Clinical Neurology and Neurosurgery 120 (2014) 45–48
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Clinical Neurology and Neurosurgery journal homepage: www.elsevier.com/locate/clineuro
Association between promoter region of the uPAR (rs344781) gene polymorphism in genetic susceptibility to migraine without aura in three Iranian hospitals Alireza Zandifar a,b , Samira Soleimani a,b , Niloufar Iraji a,b , Faraidoon Haghdoost a,b , Mohamadhasan Tajaddini a,c , Shaghayegh Haghjooy Javanmard a,∗ a
Physiology Research Center, Department of Physiology, Isfahan University of Medical Sciences, Isfahan, Iran Medical Student Research Center, Isfahan University of Medical Sciences, Isfahan, Iran c School of Pharmacy and Isfahan Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran b
a r t i c l e
i n f o
Article history: Received 14 April 2013 Received in revised form 14 January 2014 Accepted 8 February 2014 Available online 17 February 2014 Keywords: Migraine without aura Urokinase plasminogen activator (uPA) Urokinase plasminogen activator receptor (uPAR) Gene polymorphism Headache severity
a b s t r a c t Introduction: Migraine is a chronic neurological disorder. Inflammation has a key role in migraine pathophysiology. Urokinase plasminogen activator receptor (uPAR) directly involves in inflammatory conditions by facilitating migration of inflammatory cells to different tissues. The aim of this study was to investigate whether uPAR rs344781, common genetic polymorphism in the uPAR promoter region, might be associated with migraine without aura susceptibility in an Iranian population. Methods: We enrolled 103 newly diagnosed patients with migraine and 100 healthy controls. Peripheral blood sample was used for DNA extraction and uPAR rs344781 gene polymorphism was determined. Patients filled HIT-6 as a tool to evaluate headache severity. Results: The genotype frequency of uPAR is significantly different between migraine patients and control subjects. Heterozygote genotype (AG) was statistically more frequent in the patients than the controls (P = 0.001; OR = 2.67, 95% CI = 1.51–4.7). Also G allele was more frequent in the patients. Total HIT-6 score was not significantly different between heterozygote and homozygote patients (55.50 ± 2.22 vs. 49.60 ± 3.68 respectively, P = 0.075). Conclusion: In conclusion, our study showed a significant association between uPAR rs344781 gene promoter polymorphism and migraine without aura susceptibility but not with headache severity. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Migraine is a chronic neurological disorder affecting about 12% of the adult population (males 5.6%, females 17.1%) [1]. World Health Organization rated migraine as one of the most disabling chronic disorders [2]. The clinical presentation includes recurrent headache attacks frequently associated with nausea, vomits, and sensitivity to light and loud sound exposure [3]. Meningeal blood vessels are innervated by C and A␦ fibers that originate in the trigeminal and C1-3 dorsal root ganglions [4]. Activation of the trigeminovascular system stimulates nociceptive neurons on meningeal blood vessels to release vasoactive
∗ Corresponding author at: Physiology Research Center, Isfahan University of Medical Sciences and Health Services, Hezar-Jerib Street, Isfahan, Iran. Tel.: +98 311 7922295; fax: +98 311 7922295. E-mail addresses: sh [email protected], [email protected] (S.H. Javanmard). http://dx.doi.org/10.1016/j.clineuro.2014.02.003 0303-8467/© 2014 Elsevier B.V. All rights reserved.
neuropeptides such as substance P (SP), calcitonin gene-related peptide (CGRP), neurokinin A (NKA), and nitric oxide (NO) [5]. These neuropeptides cause vasodilatation of meningeal blood vessels, extravasattion of inflammatory chemicals and sterile inflammation. Then information is relayed to the thalamus and cortex that generates pain of migraine [6]. Urokinase-type plasminogen activator (uPA) is one of the two physiological serine proteases responsible for the activation of plasminongen to plasmin. uPA activity is regulated by its inhibitors (PAI-1 and PAI-2), its receptor (uPAR) and an expanding list of their interacting proteins. In addition to plasminogen activation, this system also plays important roles in the regulation of many cellular processes including cell proliferation, adhesion and migration [7]. The urokinase plasminogen activator receptor (uPAR; CD87 antigen) is expressed on different cell types including neutrophils, lymphocytes, monocytes, macrophages and vascular endothelial cells. uPAR take parts in several immune functions including migration, adhesion, angiogenesis, fibrinolysis and cell proliferation. uPAR interacts with urokinase plasminogen activator (uPA),
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extracellular matrix (ECM) protein vitronectin and intracellular signaling molecules such as integrins and a G-coupled receptor [8]. Also uPAR directly involves in inflammatory conditions by facilitating migration of inflammatory cells to different tissues [9]. The human uPAR gene has been mapped to chromosome 19 (19q13.2) which contains 7 exons and 6 introns. One of the singlenucleotide polymorphisms (SNPs) that have been identified in the active promoter region of the uPAR gene is A-to-G transition at position –465. (uPAR –465 A G, rs344781), and influence uPAR gene expression [8]. Regard to possible role of inflammation in pathophysiology of migraine and effect of uPAR in inflammatory conditions, the aim of this study was investigation whether uPAR rs344781, common genetic polymorphism in the uPAR promoter region, might be associated with migraine without aura susceptibility in an Iranian population. 