Association of interleukin (IL)-4 gene intron 3 VNTR polymorphism with multiple sclerosis in Turkish population

Human Immunology xxx (2013) xxx–xxx

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Association of interleukin (IL)-4 gene intron 3 VNTR polymorphism with multiple sclerosis in Turkish population Nevin Karakus a,⇑, Serbulent Yigit a,⇑, Gulsum Semiha Kurt b, Betul Cevik b, Osman Demir c, Omer Ates a a

Gaziosmanpasa University, Faculty of Medicine, Department of Medical Biology, Tokat, Turkey Gaziosmanpasa University, Faculty of Medicine, Department of Neurology, Tokat, Turkey c Gaziosmanpasa University, Faculty of Medicine, Department of Biostatistics, Tokat, Turkey b

a r t i c l e

i n f o

Article history: Received 17 December 2012 Accepted 29 May 2013 Available online xxxx

a b s t r a c t Objective: Genetic risk factors are known to contribute to the etiology of multiple sclerosis (MS). Interleukin (IL)-4 gene polymorphisms have been associated with immune-mediated diseases. The aim of this study was to explore the frequency of IL-4 gene intron 3 VNTR (variable number tandem repeat) polymorphism in a cohort of Turkish patients with MS. Methods: The study included 125 patients with MS and 160 healthy controls. Genomic DNA was isolated and genotyped using polymerase chain reaction (PCR) analyses for the IL-4 gene intron 3 VNTR polymorphism. Results: The distribution of genotype and allele frequencies of IL-4 gene intron 3 VNTR polymorphism was statistically different between MS patients and control group (p = 0.003 and p = 0.002, respectively). There were no statistically significant association between IL-4 VNTR polymorphism and clinical and demographical characteristics of MS patients. Conclusion: The results of this study suggest that intron 3 VNTR polymorphism of the IL-4 gene was positively associated with predisposition to develop MS in Turkish population. Ó 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

1. Introduction Multiple sclerosis (MS) is a chronic neurodegenerative autoimmune disease of the central nervous system (CNS) characterized by inflammation, demyelination and axonal degeneration, and is believed to arise from complex interactions of both environmental and genetic risk factors [1]. Disease onset usually occurs in young adults, and it is more common in women [2]. Cytokines play a crucial role in the increasing prevalence of inflammatory diseases by an altered balance between Th1 and Th2 immune response. It has been long-established that the complex balance between Th1 and Th2 cytokines of the immune system plays an important role in the pathogenesis of MS [3]. Thus, the genes of Th2 cytokines are attractive candidates in susceptibility or disease modification or both in MS. Interleukin (IL)-4 is a typical Th2 cytokine with decisive significance in regulating Th1/Th2 cytokine balance [4,5]. In addition, IL-4 also inhibits the synthesis of interferon-c, a cytokine known to exert a number of deleterious effects in MS. ⇑ Corresponding authors. Address: Gaziosmanpasa University, Faculty of Medicine, Department of Medical Biology, 60100 Tokat, Turkey. Fax: +90 356 2133179. E-mail addresses: [email protected] (N. Karakus), [email protected] (S. Yigit).

Polymorphisms in IL-4 gene have been reported in patients with immune mediated disorders [6]. The IL-4 gene, 10 kb in size, consisting of four exons and three introns, is located on the long arm of chromosome 5 (q23–31) in a cluster of other cytokine genes (IL-3, IL-5, IL-9, IL-13, and IL-15, granulocyte colony-stimulating factor, and interferon regulatory factor) [7]. This gene contains a 70-bp variable number of tandem repeat (VNTR) polymorphism located in intron 3 associated with IL-4 production. Regarding this VNTR polymorphism, two alleles are observed: one with a 70-bp deletion and one with a 70-bp insertion which are also referred to as P1 and P2, respectively [8]. IL-4 gene VNTR P2P2 genotype is correlated with lower IL-4 concentrations. Some studies have shown evidences supporting that the P1 allele induced higher expression of IL-4 than the P2 allele and that the P2 allele may be a protective factor in some disease [8–10]. Immunologic diseases of rheumatoid arthritis (RA), immune thrombocytopenic purpura, end-stage renal disease (ESRD), systemic lupus erythematosus (SLE), bladder cancer, severe malaria (SM) type-2 diabetes, vitiligo and periodontitis were associated with IL-4 intron 3 VNTR polymorphism [10–18]. Association between MS and IL-4 gene intron 3 VNTR polymorphism has been studied in different populations [8,19,20]. However, there is no report about the relationship of IL-4 gene intron 3 VNTR polymorphism and MS in Turkish population up to now. The aim of the

