- Email: [email protected]
Association between interleukin-32 polymorphism and multiple sclerosis
Accepted Manuscript Association between interleukin-32 polymorphism and multiple sclerosis
Zaher Morsaljahan, Alireza Rafiei, Reza Valadan, Mahmoud Abedini, Masoumeh Paksersht, Rezvan Khajavi PII: DOI: Reference:
S0022-510X(17)30349-0 doi: 10.1016/j.jns.2017.05.045 JNS 15352
To appear in:
Journal of the Neurological Sciences
Received date: Revised date: Accepted date:
28 December 2016 29 April 2017 22 May 2017
Please cite this article as: Zaher Morsaljahan, Alireza Rafiei, Reza Valadan, Mahmoud Abedini, Masoumeh Paksersht, Rezvan Khajavi , Association between interleukin-32 polymorphism and multiple sclerosis. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jns(2017), doi: 10.1016/ j.jns.2017.05.045
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ACCEPTED MANUSCRIPT Association between Interleukin-32 polymorphism and Multiple Sclerosis Zaher Morsaljahan1, 2, Alireza Rafiei 2*, Reza Valadan2, Mahmoud Abedini 3, Masoumeh Paksersht1, Rezvan Khajavi2
1-M.Sc. Student of Immunology, Mazandaran University of Medical Sciences, Sari, Iran 2* -Molecular and Cell Biology Research Center, Department of Immunology, Faculty of
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Medicine, Mazandaran University of Medical Sciences, Sari, Iran
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3- Department of Neurology, Buali Sina Hospital, Faculty of Medicine, Mazandaran University
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of Medical Sciences, Sari, Iran
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*Corresponding author:
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A. Rafiei, Mailing address: Faculty of Medicine, Mazandaran University of Medical Sciences, KM 18 Khazar Blvd, Khazar Sq. Sari, Iran, Phone: +98-11-33543088, Fax: +98-11-33543614, Email: [email protected].
ACCEPTED MANUSCRIPT Abstract
Background and aim: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Environmental and genetic factors play a key role in the development of the disease. Interleukin-32 (IL-32) is a cytokine inducing crucial inflammatory cytokines such as
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TNFα, IL-6, IL-1, and MIP-2. The present study was an attempt to reveal any association
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between IL-32 levels and C/T promoter SNP with susceptibility to MS.
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Methods: This case control study recruited a total of 304 subjects including 132 MS patients and
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172 sex- and age-matched healthy controls. Clinical and epidemiological characteristics of the RRMS, PPMS, and PPMS populations were assessed. Serum levels and C/T polymorphism of
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IL-32 were determined by ELISA and RFLP-PCR methods, respectively.
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Results: Serum levels of IL-32 were significantly different between MS patients and controls. IL-32 was dramatically higher in the patients than that healthy controls (2297.4 ± 280.2 ver.
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712.9 ± 90.2, p=0.001). C allele was prominent in MS patients than the controls and might
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increase the risk of MS up to 1.6 fold (95% CI; 1.02-2.4, p=0.038). In addition, the presence of C
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allele enhanced IL-32 production drastically. Conclusion: This is the first study in which IL-32 gene promoter C/T polymorphism and its
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serum levels were investigated. The increase in serum levels of IL-32 in accordance with additive effect of the presence of C allele in MS patients might introduce IL-32 as a key player in MS pathogenesis or immunedysregulation.
Keywords: IL-32; Multiple sclerosis; Polymorphism; RRMS; SPMS
ACCEPTED MANUSCRIPT Introduction Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease which occurs due to demyelination of the central nervous cells [1]. MS is the most common disabling neurological disease in young and middle-aged people. Most people with MS have an age range between 30
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and 40 years. The outbreak of this disease in Iran is about 53 cases per 100000 people [1] but
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considering that more than half of Iran's population are people under 35 years, epidemiological
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studies show that the prevalence of the disease has increased in recent years [2]. Clinical signs
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and symptoms include: fatigue, disability, chills, severe paralysis, loss of balance, and numbness, etc [3].The incidence of MS in people who share the same gene or siblings with affected parents,
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is about 3-5%. This rate in identical twins is 25% that reflects the role of genetic factors in susceptibility to the disease [4]. However, the etiology of the disease remains unknown [1] and
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in need of further research.
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For a long time it was thought that autoreactive CD4+ T lymphocytes producing IFN-, are the
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key mediators of MS, but recent studies have shown that other cells like Th17 generating proinflammatory cytokines such as IL-1, IL-6 and IL-17, play an important role in the pathology
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of the disease [4]. Interleukin (IL-32) is a proinflammatory cytokine first found in activated
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natural killer (NK) cells and T lymphocytes and was named NK4 [5]. Recently, interleukin-32 (IL-32) has been known as a critical molecule in the pathophysiology of chronic inflammatory diseases of the immune system [6]. IL-32 is produced by T, NK, monocytes, endothelial and epithelial cells [7]. It stimulates the production of some proinflammatory cytokines such as: IL13, IL-12, IL-6 and TNF-α from dendritic and inflammatory cells that in turn leads to Th1 and Th17 activation [6]. IL-32 gene is located on chromosome 16 p13.3 containing eight exons and six isoforms. These isoforms include alpha, beta, gamma, delta, epsilon and zeta [7, 8]. Among
ACCEPTED MANUSCRIPT them, IL-32 is the most abundant isoform [9]. Recent studies have demonstrated that the expression of this cytokine is increased in many infectious and non-infectious diseases such as acute lung injury [10], cancer [11] and systemic lupus erythematosus [12]. In addition, increased expression of this cytokine has been proven in autoimmune diseases such as rheumatoid arthritis
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[13] and IBD [14, 15]. Recently, several single nucleotide polymorphisms (SNP) in IL-32 gene
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have been studied, but until now the association between these interleukin polymorphisms and
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multiple sclerosis has not been reported. Therefore, given the involvement of IL-32 in
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inflammation and its association with inflammatory and infectious diseases, we hypothesized that C/T SNP within the IL-32 promoter and also its serum levels would be associated with
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susceptibility to or outcomes in patients with multiple sclerosis. In addition, the effect of this
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SNP on disease activity and severity is also considered.
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Materials and methods
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Study subjects
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In this case-control study, a total of 303 Caucasian origin people including 132 MS patients (108 females and 24 males; aged 32.3 ± 7.4 years) and 171 unrelated healthy controls (127 females
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and 42 males; aged 31.05 ± 8.6 years) were enrolled between March 2014 and September 2015 in Buali-Sina, the second affiliated hospital of Mazandaran University of Medical Sciences, Sari, Iran. MS was diagnosed based on McDonald criteria [16] and expanded disability status scale (EDSS) was used to evaluate MS status. All patients were responded to three times in a week intramuscular
injection
of
IFN-1a
(CinnoVex,
Cinagene
Company,
Iran)
treatment.
Demographic and clinical characteristics of study population were recorded in an appropriate questionnaire. Subjects with a family history of neurodegenerative or inherited diseases were
ACCEPTED MANUSCRIPT excluded from the study. Expanded disability status scale (EDSS) index, which indicated a range of 0-10, was established according to the Kurtzke Functional Systems Scores [17]. The progression index was defined as the last EDSS divided by the disease duration. The control group consisted of healthy volunteers matched to MS patients according to age, sex,
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localization, and ethnicity. None of them had a history of inflammatory or autoimmune diseases.
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Ethical approval
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The study protocol was approved by the Ethics Committee of Mazandaran University of Medical Sciences and written informed consent was obtained from all patients and healthy control
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subjects. The study was conducted in accordance with the Declaration of Helsinki.