2. Methods and materials 2.1. Patients and settings This case-control study was conducted on 103 consecutive patients that were diagnosed as migraine without aura and a control group that were selected from people who accompanied the patients. Convenient sampling and no randomized sampling method were applied. Diagnosis of migraine was based on International Headache Society criteria (IHS) [10] from outpatient neurology clinics of three hospitals between November 2011 and June 2012 in Isfahan, Iran. Headache and socio-demographic characteristics including age, sex, frequency of headaches and family history of the subjects were asked. Patients who have not taken any medications for their migraine and also had at least a three-month history of headaches prior to the diagnoses were consecutively selected. The controls were selected from accompanying people of patients with migraine and other neurological disorders who did not have any history for migraines and also any family history of migraine in the first degree relatives. Case and control groups were matched for age and sex. An informed consent was taken from participants before entering the study. The study was approved by the Ethical Committee of Isfahan University of Medical Sciences (Grant No: 191006). 2.2. HIT-6 questionnaire Each patient filled HIT-6 questionnaire for assessment severity of headaches. HIT-6 questionnaire consists of six questions that show six dimensions: pain severity, role functioning, social functioning, vitality, emotional distress, and cognitive functioning. Each question has 5 available answers: never, rarely, sometimes, very often and always. Choices have been scored between 6 and 13 respectively. The total score is between 36 and 78 [11]. In the present study we used the Persian version of HIT-6 that has been validated in our pervious study [12]. 2.3. DNA extraction and genotyping Two mL of peripheral blood was collected from each participant. Genomic DNA samples were extracted from peripheral whole blood using the AccuPrep Genomic DNA Extraction kit (Bioneer Inc., Korea) according to the manufacturer’s protocol. The SNP rs344781 was identified by the NCBI data bank and primers were designed by Beacon Designer 7.91 to flank the coding regions (PREMIER Biosoft International, USA and synthesized by TIB MOLBIOL, Germany). The forward primer was 5 -TGGAGGGCTCAACACATTC-3 and reverse primer was 5 GTGGCTGATTATTTGGCTAACAG-3 .
Table 1 Demographic and clinical characteristics of patients with migraine. Characteristic
Mean ± S.E. or percentage
Frequency of headache per month
8.32 ± 0.81
Family history for migraine Positive Negative
77% 23%
Total HIT-6 score
53.51 ± 1.93
HIT-6: headache impact test.
Genotyping was done by high-resolution melt (HRM) assay using a Rotor-Gene 6000 instrument (Corbett Life Science, Australia). PCR reactions were carried out in duplicate in 20 l of final volume using the type-it HRM kit (Qiagen), HRM PCR buffer, HotStarTaq Plus DNA polymerase, nucleotides and EvaGreen dye, and 30 ng DNA. The PCR program consisted of an initial denaturation–activation step at 95 ◦ C for 5 min, followed by a 40-cycle program (denaturation at 95 ◦ C for 15 s, annealing conditions 55 ◦ C for 5 s, 72 ◦ C for 15 s; a HRM step from 70 to 95 ◦ C rising at 0.1 ◦ C/s. Curves for each duplicate were checked on the shape and peak height to meet reproducibility. Normalized and temperature-shifted melting curves from HRM, suggestive of single nucleotide polymorphisms (SNP), were distinguished and the samples were subjected to direct sequencing. 2.4. Data analysis The sample size was calculated using the 2 proportion Pocock’s formula. We analyzed our data with SPSS software (version 18.0, Chicago IL). An independent t-test was used for quantitative variables between two groups. Relation between polymorphism (homozygous and heterozygous) and different categorized variables (age, sex, case-control) was established using Chi-square test and calculation of odds ratio (CI = 95%). The significant level was considered as P < 0.05. 3. Results From 103 subjects with migraine and 100 healthy controls, DNA samples were analyzed for uPAR rs344781 polymorphism. In the case and control group 82.6% and 78% of participants were female respectively and the difference was not statistically significant. Also there were no significant differences in the mean ages (34.08 ± 1.01 vs. 34.77 ± 1.06 years), education level (40% vs. 36% without academic degrees), and residency (51.5% vs. 57.6% urban) between case and control groups, respectively. Subjects’ characteristics of the case group are reported in Table 1. The allele frequencies of A and G in the study population were 298 (73.4%) and 108 (26.6%) respectively. The mutant allele was more frequent in the migraine patients than the control subjects (32.6% vs. 20.5%, P = 0.003) (Table 2). The frequencies of homozygote and heterozygote genotypes in the study population were 95 (46.8%) and 108 (53.2%) respectively. The heterozygote genotype was more frequent in the migraine patients than the control subjects (65% vs. 41%, P = 0.001) (Table 2). The comparison of allele frequencies, between males and females in the case and also control group showed that there is no significant difference in the alleles distribution according to the sex (P = 0.349 and 0.230 respectively). Further analysis in the migraine subjects showed that the distribution of uPAR rs344781 gene polymorphism was not associated with the presence of family history of migraine (P = 0.985).