0198-8859/$36.00 - see front matter Ó 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humimm.2013.05.011

Please cite this article in press as: Karakus N et al. Association of interleukin (IL)-4 gene intron 3 VNTR polymorphism with multiple sclerosis in Turkish population. Hum Immunol (2013), http://dx.doi.org/10.1016/j.humimm.2013.05.011

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N. Karakus et al. / Human Immunology xxx (2013) xxx–xxx

present study was to analyze the association of IL-4 VNTR polymorphism with MS susceptibility and disease progression on MS patients in Turkish population. 2. Materials and methods 2.1. Study population The study population comprised 125 unrelated MS patients (mean age 36.86 ± 9.626 years; 34 males, 91 females) who were registered at the outpatient clinic of the Neurology Department at Gaziosmanpasa Medical Faculty and they all fulfilled the 2005 Revised McDonald multiple sclerosis criteria for classification [21]. A total of 160 unrelated hospital-based healthy subjects (mean age 36.45 ± 8.06 years; 56 males, 104 females) were recruited consecutively. All participants, patients and healthy controls, were of Turkish origin, from the inner Central Black Sea region of Turkey. All participants, patients and healthy controls, were of Turkish origin, from inner Central Black Sea region of Turkey. The healthy controls matched for age and gender with MS patients (p = 0.496 and p = 0.199, respectively) (Table 1) and free from another inflammatory-demyelinating disease. The protocol of this study was approved by the Institutional Ethics Committee, and all participants gave written informed consent before entering the study. 2.2. Genotyping DNA was extracted from 2 ml venous blood according to kit procedure (Sigma, USA) and stored at 20 °C. To detect 70 bp VNTR polymorphism (rs8179190) of IL-4 gene, polymerase chain reaction (PCR) assay as described by Mout et al. was used [22]. PCR was performed with a 25 ll reaction mixture containing 50 ng DNA, 0.8 lM of each primer, 200 lM of each dNTP, 2.5 mM MgCl2, 0.5 U Taq polymerase, 10 KCl buffer (MBI, Fermentas). Amplification was carried out using primers F 50 AGG CTG AAA GGG GGA Table 1 The demographic characteristics of patients with MS and healthy controls. Demographic characteristics

Miraine patients n = 125

Healthy controls n = 160

p

Age, mean ± SD (years) Gender, male/female, n (%)

36.86 ± 9.626 34/91 (27.2/72.8)

36.15 ± 8.015 56/104 (35.0/65.0)

0.496 0.199

Data were analyzed by analysis of variance and v2 test. MS = Multiple sclerosis, SD = standard deviation.