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Blood sampling and IL-32 amplicon amplification
Blood samples were obtained from antecubital vein and were kept in test tubes without
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anticoagulant or in test tubes containing 50 mM EDTA anticoagulant. Serum was extracted from
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collected blood by centrifuging in 1500 g for 15 min and was stored at -80 C for further analysis. Genomic DNA was extracted using modified salting-out method and was stored at -20
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C for genotyping. The quality and quantity of extracted DNA were determined using agarose
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gel electrophoresis and spectrophotometric measurement of absorbance at 260 and 280 nm by
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the Genova nanospectrophotometer (JENWAY, UK). The flanking sequence of T/C SNP (rs45499297) was amplified using PCR. A pair of primers was designed using primer premier version 6 (Premier biosoft). To amplify a 445 bp fragment of IL-32 gene, the following primers were used (forward, 5'- GATTGCTGAGACCAGTGA-3' and reverse, 5'- TCTCTGAGCCCAGGAATG3'). Amplification was performed in the final volume of 20 μl containing 100 ng of the template DNA, 250 nM of each primer, and 10 μl of 2x master mix (Amplicon, Denmark). PCR
ACCEPTED MANUSCRIPT conditions were performed in a gradient thermocycler (Eppendorf, Germany). The thermal profile was as follows, an initial denaturation step at 95˚C for 3 min followed by 35 cycles of denaturation at 94 ˚C for 30 s, annealing at 62 ˚C for 30 s, and an extension at 72 ˚C for 45 s, with a final extension step at 72 ˚C for 5 min. Amplified products were visualized by 1 % (w/v)
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agarose gel electrophoresis in TBE buffer, then they were stained with Syber Green (0.5 μg/ml),
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and were photographed under UV transillumination (Figure 1). The assays were repeated for
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20% of the samples, and the results were 100% concordant. 5% of PCR products were subjected
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to sequencing after purification.
Figure 1: Interleukin-32 (IL-32) amplification . A representative image of an interleukin-32 (IL32) amplification is shown. The IL-32 was amplified by polymerase chain reaction.
RFLP analysis
ACCEPTED MANUSCRIPT IL-32 PCR products were digested with BamHI restriction enzyme (Thermo Fisher Scientific, Massachusetts, USA). Figure 2 shows primers and restriction enzyme locations on IL-32 gene sequence. The reactions were carried out in a total volume of 15 μl containing 2 μl of the PCR products, 1 U of BamHI and 2 μl of the provided (appropriate) buffer and then incubated at 37
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°C for 16 h. 10μl of each digested product was then analyzed on a 2% agarose gel in TBE buffer
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at 90 V for 30 min and visualized by UV illumination (UVITEC) after Syber Green staining. To
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estimate the size of the fragments, a100 bp DNA ladder (Fermentas, Germany) was used in the
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gels (Figure 3).
Figure 2 is shown primer sequences to amplify a fragment of IL-32 gene that contain rs45499297 IL-32 gene.
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polymorphism. Y letter shows a T/C variation which changes restriction site of BamH1 endonuclease on
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Figure 3 Interleukin-32 genotyping. A representative image of the results of an interleukin-32 (IL-32) genotyping assay is shown. PCR products were digested with BamH1. Products were then separated by agarose gel electrophoresis. The IL-32 TT genotype was evident as 306 and 139 bp fragments, CT as 445, 306 and 139 bp fragments, whereas, CC mutant genotype was not digested with BamH1.
Cytokine measurement Serum levels of IL-32 were quantified with a quantitative sandwich enzyme immunoassay using an ELISA DuoSet kit (R&D, CA). Briefly, the plates were coated by goat anti-human IL-32 antibody as the capture antibody for 16 hours at room temperature. Subsequently, 100 μl of standards or sera were added and the procedure was performed according to the manufacturer’s
ACCEPTED MANUSCRIPT instructions. Reference concentrations of IL-32 were used to prepare assay calibration. The absorption was determined with an ELISA reader (Biotek ELX800, USA) at 450 nm. The concentrations were interpolated from standard curves expressed in pg/ml. Inter- and intra-assay coefficients of variation were below 10%. To avoid any bias, all samples were analyzed blindly
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without knowledge of the clinical status. All samples were run in duplicate with the appropriate
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standards on Nunc MaxiSorb 96-well micro plates (Sigma-Aldrich, Germany).
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Statistical analysis
In order to evaluate the quantitative data, after getting ensured that the data were normally using
Kolmogrov-Smirnov
test,
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distributed
the
quantitative
were
evaluated
using
and the qualitative data were
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independent t-test or one-way analysis of variance (ANOVA)
data
assessed applying 2 or Fisher Exact tests, appropriately. The Hardy–Weinberg equilibrium of
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genotype distribution in the control and MS patients was tested by a goodness-of-fit 2 test. Co-
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dominant, dominant, and recessive models were used to show the effect of gene dose. Multivariate logistic regression analysis was used to assess any association in the gene dosage
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between the patients and the controls by calculating odds ratios and 95% confidence intervals (CIs). Associations between the genotypes and the risk of MS among the subgroups regarding by sex, age, family history, and MS forms were further evaluated by stratification. Statistical analysis was performed using the Statistical Package Program for the Social Sciences (SPSS 17). All statistical tests were two sided and the p-values less than 0.05 were considered to be statistically significant.
ACCEPTED MANUSCRIPT Results 3-1 Demographic and disease characteristics Demographic and clinical characteristics of MS patients and healthy controls are presented in Table 1.
MS has been classified into relapse remitting MS (RRMS), secondary progressive MS
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(SPMS), and primary progressive MS (PPMS). 132 MS patients consisted of 96 RRMS, 34
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SPMS, and 2 PPMS. Since PPMS patients were very few, we combined SPMS and PPMS
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(SPMS + PPMS) in further analysis. The average age of the patients was 32.3 ± 7.4 years that
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was fully matched with control individuals (31.05 ± 8.6 years). No significant difference was found between cases and controls with regard to sex, since 81.8% and 75% of the patients and
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controls were females, respectively. However, there was just a significant difference in two study populations with regard to positive family history of MS (P<0.0001).
There were no significant
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differences between RRMS and PPMS + SPMS patients in regard to age, sex, family history of
controls,
respectively,
but none of these differences were statistically significant.
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and
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MS and tobacco smoking (Table 1). The frequency of smoking was 7.6% and 12.5% in patients
Interestingly, disease-associated criteria such as age at the onset of disease diagnosis, EDSS
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score, progressive index, and relapse were not significantly different between RRMS and SPMS
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+ PPMS (P>0.05).
Table 1 Demographic and clinical characteristics of MS patients
P-
Controls
Total MS
P-value
RRMS
PPMS + SPMS
value
Age
31.05 ± 8.6
32.3 ± 7.4
0.201
32.3 ± 7.6
32.4 ± 6.9
0.901
Female – n (%)
128 (74.9)
108 (81.8)
0.164
83 (86.5)
25 (69.4)
0.025
Positive Family history of MS
2 (1.2)
19 (14.4)
<0.0001
16 (16.7)
3 (8.3)
0.176
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150 (87.7)
122 (92.4)
91 (94.8)
31 (86.1)
0.128
0.099
21 (12.3)
10 (7.6)
5 (5.2)
5 (13.9)
Fatigue
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72 (54.5)
49 (68.1)
23 (31.9)
0.130
Sexual dysfunction
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10 (7.6)
6 (60)
4 (40)
0.274
3.4 ± 0.62
0.131
27.7 ± 8
28.1 ± 7.3
0.774
1.49 ± 0.9
1.37 ± 0.8
0. 461
2.52 ± 0.77
3.88 ± 1.6
0.461
2.32 ± 1.82
2.39 ± 2.7
0.866
1.54 ± 0.5
1.49 ± 0.51
0.574
2300.1 ± 335.4
2290.0 ± 529.2
4.4 ± 0.45
Age at MSdiagnosis
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27.8 ± 7.8
EDSS score
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1.46 ± 0.9
Progressive index
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Total Number of relapses
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IL-32 (pg/ml)*
2.34 ± 2.07
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1.53 ± 0.5
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2.89 ± 0.72
712.9 ± 90.2
2297.4 ± 280.2
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No. of relapses in last 2 y
4.8 ± 0.5
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Duration of illness*
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0.001
0.980
% of total for each group is shown in parenthesis.
