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Association between fibromyalgia syndrome and polymorphism of the IL-4 gene in a Turkish population
Gene 527 (2013) 62–64
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Association between fibromyalgia syndrome and polymorphism of the IL-4 gene in a Turkish population Serbulent Yigit a,⁎, Ahmet Inanir b, Akın Tekcan c, Sema Inanir d, Sengul Tural c, Omer Ates a a
Gaziosmanpasa University, Faculty of Medicine, Department of Medical Biology, Tokat, Turkey Gaziosmanpasa University, Faculty of Medicine, Department of Physical Therapy and Rehabilitation, Tokat, Turkey Ondokuz Mayis University, Faculty of Medicine, Department of Medical Biology, Samsun, Turkey d Gaziosmanpasa University, Faculty of Medicine, Department of Psychiatry, Tokat, Turkey b c
a r t i c l e
i n f o
Article history: Accepted 3 April 2013 Available online 2 May 2013 Keywords: Fibromyalgia syndrome Interleukin-4 gene (IL-4) Polymorphism
a b s t r a c t Purpose: Fibromyalgia (FM) syndrome is a form of non-articular rheumatism characterized by long term and widespread musculoskeletal pain, morning stiffness, sleep disturbance, paresthesia, and pressure hyperalgesia at characteristic sites, called soft tissue tender points. The etiology of FM is still obscure. Genetic factors may predispose individuals to FM. Cytokines may play a role in the pathophysiology of FM. The aim of this study was to investigate the interleukin-4 (IL-4) 70 bp VNTR variations in Turkish patients with FM and evaluate if there was an association with clinical features, especially between these polymorphisms. Methods: The study included 300 patients with FM and 270 healthy controls. Genomic DNA was isolated and genotyped using polymerase chain reaction (PCR) for the IL-4 gene 70 bp VNTR polymorphisms. Results: There was statistically significant difference between the groups with respect to IL-4 genotype distribution and allele frequencies (p b 0.0001). The homozygous P1P1 genotype and P1 allele were significantly higher in FM patients than in healthy controls (p = 0.04; OR: 3.25, 95% CI: 1–10, p b 0.0001; OR:4.84, 95% CI:3–7.7). There was not any difference between the groups respect to IL-4 genotype distribution and allele frequencies (p > 0.05) and clinical characteristics. Conclusion: Our findings suggest that there is an association of IL-4 gene 70 bp VNTR polymorphism with susceptibility of a person for development of FM. As a result, further studies are necessary to determine whether IL-4 may be a genetic marker for FM in the Turkish population. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Fibromyalgia (FM) syndrome is a form of non-articular rheumatism characterized by long term and widespread musculoskeletal pain, morning stiffness, sleep disturbance, paresthesia, and pressure hyperalgesia at characteristic sites, called soft tissue tender points (Barth et al., 2001; Gür et al., 2002; Ortega et al., 2009; Su et al., 2007). Chronic widespread pain is a common medical condition which is difficult to treat (Wolfe et al., 1995). One major subgroup of patients with chronic widespread pain is classified as having fibromyalgia (FM) according to the criteria of the American College of Rheumatology (ACR) (Üçeyler et al., 2006; Wolfe et al., 1990). FM
Abbreviations: FMF, Familial Mediterranean fever; MEFV, Mediterranean fever; EDTA, Ethylendiamine-tetraacetate; PCR, Polymerase chain reaction; RFLP, Restriction fragment length polymorphism; IL-4, Interleukin-4; FM, Fibromyalgia; ACR, American College of Rheumatology; CNS, Central nervous system; IL-1a, Interleukin 1a; IL-1b, Interleukin 1b; IL-2, Interleukin 2; IL-6, Interleukin 6; TNF-a, Tumor necrosis factor a; SD, Standard deviation; IL-8, Interleukin 8. ⁎ Corresponding author at: Gaziosmapasa University, Faculty of Medicine, Department of Medical Biology, 60100, Tokat, Turkey. Tel.: +90 356 2129500; fax: +90 356 2133179. E-mail address: [email protected] (S. Yigit). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.04.020
usually appears at the age of 30–40 years (after the age of 55 years is rare) (Ortega et al., 2009). FM syndrome consists of different subgroups: FM is categorized as primary or secondary FM. While primary FM does not reveal any definitive organic factor, underlying diseases of secondary FM, such as inflammatory rheumatic processes, are frequently diagnosed (Ortega et al., 2009). The prevalence of FM is estimated to be 2% of the population of industrialized Western countries and the US (Üçeyler et al., 2006; Wolfe et al., 1995). The etiology of FM is still obscure (Gür et al., 2002). Genetic factors may predispose individuals to FM. FM in family clusters suggests a genetic predisposition (Di Franco et al., 2010). Neurochemicals may have a key role in FM. In several controlled studies, it has been discovered that increased levels of the inflammatory transmitter Substance P are found in the spinal fluid of FM patients (Ortega et al., 2009; Su et al., 2007; Torpy et al., 2000; Wang et al., 2008; Wolfe et al., 1990). SP is closely associated with pain (Ortega et al., 2009). Recent data highlight the putative role of cytokines in the pathogenesis of FM (Di Franco et al., 2010; Üçeyler et al., 2006; Wang et al., 2008). Cytokines are immunomodulating proteins that have a wide range of biological activities. Cytokines have been identified as pro-inflammatory or antiinflammatory. The major pro-inflammatory cytokines are interleukin 1a (IL-1a), interleukin 1b (IL-1b), interleukin 2 (IL-2), interleukin 6 (IL-6),
