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Neuroscience Letters 545 (2013) 46– 49

Contents lists available at SciVerse ScienceDirect

Neuroscience Letters

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elationship between NF-�B1 −94 ins/del ATTG polymorphism andusceptibility of multiple sclerosis in Iranian MS patients

amid Zahednasaba, Seyed Alireza Mesbah-Namina,∗, Mohammad Ali Sahraianb,ohammad Balooda, Rozita Doostib

Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IranSina MS Research Center, Brain and Spinal Injury Research Center, Tehran University of Medical Sciences, Tehran, Iran

i g h l i g h t s

We investigated the −94 ins/del NF-�B1 gene polymorphism.This polymorphism was associated with other auto-immune diseases.This is the first time this polymorphism has been studied in Iranian MS patients.

a r t i c l e i n f o

rticle history:eceived 19 March 2013ccepted 13 April 2013

eywords:ultiple sclerosis

a b s t r a c t

Multiple sclerosis (MS) is one of the most common neurological diseases of the central nervous system(CNS) which is mediated by the autoimmune reactions against myelin sheath. Both genetic and environ-mental factors are thought to be involved in the pathogenesis of MS. NF-�B1 is one of the most importantmolecules which regulates the immune functions. NF-�B1 −94 ins/del ATTG promoter polymorphismis a well-studied region in NF-�B1 gene associated with several common autoimmune diseases such as

F-�B1olymorphism

systemic lupus erythematosus (SLE). Our hypothesis was aimed to address the potential association ofNF-�B polymorphism and MS. Therefore, we analyzed 200 sex and age matched MS patients along with200 healthy individuals using PCR–RFLP. The data revealed no significant differences in the frequency ofthe −94 ins/del ATTG polymorphism in multiple sclerosis patients compared with the control group. Toconclude, our study showed no association between −94 ins/del ATTG polymorphism and risk of multiple

ts.

sclerosis in Iranian patien

. Introduction

Multiple sclerosis (MS) is one of the most debilitating neu-ological diseases of the central nervous system (CNS) affectingoung adults with an age range of 20–40 years old [18]. Theisease is characterized by demyelination and hard axonal dam-ge due to immune reactions against myelin sheath [18]. About0% of MS patients are diagnosed as a relapsing–remitting MSRRMS) transforming into a secondary progressive MS coursever a decade upon the diagnosis of the disease. In a smallerroup of patients (20%), multiple sclerosis begins with a pri-

ary progressive phase [14]. To date, the precise etiology of MS

isease remains elusive. Several lines of evidence show that inter-lay between genetic and environmental factors may be involved

∗ Corresponding author at: Jalal Ale Ahmad Highway, Nasr Bridge, Tarbiatodares University, P.O. Box 14115-111, Tehran, Iran. Tel.: +98 2182883570;

ax: +98 2182884555.E-mail address: mesbahna@modares.ac.ir (S.A. Mesbah-Namin).

304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.neulet.2013.04.014

© 2013 Elsevier Ireland Ltd. All rights reserved.

in the pathology of MS disease [23]. Among environmental factors,Epstein-Barr Virus (EBV), smoking and vitamin D deficiency havebeen highly followed by recent research studies [23].

Among the genetic loci, MHC class II, is the most well studiedlocus thought to be strongly associated with the disease suscepti-bility. A large body of scientific research indicates that 50% of MSpatients share HLADRB1*1501 [9,23]. Furthermore, genome-wideassociation studies (GWAS) show that multiple non-HLA genes arealso involved in the susceptibility of MS. They have shown thatsome variants within IL-7R� along with IL-2R� are significantlyassociated with the disease susceptibility [25]. In accordance toabove statements and similar studies performed in this regard,some non-HLA genes have undoubtedly been linked to the immunefunction in the pathogenesis of multiple sclerosis. However, thesefindings are still open to the question that how the unique geneticfeatures might contribute to the immune function in MS.

Nuclear factor kappa B (NF-�B) is a family of critical transcrip-tional factors involved in the regulation of major inflammatoryresponses including innate and adaptive immune responses, cellgrowth, apoptosis and tissue differentiation [3]. The NF-�B has also

science Letters 545 (2013) 46– 49 47

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The −94 ins/del ATTG allele frequency was approximately simi-lar in multiple sclerosis patients and control groups (Table 1), withno significant statistical difference. No difference was found in the

Table 1Clinical features of MS patients and controls.