A. Zandifar et al. / Clinical Neurology and Neurosurgery 120 (2014) 45–48
47
Table 2 Distribution of allele and genotype of rs344781 gene polymorphism in the case and control groups. Case Alleles A G
Control 139 (67.4%) 67 (32.6%)
Genotype Homozygote (AA) Heterozygote (AG)
36 (35%) 67 (65%)
159 (79.5%) 41 (20.5%) 59 (59%) 41 (41%)
Comparison of headache frequency per month and total HIT6 score between the homozygote and heterozygote patients are shown in Table 3. The analyses showed that the mentioned factors are not significantly different between the homozygote and heterozygote patients. 4. Discussion The present study showed that the uPAR rs344781 gene promoter polymorphism is associated with migraine without aura susceptibility in an Iranian population. Our data demonstrated high risk for migraine in individuals carrier for G allele in comparison with A allele carriers. Also subjects with the AG genotype showed an increased risk for migraine compared with carriers of the AA genotype. Also we found no significant difference in severity of headache between heterozygote and homozygote patients. Several evidences showed the plasminogen activator (PA) system in addition to its function in thrombolysis or extracellular matrix degeneration, plays a key role in cellular migration, adhesion, differentiation and proliferation through intracellular signaling [13]. uPAR as a glycosylphosptatidylinositol (GPI)-linked receptor of uPA, precipitates in inflammatory cytokines secretion such as TNFa, IL-1b, IL-6 and IL-8 from mononuclear cells. On the other hand some studies reported increase of IL-6, IL-2 and TNF in migraineurs that established the role of inflammation in pathophysiology of migraine [14]. Several groups of proteases, inhibitors, receptors and modulators are affected by the PA system; Urokinase and uPA-generated plasmin can release MMPs, as well as growth factors such as VEGF, bFGF, HGF, TGFb, and PDGF [15,16]. Imamura et al. showed increased plasma matrix metalloproteinase-9 levels in patients with migraine [17]. In another study Ishizaki et al. showed TGFbeta1 in Platelet poor plasma was significantly increased in patients with migraine during headache-free periods. This study suggests TGF-beta1 may play some role in the development of migraine headache [18]. uPAR is expressed on different cells that involve in immunologic processes including monocytes, activated T cells and macrophages in addition is present on endothelial cells, keratinocytes, fibroblasts, smooth muscle cells, megakaryocytes and certain tumor cells [9]. Also several studies established significant effects of
Table 3 Comparison of headache frequency per month and total HIT-6 score between homozygote and heterozygote patients. Patients Homozygote (AA) Frequency of headache per month
7.71 ± 1.61
Total HIT-6 score
49.60 ± 3.68
Data are given as mean ± S.E.
P-value Heterozygote (AG) 8.63 ± 0.93
0.301
55.50 ± 2.22
0.075
P-Value
OR (95% confidence interval)
0.003
1.86 (1.19–2.93)
0.001
2.67 (1.51–4.72)
endothelial dysfunction and vascular smooth muscles dysfunction in pathogenesis of migraine [19,20]. These mechanisms could explain high prevalence of cerebrovascular disease such as stroke in migraineur specifically migraine with aura [21]. In the other hand the uPA/uPAR system plays a key role in the pathogenesis of vascular diseases [22]. However the therapeutics effect of Intravenous recombinant tissue type PA (rtPA, alteplase) is approved for the treatment of acute ischemic stroke [23] but some animal studies showed that both t-PA-deficiency and its inhibition with the natural t-PA inhibitor are associated with significant neuronal survival and decreases infarct size in comparison with control group [24,25]. Also studies indicated that increase of t-PA activity occurs following cerebral ischemia, and high levels of both t-PA and PAI-1 reported in patients with a history of stroke [26]. Evidences showed that uPAR or uPAR-associated pathways are involved in pathological conditions of the central nervous system such as epileptic disorders, stroke, multiple sclerosis, alzheimer’s disease, cerebral malaria, HIV-associated leukoencephalopathy and encephalitis [27]. In the recent years various studies focused on finding an association between different gene polymorphisms and migraine [28,29]. Regard to substantial role of uPAR in several pathological processes that might be effective in pathogenesis of migraine we anticipated there was an association between uPAR polymorphism and susceptibly to migraine. Results of present study showed that risk of having migraine in subjects who are carrier of G allele (the variant allele) is 2.67 times more than noncarriers and the G allele can be a risk factor for migraine. There are several studies that have investigated the uPAR polymorphism in different inflammatory conditions. Some studies have investigated the association of uPAR rs344781 genetic polymorphism and some malignant diseases such as nonsmall cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC). Shih et al. reported that individuals with uPAR variant polymorphic homozygote had a higher risk of having NSCLC compared with wild homozygote and heterozygote individuals. But study of Weng et al. could not find any significant relation between uPAR TT or CC genotypes with HCC but their results suggested that the predicted effect of the uPAR T/C variant on the risk of HCC is age dependent, and the increase of gene sensitivity in older individuals with uPAR gene