S. Yigit et al. / Gene 527 (2013) 62–64
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and tumor necrosis factor a (TNF-a); the major anti-inflammatory cytokines are interleukin 4 (IL-4), interleukin 10 (IL-10), and interleukin 13 (IL-13). Cytokines affect the functions of the central nervous system (CNS) through autonomic, neuroendocrine, and behavioral mechanisms (Menzies and Lyon, 2010). IL-4 induces and up-regulates the transcription of μ- and δ-opioid receptors, one mechanism by which it produces hypoalgesia may be through the endogenous opioid system. Signals arising through physiologic stimulation of the opioid receptor system may be reduced in patients with low IL-4 levels, with subsequent increase in pain perception and the common relative opioid resistance seen in patients with chronic widespread pain (Üçeyler et al., 2006). IL-4 70 bp VNTR polymorphism have not yet sufficiently been investigated in FM patients. Therefore, we investigated whether the IL-4 gene 70 bp VNTR polymorphism might be involved in the pathogenesis of FM.
2.3. Statistical analysis
2. Materials and methods
Demographic variables and baseline characteristics of patients were given according to the presence (carrier) or absence (non-carrier) of the patients in Table 1. So many clinic and demographic features (gender, age, height, weight, BMI, sleep disturbances, fatigue, difficulty concentrating, headache, paresthesia, feeling of stiffness, feeling of swelling in soft tissues, morning fatigue, irritable bowel syndrome, dysmenorrhea, dryness of eye, Raynaud's syndrome, dysuria, restless legs, dryness of mouth) were analyzed. Among the demographic and clinical characteristic of FM patients, there were not found any statistically difference between carriers and non-carriers of IL-4 70 bp VNTR polymorphism (Table 2). The mean age ± standard deviation (SD) was 43.05 ± 10.64 in patients and 39.97 ± 12.17 in control group respectively. Table 2 presents the distribution of IL-4 70 bp VNTR polymorphism genotypes in patients and control groups. There were statistically significant differences of the IL-4 gene mutation carrier rates and allele frequencies between FM patients and healthy controls (p b 0.001). IL-4 gene P2 allele frequency was indicated as 80.16% in the patients and 94.25% in the control group. P1 allele was 19.83% in the patients
2.1. Study population This study included 300 FM patients (297 women, 3 men) and 270 controls (258 women, 12 men) recruited from the department of Physical Medicine and Rehabilitation, Gazi Osmanpaşa University in Tokat, Turkey. Informed consent was in accordance with the study protocol, approved by the ethics committee of the Medical Faculty. All patients fulfilled the American College of Rheumatology classification criteria for FM (widespread pain present for > 3 months, and tenderness in at least 11 of 18 tender point sites). All patients and controls signed a written consent form after being informed about the details of the study. A complete clinical evaluation was done for all patients. The controls were selected by excluding the diagnosis of FM. All the individuals in the control group were healthy. All participants, patients and healthy controls were of Turkish origin. Data collection sheet included information such as age, disease duration, deformed and tender joint counts. Individual features of patients with FM are summarized in Table 1.
Analysis of the data was performed using the computer software SPSS 15.0 (SPSS, Chicago, IL, USA) and OpenEpi Info software package program (www.openepi.com). Continuous data were given as mean ± SD (standard deviation) and (min–max). The frequencies of the alleles and genotypes (Hardy–Weinberg equilibrium) in patients and controls were compared with χ2analysis. Odds ratio (OR) and 95% confidence intervals (CIs) were calculated. p value less than 0.05 (two-tailed) were regarded as statistically significant. Power analysis was made by using Minitab 15.0 package program. Hardy–Weinberg equilibrium was assessed by chi-square analysis.
3. Results
Table 2 IL-4 genotype frequencies according to the clinical characteristics in FM patients.
2.2. Genotype determination DNA was extracted from 2 mL venous blood according to the kit procedure (Sigma, USA) and stored at − 20 °C. To detect 70 bp VNTR polymorphism of IL-4 gene PCR assay as described by Mout et al. (1991) was used. PCR was performed with a 25 μl 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, KCl buffer (MBI, Fermentas). Amplification was carried out using primers F. 5′ TAGGCTGAAAGGGGGAAAGC-3′, R. 5′-CTG TTCACCTCAACTGCTCC-3′ 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 included internal controls for each genotype. Second PCR was performed to confirm samples whose results were not clear. Also, to confirm the accuracy of the genotyping, repeated analysis was performed on randomly selected samples.