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Result in all quantitative variables were presented as mean ± SD, but strike- marked (*) variables represented mean ±SE.
2- Soluble levels of IL-32 in MS patients IL-32 is a proinflammatory cytokine that is expressed in a variety of cells and is also secreted into serum. The serum was evaluated levels of IL-32 in the two study groups. As it was shown in Table 1, serum levels of IL-32 were significantly higher in MS patients than healthy controls
ACCEPTED MANUSCRIPT (2297.4 ± 280.2 ver. 712.9 ± 90.2, p=0.001). However, there was no significant difference between RRMS and SPMS + PPMS (p=0.98). 2- The effect of IL-32 T/C genotypes on IL-32 production and MS occurrence
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In this study, T/C polymorphism (rs45499297) of IL-32 was successfully genotyped in all MS
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patients and healthy controls and the genotypes were subjected to DNA sequencing (fig. 4).
Figure 4 Chromatogram of IL-32 sequence. It shows the substitution of T to C which changes restriction site of BamH1 in mutant allele.
Distribution of genotypes in MS patients and controls was in accordance with those expected by Hardy-Weinberg equilibrium (P>0.05). As it was shown in Table 2, there were no profound significant differences in genotype frequency, and in co-dominant or C allele dominant manner, between MS patients and controls, although mutant homozygous genotype (C/C) was slightly
ACCEPTED MANUSCRIPT more common in patients than controls (5.3% ver. 1.7%, p=0.064). However, C allele frequency was more prominent in MS patients than controls (19.7% ver. 13.4%, p=0.038). When two study populations were adjusted according to cofounders such as family history of MS, age, and sex, multivariate logistic regression analysis revealed that the presence of C allele might impact the
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risk of disease susceptibility up to 1.6 fold ( 95% CI; 1.02-2.44). In order to determine the impact
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of gender on allelic distribution of T/C SNP, we determined allele and genotype frequencies
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between patients and controls of male and female, separately. There was no significant difference between allele and genotype frequencies of T/C polymorphism in IL-32 between the
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patient and control groups (data not shown).
CC+CT TT
Alleles
95% CI
P value
87 (65.9) 38 (28.8) 7 (5.3)
128 (74.9) 40 (23.4) 3 (1.7)
1 1.43 3.4
0.85-2.42 0.86-13.64
0.206 0.064
45 (34.1) 87 (65.9)
43 (25.2) 128 (74.8)
1.54
0.93-2.54
0.089
212 (80.3) 52 (19.7)
296 (86.6) 47 (13.4)
1.6
1.02-2.44
0.0384
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T C
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Dominant
TT CT CC
Controls n=171
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Co-dominant
MS n=132
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IL-32 T/C SNP
Model
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Table 2 Genotype and allele frequencies of T/C polymorphism of IL-32 in MS patients and controls.
In the next step, to reveal the effect of T/C SNP on IL-32 production among two study populations, we stratified the population according to T/C genotypes and compared IL-32 levels. According to T/C genotype default, in overall, there was a significant difference between MS patients and controls in IL-32 production (p=0.016). On the other hand, harboring CC genotype
ACCEPTED MANUSCRIPT significantly increased IL-32 levels in both groups (Fig 5).
In controls, Tukey’s post hoc test
revealed a significant difference in IL-32 production between CC harboring genotype and TT genotypes (1361.1 ± 432.9 ver. 548.3 ± 105.6, p=0.035). However, this significant value was not
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detected among any genotypes of IL-32 T/C SNP in MS patients (p>0.05).
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Figure 5 Serum levels of IL-32 in MS patients and control subjects according to T/C SNP. As seen in the picture, the IL-32 concentration in patients with MS compared with controls was higher in T allele subjects. Also
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(p=0.035).
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the levels of IL-32, just only significant in controls with C/C mutant genotype compared to T/T genotype
3- Association of IL-32 T/C genotypes with clinical findings As some MS symptoms are more prevalent in MS patients, this study aimed to determine any association between the occurrence of MS clinical findings and the presence of IL-32 genotypes.
ACCEPTED MANUSCRIPT Since the mean age at disease onset in our patients was lower than that worldwide, we assessed the impact of C allele on the age at disease occurrence. One way of analysis of variance (ANOVA) revealed a significance difference between age at disease onset and IL-32 T/C genotypes (p=0.041). On the other hand, Tukey’s Post Hoc test was shown a significant
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decreased in age at disease onset in CC genotype compared to CT genotype (20.7 ± 2.9 vs. 28.9
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± 7.8, p=0.031) (Figure 6). In addition, C allele carriage patients were significantly younger than
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the T allele carrier patients (p=0.018).
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Figure 6. The effect of T/C variation of IL-32 on the age at disease onset in MS patients. CC carrier patients were significantly younger than TC heterozygous MS patients.. In the next step, we combined C allele carriage (CC+CT) in order to clarify the association between the presence of C allele and occurrence of any MS symptom. Table 3 shows sensory, visual impairment and fatigue as more prominent symptoms, and depression and sexual dysfunction as were less common symptoms among MS patients. Comparison of MS symptoms in wild IL-32 genotype carriages with mutant C allele harboring subjects revealed no significant difference.
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Table 3 Association of IL-32 T/C polymorphism and MS symptoms CC+CT
TT
OR
95% CI
P-value
Visual impairment
72 (54.5)
29 (40.3)
43 (59.7)
1.8
0.86-3.8
0.081
Fatigue
72 (54.5)
28 (38.9)
44 (61.1)
1.6
0.77-3.4
0.138
38 (29)
9 (23.7)
29 (76.3)
0.51
Sensory
77 (58.3)
30 (39)
47 (61)
1.7
Pain
41 (31.1)
14 (34.1)
27 (65.9)
Movement disorientation
46 (35.1)
18 (39.1)
28 (60.9)
Bladder dysfunction
37 (28)
15 (40.5)
Sexual dysfunction
10 (7.6)
3 (30)
26 (19.7)
10 (38.5)
Depression
0.091
0.76-3.6
0.113
1.6
0.8-3.3
0.138
1.4
0.65-2.9
0.255
22 (59.5)
1.5
0.67-3.2
0.219
7 (70)
0.82
0.2-3.3
0.538
16 (61.5)
1.3
0.52-3.1
0.379
0.22-1.2
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Muscle plastic motor
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Total
Sign
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% of total for each group is shown in parenthesis
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Discussion
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RRMS: Relapse-remitting Multiple sclerosis, PPMS: Primary progressive multiple sclerosis, SPMS: Secondary progressive multiple sclerosis
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In this study, for the first time, we revealed the association of a T/C polymorphism in the promoter of IL-32 gene with increased MS susceptibility. Substitution of T to C might evolve into a change in genetic risk that would increase disease susceptibility up to 1.6 fold. In addition, this genetic variation may provide a situation that age of the disease is reduced.
An increased
production of IL-32 was seen in MS patients compared to healthy controls. The presence of C allele in promoter region of IL-32 has an additive effect on the cytokine production.
