

Gado

K.H.

Gado

T.H.

Samie

R.M.A.

Khalil

N.M.

Emam

S.L.

and Fouad

H.H.

, Clinical significance of vitamin D deficiency and receptor gene polymorphism in systemic lupus erythematosus patients, The Egyptian Rheumatologist 39 (2017), 159–164.

Brownlee

W.J.

Hardy

T.A.

Fazekas

and Miller

D.H.

, Diagnosis of multiple sclerosis: progress and challenges, The Lancet 389 (2017), 1336–1346.

Shin

You

Lee

G.Y.

Son

and Han

S.N.

, The effects of 1, 25 (OH) 2D3 treatment on metabolic reprogramming and maturation in bone marrow-derived dendritic cells from control and diabetic mice, The Journal of Steroid Biochemistry and Molecular Biology 225 (2023), 106197.

Hasan

H.A.

Ra’ed

O.A.

Muda

W.A.M.B.W.

Mohamed

H.J.B.J.

and Samsudin

A.R.

, Association of Vitamin D receptor gene polymorphisms with metabolic syndrome and its components among adult Arabs from the United Arab Emirates, Diabetes & Metabolic Syndrome: Clinical Research & Reviews 11 (2017), S531-S537.

Ihim

S.A.

Abubakar

S.D.

Zian

Sasaki

Saffarioun

Maleknia

and Azizi

, Interleukin-18 cytokine in immunity, inflammation, and autoimmunity: Biological role in induction, regulation, and treatment, Frontiers in Immunology 13 (2022), 919973.

Al-Naseri

M.A.

Salman

E.D.

Ad’hiah

A.H.

and Al-Mashtah

S.A.

, The impact of IL1ASNPat889 position on IL-1α serum level in multiple sclerosis Iraqi patients, Current Research in Microbiology and Biotechnology 4 (2016), 933-937.

Mayo

Quintana

F.J.

and Weiner

H.L.

, The innate immune system in demyelinating disease, Immunological Reviews 248 (2012), 170–187.

10.

Glatigny

and Bettelli

, Experimental autoimmune encephalomyelitis (EAE) as animal models of multiple sclerosis (MS), Cold Spring Harbor Perspectives in Medicine 8 (2018).

11.

Sedimbi

S.K.

Hägglöf

and Karlsson

M.C.

, IL-18 in inflammatory and autoimmune disease, Cellular and Molecular Life Sciences 70 (2013), 4795–4808.

12.

Lateef

A.N.

and Mohammed

B.J.

, Effect of age on apoptosis and necrosis of peripheral blood lymphocytes in sample of Iraqi type 2 diabetes patients, Iraqi Journal of Biotechnology 22 (2023).

13.

Mohammed

B.J.

, Investigation on the effect of different concentrations of chlorine drinking water on mice livers, Biochemical & Cellular Archives 18 (2018).

14.

Mohammed

B.J.

, Effect of gasoline inhalation on tumor necrosis factor-alpha (TNF-alpha) expression in liver of rats, Bioscience Research 14 (2017), 566–573.

15.

SAS

, Statistical Analysis System, User’s Guide. Statistical. Version 9, SAS. Inst. Inc. Cary (2018), NC USA.

16.

Greer

J.M.

and McCombe

P.A.

, Role of gender in multiple sclerosis: clinical effects and potential molecular mechanisms, Journal of Neuroimmunology 234 (2011), 7–18.

17.

Al-Araji

and Mohammed

A.I.

, Multiple sclerosis in Iraq: does it have the same features encountered in Western countries? Journal of the Neurological Sciences 234 (2005), 67–71.

18.

Mohammed

B.J.

, TNF-alpha gene polymorphism and its relation to vitamin D, calcium, alkaline phosphatase and ferritin status in Iraqi beta thalassemia patients, Biomedicine 42 (2022), 906–911.

19.

Al-Hamadani

H.A.

, The role of gender in early onset relapsing remitting multiple sclerosis, Iraqi Postgraduate Medical Journal 14 (2015).

20.

Hammood

F.S.

and Mohammed

B.J.

, Genetic Identifications for Sample of Multiple Sclerosis Iraqi Patients, Iraqi Journal of Biotechnology 21 (2022), 225–235.

21.