Clinical
IL-4 genotypes
Characteristics
Status
P1/P1
P1/P2
P2/P2
p value
Sleep disturbances
Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No
8 (2.7) 6 (2) 11 (3.7) 3 (1) 5 (1.7) 9 (3) 7 (2.3) 7 (2.3) 6 (2) 8 (2.7) 8 (2.7) 6 (2) 8 (2.7) 6 (2) 10 (3.3) 4 (1.3) 5 (1.7) 9 (3) 5 (1.7) 9 (3) 5 (1.7) 9 (3) 0 14 (4.7) 2 (0.7) 12 (4) 7 (2.3) 7 (2.3) 7 (2.3) 7 (2.3)
53 (17.7) 38 (12.7) 61 (20.3) 30 (10) 44 (14.7) 47(15.7) 66 (22) 25 (8.3) 19 (6.3) 22 (24) 46 (15.3) 45 (15) 45 (15) 46 (15.3) 65 (21.7) 26 (8.7) 42 (14) 49 (16.3) 21 (7.1) 69 (23.2) 28(9.3) 63 (21) 2 (0.7) 89 (29.7) 6 (2) 85 (28.3) 47 (15.7) 44 (14.7) 48 (16) 43 (14.3)
122 (40.7) 73 (24.3) 120 (40) 75 (25) 76 (25.3) 119(39.7) 135 (45) 60 (20) 41 (13.7) 154 (51.3) 109 (36.3) 86 (28.7) 108 (36) 87 (29) 140 (46.7) 55 (18.3) 77 (25.7) 118 (39.3) 49 (16.5) 144 (48.5) 71 (23.7) 124 (41.3) 15 (5) 180 (60) 21 (7) 174 (58) 96 (32) 99 (33) 104 (34.7) 91 (30.3)
0.74
Fatigue Concentration difficulties Headache Parasthesia Feeling of stiffness Feeling of swelling in the soft tissues Morning fatigue Irritable bowel syndrome Dysmenorrhea Dryness of eye
Table 1 Demographic variables and baseline characteristics of the patients.
Raynaud's syndrome
Characteristics
Study groups
Average age of patients (n = 300) Average weight of patients Average height of patients Body mass index
43.05 74.21 161.49 28.51
± ± ± ±
10.64 10.51 6.05 4.17
Dysuria Restless legs Dryness of mouth
0.33 0.29 0.23 0.15 0.68 0.62 0.99 0.51 0.61 0.64 0.11 0.45 0.93 0.97
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S. Yigit et al. / Gene 527 (2013) 62–64
Table 3 Distribution of IL-4 gene 70 bp VNTR polymorphism and allele frequencies between FM patients and controls. Genotype
Patients (n = 300) (%)
Controls (n = 270) (%)
P1P1 P1P2 P2P2 P1P1 + P1P2:P2P2 I P1P2 + P2P2:P1P1
14 (4.66) 91 (30.33) 195 (65) 105:195 14:286
4 (1.48) 23 (8.51) 243 (90) 27:243 4:266
Allele frequency P1 P2
119 (19.83) 481 (80.16)
31 (5.74) 509 (94.25)
a
χ2
p value
49.937a
b0.0001a
OR (95%CI)
b0.0001a 0.04a
4.84 (3.05–7.7) 3.25 (1–10)
b0.0001a
4.06 (2.68–6.14)
The results that are statistically significant.
and 5.74% in the control group (Table 3). It was concluded that IL-4 70 bp VNTR polymorphism may cause FM especially in P1 allele form.
FM in a Taiwanese population (Su et al., 2007). There was no concordance between our results and the results of the Su et al. study.