polymorphism could be a possible cause [30,31]. Another study showed that uPAR rs344781 gene variant is associated with the systemic sclerosis (SSc) vascular phenotypes. The results of this study confirm the fact that uPAR plays a key roles in angiogenesis, cardiovascular disorders, and vascular remodeling and also is confirming our results in which we know migraine is a vascular disorder [8]. To the best of our knowledge, this is the first study to demonstrate the association between migraine and the uPAR gene promoter polymorphism. However the small sample size may be a limitation of the present study. Therefore this is a preliminary conclusion. Further studies with a larger sample size from a more diverse ethnic population are needed to confirm these findings. In conclusion our study demonstrated a significant association between uPAR rs344781 gene promoter polymorphism and
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migraine without aura susceptibility but we found no association between severity of headache and uPAR gene promoter polymorphism. Declaration of interest All authors have read and approved the content of the manuscript. The authors declare no conflict of interest. Authors’ contributions SS, NI, FH and MT contributed in data collection and analysis. They also contributed in drafting the manuscript. AZ and SHJ had substantial contributions to conception and design of the study, analysis of the data and drafting the manuscript. All authors read and approved the final manuscript. Acknowledgements This study was supported by Isfahan University of Medical Sciences, Isfahan, Iran (Grant # 191006). Authors would like to appreciate generous collaborations of Prof. Mohammad Saadatnia, Prof. Mohammad Reza Najafi and Prof. Abbas Ghorbani (Department of Neurology and Isfahan Neurosciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran) for diagnosing the patients. References [1] Lipton RB, Bigal ME, Diamond M, Freitag F, Reed ML, Stewart WF. Migraine prevalence, disease burden, and the need for preventive therapy. Neurology 2007;68:343–9. [2] Stewart WF, Ricci JA, Chee E, Morganstein D, Lipton R. Lost productive time and cost due to common pain conditions in the US workforce. JAMA: The Journal of the AMERICAN Medical Association 2003;290:2443–54. [3] Bigal ME, Ferrari M, Silberstein SD, Lipton RB, Goadsby PJ. Migraine in the triptan era: lessons from epidemiology, pathophysiology, and clinical science. Headache 2009;49(Suppl. 1):S21–33. [4] Bernstein C, Burstein R. Sensitization of the trigeminovascular pathway: perspective and implications to migraine pathophysiology. Journal of Clinical Neurology (Seoul, Korea) 2012;8:89–99. [5] Moulton EA, Burstein R, Tully S, Hargreaves R, Becerra L, Borsook D. Interictal dysfunction of a brainstem descending modulatory center in migraine patients. PLoS ONE 2008;3:e3799. [6] Richter F, Lehmenkuhler A. Cortical spreading depression (CSD): a neurophysiological correlate of migraine aura. Schmerz 2008;22:544–6, 548–550. [7] Ngo JC, Jiang L, Lin Z, Yuan C, Chen Z, Zhang X, et al. Structural basis for therapeutic intervention of uPA/uPAR system. Current Drug Targets 2011;12:1729–43. [8] Manetti M, Allanore Y, Revillod L, Fatini C, Guiducci S, Cuomo G, et al. A genetic variation located in the promoter region of the UPAR (CD87) gene is associated with the vascular complications of systemic sclerosis. Arthritis and Rheumatism 2011;63:247–56. [9] Thuno M, Macho B, Eugen-Olsen J. suPAR: the molecular crystal ball. Disease Markers 2009;27:157–72.
[10] The International Classification of Headache Disorders: 2nd edition. Cephalalgia 2004;24(Suppl. 1):9–160. [11] Kosinski M, Bayliss MS, Bjorner JB, Ware Jr JE, Garber WH, Batenhorst A, et al. A six-item short-form survey for measuring headache impact: the HIT-6. Quality of Life Research 2003;12:963–74. [12] Zandifar A, Banihashemi M, Haghdoost F, Masjedi SS, Manouchehri N, Asgari F, et al. Reliability and Validity of the Persian HIT-6 Questionnaire in Migraine and Tension-type Headache. Pain Practice: The Official Journal of World Institute of Pain 2013, in press. [13] Blasi F, Carmeliet P. uPAR: a versatile signalling orchestrator. Nature Reviews: Molecular Cell Biology 2002;3:932–43. [14] Yilmaz IA, Ozge A, Erdal ME, Edgunlu TG, Cakmak SE, Yalin OO. Cytokine polymorphism in patients with migraine: some suggestive clues of migraine and inflammation. Pain Medicine 2010;11:492–7. [15] Kuzuya M, Iguchi A. Role of matrix metalloproteinases in vascular remodeling. Journal of Atherosclerosis and Thrombosis 2003;10:275–82. [16] Tkachuk VA, Plekhanova OS, Parfyonova YV. Regulation of arterial remodeling and angiogenesis by urokinase-type plasminogen activator. Canadian Journal of Physiology and Pharmacology 2009;87:231–51. [17] Imamura K, Takeshima T, Fusayasu E, Nakashima K. Increased plasma matrix metalloproteinase-9 levels in migraineurs. Headache 2008;48:135–9. [18] Ishizaki K, Takeshima T, Fukuhara Y, Araki H, Nakaso K, Kusumi M, et al. Increased plasma transforming growth factor-beta1 in migraine. Headache 2005;45:1224–8. [19] Napoli R, Guardasole V, Zarra E, Matarazzo M, D’Anna C, Sacca F, et al. Vascular smooth muscle cell dysfunction in patients with migraine. Neurology 2009;72:2111–4. [20] Lee ST, Chu K, Jung KH, Kim DH, Kim EH, Choe VN, et al. Decreased number and function of endothelial progenitor cells in patients with migraine. Neurology 2008;70:1510–7. [21] Milhaud D, Bogousslavsky J, van Melle G, Liot P. Ischemic stroke and active