AAG C-30 , R 50 -CTG TTC ACC TCA ACT GCT CC-30 with initial denaturation at 95 °C for 5 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 45 s, extension at 72 °C for 1 min and final extension at 72 °C for 10 min. The PCR products were visualized on 3% agarose gel stained with ethidium bromide. PCR product was of 183 bp for P1 allele and 253 bp for P2 allele. In order to validate the accuracy and reproducibility of this method, each PCR reaction was included internal controls for each genotype. 2.3. Statistical analysis Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS 13.0) and the OpenEpi Info software package version 3.01 (www.openepi.com). Results were given as mean ± standard deviation (S.D.). The Chi-square (v2) test was used to evaluate the Hardy–Weinberg equilibrium for the distribution of the genotypes of the patients and the controls. Post power test was performed and the power was detected as 0.86. The relationships between IL-4 VNTR polymorphism and the clinical and demographics features were analyzed by using v2 test or analysis of variance (ANOVA) statistics. v2 test and Fisher’s exact test were used to compare categorical variables appropriately, and odds ratio (OR) and 95% confidence interval (CI) were used for the assessment of risk factors. All p values were 2-tailed and p values less than 0.05 were considered as significant. In addition, the generalized odds ratio (ORG) was calculated for VNTR polymorphism [23]. Bonferroni correction was used for the genotype based analyses between patients and controls. 3. Results Baseline clinical and demographics features of the study patients with MS were shown in Table 2. Gender, age, age of onset, disease duration, EDSS (expanded disability status scale) score, mean times to reach EDSS 3 and 6, MS types, BDI (Beck Depression Inventory) score, RLS (restless legs syndrome) and family history of MS patients were analyzed. Age and gender were not different between patient and control groups (p = 0.496 and 0.199, respectively). However BDI and RLS were significantly different between patient and control groups (p 6 0.0001 and p = 0.001, respectively). The three genotypes of intron 3 VNTR polymorphism of IL-4 gene were classified as: P1P1 (183 bp–two 70 bp repeat allele), P2P2 (253 bp–three 70 bp repeat allele) and P1P2 (both 183 and 253 bp fragments). The frequencies of P1P1, P2P2, and P1P2 genotypes of intron 3 VNTR polymorphism in the patients were 1.6%, 73.6%, and 24.8%; in the controls were 1.3%, 88.8%, and

Table 2 Clinical and demographical characteristics of patients stratified according to IL-4 gene intron 3 VNTR polymorphism. Characteristics

Total n = 125

P2P2 n = 92

P1P2 + P1P1 n = 33

p value

Gender, male/female, n (%) Age (years) Age of onset (years) Disease duration (years) EDSS score (years) Mean time to reach EDSS 3 (years) (n = 76) Mean time to reach EDSS 6 (years) (n = 49) MS types, n (%) RRMS + SPMS PPMS BDI score (years) Restless legs Syndrome (RLS), n (%) Family history, n (%)

34/91 (27.2/72.8) 36.86 ± 9.626 29.26 ± 8.865 7.58 ± 6.661 3.06 ± 2.015 5.91 ± 5.637 10.18 ± 7.325

24/68 (26.1/73.9) 36.45 ± 9.945 29.31 ± 8.775 7.22 ± 6.707 2.96 ± 1.925 5.32 ± 5.469 10.04 ± 7.424

10/23 (30.3/69.7) 38.03 ± 8.709 29.42 ± 9.247 8.59 ± 6.526 3.33 ± 2.256 7.45 ± 5.912 10.58 ± 7.317

0.653 0.419 0.904 0.312 0.359 0.141 0.827 1.000

121 (96.8) 4 (3.2) 16.70 ± 11.552 27/98 (27.6) 5 (4.0)

89 (73.6) 3 (75.0) 16.80 ± 11.893 19 (70.4) 4 (80.0)

32 (26.4) 1 (25.0) 16.42 ± 10.665 8 (29.6) 1 (20.0)

0.889 0.599 1.000

Data were analyzed by analysis of variance and v2 test. Mean plus standard deviation values are presented for all variables, except gender, family history, MS types and RLS. MS, multiple sclerosis; EDSS, expanded disability status scale; RRMS, relapsing-remitting; SPMS, secondary progressive; PPMS, primary progressive; BDI, Beck depression inventory.