ACCEPTED MANUSCRIPT Despite the evolution of science and technology in recent years, MS has become one of the important clinical problems. Failure to identify various factors involved in disease mechanisms, unpredictability and lack of significant treatment, causing MS to be more considered by researchers. Like many developing countries, the prevalence of this disease is increasing in Iran
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[18]. The results showed that the severity of the disease is mild to moderate in most patients. So
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that the disability index of the patients in this study was 1.6, which is in accordance with the
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findings of other studies in Iran [19]. These findings suggest that the progression of the disease is mild in most patients with MS. Due to the patient registration system on the one hand and
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enhanced experience of physicians in the diagnosis of the disease on the other hand, the most of
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patients are diagnosed at early stages. The average age of patients in this study was lower than worldwide at the time of diagnosis which showed that the age of disease onset is reduced. This is
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caused by the multiple social and psychological factors and in some cases by stressors. In
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addition, the results demonstrated that females are four times more susceptible to have MS than the males. This finding is in line with other reports which have shown female to male ratio is
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higher in RRMS patients [20].
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In recent years, a lot of studies on the etiology and risk factors for MS susceptibility have been
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done which indicate the role of the immune system and inflammatory cytokines in this disease. IL-32, a pluripotent proinflammatory cytokine, may have a role in intensifying the inflammation. It has the double effects on inflammation through augmentation of secretion various inflammatory cytokines as well as enhancing cells migration to the site of inflammation [21]. It has fund that it could induce expression of IL-1𝛽, IL-6, IL-8, IL-21, and macrophage inflammatory protein-2 (MIP-2) expression by activating AP-1, NF-κβ, p38MAPK signal pathways, has a fundamental role on pathogenesis of various immune-mediated chronic
ACCEPTED MANUSCRIPT inflammatory diseases such as rheumatoid arthritis [22, 23] , myasthenia gravis [24], and cancer [24-28]. Increased serum levels of IL-32 in MS patients compared to healthy controls might dictate inflammatory status of MS and confirms this speculation. However, there are scarcely reports on the role of IL-32 in neurodegenerative disorders. Infection with various pathogens Recently,
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such as Epstein-Barr virus (EBV) has been reported to induce IL-32 expression [29].
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it has been demonstrated that EBV latent membrane protein 1 (LMP1) is a potent inducer of IL-
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32 expression [30]. Up to three-fold increase in IL-32 production in our MS patients might be
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interpreted by the role of EBV in augmenting IL-32 expression as it has been reported that EBV is an environmental risk factor for MS [31]. Subsequently, IL-32 may play a critical role in the
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initiation and perpetuation of the pathological processes characteristic of MS. However, the present findings were not in line with the only study reported by Wang et al in 2013 [32]. They
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compared serum levels of this cytokine in patients with NMO and MS, and found increased
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levels of IL-32 in NMO but not in MS patients. This discrepancy might be due to the study population and sampling, ethnicity, disease stage and severity, and the accuracy of measurement.
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IL-32 has more than six transcripts, of which IL-32α, IL-32β, and IL-32γ are more common
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transcripts in humans. Their sample size was too low and they only recruited RRMS and measured IL-32, whereas we measured all human potent IL-32 isoforms (, , ) in a large
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sample size which contained all MS forms. Also, each isoform, has a specific biological activity. Of these, IL-32β has been found to enhance expression of the adhesion molecules on the endothelial cells [21]. Gamma isoform is the most biological active isoform of IL-32. However, as this study measured all three the most common isoforms totally, it remained unclear which isoform contributed to the increased level of IL-32 in MS. Therefore, further are warranted to evaluate precise role of each IL-32 isoform in the pathogenesis of MS.
ACCEPTED MANUSCRIPT In addition to some environmental factors, there is an undeniable impact of genetic background on MS. The history of the disease in first-degree relatives and the percentage of patients in identical twins have confirmed this claim. In line with this literature background, our findings also showed that near 15% of MS-affected patients had positive familial history of the disease.
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These results might confirm the role of genetic factors in MS pathogenesis and its frequency is in
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agreement with other studies reported form Iran [33]. On the other hand, besides HLA-
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DRB1*1501 and DQB1* 0602 genes [18, 34, 35], non-HLA genes have been reported for contributing to susceptibility to MS [36]. Because of the role of inflammation in the initiation of
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demyelination, genetic variation in proinflammatory cytokines are the most interesting research
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field in case-control association studies. In this study, we found for the first time, that the presence of C allele in IL-32 promoter was associated with susceptibility to MS. On the other
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hand, C allele carriage may increase the risk of MS up to 1.6 fold. CC homozygous genotype
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was also marginally increased the risk of MS (OR: 3.4, 95% CI: 0.86-13.64; p=0.064). These findings are in agreement with other reports that showed the role of IL-32 SNPs in chronic
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inflammatory diseases such as RA and ankylosing spondylitis [37]. The role of IL-32 SNPs has
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been also demonstrated in acute lung injury [10], endometrial [38], and thyroid cancer [11].
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Genetic factors, such as SNPs, have been reported to affect the corresponding protein expression. In order to explore whether different IL-32 genotypes influence IL-32 production, association between IL-32 T/C SNP and the corresponding serum levels in MS patients and healthy controls were determined. Interestingly, there was a close direct association between the presence of C allele and serum levels of IL-32. In fact, C carriage in homozygous pattern (CC) enhances IL-32 levels more than two times compared to the background (in the absence of C allele) in control group. Although this increasing trend was seen in MS patients who harbored C allele in hetro- or
ACCEPTED MANUSCRIPT homozygous form,
it was not statistically significant. It is probably due to clinically
heterogeneous situation of MS patients who received various treatment regimens. As this SNP is located on IL-32 promoter, it may be a functional SNP. Therefore, higher prevalence of CC mutant genotype in MS patients compared to healthy controls emphasizes the role of IL-32 in
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MS pathogenesis. Association between the presence of C allele in IL-32 T/C SNP and reducing
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age of disease may be explained by the findings have shown that IL-32 stimulates NF-KB
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pathway via enhancing activation of metaloprotenase (MMP-) 2 and 9 [39]. The roles of MMP-2 and MMP-9 in pathogenesis of MS have been demonstrated through studies which showed these
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enzymes have ability to damage blood brain barrier integrity [40, 41]. Therefore, the presence
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high IL-32 producer genotype in MS patients is accordance by its ability to stimulate MMP-2 and 9 expression/ activation. However, there was no significant correlation between C allele and
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relapses in any forms of the disease. These findings may lead the speculation that IL-32 by
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enhancing inflammatory condition has a major role in induction phase of disease.
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Despite our interesting findings, the study has some limitations due to the fact that the majority of MS population recruited in this study was in the early of disease or had RRMS with a mild
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disability status (EDSS 1.5). Therefore, the results of this study need to be confirmed in large
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samples including all forms of MS, especially advanced forms. In general, to be the best of our knowledge, this is the first study in which IL-32 gene promoter T/C polymorphism has been investigated. The increase in serum levels of IL-32 in accordance with additive effect of the presence of C allele in MS patients might present IL-32 as a key player in MS pathogenesis
Acknowledgments
ACCEPTED MANUSCRIPT We thank all patients and healthy subjects who allowed us to conduct this study. We also have a great thanks to Maedeh Ghobadi for providing us an opportunity to review medical records of the patients. This work was MSc thesis of Morsaljahan Z and was supported by a
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grant from Research Affairs of Mazandaran University of Medical Sciences.
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Declaration of interest
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The authors report no declarations of interest.
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ACCEPTED MANUSCRIPT Highlights
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Multiple sclerosis patients had a higher chance of carrying C allele at IL-32 T/C SNP than controls Harboring C allele or having CC genotype of IL-32 is potentially associated to reduce age at onset. The presence of C allele significantly increases IL-32 expression/production in the patients or controls IL-32, a proinflammatory cytokine, may enhancing inflammatory condition and has a major role in induction phase of MS.