migraine. Neurology 2001;57:1805–11. [22] Nicholl SM, Roztocil E, Davies MG. Plasminogen activator system and vascular disease. Current Vascular Pharmacology 2006;4:101–16. [23] Albers GW. Management of acute ischemic stroke. An update for primary care physicians. Western Journal of Medicine 1997;166:253–62. [24] Wang YF, Tsirka SE, Strickland S, Stieg PE, Soriano SG, Lipton SA. Tissue plasminogen activator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice. Nature Medicine 1998;4:228–31. [25] Nagai N, De Mol M, Lijnen HR, Carmeliet P, Collen D. Role of plasminogen system components in focal cerebral ischemic infarction: a gene targeting and gene transfer study in mice. Circulation 1999;99:2440–4. [26] Lindgren A, Lindoff C, Norrving B, Astedt B, Johansson BB. Tissue plasminogen activator and plasminogen activator inhibitor-1 in stroke patients. Stroke 1996;27:1066–71. [27] Bruneau N, Szepetowski P. The role of the urokinase receptor in epilepsy, in disorders of language, cognition, communication and behavior, and in the central nervous system. Current Pharmaceutical Design 2011;17:1914–23. [28] Bhaskar S, Abdullah JM, Ghazali MM. Coexistence of CACNA1A, ATP1A2, and KCNN3 gene mutation in migraine patients with human platelet polymorphism. Neurosciences (Riyadh, Saudi Arabia) 2008;13:356–8. [29] Bhaskar S, Abdullah JM. Platelet alloantigen polymorphism and migraine headaches. Neurosciences (Riyadh, Saudi Arabia) 2013;18:185–6. [30] Shih CM, Kuo WH, Lin CW, Chen W, Cheng WE, Chen SC, et al. Association of polymorphisms in the genes of the urokinase plasminogen activation system with susceptibility to and severity of non-small cell lung cancer. Clinica Chimica Acta; International Journal of Clinical Chemistry 2011;412:194–8. [31] Weng CJ, Tsai CM, Chen YC, Hsieh YH, Lin CW, Liu YF, et al. Evaluation of the association of urokinase plasminogen activator system gene polymorphisms with susceptibility and pathological development of hepatocellular carcinoma. Annals of Surgical Oncology 2010;17:3394–401.
Contents lists available at ScienceDirect
Clinical Neurology and Neurosurgery journal homepage: www.elsevier.com/locate/clineuro
Association between promoter region of the uPAR (rs344781) gene polymorphism in genetic susceptibility to migraine without aura in three Iranian hospitals Alireza Zandifar a,b , Samira Soleimani a,b , Niloufar Iraji a,b , Faraidoon Haghdoost a,b , Mohamadhasan Tajaddini a,c , Shaghayegh Haghjooy Javanmard a,∗ a
Physiology Research Center, Department of Physiology, Isfahan University of Medical Sciences, Isfahan, Iran Medical Student Research Center, Isfahan University of Medical Sciences, Isfahan, Iran c School of Pharmacy and Isfahan Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran b
a r t i c l e
i n f o
Article history: Received 14 April 2013 Received in revised form 14 January 2014 Accepted 8 February 2014 Available online 17 February 2014 Keywords: Migraine without aura Urokinase plasminogen activator (uPA) Urokinase plasminogen activator receptor (uPAR) Gene polymorphism Headache severity
a b s t r a c t Introduction: Migraine is a chronic neurological disorder. Inflammation has a key role in migraine pathophysiology. Urokinase plasminogen activator receptor (uPAR) directly involves in inflammatory conditions by facilitating migration of inflammatory cells to different tissues. The aim of this study was to investigate whether uPAR rs344781, common genetic polymorphism in the uPAR promoter region, might be associated with migraine without aura susceptibility in an Iranian population. Methods: We enrolled 103 newly diagnosed patients with migraine and 100 healthy controls. Peripheral blood sample was used for DNA extraction and uPAR rs344781 gene polymorphism was determined. Patients filled HIT-6 as a tool to evaluate headache severity. Results: The genotype frequency of uPAR is significantly different between migraine patients and control subjects. Heterozygote genotype (AG) was statistically more frequent in the patients than the controls (P = 0.001; OR = 2.67, 95% CI = 1.51–4.7). Also G allele was more frequent in the patients. Total HIT-6 score was not significantly different between heterozygote and homozygote patients (55.50 ± 2.22 vs. 49.60 ± 3.68 respectively, P = 0.075). Conclusion: In conclusion, our study showed a significant association between uPAR rs344781 gene promoter polymorphism and migraine without aura susceptibility but not with headache severity. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Migraine is a chronic neurological disorder affecting about 12% of the adult population (males 5.6%, females 17.1%) [1]. World Health Organization rated migraine as one of the most disabling chronic disorders [2]. The clinical presentation includes recurrent headache attacks frequently associated with nausea, vomits, and sensitivity to light and loud sound exposure [3]. Meningeal blood vessels are innervated by C and A␦ fibers that originate in the trigeminal and C1-3 dorsal root ganglions [4]. Activation of the trigeminovascular system stimulates nociceptive neurons on meningeal blood vessels to release vasoactive
∗ Corresponding author at: Physiology Research Center, Isfahan University of Medical Sciences and Health Services, Hezar-Jerib Street, Isfahan, Iran. Tel.: +98 311 7922295; fax: +98 311 7922295. E-mail addresses: sh [email protected], [email protected] (S.H. Javanmard). http://dx.doi.org/10.1016/j.clineuro.2014.02.003 0303-8467/© 2014 Elsevier B.V. All rights reserved.