Please cite this article in press as: Karakus N et al. Association of interleukin (IL)-4 gene intron 3 VNTR polymorphism with multiple sclerosis in Turkish population. Hum Immunol (2013), http://dx.doi.org/10.1016/j.humimm.2013.05.011

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N. Karakus et al. / Human Immunology xxx (2013) xxx–xxx Table 3 Genotype and allele frequencies of intron 3 VNTR of IL-4 gene in patient and control groups. IL-4 (intron 3 VNTR)

MS patients, n = 125

Healthy controls, n = 160

p

ORG (95% CI); OR (CI 95%)

Genotypes P2P2 P1P2 P1P1 P2P2 + P1P2 : P1P1 P2P2: P1P2 + P1P1

92 (73.6%) 31 (24.8%) 2 (1.6%) 123:2 92:33

142 (88.8%) 16 (10.0%) 2 (1.3%) 158:2 142:18

0.003

ORG = 0.36 (0.20–0.68)

– 0.0009

– OR = 2.82 (1.51–5.39)

Alleles P2 P1

215 (86.0%) 35 (14.0%)

300 (93.7%) 20 (6.3%)

0.002

OR = 2.44 (1.38–4.41)

The results that are statistically significant are shown in boldface.

10.0%. P1 and P2 allele frequencies were 14.0% and 86.0% in patient group and 6.3% and 93.7% in control group (Table 3). The distribution of genotype and allele frequencies of IL-4 gene intron 3 VNTR polymorphism was statistically different between MS patients and control group (p = 0.003 and p = 0.002, respectively) (Table 3). The association of VNTR polymorphism remained significant after Bonferroni correction. There were no statistically significant association between IL-4 VNTR polymorphism and clinical and demographical characteristics of MS patients (Table 2). We analyzed the probability of MS patients to reach EDSS: 3 or 6 during the disease course in relation to the IL-4 genotypes and no association was found (Table 2). Intron 3 VNTR polymorphism in the IL-4 gene was found to be associated with MS susceptibility in Turkish population. The observed and expected frequencies of the polymorphism were in Hardy–Weinberg equilibrium in control group but not in patient group.

4. Discussion Our data showed that P1 allele of the IL-4 gene intron 3 VNTR polymorphism was associated with MS in Turkish population. Association between MS and IL-4 gene intron 3 VNTR polymorphism has been studied in different populations [8,19,20] and diverse results have been found. Vandenbroeck et al. (1997) found similar genotype frequencies of the IL-4 gene intron 3 VNTR polymorphism between 256 sporadic MS patients and 146 healthy controls in Italy [8]. When they separated the patients in 164 Caucasians and 92 Sardinians, heterozygote frequency was 24% controls vs. 15% patients in Sardinians and 19% controls vs. 24% patients in Caucasians. In the present study the heterozygote frequency was found 10.0% in controls and 24.8% in MS patients. Our results were similar with Caucasian population. However the heterozygote frequency in control group of Caucasian population was smaller than ours, maybe because of the small sample size (n = 64) of Caucasian population. Vandenbroeck et al. (1997) reported that the carriage rate of the P1 allele was associated with older age of onset. We did not replicate this result, as no associa-

tion could be found among the age of onset and any genotype in Turkey. Also, this was not confirmed in another study on sporadic American MS cases [19], however they found a trend for association between the homozygote state for this allele and older age of onset. Kantarci et al. (2003) showed that IL-4 gene intron 3 VNTR polymorphism was associated with susceptibility to MS (p = 0.004) due to minority of heterozygotes in patients (29/122; 23.8%) compared to controls (91/244; 37.3%) in a North American population [19]. Although Kantarci et al. (2003) found association between IL4 gene VNTR polymorphism and MS like our study; this was a bit different from our results. They found higher heterozygote frequency (37.3%) in control group, but we found higher heterozygote frequency (24.8%) in patient group. Their results suggested that carriage of P2 allele was associated with more severe disease. However our results suggest that P1 allele is associated with disease. The third study of Suppiah et al. (2005) did not confirm these findings in two other European populations (Spanish-Basque and Northern Irish) [20]. They did not find any association between IL-4 gene intron 3 VNTR polymorphism and MS. According to their results the heterozygote frequency was 23.11% (49/190) in patients and 20.51% (25/94) in controls in Spanish-Basque population; 22.9% in patients (68/267) and 18.45% (38/179) in controls in Northern Irish population. These studies from different populations showed that the frequency of P1P2 genotype of IL-4 gene intron 3 VNTR polymorphism demonstrated significant differences between populations. The results of our population were more similar to the results of Caucasian population. Even though the heterozygote frequencies of patient groups were higher than control groups in four populations (Spanish-Basque, Northern Irish, Caucasian and Turkish), the heterozygote frequencies of patient groups were lower than control groups in two populations (Sardinian and American). Although heterozygote frequencies of patient groups of these different populations were similar (except Sardinian), the heterozygote frequencies of control groups of these populations were very different. Three previous studies and the present study that investigated the relationship between IL-4 gene intron 3 VNTR polymorphism and MS are identified in Table 4. Overall analysis of the VNTR polymorphism in 961 patients and