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Zaher Morsaljahan, Alireza Rafiei, Reza Valadan, Mahmoud Abedini, Masoumeh Paksersht, Rezvan Khajavi PII: DOI: Reference:
S0022-510X(17)30349-0 doi: 10.1016/j.jns.2017.05.045 JNS 15352
To appear in:
Journal of the Neurological Sciences
Received date: Revised date: Accepted date:
28 December 2016 29 April 2017 22 May 2017
Please cite this article as: Zaher Morsaljahan, Alireza Rafiei, Reza Valadan, Mahmoud Abedini, Masoumeh Paksersht, Rezvan Khajavi , Association between interleukin-32 polymorphism and multiple sclerosis. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jns(2017), doi: 10.1016/ j.jns.2017.05.045
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ACCEPTED MANUSCRIPT Association between Interleukin-32 polymorphism and Multiple Sclerosis Zaher Morsaljahan1, 2, Alireza Rafiei 2*, Reza Valadan2, Mahmoud Abedini 3, Masoumeh Paksersht1, Rezvan Khajavi2
1-M.Sc. Student of Immunology, Mazandaran University of Medical Sciences, Sari, Iran 2* -Molecular and Cell Biology Research Center, Department of Immunology, Faculty of
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Medicine, Mazandaran University of Medical Sciences, Sari, Iran
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3- Department of Neurology, Buali Sina Hospital, Faculty of Medicine, Mazandaran University
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of Medical Sciences, Sari, Iran
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*Corresponding author:
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A. Rafiei, Mailing address: Faculty of Medicine, Mazandaran University of Medical Sciences, KM 18 Khazar Blvd, Khazar Sq. Sari, Iran, Phone: +98-11-33543088, Fax: +98-11-33543614, Email: [email protected].
ACCEPTED MANUSCRIPT Abstract
Background and aim: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Environmental and genetic factors play a key role in the development of the disease. Interleukin-32 (IL-32) is a cytokine inducing crucial inflammatory cytokines such as
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TNFα, IL-6, IL-1, and MIP-2. The present study was an attempt to reveal any association
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between IL-32 levels and C/T promoter SNP with susceptibility to MS.
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Methods: This case control study recruited a total of 304 subjects including 132 MS patients and
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172 sex- and age-matched healthy controls. Clinical and epidemiological characteristics of the RRMS, PPMS, and PPMS populations were assessed. Serum levels and C/T polymorphism of
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IL-32 were determined by ELISA and RFLP-PCR methods, respectively.
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Results: Serum levels of IL-32 were significantly different between MS patients and controls. IL-32 was dramatically higher in the patients than that healthy controls (2297.4 ± 280.2 ver.
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712.9 ± 90.2, p=0.001). C allele was prominent in MS patients than the controls and might
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increase the risk of MS up to 1.6 fold (95% CI; 1.02-2.4, p=0.038). In addition, the presence of C
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allele enhanced IL-32 production drastically. Conclusion: This is the first study in which IL-32 gene promoter C/T polymorphism and its
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serum levels were investigated. The increase in serum levels of IL-32 in accordance with additive effect of the presence of C allele in MS patients might introduce IL-32 as a key player in MS pathogenesis or immunedysregulation.
Keywords: IL-32; Multiple sclerosis; Polymorphism; RRMS; SPMS
ACCEPTED MANUSCRIPT Introduction Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease which occurs due to demyelination of the central nervous cells [1]. MS is the most common disabling neurological disease in young and middle-aged people. Most people with MS have an age range between 30
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and 40 years. The outbreak of this disease in Iran is about 53 cases per 100000 people [1] but
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considering that more than half of Iran's population are people under 35 years, epidemiological
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studies show that the prevalence of the disease has increased in recent years [2]. Clinical signs
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and symptoms include: fatigue, disability, chills, severe paralysis, loss of balance, and numbness, etc [3].The incidence of MS in people who share the same gene or siblings with affected parents,
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is about 3-5%. This rate in identical twins is 25% that reflects the role of genetic factors in susceptibility to the disease [4]. However, the etiology of the disease remains unknown [1] and
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in need of further research.
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For a long time it was thought that autoreactive CD4+ T lymphocytes producing IFN-, are the
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key mediators of MS, but recent studies have shown that other cells like Th17 generating proinflammatory cytokines such as IL-1, IL-6 and IL-17, play an important role in the pathology
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of the disease [4]. Interleukin (IL-32) is a proinflammatory cytokine first found in activated
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natural killer (NK) cells and T lymphocytes and was named NK4 [5]. Recently, interleukin-32 (IL-32) has been known as a critical molecule in the pathophysiology of chronic inflammatory diseases of the immune system [6]. IL-32 is produced by T, NK, monocytes, endothelial and epithelial cells [7]. It stimulates the production of some proinflammatory cytokines such as: IL13, IL-12, IL-6 and TNF-α from dendritic and inflammatory cells that in turn leads to Th1 and Th17 activation [6]. IL-32 gene is located on chromosome 16 p13.3 containing eight exons and six isoforms. These isoforms include alpha, beta, gamma, delta, epsilon and zeta [7, 8]. Among
ACCEPTED MANUSCRIPT them, IL-32 is the most abundant isoform [9]. Recent studies have demonstrated that the expression of this cytokine is increased in many infectious and non-infectious diseases such as acute lung injury [10], cancer [11] and systemic lupus erythematosus [12]. In addition, increased expression of this cytokine has been proven in autoimmune diseases such as rheumatoid arthritis
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[13] and IBD [14, 15]. Recently, several single nucleotide polymorphisms (SNP) in IL-32 gene
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have been studied, but until now the association between these interleukin polymorphisms and
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multiple sclerosis has not been reported. Therefore, given the involvement of IL-32 in
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inflammation and its association with inflammatory and infectious diseases, we hypothesized that C/T SNP within the IL-32 promoter and also its serum levels would be associated with
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susceptibility to or outcomes in patients with multiple sclerosis. In addition, the effect of this
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SNP on disease activity and severity is also considered.
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Materials and methods
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Study subjects
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In this case-control study, a total of 303 Caucasian origin people including 132 MS patients (108 females and 24 males; aged 32.3 ± 7.4 years) and 171 unrelated healthy controls (127 females
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and 42 males; aged 31.05 ± 8.6 years) were enrolled between March 2014 and September 2015 in Buali-Sina, the second affiliated hospital of Mazandaran University of Medical Sciences, Sari, Iran. MS was diagnosed based on McDonald criteria [16] and expanded disability status scale (EDSS) was used to evaluate MS status. All patients were responded to three times in a week intramuscular
injection
of
IFN-1a
(CinnoVex,
Cinagene
Company,
Iran)
treatment.
Demographic and clinical characteristics of study population were recorded in an appropriate questionnaire. Subjects with a family history of neurodegenerative or inherited diseases were
ACCEPTED MANUSCRIPT excluded from the study. Expanded disability status scale (EDSS) index, which indicated a range of 0-10, was established according to the Kurtzke Functional Systems Scores [17]. The progression index was defined as the last EDSS divided by the disease duration. The control group consisted of healthy volunteers matched to MS patients according to age, sex,
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localization, and ethnicity. None of them had a history of inflammatory or autoimmune diseases.
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Ethical approval
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The study protocol was approved by the Ethics Committee of Mazandaran University of Medical Sciences and written informed consent was obtained from all patients and healthy control
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subjects. The study was conducted in accordance with the Declaration of Helsinki.