4. Discussion
5. Conclusion
The pathophysiology of fibromyalgia involves a number of factors, including abnormalities in the neuroendocrine and autonomic nervous systems, genetic factors, psychosocial variables, and environmental stressors (Bradley, 2009). Studies have been focused on roles of polymorphism of the genes in the serotoninergic, catecholaminergic, and dopaminergic systems in fibromyalgia (Lee et al., 2012). There are many studies that are investigating relationship between FM and cytokine. But, these studies have focused on proteins and their receptors. There are only a few studies that are investigating the association between FM and IL-4 70 bp VNTR polymorphisms. To our knowledge, this is the first study to evaluate simultaneously the prevalence of IL-4 VNTR polymorphisms among Turkish FM patients. The sources of inflammation triggering the FM syndrome remain to be elucidated. It has been proposed that FM is due to neurogenic inflammatory response to allergens, infectious agents, chemicals or emotional stress (Omoigui, 2007; Ortega et al., 2009). IL-4 is considered to be a pleiotropic anti-inflammatory cytokine produced by activated CD4-T cells, mast cells, eosinophils, and basophils. It affects T and B lymphocytes, natural killer cells, mast cells, synoviocytes, and endothelial cells, it activates and supports the growth of B cells, and triggers the production of IgG and IgE (Üçeyler et al., 2006). So, it may be said that IL-4 is enabled in inflammatory, allergic or infectious processes. It has been emphasized that the cytokines may play a role in the pathophysiology of FM in the different studies (Gür et al., 2002; Lee et al., 2012; Ortega et al., 2009; Üçeyler et al., 2006; Wang et al., 2008). In the present study, we analyzed the frequencies of 70 bp VNTR polymorphisms at intron 3 of anti-inflammatory cytokine IL-4 in FM patients in a Turkish population. Our result show that there was statistical significance between the groups with respect to IL-4 genotype distribution (p b 0.001) and allele frequencies (p b 0.001). This study indicates that the percentage of IL-4 70 bp VNTR polymorphism allele and the distribution of genotypes are significantly different between patients and controls. Wang et al. has reported that the IL-8 and TNF-α are increased, and that IL-4 and IL-10 are not different in the serum of FM patients (Wang et al., 2008). Gür et al. and Ortega et al. have published correlated results and observed increased serum IL-8 levels to compare with control groups in FM patients (Gür et al., 2002; Ortega et al., 2009). And, they concluded that IL-8 might be related to pain sensitization in FM patients. In another study, Uçeyler et al. have observed decrease of mRNA levels of IL-4 and IL-10, which are anti-inflammatory cytokines. Also, they indicated that the protein concentrations of IL-4 and IL-10 cytokines are decreased in FM patients (Üçeyler et al., 2006). In Su et al.'s single study assessing the relationship between IL-4 polymorphism and FM in the literature, they presented that IL-4 VNTR polymorphism was not shown to be associated with increased or decreased risk of
Our findings indicate that there is an association of IL-4 gene 70 bp VNTR polymorphism with susceptibility of a person to develop FM. Furthermore, 70 bp VNTR polymorphism of the IL-4 gene may constitute a risk for developing FM. Further studies with larger populations will be required to confirm these findings in different study populations. Conflict of interest We wish to draw the attention of the Editor to the following facts which may be considered as potential conflicts of interest and to significant financial contributions to this work. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. References Barth, H., Klein, R., Berg, P.A., 2001. L-tryptophan contaminant ‘peak E’ induces the release of IL-5 and IL-10 by peripheral blood mononuclear cells from patients with functional somatic syndromes. Clin. Exp. Immunol. 126 (2), 187–192 (Nov). Bradley, L.A., 2009. Pathophysiology of fibromyalgia. Am. J. Med. 122 (12 Suppl.), 22–30 (Dec). Di Franco, M., Iannuccelli, C., Valesini, G., 2010. Neuroendocrine immunology of fibromyalgia. Ann. N. Y. Acad. Sci. 1193, 84–90 (Apr). Gür, A., et al., 2002. Cytokines and depression in cases with fibromyalgia. J. Rheumatol. 29 (2), 358–361 (Feb). Lee, Y.H., Choi, S.J., Ji, J.D., Song, G.G., 2012. Candidate gene studies of fibromyalgia: a systematic review and meta-analysis. Rheumatol. Int. 32, 417–426. Menzies, V., Lyon, D.E., 2010. Integrated review of the association of cytokines with fibromyalgia and fibromyalgia core symptoms. Biol. Res. Nurs. 11 (4), 387–394 (Apr). Mout, R., Willemze, R., Landegent, J.E., 1991. Repeat polymorphisms in the interleukin4 gene. Nucleic Acids Res. 19, 3763. Omoigui, S., 2007. The biochemical origin of pain: the origin of all pain is inflammation and the inflammatory response. Part 2 of 3—inflammatory profile of pain syndromes. Med. Hypotheses 69 (6), 1169–1178. Ortega, E., et al., 2009. Exercise in fibromyalgia and related inflammatory disorders: known effects and unknown chances. Exerc. Immunol. Rev. 15, 42–65. Su, S.Y., Chen, J.J., Lai, C.C., Chen, C.M., Tsai, F.J., 2007. The association between fibromyalgia and polymorphism of monoamine oxidase A and interleukin-4. Clin. Rheumatol. 26 (1), 12–16 (Jan). Torpy, D.J., Papanicolaous, D.A., Lotsikas, A.J., Wilder, R.L., Chrousos, G.P., Pillemer, S.R., 2000. Responses of the sympathetic nervous system and the hypothalamicpituitary-adrenal axis to interleukin-6. Arthritis Rheum. 43, 872–880. Üçeyler, N., Valenza, R., Stock, M., Schedel, R., Sprotte, G., Sommer, C., 2006. Reduced levels of antiinflammatory cytokines in patients with chronic widespread pain. Arthritis Rheum. 54 (8), 2656–2664 (Aug). Wang, H., Moser, M., Schıltenwolf, M., Buchner, M., 2008. Circulating cytokine levels compared to pain in patients with fibromyalgia—a prospective longitudinal study over 6 months. J. Rheumatol. 35, 1366–1370. Wolfe, F., et al., 1990. The American College of Rheumatology 1990 criteria for the classification of fibromyalgia: report of the multicenter criteria committee. Arthritis Rheum. 33, 160–172. Wolfe, F., Ross, K., Anderson, J., Russell, I.J., Hebert, L., 1995. The prevalence and characteristics of fibromyalgia in the general population. Arthritis Rheum. 38, 19–28.