Ershadinia

Mortazavinia

Babaniamansour

Najafi-Nesheli

Babaniamansour

and Aliniagerdroudbari

, The prevalence of autoimmune diseases in patients with multiple sclerosis: A cross-sectional study in Qom, Iran, in 2018, Current Journal of Neurology 19 (2020), 98.

22.

Al-hamadani

H.A.

Marah

H.A.

and Al-Saffar

, Comparison of familial and sporadic multiple sclerosis in Iraqi patients, Journal of the Faculty of Medicine Baghdad 54 (2012), 1–6.

23.

Simon

K.C.

Munger

K.L.

Yang

and Ascherio

, Polymorphisms in vitamin D metabolism related genes and risk of multiple sclerosis, Multiple Sclerosis Journal 16 (2010), 133–138.

24.

Hadi

S.M.

and Al-Zubaidy

, Genotyping of vitamin D receptor FOKI polymorphism as a predictor for type 2 diabetes mellitus by a tetra primer-ARMS-PCR assay, Gene Reports 15 (2019), 100362.

25.

Abdulhameed

S.A.

and Mohammed

B.J.

, The relationship of gene expression between TNF and TNF-Like cytokine 1A genes in sample of multiple sclerosis iraqi patients, Iraqi Journal of Biotechnology 21 (2022), 88–95.

26.

Smolders

Damoiseaux

Menheere

Tervaert

J.W.C.

and Hupperts

, Fok-I vitamin D receptor gene polymorphism (rs10735810) and vitamin D metabolism in multiple sclerosis, Journal of Neuroimmunology 207 (2009), 117–121.

27.

Criswell

L.A.

Pfeiffer

K.A.

Lum

R.F.

Gonzales

Novitzke

Kern

Moser

K.L.

Begovich

A.B.

Carlton

V.E.

and Li

, Analysis of families in the multiple autoimmune disease genetics consortium (MADGC) collection: the PTPN22 620W allele associates with multiple autoimmune phenotypes, The American Journal of Human Genetics 76 (2005), 561–571.

28.

Banerjee

and Saxena

, Genetic polymorphisms of cytokine genes in type 2 diabetes mellitus, World Journal of Diabetes 5 (2014), 493.

29.

Al-Naseri

M.A.

Salman

E.D.

and Ad’hiah

A.H.

, Serum level and single nucleotide gene polymorphism of interleukin-1receptor antagonist in a sample of Iraq multiple sclerosis patients, Bio Genet J 4 (2016), 91–95.

30.

Losy

and Niezgoda

, IL-18 in patients with multiple sclerosis, Acta Neurologica Scandinavica 104 (2001), 171–173.

31.

Jahanbani-Ardakani

Alsahebfosoul

Etemadifar

and Abtahi

S.-H.

, Interleukin 18 polymorphisms and its serum level in patients with multiple sclerosis, Annals of Indian Academy of Neurology 22 (2019), 474.

32.

Kiran

, To evaluate the vitamin d status, vitamin d receptor polymorphism and cathelicidin antimicrobial peptide in association with tuberculosis: a case – control study? SRM University (2017).

33.

Tsoi

L.C.

Gharaee-Kermani

Berthier

C.C.

Nault

Hile

G.A.

Estadt

S.N.

Patrick

M.T.

Wasikowski

Billi

A.C.

and Lowe

, IL18-containing 5-gene signature distinguishes histologically identical dermatomyositis and lupus erythematosus skin lesions, JCI insight 5 (2020).

34.

Hruz

Wyss

Docquier

Pfaffl

M.W.

Masanetz

Borghi

Verbrugghe

Kalaydjieva

Bleuler

and Laule

, RefGenes: identification of reliable and condition specific reference genes for RT-qPCR data normalization, BMC Genomics 12 (2011), 1–14.

Relationship between vitamin D receptor genotypes (FOK1rs2228570) and IL18 gene expression in sample of multiple sclerosis Iraqi patients

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Material and methods

Table 1 The VDR (rs2228570) primer sequences for Tetra-ARMS PCR

Table 5 PCR program for IL18 gene amplification fragments

5. Conclusion

Author contributions

Availability of data and materials

Ethical considerations

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
The VDR (rs2228570) primer sequences for Tetra-ARMS PCR

Table 5
PCR program for IL18 gene amplification fragments