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Blood sampling and IL-32 amplicon amplification
Blood samples were obtained from antecubital vein and were kept in test tubes without
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anticoagulant or in test tubes containing 50 mM EDTA anticoagulant. Serum was extracted from
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collected blood by centrifuging in 1500 g for 15 min and was stored at -80 C for further analysis. Genomic DNA was extracted using modified salting-out method and was stored at -20
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C for genotyping. The quality and quantity of extracted DNA were determined using agarose
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gel electrophoresis and spectrophotometric measurement of absorbance at 260 and 280 nm by
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the Genova nanospectrophotometer (JENWAY, UK). The flanking sequence of T/C SNP (rs45499297) was amplified using PCR. A pair of primers was designed using primer premier version 6 (Premier biosoft). To amplify a 445 bp fragment of IL-32 gene, the following primers were used (forward, 5'- GATTGCTGAGACCAGTGA-3' and reverse, 5'- TCTCTGAGCCCAGGAATG3'). Amplification was performed in the final volume of 20 μl containing 100 ng of the template DNA, 250 nM of each primer, and 10 μl of 2x master mix (Amplicon, Denmark). PCR
ACCEPTED MANUSCRIPT conditions were performed in a gradient thermocycler (Eppendorf, Germany). The thermal profile was as follows, an initial denaturation step at 95˚C for 3 min followed by 35 cycles of denaturation at 94 ˚C for 30 s, annealing at 62 ˚C for 30 s, and an extension at 72 ˚C for 45 s, with a final extension step at 72 ˚C for 5 min. Amplified products were visualized by 1 % (w/v)
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agarose gel electrophoresis in TBE buffer, then they were stained with Syber Green (0.5 μg/ml),
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and were photographed under UV transillumination (Figure 1). The assays were repeated for
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20% of the samples, and the results were 100% concordant. 5% of PCR products were subjected
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to sequencing after purification.
Figure 1: Interleukin-32 (IL-32) amplification . A representative image of an interleukin-32 (IL32) amplification is shown. The IL-32 was amplified by polymerase chain reaction.
RFLP analysis
ACCEPTED MANUSCRIPT IL-32 PCR products were digested with BamHI restriction enzyme (Thermo Fisher Scientific, Massachusetts, USA). Figure 2 shows primers and restriction enzyme locations on IL-32 gene sequence. The reactions were carried out in a total volume of 15 μl containing 2 μl of the PCR products, 1 U of BamHI and 2 μl of the provided (appropriate) buffer and then incubated at 37
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°C for 16 h. 10μl of each digested product was then analyzed on a 2% agarose gel in TBE buffer
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at 90 V for 30 min and visualized by UV illumination (UVITEC) after Syber Green staining. To
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estimate the size of the fragments, a100 bp DNA ladder (Fermentas, Germany) was used in the
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gels (Figure 3).
Figure 2 is shown primer sequences to amplify a fragment of IL-32 gene that contain rs45499297 IL-32 gene.
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polymorphism. Y letter shows a T/C variation which changes restriction site of BamH1 endonuclease on
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Figure 3 Interleukin-32 genotyping. A representative image of the results of an interleukin-32 (IL-32) genotyping assay is shown. PCR products were digested with BamH1. Products were then separated by agarose gel electrophoresis. The IL-32 TT genotype was evident as 306 and 139 bp fragments, CT as 445, 306 and 139 bp fragments, whereas, CC mutant genotype was not digested with BamH1.
Cytokine measurement Serum levels of IL-32 were quantified with a quantitative sandwich enzyme immunoassay using an ELISA DuoSet kit (R&D, CA). Briefly, the plates were coated by goat anti-human IL-32 antibody as the capture antibody for 16 hours at room temperature. Subsequently, 100 μl of standards or sera were added and the procedure was performed according to the manufacturer’s
ACCEPTED MANUSCRIPT instructions. Reference concentrations of IL-32 were used to prepare assay calibration. The absorption was determined with an ELISA reader (Biotek ELX800, USA) at 450 nm. The concentrations were interpolated from standard curves expressed in pg/ml. Inter- and intra-assay coefficients of variation were below 10%. To avoid any bias, all samples were analyzed blindly
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without knowledge of the clinical status. All samples were run in duplicate with the appropriate
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standards on Nunc MaxiSorb 96-well micro plates (Sigma-Aldrich, Germany).
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Statistical analysis
In order to evaluate the quantitative data, after getting ensured that the data were normally using
Kolmogrov-Smirnov
test,
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distributed
the
quantitative
were
evaluated
using
and the qualitative data were
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independent t-test or one-way analysis of variance (ANOVA)
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assessed applying 2 or Fisher Exact tests, appropriately. The Hardy–Weinberg equilibrium of
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genotype distribution in the control and MS patients was tested by a goodness-of-fit 2 test. Co-
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dominant, dominant, and recessive models were used to show the effect of gene dose. Multivariate logistic regression analysis was used to assess any association in the gene dosage
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between the patients and the controls by calculating odds ratios and 95% confidence intervals (CIs). Associations between the genotypes and the risk of MS among the subgroups regarding by sex, age, family history, and MS forms were further evaluated by stratification. Statistical analysis was performed using the Statistical Package Program for the Social Sciences (SPSS 17). All statistical tests were two sided and the p-values less than 0.05 were considered to be statistically significant.
ACCEPTED MANUSCRIPT Results 3-1 Demographic and disease characteristics Demographic and clinical characteristics of MS patients and healthy controls are presented in Table 1.
MS has been classified into relapse remitting MS (RRMS), secondary progressive MS
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(SPMS), and primary progressive MS (PPMS). 132 MS patients consisted of 96 RRMS, 34
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SPMS, and 2 PPMS. Since PPMS patients were very few, we combined SPMS and PPMS
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(SPMS + PPMS) in further analysis. The average age of the patients was 32.3 ± 7.4 years that
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was fully matched with control individuals (31.05 ± 8.6 years). No significant difference was found between cases and controls with regard to sex, since 81.8% and 75% of the patients and
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controls were females, respectively. However, there was just a significant difference in two study populations with regard to positive family history of MS (P<0.0001).
There were no significant
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differences between RRMS and PPMS + SPMS patients in regard to age, sex, family history of
controls,
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but none of these differences were statistically significant.
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and
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MS and tobacco smoking (Table 1). The frequency of smoking was 7.6% and 12.5% in patients
Interestingly, disease-associated criteria such as age at the onset of disease diagnosis, EDSS
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score, progressive index, and relapse were not significantly different between RRMS and SPMS
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+ PPMS (P>0.05).
Table 1 Demographic and clinical characteristics of MS patients
P-
Controls
Total MS
P-value
RRMS
PPMS + SPMS
value
Age
31.05 ± 8.6
32.3 ± 7.4
0.201
32.3 ± 7.6
32.4 ± 6.9
0.901
Female – n (%)
128 (74.9)
108 (81.8)
0.164
83 (86.5)
25 (69.4)
0.025
Positive Family history of MS
2 (1.2)
19 (14.4)
<0.0001
16 (16.7)
3 (8.3)
0.176
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150 (87.7)
122 (92.4)
91 (94.8)
31 (86.1)
0.128
0.099
21 (12.3)
10 (7.6)
5 (5.2)
5 (13.9)
Fatigue
-
72 (54.5)
49 (68.1)
23 (31.9)
0.130
Sexual dysfunction
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10 (7.6)
6 (60)
4 (40)
0.274
3.4 ± 0.62
0.131
27.7 ± 8
28.1 ± 7.3
0.774
1.49 ± 0.9
1.37 ± 0.8
0. 461
2.52 ± 0.77
3.88 ± 1.6
0.461
2.32 ± 1.82
2.39 ± 2.7
0.866
1.54 ± 0.5
1.49 ± 0.51
0.574
2300.1 ± 335.4
2290.0 ± 529.2
4.4 ± 0.45
Age at MSdiagnosis
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27.8 ± 7.8
EDSS score
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1.46 ± 0.9
Progressive index
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Total Number of relapses
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IL-32 (pg/ml)*
2.34 ± 2.07
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1.53 ± 0.5
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2.89 ± 0.72
712.9 ± 90.2
2297.4 ± 280.2
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No. of relapses in last 2 y
4.8 ± 0.5
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Duration of illness*
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Yes
0.001
0.980
% of total for each group is shown in parenthesis.