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Association between fibromyalgia syndrome and polymorphism of the IL-4 gene in a Turkish population Serbulent Yigit a,⁎, Ahmet Inanir b, Akın Tekcan c, Sema Inanir d, Sengul Tural c, Omer Ates a a
Gaziosmanpasa University, Faculty of Medicine, Department of Medical Biology, Tokat, Turkey Gaziosmanpasa University, Faculty of Medicine, Department of Physical Therapy and Rehabilitation, Tokat, Turkey Ondokuz Mayis University, Faculty of Medicine, Department of Medical Biology, Samsun, Turkey d Gaziosmanpasa University, Faculty of Medicine, Department of Psychiatry, Tokat, Turkey b c
a r t i c l e
i n f o
Article history: Accepted 3 April 2013 Available online 2 May 2013 Keywords: Fibromyalgia syndrome Interleukin-4 gene (IL-4) Polymorphism
a b s t r a c t Purpose: Fibromyalgia (FM) syndrome is a form of non-articular rheumatism characterized by long term and widespread musculoskeletal pain, morning stiffness, sleep disturbance, paresthesia, and pressure hyperalgesia at characteristic sites, called soft tissue tender points. The etiology of FM is still obscure. Genetic factors may predispose individuals to FM. Cytokines may play a role in the pathophysiology of FM. The aim of this study was to investigate the interleukin-4 (IL-4) 70 bp VNTR variations in Turkish patients with FM and evaluate if there was an association with clinical features, especially between these polymorphisms. Methods: The study included 300 patients with FM and 270 healthy controls. Genomic DNA was isolated and genotyped using polymerase chain reaction (PCR) for the IL-4 gene 70 bp VNTR polymorphisms. Results: There was statistically significant difference between the groups with respect to IL-4 genotype distribution and allele frequencies (p b 0.0001). The homozygous P1P1 genotype and P1 allele were significantly higher in FM patients than in healthy controls (p = 0.04; OR: 3.25, 95% CI: 1–10, p b 0.0001; OR:4.84, 95% CI:3–7.7). There was not any difference between the groups respect to IL-4 genotype distribution and allele frequencies (p > 0.05) and clinical characteristics. Conclusion: Our findings suggest that there is an association of IL-4 gene 70 bp VNTR polymorphism with susceptibility of a person for development of FM. As a result, further studies are necessary to determine whether IL-4 may be a genetic marker for FM in the Turkish population. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Fibromyalgia (FM) syndrome is a form of non-articular rheumatism characterized by long term and widespread musculoskeletal pain, morning stiffness, sleep disturbance, paresthesia, and pressure hyperalgesia at characteristic sites, called soft tissue tender points (Barth et al., 2001; Gür et al., 2002; Ortega et al., 2009; Su et al., 2007). Chronic widespread pain is a common medical condition which is difficult to treat (Wolfe et al., 1995). One major subgroup of patients with chronic widespread pain is classified as having fibromyalgia (FM) according to the criteria of the American College of Rheumatology (ACR) (Üçeyler et al., 2006; Wolfe et al., 1990). FM
Abbreviations: FMF, Familial Mediterranean fever; MEFV, Mediterranean fever; EDTA, Ethylendiamine-tetraacetate; PCR, Polymerase chain reaction; RFLP, Restriction fragment length polymorphism; IL-4, Interleukin-4; FM, Fibromyalgia; ACR, American College of Rheumatology; CNS, Central nervous system; IL-1a, Interleukin 1a; IL-1b, Interleukin 1b; IL-2, Interleukin 2; IL-6, Interleukin 6; TNF-a, Tumor necrosis factor a; SD, Standard deviation; IL-8, Interleukin 8. ⁎ Corresponding author at: Gaziosmapasa University, Faculty of Medicine, Department of Medical Biology, 60100, Tokat, Turkey. Tel.: +90 356 2129500; fax: +90 356 2133179. E-mail address: [email protected] (S. Yigit). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.04.020
usually appears at the age of 30–40 years (after the age of 55 years is rare) (Ortega et al., 2009). FM syndrome consists of different subgroups: FM is categorized as primary or secondary FM. While primary FM does not reveal any definitive organic factor, underlying diseases of secondary FM, such as inflammatory rheumatic processes, are frequently diagnosed (Ortega et al., 2009). The prevalence of FM is estimated to be 2% of the population of industrialized Western countries and the US (Üçeyler et al., 2006; Wolfe et al., 1995). The etiology of FM is still obscure (Gür et al., 2002). Genetic factors may predispose individuals to FM. FM in family clusters suggests a genetic predisposition (Di Franco et al., 2010). Neurochemicals may have a key role in FM. In several controlled studies, it has been discovered that increased levels of the inflammatory transmitter Substance P are found in the spinal fluid of FM patients (Ortega et al., 2009; Su et al., 2007; Torpy et al., 2000; Wang et al., 2008; Wolfe et al., 1990). SP is closely associated with pain (Ortega et al., 2009). Recent data highlight the putative role of cytokines in the pathogenesis of FM (Di Franco et al., 2010; Üçeyler et al., 2006; Wang et al., 2008). Cytokines are immunomodulating proteins that have a wide range of biological activities. Cytokines have been identified as pro-inflammatory or antiinflammatory. The major pro-inflammatory cytokines are interleukin 1a (IL-1a), interleukin 1b (IL-1b), interleukin 2 (IL-2), interleukin 6 (IL-6),
S. Yigit et al. / Gene 527 (2013) 62–64
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and tumor necrosis factor a (TNF-a); the major anti-inflammatory cytokines are interleukin 4 (IL-4), interleukin 10 (IL-10), and interleukin 13 (IL-13). Cytokines affect the functions of the central nervous system (CNS) through autonomic, neuroendocrine, and behavioral mechanisms (Menzies and Lyon, 2010). IL-4 induces and up-regulates the transcription of μ- and δ-opioid receptors, one mechanism by which it produces hypoalgesia may be through the endogenous opioid system. Signals arising through physiologic stimulation of the opioid receptor system may be reduced in patients with low IL-4 levels, with subsequent increase in pain perception and the common relative opioid resistance seen in patients with chronic widespread pain (Üçeyler et al., 2006). IL-4 70 bp VNTR polymorphism have not yet sufficiently been investigated in FM patients. Therefore, we investigated whether the IL-4 gene 70 bp VNTR polymorphism might be involved in the pathogenesis of FM.