neuropeptides such as substance P (SP), calcitonin gene-related peptide (CGRP), neurokinin A (NKA), and nitric oxide (NO) [5]. These neuropeptides cause vasodilatation of meningeal blood vessels, extravasattion of inflammatory chemicals and sterile inflammation. Then information is relayed to the thalamus and cortex that generates pain of migraine [6]. Urokinase-type plasminogen activator (uPA) is one of the two physiological serine proteases responsible for the activation of plasminongen to plasmin. uPA activity is regulated by its inhibitors (PAI-1 and PAI-2), its receptor (uPAR) and an expanding list of their interacting proteins. In addition to plasminogen activation, this system also plays important roles in the regulation of many cellular processes including cell proliferation, adhesion and migration [7]. The urokinase plasminogen activator receptor (uPAR; CD87 antigen) is expressed on different cell types including neutrophils, lymphocytes, monocytes, macrophages and vascular endothelial cells. uPAR take parts in several immune functions including migration, adhesion, angiogenesis, fibrinolysis and cell proliferation. uPAR interacts with urokinase plasminogen activator (uPA),
46
A. Zandifar et al. / Clinical Neurology and Neurosurgery 120 (2014) 45–48
extracellular matrix (ECM) protein vitronectin and intracellular signaling molecules such as integrins and a G-coupled receptor [8]. Also uPAR directly involves in inflammatory conditions by facilitating migration of inflammatory cells to different tissues [9]. The human uPAR gene has been mapped to chromosome 19 (19q13.2) which contains 7 exons and 6 introns. One of the singlenucleotide polymorphisms (SNPs) that have been identified in the active promoter region of the uPAR gene is A-to-G transition at position –465. (uPAR –465 A G, rs344781), and influence uPAR gene expression [8]. Regard to possible role of inflammation in pathophysiology of migraine and effect of uPAR in inflammatory conditions, the aim of this study was investigation whether uPAR rs344781, common genetic polymorphism in the uPAR promoter region, might be associated with migraine without aura susceptibility in an Iranian population. 2. Methods and materials 2.1. Patients and settings This case-control study was conducted on 103 consecutive patients that were diagnosed as migraine without aura and a control group that were selected from people who accompanied the patients. Convenient sampling and no randomized sampling method were applied. Diagnosis of migraine was based on International Headache Society criteria (IHS) [10] from outpatient neurology clinics of three hospitals between November 2011 and June 2012 in Isfahan, Iran. Headache and socio-demographic characteristics including age, sex, frequency of headaches and family history of the subjects were asked. Patients who have not taken any medications for their migraine and also had at least a three-month history of headaches prior to the diagnoses were consecutively selected. The controls were selected from accompanying people of patients with migraine and other neurological disorders who did not have any history for migraines and also any family history of migraine in the first degree relatives. Case and control groups were matched for age and sex. An informed consent was taken from participants before entering the study. The study was approved by the Ethical Committee of Isfahan University of Medical Sciences (Grant No: 191006). 2.2. HIT-6 questionnaire Each patient filled HIT-6 questionnaire for assessment severity of headaches. HIT-6 questionnaire consists of six questions that show six dimensions: pain severity, role functioning, social functioning, vitality, emotional distress, and cognitive functioning. Each question has 5 available answers: never, rarely, sometimes, very often and always. Choices have been scored between 6 and 13 respectively. The total score is between 36 and 78 [11]. In the present study we used the Persian version of HIT-6 that has been validated in our pervious study [12]. 2.3. DNA extraction and genotyping Two mL of peripheral blood was collected from each participant. Genomic DNA samples were extracted from peripheral whole blood using the AccuPrep Genomic DNA Extraction kit (Bioneer Inc., Korea) according to the manufacturer’s protocol. The SNP rs344781 was identified by the NCBI data bank and primers were designed by Beacon Designer 7.91 to flank the coding regions (PREMIER Biosoft International, USA and synthesized by TIB MOLBIOL, Germany). The forward primer was 5 -TGGAGGGCTCAACACATTC-3 and reverse primer was 5 GTGGCTGATTATTTGGCTAACAG-3 .
Table 1 Demographic and clinical characteristics of patients with migraine. Characteristic
Mean ± S.E. or percentage
Frequency of headache per month
8.32 ± 0.81
Family history for migraine Positive Negative
77% 23%
Total HIT-6 score
53.51 ± 1.93
HIT-6: headache impact test.