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Result in all quantitative variables were presented as mean ± SD, but strike- marked (*) variables represented mean ±SE.
2- Soluble levels of IL-32 in MS patients IL-32 is a proinflammatory cytokine that is expressed in a variety of cells and is also secreted into serum. The serum was evaluated levels of IL-32 in the two study groups. As it was shown in Table 1, serum levels of IL-32 were significantly higher in MS patients than healthy controls
ACCEPTED MANUSCRIPT (2297.4 ± 280.2 ver. 712.9 ± 90.2, p=0.001). However, there was no significant difference between RRMS and SPMS + PPMS (p=0.98). 2- The effect of IL-32 T/C genotypes on IL-32 production and MS occurrence
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In this study, T/C polymorphism (rs45499297) of IL-32 was successfully genotyped in all MS
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patients and healthy controls and the genotypes were subjected to DNA sequencing (fig. 4).
Figure 4 Chromatogram of IL-32 sequence. It shows the substitution of T to C which changes restriction site of BamH1 in mutant allele.
Distribution of genotypes in MS patients and controls was in accordance with those expected by Hardy-Weinberg equilibrium (P>0.05). As it was shown in Table 2, there were no profound significant differences in genotype frequency, and in co-dominant or C allele dominant manner, between MS patients and controls, although mutant homozygous genotype (C/C) was slightly
ACCEPTED MANUSCRIPT more common in patients than controls (5.3% ver. 1.7%, p=0.064). However, C allele frequency was more prominent in MS patients than controls (19.7% ver. 13.4%, p=0.038). When two study populations were adjusted according to cofounders such as family history of MS, age, and sex, multivariate logistic regression analysis revealed that the presence of C allele might impact the
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risk of disease susceptibility up to 1.6 fold ( 95% CI; 1.02-2.44). In order to determine the impact
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of gender on allelic distribution of T/C SNP, we determined allele and genotype frequencies
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between patients and controls of male and female, separately. There was no significant difference between allele and genotype frequencies of T/C polymorphism in IL-32 between the
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patient and control groups (data not shown).
CC+CT TT
Alleles
95% CI
P value
87 (65.9) 38 (28.8) 7 (5.3)
128 (74.9) 40 (23.4) 3 (1.7)
1 1.43 3.4
0.85-2.42 0.86-13.64
0.206 0.064
45 (34.1) 87 (65.9)
43 (25.2) 128 (74.8)
1.54
0.93-2.54
0.089
212 (80.3) 52 (19.7)
296 (86.6) 47 (13.4)
1.6
1.02-2.44
0.0384
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Dominant
TT CT CC
Controls n=171
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Co-dominant
MS n=132
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IL-32 T/C SNP
Model
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Table 2 Genotype and allele frequencies of T/C polymorphism of IL-32 in MS patients and controls.
In the next step, to reveal the effect of T/C SNP on IL-32 production among two study populations, we stratified the population according to T/C genotypes and compared IL-32 levels. According to T/C genotype default, in overall, there was a significant difference between MS patients and controls in IL-32 production (p=0.016). On the other hand, harboring CC genotype
ACCEPTED MANUSCRIPT significantly increased IL-32 levels in both groups (Fig 5).
In controls, Tukey’s post hoc test
revealed a significant difference in IL-32 production between CC harboring genotype and TT genotypes (1361.1 ± 432.9 ver. 548.3 ± 105.6, p=0.035). However, this significant value was not
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detected among any genotypes of IL-32 T/C SNP in MS patients (p>0.05).
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Figure 5 Serum levels of IL-32 in MS patients and control subjects according to T/C SNP. As seen in the picture, the IL-32 concentration in patients with MS compared with controls was higher in T allele subjects. Also
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(p=0.035).
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the levels of IL-32, just only significant in controls with C/C mutant genotype compared to T/T genotype
3- Association of IL-32 T/C genotypes with clinical findings As some MS symptoms are more prevalent in MS patients, this study aimed to determine any association between the occurrence of MS clinical findings and the presence of IL-32 genotypes.
ACCEPTED MANUSCRIPT Since the mean age at disease onset in our patients was lower than that worldwide, we assessed the impact of C allele on the age at disease occurrence. One way of analysis of variance (ANOVA) revealed a significance difference between age at disease onset and IL-32 T/C genotypes (p=0.041). On the other hand, Tukey’s Post Hoc test was shown a significant
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decreased in age at disease onset in CC genotype compared to CT genotype (20.7 ± 2.9 vs. 28.9
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± 7.8, p=0.031) (Figure 6). In addition, C allele carriage patients were significantly younger than
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the T allele carrier patients (p=0.018).
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Figure 6. The effect of T/C variation of IL-32 on the age at disease onset in MS patients. CC carrier patients were significantly younger than TC heterozygous MS patients.. In the next step, we combined C allele carriage (CC+CT) in order to clarify the association between the presence of C allele and occurrence of any MS symptom. Table 3 shows sensory, visual impairment and fatigue as more prominent symptoms, and depression and sexual dysfunction as were less common symptoms among MS patients. Comparison of MS symptoms in wild IL-32 genotype carriages with mutant C allele harboring subjects revealed no significant difference.
ACCEPTED MANUSCRIPT
Table 3 Association of IL-32 T/C polymorphism and MS symptoms CC+CT
TT
OR
95% CI
P-value
Visual impairment
72 (54.5)
29 (40.3)
43 (59.7)
1.8
0.86-3.8
0.081
Fatigue
72 (54.5)
28 (38.9)
44 (61.1)
1.6
0.77-3.4
0.138
38 (29)
9 (23.7)
29 (76.3)
0.51
Sensory
77 (58.3)
30 (39)
47 (61)
1.7
Pain
41 (31.1)
14 (34.1)
27 (65.9)
Movement disorientation
46 (35.1)
18 (39.1)
28 (60.9)
Bladder dysfunction
37 (28)
15 (40.5)
Sexual dysfunction
10 (7.6)
3 (30)
26 (19.7)
10 (38.5)
Depression
0.091
0.76-3.6
0.113
1.6
0.8-3.3
0.138
1.4
0.65-2.9
0.255
22 (59.5)
1.5
0.67-3.2
0.219
7 (70)
0.82
0.2-3.3
0.538
16 (61.5)
1.3
0.52-3.1
0.379
0.22-1.2
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Muscle plastic motor
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% of total for each group is shown in parenthesis
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Discussion
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RRMS: Relapse-remitting Multiple sclerosis, PPMS: Primary progressive multiple sclerosis, SPMS: Secondary progressive multiple sclerosis
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In this study, for the first time, we revealed the association of a T/C polymorphism in the promoter of IL-32 gene with increased MS susceptibility. Substitution of T to C might evolve into a change in genetic risk that would increase disease susceptibility up to 1.6 fold. In addition, this genetic variation may provide a situation that age of the disease is reduced.
An increased
production of IL-32 was seen in MS patients compared to healthy controls. The presence of C allele in promoter region of IL-32 has an additive effect on the cytokine production.