Table 4 Characteristics of the individual studies included in the systematic review and overall analysis. Study (reference)

Vandenbroeck et al. (1997) [8] Kantarci et al. (2003) [19] Suppiah et al. (2005) [20] Present study Total studies

Population

Italy (total) (Caucasians and Sardinians) America Spanish-Basque and Northern Irish Turkish Overall

MS patients (P) control subjects (C)

256 146 122 458 125 961

(P) (C) (P) 244 (P) 273 (P) 160 (P) 823

(C) (C) (C) (C)

Genotype frequency N (%) P2P2

P1P2 + P1P1

199 (77.3) 114 (78.1) 88 (72.1) 151 (61.9) 336 (73.4) 206 (75.5) 92 (73.6) 142 (88.8) 715 (74.6) 613 (74.5)

55 (21.7) 32 (21.9) 34 (27.9) 93 (38.1) 122 (26.6) 67 (24.5) 33 (26.4) 18 (11.2) 244 (25.4) 210 (25.5)

p

0.9508 0.0522 0.5314 0.0009 0.9718

The results that are statistically significant are shown in boldface.

Please cite this article in press as: Karakus N et al. Association of interleukin (IL)-4 gene intron 3 VNTR polymorphism with multiple sclerosis in Turkish population. Hum Immunol (2013), http://dx.doi.org/10.1016/j.humimm.2013.05.011

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N. Karakus et al. / Human Immunology xxx (2013) xxx–xxx

823 controls revealed no significant association between MS and IL-4 gene VNTR polymorphism (p = 0.9718). So far results from genome wide association studies (GWAS) have not implicated IL4 polymorphisms in MS [24]. However, IL-4 gene could have been lost in GWAS and could be confirmed in a later GWAS. It was suggested that a distinct number of VNTR copies might affect the transcriptional activity of the IL-4 gene [25]. Some studies have shown evidences supporting that the P1 allele induced higher expression of IL-4 than the P2 allele and that the P1 allele may be a susceptibility factor in some diseases [8–10]. In the present study, the frequency of P1 allele, which was supposed to lead to overexpression of IL-4, was found higher in patients than control group. This is incompatible with the fact that intracellular IL-4 is decreased in patients with MS [26]. We suppose that, different from the other suggestions, P1 allele of IL-4 gene intron 3 VNTR polymorphism induce lower expression of IL-4 according to our results. In conclusion, although there are reports that evaluated the association of VNTR polymorphisms of IL-4 gene with MS, to our knowledge, this is the first association study that attempted to evaluate the association of IL-4 gene VNTR polymorphism on MS in Turkish population. Our findings suggest that IL-4 gene intron 3 VNTR polymorphism may affect the expression or function of the protein and might influence the susceptibility to or course and severity of MS. Given the limited knowledge of phenotypic effects of this polymorphism, we cannot easily explain this observation on a biological basis. As a result further studies on the role of IL-4 gene VNTR polymorphism in relation to MS are warranted with large cohort on different ethnic backgrounds. Furthermore, the role and effect of IL-4 gene VNTR polymorphism needs to be confirmed through in vivo expression on animal studies so that the function of each allele could be explained and understood.

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[9]

[10]

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Please cite this article in press as: Karakus N et al. Association of interleukin (IL)-4 gene intron 3 VNTR polymorphism with multiple sclerosis in Turkish population. Hum Immunol (2013), http://dx.doi.org/10.1016/j.humimm.2013.05.011