Patients, n = 200 Healthy individuals, n = 200

Female:male 143:57 146:54Age (mean years ± SD) 32 ± 9.14 31 ± 7.95RR 158 –SP 32 –

H. Zahednasab et al. / Neuro

key role in the activation of T cells and other immune cells includ-ng B cells, dendritic cells (DCs), macrophages and CNS resident glia

hich are all thought to be involved in the pathogenesis of MS [22].In mammals, five members of NF-�B family have been identi-

ed: p50/p105, p52/p100, p65/RelA, RelB, and c-Rel [3]. The majororm is a heterodimer of the p50/p105 and p65/RelA subunitsncoded by NF-�B1 and RelA genes, respectively [6]. In a study per-ormed by Eggert and colleagues [10] they found elevated levels ofNA binding p50, in a group consisting of 5 patients with RR-MS,0 with SP-MS and 5 with PP-MS, compared to 24 healthy controlsne of the most prominent polymorphisms identified within theromoter of this gene, is −94 ins/del ATTG which is able to reg-late the expression of NF-�B1 [12]. It has also been shown thathe aberrant expression and impaired NF-�B1 signaling are bothnvolved in the human autoimmune diseases such as SLE [11] andrave’s disease [13]. Considering the fact that NF-�B plays a critical

ole in inflammation, apoptosis and other autoimmune diseases, weypothesized that a functional polymorphism in the NFKB1 pro-oter region might play an important role in the development

f multiple sclerosis. We therefore investigated the −94 inser-ion/deletion ATTG promoter polymorphism of NFKB1 in patientsith MS and healthy controls.

. Materials and methods

.1. Study population

We studied 200 MS patients with definite MS accordingo McDonald’s [21] criteria (143 women, 57 men) along withealthy individuals (146 women, 54 men). These patients hadelapsing/remitting (RR), secondary progressive (SP) or primaryrogressive (PP) course. The mean age of the patients was 32 ± 9.14ears, and the mean EDSS [16] was 3.5 ± 1.28 (range 0–7). The con-rol group was selected from healthy volunteers matched based onex and age. They had no evidence or family history of autoimmuneiseases or cancers. The study was approved by the local ethicalommittee and all the participants signed an informed consent forhe study.

.2. Genotyping

Genomic DNA was extracted from peripheral blood samplesollected on EDTA anticoagulant using spin column technique ofIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany) accord-

ng to the manufacturer’s instructions, eluted in 50 �L of waternd subsequently quantified using Nanodrop spectrophotome-er (UV-Visible NanoDrop 1000; Thermo Fisher Scientific Inc.)nd standardized to100 ng/mL. The polymerase chain reactionPCR)–restriction fragment length polymorphism (RFLP) methodas used based on a protocol presented by Ota et al. [19] for geno-

yping the −94 ins/del ATTG polymorphism.Primers were designed on both ends of the poly-

orphism allele 5′-TGGGCACAAGTCGTTTATGA-3′ and′-CTGGAGCCGGTAGGGAAG-3′, amplifying a fragment of 281 bpsr 285 bps [4]. Genomic DNA (100 ng) was amplified in a 25 mLCR reaction under the following cycling conditions: denaturationt 95 ◦C for 3 min, followed by 35 cycles at 95 ◦C for 1 min, 56.8 ◦Cor 1 min, and 72 ◦C for 1 min, with a final extension at 72 ◦C for

min. Amplifications were performed in a MyCycler thermalycler (Bio-Rad).

The presence of ATTG allele provides a restriction enzyme site

or Van91I enzyme (Promega) and yields 240 and 45 bp fragments.ive microliters of the PCR products were digested with 1U ofan91I at 37 ◦C for 4 h. The digestion products were analyzed on a% agarose gels (Sigma) containing ethidium bromide (0.5 mg/mL)

Fig. 1. The pattern of enzyme digestion: A, C, D and F are ins/del. B and E are del/deland ins/ins respectively.

(Sigma) and visualized under UV illumination using the Gel DocXR (Bio-Rad). The pattern of enzyme digestion has been shown(Fig. 1).

2.3. Statistical analysis

Statistical calculations were performed by means of the SPSSv.18.0 (SPSS Inc., Chicago, IL, USA). Hardy–Weinberg analysiswas applied to compare the observed and expected genotypefrequencies using the chi-square test. Logistic regression wasperformed to evaluate the association of genotypes and allele fre-quencies with disease. Moreover, odds ratios and 95% confidenceinterval (CI) were estimated for both the patients and healthycontrols.

3. Results

3.1. Characteristics of the study population

The characteristics of the MS patients and control groups arepresented in Table 1. The cases and controls appear well matchedin age and sex.