ACCEPTED MANUSCRIPT Despite the evolution of science and technology in recent years, MS has become one of the important clinical problems. Failure to identify various factors involved in disease mechanisms, unpredictability and lack of significant treatment, causing MS to be more considered by researchers. Like many developing countries, the prevalence of this disease is increasing in Iran
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[18]. The results showed that the severity of the disease is mild to moderate in most patients. So
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that the disability index of the patients in this study was 1.6, which is in accordance with the
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findings of other studies in Iran [19]. These findings suggest that the progression of the disease is mild in most patients with MS. Due to the patient registration system on the one hand and
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enhanced experience of physicians in the diagnosis of the disease on the other hand, the most of
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patients are diagnosed at early stages. The average age of patients in this study was lower than worldwide at the time of diagnosis which showed that the age of disease onset is reduced. This is
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caused by the multiple social and psychological factors and in some cases by stressors. In
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addition, the results demonstrated that females are four times more susceptible to have MS than the males. This finding is in line with other reports which have shown female to male ratio is
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higher in RRMS patients [20].
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In recent years, a lot of studies on the etiology and risk factors for MS susceptibility have been
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done which indicate the role of the immune system and inflammatory cytokines in this disease. IL-32, a pluripotent proinflammatory cytokine, may have a role in intensifying the inflammation. It has the double effects on inflammation through augmentation of secretion various inflammatory cytokines as well as enhancing cells migration to the site of inflammation [21]. It has fund that it could induce expression of IL-1𝛽, IL-6, IL-8, IL-21, and macrophage inflammatory protein-2 (MIP-2) expression by activating AP-1, NF-κβ, p38MAPK signal pathways, has a fundamental role on pathogenesis of various immune-mediated chronic
ACCEPTED MANUSCRIPT inflammatory diseases such as rheumatoid arthritis [22, 23] , myasthenia gravis [24], and cancer [24-28]. Increased serum levels of IL-32 in MS patients compared to healthy controls might dictate inflammatory status of MS and confirms this speculation. However, there are scarcely reports on the role of IL-32 in neurodegenerative disorders. Infection with various pathogens Recently,
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such as Epstein-Barr virus (EBV) has been reported to induce IL-32 expression [29].
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it has been demonstrated that EBV latent membrane protein 1 (LMP1) is a potent inducer of IL-
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32 expression [30]. Up to three-fold increase in IL-32 production in our MS patients might be
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interpreted by the role of EBV in augmenting IL-32 expression as it has been reported that EBV is an environmental risk factor for MS [31]. Subsequently, IL-32 may play a critical role in the
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initiation and perpetuation of the pathological processes characteristic of MS. However, the present findings were not in line with the only study reported by Wang et al in 2013 [32]. They
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compared serum levels of this cytokine in patients with NMO and MS, and found increased
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levels of IL-32 in NMO but not in MS patients. This discrepancy might be due to the study population and sampling, ethnicity, disease stage and severity, and the accuracy of measurement.
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IL-32 has more than six transcripts, of which IL-32α, IL-32β, and IL-32γ are more common
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transcripts in humans. Their sample size was too low and they only recruited RRMS and measured IL-32, whereas we measured all human potent IL-32 isoforms (, , ) in a large
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sample size which contained all MS forms. Also, each isoform, has a specific biological activity. Of these, IL-32β has been found to enhance expression of the adhesion molecules on the endothelial cells [21]. Gamma isoform is the most biological active isoform of IL-32. However, as this study measured all three the most common isoforms totally, it remained unclear which isoform contributed to the increased level of IL-32 in MS. Therefore, further are warranted to evaluate precise role of each IL-32 isoform in the pathogenesis of MS.
ACCEPTED MANUSCRIPT In addition to some environmental factors, there is an undeniable impact of genetic background on MS. The history of the disease in first-degree relatives and the percentage of patients in identical twins have confirmed this claim. In line with this literature background, our findings also showed that near 15% of MS-affected patients had positive familial history of the disease.
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These results might confirm the role of genetic factors in MS pathogenesis and its frequency is in
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agreement with other studies reported form Iran [33]. On the other hand, besides HLA-
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DRB1*1501 and DQB1* 0602 genes [18, 34, 35], non-HLA genes have been reported for contributing to susceptibility to MS [36]. Because of the role of inflammation in the initiation of
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demyelination, genetic variation in proinflammatory cytokines are the most interesting research
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field in case-control association studies. In this study, we found for the first time, that the presence of C allele in IL-32 promoter was associated with susceptibility to MS. On the other
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hand, C allele carriage may increase the risk of MS up to 1.6 fold. CC homozygous genotype
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was also marginally increased the risk of MS (OR: 3.4, 95% CI: 0.86-13.64; p=0.064). These findings are in agreement with other reports that showed the role of IL-32 SNPs in chronic
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inflammatory diseases such as RA and ankylosing spondylitis [37]. The role of IL-32 SNPs has
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been also demonstrated in acute lung injury [10], endometrial [38], and thyroid cancer [11].
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Genetic factors, such as SNPs, have been reported to affect the corresponding protein expression. In order to explore whether different IL-32 genotypes influence IL-32 production, association between IL-32 T/C SNP and the corresponding serum levels in MS patients and healthy controls were determined. Interestingly, there was a close direct association between the presence of C allele and serum levels of IL-32. In fact, C carriage in homozygous pattern (CC) enhances IL-32 levels more than two times compared to the background (in the absence of C allele) in control group. Although this increasing trend was seen in MS patients who harbored C allele in hetro- or
ACCEPTED MANUSCRIPT homozygous form,
it was not statistically significant. It is probably due to clinically
heterogeneous situation of MS patients who received various treatment regimens. As this SNP is located on IL-32 promoter, it may be a functional SNP. Therefore, higher prevalence of CC mutant genotype in MS patients compared to healthy controls emphasizes the role of IL-32 in
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MS pathogenesis. Association between the presence of C allele in IL-32 T/C SNP and reducing
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age of disease may be explained by the findings have shown that IL-32 stimulates NF-KB
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pathway via enhancing activation of metaloprotenase (MMP-) 2 and 9 [39]. The roles of MMP-2 and MMP-9 in pathogenesis of MS have been demonstrated through studies which showed these
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enzymes have ability to damage blood brain barrier integrity [40, 41]. Therefore, the presence
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high IL-32 producer genotype in MS patients is accordance by its ability to stimulate MMP-2 and 9 expression/ activation. However, there was no significant correlation between C allele and
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relapses in any forms of the disease. These findings may lead the speculation that IL-32 by
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enhancing inflammatory condition has a major role in induction phase of disease.
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Despite our interesting findings, the study has some limitations due to the fact that the majority of MS population recruited in this study was in the early of disease or had RRMS with a mild
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disability status (EDSS 1.5). Therefore, the results of this study need to be confirmed in large
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samples including all forms of MS, especially advanced forms. In general, to be the best of our knowledge, this is the first study in which IL-32 gene promoter T/C polymorphism has been investigated. The increase in serum levels of IL-32 in accordance with additive effect of the presence of C allele in MS patients might present IL-32 as a key player in MS pathogenesis
Acknowledgments
ACCEPTED MANUSCRIPT We thank all patients and healthy subjects who allowed us to conduct this study. We also have a great thanks to Maedeh Ghobadi for providing us an opportunity to review medical records of the patients. This work was MSc thesis of Morsaljahan Z and was supported by a
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grant from Research Affairs of Mazandaran University of Medical Sciences.
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Declaration of interest
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The authors report no declarations of interest.
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Multiple sclerosis patients had a higher chance of carrying C allele at IL-32 T/C SNP than controls Harboring C allele or having CC genotype of IL-32 is potentially associated to reduce age at onset. The presence of C allele significantly increases IL-32 expression/production in the patients or controls IL-32, a proinflammatory cytokine, may enhancing inflammatory condition and has a major role in induction phase of MS.
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