2.3. Statistical analysis
2. Materials and methods
Demographic variables and baseline characteristics of patients were given according to the presence (carrier) or absence (non-carrier) of the patients in Table 1. So many clinic and demographic features (gender, age, height, weight, BMI, sleep disturbances, fatigue, difficulty concentrating, headache, paresthesia, feeling of stiffness, feeling of swelling in soft tissues, morning fatigue, irritable bowel syndrome, dysmenorrhea, dryness of eye, Raynaud's syndrome, dysuria, restless legs, dryness of mouth) were analyzed. Among the demographic and clinical characteristic of FM patients, there were not found any statistically difference between carriers and non-carriers of IL-4 70 bp VNTR polymorphism (Table 2). The mean age ± standard deviation (SD) was 43.05 ± 10.64 in patients and 39.97 ± 12.17 in control group respectively. Table 2 presents the distribution of IL-4 70 bp VNTR polymorphism genotypes in patients and control groups. There were statistically significant differences of the IL-4 gene mutation carrier rates and allele frequencies between FM patients and healthy controls (p b 0.001). IL-4 gene P2 allele frequency was indicated as 80.16% in the patients and 94.25% in the control group. P1 allele was 19.83% in the patients
2.1. Study population This study included 300 FM patients (297 women, 3 men) and 270 controls (258 women, 12 men) recruited from the department of Physical Medicine and Rehabilitation, Gazi Osmanpaşa University in Tokat, Turkey. Informed consent was in accordance with the study protocol, approved by the ethics committee of the Medical Faculty. All patients fulfilled the American College of Rheumatology classification criteria for FM (widespread pain present for > 3 months, and tenderness in at least 11 of 18 tender point sites). All patients and controls signed a written consent form after being informed about the details of the study. A complete clinical evaluation was done for all patients. The controls were selected by excluding the diagnosis of FM. All the individuals in the control group were healthy. All participants, patients and healthy controls were of Turkish origin. Data collection sheet included information such as age, disease duration, deformed and tender joint counts. Individual features of patients with FM are summarized in Table 1.
Analysis of the data was performed using the computer software SPSS 15.0 (SPSS, Chicago, IL, USA) and OpenEpi Info software package program (www.openepi.com). Continuous data were given as mean ± SD (standard deviation) and (min–max). The frequencies of the alleles and genotypes (Hardy–Weinberg equilibrium) in patients and controls were compared with χ2analysis. Odds ratio (OR) and 95% confidence intervals (CIs) were calculated. p value less than 0.05 (two-tailed) were regarded as statistically significant. Power analysis was made by using Minitab 15.0 package program. Hardy–Weinberg equilibrium was assessed by chi-square analysis.
3. Results
Table 2 IL-4 genotype frequencies according to the clinical characteristics in FM patients.
2.2. Genotype determination DNA was extracted from 2 mL venous blood according to the kit procedure (Sigma, USA) and stored at − 20 °C. To detect 70 bp VNTR polymorphism of IL-4 gene PCR assay as described by Mout et al. (1991) was used. PCR was performed with a 25 μl 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, KCl buffer (MBI, Fermentas). Amplification was carried out using primers F. 5′ TAGGCTGAAAGGGGGAAAGC-3′, R. 5′-CTG TTCACCTCAACTGCTCC-3′ 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 included internal controls for each genotype. Second PCR was performed to confirm samples whose results were not clear. Also, to confirm the accuracy of the genotyping, repeated analysis was performed on randomly selected samples.