Genotyping was done by high-resolution melt (HRM) assay using a Rotor-Gene 6000 instrument (Corbett Life Science, Australia). PCR reactions were carried out in duplicate in 20 l of final volume using the type-it HRM kit (Qiagen), HRM PCR buffer, HotStarTaq Plus DNA polymerase, nucleotides and EvaGreen dye, and 30 ng DNA. The PCR program consisted of an initial denaturation–activation step at 95 ◦ C for 5 min, followed by a 40-cycle program (denaturation at 95 ◦ C for 15 s, annealing conditions 55 ◦ C for 5 s, 72 ◦ C for 15 s; a HRM step from 70 to 95 ◦ C rising at 0.1 ◦ C/s. Curves for each duplicate were checked on the shape and peak height to meet reproducibility. Normalized and temperature-shifted melting curves from HRM, suggestive of single nucleotide polymorphisms (SNP), were distinguished and the samples were subjected to direct sequencing. 2.4. Data analysis The sample size was calculated using the 2 proportion Pocock’s formula. We analyzed our data with SPSS software (version 18.0, Chicago IL). An independent t-test was used for quantitative variables between two groups. Relation between polymorphism (homozygous and heterozygous) and different categorized variables (age, sex, case-control) was established using Chi-square test and calculation of odds ratio (CI = 95%). The significant level was considered as P < 0.05. 3. Results From 103 subjects with migraine and 100 healthy controls, DNA samples were analyzed for uPAR rs344781 polymorphism. In the case and control group 82.6% and 78% of participants were female respectively and the difference was not statistically significant. Also there were no significant differences in the mean ages (34.08 ± 1.01 vs. 34.77 ± 1.06 years), education level (40% vs. 36% without academic degrees), and residency (51.5% vs. 57.6% urban) between case and control groups, respectively. Subjects’ characteristics of the case group are reported in Table 1. The allele frequencies of A and G in the study population were 298 (73.4%) and 108 (26.6%) respectively. The mutant allele was more frequent in the migraine patients than the control subjects (32.6% vs. 20.5%, P = 0.003) (Table 2). The frequencies of homozygote and heterozygote genotypes in the study population were 95 (46.8%) and 108 (53.2%) respectively. The heterozygote genotype was more frequent in the migraine patients than the control subjects (65% vs. 41%, P = 0.001) (Table 2). The comparison of allele frequencies, between males and females in the case and also control group showed that there is no significant difference in the alleles distribution according to the sex (P = 0.349 and 0.230 respectively). Further analysis in the migraine subjects showed that the distribution of uPAR rs344781 gene polymorphism was not associated with the presence of family history of migraine (P = 0.985).
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Table 2 Distribution of allele and genotype of rs344781 gene polymorphism in the case and control groups. Case Alleles A G
Control 139 (67.4%) 67 (32.6%)
Genotype Homozygote (AA) Heterozygote (AG)
36 (35%) 67 (65%)
159 (79.5%) 41 (20.5%) 59 (59%) 41 (41%)
Comparison of headache frequency per month and total HIT6 score between the homozygote and heterozygote patients are shown in Table 3. The analyses showed that the mentioned factors are not significantly different between the homozygote and heterozygote patients. 4. Discussion The present study showed that the uPAR rs344781 gene promoter polymorphism is associated with migraine without aura susceptibility in an Iranian population. Our data demonstrated high risk for migraine in individuals carrier for G allele in comparison with A allele carriers. Also subjects with the AG genotype showed an increased risk for migraine compared with carriers of the AA genotype. Also we found no significant difference in severity of headache between heterozygote and homozygote patients. Several evidences showed the plasminogen activator (PA) system in addition to its function in thrombolysis or extracellular matrix degeneration, plays a key role in cellular migration, adhesion, differentiation and proliferation through intracellular signaling [13]. uPAR as a glycosylphosptatidylinositol (GPI)-linked receptor of uPA, precipitates in inflammatory cytokines secretion such as TNFa, IL-1b, IL-6 and IL-8 from mononuclear cells. On the other hand some studies reported increase of IL-6, IL-2 and TNF in migraineurs that established the role of inflammation in pathophysiology of migraine [14]. Several groups of proteases, inhibitors, receptors and modulators are affected by the PA system; Urokinase and uPA-generated plasmin can release MMPs, as well as growth factors such as VEGF, bFGF, HGF, TGFb, and PDGF [15,16]. Imamura et al. showed increased plasma matrix metalloproteinase-9 levels in patients with migraine [17]. In another study Ishizaki et al. showed TGFbeta1 in Platelet poor plasma was significantly increased in patients with migraine during headache-free periods. This study suggests TGF-beta1 may play some role in the development of migraine headache [18]. uPAR is expressed on different cells that involve in immunologic processes including monocytes, activated T cells and macrophages in addition is present on endothelial cells, keratinocytes, fibroblasts, smooth muscle cells, megakaryocytes and certain tumor cells [9]. Also several studies established significant effects of
Table 3 Comparison of headache frequency per month and total HIT-6 score between homozygote and heterozygote patients. Patients Homozygote (AA) Frequency of headache per month
7.71 ± 1.61
Total HIT-6 score
49.60 ± 3.68
Data are given as mean ± S.E.