3.2. No association of the −94 ins/del ATTG polymorphism withmultiple sclerosis

PP 10 –EDSS (mean ± SD) 3.5 ± 1.28 –

EDSS, Expanded Disability Status Scale; RR, relapsing–remitting; SP, secondary pro-gressive; PP, primary progressive.

48 H. Zahednasab et al. / Neuroscience Letters 545 (2013) 46– 49

Table 2The −94 ins/del ATTG genotype and allele frequencies distribution in 200 control group and in 200 MS patients.

Genotype Patients, n = 200 (%) Control n = 200 (%) OR CI P-value

del/del 15 (7.5%) 18 (9%) 1.0 (reference) – –del/ins 153 (76.5%) 144 (72%) 1.27 0.59–2.78 0.51ins/ins 32 (16%) 38 (19%) 1.01 0.41–2.52 0.98Alleles

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Del 183 (45.75%) 180 (45%)

Ins 217 (54.25%) 220 (55%)

R, odds ratio; CI, confidence interval.

istribution of genotype frequencies between multiple sclerosisases and controls (Table 2).

. Discussion

The effective role of genetics in MS susceptibility is undeni-ble. Epidemiological studies indicate that the risk of MS wouldncrease 300-fold for monozygotic twins to 20-40-fold for biolog-cal first-degree relatives [24]. Genome-wide association studiesGWASs) have identified some susceptibility loci associated withmmune-related risk conferring genes in multiple sclerosis [2,27].

oreover, it has been shown that dysfunctions in the apoptosis’egulatory genes are involved in several autoimmune diseases suchs multiple sclerosis [8]. One of those well-studied genes is NF-�B1hat plays a central role in immune function [3]. The NF-�B1 reg-lates some target genes encoding cytokines, growth factors, celldhesion molecules and anti- and pro-apoptotic proteins [22]. Poly-orphisms in the promoter regions of NF-�B1 results in alteredF-�B1 expression, which in turn results in altered transcriptionf the inflammatory cytokines, and may explain the overexpress-on of these cytokines and increased serum concentrations in MS.n addition, the role of NF-�B1 in the regulation of apoptosis-elated factors and death receptors, giving rise to apoptosis, haseen shown [26]. On the other hand, NF-�B1, when present in

ts active form, results in increased production of anti-apoptoticolecules and some inflammatory cytokines. Alterations of these

roteins might lead to resistance of apoptosis by Th1 lymphocytes,hich could be responsible for the prolonged state of inflammation

bserved in patients afflicted with MS [15]. We did not find anyssociation between MS patients and healthy individuals when weompared genotypic and allele frequencies and it seems that aber-ation in NF-�B1 expression may be independent of this commonolymorphic site. Both genetic and environmental factors might be

n charge of NF-�B1 overexpression. (i) Genetic: other polymorphicites in the promoter region of NF-�B1 that can alter the expres-ion of NF-�B1. In addition, dysregulation of NF-�BIA, the inhibitorf NF-�B, can lead to the overexpression of NF-�B1 which is seenn some pathological conditions [20]. (ii) Environmental factor(s):t has been shown that Epstein-Barr Virus (EBV), 99% seropositivityn MS patients [17], is capable of causing an aberration in NF-�Bxpression both in lytic and latent phase. A study performed byriza et al. [1] showed that the EBV-encoded dUTPase activates NF-B thereby TLR2 and MyD88-dependent signaling pathway. Thishenomenon occurs during the lytic replication phase of the EBV

ife cycle. Another study carried out by Cahir et al. [5] indicated thatatent membrane protein-1 (LMP1) recruits TRAFs and TRADDs toirectly activate NF-�B. Another explanatory reason for the aber-ation of NF-�B expression may be due to vitamin D deficiencyeen in MS patients. It has been shown that vitamin D up-regulateskappaBalpha levels by increasing mRNA stability and decreasingkappaBalpha phosphorylation. The increase in IkappaBalpha levels

educes nuclear translocation of NF-�B and consequently down-rades its activity [7].

To the best of our knowledge, this is the first study focusingn the association of NF-�B1 −94 ins/del ATTG functional four

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1.0 (reference) – –0.97 0.73–1.29 0.83

base-pair with MS disease. The limitation of our study was smallsample size. Further investigations are warranted to illuminate theassociation of −94 ins/del ATTG with MS susceptibility.

Acknowledgments

The financial support of this study has been provided by Tar-biat Modares University. We thank Dr. Manshadi’s lab for providingnormal samples.

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