Clinical
IL-4 genotypes
Characteristics
Status
P1/P1
P1/P2
P2/P2
p value
Sleep disturbances
Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No
8 (2.7) 6 (2) 11 (3.7) 3 (1) 5 (1.7) 9 (3) 7 (2.3) 7 (2.3) 6 (2) 8 (2.7) 8 (2.7) 6 (2) 8 (2.7) 6 (2) 10 (3.3) 4 (1.3) 5 (1.7) 9 (3) 5 (1.7) 9 (3) 5 (1.7) 9 (3) 0 14 (4.7) 2 (0.7) 12 (4) 7 (2.3) 7 (2.3) 7 (2.3) 7 (2.3)
53 (17.7) 38 (12.7) 61 (20.3) 30 (10) 44 (14.7) 47(15.7) 66 (22) 25 (8.3) 19 (6.3) 22 (24) 46 (15.3) 45 (15) 45 (15) 46 (15.3) 65 (21.7) 26 (8.7) 42 (14) 49 (16.3) 21 (7.1) 69 (23.2) 28(9.3) 63 (21) 2 (0.7) 89 (29.7) 6 (2) 85 (28.3) 47 (15.7) 44 (14.7) 48 (16) 43 (14.3)
122 (40.7) 73 (24.3) 120 (40) 75 (25) 76 (25.3) 119(39.7) 135 (45) 60 (20) 41 (13.7) 154 (51.3) 109 (36.3) 86 (28.7) 108 (36) 87 (29) 140 (46.7) 55 (18.3) 77 (25.7) 118 (39.3) 49 (16.5) 144 (48.5) 71 (23.7) 124 (41.3) 15 (5) 180 (60) 21 (7) 174 (58) 96 (32) 99 (33) 104 (34.7) 91 (30.3)
0.74
Fatigue Concentration difficulties Headache Parasthesia Feeling of stiffness Feeling of swelling in the soft tissues Morning fatigue Irritable bowel syndrome Dysmenorrhea Dryness of eye
Table 1 Demographic variables and baseline characteristics of the patients.
Raynaud's syndrome
Characteristics
Study groups
Average age of patients (n = 300) Average weight of patients Average height of patients Body mass index
43.05 74.21 161.49 28.51
± ± ± ±
10.64 10.51 6.05 4.17
Dysuria Restless legs Dryness of mouth
0.33 0.29 0.23 0.15 0.68 0.62 0.99 0.51 0.61 0.64 0.11 0.45 0.93 0.97
64
S. Yigit et al. / Gene 527 (2013) 62–64
Table 3 Distribution of IL-4 gene 70 bp VNTR polymorphism and allele frequencies between FM patients and controls. Genotype
Patients (n = 300) (%)
Controls (n = 270) (%)
P1P1 P1P2 P2P2 P1P1 + P1P2:P2P2 I P1P2 + P2P2:P1P1
14 (4.66) 91 (30.33) 195 (65) 105:195 14:286
4 (1.48) 23 (8.51) 243 (90) 27:243 4:266
Allele frequency P1 P2
119 (19.83) 481 (80.16)
31 (5.74) 509 (94.25)
a
χ2
p value
49.937a
b0.0001a
OR (95%CI)
b0.0001a 0.04a
4.84 (3.05–7.7) 3.25 (1–10)
b0.0001a
4.06 (2.68–6.14)
The results that are statistically significant.
and 5.74% in the control group (Table 3). It was concluded that IL-4 70 bp VNTR polymorphism may cause FM especially in P1 allele form.
FM in a Taiwanese population (Su et al., 2007). There was no concordance between our results and the results of the Su et al. study.
4. Discussion
5. Conclusion
The pathophysiology of fibromyalgia involves a number of factors, including abnormalities in the neuroendocrine and autonomic nervous systems, genetic factors, psychosocial variables, and environmental stressors (Bradley, 2009). Studies have been focused on roles of polymorphism of the genes in the serotoninergic, catecholaminergic, and dopaminergic systems in fibromyalgia (Lee et al., 2012). There are many studies that are investigating relationship between FM and cytokine. But, these studies have focused on proteins and their receptors. There are only a few studies that are investigating the association between FM and IL-4 70 bp VNTR polymorphisms. To our knowledge, this is the first study to evaluate simultaneously the prevalence of IL-4 VNTR polymorphisms among Turkish FM patients. The sources of inflammation triggering the FM syndrome remain to be elucidated. It has been proposed that FM is due to neurogenic inflammatory response to allergens, infectious agents, chemicals or emotional stress (Omoigui, 2007; Ortega et al., 2009). IL-4 is considered to be a pleiotropic anti-inflammatory cytokine produced by activated CD4-T cells, mast cells, eosinophils, and basophils. It affects T and B lymphocytes, natural killer cells, mast cells, synoviocytes, and endothelial cells, it activates and supports the growth of B cells, and triggers the production of IgG and IgE (Üçeyler et al., 2006). So, it may be said that IL-4 is enabled in inflammatory, allergic or infectious processes. It has been emphasized that the cytokines may play a role in the pathophysiology of FM in the different studies (Gür et al., 2002; Lee et al., 2012; Ortega et al., 2009; Üçeyler et al., 2006; Wang et al., 2008). In the present study, we analyzed the frequencies of 70 bp VNTR polymorphisms at intron 3 of anti-inflammatory cytokine IL-4 in FM patients in a Turkish population. Our result show that there was statistical significance between the groups with respect to IL-4 genotype distribution (p b 0.001) and allele frequencies (p b 0.001). This study indicates that the percentage of IL-4 70 bp VNTR polymorphism allele and the distribution of genotypes are significantly different between patients and controls. Wang et al. has reported that the IL-8 and TNF-α are increased, and that IL-4 and IL-10 are not different in the serum of FM patients (Wang et al., 2008). Gür et al. and Ortega et al. have published correlated results and observed increased serum IL-8 levels to compare with control groups in FM patients (Gür et al., 2002; Ortega et al., 2009). And, they concluded that IL-8 might be related to pain sensitization in FM patients. In another study, Uçeyler et al. have observed decrease of mRNA levels of IL-4 and IL-10, which are anti-inflammatory cytokines. Also, they indicated that the protein concentrations of IL-4 and IL-10 cytokines are decreased in FM patients (Üçeyler et al., 2006). In Su et al.'s single study assessing the relationship between IL-4 polymorphism and FM in the literature, they presented that IL-4 VNTR polymorphism was not shown to be associated with increased or decreased risk of
Our findings indicate that there is an association of IL-4 gene 70 bp VNTR polymorphism with susceptibility of a person to develop FM. Furthermore, 70 bp VNTR polymorphism of the IL-4 gene may constitute a risk for developing FM. Further studies with larger populations will be required to confirm these findings in different study populations. Conflict of interest We wish to draw the attention of the Editor to the following facts which may be considered as potential conflicts of interest and to significant financial contributions to this work. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. References Barth, H., Klein, R., Berg, P.A., 2001. L-tryptophan contaminant ‘peak E’ induces the release of IL-5 and IL-10 by peripheral blood mononuclear cells from patients with functional somatic syndromes. Clin. Exp. Immunol. 126 (2), 187–192 (Nov). Bradley, L.A., 2009. Pathophysiology of fibromyalgia. Am. J. Med. 122 (12 Suppl.), 22–30 (Dec). Di Franco, M., Iannuccelli, C., Valesini, G., 2010. Neuroendocrine immunology of fibromyalgia. Ann. N. Y. Acad. Sci. 1193, 84–90 (Apr). Gür, A., et al., 2002. Cytokines and depression in cases with fibromyalgia. J. Rheumatol. 29 (2), 358–361 (Feb). Lee, Y.H., Choi, S.J., Ji, J.D., Song, G.G., 2012. Candidate gene studies of fibromyalgia: a systematic review and meta-analysis. Rheumatol. Int. 32, 417–426. Menzies, V., Lyon, D.E., 2010. Integrated review of the association of cytokines with fibromyalgia and fibromyalgia core symptoms. Biol. Res. Nurs. 11 (4), 387–394 (Apr). Mout, R., Willemze, R., Landegent, J.E., 1991. Repeat polymorphisms in the interleukin4 gene. Nucleic Acids Res. 19, 3763. Omoigui, S., 2007. The biochemical origin of pain: the origin of all pain is inflammation and the inflammatory response. Part 2 of 3—inflammatory profile of pain syndromes. Med. Hypotheses 69 (6), 1169–1178. Ortega, E., et al., 2009. Exercise in fibromyalgia and related inflammatory disorders: known effects and unknown chances. Exerc. Immunol. Rev. 15, 42–65. Su, S.Y., Chen, J.J., Lai, C.C., Chen, C.M., Tsai, F.J., 2007. The association between fibromyalgia and polymorphism of monoamine oxidase A and interleukin-4. Clin. Rheumatol. 26 (1), 12–16 (Jan). Torpy, D.J., Papanicolaous, D.A., Lotsikas, A.J., Wilder, R.L., Chrousos, G.P., Pillemer, S.R., 2000. Responses of the sympathetic nervous system and the hypothalamicpituitary-adrenal axis to interleukin-6. Arthritis Rheum. 43, 872–880. Üçeyler, N., Valenza, R., Stock, M., Schedel, R., Sprotte, G., Sommer, C., 2006. Reduced levels of antiinflammatory cytokines in patients with chronic widespread pain. Arthritis Rheum. 54 (8), 2656–2664 (Aug). Wang, H., Moser, M., Schıltenwolf, M., Buchner, M., 2008. Circulating cytokine levels compared to pain in patients with fibromyalgia—a prospective longitudinal study over 6 months. J. Rheumatol. 35, 1366–1370. Wolfe, F., et al., 1990. The American College of Rheumatology 1990 criteria for the classification of fibromyalgia: report of the multicenter criteria committee. Arthritis Rheum. 33, 160–172. Wolfe, F., Ross, K., Anderson, J., Russell, I.J., Hebert, L., 1995. The prevalence and characteristics of fibromyalgia in the general population. Arthritis Rheum. 38, 19–28.