P-value Heterozygote (AG) 8.63 ± 0.93
0.301
55.50 ± 2.22
0.075
P-Value
OR (95% confidence interval)
0.003
1.86 (1.19–2.93)
0.001
2.67 (1.51–4.72)
endothelial dysfunction and vascular smooth muscles dysfunction in pathogenesis of migraine [19,20]. These mechanisms could explain high prevalence of cerebrovascular disease such as stroke in migraineur specifically migraine with aura [21]. In the other hand the uPA/uPAR system plays a key role in the pathogenesis of vascular diseases [22]. However the therapeutics effect of Intravenous recombinant tissue type PA (rtPA, alteplase) is approved for the treatment of acute ischemic stroke [23] but some animal studies showed that both t-PA-deficiency and its inhibition with the natural t-PA inhibitor are associated with significant neuronal survival and decreases infarct size in comparison with control group [24,25]. Also studies indicated that increase of t-PA activity occurs following cerebral ischemia, and high levels of both t-PA and PAI-1 reported in patients with a history of stroke [26]. Evidences showed that uPAR or uPAR-associated pathways are involved in pathological conditions of the central nervous system such as epileptic disorders, stroke, multiple sclerosis, alzheimer’s disease, cerebral malaria, HIV-associated leukoencephalopathy and encephalitis [27]. In the recent years various studies focused on finding an association between different gene polymorphisms and migraine [28,29]. Regard to substantial role of uPAR in several pathological processes that might be effective in pathogenesis of migraine we anticipated there was an association between uPAR polymorphism and susceptibly to migraine. Results of present study showed that risk of having migraine in subjects who are carrier of G allele (the variant allele) is 2.67 times more than noncarriers and the G allele can be a risk factor for migraine. There are several studies that have investigated the uPAR polymorphism in different inflammatory conditions. Some studies have investigated the association of uPAR rs344781 genetic polymorphism and some malignant diseases such as nonsmall cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC). Shih et al. reported that individuals with uPAR variant polymorphic homozygote had a higher risk of having NSCLC compared with wild homozygote and heterozygote individuals. But study of Weng et al. could not find any significant relation between uPAR TT or CC genotypes with HCC but their results suggested that the predicted effect of the uPAR T/C variant on the risk of HCC is age dependent, and the increase of gene sensitivity in older individuals with uPAR gene polymorphism could be a possible cause [30,31]. Another study showed that uPAR rs344781 gene variant is associated with the systemic sclerosis (SSc) vascular phenotypes. The results of this study confirm the fact that uPAR plays a key roles in angiogenesis, cardiovascular disorders, and vascular remodeling and also is confirming our results in which we know migraine is a vascular disorder [8]. To the best of our knowledge, this is the first study to demonstrate the association between migraine and the uPAR gene promoter polymorphism. However the small sample size may be a limitation of the present study. Therefore this is a preliminary conclusion. Further studies with a larger sample size from a more diverse ethnic population are needed to confirm these findings. In conclusion our study demonstrated a significant association between uPAR rs344781 gene promoter polymorphism and
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migraine without aura susceptibility but we found no association between severity of headache and uPAR gene promoter polymorphism. Declaration of interest All authors have read and approved the content of the manuscript. The authors declare no conflict of interest. Authors’ contributions SS, NI, FH and MT contributed in data collection and analysis. They also contributed in drafting the manuscript. AZ and SHJ had substantial contributions to conception and design of the study, analysis of the data and drafting the manuscript. All authors read and approved the final manuscript. Acknowledgements This study was supported by Isfahan University of Medical Sciences, Isfahan, Iran (Grant # 191006). Authors would like to appreciate generous collaborations of Prof. Mohammad Saadatnia, Prof. Mohammad Reza Najafi and Prof. Abbas Ghorbani (Department of Neurology and Isfahan Neurosciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran) for diagnosing the patients. References [1] Lipton RB, Bigal ME, Diamond M, Freitag F, Reed ML, Stewart WF. Migraine prevalence, disease burden, and the need for preventive therapy. Neurology 2007;68:343–9. [2] Stewart WF, Ricci JA, Chee E, Morganstein D, Lipton R. Lost productive time and cost due to common pain conditions in the US workforce. JAMA: The Journal of the AMERICAN Medical Association 2003;290:2443–54. [3] Bigal ME, Ferrari M, Silberstein SD, Lipton RB, Goadsby PJ. Migraine in the triptan era: lessons from epidemiology, pathophysiology, and clinical science. Headache 2009;49(Suppl. 1):S21–33. [4] Bernstein C, Burstein R. Sensitization of the trigeminovascular pathway: perspective and implications to migraine pathophysiology. Journal of Clinical Neurology (Seoul, Korea) 2012;8:89–99. [5] Moulton EA, Burstein R, Tully S, Hargreaves R, Becerra L, Borsook D. Interictal dysfunction of a brainstem descending modulatory center in migraine patients. PLoS ONE 2008;3:e3799. [6] Richter F, Lehmenkuhler A. Cortical spreading depression (CSD): a neurophysiological correlate of migraine aura. Schmerz 2008;22:544–6, 548–550. [7] Ngo JC, Jiang L, Lin Z, Yuan C, Chen Z, Zhang X, et al. Structural basis for therapeutic intervention of uPA/uPAR system. Current Drug Targets 2011;12:1729–43. [8] Manetti M, Allanore Y, Revillod L, Fatini C, Guiducci S, Cuomo G, et al. A genetic variation located in the promoter region of the UPAR (CD87) gene is associated with the vascular complications of systemic sclerosis. Arthritis and Rheumatism 2011;63:247–56. [9] Thuno M, Macho B, Eugen-Olsen J. suPAR: the molecular crystal ball. Disease Markers 2009;27:157–72.
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